HIF-1-dependent heme synthesis promotes gemcitabine resistance in human non-small cell lung cancers via enhanced ABCB6 expression.

Gemcitabine is commonly used to treat various cancer types, including human non-small cell lung cancer (NSCLC). However, even cases that initially respond rapidly commonly develop acquired resistance, limiting our ability to effectively treat advanced NSCLC. To gain insight for developing a strategy to overcome gemcitabine resistance, the present study investigated the mechanism of gemcitabine resistance in NSCLC according to the involvement of ATP-binding cassette subfamily B member 6 (ABCB6) and heme biosynthesis. First, an analysis of ABCB6 expression in human NSCLCs was found to be associated with poor prognosis and gemcitabine resistance in a hypoxia-inducible factor (HIF)-1-dependent manner. Further experiments showed that activation of HIF-1α/ABCB6 signaling led to intracellular heme metabolic reprogramming and a corresponding increase in heme biosynthesis to enhance the activation and accumulation of catalase. Increased catalase levels diminished the effective levels of reactive oxygen species, thereby promoting gemcitabine-based resistance. In a mouse NSCLC model, inhibition of HIF-1α or ABCB6, in combination with gemcitabine, strongly restrained tumor proliferation, increased tumor cell apoptosis, and prolonged animal survival. These results suggest that, in combination with gemcitabine-based chemotherapy, targeting HIF-1α/ABCB6 signaling could result in enhanced tumor chemosensitivity and, thus, may improve outcomes in NSCLC patients.

The compound effect of irradiation and familial pseudohyperkalemia on potassium leak from red blood cells.

BACKGROUND: Familial pseudohyperkalemia (FP) is a rare asymptomatic condition characterized by an increased rate of potassium leak from red blood cells (RBC) on refrigeration. Gamma irradiation compromises RBC membrane integrity and accelerates potassium leakage. Here, we compared the effect of irradiation, applied early or late in storage, on FP versus non-FP RBC. STUDY DESIGN: Five FP and 10 non-FP individuals from the National Institute for Health Research Cambridge BioResource, UK, and three FP and six non-FP individuals identified by Australian Red Cross Lifeblood consented to the study. Blood was collected according to standard practice in each center, held overnight at 18-24°C, leucocyte-depleted, and processed into red cell concentrates (RCC) in Saline Adenine Glucose Mannitol. On Day 1, RCC were split equally into six Red Cell Splits (RCS). Two RCS remained non-irradiated, two were irradiated on Day 1 and two were irradiated on Day 14. RBCs were tested over cold storage for quality parameters. RESULTS: As expected, non-irradiated FP RCS had significantly higher supernatant potassium levels than controls throughout 28 days of storage (p < .001). When irradiated early, FP RCS released potassium at similar rates to control. When irradiated late, FP RCS supernatants had higher initial post-irradiation potassium concentration than controls but were similar to controls by the end of storage (14 days post-irradiation). No other parameters studied showed a significant difference between FP and control. DISCUSSION: FP does not increase the rate of potassium leak from irradiated RBCs. Irradiation may cause a membrane defect similar to that in FP RBCs.

Familial pseudohyperkalemia induces significantly higher levels of extracellular potassium in early storage of red cell concentrates without affecting other standard measures of quality: A case control and allele frequency study.

BACKGROUND: Familial pseudohyperkalemia (FP) is characterized by an increased rate of potassium leakage in refrigerated red cells and is associated with the minor allele of the single nucleotide polymorphism rs148211042 (R723Q) in the ABCB6 gene. The study aims were to obtain the minor allele frequencies of ABCB6 variants and to measure supernatant potassium accumulation, and other red cell storage parameters, in red cell concentrates (RCC) from carriers of variant rs148211042 under standard blood bank conditions. STUDY DESIGN: Whole blood units were collected from 6 FP individuals and 11 controls and processed into RCC in additive solution. RCC were sampled and tested over cold storage for full blood count, extracellular potassium, glucose, lactate, microvesicle release, deformability, hemolysis, pH, adenosine triphosphate, and 2,3-diphosphoglycerate. RESULTS: Screening of genotyped cohorts identified that variant rs148211042 is present in 1 in 394 British citizens of European ancestry. FP RCC had significantly higher supernatant potassium at all time points from day 3 onwards (p < .001) and higher mean cell volume (p = .032) than controls. The initial rate of potassium release was higher in FP RCC; supernatant potassium reached 46.0 (23.8-57.6) mmol/L (mean [range]) by day 5, increasing to 68.9 (58.8-73.7) mmol/L by day 35. Other quality parameters were not significantly different between FP RCC and controls. CONCLUSION: These data suggest that if a blood donor has FP, reducing the RCC shelf-life to 5 days may be insufficient to reduce the risk of hyperkalemia in clinical scenarios such as neonatal large volume transfusion.

