Substrate Profiling of Cysteine Proteases in Physiology and Pathology

蛋白酶通过有限的、特定位点的蛋白质加工和蛋白质降解来塑造蛋白质组。对于大多数蛋白酶，几乎没有已知的体内底物。对于半胱氨酸组织蛋白酶，这与不断扩大的生理和病理功能形成对比，包括表皮稳态（组织蛋白酶L）和肿瘤发生（组织蛋白酶B）。通过系统范围的底物分析，我们提供了组织蛋白酶功能的分子基础。除了活性位点特异性测定外，我们还利用液相色谱/串联质谱技术鉴定了复杂蛋白质组中的底物和裂解位点。组织蛋白酶依赖的蛋白质降解是通过时间分辨脉冲追逐策略检查。底物分析的实验系统包括皮肤、肝脏和肿瘤背景下组织蛋白酶的小鼠模型，以及体外、细胞背景和体内分析。候选底物需要经过多个阶段的验证，包括裂解动力学、蛋白酶和底物的原位共定位以及加工底物在细胞环境中的功能表征。我们通过在蛋白酶和底物缺乏的细胞中转基因表达改变或加工的底物来重建体内的蛋白水解途径。总之，我们建立了一个蛋白酶底物分析平台，旨在探索蛋白酶生物学的新视角。

Proteases mold proteomes through limited, site-specific protein processing and protein degradation. For most proteases almost no in vivo substrates are known. For cysteine cathepsins, this contrasts an expanding list of physiological and pathological functions, including epidermal homeostasis (cathepsin L) and tumorigenesis (cathepsin B). Through system-wide substrate profiling, we provide the molecular basis for cathepsin function. Along with active site specificity determination, we identify substrates and cleavage sites in complex proteomes by liquid chromatography/tandem mass spectrometry based strategies. Cathepsin-dependent protein degradation is examined by time-resolved pulse-chase strategies. Experimental systems for substrate profiling include murine models of cathepsin biology in skin, liver, and tumor context with in vitro, cell contextual, and in vivo assays. Substrate candidates undergo multi-stage validation including cleavage kinetics, in situ co-localization of protease and substrates, and functional characterization of processed substrates in the cellular context. We reconstruct proteolytic pathways in vivo by transgenic expression of altered or processed substrates in protease- and substrate-deficient cells. In summary, we establish a platform for protease substrate profiling with the aim to explore new perspectives in protease biology.