RESUMO
ABCA4 is an ATP-binding cassette (ABC) transporter that prevents the buildup of toxic retinoid compounds by facilitating the transport of N-retinylidene-phosphatidylethanolamine across membranes of rod and cone photoreceptor cells. Over 1500 missense mutations in ABCA4, many in the nucleotide binding domains (NBDs), have been genetically linked to Stargardt disease (STGD1). Here, we show by Cryo-electron microscopy that ABCA4 is converted from an open outward conformation to a closed conformation upon the binding of AMP-PNP. Structural information and biochemical studies were used to further define the role of the NBDs in the functional properties of ABCA4 and the mechanisms by which mutations lead to the loss in activity. We show that ATPase activity in both NBDs is required for the functional activity of ABCA4. Mutations in Walker A asparagine residues cause a severe reduction in substrate-activated ATPase activity due to the loss in polar interactions with residues within the D-loops of the opposing NBD. The structural basis for how disease mutations in other NBD residues including the R1108C, R2077W, R2107H and L2027F affect the structure and function of ABCA4 is described. Collectively, our studies provide insight into the structure and function of ABCA4 and mechanisms underlying STGD1.
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Synaptic refinement is a critical physiological process that removes excess synapses to establish and maintain functional neuronal circuits. Recent studies have shown that focal exposure of phosphatidylserine (PS) on synapses acts as an "eat me" signal to mediate synaptic pruning. However, the molecular mechanism underlying PS externalization at synapses remains elusive. Here, we find that murine CDC50A, a chaperone of phospholipid flippases, localizes to synapses, and that its expression depends on neuronal activity. Cdc50a knockdown leads to phosphatidylserine exposure at synapses and subsequent erroneous synapse removal by microglia partly via the GPR56 pathway. Taken together, our data support that CDC50A safeguards synapse maintenance by regulating focal phosphatidylserine exposure at synapses.
Assuntos
Proteínas de Membrana/genética , Microglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fosfatidilserinas/farmacologia , Receptores Acoplados a Proteínas G/genética , Sinapses/efeitos dos fármacos , Animais , Regulação da Expressão Gênica , Genes Reporter , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/citologia , Microglia/metabolismo , Plasticidade Neuronal , Neurônios/citologia , Neurônios/metabolismo , Fosfatidilserinas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sinapses/genética , Sinapses/metabolismo , Transmissão Sináptica , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato , Proteína Vermelha FluorescenteRESUMO
G protein-coupled receptor (GPCR) signaling is ubiquitous. As an archetype of this signaling motif, rod phototransduction has provided many fundamental, quantitative details, including a dogma that one active GPCR molecule activates a substantial number of downstream G protein/enzyme effector complexes. However, rod phototransduction is light-activated, whereas GPCR pathways are predominantly ligand-activated. Here, we report a detailed study of the ligand-triggered GPCR pathway in mammalian olfactory transduction, finding that an odorant-receptor molecule when (one-time) complexed with its most effective odorants produces on average much less than one downstream effector. Further experiments gave a nominal success probability of tentatively â¼10-4 (more conservatively, â¼10-2 to â¼10-5). This picture is potentially more generally representative of GPCR signaling than is rod phototransduction, constituting a paradigm shift.
