Nitric oxide co-ordinates early defence responses through mitochondrial regulation

Lead Research Organisation: University of Sheffield
Department Name: Molecular Biology and Biotechnology

Abstract

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Technical Summary

Nitric oxide (NO) plays an important role as an inter- and intra-cellular messenger, acting in the cardiovascular system, in the nervous system and as a component of the immune system. NO is a diatomic free radical which is a gas at room temperature, making it highly diffusible within the vasculature. One of its major targets is the soluble guanylyl cyclase (sGC). The binding of NO to Fe2+ in the haem group of sGC activates the enzyme and increases the concentration of cGMP (guanosine 3?, 5?-cyclic monophosphate). For many years we have focused on NO/cGMP signalling and the importance of this pathway to a wide range of biological systems, including blood pressure regulation, platelet aggregation, smooth muscle relaxation and peripheral and central neurotransmission. More recently we have been involved in understanding how NO can signal via a completely different pathway that has equally important pleiotropic biological consequences. We have shown that NO, in the nanomolar concentration range that activates sGC, can also bind to the mitochondrial enzyme cytochrome c oxidase (complex IV) and inhibit it in a manner that is reversible and in competition with oxygen. This inhibition can prevent the enzyme from using any available oxygen, resulting in what we have termed ?metabolic hypoxia?. We aim to further our understanding of how this NO-mediated inhibition of cellular respiration results in signalling to transduction pathways that ultimately control the fate of the cell. In order to do this we have developed a series of biochemical techniques, cell lines and reagents that permit the highly regulated generation of NO in a dose- and time?dependent manner.
 
Description From Targets to Novel Drugs
Amount £43,100 (GBP)
Organisation Bayer 
Sector Private
Country Germany
Start 09/2009 
End 08/2011
 
Title Overexpress S127R-PCSK9 stable cell line 
Description we generated a human T-Rex-293 stable cell line that overexpressed Flag-tagged S127R-PCSK9. 
Type Of Material Cell line 
Provided To Others? No  
Impact It is very useful for study mutation form S127R-PCSK9 function and purify protein 
 
Title Overexpressed D374Y-PCSk9 stable cell line 
Description We generated a human-T-Rex-293 stable cell line that overexpressed Flag-tagged D374Y mutation. 
Type Of Material Cell line 
Provided To Others? No  
Impact It is very useful for study mutation form D374Y-PCSK9 and purify protein 
 
Title Overexpressed F216L-PCSK9 stable cell line 
Description we generated a human T-Rex-293 stable cell line that overexpressed FLAG-tagged F216L-PCSK9 
Type Of Material Cell line 
Provided To Others? No  
Impact It is very useful to study mutation form F216L-PCSK9 function and purify protein 
 
Title Wild-type PCSK9 overexpressing stable cell line 
Description We have generated a human-T-Rex-293 stable cell line that over-expressed Flag-tagged wild-typ PCSK9 
Type Of Material Cell line 
Provided To Others? No  
Impact It can be used to study PCSK9 function and purify PCSK9 protein 
 
Title anti-GRP78 scFv antibody 
Description Specific for GRP78 protein, potentially for caner diagnosis and treatment 
Type Of Material Antibody 
Provided To Others? No  
Impact This is a potentially therapeutic humanized antibody fragment, targeting cancer cell surface. It is subjected to ongoing patent application 
 
Description Develop of novel inhibitors of PCSK9 for treatment of hypercholesterolaemia 
Organisation MRC-Technology
Country United Kingdom 
Sector Academic/University 
PI Contribution Our group has identified a novel pathway to regulate cholesterol uptake by regulating PCSK9 and set up the screening assay. MRC Technology supplied us the NINDS compounds library. The high-throughput screening facility is provided by the Dept of Cardiovascular Science, University of Sheffield
Collaborator Contribution The MRCT provide us the NINDS compounds library, a collection of known drugs and pharmcologically activi compounds. The Department provide high-throughput screening facility and experienced HTS expert to help us screening compounds library.
Impact We have just started to optimizing the HTS screening conditions and will produce results soon.
Start Year 2010
 
