InteractiveFly: GeneBrief

bendless: Biological Overview | References

BIOLOGICAL OVERVIEW

The signaling mechanisms that allow the conversion of a growth cone into a mature and stable synapse are yet to be completely understood. Ubiquitination plays key regulatory roles in synaptic development and may be involved in this process. Previous studies identified the Drosophila ubiquitin conjugase bendless (ben) as important for central synapse formation, but the precise role it plays has not been elucidated. These studies indicate that Ben plays a pivotal role in synaptic growth and maturation. An incipient synapse is present with a high penetrance in ben mutants, suggesting that Ben is required for a developmental step after target recognition. Cell-autonomous rescue experiments were used to show that Ben has a presynaptic role in synapse growth. The TARGET system was harnessed to transiently express UAS-ben in a ben mutant background and a well defined critical period for Ben function was identified in establishing a full-grown, mature synaptic terminal. The protein must be present at a time point before but not during the actual growth process. Phenotypic evidence is provided demonstrating that Ben is not a part of the signal transduction pathway involving the well characterized ubiquitin ligase Highwire. It is concluded that Bendless functions as a novel developmental switch that permits the transition from axonal growth and incipient synapse formation to synaptic growth and maturation in the CNS (Uthaman, 2008).

The results from this study have given new insights into how ubiquitin system components establish functional synaptic connections. The temporal analysis of Bendless has been critical in illustrating its role as a developmental 'switch' in converting a growth cone into a mature synapse. As mentioned previously, the ben mutation is the result of a single amino acid change in the conserved catalytic core of the conjugase domain of the protein. This highlights the fact that the conjugase activity of the protein is necessary for the observed synaptic phenotype. Analysis of synaptic growth in Drosophila has primarily been done at the peripheral synapse of the NMJ. Components of the ubiquitin system, such as the ubiquitin ligase hiw, the deubiquitinating protease faf, and the synapse-associated E3 ligase PDZRN3, are known to play important roles in the growth and function of the fly NMJ. Significant studies have been performed with particular regard to the conserved family of hiw ubiquitin ligases. In Drosophila, hiw functions as a negative regulator of synapse development as mutants exhibit dramatic synaptic overgrowth at the larval NMJ. In Caenorhabditis elegans, loss of function of the hiw homolog rpm-1 results in multiple phenotypes at the NMJ as well as in the CNS. At the NMJ, some NMJs exhibit enlarged presynaptic terminals containing multiple active zones, whereas others contain underdeveloped or absent presynaptic terminals. In the worm mechanosensory circuit, the sensory neurons were found to retract synaptic branches, extend ectopic axons, and fail to accumulate synaptic vesicles, whereas some of the motor neurons exhibited phenotypes such as altered synaptic organization, branching, and overgrowth. Downstream signaling components have been isolated for both hiw and rpm-1 in Drosophila and C. elegans, respectively, and a number of conserved elements have been identified. Mutations in homologs of hiw in zebrafish and mice are also known to cause a variety of synaptic disruptions (Uthaman, 2008).

Ben and Hiw play distinct roles in synapse growth. This study has analyzed the novel roles played by these ubiquitin system components at the giant fiber system (GFS) central synapse. ben and hiw loss of function result in very different phenotypes, with ben specimens exhibiting synaptic undergrowth and hiw specimens exhibiting synaptic overgrowth. Ben function does not involve JNK, a well characterized downstream signaling partner identified for Hiw in Drosophila. It is also interesting to compare and contrast the role Hiw plays at a peripheral synapse with the role Ben plays at a central synapse. hiw mutants exhibit a presynaptic overgrowth phenotype at the NMJ, whereas ben mutants exhibit a reduction in presynaptic growth in the CNS. Also, Hiw does not localize to the nucleus and was found to regulate synaptic growth throughout development, whereas Ben has nuclear as well as cytosolic localization and only functions in a critical time period. Finally, Hiw activity is associated with the bone morphogenetic protein (BMP) retrograde signaling pathway that is known to be dependent on the retrograde motor. No evidence was found that Bendless function is dependent on the retrograde motor. All these data underline the fact that there are distinct targets for the ubiquitination cascades involving Ben and Hiw (Uthaman, 2008).

Functional neuronal circuits are established through a series of events: neurite outgrowth, axon guidance, target recognition, synapse formation, and synaptic growth and maturation. When the bendless mutant was originally characterized, Ben was thought to play an important role in either axon guidance or target recognition (Muralidhar, 1993; Oh, 1994). Analysis of the ben mutant clearly shows that Ben has an important role in synaptic growth. A number of specimens exhibit dye coupling between the GF and the motorneuron dendrite demonstrating that an incipient synapse is still formed and that the mutant phenotype arises from a failure of this immature connection to grow into a mature synapse. In addition, both gap junctional and chemical components are present at ben mutant terminals with synaptic vesicle marker localization as well as ultrastructural analyses (Uthaman, 2008).

The current view of synapse formation is that a nascent synapse can be rapidly assembled from material present in a growth cone in prepackaged vesicles and packets. After this primary rapid assembly of a nascent synapse, a secondary slower growth and maturation process takes place to result in a stable mature synapse. An insightful study on the Drosophila kinesin immaculate connections (imac) has shown it to be a permissive regulator of presynaptic maturation at the larval NMJ. Imac was found to be involved in the anterograde transport of synaptic vesicle precursors to the tip of the growth cone, an initial stage of synaptogenesis. In ben specimens, synaptic vesicles are still transported all the way down to the tip of the truncated terminal as evidenced by the localization of GFP-tagged synaptotagmin and synaptobrevin. The data strongly suggest that the ben mutant phenotype is resultant at a point after synaptic vesicular transport. Hence, it is concluded that the bendless terminal is an incipient synapse that fails to grow and mature and that Ben is a permissive regulator whose function is required for the initiation of a secondary process in presynaptic growth (Uthaman, 2008).

It is counterintuitive that, although Bendless is required for synaptic growth and maturation, the data show that it is not required during the growth process. This highlights the important role Ben plays as a developmental switch. Transient expression of UAS-ben before the growth of the presynaptic terminal was sufficient to rescue the ben phenotype anatomically and physiologically, but expression during the growth period had no effect. This suggests that Bendless is not involved in the actual growth process but rather has to be present in advance to alter signaling and initiate changes that allow growth to take place. Here it is essential to differentiate between axonal and synaptic growth, because it has been determined previously that axonal growth is unaffected in ben mutants (Muralidhar, 1993). Hence, Ben function is required to permit axonal growth to switch to synaptic growth (Uthaman, 2008).

The molecules in the signaling pathway of this novel mechanism remain to be further investigated. In conclusion, tight spatial and temporal control of synaptic connectivity in the nervous system is undoubtedly crucial to normal function. Determining how exactly Bendless regulates the formation of a mature synapse will give future novel insights into this phenomenon (Uthaman, 2008).

no poles encodes a predicted E3 ubiquitin ligase required for early embryonic development of Drosophila

In a screen for cell-cycle regulators, a Drosophila maternal effect-lethal mutant was identified named 'no poles' (nopo). Embryos from nopo females undergo mitotic arrest with barrel-shaped, acentrosomal spindles during the rapid S-M cycles of syncytial embryogenesis. CG5140, which encodes a candidate RING domain-containing E3 ubiquitin ligase, was identified as the nopo gene. A conserved residue in the RING domain is altered in the EMS-mutagenized allele of nopo, suggesting that E3 ligase activity is crucial for NOPO function. Mutation of a DNA checkpoint kinase, CHK2, suppresses the spindle and developmental defects of nopo-derived embryos, revealing that activation of a DNA checkpoint operational in early embryos contributes significantly to the nopo phenotype. CHK2-mediated mitotic arrest has been shown to occur in response to mitotic entry with DNA damage or incompletely replicated DNA. Syncytial embryos lacking NOPO exhibit a shorter interphase during cycle 11, suggesting that they may enter mitosis prior to the completion of DNA replication. Bendless (Ben), an E2 ubiquitin-conjugating enzyme, interacts with NOPO in a yeast two-hybrid assay; furthermore, ben-derived embryos arrest with a nopo-like phenotype during syncytial divisions. These data support the model that an E2-E3 ubiquitination complex consisting of Ben-Uev1A (E2 heterodimer) and Nopo (E3 ligase) is required for the preservation of genomic integrity during early embryogenesis (Merkle, 2009).

