Copper resistance in the cold: Genome analysis and characterisation of a PIB-1 ATPase in Bizionia argentinensis.

Copper homeostasis is a fundamental process in organisms, characterised by unique pathways that have evolved to meet specific needs while preserving core resistance mechanisms. While these systems are well-documented in model bacteria, information on copper resistance in species adapted to cold environments is scarce. This study investigates the potential genes related to copper homeostasis in the genome of Bizionia argentinensis (JUB59-T), a psychrotolerant bacterium isolated from Antarctic seawater. We identified several genes encoding proteins analogous to those crucial for copper homeostasis, including three sequences of copper-transport P1B-type ATPases. One of these, referred to as BaCopA1, was chosen for cloning and expression in Saccharomyces cerevisiae. BaCopA1 was successfully integrated into yeast membranes and subsequently extracted with detergent. The purified BaCopA1 demonstrated the ability to catalyse ATP hydrolysis at low temperatures. Structural models of various BaCopA1 conformations were generated and compared with mesophilic and thermophilic homologous structures. The significant conservation of critical residues and structural similarity among these proteins suggest a shared reaction mechanism for copper transport. This study is the first to report a psychrotolerant P1B-ATPase that has been expressed and purified in a functional form.

AP-1γ2 is an adaptor protein 1 variant required for endosome-to-Golgi trafficking of the mannose-6-P receptor (CI-MPR) and ATP7B copper transporter.

Selective retrograde transport from endosomes back to the trans-Golgi network (TGN) is important for maintaining protein homeostasis, recycling receptors, and returning molecules that were transported to the wrong compartments. Two important transmembrane proteins directed to this pathway are the Cation-Independent Mannose-6-phosphate receptor (CI-MPR) and the ATP7B copper transporter. Among CI-MPR functions is the delivery of acid hydrolases to lysosomes, while ATP7B facilitates the transport of cytosolic copper ions into organelles or the extracellular space. Precise subcellular localization of CI-MPR and ATP7B is essential for the proper functioning of these proteins. This study shows that both CI-MPR and ATP7B interact with a variant of the clathrin adaptor 1 (AP-1) complex that contains a specific isoform of the γ-adaptin subunit called γ2. Through synchronized anterograde trafficking and cell-surface uptake assays, we demonstrated that AP-1γ2 is dispensable for ATP7B and CI-MPR exit from the TGN while being critically required for ATP7B and CI-MPR retrieval from endosomes to the TGN. Moreover, AP-1γ2 depletion leads to the retention of endocytosed CI-MPR in endosomes enriched in retromer complex subunits. These data underscore the importance of AP-1γ2 as a key component in the sorting and trafficking machinery of CI-MPR and ATP7B, highlighting its essential role in the transport of proteins from endosomes.

CtpB is a plasma membrane copper (I) transporting P-type ATPase of Mycobacterium tuberculosis.

BACKGROUND: The intracellular concentration of heavy-metal cations, such as copper, nickel, and zinc is pivotal for the mycobacterial response to the hostile environment inside macrophages. To date, copper transport mediated by P-type ATPases across the mycobacterial plasma membrane has not been sufficiently explored. RESULTS: In this work, the ATPase activity of the putative Mycobacterium tuberculosis P1B-type ATPase CtpB was associated with copper (I) transport from mycobacterial cells. Although CtpB heterologously expressed in M. smegmatis induced tolerance to toxic concentrations of Cu2+ and a metal preference for Cu+, the disruption of ctpB in M. tuberculosis cells did not promote impaired cell growth or heavy-metal accumulation in whole mutant cells in cultures under high doses of copper. In addition, the Cu+ ATPase activity of CtpB embedded in the plasma membrane showed features of high affinity/slow turnover ATPases, with enzymatic parameters KM 0.19 ± 0.04 µM and Vmax 2.29 ± 0.10 nmol/mg min. In contrast, the ctpB gene transcription was activated in cells under culture conditions that mimicked the hostile intraphagosomal environment, such as hypoxia, nitrosative and oxidative stress, but not under high doses of copper. CONCLUSIONS: The overall results suggest that M. tuberculosis CtpB is associated with Cu+ transport from mycobacterial cells possibly playing a role different from copper detoxification.

Substrain-related dependence of Cu(I)-ATPase activity among prion protein-null mice.

