Identifying the neuronal substrates of the depalmitoylating enzyme PPT1

 描述（由申请人提供）：棕榈酰化是C16脂肪酸与蛋白质的共价连接。棕榈酰化是一种动态的翻译后修饰，与其他稳定的脂质修饰（异戊二烯化，法尼基化）不同。去除这种脂质修饰的主要酶之一是棕榈酰蛋白硫酯酶1（PPT 1）。PPT 1的隐性功能丧失突变导致神经元蜡样脂褐质沉积症（NCL），这是一种伴有溶酶体病理学的进行性神经退行性疾病。PPT 1酶活性的完全丧失导致疾病的婴儿形式，而残留活性（5-10%）导致成人发作的NCL。患者积累脂化肽，表明缺乏脱棕榈酰化导致蛋白质降解受损。这些临床研究结果强烈表明，PPT 1活性对神经元功能和健康至关重要。尽管它的重要性，底物的PPT 1仍有待确定。在这个建议中，我们的目标是确定PPT 1基板的基础上，一个公正的蛋白质组学筛选比较野生型和PPT 1基因敲除大脑的棕榈酸。然后，我们将研究这些底物中的哪一种在神经元活动中被去棕榈酰化。这些实验将描绘一个重要的，但研究不足，神经元细胞生物学方面，并阐明NCL的机制。实现这些目标对人类健康至关重要，因为PPT 1和NCL之间存在直接联系，以及涉及蛋白质棕榈酰化的广泛神经退行性疾病。

 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.