The human ABCB6 protein is the functional homologue of HMT-1 proteins mediating cadmium detoxification.

ABCB6 belongs to the family of ATP-binding cassette (ABC) transporters, which transport various molecules across extra- and intra-cellular membranes, bearing significant impact on human disease and pharmacology. Although mutations in the ABCB6 gene have been linked to a variety of pathophysiological conditions ranging from transfusion incompatibility to pigmentation defects, its precise cellular localization and function is not understood. In particular, the intracellular localization of ABCB6 has been a matter of debate, with conflicting reports suggesting mitochondrial or endolysosomal expression. ABCB6 shows significant sequence identity to HMT-1 (heavy metal tolerance factor 1) proteins, whose evolutionarily conserved role is to confer tolerance to heavy metals through the intracellular sequestration of metal complexes. Here, we show that the cadmium-sensitive phenotype of Schizosaccharomyces pombe and Caenorhabditis elegans strains defective for HMT-1 is rescued by the human ABCB6 protein. Overexpression of ABCB6 conferred tolerance to cadmium and As(III) (As2O3), but not to As(V) (Na2HAsO4), Sb(V), Hg(II), or Zn(II). Inactivating mutations of ABCB6 abolished vacuolar sequestration of cadmium, effectively suppressing the cadmium tolerance phenotype. Modulation of ABCB6 expression levels in human glioblastoma cells resulted in a concomitant change in cadmium sensitivity. Our findings reveal ABCB6 as a functional homologue of the HMT-1 proteins, linking endolysosomal ABCB6 to the highly conserved mechanism of intracellular cadmium detoxification.

A Case-Control Study of ABCB1, ABCB6, and ABCG1 Polymorphisms and Schizophrenia in a Han Chinese Population.

BACKGROUND: Schizophrenia (SCZ) is a complex, heritable, and devastating psychiatric disorder. Mutations in the members of ABC transporters have been associated with psychiatric illnesses. AIMS: In this study, we investigated whether 9 SNPs in ABCB1 (rs6946119, rs28401781, rs4148739, and rs3747802), ABCB6 (rs1109866, rs1109867, rs3731885, and rs3755047), and ABCG1 (rs182694) contribute to the risk of SCZ in a Han Chinese population. METHODS: We conducted a case-control study in a Han Chinese population, involving 1,034 SCZ patients and 1,034 unrelated healthy controls to genotype 9 SNPs. RESULTS: The analysis demonstrated that rs182694 of ABCG1 was significantly different between SCZ patients and controls as to allele (rs182694: p = 0.0070, χ2 = 7.27) and genotype frequencies (rs182694: p = 0.0013, χ2 = 13.35). CONCLUSIONS: Our findings support an association between ABCG1 polymorphism and SCZ in a Han Chinese population.

Leishmania panamensis infection and antimonial drugs modulate expression of macrophage drug transporters and metabolizing enzymes: impact on intracellular parasite survival.

