Picky ABCG5/G8 and promiscuous ABCG2 ‐ a tale of fatty diets and drug toxicity

Khunweeraphong, Narakorn; Mitchell-White, James; Szöllősi, Dániel; Hussein, Toka O. K.; Kuchler, Karl; Kerr, Ian D.; Stockner, Thomas; Lee, Jyh-Yeuan

doi:10.1002/1873-3468.13938

Cited by 20 publications

(27 citation statements)

References 187 publications

(259 reference statements)

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“…Using the CHS-coupled ATPase activity as the functional readout, we initiated studies in the catalytic mechanism of ABCG5/G8 by exploiting the transporter’s missense mutations that undergo proper trafficking to post-ER cell membranes (ER-escaped mutants). In this study, we used Pichia pastoris yeast and expressed recombinant proteins of G8-G216D, a catalytically deficient mutant [ 18 ], ABCG5 E146Q /G8 WT (G5-E146Q) and ABCG5 WT /G8 R543S (G8-R543S), two loss-of-function/sitosterolemia missense mutants [ 22 , 37 ], and ABCG5 A540F /G8 WT (G5-A540F), a loss-of-function mutant with putative sterol-binding defect [ 21 ] ( Figure 1 B and Figure S1 ).The purified mutants were preincubated with E. coli polar lipids and sodium cholate as described above. As shown in Figure 5 , when compared with WT, the sitosterolemia missense mutants, G5-E146Q and G8-R543S, showed a ~80% reduction of the specific activity in CHS-coupled ATP hydrolysis (160 ± 15 nmol/min/mg and 150 ± 5 nmol/min/mg, respectively).…”

Section: Resultsmentioning

confidence: 99%

Transmembrane Polar Relay Drives the Allosteric Regulation for ABCG5/G8 Sterol Transporter

Xavier

Zein

Venes

et al. 2020

IJMS

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The heterodimeric ATP-binding cassette (ABC) sterol transporter, ABCG5/G8, is responsible for the biliary and transintestinal secretion of cholesterol and dietary plant sterols. Missense mutations of ABCG5/G8 can cause sitosterolemia, a loss-of-function disorder characterized by plant sterol accumulation and premature atherosclerosis. A new molecular framework was recently established by a crystal structure of human ABCG5/G8 and reveals a network of polar and charged amino acids in the core of the transmembrane domains, namely, a polar relay. In this study, we utilize genetic variants to dissect the mechanistic role of this transmembrane polar relay in controlling ABCG5/G8 function. We demonstrated a sterol-coupled ATPase activity of ABCG5/G8 by cholesteryl hemisuccinate (CHS), a relatively water-soluble cholesterol memetic, and characterized CHS-coupled ATPase activity of three loss-of-function missense variants, R543S, E146Q, and A540F, which are respectively within, in contact with, and distant from the polar relay. The results established an in vitro phenotype of the loss-of-function and missense mutations of ABCG5/G8, showing significantly impaired ATPase activity and loss of energy sufficient to weaken the signal transmission from the transmembrane domains. Our data provide a biochemical evidence underlying the importance of the polar relay and its network in regulating the catalytic activity of ABCG5/G8 sterol transporter.

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Section: Resultsmentioning

confidence: 99%

Transmembrane Polar Relay Drives the Allosteric Regulation for ABCG5/G8 Sterol Transporter

Xavier

Zein

Venes

et al. 2020

IJMS

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show abstract

“…Using the CHS-coupled ATPase activity as the functional readout, we initiated studies in the catalytic mechanism of ABCG5/G8 by exploiting the transporter’s missense mutations that undergo proper trafficking to post-ER cell membranes (ER-escaped mutants). In this study, we have used Pichia pastoris yeast and expressed recombinant proteins of G8-G216D, a catalytically deficient mutant [18], ABCG5 EI46Q /G8 WT (G5-E146Q) and ABCG5 WT /G8 R543S (G8-R543S), two loss-of-function/sitosterolemia missense mutants [22,37], and ABCG5 A54OF /G8 WT (G5-A540F), a loss-of-function mutant with putative sterol-binding defect [21] ( Figure 1B & Supplementary Figure 1 ).The purified mutants were preincubated with E. coli polar lipids and sodium cholate as described above. As shown in Figure 5 , when compared with WT, the sitosterolemia missense mutants, G5-E146Q and G8-R543S, show a ~80% reduction of the specific activity in CHS-coupled ATP hydrolysis (160±15 nmol/min/mg and 150 ± 5 nmol/min/mg, respectively).…”

