The copper transporter, SLC31A1, transcriptionally activated by ELF3, imbalances copper homeostasis to exacerbate cisplatin-induced acute kidney injury through mitochondrial dysfunction.

Acute kidney injury (AKI) is a common complication of cisplatin chemotherapy, which greatly limits its clinical effect and application. This study explored the function of solute Carrier Family 31 Member 1 (SLC31A1) in cisplatin-induced AKI and its possible mechanism. Mice and HK-2 cells were exposed to cisplatin to establish the in vivo and in vitro AKI models. Cell viability was detected by CCK-8. Mitochondrial and oxidative damage was determined by Mito-Tracker Green staining, mtROS level, ATP production, mitochondrial membrane potential, MDA content and CAT activity. AKI was evaluated by renal function and histopathological changes. Apoptosis was detected by TUNEL and caspase-3 expression. Molecule expression was measured by RT-qPCR, Western blotting, and immunohistochemistry. Molecular mechanism was studied by luciferase reporter assay and ChIP. SLC31A1 level was predominantly increased by cisplatin exposure in AKI models. Notably, copper ion (Cu+) level was enhanced by cisplatin challenge. Moreover, Cu+ supplementation intensified cisplatin-induced cell death, mitochondrial dysfunction, and oxidative stress in HK-2 cells, indicating the involvement of cuproptosis in cisplatin-induced AKI, whereas these changes were partially counteracted by SLC31A1 knockdown. E74 like ETS transcription factor 3 (ELF3) could directly bind to SLC31A1 promoter and promote its transcription. ELF3 was up-regulated and positively correlated with SLC31A1 expression upon cisplatin-induced AKI. SLC31A1 silencing restored renal function, alleviated mitochondrial dysfunction, and apoptosis in cisplatin-induced AKI mice. ELF3 transcriptionally activated SLC31A1 to trigger cuproptosis that drove cisplatin-induced AKI through mitochondrial dysfunction, indicating that SLC31A1 might be a promising therapeutic target to mitigate AKI during cisplatin chemotherapy.

Copper transporter gene ATP7A: A predictive biomarker for immunotherapy and targeted therapy in hepatocellular carcinoma.

BACKGROUND: ATP7A is an important copper transporter that regulates numerous cellular biological processes. However, the role of ATP7A in immunotherapy and targeted therapy, especially for hepatocellular carcinoma (HCC), remains unknown. METHODS: We analyzed ATP7A expression and its effect on digestive system tumor prognoses, assessed its expression in tissue microarrays from 319 HCC patients, and investigated the relationship between ATP7A expression and tumor immunity. Specifically, we evaluated the possible association between ATP7A and programmed death ligand 1 (PD-L1) expression in human HCC tissues. Finally, we analyzed the effect of ATP7A on sorafenib efficacy in HCC. RESULTS: ATP7A is generally highly expressed in digestive system tumors but related to poor prognosis only in HCC. ATP7A levels are positively associated with immune cell infiltration and immune checkpoint expression (especially PD-L1). HCC patients coexpressing APT7A and PD-L1 demonstrate poor prognoses. Moreover, HCC patients with high ATP7A levels were more sensitive to sorafenib and demonstrated higher survival rates after sorafenib treatment. CONCLUSIONS: This study provides insights into the correlation between ATP7A levels and tumor immune infiltration and immune checkpoint function in HCC, sheds light on the significance of ATP7A in cancer progression, and provides guidance for more effective and general therapeutic strategies.

Biallelic pathogenic variants in COX11 are associated with an infantile-onset mitochondrial encephalopathy.

Primary mitochondrial diseases are a group of genetically and clinically heterogeneous disorders resulting from oxidative phosphorylation (OXPHOS) defects. COX11 encodes a copper chaperone that participates in the assembly of complex IV and has not been previously linked to human disease. In a previous study, we identified that COX11 knockdown decreased cellular adenosine triphosphate (ATP) derived from respiration, and that ATP levels could be restored with coenzyme Q10 (CoQ10 ) supplementation. This finding is surprising since COX11 has no known role in CoQ10 biosynthesis. Here, we report a novel gene-disease association by identifying biallelic pathogenic variants in COX11 associated with infantile-onset mitochondrial encephalopathies in two unrelated families using trio genome and exome sequencing. Functional studies showed that mutant COX11 fibroblasts had decreased ATP levels which could be rescued by CoQ10 . These results not only suggest that COX11 variants cause defects in energy production but reveal a potential metabolic therapeutic strategy for patients with COX11 variants.

