SIGNAL PEPTIDASE STRUCTURE AND FUNCTION

信号肽酶是一种膜结合蛋白水解酶， 来自分泌蛋白质的肽靶向内质网腔 网状细胞。 该酶的活性与一个从两个到三个的复合物有关。 5种在管腔上具有球状结构域的膜蛋白 在内质网的一侧。 这种酶是维持生命所必需的 在酵母中，被认为是所有真核生物的必需基因 有机体 分泌蛋白中的突变影响细胞分裂， 已经描述了由信号肽酶产生信号肽， 描述导致血友病（凝血因子X），家族性 神经垂体性尿崩症（加压素）和一种家族性 甲状旁腺激素（parathyroid hormone） 许多其他继承的 可能存在缺陷，但未被识别。 因为 这种酶在蛋白质合成和分泌中的中心作用， 重要是要充分了解其功能和蛋白质中的作用 转移到内质网中。 的催化机理 微粒体信号肽酶似乎是新的，许多定义了一个新的 机械蛋白酶类。 已知信号的氨基酸序列 肽酶蛋白质与任何公认的蛋白酶无关 家庭 该项目的主要目标是确定结构和 内质网信号肽酶的功能特性 动物细胞。 该项目有三个具体目标：1）确定 催化功能性氨基酸残基; 2）定义延伸的 底物切割特异性;和3）鉴定蛋白质组分 微粒体信号肽酶是信号肽 处理. 信号肽酶活性的特异性不可逆抑制剂 将被用于定位和鉴定所需的氨基酸残基， 催化作用 被抑制的肽酶的修饰的蛋白质和肽片段 将通过高效液相色谱法进行纯化，并通过 氨基酸序列分析和/或电喷雾电离质谱 光谱法 另外，更高特异性的信号抑制剂 将制备肽酶。 模型前蛋白信号肽酶底物 将通过定点诱变进行修饰，并用于揭示 信号肽的结构决定簇， 底物结合位点。 微粒体信号肽酶将在 组织培养和细菌细胞来鉴定单个蛋白质 识别和切割信号所必需和充分的组分 体内肽。 结构和机制研究将大大 通过产生和纯化酶活性的能力来促进， 重组信号肽酶复合物。 实现这一目标将使 精确确定这种不寻常的膜的作用机制 结合蛋白酶

Signal peptidase is a membrane-bound proteolytic enzyme that cleaves signal peptides from secretory proteins targeted to the lumen of the endoplasmic reticulum. The enzyme activity is associated with a complex of from two to five integral membrane proteins with their globular domains on the lumenal side of the endoplasmic reticulum. The enzyme is essential for viability in yeast and is presumed to be an essential gene of all eukaryotic organisms. Mutations in secretory proteins affecting cleavage of the signal peptide by signal peptidase have been described that have been described that result in hemophilia (coagulation factor X), familial neurohypophyseal diabetes insipidus (vasopressin), and a form of familial hypoparathyroidism (parathyroid hormone). Many other such inherited defects probably exist but have not been recognized. Because of the central role of this enzyme in protein biosynthesis and secretion, it is important to fully understand its function and role in protein translocation into the endoplasmic reticulum. The catalytic mechanism of microsomal signal peptidase appears to be novel and many define a new mechanistic proteinase class. The amino acid sequences of the known signal peptidase proteins are unrelated to any of the well established proteinase families. The major goal of this project is to define the structural and functional properties of the signal peptidase of the endoplasmic reticulum of animal cells. The project has three specific aims: 1) to identify catalytically functional amino acid residues; 2) to define the extended substrate cleavage specificity; and 3) to identify the protein components of microsomal signal peptidase that are essential for signal peptide processing. Specific, irreversible inhibitors of signal peptidase activity will be used to localize and identify amino acid residues required for catalysis. Modified proteins and peptide fragments of inhibited peptidase will be purified by high performance liquid chromatography and analyzed by animo acid sequence analysis and/or electrospray ionization mass spectrometry. Additional, more highly specific inhibitors of signal peptidase will be prepared. A model preprotein signal peptidase substrate will be modified by site-directed mutagenesis and used to reveal the structural determinants of signal peptides that define the extended substrate binding site. Microsomal signal peptidase will be expressed in tissue culture and bacterial cells to identify the individual protein components necessary and sufficient for recognition and cleavage of signal peptides in vivo. Structural and mechanistic studies will be greatly facilitated by the ability to produce and purify an enzymatically active, recombinant signal peptidase complex. Achievement of this goal will permit precise determination of the mechanism of action of this unusual membrane- bound proteinase.