ABSTRACT
Ecm29 is a 200-kDa HEAT repeat protein that binds the 26 S proteasome. Genome-wide two-hybrid screens and mass spectrometry have identified molecular motors, endosomal components, and ubiquitin-proteasome factors as Ecm29-interacting proteins. The C-terminal half of human Ecm29 binds myosins and kinesins; its N-terminal region binds the endocytic proteins, Vps11, Rab11-FIP4, and rabaptin. Whereas full-length FLAG-Ecm29, its C-terminal half, and a small central fragment of Ecm29 remain bound to glycerol-gradient-separated 26 S proteasomes, the N-terminal half of Ecm29 does not. Confocal microscopy showed that Ecm-26 S proteasomes are present on flotillin-positive endosomes, but they are virtually absent from caveolin- and clathrin-decorated endosomes. Expression of the small central fragment of Ecm29 markedly reduces proteasome association with flotillin-positive endosomes. Identification of regions within Ecm29 capable of binding molecular motors, endosomal proteins, and the 26 S proteasome supports the hypothesis that Ecm29 serves as an adaptor for coupling 26 S proteasomes to specific cellular compartments.
Subject(s)
Endosomes/metabolism , Molecular Motor Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Animals , Endosomes/genetics , HeLa Cells , Humans , Mice , Molecular Motor Proteins/genetics , Proteasome Endopeptidase Complex/genetics , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/metabolism , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolismABSTRACT
Protein degradation is a critical factor in controlling cellular protein abundance. Here, we compare classical methods for determining protein degradation rates to a novel GFP (green fluorescent protein) fusion protein based method that assesses the intrinsic stability of cloned cDNA library products by flow cytometry [Yen et al. (2008) Science 322, 918]. While no method is perfect, we conclude that chimeric gene reporter approaches, though powerful, should be applied cautiously, due principally to GFP (or other reporter tag) interference with protein organelle targeting or incorporation into macromolecular assemblies, both of which cause spuriously high degradation rates.
Subject(s)
Biochemistry/methods , Proteins/metabolism , Animals , Cloning, Molecular/methods , Flow Cytometry/methods , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Proteins/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
Disease-causing mutations of genes encoding small MW heat shock proteins (sHSPs) constitute a growing family of inherited myofibrillar disorders. In the present work, we found that three structurally-distinct CryAB mutants R120G, 450delA and 464delCT are mostly present in the detergent-insoluble fractions when overexpressed in H9c2 rat heart cells. We found that either over-expression or knockdown of HSPB1, a related sHSP, affects the solubility, stability, and degradation of aggregation-prone CryAB mutants. HSPB1 overexpression has negligible effects on the solubility and protein aggregates of either R120G and/or 450delA but increased the solubility and prevented formation of 464delCT aggregates. HSPB1 knockdown decreased solubility and increased protein aggregates of all CryAB mutants, indicating a key role for HSPB1 in clearance of CryAB mutants under basal conditions. We provide four lines of evidence that such selective clearance of R120G, 450delA and 464delCT mutants by HSPB1 is mediated by the ubiquitin-proteasome system (UPS). First, we found that treatment with the proteasome inhibitors increased the levels of all CryAB mutants. Second, R120G and 450delA overexpression corresponded to the accumulation of their specific ubiquitin conjugates in H9c2 cells. Third, HSPB1 knockdown directly increased the levels of all polyubiquitin conjugates. And fourth, the selective attenuation of 464delCT expression by HSPB1 over-expression was abrogated by the proteasome inhibition. We conclude that such selective interactions between CryAB mutants and HSPB1 overexpression might have important implications for the clinical manifestations and potential treatment.
