Mitochondrial ATP-binding Cassette Transporter Research Articles

Mitochondrial Inner Membrane ABC Transporter Bcmdl1 Is Involved in Conidial Germination, Virulence, and Resistance to Anilinopyrimidine Fungicides in Botrytis cinerea.

Botrytis cinerea causes gray mold on thousands of plants, leading to huge losses in production. Anilinopyrimidine (AP) fungicides have been applied to control B. cinerea since the 1990s. Although resistance to AP fungicides was detected soon after their application, the mechanism of AP resistance remains to be elucidated. In this study, a sexual cross between resistant and sensitive isolates was performed, and the genomes of parental isolates and progenies were sequenced to identify resistance-related single nucleotide polymorphisms (SNPs). After screening and verification, mutation E407K in the Bcmdl1 gene was identified and confirmed to confer resistance to AP fungicides in B. cinerea. Bcmdl1 was predicted to encode a mitochondrial protein that belonged to a half-type ATP-binding cassette (ABC) transporter. Although Bcmdl1 was a transporter, it did not mediate resistance to multiple fungicides but mediated resistance specifically to AP fungicides. On the other hand, reductions in conidial germination and virulence were observed in Bcmdl1 knockout transformants compared to the parental isolate and complemented transformants, illustrating the biological functions of Bcmdl1. Subcellular localization analysis indicated that Bcmdl1 was localized in mitochondria. Interestingly, the production of ATP was reduced after cyprodinil treatment in Bcmdl1 knockout transformants, suggesting that Bcmdl1 was involved in ATP synthesis. Since Mdl1 could interact with ATP synthase in yeast, we hypothesize that Bcmdl1 forms a complex with ATP synthase, which AP fungicides might target, thereby interfering with the metabolism of energy. IMPORTANCE Gray mold, caused by B. cinerea, causes huge losses in the production of many fruits and vegetables. AP fungicides have been largely adopted to control this disease since the 1990s, and the development of resistance to AP fungicides initiates new problems for disease control. Due to the unknown mode of action, information on the mechanism of AP resistance is also limited. Recently, mutations in mitochondrial genes were reported to be related to AP resistance. However, the mitochondrial process of these genes remains to be elucidated. In this study, we identified several AP resistance-related mutations by quantitative trait locus sequencing (QTL-seq) and confirmed that mutation E407K in Bcmdl1 conferred AP resistance. We further characterized the expression patterns, biological functions, subcellular localization, and mitochondrial processes of the Bcmdl1 gene. This study deepens our understanding of the mechanism of resistance to and mode of action of AP fungicides.

Production of a human mitochondrial ABC transporter in E. coli

Membrane proteins play important roles in health and disease. Despite their importance, the study of membrane proteins has been significantly limited by the difficulties inherent to their successful expression, purification, and stabilization once they have been extracted from the cell membrane. In addition, expression of human membrane proteins commonly requires the use of expensive and/or time-consuming eukaryotic systems, hence their successful expression in bacteria will be obviously beneficial for experimental research. Furthermore, since lipids can have critical effects on the activity of membrane proteins and given the composition similarities between the inner mitochondrial membrane and the bacterial plasma membrane, production of mitochondrial membrane proteins in E. coli represents a logical choice. Here, we present a novel protocol to produce a human mitochondrial ATP-Binding Cassette (ABC) transporter in E. coli. The function of the three known human mitochondrial ABC transporters is not fully understood, but X-ray crystallography models of ABCB10 produced in insect cells are available. We have successfully expressed and purified ABCB10 from E. coli. The yield is close to that of another bacterial ABC transporter routinely produced in our laboratory under similar conditions. In addition, we can efficiently reconstitute detergent purified ABCB10 into lipid nanodiscs. Measurements of ATPase activity of ABCB10 produced in E. coli show an ATP hydrolysis rate similar to other human ABC transporters. This novel protocol facilitates the production of this human mitochondrial transporter for biochemical, structural, and functional analysis, and can likely be adjusted for production of other mitochondrial transporters.

