Cuproptosis: Cellular and molecular mechanisms underlying copper-induced cell death

Tsvetkov et al., 2022Tsvetkov P. Coy S. Petrova B. Dreishpoon M. Verma A. Abdusamad M. Rossen J. Joesch-Cohen L. Humeidi R. Spangler R.D. et al.Copper induces cell death by targeting lipoylated TCA cycle proteins.Science. 2022; 375: 1254-1261https://doi.org/10.1126/science.abf0529Crossref PubMed Scopus (934) Google Scholar discovered a new form of cell death triggered by targeted accumulation of Cu in mitochondria that drives lipoylated TCA cycle enzyme aggregation via direct Cu binding. Tsvetkov et al., 2022Tsvetkov P. Coy S. Petrova B. Dreishpoon M. Verma A. Abdusamad M. Rossen J. Joesch-Cohen L. Humeidi R. Spangler R.D. et al.Copper induces cell death by targeting lipoylated TCA cycle proteins.Science. 2022; 375: 1254-1261https://doi.org/10.1126/science.abf0529Crossref PubMed Scopus (934) Google Scholar discovered a new form of cell death triggered by targeted accumulation of Cu in mitochondria that drives lipoylated TCA cycle enzyme aggregation via direct Cu binding. The acquisition, distribution, and elimination of metal micronutrients, like iron (Fe) and copper (Cu) is essential for life (Andrews, 2000Andrews N.C. Iron metabolism: iron deficiency and iron overload.Annu. Rev. Genomics Hum. Genet. 2000; 1: 75-98https://doi.org/10.1146/annurev.genom.1.1.75Crossref PubMed Scopus (147) Google Scholar; Tsang et al., 2021Tsang T. Davis C.I. Brady D.C. Copper biology.Curr. Biol. 2021; 31: R421-R427https://doi.org/10.1016/j.cub.2021.03.054Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Redox active Cu functions as a catalytic or structural cofactor necessary for a diverse array of biological processes, including mitochondrial respiration, antioxidant defense, and bio-compound synthesis. Despite the physiologic requirement for Cu, it can be cytotoxic. Excess Cu buildup triggers disruption of iron-sulfur cofactors and stimulates damaging reactive oxygen species generated by Cu-driven Fenton reactions. Unlike the molecular mechanisms and intracellular consequences downstream of Fe accumulation that ultimately induce ferroptotic cell death (Dixon et al., 2012Dixon S.J. Lemberg K.M. Lamprecht M.R. Skouta R. Zaitsev E.M. Gleason C.E. Patel D.N. Bauer A.J. Cantley A.M. Yang W.S. et al.Ferroptosis: an iron-dependent form of nonapoptotic cell death.Cell. 2012; 149: 1060-1072https://doi.org/10.1016/j.cell.2012.03.042Abstract Full Text Full Text PDF PubMed Scopus (6966) Google Scholar), the known cellular machinery and enzymatic targets for Cu cannot fully explain the response of cells to Cu toxicity. Tsvetkov et al. demonstrated in Science an unexpected mechanism by which cell death is triggered by targeting Cu to mitochondria: cuproptosis (Tsvetkov et al., 2022Tsvetkov P. Coy S. Petrova B. Dreishpoon M. Verma A. Abdusamad M. Rossen J. Joesch-Cohen L. Humeidi R. Spangler R.D. et al.Copper induces cell death by targeting lipoylated TCA cycle proteins.Science. 2022; 375: 1254-1261https://doi.org/10.1126/science.abf0529Crossref PubMed Scopus (934) Google Scholar). This discovery raises exciting questions about mitochondrial Cu homeostasis in normal and altered physiologies and its exploitation as a cancer therapeutic. Tsvetkov et al. showed elesclomol mediated toxicity in cancer cells was linked to ferrodoxin-1 (FDX1) levels, elevated mitochondrial respiration rate, and was dependent on Cu availability (Tsvetkov et al., 2019Tsvetkov P. Detappe A. Cai K. Keys H.R. Brune Z. Ying W. Thiru P. Reidy M. Kugener G. Rossen J. et al.Mitochondrial metabolism promotes adaptation to proteotoxic stress.Nat. Chem. Biol. 2019; 15: 681-689https://doi.org/10.1038/s41589-019-0291-9Crossref PubMed Scopus (186) Google Scholar, Tsvetkov et al., 2022Tsvetkov P. Coy S. Petrova B. Dreishpoon M. Verma A. Abdusamad M. Rossen J. Joesch-Cohen L. Humeidi R. Spangler R.D. et al.Copper induces cell death by targeting lipoylated TCA cycle proteins.Science. 