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Targeted expression of dominant negative proteasome mutants in Drosophila melanogaster
... Degradation by the proteasome could be a means to regulate Pk levels. We examined this hypothesis by first co-expressing a dominant negative (DN) form of the proteasome 20S β2 subunit, Prosbeta2 [42][43][44] along with Pk under sev-Gal4 control and analyzing the adult phenotype. DNProsbeta2 expression synergized with the Pk GOF phenotype to enhance chirality defects, particularly symmetrical clusters (Fig 5C, 5D and 5G). ...
... DNProsbeta2 expression synergized with the Pk GOF phenotype to enhance chirality defects, particularly symmetrical clusters (Fig 5C, 5D and 5G). In a complementary approach, we used the milder act-EGFP-Pk phenotype and examined the effect of another DN proteasome component-this time β6 (Prosbeta6) [42][43][44], under the control of GMR-Gal4, which is expressed in all post-mitotic, differentiating cells in the eye. We again saw an increase in Only the genotype of the R3-R4 pair was scored in pairs in which R3 and R4 were of different genotypes. ...
... sevGal4, UAS-Pk; sevGal4, UAS-Sple [14,28]; sev-Sple-WT [14,28]; UAS-Nmo, [24], nubGal4 [25]. UAS-DNProsβ2 and UAS-DNProsβ6 (also called Dts5) [42,56] flies were as described (see refs above). ...
Planar cell polarity (PCP) instructs tissue patterning in a wide range of organisms from fruit flies to humans. PCP signaling coordinates cell behavior across tissues and is integrated by cells to couple cell fate identity with position in a developing tissue. In the fly eye, PCP signaling is required for the specification of R3 and R4 photoreceptors based upon their positioning relative to the dorso-ventral axis. The ‘core’ PCP pathway involves the asymmetric localization of two distinct membrane-bound complexes, one containing Frizzled (Fz, required in R3) and the other Van Gogh (Vang, required in R4). Inhibitory interactions between the cytosolic components of each complex reinforce asymmetric localization. Prickle (Pk) and Spiny-legs (Pk-Sple) are two antagonistic isoforms of the prickle (pk) gene and are cytoplasmic components of the Vang complex. The balance between their levels is critical for tissue patterning, with Pk-Sple being the major functional isoform in the eye. Here we uncover a post-translational role for Nemo kinase in limiting the amount of the minor isoform Pk. We identified Pk as a Nemo substrate in a genome-wide in vitro band-shift screen. In vivo, nemo genetically interacts with pkpk but not pksple and enhances PCP defects in the eye and leg. Nemo phosphorylation limits Pk levels and is required specifically in the R4 photoreceptor like the major isoform, Pk-Sple. Genetic interaction and biochemical data suggest that Nemo phosphorylation of Pk leads to its proteasomal degradation via the Cullin1/SkpA/Slmb complex. dTAK and Homeodomain interacting protein kinase (Hipk) may also act together with Nemo to target Pk for degradation, consistent with similar observations in mammalian studies. Our results therefore demonstrate a mechanism to maintain low levels of the minor Pk isoform, allowing PCP complexes to form correctly and specify cell fate.
... The following strains were used in this study: Canton-S (wild-type), hiw ND8 (Wan et al., 2000), hiw ΔN , UAS-hiw and UAS-hiw-ΔRING from (Wu et al., 2005), OK6-Gal4 (McCabe et al., 2004), BG380-Gal4 (Budnik et al., 1996) m12-Gal4 (P(GAL4) 5053A ) (Ritzenthaler et al., 2000), ppk-Gal4 (Kuo et al., 2005), Or47b-Gal4 (Vosshall et al., 2000), UAS-UBP2 (DiAntonio et al., 2001), UAS-DTS5, and UAS-DTS7 from (Belote and Fortier, 2002), wnd 1 , wnd 3 , and UAS-wnd from . UAS-HA::nmnat (Zhai et al., 2006), UAS-WldS (Hoopfer et al., 2006), were acquired from the Vienna RNAi center (Dietzl et al., 2007). ...
... Inhibition of the proteasome by addition of MG132, using several different concentrations and periods of time that affect known targets to the UPS (Materials and Methods), did not affect the down-regulation of Nmnat by Hiw in S2R+ cells ( Figure 4.13.A). To inhibit the proteasome in vivo we co-expressed dominant-negative proteasome subunit mutations, DTS5 and DTS7, which in previous studies had been shown to lead to allow targets of the UPS to accumulate (Belote and Fortier, 2002;Pandey et al., 2007;Speese et al., 2003). This led to only minor (7%) changes in the levels of HA-Nmnat in sensory neuron terminals (Figure 4.13.B). ...
... (C) Hiw and the UPS may influence Nmnat levels cooperatively. Total protein from third instar larval brains or young adult heads processed for Western blot from animals coexpressing DTS5 and DTS7 (Belote and Fortier, 2002) to inhibit the proteasome (raised at 25 °C), and compared to animals co-expressing two control UAS-PKC transgenes. Relative levels of HA-Nmnat protein, compared to the β-catenin standard, were measured on the LiCor Odyssey system. ...
Axons allow neurons to communicate over long distances, and their long length makes them vulnerable to injury. Neurons make several important responses to axonal injury. One is that the axons that are still connected to the cell body can initiate new axonal growth. This requires transcriptional reprogramming, and an important question is how this nuclear event can be induced by an injury in a distal site of the axon. Another response is that the distal stump, which is disconnected from the cell body, degenerates through a process called Wallerian degeneration. This self-destruction process is poorly understood, but is misregulated in some neurodegenerative diseases. To study these responses in a model organism, I developed an axonal injury assay in Drosophila. I have characterized the function an axonal signaling pathway, which is regulated by the ubiquitin ligase Hiw, and its conserved axonal target, Wnd. I found that Hiw and Wnd, through a downstream signaling cascade, regulate several important responses to axonal injury. First, the Hiw/Wnd signaling pathway is activated by axonal injury and mediates the transcriptional changes that facilitate new axonal growth from the proximal stump. Second, this signaling pathway also inhibits Wallerian degeneration, allowing for neurons that have been injured once to have increased resilience to a second injury. This demonstrates that Hiw and Wnd control remarkable plasticity in the axonal degeneration process. Third, Hiw performs an additional role in degeneration by regulating the levels and localization of NMNAT. This NAD+ synthesizing enzyme plays an essential role in dramatic inhibition to degeneration when hiw is mutant. Since the Hiw/DLK pathway is highly conserved, these findings may ultimately be of clinical interest for understanding and treating nerve damage in humans.
... Male flies, 2-4 days post-eclosion were used for all experiments. Transgenic UAS lines expressing the "degron" CL1 (Pandey et al., 2007) and the dominant negative proteasomal subunits Pros26 and Prosβ2 (Belote et al., 2002) were gifts of Paul Taylor (U. Penn) and John Belote (Syracuse U.), respectively. ...
... Conjugation to ubiquitin can have multiple effects including targeting of proteins to the proteasome or autophagosome, regulation of endocytosis, maintenance of synaptic function and alterations in mitochondrial dynamics (Chin et al., 2010, DiAntonio et al., 2004, Schapira, 2011, Schmidt et al., 2014. To test whether proteasome inhibition would mimic the neurotoxic effects of E1 ligase inhibition, we used two previously characterized dominant-negative proteasome subunits, Pros26 and Prosβ2 (Belote et al., 2002). Both constructs are temperature sensitive and show inhibitory effects at the non-permissive temperature (30°C) but not at lower temperatures (e.g. ...
... Both constructs are temperature sensitive and show inhibitory effects at the non-permissive temperature (30°C) but not at lower temperatures (e.g. 18°C) (Belote et al., 2002). Flies constitutively expressing one copy of Ddc-Gal4 and each of the Pros subunits consistently died as 3rd instar larva (not shown). ...
... Male flies, 2-4 days post-eclosion were used for all experiments. Transgenic UAS lines expressing the ''degron'' CL1 (Pandey et al., 2007) and the dominant negative proteasomal subunits Pros26 and Prosb2 (Belote and Fortier, 2002) were gifts of Paul Taylor (U. Penn) and John Belote (Syracuse U.), respectively. ...
... Conjugation to ubiquitin can have multiple effects including targeting of proteins to the proteasome or autophagosome, regulation of endocytosis, maintenance of synaptic function and alterations in mitochondrial dynamics (Chin et al., 2010;DiAntonio and Hicke, 2004;Schapira, 2011;Schmidt and Finley, 2014). To test whether proteasome inhibition would mimic the neurotoxic effects of E1 ligase inhibition, we used two previously characterized dominant-negative proteasome subunits, Pros26 and Prosb2 (Belote and Fortier, 2002). Both constructs are temperature sensitive and show inhibitory effects at the nonpermissive temperature (30 8C) but not at lower temperatures (e.g. ...
... Both constructs are temperature sensitive and show inhibitory effects at the nonpermissive temperature (30 8C) but not at lower temperatures (e.g. 18 8C) (Belote and Fortier, 2002). Flies constitutively expressing one copy of Ddc-Gal4 and each of the Pros subunits consistently died as 3rd instar larva (not shown). ...
The model genetic organism Drosophila melanogaster, commonly known as the fruit fly, uses many of the same neurotransmitters as mammals and very similar mechanisms of neurotransmitter storage, release and recycling. This system offers a variety of powerful molecular-genetic methods for the study of transporters, many of which would be difficult in mammalian models. We review here progress made using Drosophila to understand the function and regulation of neurotransmitter transporters and discuss future directions for its use.
... The UAS-127Q, UAS-MJDtr-Q78(S), UAS-httex1p Q93, UAS-hsrv-RNAi 2 , UAS-hsrv-RNAi 3 , EP93D, and EP3037 transgenic lines have been described previously (Mallik and Lakhotia 2009a). The GMR-GAL4 (Hay et al. 1994), P{Act5C-GAL4}25F01/CyO (Ekengren et al. 2001), and UAS-Pros26 1 .B; UAS-Prosb2 1 (Belote and Fortier 2002) stocks were obtained from the Bloomington Stock Center. UAS-CBP FL-AD (acetylase dead version, F2161A), UAS-CBP RNAi, UAS-CBP DNZK, UAS-CBP DQ, and UAS-CBP DBHQ, all homozygous viable stocks, were provided by J. P. Kumar (Kumar et al. 2004;Anderson et al. 2005). ...
... In the first approach, we used the UAS-Pros26 1 .B and UAS-Prosb2 1 transgenes that carry temperature-sensitive missense mutations in the 20S proteasome subunits b6 and b2, respectively (Belote and Fortier 2002). Likewise, immunoprecipitation with CBP also pulled down Hrb57A from GMR-GAL4/GMR-GAL4; hsrv-RNAi 3 /hsrv-RNAi 3 eye discs (lane 4). ...
... Although these temperature-sensitive mutant alleles have maximum effect at 29° (Belote and Fortier 2002), we reared the larvae and flies carrying these transgenes at 25°since expression of the mutant proteasome subunits even at 25°affects eye structure but the additional poly(Q) toxicity due to elevated temperature is avoided. ...
... Inhibition of the proteasome by addition of MG132, using several different concentrations and periods of time that affect known targets to the UPS (Materials and Methods) [43,44], did not affect the down-regulation of Nmnat by Hiw in S2R+ cells (Figure S4A). To inhibit the proteasome in vivo we co-expressed dominant-negative proteasome subunit mutations , DTS5 and DTS7, which in previous studies had been shown to lead to allow targets of the UPS to accumulate454647. This led to only minor (7%) changes in the levels of HA-Nmnat in sensory neuron terminals (Figure S4B). ...
... The following strains were used in this study: Canton-S (wildtype ), hiw ND8 [8], hiw DN , UAS-hiw and UAS-hiw-DRING from [67], OK6-Gal4 [68], BG380-Gal4 [69] m12-Gal4 (P(GAL4) 5053A ) [70], ppk-Gal4 [71], Or47b-Gal4 [72], UAS-UBP2 [41], UAS-DTS5, and UAS-DTS7 from [45], wnd 1 , wnd 3 , and UAS-wnd from [16] . UAS- HA::nmnat [25], UAS-WldS [2] , UAS-mNmnat1::myc, UAS-mNmnat2::- myc, and UAS-mNmnat3::myc [51,52], and UAS-Dcr2 were gifts from Grace Zhai, Liqun Luo, Marc Freeman, and Stephan Thor. ...
... (C) Hiw and the UPS may influence Nmnat levels cooperatively. Total protein from third instar larval brains or young adult heads processed for Western blot from animals coexpressing DTS5 and DTS7 [45] to inhibit the proteasome (raised at 25uC), and compared to animals co-expressing two control UAS-PKC transgenes. Relative levels of HA-Nmnat protein, compared to the b-catenin standard, were measured on the LiCor Odyssey system. ...
