The N-terminus of the human copper transporter 1 (hCTR1) is localized extracellularly, and interacts with itself.
Ontology highlight
ABSTRACT:
SUBMITTER: Klomp AE
PROVIDER: S-EPMC1223224 | biostudies-other | 2003 Mar
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Copper(I) is an essential metal for all life forms. Though Cu(II) is the most abundant and stable state, its reduction to Cu(I) via an unclear mechanism is prerequisite for its bioutilization. In eukaryotes, the copper transporter-1 (CTR1) is the primary high-affinity copper importer, although its mechanism and role in Cu(II) reduction remain uncharacterized. Here we show that extracellular amino-terminus of human CTR1 contains two methionine-histidine clusters and neighboring aspartates that distinctly bind Cu(I) and Cu(II) preceding its import. We determined that hCTR1 localizes at the basolateral membrane of polarized MDCK-II cells and that its endocytosis to Common-Recycling-Endosomes is regulated by reduction of Cu(II) to Cu(I) and subsequent Cu(I) coordination by the methionine cluster. We demonstrate the transient binding of both Cu(II) and Cu(I) during the reduction process is facilitated by aspartates that also act as another crucial determinant of hCTR1 endocytosis. Mutating the first Methionine cluster (7Met-Gly-Met9) and Asp13 abrogated copper uptake and endocytosis upon copper treatment. This phenotype could be reverted by treating the cells with reduced and nonreoxidizable Cu(I). We show that histidine clusters, on other hand, bind Cu(II) and are crucial for hCTR1 functioning at limiting copper. Finally, we show that two N-terminal His-Met-Asp clusters exhibit functional complementarity, as the second cluster is sufficient to preserve copper-induced CTR1 endocytosis upon complete deletion of the first cluster. We propose a novel and detailed mechanism by which the two His-Met-Asp residues of hCTR1 amino-terminus not only bind copper, but also maintain its reduced state, crucial for intracellular uptake.
| S-EPMC8867124 | biostudies-literature
Project description:High-affinity cellular copper uptake is mediated by the CTR (copper transporter) 1 family of proteins. The highly homologous hCTR (human CTR) 2 protein has been identified, but its function in copper uptake is currently unknown. To characterize the role of hCTR2 in copper homoeostasis, epitope-tagged hCTR2 was transiently expressed in different cell lines. hCTR2-vsvG (vesicular-stomatitis-virus glycoprotein) predominantly migrated as a 17 kDa protein after imunoblot analysis, consistent with its predicted molecular mass. Chemical cross-linking resulted in the detection of higher-molecular-mass complexes containing hCTR2-vsvG. Furthermore, hCTR2-vsvG was co-immunoprecipitated with hCTR2-FLAG, suggesting that hCTR2 can form multimers, like hCTR1. Transiently transfected hCTR2-eGFP (enhanced green fluorescent protein) was localized exclusively to late endosomes and lysosomes, and was not detected at the plasma membrane. To functionally address the role of hCTR2 in copper metabolism, a novel transcription-based copper sensor was developed. This MRE (metal-responsive element)-luciferase reporter contained four MREs from the mouse metallothionein 1A promoter upstream of the firefly luciferase open reading frame. Thus the MRE-luciferase reporter measured bioavailable cytosolic copper. Expression of hCTR1 resulted in strong activation of the reporter, with maximal induction at 1 muM CuCl2, consistent with the K(m) of hCTR1. Interestingly, expression of hCTR2 significantly induced MRE-luciferase reporter activation in a copper-dependent manner at 40 and 100 microM CuCl2. Taken together, these results identify hCTR2 as an oligomeric membrane protein localized in lysosomes, which stimulates copper delivery to the cytosol of human cells at relatively high copper concentrations. This work suggests a role for endosomal and lysosomal copper pools in the maintenance of cellular copper homoeostasis.