OBJECTIVES: Treatment failure is multifactorial. Despite the importance of host cell drug transporters and metabolizing enzymes in the accumulation, distribution and metabolism of drugs targeting intracellular pathogens, their impact on the efficacy of antileishmanials is unknown. We examined the contribution of pharmacologically relevant determinants in human macrophages in the antimony-mediated killing of intracellular Leishmania panamensis and its relationship with the outcome of treatment with meglumine antimoniate. METHODS: Patients with cutaneous leishmaniasis who failed (n = 8) or responded (n =8) to treatment were recruited. Gene expression profiling of pharmacological determinants in primary macrophages was evaluated by quantitative RT-PCR and correlated to the drug-mediated intracellular parasite killing. Functional validation was conducted through short hairpin RNA gene knockdown. RESULTS: Survival of L. panamensis after exposure to antimonials was significantly higher in macrophages from patients who failed treatment. Sixteen macrophage drug-response genes were modulated by infection and exposure to meglumine antimoniate. Correlation analyses of gene expression and intracellular parasite survival revealed the involvement of host cell metallothionein-2A and ABCB6 in the survival of Leishmania during exposure to antimonials. ABCB6 was functionally validated as a transporter of antimonial compounds localized in both the cell and phagolysosomal membranes of macrophages, revealing a novel mechanism of host cell-mediated regulation of intracellular drug exposure and parasite survival within phagocytes. CONCLUSIONS: These results provide insight into host cell mechanisms regulating the intracellular exposure of Leishmania to antimonials and variations among individuals that impact parasite survival. Understanding of host cell determinants of intracellular pharmacokinetics/pharmacodynamics opens new avenues to improved drug efficacy for intracellular pathogens.

Identification of novel genetic variations in ABCB6 and GRN genes associated with HIV-associated lipodystrophy.

Protease inhibitors (PIs) are associated with an incidence of lipodystrophy among people living with HIV(PLHIV). Lipodystrophiesare characterised by the loss of adipose tissue. Evidence suggests that a patient's lipodystrophy phenotype is influenced by genetic mutation, age, gender, and environmental and genetic factors, such as single-nucleotide variants (SNVs). Pathogenic variants are considered to cause a more significant loss of adipose tissue compared to non-pathogenic. Lipid metabolising enzymes and transporter genes have a role in regulating lipoprotein metabolism and have been associated with lipodystrophy in HIV-infected patients (LDHIV). The long-term effect of the lipodystrophy syndrome is related to cardiovascular diseases (CVDs). Hence, we determined the SNVs of lipid metabolising enzymes and transporter genes in a total of 48 patient samples, of which 24 were with and 24 were without HIV-associated lipodystrophy (HIVLD) using next-generation sequencing. A panel of lipid metabolism, transport and elimination genes were sequenced. Three novel heterozygous non-synonymous variants at exon 8 (c.C1400A:p.S467Y, c.G1385A:p.G462E, and c.T1339C:p.S447P) in the ABCB6 gene were identified in patients with lipodystrophy. One homozygous non-synonymous SNV (exon5:c.T358C:p.S120P) in the GRN gene was identified in patients with lipodystrophy. One novelstop-gain SNV (exon5:c.C373T:p.Q125X) was found in the GRN gene among patients without lipodystrophy. Patients without lipodystrophy had one homozygous non-synonymous SNV (exon9:c.G1462T:p.G488C) in the ABCB6 gene. Our findings suggest that novel heterozygous non-synonymous variants in the ABCB6 gene may contribute to defective protein production, potentially intensifying the severity of lipodystrophy. Additionally, identifying a stop-gain SNV in the GRN gene among patients without lipodystrophy implies a potential role in the development of HIVLD.

Marked hyperkalemia due to inappropriate blood sample storage in two suspected cases of familial pseudohyperkalemia.

We herein report two suspected cases of pseudohyperkalemia who presented with severe hyperkalemia examined at small primary care clinics; however, re-exams at a tertiary care hospital showed normal potassium levels. We reproduced the laboratory examination conditions of the clinics and found that hyperkalemia was due to sampling/storage condition of serum, which is strongly suggestive of familial pseudohyperkalemia (FP). FP is a possible but under-appreciated cause of hyperkalemia, which does not require treatment, so it is important to include FP in the differential diagnosis of hyperkalemia especially in cases with discrepant of serum potassium levels at different settings.

Association of UBE2L6 and ABCB6 Expression With Platinum Resistance in Serous Ovarian Carcinoma.

