Abstract
Nutrient availability strongly affects intestinal homeostasis. Here, we report that low-protein (LP) diets decrease amino acids levels, impair the DNA damage response (DDR), cause DNA damage and exacerbate inflammation in intestinal tissues of male mice with inflammatory bowel disease (IBD). Intriguingly, loss of nuclear fragile X mental retardation-interacting protein 1 (NUFIP1) contributes to the amino acid deficiency-induced impairment of the DDR in vivo and in vitro and induces necroptosis-related spontaneous enteritis. Mechanistically, phosphorylated NUFIP1 binds to replication protein A2 (RPA32) to recruit the ataxia telangiectasia and Rad3-related (ATR)–ATR-interacting protein (ATRIP) complex, triggering the DDR. Consistently, both reintroducing NUFIP1 but not its non-phospho-mutant and inhibition of necroptosis prevent bowel inflammation in male Nufip1 conditional knockout mice. Intestinal inflammation and DNA damage in male mice with IBD can be mitigated by NUFIP1 overexpression. Moreover, NUFIP1 protein levels in the intestine of patients with IBD were found to be significantly decreased. Conclusively, our study uncovers that LP diets contribute to intestinal inflammation by hijacking NUFIP1–DDR signalling and thereby activating necroptosis.
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Data availability
Phosphorylation sites were found in the protein PTM database (https://www.phosphosite.org). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD053976. The dataset GSE111889 was downloaded from the GEO database45 (https://www.ncbi.nlm.nih.gov/geo/). Source data are provided with this paper.
Code availability
No custom codes were used in this study.
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Acknowledgements
We thank members of the Lei laboratory for discussion throughout this study and the Biomedical Core Facility of Fudan University for technical support. We thank S. Bing (Shanghai Institute of Immunology, Department of Immunology and Microbiology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China) for offering the ΔDEPDC5 stable HeLa cell line. This work was supported by the National Key R&D Program of China (2020YFA0803402 and 2019YFA0801703 to Q.-Y.L.), the Natural Science Foundation of China (82121004, 82330092 and 81790250 to Q.-Y.L.; 82103116 to H.M.; 82472873 to M.Y.), the Innovation Program of Shanghai Municipal Education Commission (2023ZKZD11), the Shanghai Municipal Science and Technology Major Project and the New Cornerstone Science Foundation (to Q.-Y.L.).
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H.M., J.T., M.Y. and Q.-Y.L. designed experiments, performed data analyses and wrote the manuscript. H.M. and J.T. performed the most experiments. S.-Y.C., C.-P.Y., Y.-T.Q., C.W., Y.L., L.Z. and J.Y. helped with the methodology. Q.-Y.L. and M.Y. supervised the study.
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Extended data
Extended Data Fig. 1 Low-protein (LP) diets aggravate intestinal DNA damage and inflammation.
a, Representative IHC images of colon tissues from mice fed with AIN-93G, 6% protein, or 60% protein (high-protein, HP) diets and water containing 2% DSS. b, Quantification of IHC of γH2A.X in crypts of colon tissues from mice fed with AIN-93G or 60% protein diets and water containing 2% DSS. n = 73 (AIN-93G) and 69 (60% protein) crypts. 3 independent experiments. c, Quantification of IHC of inflammatory cells markers in colon tissues from mice in (b). Sample size (n) see SourceData_Extended_Fig1. d, Representative IHC images of p-ATR and γH2A.X in colon tissues from mice fed with AIN-93G or LP diets. DDR was induced by intraperitoneal injection of 5-FU. n = 3 mice. e, Western blot of p-CHK1 and γH2A.X in HeLa cells in response to HU treatment and deprivation of individual amino acid. n = 3 independent experiments. f, Relative quantification of p-CHK1 in HeLa cells in response to HU treatment and deprivation of individual amino acid. Normal culture medium as control. n = 3 independent experiments. g, Relative quantification of γH2A.X level in HeLa cells in response to HU treatment and deprivation of individual amino acid. Normal culture medium as control. n = 3 independent experiments. h, Western blot of p-ATR, p-CHK1 and γH2A.X in HeLa cells. 2 mM HU was added to control or GCN2 knockout HeLa cells with or without amino acids starvation. n = 3 independent experiments. i, j, Knockout of GCN1 (i) (n = 3 independent experiments.) or DEPDC5 (j) (n = 3 independent experiments) does not rescue the levels of DDR markers downregulated by amino acids deprivation in HeLa cells. Western blot analyses of levels of p-ATR, p-CHK1 and γH2A.X in cells in response to HU treatment with or without amino acids starvation. Scale bar, 50 µm. Error bars represented mean ± s.d. Statistical comparisons were made using Mann–Whitney U-test (b, c) or one-way ANOVA test (f, g, h, j). NS, not significant.
