Shifting the Paradigm: The Putative Mitochondrial Protein ABCB6 Resides in the Lysosomes of Cells and in the Plasma Membrane of Erythrocytes

ABCB6, a member of the adenosine triphosphate–binding cassette (ABC) transporter family, has been proposed to be responsible for the mitochondrial uptake of porphyrins. Here we show that ABCB6 is a glycoprotein present in the membrane of mature erythrocytes and in exosomes released from reticulocytes during the final steps of erythroid maturation. Consistent with its presence in exosomes, endogenous ABCB6 is localized to the endo/lysosomal compartment, and is absent from the mitochondria of cells. Knock-down studies demonstrate that ABCB6 function is not required for de novo heme biosynthesis in differentiating K562 cells, excluding this ABC transporter as a key regulator of porphyrin synthesis. We confirm the mitochondrial localization of ABCB7, ABCB8 and ABCB10, suggesting that only three ABC transporters should be classified as mitochondrial proteins. Taken together, our results challenge the current paradigm linking the expression and function of ABCB6 to mitochondria

An inhibitor of oxidative phosphorylation exploits cancer vulnerability.

Metabolic reprograming is an emerging hallmark of tumor biology and an actively pursued opportunity in discovery of oncology drugs. Extensive efforts have focused on therapeutic targeting of glycolysis, whereas drugging mitochondrial oxidative phosphorylation (OXPHOS) has remained largely unexplored, partly owing to an incomplete understanding of tumor contexts in which OXPHOS is essential. Here, we report the discovery of IACS-010759, a clinical-grade small-molecule inhibitor of complex I of the mitochondrial electron transport chain. Treatment with IACS-010759 robustly inhibited proliferation and induced apoptosis in models of brain cancer and acute myeloid leukemia (AML) reliant on OXPHOS, likely owing to a combination of energy depletion and reduced aspartate production that leads to impaired nucleotide biosynthesis. In models of brain cancer and AML, tumor growth was potently inhibited in vivo following IACS-010759 treatment at well-tolerated doses. IACS-010759 is currently being evaluated in phase 1 clinical trials in relapsed/refractory AML and solid tumors

Screening the Expression of ABCB6 in Erythrocytes Reveals an Unexpectedly High Frequency of Lan Mutations in Healthy Individuals

Lan is a high-incidence blood group antigen expressed in more than 99.9% of the population. Identification of the human ABC transporter ABCB6 as the molecular basis of Lan has opened the way for studies assessing the relation of ABCB6 function and expression to health and disease. To date, 34 ABCB6 sequence variants have been described in association with reduced ABCB6 expression based on the genotyping of stored blood showing weak or no reactivity with anti-Lan antibodies. In the present study we examined the red blood cell (RBC) surface expression of ABCB6 by quantitative flow cytometry in a cohort of 47 healthy individuals. Sequencing of the entire coding region of the ABCB6 gene in low RBC ABCB6 expressors identified a new allele (IVS9+1G>A, affecting a putative splice site at the boundary of exon 9) and two nonsynonymous SNPs listed in the SNP database (R192Q (rs150221689) and G588 S (rs145526996)). The R192Q mutation showed co-segregation with reduced RBC ABCB6 expression in a family, and we found the G588 S mutation in a compound heterozygous individual with undetectable ABCB6 expression, suggesting that both mutations result in weak or no expression of ABCB6 on RBCs. Analysis of the intracellular expression pattern in HeLa cells by confocal microscopy indicated that these mutations do not compromise overall expression or the endolysosomal localization of ABCB6. Genotyping of two large cohorts, containing 235 and 1039 unrelated volunteers, confirmed the high allele frequency of Lan-mutations. Our results suggest that genetic variants linked to lower or absent cell surface expression of ABCB6/Langereis may be more common than previously thought.This work was supported by the Lendulet Program of the Hungarian Academy of Sciences (GS), OTKA 83533 and by the Polish POIG grant 01.01.02-10-005/08 TESTOPLEK, supported by the EU through the European Regional Development Fund. Hajnalka Andrikovics is a recipient of the Janos Bolyai Research Scholarship from the Hungarian Academy of Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Dr. Camilo Toro and Dr. William Gahl of the NIH Undiagnosed Diseases Program for an affected patient specimen; that work was supported by the Intramural Research Program of the National Human Genome Research Institute and the Office of the Director of the NIH. We thank Lionel Arnaud (National Institute of Blood Transfusion (INTS), Paris, France) for helpful discussions

