The Experts below are selected from a list of 939 Experts worldwide ranked by ideXlab platform
Pernilla Wittung-stafshede - One of the best experts on this subject based on the ideXlab platform.
-
Evaluation of copper chaperone ATOX1 as prognostic biomarker in breast cancer
Breast Cancer, 2020Co-Authors: Stéphanie Blockhuys, Donita C. Brady, Pernilla Wittung-stafshedeAbstract:Copper is involved in different hallmarks of cancer, including metastasis, but responsible copper-binding proteins and pathways are not clear. The copper chaperone ATOX1 was recently shown to play a role in breast cancer cell migration, which is a key step in metastasis. Since most cancer-related deaths are due to metastasis, we hypothesized that ATOX1 mRNA expression may be associated with breast cancer disease progression and thus, a prognostic biomarker in breast cancer. We therefore studied the association of ATOX1 expression levels with clinicopathological parameters and survival for 1904 breast cancer patients using the METABRIC data set. Our results indicate ATOX1 expression levels as a potential prognostic biomarker for ER-positive subtypes and early stages of breast cancer. Pre-clinical studies and clinical trials are desired to identify the molecular roles of ATOX1 in these conditions.
-
Interaction between Copper Chaperone ATOX1 and Parkinson's Disease Protein α-Synuclein Includes Metal-Binding Sites and Occurs in Living Cells.
ACS Chemical Neuroscience, 2019Co-Authors: Istvan Horvath, Stéphanie Blockhuys, Darius Šulskis, Stellan Holgersson, Ranjeet Kumar, Björn M. Burmann, Pernilla Wittung-stafshedeAbstract:Alterations in copper ion homeostasis appear coupled to neurodegenerative disorders, but mechanisms are unknown. The cytoplasmic copper chaperone ATOX1 was recently found to inhibit amyloid formation in vitro of α-synuclein, the amyloidogenic protein in Parkinson's disease. As α-synuclein may have copper-dependent functions, and free copper ions promote α-synuclein amyloid formation, it is important to characterize the ATOX1 interaction with α-synuclein on a molecular level. Here we applied solution-state nuclear magnetic resonance spectroscopy, with isotopically labeled α-synuclein and ATOX1, to define interaction regions in both proteins. The α-synuclein interaction interface includes the whole N-terminal part up to Gln24; in ATOX1, residues around the copper-binding cysteines (positions 11-16) are mostly perturbed, but additional effects are also found for residues elsewhere in both proteins. Because α-synuclein is N-terminally acetylated in vivo, we established that ATOX1 also inhibits amyloid formation of this variant in vitro, and proximity ligation in human cell lines demonstrated α-synuclein-ATOX1 interactions in situ. Thus, this interaction may provide the direct link between copper homeostasis and amyloid formation in vivo.
-
Copper relay path through the N-terminus of Wilson disease protein, ATP7B.
Metallomics, 2019Co-Authors: Kumaravel Ponnandai Shanmugavel, Pernilla Wittung-stafshedeAbstract:In human cells, copper (Cu) ions are transported by the cytoplasmic Cu chaperone ATOX1 to the Wilson disease protein (ATP7B) in the Golgi for loading of Cu-dependent enzymes. ATP7B is a membrane-spanning protein which, in contrast to non-mammalian homologs, has six cytoplasmic metal-binding domains (MBDs). To address the reason for multiple MBDs, we introduced strategic mutations in which one, two or three MBDs had been blocked for Cu binding via cysteine-to-serine mutations (but all six MBDs are present in all) in a yeast system that probes Cu flow through ATOX1 and ATP7B. The results, combined with earlier work, support a mechanistic model in which MBD1-3 forms a regulatory unit of ATP7B Cu transport. Cu delivery via ATOX1 to this unit, followed by loading of Cu in MBD3, promotes release of inhibitory interactions. Whereas the Cu site in MBD4 can be mutated without a large effect, an intact Cu site in either MBD5 or MBD6 is required for Cu transport. All MBDs, expressed as single-domain proteins, can replace ATOX1 and deliver Cu to full-length ATP7B. However, only MBD6 can deliver Cu to truncated ATP7B where all six MBDs are removed, suggesting a docking role for this structural unit.
-
Copper chaperone blocks amyloid formation via ternary complex.
Quarterly Reviews of Biophysics, 2018Co-Authors: Istvan Horvath, Ranjeet Kumar, Tony Werner, Pernilla Wittung-stafshedeAbstract:Protein misfolding in cells is avoided by a network of protein chaperones that detect misfolded or partially folded species. When proteins escape these control systems, misfolding may result in protein aggregation and amyloid formation. We here show that aggregation of the amyloidogenic protein alpha-synuclein (alpha S), the key player in Parkinson's disease, is controlled by the copper transport protein ATOX1 in vitro. Copper ions are not freely available in the cellular environment, but when provided by ATOX1, the resulting copper-dependent ternary complex blocks aS aggregation. Because the same inhibition was found for a truncated version of alpha S, lacking the C-terminal part, it appears that ATOX1 interacts with the N-terminal copper site in alpha S. Metal-dependent chaperoning may be yet another manner in which cells control its proteome.
-
Copper Chaperone ATOX1 Interacts with Cell Cycle Proteins.
