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Fumarate suppresses B-cell activation and function through direct inactivation of LYN

Abstract

Activated B cells increase central carbon metabolism to fulfill their bioenergetic demands, yet the mechanistic basis for this, as well as metabolic regulation in B cells, remains largely unknown. Here, we demonstrate that B-cell activation reprograms the tricarboxylic acid cycle and boosts the expression of fumarate hydratase (FH), leading to decreased cellular fumarate abundance. Fumarate accumulation by FH inhibition or dimethyl-fumarate treatment suppresses B-cell activation, proliferation and antibody production. Mechanistically, fumarate is a covalent inhibitor of tyrosine kinase LYN, a key component of the BCR signaling pathway. Fumarate can directly succinate LYN at C381 and abrogate LYN activity, resulting in a block to B-cell activation and function in vitro and in vivo. Therefore, our findings uncover a previously unappreciated metabolic regulation of B cells, and reveal LYN is a natural sensor of fumarate, connecting cellular metabolism to B-cell antigen receptor signaling.

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Fig. 1: Identification of fumarate as a critical metabolic regulator of B-cell activation.
Fig. 2: Fumarate inhibits B-cell differentiation and function in vivo.
Fig. 3: Fumaric esters succinate LYN and restrain its activity.
Fig. 4: Fumarate suppression of LYN abolishes BCR signaling in vitro and in vivo.
Fig. 5: LYN-C381 is critical for BCR signaling and B-cell activation in vivo.
Fig. 6: LynC381S/C381S transgenic mice display suppressed LYN activity and B-cell function.

Data availability

The source data underlying Figs. 16 as well as Extended Data Figs. 18 are provided as a source data file with this paper. All the other data supporting the findings of this study are available within the article and its supplementary information files, and from the corresponding author on reasonable request. A reporting summary for this article is available as a Supplementary Information file. The FACS gating strategies are described in Supplementary Fig. 1. LYN crystal structure (PDB ID 5XY1) is available on the RCSB PDB. Proteome database (mouse, Mus musculus, UP000000589) is available on the UniProt (https://www.uniprot.org/proteomes/UP000000589).

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Acknowledgements

We thank C. Li at Peking university for his help with statistical analysis. We thank J. Hu at Peking University for insightful discussion and helpful comments. We thank H. Qi, Z. Dong and W. Liu at Tsinghua University for materials and/or comments. We thank G. Li and J. Wu at the Jiang laboratory for their technical assistance and/or discussions. We thank Y. Liu and the Tsinghua University Branch of China National Center for Protein Sciences (Beijing) and Tsinghua University Technology Center for Protein Research for the Cell Function Analyzing Facility support. We thank Y. Zhang and the imaging Core Facility, Technology Center for Protein Sciences of Tsinghua University for assistance using Dragonfly. This research was supported by National Natural Science Foundation of China (grant nos. 82125030 and 81930082) to P.J., National Natural Science Foundation of China (grant nos. 22076003 and 21775006) to X.Z., and the Tsinghua-Peking Center for Life Sciences and National Key R&D Program of China (grant no. 2019YFA0801701) to P.J. J.C. was supported by the Tsinghua-Peking Center for Life Sciences and the China Postdoctoral Science Foundation (grant no. 2020TQ0175). The graphical abstract and working model were created with BioRender.com.

Author information

Authors and Affiliations

Authors

Contributions

J.C. and P.J. designed the experiments. J.C. and J.Y. performed most of the experiments. Y.L. performed the identification of fumarate succination target(s) and measurement of metabolites under the supervision of X.Z. L.Z., Y.Z., X.M. and Yining Chen provided technical assistance. X.S., Yunan Chen and Y.L. performed the molecular dynamic simulation under the supervision of X.Z. and Y.Z. J.C. organized and analyzed the data. P.J. supervised the research. J.C. and P.J. wrote the manuscript. All authors commented on the manuscript.

Corresponding authors

Correspondence to Xinxiang Zhang or Peng Jiang.

