MOLECULAR CELL BIOLOGY OF ALZHEIMER AMYLOIDOGENESIS

阿尔茨海默病以临床痴呆为特征 大脑蛋白质的病理改变。结构性损害包括 细胞外β/A4-淀粉样蛋白沉积。β/A4-淀粉样蛋白是通过 一种大的跨膜前体--β/A4-淀粉样蛋白的蛋白分解 前体蛋白(APP)。APP编码序列的突变为 与家族性脑淀粉样变性有关，指出了 APP在淀粉样变性发病机制中的作用不同国家之间的联系 APP突变基因与脑淀粉样变性表型的相关性 提示另一种淀粉样蛋白分解可能是一种最终常见的 家族性和散发性脑淀粉样变性疾病的通路， 包括阿尔茨海默氏症。 大约30%的APP分子的标准蛋白分解途径(在PC-12中 细胞)在β/A44-淀粉样蛋白结构域内裂解，排除淀粉样蛋白 队形。因此，β/A4-淀粉样蛋白的产生必须通过一种 另一种蛋白质分解途径。替代方案存在的证据 在对人类的研究中，已经出现了几个实验室的路径 脑血管、脑和细胞连续培养。细胞培养 产生APP微异相蛋白分解的系统包括 大鼠PC-12细胞系(在超生理蛋白条件下 磷酸化)，猴成纤维细胞(跟随人的过表达 重组痘苗病毒中的APP)，人APP转染人293 细胞，重组人APP杆状病毒感染Sf9细胞。Sf9 系统之所以特别吸引人，是因为它的超高级别 重组蛋白的表达，为纯化提供了方便 以及对感兴趣的物种进行排序。Sf9细胞忠实地重述 哺乳动物细胞的许多生物学特性。当人工应用程序 在Sf9细胞中表达，APP分子的一部分被切割在一个位置 与人类APP蛋白水解酶的主要切割位点完全相同 细胞，从而为使用Sf9细胞作为模型系统提供有效性 用于APP蛋白分解。 在感染的高复数时，除了APP在 这个主要的保守位点(产生14-15KDA的羧基末端 片段)，Sf9细胞产生离散且数量有限的其他APP 羧基末端片段，包括16-、17-和25-KDA的种类。通过 抗原性分析，17-KDA物种已被证明包含 β/A4-淀粉样域的氨基末端表位，因此是一种 推测为淀粉样变性片段。虽然有越来越多的免疫化学物质 这种推测的淀粉样变原片段的证据，直接蛋白质 对这样一个物种的测序还没有实现，这是主要目标 使用杆状病毒/Sf9表达系统。在……里面 此外，推测的淀粉样变异体APP分子(来自家族性 大脑淀粉样变性)将在杆状病毒中过表达，其 蛋白水解性片段的鉴定、纯化和测序。这个 APP蛋白分解的淀粉样蛋白生成途径的最终鉴定是 对淀粉样蛋白形成的成功解剖和设计至关重要 淀粉样蛋白发生的体外模型的策略。

Alzheimer's disease is characterized by clinical dementia in association with pathologic alterations in brain proteins. Structural lesions include extracellular beta/A4-amyloid deposits. Beta/A4-amyloid is derived through proteolysis of a large, transmembrane precursor, the beta/A4-amyloid precursor protein (APP). Mutations in the coding sequence of APP are associated with familial cerebral amyloidoses, pointing to the importance of APP in the pathogenesis of amyloidosis. The association of different APP-mutation genotypes with the cerebral amyloidosis phenotype strongly suggests that alternative amyloidogenic proteolysis may be a final common pathway in both familial and sporadic cerebral amyloidotic diseases, including Alzheimer's disease. A standard pathway for proteolysis of about 30% of APP molecules (in PC-12 cells) cleaves within the beta/A44- amyloid domain, precluding amyloid formation. The generation of beta/A4-amyloid must therefore occur via an alternative proteolytic pathway. Evidence for the existence of alternative pathways has emerged from several laboratories, in studies of human cerebral vessels, brain, and cells in continuous culture. Cell culture systems which generate microheterogeneous proteolysis of APP include the rat PC-12 line (under conditions of supraphysiological protein phosphorylation), the monkey fibroblast (following overexpression of human APP in recombinant vaccinia virus), the human APP-transfected human 293 cell, and the recombinant human APP-baculovirus infected Sf9 cell. The Sf9 system is particularly attractive because of the extraordinarily high-level expression of recombinant protein, providing convenience for purification and sequencing of species of interest. Sf9 cells faithfully recapitulate many of the biological properties of mammalian cells. When human APP is expressed in Sf9 cells, a portion of APP molecules is cleaved in a position exactly identical to the major cleavage site for proteolyzing APP in human cells, thus providing validity to the use of the Sf9 cell as a model system for APP proteolysis. At high multiplicities-of-infection, in addition to the cleavage of APP at this major conserved site (which generates a 14-15 Kda carboxyl-terminal fragment), Sf9 cells produce a discrete and limited number of other APP carboxyl-terminal fragments, including species of 16-, 17- and 25-Kda. By antigenic analysis, the 17-Kda species has been demonstrated to incorporate amino-terminal epitopes of the beta/A4-amyloid domain and is thus a putative amyloidogenic fragment. While there is mounting immunochemical evidence for such putative amyloidogenic fragments, direct protein sequencing of such a species has not been achieved, and is the primary goal of this proposal, using the baculoviral/Sf9 expression system. In addition, putative amyloidogenic mutant APP molecules (from familial cerebral amyloidoses) will be overexpressed in baculoviruses, and their proteolytic fragments characterized, purified and sequenced. The definitive identification of amyloidogenic pathways for APP proteolysis is crucial to the successful dissection of amyloidogenesis and to the design of strategies for in vitro models of amyloidogenesis.