Extended Data Fig. 2 NUFIP1 is essential for amino acid-regulated DDR.
a, Gene Ontology (GO) analysis of biological processes of chromatin-bound proteins in HeLa cells. The selected proteins were enriched more than 1.5 times in HU treatment group than in NC group. The enriched terms are ranked by -log10 (p values). n = 1. b, c, Western blot of p-ATR, p-CHK1 and γH2A.X in NUFIP1 knockdown (b) and knockout (c) HeLa cells. Cells were treated with 2 mM HU. n = 3 independent experiments. d, e, Representative IHC images of p-ATR (d) and γH2A.X (e) in colon tissues from mice fed with AIN-93G or LP diets. EV or NUFIP1 was overexpressed in colon tissues of mice fed with LP diets using AAV delivery system. DDR was induced by 5-FU treatment. Scale bars, 50 µm. n = 4 mice. Error bars represented mean ± s.d. p values were determined by one-way ANOVA test.
Extended Data Fig. 3 The loss of NUFIP1 triggers spontaneous enteritis.
a, b, Mouse model (a) and schematic representation (b). One dose (400 mg/kg) of tamoxifen was used. c, NUFIP1 is deleted in intestinal epithelium Nufip1(Δ/Δ) iIEC mice. Western blot of NUFIP1 protein level in ileum and colon tissues from Nufip1fl/fl, Nufip1(+/Δ) iIEC and Nufip1(Δ/Δ) iIEC mice at 7 dpi of tamoxifen treatment. Representative results were from two independent mice. d, Nufip1fl/fl, Nufip1(+/Δ) iIEC and Nufip1(Δ/Δ) iIEC mice weight change after tamoxifen treatment. Data were represented as mean±s.d. p values were derived from two-way ANOVA test. n = 6 mice. e, Representative IHC images of Ki-67 and γH2A.X in ileum tissues from Nufip1fl/fl and Nufip1(Δ/Δ) iIEC mice at 7dpi of tamoxifen treatment. n = 6 mice. f, Representative IHC images of Ki-67 and γH2A.X in colon tissues from Nufip1fl/fl and Nufip1(Δ/Δ) iIEC mice at 7dpi of tamoxifen treatment. n = 6 mice. g, Representative IHC images of inflammatory cells markers in ileum tissues from Nufip1fl/fl and Nufip1(Δ/Δ) iIEC mice at 7 dpi of tamoxifen treatment. n = 6 mice. h, Representative IHC images of inflammatory cells markers in colon tissues of Nufip1fl/fl and Nufip1(Δ/Δ) iIEC mice at 7 dpi of tamoxifen treatment. n = 6 mice. i, Western blot analysis of NUFIP1 in ileum and colon tissues from Nufip1fl/fl, Nufip1(+/Δ) iIEC and Nufip1(Δ/Δ) iIEC mice at 28 dpi of tamoxifen treatment. Representative results were from two independent mice. j, Representative H&E staining images of ileum and colon sections from Nufip1fl/fl and Nufip1(Δ/Δ) iIEC mice at 28 dpi of tamoxifen treatment. n = 3 mice. Scale bar, 50 µm.
