RUI: Characterization of Porcupine membrane topology and palmitoyl acyltransferase activity

RUI：豪猪膜拓扑结构和棕榈酰酰基转移酶活性的表征

• 批准号: 1244602
• 负责人: Laura Burrus
• 金额: $ 58.16万
• 依托单位: San Francisco State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1244602&HistoricalAwards=false
• 关键词: RUI; Characterization; Porcupine; membrane; topology

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.

智力优点：蛋白质经​​常经历对其功能至关重要的翻译后修饰。脂质部分的共价附着（例如棕榈酰基组）就是这样的修饰。蛋白质可以在N末端残基（N-连接），半胱氨酸残基（S-链接）和/或丝氨酸残基（O链接）上酰化。胞质蛋白和跨膜蛋白的细胞内结构域的S-顶酰化是一种可逆过程，可调节蛋白质稳定性，膜束缚，膜运输和神经元传播。相对较新的发现，棕榈酰化是棕榈酰化的，它提出了许多问题。例如，棕榈酰化如何调节形态的分布和活性？催化这种重要修饰的酶的结构如何影响其功能？猪（Porcn）是膜结合的O-酰基转移酶（MBOAT）家族的成员，将O连接的棕榈酸酯添加到Wnt蛋白中的保守丝氨酸中是必需的。尽管PORCN在调节Wnt活性中的核心作用，但实际上，该酶的结构或酶促功能几乎没有任何了解。缺乏生化洞察力扩展到几乎所有棕榈酰酰基转移酶，这在很大程度上是由于缺乏快速敏感的定量测定法来测量棕榈酰化。该项目的总体目的是定义PORCN的膜拓扑，并开发和采用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。除了获得有关PORCN结构和功能的特定见解外，该项目还将提供酰基转移酶活性的测定法，这将导致所有蛋白质棕榈酰酰基转移酶的这一技术的显着进步。该项目将为未来的比较研究奠定基础，以了解S-和O-acyl转移酶之间的机械差异以及控制棕榈酰化和MBOAT的DHHC酶之间的相似性和差异。Baroader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导致该项目的全面培训，并且具有较少的挑战性培训，并且在许多方面具有挑战性的培训，并且在许多方面具有挑战性的培训，并且具有挑战性的人，并且具有较低的挑战性，并且具有挑战性的范围，并且具有挑战性的范围，并且具有挑战性的范围，并且具有挑战性的培训，并且具有挑战性的培训，并且具有挑战性的培训，并且具有较低的挑战性，并且具有挑战性的培训，并且具有较低的挑战性，并且具有成熟的范围，并且具有成熟的范围，并且具有挑战性的培训。先前的研究经验。学生（每年2个本科生和2名硕士学生）将接受培训，以发展其批判性思维技能，演示能力和动手技术知识，从而受益于与PI的密切指导关系。参与该实验室的尖端主要研究将在旧金山国立大学（SFSU）的不同阶段的女性和代表性不足的少数族裔学生的职业发展，这是历史上代表性不足的少数族裔（URM） - 服务机构。 SFSU为该地区最多样化的学生群体之一提供服务，其中35％的URM和58％的女性。