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Martin Rechsteiner

Publications and source records attributed to Martin Rechsteiner.

14 recordsLinked to original sources
PubMed2006

Proteasomes and antigen presentation: evidence that a KEKE motif does not promote presentation of the class I epitope SIINFEKL.

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.

Amino Acid Motifs↗
PubMed2005

Proteasome impairment does not contribute to pathogenesis in R6/2 Huntington's disease mice: exclusion of proteasome activator REGgamma as a therapeutic target.

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.

Analysis of Variance↗
PubMed2005

The axial channel of the 20S proteasome opens upon binding of the PA200 activator.

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.

Allosteric Site↗
PubMed2005

Mobilizing the proteolytic machine: cell biological roles of proteasome activators and inhibitors.

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.

Animals↗
PubMed2005

Purification and assay of proteasome activator PA200.

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.

Animals↗
PubMed2004

Characterization of mammalian Ecm29, a 26 S proteasome-associated protein that localizes to the nucleus and membrane vesicles.

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.

Amino Acid Sequence↗
PubMed2004

Purification procedures determine the proteasome activation properties of REG gamma (PA28 gamma).

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.

Ammonium Sulfate↗
PubMed2004

New HEAT-like repeat motifs in proteins regulating proteasome structure and function.

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.

Amino Acid Motifs↗
PubMed2003

Evidence that DNA replication is not regulated by ubiquitin-dependent proteolysis in Xenopus egg extract.

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.

Animals↗
PubMed2003

Are Huntington's and polyglutamine-based ataxias proteasome storage diseases?

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.

Animals↗
PubMed2002

PA200, a nuclear proteasome activator involved in DNA repair.

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.

Amino Acid Sequence↗
PubMed2002

Protein surveillance machinery in brains with spinocerebellar ataxia type 3: redistribution and differential recruitment of 26S proteasome subunits and chaperones to neuronal intranuclear inclusions.

Intracellular aggregates commonly forming neuronal intranuclear inclusions are neuropathological hallmarks of spinocerebellar ataxia type 3 and of other disorders characterized by expanded polyglutamine-(poly-Q) tracts. To characterize cellular responses to these aggregates, we performed an immunohistochemical analysis of neuronal intranuclear inclusions in pontine neurons of patients affected by spinocerebellar ataxia type 3, using a panel of antibodies directed against chaperones and proteasome subunits. A subset of the neuronal intranuclear inclusions stained positively for the chaperones Hsp90alpha and HDJ-2, a member of the Hsp40 family. Most neuronal intranuclear inclusions were ubiquitin positive, suggesting degradation by ubiquitin-dependent proteasome pathways. Surprisingly, only a fraction of neuronal intranuclear inclusions were immunopositive for antibodies directed against subunits of the 20S proteolytic core, whereas most inclusions were stained by antibodies directed against subunits of the 11S and 19S regulatory particles. These results suggest that the proteosomal proteolytic machinery that actively degrades neuronal intranuclear inclusions is assembled in only a fraction of pontine neurons in end stage spinocerebellar ataxia type 3. The dissociation between regulatory subunits and the proteolytic core and the changes in subcellular subunit distribution suggest perturbations of the proteosomal machinery in spinocerebellar ataxia type 3 brains.

Adenosine Triphosphatases↗