Axonal degeneration is a hallmark of many neuropathies, neurodegenerative diseases, and injuries. Here, using a Drosophila injury model, we have identified a highly conserved E3 ubiquitin ligase, Highwire (Hiw), as an important regulator of axonal and synaptic degeneration. Mutations in hiw strongly inhibit Wallerian degeneration in multiple neuron types and developmental stages. This new phenotype is mediated by a new downstream target of Hiw: the NAD+ biosynthetic enzyme nicotinamide mononucleotide adenyltransferase (Nmnat), which acts in parallel to a previously known target of Hiw, the Wallenda dileucine zipper kinase (Wnd/DLK) MAPKKK. Hiw promotes a rapid disappearance of Nmnat protein in the distal stump after injury. An increased level of Nmnat protein in hiw mutants is both required and sufficient to inhibit degeneration. Ectopically expressed mouse Nmnat2 is also subject to regulation by Hiw in distal axons and synapses. These findings implicate an important role for endogenous Nmnat and its regulation, via a conserved mechanism, in the initiation of axonal degeneration. Through independent regulation of Wnd/DLK, whose function is required for proximal axons to regenerate, Hiw plays a central role in coordinating both regenerative and degenerative responses to axonal injury. Citation: Xiong X, Hao Y, Sun K, Li J, Li X, et al. (2012) The Highwire Ubiquitin Ligase Promotes Axonal Degeneration by Tuning Levels of Nmnat Protein. PLoS Biol 10(12): e1001440.
... The development of these muscles is highly stereotyped, with each muscle cell achieving a specific orientation, a standard number of nuclei and innervation at NMJs (neuromuscular junctions) of exacting size and placement (Bate, 1990; Abmayr et al., 1995; Beckett and Baylies, 2006). Transgenic expression of mutant proteasome β subunits has been used to specifically inhibit the Drosophila proteasome (Schweisguth, 1999; Belote and Fortier, 2002; Speese et al., 2003; Neuburger et al., 2006). These mutant proteasome subunits can be spatially targeted only to muscles using the GAL4-UAS system (where UAS stands for up-stream activating sequence) (Brand and Perrimon, 1993), and temporally targeted using the conditional GS (GeneSwitch) system (Osterwalder et al., 2001). ...
... The mutant subunits act in a dominant-negative manner to interfere with proteasome function; in many previous studies, these mutant subunits have been shown to inhibit UPS-mediated protein degradation of known proteasome substrates (Schweisguth, 1999; Speese et al., 2003; Neuburger et al., 2006). Transgenic co-expression of both proteasome subunit mutants in the Drosophila eye using the GAL4-UAS system has a synergistic effect in blocking proteasome function (Belote and Fortier, 2002). Both mutant subunits were therefore co-expressed in the musculature in the present study, and this double mutant strain will hence be referred to as DTS. ...
... For the inducible UAS transgene expression to interfere with proteasome function, the GS system was utilized (Osterwalder et al., 2001). The homozygous UAS-DTS5 2B(2), DTS7 1B(3) line [generously provided by Dr John Belote, Department of Biology, Syracuse University, Syracuse NY, U.S.A. (Belote and Fortier, 2002)] was crossed with the homozygous muscle-specific MHC GS-GAL4 line [generously provided by Dr Haig Keshishian, Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, U.S.A. (Osterwalder et al., 2001)] to generate heterozygote GS-MHC-GAL4/UAS-DTS 5,7 flies containing a single copy of the UAS transgene and a single copy of the GAL4 driver. The GS system uses a RU486-dependent-GAL4-progesterone-receptor fusion protein. ...
Background information:
Protein degradation via the UPS (ubiquitin-proteasome system) plays critical roles in muscle metabolism and signalling pathways. The present study investigates temporal requirements of the UPS in muscle using conditional expression of mutant proteasome beta subunits to cause targeted inhibition of proteasome function.
Results and conclusions:
The Drosophila GeneSwitch system was used, with analyses of the well-characterized larval somatic body wall muscles. This method acutely disrupts proteasome function and causes rapid accumulation of polyubiquitinated proteins, specifically within the muscle. Within 12 h of transgenic proteasome inhibition, there was a gross disorganization of muscle architecture and prominent muscle atrophy, progressing to the arrest of all co-ordinated movement by 24 h. Progressive muscle architecture changes include rapid loss of sarcomere organization, loss of nuclei spacing/patterning, vacuole formation and the accumulation of nuclear and cytoplasmic aggregates at the ultrastructural level. At the neuromuscular junction, the highly specialized muscle membrane folds of the subsynaptic reticulum were rapidly lost. Within 24 h after transgenic proteasome inhibition, muscles contained numerous autophagosomes and displayed highly elevated expression of the endoplasmic reticulum chaperone GRP78 (glucose-regulated protein of 78 kDa), indicating that the loss of muscle maintenance correlates with induction of the unfolded protein response. Taken together, these results demonstrate that the UPS is acutely required for maintenance of muscle and neuromuscular junction architecture, and provides a Drosophila genetic model to mechanistically evaluate this requirement.
... RNAi-mediated knockdown of two wellconserved 19S proteasome regulatory particle subunit genes, Mov34 and Rpn6 [37], resulted in significant accumulation of CiFL protein ( Figure 2F; data not shown). Similarly, when the function of 20S proteasome core particle b6 (Pros26) or b2 (Prosbeta2) subunit was disrupted in the dorsal compartment of the wing disc by the expression of dominant negative temperature-sensitive mutants DTS5 or DTS7 [38], CiFL protein was stabilized in more anterior cells ( Figure 2G; data not shown). In contrast, when the essential lysosomal components, car (carnation) [39], dor (deep orange) [40], Hrs (Hepatocyte growth factor regulated tyrosine kinase substrate) or Stam (Signal transducer adaptor molecule) [41], [42], were specifically knocked down by RNAi, little if any effect on CiFL stability was observed ( Figure 2H; data not shown). ...
... For Figure 2E and F, UAS-ubp (gift of Liqun Luo) [34], UAS-Mov34 RNAi (V26183) or UAS-Rpn6 RNAi (V18021) was crossed with MS1096-Gal4 at 18uC. For Figure 2G, UAS-DTS5 or UAS-DTS7 [38] was crossed with MS1096-Gal4 at 29uC. For Figure 2H Figure 4E-H, UAS-CG13343 RNAi lines were crossed with ap-Gal4, UAS-mCD8-gfp at 29uC. ...
... The 19S regulatory particle is composed of at least nineteen subunits involved in recognition of ubiquitin-conjugated substrates, ATP hydrolysis, de-ubiquitination, protein unfolding and feeding of the substrates into the 20S catalytic core for degradation [355]. Genetic studies in yeast and Drosophila have revealed that mutations in many subunits of the 20S core and the 19S regulatory domains impair proteasome function [358,359]. Genetic analysis of proteasome function is also of clinical importance as proteasome inhibition may be used as potential anti-tumor strategy, especially for treatment of multiple myeloma [360][361][362][363]. ...
... This is the first report in which a clonal analysis of proteasome mutants was performed. Usually, dominant temperature sensitive (DTS) alleles of proteasome subunits (DTS5, DTS7, etc.), RNAi or pharmacological inhibition have been used to study proteasome function [358,372,377,390,395,402]. In other approaches, whole embryos mutant for proteasome subunits were characterized for defects in dendrite pruning in sensory neurons in Drosophila [193,403,404]. ...
Apoptosis is a programmed cell death mechanism that is evolutionary conserved from worms to humans. Apoptosis is mediated by initiator and effector caspases. The initiator caspases carry long pro-domains for their interaction with scaffolding proteins to form a cell-death platform, which is essential for their activation. Activated initiator caspases then cleave effector caspases that execute cell death through cleaving downstream targets. In addition to their apoptotic function, caspases also participate in events where caspase activity is not required for cell killing, but for regulating other functions, so-called non-apoptotic functions of caspases. The Drosophila initiator caspase Dronc, the ortholog of mammalian caspase-2 and caspase-9 has a CARD domain that is essential for its interaction with the scaffolding protein Dark to form the apoptosome. Apoptosome formation is crucial for activation of Dronc. Activity of both initiator and effector caspases are further kept in control by the ubiquitin system to avoid inappropriate caspase activity. However, mechanistic details of how the ubiquitin system regulates activation of Dronc are not clear. Therefore, I investigated the ubiquitylation status of Dronc and its function in Drosophila. I found that Dronc is mono-ubiquitylated at Lys78 (K78) in its CARD domain, which blocks its interaction with Dark and formation of the apoptosome. Furthermore, I demonstrated that K78 mono-ubiquitylation plays an inhibitory role in Dronc’s non-apoptotic functions, which may not require its catalytic activity but may be important for the survival of the fly. This thesis study unveils the link between the ubiquitin system and caspases through a regulatory mechanism where a single mono-ubiquitylation event could inhibit both apoptotic and non-apoptotic functions of a caspase.
... 5 Genetic studies in yeast and Drosophila have revealed that mutations in many subunits of the 20S core and the 19S regulatory domains impair proteasome function. 8,9 Genetic analysis of proteasome function is also of clinical importance as proteasome inhibition may be used as potential antitumor strategy, especially for treatment of multiple myeloma. [10][11][12][13] Autophagy is characterized by the formation of doublemembrane vesicles termed autophagosomes. ...
... Usually, dominant temperature sensitive (DTS) alleles of proteasome subunits (DTS5, DTS7, etc.), RNAi or pharmacological inhibition have been used to study proteasome function. 8,22,27,51,57,64 In other approaches, whole embryos mutant for proteasome subunits were characterized for defects in dendrite pruning in sensory neurons in Drosophila. [65][66][67] However, a specific analysis characterizing recessive alleles for defects in proteasome activity has not been reported. ...
A major function of ubiquitylation is to deliver target proteins to the proteasome for degradation. In the apoptotic pathway in Drosophila, the inhibitor of apoptosis protein 1 (Diap1) regulates the activity of the initiator caspase Dronc (death regulator Nedd2-like caspase; caspase-9 ortholog) by ubiquitylation, supposedly targeting Dronc for degradation by the proteasome. Using a genetic approach, we show that Dronc protein fails to accumulate in epithelial cells with impaired proteasome function suggesting that it is not degraded by the proteasome, contrary to the expectation. Similarly, decreased autophagy, an alternative catabolic pathway, does not result in increased Dronc protein levels. However, combined impairment of the proteasome and autophagy triggers accumulation of Dronc protein levels suggesting that autophagy compensates for the loss of the proteasome with respect to Dronc turnover. Consistently, we show that loss of the proteasome enhances endogenous autophagy in epithelial cells. We propose that enhanced autophagy degrades Dronc if proteasome function is impaired.Cell Death and Differentiation advance online publication, 22 April 2016; doi:10.1038/cdd.2016.40.
... The decrease, however, was completely blocked in the presence of MG132 (Fig. 4B). We also tested the involvement of the proteasome in vivo, using transgenic lines that over-express Pros26 1 and Prosβ 2 , dominant-negative mutant forms of the 20S proteasome subunits β6 and β2, respectively [32]. Flies expressing Pros26 1 and/or Prosβ 2 alleles have been shown to exhibit normal proteasome function at 18°C, but severely impaired function when shifted to 29°C [33,34]. ...
... We used previously generated transgenic lines: UAS-GFP-K v 4 [67,68], UAS-Aβ40 and UAS-Aβ42/CyO [16][17][18], GFP.S65T.T10 [30,31], UAS-Pros26 1 ;UAS-Prosβ 2 [32], and UAS-EKO [40]. For UAS-K v 4, the wild-type Shal2 isoform was subcloned into the pENTR1A vector (Gateway pENTR vectors, Invitrogen), then recombined in vitro using lambda integrase into the pTW destination vector (Drosophila Gateway Vector Collection, available through the Drosophila Genomics Resource Center), generating the pUAST-Shal2 transformation vector. ...
Alzheimer's disease (AD) is the most prevalent form of dementia in the elderly. β-amyloid (Aβ) accumulation in the brain is thought to be a primary event leading to eventual cognitive and motor dysfunction in AD. Aβ has been shown to promote neuronal hyperactivity, which is consistent with enhanced seizure activity in mouse models and AD patients. Little, however, is known about whether, and how, increased excitability contributes to downstream pathologies of AD. Here, we show that overexpression of human Aβ42 in a Drosophila model indeed induces increased neuronal activity. We found that the underlying mechanism involves the selective degradation of the A-type K+ channel, Kv4. An age-dependent loss of Kv4 leads to an increased probability of AP firing. Interestingly, we find that loss of Kv4 alone results in learning and locomotion defects, as well as a shortened lifespan. To test whether the Aβ42-induced increase in neuronal excitability contributes to, or exacerbates, downstream pathologies, we transgenically over-expressed Kv4 to near wild-type levels in Aβ42-expressing animals. We show that restoration of Kv4 attenuated age-dependent learning and locomotor deficits, slowed the onset of neurodegeneration, and partially rescued premature death seen in Aβ42-expressing animals. We conclude that Aβ42-induced hyperactivity plays a critical role in the age-dependent cognitive and motor decline of this Aβ42-Drosophila model, and possibly in AD.
