Chemical methods to study protein palmitoylation pathways

描述（由申请人提供）：蛋白质s -棕榈酰化是一种翻译后修饰（PTM），其中脂肪酰基部分（饱和16C棕榈酸酯）通过硫酯键连接到目标蛋白质上的半胱氨酸残基。与不可逆的肉豆蔻酰化和戊烯酰化不同，棕榈酰化是动态可逆的。被称为蛋白质酰基转移酶（PATs）的酶将棕榈酸基团连接到蛋白质上，而酰基蛋白硫酯酶（APTs）通过水解硫酯键来去除修饰。尽管棕榈酰化蛋白是一种调控一系列动态过程的PTM，包括细胞信号传导、细胞分裂和突触形成，但很少有文献记载可逆棕榈酰化蛋白的例子，这反映了研究这一动态过程的工具普遍缺乏。我们最近发现了一类化合物，可以增强寄生虫病原体弓形虫入侵宿主细胞的过程。我们已经确定这些化合物通过结合和抑制人类酰基蛋白硫酯酶1 （APT1）的寄生虫同源物起作用，APT1是一种水解酶，参与一系列信号蛋白的去棕榈酰化，包括Ras， eNOS和G蛋白。我们的化合物直接阻断这种酶的功能，导致棕榈酰化底物的积累，改变寄生虫的运动和细胞器分泌。此外，同源性搜索和最近的报道表明，寄生虫可能表达另外三种酰基蛋白硫酯酶。我们假设可逆棕榈酰化是弓形虫和其他可能的人类病原体用来调节重要过程的关键调节过程，并且了解如何调节去除特定底物上的棕榈酸基团将揭示可能被破坏以获得治疗效果的途径。因此，我们建议：1)确定弓形虫的去棕榈酰化酶库，并开发小分子抑制剂来研究它们的功能；2)使用化学蛋白质组学策略来鉴定受动态棕榈酰化调节的候选蛋白质底物；3)开发化学工具来验证特异性去棕榈酰化事件的重要性。本方案利用多种化学、生物化学、蛋白质组学和细胞生物学方法来实现这些目标。

DESCRIPTION (provided by applicant): Protein S-palmitoylation is a post-translational modification (PTM) where a fatty acyl moiety (saturated 16C palmitate) is linked via a thioester bond to a cysteine residue on target proteins. Unlike myristoylation and prenylation, which are irreversible processes, palmitoylation is dynamic and reversible. Enzymes termed protein acyltransferases (PATs) attach the palmitate group to proteins, while acyl-protein thioesterases (APTs) remove the modification by hydrolysis of the thioester bond. Despite being a PTM that regulates a range of dynamic process including cell signaling, cell division and synapse formation, very few examples of reversibly palmitoylated proteins have been documented, reflecting the general lack of tools available for studying this dynamic process. We recently identified a class of compounds that enhance the process of host cell invasion by the parasite pathogen Toxoplasma gondii. We have determined that these compounds function by binding and inhibiting the parasite homolog of human acyl-protein thioesterase 1 (APT1), a hydrolase involved in depalmitoylation of a range of signaling proteins including Ras, eNOS and G proteins. Our compounds directly block the function of this enzyme, resulting in accumulation of palmitoylated substrates and alteration of parasite motility and organelle secretion. In addition, homology search and recent reports indicate that parasites may express three additional acyl-protein thioesterases. We hypothesize that reversible palmitoylation is a key regulatory process used by T. gondii and likely other human pathogens to regulate important processes and that understanding how regulated removal of palmitate groups on specific substrates will shed light on pathways that can be disrupted for therapeutic gain. Therefore, we propose to 1) determine the repertoire of depalmitoylating enzymes in T. gondii and develop small molecule inhibitors to study their function 2) Use a chemical proteomics strategy to identify candidate protein substrates regulated by dynamic palmitoylation and 3) develop chemical tools to validate the importance of specific depalmitoylation events. This proposal makes use of diverse chemical, biochemical, proteomic and cell biological methods to accomplish these aims.