To ensure faithful transmission of the genome upon cell division, eukaryotic cells have developed checkpoints, regulatory pathways that delay cell-cycle progression until completion of prior events. The DNA damage/replication checkpoint plays a crucial role in preserving genomic integrity. Upon detection of DNA defects, the kinases ATM (ataxia telangiectasia mutated) and ATR (ATM-Rad3-related) are recruited to sites of damage and activated. ATM and ATR substrates include checkpoint kinases CHK1 and CHK2, which phosphorylate proteins that mediate cell-cycle arrest. The ensuing delay, resulting from engagement of this checkpoint, presumably allows cells time to correct defects (Merkle, 2009).

Research over the past decade has highlighted major roles for protein ubiquitination in regulating cellular responses to DNA damage. This post-translational modification, which involves covalent linkage of one or more ubiquitin molecules to another protein, regulates many fundamental cellular processes. Ubiquitination may alter the fate of a protein in numerous ways, such as targeting it for destruction by the 26S proteasome, changing its subcellular location, or changing its protein-protein interactions (Merkle, 2009).

Ubiquitination is a highly dynamic, multi-step process that requires three components: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2 or Ubc) and ubiquitin ligase (E3). E3s can be divided into two main classes: HECT and RING domain-containing proteins. RING-type E3 ubiquitin ligases contain a specialized motif of 40 to 60 residues that binds two zinc atoms. Many RING-type E3s bind to partnering E2 conjugating enzymes via their RING domains. Database searches of the Drosophila genome predict that it contains one E1, 36 E2s and ~130 E3s, which represents ~40% of the ubiquitination machinery in humans (Merkle, 2009).

Significant insights into the roles of many cell-cycle regulators have come from studying their functions in Drosophila. Drosophila is well suited for studying cell-cycle regulation during the formation of a multicellular organism, in large part because of its developmental use of cell cycles that differ in structure from canonical G1-S-G2-M cycles and the availability of genetic tools. The first thirteen cell cycles of Drosophila embryogenesis involve nearly synchronous nuclear divisions driven by stockpiles of maternally expressed mRNA and protein. These rapid cycles (~10 minutes in length) consist of oscillating S-M (DNA replication-mitosis) phases without intervening gap phases or cytokinesis. Minimal gene transcription occurs during this developmental stage, so cell cycles are regulated by post-transcriptional mechanisms. At cycle 14, the embryo cellularizes and initiates zygotic transcription at the midblastula transition (MBT) (Merkle, 2009).

This study reports the identification and characterization of a Drosophila maternal-effect lethal mutant 'no poles' (nopo). Embryos from nopo females undergo mitotic arrest with acentrosomal, barrel-shaped spindles during syncytial divisions. The results indicate that this arrest is secondary to the activation of a CHK2-mediated DNA checkpoint in early embryos. Nopo, a predicted E3 ubiquitin ligase, interacts with an E2 component, Ben. ben females are sterile, producing embryos with nopo-like defects. It is proposed that Ben-Uev1A and Nopo function together as an E2-E3 complex required for genomic integrity during Drosophila embryogenesis (Merkle, 2009).

nopo encodes a predicted protein of 435 amino acids containing an N-terminal RING domain. The putative mammalian homolog of Nopo was named 'TRAF-interacting protein' (TRIP) based on its ability to bind tumor necrosis factor (TNF) receptor-associated factors (TRAFs). Mammalian TRIP was recently demonstrated to have RING-dependent E3 ubiquitin ligase activity in an auto-ubiquitination assay. Drosophila Nopo and human TRIP are 20% identical and 34% similar overall, with 47% identity and 65% similarity in their RING domains. Importantly, nopo^Z1447 causes a glutamic acid to lysine change in the RING domain at position 11 of the predicted protein, a residue that is invariantly negatively charged across species (Merkle, 2009).

A model is proposed in which Nopo interacts with the Ben-Uev1A heterodimer to form a functional E2-E3 ubiquitin ligase complex required during syncytial embryogenesis for genomic integrity, cell-cycle progression, and the continuation of development. In the absence of Nopo, a lack of ubiquitination of, as yet unidentified, Nopo targets results in the truncation of S-phase and/or spontaneous DNA damage. Mitotic entry with unreplicated and/or damaged DNA triggers the activation of a CHK2-mediated checkpoint that leads to changes in spindle morphology, mitotic arrest and failure of nopo-derived embryos to develop to cellularization (Merkle, 2009).

The idea is favored thatNopo regulates the timing of S-M transitions in syncytial embryos to ensure that S-phase is of sufficient length to allow the completion of DNA replication prior to mitotic entry. The inhibition of DNA replication in syncytial embryos (e.g. via aphidicolin injection) leads to chromatin bridging in subsequent mitoses and CHK2 activation, both of which occur in nopo-derived embryos, presumably because of mitotic entry with unreplicated chromosomes. The mechanism by which Nopo coordinates S-M transitions is unknown. The data suggest that nopo may alter the timing of these transitions independently of CDK1-Cyclin B, although localized changes in the levels and/or activities of these regulators not detectable by immunoblotting of whole-embryo lysates could play a crucial role. It is unclear why the MEI-41/GRP-dependent checkpoint, which appears to be functional in nopo-derived embryos, is not sufficient to slow mitotic entry (Merkle, 2009).

The punctate nuclear localization observed for Nopo and its human homolog, TRIP, expressed in HeLa cells may indicate a direct role for these proteins in the regulation of chromatin structure. Furthermore, the G2 phase-specific localization that was observe for Nopo/TRIP in transfected HeLa cells may be consistent with a role for Nopo in slowing S-M transitions in syncytial embryos; in the absence of nopo, embryos that enter mitosis prematurely would probably do so without finishing DNA replication because of a lack of gap phases (Merkle, 2009).

An alternative explanation for CHK2 activation in nopo-derived embryos is that they might incur elevated levels of spontaneous DNA damage. Syncytial embryos are considered to be unusual in that they activate CHK2 but not CHK1 in response to DNA-damaging agents. Thus, spontaneous DNA damage would not be predicted to elicit the MEI-41/GRP-mediated replication checkpoint but would cause CHK2-dependent centrosomal inactivation during mitosis. Such a model would be consistent with the apparent lack of activation of the MEI-41/GRP-dependent checkpoint in nopo-derived embryos, although it would not explain why interphase 11 is shortened (Merkle, 2009).

Syncytial embryos from microcephalin (mcph1) mutant females undergo mitotic arrest with a phenotype similar to that described for nopo (Rickmyre, 2007). Like nopo, CHK2-mediated centrosomal inactivation causes mitotic arrest in embryos lacking mcph1. nopo and mcph1 are unique among maternal-effect lethal mutants in which CHK2-mediated centrosomal inactivation has been reported (e.g., grp, mei-41, wee1) in that their phenotypes appear to be more severe: centrosomes typically detach from spindles, and mitotic arrest occurs earlier, during precortical syncytial divisions. The underlying defects in nopo and mcph1 mutants may be distinct, however, because mnk mcph1-derived embryos (referring to maternal nuclear kinase (mnk), also known as loki, which encodes Drosophila CHK2) exhibit normal cycle 11 interphase length, which is truncated in mnk nopo-derived embryos (Rickmyre, 2007). Furthermore, no genetic interaction was detected between nopo and mcph1 (Merkle, 2009).

Mammalian TRIP was identified in a yeast two-hybrid screen for tumor necrosis factor (TNF) receptor-associated factor (TRAF) interactors. TRAFs transduce signals from members of the tumor necrosis factor (TNF)/tumor necrosis factor receptor (TNFR) superfamily, which elicit diverse cellular responses in the immune and inflammatory systems. TRIP has been reported to inhibit TRAF2-mediated NFkappaB activation; the RING domain of TRIP, however, was not required for inhibition. By contrast, the current analysis of nopo^Z1447 indicates that this motif is essential for Nopo function in Drosophila embryogenesis, probably by mediating its interactions with E2 components, as has been shown for other E3 ligases. Drosophila Eiger (TNF ligand) and Wengen (TNF receptor) play roles in dorsal closure, neuroblast divisions, and the response to fungal pathogens. A role for TNF signaling in early Drosophila embryogenesis has not been reported (Merkle, 2009).

