Final Report Summary - NUCLEIC ACIDS SENSOR (Molecular basis of the recognition of foreign nucleic acids in innate immunity)
DNA Sensors International Reintegration Grant 231000
Publishable summary
In vertebrate cells sensing of pathogen associated molecular patterns (PAMPs) initiates a variety of innate immune responses which often lead to apoptotic cell death. Receptor proteins for PAMPs can either sense the extracellular environment in which case are usually transmembrane proteins (e. g. Toll-like receptors) or the cytoplasmic environment by soluble receptors. Central role in such pathogen recognition have proteins that recognise foreign nucleic acids. In the cytoplasm of vertebrate cells RNA helicases like RIG I and MDA 5 recognise viral RNAs and lead to the production of type I interferons, while effector proteins, like PKR, recognise viral dsRNA and block cellular translation. The last years several distinct pathways have been partially characterised for the recognition of dsDNA in the cytoplasm. DAI is the first of these cytoplasmic receptors for dsDNA implicated in the activation of type I interferons. DAI belongs to a small family of proteins that contain Zalpha domains all involved in interferon response. Zalpha domains recognise and specifically bind purine/pyrimidine repeats in the left handed conformation known as Z-DNA. Other members of this protein family are the interferon inducible RNA editing enzyme ADAR1, the viral inhibitor of interferon response E3L and PKZ: a PKR like protein that recognises Z-DNA instead of dsRNA.
Understanding the protein nucleic acids interactions involved in this interferon response pathway and mediated by Zalpha domains will reveal the features that allow distinction between pathogen and self-nucleic acids in disease as well as in erroneous responses against self that cause auto-inflammatory diseases. The study of such interactions has been the goal of the newly established structural biology lab at the Instituto Gulbenkian de Ci?ncia with the support of the Marie Curie program.
The Marie Curie IRG grant enabled first the establishment of the first structural biology lab at IGC and then our work that has led to an extensive characterisation of the interactions of Zalpha domains with DNA by determining relevant crystal structures of DAI, ADAR1, PKZ and ORF112 Zalpha domains all but one in complex with DNA and at high resolution.
The structure of Zalpha domains bound to a Z-Z junction demonstrated the ability of Zalpha domains to interact with a wide range of sequences common in genomes and revealed the configuration of such interactions. Moreover provided significant clues regarding the role of Z-DNA formation in genomic instability associated with rearrangements found in many cancers (de Rosa et al, PNAS 2010).
We discovered and characterised biochemically as well as structurally a novel member of the Zalpha domain proteins for the first time from a Herpes virus that infects lower-vertebrate species. This protein (ORF112) is a competitive inhibitor of cellular activators of the interferon response as PKZ and DAI, at least in vitro. ORF112 revealed a domain swapping mode of dimerisation of Zalpha domains not previously seen. (AR. Tome et al, Journal of Virology, in Press, February 2013). As Cyprinid Herpesvirus 3 has emerged as a highly lethal pathogen affecting aquacultures of worldwide, the discovery of a potentially crucial immune system evasion protein may contribute in combating the disease. This study has been complemented with the characterisation of the Zalpha domains of the host protein PKZ from zebrafish that is being inhibited by ORF112 (manuscript in preparation).
Furthermore, we determined the crystal structure of the DAI Zab fragment in complex with T (CG) 3 DNA at 2. 4 ? resolution. Unfortunately the structure revealed that the Zab fragment was proteolytically cleaved leaving a protein that roughly corresponds to the Zbeta domain and thus less informative than the intact double Zalpha domain protein (manuscript in preparation).
Overall, the structural analysis of Zalpha-domain complexes allowed us to devise for the first time a model for the mechanism by which Zalpha domains stabilise the high energy structure of DNA known as Z-DNA. At the same time it permitted to devise the profile of the DNA that is being recognised by the nucleic acid sensors in the cytoplasm of vertebrate cells.
The establishment of the first structural biology lab at the Instituto Gulbenkian de Ci?ncia allowed us to develop collaborative projects with other groups of the institute (Santos et. al, Mol Biol Evol 2011) and promoted the knowledge of molecular and structural among the institute's students through intense series of classes on structural biology within the framework of the PhD program.