ABCB7 is a gatekeeper of both programmed cell death, apoptotic and non-apoptotic cell death

Abstract Background The ATP-binding cassette (ABC) transporters are superfamily proteins of cellular translocation machinery. Many ABC transporters release drugs to give multidrug resistance. However, ABC transporters existing in intracellular compartments give drug resistance by regulating biological processes, rather than direct release of drugs. The current study aimed to investigate the molecular mechanism that causes the drug resistance of the mitochondrial ABC transporters in cancer cells. Methods RNA-sequencing and bioinformatics analyses were performed to identify key mitochondrial ABC transporters and ABC transporter subfamily B member 7 (ABCB7)-mediated transcriptomic alterations. We investigated role of ABCB7 in iron homeostasis by examining iron concentration and reactive oxygen species (ROS) generation. Drug resistance and cellular survival mechanisms were understood by comparing the type and degree of cell death according to the ABCB7 expression. Results ABCB7 is highly expressed in patients with glioblastoma and has a negative correlation with prognosis. The increase in ABCB7 gives resistance to drug and the loss of ABCB7 leads to cell death. ABCB7 increases the expression of hypoxia-inducible factor 1 alpha (HIF1α) and activates HIF1α signaling by regulating iron concentration. When ABCB7 expression is maintained high in cancer cells, hypoxia-independent activation of HIF1α inhibits cell death by suppressing leucine zipper downregulated in cancer 1 (LDOC1). However, when ABCB7 is decreased, non-apoptotic cell death is induced by the change of iron concentration, and apoptosis which is inhibited by HIF1α signal is also induced. Additionally, because ABCB7 is essential for survival of universal cancer cells, it is presented as a pan-cancer core fitness gene. Conclusions Our findings demonstrate the ABCB7 inhibits both apoptotic and non-apoptotic cell death via maintenance of iron homeostasis and hypoxia-independent HIF1α signaling. Editorial acknowledgement Cancer Growth Regulation Laboratory for their discussion and technical assistance. Legal entity responsible for the study The authors. Funding National Research Foundation of Korea (NRF) [grant numbers: 2015R1A5A1009024 and 2017R1E1A1A01074205], the School of Life Sciences and Biotechnology for BK21 Plus, Korea University and Institute of Animal Molecular Biotechnology Korea University. Disclosure All authors have declared no conflicts of interest.

Mitochondrial membrane transporters and metabolic switch in heart failure.

Mitochondrial dysfunction is widely recognized as a major factor for the progression of cardiac failure. Mitochondrial uptake of metabolic substrates and their utilization for ATP synthesis, electron transport chain activity, reactive oxygen species levels, ion homeostasis, mitochondrial biogenesis, and dynamics as well as levels of reactive oxygen species in the mitochondria are key factors which regulate mitochondrial function in the normal heart. Alterations in these functions contribute to adverse outcomes in heart failure. Iron imbalance and oxidative stress are also major factors for the evolution of cardiac hypertrophy, heart failure, and aging-associated pathological changes in the heart. Mitochondrial ATP-binding cassette (ABC) transporters have a key role in regulating iron metabolism and maintenance of redox status in cells. Deficiency of mitochondrial ABC transporters is associated with an impaired mitochondrial electron transport chain complex activity, iron overload, and increased levels of reactive oxygen species, all of which can result in mitochondrial dysfunction. In this review, we discuss the role of mitochondrial ABC transporters in mitochondrial metabolism and metabolic switch, alterations in the functioning of ABC transporters in heart failure, and mitochondrial ABC transporters as possible targets for therapeutic intervention in cardiac failure.

Mitochondrial ABC Transporters and Iron Metabolism

Mitochondrial are a key organelle in iron metabolism and many metabolic processes involved in iron homeostasis occur in the mitochondria. Eukaryotic cells have developed different transport mechanisms to deal with coordinating movement of iron and iron-related molecules across membranes. Some of those transport mechanisms involve ATP-binding cassette (ABC) transporters. There are four mitochondrial ABC transporters Abcb6, Abcb7, Abcb8 and Abcb10. Abcb6 is localized to the outer membrane of mitochondria where it is involved in porphyrin transport. Abcb7, Abcb8 and Abcb10 are localized to the inner mitochondrial membrane and the exact molecule transported by each is still unclear. Here, we provide a brief review of what is known about each transporter and its role in mitochondrial iron homeostasis. We describe the human diseases associated with known mutations in the genes encoding these proteins and discuss the possible importance of these transporters in immune cell function.

