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An infection-induced oxidation site regulates legumain processing and tumor growth

Nature Chemical Biology volume 18, pages 698–705 (2022)Cite this article

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Abstract

Oxidative stress is a defining feature of most cancers, including those that stem from carcinogenic infections. Reactive oxygen species can drive tumor formation, yet the molecular oxidation events that contribute to tumorigenesis are largely unknown. Here we show that inactivation of a single, redox-sensitive cysteine in the host protease legumain, which is oxidized during infection with the gastric cancer-causing bacterium Helicobacter pylori, accelerates tumor growth. By using chemical proteomics to map cysteine reactivity in human gastric cells, we determined that H. pylori infection induces oxidation of legumain at Cys219. Legumain oxidation dysregulates intracellular legumain processing and decreases the activity of the enzyme in H. pylori-infected cells. We further show that the site-specific loss of Cys219 reactivity increases tumor growth and mortality in a xenograft model. Our findings establish a link between an infection-induced oxidation site and tumorigenesis while underscoring the importance of cysteine reactivity in tumor growth.

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Fig. 1: Legumain Cys219 is oxidized during H. pylori infection.
Fig. 2: H. pylori infection alters prolegumain processing, localization and ubiquitination.
Fig. 3: Legumain Cys219 is required for intracellular prolegumain processing.
Fig. 4: Legumain C219S enhances tumor growth.

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Data availability

Supplementary information is available for this paper. Mass-spectrometry proteomics data that support the findings of this study have been deposited in ProteomeXchange via the PRIDE partner repository with the dataset identifier PXD025841. Source data are provided with this paper.

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Acknowledgements

We thank V. Muthusamy (Center for Precision Cancer Modeling, Yale Cancer Center) and J. Nikolaus (West Campus Imaging Core, Yale University) for experimental assistance. We also thank R. Gaudet, J. MacMicking, R. Morris, R. Wasko, C. Takacs, S. Simon and M. Bogyo for reagents and/or technical support. We are grateful to the Hatzios lab, C. Crews and A. Goodman for comments on the manuscript, and to D. Monack and M. Waldor for helpful discussions. This work was supported by National Institutes of Health (NIH) Training Grant T32 GM067543 to Y.K. and E.M.G., and T32 AI007281 to J.H.B.S.; a Gruber Science Fellowship to E.M.G.; NSF Graduate Research Fellowship DGE 1147470 and a Stanford Graduate Fellowship to C.F.; NIH AI118932, CA116087 and Department of Veterans Affairs BX004447 to T.L.C.; Novo Nordisk Foundation Challenge Programme NNF19OC0056411 to M.R.A.; NIH R35GM134964 to E.W.; NIH R35GM137952, American Cancer Society Institutional Research Grant #IRG 17-172-57, a Pilot Grant from the Yale Cancer Center and a Conquer Cancer Now Award from the Concern Foundation to S.K.H.; and NIH Research Grant CA-16359 from the National Cancer Institute.

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Authors and Affiliations

  1. Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT, USA

    Yekaterina Kovalyova, Elizabeth M. Gordon & Stavroula K. Hatzios

  2. Microbial Sciences Institute, Yale University, West Haven, CT, USA

    Yekaterina Kovalyova, Elizabeth M. Gordon & Stavroula K. Hatzios

  3. Department of Chemistry, Yale University, New Haven, CT, USA

    Yekaterina Kovalyova & Stavroula K. Hatzios

  4. Department of Chemistry, Boston College, Chestnut Hill, MA, USA

    Daniel W. Bak & Eranthie Weerapana

  5. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA

    Connie Fung & Manuel R. Amieva

  6. Department of Pathology, Microbiology and Immunology, Vanderbilt University, Nashville, TN, USA

    Jennifer H. B. Shuman & Timothy L. Cover

  7. Department of Medicine, Vanderbilt University School of Medicine, and Veterans Affairs Tennessee Valley Healthcare System, Nashville, TN, USA

    Timothy L. Cover

  8. Department of Pediatrics, Division of Infectious Diseases, Stanford University School of Medicine, Stanford, CA, USA

    Manuel R. Amieva

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Contributions

Y.K. and S.K.H. developed the concept for the study. Y.K. and D.W.B. carried out formal analysis of the results. Y.K., D.W.B. and E.M.G. undertook the investigation. C.F., J.H.B.S., T.L.C., M.R.A. and E.W. were responsible for the study resources. Y.K. and D.W.B. curated the data. Y.K. and S.K.H. wrote the original draft of the manuscript. All authors reviewed and edited the manuscript. Y.K., D.W.B., E.M.G. and S.K.H. visualized the data. S.K.H. supervised the project. E.W. and S.K.H. undertook administration of the project. T.L.C., M.R.A., E.W. and S.K.H. acquired funding for the work.