Description Develop of novel inhibitors of PCSK9 for treatment of hypercholesterolaemia 
Organisation University of Sheffield
Department Department of Cardiovascular Science
Country United Kingdom 
Sector Academic/University 
PI Contribution Our group has identified a novel pathway to regulate cholesterol uptake by regulating PCSK9 and set up the screening assay. MRC Technology supplied us the NINDS compounds library. The high-throughput screening facility is provided by the Dept of Cardiovascular Science, University of Sheffield
Collaborator Contribution The MRCT provide us the NINDS compounds library, a collection of known drugs and pharmcologically activi compounds. The Department provide high-throughput screening facility and experienced HTS expert to help us screening compounds library.
Impact We have just started to optimizing the HTS screening conditions and will produce results soon.
Start Year 2010
 
Description Targeting GRP78 for cancer diagnosis and treatment 
Organisation Bayer
Country Germany 
Sector Private 
PI Contribution We have identified that nitric oxide upregulates GRP78 protein through coupling ER stress and mitochondria respiration. As GRP78 is a good target for cancer diagnosis and treatment, we have developed specific antibody. University of Newcastle has provided us recombinant protein. We have carried out screening phage library and isolated antibody has been used to in nude mice animal model(a grant supported by Bayer Healthcare and in situ hybridization for breast cancer detection(Patholgy analysis has been helped by UCL, Division of Surgery.
Collaborator Contribution Purify functional proteinProfessor Nicola Brown is helping us to develop animal modelDr. Marilena Loizidou is helping us to analyze the breast cancer in situ hybridization results.
Impact The collaboration with university of Newcastle has been successful, resulted in a publication in JBC, PMID 16418174. The animal model is running and will produce results soon
Start Year 2006
 
Description Targeting GRP78 for cancer diagnosis and treatment 
Organisation Newcastle University
Department Institute for Cell and Molecular Biosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We have identified that nitric oxide upregulates GRP78 protein through coupling ER stress and mitochondria respiration. As GRP78 is a good target for cancer diagnosis and treatment, we have developed specific antibody. University of Newcastle has provided us recombinant protein. We have carried out screening phage library and isolated antibody has been used to in nude mice animal model(a grant supported by Bayer Healthcare and in situ hybridization for breast cancer detection(Patholgy analysis has been helped by UCL, Division of Surgery.
Collaborator Contribution Purify functional proteinProfessor Nicola Brown is helping us to develop animal modelDr. Marilena Loizidou is helping us to analyze the breast cancer in situ hybridization results.
Impact The collaboration with university of Newcastle has been successful, resulted in a publication in JBC, PMID 16418174. The animal model is running and will produce results soon
Start Year 2006
 
Description Targeting GRP78 for cancer diagnosis and treatment 
Organisation University College London
Department Division of Surgery & Interventional Science
Country United Kingdom 
Sector Academic/University 
PI Contribution We have identified that nitric oxide upregulates GRP78 protein through coupling ER stress and mitochondria respiration. As GRP78 is a good target for cancer diagnosis and treatment, we have developed specific antibody. University of Newcastle has provided us recombinant protein. We have carried out screening phage library and isolated antibody has been used to in nude mice animal model(a grant supported by Bayer Healthcare and in situ hybridization for breast cancer detection(Patholgy analysis has been helped by UCL, Division of Surgery.
Collaborator Contribution Purify functional proteinProfessor Nicola Brown is helping us to develop animal modelDr. Marilena Loizidou is helping us to analyze the breast cancer in situ hybridization results.
Impact The collaboration with university of Newcastle has been successful, resulted in a publication in JBC, PMID 16418174. The animal model is running and will produce results soon
Start Year 2006
 
Description Targeting GRP78 for cancer diagnosis and treatment 
Organisation University of Sheffield
Department Department of Oncology and Metabolism
Country United Kingdom 
Sector Academic/University 
PI Contribution We have identified that nitric oxide upregulates GRP78 protein through coupling ER stress and mitochondria respiration. As GRP78 is a good target for cancer diagnosis and treatment, we have developed specific antibody. University of Newcastle has provided us recombinant protein. We have carried out screening phage library and isolated antibody has been used to in nude mice animal model(a grant supported by Bayer Healthcare and in situ hybridization for breast cancer detection(Patholgy analysis has been helped by UCL, Division of Surgery.
Collaborator Contribution Purify functional proteinProfessor Nicola Brown is helping us to develop animal modelDr. Marilena Loizidou is helping us to analyze the breast cancer in situ hybridization results.
Impact The collaboration with university of Newcastle has been successful, resulted in a publication in JBC, PMID 16418174. The animal model is running and will produce results soon
Start Year 2006