... Proteasome impairment leads to caspase activation and cell death in cell lines. 18 To test whether proteasome impairment leads to early cell death in vivo, we expressed an inducible dominant temperature-sensitive mutant allele of the b2 subunit of the 20S proteasome (UAS-Dts7 2c ) 35 specifically in salivary gland cells, using the salivary gland-specific GAL4 driver fkh-GAL4. To impair proteasome function before the onset of salivary gland cell death, animals expressing Dts7 2c in salivary gland cells were shifted from permissive to restrictive temperature at puparium formation (0 h) to 8 h APF. ...
... For ectopic proteasome impairment studies, UAS-Dts7 2c on III was used. 35 To monitor proteasome impairment, UAS-M-GFP was used as the positive control, and UAS-GFP-CL1 was used as the experimental stock. [32][33][34] For ectopic p35 expression, UAS-p35 was used. ...
Proteasome inhibitors induce cell death and are used in cancer therapy, but little is known about the relationship between proteasome impairment and cell death under normal physiological conditions. Here, we investigate the relationship between proteasome function and larval salivary gland cell death during development in Drosophila. Drosophila larval salivary gland cells undergo synchronized programmed cell death requiring both caspases and autophagy (Atg) genes during development. Here, we show that ubiquitin proteasome system (UPS) function is reduced during normal salivary gland cell death, and that ectopic proteasome impairment in salivary gland cells leads to early DNA fragmentation and salivary gland condensation in vivo. Shotgun proteomic analyses of purified dying salivary glands identified the UPS as the top category of proteins enriched, suggesting a possible compensatory induction of these factors to maintain proteolysis during cell death. We compared the proteome following ectopic proteasome impairment to the proteome during developmental cell death in salivary gland cells. Proteins that were enriched in both populations of cells were screened for their function in salivary gland degradation using RNAi knockdown. We identified several factors, including trol, a novel gene CG11880, and the cop9 signalsome component cop9 signalsome 6, as required for Drosophila larval salivary gland degradation.Cell Death and Differentiation advance online publication, 31 August 2012; doi:10.1038/cdd.2012.110.
... RNAi-mediated knockdown of two wellconserved 19S proteasome regulatory particle subunit genes, Mov34 and Rpn6 [37], resulted in significant accumulation of CiFL protein ( Figure 2F; data not shown). Similarly, when the function of 20S proteasome core particle b6 (Pros26) or b2 (Prosbeta2) subunit was disrupted in the dorsal compartment of the wing disc by the expression of dominant negative temperature-sensitive mutants DTS5 or DTS7 [38], CiFL protein was stabilized in more anterior cells ( Figure 2G; data not shown). In contrast, when the essential lysosomal components, car (carnation) [39], dor (deep orange) [40], Hrs (Hepatocyte growth factor regulated tyrosine kinase substrate) or Stam (Signal transducer adaptor molecule) [41], [42], were specifically knocked down by RNAi, little if any effect on CiFL stability was observed ( Figure 2H; data not shown). ...
... For Figure 2E and F, UAS-ubp (gift of Liqun Luo) [34], UAS-Mov34 RNAi (V26183) or UAS-Rpn6 RNAi (V18021) was crossed with MS1096-Gal4 at 18uC. For Figure 2G, UAS-DTS5 or UAS-DTS7 [38] was crossed with MS1096-Gal4 at 29uC. For Figure 2H Figure 4E-H, UAS-CG13343 RNAi lines were crossed with ap-Gal4, UAS-mCD8-gfp at 29uC. ...
Hedgehog (Hh) signaling is highly conserved in all metazoan animals and plays critical roles in many developmental processes. Dysregulation of the Hh signaling cascade has been implicated in many diseases, including cancer. Although key components of the Hh pathway have been identified, significant gaps remain in our understanding of the regulation of individual Hh signaling molecules. Here, we report the identification of novel regulators of the Hh pathway, obtained from an in vivo RNA interference (RNAi) screen in Drosophila. By selectively targeting critical genes functioning in post-translational modification systems utilizing ubiquitin (Ub) and Ub-like proteins, we identify two novel genes (dUba3 and dUbc12) that negatively regulate Hh signaling activity. We provide in vivo and in vitro evidence illustrating that dUba3 and dUbc12 are essential components of the neddylation pathway; they function in an enzyme cascade to conjugate the ubiquitin-like NEDD8 modifier to Cullin proteins. Neddylation activates the Cullin-containing ubiquitin ligase complex, which in turn promotes the degradation of Cubitus interruptus (Ci), the downstream transcription factor of the Hh pathway. Our study reveals a conserved molecular mechanism of the neddylation pathway in Drosophila and sheds light on the complex post-translational regulations in Hh signaling.
... All stocks were maintained at 25°C on standard cornmeal medium supplemented with dry yeast. The homozygous UAS-DTS5 2B(2), DTS7 1B(3) line (generously provided by Dr. John Belote, Syracuse University (Belote JM and Fortier, 2002)) was crossed with the homozygous muscle-specific myosin heavy chain (MHC)-GAL4 driver line to generate heterozygote MHC-GAL4/UAS-DTS 5,7 larvae containing a single copy of the UAS transgene and a single copy of the GAL4 driver. To generate heterozygote controls, UAS-DTS 5,7 and MHC-GAL4 lines were crossed with the wild-type Oregon-R (+/+) line. ...
... Mutant subunits act in a dominant negative manner to interfere with proteasome function; in multiple previous studies, they have been shown to inhibit UPS-mediated degradation of known proteasome substrates (Schweisguth, 1999;Speese et al., 2003;Neuburger et al., 2006). Transgenic co-expression of both proteasome subunit mutants in the Drosophila eye using the UAS-GAL4 system has a synergistic effect (Belote JM and Fortier, 2002). Previously, it was shown in our laboratory that the proteasome is highly localized at the Drosophila NMJ synapse and that presynaptic inhibition of proteasome function rapidly leads to changes in synaptic transmission that are likely mediated through degradation of the synaptic vessel priming protein DUNC-13 Speese et al., 2003). ...
The ubiquitin-proteasome system (UPS) actively controls protein dynamics and local abundance via regulated protein degradation. This study investigates UPS' roles in the regulation of postsynaptic function and molecular composition in the Drosophila neuromuscular junction (NMJ) genetic system. To specifically impair UPS function postsynaptically, the UAS/GAL4 transgenic method was employed to drive postsynaptic expression of proteasome beta2 and beta6 subunit mutant proteins, which operate through a dominant negative mechanism to block proteasome function. When proteasome mutant subunits were constitutively expressed, excitatory junctional current (EJC) amplitudes were increased, demonstrating that postsynaptic proteasome function limits neurotransmission strength. Interestingly, the alteration in synaptic strength was calcium-dependent and miniature EJCs had significantly smaller mean amplitudes and more rapid mean decay rates. Postsynaptic levels of the Drosophila PSD-95/SAP97 homologue, discs large (DLG), and the GluRIIB-containing glutamate receptor were increased, but GluRIIA-containing receptors were unaltered. With acute postsynaptic proteasome inhibition using an inducible transgenic system, neurotransmission was similarly elevated with the same specific increase in postsynaptic GluRIIB abundance. These findings demonstrate postsynaptic proteasome regulation of glutamatergic synaptic function that is mediated through specific regulation of GluRIIB-containing glutamate receptors.
... However, MG132 fed larvae show no change in Marf:: HA levels between wildtype and lrpprc2 A mutant clones (Fig 2A-2A" and 2C-2C"). We further expressed a dominant negative form of Prosβ6 to inhibit UPS activity [54] and tested its effect on Marf::HA levels in lrpprc2 A mutant clones. Similar to MG132 treatment, we found that Marf::HA levels were restored in lrpprc2 A clones upon Prosβ6 1 overexpression (Fig 2D-2D"). ...
Cells under mitochondrial stress often co-opt mechanisms to maintain energy homeostasis, mitochondrial quality control and cell survival. A mechanistic understanding of such responses is crucial for further insight into mitochondrial biology and diseases. Through an unbiased genetic screen in Drosophila, we identify that mutations in lrpprc2, a homolog of the human LRPPRC gene that is linked to the French-Canadian Leigh syndrome, result in PINK1-Park activation. While the PINK1-Park pathway is well known to induce mitophagy, we show that PINK1-Park regulates mitochondrial dynamics by inducing the degradation of the mitochondrial fusion protein Mitofusin/Marf in lrpprc2 mutants. In our genetic screen, we also discover that Bendless, a K63-linked E2 conjugase, is a regulator of Marf, as loss of bendless results in increased Marf levels. We show that Bendless is required for PINK1 stability, and subsequently for PINK1-Park mediated Marf degradation under physiological conditions, and in response to mitochondrial stress as seen in lrpprc2. Additionally, we show that loss of bendless in lrpprc2 mutant eyes results in photoreceptor degeneration, indicating a neuroprotective role for Bendless-PINK1-Park mediated Marf degradation. Based on our observations, we propose that certain forms of mitochondrial stress activate Bendless-PINK1-Park to limit mitochondrial fusion, which is a cell-protective response.
... activity by altering the β2-β6 interfacing regions 75, 76 . Importantly, previous studies have associated β5 and β6 proteasome-subunit deregulation with disrupted Drosophila eye and wing development 69,77,78 . In accordance to our findings, data from transgenic mice have shown that reduction in β5-associated chymotrypsin-like activity leads to multiple early-aging phenotypes and shortened lifespan 79 . ...
Drosophila brain has emerged as a powerful model system for the investigation of genes being related to neurological pathologies. To map the proteomic landscape of fly brain, in a high-resolution scale, we herein employed a nano liquid chromatography-tandem mass spectrometry technology, and high-content catalogues of 7,663 unique peptides and 2,335 single proteins were generated. Protein-data processing, through UniProt, DAVID, KEGG and PANTHER bioinformatics subroutines, led to fly brain-protein classification, according to sub-cellular topology, molecular function, implication in signaling and contribution to neuronal diseases. Given the importance of Ubiquitin Proteasome System (UPS) in neuropathologies and by using the almost completely reassembled UPS, we genetically targeted genes encoding components of the ubiquitination-dependent protein-degradation machinery. This analysis showed that driving RNAi toward proteasome components and regulators, using the GAL4-elav.L driver, resulted in changes to longevity and climbing-activity patterns during aging. Our proteomic map is expected to advance the existing knowledge regarding brain biology in animal species of major translational-research value and economical interest.
... We show that mutation of dHDAC6 notably affects the key features of PD in the ␣-synuclein modeled flies, namely selective loss of DA neurons, formation of ␣-synuclein-containing inclusions in the brain, decrease of locomotion ability, and shortening of life span. In addition, our genetic experiments show that dHDAC6 also functions to prevent the Figure S1 mutation of dHDAC6 leads to a more severe rough eye phenotype characterizing impaired ubiquitin proteasome system (UPS) (Belote and Fortier, 2002), suggesting that dHDAC6 may play a compensatory role in the impaired UPS. Together, these lines of evidence indicate multiple roles of dHDAC6 in protein quality control machinery in vivo. ...
Parkinson's disease (PD) is associated with progressive degeneration of dopaminergic (DA) neurons. We report for the first time that the Drosophila histone deacetylase 6 (dHDAC6) plays a critical role in the protection of DA neurons and the formation of-synuclein inclusions by using a Drosophila PD model constructed by ectopic expression of human-synuclein. Depletion of dHDAC6 significantly enhances the effects caused by ectopic expression of-synuclein, namely, loss of DA neurons, retinal degeneration, and locomotor dysfunction. Expression of-synuclein in the DA neurons leads to fewer inclusions in the brains of dHDAC6 mutant flies than in wild-type flies. Conversely, overexpres-sion of dHDAC6 is able to suppress the-synuclein-induced DA neuron loss and retinal degeneration and promote inclusion formation. Furthermore, mutation of dHDAC6 reinforces the accumulation of oligomers that are suggested to be a toxic form of-synuclein. We propose that-synuclein inclusion formation in the presence of dHDAC6 protects DA neurons from being damaged by oligomers, which may uncover a common mechanism for synucleinopathies.
... Inhibiting the proteasome activity by overexpressing DTS-7 (dominant temperature-sensitive mutation of the proteasome β2 subunit; Schweisguth, 1999;Belote and Fortier, 2002) and exposing the animals to the restrictive temperature (29°C) produced large ubiquitin-positive inclusion in muscles (Supplemental Figure S10, C and D). This indicated that DTS-7 can efficiently suppress proteasome activity. ...
