RESUMEN
The copper (Cu) transporters ATPase copper-transporting alpha (ATP7A) and ATPase copper-transporting beta (ATP7B) are essential for the normal function of the mammalian central nervous system. Inactivation of ATP7A or ATP7B causes the severe neurological disorders, Menkes disease and Wilson disease, respectively. In both diseases, Cu imbalance is associated with abnormal levels of the catecholamine-type neurotransmitters dopamine and norepinephrine. Dopamine is converted to norepinephrine by dopamine-ß-hydroxylase (DBH), which acquires its essential Cu cofactor from ATP7A. However, the role of ATP7B in catecholamine homeostasis is unclear. Here, using immunostaining of mouse brain sections and cultured cells, we show that DBH-containing neurons express both ATP7A and ATP7B. The two transporters are located in distinct cellular compartments and oppositely regulate the export of soluble DBH from cultured neuronal cells under resting conditions. Down-regulation of ATP7A, overexpression of ATP7B, and pharmacological Cu depletion increased DBH retention in cells. In contrast, ATP7B inactivation elevated extracellular DBH. Proteolytic processing and the specific activity of exported DBH were not affected by changes in ATP7B levels. These results establish distinct regulatory roles for ATP7A and ATP7B in neuronal cells and explain, in part, the lack of functional compensation between these two transporters in human disorders of Cu imbalance.
Asunto(s)
Encéfalo/enzimología , ATPasas Transportadoras de Cobre/biosíntesis , Dopamina beta-Hidroxilasa/metabolismo , Regulación Enzimológica de la Expresión Génica , Neuronas/enzimología , Animales , Encéfalo/citología , Cobre/metabolismo , ATPasas Transportadoras de Cobre/genética , Dopamina beta-Hidroxilasa/genética , Ratones , Neuronas/citología , ProteolisisRESUMEN
ATP7B is a copper-transporting P1B-type ATPase (Cu-ATPase) with an essential role in human physiology. Mutations in ATP7B cause the potentially fatal Wilson disease, and changes in ATP7B expression are observed in several cancers. Despite its physiologic importance, the biochemical information about ATP7B remains limited because of a complex multidomain organization of the protein. By analogy with the better characterized prokaryotic Cu-ATPases, ATP7B is assumed to be a single-chain monomer. We show that in eukaryotic cells, human ATP7B forms dimers that can be purified following solubilization. Deletion of the four N-terminal metal-binding domains, characteristic for human ATP7B, does not disrupt dimerization, i.e. the dimer interface is formed by the domains that are conserved among Cu-ATPases. Unlike the full-length ATP7B, which is targeted to the trans-Golgi network, 1-4ΔMBD-7B is targeted primarily to vesicles. This result and the analysis of differentially tagged ATP7B variants indicate that the dimeric structure is retained during ATP7B trafficking between the intracellular compartments. Purified dimeric species of 1-4ΔMBD-7B were characterized by a negative stain electron microscopy in the presence of ADP/MgCl2 Single-particle analysis yielded a low-resolution 3D model that provides the first insight into an overall architecture of a human Cu-ATPase, positions of the main domains, and a dimer interface.
Asunto(s)
ATPasas Transportadoras de Cobre/química , ATPasas Transportadoras de Cobre/metabolismo , Multimerización de Proteína , Cobre/metabolismo , ATPasas Transportadoras de Cobre/genética , Cristalografía por Rayos X , Células HEK293 , Degeneración Hepatolenticular/genética , Degeneración Hepatolenticular/metabolismo , Humanos , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica , Dominios Proteicos , Estabilidad Proteica , Transporte de ProteínasRESUMEN
Wilson disease (WD) is a monogenic autosomal-recessive disorder of copper accumulation that leads to liver failure and/or neurological deficits. WD is caused by mutations in ATP7B, a transporter that loads Cu(I) onto newly synthesized cupro-enzymes in the trans-Golgi network (TGN) and exports excess copper out of cells by trafficking from the TGN to the plasma membrane. To date, most WD mutations have been shown to disrupt ATP7B activity and/or stability. Using a multidisciplinary approach, including clinical analysis of patients, cell-based assays, and computational studies, we characterized a patient mutation, ATP7B(S653Y), which is stable, does not disrupt Cu(I) transport, yet renders the protein unable to exit the TGN. Bulky or charged substitutions at position 653 mimic the phenotype of the patient mutation. Molecular modeling and dynamic simulation suggest that the S653Y mutation induces local distortions within the transmembrane (TM) domain 1 and alter TM1 interaction with TM2. S653Y abolishes the trafficking-stimulating effects of a secondary mutation in the N-terminal apical targeting domain. This result indicates a role for TM1/TM2 in regulating conformations of cytosolic domains involved in ATP7B trafficking. Taken together, our experiments revealed an unexpected role for TM1/TM2 in copper-regulated trafficking of ATP7B and defined a unique class of WD mutants that are transport-competent but trafficking-defective. Understanding the precise consequences of WD-causing mutations will facilitate the development of advanced mutation-specific therapies.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Cobre/metabolismo , Degeneración Hepatolenticular/genética , Mutación , Adenosina Trifosfatasas/química , Secuencia de Aminoácidos , Animales , Proteínas de Transporte de Catión/química , Membrana Celular/metabolismo , ATPasas Transportadoras de Cobre , Aparato de Golgi/metabolismo , Humanos , Hígado/metabolismo , Modelos Moleculares , Modelos Teóricos , Datos de Secuencia Molecular , Transporte de Proteínas , Homología de Secuencia de AminoácidoRESUMEN
