EPR Spectroscopy Shows that the Blood Carrier Protein, Human Serum Albumin, Closely Interacts with the N-Terminal Domain of the Copper Transporter, Ctr1

Shenberger, Yulia; Shimshi, Amit; Ruthstein, Sharon

doi:10.1021/acs.jpcb.5b00091

Cited by 32 publications

(45 citation statements)

References 51 publications

(105 reference statements)

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“…The native-sequence peptide, Ctr1-14, has been investigated previously [5, 7, 20]. This native-sequence peptide contains the ATCUN and bis -His motifs discussed above, which participate in Cu(II) acquisition from human serum albumin and copper reduction in the presence of ascorbate.…”

Section: Resultsmentioning

confidence: 99%

“…Recent evidence demonstrates that the extracellular copper carrier, human serum albumin, can directly interact with the Ctr1 amino terminus [5] and can transfer Cu(II) to the extracellular domain of human Ctr1 [6]. The extracellular domain of human Ctr1 binds to Cu(II) with an amino terminal high affinity Cu(II), Ni(II) ATCUN binding site, comprised of a histidine in the third position from the free amino terminal [7-9].…”

Section: Introductionmentioning

confidence: 99%

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Sequence proximity between Cu(II) and Cu(I) binding sites of human copper transporter 1 model peptides defines reactivity with ascorbate and O2

Schwab

Shearer

Conklin

et al. 2016

Journal of Inorganic Biochemistry

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The critical nature of the copper transporter 1 (Ctr1) in human health has spurred investigation of Ctr1 structure and function. Ctr1 specifically transports Cu(I), the reduced form of copper, across the plasma membrane. Thus, extracellular Cu(II) must be reduced prior to transport. Unlike yeast Ctr1, mammalian Ctr1 does not rely on any known mammalian reductase. Previous spectroscopic studies of model peptides indicate that human Ctr1 could serve as both copper reductase and transporter. Ctr1 peptides bind Cu(II) at an amino terminal high-affinity Cu(II), Ni(II) (ATCUN) site. Ascorbate-dependent reduction of the Cu(II)-ATCUN complex is possible by virtue of an adjacent HH (bis-His), as this bis-His motif and one methionine ligand constitute a high affinity Ctr1 Cu(I) binding site. Here, we synthetically varied the distance between the ATCUN and bis-His motifs in a series of peptides based on the human Ctr1 amino terminal, with general sequence MDHAnHHMGMSYMDS, where n = 0-4. We tested the ability of each peptide to reduce Cu(II) with ascorbate and stabilize Cu(I) under ambient conditions (20% O2). This study reveals that significant differences in coordination structure and chemical behavior with ascorbate and O2 result from changes in the sequence proximity of ATCUN and bis-His. Peptides that deviate from the native Ctr1 pattern were less effective at forming stable Cu(I)-peptide complexes and/or resulted in O2-dependent oxidative damage to the peptide.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Sequence proximity between Cu(II) and Cu(I) binding sites of human copper transporter 1 model peptides defines reactivity with ascorbate and O2

Schwab

Shearer

Conklin

et al. 2016

Journal of Inorganic Biochemistry

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show abstract

“…Recent evidence suggests that human serum albumin can directly interact with a model peptide of the Ctr1 N-terminal (designated Ctr1−14: MDHSHHMGMSYMDS), 39 and further evidence demonstrates that serum albumin can transfer Cu(II) to the extracellular domain of Ctr1. 40 Because Ctr1 specifically transports Cu(I) across the plasma membrane, extracellular Cu(II) must therefore be reduced to Cu(I) prior to Ctr1-dependent transport.…”

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confidence: 99%

Model Peptide Studies Reveal a Mixed Histidine-Methionine Cu(I) Binding Site at the N-Terminus of Human Copper Transporter 1

et al. 2015

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Copper is a vital metal cofactor in enzymes that are essential to myriad biological processes. Cellular acquisition of copper is primarily accomplished through the Ctr family of plasma membrane copper transport proteins. Model peptide studies indicate that the human Ctr1 N-terminus binds to Cu(II) with high affinity through an amino terminal Cu(II), Ni(II) (ATCUN) binding site. Unlike typical ATCUN-type peptides, the Ctr1 peptide facilitates the ascorbate-dependent reduction of Cu(II) bound in its ATCUN site by virtue of an adjacent HH (bis-His) sequence in the peptide. It is likely that the Cu(I) coordination environment influences the redox behavior of Cu bound to this peptide; however, the identity and coordination geometry of the Cu(I) site has not been elucidated from previous work. Here, we show data from NMR, XAS, and structural modeling that sheds light on the identity of the Cu(I) binding site of a Ctr1 model peptide. The Cu(I) site includes the same bis-His site identified in previous work to facilitate ascorbate-dependent Cu(II) reduction. The data presented here are consistent with a rational mechanism by which Ctr1 provides coordination environments that facilitate Cu(II) reduction prior to Cu(I) transport.

