Intracellular Processing, Activation, and Sorting of Lysosomal Cathepsins

溶酶体组织蛋白酶被认为在细胞内蛋白降解中起重要作用。与大多数溶酶体酸水解酶一样，组织蛋白酶在内质网的膜结合核糖体上作为糖基化的前酶合成，高分子量的前体随后通过有限的蛋白质水解转化为翻译后的成熟酶。在我的研究中，我发现了以下证据：(1)通过免疫印迹分析，在大鼠肝微粒体管腔中鉴定了组织蛋白酶B、H、L和D的潜伏形式，并对肝微粒体内容物进行体外活化实验，发现潜伏的原组织蛋白酶在酸性条件下转化为成熟酶的活性形式。(2)蛋白酶抑制蛋白的水解过程和酶的激活，表明溶酶体中主要的天冬氨酸蛋白酶组织蛋白酶D参与了原蛋白酶的前肽加工过程。(3)为了进一步表征溶酶体组织蛋白酶的细胞内加工和激活机制，用Con A-Sepharose、Sepharose- gly - ph - glysc和抗组织蛋白酶B IgG Sepharose高度纯化微粒体原组织蛋白酶B，然后在几种蛋白酶抑制剂的存在下，将该前酶与大鼠肝脏三体内容物孵育。结果证实溶酶体cathepsin D确实是原athepsin b的加工蛋白酶，并分离出用cathepsin D消化后的原athepsin加工形式，测定其分子nh_2末端序列。测定的原athepsin B的nh_2末端序列为Pro-66，位于成熟组织蛋白酶B的nh_2末端上游14个氨基酸，表明原athepsin B在细胞内转运过程中经历了多个加工步骤。(4)将含有组织蛋白酶L cDNA的多拷贝质粒引入酵母中，以确定溶酶体组织蛋白酶L的细胞内分选机制。这些体内和体外研究将帮助我阐明溶酶体组织蛋白酶的细胞外加工和激活机制。少

Lysosomal cathepsins are considered to play nd important role in intracellular protein degradation. Like most lysosomal acid hydrolases, cathepsins are synthesized as the glycosylated preproenzymens on membrane-bound ribosomes of endoplasmic reticulum and the higher-molecular-weight precursors are subsequently converted into the mature enzymes posttranslationally by limited proteolysis.In my studies, I have found the following evidence : (1) The latent proforms of cathepsins B, H, L, and D were identified by immunoblot analysis in the rat liver microsomal lumen, and the in vitro activation experiments with liver microsomal contents revealed that latent procathepsins are converted to the active forms of mature enzymes under acidic conditions. (2) The proteolytic processing events, and the activation of the enzymes were strongly inhibited by pepstatin, therefore, suggesting that cathepsin D, a major lysosomal aspartic proteinase is involved in the propeptide-processing of procathepsins. … More (3) To further characterize these intracellular processing and activation mechanisms for the lysosomal cathepsins, microsomal procathepsin B was highly purified by the Con A-Sepharose, Sepharose-Gly-Phe-GlySc, and the anti-cathepsin B IgG Sepharose and then this proenzyme was incubated with rat liver tritosomal contents in the presence of several proteinase inhibitors. The result confirmed that the lysosomal cathepsin D is indeed a processing proteinase of procathepsin B. Furthermore, the processed form of procathepsin after the digestion with cathepsin D was isolated and subjected to the determination of the NH_2-terminal sequence of the molecule. The determined NH_2-terminal sequence of procathepsin B revealed to be Pro-66 which is 14 amino acids upstream of the NH_2-terminus of the mature cathepsin B, suggesting that the procathepsin B would undergo multiple processing step during intracellular transport. (4) The multicopy plasmid containing the cathepsin L cDNA has been introduced into the yeast to determine the intracellular sorting machinery of lysosomal cathepsin L. These in vivo and in vitro studies will help me to clarify the extracellular processing and activation mechanisms lysosomal cathepsins. Less