Nonsynonymous single-nucleotide polymorphisms in the G6PC2 gene affect protein expression, enzyme activity, and fasting blood glucose

Overway, Emily M.; Bosma, Karin J.; Claxton, Derek; Oeser, James K.; Singh, Kritika; Breidenbach, Lindsay B.; Mchaourab, Hassane S.; Davis, Lea K.; O’Brien, Richard M.

doi:10.1016/j.jbc.2021.101534

Cited by 10 publications

(9 citation statements)

References 66 publications

(144 reference statements)

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“…Likewise, K76N, G118S, H119L, R170Q, and T255I were catalytically inactive despite similar expression as WT. We further confirmed the impaired activity of K76N and H119L with an in situ luciferase reporter assay that indirectly measures G6Pase activity within intact cells ( 57 , 58 ). This assay capitalized on a cell line derived from rat islets, 832/13, which displays limited endogenous G6Pase activity.…”

Section: Resultssupporting

confidence: 60%

Integrative analysis of pathogenic variants in glucose-6-phosphatase based on an AlphaFold2 model

Sinclair,

Stein,

Sheehan

et al. 2024

PNAS Nexus

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Mediating the terminal reaction of gluconeogenesis and glycogenolysis, the integral membrane protein G6PC1 regulates hepatic glucose production by catalyzing hydrolysis of glucose-6-phosphate (G6P) within the lumen of the endoplasmic reticulum. Consistent with its vital contribution to glucose homeostasis, inactivating mutations in G6PC1 cause glycogen storage disease (GSD) type 1a characterized by hepatomegaly and severe hypoglycemia. Despite its physiological importance, the structural basis of G6P binding to G6PC1 and the molecular disruptions induced by missense mutations within the active site that give rise to GSD type 1a are unknown. Here, we determine the atomic interactions governing G6P binding as well as explore the perturbations imposed by disease-linked missense variants by subjecting an AlphaFold2 G6PC1 structural model to molecular dynamics simulations and in silico predictions of thermodynamic stability validated with robust in vitro and in situ biochemical assays. We identify a collection of side chains, including conserved residues from the signature phosphatidic acid phosphatase motif, that contribute to a hydrogen bonding and van der Waals network stabilizing G6P in the active site. Introduction of GSD type 1a mutations modified the thermodynamic landscape, altered side chain packing and substrate binding interactions, and induced trapping of catalytic intermediates. Our results, which corroborate the high quality of the AF2 model as a guide for experimental design and to interpret outcomes, not only confirm active site structural organization but also suggest novel mechanistic contributions of catalytic and non-catalytic side chains.

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Section: Resultssupporting

confidence: 60%

Integrative analysis of pathogenic variants in glucose-6-phosphatase based on an AlphaFold2 model

Sinclair,

Stein,

Sheehan

et al. 2024

PNAS Nexus

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View full text Add to dashboard Cite

show abstract

“…Likewise, K76N, G118S, H119L and R170Q were catalytically inactive despite similar expression as WT. We further confirmed the impaired activity of K76N and H119L with an in situ luciferase reporter assay that indirectly measures G6Pase activity within intact cells (50, 51). This assay capitalized on a cell line derived from rat islets, 832/13, which displays limited endogenous G6Pase activity.…”

Section: Resultssupporting

confidence: 60%

Molecular mechanisms of catalytic inhibition for active site mutations in glucose-6-phosphatase catalytic subunit 1 linked to glycogen storage disease

Sinclair¹,

Sheehan²,

Hawes³

et al. 2023

Preprint

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Mediating the terminal reaction of gluconeogenesis and glycogenolysis, the integral membrane protein G6PC1 regulates hepatic glucose production by catalyzing hydrolysis of glucose-6-phosphate within the lumen of the endoplasmic reticulum. Because G6PC1 function is essential for blood glucose homeostasis, inactivating mutations cause glycogen storage disease (GSD) type 1a, which is characterized by severe hypoglycemia. Despite its physiological importance, the structural basis of G6P binding to G6PC1 and the molecular disruptions induced by missense mutations within the active site that give rise to GSD type 1a are unknown. Exploiting a computational model of G6PC1 derived from the groundbreaking structure prediction algorithm AlphaFold2 (AF2), we combine molecular dynamics (MD) simulations and computational predictions of thermodynamic stability with a robust in vitro screening platform to define the atomic interactions governing G6P binding within the active site as well as explore the energetic perturbations imposed by disease-linked variants. From over 15 μs of MD simulations, we identify a collection of side chains, including conserved residues from the signature phosphatidic acid phosphatase motif, that contribute to a hydrogen bonding and van der Waals network that stabilize G6P in the active site. Introduction of GSD type 1a mutations into the G6PC1 sequence causes changes in G6P binding energy, thermodynamic stability and structural properties, suggesting multiple mechanisms of catalytic impairment. Our results, which corroborate the high quality of the AF2 model as a guide for experimental design and to interpret outcomes, not only confirm active site structural organization but also suggest novel mechanistic contributions of catalytic side chains.

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“…Several genome-wide association studies (GWAS) have identified multiple genetic variants in G6PC2 that are associated with FBG levels ( 38 , 39 ), with studies suggesting that single-nucleotide polymorphisms (SNPs) in G6PC2 affect RNA splicing ( 40 ), protein expression ( 41 , 42 ), and DNA methylation ( 43 ). Consistent with previous findings ( 4 ), our results here show that knockdown of G6PC2 increases insulin secretion at submaximal glucose concentrations.…”

Section: Discussionmentioning

confidence: 99%

“…Microsome isolation and in vitro G6Pase activity measurements were performed as previously described ( 42 ). Briefly, βTC3 WT or KO cells in six 3.5-cm diameter dishes were harvested and resuspended in 1 ml of solution containing 50 mM Tris HCl (pH 8.0), 50 mM NaCl, 0.5 mM EDTA, and 10% glycerol at 4 °C.…”

Section: Methodsmentioning

confidence: 99%

Glucose-6-phosphatase catalytic subunit 2 negatively regulates glucose oxidation and insulin secretion in pancreatic β-cells

Rahim

Nakhe

Banerjee

et al. 2022

Journal of Biological Chemistry

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Nonsynonymous single-nucleotide polymorphisms in the G6PC2 gene affect protein expression, enzyme activity, and fasting blood glucose

Cited by 10 publications

References 66 publications

Integrative analysis of pathogenic variants in glucose-6-phosphatase based on an AlphaFold2 model

Integrative analysis of pathogenic variants in glucose-6-phosphatase based on an AlphaFold2 model

Molecular mechanisms of catalytic inhibition for active site mutations in glucose-6-phosphatase catalytic subunit 1 linked to glycogen storage disease

Glucose-6-phosphatase catalytic subunit 2 negatively regulates glucose oxidation and insulin secretion in pancreatic β-cells

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