A L Hubbard

Johns Hopkins Medicine, Baltimore, Maryland, United States

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Publications (79)524.93 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The Wilson Disease protein ATP7B exhibits Cu-dependent trafficking. In high Cu, ATP7B exits the trans-Golgi network (TGN) and moves to the apical domain of hepatocytes, where it facilitates elimination of excess Cu through the bile. Cu levels also affect ATP7B phosphorylation. ATP7B is basally phosphorylated in low Cu and becomes more phosphorylated (″hyperphosphorylated″) in elevated Cu. The functional significance of hyperphosphorylation remains unclear. We showed that hyperphosphorylation occurs even when ATP7B is restricted to the TGN. We performed comprehensive phosphoproteomics of ATP7B in low versus high Cu, which revealed 24 Ser/Thr residues in ATP7B could be phosphorylated, and only four of which were Cu-responsive. Most of the phosphorylated sites were found in the N- and C-terminal cytoplasmic domains. Using truncation and mutagenesis, we showed that inactivation or elimination of all 6 N-terminal metal binding domains (MBDs) did not block Cu-dependent, reversible, apical trafficking but did block hyperphosphorylation in hepatic cells. We showed that 9 of 15 of Ser/Thr in the C-terminal domain were phosphorylated. Inactivation of 13 C-terminal phosphorylation sites reduced basal phosphorylation and eliminated hyperphosphorylation, suggesting that Cu binding at the N-terminus propagates to the ATP7B C-terminal region. C-terminal mutants with either inactivating or phosphomimetic substitutions showed little effect upon Cu-stimulated trafficking, indicating that trafficking does not depend on phosphorylation at these sites. Thus, our studies revealed Cu-dependent conformational changes in the N-terminal region lead to hyperphosphorylation at C-terminal sites, which seem not to affect trafficking and may instead fine-tune Cu sequestration. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 02/2015; 290(14). DOI:10.1074/jbc.M114.627414 · 4.57 Impact Factor
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    Lydia K. Nyasae · Michael J. Schell · Ann L. Hubbard
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    ABSTRACT: Physiologic Cu levels regulate the intracellular location of the Cu ATPase ATP7B. Here, we determined the routes of Cu-directed trafficking of endogenous ATP7B in the polarized hepatic cell line WIF-B and in the liver in vivo. Copper (10 μM) caused ATP7B to exit the trans-Golgi network (TGN) in vesicles, which trafficked via large basolateral endosomes to the apical domain within one hour. Although perturbants of luminal acidification had little effect on the TGN localization of ATP7B in low Cu, they blocked delivery to the apical membrane in elevated Cu. If the vesicular proton-pump inhibitor bafilomycin-A1 (Baf) was present with Cu, ATP7B still exited the TGN, but accumulated in large endosomes located near the coverslip, in the basolateral region. Baf washout restored ATP7B trafficking to the apical domain. If ATP7B was staged apically in high Cu, Baf addition promoted the accumulation of ATP7B in subapical endosomes, indicating a blockade of apical recycling, with concomitant loss of ATP7B at the apical membrane. The retrograde pathway to the TGN, induced by Cu removal, was far less affected by Baf than the anterograde (Cu-stimulated) case. Overall, loss of acidification impaired Cu-regulated trafficking of ATP7B at two main sites: 1) sorting and exit from large basolateral endosomes and 2) recycling via endosomes near the apical membrane.
    Traffic 09/2014; 15(12). DOI:10.1111/tra.12229 · 4.71 Impact Factor
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    ABSTRACT: The intestinal brush border (BB) Na(+)/H(+) exchanger, NHE3, is acutely regulated through changes in its endocytosis/exocytosis. Myosin VI, a minus-end directed motor, has been implicated in endocytosis at the inter-microvillar (MV) cleft and vesicle remodeling in the terminal web. We asked if myosin VI also regulates NHE3 movement down MV. Basal NHE3 activity and surface amount, determined by BCECF/fluorometry and biotinylation, respectively, were increased in myosin VI knock-down (KD) Caco-2/Bbe cells. Carbachol (CCH) and forskolin (FSK) stimulated NHE3 endocytosis in control but not in myosin VI KD cells. Importantly, immuno-EM results showed NHE3 preferentially localized in the basal half of MV in control but in the distal half of myosin VI KD cells. Dynasore duplicated some aspects of myosin VI KD: it increased basal surface NHE3 activity and prevented FSK-induced NHE3 endocytosis; but NHE3's distribution along the MV was intermediate in dynasore-treated as compared to either myosin VI KD or untreated cells. We conclude that myosin VI is required for basal and stimulated endocytosis of NHE3 in intestinal cells and suggest that myosin VI also moves NHE3 down MV.
