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International journal of molecular sciences2019; 21(1); 105; doi: 10.3390/ijms21010105

Screening and Identification of Novel cGAS Homologues Using a Combination of in Vitro and In Vivo Protein Synthesis.

Abstract: The cyclic GMP-AMP synthase (cGAS) catalyzes the synthesis of the multifunctional second messenger, cGAMP, in metazoans. Although numerous cGAS homologues are predicted in protein databases, the catalytic activity towards cGAMP synthesis has been proven for only four of them. Therefore, we selected five novel and yet uncharacterized cGAS homologues, which cover a broad range in the field of vertebrates. Cell-free protein synthesis (CFPS) was used for a pre-screening to investigate if the cGAS genes originating from higher organisms can be efficiently expressed in a bacterial expression system. As all tested cGAS variants were expressible, enzymes were synthesized in vivo to supply higher amounts for a subsequent in vitro activity assay. The assays were carried out with purified enzymes and revealed vast differences in the activity of the homologues. For the first time, the cGAS homologues from the Przewalski's horse, naked mole-rat, bald eagle, and zebrafish were proven to catalyze the synthesis of cGAMP. The extension of the list of described cGAS variants enables the acquisition of further knowledge about the structural and molecular mechanism of cGAS, potentially leading to functional improvement of the enzyme.
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Publication Date: 2019-12-22 PubMed ID: 31877895PubMed Central: PMC6981698DOI: 10.3390/ijms21010105Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research article is about the screening and identification of new versions, or homologues, of the cGAS enzyme. The researchers selected five potential homologues in various vertebrate creatures, tested their expression in a bacterial system, and evaluated their activity, discovering that four of them can indeed catalyze the synthesis of cGAMP.

Objectives of the Research

  • The main aim of the research was to identify and analyze new homologues of the cyclic GMP-AMP synthase (cGAS) enzyme in vertebrates beyond what has been proven in protein databases.

Methods Used in the Research

  • The researchers selected five uncharacterized cGAS homologues that represent a broad range in the field of vertebrates. The organisms they chose include the Przewalski’s horse, naked mole-rat, bald eagle, and zebrafish.
  • The team first used Cell-free protein synthesis (CFPS) for pre-screening purposes. CFPS allowed them to see if the cGAS genes from these creatures could be efficiently expressed in a bacterial expression system.
  • Once the team established that all tested cGAS variants were expressible, they synthesized these enzymes in vivo to generate greater amounts for a follow-up in vitro activity assay.

Findings of the Research

  • Using in vitro activity assays, which were carried out with the purified enzymes, the researchers found significant differences in the activity levels of the homologues.
  • The study established that the cGAS homologues from the Przewalski’s horse, naked mole-rat, bald eagle, and zebrafish could catalyze the synthesis of cGAMP. This was the first time this ability was proven for these organisms.

Implications of the Research

  • The identification and characterization of these new cGAS variants contribute to a broader understanding of the structural and molecular mechanism of cGAS.
  • The findings may potentially lead to functional improvements of the cGAS enzyme, which can have significant implications for several biochemical applications and possibly related health conditions.

Cite This Article

APA
Rolf J, Siedentop R, Lütz S, Rosenthal K. (2019). Screening and Identification of Novel cGAS Homologues Using a Combination of in Vitro and In Vivo Protein Synthesis. Int J Mol Sci, 21(1), 105. https://doi.org/10.3390/ijms21010105

Publication

International journal of molecular sciences
ISSN: 1422-0067
NlmUniqueID: 101092791
Country: Switzerland
Language: English
Volume: 21
Issue: 1
PII: 105

Researcher Affiliations

Rolf, Jascha
  • Chair for Bioprocess Engineering, Department of Biochemical and Chemical Engineering, TU Dortmund University, D-44227 Dortmund, Germany.
Siedentop, Regine
  • Chair for Bioprocess Engineering, Department of Biochemical and Chemical Engineering, TU Dortmund University, D-44227 Dortmund, Germany.
Lütz, Stephan
  • Chair for Bioprocess Engineering, Department of Biochemical and Chemical Engineering, TU Dortmund University, D-44227 Dortmund, Germany.
Rosenthal, Katrin
  • Chair for Bioprocess Engineering, Department of Biochemical and Chemical Engineering, TU Dortmund University, D-44227 Dortmund, Germany.

MeSH Terms

  • Animals
  • Biocatalysis
  • Cell-Free System
  • Eagles / genetics
  • Eagles / metabolism
  • Gene Expression Regulation, Enzymologic
  • Horses / genetics
  • Horses / metabolism
  • Humans
  • Isoenzymes / genetics
  • Isoenzymes / metabolism
  • Mole Rats / genetics
  • Mole Rats / metabolism
  • Nucleotides, Cyclic / metabolism
  • Nucleotidyltransferases / genetics
  • Nucleotidyltransferases / metabolism
  • Protein Biosynthesis
  • Recombinant Proteins / metabolism
  • Species Specificity
  • Zebrafish / genetics
  • Zebrafish / metabolism

Conflict of Interest Statement

The authors declare no conflict of interest.

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Citations

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