Computational and Structural Biotechnology Journal, 2018Co-Authors: Maria Matson Dzebo, Emanuele Celauro, Stéphanie Blockhuys, Sebastian Valenzuela, Elin K. Esbjörner, Pernilla Wittung-stafshedeAbstract:The anaphase-promoting complex (APC) is involved in several processes in the cell cycle, most prominently it facilitates the separation of the sister chromatids during mitosis, before cell division. Because of the key role in the cell cycle, APC is suggested as a putative target for anticancer agents. We here show that the copper chaperone ATOX1, known for shuttling copper in the cytoplasm from Ctr1 to ATP7A/B in the secretory pathway, interacts with several APC subunits. ATOX1 interactions with APC subunits were discovered by mass spectrometry of co-immunoprecipitated samples and further confirmed using proximity ligation assays in HEK293T cells. Upon comparing wild-type cells with those in which the ATOX1 gene had been knocked out, we found that in the absence of ATOX1 protein, cells have prolonged G2/M phases and a slower proliferation rate. Thus, in addition to copper transport for loading of copper-dependent enzymes, ATOX1 may modulate the cell cycle by interacting with APC subunits.
Tohru Fukai - One of the best experts on this subject based on the ideXlab platform.
-
nuclear translocation of ATOX1 potentiates activin a induced cell migration and colony formation in colon cancer
PLOS ONE, 2020Co-Authors: Arundhati Jana, Archita Das, Tohru Fukai, Nancy Krett, Grace Guzman, Alexandra L Thomas, Georgina Mancinelli, Jessica Bauer, Masuko Ushiofukai, Barbara JungAbstract:BACKGROUND Colorectal cancer remains a deadly cancer due to metastatic disease. To understand the molecular mechanisms of metastasis in colon cancer, we investigated whether the copper chaperone antioxidant-1 (ATOX1) protein plays a role in this process. Recent findings indicate that ATOX1 protein has transcription factor activities and plays a vital role in cell proliferation in cancer cells. However, the role of ATOX1 in metastasis has not been examined. METHODS ATOX1 expression was determined by immunofluorescence in a tissue microarray generated from a spectrum of CRC patients. Subcellular fractionation of colon cancer cell lines SW480 and SW620 cells was used to examine the cellular location of ATOX1 in the face of activin A, a cytokine that stimulates colon cancer metastasis. ATOX1 expression was genetically manipulated and cellular migration measured through trans-well assay and proliferation measured by colony formation assays. RESULTS Here we demonstrate that in patients with metastatic colon cancer, there is a significant increase in the expression of nuclear ATOX1. Interestingly, the metastatic CRC cell line SW620 has increased nuclear localization of ATOX1 compared to its related non-metastatic cell line SW480. Further, inhibition of endogenous ATOX1 by siRNA in SW620 decreased colony formation and reactive oxygen species generation via decreased expression of ATOX1 targets cyclin D1 and NADPH oxidase subunit p47 phox, respectively. Additionally, overexpression of nuclear-targeted but not copper binding domain-mutated ATOX1 in SW480 cells increased colony formation and cell migration that was further augmented by activin A stimulation, a known enhancer of colon cancer metastasis. CONCLUSIONS Our findings suggest that nuclear ATOX1 might be a new therapeutic target as well as a new biomarker for metastatic colorectal cancer.
-
Abstract 15194: Antioxidant-1, Proinflammatory Cu-Dependent Transcription Factor, Mediates Disturbed Flow-Induced Endothelial Cell Inflammation and Atherosclerosis
Circulation, 2016Co-Authors: Sudhahar Varadarajan, Archita Das, Masuko Ushio-fukai, Bayasgalan Surenkhuu, Asahi Kato, Ken Shibata Lizama, Anuja Waghulkar, Tohru FukaiAbstract:Introduction: Disturbed flow (d-flow) including oscillatory shear (OS) causes endothelial dysfunction, leading to atherosclerosis. SUMOylation alters localization and transcriptional activity of protein. Copper (Cu), an essential micronutrient, plays a pivotal role in inflammation. We recently reported that Cu transport protein, antioxidant 1 (ATOX1) functions not only as Cu chaperone to activate secretory Cu enzymes via Cu exporter ATP7A, but also as Cu-dependent transcription factor for inflammatory genes. Hypothesis: ATOX1 is involved in OS-induced endothelial cells (ECs) inflammation and atherosclerosis. Results: We produced a region of d-flow in ApoE -/- mice using partial ligation of left common carotid artery (LCA) and high-fat feeding for 3 weeks. Atherosclerotic lesion (78 ±3.8%; Oil red O) were decreased in ATOX1 -/- /ApoE -/- mice which was associated with decreased ICAM1/VCAM1 expression (p -/- mice. Aortic EC located in d-flow exposed areas in ApoE -/- mice visualized by en face staining, and cultured ECs exposed OS (±5 dyne/cm 2 ), but not laminar shear stress, showed increased ATOX1 nuclear translocation and increased ICAM1 (3.9±1.1 fold)/VCAM1 (1.8 ± 0.3 fold) expression, which was rescued by ATOX1 knockdown. Mechanistically, OS promoted ATOX1 SUMOylation. Importantly, OS-induced ATOX1 nuclear translocation was inhibited by SUMOylation-defective mutant ATOX1-K3R, Cu chelator or Cu importer CTR1 siRNA. Furthermore, OS promoted downregulation of Cu transporter ATP7A via binding to COMMD1, a key regulator of CuATPase proteasomal degradation, thereby switching ATOX1 from the Cu chaperone for ATP7A to the Cu-dependent transcription factor. Indeed, COMMD1 knockdown rescued ATP7A expression reduction (63±7.4%) and ATOX1 nuclear translocation induced by OS. Conclusion: OS-induced downregulation of ATP7A via COMMD1 promoted ATOX1 nuclear translocation mediated through SUMOylation, thereby enhancing ATOX1-dependent adhesion molecule expression in ECs. Thus, ATOX1 is a novel Cu-dependent mechanosensitive transcription factor that senses OS-induced signal to promote EC inflammation and atherosclerosis.
