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Proteolytic elimination of N-myristoyl modifications by the Shigella virulence factor IpaJ

Nature volume 496, pages 106–109 (2013)Cite this article

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Abstract

Protein N-myristoylation is a 14-carbon fatty-acid modification that is conserved across eukaryotic species and occurs on nearly 1% of the cellular proteome1,2. The ability of the myristoyl group to facilitate dynamic protein–protein and protein–membrane interactions (known as the myristoyl switch) makes it an essential feature of many signal transduction systems3. Thus pathogenic strategies that facilitate protein demyristoylation would markedly alter the signalling landscape of infected host cells. Here we describe an irreversible mechanism of protein demyristoylation catalysed by invasion plasmid antigen J (IpaJ), a previously uncharacterized Shigella flexneri type III effector protein with cysteine protease activity. A yeast genetic screen for IpaJ substrates identified ADP-ribosylation factor (ARF)1p and ARF2p, small molecular mass GTPases that regulate cargo transport through the Golgi apparatus4. Mass spectrometry showed that IpaJ cleaved the peptide bond between N-myristoylated glycine-2 and asparagine-3 of human ARF1, thereby providing a new mechanism for host secretory inhibition by a bacterial pathogen5,6. We further demonstrate that IpaJ cleaves an array of N-myristoylated proteins involved in cellular growth, signal transduction, autophagasome maturation and organelle function. Taken together, these findings show a previously unrecognized pathogenic mechanism for the site-specific elimination of N-myristoyl protein modification.

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Figure 1: Shigella IpaJ and VirA disrupt Golgi morphology.
Figure 2: IpaJ belongs to the C39-like family of cysteine proteases and targets ARF-family GTPases.
Figure 3: IpaJ cleaves the N-myristoylated glycine of lipidated substrates.
Figure 4: Conformational-dependent cleavage of lipidated substrates by IpaJ.

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References

  1. Farazi, T. A., Waksman, G. & Gordon, J. I. The biology and enzymology of protein N-myristoylation. J. Biol. Chem. 276, 39501–39504 (2001)

    Article  CAS  Google Scholar 

  2. Maurer-Stroh, S. et al. MYRbase: analysis of genome-wide glycine myristoylation enlarges the functional spectrum of eukaryotic myristoylated proteins. Genome Biol. 5, R21 (2004)

    Article  Google Scholar 

  3. McLaughlin, S. & Aderem, A. The myristoyl-electrostatic switch: a modulator of reversible protein-membrane interactions. Trends Biochem. Sci. 20, 272–276 (1995)

    Article  CAS  Google Scholar 

  4. Kahn, R. A. Toward a model for Arf GTPases as regulators of traffic at the Golgi. FEBS Lett. 583, 3872–3879 (2009)

    Article  CAS  Google Scholar 

  5. Dong, N. et al. Structurally distinct bacterial TBC-like GAPs link Arf GTPase to Rab1 inactivation to counteract host defenses. Cell 150, 1029–1041 (2012)

    Article  CAS  Google Scholar 

  6. Mounier, J. et al. Shigella effector IpaB-induced cholesterol relocation disrupts the Golgi complex and recycling network to inhibit host cell secretion. Cell Host Microbe 12, 381–389 (2012)

    Article  CAS  Google Scholar 

  7. Selyunin, A. S. et al. The assembly of a GTPase-kinase signalling complex by a bacterial catalytic scaffold. Nature 469, 107–111 (2011)

    Article  ADS  CAS  Google Scholar 

  8. Ashida, H. et al. Shigella are versatile mucosal pathogens that circumvent the host innate immune system. Curr. Opin. Immunol. 23, 448–455 (2011)

    Article  CAS  Google Scholar 

  9. Way, S. S., Borczuk, A. C., Dominitz, R. & Goldberg, M. B. An essential role for gamma interferon in innate resistance to Shigella flexneri infection. Infect. Immun. 66, 1342–1348 (1998)

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Buysse, J. M., Dunyak, D. S., Hartman, A. B. & Venkatesan, M. M. Identification and molecular characterization of a 27 kDa Shigella flexneri invasion plasmid antigen, IpaJ. Microb. Pathog. 23, 357–369 (1997)

    Article  CAS  Google Scholar 

  11. Liu, M. et al. Sequence analysis and characterization of plasmid pSFD10 from Salmonella choleraesuis. Plasmid 48, 59–63 (2002)

