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TmcA functions as a lysine 2-hydroxyisobutyryltransferase to regulate transcription

Nature Chemical Biology volume 18, pages 142–151 (2022)Cite this article

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Abstract

Protein lysine 2-hydroxyisobutyrylation (Khib) has recently been shown to play a critical role in the regulation of cellular processes. However, the mechanism and functional consequence of Khib in prokaryotes remain unclear. Here we report that TmcA, an RNA acetyltransferase, functions as a lysine 2-hydroxyisobutyryltransferase in the regulation of transcription. We show that TmcA can effectively catalyze Khib both in vitro and intracellularly, and that R502 is a key site for the Khib catalytic activity of TmcA. Using quantitative proteomics, we identified 467 endogenous candidates targeted by TmcA for Khib in Escherichia coli. Interestingly, we demonstrate that TmcA can specifically modulate the DNA-binding activity of H-NS, a nucleoid-associated protein, by catalysis of Khib at K121. Furthermore, this TmcA-targeted Khib regulates transcription of acid-resistance genes and enhances E. coli survival under acid stress. Our study reveals transcription regulation mediated by TmcA-catalyzed Khib for bacterial acid resistance.

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Fig. 1: Identification of TmcA and YiaC as potential candidates for lysine 2-hyroxyisobutyryltransferase.
Fig. 2: R502 of TmcA is a key site for catalysis of Khib by TmcA.
Fig. 3: Profiling endogenous substrate proteins for Khib by TmcA in E. coli.
Fig. 4: TmcA specifically catalyzes K121hib of H-NS.
Fig. 5: TmcA regulates K121hib of H-NS that influences the DNA-binding ability of H-NS and survival under acid stress.
Fig. 6: TmcA regulates K121hib of H-NS to promote the transcription of acid-resistance-related genes under acid stress.

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Data availability

All data required to evaluate the conclusions are present in the paper and in Supplementary information. The proteomics MS data have been deposited with the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository, with the dataset identifiers PXD024897 and PXD024901. RNA-seq data can be obtained at the Gene Expression Omnibus database under accession no. GSE169499. Source data are provided with this paper.

References

  1. Park, J. et al. SIRT5-mediated lysine desuccinylation impacts diverse metabolic pathways. Mol. Cell 50, 919–930 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Rardin, M. J. et al. SIRT5 regulates the mitochondrial lysine succinylome and metabolic networks. Cell Metab. 18, 920–933 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Choudhary, C., Weinert, B. T., Nishida, Y., Verdin, E. & Mann, M. The growing landscape of lysine acetylation links metabolism and cell signalling. Nat. Rev. Mol. Cell Biol. 15, 536–550 (2014).

    Article  CAS  PubMed  Google Scholar 

  4. Zhao, S. et al. Regulation of cellular metabolism by protein lysine acetylation. Science 327, 1000–1004 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Harmel, R. & Fiedler, D. Features and regulation of non-enzymatic post-translational modifications. Nat. Chem. Biol. 14, 244–252 (2018).

    Article  CAS  PubMed  Google Scholar 

  6. Sreelatha, A. et al. Protein AMPylation by an evolutionarily conserved pseudokinase. Cell 175, 809–821 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Chambers, K. A. & Scheck, R. A. Bacterial virulence mediated by orthogonal post-translational modification. Nat. Chem. Biol. 16, 1043–1051 (2020).

    Article  CAS  PubMed  Google Scholar 

  8. Sabari, B. R., Zhang, D., Allis, C. D. & Zhao, Y. Metabolic regulation of gene expression through histone acylations. Nat. Rev. Mol. Cell Biol. 18, 90–101 (2017).

    Article  CAS  PubMed  Google Scholar 

  9. Wagner, E. J. & Carpenter, P. B. Understanding the language of Lys36 methylation at histone H3. Nat. Rev. Mol. Cell Biol. 13, 115–126 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Zhang, D. et al. Metabolic regulation of gene expression by histone lactylation. Nature 574, 575–580 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Semer, M. et al. DNA repair complex licenses acetylation of H2AZ1 by KAT2A during transcription. Nat. Chem. Biol. 15, 992–1000 (2019).

    Article  PubMed  CAS  Google Scholar 

  12. Browning, D. F., Grainger, D. C. & Busby, S. J. Effects of nucleoid-associated proteins on bacterial chromosome structure and gene expression. Curr. Opin. Microbiol. 13, 773–780 (2010).

    Article  CAS  PubMed  Google Scholar 

  13. Dillon, S. C. & Dorman, C. J. Bacterial nucleoid-associated proteins, nucleoid structure and gene expression. Nat. Rev. Microbiol. 8, 185–195 (2010).

