Ubiquitin activating enzyme 1: Biological Overview | References
Gene name - Ubiquitin activating enzyme 1
Cytological map position - 46A1-46A1
Function - enzyme
Symbol - Uba1
FlyBase ID: FBgn0023143
Genetic map position - chr2R:5575792-5580833
Classification - ubiquitin-activating enzyme E1
Cellular location - cytoplasmic
Ubiquitination is an essential process regulating turnover of proteins for basic cellular processes such as the cell cycle and cell death (apoptosis). Ubiquitination is initiated by ubiquitin-activating enzymes (E1), which activate and transfer ubiquitin to ubiquitin-conjugating enzymes (E2). Conjugation of target proteins with ubiquitin is then mediated by ubiquitin ligases (E3). Ubiquitination has been well characterized using mammalian cell lines and yeast genetics. However, the consequences of partial or complete loss of ubiquitin conjugation in a multi-cellular organism are not well understood. This study reports the characterization of Uba1, the only E1 in Drosophila. Weak and strong Uba1 alleles behave genetically differently with sometimes opposing phenotypes. Whereas weak Uba1 alleles protect cells from cell death, clones of strong Uba1 alleles are highly apoptotic. Strong Uba1 alleles cause cell cycle arrest which correlates with failure to reduce cyclin levels. Surprisingly, clones of strong Uba1 mutants stimulate neighboring wild-type tissue to undergo cell division in a non-autonomous manner giving rise to overgrowth phenotypes of the mosaic fly. It was demonstrated that the non-autonomous overgrowth is caused by failure to downregulate Notch signaling in Uba1 mutant clones. In summary, the phenotypic analysis of Uba1 demonstrates that impaired ubiquitin conjugation has significant consequences for the organism, and may implicate Uba1 as a tumor suppressor gene (Lee, 2008).
Ubiquitination refers to the covalent attachment of the small protein ubiquitin to target proteins. This modification usually targets ubiquitinated proteins for proteolytic degradation by the proteasome. In this capacity, ubiquitination is essential for turnover of proteins involved in many cellular processes including the cell cycle, cell death, signal transduction, etc. For example, ubiquitin-mediated degradation of cyclins is essential for progression through the cell cycle. Inhibitor of apoptosis proteins (IAPs) need to be ubiquitinated and degraded in cells undergoing apoptosis. However, ubiquitin-mediated degradation of caspases, the principal executioners of apoptosis, has been reported to protect cells from apoptosis. In addition, non-traditional functions of ubiquitination, which do not target proteins for proteolysis, have been reported. In this regard, it is noteworthy that activated cell surface signaling receptors are ubiquitinated, usually mono-ubiquitinated, for endocytosis and protein sorting at the early endosome (Lee, 2008 and references therein).
Alterations in the ubiquitination machinery are often associated with human diseases such as cancer, neurodegenerative disorders and inflammation. On the other hand, targeting the ubiquitination system for therapeutic purposes holds promise in the clinic. Thus, a detailed understanding of the role of ubiquitination for proper homeostasis and physiology of multi-cellular organisms is critical (Lee, 2008).
E1 ubiquitin-activating enzymes catalyze the first step in the ubiquitination cycle, the ATP-dependent formation of a thioester bond between the C-terminal glycine residue of ubiquitin and the active site cysteine of the E1 (Haas, 1997; Pickart, 2001). This is followed by the transfer of ubiquitin from the E1 to a ubiquitin-conjugating enzyme (E2). The final step is the conjugation of ubiquitin to target proteins mediated by ubiquitin ligases (E3). The specificity of the ubiquitination process is conferred to by E3 ubiquitin ligases. The genomes of eukaryotic organisms contain hundreds of different E3-encoding genes required for the regulated protein turnover in many cellular processes. By contrast, there are considerably fewer E1 and E2 enzymes. For example, the Drosophila genome encodes only one E1 enzyme, termed Uba1 (Watts, 2003). This low complexity suggests that the primary function of the E1 enzyme is to provide activated ubiquitin for all ubiquitin-dependent reactions. This has indeed been observed in yeast. Genetic inactivation of the yeast gene Uba1 blocks most, if not all ubiquitin conjugation (see Ghaboosi, 2007). There are mammalian cell lines containing temperature-sensitive alleles of E1. These cell lines have been of great importance for understanding the role of ubiquitin-mediated degradation of cyclins for progression through the cell cycle, and have further suggested an essential function of E1 enzymes to provide activated ubiquitin for conjugation of target proteins (Lee, 2008).
However, despite these valuable analyses of ubiquitin conjugation in single cell organisms and cell lines, a systematic analysis of partial or complete loss of ubiquitin conjugation in multi-cellular organisms has not been reported. This can be accomplished by reducing the activity of the only E1 enzyme in Drosophila, Uba1 (Watts, 2003). To date, a role of Drosophila Uba1 (from now on referred to as Uba1) has only been reported for axon pruning in the nervous system, and the precise mechanistic function of Uba1 in this process is unknown (Kuo, 2006; Watts, 2003). This study reports the isolation and characterization of weak and strong alleles of Uba1 in Drosophila. Depending on the strength of the Uba1 allele, different and sometimes opposing phenotypes are observed. For example, weak Uba1 alleles protect cells from apoptosis, whereas mutant clones of strong alleles are highly apoptotic. Strong Uba1 alleles which affect significantly ubiquitin conjugation, cause cell cycle arrest that correlates with increased cyclin levels. Unexpectedly, clones of strong Uba1 alleles induce cell proliferation in neighboring tissue, triggering non-autonomous overgrowth. These Uba1 clones fail to downregulate Notch activity which stimulates Jak/STAT signaling, and thus growth, in neighboring wild-type tissue. In summary, this analysis demonstrates that the lack of ubiquitin conjugation has significant consequences for the organism, and may implicate Uba1 as a tumor suppressor gene (Lee, 2008).
Before this study, E1 ubiquitin-activating enzymes have only been characterized in yeast and in mammalian cell lines. This study analyzed the only E1 gene in Drosophila, Uba1, and uncovered two unexpected mutant phenotypes. First, while partial loss of ubiquitin conjugation caused by weak Uba1 alleles inhibits cell death, strong Uba1 alleles are highly apoptotic. Second, while strong Uba1 clones are cell cycle arrested, they do stimulate neighboring wild-type cells to undergo cell proliferation and induce non-autonomous overgrowth. It was also found that photoreceptor differentiation occurs both in clones of weak and strong Uba1 alleles. However, the onset of photoreceptor differentiation is slightly delayed in clones of strong alleles. Similar observations have been made in a different study (Pfleger, 2007; Lee, 2008).
This study has identified Uba1 alleles as suppressors of the apoptotic phenotype caused by GMR-hid, and showed that Uba1 is also required for normal developmental cell death. This requirement is probably mediated through the control of Diap1 protein levels which in turn mediates ubiquitination of the caspase Dronc (Chai, 2003; Wilson, 2002). However, the GMR-hid-suppressing Uba1 alleles are weak. They affect overall ubiquitin conjugation only mildly suggesting that ubiquitin-mediated degradation can still occur in an almost normal manner. In accord, the increased protein levels of Diap1 are even able to reduce the protein levels of Dronc in clones expressing weak Uba1 alleles (Lee, 2008).
It is interesting to note that whereas Diap1 protein levels are increased in clones expressing weak Uba1 alleles, other proteins such as Ci, Arm or Dronc are normal in abundance or even reduced, respectively. This suggests that some proteins such as Diap1 respond in a very sensitive manner to partial loss of activated ubiquitin, whereas other proteins do not. Because Diap1 has a fairly short half-life (~30-40 minutes) compared to Dronc (~3 hours), the requirement of a fully functional ubiquitination machinery may be much stricter for Diap1, providing an explanation for why Diap1 responds so sensitively to a small reduction of activated ubiquitin for protein conjugation (Lee, 2008).