Assuntos
Ligantes , Odorantes , Receptores Acoplados a Proteínas G , Receptores Odorantes , Transdução de Sinais , Olfato , Animais , Transdução de Sinal Luminoso , Mamíferos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Odorantes/metabolismo , Células Fotorreceptoras Retinianas BastonetesRESUMO
N-retinylidene-phosphatidylethanolamine (N-Ret-PE), the Schiff-base conjugate formed through the reversible reaction of retinal (Vitamin A-aldehyde) and phosphatidylethanolamine, plays a crucial role in the visual cycle and visual pigment photoregeneration. However, N-Ret-PE can react with another molecule of retinal to form toxic di-retinoids if not removed from photoreceptors through its transport across photoreceptor membranes by the ATP-binding-cassette transporter ABCA4. Loss-of-function mutations in ABCA4 are known to cause Stargardt disease (STGD1), an inherited retinal degenerative disease associated with the accumulation of fluorescent di-retinoids and severe loss in vision. A larger assessment of retinal-phospholipid Schiff-base conjugates in photoreceptors is needed, along with further investigation of ABCA4 residues important for N-Ret-PE binding. In this study we show that N-Ret-PE formation is dependent on pH and phospholipid content. When retinal is added to liposomes or photoreceptor membranes, 40 to 60% is converted to N-Ret-PE at physiological pH. Phosphatidylserine and taurine also react with retinal to form N-retinylidene-phosphatidylserine and N-retinylidene-taurine, respectively, but at significantly lower levels. N-retinylidene-phosphatidylserine is not a substrate for ABCA4 and reacts poorly with retinal to form di-retinoids. Additionally, amino acid residues within the binding pocket of ABCA4 that contribute to its interaction with N-Ret-PE were identified and characterized using site-directed mutagenesis together with functional and binding assays. Substitution of arginine residues and hydrophobic residues with alanine or residues implicated in STGD1 significantly reduced or eliminated substrate-activated ATPase activity and substrate binding. Collectively, this study provides important insight into conditions which affect retinal-phospholipid Schiff-base formation and mechanisms underlying the pathogenesis of STGD1.
Assuntos
Fosfolipídeos , Doença de Stargardt , Humanos , Transportadores de Cassetes de Ligação de ATP/metabolismo , Fosfatidilserinas , Retinoides/metabolismo , Doença de Stargardt/metabolismoRESUMO
ATPase, class 1, type 8 A, member 2 (ATP8A2) is a P4-ATPase with a critical role in phospholipid translocation across the plasma membrane. Pathogenic variants in ATP8A2 are known to cause cerebellar ataxia, impaired intellectual development, and disequilibrium syndrome 4 (CAMRQ4) which is often associated with encephalopathy, global developmental delay, and severe motor deficits. Here, we present a family with two siblings born from a consanguineous, first-cousin union from Sudan presenting with global developmental delay, intellectual disability, spasticity, ataxia, nystagmus, and thin corpus callosum. Whole exome sequencing revealed a homozygous missense variant in the nucleotide binding domain of ATP8A2 (p.Leu538Pro) that results in near complete loss of protein expression. This is in line with other missense variants in the same domain leading to protein misfolding and loss of ATPase function. In addition, by performing diffusion-weighted imaging, we identified bilateral hyperintensities in the posterior limbs of the internal capsule suggesting possible microstructural changes in axon tracts that had not been appreciated before and could contribute to the sensorimotor deficits in these individuals.
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INTRODUCTION: Conclusive molecular genetic diagnoses in inherited retinal diseases remains a major challenge due to the large number of variants of uncertain significance (VUS) identified in genetic testing. Here, we determined the genotypic and phenotypic spectrum of ABCA4 gene variants in a cohort of Canadian inherited retinal dystrophy subjects. METHODS: This retrospective study evaluated 64 subjects with an inherited retinal dystrophy diagnosis with variants in the ABCA4 gene. Pathogenicity of variants was assessed by comparison to genetic databases and in silico modelling. ABCA4 variants classified as VUS were further evaluated using a cryo-electron structural model of the ABCA4 protein to predict impact on protein function and were also assessed for evolutionary conservation. RESULTS: Conclusive disease-causing biallelic ABCA4 variants were detected in 52 subjects with either Stargardt's disease, cone-rod dystrophy, macular dystrophy, or pattern dystrophy. A further 14 variants were novel comprising 1 nonsense, 1 frameshift, 3 splicing, and 9 missense variants. Based on in silico modelling, protein modelling and evolutionary conservation from human to zebrafish, we re-classified 5 of these as pathogenic and a further 3 as likely pathogenic. We also added to the ABCA4 phenotypic spectrum seen with four known pathogenic variants (c.2161-2A>G; Leu296Cysfs*4; Arg1640Gln; and Pro1380Leu). CONCLUSIONS: This study expands the genotypic and phenotypic spectrum of ABCA4 disease-associated variants. By panel-based genetic testing, we identified 14 novel ABCA4 variants of which 8 were determined to be disease-causing or likely disease-causing. These methodologies could circumvent somewhat the need for labour intensive in vitro and in vivo assessments of novel ABCA4 variants.
