Proper functioning of the cell cycle and its regulation in response to both internal and external stimuli is essential for the normal growth, division, and differentiation of cells and tissues. Major control over cell cycle progression is exerted by ubiquitin-mediated proteolysis, particularly during the exit from mitosis and in G1. The initiation of chromosome separation (anaphase) and completion of the cell cycle require the degradation of a number of cell cycle regulatory proteins such as cyclins, securin (which regulates the protein holding sister chromosomes together), and microtubule motor proteins. The ubiquitin ligase (or E3) for the degradation of these proteins is the Anaphase Promoting Complex (APC). APC substrates contain degradation motifs (typically Destruction Boxes and KEN Boxes) and are targeted to the APC by two substrate recognition proteins, Cdc20 and Cdh1, active during late mitosis and in G1, respectively. Our long-term goal is to understand the ubiquitination of APC substrates at a mechanistic level. Although the components in this process are well known, there are still some significant gaps in our understanding. We will address these points in Saccharomyces cerevisiae using a combination of molecular genetics, studies in vivo, and biochemical assays in vitro. We expect that tools that we develop as well as conceptual advances will be of significant benefit to the field. Since APC function and regulation are highly conserved, insights we gain into the functions of the yeast system should be applicable to humans, as well. To further our understanding of APC function, we propose the following Specific Aims: 1) to determine how substrates are released from the APC: The role of ubiquitination and the origin of processivity. Substrates containing single sites of ubiquitination will be used in single-encounter assays to determine the requirements for dissociation of the ubiquitinated substrate from Cdh1 and/or the APC. We will explore the nature of substrate processivity, and characterize the interaction between a substrate and the Doc1 subunit of the APC. 2) To characterize the roles of deubiquitinating enzymes (DUBs) in regulating APC substrate degradation. Compared to substrate ubiquitination, comparatively little is known about the deubiquitinating enzymes (DUBs) that act on these proteins. We will identify and determine the functions of DUBs acting on important APC substrates affecting the spindle checkpoint and analyze a DUB that we hypothesize may play a proofreading function during ubiquitination. APC regulation and mis-regulation play important roles in the responses of cells to many anti-cancer agents and in the mis-segregation of chromosomes leading to aneuploidy and the development of tumors. Our studies will further our understanding of this essential cell cycle regulatory process and, in the longer term, provide targets for therapeutic intervention, directed towards the APC itself or the DUBs acting on its substrates.

Public Health Relevance

Proper functioning of the cell cycle requires, among other things, that certain regulatory proteins are degraded during or just following cell division;error in this process can lead to many abnormalities, most notably cancer as a result of the incorrect segregation of chromosomes to daughter cells. We are studying the mechanisms by which the cell attaches and remove a tag to proteins to signal that they should be degraded. Though basic in nature, the proposed experiments could direct future efforts to modulate this machinery for therapeutic benefit.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM101044-01A1
Application #
8435719
Study Section
Cellular Signaling and Regulatory Systems Study Section (CSRS)
Program Officer
Hamlet, Michelle R
Project Start
2013-08-01
Project End
2017-05-31
Budget Start
2013-08-01
Budget End
2014-05-31
Support Year
1
Fiscal Year
2013
Total Cost
$313,822
Indirect Cost
$123,822
Name
Yale University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520