Biophysical and functional properties of purified glucose-6-phosphatase catalytic subunit 1

Claxton, Derek; Overway, Emily M.; Oeser, James K.; O’Brien, Richard M.; Mchaourab, Hassane S.

doi:10.1016/j.jbc.2021.101520

Cited by 12 publications

(35 citation statements)

References 84 publications

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“…Moreover, a murine model of GSD type 1a (G6PC1 -/-) recapitulates a similar phenotype as human patients (34,35). Importantly, detergent-solubilized mouse G6PC1 demonstrates elevated structural and catalytic stability relative to human G6PC1 in biochemical assays (33), supporting an unequivocal interpretation of our in vitro studies. Thus, mouse G6PC1, subsequently referred to simply as G6PC1, is an appropriate model for computational studies of substrate binding and experimental evaluation of structural and functional perturbations associated with disease-linked mutations.…”

Section: Structure and Stability Of G6pc1 In A Model Lipid Bilayersupporting

confidence: 75%

“…Measurements of expression and G6P hydrolysis following transfection of adherent HEK293SG cells (Nacetylglucosaminyl-transferase I-negative; ATCC CRL-3022) followed the previously published methods (33).…”

Section: Screening G6pc1 Expression and Activity In Vitromentioning

confidence: 99%

“…Expression and purification of G6PC1 from Sf9 insect cells Heterologous expression and purification of Q14R and D38V followed the previously published protocols with minor adjustments (33). Briefly, G6PC1 in the pFastBac1 vector was expressed as an EGFP fusion that contains a C-terminal His8 tag via baculovirus transduction of Sf9 insect cells for 72 hrs at 27 C.…”

Section: Screening G6pc1 Expression and Activity In Vitromentioning

confidence: 99%

“…The Pi release rate was determined from titration of 0.1 g (2.4 pmol) G6PC1 with G6P at 30 C for 1 min at pH 6.5 as previously described (33). A linear [G6P]-dependent curve collected on ice was subtracted from reaction carried out at 30 C.…”

Section: Measurement Of G6p Hydrolysis and Thermostability With Purif...mentioning

confidence: 99%

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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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Section: Structure and Stability Of G6pc1 In A Model Lipid Bilayersupporting

confidence: 75%

Section: Screening G6pc1 Expression and Activity In Vitromentioning

confidence: 99%

Section: Screening G6pc1 Expression and Activity In Vitromentioning

confidence: 99%

Section: Measurement Of G6p Hydrolysis and Thermostability With Purif...mentioning

confidence: 99%

See 2 more Smart Citations

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

Self Cite

View full text Add to dashboard Cite

show abstract

“…Several genes were found to be upregulated, with members of the solute carrier (SLC) transporter superfamily such as Slc22a7 , Slc01b3 , Slc30a10 , and acetyl‐CoA synthetases such as Acsm2a, Acsm2b being the most significantly expressed (L. Lin et al, 2015; Watkins et al, 2007). Yet another highly upregulated gene was the G6pc1 gene which encodes for the catalytic subunit of Glucose 6‐phosphatase that catalyzes the hydrolysis of glucose 6‐phosphate (G6P) to glucose and inorganic phosphate, and thus, being involved in the last step of gluconeogenesis and glycogenolytic pathways (Claxton et al, 2022). This agrees with previous reports that suggest circadian regulation of glucose metabolism in the liver (Lamia et al, 2008).…”

Section: Resultsmentioning

confidence: 99%

Influence of circadian rhythm on drug metabolism in 3D hepatic spheroids

Narayanan

Rodrigues

Dordick

2022

Biotech & Bioengineering

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Circadian rhythms are characterized as oscillations that fluctuate based on a 24 h cycle and are responsible for regulation of physiological functions. While the internal clock synchronizes gene expression using external cues like light, a similar synchronization can be induced in vitro by incubating the cells with an increased percentage of serum followed by its rapid removal. Previous studies have suggested that synchronization of HepG2 cell line induced the rhythmic expression of drugmetabolizing enzymes (DME) most specifically the cytochrome P450 enzymes.However, there is a lack of evidence demonstrating the influence of threedimensional microenvironment on the rhythmicity of these genes. To understand this interplay, gene expression of the circadian machinery and CYP450s were compared using the model human hepatocarcinoma cell line, HepG2. Upon serum shock synchronization, gene and protein expression of core clock regulators was assessed and rhythmic expression of these genes was demonstrated. Further insight into the interrelations between various gene pairs was obtained using statistical analysis. Using RNA sequencing, an in-depth understanding of the widespread effects of circadian regulation on genes involved in metabolic processes in the liver was obtained. This study aids in the better understanding of chronopharmacokinetic events in humans using physiologically relevant 3D culture systems.

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Combined toxic effects of fluxapyroxad and multi-walled carbon nanotubes in Xenopus laevis larvae

Zhao,

Luo,

Jiao

et al. 2024

Chemosphere

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Biophysical and functional properties of purified glucose-6-phosphatase catalytic subunit 1

Cited by 12 publications

References 84 publications

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

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

Influence of circadian rhythm on drug metabolism in 3D hepatic spheroids

Combined toxic effects of fluxapyroxad and multi-walled carbon nanotubes in Xenopus laevis larvae

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