Copper (Cu) transporting ATPases represent a highly conserved subclass of P-type ATPases with critical roles in Cu export and metalloenzyme synthesis. Despite their important biological roles and association with a wide range of human diseases, no high-affinity small-molecule inhibitors have been described. Here, we identify MKV3 as a small molecule inhibitor of Cu-transporting P-type ATPases that targets a conserved Cu+ entry site to the translocation pathway. In silico docking against the Xenopus ATP7B structure revealed a highly conserved pocket suitable for pharmacological inhibition. MKV3 bound human ATP7A and ATP7B with nanomolar affinity, competed with N-terminal metal-binding domains for access to the Cu+ entry site, and selectively inhibited Escherichia coli CopA ATPase activity and Cu+ transport. Mechanistically, MKV3 blocked chaperone-mediated Cu+ delivery to the intramembranous CPC site of CopA that is essential for its transport function. We further identified a single charged P-domain residue that governed MKV3 affinity and potency across species. Functionally, MKV3 phenocopied the genetic loss of Cu+-ATPases in bacteria, fungi, plants, zebrafish, and mammals, impairing copper-dependent enzymes, transporter trafficking, and copper tolerance. These findings establish a conserved, druggable vulnerability in Cu+-ATPases and introduce MKV3 as a broadly active chemical tool to modulate copper homeostasis across biological kingdoms.

Abstract Copper (Cu) is an essential trace element that serves as a cofactor for enzymes involved in diverse metabolic pathways and processes across multiple subcellular compartments. Mutations that cause systemic or subcellular Cu deficiency result in fatal pediatric disorders, including Menkes disease and related mitochondrial disorders. Treatment of these disorders requires pharmacological agents that can transport Cu across membranes; however, the only approved drug, Cu histidinate, shows limited efficacy. Using genetic models of Menkes disease, we recently demonstrated that the Cu ionophore elesclomol (ES) efficiently restores the activity of mitochondrial and cytosolic cuproenzymes, but its ability to metalate cuproenzymes within secretory compartments is low. To overcome this limitation, we investigated Cu nanoparticles (Cu-NPs) as a facile means of delivering Cu to multiple subcellular compartments simultaneously. Cu-NPs were synthesized by chemical reduction to produce spherical nanoparticles with an average size of 80 nm. Biophysical characterization of Cu-NPs revealed zero-valent Cu and a neutral zeta potential, compatible with biological interactions and in vivo circulation. In cellular models of Cu deficiency, Cu-NPs were shown to internalize via clathrin-mediated endocytosis and restore the activity of cuproenzymes in mitochondrial, cytosolic, and secretory compartments. Importantly, biomarkers of Cu toxicity were not activated under these conditions, indicating a favorable therapeutic index. Overall, these findings highlight Cu-NPs as versatile Cu delivery agents with therapeutic potential for Cu deficiency disorders.

Copper (Cu) is an essential cofactor for cytochrome c oxidase (CcO), a mitochondrial respiratory chain enzyme that is metalated in the intermembrane space (IMS) primarily using Cu derived from the mitochondrial matrix pool. While Cu import into the matrix depends on the inner membrane carrier SLC25A3, the route by which matrix Cu is exported to the IMS for insertion into CcO has remained a major, unresolved step in intramitochondrial Cu trafficking. Here, we leveraged our recent discovery that the Cu ionophore elesclomol (ES) releases Cu directly into the mitochondrial matrix to show that SLC25A3 is required for exporting Cu to the IMS for CcO metalation. Loss of SLC25A3 decreases mitochondrial Cu content and CcO activity as expected. Strikingly, bypassing the loss of SLC25A3 with ES-mediated Cu delivery to the matrix fails to restore CcO function; rather, it drives toxic Cu retention and triggers cuproptosis, revealing that SLC25A3-facilitated Cu export is the limiting determinant of CcO metalation. Heterologous expression in Lactococcus lactis confirms that SLC25A3 can mediate Cu export. These results suggest that SLC25A3 is the long-sought mitochondrial Cu exporter with a dual role in enabling CcO metalation and gating susceptibility to cuproptosis.