BACKGROUND/AIM: Platinum-based drugs are the standard treatment for ovarian cancer, and platinum resistance is a major problem. A previous study has reported that the UBE2L6 expression is elevated in cisplatin-resistant cells, which in turn leads to cisplatin resistance by modulating the transcriptional expression of ABCB6. The present study aimed to investigate the expression of UBE2L6 and ABCB6 in ovarian carcinoma and to evaluate the association between these markers and platinum resistance. PATIENTS AND METHODS: Ninety-two patients diagnosed with serous ovarian carcinoma (SOC) were enrolled in this study. Tissue samples were collected from these patients and analysed using immunohistochemistry to assess the expression of UBE2L6 and ABCB6. RESULTS: UBE2L6 and ABCB6 staining was positive in 41 (44.6%) and 46 (50.0%) cases, respectively. UBE2L6 expression was statistically significantly associated with International Federation of Gynecology and Obstetrics (FIGO) stage (p=0.008). Both UBE2L6 and ABCB6 were significantly associated with platinum sensitivity (p<0.001 and p<0.001). A positive correlation was observed between the expression levels of UBE2L6 and ABCB6 (r=0.673, p<0.001). Progression-free survival (PFS) was significantly longer in the UBE2L6 negative group than that in the positive group (median PFS, 31.4 vs. 11.1 months, p<0.01) and in the ABCB6 negative group than that in the positive group (median PFS, 29.6 vs. 12.2 months, p<0.01). CONCLUSION: UBE2L6 and ABCB6 expression is associated with the prognosis of SOC. UBE2L6 and ABCB6 may be potential biomarkers of platinum-resistant ovarian cancer.

Protoporphyrin IX (PpIX) Fluorescence during Meningioma Surgery: Correlations with Histological Findings and Expression of Heme Pathway Molecules.

Background: The usefulness of 5-ALA-mediated fluorescence-guided resection (FGR) in meningiomas is controversial, and information on the molecular background of fluorescence is sparse. Methods: Specimens obtained during 44 FGRs of intracranial meningiomas were analyzed for the presence of tumor tissue and fluorescence. Protein/mRNA expression of key transmembrane transporters/enzymes involved in PpIX metabolism (ABCB6, ABCG2, FECH, CPOX) were investigated using immunohistochemistry/qPCR. Results: Intraoperative fluorescence was observed in 70 of 111 specimens (63%). No correlation was found between fluorescence and the WHO grade (p = 0.403). FGR enabled the identification of neoplastic tissue (sensitivity 84%, specificity 67%, positive and negative predictive value of 86% and 63%, respectively, AUC: 0.75, p < 0.001), and was improved in subgroup analyses excluding dura specimens (86%, 88%, 96%, 63% and 0.87, respectively; p < 0.001). No correlation was found between cortical fluorescence and tumor invasion (p = 0.351). Protein expression of ABCB6, ABCG2, FECH and CPOX was found in meningioma tissue and was correlated with fluorescence (p < 0.05, each), whereas this was not confirmed for mRNA expression. Aberrant expression was observed in the CNS. Conclusion: FGR enables the intraoperative identification of meningioma tissue with limitations concerning dura invasion and due to ectopic expression in the CNS. ABCB6, ABCG2, FECH and CPOX are expressed in meningioma tissue and are related to fluorescence.

Targeting ABCB6 with nitidine chloride inhibits PI3K/AKT signaling pathway to promote ferroptosis in multiple myeloma.

Since multiple myeloma (MM) remains a cureless malignancy of plasma cells to date, it becomes imperative to develop novel drugs and therapeutic targets for MM. We screened a small molecule library comprising 3633 natural product drugs, which demonstrated that Nitidine Chloride (NC), an extract from traditional Chinese medicine Zanthoxylum nitidum. We used Surface Plasmon Resonance-High Performance Liquid Chromatography-Protein Mass Spectrometry (SPR-HPLC-MS), Cellular Thermal Shift Assay (CETSA), molecular docking, and SPR assay to identify the potential targets of NC, in which ABCB6 was the unique target of NC. The effects of ABCB6 on cellular proliferation and drug resistance were determined by CCK8, western blot, flow cytometry, site-mutation cells, transmission electron microscopy, immunohistochemistry staining and xenograft model in vitro and in vivo. NC induced MM cell death by promoting ferroptosis. ABCB6 is the direct target of NC. ABCB6 expression was increased in MM samples compared to normal controls, which was significantly associated with MM relapse and poor outcomes. VGSK was the inferred binding epitope of NC on the ABCB6 protein. In the ABCB6-mutated MM cells, NC did not display cancer resistance, implying the vital role of ABCB6 in NC's bioactivity. Moreover, the silencing of ABCB6 significantly inhibited MM cell growth. Mechanistically, the direct binding of NC to ABCB6 suppressed PI3K/AKT signaling pathway to promote ferroptosis. In conclusion, ABCB6 can be a potential therapeutic target and prognostic biomarker in MM, while NC can be considered a novel drug for MM treatment.

