Ufmylation of UFBP1 Is Dispensable for Endoplasmic Reticulum Stress Response, Embryonic Development, and Cardiac and Intestinal Homeostasis

Abstract

Protein modification by ubiquitin fold modifier 1 (UFM1), termed ufmylation, regulates various physiological and pathological processes. Among emerging UFM1 targets, UFM1 binding protein 1 (UFBP1) is the first identified ufmylation substrate. Recent clinical and animal studies have demonstrated the pivotal roles of UFBP1 in development, hematopoiesis, intestinal homeostasis, chondrogenesis, and neuronal development, which has been linked to its function in maintaining endoplasmic reticulum (ER) homeostasis. However, the importance of UFBP1 ufmylation in these cellular and physiological processes has yet to be determined. It has been proposed that ufmylation of lysine 268 (267 in humans) in UFBP1 plays a critical role in mediating the effects of the ufmylation pathway. In this study, we for the first time probe the pathophysiological significance of UFBP1 ufmylation in vivo by creating and characterizing a mouse UFBP1 knockin (KI) model in which the lysine 268 of UFBP1, the amino acid accepting UFM1, was mutated to arginine. Our results showed that the K268R mutation reduced the total ufmylated proteins without altering the expression levels of individual ufmylation enzymes in mouse embryonic fibroblasts. The K268R mutation did not alter ER stress-stimuli-induced ER stress signaling or cell death in mouse embryonic fibroblasts. The homozygous KI mice were viable and morphologically indistinguishable from their littermate wild-type controls up to one year of age. Serial echocardiography revealed no cardiac functional impairment of the homozygous KI mice. Furthermore, the homozygous KI mice exhibited the same susceptibility to dextran sulfate sodium (DSS) -induced colitis as wild-type mice. Taken together, these results suggest that UFBP1 K268 is dispensable for ER stress response, embryonic development, cardiac homeostasis under physiological conditions, and intestinal homeostasis under pathological conditions. Our studies call for future investigations to understand the biological function of UFBP1 ufmylation and offer a new mouse model to determine the roles of UFBP1 ufmylation in different tissues under stress conditions.

Keywords: ER stress response; UFBP1; UFM1; heart failure; intestine; ufmylation.

Figure 1

Creation of UFBP1 K268R knockin (KI) mice. (A) Schematics of the generation of UFBP1 K268R mice via CRISPR–Cas9 gene editing strategy. (B) Validation of K268R mutation in the KI allele by genomic DNA sequencing. (C) Western blot analysis of UFBP1 protein levels in different organs from seven-month-old WT and homozygous (KI) mice.

Figure 2

The impact of UFBP1 K268R KI on ufmylation. (A,B) Western blot analysis (A) and quantification (B) of UFM1 and UFM1 conjugates in total protein lysates from WT and homozygous KI MEFs, n = 3. (C,D) Western blot analysis (C) and quantification (D) of ufmylation pathway components in MEFs, n = 3. *** p < 0.001; **** p < 0.0001. Error bars indicate SEM. Two-way ANOVA, followed by multiple comparisons, was performed.

Figure 3

K268R mutation has no impact on ER stress response. WT and homozygous KI MEFs were treated with tunicamycin (1µg/mL) for 6 h (A,B) or 24 h (C,D) before the harvest for subsequent analyses. (A) The qPCR analyses of indicated ER stress effectors, n = 4. (B) Western blot (B) and quantification of indicated proteins, n = 2. (C) Lactate dehydrogenase (LDH) activity assay, n = 2. Live (green)–Dead (red) viability/cytotoxicity assay (D,E). (D) Representative images of live and dead cell staining. Nuclei were counterstained with Hoechst 33342 (blue). White dotted line indicates the zoom image of selected area. The yellow arrowheads indicate dead cells. Scale bar, 10 µm and quantification of percentage viable cell (E). 7 areas, ~1000 cells per group were counted. Unpaired t–test. * p < 0.05 considered significant; *** p < 0.001. Data mean ± SEM.

Figure 4

K268R mutation does not influence embryonic development. (A) Gross morphology of indicated mouse neonates at postnatal Day 3. (B) Gross morphology of indicated mice at 2 weeks of age. (C) Bodyweight at seven months old, n = +/+ = 9, KI/+ = 9, and KI/KI = 11. (D) Major organs—lung, liver, and kidney–weight normalized to tibia length. One–way ANOVA followed by post hoc Tukey’s multiple comparison test was performed. p < 0.05 considered significant. Data mean ± SEM.

Figure 5

K268R mutation does not cause cardiac dysfunction during aging. (A) Gross morphology of hearts from seven–month–old hearts. (B) Heart weight to body weight ratio of hearts at seven months of age, n = +/+ = 8, KI/+ = 8, and KI/KI = 10. (C) Quantification of echocardiographic data at seven months of age. EF, ejection fraction; FS, fractional shortening; LVPWs, systolic left ventricular posterior wall thickness; LVIDd, diastolic left ventricular internal diameter, n = +/+ = 10, KI/+ = 12, and KI/KI = 14. One–way ANOVA followed by post hoc Tukey’s multiple comparison test was performed. p < 0.05 considered significant. Data mean ± SEM.

Figure 6

K268R mutation does not exacerbate DSS–induced colitis. (A) Scheme of experimental design. Seven–month–old mice were treated with 5% DSS in drinking water for 5 consecutive days and traced for an additional 2 days. (B) Changes in body weight over time. The percentage of body weight loss was calculated relative to the body weight of the individual mouse group at Day 0. (C,D) Clinical scores of anal appearances (C) and diarrhea (D) of DSS–treated mice, n = +/+ = 6, KI/+ = 6, and KI/KI = 7. (E) Representative gross morphology of the colon with and without DSS in each group. (F) Quantification of gastrointestinal length. One–way ANOVA followed by post hoc Tukey’s multiple comparison test was performed. Data mean ± SEM. *** p < 0.001.