Subject(s)
HSP27 Heat-Shock Proteins/metabolism , Mutant Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Processing, Post-Translational , Ubiquitin/metabolism , alpha-Crystallin B Chain/chemistry , alpha-Crystallin B Chain/metabolism , Amino Acid Sequence , Animals , Autophagy , Cytoplasm/metabolism , Down-Regulation , Gene Knockdown Techniques , Heat-Shock Proteins , Humans , Mice , Molecular Chaperones , Molecular Sequence Data , Mutant Proteins/chemistry , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Polyubiquitin/metabolism , Protein Structure, Quaternary , Rats , Signal Transduction , Solubility , Subcellular Fractions/metabolismABSTRACT
Proteasomes perform the majority of proteolysis that occurs in the cytosol and nucleus of eukaryotic cells and, thereby, perform crucial roles in cellular regulation and homeostasis. Isolated proteasomes are inactive because substrates cannot access the proteolytic sites. PA28 and PA200 are activators that bind to proteasomes and stimulate the hydrolysis of peptides. Several protein inhibitors of the proteasome have also been identified, and the properties of these activators and inhibitors have been characterized biochemically. By contrast, their physiological roles--which have been reported to include production of antigenic peptides, proteasome assembly and DNA repair--are controversial. In this article, we briefly review the biochemical data and discuss the possible biological roles of PA28, PA200 and proteasome inhibitors.
Subject(s)
Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors , Animals , Apoptosis , Autoantigens/immunology , Autoantigens/physiology , Enzyme Activation , Humans , Immune System , Muscle Proteins/immunology , Muscle Proteins/physiology , Proteasome Endopeptidase Complex/chemistry , Proteasome Endopeptidase Complex/immunology , Proteasome Endopeptidase Complex/physiologyABSTRACT
Previously we proposed that stretches of alternating Lys(K) and Glu(E) in polypeptides promote the expression of nearby sequences on Class I molecules [Realini, C., Rogers, S.W., Rechsteiner, M., 1994. KEKE motifs. Proposed roles in protein-protein association and presentation of peptides by MHC class I receptors. FEBS Lett. 348, 109-113]. As a test of the KEKE hypothesis we have employed osmotic lysis of pinosomes and transfection to introduce or express various ubiquitin peptide fusion proteins in the cytosol of wild type and PA28alphabetagamma- mouse embryo fibroblasts. KEKE or non-KEKE motifs were placed between ubiquitin and the OVA epitope SIINFEKL that was at or near the C-termini of the various fusion proteins. Measurements of surface Kb-SIINFEKL complexes using the monoclonal antibody 25.-D1.16 allowed us to assess the effects of upstream KEKE motifs and PA28 status on SIINFEKL surface presentation. KEKE motifs did not enhance presentation of the OVA epitope. However, our studies did confirm that PA28alphabeta is needed for efficient SIINFEKL surface expression when hsp90 is inhibited.
Subject(s)
Amino Acid Motifs , Antigen Presentation , Epitopes , Histocompatibility Antigens Class I/chemistry , Proteasome Endopeptidase Complex/chemistry , Amino Acid Sequence , Animals , Antibodies, Monoclonal/metabolism , Cell Line , Cytosol/immunology , Embryo, Mammalian/cytology , Fibroblasts/cytology , Fibroblasts/immunology , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/immunology , Mice , Molecular Sequence Data , Proteins/immunology , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/immunology , Ubiquitin/immunology , Ubiquitin/metabolismABSTRACT
Spinal and bulbar muscular atrophy (SBMA) is caused by expression of a polyglutamine (polyQ)-expanded androgen receptor (AR). The inefficient nuclear proteasomal degradation of the mutant AR results in the formation of nuclear inclusions containing amino-terminal fragments of the mutant AR. PA28γ (also referred to as REGγ) is a nuclear 11S-proteasomal activator with limited proteasome activation capabilities compared to its cytoplasmic 11S (PA28α, PA28ß) counterparts. To clarify the role of REGγ in polyQ-expanded AR metabolism, we carried out genetic and biochemical studies in cell models of SBMA. Overexpression of REGγ in a PC12 cell model of SBMA increased polyQ-expanded AR aggregation and contributed to polyQ-expanded AR toxicity in the presence of dihydrotestosterone (DHT). These effects of REGγ were independent of its association with the proteasome and may be due, in part, to the decreased binding of polyQ-expanded AR by the E3 ubiquitin-ligase MDM2. Unlike its effects in PC12 cells, REGγ overexpression rescued transgenic SBMA motor neurons from DHT-induced toxicity in a proteasome binding-dependent manner, suggesting that the degradation of a specific 11S proteasome substrate or substrates promotes motor neuron viability. One potential substrate that we found to play a role in mutant AR toxicity is the splicing factor SC35. These studies reveal that, depending on the cellular context, two biological roles for REGγ impact cell viability in the face of polyQ-expanded AR; a proteasome binding-independent mechanism directly promotes mutant AR aggregation while a proteasome binding-dependent mechanism promotes cell viability. The balance between these functions likely determines REGγ effects on polyQ-expanded AR-expressing cells.