| S-EPMC2267400 | biostudies-other
Project description:Human copper transporter 1 (hCTR1) is a homotrimer of a 190-amino acid monomer having three transmembrane domains believed to form a pore for copper permeation through the plasma membrane. The hCTR1-mediated copper transport mechanism is not well understood, nor has any measurement been made of the rate at which copper ions are transported by hCTR1. In this study, we estimated the rate of copper transport by the hCTR1 trimer in cultured cells using (64)Cu uptake assays and quantification of plasma membrane hCTR1. For endogenous hCTR1, we estimated a turnover number of about 10 ions/trimer/s. When overexpressed in HEK293 cells, a second transmembrane domain mutant of hCTR1 (H139R) had a 3-fold higher Km value and a 4-fold higher turnover number than WT. Truncations of the intracellular C-terminal tail and an AAA substitution of the putative metal-binding HCH C-terminal tripeptide (thought to be required for transport) also exhibited elevated transport rates and Km values when compared with WT hCTR1. Unlike WT hCTR1, H139R and the C-terminal mutants did not undergo regulatory endocytosis in elevated copper. hCTR1 mutants combining methionine substitutions that block transport (M150L,M154L) on the extracellular side of the pore and the high transport H139R or AAA intracellular side mutations exhibited the blocked transport of M150L,M154L, confirming that Cu(+) first interacts with the methionines during permeation. Our results show that hCTR1 elements on the intracellular side of the hCTR1 pore, including the carboxyl tail, are not essential for permeation, but serve to regulate the rate of copper entry.
| S-EPMC3689948 | biostudies-literature
Project description:Copper uptake proteins (CTRs), mediate cellular acquisition of the essential metal copper in all eukaryotes. Here, we report the structure of the human CTR1 protein solved by electron crystallography to an in plane resolution of 7 A. Reminiscent of the design of traditional ion channels, trimeric hCTR1 creates a pore that stretches across the membrane bilayer at the interface between the subunits. Assignment of the helices identifies the second transmembrane helix as the key element lining the pore, and reveals how functionally important residues on this helix could participate in Cu(I)-coordination during transport. Aligned with and sealing both ends of the pore, extracellular and intracellular domains of hCTR1 appear to provide additional metal binding sites. Consistent with the existence of distinct metal binding sites, we demonstrate that hCTR1 stably binds 2 Cu(I)-ions through 3-coordinate Cu-S bonds, and that mutations in one of these putative binding sites results in a change of coordination chemistry.
| S-EPMC2647337 | biostudies-literature
Project description:The eukaryotic RNA exosome is a ribonucleolytic complex involved in RNA processing and turnover. It consists of a nine-subunit catalytically inert core that serves a structural function and participates in substrate recognition. Best defined in Saccharomyces cerevisiae, enzymatic activity comes from the associated subunits Dis3p (Rrp44p) and Rrp6p. The former is a nuclear and cytoplasmic RNase II/R-like enzyme, which possesses both processive exo- and endonuclease activities, whereas the latter is a distributive RNase D-like nuclear exonuclease. Although the exosome core is highly conserved, identity and arrangements of its catalytic subunits in different vertebrates remain elusive. Here, we demonstrate the association of two different Dis3p homologs--hDIS3 and hDIS3L--with the human exosome core. Interestingly, these factors display markedly different intracellular localizations: hDIS3 is mainly nuclear, whereas hDIS3L is strictly cytoplasmic. This compartmental distribution reflects the substrate preferences of the complex in vivo. Both hDIS3 and hDIS3L are active exonucleases; however, only hDIS3 has retained endonucleolytic activity. Our data suggest that three different ribonucleases can serve as catalytic subunits for the exosome in human cells.
| S-EPMC2910271 | biostudies-literature
Project description:Copper is an essential micronutrient. Following entry via the human copper transporter 1 (hCTR1), copper is delivered to several copper chaperones, which subsequently transfer the metal to specific targets via protein:protein interactions. It is has been assumed, but not demonstrated, that chaperones acquire copper directly from hCTR1. However, some reports have pointed to an intermediary role for glutathione (GSH), an abundant copper-binding tri-peptide. To address the issue of how transported copper is acquired by the copper chaperones in vivo, we measured the initial rate of (64)Cu uptake in cells in which the cellular levels of copper chaperones or GSH were substantially depleted or elevated. Knockdown or overexpression of copper chaperones ATOX1, CCS, or both had no effect on the initial rate of (64)Cu entry into HEK293 cells having endogenous or overexpressed hCTR1. In contrast, depleting cellular GSH using L-buthionine-sulfoximine (BSO) caused a 50% decrease in the initial rate of (64)Cu entry in HEK293 cells and other cell types. This decrease was reversed by washout of BSO or GSH replenishment with a permeable ester. BSO treatment under our experimental conditions had no significant effects on the viability, ATP levels, or metal content of the cells. Attenuated (64)Cu uptake in BSO was not due to oxidation of the cysteine in the putative metal-binding motif (HCH) at the intracellular hCTR1 COOH terminus, because a mutant lacking this motif was fully active, and (64)Cu uptake was still reduced by BSO treatment. Our data suggest that GSH plays an important role in copper handling at the entry step.
