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A metazoan ortholog of SpoT hydrolyzes ppGpp and functions in starvation responses

Nature Structural & Molecular Biology volume 17, pages 1188–1194 (2010)Cite this article

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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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Figure 1: Identification of SpoT homologs in metazoa.
Figure 2: Mesh1 compared with bacterial ppGpp hydrolase shows a conserved structure and similar enzymatic activity.
Figure 3: Mesh1 hydrolyzes ppGpp in vivo.
Figure 4: Mesh1 null mutant Drosophila shows retarded body growth and impaired starvation resistance.

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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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Author notes

  1. Dawei Sun, Gina Lee and Jun Hee Lee: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Magnetic Resonance, Korea Basic Science Institute, Chungbuk, South Korea

    Dawei Sun, Hye-Yeon Kim & Young Ho Jeon

  2. Department of Bio-Analytical Science, University of Science and Technology, Daejeon, South Korea

    Dawei Sun & Young Ho Jeon

  3. Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, South Korea

    Gina Lee, Jun Hee Lee, Seung-Yeol Park, Yongsung Kim, Chankyu Park, Jung Hoe Kim & Jongkyeong Chung

  4. National Creative Research Initiatives Center and School of Biological Sciences, Seoul National University, Seoul, South Korea

    Gina Lee, Yongsung Kim & Jongkyeong Chung

  5. Department of Chemistry, Seoul National University, Seoul, South Korea

    Hyun-Woo Rhee & Jong-In Hong

  6. Pohang Accelerator Laboratory, Pohang University of Science and Technology, Pohang, South Korea

    Kyung-Jin Kim

  7. Korea Research Institute of Bioscience and Biotechnology, Chungbuk, South Korea

    Bo Yeon Kim

  8. Department of Microbiology, Chonnam National University Medical College, Kwangju, South Korea

    Hyon E Choy

  9. Institute of Molecular Biology and Genetics, Seoul National University, Seoul, South Korea

    Jongkyeong Chung

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Contributions

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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Correspondence to Young Ho Jeon or Jongkyeong Chung.

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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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