ABSTRACT
Superoxide dismutase (SOD) 3, a copper (Cu)-containing anti-oxidative enzyme, plays a key role in extracellular redox homeostasis. Cu chaperone antioxidant-1 (Atox-1) not only delivers Cu ions to SOD3 at the trans-Golgi network, it also functions as a transcription factor of SOD3; however, the role of Atox-1 in the regulation of SOD3 during the monocytic differentiation of THP-1 cells has not yet been elucidated. A treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced the expression of the Cu transport protein ATP7A in THP-1 cells. On the other hand, the nuclear translocation of Atox-1 was detected in TPA-treated THP-1 cells, and was suppressed in the presence of the Cu chelator, bathocuproinedisulfonic acid. Furthermore, Atox-1 bound to the SOD3 promoter region in TPA-treated THP-1 cells. The overexpression of Atox-1 in THP-1 cells significantly enhanced TPA-elicited SOD3 expression, whereas its knockdown suppressed this induction. The present results demonstrate that Atox-1 functions as a key molecule in TPA-elicited SOD3 expression.
Subject(s)
Copper-Transporting ATPases/genetics , Copper/metabolism , Metallochaperones/genetics , Monocytes/drug effects , Superoxide Dismutase/genetics , Cell Differentiation/drug effects , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Chelating Agents/pharmacology , Copper Transport Proteins , Copper-Transporting ATPases/metabolism , Gene Expression Regulation , Humans , Metallochaperones/antagonists & inhibitors , Metallochaperones/metabolism , Molecular Chaperones , Monocytes/cytology , Monocytes/metabolism , Oxidation-Reduction , Phenanthrolines/pharmacology , Promoter Regions, Genetic , Protein Binding , Protein Transport , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction , Superoxide Dismutase/metabolism , THP-1 Cells , Tetradecanoylphorbol Acetate/analogs & derivatives , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
Viperin (RSAD2) is an interferon-stimulated antiviral protein that belongs to the radical S-adenosylmethionine (SAM) enzyme family. Viperin's iron-sulfur (Fe/S) cluster is critical for its antiviral activity against many different viruses. CIA1 (CIAO1), an essential component of the cytosolic iron-sulfur protein assembly (CIA) machinery, is crucial for Fe/S cluster insertion into viperin and hence for viperin's antiviral activity. In the CIA pathway, CIA1 cooperates with CIA2A, CIA2B, and MMS19 targeting factors to form various complexes that mediate the dedicated maturation of specific Fe/S recipient proteins. To date, however, the mechanisms of how viperin acquires its radical SAM Fe/S cluster to gain antiviral activity are poorly understood. Using co-immunoprecipitation and 55Fe-radiolabeling experiments, we therefore studied the roles of CIA2A, CIA2B, and MMS19 for Fe/S cluster insertion. CIA2B and MMS19 physically interacted with the C terminus of viperin and used CIA1 as the primary viperin-interacting protein. In contrast, CIA2A bound to viperin's N terminus in a CIA1-, CIA2B-, and MMS19-independent fashion. Of note, the observed interaction of both CIA2 isoforms with a single Fe/S target protein is unprecedented in the CIA pathway. 55Fe-radiolabeling experiments with human cells depleted of CIA1, CIA2A, CIA2B, or MMS19 revealed that CIA1, but none of the other CIA factors, is predominantly required for 55Fe/S cluster incorporation into viperin. Collectively, viperin maturation represents a novel CIA pathway with a minimal requirement of the CIA-targeting factors and represents a new paradigm for the insertion of the Fe/S cofactor into a radical SAM protein.
Subject(s)
Carrier Proteins/metabolism , Iron-Sulfur Proteins/metabolism , Metallochaperones/metabolism , Models, Biological , Nuclear Proteins/metabolism , Proteins/metabolism , Transcription Factors/metabolism , Amino Acid Substitution , Apoproteins/chemistry , Apoproteins/genetics , Apoproteins/metabolism , Carrier Proteins/antagonists & inhibitors , Carrier Proteins/chemistry , Carrier Proteins/genetics , HEK293 Cells , Humans , Immunoprecipitation , Iron/chemistry , Iron/metabolism , Iron Radioisotopes , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/genetics , Metallochaperones/antagonists & inhibitors , Metallochaperones/chemistry , Metallochaperones/genetics , Metalloproteins , Mutation , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Oxidoreductases Acting on CH-CH Group Donors , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Interaction Domains and Motifs , Proteins/chemistry , Proteins/genetics , RNA Interference , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Transcription Factors/antagonists & inhibitors , Transcription Factors/chemistry , Transcription Factors/geneticsABSTRACT
Copper is a transition metal that plays critical roles in many life processes. Controlling the cellular concentration and trafficking of copper offers a route to disrupt these processes. Here we report small molecules that inhibit the human copper-trafficking proteins Atox1 and CCS, and so provide a selective approach to disrupt cellular copper transport. The knockdown of Atox1 and CCS or their inhibition leads to a significantly reduced proliferation of cancer cells, but not of normal cells, as well as to attenuated tumour growth in mouse models. We show that blocking copper trafficking induces cellular oxidative stress and reduces levels of cellular ATP. The reduced level of ATP results in activation of the AMP-activated protein kinase that leads to reduced lipogenesis. Both effects contribute to the inhibition of cancer cell proliferation. Our results establish copper chaperones as new targets for future developments in anticancer therapies.
Subject(s)
Cell Proliferation/drug effects , Copper/metabolism , Metallochaperones/antagonists & inhibitors , Molecular Chaperones/antagonists & inhibitors , Neoplasms/metabolism , Amino Acid Sequence , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Copper Transport Proteins , Drug Discovery , Gene Knockdown Techniques , Humans , Metallochaperones/chemistry , Metallochaperones/genetics , Metallochaperones/metabolism , Mice , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Molecular Sequence Data , Oxidative Stress/drug effects , Sequence Alignment , Xenograft Model Antitumor AssaysABSTRACT
Tetrathiomolybdate (TM) is an orally active agent for treatment of disorders of copper metabolism. Here we describe how TM inhibits proteins that regulate copper physiology. Crystallographic results reveal that the surprising stability of the drug complex with the metallochaperone Atx1 arises from formation of a sulfur-bridged copper-molybdenum cluster reminiscent of those found in molybdenum and iron sulfur proteins. Spectroscopic studies indicate that this cluster is stable in solution and corresponds to physiological clusters isolated from TM-treated Wilson's disease animal models. Finally, mechanistic studies show that the drug-metallochaperone inhibits metal transfer functions between copper-trafficking proteins. The results are consistent with a model wherein TM can directly and reversibly down-regulate copper delivery to secreted metalloenzymes and suggest that proteins involved in metal regulation might be fruitful drug targets.
