Liver as a key organ in the supply, storage, and excretion of copper1–4

Eve A Roberts and Bibudhendra Sarkar

ABSTRACT The majority (95%) of in blood is incorporated in Downloaded from https://academic.oup.com/ajcn/article/88/3/851S/4649166 by guest on 04 October 2021 The liver plays an important role in the disposition of copper. Most , which is itself an acute-phase reactant and there- dietary copper passes through the liver where it can be used for fore subject to variation independent of total body copper. It is and energy production or excreted through the biliary route. not routine to measure serum copper- or copper- Because copper is a prooxidant, its intracellular handling is tightly histidine directly, and estimates of non–ceruloplasmin-bound managed. In Wilson , in which synthesis of ceruloplasmin copper are faulty unless ceruloplasmin is measured as ferroxi- and biliary excretion of copper are defective, copper accumulates in dase activity. Basal 24-h urinary copper excretion reflects non– the liver and leads to progressive liver damage. The features of ceruloplasmin-bound copper and provides valuable diagnostic hepatic Wilson disease are highly variable. The spectrum of liver information in Wilson disease (WD). Very low plasma concen- disease includes mild , fatty liver, an autoimmune dis- trations of ceruloplasmin, if measured enzymatically, may char- order, and . Wilson disease thus resembles drug hepatotox- acterize copper insufficiency in patients with WD, who have icity, and indeed it can be regarded as a prototypic example of been too aggressively treated (2). endogenous hepatotoxicity. Biomarkers developed for detecting drug hepatotoxicity may be relevant to Wilson disease. Biomarkers developed through metalloproteomics, which for copper seeks to COPPER IN THE LIVER define a set of that have copper-binding capacity, or through genomic studies may also be relevant to Wilson disease and other Dietary copper loosely bound to albumin or histidine reaches disorders of copper handling, whether copper is deficient or the liver by the portal vein and is taken up into hepatocytes across overloaded. Am J Clin Nutr 2008;88(suppl):851S–4S. the sinusoidal plasma membrane (Figure 1). Because copper associated with these transporters is Cu(II), it must be reduced to Cu(I) before hepatocellular uptake. After the available copper is reduced either by a reductase on the outer aspect of the hepato- INTRODUCTION cyte membrane or possibly by dietary reductants, it is transported Copper is essential for the action of diverse , perform- into the hepatocyte by hCTR1, a member of the solute ligand ing a broad range of physiologic functions. An ordinary diet carrier superfamily, encoded by the SLC31A1 (3). hCTR1 provides sufficient copper because typical daily intake of copper appears to exist as a trimer, forming a channel in the hepatocel- ranges from 1 to 10 mg, usually Ȃ2–5 mg/d, depending on the lular plasma membrane. Analogous to the Wilson adenosine amount of meat, legumes, shellfish, and chocolate consumed. 5Ј-triphosphatase (ATPase), hCTR1 has copper-binding do- The recommended daily intake is 0.9 mg/d. Efficiency of intes- mains near the amino terminus, but these domains consist of a tinal absorption is high (55–75%) and intake is not regulated; methionine cluster motif (MXXM), as opposed to a cysteine normal copper balance is maintained by regulation of excretion cluster (CXXC). Copper uptake may be linked to potassium for which the predominant route is hepatobiliary. Most (85%) of transport. Although hCTR1 is not the regulatory control point for dietary copper is excreted. The renal pathway accounts for 5% copper , it is degraded when copper concentrations of copper excretion, unless renal tubular reabsorption capacity is are high. A second copper transporter, known as hCTR2, may exceeded. Dietary copper, as well as copper found in saliva and gastric 1 From the Genetics and Genome Biology Program (EAR) and Molecular and pancreatic juices, is absorbed in the proximal small intestine. Structure and Function Program (BS), Hospital for Sick Children Research Absorption is probably by hCTR1 expressed on enterocytes, Institute, and the Departments of Paediatrics (EAR), Medicine (EAR), Phar- although the divalent cation transporter (DMT1), which is macology (EAR), and Biochemistry (BS), University of Toronto, Toronto, mainly involved in iron uptake, may play a limited role. Once ON, Canada. 2 absorbed, copper is in an exchangeable pool, bound reversibly to Presented at the symposium “Molecular Biomarkers of Copper Ho- and to various amino acids, histidine being the meostasis,” held in Vin˜a del Mar, Chile, September 26–29, 2007. 3 Supported by operating grants from the Canadian Institutes of Health most important. Copper-albumin and copper-histidine distribute Research and the Canadian Liver Foundation. copper to various tissues, mainly to the liver; copper loosely 4 Reprints not available. Address correspondence to EA Roberts, Division bound to amino acids is filtered in the kidneys and avidly reab- of Gastroenterology, Hepatology, and Nutrition, Room 8263, Black Wing, sorbed in the tubules. Copper excreted into bile is in a complex The Hospital for Sick Children, 555 University Avenue, Toronto, ON M5G not available for reabsorption (1). 1X8, Canada. E-mail: [email protected].

