Cullin-ring based ubiquitin ligases (CRLs) constitute one of the largest classes of E3 ubiquitin ligases in mammalian cells, yet many aspects of the mechanisms by which these E3s are regulates and how they their substrates ultimately are degraded by the proteasome remain unknown. In the previous funding cycle, we have explored the functions of Cul3-BTB protein and Cul4-Ddb1DCAF E3s through structural, mechanistic, and genetic approaches. In this renewal, we seek to examine two emerging themes in the ubiquitination field. Theme 1 concerns the question of how one goes about defining the entire repertoire of genes that are require to signal ubiquitination of a substrate, perform the ubiquitination event, and then target the protein to the proteasome. This question is being analyzed in the context of the replication licensing protein Cdt1 and its DNA damage-dependent turnover via the Cul4-Ddb1Cdt2 E3. We have recently completed a genome wide screen for genes whose depletion by RNAi blocks UV-dependent Cdt1 turnover. This screen has revealed 600 candidate genes currently being validated, including near saturation of the known genes involved directly in Cdt1 ubiquitination and proteolysis. A number of known and candidate DNA damage genes were identified, allowing us to place these genes in a hypothetic pathway upstream and downstream of Cdt1 ubiquitination. Through a series of secondary assays, Aim 1 will order validated genes into a signaling pathway, and experiments in mammalian cells and in Xenopus egg extracts will define the mechanisms involved for a subset of genes acting at distinct points in the pathway, including components that may link ubiquitinated Cdt1 to the proteasome. Theme 2 addresses the general question of how ubiquitinated substrates are targeted to the proteasome. There is accumulating evidence in the field indicating that "facilitator" proteins, including proteins with various sorts of ubiquitin binding domains, promote the recognition of ubiquitinated proteins by the proteasome. We have adapted and further developed emerging technologies which allow us to measure the relative rates of turnover of individual substrate in the presence and absence of RNAi targeting components of the degradation machinery. Using this technology in the context of a panel of substrates of the SCF2-TRCP complex, Aim 2 will perform a combinatorial genetic analysis of candidate "facilitator" proteins in order to uncover relevant and potentially redundant "facilitator" proteins for particular substrates, thereby revealing the underlying specificity in substrate recruitment to the proteasome. Using the Global Protein Stability (GPS) system, we will identify targets of the proteasome-associated Rpn10 "facilitator" protein from a library of cells expressing 12,000 human genes, thereby defining the repertoire of ubiquitination targets that require Rpn10 for their turnover. In vitro reconstitution experiments will attempt to recapitulate in vivo specificity of facilitators in an in vitro setting.

Public Health Relevance

Modification of proteins by ubiquitin constitutes a primary mode of protein regulation in cells and underlies diverse signaling pathways. Many human diseases reflect disruption in the pathways that control turnover of cellular proteins. This proposal seeks to understand in greater detail the pathways that control protein ubiquitination and how substrates are targeted to the proteasome.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM070565-08
Application #
8290507
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Hamlet, Michelle R
Project Start
2005-03-01
Project End
2013-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
8
Fiscal Year
2012
Total Cost
$315,464
Indirect Cost
$124,344
Name
Harvard University
Department
Pathology
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Mancias, Joseph D; Wang, Xiaoxu; Gygi, Steven P et al. (2014) Quantitative proteomics identifies NCOA4 as the cargo receptor mediating ferritinophagy. Nature 509:105-9
Monda, Julie K; Scott, Daniel C; Miller, Darcie J et al. (2013) Structural conservation of distinctive N-terminal acetylation-dependent interactions across a family of mammalian NEDD8 ligation enzymes. Structure 21:42-53
Sarraf, Shireen A; Raman, Malavika; Guarani-Pereira, Virginia et al. (2013) Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization. Nature 496:372-6
Raman, Malavika; Havens, Courtney G; Walter, Johannes C et al. (2011) A genome-wide screen identifies p97 as an essential regulator of DNA damage-dependent CDT1 destruction. Mol Cell 44:72-84
Lee, Peter C W; Sowa, Mathew E; Gygi, Steven P et al. (2011) Alternative ubiquitin activation/conjugation cascades interact with N-end rule ubiquitin ligases to control degradation of RGS proteins. Mol Cell 43:392-405
Behrends, Christian; Sowa, Mathew E; Gygi, Steven P et al. (2010) Network organization of the human autophagy system. Nature 466:68-76
O'Connell, Brenda C; Adamson, Britt; Lydeard, John R et al. (2010) A genome-wide camptothecin sensitivity screen identifies a mammalian MMS22L-NFKBIL2 complex required for genomic stability. Mol Cell 40:645-57
Svendsen, Jennifer M; Smogorzewska, Agata; Sowa, Mathew E et al. (2009) Mammalian BTBD12/SLX4 assembles a Holliday junction resolvase and is required for DNA repair. Cell 138:63-77
Schulman, Brenda A; Harper, J Wade (2009) Ubiquitin-like protein activation by E1 enzymes: the apex for downstream signalling pathways. Nat Rev Mol Cell Biol 10:319-31
Zheng, Gang; Schweiger, Michal-Ruth; Martinez-Noel, Gustavo et al. (2009) Brd4 regulation of papillomavirus protein E2 stability. J Virol 83:8683-92

Showing the most recent 10 out of 11 publications