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Nature Chemical Biology thanks James Galligan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Fumarate depletion by FH upregulation is essential for B-cell activation.

a, Schematic representation of TCA cycle. b and c, Mouse naive B cells were stimulated by 10 µg/ml (unless otherwise indicated) F(ab’)2 fragments of antimouse IgM antibody (hereafter referred to as anti-IgM) for indicated times or left unstimulated (no stim). Cells were analyzed by western blot (b) and LC-MS for metabolites (c). Cit, Citrate; α-KG, alpha-ketoglutarate; Suc, succinate; Fum, fumarate; Mal, malate; OAA, oxaloacetate. Stim stands for stimulation (n = 3 independent wells). d, Mouse naive B cells were treated with DMSO (Ctrl), indicated metabolites (1 mM) or 50 µM fumarate hydratase-IN-1 (FHi) for 1 hour prior to 24 hours of anti-IgM antibody treatment or left untreated (no stim). Surface expression of CD86+ and CD69+ on B cells were measured by FACS analysis (n = 3 independent wells). e, Mouse naive B cells were treated with DMSO, 50 µM FHi or 100 µM DMF as indicated for 1 hour before activation for indicated times. The percentage of CD86+ and CD69+ B cells was measured (n = 3 independent wells). f and g, Mouse naive B cells were treated with DMSO or increasing amounts of FHi or DMF for 1 hour prior to stimulation without (no stim) or with anti-IgM antibody for 24 hours. The MFI (f) and percentage (g) of CD86+ B cells and CD69+ B cells were measured (n = 3 independent wells). h and i, Mouse naive B cells expressing shFH or shCtrl were stimulated with anti-IgM antibody. The levels of metabolites (i), MFI and percentage of CD86+ and CD69+ B cells, and FH expression were measured (h). n = 3 independent wells. The data are normalized to the average of no stimulation samples(c) or the average of control samples (i). The data in c, g, h, and i are normally distributed, and the differences were evaluated by using 2-tailed Student’s t test. The data in d, e, and f are non-Gaussian distributed, and the differences were evaluated by using 2-tailed Mann-Whitney U test. Data are the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, based on Student’s t-test or Mann-Whitney U test.

Source data

Extended Data Fig. 2 Effect of exogenous fumarate on B-cell death during activation.

a, Mouse naive B cells were stimulated with 10 µg/ml F(ab’)2 fragments of anti-mouse IgM antibody for 1 hour and then treated with DMSO (Ctrl), FHi (50 µM) or DMF (100 µM) for another 24 hours. The percentage of CD86+ B cells and CD69+ B cells was measured by FACS analysis (n = 3 independent wells). Stim stands for stimulation. b, Mouse naive B cells were treated as in Extended Data Fig. 1d. Cell size was measured by FACS analysis. c, Mouse naive B cells were treated as in Fig. 1c. Cell size was measured by FACS analysis. d, Mouse naive B cells were treated as in Extended Data Fig. 1f, g. Cell size was measured by FACS analysis. e, Mouse naive B cells treated with DMSO (Ctrl) or increasing amounts of FHi, DMF or for 1 hour were stimulated with 10 µg/ml F(ab’)2 fragments of anti-mouse IgM antibody for another 48 hours, and cell number was counted (n = 3 independent wells). f, Mouse naive B cells treated with FHi (50 µM), DMF (100 µM) or DMSO (Ctrl) for 1 hour were activated with 10 µg/ml F(ab’)2 fragments of anti-mouse IgM antibody for 48 hours. Cell death was determined by 7AAD staining and FACS analysis. g, Mouse naive B cells were treated as in Extended Data Fig. 1d. Cell were stained with DAPI and analyzed by FACS. h, Mouse naive B cells were treated as in Fig. 1c. Cell were stained with DAPI and analyzed by FACS. i, Mouse naive B cells were treated as in Extended Data Fig. 1f, g. Cell were stained with DAPI and analyzed by FACS. The data in e are normalized to the average of control samples. The data in e are normally distributed, and the differences were evaluated by using 2-tailed Student’s t test. The data in a are non-Gaussian distributed, and the differences were evaluated by using 2-tailed Mann-Whitney U test. Data are the mean ± SD. Each experiment was performed three independent times. *P < 0.05, **P < 0.01, ***P < 0.001, based on Student’s t-test or Mann-Whitney U test.

Source data

Extended Data Fig. 3 Identification of LYN as a succination target for fumarate.

a, The schematic representation of proteome labelling, enrichment, and digestions to provide probe-labelled peptides for MS analysis. LC-MS/MS, liquid-chromatography-MS/MS. b, Gene Ontology (GO) terms enrichment of functional pathways by competitive activity-based protein profiling (P < 0.05). c, B cell lysates were labeled with IA-alkyne probe without (-) or with DMF (100 µM) competition. Then the enriched proteins as well as input proteins were immunoblotted with LYN antibody. d, Related to Fig. 3d. Purified LYN proteins was treated with DMSO (Ctrl) or increasing concentrations of DMF for 30 minutes and LYN kinase activity was measured by ELISA. e and f, Recombinant LYN with DMSO (up left panel), FA (1 mM), MMF (200 µM), or DMF (100 µM) treatment for 30 minutes was digested with trypsin and analyzed by LC-MS/MS for the position(s) of succination (f). Sequences for all the six detected peptide species covering the succinated residues are shown (e). The MASCOT score obtained for each peptide is listed accordingly. g, Purified wild-type (WT) LYN and LYN-C381S proteins were analyzed by SDS-PAGE followed by Coomassie Blue staining (left panel). The proteins were incubated with DMSO (-) or DMF (100 µM) for 30 minutes to assay LYN kinase activity by ELISA (right and bottom panels). The data in d and g are normally distributed, and the differences were evaluated by using 2-tailed Student’s t test. Data are the mean ± SD. Each experiment was performed three independent times. *P < 0.05, **P < 0.01, ***P < 0.001, based on Student’s t-test.

