Metaobolomics of Neurodegenerative Disorders Caused by Hydrolase Deficiencies

水解酶缺陷引起的神经退行性疾病的代谢组学

• 批准号: 7741199
• 负责人: Brent Randall Martin
• 金额: $ 4.11万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-12-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7741199
• 关键词: Acyl Coenzyme A; Age; Analytical Biochemistry; Biochemical; Birth; Blindness; Brain; Catalytic Domain; Cell Line; Cells; Cessation of life; Computer software; Coupled; Cysteine; Data Set; Deposition; Disease; Disease Progression; Enzymes; Equipment and supply inventories; Event; Goals; Harvest; Human; Hydrolase; In Vitro; Individual; Infantile neuronal ceroid lipofuscinosis; Knockout Mice; Label; Link; Maps; Mediating; Membrane Proteins; Metabolic; Methods; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Pathogenesis; Pathway interactions; Patients; Peptides; Physiologic pulse; Play; Precursor B-Lymphoblast; Relative (related person); Role; Sampling; Scanning; Serine Hydrolase; Signal Pathway; Staging; Tissues; United States; comparative; immortalized cell; in vivo; instrument; metabolomics; palmitoylation; preference; programs; protein metabolite; small molecule; thioester; thioesterase PPT1 gene product

DESCRIPTION (provided by applicant): Protein palmitoyl thioesterase 1 (PPT1) deficiency, or infantile neuronal ceroid lipofuscinosis (INCL), is a devastating human neurodegenerative disease characterized by lysosomal accumulation of autofluorescent granular osmophilic deposits. INCL is the most common class of several NCL diseases, which occur in 1:12,500 births in the United States. Individuals with mutations in the conserved serine hydrolase catalytic domain of PPT1 are subject to early blindness and progressive retardation, culminating in death by age 8-11 years. PPT1 catalyses the release of fatty-acyl chains from S-acylated substrates in vitro, but has little preference for S-acylated cysteine over acyl-CoA. Although dynamic palmitoylation is clearly important for many cytosolic and membrane proteins, PPT1 is believed to be exclusively lysosomal, implying it does not play a direct role in these well-characterized signaling pathways. Metabolic pulse-chase cysteine labeling has revealed the presence of accumulated lysosomal organic-soluble cysteine thioester metabolites in human immortalized PPT1-/- B-lymphoblast cell lines derived from INCL patients, yet a decade later, no detailed in vivo biochemical functions for this enzyme have been shown. In order to gain a better understanding of INCL pathogenesis, I propose to profile and chemically identify PPT1 substrates by comparative metabolomics. Using methods first developed in my sponsoring lab, metabolites (small molecules and peptides) will be harvested from cells and tissues by organic extraction, and then analyzed in broad scanning mode by LC-MS. PPT1+/+ and PPT1-/- metabolomes from patient-derived immortalized cell lines or PPT1 knockout (-/-) mice will be metabolically labeled with L-[13C3,15N]-cysteine, separating primary cysteine-thioester substrates from other secondary metabolites. Brain samples from PPT1-/- mice collected at progressive stages of neurodegeneration will be profiled and grouped into common pathways linked to PPT1-/- pathogenesis. The goal of this proposal is first identify PPT1 substrates, then to map the cascading secondary metabolite changes to identify important nodes critical for disease progression. Individuals with mutations in protein palmitoyl thioesterase 1 (PPT1) are subject to early blindness and progressive retardation, culminating in death by age 8-11 years. Significant effort has been focused on understanding the key events leading to PPT1-mediated neurodegeneration, yet no detailed in vivo biochemical functions for the enzyme are known. Using advanced analytical biochemistry, the primary substrates and secondary metabolite changes associated with disease pathogenesis will be identified.

描述（由申请人提供）：蛋白质棕榈酰硫酯酶1（PPT1）缺乏症或婴儿神经蛋白神经蛋白粘膜脂肪促脂肪促脂肪肌cin（含），是一种毁灭性的人类神经退行性疾病，其特征是自身荧光荧光荧光颗粒颗粒颗粒渗透渗透渗透渗透渗透性渗透性溶质体的溶酶体积累。包括几种NCL疾病中最常见的类别，在美国的1：12,500分中发生。 PPT1的保守丝氨酸水解酶催化结构域中突变的个体受早期失明和进行性迟缓的影响，最终在8-11岁时死亡。 PPT1在体外从S-酰基底物中释放脂肪酰基链的释放，但对S-酰基的半胱氨酸而不是酰基-COA的偏爱。尽管动态棕榈酰化对于许多胞质和膜蛋白显然很重要，但PPT1被认为仅是溶酶体，这意味着它在这些特征良好的信号传导途径中并不直接起作用。代谢脉搏疗法半胱氨酸标记表明，在人类不朽的PPT1 - / - B溶血细胞系中存在累积的溶酶体有机溶性半胱氨酸半胱氨酸硫代代谢物，这些细胞是来自含有的患者，但十年后，却没有十年后，但尚未显示出详细的bioChemachical in vivo biochemical功能。为了更好地了解包含发病机理，我建议通过比较代谢组学对PPT1底物进行化学概况。使用我的赞助实验室中首次开发的方法，将通过有机提取从细胞和组织中收集代谢物（小分子和肽），然后通过LC-MS在广泛的扫描模式下进行分析。来自患者衍生的永生细胞系或PPT1敲除（ - / - ）小鼠的PPT1+/+和PPT1 - / - 代谢组将用L- [13C3,15N] - 半胱氨酸代谢标记，将原代胞和thio thioester底物分离出来，将其分离出来。在神经退行性阶段收集的PPT1 - / - 小鼠的脑样品将被分类并分组为与PPT1 - / - 发病机理相关的常见途径。该提案的目的是首先识别PPT1底物，然后绘制级联的二级代谢产物变化，以识别重要的节点对疾病进展至关重要。蛋白质棕榈酰硫代酶1（PPT1）中具有突变的个体受早期失明和进行性迟缓的影响，最终在8-11岁时死亡。重大努力集中在理解导致PPT1介导的神经变性的关键事件上，但尚无详细的体内生化功能的酶。使用晚期分析生物化学，将确定与疾病发病机理相关的主要底物和二级代谢产物变化。