The Interactive Fly
Zygotically transcribed genes
Ubiquitin (Ub) is a member of a family of conserved polypeptides that are covalently attached to protein substrates. Multiple rounds of modification create a poly(Ub) chain on the substrate that targets the substrate for degradation by the proteasome. The transfer of free Ub onto a protein substrate is a multistep process. E1 activates free Ub at the expense of ATP. Ub is then transferred to an E2 (or ubiquitin protein-conjugating enzyme). It is believed that each E2 is responsible for ubiquitinating distinct substrates. Although a free E2 enzyme may directly transfer Ub onto a substrate in a purified system, this reaction is promoted by additional proteins referred to as E3s or ubiquitin protein ligases. Some E3s act as intermediary Ub carriers in the transfer of Ub from E2 to substrate. Other E3s act as adapters, tethering E2 to E2's substrates. It turns out that a variety of structurally distinct E3 proteins each serve to regulate the interaction between E2 proteins and various distinct substrates.
Proteasome-dependent and autophagy-mediated degradation of eukaryotic cellular proteins represent the two major proteostatic mechanisms that are critically implicated in a number of signaling pathways and cellular processes. Deregulation of functions engaged in protein elimination frequently leads to development of morbid states and diseases. In this context, and through the utilization of GAL4/UAS genetic tool, this study examined the in vivo contribution of proteasome and autophagy systems in Drosophila eye and wing morphogenesis. By exploiting the ability of GAL4-ninaE. GMR and P{GawB}Bx(MS1096) genetic drivers to be strongly and preferentially expressed in the eye and wing discs, respectively, this study proved that proteasomal integrity and ubiquitination proficiency essentially control fly's eye and wing development. Indeed, subunit- and regulator-specific patterns of severe organ dysmorphia were obtained after the RNAi-induced downregulation of critical proteasome components (Rpn1, Rpn2, alpha5, beta5 and beta6) or distinct protein-ubiquitin conjugators (UbcD6, but not UbcD1 and UbcD4). Proteasome deficient eyes presented with either rough phenotypes or strongly dysmorphic shapes, while transgenic mutant wings were severely folded and carried blistered structures together with loss of vein differentiation. Moreover, transgenic fly eyes overexpressing the UBP2-yeast deubiquitinase enzyme were characterized by an eyeless-like phenotype. Therefore, the proteasome/ubiquitin proteolytic activities are undoubtedly required for the normal course of eye and wing development. In contrast, the RNAi-mediated downregulation of critical Atg (1, 4, 7, 9 and 18) autophagic proteins revealed their non-essential, or redundant, functional roles in Drosophila eye and wing formation under physiological growth conditions, since their reduced expression levels could only marginally disturb wing's, but not eye's, morphogenetic organization and architecture. However, Atg9 proved indispensable for the maintenance of structural integrity of adult wings in aged flies. In all, these findings clearly demonstrate the gene-specific fundamental contribution of proteasome, but not autophagy, in invertebrate eye and wing organ development (Velentzas, 2013).
Ubiquitination and the reverse process deubiquitination regulate protein stability and function during animal development. The Drosophila USP5 homolog Leon functions as other family members of unconventional deubiquitinases, disassembling free, substrate-unconjugated polyubiquitin chains to replenish the pool of mono-ubiquitin, and maintaining cellular ubiquitin homeostasis. However, the significance of Leon/USP5 in animal development is still unexplored. This study generated leon mutants to show that Leon is essential for animal viability and tissue integrity during development. Both free and substrate-conjugated polyubiquitin chains accumulate in leon mutants, suggesting that abnormal ubiquitin homeostasis caused tissue disorder and lethality in leon mutants. Further analysis of protein expression profiles in leon mutants shows that the levels of all proteasomal subunits were elevated. Also, proteasomal enzymatic activities were elevated in leon mutants. However, proteasomal degradation of ubiquitinated substrates was impaired. Thus, aberrant ubiquitin homeostasis in leon mutants disrupts normal proteasomal degradation, which is compensated by elevating the levels of proteasomal subunits and activities. Ultimately, the failure to fully compensate the dysfunctional proteasome in leon mutants leads to animal lethality and tissue disorder (Wang, 2014).
Synapse formation and growth are tightly controlled processes. How synaptic growth is terminated after reaching proper size remains unclear. This study shows that Leon, the Drosophila USP5 deubiquitinase, controls postsynaptic growth. In leon mutants, postsynaptic specializations of neuromuscular junctions are dramatically expanded, including the subsynaptic reticulum, the postsynaptic density, and the glutamate receptor cluster. Expansion of these postsynaptic features is caused by a disruption of ubiquitin homeostasis with accumulation of free ubiquitin chains and ubiquitinated substrates in the leon mutant. Accumulation of Ubiquilin (Ubqn), the ubiquitin receptor whose human homolog ubiquilin 2 is associated with familial amyotrophic lateral sclerosis, also contributes to defects in postsynaptic growth and ubiquitin homeostasis. Importantly, accumulations of postsynaptic proteins cause different aspects of postsynaptic overgrowth in leon mutants. Thus, the deubiquitinase Leon maintains ubiquitin homeostasis and proper Ubqn levels, preventing postsynaptic proteins from accumulation to confine postsynaptic growth (Wang, 2017).
A synapse is a specialized structure where signals are transmitted from a neuron to another neuron or other target cells such as muscles. Proper synapse formation is prerequisite to building functional synapses and constructing neuronal circuits. Synapse abnormalities are suggested to induce neurological and psychological disorders such as autism spectrum disorders and fragile X syndrome. Formation of postsynapses requires coordinated formation of several specialized structures. One prominent postsynaptic feature at neuromuscular junctions (NMJs) is the extensively folded muscular membranes. Specialized folding of postjunctional membranes is thought to increase the area exposed to the synaptic cleft and ensure the effectiveness of neuromuscular transmission. In addition to membrane specializations, the postsynaptic density (PSD) is also a common element whose size requires proper control. The PSD contains scaffolding proteins that recruit signaling protein complexes and neurotransmitter receptors, matching precisely the presynaptic active zones. Formations of postsynaptic membrane and PSD are tightly controlled and coordinated yet these processes remain elusive (Wang, 2017).
The Drosophila NMJ is a model to study synapse formation and activity-dependent synapse remodeling. Synaptic boutons are swollen structures of axonal terminals embedded in highly folded muscular membranes called the subsynaptic reticulum (SSR) and each bouton contains tens of neurotransmitter release sites paired with PSDs. During larval development, the SSR and the PSD concomitantly form and gradually increase their sizes. Two crucial factors, postsynaptic density protein-95/Discs large (Dlg) localized at the SSR and Drosophila p21-activating kinase (dPak) localized at the PSD, regulate SSR formation. At the PSD, two types of localized glutamate receptors (GluRs), IIA and IIB, appear in distinct GluR clusters. The abundance of GluRIIA at the PSD is regulated by PSD-localized dPak and the SSR-localized NF-κB complex, NF-κB/Dorsal (Dl), IκB/Cactus (Cact) and IRAK/Pelle (Pll) (Zhou, 2015 and references therein). Thus, the postsynaptic protein could localize at either SSR or PSD, and confer growth regulation on SSR, PSD or both (Wang, 2017).
Ubiquitination plays essential roles in various cellular processes including synaptic growth. Ubiquitin species are dynamically balanced among free and substrate-conjugated forms of mono-ubiquitin and ubiquitin chains. Ubiquitin homeostasis, i.e. the maintenance of diverse ubiquitin species in proper proportions and levels, is regulated in cellular growth and differentiation, a large superfamily of ubiquitin regulators, participate in the dynamic equilibrium of ubiquitin species. While some DUBs process newly synthesized ubiquitin precursors for ubiquitin supply, others recycle ubiquitin by cleaving ubiquitin chains from protein substrates prior to proteasomal degradation. USP5, the focus of this study, is dedicated to disassembly of free ubiquitin chains for recycling. Physiologically, heat shock stress in yeast causes a reduction of the mono-ubiquitin level. To compensate for ubiquitin depletion, the level of the DUB Doa4 is elevated, leading to an increase in the mono-ubiquitin level by cleaving free ubiquitin chains. The ataxia mice axJ, carrying mutations in the DUB USP14, displayed nerve swelling and abnormal neurotransmission at NMJs. The defects are caused by a reduction in the ubiquitin level as lower ubiquitin levels were detected in the mutant mice and introducing an ubiquitin transgene suppressed the axJ phenotypes. Thus, regulation of the ubiquitin level is a critical step in synapse development and for preventing neurological disorders (Wang, 2017).
Drosophila USP5/Leon is essential to maintain ubiquitin homeostasis during tissue formation and controls activation of apoptosis and the JNK pathway during eye development. This study characterized the role of Leon in postsynaptic growth after synapse formation. In leon mutants, while the presynapse maintains normal morphology, the postsynapse overelaborates, displaying expanded SSR, enlarged PSD and excess PSD-localized GluR clusters. Free ubiquitin chains and ubiquitinated substrates accumulate in leon postsynapses, revealing defects in ubiquitin homeostasis. Genetic analysis shows that accumulations of several postsynaptic proteins accounts for overelaborated postsynaptic structures. The ubiquitin receptor Ubiquilin (Ubqn) recognizes and transfers ubiquitinated substrates to the proteasome for degradation. The Ubqn level is elevated in leon postsynapses and reducing the Ubqn level suppresses leon mutant phenotypes. Importantly, co-overexpression of free ubiquitin chains and Ubqn promotes expansion of these postsynaptic features. Thus, ubiquitin homeostasis such as disassembly of free ubiquitin chains, timely degradation of proteins, and normal function of the ubiquitin receptor Ubqn are compromised in leon mutants, leading to postsynaptic overgrowth (Wang, 2017).
Sleep is an ancient animal behavior that is regulated similarly in species ranging from flies to humans. Various genes that regulate sleep have been identified in invertebrates, but whether the functions of these genes are conserved in mammals remains poorly explored. Drosophila insomniac (inc) mutants exhibit severely shortened and fragmented sleep. Inc protein physically associates with the Cullin-3 (Cul3) ubiquitin ligase, and neuronal depletion of Inc or Cul3 strongly curtails sleep, suggesting that Inc is a Cul3 adaptor that directs the ubiquitination of neuronal substrates that impact sleep. Three proteins similar to Inc exist in vertebrates-KCTD2, KCTD5, and KCTD17-but are uncharacterized within the nervous system and their functional conservation with Inc has not been addressed. This study shows that Inc and its mouse orthologs exhibit striking biochemical and functional interchangeability within Cul3 complexes. Remarkably, KCTD2 and KCTD5 restore sleep to inc mutants, indicating that they can substitute for Inc in vivo and engage its neuronal targets relevant to sleep. Inc and its orthologs localize similarly within fly and mammalian neurons and can traffic to synapses, suggesting that their substrates may include synaptic proteins. Consistent with such a mechanism, inc mutants exhibit defects in synaptic structure and physiology, indicating that Inc is essential for both sleep and synaptic function. These findings reveal that molecular functions of Inc are conserved through ~600 million years of evolution and support the hypothesis that Inc and its orthologs participate in an evolutionarily conserved ubiquitination pathway that links synaptic function and sleep regulation (Li, 2017).
The presence of sleep states in diverse animals has been suggested to reflect a common purpose for sleep and the conservation of underlying regulatory mechanisms. This study has shown that attributes of the Insomniac protein likely to underlie its impact on sleep in Drosophila-its ability to function as a multimeric Cul3 adaptor and engage neuronal targets that impact sleep-are functionally conserved in its mammalian orthologs. This comparative analysis of Inc family members in vertebrate and invertebrate neurons furthermore reveals that these proteins can traffic to synapses and that Inc itself is essential for normal synaptic structure and excitability. These findings support the hypothesis that Inc family proteins serve as Cul3 adaptors and direct the ubiquitination of conserved neuronal substrates that impact sleep and synaptic function (Li, 2017).
The ability of KCTD2 and KCTD5 to substitute for Inc in the context of sleep is both surprising and notable given the complexity of sleep-wake behavior and the likely functions of these proteins as Cul3 adaptors. Adaptors are multivalent proteins that self-associate, bind Cul3, and recruit substrates, and these interactions are further regulated by additional post-translational mechanisms. The findings indicate that KCTD2 and KCTD5 readily substitute for Inc within oligomeric Inc-Cul3 complexes, and strongly suggest that these proteins recapitulate other aspects of Inc function in vivo including the ability to engage neuronal targets that impact sleep. The simplest explanation for why KCTD2 and KCTD5 have retained the apparent ability to engage Inc targets despite the evolutionary divergence of Drosophila and mammals is that orthologs of Inc targets are themselves conserved in mammals. This inference draws support from manipulations of Drosophila Roadkill/HIB and its mammalian ortholog SPOP, Cul3 adaptors of the MATH-BTB family that regulate the conserved Hedgehog signaling pathway. While the ability of SPOP to substitute for HIB has not been assessed by rescue at an organismal level, clonal analysis in Drosophila indicates that ectopically expressed mouse SPOP can degrade the endogenous HIB substrate Cubitus Interruptus (Ci), and conversely, that HIB can degrade mammalian Gli proteins that are the conserved orthologs of Ci and substrates of SPOP. By analogy, Inc targets that impact sleep are likely to have orthologs in vertebrates that are recruited by KCTD2 and KCTD5 to Cul3 complexes. While the manipulations do not resolve whether KCTD17 can substitute for Inc in vivo, the ability of KCTD17 to assemble with fly Inc and Cul3 suggests that functional divergence among mouse Inc orthologs may arise outside of the BTB domain, and in particular may reflect properties of their C-termini including the ability to recruit substrates (Li, 2017).
The finding that Inc can transit to synapses and is required for normal synaptic function is intriguing in light of hypotheses that invoke synaptic homeostasis as a key function of sleep. While ubiquitin-dependent mechanisms contribute to synaptic function and plasticity and sleep is known to influence synaptic remodeling in both vertebrates and invertebrates, molecular links between ubiquitination, synapses, and sleep remain poorly explored. Other studies in flies have indicated that regulation of RNA metabolism may similarly couple synaptic function and the control of sleep. Alterations in the activity of the Fragile X mental retardation protein (FMR), a regulator of mRNA translation, cause defects in the elaboration of neuronal projections and the formation of synapses as well as changes in sleep duration and consolidation. Loss of Adar, a deaminase that edits RNA, leads to increased sleep through altered glutamatergic synaptic function. Like Inc, these proteins are conserved in mammals, suggesting that further studies in flies may provide insights into diverse mechanisms by which sleep influences synaptic function and conversely, how changes in synapses may impact the regulation of sleep (Li, 2017).
These findings at a model synapse suggest that the impact of Inc on synaptic function may be intimately linked to its influence on sleep but do not yet resolve important aspects of such a mechanism. The synaptic phenotypes of inc mutants-increased synaptic growth, decreased evoked neurotransmitter release, and modest effects on spontaneous neurotransmission-are qualitatively distinct from those of other short sleeping mutants. Shaker (Sh) and Hyperkinetic (Hk) mutations decrease sleep in adults but increase both excitability and synaptic growth at the NMJ, suggesting that synaptic functions of Inc may affect sleep by a mechanism different than broad neuronal hyperexcitability. While a parsimonious model is that Inc directs the ubiquitination of a target critical for synaptic transmission both at the larval NMJ and in neuronal populations that promote sleep, this hypothesis awaits the elucidation of Inc targets, definition of the temporal requirements of Inc activity, and further mapping of the neuronal populations through which Inc impacts sleep. Finally, determining the localization of endogenous Inc within neurons is essential to distinguish possible presynaptic and postsynaptic functions of Inc and whether Inc engages local synaptic proteins or extrasynaptic targets that ultimately influence synaptic function (Li, 2017).
A clear implication of these findings is that neuronal targets and synaptic functions of Inc may be conserved in other animals. While the impact of Inc orthologs on sleep in vertebrates is as yet unknown, findings from C. elegans support the notion that conserved molecular functions of Inc and Cul3 may underlie similar behavioral outputs in diverse organisms. INSO-1/C52B11.2, the only C. elegans ortholog of Inc, interacts with Cul3, and RNAi against Cul3 and INSO-1 reduces the duration of lethargus, a quiescent sleep-like state, suggesting that effects of Cul3- and Inc-dependent ubiquitination on sleep may be evolutionarily conserved. The functions of Inc orthologs and Cul3 in the mammalian nervous system await additional characterization, but emerging data suggest functions relevant to neuronal physiology and disease. Human mutations at the KCTD2/ATP5H locus are associated with Alzheimer's disease, and mutations of KCTD17 with myoclonic dystonia. Cul3 lesions have been associated in several studies with autism spectrum disorders and comorbid sleep disturbances. More generally, autism spectrum disorders are commonly associated with sleep deficits and are thought to arise in many cases from altered synaptic function, but molecular links to sleep remain fragmentary. Studies of Inc family members and their conserved functions in neurons are likely to broaden understanding of how ubiquitination pathways may link synaptic function to the regulation of sleep and other behaviors (Li, 2017).
At the Drosophila neuromuscular junction, inhibition of postsynaptic glutamate receptors activates retrograde signaling that precisely increases presynaptic neurotransmitter release to restore baseline synaptic strength. However, the nature of the underlying postsynaptic induction process remains enigmatic. In this study a forward genetic screen is described to discover factors in the postsynaptic compartment necessary to generate retrograde homeostatic signaling. This approach identified insomniac (inc), a putative adaptor for the Cullin-3 (Cul3) ubiquitin ligase complex, which together with Cul3 is essential for normal sleep regulation. Interestingly, it was found that Inc and Cul3 rapidly accumulate at postsynaptic compartments following acute receptor inhibition and are required for a local increase in mono-ubiquitination. Finally, it was shown that Peflin, a Ca(2+)-regulated Cul3 co-adaptor, is necessary for homeostatic communication, suggesting a relationship between Ca(2+) signaling and control of Cul3/Inc activity in the postsynaptic compartment. This study suggests that Cul3/Inc-dependent mono-ubiquitination, compartmentalized at postsynaptic densities, gates retrograde signaling and provides an intriguing molecular link between the control of sleep and homeostatic plasticity at synapses (Kikuma, 2019).
