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Transglutaminase in disease: a novel therapeutic target?

Final Report Summary - TRANSPATH (Transglutaminase in disease: a novel therapeutic target?)

TRANSPATH is a multisectorial Network designed to facilitate the exchange of knowledge and technology between 7 leading European research teams and 3 SME partners and 2 associate SME Partners each involved in basic and applied research into transglutaminase (TG) related human diseases. Transglutaminases (TGs) are a widely distributed group of multifunctional enzymes that catalyse the post-translational modification of proteins by the formation of stable isopeptide bonds or by deamidation of proteins. The ambitions of TRANSPATH are to accelerate the development of new strategies for the treatment and diagnosis of a number of human diseases where transglutaminase activity is reported to play a role ,including metastatic cancer, neurodegeneration and celiac disease. The subject is highly relevant as deregulation of tissue transglutaminase activity is observed in a number of these diseases while the exact mechanisms are still largely unknown. The project targets this unmet clinical need.

The main objectives are:
1) to establish the molecular nature of the role of transglutaminases in the pathogenesis of diseases which are known to involve these multifunctional enzymes with a view to developing novel specific inhibitors and new therapeutic approaches which will have a major impact on their treatment;
2) provide an extensive cross sectorial scientific and supporting training network that will increase the capabilities of highly skilled researchers for the European biotechnology industry and academia.

Societal Significance of the Project

The chosen diseases, neurodegeneration, cancer and coeliac disease studied in this project are all very important with respect to human health and quality of life at the European level. The worldwide incidence of cancer is around 12 million; with around 3m cases in Europe, causing 19% of all deaths in Europe. Although the mortality rate has decreased significantly for some cancers, overall the change has been relatively small since the 1970s and new therapeutic avenues are urgently needed. Neurodegenerative disorders are a group of increasingly important diseases with significant competitive opportunities for European biotechnology. The cost of illness due to dementia in the EU in 2008 was estimated to be €160bn with a total world cost of over $600bn. An estimated 380,000 individuals are affected by Multiple Sclerosis in Europe. In 2005 the estimated annual direct cost to Europe was €6bn. Total drug sales for all neurodegenerative diseases in 2008 was $18.5bn and predicted to increase to $40bn by 2015. However there is still an urgent need for more effective drugs. Celiac disease occurs, on average, once in every 1,000 live births and is still one of most undiagnosed diseases and even though point of care kits are available their present accuracy still requires invasive surgery to confirm diagnosis. Currently, management of this disease is through diet alone, any therapy that removes the need for a strict gluten-free diet would have significant uptake. The celiac market is expected to be $8bn by 2019 with no candidate drug therapies currently passing Phase II

The project has been divided into 6 major scientific work packages (WPs) plus a Training ,Management and Dissemination WP each with their own individual Objectives.

Work Package WP1: To identify the factors that govern the extracellular localisation of TG2 at the cell surface and in the extracellular matrix and their subsequent effects on matrix assembly and crosslinking.

Work Package WP2: Development of novel chemical compounds and antibodies antagonistic to TG2

Work Package WP3: Transglutaminase 2 (TG2) a potential therapeutic target in the treatment of highly malignant tumours.

Work Package WP4: Dissecting the role of TG2 in the pathogenesis of neurodegenerative diseases using Huntington’s disease (HD) as a model.

Work Package WP5: Dissecting the involvement of TG2 in celiac disease pathogenesis.

Work Package WP6: Dissecting the role of TG2 in infiltration and migration of monocytes in
Multiple Sclerosis.

Work package 7 Training

Work Package 8 Management of the Project .

