Cloning and characterization of Pros45, the Drosophila SUG1 proteasome subunit homolog

The proteasome plays essential roles in a variety of cellular processes, including degradation of the bulk of cellular proteins, degradation of short-lived proteins such as cell cycle regulators, generation of antigenic peptides, and mediating programmed cell death. One of the best characterized subunits of the 26S proteasome is encoded by the yeast gene SUG1. We report here the cloning and characterization of the Drosophila homolog of this gene, Pros45. At the protein level, Pros45 is highly conserved with respect to its homologs in a variety of taxa: it shows 74% identity to yeast Sug1; 86% to mouse m56/mSug1/FZA-B; 87% to human Trip1; and 97% to moth 18-56. Using a genomic clone as a probe for in situ hyridization to polytene chromesomes, we demonstrated that Pros45 maps to 19F, near the base of the X chromosome. Use of a pros45 cDNA clone as a probe revealed a second site of hybridization at 99CD. Pros45 mRNA is found in the unfertilized egg and in all cells of the early embryo. By the end of embryogenesis, Pros45 is expressed predominantly in the central nervous system. Targeted expression of Pros45 in a variety of different cells using the Gal4 UAS P-element system failed to generate an overt phenotype. This study provides the foundation for further examination of the role of the 26S proteasome in homeostasis and development in Drosophila.     

ASK1 Negatively Regulates the 26 S Proteasome

The 26 S proteasome, composed of the 20 S core and 19 S regulatory particle, plays a central role in ubiquitin-dependent proteolysis. Disruption of this process contributes to the pathogenesis of the various diseases; however, the mechanisms underlying the regulation of 26 S proteasome activity remain elusive. Here, cell culture experiments and in vitro assays demonstrated that apoptosis signal-regulating kinase 1 (ASK1), a member of the MAPK kinase kinase family, negatively regulated 26 S proteasome activity. Immunoprecipitation/Western blot analyses revealed that ASK1 did not interact with 20 S catalytic core but did interact with ATPases making up the 19 S particle, which is responsible for recognizing polyubiquitinated proteins, unfolding them, and translocating them into the 20 S catalytic core in an ATP-dependent process. Importantly, ASK1 phosphorylated Rpt5, an AAA ATPase of the 19 S proteasome, and inhibited its ATPase activity, an effect that may underlie the ability of ASK1 to inhibit 26 S proteasome activity. The current findings point to a novel role for ASK1 in the regulation of 26 S proteasome and offer new strategies for treating human diseases caused by proteasome malfunction.     

Localisation of 26S proteasomes with different subunit composition in insect muscles undergoing programmed cell death

The 26S proteasome is a large multisubunit complex involved in degrading both cytoplasmic and nuclear proteins. We have investigated the subcellular distribution of four regulatory ATPase subunits (S6 (TBP7/MS73), S6' (TBP1), S7 (MSS1), and S10b (SUG2)) together with components of 20S proteasomes in the intersegmental muscles (ISM) of Manduca sexta during developmentally programmed cell death (PCD). Immunogold electron microscopy shows that S6 is located in the heterochromatic part of nuclei of ISM fibres. S6' is present in degraded material only outside intact fibres. S7 can be detected in nuclei, cytoplasm and also in degraded material. S10b, on the other hand, is initially found in nuclei and subsequently in degraded cytoplasmic locations during PCD. 20S proteasomes are present in all areas where ATPase subunits are detected, consistent with the presence of intact 26S proteasomes. These results are discussed in terms of heterogeneity of 26S proteasomes, 26S proteasome disassembly and the possible role of ATPases in non-proteasome complexes in the process of PCD. Cell Death and Differentiation (2000) 7, 1210 - 1217.     

The RAD23 Family Provides an Essential Connection between the 26S Proteasome and Ubiquitylated Proteins in Arabidopsis

The ubiquitin (Ub)/26S proteasome system (UPS) directs the turnover of numerous regulatory proteins, thereby exerting control over many aspects of plant growth, development, and survival. The UPS is directed in part by a group of Ub-like/Ub-associated (UBL/UBA) proteins that help shuttle ubiquitylated proteins to the 26S proteasome for breakdown. Here, we describe the collection of UBL/UBA proteins in Arabidopsis thaliana, including four isoforms that comprise the RADIATION SENSITIVE23 (RAD23) family. The nuclear-enriched RAD23 proteins bind Ub conjugates, especially those linked internally through Lys-48, via their UBA domains, and associate with the 26S proteasome Ub receptor RPN10 via their N-terminal UBL domains. Whereas homozygous mutants individually affecting the four RAD23 genes are without phenotypic consequences (rad23a, rad23c, and rad23d) or induce mild phyllotaxy and sterility defects (rad23b), higher-order mutant combinations generate severely dwarfed plants, with the quadruple mutant displaying reproductive lethality. Both the synergistic effects of a rad23b-1 rpn10-1 combination and the response of rad23b plants to mitomycin C suggest that RAD23b regulates cell division. Taken together, RAD23 proteins appear to play an essential role in the cell cycle, morphology, and fertility of plants through their delivery of UPS substrates to the 26S proteasome.     