TRIP was recently reported to be an essential factor in mice. TRIP-deficient mice die soon after implantation as a result of defects in early embryonic development. Compared with wild-type littermates, TRIP^-/- embryos are smaller in size with a reduced cell number. TRAF2 does not appear to be required until later in development, suggesting that TRIP has TRAF2-independent roles in early embryos. It will be interesting to see whether mammalian TRIP, by analogy to Drosophila Nopo, is required for genomic integrity during embryonic development (Merkle, 2009).

The data support a model in which Nopo ubiquitin ligase acts in concert with Ben-Uev1A heterodimers to regulate Drosophila syncytial embryogenesis. The yeast two-hybrid interaction and co-localization of Nopo and Ben led to the identification of an unanticipated role for Ben in early embryogenesis and additional roles for Nopo in synapse formation and innate immunity. Although the spindle defects of ben-derived embryos are strikingly similar to those of nopo mutants, they typically arrest earlier in syncytial development, suggesting that another E3 ligase that requires Ben may function in parallel with Nopo. Although nopo egg chambers appear normal, a possible requirement for Ben-Uev1A-Nopo complexes during oogenesis has not been ruled out; some defects in nopo- and ben-derived embryos could be a secondary consequence of previous defects during oogenesis (Merkle, 2009).

K63-linked ubiquitin chains are thought to act as non-proteolytic signals (e.g. affecting protein localization and/or interactions), whereas K48-linked ubiquitin chains have established roles in targeting proteins for proteasome-mediated degradation. Ben-Uev1A E2 homologs in budding yeast (Ubc13-Mms2p) mediate K63-linked polyubiquitination of PCNA during postreplicative repair. In mammalian cells, the E2 heterodimer Ubc13-Mms2 mediates DNA damage repair, while Ubc13-Uev1A promotes NFkappaB activation; both E2 complexes regulate these processes by mediating K63 ubiquitin chain assembly on target proteins. It is proposed that Ben-Uev1A-Nopo (E2-E3) complexes mediate the assembly of K63-linked ubiquitin chains on proteins that preserve genomic integrity in early Drosophila embryogenesis (Merkle, 2009).

Src42A modulates tumor invasion and cell death via Ben/dUev1a-mediated JNK activation in Drosophila

Loss of the cell polarity gene could cooperate with oncogenic Ras to drive tumor growth and invasion, which critically depends on the c-Jun N-terminal Kinase (JNK) signaling pathway in Drosophila. By performing a genetic screen, this study identified Src42A, the ortholog of mammalian Src, as a key modulator of both Ras^V12/lgl ^-/-triggered tumor invasion and loss of cell polarity gene-induced cell migration. The genetic study further demonstrated that the Bendless (Ben)/dUev1a ubiquitin E2 complex is an essential regulator of Src42A-induced, JNK-mediated cell migration. Furthermore, this study showed that ectopic Ben/dUev1a expression induced invasive cell migration along with increased MMP1 production in wing disc epithelia. Moreover, Ben/dUev1a could cooperate with Ras^V12 to promote tumor overgrowth and invasion. In addition, it was found that the Ben/dUev1a complex is required for ectopic Src42A-triggered cell death and endogenous Src42A-dependent thorax closure. These data not only provide a mechanistic insight into the role of Src in development and disease but also propose a potential oncogenic function for Ubc13 and Uev1a, the mammalian homologs of Ben and dUev1a (Ma, 2013a).

Bendless modulates JNK-mediated cell death and migration in Drosophila

The TNF-JNK pathway is a highly conserved signaling pathway that regulates a wide spectrum of biological processes including cell death and migration. To further delineate this pathway, a genetic screen was carried out for dominant modifiers of the cell death phenotype triggered by ectopic expression of Eiger (Egr), the Drosophila TNF ortholog. This study shows that Bendless (Ben), an E2 ubiquitin-conjugating enzyme, modulates Egr-induced JNK activation and cell death through dTRAF2. Furthermore, Ben physically interacts with dTRAF2 and regulates Egr-induced dTRAF2 polyubiquitination. Finally, Ben is required for JNK-dependent tumor progression, cell migration, oxidative stress resistance and longevity. These results indicate that Ben constitutes an essential component of the evolutionarily conserved TNF-JNK pathway that modulates cell death and invasion, tumor progression, stress response and lifespan in metazoans (Ma, 2013b)

Innate immune signaling in Drosophila is regulated by TGFbeta-activated kinase (Tak1)-triggered ubiquitin editing

Coordinated regulation of innate immune responses is necessary in all metazoans. In Drosophila, the Imd pathway detects gram-negative bacterial infections through recognition of DAP-type peptidoglycan and activation of the NF-kappaB precursor Relish, which drives robust antimicrobial peptide (AMP) gene expression. Imd is a receptor-proximal adaptor protein homologous to mammalian RIP1 that is regulated by proteolytic cleavage and K63-polyubiquitination. However, the precise events and molecular mechanisms that control the post-translational modification of Imd remain unclear. This study demonstrates that Imd is rapidly K63-polyubiquitinated at lysine residues 137 and 153 by the sequential action of two E2 enzymes, Ubc5 (Effete) and Ubc13 (Bendless)-Uev1a, in conjunction with the E3 ligase Diap2. K63-ubiquitination activates the TGFβ-activated kinase (Tak1), which feeds back to phosphorylate Imd, triggering the removal of K63-chains and the addition of K48-polyubiquitin. This ubiquitin editing process results in the proteosomal degradation of Imd, which is proposed to function to restore homeostasis to the Drosophila immune response (Chen, 2017).

Previous work has demonstrated that Imd is cleaved, K63-polyubiquitinated and phosphorylated upon immune stimulation (Paquette, 2010). While this earlier study did not find K48- polyubiquitin chains, others have reported evidence of both K63- and K48-Imd modifications (Thevenon, 2009). However, the overall dynamics of and interconnections between these IMD post-translational modifications remained unclear. This study showed that peptidoglycan (PGN) from bacterial cell walls stimulation of S2 cells leads to five different Imd modifications: proteolytic cleavage, K63-polyubiquitination, phosphorylation, K63-deubiquitination and K48-polyubiquitination, which leads to degradation of Imd through proteasome. These immune triggered signaling events are robust and incredibly rapid, with Imd cleavage and K63-polyubiquitination occurring as early as 2 minutes after PGN stimulation. While K63-modification peaks early and then steadily declines, K48-conjugation appears later, along with phosphorylation, and declines in proteasome-dependent manner. These kinetics argue that Imd is sequentially conjugated with K63 then K48 ubiquitin, so-called ubiquitin editing, as has been reported for IRAK1 and RIP1 in mammalian innate immune signaling pathways (Chen, 2017).

In addition to ubiquitination, two slow-migrating species of Imd were detected and shown to be phosphorylated forms. Judging by their size, these two phospho-forms appear to be derived from either full-length Imd (upper) or cleaved Imd (lower). Interestingly, persistence of phosphorylated Imd was observed in the proteasome-inhibited samples, suggesting that Imd is both K48-polyubiquitinated and phosphorylated before entering proteasome. Tak1 is required for these phosphorylation events as well as for ubiquitin editing, demonstrating key role for this MAP3K in a negative feedback loop (Chen, 2017).