-
Abstract 13113: Identification of Novel Copper-dependent Wound Repair Mechanism: Role of Copper Transport Protein Antioxidant 1
Circulation, 2015Co-Authors: Archita Das, Gin-fu Chen, Ha Won Kim, Seock-won Youn, Sudhahar Varadarajan, Lydia Finney, Masuko Ushio-fukai, Jay Yang, Tohru FukaiAbstract:Copper (Cu) facilitates wound healing and angiogenesis with unknown mechanism. Bioavailability of Cu is controlled by transport-proteins, including cytosolic Cu-chaperone ATOX1, which is required for activation of secretory Cu enzymes. ATOX1 also functions as a Cu-dependent transcription factor, but its role in wound healing is unknown. Using mouse skin puncture model, here we show that ATOX1 protein (8-fold) and Cu-level (by X-ray Fluorescence Microscopy; 2.5-fold) were increased in wounding tissue in wild type (WT) mice at day 7 when ATOX1 was localized in nucleus of dermal endothelial cells (ECs) as well as cytosol of epidermal cells, granulation tissue. Furthermore, topical Cu treatment enhanced (20% vs PBS), but specific Cu chelator BCS reduced wound repair in WT mice. Importantly, ATOX1 knockout (KO) mice showed abolished topical Cu-induced wound repair or impaired endogenous wound healing vs. WT mice, which was associated with decreased angiogenesis (CD31+, 45% ), proliferation (BrdU+, 60%), ROS-production (DHE+), collagen formation (Masson9s Trichrome), and infiltration of macrophage (Mac3+,40%) which secrets angiogenic cytokines VEGF and SDF-1α. Mechanistically, ATOX1KO mice exhibited reduced wounding-induced expression of ATOX1 target proteins such as p47phox NADPH oxidase and cyclin D1 as well as extracellular matrix secretory Cu-enzyme lysyl oxidase activity. Finally, bone marrow (BM) transplantation revealed that ATOX1 in both BM and tissue resident cells are required for wound repair. Consistently, ATOX1 protein and Cu were markedly increased in WT-BM tissue after wounding. Impaired wound healing in ATOX1 KO mice was rescued by both ATOX1-gene transfer and WT-BM topical treatment in wound tissues. In summary, ATOX1 senses Cu to accelerate wound healing/repair by promoting angiogenesis, inflammatory cell recruitment, proliferation, extracellular matrix maturation as well as BM cell function. Taken together, Cu-ATOX1-based therapy may represent a novel therapeutic strategy to promote dermal wound healing and tissue regeneration.
-
Abstract 13097: Novel Interaction of Antioxidant-1 With NADPH Oxidase p47phox and TRAF4: Role in TNFα-Induced Inflammation and Atherosclerosis
Circulation, 2015Co-Authors: Archita Das, Gin-fu Chen, Sudhahar Varadarajan, Masuko Ushio-fukai, Bayasgalan Surenkhuu, Jun Tian, Tohru FukaiAbstract:Tumor necrosis factor-TNFα plays a key role in atherosclerosis via NADPH oxidase-derived reactive oxygen species (ROS) and its downstream redox-sensitive inflammatory responses. Copper (Cu) has been implicated in atherosclerosis and inflammation with unknown mechanism. Bioavailability of Cu is controlled by transport-proteins including Cu-chaperone ATOX1 which obtains Cu via Cu-importer CTR1 localized at plasma membrane. We recently found that ATOX1 also functions as a Cu-dependent transcription factor for NADPH oxidase p47phox. However, role of ATOX1 in atherosclerosis is unknown. Here we show that ApoE-/-ATOX1-/- mice with 16 weeks high-fat-diet (HFD) showed decreased atherosclerotic lesion (40%, Oil-O-red staining). inflammatory cell recruitment (50% decrease, Mac3+) and extracellular matrix deposition (Masson’s Trichrome) compared to ApoE-/-HFD. Mechanistically, in cultured endothelial cells (ECs), ATOX1 knockdown with siRNA, or Cu-chelator BCS inhibited TNFα-induced ROS production (70%) at later phase (16hrs) and its downstream VCAM1/ICAM1 expression and monocyte adhesion by reducing transcription of p47phox. Unexpectedly, TNFα stimulation rapidly promoted ATOX1/p47phox binding in a PKCδ-dependent and Cu-“independent” manner, which is required for p47phox membrane translocation involved in early phase of (5min) ROS production and subsequent Cys oxidation of Cu-importer CTR1 detected by a biotin-labeled Cys-OH trapping reagent. This was followed by CTR1/Cu-dependent ATOX1 binding to TNFα receptor associated factor (TRAF)4, which promoted ATOX1 nuclear translocation to increase p47phox expression leading to late-phase of ROS production, facilitating further monocyte adhesion to ECs. In summary, ATOX1 plays an important role in activating p47phox-based NADPH oxidase and inflammatory responses in ECs by binding to p47phox that induces early phase of ROS to promote CTR1 oxidation in Cu-independent manner. This is followed by Cu-dependent ATOX1/TRAF4 binding required for ATOX1 nuclear translocation to function as a transcription factor for p47phox, leading to late-phase of ROS production, which contributes to acceleration of atherosclerosis in vivo.
