The covalent attachment of SUMO (small ubiquitin-like protein modifier) to target proteins results in modifications in their activity, binding interactions, localization or half-life. The reversal of this modification is catalysed by SENPs (SUMO-specific processing proteases). Mammals contain four SUMO paralogues and six SENP enzymes. In the present paper, we describe a systematic analysis of human SENPs, integrating estimates of relative selectivity for SUMO1 and SUMO2, and kinetic measurements of recombinant C-terminal cSENPs (SENP catalytic domains). We first characterized the reaction of each endogenous SENP and cSENPs with HA–SUMO-VS [HA (haemagglutinin)-tagged SUMO-vinyl sulfones], active-site-directed irreversible inhibitors of SENPs. We found that all cSENPs and endogenous SENP1 react with both SUMO paralogues, whereas all other endogeneous SENPs in mammalian cells and tissues display high selectivity for SUMO2-VS. To obtain more quantitative data, the kinetic properties of purified cSENPs were determined using SUMO1- or SUMO2-AMC (7-amino-4-methylcoumarin) as substrate. All enzymes bind their respective substrates with high affinity. cSENP1 and cSENP2 process either SUMO substrate with similar affinity and catalytic efficiency; cSENP5 and cSENP6 show marked catalytic specificity for SUMO2 as measured by Km and kcat, whereas cSENP7 works only on SUMO2. Compared with cSENPs, recombinant full-length SENP1 and SENP2 show differences in SUMO selectivity, indicating that paralogue specificity is influenced by the presence of the variable N-terminal domain of each SENP. Our data suggest that SUMO2 metabolism is more dynamic than that of SUMO1 since most SENPs display a marked preference for SUMO2.
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Research Article|
August 13 2010
Distribution and paralogue specificity of mammalian deSUMOylating enzymes
Nagamalleswari Kolli;
Nagamalleswari Kolli
*Department of Biochemistry, Emory University, Atlanta, GA 30322, U.S.A.
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Jowita Mikolajczyk;
Jowita Mikolajczyk
†Burnham Institute for Medical Research, La Jolla, CA 92037, U.S.A.
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Marcin Drag;
Marcin Drag
†Burnham Institute for Medical Research, La Jolla, CA 92037, U.S.A.
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Debaditya Mukhopadhyay;
Debaditya Mukhopadhyay
‡Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, U.S.A.
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Nela Moffatt;
Nela Moffatt
*Department of Biochemistry, Emory University, Atlanta, GA 30322, U.S.A.
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Mary Dasso;
Mary Dasso
‡Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, U.S.A.
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Guy Salvesen;
Guy Salvesen
†Burnham Institute for Medical Research, La Jolla, CA 92037, U.S.A.
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Keith D. Wilkinson
Keith D. Wilkinson
1
*Department of Biochemistry, Emory University, Atlanta, GA 30322, U.S.A.
1To whom correspondence should be addressed (email keith.wilkinson@emory.edu).
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Publisher: Portland Press Ltd
Received:
April 01 2010
Revision Received:
June 09 2010
Accepted:
June 30 2010
Accepted Manuscript online:
June 30 2010
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2010 Biochemical Society
2010
Biochem J (2010) 430 (2): 335–344.
Article history
Received:
April 01 2010
Revision Received:
June 09 2010
Accepted:
June 30 2010
Accepted Manuscript online:
June 30 2010
Citation
Nagamalleswari Kolli, Jowita Mikolajczyk, Marcin Drag, Debaditya Mukhopadhyay, Nela Moffatt, Mary Dasso, Guy Salvesen, Keith D. Wilkinson; Distribution and paralogue specificity of mammalian deSUMOylating enzymes. Biochem J 1 September 2010; 430 (2): 335–344. doi: https://doi.org/10.1042/BJ20100504
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