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“…To transport copper into the cells, albumin or transcuprein delivers copper to the extracellular domain of the plasma membrane transporter CTR-1, which then imports copper across the cell membrane and further deliver to copper chaperone proteins [82,83]. Copper(II) is believed to be reduced to Cu(I) during this stage by metalloreductases, but the exact reduction process and maintenance of Cu(I) with the presence of oxygen before reaching CTR-1 remains unclear [76].…”

Section: Copper Metabolismmentioning

confidence: 99%

Hypoxia imaging and theranostic potential of [64Cu][Cu(ATSM)] and ionic Cu(II) salts: a review of current evidence and discussion of the retention mechanisms

et al. 2020

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Background: Tumor hypoxia (low tissue oxygenation) is an adverse condition of the solid tumor environment, associated with malignant progression, radiotherapy resistance, and poor prognosis. One method to detect tumor hypoxia is by positron emission tomography (PET) with the tracer [ 64 Cu][Cu-diacetyl-bis(N(4)methylthiosemicarbazone)] ([ 64 Cu][Cu(ATSM)]), as demonstrated in both preclinical and clinical studies. In addition, emerging studies suggest using [ 64 Cu][Cu(ATSM)] for molecular radiotherapy, mainly due to the release of therapeutic Auger electrons from copper-64, making [ 64 Cu][Cu(ATSM)] a "theranostic" agent. However, the radiocopper retention based on a metal-ligand dissociation mechanism under hypoxia has long been controversial. Recent studies using ionic Cu(II) salts as tracers have raised further questions on the original mechanism and proposed a potential role of copper itself in the tracer uptake. We have reviewed the evidence of using the copper radiopharmaceuticals [ 60/61/62/64 Cu][Cu(ATSM)]/ionic copper salts for PET imaging of tumor hypoxia, their possible therapeutic applications, issues related to the metal-ligand dissociation mechanism, and possible explanations of copper trapping based on studies of the copper metabolism under hypoxia. Results:We found that hypoxia selectivity of [ 64 Cu][Cu(ATSM)] has been clearly demonstrated in both preclinical and clinical studies. Preclinical therapeutic studies in mice have also demonstrated promising results, recently reporting significant tumor volume reductions and improved survival in a dose-dependent manner. Cu(II)-[Cu(ATSM)] appears to be accumulated in regions with substantially higher CD133 + expression, a marker for cancer stem cells. This, combined with the reported requirement of copper for activation of the hypoxia inducible factor 1 (HIF-1), provides a possible explanation for the therapeutic effects of [ 64 Cu][Cu(ATSM)]. Comparisons between [ 64 Cu][Cu(ATSM)] and ionic Cu(II) salts have showed similar results in both imaging and therapeutic studies, supporting the argument for the central role of copper itself in the retention mechanism.Conclusions: We found promising evidence of using copper-64 radiopharmaceuticals for both PET imaging and treatment of hypoxic tumors. The Cu(II)-[Cu(ATSM)] retention mechanism remains controversial and future mechanistic studies should be focused on understanding the role of copper itself in the hypoxic tumor metabolism.

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EPR Spectroscopy Shows that the Blood Carrier Protein, Human Serum Albumin, Closely Interacts with the N-Terminal Domain of the Copper Transporter, Ctr1

Cited by 32 publications

References 51 publications

Sequence proximity between Cu(II) and Cu(I) binding sites of human copper transporter 1 model peptides defines reactivity with ascorbate and O2

Sequence proximity between Cu(II) and Cu(I) binding sites of human copper transporter 1 model peptides defines reactivity with ascorbate and O2

Model Peptide Studies Reveal a Mixed Histidine-Methionine Cu(I) Binding Site at the N-Terminus of Human Copper Transporter 1

Hypoxia imaging and theranostic potential of [64Cu][Cu(ATSM)] and ionic Cu(II) salts: a review of current evidence and discussion of the retention mechanisms

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