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，或婴儿神经元蜡样脂褐素沉着症(INCL)，是一种毁灭性的人类神经退行性疾病，其特征是溶酶体积聚自体荧光颗粒嗜渗沉淀物。INCL是几种NCL疾病中最常见的一种，在美国发生在1：12,500的新生儿中。在PPT1保守的丝氨酸水解酶催化结构域发生突变的个体会出现早期失明和进行性发育迟缓，最终导致8-11岁的死亡。在体外，PPT1催化S酰化的底物释放脂肪酰链，但对S酰化的半胱氨酸几乎没有选择性。虽然动态棕榈酰化对许多胞浆和膜蛋白很重要，但PPT1被认为是唯一的溶酶体，这意味着它在这些特征明确的信号通路中不起直接作用。代谢脉冲追逐半胱氨酸标记显示，在INL患者来源的永生化的人PPT1-/-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酸酯代谢物，但10年后，这种酶的体内生化功能尚未被详细显示。为了更好地了解INCL的发病机制，我建议通过比较代谢组学来分析和化学鉴定PPT1底物。使用我的赞助实验室首次开发的方法，将通过有机提取从细胞和组织中获取代谢物(小分子和多肽)，然后用LC-MS进行广泛扫描模式的分析。来自患者来源的永生化细胞系或PPT1基因敲除(-/-)小鼠的PPT1+/+和PPT1-/-代谢物将被代谢标记为L-[13C3，15N]-半胱氨酸，将半胱氨酸硫酯的初级底物与其他次生代谢物分离。在神经退行性变的进行性阶段收集的PPT1-/-小鼠的大脑样本将被描述并分组到与PPT1-/-发病相关的共同途径中。这项建议的目标是首先确定PPT1底物，然后绘制级联的次生代谢物变化图，以确定对疾病进展至关重要的重要节点。具有蛋白棕榈酰硫酯酶1(PPT1)突变的个体会出现早期失明和进行性发育迟缓，最终导致8-11岁的死亡。人们一直致力于了解导致PPT1介导的神经变性的关键事件，但还不知道PPT1酶在体内的详细生化功能。利用先进的分析生物化学，将确定与疾病发病机制相关的主要底物和次生代谢物变化。

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.