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。脂质部分如棕榈酰基的共价连接是一种这样的修饰。蛋白质可以在N-末端残基（N-连接的）、半胱氨酸残基（S-连接的）和/或丝氨酸残基（O-连接的）上酰化。胞质蛋白和跨膜蛋白的胞内结构域的S-棕榈酰化是调节蛋白稳定性、膜束缚、膜运输和神经元传递的可逆过程。相对较新的发现，即Wnt形态发生蛋白是棕榈酰化的，这一发现提出了与它所回答的问题一样多的问题。例如，棕榈酰化是如何调节形态原的分布和活性的？催化这种重要修饰的酶的结构如何影响它们的功能？Porcupine（Porcn）是膜结合O-酰基转移酶（MBOAT）家族的一员，在Wnt蛋白的保守丝氨酸上添加O-连接的棕榈酸酯是必需的。尽管Porcn在调节Wnt活性中起着核心作用，但实际上对这种酶的结构或酶功能一无所知。这种缺乏生物化学的洞察力延伸到几乎所有的棕榈酰酰基转移酶，在很大程度上，由于缺乏快速敏感的定量测定来测量棕榈酰化。该项目的总体目标是定义Porcn的膜拓扑结构，并开发和应用定量体外方法来评估已知对Porcn功能至关重要的残基的作用。除了获得有关Porcn结构和功能的具体见解外，该项目还将提供酰基转移酶活性的测定，这将导致所有蛋白质棕榈酰酰基转移酶的这项技术的重大进步。该项目将为未来的比较研究奠定基础，旨在了解S-和O-酰基转移酶之间的机制差异，以及DHHC酶控制棕榈酰化和MBOATs之间的相似性和差异。更广泛的影响：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和具有智力挑战性的科学培训经验，其中许多人进入实验室之前很少或根本没有研究经验。学生（每年2名本科生和2名硕士生）将接受培训，以发展他们的批判性思维能力，演讲技巧和实践技术知识，并受益于与PI的密切导师关系。在这个实验室的前沿主要研究的参与将大大有助于女性和代表性不足的少数民族学生在其职业生涯的不同阶段在旧金山弗朗西斯科州立大学（SFSU），历史上代表性不足的少数民族（URM）服务机构的职业发展。SFSU为该地区文化最多元化的学生群体之一提供服务，其中35%为URM，58%为女性。

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.