Am J Clin Nutr 2008;88(suppl):851S–4S. Printed in USA. © 2008 American Society for Nutrition 851S 852S ROBERTS AND SARKAR

Cu ATOX1 CO MMD1 Cu ATP7B Cu-GSH (Wilson ATPase) COX17 CTR1 CCS MRP2 BC SOD1 Downloaded from https://academic.oup.com/ajcn/article/88/3/851S/4649166 by guest on 04 October 2021 Metallothionein Cu Ceruloplasmin

hepatocytes FIGURE 1. Cartoon depicting the network of proteins involved in copper disposition within the hepatocyte. A doublet of hepatocytes is shown, with the specialized bile canalicular membrane located between 2 tight junctions (pointed octagons). Copper (Cu; small circle) is taken up by the sinusoidal plasma membrane, deployed within the hepatocyte by metallochaperones, used in ceruloplasmin production, stored or detoxified, or excreted into bile. The uptake mechanism involves a reductase (cross) and CTR1 (square), the principal plasma membrane copper transporter. Metallochaperones target copper to specific sites: ATOX1 (square) to Wilson adenosine 5Ј-triphosphatase (ATPase; oval with directional arrow) in trans-Golgi network; Cox17 (hatched ellipse) to mitochondria; CCS (cross-hatched ellipse) to SOD1 in . Glutathione (GSH; horseshoe) mediates other intracellular transfer, including incorporation into metallothionein. When intracellular copper concentrations are low or normal, the Wilson ATPase participates in production of holoceruloplasmin (inverted trapezoid containing copper) in the ; holoceruloplasmin is then secreted into the blood. When intracellular copper concentrations are elevated, the Wilson ATPase expedites biliary excretion of copper by a process that may also involve COMMD1. Biliary copper excretion can also take place by the transporter MRP2. The complete network of proteins involved in hepatocellular copper disposition has not yet been identified. BC, bile canaliculus. mediate low-affinity copper uptake. The role of DMT1 in hep- domains (GMXCXXC); 8 transmembrane segments forming a atocellular copper uptake is controversial. pore, including the distinctive CPC motif in segment 6; and an Copper does not exist in ionic form within cells. In hepatocytes ATPase region. Its structure has been determined by and other cells it is always bound to low-molecular-weight pro- mapping with the use of the sarcoplasmic Ca2ѿ P-type ATPase teins, called metallochaperones, which each deliver copper to Serca1 as model (8), and structures of portions of the protein have different specific target molecules within the cell (4). In all like- also been solved (5). Conformational changes associated with lihood, the entire fleet of copper-metallochaperones has not yet copper binding may influence Wilson ATPase function; accord- been identified. CCS1 guides copper to SOD1, a principally ingly, the N-terminal region may play a regulatory role. Recent cytoplasmic defense against oxidant stress. ATOX1 directs cop- functional studies were performed with a truncated human Wil- per to the Wilson ATPase. It has 1 CXXC copper-binding do- son ATPase lacking the copper-binding domains 1–5 (9). main, similar to the 6 founding the Wilson ATPase. It interacts Mechanistic details of the intracellular action of the Wilson AT- with the Wilson ATPase (5) and with copper (6). Metallothio- Pase remain unclear. Positioned in the trans-Golgi network, it par- neins and glutathione are also found in the cytoplasm; they in- ticipates in the synthesis of holoceruloplasmin, apparently by mak- teract with copper but are not metallochaperones. ing copper available for incorporation into the nascent holoprotein. The role of copper in normal mitochondrial prin- Remarkably, when intracellular copper concentrations are elevated, cipally involves cytochrome-c oxidase (complex IV in the it moves from the trans-Golgi region to the apical (bile canalicular) OX-PHOS chain) and associated proteins such as Cox17, Cox19, membrane of the hepatocyte (10). The mechanism of biliary excre- Sco1, and