    Article  CAS  Google Scholar 

  12. Michiels, J., Dirix, G., Vanderleyden, J. & Xi, C. Processing and export of peptide pheromones and bacteriocins in Gram-negative bacteria. Trends Microbiol. 9, 164–168 (2001)

    Article  CAS  Google Scholar 

  13. Slagowski, N. L., Kramer, R. W., Morrison, M. F., LaBaer, J. & Lesser, C. F. A functional genomic yeast screen to identify pathogenic bacterial proteins. PLoS Pathog. 4, e9 (2008)

    Article  Google Scholar 

  14. Herman, P. K., Stack, J. H., DeModena, J. A. & Emr, S. D. A novel protein kinase homolog essential for protein sorting to the yeast lysosome-like vacuole. Cell 64, 425–437 (1991)

    Article  CAS  Google Scholar 

  15. Sze, S. K., Ge, Y., Oh, H. & McLafferty, F. W. Top-down mass spectrometry of a 29-kDa protein for characterization of any posttranslational modification to within one residue. Proc. Natl Acad. Sci. USA 99, 1774–1779 (2002)

    Article  ADS  CAS  Google Scholar 

  16. Kelleher, N. L. Top-down proteomics. Anal. Chem. 76, 197A–203A (2004)

    Article  Google Scholar 

  17. Charron, G. et al. Robust fluorescent detection of protein fatty-acylation with chemical reporters. J. Am. Chem. Soc. 131, 4967–4975 (2009)

    Article  CAS  Google Scholar 

  18. Barr, F. A., Puype, M., Vandekerckhove, J. & Warren, G. GRASP65, a protein involved in the stacking of Golgi cisternae. Cell 91, 253–262 (1997)

    Article  CAS  Google Scholar 

  19. Kamps, M. P., Buss, J. E. & Sefton, B. M. Mutation of NH2-terminal glycine of p60src prevents both myristoylation and morphological transformation. Proc. Natl Acad. Sci. USA 82, 4625–4628 (1985)

    Article  ADS  CAS  Google Scholar 

  20. Stumpo, D. J., Graff, J. M., Albert, K. A., Greengard, P. & Blackshear, P. J. Molecular cloning, characterization, and expression of a cDNA encoding the “80- to 87-kDa” myristoylated alanine-rich C kinase substrate: a major cellular substrate for protein kinase C. Proc. Natl Acad. Sci. USA 86, 4012–4016 (1989)

    Article  ADS  CAS  Google Scholar 

  21. Sun, Q. et al. Identification of Barkor as a mammalian autophagy-specific factor for Beclin 1 and class III phosphatidylinositol 3-kinase. Proc. Natl Acad. Sci. USA 105, 19211–19216 (2008)

    Article  ADS  CAS  Google Scholar 

  22. Franco, M., Chardin, P., Chabre, M. & Paris, S. Myristoylation-facilitated binding of the G protein ARF1GDP to membrane phospholipids is required for its activation by a soluble nucleotide exchange factor. J. Biol. Chem. 271, 1573–1578 (1996)

    Article  CAS  Google Scholar 

  23. Liu, Y., Kahn, R. A. & Prestegard, J. H. Structure and membrane interaction of myristoylated ARF1. Structure 17, 79–87 (2009)

    Article  CAS  Google Scholar 

  24. Liu, Y., Kahn, R. A. & Prestegard, J. H. Dynamic structure of membrane-anchored Arf*GTP. Nature Struct. Mol. Biol. 17, 876–881 (2010)

    Article  CAS  Google Scholar 

  25. Shao, F., Merritt, P. M., Bao, Z., Innes, R. W. & Dixon, J. E. A Yersinia effector and a Pseudomonas avirulence protein define a family of cysteine proteases functioning in bacterial pathogenesis. Cell 109, 575–588 (2002)

    Article  CAS  Google Scholar 

  26. Choy, A. et al. The Legionella effector RavZ inhibits host autophagy through irreversible Atg8 deconjugation. Science 338, 1072–1076 (2012)

    Article  ADS  CAS  Google Scholar 

  27. Phalipon, A. & Sansonetti, P. J. Shigellosis: innate mechanisms of inflammatory destruction of the intestinal epithelium, adaptive immune response, and vaccine development. Crit. Rev. Immunol. 23, 371–401 (2003)

    Article  Google Scholar 

  28. Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000)