Conjugation of ubiquitin usually occurs on lysine side chains of target proteins, and mass spectrometry of immuno-purified endogenously expressed Imd identified K137 and K153 as the sites of ubiquitin linkage. Note, the mass spec analysis includes 50% coverage of Imd, and a cluster of four lysine residues at the very C-terminus were not observed. Substitution of K137 and K153 residue with Arginine prevented signal- induced ubiquitination of Imd in S2 cells and reduced expression of the AMP gene Diptericin in both cells and flies. These results demonstrate that both lysine residues are required for K63- polyubiquitination and downstream signaling events. In S2 cells, mutation of single lysine residue led to a partial reduction of K63- ubiquitination and a partial reduction of AMP gene induction. Surprisingly, single lysine mutation did not correspondingly reduce Imd K48- polyubiquitination, while the double lysine mutation completely blocked it. These results suggest that even the reduced signal, mediated by a single K63-chain, is sufficient to trigger a robust feedback response with K48-chain formation. On the other hand, a complete block in K63-chains prevents Tak1 activation, which in turn fails to promote the ubiquitin editing of Imd. These findings are consistent with results observed with knockdown of Ubc5, Ubc13 and Uev1a. Ubc5 depletion prevented all K63 ubiquitination and signaling (as measured by Diptericin induction), and subsequent K48 modification was absent, while the Ubc13 and/or Uev1a knockdown showed residual K63 chains and greatly reduced Diptericin expression but robust K48- ubiquitination. These results also suggest that K48-polyubiquitination may occur on lysine residues beyond K137 and K153, although more detailed mass spectrometric analyses is required to map these sites more thoroughly (Chen, 2017).

On the other hand, only double lysine mutation leads to significant reduction of AMP expression in adult flies, and this is reduction is not as robust as in cultured cells. This pattern suggests that activation of the NF-κB protein Relish and its transcriptional targets do not solely rely on ubiquitination of Imd K137/153. Other possible ubiquitination targets include the upstream caspase Dredd, which has been shown to be critical for signaling (26), or the E3 ligase Diap2. This redundancy may represent multiple parallel mechanisms that contribute to the NF-κB activation. Furthermore, tissue specific immune differences may contribute to the discrepancy in Diptericin induction between macrophage-derived S2 cells and whole flies, in which the fat body rather than hemocytes is the major organ for inducible expression of AMPs (Chen, 2017).

Previous work has suggested that Diap2 is the E3 ligase for Imd ubiquitination. With the advantage of ubiquitin linkage specific antibodies, data presented in this study show that Diap2 is required for Imd K63-polyubiquitination and signaling, as measured by induction level of Diptericin. Moreover, the accumulation of cleaved but non-ubiquitinated Imd, in the Diap2 depleted cells and flies, provides further evidence that ubiquitination is downstream of Imd cleavage and highlights the role of Diap2 as the critical E3 in the K63- modification of Imd. In addition, Imd is no longer K48-modified when Diap2 is removed, suggesting that either Diap2 is also involved in K48- conjugation, or the failure of K63- polyubiquitination leads to the loss of K48- polyubiquitination. Given the role of Tak1 in this ubiquitin editing event, the latter hypothesis is favored, and another E3 is likely involved in the K48 conjugation (Chen, 2017).

In addition to E3 ligases, E2 ubiquitin conjugating enzymes are the other key factors in the ubiquitin conjugation reaction. Previously work has shown that Ubc5 and Ubc13-Uev1a are all involved in Imd ubiquitination. However, the mechanism by which these E2s collaborated was unclear. Results from in vitro reconstituted ubiquitination assays suggested a two-step reaction model for ubiquitin conjugation with different E2s. In particular, it was shown that some E2s, such as Ubc5, are effective at the initial ubiquitination of substrates but are ineffective at generating long chains, while other E2s, like Ubc13/Uev1a, are efficient at generating long ubiquitin chains but fail to conjugate substrate proteins. Thus, these two types of E2s can work together to generate long ubiquitin chains conjugated to target proteins. It is proposed that Imd undergoes ubiquitin chain initiation and elongation catalyzed by two separate E2s. Once cleaved, Imd interacts with Diap2 through its BIR repeats and is first modified by Ubc5-mediated substrate ubiquitination on lysines 137 and K153. Subsequently, the E2 complex of Ubc13-Uev1a pairs with an E3 (possibly Diap2 although another unidentifed E3 is not excluded) and switches the reaction to chain elongation mode, during which additional ubiquitin molecules are attached to the substrate-linked ubiquitin in a K63-specific manner. In the absence of Ubc13/Uev1a, Ubc5 alone is still able to elongate the polyubiquitin chains, but less efficiently and with unknown linkages (Chen, 2017).

Induction of Diptericin expression generally tracks with the K63-polyubiquitination signal (but not the total Ub signal) observed in various E2 knockdown cells. The one exception is the samples in which Ubc13 and Uev1a are both knocked down, and Ubc5 is still available. These samples display a similar K63 intensity as the single Ubc13 or Uev1a RNAi lanes, but Diptericin induction is lower, close to background levels. Ubc5 alone is known to conjugate ubiquitin without linkage specificity, a random polyubiquitin chain that consist of all seven types lysine linkage. Since the K63 antibody recognizes K63-linked diubiquitin, it is possible that Ubc5-mediated random ubiquitin chain elongation generates some K63 di-ubiquitin linkages which are detected by this antibody, but are unable support signaling due to their altered topology and limited amounts of K63-linkages. More detailed biochemical characterization of these Ubc5-catalyzed chains is required to confirm this hypothesis (Chen, 2017).

K48 modification of Imd shows a subtle difference relative to the K63 chains. Again, knock down of Ubc5 causes complete blocking of K48 conjugation, but depletion of Ubc13/Uev1a has no effect. The failure of K48 modification in the Ubc5 depleted cells may have two possible underlying causes. Firstly, the complete lack of K63 chains will fail to activate Tak1 and the subsequent ubiquitin editing feedback loop, while the Ubc13 and/or Uev1a RNAi display some residual K63 activity and thus can trigger Tak1 and the feedback response. Alternatively, Ubc5 might be directly required for Imd K48-polyubiquitination as shown in the degradation of proteins in multiple Drosophila pathways including eye development, maintenance of germline stem cells and apoptosis. These are not mutually exclusive possibilities (Chen, 2017).

Phosphorylation of Imd appears to be a major regulator of these ubiquitin-editing events. Knockdown of Tak1 prevents Imd phosphorylation in S2 cells and in adult flies. Moreover, immune-purified Tak1 can directly phosphorylate recombinant Imd in vitro, while neither JNKK nor IKK are required for phosphorylation of cleaved Imd, strongly arguing that Tak1 directly modifies Imd. RNAi-depletion or drug inhibition of Tak1 prevents K63-deubiquitination and the subsequent K48-polyubiquitination/proteasome-mediated degradation, leading to accumulation of cleaved but unphosphorylated Imd, presumably an intermediate during chain editing. From these results, it is inferred that Tak1-mediated phosphorylation of Imd is required for ubiquitin editing. Future studies will reveal the underlying mechanisms by which phosphorylation triggers K63-deubiquitination and K48 chain conjugation. Nonetheless, these results are consistent with earlier reports of Imd regulation by K63-deubiquitination and degradation (Chen, 2017).

Considering the results presented in this studdy together with earlier studies, the following model of Imd signal activation and subsequent down-regulation is proposed. One of the earliest events after PGN-stimulation is the rapid cleavage of Imd by the caspase-8 homolog DREDD at D30. Cleaved Imd then interacts with BIR-repeats of the E3 ligase Diap2 and is K63-polyubiquitinated through the sequential action of Ubc5, for substrate conjugation, and Ubc13-Uev1a, for catalyzing long K63 chains. These K63-polyubiquitin chains are then likely to activate the Tak1/Tab2 kinase complex through the conserved K63-binding motif in Tab2, which in turns signals through IKK complex to activate the NF-κB precursor Relish. Relish is central for the robust induction of AMP gene transcription. Meanwhile, Tak1 also mediates a retrograde signal that phosphorylates Imd and triggers ubiquitin editing, and leads to the degradation of Imd through proteasome. This regulatory interaction between Tak1 and Imd represents a novel homeostatic loop whereby the Drosophila immune response is rapidly activated but also quickly shutdown. Future studies are necessary to determine function of this feedback loop relative to other feedback mechanisms reported for the Imd pathway (Chen, 2017).

