The control of protein levels in the cell was once thought to be almost entirely due to changes in transcription and translation. Today we understand that a significant part of this control occurs by regulating protein stability through the ubiquitin-proteasome system. This is particularly clear in the cell cycle, where transitions from one state to another are mediated by rapid changes in protein stability, but the general importance of post-translational modifications that affect protein stability is turning out to be quite common. In this grant, we propose to use protein microarrays to develop a general method for measuring post-translational modifications quantitatively, initially focusing on ubiquitination and sumoylation. The key feature of this approach is the use of concentrated cell extracts that are stably arrested in specific states of the cell cycle and that can perform cell cycle phase-appropriate reactions. This should provide a new approach to understanding regulation via post-translational modification. We also propose to investigate in detail the order in which a given E3 enzyme, particularly the anaphase promoting complex (APC), ubiquitinates its substrates. Ordering is a product of the complex kinetics of protein modification, including multistep polyubiquitination, deubiquitination, binding to carrier proteins, and proteasomal degradation. We are taking two complementary approaches to studying and modeling this network of reactions. The first is to develop simplified systems in which ubiquitination can only occur at a single site, or a few defined sites, and the kinetics of each reaction can therefore be fully understood. The second is to biochemically reconstitute the complete system, allowing us to ascertain the role of the proteasome in affecting the kinetics of ubiquitination and the ordering of substrate degradation and identify any new components that may previously have been missed. These two approaches will be connected via a mathematical model. Finally, we propose to study whether tight control of protein levels via active transcriptional, translational, and proteolytic mechanisms is frequent, or rare. Whether protein levels are controlled or variable has important implications for understanding disease susceptibility and drug efficacy, as well as for evolution

Public Health Relevance

Protein degradation is one of the most highly regulated processes in cells and is involved in cell proliferation and cancer, inflammation, and neurodegenerative disease. As such, it is a promising target for pharmacologic intervention. This proposal studies which proteins are signaled out for regulated degradation and on a molecular level how the particular choices are made.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM039023-26
Application #
8245216
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Hamlet, Michelle R
Project Start
1993-09-01
Project End
2014-03-31
Budget Start
2012-04-01
Budget End
2014-03-31
Support Year
26
Fiscal Year
2012
Total Cost
$652,650
Indirect Cost
$265,121
Name
Harvard University
Department
Biology
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
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Lu, Ying; Wang, Weiping; Kirschner, Marc W (2015) Specificity of the anaphase-promoting complex: a single-molecule study. Science 348:1248737
Lu, Ying; Lee, Byung-hoon; King, Randall W et al. (2015) Substrate degradation by the proteasome: a single-molecule kinetic analysis. Science 348:1250834
Wang, Weiping; Wu, Tao; Kirschner, Marc W (2014) The master cell cycle regulator APC-Cdc20 regulates ciliary length and disassembly of the primary cilium. Elife 3:e03083
Zhao, Rui; Deibler, Richard W; Lerou, Paul H et al. (2014) A nontranscriptional role for Oct4 in the regulation of mitotic entry. Proc Natl Acad Sci U S A 111:15768-73
Wan, Lixin; Tan, Mingjia; Yang, Jie et al. (2014) APC(Cdc20) suppresses apoptosis through targeting Bim for ubiquitination and destruction. Dev Cell 29:377-91
Merbl, Yifat; Kirschner, Marc W (2014) Post-Translational Modification Profiling--a High-Content Assay for Identifying Protein Modifications in Mammalian Cellular Systems. Curr Protoc Protein Sci 77:27.8.1-13
Fukushima, Hidefumi; Ogura, Kohei; Wan, Lixin et al. (2013) SCF-mediated Cdh1 degradation defines a negative feedback system that coordinates cell-cycle progression. Cell Rep 4:803-16
Wang, Weiping; Kirschner, Marc W (2013) Emi1 preferentially inhibits ubiquitin chain elongation by the anaphase-promoting complex. Nat Cell Biol 15:797-806
Merbl, Yifat; Refour, Phillipe; Patel, Hevan et al. (2013) Profiling of ubiquitin-like modifications reveals features of mitotic control. Cell 152:1160-72

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