Human copper transporter 2 is localized in late endosomes and lysosomes and facilitates cellular copper uptake.

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.

Renal proximal tubular cells acquire resistance to cell death stimuli in mice with hereditary tyrosinemia type 1.

BACKGROUND: Hereditary tyrosinemia type 1 (HT1), which is associated with severe liver and kidney damage, is caused by deficiency of fumarylacetoacetate hydrolase (FAH), the last enzyme of the tyrosine breakdown cascade. HT1-associated liver and kidney failure can be prevented by blocking an enzyme upstream of FAH in the tyrosine breakdown pathway with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC). FAH knockout mice develop the HT1 phenotype when NTBC treatment is discontinued. METHODS: The occurrence of cell death was investigated in kidneys of Fah(-/-) mice on and off NTBC either unchallenged or injected with 800 mg/kg of homogentisic acid (HGA), an intermediate of tyrosine breakdown. RESULTS: No cell death could be detected in kidneys of Fah(-/-) mice on NTBC. A slight increase of cleaved caspase-3 was the only apoptosis-related feature that could be detected in kidneys of Fah(-/-) mice off NTBC. Challenge of Fah(-/-) mice on NTBC with HGA led to massive death of renal proximal tubular cells, with positive terminal deoxynucleotidyl transferase-mediated deoxyuridine diphosphate (dUDP) nick-end labeling (TUNEL) and DNA fragmentation assays, but hardly any cleavage of caspase-9 and caspase-3. Fah(-/-) mice off NTBC acquired resistance to HGA-induced renal cell death and the kidneys exhibited relatively few features of apoptosis upon challenge with HGA, with a small increase in expression of cleaved caspase-9 and caspase-3. CONCLUSION: Kidneys of adult Fah(-/-) mice, withdrawn from NTBC for 15 days, reveal limited characteristics of apoptosis, and have acquired resistance to a caspase-9- and caspase-3-independent form of cell death provoked by HGA.