Novel Mechanism of Glycosylation Regulating Pancreatic Cancer Reveled
Pancreatic cancer is a highly malignant digestive system tumor, and the five-year survival rate of patients after diagnosis is only about 10%. Changes in metabolism are one of the important features of tumor cells. Tumor cells undergo metabolic reprogramming to generate the substances, energy, and redox forces required for their rapid proliferation. The proliferation of pancreatic ductal adenocarcinoma (PDAC, the most common pathological type in pancreatic cancer) with mutations in the oncogene Kras is highly dependent on the glutamine (Gln) catabolic pathway. In this pathway, Gln is first converted to aspartate (Asp), followed by oxaloacetate (OAA) catalyzed by aspartate aminotransferase (GOT1), which is further converted to malate catalyzed by malate dehydrogenase (MDH1), and then oxidized by malate (ME1) to finally form pyruvate and NADPH; the latter provides reducing equivalents and maintains intracellular redox balance. This pathway is essential for the proliferation and survival of PDAC cells, so a deep understanding of the regulatory mechanism of this pathway is helpful to provide new ideas and targets for the clinical treatment of PDAC.
O-GlcNAc glycosylation is a post-translational modification in which N-acetylglucosamine is covalently linked to the serine (Ser) or threonine (Thr) hydroxyl group of proteins in the form of a β-glycosidic bond. This modification is a highly dynamic modification that changes in response to intracellular nutritional status and extracellular stimuli. This modification occurs widely on proteins in cells and regulates important biological processes such as gene transcription, signal transduction, protein synthesis and metabolic reprogramming. Previous studies have shown that the level of O-GlcNAc glycosylation is abnormally increased in PDAC. However, the molecular mechanism by which O-GlcNAc glycosylation regulates the occurrence and development of PDAC remains unclear.
Recently, Professor Yi Wen's research group and Professor Zhou Ruhong's research group from the School of Life Sciences, Zhejiang University jointly published a research paper titled: O-GlcNAcylation promotes pancreatic tumor growth by regulating malate dehydrogenase 1 in Nature Chemical Biology.
This study revealed the molecular mechanism of O-GlcNAc glycosylation regulating glutamine metabolism and promoting the growth of PDAC through the interdisciplinary approach of glycochemical biology, tumor biology and computational biology.
The study first found that the expression of OGT, a glycosyltransferase that mediates O-GlcNAc modification, was significantly increased in pancreatic cancer patient tissues; knockdown of OGT significantly inhibited the Gln metabolism of PDAC cells and inhibited the proliferation of PDAC cells.
Further studies found that O-GlcNAc modification had a significant effect on the function of MDH1, a key enzyme in the Gln metabolic pathway. Ser189 was identified as the glycosylation modification site of MDH1 by high-resolution mass spectrometry combined with point mutation. In vitro enzyme activity experiments showed that glycosylation at Ser189 could enhance its enzyme activity. Targeted metabolomics experiments confirmed that MDH1 glycosylation promotes Gln metabolism and NADPH production.
In addition, MDH1 is involved in the intracellular malate-aspartate shuttle pathway that coordinates glycolysis and mitochondrial respiration; MDH1 glycosylation will facilitate the regeneration of NADH, thereby promoting mitochondrial respiration. In vivo tumorigenesis experiments in mice and analysis of clinical samples further verified that the glycosylation modification at Ser189 of MDH1 promotes the growth of PDAC.
In order to further understand the molecular mechanism by which O-GlcNAc glycosylation enhances MDH1 enzymatic activity, the authors used all-atom molecular dynamics simulations to investigate the kinetic behavior of MDH1 monomer-substrate (NADH, MAK) complexes in the glycosylated and non-glycosylated states at Ser189. The results showed that the substrate was more stable in the case of glycosylation and had a higher probability of binding. This is due to the protection of the substrate binding pocket by Ser189 O-GlcNAc, prompting enhanced interactions between MDH1 and substrate, and between substrate and substrate; enhanced interactions between MDH1 and substrate mainly arise from the contact contribution of Ser189 O-GlcNAc to protein residues Gln228, Gln229, and Arg98. The corresponding triad mutation (Q228A/Q229A/R98A) activity assay experiments also confirmed this observation.
Furthermore, unlike monomers, MDH1 dimers did not show significant differences in substrate binding in the non-glycosylated and glycosylated states. This may be due to the fact that the helical regions involved in the substrate binding pocket are constrained by dimer interface interactions and are not as flexible as in monomers. Overall, Ser189 O-GlcNAc can act as a 'molecular glue' to improve the substrate by stabilizing the substrate-binding pocket on the MDH1 monomer and enhancing protein-substrate, and substrate-substrate interactions. The binding and stability of the compound ultimately promote the enzymatic activity of MDH1.
Taken together, this study revealed an important function of the OGT-MDH1 axis in the occurrence and development of PDAC. Given that MDH1 is highly expressed in PDAC, and the level of MDH1 glycosylation is positively correlated with PDAC progression, our findings suggest that intervention in MDH1 glycosylation can be a potential strategy for targeting PDAC.
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