Periodontal health of an adult population in Hungary: Findings of a national survey

Objectives: To estimate the levels of periodontal health conditions of Hungarian adults. Material and Methods: Periodontal data on 4153 adults in 304 survey locations from all Hungarian regions were analysed. The Community Periodontal Index (CPI) was used to report the occurrence of probing pocket depth, calculus, and gingival inflammation. Age, gender, socioeconomic and health status, oral hygiene and lifestyle habits, dental office attendance, level of education, and fixed partial denture (FPD) treatment were evaluated for their association with periodontal conditions. CPI score as an outcome was dichotomized using an accepted threshold as low (<3) and high (3, 4) for multiple logistic regression modelling Results: CPI2 was the most prevalent score in all age groups. CPI scores were also strongly associated with the independent variables. Approximately 66% of subjects visited a dentist only in the case of an emergency. Lack of periodontal aspects of restorative care was demonstrated by the result of CPI0 among 16% of non-FPD wearers compared with only 9% of individuals treated with FPD. Conclusion: The present survey indicates that oral hygiene standards and periodontal health conditions need improvement in Hungary. Effective intervention programme for the prevention and control of periodontal disease are recommended at a national level

Functional expression of the 11 human Organic Anion Transporting Polypeptides in insect cells reveals that sodium fluorescein is a general OATP substrate

Organic Anion Transporting Polypeptides (OATPs), encoded by genes of the Solute Carrier Organic Anion (SLCO) family, are transmembrane proteins involved in the uptake of various compounds of endogenous or exogenous origin. In addition to their physiological roles, OATPs influence the pharmacokinetics and drug-drug interactions of several clinically relevant compounds. To examine the function and molecular interactions of human OATPs, including several poorly characterized family members, we expressed all 11 human OATPs at high levels in the baculovirus-Sf9 cell system. We measured the temperature- and inhibitor-sensitive cellular accumulation of sodium fluorescein and fluorescein-methotrexate, two fluorescent substrates of the OATPs, OATP1B1 and 1B3. OATP1B1 and 1B3 were functional in Sf9 cells, showing rapid uptake (t1/2(fluorescein-methotrexate) 2.64 and 4.16min, and t1/2(fluorescein) 6.71 and 5.58min for OATP1B1 and 1B3, respectively) and high-affinity transport (Km(fluorescein-methotrexate) 0.23 and 0.53μM, and Km(fluorescein) 25.73 and 38.55μM for OATP1B1 and 1B3, respectively) of both substrates. We found that sodium fluorescein is a general substrate of all human OATPs: 1A2, 1B1, 1B3, 1C1, 2A1, 2B1, 3A1, 4A1, 4C1, 5A1 and 6A1, while fluorescein-methotrexate is only transported by 1B1, 1B3, 1A2 and 2B1. Acidic extracellular pH greatly facilitated fluorescein uptake by all OATPs, and new molecular interactions were detected (between OATP2B1 and Imatinib, OATP3A1, 5A1 and 6A1 and estradiol 17-β-d-glucuronide, and OATP1C1 and 4C1 and prostaglandin E2). These studies demonstrate, for the first time, that the insect cell system is suitable for the functional analysis of the entire human OATP family, and for drug-OATP interaction screening

ABCB6 is not present in the purified mitochondrial fraction of K562 cells.