Mitochondria dysfunction is considered as a hallmark of multiple neuro-degenerative diseases, including Parkinson's disease (PD). PD familial genes, Pink1 and parkin function in a conserved pathway that regulates mitochondrial function including dynamics (fusion and fission). Mammalian cell culture studies suggested that pink1/ parkin pathway promotes mitophagy (mitochondrial autophagy). Mitophagy through mitochondrial fission and auto-lysosomal recycling was considered as a quality control system in organelle level. Whether this quality control machinery involves in PD pathogenesis in vivo, remains elusive. Here, we found elevating autophagy by Atg1 over-expression can significantly rescue mitochondrial defects and apoptotic cell death in pink1 and parkin mutants in Drosophila. Surprisingly, the rescue effect relied both on the autophagy-lysosome machinery and on drp1, a mitochondrial fission molecule. We further showed that Atg1 promotes mitochondrial fission by post-transcriptional increasing of Drp1 protein level. In contrast, increasing fission (by drp1 overexpression) or inhibiting fusion (by mfn knocking down) rescue pink1 mutants when lysosomal or proteasomal machinery were impaired. Taken together, our results identified atg1 as a dual function node to control mitochondrial quality by promoting mitochondria fission and autophagy, which makes it a potential therapeutic target for treatment of mitochondrial dysfunction related diseases, including PD.
... shorten life span. First, cross talk has been observed between the different degradation pathways, and damage to one pathway could be compensated for by the others (46,62). Second, the short life spans of Ab42-expressing flies were further shortened when both Pros26 and ATG1 were knocked down. ...
The endosomal-lysosomal system (ELS), autophagy, and ubiquitin-proteasome system (UPS) are cellular degradation pathways that each play a critical role in the removal of misfolded proteins and the prevention of the accumulation of abnormal proteins. Recent studies on Alzheimer's disease (AD) pathogenesis have suggested that accumulation of aggregated β-amyloid (Aβ) peptides in the AD brain results from a dysfunction in these cellular clearance systems. However, the specific roles of these pathways in the removal of Aβ peptides and the pathogenesis underlying AD are unclear. Our in vitro and in vivo genetic approaches revealed that ELS mainly removed monomeric β-amyloid42 (Aβ42), while autophagy and UPS clear oligomeric Aβ42. Although overproduction of phosphatidylinositol 4-phosphate-5 increased Aβ42 clearance, it reduced the life span of Aβ42 transgenic flies. Our behavioral studies further demonstrated impaired autophagy and UPS-enhanced Aβ42-induced learning and memory deficits, but there was no effect on Aβ42-induced reduction in life span. Results from genetic fluorescence imaging showed that these pathways were damaged in the following order: UPS, autophagy, and finally ELS. The results of our study demonstrate that different degradation pathways play distinct roles in the removal of Aβ42 aggregates and in disease progression. These findings also suggest that pharmacologic treatments that are designed to stimulate cellular degradation pathways in patients with AD should be used with caution.-Ji, X.-R., Cheng, K.-C., Chen, Y.-R., Lin, T.-Y., Cheung, C. H. A., Wu, C.-L., Chiang, H.-C. Dysfunction of different cellular degradation pathways contributes to specific β-amyloid42-induced pathologies.
... It is important to note that the increase in bouton number in the ctp/y mutant is mostly due to an increase in the number of supernumerary 'satellite' boutons that concentrate distally along the synaptic branch (Dickman et al., 2006) (Fig. 8Ai,ii,Bi,ii). This synaptic growth phenotype is also seen at NMJs in larvae expressing a dominant-negative temperature-sensitive proteasome mutation known to block proteasome function in the motor neuron (C155-Gal4; UAS-DTS7) (Fig. 8E) (Belote and Fortier, 2002;Smyth and Belote, 1999). Quantification of total bouton numbers supports that both ctp mutations and proteasome inhibition results in similar increases in synaptic growth compared to that seen in controls (Fig. 8D,E). ...
Because of their functional polarity and elongated morphologies, microtubule-based transport of proteins and organelles is critical for normal neuronal function. The proteasome is required throughout the neuron for the highly regulated degradation of a broad set of protein targets whose functions underlie key physiological responses including synaptic plasticity and axonal degeneration. Molecularly, the relationship between proteasome transport and the transport of the targets of proteasomes is unclear. The dynein motor complex is required for the microtubule-based motility of numerous proteins and organelles in neurons. Here we demonstrate that microtubule-based transport of proteasomes within the neuron utilizes a distinct dynein light chain compared to synaptic proteins. Live imaging of proteasomes and synaptic vesicle proteins in axons and synapses finds that these cargoes traffic independently and that proteasomes exhibit significantly reduced retrograde transport velocities compared to synaptic vesicle proteins. Genetic and biochemical analyses reveals that the Drosophila homologue of the LC8 dynein light chain Cut-up binds proteasomes and functions specifically during their transport. These data support the model that Cut-up functions to specify the dynein-mediated transport of neuronal proteasomes.
... Another method, useful in organisms such as drosophila (fruitfly) is to induce mutations in a large population and then screen the progeny for the desired mutation. A similar process can be used in both plants and prokaryotes [4][5][6][7]. ...
... Since complete knockout of the β5 subunit was found to be lethal in various model systems (Heinemeyer et al., 1993;Belote & Fortier, 2002), our model system impairs proteasome function by incorporation of non-functional β5 subunits into the proteasome, with the expression of endogenous β5 subunits still intact. We did not investigate the relative levels of exogenous β5 subunit incorporation compared to endogenous β5 into proteasome complexes as there is no reliable commercially-available antibody that effectively recognizes endogenous β5 subunits. ...
... The difference between the p53 mRNA and protein levels raised the possibility that p53 protein may be targeted for degradation in endocycling cells. To test this, we inhibited proteasome function in larval salivary glands by using Fkh:GAL4 to drive expression of two temperature-sensitive, dominantnegative subunits of the proteasome, P{UAS-Pros26 1 } and P{UAS-Prosbeta2 1 }, created by the Belote lab [62]. We then compared the levels of p53 protein in B-D and SG cells from the same animals at the permissive (25uC) and non-permissive (29uC) temperature for proteasome function in SG cells. ...
Apoptotic cell death is an important response to genotoxic stress that prevents oncogenesis. It is known that tissues can differ in their apoptotic response, but molecular mechanisms are little understood. Here, we show that Drosophila polyploid endocycling cells (G/S cycle) repress the apoptotic response to DNA damage through at least two mechanisms. First, the expression of all the Drosophila p53 protein isoforms is strongly repressed at a post-transcriptional step. Second, p53-regulated pro-apoptotic genes are epigenetically silenced in endocycling cells, preventing activation of a paused RNA Pol II by p53-dependent or p53-independent pathways. Over-expression of the p53A isoform did not activate this paused RNA Pol II complex in endocycling cells, but over-expression of the p53B isoform with a longer transactivation domain did, suggesting that dampened p53B protein levels are crucial for apoptotic repression. We also find that the p53A protein isoform is ubiquitinated and degraded by the proteasome in endocycling cells. In mitotic cycling cells, p53A was the only isoform expressed to detectable levels, and its mRNA and protein levels increased after irradiation, but there was no evidence for an increase in protein stability. However, our data suggest that p53A protein stability is regulated in unirradiated cells, which likely ensures that apoptosis does not occur in the absence of stress. Without irradiation, both p53A protein and a paused RNA pol II were pre-bound to the promoters of pro-apoptotic genes, preparing mitotic cycling cells for a rapid apoptotic response to genotoxic stress. Together, our results define molecular mechanisms by which different cells in development modulate their apoptotic response, with broader significance for the survival of normal and cancer polyploid cells in mammals.
... Strikingly, Ub K48 -Imd accumulation seen in Usp2-silenced flies was strongly reduced in infected flies compared to uninfected flies indicating that the linkage of Ub K48 on Imd is actively prevented in response to immune challenge, thus allowing for Imd stabilisation ( Figure 6D). To investigate whether accumulation of Imd protein could be sufficient to induce ectopic activation of the Imd pathway in vivo, we blocked proteasome function in flies expressing two conditional dominant-negative proteasome subunits (UAS-pros26 1ts ; UAS-prosβ2 1ts ) [28]. Flies kept at the restrictive temperature (30°C) for 72 hours accumulated Ub K48 -linked and full length Imd ( Figure 6E) and concomitantly, they displayed significant activation of the AttA and Dpt expression ( Figure 6F). ...
Background:
Rapid activation of innate immune defences upon microbial infection depends on the evolutionary conserved NF-κB dependent signals which deregulation is frequently associated with chronic inflammation and oncogenesis. These signals are tightly regulated by the linkage of different kinds of ubiquitin moieties on proteins that modify either their activity or their stability. To investigate how ubiquitin specific proteases (USPs) orchestrate immune signal regulation, we created and screened a focused RNA interference library on Drosophila NF-κB-like pathways Toll and Imd in cultured S2 cells, and further analysed the function of selected genes in vivo.
Results:
We report here that USP2 and USP34/Puf, in addition to the previously described USP36/Scny, prevent inappropriate activation of Imd-dependent immune signal in unchallenged conditions. Moreover, USP34 is also necessary to prevent constitutive activation of the Toll pathway. However, while USP2 also prevents excessive Imd-dependent signalling in vivo, USP34 shows differential requirement depending on NF-κB target genes, in response to fly infection by either Gram-positive or Gram-negative bacteria. We further show that USP2 prevents the constitutive activation of signalling by promoting Imd proteasomal degradation. Indeed, the homeostasis of the Imd scaffolding molecule is tightly regulated by the linkage of lysine 48-linked ubiquitin chains (K48) acting as a tag for its proteasomal degradation. This process is necessary to prevent constitutive activation of Imd pathway in vivo and is inhibited in response to infection. The control of Imd homeostasis by USP2 is associated with the hydrolysis of Imd linked K48-ubiquitin chains and the synergistic binding of USP2 and Imd to the proteasome, as evidenced by both mass-spectrometry analysis of USP2 partners and by co-immunoprecipitation experiments.
Conclusion:
Our work identified one known (USP36) and two new (USP2, USP34) ubiquitin specific proteases regulating Imd or Toll dependent immune signalling in Drosophila. It further highlights the ubiquitin dependent control of Imd homeostasis and shows a new activity for USP2 at the proteasome allowing for Imd degradation. This study provides original information for the better understanding of the strong implication of USP2 in pathological processes in humans, including cancerogenesis.
... The following mutations were used in this study: fzy 1 , fzy 4 and fzy AC-10 (Nüsslein-Volhard and Wieschaus, 1980;Ashburner et al., 1990), fzy tDa (Dawson et al., 1995), dredd B118 (Leulier et al., 2000), atg1 mutant alleles (unc-51 3 and unc-51 25 ) (Toda et al., 2008), apc2 mutant alleles (mr 3 and mr 4 ) (Reed and Orr-Weaver, 1997), apc11 (lmg 138 ) (Nagy et al., 2012), aif (aif KO ) (Joza et al., 2008), bsk 1 (Nüsslein-Volhard and Wieschaus, 1980), dronc (Nc 51 ) (Chew et al., 2004), ark 82 (Akdemir et al., 2006), apc5 (ida B4 and ida D14 ) (Vaskova et al., 2000), pink1 B8 (Park et al., 2006), cav 1 (Cenci et al., 2003), cav 2248 (Li et al., 2011), Su(var)205 4 and Su(var)205 5 (Eissenberg et al., 1992), mir-8 Δ3 (Karres et al., 2007), p53 5A-1-4 and p53 11-1B-1 (Rong et al., 2002). The following transgenic fly lines were used in this study puc-lacZ (puc E69 ) (Ring and Martinez Arias, 1993), p53-RE-gfp (Brodsky et al., 2000), UAS-GCaMP3.0 (Tian et al., 2009), UAS-dTak1 and UAS-dTak1 DN (Takatsu et al., 2000), UAS-hep (Boutros et al., 1998), UAS-hep CA (Adachi-Yamada et al., 1999), UAS-ask1 (Kuranaga et al., 2002), UAS-p35 (Hay et al., 1994), GUS-p53 and GUS-p53 DN (Brodsky et al., 2000), UAS-wee (Price et al., 2002), UAS-tdp43 Δnls and UAS-vca R152H (Ritson et al., 2010), UAS-cycA Δ170 , UAS-pim, UAS-pim dba -myc and UAS-pim kena -myc (Leismann et al., 2000;Leismann and Lehner, 2003), hs-cycB s (Su and O'Farrell, 1997), UAS-pros26β6 1 ; UAS-prosβ2 1 (Belote and Fortier, 2002). The following stocks were obtained from the Bloomington Drosophila Stock Center, apc10 e01070 , apc6 (cdc16 MB09129 ), fen1 EY12786 , rod X-1 , asp 1 and mit (1) ...