ABSTRACT
Proteasomes consist of a proteolytic core called the 20 S particle and ancillary factors that regulate its activity in various ways. PA200 has been identified as a large (200 kDa) nuclear protein that stimulates proteasomal hydrolysis of peptides. To characterize its interaction with the 20 S core, we have visualized PA200-20 S complexes by electron microscopy. Monomers of PA200 bind to one or both ends of the 20 S core. Reconstructed in three dimensions to 23 A resolution from cryo-electron micrographs of the singly bound complex, PA200 has an asymmetric dome-like structure with major and minor lobes. Taking into account previous bioinformatic analysis, it is likely to represent an irregular folding of an alpha-helical solenoid composed of HEAT-like repeats. PA200 makes contact with all alpha-subunits except alpha7, and this interaction induces an opening of the axial channel through the alpha-ring. Thus, the activation mechanism of PA200 is expressed via its allosteric effects on the 20 S core particle, perhaps facilitating release of digestion products or the entrance of substrates.
Subject(s)
Allosteric Site , Ion Channels/metabolism , Proteasome Endopeptidase Complex/metabolism , Repetitive Sequences, Amino Acid/physiology , Catalytic Domain , Ion Channels/chemistry , Ion Channels/ultrastructure , Proteasome Endopeptidase Complex/chemistry , Proteasome Endopeptidase Complex/ultrastructure , Protein Binding , Protein Conformation , Protein FoldingABSTRACT
PA200, the most recently discovered activator of the 20S proteasome, is a nuclear protein thought to play a role in DNA repair. Homologs of PA200 have been found in rat, frog, birds, worms, and budding yeast, where it is called Blm3p (now known as Blm10p), but not in Drosophila or fission yeast. Western blots of SDS-PAGE transfers reveal 160 and 200K forms of mammalian PA200, and organ surveys demonstrate that the 200K species is highest in testis. PA200 purified from bovine testis binds the ends of the cylindrical 20S proteasome, forming volcano-shaped structures in negatively stained EM images. In vitro assays demonstrate that binding of PA200 activates peptide hydrolysis by the 20S proteasome. This chapter describes the purification and assay of bovine testis PA200.
Subject(s)
Nuclear Proteins/isolation & purification , Nuclear Proteins/physiology , Proteasome Endopeptidase Complex/metabolism , Testis/chemistry , Animals , Cattle , Centrifugation, Density Gradient/methods , Chromatography, DEAE-Cellulose/methods , Chromatography, Gel/methods , Electrophoresis, Polyacrylamide Gel/methods , Enzyme Activation/physiology , Male , Nuclear Proteins/analysis , Proteins , Testis/metabolismABSTRACT
To date, 10 neurological diseases, including Huntington's and several ataxias, are caused by a lengthening of glutamine (Q) tracts in various proteins. Even though the Q expansions arise in unrelated proteins, the diseases share three striking features: (1) 35 contiguous glutamines constitutes the pathological threshold for 9 of the 10 diseases; (2) the Q-expanded proteins are expressed in many tissues, yet pathology is largely restricted to neurons; and (3) the Q-expanded proteins or fragments thereof form nuclear inclusions that also contain ubiquitin, proteasomes and chaperones. Our studies of the proteasome activator REGgamma suggest a possible explanation for these shared properties. REGgamma is highly expressed in brain, located in the nucleus and actually suppresses the proteasome active sites principally responsible for cleaving glutamine-MCA bonds. These observations coupled with reports that peptides longer than 35 residues, the polyQ pathology threshold, are unable to diffuse out of the proteasome suggest the following hypothesis. Proteins containing long glutamine tracts are efficiently pumped into REGgamma-capped 26S proteasomes, but REGgamma suppression of cleavage after glutamine produces polyQ fragments too long to diffuse out of the 20S proteolytic core thereby inactivating the 26S proteasome. In effect, we hypothesize that the polyQ pathologies may be proteasomal storage diseases analogous to disorders of lysosome catabolism.
