Bokoch G M, Prossnitz V
Department of Immunology, Research Institute of Scripps Clinic, La Jolla, California 92037.
J Clin Invest. 1992 Feb;89(2):402-8. doi: 10.1172/JCI115599.
The formation of oxygen radicals by phagocytic cells occurs through the activation of a multiple-component NADPH oxidase system. An unidentified low molecular weight GTP-binding protein has been proposed to modulate the activity of the NADPH oxidase. The low molecular weight GTP-binding proteins undergo posttranslational processing, including an initial covalent incorporation of an isoprenyl group. To test whether such an isoprenylation reaction might be required for the activity of the oxidase, we utilized compactin and lovastatin as inhibitors of the isoprenylation pathway. Treatment of DMSO-differentiated HL-60 cells with compactin produced a concentration-dependent inhibition of O2- formation in response to FMLP or phorbol myristate acetate. Cell viability was not affected nor was normal differentiation of the HL-60 cells into a neutrophil-like cell. The inhibitory effect of compactin was specifically prevented by addition of exogenous mevalonic acid to the HL-60 cells, indicating that the inhibitory effects of the drug were due to blockade of the pathway leading to isoprenoid synthesis. Addition of cholesterol, ubiquinone, or dolichol, which are also downstream products of the isoprenoid pathway, did not override the inhibitory effects of the drug. Subcellular fractions were prepared from compactin-treated cells, and the location of the compactin-sensitive factor was determined by complementation analysis in a cell-free NADPH oxidase system. The inhibited factor was localized to the HL-60 cytosol. These data suggest that an isoprenoid pathway intermediate is necessary for activation of the phagocyte NADPH oxidase. This is likely to represent the requirement for an isoprenoid moiety in the posttranslational modification of a low molecular weight GTP-binding protein. Our studies provide support for the involvement of such a low molecular weight GTP-binding protein in NADPH oxidase activation.
吞噬细胞中氧自由基的形成是通过多组分NADPH氧化酶系统的激活而发生的。一种未鉴定的低分子量GTP结合蛋白被认为可调节NADPH氧化酶的活性。低分子量GTP结合蛋白会经历翻译后加工，包括异戊二烯基的初始共价掺入。为了测试这种异戊二烯化反应是否可能是氧化酶活性所必需的，我们使用洛伐他汀和辛伐他汀作为异戊二烯化途径的抑制剂。用辛伐他汀处理二甲基亚砜分化的HL-60细胞，可产生对FMLP或佛波酯肉豆蔻酸酯刺激的O2-形成的浓度依赖性抑制。细胞活力未受影响，HL-60细胞向中性粒细胞样细胞的正常分化也未受影响。向HL-60细胞中添加外源性甲羟戊酸可特异性地阻止辛伐他汀的抑制作用，表明该药物的抑制作用是由于阻断了导致类异戊二烯合成的途径。添加类异戊二烯途径的下游产物胆固醇、泛醌或多萜醇并不能克服该药物的抑制作用。从辛伐他汀处理的细胞中制备亚细胞组分，并通过无细胞NADPH氧化酶系统中的互补分析确定辛伐他汀敏感因子的位置。受抑制的因子定位于HL-60细胞质溶胶中。这些数据表明类异戊二烯途径中间体是吞噬细胞NADPH氧化酶激活所必需的。这可能代表了低分子量GTP结合蛋白翻译后修饰中对类异戊二烯部分的需求。我们的研究为这种低分子量GTP结合蛋白参与NADPH氧化酶激活提供了支持。