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ABCA4-related retinopathy is the most common inherited Mendelian eye disorder worldwide, caused by biallelic variants in the ATP-binding cassette transporter ABCA4. To date, over 2200 ABCA4 variants have been identified, including missense, nonsense, indels, splice site and deep intronic defects. Notably, more than 60% are missense variants that can lead to protein misfolding, mistrafficking and degradation. Currently no approved therapies target ABCA4. In this study, we demonstrate that ABCA4 misfolding variants are temperature-sensitive and reduced temperature growth (30 °C) improves their traffic to the plasma membrane, suggesting the folding of these variants could be rescuable. Consequently, an in vitro platform was developed for the rapid and robust detection of ABCA4 traffic to the plasma membrane in transiently transfected cells. The system was used to assess selected candidate small molecules that were reported to improve the folding or traffic of other ABC transporters. Two candidates, 4-PBA and AICAR, were identified and validated for their ability to enhance both wild-type ABCA4 and variant trafficking to the cell surface in cell culture. We envision that this platform could serve as a primary screen for more sophisticated in vitro testing, enabling the discovery of breakthrough agents to rescue ABCA4 protein defects and mitigate ABCA4-related retinopathy.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Dobramento de Proteína , Transporte Proteico , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Humanos , Dobramento de Proteína/efeitos dos fármacos , Células HEK293 , Membrana Celular/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Over 1200 variants in the ABCA4 gene cause a wide variety of retinal disease phenotypes, the best known of which is autosomal recessive Stargardt disease (STGD1). Disease-causing variation encompasses all mutation categories, from large copy number variants to very mild, hypomorphic missense variants. The most prevalent disease-causing ABCA4 variant, present in ~ 20% of cases of European descent, c.5882G > A p.(Gly1961Glu), has been a subject of controversy since its minor allele frequency (MAF) is as high as ~ 0.1 in certain populations, questioning its pathogenicity, especially in homozygous individuals. We sequenced the entire ~140Kb ABCA4 genomic locus in an extensive cohort of 644 bi-allelic, i.e. genetically confirmed, patients with ABCA4 disease and analyzed all variants in 140 compound heterozygous and 10 homozygous cases for the p.(Gly1961Glu) variant. A total of 23 patients in this cohort additionally harbored the deep intronic c.769-784C > T variant on the p.(Gly1961Glu) allele, which appears on a specific haplotype in ~ 15% of p.(Gly1961Glu) alleles. This haplotype was present in 5/7 of homozygous cases, where the p.(Gly1961Glu) was the only known pathogenic variant. Three cases had an exonic variant on the same allele with the p.(Gly1961Glu). Patients with the c.[769-784C > T;5882G > A] complex allele exhibit a more severe clinical phenotype, as seen in compound heterozygotes with some more frequent ABCA4 mutations, e.g. p.(Pro1380Leu). Our findings indicate that the c.769-784C > T variant is major cis-acting modifier of the p.(Gly1961Glu) allele. The absence of such additional allelic variation on most p.(Gly1961Glu) alleles largely explains the observed paucity of affected homozygotes in the population.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Alelos , Frequência do Gene , Humanos , Mutação , Penetrância , Fenótipo , Doença de Stargardt/genéticaRESUMO
BACKGROUND: ABCA4, the gene implicated in Stargardt disease (STGD1), contains 50 exons, of which 17 contain multiples of three nucleotides. The impact of in-frame exon skipping is yet to be determined. Antisense oligonucleotides (AONs) have been investigated in Usher syndrome-associated genes to induce skipping of in-frame exons carrying severe variants and mitigate their disease-linked effect. Upon the identification of a STGD1 proband carrying a novel exon 17 canonical splice site variant, the activity of ABCA4 lacking 22 amino acids encoded by exon 17 was examined, followed by design of AONs able to induce exon 17 skipping. METHODS: A STGD1 proband was compound heterozygous for the splice variant c.2653+1G>A, that was predicted to result in in-frame skipping of exon 17, and a null variant [c.735T>G, p.