The Interactive Fly

Zygotically transcribed genes

Ubiquitination and protein degradation

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, but not autophagy, disruption results in severe eye and wing dysmorphia: a subunit- and regulator-dependent process in Drosophila

The deubiquitinase Leon/USP5 regulates ubiquitin homeostasis during Drosophila development

USP5/Leon deubiquitinase confines postsynaptic growth by maintaining ubiquitin homeostasis through Ubiquilin

Pri sORF peptides induce selective proteasome-mediated protein processing

Variation in Dube3a expression affects neurotransmission at the Drosophila neuromuscular junction

Quantitative proteomics reveals extensive changes in the ubiquitinome after perturbation of the proteasome by targeted dsRNA mediated subunit knockdown in Drosophila

A proteomics approach to identify targets of the ubiquitin-like molecule Urm1 in Drosophila melanogaster

Ubiquitin ligases
Anaphase promoting complex and its regulators
Proteins that regulate Tramtrack degradation
Other proteins that regulate protein degradation
Ubiquitin activating enzymes

Ubiquitin conjugating enzymes

Proteasome, but not autophagy, disruption results in severe eye and wing dysmorphia: a subunit- and regulator-dependent process in Drosophila

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).

The deubiquitinase Leon/USP5 regulates ubiquitin homeostasis during Drosophila development

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).

USP5/Leon deubiquitinase confines postsynaptic growth by maintaining ubiquitin homeostasis through Ubiquilin

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).

Pri sORF peptides induce selective proteasome-mediated protein processing

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).

Variation in Dube3a expression affects neurotransmission at the Drosophila neuromuscular junction

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).

Quantitative proteomics reveals extensive changes in the ubiquitinome after perturbation of the proteasome by targeted dsRNA mediated subunit knockdown in Drosophila

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).

A proteomics approach to identify targets of the ubiquitin-like molecule Urm1 in Drosophila melanogaster

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).


Chanut-Delalande, H., Hashimoto, Y., Pelissier-Monier, A., Spokony, R., Dib, A., Kondo, T., Bohere, J., Niimi, K., Latapie, Y., Inagaki, S., Dubois, L., Valenti, P., Polesello, C., Kobayashi, S., Moussian, B., White, K. P., Plaza, S., Kageyama, Y. and Payre, F. (2014). Pri peptides are mediators of ecdysone for the temporal control of development. Nat Cell Biol 16: 1035-1044. PubMed ID: 25344753

Khoshnood, B., Dacklin, I. and Grabbe, C. (2017). A proteomics approach to identify targets of the ubiquitin-like molecule Urm1 in Drosophila melanogaster. PLoS One 12(9): e0185611. PubMed ID: 28953965

Kondo, T., Plaza, S., Zanet, J., Benrabah, E., Valenti, P., Hashimoto, Y., Kobayashi, S., Payre, F. and Kageyama, Y. (2010). Small peptides switch the transcriptional activity of Shavenbaby during Drosophila embryogenesis. Science 329: 336-339. PubMed ID: 20647469

Sap, K. A., Bezstarosti, K., Dekkers, D. H., Voets, O. and Demmers, J. A. (2017). Quantitative proteomics reveals extensive changes in the ubiquitinome after perturbation of the proteasome by targeted dsRNA mediated subunit knockdown in Drosophila. J Proteome Res. PubMed ID: 28665616

Valdez, C., Scroggs, R., Chassen, R. and Reiter, L.T. (2015). Variation in Dube3a expression affects neurotransmission at the Drosophila neuromuscular junction. Biol Open 4: 776-782. PubMed ID: 25948754

Velentzas, P. D., Velentzas, A. D., Pantazi, A. D., Mpakou, V. E., Zervas, C. G., Papassideri, I. S. and Stravopodis, D. J. (2013). Proteasome, but not autophagy, disruption results in severe eye and wing dysmorphia: a subunit- and regulator-dependent process in Drosophila. PLoS One 8: e80530. PubMed ID: 24282550

Wang, C. H., Chen, G. C. and Chien, C. T. (2014). The deubiquitinase Leon/USP5 regulates ubiquitin homeostasis during Drosophila development. Biochem Biophys Res Commun. PubMed ID: 25152394

Wang, C. H., Huang, Y. C., Chen, P. Y., Cheng, Y. J., Kao, H. H., Pi, H. and Chien, C. T. (2017). USP5/Leon deubiquitinase confines postsynaptic growth by maintaining ubiquitin homeostasis through Ubiquilin. Elife 6. PubMed ID: 28489002

Zanet, J., Benrabah, E., Li, T., Pelissier-Monier, A., Chanut-Delalande, H., Ronsin, B., Bellen, H. J., Payre, F. and Plaza, S. (2015). Pri sORF peptides induce selective proteasome-mediated protein processing. Science 349: 1356-1358. PubMed ID: 26383956

date revised: 2 March 2016

Zygotically transcribed genes

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