Subject(s)
Ataxia/metabolism , Cysteine Endopeptidases/metabolism , Glutamine/metabolism , Huntington Disease/metabolism , Multienzyme Complexes/metabolism , Animals , Ataxia/therapy , Autoantigens , Huntington Disease/therapy , Mice , Nuclear Proteins/metabolism , Proteasome Endopeptidase Complex , Proteins/metabolism , Ubiquitin/metabolismABSTRACT
Ubiquitin is a small, evolutionarily conserved eukaryotic protein that can be attached to a wide variety of intracellular proteins including itself. Covalent attachment of ubiquitin to other proteins serves various functions, but its major role is to target cellular proteins for destruction. Cellular components that activate, transfer, remove, or simply recognize ubiquitin number in the hundreds, perhaps even in the thousands. In light of this complexity the ubiquitin pathway is ideal for a systems biology approach.
Subject(s)
Cysteine Endopeptidases/metabolism , Models, Biological , Multienzyme Complexes/metabolism , Muscle Proteins/metabolism , Peptide Hydrolases/metabolism , Proteins/metabolism , Signal Transduction/physiology , Ubiquitin/metabolism , Cysteine Endopeptidases/chemistry , Hydrolysis , Multienzyme Complexes/chemistry , Muscle Proteins/chemistry , Peptide Fragments/biosynthesis , Peptide Hydrolases/chemistry , Proteasome Endopeptidase Complex , Proteins/chemistry , Systems Analysis , Ubiquitin/chemistryABSTRACT
Toll-like receptor 3 (TLR3) recognizes viral double-stranded RNA and stimulates the innate immune response. We found that depletion of extracellular mutant 29 (Ecm29), an adaptor protein that binds to a subset of 26S proteasomes (Ecm proteasomes), increased the abundance of TLR3 in human embryonic kidney-293 and HeLa cells. Loss of Ecm29 also increased the amounts of LC3ß and p62, two proteins that mediate autophagy. The absence of Ecm29 enhanced TLR3 signaling, which was characterized by the increased abundance of the adaptor protein and E3 ubiquitin ligase tumor necrosis factor receptor-associated factor 3, increased phosphorylation and activation of effector kinases downstream of TLR3, increased nuclear localization of the transcription factor interferon regulatory factor 3, and the accumulation of signaling molecules at juxtanuclear recycling endosomes. We conclude that Ecm proteasomes play a previously uncharacterized role in mediating autophagy, trafficking of TLR3, and attenuation of TLR3-dependent signaling.
Subject(s)
Autophagy/immunology , Proteasome Endopeptidase Complex/immunology , Signal Transduction/immunology , Toll-Like Receptor 3/immunology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/immunology , Adaptor Proteins, Signal Transducing/metabolism , Autophagy/genetics , HEK293 Cells , HeLa Cells , Humans , Microtubule-Associated Proteins/biosynthesis , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/immunology , Phosphorylation/genetics , Phosphorylation/immunology , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/metabolism , Sequestosome-1 Protein , Signal Transduction/genetics , Toll-Like Receptor 3/biosynthesis , Toll-Like Receptor 3/genetics , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/immunology , Ubiquitin-Protein Ligases/metabolismABSTRACT
Eukaryotic proteasomes have been reported to cleave only once within polyglutamine tracts and then only after the N-terminal glutamine (Venkatraman, P., Wetzel, R., Tanaka, M., Nukina, N., and Goldberg, A. L. (2004) Mol. Cell 14, 95-104). We have obtained results that directly conflict with that report. In the presence of the proteasome activator PA28gamma(K188E) human red cell proteasomes progressively degraded fluorescein-GGQ(10)RR or fluorescein-HPHQ(10)RR into small fragments as shown by size exclusion chromatography and mass spectrometry. MALDI-TOF mass spectrometry revealed that proteolytic products arose from cleavage after every glutamine in fluorescein-HPHQ(10)RR, and mass accuracy rules out deamidation of glutamine to glutamic acid as an explanation for peptide degradation. Moreover, degradation cannot be attributed to a contaminating protease because peptide hydrolysis was completely blocked by the proteasome-specific inhibitors, lactacystin and epoxomicin. We conclude that proteasomes cleave repetitively anywhere within a stretch of ten glutamine residues. Thus our results cast doubt on the idea that mammalian proteasomes cannot degrade glutamine-expanded regions within pathogenic polyQ-expanded proteins, such as Huntingtin.
