ABCMdb

ABCG2 p.Gly188Glu

Predicted by SNAP2:	A: D (63%), C: N (61%), D: D (85%), E: D (85%), F: D (80%), H: D (80%), I: D (75%), K: D (53%), L: D (53%), M: D (71%), N: D (71%), P: D (75%), Q: D (75%), R: N (53%), S: D (59%), T: D (66%), V: D (71%), W: D (80%), Y: D (80%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Gout-causing Q141K mutation in ABCG2 leads to inst... Proc Natl Acad Sci U S A. 2013 Mar 26;110(13):5223-8. doi: 10.1073/pnas.1214530110. Epub 2013 Mar 14. Woodward OM, Tukaye DN, Cui J, Greenwell P, Constantoulakis LM, Parker BS, Rao A, Kottgen M, Maloney PC, Guggino WB
Gout-causing Q141K mutation in ABCG2 leads to instability of the nucleotide-binding domain and can be corrected with small molecules.
Proc Natl Acad Sci U S A. 2013 Mar 26;110(13):5223-8. doi: 10.1073/pnas.1214530110. Epub 2013 Mar 14., [PMID:23493553]

Abstract [show]
The multidrug ATP-binding cassette, subfamily G, 2 (ABCG2) transporter was recently identified as an important human urate transporter, and a common mutation, a Gln to Lys substitution at position 141 (Q141K), was shown to cause hyperuricemia and gout. The nature of the Q141K defect, however, remains undefined. Here we explore the Q141K ABCG2 mutation using a comparative approach, contrasting it with another disease-causing mutation in an ABC transporter, the deletion of Phe-508 (DeltaF508) in the cystic fibrosis transmembrane conductance regulator (CFTR). We found, much like in DeltaF508 CFTR, that the Q141K mutation leads to instability in the nucleotide-binding domain (NBD), a defect that translates to significantly decreased protein expression. However, unlike the CFTR mutant, the Q141K mutation does not interfere with the nucleotide-binding domain/intracellular loop interactions. This investigation has also led to the identification of critical residues involved in the protein-protein interactions necessary for the dimerization of ABCG2: Lys-473 (K473) and Phe-142 (F142). Finally, we have demonstrated the utility of using small molecules to correct the Q141K defect in expression and function as a possible therapeutic approach for hyperuricemia and gout.

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No. Sentence Comment
71 We separately mutated two residues in the Q141K background-a Gly to Glu substitution at position 188 (G188E) and an Arg to Lys substitution at 193 (R193K) (homologous to the Gly-550 and Arg-555 residues of CFTR, shown in Fig. 3A)-and found that the G188E mutation acted as a suppressor mutation, significantly increased the amount of the Q141K total protein (Fig. 3 B-E and Fig. S4 E and G), and increased the dimer protein (Fig. 3 C and E and Fig. S4 F and H) and the surface protein (Fig. 3F); the R193K mutation did not act as a suppressor (P < 0.41; n = 10). Login to comment
80 G188E mutation introduced into the wild-type protein produced no change in expression (Fig. 3G) and did not recue the ƊF142 mutation (Fig. S2 E and F), supporting the hypothesis that the F142 deletion disrupts both stability and interdomain interactions. Login to comment
107 ABCG2 WT 141K 188E -- -- 188E 76k ABCG2 GAPDH surface ABCG2 Na/K atpase WT 141K 188E 193K -- WT 141K 188E 193K -- 76k ABCG2 GAPDH ABCG2 monomer ABCG2 dimer 140k WT 141K 188E 193K -- A B C 0.0 0.5 1.0 Normalized ABCG2 protein WT 141K G188E R193K -- ** 0.0 0.5 1.0 1.5 2.0 2.5 Normalized ABCG2 dimer protein WT 141K G188E R193K -- * E F G D CFTR Fig. 3. Login to comment
114 (F) Surface expression of biotinylated ABCG2 and Q141K variants and Na+ /K+ ATPase, demonstrating strong rescue with the Q141K G188E mutation (n = 6). Login to comment
115 (G) The G188E mutation does not affect WT ABCG2 protein expression (n = 3). Login to comment
121 Interestingly, we found that 4-PBA was also able to increase the amount of Q141K/G188E protein (Fig. 5 A-C), demonstrating that the G188E suppressor mutation is not a full rescue and that some protein is still targeted for degradation. Login to comment
158 Here we tested homologous NBD mutations in Q141K ABCG2 and found the G188E mutation rescued Q141K expression, suggesting the Q141K mutation results in increased ABCG2 NBD instability. Login to comment

[hide] Effects of the gout-causing Q141K polymorphism and... Biochem Biophys Res Commun. 2013 Jul 19;437(1):140-5. doi: 10.1016/j.bbrc.2013.06.054. Epub 2013 Jun 22. Saranko H, Tordai H, Telbisz A, Ozvegy-Laczka C, Erdos G, Sarkadi B, Hegedus T
Effects of the gout-causing Q141K polymorphism and a CFTR DeltaF508 mimicking mutation on the processing and stability of the ABCG2 protein.
Biochem Biophys Res Commun. 2013 Jul 19;437(1):140-5. doi: 10.1016/j.bbrc.2013.06.054. Epub 2013 Jun 22., [PMID:23800412]