Alternatively, it is also possible that the Uba1 alleles isolated in this study specifically affect the interaction with UbcD1, the E2-conjugating-enzyme which targets Diap1 for ubiquitin-mediated degradation. Thus, the interaction with other E2 enzymes may be normal, so that ubiquitin conjugation and degradation of other proteins may be normal. Which of these two possibilities applies has not been tested (Lee, 2008).
Strong Uba1 alleles, which significantly reduce ubiquitin conjugation, affect the levels of all proteinsanalyzed. For example, although Diap1 levels are increased with strong Uba1 alleles, Dronc is no longer efficiently degraded. Instead, Dronc protein accumulates, suggesting that activated ubiquitin required for conjugation and degradation is no longer available. However, it is unclear why cells in Uba1 clones die. Dronc needs to be cleaved for activation, and Diap1 can directly bind to and inhibit caspases without degradation, at least in vitro. Thus, the increased Diap1 levels should still be able to inhibit the accumulated Dronc protein. Mutants in ark (also known as D-Apaf-1, hac-1 and dark), which encodes an adaptor protein required for Dronc activation, block cell death in Uba1, suggesting that cell death in Uba1 mutants is indeed mediated via Dronc. Thus, simple binding of Diap1 to Dronc may not be sufficient to completely inhibit Dronc activity. Instead, ubiquitination may be required for full inactivation of Dronc (Lee, 2008).
Consistent with the expectation, loss of ubiquitin conjugation in strong Uba1 alleles causes cell cycle arrest. This correlates with increased protein levels of Cyclins A and B, the ubiquitin-dependent degradation of which is required for cell cycle progressio (Lee, 2008).
However, the non-autonomous overgrowth phenotype was unexpected. Strong Uba1 clones appear to be able to secrete a growth factor that promotes cell proliferation and overgrowth in adjacent wild-type tissue. In this capacity, Uba1 qualifies as a tumor suppressor gene (Lee, 2008).
It is interesting to note that the phenotypes observed for Uba1 are very similar to those of vps23 and vps25. In both cases, Notch signaling is inappropriately increased. Notch triggers Jak/STAT signaling in neighboring wild-type tissue, presumably through secretion of Unpaired, which encodes an Interleukin-like factor and acts as the ligand of the receptor of the Jak/STAT signaling pathway. However, the ultimate cause of Notch activation may be different. In the case of vps23 and vps25, Notch is internalized via endocytosis, however, endosomal protein sorting is impaired, thus turnover of Notch is affected. In the case of Uba1, it is not clear whether the lack of ubiquitination affects endocytosis of membrane-localized Notch or ubiquitin-mediated degradation of intracellular Notch in the nucleus. Failure of either may cause inappropriate signaling. The accumulation of Notch in Uba1 clones is not as striking as in vps25 clones, making it difficult to identify the subcellular localization of accumulated Notch (Lee, 2008).
Another interesting observation is the fact that increased Notch activity is not observed in clones of Uba1D6 at 25°C, at which temperature non-autonomous cell proliferation is not observed. Consistently, no increased STAT signaling was detected under these conditions. If Notch signaling is increased at 25°C, why does this not induce non-autonomous proliferation? One potential reason may lie in the fact that Uba1 clones at 25°C are protected from apoptosis, whereas at 29#176;C they are apoptotic. Thus, an apoptotic environment may be necessary for the induction of non-autonomous proliferation. A similar phenomenon, referred to as apoptosis-induced compensatory proliferation, has recently been reported. In these studies, apoptotic cells trigger the secretion of Dpp and Wg which promote proliferation in neighboring cells. An involvement of Notch was not reported. However, in the aforementioned studies, apoptosis-induced compensatory proliferation is only detectable if cell death is simultaneously blocked. In the case of Uba1, vps23 and vps25, overgrowth occurs without inhibition of apoptosis. Therefore, there may be different forms of compensatory proliferation in response to different apoptotic triggers (Lee, 2008).
In summary, this study has largely focused on the effects of loss of ubiquitin conjugation for apoptosis and cell proliferation. This analysis demonstrates that the loss of ubiquitin conjugation has significant consequences for the organism, and may implicate Uba1 as a tumor suppressor gene in Drosophila. The Uba1 alleles identified in this study will be of further use to analyze a general requirement of ubiquitination for other cellular processes as well (Lee, 2008).
Ras signaling can promote proliferation, cell survival and differentiation. Mutations in components of the Ras pathway are found in many solid tumors and are associated with developmental disorders. This study demonstrates that Drosophila tissues containing hypomorphic mutations in E1, the most upstream enzyme in the ubiquitin pathway, display cell-autonomous upregulation of Ras-ERK activity and Ras-dependent ectopic proliferation. Ubiquitylation is widely accepted to regulate receptor tyrosine kinase (RTK) endocytosis upstream of Ras. However, although the ectopic proliferation of E1 hypomorphs is dramatically suppressed by removing one copy of Ras, removal of the more upstream components Egfr, Grb2 or sos shows no suppression. Thus, decreased ubiquitylation may lead to growth-relevant Ras-ERK activation by failing to regulate a step downstream of RTK endocytosis. This study further demonstrates that Drosophila Ras is ubiquitylated. These findings suggest that Ras ubiquitylation restricts growth and proliferation in vivo. An intriguing observation is that complete inactivation of E1 causes non-autonomous activation of Ras-ERK in adjacent tissue, mimicking oncogenic Ras overexpression. Maintaining sufficient E1 function is required both cell autonomously and non-cell autonomously to prevent inappropriate Ras-ERK-dependent growth and proliferation in vivo and may implicate loss of Ras ubiquitylation in developmental disorders and cancer (Yan, 2009).
Therefore, impaired ubiquitin pathway function due to mutation in E1 results in a growth-relevant, cell-autonomous increase in Ras-ERK activity. It is widely accepted that RTK endocytosis is regulated by ubiquitylation and that a failure of RTK ubiquitylation promotes increased signaling through Ras. Contributions from upstream regulators of Ras to the phenotypes of E1 mutants in the current system cannot be ruled out; however, mutation in Ras dominantly suppress the increased proliferation and pupal lethality of E1 hypomorphs strongly, whereas mutations in Egfr, drk and sos did not. One possible explanation is that multiple upstream steps that converge on Ras are regulated by ubiquitylation. Alternatively, it is possible that an as-yet-unidentified regulator of Ras is regulated by ubiquitylation. However, the simplest model to explain the current findings is that the cell-autonomous increase in Ras activity may be independent of Egfr and Grb2/sos and occurs at the step of Ras. Indeed, this study has demonstrated that Drosophila Ras is ubiquitylated. These findings suggest the exciting model that decreased ubiquitylation of Ras itself causes increased activation of ERK. This may be a mechanism highly conserved between Drosophila and mammals, because a recent study reports di-ubiquitylation of H-Ras and N-Ras in vitro (Jura, 2006). Whereas Jura established ubiquitylation of H-Ras and N-Ras in a tissue-culture context, the physiological relevance of Ras ubiquitylation has not been investigated. These Drosophila studies demonstrate that in vivo, the activation of Ras is highly sensitive to ubiquitylation. Impairing ubiquitylation leads to increased Ras-ERK activation that promotes ectopic cell proliferation and confers increased resistance to cell death in vivo in a developmental context (Yan, 2009).
How does Ras ubiquitylation restrict signaling through downstream effectors? It is possible that ubiquitylated Ras adopts a conformation that no longer interacts with Raf. Alternatively, ubiquitylation may alter Ras localization, thus isolating it from downstream effectors. Indeed, Jura (2006) showed that a construct of H-Ras fused to ubiquitin (to mimic constitutively ubiquitylated Ras) preferentially localizes to the endosomes (Yan, 2009).