| S-EPMC3625801 | biostudies-literature
Project description:Matrilin-2 is a component of extracellular filamentous networks. To study the interactions by which it can be integrated into such assemblies, full-length and truncated forms of matrilin-2 were recombinantly expressed in HEK-293 cells and purified from conditioned medium. The recombinant proteins, when used in interaction assays, showed affinity to matrilin-2 itself, but also to other collagenous and non-collagenous extracellular matrix proteins. The interaction between matrilin-2 and collagen I was studied in greater detail and could be shown to occur at distinct sites on the collagen I molecule and to have a K (D) of about 3 x 10(-8) M. Interactions with some non-collagenous protein ligands were even stronger, with matrilin-2 binding to fibrillin-2, fibronectin and laminin-1-nidogen-1 complexes, with K (D) values in the range of 10(-8)-10(-11) M. Co-localization of matrilin-2 with these ligands in the dermal-epidermal basement membrane, in the microfibrils extending from the basement membrane into the dermis, and in the dermal extracellular matrix, indicates a physiological relevance of the interactions in the assembly of supramolecular extracellular matrix structures.
| S-EPMC1222949 | biostudies-other
Project description:Polypeptides present remarkable selectivity challenges for chemical methods. Amino groups are ubiquitous in polypeptide structure, yet few paradigms exist for reactivity and selectivity in arylation of amine groups. This communication describes the utilization of boronic acid reagents bearing certain o-electron withdrawing groups for copper-mediated amine arylation of the N-terminus under mild conditions and primarily aqueous solvent. The method adds to the toolkit of boronic acid reagents for polypeptide modification under mild conditions in water that shows complete selectivity for the N-terminus in the presence of lysine side chains.
| S-EPMC8162437 | biostudies-literature
Project description:Uptake of copper (Cu) ions into human cells is mediated by the plasma membrane protein Ctr1 and is followed by Cu transfer to cytoplasmic Cu chaperones for delivery to Cu-dependent enzymes. The C-terminal cytoplasmic tail of Ctr1 is a 13-residue peptide harboring an HCH motif that is thought to interact with Cu. We here employ biophysical experiments under anaerobic conditions in peptide models of the Ctr1 C-terminus to deduce Cu-binding residues, Cu affinity, and the ability to release Cu to the cytoplasmic Cu chaperone Atox1. Based on NMR assignments and bicinchoninic acid competition experiments, we demonstrate that Cu interacts in a 1:1 stoichiometry with the HCH motif with an affinity, KD, of ?10(-14) M. Removing either the Cys residue or the two His residues lowers the Cu-peptide affinity, but site specificity is retained. The C-terminal peptide and Atox1 do not interact in solution in the absence of Cu. However, as directly demonstrated at the residue level via NMR spectroscopy, Atox1 readily acquires Cu from the Cu-loaded peptide. We propose that Cu binding to the Ctr1 C-terminal tail regulates Cu transport into the cytoplasm such that the metal ion is only released to high-affinity Cu chaperones.
| S-EPMC4805863 | biostudies-other
Project description:The selective estrogen receptor modulator tamoxifen increases extracellular dopamine in vivo and acts as a neuroprotectant in models of dopamine neurotoxicity. We investigated the effect of tamoxifen on dopamine transporter (DAT)-mediated dopamine uptake, dopamine efflux, and [3H]WIN 35,428 [(-)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane] binding in rat striatal tissue. Tamoxifen dose-dependently blocked dopamine uptake (54% reduction at 10 μM) and amphetamine-stimulated efflux (59% reduction at 10 μM) in synaptosomes. It also produced a small but significant reduction in [3H]WIN 35,428 binding in striatal membranes, indicating a weak interaction with the substrate binding site in the DAT. Biotinylation and cysteine accessibility studies indicated that tamoxifen stabilizes the outward-facing conformation of the DAT in a cocaine-like manner and does not affect surface expression of the DAT. Additional studies with mutant DAT constructs D476A and I159A suggested a direct interaction between tamoxifen and a secondary substrate binding site of the transporter. Locomotor studies revealed that tamoxifen attenuates amphetamine-stimulated hyperactivity in rats but has no depressant or stimulant activity in the absence of amphetamine. These results suggest a complex mechanism of action for tamoxifen as a regulator of the DAT. Due to its effectiveness against amphetamine actions and its central nervous system permeant activity, the tamoxifen structure represents an excellent starting point for a structure-based drug-design program to develop a pharmacological therapeutic for psychostimulant abuse.
| S-EPMC7250473 | biostudies-literature