By screening >300 genes with putative functions at synapses, this study has identified inc as a key postsynaptic regulator of retrograde homeostatic signaling at the Drosophila NMJ. The data suggest that Inc and Cul3 are recruited to the postsynaptic compartment within minutes of glutamate receptor perturbation, where they promote local mono-ubiquitination. Inc/Cul3 appear to function downstream of or in parallel to CaMKII and upstream of retrograde signaling during PHP. Pef was identified as a putative co-adaptor that may work with Inc/Cul3 to link Ca2+ signaling in the postsynaptic compartment with membrane trafficking and retrograde communication. Altogether, these findings implicate a post translational signaling system involving mono-ubiquitination in the induction of retrograde homeostatic signaling at postsynaptic compartments (Kikuma, 2019).
Although forward genetic screens have been very successful in identifying genes required in the presynaptic neuron for the expression of PHP, these screens have provided less insight into the postsynaptic mechanisms that induce retrograde homeostatic signaling. It seems clear that many genes acting presynaptically are individually required for PHP, with loss of any one completely blocking PHP expression. Indeed, ~25 genes that function in neurons have thus far been implicated in PHP expression. In contrast, forward genetic screens have largely failed to uncover new genes functioning in the postsynaptic muscle during PHP, implying some level of redundancy. The specific postsynaptic induction mechanisms driving retrograde PHP signaling have therefore remained unclear, and are further complicated by cap-dependent translation and metabolic pathways that contribute to sustaining PHP expression over chronic, but not acute, time scales. Therefore, it is perhaps not surprising that despite screening hundreds of mutants, this study found only a single gene, insomniac, to be required for PHP induction. Inc is expressed in the nervous system and can traffic to the presynaptic terminals of motor neuron. In the context of PHP signaling, however, inc was found to be required in the postsynaptic compartment, where it functions downstream of or in parallel to CaMKII. One attractive possibility is that a reduction in CaMKII-dependent phosphorylation of postsynaptic targets enables subsequent ubiquitination by Cul3-Inc complexes, and that this modification ultimately drives retrograde signaling during PHP. Indeed, reciprocal influences of phosphorylation and ubiquitination on shared targets are a common regulatory feature in a variety of signaling systems. The dynamic interplay of phosphorylation and ubiquitination in the postsynaptic compartment may enable a sensitive and tunable mechanism for controlling the timing and calibrating the amplitude of retrograde signaling at the NMJ (Kikuma, 2019).
The substrates targeted by Inc and Cul3 during PHP induction are not known, but the identification of mono-ubiquitination in the postsynaptic compartment during PHP signaling and the putative Cul3 co-adaptor Peflin provides a foundation from which to assess possible candidates and pathways. In mammals, Pef forms a complex with another Ca2+ binding protein, ALG2, to confer Ca2+ regulation to membrane trafficking pathways. Moreover, Pef/ALG2 were recently found to serve as target-specific co-adaptors for Cul3-KLHL12. In particular, SEC31 and other components involved in ER-mediated membrane trafficking pathways were shown to be targeted for mono-ubiquitination, which in turn modulate Collagen secretion. One attractive possibility, therefore, is that Cul3/Inc could respond to changes in Ca2+ in the postsynaptic compartment through regulation by Pef during PHP signaling to control membrane trafficking pathways. Importantly, the subsynaptic reticulum (SSR) is a complex and membraneous network at the Drosophila NMJ, where electrical, Ca2+-dependent, and membrane trafficking pathways in the postsynaptic compartment are integrated (Teodoro, 2013; Nguyen, 2016). Indeed, Multiplexin, a fly homolog of Collagen XV/XVIII and a proposed retrograde signal, is secreted into the synaptic cleft and is required for trans-synaptic retrograde signaling during PHP (Wang, 2014). In addition, another proposed retrograde signal and secreted protein, Semaphorin 2B, was recently shown to function postsynaptically in retrograde PHP signaling (Orr, 2017). However, inc does not appear to be the closest Drosophila ortholog to KLHL12, and it is therefore possible that Pef and Cul3/Inc regulate postsynaptic PHP signaling through a more indirect mechanism (Kikuma, 2019).
While the precise relationships between CaMKII, Inc, Cul3, and Pef are currently unclear, the activity of membrane trafficking pathways could ultimately be targeted for modulation by Ca2+- and Cul3/Inc-dependent signaling during PHP induction. First, a role for postsynaptic membrane trafficking and elaboration during PHP signaling has already been suggested. In addition, extracellular Ca2+ does not appear to be involved in rapid PhTx-dependent PHP induction. It is therefore tempting to speculate that Ca2+ release from the postsynaptic SSR during rapid PHP signaling may influence Cul3/Inc activity through Pef-dependent regulation, as transient changes in ER-derived Ca2+-signaling controls Pef-dependent recruitment of Cul3 (McGourty, 2016). Alternatively, postsynaptic scaffolds and/or glutamate receptors themselves may be targeted by Cul3/Inc at the Drosophila NMJ, given that these proteins are involved in ubiquitin-mediated signaling and remodeling at dendritic spines. Consistent with this idea, there is evidence that signaling complexes composed of neurotransmitter receptors, CaMKII, and membrane-associated guanylate kinases are intimately associated at postsynaptic densities in Drosophila, as they are in the mammalian central nervous system. There has been speculation that these complexes are targets for modulation during PHP signaling. Although these models are not mutually exclusive, further studies will be required to determine the specific substrates and signal transduction mechanisms through which Cul3/Inc and Pef initiate and sustain retrograde homeostatic communication in postsynaptic compartments (Kikuma, 2019).
While it is well established that the ubiquitin proteasome system can sculpt and remodel synaptic architecture, the importance of mono-ubiquitination at synapses is less studied. Ubiquitin-dependent pathways play key roles in synaptic structure, function, and degeneration, and also contribute to activity-dependent dendritic growth. However, the fact that some proteins persist for long periods at synapses suggests that modification of these proteins by ubiquitin likely include non-degredative and reversible mechanisms. Indeed, a recent study revealed a remarkable heterogeneity in the stability of synaptic proteins, with some short lived and rapidly turned over, while others persisting for long time scales, with half lives of months or longer. At the Drosophila NMJ, rapid ubiquitin-dependent proteasomal degradation at presynaptic terminals is necessary for the expression of PHP through modulation of the synaptic vesicle pool (Wentzel, 2018). In contrast, postsynaptic proteasomal degradation does not appear to be involved in rapid PHP signaling, suggesting that ubiquitin-dependent pathways in the postsynaptic compartment contribute to PHP signaling by non-degradative mechanisms. The current data demonstrate that Cul3, Inc, and Pef function in muscle to enable retrograde PHP signaling, and suggest that Cul3/Inc rapidly trigger mono-ubiquitination at postsynaptic densities following glutamate receptor perturbation. Interestingly, synaptic proteins can be ubiquitinated in <15 s following depolarization-induced Ca2+ influx at synapses (Chen, 2003) and changes in intracellular Ca2+ can activate Pef and Cul3 signaling with similar rapidity. Therefore, both poly- and mono-ubiquination may function in combination with other rapid and reversible processes, including phosphorylation at postsynaptic compartments to enable robust and diverse signaling outcomes during the induction of homeostatic plasticity (Kikuma, 2019).
A prominent hypothesis postulates that a major function of sleep is to homeostatically regulate synaptic strength following experience-dependent changes that accrue during wakefulness. Several studies have revealed changes in neuronal firing rates and synapses during sleep/wake behavior, yet few molecular mechanisms that directly associate the electrophysiological process of homeostatic synaptic plasticity and sleep have been identified. The finding that inc is required for the homeostatic control of synaptic strength provides an intriguing link to earlier studies, which implicate inc in the regulation of sleep. It remains to be determined to what extent the role of inc in controlling PHP signaling at the NMJ is related to the impact of inc on sleep and, if so, whether Inc targets the same substrates to regulate these processes. Interestingly, virtually all neuropsychiatric disorders are associated with sleep dysfunction, including those associated with homeostatic plasticity and Fragile X Syndrome, and sleep behavior is also disrupted by mutations in the Drosophila homolog of FMRP, dfmr1. Further investigation of this intriguing network of genes involved in the homeostatic control of sleep and synaptic plasticity may help solve the biological mystery that is sleep and also shed light on the etiology of neuropsychiatric diseases (Kikuma, 2019).
A wide variety of RNAs encode small open-reading-frame (smORF/sORF) peptides, but their functions are largely unknown. This study shows that Drosophila polished-rice (pri) sORF peptides trigger proteasome-mediated protein processing, converting the Shavenbaby (Svb) transcription repressor into a shorter activator. A genome-wide RNA interference screen identifies an E2-E3 ubiquitin-conjugating complex, UbcD6-Ubr3, which targets Svb to the proteasome in a pri-dependent manner. Upon interaction with Ubr3, Pri peptides promote the binding of Ubr3 to Svb. Ubr3 can then ubiquitinate the Svb N terminus, which is degraded by the proteasome. The C-terminal domains protect Svb from complete degradation and ensure appropriate processing. These data show that Pri peptides control selectivity of Ubr3 binding, which suggests that the family of sORF peptides may contain an extended repertoire of protein regulators (Zanet, 2015).
Eukaryotic genomes encode many noncoding RNAs (ncRNAs) that lack the classical hallmarks of protein-coding genes. However, both ncRNAs and mRNAs often contain small open reading frames (sORFs), and there is growing evidence that they can produce peptides, from yeast to plants or humans. The polished rice or tarsal-less (pri) RNA contains four sORFs that encode highly related 11- to 32-amino acid peptides, required for embryonic development across insect species. In flies, pri is essential for the differentiation of epidermal outgrowths called trichomes. Trichome development is governed by the Shavenbaby (Svb) transcription factor; however, only in the presence of pri can Svb turn on the program of trichome development, i.e., activate expression of cellular effectors. Indeed, the Svb protein is translated as a large repressor, pri then induces truncation of its N-terminal region, which leads to a shorter activator (Kondo, 2010). Thereby, pri defines the developmental timing of epidermal differentiation, in a direct response to systemic ecdysone hormonal signaling (Chanut-Delalande, 2014). Although there is currently a clear framework for the developmental functions of pri, how these small peptides can trigger Svb processing is unknown (Zanet, 2015).
To identify factors required for Svb processing in response to pri, a genome-wide RNA interference (RNAi) screen was performed in a cell line coexpressing green fluorescent protein (GFP)-tagged Svb and pri. An automated assay was set up quantifying Svb processing for each of the Drosophila genes, with an inhibitory score reflecting the proportion of cells unable to cleave off the Svb N terminus. pri RNAi displayed the highest score, which validated this approach to identifying molecular players in Svb processing. Methods used to evaluate results from genome-wide screening all converged on a key role for the proteasome. For instance, COMPLEAT, a bioinformatic framework based on protein complex analysis, identified the proteasome in 66 out of the 71 top predictions. A survey of individual proteasome subunits indicated that both the 20S catalytic core and the 19S regulatory particles are required for Svb processing. Chemical proteasome inhibitors independently confirmed this conclusion, because they also prevented pri-induced Svb processing. These data thus provide compelling evidence that Svb processing results from a pri-dependent proteolysis by the proteasome (Zanet, 2015).
To investigate how pri regulates proteolysis of Svb, the protein region(s) in Svb were identified that are involved in pri-dependent processing. Systematic deletions demonstrated the importance of the Svb N terminus for pri response and restricted the minimal motif to the N-terminal 31 amino acids. Deletion of this motif within an otherwise full-length protein (Δ31) made Svb refractory to pri. Conversely, the Svb N terminus when fused to GFP (1s::GFP) was sufficient to transform this protein into a pri target and to make GFP sensitive to pri. Unlike Svb, however, 1s::GFP was completely degraded by the proteasome upon pri expression (Zanet, 2015).
Recent studies have shown that structural features of proteins influence their degradation by the proteasome: Whereas unstructured substrates, such as intrinsically disordered regions, favor degradation, tightly folded domains can resist proteasome progression. Analysis of Svb sequences predicted intrinsically disordered features throughout its N-terminal moiety, which is degraded. By contrast, the proteasome-resistant C-terminal moiety comprises two folded regions: the transcriptional activation and zinc finger domains. Within the transcriptional activation region, amino acids 532 to 701 protected Svb from complete degradation. Indeed, the C-terminally truncated mutants of 1 to 701 amino acids (and longer) were still processed, whereas mutants shortened by 1 to 532 amino acids (and shorter) were fully degraded. Whether other folded domains would also protect Svb from complete degradation was tested and it was found that attaching zinc fingers to short Svb mutants-otherwise degraded upon pri expression-was sufficient to restore processing. Likewise, the DNA binding domain of Gal4 protected against degradation, which indicated that even a heterologous protein domain with strong structure can protect Svb from full degradation in response to pri. Hence, distinct regions of Svb mediate its processing by the proteasome: the 31 N-terminal residues act as a pri-dependent degradation signal, or degron, and C-terminal domains act as stabilizing features that prevent complete degradation (Zanet, 2015).
Proteins are targeted to the proteasome by the covalent attachment of ubiquitin to Lys residues. The Svb N terminus is highly conserved from insects to human; it comprises two invariant Lys residues (K3 and K8) and a third one at a less constrained position (K28 in Drosophila). Individual Lys substitutions had only a weak effect or no effect, whereas simultaneous mutation of all three Lys (3Kmut) abolished Svb processing. Furthermore, strong pri-dependent ubiquitination of Svb was detected when the proteasome was inhibited. By contrast, this was no longer seen in the 3Kmut variant, which demonstrated the key role of these three Lys in ubiquitin-dependent Svb processing (Zanet, 2015).
Ubiquitin conjugation requires three enzymes (E1, E2, and E3); specificity is generally conferred by the E3 ubiquitin ligases that recognize and bind to substrates. A prominent hit from the RNAi screen was Ubr3 (7 hits out of the top 15), which encodes an E3. Ranking all Drosophila ubiquitin enzymes by their inhibitory score confirmed that Ubr3 was the major E3 required for Svb processing and identified UbcD6 (Rad6) as its associated E2, consistent with evidence that human Ubr3 also forms a complex with UbcD6. Like many proteasome factors, Ubr3 has a broad subcellular distribution in cytoplasm and nuclei, whereas Svb and UbcD6 are nuclear proteins. Svb processing still occurred normally when nuclear export was impaired, which indicated that the proteolytic activation of Svb takes place within the nucleus (Zanet, 2015).
Several additional lines of evidence support the conclusion that Ubr3 mediates the function of pri for Svb ubiquitination. First, Ubr3 coimmunoprecipitated with Svb in a pri-dependent manner and ubiquitinated Svb was found in a complex with Ubr3 upon proteasome inhibition. Second, the N terminus of Svb was sufficient for Ubr3 binding in response to pri. Note that a functional N-terminal degron in Svb was required for its interaction with Ubr3, because the ubiquitin-resistant 3Kmut variant no longer bound Ubr3. Third, in protein extracts from cells that do not express pri, addition of synthetic Pri peptide was sufficient to promote Ubr3-Svb interaction in vitro, in a dose-dependent manner. By contrast, a peptide of the same composition but in a 'scrambled' sequence lacked activity (Zanet, 2015).
Although critical for the binding of Ubr3 to the Svb N terminus, Pri peptides are, however, not indispensable for Ubr3 activity. pri did not influence the binding of Ubr3 to Ape1 (Rrp1), a factor involved in DNA repair and regulated by Ubr3-dependent proteasome degradation. Also, the interaction of Ubr3 with DIAP1, which inhibits apoptosis, occurred with or without pri. Moreover, Pri peptides interacted with Ubr3, even in the absence of Svb. Finally, the isolated UBR-box of Ubr3 no longer required Pri peptides to bind Svb, which suggested that other Ubr3 motifs prevent Svb interaction in the absence of pri. It is therefore concluded that Pri peptides directly regulate the selectivity of Ubr3 for binding to the Svb N terminus and, thereby, trigger Svb ubiquitination and processing by the proteasome (Zanet, 2015).
Recently a Ubr3 loss-of-function allele was isolated, and its phenotype in the differentiation of epidermal cells was assayed. As observed for pri mutants, embryos lacking Ubr3 were unable to differentiate trichomes and to process Svb. Moreover, inactivation of either UbcD6 or Ubr3 prevented formation of adult trichomes in mosaic animals. When compared with their wild-type neighbors, Ubr3-null cells accumulated the repressor form of Svb, which demonstrated Ubr3's essential role for Svb processing in vivo (Zanet, 2015).
Taken together, thes data show that Pri peptides control the binding of the Ubr3 ubiquitin ligase to Svb and activate its processing by the proteasome. In the absence of Pri, Ubr3 nonetheless recognizes other substrates, which shows that a main role for Pri peptides is to modify the binding selectivity of Ubr3. This could potentially be achieved through a conformational change in Ubr3 protein, as proposed for Ubr1, that unmasked the recognition site for Svb upon Pri peptide binding to Ubr3 (Zanet, 2015).
Although recent work has uncovered thousands of novel sORF peptides, only a handful of their molecular targets have yet been identified. sORF peptides have recently been found to bind and regulate the Ca2+ uptake SERCA protein, the heterotrimeric guanine nucleotide-binding protein coupled signaling APJ (Apelin), and the DNA repair protein Ku. Protein-protein interactions often involve small protein regions, and artificial peptides that mimic these binding surfaces have been proven to be potent modulators of protein complexes. It is proposed that sORF-encoded peptides provide an unexplored reservoir of protein-binding interfaces, well suited to regulate the activity of a wide range of cellular factors (Zanet, 2015).
