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
  • A new Drosophila model of Ubiquilin knockdown shows the effect of impaired proteostasis on locomotive and learning abilities
  • Mask mitigates MAPT- and FUS-induced degeneration by enhancing autophagy through lysosomal acidification
  • Expression and regulation of deubiquitinase-resistant, unanchored ubiquitin chains in Drosophila


    Ubiquitin ligases
      Bre1
      E3 ubiquitin ligase required for the monoubiquitination of histone H2B and, indirectly, for H3K4 methylation -
      directs histone modification that is critical for the transcription of Notch target genes

      Cbl
      Src homology 2 and RING finger domain protein - ubiquitin ligase - positively regulates receptor tyrosine ubiquitination
      accelerating degradation of ligand-receptor complexes

      degringolade
      SUMO-targeted ubiquitin ligase that acts as a negative regulator of the repressor Hairy and its corepressor Groucho
      during embryonic segmentation and neurogenesis - acts as a regulator in sex determination by interfering
      with the repressive activities of Deadpan and Groucho
      highwire
      RING-H2 finger E3 ubiquitin ligases - controls synaptic growth

      hyperplastic discs
      a HECT domain E3 ubiquitin ligase that is likely to act by targeting Cubitus interruptus and an unknown activator of hedgehog expression for proteolysis

      mind-bomb
      regulates endocytosis of Delta and Serrate

      parkin
      RING-type E3 ubiquitin-protein ligase - degrades proteins with aberrant conformations - mutants exhibit muscle degeneration, male sterility, and defects in brain dopaminergic cells

      neuralized
      RING finger domain acts as an E3 ubiquitin ligase - Target components of the Notch-Delta pathway for ubiquitination

      no poles
      E3 ubiquitin ligase required for the preservation of genomic integrity during early embryogenesis

      Ubiquitin protein ligase E3A
      Ubiquitination - protein degradation - model for Angelman syndrome - neuromuscular junction -
      regulation of the actin cytoskeleton, the formation of terminal dendritic branches, and dopamine/serotonin synthesis

      Smurf1
      HECT domain ubiquitin-protein ligases that regulates Decapentaplegic pathway signaling by targeting Mothers against Dpp for degradation

      seven in absentia
      a C3HC4 RING zinc-finger motif protein that interacts physically with UBCD1, a component of the ubiquitin-dependent protein degradation pathway -
      partners Phyllopod in the regulation of Tramtrack levels

      thread (common alternative name: Diap1)
      ubiquitin ligase that functions as an inhibitor of apoptosis - Thread inhibition of caspase function is counteracted by proapoptotic proteins Hid, Grim and Reaper

      von Hippel-Lindau
      E3 ubiquitin ligase - functions in follicular epithelial morphogenesis to stabilize microtubule bundles and aPKC -
      in trachea controls the HIF-1 α/sima hypoxia response pathway

      Other ubiquitin ligases

    Anaphase promoting complex and its regulators
      Cdc27 (common alternative name makos)
      subunit of anaphase-promoting complex (APC) - involved in regulating cell cycle

      effete
      E2 ubiquitin-conjugating enzyme - mediates anaphase-promoting complex degradation - controls germ-line stem cell maintenance
      by targeting Cyclin A - regulates programmed cell death and neuronal process remodeling
      fizzy
      WD40 domain protein - required for the activity of the anaphase promoting complex in mitosis - functions to
      target the three mitotic cyclins A, B and B3 for destruction in the egg and drive anaphase progression during meiosis

      fizzy related - alternative name for retina aberrant in pattern
      Trp-Asp repeat (WD-repeat) protein - negatively regulates the levels of cyclins A, B and B3 thus promoting mitosis

      imaginal discs arrested
      homolog of APC5, a subunit of the anaphase-promoting complex - required for proliferation of imaginal disc cells -
      required for the localization of Miranda and its associated cargo proteins to the neuroblast basal cortex

      morula
      APC2 subunit of the anaphase promoting complex - cullin-homology domain protein - regulates protein degradation and cell cycle

      retina aberrant in pattern
      Trp-Asp repeat (WD-repeat) protein - negatively regulates the levels of cyclins A, B and B3 thus promoting mitosis

      vihar
      E2 ubiquitin-conjugating enzyme - contributes to the spatiotemporal control of Cyclin B degradation that occurs first during mitosis at the spindle poles