Section: Resultsmentioning

confidence: 99%

“…(G5-E146Q) and ABCG5WT/G8R543S (G8-R543S), two loss-of-function/sitosterolemia missense mutants [22,37], and ABCG5A540F/G8WT (G5-A540F), a loss-of-function mutant with putative sterol-binding defect [21] ( Figure 1B & Supplementary Figure 1). The purified mutants were preincubated with E.…”

Section: Missense Mutants Impair Chs-coupled Atpase Activity Of Abcg5mentioning

confidence: 99%

Transmembrane polar relay drives the allosteric regulation for ABCG5/G8 sterol transporter

Xavier

Zein

Venes

et al. 2020

Preprint

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The heterodimeric ATP-binding cassette (ABC) sterol transporter, ABCG5/G8, is responsible for the biliary and transintestinal secretion of cholesterol and dietary plant sterols. Missense mutations of ABCG5/G8 can cause sitosterolemia, a loss-of-function disorder characterized by plant sterol accumulation and premature atherosclerosis. A new molecular framework was recently established by a crystal structure of human ABCG5/G8 and reveals a network of polar and charged amino acids in the core of the transmembrane domains, namely polar relay. In this study, we utilize genetic variants to dissect the mechanistic role of this transmembrane polar relay in controlling ABCG5/G8 function. We demonstrated a sterol-coupled ATPase activity of ABCG5/G8 by cholesteryl hemisuccinate (CHS), a relatively water-soluble cholesterol memetic, and characterized CHS-coupled ATP catalysis by using three loss-of-function missense variants, two sitosterolemia mutations (E146Q and R543S; within polar relay) and one sterol-binding mutation (A540F; distant from the polar relay). The results established an in vitro phenotype of the loss-of-function and missense mutations of ABCG5/G8, showing significantly impaired ATPase activity and loss of energy sufficient to weaken the polar relay network by all three mutants. Our data herein provide a biochemical evidence underlying the importance of the polar relay in regulating the catalytic activity of ABCG5/G8 sterol transporter.

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“…In a recent review [ 24 ], it was noted that composition of residues in the polar relay of ABCG2 and of ABCG5/G8 differs, with ABCG8 having a relatively high number of charged residues and ABCG2 a relatively low number. In residues with the conservation pattern (ABCG1, ABCG4), (ABCG2), (ABCG5), (ABCG8) this pattern is reiterated, ABCG8 having seven charged residues, ABCG5 five and ABCG2 one ( Figure 5 b).…”

Section: Resultsmentioning

confidence: 99%

Analysis of Sequence Divergence in Mammalian ABCGs Predicts a Structural Network of Residues That Underlies Functional Divergence

Mitchell-White

Stockner

Holliday

et al. 2021

IJMS

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The five members of the mammalian G subfamily of ATP-binding cassette transporters differ greatly in their substrate specificity. Four members of the subfamily are important in lipid transport and the wide substrate specificity of one of the members, ABCG2, is of significance due to its role in multidrug resistance. To explore the origin of substrate selectivity in members 1, 2, 4, 5 and 8 of this subfamily, we have analysed the differences in conservation between members in a multiple sequence alignment of ABCG sequences from mammals. Mapping sets of residues with similar patterns of conservation onto the resolved 3D structure of ABCG2 reveals possible explanations for differences in function, via a connected network of residues from the cytoplasmic to transmembrane domains. In ABCG2, this network of residues may confer extra conformational flexibility, enabling it to transport a wider array of substrates.

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Picky ABCG5/G8 and promiscuous ABCG2 ‐ a tale of fatty diets and drug toxicity

Cited by 20 publications

References 187 publications

Transmembrane Polar Relay Drives the Allosteric Regulation for ABCG5/G8 Sterol Transporter

Transmembrane Polar Relay Drives the Allosteric Regulation for ABCG5/G8 Sterol Transporter

Transmembrane polar relay drives the allosteric regulation for ABCG5/G8 sterol transporter

Analysis of Sequence Divergence in Mammalian ABCGs Predicts a Structural Network of Residues That Underlies Functional Divergence

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