ABCMdb

ABCB1 p.Ser23Cys

Predicted by SNAP2:	A: N (78%), C: N (57%), D: N (78%), E: N (87%), F: N (53%), G: N (72%), H: N (82%), I: N (66%), K: N (93%), L: N (72%), M: N (78%), N: N (87%), P: N (78%), Q: N (87%), R: N (82%), T: N (87%), V: N (72%), W: N (53%), Y: N (53%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, T: N, V: N, W: N, Y: N,

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[hide] Biochemical characterization of domains in the mem... Biochemistry. 2005 Feb 22;44(7):2661-70. Kaur P, Rao DK, Gandlur SM
Biochemical characterization of domains in the membrane subunit DrrB that interact with the ABC subunit DrrA: identification of a conserved motif.
Biochemistry. 2005 Feb 22;44(7):2661-70., 2005-02-22 [PMID:15709779]

Abstract [show]
DrrA and DrrB proteins confer resistance to the commonly used anticancer agents daunorubicin and doxorubicin in the producer organism Streptomyces peucetius. The drrAB locus has previously been cloned in Escherichia coli, and the proteins have been found to be functional in this host. DrrA, a soluble protein, belongs to the ABC family of proteins. It forms a complex with the integral membrane protein DrrB. Previous studies suggest that the function and stability of DrrA and DrrB are biochemically coupled. Thus, DrrA binds ATP only when it is in a complex with DrrB in the membrane. Further, DrrB is completely degraded if DrrA is absent. In the present study, we have characterized domains in DrrB that may be directly involved in interaction with DrrA. Several single-cysteine substitutions in DrrB were made. Interaction between DrrA and DrrB was studied by using a cysteine to amine chemical cross-linker that specifically cross-links a free sulfhydryl group in one protein (DrrB) to an amine in another (DrrA). We show here that DrrA cross-links with both the N- and the C-terminal ends of the DrrB protein, implying that they may be involved in interaction. Furthermore, this study identifies a motif within the N-terminal cytoplasmic tail of DrrB, which is similar to a motif recently shown by crystal structure analysis in BtuC and previously shown by sequence analysis to be also present in exporters, including MDR1. We propose that the motif present in DrrB and other exporters is actually a modified version of the EAA motif, which was originally believed to be present only in the importers of the ABC family. The present work is the first report where domains of interaction in the membrane component of an ABC drug exporter have been biochemically characterized.