(Tyr245*)]. Clinical characteristics of this proband were studied using multi-modal imaging and complete ophthalmological examination. The aberrant splicing of c.2653+1G>A was investigated in vitro in HEK293T cells with wild-type and mutant midigenes. The residual activity of the mutant ABCA4 protein lacking Asp864-Gly885 encoded by exon 17 was analyzed with all-trans-retinal-activated ATPase activity assay, along with its subcellular localization. To induce exon 17 skipping, the effect of 40 AONs was examined in vitro in WT WERI-Rb-1 cells and 3D human retinal organoids. RESULTS: Late onset STGD1 in the proband suggests that c.2653+1G>A does not have a fully deleterious effect. The in vitro splice assay confirmed that this variant leads to ABCA4 transcripts without exon 17. ABCA4 Asp864_Gly863del was stable and retained 58% all-trans-retinal-activated ATPase activity compared to WT ABCA4. This sequence is located in an unstructured linker region between transmembrane domain 6 and nucleotide-binding domain-1 of ABCA4. AONs were designed to possibly reduce pathogenicity of severe variants harbored in exon 17. The best AON achieved 59% of exon 17 skipping in retinal organoids. CONCLUSIONS: Exon 17 deletion in ABCA4 does not result in the absence of protein activity and does not cause a severe STGD1 phenotype when in trans with a null allele. By applying AONs, the effect of severe variants in exon 17 can potentially be ameliorated by exon skipping, thus generating partial ABCA4 activity in STGD1 patients.
Assuntos
Adenosina Trifosfatases , Retinaldeído , Humanos , Doença de Stargardt/genética , Células HEK293 , Éxons/genética , Proteínas Mutantes , Transportadores de Cassetes de Ligação de ATP/genéticaRESUMO
Adenosine Triphosphatase (ATPase) Phospholipid Transporting 11C gene (ATP11C) encodes the major phosphatidylserine (PS) flippase in human red blood cells (RBCs). Flippases actively transport phospholipids (e.g., PS) from the outer to the inner leaflet to establish and maintain phospholipid asymmetry of the lipid bilayer of cell membranes. This asymmetry is crucial for survival since externalized PS triggers phagocytosis by splenic macrophages. Here we report on pathophysiological consequences of decreased flippase activity, prompted by a patient with hemolytic anemia and hemizygosity for a novel c.2365C > T p.(Leu789Phe) missense variant in ATP11C. ATP11C protein expression was strongly reduced by 58% in patient-derived RBC ghosts. Furthermore, functional characterization showed only 26% PS flippase activity. These results were confirmed by recombinant mutant ATP11C protein expression in HEK293T cells, which was decreased to 27% compared to wild type, whereas PS-stimulated ATPase activity was decreased by 57%. Patient RBCs showed a mild increase in PS surface exposure when compared to control RBCs, which further increased in the most dense RBCs after RBC storage stress. The increase in PS was not due to higher global membrane content of PS or other phospholipids. In contrast, membrane lipid lateral distribution showed increased abundance of cholesterol-enriched domains in RBC low curvature areas. Finally, more dense RBCs and subtle changes in RBC morphology under flow hint toward alterations in flow behavior of ATP11C-deficient RBCs. Altogether, ATP11C deficiency is the likely cause of hemolytic anemia in our patient, thereby underlining the physiological role and relevance of this flippase in human RBCs.
Assuntos
Anemia Hemolítica Congênita , Fosfatidilserinas , Humanos , Fosfatidilserinas/metabolismo , Células HEK293 , Eritrócitos/metabolismo , Anemia Hemolítica Congênita/genética , Anemia Hemolítica Congênita/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Fosfolipídeos/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismoRESUMO
The outer segment (OS) organelle of vertebrate photoreceptors is a highly specialized cilium evolved to capture light and initiate light response. The plasma membrane which envelopes the OS plays vital and diverse roles in supporting photoreceptor function and health. However, little is known about the identity of its protein constituents, as this membrane cannot be purified to homogeneity. In this study, we used the technique of protein correlation profiling to identify unique OS plasma membrane proteins. To achieve this, we used label-free quantitative MS to compare relative protein abundances in an enriched preparation of the OS plasma membrane with a preparation of total OS membranes. We have found that only five proteins were enriched at the same level as previously validated OS plasma membrane markers. Two of these proteins, TMEM67 and TMEM237, had not been previously assigned to this membrane, and one, embigin, had not been identified in photoreceptors. We further showed that embigin associates with monocarboxylate transporter MCT1 in the OS plasma membrane, facilitating lactate transport through this cellular compartment.