-
Abstract 16637: Antioxidant-1, a Novel Cu-Dependent Transcription Factor for NADPH Oxidase p47phox, Promotes Wound Healing by Regulating Inflammation and Angiogenesis
Circulation, 2014Co-Authors: Archita Das, Gin-fu Chen, Ha Won Kim, Seock-won Youn, Sudhahar Varadarajan, Ronald D. Mckinney, Lydia Finney, Masuko Ushio-fukai, Tohru FukaiAbstract:Background: Wound healing is important repair process after injury associated with inflammation and angiogenesis. Copper (Cu) plays an important role in wound healing and angiogenesis; however, underlying mechanism is unknown. Bioavailability of Cu is tightly controlled by transport proteins (Cu importer CTR1 and Cu exporter ATP7A) and chaperone (antioxidant-1, ATOX1) which transfers Cu to ATP7A. We reported that ATOX1 also functions as a Cu-dependent transcription factor; however, its role in wound healing is unclear. Results: Using mouse skin puncture model, here we show that, Atox 1 protein (8 fold) and Cu level (by X-ray Fluorescence Microscope; 2.5 fold) increased in skin after wounding in WT mice at day 7 when ATOX1 was localised in the nucleus of dermis endothelial cell (ECs) and ATP7A was markedly decreased. ATP7A transgenic mice showed impaired wound healing. ATOX1 knockout (KO) mice exhibited decrease in wound repair (40% inhibition vs. WT, day 7) which was rescued by ATOX1 gene transfer by lentivirus. Macrophage which secrets angiogenic cytokines/chemokines was significantly reduced which resulted in decreased VEGF and SDF-1α protein and angiogenesis at day 7. In cultured human ECs, inflammatory cytokine TNF-stimulation significantly decreased ATP7A protein (80%) and increased intracellular Cu and ATOX1 in nucleus. ATP7A knockdown with siRNA in ECs increased intracellular Cu and promoted nuclear ATOX1, which were rescued by Cu importer CTR1 siRNA or Cu chelator BCS, indicating ATP7A downregulation-induced Cu accumulation is required for ATOX1 nuclear translocation. By screening ATOX1 target genes, we found that NADPH oxidase p47phox promoter contains ATOX1 binding responsible element. p47phox protein expression was significantly increased by wounding or overexpression of nuclear-targeted ATOX1, which was markedly decreased in ATOX1 KO mice with its downstream VCAM1/ICAM1. Conclusions: Cu chaperone ATOX1 functions as a transcription factor by sensing ATP7A downregulation-induced intracellular Cu to upregulate NADPH oxidase p47phox and its downstream redox-sensitive VCAM1/ICAM1 expression in response to injury. This in turn promotes inflammatory cell recruitment and angiogenesis, and thus stimulating wound repair.
Masuko Ushio-fukai - One of the best experts on this subject based on the ideXlab platform.
-
Abstract 15194: Antioxidant-1, Proinflammatory Cu-Dependent Transcription Factor, Mediates Disturbed Flow-Induced Endothelial Cell Inflammation and Atherosclerosis
Circulation, 2016Co-Authors: Sudhahar Varadarajan, Archita Das, Masuko Ushio-fukai, Bayasgalan Surenkhuu, Asahi Kato, Ken Shibata Lizama, Anuja Waghulkar, Tohru FukaiAbstract:Introduction: Disturbed flow (d-flow) including oscillatory shear (OS) causes endothelial dysfunction, leading to atherosclerosis. SUMOylation alters localization and transcriptional activity of protein. Copper (Cu), an essential micronutrient, plays a pivotal role in inflammation. We recently reported that Cu transport protein, antioxidant 1 (ATOX1) functions not only as Cu chaperone to activate secretory Cu enzymes via Cu exporter ATP7A, but also as Cu-dependent transcription factor for inflammatory genes. Hypothesis: ATOX1 is involved in OS-induced endothelial cells (ECs) inflammation and atherosclerosis. Results: We produced a region of d-flow in ApoE -/- mice using partial ligation of left common carotid artery (LCA) and high-fat feeding for 3 weeks. Atherosclerotic lesion (78 ±3.8%; Oil red O) were decreased in ATOX1 -/- /ApoE -/- mice which was associated with decreased ICAM1/VCAM1 expression (p -/- mice. Aortic EC located in d-flow exposed areas in ApoE -/- mice visualized by en face staining, and cultured ECs exposed OS (±5 dyne/cm 2 ), but not laminar shear stress, showed increased ATOX1 nuclear translocation and increased ICAM1 (3.9±1.1 fold)/VCAM1 (1.8 ± 0.3 fold) expression, which was rescued by ATOX1 knockdown. Mechanistically, OS promoted ATOX1 SUMOylation. Importantly, OS-induced ATOX1 nuclear translocation was inhibited by SUMOylation-defective mutant ATOX1-K3R, Cu chelator or Cu importer CTR1 siRNA. Furthermore, OS promoted downregulation of Cu transporter ATP7A via binding to COMMD1, a key regulator of CuATPase proteasomal degradation, thereby switching ATOX1 from the Cu chaperone for ATP7A to the Cu-dependent transcription factor. Indeed, COMMD1 knockdown rescued ATP7A expression reduction (63±7.4%) and ATOX1 nuclear translocation induced by OS. Conclusion: OS-induced downregulation of ATP7A via COMMD1 promoted ATOX1 nuclear translocation mediated through SUMOylation, thereby enhancing ATOX1-dependent adhesion molecule expression in ECs. Thus, ATOX1 is a novel Cu-dependent mechanosensitive transcription factor that senses OS-induced signal to promote EC inflammation and atherosclerosis.