Source data

Extended Data Fig. 4 Structural analysis of possible conformational change caused by C381S mutation in LYN.

a, Structure of the kinase domain containing C381 and ADFG motif of LYN. b, Site C381 (red) is close in sequence to ADFG (residues 384-387, orange). c, Superimposition of LYN (magneta) and LYN-C381S(green). Mutation of C381 (red) may affect the conformation of ADFG motif (orange). d, Comparison of ADFG motif conformations in LYN (magneta) and LYN-C381S (green). e, Ramachandran plot of LYN and LYN-C381S. The dihedral angle scattering of the D site in LYN-C381S is broader than that in LYN. More information please see Methods.

Extended Data Fig. 5 C381 residue structurally controls LYN activity.

a-c, Purified WT and mutant LYN were analyzed by SDS-PAGE followed by Coomassie Blue staining (a). The LYN proteins were incubated with DMSO (Ctrl) or with DMF (100 µM) for 30 minutes. LYN kinase activity was analyzed by ELISA (b), and levels of LYN and phosphorylated LYN-Y396 were determined by western blot (c). d, Ramachandran plot of various LYN-C381 mutants. The data in b is non-Gaussian distributed, and the differences were evaluated by using 2-tailed Mann-Whitney U test. Data are the mean ± SD. Each experiment was performed three independent times. *P < 0.05, **P < 0.01, ***P < 0.001, based on Mann-Whitney U test.

Source data

Extended Data Fig. 6 Fumarate accumulation attenuates BCR signaling.

a, The BCR signaling pathway is illustrated. b, Mouse naive B cells were treated with DMSO (Ctrl), or increasing amounts of FHi or DMF for 1 hour followed by stimulation with 10 µg/ml of F(ab’)2 fragment of anti-mouse IgM antibody (anti-IgM) for 5 minutes or no stimulation (no stim). Cell lysates were prepared and used for immunoblotting with indicated antibodies. c, Mouse naive B cells pretreated with DMSO (Ctrl), 50 µM FHi or 100 µM DMF for 1 hour were stimulated with 10 µg/ml F(ab’)2 fragments of anti-mouse IgM antibody (anti-IgM) for 6 minutes or left unstimulated (no stim). The phosphorylation levels of AKT-S473 and p44/42-T202/204 were analyzed by FACS (n = 3 independent wells). d-f, Mouse naive B cells pretreated with DMSO (Ctrl), FHi (50 µM) or DMF (50 µM) for 30 minutes were stimulated with 10 µg/ml F(ab’)2 fragments of anti-mouse IgM antibody for indicated times, or left unstimulated (no stim). Then cells were subjected to immunostaining with the indicated antibodies followed by confocal immunofluorescence imaging. Scale bars, 30 µm. g, Related to Fig. 4e. Mouse naive B cells were infected with lentivirus expressing LYN shRNA (shLYN) or scramble shRNA (shCtrl) were treated with DMSO (Ctrl), 100 µM DMF, or 50 µM FHi for 1 hour. Then cells were stimulated with 10 µg/ml F(ab’)2 fragments of anti-mouse IgM antibody for 24 hours or left unstimulated (no stim). The percentage of CD86+ B cells and CD69+ B cells was measured by FACS analysis (n = 3 independent wells). The data in c are normally distributed, and the differences were evaluated by using 2-tailed Student’s t test. The data in d-g are non-Gaussian distributed, and the differences were evaluated by using 2-tailed Mann-Whitney U test. Data are the mean ± SD. Each experiment was performed three independent times. *P < 0.05, **P < 0.01, ***P < 0.001, based on Student’s t-test or Mann-Whitney U test.