It is generally assumed that E1 activity is not limiting; decreasing E1 activity so it becomes limiting could amplify substrate specificities such that some ubiquitin-mediated processes are affected early and dramatically whereas others are affected to a lesser extent or at a later time. The extreme sensitivity of E1 phenotypes to Ras gene dosage strongly supports the argument that Ras regulation is affected early and/or dramatically upon a decrease in E1 function and implies that maintaining sufficient activity of the ubiquitin pathway is crucial to prevent inappropriate Ras-ERK activation in vivo (Yan, 2009).
This paper also presents intriguing observation that that there is growth-relevant Ras-ERK activation in cells adjacent to E1 null clones, and this Ras activation mimics oncogenic Ras. What is the mechanism underlying non-autonomous Ras activation? Given the pleiotropic effects caused by the global loss of ubiquitylation, elucidating this experimentally is difficult. Previous work in mammalian systems reports that Ras activation increases the release of EGF-like ligands, and this study has demonstrated that a cell-autonomous increase in Ras signaling through ERK is sufficient to promote activation of Ras in neighboring cells. Thus, it is possible that the cell-autonomous increase in Ras activation in E1 null cells promotes the non-autonomous Ras activation. Investigating the role of ubiquitylation in preventing non-autonomous Ras activation in the future will be exciting and may elucidate the ability of stromal cells to promote growth and invasiveness of adjacent tumor cells (Yan, 2009).
By demonstrating that maintaining sufficient ubiquitin pathway activity is crucial for Ras regulation both cell-autonomously and non-autonomously, this study provides further support for the previous suggestion that E1 may be a tumor suppressor gene (Pfleger, 2007). In fact, one study using comparative genomic hybridization reports a loss in DNA copy number of the human E1 chromosomal region in breast cancer lines and tumors. Microarray and/or serial analysis of gene expression (SAGE) methods reveal significantly decreased E1 RNA levels in many cancer cell lines and tumors. Previous SAGE studies have shown that E1 levels drop dramatically in the leukocytes and luminal epithelial cells of invasive ductal carcinomas compared to those of normal breast tissue and ductal carcinomas in situ, potentially implicating E1 loss in breast cancer progression. Given these reports and the findings of Ras-dependent overgrowth due to mutation of E1 in vivo, it is proposed that downregulating E1, either by mutation or other means, could be a mechanism employed by tumor cells to achieve cell death resistance and Ras activation. Identification of the ubiquitin ligase or ligases targeting Ras is of high importance, as such ligase(s) may play a crucial role in normal proliferation and may be dysregulated in developmental disorders and in cancer (Yan, 2009).
Autophagy is a conserved process that delivers components of the cytoplasm to lysosomes for degradation. The E1 and E2 enzymes encoded by Atg7 and Atg3 are thought to be essential for autophagy involving the ubiquitin-like protein Atg8. This study describes an Atg7- and Atg3-independent autophagy pathway that facilitates programmed reduction of cell size during intestine cell death. Although multiple components of the core autophagy pathways, including Atg8, are required for autophagy and cells to shrink in the midgut of the intestine, loss of either Atg7 or Atg3 function does not influence these cellular processes. Rather, Uba1, the E1 enzyme used in ubiquitylation, is required for autophagy and reduction of cell size. These data reveal that distinct autophagy programs are used by different cells within an animal, and disclose an unappreciated role for ubiquitin activation in autophagy (Chang, 2013).
Macroautophagy (autophagy) is a system that is used to transfer cytoplasmic material, including proteins and organelles, to lysosomes by all eukaryotic cells. Autophagy is augmented during cell stress to reduce damage to enable cell survival, and is also associated with the death of animal cells. Although most studies of this process have focused on stress-induced autophagy, such as nutrient deprivation, autophagy is also a normal aspect of animal development where it is required for proper death and removal of cells and tissues. Defects in autophagy lead to accumulation of protein aggregates and damaged organelles, as well as human disorders. Most of the knowledge about the genes controlling autophagy is based on pioneering studies in the yeast Saccharomyces cerevisiae, and it is not clear whether cells that exist in extremely different contexts within multi-cellular organisms could use alternative factors to regulate this catabolic process (Chang, 2013).
Atg genes that are conserved from yeast to humans are required for autophagy, and include the Atg1 and Vps34 regulatory complexes, as well as two ubiquitin-like conjugation pathways. The two ubiquitin-like molecules, named Atg8 (LC3 and GABARAP in mammals) and Atg12, become associated with the isolation membranes that form autophagosomes through the activity of the E1 enzyme Atg7. Atg3 functions as the E2-conjugating enzyme for Atg8, and Atg10 functions as the E2 for Atg12. Atg12 associates with Atg5 and Atg16 during the formation of the autophagosome, and Atg8 is conjugated to the lipid phosphatidyl-ethanolamine enabling this protein to associate with the isolation membrane and autophagosome. Lipidated Atg8 remains associated with autophagosomes until fusion with lysosomes to form autolysosomes where cargoes are degraded by lysosomal enzymes (Chang, 2013).
Degradation of the midgut of the Drosophila melanogaster intestine involves a large change in midgut length, has elevated autophagy and markers of caspases associated with it, requires autophagy, and seems to be caspase independent (Denton, 2009, Lee, 2002; Micchelli, 2011). This study shows that autophagy is required for programmed reduction in cell size at the onset of intestine cell death in Drosophila. Atg genes encoding components of the Atg1 and Vps34 complexes are required for midgut cell autophagy and reduction in size. Surprisingly, although Atg8a is required for autophagy and programmed cell size reduction, the evolutionarily conserved E1-activating enzyme Atg7 and E2-conjugating enzyme Atg3 are not required for these cellular events. This study screened the E1-activating enzymes encoded by the fly genome and identified Uba1 as being required for autophagy and reduction of cell size during midgut cell death. Although the genes that control autophagy are conserved throughout eukaryotes, the current data provide evidence indicating that the core autophagy machinery may not be identical in all cells within an organism (Chang, 2013).
Autophagy has been shown to influence cell size during growth factor and nutrient restriction in mammalian cells lines, but this study indicates that autophagy controls cell size as part of a normal developmental program. The discovery that Atg7 and Atg3 are not required for autophagy and cell size reduction in dying midgut cells in Drosophila is surprising. Although an Atg5, Atg7- and LC3-independent autophagy pathway has been reported (Nishida, 2009), this study describes autophagy that requires Atg8 (LC3) and does not require Atg7 and Atg3. It has been assumed that components of the core Atg8 (LC3) and Atg12 conjugation pathways are used by all eukaryotic cells, but this study provides evidence that alternative factors can function to regulate autophagy in a cell-context-specific manner (Chang, 2013).
This study highlights that autophagy may have different regulatory mechanisms in distinct cell types within an animal. Different forms of autophagy could involve either unique regulatory pathways , different amounts and rates of autophagy or alternative cargo selection mechanisms, and these are not mutually exclusive. Another possibility is that differences in cargo selection alone, perhaps based on specific cargo adaptor proteins, could mediate a distinct type of autophagy (Chang, 2013).
This paper reports that an E1 enzyme other than Atg7 is required for Atg8 and Atg5 puncta formation, and clearance of ubiquitin-binding protein p62 and mitochondria. The studies indicate that Uba1 fails to function in place of Atg7, as expected on the basis of the unique architecture and use of ubiquitin-like proteins and E2-binding domains in these highly divergent E1 enzymes. Although the possibility cannot be excluded that Atg8a is activated by unknown factors, the simplest model to explain the data positions Uba1 function at a different stage of the autophagy process that depends on ubiquitin conjugation. Previous work in a mammalian cell line indicated that Uba1 is required for protein degradation by lysosomes, but this was not because of decreased autophagosome formation (Lenk, 1992). In addition, recent work in Drosophila implicated the de-ubiquitylation enzyme USP36 in autophagy (Taillebourg, 2012). However, the inability of Atg5 knockdown to suppress the USP36 mutant phenotype, as well as the accumulation of both GFP-Atg8a and ubiquitin-binding protein p62 in USP36 mutant cells, suggests a defect in autophagic flux rather than a defect in the formation of autophagosomes. p62 and other ubiquitin-binding proteins are known to facilitate recruitment of ubiquitylated cargoes into autophagosomes (Johansen, 2011). In addition, p62 was recently shown to accumulate at sites of autophagosome formation even when autophagosome formation is blocked (Itakura, 2011). Thus, it is possible that Uba1 promotes cargo recruitment to the sites of autophagosome formation to facilitate autophagy. However, it is also possible that Uba1 could function at multiple stages in the regulation of autophagy (Chang, 2013).
