Contribution of Proteasomal β-Subunits to the Cleavage of Peptide Substrates Analyzed with Yeast Mutants

Abstract

Proteasomes generate peptides that can be presented by major histocompatibility complex (MHC) class I molecules in vertebrate cells. Using yeast 20 S proteasomes carrying different inactivated ␤-subunits, we investigated the specificities and contributions of the different ␤-subunits to the degradation of polypeptide substrates containing MHC class I ligands and addressed the question of additional proteolytically active sites apart from the active ␤-subunits. We found a clear correlation between the contribution of the different subunits to the cleavage of fluorogenic and long peptide substrates, with ␤5/Pre2 cleaving after hydrophobic, ␤2/Pup1 after basic, and ␤1/Pre3 after acidic residues, but with the exception that ␤2/Pup1 and ␤1/Pre3 can also cleave after some hydrophobic residues. All proteolytic activities including the "branched chain amino acid-preferring" component are associated with ␤5/Pre2, ␤1/Pre3, or ␤2/ Pup1, arguing against additional proteolytic sites. Because of the high homology between yeast and mammalian 20 S proteasomes in sequence and subunit topology and the conservation of cleavage specificity between mammalian and yeast proteasomes, our results can be expected to also describe most of the proteolytic activity of mammalian 20 S proteasomes leading to the generation of MHC class I ligands.

Integration of the ubiquitin-proteasome pathway with a cytosolic oligopeptidase activity

“…Notwithstanding selection of adapted cells on an inhibitor that completely blocks chymotrypsin-like activity (Fig. 2B), the remaining active Z subunits must be capable of cleaving C terminal of leucine, although less efficiently than the X͞LMP7 subunits (24,29). Thus, adapted cells possess partially functional proteasomes that can cleave polypeptides with surprisingly similar specificity but with reduced efficiency.…”

“…In vitro proteasome digestion reactions containing 0.5 g of purified proteasome and 20 g of peptide DPVHGEFAPGNYPALWSYAMG (Sendai NP 317-338 ) were performed as described (24). Digestion reactions were analyzed by C18 (Waters Delta Pak 3.9 mm ϫ 150 mm) reverse phase-HPLC (Hewlett-Packard HP-1100).…”

Re-evaluating the Generation of a “Proteasome-Independent” MHC Class I-Restricted CD8 T Cell Epitope

“…Subsequent reports described a similar presentation phenotype for several other epitopes (16 -22). In addition, cell-free proteasome digests do not always result in the generation of epitopes produced in, and presented by, intact cells (14,23,24), suggesting that other proteases may be involved in the generation of some epitopes. NP 147-155 is destroyed rather than generated by the proteasome during such digests (14,23), a result that is greatly accentuated by the A146P mutation (14).…”

“…In addition, cell-free proteasome digests do not always result in the generation of epitopes produced in, and presented by, intact cells (14,23,24), suggesting that other proteases may be involved in the generation of some epitopes. NP 147-155 is destroyed rather than generated by the proteasome during such digests (14,23), a result that is greatly accentuated by the A146P mutation (14). Therefore, NP 147-155 production may involve the action of proteases operating in parallel with the proteasome that are effective only in the absence of fully functional proteasome (which could serve to destroy the epitope).…”

Proteasome inhibitors mechanism; source for design of newer therapeutic agents

“…35 Similarly, epoxomicin was chemically converted into an affinity label by attachment of a biotin moiety. 34 Both of these labeled compounds Proteasome inhibitors; newer therapeutic agents SL Harer et al were crucial for the identification of the proteasome as their primary protein target. 31,35 In addition to the obvious utility of these reagents for target identification, both classes of affinity probes can also be used to rapidly monitor proteasome activity under different physiological conditions.…”