Copper (Cu) transporting ATPases represent a highly conserved subclass of P-type ATPases with critical roles in Cu export and metalloenzyme synthesis. Despite their important biological roles and association with a wide range of human diseases, no high-affinity small-molecule inhibitors have been described. Here, we identify MKV3 as a first-in-class inhibitor of Cu-transporting P-type ATPases that targets a conserved Cu+ entry site to the translocation pathway. In silico docking against the Xenopus ATP7B structure revealed a highly conserved pocket suitable for pharmacological inhibition. MKV3 bound human ATP7A and ATP7B with nanomolar affinity, competed with N-terminal metal-binding domains for access to the Cu+ entry site, and selectively inhibited Escherichia coli CopA ATPase activity and Cu+ transport. Mechanistically, MKV3 blocked chaperone-mediated Cu+ delivery to the intramembranous CPC site of CopA that is essential for its transport function. We further identified a single charged P-domain residue that governed MKV3 affinity and potency across species. Functionally, MKV3 phenocopied the genetic loss of Cu+-ATPases in bacteria, fungi, plants, zebrafish, and mammals, impairing copper-dependent enzymes, transporter trafficking, and copper tolerance. These findings establish a conserved, druggable vulnerability in Cu+-ATPases and introduce MKV3 as a broadly active chemical tool to modulate copper homeostasis across biological kingdoms.

Purpose:Radiation therapy (RT) for head and neck cancer commonly causes fibrosis of the salivary gland (SG) and loss of secretory function. We previously demonstrated that transdermal injection of the copper chelator tetrathiomolybdate into irradiated submandibular glands reduced the early deposition of fibrillar collagen and preserved their integrity and function. Although these studies identified copper metabolism as a novel therapeutic target to control RT-induced damage to the SG, local delivery of copper chelators in human SG presents clinical hurdles including specialized personnel and equipment needed for administration together with the discomfort felt by patients upon receiving this treatment. To overcome these issues, the current study investigated whether systemic administration of tetrathiomolybdate (TTM) via drinking water could similarly reduce collagen deposition and preserve secretory function. Methods and Materials:Mice received TTM in drinking water (0.1 mg/mL) beginning 7 days before and continuing through 7 days after a single 15-Gy dose of RT to the neck. Systemic TTM exposure and copper bioavailability were assessed by measuring molybdenum and copper levels and serum ceruloplasmin activity. RT-induced SG injury was evaluated by collagen deposition, histopathology, epithelial integrity, and stimulated saliva secretion. Results:Systemically administered TTM reached the submandibular gland and reversibly reduced copper bioavailability, as demonstrated by increased molybdenum levels, reduced copper-to-molybdenum ratios, and suppression of serum ceruloplasmin activity. Moreover, TTM treatment attenuated RT-induced collagen deposition and epithelial damage and promoted recovery of ZO-1 expression. Finally, at 30 days after RT, stimulated saliva flow was significantly higher in TTM-treated irradiated mice than in irradiated mice receiving regular water. Conclusion:Systemic TTM administration reduces copper bioavailability, attenuates RT-induced collagen deposition and epithelial injury, and preserves SG secretory function. These findings support systemic copper chelation as a less invasive alternative to local TTM delivery for limiting RT-induced SG damage.

Copper is an essential but potentially toxic nutrient required for a variety of biological functions. Mammalian cells use a complex network of copper transporters and metallochaperones to maintain copper homeostasis. Previous work investigating the role of copper in various disease states has highlighted the importance of copper transporters and metallochaperones. However, questions remain about how copper distribution changes under dynamic conditions like tissue differentiation. We previously reported that the copper exporter ATP7A is required for skeletal myoblast differentiation and that its expression changes in a differentiation dependent manner. Here, we sought to further understand the ATP7A-mediated copper export pathway by examining ATOX1, the copper chaperone that delivers copper to ATP7A. To investigate the role of ATOX1 in a dynamic cellular context, we characterized its protein-protein interactions during myoblast differentiation using the proximity labeling protein APEX2 to biotinylate proteins near ATOX1. We discovered that the ATOX1 interactome undergoes dramatic changes as myoblasts differentiate. These dynamic interactions correlate with distinct phenotypes of ATOX1 deficiency in proliferating and differentiated cells. Together, our results highlight the dynamic interactome of ATOX1 and its contribution to myoblast differentiation.

