Periodic Reporting for period 1 - NEMoCuRe (Role of S-Nitrosylation of epigenetic modifiers in vascular regeneration)
Reporting period: 2019-01-01 to 2020-12-31
What is the problem/issue being addressed?
Humans have a limited capacity to regenerate and restore tissues and organs compared to lower vertebrates, such as zebrafish (Danio rerio). Understanding molecular mechanisms underpinning regenerative processes would pave the way for new therapies in humans. S-nitrosylation, the covalent attachment of a nitric oxide (NO) group to the thiol side chain of the cysteine, has emerged as an important mechanism for dynamic, posttranslational regulation of most or all main classes of protein. In addition, hypo- or hyper-S-nitrosylation of specific protein targets have been shown to be directly implicated in the etiology and symptomatology of an increasing number of cardiovascular disease. As for other posttranslational modifications, S-nitrosylation affects proteins in all cellular compartments. However, less is known about S-nitrosylation of chromatin modifiers. These are enzymes that modify the epigenome directly through DNA methylation or modifications of histones. S-nitrosylation could alter the activity of these chromatin modifiers and could promote an open chromatin state that is required for gene transcription during tissue repair. S-nitrosylation also plays a major role in the innate immune response to stress or injury, and pathogens. Therefore, modulating the immune system and its downstream pathways including iNOS, that catalyzes the NO production, could cause changes in S-nitrosylation, in particular of epigenetic modifiers, and eventually might be therapeutic in human disorders. The principle aim of this proposal is to explore mechanisms involving protein S-nitrosylation and their impact on tissue repair, specifically in vascular repair and regeneration. In particular, the adult zebrafish caudal fin completely regrows within a month upon amputation. Furthermore the vascular regeneration in this model can be easily followed under fluorescent microscopy using the transgenic line Tg(fli1:EGFP), where green fluorescent protein is expressed under control of an endothelial marker. Therefore, it will be adopted as a model of tissue injury/regeneration.
Why is it important for society?
The role of S-nitrosylation in the regulation of nuclear proteins, in particular of epigenetic modifiers, is almost completely unknown. This innovative project aims to establish whether S-nitrosylation of chromatin modifiers can create open epigenetic states that in turn favor cellular plasticity and promotes tissue regeneration. Insights from this proposal may lead to novel therapeutic approaches targeting cardiovascular disease by promoting vascular regeneration via mechanisms modulating the nitrosylation of key chromatin modifiers.
What are the overall objectives?
1) Are there any changes in S-nitrosylation of epigenetic modifiers in zebrafish tailfin after injury?
2) Does S-nitrosylation of epigenetic modifiers affects vascular regeneration?
3) Is there any interaction between immune system, iNOS and S-nitrosylation of epigenetic modifiers?
Humans have a limited capacity to regenerate and restore tissues and organs compared to lower vertebrates, such as zebrafish (Danio rerio). Understanding molecular mechanisms underpinning regenerative processes would pave the way for new therapies in humans. S-nitrosylation, the covalent attachment of a nitric oxide (NO) group to the thiol side chain of the cysteine, has emerged as an important mechanism for dynamic, posttranslational regulation of most or all main classes of protein. In addition, hypo- or hyper-S-nitrosylation of specific protein targets have been shown to be directly implicated in the etiology and symptomatology of an increasing number of cardiovascular disease. As for other posttranslational modifications, S-nitrosylation affects proteins in all cellular compartments. However, less is known about S-nitrosylation of chromatin modifiers. These are enzymes that modify the epigenome directly through DNA methylation or modifications of histones. S-nitrosylation could alter the activity of these chromatin modifiers and could promote an open chromatin state that is required for gene transcription during tissue repair. S-nitrosylation also plays a major role in the innate immune response to stress or injury, and pathogens. Therefore, modulating the immune system and its downstream pathways including iNOS, that catalyzes the NO production, could cause changes in S-nitrosylation, in particular of epigenetic modifiers, and eventually might be therapeutic in human disorders. The principle aim of this proposal is to explore mechanisms involving protein S-nitrosylation and their impact on tissue repair, specifically in vascular repair and regeneration. In particular, the adult zebrafish caudal fin completely regrows within a month upon amputation. Furthermore the vascular regeneration in this model can be easily followed under fluorescent microscopy using the transgenic line Tg(fli1:EGFP), where green fluorescent protein is expressed under control of an endothelial marker. Therefore, it will be adopted as a model of tissue injury/regeneration.
