Identifying the neuronal substrates of the depalmitoylating enzyme PPT1

 描述(申请人提供)：棕榈酰化是C16脂肪酸与蛋白质的共价结合。棕榈酰化是一种动态的翻译后修饰，不同于其他稳定的脂质修饰(异丙二烯基化、法尼化)。棕榈酰蛋白硫酯酶1(PPT1)是去除这种脂质修饰的主要酶之一。PPT1的隐性功能丧失突变导致神经性蜡样脂褐素沉着症(NCL)，这是一种具有溶酶体病理的进行性神经退行性疾病。PPT1酶活性的完全丧失会导致这种疾病的婴儿形式，而残留的活性(5%-10%)会导致成人发病的NCL。患者积累了脂化肽，这表明脱氨丝裂作用不足会导致蛋白质降解受损。这些临床研究结果有力地表明，PPT1活性对神经元功能和健康至关重要。尽管PPT1很重要，但其底物仍有待确定。在这项提案中，我们的目标是基于比较野生型和PPT1基因敲除脑的棕榈体的无偏见蛋白质组筛选来识别PPT1底物。然后，我们将检查这些底物中的哪一种是对神经元活动的反应而去乙酰化的。这些实验将勾勒出神经细胞生物学的一个重要但尚未被研究的方面，并阐明NCL的机制。考虑到PPT1和NCL之间的直接联系，以及涉及蛋白质棕榈酰化的广泛神经退行性疾病，实现这些目标对人类健康非常重要。

 DESCRIPTION (provided by applicant): Palmitoylation is the covalent attachment of C16 fatty acids to proteins. Palmitoylation is a dynamic post- translational modification and unlike other stable lipid modifications (isoprenylation, farnesylation). One of the major enzymes that remove this lipid modification is palmitoyl protein thioesterase 1 (PPT1). Recessive loss-of-function mutations in PPT1 cause Neuronal Ceroid Lipofuscinosis (NCL), a progressive neurodegenerative disease with lysosomal pathology. Complete loss of PPT1 enzymatic activity leads to an infantile form of the disease, while residual activity (5-10%) leads to adult-onset NCL. Patients accumulate lipidated peptides suggesting that deficient depalmitoylation leads to compromised protein degradation. These clinical findings strongly suggest that PPT1 activity is critical for neuronal function and health. Despite its importance, the substrates of PPT1 remain to be defined. In this proposal, we aim to identify PPT1 substrates based on an unbiased proteomic screen comparing the palmitomes of wildtype and PPT1 knockout brains. Then, we will examine which of these substrates are depalmitoylated in response to neuronal activity. These experiments will delineate an important, yet understudied, aspect of neuronal cell biology and elucidate the mechanisms of NCL. Achieving these goals is important for human health, given the direct links between PPT1 and NCL as well as the wide range of neurodegenerative disorders that involve protein palmitoylation.