Basic understanding of how components of the enzyme NADPH oxidase work together to help kill microbes is fundamental to developing new drug treatments for CGD. The CGD Society has funded over £343,000 worth of projects led by world-renowned scientists. The work has resulted in 13 publications.
Here we summarise the grants we have funded and their outcomes.
Synthesis of the enzyme affected in CGD
Grant awarded to: Professor Marie-José Stasia and Professor Marie-Claire Dagher, Université Joseph Fourier, Grenoble, France
Amount: £25,000 over one year ending in 2011
Official title: ‘Xminus chronic granulomatous disease variants, powerful models to study the cytochrome b558 synthesis process in neutrophils’
Aim: To study mutations in a rare form of CGD, collectively known as X minus CGD variants. Patients with this form of CGD have defects in the gp91phox component of NADPH oxidase that leads to them having only partial or no oxidase activity to fight infections.
Outcomes and benefits: Fifteen cell lines were produced from patients with different mutations in the oxidase enzyme subunit gp91phox. The cell lines were generated as resources to examine the structural and functional effects of the different mutations and to improve knowledge of how gp91phox is made and works.
This study may help in the development of a therapy for CGD based on delivery of the CGD enzyme in liposomes.
Publications resulting from this work:
Regulation of NADPH oxidase activity in phagocytes: relationship between FAD/NADPH binding and oxidase complex assembly
Debeurme F, Picciocchi A, Dagher MC, Grunwald D, Beaumel S, Fieschi F, Stasia MJ.
Journal of Biological Chemistry, 2010 Oct 22; 285(43): 33197–208.
http://www.ncbi.nlm.nih.gov/pubmed/20724480Role of putative second transmembrane region of Nox2 protein in the structural stability and electron transfer of the phagocytic NADPH oxidase
Picciocchi A, Debeurme F, Beaumel S, Dagher MC, Grunwald D, Jesaitis AJ, Stasia MJ. Journal of Biological Chemistry, 2011 Aug 12; 286(32): 28357–69.
http://www.ncbi.nlm.nih.gov/pubmed/21659519Rare duplication or deletion of exons 6, 7 and 8 in CYBB leading to X-linked chronic granulomatous disease in two patients from different families
Stasia MJ, van Leeuwen K, de Boer M, Martel C, Mollin M, Thuret I, Michel G, Hanson C, Augustine NH, Coutton C, Satre V, Wittwer CT, Hill H, Roos D.
Journal of Clinical Immunology, 2012 Aug; 32(4): 653–62.
http://www.ncbi.nlm.nih.gov/pubmed/22382877Clinical, functional and genetic analysis of twenty-four patients with chronic granulomatous disease – identification of eight novel mutations in CYBB and NCF2 genes
Martel C, Mollin M, Beaumel S, Brion JP, Coutton C, Satre V, Vieville G, Callanan M, Lefebvre C, Salmon A, Pagnier A, Plantaz D, Bost-Bru C, Eitenschenck L, Durieu I, Floret D, Galambrun C, Chambost H, Michel G, Stephan JL, Hermine O, Blanche S, Blot N, Rubié H, Pouessel G, Drillon-Haus S, Conrad B, Posfay-Barbe KM, Havlicekova Z, Voskresenky-Baricic T, Jadranka K, Arriazu MC, Garcia LA, Sfaihi L, Bordigoni P, Stasia MJ.
Journal of Clinical Immunology, 2012 Oct; 32(5): 942–58.
http://www.ncbi.nlm.nih.gov/pubmed/22562447Mapping the important areas of the enzyme affected in CGD
Grant awarded to: Professor Gary Bokoch and Dr Yu Kao, The Scripps Research Institute, La Jolla, California, USA
Amount: £25,420 over one year ending in 2007
Official title: ‘Regulatory roles of Rac2 GTPase in CGD genetic defects of NADPH oxidase function’
Aim: The oxidase enzyme affected in CGD is a complex molecule made up of five different parts that interact together to help kill bacteria and fungi. This study aimed to find out more about how the complex fits together to form an active complex.
Outcomes and benefits: The project mapped important areas of the oxidase enzyme affected in people with CGD who have the full complement of enzyme parts, but who have mutations that prevent it from working effectively. By finding out that key interactions are missing and involved in activation of the oxidase enzyme, the work gave insights into strategies to help reactivate the enzyme affected in CGD so that it can work properly and fight infections.
Publications resulting from this work:
Identification of a conserved Rac-binding site on NADPH oxidases supports a direct GTPase regulatory mechanism
Kao Y-Y, Gianni D, Bohl B, Taylor RM, Bokoch GM.
Journal of Biological Chemistry, 2008 May 9; 283(19): 12736–46. www.ncbi.nlm.nih.gov/pmc/articles/PMC2442356/?tool=pubmedRegulation of NADPH oxidases by RAC GTPase
Bokoch GM, Gianni D, Kim J-S, Kao Y-Y. Handbook of Cell Signaling (2).Detecting oxidase activity within cells
Grant awarded to: Dr Cees Otto and Professor Dirk Roos, University of Twente, the Netherlands
Amount: £59,000 over one year ending in 2006
Official title: ‘Multidimensional microscopy of defective NADPH oxidase activation in CGD’
Aim: To develop a new technique to measure the precise activity of the oxidase enzyme in single cells.
