Midline-1 (MID1) is a member of a large family of tripartite RBCC motif (TRIM) proteins found in all vertebrates. It associates with microtubules and facilitates the ubiquitination and subsequent degradation of the catalytic subunit of the most abundant Ser/Thr protein phosphatase (PP2Ac). PP2Ac regulates a vast array of intracellular processes that affect essentially every aspect of the cell's life cycle. Consequently, MID1 appears to regulate epithelial-mesenchyme cellular differentiation along the ventral midline in vertebrates. This project aims to establish the structure and mechanism of function of MID1's three N-terminal zinc-binding domains, Ring Finger (RF) and two B-box domains, which are essential for MID1's functions. RF mediates PP2Ac ubiquitination, while B-boxes 1 and 2 target alpha-4, a regulator of PP2Ac. The structure of B-box 1, with a canonical RF-fold, suggests a novel mechanism for ubiquitin E3 ligase activity and specificity. This project will employ NMR spectroscopy and pull-down assays coupled with mutagenesis to demonstrate that MID1 represents a new class of Ub E3 ligase in which both RF and B-box domains are required to target PP2Ac for degradation.
Knowledge of the mechanism of MID1's role in the ubiquitination of PP2Ac is significant to elucidate signal transduction pathways associated with cellular differentiation during embryogenesis. Mechanistic studies of MID1 will yield an essential model for understanding the structure-function relationship of similar proteins in the TRIM family, for which no information is available. TRIM proteins are involved in signal transduction pathways critical to vertebrate development. This project will allow the PI to offer innovative and novel research opportunities for students. Fifteen undergraduates that included eleven females, two African-Americans, and one Native American have been involved in many aspects of research in the PI's lab. As a minority and a protein NMR spectroscopist in Oklahoma, the PI is in a unique position to attract and recruit students of various ethnic and racial backgrounds. The PI has presented seminars and participated in recruitment activities at annual symposia by the Oklahoma Academy of Sciences that focused on undergraduate research from universities across Oklahoma, and the Louis Stokes Alliances for Minority Participation (LSAMP) Program. LSAMP includes students from Langston University, a historically African-American undergraduate institution located ~20 miles away from Oklahoma State University. The PI's extensive experience with mentoring five Meyerhoff minority students at the University of Maryland Baltimore County and two at Johns Hopkins School of Medicine are consistent with the PI's commitment to not just increase the number of minorities in Biochemistry but also to mentor them. The PI has made a commitment to continue making regular recruiting trips to academic institutions throughout Oklahoma.
