20S Core Research Articles

Characterization of a new series of non-covalent proteasome inhibitors with exquisite potency and selectivity for the 20S β5-subunit

The mammalian 26S proteasome is a 2500 kDa multi-catalytic complex involved in intracellular protein degradation. We describe the synthesis and properties of a novel series of non-covalent di-peptide inhibitors of the proteasome used on a capped tri-peptide that was first identified by high-throughput screening of a library of approx. 350000 compounds for inhibitors of the ubiquitin–proteasome system in cells. We show that these compounds are entirely selective for the β5 (chymotrypsin-like) site over the β1 (caspase-like) and β2 (trypsin-like) sites of the 20S core particle of the proteasome, and over a panel of less closely related proteases. Compound optimization, guided by X-ray crystallography of the liganded 20S core particle, confirmed their non-covalent binding mode and provided a structural basis for their enhanced in vitro and cellular potencies. We demonstrate that such compounds show low nanomolar IC50 values for the human 20S β5 site in vitro, and that pharmacological inhibition of this site in cells is sufficient to potently inhibit the degradation of a tetra-ubiquitin–luciferase reporter, activation of NFκB (nuclear factor κB) in response to TNF-α (tumour necrosis factor-α) and the proliferation of cancer cells. Finally, we identified capped di-peptides that show differential selectivity for the β5 site of the constitutively expressed proteasome and immunoproteasome in vitro and in B-cell lymphomas. Collectively, these studies describe the synthesis, activity and binding mode of a new series of non-covalent proteasome inhibitors with unprecedented potency and selectivity for the β5 site, and which can discriminate between the constitutive proteasome and immunoproteasome in vitro and in cells.

Four-Dimensional Orthogonal Electrophoresis System for Screening Protein Complexes and Protein−Protein Interactions Combined with Mass Spectrometry

Most current approaches for purification and identification of protein complexes adopt affinity purifications combined with mass spectrometry, such as co-immunoprecipitation and tandem affinity purification. Herein, we propose a new approach, termed as the four-dimensional orthogonal electrophoresis (4-DE) system, to find and analyze the cytoplasmic protein complexes. 4-DE system is composed of two parts: nondenaturing part (Part I) and denaturing part (Part II). Through Part I and decision procedure separations, six protein complex candidates 20S core particle of proteasome (CP), hemoglobin (Hb, α2β2), Hb (α2δ2), peroxiredoxin-2 (PRDX2), carbonic anhydrase-1 (CAH1), and heat shock protein 60 (HSP60) were separated. CP, Hb (α2β2), PRDX2, and HSP60 with different MWs and pI's were chosen for Part II proteomic analysis. The results indicate that 4-DE is not only suitable for studying protein complexes and protein-protein interactions as well as structural proteomics from complex biological samples, but can also be easy to separate and concentrate intact protein complexes from dilute complex samples.

Dissection of the assembly pathway of the proteasome lid in Saccharomyces cerevisiae

The 26S proteasome is a highly conserved multisubunit protease that degrades ubiquitinated proteins in eukaryotic cells. It comprises a 20S core particle and two 19S regulatory particles that are further divided into the lid and base complexes. The lid is a nine subunits complex that is structurally related to the COP9 signalosome and the eukaryotic initiation factor 3. Although the assembly pathway of the 20S and the base are well described, that of the lid is still unclear. In this study, we dissected the lid assembly using yeast lid mutant cells, rpn7-3, Δ rpn9, and rpn12-1. Using mass spectrometry, we identified a number of lid subassemblies, such as Rpn3–Rpn7 pair and a lid-like complex lacking Rpn12, in the mutants. Our analysis suggests that the assembly of the lid is a highly ordered and multi-step process; first, Rpn5, 6, 8, 9, and 11 are assembled to form a core module, then a second module, consisting of Rpn3, 7, and Sem1, is attached, followed by the incorporation of Rpn12 to form the lid complex.

Abstract LB-295: Identification of the first class of proteasome inhibitors that impair 26S proteasome assembly in vitro.