Copper chelation therapy inhibits renal fibrosis by modulating copper transport proteins.

The copper (Cu) transporter proteins play an important role in the maintenance of the Cu homeostasis in the body. Lysyl oxidase (LOX) proteins are involved in crosslinking of collagens and elastin molecules resulting in the establishment of extracellular matrix (ECM) and require Cu for their functional activity. Although there are few reports showing the protective effects of Cu chelators, the mechanism behind protection remains unknown. The present study investigated the role of Cu transporter proteins in renal fibrosis. We used tubular epithelial cells and three different animal models of renal injury to investigate the induction of Cu transporter proteins in renal injury with different etiology. We used disulfiram, clioquinol as two Cu chelators and ammonium tetrathiomolybdate as a standard Cu chelator. In addition, ß-aminopropionitrile (BAPN) was used as a standard LOX inhibitor. We demonstrated that renal fibrosis is associated with the induction of Cu transporter proteins such as ATP7A and Copper Transporter 1 (CTR1) but the Cu overload did not induce renal fibrosis. In addition, the Cu chelators inhibited renal fibrosis by inhibiting the Cu transporter proteins.

The Crossroads between Host Copper Metabolism and Influenza Infection.

Three main approaches are used to combat severe viral respiratory infections. The first is preemptive vaccination that blocks infection. Weakened or dead viral particles, as well as genetic constructs carrying viral proteins or information about them, are used as an antigen. However, the viral genome is very evolutionary labile and changes continuously. Second, chemical agents are used during infection and inhibit the function of a number of viral proteins. However, these drugs lose their effectiveness because the virus can rapidly acquire resistance to them. The third is the search for points in the host metabolism the effect on which would suppress the replication of the virus but would not have a significant effect on the metabolism of the host. Here, we consider the possibility of using the copper metabolic system as a target to reduce the severity of influenza infection. This is facilitated by the fact that, in mammals, copper status can be rapidly reduced by silver nanoparticles and restored after their cancellation.

Molecular characterization of the COPT/Ctr-type copper transporter family under heavy metal stress in alfalfa.

In nature, heavy metals significantly affect crop growth and quality. Among various heavy metals, copper (Cu) is both essential and toxic to plants depending on the concentration and complex homeostatic networks. The Cu transporter family (COPT) plays important roles in Cu homeostasis, including absorption, transportation, and growth in plants; however, this gene family is still poorly understood in alfalfa (Medicago sativa L.). In this study, a total of 12 MsCOPTs were identified and characterized. Based on the conserved motif and phylogenetic analysis, MsCOPTs could be divided into four subgroups (A1, A2, A3, and B). Gene structure, chromosomal location, and synteny analyses of MsCOPTs showed that segmental and tandem duplications likely contributed to their evolution. Tissue-specific expression analysis of MsCOPT genes indicated diverse spatiotemporal expression patterns. Most MsCOPT genes had high transcription levels in roots and nodules, indicating that these genes may play vital roles in the absorption and transport of Cu through root. The complementary heterologous expression function of yeast once again indicates that root-specific COPT can supplement the growth of defective yeast strains on YPEG medium, suggesting that these genes are Cu transporters. In summary, for the first time, our research identified COPT family genes at the whole-genome level to provide guidance for effectively improving the problem of Cu deficiency in the grass-livestock chain and provide theoretical support for the subsequent development of grass and animal husbandry.

Caveolin-1 stabilizes ATP7A, a copper transporter for extracellular SOD, in vascular tissue to maintain endothelial function.

Caveolin-1 (Cav-1) is a scaffolding protein and a major component of caveolae/lipid rafts. Previous reports have shown that endothelial dysfunction in Cav-1-deficient (Cav-1-/-) mice is mediated by elevated oxidative stress through endothelial nitric oxide synthase (eNOS) uncoupling and increased NADPH oxidase. Oxidant stress is the net balance of oxidant generation and scavenging, and the role of Cav-1 as a regulator of antioxidant enzymes in vascular tissue is poorly understood. Extracellular SOD (SOD3) is a copper (Cu)-containing enzyme that is secreted from vascular smooth muscle cells/fibroblasts and subsequently binds to the endothelial cells surface, where it scavenges extracellular [Formula: see text] and preserves endothelial function. SOD3 activity is dependent on Cu, supplied by the Cu transporter ATP7A, but whether Cav-1 regulates the ATP7A-SOD3 axis and its role in oxidative stress-mediated vascular dysfunction has not been studied. Here we show that the activity of SOD3, but not SOD1, was significantly decreased in Cav-1-/- vessels, which was rescued by re-expression of Cav-1 or Cu supplementation. Loss of Cav-1 reduced ATP7A protein, but not mRNA, and this was mediated by ubiquitination of ATP7A and proteasomal degradation. ATP7A bound to Cav-1 and was colocalized with SOD3 in caveolae/lipid rafts or perinucleus in vascular tissues or cells. Impaired endothelium-dependent vasorelaxation in Cav-1-/- mice was rescued by gene transfer of SOD3 or by ATP7A-overexpressing transgenic mice. These data reveal an unexpected role of Cav-1 in stabilizing ATP7A protein expression by preventing its ubiquitination and proteasomal degradation, thereby increasing SOD3 activity, which in turn protects against vascular oxidative stress-mediated endothelial dysfunction.

