Eckhoff Julia, Dohmen R Jürgen
Institute for Genetics, Biocenter, University of Cologne, 50674, Cologne, Germany.
Methods Mol Biol. 2016;1475:123-35. doi: 10.1007/978-1-4939-6358-4_9.
SUMO-specific proteases, known as Ulps in baker's yeast and SENPs in humans, have important roles in controlling the dynamics of SUMO-modified proteins. They display distinct modes of action and specificity, in that they may act on the SUMO precursor, mono-sumoylated, and/or polysumoylated proteins, and they might be specific for substrates with certain SUMO paralogs. SUMO chains may be dismantled either by endo or exo mechanisms. Biochemical characterization of a protease usually requires purification of the protein of interest. Developing a purification protocol, however, can be very difficult, and in some cases, isolation of a protease in its pure form may go along with a substantial loss of activity. To characterize the reaction mechanism of Ulps, we have developed an in vitro assay, which makes use of substrates endowed with artificial poly-SUMO chains of defined lengths, and S. cerevisiae Ulp enzymes in crude extract from E. coli. This fast and economic approach should be applicable to SUMO-specific proteases from other species as well.
SUMO特异性蛋白酶，在面包酵母中称为Ulp，在人类中称为SENP，在控制SUMO修饰蛋白的动态过程中发挥着重要作用。它们表现出不同的作用方式和特异性，因为它们可能作用于SUMO前体、单SUMO化和/或多SUMO化蛋白，并且它们可能对具有某些SUMO旁系同源物的底物具有特异性。SUMO链可以通过内切或外切机制拆解。蛋白酶的生化特性通常需要纯化目标蛋白。然而，开发纯化方案可能非常困难，在某些情况下，以纯形式分离蛋白酶可能会伴随着活性的大量损失。为了表征Ulp的反应机制，我们开发了一种体外测定法，该方法利用具有确定长度的人工多SUMO链的底物以及来自大肠杆菌粗提物中的酿酒酵母Ulp酶。这种快速且经济的方法也应该适用于其他物种的SUMO特异性蛋白酶。