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76 Primers used for making S23C mutation are shown here as an example: Single cysteine substitution mutants were created at amino acid positions 4, 15, 23, 35, 44, 53, 70, 80, 92, 107, 116, 129, 149, 160, 173, 213, 236, 249, 270, and 282 in DrrB. Login to comment
77 The mutants were named S4C, S15C, S23C, S35C, A44C, C260S up: 5'-GGCCTGGTCCTGTCCGTGTCGGCAGGG-3' C260S dn: 5'-CCCTGCCGACACGGACAGGACCAGGCC-3' S23C up: 5'- CGGACGGTGCTGTGCGCGGGTGAACGG-3' S23C dn: 5'- CCGTTCACCCGCGCACAGCACCGTCCG-3' V53C, T70C, S80C, V92C, S107C, V116C, A129C, T149C, A160C, V173C, S213C, S236C, T249C, A270C, and A282C, respectively. Login to comment
156 FIGURE 3: Chemical cross-linking between wild-type DrrA and DrrB containing cysteine substitution C260S or S23C. Login to comment
157 The cell membrane fraction containing the DrrA and DrrB (C260S or S23C) proteins was subjected to chemical cross-linking using different concentrations of GMBS, as described in Materials and Methods. Login to comment
163 (E) A reaction containing the cross-linked S23C sample was analyzed by SDS-PAGE, as described above. Login to comment
166 The lane containing the S23C sample was vertically spliced into halves. Each half was then probed with either the anti-DrrA or anti-DrrB antibodies. Detection was done by the chemiluminescence method. Login to comment
174 The N-terminal mutant S23C was the first mutant where a cross-linked product of DrrA and DrrB was identified (Figure 3). Login to comment
180 The 60 kDa cross-linked species did not appear in the cysteine-less (C260S) sample on addition of the cross-linker (Figure 3A,C) or in the S23C control sample where DMSO alone was added instead of the cross-linker (Figure 3B,D, lane 1). Login to comment
181 To further verify if the same cross-linked species was detected by both anti-DrrA and anti-DrrB antibodies, a lane containing the cross-linked S23C sample was spliced vertically into halves. Each half was probed with either the FIGURE 4: Chemical cross-linking between wild-type DrrA and DrrB containing cysteine substitutions in the N-terminal cytoplasmic tail, the transmembrane domains TM1 and TM2, and the cytoplasmic loop C1. Login to comment
193 The location of the cross-linked species is marked as A+B. (A) S23C (positive control), A129C, T149C, A160C. Login to comment
196 Table 1: Doxorubicin Resistance of E. coli N43 Cells Expressing Wild Type DrrA with DrrB Containing Different Cysteine Substitutionsa amt of dox, µg/mLdomain of DrrB location of cysteine 0 4 6 8 (wild type) C260 +++ +++ +++ ++ N-terminus S15C +++ +++ +++ ++ N-terminus S23C +++ +++ +++ ++ N-terminus S35C +++ +++ ++ ++ N-terminus A44C +++ +++ ++ + TM 1 V53C +++ ++ ++ - TM 1 T70C +++ + + + P 1 S80C +++ ++ + - TM 2 V92C +++ ( - - TM 2 S107C +++ +++ ++ ++ C 1 V116C +++ +++ +++ ++ TM 3 A129C +++ ++ + - TM 4 T149C +++ ++ ++ + C 2 A160C +++ +++ ++ + TM 5 V173C +++ +++ ++ + TM 6 S213C +++ +++ ++ + C 3 S236C +++ ++ + - TM 7 S249C +++ + + - TM 8 A270C +++ +++ ++ ++ C-terminus A282C +++ +++ ++ ++ cysteine-less DrrB C260S +++ +++ +++ +++ vector only pSU2718 ++++ ( - - a Legend: +++, very good growth; ++, good growth; +, some growth; -, no growth. Login to comment
205 It should be pointed out that the disappearance of the 43 kDa band happens in all the samples, irrespective of the presence (S23C) or absence (C260S) of a cysteine in DrrB (Figure 3A). Login to comment
206 Since the 60 kDa cross-linked species is not seen in C260S (even though the 43 kDa band disappears), it clearly indicates that the cross-linked species seen in S23C is not the result of cross-linking between DrrB and this 43 kDa unrelated protein. Login to comment
209 S23C, which showed a specific 60 kDa cross-linked species of DrrA and DrrB (described above), was used as a positive control in further cross-linking experiments. Login to comment
211 As seen with S23C, all the other cysteine substitution mutants tested in the N-terminal tail, S4C, S15C, S35C, and S44C, also showed the appearance of the 60 kDa species on cross-linking with GMBS by Western blot analysis (Figure 4A,B). Login to comment

[hide] Gene polymorphism influencing treatment response i... J Psychiatr Res. 2008 Sep;42(11):884-93. Epub 2007 Dec 20. Alenius M, Wadelius M, Dahl ML, Hartvig P, Lindstrom L, Hammarlund-Udenaes M
Gene polymorphism influencing treatment response in psychotic patients in a naturalistic setting.
J Psychiatr Res. 2008 Sep;42(11):884-93. Epub 2007 Dec 20., [PMID:18086475]

Abstract [show]
RATIONALE: Many patients with psychotic symptoms respond poorly to treatment. Factors possibly affecting treatment response include the presence of polymorphisms in genes coding for various receptor populations, drug-metabolizing enzymes or transport proteins. OBJECTIVES: To investigate whether genetic polymorphisms could be indicators of treatment response to antipsychotic drugs. The genes of interest were the dopamine D2 receptor gene (DRD2), the serotonin 2A and 2C receptor genes (HTR2A and HTR2C), the P-glycoprotein gene (ABCB1 or MDR1) and the drug-metabolizing cytochrome P450 2D6 gene (CYP2D6). MATERIAL AND METHODS: Data for this naturalistic, cross-sectional study of patients requiring antipsychotic drugs and attending the Psychosis Outpatient Care clinic in Jonkoping, Sweden were obtained from patient interviews, blood samples and information from patient files. Blood samples were genotyped for DRD2 Taq1 A, Ins/Del and Ser311Cys, HTR2A T102C, HTR2C Cys23Ser, ABCB1 1236C>T, 2677G>T/A, 3435C>T and genetic variants of CYP2D6. The patients (n=116) were grouped according to the CANSEPT method regarding significant social and clinical needs and significant side effects. RESULTS: Patients on olanzapine homozygous for ABCB1 3435T, had more significant social and clinical needs than others. Patients with one or two DRD2 Taq1 A1 alleles had a greater risk of significant side effects, particularly if they were male, Caucasian, had a schizophrenic or delusional disorder or were taking strong dopamine D2-receptor antagonistic drugs. CONCLUSION: If these results are confirmed, patients carrying the DRD2 Taq1 A1 allele would benefit from using drugs without strong dopamine D2 receptor antagonistic properties.

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127 The second polymorphism studied was the change from serine to cysteine at residue 23 of the serotonin receptor 2C gene (HTR2C) located on the X-chromosome. Login to comment