We have developed specific inhibitors of, and substrates for, deubiquitinating enzymes (DUBs) acting upon ubiquitin, NeddS, SUMO-1, SUMO-2/3, and ISG15 (collectively referred to as ubiquitin-like or UBL proteins). We refer to these as the UBL-X panel of reagents. The specificity of these reagents is outstanding and methods to assess cross reactivity between DUB homologs and UBL paralogs have been developed. These reagents have allowed us to define the likely specificity of a number of individual DUBs, led to some surprising conclusions regarding the spectrum of activities exhibited by different classes of DUBs, and suggested new opportunities for developing and characterizing inhibitors that specifically interfere with selected pathways of UBL protein metabolism. In the coming period: We will use the UBL-X panel of reagents in quantitative labeling and kinetic approaches to determine the amounts and paralog specificity of active desumoylating enzymes in lysates from control and stressed cells. The specificity will be compared to that of the purified catalytic domains. We will test the hypothesis that changes in cellular levels of sumoylated proteins in response to cellular stress are controlled by levels of active desumoylating enzymes. We will determine the enzymatic specificity of the DUB activity exhibited by the SARS virus papain-like protease using the panel of UBL-X reagents. This information will suggest possible cellular substrates and be used to mount a high throughput drug screen to identify antiviral agents that may be useful in limiting the spread of coronavirus infections. We will use the panel of UBL-X reagents to evaluate the specificity of inhibitors that have been reported to inhibit deubiquitinating enzymes. This will provide a rapid and selective method to evaluate the global effects of """"""""specific"""""""" inhibitors on the spectrum of deubiquitinating enzymes. In the broadest sense, these studies will contribute to our understanding of UBL protein metabolism and guide development of inhibitors and drugs for the treatment of cancers and microbial infections.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM066355-08
Application #
7667456
Study Section
Special Emphasis Panel (ZRG1-CB-B (02))
Program Officer
Jones, Warren
Project Start
2002-08-01
Project End
2010-07-31
Budget Start
2009-08-01
Budget End
2010-07-31
Support Year
8
Fiscal Year
2009
Total Cost
$315,697
Indirect Cost
Name
Emory University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
066469933
City
Atlanta
State
GA
Country
United States
Zip Code
30322
Eletr, Ziad M; Wilkinson, Keith D (2014) Regulation of proteolysis by human deubiquitinating enzymes. Biochim Biophys Acta 1843:114-28
Chernova, Tatiana A; Romanyuk, Andrey V; Karpova, Tatiana S et al. (2011) Prion induction by the short-lived, stress-induced protein Lsb2 is regulated by ubiquitination and association with the actin cytoskeleton. Mol Cell 43:242-52
Kolli, Nagamalleswari; Mikolajczyk, Jowita; Drag, Marcin et al. (2010) Distribution and paralogue specificity of mammalian deSUMOylating enzymes. Biochem J 430:335-44
Wang, Tao; Yin, Luming; Cooper, Eric M et al. (2009) Evidence for bidentate substrate binding as the basis for the K48 linkage specificity of otubain 1. J Mol Biol 386:1011-23
Shanks, John; Burtnick, Mary N; Brett, Paul J et al. (2009) Burkholderia mallei tssM encodes a putative deubiquitinase that is secreted and expressed inside infected RAW 264.7 murine macrophages. Infect Immun 77:1636-48
Wang, Yonggang; Mukhopadhyay, Debaditya; Mathew, Smita et al. (2009) Identification and developmental expression of Xenopus laevis SUMO proteases. PLoS One 4:e8462
Griffiths, Lyra M; Swartzlander, Dan; Meadows, Kellen L et al. (2009) Dynamic compartmentalization of base excision repair proteins in response to nuclear and mitochondrial oxidative stress. Mol Cell Biol 29:794-807
Wilkinson, Keith D (2009) DUBs at a glance. J Cell Sci 122:2325-9
Drag, Marcin; Mikolajczyk, Jowita; Bekes, Miklos et al. (2008) Positional-scanning fluorigenic substrate libraries reveal unexpected specificity determinants of DUBs (deubiquitinating enzymes). Biochem J 415:367-75
Yun, Chawon; Wang, Yonggang; Mukhopadhyay, Debaditya et al. (2008) Nucleolar protein B23/nucleophosmin regulates the vertebrate SUMO pathway through SENP3 and SENP5 proteases. J Cell Biol 183:589-95

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