It is critical to understand the mechanisms that regulate autophagy given the interest in this catabolic process as a therapeutic target for multiple age-associated disorders, including cancer and neurodegeneration. Significantly, these studies illuminate that autophagy has different regulatory mechanisms in distinct cell types within an animal, and highlight the importance of studying core autophagy genes in specific cell types under physiological conditions (Chang, 2013).
Hedgehog transduces signal by promoting cell surface expression of the seven-transmembrane protein Smoothened (Smo) in Drosophila, but the underlying mechanism remains unknown. This study demonstrates that Smo is downregulated by ubiquitin-mediated endocytosis and degradation, and that Hh increases Smo cell surface expression by inhibiting its ubiquitination. Smo is ubiquitinated at multiple Lysine residues including those in its autoinhibitory domain (SAID), leading to endocytosis and degradation of Smo by both lysosome- and proteasome-dependent mechanisms. Hh inhibits Smo ubiquitination via PKA/CK1-mediated phosphorylation of SAID, leading to Smo cell surface accumulation. Inactivation of the ubiquitin activating enzyme Uba1 or perturbation of multiple components of the endocytic machinery leads to Smo accumulation and Hh pathway activation. In addition, this study found that the non-visual beta-arrestin Kurtz (Krz) interacts with Smo and acts in parallel with ubiquitination to downregulate Smo. Finally, it was shown that Smo ubiquitination is counteracted by the deubiquitinating enzyme UBPY/USP8. Gain and loss of UBPY lead to reciprocal changes in Smo cell surface expression. Taken together, these results suggest that ubiquitination plays a key role in the downregulation of Smo to keep Hh pathway activity off in the absence of the ligand, and that Hh-induced phosphorylation promotes Smo cell surface accumulation by inhibiting its ubiquitination, which contributes to Hh pathway activation (Li, 2012).
Neurodegenerative diseases cause tremendous suffering for those afflicted and their families. Many of these diseases involve accumulation of mis-folded or aggregated proteins thought to play a causal role in disease pathology. Ubiquitinated proteins are often found in these protein aggregates, and the aggregates themselves have been shown to inhibit the activity of the proteasome. These and other alterations in the Ubiquitin Pathway observed in neurodegenerative diseases have led to the question of whether impairment of the Ubiquitin Pathway on its own can increase mortality or if ongoing neurodegeneration alters Ubiquitin Pathway function as a side-effect. To address the role of the Ubiquitin Pathway in vivo, loss-of-function mutations was studied in the Drosophila Ubiquitin Activating Enzyme, Uba1 or E1, the most upstream enzyme in the Ubiquitin Pathway. Loss of only one functional copy of E1 caused a significant reduction in adult lifespan. Rare homozygous hypomorphic E1 mutants reached adulthood. These mutants exhibit further reduced lifespan and show inappropriate Ras activation in the brain. Removing just one functional copy of Ras restores the lifespan of heterozygous E1 mutants to that of wild-type flies and increases the survival of homozygous E1 mutants. E1 homozygous mutants also showed severe motor impairment. These findings suggest that processes that impair the Ubiquitin Pathway are sufficient to cause early mortality. Reduced lifespan and motor impairment are seen in the human disease X-linked Infantile Spinal Muscular Atrophy, which is associated with mutation in human E1 warranting further analysis of these mutants as a potential animal model for study of this disease (Liu, 2013).
In humans, E1 is encoded by the gene Ube1 on the X chromosome. Given the high conservation of genes in the Ubiquitin Pathway, this could mean that women carrying one mutant copy of E1 might be at risk for reduced lifespan. How does loss of only one copy of E1 cause such a change in lifespan? The Ubiquitin Pathway controls a number of crucial cellular activities including signal transduction, apoptosis, and proteasome-mediated protein degradation. Proteasome activity and assembly decline with increased age. Therefore, it is possible that at a young age, the threshold of E1 is easily met by only one functional genomic copy, but that as age advances and the proteasome becomes more limiting, that one copy of E1 is no longer sufficient to allow for clearance of misfolded or aggregating proteins. Thus, one possible explanation is that increased protein aggregation in flies with only one functional copy of E1 could cause increased mortality (Liu, 2013).
Disease-associated mutations in specific genes have been identified in familial forms of a number of neurodegenerative diseases including Huntington's Disease (HD), Alzheimer's Disease (AD), and Parkinson's Disease (PD). In HD, the length of the expanded polyQ region in part determines the age of onset of the disease; longer repeats often result in onset of symptoms at an earlier age. Intriguingly, however, patients with the same polyQ length do not always exhibit the same time of onset and course of the disease. Therefore, polyQ length alone cannot explain all differences in disease presentation. Environmental factors and genetic background likely also contribute to variations in disease progression. It will be exciting to explore if human E1 variants could create sensitive genetic backgrounds with adverse effects on the course of disease progression in patients suffering from HD. Moreover, there are familial cases of other neurodegenerative diseases in which causal mutations have not been identified. In addition, for some diseases, there are sporadic cases with no family history. In fact, sporadic AD is far more prevalent than familial AD, and the causes of sporadic AD also remain unclear. Thus, it is highly likely that there are a number of genes whose mutation or dysregulation serve as risk factors or even causes of sporadic AD cases. It is speculated that human E1 variants may serve as risk factors for the age-related decline in AD and other diseases. In the future, it will be important to address how loss of E1 affects lifespan in Drosophila neurodegeration models including models of HD and AD (Liu, 2013).
The Ubiquitin Pathway also regulates a number of signaling pathways including (but not limited to) Ras signaling. Upstream RTKs are down-regulated by ubiquitination. Therefore, another possibility is that upon aging, specific signaling pathways are dysregulated and contribute to reduced lifespan. In fact, examination of the brains of AD patients found evidence of increased Ras signaling. Also, expressing activated Ras in neurons causes AD-type phenotypes in neurons in culture. Importantly, This study has shown that reducing the gene dosage of Ras in flies carrying only one mutant copy of E1 restores lifespan to that of wild-type controls (Liu, 2013).
There are a number of variants reported for human E1 including loss-of-function alleles. In humans, the E1 gene Ube1 is located on the X chromosome and has been lost from the Y chromosome, so a male inheriting a loss of function variant in E1 would have no wild-type copy. Some human E1 variants are associated with X-linked Infantile Spinal Muscular Atrophy (XL-SMA), a rare and severe form of Spinal Muscular Atrophy. XL-SMA is a tragic condition in which males who inherit a mutant copy of E1 typically live less than two years and during which time they suffer terribly. Mothers who are carriers for a mutant copy of E1 often have a history of miscarriages presumably because many of their affected male children do not make it to term. XL- SMA has a similar presentation to the severe Type 1 SMA caused by mutation in the SMN1 gene, but also presents with congenital contractures (Liu, 2013).
This study reports that flies homozygous for null mutations in E1 do not survive, but flies homozygous for hypomorphic E1 mutations can survive to adulthood at a very reduced rate, and these flies show a number of patterning abnormalities and severe motor impairment. Their lifespan is dramatically reduced compared to heterozygous mutants and wild-type controls (Liu, 2013).