Sco2 (7). Cox17 is the main copper chaperone for tion of copper is uncertain. Recent studies have shown that targeting delivery of copper to mitochondria; Cox19 is required COMMD1 is abnormal in Bedlington terrier copper toxicosis, a for complex IV expression and may have copper-transport func- hepatic disorder of copper disposition distinct from WD, in which tions. Sco1 and Sco2 are assembly factors for complex IV. biliary copper excretion is defective. COMMD1, as well as several Thioredoxin-2 (Trx-2) and some metallothioneins are also found other members of the COMMD family, can interact with the Wilson in the mitochondrial matrix. ATPase (11). COMMD1 binds Cu(II) (12), but exactly how this In hepatocytes the Wilson ATPase plays a pivotal role in relates to its intracellular function is not yet determined. COMMD1 copper disposition. It is an intracellular copper-transporting has regulatory functions relating to nuclear ␬B P-type ATPase with molecular weight Ȃ165 kDa (1411 amino (NF-␬B) and other transporters (13). The X-linked inhibitor of ap- acids). Like other P-type ATPases, it has a cation channel and optosis interacts with COMMD1; the X-linked inhibitor of apopto- phosphorylation domain with a highly conserved DKTGT motif sis can also bind copper and appears to become nonfunctional by in which the aspartate residue is transiently phosphorylated dur- doing so (14). This may also have a bearing on propensity to apo- ing the transport cycle. The Wilson ATPase has 6 copper-binding ptosis in WD. LIVER AND COPPER 853S

WILSON DISEASE outside inside WD is a disorder of hepatic copper disposition, first described hormones in 1912. It is due to mutations in the gene ATP7B. Because the Wilson ATPase has 2 main intracellular actions (promoting in- drugs corporation of copper into apoceruloplasmin and expediting ex- normal chemicals cretion of excess copper into bile), patients characteristically Liver damage mishandled have hepatic copper overload, with low serum concentrations of : fructose enzymatically active ceruloplasmin and thus proportionally low xenobiotics (aldolase B defic) serum concentrations of copper, except when there are high se- tyrosine (FAH rum concentrations of non–ceruloplasmin-bound copper. More deficiency) than 300 mutations in ATP7B have been identified. Most of them environmental toxins abnormal proteins: are low-abundance mutations, and most patients are compound α 1-antitrypsin heterozygotes. WD occurs worldwide; the average incidence is : Ȃ30 affected persons per million population. WD can lead to Fe, Cu Downloaded from https://academic.oup.com/ajcn/article/88/3/851S/4649166 by guest on 04 October 2021 liver disease, progressive neurologic disorder, or psychiatric ill- FIGURE 2. Wilson disease as a disorder of endogenous hepatotoxicity. ness. The hepatic presentation usually occurs at younger ages. As Copper is an essential and normal component of living organisms, but in yet, clear-cut genotype-phenotype patterns remain elusive. Dif- Wilson disease it is not handled properly; thus, it accumulates and becomes ferent mutations have differing effects on the structure or func- hepatotoxic. Other disorders can also act like endogenous hepatotoxicity tion of the Wilson ATPase; eg, some mutants fail to relocate disorders; this may involve a genetically disordered metabolic pathway that generates an overload of a toxic intermediate from an otherwise nontoxic intracellularly when copper concentration is elevated. WD is substrate (such as galactose or fructose) or a mutation in a protein that renders ␣ fatal if not treated, but with effective treatment, especially if it hepatotoxic ( 1-antitrypsin). FAH, fumarylacetoacetate ; defic, commenced early, the outlook for a normal healthy life is excel- deficiency. lent. Treatment is lifelong with either an oral chelating agent or salts; liver transplantation is reserved for patients who do not METALLOPROTEOMICS respond to medical