Protein substrates of the proteasome must apparently be unfolded and translocated through a narrow channel to gain access to the proteolytic active sites of the enzyme. Protein folding in vivo is mediated by molecular chaperones. Here, to test for chaperone activity of the proteasome, we assay the reactivation of denatured citrate synthase. Both human and yeast proteasomes stimulate the recovery of the native structure of citrate synthase. We map this chaperone-like activity to the base of the regulatory particle of the proteasome, that is, to the ATPase-containing assembly located at the substrate-entry ports of the channel. Denatured but not native citrate synthase is bound by the base complex. Ubiquitination of citrate synthase is not required for its binding or refolding by the base complex of the proteasome. These data suggest a model in which ubiquitin-protein conjugates are initially tethered to the proteasome by specific recognition of their ubiquitin chains; this step is followed by a nonspecific interaction between the base and the target protein, which promotes substrate unfolding and translocation.

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Proteasome-catalyzed peptide splicing (PCPS) represents an additional activity of mammalian 20S proteasomes recently identified in connection with antigen presentation. We show here that PCPS is not restricted to mammalians but that it is also a feature of yeast 20S proteasomes catalyzed by all three active site ␤ subunits. No major differences in splicing efficiency exist between human 20S standard-and immuno-proteasome or yeast 20S proteasome. Using H 2 18 O to monitor the splicing reaction we also demonstrate that PCPS occurs via direct transpeptidation that slightly favors the generation of peptides spliced in cis over peptides spliced in trans. The 20S proteasome with its proteolytically active site ␤-subunits (␤1, ␤2, and ␤5) is a N-terminal nucleophilic hydrolase, widely conserved during evolution from yeast to mammals. It is the central proteolytic machinery of the ubiquitin proteasome system and the catalytic core of the 26S proteasome that is built by the association of 19S regulator complexes with the 20S proteasome. As part of the 26S proteasome the 20S core degrades poly-ubiquitylated proteins to peptides of 3 to 20 residues in length (1). A small percentage of these peptides is transported to the endoplasmic reticulum, bound by major histocompatibility complex (MHC) 1 class I molecules, and presented at the cell surface to CD8ϩ cytotoxic T lymphocyte for immune recognition. This antigen presentation pathway is usually restricted to the proteasome-dependent processing of self-and viral-proteins (2). Antigen presentation is generally increased after IFN-␥ stimuli because it induces, among others, the synthesis of alternative catalytic subunits (␤1i, ␤2i, and ␤5i) and the concomitant formation of immunoproteasomes (i-proteasomes) (2).All active ␤ subunits carry an N-terminal threonine residue as reactive nucleophile. Therefore, their distinct cleavage preferences are determined by the structural features of the substrate binding pockets. In particular, the nonprimed substrate binding site of the active site ␤ subunits binds the residues of the peptide substrate that are located at the N-terminal side of the cleaved residue. The residues of the peptide located C-terminally of the cleavage site are bound by the primed substrate binding site. The binding to both substrate binding sites of the active site ␤ subunit provides the stability and the orientation of the substrate, which is mandatory to carry out the proteolytic cleavage (3).Peptides can be produced by proteasomes during the degradation of proteins or polypeptides by conventional peptide bond hydrolysis or by proteasome-catalyzed peptide splicing (PCPS). The latter has been demonstrated in vivo so far only for four MHC class I-restricted epitopes (4 -8), leading to the assumption that PCPS is most likely a rare event that lacks any wider functional importance (9). PCPS was suggested to occur in a direct transpeptidation reaction, in either cis or trans, by linking two proteasomal cleavage products (PCPs) derived either from the same or from two ...

From the thermoacidophilic archaebacterium, Thermopiasma acidophilum, a proteolytically active particle has been isolated which is almost identical in size. and shape with the multicatalytic proteinase (prosome) from rat. This result indicates that prosomes have been developed early in evolution and that they possibly serve functions common to all living cells.