    Journal of Cell Science 06/2014; 127(16). DOI:10.1242/jcs.149930 · 5.33 Impact Factor
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    ABSTRACT: 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.
    Proceedings of the National Academy of Sciences 03/2014; 111. DOI:10.1073/pnas.1314161111 · 9.81 Impact Factor
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    Molecular Cytogenetics 01/2014; 7(Suppl 1):P68. DOI:10.1186/1755-8166-7-S1-P68 · 2.66 Impact Factor
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    ABSTRACT: Life-threatening intestinal and systemic effects of the Shiga toxins produced by enterohemorrhagic Escherichia coli (EHEC) require toxin uptake and transcytosis across intestinal epithelial cells. We have recently demonstrated that EHEC infection of intestinal epithelial cells stimulates toxin macropinocytosis, an actin-dependent endocytic pathway. Host actin rearrangement necessary for EHEC attachment to enterocytes is mediated by the type 3 secretion system which functions as a molecular syringe to translocate bacterial effector proteins directly into host cells. Actin-dependent EHEC attachment also requires the outer membrane protein intimin, a major EHEC adhesin. Here, we investigate the role of type 3 secretion in actin turnover occurring during toxin macropinocytosis. Toxin macropinocytosis is independent of EHEC type 3 secretion and intimin attachment. EHEC soluble factors are sufficient to stimulate macropinocytosis and deliver toxin into enterocytes in vitro and in vivo; intact bacteria are not required. Intimin-negative enteroaggregative Escherichia coli (EAEC) O104:H4 robustly stimulate Shiga toxin macropinocytosis into intestinal epithelial cells. The apical macropinosomes formed in intestinal epithelial cells move through the cells and release their cargo at these cells' basolateral sides. Further analysis of EHEC secreted proteins shows that a serine protease EspP alone is able to stimulate host actin remodeling and toxin macropinocytosis. The observation that soluble factors, possibly serine proteases including EspP, from each of two genetically distinct toxin-producing strains, can stimulate Shiga toxin macropinocytosis and transcellular transcytosis alters current ideas concerning mechanisms whereby Shiga toxin interacts with human enterocytes. Mechanisms important for this macropinocytic pathway could suggest new potential therapeutic targets for Shiga toxin-induced disease.
    PLoS ONE 07/2013; 8(7):e69196. DOI:10.1371/journal.pone.0069196 · 3.23 Impact Factor
  • Inflammatory Bowel Diseases 12/2011; 17:S85. DOI:10.1097/00054725-201112002-00281 · 5.48 Impact Factor
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    ABSTRACT: Gastrointestinal infection with Shiga toxins producing enterohemorrhagic Escherichia coli causes the spectrum of gastrointestinal and systemic complications, including hemorrhagic colitis and hemolytic uremic syndrome, which is fatal in ∼10% of patients. However, the molecular mechanisms of Stx endocytosis by enterocytes and the toxins cross the intestinal epithelium are largely uncharacterized. We have studied Shiga toxin 1 entry into enterohemorrhagic E. coli-infected intestinal epithelial cells and found that bacteria stimulate Shiga toxin 1 macropinocytosis through actin remodeling. This enterohemorrhagic E. coli-caused macropinocytosis occurs through a nonmuscle myosin II and cell division control 42 (Cdc42)-dependent mechanism. Macropinocytosis of Shiga toxin 1 is followed by its transcytosis to the basolateral environment, a step that is necessary for its systemic spread. Inhibition of Shiga toxin 1 macropinocytosis significantly decreases toxin uptake by intestinal epithelial cells and in this way provides an attractive, antibiotic-independent strategy for prevention of the harmful consequences of enterohemorrhagic E. coli infection.