There is currently no animal model in which to study XL-SMA. This study shows that Drosophila E1 homozygous mutants recapitulate some aspects of human XL-SMA such as motor impairment and reduced lifespan. Thus, these Drosophila mutants warrant further study to determine if they recapitulate other aspects of this disease, such as degeneration of motorneurons reminiscent of the loss of anterior horn cells in XL-SMA, to establish if they could serve as an animal model to increase understanding of this devastating disease. It was previously shown that reducing the gene dosage of Ras in homozygous E1 mutants increases their survival to adulthood Yan, 2009), and the current investigation reported that it also extends their adult lifespan. If Ras signaling contributes to XL-SMA pathology in humans as it does to reduced lifespan in Drosophila E1 mutants, targeting Ras may serve as a potential therapeutic strategy for XL-SMA (Liu, 2013).
The ubiquitin-proteasome system is one of the main proteolytic pathways. It inhibits apoptosis by degrading pro-apoptotic regulators, such as caspases or the tumor suppressor p53. However, it also stimulates cell death by degrading pro-survival regulators, including IAPs. In Drosophila, the control of apoptosis by Bcl-2 family members is poorly documented. Using a genetic modifier screen designed to identify regulators of mammalian bax-induced apoptosis in Drosophila, this study identified the ubiquitin activating enzyme Uba1 as a suppressor of bax-induced cell death. Uba1 was demonstrated to regulate apoptosis induced by Debcl, the only counterpart of Bax in Drosophila. Furthermore, these apoptotic processes were shown to involve the same multimeric E3 ligase-an SCF complex consisting of three common subunits and a substrate-recognition variable subunit identified in these processes as the Slimb F-box protein. Thus, Drosophila Slimb, the homologue of beta-TrCP targets Bax and Debcl to the proteasome. These new results shed light on a new aspect of the regulation of apoptosis in fruitfly that identifies the first regulation of a Drosophila member of the Bcl-2 family (Colin, 2014).
This paper reports the regulation of bax- and debcl-induced apoptosis by the ubiquitin-proteasome pathway. The stimulation of this pathway by overexpressing Uba1, which encodes the ubiquitin activating enzyme, leads to an almost complete loss of bax-induced cell death. This regulation seems conserved through evolution, as debcl-induced apoptosis is also regulated in this way. However, since Bax seems to necessitate Debcl in order to kill Drosophila eye cells, one could wonder whether suppression of Bax-induced cell death depends on the direct effect of Uba1 on Debcl. Nevertheless, since both Bax and Debcl proteins are degraded when Uba1 is overexpressed, this seems unlikely unless Debcl stabilizes Bax (Colin, 2014).
Since Buffy inhibits autophagy in response to starvation, it is hypothesized that Debcl induces an autophagic cell death. Autophagy was monitored by using a UAS-Atg8-GFP transgene. It was found that actually no autophagy could be detected upon Debcl expression. Uba1 has been shown in the literature to be required for autophagy and reduction of cell size in the intestine. This study shows that Debcl-induced cell death in the wing disc is not only suppressed by Uba1 but also by proteasome mutants. These data suggest that the UPS pathway is the main proteolytic pathway involved in the suppression of Bax and Debcl-induced apoptosis by Uba1. However, given that slmb has been shown to regulate Wg and Dpp pathways, it cannot be excluded that these pathways are partially involved in phenotypic suppression (Colin, 2014).
Studies of the proteasome-dependent regulation of members of the Bcl-2 family in mammals have only rarely led to the identification of the specific E3 ligases. This study identified an SCFSlmb complex as the E3 ubiquitin ligase that regulates the Debcl pathway and may target it to proteasomal degradation. It would be interesting to determine whether the mammalian homologue of Slmb, β-TrCP, targets Bax to the proteasome in mammalian cells (Colin, 2014).
This study has show Debcl is a target of the UPS, thus finding a new regulation of apoptosis that differs from the control of Dronc, Drice and RHG protein levels by Diap1. Indeed, Diap1 is a key enzyme that decides of cell fate by degrading either pro-apoptotic regulators or itself, leading to either cell survival or apoptosis. Since Diap1 levels are downregulated by the F-box protein Morgue in presence of Rpr or Grim, it could be hypothesized that the Morgue/Diap1 pathway is involved in bax- and debcl-induced apoptosis regulation. This does not seem to be the case because a hypomorphic allele of morgue did not increase bax- and debcl-induced apoptosis. Furthermore, overexpression of diap1 does not inhibit bax- and debcl-induced apoptosis. Thus, the proteasome-dependent regulation that this study has identified is independent of Diap1 and differs from the Morgue/Diap1 regulation of cell death. The existence of different UPS-modulated cell death pathways is also supported by the reported absence of genetic interaction between RHG pathway components and Debcl (Colin, 2014).
The results indicate an anti-apoptotic role of Uba1 in the wing tissue. However, two other studies revealed a pro-apoptotic role of Uba1 in the eye; strong Uba1 loss-of-function alleles lead to apoptosis and compensatory proliferation in the developing eye. As previously shown in other systems, these processes seem to involve the RHG/Diap1/Dronc pathway. Hypomorphic alleles of Uba1 have shown opposite effects as they suppressed hid- or grim-induced apoptosis in the eye. These results are consistent with previous data indicating that Uba1 overexpression, using the Uba1 EP2375 allele, increases RHG-induced apoptosis. In principle, this apparent contradiction may result from either the cell death signal or the studied tissue. The use of a GMR-Gal4 driver shows that Uba1 overexpression also inhibits debcl-induced apoptosis in the eye tissue, which suggests that the Uba1 effect is specific of debcl-induced apoptosis. In contrast, rpr-induced cell death is enhanced by Uba1 overexpression in the eye, whereas it is suppressed by Uba1 in the wing. These results suggest that Debcl could be involved in rpr-induced cell death in the wing but not in the eye. RHG proteins are known to mediate their pro-apoptotic function by stimulating Diap1 degradation by the UPS while Debcl is a direct target of ubiquitination. Therefore, RHG-induced degradation of Diap1 through its ubiquitination by Morgue could explain the pro-apoptotic role of Uba1 in the eye whereas the anti-apoptotic Uba1 function mediated by Slmb in Debcl-induced cell death would rely on Debcl degradation. By showing that different pro-apoptotic pathways are regulated by the UPS in Drosophila, this work suggests that the tissue-dependent effect of the pleiotropic enzyme Uba1 must result from a change in the balance between UPS pro- and anti-apoptotic effects. It is proposed that this change relies on the availability in E3 enzymes, the incoming signals and relative amounts of pro- and anti-apoptotic regulators of cell fate (Colin, 2014).
Protein ubiquitination has been shown to regulate a wide variety of cellular process including cell cycle progression, protein trafficking and apoptosis. Most regulation of ubiquitination occurs at the level of E2 or E3 enzymes and their interactions with specific substrates. In a screen for mutations that cause tissue overgrowth, this study recovered multiple mutations in the Drosophila Uba1 gene that encodes the E1 enzyme that is required for the first step of most, if not all, ubiquitination reactions. Previous studies with yeast and mammalian cells have shown that disrupting E1 function results in a cell-cycle arrest. This study shows that in the developing Drosophila eye, clones of cells that are homozygous for partial loss of function alleles of Uba1 show defects in apoptosis. Moreover, clones homozygous for stronger or complete loss of function alleles of Uba1, that are predicted to have a global defect on ubiquitination, survive poorly but are able to stimulate the overgrowth of adjacent wild-type tissue. Experiments with mammalian cells show that reducing the level of RNA of the mammalian Uba1 ortholog, UBE1, also results in increased expression of specific growth factor genes. These studies show that a reduction in E1 activity can promote tissue growth in a multicellular organism and raise the possibility that changes in E1 activity may occur during normal development or in cancer (Pfleger, 2007).