Abstract The Ubiquitin-Proteasome System (UPS) is the main non-lysosomial proteolytic machinery in the eukaryotic cells. Prior to their degradation proteins are adressed to the catalytic 26S proteasome complex composed of a 20S “core” that contains proteolytic activities, assembled with one or two 19S regulatory particles that bind proteins to be degraded. Due to its central role of in regulating cell cycle progression and signal transduction the UPS has been proposed as a suitable target for antineoplastic strategies. To date only catalytic inhibitors of proteasome, targeting proteolytic activities of the 20S core particle, have been characterized and/or have emerged as potent anticancer drugs (1). However, the capacity of the UPS to degrade proteins not only depends of the catalytic activities of the 20S core, but also of the 26S proteasome assembly, a dynamic and tightly controlled cellular process. Thus, compounds impairing 26S proteasome assembly could represent a novel class of proteasome inhibitors. With the aim of identifying such molecules we first set up a robust biochemical screening assay allowing us to follow 26S proteasome assembly in vitro. First, 26S proteasome was purified from Hela cells using affinity chromatography followed by gel filtration. Then, experimental conditions were determined for in vitro 26S disassembly into its constitutive sub-complexes (19S+20S) ands its re-assembly as an active and functional 26S entity. The differential CTL-activity between 26S and 20S proteasomes (2) was then used as the readout to follow the reassembly of 26S in vitro, prior to be confirmed using in gel overlay experiments and western-blotting. Using this biochemical assay: 1) we screened naturals compounds endowed with antiproliferative properties and described as inhibitors of the ubiquitine-proteasome pathway (3). 2) we identified structurally-related natural molecules, epidithiodioxopiperazine and diketodithiopiperazine derivatives, as the first class of molecules inhibiting proteasome assembly without significantly affecting neither 26S nor 20S catalytic activity in vitro. Our research efforts now concentrate in showing that: 1) these molecules inhibit proteasome assembly in cells, and that 2) this inhibition can be correlated with their antiproliferative effects. References Sterz J. et al. Extert Opin. Invest. Drugs, 2008, 17 Rivett AJ et al., Experimental Gerontology, 2002, 37 Vandenberghe I. et al., Biochem. Pharmacol., 2008, 76 Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-295.

Pup grows up: in vitro characterization of the degradation of pupylated proteins

By reconstituting the recently discovered prokaryotic ubiquitin-like protein (Pup)--proteasome degradation system in vitro, Weber-Ban and colleagues (Striebel et al, 2010) elucidate its mechanism and describe a surprising variation on the established principles of protease targeting. Nevertheless, their findings suggest that the bacterial and eukaryotic systems follow the same overall principles even if the details differ.

Exploring the Dynamics of the Proteasome Interaction Network by Quantitative Proteomics

Most cellular processes in a cell are mediated by large protein assemblies or multi‐protein complexes, not by individual proteins. The protein complexes resemble molecular machines with highly ordered dynamic structures that assemble, store and transduce biological information. Systematic identification and characterization of these macromolecular machines will provide the essential knowledge base and set the stage for linking proteome dynamics and architecture to cellular functions. The 26S proteasome is a macromolecular machine existing in all eukaryotes and is responsible for ubiquitin/ATP dependent protein degradation in both cytosol and nucleus. It consists of two subcomplexes, the 20S core particle (CP) and the 19S regulatory complex (RP). The 20S CP is responsible for various catalytic activities, while the 19S RP is involved in several biochemical functions including recognition and unfolding of polyubiquitinated substrates, assisting in opening the gate of the 20S chamber and subsequently translocating unfolded substrates into the 20S for degradation. Selective degradation in cells is tightly controlled and plays an important role for regulating cell cycle progression, signal transduction and maintaining genome stability, etc. Disruption of normal proteolytic destruction pathways can lead to a wide range of human disease, including cancer and neurodegenerative disorders. Despite intensive research, the unknown still exceeds what are currently known on ubiquitin/proteasome dependent degradation. In order to understand the regulation of the 26S proteasome function, we have developed an integrated proteomic approaches to comprehensively characterize the 26S proteasome complexes. In addition, the QTAX strategy, quantitative identification of tandem‐affinity purified cross‐linked (x) protein complexes was developed to capture protein interaction of all natures in living cells, which has been successfully applied to map the dynamic interaction network of the 26S proteasome under various physiological conditions. Coupled with protein interaction network analysis, validity of the identified proteasome interactions have been confirmed and the biological significance of the identified interactions has been suggested. The methodologies presented here can be applied for the study of other protein complexes and their interaction networks.