    Article  CAS  PubMed  Google Scholar 

  14. Sakatos, A. et al. Posttranslational modification of a histone-like protein regulates phenotypic resistance to isoniazid in mycobacteria. Sci. Adv. 4, eaao1478 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Carabetta, V. J., Greco, T. M., Cristea, I. M. & Dubnau, D. YfmK is an N(ε)-lysine acetyltransferase that directly acetylates the histone-like protein HBsu in Bacillus subtilis. Proc. Natl Acad. Sci. USA 116, 3752–3757 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Dai, L. et al. Lysine 2-hydroxyisobutyrylation is a widely distributed active histone mark. Nat. Chem. Biol. 10, 365–370 (2014).

    Article  CAS  PubMed  Google Scholar 

  17. Huang, H. et al. p300-Mediated lysine 2-hydroxyisobutyrylation regulates glycolysis. Mol. Cell 70, 663–678 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Huang, H. et al. Landscape of the regulatory elements for lysine 2-hydroxyisobutyrylation pathway. Cell Res. 28, 111–125 (2018).

    Article  CAS  PubMed  Google Scholar 

  19. Huang, J. et al. 2-Hydroxyisobutyrylation on histone H4K8 is regulated by glucose homeostasis in Saccharomyces cerevisiae. Proc. Natl Acad. Sci. USA 114, 8782–8787 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Dong, H. et al. Systematic identification of lysine 2-hydroxyisobutyrylated proteins in Proteus mirabilis. Mol. Cell. Proteomics 17, 482–494 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Dong, H. et al. Protein lysine de-2-hydroxyisobutyrylation by CobB in prokaryotes. Sci. Adv. 5, eaaw6703 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Sabari, B. R. et al. Intracellular crotonyl-CoA stimulates transcription through p300-catalyzed histone crotonylation. Mol. Cell 58, 203–215 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Dancy, B. M. & Cole, P. A. Protein lysine acetylation by p300/CBP. Chem. Rev. 115, 2419–2452 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Christensen, D. G. et al. Mechanisms, detection, and relevance of protein acetylation in prokaryotes. mBio 10, e02708-18 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Christensen, D. G. et al. Identification of novel protein lysine acetyltransferases in Escherichia coli. mBio 9, e01905-18 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Ikeuchi, Y., Kitahara, K. & Suzuki, T. The RNA acetyltransferase driven by ATP hydrolysis synthesizes N4-acetylcytidine of tRNA anticodon. EMBO J. 27, 2194–2203 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Chimnaronk, S. et al. RNA helicase module in an acetyltransferase that modifies a specific tRNA anticodon. EMBO J. 28, 1362–1373 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Dorman, C. J. H-NS: a universal regulator for a dynamic genome. Nat. Rev. Microbiol. 2, 391–400 (2004).

    Article  CAS  PubMed  Google Scholar 

  29. Tendeng, C. & Bertin, P. N. H-NS in Gram-negative bacteria: a family of multifaceted proteins. Trends Microbiol. 11, 511–518 (2003).

    Article  CAS  PubMed  Google Scholar 

  30. Weinert, B. T. et al. Lysine succinylation is a frequently occurring modification in prokaryotes and eukaryotes and extensively overlaps with acetylation. Cell Rep. 4, 842–851 (2013).

    Article  CAS  PubMed  Google Scholar 

  31. Weinert, B. T. et al. Acetyl-phosphate is a critical determinant of lysine acetylation in E. coli. Mol. Cell 51, 265–272 (2013).

    Article  CAS  PubMed  Google Scholar 

  32. Dilweg, I. W. & Dame, R. T. Post-translational modification of nucleoid-associated proteins: an extra layer of functional modulation in bacteria? Biochem. Soc. Trans. 46, 1381–1392 (2018).

    Article  CAS  PubMed  Google Scholar 

  33. Hommais, F. et al. Large-scale monitoring of pleiotropic regulation of gene expression by the prokaryotic nucleoid-associated protein, H-NS. Mol. Microbiol. 40, 20–36 (2001).

  34. Gao, X. et al. Engineered global regulator H-NS improves the acid tolerance of E. coli. Microb. Cell Fact. 17, 118 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. Krin, E., Danchin, A. & Soutourina, O. Decrypting the H-NS-dependent regulatory cascade of acid stress resistance in Escherichia coli. BMC Microbiol. 10, 273 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. Wang, W., Li, G. W., Chen, C., Xie, X. S. & Zhuang, X. Chromosome organization by a nucleoid-associated protein in live bacteria. Science 333, 1445–1449 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Dame, R. T., Rashid, F. Z. M. & Grainger, D. C. Chromosome organization in bacteria: mechanistic insights into genome structure and function. Nat. Rev. Genet. 21, 227–242 (2020).