treatment or present initially with Wilsonian Proteomics encompasses protein expression, including fulminant hepatic failure (15). structure-function relations, physiologically and in disease Liver damage in WD appears to involve oxidant stress. Copper states. An organism’s versatility and variability—reflecting its is a prooxidant because it can exist in 2 different valence states; developmental program as well as its cumulative responses to indeed, this is the basis of its metabolic utility. It participates in external stimuli or stressors—is indicated in protein expression, the Haber-Weiss reaction; its ability to generate reactive oxygen reflecting its genome but subject to additional posttranslational species has been shown in HepG2 cells (16). Apoptosis is prom- modifications. To analyze protein expression, we may limit it by inent in Wilsonian liver damage. Recent studies suggest that one restricting our examination to proteins of a single organ or cell mechanism leading to apoptosis is activation of acid sphingo- type or subcellular organelle, by examining only a physiologic myelinase and consequent production of ceramide, which can state or a disease, or by specifying a time point or developmental initiate apoptosis (17); increased plasma acid sphingomyelinase stage. We have modified standard proteomics strategies to ex- activity was proposed as a biomarker of WD. amine proteins interacting with a ; we call this “metallo- One of the striking features about WD is its clinical diversity proteomics” (19). In defining a metalloproteome, we seek to as liver disease. The pattern of the liver disease is highly variable: determine the set of proteins that have metal-binding capacity, by an acute illness resembling viral or autoimmune hepatitis, fatty virtue either of being or of having metal-binding liver, cirrhosis, or simply asymptomatic elevation of serum ami- sites. We are interested in the metalloproteome for copper, but notransferases. WD may present as fulminant hepatic failure equally a metalloproteome could be defined for any metal. with acute intravascular hemolysis and rapidly progress to renal We examined the copper metalloproteome in HepG2 cell ly- failure; this is apparently due to widespread hepatocellular apo- sates to determine more completely the proteins involved in ptosis in an already damaged liver. Hepatocellular carcinoma is hepatocellular handling of copper. Our initial strategy was to use uncommon. This clinical diversity is highly reminiscent of drug- a copper-loaded immobilized metal affinity column to capture induced hepatotoxicity. Drug-induced liver disease can resemble copper-binding proteins; thus, we identified 38 high-abundance almost any type of liver disease described, and notably a single proteins, in addition to some nonspecific proteins. Some of the drug can lead to more than one pattern of liver injury. Accord- proteins of interest were known to have copper-binding capabil- ingly, we propose that WD can be regarded as a prototype of ity, but others were not previously known to be able to bind endogenous hepatotoxicity (Figure 2). In WD amounts of cop- copper. We also examined copper depletion and found a novel per usually not toxic are mishandled and become toxic to the variation in posttranslational modification of elongation factor liver. A similar situation can exist with iron in genetic hemo- 1␣, depending on whether HepG2 cells were copper depleted chromatosis disorders, and other genetic-metabolic fit (20). A more focused examination of HepG2 and micro- into this scheme in that toxic intermediates are formed in the liver somes showed a broader spectrum of copper-binding proteins because of abnormal metabolic pathways. In fact WD may be an (21). We have shown that protein disulfide , a highly excellent paradigm for studying –asso- abundant classic chaperone protein of the endoplasmic reticu- ciated hepatotoxicity. The important operational