Dimerization of ABCG2 Analysed by Bimolecular Fluorescence Complementation

ABCG2 is one of three human ATP binding cassette transporters that are functionally capable of exporting a diverse range of substrates from cells. The physiological consequence of ABCG2 multidrug transport activity in leukaemia, and some solid tumours is the acquisition of cancer multidrug resistance. ABCG2 has a primary structure that infers that a minimal functional transporting unit would be a homodimer. Here we investigated the ability of a bimolecular fluorescence complementation approach to examine ABCG2 dimers, and to probe the role of individual amino acid substitutions in dimer formation. ABCG2 was tagged with fragments of venus fluorescent protein (vYFP), and this tagging did not perturb trafficking or function. Co-expression of two proteins bearing N-terminal and C-terminal fragments of YFP resulted in their association and detection of dimerization by fluorescence microscopy and flow cytometry. Point mutations in ABCG2 which may affect dimer formation were examined for alterations in the magnitude of fluorescence complementation signal. Bimolecular fluorescence complementation (BiFC) demonstrated specific ABCG2 dimer formation, but no changes in dimer formation, resulting from single amino acid substitutions, were detected by BiFC analysis

Identification of residues in ABCG2 affecting protein trafficking and drug transport, using co-evolutionary analysis of ABCG sequences

ABCG2 is an ABC (ATP-binding cassette) transporter with a physiological role in urate transport in the kidney and is also implicated in multi-drug efflux from a number of organs in the body. The trafficking of the protein and the mechanism by which it recognizes and transports diverse drugs are important areas of research. In the current study, we have made a series of single amino acid mutations in ABCG2 on the basis of sequence analysis. Mutant isoforms were characterized for cell surface expression and function. One mutant (I573A) showed disrupted glycosylation and reduced trafficking kinetics. In contrast with many ABC transporter folding mutations which appear to be ‘rescued’ by chemical chaperones or low temperature incubation, the I573A mutation was not enriched at the cell surface by either treatment, with the majority of the protein being retained in the endoplasmic reticulum (ER). Two other mutations (P485A and M549A) showed distinct effects on transport of ABCG2 substrates reinforcing the role of TM helix 3 in drug recognition and transport and indicating the presence of intracellular coupling regions in ABCG2

Infant feeding practices at routine PMTCT sites, South Africa: results of a prospective observational study amongst HIV exposed and unexposed infants - birth to 9 months

<p>Abstract</p> <p>Background</p> <p>We sought to investigate infant feeding practices amongst HIV-positive and -negative mothers (0-9 months postpartum) and describe the association between infant feeding practices and HIV-free survival.</p> <p>Methods</p> <p>Infant feeding data from a prospective observational cohort study conducted at three (of 18) purposively-selected routine South African PMTCT sites, 2002-2003, were analysed. Infant feeding data (previous 4 days) were gathered during home visits at 3, 5, 7, 9, 12, 16, 20, 24, 28, 32 and 36 weeks postpartum. Four feeding groups were of interest, namely exclusive breastfeeding, mixed breastfeeding, exclusive formula feeding and mixed formula feeding. Cox proportional hazards models were fitted to investigate associations between feeding practices (0-12 weeks) and infant HIV-free survival.</p> <p>Results</p> <p>Six hundred and sixty five HIV-positive and 218 HIV-negative women were recruited antenatally and followed-up until 36 weeks postpartum. Amongst mothers who breastfed between 3 weeks and 6 months postpartum, significantly more HIV-positive mothers practiced exclusive breastfeeding compared with HIV-negative: at 3 weeks 130 (42%) versus 33 (17%) (p < 0.01); this dropped to 17 (11%) versus 1 (0.7%) by four months postpartum. Amongst mothers practicing mixed breastfeeding between 3 weeks and 6 months postpartum, significantly more HIV-negative mothers used commercially available breast milk substitutes (p < 0.02) and use of these peaked between 9 and 12 weeks. The probability of postnatal HIV or death was lowest amongst infants living in the best resourced site who avoided breastfeeding, and highest amongst infants living in the rural site who stopped breastfeeding early (mean and standard deviations: 10.7% ± 3% versus 46% ± 11%).</p> <p>Conclusions</p> <p>Although feeding practices were poor amongst HIV-positive and -negative mothers, HIV-positive mothers undertake safer infant feeding practices, possibly due to counseling provided through the routine PMTCT programme. The data on differences in infant outcome by feeding practice and site validate the WHO 2009 recommendations that site differences should guide feeding practices amongst HIV-positive mothers. Strong interventions are needed to promote exclusive breastfeeding (to 6 months) with continued breastfeeding thereafter amongst HIV-negative motherswho are still the majority of mothers even in high HIV prevalence setting like South Africa.</p

BiFC principle and ABCG2 BiFC constructs employed.