Distinct roles of Bendless in regulating FSC niche competition and daughter cell differentiation

A major goal in the study of adult stem cells is to understand how cell fates are specified at the proper time and place to facilitate tissue homeostasis. This study found that an E2 ubiquitin ligase, Bendless (Ben), has multiple roles in the Drosophila ovarian epithelial follicle stem cell (FSC) lineage. First, Ben is part of the JNK signaling pathway, and it, as well as other JNK pathway genes, were found to be essential for differentiation of FSC daughter cells. The data suggest that JNK signaling promotes differentiation by suppressing the activation of the EGFR effector, ERK. Loss of ben, but not the JNK kinase hemipterous, resulted in an upregulation of hedgehog signaling, increased proliferation and increased niche competition. Lastly, it was demonstrate that the hypercompetition phenotype caused by loss of ben is suppressed by decreasing the rate of proliferation or knockdown of the hedgehog pathway effector, Smoothened (Smo). Taken together, these findings reveal a new layer of regulation in which a single gene influences cell signaling at multiple stages of differentiation in the early FSC lineage (Tatapudy, 2021).

The segregation of FSC and daughter cell fates requires the orchestration of multiple developmental signaling pathways to ensure that cell fate specification and proliferation occurs at the correct time and place. This study has identified a new layer of regulation in which a single gene, ben, has multiple distinct roles in promoting differentiation and proliferation in the early FSC lineage (Tatapudy, 2021).

First, the findings demonstrate that ben functions along with the rest of the JNK signaling pathway to promote pFC (posterior follicle cell) differentiation. The phenotypes observed in JNK pathway mutants strongly suggest that the primary defect is a failure to differentiate into mature stalk cells. The range of tube-like and expanded stalk morphological phenotypes that were observed could both be explained by an inability of cells in the stalk regions between follicles to facilitate the pinching off of follicles from the germarium or to intercalate into a single row after the pinching off process has been initiated. The possibility cannot be ruled out that differentiation toward the polar cell fate or main body cell fate are also impaired upon loss of JNK signaling, but no evidence was seen for this with the markers used. Notably, although RNAi knockdown of ben or hep impaired the activity of a reporter for Notch signaling, NRE-GFP, in Region 2b pFCs and mature polar cells, this did not appear to interfere with the ability of mutant pFCs to differentiate into polar cells, so the significance of this effect on cell fate specification is unclear. Additional reporters of Notch signaling may help to determine the extent to which Notch signaling is disrupted in these mutant conditions. It was also found that pERK is retained in pFCs of ovarioles with impaired JNK signaling, and that constitutive activation of pERK phenocopies the differentiation defects observed in JNK pathway mutants. These observations suggest that JNK signaling promotes pFC differentiation into stalk cells by inhibiting the activation of ERK in pFCs (Tat in a JNK-independent manner to help shape the gradient of Hh signaling in FSCs and pFCs. Indeed, it was found that levels of Ptc-pelican-GFP, indicative of Hh signaling, are higher in ben mutant FSCs compared with wild type, and the Ptc-pelican-GFP reporter remains detectable throughout Regions 2b and 3 of tapudy, 2021).

Second, ben functions in the germarium. Likewise, it was found that RNAi knockdown of ben caused the main body follicle cells to retain zfh1 expression, a downstream target of Hh signaling, throughout the germarium and even into Stage 2 in some cases, where it is completely absent in wild-type tissue. However, it was also found that ben mutant main body follicle cells were able to mature into a Cas+, Eya- state, indicating proper differentiation with respect to these two markers. Thus, although loss of ben does not fully impair main body follicle cell differentiation, it may influence the process through its role as an upstream regulator of zfh1 expression. Further study will be required to understand the functional significance of decreased cas and zfh1 expression during main body follicle cell differentiation (Tatapudy, 2021).

JNK signaling has been found to be involved in a wide variety of biological processes in a cell type- and context-dependent manner. Some of the most well-known functions of JNK signaling involve responses to non-homeostatic conditions, such as stress, cellular damage, infection and tumor growth. However, JNK signaling has also been found to be important for processes that occur during normal development or adult homeostasis. Indeed, hep mutants were originally described for their role in dorsal closure of the epidermis in the embryo, and subsequent studies have clearly established the importance of the JNK signaling pathway in this process. Likewise, JNK signaling is also important during adult homeostasis in mammalian stem cell lineages, for example to promote self-renewal of human hematopoietic stem cells and differentiation in the mouse intestinal stem cell and human neural stem cell lineages. In line with this type of role for JNK signaling, the current findings reveal new pathway interactions for JNK signaling in the regulation of cell fate decisions in an epithelial stem cell lineage (Tatapudy, 2021).

In addition to regulating cell fate decisions, a JNK-independent role was identified for Ben in the regulation of proliferation rate and FSC niche competition. Previous studies have established that increased proliferation causes hypercompetition, and that the proliferative response to Hh signaling is the key mediator of the FSC niche competition phenotypes in Hh pathway mutants. Therefore, the hypercompetition phenotype of ben mutant clones is likely caused, in large part, by the increased Hh signaling levels and proliferation rates in these mutants. The findings that overexpression of dap or RNAi knockdown of smo was sufficient to suppress the hypercompetition phenotype of benA mutant clones are consistent with this. It is possible that delayed differentiation in newly-produced ben mutant pFCs also contributes to the hypercompetition phenotype. However, the observation that hep^G0107 mutant clones are not hypercompetitive indicates that the hypercompetition phenotype of ben mutant clones is not caused by a block in the JNK-dependent functions of Ben in pFC differentiation (Tatapudy, 2021).

Taken together, these findings indicate that Ben has JNK-dependent and -independent roles in regulating pFC cell fate decisions as they differentiate into polar, stalk and main body follicle cells. Specifically, the findings support a model in which JNK signaling promotes the differentiation of pFCs during normal homeostasis by helping to downregulate EGFR signaling in early pFCs. Previous studies found that pERK inhibits Groucho and that Groucho is required for the upregulation of Notch signaling in pFCs. Thus, the retention of pERK in pFCs may explain why Notch signaling is not upregulated in JNK pathway mutants. Separately, Ben inhibits Hh signaling in a JNK-independent manner, thereby controlling pFC proliferation, regulating FSC niche competition, and possibly contributing to main body follicle cell differentiation. Thus, this study has revealed a new layer of regulation within the signaling network that controls cell behavior in the early FSC lineage. The recently published single-cell atlases of the adult Drosophila ovary revealed a continuum of changes in gene expression that occur during cellular differentiation in the early FSC lineage, and provide the tools for characterizing this signaling network in a cell-type specific manner. In future studies, it will be interesting to apply these new single-cell approaches to mutants such as those described in this study to further elucidate the molecular basis of cell fate decisions in the FSC lineage (Tatapudy, 2021).

The role of ubiquitination in Drosophila innate immunity: Signaling upstream of Relish

Infection of Drosophila by Gram-negative bacteria triggers a signal transduction pathway (the IMD pathway) culminating in the expression of genes encoding antimicrobial peptides. A key component in this pathway is a Drosophila IkappaB kinase (DmIKK) complex, which stimulates the cleavage and activation of the NF-kappaB transcription factor Relish. Activation of the DmIKK complex requires the MAP3K dTAK1, but the mechanism of dTAK1 activation is not understood. In human cells, the activation of TAK1 and IKK requires the human ubiquitin-conjugating enzymes Ubc13 and UEV1a. This study demonstrates that the Drosophila homologs of Ubc13, (Bendless) and UEV1a, are similarly required for the activation of dTAK1 and the DmIKK complex. Surprisingly, the Drosophila caspase DREDD and its partner dFADD are required for the activation of DmIKK and JNK, in addition to their role in Relish cleavage. These studies reveal an evolutionarily conserved role of ubiquitination in IKK activation, and provide new insights into the hierarchy of signaling components in the Drosophila antibacterial immunity pathway (Zhou, 2005).

A cell culture system was established to study the IMD and Toll signaling pathways in S2 cells. The IMD pathway is activated by treating the cells with Gram-negative peptidoglycan (which is present in crude preparations of lipopolysaccharides), whereas activation of the Toll pathway is achieved by treating the cells with the Spätzle ligand. Active Spätzle is produced from a cell line stably transfected with a plasmid containing the copper-inducible metallothionein promoter driving the expression of active Spätzle C-106. When these cells are treated with copper, active SPZ is secreted into the medium, and this conditioned media can be used to activate naïve cells. Using the RNAi-mediated gene inactivation method, it was found that SPZ-induced Drosomycin gene activation in S2 cells requires the Drosophila Rel proteins Dif and Dorsal, as well as Toll, dMyD88, Tube, Pelle, and Slimb, as expected. In sharp contrast, these dsRNAs do not block the expression of antimicrobial peptide genes induced by peptidoglycan. Instead, RNAi studies demonstrate that peptidoglycan-induced gene expression requires all known components of the IMD pathway. Therefore this RNAi approach was used to determine the roles of candidate signaling components in the IMD and Toll signaling pathways (Zhou, 2005).

