Copper is essential throughout the body, and slight variations from an acceptable range (between approximately 50 and 80 mg) can have catastrophic effects. With a range so small, it is clear that copper is a tightly regulated molecule in the body with very specific functions. Copper deficiency and copper excess are both implicated in serious conditions that can have deadly consequences. It is for this reason that modulating copper concentrations throughout the body is so vastly important for human survival. Due to its presence in the mitochondria, copper is typically concentrated in organs and tissues that have high metabolic demands, such as the kidney, liver, and brain [54]. When There is an excessive amount of copper in the liver, copper can, under normal conditions, be redistributed for excretion through the action of several intracellular proteins. In the liver specifically, there exists a biliary excretion pathway that redirects cellular copper to bile [12].
Copper cannot be synthesized in the body, and is instead received primarily through food. Organ meat, such as animal liver, is very rich in copper. This is due to the high metabolic demands of cells in the liver, and the very involved role of the liver in copper metabolism. Beef liver is especially high in copper, with concentrations reported as high as 157 mg/kg. Some nuts and seeds have also been reported to have high copper content, such as pecans and sunflower seeds [29]. The recommended daily copper intake per individual is 0.9 mg per day, though actual copper intake may range from 1-10 mg per day due to modern dietary habits [41, 42]. In some places, drinking water is a significant source of copper [50].
When foods containing copper are consumed, copper is absorbed in the small intestine by enterocytes, a process largely mediated by copper transporter CTR1 [19, 31, 41]. After it is absorbed, copper is transported to the liver through the portal vein, loosely bound to serum proteins such as albumin [7, 19]. The liver serves as a center for copper metabolism; the majority of absorbed copper must travel through the liver before it is excreted or circulated through the rest of the body, as the major copper-carrying protein in the blood, ceruloplasmin, is expressed primarily in hepatocytes. Most dietary copper is excreted and quickly removed from circulation [42].
Copper is essential for many functions in the human body. It has been implicated as a major player in redox reactions as an electron transfer intermediate. With two common oxidation states, copper serves as a cofactor for various oxidase and reductase enzymes, many of which are located in the mitochondria and are often discussed in the context of oxidative damage.
One such enzyme that uses copper is superoxide dismutase, which requires copper and cannot function with other metals. Superoxide dismutase is a powerful antioxidant enzyme that has a protective function against oxygen radicals in the cell. It is very important in limiting oxidative damage within the cell, as it catalyzes the reaction that converts a superoxide radical into molecular oxygen and hydrogen peroxide.
Thus, copper is an essential component of many metalloenzymes that drive functions including respiration and protection from free radicals. It has even been shown to have a key role in connective tissue synthesis, melanin synthesis, and in gene regulation and expression [54]. Other proteins have been shown to be copper-carrying proteins, such as ceruloplasmin and albumin as well as some amino acids [7].
Copper may have a role in apoptotic pathways, where increased concentrations of copper reduce the activity of XIAP, a pro-survival protein that inhibits caspase-3. Through direct binding of copper, XIAP undergoes a conformational change which significantly reduces its effectiveness in inhibiting caspase-3 [33]. So, at high copper concentrations, there is a remarkable decrease in caspase-3 inhibition accompanied by deregulation of apoptotic pathways. In this way, copper is involved in regulating cell death.
ATP7B is a major player in copper metabolism, and is the focus of an inborn error of copper metabolism, Wilson Disease. ATP7B is most expressed in the liver, kidney, and placenta, and various splice isoforms have been identified in the brain as well as in the kidney and placenta. In the healthy body, this copper transporter is responsible for directing cellular copper throughout the cell and for copper excretion through bile from hepatocytes. It is localized to the trans-golgi network (TGN), and interacts with copper metallochaperones to bind and release copper.
When there is an excess of copper, ATP7B is released from the TGN through vesicular transport and mediates the excretion of excess cytoplasmic copper via bile. Lysosomes serve as an intermediate for copper excretion, and the copper is transported to the bile canaliculi of the hepatocyte. As a result, the copper can be removed and copper does not accumulate in hepatocytes. ATP7B receives copper from copper metallochaperones, namely intracellular copper carriers. Serum copper is transported into the cell through the function of membrane copper transporter CTR1. Once in the cytoplasm, copper is bound to metallochaperone ATOX1, which has been shown to interact directly with the metal-binding domains of ATP7B [16, 31]. It is through this interaction that ATP7B binds copper, and initiates its own transporter functions.
