重组人亲环素J的生化特性

Biochemical Features of Recombinant Human Cyclophilin J.

作者信息

Chen Jian, Liefke Robert, Jiang Lixin, Wang Jiahui, Huang Chaoqun, Gong Zhaohua, Schiene-Fischer Cordelia, Yu Long

机构信息

Department of Oncology, Yantai Yuhuangding Hospital, Affiliated Hospital of Qingdao University, Yantai, Shandong, P.R. China State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, P.R. China.

MPG-Research-Unit Enzymology of Protein Folding, Halle/Saale, Germany.

出版信息

Anticancer Res. 2016 Mar;36(3):1175-80.

PMID:26977013

Abstract

AIM

To characterize the biochemical features of the newest member of cyclophilin family of peptidyl-prolyl cis/trans-isomerases (PPIases), cyclophilin J (CYPJ).

MATERIALS AND METHODS

PPIase assays were performed on purified hCYPJ and its mutated variants. The substrate specificity, half-maximal inhibitory concentration (IC50) of cyclosporin A (CsA) inhibition and circular dichroism (CD) spectrum of CYPJ were measured. Mercury pathway profiling luciferase assays were also performed.

RESULTS

The catalytic number/Michaelis constant (kcat/KM) value of CYPJ was 9.5×10(4) s(-1)M(-1). CYPJ additionally catalyzed norleucine-proline, isoleucine-proline and glutamine-proline peptides compared to CYPA and Escherichia coli PPIases. CYPJ was inhibited by CsA in a dose-dependent manner with IC50 of 12.1±0.9 μM. The CD spectrum of CYPJ was similar to CYPA. CYPJ significantly up-regulated the transcription of E-box, E2F, retinoblastoma (Rb), p53, activator protein 1 (AP1), NF-κB and phospho-cAMP response element (CRE) cis-response element in 293T cells.

CONCLUSION

CYPJ structurally resembles CYPA. It is sensitive to inhibition by CsA and plays a role in regulating cell growth, proliferation, and apoptosis.

摘要

目的

表征肽基脯氨酰顺/反异构酶（PPIase）亲环蛋白家族最新成员亲环蛋白J（CYPJ）的生化特性。

材料与方法

对纯化的人CYPJ及其突变变体进行PPIase测定。测量了CYPJ的底物特异性、环孢素A（CsA）抑制的半数最大抑制浓度（IC50）和圆二色性（CD）光谱。还进行了汞途径分析荧光素酶测定。

结果

CYPJ的催化常数/米氏常数（kcat/KM）值为9.5×10⁴ s⁻¹M⁻¹。与亲环蛋白A（CYPA）和大肠杆菌PPIase相比，CYPJ还催化了正亮氨酸-脯氨酸、异亮氨酸-脯氨酸和谷氨酰胺-脯氨酸肽。CYPJ被CsA以剂量依赖性方式抑制，IC50为12.1±0.9 μM。CYPJ的CD光谱与CYPA相似。CYPJ显著上调了293T细胞中E盒、E2F、视网膜母细胞瘤（Rb）、p53、活化蛋白1（AP1）、核因子κB（NF-κB）和磷酸化环磷酸腺苷反应元件（CRE）顺式反应元件的转录。