PDF(Pubmed)
Abstract：
Proteins delivered by endocytosis or autophagy to lysosomes are degraded by exo- and endoproteases. In humans 15 lysosomal cathepsins (CTS) act as important physiological regulators. The cysteine proteases CTSB and CTSL and the aspartic protease CTSD are the most abundant and functional important lysosomal proteinases. Whereas their general functions in proteolysis in the lysosome, their individual substrate, cleavage specificity, and their possible sequential action on substrate proteins have been previously studied, their functional redundancy is still poorly understood. To address a possible common role of highly expressed and functional important CTS proteases, we generated CTSB-, CTSD-, CTSL-, and CTSBDL-triple deficient (KO) human neuroblastoma-derived SH-SY5Y cells and CTSB-, CTSD-, CTSL-, CTSZ and CTSBDLZ-quadruple deficient (KO) HeLa cells. These cells with a combined cathepsin deficiency exhibited enlarged lysosomes and accumulated lipofuscin-like storage material. The lack of the three (SH-SY5Y) or four (HeLa) major CTSs caused an impaired autophagic flux and reduced degradation of endocytosed albumin. Proteome analyses of parental and CTS-depleted cells revealed an enrichment of cleaved peptides, lysosome/autophagy-associated proteins, and potentially endocytosed membrane proteins like the amyloid precursor protein (APP), which can be subject to endocytic degradation. Amino- and carboxyterminal APP fragments accumulated in the multiple CTS-deficient cells, suggesting that multiple CTS-mediated cleavage events regularly process APP. In summary, our analyses support the idea that different lysosomal cathepsins act in concert, have at least partially and functionally redundant substrates, regulate protein degradation in autophagy, and control cellular proteostasis, as exemplified by their involvement in the degradation of APP fragments.
摘要：
通过胞吞或自噬递送至溶酶体的蛋白质被外切和内切蛋白酶降解。在人类中,15个溶酶体组织蛋白酶(CTS)充当重要的生理调节物。半胱氨酸蛋白酶CTSB和CTSL以及天冬氨酸蛋白酶CTSD是最丰富和功能最重要的溶酶体蛋白酶。而它们在溶酶体中的蛋白水解中的一般功能，他们的个体底物，裂解特异性，它们对底物蛋白可能的顺序作用已经被研究过，它们的功能冗余仍然知之甚少。为了解决高表达和功能重要的CTS蛋白酶可能的共同作用，我们产生了CTSB-，CTSD-,CTSL-,和CTSBDL-三重缺陷（KO）人类神经母细胞瘤来源的SH-SY5Y细胞和CTSB-，CTSD-,CTSL-,CTSZ和CTSBDLZ-四重缺陷(KO)HeLa细胞。这些合并有组织蛋白酶缺陷的细胞表现出扩大的溶酶体和积累的脂褐素样储存材料。缺乏三种（SH-SY5Y）或四种（HeLa）主要CTSs会导致自噬通量受损，并减少内吞白蛋白的降解。亲本和CTS耗尽细胞的蛋白质组分析揭示了裂解肽的富集，溶酶体/自噬相关蛋白，和潜在的内吞膜蛋白，如淀粉样蛋白前体蛋白（APP），可能会被内吞降解。在多个CTS缺陷细胞中积累的氨基和羧基末端APP片段，提示多个CTS介导的裂解事件有规律地处理APP。总之,我们的分析支持不同溶酶体组织蛋白酶协同作用的观点，具有至少部分和功能冗余的基板，调节自噬中的蛋白质降解，并控制细胞的蛋白质停滞，例如它们参与APP片段的降解。