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Correspondence to Stavroula K. Hatzios.

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Extended data

Extended Data Fig. 1 H. pylori infection increases ROS accumulation, decreases GSH levels, and enhances protein cysteine sulfenylation in AGS cells.

(a) Intracellular ROS in AGS cells incubated with H. pylori G27MA (MOI 50, 18 h), 5 mM hydrogen peroxide for 1 h, or media alone were quantified using the fluorogenic ROS indicator CM-H2DCFDA (left). Fluorescence measurements were normalized by the total number of live cells per condition (right). (b) Intracellular GSH levels in H. pylori-infected (H. pylori G27MA, MOI 50, 18 h) and uninfected AGS cells (left), normalized by the total number of live cells per condition (right). Data represent three independent experiments. Each circle represents an independent experiment. Bars represent means ± SD. *P = 0.03; **P = 0.007; ***P = 0.005; ****P < 0.0001; n.s., not significant. Two-way analysis of variance (ANOVA) with Šídák’s multiple comparisons test was used for (a); two-tailed unpaired t-test was used for (b). (c) Western blot analysis of biotinylated proteins in H. pylori-infected (H. pylori G27MA, MOI 25, 18 h) and uninfected AGS cells. Proteins labeled with the sulfenic acid-specific probe DYn-2 were conjugated with diazo biotin azide and enriched on streptavidin beads prior to Western blot analysis. Western blot analysis was performed twice with consistent results.

Source data

Extended Data Fig. 2 Supplementary data for Fig. 1b,c.

(a) L/H ratios of peptides identified in IA-enriched samples from H. pylori-infected (H. pylori G27MA, MOI 50, 18 h) and uninfected AGS cells. (b) L/H ratios of peptides identified in unenriched lysates from H. pylori-infected (H. pylori G27MA, MOI 50, 18 h) and uninfected AGS cells. (c) Representative light (red) and heavy (blue) extracted ion chromatographs (EICs) (top) and isotopic envelopes (bottom) of cysteine-containing peptides from IA-enriched samples with protein abundance-corrected L/H ≤ 0.5. The L/H ratio of each peptide is shown above the corresponding EIC. The average L/H ratios (n = 3) of peptides are shown in (a) and (b). Representative L/H ratios of peptides are shown in (c).

Source data

Extended Data Fig. 3 Legumain consistently exhibits reduced cysteine reactivity in H. pylori-infected cells under various infection conditions.

(a) Western blot analysis of legumain in H. pylori-infected (H. pylori G27MA, MOI 50, 18 h) and uninfected KATO III cells before (input) and after IA enrichment. (b) Western blot analysis of legumain in uninfected AGS cells and AGS cells infected with the H. pylori strains G27 or PMSS1 (MOI 50, 18 h) before (input) and after IA enrichment. (c) Western blot analysis of legumain in uninfected AGS cells and AGS cells infected with H. pylori G27MA for 18 h at MOI 25 or 50 before (input) and after IA enrichment. (d) Quantification of AGS cell viability following infection with H. pylori G27MA (MOI 25, 18 h) or incubation with media alone for 18 h. Data represent three independent experiments. Bars represent means ± SD. Each circle represents an independent experiment. n.s., not significant, by two-tailed unpaired t-test. Western blot analyses were performed two (c) or three (a, b) times with consistent results. Band intensities of mature legumain and ACAT1 were normalized by the corresponding band intensities in the uninfected sample.

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Extended Data Fig. 4 Biochemical validation of legumain-deficient KO cells generated via CRISPR/Cas9 genome editing.

Western blot analysis of legumain and actin in WT AGS and KO cells. Western blot analysis was performed three times with consistent results.