Predictive Structure and Topology of Peroxisomal ATP-Binding Cassette (ABC) Transporters.

The peroxisomal ATP-binding Cassette (ABC) transporters, which are called ABCD1, ABCD2 and ABCD3, are transmembrane proteins involved in the transport of various lipids that allow their degradation inside the organelle. Defective ABCD1 leads to the accumulation of very long-chain fatty acids and is associated with a complex and severe neurodegenerative disorder called X-linked adrenoleukodystrophy (X-ALD). Although the nucleotide-binding domain is highly conserved and characterized within the ABC transporters family, solid data are missing for the transmembrane domain (TMD) of ABCD proteins. The lack of a clear consensus on the secondary and tertiary structure of the TMDs weakens any structure-function hypothesis based on the very diverse ABCD1 mutations found in X-ALD patients. Therefore, we first reinvestigated thoroughly the structure-function data available and performed refined alignments of ABCD protein sequences. Based on the 2.85 Å resolution crystal structure of the mitochondrial ABC transporter ABCB10, here we propose a structural model of peroxisomal ABCD proteins that specifies the position of the transmembrane and coupling helices, and highlight functional motifs and putative important amino acid residues.

Mitochondrial ABC Transporter ATM3 Is Essential for Cytosolic Iron-Sulfur Cluster Assembly.

The mitochondrial ATP-binding cassette transporter ATM3 has been studied in Arabidopsis. Its function, however, is poorly understood in other model plant species. This study reports that the ATM3 is required for cytosolic iron-sulfur cluster assembly and is essential for meristem maintenance in rice (Oryza sativa). The loss of function of OsATM3 is lethal in rice at the four-leaf stage. In the osatm3 T-DNA insertion mutant, the fourth leaf fails to develop and the lateral roots are short. Cytosolic iron-sulfur protein activities were significantly reduced in both osatm3 and RNA interference transgenic lines. The expression profiles of many iron metabolism genes were altered in the osatm3 and RNA interference lines. Glutathione metabolism was impaired and reactive oxygen species, particularly superoxide, accumulated in osatm3 Promoter-β-glucuronidase staining of the transgenic line indicated that OsATM3 is highly expressed in lateral root primordia, root tip meristem zones, and shoot apical meristem regions. The average cell size was significantly greater in osatm3 than in the wild type. Massive cell death occurred in the osatm3 root tip meristem zone. Quantitative RT-PCR revealed transcriptional reprogramming of the genes in the osatm3 and RNAi lines involved in DNA repair and cell cycle arrest. Our results suggest that the mitochondrial ATM3 is essential for iron homeostasis in rice.

LmABCB3, an atypical mitochondrial ABC transporter essential for Leishmania major virulence, acts in heme and cytosolic iron/sulfur clusters biogenesis.

BackgroundMitochondria play essential biological functions including the synthesis and trafficking of porphyrins and iron/sulfur clusters (ISC), processes that in mammals involve the mitochondrial ATP-Binding Cassette (ABC) transporters ABCB6 and ABCB7, respectively. The mitochondrion of pathogenic protozoan parasites such as Leishmania is a promising goal for new therapeutic approaches. Leishmania infects human macrophages producing the neglected tropical disease known as leishmaniasis. Like most trypanosomatid parasites, Leishmania is auxotrophous for heme and must acquire porphyrins from the host.MethodsLmABCB3, a new Leishmania major protein with significant sequence similarity to human ABCB6/ABCB7, was identified and characterized using bioinformatic tools. Fluorescent microscopy was used to determine its cellular localization, and its level of expression was modulated by molecular genetic techniques. Intracellular in vitro assays were used to demonstrate its role in amastigotes replication, and an in vivo mouse model was used to analyze its role in virulence. Functional characterization of LmABCB3 was carried out in Leishmania promastigotes and Saccharomyces cerevisiae. Structural analysis of LmABCB3 was performed using molecular modeling software.ResultsLmABCB3 is an atypical ABC half-transporter that has a unique N-terminal extension not found in any other known ABC protein. This extension is required to target LmABCB3 to the mitochondrion and includes a potential metal-binding domain. We have shown that LmABCB3 interacts with porphyrins and is required for the mitochondrial synthesis of heme from a host precursor. We also present data supporting a role for LmABCB3 in the biogenesis of cytosolic ISC, essential cofactors for cell viability in all three kingdoms of life. LmABCB3 fully complemented the severe growth defect shown in yeast lacking ATM1, an orthologue of human ABCB7 involved in exporting from the mitochondria a gluthatione-containing compound required for the generation of cytosolic ISC. Indeed, docking analyzes performed with a LmABCB3 structural model using trypanothione, the main thiol in this parasite, as a ligand showed how both, LmABCB3 and yeast ATM1, contain a similar thiol-binding pocket. Additionally, we show solid evidence suggesting that LmABCB3 is an essential gene as dominant negative inhibition of LmABCB3 is lethal for the parasite. Moreover, the abrogation of only one allele of the gene did not impede promastigote growth in axenic culture but prevented the replication of intracellular amastigotes and the virulence of the parasites in a mouse model of cutaneous leishmaniasis.ConclusionsAltogether our results present the previously undescribed LmABCB3 as an unusual mitochondrial ABC transporter essential for Leishmania survival through its role in the generation of heme and cytosolic ISC. Hence, LmABCB3 could represent a novel target to combat leishmaniasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1284-5) contains supplementary material, which is available to authorized users.