<p><b>A</b> The underlying principle of BiFC is the refolding and maturation of a chromophore (here vYFP) that occurs upon the interaction of two proteins (here shown as cylinders) bearing complementary fragments of the YFP protein (red and blue interlocking shapes). <b>B</b> In the current manuscript, ABCG2 comprised of a N-terminal nucleotide binding domain (orange) and a C-terminal TMD (olive green) is tagged with either full length YFP variants (yellow circles), or with complementing fragments of YFP variants, shown as red and blue hemispheres. The tagging of YFP and fragments was at either the 5′ (N-terminus) or the 3′ (C-terminus) of the ABCG2 cDNA.</p

Oligonucleotide primers employed.

<p>Oligonucleotide primers employed.</p

N-terminal tagging with ½ YFP molecules does not affect function of the ABCG2 protein.

<p>HEK293T cells were transiently transfected with YFP_ABCG2 constructs (C-E) or negative control (empty pcDNA vector; A) and positive control (His₁₂-ABCG2) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025818#s2" target="_blank">Methods</a> and Materials. Following transfection aliquots of cells were incubated with the ABCG2 substrate mitoxantrone in the presence or absence of the inhibitor fumitremorgin C (FTC) and cellular fluorescence determined by flow cytometry. Blue filled histograms represent the cellular fluorescence in the absence of FTC, and the rightward shift in the presence of the inhibitor (black lines) demonstrates functional competence at a level similar to those of the characterized control His₁₂-ABCG2 (B). The apparent large number of cells with zero fluorescence in the eYFP-ABCG2 panel (C) is a fluorescence artefact due to interference from the YFP fluorescence of this construct. The graphs are representative of >5 independent experiments.</p

ABCG2 BiFC quantification by high content screening.

<p>HEK293T cells were transfected in 96-well plates as described in the methods, with pairs of constructs expressing vYN-ABCG2 and vYC-ABCG2 (panels <b>B, C, D</b>) or with a single construct expressing only vYC-ABCG2 (<b>A</b>). ABCG2 isoforms expressed were wild type (<b>A, B</b>), E211Q (<b>C</b>) and C603A (<b>D</b>). For each transfection two representative images from multi-well confocal acquisition are shown, with and without the Hoechst 33342 stained nuclei for clarity. Images were taken with a 40X objective on a Molecular Devices IX Micro plate reader. <b>E</b> The percentage BiFC positive cells was determined as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025818#s2" target="_blank">Methods</a>, employing negative (single transfections) to set thresholds for fluorescence determination above background. <b>F</b> Mean intensity fluorescence levels were detected for YFP complementation for each ABCG2 isoform, and the data was normalised within each experiment to the mean fluorescence intensity observed for wild type ABCG2 BiFC. Data in <b>E</b> and <b>F</b> are the mean (± s.e.m.) of 4-7 independent experiments.</p

ABCG2 bimolecular fluorescence complementation is specific.

<p>HEK293T cells were transfected with constructs expressing either eYFP-ABCG2 (as a positive fluorescence control, <b>A</b>), complementing halves of vYFP tagged to ABCG2 (<b>B</b>) or with vYN-ABCG2 and a non-specific interaction partner β2 adrenergic receptor (β2AR) with a C-terminal vYC fragment (<b>C</b>). Live cell imaging (left hand panel) shows that all three transfections result in cellular fluorescence indicating the complementation of both the cognate (ABCG2:ABCG2) and non cognate (ABCG2: β2AR) protein pairs. However, cells fixed and counter stained with DAPI (middle panel) show that the BiFC signal arising from ABCG2:ABCG2 interaction is membrane localized, in contrast to the BiFC signal from the ABCG2: β2AR interaction which shows cytoplasmic retention. Immunofluorescence of cells with anti ABCG2 antibodies (right panel) further demonstrates that the ABCG2: β2AR interaction results in intracellular retention of ABCG2. Data are representative of at least 4 independent experiments.</p

Complementation of YFP-ABCG2 constructs occurs at the plasma membrane.

<p>Constructs expressing half-tagged vYFP-ABCG2 individually (<b>A, B</b>) or together (<b>C</b>) were transfected into HEK293T cells as indicated schematically to the left. Constructs reached the plasma membrane as confirmed by immuno-staining with 5D3 antibodies (left hand panel). No fluorescence could be detected at 515 nm by flow cytometry of cells transfected with the vYN-ABCG2 or vYC-ABCG2 construct, but in co-transfected cells a YFP fluorescence was detected (middle panel). Confirmation of the BiFC interaction was obtained from imaging fixed cells by fluorescence microscopy (right hand panel). DAPI was employed as a nuclear counter-stain. Individual transfections resulted in only DAPI labelled nuclei in such experiments. Data are representative of at least 4 independent experiments.</p