Modulating Lysine Crotonylation in Ulcerative Colitis Maintains Mitochondrial Homeostasis: Modulating Crotonylation in Ulcerative Colitis

Abstract

Ulcerative colitis (UC) is a chronic and persistent clinical condition that is challenging to cure. Lysine crotonylation (KCr), a recently discovered post-translational modification (PTM), alters protein structure, stability, localization and activity in a variety of processes including cell differentiation and organism development. This study was designed to elucidate the pathophysiological relevance of KCr in UC and uncover potential underlying mechanisms involved. PTM proteomics was employed to track dynamic alterations in KCr sites and protein level in the colon tissue of dextran sulfate sodium (DSS)-induced UC model mice. Following the validation of differentially crotonylated proteins via Western blot assay, functional and mechanistic analyses of specific KCr sites were conducted in vitro. Gain-of-function or loss-of-function mutations were implemented at selected protein KCr sites. The differentially crotonylated proteins including citrate synthetase (CS) between the colon tissue of DSS-induced mice and control mice were predominantly associated with the tricarboxylic acid (TCA) cycle, as evidenced by significant enrichment in the KEGG pathway analysis. These proteins were primarily localized in mitochondria, suggesting a potential link among UC pathogenesis, mitochondria and the TCA cycle. Collectively, increased KCr restricts inflammasome activation by inducing mitophagy, thereby maintaining mitochondrial homeostasis, reducing oxidative stress and inhibiting apoptosis in UC. KCr represents a potential promising therapeutic target for the treatment of UC.

Keywords: Citrate synthetase; Lysine crotonylation; Mitophagy; Ulcerative colitis.

FIGURE 1

Lower KCr level of enzymes in the TCA cycle is associated with UC. (A) Schematic overview of the DSS‐induced UC model. (B) Representative pictures of mice colon. (C) Representative Western blot images of PTMs in the colon tissue between control and DSS‐induced UC mice. (D) Two‐dimensional scatter plot of PCA distribution. (E) Box plot of RSD distribution. (F) Heatmap of PCC from all quantified proteins. (G) Differential protein sublocalization. (H) KEGG pathway enrichment. ^### p < 0.001 versus control group (n = 6).

FIGURE 2

NaCr promotes KCr to impede the inflammatory progression of UC. (A) Timeline of the animal experiment. Female mice in NaCr group were treated with NaCr (20 mg kg⁻¹). (B) Body weight changes (n = 12). (C) DAI scores (n = 12). (D) Length of colon tissues (n = 6). (E) Liver index, spleen index and thymus index (n = 6). (F) H&E‐staining. (G) Pathological scores (n = 6). The blue arrows represent the complete destruction of ulcers and crypt structure. The red arrow indicates mucosal repair and reduced crypt damage. (H) The contents of IFN‐γ, TNF‐α, IL‐2, IL‐6, IL‐9, IL‐17A, IL‐4, and IL‐10 in serum were detected by ELISA assay (n = 6). (I) NCM460 cells viability was detected using CCK8 assay at 48 h after NaCr treatment. (J) NCM460 cells were treated with LPS (2 µg mL⁻¹). The mRNA expression of proinflammatory cytokines (IL‐6, IL‐1β, TNF‐α, and IL‐4) was determined by qPCR (n = 3). Data were presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 versus DSS group; ^## p < 0.01, ^### p < 0.001 versus control group.

FIGURE 3

Increased KCr in CS enhances the production of citrate catalyzed by CS. (A) Metabolites in TCA cycle. (B) Concentration of citrate catalyzed by CS (n = 6). (C) Concentration of α‐ketoglutarate (n = 6). (D) Concentration of fumarate (n = 6). (E) Concentration of malate (n = 6). (F) Concentration of 2‐hydroxyglutarate (n = 6). (G) Concentration of l‐lactate (n = 6). Mice in NaCr group were treated with NaCr (20 mg kg⁻¹). Data were presented as the mean ± SD. ^* p < 0.05 versus DSS group; ^## p < 0.01 versus control group.

