MICROSOMAL SIGNAL PEPTIDE PROCESSING

内质网（ER）是合成几乎所有 分泌的蛋白质疏水信号序列的氨基末端 分泌蛋白是它们进入ER的关键。越来越 大量证据表明，细胞质中的特定蛋白质和 ER膜在蛋白质合成过程中与信号序列结合， 跨内质网膜转运。信号序列的去除 前体蛋白在信号肽酶的作用下释放成熟的 蛋白质到ER腔。切割的信号肽的命运很差， 这是可以理解的，但是由于信号肽被认为与组分结合， 易位装置的去除和/或降解可以是 重要的是保持易位装置的可用性 进行连续的蛋白质运输。现在很明显，在 至少一些信号肽经历特异性蛋白水解加工事件 通过微粒体信号肽肽酶（MSPP）。初步数据表明 MSPP可能与E.大肠杆菌蛋白酶 四.该提案的长期目标是将MSPP从 哺乳动物粗糙微粒体，并建立一个无细胞系统， 酵母，酿酒酵母，使未来的遗传研究， MSPP的作用。使用溶解的微粒体膜， 提出了具体的目标：（1）表征哺乳动物的溶解性 MSPP和优化其活性的直接和间接测定。(2)净化 来自哺乳动物微粒体的MSPP。(3)建立MSPP的无细胞测定法 在酵母中的活性，酿酒酵母。MSPP很可能是一个 重要组成部分的易位装置，并可能参与 其他细胞蛋白水解过程。这些研究至关重要 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 特异性分泌蛋白（如激素、凝血因子）已被 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在细胞中积累， 它们是合成的，可能导致细胞功能被破坏， 特定蛋白质分泌不足。因此， 正常的信号序列脂肪和功能是重要的，不仅对我们的 基本了解分泌蛋白的生物合成，但对于 了解异常信号序列的全部医学含义 功能此外，了解基本的差异， 真核生物和细菌的分泌机制将确定目标， 新抗生素的未来发展。

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.