Subject(s)
Peptides/metabolism , Proteasome Endopeptidase Complex/metabolism , Enzyme Activation , Humans , Ions/chemistry , Mass Spectrometry , Mutation/genetics , Peptides/chemistry , Proteasome Endopeptidase Complex/geneticsABSTRACT
Huntington's disease (HD) is one of a group of neurodegenerative disorders caused by the pathological expansion of a glutamine tract. A hallmark of these so-called polyglutamine diseases is the presence of ubiquitylated inclusion bodies, which sequester various components of the 19S and 20S proteasomes. In addition, the ubiquitin-proteasome system (UPS) has been shown to be severely impaired in vitro in cells overexpressing mutant huntingtin. Thus, because of its fundamental housekeeping function, impairment of the UPS in neurons could contribute to neurotoxicity. We have recently proposed that the proteasome activator REGgamma could contribute to UPS impairment in polyglutamine diseases by suppressing the proteasomal catalytic sites responsible for cleaving Gln-Gln bonds. Capping of proteasomes with REGgamma could therefore contribute to a potential 'clogging' of the proteasome by pathogenic polyglutamines. We show here that genetic reduction of REGgamma has no effect on the well-defined neurological phenotype of R6/2 HD mice and does not affect inclusion body formation in the R6/2 brain. Surprisingly, we observe increased proteasomal 'chymotrypsin-like' activity in 13-week-old R6/2 brains relative to non-R6/2, irrespective of REGgamma levels. However, assays of 26S proteasome activity in mouse brain extracts reveal no difference in proteolytic activity regardless of R6/2 or REGgamma genotype. These findings suggest that REGgamma is not a viable therapeutic target in polyglutamine disease and that overall proteasome function is not impaired by trapped mutant polyglutamine in R6/2 mice.
Subject(s)
Autoantigens/metabolism , Brain/metabolism , Huntington Disease/metabolism , Neurons/metabolism , Peptides/metabolism , Proteasome Endopeptidase Complex/metabolism , Analysis of Variance , Animals , Blotting, Western , Crosses, Genetic , DNA Primers , Exploratory Behavior/physiology , Genotype , Immunohistochemistry , Inclusion Bodies/pathology , Mice , Mice, Mutant Strains , Rotarod Performance TestABSTRACT
The selectivity underlying the recognition of oxidized calmodulin (CaM) by the 20S proteasome in complex with Hsp90 was identified using mass spectrometry. We find that degradation of oxidized CaM (CaMox) occurs in a multistep process, which involves an initial cleavage that releases a large N-terminal fragment (A1-F92) as well as multiple smaller carboxyl-terminus peptides ranging from 17 to 26 amino acids in length. These latter small peptides are enriched in methionine sulfoxides (MetO), suggesting a preferential degradation around MetO within the carboxyl-terminal domain. To confirm the specificity of CaMox degradation and to identify the structural signals underlying the preferential recognition and degradation by the proteasome/Hsp90, we have investigated how the oxidation of individual methionines affect the degradation of CaM using mutants in which all but selected methionines in CaM were substituted with leucines. Substitution of all methionines with leucines except Met144 and Met145 has no detectable effect on the structure of CaM, permitting a determination of how site-specific substitutions and the oxidation of Met144 and Met145 affects the recognition and degradation of CaM by the proteasome/Hsp90. Comparable rates of degradation are observed upon the selective oxidation of Met144 and Met145 in CaM-L7 relative to that observed upon oxidation of all nine methionines in wild-type CaM. Substitution of leucines for either Met144 or Met145 promotes a limited recognition and degradation by the proteasome that correlates with decreases in the helical content of CaM. The specific oxidation of Met144 has little effect on rates of proteolytic degradation by the proteasome/Hsp90 or the structure of CaM. In contrast, the specific oxidation of Met145 results in both large increases in the rate of degradation by the proteasome/Hsp90 and significant circular dichroic spectral shape changes that are indicative of changes in tertiary rather than secondary structure. Thus, tertiary structural changes resulting from the site-specific oxidation of a single methionine (i.e., Met145) promote the degradation of CaM by the proteasome/Hsp90, suggesting a mechanism to regulate cellular metabolism through the targeted modulation of CaM abundance in response to oxidative stress.