Changes in UBE3A expression levels in neurons can cause neurogenetic disorders ranging from Angelman syndrome (AS) (decreased levels) to autism (increased levels). This study investigated the effects on neuronal function of varying UBE3A levels using the Drosophila neuromuscular junction as a model for both of these neurogenetic disorders. Stimulations that evoked excitatory junction potentials (EJPs) at 1 Hz intermittently failed to evoke EJPs at 15 Hz in a significantly higher proportion of Dube3a over-expressors using the pan neuronal GAL4 driver C155-GAL4 (C155-GAL4>UAS-Dube3a) relative to controls (C155>+ alone). However, in the Dube3a over-expressing larval neurons with no failures, there was no difference in EJP amplitude at the beginning of the train, or the rate of decrease in EJP amplitude over the course of the train compared to controls. In the absence of tetrodotoxin (TTX), spontaneous EJPs were observed in significantly more C155-GAL4>UAS-Dube3a larva compared to controls. In the presence of TTX, spontaneous and evoked EJPs were completely blocked and mEJP amplitude and frequency did not differ among genotypes. These data suggest that over-expression of wild type Dube3a, but not a ubiquitination defective Dube3a-C/A protein, compromises the ability of motor neuron axons to support closely spaced trains of action potentials, while at the same time increasing excitability. EJPs evoked at 15 Hz in the absence of Dube3a (Dube3a15b homozygous mutant larvae) decay more rapidly over the course of 30 stimulations compared to w1118 controls, and Dube3a15b larval muscles have significantly more negative resting membrane potentials (RMP). However, these results could not be recapitulated using RNAi knockdown of Dube3a in muscle or neurons alone, suggesting more global developmental defects contribute to this phenotype. These data suggest that reduced UBE3A expression levels may cause global changes that affect RMP and neurotransmitter release from motorneurons at the neuromuscular junction. Similar affects of under- and over-expression of UBE3A on membrane potential and synaptic transmission may underlie the synaptic plasticity defects observed in both AS and autism (Valdez, 2015).
Angelman syndrome (AS) is a devastating human neurological disorder characterized by cognitive and behavioral defects, muscle hypotonia as well as jerky limb movements and a debilitating ataxic gait. Mouse models of UBE3A maternal loss of function exhibit deficits in learning, hippocampal long term potentiation, and experience-dependent maturation of the neocortex, which may represent alterations in calcium/calmodulin-dependent protein kinase II, properties of axonal initial segment, postsynaptic regulation of glutamatergic signaling, and dendrite morphogenesis. The ataxic gait phenotype of AS is clearly recapitulated in mice deficient for Ube3a as demonstrated by rotarod performance, gait analysis, and cerebellar controlled licking behavior. Although these gait phenotypes appear to be primarily due to a decrease in inhibitory signals in the cerebellum, a comprehensive analysis of motor neuron function in the absence of UBE3A has not yet been performed and rescue of Ube3a levels in the cerebellum of Ube3a deficient mice does not always rescue the ataxic gait phenotype (Valdez, 2015).
Duplications of the same region deleted in the majority of individuals with AS are the second most common genetic lesion (3-5% of cases) found in autism. Just as maternal deletion is required for an AS phenotype, maternal duplications of 15q are specifically associated with increased autism risk. A mouse model with a duplication syntenic to human interstitial duplications of 15q11.2-q13, displayed behavioral deficits characteristic of autism, possibly caused by a deficit in 5-HT2c receptor signaling. These data support the hypothesis that the level of UBE3A expressed from the maternal allele in neurons is critical to neuronal development and function; deficiency for maternal UBE3A resulting in Angelman syndrome and duplication of maternal UBE3A driving increased autism risk (Valdez, 2015).
Drosophila models of Dube3a deficiency [the orthologue to UBE3A in flies have revealed that the loss of Dube3a in neurons results in decreased dendritic arborization in larval peripheral neurons, decreased dopamine levels in adult fly brain, and a clearly measurable defect in climbing ability in adult flies. Adult flies deficient for Dube3a or expressing wild type Dube3a in neurons showed significant defects in climbing ability that were ubiquitin ligase dependent, implying an underlying defect at the neuromuscular junction that may also depend on Dube3a ubiquitination. Previous work has shown that Dube3a loss of function causes changes in the expression of various protein components of the actin cytoskeleton eventually leading to a measurable loss of filamentous actin in the larval muscle wall, so this effect may also be due to muscle developmental defects (Valdez, 2015).
The fly neuromuscular junction (NMJ) is an excellent model for examination of genes involved in synapse formation, function and regulation, but can also be used to examine the effects post-synaptic defects in larval muscle on neurophysiology. Studies of mammalian synapses in the brain have pointed to a pivotal role for the ubiquitin proteasome system in both pre and post-synaptic regulation and this is also true for the development and function of the fly NMJ. To find out how changes in Dube3a levels affected neuronal function (both axonal and synaptic) at the NMJ this study examined synaptic transmission at 3rd instar larval NMJ under conditions of both loss and over-expression of Dube3a. Defects were identifed in axonal propagation of action potentials and synaptic transmission associated with changes in Dube3a in motor neurons. This study provides evidence that the phenotypes observed in humans and mice with decreased or elevated Ube3a may be at least in part related to defects in axonal and synaptic function (Valdez, 2015).
This study demonstrates that both over-expression and deficiency for Dube3a, the fly orthologue of human UBE3A, alters neurotransmission at the neuromuscular junction in Drosophila melanogaster 3rd instar larvae. In a significant proportion of larvae expressing elevated levels of Dube3a in neurons, rapid stimulation of motor nerves intermittently fails to evoke an EJP, and spontaneous depolarizations resembling evoked EJPs are frequently observed in the absence of TTX. However, the amplitude of the first EJP in the train of evoked EJPs and the amplitude and frequency of mEJPs does not vary between any of the genotypes, indicating that this is an axonal rather than vesicle recycling issue. Also, in over-expressors that do not exhibit evoked EJP failure, EJP amplitude does not change more than controls during rapid stimulation. Finally, the spontaneous depolarizations are not observed in larvae over-expressing a ubiquitination defective form of Dube3a (Dube3a-C/A) indicating that the phenomena is dependent on the ubiquitin ligase function of the Dube3a protein. These data could be explained by assuming that evoked EJP failure and spontaneous depolarizations result from regulation of Dube3a ubiquitin target(s) in motor neuron axons rather than directly on the release of neurotransmitter at the synapse. One possible explanation for the spontaneous depolarizations and failures is that Dube3a over-expression results in a depolarization of the RMP of the motor neurons. It is possible that a depolarized membrane potential could result in inactivation of Na+ channels, which could lead to inability of the axons to conduct closely spaced action potentials. At the same time, depolarization of the membrane potential could increase excitability of the axon by bringing it closer to the potential where large numbers of Na+ channels begin to activate. Under this condition any minor perturbation of the axon membrane potential could trigger an action potential in the motor neuron and subsequent EJP in the targeted muscle. The spontaneous depolarizations often appear as bursts, the termination of which might be also be explained by Na+ channel inactivation, similar to the intermittent failures observed at rapid stimulation rates. There was no significant difference in muscle RMP in Dube3a over-expressors versus controls, which is expected since the C155-GAL4 driver employed selectively targets neurons and not muscle (Valdez, 2015). Complete loss of Dube3a expression in the mutant resulta in a different pattern of effects from over-expression. In w1118; Dube3a15b/Dube3a15b larvae, which make no functional Dube3a protein, the EJP decreases more rapidly in response to rapid stimulation compared to their w1118 controls. This is typically referred to as short term depression (STD). The observation of apparent STD in Dube3a15b larvae could be related to the observation that short term facilitation (STF) is less frequently observed in Dube3a15b versus their w1118 controls. STD is thought to be due to a depletion of the readily releasable pool of synaptic vesicles, while STF is thought to be the result of Ca2+ build up in the terminal due to rapid successive depolarizations. At the stimulation rate of 15 Hz, the overall change in EJP amplitude could be a balance between STF and STD. Possibly, a deficit in STF in Dube3a15b larvae could have led to an overall faster decrease in EJP amplitude relative to w1118 controls (Valdez, 2015). Also, the RMP in the muscles of Dube3a15b mutants is significantly more negative than their w1118 controls. These data may reflect a deficit in one or more of the processes or elements involved in maintenance of the RMP. A recent study suggests that Na+/K+ ATPase is ubiquitinated in a Dube3a dependent manner. One might expect that if the loss of Dube3a is causing the more negative RMP in muscle via an effect on Na+/K+ ATPase levels or activity, then the motor neurons may also be affected because regulation of muscle and nerve cell membrane potential both depend on the Na+/K+ ATPase. Nevertheless, changes in resting K+ levels due to leakage across the membrane could also explain these findings. However, it may be more than a coincidence that the effects of over-expression of Dube3a results in increased evoked EJP failures and increased spontaneous, both of which may be indications of a depolarized RMP in motor neurons in the corresponding larvae. Over-expression of Dube3a may have the opposite effects on RMP as loss of Dube3a via opposing actions on this ubiquitin target (Valdez, 2015). The data on the structure of the synaptic active zones suggests that C155>Dube3a-27 and C155>Dube3a-51 larvae have fewer active zones and that C155>Dube3a-51 also have smaller synaptic vesicles relative to the other genotypes. It was also shown that there is a slight increase in synaptic zone density by NC82 staining, however these results did not reach significance despite the large dataset analyzed. These effects of altered Dube3a expression do not seem to explain the effects of over-expression or deficiency on the electrophysiological paradigms employed. However, they may later prove to be important observations that explain deficits in synaptic transmission not tested in the present study. In mouse models of both Angelman syndrome (decreased Ube3a) and Duplication 15q autism (elevated Ube3a) there are defects in glutamatergic synaptic transmission. This study shows that these defects in glutamatergic signaling can be recapitulated in the fly models for both syndromes as well, validating the fly model system for both syndromes. Thus, in a simple and easy to manipulate model system, the Drosophila NMJ, one can now investigate the downstream effects of changes in Dube3a levels on potential ubiquitin targets in the context of neuronal function. Some putative Ube3a protein targets such as Arc and CamKII have been known for some time, while an entirely new set of potential Dube3a targets has been recently identified through a proteomic screen in flies. It can be anticipated that by manipulating the putative targets of Dube3a in the fly NMJ system through shRNAi knock down or mutations in these genes one can begin to unravel the molecular mechanism behind the neurological defects observed in humans with both AS and Duplication 15q autism (Valdez, 2015).
The ubiquitin-proteasome system (UPS), a highly regulated mechanism including the active marking of proteins by ubiquitin in order to be degraded, is critical in regulating proteostasis. Dysfunctioning of the UPS has been implicated in diseases such as cancer and neurodegenerative disorders. This study investigated the effects of proteasome malfunctioning on global proteome and ubiquitinome dynamics using SILAC proteomics in Drosophila S2 cells. dsRNA mediated knockdown of specific proteasome target subunits is used to inactivate the proteasome. Upon this perturbation, both the global proteome and the ubiquitinome become modified to a great extent, the overall impact on the ubiquitinome being most dramatic. The abundances of approx. 10% of all proteins are increased, while the abundances of the far majority of over 14 thousand detected diGly peptides are increased, suggesting that the pool of ubiquitinated proteins is highly dynamic. Remarkably, several proteins show heterogeneous ubiquitination dynamics, with different lysine residues on the same protein showing either increased or decreased ubiquitination. This suggests the occurrence of simultaneous and functionally different ubiquitination events. This strategy offers a powerful tool to study the response of the ubiquitinome upon interruption of normal UPS activity by targeted interference and opens up new avenues for the dissection of the mode of action of individual components of the proteasome. Since this is the first comprehensive ubiquitinome screen upon proteasome malfunctioning in a fruit fly cell system, this data set will serve as a valuable repository for the Drosophila community (Sap, 2017).
By covalently conjugating to target proteins, ubiquitin-like modifiers (UBLs) act as important regulators of target protein localization and activity. The most ancient and one of the least studied UBLs is Urm1, a dual-function protein that in parallel to performing similar functions as its prokaryotic ancestors in tRNA modification. Affinity purification followed by mass spectrometry were used to identify putative targets of Urm1 conjugation (urmylation) at three developmental stages of the Drosophila melanogaster lifecycle. Altogether 79 Urm1-interacting proteins were recovered in Drosophila, which include the already established Urm1 binding partners Prx5 and Uba4, together with 77 candidate urmylation targets that are completely novel in the fly. Among these, the majority was exclusively identified during either embryogenesis, larval stages or in adult flies. Biochemical evidence is presented that four of these proteins are covalently conjugated by Urm1, whereas the fifth verified Urm1-binding protein appears to interact with Urm1 via non-covalent means. Besides recapitulating the previously established roles of Urm1 in tRNA modification and during oxidative stress, functional clustering of the newly identified Urm1-associated proteins further positions Urm1 in protein networks that control other types of cellular stress, such as immunological threats and DNA damage. In addition, the functional characteristics of several of the candidate targets strongly match the phenotypes displayed by Urm1n123 null animals, including embryonic lethality, reduced fertility and shortened lifespan. In conclusion, this identification of candidate targets of urmylation significantly increases the knowledge of Urm1 and presents an excellent starting point for unravelling the role of Urm1 in the context of a complex living organism (Khoshnood, 2017).
Ubiquilin(UBQLN) plays a crucial role in cellular proteostasis through its involvement in the ubiquitin proteasome system and autophagy. Mutations in the UBQLN2 gene have been implicated in amyotrophic lateral sclerosis (ALS) and ALS with frontotemporal lobar dementia (ALS/FTLD). Previous studies reported a key role for UBQLN in Alzheimer's disease (AD); however, the mechanistic involvement of UBQLN in other neurodegenerative diseases remains unclear. The genome of Drosophila contains a single UBQLN homolog (dUbqn) that shows high similarity to UBQLN1 and UBQLN2; therefore, the fly is a useful model for characterizing the role of UBQLN in vivo in neurological disorders affecting locomotion and learning abilities. This study performed a phenotypic and molecular characterization of diverse dUbqn RNAi lines. The depletion of dUbqn induced the accumulation of polyubiquitinated proteins and caused morphological defects in various tissues. The results showed that structural defects in larval neuromuscular junctions, abdominal neuromeres, and mushroom bodies correlated with limited abilities in locomotion, learning, and memory. These results contribute to understanding of the impact of impaired proteostasis in neurodegenerative diseases and provide a useful Drosophila model for the development of promising therapies for ALS and FTLD (Jantrapirom, 2018).
This study shows that Mask, an Ankyrin-repeat and KH-domain containing protein, plays a key role in promoting autophagy flux and mitigating degeneration caused by protein aggregation or impaired ubiquitin-proteasome system (UPS) function. In Drosophila eye models of human tauopathy or amyotrophic lateral sclerosis diseases, loss of Mask function enhanced, while gain of Mask function mitigated, eye degenerations induced by eye-specific expression of human pathogenic MAPT/TAU or FUS proteins. The fly larval muscle, a more accessible tissue, was then used to study the underlying molecular mechanisms in vivo. Mask was found to modulate the global abundance of K48- and K63-ubiquitinated proteins by regulating macroautophagy/autophagy-lysosomal-mediated degradation, but not UPS function. Indeed, upregulation of Mask compensated the partial loss of UPS function. It was further demonstrated that Mask promotes autophagic flux by enhancing lysosomal function, and that Mask is necessary and sufficient for promoting the expression levels of the proton-pumping vacuolar (V)-type ATPases in a TFEB-independent manner. Moreover, the beneficial effects conferred by Mask expression on the UPS dysfunction and neurodegenerative models depend on intact autophagy-lysosomal pathway. These findings highlight the importance of lysosome acidification in cellular surveillance mechanisms and establish a model for exploring strategies to mitigate neurodegeneration by boosting lysosomal function (Zhu, 2017).
Misfolded protein aggregates in and outside of cells in the central nervous system are pathological hallmarks of many neurodegenerative disorders including Alzheimer (AD), Parkinson (PD), Huntington (HD) diseases and amyotrophic lateral sclerosis (ALS). Interestingly, many of the aggregated proteins (such as MAPT (TAU) and APP for Alzheimer disease, SNCA/α-synuclein for Parkinson disease, HTT (Huntingtin) for Huntington disease, FUS, SOD1 and TARDBP/TDP-43 for ALS) can serve as seeds for 'prion-like' spreading of the aggregation within and among cells. It is not entirely clear whether these aggregates are the causes or the results of progressive and cell-type-specific neurodegeneration. However, mounting evidence suggests that clearance and prevention of these toxic protein aggregates are beneficial for meliorating degeneration (Zhu, 2017).
Two major pathways collaborate in regulating intracellular protein degradation: the ubiquitin-proteasome system (UPS) and the autophagy-lysosomal system. Under the normal conditions, UPS serves as the primary route for rapid protein turnover while autophagy mainly degrades long-lived proteins and large cellular organelles under basal conditions and can be robustly induced in face of stresses such as starvation, organelle damage or accumulation of misfolded proteins. However when it comes to degradation of damaged proteins in diseased states, autophagy has been shown to play at least an equally important role as UPS.5. Many of the neurodegenerative disease-related proteins are delivered to autophagic vacuoles and degraded by the autophagy pathway. Meanwhile, impairment of autophagy in the mouse brain causes neurodegeneration associated with ubiquitin-positive protein aggregation. These data suggest that UPS and autophagy are both indispensable in maintaining cellular protein homeostasis. Furthermore, recent studies indicate that UPS and autophagy pathways coordinate with each other to prevent accumulation of toxic protein aggregates, so that enhanced activity of one pathway can compensate if the other is compromised (Zhu, 2017).
Both UPS and autophagy degradation systems are complex processes consisting of chains of sequential events orchestrated by a large group of proteins. To understand their coordinated action, it is necessary to identify novel players that are necessary and sufficient to mediate the compensatory function between the two systems. This study shows Mask, a conserved protein with Ankyrin repeats and a KH domain, as a novel and critical player in such a context. Initially identified as a modulator of receptor tyrosine signaling during Drosophila development (Smith, 2002), Mask has recently been shown to function as a cofactor of the Hippo pathway effector Yorkie and together they regulate target gene transcription with another transcription cofactor (Scalloped) during cell proliferation (Sansores-Garcia, 2013; Sidor, 2013). The human ortholog of Mask, ANKHD1, is highly expressed in several cancer cell lines. Loss of mask function rescues the mitochondrial defects and muscle degeneration observed with pink1 and park mutants (Zhu, 2015). This study shows that in MAPT- and FUS-induced eye degeneration fly models, loss of Mask function enhances degeneration, while gain of Mask function suppresses degeneration. By enhancing V-type ATPase expression, Mask promotes lysosome acidification and autophagic flux; Mask is necessary and sufficient to mediate a compensatory effect for partial loss of UPS function, to increase clearance of ubiquitinated proteins, and to protect against degeneration induced by aggregation-prone mutations (Zhu, 2017).