-
Abstract 13113: Identification of Novel Copper-dependent Wound Repair Mechanism: Role of Copper Transport Protein Antioxidant 1
Circulation, 2015Co-Authors: Archita Das, Gin-fu Chen, Ha Won Kim, Seock-won Youn, Sudhahar Varadarajan, Lydia Finney, Masuko Ushio-fukai, Jay Yang, Tohru FukaiAbstract:Copper (Cu) facilitates wound healing and angiogenesis with unknown mechanism. Bioavailability of Cu is controlled by transport-proteins, including cytosolic Cu-chaperone ATOX1, which is required for activation of secretory Cu enzymes. ATOX1 also functions as a Cu-dependent transcription factor, but its role in wound healing is unknown. Using mouse skin puncture model, here we show that ATOX1 protein (8-fold) and Cu-level (by X-ray Fluorescence Microscopy; 2.5-fold) were increased in wounding tissue in wild type (WT) mice at day 7 when ATOX1 was localized in nucleus of dermal endothelial cells (ECs) as well as cytosol of epidermal cells, granulation tissue. Furthermore, topical Cu treatment enhanced (20% vs PBS), but specific Cu chelator BCS reduced wound repair in WT mice. Importantly, ATOX1 knockout (KO) mice showed abolished topical Cu-induced wound repair or impaired endogenous wound healing vs. WT mice, which was associated with decreased angiogenesis (CD31+, 45% ), proliferation (BrdU+, 60%), ROS-production (DHE+), collagen formation (Masson9s Trichrome), and infiltration of macrophage (Mac3+,40%) which secrets angiogenic cytokines VEGF and SDF-1α. Mechanistically, ATOX1KO mice exhibited reduced wounding-induced expression of ATOX1 target proteins such as p47phox NADPH oxidase and cyclin D1 as well as extracellular matrix secretory Cu-enzyme lysyl oxidase activity. Finally, bone marrow (BM) transplantation revealed that ATOX1 in both BM and tissue resident cells are required for wound repair. Consistently, ATOX1 protein and Cu were markedly increased in WT-BM tissue after wounding. Impaired wound healing in ATOX1 KO mice was rescued by both ATOX1-gene transfer and WT-BM topical treatment in wound tissues. In summary, ATOX1 senses Cu to accelerate wound healing/repair by promoting angiogenesis, inflammatory cell recruitment, proliferation, extracellular matrix maturation as well as BM cell function. Taken together, Cu-ATOX1-based therapy may represent a novel therapeutic strategy to promote dermal wound healing and tissue regeneration.
-
Abstract 13097: Novel Interaction of Antioxidant-1 With NADPH Oxidase p47phox and TRAF4: Role in TNFα-Induced Inflammation and Atherosclerosis
Circulation, 2015Co-Authors: Archita Das, Gin-fu Chen, Sudhahar Varadarajan, Masuko Ushio-fukai, Bayasgalan Surenkhuu, Jun Tian, Tohru FukaiAbstract:Tumor necrosis factor-TNFα plays a key role in atherosclerosis via NADPH oxidase-derived reactive oxygen species (ROS) and its downstream redox-sensitive inflammatory responses. Copper (Cu) has been implicated in atherosclerosis and inflammation with unknown mechanism. Bioavailability of Cu is controlled by transport-proteins including Cu-chaperone ATOX1 which obtains Cu via Cu-importer CTR1 localized at plasma membrane. We recently found that ATOX1 also functions as a Cu-dependent transcription factor for NADPH oxidase p47phox. However, role of ATOX1 in atherosclerosis is unknown. Here we show that ApoE-/-ATOX1-/- mice with 16 weeks high-fat-diet (HFD) showed decreased atherosclerotic lesion (40%, Oil-O-red staining). inflammatory cell recruitment (50% decrease, Mac3+) and extracellular matrix deposition (Masson’s Trichrome) compared to ApoE-/-HFD. Mechanistically, in cultured endothelial cells (ECs), ATOX1 knockdown with siRNA, or Cu-chelator BCS inhibited TNFα-induced ROS production (70%) at later phase (16hrs) and its downstream VCAM1/ICAM1 expression and monocyte adhesion by reducing transcription of p47phox. Unexpectedly, TNFα stimulation rapidly promoted ATOX1/p47phox binding in a PKCδ-dependent and Cu-“independent” manner, which is required for p47phox membrane translocation involved in early phase of (5min) ROS production and subsequent Cys oxidation of Cu-importer CTR1 detected by a biotin-labeled Cys-OH trapping reagent. This was followed by CTR1/Cu-dependent ATOX1 binding to TNFα receptor associated factor (TRAF)4, which promoted ATOX1 nuclear translocation to increase p47phox expression leading to late-phase of ROS production, facilitating further monocyte adhesion to ECs. In summary, ATOX1 plays an important role in activating p47phox-based NADPH oxidase and inflammatory responses in ECs by binding to p47phox that induces early phase of ROS to promote CTR1 oxidation in Cu-independent manner. This is followed by Cu-dependent ATOX1/TRAF4 binding required for ATOX1 nuclear translocation to function as a transcription factor for p47phox, leading to late-phase of ROS production, which contributes to acceleration of atherosclerosis in vivo.