Structures and functions of mitochondrial ABC transporters.

A small number of physiologically important ATP-binding cassette (ABC) transporters are found in mitochondria. Most are half transporters of the B group forming homodimers and their topology suggests they function as exporters. The results of mutant studies point towards involvement in iron cofactor biosynthesis. In particular, ABC subfamily B member 7 (ABCB7) and its homologues in yeast and plants are required for iron-sulfur (Fe-S) cluster biosynthesis outside of the mitochondria, whereas ABCB10 is involved in haem biosynthesis. They also play a role in preventing oxidative stress. Mutations in ABCB6 and ABCB7 have been linked to human disease. Recent crystal structures of yeast Atm1 and human ABCB10 have been key to identifying substrate-binding sites and transport mechanisms. Combined with invitro and invivo studies, progress is being made to find the physiological substrates of the different mitochondrial ABC transporters.

Functional Coupling of ATP-binding Cassette Transporter Abcb6 to Cytochrome P450 Expression and Activity in Liver

Although endogenous mechanisms that negatively regulate cytochrome P450 (P450) monooxygenases in response to physiological and pathophysiological signals are not well understood, they are thought to result from alterations in the level of endogenous metabolites, involved in maintaining homeostasis. Here we show that homeostatic changes in hepatic metabolite profile in Abcb6 (mitochondrial ATP-binding cassette transporter B6) deficiency results in suppression of a specific subset of hepatic P450 activity. Abcb6 null mice are more susceptible to pentobarbital-induced sleep and zoxazolamine-induced paralysis, secondary to decreased expression and activity of Cyp3a11 and Cyp2b10. The knock-out mice also show decrease in both basal and xeno-inducible expression and activity of a subset of hepatic P450s that appear to be related to changes in hepatic metabolite profile. These data, together with the observation that liver extracts from Abcb6-deficient mice suppress P450 expression in human primary hepatocytes, suggest that this mouse model may provide an opportunity to understand the physiological signals and the mechanisms involved in negative regulation of P450s.

PAAT, a novel ATPase and trans-regulator of mitochondrial ABC transporters, is critically involved in the maintenance of mitochondrial homeostasis.

ATP-binding cassette (ABC) transporters are implicated in a diverse range of physiological and pathophysiological processes, such as cholesterol and lipid transportation and multidrug resistance. Despite the considerable efforts made in understanding of the cellular function of ABC proteins, the regulation mechanism of this type of protein is still poorly defined. Here we report the identification and functional characterization of a novel ATPase protein, protein associated with ABC transporters (PAAT), in humans. PAAT contains a nucleotide-binding domain (NBD)-like domain and a signal for intramitochondrial sorting. We showed that PAAT is localized in both the cytoplasm and the mitochondria and has an intrinsic ATPase activity. PAAT physically interacts with the 3 known mitochondrial inner membrane ABC proteins, ABCB7, ABCB8, and ABCB10, but not ABCB1, ABCB6, or ABCG2, and functionally regulates the transport of ferric nutrients and heme biosynthesis. Significantly, PAAT deficiency promotes cell death, reduces mitochondrial potential, and sensitizes mitochondria to oxidative stress-induced DNA damages. Our experiments revealed that PAAT is a novel ATPase and a trans-regulator of mitochondrial ABC transporters that plays an important role in the maintenance of mitochondrial homeostasis and cell survival.