Previous studies have shown that activation of the mammalian IKK complex requires a ubiquitination step. In particular, TRAF6-mediated IKK activation was shown to require a dimeric ubiquitin-conjugating enzyme composed of the Ubc13 and UEV1a proteins. Bendless and dUEV1a are the Drosophila homologs of Ubc13 and UEV1a, respectively. Bendless and dUEV1a, like their mammalian counterparts, associate with each other in vivo. To investigate whether Bendless and dUEV1a are required for antibacterial gene expression in response to peptidoglycan, the RNAi-mediated gene inactivation method was used. S2 cells were transfected with various dsRNAs. After 48 h, cells were first treated with 20-hydroxy-ecdysone for 24 h to enhance their competence to induce antimicrobial genes in response to immune challenge, and then treated with peptidoglycan or SPZ to activate the IMD or Toll signaling pathways, respectively. Total RNA was isolated from these cells and subjected to Northern blotting analysis using cDNA fragments corresponding to Diptericin or Drosomycin as probes, to examine the activation of the IMD and Toll pathways, respectively. Both Bendless and dUEV1a are required for maximal levels of antibacterial peptide gene expression in response to peptidoglycan treatment. In fact, when both Bendless and dUEV1a are both targeted by RNAi, Diptericin induction is reduced to near background levels. (The partial effect of Bendless or dUEV1a RNAi alone is likely because of the fact that RNAi often does not generate a complete null phenotype.) By contrast, the induction of Drosomycin by Toll activation is unaffected by the RNAi-mediated knock-down of Bendless and dUEV1a. Note that the same cells were stimulated with either peptidoglycan or SPZ in. As a control, S2 cells treated with DmIKKgamma or Toll dsRNA showed significantly reduced peptidoglycan-induced Diptericin or SPZ-induced Drosomycin gene expression, respectively. As a control, mRNA and/or protein levels of targeted genes were examined by RT-PCR and/or Western blotting to confirm the effectiveness of RNAi (Zhou, 2005).

To provide further evidence that Bendless is involved in the IMD pathway, a dominant negative mutant Bendless was used to determine whether peptidoglycan-induced antibacterial gene activation can be blocked. Stable S2 cell lines were generated that express either wild-type or C87A Bendless under the control of the metallothionein promoter. The cysteine to alanine mutation at position 87 creates a dominant-negative mutant because this residue, located in the catalytic pocket in ubiquitin-conjugating enzymes, is crucial for the catalytic activity of E2s. These cells were then treated with various combinations of peptidoglycan and copper, and Northern blotting was employed to examine the expression of antibacterial genes including Attacin, Cecropin and Diptericin. Overexpression of wild-type Bendless has no effect on peptidoglycan-induced antibacterial gene activation, since similar levels of antibacterial peptide gene expression were detected in cells treated with or without copper. In contrast, overexpression of Bendless C87A leads to a significant reduction in peptidoglycan-activated expression of antibacterial peptide genes (Zhou, 2005). Bendless flies have been identified which carry a proline to serine substitution at position 97 within the strictly conserved active site region of E2s. In order to determine whether bendless flies are defective in response to Gram-negative bacterial infection, both wild-type and bendless flies were subjected to E. coli infection and Diptericin gene activation was examined by Northern blotting. bendless flies display significantly weaker Diptericin gene activation compared with wild-type flies. These results indicate that the Bendless-dUEV1a E2 complex is required for signaling by the IMD pathway (Zhou, 2005).

Experiments were carried out to determine whether Bendless and dUEV1a are required for peptidoglycan-induced activation of the DmIKK complex. Previous studies have shown that peptidoglycan treatment induces the kinase activity of the endogenous DmIKK complex in S2 cells. Cells were transfected with dsRNAs corresponding to various mRNAs. After 48 h, these cells were treated with peptidoglycan for 15 min, and the endogenous DmIKK complex was immunoprecipitated and subjected to in vitro kinase assays using recombinant Relish protein as substrate. Bendless or dUEV1a dsRNA treatment leads to a significant decrease in peptidoglycan-induced DmIKK kinase activity, suggesting that Bendless and dUEV1a are required for peptidoglycan-induced DmIKK activation. As a control, DmIKKgamma dsRNA treatment completely abolished peptidoglycan-induced DmIKK activation. It is concluded that the ubiquitin-conjugating enzymes Bendless and dUEV1a are specifically involved in the Drosophila IMD pathway, and they play a role upstream of the DmIKK complex (Zhou, 2005).

Overexpression of IMD in Drosophila results in the activation of antibacterial genes in the absence of bacterial infection. IMD overexpression, under control of the copper-inducible metallothionein promoter, can also strongly activate expression of the Diptericin gene in S2 cells. This stable cell line therefore provides a useful tool to perform an epistatic analysis to determine the position of Bendless/dUEV1a complex relative to IMD in the Drosophila antibacterial signaling pathway. The IMD stable cells were first transfected with dsRNAs derived from various genes and then stimulated with copper or peptidoglycan, and IMD- and peptidoglycan-induced Diptericin gene activation was examined. Overexpression of IMD, via the addition of copper, leads to strong activation of the Diptericin gene. In fact, copper-induced IMD expression is as potent as peptidoglycan in driving diptericin expression. Cells transfected with LacZ dsRNA show a similar Diptericin expression profile compared with cells that were mock-treated. Consistent with the observation that the DmIKK complex functions downstream of IMD, cells transfected with DmIKKgamma dsRNA are severely defective in both IMD- and peptidoglycan-induced Diptericin expression. Furthermore, cells treated with dsRNAs derived from Bendless and dUEV1a genes display a significant reduction in both peptidoglycan- and IMD-mediated Diptericin gene activation. These results indicate that the ubiquitin conjugating enzymes Bendless and dUEV1a function downstream of IMD in this signaling pathway (Zhou, 2005).

Thus, by differentially activating the IMD and Toll signaling pathways in Drosophila S2 cells, this study shows that Bendless (dUbc13) and dUEV1a are required for the Drosophila IMD signaling pathway. Using RNAi to target Bendless and/or dUEV1a significantly reduces the levels of peptidoglycan-induced antibacterial peptide gene expression and activation of the Drosophila IKK complex. This mechanism of IKK activation is highly conserved; in mammals Ubc13 and UEV1a are required for TNFalpha-, IL-1β-, and TCR-mediated IKK and NF-kappaB activation (Zhou, 2005).

This ubiquitin-dependent kinase activation does not involve proteasome-mediated degradation. Proteasome inhibitors do not block IKK activation, in flies or humans. Moreover, ubiquitination without degradation has been shown to activate the human IKK complex, and a similar Drosophila activity has been identified, The primary sequence of the mammalian Ubc13/UEV1a and those of the Drosophila Bendless/dUEV1a are highly conserved (90% similarity for Ubc13, 79% for UEV1a). The crystal structures of the yeast and human Ubc13/UEV1a (Mms2) have shown clearly that this E2 complex can make only K63-linked polyubiquitin chains. Thus, it is likely that the Drosophila Bendless/dUEV1a E2 catalyzes the formation of K63-linked ubiquitin chains (Zhou, 2005).

In a cell-free system, human TRAF6 was shown to be an E3 ligase that auto-ubiquitinates in conjunction with Ubc13/UEV1a. This results in the activation of TRAF6 and, in turn, the activation of TAK1. Activated TAK1 phosphorylates key serine residues in the activation loop of IKKβ, resulting in the activation of IKKβ. It is suspected that similar mechanisms are involved in the Drosophila IMD pathway. This study demonstrates that the Drosophila TAK1 homolog functions downstream of Bendless and dUEV1a. Furthermore, a Drosophila homolog of TAB2/TAB3 is also required for the IMD pathway. Interestingly, the C-terminal zinc finger domain of TAB2, which is conserved in the Drosophila protein, has recently been shown to bind specifically to K63-polyubiquitin chains. Strikingly, it has found that a galere/dTAB2 mutant, which is defective in the IMD pathway, carries a mutation in this zinc finger domain (Zhou, 2005).

