Oxidant-induced activation of protein kinase A: from biochemistry to the cardiovascular physiology of a novel 'redox-dead' knock-in mouse

Lead Research Organisation: King's College London
Department Name: Cardiovascular

Abstract

Free radicals and oxidising chemicals are produced in cells and tissues. Historically these have been primarily associated with disease or dysfunction. However, over the past decade there has been a growing realisation that these species are not always harmful, and in fact they can play important regulatory roles. We have found in preliminary studies that a protein (known as protein kinase A), which is very important for how the heart is regulated is chemically modified by oxidants and this directly activates it. Here we outline studies that will help fully define the importance of this modification and activation of protein kinase A, both in health and in disease. We will utilise many experimental approaches in this work, including generation of a genetically modified mouse that has been altered so that it cannot be oxidised and directly activated by oxidants. The availability of such mice would be an invaluable resource that will allow us to study fully the importance of oxidant-induced protein kinase A activation in cells, tissues, organs and the animal as a whole.

Technical Summary

We have recently found that Type I PKA is subject to reversible cysteine-targeted oxidation, a modification that regulates the activity of this kinase. This oxidative cysteine modification involves a disulfide bond forming between the two regulatory (RIalpha) subunits in the Type I PKA holoenzyme. In this application, which is aimed at further understanding the significance of this oxidative modification of PKA, we outline two primary lines of investigation. (1) A program of experiments that will further characterise the fundamental nature of this mode of kinase activation. (2) A characterisation of a novel ?redox-dead? knock-in mouse line we have generated, in which wild-type RI is replaced with a mutant protein lacking the cysteines required for kinase disulfide bond formation. This Cys17Ser RI PKA mouse is a unique model and will allow us to understand the importance of PKA RI oxidation in health and disease.

Publications

10 25 50
 
Description British Heart Foundation Novel approaches to understanding oxidant sensing and signalling in the cardiovascular system
Amount £193,642 (GBP)
Funding ID FS/11/45/28859 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2012 
End 07/2015
 
Description Disulfide activated protein kinase G Ialpha as a new therapeutic target in sepsis
Amount £502,927 (GBP)
Funding ID MR/L009684/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 03/2014 
End 02/2018
 
Description Does redox state of PKG1alpha control hypoxic vasoconstriction and remodelling in the pulmonary vasculature?
Amount £381,870 (GBP)
Funding ID FS/14/57/31138 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2015 
End 03/2019
 
Description Does redox state of PKG1alpha control hypoxic vasoconstriction and remodelling in the pulmonary vasculature?
Amount £381,870 (GBP)
Funding ID FS/14/57/31138 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2015 
End 03/2019
 
Description Does redox state of PKG1alpha control hypoxic vasoconstriction and remodelling in the pulmonary vasculature?
Amount £381,870 (GBP)
Funding ID FS/14/57/31138 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2015 
End 03/2019
 
Description ERC Advanced - CARDIOREDOX
Amount € 2,255,659 (EUR)
Funding ID 339095 
Organisation European Research Council (ERC) 
Sector Public
Country Belgium
Start 12/2013 
End 11/2018
 
Description Expanding our understanding of the redox regulation of soluble Epoxide Hydrolase in cardiovascular health and disease
Amount £253,143 (GBP)
Funding ID PG/15/26/31373 
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2015 
End 03/2018
 
Description Fondation Leducq, Transatlantic Network of Excellence
Amount £300,000 (GBP)
Organisation Transatlantic Networks of Excellence in Cardiovascular Research Program 
Sector Charity/Non Profit
Country France
Start 01/2010 
End 12/2013
 
Description MRC project grant
Amount £502,927 (GBP)
Funding ID MR/L009684/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 02/2014 
End 01/2018
 
Description Protein kinase GIa disulfide oxidation
Amount £151,405 (GBP)
Funding ID MR/K003232/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2012 
End 09/2013
 
Title Cys17Ser RI PKA redox-dead mouse 
Description Novel mouse line that contains PKA protein that is 'redox-dead' 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2011 
Provided To Others? Yes  
Impact work in progress 
 
Title PEG-switch 
Description Allso reversible proetin oxidation in candidate proteins to be monitored 
Type Of Material Technology assay or reagent 
Year Produced 2013 
Provided To Others? Yes  
Impact People can more easily and routeinely monitor protein thiol redox state 
 
Title anti-sulforaphane protein adduct antibody 
Description Allows sulforaphane-modified proteins to be monitored and identified. 
Type Of Material Antibody 
Year Produced 2013 
Provided To Others? Yes  
Impact Has allowed novel targets of sulforaphane to be identified. 
 
Description KCL BHF Centre 
Organisation King's College London
Department BHF Centre of Research Excellence
Country United Kingdom 
Sector Academic/University 
PI Contribution The KCL BHF centre of Resaerch Excellence has provided us funding as part of their mission. Our research using this funding has aided this mission.
Collaborator Contribution They provided funds and intelelctuall support.
Impact Helped gain pilot data for follow-on funding
Start Year 2010
 
Description Media interest 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact After we published a paper titled: Protection from hypertension in mice by the Mediterranean diet is mediated by nitro fatty acid inhibition of soluble epoxide hydrolase, we had a lot of press interest and phonecalls with jouralsits froma round the work. Here are a very few examples of the news agencies that cited the work.

http://www.dailymail.co.uk/health/article-2632932/Tossing-lettuce-olive-oil-sprinkling-nuts-avocado-boosts-heart-health.html

http://www.bbc.co.uk/news/health-27470115

http://www.express.co.uk/news/health/476945/Why-a-Mediterranean-diet-is-good-for-your-health

http://www.telegraph.co.uk/health/healthnews/10840283/Olive-oil-on-salad-may-save-your-life.html

http://www.independent.co.uk/life-style/health-and-families/health-news/why-is-a-mediterranean-diet-so-good-for-you-scientists-think-they-know-9400889.html


Apart from substantive interest from all types of news agencies, we even had members of the public get in contact to discuss the findings.
Year(s) Of Engagement Activity 2014
URL http://www.bbc.co.uk/news/health-27470115