Degradation of type I membrane proteins involves their extraction from the membrane, transfer to the cytosol, and destruction by the proteasome. This pathway is exemplified by the degradation of MHC class I molecules in cells that express the HCMV US2 or US11 products, both of which dramatically enhance the rate of degradation. We shall develop a chemistry-based approach to dissect this pathway in more detail. The generation of a chemically charged suppressor tRNA, equipped with a cleavable, photoreactive radiolabeled amino acid derivative should allow site-specific incorporation of the photoprobe into protein in membrane-supplemented in vitro translation systems. Photocrosslinking and subsequent reduction should result in transfer-of-label and may identify interacting partners in the US2-mediated degradation pathway of MHC Class I molecules. The next step in degradation is the removal of N-linked glycans by a peptide N-glycanase, of unknown identity and most likely confined to the cytosol. As possible inhibitors of this activity, electrophilic derivatives of G1cNAc will be prepared, also in radiolabeled or biotinylated form to assist in identification and purification of the target enzymes for which a novel assay has been developed. This reaction is followed by conjugation of the substrate with ubiquitin (Ub) to target the substrate to the proteasome. Prior to proteasomemediated proteolysis, the Ub moieties must be removed in a reaction catalyzed by members of the Ubisopeptidase multigene family, about whose specificity and control relatively little is known. We have synthesized an Ub-vinylsulfone that acts as an irreversible, covalent inhibitor of at least some of these isopeptidases. This class of compounds will be developed further to probe the in vitro dislocation of MHC Class I molecules. Proteolysis, finally, is carried out by the proteasome. We shall synthesize proteasome substrates that are heterobifunctional compounds containing a photoreactive moiety and an electrophile, to probe more accurately interactions of substrates -and potentially cellular proteins- with the proteasome. In the aggregate, the resulting tools are expected to shed new light on this poorly understood pathway of membrane protein turnover. The chemical-biological approach should allow development of more widely applicable molecular probes that could be used to manipulate their targets in health and disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM062502-04
Application #
6770193
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Shapiro, Bert I
Project Start
2001-07-01
Project End
2005-08-31
Budget Start
2004-07-01
Budget End
2005-08-31
Support Year
4
Fiscal Year
2004
Total Cost
$301,860
Indirect Cost
Name
Harvard University
Department
Pathology
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Kirak, Oktay; Frickel, Eva-Maria; Grotenbreg, Gijsbert M et al. (2010) Transnuclear mice with predefined T cell receptor specificities against Toxoplasma gondii obtained via SCNT. Science 328:243-8
Love, Kerry Routenberg; Pandya, Renuka K; Spooner, Eric et al. (2009) Ubiquitin C-terminal electrophiles are activity-based probes for identification and mechanistic study of ubiquitin conjugating machinery. ACS Chem Biol 4:275-87
Monach, Paul A; Hueber, Wolfgang; Kessler, Benedikt et al. (2009) A broad screen for targets of immune complexes decorating arthritic joints highlights deposition of nucleosomes in rheumatoid arthritis. Proc Natl Acad Sci U S A 106:15867-72
Ernst, Robert; Mueller, Britta; Ploegh, Hidde L et al. (2009) The otubain YOD1 is a deubiquitinating enzyme that associates with p97 to facilitate protein dislocation from the ER. Mol Cell 36:28-38
Park, Boyoun; Brinkmann, Melanie M; Spooner, Eric et al. (2008) Proteolytic cleavage in an endolysosomal compartment is required for activation of Toll-like receptor 9. Nat Immunol 9:1407-14
Vyas, Jatin M; Van der Veen, Annemarthe G; Ploegh, Hidde L (2008) The known unknowns of antigen processing and presentation. Nat Rev Immunol 8:607-18
Altun, Mikael; Edstrom, Erik; Spooner, Eric et al. (2007) Iron load and redox stress in skeletal muscle of aged rats. Muscle Nerve 36:223-33
Borodovsky, Anna; Ovaa, Huib; Meester, Wim J N et al. (2005) Small-molecule inhibitors and probes for ubiquitin- and ubiquitin-like-specific proteases. Chembiochem 6:287-91
Altun, Mikael; Galardy, Paul J; Shringarpure, Reshma et al. (2005) Effects of PS-341 on the activity and composition of proteasomes in multiple myeloma cells. Cancer Res 65:7896-901
van Swieten, Paul F; Maehr, Rene; van den Nieuwendijk, Adrianus M C H et al. (2004) Development of an isotope-coded activity-based probe for the quantitative profiling of cysteine proteases. Bioorg Med Chem Lett 14:3131-4

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