The ubiquitin-proteasome system is a major regulator of cellular processes in which speed, specificity, and timing are critical (e.g., cell cycle progression, gene transcription, cell differentiation, apoptosis, and signal transduction). Ubiquitin-mediated proteolysis of key substrates is mediated by multiple machines encompassing two functions: ubiquitin-tagging (i.e. ubiquitin conjugating enzymes and ubiquitin ligases) and protein degradation (i.e. the proteasome). Ubiquitin ligases, which contribute to transfer ubiquitin to proteins destined for degradation, are often composed of several subunits (e.g., the SCF ubiquitin ligase complexes) that impart a high degree of precision to the process. In humans, there are 69 SCF ligases, each characterized by invariable, structural elements (Skp1, Cul1, and Rbx1) and a variable F- box protein subunit that provides specificity by directly recruiting the substrate to the core of the ligase. Notably, only 9 of the 69 human SCF ligases (containing the F-box proteins 2TrCP1, 2TrCP2, Fbxw7, Skp2, Fbxl3, Fbxl5, Fbxo1, Fbxo4, and Fbxo6) have well-established/accepted substrates and functions. The remaining 60 F-box proteins are considered "orphans," and their substrates still await discovery. Fbxo15 is an orphan F-box protein that our preliminary data and published findings suggest to be involved in the control of stem cell self-renewal and differentiation. Our laboratory has successfully utilized two techniques for unbiased identification of F-box protein substrates. Using traditional tandem affinity purifications, we have identified novel SCF substrates, but we also developed a novel immunoaffinity/enzymatic assay that enriches for ubiquitylated substrates based on the ability of SCF complexes to ubiquitylate co-purified substrates in vitro. Based on our previous success in identifying and characterizing F-box protein substrates, we propose the following two aims. We will identify biologically significant substrates of human Fbxo15 (Specific Aim 1) and validate the Fbxo15-dependent regulation of these substrates (Specific Aim 2).

Public Health Relevance

Many proliferative diseases, such as cancer, develop due to defects in differentiation processes. Preliminary data suggests that the ubiquitin-mediated proteolysis of Fbxo15 substrates is important in controlling the differentiation state of mammalian cells, and this team is committed to the integration of its basic research into the molecular mechanisms of Fbxo15 substrate degradation with an understanding of malignant transformation. A detailed understanding of the pathways that regulate the self-renewal and differentiation of cells may lead to novel therapies for cancer.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21CA161108-02
Application #
8274655
Study Section
Molecular Oncogenesis Study Section (MONC)
Program Officer
Hildesheim, Jeffrey
Project Start
2011-07-01
Project End
2014-06-29
Budget Start
2012-07-01
Budget End
2014-06-29
Support Year
2
Fiscal Year
2012
Total Cost
$183,788
Indirect Cost
$75,038
Name
New York University
Department
Pathology
Type
Schools of Medicine
DUNS #
121911077
City
New York
State
NY
Country
United States
Zip Code
10016
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