Posters
The Interplay between O-GlcNAcylation and Phosphorylation in diabetic heart
Presentation Type
Poster
Discipline Track
Biomedical ENGR/Technology/Computation
Abstract Type
Research/Clinical
Abstract
Background: Diabetes mellitus prevalence has reached pandemic proportions, and diabetic cardiomyopathy (DC) is a significant and frequently (60%) associated complication. Alteration of myofilament site-specific phosphorylation stoichiometry is evident in experimental and human failing hearts. Another important post-translational modification (PTM) associated with diabetes is OGlcNAcylation which is linked to glucose metabolism.
Methods: In order to find the missing link between hyperglycemia and abnormal cardiac function in diabetic, we perform global myofilament site-specific O-GlcNAcylation and Phosphorylation mapping and quantification in normal and T2D hearts. This data was generated for 3 wild type and 3 diabetic heart challenged with force-frequency response, and one wild type and one diabetic heart challenged with isoproterenol. We studied the mature male ob/ob mice (age 3-4 months) as a model for type 2 diabetes, with agematched C57BL/6J mice as controls. Myofilament proteins were isolated and protein concentration were determined by the Lowry assay and equal amount of proteins (~200 µg) were reduced with 5 mM DTT, alkylated with 15 mM iodoacetamide, and digested by trypsin (trypsin/protein ratio = 1/50). Tryptic peptides were labeled for quantitation with TMT 10plex labels following the manufactures guidelines. The labeled peptides were combined and fractioned offline using XBridge HPLC column. Resulting 96 fractions were combined into 24 fractions for LC-MS/MS runs, while only reserving 10% for the protein abundance analysis, and combining the remaining 45% for BEMAD for O-GlcNAc peptide enrichment and the other 45% for TiO2 enrichment for Phosphorylation.
Results: we present the result of our investigation in the interplay between O-GlcNAcylation and phosphorylation in the diabetic cardiomyopathy. Our results show that there is significantly high correlation between the fold change of phosphorylation and OGlcNAcylation of the same residue in the protein, and among proteins in the same biological pathways. We also investigate whether the proximity on the protein sequence has any effect on the correlation between phosphorylation and O- GlcNAcylation of two intra-protein residues.
Conclusion: There is an interplay between O- GlcNAcylation and phosphorylation among the proteins in the same biological pathways. Also, our result suggests that the closely positioned intra-protein residues have higher correlation between the phosphorylation and O-GlcNAcylation.
Recommended Citation
Das, Amit; Garza Rodriguez, Maria de Lourdes; Ramírez-Correa, Genaro A.; and Ayati, Marzieh, "The Interplay between O-GlcNAcylation and Phosphorylation in diabetic heart" (2023). Research Symposium. 81.
https://scholarworks.utrgv.edu/somrs/2022/posters/81
The Interplay between O-GlcNAcylation and Phosphorylation in diabetic heart
Background: Diabetes mellitus prevalence has reached pandemic proportions, and diabetic cardiomyopathy (DC) is a significant and frequently (60%) associated complication. Alteration of myofilament site-specific phosphorylation stoichiometry is evident in experimental and human failing hearts. Another important post-translational modification (PTM) associated with diabetes is OGlcNAcylation which is linked to glucose metabolism.
Methods: In order to find the missing link between hyperglycemia and abnormal cardiac function in diabetic, we perform global myofilament site-specific O-GlcNAcylation and Phosphorylation mapping and quantification in normal and T2D hearts. This data was generated for 3 wild type and 3 diabetic heart challenged with force-frequency response, and one wild type and one diabetic heart challenged with isoproterenol. We studied the mature male ob/ob mice (age 3-4 months) as a model for type 2 diabetes, with agematched C57BL/6J mice as controls. Myofilament proteins were isolated and protein concentration were determined by the Lowry assay and equal amount of proteins (~200 µg) were reduced with 5 mM DTT, alkylated with 15 mM iodoacetamide, and digested by trypsin (trypsin/protein ratio = 1/50). Tryptic peptides were labeled for quantitation with TMT 10plex labels following the manufactures guidelines. The labeled peptides were combined and fractioned offline using XBridge HPLC column. Resulting 96 fractions were combined into 24 fractions for LC-MS/MS runs, while only reserving 10% for the protein abundance analysis, and combining the remaining 45% for BEMAD for O-GlcNAc peptide enrichment and the other 45% for TiO2 enrichment for Phosphorylation.
Results: we present the result of our investigation in the interplay between O-GlcNAcylation and phosphorylation in the diabetic cardiomyopathy. Our results show that there is significantly high correlation between the fold change of phosphorylation and OGlcNAcylation of the same residue in the protein, and among proteins in the same biological pathways. We also investigate whether the proximity on the protein sequence has any effect on the correlation between phosphorylation and O- GlcNAcylation of two intra-protein residues.
Conclusion: There is an interplay between O- GlcNAcylation and phosphorylation among the proteins in the same biological pathways. Also, our result suggests that the closely positioned intra-protein residues have higher correlation between the phosphorylation and O-GlcNAcylation.