Proteasomes and antigen presentation: evidence that a KEKE motif does not promote presentation of the class I epitope SIINFEKL.
Previously we proposed that stretches of alternating Lys(K) and Glu(E) in polypeptides promote the expression of nearby sequences on Class I molecules [Realini, C., Rogers, S.W., Rechsteiner, M., 1994. KEKE motifs. Proposed roles in protein-protein association and presentation of peptides by MHC class I receptors. FEBS Lett. 348, 109-113]. As a test of the KEKE hypothesis we have employed osmotic lysis of pinosomes and transfection to introduce or express various ubiquitin peptide fusion proteins in the cytosol of wild type and PA28alphabetagamma- mouse embryo fibroblasts. KEKE or non-KEKE motifs were placed between ubiquitin and the OVA epitope SIINFEKL that was at or near the C-termini of the various fusion proteins. Measurements of surface Kb-SIINFEKL complexes using the monoclonal antibody 25.-D1.16 allowed us to assess the effects of upstream KEKE motifs and PA28 status on SIINFEKL surface presentation. KEKE motifs did not enhance presentation of the OVA epitope. However, our studies did confirm that PA28alphabeta is needed for efficient SIINFEKL surface expression when hsp90 is inhibited.
Gonciarz-Swiatek
Malgorzata
M
Department of Biochemistry, University of Utah, School of Medicine, 15 North Medical Drive East RM 4100, Salt Lake City, UT 84112-5650, USA.
Rechsteiner
Martin
M
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D017951:Antigen Presentation; D002460:Cell Line; D003600:Cytosol / Q000276:immunology; D004622:Embryo, Mammalian / Q000166:cytology; D000939:Epitopes; D005347:Fibroblasts / Q000166:cytology / Q000276:immunology; D015395:Histocompatibility Antigens Class I / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry*; D011506:Proteins / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology; D025801:Ubiquitin / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D015395:Histocompatibility Antigens Class I; D011506:Proteins; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex; C486213:Psme1 protein, mouse