Flavonoids as Potential Drugs for VPS13-Dependent Rare Neurodegenerative Diseases.

Several rare neurodegenerative diseases, including chorea acanthocytosis, are caused by mutations in the VPS13A-D genes. Only symptomatic treatments for these diseases are available. Saccharomyces cerevisiae contains a unique VPS13 gene and the yeast vps13Δ mutant has been proven as a suitable model for drug tests. A library of drugs and an in-house library of natural compounds and their derivatives were screened for molecules preventing the growth defect of vps13Δ cells on medium with sodium dodecyl sulfate (SDS). Seven polyphenols, including the iron-binding flavone luteolin, were identified. The structure-activity relationship and molecular mechanisms underlying the action of luteolin were characterized. The FET4 gene, which encodes an iron transporter, was found to be a multicopy suppressor of vps13Δ, pointing out the importance of iron in response to SDS stress. The growth defect of vps13Δ in SDS-supplemented medium was also alleviated by the addition of iron salts. Suppression did not involve cell antioxidant responses, as chemical antioxidants were not active. Our findings support that luteolin and iron may target the same cellular process, possibly the synthesis of sphingolipids. Unveiling the mechanisms of action of chemical and genetic suppressors of vps13Δ may help to better understand VPS13A-D-dependent pathogenesis and to develop novel therapeutic strategies.

Expanding Toolbox for Genes Expression of Yarrowia lipolytica to Include Novel Inducible, Repressible, and Hybrid Promoters.

Promoters are critical tools to precisely control gene expression for both synthetic biology and metabolic engineering. Although Yarrowia lipolytica has demonstrated many industrially relevant advantages, promoter discovery efforts on this non-conventional yeast are limited due to the challenge in finding suitable inducible and repressible promoters. Six copper-inducible promoters and five repressible promoters were isolated in this work. Especially, Cu2+-repressible promoters showed relatively high activity under non-repressing conditions compared with a constitutive promoter, but the strength could be almost fully repressed by a supplement of a low content of Cu2+. The six Cu2+-inducible promoters were engineered to improve their dynamic regulation range with a tandem upstream activation sequence. An engineered promoter was successfully used to construct a more productive pathway for production of a novel bioproduct, wax ester, than that used for both Cu2+-inducible promoter and constitutive promoter. This study provides effective tools applicable to fine-tune the gene expression in this microbial host.

The Caenorhabditis elegans homolog of human copper chaperone Atox1, CUC-1, aids in distal tip cell migration.

Cell migration is a fundamental biological process involved in for example embryonic development, immune system and wound healing. Cell migration is also a key step in cancer metastasis and the human copper chaperone Atox1 was recently found to facilitate this process in breast cancer cells. To explore the role of the copper chaperone in other cell migration processes, we here investigated the putative involvement of an Atox1 homolog in Caenorhabditis elegans, CUC-1, in distal tip cell migration, which is a key process during the development of the C. elegans gonad. Using knock-out worms, in which the cuc-1 gene was removed by CRISPR-Cas9 technology, we probed life span, brood size, as well as distal tip cell migration in the absence or presence of supplemented copper. Upon scoring of gonads, we found that cuc-1 knock-out, but not wild-type, worms exhibited distal tip cell migration defects in approximately 10-15% of animals and, had a significantly reduced brood size. Importantly, the distal tip cell migration defect was rescued by a wild-type cuc-1 transgene provided to cuc-1 knock-out worms. The results obtained here for C. elegans CUC-1 imply that Atox1 homologs, in addition to their well-known cytoplasmic copper transport, may contribute to developmental cell migration processes.

Copper promotes sheep pancreatic duct organoid growth by activation of an antioxidant protein 1-dependent MEK-ERK pathway.