The prion protein (PrPC) binds copper and affects copper metabolism, albeit among a poorly understood functional landscape. Much of the data on physiological roles of PrPC were obtained in mice of mixed genetic background deficient of the PrPC-coding gene Prnp. This strategy is currently under scrutiny due to the flanking gene problem, in particular related with a polymorphism, typical of both the 129Sv and 129Ola mouse substrains, in the Sirpa gene located in the vicinity of Prnp. Here we report an investigation of biochemical properties of Cu(I)-ATPases as a function of genotype in two strains of PrPC-deficient mice. We found that both the brain and liver of Prnp-null mice of mixed B6;129Sv background had diminished activity, accompanied by increased catalytic phosphorylation of Cu(I)-ATPase, as compared with the respective wild-type animals. However, no such differences were found between Prnp-null and wild-type mice of a B10;129Ola background. Activity of Cu(I)-ATPase was strongly reduced in brain tissue from mice of 129Sv strain, when compared with wild-type either of B6;129Sv, and especially of mice of the B6 strain. No differences between wild-type and Prnp-null brain tissue were noted in the expression of either Atp7a or b genes, and RFLP analysis indicated that the Sirpa129 polymorphism was present in both the B6;129Sv and B10;129Ola Prnp-null mouse colonies used in this study. The results suggest a novel substrain-dependent effect of 129Sv, but not 129Ola, genotype upon the regulation of the Cu(I)-ATPase catalytic cycle in Prnp-null mice, rather than either a Prnp-dependent, or a 129 strain-dependent effect.

CtpB is a plasma membrane copper (I) transporting P-type ATPase of Mycobacterium tuberculosis

BACKGROUND: The intracellular concentration of heavy-metal cations, such as copper, nickel, and zinc is pivotal for the mycobacterial response to the hostile environment inside macrophages. To date, copper transport mediated by P-type ATPases across the mycobacterial plasma membrane has not been sufficiently explored. RESULTS: In this work, the ATPase activity of the putative Mycobacterium tuberculosis P1B-type ATPase CtpB was associated with copper (I) transport from mycobacterial cells. Although CtpB heterologously expressed in M. smegmatis induced tolerance to toxic concentrations of Cu2+ and a metal preference for Cu+, the disruption of ctpB in M. tuberculosis cells did not promote impaired cell growth or heavy-metal accumulation in whole mutant cells in cultures under high doses of copper. In addition, the Cu+ ATPase activity of CtpB embedded in the plasma mem-brane showed features of high affinity/slow turnover ATPases, with enzymatic parametersKM 0.19 ± 0.04 µM and Vmax 2.29 ± 0.10 nmol/mg min. In contrast, the ctpB gene transcription was activated in cells under culture conditions that mimicked the hostile intraphagosomal environment, such as hypoxia, nitrosative and oxidative stress, but not under high doses of copper. CONCLUSIONS: The overall results suggest that M. tuberculosis CtpB is associated with Cu+ transport from mycobacterial cells possibly playing a role different from copper detoxification.

Life-time exposure to waterborne copper II: Patterns of tissue accumulation and gene expression of the metal-transport proteins ctr1 and atp7b in the killifish Poecilia vivipara.

The involvement of transporting proteins on copper (Cu) bioaccumulation was evaluated in the killifish Poecilia vivipara chronically exposed to environmentally relevant concentrations of waterborne Cu. Fish (<24 h-old) were maintained under control condition or exposed to different waterborne Cu concentrations (5, 9 and 20 µg/L) for 28 and 345 days in saltwater. Following exposure periods, Cu accumulation and the expression of genes encoding for the high affinity Cu-transporter (ctr1) and the P-type Cu-ATPase (atp7b) were evaluated. Whole-body metal accumulation and gene expression were evaluated in fish exposed to 28 days. Similarly, in fish exposed to 345 days, liver, gills and gut were also evaluated. No fish survival was observed after exposure to 20 µg/L for 345 days. Whole-body Cu accumulation was significantly higher in fish exposed to 20 µg/L Cu for 28 days and in fish exposed to 9 µg/L for 345 days in comparison to control animals. Similarly, tissue Cu accumulation was significantly higher in fish exposed to 9 µg/L for 345 days in comparison to control animal. However, no significant accumulation was observed in fish muscle. Following exposure for 28 days, whole-body ctr1 expression was slightly induced in fish exposed to 9 µg/L. In turn, no significant change in ctr1 expression was observed following exposure to Cu for 345 days. Differently, whole-body atp7b expression was markedly up-regulated in the whole-body of fish exposed Cu for 28 days and in tissues of fish exposed to Cu for 345 days. These findings indicate the expression of atp7b is more responsive to Cu accumulation in P. vivipara than ctr1 expression and, therefore, more suitable to be used as a biomarker of exposure to this metal. Also, we argue that the expression of atp7b is sustained at elevated levels for as much time as fish are maintained in Cu contaminated water.