Yeast Cycloheximide-resistant crl Mutants Are Proteasome Mutants Defective in Protein Degradation

In 1988 McCusker and Haber generated a series of mutants which are resistant to the minimum inhibitory concentration of the protein synthesis inhibitor cycloheximide. These cycloheximide-resistant, temperature-sensitive (crl) mutants, in addition, exhibited other pleiotropic phenotypes, e.g., incorrect response to starvation, hypersensitivity against amino acid analogues, and other protein synthesis inhibitors. Temperature sensitivity of one of these mutants, crl3–2, had been found to be suppressed by a mutation, SCL1–1, which resided in an α-type subunit of the 20S proteasome. We cloned the CRL3 gene by complementation and found CRL3 to be identical to the SUG1/CIM3 gene coding for a subunit of the 19S cap complex of the 26S proteasome. Another mutation, crl21, revealed to be allelic with the 20S proteasomal gene PRE3. crl3–2 and crl21 mutant cells show significant defects in proteasome-dependent proteolysis, whereas the SCL1–1 suppressor mutation causes partial restoration of crl3–2-induced proteolytic defects. Notably, cycloheximide resistance was also detected for other proteolytically deficient proteasome mutants (pre1–1, pre2–1, pre3–1, pre4–1). Moreover, proteasomal genes were found within genomic sequences of 9 of 13 chromosomal loci to which crl mutations had been mapped. We therefore assume that most if not all crl mutations reside in the proteasome and that phenotypes found are a result of defective protein degradation.     

Cloning and characterization of Pros45, the Drosophila SUG1 proteasome subunit homolog

The proteasome plays essential roles in a variety of cellular processes, including degradation of the bulk of cellular proteins, degradation of short-lived proteins such as cell cycle regulators, generation of antigenic peptides, and mediating programmed cell death. One of the best characterized subunits of the 26S proteasome is encoded by the yeast gene SUG1. We report here the cloning and characterization of the Drosophila homolog of this gene, Pros45. At the protein level, Pros45 is highly conserved with respect to its homologs in a variety of taxa: it shows 74% identity to yeast Sug1; 86% to mouse m56/mSug1/FZA-B; 87% to human Trip1; and 97% to moth 18-56. Using a genomic clone as a probe for in situ hyridization to polytene chromesomes, we demonstrated that Pros45 maps to 19F, near the base of the X chromosome. Use of a pros45 cDNA clone as a probe revealed a second site of hybridization at 99CD. Pros45 mRNA is found in the unfertilized egg and in all cells of the early embryo. By the end of embryogenesis, Pros45 is expressed predominantly in the central nervous system. Targeted expression of Pros45 in a variety of different cells using the Gal4 UAS P-element system failed to generate an overt phenotype. This study provides the foundation for further examination of the role of the 26S proteasome in homeostasis and development in Drosophila. Received: 23 October 1997 / Accepted: 6 March 1998     

NADH Binds and Stabilizes the 26S Proteasomes Independent of ATP

The 26S proteasome is the end point of the ubiquitin- and ATP-dependent degradation pathway. The 26S proteasome complex (26S PC) integrity and function has been shown to be highly dependent on ATP and its homolog nucleotides. We report here that the redox molecule NADH binds the 26S PC and is sufficient in maintaining 26S PC integrity even in the absence of ATP. Five of the 19S proteasome complex subunits contain a putative NADH binding motif (GxGxxG) including the AAA-ATPase subunit, Psmc1 (Rpt2). We demonstrate that recombinant Psmc1 binds NADH via the GxGxxG motif. Introducing the ΔGxGxxG Psmc1 mutant into cells results in reduced NADH-stabilized 26S proteasomes and decreased viability following redox stress induced by the mitochondrial inhibitor rotenone. The newly identified NADH binding of 26S proteasomes advances our understanding of the molecular mechanisms of protein degradation and highlights a new link between protein homeostasis and the cellular metabolic/redox state.     