Rare inherited diseases caused by mutations in the copper transporters SLC31A1 (CTR1) or ATP7A induce copper deficiency in the brain, causing seizures and neurodegeneration in infancy through poorly understood mechanisms. Here, we used multiple model systems to characterize the molecular mechanisms by which neuronal cells respond to copper deficiency. Targeted deletion of CTR1 in neuroblastoma cells produced copper deficiency that produced a metabolic shift favoring glycolysis over oxidative phosphorylation. Proteomic and transcriptomic analysis of CTR1 knockout (KO) cells revealed simultaneous up-regulation of mTORC1 and S6K signaling and reduced PERK signaling. Patterns of gene and protein expression and pharmacogenomics show increased activation of the mTORC1-S6K pathway as a prosurvival mechanism, ultimately resulting in increased protein synthesis. Spatial transcriptomic profiling of Atp7aflx/Y :: Vil1Cre/+ mice identified up-regulated protein synthesis machinery and mTORC1-S6K pathway genes in copper- deficient Purkinje neurons in the cerebellum. Genetic epistasis experiments in Drosophila demonstrated that copper deficiency dendritic phenotypes in class IV neurons are improved or rescued by increased S6k expression or 4E-BP1 (Thor) RNAi, while epidermis phenotypes are exacerbated by Akt, S6k, or raptor RNAi. Overall, we demonstrate that increased mTORC1-S6K pathway activation and protein synthesis is an adaptive mechanism by which neuronal cells respond to copper deficiency.

Radiation therapy is a first-line treatment for head and neck cancer; however, it typically leads to hyposalivation stemming from fibrosis of the salivary gland. Current strategies to restore glandular function are dependent on the presence of residual functional salivary gland tissue, a condition commonly not met in patients with extensive fibrotic coverage of the salivary gland resulting from radiation therapy. Fibrosis is defined by the pathological accumulation of connective tissue (i.e., extracellular matrix) and excessive deposition of crosslinked (fibrillar) collagen that can impact a range of tissues and given that collagen crosslinking is necessary for fibrosis formation, inhibiting this process is a reasonable focus for developing anti-fibrotic therapies. Collagen crosslinking is catalyzed by the lysyl oxidase family of secreted copper-dependent metalloenzymes, and since that copper is an essential cofactor in all lysyl oxidase family members, we tested whether localized delivery of a copper chelator into the submandibular gland of irradiated mice could suppress collagen deposition and preserve the structure and function of this organ. Our results demonstrate that transdermal injection of tetrathiomolybdate into salivary glands significantly reduced the early deposition of fibrillar collagen in irradiated mice and preserved the integrity and function of submandibular gland epithelial tissue. Together, these studies identify copper metabolism as a novel therapeutic target to control radiation induced damage to the salivary gland and the current findings further indicate the therapeutic potential of repurposing clinically approved copperchelators as neoadjuvant treatments for radiation therapy

Copper promotes tumor growth and metastasis through a variety of mechanisms, most notably as a cofactor within the lysyl oxidase (LOX) family of secreted cuproenzymes. Members of this family, which include LOX and LOX-like enzymes LOXL1-4, catalyze the copper-dependent crosslinking of collagens and elastin within the extracellular matrix (ECM). Elevated LOX expression is associated with higher incidence and worse prognosis in multiple cancers, including colorectal, breast, pancreatic, and head and neck. In this study, we demonstrated that elevated LOX expression correlates with decreased overall survival and shorter median time to first progression in patients with lung cancer. Previous studies have demonstrated that LOX secreted from tumors is critical for pre-metastatic niche formation by promoting ECM remodeling and the recruitment of immune cells and endothelial precursors. Here, we demonstrated that ablation of the LOX gene in Lewis lung carcinoma (LLC) cells diminishes tumor growth and metastasis compared to wild-type LLC cells in a syngeneic mouse model. Although the role of tumor-derived LOX in tumor formation and metastasis is well established, little is known regarding the possible contribution of LOX produced by the parenchymal tissue of metastatic organs. Thus, this report describes our findings that host-derived LOX produced by the lung contributes to the pulmonary metastasis of LLC cells in mice. The suppression of pulmonary lysyl oxidase expression reduces the metastatic potential of Lewis Lung Carcinoma cells in mice, revealing a previously unknown influence of LOX expression in the parenchymal tissue of metastatic target organs on the seeding of tumor cells.

