The Ubiquitin Proteasome Pathway (UPP) controls the activities of signaling systems through reversible conjugation of ubiquitin and ubiquitin like proteins (Ubls). The UPP system contains more than 900 genes and functions in virtually all aspects of biology, including aging, neurodegeneration and cancer, yet the functions of only a subset of these genes are known. This proposal seeks to continue our development a systems-based approach for identifying functions and targets of UPP components in the cell division cycle and checkpoint pathways in mammalian cells. The platform we are developing employs global analysis of the UPP in these processes using loss of function genetics, proteomics, and newly established genetic approaches for the identification of substrates for E3 ubiquitin ligases. The integration of these systems with a focus on cell cycle and checkpoint controls provides a powerful approach to gene and pathway discovery. Using these systems, we have identified several new genes that control the spindle checkpoint controlling chromosome segregation. One of these, the deubiquitinating enzyme Usp44, controls the ubiquitination status of the APC regulatory protein Cdc20, a recently identified step in APC activation that promotes dissociation of Mad2. Usp44 therefore functions to maintain Cdc20 in its Mad2-inhibited state. Usp44 itself is also regulated during the cell cycle and in response to checkpoint activation.
In Aim 1, we will continue to elucidate how Usp44 is regulated and how its activity controls the checkpoint. In addition, we will perform a systematic mechanistic analysis of 4 additional deubiquitinating enzymes we have already validated as being required for the checkpoint.
In Aim 2, we propose to validate and analyze candidate genes from a recently performed RNAi screen to identify UPP genes involved in the response of cells to ionizing radiation.
In Aim 3, we will systematically analyze the role of the UPP in cell proliferation using new barcode-based shRNA screening capabilities coupled with high throughput flow cytometry.
In Aim 4, we continue to develop a general solutions to the problem of finding substrates for E3s. We have devised a novel flow-cytometry based system that allows degradation substrates for particular E3s to be identified using microarray hybridization and can also be used to identify proteins whose abundance is altered in response to a particular stimulus.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Research Project (R01)
Project #
5R01AG011085-19
Application #
8220902
Study Section
Special Emphasis Panel (ZRG1-CSRS-N (01))
Program Officer
Velazquez, Jose M
Project Start
1997-04-01
Project End
2013-08-31
Budget Start
2012-02-15
Budget End
2013-08-31
Support Year
19
Fiscal Year
2012
Total Cost
$424,049
Indirect Cost
$173,873
Name
Harvard University
Department
Pathology
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Rose, Christopher M; Isasa, Marta; Ordureau, Alban et al. (2016) Highly Multiplexed Quantitative Mass Spectrometry Analysis of Ubiquitylomes. Cell Syst 3:395-403.e4
Harper, J Wade; Bennett, Eric J (2016) Proteome complexity and the forces that drive proteome imbalance. Nature 537:328-38
Brown, Nicholas G; VanderLinden, Ryan; Watson, Edmond R et al. (2016) Dual RING E3 Architectures Regulate Multiubiquitination and Ubiquitin Chain Elongation by APC/C. Cell 165:1440-53
Nadarajan, Saravanapriah; Mohideen, Firaz; Tzur, Yonatan B et al. (2016) The MAP kinase pathway coordinates crossover designation with disassembly of synaptonemal complex proteins during meiosis. Elife 5:e12039
Willis, Nicholas A; Zhou, Chunshui; Elia, Andrew E H et al. (2016) Identification of S-phase DNA damage-response targets in fission yeast reveals conservation of damage-response networks. Proc Natl Acad Sci U S A 113:E3676-85
Zhou, Chunshui; Elia, Andrew E H; Naylor, Maria L et al. (2016) Profiling DNA damage-induced phosphorylation in budding yeast reveals diverse signaling networks. Proc Natl Acad Sci U S A 113:E3667-75
Bui, Duyen Amy; Lee, Wendy; White, Anne E et al. (2016) Cytokinesis involves a nontranscriptional function of the Hippo pathway effector YAP. Sci Signal 9:ra23
Scott, Daniel C; Rhee, David Y; Duda, David M et al. (2016) Two Distinct Types of E3 Ligases Work in Unison to Regulate Substrate Ubiquitylation. Cell 166:1198-1214.e24
Gallegos, Lisa Leon; Ng, Mei Rosa; Sowa, Mathew E et al. (2016) A protein interaction map for cell-cell adhesion regulators identifies DUSP23 as a novel phosphatase for β-catenin. Sci Rep 6:27114
Raman, Malavika; Sergeev, Mikhail; Garnaas, Maija et al. (2015) Systematic proteomics of the VCP-UBXD adaptor network identifies a role for UBXN10 in regulating ciliogenesis. Nat Cell Biol 17:1356-69

Showing the most recent 10 out of 120 publications