Abstract [show]
ABCG2 is an important multidrug transporter involved also in urate transport, thus its mutations can lead to the development of gout and may also alter general drug absorption, distribution and excretion. The frequent ABCG2 polymorphism, Q141K, is associated with an elevated risk of gout and has been controversially reported to reduce the plasma membrane expression and/or the transport function of the protein. In the present work we examined the stability and cellular processing of the Q141K ABCG2 variant, as well as that of the DeltaF142 ABCG2, corresponding to the DeltaF508 mutation in the CFTR (ABCC7) protein, causing cystic fibrosis. The processing and localization of full length ABCG2 variants were investigated in mammalian cells, followed by Western blotting and confocal microscopy, respectively. Folding and stability were examined by limited proteolysis of Sf9 insect cell membranes expressing these ABCG2 constructs. Stability of isolated nucleotide binding domains, expressed in and purified from bacteria, was studied by CD spectroscopy. We find that the Q141K variant has a mild processing defect which can be rescued by low temperature, a slightly reduced activity, and a mild folding defect, especially affecting the NBD. In contrast, the DeltaF142 mutant has major processing and folding defects, and no ATPase function. We suggest that although these mutations are both localized within the NBD, based on molecular modeling their contribution to the ABCG2 structure and function is different, thus rescue strategies may be devised accordingly.

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No. Sentence Comment
20 http://dx.doi.org/10.1016/j.bbrc.2013.06.054 Abbreviations: CFTR, cystic fibrosis transmembrane conductance regulator, ABCC7; HEK, human embryonic kidney 293 cells; NBD, nucleotide binding domain; MBP, maltose binding protein; TMD, transmembrane domain; PBA, 4-phenylbutyrate; Sf9, Spodoptera frugiperda cells; SNP, single nucleotide polymorphism; 3R, G188E, R191Q, and R193K rescue mutations in ABCG2-NBD. Login to comment
113 Therefore all these three mutations were introduced into the corresponding regions of the ABCG2 DF142 construct (3R: G188E, R191Q, and R193K). Login to comment
120 Interestingly, one of the rescue mutations, G188E has been reported to promote Q141K maturation [11]. Login to comment
122 While this G188E mutation does not increase the maturation of WT ABCG2 [11], the analogous G550E mutation promotes the maturation of WT CFTR [21] that again suggest fundamental differences between the NBDs of the two proteins. Login to comment

[hide] ABCG2: the molecular mechanisms of urate secretion... Am J Physiol Renal Physiol. 2015 Sep 15;309(6):F485-8. doi: 10.1152/ajprenal.00242.2015. Epub 2015 Jul 1. Woodward OM
ABCG2: the molecular mechanisms of urate secretion and gout.
Am J Physiol Renal Physiol. 2015 Sep 15;309(6):F485-8. doi: 10.1152/ajprenal.00242.2015. Epub 2015 Jul 1., [PMID:26136557]

Abstract [show]
The human propensity for high levels of serum uric acid (SUA) is a trait that has defied explanation. Is it beneficial? Is it pathogenic? Its role in the human diseases like gout and kidney stones was discovered over a century ago [Richette P, Bardin T. Lancet 375: 318-328, 2010; Rivard C, Thomas J, Lanaspa MA, Johnson RJ. Rheumatology (Oxford) 52: 421-426, 2013], but today emerging new genetic and epidemiological techniques have revived an age-old debate over whether high uric acid levels (hyperuricemia) independently increase risk for diseases like hypertension and chronic kidney disease [Feig DI. J Clin Hypertens (Greenwich) 14: 346-352, 2012; Feig DI, Madero M, Jalal DI, Sanchez-Lozada LG, Johnson RJ. J Pediatr 162: 896-902, 2013; Feig DI, Soletsky B, Johnson RJ. JAMA 300: 924-932, 2008; Wang J, Qin T, Chen J, Li Y, Wang L, Huang H, Li J. PLoS One 9: e114259, 2014; Zhu P, Liu Y, Han L, Xu G, Ran JM. PLoS One 9: e100801, 2014]. Part of the mystery of the role uric acid plays in human health stems from our lack of understanding of how humans regulate uric acid homeostasis, an understanding that could shed light on the historic role of uric acid in human adaptation and its present role in human pathogenesis. This review will highlight the recent work to identify the first important human uric acid secretory transporter, ABCG2, and the identification of a common causal ABCG2 variant, Q141K, for hyperuricemia and gout.

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74 Using either small molecules like the drug VRT-325 (11), or by using the suppressor mutation G188E to enhance NBD sandwich formation (25), we were able to rescue expression, trafficking, and function (32). Login to comment