CHIP promotes the degradation of mutant SOD1 by reducing its interaction with VCP and S6/S6′ subunits of 26S proteasome

Previously we showed that CHIP, a co-chaperone of Hsp70 and E3 ubiquitin ligase, can promote the degradation of mutant SOD1 linked to familial amyotrophic lateral sclerosis (fALS) via a mechanism not involving SOD1 ubiquitylation. Here we present evidence that CHIP functions in the interaction of mutant SOD1 with 26S proteasomes. Bag-1, a coupling factor between molecular chaperones and the proteasomes, formed a complex with SOD1 in an hsp70-dependent manner but had no direct effect on the degradation of mutant SOD1. Instead, Bag-1 stimulated interaction between CHIP and the proteasome-associated protein VCP (p97), which do not associate normally. Over-expressed CHIP interfered with the association between mutant SOD1 and VCP. Conversely, the binding of CHIP to mutant SOD1 was inhibited by VCP, implying that the chaperone complex and proteolytic machinery are competing for the common substrates. Finally we observed that mutant SOD1 strongly associated with the 19S complex of proteasomes and CHIP over-expression specifically reduced the interaction between S6/S6′ ATPase subunits and mutant SOD1. These results suggest that CHIP, together with ubiquitin-binding proteins such as Bag-1 and VCP, promotes the degradation of mutant SOD1 by facilitating its translocation from ATPase subunits of 19S complex to the 20S core particle.

Phosphorylation and Methylation of Proteasomal Proteins of the HaloarcheonHaloferax volcanii

Proteasomes are composed of 20S core particles (CPs) of α- and β-type subunits that associate with regulatory particle AAA ATPases such as the proteasome-activating nucleotidase (PAN) complexes of archaea. In this study, the roles and additional sites of post-translational modification of proteasomes were investigated using the archaeon Haloferax volcanii as a model. Indicative of phosphorylation, phosphatase-sensitive isoforms of α1 and α2 were detected by 2-DE immunoblot. To map these and other potential sites of post-translational modification, proteasomes were purified and analyzed by tandem mass spectrometry (MS/MS). Using this approach, several phosphosites were mapped including α1 Thr147, α2 Thr13/Ser14 and PAN-A Ser340. Multiple methylation sites were also mapped to α1, thus, revealing a new type of proteasomal modification. Probing the biological role of α1 and PAN-A phosphorylation by site-directed mutagenesis revealed dominant negative phenotypes for cell viability and/or pigmentation for α1 variants including Thr147Ala, Thr158Ala and Ser58Ala. An H. volcanii Rio1p Ser/Thr kinase homolog was purified and shown to catalyze autophosphorylation and phosphotransfer to α1. The α1 variants in Thr and Ser residues that displayed dominant negative phenotypes were significantly reduced in their ability to accept phosphoryl groups from Rio1p, thus, providing an important link between cell physiology and proteasomal phosphorylation.

Inhibition of 19S proteasomal regulatory complex subunit PSMD8 increases polyspermy during porcine fertilization in vitro

