PROTEIN TRANSLOCATION AND PROCESSING IN OVIDUCTS

该项目将专注于信号的结构和酶学 多肽酶，一种膜结合的蛋白水解酶，可去除 来自新生分泌蛋白的氨基末端信号肽 被运输到内质网的管腔中。这个 该酶的作用机制尚未确定。 尽管这种酶在分泌物的生物合成中起着核心作用 蛋白质。信号肽的催化机制可能定义了一种新的 蛋白水解酶的一类。信号肽序列的突变 直接影响信号肽酶的物质已被发现并被证明 会导致人类疾病。构成这两种蛋白质的性质 将对母鸡输卵管微粒体信号肽酶进行检测，并对其 定义的函数。酶最低需要量的蛋白质(S) 活动将被确定。合成肽类似物含 共价连接的光活化交联点将被用于 确定蛋白质亚基在催化作用中的作用 信号肽酶。自然发生的基因突变的影响 凝血因子X和凝血因子X这两种人类蛋白质的信号肽区 血清白蛋白，将被检查以提供详细的描述 这些基因突变对这些化合物生物合成的影响 蛋白质。研究基因突变效应的一般方法 将开发信号肽酶上的信号肽。一个cdna克隆 编码母鸡输卵管信号肽酶的19 kDa蛋白将是 进行了克隆和鉴定。克隆19个人的一个重要目标 KDA亚基是决定是否还有两个不同的基因 编码这种大小的相关母鸡输卵管信号肽酶蛋白，如 已经在犬类信号肽酶中得到证实。CDNA克隆 编码信号肽蛋白亚基将在一个 异源细胞系统产生具有酶活性的蛋白质(S)。 这一方法的成功开发将导致未来的研究 利用定点诱变技术探讨其酶机制 蛋白水解酶。这些研究的完成将产生一份详细的 对微体信号结构和功能的认识 在生物合成的早期阶段起关键作用的多肽酶 真核生物中的分泌蛋白质。

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.