A cartoon from Wu et al. illustrating copper transport into, within, and out of hepatocytes. Copper initially enters hepatocytes through hCTR1, a transmembrane protein that transports copper into the cytoplasm of cells. Copper is bound to ATOX1, a copper metallochaperone that carries copper to ATP7B in the trans-golgi network (TGN). ATP7B pumps copper across the TGN membrane and into the inside, a space called the lumen. It is there that copper is bound to ceruloplasmin, which safely carries through the blood. When copper builds up in the cytoplasm, ATP7B is able to leave the TGN via vesicular trafficking, and eventually interacts with lysosomes to excrete the toxic cytoplasmic copper into the bile duct. The mechanism by which ATP7B interacts with the lysosome is unknown, though recent research indicates that COMMD1 may play a role [59].
The ATP7B gene is approximately 80 kb, with 21 exons that encode a 7.5 kb transcript. This transcript yields a 1465 amino acid P1B ATPase, a subset of P-type ATPases [53]. The protein is composed of six metal-binding domains, eight transmembrane helices, and phosphorylation (P-domain), phosphatase (A-domain), and ATP-binding domains (N-domain).
There are six metal binding sites located in the cytosolic N-terminus of the protein, each composed of approximately 70 residues, and contain the conserved GM(T/H)CxSCxxxIE motif that binds Cu(I) through the action of cysteine residues [61]. The positive role of sulfur in copper binding has implications for chelating compounds, which will be further discussed in page 4 in the context of heavy metal toxicity treatments [11, 48]. The first 4 MBDs are believed to be regulatory, and the MBD 5 and 6, which are most proximal to the membrane, are essential to ATP7B function [32, 61].
A cartoon from Bie et al. that shows the general copper translocation cycle of copper transporter ATPases. Copper binds to the MBD of the protein and ATP binds to the N-domain. It is believed in ATP7B that the copper binding to the MBD must be moved within the protein before it is transported, as there is a general lack of essential function in MBD 1-4. The P-domain is autophosphorylated upon hydrolysis of ATP, releasing ADP and inducing a conformational change that allows for the shuttling of copper across the TGN membrane. The A-domain removes the phosphate from the P-domain, resetting the cycle for more copper to be transported into the TGN lumen [4].
The N-domain is exceptionally important for ATP7B function because it is responsible for binding ATP. The domain is characterized by a highly conserved SEHPL domain, wherein the histidine is an invariant residue with connections to Wilson Disease pathogenesis (See Page 3) [10]. The P-domain is essential for auto-phosphorylation of an aspartate residue in the conserved DKTGT motif. Phosphorylation of this residue requires the binding of ATP and inter-domain interaction with the N-domain. ATP hydrolysis and resultant phosphorylation induces a conformational change in the enzyme that drives copper transport across the membrane. The A-domain exhibits phosphatase activity by dephosphorylating the P-domain after copper transport. This function allows for a reset of the transportation cycle and a return to the original ATP7B conformation [4]. The full reaction requires ATP, H2O, and Cu(I), and the products of the reaction are ADP, phosphate, and Cu(I) (translocated across the membrane and into the TGN lumen). Once transported across the TGN membrane, copper is able to be incorporated into ceruloplasmin and other proteins that require copper to function.
Hi Alex – great job on this page. We talk about Wilson’s disease in medical school, but surprisingly spend WAY less time talking about the physiologic functions of copper in a non-disease state!
You mentioned the CTR1 channel as being used for cellular uptake, both from the serum and from the gut lumen via enterocytes. I’m interested if you had come across any processes that regulate CTR1 function (i.e. expression or membrane translocation, or something)? Does the body control the rate at which it absorbs copper from food in any way?
Hi Elliott,
Thank you for the comment. When I was doing my research for this project, I had not come across any processes that regulate CTR1 function, as the more specific roles of this transporter are not fully understood. However, I did read that as copper consumption increases, absorption decreases, which makes sense physiologically as it limits the accumulation of copper. So, it is possible that a high extracellular copper concentration may regulate the function of CTR1, but that is just my educated opinion and I have not seen any published data about it. The fact that copper absorption decreases as copper consumption increases does tell us that the body has mechanisms in place to control that rate at which we absorb copper, but unfortunately the majority of these processes are unknown. In responding to your comment, however, I did come across an interesting paper that explored interactions between CTR1 and VEGFR2, where VEGF promotes the binding of CTR1 and VEGFR2 and promotes neovascularization in an ROS-dependent manner (https://www.ahajournals.org/doi/abs/10.1161/circ.140.suppl_1.10910). This indicates that CTR1 may be involved in other pathways, including neovascularization and wound healing. So, in this way it seems that CTR1 function can potentially be regulated by VEGF and by ROS.
Very interesting article Alex! The cellular responsibilities of copper extend much farther than my lil old brain ever could have imagined.
You said that the liver functions as the primary conduit through which copper metabolises into the body. For those with liver disease, are there complications that stem specifically from the inhibition of this function, or is copper versatile enough to find other ways into our cells?