Efficient Purification and Reconstitution of ATP Binding Cassette Transporter B6 (ABCB6) for Functional and Structural Studies

The mitochondrial ATP binding cassette transporter ABCB6 has been associated with a broad range of physiological functions, including growth and development, therapy-related drug resistance, and the new blood group system Langereis. ABCB6 has been proposed to regulate heme synthesis by shuttling coproporphyrinogen III from the cytoplasm into the mitochondria. However, direct functional information of the transport complex is not known. To understand the role of ABCB6 in mitochondrial transport, we developed an in vitro system with pure and active protein. ABCB6 overexpressed in HEK293 cells was solubilized from mitochondrial membranes and purified to homogeneity. Purified ABCB6 showed a high binding affinity for MgATP (Kd = 0.18 μM) and an ATPase activity with a Km of 0.99 mM. Reconstitution of ABCB6 into liposomes allowed biochemical characterization of the ATPase including (i) substrate-stimulated ATPase activity, (ii) transport kinetics of its proposed endogenous substrate coproporphyrinogen III, and (iii) transport kinetics of substrates identified using a high throughput screening assay. Mutagenesis of the conserved lysine to alanine (K629A) in the Walker A motif abolished ATP hydrolysis and substrate transport. These results suggest a direct interaction between mitochondrial ABCB6 and its transport substrates that is critical for the activity of the transporter. Furthermore, the simple immunoaffinity purification of ABCB6 to near homogeneity and efficient reconstitution of ABCB6 into liposomes might provide the basis for future studies on the structure/function of ABCB6.

The DUF59 Family Gene AE7 Acts in the Cytosolic Iron-Sulfur Cluster Assembly Pathway to Maintain Nuclear Genome Integrity in Arabidopsis

Eukaryotic organisms have evolved a set of strategies to safeguard genome integrity, but the underlying mechanisms remain poorly understood. Here, we report that asymmetric leaves1/2 enhancer7 (AE7), an Arabidopsis thaliana gene encoding a protein in the evolutionarily conserved Domain of Unknown Function 59 family, participates in the cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) pathway to maintain genome integrity. The severe ae7-2 allele is embryo lethal, whereas plants with the weak ae7 (ae7-1) allele are viable but exhibit highly accumulated DNA damage that activates the DNA damage response to arrest the cell cycle. AE7 is part of a protein complex with CIA1, NAR1, and MET18, which are highly conserved in eukaryotes and are involved in the biogenesis of cytosolic and nuclear Fe-S proteins. ae7-1 plants have lower activities of the cytosolic [4Fe-4S] enzyme aconitase and the nuclear [4Fe-4S] enzyme DNA glycosylase ROS1. Additionally, mutations in the gene encoding the mitochondrial ATP binding cassette transporter ATM3/ABCB25, which is required for the activity of cytosolic Fe-S enzymes in Arabidopsis, also result in defective genome integrity similar to that of ae7-1. These results indicate that AE7 is a central member of the CIA pathway, linking plant mitochondria to nuclear genome integrity through assembly of Fe-S proteins.

Polycyclic Aromatic Hydrocarbons (PAHs) Mediate Transcriptional Activation of the ATP Binding Cassette Transporter ABCB6 Gene via the Aryl Hydrocarbon Receptor (AhR)