During neuronal maturation, dendrites develop from immature neurites into mature arbors. In response to changes in the environment, dendrites from certain mature neurons can undergo large-scale morphologic remodeling. A group of Drosophila peripheral sensory neurons, the class IV dendritic arborization (C4da) neurons completely degrade and regrow their elaborate dendrites. Larval dendrites of C4da neurons are first severed from the soma and subsequently degraded during metamorphosis. This process is controlled by both intracellular and extracellular mechanisms: The ecdysone pathway and ubiquitin-proteasome system (UPS) are cell-intrinsic signals that initiate dendrite breakage, and extracellular matrix metalloproteases are required to degrade the severed dendrites. Surprisingly, C4da neurons retain their axonal projections during concurrent dendrite degradation, despite activated ecdysone and UPS pathways. These results demonstrate that, in response to environmental changes, certain neurons have cell-intrinsic abilities to completely lose their dendrites but keep their axons and subsequently regrow their dendritic arbors (Kuo, 2005).
To visualize abdominal C4da neurons during Drosophila metamorphosis, a pickpocket (ppk)-EGFP reporter line was used. Filleted white pupae (WP), at the onset of metamorphosis, were stained with an anti-EGFP antibody to reveal three C4da neurons, vdaB (V), v'ada (V'), and ddaC (D), in each hemisegment. Because the soma and dendritic projections of these neurons remained very close to the body surface during pupariation, live imaging was used to follow these neurons throughout metamorphosis (Kuo, 2005).
Initially at the WP stage, the C4da neurons exhibited intact, complex class IV dendritic branches that covered much of the pupal surface. Shortly after the white pupal stage, 2 h after puparium formation (APF), fine terminal dendritic branches began to disappear. By 10 h APF, most major dendritic branches were severed from the soma. This severing of dendrites has also been observed in a recent study of da neuron remodeling. During the next 8 h, which coincided with head eversion during metamorphosis, these severed and blebbing dendrites are degraded. By 20 h APF, the process of larval dendrite removal is complete, leaving C4da neurons with their axonal projections but devoid of larval dendrites. Axons from all three C4da neurons project into the VNC. By this time, V' and D neurons begin to extend fine dendritic projections. The V neurons, which do not show new dendritic projections, disappear between 30 and 35 h APF, leaving V' as the surviving neuron in the ventral hemisegment (Kuo, 2005).
Compared with the rapid sequence of larval dendritic pruning, the process of pupal dendrite regrowth is slow. By 70 h APF, both V' and D neurons begin to take on the shape of their respective adult neurons. By 95 h APF, shortly before adult eclosion, the dendritic patterns of abdominal V' neurons closely resemble larval C4da neurons before pupariation. In contrast, the D neurons take on a more elongated dendritic field, perhaps reflecting a functional divergence between these two neurons in the adult fly. These results show that C4da neurons can completely degrade their elaborate larval dendrites during early metamorphosis, survive these changes, and subsequently regrow their dendritic arbors (Kuo, 2005).
During Drosophila metamorphosis, most larval organs are replaced by adult structures. To understand the cellular environment during C4da dendrite degradation, the expression of Armadillo, an adhesive junction protein that outlines the epithelial monolayer during early metamorphosis, was examined. High-level Armadillo staining at the WP stage is completely abolished by 13 h APF but subsequently returns at 20 h APF when the pupal epithelium is reformed. Thus, the pruning of C4da neuron dendrites occurs concurrently with epithelial remodeling during metamorphosis. To determine whether the degradation of larval dendrites is a result of local tissue remodeling or neuron-intrinsic signaling, focus was placed on enzymes that are important for tissue remodeling (Kuo, 2005).
Drosophila matrix metalloproteases (metalloproteinases) Mmp1 and Mmp2 regulate tissue remodeling during metamorphosis. The weaker alleles of both genes, Mmp1Q273 and Mmp2W307, survive past head eversion to midpupariation, making it possible to visualize dendritic pruning of ppk-EGFP-expressing C4da neurons. Remarkably, there were abundant C4da neuron larval dendrites in both Mmp1 and Mmp2 mutants after head eversion. Whereas in WT pupae at 20 h APF all larval dendrites from C4da neurons were cleared from the extracellular space, in both Mmp1 and Mmp2 mutants, larval dendrites that are severed from the soma remain. These larval dendrites persist to much later stages at 50 and 35 h APF, just before the lethal phases of Mmp1Q273 and Mmp2W307 mutants, respectively. The ineffective removal of larval dendrites in Mmp mutants is not caused by a generalized delay in metamorphosis because Mmp mutant pupae had completed head eversion and epidermal remodeling, thus indicating a specific defect in dendrite degradation. Because Mmp1;Mmp2 double mutants do not survive to pupariation, it was not possible to look at dendritic pruning in the double mutant background (Kuo, 2005).
To determine whether Mmps functions on the cell surface of dendrites to regulate degradation, an Mmp inhibitor was expressed in C4da neurons to see whether the survival of larval dendrites can be prolonged. Using the ppk promoter to express transcriptional activator Gal4 (ppk-Gal4), the UAS-Gal4 system was used to express the Drosophila tissue inhibitor of metalloproteases (TIMP) in C4da neurons. Fly TIMP is closely related to mammalian TIMP-3, which associates with extracellular membrane surfaces to modulate Mmp activities. In control animals expressing GFP at 20 h APF, identical pruning of larval dendrites as in ppk-EGFP flies was seen. In contrast, when TIMP is overexpressed in C4da neurons, larval dendrites remain in the extracellular space at 20 h APF (Kuo, 2005).
The fact that TIMP inhibition can successfully delay the degradation of larval dendrites confirms Mmp's involvement in this process. But these enzymes could be synthesized by either the C4da neurons or by the surrounding cells. To identify the source of this Mmp activity, MARCM studies were performed to generate C4da clones that in both Mmps. Mmp1Q112Mmp2W307 double mutant C4da clones not only show dendritic branching patterns similar to WT clones during early pupariation, but live time-lapse imaging revealed complete larval dendrite removal after head eversion at 20 h APF, just like WT controls. These results show that cell-intrinsic Mmps are not required for dendritic pruning and that extracellular Mmp activity is sufficient for degrading severed larval dendrites during metamorphosis. A possible source of this extracellular activity could be phagocytic blood cells, because they have been shown to engulf dendritic debris during metamorphosis (Kuo, 2005).
Whereas removal of severed dendrites requires extrinsic metalloproteases, C4da neurons in Mmp mutants still retain their ability to sever larval dendrites from the soma during metamorphosis. To look for cell-intrinsic pathways in cleaving larval dendrites, the role of ecdysone, a steroid hormone that regulates much of Drosophila metamorphosis, was examined. Binding of ecdysone to its nuclear receptor heterodimers, consisting of Ultraspiracle (Usp) and one of three EcR isoforms (EcR-A, EcR-B1, and EcR-B2), mediates a transcriptional hierarchy that regulates tissue responses during metamorphosis. To determine whether EcR signaling plays a role in initiating dendritic pruning, EcR expression patterns were examined in the ppk-EGFP transgenic line that specifically labels C4da neurons (Kuo, 2005).
Staining with the EcR-C antibody, which recognizes the common regions of EcR family members, and staining with EcR-A and EcR-B1 specific antibodies during different stages of late larval through early pupal development, revealed that all three C4da neurons exhibit similar staining patterns. In third-instar larvae, when the ecdysone level is low before the onset of pupariation, EcR expression in C4da neurons is relatively low when compared with surrounding cells that already exhibit a high level of nuclear EcR. At the WP stage, with a transient rise in ecdysone level, EcR in C4da neurons becomes concentrated in the nucleus. Over the next 7 h, EcR gradually redistributes throughout the soma of C4da neurons, which corresponds to a rapid drop-off in ecdysone levels in the pupae after initiation of metamorphosis. Strong nuclear EcR localization in C4da neurons returns at 20 h APF, correlating with the onset of midpupal ecdysone release. Antibodies specific to either EcR-A or B1 show that whereas EcR-A expression is diffuse and weak throughout metamorphosis, EcR-B1 expression in C4da neurons corresponds to the dynamic nuclear localization patterns seen with the EcR-C antibody (Kuo, 2005).