    AJP Cell Physiology 08/2011; 301(5):C1140-9. DOI:10.1152/ajpcell.00036.2011 · 3.67 Impact Factor
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    ABSTRACT: ClC-5, a chloride/proton exchanger, is predominantly expressed and localized in subapical endosomes of the renal proximal tubule. Mutations of the CLCN5 gene cause Dent disease. The symptoms of Dent disease are replicated in Clcn5 knock-out mice. Absence of ClC-5 in mice is associated with reduced surface expression of NHE3 in proximal tubules. The molecular basis for this change is not fully understood. In this study, we investigated the mechanisms by which ClC-5 regulates trafficking of NHE3. Whether ClC-5-dependent endocytosis, exocytosis, or both contributed to the altered distribution of NHE3 was examined. First, NHE3 activity in proximal tubules of wild type (WT) and Clcn5 KO mice was determined by two-photon microscopy. Basal and dexamethasone-stimulated NHE3 activity of Clcn5 KO mice was decreased compared with that seen in WT mice, whereas the degree of inhibition of NHE3 activity by increasing cellular concentration of cAMP (forskolin) or Ca(2+) (A23187) was not different in WT and Clcn5 KO mice. Second, NHE3-dependent absorption of HCO(3)(-), measured by single tubule perfusion, was reduced in proximal tubules of Clcn5 KO mice. Third, by cell surface biotinylation, trafficking of NHE3 was examined in short hairpin RNA (shRNA) plasmid-transfected opossum kidney cells. Surface NHE3 was reduced in opossum kidney cells with reduced expression of ClC-5, whereas the total protein level of NHE3 did not change. Parathyroid hormone decreased NHE3 surface expression, but the extent of decrease and the rate of endocytosis observed in both scrambled and ClC-5 knockdown cells were not significantly different. However, the rates of basal and dexamethasone-stimulated exocytosis of NHE3 were attenuated in ClC-5 knockdown cells. These results show that ClC-5 plays an essential role in exocytosis of NHE3.
    Journal of Biological Chemistry 05/2011; 286(26):22833-45. DOI:10.1074/jbc.M111.224998 · 4.57 Impact Factor
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    ABSTRACT: 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.
    Proceedings of the National Academy of Sciences 03/2011; 108(13):5390-5. DOI:10.1073/pnas.1014959108 · 9.81 Impact Factor
  • L Braiterman · L Nyasae · F Leves · A L Hubbard
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    ABSTRACT: ATP7A and ATP7B are copper-transporting P-type ATPases that are essential to eukaryotic copper homeostasis and must traffic between intracellular compartments to carry out their functions. Previously, we identified a nine-amino acid sequence (F37-E45) in the NH(2) terminus of ATP7B that is required to retain the protein in the Golgi when copper levels are low and target it apically in polarized hepatic cells when copper levels rise. To understand further the mechanisms regulating the intracellular dynamics of ATP7B, using multiple functional assays, we characterized the protein phenotypes of 10 engineered and Wilson disease-associated mutations in the ATP7B COOH terminus in polarized hepatic cells and fibroblasts. We also examined the behavior of a chimera between ATP7B and ATP7A. Our results clearly demonstrate the importance of the COOH terminus of ATP7B in the protein's copper-responsive apical trafficking. L1373 at the end of transmembrane domain 8 is required for protein stability and Golgi retention in low copper, the trileucine motif (L1454-L1456) is required for retrograde trafficking, and the COOH terminus of ATP7B exhibits a higher sensitivity to copper than does ATP7A. Importantly, our results demonstrating that four Wilson disease-associated missense mutations behaved in a wild-type manner in all our assays, together with current information in the literature, raise the possibility that several may not be disease-causing mutations.
    AJP Gastrointestinal and Liver Physiology 03/2011; 301(1):G69-81. DOI:10.1152/ajpgi.00038.2011 · 3.74 Impact Factor
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    ABSTRACT: To identify additional potential functions for the multi-PDZ domain containing protein Na+/H+ exchanger regulatory factor 2 (NHERF2), which is present in the apical domain of intestinal epithelial cells, proteomic studies of mouse jejunal villus epithelial cell brush border membrane vesicles compared wild-type to homozygous NHERF2 knockout FVB mice by a two-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS)-iTRAQ approach. Jejunal architecture appeared normal in NHERF2 null in terms of villus length and crypt depth, Paneth cell number, and microvillus structure by electron microscopy. There was also no change in proliferative activity based on BrdU labeling. Four brush border membrane vesicles (BBMV) preparations from wild-type mouse jejunum were compared with four preparations from NHERF2 knockout mice. LC-MS/MS identified 450 proteins in both matched wild-type and NHERF2 null BBMV; 13 proteins were changed in two or more separate BBMV preparations (9 increased and 4 decreased in NHERF2 null mice), while an additional 92 proteins were changed in a single BBMV preparation (68 increased and 24 decreased in NHERF2 null mice). These proteins were categorized as 1) transport proteins (one increased and two decreased in NHERF2 null); 2) signaling molecules (2 increased in NHERF2 null); 3) cytoskeleton/junctional proteins (4 upregulated and 1 downregulated in NHERF2 null); and 4) metabolic proteins/intrinsic BB proteins) (2 upregulated and 1 downregulated in NHERF2 null). Immunoblotting of BBMV was used to validate or extend the findings, demonstrating increase in BBMV of NHERF2 null of MCT1, coronin 3, and ezrin. The proteome of the NHERF2 null mouse small intestinal BB demonstrates up- and downregulation of multiple transport proteins, signaling molecules, cytoskeletal proteins, tight junctional and adherens junction proteins, and proteins involved in metabolism, suggesting involvement of NHERF2 in multiple apical regulatory processes and interactions with luminal contents.