    Article  ADS  CAS  Google Scholar 

  29. Pei, J., Kim, B. H. & Grishin, N. V. PROMALS3D: a tool for multiple protein sequence and structure alignments. Nucleic Acids Res. 36, 2295–2300 (2008)

    Article  CAS  Google Scholar 

  30. Roth, M. J. et al. Sensitive and reproducible intact mass analysis of complex protein mixtures with superficially porous capillary reversed-phase liquid chromatography mass spectrometry. Anal. Chem. 83, 9586–9592 (2011)

    Article  CAS  Google Scholar 

  31. Shima, D. T., Haldar, K., Pepperkok, R., Watson, R. & Warren, G. Partitioning of the Golgi apparatus during mitosis in living HeLa cells. J. Cell Biol. 137, 1211–1228 (1997)

    Article  CAS  Google Scholar 

  32. Steele-Mortimer, O. Meresse, S. Gorvel, J. P., Toh, B. H. & Finlay, B. B. Biogenesis of Salmonella typhimurium-containing vacuoles in epithelial cells involves interactions with the early endocytic pathway. Cell. Microbiol. 1, 33–49 (1999)

    Article  CAS  Google Scholar 

  33. Shiloh, M. U. et al. Phenotype of mice and macrophages deficient in both phagocyte oxidase and inducible nitric oxide synthase. Immunity 10, 29–38 (1999)

    Article  CAS  Google Scholar 

  34. Rivera, V. M. et al. Regulation of protein secretion through controlled aggregation in the endoplasmic reticulum. Science 287, 826–830 (2000)

    Article  ADS  CAS  Google Scholar 

  35. Soding, J., Biegert, A. & Lupas, A. N. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res. 33, W244–W248 (2005)

    Article  Google Scholar 

  36. Alto, N. M. & Dixon, J. E. Analysis of Rho-GTPase mimicry by a family of bacterial type III effector proteins. Methods Enzymol. 439, 131–143 (2008)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank our colleagues at University of Texas Southwestern Medical Center, specifically K. Orth, Y. M. Chook and J. Seeman, for discussions in preparing this manuscript. We are particularly indebted to L. Kinch, N. Grishin, D. Mitchel, X. Guo, D. Trudgian and S. Perelman for their contributions. We acknowledge the services of the University of Texas Southwestern Medical Center proteomics core, supported by a Cancer Prevention and Research Institute of Texas grant RP120613. A.S.S. was supported by the Howard Hughes Medical Institute International Student Research fellowship. B.A.W. was supported by a National Institutes of Health training grant (NIAID; 5T32AI007520) and S.S.W. by the Burroughs Wellcome Fund. This work was supported by grants from the National Institutes of Health (NIAID; RO1AI083359 and NIGMS; R01GM100486), the Welch Foundation (I-1704) and the Burroughs Wellcome Fund to N.M.A.

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Authors and Affiliations

  1. Department of Microbiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-8816, USA,

    Nikolay Burnaevskiy, Bethany A. Weigele, Andrey S. Selyunin & Neal M. Alto

  2. Department of Pediatrics, Section of Pediatric Infectious Disease, Indiana University School of Medicine, 705 Riley Hospital Drive, ROC 4380, Indianapolis, Indiana 46202, USA,

    Thomas G. Fox

  3. Department of Pathology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-8816, USA,

    Daniel A. Plymire & Steven M. Patrie

  4. Division of Infectious Diseases, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, MLC 7017, Cincinnati, Ohio 45229-3039, USA,

    James M. Ertelt & Sing Sing Way

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  1. Nikolay Burnaevskiy
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Contributions

N.B. and N.M.A. conceived the general ideas for this work. N.B. and N.M.A. planned, performed and interpreted experiments. T.G.F. and A.S.S. generated bacterial knockout strains and performed GTPase assays, respectively. J.M.E. and S.S.W. performed mouse infection studies. B.A.W. performed the bioinformatics studies on IpaJ. D.A.P. and S.M.P. performed top-down mass spectrometry. N.M.A. and N.B. wrote the manuscript and all authors provided editorial input.

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Correspondence to Neal M. Alto.

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The authors declare no competing financial interests.

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Burnaevskiy, N., Fox, T., Plymire, D. et al. Proteolytic elimination of N-myristoyl modifications by the Shigella virulence factor IpaJ. Nature 496, 106–109 (2013). https://doi.org/10.1038/nature12004

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