    Proteins that regulate Tramtrack degradation
      ebi
      a Trp-Asp (WD) repeats protein that targets Tramtrack during eye development - also independently limits S phase entry

      phyllopod
      novel protein that interacts physically with Seven in absential (Sina) and Ebi

      seven in absentia
      a C3HC4 RING zinc-finger motif protein that interacts physically with UBCD1, a component of the ubiquitin-dependent protein degradation pathway -
      partners Phyllopod in the regulation of Tramtrack levels


    Other proteins that regulate protein degradation
      archipelago
      F box component of an SCF-ubiquitin ligase - negatively regulates the levels and activity of Drosophila Myc by linking Myc to a protein degradation pathway -
      also negatively regulates Cyclin E and Notch

      Bruce
      a large protein that contains an N-terminal baculovirus IAP repeat (BIR) and a C-terminal ubiquitin conjugation domain -
      participates in regulation of apoptosis and autophagy

      cut up
      regulates trafficking of proteasomes and synaptic vesicle proteins in axons

      CYLD
      deubiquitinating enzyme that deubiquitinates dTRAF2 and prevents dTRAF2 from ubiquitin-mediated proteolytic degradation -
      involved in stress response - required for Eiger-triggered JNK activation and cell death

      COP9 complex homolog subunit 5
      Component of COP9 signalsome - regulates ubiquitin-dependent protein degradation - Jab1/MPN domain metalloenzyme motif

      cul4
      cullin family protein that regulates cell cycle by controlling degradation of Dacapo and Cyclin E

      Cullin-3
      Cullen-3 homolog acting in protein degradation - loss of function has pleiotropic effects during development, including defects
      in external sensory organ development, pattern formation and cell growth and survival - targets Cubitus interruptus

      debra
      involved in the Hedgehog signaling pathway as a mediator of protein degradation by the lysosome -
      required in a physiological, nondevelopmental aspect of long-term memory

      dodo
      peptidylprolyl isomerase - facilitates the degradation of the transcription factor CF2, which regulates expression
      of the rhomboid gene in follicle cells

      encore
      During oogenesis Encore physically interacts with the proteasome, protein degradation factor Cul1 and Cyclin E -
      In encore mutant germaria Cyclin E degradation is compromised, thus impairing exit from cystoblast cell divison

      fat facets
      a deubiquitinating enzyme that targets Liquid facets during eye development

      GDI interacting protein 3 (common alternative name: Armless)
      ubiquitin-like protein that functions in protein degradation - required for Wingless target gene expression

      Hepatocyte growth factor regulated tyrosine kinase substrate (common alternative name: Hrs)
      endosomal sorting of ubiquitinated receptors - targeting of EGFR, PVR, Notch, Hedgehog and Dpp receptors for degradation

      HDAC6
      a microtubule-associated deacetylase - interacts with polyubiquitinated proteins to stimulate autophagy as a compensatory degradation system
      when the ubiquitin-proteasome system is impaired

      jetlag
      an F-box protein with leucine-rich repeats (LRRs), a putative component of a SCF E3 ubiquitin ligase complex -
      essential for resetting the clock by transmitting light signals from Cryptochrome to Timeless resulting in the degredation of Timeless

      lin-19-like (common alternative name: Cullin1)
      a major component of a series of multimeric ubiquitin ligases that control the degradation of a broad range of proteins

      puffyeye
      a ubiquitin-specific protease that regulates Myc levels and function - regulates both the ubiquitin ligase Archipelago and its cell cycle substrate Cyclin E

      roadkill
      a member of a conserved family of proteins that serve as substrate adaptors for Cullin3-mediated ubiquitylation -
      regulates Cubitus interruptus levels by targeting it for degradation

      scotti
      pseudosubstrate inhibitor of a Cullin-3-based E3 ubiquitin ligase complex required for caspase activation during Drosophila spermatid terminal differentiation

      scrawny
      protein deubiquitinase required in germline, epithelial, and intestinal stem cells - acts to repress differentiation genes - deubiquitylates histone H2B and functions in gene silencing

      skpA
      one of six Skp1 genes in Drosophila - SkpA is a component of a series of multimeric ubiquitin ligases - regulates centrosome duplication,
      chromatin condensation, cell cycle progression and endoreduplication

      morgue
      F box and a ubiquitin E2 conjugase domain - involved in degradation of Thread/DIAP1