Abstract Cu (Cu) is essential for several biochemical pathways due to its role as a catalytic cofactor or allosteric regulator of enzymes. Its import and distribution are tightly controlled by transporters and metallochaperones and Cu homeostasis is maintained by balancing Cu uptake and export. Genetic diseases are caused by impaired Cu transporters CTR1, ATP7A, or ATP7B but little is known about the regulatory mechanisms by which these proteins meet the fluctuating demands of Cu in specific tissues. Cu is required for differentiation of skeletal myoblasts to myotubes. Here, we demonstrate that ATP7A is needed for myotube formation and that its increased abundance during differentiation is mediated by stabilization of Atp7a mRNA via the 3′ untranslated region. Increased ATP7A levels during differentiation resulted in increased Cu delivery to lysyl oxidase, a secreted cuproenzyme that needed for myotube formation. These studies identify a previously unknown role for Cu in regulating muscle differentiation and have broad implications for understanding Cu-dependent differentiation in other tissues.

Mitochondria influence cellular function through both cell-autonomous and non-cell autonomous mechanisms, such as production of paracrine and endocrine factors. Here, we demonstrate that mitochondrial regulation of the secretome is more extensive than previously appreciated, as both genetic and pharmacological disruption of the electron transport chain caused upregulation of the Alzheimer's disease risk factor apolipoprotein E (APOE) and other secretome components. Indirect disruption of the electron transport chain by gene editing of SLC25A mitochondrial membrane transporters as well as direct genetic and pharmacological disruption of either complexes I, III, or the copper-containing complex IV of the electron transport chain elicited upregulation of APOE transcript, protein, and secretion, up to 49-fold. These APOE phenotypes were robustly expressed in diverse cell types and iPSC-derived human astrocytes as part of an inflammatory gene expression program. Moreover, age- and genotype-dependent decline in brain levels of respiratory complex I preceded an increase in APOE in the 5xFAD mouse model. We propose that mitochondria act as novel upstream regulators of APOE-dependent cellular processes in health and disease.

Recent studies have uncovered the therapeutic potential of elesclomol (ES), a copper-ionophore, for copper deficiency disorders. However, we currently do not understand the mechanism by which copper brought into cells as ES-Cu(II) is released and delivered to cuproenzymes present in different subcellular compartments. Here, we have utilized a combination of genetic, biochemical, and cell-biological approaches to demonstrate that intracellular release of copper from ES occurs inside and outside of mitochondria. The mitochondrial matrix reductase, FDX1, catalyzes the reduction of ES-Cu(II) to Cu(I), releasing it into mitochondria where it is bioavailable for the metalation of mitochondrial cuproenzyme- cytochrome c oxidase. Consistently, ES fails to rescue cytochrome c oxidase abundance and activity in copper-deficient cells lacking FDX1. In the absence of FDX1, the ES-dependent increase in cellular copper is attenuated but not abolished. Thus, ES-mediated copper delivery to nonmitochondrial cuproproteins continues even in the absence of FDX1, suggesting alternate mechanism(s) of copper release. Importantly, we demonstrate that this mechanism of copper transport by ES is distinct from other clinically used copper-transporting drugs. Our study uncovers a unique mode of intracellular copper delivery by ES and may further aid in repurposing this anticancer drug for copper deficiency disorders.