Cancer stem cells likely survive chemotherapy or radiotherapy by acquiring mutations that inactivate the endogenous apoptotic machinery or by cycling slowly. Thus, knowledge about the mechanisms linking the activation of an alternative cell death modality and the cell cycle machinery could have a transformative impact on the development of new cancer therapies, but the mechanisms remain completely unknown. We investigated the regulation of alternative cell death in Drosophila larval brain neural stem cells (neuroblasts) in which apoptosis is normally repressed. From a screen, we identified two novel loss-of-function alleles of the Cdc20/fizzy (fzy) gene that lead to premature brain neuroblast loss without perturbing cell proliferation in other diploid cell types. Fzy is an evolutionarily conserved regulator of anaphase promoting complex/cyclosome (APC/C). Neuroblasts carrying the novel fzy allele or exhibiting reduced APC/C function display hallmarks of necrosis. By contrast, neuroblasts overexpressing the non-degradable form of canonical APC/C substrates required for cell cycle progression undergo mitotic catastrophe. These data strongly suggest that Fzy can elicit a novel pro-survival function of APC/C by suppressing necrosis. Neuroblasts experiencing catastrophic cellular stress, or overexpressing p53, lose Fzy expression and undergo necrosis. Co-expression of fzy suppresses the death of these neuroblasts. Consequently, attenuation of the Fzy-dependent survival mechanism functions downstream of catastrophic cellular stress and p53 to eliminate neuroblasts by necrosis. Strategies that target the Fzy-dependent survival mechanism might lead to the discovery of new treatments or complement the pre-existing therapies to eliminate apoptosis-resistant cancer stem cells by necrosis.
... Protein carrying lysine-48 linked poly-ubiquitin chains are targeted to the proteasome for degradation. To determine if midgut cell shrinking was influenced by altered proteasome function, we expressed Dts7, a dominant temperature sensitive mutant of the β2 subunit of the proteasome 18 , in clones of cells in the midgut. We also monitored the activity of the proteasome using CL1-GFP as a reporter 19 . ...
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. Here, we describe 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. Our data reveal that distinct autophagy programs are used by different cells within an animal, and disclose an unappreciated role for ubiquitin activation in autophagy.
... We show that mutation of dHDAC6 notably affects the key features of PD in the ␣-synuclein modeled flies, namely selective loss of DA neurons, formation of ␣-synuclein-containing inclusions in the brain, decrease of locomotion ability, and shortening of life span. In addition, our genetic experiments show that dHDAC6 also functions to prevent the Figure S1 mutation of dHDAC6 leads to a more severe rough eye phenotype characterizing impaired ubiquitin proteasome system (UPS) (Belote and Fortier, 2002), suggesting that dHDAC6 may play a compensatory role in the impaired UPS. Together, these lines of evidence indicate multiple roles of dHDAC6 in protein quality control machinery in vivo. ...
Parkinson's disease (PD) is associated with progressive degeneration of dopaminergic (DA) neurons. We report for the first time that the Drosophila histone deacetylase 6 (dHDAC6) plays a critical role in the protection of DA neurons and the formation of alpha-synuclein inclusions by using a Drosophila PD model constructed by ectopic expression of human alpha-synuclein. Depletion of dHDAC6 significantly enhances the effects caused by ectopic expression of alpha-synuclein, namely, loss of DA neurons, retinal degeneration, and locomotor dysfunction. Expression of alpha-synuclein in the DA neurons leads to fewer inclusions in the brains of dHDAC6 mutant flies than in wild-type flies. Conversely, overexpression of dHDAC6 is able to suppress the alpha-synuclein-induced DA neuron loss and retinal degeneration and promote inclusion formation. Furthermore, mutation of dHDAC6 reinforces the accumulation of oligomers that are suggested to be a toxic form of alpha-synuclein. We propose that alpha-synuclein inclusion formation in the presence of dHDAC6 protects DA neurons from being damaged by oligomers, which may uncover a common mechanism for synucleinopathies.
... AKT activation in mammalian cells has been shown to lead to low FOXO level due to ubiquitination and proteosomal degradation of FOXO [42]. To address this possibility in Drosophila, we reduced ubiquitin mediated proteosomal degradation in dcerk 1 using a dominant temperature sensitive (DTS) mutation, DTS5 that affects the b6 proteosomal subunit [43]. Since ubiquitous expression of DTS5 resulted in lethality, we overexpressed DTS5 in photoreceptors using the GMR GAL4 driver and measured FOXO transcript level in heads. ...
The sphingolipid ceramide elicits several stress responses, however, organisms survive despite increased ceramide but how they do so is poorly understood. We demonstrate here that the AKT/FOXO pathway regulates survival in increased ceramide environment by metabolic adaptation involving changes in glycolysis and lipolysis through novel downstream targets. We show that ceramide kinase mutants accumulate ceramide and this leads to reduction in energy levels due to compromised oxidative phosphorylation. Mutants show increased activation of Akt and a consequent decrease in FOXO levels. These changes lead to enhanced glycolysis by upregulating the activity of phosphoglyceromutase, enolase, pyruvate kinase, and lactate dehydrogenase to provide energy. A second major consequence of AKT/FOXO reprogramming in the mutants is the increased mobilization of lipid from the gut through novel lipase targets, CG8093 and CG6277 for energy contribution. Ubiquitous reduction of these targets by knockdown experiments results in semi or total lethality of the mutants, demonstrating the importance of activating them. The efficiency of these adaptive mechanisms decreases with age and leads to reduction in adult life span of the mutants. In particular, mutants develop cardiac dysfunction with age, likely reflecting the high energy requirement of a well-functioning heart. The lipases also regulate physiological triacylglycerol homeostasis and are important for energy metabolism since midgut specific reduction of them in wild type flies results in increased sensitivity to starvation and accumulation of triglycerides leading to cardiac defects. The central findings of increased AKT activation, decreased FOXO level and activation of phosphoglyceromutase and pyruvate kinase are also observed in mice heterozygous for ceramide transfer protein suggesting a conserved role of this pathway in mammals. These data reveal novel glycolytic and non-autonomous lipolytic
... Specifically, expression of temperature sensitive dominant negative mutant for the 20S proteasome β2 and β6 subunits causes a rough, reduced eye (Belote and Fortier, 2002). Expression of RNAi against dTNKS further enhanced this phenotype, similarly to what was previously observed for reduction of DmPI31 function (Figures 3C-E) (Bader et al., 2011). ...
Protein degradation by the ubiquitin-proteasome system is central to cell homeostasis and survival. Defects in this process are associated with diseases such as cancer and neurodegenerative disorders. The 26S proteasome is a large protease complex that degrades ubiquitinated proteins. Here, we show that ADP-ribosylation promotes 26S proteasome activity in both Drosophila and human cells. We identify the ADP-ribosyltransferase tankyrase (TNKS) and the 19S assembly chaperones dp27 and dS5b as direct binding partners of the proteasome regulator PI31. TNKS-mediated ADP-ribosylation of PI31 drastically reduces its affinity for 20S proteasome α subunits to relieve 20S repression by PI31. Additionally, PI31 modification increases binding to and sequestration of dp27 and dS5b from 19S regulatory particles, promoting 26S assembly. Inhibition of TNKS by either RNAi or a small-molecule inhibitor, XAV939, blocks this process to reduce 26S assembly. These results unravel a mechanism of proteasome regulation that can be targeted with existing small-molecule inhibitors.
... To investigate the cause of MyoV loss observed in crb mutant photoreceptors, we aimed to prevent the loss of MyoV by overexpression of a dominant-negative proteasome subunit Pros26 1 2B (Belote and Fortier, 2002). This resulted in a marked increase in MyoV staining (compare Fig. 2 A with Fig. 2 D). ...
The evolutionarily conserved Crumbs (Crb) complex is crucial for photoreceptor morphogenesis and homeostasis. Loss of Crb results in light-dependent retinal degeneration, which is prevented by feeding mutant flies carotenoid-deficient medium. This suggests a defect in rhodopsin 1 (Rh1) processing, transport, and/or signaling, causing degeneration; however, the molecular mechanism of this remained elusive. In this paper, we show that myosin V (MyoV) coimmunoprecipitated with the Crb complex and that loss of crb led to severe reduction in MyoV levels, which could be rescued by proteasomal inhibition. Loss of MyoV in crb mutant photoreceptors was accompanied by defective transport of the MyoV cargo Rh1 to the light-sensing organelle, the rhabdomere. This resulted in an age-dependent accumulation of Rh1 in the photoreceptor cell (PRC) body, a well-documented trigger of degeneration. We conclude that Crb protects against degeneration by interacting with and stabilizing MyoV, thereby ensuring correct Rh1 trafficking. Our data provide, for the first time, a molecular mechanism for the light-dependent degeneration of PRCs observed in crb mutant retinas.
... First we analyzed whether expression of DmPI31 had an effect on phenotypes caused by impaired proteasome function. Targeted expression of the dominant temperature sensitive proteasome alleles UAS-DTS5 and UAS-DTS7 with GMR-Gal4 at 29°C causes a small, rough eye phenotype (Belote and Fortier, 2002) (Figure 5B and 5C). Co-expression of DmPI31 with these constructs suppressed this phenotype ( Figure 5D). ...
The ubiquitin-proteasome system catalyzes the degradation of intracellular proteins. Although ubiquitination of proteins determines their stabilities, there is growing evidence that proteasome function is also regulated. We report the functional characterization of a conserved proteasomal regulatory complex. We identified DmPI31 as a binding partner of the F box protein Nutcracker, a component of an SCF ubiquitin ligase (E3) required for caspase activation during sperm differentiation in Drosophila. DmPI31 binds Nutcracker via a conserved mechanism that is also used by mammalian FBXO7 and PI31. Nutcracker promotes DmPI31 stability, which is necessary for caspase activation, proteasome function, and sperm differentiation. DmPI31 can activate 26S proteasomes in vitro, and increasing DmPI31 levels suppresses defects caused by diminished proteasome activity in vivo. Furthermore, loss of DmPI31 function causes lethality, cell-cycle abnormalities, and defects in protein degradation, demonstrating that DmPI31 is physiologically required for normal proteasome activity.
... Previous studies have suggested that the mechanisms underlying the toxicity of TDP-43 include hyperphosphorylation of the mutated variants, followed by excessive targeting to the proteasome, which may eventually become functionally overwhelmed (3,30). To test whether the proteasome contributes to TDP-43 neurotoxicity in vivo, we used a dominantnegative form of the b2 subunit of the 20S proteasome complex, namely prosb (42). Overexpression of prosb alone in the eye does not produce a visible phenotype (Fig. 7I); however, when coexpressed with wild-type and A315T mutant hTDP-43, it enhanced the depigmentation phenotype due to TDP-43 overexpression (Fig. 7A and E with B and F, respectively). ...
The RNA-binding protein TDP-43 has been linked to amyotrophic lateral sclerosis (ALS) both as a causative locus and as a marker of pathology. With several missense mutations being identified within TDP-43, efforts have been directed towards generating animal models of ALS in mouse, zebrafish, Drosophila and worms. Previous loss of function and overexpression studies have shown that alterations in TDP-43 dosage recapitulate hallmark features of ALS pathology, including neuronal loss and locomotor dysfunction. Here we report a direct in vivo comparison between wild-type and A315T mutant TDP-43 overexpression in Drosophila neurons. We found that when expressed at comparable levels, wild-type TDP-43 exerts more severe effects on neuromuscular junction architecture, viability and motor neuron loss compared with the A315T allele. A subset of these differences can be compensated by higher levels of A315T expression, indicating a direct correlation between dosage and neurotoxic phenotypes. Interestingly, larval locomotion is the sole parameter that is more affected by the A315T allele than wild-type TDP-43. RNA interference and genetic interaction experiments indicate that TDP-43 overexpression mimics a loss-of-function phenotype and suggest a dominant-negative effect. Furthermore, we show that neuronal apoptosis does not require the cytoplasmic localization of TDP-43 and that its neurotoxicity is modulated by the proteasome, the HSP70 chaperone and the apoptosis pathway. Taken together, our findings provide novel insights into the phenotypic consequences of the A315T TDP-43 missense mutation and suggest that studies of individual mutations are critical for elucidating the molecular mechanisms of ALS and related neurodegenerative disorders.
... To study whether the proteasome is involved in Cul3 protein degradation, we used dominant temperature-sensitive (DTS) proteasome mutant flies. Prosβ2 1 has missense mutations in the 20S proteasome subunits β2 and the expression of UAS-Prosβ2 1 represses proteasome function at the restrictive temperature (29°C) (Belote and Fortier, 2002;Schweisguth, 1999). Repression of proteasome function by expressing UAS-Prosβ2 1 with the ubiquitous Da-GAL4 driver resulted in the accumulation of unneddylated Cul3 protein at the restrictive temperature (Fig. 5F, lanes 1 and 2). ...