Work Package 9 Dissemination and Outreach

Summary of the Project achievements

TRANSPATH has made a number of achievements during the life of the project facilitated by the sharing and transfer of knowledge between its academic and commercial partners which are in line with its original objectives.Of particular note has been the development of models to study the mechanism of export of tissue transglutaminase (TG2) onto the cell surface and into the extracellular matrix(ECM) where many of its pathological actions occur.This includes development of different TG2 mutants at sites in TG2 which explore the binding of the enzyme to its different extracellular binding partners. Identification of the heparin binding sites on TG2 has shown their importance in TG2 secretion to the extracellular matrix under normal and stress conditions. We have shown that inhibitory TG2 heparin binding mimic peptides and monoclonal antibodies raised to the heparin binding site on TG2 can block the deposition of TG2 into the ECM which offers itself as another therapeutic avenue in the treatment of extracellular TG2 related diseases.The application of in silico modelling to produce highly specific small molecule inhibitors of TG2 has resulted in highly potent, non toxic and specific inhbitios of the enzyme that have shown efficacy in both cell and animal models of TG2 associated human disease eg cancer ,multiple sclerosis and nephrosclerosis .In addition we have demonstrated by using fluorescent inhibitor probes that these inhibitors can also inactivate intracellular TG2 leading to inhibition of its deposition into the ECM.Importantly new assays for the screening of other transglutaminases for assessing the specificity of these highly potent TG2 specific inhibitors have been developed.

Elucidation of the TG2 binding site on fibronectin has been achieved and recombinant production of this fibronectin binding fragment has been undertaken with the view to its use as an antagonist of cell adhesion and migration and as a probe for the staining of cell surface TG2 . Using phage antibody library technology a panel of 30 antibodies to humanTG2 have been isolated which have been screened for their effects on TG2 function. The recombinant production of an inhibitory antibody to TG2 has been achieved and its inhibitory action in cell models has confirmed its effectiveness in blocking TG2 function.

In kidney tumour cells studies have shown that TG2 plays a major role in cell migration and invasion and in epithelial mesenchymal transition which is required for tumour progression . New TG2 binding partners have also been isolated e.g. DNAJ A1/HSP 40 that could be important in the mechanisms that TG2 uses in tumour migration .

The role of TG2 in autophagy has been established and the importance of this in neurodegenerative diseases has been confirmed .Cell models to establish the importance of TG2 in protein aggregate formation in neurodegenerative diseases have been developed .The effects of small compound TG2 inhibitors and mutant TG2 that that has isopeptidase activity ,on the formation of these aggregates has been investigation but no obvious effects were noted on aggregate formation.The TG2-mediated cross-linking of pathological huntingtin aggregates was also analysed but was found not to occur in cells transfected with active transglutaminase and when subjected to autophagy or proteasome inhibition. Our studies therefore contradict and raise questions about previous hypotheses which highlighted the role of TG2 in the aggregate formation leading to the aggravation of Huntington’s disease.

The major epitope of extracellular recognised TG2 has been identified as overlapping the fibronectin binding site and N-terminal region.The antibody raised against this site preserves the oxidation of TG2 within the ECM and increases its activation by Ca2+ which may have implications in the pathology generated by TG2 autoantibodies in celiac disease.
The clinical symptoms of EAE (experimental autoimmune encephalomyelitis).in animal models for multiple sclerosis were reduced in TG2 knock-out mice supporting the hypothesis that TG2 plays a role in the pathogenesis of EAE. The contribution of TG2 to human monocyte migration has been established using specific TG2 inhibitors and TG2 silencing and it has been shown that the major inflammatory mediator Il-4 regulates TG2 expression and activity in these cells. Moreover, TG2 has been shown to be important in driving macrophages into an M2a differentiation status during the progression of EAE.

Research Impact

The project has focused on the skill exchange and transfer and use of knowledge between the academic ,health and industrial sectors and also between disciplines to achieve the desired capabilities and working practices within the partnership. This has been achieved via Knowledge and Technology transfer and exchange between Academics and Industry partners via short visits and secondments, through interdisciplinary scientific research and high quality training programmes in generic and transferable skills and in state of the art techniques. The latter of which was taught by a combination of both academic and industrial partners. The project also proposes to provide a pool of highly knowledgeable researchers with PhD qualifications trained in generic and transferable skills relevant to the European Biotechnology sector. These will include: business skills, and state of the art science such as molecular modelling and drug development, systems biology, clinical trials, animal models, and diagnostic development.