Cathepsin L Sites Involved in Processing and Lysosomal Sorting

溶酶体蛋白酶在细胞蛋白质周转中具有活性， 通过吞噬作用进入细胞的分子的降解。 像 分泌蛋白，它们在膜结合核糖体上合成。 存在两种途径用于它们随后从分泌蛋白中分选 并转运至溶酶体。 在成纤维细胞中，磷酸转移酶 磷酸转化为甘露糖残基，形成甘露糖-6-磷酸 识别标记，使溶酶体蛋白反应， 将它们携带到溶酶体的特异性受体。 肾脏和肝脏中 细胞，存在替代途径，但细胞上的分选序列 溶酶体酶和与之反应的细胞受体尚未被发现。 表征了 因此，转运到溶酶体不是默认途径 而是需要溶酶体酶的生物合成形式， 由特定的细胞酶以复杂的序列识别。 而 溶酶体半胱氨酸蛋白酶的活性位点已经被仔细地 研究，几乎没有什么是已知的结构图案， 在溶酶体上构成加工酶或受体结合位点 内切酶 我们将定义这些表面序列的溶酶体 半胱氨酸蛋白酶组织蛋白酶L的偶联分析 分子生物学技术。 将构建突变酶 通过位点特异性和/或饱和诱变技术， 通过电穿孔进入真核细胞。 适当的细胞分离 将通过免疫荧光、脉冲追踪 表达蛋白的生物合成分析，以及细胞 分馏 通过这种方法，我们希望确定特定的氨基酸 由磷酸转移酶识别的酸序列，并鉴定 对组织蛋白酶L的识别至关重要的表面序列 负责前肽去除的加工蛋白酶，不对称 裂解成轻链和重链，并除去羧基末端 个氨基酸 这项研究解决了一个重要的和及时的问题，在现代 细胞生物学，也就是说， 细胞或分泌蛋白的正确分选。 取得成功结果 这项工作的完成将增加我们对蛋白质-蛋白质部分的了解 相互作用和识别特异性的基础。

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.