-
Abstract 16637: Antioxidant-1, a Novel Cu-Dependent Transcription Factor for NADPH Oxidase p47phox, Promotes Wound Healing by Regulating Inflammation and Angiogenesis
Circulation, 2014Co-Authors: Archita Das, Gin-fu Chen, Ha Won Kim, Seock-won Youn, Sudhahar Varadarajan, Ronald D. Mckinney, Lydia Finney, Masuko Ushio-fukai, Tohru FukaiAbstract:Background: Wound healing is important repair process after injury associated with inflammation and angiogenesis. Copper (Cu) plays an important role in wound healing and angiogenesis; however, underlying mechanism is unknown. Bioavailability of Cu is tightly controlled by transport proteins (Cu importer CTR1 and Cu exporter ATP7A) and chaperone (antioxidant-1, ATOX1) which transfers Cu to ATP7A. We reported that ATOX1 also functions as a Cu-dependent transcription factor; however, its role in wound healing is unclear. Results: Using mouse skin puncture model, here we show that, Atox 1 protein (8 fold) and Cu level (by X-ray Fluorescence Microscope; 2.5 fold) increased in skin after wounding in WT mice at day 7 when ATOX1 was localised in the nucleus of dermis endothelial cell (ECs) and ATP7A was markedly decreased. ATP7A transgenic mice showed impaired wound healing. ATOX1 knockout (KO) mice exhibited decrease in wound repair (40% inhibition vs. WT, day 7) which was rescued by ATOX1 gene transfer by lentivirus. Macrophage which secrets angiogenic cytokines/chemokines was significantly reduced which resulted in decreased VEGF and SDF-1α protein and angiogenesis at day 7. In cultured human ECs, inflammatory cytokine TNF-stimulation significantly decreased ATP7A protein (80%) and increased intracellular Cu and ATOX1 in nucleus. ATP7A knockdown with siRNA in ECs increased intracellular Cu and promoted nuclear ATOX1, which were rescued by Cu importer CTR1 siRNA or Cu chelator BCS, indicating ATP7A downregulation-induced Cu accumulation is required for ATOX1 nuclear translocation. By screening ATOX1 target genes, we found that NADPH oxidase p47phox promoter contains ATOX1 binding responsible element. p47phox protein expression was significantly increased by wounding or overexpression of nuclear-targeted ATOX1, which was markedly decreased in ATOX1 KO mice with its downstream VCAM1/ICAM1. Conclusions: Cu chaperone ATOX1 functions as a transcription factor by sensing ATP7A downregulation-induced intracellular Cu to upregulate NADPH oxidase p47phox and its downstream redox-sensitive VCAM1/ICAM1 expression in response to injury. This in turn promotes inflammatory cell recruitment and angiogenesis, and thus stimulating wound repair.
-
Abstract 16440: Cu Transporter ATP7A Protects Against Fibrosis Through Limiting Endothelial to Mesenchymal Transition
Circulation, 2014Co-Authors: Seock-won Youn, Archita Das, Sudhahar Varadarajan, Ronald D. Mckinney, Tohru Fukai, Masuko Ushio-fukaiAbstract:Background: Endothelial to mesenchymal transition (EndMT) is induced by inflammation and contributes to fibrosis; however, underlying mechanism is poorly understood. Cu plays an important role in physiological processes and pathophysiologies associated with inflammatory diseases. Since excess Cu is toxic, bioavailability of Cu is tightly controlled by Cu exporter ATP7A, which obtains Cu via Cu chaperone, ATOX1, and exclude Cu. We reported that ATOX1 also functions as a Cu dependent transcription factor. However, role of Cu transport proteins in EndMT is entirely unknown.[[Unable to Display Character: ]] Results: Here we show that TNFα stimulation for 24hr in HUVEC significantly decreased ATP7A protein (80%) and increased intracellular Cu and ATOX1 in nucleus, which was associated with shape change forming EndMT. ATP7A depletion with shRNA in EC significantly reduced EC markers (VE-cadherin and VEGFR2) and increased mesenchymal markers (αSMA, Calponin, SM22α, Collagen I/II). ATP7A siRNA also increas...
Sharon Ruthstein - One of the best experts on this subject based on the ideXlab platform.
-
Cu(I) Controls Conformational States in Human ATOX1 Metallochaperone: An EPR and Multiscale Simulation Study
The Journal of Physical Chemistry B, 2020Co-Authors: Ortal Perkal, Zena Qasem, Meital Turgeman, Renana Schwartz, Lada Gevorkyan-airapetov, Matic Pavlin, Alessandra Magistrato, Dan Thomas Major, Sharon RuthsteinAbstract:ATOX1 is a human copper metallochaperone that is responsible for transferring copper ions from the main human copper transporter, hCtr1, to ATP7A/B in the Golgi apparatus. ATOX1 interacts with the Ctr1 C-terminal domain as a dimer, although it transfers the copper ions to ATP7A/B in a monomeric form. The copper binding site in the ATOX1 dimer involves Cys12 and Cys15, while Lys60 was also suggested to play a role in the copper binding. We recently showed that ATOX1 can adopt various conformational states, depending on the interacting protein. In the current study, we apply EPR experiments together with hybrid quantum mechanics-molecular mechanics molecular dynamics simulations using a recently developed semiempirical density functional theory approach, to better understand the effect of ATOX1's conformational states on copper coordination. We propose that the flexibility of ATOX1 occurs owing to protonation of one or more of the cysteine residues, and that Cys15 is an important residue for ATOX1 dimerization, while Cys12 is a critical residue for Cu(I) binding. We also show that Lys60 electrostatically stabilizes the Cu(I)-ATOX1 dimer.
-
The structural flexibility of the human copper chaperone ATOX1: Insights from combined pulsed EPR studies and computations.
