Protein engineering to develop a redox insensitive endothelial nitric oxide synthase

Ruslan Rafikov, Sanjiv Kumar, Saurabh Aggarwal, Daniel Pardo, Fabio V. Fonseca, Jessica Ransom, Olga Rafikova, Qiumei Chen, Matthew L. Springer, Stephen Matthew Black

Research output: Contribution to journalArticle

3 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)156-164
Number of pages9
JournalRedox Biology
Volume2
Issue number1
DOIs
StatePublished - Jan 1 2014

Fingerprint

Protein Engineering
Nitric Oxide Synthase Type III
Oxidation-Reduction
Dimers
Endothelial cells
Proteins
Cysteine
Endothelial Cells
Mutagenesis
Oxidative stress
Mutant Proteins
Site-Directed Mutagenesis
Oxidants
Workplace
Hydrogen Peroxide
Vascular Endothelial Growth Factor A
Arginine
Monolayers
Assays
Oxidative Stress

Keywords

  • Endothelial nitric oxide synthase
  • Protein engineering
  • Redox stability
  • Zinc tetrathiolate cluster

ASJC Scopus subject areas

  • Biochemistry
  • Organic Chemistry

Cite this

Rafikov, R., Kumar, S., Aggarwal, S., Pardo, D., Fonseca, F. V., Ransom, J., ... Black, S. M. (2014). Protein engineering to develop a redox insensitive endothelial nitric oxide synthase. Redox Biology, 2(1), 156-164. https://doi.org/10.1016/j.redox.2013.12.015

Protein engineering to develop a redox insensitive endothelial nitric oxide synthase. / Rafikov, Ruslan; Kumar, Sanjiv; Aggarwal, Saurabh; Pardo, Daniel; Fonseca, Fabio V.; Ransom, Jessica; Rafikova, Olga; Chen, Qiumei; Springer, Matthew L.; Black, Stephen Matthew.

In: Redox Biology, Vol. 2, No. 1, 01.01.2014, p. 156-164.

Research output: Contribution to journalArticle

Rafikov, R, Kumar, S, Aggarwal, S, Pardo, D, Fonseca, FV, Ransom, J, Rafikova, O, Chen, Q, Springer, ML & Black, SM 2014, 'Protein engineering to develop a redox insensitive endothelial nitric oxide synthase', Redox Biology, vol. 2, no. 1, pp. 156-164. https://doi.org/10.1016/j.redox.2013.12.015
Rafikov, Ruslan ; Kumar, Sanjiv ; Aggarwal, Saurabh ; Pardo, Daniel ; Fonseca, Fabio V. ; Ransom, Jessica ; Rafikova, Olga ; Chen, Qiumei ; Springer, Matthew L. ; Black, Stephen Matthew. / Protein engineering to develop a redox insensitive endothelial nitric oxide synthase. In: Redox Biology. 2014 ; Vol. 2, No. 1. pp. 156-164.
@article{520057f044814f728422ba10f4463ce0,
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.",
keywords = "Endothelial nitric oxide synthase, Protein engineering, Redox stability, Zinc tetrathiolate cluster",
author = "Ruslan Rafikov and Sanjiv Kumar and Saurabh Aggarwal and Daniel Pardo and Fonseca, {Fabio V.} and Jessica Ransom and Olga Rafikova and Qiumei Chen and Springer, {Matthew L.} and Black, {Stephen Matthew}",
year = "2014",
month = "1",
day = "1",
doi = "10.1016/j.redox.2013.12.015",
language = "English (US)",
volume = "2",
pages = "156--164",
journal = "Redox Biology",
issn = "2213-2317",
publisher = "Elsevier BV",
number = "1",

}

TY - JOUR

T1 - Protein engineering to develop a redox insensitive endothelial nitric oxide synthase

AU - Rafikov, Ruslan

AU - Kumar, Sanjiv

AU - Aggarwal, Saurabh

AU - Pardo, Daniel

AU - Fonseca, Fabio V.

AU - Ransom, Jessica

AU - Rafikova, Olga

AU - Chen, Qiumei

AU - Springer, Matthew L.

AU - Black, Stephen Matthew

PY - 2014/1/1

Y1 - 2014/1/1

N2 - 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.

AB - 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.

KW - Endothelial nitric oxide synthase

KW - Protein engineering

KW - Redox stability

KW - Zinc tetrathiolate cluster

UR - http://www.scopus.com/inward/record.url?scp=84892866489&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84892866489&partnerID=8YFLogxK

U2 - 10.1016/j.redox.2013.12.015

DO - 10.1016/j.redox.2013.12.015

M3 - Article

VL - 2

SP - 156

EP - 164

JO - Redox Biology

JF - Redox Biology

SN - 2213-2317

IS - 1

ER -