implication of lum, is a copper-binding protein with the classic CXXC binding this concept of WD is that genomic and proteomic techniques motif. Its copper-binding properties were confirmed by exten- being used to develop biomarkers for drug hepatotoxicity may be sive biophysical analysis (22). Others have shown that it is up- relevant to WD (18). regulated in the liver of copper-challenged North Ronaldsay 854S ROBERTS AND SARKAR sheep (23). We have also examined expression of some of the of an enzymatic assay for the determination of serum ceruloplasmin. proteins we found in the copper metalloproteome in the toxic J Lab Clin Med 2004;144:294–301. 3. Petris MJ. The SLC31 (Ctr) copper transporter family. Pflugers Arch milk mouse, a model for WD. Noting that peroxiredoxin-1 was 2004;447:752–5. identified in the copper-metalloproteome, we found that the mi- 4. Field LS, Luk E, Culotta VC. Copper chaperones: personal escorts for tochondrial protein peroxiredoxin-3 was indeed up-regulated in metal ions. J Bioenerg Biomembr 2002;34:373–9. hepatic mitochondria as the liver disease progresses during the 5. Singleton C, Le Brun NE. Atx1-like chaperones and their cognate P-type first 6 mo of life. Similarly, the mitochondrial protein Trx-2 was ATPases: copper-binding and transfer. Biometals 2007;20:275–89. 6. Narindrasorasak S, Zhang X-F, Roberts E, Sarkar B. Comparative anal- also up-regulated (24). Although Trx-2 has the canonical CXXC ysis of metal binding characteristics of copper chaperone proteins, Atx1 primary sequence, we expect that preoxiredoxin-3 forms a “vir- and Atox1. Bioinorg Chem Appl 2004;2:105–23. tual” CXXC copper-binding domain through dimerization as 7. Horng YC, Leary SC, Cobine PA, et al. Human Sco1 and Sco2 function peroxiredoxin-1 does. Thus, a proteomics strategy can identify as copper-binding proteins. J Biol Chem 2005;280:34113–22. 8. Fatemi N, Sarkar B. Structural and functional insights of Wilson disease functional copper-binding proteins, in addition to those predicted copper-transporting ATPase. J Bioenerg Biomembr 2002;34:339–49. by their primary sequence. Quantitative assessment of the met- 9. Portmann R, Solioz M. Purification and functional reconstitution of the alloproteome can be attained by adding an isotope-coded affinity human Wilson copper ATPase, ATP7B. FEBS Lett 2005;579:3589–95. Downloaded from https://academic.oup.com/ajcn/article/88/3/851S/4649166 by guest on 04 October 2021 tag component to the method, followed by mass spectrometric 10. La Fontaine S, Mercer JF. Trafficking of the copper-ATPases, ATP7A analysis. and ATP7B: role in copper homeostasis. Arch Biochem Biophys 2007; 463:149–67. Other groups have taken some different method approaches. 11. Tao TY, Liu F, Klomp L, Wijmenga C, Gitlin JD. The copper toxicosis Protein-chip plus the surface-enhanced laser desorption ioniza- gene product Murr1 directly interacts with the . tion method was used to investigate copper-binding proteins in J Biol Chem 2003;278:41593–6. HepG2 cells that were incubated with either physiologic or ele- 12. Narindrasorasak S, Kulkarni P, Deschamps P, She YM, Sarkar B. Char- ␮ acterization and copper binding properties of human COMMD1 vated (100 mol/L) concentrations of copper. This method has (MURR1). Biochemistry 2007;46:3116–28. advantages relating to reproducibility and high throughput; how- 13. de Bie P, van de Sluis B, Klomp L, Wijmenga C. The many faces of the ever, lack of immediate identification of the proteins detected is copper metabolism protein MURR1/COMMD1. J Hered 2005;96:803– a drawback. Among the proteins found, various metallothioneins 11. and hCTR2 were identified (25). In a non-Wilsonian hereditary 14. Mufti AR, Burstein E, Csomos RA, et al. XIAP is a copper binding protein deregulated in Wilson’s disease and other copper toxicosis dis- copper toxicosis in sheep, a proteomic strategy showed impor- orders. Mol Cell 2006;21:775–85. tant changes in