The average adult male contains only ~100 mg of Cu .

1. The average adult male contains only ~100 mg of Cu. • Cu1+ prefers sulfur donor ligands (cysteine or methionine), whereas Cu2+ prefers nitrogen donors (histidine) or oxygen donors (glutamate or aspartate). • Although more than 90% of the serum Cu is bound by ceruloplasmin, mice lacking ceruloplasmin have no apparent defect in Cu absorption or distribution. • alterations in Cu balance have been linked, but not causally associated, to changes in senile plaque deposition in Alzheimer’s disease. • Cu binding to α-synuclein has been linked to the aggregation of this protein, which is observed in Parkinson’s disease. • N-terminal octapeptide repeats in the prion protein (PrP) serves as high-affinity Cu2+ binding sites that may alter the structure of PrP to a form associated with prion disease

2. Examples of Cu binding and Cu homeostasis proteins Amyloid precursor protein (APP) Protein involved in neuronal development and potentially Cu metabolism; (Alzheimer’s disease) Atox1 Metallochaperone that delivers Cu to ATP7A and ATP7B Cu1+ transporters ATP7A Cu1+-transporting P-type ATPase expressed in all tissues except liver ATP7B Cu1+-transporting P-type ATPase expressed primarily in the liver Carbon monoxide dehydrogenase to acetyl-CoA synthase Moorella thermoacetica; reduces CO2 to CO and assembly of acetyl-CoA Ceruloplasmin Serum ferroxidase that functions in Fe3+ loading onto transferrin Coagulation factors V and VIII Homologous pro-coagulants present on the surface of platelets (blood coagulation). CCS Metallochaperone that delivers Cu to Cu/Zn SOD CopZArchaeoglobus fulgidus [2Fe-2S] and Zn2+-containing Cu chaperone Cox17 Metallochaperone that transfers Cu to Sco1 and Cox11 for cytochrome oxidase Cu loading in mitochondria Ctr1 High-affinity Cu1+ transporter involved in cellular Cu uptake Cu/Zn SOD (SOD1): Antioxidant enzyme Cytochrome c oxidase: mitochondrial respiratory chain. Dopamine â-hydroxylase (DBH) Oxygenase, converts dopamine to norepinephrine

3. Ethylene receptor (ETR1): Member of a plant receptor family that uses a Cu cofactor for ethylene binding and signaling Hemocyanin: Oxygen transport in invertebrates Hephaestin: Transmembrane multi-Cu ferroxidase; iron efflux from enterocytes and macrophages Glucose oxidase: Pentose phosphate pathway oxidoreductase that catalyzes the oxidation of D-glucose into D-glucono-1, 5-lactone and hydrogen peroxide Laccase: Phenol oxidase involved in melanin production Lysyl oxidase: Catalyzes formation of aldehydes from lysine in collagen and elastin precursors for connective tissue maturation Metallothionein: Cysteine-rich small-molecular-weight metal-binding and detoxification protein Peptidylglycine-α-amidating mono-oxygenase (PAM): conversion of peptidylglycine substrates into α-amidated products; neuropeptide maturation Prion protein (PrP) Protein whose function is unclear but binds Cu via the N-terminal octapeptide repeats Steap proteins/Fre1/Fre2 Family of metalloreductases for Fe3+ and Cu2+ reduction Tyrosinase Monophenol mono-oxygenase; melanin synthesis XIAP Inhibitor of apoptosis through binding and catalytic inhibition of several caspases

5. Defects in Cu homeostasis are directly responsible for human diseases. Mutations in the ATP7A or ATP7B genes, encoding P-type Cu1+-transporting ATPase pumps expressed in extrahepatic tissues or in the liver, respectively, cause Menkes and Wilson’s diseases. Menkes disease (ATP7A) is an X-linked lethal disorder of intestinal Cu hyperaccumulation with severe Cu deficiency in peripheral tissues and concomitant deficits in Cu-dependent enzymes that lead to the clinical hallmarks of the disease. Wilson’s disease (ATP7B) is an autosomal recessive disease characterized by striking hepatic and neuronal Cu overload, hepatotoxicity, neuropsychological and other defects that require chronic therapy to enhance Cu excretion or reduce Cu absorption.

