Decreased Expression of the Slc31a1 Gene and Cytoplasmic Relocalization of Membrane CTR1 Protein in Renal Epithelial Cells: A Potent Protective Mechanism against Copper Nephrotoxicity in a Mouse Model of Menkes Disease.

Kidneys play an especial role in copper redistribution in the organism. The epithelial cells of proximal tubules perform the functions of both copper uptake from the primary urine and release to the blood. These cells are equipped on their apical and basal membrane with copper transporters CTR1 and ATP7A. Mosaic mutant mice displaying a functional dysfunction of ATP7A are an established model of Menkes disease. These mice exhibit systemic copper deficiency despite renal copper overload, enhanced by copper therapy, which is indispensable for their life span extension. The aim of this study was to analyze the expression of Slc31a1 and Slc31a2 genes (encoding CTR1/CTR2 proteins) and the cellular localization of the CTR1 protein in suckling, young and adult mosaic mutants. Our results indicate that in the kidney of both intact and copper-injected 14-day-old mutants showing high renal copper content, CTR1 mRNA level is not up-regulated compared to wild-type mice given a copper injection. The expression of the Slc31a1 gene in 45-day-old mice is even reduced compared with intact wild-type animals. In suckling and young copper-injected mutants, the CTR1 protein is relocalized from the apical membrane to the cytoplasm of epithelial cells of proximal tubules, the process which prevents copper transport from the primary urine and, thus, protects cells against copper toxicity.

Ubiquitin-independent proteasome system is required for degradation of Arabidopsis COPPER TRANSPORTER 2.

Plants have evolved sophisticated mechanisms to adjust to deficiency or excess of nutrients. Membrane transport proteins play a central role in nutrient uptake from soil. In Arabidopsis thaliana, the COPPER TRANSPORTOR (COPT) family encodes high-affinity copper transporters. COPT2 is transcriptionally regulated in response to changing levels of cellular copper. However, little is known about whether COPT2 activity is subject to multiple levels of regulation. Here, we showed that the plasma membrane-/endoplasmic reticulum-resident COPT2 protein is degraded in response to excess copper. Confocal microscopy analysis together with pharmacological treatment with a vesicle trafficking inhibitor or vacuolar ATPase inhibitor indicated that copper-mediated downregulation of COPT2 is unlikely to be controlled by endosomal recycling and vacuolar system. However, COPT2 protein is stabilized by proteasome inhibition. Through site-directed mutagenesis, we found that COPT2 cannot be ubiquitinated, and lysine residues at the C-terminus is dispensable for copper-induced degradation of COPT2 but required for copper acquisition. Altogether, our findings reveal that unlike many metal transporters in Arabidopsis, COPT2 is a substrate of ubiquitin-independent proteasomal degradation but not of vacuolar proteases. These findings highlight the mechanistic diversity and complexity of plasma membrane transporter degradation.

Altered copper homeostasis underlies sensitivity of hepatocellular carcinoma to copper chelation.

Hepatocellular carcinoma (HCC), the most common primary liver cancer, of which ∼800 000 new cases will be diagnosed worldwide this year, portends a five-year survival rate of merely 17% in patients with unresectable disease. This dismal prognosis is due, at least in part, from the late stage of diagnosis and the limited efficacy of systemic therapies. As a result, there is an urgent need to identify risk factors that contribute to HCC initiation and provide targetable vulnerabilities to improve patient survival. While myriad risk factors are known, elevated copper (Cu) levels in HCC patients and the incidence of hepatobiliary malignancies in Wilson disease patients, which exhibit hereditary liver Cu overload, suggests the possibility that metal accumulation promotes malignant transformation. Here we found that expression of the Cu transporter genes ATP7A, ATP7B, SLC31A1, and SLC31A2 was significantly altered in liver cancer samples and were associated with elevated Cu levels in liver cancer tissue and cells. Further analysis of genomic copy number data revealed that alterations in Cu transporter gene loci correlate with poorer survival in HCC patients. Genetic loss of the Cu importer SLC31A1 (CTR1) or pharmacologic suppression of Cu decreased the viability, clonogenic survival, and anchorage-independent growth of human HCC cell lines. Mechanistically, CTR1 knockdown or Cu chelation decreased glycolytic gene expression and downstream metabolite utilization and as a result forestalled tumor cell survival after exposure to hypoxia, which mimics oxygen deprivation elicited by transarterial embolization, a standard-of-care therapy used for patients with unresectable HCC. Taken together, these findings established an association between altered Cu homeostasis and HCC and suggest that limiting Cu bioavailability may provide a new treatment strategy for HCC by restricting the metabolic reprogramming necessary for cancer cell survival.

Designing yeast as plant-like hyperaccumulators for heavy metals.

Hyperaccumulators typically refer to plants that absorb and tolerate elevated amounts of heavy metals. Due to their unique metal trafficking abilities, hyperaccumulators are promising candidates for bioremediation applications. However, compared to bacteria-based bioremediation systems, plant life cycle is long and growing conditions are difficult to maintain hindering their adoption. Herein, we combine the robust growth and engineerability of bacteria with the unique waste management mechanisms of plants by using a more tractable platform-the common baker's yeast-to create plant-like hyperaccumulators. Through overexpression of metal transporters and engineering metal trafficking pathways, engineered yeast strains are able to sequester metals at concentrations 10-100 times more than established hyperaccumulator thresholds for chromium, arsenic, and cadmium. Strains are further engineered to be selective for either cadmium or strontium removal, specifically for radioactive Sr90. Overall, this work presents a systematic approach for transforming yeast into metal hyperaccumulators that are as effective as their plant counterparts.

Copper transporter COPT5 participates in the crosstalk between vacuolar copper and iron pools mobilisation.

Copper (Cu) deficiency affects iron (Fe) homeostasis in several plant processes, including the increased Fe requirements due to cuproprotein substitutions for the corresponding Fe counterpart. Loss-of-function mutants from Arabidopsis thaliana high affinity copper transporter COPT5 and Fe transporters NATURAL RESISTANCE-ASSOCIATED MACROPHAGE PROTEIN 3/4 (NRAMP3 and NRAMP4) were used to study the interaction between metals internal pools. A physiological characterisation showed that the copt5 mutant is sensitive to Fe deficiency, and that nramp3nramp4 mutant growth was severely affected under limiting Cu. By a transcriptomic analysis, we observed that NRAMP4 expression was highly induced in the copt5 mutant under Cu deficiency, while COPT5 was overexpressed in the nramp3nramp4 mutant. As a result, an enhanced mobilisation of the vacuolar Cu or Fe pools, when the other metal export through the tonoplast is impaired in the mutants, has been postulated. However, metals coming from internal pools are not used to accomplish the increased requirements that derive from metalloprotein substitution under metal deficiencies. Instead, the metal concentrations present in aerial parts of the copt5 and nramp3nramp4 mutants conversely show compensated levels of these two metals. Together, our data uncover an interconnection between Cu and Fe vacuolar pools, whose aim is to fulfil interorgan metal translocation.