certain hepatic proteins with copper challenge in 15. Roberts EA, Schilsky ML. A practice guideline on Wilson disease. the copper-sensitive North Ronaldsay breed. Up-regulated pro- Hepatology 2003;37:1475–92. teins included NADPѿ-dependent isocitrate dehydrogenase, 16. Seth R, Yang S, Choi S, Sabean M, Roberts EA. In vitro assessment of copper-induced toxicity in the human hepatoma line, Hep G2. Toxicol In retinol-binding protein, and protein disulfide isomerase (23). Vitro 2004;18:50109. Combination of results relating to copper disposition in a variety 17. Lang PA, Schenck M, Nicolay JP, et al. Liver cell death and anemia in of experimental models or diseases or both may help to focus on Wilson disease involve acid sphingomyelinase and ceramide. Nat Med candidate biomarkers. profiling of copper- 2007;13:164–70. loaded HepG2 cells has shown similar changes to those identified 18. Gatzidou ET, Zira AN, Theocharis SE. Toxicogenomics: a pivotal piece in the puzzle of toxicological research. J Appl Toxicol 2007;27:302–9. by proteomics, also with relating to drug metabolism and 19. Kulkarni PP, She YM, Smith SD, Roberts EA, Sarkar B. Proteomics of immune response (26). Examination of the transcriptome in the metal transport and metal-associated diseases. Chemistry 2006;12: Atp7b knockout mouse has shown changes in lipid metabolism, 2410–22. metallothioneins, and various proteins controlling the cell cycle 20. She YM, Narindrasorasak S, Yang S, Spitale N, Roberts EA, Sarkar B. Identification of metal-binding proteins in human hepatoma lines by (27). immobilized metal affinity chromatography and mass spectrometry. In conclusion, our knowledge of how copper is handled in Mol Cell Proteomics 2003;2:1306–18. hepatocytes has progressed greatly because the identification of 21. Smith SD, She YM, Roberts EA, Sarkar B. Using immobilized metal the gene abnormal in WD. Metalloproteomics offers a strategy affinity chromatography, two-dimensional electrophoresis and mass for identifying other proteins involved in copper disposition and spectrometry to identify hepatocellular proteins with copper-binding ability. J Proteome Res 2004;3:834–40. can provide candidate biomarkers. Some of these biomarkers 22. Narindrasorasak S, Yao P, Sarkar B. Protein disulfide isomerase, a may be valuable for diagnosing WD, which is often difficult to multifunctional protein chaperone, shows copper-binding activity. Bio- diagnose by currently available clinical and genetic methods. chem Biophys Res Commun 2003;311:405–14. 23. Simpson DM, Beynon RJ, Robertson DH, Loughran MJ, Haywood S. We thank S Yang and Y-M She for expert assistance and thank Dr John Copper-associated liver disease: a proteomics study of copper challenge Gollan, University of Nebraska School of Medicine, for sharing the earliest in a sheep model. Proteomics 2004;4:524–36. draft of the hepatocyte cartoon. 24. Roberts EA, Robinson BH, Yang S. Mitochondrial structure and func- The author’s responsibilities were as follows—EAR: organized and wrote tion in the untreated Jackson toxic milk (tx-j) mouse, a model for Wilson the first draft, provided key concepts and the figures, and managed all revi- disease. Mol Genet Metab 2008;93:54–65. 25. Roelofsen H, Balgobind R, Vonk RJ. Proteomic analyzes of copper sions of the manuscript; BS: reviewed the entire manuscript in its original and metabolism in an in vitro model of Wilson disease using surface en- revised forms and clarified all technical points relating to the bioinorganic hanced laser desorption/ionization-time of flight-mass spectrometry. chemistry of copper. None of the authors had a personal or financial conflict J Cell Biochem 2004;93:732–40. of interest. 26. Muller P, van Bakel H, van de Sluis B, Holstege F, Wijmenga C, Klomp LW. Gene expression profiling of liver cells after copper overload in vivo and in vitro reveals new copper-regulated genes. 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