Tyrosinase (TYR) and tyrosinase-related proteins 1 and 2 (TYRP1 and TYRP2) are essential for pigmentation. They are generally classified as type-3 copper proteins, with binuclear copper active sites. Although there is experimental evidence for a copper cofactor in TYR, delivered via the copper transporter, ATP7A, the presence of copper in TYRP1 and TYRP2 has not been demonstrated. Here, we report that the expression and function of TYRP1 requires zinc, mediated by ZNT5-ZNT6 heterodimers (ZNT5-6) or ZNT7-ZNT7 homodimers (ZNT7). Loss of ZNT5-6 and ZNT7 function results in hypopigmentation in medaka fish and human melanoma cells, and is accompanied by immature melanosomes and reduced melanin content, as observed in TYRP1 dysfunction. The requirement of ZNT5-6 and ZNT7 for TYRP1 expression is conserved in human, mouse, and chicken orthologs. Our results provide novel insights into the pigmentation process and address questions regarding metalation in tyrosinase protein family.

PURPOSE. To develop a mouse model of human dry eye disease (DED) for investigation of sex differences in autoimmune-associated dry eye pathology.METHODS. Ocular surface disease was assessed by quantifying corneal epithelial damage with lissamine green stain in the NOD.H-2h4,IFN gamma -/-,CD28-/-(NOD.H-2h4 DKO) mouse model of Sjogren's syndrome (SS). Lacrimal gland function was assessed by tear volume quantification with phenol red thread and lacrimal gland inflammation (i.e., dacryoad-enitis) was assessed by quantification of immune cell foci, flow cytometric analysis of immune cell composition, and expression of proinflammatory markers.RESULTS. The NOD.H-2h4 DKO mouse model of SS exhibits greater age-dependent increases in corneal damage than in NOD.H-2h4 parental mice and demonstrates an earlier disease onset in females compared to males. The severity of ocular surface disease correlates with loss of goblet cell density, increased conjunctivitis, and dacryoadenitis that is more pronounced in NOD.H-2h4 DKO than NOD.H-2h4 mice. B cells dominate lacrimal infiltrates in 16-week-old NOD.H-2h4 and NOD.H-2h4 DKO mice, but T helper cells and macrophages are also present. Lacrimal gland expression of proinflammatory genes, including the P2X7 and P2Y2 purinergic receptors, is greater in NOD.H-2h4 DKO than NOD.H-2h4 mice and correlates with dacryoadenitis.CONCLUSIONS. Our results demonstrate for the first time that autoimmune dry eye disease occurs in both sexes of NOD.H-2h4 DKO and NOD.H-2h4 mice, with earlier onset in female NOD.H-2h4 DKO mice when compared to males of the same strain. This study demonstrates that both NOD.H-2h4 and NOD.H-2h4 DKO mice are novel models that closely resemble SS-related and sex-dependent DED.

Copper is an essential nutrient whose redox properties make it both beneficial and toxic to the cell. Recent progress in studying transition metal signalling has forged new links between researchers of different disciplines that can help translate basic research in the chemistry and biology of copper into clinical therapies and diagnostics to exploit copper-dependent disease vulnerabilities. This concept is particularly relevant in cancer, as tumour growth and metastasis have a heightened requirement for this metal nutrient. Indeed, the traditional view of copper as solely an active site metabolic cofactor has been challenged by emerging evidence that copper is also a dynamic signalling metal and metalloallosteric regulator, such as for copper-dependent phosphodiesterase 3B (PDE3B) in lipolysis, mitogen-activated protein kinase kinase 1 (MEK1) and MEK2 in cell growth and proliferation and the kinases ULK1 and ULK2 in autophagy. In this Perspective, we summarize our current understanding of the connection between copper and cancer and explore how challenges in the field could be addressed by using the framework of cuproplasia, which is defined as regulated copper-dependent cell proliferation and is a representative example of a broad range of metalloplasias. Cuproplasia is linked to a diverse array of cellular processes, including mitochondrial respiration, antioxidant defence, redox signalling, kinase signalling, autophagy and protein quality control. Identifying and characterizing new modes of copper-dependent signalling offers translational opportunities that leverage disease vulnerabilities to this metal nutrient.