Proper amounts of copper supplemented in livestock feed improve the physical growth and traits of farm animals. The pancreas is an important organ with both exocrine and endocrine portions. To investigate the role and mechanism of copper in the sheep pancreas, we first established sheep pancreatic duct organoids (sPDOs). We found that an appropriate amount of copper benefited the formation and growth of sPDOs, whereas excess or deficient copper damaged sPDOs. We found that the proliferation-stimulating effect of copper was related to the copper chaperone antioxidant protein 1 (ATOX1)-dependent activation of MEK-ERK1/2 signaling. Atox1 knockdown suppressed the cell proliferation of sPDOs, even in the presence of the MEK activator. These results indicate that moderate concentrations of copper promote sPDO growth through ATOX1-regulated cell proliferation by activation of MEK-ERK. Moreover, our study indicates that organoids may be a useful model to study organ growth mechanisms in livestock.

Endothelin-1 Mediated Decrease in Mitochondrial Gene Expression and Bioenergetics Contribute to Neurodegeneration of Retinal Ganglion Cells.

Endothelin-1 (ET-1) is a vasoactive peptide that is elevated in aqueous humor as well as circulation of primary open angle glaucoma (POAG) patients. ET-1 has been shown to promote degeneration of optic nerve axons and apoptosis of retinal ganglion cells (RGCs), however, the precise mechanisms are still largely unknown. In this study, RNA-seq analysis was used to assess changes in ET-1 mediated gene expression in primary RGCs, which revealed that 23 out of 156 differentially expressed genes (DEGs) had known or predicted mitochondrial function, of which oxidative phosphorylation emerged as the top-most enriched pathway. ET-1 treatment significantly decreased protein expression of key mitochondrial genes including cytochrome C oxidase copper chaperone (COX17) and ATP Synthase, H+ transporting, Mitochondrial Fo Complex (ATP5H) in primary RGCs and in vivo following intravitreal ET-1 injection in rats. A Seahorse ATP rate assay revealed a significant decrease in the rate of mitochondrial ATP production following ET-1 treatment. IOP elevation in Brown Norway rats showed a trend towards decreased expression of ATP5H. Our results demonstrate that ET-1 produced a decrease in expression of vital components of mitochondrial electron transport chain, which compromise bioenergetics and suggest a mechanism by which ET-1 promotes neurodegeneration of RGCs in glaucoma.

Copper trafficking in eukaryotic systems: current knowledge from experimental and computational efforts.

Copper plays a vital role in fundamental cellular functions, and its concentration in the cell must be tightly regulated, as dysfunction of copper homeostasis is linked to severe neurological diseases and cancer. This review provides a compendium of current knowledge regarding the mechanism of copper transfer from the blood system to the Golgi apparatus; this mechanism involves the copper transporter hCtr1, the metallochaperone Atox1, and the ATPases ATP7A/B. We discuss key insights regarding the structural and functional properties of the hCtr1-Atox1-ATP7B cycle, obtained from diverse studies relying on distinct yet complementary biophysical, biochemical, and computational methods. We further address the mechanistic aspects of the cycle that continue to remain elusive. These knowledge gaps must be filled in order to be able to harness our understanding of copper transfer to develop therapeutic approaches with the capacity to modulate copper metabolism.

In-Cell NMR in Human Cells: Direct Protein Expression Allows Structural Studies of Protein Folding and Maturation.

