Natasha E. Zachara

Stress granules are transient biomolecular condensates of proteins and RNA formed during stress. Stress granules support cell survival by sequestering components of the translational machinery, suppressing pro-apoptotic signaling, and conserving cellular resources. O-GlcNAcylation, a dynamic modification of cytoplasmic proteins, is an important regulator of stress responses, including stress-granule assembly. O-GlcNAcylation is protective in acute myocardial stress; however, whether O-GlcNAcylation regulates stress granules to protect cardiomyocytes remains unclear. Here, we investigated whether O-GlcNAcylation regulates stress granule formation in cardiomyocytes exposed to sodium arsenite stress or ischemia/reperfusion (I/R) injury. G3BP1 is an RNA-binding protein that is essential in stress granule formation. Using an EGFP–G3BP1 reporter, we found that OGT, the enzyme responsible for installing O-GlcNAc on target proteins, is necessary for efficient stress-granule formation in cardiomyocytes. Importantly, we identified T268 of G3BP1 as an O-GlcNAcylation site in these cells and demonstrated by mutagenesis its functional importance for stress granule assembly. In primary cardiomyocytes subjected to simulated I/R, stress granules formed transiently during ischemia, and this response was decreased by OGT-knockdown. Similarly, stress granules were transiently detected at the ischemic phase of I/R injury, and their numbers were reduced in OGT-deficient hearts. Furthermore, pretreatment with sodium arsenite before I/R was cardioprotective. Likewise, pretreatment with sodium arsenite protected cells from staurosporine-induced death, and this effect was decreased by OGT knockdown. Together, these findings identify OGT and O-GlcNAcylation as important regulators of stress-granule assembly in cardiomyocytes, define G3BP1 as an O-GlcNAc-modified effector, and demonstrate that stress granules can act as cytoprotective mediators during acute cardiac injury.

The treatment of defective glycosylation in clinical practice has been limited to patients with rare and severe phenotypes associated with congenital disorders of glycosylation (CDG). Carried by approximately 5% of the human population, the discovery of the highly pleiotropic, missense mutation in a manganese transporter ZIP8 has exposed under-appreciated roles for Mn homeostasis and aberrant Mn-dependent glycosyltransferases activity leading to defective N-glycosylation in complex human diseases. Here, we test the hypothesis that aberrant N-glycosylation contributes to disease pathogenesis of ZIP8 A391T-associated Crohn’s disease. Analysis of N-glycan branching in intestinal biopsies demonstrates perturbation in active Crohn’s disease and a genotype-dependent effect characterized by increased truncated N-glycans. A mouse model of ZIP8 391-Thr recapitulates the intestinal glycophenotype of patients carrying mutations in ZIP8. Borrowing from therapeutic strategies employed in the treatment of patients with CDGs, oral monosaccharide therapy with N-acetylglucosamine ameliorates the epithelial N-glycan defect, bile acid dyshomeostasis, intestinal permeability, and susceptibility to chemical-induced colitis in a mouse model of ZIP8 391-Thr. Together, these data support ZIP8 391-Thr alters N-glycosylation to contribute to disease pathogenesis, challenging the clinical paradigm that CDGs are limited to patients with rare diseases. Critically, the defect in glycosylation can be targeted with monosaccharide supplementation, providing an opportunity for genotype-driven, personalized medicine.

Background: O-GlcNAcylation is a post-translational modification catalyzed by the enzyme O-GlcNAc transferase, which transfers a single N-acetylglucosamine sugar from UDP-GlcNAc to the protein on serine and threonine residues on proteins. Another enzyme, O-GlcNAcase (OGA), removes this modification. O-GlcNAcylation plays an important role in pathophysiology. Here, we report that O-GlcNAcylation is essential for hepatocyte differentiation, and chronic loss results in fibrosis and HCC. Methods: Single-cell RNA-sequencing (RNA-seq) was used to investigate hepatocyte differentiation in hepatocyte-specific O-GlcNAc transferase-knockout (OGT-KO) mice with decreased hepatic O-GlcNAcylation and in O-GlcNAcase-KO mice with increased O-GlcNAcylation in hepatocytes. Patients HCC samples and the diethylnitrosamine-induced HCC model were used to investigate the effect of modulation of O-GlcNAcylation on the development of liver cancer. Results: Loss of hepatic O-GlcNAcylation resulted in disruption of liver zonation. Periportal hepatocytes were the most affected by loss of differentiation, characterized by dysregulation of glycogen storage and glucose production. O-GlcNAc transferase-KO mice exacerbated diethylnitrosamine-induced HCC development with increased inflammation, fibrosis, and YAP signaling. Consistently, O-GlcNAcase -KO mice with increased hepatic O-GlcNAcylation inhibited diethylnitrosamine-induced HCC. A progressive loss of O-GlcNAcylation was observed in patients with HCC. Conclusions: Our study shows that O-GlcNAcylation is a critical regulator of hepatic differentiation, and loss of O-GlcNAcylation promotes hepatocarcinogenesis. These data highlight increasing O-GlcNAcylation as a potential therapy in chronic liver diseases, including HCC. Export