The Drosophila E3 ligase, analogous to human TRAF2 or TRAF6, which functions with Bendless and dUEV1a in the activation of dTAK1 and DmIKK remains to be identified. In Drosophila, the dTRAF2 protein is the closest homolog of mammalian TRAF6, and it is the only Drosophila TRAF protein that contains the RING domain, typical of E3 ligases. However, RNAi knockdown and dominant-negative studies suggest that dTRAF2 is not involved in either the IMD or the Toll signaling pathways in S2 cells. In fact this gene is expressed at undetectably low levels in S2 cells. In one previous study dTRAF2 was reported to interact physically and functionally with Pelle, a key signaling component in the Toll signaling pathway that controls the antifungal immune response. However, these studies were based on overexpression experiments and in vitro binding assays, which might not reflect the physiological role of dTRAF2. In contrast, a recent publication demonstrated that dTRAF2 mutants are not fully able to induce antimicrobial peptide genes following E. coli infection. However, these studies did not clearly determine whether dTRAF2 is involved in the Toll or IMD pathways. The data suggest that dTRAF2 is not a critical component of the IMD pathway in S2 cells. Further studies, in cells and in flies, are necessary to elucidate the role of dTRAF2 in Drosophila immunity (Zhou, 2005).

The possibility is considered that other Drosophila RING-containing proteins might be involved in Drosophila immunity. However, RNAi knockdown studies with 10 different RING domain-encoding genes failed to block either the Toll or IMD pathways. Finally, since the structure of the RING domain of Rad5 has been successfully modeled to fit into the structure of the dimeric ubiquitin-conjugating enzyme complex Ubc13/UEV1a, it was reasoned that the potential ubiquitin ligase involved in the IMD pathway might physically interact with Bendless and dUEV1a. Therefore yeast two-hybrid screens were performed using Bendless and dUEV1a as baits in an effort to identify their protein interaction partners. A Drosophila RING protein, CG14435, was identified in such screens. CG14435 interacts robustly with both Bendless and dUEV1a in yeast two-hybrid assays. Furthermore, the CG14435-Bendless and CG14435-dUEV1a interaction was confirmed by co-immunoprecipitation of overexpressed proteins in S2 cells. However, RNAi-based studies suggest that CG14435 is not involved in the Drosophila innate immunity signaling pathways. It has been shown that bendless flies display defective synaptic connectivity and abnormal morphology within the visual system, suggesting Bendless functions in a variety of developmental processes. In addition, the Saccharomyces cerevisiae homologs of Bendless and dUEV1a have been implicated in DNA damage repair. Therefore it is possible that CG14435 is involved in some cellular processes other than immunity which require Bendless and dUEV1a. Further studies are necessary to elucidate the physiological role of CG14435 and to identify the ubiquitin ligase activity required for ubiquitination-dependent DmIKK activation (Zhou, 2005).

As in the TRAF6 pathway, dTRAF2 and/or other E3 ligases that function with Bendless and dUEV1a in the IMD pathway may be the target(s) of K63 polyubiquitination. Another possible target of Bendless/dUEV1a-mediated ubiquitination is the Drosophila IKKgamma subunit (also known as NEMO in mammals) of the IKK complex. In mammals, it has recently been shown that NEMO is K63 polyubiquitinated by the Ubc13/UEV1a complex in response to Bcl10 expression or T-cell activation. Other possible targets of ubiquitination by Bendless and dUEV1a in the IMD pathway include the Drosophila TAB2 homolog and IMD. Recently, it was shown that the two mammalian homologs of TAB2 and TAB3 were ubiquitinated or associated with other ubiquitinated proteins. In addition, the mammalian RIP1, which is homologous to IMD protein especially in its death domain, has recently been shown to be K63 polyubiquitinated and associated with TAB2 in a TNFalpha-dependent manner. In any case, K63 polyubiquitin chains likely function to recruit the Drosophila TAK1/TAB2 complex, via the TAB2 K63 polyubiquitin binding domain, to either (or both) the upstream activators, such as IMD, and/or the downstream target of dTAK1 kinase activity, the Drosophila IKK complex (Zhou, 2005).

As expected, the epistatic analyses presented in this study demonstrate that IMD functions upstream of all other components in the pathway except the receptor PGRP-LC, and is required for IKK activation. Moreover, Bendless and dUEV1a function downstream of IMD and upstream of dTAK1, as predicted from the model for Ubc13 and UEV1a in mammals. dTAK1 is required for activation of the Drosophila IKK complex and likely functions as the IKK kinase (Zhou, 2005).

Although it is established that dFADD and DREDD are required for the IMD pathway, previous experiments have suggested that they function downstream of the DmIKK complex. For example, DREDD overexpression in flies leads to Diptericin gene expression in the absence of Gram-negative bacterial infection, and DREDD-mediated Diptericin gene activation requires neither the DmIKK complex nor dFADD. Also, recent studies have shown that DREDD interacts with Relish, and that a caspase-cleavage site within Relish is required for peptidoglycan-induced Relish activation. Therefore, it was speculated that DREDD functions downstream of the DmIKK complex by directly cleaving DmIKK-phosphorylated Relish. This possibility is consistent with the observation that DREDD and dFADD are required for dTAK1Delta-mediated Diptericin gene activation. Surprisingly, dFADD and DREDD are required for peptidoglycan-induced DmIKK activation, arguing that DREDD and dFADD function upstream in the pathway. In addition, DREDD is also required for peptidoglycan-induced JNK activation, but neither DREDD nor dFADD are required for dTAK1- or dTAK1Delta-mediated DmIKK activation, suggesting that dFADD and DREDD act at a step upstream of dTAK1 in response to peptidoglycan. Based on these observations, it is proposed that dFADD and DREDD play dual roles in the Drosophila antibacterial signaling pathway. On the one hand, dFADD transduces signals from IMD to DREDD, resulting in DREDD activation and enabling Relish cleavage; in contrast, dFADD and DREDD contribute to peptidoglycan-induced DmIKK activation through a mechanism that remains to be elucidated. DREDD may function similarly to human Caspase-8, which is a DED-containing apical caspase similar to DREDD. Caspase-8 has recently been shown to be required for NF-kappaB activation in response TCR-signaling. This role of Caspase-8 requires the enzymatic activity of full-length protein Caspase-8 and is involved in recruiting the IKK complex to the upstream signaling complex of CARMA1, Bcl10, and MALT1. Interestingly MALT1 is also a caspase-like gene (sometimes referred to as a paracaspase), and it is thought to function as an E3-ligase accessory factor with Ubc13 and UEV1a in TCR-mediated NF-kappaB activation. DREDD may similarly function as E3-ligase accessory factors with Bendless and dUEV1a as the E2, in the IMD pathway (Zhou, 2005).

The following scheme is proposed for the Drosophila antibacterial signaling pathway. Peptidoglycan treatment or Gram-negative bacterial infection leads to the activation of the membrane-bound peptidoglycan-recognition protein, PGRP-LC. Activated PGRP-LC in turn transduces signal to IMD. IMD, in turn, interacts with dFADD and subsequently DREDD. It is proposed that IMD, dFADD and DREDD form a complex that contributes to dTAK1 activation, perhaps as part of an E3 ligase. This complex is likely to function in conjunction with Bendless/dUEV1a to activate dTAK1 and then DmIKK. Once activated, the Drosophila IKK complex phosphorylates Relish, which is subsequently cleaved. The N-terminal Relish cleavage product, an NF-kappaB transcription factor, then translocates to the nucleus where it activates antimicrobial peptide gene expression. In addition to their role in IKK activation, DREDD and dFADD are also proposed to function downstream in this pathway, in the signal-induced cleavage of phospho-Relish (Zhou, 2005).

bendless, a Drosophila gene affecting neuronal connectivity, encodes a ubiquitin-conjugating enzyme homolog

The Drosophila bendless (ben) gene was originally isolated as a mutation affecting the escape jump response (Thomas, 1982; Thomas, 1984). This behavioral defect was ascribed to a single lesion affecting the connectivity between the giant fiber and the tergotrochanter motor neuron. A closer examination of the ben phenotype suggests that ben activity is broader and affects a variety of other neurons including photoreceptor cells and their axons. Mosaic analysis indicates that the focus of ben activity is presynaptic. This study reports the cloning of the ben gene through a chromosomal walk, and shows that it is homologous to a class of ubiquitin-conjugating enzymes. The major role of ubiquitination in the protein degradative pathway suggests that ben regulates neural developmental processes such as growth cone guidance by targeting specific proteins for degradation (Oh, 1994).

