Cellular copper levels determine the phenotype of the Arg875 variant of ATP7B/Wilson disease protein

In human disorders, the genotype-phenotype relationships are often complex and influenced by genetic and/or environmental factors. Wilson disease (WD) is a monogenic disorder caused by mutations in the copper-transporting P-type ATPase ATP7B. WD shows significant phenotypic diversity even in patients carrying identical mutations; the basis for such diverse manifestations is unknown. We demonstrate that the 2623A/G polymorphism (producing the Gly(875) → Arg substitution in the A-domain of ATP7B) drastically alters the intracellular properties of ATP7B, whereas copper reverses the effects. Under basal conditions, the common Gly(875) variant of ATP7B is targeted to the trans-Golgi network (TGN) and transports copper into the TGN lumen. In contrast, the Arg(875) variant is located in the endoplasmic reticulum (ER) and does not deliver copper to the TGN. Elevated copper corrects the ATP7B-Arg(875) phenotype. Addition of only 0.5-5 μM copper triggers the exit of ATP7B-Arg(875) from the ER and restores copper delivery to the TGN. Analysis of the recombinant A-domains by NMR suggests that the ER retention of ATP7B-Arg(875) is attributable to increased unfolding of the Arg(875)-containing A-domain. Copper is not required for the folding of ATP7B-Arg(875) during biosynthesis, but it stabilizes protein and stimulates its activity. A chemotherapeutical drug, cisplatin, that mimics a copper-bound state of ATP7B also corrects the "disease-like" phenotype of ATP7B-Arg(875) and promotes its TGN targeting and transport function. We conclude that in populations harboring the Arg(875) polymorphism, the levels of bioavailable copper may play a vital role in the manifestations of WD.     

Suppression of peroxisomal membrane protein defects by peroxisomal ATP binding cassette (ABC) proteins

X-Linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder characterized by reduced peroxisomal very long chain fatty acid (VLCFA) beta-oxidation. The X - ALD gene product (ALDP) is a peroxisomal transmembrane protein with an ATP binding cassette (ABC). ALDP and three other ABC proteins (PMP70, ALDR, P70R) localize to the peroxisomal membrane. The function of this family of peroxisomal membrane proteins is unknown. We used complementation studies to begin analysis of their role in VLCFA beta-oxidation and on the peroxisomal membrane. Expression of either ALDP or PMP70 restores VLCFA beta- oxidation in X-ALD fibroblasts, indicating overlapping functions. Their expression also restores peroxisome biogenesis in cells that are deficient in the peroxisomal membrane protein Pex2p. Thus it is likely that complex protein interactions are involved in the function and biogenesis of peroxisomal membranes that may contribute to disease heterogeneity.      

Distinct phenotype of a Wilson disease mutation reveals a novel trafficking determinant in the copper transporter ATP7B

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.Copper is essential for the normal development and function of human cells because it serves as a cofactor for many important metabolic enzymes. However, intracellular levels of copper must be tightly regulated (1, 2) because excess copper is toxic. Inborn mutations in the Cu(I)-ATPases, ATP7A [Online Mendelian Inheritance in Man (OMIM) accession no.*606882] or ATP7B (OMIM *300011) result in either systemic copper deficiency or copper accumulation in several tissues, causing Menkes disease or Wilson disease (WD), respectively. WD (OM#277900) is an autosomal-recessive disorder with a heterogeneous clinical presentation; in the absence of family history, diagnosis of WD requires multiple clinical and laboratory studies (3). The large number of rare mutations in the ATP7B gene [>500, (www.hgmd.cf.ac.uk/ac/gene.php?gene=ATP7B)] contribute to the difficulty in making genotype-phenotype associations. Presently, about two dozen mutations found in WD patients have been characterized in detail (4–7). These studies revealed that the most common effect of a WD mutation is ATP7B misfolding, which results in retention of newly synthesized ATP7B in the endoplasmic reticulum (ER), a marked decrease in protein stability, and loss of Cu(I)-transport activity (8, 9). Destabilization and inactivation of ATP7B explain the common phenotypic manifestations in WD, such as impaired copper export from the liver and the lack of copper incorporation into secreted cuproenzymes, such as ceruloplasmin (CPN).ATP7B and the highly homologous ATP7A (Menkes disease protein) play central roles in maintaining copper levels in cells. These proteins belong to the evolutionarily conserved family of P_1B-ATPases, which use the energy of ATP hydrolysis to transport copper from the cytosol across cellular membranes (Fig. 1A). ATP7A and ATP7B load copper onto newly synthesized cupro-proteins in the late Golgi and remove excess copper from the cytosol after relocating to vesicles, which in turn traffic to the plasma membrane to release copper into the extracellular milieu. In low and basal copper, ATP7A and ATP7B are located predominantly in a subcompartment of the trans-Golgi network (TGN) marked by syntaxin 6 (10). When copper levels increase, ATP7A and ATP7B exit the TGN in distinct vesicles; ATP7B vesicles move to the apical region in polarized epithelia, whereas ATP7A vesicles move to the basolateral region. Because copper-dependent ATP7B trafficking is a complex process, the precise sequence of events and the function of trafficking determinants in ATP7B’s structure are yet to be fully understood.Open in a separate windowFig. 1.Hypothetical ATP7B model and multiple species alignment of the conserved regions, amino acids 621–668, in the two Cu-ATPases. (A) A hypothetical ATP7B ribbon model, generated by UCSF Chimera, showing the conserved core organization (20). The two large cytoplasmic loops in the core structure are: the A domain (actuator, green), between TM4 and TM5, which contains the phosphatase activity; and the N and P domains (nucleotide binding and phosphorylation, red) between TM6 and TM7, which bind ATP (N), catalyzing formation of a phosphorylated intermediate (P) as part of the catalytic cycle. The eight TMs (yellow are): TM1 (amino acids 645–670), TM2 (including the platform helix, amino acids 694–722), TM3 (amino acids 729–749), TM4 (amino acids 765–786), TM5 (amino acids 916–942), TM6 (amino acids 967–1004), TM7 (amino acids 1307–1345), and TM8 (amino acids 1352–1373). The six N-terminal MBDs (blue, N-MBDs, also referred to as the N-terminal domain of ATP7B, N-ATP7B) (61, 62) were manually positioned onto the published model. The box approximates the region of the multiple species alignment shown in B. (B) A multiple species alignment of human ATP7B sequence 621–668 (Upper) and ATP7A sequence 621–668 (Lower). WD patient mutations are underlined and in bold. The ATP7B S653 position is marked with an asterisk (bold). Portions of MBD6 and TMD 1 are bracketed. In ATP7A, the bracketed sequence shows the deleted region that is replaced with two amino acids (IR) in a patient with Occipital Horn Syndrome. The deleted sequence of ATP7A is underlined and in bold (35). Alignments were obtained using ClustalW (63). Amino acids that are identical (*), conserved (:), and semiconserved (.) are shown.We previously developed a comprehensive set of cell-based assays that use both polarized hepatic cells and fibroblasts lacking ATP7B and ATP7A (derived from a Menkes disease patient) to evaluate the activity, stability, and trafficking of ATP7B and its mutants (11, 12). In this study, we combined these assays with additional mutational analysis and computational studies to dissect the molecular phenotype of WD mutations found in a highly conserved region of ATP7B, G⁶²¹-S⁶⁶⁸. We demonstrate that the S653Y mutation has a distinct “transport-competent/trafficking-defective” phenotype. We also show that the transmembrane segment (TM) that harbors S653 has an important and previously unanticipated role in regulating exit of ATP7B from the TGN in response to copper elevation.     