2022; 375: 1254-1261https://doi.org/10.1126/science.abf0529Crossref PubMed Scopus (934) Google Scholar). This form of Cu-dependent, mitochondrially induced cell death was distinct from apoptosis, ferroptosis, or necroptosis, but the mechanism of sensitivity remained elusive. FDX1 functions as a direct binder of elesclomol that reduces cupric ions to cuprous ions releasing them within the mitochondrial matrix (Tsvetkov et al., 2019Tsvetkov P. Detappe A. Cai K. Keys H.R. Brune Z. Ying W. Thiru P. Reidy M. Kugener G. Rossen J. et al.Mitochondrial metabolism promotes adaptation to proteotoxic stress.Nat. Chem. Biol. 2019; 15: 681-689https://doi.org/10.1038/s41589-019-0291-9Crossref PubMed Scopus (186) Google Scholar). Whole-genome and metabolism-focused CRISPR screens revealed FDX1 and the metabolic enzymes necessary for lipoic acid synthesis and known protein targets of lipoylation DLAT, PDHA1, and PDHB within the pyruvate dehydrogenase (PDH) complex as mediators of Cu ionophore toxicity. Lipoylation is a posttranslational modification where lipoic acid is attached to proteins that are involved in regulation of metabolic flux in mitochondria. Inhibitors of the mitochondrial pyruvate carrier reduced Cu-induced cell death, supporting the importance of the PDH complex to cuproptosis and cementing genetic connections between Cu-induced cell death, FDX1, TCA cycle enzyme lipoylation, and the synthesis of lipoic acid. Using genetic co-dependency mapping, Tsvetkov et al. discovered that cancer cells dependent on FDX1 also require components of the lipoic acid pathway for viability. FDX1 donates an electron to lipoate synthase and as a result, FDX1 loss promotes accumulation and depletion of TCA cycle intermediates that correspond to specific reductions in metabolic enzyme lipoylation. Thus, tying together multiple observations regarding susceptibility to elesclomol. The mechanism as to why the aggregation of these proteins induces cuproptosis is unresolved. The discovery of cuproptosis opens new areas of investigation into mitochondrial Cu. Under physiological conditions, mitochondria have a store of Cu in the matrix that is imported via the mitochondrial carrier family protein, SLC25A3, for the assembly of the cytochrome c oxidase of the electron transport chain (Zhu et al., 2021Zhu X. Boulet A. Buckley K.M. Phillips C.B. Gammon M.G. Oldfather L.E. Moore S.A. Leary S.C. Cobine P.A. Mitochondrial copper and phosphate transporter specificity was defined early in the evolution of eukaryotes.Elife. 2021; 10https://doi.org/10.7554/elife.64690Crossref Google Scholar). After import to the matrix, there are no known protein binding partners, in fact the Cu has been shown to exist in a stable complex with a non-proteinaceous ligand. This Cu is exchangeable with proteins and could participate in binding to other targets. Cuproptosis initiated in this compartment expands the physiological consequences for this Cu pool. The regulation and maintenance of this pool is relatively poorly understood due to a lack of targets. However, Cu deficiencies in mammals have severe developmental defects that can be reversed by elesclomol (Guthrie et al., 2020Guthrie L.M. Soma S. Yuan S. Silva A. Zulkifli M. Snavely T.C. Greene H.F. Nunez E. Lynch B. de Ville C. et al.Elesclomol alleviates Menkes pathology and mortality by escorting Cu to cuproenzymes in mice.Science. 2020; 368: 620-625https://doi.org/10.1126/science.aaz8899Crossref PubMed Scopus (43) Google Scholar). It may be possible that during deficiency processes such as cuproptosis are disrupted. In that scenario, cuproptosis, like apoptosis, could then have roles in regulating those developmental programs and pathways. Cu metalloallostery is the binding of Cu in non-catalytic sites in proteins to facilitate regulation of pathways such as proliferation, autophagy, and lipolysis (reviewed in Tsang et al., 2021Tsang T. Davis C.I. Brady D.C. Copper biology.Curr. Biol. 2021; 31: R421-R427https://doi.org/10.1016/j.cub.2021.03.054Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). However, adventitious binding of Cu to nonspecific sites has long been recognized as a potential mechanism of Cu toxicity and is often invoked as a critique of metalloallostery. Cuproptosis induced by the unexpected binding of Cu to lipoylated proteins is another example of specific binding to previously unrecognized sites adding cell death to the list of processes linked to Cu biology. Tsvetkov et al. demonstrated that Wilson disease hepatocytes, which hyper accumulate Cu due to mutations in ATP7B, have reduced iron sulfur cluster and lipoylated protein expression, suggesting that cuproptosis may contribute to the disease pathology. However, elevated Cu levels in Wilson disease hepatocytes engages Cu metalloallostery to directly upregulate autophagy and lysosome biogenesis as a means to clear damaged mitochondria and likely dampen cuproptosis (Polishchuk et al., 2019Polishchuk E.V. Merolla A. Lichtmannegger J. Romano A. Indrieri A. Ilyechova E.Y. Concilli M. De Cegli R. Crispino R. Mariniello M. et al.Activation of autophagy, observed in Liver Tissues from Patients with Wilson disease and from ATP7B-deficient Animals, Protects hepatocytes from copper-induced apoptosis.Gastroenterology. 