Department of Pharmaceutics and Medicinal Chemistry, Thomas J. Long School of Pharmacy and Health Sciences, University of the Pacific, Stockton, CA 95211, USA.
Anal Biochem. 2011 Mar 15;410(2):257-65. doi: 10.1016/j.ab.2010.12.007. Epub 2010 Dec 10.
Cyclophilin-40 (CyP40) is part of the immunophilin family and is found in Hsp90-containing protein complexes. We were interested in identifying proteins that interact with CyP40. CyP40-interacting proteins in HeLa cells were identified using the tandem affinity purification approach. Adenovirus expressing human CyP40 protein (Ad-CyP40), fused with streptavidin and calmodulin binding peptides at the N terminus, was generated. Proteins were separated on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel after tandem affinity purification. Here 10 silver-stained protein bands that were enriched in the Ad-CyP40-infected lysate and the corresponding regions in the control lysate were excised, digested by trypsin, and identified by tandem mass spectrometric analysis. Of 11 interacting proteins that were identified, 4 (RACK1, Ku70, RPS3, and NF45) were expressed in rabbit reticulocyte lysate, bacteria, and MCF-7 cells. We confirmed that these proteins interact with CyP40. We observed that RACK1 suppressed the cobalt chloride-induced, hypoxia response element-dependent luciferase activity in MCF-7 cells but not in MCF-7 stable cells expressing approximately 10% of the cellular CyP40 content. In addition, RACK1 reduced the HIF-1α protein accumulation after cobalt chloride treatment, which was not observed when the CyP40 content was down-regulated. Collectively, we conclude that reduction of the HIF-1 α protein by RACK1 is CyP40-mediated.
亲环蛋白 40（CyP40）是免疫亲和素家族的一部分，存在于含有热休克蛋白 90 的蛋白复合物中。我们有兴趣确定与 CyP40 相互作用的蛋白质。使用串联亲和纯化方法鉴定 HeLa 细胞中的 CyP40 相互作用蛋白。生成了表达人 CyP40 蛋白（Ad-CyP40）的腺病毒，该蛋白在 N 端融合了链亲和素和钙调蛋白结合肽。在串联亲和纯化后，将蛋白质在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳凝胶上分离。在此，在 Ad-CyP40 感染的裂解物中富集的 10 个银染蛋白条带和对照裂解物中的相应区域被切除，用胰蛋白酶消化，并通过串联质谱分析鉴定。在鉴定的 11 个相互作用蛋白中，有 4 个（RACK1、Ku70、RPS3 和 NF45）在兔网织红细胞裂解物、细菌和 MCF-7 细胞中表达。我们证实这些蛋白质与 CyP40 相互作用。我们观察到 RACK1 抑制了 MCF-7 细胞中钴诱导的缺氧反应元件依赖性荧光素酶活性，但在表达约 10%细胞 CyP40 含量的 MCF-7 稳定细胞中则没有。此外，在下调 CyP40 含量时，RACK1 减少了钴处理后 HIF-1α 蛋白的积累。综上所述，我们得出结论，RACK1 通过 CyP40 介导减少 HIF-1α 蛋白。
