title: Protein engineering to develop a redox insensitive endothelial nitric oxide synthase.
abstract: The zinc tetrathiolate (ZnS4) cluster is an important structural feature of endothelial nitric oxide synthase (eNOS). The cluster is located on the dimeric interface and four cysteine residues (C94 and C99 from two adjacent subunits) form a cluster with a Zn ion in the center of a tetrahedral configuration. Due to its high sensitivity to oxidants this cluster is responsible for eNOS dimer destabilization during periods of redox stress. In this work we utilized site directed mutagenesis to replace the redox sensitive cysteine residues in the ZnS4 cluster with redox stable tetra-arginines. Our data indicate that this C94R/C99R eNOS mutant is active. In addition, this mutant protein is insensitive to dimer disruption and inhibition when challenged with hydrogen peroxide (H2O2). Further, the overexpression of the C94R/C99R mutant preserved the angiogenic response in endothelial cells challenged with H2O2. The over-expression of the C94R/C99R mutant preserved the ability of endothelial cells to migrate towards vascular endothelial growth factor (VEGF) and preserved the endothelial monolayer in a scratch wound assay. We propose that this dimer stable eNOS mutant could be utilized in the treatment of diseases in which there is eNOS dysfunction due to high levels of oxidative stress.
authors.affiliation: Pulmonary Vascular Disease, Vascular Biology Center, Georgia Regents University, Augusta, GA, USA.
authors.forename: Ruslan
authors.initials: R
authors.lastname: Rafikov
authors.affiliation: Pulmonary Vascular Disease, Vascular Biology Center, Georgia Regents University, Augusta, GA, USA.
authors.forename: Sanjiv
authors.initials: S
authors.lastname: Kumar
authors.affiliation: Pulmonary Vascular Disease, Vascular Biology Center, Georgia Regents University, Augusta, GA, USA.
authors.forename: Saurabh
authors.initials: S
authors.lastname: Aggarwal
authors.affiliation: Pulmonary Vascular Disease, Vascular Biology Center, Georgia Regents University, Augusta, GA, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Pardo
authors.affiliation: Pulmonary Vascular Disease, Vascular Biology Center, Georgia Regents University, Augusta, GA, USA.
authors.forename: Fabio V
authors.initials: FV
authors.lastname: Fonseca
authors.affiliation: Pulmonary Vascular Disease, Vascular Biology Center, Georgia Regents University, Augusta, GA, USA.
authors.forename: Jessica
authors.initials: J
authors.lastname: Ransom
authors.affiliation: Pulmonary Vascular Disease, Vascular Biology Center, Georgia Regents University, Augusta, GA, USA.
authors.forename: Olga
authors.initials: O
authors.lastname: Rafikova
authors.affiliation: The Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA, USA.
authors.forename: Qiumei
authors.initials: Q
authors.lastname: Chen
authors.affiliation: The Division of Cardiology, University of California, San Francisco, San Francisco, CA, USA.
authors.forename: Matthew L
authors.initials: ML
authors.lastname: Springer
authors.affiliation: Pulmonary Vascular Disease, Vascular Biology Center, Georgia Regents University, Augusta, GA, USA. Electronic address: sblack@gru.edu.
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Black
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry*; D020125:Mutation, Missense; D052250:Nitric Oxide Synthase Type III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D006861:Hydrogen Peroxide; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III
keywords: Endothelial nitric oxide synthase; Protein engineering; Redox stability; Zinc tetrathiolate cluster