Extended Data Fig. 4 Phosphorylation of NUFIP1 determines its function on DDR signaling.
a, Phos-tag blot analysis of NUFIP1. n = 3 independent experiments. b, Phos-tag blot analysis of NUFIP1WT, NUFIP1S292A, NUFIP1S403A and NUFIP1S292/403A. n = 3 independent experiments. c, Anti-p-NUFIP1 (S292) antibody specifically recognizes site-specific (S292) phospho-peptide of NUFIP1. n = 3 independent experiments. d, NUFIP1S292A mutant decreases phosphorylation level of p-NUFIP1. n = 3 independent experiments. e, Phos-tag blot analysis of NUFIP1 in HeLa cells treated with kinase inhibitors. No. 1 VE822. No. 2 AZ20. No. 3 RGB-286638. No. 4 AZD-5438. No. 5 THZ531. No. 6 Ralimetinib dimesylate. No. 7 AZ304. No. 8 PF-04691502. No.9 CLK-IN-T3. n = 3 independent experiments. f, Phos-tag blot analysis of NUFIP1WT, NUFIP1S292A, or NUFIP1S403A in HeLa cells treated with RGB-286638 or AZD-5438. n = 3 independent experiments. g, Detection of p-S292 level using anti-pS292 antibody after overexpression of kinases. FLAG-NUFIP1 was obtained from HEK293T cells by immunoprecipitation. n = 3 independent experiments. h, Quantification of p-NUFIP1 after overexpression kinases. i, Detection of NUFIP1 p-S292 level with p-S292 antibody after overexpression of CDK9WT or CDK9D167N. n = 3 independent experiments. j, Western blot of p-NUFIP1 based on in vitro kinase assay. n = 3 independent experiments. k, Western blot of p-NUFIP1, p-ATR, p-CHK1 and γH2A.X in HeLa cells. Control and CDK9 knockdown cells were treated with HU. n = 3 independent experiments. l, CDK9 protein level in chromatin fraction. n = 3 independent experiments. m, Phos-tag blot analysis of NUFIP1WT and NUFIP1S283A. n = 3 independent experiments. n, Colon tissue was obtained from Nufip1fl/fl and AAV-infected Nufip1(Δ/Δ) iIEC mice at 7dpi of tamoxifen treatment. Representative results were from two independent mice. o, Representative IHC images of Ki-67 and γH2A.X in colon tissue from mice in (n). n = 6 mice. p, Representative IHC images of inflammatory cells markers in colon tissues from mice in (n). n = 6 mice. Scale bar, 50 µm. The data were represented as mean±s.d. p values in (e, h, i, k, l) were determined by one-way ANOVA test. p values in (j) were determined by two-tailed unpaired t-test.
Extended Data Fig. 5 LC3B-interacting regions (LIRs) of NUFIP1 is irrelevant to its function on DDR regulation.
a, Western blot of p-ATR, p-CHK1 and γH2A.X in HEK293T cells. 2 mM HU was added to cells with or without amino acids starvation. n = 3 independent experiments. b, Schematic representation of AAV-infected Nufip1(Δ/Δ) iIEC mice. Mice was fed with 6% protein diets and DDR was induced by 5-FU treatment. c, Proteins were isolated from colon tissue of mice in (b) and examined with indicated antibodies. Representative results were from three independent mice. d, The representative IHC images of p-ATR and γH2A.X in colon tissues from mice in (b). n = 3 mice. e, Quantification of IHC of p-ATR and γH2A.X in colonic crypts from mice in (b). n = 61 crypts. 3 independent experiments. Scale bar, 50 µm. The data were represented as mean±s.d. Statistical comparisons were made using one-way ANOVA test (a) or Kolmogorov-Smirnov test (e).