Role of very-long-chain acyl-coenzyme A synthetase in X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is characterized biochemically by decreased ability of cells to activate (via very-long-chain acyl-coenzyme A synthetase [VLCS]) and subsequently degrade very-long-chain fatty acids in peroxisomes. It is noteworthy that the gene defective in X-ALD encodes ALDP, a peroxisomal membrane protein unrelated to VLCS. We cloned human VLCS (hVLCS) and found that peroxisomes from X-ALD fibroblasts contained immunoreactive hVLCS, refuting the earlier hypothesis that ALDP is required to anchor VLCS to the peroxisomal membrane. Furthermore, hVLCS was topographically oriented facing the peroxisomal matrix in both control and X-ALD fibroblasts, contradicting the alternative hypothesis that ALDP is required to translocate VLCS into peroxisomes. However, overexpression of both hVLCS and ALDP in X-ALD fibroblasts synergistically increased very-long-chain fatty acid beta-oxidation, indicating that these proteins interact functionally.     

Sequence, tissue distribution, and differential expression of mRNA for a putative insulin-responsive glucose transporter in mouse 3T3-L1 adipocytes.

The cDNAs for two putative glucose transporters from mouse 3T3-L1 adipocytes were isolated and sequenced. One of these cDNAs encodes the murine homolog of the human hepG2/erythrocyte glucose transporter, termed GT1. GT1 mRNA is most abundant in mouse brain and is expressed in both 3T3-L1 preadipocytes and adipocytes. The other cDNA encodes a glucose transporter-like protein, termed GT2, that has a unique amino acid sequence and tissue distribution. GT2 cDNA encodes a protein with 63% amino acid sequence identity and a similar structural organization to GT1. GT2 mRNA is found at high levels in mouse skeletal muscle, heart, and adipose tissue, all of which exhibit insulin-stimulated glucose uptake. GT2 mRNA is absent from 3T3-L1 preadipocytes but is induced dramatically during differentiation into adipocytes. This increase in mRNA content correlates closely with the acquisition of insulin-stimulated glucose uptake. We propose that GT2 is an insulin-regulated glucose transporter.     

Peroxisomal very long chain fatty acid beta-oxidation activity is determined by the level of adrenodeukodystrophy protein (ALDP) expression

Impaired peroxisomal beta-oxidation of saturated very long chain fatty acids (VLCFA, >/=C22:0) results in increased VLCFA levels in the tissues and body fluids of patients with disorders of peroxisomal biogenesis (i.e., Zellweger syndrome and neonatal adrenoleukodystrophy) and single peroxisomal protein defects (i.e., X-linked adrenoleukodystrophy (X-ALD) and acyl-CoA oxidase deficiency). We show that SV40T transformation also results in impaired peroxisomal beta-oxidation and VLCFA accumulation despite the presence of abundant peroxisomes. To explore the mechanism responsible for this observation, we have examined expression of key components of peroxisomal VLCFA beta-oxidation. We found that expression of both acyl-CoA oxidase, the rate limiting enzyme of peroxisomal VLCFA beta-oxidation and the adrenoleukodystrophy protein (ALDP), the defective gene product in X-ALD, are reduced after SV40T transformation. Surprisingly, ALDP overexpression by itself restores peroxisomal VLCFA beta-oxidation in SV40T-transformed control and X-ALD cells. These results demonstrate that ALDP is a fundamental component in VLCFA peroxisomal beta-oxidation and may serve as a "gatekeeper" for VLCFA homeostasis.