Assuntos
Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Simportadores/metabolismo , Animais , Bovinos , Camundongos Endogâmicos C57BLRESUMO
Phospholipid flippases (P4-ATPases) utilize ATP to translocate specific phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of biological membranes, thus generating and maintaining transmembrane lipid asymmetry essential for a variety of cellular processes. P4-ATPases belong to the P-type ATPase protein family, which also encompasses the ion transporting P2-ATPases: Ca2+-ATPase, Na+,K+-ATPase, and H+,K+-ATPase. In comparison with the P2-ATPases, understanding of P4-ATPases is still very limited. The electrogenicity of P4-ATPases has not been explored, and it is not known whether lipid transfer between membrane bilayer leaflets can lead to displacement of charge across the membrane. A related question is whether P4-ATPases countertransport ions or other substrates in the opposite direction, similar to the P2-ATPases. Using an electrophysiological method based on solid supported membranes, we observed the generation of a transient electrical current by the mammalian P4-ATPase ATP8A2 in the presence of ATP and the negatively charged lipid substrate phosphatidylserine, whereas only a diminutive current was generated with the lipid substrate phosphatidylethanolamine, which carries no or little charge under the conditions of the measurement. The current transient seen with phosphatidylserine was abolished by the mutation E198Q, which blocks dephosphorylation. Likewise, mutation I364M, which causes the neurological disorder cerebellar ataxia, mental retardation, and disequilibrium (CAMRQ) syndrome, strongly interfered with the electrogenic lipid translocation. It is concluded that the electrogenicity is associated with a step in the ATPase reaction cycle directly involved in translocation of the lipid. These measurements also showed that no charged substrate is being countertransported, thereby distinguishing the P4-ATPase from P2-ATPases.
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Adenosina Trifosfatases/genética , Transporte Biológico/genética , Lipídeos de Membrana/genética , Proteínas de Transferência de Fosfolipídeos/genética , Fosfolipídeos/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Animais , ATPases Transportadoras de Cálcio/química , ATPases Transportadoras de Cálcio/genética , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Ataxia Cerebelar/genética , Citoplasma/genética , Citoplasma/metabolismo , Fenômenos Eletrofisiológicos/genética , ATPase Trocadora de Hidrogênio-Potássio/química , ATPase Trocadora de Hidrogênio-Potássio/genética , Humanos , Deficiência Intelectual/genética , Lipídeos de Membrana/metabolismo , Mutação/genética , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fosfolipídeos/genética , Especificidade por Substrato/genéticaRESUMO
ATP8A2 is a P4-ATPase that flips phosphatidylserine across membranes to generate and maintain transmembrane phospholipid asymmetry. Loss-of-function variants cause severe neurodegenerative and developmental disorders. We have identified three ATP8A2 variants in unrelated Iranian families that cause intellectual disability, dystonia, below-average head circumference, mild optic atrophy, and developmental delay. Additionally, all the affected individuals displayed tooth abnormalities associated with defects in teeth development. Two variants (p.Asp825His and p.Met438Val) reside in critical functional domains of ATP8A2. These variants express at very low levels and lack ATPase activity. Inhibitor studies indicate that these variants are misfolded and degraded by the cellular proteasome. We conclude that Asp825, which coordinates with the Mg2+ ion within the ATP binding site, and Met438 are essential for the proper folding of ATP8A2 into a functional flippase. We also provide evidence on the association of tooth abnormalities with defects in ATP8A2, thereby expanding the clinical spectrum of the associated disease.
