Molecular Mechanisms of ApoE4 Proteolysis in Alzheimer's Disease

阿尔茨海默病中 ApoE4 蛋白水解的分子机制

• 批准号: 8488281
• 负责人: TROY T ROHN
• 金额: $ 28.45万
• 依托单位: BOISE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8488281
• 关键词: Accounting; Affinity; Age; Alleles; Alzheimer' s Disease; Alzheimer' s disease risk; Amino Acid Sequence; Amino Acid Substitution; Amino Acids; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Antibodies; Apolipoprotein E; Apoptosis; Biological Assay; Biological Models; Biomedical Research; Brain; Caspase; Cathepsins; Cell Count; Cell-Free System; Cholesterol; Circular Dichroism; Cleaved cell; Complex; Consensus; Data; Development; Disease; Environmental Risk Factor; Escherichia coli; Event; Genetic; Glycoproteins; Human; In Situ; In Vitro; Individual; Late Onset Alzheimer Disease; Length; Lipoproteins; Mass Spectrum Analysis; Mediator of activation protein; Metalloproteases; Microscopy; Molecular; Movement; Nature; Nerve Degeneration; Neurodegenerative Disorders; Neurofibrillary Tangles; Pathogenesis; Pathway interactions; Peptide Hydrolases; Play; Positioning Attribute; Predisposition; Process; Production; Protein Isoforms; Proteins; Proteolysis; Recombinants; Relative (related person); Risk; Role; Sampling; Sedimentation process; Serine Protease; Site; Testing; Tissues; Universities; Validation; Variant; Western Blotting; Work; apolipoprotein E-3; apolipoprotein E-4; base; caspase-3; cell type; chymotrypsin; design; genetic risk factor; high risk; in vitro Assay; insight; light scattering; loss of function; molecular pathology; neurofibrillary tangle formation; novel; public health relevance; research study; tau Proteins

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is a progressive neurodegenerative disease likely caused by a combination of both genetic and environmental factors. Of the genetic risk factors identified, the 34 kDa protein, apolipoprotein (apo) E4, is of significant importance as apoeE4 carriers account for 65-80 percent of all AD cases. Although apoE4 plays a normal role in lipoprotein transport, how it contributes to AD pathogenesis is currently unknown. Emerging data suggests that apoE4 is sensitive to proteolytic cleavage and thus contributes to the underlying molecular pathology associated with AD possibly through a loss of function. However, the molecular mechanisms underlying the proteolytic cleavage of apoE4, including the identity of the protease involve, has not been clarified. The purpose of this study is to identify the protease responsible for ApoE4 cleavage and to determine whether or not this cleavage event occurs in the AD brain. Using a site-directed antibody to cleaved apoE4 we have recently determined a major cleavage fragment of apoE4 of 18 KDa is present in recombinant forms following purification of apoE4 from E. coli and is present in situ in AD brain sections. We hypothesize that this proteolytic event represents a novel pathway for apoE4 cleavage and that caspases are responsible for this cleavage in the AD brain. Experiments described in Aim 1 will rigorously test the hypothesis that apoE4 is cleaved by caspases to generate a 18 kDa amino-terminal fragment. These experiments will characterize the cleavage event by analyzing the production of the apoE4 fragment following incubation of purified apoE4 or recombinant forms with caspase-3 in a cell-free system or following caspase activation in a model system of apoptosis. These experiments will be aided through the development of a site-directed cleavage antibody that specifically detects the amino-terminal 18 kDa fragment of apoE4 following cleavage at D172. Experiments outlined in Aim 2 will determine whether proteolytic cleavage is unique to only the E4 isoform, and not other known isoforms including apoE2 or apoE3. We hypothesize that only apoE4 will be uniquely sensitive to proteolytic cleavage compared to other isoforms and thus, this will serve as an underlying event connecting apoE4 proteolysis to an enhanced risk of late-onset AD. Using our site-directed cleavage antibodies, experiments in Aim 3 will determine if this same apoE4 fragment is generated in the AD brain and if so, what cell type. Due to the role of caspases in cleaving tau and contributing to the formation of neurofibrillary tangles, we hypothesize this 18 kDa fragment of apoE4 will localize within neurofibrillary tangles of the AD brain. Both immunohistochemical analysis using fixed post mortem brain sections as well as Western blot analysis from AD samples will be performed and compared to age-matched controls.

描述（由申请人提供）：阿尔茨海默病（AD）是一种进行性神经退行性疾病，可能由遗传和环境因素共同引起。在已确定的遗传风险因素中，34 kDa蛋白质载脂蛋白（apo）E4是 apoeE 4携带者占所有AD病例的65- 80%。虽然apoE 4在脂蛋白转运中起着正常的作用，但它如何参与AD的发病机制目前尚不清楚。新出现的数据表明，apoE 4是敏感的蛋白水解裂解，从而有助于潜在的分子病理学与AD可能通过功能丧失。然而，潜在的apoE 4的蛋白水解切割的分子机制，包括蛋白酶的身份参与，尚未澄清。本研究的目的是鉴定负责ApoE 4切割的蛋白酶，并确定这种切割事件是否发生在AD脑中。我们最近用一种切割apoE 4的定点抗体测定了从大肠杆菌中纯化apoE 4后，apoE 4的一个18 KDa的主要切割片段以重组形式存在。大肠杆菌中表达，并原位存在于AD脑切片中。我们推测，这种蛋白水解事件代表了一种新的途径apoE 4裂解和半胱天冬酶是负责这种分裂在AD脑。目的1中描述的实验将严格检验apoE 4被半胱天冬酶切割以产生18 kDa氨基末端片段的假设。这些实验将通过分析纯化的apoE 4或重组形式与半胱天冬酶-3在无细胞系统中孵育后或在凋亡模型系统中半胱天冬酶活化后apoE 4片段的产生来表征切割事件。这些实验将通过开发定点切割抗体来辅助，该抗体特异性检测在D172处切割后apoE 4的氨基末端18 kDa片段。目的2中概述的实验将确定蛋白水解切割是否仅对E4同种型是独特的，而不是其他已知的同种型，包括apoE 2或apoE 3。我们假设，只有apoE 4将是唯一敏感的蛋白水解裂解相比，其他亚型，因此，这将作为一个潜在的事件连接apoE 4蛋白水解的晚发型AD的风险增加。使用我们的定点切割抗体，目标3中的实验将确定是否在AD脑中产生相同的apoE 4片段，如果是，则是什么细胞类型。由于半胱天冬酶在切割tau蛋白中的作用， 我们推测apoE 4的18 kDa片段将定位于AD脑的神经元缠结内。将使用固定的死后脑切片进行免疫组织化学分析以及AD样本的蛋白质印迹分析，并与年龄匹配的对照进行比较。