Reconstitution and purification of the D-glucose transporter from human erythrocytes.

Kasahara, Michihiro; Hinkle, Peter C.

doi:10.1016/s0021-9258(19)66976-0

Cited by 597 publications

(125 citation statements)

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“…Glucose transporter 1 (GLUT1) is the first glucose transporter discovered and one of the glucose transporters with the greatest affinity for glucose (1). GLUT1 is encoded by the solute carrier family 2A1 (SLC2A1) gene (2), and its crystal structure was analyzed for the first time in 2014 (3).…”

Section: Introductionmentioning

confidence: 99%

Comprehensive Analysis of GLUT1 Immune Infiltrates and ceRNA Network in Human Esophageal Carcinoma

Liu

Gao

et al. 2021

Front. Oncol.

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BackgroundGlucose transporter 1 (GLUT1) is encoded by the solute carrier family 2A1 (SLC2A1) gene and is one of the glucose transporters with the greatest affinity for glucose. Abnormal expression of GLUT1 is associated with a variety of cancers. However, the biological role of GLUT1 in esophageal carcinoma (ESCA) remains to be determined.MethodsWe analyzed the expression of GLUT1 in pan-cancer and ESCA as well as clinicopathological analysis through multiple databases. Use R and STRING to perform GO/KEGG function enrichment and PPI analysis for GLUT1 co-expression. TIMER and CIBERSORT were used to analyze the relationship between GLUT1 expression and immune infiltration in ESCA. The TCGA ESCA cohort was used to analyze the relationship between GLUT1 expression and m6A modification in ESCA, and to construct a regulatory network in line with the ceRNA hypothesis.ResultsGLUT1 is highly expressed in a variety of tumors including ESCA, and is closely related to histological types and histological grade. GO/KEGG functional enrichment analysis revealed that GLUT1 is closely related to structural constituent of cytoskeleton, intermediate filament binding, cell-cell adheres junction, epidermis development, and P53 signaling pathway. PPI shows that GLUT1 is closely related to TP53, GIPC1 and INS, and these three proteins all play an important role in tumor proliferation. CIBERSORT analysis showed that GLUT1 expression is related to the infiltration of multiple immune cells. When GLUT1 is highly expressed, the number of memory B cells decreases. ESCA cohort analysis found that GLUT1 expression was related to 7 m6A modifier genes. Six possible crRNA networks in ESCA were constructed by correlation analysis, and all these ceRNA networks contained GLUT1.ConclusionGLUT1 can be used as a biomarker for the diagnosis and treatment of ESCA, and is related to tumor immune infiltration, m6A modification and ceRNA network.

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Section: Introductionmentioning

confidence: 99%

Comprehensive Analysis of GLUT1 Immune Infiltrates and ceRNA Network in Human Esophageal Carcinoma

Liu

Gao

et al. 2021

Front. Oncol.

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“…ST-NhaA was added to the lipid-detergent suspension at a lipid/ protein mass ratio of 50:1, and the solution was incubated for 1 h. Detergent was removed by addition of 30% (v/v) BioBeads shaking overnight. The next morning, BioBeads were settled and exchanged for a fresh aliquot, and the dispersion was shaken for 1 h. After removal of the BioBeads, two freeze-thaw cycles followed to obtain more unilamellar vesicles (40). The proteoliposomes were consequently extruded 51 times through a 100 nm polycarbonate membrane (LFM-100; Avestin, Ottawa, Canada).…”

Section: Reconstitution and Functional Assaymentioning

confidence: 99%

Stairway to Asymmetry: Five Steps to Lipid-Asymmetric Proteoliposomes

Markones

Fippel

Kaiser

et al. 2020

Biophysical Journal

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Membrane proteins are embedded in a complex lipid environment that influences their structure and function. One key feature of nearly all biological membranes is a distinct lipid asymmetry. However, the influence of membrane asymmetry on proteins is poorly understood, and novel asymmetric proteoliposome systems are beneficial. To our knowledge, we present the first study on a multispanning protein incorporated in large unilamellar liposomes showing a stable lipid asymmetry. These asymmetric proteoliposomes contain the Na þ /H þ antiporter NhaA from Salmonella Typhimurium. Asymmetry was introduced by partial, outside-only exchange of anionic phosphatidylglycerol (PG), mimicking this key asymmetry of bacterial membranes. Outer-leaflet and total fractions of PG were determined via z-potential (z) measurements after lipid exchange and after scrambling of asymmetry. z-Values were in good agreement with exclusive outside localization of PG. The electrogenic Na þ /H þ antiporter was active in asymmetric liposomes, and it can be concluded that reconstitution and generation of asymmetry were successful. Lipid asymmetry was stable for more than 7 days at 23 C and thus enabled characterization of the Na þ /H þ antiporter in an asymmetric lipid environment. We present and validate a simple five-step protocol that addresses key steps to be taken and pitfalls to be avoided for the preparation of asymmetric proteoliposomes: 1) optimization of desired lipid composition, 2) detergent-mediated protein reconstitution with subsequent detergent removal, 3) generation of lipid asymmetry by partial exchange of outer-leaflet lipid, 4) verification of lipid asymmetry and stability, and 5) determination of protein activity in the asymmetric lipid environment. This work offers guidance in designing asymmetric proteoliposomes that will enable researchers to compare functional and structural properties of membrane proteins in symmetric and asymmetric lipid environments.

