PROTEIN TRANSLOCATION AND PROCESSING IN OVIDUCTS

该项目将侧重于信号的结构和酶学 肽酶，一种膜结合蛋白水解酶， 来自新生分泌蛋白的氨基末端信号肽， 被输送到内质网的内腔。 的 这种蛋白酶的作用机制尚未确定， 尽管这种酶在分泌的 proteins. 信号肽酶的催化机制可能定义了一种新的 蛋白水解酶类。 信号肽序列突变 直接影响信号肽酶已经被发现并显示， 导致人类疾病。 组成这两种蛋白质的性质 将检查母鸡输卵管微粒体信号肽酶， 功能定义。 酶促反应所需的最低蛋白质 活动将被确定。 合成肽类似物， 共价连接的可光活化的交联位点将用于 确定蛋白质亚基在催化作用中的作用， 信号肽酶 基因组中自然发生的突变 两种人类蛋白质，凝血因子X和 血清白蛋白，将进行检查，以提供详细的描述， 这些基因突变对这些生物合成的影响 proteins. 一种研究基因突变效应的一般方法 将开发信号肽对信号肽酶作用。 的cDNA克隆 编码19 kDa蛋白的鸡输卵管信号肽酶将被 克隆并鉴定。 克隆的一个重要目标是 kDa亚基是为了确定是否也有两个不同的基因 编码这种大小的相关母鸡输卵管信号肽酶蛋白，如 已经在犬信号肽酶中得到证实。cDNA克隆 编码信号肽酶蛋白亚基的蛋白质将以 异源细胞系统以产生酶活性蛋白。 这种方法的成功发展将导致未来的研究 使用定点诱变来探测这种酶的机制， 蛋白酶。 完成这些研究后， 了解微粒体信号的结构和功能 在生物合成早期起关键作用的肽酶 真核生物分泌蛋白质。

The project will focus on the structure and enzymology of signal peptidase, a membrane-bound proteolytic enzyme that removes amino-terminal signal peptides from nascent secretory proteins as they are transported into the lumen of the endoplasmic reticulum. The mechanism of action of this proteinase has not yet been determined in spite of the central role of this enzyme in the biosynthesis of secreted proteins. The catalytic mechanism of signal peptidase may define a new class of proteolytic enzymes. Mutations in signal peptide sequences that directly affect signal peptidase have been discovered and shown to result in human disease. The nature of the two proteins that constitute hen oviduct microsomal signal peptidase will be examined and their functions defined. The protein(s) minimally required for enzymatic activity will be determined. Synthetic peptide analogs bearing covalently attached photoactivatible cross linking sites will be used to identify the roles of the protein subunits in the catalytic action of signal peptidase. The effect of naturally occurring mutations in the signal peptide regions of two human proteins, coagulation factor X and serum albumin, will be examined to provide a detailed description of the effects of these genetic mutations on the biosynthesis of these proteins. A general approach for studying the effect of mutations of signal peptides on signal peptidase will be developed. A cDNA clone encoding the 19 kDa protein of hen oviduct signal peptidase will be cloned and characterized. An important goal of the cloning of the 19 kDa subunit is to determine whether there are also two distinct genes encoding related hen oviduct signal peptidase proteins of this size, as has been demonstrated in the canine signal peptidase. cDNA clones encoding signal peptidase protein subunits will be expressed in a heterologous cell system to produce enzymatically active protein(s). Successful development of this approach will lead to future studies using site-directed mutagenesis to probe the enzymatic mechanism this proteinase. Completion of these studies will yield a detailed understanding of the structure and function of microsomal signal peptidase which plays a key role in the early phase of the biosynthesis of secreted proteins in eukaryotes.