Regulated intramembrane proteolysis (RIP) describes the protease-dependent cleavage of transmembrane proteins within the hydrophobic core of cellular membranes. Intramembrane-cleaving proteases (I-CliPs) that catalyze these reactions are found in all kingdoms of life and are involved in a wide range of cellular processes, including signaling and protein homeostasis. I-CLiPs are multispanning membrane proteins and represent challenging targets in structural and enzyme biology. Here we introduce iCLiPSpy, a simple assay to study I-CLiPs in vivo. To allow easy detection of enzyme activity, we developed a heme-binding reporter based on TNFα that changes color after I-CLiP-mediated proteolysis. Co-expression of the protease and reporter in Escherichia coli (E. coli) results in white or green colonies, depending on the activity of the protease. As a proof of concept, we use this assay to study the bacterial intramembrane-cleaving zinc metalloprotease RseP in vivo. iCLiPSpy expands the methodological repertoire for identifying residues important for substrate binding or activity of I-CLiPs and can in principle be adapted to a screening assay for the identification of inhibitors or activators of I-CLiPs, which is of great interest for proteases being explored as biomedical targets.

受调节的膜内蛋白水解 (RIP) 描述了细胞膜疏水核心内跨膜蛋白的蛋白酶依赖性裂解。催化这些反应的膜内切割蛋白酶 (I-Clip) 存在于所有生命王国中，并参与广泛的细胞过程，包括信号传导和蛋白质稳态。I-CLiPs 是多跨膜蛋白，代表了结构和酶生物学中具有挑战性的目标。在这里，我们介绍 iCLiPSpy，这是一种在体内研究 I-CLiP 的简单测定法。为了便于检测酶活性，我们开发了一种基于 TNFα 的血红素结合报告基因，它在 I-CLiP 介导的蛋白水解后会改变颜色。蛋白酶和报告基因在大肠杆菌中的共表达( E. coli) 产生白色或绿色菌落，具体取决于蛋白酶的活性。作为概念证明，我们使用该测定法在体内研究细菌膜内裂解锌金属蛋白酶 RseP。iCLiPSpy 扩展了用于鉴定对底物结合或 I-CLiP 活性重要的残基的方法学库，并且原则上可以适用于鉴定 I-CLiP 抑制剂或激活剂的筛选试验，这对于正在探索的蛋白酶非常有意义生物医学目标。