Liver is endowed with a mechanism to induce hepatic cytochromes P450 (CYP450s) in response to therapeutic drugs and environmental contaminants, leading to increased detoxification and elimination of the xenobiotics. Each CYP450 is composed of an apoprotein moiety and a heme prosthetic group, which is required for CYP450 activity. Thus, under conditions of CYP450 induction, there is a coordinate increase in heme biosynthesis to compensate for the increased expression of CYP450s. ABCB6, a mitochondrial ATP binding cassette transporter, which regulates coproporphyrinogen transport from the cytoplasm into the mitochondria to complete heme biosynthesis, represents a previously unrecognized rate-limiting step in heme biosynthesis. However, it is not known if exposure to drugs and environmental contaminants induces ABCB6 expression, to assure an adequate and apparently coordinated supply of heme for the generation of functional cytochrome holoprotein. In the present study, we demonstrate that polycyclic aromatic hydrocarbons (PAHs), the widely distributed environmental toxicants shown to induce porphyrin accumulation causing hepatic porphyria, up-regulate ABCB6 expression in both mice and humans. Using siRNA technology and Abcb6 knock-out mice, we demonstrate that PAH-mediated increase in hepatic porphyrins is compromised in the absence of ABCB6. Moreover, in vivo studies in aryl hydrocarbon receptor (AhR) knock-out mice demonstrate that PAH induction of ABCB6 is mediated by AhR. Promoter activation studies combined with electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrate direct interactions between the AhR binding sites in the ABCB6 promoter and the AhR receptor, implicating drug activation mechanisms for ABCB6 similar to those found in inducible cytochrome P450s. These studies are the first to describe direct transcriptional activation of both mouse and human ABCB6 by xenobiotics.

Systematic molecular genetic analysis of congenital sideroblastic anemia: Evidence for genetic heterogeneity and identification of novel mutations

Sideroblastic anemias are heterogeneous congenital and acquired bone marrow disorders characterized by pathologic iron deposits in mitochondria of erythroid precursors. Among the congenital sideroblastic anemias (CSAs), the most common form is X-linked sideroblastic anemia, due to mutations in 5-aminolevulinate synthase (ALAS2). A novel autosomal recessive CSA, caused by mutations in the erythroid specific mitochondrial transporter SLC25A38, was recently defined. Other known etiologies include mutations in genes encoding the thiamine transporter SLC19A2, the RNA-modifying enzyme pseudouridine synthase 1 (PUS1), a mitochondrial ATP-binding cassette transporter (ABCB7), glutaredoxin 5 (GLRX5), as well as mitochondrial DNA deletions. Despite these known diverse causes, in a substantial portion of CSA cases a presumed genetic defect remains unknown. In the context of the recent discovery of SLC25A38 as a major novel cause, we systematically analyzed a large cohort of previously unreported CSA patients. Sixty CSA probands (28 females, 32 males) were examined for ALAS2, SLC25A38, PUS1, GLRX5, and ABCB7 mutations. SLC19A2 and mitochondrial DNA were only analyzed if characteristic syndromic features were apparent. Twelve probands had biallelic mutations in SLC25A38. Seven ALAS2 mutations were detected in eight sporadic CSA cases, two being novel. We also identified a novel homozygous null PUS1 mutation and novel mitochondrial DNA deletions in two patients with Pearson syndrome. No mutations were encountered in GLRX5, ABCB7, or SLC19A2. The remaining undefined probands (43%) can be grouped according to gender, family, and clinical characteristics, suggesting novel X-linked and autosomal recessive forms of CSA.

Cell Survival under Stress Is Enhanced by a Mitochondrial ATP-Binding Cassette Transporter That Regulates Hemoproteins

The ATP-binding cassette (ABC) transporter ABCB6 localizes to the mitochondria, where it imports porphyrins and up-regulates de novo porphyrin synthesis. If ABCB6 also increases the intracellular heme concentration, it may broadly affect the regulation and physiology of cellular hemoproteins. We tested whether the ability of ABCB6 to accelerate de novo porphyrin biosynthesis alters mitochondrial and extramitochondrial heme levels. ABCB6 overexpression increased the quantity of cytosolic heme but did not affect mitochondrial heme levels. We then tested whether the increased extramitochondrial heme would increase the concentration and/or activity of cellular hemoproteins (hemoglobin, catalase, and cytochrome c oxidase). ABCB6 overexpression increased the activity and quantity of hemoproteins found in several subcellular compartments, and reduction of ABCB6 function (by small interfering RNA or knockout) reversed these findings. In complementary studies, suppression of ABCB6 expression sensitized cells to stress induced by peroxide and cyanide, whereas overexpression of ABCB6 protected against both stressors. Our findings show that the ability of ABCB6 to increase cytosolic heme levels produces phenotypic changes in hemoproteins that protect cells from certain stresses. Collectively, these findings have implications for the health and survival of both normal and abnormal cells, which rely on heme for multiple cellular processes.