Genetic Evidence Linking Age-Dependent Attenuation of the 26S Proteasome with the Aging Process

The intracellular accumulation of unfolded or misfolded proteins is believed to contribute to aging and age-related neurodegenerative diseases. However, the links between age-dependent proteotoxicity and cellular protein degradation systems remain poorly understood. Here, we show that 26S proteasome activity and abundance attenuate with age, which is associated with the impaired assembly of the 26S proteasome with the 19S regulatory particle (RP) and the 20S proteasome. In a genetic gain-of-function screen, we characterized Rpn11, which encodes a subunit of the 19S RP, as a suppressor of expanded polyglutamine-induced progressive neurodegeneration. Rpn11 overexpression suppressed the age-related reduction of the 26S proteasome activity, resulting in the extension of flies'' life spans with suppression of the age-dependent accumulation of ubiquitinated proteins. On the other hand, the loss of function of Rpn11 caused an early onset of reduced 26S proteasome activity and a premature age-dependent accumulation of ubiquitinated proteins. It also caused a shorter life span and an enhanced neurodegenerative phenotype. Our results suggest that maintaining the 26S proteasome with age could extend the life span and suppress the age-related progression of neurodegenerative diseases.Ubiquitin-conjugated, misfolded protein aggregates are observed in the brain during normal aging and in late-onset human neurodegenerative diseases, such as Alzheimer''s, Parkinson''s, and polyglutamine diseases (e.g., Huntington''s disease or spinocerebellar ataxias) (9). Many of the mutations that cause dominantly inherited neurodegenerative diseases dramatically increase the amount of protein aggregates in vitro and in vivo, supporting the widely accepted hypothesis that proteotoxicity caused by the aggregates underlies the pathogenesis of many neurodegenerative diseases (32). Proteotoxicity can have many effects, including disruption of microtubule-dependent axonal transport (10), perturbation of membrane permeability (23), and impaired function of the ubiquitin-proteasome system (UPS) (1, 17). Aggregation-mediated toxicity has also been suggested in normal aging, because recent reports show that the impairment of autophagy in the central nervous system causes accumulation of ubiquitinated proteins and leads to neurodegenerative diseases (12, 21). These observations suggest that the continuous clearance of misfolded proteins through cellular degradation systems, including the UPS and autophagy, is important for preventing aggregation-mediated proteotoxicity both in age-related neurodegenerative diseases and in normal aging.Clinical symptoms of neurodegenerative diseases generally do not appear or progress until advanced ages, not only in sporadic forms but also in inherited forms of neurodegenerative diseases (26). These observations suggest that aggregation-mediated toxicity appears in a late-onset manner both in normal aging and in neurodegenerative diseases. Furthermore, a link between the aging process and aggregation-mediated proteotoxicity has been suggested by evidence that Huntington''s disease-associated proteotoxicity was ameliorated when the aging process slowed, that is, the life span extension via decreased insulin/insulin growth factor-1-like signaling in Caenorhabditis elegans (13, 31).A possible mechanism for the late onset of aggregation-mediated toxicity is age-related impairment of the UPS, because loss-of-function mutations in genes encoding UPS components can enhance the cytotoxicity of protein aggregation in dominantly inherited neurodegenerative diseases (4, 5, 18). In addition, an age-related decline of proteasome activity has been observed in the tissues of humans and other mammals (8) and in aged flies (36). Considering the role of the proteasome in neuroprotection and the age dependence of most neurodegenerative diseases, the age-related decline of proteasome activity could well be a key factor both in normal aging and in the late onset and/or progression of neurodegenerative diseases. However, the mechanism underlying the age-related decline of proteasome activity remains to be elucidated, and there is no direct genetic evidence showing that the age-related decline of proteasome activity causes age-related aggregation-mediated toxicity in normal aging and in age-related neurodegenerative diseases.Here, we studied the age-related decline of proteasome activity by using Drosophila melanogaster and found age-related attenuation of the 26S proteasome activity and abundance that was associated with impaired assembly of the 26S proteasome with the 19S regulatory particle (RP) and the 20S proteasome. In a genetic gain-of-function screen, we identified Rpn11, which encodes one of the lid subunits in the 19S RP, as a suppressor of the age-dependent progression of a polyglutamine-induced neurodegenerative phenotype. The overexpression of Rpn11 prevented the age-related reduction of the 26S proteasome activity, which suppressed the age-dependent accumulation of ubiquitinated proteins and extended the life span. On the other hand, the loss of function of Rpn11 enhanced the age-related reduction of 26S proteasome activity, leading to a shorter life span, a premature age-dependent accumulation of ubiquitinated proteins, and an early onset of a neurodegenerative phenotype. Our results demonstrate for the first time that the age-related reduction of the 26S proteasome activity is a key factor in the induction of certain age-related biological changes and in the increased risk for the onset or progression of neurodegenerative diseases. Our findings imply that improving the amount and/or activity of the 26S proteasome by overexpressing a lid subunit, such as Rpn11, could provide an extension to the mean life span and prevent the age-dependent onset or progression of neurodegeneration.     

How does the plant proteasome control leaf size?