    Article  CAS  PubMed  Google Scholar 

  38. Zhao, S., Zhang, X. & Li, H. Beyond histone acetylation-writing and erasing histone acylations. Curr. Opin. Struct. Biol. 53, 169–177 (2018).

    Article  CAS  PubMed  Google Scholar 

  39. Kaczmarska, Z. et al. Structure of p300 in complex with acyl-CoA variants. Nat. Chem. Biol. 13, 21–29 (2017).

    Article  CAS  PubMed  Google Scholar 

  40. Liu, X. et al. NAT10 regulates p53 activation through acetylating p53 at K120 and ubiquitinating Mdm2. EMBO Rep. 17, 349–366 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Ito, S. et al. Human NAT10 is an ATP-dependent RNA acetyltransferase responsible for N4-acetylcytidine formation in 18 S ribosomal RNA (rRNA). J. Biol. Chem. 289, 35724–35730 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Dame, R. T., Noom, M. C. & Wuite, G. J. Bacterial chromatin organization by H-NS protein unravelled using dual DNA manipulation. Nature 444, 387–390 (2006).

    Article  CAS  PubMed  Google Scholar 

  43. Krulwich, T. A., Sachs, G. & Padan, E. Molecular aspects of bacterial pH sensing and homeostasis. Nat. Rev. Microbiol. 9, 330–343 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Morris, G. M. et al. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J. Comput. Chem. 30, 2785–2791 (2009).

  45. Pettersen, E. F. et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  46. Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

    PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by funding from the National Natural Science Foundation of China to K.Z. (nos. 21874100 and 22074103), to H.D. (no. 32101023), to G.Z. (no. 21904097) and to X.B. (no. 22004091); and from the Talent Excellence Program from Tianjin Medical University, to K.Z.

Author information

Authors and Affiliations

  1. The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Key Laboratory of Breast Cancer Prevention and Therapy (Ministry of Education), Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University Cancer Institute and Hospital, Tianjin Medical University General Hospital, Tianjin Medical University, Tianjin, China

    Hanyang Dong, Yujie Zhao, Yue Han, Jianji Zhang, Xue Bai, Guijin Zhai, Hui Zhang, Shanshan Tian & Kai Zhang

  2. The Key Laboratory of Molecular Biology for High Cancer Incidence Coastal Chaoshan Area, Department of Biochemistry and Molecular Biology, Shantou University Medical College, Shantou, China

    Hanyang Dong & Liyan Xu

  3. Tianjin Key Laboratory of Radiation Medicine and Molecular Nuclear Medicine, Institute of Radiation Medicine, Peking Union Medical College & Chinese Academy of Medical Sciences, Tianjin, China

    Changfen Bi

  4. Tianjin Key Laboratory of Medical Epigenetics, Department of Cell Biology, Tianjin Medical University, Tianjin, China

    Deqing Hu

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Contributions

K.Z. supervised experiments. H.D. and K.Z. designed experiments, analyzed the data and wrote the manuscript. H.D., J.Z. and Y.Z. carried out cell culture, enzymatic activity assay and molecular biological experiments. H.D., G.Z. and X.B. carried out proteomic survey. C.B. and H.D. carried out chemical synthesis. H.D., Y.H., S.T., D.H., L.X. and K.Z. carried out data collection, analysis and interpretation. All authors discussed the results and commented on the manuscript.

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Correspondence to Kai Zhang.

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Peer review information Nature Chemical Biology thanks the anonymous reviewers for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Identification TmcA as potential lysine 2-hyroxyisobutyryltransferase, not a lysine acetyltransferase.

a, Overexpression of TmcA and YiaC increase Khib in E. coli. E. coli BL21 were transferred with empty vector (pET28a) as control and GNAT-pET28a vectors for GNAT overexpressing strains, the Khib level of whole cell lysates were analyzed by western blotting. b, Overexpression of TmcA has no obvious effect on Kac in E. coli. E. coli BL21 were transferred with empty vector (pET28a) as control and TmcA-pET28a vector overexpressing strains, the Kac level of whole cell lysates were analyzed by western blotting. c, TmcA KO has no obvious effect on Kac in E. coli. Kac levels of TmcA KO E. coli MG1655 were analyzed by western blotting, WT as control. And each group had three biological repetitions. All western experiments had three biological repetitions with similar result.

