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）：阿尔茨海默氏病（AD）是一种可能由遗传因素和环境因素的组合引起的进行性神经退行性疾病。在确定的遗传危险因素中，34 kDa蛋白载脂蛋白（APO）E4为 apoee4载体占所有广告案例的65-80％。尽管APOE4在脂蛋白转运中起正常的作用，但目前未知它如何促进AD发病机理。新兴数据表明APOE4对蛋白水解裂解敏感，因此有助于通过功能丧失与AD相关的潜在分子病理。但是，尚未阐明APOE4蛋白水解裂解的基础机制，包括蛋白酶的鉴定。这项研究的目的是确定负责APOE4裂解的蛋白酶，并确定该裂解事件是否发生在AD大脑中。使用位于裂解APOE4的位置定向的抗体，我们最近确定了从大肠杆菌纯化APOE4后，以重组形式存在重组形式的APOE4的主要裂解片段，并且在AD脑切片的原位存在。我们假设这种蛋白水解事件代表了APOE4裂解的新途径，并且胱天蛋白酶酶在AD大脑中的这种裂解负责。 AIM 1中描述的实验将严格检验以下假设：caspase裂解APOE4以产生18 kDa氨基末端片段。这些实验将通过分析纯化的APOE4或重组形式与无细胞系统中的caspase-3孵育后，通过分析APOE4碎片的产生来表征裂解事件，或者在无细胞系统中的caspase激活中孵育caspase-3。这些实验将通过开发位点定向的切割抗体的开发，该抗体特异性检测D172裂解后APOE4的氨基末端18 kDa片段。 AIM 2中概述的实验将确定蛋白水解裂解是否仅是E4同工型独有的，而不是包括APOE2或APOE3在内的其他已知同工型。我们假设与其他同工型相比，只有APOE4将对蛋白水解裂解具有唯一的敏感性，因此，这将作为将APOE4蛋白水解与增强迟发性AD风险相连的基本事件。使用我们的位置定向的切割抗体，AIM 3中的实验将确定是否在AD大脑中产生了相同的APOE4片段，如果是，则是哪种细胞类型。由于胱天蛋白酶在切割tau中的作用并有助于 神经原纤维缠结的形成，我们假设APOE4的18 kDa片段将位于AD脑的神经原纤维缠结中。使用固定的Mortem脑部切片以及来自AD样品的蛋白质印迹分析的免疫组织化学分析，并将其与年龄匹配的对照进行比较。