Cellular structural biology methods are needed to characterize biological processes at atomic resolution in the physiological environment of the cell. Toward this goal, solution in-cell NMR is a powerful approach because it provides structural and dynamic data on macromolecules inside living cells. Several approaches have been developed for in-cell NMR in cultured human cells, which are needed to study processes related to human diseases that rely on the delivery of exogenous macromolecules to the cells. Such strategies, however, may not be applicable to proteins that are sensitive to the external environment or prone to aggregate and can introduce artifacts during protein purification or delivery. As a complementary approach, direct protein expression for in-cell NMR in human cells was developed. This strategy is especially useful when studying processes like protein folding, maturation, and post-translational modification, starting right after protein synthesis. Compared with the protein expression techniques in mammalian cells commonly used in cellular biology, the low sensitivity of NMR requires higher protein levels. Among the cell lines used for high-yield protein expression, the HEK293T cell line was chosen, as it can be efficiently transfected with a cost-effective reagent. A vector originally designed for secreted proteins allows high-level cytosolic protein expression. For isotopic labeling, commercially available or homemade labeled media are employed. Uniform or amino acid type-selective labeling strategies are possible. Protein expression can be targeted to specific organelles (e.g., mitochondria), allowing for in organello NMR applications. A variant of the approach was developed that allows the sequential expression of two or more proteins, with only one selectively labeled. Protein expression in HEK293T cells was applied to recapitulate the maturation steps of intracellular superoxide dismutase 1 (SOD1) and to study the effect of mutations linked to familial amyotrophic lateral sclerosis (fALS) by in-cell NMR. Intracellular wild-type SOD1 spontaneously binds zinc, while it needs the copper chaperone for superoxide dismutase (CCS) for copper delivery and disulfide bond formation. Some fALS-linked mutations impair zinc binding and cause SOD1 to irreversibly unfold, likely forming the precursor of cytotoxic aggregates. The SOD-like domain of CCS acts as a molecular chaperone toward mutant SOD1, stabilizing its folding and allowing zinc binding and correct maturation. Changes in protein redox state distributions can also be investigated by in-cell NMR. Mitochondrial proteins require the redox-regulating partners glutaredoxin 1 (Grx1) and thioredoxin (Trx) to remain in the reduced, import-competent state in the cytosol, whereas SOD1 requires CCS for disulfide bond formation. In both cases, the proteins do not equilibrate with the cytosolic redox pool. Cysteine oxidation in response to oxidative stress can also be monitored. In the near future, in-cell NMR in human cells will likely benefit from technological advancements in NMR hardware, the development of bioreactor systems for increased sample lifetime, the application of paramagnetic NMR to obtain structural restraints, and advanced tools for genome engineering and should be increasingly integrated with advanced cellular imaging techniques.

The Involvement of Cytochrome c Oxidase in Mitochondrial Fusion in Primary Cultures of Neonatal Rat Cardiomyocytes.

Cytochrome c oxidase (CCO) is a copper-dependent enzyme of mitochondrial respiratory chain. In pressure overload-induced cardiac hypertrophy, copper level and CCO activity are both depressed, along with disturbance in mitochondrial fusion and fission dynamics. Copper repletion leads to recovery of CCO activity and normalized mitochondrial dynamics. The present study was undertaken to define the link between CCO activity and mitochondrial dynamic changes. Primary cultures of neonatal rat cardiomyocytes were treated with phenylephrine to induce cell hypertrophy. Hypertrophic cardiomyocytes were then treated with copper to reverse hypertrophy. In the hypertrophic cardiomyocytes, CCO activity was depressed and mitochondrial fusion was suppressed. Upon copper repletion, CCO activity was recovered and mitochondrial fusion was reestablished. Depression of CCO activity by siRNA targeting CCO assembly homolog 17 (COX17), a copper chaperone for CCO, led to fragmentation of mitochondria, which was not recoverable by copper supplementation. This study thus demonstrates that copper-dependent CCO is critical for mitochondrial fusion in the regression of cardiomyocyte hypertrophy.

Copper(I)-binding properties of de-coppering drugs for the treatment of Wilson disease. α-Lipoic acid as a potential anti-copper agent.

Wilson disease is an autosomal recessive genetic disorder caused by loss-of-function mutations in the P-type copper ATPase, ATP7B, which leads to toxic accumulation of copper mainly in the liver and brain. Wilson disease is treatable, primarily by copper-chelation therapy, which promotes copper excretion. Although several de-coppering drugs are currently available, their Cu(I)-binding affinities have not been quantitatively characterized. Here we determined the Cu(I)-binding affinities of five major de-coppering drugs - D-penicillamine, trientine, 2,3-dimercapto-1-propanol, meso-2,3-dimercaptosuccinate and tetrathiomolybdate - by exploring their ability to extract Cu(I) ions from two Cu(I)-binding proteins, the copper chaperone for cytochrome c oxidase, Cox17, and metallothionein. We report that the Cu(I)-binding affinity of these drugs varies by four orders of magnitude and correlates positively with the number of sulfur atoms in the drug molecule and negatively with the number of atoms separating two SH groups. Based on the analysis of structure-activity relationship and determined Cu(I)-binding affinity, we hypothesize that the endogenous biologically active substance, α-lipoic acid, may be suitable for the treatment of Wilson disease. Our hypothesis is supported by cell culture experiments where α-lipoic acid protected hepatic cells from copper toxicity. These results provide a basis for elaboration of new generation drugs that may provide better therapeutic outcomes.

Effective Inhibition of Cellular ROS Production by MXCXXC-Type Peptides: Potential Therapeutic Applications in Copper-Homeostasis Disorders.