Protein Science, 2017Co-Authors: Ariel R. Levy, Meital Turgeman, Lada Gevorkyan-aiapetov, Sharon RuthsteinAbstract:Metallochaperones are responsible for shuttling metal ions to target proteins. Thus, a metallochaperone's structure must be sufficiently flexible both to hold onto its ion while traversing the cytoplasm and to transfer the ion to or from a partner protein. Here, we sought to shed light on the structure of ATOX1, a metallochaperone involved in the human copper regulation system. ATOX1 shuttles copper ions from the main copper transporter, Ctr1, to the ATP7b transporter in the Golgi apparatus. Conventional biophysical tools such as X-ray or NMR cannot always target the various conformational states of metallochaperones, owing to a requirement for crystallography or low sensitivity and resolution. Electron paramagnetic resonance (EPR) spectroscopy has recently emerged as a powerful tool for resolving biological reactions and mechanisms in solution. When coupled with computational methods, EPR with site-directed spin labeling and nanoscale distance measurements can provide structural information on a protein or protein complex in solution. We use these methods to show that ATOX1 can accommodate at least four different conformations in the apo state (unbound to copper), and two different conformations in the holo state (bound to copper). We also demonstrate that the structure of ATOX1 in the holo form is more compact than in the apo form. Our data provide insight regarding the structural mechanisms through which ATOX1 can fulfill its dual role of copper binding and transfer.
-
Ctr1 Intracellular Loop Is Involved in the Copper Transfer Mechanism to the ATOX1 Metallochaperone.
The Journal of Physical Chemistry B, 2016Co-Authors: Ariel R. Levy, Matan Nissim, Netanel Mendelman, Jordan H. Chill, Sharon RuthsteinAbstract:Understanding the human copper cycle is essential to understand the role of metals in promoting neurological diseases and disorders. One of the cycles controlling the cellular concentration and distribution of copper involves the copper transporter, Ctr1; the metallochaperone, ATOX1; and the ATP7B transporter. It has been shown that the C-terminus of Ctr1, specifically the last three amino acids, HCH, is involved in both copper coordination and the transfer mechanism to ATOX1. In contrast, the role of the intracellular loop of Ctr1, which is an additional intracellular segment of Ctr1, in facilitating the copper transfer mechanism has not been investigated yet. Here, we combine various biophysical methods to explore the interaction between this Ctr1 segment and metallochaperone ATOX1 and clearly demonstrate that the Ctr1 intracellular loop (1) can coordinate Cu(I) via interactions with the side chains of one histidine and two methionine residues and (2) closely interacts with the ATOX1 metallochaperone. Our findings are another important step in elucidating the mechanistic details of the eukaryotic copper cycle.
-
Ctr1 Intracellular Loop Is Involved in the Copper Transfer Mechanism to the ATOX1 Metallochaperone
2016Co-Authors: Ariel R. Levy, Netanel Mendelman, Matan Nissim, Jordan H. Chill, Sharon RuthsteinAbstract:Understanding the human copper cycle is essential to understand the role of metals in promoting neurological diseases and disorders. One of the cycles controlling the cellular concentration and distribution of copper involves the copper transporter, Ctr1; the metallochaperone, ATOX1; and the ATP7B transporter. It has been shown that the C-terminus of Ctr1, specifically the last three amino acids, HCH, is involved in both copper coordination and the transfer mechanism to ATOX1. In contrast, the role of the intracellular loop of Ctr1, which is an additional intracellular segment of Ctr1, in facilitating the copper transfer mechanism has not been investigated yet. Here, we combine various biophysical methods to explore the interaction between this Ctr1 segment and metallochaperone ATOX1 and clearly demonstrate that the Ctr1 intracellular loop (1) can coordinate Cu(I) via interactions with the side chains of one histidine and two methionine residues and (2) closely interacts with the ATOX1 metallochaperone. Our findings are another important step in elucidating the mechanistic details of the eukaryotic copper cycle
-
Probing the Structural Flexibility of the Human Copper Metallochaperone ATOX1 Dimer and Its Interaction with the CTR1 C-Terminal Domain
The Journal of Physical Chemistry B, 2014Co-Authors: Ariel R. Levy, Valeria Yarmiayev, Yoni Moskovitz, Sharon RuthsteinAbstract:Both the essentiality and the toxicity of copper in human, yeast, and bacteria cells require precise mechanisms for acquisition, intimately linked to controlled distribution, which have yet to be fully understood. This work explores one aspect in the copper cycle, by probing the interaction between the human copper chaperone ATOX1 and the c-terminal domain of the copper transporter, CTR1, using electron paramagnetic resonance (EPR) spectroscopy and circular dichroism (CD). The data collected here shows that the ATOX1 keeps its dimer nature also in the presence of the CTR1 c-terminal domain; however, two geometrical states are assumed by the ATOX1. One is similar to the geometrical state reported by the crystal structure, while the latter has not yet been constructed. In the presence of the CTR1 c-terminal domain, both states are assumed; however, the structure of ATOX1 is more restricted in the presence of the CTR1 c-terminal domain. This study also shows that the last three amino acids of the CTR1 c-term...
Svetlana Lutsenko - One of the best experts on this subject based on the ideXlab platform.
-
The metal chaperone ATOX1 regulates the activity of the human copper transporter ATP7B by modulating domain dynamics.