      Myelodysplasia/myeloid leukemia factor
      interacts with various factors involved in transcriptional regulation, involved in protein stabilization, regulates assembly of the COP9 signalosome complex, regulates Lozenge during hematopoetic development

      Ornithine decarboxylase antizyme (common alternative name: gutfeeling)
      no documented role in protein degradation in Drosophila - in mammals Antizyme and ubiquitin are respectively two types of proteasome targeting devices
      that mark proteins for both ubiquitin-independent and ubiquitin-dependent degradation by the 26 S proteasome

      supernumerary limbs
      beta-transducin family Trp-Asp repeats family - subunit of a multi-protein complex that targets proteins for degradation by the
      ubiquitin-proteasome pathway - an important regulator of Wingless, Hedgehog and Dorsal pathways

      Suppressor of cytokine signaling at 36E
      reduces JAK/STAT pathway activity in response to ligand stimulation--a process that involves endocytic trafficking and lysosomal degradation of the Domeless (Dome) receptor

      Ter94 (common alternative name: Valosin Containing Protein or VCP)
      AAA family ATPase, CDC48 subfamily - modulation of proteolytic degradation - Hedgehog pathway -Wingless pathway - dendritic pruning -
      motor neuron degeneration - ER stress response - maintenance of paternal chromosome integrity in the Drosophila zygote
      tribbles
      induces degradation of the CDC25 mitotic activators String and Twine via the proteosome pathway - required for turnover of fly C/EBP during cell migration


    Ubiquitin activating enzymes

    Ubiquitin conjugating enzymes
      FlyBase link: BcDNA:GH02435
      FlyBase link: BG:DS01486.1
      FlyBase link: BcDNA:LD22087
      bendless
      ubiquitin-conjugating enzyme that functions as a developmental switch that permits the transition from axonal growth and incipient synapse
      formation to synaptic growth and maturation - required in the IMD immune response signaling pathway for IKK activation leading to NFkappaB activation
      FlyBase link: Bruce
      FlyBase link: CG2574
      FlyBase link: CG5440
      FlyBase link: CG5823
      FlyBase link: CG7220
      FlyBase link: CG7375
      FlyBase link: CG7656
      FlyBase link: CG8188
      FlyBase link: CG10682
      FlyBase link: CG14739
      FlyBase link: CG17030
      courtless
      FlyBase link: crl
      FlyBase link: EG:25E8.2
      lesswright (common alternative names: Ubc9 and Semushi) -
      SUMO E2-conjugating enzyme - stabilizes proteins against ubiquitinization and subsequent degradation
      FlyBase link: UbcD2
      FlyBase link: UbcD4
      FlyBase link: UbcD6
      FlyBase link: UbcD10
      FlyBase link: Ubc-E2H
      FlyBase link: Ubc14B
      FlyBase link: Ubc47D
      FlyBase link: Ubc84D
      FlyBase link: Ubc84D
      FlyBase link: Ubc87F
      FlyBase link: Uev1A

    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).

    A new Drosophila model of Ubiquilin knockdown shows the effect of impaired proteostasis on locomotive and learning abilities

    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).

    Mask mitigates MAPT- and FUS-induced degeneration by enhancing autophagy through lysosomal acidification

    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).

    Expression and regulation of deubiquitinase-resistant, unanchored ubiquitin chains in Drosophila

    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).

    References

    Blount, J. R., Libohova, K., Marsh, G. B., Sutton, J. R. and Todi, S. V. (2018). Expression and regulation of deubiquitinase-resistant, unanchored ubiquitin chains in Drosophila. Sci Rep 8(1): 8513. PubMed ID: 29855490

    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

    Jantrapirom, S., Lo Piccolo, L., Yoshida, H. and Yamaguchi, M. (2018). A new Drosophila model of Ubiquilin knockdown shows the effect of impaired proteostasis on locomotive and learning abilities. Exp Cell Res 362(2): 461-471. PubMed ID: 29247619

    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

    Zhu, M., Zhang, S., Tian, X. and Wu, C. (2017). Mask mitigates MAPT- and FUS-induced degeneration by enhancing autophagy through lysosomal acidification. Autophagy 14:1-15. PubMed ID: 28806139


    date revised: 2 March 2016

    Zygotically transcribed genes

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