Subject(s)
Calmodulin/chemistry , HSP90 Heat-Shock Proteins/chemistry , Methionine/chemistry , Proteasome Endopeptidase Complex/chemistry , Amino Acid Substitution , Animals , Cattle , Kinetics , Oxidation-Reduction , Proteasome Endopeptidase Complex/analysis , Protein Denaturation , Protein Structure, Tertiary , Radioisotopes/chemistry , Structure-Activity RelationshipABSTRACT
We have identified a novel 200 kDa nuclear protein that activates the proteasome. The protein, which we call PA200, has been purified to homogeneity from bovine testis and has been shown to activate proteasomal hydrolysis of peptides, but not proteins. Following gamma-irradiation of HeLa cells the uniform nuclear distribution of PA200 changes to a strikingly punctate pattern, a behavior characteristic of many DNA repair proteins. Homologs of PA200 are present in worms, plants and yeast. Others have shown that mutation of yeast PA200 results in hypersensitivity to bleomycin, and exposure of yeast to DNA damaging agents induces the PA200 message. Taken together, these findings implicate PA200 in DNA repair, possibly by recruiting proteasomes to double strand breaks.
Subject(s)
Cysteine Endopeptidases/metabolism , Multienzyme Complexes/metabolism , Nuclear Proteins/physiology , Amino Acid Sequence , Animals , Cattle , Cell Nucleus/chemistry , DNA Damage , DNA Repair , DNA, Complementary/genetics , Enzyme Activation , Gamma Rays , HeLa Cells/enzymology , HeLa Cells/radiation effects , Humans , Male , Mice , Molecular Sequence Data , Molecular Weight , Neoplasm Proteins/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/isolation & purification , Organ Specificity , Peptides/metabolism , Proteasome Endopeptidase Complex , Protein Isoforms/chemistry , Protein Isoforms/isolation & purification , Rabbits , Species Specificity , Testis/chemistryABSTRACT
The Xenopus early embryonic cell cycle consists of rapid oscillations between mitosis and DNA synthesis. We used ubiquitin (Ub)-dependent proteolysis inhibitors to determine whether Ub-mediated proteolysis regulates the initiation of DNA replication in Xenopus egg extract. Methylated Ub, a chemically modified Ub that cannot form chains, and S5a, a Ub chain-binding subunit of the 26S proteasome, were added to extract at concentrations known to inhibit cyclin B proteolysis and their effects on cell cycle progression and DNA replication were examined. DNA replication initiated concomitant with controls and proceeded in a semiconservative fashion in the presence of both methylated Ub and S5a. However, mitotic progression was halted, showing that the inhibitors were functional. We conclude that initiation of DNA replication is not regulated by Ub-dependent proteolysis in the early Xenopus cell cycle.
Subject(s)
DNA Replication , Oocytes/metabolism , Ubiquitin/metabolism , Animals , Aphidicolin/metabolism , Carrier Proteins/metabolism , Cyclin B/metabolism , Cyclin B2 , Enzyme Inhibitors/metabolism , Humans , Methylation , Proteasome Endopeptidase Complex , RNA-Binding Proteins , Tissue Extracts/metabolism , Ubiquitin/chemistry , XenopusABSTRACT
The proteasome activation properties of recombinant REG gamma molecules depend on purification procedures. Prior to ammonium sulfate precipitation recombinant REG gamma activates the trypsin-like catalytic subunit of the proteasome; afterwards it activates all three catalytic subunits. The expanded activation specificity is accompanied by reduced stability of the REG gamma heptamer providing support for the idea that a "tight" REG gamma heptamer suppresses the proteasome's chymotrypsin-like and postglutamyl-preferring active sites. In an attempt to determine whether REG gamma synthesized in mammalian cells also exhibits restricted activation properties, extracts were prepared from several mammalian organs and cell lines. Surprisingly, endogenous REG gamma was found to be largely monomeric. In an alternate approach, COS7 cells were cotransfected with plasmids expressing FLAG-REG gamma and REG gamma. The expressed FLAG-REG gamma molecules were shown to form oligomers with untagged REG gamma subunits, and the mixed oligomers preferentially activated the proteasome's trypsin-like subunit. Thus, REG gamma molecules synthesized in mammalian cells also exhibit restricted activation properties.