    Physiological Genomics 03/2011; 43(11):674-84. DOI:10.1152/physiolgenomics.00258.2010 · 2.81 Impact Factor
  • Gastroenterology 01/2011; 140(5). DOI:10.1016/S0016-5085(11)60035-8 · 13.93 Impact Factor
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    ABSTRACT: Na/H exchanger regulatory factor 1 (NHERF1) is a scaffold protein made up of two PDZ domains and an ERM binding domain. It is in the brush border of multiple epithelial cells where it modulates 1) Na absorption by regulating NHE3 complexes and cytoskeletal association, 2) Cl secretion through trafficking of CFTR, and 3) Na-coupled phosphate absorption through membrane retention of NaPi2a. To further understand the role of NHERF1 in regulation of small intestinal Na absorptive cell function, with emphasis on apical membrane transport regulation, quantitative proteomic analysis was performed on brush border membrane vesicles (BBMV) prepared from wild-type (WT) and homozygous NHERF1 knockout mouse jejunal villus Na absorptive cells. Jejunal architecture appeared normal in NHERF1 null; however, there was increased proliferative activity, as indicated by increased crypt BrdU staining. LC-MS/MS analysis using iTRAQ to compare WT and NHERF1 null BBMV identified 463 proteins present in both WT and NHERF1 null BBMV of simultaneously prepared and studied samples. Seventeen proteins had an altered amount of expression between WT and NHERF1 null in two or more separate preparations, and 149 total proteins were altered in at least one BBMV preparation. The classes of the majority of proteins altered included transport proteins, signaling and trafficking proteins, and proteins involved in proliferation and cell division. Affected proteins also included tight junction and adherens junction proteins, cytoskeletal proteins, as well as metabolic and BB digestive enzymes. Changes in abundance of several proteins were confirmed by immunoblotting [increased CEACAM1, decreased ezrin (p-ezrin), NHERF3, PLCβ3, E-cadherin, p120, β-catenin]. The changes in the jejunal BBMV proteome of NHERF1 null mice are consistent with a more complex role of NHERF1 than just forming signaling complexes and anchoring proteins to the apical membrane and include at least alterations in proteins involved in transport, signaling, and proliferation.
    Physiological Genomics 11/2010; 42A(3):200-10. DOI:10.1152/physiolgenomics.00001.2010 · 2.81 Impact Factor
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    ABSTRACT: Manganese is a trace element that is an essential co-factor in many enzymes critical to diverse biological pathways. However, excess Mn(2+) leads to neurotoxicity, with psychiatric and motor dysfunction resembling parkinsonism. The liver is the main organ for Mn(2+) detoxification by excretion into bile. Although many pathways of cellular Mn(2+) uptake have been established, efflux mechanisms remain essentially undefined. In this study, we evaluated a potential role in Mn(2+) detoxification by the Secretory Pathway Ca(2+), Mn(2+)-ATPase in rat liver and a liver-derived cell model WIF-B that polarizes to distinct bile canalicular and sinusoidal domains in culture. Of two known isoforms, only secretory pathway Ca(2+)-ATPase isoform 1 (SPCA1) was expressed in liver and WIF-B cells. As previously observed in non-polarized cells, SPCA1 showed overlapping distribution with TGN38, consistent with Golgi/TGN localization. However, a prominent novel localization of SPCA1 to an endosomal population close to, but not on the basolateral membrane was also observed. This was confirmed by fractionation of rat liver homogenates which revealed dual distribution of SPCA1 to the Golgi/TGN and a fraction that included the early endosomal marker, EEA1. We suggest that this novel pool of endosomes may serve to sequester Mn(2+) as it enters from the sinusoidal/basolateral domains. Isoform-specific partial knockdown of SPCA1 delayed cell growth and formation of canalicular domain by about 30% and diminished viability upon exposure to Mn(2+). Conversely, overexpression of SPCA1 in HEK 293T cells conferred tolerance to Mn(2+) toxicity. Taken together, our findings suggest a role for SPCA1 in Mn(2+) detoxification in liver.
