Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Proteomics. 2011 Aug;11(16):3359-68. doi: 10.1002/pmic.201100001.
Interactions of microsomal cytochromes P450 (CYPs) with other proteins in the microsomal membrane are important for their function. In addition to their redox partners, CYPs have been reported to interact with other proteins not directly involved in their enzymatic function. In this study, proteins were identified that interact with CYP2C2 in vivo in mouse liver. Flag-tagged CYP2C2 was expressed exogenously in mouse liver and was affinity purified, along with associated proteins which were identified by MS and confirmed by Western blotting. Over 20 proteins reproducibly copurified with CYP2C2. The heterogeneous sedimentation velocity of CYP2C2 and associated proteins by centrifugation in sucrose gradients and sequential immunoprecipitation analysis were consistent with multiple CYP2C2 complexes of differing composition. The abundance of CYPs and other drug metabolizing enzymes and NAD/NADP requiring enzymes associated with CYP2C2 suggest that complexes of these proteins may improve enzymatic efficiency or facilitate sequential metabolic steps. Chaperones, which may be important for maintaining CYP function, and reticulons, endoplasmic reticulum proteins that shape the morphology of the endoplasmic reticulum and are potential endoplasmic reticulum retention proteins for CYPs, were also associated with CYP2C2.
微粒体细胞色素 P450（CYPs）与微粒体膜中其他蛋白质的相互作用对于它们的功能很重要。除了其氧化还原伴侣外，据报道 CYP 还与其他不直接参与其酶功能的蛋白质相互作用。在这项研究中，鉴定了在小鼠肝脏中与 CYP2C2 体内相互作用的蛋白质。在小鼠肝脏中外源性表达了带有 Flag 标签的 CYP2C2，并通过 MS 鉴定和 Western blot 验证，对与其相关的蛋白进行了亲和纯化。有超过 20 种蛋白质可重复性地与 CYP2C2 共纯化。通过蔗糖梯度离心和顺序免疫沉淀分析，CYP2C2 和相关蛋白的异质沉降速度与不同组成的多个 CYP2C2 复合物一致。与 CYP2C2 相关的 CYP 和其他药物代谢酶以及 NAD/NADP 所需酶的丰度表明，这些蛋白质的复合物可能会提高酶的效率或促进连续的代谢步骤。伴侣蛋白对于维持 CYP 功能很重要，内质网蛋白 reticulons 也与 CYP2C2 相关，它可以塑造内质网的形态，并且可能是 CYP 的内质网保留蛋白。