Subject(s)
Cysteine Endopeptidases/isolation & purification , Cysteine Endopeptidases/metabolism , Multienzyme Complexes/isolation & purification , Multienzyme Complexes/metabolism , Nuclear Proteins/isolation & purification , Nuclear Proteins/metabolism , Ammonium Sulfate/chemistry , Animals , Autoantigens , COS Cells , Chlorocebus aethiops , Cysteine Endopeptidases/chemistry , Enzyme Activation , Fractional Precipitation , Multienzyme Complexes/chemistry , Nuclear Proteins/chemistry , Proteasome Endopeptidase Complex , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Substrate SpecificityABSTRACT
We have identified repeat motifs in the large proteasome-binding proteins PA200 and Ecm29 by applying a sensitive sequence profile method. These repeat motifs, especially those of PA200, resemble HEAT/ARM repeats in length and other properties but differ from them in the occupancy of certain positions. The HEAT motif consists of two alpha-helices and two turns: molecular modeling suggests that in the PA200 and Ecm29 repeats, the alpha-helices may be slightly turned relative to their orientations in typical HEAT repeats. Both PA200 and Ecm29 are composed almost entirely of such repeats, and therefore are likely to have alpha-helical solenoid structures. These observations lead us to speculate on how PA200 and Ecm29 may associate with proteasomes.
Subject(s)
Nuclear Proteins/chemistry , Proteasome Endopeptidase Complex/chemistry , Amino Acid Motifs , Amino Acid Sequence , Computational Biology , Consensus Sequence , Proteasome Endopeptidase Complex/physiology , Protein Binding , Protein Structure, Secondary , Repetitive Sequences, Nucleic AcidABSTRACT
In addition to its thirty or so core subunits, a number of accessory proteins associate with the 26 S proteasome presumably to assist in substrate degradation or to localize the enzyme within cells. Among these proteins is ecm29p, a 200-kDa yeast protein that contains numerous HEAT repeats as well as a putative VHS domain. Higher eukaryotes possess a well conserved homolog of yeast ecm29p, and we produced antibodies to three peptides in the human Ecm29 sequence. The antibodies show that Ecm29 is present exclusively on 26 S proteasomes in HeLa cells and that Ecm29 levels vary markedly among mouse organs. Confocal immunofluorescence microscopy localizes Ecm29 to the centrosome and a subset of secretory compartments including endosomes, the ER and the ERGIC. Ecm29 is up-regulated 2-3-fold in toxinresistant mutant CHO cells exhibiting increased rates of ER-associated degradation. Based on these results we propose that Ecm29 serves to couple the 26 S proteasome to secretory compartments engaged in quality control and to other sites of enhanced proteolysis.
Subject(s)
Cell Nucleus/chemistry , Cytoplasmic Vesicles/chemistry , Nuclear Proteins/analysis , Proteasome Endopeptidase Complex/chemistry , Proteasome Endopeptidase Complex/metabolism , Amino Acid Sequence , Animals , Antibodies , Brain Chemistry , CHO Cells , Cattle , Cell Line , Centrosome/chemistry , Cricetinae , Cricetulus , Cytoplasm/chemistry , Endoplasmic Reticulum/metabolism , Endosomes/chemistry , Fluorescent Antibody Technique , HeLa Cells , Humans , Male , Mice , Molecular Sequence Data , Nuclear Proteins/chemistry , Nuclear Proteins/physiology , Peptide Fragments/chemistry , Peptide Fragments/immunology , ProstateABSTRACT
The 20 S proteasome has been suggested to play a critical role in mediating the degradation of abnormal proteins under conditions of oxidative stress and has been found in tight association with the molecular chaperone Hsp90. To elucidate the role of Hsp90 in promoting the degradation of oxidized calmodulin (CaM(ox)), we have purified red blood cell 20 S proteasomes free of Hsp90 and assessed their ability to degrade CaM(ox) in the absence or presence of Hsp90. Purified 20 S proteasome does not degrade CaM(ox) unless Hsp90 is added. CaM(ox) degradation is sensitive to both proteasome and Hsp90-specific inhibitors and is further enhanced in the presence of 2 mm ATP. Irrespective of the presence of Hsp90, we find that unoxidized CaM is not significantly degraded. Direct binding measurements demonstrate that Hsp90 selectively associates with CaM(ox); essentially no binding is observed between Hsp90 and unoxidized CaM. These results indicate that Hsp90 in association with the 20 S proteasome can selectively associate with oxidized and partially unfolded CaM to promote degradation by the proteasome.