Miriam Suazo

Metallothionein is crucial for safe intracellular copper storage and cell survival at normal and supra-physiological exposure levels.

MTs (metallothioneins) increase the resistance of cells to exposure to high Cu (copper) levels. Characterization of the MT-Cu complex suggests that MT has an important role in the cellular storage and/or delivery of Cu ions to cuproenzymes. In this work we investigate how these properties contribute to Cu homoeostasis by evaluating the uptake, accumulation and efflux of Cu in wild-type and MT I/II null rat fibroblast cell lines. We also assessed changes in the expression of Cu metabolism-related genes in response to Cu exposure. At sub-physiological Cu levels (0.4 microM), the metal content was not dependent on MT; however, when extracellular Cu was increased to physiological levels (10 microM), MTs were required for the cells ability to accumulate the metal. The subcellular localization of the accumulated metal in the cytoplasm was MT-dependent. Following supra-physiological Cu exposure (>50 microM), MT null cells had a decreased capacity for Cu storage and an elevated sensitivity to a minor increment in intracellular metal levels, suggesting that intracellular Cu toxicity is due not to the metal content but to the interactions of the metal with cellular components. Moreover, MT null cells failed to show increased levels of mRNAs encoding MT I, SOD1 (superoxide dismutase 1) and Ccs1 (Cu chaperone for SOD) in response to Cu exposure. These results support a role for MT in the storage of Cu in a safe compartment and in sequestering an intracellular excess of Cu in response to supra-physiological Cu exposure. Gene expression analysis suggests the necessity of having MT as part of the signalling pathway that induces gene expression in response to Cu.

The N-terminal copper-binding domain of the amyloid precursor protein protects against Cu2+ neurotoxicity in vivo

The amyloid precursor protein (APP) contains a Cu binding domain (CuBD) localized between amino acids 135 and 156 (APP135‐156), which can reduce Cu2+ to Cu1+ in vitro. The physiological function of this APP domain has not yet being established; nevertheless several studies support the notion that the CuBD of APP is involved in Cu homeostasis. We used APP synthetic peptides to evaluate their protective properties against Cu2+ neurotoxicity in a bilateral intra‐hippocampal injection model. We found that human APP135‐156 protects against Cu2+‐induced neurotoxic effects, such as, impairment of spatial memory, neuronal cell loss, and astrogliosis. APP135‐156 lacking two histidine residues showed protection against Cu2+; however, APP135‐156 mutated in cysteine 144, a key residue in the reduction of Cu2+ to Cu1+, did not protect against Cu2+ neurotoxicity. In accordance with recent reports, the CuBD of the Caenorhabditis elegans, APL‐1, protected against Cu2+ neurotoxicity in vivo. We also found that Cu2+ neurotoxicity is associated with an increase in nitrotyrosine immunofluorescence as well as with a decrease in Cu2+ uptake. The CuBD of APP therefore may play a role in the detoxification of brain Cu.

Overexpression of amyloid precursor protein increases copper content in HEK293 cells

Amyloid precursor protein (APP) is a transmembrane glycoprotein widely expressed in mammalian tissues and plays a central role in Alzheimers disease. However, its physiological function remains elusive. Cu(2+) binding and reduction activities have been described in the extracellular APP135-156 region, which might be relevant for cellular copper uptake and homeostasis. Here, we assessed Cu(2+) reduction and (64)Cu uptake in two human HEK293 cell lines overexpressing APP. Our results indicate that Cu(2+) reduction increased and cells accumulated larger levels of copper, maintaining cell viability at supra-physiological levels of Cu(2+) ions. Moreover, wild-type cells exposed to both Cu(2+) ions and APP135-155 synthetic peptides increased copper reduction and uptake. Complementation of function studies in human APP751 transformed Fre1 defective Saccharomyces cerevisiae cells rescued low Cu(2+) reductase activity and increased (64)Cu uptake. We conclude that Cu(2+) reduction activity of APP facilitates copper uptake and may represent an early step in cellular copper homeostasis.

Assessing chaperone for Zn, Cu-superoxide dismutase as an indicator of copper deficiency in malnourished children

UNLABELLED It is not clear how frequent is copper deficiency in humans. Current copper markers are not sensitive enough to detect early copper deficiency and new markers are needed. CCS is a candidate to become a copper biomarker. OBJECTIVE Measuring CCS mRNA relative expression in malnourished children and compare results (a) with those of the same children after nutritional recovery and (b) with well-nourished children. METHOD On admission to the protocol and after 15 day nutritional treatment, severely (G1=18) and moderately (G2=10) malnourished children were compared with well-nourished healthy controls (G3=15), measuring anthropometric indicators, blood biochemistry, Cu, Fe and Zn serum concentrations, ceruloplasmin, C Reactive protein and mRNA abundance of CCS, SOD and MT2 in peripheral mononuclear cells. RESULT In malnourished groups, mean serum copper concentration was below the cut-off on admission to hospital and increased after 15 days (t-test, p<0.01). On admission to protocol, CCS mRNA abundance in G1 and G2 was higher than in G3 (one way ANOVA, p<0.001). After 15 days, CCS expression decreased as expected (t-test, p<0.001). Initial SOD mRNA relative abundance was higher in study groups than controls and also between G1 and G2 (One way ANOVA, both p<0.01); after 15 days, G1 and G2 were not different (t-test, NS). MT2A abundance of transcripts did not follow a clear change pattern. CONCLUSION CCS mRNA abundance responded as expected, being higher in malnourished children than in controls; nutritional recovery in these latter resulted in decreasing expression of the chaperone, supporting the hypothesis that CCS may be a copper biomarker.

Identification of reference genes for quantitative real-time PCR studies in human cell lines under copper and zinc exposure

Accurate quantification depends on normalization of the measured gene expression data. In particular, gene expression studies with exposure to metals are challenging due their toxicity and redox-active properties. Here, we assessed the stability of potential reference genes in three cell lines commonly used to study metal cell metabolism: Caco-2 (colon), HepG2 (liver) and THP-1 (peripheral blood) under copper (Cu) or zinc (Zn) exposure. We used combined statistical tools to identify the best reference genes from a set of eleven candidates, which included traditional “housekeeping” genes such as GAPDH and B-ACTIN, in cell lines exposed to high and low, Zn and Cu concentrations. The expression stabilities of ATP5B (ATP synthase) and CYC1 (subunits of the cytochrome) were the highest considering the effect of Zn and Cu treatments whereas SDHA (succinate dehydrogenase) was found to be the most unstable gene. Even though the transcriptional effect of Zn and Cu is very different in term of redox properties, the same best reference genes were identified when Zn or Cu treatments were analyzed together. Our results indicate that ATP5B/CYC1 are the best candidates for reference genes after metal exposure, which can be used as a suitable starting point to evaluate gene expression with other metals or in different cell types in human models.