The Drosophila bendless gene encodes a neural protein related to ubiquitin-conjugating enzymes

The bendless (ben) mutation of Drosophila has been shown to alter synaptic connectivity between a subset of CNS neurons. ben also causes morphological abnormalities within the visual system, suggesting that ben functions in a number of different developmental processes. This study shows that the ben gene encodes a protein which is closely related to ubiquitin-conjugating enzymes and that a missense mutation in the highly conserved active site region is associated with the ben mutation. High levels of ben expression are restricted to the nervous system during development. These results suggest a role for ubiquitin-mediated protein modification in nervous system development, including, but not exclusive to, the regulation of synaptic connectivity (Muralidhar, 1993).

Bendless alters thoracic musculature in Drosophila

bendless (ben) is an X chromosome mutation in Drosophila melanogaster, known to alter patterns of connections in the CNS and thus modify behavior. This study reports that in addition to its CNS effects, ben^- has pleiotropic phenotypes affecting thoracic muscle patterning, pupal mortality, and post-eclosional mobility. The tergal depressor of the trochanter (TDT) normally attaches ventrally to an apodeme on the trochanter and dorsally to the lateral scutum just posterior to the intrascutal suture. In ben^- individuals, TDT may attach anywhere within the boundaries of the attachment areas for TDT and dorsoventral muscles I (DVM I) and II (DVM II). Furthermore, TDT may completely lack a dorsal attachment, although it still maintains a ventral attachment. DVMs may also attach abnormally to dorsal sites normally occupied by an adjacent DVM, or may be entirely eliminated. DVM loss occurs independently of the position or presence of TDT dorsal attachment. The cytology of ben^- TDT is altered. Muscles may have fibers that are swollen and stain abnormally. Other fibers may have large, axially aligned holes. ben^- flies have an increased likelihood of failing to eclose and, upon eclosion, show impaired mobility. Several possible mechanisms are described for the ben^- developmental defects (Edgecomb, 1993).

Mutations altering synaptic connectivity between identified neurons in Drosophila

By studying the effects of mutations on a simple circuit of identified neurons in Drosophila, genes were found whose proper functioning is necessary to produce normal synaptic connections between the neurons. These neurons comprise the giant fiber (GF) system; the GFs are command neurons activated by a light-off stimulus and evoke a stereotyped pattern of activity in the thoracic muscles producing an escape jump. Each GF monosynaptically drives a motor neuron innervating the tergotrochanteral muscle (jump muscle, TTM). Each GF also disynaptically drives the motor neurons innervating the dorsal longitudinal flight muscle (DLM) via the peripherally synapsing interneuron (PSI). A search was made for mutations affecting these identified synapses. Fifty thousand mutagenized flies were screened for nonjumping behavior to the light-off stimulus. Fifty-seven nonjumping mutant lines were established from individuals selected in the screen. Members of the lines were then tested for abnormal GF motor output to the TTM and DLM. From these lines, four X-linked mutations (representing three complementation groups) were isolated which affect the circuit. The mutations differentially disrupt specific synapses within the GF system. One mutation, bendless, disrupts synaptic transmission between the GF and the TTM motor neuron. Another, gfA, disrupts the synaptic connections of the PSI, and a third mutation, passover, disrupts transmission in both pathways (Thomas, 1984).

Search PubMed for articles about Drosophila Bendless

Chen, L., Paquette, N., Mamoor, S., Rus, F., Nandy, A., Leszyk, J., Shaffer, S. A. and Silverman, N. (2017). Innate immune signaling in Drosophila is regulated by TGFbeta-activated kinase (Tak1)-triggered ubiquitin editing. J Biol Chem [Epub ahead of print]. PubMed ID: 28377500

Edgecomb, R. S., Ghetti, C. and Schneiderman, A. M. (1993). Bendless alters thoracic musculature in Drosophila. J. Neurogenet. 8(4): 201-19. PubMed ID: 8320599

Ma, X., Shao, Y., Zheng, H., Li, M., Li, W. and Xue, L. (2013a). Src42A modulates tumor invasion and cell death via Ben/dUev1a-mediated JNK activation in Drosophila. Cell Death Dis 4: e864. PubMed ID: 24136228

Ma, X., Li, W., Yu, H., Yang, Y., Li, M., Xue, L. and Xu, T. (2013b). Bendless modulates JNK-mediated cell death and migration in Drosophila. Cell Death Differ. PubMed ID: 24162658

Merkle, J. A., et al. (2009). no poles encodes a predicted E3 ubiquitin ligase required for early embryonic development of Drosophila. Development 136(3): 449-59. PubMed ID: 19141674

Muralidhar, M. G. and Thomas, J. B. (1993). The Drosophila bendless gene encodes a neural protein related to ubiquitin-conjugating enzymes. Neuron 11(2): 253-66. PubMed ID: 8394720

Rickmyre, J. L., Dasgupta, S., Ooi, D. L., Keel, J., Lee, E., Kirschner, M. W., Waddell, S. and Lee, L. A. (2007). The Drosophila homolog of MCPH1, a human microcephaly gene, is required for genomic stability in the early embryo. J. Cell Sci. 120: 3565-3577. PubMed ID: 17895362

Oh, C. E., McMahon, R., Benzer, S. and Tanouye, M. A. (1994). bendless, a Drosophila gene affecting neuronal connectivity, encodes a ubiquitin-conjugating enzyme homolog. J. Neurosci. 14(5 Pt 2): 3166-79. PubMed ID: 8182464

Paquette, N., Broemer, M., Aggarwal, K., Chen, L., Husson, M., Erturk-Hasdemir, D., Reichhart, J. M., Meier, P., and Silverman, N. (2010) Caspase-mediated cleavage, IAP binding, and ubiquitination: linking three mechanisms crucial for Drosophila NF- kappaB signaling. Mol Cell 37: 172-182. PubMed ID: 20122400

Tatapudy, S., Peralta, J. and Nystul, T. (2021). Distinct roles of Bendless in regulating FSC niche competition and daughter cell differentiation. Development 148(22). PubMed ID: 35020878

Thevenon, D., Engel, E., Avet-Rochex, A., Gottar, M., Bergeret, E., Tricoire, H., Benaud, C., Baudier, J., Taillebourg, E., and Fauvarque, M. O. (2009) The Drosophila ubiquitin- specific protease dUSP36/Scny targets IMD to prevent constitutive immune signaling. Cell Host Microbe 6: 309-320. PubMed ID: 19837371

Thomas, J. B. and Wyman, R. J. (1982). A mutation in Drosophila alters normal connectivity between two identified neurons. Nature 298: 650-651. PubMed ID: 6808394

Thomas, J. B. and Wyman, R. J. (1984). Mutations altering synaptic connectivity between identified neurons in Drosophila. J. Neurosci. 4: 530-538. PubMed ID: 6699687

Uthaman, S. B., Godenschwege, T. A. and Murphey, R. K. (2008). A mechanism distinct from highwire for the Drosophila ubiquitin conjugase bendless in synaptic growth and maturation. J. Neurosci. 28(34): 8615-23. PubMed ID: 18716220

Zhou, R., Silverman, N., Hong, M., Liao, D. S., Chung, Y., Chen, Z. J. and Maniatis, T. (2005). The role of ubiquitination in Drosophila innate immunity. J. Biol. Chem. 280: 34048-34055. PubMed ID: 16081424

date revised: 22 February 2022

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