The ubiquitin/26S proteasome pathway plays a central role in the degradation of short-lived regulatory proteins to control many cellular events. The Arabidopsis genome contains two genes, AtRPT2a and AtRPT2b, which encode paralog molecules of the RPT2 subunit of 19S proteasome. We demonstrated that mutation of the AtRPT2a gene causes a specific phenotype of enlarged leaves due to increased cell size in correlation with expanded endoreduplication. This phenotype was also observed in the knockout mutant of AtRPT5a, which encodes one of the paralogs of the RPT5 subunit. Taken together, this suggests that a cell size-specific proteasome consisting of AtRPT2a and AtRPT5a is involved in controlling cell size during leaf development.Key words: 26S proteasome, endoreduplication, leaf size, RPT2a, RPT5a     

Mouse homologue of yeast Prp19 interacts with mouse SUG1, the regulatory subunit of 26S proteasome

Yeast Prp19 has been shown to involve in pre-mRNA splicing and DNA repair as well as being an ubiquitin ligase. Mammalian homologue of yeast Prp19 also plays on similar functional activities in cells. In the present study, we isolated mouse SUG1 (mSUG1) as binding partner of mouse Prp19 (mPrp19) by the yeast two-hybrid system. We confirmed the interaction of mPrp9 with mSUG1 by GST pull-down assay and co-immunoprecipitation assay. The N-terminus of mPrp19 including U-box domain was associated with the C-terminus of mSUG1. Although, mSUG1 is a regulatory subunit of 26S proteasome, mPrp19 was not degraded in the proteasome-dependent pathway. Interestingly, GFP-mPrp19 fusion protein was co-localized with mSUG1 protein in cytoplasm as the formation of the speckle-like structures in the presence of a proteasome inhibitor MG132. In addition, the activity of proteasome was increased in cells transfected with mPrp19. Taken together, these results suggest that mPrp19 involves the regulation of protein turnover and may transport its substrates to 26S proteasome through mSUG1 protein.     

Nitric oxide regulates the 26S proteasome in vascular smooth muscle cells

It is well established that nitric oxide (NO) inhibits vascular smooth muscle cell (VSMC) proliferation by modulating cell cycle proteins. The 26S proteasome is integral to protein degradation and tightly regulates cell cycle proteins. Therefore, we hypothesized that NO directly inhibits the activity of the 26S proteasome. The three enzymatic activities (chymotrypsin-like, trypsin-like and caspase-like) of the 26S proteasome were examined in VSMC. At baseline, caspase-like activity was approximately 3.5-fold greater than chymotrypsin- and trypsin-like activities. The NO donor S-nitroso-N-acetylpenicillamine (SNAP) significantly inhibited all three catalytically active sites in a time- and concentration-dependent manner (P < 0.05). Caspase-like activity was inhibited to a greater degree (77.2% P < 0.05). cGMP and cAMP analogs and inhibitors had no statistically significant effect on basal or NO-mediated inhibition of proteasome activity. Dithiothreitol, a reducing agent, prevented and reversed the NO-mediated inhibition of the 26S proteasome. Nitroso-cysteine analysis following S-nitrosoglutathione exposure revealed that the 20S catalytic core of the 26S proteasome contains 10 cysteines which were S-nitrosylated by NO. Evaluation of 26S proteasome subunit protein expression revealed differential regulation of the α and β subunits in VSMC following exposure to NO. Finally, immunohistochemical analysis of subunit expression revealed distinct intracellular localization of the 26S proteasomal subunits at baseline and confirmed upregulation of distinct subunits following NO exposure. In conclusion, NO reversibly inhibits the catalytic activity of the 26S proteasome through S-nitrosylation and differentially regulates proteasomal subunit expression. This may be one mechanism by which NO exerts its effects on the cell cycle and inhibits cellular proliferation in the vasculature.     

The 26S proteasome drives trinucleotide repeat expansions

Trinucleotide repeat (TNR) expansion is the causative mutation for at least 17 inherited neurological diseases. An important question in the field is which proteins drive the expansion process. This study reports that the multi-functional protein Sem1 is a novel driver of TNR expansions in budding yeast. Mutants of SEM1 suppress up to 90% of expansions. Subsequent analysis showed that Sem1 facilitates expansions via its function in the 26S proteasome, a highly conserved multi-subunit complex with both proteolytic and non-proteolytic functions. The proteolytic function of the 26S proteasome is relevant to expansions, as mutation of additional proteasome components or treatment of yeast with a proteasome inhibitor suppressed CTG•CAG expansions. The 26S proteasome also drives expansions in human cells. In a human astrocytic cell line, siRNA-mediated knockdown of 26S proteasome subunits PSMC5 or PSMB3 reduced expansions. This expansion phenotype, both in yeast and human cells, is dependent on the proteolytic activity of the proteasome rather than a stress response owing to depletion of free ubiquitin. Thus, the 26S proteasome is a novel factor that drives expansions in both yeast and human cells by a mechanism involving protein degradation.