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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - Za-AV (Determination of physiologically relevant RNA substrate(s) and design of small-molecule inhibitors for ZÎ± domains as potential intervention strategies for viral infections and autoimmunity)

Teaser

Summary

The problems being addressed by the actions pertained to finding out the substrate specificity of Z-alpha domains using RNA-seq analysis and the subsequent biophysical characterisation of the protein and its cognate substrate/s.This issue is important to society because viral infections cause a lot of economic and social hardships. Viruses are recognized by host immune system that activates interferon response pathway to bring about viral clearance. However, the magnitude of the response is modulated in order to prevent infliction of self injury. Recognition of Z-conformation of nucleic acids by protein domains called Z-alpha play an important role in this balance between self and non-self discrimination. Insights from this work will provide critical insights into design of antiviral strategies.

The overall objective of the actions was to determine the substrate nucleic acids recognized by Z-alpha domain and attempts at designing small-molecule binders for these protein domains. The current project, after an year since its inception, has been successful in standardizing RNA-seq experiments. The RNA-seq pipeline was standardized in E.coli and the results were analyzed. Contrary to previous reports, analysis of the resultant RNA-seq data showed that ZÎ± domain of ADAR1 preferentially binds to 5S rRNA from E. coli. Subsequently, the analysis was carried on polyA and non poly A enriched RNA fractions from A549 cells mammalian cell lines pulled down employing the wild-type and nucleic acid binding deficient mutant of ZÎ±Î² domains from ZBP1. The analysis of the pulldown data provides us with a list of preferential RNA substrates pulled down by the wild-type protein vis-Ã -vis the mutant variant. We had hypothesized that, since the ZÎ± domain is a part of the multidomain protein ADAR1 that is involved in A to I editing, it is highly likely that inosine containing sequences having a propensity for adopting Z conformation might be a recognizable motif for ZÎ± domain to bind to specific nucleic acid substrates. Further, haplotype-based variant analysis shows a slight enrichment of A2G mutations (indicative of A to I editing) in the RNA pool pulled-down by wild-type vis-Ã -vis mutant. However, this conclusion needs further experiments to validate.
Furthermore, in support of the hypothesis that inosine containing oligonucleotides form the preferential substrate for ZÎ± domains, we have demonstrated the binding of ZÎ±Î² domains from ZBP1 to oligonucleotides enriched in TI and CI containing repeats using electrophoretic mobility gel shift assays. Additionally, the study has also attempted to qualitatively model the isothermal titration calorimetry data of ZÎ± domain binding to the oligonucleotides T(CG)3 and T(CG)6 constrained by the assumption that the protein domain can bind only to the Z-conformation. Further experiments are needed to model the data quantitatively and to eliminate the possibility of an induced fit to Z-conformation rather than a lock and key picture. Further, as part of effort to discover novel small-molecules binding to ZÎ± domain, we had projected to carry out virtual ligand screening and high-throughput experimental screening. Before experimental screening, successful virtual ligand screening (VLS) was carried out using hybrid next-generation methodologies combining the principles of both structure-based and ligand-based screening approaches. FINDSITEcomb, a fold-based approach, and PoLi, a pocket-based approach, were employed to predict small-molecule binders for the ZÎ± domain of ADAR1 and ZÎ±Î² domain of ZBP1.

This project was pivotal for my training in both scientific and transferable skills, for expanding my network, for giving me exposure into project management and for contributing holistically to advancing my overall scientific temperament. I could not only pursue my science but attended training workshops in science communication, use of social media for effective science outreach and RNA-seq analysi

Work performed

1. RNA-seq analysis has been carried out to understand potential RNA substrate molecules that are prefered by the Wild-type Z-alpha,beta domain of ZBP1 protein.
2. Analysis has been carried out to find out enrichment of inosines in the wild-type pooled down pool of RNA.
3. EMSA analysis has been carried out to show that the protein domains Z-alpha,beta ZBP1 binds to insoine containing oligonucleotides.
4. Analysis of isothermal titration calorimetry data has been carried out to tease apart possible factors determining substrate recognition and binding by Z-alpha domains.
5. Analysis of Mass-spectrometry proteomics data has been carried out to understand possible interaction partners for this protein domain.
6. Virtual ligand screening has been performed on all permutations and combinations of Z-alpha, beta domains to understand small-molecule binders for these domains.
7. Appropriate single and double mutant constructs have been generated to understand cooperativity and synergy in substrate recognition by single domain and tandem Z-alpha domains.

Final results

Progress beyond the state of the art:
1. Preliminary insights into substrate recognition by Z-alpha domains.
2. Predictions of small-molecule that can bind to the protein domain.
3. Analysis of proteomics data to understand interacting partners for this domain.
4. Generation of appropriate mutant constructs to understand cooperativity and synergy.

expected results:
1. Substrates recognized by the protein domain.
2. Small molecule binders/inhibitors for this protein domain.

potential impacts:
1. Further insights into interferon response and how viruses regulate host interferon response.
2. Small molecule binders/inhibitors for Z-alpha domain as potential antiviral therapy.