Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Room: Virginia B
Carolina Muniz - Howard University
Co-Author(s): Nida Alsaffar, Eric Walters. Department of Biochemistry and Molecular Biology, Howard UniversityCollege of Medicine, Washington, D.C. 20059.
Glutathione S-transferases (GSTs) are enzymes that catalyze conjugation of reduced glutathione (GSH) to a wide range of substrates. S-glutathionylation is the posttranslational modification of protein cysteine residues by the addition of glutathione. GST enzymes participate in S-glutathionylation ofproteins that regulate transport, membrane stabilization, and signal transduction mechanisms in eukaryotic cells. The Dictyostelium discoideum slime mold is an excellent model organism that, when starved, initiates a program of chemotaxisand multi-cellular aggregation that culminates in the formation of fruiting bodies. This developmental morphogenesis is governed by a GSH-sensitive YakA-mediated signaling pathway that secretes cAMP as a primary chemosensor. Recent findings in our laboratory implicate D. discoideum GSTs (DdGST)enzymes as important mediators that affect homeostasis and development of the organism. Their specific mechanism(s) of action and role in S-glutathionylation of YakA signaling remains unclear. In this study, we employed a bioinformatics, web-based program (GSHSite: www. http://csb.cse.yzu.edu.tw/GSHSite/)to survey potential S-glutathionylation sites among members of the D. discoideum YakA signaling pathway (YakA, puF, Aca, PKaC, cAr1, PDE/Reg). GSHSite analysis revealed high probability for S-glutathionylation of the PKaC at amino residue492Cys (Score = 0.556672), whereas other members of the YakA pathway displayed zero probability for S-glutathionylation. These results suggest that DdGSTs enzymesmay mediate cAMP synthesis through direct S-glutathionylation of PKaC. This hypothesis is consistent with preliminary observations in our laboratory where RNAi-mediatedknockdown of the DdGSTA2 isozyme expression disrupts development in D. discoideum. In addition, utilization of theweb-based NetPhos 3.1 program (http://www.cbs.dtu.dk/services/NetPhos/ ) revealed multiple sites for Ser/Thr/Tyr phosphorylation across the DdGSTA2 isozyme (threshold scores of 0.5 or higher): PKC (21.05%) ,INSR (10.52%) , PKA (15.78%) , DNAPK (5.26%) , cdc2 (15.78%), EGFR (5.26%), CKII (10.52%), CKI (10.52%). The NetPhos 3.1 analysis suggests that DdGSTA2 enzymatic and non-enzymatic function(s) may be conferredphosphorylation/de-phosphorylation of the enzyme via PKC. Collectively, the findings in this study suggest cooperative mechanisms of S-glutathionylation and phosphorylation between DdGST enzymes and members of the YakA pathway to regulate cell signaling and development in D. discoideum.
Funder Acknowledgement(s): Dr. Eric Walters
Faculty Advisor: Dr. Eric Walters, firstname.lastname@example.org
Role: I conducted calculations of web based amino acids analysis of netphos and glutathionylAtion. In addition, conducted wet lab gsh pull down assays to isolate gst enzymes Finally, I performed sds gel electrophoresis of the gst proteins ( including Gsta2) in attempts to characterize its phosphorylation.