Autophagy, an evolutionarily conserved cellular mechanism that preserves metabolic homeostasis during nutrient unavailability, is traditionally regarded as a self-eating degradative process with limited selectivity. However, mounting evidence suggests that both micro- and macro-autophagy can play cytoprotective roles to specifically target damaged and toxic organelles and proteins for clearance under pathological conditions. The mechanism of selective autophagy is unclear. There is some evidence that autophagy receptors can recognize ubiquitin-dependent and ubiquitin-independent signals for selective degradation. Autophagy is a multistep process including nucleation, autophagosome formation and fusion with lysosomes and each step can be regulated to enhance degradation of damaged cellular components. Research has emerged showing TFEB is a potent regulator of the autophagy-lysosomal pathway whose activation can promote lysosomal function and mitigate disease in a range of neurodegenerative disorders. This study shows that Mask acts in a TFEB-independent manner to boost the expression of V-ATPase subunits. This study provides novel evidence that lysosome function is not only required for the normal clearance of ubiquitinated and misfolded proteins, but its activity can also be boosted potential through enhanced lysosomal acidification, to mitigate cellular degeneration caused by toxic protein aggregation (Zhu, 2017).
Mask is well positioned to regulate lysosome-mediated clearance of ubiquitinated and misfolded proteins. As a positive regulator of several V-type ATPase V1 subunits expression, Mask function is necessary and sufficient to promote lysosomal acidification and autophagosome degradation in a cell-autonomous manner. When the UPS function is impaired, increased Mask expression is sufficient to increase autophagic flux, which in turn compensates the partial loss of the proteasome-mediated degradation. Interestingly, even when UPS function is intact, levels of Mask activity impact the abundance of UPS-dependent (K48) and -independent (such as K63) ubiquitin-conjugated proteins, suggesting that autophagy and lysosome-mediated degradation plays an important role for basal protein homeostasis. Under pathological conditions such as UPS inactivation or excessive accumulation of disease proteins, upregulation of Mask activity substantially suppressed the cellular degeneration phenotypes in both muscles and photoreceptors, potentially through Mask-mediated increase of autophagy and lysosome activities and subsequent degradation of harmful protein aggregates, as suggested by the current biochemical and genetic analyses. In support of this notion, upregulation of Mask promotes autophagic flux in larval muscles, adult eyes and adult brains (Zhu, 2017).
This work in the Drosophila model organism yielded new insight into Mask-mediated cellular protective mechanisms that regulate lysosomal function in normal and stressed conditions caused by misfolding-prone disease proteins or impaired UPS. Such mechanisms may provide a therapeutic approach for the treatment of a group of neurodegenerative disorders caused by intracellular inclusions (Zhu, 2017).
The modifier protein, ubiquitin (Ub) regulates various cellular pathways by controlling the fate of substrates to which it is conjugated. Ub moieties are also conjugated to each other, forming chains of various topologies. In cells, poly-Ub is attached to proteins and also exists in unanchored form. Accumulation of unanchored poly-Ub is thought to be harmful and quickly dispersed through dismantling by deubiquitinases (DUBs). This study asked whether disassembly by DUBs is necessary to control unanchored Ub chains in vivo. Drosophila melanogaster lines were generated that express Ub chains non-cleavable into mono-Ub by DUBs. These chains are rapidly modified with different linkages and represent various types of unanchored species. Unanchored poly-Ub is not devastating in Drosophila, under normal conditions or during stress. The DUB-resistant, free Ub chains are degraded by the proteasome, at least in part through the assistance of VCP and its cofactor, p47. Also, unanchored poly-Ub that cannot be cleaved by DUBs can be conjugated en bloc, in vivo. These results indicate that unanchored poly-Ub species need not be intrinsically toxic; they can be controlled independently of DUB-based disassembly by being degraded, or through conjugation onto other proteins (Blount, 2018).
The ubiquitin-proteasome pathway (UPP) is central to proteostasis network (PN) functionality and proteome quality control. Yet, the functional implication of the UPP in tissue homeodynamics at the whole organism level and its potential cross-talk with other proteostatic or mitostatic modules are not well understood. This study shows that knock down (KD) of proteasome subunits in Drosophila flies, induced, for most subunits, developmental lethality. Ubiquitous or tissue specific proteasome dysfunction triggered systemic proteome instability and activation of PN modules, including macroautophagy/autophagy, molecular chaperones and the antioxidant cncC (the fly ortholog of NFE2L2/Nrf2) pathway. Also, proteasome KD increased genomic instability, altered metabolic pathways and severely disrupted mitochondrial functionality, triggering a cncC-dependent upregulation of mitostatic genes and enhanced rates of mitophagy. Whereas, overexpression of key regulators of antioxidant responses (e.g., cncC or foxo) could not suppress the deleterious effects of proteasome dysfunction; these were alleviated in both larvae and adult flies by modulating mitochondrial dynamics towards increased fusion or by enhancing autophagy. These findings reveal the extensive functional wiring of genomic, proteostatic and mitostatic modules in higher metazoans. Also, they support the notion that age-related increase of proteotoxic stress due to decreased UPP activity deregulates all aspects of cellular functionality being thus a driving force for most age-related diseases (Tsakiri, 2019).
Cell competition allows winner cells to eliminate less fit loser cells in tissues. In Minute cell competition, cells with a heterozygous mutation in ribosome genes, such as RpS3(+/-) cells, are eliminated by wild-type cells. How cells are primed as losers is partially understood and it has been proposed that reduced translation underpins the loser status of ribosome mutant, or Minute, cells. Using Drosophila this study shows that reduced translation does not cause cell competition. Instead, proteotoxic stress was identified as the underlying cause of the loser status for Minute competition and competition induced by mahjong, an unrelated loser gene. RpS3(+/-) cells exhibit reduced autophagic and proteasomal flux, accumulate protein aggregates and can be rescued from competition by improving their proteostasis. Conversely, inducing proteotoxic stress is sufficient to turn otherwise wild-type cells into losers. Thus, it is proposed that tissues may preserve their health through a proteostasis-based mechanism of cell competition and cell selection (Baumgartner, 2021).
cAMP Responsible Element Binding Protein (CREB) is an evolutionarily conserved transcriptional factor that regulates cell growth, synaptic plasticity and so on. This study unexpectedly found proteasome inhibitors, such as MLN2238, robustly increase CREB activity in adult flies through a large-scale compound screening. Mechanistically, reactive oxidative species (ROS) generated by proteasome inhibition are required and sufficient to promote CREB activity through c-Jun N-terminal kinase (JNK). In 293 T cells, JNK activation by MLN2238 is also required for increase of CREB phosphorylation at Ser(133). Meanwhile, transcriptome analysis in fly intestine identified a group of genes involved in redox and proteostatic regulation are augmented by overexpressing CRTC (CREB-regulated transcriptional coactivator). Intriguingly, CRTC overexpression in muscles robustly restores protein folding and proteasomal activity in a fly Huntington's disease (HD) model, and ameliorates HD related pathogenesis, such as protein aggregates, motility, and lifespan. Moreover, CREB activity increases during aging, and further enhances its activity can suppress protein aggregates in aged muscles. Together, these results identified CRTC/CREB downstream ROS/JNK signaling as a conserved sensor to tackle oxidative and proteotoxic stresses. Boosting CRTC/CREB activity is a potential therapeutic strategy to treat aging related protein aggregation diseases (Yin, 2022).
Ubiquitination is a post-translational modification that regulates cellular processes by altering the interactions of proteins to which ubiquitin, a small protein adduct, is conjugated. Ubiquitination yields various products, including mono- and poly-ubiquitinated substrates, as well as unanchored poly-ubiquitin chains whose accumulation is considered toxic. Previous work has shown that transgenic, unanchored poly-ubiquitin is not problematic in Drosophila melanogaster. In the fruit fly, free chains exist in various lengths and topologies and are degraded by the proteasome; they are also conjugated onto other proteins as one unit, eliminating them from the free ubiquitin chain pool. To further explore the notion of unanchored chain toxicity, this study examined when free poly-ubiquitin might become problematic. It was found that unanchored chains can be highly toxic if they resemble linear poly-ubiquitin that cannot be modified into other topologies. These species upregulate NF-κB signaling, and modulation of the levels of NF-κB components reduces toxicity. In additional studies,toxicity from untethered, linear chains was shown to be regulated by isoleucine 44, which anchors a key interaction site for ubiquitin. It is concluded that free ubiquitin chains can be toxic, but only in uncommon circumstances, such as when the ability of cells to modify and regulate them is markedly restricted (Blount, 2020).
Ubiquitination-mediated protein degradation is the selective degradation of diverse forms of damaged proteins that are tagged with ubiquitin, while deubiquitinating enzymes reverse ubiquitination-mediated protein degradation by removing the ubiquitin chain from the target protein. The interactions of ubiquitinating and deubiquitinating enzymes are required to maintain protein homeostasis. The ubiquitin-specific protease USP7 is a deubiquitinating enzyme that indirectly plays a role in repairing DNA damage and development. However, the mechanism of its participation in aging has not been fully explored. Regarding this issue, this study found that USP7 was necessary to maintain the normal lifespan of Drosophila melanogaster, and knockdown of dusp7 shortened the lifespan and reduced the ability of Drosophila to cope with starvation, oxidative stress and heat stress. Furthermore, this study showed that the ability of USP7 to regulate aging depends on the autophagy and ubiquitin signaling pathways. Furthermore, 2,5-dimethyl-celecoxib (DMC), a derivative of celecoxib, can partially restore the shortened lifespan and aberrant phenotypes caused by dusp7 knockdown. These results suggest that USP7 is an important factor involved in the regulation of aging, and related components in this regulatory pathway may become new targets for anti-aging treatments (Cui, 2020).
The accumulation of protein aggregates and dysfunctional organelles as organisms age has led to the hypothesis that aging involves general breakdown of protein quality control. This hypothesis was tested using a proteomic and informatic approach in the fruit fly Drosophila melanogaster. Turnover of most proteins was markedly slower in old flies. However, ribosomal and proteasomal proteins maintained high turnover rates, suggesting that the observed slowdowns in protein turnover might not be due to a global failure of quality control. As protein turnover reflects the balance of protein synthesis and degradation, whether decreases in synthesis or decreases in degradation would best explain the observed slowdowns in protein turnover was investigated. It was found that while many individual proteins in old flies showed slower turnover due to decreased degradation, an approximately equal number showed slower turnover due to decreased synthesis, and enrichment analyses revealed that translation machinery itself was less abundant. Mitochondrial complex I subunits and glycolytic enzymes were decreased in abundance as well, and proteins involved in glutamine-dependent anaplerosis were increased, suggesting that old flies modify energy production to limit oxidative damage. Together, these findings suggest that age-related proteostasis changes in Drosophila represent a coordinated adaptation rather than a systems collapse (Vincow, 2021).
Linear ubiquitination is an atypic ubiquitination process that directly connects the N- and C-termini of ubiquitin and is catalyzed by HOIL-1-interacting protein (HOIP). It is involved in the immune response or apoptosis by activating the nuclear factor-κB pathway and is associated with polyglucosan body myopathy 1, an autosomal recessive disorder with progressive muscle weakness and cardiomyopathy. However, little is currently known regarding the function of linear ubiquitination in muscles. This study investigated the role of linear ubiquitin E3 ligase (LUBEL), a Drosophila HOIP ortholog, in the development and aging of muscles. The muscles of the flies with down-regulation of LUBEL or its downstream factors, kenny and Relish, developed normally, and there were no obvious abnormalities in function in young flies. However, the locomotor activity of the LUBEL RNAi flies was reduced compared to age-matched control, while LUBEL RNAi did not affect the increased mitochondrial fusion or myofiber disorganization during aging. Interestingly, the accumulation of polyubiquitinated protein aggregation during aging decreased in muscles by silencing LUBEL, kenny, or Relish. Meanwhile, the levels of autophagy and global translation, which are implicated in the maintenance of proteostasis, did not change due to LUBEL down-regulation. In conclusion, a new role of linear ubiquitination is proposed in proteostasis in the muscle aging (Lee, 2021).
Ariadne-1 (Ari-1) is an E3 ubiquitin-ligase essential for neuronal development, but whose neuronal substrates are yet to be identified. To search for putative Ari-1 substrates, this study used an in vivo ubiquitin biotinylation strategy coupled to quantitative proteomics of Drosophila heads. Sixteen candidates were identified that met the established criteria: a significant change of at least two-fold increase on ubiquitination, with at least two unique peptides identified. Amongst those candidates, Comatose (Comt), the homologue of the N-ethylmaleimide sensitive factor (NSF), which is involved in neurotransmitter release, was identified. Using a pulldown approach that relies on the overexpression and stringent isolation of a GFP-fused construct, Comt/NSF was validated to be an ubiquitination substrate of Ari-1 in fly neurons, resulting in the preferential monoubiquitination of Comt/NSF. The possible functional relevance of this modification was tested using Ari-1 loss of function mutants, which displayed a lower rate of spontaneous neurotransmitter release due to failures at the pre-synaptic side. By contrast, evoked release in Ari-1 mutants was enhanced compared to controls in a Ca(2+) dependent manner without modifications in the number of active zones, indicating that the probability of release per synapse is increased in these mutants. This phenotype distinction between spontaneous versus evoked release suggests that NSF activity may discriminate between these two types of vesicle fusion. These results thus provide a mechanism to regulate NSF activity in the synapse through Ari-1-dependent ubiquitination (Ramirez, 2021).
Neurotransmitter release is mediated by a set of protein-protein interactions that include the N-ethylmaleimide sensitive factor (NSF), soluble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs). These proteins assemble into a tripartite complex in order to elicit synaptic vesicle fusion, which is formed by one synaptic vesicle membrane SNARE protein (v-SNARE), Synaptobrevin, and two plasma membrane SNARE proteins (t-SNAREs), Syntaxin and the 25-kDa synaptosome-associated protein. Following vesicle fusion, the tripartite SNARE complex disassembles by the activities of NSF and SNAPs. Free t-SNAREs from the plasma membrane can then participate in new priming reactions, while the v-SNAREs can be incorporated into recycled synaptic vesicles. These interactions, also routinely used for intracellular vesicle trafficking in all cell types, are conserved across species, including Drosophila (Ramirez, 2021).
Deviations on the rate of neurotransmitter release are at the origin of multiple neural diseases, including Parkinson's disease. Under physiological conditions, the Leucine-rich repeat Serine/Threonine-protein kinase 2 (LRRK2) phosphorylates NSF to enhance its ATPase activity, which facilitates the disassembly of the SNARE complex. However, the most common Parkinson's disease mutation in LRRK2 causes an excess of kinase activity that interferes with the vesicle recycling. Similarly, α-Synuclein, another Parkinson's disease protein, alters neurotransmitter release by preventing the v-SNARE vesicle-associated membrane protein (VAMP)-2, also known as Synaptobrevin-2, from joining the SNARE complex cycle. Correct neural functioning, therefore, requires delicate regulation in vesicle trafficking. This regulation can be achieved by posttranslational modifications, such as ubiquitination. In fact, ubiquitination of certain proteins can affect their activity or life span. At the presynaptic side, for example, increased neurotransmitter release correlates with decreased protein ubiquitination. Similarly, acute pharmacological proteasomal inhibition causes rapid strengthening of neurotransmission (Ramirez, 2021).
Ariadne 1 is an E3 ubiquitin-ligase, first identified in Drosophila, from a conserved gene family defined by two C3HC4 Ring fingers separated by a C6HC in-Between-Rings domain (the RBR motif). Ari-1 had been described to be essential for neuronal development, and its mutants reported to exhibit reduced eye rhabdomere surface and endoplasmic reticulum, as well as aberrant axonal pathfinding. However, despite its importance, no neuronal substrates have been reported so far. Only three Ari-1 substrates have been postulated, either in cultured cells or in vitro, while three Parkin substrates were reported to interact with Ari-1 in COS-1 cells. For this reason, with the aim to identify neuronal Ari-1 substrates, advantage was taken of two methodologies. The first one, the bioUb strategy, allows the identification of hundreds of ubiquitinated proteins from neuronal tissues. The system relies on the overexpression of a tagged ubiquitin that bears a 16 amino acid long biotinylatable peptide, which can be biotinylated by the Escherichia coli biotin holoenzyme synthetase enzyme (BirA) in neurons in vivo. Remarkably, this approach can be efficiently applied to identify neuronal E3 ligase substrates. In contrast, the second methodology favors the isolation of GFP-tagged proteins under denaturing conditions to further characterize their ubiquitination pattern under the presence or absence of an E3 ligase (Ramirez, 2021).
This study has combined the bioUb strategy with the overexpression of Ari-1 and identified 16 putative neuronal substrates of Ari-1. Among those, focus was placed on Comatose (Comt), the fly NSF orthologue, due to its relevance in normal and pathological function at the synapse. By the isolation of GFP-tagged Comt from Drosophila photoreceptor neurons overexpressing Ari-1, this study confirmed Comt/NSF as an Ari-1 ubiquitin substrate and showed that it is mostly monoubiquitinated. Furthermore, Ari-1 loss-of-function mutants displayed lower rate of spontaneous neurotransmitter release, but enhanced evoked release, due to failures at the presynaptic side. These defects in the mutants are compatible with a deregulation of Com/NSF activity. Altogether, these data show that Ari-1 regulates neurotransmitter release by controlling Comt/NSF activity through ubiquitination (Ramirez, 2021).
This study identified sixteen novel putative substrates of Ari-1 in Drosophila photoreceptor neurons in vivo by means of an unbiased proteomic approach. Remarkably, despite Ari-1 being recently shown to regulate the positioning of the cell nucleus in muscles via a direct interaction with Parkin (Tan, 2018), as well as to interact with some Parkin substrates, there is no overlap between the substrates identified for Ari-1 and those previously identified for Parkin in flies. Taken together, the available data suggest that the 16 targets identified in this study are specifically regulated by Ari-1 in Drosophila photoreceptor neurons and that this E3 ligase has a wide functional repertoire (Ramirez, 2021).
This study focused on Comt, an ATPase required for the maintenance of the neurotransmitter release. Ubiquitination of proteins involved in vesicle trafficking and neurotransmitter release had been previously reported. Similarly, the importance of the ubiquitination machinery for the proper neuronal function has also been demonstrated. The alterations produced on synaptic transmission by ubiquitination are typically attributed to an acute control of synaptic protein turnover. However, many of these presynaptic proteins have been reported to be mainly mono- or di-ubiquitinated, a type of ubiquitin modification that is not usually associated with protein degradation. In line with this, the results showed that Comt/NSF is preferentially monoubiquitinated by Ari-1/ARIH1, suggesting that Ari-1/ARIH1 could be regulating Comt/NSF activity, rather than its life span or expression levels (Ramirez, 2021).