Cullin-RING ubiquitin ligases (CRLs), which comprise the largest class of E3 ligases, regulate diverse cellular processes by targeting numerous proteins. Conjugation of the ubiquitin-like protein Nedd8 with Cullin activates CRLs. Cullin-associated and neddylation-dissociated 1 (Cand1) is known to negatively regulate CRL activity by sequestering unneddylated Cullin1 (Cul1) in biochemical studies. However, genetic studies of Arabidopsis have shown that Cand1 is required for optimal CRL activity. To elucidate the regulation of CRLs by Cand1, we analyzed a Cand1 mutant in Drosophila. Loss of Cand1 causes accumulation of neddylated Cullin3 (Cul3) and stabilizes the Cul3 adaptor protein HIB. In addition, the Cand1 mutation stimulates protein degradation of Cubitus interruptus (Ci), suggesting that Cul3-RING ligase activity is enhanced by the loss of Cand1. However, the loss of Cand1 fails to repress the accumulation of Ci in Nedd8(AN015) or CSN5(null) mutant clones. Although Cand1 is able to bind both Cul1 and Cul3, mutation of Cand1 suppresses only the accumulation of Cul3 induced by the dAPP-BP1 mutation defective in the neddylation pathway, and this effect is attenuated by inhibition of proteasome function. Furthermore, overexpression of Cand1 stabilizes the Cul3 protein when the neddylation pathway is partially suppressed. These data indicate that Cand1 stabilizes unneddylated Cul3 by preventing proteasomal degradation. Here, we propose that binding of Cand1 to unneddylated Cul3 causes a shift in the equilibrium away from the neddylation of Cul3 that is required for the degradation of substrate by CRLs, and protects unneddylated Cul3 from proteasomal degradation. Cand1 regulates Cul3-mediated E3 ligase activity not only by acting on the neddylation of Cul3, but also by controlling the stability of the adaptor protein and unneddylated Cul3.
... In parallel experiments in intact wing discs, crb i is able to deplete levels of an Ex:GFP fusion protein [22] (Fig. 3G). Genetic reduction of proteasome activity with a dominant-negative allele of the proteasomal subunit Pros2β [23] also elevates levels of Ex:GFP levels in normal wing disc cells and partially restores Ex:GFP levels in discs that also express crb i (Fig. 3G). Expression of crb i did not stimulate endolysosomal routing of Ex:GFP as measured by the effect of treatment with the lysosomal inhibitor chloroquine on Ex:GFP localization (Fig. S3). ...
Altered expression of apicobasal polarity factors is associated with cancer in vertebrates and tissue overgrowth in invertebrates, yet the mechanisms by which these factors affect growth-regulatory pathways are not well defined. We have tested the basis of an overgrowth phenotype driven by the Drosophila protein Crumbs (Crb), which nucleates an apical membrane complex that functionally interacts with the Par6/Par3/aPKC and Scrib/Dlg/Lgl apicobasal polarity complexes.
We find that Crb-driven growth is dependent upon the Salvador/Warts/Hippo (SWH) pathway and its transcriptional effector Yorkie (Yki). Expression of the Crb intracellular domain elevates Yki activity, and this correlates in tissues and cultured cells with loss of Expanded (Ex), an apically localized SWH component that inhibits Yki. Reciprocally, loss of crb elevates Ex levels, although this excess Ex does not concentrate to its normal location at apical junctions. The Ex-regulatory domain of Crb maps to the juxtamembrane FERM-binding motif (JM), a cytoskeletal interaction domain distinct from the PDZ-binding motif (PBM) through which Crb binds polarity factors. Expression of Crb-JM drives Yki activity and organ growth with little effect on tissue architecture, while Crb-PBM reciprocally produces tissue architectural defects without significant effect on Yki activity.
These studies identify Crb as a novel SWH regulator via JM-dependent effects on Ex levels and localization and suggest that discrete domains within Crb may allow it to integrate junctional polarity signals with a conserved growth pathway.
... We tested whether reducing ubiquitinmediated proteosomal degradation in vivo would allow us to detect NORPA in dcerk 1 . We used a dominant temperature-sensitive (DTS) mutation, DTS5, that affects the 6 proteosomal subunit (25). If NORPA is targeted for degradation in dcerk 1 , then expressing DTS5 in dcerk 1 photoreceptors should result in restoration of NORPA. ...
Phosphoinositide-specific phospholipase C (PLC) is a central effector for many biological responses regulated by G-protein-coupled receptors including Drosophila phototransduction where light sensitive channels are activated downstream of NORPA, a PLCbeta homolog. Here we show that the sphingolipid biosynthetic enzyme, ceramide kinase, is a novel regulator of PLC signaling and photoreceptor homeostasis. A mutation in ceramide kinase specifically leads to proteolysis of NORPA, consequent loss of PLC activity, and failure in light signal transduction. The mutant photoreceptors also undergo activity-dependent degeneration. Furthermore, we show that a significant increase in ceramide, resulting from lack of ceramide kinase, perturbs the membrane microenvironment of phosphatidylinositol 4, 5, bisphosphate (PIP(2)), altering its distribution. Fluorescence image correlation spectroscopic studies on model membranes suggest that an increase in ceramide decreases clustering of PIP(2) and its partitioning into ordered membrane domains. Thus ceramide kinase-mediated maintenance of ceramide level is important for the local regulation of PIP(2) and PLC during phototransduction.
... Since studies with yeast (Heinemeyer et al. 1993) and Drosophila (Belote and Fortier 2002) demonstrated that a functional b5 subunit is essential for life, we reasoned that a b5 knock-out would also confer a lethal effect on the mouse neuronal HT4 cells. To avoid a complete elimination of the chymotrypsin-like activity of the proteasome in HT4 cells, Ub-conjugates and synuclein co-localize in aggregates in Mutaß5 stable transfectants treated with Cd 2+ . ...
Accumulation of ubiquitinated proteins in inclusions is common to various neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis, although it occurs in selective neurons in each disease. The mechanisms generating such abnormal aggregates and their role in neurodegeneration remain unclear. Inclusions appear in familial and non-familial cases of neurodegenerative disorders, suggesting that factors other than particular mutations contribute to protein accumulation and aggregation. Proteasome impairment triggered by aging or conditions such as oxidative stress may contribute to protein accumulation and aggregation in neurodegeneration. To test this hypothesis in mouse neuronal cells, we overexpressed a 20S proteasome beta5 subunit with an active site mutation. The N-terminal threonine to alanine substitution resulted in impairment of the chymotrypsin-like activity, which is a rate-limiting step in protein degradation by the proteasome. The Thr1Ala mutation was not lethal under homeostatic conditions. However, this single amino acid substitution significantly hypersensitized the cells to oxidative stress, triggering not only the accumulation and aggregation of ubiquitinated proteins, including synuclein, but also cell death. Our results demonstrate that this genetic manipulation of proteasome activity involving a single amino acid substitution causes the formation of protein aggregates in stressed neuronal cells independently of the occurrence of mutations in other cellular proteins. These results support the notion that proteasome disruption may be central to the development of familial as well as sporadic cases of neurodegeneration.
... This approach has been quite productive in Drosophila and is still in wide use (e.g. (23)(24)(25) ). Dominant negative alleles will typically contain mutations that allow for the assembly of the altered protein into multimeric complexes, but leave the protein functionally inactive, thereby poisoning the entire complex. ...
In Drosophila, the genetic approach is still the method of choice for answering fundamental questions on cell biology, signal transduction, development, physiology and behavior. In this approach, a gene's function is ascertained by altering either the amount or quality of the gene product, and then observing the consequences. The genetic approach is itself polymorphous, encompassing new and more complex techniques that typically employ the growing collections of transgenes. The keystone of these modern Drosophila transgenic techniques has been the Gal4 binary system. Recently, several new techniques have modified this binary system to offer greater control over the timing, tissue specificity and magnitude of gene expression. Additionally, the advances in post-transcriptional gene silencing, or RNAi, have greatly expanded the ability to knockdown almost any gene's function. Regardless of the growing experimental intricacy, the application of these advances to modify gene activity still obeys the fundamental principles of genetic analysis. Several of these transgenic techniques, which offer more precise control over a gene's activity, will be reviewed here with a discussion on how they may be used for determining a gene's function.
... If this were the case RNAi with Pros26.4 might interfere with all these processes. Interestingly, impeding the activity of the central core subunits 6 or 2 causes superficially very similar phenotypes to depletion of Pros26.4 (Belote and Fortier, 2002). The authors mis-expressed respective dominant negative mutants DTS5 and DTS7 in the photoreceptor cells. ...
The proteasome is the major degradation machinery of the cell that regulates multiple cellular processes as diverse as cell cycle, signal transduction and gene expression. Recognition and unfolding of target proteins involves the regulatory cap whose base contains six AAA-ATPases that display reverse chaperone activity. One of them, Rpt2 (also known as S4), has an essential role in gating the degradative central core. We have isolated the orthologous gene Pros26.4 from Drosophila melanogaster as a molecular interaction partner of Hairless. Hairless plays a major role as antagonist of Notch signalling in Drosophila, prompting our interest in the Hairless-Pros26.4 interaction. We find that Pros26.4 negatively regulates Hairless at the genetic and molecular level. Depletion of Pros26.4 by using tissue-specific RNA interference (RNAi) resulted in a specific stabilization of the Hairless protein, but not in stabilization of the intracellular domain of Notch or the effector protein Suppressor of Hairless. Thus, the Hairless-Pros26.4 interaction provides a novel mechanism of positive regulation of Notch signalling.
... Because Pros26 1 and Prosb2 1 act in a dominant-negative manner, it is also possible to target their effects to particular cells or tissues using the UAS/GAL4 binary system of Brand and Perrimon (1993). A number of UAS-Pros26 1 and UAS-Prosb2 1 transgenic lines have been generated and used for this purpose (Schweisguth 1999; Belote and Fortier 2002; Chan et al. 2002; Khush et al. 2002; Speese et al. 2003; Shulman and Feany 2003; Tang et al. 2005). These transgenic lines provide a complementary approach to the use of proteasome inhibitors (Myung et al. 2001) to investigate the roles that proteasomerelated proteolysis plays during development. ...
Two dominant temperature-sensitive (DTS) lethal mutants of Drosophila melanogaster are Pros26(1) and Prosbeta2(1), previously known as DTS5 and DTS7. Heterozygotes for either mutant die as pupae when raised at 29 degrees , but are normally viable and fertile at 25 degrees . Previous studies have identified these as missense mutations in the genes encoding the beta6 and beta2 subunits of the 20S proteasome, respectively. In an effort to isolate additional proteasome-related mutants a screen for dominant suppressors of Pros26(1) was carried out, resulting in the identification of Pros25(SuDTS) [originally called Su(DTS)], a missense mutation in the gene encoding the 20S proteasome alpha2 subunit. Pros25(SuDTS) acts in a dominant manner to rescue both Pros26(1) and Prosbeta2(1) from their DTS lethal phenotypes. Using an in vivo protein degradation assay it was shown that this suppression occurs by counteracting the dominant-negative effect of the DTS mutant on proteasome activity. Pros25(SuDTS) is a recessive polyphasic lethal at ambient temperatures. The effects of these mutants on larval neuroblast mitosis were also examined. While Prosbeta2(1) shows a modest increase in the number of defective mitotic figures, there were no defects seen with the other two mutants, other than slightly reduced mitotic indexes.
... In the following steps, the caspase-like and the trypsin-like activities further breakdown the fragments generated in the initial step. Studies with yeast (Heinemeyer et al., 1993) and Drosophila (Belote & Fortier, 2002) demonstrated that a functional β5 subunit is essential for survival. ...
In all cells, protein degradation is a constant, ongoing process that is critical for cell survival and repair. The ubiquitin/proteasome pathway (UPP) is the major proteolytic pathway that degrades intracellular proteins in a regulated manner. It plays critical roles in many cellular processes and diseases. Disruption of the UPP is particularly relevant to pathophysiological conditions that provoke the accumulation of aberrant proteins, such as in aging as well as in a variety of neurodegenerative disorders including Alzheimer's and Parkinson's diseases. For unknown reasons, most of these neurodegenerative disorders that include familial and sporadic cases exhibit a late onset. It is possible that these neurodegenerative conditions exhibit a late onset because proteasome activity decreases with aging. Aging-dependent impairment in proteolysis mediated by the proteasome may have profound ramifications for cell viability. It can lead to the accumulation of modified, potentially toxic proteins in cells and can cause cell injury or premature cell death by apoptosis or necrosis. While it is accepted that aging affects UPP function, the question is why does aging cause a decline in regulated protein degradation by the UPP? Herein, we review some of the properties of the UPP and mechanisms mediating its age-dependent impairment. We also discuss the relevance of these findings leading to a model that proposes that UPP dysfunction may be one of the milestones of aging.
