Distribution and paralogue specificity of mammalian deSUMOylating enzymes.
Distribution and paralogue specificity of mammalian deSUMOylating enzymes.
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DOI：
10.1042/bj20100504
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发表时间：
2010-09-01
期刊：
影响因子：
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通讯作者：
Wilkinson KD
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作者：
Kolli N;Mikolajczyk J;Drag M;Mukhopadhyay D;Moffatt N;Dasso M;Salvesen G;Wilkinson KD
小泛素样蛋白修饰物(SUMO)与靶蛋白的共价结合导致其活性、结合作用、定位或半衰期的改变。这种修饰的逆转是由相扑特异性加工酶(SENPs)催化的。哺乳动物含有4个相扑对虾和6个SENP酶。我们的研究描述了对人类SENP的系统分析，整合了对SUMO1和SUMO2相对选择性的估计，以及重组C末端SENP催化结构域(CSENPs)的动力学测量。我们首次表征了每个内源性SENP及其催化结构域(CSENP)与HA标记的SUMO1和SUMO2乙烯基砜(HA-SUMO-VS)的反应，这是SENP的活性位点定向不可逆抑制剂。我们发现，所有cSENPs和内源性SENP1都与两个相扑类似物反应，而哺乳动物细胞和组织中的所有其他内源性SENPs对SUMO2-VS都表现出高选择性。为了获得更多的定量数据，以SUMO1或SUMO2-氨甲基香豆素(SUMO-AMC)为底物，测定了纯化的cSENPs的动力学性质。所有的酶都以高亲和力结合各自的底物。CSENP1和cSENP2处理SUMO底物具有相似的亲和力和催化效率；cSENP5和cSENP6对SUMO2显示出显著的催化专一性，Km和kcat测量，而cSENP7仅对SUMO2起作用。与cSENPs相比，重组全长SENP1和SENP2在相扑选择性方面存在差异，表明副对数特异性受到每个SENP可变N-末端结构域的存在的影响。我们的数据表明，SUMO2的代谢比SUMO1更动态，因为大多数SENP都表现出对SUMO2的明显偏好。
The covalent attachment of the small ubiquitin-like protein modifier (SUMO) to target proteins results in modifications in their activity, binding interactions, localization or half-life. The reversal of this modification is catalyzed by SUMO-specific processing proteases (SENPs). Mammals contain four SUMO paralogs and six SENP enzymes. Our studies describe a systematic analysis of human SENPs, integrating estimates of relative selectivity for SUMO1 and SUMO2, and kinetic measurements of recombinant C-terminal SENP catalytic domains (cSENPs). We first characterized the reaction of each endogenous SENP and their catalytic domains (cSENP) with HA-tagged SUMO1 and SUMO2 vinyl sulfones (HA-SUMO-VS), active site-directed irreversible inhibitors of SENPs. We found that all cSENPs and endogenous SENP1 react with both SUMO paralogs, while 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-amidomethyl coumarin (SUMO-AMC) 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 while cSENP7 works only on SUMO2. Compared to cSENPs, recombinant full-length SENP1 and SENP2 show differences in SUMO selectivity indicating that paralog specificity is influenced by the presence of the variable N-terminal domain of each SENP. Our data suggests that SUMO2 metabolism is more dynamic than that of SUMO1 since most SENPs display a marked preference for SUMO2.