Ari-1 mutations result in abnormal synaptic function at the larval stage, a result consistent with a regulatory function of NSF. All mutant alleles examined exhibit a reduced frequency of spontaneous synaptic release. In addition, ari-12 mutants exhibit a large calcium-dependent evoked release. Analysis of the mechanism for enhanced evoked release in ari-12 suggests that the primary defect consists in an increased probability of vesicle fusion in response to calcium entry in the presynaptic side. First, by comparing the amplitude and time course of spontaneously occurring postsynaptic events in mutant and control animals, the possibility of a postsynaptic modification was ruled out. Since no significant differences were found, it is concluded that the receptor field size and kinetic properties of postsynaptic receptors are normal in the mutant (Ramirez, 2021).
The ari-1 functional defects could result from alterations of synaptic transmission during development; therefore, the number of synaptic contacts was quantified, assuming that most release sites occur within varicosities. No significant difference was observed between mutant and control. Although this study does not include electron microscopy quantification of synaptic vesicles, th confocal microscopy and electrophysiology data point toward an upregulation of release from single synapses (Ramirez, 2021).
The failure analysis from single varicosities represents direct evidence that, at relatively low calcium concentrations, mutant terminals release more quanta than controls in response to an action potential. Whether increased quantal release could be explained on the basis of more release sites being concentrated on mutant terminals was examined. The focal recordings using saturating calcium concentrations argue against this possibility. When mutant terminals are exposed to high calcium, in order to increase the likelihood that all active zones within the bouton will release a quantum, EJP amplitudes in the mutant are indistinguishable from that of controls. These data suggest that the number of release sites in mutant and control terminals is similar and favor the hypothesis that, at physiological calcium concentrations, the probability of vesicle fusion upon calcium entry is increased in the mutant (Ramirez, 2021).
It was found that ari-1 mutants have opposite effects on spontaneous and evoked release. Classically, the two modes of vesicular release have been considered to represent a single exocytotic process that functions at different rates depending on the Ca2+ concentration. However, recent work challenges this idea and supports the alternative model where spontaneous and evoked response might come from different vesicles pools. Several experimental evidences indicate that both forms of release may represent separate fusion pathways. Employing a state-of-the-art optical imaging in larval NMJ, it has been shown that evoked and spontaneous release can be segregated across active zones. Thus, three types of active zones could be defined: those that only release vesicles in response to a rise of intracellular calcium (evoked release), a second population that only participates in spontaneous release, and a third small proportion (around 4%) that participates in both evoked and spontaneous release. This result advocates for a different molecular and spatial segregation of both modes of release (Ramirez, 2021).
Differential content or activity of regulatory SNARE binding proteins could discriminate between spontaneous and evoked release. It has been shown that the presence of the Vamp-7 isoform could participate in this differential release. Vamp-7 preferentially labels vesicles unresponsive to stimulation, and it colocalizes only partially with the endogenous synaptic vesicle glycoprotein Sv2 and the vesicular glutamate transporter Vglut1, suggesting that this vesicle pool does not support evoked transmitter release. Recently, it was shown that the double knockout mouse for Synaptobrevin genes, syb1 and syb2, results in a total block of evoked release, while spontaneous release was increased in both frequency and quantal size without changes in the number of docked vesicles at the active zone, confirming the idea that evoked and spontaneous releases are differentially regulated. Interestingly, Vamp-7 was found by MS to be less ubiquitinated when Ari-1 is overexpressed, suggesting that Ari-1 mutants could be favoring evoked release through NSF and reducing spontaneous release through Vamp7. Thus, Ari-1 could be acting as a repressor and activator of evoked and spontaneous release, respectively. All together, these results evidence a new layer of complexity over the actual fine-tuning of synaptic transmission. A physiological regulatory mechanism for both types of release has been recently demonstrated for inhibitory synapses at the trapezoid body, an important brain area in auditory integration. In this nucleus, activation of metabotropic glutamate receptor mGluR1 differentially modulates both spontaneous and evoked release in both GABAergic and Glycynergic synapses (Ramirez, 2021).
Early functional studies of NSF employing the fly thermo-sensitive mutant allele comtts17, have reported a reversible reduction of synaptic transmission. Consistent with a role of NSF on SNARE dissociation, this inhibition parallels an increase in the number of synaptic vesicles at the presynaptic terminal. At this point, it can only be speculated how specifically Ari-1/ARIH1 regulates Comt/NSF activity within the presynaptic terminal. Opposite to the role of NSF mutant comtts17, which impairs SNARE complex disassembly, a change that enhances NSF functionality due to the lack of its ubiquitination would favor the dissociation of the so-called trans-SNARE complex. Further, this would build up the number of SNARE complexes assembled per vesicle, thus increasing the efficiency of fusion machinery in a Ca2+-dependent manner. Consistent with this interpretation, it has been shown that fast release of a synaptic vesicle requires at least three SNARE complexes, whereas slower release may occur with fewer complexes (Ramirez, 2021).
Interestingly, some of the additional putative substrates identified are also related to synapse physiology and neurotransmitter release. PPO1 is an enzyme with L-DOPA monooxygenase activity, hence, may be involved in the metabolism of dopamine neurotransmitter. Similarly, GstO3 is involved in glutathione metabolism, another type of neurotransmitter. Vha44 and Vha68-1 are components of the vacuolar proton-pump ATPase, whose mutations have been reported to impair neurotransmitter release. Vha44 has also been described as an enhancer of Tau-induced neurotoxicity, and CG15117, orthologue of human GUSB, has been associated with neuropathological abnormalities. The long recovery time from paralysis observed in comt6 ari2/comt6 females could result from the role of Ari-1 in the ubiquitination of these additional substrates, in addition to the role of Comt in tissues other than the nervous system.
The data reported in this study may be relevant in the context of Parkinson's disease. It should be noted that most Parkinson's-related genes encode proteins involved in vesicle recycling and neurotransmitter release at the synapse. Thus, the kinase LRRK2 phosphorylates NSF to enhance its ATPase activity upon the SNARE complex and facilitate its disassembly. Pathological mutations in this protein, such as G2019S, cause an excess of kinase activity that interferes with vesicle recycling. Deregulated synaptic aggregates of α-Synuclein may target VAMP-2 hampering the
formation of the SNARE complex. Parkin is a structural relative of Ari-1, based on their common Cysteine rich C3HC4 motif, which is also at the origin of some forms of Parkinson's disease. All these genes and their corresponding mechanisms of activity sustain the scenario in which several types of Parkinson's disease seem to result from a defective activity of the synapse. In this context, the role of Ari-1/ARIH1 emerges as a mechanism to regulate a key component of the SNARE complex, Comt/NSF. Conceivably, Ari-1/ARIH1 may become a suitable target for either diagnosis or pharmacological treatment of Parkinson's and related diseases (Ramirez, 2021).
Neurodegeneration in the central nervous system (CNS) is a defining feature of organismal aging that is influenced by peripheral tissues. Clinical observations indicate that skeletal muscle influences CNS aging, but the underlying muscle-to-brain signaling remains unexplored. In Drosophila, this study found that moderate perturbation of the proteasome in skeletal muscle induces compensatory preservation of CNS proteostasis during aging. Such long-range stress signaling depends on muscle-secreted Amyrel amylase. Mimicking stress-induced Amyrel upregulation in muscle reduces age-related accumulation of poly-ubiquitinated proteins in the brain and retina via chaperones. Preservation of proteostasis stems from the disaccharide maltose, which is produced via Amyrel amylase activity. Correspondingly, RNAi for SLC45 maltose transporters reduces expression of Amyrel-induced chaperones and worsens brain proteostasis during aging. Moreover, maltose preserves proteostasis and neuronal activity in human brain organoids challenged by thermal stress. Thus, proteasome stress in skeletal muscle hinders retinal and brain aging by mounting an adaptive response via amylase/maltose (Rai, 2021).
Polycomb-group (PcG) genes were first identified in Drosophila for their roles in maintaining correct expression patterns of homeotic genes. PcG-mediated transcription silencing was later proved to be a well-conserved regulatory mechanism throughout metazoans. Classical PcG targets, such as Hox genes, play important roles in biological processes ranging from stem cell maintenance to genomic imprinting. Recent genome-wide studies unveiled additional PcG targets, many of which encode transcription factors and cell-signaling proteins that regulate a diverse array of downstream effectors. Thus, PcG may act in a much broader spectrum of cellular processes than previously anticipated (Du, 2016).
PcG silencing depends primarily on the activities of two Polycomb repressive complexes (PRC). In Drosophila, PRC1 is composed of Pc (Polycomb), Ph (Polyhomeotic), Psc (Posterior sex combs), and Sce (Sex combs extra). The main subunits of the PRC2 include Esc (Extra sex combs), E(z) (Enhancer of zeste), Su(z)12 (Suppressor of zeste 12) and Caf1 (Chromatin assembly factor 1). Relying on the presence of a conserved enzymatic SET domain in E(z), PRC2 catalyzes tri-methylation of histone H3 at Lys 27 (H3K27me3). Pc then employs its chromo domain to recognize H3K27me3 mark, resulting in recruitment of PRC1 to PcG targets. Mechanisms utilized by PRC1 to silence target genes include histone H2A mono-ubiquitination, chromatin compaction, and direct interaction with the general transcription machinery (Du, 2016).
While intensive studies have been focused on uncovering mechanisms by which PcG proteins epigenetically repress target gene expression, few are devoted to define how the PcG activities are regulated. Nevertheless, several transcription factors and microRNAs are known to directly modulate PcG expression. Feedback regulatory loops may also be important to maintain proper expression of PcG, which themselves are subject to epigenetic repression. Furthermore, post-translational modifications on several PcG proteins have been reported, and the importance of such modifications has only been revealed recently. For example, SUMOylation is shown to modulate PcG activity by affecting chromatin targeting of the Pc protein, and O-GlcNAcylation has been demonstrated to prevent aggregation of PRC1 subunit Ph in Drosophila (Du, 2016).
This report describe that a Drosophila gene CG32676, which was named stuxnet (stx), functions through ubiquitin-independent degradation (UID) to control Pc protein stability and thereby PcG-mediated epigenetic repression. This study shows further that vertebrate Stx regulates orthologous Pc protein in the same fashion. Together, these results highlight a conserved regulatory mechanism for Pc, the founding member of the PcG family of proteins (Du, 2016).
Taking advantage of genetic tools available in Drosophila, the function of a UBL-domain-containing protein, Stx, was examined, and its unexpected role of regulated Pc protein degradation in epigenetic repression. These analyses on classical PcG targets demonstrate that Stx functions as a Pc-specific regulator that negatively modulates the PcG activity. Importantly, this mode of regulation was found to be conserved from flies to vertebrates (Du, 2016).
stx activity is essential for Drosophila development. The fact that pupal lethal phenotype associated with loss-of-function stx mutations can be rescued by removing 50% of Pc activity strongly supports that modulating the Pc expression is the major developmental process regulated by stx. Stx might not be a constitutive component of the canonical PRC1. However, the ability of Stx to reduce Pc recruitment to target gene loci argues that Stx may act as a gatekeeper for control of Pc availability to form highly dynamic PRC complexes on target chromatin. As stx activity is necessary for PcG target expression, Stx could function in an intrinsic machinery to regulate Pc protein homeostasis. Stx directly binds Pc through a serine-rich PcB domain and interacts with the proteasome through the UBL domain. As Pc protein degradation does not rely on ubiquitination, the UBL domain in Stx, upon interaction with Pc, could serve as a recognition signal that marks Pc protein for degradation in the proteasome. Thus, a model is proposed in which Stx acts first as an adapter and then a chaperone-like protein to facilitate proteasomal degradation of Pc, resulting in altered PcG activity in animal development. Intriguingly, upon inspection of modENCODE database, multiple binding sites were found for PcG components, including Pc, Psc, Sce, and Pho, and Ubx, which is itself a PcG target, thus pointing to the existence of a potential feedback loop between Stx and PcG activity (Du, 2016).
Altered Pc protein abundance has been noted in several biological processes. In the Sce mutant fly embryos, the bulk level of Pc protein is significantly reduced, but Ph and Psc are not affected. Similar results have been reported in mouse ES cells for RING1B and Cbx4, mammalian orthologs of Sce and Pc. However, the significance of such regulation was not understood. It is suspected that binding with Sce might stabilize Pc, which is crucial for PRC1 assembly. It is interesting to note that the level of Pc changes rapidly in the cell cycle. The oscillation of Pc protein during the cell cycle is thought to be important for establishment and maintenance of cellular epigenetic memory. The observation of the reciprocal expression pattern of Pc and Stx as well as the ability of Stx to control Pc abundance in cell cycle are in favor of a notion that regulated Pc protein stability may be one way to dynamically control Pc activity in physiological contexts. How Stx participates in such regulation is an interesting question that awaits further exploration (Du, 2016).
The PRC1 is composed of four core subunits, each of which has unique molecular activities non-exchangeable among each other. However, the loss-of-function phenotypes of individual PRC1 subunits in Drosophila only partially overlap, revealing the complexity of PRC1 regulation in various cellular processes. The differential requirement of PRC1 subunits in development might be due to the presence of distinct PRC complexes in a temporal and tissue-specific manner. This view is further complicated in vertebrates by partially redundant orthologous PRC1 proteins and the formation of multiple non-canonical complexes. Thus, it will be necessary to explore the regulatory machineries utilized by individual PRC1 components to better understand how PRC complexes exert versatile functions in vivo. This study has shown that Stx targets Pc for proteasomal degradation, but whether parallel regulators exist for other PRC1 components is still unknown (Du, 2016).
This study of Stx regulation on Pc stability reveals that the activity of Pc protein, the founding member of the PRC complexes, can be controlled through regulated protein degradation. Surprisingly, it was found that fly Pc protein is largely regulated by UID. The list of substrates that undergo UID has expanded rapidly in recent years. Intriguingly, many UID substrates are localized to the nucleus, including transcription factors and chromatin remodeling factors. The addition of Pc, a key epigenetic regulator, to this list leads to the belief that UID in the nucleus may participate in the control of gene expression (Du, 2016).
Consistent with a role of Stx on Pc stability in Drosophila development, proteasomal degradation has been reported to affect the stability of several PcG components in cultured vertebrate cells, including three PRC1 proteins BMI1, RING1B, and PHC, and one PRC2 protein EZH2. It is thus highly likely that protein degradation may play a general role in regulating PcG activity (Du, 2016).
Appropriate PcG activity is essential for stem cell maintenance and lineage specification in vertebrates. Altered PcG activity is associated with malignant human diseases, including cancer. Furthermore, dysregulated stx expression and Stx mutations are reported in several forms of cancer in the COSMIC database. Consistently, genes co-expressed with stx shown in COXPRESdb are clustered into pathways in cancer as well as Notch and MAPK signaling pathways. Very recently, Stx mutations were found in patients with autism spectrum disorders (ASD) by whole-exome sequencing. Given the strong connection between PcG and ASD, Stx may play a role in ASD through its regulation of PcG activity. Thus, the identification of regulators of PcG activity, such as Stx, may provide additional therapeutic targets for relevant diseases (Du, 2016).
Germ cells undergo distinct nuclear processes as they differentiate into gametes. While these events must be coordinated to ensure proper maturation, the stage-specific transport of proteins in and out of germ cell nuclei remains incompletely understood. Efforts to genetically characterize Drosophila genes that exhibit enriched expression in germ cells led to the finding that loss of the highly-conserved Importin β/karyopherin family member Importin-9 (Ipo9) results in female and male sterility. Immunofluorescence and fluorescent in situ hybridization (FISH) revealed that Ipo9 (KO) mutants display chromosome condensation and segregation defects during meiosis. In addition, Ipo9 (KO) mutant males form abnormally structured sperm and fail to properly exchange histones for protamines. Ipo9 physically interacts with proteasome proteins and Ipo9 mutant males exhibit disruption of the nuclear localization of several proteasome components. Thus, Ipo9 coordinates the nuclear import of functionally related factors necessary for the completion of gametogenesis (Palacios, 2021).
Subcellular compartmentalization allows for complex modes of gene regulation in eukaryotic cells. The regulated and active transport of macromolecules between different compartments promotes cellular homeostasis and often drives differentiation. Transport of molecules from the cytoplasm to the nucleus depends on a family of proteins called karyopherins, also known as importins. The karyopherin superfamily of transporters consists of importin α and importin β subgroups. All the proteins within this karyopherin superfamily share tandem huntingtin, elongation factor 3, protein phosphatase 2A and mechanistic target of rapamycin (HEAT) repeats. These repeats allow these proteins to bind to various cargo proteins, which often, but not always, contain a nuclear localization signal within their peptide sequence. Karyopherins then transport these cargoes into the nucleus through nuclear pores (Palacios, 2021).
Another key component of the transport machinery is the small GTPase Ran (Cautain, 2015). Cytoplasmic Ran is typically maintained in a GDP-bound state, whereas nuclear Ran binds GTP. This concentration gradient of GDP and GTP bound Ran provides a directional cue for the transport of proteins between the cytoplasm and nucleus. Once importins enter the nucleus, high affinity interactions with RanGTP cause karyopherins to release their cargoes and recycle back to the cytoplasm (Palacios, 2021).
Accumulating evidence suggests that β-karyopherins do not simply function as constitutive and redundant housekeeping proteins. Interactions between different β-karyopherins with specific cargoes depend not only on their overlapping expression patterns in time and space, but also on clear differences in the affinities of the physical interactions. For example, histones can bind to multiple β-karyopherins, but their affinities vary. For example, Kapβ2 and Imp5 exhibit very strong affinity for Histone H3, whereas Impβ, Imp4, Imp7, Imp9 and Impα display weaker interactions. Additionally, a previous study identified a group of 468 cargoes for 12 β-karyopherins. Three hundred and thirty two of these cargoes were unique to one β-karyopherin family member, suggesting a division of function among these transporters. Several β-karyopherin family members have been associated with specific diseases. Accumulating evidence shows that β-karyopherins are overexpressed in multiple tumors including melanoma, pancreatic, breast, colon, gastric, prostate, esophageal, lung cancer and lymphomas. Additionally, specific karyopherin-β proteins, such as exportin-1, have been implicated in drug resistance in cancer (Palacios, 2021).