Extended Data Fig. 6 NUFIP1 facilitates the recruitment of ATRIP/ATR complex to chromatin upon DNA damage.
a, Chromatin fraction was obtained from normal or NUFIP1 knockdown HeLa cells treated with HU. Proteins were detected using indicated antibodies. n = 3 independent experiments. b, Chromatin fraction was obtained from NUFIP1-silenced HeLa cells with NUFIP1WT or NUFIP1S292A putback. Proteins were detected with indicated antibodies. n = 3 independent experiments. c,d, Representative immunofluorescent staining images of ATR (c) and ATRIP (d) in normal or NUFIP1 knockdown HeLa cells treated with HU. The nucleus was circled by the dashed line. RPA32 as the negative control. Blue, DAPI. Green, RPA32. Red, ATR/ATRIP. Scale bar, 10 µm. n = 3 independent experiments. e, Proteins were obtained from HU-treated HeLa cells overexpressing FLAG-NUFIP1 by immunoprecipitation and examined using indicated antibodies. FLAG-HNRNPU as the negative control. n = 3 independent experiments.
Extended Data Fig. 7 Loss of NUFIP1 or amino acids deficiency enhances necroptosis in intestine.
a, Representative images and statistical analysis of immunofluorescent staining of Cleaved Caspase-3 (CC3) in ileum and colon tissues from Nufip1fl/fl and Nufip1(Δ/Δ) iIEC mice at 7-dpi. Blue, DAPI. Red, CC3. Sample size (n) see SourceData_Extended_Fig7. b, PI tracing of intestinal organoids isolated from Villin-creERT2 Nufip1fl/fl mice. Organoids were treated with 4-OHT in presence or absence of zVAD-fmk, as indicated. The right panel is the statistical analysis of relative PI area per organoid (n = 3 independent experiments. Representative results are from 9 organoids). c, PI tracing was performed with intestinal organoid from Villin-creETR2 Nufip1fl/fl mice. Organoids were treated with 4-OHT in presence or absence of GSK′872, as indicated. The right panel is the statistical analysis of relative PI area per organoid (n = 3 independent experiments. Representative results are from 9 organoids). d, Western blot of RIPK3. Proteins from colon tissues of AAV infected Nufip1(Δ/Δ) iIEC mice were examined using the indicated antibodies. Representative results were from three independent mice. e, Representative IHC images of p-RIPK3 and p-MLKL in colon tissues from AAV-infected Nufip1(Δ/Δ) iIEC mice. Area within the black box was the enlarged view of dashed line area. n = 3 mice. f, Representative IHC images of inflammatory cells markers in colon tissues from AAV-infected Nufip1(Δ/Δ) iIEC mice. Area within black box was the enlarged view of dashed line area. n = 3 mice. g, Representative images of TUNEL assays in colon tissues of mice fed with AIN-93G or 6% protein diets and water containing 2% DSS. Area within the white box was the enlarged view of dashed line area. n = 3 mice. h, Representative IHC images of p-MLKL and p-RIPK3 in colon tissues from mice in (g). Area within the black box was the enlarged view of dashed line area. The red arrowheads denoted dead cells. n = 3 mice. Scale bar, 50 µm. Data were represented as mean ± s.d. Statistical comparisons were made using Mann–Whitney U-test (a) or one-way ANOVA test (b, c) or two-tailed unpaired t test (d).
Extended Data Fig. 8 Enhancing NUFIP1 expression alleviates intestinal inflammation in IBD mouse model.
a, Representative H&E and IHC images of colon tissues from mice infected with AAV and fed with water containing 1.5% DSS. Area within the black box was the enlarged view of dashed line area. Scale bar, 50 µm. n = 4 mice.
Supplementary information
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Ming, H., Tan, J., Cao, SY. et al. NUFIP1 integrates amino acid sensing and DNA damage response to maintain the intestinal homeostasis. Nat Metab (2025). https://doi.org/10.1038/s42255-024-01179-5
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DOI: https://doi.org/10.1038/s42255-024-01179-5