Analysis of the regulation and function of NF- B1 p105, TPL-2 and ABIN-2 in immune responses

Lead Research Organisation: MRC National Inst for Medical Research

Abstract

Rheumatoid arthritis (RA) is a common human autoimmune disease, which is characterized by chronic inflammation of the synovial joints and progressive destruction of cartilage and bone. Localized inflammatory responses, in this case in blood vessel walls, are similarly important in atherosclerosis, a leading cause of morbidity and mortality in industrialized countries. An understanding of the mechanism by which inflammatory responses are controlled is important for development of new therapies for these major diseases.The initiation of inflammatory responses depends on the NF-?B family of intracellular proteins, which regulate the expression of multiple genes that are switched on in both RA and atherosclerosis. The activation of NF-?B involves several distinct signalling pathways and my laboratory concentrates on one of these that is dependant on the protein NF-B1 p105. Our recent work has shown that this controls not only the activation of NF-B proteins but also of another signalling protein, TPL-2. We are currently investigating in more detail how p105 regulates TPL-2 activation and how both of these proteins control immune and inflammatory responses. It is hoped that our work might identify novel targets for the development of anti-inflammatory drugs for the treatment of inflammatory diseases such as RA and atherosclerosis.

Technical Summary

Objectives: Research in my laboratory concentrates on the role of signal-induced proteolysis of the NF-?B inhibitory protein NF-?B1 p105 in immune responses and also the regulation and function of the p105-associated proteins, TPL-2 (COT) kinase and ABIN-2. This works may identify novel targets for pharmacological manipulation of the immune system in inflammatory diseases such rheumatoid arthritis and atherosclerosis. Methodology: My research involves a variety of molecular biology techniques, analytical and preparative protein chemistry, in vitro cell culture and confocal microscopy. Summary: My major research achievements during the last five years are summarized below: Demonstration that NF-?B1 p105 negatively regulates the MEK kinase activity of TPL-2 Demonstration that TLR4 activation of ERK MAP kinase in macrophages is dependent on IKK-triggered proteolysis of NF-?B1 p105Demonstration that ABIN-2 forms a ternary complex with NF-?B1 p105 / TPL-2 and is required to maintain TPL-2 protein stability Demonstration that ABIN-2 is required for optimal activation of the TPL-2 / ERK signalling pathway in innate immune responses

Publications

10 25 50
 
Description ARC Project Grant
Amount £168,725 (GBP)
Funding ID 18864 
Organisation Versus Arthritis 
Start 02/2009 
End 02/2012
 
Description Development Gap Funding
Amount £119,000 (GBP)
Organisation MRC-Technology 
Sector Academic/University
Country United Kingdom
Start 03/2012 
End 03/2014
 
Description Leukaemia Research Project Grant 2007
Amount £158,751 (GBP)
Organisation Leukaemia and Lymphoma Research 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2007 
End 03/2012
 
Description MRCT Development Gap Funding
Amount £25,046 (GBP)
Organisation MRC-Technology 
Sector Academic/University
Country United Kingdom
Start 01/2009 
End 01/2010
 
Title VP35 
Description We have developed a new method for production of recombinant proteins in human HEK293 cells. This involves co-expressing Ebola virus VP35, which blocks DNA plasmid activation of PKR, increasing protein translation and substantially increasing protein yield. 
Type Of Material Technology assay or reagent 
Year Produced 2012 
Provided To Others? Yes  
Impact This new method has allowed my group to produce sufficient TPL-2 complex to carry out a high throughput screen for TPL-2 inhibitors with MRC Technology. 
 
