RUI: Characterization of Porcupine membrane topology and palmitoyl acyltransferase activity

San Francisco State University, San Francisco CA

Investigators

Abstract

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.