Familial sideroblastic anemia associated with cardiac atrial septal defect

Sideroblastic anemia (SA) is defined by the presence of ringed sideroblasts in the bone marrow, and may be due to both hereditary and acquired causes. The most common hereditary form is X-linked SA (XLSA), which is due to mutations in the erythroid-specific 5-aminolevulinate synthase gene (ALAS2) [1,2] and occurs predominantly in men [3]. Another form of XLSA, X-linked SA and ataxia, is due to mutations in the mitochondrial ATP binding cassette transporter ABCB7 [4,5]. Other syndromic forms are inherited in an autosomal recessive manner (thiamine-responsive megaloblastic anemia with diabetes and deafness [6]; mitochondrial myopathy, lactic acidosis and SA [7,8]) or result from sporadic congenital defects in mitochondrial DNA (Pearson marrow pancreas syndrome) [9].

ABC-Me (ABCB10) Is Required for Erythroid Development in the Mouse Embryo and Is Protective against Mitochondrial Oxidative Stress

We previously described that mitochondrial ATP-Binding Cassette transporter, ABCme (ABCB10) is induced by GATA-1 in erythroid cells and that its over-expression leads to increased heme production. However, the role of ABCme in vivo and the hematologic phenotype of its deficiency have not been described thus far. Here we report for the first time that ABCme is essential for embryonic blood development in vivo. ABCme deficient mouse was generated on 129SvEvBrd/C57BL6J background and backcrossed to n = 4 on the C57BL6J. The homozygous KO mouse is embryonic lethal. ABCme KO dies at 12.5 days of gestation and displayed aplastic embryonic anemia by 10.5 days of gestation, manifested as improper blood development as well as erythroid progenitor apoptosis (Figure 1). Ex vivo analysis of maturation of isolated yolk sac blood determined that ABCme KO erythroid cells fail to differentiate beyond the stage of basophilic erythroblast and exhibit 55% of apoptosis in CD71 positive cells in comparison with 29.25% in ABCme HET, and 17.5% in WT. Erythroid colony assay indicated 50% reduction in the formation of CFU-E and an 80% reduction in BFU-E in comparison with WT. Disruption of heme synthesis may result in accumulation of heme intermediates that are pro-oxidants. To test the hypothesis that apoptosis is induced by oxidative damage we tested both mitochondrial oxidation and the rescue effects of mitochondrial antioxidants in the ABCme KO. Isolated mitochondria of ABCme KO erythroid cells demonstrated significantly elevated levels (5 fold) of oxidized protein in comparison to both WT and HET cells. This elevated level of superoxide were confirmed and identified to be mitochondrial in nature by FACS analysis of superoxide sensitive dye MitoSox. Using antioxidants we tested the patho-physiological role of the elevated ROS in the hematologic phenotype. Treatment of ABCme KO cells with the SOD2 mimetic TBAP resulted in partial and complete rescue of the apoptotic phenotypes of the KO and the HET respectively. Our findings suggest that ABCme is required for erythroid development in vivo and that its deficiency results in impaired erythroid maturation, hemoglobinzation, and increased mitochondrial oxidative damage. [Display omitted] Light microscopy of Day 10.5 p.c. Embryo and Yolk Sac (Top). At this stage KO embryos are still alive as denoted by heartbeat as well as normal development of other organ systems. However, lack of red cell development is apparent. H/E staining of day 10.5 p.c. yolk sac blood islands (Bottom). KO blood islands demonstrate reduced number, increased nuclear fragmentation associated with apoptosis, and a lack of developed erythroid progenitors. (WT= Wildtype mice, KO= ABCme −/− mice.)

