Biochemical Features of Recombinant Human Cyclophilin J

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.

Keywords: CYPJ; Cyclophilin J; PPIL3; PPIase activity; biochemistry.