The 26S proteoasome is a multi-subunit protease specific to ubiquitinated substrate proteins. It is composed of a 20S proteasomal core with substrate degradation activity, and a 19S regulatory complex that acts in substrate recognition, deubiquitination, priming and transport to the 20S core. Inhibition of proteolytic activities associated with the sperm acrosome-borne 20S core prevents fertilization in mammals, ascidians and echinoderms. Less is known about the function of the proteasomal 19S complex during fertilization. The present study examined the role of PSMD8, an essential non-ATPase subunit of the 19S complex, in sperm-ZP penetration during porcine fertilization in vitro (IVF). Immunofluorescence localized PSMD8 to the outer acrosomal membrane, acrosomal matrix and the inner acrosomal membrane. Colloidal gold transmission electron microscopy detected PSMD8 on the surface of vesicles in the acrosomal shroud, formed as a result of zona pellucida-induced acrosomal exocytosis. Contrary to the inhibition of fertilization by blocking of the 20S core activities, fertilization and polyspermy rates were increased by adding anti-PSMD8 antibody to fertilization medium. This observation is consistent with a possible role of PSMD8 in substrate deubiquitination, a process which when blocked, may actually accelerate substrate proteolysis by the 26S proteasome. Subunit PSMD8 co-immunoprecipitated with acrosomal surface-associated spermadhesin AQN1. This association indicates that the sperm acrosome-borne proteasomes become exposed onto the sperm surface following the acrosomal exocytosis. Since immunological blocking of subunit PSMD8 increases the rate of polyspermy during porcine fertilization, the activity of the 19S complex may be a rate-limiting factor contributing to anti-polyspermy defense during porcine fertilization.

Proteomic Characterization of Human Erythrocyte 20S Proteasome and Analysis of Species-dependent 20S Proteasome Heterogeneity

Abstract A method for the purification of 20S proteasome (20S core particle, CP) was developed by combining differential centrifugations with nondenaturing polyacrylamide gel electrophoresis (native-PAGE), irrespective of species origins of CPs. CP purified from human erythrocytes was subjected to proteomic analysis by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), revealing 33 spots of subunit isoforms with different molecular weights and isoelectric points. Furthermore, other four CPs were purified from yeast, mouse liver, and two pancreatic cancer cell lines SW1990 and PANC-1 using the method mentioned above and subjected to proteasome heterogeneity analysis by two-dimensional native/SDS-PAGE (native/sodium dodecyl sulphate polyacrylamide gel electrophoresis), in combination with CP from erythrocytes. In conclusion, the method described acts as a rapid and effective tool for CP isolations, and the results obtained may serve as a footstone for the investigations of species-dependent proteasome heterogeneity.

Proteasome inhibitor-induced apoptosis in acute myeloid leukemia: A correlation with the proteasome status

The proteasome plays a critical role in the regulation of many cellular processes, including the cell cycle and tumor growth. The proteasome inhibitor bortezomib has recently been approved for the treatment of relapsed and refractory multiple myeloma. In this study, we investigated the induction of apoptosis by proteasome inhibitors in several human acute myeloid leukemia (AML) cell lines and in primary cells from patients. We demonstrate that these drugs induce a high level of apoptosis in the KG1a cell line, in which the therapeutic drug daunorubicin is poorly active, compared to other AML cell lines. In parallel, we found that significantly different levels of apoptosis were induced in primary cells from patients depending on the FAB-based differentiation status of these cells. Moreover, the level of 20S proteasome in KG1a cells was also high compared to other AML cell lines, suggesting a relationship between the high sensitivity to proteasome inhibitors and an elevated amount of 20S proteasome. In good accordance, we identified two groups of patient cells expressing high and low levels of 20S proteasome, with respective high and low sensitivity to proteasome inhibitors. Further comparison of the proteasome status in KG1a and U937 cells also suggests that a high proportion of the 19S regulatory complex in U937 cells compared to the 20S core complex may explain an increased proteasome activity. Altogether, our results suggest that various AML subtypes may present different responses to proteasome inhibitors, that these molecules can be potentially considered as interesting therapeutic alternatives for these pathologies, and that the amount of 20S proteasome in AML cells may be predictive of the cellular response to these inhibitors.

RNAi suppression of RPN12a decreases the expression of type-A ARRs, negative regulators of cytokinin signaling pathway, in Arabidopsis.

