MICROSOMAL SIGNAL PEPTIDE PROCESSING

内质网(ER)是合成几乎所有 分泌的蛋白质。氨基末端的疏水信号序列 分泌蛋白是它们进入内质网的关键。不断增加的 大量证据表明，胞浆中的特定蛋白质和 在蛋白质合成过程中，ER膜与信号序列结合 在内质网膜上的移位。将信号序列从 前体蛋白在信号肽的作用下释放成熟蛋白 蛋白质进入内质网管腔。被切割的信号肽的命运很糟糕。 可以理解，但由于信号肽被认为与成分结合 对于移位装置，其移除和/或降解可以 在维持移位设备的可用性方面很重要 用来继续蛋白质的运输。现在很明显，在 至少一些信号肽经历特定的蛋白水解物处理事件 通过微体信号肽酶(MSPP)。初步数据显示 该MSPP可能与一种来自大肠杆菌的类似肽--蛋白酶有关 这项提案的长期目标是净化MSPP 哺乳动物粗大微粒体和建立无细胞系统 酵母，酿酒酵母，以使未来的遗传研究 MSPP的作用。使用溶解的微粒体膜， 具体目标是：(1)表征哺乳动物的溶解度 MSPP，并优化其活性的直接和间接测定。(2)净化 哺乳动物微生物体的MSPP。(3)建立MSPP的无细胞检测方法 酵母中的活性，酿酒酵母。MSPP很可能是一种 转位装置的重要组成部分，并可能参与 其他细胞蛋白分解过程也是如此。这些研究是必不可少的 为了阐明这种新描述的酶在真核细胞中的作用 生物学。 越来越多的人类疾病的特点是缺乏 特定的分泌蛋白(如激素、凝血因子) 归因于缺陷蛋白中信号序列功能的缺陷。 在某些情况下，未切割的蛋白质可能会在细胞中积聚，在这种情况下 它们是合成的，可能导致细胞功能中断以及 分泌不充分的特定蛋白质。因此，对 正常的信号序列脂肪和功能不仅对我们的 对分泌蛋白生物发生的基本了解，但对于 理解异常信号序列的全部医学含义 功能。此外，对两者的基本区别的理解 真核细胞和细菌分泌机器将识别靶标 新型抗生素的未来发展。

The endoplasmic reticulum (ER) is the site of synthesis of virtually all secreted proteins. Hydrophobic signal sequences at the amino terminal of secretory proteins are the key to their entry to the ER. An increasing body of evidence demonstrates that specific proteins in the cytosol and the ER membrane bind to signal sequences during the process of protein translocation across the ER membrane. Removal of the signal sequence from the precursor protein by the action of signal peptidase release the mature protein to the ER lumen. The fate of the cleaved signal peptide is poorly understood, but since the signal peptide is believed to bind to components of the translocation apparatus, its removal and or degradation may be important in maintaining the availability of the translocation apparatus for continued rounds of protein transport. It is now apparent that at least some signal peptides undergo a specific proteolytic processing event by a microsomal signal peptide peptidase (MSPP). Preliminary data suggest that MSPP may be related to an analogous peptide from E. coli, protease IV. The long-term objectives of this proposal are to purify MSPP from mammalian rough microsomes and to establish a cell-free system in the yeast, Saccharomyces cerevesiae, to enable future genetic studies on the role of MSPP. Using solubilized microsomal membranes, the following specific aims are proposed: (1) Characterize the solubility of mammalian MSPP and optimize direct and indirect assays for its activity. (2) Purify MSPP from mammalian microsomes. (3) Establish cell-free assays for MSPP activity in the yeast, Saccharomyces cerevisiae. MSPP is likely to be an important component of the translocation apparatus and may be involved in other cellular proteolytic processes as well. These studies are essential for elucidating the role of this newly described enzyme in eukaryotic cell biology. An increasing number of human diseases characterized by deficiencies in specific secretory proteins (e.g. hormones, clotting factors) have been ascribed to defects in signal sequence function in the deficient protein. In some cases the uncleaved proteins may accumulate in the cell in which they are synthesized, and may result in disrupted cell function as well as insufficient secretion of the specific protein. Thus, an understanding of normal signal sequence fat and function is important not only for our fundamental understanding off secretory protein biogenesis, but for understanding the full medical implications of abnormal signal sequence function. Moreover, an understanding of basic differences between eukaryotic and bacterial secretory machinery will identify targets for the future development of novel antibiotics.