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Browsing by Author "Chikwana, Vimbai M."
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Item Brain glycogen serves as a critical glucosamine cache required for protein glycosylation(Elsevier, 2021) Sun, Ramon C.; Young, Lyndsay E.A.; Bruntz, Ronald C.; Markussen, Kia H.; Zhou, Zhengqiu; Conroy, Lindsey R.; Hawkinson, Tara R.; Clarke, Harrison A.; Stanback, Alexandra E.; Macedo, Jessica K.A.; Emanuelle, Shane; Brewer, M. Kathryn; Rondon, Alberto L.; Mestas, Annette; Sanders, William C.; Mahalingan, Krishna K.; Tang, Buyun; Chikwana, Vimbai M.; Segvich, Dyann M.; Contreras, Christopher J.; Allenger, Elizabeth J.; Brainson, Christine F.; Johnson, Lance A.; Taylor, Richard E.; Armstrong, Dustin D.; Shaffer, Robert; Waechter, Charles J.; Vander Kooi, Craig W.; DePaoli-Roach, Anna A.; Roach, Peter J.; Hurley, Thomas D.; Drake, Richard R.; Gentry, Matthew S.; Biochemistry and Molecular Biology, School of MedicineGlycosylation defects are a hallmark of many nervous system diseases. However, the molecular and metabolic basis for this pathology is not fully understood. In this study, we found that N-linked protein glycosylation in the brain is metabolically channeled to glucosamine metabolism through glycogenolysis. We discovered that glucosamine is an abundant constituent of brain glycogen, which functions as a glucosamine reservoir for multiple glycoconjugates. We demonstrated the enzymatic incorporation of glucosamine into glycogen by glycogen synthase, and the release by glycogen phosphorylase by biochemical and structural methodologies, in primary astrocytes, and in vivo by isotopic tracing and mass spectrometry. Using two mouse models of glycogen storage diseases, we showed that disruption of brain glycogen metabolism causes global decreases in free pools of UDP-N-acetylglucosamine and N-linked protein glycosylation. These findings revealed fundamental biological roles of brain glycogen in protein glycosylation with direct relevance to multiple human diseases of the central nervous system.Item Discovery and Development of Small-Molecule Inhibitors of Glycogen Synthase(ACS, 2020-03) Tang, Buyun; Frasinyuk, Mykhaylo S.; Chikwana, Vimbai M.; Mahalingan, Krishna K.; Morgan, Cynthia A.; Segvich, Dyann M.; Bondarenko, Svitlana P.; Mrug, Galyna P.; Wyrebek, Przemyslaw; Watt, David S.; DePaoli-Roach, Anna A.; Roach, Peter J.; Hurley, Thomas D.; Biochemistry and Molecular Biology, School of MedicineThe overaccumulation of glycogen appears as a hallmark in various glycogen storage diseases (GSDs), including Pompe, Cori, Andersen, and Lafora disease. Accumulating evidence suggests that suppression of glycogen accumulation represents a potential therapeutic approach for treating these GSDs. Using a fluorescence polarization assay designed to screen for inhibitors of the key glycogen synthetic enzyme, glycogen synthase (GS), we identified a substituted imidazole, (rac)-2-methoxy-4-(1-(2-(1-methylpyrrolidin-2-yl)ethyl)-4-phenyl-1H-imidazol-5-yl)phenol (H23), as a first-in-class inhibitor for yeast GS 2 (yGsy2p). Data from X-ray crystallography at 2.85 Å, as well as kinetic data, revealed that H23 bound within the uridine diphosphate glucose binding pocket of yGsy2p. The high conservation of residues between human and yeast GS in direct contact with H23 informed the development of around 500 H23 analogs. These analogs produced a structure–activity relationship profile that led to the identification of a substituted pyrazole, 4-(4-(4-hydroxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl)pyrogallol, with a 300-fold improved potency against human GS. These substituted pyrazoles possess a promising scaffold for drug development efforts targeting GS activity in GSDs associated with excess glycogen accumulation.Item Incorporation of phosphate into glycogen by glycogen synthase(Elsevier, 2016-05-01) Contreras, Christopher J.; Segvich, Dyann M.; Mahalingan, Krishna; Chikwana, Vimbai M.; Kirley, Terence L.; Hurley, Thomas D.; DePaoli-Roach, Anna A.; Roach, Peter J.; Department of Biochemistry & Molecular Biology, IU School of MedicineThe storage polymer glycogen normally contains small amounts of covalently attached phosphate as phosphomonoesters at C2, C3 and C6 atoms of glucose residues. In the absence of the laforin phosphatase, as in the rare childhood epilepsy Lafora disease, the phosphorylation level is elevated and is associated with abnormal glycogen structure that contributes to the pathology. Laforin therefore likely functions in vivo as a glycogen phosphatase. The mechanism of glycogen phosphorylation is less well-understood. We have reported that glycogen synthase incorporates phosphate into glycogen via a rare side reaction in which glucose-phosphate rather than glucose is transferred to a growing polyglucose chain (Tagliabracci et al. (2011) Cell Metab13, 274-282). We proposed a mechanism to account for phosphorylation at C2 and possibly at C3. Our results have since been challenged (Nitschke et al. (2013) Cell Metab17, 756-767). Here we extend the evidence supporting our conclusion, validating the assay used for the detection of glycogen phosphorylation, measurement of the transfer of (32)P from [β-(32)P]UDP-glucose to glycogen by glycogen synthase. The (32)P associated with the glycogen fraction was stable to ethanol precipitation, SDS-PAGE and gel filtration on Sephadex G50. The (32)P-signal was not affected by inclusion of excess unlabeled UDP before analysis or by treatment with a UDPase, arguing against the signal being due to contaminating [β-(32)P]UDP generated in the reaction. Furthermore, [(32)P]UDP did not bind non-covalently to glycogen. The (32)P associated with glycogen was released by laforin treatment, suggesting that it was present as a phosphomonoester. The conclusion is that glycogen synthase can mediate the introduction of phosphate into glycogen, thereby providing a possible mechanism for C2, and perhaps C3, phosphorylation.