Description Analysis of Abin2 knockout mice 
Organisation Medical Research Council (MRC)
Department MRC National Institute for Medical Research (NIMR)
Country United Kingdom 
Sector Public 
PI Contribution Analysis of Abin2 knockout mice.
Impact 16633345
 
Description Arrestin-2 regulation of NF-kB1 p105 
Organisation Thomas Jefferson University
Country United States 
Sector Academic/University 
PI Contribution Provided advice on the regulation of the TPL-2/ERK signalling pathway.
Impact 16980301
 
Description Characterisation of the TPL-2/NF-kB1 p105/ABIN-2 complex 
Organisation GlaxoSmithKline (GSK)
Department Crick-GSK LinkLabs
Country United Kingdom 
Sector Private 
PI Contribution Knowledge about the regulation and function of the TPL-2 complex in inflammation. Provided purified TPL-2/NF-kB1 p105/ABIN-2 complex for enzyme kinetic experiments.
Collaborator Contribution Funded 3-year Post-Doctoral Research Associate to work on the characterisation of the TPL-2/NF-kB1 p105/ABIN-2 complex.
Impact Assaying kinase activity of the TPL-2/NF-?B1 p105/ABIN-2 complex using an optimal peptide substrate. Kümper S, Gantke T, Chen CS, Soneji Y, Pattison MJ, Chakravarty P, Kjær S, Thomas D, Haslam C, Leavens BJ, House D, Powell DJ, Ley SC. Biochem J. 2018 Jan 11;475(1):329-340. doi: 10.1042/BCJ20170579. PMID: 29229763
Start Year 2015
 
Description HIF and NF-kB 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution Provided advice on NF-kB signalling and reagents for experiments.
Impact 17003112
 
Description Production of Abin2 knockout mice 
Organisation Medical Research Council (MRC)
Department MRC National Institute for Medical Research (NIMR)
Country United Kingdom 
Sector Public 
PI Contribution Generation and analysis of ABIN-2 deficient mice.
Impact 16633345
 
Description Production of Nfkb1[SS/AA] knockin mice. 
Organisation Medical Research Council (MRC)
Department MRC National Institute for Medical Research (NIMR)
Country United Kingdom 
Sector Public 
PI Contribution Generated and analysed Nfkb1[SS/AA] knockin mice.
Impact 19060899
 
Description Regulation of IFNb production by TPL-2. 
Organisation Medical Research Council (MRC)
Department MRC National Institute for Medical Research (NIMR)
Country United Kingdom 
Sector Public 
PI Contribution Generated some of initial data showing that the TPL-2/ERK pathway regulated IFNb production.
Impact 19667062
 
Description Regulation of TNF production by TPL-2 
Organisation Cancer Research UK
Department Proteomics Laboratory, LRI
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Provided data showing the effects of TPL-2 deficiency on TNF production by macrophages.
Collaborator Contribution Mass spectrometric analysis of protein phosphorylation
Impact 18187448
Start Year 2012
 
Description Regulation of TNF production by TPL-2 
Organisation University of Dundee
Country United Kingdom 
Sector Academic/University 
PI Contribution Provided data showing the effects of TPL-2 deficiency on TNF production by macrophages.
Collaborator Contribution Mass spectrometric analysis of protein phosphorylation
Impact 18187448
Start Year 2012
 
Description TPL-2 and lymphoma / leukaemia 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution Initiated project after discussion with Ming Du
Impact 17324460
 
Description TPL-2 complex production 
Organisation MRC-Technology
Country United Kingdom 
Sector Academic/University 
PI Contribution > Developed method for production of TPL-2/p105/ABIN-2 complex
Collaborator Contribution > Developed assays to measure catalytic activity of TPL-2
Impact NA
Start Year 2011
 
Description p38 gamma/delta regulation of TPL-2 signalling 
Organisation Spanish National Centre for Biotechnology
Department Department of Immunology and Oncology
Country Spain 
Sector Public 
PI Contribution > Advice on TPL-2 signalling in macrophages and dendritic cells > Retroviral expression of p38 gamma in Tpl2-/- macrophages
Collaborator Contribution > Demonstrated requirment for p38 gamma/delta to maintain steady-state levels of TPL-2 in myeloid cells
Impact Risco, A., Del Fresno, C., Mambol, A., Alsina-Beauchamp, D. Mackenzie, K. F., Yang, H. T., Barber, D. F., Morcelle, C., Arthur, J. S., Ley, S. C., Ardavin, C., Cuenda, A. (2012) p38? and p38d kinases regulate the Toll-like receptor 4 (TLR4)-induced cytokine production by controlling ERK1/2 protein kinase pathway activation. Proc. Natl. Acad. Sci. USA 109, 11200 - 11205.
Start Year 2010