Wilson disease (WD) is an autosomal-recessive disorder caused by mutations in the copper (Cu)-transporter ATP7B. Thus far, studies of WD mutations have been limited to analysis of ATP7B mutants in the homozygous states. However, the majority of WD patients are compound-heterozygous, and how different mutations on two alleles impact ATP7B properties is unclear. We characterized five mutations identified in Indian WD patients, first by expressing each alone and then by co-expressing two mutants with dissimilar properties. Mutations located in the regulatory domains of ATP7B-A595T, S1362A, and S1426I-do not affect ATP7B targeting to the trans-Golgi network (TGN) but reduce its Cu-transport activity. The S1362A mutation also inhibits Cu-dependent trafficking from the TGN. The G1061E and G1101R mutations, which are located within the ATP-binding domain, cause ATP7B retention in the endoplasmic reticulum, inhibit Cu-transport, and lower ATP7B protein abundance. Co-expression of the A595T and G1061E mutations, which mimics the compound-heterozygous state of some WD patients, revealed an interaction between these mutants that altered their intracellular localization and trafficking under both low and high Cu conditions. These findings highlight the need to study WD variants in both the homozygous and compound-heterozygous states to better understand the genotype-phenotype correlations and incomplete penetrance observed in WD.
Asunto(s)
ATPasas Transportadoras de Cobre/genética , Degeneración Hepatolenticular/genética , Adenosina Trifosfatasas/metabolismo , Alelos , Proteínas de Transporte de Catión/genética , Cobre/metabolismo , ATPasas Transportadoras de Cobre/metabolismo , Retículo Endoplásmico/metabolismo , Estudios de Asociación Genética , Células HEK293 , Humanos , Mutación , Transporte de Proteínas , Red trans-Golgi/genética , Red trans-Golgi/metabolismoRESUMEN
Correction for 'Single nucleotide polymorphisms in the human ATP7B gene modify the properties of the ATP7B protein' by Courtney J. McCann et al., Metallomics, 2019, 11, 1128-1139.
RESUMEN
Single nucleotide polymorphisms (SNPs) are the largest source of sequence variation in the human genome. However, their functional significance is not well understood. We show that SNPs in the Wilson disease gene, ATP7B, that produce amino-acid substitutions K832R and R952K, modulate ATP7B properties in vitro and influence serum copper (Cu) status in vivo. The presence of R832 is associated with a lower ATP7B abundance and a diminished trafficking in response to elevated Cu. The K832R substitution alters surface exposure of amino acid residues in the actuator domain and increases its conformational flexibility. All SNP-related ATP7B variants (R832/R952, R832/K952, K832/K952, and K832/R952) have Cu-transport activity. However, the activity of ATP7B-K832/K952 is lower compared to other variants. In humans, the presence of K952 is associated with a higher fraction of exchangeable Cu in serum. Thus, SNPs may modulate the properties of ATP7B and the organism Cu status.
Asunto(s)
ATPasas Transportadoras de Cobre/genética , ATPasas Transportadoras de Cobre/metabolismo , Cobre/metabolismo , Polimorfismo de Nucleótido Simple , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Cobre/sangre , ATPasas Transportadoras de Cobre/química , Células HEK293 , Degeneración Hepatolenticular/sangre , Degeneración Hepatolenticular/genética , Degeneración Hepatolenticular/metabolismo , Humanos , Simulación de Dinámica Molecular , Conformación Proteica , Transporte de ProteínasRESUMEN
Heavy metal P1B-type ATPases play a critical role in cell survival by maintaining appropriate intracellular metal concentrations. Archaeoglobus fulgidus CopB is a member of this family that transports Cu(II) from the cytoplasm to the exterior of the cell using ATP as energy source. CopB has a 264 amino acid ATPBD (ATP-binding domain) that is essential for ATP binding and hydrolysis as well as ultimately transducing the energy to the transmembrane metal-binding site for metal occlusion and export. The relevant conformations of this domain during the different steps of the catalytic cycle are still under discussion. Through crystal structures of the apo- and phosphate-bound ATPBDs, with limited proteolysis and fluorescence studies of the apo- and substrate-bound states, we show that the isolated ATPBD of CopB cycles from an open conformation in the apo-state to a closed conformation in the substrate-bound state, then returns to an open conformation suitable for product release. The present work is the first structural report of an ATPBD with its physiologically relevant product (phosphate) bound. The solution studies we have performed help resolve questions on the potential influence of crystal packing on domain conformation. These results explain how phosphate is co-ordinated in ATPase transporters and give an insight into the physiologically relevant conformation of the ATPBD at different steps of the catalytic cycle.