    Biology of Metals 10/2010; 24(1):159-70. DOI:10.1007/s10534-010-9384-3 · 2.69 Impact Factor
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    ABSTRACT: Copper plays an indispensable role in the physiology of the human central nervous system (CNS). As a cofactor of dopamine-β-hydroxylase, peptidyl-α-monooxygenase, superoxide dismutases, and many other enzymes, copper is a critical contributor to catecholamine biosynthesis, activation of neuropeptides and hormones, protection against reactive oxygen species, respiration and other processes essential for normal CNS function. Copper content in the CNS is tightly regulated, and changes in copper levels in the brain are associated with a wide spectrum of pathologies. However, the mechanistic understanding of copper transport in the CNS is still in its infancy. Little is known about copper distribution among various cell types or cell-specific regulation of copper homeostasis, despite the fact that the molecules mediating copper transport and distribution in the brain (CTR1, Atox1, CCS, ScoI/II, ATP7A and ATP7B) have been identified and their importance in CNS function increasingly understood. In this review, we summarize current knowledge about copper levels and uses in the CNS and describe the molecules involved in maintaining copper homeostasis in the brain.
    Metallomics 09/2010; 2(9):596-608. DOI:10.1039/c0mt00006j · 3.98 Impact Factor
  • Gastroenterology 05/2010; 138(5). DOI:10.1016/S0016-5085(10)60214-4 · 13.93 Impact Factor
  • Lelita T. Braiterman · Ann L. Hubbard
    The Liver: Biology and Pathobiology, Fifth Edition, 10/2009: pages 73 - 105; , ISBN: 9780470747919
  • Gastroenterology 05/2009; 136(5). DOI:10.1016/S0016-5085(09)63285-6 · 13.93 Impact Factor
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    ABSTRACT: Human Cu-ATPases ATP7A and ATP7B maintain copper homeostasis through regulated trafficking between intracellular compartments. Inactivation of these transporters causes Menkes disease and Wilson disease, respectively. In Menkes disease, copper accumulates in kidneys and causes tubular damage, indicating that the renal ATP7B does not compensate for the loss of ATP7A function. We show that this is likely due to a kidney-specific regulation of ATP7B. Unlike ATP7A (or hepatic ATP7B) which traffics from the TGN to export copper, renal ATP7B does not traffic and therefore is unlikely to mediate copper export. The lack of ATP7B trafficking is not on account of the loss of a kinase-mediated phosphorylation or simultaneous presence of ATP7A in renal cells. Rather, the renal ATP7B appears 2-3 kDa smaller than hepatic ATP7B. Recombinant ATP7B expressed in renal cells is similar to hepatic protein in size and trafficking. The analysis of ATP7B mRNA revealed a complex behavior of exon 1 upon amplification, suggesting that it could be inefficiently translated. Recombinant ATP7B lacking exon 1 traffics differently in renal and hepatic cells, but does not fully recapitulate the endogenous phenotype. We discuss factors that may contribute to cell-specific behavior of ATP7B and propose a role for renal ATP7B in intracellular copper storage.
    Traffic 03/2009; 10(6):767-79. DOI:10.1111/j.1600-0854.2009.00901.x · 4.71 Impact Factor

Publication Stats

4k Citations
524.93 Total Impact Points

Institutions

  • 1983–2015
    • Johns Hopkins Medicine
      Baltimore, Maryland, United States
  • 1984–2014
    • Johns Hopkins University
      • • Department of Cell Biology
      • • Department of Biology
      • • Department of Medicine
      Baltimore, Maryland, United States
  • 1988
    • Howard University
      • Department of Anatomy
      Washington, WV, United States
  • 1980
    • Yale University
      New Haven, Connecticut, United States