Systematic Analysis of Known Candidate Genes in 58 Probands with Previously Uncharacterized Congenital Sideroblastic Anemia: Evidence for Genetic Heterogeneity and Identification of Novel Mutations in ALAS2 and PUS1

Sideroblastic anemias are a heterogeneous group of congenital and acquired bone marrow disorders characterized by pathologic iron accumulation in the mitochondria of erythroid precursors. Whereas the molecular ontology of acquired sideroblastic anemia is largely obscure, the genetic etiology is known for many patients with congenital sideroblastic anemia (CSA). Mutations in the heme biosynthetic enzyme 5-aminolevulinate synthase 2 gene (ALAS2)—associated with the classic, X-linked form of the disease—or mitochondrial DNA mutations/deletions (as in Pearson syndrome) have been reported in many cases. Rare cases are associated with mutations in the genes encoding a thiamine transporter (SLC11A2), pseudouridine synthase 1 (PUS1), a mitochondrial ATP-binding cassette transporter (ABCB7), and glutaredoxin 5 (GLRX5). Nonetheless, the molecular defect in a majority of cases of CSA remains unknown and there appears to be a significant bias in underreporting those cases that go genetically uncharacterized. Given this, we undertook a systematic genetic investigation of a large series of previously unreported CSA patients.METHODS AND RESULTS: 58 probands (26 female, 32 male) with CSA were studied. This cohort was comprised of 49 singleton patients and 10 families, including 8 families with at least 2 affected full siblings (1 set of monozygotic male twins, 1 each male or female sibling pairs, 2 male/female sibling pairs, 1 set each of 3 female or 3 male siblings, and one set of 3 siblings of unknown sex), and 2 families in which there was a history of CSA in a prior generation. In the great majority, CSA was the only clinical phenotype. We employed a sequential sequencing strategy to identify variants in ALAS2, PUS1, and GLRX5; ABCB7, SLC19A2, and mitochondrial DNA variants were not included in our analysis due to the absence of associated syndromic phenotypes in the cohort. To begin, ALAS2 was characterized by sequencing the coding region, intron-exon boundaries, proximal promoter, and intron 8, which contains an erythroid-specific enhancer, in all subjects. We detected 7 different missense mutations in 8 of the 58 probands, each of which was a singleton patient. No ALAS2 mutations were found in the promoter or intron 8. Two of the ALAS2 mutations were novel: V301A in a male patient and R517G in a female patient with a skewed X chromosome inactivation pattern in peripheral blood leukocytes as assessed by the human androgen receptor assay (HUMARA). The coding regions and intron-exon boundaries of PUS1 and GLRX5 were sequenced in all probands lacking ALAS2 mutations. Among these, a novel homozygous null (Q154X) PUS1 mutation was found in one familial case; only known polymorphisms in GLRX5 were detected. In 48 probands, including the 9 remaining familial cases, we did not identify a disease-causing mutation. However linkage studies in three families with affected males only were consistent with linkage to the ALAS2 locus in each case. CONCLUSIONS: These results demonstrate that:ALAS2 mutations are commonly associated with CSA,ALAS2 mutations and non-random X inactivation in affected females underscore the importance of ALAS2 analysis in women as well as in men with CSA,many potentially X-linked cases of CSA may have either ALAS2 mutations not detected by conventional sequencing approaches or mutations in other X-linked genes,PUS1 and GLRX5 variants are unusual causes of CSA, andthere is strong genetic evidence that other, autosomal recessive forms of CSA exist.

Switching of the homooligomeric ATP‐binding cassette transport complex MDL1 from post‐translational mitochondrial import to endoplasmic reticulum insertion

The ATP-binding cassette transporter MDL1 of Saccharomyces cerevisiae has been implicated in mitochondrial quality control, exporting degradation products of misassembled respiratory chain complexes. In the present study, we identified an unusually long leader sequence of 59 amino acids, which targets MDL1 to the inner mitochondrial membrane with its nucleotide-binding domain oriented to the matrix. By contrast, MDL1 lacking this leader sequence is directed into the endoplasmic reticulum membrane with the nucleotide-binding domain facing the cytosol. Remarkably, in both targeting routes, the ATP-binding cassette transporter maintains its intrinsic properties of membrane insertion and assembly, leading to homooligomeric complexes with similar activities in ATP hydrolysis. The physiological consequences of both targeting routes were elucidated in cells lacking the mitochondrial ATP-binding cassette transporter ATM1, which is essential for biogenesis of cytosolic iron-sulfur proteins. The mitochondrial MDL1 complex can complement ATM1 function, whereas the endoplasmic reticulum-targeted version, as well as MDL1 mutants deficient in ATP binding and hydrolysis, cannot overcome the Deltaatm1 growth phenotype.