The 26S proteasome is a 2-MDa complex with a central role in protein turn over. The 26S proteasome is comprised of one 20S core particle and two 19S regulatory particles (RPs). The RPN12a protein, a non-ATPase subunit of the 19S RP, was previously shown to be involved in cytokinin signaling in Arabidopsis. To further investigate cellular roles of RPN12a, RNAi transgenic plants of RPN12a were constructed. As expected, the 35S:RNAi-RPN12a plants showed cytokinin signaling defective phenotypes, including abnormal formation of leaves and inflorescences. Furthermore, RNAi knock-down transgenic plants exhibited additional unique phenotypes, including concave and heart-shape cotyledons, triple cotyledons, irregular and clustered guard cells, and defects in phyllotaxy, all of which are typical for defective cytokinin signaling. We next examined the mRNA level of cytokinin signaling components, including type-A ARRs, type-B ARRs, and CRFs. The expression of type-A ARRs, encoding negative regulators of cytokinin signaling, was markedly reduced in 35S:RNAi-RPN12a transgenic plants relative to that in wild type plants, while type-B ARRs and CRFs were unaffected. Our results also indicate that in vivo stability of the ARR5 protein, a negative regulator of cytokinin signaling, is mediated by the 26S proteasome complex. These results suggest that RPN12a participates in feedback inhibitory mechanism of cytokinin signaling through modulation of the abundance of ARR5 protein in Arabidopsis.

Inhibition of Human 20S Proteasome by Compounds from Seeds of Psoralea corylifolia

The 26S pro-teasome is composed of 20S core and 19S regulator. Since pro-teasomes interact primarily with endogenous proteins, inhi-bition of the proteolytic action of the proteasome may block the signaling action of the transcription factor NF- κB and, thus, inhibit the completion of the cell cycle and hence the mitotic proliferation of cancerous cells, leading to cell death by apopto-sis and inhibition of angiogenesis and metastasis.

An atomic model AAA-ATPase/20S core particle sub-complex of the 26S proteasome

The 26S proteasome is the most downstream element of the ubiquitin–proteasome pathway of protein degradation. It is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). The RP consists of 6 AAA-ATPases and at least 13 non-ATPase subunits. Based on a cryo-EM map of the 26S proteasome, structures of homologs, and physical protein–protein interactions we derive an atomic model of the AAA-ATPase–CP sub-complex. The ATPase order in our model (Rpt1/Rpt2/Rpt6/Rpt3/Rpt4/Rpt5) is in excellent agreement with the recently identified base–precursor complexes formed during the assembly of the RP. Furthermore, the atomic CP–AAA-ATPase model suggests that the assembly chaperone Nas6 facilitates CP–RP association by enhancing the shape complementarity between Rpt3 and its binding CP alpha subunits partners.

Insights into the molecular architecture of the 26S proteasome

Cryo-electron microscopy in conjunction with advanced image analysis was used to analyze the structure of the 26S proteasome and to elucidate its variable features. We have been able to outline the boundaries of the ATPase module in the "base" part of the regulatory complex that can vary in its position and orientation relative to the 20S core particle. This variation is consistent with the "wobbling" model that was previously proposed to explain the role of the regulatory complex in opening the gate in the alpha-rings of the core particle. In addition, a variable mass near the mouth of the ATPase ring has been identified as Rpn10, a multiubiquitin receptor, by correlating the electron microscopy data with quantitative mass spectrometry.

Impaired nuclear functions lead to increased senescence and inefficient differentiation in human myoblasts with a dominant p.R545C mutation in the LMNA gene