Cyclic and acyclic peptides with sequences derived from metallochaperone binding sites, but differing at position 2, were analyzed for their inhibitory reactivity towards cellular ROS (reactive oxygen species) formation and catalytic activity towards oxidation with H2 O2 , in comparison with three commercial drugs clinically employed in chelation therapy for Wilson's disease. Acyclic peptides were more effective inhibitors than the cyclic ones, with one leading peptide with threonine at position 2 systematically showing the highest efficiency in reducing cellular ROS levels and in inhibiting Cu oxidation. This peptide was more effective than all commercial drugs in all aspects analyzed, and showed no toxicity towards human colon HT-29 cancer cells at concentrations 10-100 times higher than the IC50 of the commercial drugs, corroborating its high medicinal potential.

Reduced glutathione biosynthesis in Drosophila melanogaster causes neuronal defects linked to copper deficiency.

Glutathione (GSH) is a tripeptide often considered to be the master antioxidant in cells. GSH plays an integral role in cellular redox regulation and is also known to have a role in mammalian copper homeostasis. In vitro evidence suggests that GSH is involved in copper uptake, sequestration and efflux. This study was undertaken to further investigate the roles that GSH plays in neuronal copper homeostasis in vivo, using the model organism Drosophila melanogaster. RNA interference-mediated knockdown of the Glutamate-cysteine ligase catalytic subunit gene (Gclc) that encodes the rate-limiting enzyme in GSH biosynthesis was utilised to genetically deplete GSH levels. When Gclc was knocked down in all neurons, this caused lethality, which was partially rescued by copper supplementation and was exacerbated by additional knockdown of the copper uptake transporter Ctr1A, or over-expression of the copper efflux transporter ATP7. Furthermore, when Gclc was knocked down in a subset of neuropeptide-producing cells, this resulted in adult progeny with unexpanded wings, a phenotype previously associated with copper dyshomeostasis. In these cells, Gclc suppression caused a decrease in axon branching, a phenotype further enhanced by ATP7 over-expression. Therefore, we conclude that GSH may play an important role in regulating neuronal copper levels and that reduction in GSH may lead to functional copper deficiency in neurons in vivo. We provide genetic evidence that glutathione (GSH) levels influence Cu content or distribution in vivo, in Drosophila neurons. GSH could be required for binding Cu imported by Ctr1A and distributing it to chaperones, such as Mtn, CCS and Atox1. Alternatively, GSH could modify the copper-binding and transport activities of Atox1 and the ATP7 efflux protein via glutathionylation of copper-binding cysteines.

Soluble Moringa oleifera leaf extract reduces intracellular cadmium accumulation and oxidative stress in Saccharomyces cerevisiae.

Moringa oleifera leaves are a well-known source of antioxidants and traditionally used for medicinal applications. In the present study, the protective action of soluble M. oleifera leaf extract (MOLE) against cadmium toxicity was investigated in the model eukaryote Saccharomyces cerevisiae. The results showed that this extract exhibited a protective effect against oxidative stress induced by cadmium and H2O2 through the reduction of intracellular reactive oxygen species. Interestingly, not only the co-exposure of soluble MOLE with cadmium but also pretreatment of this extract prior to cadmium exposure significantly reduced the cadmium uptake through an inhibition of Fet4p, a low-affinity iron(II) transporter. In addition, the supplementation of soluble MOLE significantly reduced intracellular iron accumulation in a Fet4p-independent manner. Our findings suggest the potential use of soluble extract from M. oleifera leaves as a dietary supplement for protection against cadmium accumulation and oxidative stress.

Ultramild protein-mediated click chemistry creates efficient oligonucleotide probes for targeting and detecting nucleic acids.

Functionalized synthetic oligonucleotides are finding growing applications in research, clinical studies, and therapy. However, it is not easy to prepare them in a biocompatible and highly efficient manner. We report a new strategy to synthesize oligonucleotides with promising nucleic acid targeting and detection properties. We focus in particular on the pH sensitivity of these new probes and their high target specificity. For the first time, human copper(I)-binding chaperon Cox17 was applied to effectively catalyze click labeling of oligonucleotides. This was performed under ultramild conditions with fluorophore, peptide, and carbohydrate azide derivatives. In thermal denaturation studies, the modified probes showed specific binding to complementary DNA and RNA targets. Finally, we demonstrated the pH sensitivity of the new rhodamine-based fluorescent probes in vitro and rationalize our results by electronic structure calculations.