Cathepsin L Sites Involved in Processing and Lysosomal Sorting

溶酶体蛋白水解酶在细胞蛋白质的周转和通过吞噬进入细胞的分子的降解中起作用。像分泌蛋白一样，它们是在膜结合的核糖体上合成的。它们随后从分泌蛋白中分选并运输到溶酶体有两条途径。在成纤维细胞中，磷酸转移酶将磷酸加到甘露糖残基上，形成甘露糖-6-磷酸识别标记，使溶酶体蛋白与特定的受体发生反应，后者将其携带到溶酶体。在肾脏和肝脏细胞中，存在另一种途径，但溶酶体酶及其与之反应的细胞受体的分类序列尚未确定。因此，运输到溶酶体并不是一个默认的途径，而是需要特定的细胞酶以复杂的序列识别生物合成形式的溶酶体酶。虽然溶酶体半胱氨酸蛋白酶的活性部位已经被仔细研究，但几乎对构成加工酶的结构基序或溶酶体酶上的受体结合部位知之甚少。我们将通过蛋白质结构分析和分子生物学技术相结合的方法，为溶酶体半胱氨酸蛋白酶L确定这些表面序列。突变酶将通过定点和/或饱和突变技术构建，并通过电穿孔导入真核细胞。突变酶的适当细胞分离将通过免疫荧光、表达蛋白生物合成的脉冲追逐分析和细胞分级来检测。通过这种方法，我们希望通过处理负责前肽去除、不对称裂解成轻链和重链以及去除羧基末端氨基酸的酶来鉴定磷酸转移酶识别的特定氨基酸序列和识别组织蛋白酶L的关键表面序列。这项研究解决了现代细胞生物学中一个重要而及时的问题，即负责细胞或分泌蛋白质正确分选的机制是什么。这项工作的成功结果将增加我们对截面蛋白质-蛋白质相互作用的理解，并为识别特异性的基础。

Lysosomal proteases are active in cellular protein turnover and degradation of molecules which enter the cell by phagocytosis. Like secretory proteins, they are synthesized on membrane-bound ribosomes. Two pathways exist for their subsequent sorting from secretory proteins and transport to lysosomes. In fibroblasts, a phosphotransferase adds phosphate to mannose residues, forming the mannose-6-phosphate recognition marker which enables lysosomal proteins to react with specific receptors which carry them to lysosomes. In kidney and liver cells, an alternative pathway exists but the sorting sequences on the lysosomal enzymes and the cellular receptor it reacts with have not been characterized. Thus transport to the lysosome is not a default pathway but rather requires that biosynthetic forms of the lysosomal enzymes be recognized by specific cellular enzymes in a complex sequence. While the active sites of lysosomal cysteine proteases have been carefully studied, virtually nothing is known about the structural motifs which constitute processing enzyme or receptor binding sites on lysosomal enzymes. We will define these surface sequences for the lysosomal cysteine protease cathepsin L by coupling analysis of protein structure with molecular biology technology. Mutant enzymes will be constructed by site-specific and/or saturation mutagenesis techniques and introduced into eukaryotic cells by electroporation. Proper cellular segregation of the mutant enzymes will be assayed by immunofluorescence, pulse-chase analysis of the biosynthesis of the expressed protein, and cell fractionation. By this method we hope to identify the specific amino acid sequences recognized by the phosphotransferase and to identify surface sequences critical to the recognition of cathepsin L by processing proteases responsible for propeptide removal, asymmetric cleavage into light and heavy chains, and removal of carboxyl terminal amino acids. This research addresses an important and timely problem in modern cell biology, namely, what are the mechanisms responsible for the correct sorting of cellular or secretory proteins. A successful outcome of this work will increase our understanding of section protein-protein interactions and the basis of recognition specificity.