Source data

Extended Data Fig. 5 Supplementary data for Fig. 2b.

Western blot analysis of (a) HA (top), protein markers of various subcellular compartments (S6K1, cytosol; VDAC1, mitochondria; CALR, endoplasmic reticulum), and (b) cathepsins B (left) and D (right) before (input) and after (anti-HA IP) lysosome enrichment of H. pylori-infected (H. pylori G27MA, MOI 25, 18 h) and uninfected AGS cells. Western blot analyses were performed three times with consistent results.

Source data

Extended Data Fig. 6 Supplementary data for Fig. 2h.

Western blot analysis of legumain, HA-tagged ubiquitin, and Myc-tagged TRAF6 before (left, input) and after (right, IP) anti-FLAG IP of lysates from H. pylori-infected and uninfected HEK293T cells transiently transfected with FLAG-tagged LGMN, HA-tagged UBB, and/or Myc-tagged TRAF6. Cells were infected with H. pylori G27MA at MOI 25 for 15 h followed by a 3-h incubation in serum-deficient medium. Western blot analyses were performed three times with consistent results.

Source data

Extended Data Fig. 7 Intracellular legumainC219A is not processed to the mature enzyme.

Western blot analysis of legumain in KO cells transfected with WT LGMN or LGMNC219A. Western blot analysis was performed three times with consistent results.

Source data

Extended Data Fig. 8 KO cells expressing WT legumain or legumainC219S exhibit similar NF-κB activity and IL-8 expression during H. pylori infection.

(a) Luminescence of H. pylori-infected (H. pylori G27MA, MOI 25, 18 h) and uninfected KO cells transduced with WT LGMN or LGMNC219S and transiently transfected with the NF-κB-inducible luciferase reporter pNiFty2-Luc. (b) RT-qPCR analysis of IL-8 expression normalized to GAPDH expression in H. pylori-infected (H. pylori G27MA, MOI 25, 18 h) and uninfected KO cells transduced with WT LGMN or LGMNC219S. Data represent three independent experiments. Each circle represents an independent experiment. Bars represent means ± SD. n.s., not significant. Two-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test was used in (a); two-tailed unpaired t-test was used in (b).

Source data

Extended Data Fig. 9 Legumain is expressed in the stomach tissue of H. pylori-infected gerbils.

Representative confocal micrographs with immunofluorescent detection of legumain (LGMN; green) in gastric tissue sections from H. pylori-infected gerbils. Sections with labeled legumain (top) or stained with secondary antibody alone (bottom) are shown. Tissue sections were counterstained with DAPI (blue) and wheat germ agglutinin (WGA; red) to detect nuclei and cellular glycoconjugates, respectively. Sections from six gerbils, using at least two different fields of view per section, were analyzed with consistent results. Scale bar, 40 µm.

Extended Data Fig. 10 Supplementary data for Fig. 4.

(a) Western blot analysis of legumain in two independently derived clonal lines of KO cells transduced with WT LGMN (WT#1 and WT#2) or LGMNC219S (C219S#1 and C219S#2). Western blot analysis was performed three times with consistent results. (b) Cell viability of WT AGS cells and WT#1, WT#2, C219S#1, and C219S#2 cells quantified by alamarBlue fluorescence. Data represent three independent experiments. Error bars represent means ± SD. (c) Average tumor volume of Rag2−/− IL2RG−/− mice subcutaneously implanted with KO cells transduced with WT LGMN or LGMNC219S. These data represent a second independent experiment performed using n = 12 mice and a single clonal cell line per condition (WT#1 and C219S#1). Error bars represent means ± SEM. ****P < 0.0001 by two-tailed unpaired t-test. (d) Survival curve of mice in (c). Tick marks represent censored events. ****P < 0.0001 by Mantel-Cox test.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1−3.

Reporting Summary

Supplementary Data 1

Cysteine peptides quantified by reactivity profiling and total protein abundance quantified by SILAC mass-spectrometry analysis in H. pylori-infected versus uninfected human gastric cells.

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Kovalyova, Y., Bak, D.W., Gordon, E.M. et al. An infection-induced oxidation site regulates legumain processing and tumor growth. Nat Chem Biol 18, 698–705 (2022). https://doi.org/10.1038/s41589-022-00992-x

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