Outcomes and benefits: This project developed a new technique to measure the precise activity of the oxidase enzyme in single cells. The advantage of this method is that it can measure the activity of the oxidase enzyme without the need for other labelling techniques. The procedure can be done very quickly, at about one second per cell, allowing rapid screening of large numbers of cells.
This single-cell detection method could be of great benefit to assessing the efficacy of gene therapy.
Publications resulting from this work:
Single-cell optical imaging of the phagocyte NADPH oxidase
van Manen HJ, van Bruggen R, Roos D, Otto C.
Antioxidants & Redox Signaling, 2006 Sep–Oct; 8(9–10): 1509–22.
http://www.ncbi.nlm.nih.gov/pubmed/16987007Single-cell Raman and fluorescence microscopy reveal the association of lipid bodies with phagosomes in leukocytes
van Manen HJ, Kraan YM, Roos D, Otto C.
Proceedings of the National Academy of Sciences of the United States of America, 2005 Jul 19; 102(29): 10159–64
www.ncbi.nlm.nih.gov/pmc/articles/PMC1177376/?tool=pubmedRaman microscopy of phagocytosis: shedding light on macrophage foam cell formation van Manen HJ, Uzunbajakava N, van Bruggen R, Roos D, Otto C.
Journal of the American Chemical Society, 2003 Oct 8; 125(40): 12112–3.Finding out how the enzyme affected in CGD works
Grant awarded to: Dr Miriam Hirshberg, University of Cambridge; Professor Edgar Pick, Sackler School of Medicine, Tel Aviv, Israel
Amount: £33,930 over one year ending in 2005
Official title: ‘Uncovering the ways the marriage between p67phox and Rac leads to NADPH oxidase activation and ultimate killing action’
Aim: To study in great detail how two components of the enzyme affected in CGD work together.
Outcomes and benefits: The work focused on the crucial role p67 phox and the protein Rac1 play in the activation of gp91 phox that starts the process of producing chemical bleach in cells to kill microbes. Detailed structural knowledge was obtained, using the most sophisticated and up-to-date methods.
This study helped pinpoint and map important functional parts of the enzyme so that therapeutic drugs can be better designed in the future.
Publications resulting from this work:
Activation of the phagocyte NADPH oxidase by Rac guanine nucleotide exchange factors in conjunction with ATP and nucleoside diphosphate kinase
Mizrahi A, Molshanski-Mor S, Weinbaum C, Zheng Y, Hirshberg M, Pick E.
Journal of Biological Chemistry, 2005 Feb 4; 280(5):3 802–11. Erratum in: Journal of Biological Chemistry, 2005 Mar 25; 280(12): 12064.
http://www.jbc.org/content/280/5/3802.longUnderstanding mechanisms in CGD-affected cells
Grant awarded to: Professor Dirk Roos and Dr Cees Otto, Department of Experimental Immunohematology, Sanquin Research, Amsterdam, and Department of Science and Technology, Biophysical Engineering Group, University of Twente, the Netherlands
Amount: £124,300 over three years ending in 2004
Official title: ‘Analysis of functional defects in CGD by confocal microscopy’
Aim: The aim of this project was to improve understanding of how CGD mutations affect the working of the oxidase enzyme.
Outcomes and benefits: The functional defects caused by CGD mutations were studied using confocal microscopy. This method makes it possible to focus on a 3-dimensional scan of a single cell and to study the response of single molecules within it. By imaging the different parts of the enzyme affected in CGD, more knowledge was gained on how the enzyme is assembled and activated in healthy cells and how these processes are different in CGD.
The information gained will have long-term benefit and add to increased knowledge of the CGD enzyme defect.
Publications resulting from this work:
Resonance Raman imaging of the NADPH oxidase subunit cytochrome b558 in single neutrophilic granulocytes
van Manen HJ, Uzunbajakava N, van Bruggen R, Roos D, Otto C.
Journal of the American Chemical Society, 2003 Oct 8; 125(40): 12112–3. http://www.ncbi.nlm.nih.gov/pubmed/14518995Continuous translocation of Rac2 and the NADPH oxidase component p67phox during phagocytosis
van Bruggen R, Anthony E, Fernandez-Borja M, Roos D.
Journal of Biological Chemistry, 2004; 279(10): 9097–102.www.jbc.org/content/279/10/9097.long
Intracellular chemical imaging of heme-containing enzymes involved in innate immunity using resonance Raman microscopy
van Manen HJ, Kraan YM, Roos D, Otto C.
Journal of Physical Chemistry B, 2004; 108, 18762–18771.Improving diagnostic testing for variant CGD
Grant awarded to: Professor Anthony Segal, Dr Andrew Cross, Louise Morton, CGD Society-funded CGD Clinical Specialist Nurse, Centre for Molecular Medicine, University College London
Amount: £75,472 over two years ending in 2005
Official title: ‘Characterisation of genotype/phenotype relationships of UK patients with CGD’
Aim: Variant CGD, where the proteins responsible for fighting infection may be able to function partially but not at a level sufficient to fight off infection, can sometimes be misdiagnosed. This project aimed to improve diagnostic testing for variant CGD by setting up new diagnostic methods.
Outcomes and benefits: A cell-free assay was established to identify the abnormal protein from people with variant CGD. Correct diagnosis is important for counselling and supporting the patient and their family, and for deciding what therapies would be of long-term benefit.