To examine the functional significance of EcR expression, attempts were made to disrupt ecdysone signaling specifically in C4da neurons. EcR mutants either do not survive to the pupal stage or die shortly after the onset of metamorphosis; therefore, it is not possible to look at dendritic remodeling in those mutants. The cytological location of EcR genes also precludes MARCM studies; therefore, use was made of a set of dominant-negative (DN) EcR proteins to inhibit EcR activity. When ecdysone signaling is inhibited by EcR-DN proteins, C4da neurons lose their ability to initiate larval dendrite pruning at 20 h APF. To determine whether the defects are specific to the ecdysone signaling pathway, attempts were made to rescue the EcR-DN phenotype. Coexpression of both EcR-DN and WT EcR-B1 proteins in C4da neurons resulted in complete rescue of dendritic pruning defects in all three neurons. This rescue is complete with two copies of ppk-Gal4 in C4da neurons, showing that the rescue is not caused by reduced expression of DN protein in the coexpression experiments (Kuo, 2005).
Because dimerization of EcR-B1 with its obligatory hormone receptor partner Usp is essential for transcriptional regulation, the involvement of Usp in dendrite remodeling was examined. Usp mutants do not survive to metamorphosis; however, it was possible to generate Usp MARCM clones for analysis. At 20 h APF, Usp mutant C4da clones fail to prune their larval dendrites, and this genetic mutation shows an identical phenotype to the EcR-DN experiments. Given the severity and full penetrance of this phenotype, together with the timing of EcR-B1 nuclear localization, it is concluded that the ecdysone signaling pathway plays an important cell-intrinsic role in initiating dendritic pruning in C4da neurons during metamorphosis (Kuo, 2005).
What might be the cellular machineries that carry out dendrite pruning in C4da neurons? One attractive model is a caspase-mediated local digestion and degradation of dendrites. However, overexpression of p35, an effective inhibitor of fly caspases, in C4da neurons did not prevent or delay larval dendrite degradation during metamorphosis. Another protein degradation pathway, the ubiquitin protease system (UPS), has been shown to regulate both axon and dendrite pruning of mushroom body neurons during fly metamorphosis. To test the involvement of UPS in C4da neuron remodeling, use was made of ppk-Gal4 to overexpress UBP2, a yeast ubiquitin protease, in the C4da neurons. By reversing the process of substrate ubiquination, UBP2 is an effective UPS inhibitor in the fly. Some of the C4da neurons expressing UBP2 aberrantly retained their larval dendritic arbors. Note that this pruning defect is very different from that seen in Mmp mutants. Whereas Mmp mutants accumulated severed larval dendrites in the extracellular space, UBP2 inhibition prevented efficient severing of dendrites from the soma (Kuo, 2005).
To further examine the involvement of the UPS machinery in dendritic pruning, the MARCM system was used to generate C4da clones that were either deficient in ubiquitin activation enzyme 1 (Uba1) or had mutation in the 19S particle of the proteasome (Mov34). Time-lapse imaging of Uba1 and Mov34 mutant C4da clones at WP stage and 20 h APF showed that, unlike WT clones, both mutant clones failed to efficiently sever their larval dendrites during metamorphosis. These results confirmed the requirement for an activated UPS in the severing of larval dendrites from C4da neurons during metamorphosis (Kuo, 2005).
To compare the defects in larval dendritic pruning caused by different mutations, the number of large (primary and secondary) dendritic branches that remain attached to C4da neuron soma was counted. In WT pupae at the start of metamorphosis, C4da neurons extended close to 20 large dendritic branches, none of which was retained after head eversion at 20 h APF. Mutations that disrupt ecdysone signaling, such as EcR-DN expression or Usp-deficient clones, result in the retention of 85%-90% of large dendritic branches after head eversion. Mutations in the UPS pathway, such as Uba1 and Mov34, resulted in the retention of 45%-49% of large dendritic branches at 20 h APF. Mmp1 or Mmp2 mutants retained only 3%-8% of large dendritic branches after head eversion, and Mmp1;Mmp2 mutant clones did not retain larval dendrites at 20 h APF. These data suggest that dendrite remodeling in C4da neurons starts with signals from ecdysone and UPS that result in the cleavage of larval dendrites from the soma, which then allows for the degradation of severed dendrites by Mmp activity in the extracellular matrix (Kuo, 2005).
It is possible that UPS is an upstream regulator of EcR and can lead to EcR misexpression in UPS mutants; however, normal EcR expression patterns are observed in both Uba1 and Mov34 C4da MARCM clones. It is also conceivable that EcR signaling is upstream of the UPS cascade, but this idea is difficult to demonstrate experimentally. It was reasoned that in this case, inhibition of EcR signaling should result in lower levels of protein ubiquination. However, staining in EcR-DN-expressing C4da neurons showed no significant differences in the level of ubiquitin/polyubiquitin between undegraded larval dendrites and WT dendrites before degradation. This finding does not rule out an EcR function upstream of UPS during dendritic remodeling, because EcR signaling may regulate critical factors in the UPS cascade after protein ubiquination at the level of ubiquitin ligases. The identities of such ligases are currently unknown (Kuo, 2005).
To test whether dendritic pruning in C4da neurons involves concurrent axonal remodeling, axon tracks of C4da neurons were examined in the Drosophila VNC during early metamorphosis. Direct live imaging of the ppk-EGFP transgenic line at the WP stage showed axon tracks from three C4da neurons. Axon tracing of EGFP-expressing C4da neurons at 6 h APF showed continuous axon tracks between all three C4da neurons and the VNC. At 10 h APF, the VNC appeared more compact, presumably as a result of the various remodeling events that occur in the nervous system during metamorphosis. At 20 h APF, axon tracks of EGFP-expressing C4da neurons can still be clearly identified at the VNC and are continuous with the soma, despite the complete removal of dendritic arbors of these same neurons (Kuo, 2005).
Drosophila peripheral sensory neurons generally have simple axon projections into the VNC that terminate locally. To visualize C4da neuron axon terminals during metamorphosis, the UAS>CD2>CD8-GFP system was used, together with ppk-Gal4, to generate single clones of surviving V' and D neurons. The V' C4da neuron was found to project its axon ipsilaterally upon entering the VNC to the segment immediately anterior during the WP stage. At 20 h APF, after complete pruning of larval dendrites, the V' C4da neuron keeps this axonal projection intact in the VNC. The D C4da neuron axon, in addition to having an ipsilateral branch that projects to the anterior segment, sends a commissural branch that crosses the midline at the segment where the axon enters the VNC. Likewise, at 20 h APF, the D C4da neuron keeps both axonal terminal branches intact. These data show that C4da neurons do not significantly modify their larval axons during concurrent dendrite degradation, despite the activated ecdysone and UPS pathways, which are known to facilitate axon remodeling and degradation (Kuo, 2005).
What might account for the dendrite-specific remodeling in C4da neurons, as opposed to the previously reported concurrent remodeling of both axons and dendrites? It is possible that local environments may play a role. A recent study in Manduca found central versus peripheral hormonal differences affecting axon versus dendrite remodeling. However, it remains to be tested whether the ecdysone levels are different in the fly epidermis and the VNC during metamorphosis. Anatomically, C4da neurons have distinct axon-dendrite polarity in that the cell bodies send out multiple primary dendritic arbors to the surrounding environment while each extends a single axon toward the VNC. This morphology is in contrast to most insect neurons, such as femoral depressor motoneurons and mushroom body gamma-neurons, which extend a single branch from the soma that later gives rise to both dendrites and axons. As such, C4da neurons may have developed separate mechanisms at the soma to remodel just the dendrites. Just what these mechanisms might include is currently unknown (Kuo, 2005).
