Preparation of SUMO proteases and kinetic analysis using endogenous substrates.
Preparation of SUMO proteases and kinetic analysis using endogenous substrates.
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DOI：
10.1007/978-1-59745-566-4_15
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Lima, Christopher D
中科院分区：
文献类型：
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作者：
Reverter, David;Lima, Christopher D
SUMO蛋白酶催化两个反应，SUMO从底物中解缀合和前体SUMO同种型的加工以制备用于缀合的SUMO。SUMO蛋白酶家族包括酵母中的两个成员（Ulp 1和Ulp 2）和人类中的多达六个成员（SENP 1 -3，SENP 5 -7）。SENP/Ulp蛋白酶各自含有催化蛋白酶活性的保守C-末端结构域。C-末端蛋白酶结构域在体外SUMO加工和解缀合过程中表现出独特的特异性。虽然有许多可用的试剂来评估这些活动，包括融合蛋白和化学修饰的SUMO亚型，我们的研究表明，底物的C-末端的易裂键的组成可以实质性地影响蛋白酶的活性。因此，我们广泛依赖于利用内源性底物（即野生型SUMO前体和SUMO缀合底物）的测定。在本章中，我们将描述纯化和表征的SUMO前体，SUMO共轭底物，和SUMO蛋白酶的方法细节。我们还将描述使用内源性底物的SUMO去缀合和成熟的动力学分析方法。
SUMO proteases catalyze two reactions, deconjugation of SUMO from substrates and processing of precursor SUMO isoforms to prepare SUMO for conjugation. The SUMO protease family includes two members in yeast (Ulp1 and Ulp2) and as many as six members in human (SENP1–3, SENP5–7). SENP/Ulp proteases each contain conserved C-terminal domains that catalyze protease activity. The C-terminal protease domains exhibit unique specificities during SUMO processing and deconjugation in vitro. While there are many available reagents to assess these activities, including fusion proteins and chemically modified SUMO isoforms, our studies have indicated that the composition of substrates C-terminal to the scissile bond can substantively influence the activity of the protease. As such, we have relied extensively on assays that utilize endogenous substrates, namely wild-type SUMO precursors and SUMO conjugated substrates. In this chapter, we will describe methodological details for purification and characterization of SUMO precursors, SUMO conjugated substrates, and SUMO proteases. We will also describe methods for kinetic analysis of SUMO deconjugation and maturation using endogenous substrates.