ABCB6 knockdown suppresses melanogenesis through the GSK3-ß/ß-catenin signaling axis in human melanoma and melanocyte cell lines.

BACKGROUND: Melanogenesis is a multistep process in which melanocytes produce melanin pigments within melanosomes. However, the roles played by the biological factors and pathways in this process are not yet fully understood. OBJECTIVE: To investigate the role of ATP-binding cassette subfamily B member 6 (ABCB6) in the regulation of melanogenesis in vitro. METHODS: Real-time PCR and western blotting were used to assess the knockdown efficiency of ABCB6 in MNT-1 and PIG1 stable cell lines. Cleavage by NaOH was used to determine melanin content, while the number of melanosomes was examined for each stage by transmission electron microscopy. Immunofluorescence microscopy was used to evaluate endogenous protein location. Differentially expressed genes were detected using RNA sequencing, and gene expression was assessed by quantitative real-time PCR. KEGG mapping was used for pathway enrichment analysis. Co-immunoprecipitation was used for protein-protein interactions analysis. RESULTS: We found that ABCB6 inhibition could impair melanocyte maturation and melanin production in human melanoma (MNT-1) and immortalized human melanocyte (PIG1) cell lines. Moreover, ABCB6 knockdown inhibited the protein expression of melanocyte inducing microphthalmia-associated transcription factor (MITF) and its three downstream melanogenic enzymes (TYR, TYRP1 and TYRP2). Mechanistically, we revealed that ABCB6 could interact with and modulate glycogen synthase kinase 3 beta (GSK3-ß) to exert its biological effect on melanogenesis. CONCLUSION: Our findings suggest that ABCB6 is a key regulator of melanogenesis via the GSK3-ß/ß-catenin signaling pathway. However, further in-depth studies are essential to uncover the relationship between ABCB6 and pigmentation disorders.

Case Report: Familial Pseudohyperkalemia Due to Red Blood Cell Membrane Leak in a Chinese Patient.

Hyperkalemia is a critical condition requiring careful evaluation and timely intervention. Many conditions could manifest as pseudohyperkalemia and it's important to differentiate them as inappropriate potassium-lowering therapy might lead to detrimental outcomes. A 56-year-old female was admitted for hyperkalemia (5.62-8.55 mmol/L). She had no symptoms or signs of hyperkalemia. A comprehensive work-up of hyperkalemia retrieved no valuable findings. Her blood samples underwent incubation tests at different temperatures and revealed temperature-dependent potassium leaks from red blood cells. Based on all test results, a diagnosis of hyperkalemia caused by red blood cell membrane defects was suspected. Whole-genome sequencing revealed a heterozygous c.1123C>T (p. R375W) mutation in the ABCB6 gene and confirmed the diagnosis of familial pseudohyperkalemia (FP). FP is an inherited benign condition in which red blood cells have increased cold-induced permeability to potassium. The patient was discharged with no additional treatment and she was suggested avoiding blood donation.

Structural Insights into Porphyrin Recognition by the Human ATP-Binding Cassette Transporter ABCB6.

Human ABCB6 is an ATP-binding cassette transporter that regulates heme biosynthesis by translocating various porphyrins from the cytoplasm into the mitochondria. Here we report the cryo-electron microscopy (cryo-EM) structures of human ABCB6 with its substrates, coproporphyrin III (CPIII) and hemin, at 3.5 and 3.7 Å resolution, respectively. Metalfree porphyrin CPIII binds to ABCB6 within the central cavity, where its propionic acids form hydrogen bonds with the highly conserved Y550. The resulting structure has an overall fold similar to the inward-facing apo structure, but the two nucleotide-binding domains (NBDs) are slightly closer to each other. In contrast, when ABCB6 binds a metal-centered porphyrin hemin in complex with two glutathione molecules (1 hemin: 2 glutathione), the two NBDs end up much closer together, aligning them to bind and hydrolyze ATP more efficiently. In our structures, a glycine-rich and highly flexible "bulge" loop on TM helix 7 undergoes significant conformational changes associated with substrate binding. Our findings suggest that ABCB6 utilizes at least two distinct mechanisms to fine-tune substrate specificity and transport efficiency.

