An alanyl-alanyl-phenylalanyl-7-amino-4-methylcoumarin-hydrolyzing protease particle copurifying with 26S proteasomes was isolated and identified as tripeptidyl peptidase II (TPPII), a cytosolic subtilisin-like peptidase of unknown function. The particle is larger than the 26S proteasome and has a rod-shaped, dynamic supramolecular structure. TPPII exhibits enhanced activity in proteasome inhibitor-adapted cells and degrades polypeptides by exo- as well as predominantly trypsin-like endoproteolytic cleavage. TPPII may thus participate in extralysosomal polypeptide degradation and may in part account for nonproteasomal epitope generation as postulated for certain major histocompatibility complex class I alleles. In addition, TPPII may be able to substitute for some metabolic functions of the proteasome.
Although a pivotal role of proteasomes in the proteolytic generation of epitopes for major histocompatibility complex (MHC) class I presentation is undisputed, their precise function is currently the subject of an active debate: do proteasomes generate many epitopes in definitive form, or do they merely generate the COOH termini, whereas the definitive NH2 termini are cleaved by aminopeptidases? We determined five naturally processed MHC class I ligands derived from HIV-1 Nef. Unexpectedly, the five ligands correspond to only three cytotoxic T lymphocyte (CTL) epitopes, two of which occur in two COOH-terminal length variants. Parallel analyses of proteasomal digests of a Nef fragment encompassing the epitopes revealed that all five ligands are direct products of proteasomes. Moreover, in four of the five ligands, the NH2 termini correspond to major proteasome cleavage sites, and putative NH2-terminally extended precursor fragments were detected for only one of the five ligands. All ligands are transported by the transporter associated with antigen processing (TAP). The combined results from these five ligands provide strong evidence that many definitive MHC class I ligands are precisely cleaved at both ends by proteasomes. Additional evidence supporting this conclusion is discussed, along with contrasting results of others who propose a strong role for NH2-terminal trimming with direct proteasomal epitope generation being a rare event.
HIV proteins contain a multitude of naturally processed cytotoxic T lymphocyte (CTL) epitopes that concentrate in clusters. The molecular basis of epitope clustering is of interest for understanding HIV immunogenicity and for vaccine design. We show that the CTL epitope clusters of HIV proteins predominantly coincide with hydrophobic regions, whereas the noncluster regions are predominantly hydrophilic. Analysis of the proteasomal degradation products of full-length HIV-Nef revealed a differential sensitivity of cluster and noncluster regions to proteasomal processing. Compared with the epitope-scarce noncluster regions, cluster regions are digested by proteasomes more intensively and with greater preference for hydrophobic P1 residues, resulting in substantially greater numbers of fragments with the sizes and COOH termini typical of epitopes and their precursors. Indeed, many of these fragments correspond to endogenously processed Nef epitopes and͞or their potential precursors. The results suggest that differential proteasomal processing contributes importantly to the clustering of CTL epitopes in hydrophobic regions. C omprehensive analyses of several HIV antigens including Nef have revealed an enormous diversity of epitopes recognized by cytotoxic T lymphocytes (CTLs) of HIV ϩ patients (see the HIV Molecular Immunology Database, http:͞͞hiv-web.lanl.gov͞ content͞immunology). Moreover, HIV CTL epitopes extensively overlap in so-called epitope clusters, whereas other regions of the HIV proteins are almost completely devoid of CTL epitopes (refs. 1-6 and HIV Molecular Immunology Database). Evidence for CTL epitope clusters exists also for non-HIV antigens (7). Although CTL epitope clusters often coincide with rather conserved regions of the HIV proteins (1, 4, 6), it has been postulated that the highly nonuniform distribution of HIV CTL epitopes may be related to antigen processing and presentation (2,4,6).Proteasomes seem to participate in the generation of many if not most CTL epitopes. In the classical class I antigen-processing pathway proteasomes generate epitopes as well as precursors that are trimmed by cytosolic and endoplasmic reticulum-resident aminopeptidases. Trimming by carboxypeptidases seems to be highly unusual. Cytosolic endoproteases other than proteasomes may be involved in the production of certain epitopes, but thus far none have been unequivocally identified (for several excellent reviews, see ref. 8 and all articles in the same issue). Important information on the role of proteasomes in antigen processing has been obtained by in vitro digestion of proteins and protein fragments with isolated proteasomes. However, thus far analyses of CTL epitopecontaining full-length antigens (ovalbumin and -galactosidase) (9, 10) and most antigen fragments focused on a single epitope and͞or related epitope precursors. In nearly all experiments with CTL epitope-containing substrates, 20S proteasomes were used, and excellent correlations with epitope recognition on antigenpresenting cells were often reporte...
We have studied polypeptide processing by purified proteasomes, with regard to proteolytic specificity and cytotoxic T-lymphocyte (CTL) epitope generation. Owing to defined preferences with respect to cleavage sites and fragment length, proteasomes degrade polypeptide substrates into cohorts of overlapping oligopeptides. Many of the proteolytic fragments exhibit structural features in common with major histocompatibility complex (MHC) class I ligands including fragment size and frequencies of amino acids at fragment boundaries. Proteasomes frequently generate definitive MHC class I ligands and/or slightly longer peptides, while substantially larger peptides are rare. Individual CTL epitopes are produced in widely varying amounts, often consistent with immunohierarchies among CTL epitopes. We further found that polypeptide processing is remarkably conserved among proteasomes of eukaryotic origin and that invertebrate proteasomes can efficiently produce known high-copy MHC class I ligands, suggesting evolutionary adaptation of the transporter associated with antigen processing and MHC class I to ancient constraints imposed by proteasomal protein degradation.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.