Many importins exhibit enriched expression in gonads and are functionally required during different stages of spermatogenesis and oogenesis across many species, including Drosophila. Drosophila ovaries are organized into discrete units called ovarioles, which contain a series of sequentially developing egg chambers. Each egg chamber is comprised of 16 germ cells, 15 nurse cells and one oocyte, surrounded by a layer of somatic follicle cells. The initiation of meiosis occurs early in oogenesis, marked by the formation of the synaptonemal complex (SC) and the generation of the programmed double strand breaks. After these first events, oocytes remain arrested in prophase 1 of meiosis until stage 12, followed by prometaphase 1 at stage 13 and metaphase 1 at stage 14 (Palacios, 2021).
The Drosophila testis is structured as a closed-end coiled tube. At the tip of the testis, 10-14 germline stem cells (GSCs) surround a small cluster of somatic cells called the hub. GSCs typically divide asymmetrically to produce another GSC and a gonialblast. Gonialblasts become enveloped by two somatic cyst cells, that function in an analogous manner to the Sertoli cells of the mammalian testis. The Drosophila gonialblast goes through four incomplete mitotic divisions to form an interconnected 16-cell spermatogonial cell cyst. Each spermatocyte within the cyst undergoes meiosis, resulting in the formation of cysts that contain 64 interconnected haploid cells. Immediately after the completion of meiosis, these cells enter the 'onion stage', which is marked by the appearance of the nebenkern, a two-stranded helical structure derived from mitochondria. Defects in meiosis can result in the appearance of fragmented nebenkern and alterations in the normal 1:1 ratio of nuclei and nebenkern (Palacios, 2021).
Spermiogenesis is marked by nuclear elongation and chromatin reorganization. Nuclear elongation is dependent on microtubules from the basal body that associate with the nucleus. Chromatin organization switches from a histone-based to protamine-based packaging in the late elongation stage. During elongation, the nuclear envelope that is in contact with the basal body forms a cavity that fills with microtubules while the nucleus takes on a 'canoe' shape. During chromatin reorganization, histones are ubiquitylated by an unknown ubiquitin ligase and subsequently degraded by the proteasome at the late canoe stage, immediately before protamines are incorporated into the chromatin. After histone removal, the transition like-protein (Tpl) is incorporated. This facilitates protamine incorporation. In Drosophila, mature sperm contain Mst35Ba (protamine A), Mst35Bb (protamine B) and Mst77F. Towards the end of spermiogenesis, sperm form their own membranes in a process called individualization (Fabian and Brill, 2012) (Palacios, 2021).
In Drosophila, mutants in several importins develop normally into adults, but exhibit various defects in fertility. Importin α2 mutant males exhibit a dramatic decrease in the formation of individualized and motile sperm, whereas mutant females produce small and deflated eggs with missing or fused dorsal appendages. Similarly, mutations in Importin α1 also cause male and female sterility, marked by egg-laying defects in females and the formation of spermatocytes with abnormally large round nuclei in males, and loss of Importin α3 leads to the arrest of oogenesis. The specific cargoes responsible for these phenotypes remain unknown (Palacios, 2021).
This study reports that null mutations in Ipo9 (also known as Ranbp9) cause disruption of chromosome segregation and condensation during meiosis in both female and male Drosophila. Previous results have shown that Ipo9 helps to traffic Actin, Histone H2A-H2B dimers and a variety of other factors into nuclei. This study confirmed that loss of Drosophila Ipo9 disrupts the accumulation of nuclear actin during oogenesis. In addition, it was found that Ipo9 promotes chromosome segregation during meiosis, and the exchange of histones for protamines during spermiogenesis. Biochemical experiments suggest that Ipo9 physically associates with proteasome components, and immunofluorescence studies show that loss of Ipo9 disrupts the normal trafficking of the proteasome into germ cell nuclei during spermiogenesis. Together, these data reveal new processes directly regulated by a specific nuclear transport factor during gametogenesis (Palacios, 2021).
This study provides evidence that Ipo9 specifically regulates a number of critical processes during Drosophila gametogenesis. Ipo9 null mutants survive to adulthood but exhibit female and male sterility. In the ovary, loss of Ipo9 results in defects in chromosome orientation and segregation during meiosis, resulting in mitotic catastrophes during early embryogenesis in progeny derived from Ipo9 homozygous mutant females. Ipo9 mutant males also exhibit numerous phenotypes during germ cell development, including defects in meiosis, and disruption of the nuclear shape changes and failure to fully exchange histones for protamines during spermiogenesis. Together, these represent a unique spectrum of phenotypes compared to other Drosophila β-karyopherin family members. Of the 12 Drosophila β-karyopherin genes that have been genetically characterized, loss-of-function alleles in seven result in lethality. Several other importin mutants do survive until adulthood, including ebomut, aplnull and artsnull. ebomut homozygotes display neuronal defects, aplnull mutants are male sterile, whereas artsnull mutant females produce smaller eggs that cannot be fertilized. In addition, a ketel dominant negative mutant (ketelD) shows a female sterile phenotype and embryos derived from these flies exhibit chromosome segregation defects somewhat similar to those displayed by Ipo9KO mutants. Thus, amongst Drosophila karyopherin family members studied to date, Ipo9 is the only gene that displays specific defects during meiosis in both females and males, and in late sperm development, when mutated (Palacios, 2021).
Transgenic rescue experiments confirm that Ipo9 functions to promote the transport of proteins from the cytoplasm to the nucleus during oogenesis and spermatogenesis. A full-length Ipo9 transgene rescues most of the sterile phenotypes exhibited by Ipo9 mutants when driven in the germline. It is suspected that the failure of the Ipo9 wild-type transgene to fully rescue the male sterility of the mutant is likely due to the failure of the vasa-gal4 driver to fully recapitulate the endogenous expression pattern of Ipo9. The N-terminal domains of β-karyopherin proteins normally promote cytoplasmic-to-nuclear trafficking by contacting the nuclear pore and helping cargoes move through the nuclear pore complex. This domain also binds to RanGTP, and thus participates in the cycling of importins back-and-forth between the cytoplasm and nucleus. Strikingly, deletion of the N-terminal karyopherin domain renders the transgene non-functional, confirming that Ipo9 acts as an essential transport factor during gametogenesis in both males and females (Palacios, 2021).
The transition from histone-based to protamine-based chromatin organization is essential for the nuclear shaping that leads to a highly compact sperm nucleus. Ipo9KO nuclei are able to incorporate protamine-B, however histone H2A and H2Av are not completely removed. These results may partially explain why Ipo9KO nuclei do not elongate properly. Evidence that the ubiquitin proteasome pathway is involved in histone removal during spermiogenesis includes the histone ubiquitylation that occurs before protamine deposition and the delay in histone removal in proteasome mutants. Interestingly, Ipo9KO nuclei do not exhibit strong nuclear ubiquitylation after protamine incorporation, even though they still retain nuclear histones. Additionally, it was observed that Ipo9KO spermatids showed a significant reduction in the nuclear localization of several proteasome proteins, including Prosα6T, Prosα3T and Prosα2, compared to the control spermatids. These results suggest that the ligase(s) responsible for histone ubiquitylation and components of proteasome that ultimately degrade ubiquitylated histones are potential cargoes of Ipo9. Interestingly, Ipo9 appears to physically associate with a number of specific ubiquitin ligases, including Hyperplastic discs and KLHL10, which have been implicated in the regulation of male germ cell development, and with several components of the proteasome. Perhaps Ipo9 has evolved to temporally coordinate the import of these functionally related proteins during late sperm development. Such specialization in nuclear import may offer an economy of scale that would not exist if the responsibility of nuclear import during this critical phase of sperm development, when the cytoplasm and nuclei of sperm are becoming highly compacted, was spread across a number of potentially redundant β-karyopherins. This type of coordination in trafficking has been proposed previously in different contexts. Thus, further study of Ipo9 cargoes during sperm development may reveal critical unknown factors that play roles in meiosis, chromosome compaction and segregation, and nuclear shape changes (Palacios, 2021).
The Hedgehog (Hh) family of secreted proteins governs embryonic development and adult tissue homeostasis by regulating the abundance, localization, and activity of the GPCR family protein Smoothened (Smo). Smo trafficking and subcellular accumulation are controlled by multiple posttranslational modifications (PTMs) including phosphorylation, ubiquitination, and sumoylation, which appears to be conserved from Drosophila to mammals. Smo ubiquitination is dynamically regulated by E3 ubiquitin ligases and deubiquitinases (dubs) and is opposed by Hh signaling. By contrast, Smo sumoylation is stimulated by Hh, which counteracts Smo ubiquitination by recruiting the dub USP8. This study describes cell-base assays for Smo ubiquitination and its regulation by Hh and the E3 ligases in Drosophila. Also described are assays for Smo sumoylation in both Drosophila and mammalian cultured cells (Han, 2022).
During the maternal-to-zygotic transition (MZT), which encompasses the earliest stages of animal embryogenesis, a subset of maternally supplied gene products is cleared, thus permitting activation of zygotic gene expression. In the Drosophila melanogaster embryo, the RNA-binding protein Smaug (SMG) plays an essential role in progression through the MZT by translationally repressing and destabilizing a large number of maternal mRNAs. The SMG protein itself is rapidly cleared at the end of the MZT by a Skp/Cullin/F-box (SCF) E3-ligase complex. Clearance of SMG requires zygotic transcription and is required for an orderly MZT. This study shows that an F-box protein, which was named Bard (encoded by CG14317), is required for degradation of SMG. Bard is expressed zygotically and physically interacts with SMG at the end of the MZT, coincident with binding of the maternal SCF proteins, SkpA and Cullin1, and with degradation of SMG. shRNA-mediated knock-down of Bard or deletion of the bard gene in the early embryo results in stabilization of SMG protein, a phenotype that is rescued by transgenes expressing Bard. Bard thus times the clearance of SMG at the end of the MZT (Cao, 2021).
JAK/STAT signaling regulates central biological functions such as development, cell differentiation and immune responses. In Drosophila, misregulated JAK/STAT signaling in blood cells (hemocytes) induces their aberrant activation. This study identified several components of the proteasome complex as negative regulators of JAK/STAT signaling in Drosophila. A selected proteasome component, Prosα6, was studied further. In S2 cells, Prosα6 silencing decreased the amount of the known negative regulator of the pathway, ET, leading to enhanced expression of a JAK/STAT pathway reporter gene. Silencing of Prosα6 in vivo resulted in activation of the JAK/STAT pathway, leading to the formation of lamellocytes, a specific hemocyte type indicative of hemocyte activation. This hemocyte phenotype could be partially rescued by simultaneous knockdown of either the Drosophila STAT transcription factor, or MAPKK in the JNK-pathway. These results suggest a role for the proteasome complex components in the JAK/STAT pathway in Drosophila blood cells both in vitro and in vivo (Jarvela-Stolting, 2021).
Oculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder characterized by progressive weakness and degeneration of specific muscles. OPMD is due to extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). Aggregation of the mutant protein in muscle nuclei is a hallmark of the disease. Previous transcriptomic analyses revealed the consistent deregulation of the ubiquitin-proteasome system (UPS) in OPMD animal models and patients, suggesting a role of this deregulation in OPMD pathogenesis. Subsequent studies proposed that UPS contribution to OPMD involved PABPN1 aggregation. This study used a Drosophila model of OPMD, expression of alanine-expanded mammalian PABPN1 in Drosophila muscles, to address the functional importance of UPS deregulation in OPMD. Through genome-wide and targeted genetic screens a large number of UPS components were identified that are involved in OPMD. Half dosage of UPS genes reduces OPMD muscle defects suggesting a pathological increase of UPS activity in the disease. Quantification of proteasome activity confirms stronger activity in OPMD muscles, associated with degradation of myofibrillar proteins. Importantly, improvement of muscle structure and function in the presence of UPS mutants does not correlate with the levels of PABPN1 aggregation, but is linked to decreased degradation of muscle proteins. Oral treatment with the proteasome inhibitor MG132 is beneficial to the OPMD Drosophila model, improving muscle function although PABPN1 aggregation is enhanced. This functional study reveals the importance of increased UPS activity that underlies muscle atrophy in OPMD. It also provides a proof-of-concept that inhibitors of proteasome activity might be an attractive pharmacological approach for OPMD (Ribot, 2022).
Ubiquitylation is critical for preventing aberrant DNA repair and for efficient maintenance of genome stability. As deubiquitylases (DUBs) counteract ubiquitylation, they must have a great influence on many biological processes, including DNA damage response. To elucidate the role of DUBs in DNA repair in Drosophila melanogaster, systematic siRNA screening was applied to identify DUBs with a reduced survival rate following exposure to ultraviolet and X-ray radiations. As a secondary validation, the direct repeat (DR)-white reporter system with which site-specific DSBs were induced was applied and the importance of the DUBs Ovarian tumor domain-containing deubiquitinating enzyme 1 (Otu1), Ubiquitin carboxyl-terminal hydrolase 5 (Usp5), and Ubiquitin carboxyl-terminal hydrolase 34 (Usp34) in DSB repair pathways were applied using Drosophila. The results indicate that the loss of Otu1 and Usp5 induces strong position effect variegation in Drosophila eye following I-SceI-induced DSB deployment. Otu1 and Usp5 are essential in DNA damage-induced cellular response, and both DUBs are required for the fine-tuned regulation of the non-homologous end joining pathway. Furthermore, the Drosophila DR-white assay demonstrated that homologous recombination does not occur in the absence of Usp34, indicating an indispensable role of Usp34 in this process (Pahi, 2022).
Ubiquitin (Ub)-mediated regulation of plasmalemmal ion channel activity canonically occurs via stimulation of endocytosis. Whether ubiquitination can modulate channel activity by alternative mechanisms remains unknown. This study shows that the transient receptor potential vanilloid 4 (TRPV4) cation channel is multiubiquitinated within its cytosolic N-terminal and C-terminal intrinsically disordered regions (IDRs). Mutagenizing select lysine residues to block ubiquitination of the N-terminal but not C-terminal IDR resulted in a marked elevation of TRPV4-mediated intracellular calcium influx, without increasing cell surface expression levels. Conversely, enhancing TRPV4 ubiquitination via expression of an E3 Ub ligase reduced TRPV4 channel activity but did not decrease plasma membrane abundance. These results demonstrate Ub-dependent regulation of TRPV4 channel function independent of effects on plasma membrane localization. Consistent with ubiquitination playing a key negative modulatory role of the channel, gain-of-function neuropathy-causing mutations in the TRPV4 gene led to reduced channel ubiquitination in both cellular and Drosophila models of TRPV4 neuropathy, whereas increasing mutant TRPV4 ubiquitination partially suppressed channel overactivity. Together, these data reveal a novel mechanism via which ubiquitination of an intracellular flexible IDR domain modulates ion channel function independently of endocytic trafficking and identify a contributory role for this pathway in the dysregulation of TRPV4 channel activity by neuropathy-causing mutations (Aisenberg, 2022).
SQSTM1/p62-type selective macroautophagy/autophagy receptors cross-link poly-ubiquitinated cargo and autophagosomal LC3/Atg8 proteins to deliver them for lysosomal degradation. Consequently, loss of autophagy leads to accumulation of polyubiquitinated protein aggregates that are also frequently seen in various human diseases, but their physiological relevance is incompletely understood. Using a genetically non-redundant Drosophila model, this study shows that specific disruption of ubiquitinated protein autophagy and concomitant formation of polyubiquitinated aggregates has hardly any effect on bulk autophagy, proteasome activity and fly healthspan. Accumulation of ref(2)P/SQSTM1 due to a mutation that disrupts its binding to Atg8a results in the co-sequestering of Keap1 and thus activates the cnc/NFE2L2/Nrf2 antioxidant pathway. These mutant flies have increased tolerance to oxidative stress and reduced levels of aging-associated mitochondrial superoxide. Interestingly, ubiquitin overexpression in ref(2)P point mutants prevents the formation of large aggregates and restores the cargo recognition ability of ref(2)P, although it does not prevent the activation of antioxidant responses. Taken together, potential detrimental effects of impaired ubiquitinated protein autophagy are compensated by the aggregation-induced antioxidant response (Bhattacharjee, 2022).
Methionine 1 (M1)-linked ubiquitination plays a key role in the regulation of inflammatory nuclear factor-κB (NF-κB) signalling and is important for clearance of pathogen infection in Drosophila melanogaster. M1-linked ubiquitin (M1-Ub) chains are assembled by the linear ubiquitin E3 ligase (LUBEL) in flies. The role of LUBEL was studied in sterile inflammation induced by different types of cellular stresses. The LUBEL was found to catalyse formation of M1-Ub chains in response to hypoxic, oxidative and mechanical stress conditions. LUBEL is shown to be important for flies to survive low oxygen conditions and paraquat-induced oxidative stress. This protective action seems to be driven by stress-induced activation of the NF-κB transcription factor Relish via the immune deficiency (Imd) pathway. In addition to LUBEL, the intracellular mediators of Relish activation, including the transforming growth factor activating kinase 1 (Tak1), Drosophila inhibitor of apoptosis (IAP) Diap2, the IκB kinase γ (IKKγ) Kenny and the initiator caspase Death-related ced-3/Nedd2-like protein (Dredd), but not the membrane receptor peptidoglycan recognition protein (PGRP)-LC, are shown to be required for sterile inflammatory response and survival. Finally, it was shown that the stress-induced upregulation of M1-Ub chains in response to hypoxia, oxidative and mechanical stress is also induced in mammalian cells and protects from stress-induced cell death. Taken together, these results suggest that M1-Ub chains are important for NF-κB signalling in inflammation induced by stress conditions often observed in chronic inflammatory diseases and cancer (Aalto, 2022).