2019; 156: 1173-1189.e5https://doi.org/10.1053/j.gastro.2018.11.032Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Ionophore-induced accumulation of metals wreaks havoc in cells as homeostasis is disrupted meaning the protein and endogenous ligands that normally control metal availability are overwhelmed. In E. coli, ionophore-supplied Cu dramatically affected protein stability of an unexpectedly high number of proteins ranging from metabolic enzymes to ribosomal proteins (Wiebelhaus et al., 2021Wiebelhaus N. Zaengle-Barone J.M. Hwang K.K. Franz K.J. Fitzgerald M.C. Protein Folding stability Changes across the Proteome reveal targets of Cu toxicity in E. coli.ACS Chem. Biol. 2021; 16: 214-224https://doi.org/10.1021/acschembio.0c00900Crossref PubMed Scopus (25) Google Scholar). Kurdistani and coworkers showed that histone H3 bound Cu and acted as a Cu reductase (Attar et al., 2020Attar N. Campos O.A. Vogelauer M. Cheng C. Xue Y. Schmollinger S. Salwinski L. Mallipeddi N.V. Boone B.A. Yen L. et al.The histone H3-H4 tetramer is a copper reductase enzyme.Science. 2020; 369: 59-64https://doi.org/10.1126/science.aba8740Crossref PubMed Scopus (41) Google Scholar). The combination of these studies further reinforced the idea that multiple additional protein binding sites exist for exchangeable Cu. An intriguing possibility that the discovery of cuproptosis has uncovered is a role for Cu binding to lipoylated proteins of mitochondria under normal conditions. Cu availability could be a transient allosteric regulation mechanism via aggregation of the proteins on smaller scales. This finely tuned system can be overwhelmed in Cu excess induced by ionophores or disease, leading to Cu-mediated aggregation and cell death. Finally, certain cancers accumulate Cu in a phenomenon recently coined cuproplasia in which pro-growth Cu-dependent pathways are engaged to drive hallmarks of cancer, and cuproptosis is avoided (Ge et al., 2022Ge E.J. Bush A.I. Casini A. Cobine P.A. Cross J.R. DeNicola G.M. Dou Q.P. Franz K.J. Gohil V.M. Gupta S. et al.Connecting copper and cancer: from transition metal signalling to metalloplasia.Nat. Rev. Cancer. 2022; 22: 102-113https://doi.org/10.1038/s41568-021-00417-2Crossref PubMed Scopus (338) Google Scholar). One hypothesis that warrants future exploration is whether lower SLC25A3 expression is a determinant of Cu-induced cell death in the context of cuproplasia. Although this accumulation of Cu could be a vulnerability that ionophores can exploit, metabolic rewiring of those cancer cells may favor Cu-dependent pro-growth signaling pathways and result in resistance. To further advance our understanding of cuproptosis and Cu biology, we must still resolve: what is the hallmark signal to readout cuproptosis? Why does Cu accumulation fail to activate established cell death pathways? How do disorders associated with elevated intracellular Cu avoid cuproptosis? How does aggregation of the lipoylated proteins induce the cascade that kills cells? What are the other roles of Cu in mitochondria? The identification of cuproptosis has opened the door to exploration of novel aspects of mitochondrial biology in cancers and normal cellular physiology. We thank the following sources for funding: NIH (GM124749 to D.C.B. and GM120211 to P.A.C.) and Ludwig Cancer Research Princeton Branch to D.C.B. D.C.B. holds ownership in Merlon Inc. and Elaeis Therapeutics, is an inventor on the patent application 20150017261 entitled “Methods of treating and preventing cancer by disrupting the binding of copper in the MAP kinase pathway,” and is a member of the Molecular Cell advisory board. P.A.C. reports no competing interests.