<p>Cellular membranes were separated by differential centrifugation (A); mitochondria were isolated using anti-TOM22 magnetic beads (B) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037378#s4" target="_blank">Materials and Methods</a>. Following separation by SDS-PAGE, organelle membranes were transferred to a PVDF membrane, which was incubated in antibodies recognizing specific organelle markers and ABCB6. <b>A</b>. Cellular fractions corresponding to total cell lysate (lane 1); nuclei and intact cells (lane 2); mitochondrial pellet of the 8000×g centrifugation (lane 3); pellet of 12.000×g centrifugation (lane 4); pellet of 20.000×g centrifugation (lane 5). <b>B</b>. Isolation of mitochondria using TOM22 immunobeads. Total cell lysate (lane 1); fraction bound to the beads after repeated washing steps (lane 2); pellet of the flow through (lane 3).</p

Human reticulocytes release ABCB6 in association with exosomes. A

<p>. 250 µL packed cells volume (PCV) of human RBCs from reticulocyte-enriched (4.5%) blood was cultured for 48 h and exosomes were collected from the medium as previously described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037378#pone.0037378-Barres1" target="_blank">[42]</a>. 0.5 µL PCV of RBCs before (t0) or after (48 h) maturation, as well as the completeness of exosomes were loaded on 10% SDS-PAGE for immunoblot analysis of the indicated proteins. The molecular mass (kDa) standards are indicated on the left. Note that the TfR is not detected in RBCs due to the low reticulocyte count but revealed in exosomes due to its concentration in the vesicles. <b>B</b>. Human RBCs from reticulocyte-enriched (10%) blood were adsorbed on cover slips pretreated with poly-L-lysine, fixed 20 min by 1% PFA and immunostained for ABCB6 (antibody Ab 7474 at; dilution 1/:50/Alexa Fluor 594; A21207) or and TfR (MoAb H68.4 at; dilution 1/:200/Alexa Fluor 488; A11029) after permeabilization, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037378#s4" target="_blank">Materials and Methods</a>. Coverslips were observed using a Leica confocal SPE and a Leica 63× ACS APO 1.33 objective. Note that none of the cells shown are positive to DAPI although contained in the mounting reagent. Arrows point to possible colocalization of ABCB6 and TfR in MVE.</p

Unshielding Multidrug Resistant Cancer through Selective Iron Depletion of P-Glycoprotein–Expressing Cells

Clinical evidence shows that following initial response to treatment, drug-resistant cancer cells frequently evolve and, eventually, most tumors become resistant to all available therapies. We compiled a focused library consisting of >500 commercially available or newly synthetized 8-hydroxyquinoline (8OHQ) derivatives whose toxicity is paradoxically increased rather than decreased by the activity of P-glycoprotein (Pgp), a transporter conferring multidrug resistance (MDR). Here, we deciphered the mechanism of action of NSC297366 that shows exceptionally strong Pgp-potentiated toxicity. Treatment of cells with NSC297366 resulted in changes associated with the activity of potent anticancer iron chelators. Strikingly, iron depletion was more pronounced in MDR cells due to the Pgp-mediated efflux of NSC297366-iron complexes. Our results indicate that iron homeostasis can be targeted by MDR-selective compounds for the selective elimination of multidrug resistant cancer cells, setting the stage for a therapeutic approach to fight transporter-mediated drug resistance. SIGNIFICANCE: Modulation of the MDR phenotype has the potential to increase the efficacy of anticancer therapies. These findings show that the MDR transporter is a "double-edged sword" that can be turned against resistant cancer

Characterization of ABCB6 mutations responsible for the Lan- blood type.

<p>RBC membrane lysates (80 ug/lane) were prepared from a compound heterozygote individual carrying the R276W and the G588S mutations (lane 1); homozygotes carrying the R192W mutation (c.574C>T, p.Arg192Trp); lanes 2, 3); or individuals carrying two wild-type alleles (lanes 4, 5). ABCB6 expression is revealed by the ABCB6-567 monoclonal antibody <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111590#pone.0111590-Paterson1" target="_blank">[9]</a>, Band III is shown as loading control.</p

Erythrocytic ABCB6 expression in 47 healthy individuals.

<p>Histogram illustrating the distribution of erythrocytic ABCB6 expression in 47 unrelated healthy individuals, as measured by a quantitative FACS assay using the OSK43 monoclonal antibody specifically recognizing a surface epitope of ABCB6 (see Methods). The four individuals exhibiting significantly lower ABCB6 levels (below the 10^th percentile) are labeled by asterisks.</p