Protein modification with the small protein ubiquitin, a process referred to as ubiquitylation, plays crucial roles in the majority of cellular processes. Ubiquitin ligases are the most complex and most important components of the ubiquitylation machinery. They confer substrate selectivity and are the main targets for regulation. The importance of ubiquitin ligases (E3 enzymes) is underscored by the vast number of proteins functioning as E3s. It is estimated that the human genome encodes 600- 1000 proteins with ubiquitin ligase activity, a number that is significantly higher than that of protein kinases. Ubiquitin ligases are poised to form a link between metabolic pathways and other cellular processes such as cell cycle, stress response, and differentiation. The importance of cross talk between metabolism and other cellular pathways is evident, and it is becoming increasingly clear that a plethora of human diseases is directly connected to misregulation at the interface between metabolism and signaling to other cellular pathways. Despite the importance of understanding how metabolism communicates with other cellular processes, our understanding at the molecular level is very limited at best. The parent grant of this proposal uses a pathway that connects sulfur amino acid metabolism with cell proliferation as a model to understand regulation by non-proteolytic ubiquitylation. The central player is the ubiquitin ligase SCFMet30, which integrates metabolism of sulfur containing metabolites with the cell cycle. This revision application proposes to significantly extend the scope of the parent grant to understand how levels of sulfur containing metabolites regulate the SCFMet30 ubiquitin ligase (specific aim 1), and to explore the hypothesis that ubiquitin ligases directly connect metabolic pathways with other cellular processes (specific aim 2). We will develop and apply mass spectrometric approaches to probe interactions of metabolites with components of the SCFMet30 pathway (specific aim 1), and other ubiquitin ligases in yeast and human cells (specific aim 2). The physiological importance of identified interactions will then be probed using mutations in the identified binding sites. Ubiquitin ligases are the most diverse group of cellular regulators and exciting findings in the plant system have demonstrated ubiquitin ligases as receptors for metabolite related small molecules. This proposal aims to define ubiquitin ligases as the molecular link between metabolism and other cellular processes in yeast and humans. Findings from these studies are likely to define new paradigms in metabolite sensing and to uncover novel disease related pathways.

Public Health Relevance

Protein modification with ubiquitin controls most if not all functions of living cells and is involved in many human diseases. This project seeks to identify interactions between the ubiquitylation machinery and cellular metabolites and connect these two fundamental cellular processes, metabolism and signaling, at a molecular level. Results from these studies will uncover new drug targets for a plethora of human diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM066164-11S1
Application #
8573095
Study Section
Special Emphasis Panel (ZGM1-CBB-0 (MI))
Program Officer
Gerratana, Barbara
Project Start
2002-08-01
Project End
2016-07-31
Budget Start
2013-08-08
Budget End
2014-07-31
Support Year
11
Fiscal Year
2013
Total Cost
$115,531
Indirect Cost
$40,531
Name
University of California Irvine
Department
Biochemistry
Type
Schools of Medicine
DUNS #
046705849
City
Irvine
State
CA
Country
United States
Zip Code
92697
Lin, Da-Wei; Chung, Benjamin P; Kaiser, Peter (2014) S-adenosylmethionine limitation induces p38 mitogen-activated protein kinase and triggers cell cycle arrest in G1. J Cell Sci 127:50-9
Kaiser, Peter; Mayor, Thibault (2011) Gold for ubiquitin in Vancouver: First Conference on Proteomics of Protein Degradation and Ubiquitin Pathways held June 6-8, 2010 in Vancouver, University of British Columbia, organized By Lan Huang, Thibault Mayor, and Peipei Ping. Mol Cell Proteomics 10:R110.003863
Flick, Karin; Kaiser, Peter (2009) Proteomic revelation: SUMO changes partners when the heat is on. Sci Signal 2:pe45
Meierhofer, David; Wang, Xiaorong; Huang, Lan et al. (2008) Quantitative analysis of global ubiquitination in HeLa cells by mass spectrometry. J Proteome Res 7:4566-76
Kaiser, Peter; Meierhofer, David; Wang, Xiaorong et al. (2008) Tandem affinity purification combined with mass spectrometry to identify components of protein complexes. Methods Mol Biol 439:309-26
Wang, Xiaorong; Chen, Chi-Fen; Baker, Peter R et al. (2007) Mass spectrometric characterization of the affinity-purified human 26S proteasome complex. Biochemistry 46:3553-65
Tagwerker, Christian; Zhang, Hongwei; Wang, Xiaorong et al. (2006) HB tag modules for PCR-based gene tagging and tandem affinity purification in Saccharomyces cerevisiae. Yeast 23:623-32
Guerrero, Cortnie; Tagwerker, Christian; Kaiser, Peter et al. (2006) An integrated mass spectrometry-based proteomic approach: quantitative analysis of tandem affinity-purified in vivo cross-linked protein complexes (QTAX) to decipher the 26 S proteasome-interacting network. Mol Cell Proteomics 5:366-78
Kaiser, Peter; Huang, Lan (2005) Global approaches to understanding ubiquitination. Genome Biol 6:233
Yen, James L; Su, Ning-Yuan; Kaiser, Peter (2005) The yeast ubiquitin ligase SCFMet30 regulates heavy metal response. Mol Biol Cell 16:1872-82