Proteomic characterization of aging cerebellum

衰老小脑的蛋白质组学特征

• 批准号: 6949990
• 负责人: CHRISTIAN SCHONEICH
• 金额: $ 28.8万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6949990
• 关键词: NMDA receptors; aging; aldehydes; biological signal transduction; cerebellum; clinical research; dihydroxyphenylalanine; electrospray ionization mass spectrometry; laboratory rat; matrix assisted laser desorption ionization; membrane proteins; molecular biology; neural transmission; oxidative stress; phosphorylation; posttranslational modifications; protein localization; protein purification; protein structure function; proteomics; synapses; technology /technique development; thiols; tyrosine analog

DESCRIPTION (provided by applicant): The overall goal of this research is a detailed characterization of age-dependent changes in the proteome of an oxidation-sensitive area of the brain, the cerebellum. This region controls vestibular function, fine motor behaviors, and some higher cognitive processes such as episodic memory and olfactory perception, all of which are frequently compromised in aged individuals. Furthermore, preliminary studies showed more markers of oxidative stress in aged rat cerebellum than any other brain region. Protein expression will quantitated and post-translational modifications identified and localized in cerebella from F344/BNF1 rats at 5, 22, and 34 months. Analysis will focus specifically on protein phosphorylation and accumulation of protein oxidative modifications such as oxidized thiols, 3-nitrotyrosine, 3, 4-dihydroxyphenylalanine, and 4-hydroxynonenal adducts. Proteomic analysis will be carried out with three cerebellar subcellular fractions involved in neurotransmission and signaling: (i) cerebellar synaptic plasma membranes, (ii) synaptic junctional complexes, and (iii) a multi-subunit glutamate receptor involved in Ca+ and nitric oxide (NO)-dependent cell signaling, the NMDA receptor complex. Analysis of these fractions will yield representative results on composition and potential functional integrity of protein complexes. Yet, selection of these subcellular fractions allows for limited sample sizes (i.e., number of proteins to be analyzed) permitting optimization of quantitative analysis and definitive localization of post-translational modifications through maximum sequence coverage of identified proteins. The Specific Aims are to 1) conduct quantitative proteomic analysis and differential display of age-dependent changes in expression of soluble and membrane proteins in cerebellum, 2) analyze age-dependent phosphorylation status of cerebellar proteins, 3) identify and localize age-dependent oxidative post-translational modifications on cerebellar proteins, and 4) develop novel technologies for enrichment, characterization, and quantitative analysis of DOPA-containing peptides and proteins. Results of these proteomic studies will contribute to emerging databases providing critical insights into brain mechanisms underlying functional decline with age.

描述（由申请人提供）：这项研究的总体目标是对年龄依赖性变化的详细表征，大脑对大脑的氧化敏感区域的蛋白质组变化。该区域控制着前庭功能，精细运动行为以及一些较高的认知过程，例如情景记忆和嗅觉感知，所有这些过程在老年人中经常受到损害。此外，初步研究显示，老年大鼠小脑的氧化应激标志物比任何其他大脑区域都要多。蛋白质表达将在5、22和34个月的F344/BNF1大鼠中定量并定位在小脑中的翻译后修饰。分析将专门集中于蛋白质磷酸化和蛋白质氧化修饰的积累，例如氧化硫醇，3-硝基酪氨酸，3、4-二羟基苯基丙氨酸和4-羟基苯胺加合物。 Proteomic analysis will be carried out with three cerebellar subcellular fractions involved in neurotransmission and signaling: (i) cerebellar synaptic plasma membranes, (ii) synaptic junctional complexes, and (iii) a multi-subunit glutamate receptor involved in Ca+ and nitric oxide (NO)-dependent cell signaling, the NMDA receptor complex.对这些馏分的分析将产生有关蛋白质复合物的组成和潜在功能完整性的代表性结果。然而，选择这些亚细胞分数允许样本量有限（即要分析蛋白质的数量），允许优化定量分析和通过鉴定蛋白的最大序列覆盖率对翻译后修饰的确定定位。具体目的是1）进行定量蛋白质组学分析，以及在小脑中依赖年龄和膜蛋白表达的年龄依赖性变化的变化显示，2）分析小脑蛋白的年龄依赖性磷酸化状态，3）识别和定位于年龄依赖于年龄的氧化后蛋白质蛋白质，并在凝固性蛋白质上局限于蛋白质的蛋白质，以及4个）蛋白质的蛋白质，以及4）4）表征和含DOPA的肽和蛋白质的定量分析。这些蛋白质组学研究的结果将有助于新兴数据库，从而为随着年龄的增长的功能下降而提供对大脑机制的关键见解。