Metaobolomics of Neurodegenerative Disorders Caused by Hydrolase Deficiencies

描述（由申请方提供）：蛋白棕榈酰硫酯酶1（PPT 1）缺乏症或婴儿神经元蜡样质脂褐质沉积症（INCL）是一种破坏性人类神经退行性疾病，其特征为自体荧光颗粒状嗜酸性沉积物的溶酶体蓄积。INCL是几种NCL疾病中最常见的一类，在美国发生率为1：12，500。在PPT 1的保守丝氨酸水解酶催化结构域中具有突变的个体易患早期失明和进行性发育迟缓，最终在8-11岁时死亡。PPT 1在体外催化脂肪酰基链从S-酰化底物中释放，但对S-酰化半胱氨酸的偏好略高于酰基辅酶A。尽管动态棕榈酰化对于许多胞质和膜蛋白显然是重要的，但认为PPT 1仅在溶酶体中，这意味着它在这些充分表征的信号传导途径中不起直接作用。代谢脉冲追踪半胱氨酸标记揭示了来自INCL患者的人永生化PPT 1-/-B淋巴母细胞系中存在累积的溶酶体有机可溶性半胱氨酸硫酯代谢物，但十年后，尚未显示该酶的详细体内生化功能。为了更好地了解INCL的发病机制，我建议通过比较代谢组学分析和化学鉴定PPT 1底物。利用我实验室开发的方法，将通过有机提取从细胞和组织中收获（小分子和肽），然后通过LC-MS以宽扫描模式进行分析。来自患者来源的永生化细胞系或PPT 1敲除（-/-）小鼠的PPT 1 +/+和PPT 1-/-代谢组将用L-[13 C3，15 N]-半胱氨酸代谢标记，将初级半胱氨酸硫酯底物与其它次级代谢物分离。将对在神经变性进行性阶段收集的来自PPT 1-/-小鼠的脑样品进行分析，并将其分组为与PPT 1-/-发病机制相关的共同途径。该提案的目标是首先确定PPT 1底物，然后绘制级联次级代谢物变化，以确定对疾病进展至关重要的重要节点。具有蛋白质棕榈酰硫酯酶1（PPT 1）突变的个体易患早期失明和进行性发育迟缓，最终在8-11岁时死亡。大量的工作集中在理解导致PPT 1介导的神经变性的关键事件上，但还没有详细的酶的体内生化功能是已知的。使用先进的分析生物化学，主要底物和次级代谢物的变化与疾病的发病机制将被确定。

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.