Differential proteomic analysis of lymphocytes treated with mycophenolic acid reveals caspase 3-induced cleavage of rho GDP dissociation inhibitor 2.

The antiproliferative immunosuppressive drug mycophenolic acid (MPA) is an uncompetitive inhibitor of inosine monophosphate dehydrogenase, a key enzyme in de novo synthesis of purine nucleotides. The latter are not only required for synthesis of DNA and RNA but also are essential for the regulation of numerous cellular signaling pathways modulated by guanine nucleotide binding proteins (G proteins). We undertook an analysis of the influence of MPA on protein expression in a T-lymphoblast cell line (CCRF-CEM), which displays concentration-dependent inhibition of proliferation by MPA to obtain insight into the influence of MPA on the cellular proteome. Cells were stimulated with phorbol myristate acetate/ionomycin and incubated in the presence or absence of MPA. Two-dimensional electrophoresis and densitometric imaging revealed 11 differentially expressed protein spots (P < 0.05) on MPA treatment, 6 with increased and 5 with decreased abundance. After in-gel tryptic digestion, proteins were identified by quadrupole time-of-flight mass spectrometry. Proteins displaying increased abundance after MPA treatment included splicing factor arginine/serine-rich 2, prostaglandin E synthase 3, peptidyl-prolyl cis-trans isomerase A, and deoxyuridine 5'-triphosphate nucleotidohydrolase. Endoplasmin, proliferating cell nuclear antigen, acidic leucine-rich nuclear phosphoprotein 32 family member A, and cofilin 1 showed decreased abundance after MPA treatment. Three separate spots (1 decreased and 2 increased abundance) were identified as Rho guanosine diphosphate dissociation inhibitor 2 (Rho GDI 2) proteins. Western blotting with a monoclonal antibody directed against the Rho GDI 2 site cleaved by caspase 3 demonstrated 1 spot with increased abundance to be the caspase 3-cleaved product of Rho GDI 2 lacking the first 19 amino acids. Rho GDI 2 plays a central regulatory role in the activation of Rho guanosine triphosphatases that function as molecular switches in cell signaling pathways affecting cell cytoskeletal dynamics and motility. Our data suggest that MPA can modulate Rho GDI 2 levels in T lymphocytes, thereby potentially disrupting cell signaling pathways important for T-cell function.

Proteomic analysis of cellular response to osmotic stress in thick ascending limb of Henle's loop (TALH) cells.

Epithelial cells of the thick ascending limb of Henle's loop (TALH cells) play a major role in the urinary concentrating mechanism. They are normally exposed to variable and often very high osmotic stress, which is particularly due to high sodium and chloride reabsorption and very low water permeability of the luminal membrane. It is already established that elevation of the activity of aldose reductase and hence an increase in intracellular sorbitol are indispensable for the osmotic adaptation and stability of the TALH cells. To identify new molecular factors potentially associated with the osmotic stress-resistant phenotype in kidney cells, TALH cells exhibiting low or high levels of resistance to osmotic stress were characterized using proteomic tools. Two-dimensional gel analysis showed a total number of 40 proteins that were differentially expressed in TALH cells under osmotic stress. Twenty-five proteins were overexpressed, whereas 15 proteins showed a down-regulation. Besides the sorbitol pathway enzyme aldose reductase, whose expression was 15 times increased, many other metabolic enzymes like glutathione S-transferase, malate dehydrogenase, lactate dehydrogenase, alpha enolase, glyceraldehyde-3-phosphate dehydrogenase, and triose-phosphate isomerase were up-regulated. Among the cytoskeleton proteins and cytoskeleton-associated proteins vimentin, cytokeratin, tropomyosin 4, and annexins I, II, and V were up-regulated, whereas tubulin and tropomyosins 1, 2, and 3 were down-regulated. The heat shock proteins alpha-crystallin chain B, HSP70, and HSP90 were found to be overexpressed. In contrast to the results in oxidative stress the endoplasmic reticulum stress proteins like glucose-regulated proteins (GRP78, GRP94, and GRP96), calreticulin, and protein-disulfide isomerase were down-regulated under hypertonic stress.