Post-translational modification of target proteins via covalent attachment of ubiquitin or ubiquitin-like modifiers is a key mechanism for cellular signaling. The central biological role of ubiquitin mediated removal of certain protein factors through targeting to the 26S proteasome has become increasingly clear. Moreover, defects in ubiquitin signaling are directly implicated in an increasing number of diseases, such as Parkinson's and other neurodegenerative disorders as well as a variety of human cancers, such as breast and ovarian cancer as well as various types of lymphoma. Coupled structural and functional analysis of the ubiquitination process is essential to understanding how this common signaling process modulates protein function in healthy tissue and is defective in diseased states. Addition of ubiquitin to target proteins is accomplished via an enzyme cascade consisting of activating (E1), conjugating (E2), and ligating (E3) enzymes. Poly-ubiquitination also can require a specialized poly-ubiquitin ligase, termed an E4 ligase. Our studies focus on the newly discovered U-box domain family of E3 ligases, which are just beginning to be characterized. Our laboratory determined the first structure of a U-box domain from an essential RNA splicing factor, Prp19, and showed that the U-box was required for E3 ligase activity.
In Aim 1, we propose extending these studies to generate a complete structural model of the intact U-box ligase, for which there are currently no examples.
Aim 2 focuses on two other members of the U-box family, CHIP and Ufd2, which alone have E3 activity but when bound to each other exhibit the unique E4 poly-ubiquitination activity. The goal of this Aim is to determine the structural and functional basis for E3 versus E4 activity. CHIP has the remarkable ability to switch between two different functions in its role as a co-chaperone of Hsp70, serving as either an E3 ligase or promoting protein refolding. Thus, CHIP appears to serve as a key factor enabling switching between refolding and degratory regimes in response to cellular stress.
Aim 3 is directed towards developing an understanding of this switching mechanism. We propose biochemical and structural characterization of the interactions of CHIP with: (i) Ubc5, its complementary E2 ligase, (ii) Bag-1, the co-chaperone that stimulates CHIP'S activity as a ubiquitin ligase, and (iii) HspBPI, the competing co-chaperone that switches CHIP to its protein refolding regime.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Special Emphasis Panel (ZRG1-BCMB-B (02))
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Smith, Ward
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Vanderbilt University Medical Center
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Bhuripanyo, Karan; Wang, Yiyang; Liu, Xianpeng et al. (2018) Identifying the substrate proteins of U-box E3s E4B and CHIP by orthogonal ubiquitin transfer. Sci Adv 4:e1701393
Topolska-Wo?, Agnieszka M; Shell, Steven M; Kila?czyk, Ewa et al. (2015) Dimerization and phosphatase activity of calcyclin-binding protein/Siah-1 interacting protein: the influence of oxidative stress. FASEB J 29:1711-24
Soss, Sarah E; Rose, Kristie L; Hill, Salisha et al. (2015) Biochemical and Proteomic Analysis of Ubiquitination of Hsc70 and Hsp70 by the E3 Ligase CHIP. PLoS One 10:e0128240
Soss, Sarah E; Klevit, Rachel E; Chazin, Walter J (2013) Activation of UbcH5c~Ub is the result of a shift in interdomain motions of the conjugate bound to U-box E3 ligase E4B. Biochemistry 52:2991-9
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Nordquist, Kyle A; Dimitrova, Yoana N; Brzovic, Peter S et al. (2010) Structural and functional characterization of the monomeric U-box domain from E4B. Biochemistry 49:347-55

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