Cells under mitochondrial stress often co-opt mechanisms to maintain energy homeostasis, mitochondrial quality control and cell survival. A mechanistic understanding of such responses is crucial for further insight into mitochondrial biology and diseases. Through an unbiased genetic screen in Drosophila, we identify that mutations in lrpprc2, a homolog of the human LRPPRC gene that is linked to the French-Canadian Leigh syndrome, results in PINK1-Park activation. While the PINK1-Park pathway is well known to induce mitophagy, we show that in the case of lrpprc2 mutants, PINK1-Park regulates mitochondrial dynamics by inducing degradation of the mitochondrial fusion protein Mitofusin/Marf. We also discover that Bendless, a K63-linked E2 conjugase, is a regulator of Marf, as loss of bendless results in increased Marf levels. We show that Bendless is required for PINK1 stability, and subsequently for PINK1-Park mediated Marf degradation under physiological conditions, and in response to mitochondrial stress as seen in lrpprc2. Additionally, we show that loss of Bendless in lrpprc2 mutant eye results in photoreceptor degeneration, indicating a neuroprotective role for Bendless-PINK1-Park mediated Marf degradation. Based on our observations, we propose that certain forms of mitochondrial stress activate Bendless-PINK1-Park to limit mitochondrial fusion, which is a cell-protective response.
Sarcomeres, the fundamental contractile units of muscles, are conserved structures composed of actin thin filaments and myosin thick filaments. How sarcomeres are formed and maintained is not well understood. Here, we show that knockdown of Drosophila cofilin (DmCFL), an actin depolymerizing factor, disrupts both sarcomere structure and muscle function. The loss of DmCFL also results in the formation of sarcomeric protein aggregates and impairs sarcomere addition during growth. The activation of the proteasome delays muscle deterioration in our model. Furthermore, we investigate how a point mutation in CFL2 that causes nemaline myopathy (NM) in humans affects CFL function and leads to the muscle phenotypes observed in vivo. Our data provide significant insights to the role of CFLs during sarcomere formation, as well as mechanistic implications for disease progression in NM patients.
Speckled (Spc), an X-ray-induced lethal mutant of Bombyx mori, exhibits a mosaic dark-brown-spotted larval epidermis in both sexes and egg-laying problems only in females. Here, we report the morphological characterization and molecular mapping of the Spc mutant. Morphological investigations revealed that the epidermal ultrastructure of the small, dark-brown spots was more dense than that of the white regions in both Spc/+ mutants and wild type, and that the lethality of the Spc/Spc mutants occurred during early embryogenesis. Furthermore, the ovarioles and ovipositor were disconnected in approximately 85.5% of Spc/+ females, a further 2.5% had a connection between the ovarioles and ovipositor that was too narrow to lay eggs. The remaining females showed a normal connection similar to that of the wild type. We successfully narrowed down the location of the Spc mutation to a region on chromosome 4 that was ∼1041 kb long. Gene-prediction analysis identified 25 candidate genes in this region. Chromosome structure analysis indicated that a ∼305 kb deletion was included in the mapping region. Temporal and spatial reverse transcription PCR (RT-PCR) analysis showed that several genes in the mapped region are associated with the Spc mutant. Although the genes responsible for the Spc mutation were not definitively identified, our results further the current understanding of the complex mechanism underlying the multiple morphological defects in Spc mutants.
Autophagy is a cellular process that delivers cytoplasmic materials for degradation by the lysosomes. Autophagy-related (Atg) genes were identified in yeast genetic screens for vehicle formation under stress conditions, and Atg genes are conserved from yeast to human. When cells or animals are under stress, autophagy is induced and Atg8 (LC3 in mammal) is activated by E1 activating enzyme Atg7. Atg8-containing membranes form and surround cargos, close and mature to become the autophagosomes. Autophagosomes fuse with lysosomes, and cargos are degraded by lysosomal enzymes to sustain cell viability. Therefore, autophagy is most frequently considered to function in cell survival. Whether the Atg gene regulatory pathway that was defined in yeast is utilized for all autophagy in animals, as well as if autophagy could function in a cell death scenario, are less understood.
The Drosophila larval digestive tissues, such as the midgut of the intestine and the salivary gland, are no longer required for the adult animal and are degraded during the pupal stage of development. Cells stop growing at the end of larval development, and proper cell growth arrest is required for midgut degradation. Ectopic activation of the PI3K/Akt signaling induces cell growth and inhibits autophagy and midgut degradation. Down regulating PI3K/Akt pathway by Pten mis-expression activates autophagy. In addition, mis-expression of autophagy initiator Atg1 inhibits cell growth and knocking down autophagy restore PI3K/Akt activity. Together, these results indicate that autophagy and growth signaling mutually inhibit each other.
Midgut destruction relies on the autophagy gene Atg18, but not caspase activation. The intestine length shortens and the cells undergo programmed cell size reduction, a phenomenon that also requires Atg18, before cell death occurs during midgut destruction. To further investigate whether cell size reduction is cell autonomous and requires other Atg genes, we reduced the function of Atg genes in cell clones using either gene mutations or RNAi knockdowns. Indeed, many Atg genes, including Atg8, are required for autophagy and cell size reduction in a cell autonomous manner. Surprisingly, Atg7 is not required for midgut cell size reduction and autophagy even though this gene is essential for stress-induced autophagy. Therefore, we screened for known E1 enzymes that may function in the midgut, and discovered that Uba1 is required for autophagy, size reduction and clearance of mitochondria. Uba1 does not enzymatically substitute for Atg7, and Ubiquitin phenocopies Uba1, suggesting Uba1 functions through ubiquitination of unidentified molecule(s) to regulate autophagy.
In conclusion, this thesis describes: First, autophagy participates in midgut degradation and cell death. Second it reveals a previously un-defined role of Uba1 in autophagy regulation. Third it shows that the Atg genes are not functionally conserved and the requirement of some Atg genes can be context dependent.
The innate immune system relies on the recognition of “non-self” and on the activation of adapted responses, among which NF-κB signaling pathways play a crucial role. These pathways are tightly regulated, in order to prevent an excessive and sustained immune response, responsible for several pathologies, such as autoimmune and pro-inflammatory diseases. During my PhD thesis, I elucidated some Drosophila regulatory mechanisms of NF-κB pathways, Toll and IMD, which rely on protein ubiquitination and their subsequent degradation by the endocytic pathway or proteasome. Reversible ubiquitination of proteins is a post-translational modification, regulating their activity, their stability and the subcellular localization. In particular, ubiquitination of membrane receptors could trigger their internalization and their subsequent lysosomal degradation. In Drosophila, the PGRP-LC receptor specifically recognizes diaminopimelic acid containing peptidoglycan (PGN) and induces the IMD signaling pathway. I proved that PGRP-LC receptor is ubiquitinated, internalized and degraded by the endocytic pathway. In this process, I identified the major role of the USP8 deubiquitinating enzyme, which controls the degradation of ubiquitinated PGRP-LC. Besides, I showed that the IMD stimulation by PGN enhances the PGRP-LC internalization and its degradation, ensuring receptors elimination once the IMD pathway has been activated. Moreover, I took part to studies, aiming to understand the role of USP2, USP34 and USP36, previously selected by the team as negative regulators of the IMD and/or Toll pathways. In particular, my results showed that USP2 principally acts at the Imd level, allowing for the hydrolysis of its K48 poly-ubiquitin chains and its proteasomal degradation. Finally, I observed that USP2 also interacts with PGRP-LC and favors the hydrolysis of PGRP-LC associated K48 chains, whereas the degradation of K48 poly-ubiquitinated PGRP-LC is independent from the proteasome, but rather depends on the Hrs and Rab5 endocytic proteins and on the USP8 deubiquitinating enzyme.
Ubiquitylated developmental membrane signaling proteins are often internalized for endocytic trafficking, through which endosomal sorting complexes required for transport (ESCRT) act sequentially to deliver internalized cargos to lysosomes. The ESCRT function in endocytic sorting is well established; however, it is not fully understood how the sorting machinery itself is regulated. Here, we show that Ubiquitin isopeptidase Y (Ubpy) plays a conserved role in vivo in the homeostasis of an essential ESCRT-0 complex component Hrs. We find that, in the absence of Drosophila Ubpy, multiple membrane proteins that are essential components of important signaling pathways accumulate in enlarged, aberrant endosomes. We further demonstrate that this phenotype results from endocytic pathway defects. We provide evidence that Ubpy interacts with and deubiquitylates Hrs. In Ubpy-null cells, Hrs becomes ubiquitylated and degraded in lysosomes, thus disrupting the integrity of ESCRT sorting machinery. Lastly, we find that signaling proteins are enriched in enlarged endosomes when Hrs activity is abolished. Together, our data support a model in which Ubpy plays a dual role in both cargo deubiquitylation and the ESCRT-0 stability during development.
Several dominant neurodegenerative disorders result from alleles carrying expanded stretch of polyglutamine (polyQ) tracts in the encoded proteins, which become toxic and form insoluble cytoplasmic and/or nuclear aggregates or “inclusion bodies” in the affected neuronal cells. Unlike the known modulatory roles of molecular chaperones like Hsp90, Hsp70, Hsp40 etc in mis-folding and aggregation of the toxic proteins, little is known about role of the Hsp60 family chaperones in polyQ protein metabolism. Present study identified Hsp60D, a member of the Drosophila Hsp60 family, as a novel modifier of neurodegeneration in fly models of polyQ disorders, viz., Spinocerebellar Ataxia type 3 (SCA3, caused by mutated MJDtr-Q78 allele) or the 127Q model. We showed that the reduction in the cellular levels of Hsp60D protein through directed RNAi in the polyQ expressing developing eye cells not only improved external eye morphology, retinal structure and vision, but also reduced the number of inclusion bodies and the associated expression of Hsp70. Further, Hsp60D-RNAi suppressed the organismal lethality in flies expressing the pathogenic polyQ proteins pan-neuronally. The suppression of polyQ phenotype by Hsp60D-RNAi is largely independent of proteasomal and SUMO activities but appears to require DIAP1. Our results suggest that Hsp60D may be required for folding of polypeptides with polyQ stretches so that in its absence, due to targeted RNAi, the expanded polyQ polypeptides fail to fold in a manner that can produce the toxic inclusion bodies. Inhibition of caspase activity and apoptosis following Hsp60D-RNAi, reported by us earlier, may also help survival of the expanded polyQ expressing neuronal cells.
Following earlier reports on modulation of poly(Q) toxicity in Drosophila by the developmentally active and stress-inducible noncoding hsromega gene, we investigated possible mediators of this modulation. RNAi-mediated downregulation of the large nuclear hsromega-n transcript, which organizes the nucleoplasmic omega speckles, suppressed the enhancement of poly(Q) toxicity brought about by reduced availability of the heterogeneous nuclear ribonucleoprotein (hnRNP) Hrb87F and of the transcriptional regulator, cAMP response element binding (CREB) binding protein (CBP). Levels of CBP RNA and protein were reciprocally affected by hsromega transcript levels in eye disc cells. Our data suggest that CBP and hnRNPs like Hrb57A and Hrb87F physically interact with each other. In addition, downregulation of hsromega transcripts partially rescued eye damage following compromised proteasome activity, while overexpression of hsromega and/or poly(Q) proteins disrupted the proteasomal activity. Rescue of poly(Q) toxicity by hsromega-RNAi required normal proteasomal function. We suggest that hsromega-RNAi suppresses poly(Q) toxicity by elevating cellular levels of CBP, by enhancing proteasome-mediated clearance of the pathogenic poly(Q) aggregates, and by inhibiting induced apoptosis. The direct and indirect interactions of the hsromega transcripts with a variety of regulatory proteins like hnRNPs, CBP, proteasome, Drosophila inhibitor of apoptosis protein 1 (DIAP1), etc., reinforce the view that the noncoding hsromega RNA functions as a "hub" in cellular networks to maintain homeostasis by coordinating the functional availability of crucial cellular regulatory proteins.
A Corrigendum to this paper has been published at https://www.genetics.org/content/genetics/suppl/2019/06/25/genetics.109.113696.DC1/Corrigendum_for_Mallik_and_Lakhotia_FINAL.pdf
The ubiquitin proteasome system (UPS) mediates regulated protein degradation and provides a mechanism for closely controlling protein abundance in spatially restricted domains within cells. We hypothesized that the UPS may acutely determine the local concentration of key regulatory proteins at neuronal synapses as a means for locally modulating synaptic efficacy and the strength of neurotransmission communication.
We investigated this hypothesis at the Drosophila neuromuscular synapse by using an array of genetic and pharmacological tools. This study demonstrates that UPS components are present in presynaptic boutons and that the UPS functions locally in the presynaptic compartment to rapidly eliminate a conditional transgenic reporter of proteasome activity. We assayed a panel of synaptic proteins to determine whether the UPS acutely regulates the local abundance of native synaptic targets. Both acute pharmacological inhibition of the proteasome (<1 hr) and targeted genetic perturbation of proteasome function in the presynaptic neuron cause the specific accumulation of the essential synaptic vesicle-priming protein DUNC-13. Most importantly, acute pharmacological inhibition of the proteasome (<1 hr) causes a rapid strengthening of neurotransmission (an approximately 50% increase in evoked amplitude) because of increased presynaptic efficacy. The proteasome-dependent regulation of presynaptic protein abundance, both of the exogenous reporter and native DUNC-13, and the modulation of presynaptic neurotransmitter release occur on an intermediate, rapid (tens of minutes) timescale.
