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质降解中起重要作用。与大多数溶酶体酸性水解酶一样，组织蛋白酶是在内质网膜结合核糖体上合成的糖基化前体，高分子量的前体随后通过有限的蛋白水解后转化为成熟的酶。（1）通过免疫印迹分析在大鼠肝微粒体管腔中鉴定组织蛋白酶B、H、L和D的潜伏前体，并且用肝微粒体内容物进行的体外活化实验揭示，在酸性条件下，潜伏的组织蛋白酶原被转化为成熟酶的活性形式。(2)胃蛋白酶抑制剂强烈抑制蛋白水解加工事件和酶的活化，因此，表明组织蛋白酶D，一种主要的溶酶体天冬氨酸蛋白酶参与前肽加工的前体蛋白酶。 ...更多信息 (3)为了进一步表征溶酶体组织蛋白酶的这些细胞内加工和活化机制，通过Con A-Sepharose、Sepharose-Gly-Phe-GlySc和抗组织蛋白酶B IgG Sepharose高度纯化微粒体组织蛋白酶原B，然后在存在几种蛋白酶抑制剂的情况下将该酶原与大鼠肝tritosomal内容物孵育。结果证实溶酶体组织蛋白酶D确实是组织蛋白酶原B的加工蛋白酶。此外，还分离了经组织蛋白酶D消化后的蛋白酶原，并测定了其氨基端序列。组织蛋白酶原B NH_2-端序列测定结果表明，组织蛋白酶原B NH_2-端上游14个氨基酸为Pro-66，提示组织蛋白酶原B在胞内转运过程中可能经历多个加工步骤。(4)将含有组织蛋白酶L cDNA的多拷贝质粒导入酵母，以确定溶酶体组织蛋白酶L的细胞内分选机制。这些在体内和体外的研究将帮助我阐明细胞外加工和激活机制溶酶体组织蛋白酶。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