Driving Forces of Proteasome-catalyzed Peptide Splicing in Yeast and Humans

Abstract

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 ...

Molecular alterations in proteasomes of rat liver during aging result in altered proteolytic activities

“…However, we did not detect a clear impact of polypeptide length on the degradation rate by 20S proteasomes in agreement with our previous observation (Mishto et al 2008) and the degradation rates of polypeptides with similar length but different sequence, such as pp89 and gp100 , were differently affected by the age of the animals. Previously, we have also shown that the impact of the six proteasomal catalytic sites varies between these latter substrates (Mishto et al 2012). These two facts suggest that rather the sequence than the length of the substrate polypeptides is the reason for the agedependent differences in proteasomal degradation rate.…”

Definition of Proteasomal Peptide Splicing Rules for High-Efficiency Spliced Peptide Presentation by MHC Class I Molecules

“….S n depending on their proximity to the scissile peptide bond, and C-terminal ligation partners that may fit the primed proteasomal pockets S 1 9, S 2 9, S 3 9. . .S n 9, but have also been postulated to bind to a distinct proteasomal splicing product binding site (11). Amino acid residues in both ligation partners that interact with these proteasomal substrate binding pockets are here referred to as P 1 , P 2 , P 3 .…”

“…Because spliced Ags may mediate T cell responses to cancer, transplants, and viral infection, predicting their generation would be important for the development of vaccines and immunotherapies or to pharmacologically suppress T cell responses (12). Although proteasomal peptide ligation can readily be detected in vitro (11,15), identifying spliced Ags in vivo has proven to be difficult with only five immunomodulatory spliced epitopes identified (5)(6)(7)(8)(9). Classically, novel epitopes are identified by cell surface elution and mass spectroscopy (MS) analysis, followed by matching against protein databases (16,17).…”

“…However, because spliced Ags are not contiguous, they neither match existing databases nor can they be predicted from contiguous protein sequences. Knowledge of the rules-if anythat govern the production of noncontiguous Ags would facilitate their prediction and identification, but it is currently not known whether proteasome splicing is determined by explicit rules or whether it is a random event (5,10,11).…”

“…This reversed cleavage has also been shown for other proteases including the proteasome that then would yield new spliced Ags that are not genetically coded yet can be presented to the immune system (5)(6)(7)(8)(9). To date, five spliced Ags that are immunogenic in vivo have been described: [RTK][QLYPEW] (5), [NTYAS][PRFK] (6), [SLPRGT] [STPK] (7), [IYMDGT][ADFSF] (8), and [RSYVPLAH][R] (9), all of which are produced by the proteasome through a transpeptidation mechanism (5,7,10,11). During normal proteasomal peptide hydrolysis, the N-terminal threonine residue of a catalytically active subunit reacts with the scissile peptide bond to form an O-acyl enzyme intermediate, resulting in the release of the C-terminal part of the peptide.…”