Substrate Profiling of Cysteine Proteases in Physiology and Pathology

蛋白酶通过有限的、位点特异性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列表形成对比，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。沿着活性位点特异性的测定，我们通过液相色谱/串联质谱法确定了复杂蛋白质组中的底物和切割位点。组织蛋白酶依赖性蛋白质降解的时间分辨脉冲追踪策略检查。用于底物分析的实验系统包括皮肤、肝脏和肿瘤环境中组织蛋白酶生物学的鼠模型，以及体外、细胞环境和体内测定。底物候选物经过多阶段验证，包括裂解动力学、蛋白酶和底物的原位共定位以及细胞环境中加工底物的功能表征。我们重建蛋白水解途径在体内的蛋白酶和底物缺陷细胞的转基因表达的改变或加工基板。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.