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Adenosina Trifosfatases , Fosfolipídeos , Adenosina Trifosfatases/química , Citoplasma/metabolismo , Humanos , Irã (Geográfico) , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fosfolipídeos/metabolismo , Domínios ProteicosRESUMO
In mammalian olfactory transduction, odorants activate a cAMP-mediated signaling pathway that leads to the opening of cyclic nucleotide-gated (CNG), nonselective cation channels and depolarization. The Ca2+ influx through open CNG channels triggers an inward current through Ca2+-activated Cl channels (ANO2), which is expected to produce signal amplification. However, a study on an Ano2-/- mouse line reported no elevation in the behavioral threshold of odorant detection compared with wild type (WT). Subsequent studies by others on the same Ano2-/- line, nonetheless, found subtle defects in olfactory behavior and some abnormal axonal projections from the olfactory receptor neurons (ORNs) to the olfactory bulb. As such, the question regarding signal amplification by the Cl current in WT mouse remains unsettled. Recently, with suction-pipette recording, we have successfully separated in frog ORNs the CNG and Cl currents during olfactory transduction and found the Cl current to predominate in the response down to the threshold of action-potential signaling to the brain. For better comparison with the mouse data by others, we have now carried out similar current-separation experiments on mouse ORNs. We found that the Cl current clearly also predominated in the mouse olfactory response at signaling threshold, accounting for â¼80% of the response. In the absence of the Cl current, we expect the threshold stimulus to increase by approximately sevenfold.
Assuntos
Anoctaminas/fisiologia , Encéfalo/fisiologia , Cálcio/farmacologia , Cloretos/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/fisiologia , Neurônios Receptores Olfatórios/fisiologia , Animais , Encéfalo/citologia , AMP Cíclico/farmacologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Knockout , Neurônios Receptores Olfatórios/citologia , Neurônios Receptores Olfatórios/efeitos dos fármacos , Técnicas de Patch-Clamp , Transdução de Sinais/efeitos dos fármacos , Olfato/efeitos dos fármacosRESUMO
Stargardt macular degeneration (Stargardt disease 1 [STGD1]) is caused by mutations in the gene encoding ABCA4, an ATP-binding cassette protein that transports N-retinylidene-phosphatidylethanolamine (N-Ret-PE) across photoreceptor membranes. Reduced ABCA4 activity results in retinoid accumulation leading to photoreceptor degeneration. The disease onset and severity vary from severe loss in visual acuity in the first decade to mild visual impairment late in life. We determined the effect of 22 disease-causing missense mutations on the expression and ATPase activity of ABCA4 in the absence and presence of N-Ret-PE. Three classes were identified that correlated with the disease onset in homozygous STGD1 individuals: Class 1 exhibited reduced ABCA4 expression and ATPase activity that was not stimulated by N-Ret-PE; individuals homozygous for these variants had an early disease onset (≤13 years); Class 2 showed reduced ATPase activity with limited stimulation by N-Ret-PE; these correlated with moderate disease onset (14-40 years); and Class 3 displayed high expression and ATPase activity that was strongly activated by N-Ret-PE; these were associated with late disease onset (>40 years). On the basis of our results, we introduce a functionality index for gauging the effect of missense mutations on STGD1 severity. Our studies support the mild phenotype exhibited by the p.Gly863Ala, p.Asn1868Ile, and p.Gly863Ala/p.Asn1868Ile variants.
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Transportadores de Cassetes de Ligação de ATP/genética , Doença de Stargardt/genética , Adolescente , Adulto , Criança , Células HEK293 , Homozigoto , Humanos , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Fenótipo , Fosfatidiletanolaminas , Retinoides , Adulto JovemRESUMO
ATP-dependent phospholipid flippase activity crucial for generating lipid asymmetry was first detected in red blood cell (RBC) membranes, but the P4-ATPases responsible have not been directly determined. Using affinity-based MS, we show that ATP11C is the only abundant P4-ATPase phospholipid flippase in human RBCs, whereas ATP11C and ATP8A1 are the major P4-ATPases in mouse RBCs. We also found that ATP11A and ATP11B are present at low levels. Mutations in the gene encoding ATP11C are responsible for blood and liver disorders, but the disease mechanisms are not known. Using heterologous expression, we show that the T415N substitution in the phosphorylation motif of ATP11C, responsible for congenital hemolytic anemia, reduces ATP11C expression, increases retention in the endoplasmic reticulum, and decreases ATPase activity by 61% relative to WT ATP11C. The I355K substitution in the transmembrane domain associated with cholestasis and anemia in mice was expressed at WT levels and trafficked to the plasma membrane but was devoid of activity. We conclude that the T415N variant causes significant protein misfolding, resulting in low protein expression, cellular mislocalization, and reduced functional activity. In contrast, the I355K variant folds and traffics normally but lacks key contacts required for activity. We propose that the loss in ATP11C phospholipid flippase activity coupled with phospholipid scramblase activity results in the exposure of phosphatidylserine on the surface of RBCs, decreasing RBC survival and resulting in anemia.