Novel missense mutation of SASH1 in a Chinese family with dyschromatosis universalis hereditaria.

BACKGROUND: Dyschromatosis universalis hereditaria (DUH) is a pigmentary dermatosis characterized by generalized mottled macules with hypopigmention and hyperpigmention. ABCB6 and SASH1 are recently reported pathogenic genes related to DUH, and the aim of this study was to identify the causative mutations in a Chinese family with DUH. METHODS: Sanger sequencing was performed to investigate the clinical manifestation and molecular genetic basis of these familial cases of DUH, bioinformatics tools and multiple sequence alignment were used to analyse the pathogenicity of mutations. RESULTS: A novel missense mutation, c.1529G>A, in the SASH1 gene was identified, and this mutation was not found in the National Center for Biotechnology Information Database of Short Genetic Variation, Online Mendelian Inheritance in Man, ClinVar, or 1000 Genomes Project databases. All in silico predictors suggested that the observed substitution mutation was deleterious. Furthermore, multiple sequence alignment of SASH1 revealed that the p.S510N mutation was highly conserved during evolution. In addition, we reviewed the previously reported DUH-related gene mutations in SASH1 and ABCB6. CONCLUSION: Although the affected family members had identical mutations, differences in the clinical manifestations of these family members were observed, which reveals the complexity of the phenotype-influencing factors in DUH. Our findings reveal the mutation responsible for DUH in this family and broaden the mutational spectrum of the SASH1 gene.

Cryo-electron microscopy structure of human ABCB6 transporter.

Human ATP-binding cassette transporter 6 of subfamily B (ABCB6) is an ABC transporter involved in the translocation toxic metals and anti-cancer drugs. Using cryo-electron microscopy, we determined the molecular structure of full-length ABCB6 in an apo state. The structure of ABCB6 unravels the architecture of a full-length ABCB transporter that harbors two N-terminal transmembrane domains which is indispensable for its ATPase activity in our in vitro assay. A slit-like substrate binding pocket of ABCB6 may accommodate the planar shape of porphyrins, and the existence of a secondary cavity near the mitochondrial intermembrane space side would further facilitate substrate release. Furthermore, the ATPase activity of ABCB6 stimulated with a variety of porphyrin substrates showed different profiles in the presence of glutathione (GSH), suggesting the action of a distinct substrate translocation mechanism depending on the use of GSH as a cofactor.

Systematic analysis of the ABC transporter family in hepatocellular carcinoma reveals the importance of ABCB6 in regulating ferroptosis.

AIMS: ATP-binding cassette (ABC) transporters constitute one of the largest families of membrane proteins in most organisms; however, their functions in hepatocellular carcinoma (HCC) remain unclear. MAIN METHODS: A set of bioinformatic tools was integrated to analyze the expression of 49 members of the ABC transporter family. The function of members which had prognostic values in HCC was explored by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. KEY FINDINGS: ABCA8 and ABCA9 were significantly down-regulated in HCC. Prognostic analysis indicated that HCC patients with low expression of ABCA8 and ABCA9 had significantly shorter survival time. On the contrary, ABCB6 was over-expressed in the disease and high expression of ABCB6 was associated with worse prognosis. Co-expression analysis, and subsequently GO and KEGG analysis indicated that ABCA8 and ABCA9 might participate in the catabolic processes of multiple metabolites, while ABCB6 might regulate ferroptosis. SIGNIFICANCE: This study reveals a previously unrecognized function of ABCB6 in HCC, by regulating ferroptosis. Since ABCB6 is over-expressed in HCC and ferroptosis involves in cancer development, ABCB6 might be a promising therapeutic target in the disease.

Identification of a Novel Mutation in SASH1 Gene in a Chinese Family With Dyschromatosis Universalis Hereditaria and Genotype-Phenotype Correlation Analysis.

