Cathepsin L Sites Involved in Processing and Lysosomal Sorting

组织蛋白酶 L 参与加工和溶酶体分选的位点

• 批准号: 8908842
• 负责人: Ann Erickson
• 金额: $ 14万
• 依托单位: University of North Carolina at Chapel Hill
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-09-01 至 1992-02-29
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=8908842&HistoricalAwards=false
• 关键词: Cathepsin; Sites; Involved; Processing; Lysosomal

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.

溶酶体蛋白酶在细胞蛋白周转和通过吞噬作用进入细胞的分子的降解中起作用。像分泌蛋白一样，它们在膜结合核糖体上合成。它们随后从分泌蛋白中分选并转运到溶酶体存在两种途径。在成纤维细胞中，磷酸转移酶将磷酸盐添加到甘露糖残基上，形成甘露糖-6-磷酸识别标记物，使溶酶体蛋白与携带它们到溶酶体的特定受体发生反应。在肾和肝细胞中，存在另一种途径，但溶酶体酶的分选序列及其与之反应的细胞受体尚未被表征。因此，转运到溶酶体并不是一个默认的途径，而是需要溶酶体酶的生物合成形式被特定的细胞酶以复杂的序列识别。虽然对溶酶体半胱氨酸蛋白酶的活性位点已经进行了仔细的研究，但实际上对构成溶酶体酶加工酶或受体结合位点的结构基序一无所知。我们将通过蛋白质结构与分子生物学技术的耦合分析来确定溶酶体半胱氨酸蛋白酶组织蛋白酶L的表面序列。突变酶将通过位点特异性和/或饱和诱变技术构建，并通过电穿孔引入真核细胞。将通过免疫荧光、脉冲追踪分析表达蛋白的生物合成和细胞分离来检测突变酶的适当细胞分离。通过这种方法，我们希望通过处理蛋白酶来识别磷酸转移酶识别的特定氨基酸序列，并识别对组织蛋白酶L识别至关重要的表面序列，这些蛋白酶负责前肽的去除，不对称切割成轻链和重链，以及羧基末端氨基酸的去除。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，即什么是负责细胞或分泌蛋白正确分选的机制。这项工作的成功结果将增加我们对片段蛋白-蛋白相互作用和识别特异性基础的理解。