In cells, a majority of unwanted and harmful proteins are eliminated through a process called the ubiquitin-proteasome pathway. In this process, a target protein is tagged with a chain of ubiquitin molecules and the resulting marked protein is then recognized by the proteasome, which drives protein degradation. Proper functioning of the ubiquitin tagging and proteasomal activities is of key importance for the function of a cell and an organism. Defective regulation of the ubiquitin-proteasome system is manifest in human diseases including cancers. Our long-range goal is to understand the precise functioning of the SCF E3 ubiquitin ligase complex, which is the key machinery in cells responsible for tagging a large set of proteins with a chain of ubiquitin for degradation. The short-term objective of our project is to determine how a chain of ubiquitin is built on a protein, which signals degradation. A protein target has a built-in "degradation code" that dictates its destruction. In an ubiquitin molecule, "conjugation codes" are present to direct the formation of an ubiquitin chain, or a linkage that connects an ubiquitin to the protein target. The ubiquitin tagging reaction can be viewed as a process, beginning with the "reading" of the "degradation code" on the protein target, and ubiquitin "conjugation codes," by protein facilitators that include SCF E3 and enzymes such as UbcH5 and Cdc34. Subsequently, SCF, UbcH5, and Cdc34 act to "build" a large protein network that links the protein target with a chain of ubiquitin. However, it remains poorly understood regarding the nature of the ubiquitin "conjugation codes," how these codes are "read," as well as the network "building" process necessary for ubiquitin tagging. Using the tools of biochemistry, molecular biology and cell biology, our project is to: 1) identify new molecular components that help form the complex of a protein target and an SCF E3, which triggers the ubiquitin tagging process;2) understand how SCF, UbcH5, and Cdc34 build protein networks for tagging a target protein with ubiquitin;3) identify the ubiquitin "conjugation codes" and uncover the mystery of how Cdc34 reads these "codes."

Public Health Relevance

This project intends to uncover the mystery of tagging a protein target with a chain of ubiquitin molecules, which leads to protein degradation. Knowledge gained in this study will advance understanding of this ubiquitin tagging process that is fundamental to the function of a cell and an organism. Importantly, the molecular insights revealed in this project will improve the designing of drugs effective for treating human diseases that include cancer.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM061051-10
Application #
8319308
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Anderson, Vernon
Project Start
2000-04-01
Project End
2015-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
10
Fiscal Year
2012
Total Cost
$319,413
Indirect Cost
$130,413
Name
Icahn School of Medicine at Mount Sinai
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
078861598
City
New York
State
NY
Country
United States
Zip Code
10029
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Scheufele, Florian; Wolf, Benjamin; Kruse, Michael et al. (2014) Evidence for a regulatory role of Cullin-RING E3 ubiquitin ligase 7 in insulin signaling. Cell Signal 26:233-9
Kovacev, Jordan; Wu, Kenneth; Spratt, Donald E et al. (2014) A snapshot of ubiquitin chain elongation: lysine 48-tetra-ubiquitin slows down ubiquitination. J Biol Chem 289:7068-81
Chong, Robert A; Wu, Kenneth; Spratt, Donald E et al. (2014) Pivotal role for the ubiquitin Y59-E51 loop in lysine 48 polyubiquitination. Proc Natl Acad Sci U S A 111:8434-9
Spratt, Donald E; Wu, Kenneth; Kovacev, Jordan et al. (2012) Selective recruitment of an E2~ubiquitin complex by an E3 ubiquitin ligase. J Biol Chem 287:17374-85
Wu, Kenneth; Yan, Hua; Fang, Lei et al. (2011) Mono-ubiquitination drives nuclear export of the human DCN1-like protein hDCNL1. J Biol Chem 286:34060-70
Sarikas, Antonio; Hartmann, Thomas; Pan, Zhen-Qiang (2011) The cullin protein family. Genome Biol 12:220
Tan, Mingjia; Zhu, Yueming; Kovacev, Jordan et al. (2010) Disruption of Sag/Rbx2/Roc2 induces radiosensitization by increasing ROS levels and blocking NF-kappaB activation in mouse embryonic stem cells. Free Radic Biol Med 49:976-83
Choi, Yun-Seok; Wu, Kenneth; Jeong, Kwiwan et al. (2010) The human Cdc34 carboxyl terminus contains a non-covalent ubiquitin binding activity that contributes to SCF-dependent ubiquitination. J Biol Chem 285:17754-62
Wu, Kenneth; Kovacev, Jordan; Pan, Zhen-Qiang (2010) Priming and extending: a UbcH5/Cdc34 E2 handoff mechanism for polyubiquitination on a SCF substrate. Mol Cell 37:784-96

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