title: Characterization of Peptidyl-Prolyl Cis-Trans Isomerase- and Calmodulin-Binding Activity of a Cytosolic Arabidopsis thaliana Cyclophilin AtCyp19-3.
abstract: Cyclophilins, which bind to immunosuppressant cyclosporin A (CsA), are ubiquitous proteins and constitute a multigene family in higher organisms. Several members of this family are reported to catalyze cis-trans isomerisation of the peptidyl-prolyl bond, which is a rate limiting step in protein folding. The physiological role of these proteins in plants, with few exceptions, is still a matter of speculation. Although Arabidopsis genome is predicted to contain 35 cyclophilin genes, biochemical characterization, imperative for understanding their cellular function(s), has been carried only for few of the members. The present study reports the biochemical characterization of an Arabidopsis cyclophilin, AtCyp19-3, which demonstrated that this protein is enzymatically active and possesses peptidyl-prolyl cis-trans isomerase (PPIase) activity that is specifically inhibited by CsA with an inhibition constant (Ki) of 18.75 nM. The PPIase activity of AtCyp19-3 was also sensitive to Cu(2+), which covalently reacts with the sulfhydryl groups, implying redox regulation. Further, using calmodulin (CaM) gel overlay assays it was demonstrated that in vitro interaction of AtCyp19-3 with CaM is Ca(2+)-dependent, and CaM-binding domain is localized to 35-70 amino acid residues in the N-terminus. Bimolecular fluorescence complementation assays showed that AtCyp19-3 interacts with CaM in vivo also, thus, validating the in vitro observations. However, the PPIase activity of the Arabidopsis cyclophilin was not affected by CaM. The implications of these findings are discussed in the context of Ca(2+) signaling and cyclophilin activity in Arabidopsis.
authors: Department of Biotechnology, Guru Nanak Dev University, Amritsar-143005, Punjab, India.
authors: Gundeep
authors: G
authors: Kaur
authors: Department of Biotechnology, Guru Nanak Dev University, Amritsar-143005, Punjab, India.
authors: Supreet
authors: S
authors: Singh
authors: Department of Biotechnology, Guru Nanak Dev University, Amritsar-143005, Punjab, India; Department of Bioinformatics, Hans Raj Mahila Maha Vidayalaya, Jalandhar, Punjab, India.
authors: Harpreet
authors: H
authors: Singh
authors: Interdiscipinary Center for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi, India.
authors: Mrinalini
authors: M
authors: Chawla
authors: Department of Biotechnology, Guru Nanak Dev University, Amritsar-143005, Punjab, India.
authors: Tanima
authors: T
authors: Dutta
authors: Department of Biotechnology, Guru Nanak Dev University, Amritsar-143005, Punjab, India.
authors: Harsimran
authors: H
authors: Kaur
authors: Department of Biology, Queen's University, Kingston, Ontario, Canada.
authors: Kyle
authors: K
authors: Bender
authors: Department of Biology, Queen's University, Kingston, Ontario, Canada.
authors: W A
authors: WA
authors: Snedden
authors: Interdiscipinary Center for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi, India.
authors: Sanjay
authors: S
authors: Kapoor
authors: Stress Physiology and Molecular Biology Laboratory, School of Life Sciences, Jawaharlal, Nehru University, New Delhi, India.
authors: Ashwani
authors: A
authors: Pareek
authors: Department of Biotechnology, Guru Nanak Dev University, Amritsar-143005, Punjab, India.
authors: Prabhjeet
authors: P
authors: Singh
mesh_terms: D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D003300:Copper / Q000494:pharmacology; D021983:Cyclophilins / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary
chemical_list: D029681:Arabidopsis Proteins; D002147:Calmodulin; D003300:Copper; D021983:Cyclophilins