Journal of Biological Chemistry, 2017Co-Authors: Nan Yang, Svetlana Lutsenko, Jameson R. Bothe, Marco Tonelli, Sergiy Nokhrin, Natalia V. Dolgova, Lelita T. Braiterman, Oleg Y. DmitrievAbstract:The human transporter ATP7B delivers copper to the biosynthetic pathways and maintains copper homeostasis in the liver. Mutations in ATP7B cause the potentially fatal hepatoneurological disorder Wilson disease. The activity and intracellular localization of ATP7B are regulated by copper, but the molecular mechanism of this regulation is largely unknown. We show that the copper chaperone ATOX1, which delivers copper to ATP7B, and the group of the first three metal-binding domains (MBD1-3) are central to the activity regulation of ATP7B. ATOX1-Cu binding to ATP7B changes domain dynamics and interactions within the MBD1-3 group and activates ATP hydrolysis. To understand the mechanism linking ATOX1-MBD interactions and enzyme activity, we have determined the MBD1-3 conformational space using small angle X-ray scattering and identified changes in MBD dynamics caused by apo-ATOX1 and ATOX1-Cu by solution NMR. The results show that copper transfer from ATOX1 decreases domain interactions within the MBD1-3 group and increases the mobility of the individual domains. The N-terminal segment of MBD1-3 was found to interact with the nucleotide-binding domain of ATP7B, thus physically coupling the domains involved in copper binding and those involved in ATP hydrolysis. Taken together, the data suggest a regulatory mechanism in which ATOX1-mediated copper transfer activates ATP7B by releasing inhibitory constraints through increased freedom of MBD1-3 motions.
-
The Role of Copper Chaperone ATOX1 in Coupling Redox Homeostasis to Intracellular Copper Distribution
Antioxidants, 2016Co-Authors: Yuta Hatori, Svetlana LutsenkoAbstract:Human antioxidant protein 1 (ATOX1) is a small cytosolic protein with an essential role in copper homeostasis. ATOX1 functions as a copper carrier facilitating copper transfer to the secretory pathway. This process is required for activation of copper dependent enzymes involved in neurotransmitter biosynthesis, iron efflux, neovascularization, wound healing, and regulation of blood pressure. Recently, new cellular roles for ATOX1 have emerged. Changing levels of ATOX1 were shown to modulate response to cancer therapies, contribute to inflammatory response, and protect cells against various oxidative stresses. It has also become apparent that the activity of ATOX1 is tightly linked to the cellular redox status. In this review, we summarize biochemical information related to a dual role of ATOX1 as a copper chaperone and an antioxidant. We discuss how these two activities could be linked and contribute to establishing the intracellular copper balance and functional identity of cells during differentiation.
-
an expanding range of functions for the copper chaperone antioxidant protein ATOX1
Antioxidants & Redox Signaling, 2013Co-Authors: Yuta Hatori, Svetlana LutsenkoAbstract:Abstract Significance: Antioxidant protein 1 (ATOX1 in human cells) is a copper chaperone for the copper export pathway with an essential role in cellular copper distribution. In vitro, ATOX1 binds and transfers copper to the copper-transporting ATPases, stimulating their catalytic activity. Inactivation of ATOX1 in cells inhibits maturation of secreted cuproenzymes as well as copper export from cells. Recent Advances: Accumulating data suggest that cellular functions of ATOX1 are not limited to its copper-trafficking role and may include storage of labile copper, modulation of transcription, and antioxidant defense. The conserved metal binding site of ATOX1, CxGC, differs from the metal-binding sites of copper-transporting ATPases and has a physiologically relevant redox potential that equilibrates with the GSH:GSSG pair. Critical Issues: Tight relationship appears to exist between intracellular copper levels and glutathione (GSH) homeostasis. The biochemical properties of ATOX1 place it at the intersect...
-
An expanding range of functions for the copper chaperone/antioxidant protein ATOX1.
Antioxidants & Redox Signaling, 2013Co-Authors: Yuta Hatori, Svetlana LutsenkoAbstract:Abstract Significance: Antioxidant protein 1 (ATOX1 in human cells) is a copper chaperone for the copper export pathway with an essential role in cellular copper distribution. In vitro, ATOX1 binds and transfers copper to the copper-transporting ATPases, stimulating their catalytic activity. Inactivation of ATOX1 in cells inhibits maturation of secreted cuproenzymes as well as copper export from cells. Recent Advances: Accumulating data suggest that cellular functions of ATOX1 are not limited to its copper-trafficking role and may include storage of labile copper, modulation of transcription, and antioxidant defense. The conserved metal binding site of ATOX1, CxGC, differs from the metal-binding sites of copper-transporting ATPases and has a physiologically relevant redox potential that equilibrates with the GSH:GSSG pair. Critical Issues: Tight relationship appears to exist between intracellular copper levels and glutathione (GSH) homeostasis. The biochemical properties of ATOX1 place it at the intersect...
-
Functional partnership of the copper export machinery and glutathione balance in human cells.
Journal of Biological Chemistry, 2012Co-Authors: Yuta Hatori, Sara Clasen, Nesrin M. Hasan, Amanda N. Barry, Svetlana LutsenkoAbstract:Abstract Cells use the redox properties of copper in numerous physiologic processes, including antioxidant defense, neurotransmitter biosynthesis, and angiogenesis. Copper delivery to the secretory pathway is an essential step in copper utilization and homeostatic maintenance. We demonstrate that the glutathione/glutathione disulfide (GSH/GSSG) pair controls the copper transport pathway by regulating the redox state of a copper chaperone ATOX1. GSSG oxidizes copper-coordinating cysteines of ATOX1 with the formation of an intramolecular disulfide. GSH alone is sufficient to reduce the disulfide, restoring the ability of ATOX1 to bind copper; glutaredoxin 1 facilitates this reaction when GSH is low. In cells, high GSH both reduces ATOX1 and is required for cell viability in the absence of ATOX1. In turn, ATOX1, which has a redox potential similar to that of glutaredoxin, becomes essential for cell survival when GSH levels decrease. ATOX1+/+ cells resist short term glutathione depletion, whereas ATOX1−/− cells under the same conditions are not viable. We conclude that GSH balance and copper homeostasis are functionally linked and jointly maintain conditions for copper secretion and cell proliferation.