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Adenosina Trifosfatases/metabolismo , Eritrócitos/enzimologia , Fosfolipídeos/metabolismo , Adenosina Trifosfatases/genética , Animais , Membrana Eritrocítica/enzimologia , Membrana Eritrocítica/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fosforilação , Dobramento de ProteínaRESUMO
The P-type ATPase protein family includes, in addition to ion pumps such as Ca2+-ATPase and Na+,K+-ATPase, also phospholipid flippases that transfer phospholipids between membrane leaflets. P-type ATPase ion pumps translocate their substrates occluded between helices in the center of the transmembrane part of the protein. The large size of the lipid substrate has stimulated speculation that flippases use a different transport mechanism. Information on the functional importance of the most centrally located helices M5 and M6 in the transmembrane domain of flippases has, however, been sparse. Using mutagenesis, we examined the entire M5-M6 region of the mammalian flippase ATP8A2 to elucidate its possible function in the lipid transport mechanism. This mutational screen yielded an informative map assigning important roles in the interaction with the lipid substrate to only a few M5-M6 residues. The M6 asparagine Asn-905 stood out as being essential for the lipid substrate-induced dephosphorylation. The mutants N905A/D/E/H/L/Q/R all displayed very low activities and a dramatic insensitivity to the lipid substrate. Strikingly, Asn-905 aligns with key ion-binding residues of P-type ATPase ion pumps, and N905D was recently identified as one of the mutations causing the neurological disorder cerebellar ataxia, mental retardation, and disequilibrium (CAMRQ) syndrome. Moreover, the effects of substitutions to the adjacent residue Val-906 (i.e. V906A/E/F/L/Q/S) suggest that the lipid substrate approaches Val-906 during the translocation. These results favor a flippase mechanism with strong resemblance to the ion pumps, despite a location of the translocation pathway in the periphery of the transmembrane part of the flippase protein.
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Adenosina Trifosfatases/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Substituição de Aminoácidos , Animais , Asparagina/química , Asparagina/genética , Asparagina/metabolismo , Bovinos , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/genética , FosforilaçãoRESUMO
ABCA4 is a member of the superfamily of ATP-binding cassette (ABC) proteins that transports N-retinylidene-phosphatidylethanolamine (N-Ret-PE) across outer segment disc membranes thereby facilitating the removal of potentially toxic retinoid compounds from photoreceptor cells. Mutations in the gene encoding ABCA4 are responsible for Stargardt disease (STGD1), an autosomal recessive retinal degenerative disease that causes severe vision loss. To define the molecular basis for STGD1 associated with the p.Asn965Ser (N965S) mutation in the Walker A motif of nucleotide binding domain 1 (NBD1), we generated a p.Asn965Ser knockin mouse and compared the subcellular localization and molecular properties of the disease variant with wild-type (WT) ABCA4. Here, we show that the p.Asn965Ser ABCA4 variant expresses at half the level of WT ABCA4, partially mislocalizes to the endoplasmic reticulum (ER) of photoreceptors, is devoid of N-Ret-PE activated ATPase activity, and causes an increase in autofluorescence and the bisretinoid A2E associated with lipofuscin deposits in retinal pigment epithelial cells as found in Stargardt patients and Abca4 knockout mice. We also show for the first time that a significant fraction of WT ABCA4 is retained in the inner segment of photoreceptors. On the basis of these studies we conclude that loss in substrate-dependent ATPase activity and protein misfolding are mechanisms underlying STGD1 associated with the p.Asn965Ser mutation in ABCA4. Functional and molecular modeling studies further suggest that similar pathogenic mechanisms are responsible for Tangiers disease associated with the p.Asn935Ser (N935S) mutation in the NBD1 Walker A motif of ABCA1.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Degeneração Macular/congênito , Animais , Transporte Biológico , Técnicas de Introdução de Genes , Variação Genética , Degeneração Macular/genética , Degeneração Macular/metabolismo , Camundongos , Mutação , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Doença de StargardtRESUMO
