Hizukuri Y, Akiyama K, Akiyama Y
Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan.
Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan.
Methods Enzymol. 2017;584:1-33. doi: 10.1016/bs.mie.2016.09.044. Epub 2016 Oct 31.
Intramembrane-cleaving proteases (I-CLiPs) are a group of membrane-associated proteases with a unique feature: they are believed to cleave their substrate within the hydrophobic lipid bilayer, even though peptide bond hydrolysis requires a water molecule. Escherichia coli RseP, which belongs to the S2P zinc metalloprotease family of I-CLiPs, plays an essential role in activation of a cell envelope stress response through cleavage of anti-σ protein RseA, a single-span transmembrane protein. A recent study showed that it also cleaves remnant signal peptides generated upon membrane translocation of secretory proteins. Here, we describe several methods for characterization of the proteolytic functions and structure of RseP mainly in vivo, including a proteolytic activity assay using model substrates, an in vitro analysis of cleavage of signal peptides in a detergent solution and in the membrane vesicles, structural analysis of membrane-embedded RseP based on the thiol modifiability of introduced cysteine residues, and the protein interaction analysis by in vivo cross-linking protocols.
膜内裂解蛋白酶（I-CLiPs）是一类与膜相关的蛋白酶，具有独特的特性：尽管肽键水解需要水分子，但它们被认为能在疏水脂质双层内切割其底物。大肠杆菌RseP属于I-CLiPs的S2P锌金属蛋白酶家族，通过切割单跨膜蛋白抗σ蛋白RseA，在细胞包膜应激反应的激活中起重要作用。最近的一项研究表明，它还能切割分泌蛋白跨膜转运时产生的残余信号肽。在这里，我们描述了几种主要在体内表征RseP蛋白水解功能和结构的方法，包括使用模型底物的蛋白水解活性测定、在去污剂溶液和膜囊泡中对信号肽切割的体外分析、基于引入的半胱氨酸残基的硫醇可修饰性对膜嵌入RseP的结构分析，以及通过体内交联方案进行的蛋白质相互作用分析。