The mitochondrial kinase PTEN-induced kinase 1 (PINK1) and cytosolic ubiquitin ligase (E3) Parkin/PRKN are involved in mitochondrial quality control responses. PINK1 phosphorylates ubiquitin and the Parkin ubiquitin-like (Ubl) domain at serine 65 and promotes Parkin activation and translocation to damaged mitochondria. Upon Parkin activation, the Ubl domain is ubiquitinated at lysine (K) 27 and K48 residues. However, contribution of K27/K48 ubiquitination towards Parkin activity remains unclear. In this study, ubiquitination of K56 (corresponding to K27 in the human), K77 (K48 in the human), or both, was blocked by generating Drosophila Parkin (dParkin) mutants to examine the effects of Parkin Ubl domain ubiquitination on Parkin activation in Drosophila. The dParkin, in which K56 was replaced with arginine (dParkin K56R), rescued pupal lethality in flies by co-expression with PINK1, whereas dParkin K77R could not. The dParkin K56R exhibited reduced abilities of mitochondrial fragmentation and motility arrest, which are mediated by degrading Parkin E3 substrates Mitofusin and Miro, respectively. Pathogenic dParkin K56N, unlike dParkin K56R, destabilized the protein, suggesting that not only was dParkin K56N non-ubiquitin-modified at K56 but also the structure of the Ubl domain for activation was largely affected. Ubiquitin attached to K27 of the Ubl domain during PINK1-mediated Parkin activation was likely to be phosphorylated because human Parkin K27R weakened Parkin self-binding and activation in trans. Therefore, these findings suggest a new mechanism of Parkin activation, where an activation complex is formed through phospho-ubiquitin attachment on the K27 residue of the Parkin Ubl domain (Liu, 2022).
Ubiquitylation of the ligands and the receptor plays an important part in the regulation of the activity of the evolutionary conserved Notch signalling pathway. However, its function for activation of Notch is not completely understood, despite the identification of several E3 ligases devoted to the receptor. This study analysed a variant of the Notch receptor where all lysines in its intracellular domain are replaced by arginines. A analysis of this variant revealed that ubiquitylation of Notch is not essential for its endocytosis. Two functions were identifiee for ubiquitylation of lysines in the Notch receptor. First, it is required for the degradation of free Notch intracellular domain (NICD) in the nucleus, which prevents a prolonged activation of the pathway. More importantly, it is also required for the incorporation of Notch into intraluminal vesicles of maturing endosomes to prevent ligand-independent activation of the pathway from late endosomal compartments. These findings clarify the role of lysine-dependent ubiquitylation of the Notch receptor and indicate that Notch is endocytosed by several independent operating mechanisms (Schnute, 2022).
AGO/miRNA-mediated gene silencing and ubiquitin-mediated protein quality control represent two fundamental mechanisms that control proper gene expression. This study unexpectedly discovered that fly and human AGO proteins (see Drosophila Ago1), which are key components in the miRNA pathway, undergo lipid-mediated phase separation and condense into RNP granules on the endoplasmic reticulum (ER) membrane to control protein production. Phase separation on the ER is mediated by electrostatic interactions between a conserved lipid-binding motif within the AGOs and the lipid PI(4,5)P(2). The ER-localized AGO condensates recruit the E3 ubiquitin ligase Ltn1 to catalyze nascent-peptide ubiquitination and coordinate with the VCP-Ufd1-Npl4 complex to process unwanted protein products for proteasomal degradation. Collectively, this study provides insight into the understanding of post-transcription-translation coupling controlled by AGOs via lipid-mediated phase separation (Gao, 2022).
AGO proteins play principal roles in regulating small-RNA-mediated gene silencing. Interestingly, AGO proteins are present in cytoplasmic RNA granules and associate with membrane-bound organelles (e.g., ER). However, the functional importance of membrane-associated AGO proteins has long been underestimated. This study focused on the membrane function of AGO proteins. Investigation of the dmAGO1 revealed a mechanism where ER-localized dmAGO1 forms a complex with the E3 ubiquitin ligase Ltn1 to catalyze the ubiquitination of nascent peptides and then coordinates with the VCP-Ufd1-Npl4 complex to process unwanted new protein products, which are ultimately degraded by the proteasome. Thus, in addition to facilitating miRNA-guided repression of RNA translation, dmAGO1 also acts in concert with the ribosome quality control machinery to ensure efficient repression of gene products. Given that AGO proteins play evolutionarily conserved roles in gene expression, this study provides a critical starting point toward mechanistic understandings of post-transcription-translation coupling controlled by AGO proteins (Gao, 2022).
AGO proteins associate with cellular membranes (e.g., ER membrane). However, the functional role of AGO proteins' association with the ER membrane and how this association is regulated have remained unresolved. Prior structural studies demonstrated that AGO proteins have six well-defined domains. The PAZ domain and the MID-PIWI domains function to anchor the 3′ and 5′ ends of the small RNA guide, respectively. This study showed that lipid binding might be a fundamental function of the N domain of dmAGO1 and hsAGO2. Sequence analysis revealed that these AGO proteins do not have any transmembrane domain. However, comparison assays between the N domain of AGOs and the PH domain from phospholipase C-δ1 suggested that the two domains display a striking similarity in topological patterns. Moreover, in vitro protein-lipid binding assays showed that AGOs selectively interacted with PI(4.5)P2. Finally, this study showed that vcp knockdown caused a significant increase in PI(4,5)P2 levels in ER membranes. The increased levels of PI(4,5)P2 serve as docking modules for AGO proteins to access the ER membrane and execute their function. Consistently, expression of BiP-Flag-AGOs-KDEL enhanced the gene silencing activity when compared with wild-type AGOs. It is worth noting that the 4-residue (KDEL) extension at the C terminal of AGOs (dmAGO1 or hsAGO2) did not affect the slicer activities to cleave the targets. Collectively, these findings revealed that lipid-mediated condensation of AGOs provides a primary mechanism for function of AGOs on the ER membrane, where AGOs repress target mRNA translation and control nascent-peptide ubiquitination (Gao, 2022).
Human AGO2 plays a compelling role in tumorigenesis and cancer aggressiveness; however, the mechanisms underlying the action of hsAGO2 in cancer remain elusive. Given that lipid binding is important for AGOs' function on cellular membrane and that the LBM contains two cancer-related mutations (based on COSMIC database), it would be interesting to study whether the lipid-mediated membrane function of hsAGO2 is related to cancer in future (Gao, 2022).
It is well documented that AGOs form a complex with miRNAs and utilize miRNAs to bind the target mRNAs for post-transcriptional repression. This study uncovered a mechanism by which AGOs form a complex with Ltn1 on the ER, where AGOs facilitate Ltn1 to catalyze nascent peptides for ubiquitination, thus ensuring efficient gene silencing. Although this study identified more than one hundred high-confident targets for dmAGO1/Ltn1, they were not analyze in depth in this study. The identified targets are of potential interest because they are involved in a variety of biological processes. For example, a significant portion of targets of dmAGO1/Ltn1 is involved in the secretion pathway, which is consistent with the previous findings. Interestingly, this study also found that a number of targets of dmAGO1/Ltn1 are mitochondrial proteins; this finding raises an intriguing possibility that dmAGO1/Ltn1-mediated PQC might contribute to the homeostasis and function of mitochondria. It would be important to solve this issue in the future studies (Gao, 2022).
E3 ubiquitin ligase, HOIL1-interacting protein (HOIP), forms the linear ubiquitin chain assembly complex (LUBAC) with HOIL and SHANK-associated RH domain interactor and catalyzes linear ubiquitination, directly linking the N- and C-termini of ubiquitin. Recently, several studies have implicated linear ubiquitination in aging and Alzheimer disease (AD). However, little is currently known about the roles of HOIP in brain aging and AD pathology. This studyinvestigated the role of linear ubiquitin E3 ligase (LUBEL), a Drosophila HOIP ortholog, in brain aging and amyloid β (A:beta;) pathology in a Drosophila AD model. DNA double-strand breaks (DSBs) were increased in the aged brains of neuron-specific LUBEL-knockdown flies compared to the age-matched controls. Silencing of LUBEL in the neuron of AD model flies increased the neuronal apoptosis and neurodegeneration, whereas silencing in glial cells had no such effect. A&bet; aggregation levels and DSBs were also increased in the LUBEL-silenced AD model fly brains, but autophagy and proteostasis were not affected by LUBEL silencing. Collectively, these results suggest that LUBEL protects neurons from aging-induced DNA damage and Aβ neurotoxicity (Choi, 2022).
Target-directed microRNA (miRNA) degradation (TDMD), which is mediated by the protein ZSWIM8 (mammalian homolog of Dorado), plays a widespread role in shaping miRNA abundances across bilateria. Some endogenous small interfering RNAs (siRNAs) of Drosophila cells have target sites resembling those that trigger TDMD, raising the question as to whether they too might undergo such regulation by Dorado, the Drosophila ZSWIM8 homolog. This study finds that some of these siRNAs are indeed sensitive to Dora when loaded into Ago1, the Argonaute paralog that preferentially associates with miRNAs. Despite this sensitivity when loaded into Ago1, these siRNAs are not detectably regulated by target-directed degradation because most molecules are loaded into Ago2, the Argonaute paralog that preferentially associates with siRNAs, and with siRNAs and miRNAs loaded into Ago2 were found to be insensitive to Dora. One explanation for the protection of these small RNAs loaded into Ago2 is that these small RNAs are 2'-O-methylated at their 3' termini. However, 2'-O-methylation does not protect these RNAs from Dora-mediated target-directed degradation, which indicates that their protection is instead conferred by features of the Ago2 protein itself. Together, these observations clarify the requirements for regulation by target-directed degradation and expand understanding of the role of 2'-O-methylation in small-RNA biology (Kingston, 2021).
Analyses of siRNAs in both wild-type and dora Drosophila S2 cells demonstrated that this class of small RNAs undergoes little regulation by conventional target-directed degradation (TDD). This finding does not support the suggestion that siRNAs loaded into Ago1 instead of Ago2 might be 'purified,' or removed from the cell, by TDD-a model put forth to help explain the high steady-state enrichment of siRNAs within Ago2. Although TDD of some Ago1-loaded siRNAs is seen, most siRNAs loaded in Ago1 escape such regulation. Furthermore, even for the TDD-sensitive siRNAs, up-regulation upon loss of Dora was undetectable when examining total-sRNA samples, because, for each siRNA, the Ago1-loaded fraction was minimal when compared to the Ago2-loaded fraction, and thus any increase in the Ago1-loaded fraction negligibly affected total siRNA levels. Thus, the known TDD pathway, which requires Dora, does not appear to be a major driving force in shaping the siRNA content of Drosophila S2 cells (Kingston, 2021).
Although methylation of small RNAs is proposed to protect these RNAs from TDD, this study observed both that loss of methylation does not make Ago2-loaded species susceptible to the known TDD pathway and that gained methylation does not protect Ago1-loaded species from this pathway. These observations indicate that features of Ago proteins, rather than modifications of small RNAs, dictate the ability of Ago-RNA complexes to be regulated by TDD. This importance of the Ago protein concurs with the new model for TDD, in which Ago proteins must interact with and be ubiquitinated by the ZSWIM8/Dora for TDD to occur (Han. 2020; Shi; 2020). Whereas Ago1 can engage with Dora in a TDD-competent manner, low sequence similarity between Ago2 and Ago1 supports the idea that Ago2 might lack the features necessary for Dora recognition and polyubiquitination. With respect to the sites of polyubiquitination, studies of human AGO2 implicate K493 and at least one other lysine within a cluster of 17 surface lysines as required for maximal ZSWIM8-mediated regulation, of which K493 and 12 of the other candidates sites are conserved in Drosophila Ago1, whereas K493 and all but two of the other candidates sites are not conserved in Drosophila Ago2. By analogy, it is speculated that if piRNAs are also protected from TDD, then this protection would also be conferred by the inability of PIWI proteins to interact with and be ubiquitinated by the ZSWIM8/Dora (Kingston, 2021).
Across many species, 2'-O-methylation occurs on guide RNAs that have extensive pairing to their targets, such as plant miRNAs, but not on guide RNAs that lack extensive pairing to most of their targets, such as metazoan miRNAs. Loss of this methylation leads to increased tailing and trimming that, at least for plant miRNAs, Tetrahymena piRNAs, nematode 26G siRNAs, and some Drosophila siRNAs, is associated with small-RNA destabilization. The realization that the identity of the Ago protein rather than the methylation status of the small RNA dictates susceptibility to TDD reopens the mystery as to why the tendency to be methylated correlates with the degree of complementarity of typical sites for a given class of small RNA (Kingston, 2021).
As a new solution to this mystery, it is suggested that these classes of small regulatory RNAs with highly complementary sites reside in Ago/PIWI proteins that have intrinsically weaker interactions with the 3' termini of their guide RNAs. This weaker intrinsic binding to guide-RNA 3' termini is expected for these proteins because it would favor formation of extensive target pairing, as release of the 3' terminus appears to be required to accommodate pairing to the central region of the guide RNA. In contrast, stronger intrinsic binding to guide-RNA 3' termini is expected for proteins that associate with metazoan miRNAs, as release of the 3' terminus is not required to accommodate target recognition typical of these small RNAs, i.e., seed pairing or seed pairing plus conventional 3'-supplementary pairing. The weaker intrinsic binding proposed for Ago/PIWI proteins with guide RNAs that recognize highly complementarity sites would presumably leave the 3' termini of their guide RNAs constitutively vulnerable to tailing and trimming even when they are not paired to a target, thereby explaining the benefit of terminal 2'-O-methylation. This Hen1-mediated methylation, found in plants and animals, presumably emerged early in eukaryotic evolution and thus would have been available for incorporation into the nascent metazoan miRNA pathway. Indeed, some methylation has been reported on most Nematostella miRNAs. Perhaps, however, as exemplified by the bilaterian lineage, as the miRNA-associated Ago proteins adapted to recognize less extensively paired sites, they acquired greater affinity to their guide-RNA 3' termini, which reduced vulnerability to trimming and tailing thereby obviating a benefit for their methylation (Kingston, 2021).
Several observations support aspects of this model. First, mutations within human Ago2 that reduce binding to miRNA 3' termini promote tailing and trimming even in the absence of an extensively paired target, which confirms the assumption that weaker binding to small-RNA 3' termini imparts constitutive vulnerability to tailing and trimming. Second, the 3' termini of piRNAs and metazoan siRNAs are 2'-O-methylated after these guide RNAs are loaded into Ago/PIWI, implying that the methylation machinery has at least intermittent access to the guide-RNA 3' termini, as would be expected if these proteins have relatively weak binding to the 3' termini of their guide RNAs. Third, loss of Hen1 led to increased tailing and trimming of Ago2-associated siRNAs that were Dora-insensitive when associated with Ago1, supporting the conjecture that Ago2-associated RNAs are vulnerable to tailing and trimming even in the absence of highly complementary sites able to trigger TDD (Kingston, 2021).
The notion that small RNAs with 3' termini not stably protected within Ago are susceptible to increased tailing and trimming might also help explain the observation that methylated miR-7, when loaded in Ago1, undergoes increased trimming relative to unmethylated miR-7. Perhaps the terminal methyl group is not well-accommodated by Ago1, which is typically loaded with unmethylated miRNAs, leading to increased exposure of the 3' terminus of the methylated miR-7. Indeed, conformations of human AGO2 represented by the crystal structures would not accommodate a terminal methyl group, implying that terminal methyl modifications might similarly clash with the ground-state structure of Drosophila Ago1. Although methylation is thought to protect small RNAs from trimming in addition to tailing, the observation that trimming of methylated miR-7 is Nibbler sensitive suggests that, at least in the context of Drosophila Ago1, methylated species can still be trimmed (Kingston, 2021).
In summary, the observation that some classes of small regulatory RNAs are methylated and some are not can be at least partly explained without invoking a TDD phenomenon: Because piRNAs, siRNAs, and plant miRNAs must efficiently pair to targets throughout their length, their corresponding Ago/PIWI proteins might have reduced affinity to guide-RNA 3' termini, and this reduced affinity would render these guide RNAs more susceptible to tailing and trimming even when they are not paired to target-unless they are 2'-O-methylated (Kingston, 2021).
MicroRNAs (miRNA) load onto AGO proteins to target mRNAs for translational repression or degradation. However, miRNA degradation can be triggered when extensively base-paired with target RNAs, which induces confirmational change of AGO and recruitment of ZSWIM8 ubiquitin ligase to mark AGO for proteasomal degradation. This target RNA-directed miRNA degradation (TDMD) mechanism appears to be evolutionarily conserved, but recent studies have focused on mammalian systems. This study performed AGO1-CLASH in Drosophila S2 cells, with Dora (ortholog of vertebrate ZSWIM8) knockout mediated by CRISPR-Cas9 to identify five TDMD triggers (sequences that can induce miRNA degradation). Interestingly, one trigger in the 3' UTR of AGO1 mRNA induces miR-999 degradation. CRISPR-Cas9 knockout of the AGO1 trigger in S2 cells and in Drosophila specifically elevates miR-999, with concurrent repression of the miR-999 targets. AGO1 trigger knockout flies respond poorly to hydrogen peroxide-induced stress, demonstrating the physiological importance of this TDMD event (Sheng, 2023).
In mitosis and meiosis, chromosome segregation is triggered by the Anaphase-Promoting Complex/Cyclosome (APC/C), a multi-subunit ubiquitin ligase that targets proteins for degradation, leading to the separation of chromatids. APC/C activation requires phosphorylation of its APC3 and APC1 subunits, which allows the APC/C to bind its co-activator Cdc20. The identity of the kinase(s) responsible for APC/C activation in vivo is unclear. Cyclin B3 (CycB3) is an activator of the Cyclin-Dependent Kinase 1 (Cdk1) that is required for meiotic anaphase in flies, worms and vertebrates. It has been hypothesized that CycB3-Cdk1 may be responsible for APC/C activation in meiosis but this remains to be determined. Using Drosophila, this study found that mutations in CycB3 genetically enhance mutations in tws, which encodes the B55 regulatory subunit of Protein Phosphatase 2A (PP2A) known to promote mitotic exit. Females heterozygous for CycB3 and tws loss-of-function alleles lay embryos that arrest in mitotic metaphase in a maternal effect, indicating that CycB3 promotes anaphase in mitosis in addition to meiosis. This metaphase arrest is not due to the Spindle Assembly Checkpoint (SAC) because mutation of mad2 that inactivates the SAC does not rescue the development of embryos from CycB3-/+, tws-/+ females. Moreover, CycB3 was found to promote APC/C activity and anaphase in cells in culture. CycB3 physically associates with the APC/C, is required for phosphorylation of APC3, and promotes APC/C association with its Cdc20 co-activators Fizzy and Cortex. These results strongly suggest that CycB3-Cdk1 directly activates the APC/C to promote anaphase in both meiosis and mitosis (Garrido, 2020).