Protective effects of neocuproine copper chelator against oxidative damage in NSC34 cells.

In this work, we aim to provide evidence for the protective effect of a copper chelator, neocuproine (NeoCu), against the oxidative stress in NSC34 cells, which inhibits biomolecule oxidation and cell death. Results obtained with the comet assay allowed to determine the increase in oxidized purines and pyrimidines by H2O2 exposure, and their changes after the addition of NeoCu. We also observed a higher ATP7b activity in nuclei and a higher Cu concentration inside the cells, proving that the NeoCu acts directly in DNA to promote cell recovery in oxidative stress conditions, also observed in Reactive Oxygen Species (ROS) detection assay by Flow Cytometry. Based on these results, we propose that NeoCu is a promising drug for the protection of motor neuron cells during oxidative stress caused by neurodegenerative diseases in this system.

Metal accumulation and expression of genes encoding for metallothionein and copper transporters in a chronically exposed wild population of the fish Hyphessobrycon luetkenii.

In the present study, metal (As, Cd, Cu, Fe, Mn, Pb and Zn) accumulation and expression of genes involved in metal metabolism (metallothioneins, ATP7A and CTR1) were evaluated in gills and muscle of the fish Hyphessobrycon luetkenii living in the João Dias creek, a site historically (~1870-1996) contaminated with a metal mixture associated with copper mining (Minas do Camaquã, southern Brazil). Fish were collected in a metal impacted site of the João Dias creek and kept in a cage at this site (PP fish) or translocated to a non-metal impacted reach of this creek (PC fish). Gill metal concentrations and metallothionein gene expression were lower in PC fish than in PP fish at any experimental time (24, 48 and 72 h). In muscle, no significant changes were observed. These findings indicate that metal accumulated in gills of wild fish chronically exposed to the metal mixture are more easily excreted than those accumulated in the muscle. In this case, expression of gene encoding for metallothionein is shown to play a key role in the regulation of metal accumulation in gills of H. luetkenii living in an area historically contaminated with a metal mixture associated with copper mining.

Inhibiting ERK/Mnk/eIF4E broadly sensitizes ovarian cancer response to chemotherapy.

PURPOSE: To investigate whether ERK/MNK/eIF4E contributes chemoresistance in ovarian cancer. METHODS: The phosphorylated levels of Erk, Mnk, and eIF4E were systematically analyzed in ovarian cancer patients before and after chemotherapy, and ovarian cancer cells exposed to short- and long-term chemo-agent treatment. The roles of Erk/Mnk/eIF4E were investigated using pharmacological and genetic approaches. RESULTS: Increased phosphorylation levels of ERK, Mnk1, and eIF4E were observed in ovarian cancer cell exposed to chemotherapeutic agents, and paclitaxel-resistant SK-OV-3-r cells, and is a common response of ovarian cancer patients undergoing chemotherapy. MEK inhibitor U0126 inhibits basal and chemodrug-induced phosphorylation of ERK as well as Mnk1 and eIF4E, suggesting that Mnk1/eIF4E are the downstream signaling of ERK pathway and chemotherapy agents activate ERK/MNK/eIF4E in a MEK-dependent manner. eIF4E overexpression promotes ovarian cancer cell growth without affecting migration. In addition, ovarian cancer cells with eIF4E overexpression are more resistant to chemotherapeutic agents in aspect of growth inhibition and apoptosis induction compared to control cells. In contrast, eIF4E depletion augments chemotherapeutic agents' effect in ovarian cancer cells. These demonstrate that eIF4E play roles in growth and chemoresistance in ovarian cancer. MEK inhibitor U0126 also significantly enhances chemotherapeutic agents' inhibitory effects. CONCLUSIONS: Our work shows that ERK/Mnk/eIF4E activation is critically involved in ovarian cancer chemoresistance and inhibiting ERK/Mnk/eIF4E broadly sensitizes ovarian cancer response to chemotherapy.