title: The origin of proteasome-inhibitor resistant HLA class I peptidomes: a study with HLA-A*68:01.
abstract: Some HLA class I molecules bind a significant fraction of their constitutive peptidomes in the presence of proteasome inhibitors. In this study, A*68:01-bound peptides, and their parental proteins, were characterized through massive mass spectrometry sequencing to refine its binding motif, including the nearly exclusive preference for C-terminal basic residues. Stable isotope tagging was used to distinguish proteasome-inhibitor sensitive and resistant ligands. The latter accounted for less than 20% of the peptidome and, like in HLA-B27, arose predominantly from small and basic proteins. Under the conditions used for proteasome inhibition in vivo, epoxomicin and MG-132 incompletely inhibited the hydrolysis of fluorogenic substrates specific for the tryptic or for both the tryptic and chymotryptic subspecificities, respectively. This incomplete inhibition was also reflected in the cleavage of synthetic peptide precursors of A*68:01 ligands. For these substrates, the inhibition of the proteasome resulted in altered cleavage patterns. However these alterations did not upset the balance between cleavage at peptide bonds resulting in epitope destruction and those leading to their generation. The results indicate that inhibitor-resistant HLA class I ligands are not necessarily produced by non-proteasomal pathways. However, their generation is not simply explained by decreased epitope destruction upon incomplete proteasomal inhibition and may require additional proteolytic steps acting on incompletely processed proteasomal products.
authors.affiliation: Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid), c/ Nicolas Cabrera N.1, Universidad Autónoma, 28049 Madrid, Spain.
authors.forename: Noel
authors.initials: N
authors.lastname: García-Medel
authors.affiliation: Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid), c/ Nicolas Cabrera N.1, Universidad Autónoma, 28049 Madrid, Spain.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Sanz-Bravo
authors.affiliation: Faculty of Biology, Technion - Israel Institute of Technology, Haifa 32000, Israel.
authors.forename: Eilon
authors.initials: E
authors.lastname: Barnea
authors.affiliation: Faculty of Biology, Technion - Israel Institute of Technology, Haifa 32000, Israel.
authors.forename: Arie
authors.initials: A
authors.lastname: Admon
authors.affiliation: Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid), c/ Nicolas Cabrera N.1, Universidad Autónoma, 28049 Madrid, Spain. Electronic address: aldecastro@cbm.uam.es.
authors.forename: José A
authors.initials: JA
authors.lastname: López de Castro
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D015234:HLA-A Antigens / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008024:Ligands; D009842:Oligopeptides / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects*
chemical_list: D015853:Cysteine Proteinase Inhibitors; D015234:HLA-A Antigens; C053029:HLA-A*68 antigen; D007976:Leupeptins; D008024:Ligands; D009842:Oligopeptides; D010455:Peptides; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; C078846:epoxomicin