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“…Solute carrier (SLC) transporters for sugars include SLC2 and SLC5 families, which regulate cellular concentrations of sugars 7,8 . hGLUT‐1, a widely distributed member of the facilitative SLC2 family, transports glucose down its concentration gradient 9 .…”

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confidence: 99%

Tyrosine Kinase Inhibitors Reduce Glucose Uptake by Binding to an Exofacial Site on hGLUT‐1: Influence on ¹⁸F‐FDG PET Uptake

Damaraju

Aminpour

Kuzma

et al. 2021

Clinical Translational Sci

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Positron emission tomography (PET) using 2-deoxy-2-[ 18 F]fluoro-D-glucose ([ 18 F]FDG), a marker of energy metabolism and cell proliferation, is routinely used in the clinic to assess patient response to chemotherapy and to monitor tumor growth. Treatment with some tyrosine kinase inhibitors (TKIs) causes changes in blood glucose levels in both non-diabetic and diabetic patients. We evaluated the interaction of several classes of TKIs with human glucose transporter-1 (hGLUT-1) in FaDu and GIST-1 cells by measuring [ 3 H]2-deoxy-D-glucose ([ 3 H]2-DG) and [ 3 H]FDG uptake. Uptake of both was inhibited to varying extents by the TKIs, and representative TKIs from each class showed competitive inhibition of [ 3 H]2-DG uptake. In GIST-1 cells, [ 3 H]FDG uptake inhibition by temsirolimus and nilotinib was irreversible, whereas inhibition by imatinib, gefitinib, and pazopanib was reversible. Molecular modeling studies showed that TKIs form multiple hydrogen bonds with polar residues of the sugar binding site (i.e., Q161, Q282, Q283, N288, N317, and W388), and van der Waals interactions with the H-pocket site. Our results showed interaction of TKIs with amino acid residues at the glucose binding site to inhibit glucose uptake by hGLUT-1. We showed that inhibition of hGLUT-1 by TKIs could alter glucose levels in patients treated with TKIs, leading to hypoglycemia and fatigue, although further studies are required to evaluate roles of other SLC2 and SLC5 members. In addition, TKIs could affect tumor [ 18 F]FDG uptake, increasingly used as a marker of tumor response. hGLUT-1 inhibition by TKIs may have implications for routine [ 18 F]FDG-PET monitoring of tumor response in patients.

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Reconstitution and purification of the D-glucose transporter from human erythrocytes.

Cited by 597 publications

References 30 publications

Comprehensive Analysis of GLUT1 Immune Infiltrates and ceRNA Network in Human Esophageal Carcinoma

Comprehensive Analysis of GLUT1 Immune Infiltrates and ceRNA Network in Human Esophageal Carcinoma

Stairway to Asymmetry: Five Steps to Lipid-Asymmetric Proteoliposomes

Tyrosine Kinase Inhibitors Reduce Glucose Uptake by Binding to an Exofacial Site on hGLUT‐1: Influence on ¹⁸F‐FDG PET Uptake

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Reconstitution and purification of the D-glucose transporter from human erythrocytes.

Cited by 597 publications

References 30 publications

Comprehensive Analysis of GLUT1 Immune Infiltrates and ceRNA Network in Human Esophageal Carcinoma

Comprehensive Analysis of GLUT1 Immune Infiltrates and ceRNA Network in Human Esophageal Carcinoma

Stairway to Asymmetry: Five Steps to Lipid-Asymmetric Proteoliposomes

Tyrosine Kinase Inhibitors Reduce Glucose Uptake by Binding to an Exofacial Site on hGLUT‐1: Influence on 18F‐FDG PET Uptake

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Tyrosine Kinase Inhibitors Reduce Glucose Uptake by Binding to an Exofacial Site on hGLUT‐1: Influence on ¹⁸F‐FDG PET Uptake