Source data

Extended Data Fig. 7 Fumarate accumulation impairs B-cell development in CD4-depeleted mice and induces LYN succination in myeloid cells.

a-d, C57BL/6 mice were intraperitoneally (i.p.) injected with anti-CD4 antibody (clone GK1.5, 200 μg/mouse, twice a week) to deplete CD4+ T cells. The mice were then injected intraperitoneally (i.p.) with DMSO (Ctrl) or FHi (25 mg/kg mouse), or treated with DMF (50 mg/kg mouse) by oral gavage for 4 days (n = 6 mice per group), followed by immunization with NP-LPS (10 μg/mouse) for another 7 days. Percentage of B220+ cells (a), germinal center (GC) B cells (b), and memory B cells (c) in spleens, and bone marrow plasma cells (d) was analyzed by FACS analysis. GC B cells (GL7+, Fas+) were pregated on B220+ cells. SP, spleen; PC, plasma cells; BM, bone marrow; GC, germinal center; Mem B, memory B cells. e, Mouse naive B cells pretreated with DMSO (Ctrl), FHi (50 µM) or DMF (50 µM) for 1 hour were stimulated with 10 µg/ml F(ab’)2 fragments of anti-mouse IgM antibody for 5 minutes. LYN protein was immunoprecipitated using anti-LYN antibody and immunoblotted with indicated antibodies. f, Mouse non-B cells were enriched from spleens of C57BL/6 J mice and treated without (-) or with DMF (50 µM) for 1 hour. Mouse bone marrow-derived macrophages (BMDMs) were isolated from C57BL/6 J mice and cultured with 10 ng/mL M-CSF for 7 days before treated without (-) or with DMF (50 µM) for 1 hour. LYN proteins in non-B cells and BMDMs were immunoprecipitated using anti-LYN antibody and immunoblotted with indicated antibodies. The data in a-d are normally distributed, and the differences were evaluated by using 2-tailed Student’s t test. Data are the mean ± SD. Each experiment was performed three independent times. *P < 0.05, **P < 0.01, ***P < 0.001, based on Student’s t-test.

Source data

Extended Data Fig. 8 Generation of LynCS/CS (LynC381S/C381S) KI mice and effect of C381S mutation on BCR signaling.

a, Generation of LynCS/CS (LynC381S/C381S) KI mice. KI mutation was recombinated in oosphere of C57BL/6JGpt background mice by using CRISPR/Cas9 system. After amplification of mice genome, PCR products were analyzed by southern blot (right panel) and sequenced (left panel). b, Mouse naive B cells were treated as in Fig. 5c. the percentage of CD86+ B cells and CD69+ B cells was measured by FACS analysis (n = 3 independent wells). c, Mouse naive B cells enriched and isolated from LynWT/WT and LynCS/CS (LynC381S/C381S) mice were treated with DMSO (Ctrl), FHi (50 µM) or DMF (100 µM) for 1 hour. After stimulation with F(ab’)2 fragments of anti-mouse IgM antibody for 48 hours, cell death was determined by 7AAD staining and FACS analysis. d-f, Related to Fig. 6b. Mouse naive B cells enriched and isolated from LynWT/WT and LynCS/CS (LynC381S/C381S) mice were treated with DMSO (Ctrl), FHi (50 µM) or DMF (100 µM) for 1 hour. After stimulation with 10 µg/ml F(ab’)2 fragments of anti-mouse IgM antibody for 5 min, cells were fixed and penetrated for FACS analysis of the phosphorylation of p44/42-T202/204 (d) and AKT-S473 (e, f). g, A working model illustrating succination of LYN by fumarate suppressing B-cell activation and function. Increased fumarate in naive B cells due to low expression of FH directly succinates LYN at C381 and inhibits LYN activity, resulting in a block to BCR signaling activation. However, during B-cell activation, FH expression is strongly upregulated and consequently intracellular fumarate is reduced, leading to a decrease in LYN succination and activation of LYN and BCR signaling. The data in d and f are normally distributed, and the differences were evaluated by using 2-tailed Student’s t test. The data in b is non-Gaussian distributed, and the differences were evaluated by using 2-tailed Mann-Whitney U test. Data are the mean ± SD. Each experiment was performed three independent times. *P < 0.05, **P < 0.01, ***P < 0.001, based on Student’s t-test or Mann-Whitney U test.

Source data

Supplementary information

Supplementary Information

Supplementary Table 1 and Fig. 1.

Reporting Summary

Supplementary Data 1

List of proteins in A20 cell lysates enriched by using DMF and IA-alkyne for competitive activity-based protein profiling.

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Cheng, J., Liu, Y., Yan, J. et al. Fumarate suppresses B-cell activation and function through direct inactivation of LYN. Nat Chem Biol (2022). https://doi.org/10.1038/s41589-022-01052-0

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