ABCA4 is an ATP-binding cassette (ABC) transporter expressed in photoreceptors, where it transports its substrate, N-retinylidene-phosphatidylethanolamine (N-Ret-PE), across outer segment membranes to facilitate the clearance of retinal from photoreceptors. Mutations in ABCA4 cause Stargardt macular degeneration (STGD1), an autosomal recessive disorder characterized by a loss of central vision and the accumulation of bisretinoid compounds. The purpose of this study was to determine the molecular properties of ABCA4 variants harboring disease-causing missense mutations in the transmembrane domains. Thirty-eight variants expressed in culture cells were analyzed for expression, ATPase activities, and substrate binding. On the basis of these properties, the variants were divided into three classes: Class 1 (severe variants) exhibited significantly reduced ABCA4 expression and basal ATPase activity that was not stimulated by its substrate N-Ret-PE; Class 2 (moderate variants) showed a partial reduction in expression and basal ATPase activity that was modestly stimulated by N-Ret-PE; and Class 3 (mild variants) displayed expression and functional properties comparable to normal ABCA4. The p.R653C variant displayed normal expression and basal ATPase activity, but lacked substrate binding and ATPase activation, suggesting that arginine 653 contributes to N-Ret-PE binding. Our classification provides a basis for better understanding genotype-phenotype correlations and evaluating therapeutic treatments for STGD1.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transporte Biológico Ativo/genética , Doença de Stargardt/genética , Doença de Stargardt/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Animais , Células COS , Chlorocebus aethiops , Imunofluorescência , Expressão Gênica , Estudos de Associação Genética , Células HEK293 , Humanos , Modelos Moleculares , Mutação de Sentido Incorreto , Fosfatidiletanolaminas/metabolismo , Ligação Proteica , Domínios Proteicos , Doenças Retinianas/congênito , Doenças Retinianas/genética , Doenças Retinianas/metabolismo , Retinoides/metabolismo , Doença de Stargardt/enzimologiaRESUMO
ATP8A2 is a P4-ATPase (adenosine triphosphate) that actively flips phosphatidylserine and phosphatidylethanolamine from the exoplasmic to the cytoplasmic leaflet of cell membranes to generate and maintain phospholipid asymmetry. Mutations in the ATP8A2 gene have been reported to cause severe autosomal recessive neurological diseases in humans characterized by intellectual disability, hypotonia, chorea, and hyperkinetic movement disorders with or without optic and cerebellar atrophy. To determine the effect of disease-associated missense mutations on ATP8A2, we expressed six variants with the accessory subunit CDC50A in HEK293T cells. The level of expression, cellular localization, and functional activity were analyzed by western blot analysis, immunofluorescence microscopy, and ATPase activity assays. Two variants (p.Ile376Met and p.Lys429Met) expressed at normal ATP8A2 levels and preferentially localized to the Golgi-recycling endosomes, but were devoid of ATPase activity. Four variants (p.Lys429Asn, pAla544Pro, p.Arg625Trp, and p.Trp702Arg) expressed poorly, localized to the endoplasmic reticulum, and lacked ATPase activity. The expression of these variants was increased twofold by the addition of the proteasome inhibitor MG132. We conclude that the p.Ile376Met and p.Lys429Met variants fold in a native-like conformation, but lack key amino acid residues required for ATP-dependent lipid transport. In contrast, the p.Lys429Asn, pAla544Pro, p.Arg625Trp, and p.Trp702Arg variants are highly misfolded and undergo rapid proteosomal degradation.