We have studied myoblasts from a patient with a severe autosomal dominant Emery-Dreifuss muscular dystrophy (AD-EDMD) caused by an arginine 545 to cystein point mutation (p.R545C) in the carboxy-terminal domain of the lamin A/C gene. This mutation has pleiotropic cellular effects on these myoblasts as demonstrated by nuclear structural defects, exhibiting lobulations which increase with cell passages in culture. The organization of both lamin A/C and its inner nuclear membrane partner emerin are altered, eventually showing a honeycomb pattern upon immunofluorescence microscopy. In addition, the distribution of histone H3 trimethylated at lysine 27 and of phosphorylated RNA polymerase II, markers of inactive and active chromatin domains, respectively, are altered suggesting an impact on gene expression. Patient myoblasts also presented a high index of senescence in ex vivo culture. Moreover, our data show for the first time in an AD-EDMD context that the 20S core particle of the proteasome was inactivated. With cell passages, the 20S core protein progressively accumulated into discrete nuclear foci that largely colocalized with promyelocytic leukemia (PML) bodies while p21 accumulated throughout the nuclear compartment. Proteasome inactivation has been linked to normal cellular ageing. Our data indicate that it may also contribute to premature senescence in AD-EDMD patient myoblasts. Finally, when transferred to low-serum medium, patient myoblasts were deficient in ex vivo differentiation, as assessed by the absence of myotube formation and myogenin induction. Altogether, these data suggest that the LMNA mutation p.R545C impairs both proliferation and differentiation capacities of myoblasts as part of the pathogenesis of AD-EDMD.

Evidence for the Participation of Sperm Proteasomes in Zona Pellucida Degradation and Zona-Induced Acrosomal Exocytosis During Fertilization in the Domestic Pig.

Realization of importance of the ubiquitin-proteasome pathway (UPP) in mammalian fertilization is continuing to grow with every new publication. The UPP is known to participate in multiple steps of mammalian fertilization, including sperm capacitation, acrosomal exocytosis (AE), and sperm-zona pellucida (ZP) penetration. In the UPP, a chaperone protein ubiquitin binds in tandem fashion to proteins that need to be recycled, and targets them for proteolysis by a multi-subunit protease, the 26S proteasome. We have been studying the participation of the 26S proteasome in the ZP-induced AE and sperm-ZP penetration during porcine fertilization. The 26S proteasome is a ~2 MDa, complex consisting of a 19S regulatory complex (role: ubiquitinated substrate recognition, deubiquitination and unfolding) and the 20S core (role: substrate degradation). Three distinct proteolytic activities reside in the 20S core: trypsin-like, chymotrypsin-like and caspase-like. In the presence of proteasomal inhibitors, porcine sperm are unable to fertilize oocytes, which may be due either to inhibition of acrosomal exocytosis and/or degradation of the ZP matrix. To understand this, fresh, capacitated boar spermatozoa were incubated with solubilized porcine ZP proteins with or without proteasomal inhibitors MG 132 (inhibits chymotrypsin-like core activity), clasto-lactacystin β-lactone (targets trypsin-like activity) and epoxomicin (targets chymotrypsin-like activity). After two-hour incubation, soluble ZP proteins and exocytosed acrosomal ghosts were separated from sperm pellets by centrifugation. They were analyzed for evidence of ZP protein degradation by the sperm proteasomes and for the accumulation of undegraded sperm acrosomal proteins. Results show that solubilized ZP proteins readily bind to acrosomal caps of the capacitated boar spermatozoa and induce AE. Presence of proteasomal inhibitors during sperm-ZP coincubation protected the zona glycoprotein ZPB from being degraded, without affecting its deglycosylation. Additionally, release of the 26S proteasomes with the acrosomal ghosts was reduced in the presence of proteasomal inhibitors. Acrosomal ghost-associated proteins such as Sperm Adhesion Molecule 1 (SPAM1), Lactadherin/SP47, Beta-Acrosin, Zona Pellucida-Binding Protein 2 (ZPBP2/IAM38) and Proacrosin-binding Protein (SP32), were protected from proteasomal degradation and accumulated in the ghost fraction. Thus, it appears that proteasomes are released from the sperm acrosome during ZP-induced acrosomal exocytosis. However, since acrosomal ghost remains zona bound and surrounds the sperm head during early stages of sperm-ZP penetration, the resident acrosomal proteasomes could assist the spermatozoon during the formation of the fertilization slit. A second pool of proteasomes remains associated with the inner acrosomal membrane (IAM) of the exocytosed spermatozoa; it may provide the fertilizing spermatozoon with a mean to digest the zona, as it advances through the fertilization slit. Collectively these results explain why the inhibition of sperm proteasomal activity prevents porcine fertilization. This model provides tools to modulate fertilization in vitro and points to a new direction in which to contemplate non-hormonal contraceptive design. Supported by USDA-NRI grant #2007-01319 and by seed funding from the F21C Program of UM (PS). (platform)

Inhibition of 19S Proteasomal Regulatory Complex Subunit PSMD8 Increases Polyspermy During Porcine Fertilization In Vitro.