This study has provided evidence that certain mature neurons have the ability to selectively degrade their dendritic projections in vivo and regrow new ones. Although fly metamorphosis is a specialized developmental process, dendrite-specific remodeling may provide a paradigm for neurons to retain part of their connections in the neuronal circuitry while responding to environmental changes such as tissue degeneration near their dendrites. Certain conditions in mammalian systems, such as trauma and injury, can induce localized degeneration and remodeling and may mimic the active tissue remodeling during metamorphosis. In the human CNS, for example, significant reorganization of granule cell projections in the dentate gyrus after human temporal lobe epilepsy has been observed. Thus, it would be of great interest to examine whether dendritic-specific remodeling of C4da neurons in Drosophila represents an evolutionarily conserved mechanism for neurons to respond to drastic changes in their environment, and to determine whether mammalian neurons have similar capacities to remodel their dendrites (Kuo, 2005).
Axon pruning is widely used for the refinement of neural circuits in both vertebrates and invertebrates, and may also contribute to the pathogenesis of neurodegenerative diseases. However, little is known about the cellular and molecular mechanisms of axon pruning. This study used the stereotyped pruning of gamma neurons of the Drosophila mushroom bodies (MB) during metamorphosis to investigate these mechanisms. Detailed time course analyses indicate that MB axon pruning is mediated by local degeneration rather than retraction and that the disruption of the microtubule cytoskeleton precedes axon pruning. In addition, multiple lines of genetic evidence demonstrate an intrinsic role of the ubiquitin-proteasome system in axon pruning; for example, loss-of-function mutations of the ubiquitin activating enzyme (E1) or proteasome subunits in MB neurons block axon pruning. These findings suggest that some forms of axon pruning during development may share similarities with degeneration of axons in response to injury (Watts, 2003).
Search PubMed for articles about Drosophila Uba1
Chai, J., Yan, N., Huh, J. R., Wu, J. W., Li, W., Hay, B. A. and Shi, Y. (2003). Molecular mechanism of Reaper-Grim-Hid-mediated suppression of DIAP1-dependent Dronc ubiquitination. Nat Struct Biol 10: 892-898. PubMed ID: 14517550
Chang, T. K., Shravage, B. V., Hayes, S. D., Powers, C. M., Simin, R. T., Wade Harper, J. and Baehrecke, E. H. (2013). Uba1 functions in Atg7- and Atg3-independent autophagy. Nat Cell Biol 15: 1067-1078. PubMed ID: 23873149
Colin, J., Garibal, J., Clavier, A., Rincheval-Arnold, A., Gaumer, S., Mignotte, B. and Guenal, I. (2014). The Drosophila Bcl-2 family protein Debcl is targeted to the proteasome by the beta-TrCP homologue Slimb. Apoptosis 19: 1444-1456. PubMed ID: 25208640
Denton, D., Shravage, B., Simin, R., Mills, K., Berry, D. L., Baehrecke, E. H. and Kumar, S. (2009). Autophagy, not apoptosis, is essential for midgut cell death in Drosophila. Curr Biol 19: 1741-1746. PubMed ID: 19818615
Ghaboosi, N. and Deshaies, R. J. (2007). A conditional yeast E1 mutant blocks the ubiquitin-proteasome pathway and reveals a role for ubiquitin conjugates in targeting Rad23 to the proteasome. Mol Biol Cell 18: 1953-1963. PubMed ID: 17360968
Haas, A. L. and Siepmann, T. J. (1997). Pathways of ubiquitin conjugation. FASEB J 11: 1257-1268. PubMed ID: 9409544
Itakura, E. and Mizushima, N. (2011). p62 Targeting to the autophagosome formation site requires self-oligomerization but not LC3 binding. J Cell Biol 192: 17-27. PubMed ID: 21220506
Johansen, T. and Lamark, T. (2011). Selective autophagy mediated by autophagic adapter proteins. Autophagy 7: 279-296. PubMed ID: 21189453
Jura, N., Scotto-Lavino, E., Sobczyk, A. and Bar-Sagi, D. (2006). Differential modification of Ras proteins by ubiquitination. Mol Cell 21: 679-687. PubMed ID: 16507365
Kuo, C. T., Jan, L. Y. and Jan, Y. N. (2005). Dendrite-specific remodeling of Drosophila sensory neurons requires matrix metalloproteases, ubiquitin-proteasome, and ecdysone signaling. Proc. Natl. Acad. Sci. 102(42): 15230-5. 16210248
Kuo, C. T., Zhu, S., Younger, S., Jan, L. Y. and Jan, Y. N. (2006). Identification of E2/E3 ubiquitinating enzymes and caspase activity regulating Drosophila sensory neuron dendrite pruning. Neuron 51: 283-290. PubMed ID: 16880123
Lee, C. Y., Cooksey, B. A. and Baehrecke, E. H. (2002). Steroid regulation of midgut cell death during Drosophila development. Dev Biol 250: 101-111. PubMed ID: 12297099
Lee, T. V., Ding, T., Chen, Z., Rajendran, V., Scherr, H., Lackey, M., Bolduc, C. and Bergmann, A. (2008). The E1 ubiquitin-activating enzyme Uba1 in Drosophila controls apoptosis autonomously and tissue growth non-autonomously. Development 135: 43-52. PubMed ID: 18045837
Lenk, S. E., Dunn, W. A., Jr., Trausch, J. S., Ciechanover, A. and Schwartz, A. L. (1992). Ubiquitin-activating enzyme, E1, is associated with maturation of autophagic vacuoles. J Cell Biol 118: 301-308. PubMed ID: 1321157
Li, S., Chen, Y., Shi, Q., Yue, T., Wang, B. and Jiang, J. (2012). Hedgehog-regulated ubiquitination controls smoothened trafficking and cell surface expression in Drosophila. PLoS Biol 10: e1001239. PubMed ID: 22253574
Liu, H. Y. and Pfleger, C. M. (2013). Mutation in E1, the ubiquitin activating enzyme, reduces Drosophila lifespan and results in motor impairment. PLoS One 8: e32835. PubMed ID: 23382794
Micchelli, C. A., Sudmeier, L., Perrimon, N., Tang, S. and Beehler-Evans, R. (2011). Identification of adult midgut precursors in Drosophila. Gene Expr Patterns 11: 12-21. PubMed ID: 20804858
Nishida, Y., Arakawa, S., Fujitani, K., Yamaguchi, H., Mizuta, T., Kanaseki, T., Komatsu, M., Otsu, K., Tsujimoto, Y. and Shimizu, S. (2009). Discovery of Atg5/Atg7-independent alternative macroautophagy. Nature 461: 654-658. PubMed ID: 19794493
Pfleger, C. M., Harvey, K. F., Yan, H. and Hariharan, I. K. (2007). Mutation of the gene encoding the ubiquitin activating enzyme ubal causes tissue overgrowth in Drosophila. Fly (Austin) 1: 95-105. PubMed ID: 18820468
Pickart, C. M. (2001). Mechanisms underlying ubiquitination. Annu Rev Biochem 70: 503-533. PubMed ID: 11395416
Taillebourg, E., Gregoire, I., Viargues, P., Jacomin, A. C., Thevenon, D., Faure, M. and Fauvarque, M. O. (2012). The deubiquitinating enzyme USP36 controls selective autophagy activation by ubiquitinated proteins. Autophagy 8: 767-779. PubMed ID: 22622177
Watts, R. J., Hoopfer, E. D. and Luo, L. (2003). Axon pruning during Drosophila metamorphosis: evidence for local degeneration and requirement of the ubiquitin-proteasome system. Neuron 38: 871-885. PubMed ID: 12818174
Wilson, R., Goyal, L., Ditzel, M., Zachariou, A., Baker, D. A., Agapite, J., Steller, H. and Meier, P. (2002). The DIAP1 RING finger mediates ubiquitination of Dronc and is indispensable for regulating apoptosis. Nat Cell Biol 4: 445-450. PubMed ID: 12021771
Yan, H., Chin, M. L., Horvath, E. A., Kane, E. A. and Pfleger, C. M. (2009). Impairment of ubiquitylation by mutation in Drosophila E1 promotes both cell-autonomous and non-cell-autonomous Ras-ERK activation in vivo. J Cell Sci 122: 1461-1470. PubMed ID: 19366732
date revised: 20 November 2014
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