Why is it important for society?
The role of S-nitrosylation in the regulation of nuclear proteins, in particular of epigenetic modifiers, is almost completely unknown. This innovative project aims to establish whether S-nitrosylation of chromatin modifiers can create open epigenetic states that in turn favor cellular plasticity and promotes tissue regeneration. Insights from this proposal may lead to novel therapeutic approaches targeting cardiovascular disease by promoting vascular regeneration via mechanisms modulating the nitrosylation of key chromatin modifiers.
What are the overall objectives?
1) Are there any changes in S-nitrosylation of epigenetic modifiers in zebrafish tailfin after injury?
2) Does S-nitrosylation of epigenetic modifiers affects vascular regeneration?
3) Is there any interaction between immune system, iNOS and S-nitrosylation of epigenetic modifiers?
Despite the importance of nitric oxide (NO) signaling in orchestrating multiple biological processes its role in tissue regeneration remains largely unexplored. We provide evidence that inducible NO synthase (iNOS) translocates from the cytoplasm to the nucleus during regeneration of the zebrafish tailfin and that this is associated with alterations in the nuclear S-nitrosylated proteome. Treatment with iNOS inhibitors or NO scavengers reduces protein S-nitrosylation and impairs tail fin regeneration. Bioinformatic analysis of liquid chromatography/tandem mass-spec dataset reveals an increase of up to 18 folds in the number of S-nitrosylated proteins during regeneration. Among these, KDM1a, a well-known epigenetic modifier, is S-nitrosylated which affects the binding of KDM1a to the its complexes and, by turn, impairing its demethylase activity. We subsequently show this to be essential for tailfin regeneration.
This work highlights, for the first time, the role of S-NO in the regulation of a chromatin modifier, i.e. KDM1a, particularly in EC, during tissue regeneration. My findings also suggest that S-NO could be modulated in EC after injury.
My discoveries could have a great impact to the society’s wellbeing, that will emerge at short and long term. At short term, within the next 10-15 years, it could lead to novel therapeutic applications in cardiovascular diseases, through the development of new drugs, or improve the efficacy of drugs currently available, and antibody-based therapeutics. This immediately reminds to the immunotherapies that currently use monoclonal antibodies designed regardless of the protein modifications. Posttranslational modifications (PTMs) of drug targets or of the drugs themselves are important for the efficacy of the drugs, depending on the mechanism of action. New antibody-mediated therapies could be based upon PTMs antibodies, including, for example, S-nitrosylation. Over the long term, the project could lead to new diagnostic tools and disease biomarkers. For example, the S-nitrosylation pattern could be specific for each individual but also for the populations, and could encompass PTM marks that could represent risk factors for certain disease. Modulating the expression and/or activity of chromatin modifiers to increase phenotypic plasticity could open new experimental and clinical strategies for vascular regeneration in humans.
As a side, but important, aspect, the intellectual property (IP) will protect my discoveries and will give me the time to develop and produce new drugs and perform clinical trials. Commercial exploitation and IP protection will also be assessed.
My discoveries could have a great impact to the society’s wellbeing, that will emerge at short and long term. At short term, within the next 10-15 years, it could lead to novel therapeutic applications in cardiovascular diseases, through the development of new drugs, or improve the efficacy of drugs currently available, and antibody-based therapeutics. This immediately reminds to the immunotherapies that currently use monoclonal antibodies designed regardless of the protein modifications. Posttranslational modifications (PTMs) of drug targets or of the drugs themselves are important for the efficacy of the drugs, depending on the mechanism of action. New antibody-mediated therapies could be based upon PTMs antibodies, including, for example, S-nitrosylation. Over the long term, the project could lead to new diagnostic tools and disease biomarkers. For example, the S-nitrosylation pattern could be specific for each individual but also for the populations, and could encompass PTM marks that could represent risk factors for certain disease. Modulating the expression and/or activity of chromatin modifiers to increase phenotypic plasticity could open new experimental and clinical strategies for vascular regeneration in humans.
As a side, but important, aspect, the intellectual property (IP) will protect my discoveries and will give me the time to develop and produce new drugs and perform clinical trials. Commercial exploitation and IP protection will also be assessed.