The last 25 years have witnessed tremendous progress in identifying and characterizing proteins that regulate the uptake, intracellular trafficking and export of copper. Although dietary copper is required in trace amounts, sufficient quantities of this metal are needed to sustain growth and development in humans and other mammals. However, copper is also a rate-limiting nutrient for the growth and proliferation of cancer cells. Oral copper chelators taken with food have been shown to confer anti-neoplastic and anti-metastatic benefits in animals and humans. Recent studies have begun to identify specific roles for copper in pathways of oncogenic signaling and resistance to anti-neoplastic drugs. Here, we review the general mechanisms of cellular copper homeostasis and discuss roles of copper in cancer progression, highlighting metabolic vulnerabilities that may be targetable in the development of anticancer therapies.

Copper is an essential metal for virtually all organisms, yet little is known about the extracellular sources of this micronutrient. In serum, the most abundant extracellular Cu-binding molecule is the multi‑copper oxidase ceruloplasmin (Cp). Cp levels increase during infection and inflammation, and pathogens can be exposed to high Cp at sites of infection. It is not known whether Cp might serve as a Cu source for microbial pathogens and we tested this using the opportunistic fungal pathogen Candida albicans. We find that C. albicans can use whole serum as a Cu source and that this Cu is sensed by the transcription factor protein Mac1. Mac1 activates expression of Mn-SOD3 superoxide dismutase and represses Cu/Zn-SOD1 during Cu starvation and both responses are regulated by serum Cu. We also show that purified human Cp can act as a sole source of Cu for the fungus and likewise modulates the Mac1 Cu stress response. To investigate whether Cp is a Cu source in serum, we compared the ability of C. albicans to use serum from wild type versus Cp-/- mutant mice. We find that serum lacking Cp is deficient in its ability to trigger the Mac1 Cu response. C. albicans did accumulate Cu from Cp-/- serum, but this Cu was not efficiently sensed by Mac1. We conclude that Cp and non-Cp Cu sources are not equivalent and are handled differently by the fungal cell. Overall, these studies are the first to show that Cp is a preferred source of Cu for a pathogen.

The last 25 years have witnessed tremendous progress in identifying and characterizing proteins that regulate the uptake, intracellular trafficking and export of copper. Although dietary copper is required in trace amounts, sufficient quantities of this metal are needed to sustain growth and development in humans and other mammals. However, copper is also a rate-limiting nutrient for the growth and proliferation of cancer cells. Oral copper chelators taken with food have been shown to confer anti-neoplastic and anti-metastatic benefits in animals and humans. Recent studies have begun to identify specific roles for copper in pathways of oncogenic signaling and resistance to anti-neoplastic drugs. Here, we review the general mechanisms of cellular copper homeostasis and discuss roles of copper in cancer progression, highlighting metabolic vulnerabilities that may be targetable in the development of anticancer therapies.

New strategies for tissue engineering have great potential for restoring and revitalizing impaired tissues and organs, including the use of smart hydrogels that can be modified to enhance organization and functionality of the salivary glands. For instance, monomers of laminin-111 peptides chemically conjugated to fibrin hydrogel (L1pM-FH) promote cell cluster formation in vitro and salivary gland regeneration in vivo when compared with fibrin hydrogel (FH) alone; however, L1pM-FH produce only weak expression of acinar differentiation markers in vivo (e.g., aquaporin-5 and transmembrane protein 16). Since previous studies demonstrated that a greater impact can be achieved when trimeric forms were used as compared with monomeric or dimeric forms, we investigated the extent to which trimers of laminin-111 chemically conjugated to FH (L1pT-FH) can increase the expression of acinar differentiation markers and elevate saliva secretion. In vitro studies using Par-C10 acinar cells demonstrated that when compared with L1pM-FH, L1pT-FH induced similar levels of acinar-like cell clustering, polarization, lumen formation, and calcium signaling. To assess the performance of the trimeric complex in vivo, we compared the ability of L1pM-FH and L1pT-FH to increase acinar differentiation markers and restore saliva flow rate in a salivary gland wound model of C57BL/6 mice. Our results show that L1pT-FH applied to wounded mice significantly improved the expression of the acinar differentiation markers and saliva secretion when compared with the monomeric form. Together, these positive effects of L1pT-FH warrant its future testing in additional models of hyposalivation with the ultimate goal of applying this technology in humans.