FIGURE 4

Elevated KCr level maintains mitochondrial homeostasis, reduces oxidative stress and inhibits apoptosis. (A) Representative TEM images of mitochondria, autophagosomes, and lysosomes in the colon tissue from control, DSS‐induced UC, and NaCr (20 mg kg^{− 1})‐treated UC mice. Black arrows indicate mitochondrial swelling, edema and reduction or disappearance of cristae structures in the DSS‐induced group. Yellow arrows indicate lysosomes, blue arrows indicate autophagosomes, while red arrows indicate the process of mitophagy. (B) Oxidative stress markers (MDA content, SOD activity, and GSH content) in serum were tested by commercially available kits according to the manufacturer's instructions (n = 8). (C, D) Western blot and quantification of BAX, BCL2, BAD, and BAK with ImageJ (n = 4) in the colon tissue from control, DSS‐induced UC, and NaCr (20 mg kg^{− 1})‐treated UC mice. (E, F) Western blot and quantification of BAX, BCL2, BAD, and BAK with ImageJ (n = 4) in NCM460 cells following the indicated treatment. Data were presented as the mean ± SD. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 versus DSS group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 versus control group.

FIGURE 5

KCr induces mitophagy by activating the PINK1/PARKIN pathway. (A) Representative Western blot images of PINK1, PARKIN, LC3, and P62 in the colon tissue from control, DSS‐induced UC, and NaCr (20 mg kg⁻¹)‐treated UC mice. (B) Quantification of PINK1, PARKIN, LC3, and P62 with ImageJ (n = 4). (C) The mRNA expression of Pink1, Parkin, Lc3, and P62 in the colon tissue from control, DSS‐induced UC, and NaCr (20 mg kg⁻¹)‐treated UC mice were determined by qPCR (n = 3). (D) Representative immunofluorescence images and quantification of LC3 and P62 in the colon tissue from control, DSS‐induced UC, and NaCr (20 mg kg⁻¹)‐treated UC mice (n = 5). (E) Representative Western blot images in NCM460 cells following the indicated treatment. (F) Quantification with ImageJ (n = 4). (G) The mRNA expression of PINK1, PARKIN, LC3, and P62 in NCM460 cells following the indicated treatment were determined by qPCR (n = 3). Data were presented as the mean ± SD. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 versus DSS group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 versus control group.

FIGURE 6

KCr restricts NLRP3 inflammasome activation by inducing mitophagy. (A) Representative Western blot images of NLRP3 in the colon tissue from control, DSS‐induced UC, and NaCr (20 mg kg⁻¹)‐treated UC mice. (B) Quantification of NLRP3 with ImageJ (n = 4). (C) The mRNA expression of Nlrp3 in the colon tissue from control, DSS‐induced UC, and NaCr (20 mg kg⁻¹)‐treated UC mice were determined by qPCR (n = 3). (D) NLRP3 protein levels in NCM460 cells after indicated treatment. (E) Quantification of NLRP3 with ImageJ (n = 4). (F) The mRNA expression of NLRP3 in NCM460 cells following the indicated treatment (n = 3). (G) Representative Western blot images of PINK1, PARKIN, LC3, P62, and NLRP3 in NCM460 cells following the indicated treatment. (H) Quantification of PINK1, PARKIN, LC3, P62, and NLRP3 with ImageJ (n = 4). (I) The mRNA expression of PINK1, PARKIN, LC3, P62, NLRP3, IL‐1β, and TNF‐α in NCM460 cells following the indicated treatmentwere determined by qPCR (n = 3). Data were presented as the mean ± SD. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 versus LPS group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 versus control group, ^& p < 0.05, ^&& p < 0.01, ^&&& p < 0.001 versus CQ group.

FIGURE 7

CS K375 crotonylation induces mitophagy and inhibits NLRP3 inflammasome activation. (A) The level of CS in DSS‐induced mice was detected by proteomics (n = 3). (B) Representative Western blot images of CS in the colon tissue from control, DSS‐induced UC, and NaCr (20 mg kg⁻¹)‐treated UC mice. (C) Quantification of CS with ImageJ (n = 4). (D) The mRNA expression of Cs in the colon tissue from control, DSS‐induced UC, and NaCr (20 mg kg⁻¹)‐treated UC mice were determined by qPCR (n = 3). (E) Representative Western blot images of CS in NCM460 cells following the indicated treatment. (F) Quantification of CS with ImageJ (n = 4). (G) The mRNA expression of CS in NCM460 cells following the indicated treatment (n = 3). (H) The level of CS K375 crotonylation in DSS‐induced mice was detected by PTM proteomics (n = 3). (I) NCM460 cells were treated with LPS (2 µg mL⁻¹) after transfection with plasmids carrying CS with or without KCr site‐specific mutation from K to R or Q for 24 h. Representative Western blot images of PINK1, PARKIN, LC3 P62, and NLRP3 in NCM460 cells following the indicated treatment. (J) Quantification with ImageJ (n = 4). Data were presented as the mean ± SD. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 versus LPS group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 versus control group, ^& p < 0.05, ^&& p < 0.01, ^&&& p < 0.001 versus WT group.