Abstract
In nutrient-starved bacteria, RelA and SpoT proteins have key roles in reducing cell growth and overcoming stresses. Here we identify functional SpoT orthologs in metazoa (named Mesh1, encoded by HDDC3 in human and Q9VAM9 in Drosophila melanogaster) and reveal their structures and functions. Like the bacterial enzyme, Mesh1 proteins contain an active site for ppGpp hydrolysis and a conserved His-Asp–box motif for Mn2+ binding. Consistent with these structural data, Mesh1 efficiently catalyzes hydrolysis of guanosine 3′,5′-diphosphate (ppGpp) both in vitro and in vivo. Mesh1 also suppresses SpoT-deficient lethality and RelA-induced delayed cell growth in bacteria. Notably, deletion of Mesh1 (Q9VAM9) in Drosophila induces retarded body growth and impaired starvation resistance. Microarray analyses reveal that the amino acid–starved Mesh1 null mutant has highly downregulated DNA and protein synthesis–related genes and upregulated stress-responsible genes. These data suggest that metazoan SpoT orthologs have an evolutionarily conserved function in starvation responses.
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Acknowledgements
This research was supported by grants from the Korean National Creative Research Initiatives Program (2010-0018291) to J.C. and from the NMR Research Program of Korea Basic Science Institute to Y.H.J. G.L. was supported by the Priority Research Centers Program through the National Research Foundation of Korea, funded by the Ministry of Education, Science and Technology (2008-005-J00203). Y.K. and B.Y.K. were supported by World Class Institute Program of the National Research Foundation of Korea, funded by the Ministry of Education, Science and Technology. Wild-type (CF1648) and ΔrelA ΔspoT (CF1693) E. coli were kindly provided by M. Cashel (US National Institutes of Health).
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D.S. determined the structures and performed HPLC and MS analyses. G.L. and J.H.L. performed Drosophila and bacteria experiments. H.-Y.K. and K.-J.K. helped to determine the structures. H.-W.R. and J.-I.H. prepared PyDPA analyses. S.-Y.P. analyzed nucleotides from larval extracts. Y.K. performed mammalian cell culture experiments. All authors, including B.Y.K., C.P., H.E.C. and J.H.K. discussed the experimental results and the text of the manuscript. D.S., G.L., J.H.L., Y.H.J. and J.C. prepared the manuscript. J.C. and Y.H.J. developed the original research idea and supervised the research project.
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Supplementary Figures 1–6, Supplementary Tables 1–2 and Supplementary Methods (PDF 827 kb)
Supplementary Table 3
Differentially expressed genes in response to amino acid starvation in wild-type and Mesh1 null mutant flies. (a–c) GO stat analysis was performed for the downregulated and upregulated genes in response to amino acid starvation in dMesh1 null mutant (b,c) or wild-type flies (a). The affected GO categories and the P value of match are indicated. Gene name and the fold change of each gene included in each GO category are indicated. (XLS 71 kb)
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Sun, D., Lee, G., Lee, J. et al. A metazoan ortholog of SpoT hydrolyzes ppGpp and functions in starvation responses. Nat Struct Mol Biol 17, 1188–1194 (2010). https://doi.org/10.1038/nsmb.1906
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DOI: https://doi.org/10.1038/nsmb.1906
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