Metaobolomics of Neurodegenerative Disorders Caused by Hydrolase Deficiencies

描述（由申请人提供）：蛋白棕榈酰硫酯酶1 （PPT1）缺乏症，或称婴儿神经元类蜡样脂质黑素病（INCL），是一种破坏性的人类神经退行性疾病，其特征是溶酶体积聚自身荧光颗粒性亲渗沉积物。INCL是几种NCL疾病中最常见的一类，在美国出生的新生儿中有1:12 500例。PPT1保守丝氨酸水解酶催化结构域突变的个体会出现早期失明和进行性发育迟缓，最终在8-11岁时死亡。在体外实验中，PPT1催化s -酰基化底物释放脂肪酰基链，但相对于酰基辅酶a， PPT1对s -酰基化半胱氨酸的偏好较小。虽然动态棕榈酰化对许多细胞质和膜蛋白显然很重要，但PPT1被认为只存在于溶酶体中，这意味着它在这些特征明确的信号通路中没有直接作用。代谢脉冲追踪半胱氨酸标记已经揭示了来自INCL患者的人类永生化PPT1-/- b淋巴母细胞系中积累的溶酶体有机可溶性半胱氨酸硫酯代谢物的存在，然而十年后，该酶的详细体内生化功能尚未显示。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PPT1底物。使用我的赞助实验室首先开发的方法，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在宽扫描模式下进行分析。来自患者来源的永生化细胞系或PPT1敲除（-/-）小鼠的PPT1+/+和PPT1-/-代谢组将被L-[13C3,15N]-半胱氨酸代谢标记，将初级半胱氨酸硫酯底物与其他次级代谢物分离开来。在神经退行性变的进展阶段收集的PPT1-/-小鼠的脑样本将被描述并分组为与PPT1-/-发病机制相关的共同途径。本提案的目标是首先确定PPT1底物，然后绘制级联次生代谢物变化，以确定疾病进展的关键节点。蛋白棕榈酰硫酯酶1 （PPT1）突变的个体易出现早期失明和进行性发育迟缓，最终在8-11岁时死亡。重要的努力集中在了解导致ppt1介导的神经退行性变的关键事件，但没有详细的体内生化功能的酶是已知的。利用先进的分析生物化学，将确定与疾病发病机制相关的初级底物和次级代谢物变化。

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.