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RESEARCH INVESTIGATIONS ON THE PROTEOME: 1. MECHANISMS OF REGULATING PROTEIN SYNTHESIS, AND 2. GLOBAL CHARACTERIZATION OF PROTEOMIC RESPONSES TO ARSENIC EXPOSURES
Citation:
ORTIZ, P. A. RESEARCH INVESTIGATIONS ON THE PROTEOME: 1. MECHANISMS OF REGULATING PROTEIN SYNTHESIS, AND 2. GLOBAL CHARACTERIZATION OF PROTEOMIC RESPONSES TO ARSENIC EXPOSURES. Presented at Pontifical Catholic University of Puerto Rico, Ponce, PUERTO RICO, April 16, 2007.
Description:
Eukaryotic Elongation Factor 2 (eEF2) mediates translocation in protein synthesis. eEF2 is modified by two post-translational modifications: the phosphorylation of Thr57 in the G domain and a unique conversion of His699 to diphthamide at the tip of domain IV. Diphthamide is the target for Corynebacterium diphtheriae and Pseudomonas aeruginosa toxins. ADP ribosylation by these toxins inhibits eEF2 function causing cell death. Mutagenesis of the tip of domain IV was used to assess its function. Utilizing a series of mutations within the tip of domain IV, we have dissected the function of this region. Strains expressing these mutants as well as lacking the diphthamide modification genes DPH2 or DPH5 showed increased -1 frameshifting. The novel mutant eEF2 forms confer dominant diphtheria toxin resistance, which correlates with an in vivo effect on translation-linked phenotypes. The results provide a new mechanism by which the translational machinery maintains the accurate production of proteins, establishes a role for the diphthamide modification, and provides evidence for the ability to suppress the lethal effect of a toxin targeted to eEF2.
Currently, we are employing proteomic approaches to investigate the effects of environmental contaminants. Chronic exposure to arsenic has been associated with human cancers of the bladder, kidney, lung, liver, and skin. Inorganic arsenic, an environmental contaminant, is biotransformed in a stepwise manner via both a reduction and then an oxidative methylation step in which arsenic cycles between +5 and +3 oxidation states. To monitor protein-level changes associated with exposure to the individual trivalent arsenicals, a proteomic analysis of human urothelial cells (UROtsa) has been performed. We are using an UROtsa cell clone that has been shown not to biotransform arsenic, making it a good model system for the study. Arsenite (AsIII), methylarsonous acid (MMAIII) and dimethylarsinous acid (DMAIII) treated UROtsa cells are being examined by 2D difference gel electrophoresis (DIGE) at a 3-11 pH range. Protein spots with changes in intensity after AsIII exposure have been identified by LC/MS/MS. These identified proteins participate in antigen processing, metal ion binding, protein folding, apoptosis regulation, biopolymer metabolism and oxidative stress response. Understanding the effects on protein expression induced by each of the three individual trivalent arsenicals may provide information crucial to understanding the key molecular events underlying their toxicity.