Mitosis and meiosis (collectively referred to as M-phase) are distinct modes of nuclear division resulting in diploid or haploid products, respectively. In animals, both require the breakdown of the nuclear envelope, the condensation of chromosomes and their correct attachment on a microtubule-based spindle, where chromosomes are under tension and chromatids are held together by cohesins. Progression through these initial phases requires multiple phosphorylation events of various protein substrates by mitotic kinases including Cyclin-Dependent Kinases (CDKs) activated by their mitotic cyclin partners. M-phase completion from this point (mitotic exit) requires the degradation of mitotic cyclins, and the dephosphorylation of several mitotic phosphoproteins by phosphatases including Protein Phosphatase 2A (PP2A). Mitotic exit begins with the segregation of chromosomes in anaphase. In mitosis, sister chromatids segregate. In meiosis I, replicated homologous chromosomes segregate, and in the subsequent meiosis II, sister chromatids segregate. Nuclear divisions are completed with the reassembly of a nuclear envelope concomitant with the decondensation of chromosomes. How mitosis and meiosis are alike and differ in the molecular mechanisms of their exit programs is not completely understood (Garrido, 2020).
Chromosome segregation is triggered by the Anaphase-Promoting Complex/Cyclosome (APC/C), a multi-subunit E3 ubiquitin ligase. By catalysing the addition of ubiquitin chains on the separase inhibitor securin, the APC/C targets it for degradation by the proteasome. As a result, separase cleaves cohesins, allowing separated chromosomes to migrate towards opposing poles of the spindle. Activation of the APC/C in mitosis requires its recruitment of its co-factor Cdc20. This recruitment can be prevented by the Spindle-Assembly Checkpoint (SAC), a complex mechanism that allows the sequestration of Cdc20 until all chromosomes are correctly attached on the spindle. Cdc20 binding to the APC/C is also inhibited by its phosphorylation at CDK sites. Phosphatase activity is then required to dephosphorylate Cdc20 and allow its binding of the APC/C for its activation of anaphase. In addition, phosphorylation of the APC/C itself is required to allow Cdc20 binding. Phosphorylation of APC3/Cdc27 and APC1 is key to this process. Phosphorylation of APC3 at CDK sites promotes the subsequent phosphorylation of APC1, inducing a conformational change in APC1 that opens the Cdc20 binding site. However, the precise identity of the kinase(s) involved in this process in vivo is unknown (Garrido, 2020).
At least 3 types of cyclins contribute to M-phase in animals: Cyclins A, B and B3. The Cyclin A type (A1 and A2 in mammals) can activate Cdk1 or Cdk2 and is required for mitotic entry, at least in part by allowing the phosphorylation of Cdc20 to prevent its binding and activation of the APC/C. This allows mitotic cyclins to accumulate without being ubiquitinated prematurely by the APC/C and degraded. The Cyclin B type (B1 and B2 in mammals) also promotes mitotic entry and is required for mitotic progression by allowing the phosphorylation of several substrates by Cdk1. Mammalian Cyclin B3, which can associate with both Cdk1 and Cdk2, is required for meiosis but its contribution to mitosis is less clear in view of its low expression in somatic cells. Drosophila possesses a single gene for each M-phase cyclin: CycA (Cyclin A), CycB (Cyclin B) and CycB3 (Cyclin B3) that collaborate to ensure mitotic progression by activating Cdk1. Genetic and RNAi results suggest that they act sequentially, CycA being required before prometaphase, CycB before metaphase and CycB3 at the metaphase-anaphase transition. CycA is the only essential cyclin, as it is required for mitotic entry. CycB and CycB3 mutants are viable, but mutations of CycB and CycB3 are synthetic-lethal, suggesting redundant roles in mitosis. However, mutation of CycB renders females sterile due to defects in ovary development, and mutant males are also sterile (Garrido, 2020).
Drosophila CycB3 associates with Cdk1 and is required for female meiosis (Jacobs, 1998). In Drosophila, eggs normally stay arrested in metaphase I of meiosis until egg laying triggers entry into anaphase I and the subsequent meiosis II. However, CycB3 mutant eggs predominantly stay arrested in meiosis I (Bourouh, 2016). In addition, silencing CycB3 expression in early embryos delays anaphase onset during the syncytial mitotic divisions (Yuan, 2015). Cyclin B3 is also required for anaphase in female meiosis of vertebrates and worms. In mice, RNAi Knock-down of Cyclin B3 in oocytes inhibits the metaphase-anaphase transition in meiosis I. Recently, two groups independently knocked out the Cyclin B3-coding Ccnb3 gene in mice and found that they were viable but female-sterile due to a highly penetrant arrest in meiotic metaphase I. In C. elegans, the closest Cyclin B3 homolog, CYB-3 is required for anaphase in meiosis and mitosis (Garrido, 2020).
How Cyclin B3 promotes anaphase in any system is unknown. One possibility is that it is required for Cdk1 to phosphorylate the APC/C on at least one of its activating subunits, APC3 or APC1. This has not been investigated. Another possibility is that inactivation of Cyclin B3 leads to an early mitotic defect that activates the SAC. This appears to be the case in C. elegans, because inactivation of the SAC rescues normal anaphase onset in the absence of CYB-3. However, in Drosophila, inactivation of the SAC by the mutation of mad2 did not eliminate the delay in anaphase onset observed when CycB3 is silenced in syncytial embryos. Similarly, in mouse oocytes, silencing Mad2 does not rescue the meiotic metaphase arrest upon Cyclin B3 depletion. In other studies, SAC markers on kinetochores did not persist in metaphase-arrested Ccnb3 KO oocytes, and SAC inactivation by chemical inhibition of Mps1 did not restore anaphase. Finally, it is also possible that Cyclin B3 is required upstream of another event required for APC/C activation, for example the activation of a phosphatase required for Cdc20 dephosphorylation and subsequent recruitment to the APC/C (Garrido, 2020).
This study has investigated how CycB3 promotes anaphase in Drosophila. Several lines of evidence are reported indicating that CycB3 directly activates the APC/C in both meiosis and mitosis (Garrido, 2020).
Altogether, the results strongly suggest that CycB3-Cdk1 directly activates the APC/C by phosphorylation, promoting its function at the metaphase-anaphase transition in meiosis and in both maternally driven early embryonic mitoses and somatic cell divisions. This regulation is likely mediated by the phosphorylation in the activation loop of APC3 by CycB3-Cdk1 that ultimately promotes the recruitment of the Cdc20-type co-activators Fizzy and Cortex. Previous work has shown that APC3 phosphorylation and APC/C activation by cyclin-CDK complexes require their CKS subunit (see Cks30A). CKS subunits can act as processivity factors that bind phosphorylated sites to promote additional phosphorylation by the CDK. Thus, phosphorylation of APC3 would prime the binding of a cyclin-CDK-CKS complex to promote the additional phosphorylation of APC1, allowing for Cdc20 binding. It has been shown that mutation of phosphorylation sites into Asp or Glu residues cannot substitute for the presence of phosphate in the CKS binding site. Therefore, it was not possible to generate a mutation in APC3 that would have mimicked phosphorylation at S316 to enhance cyclin-CDK-CKS binding. Such a mutation in APC3, if it were possible, would have potentially rescued APC/C activity in the absence of CycB3 according to this model. However, it is likely that this analysis did not detect all phosphorylation sites in the APC/C. Thus, the possibility cannot be exclustion that other phosphorylation events, mediated by CycB3-Cdk1 or another kinase, may be required for complete APC/C activation. For example, other phosphorylation events have been proposed to regulate APC/C localization. It is even formally possible that CycB3-Cdk1 is required to activate another proline-directed kinase that phosphorylates APC3 at S316. The interdependence between CycB3 and Tws that this study uncovered may reflect a role of PP2A-Tws in the recruitment of Cdc20 co-activators to the APC/C. Cdc20 must be dephosphorylated at CDK sites before binding the APC/C, and in human cells both PP2A-B55 and PP2A-B56 promote this event (Garrido, 2020).
CycB3 is strongly required for APC/C activation in meiosis and in the early syncytial mitoses, and to a lesser extent in other mitotic divisions, despite the presence of two additional mitotic cyclins, CycA and CycB, capable of activating Cdk1. There are many possible reasons for this requirement. Overexpression of stabilized forms of CycA or CycB can block or slow down anaphase, suggesting that they may interfere with APC/C function in this transition. However, under normal expression levels, CycA or CycB or both may contribute to activate the APC/C like CycB3. CycB3 mutant flies develop until adulthood, which implies that the APC/C can be activated to induce anaphase in at least a vast proportion of mitotic cells, and this activation could be mediated by CycA and/or CycB. CycA is essential for viability and CycB mutants show strong female germline development defects, complicating the examination of potential roles for these cyclins at the metaphase-anaphase transition. Thus, in principle, the requirements for CycB3 in female meiosis, in embryos and in mitotic cells in culture could merely reflect the need for a minimal threshold of total mitotic cyclins. This possibility is considered unlikely because CycB3 is expressed at much lower levels than CycB in early embryos. Moreover, while maternal heterozygosity for mutations in CycB3 and tws causes a metaphase arrest in embryos, heterozygosity for mutations in CycB and tws does not cause embryonic defects. In fact, genetic results suggest that the function of CycB is antagonized by PP2A-Tws in embryos, while CycB3 and PP2A-Tws collaborate for APC/C activation in embryos. Thus, although it is possible that CycA and CycB can participate in APC/C activation, CycB3 probably has some unique feature that makes it particularly capable of promoting APC/C activation (Garrido, 2020).
By what mechanism could CycB3 be particularly suited for APC/C activation? Cyclins can play specific roles by contributing to CDK substrate recognition or by directing CDK activity in space and time. This study did not investigate the precise nature of the molecular recognition of the APC/C by CycB3. It may be that CycB3 possesses a specific binding site for the APC/C that is lacking in CycA and CycB. Another possibility is that differences in localization between cyclins dictate their requirements. In particular, while CycA and CycB are cytoplasmic in interphase, CycB3 is nuclear. It is surmised that the nuclear localization of CycB3 may help concentrate CycB3 in the spindle area upon germinal vesicle breakdown, when the very large oocyte enters meiosis. In future studies, it will be interesting to compare the ability of different mitotic cyclins to activate the APC/C and to determine the molecular basis of potential differences (Garrido, 2020).
In any case, the results show that CycB3 activates the APC/C and that this regulation is essential in Drosophila. Cyclin B3 has been shown to be required for anaphase in female meiosis of insects (Drosophila), worms (C. elegans) and vertebrates (mice). It is tempting to conclude that the activation of the APC/C is a function of Cyclin B3 conserved in all these species. However, in C. elegans embryos, the metaphase arrest upon CYB-3 (Cyclin B3) inactivation requires SAC activity. The underlying mechanism and whether it also occurs in other systems remain to be determined. However, CYB-3 plays roles in C. elegans that have not been detected for Cyclin B3 in flies or vertebrates, including a major role in mitotic entry, where CYB-3 mediates the inhibitory phosphorylation of Cdc20. In this regard, C. elegans CYB-3 may be more orthologous to Cyclin A. Yet, given that Cyclin B3 is required for anaphase in a SAC-independent manner in flies and mice, it seems reasonable to suggest that the direct activation of the APC/C by Cyclin B3 is conserved in vertebrates (Garrido, 2020).
The transcriptional repressor Blimp-1 is a labile protein. This characteristic is key for determining pupation timing because the timing of the disappearance of Blimp-1 affects pupation timing by regulating the expression of its target betaftz-f1. However, the molecular mechanisms that regulate the protein turnover of Blimp-1 are still unclear. This study demonstrates that Blimp-1 is regulated by the ubiquitin proteasome system. Blimp-1 degradation is inhibited by proteasome inhibitor MG132. Pupation timing was delayed in mutants of 26S proteasome subunits as well as FBXO11, which recruits target proteins to the 26S proteasome as a component of the SCF ubiquitin ligase complex by slowing down the degradation speed of Blimp-1. Delay in pupation timing in the FBXO11 mutant was suppressed by the induction of betaFTZ-F1. Furthermore, fat-body-specific knockdown of proteasomal activity was sufficient to induce a delay in pupation timing. These results suggest that Blimp-1 is degraded by the 26S proteasome and is recruited by FBXO11 in the fat body, which is important for determining pupation timing (Aly, 2018).
This study showed that Drosophila Blimp-1 is degraded by the 26S proteasome system and is recruited by FBXO11 as the substrate-recognition component of the SCF complex. Furthermore, this study showed the importance of proteasome activity in the fat body to determine pupation timing. The results are correlated with previously described results that the biological timer system for pupation is located in the fat body (Akagi, 2016; Aly, 2018 and references therein).
A delay was observed in pupation timing in all of the examined heterozygous mutants of 26S proteasome components. These results suggest gene dosage effects due to loss-of-function mutations of these 26S proteasome components. In addition, a heterozygous mutant of recruiter FBXO11 also exhibited the same level of delay in pupation timing. These results indicate that the expression level of these components is an important factor to determine pupation timing; therefore, pupation timing can be controlled by the expression level of these components. Thus, it is assumed that the UPS contributes to determine pupation timing as one of the components in the biological timer during the early prepupal period. Of note, a sudden increase in the concentration of the 26S proteasome at 0 to 4 hr APF has been reported, suggesting the importance of protein degradation in developmental control. Furthermore, RNA-Seq data in the modENCODE developmental transcriptome of D. melanogaster showed that the expression of the FBXO11 increases gradually from the 3rd instar larval stage (L3) to a moderately high level at pupation and then starts to decrease again 24 hr later. These developmental changes may allow control of the degradation speed of specific targets, including Blimp-1, among many UPS target proteins that must be degraded at appropriate time points (Aly, 2018).
This study has shown that both the Blimp-1 and βftz-f1 are induced by 20E and are temporally expressed in almost all organs), but the identified target genes are still limited in number. βFTZ-F1 has multiple functions in each organ during the mid to late prepupal period. For instance, βFTZ-F1 regulates two pupal cuticle genes that are expressed in slightly different parts of the epidermis, and it also regulates a protease that is expressed in the fat body and contributes to its morphological change. Furthermore, the expression of βFTZ-F1 in the inka cells is essential for releasing the ecdysis-triggering hormone ETH, which induces pupation in the late prepupal period, and also βFTZ-F1 expression in muscles is necessary to determine the timing of muscle apoptosis during metamorphosis. Moreover, βFTZ-F1 is a master regulator of late prepupal gene expression, which is essential for histolysis of the salivary gland cells during the early pupal period. In addition, the expression timing of βFTZ-F1 is not completely the same among different organs. In a large transcriptional profiling platform, involving 29 dissected tissues from larval, pupal, and adult stages of Drosophila, FBXO11 appeared to be expressed in many tissues and/or during development with specific upregulation in the fat body from L3 up to pupation. It is deduced that the expression levels of the 26S proteasome and FBXO11 may differ depending on tissue and contribute to the determination of timing of tissue-specific developmental events through control of the degradation speed of Blimp-1 (Aly, 2018).
In C. elegans, Blmp-1 was previously identified using RNAi-based suppressor screening to suppress dre-1 heterochronic phenotypes. A dre-1 mutant showed retarded migration of the gonad, whereas a Blmp-1 mutant showed precocious gonadal migration during L2 to L3 larva and was able to suppress the retarded phenotype of dre-1. In addition, precocious fusion and differentiation of epidermal stem cells, called seam cells, was partially suppressed by the Blmp-1 mutant in C. elegans. Moreover, similar genetic interactions were observed between DRE-1 and Blmp-1 for dauer formation. These observations suggest a conserved role of Blimp-1 degradation for the determination of developmental timing across taxa (Aly, 2018).
In most Eukaryotes, ubiquitin either exists as free monoubiquitin or as a molecule that is covalently linked to other proteins. These two forms cycle between each other and due to the concerted antagonistic activity of ubiquitylating and deubiquitylating enzymes, an intracellular ubiquitin equilibrium is maintained that is essential for normal biological function. However, measuring the level and ratio of these forms of ubiquitin has been difficult and time consuming. This paper has adapted a simple immunoblotting technique to monitor ubiquitin content and equilibrium dynamics in different developmental stages and tissues of Drosophila. The data show that the level of total ubiquitin is distinct in different developmental stages, lowest at the larval-pupal transition and in three days old adult males, and highest in first instar larvae. Interestingly, the ratio of free mono-ubiquitin remains within 30-50% range of the total throughout larval development, but peaks to 70-80% at the larval-pupal and the pupal-adult transitions. It stays within the 70-80% range in adults. In developmentally and physiologically active tissues, the ratio of free ubiquitin is similarly high, most likely reflecting a high demand for ubiquitin availability. This method was used to demonstrate the disruption of the finely tuned ubiquitin equilibrium by the abolition of proteasome function or the housekeeping deubiquitylase, Usp5. These data support the notion that the ubiquitin equilibrium is regulated by tissue- and developmental stage-specific mechanisms (Nagy, 2018).
The small protein modifier, ubiquitin regulates various aspects of cellular biology through its chemical conjugation onto proteins. Ubiquitination of proteins presents itself in numerous iterations, from a single mono-ubiquitination event to chains of poly-ubiquitin. Ubiquitin chains can be attached onto other proteins or can exist as unanchored species - i.e. free from another protein. Unanchored ubiquitin chains are thought to be deleterious to the cell and rapidly disassembled into mono-ubiquitin. A recent study examined the toxicity and utilization of unanchored poly-ubiquitin in Drosophila melanogaster. Free poly-ubiquitin species were found to be largely innocuous to flies, and free poly-ubiquitin can be controlled by being degraded by the proteasome or by being conjugated onto another protein as a single unit. To explore whether an organismal defense is mounted against unanchored chains, RNA-Seq analyses was conducted to examine the transcriptomic impact of free poly-ubiquitin in the fly. Approximately 90 transcripts were found whose expression is altered in the presence of different types of unanchored poly-ubiquitin. The set of genes identified was essentially devoid of ubiquitin-, proteasome- or autophagy-related components. The seeming absence of a large and multipronged response to unanchored poly-ubiquitin supports the conclusion that these species need not be toxic in vivo and underscores the need to reexamine the role of free ubiquitin chains in the cell (Blount, 2019).
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date revised: 25 August 2023
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