Publishable summary
In vertebrate cells sensing of pathogen associated molecular patterns (PAMPs) initiates a variety of innate immune responses which often lead to apoptotic cell death. Receptor proteins for PAMPs can either sense the extracellular environment in which case are usually transmembrane proteins (e. g. Toll-like receptors) or the cytoplasmic environment by soluble receptors. Central role in such pathogen recognition have proteins that recognise foreign nucleic acids. In the cytoplasm of vertebrate cells RNA helicases like RIG I and MDA 5 recognise viral RNAs and lead to the production of type I interferons, while effector proteins, like PKR, recognise viral dsRNA and block cellular translation. The last years several distinct pathways have been partially characterised for the recognition of dsDNA in the cytoplasm. DAI is the first of these cytoplasmic receptors for dsDNA implicated in the activation of type I interferons. DAI belongs to a small family of proteins that contain Zalpha domains all involved in interferon response. Zalpha domains recognise and specifically bind purine/pyrimidine repeats in the left handed conformation known as Z-DNA. Other members of this protein family are the interferon inducible RNA editing enzyme ADAR1, the viral inhibitor of interferon response E3L and PKZ: a PKR like protein that recognises Z-DNA instead of dsRNA.
Understanding the protein nucleic acids interactions involved in this interferon response pathway and mediated by Zalpha domains will reveal the features that allow distinction between pathogen and self-nucleic acids in disease as well as in erroneous responses against self that cause auto-inflammatory diseases. The study of such interactions has been the goal of the newly established structural biology lab at the Instituto Gulbenkian de Ci?ncia with the support of the Marie Curie program.
The Marie Curie IRG grant enabled first the establishment of the first structural biology lab at IGC and then our work that has led to an extensive characterisation of the interactions of Zalpha domains with DNA by determining relevant crystal structures of DAI, ADAR1, PKZ and ORF112 Zalpha domains all but one in complex with DNA and at high resolution.
The structure of Zalpha domains bound to a Z-Z junction demonstrated the ability of Zalpha domains to interact with a wide range of sequences common in genomes and revealed the configuration of such interactions. Moreover provided significant clues regarding the role of Z-DNA formation in genomic instability associated with rearrangements found in many cancers (de Rosa et al, PNAS 2010).
We discovered and characterised biochemically as well as structurally a novel member of the Zalpha domain proteins for the first time from a Herpes virus that infects lower-vertebrate species. This protein (ORF112) is a competitive inhibitor of cellular activators of the interferon response as PKZ and DAI, at least in vitro. ORF112 revealed a domain swapping mode of dimerisation of Zalpha domains not previously seen. (AR. Tome et al, Journal of Virology, in Press, February 2013). As Cyprinid Herpesvirus 3 has emerged as a highly lethal pathogen affecting aquacultures of worldwide, the discovery of a potentially crucial immune system evasion protein may contribute in combating the disease. This study has been complemented with the characterisation of the Zalpha domains of the host protein PKZ from zebrafish that is being inhibited by ORF112 (manuscript in preparation).
Furthermore, we determined the crystal structure of the DAI Zab fragment in complex with T (CG) 3 DNA at 2. 4 ? resolution. Unfortunately the structure revealed that the Zab fragment was proteolytically cleaved leaving a protein that roughly corresponds to the Zbeta domain and thus less informative than the intact double Zalpha domain protein (manuscript in preparation).
Overall, the structural analysis of Zalpha-domain complexes allowed us to devise for the first time a model for the mechanism by which Zalpha domains stabilise the high energy structure of DNA known as Z-DNA. At the same time it permitted to devise the profile of the DNA that is being recognised by the nucleic acid sensors in the cytoplasm of vertebrate cells.
The establishment of the first structural biology lab at the Instituto Gulbenkian de Ci?ncia allowed us to develop collaborative projects with other groups of the institute (Santos et. al, Mol Biol Evol 2011) and promoted the knowledge of molecular and structural among the institute's students through intense series of classes on structural biology within the framework of the PhD program.