The 26S proteoasome is a multi-subunit protease specific to ubiquitinated substrate proteins. It is composed of a 20S proteasomal core with substrate degradation activity, and a 19S regulatory complex that acts in substrate recognition, deubiquitination, priming and transport to the 20S core. Inhibition of proteolytic activities associated with the sperm acrosome-borne 20S core prevents fertilization in mammals, ascidians and echinoderms. Less is known about the function of the proteasomal 19S complex during fertilization. The present study examined the role of PSMD8, an essential non-ATPase subunit of the 19S complex, in sperm-ZP penetration during porcine fertilization in vitro (IVF). Immunofluorescence localized PSMD8 to the outer acrosomal membrane, acrosomal matrix and the inner acrosomal membrane. Colloidal gold transmission electron microscopy detected PSMD8 on the surface of vesicles in the acrosomal shroud, formed as a result of zona pellucida-induced acrosomal exocytosis. Contrary to the inhibition of fertilization by blocking of the 20S core activities, fertilization and polyspermy rates were increased by adding anti-PSMD8 antibody to fertilization medium. This observation is consistent with a possible role of PSMD8 in substrate deubiquitination, a process which when blocked, may actually accelerate substrate proteolysis by the 26S proteasome. Subunit PSMD8 co-immunoprecipitated with acrosomal surface-associated spermadhesin AQN1. This association indicates that the sperm acrosome-borne proteasomes become exposed onto the sperm surface following the acrosomal exocytosis. Since immunological blocking of subunit PSMD8 increases the rate of polyspermy during porcine fertilization, the activity of the 19S complex may be a rate-limiting factor contributing to anti-polyspermy defense during porcine fertilization.

A relatively simple and economical protocol for proteomic analyses of human 20S proteasome: Compatible with both scaled‐up and scaled‐down purifications

The 20S core particle (CP) is composed of 28 subunits arranged in four stacked heptameric rings (alpha7beta7beta7alpha7) forming a symmetrical barrel-shaped structure. Typically, in previous reports, the purification of CP mainly relied on the antibody, liquid chromatography and affinity tag-based strategies. In this study, we report a relatively simple and economical protocol for proteomic analyses of CP, which combines differential centrifugations with native-PAGE. Furthermore, it is compatible with both scaled-up purification from erythrocytes and scaled-down purification from low to around 3.0 x 10(7) pancreatic cancer cells, SW1990. In addition, a direct three-dimensional gel electrophoresis approach that omits the interval procedure between native-PAGE and IEF/SDS-PAGE is introduced. The results obtained in this study show that this protocol has a valuable potential for the studies of CP and other protein complexes.

Multiple Proteasome-Interacting Proteins Assist the Assembly of the Yeast 19S Regulatory Particle

The 26S proteasome is a highly conserved multisubunit protease that degrades ubiquitinated proteins in eukaryotic cells. The 26S proteasome consists of the proteolytic core particle (CP) and one or two 19S regulatory particles (RPs). Although the mechanisms of CP assembly are well described, the mechanism of RP assembly is largely unknown. Here, we show that four proteasome-interacting proteins (PIPs), Nas2/p27, Nas6/gankyrin, Rpn14/PAAF1, and Hsm3/S5b, bind specific Rpt subunits of the RP and interact each other genetically. Lack of these PIPs resulted in defective assembly of the 26S proteasome at an early stage, suggesting that these proteins are bona fide RP chaperones. Each of the RP chaperones formed distinct specific subassemblies of the base components and escorted them to mature RPs. Our results indicate that the RP assembly is a highly organized and elaborate process orchestrated by multiple proteasome-dedicated chaperones.