Taken together, these studies demonstrate that the UPS functions locally within synaptic boutons to acutely control levels of presynaptic protein and that the rate of UPS-dependent protein degradation is a primary determinant of neurotransmission strength.
Two central issues in polyglutamine-induced neurodegeneration are the influence of the normal function of the disease protein and modulation by protein quality control pathways. By using Drosophila, we now directly link host protein function and disease pathogenesis to ubiquitin pathways in the polyglutamine disease spinocerebellar ataxia type 3 (SCA3). Normal human ataxin-3--a polyubiquitin binding protein with ubiquitin protease activity--is a striking suppressor of polyglutamine neurodegeneration in vivo. This suppressor activity requires ubiquitin-associated activities of the protein and is dependent upon proteasome function. Our results highlight the critical importance of host protein function in SCA3 disease and a potential therapeutic role of ataxin-3 activity for polyglutamine disorders.
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The cyclic expression of the period (PER) and timeless (TIM) proteins is critical for the molecular circadian feedback loop
inDrosophila. The entrainment by light of the circadian clock is mediated by a reduction in TIM levels. To elucidate the mechanism of this
process, the sensitivity of TIM regulation by light was tested in an in vitro assay with inhibitors of candidate proteolytic
pathways. The data suggested that TIM is degraded through a ubiquitin-proteasome mechanism. In addition, in cultures from
third-instar larvae, TIM degradation was blocked specifically by inhibitors of proteasome activity. Degradation appeared to
be preceded by tyrosine phosphorylation. Finally, TIM was ubiquitinated in response to light in cultured cells.
In Drosophila, dominant-negative mutations in the beta2 and beta6 proteasome catalytic subunit genes have been identified as dominant temperature-sensitive (DTS) mutations. At restrictive temperature, beta2 and beta6 DTS mutations confer lethality at the pupal stage. I investigate here the role of proteasome activity in regulating cell fate decisions in the sense organ lineage at the early pupal stage. Temperature-shift experiments in beta2 and beta6 DTS mutant pupae occasionally resulted in external sense organs with two sockets and no shaft. This double-socket phenotype was strongly enhanced in conditions in which Notch signaling was up-regulated. Furthermore, conditional overexpression of the beta6 dominant-negative mutant subunit led to shaft-to-socket and to neuron-to-sheath cell fate transformations, which are both usually associated with increased Notch signaling activity. Finally, expression of the beta6 dominant-negative mutant subunit led to the stabilization of an ectopically expressed nuclear form of Notch in imaginal wing discs. This study demonstrates that mutations affecting two distinct proteasome catalytic subunits affect two alternative cell fate decisions and enhance Notch signaling activity in the sense organ lineage. These findings raise the possibility that the proteasome targets an active form of the Notch receptor for degradation in Drosophila.
even-skipped represses wingless and transforms cells that would normally secrete naked cuticle into denticle secreting cells. The GAL4 system can thus be used to study regulatory interactions during embryonic devel- opment. In adults, targeted expression can be used to generate dominant phenotypes for use in genetic screens. We have directed expression of an activated form of the Dras2 protein, resulting in dominant eye and wing defects that can be used in screens to identify other members of the Dras2 signal transduction path- way. SUMMARY
Ubiquitin is a highly conserved polypeptide found in all eukaryotes. The major function of ubiquitin is to target proteins for complete or partial degradation by a multisubunit protein complex called the proteasome. Here, the Drosophila fat facets gene, which is required for the appropriate determination of particular cells in the fly eye, was shown to encode a ubiquitin-specific protease (Ubp), an enzyme that cleaves ubiquitin from ubiquitin-protein conjugates. The Fat facets protein (FAF) acts as a regulatory Ubp that prevents degradation of its substrate by the proteasome. Flies bearing fat facets gene mutations were used to show that a Ubp is cell type--and substrate-specific and a regulator of cell fate decisions in a multicellular organism.
Out of 25,000 EMS-treated third chromosomes examined, ten dominant temperature-sensitive (DTS) lethal mutations which are lethal when heterozygous at 29 degrees C but survive at 22 degrees C were recovered. Seven of the eight mutations mapped were tested for complementation; these mutants probably define eight loci. Only DTS-2 survived in homozygous condition at 22 degrees C; homozygous DTS-2 females expressed a maternal effect on embryonic viability. Two of the mutant-bearing chromosomes, DTS-1 and DTS-6, exhibited dominant phenotypes similar to those associated with Minutes. Each of the seven mutants examined exhibited a characteristic phenotype with respect to the time of death at 29 degrees C and the temperature-sensitive period during development. Only DTS-4 exhibited dominant lethality in triploid females.
Proteasomes are multicatalytic proteinase complexes that function as a major nonlysosomal proteolytic system in all eukaryotes. These particles are made up of 13-15 nonidentical subunits, and they exhibit multiple endopeptidase activities that promote the intracellular turnover of abnormal polypeptides and short-lived regulatory proteins. Although the biochemical characterization of proteasomes has been quite extensive, and although a number of the genes encoding proteasome subunits have been cloned from various organisms, there is still much to be learned about their function in vivo and what role(s) they might play during development. Here, we report the identification of the l(3)73Ai1 allele of Drosophila melanogaster as a dominant temperature-sensitive lethal mutation in a gene encoding a component of the proteasome, thus opening the way for future genetic and developmental studies on this important proteolytic system in a higher eukaryote.
Regulation of the sterol-synthesizing mevalonate pathway occurs in part through feedback-regulated endoplasmic reticulum degradation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R). In yeast, the Hmg2p isozyme of HMG-R is regulated in this manner. We have tested the involvement of ubiquitination in the regulated degradation of Hmg2p, by using both genetic and direct biochemical approaches. Hmg2p degradation required the UBC7 gene, and Hmg2p protein was directly ubiquitinated. Hmg2p ubiquitination was dependent on UBC7 and was specific for the degraded yeast Hmg2p isozyme. Furthermore, Hmg2p ubiquitination was regulated by the mevalonate pathway in a manner consistent with regulation of Hmg2p stability. Thus, regulated ubiquitination appeared to be the mechanism by which Hmg2p stability is controlled in yeast. Finally, our data indicated that the feedback signal controlling Hmg2p ubiquitination and degradation was derived from farnesyl diphosphate, and thus implied conservation of an HMG-R degradation signal between yeast and mammals.
The work of one of us (W. B.) was supported by a grant of the Human Frontiers Science Program. We wish to thank Drs. C. P. Hill (Salt Lake City) and J. M. Flanagan (Brookhaven) for making available to us structural data prior to publication. We are grateful to Drs. F. U. Hartl and M. Kania for critically reading the manuscript.
The selective degradation of many short-lived proteins in eukaryotic cells is carried out by the ubiquitin system. In this pathway, proteins are targeted for degradation by covalent ligation to ubiquitin, a highly conserved small protein. Ubiquitin-mediated degradation of regulatory proteins plays important roles in the control of numerous processes, including cell-cycle progression, signal transduction, transcriptional regulation, receptor down-regulation, and endocytosis. The ubiquitin system has been implicated in the immune response, development, and programmed cell death. Abnormalities in ubiquitin-mediated processes have been shown to cause pathological conditions, including malignant transformation. In this review we discuss recent information on functions and mechanisms of the ubiquitin system. Since the selectivity of protein degradation is determined mainly at the stage of ligation to ubiquitin, special attention is focused on what we know, and would like to know, about the mode of action of ubiquitin-protein ligation systems and about signals in proteins recognized by these systems.
Proteasomes are multicatalytic complexes that function as the major proteolytic machinery in regulated protein degradation. The eukaryotic 20S proteasome proteolytic core structure comprises 14 different subunits: 7 alpha-type and 7 beta-type. DTS7 is a dominant temperature-sensitive (DTS) lethal mutation at 29 degrees that also acts as a recessive lethal at ambient temperatures. DTS7 maps to cytological position 71AB. Molecular characterization of DTS7 reveals that this is caused by a missense mutation in a beta-type subunit gene, beta2. A previously characterized DTS mutant, l(3)73Ai1, results from a missense mutation in another beta-type subunit gene, beta6. These two mutants share a very similar phenotype, show a strong allele-specific genetic interaction, and are rescued by the same extragenic suppressor, Su(DTS)-1. We propose that these mutants might act as "poison subunits," disrupting proteasome function in a dosage-dependent manner, and suggest how they may interact on the basis of the structure of the yeast 20S proteasome.
Two dominant temperature-sensitive (DTS) Drosophila mutants are missense mutations of proteasome genes encoding beta-type subunits beta6/C5 (DTS5) and beta2/Z (DTS7). At nonpermissive temperature (29 degrees C), heterozygotes (DTS5/+ and DTS7/+) develop normally until metamorphosis; pupae fail to mature and die before eclosion. Proteasomes were purified from wild-type (WT) and heterozygous adult flies raised at permissive temperature (25 degrees C). Two-dimensional gel electrophoresis separated at least 28 proteins, 13 of which were identified with monospecific antibodies to alpha6/C2 (five species), alpha2/C3 (three species), alpha7/C8 (three species), alpha5/zeta, and beta1/Y subunits. Both quantitative and qualitative differences were observed between WT and DTS/+ proteasomes, with DTS5/+ deviating more from WT than DTS7/+ proteasomes. In DTS5/+ there was a shift to more acidic species of C2 and C3 and a shift to less acidic species of 32-kDa subunits (#3-#7) recognized by an anti-alpha subunit monoclonal antibody (MCP222) and were losses of two 32-kDa subunits (#2 and #3), decreases in Y (25 kDa; 2-fold) and 31-kDa (#9; 2-fold) subunits, and increases in 52-kDa (#1; 1.9-fold) and 24-kDa (#13; 2.3-fold) subunits. In DTS7/+ there was a less pronounced shift to acidic species of C3 and no pI shift in C2 species and subunits #3-#7 and were decreases in #9 (2.5-fold) and #14 (3-fold) and a loss of #2. The three C8 species were similar between WT, DTS5/+, and DTS7/+ proteasomes. Qualitatively, the most dramatic difference was the appearance of a new 24-kDa subunit (#16) in DTS/+ preparations, with about a 14-fold greater amount of #16 in DTS7/+ than in DTS5/+ proteasomes. Catalytically, WT and DTS/+ proteasomes had similar peptidase activities, although the DTS/+ proteasomes were slightly more sensitive to SDS and elevated temperatures in vitro. The incorporation of DTS subunits apparently altered proteasome assembly and/or processing at permissive temperature with little effect on catalytic activities. These data suggest that at nonpermissive temperature, assembly/processing is more severely affected, producing DTS-containing complexes that lack functions essential for cellular proliferation and differentiation at metamorphosis.
Sensitization of defensive reflexes in Aplysia is a simple behavioral paradigm for studying both short- and long-term memory. In the marine mollusk, as in other animals, memory has at least two phases: a short-term phase lasting minutes and a long-term phase lasting several days or longer. Short-term memory is produced by covalent modification of pre-existing proteins. In contrast, long-term memory needs gene induction, synthesis of new protein, and the growth of new synapses. The switch from short-term (STF) to long-term facilitation (LTF) in Aplysia sensory neurons requires not only positive regulation through gene induction, but also the specific removal of several inhibitory proteins. One important inhibitory protein is the regulatory (R) subunit of the cAMP-dependent protein kinase (PKA). Degradation of R subunits, which is essential for initiating long-term stable memory, occurs through the ubiquitin-proteasome pathway.
The ubiquitin-proteasome pathway has emerged as a central player in the regulation of several diverse cellular processes. Here, we describe the important components of this complex biochemical machinery as well as several important cellular substrates targeted by this pathway and examples of human diseases resulting from defects in various components of the ubiquitin-proteasome pathway. In addition, this review covers the chemistry of synthetic and natural proteasome inhibitors, emphasizing their mode of actions toward the 20S proteasome. Given the importance of proteasome-mediated protein degradation in various intracellular processes, inhibitors of this pathway will continue to serve as both molecular probes of major cellular networks as well as potential therapeutic agents for various human diseases.
The ubiquitin/proteasome pathway is the main non-lysosomal route for intracellular protein degradation in eukaryotes. It is instrumental to various cellular processes, such as cell-cycle progression, transcription and antigen processing. Recent findings also substantiate a pivotal role of the ubiquitin/proteasome pathway in the regulation of apoptosis. Regulatory molecules that are involved in programmed cell death have been identified as substrates of the proteasome. Moreover, key regulators of apoptosis themselves seem to have an active part in the proteolytic inactivation of death executors.
The ubiquitin-proteasome pathway and proteasome inhibitors
- Myung J
- Kim KB
- Crews CM
Personal communication to FlyBase available from http
- M Freeman