Immunoproteasomes are considered to be optimised to process Ags and to alter the peptide repertoire by generating a qualitatively different set of MHC class I epitopes. Whether the immunoproteasome at the biochemical level, influence the quality rather than the quantity of the immuno-genic peptide pool is still unclear. Here, we quantified the cleavage-site usage by human standard-and immunoproteasomes, and proteasomes from immuno-subunit-deficient mice, as well as the peptides generated from model polypeptides. We show in this study that the different proteasome isoforms can exert significant quantitative differences in the cleavage-site usage and MHC class I restricted epitope production. However, independent of the proteasome isoform and substrates studied, no evidence was obtained for the abolishment of the specific cleavage-site usage, or for differences in the quality of the peptides generated. Thus, we conclude that the observed differences in MHC class I restricted Ag presentation between standard-and immunoproteasomes are due to quantitative differences in the proteasome-generated antigenic peptides.Keywords: Antigen presentation r Immunoproteasome r MHC class I restricted epitopes r Proteasome r Proteolysis See accompanying Commentary by Zanker and ChenAdditional supporting information may be found in the online version of this article at the publisher's web-site IntroductionThe 20S proteasome is the central proteolytic machinery of the ubiquitin proteasome system, being responsible for the main Correspondence: Dr. Michele Mishto e-mail: michele.mishto@charite.de part of extra-lysosomal protein degradation and generation of MHC class I restricted epitopes [1]. During evolution, the 20S proteasome retained a conserved structure of four stacked seven membered rings (α 7 β 7 β 7 α 7 ). In each β ring, the 20S standard proteasome has three catalytic standard subunits (i.e. β1s, β2s and β5s) that carry an N-terminal threonine residue as a reactive nucleophile. Based on the analysis of yeast 20S proteasome active site mutants with short fluorogenic peptide substrates C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2014. 44: 3508-3521 Antigen processing 3509 chymotryptic-, tryptic-and caspase-like activities were assigned to the β5, β2 and β1 subunits, respectively [2]. Larger polypeptide substrates bind with their residues surrounding the cleavage site, that is residues in position P4 to P1 (cleavage site) and P1 to P4 , to the non-primed and primed substrate binding sites [3] of the proteasome, respectively. This provides the stability and the orientation of the substrate, thereby determining the cleavage-site usage within a protein substrate [4]. In mammalia, the cytokine IFN-γ induces the expression of three active sites carrying alternative β1i/LMP2, β2i/MECL1 and β5i/LMP7 immuno-subunits, and in consequence the formation of the immunoproteasome isoforms [5].Since the β1i and β5i immuno-subunits are encoded within the MHC class II region in close neighbourhood to ...

The ubiquitin-proteasome system is the major pathway for intracellular protein degradation and is also deeply involved in the regulation of most basic cellular processes. Its proteolytic core, the 20S proteasome, has found to be attached also to the cell plasma membrane and certain observations are interpreted as to suggest that they may be released into the extracellular medium, e.g. in the alveolar lining fluid, epididymal fluid and possibly during the acrosome reaction. Proteasomes have also been detected in normal human blood plasma and designated circulating proteasomes; these have a comparatively low specific activity, a distinct pattern of subtypes and their exact origin is still enigmatic. In patients suffering from autoimmune diseases, malignant myeloproliferative syndromes, multiple myeloma, acute and chronic lymphatic leukaemia, solid tumour, sepsis or trauma, respectively, the concentration of circulating proteasomes has been found to be elevated, to correlate with the disease state and has even prognostic significance. Similarly, ubiquitin has been discovered as a normal component of human blood and seminal plasma and in ovarian follicular fluid. Increased concentrations were measured in diverse pathological situations, not only in blood plasma but also in cerebrospinal fluid, where it may have neuroprotective effects. As defective spermatozoa are covered with ubiquitin in the epididymal fluid, extracellular ubiquitination is proposed to be a mechanism for quality control in spermatogenesis. Growing evidence exists also for a participation of extracellular proteasomes and ubiquitin in the fertilization process.

The proteasome or multicatalytic proteinase is a high molecular mass multisubunit complex ubiquitous in eukaryotes but also found in the archaebacterial proteasome is made of two different subunits only, and yet the complexes are almost identical in size and shape. Cloning and sequencing the gene encoding the small (beta) subunit of the T. acidophilum complex completes the primary structure of the archaebacterial proteasome. The similarity of the derived amino acid sequences of 233 (alpha) and 211 (beta) residues, respectively, indicates that they arose from a common ancestral gene. All the sequences of proteasomal subunits from eukaryotes available to date can be related to either the alpha-subunit or beta-subunit of the T. acidophilum "Urproteasome", and they can be distinguished by means of a highly conserved N-terminal extension, which is characteristic for alpha-type subunits. On the basis of circumstantial evidence we suggest that the alpha-subunits have regulatory and targeting functions, while the beta-subunits carry the active sites.

A proteolytic enzyme was purified from the post-myofibrillar fraction of rat skeletal muscle. The purification procedure consisted of fractionation of the muscle extract by (NH4)2SO4, chromatography on DEAE-Sephacel, fast protein liquid chromatography on Mono Q and gel filtration on Sepharose 6B. The enzyme preparation appeared to be homogeneous as judged by disc electrophoresis in polyacrylamide gels and by immunoelectrophoresis. The isoelectric point of the proteinase is at 5.1-5.2. The enzyme has an Mr of about 650 000 and dissociates into eight subunits of Mr 25 000-32 000 when subjected to electrophoresis in sodium dodecyl sulphate/polyacrylamide gels. The proteinase contains hydrolytic activity against N-blocked tripeptide 4-methyl-7-coumarylamide substrates with an arginine or phenylalanine residue adjacent to the leaving group. Maximum activity with the first group of substrates was at pH 10.5, and this activity was inhibited by leupeptin, chymostatin and Ca2+. Maximum activity with the latter group of substrates was at pH 7.5, and was also inhibited by the two microbial inhibitors, but was activated by Ca2+ ions. By using [14C]methylcasein as a substrate, maximum activity was observed at pH9.0, and this proteolytic activity was not affected by leupeptin, was enhanced by chymostatin and inhibited by Ca2+. Similar effects were observed when benzyloxycarbonyl-Leu-Leu-Glu 2-naphthylamide was used as a substrate. These enzymic activities were abolished by p-hydroxymercuribenzenesulphonic acid or mersalyl acid, whereas a small activation was observed with cysteine or dithiothreitol.