Extended Data Fig. 2 TmcA can bind with 2-hydroxyisobutyryl-CoA.

a, Structural modeling of TmcA combining acetyl-CoA. Exhibition the crystal structure of TmcA combining acetyl-CoA by UCSF Chimera. Ac-CoA: acetyl-CoA. Related to Fig. 2a. b, Structural modeling of TmcA combining 2-hydroxyisobutyryl-CoA. Using Autodock modes the binding of 2-hydroxyisobutyryl-CoA with TmcA and exhibits the crystal structure by UCSF Chimera. Hib-CoA: 2-hydroxyisobutyryl-CoA. c, Structural modeling of TmcA combining Hib-CoA under different setting of Box. Small box:100 Å × 60 Å × 60 Å, large box:120 Å × 80 Å × 80 Å. d, The hydrogen bonds and hydrophobic interaction between TmcA and Hib-CoA are shown by LigPlot+ with large box. e-f, ITC analysis of the affinity of recombinant W504A TmcA mutant with Ac-CoA and Hib-CoA separately, and each group had three biological repetitions, each group had three biological repetitions with similar result.

Extended Data Fig. 3 LC-MS/MS detection ac4C intracellularly and in vitro.

a, ac4C levels of wild type E.coli (WT), TmcA KO (KO), complementary TmcA (TmcA) and complementary TmcA R502 (TmcA R502) strains were detected by LC-MS/MS, each with three biological repetitions (data are presented as means ± SEM). b, The synthetic tRNAMet incubated with TmcA or TmcA R502 and detected the ac4C levels by LC-MS/MS. Each group had three biological repetitions with similar result.

Extended Data Fig. 4 Profiling the the endogenous substrate proteins for acetylation by TmcA in E. coli.

a, Schematic representation of experimental workflow for the SILAC quantification of Khib in WT and TmcA KO E. coli. b, The histograms show experimentally determined relative protein abundance distributions for the samples used to analyze acetylation.

Extended Data Fig. 5 TmcA-mediated Khib specifically related to transcription and translation.

Shown is an interaction network of the TmcA-regulated Khib proteome based on the STRING database (v10). The network is visualized in Cytoscape 3.8.

Extended Data Fig. 6 The MS/MS spectrum of the intracellular tryptic peptide bearing Khib and its counterpart synthetic peptide.

a, MS/MS spectrum of ASDVK(hib)DSSIR (sythesis, intracellular and both mixture) and the extracted ion chromatograms. b, Multiple protein sequences alignment of H-NS from different species.

Extended Data Fig. 7 Volcano plots showing differentially expressed genes in different E. coli MG1655 strains.

WT: wild type E. coli MG1655, O: H-NS KO E. coli MG1655, K: H-NS KO strain harboring H-NS, Q: H-NS KO strain harboring H-NS K121Q, R: H-NS KO strain harboring H-NS K121R. FDR ANOVA < 0.01, n = 3 biological repetitions.

Extended Data Fig. 8 Heatmap of acid resistance related gene expression values of different H-NS related strains.

Rows show Z scores calculated for each group. Wt: wild type E. coli MG1655, O: H-NS KO E. coli MG1655, K: H-NS KO strain harboring H-NS, Q: H-NS KO strain harboring H-NS K121Q, R: H-NS KO strain harboring H-NS K121R.

Extended Data Fig. 9 Acid resistance related genes were increased in Q strain compared with K strain.

a, AR2, AR3 and AR4 are the different mechanisms for acid resistance in E. coli, red numbers represent the difference multiple of gene expression compared Q versus K. The red numbers represent the multiple of gene up regulation. b-e, RT-qPCR detecting mRNA levels of acid resistance related genes in WT, TmcA KO strain, overexpressing TmcA strain and overexpressing TmcA R502A strain, and each group had three biological repetitions and each biological sample had three repetitions. (data are presented as means ± SEM, Two-tailed t-test, n = 3 biological repetitions).

Supplementary information

Supplementary Information

Supplementary Figs. 1–4 and Tables 1, 2, 5 and 6.

Reporting Summary

Supplementary Tables

Supplementary Table 3. Quantification results for 2-hydroisobutyrylated proteomes with TmcA KO E. coli and WT E. coli. Supplementary Table 4. Quantification results for acetylated proteomes with TmcA KO E. coli and WT E. coli.

Source data

Source Data Fig. 4

Unprocessed immunoblots and gels for Fig. 4.

Source Data Fig. 5

Unprocessed immunoblots and gels for Fig. 5.

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Dong, H., Zhao, Y., Bi, C. et al. TmcA functions as a lysine 2-hydroxyisobutyryltransferase to regulate transcription. Nat Chem Biol 18, 142–151 (2022). https://doi.org/10.1038/s41589-021-00906-3

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