Dyschromatosis universalis hereditaria (DUH) is a rare genodermatosis characterized by mottled hyperpigmented and hypopigmented macules. SASH1 and ABCB6 have been identified as the causative genes for this disorder. We performed whole exome sequencing on a Chinese family with DUH and genotype-phenotype correlation analysis in DUH and lentiginous phenotype patients. A novel heterozygous missense mutation p.Q518P in SASH1 gene was detected in this family. A majority of patients with SASH1 mutations presented as a distinct clinical phenotype clearly different from that in patients with ABCB6 mutations. Our findings further enrich the reservoir of SASH1 mutations in DUH. The clinical phenotypic difference between SASH1 and ABCB6 variants is suggestive of a close phenotype-genotype link in DUH.

ABCB6 Resides in Melanosomes and Regulates Early Steps of Melanogenesis Required for PMEL Amyloid Matrix Formation.

Genetically inheritable pigmentation defects provide a unique opportunity to reveal the function of proteins contributing to melanogenesis. Dyschromatosis universalis hereditaria (DUH) is a rare pigmentary genodermatosis associated with mutations in the ABCB6 gene. Here we use optical and electron microscopy imaging combined with biochemical tools to investigate the localization and function of ABCB6 in pigment cells. We show that ABCB6 localizes to the membrane of early melanosomes and lysosomes of the human melanocytic cell line MNT-1. Depletion of ABCB6 by siRNA impaired PMEL amyloidogenesis in early melanosomes and induced aberrant accumulation of multilamellar aggregates in pigmented melanosomes. PMEL fibril formation and normal maturation of pigmented melanosomes could be restored by the overexpression of wild-type ABCB6 but not by variants containing an inactivating catalytic mutation (K629M) or the G579E DUH mutation. In line with the impairment of PMEL matrix formation in the absence of ABCB6, morphological analysis of the retinal pigment epithelium of ABCB6 knockout mice revealed a significant decrease of melanosome numbers. Our study extends the localization of ABCB6 to melanosomes, suggesting a potential link between the function of ABCB6 and the etiology of DUH to amyloid formation in pigment cells.

N-Terminal Extension and C-Terminal Domains Are Required for ABCB6/HMT-1 Protein Interactions, Function in Cadmium Detoxification, and Localization to the Endosomal-Recycling System in Caenorhabditis elegans.

The chronic exposure of humans to toxic metals such as cadmium from food and air causes dysfunction of vital organs, neurodegenerative conditions, and cancer. In this regard, members of the ABCB sub-family of the ATP-binding cassette (ABC) transporter superfamily, ABCB6/HMT-1, are acutely required for the detoxification of heavy metals and are present in genomes of many organisms including the nematode worm, Caenorhabditis elegans and humans. We showed previously that C. elegans ABCB6/HMT-1 detoxifies cadmium, copper, and arsenic, and is expressed in liver-like cells, the coelomocytes, head neurons and intestinal cells, which are the cell types that are affected by heavy metal poisoning in humans. The subcellular localization of ABCB6/HMT-1 proteins is unclear. ABCB6/HMT-1 proteins have a distinguishing topology: in addition to one transmembrane domain and one nucleotide-binding domain, they possess a hydrophobic N-terminal extension (NTE) domain encompassing five to six transmembrane spans. The role of the NTE domain in the function of ABCB6/HMT-1 in the native organism remains to be investigated. We used a versatile, multicellular model system, C. elegans, to establish the subcellular localization of ABCB6/HMT-1 and refine its structure-function studies in the native organism. We show that ABCB6/HMT-1 localizes mainly to the apical recycling endosomes and, in part, to early and late endosomes of intestinal cells. We also show that ABCB6/HMT-1 lacking the NTE domain is mistargeted to the plasma membrane and is unable to confer cadmium resistance. Although the NTE domain is essential for ABCB6/HMT-1 interaction with itself, the absence of NTE does not fully prevent this interaction. As a result, ABCB6/HMT-1 lacking the NTE domain, and expressed in wild-type worms or co-expressed with the full-length polypeptide, inactivates and mistargets the full-length ABCB6/HMT-1. We also show that the 43 amino acid residue stretch at the COOH-terminus is required for the ABCB6/HMT-1 interaction with itself and cadmium detoxification function. These results suggest that both NTE and COOH-terminus must be present to allow the protein to interact with itself and confer cadmium resistance. Considering that ABCB6/HMT-1 proteins are highly conserved, this study advances our understanding of how these proteins function in cadmium resistance in different species. Furthermore, these studies uncover the role of the endosomal-recycling system in cadmium detoxification.