Our research focuses on the functions and mechanisms of the mammalian N-end rule pathway, a subset of the ubiquitin system. The N-end rule pathway relates the in vivo half-life of a protein to the identity of its N-terminal residue. We have shown that i) the NTAN1-mediated N-terminal deamidation is involved in socially conditioned behavior, ii) the ATE1-mediated N-terminal arginylation is essential for cardiovascular development, iii) the N-terminal oxidation, a novel protein modification, mediates a novel oxygen-dependent protein degradation which may function as an oxygen sensor, iv) UBR1 and UBR2 are functionally overlapping downstream Ub ligases of ATE1, v) UBR1 is involved in fat metabolism and muscle protein degradation, whereas UBR2 is essential for homologous chromosome and female viability, and vi) the cooperative activity of UBR1 and UBR2 underlies ATE1-mediated cardiovascular development, and other biological processes including central nervous development, mesenchymal cell proliferation, and chromosome stability.
We are further characterizing these mutant mice to understand the molecular mechanisms underlying the functions of the N-end rule pathway. In addition, we recently developed a peptide-based pulldown assay for identifying proteins (including target substrates) that interact with the N-end rule components. Using this assay coupled with mass spectrometric analysis, we identified a 600 kDa-protein termed p600 that binds to N-terminal destabilizing residues. It is to be determined whether ubiquitylation of the N-end rule pathway is mediated by the UBR1-UBR2-p600. Another focus of our research is to develop inhibitors of the N-end rule pathway. We have previously developed protein-based bivalent inhibitors of UBR1. We now are developing the peptide-based bivalent inhibitors, for therapeutic applications.
Ubiquitin (Ub) is a 76 residue protein whose conjugation to other proteins regulates many critical biological processes. Ub-dependent proteolysis involves the marking of a target protein through covalent conjugation of Ub to a substrate, which is mediated by the E1-E2-E3 enzymatic cascade. The functions of E3 include the recognition of a substrate's degradation signal. The ubiquitylated substrates are subsequently degraded by the 26S proteasome.
Our research is focused on the functions and mechanisms of the Ub-dependent N-end rule proteolytic pathway that relates the in vivo half life of a protein to the identity of its N terminal residue. Through biochemical and genetic approaches, we have learned that this pathway is hierarchical. Briefly, N-terminal Asn and Gln (of short-lived substrates) are converted to Asp and Glu, respectively, which are then conjugated with Arg to generate a new N-terminal residue (Arg). N-terminal Arg (of short-lived substrates) and other primary destabilizing residues are directly bound by UBR box ubiquitin ligases. In addition to Asp and Glu, N-terminal Cys is destabilizing through its oxidation and subsequent Arg conjugation. This pathway is essential for many processes, including cardiovascular system and meiosis in mice.
Our ongoing projects are: (1) to identify components of this pathway by using affinity-based proteomics, (2) to elucidate the in vivo functions of specific component by using mouse knockout techniques, (3) to identify in vivo substrates of this pathway by using functional proteomics, (4) to elucidate biochemical principles underlying the E3-substrate interaction by using biochemical techniques, and (5) to apply acquired knowledge to clinical settings.
Kwon, Y.T., Z.X. Xia, J.Y. An, T. Tasaki, I. Davydov, J.W. Seo, J. Sheng, Y. Xie, and A. Varshavsky. Female lethality and apoptosis of spermatocytes in mice lacking the UBR2 ubiquitin ligase of the N-end rule pathway. (submitted)
Kwon, Y.T., A.S. Kashina, I.V. Davydov, R.G. Hu, J.Y. An, J.W. Seo, F. Du and A. Varshavsky. An essential role of N-terminal arginylation in cardiovascular development. Science 297:96-99, 2002.
Kwon, Y.T., Z.X. Xia, I.V. Davydov, S.H. Lecker and A. Varshavsky. Construction and analysis of mouse strains lacking the ubiquitin ligase UBR1 (E3α) of the N-end rule pathway. Mol. Cell. Biol. 21:8007-8021, 2001.
Kwon, Y.T., S.A. Balogh, I.V. Davydov, A.S. Kashina, J.K. Yoon, Y. Xie, V.H. Denenberg, and A. Varshavsky. Altered activity, social behavior, and spatial memory in mice lacking the NTAN1p amidase and the asparagine branch of the N-end rule pathway. Mol. Cell. Biol. 20:4135-4148, 2000.
Kwon, Y.T., A.S. Kashina and A. Varshavsky. Alternative splicing results in differential expression, activity and localization of the two forms of arginyl-tRNA-protein transferase, a component of the N-end rule pathway. Mol. Cell. Biol. 19:182-193, 1999.
Kwon, Y.T., F. Lévy and A. Varshavsky. Bivalent inhibitor of the N-end rule pathway. J. Biol. Chem. 274:18135-18139, 1999.
Kwon, Y.T., Y. Reiss, A. Hershko, V. Fried, D.K. Gonda, P.R.H. Waller, N.A. Jenkins, N.G. Copeland and A. Varshavsky. Mouse and human genes encoding the recognition component of the N-end rule pathway. Proc. Natl. Acad. Sci. USA 95:7898-7903, 1998.