RUI: Characterization of Porcupine membrane topology and palmitoyl acyltransferase activity

智力优势：蛋白质经常经历对其功能至关重要的翻译后修饰。脂类部分的共价连接，如棕榈酰基，就是这样一种修饰。蛋白质可以在N-末端残基(N-连接)、半胱氨酸残基(S连接)和/或丝氨酸残基(O-连接)上酰化。胞浆蛋白的S棕榈酰化和跨膜蛋白的胞内结构域是一个可逆的过程，调节蛋白的稳定性、膜系留、膜转运和神经元传递。相对较新的发现，WNT的形态成因是棕榈酰化的，这引发了许多问题，正如它已经回答了问题一样。例如，棕榈酰化如何调节形态原的分布和活性？催化这一重要修饰的酶的结构如何影响它们的功能？豪猪(PORCN)是膜结合O-酰基转移酶(MBOAT)家族的一员，在Wnt蛋白中需要将O-连接的棕榈酸酯加到保守的丝氨酸上。尽管PORCN在调节Wnt活性中起着核心作用，但对该酶的结构或酶功能几乎一无所知。在很大程度上，由于缺乏快速、灵敏的定量分析来测量棕榈酰化作用，这种缺乏生化洞察力的现象几乎延伸到了所有的棕榈酰转移酶。本项目的总体目标是定义PORCN的膜拓扑结构，并开发和应用一种定量的体外方法来评估已知对PORCN功能至关重要的残基的作用。除了获得对PORCN结构和功能的具体了解外，该项目还将提供酰基转移酶活性的检测，这将导致所有蛋白质棕榈酰转移酶这项技术的显著进步。该项目将为未来的比较研究奠定基础，旨在了解S-酰基转移酶和O-酰基转移酶之间的机制差异，以及控制棕榈酰化和MBOAT的六六六酶之间的异同。广泛影响：这项工作也具有重要的广泛影响，因为该项目中的实验将有助于为本科生和硕士生提供全面的、具有智力挑战的科学培训体验，他们中的许多人进入实验室之前几乎没有研究经验。学生(每年2名本科生和2名硕士研究生)将接受培训，以发展他们的批判性思维技能、陈述技能和动手技术知识，受益于与PI的密切指导关系。参与该实验室的前沿初级研究将对旧金山州立大学(SFSU)女性和代表不足的少数族裔学生在其职业生涯的不同阶段的职业发展做出重大贡献，SFSU是一所历史上代表不足的少数族裔(URM)服务机构。旧金山州立大学为该地区最具文化多样性的学生群体之一提供服务，其中35%的学生来自URM，58%的学生来自女性。

Intellectual merit: Proteins often undergo post-translational modifications that are critical for their function. The covalent attachment of lipid moieties, such as palmitoyl groups, is one such modification. Proteins can be acylated on N-terminal residues (N-linked), cysteine residues (S-linked) and/or serine residues (O-linked). The S-palmitoylation of cytosolic proteins and the intracellular domains of transmembrane proteins is a reversible process that regulates protein stability, membrane tethering, membrane trafficking, and neuronal transmission. The relatively recent discovery that Wnt morphogens are palmitoylated has raised as many questions as it has answered. For instance, how does palmitoylation regulate morphogen distribution and activity? And how does the structure of the enzymes that catalyze this important modification influence their function? Porcupine (Porcn), a member of the membrane bound O-acyl transferase (MBOAT) family, is required for the addition of O-linked palmitate to a conserved serine in Wnt proteins. Despite the central role of Porcn in regulating Wnt activity, virtually nothing is known about the structure or enzymatic function of this enzyme. This lack of biochemical insight extends to virtually all palmitoyl acyltransferases, in large part, due to the lack of fast sensitive quantitative assays to measure palmitoylation. The overall objective of this project is to define the membrane topology of Porcn and to develop and apply a quantitative in vitro approach to assess the roles of residues known to be critical for Porcn function. In addition to gaining specific insight about Porcn structure and function, the project will deliver an assay for acyl transferase activity that would result in significant advancement in this technology for all protein palmitoyl acyltransferases. The project will lay the groundwork for future comparative studies designed to understand the mechanistic differences between S- and O-acyl transferases as well as the similarities and differences between DHHC enzymes that control palmitoylation and MBOATs.Broader impact:This work also has important broad impact because the experiments in this project will contribute to a comprehensive and intellectually challenging scientific training experience for undergraduates and master's degree students, many of whom enter the lab with little or no prior research experience. Students (2 undergrads and 2 master's level students per year) will be trained to develop their critical thinking skills, presentation skills, and hands-on technical knowledge, benefiting from a close mentorship relationship with the PI. Involvement in the cutting-edge primary research of this laboratory will contribute significantly to the career development of female and underrepresented minority students at different stages of their careers at San Francisco State University (SFSU), a historically underrepresented minority (URM)-serving institution. SFSU serves one of the most culturally diverse student populations in the region with 35% URM and 58% women.