Dr. Elsie Parés
Institution: UPR Mayaguez Campus
Understanding the molecular action of potential target drugs against Ras2p during cell wall integrity signaling pathways in yeast.
Saccharomyces cerevisiae (S. cerevisiae), a budding yeast, has a plasma membrane and a cell wall that maintains cell shape and integrity. The cell wall remodels its rigid structure during vegetative growth and pheromone-induced morphogenesis which is highly regulated by five cell surface sensors, members of cell wall integrity signaling pathway (CWIP). In particular, three members of the Wsc-family of stress sensors have shown two highly conserved regions located at the C-terminal of their cytoplasmic domain. Wsc1p, together with Wsc2p and Wsc3p, belongs to a family of highly O-glycosylated cell surface proteins where the last two are less studied. Based on reported studies, the short cytoplasmic domain of Wsc1p interacts with Rom2p, a guanine nucleotide exchange factor, and their interaction causes stimulation of the small G-protein Rho1p, that subsequent activates protein kinase C (Pkc1p) and a MAP kinase cascade. The results from the mutational analysis has shown that Wsc1p has a Serine-rich region important for Rom2p interaction, which is negatively regulated by phosphorylation at the amino acid positions 319, 320, 322 and 323. Molecular models were generated using the SYBYL-X 2.1 program, and each three-dimensional structure were used in initial molecular docking studies. The results were critical to understand that these three cytoplasmic tails have the ability to interact with Ras2p and with six common antifungal compounds. Ras2p has been included in this preliminary study due to three main reasons: 1) it is a small monomeric GTP-binding protein primarily localized in the plasma membrane and 2) in the case of positive regulation of Ras2p, it requires the action of GEFs (Guanine nucleotide exchange factor) that catalyzes the transformation from an inactive GDP-bound form of Ras2p to active GTP-bound form, similar to Rho1 role. Further molecular docking experiments were run, and the results have shown that the Serine-rich regions from each cytoplasmic tail interact with Ras2p in a specific fashion, and that Ras2p can also interact with some targeted K-Ras drugs. The first specific aim is to run a Pan-Ras (S. cerevisiae) activation assay with the Wsc1p C-tail and seven serine-rich peptides to validate any formation of protein complexes with Ras2p in order to fill this gap in knowledge. The isolated protein complexes will be characterized by mass spectrometry. As a second specific aim, several antifungal susceptibility testing will be done to demonstrate whether the K-Ras (GDP/GTP) and Ras (GDP) inhibitors can interfere with Ras2p and, as a result, whether their action might block the CWIP. The long term goal of this work is to identify new antifungal compounds against Ras2p that might help to describe another mechanism for the control or abolishing of the CWIP in yeast.