MOLECULAR CELL BIOLOGY OF ALZHEIMER AMYLOIDOGENESIS

阿尔茨海默病淀粉样变性的分子细胞生物学

• 批准号: 3123433
• 负责人: SAMUEL E. GANDY
• 金额: $ 16.95万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-20 至 1995-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3123433
• 关键词: Alzheimer's disease; Baculoviridae; SDS polyacrylamide gel electrophoresis; amyloid proteins; amyloidosis; chemical kinetics; gel mobility shift assay; human genetic material tag; immunoaffinity chromatography; mass spectrometry; molecular pathology; monoclonal antibody; mutant; phosphorylation; point mutation; posttranslational modifications; protein purification; protein sequence; proteolysis; radioimmunoassay; recombinant DNA; reversed phase chromatography; synthetic peptide; tissue /cell culture; western blottings

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.

阿尔茨海默氏病的特点是临床痴呆症的关联 大脑蛋白质的病理改变 结构性病变包括 细胞外β/A4淀粉样蛋白沉积。 β/A4-淀粉样蛋白来源于 大的跨膜前体β/A4-淀粉样蛋白的蛋白水解 前体蛋白（APP）。 APP编码序列中的突变是 与家族性脑淀粉样变性相关，指出 APP在淀粉样变性发病机制中的作用。 不同的协会 APP突变基因型与脑淀粉样变表型密切相关 提示替代性淀粉样蛋白水解可能是最后一种常见的 在家族性和散发性脑淀粉样变性疾病中， 包括老年痴呆症 约30%的APP分子（在PC-12中）蛋白水解的标准途径 细胞）在β/A44-淀粉样蛋白结构域内裂解，排除淀粉样蛋白 阵 因此，β/A4-淀粉样蛋白的产生必须通过 替代蛋白水解途径。 存在替代品的证据 几个实验室已经出现了通路，在人类的研究中， 连续培养的脑血管、脑和细胞。 细胞培养 产生APP的微异相蛋白水解的系统包括 大鼠PC-12细胞系（在超生理蛋白质条件下 磷酸化），猴成纤维细胞（在人成纤维细胞过度表达后）， 重组牛痘病毒中的APP），人APP转染的人293 细胞，以及重组人APP-杆状病毒感染Sf 9细胞。 的sf 9 系统是特别有吸引力的，因为非常高的水平， 表达重组蛋白，便于纯化 以及对感兴趣的物种进行测序。 sf 9细胞忠实地再现 哺乳动物细胞的许多生物学特性。 当人类APP 在Sf 9细胞中表达，APP分子的一部分在一个位置被切割， 与人APP蛋白水解的主要切割位点完全相同 细胞，从而为使用Sf 9细胞作为模型系统提供了有效性 用于APP蛋白水解。 在高感染倍数下，除了APP的裂解外， 这一主要的保守位点（它产生一个14-15 Kda的羧基末端 片段），Sf 9细胞产生离散和有限数量的其他APP 羧基末端片段，包括16-，17-和25-Kda的种类。 通过 抗原分析，17-Kda的种类已被证明纳入 β/A4-淀粉样蛋白结构域的氨基末端表位，因此是一种 推定的淀粉样蛋白生成片段。 尽管免疫化学 这种假定的淀粉样蛋白片段，直接蛋白 这种物种的测序尚未实现，这是主要目标 该建议，使用杆状病毒/Sf 9表达系统。 在 此外，推定的淀粉样蛋白生成突变APP分子（来自家族性 脑淀粉样变性）将在杆状病毒中过表达， 对蛋白水解片段进行表征、纯化和测序。 的 确定性鉴定APP蛋白水解的淀粉样蛋白生成途径， 对于淀粉样蛋白生成的成功解剖和设计 淀粉样蛋白生成体外模型的策略。