6. Ligandi??? Assorbimento del rame

7. Struttura di Ctr-1 (?) Facilita la cattura e l’endocitosi di Crt-1 a basse concentrazioni di rame

8. Cu import, intracellular routing and biliary secretion in the liver. Cu1+ is imported at the plasma membrane by Ctr1 and routed to Cu/Zn SOD1 by CCS (Cu chaperone for SOD), to the ATP7B (Cu1+-transporting P-type ATPase) at the secretory apparatus, and at the bile canalicular membrane by Atox1. Cu movement to the mitochondria may involve one or more as-yet uncharacterized intracellular ligands (denoted L) (COX-17 ?).

9. In the intermembrane space (IMS), Cu1+ is bound by Cox17 and delivered either to Sco1, which transfers the Cu to the Cox2 subunit, or to Cox11, which delivers Cu to the Cox1 subunit of cytochrome oxidase (CCO). A novel Cu ligand (L) has been isolated from both yeast and mouse liver and may function in mitochondrial Cu delivery COX17

10. (Cu chaperone) Cu1+ delivery pathway to ATP7B that pumps excess Cu1+ into the bile for excretion. Mutation of ATP7B causes Wilson’s disease, which is characterized by a Cu overload in the liver, neurons and other tissues. A series of ligand exchange reactions between Atox1 and the metal binding motifs of ATP7B (CxxC) results in the movement of Cu1+ from Atox1 to ATP7B, followed by transport across the hepatocyte membrane into the bile. Similar Atox1 and ATP7A for Cu1+ movement across the basolateral membrane of IECs, and Atox1 and ATP7A or ATP7B for Cu1+ incorporation into secreted Cu-dependent proteins.

11. The CCS domain II structurally resembles Cu/Zn SOD and facilitates many of the CCS-SOD1 interactions. Domains I and III have CX2C and CXC Cu1+ binding domains. Heterodimerization between a monomer of Cu1+-loaded CCS and Zn-loaded SOD1 results in the transfer of Cu onto SOD1 in a process that involves oxygen-dependent intrasubunit disulfide bond formation in SOD1 catalyzed by Cu-CCS. The transient accumulation of Cu1+-loaded CCS, when all SOD1 is Cu-metallated, could expose a lysine residue for ubiquitination.

12. Human pathogen C. neoformans. Extracellular Cu, most likely reduced by the Fre family of metalloreductases, is transported into the cell as Cu1+ through the high-affinity Ctr4 transporter. The cytosolic Cu chaperone Atx1 delivers Cu to the secretory compartment via the P-type ATPase Ccc2, where it becomes incorporated into Cu-dependent enzymes including the multi-Cu ferroxidase Fet3 that, together with the Ftr1 permease, mediate high affinity iron uptake, and into laccase. Ctr4 Fre Ftr1 Fre Cuf1 The Cuf1 transcription factor regulates the expression of cellular Cu homeostasis genes.

13. Many questions remain to be answered with respect to Cu homeostasis. How do Cu chaperones obtain Cu, and is this process prioritized depending on the physiological state of cells? Can new Cu-dependent proteins be identified, and will they add to the diversity of functions already known for Cu in biology? How is the import of Cu coordinated with its mobilization from intracellular stores? What is the chemical nature of Cu ligands that ferry Cu around cells and throughout the bloodstream or across the blood brain barrier? How do cells sense when a protein is Cu-loaded and appropriately regulate protein steady state levels or subcellular trafficking? Can sensitive and high-resolution techniques be developed for the subcellular localization, quantitation and speciation of Cu? Can Cu ligands and delivery mechanisms be developed to treat diseases such as Menkes disease? Is Cu causally related to neurodegenerative diseases such as Alzheimer’s, Parkinson’s and prion disease, and if so, how can manipulation of bioavailable Cu alter the outcome of these diseases? Further investigations of the fundamental biochemistry, genetics, cell biology, physiology and chemistry of Cu will be critical to answering these questions that explore the roles of Cu in health and disease.