Heller Tanja, Asif Abdul R, Petrova Darinka Todorova, Doncheva Yuliana, Wieland E, Oellerich Michael, Shipkova Maria, Armstrong Victor William
Department of Clinical Chemistry, University Medicine Göttingen, Robert-Koch-Strasse 40, Göttingen, Germany.
Ther Drug Monit. 2009 Apr;31(2):211-7. doi: 10.1097/FTD.0b013e318196fb73.
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.
抗增殖免疫抑制药物霉酚酸（MPA）是肌苷单磷酸脱氢酶的非竞争性抑制剂，肌苷单磷酸脱氢酶是嘌呤核苷酸从头合成中的关键酶。嘌呤核苷酸不仅是DNA和RNA合成所必需的，而且对于由鸟嘌呤核苷酸结合蛋白（G蛋白）调节的众多细胞信号通路的调控也至关重要。我们分析了MPA对T淋巴母细胞系（CCRF-CEM）中蛋白质表达的影响，该细胞系显示出MPA对增殖的浓度依赖性抑制作用，以深入了解MPA对细胞蛋白质组的影响。用佛波酯/离子霉素刺激细胞，并在有或无MPA的情况下孵育。二维电泳和密度成像显示，MPA处理后有11个差异表达的蛋白质斑点（P<0.05），6个丰度增加，5个丰度降低。经胶内胰蛋白酶消化后，通过四极杆飞行时间质谱鉴定蛋白质。MPA处理后丰度增加的蛋白质包括富含精氨酸/丝氨酸的剪接因子2、前列腺素E合酶3、肽基脯氨酰顺反异构酶A和脱氧尿苷5'-三磷酸核苷酸水解酶。内质蛋白、增殖细胞核抗原、富含酸性亮氨酸的核磷蛋白32家族成员A和丝切蛋白1在MPA处理后丰度降低。三个单独的斑点（1个丰度降低，2个丰度增加）被鉴定为Rho鸟苷二磷酸解离抑制剂2（Rho GDI 2）蛋白。用针对被半胱天冬酶3切割的Rho GDI 2位点的单克隆抗体进行蛋白质印迹分析表明，1个丰度增加的斑点是缺少前19个氨基酸的Rho GDI 2的半胱天冬酶3切割产物。Rho GDI 2在Rho鸟苷三磷酸酶的激活中起核心调节作用，Rho鸟苷三磷酸酶在影响细胞细胞骨架动力学和运动性的细胞信号通路中起分子开关的作用。我们的数据表明，MPA可以调节T淋巴细胞中Rho GDI 2的水平，从而可能破坏对T细胞功能重要的细胞信号通路。
