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Tuesday, 20 November 2007

My project (13)

A critical part of the MNK and WND physiological response are their ability to change intracellular localisation in response to copper levels, relocating to sites where copper transport is required.
Under basal physiological copper levels, MNK and WND concentrate within the trans Golgi network (TGN) region, where they are postulated to pump copper into the TGN lumen for incorporatio into proteins on the secretory pathway.
When copper levels are raised, MNK has been reported to traffic to the plasma membrane and to the basolateral membrane in some polarized cell.
In response to elevated copper levels WND traffics to sub apical vesicles in some polarized cell lines, and has also been deonstrated partially at the apical membrane.
When intracellular copper levels are reduced, both transporters return via an endocytic route to the TGN.


praspowt said...

Kuo et al. (1997) determined the gene expression patterns during mouse embryonic development for the Atp7a and Atp7b genes by RNA in situ hybridization. Atp7a expression was widespread throughout development whereas Atp7b expression was more delimited. Kuo et al. (1997) suggested that Atp7a functions primarily in the homeostatic maintenance of cell copper levels, whereas Atp7b may be involved specifically in the biosynthesis of distinct cuproproteins in different tissues.

Studies in cultured cells localized the MNK protein to the final compartment of the Golgi apparatus, the trans-Golgi network (TGN). At this location, MNK is predicted to supply copper to the copper-dependent enzymes as they migrate through the secretory pathway. However, under conditions of elevated extracellular copper, the MNK protein undergoes a rapid relocalization to the plasma membrane where it functions in the efflux of copper from cells. By in vitro mutagenesis of the human ATP7A cDNA and immunofluorescence detection of mutant forms of the MNK protein expressed in cultured cells, Petris et al. (1998) demonstrated that the dileucine, L1487L1488, was essential for localization of MNK within the TGN, but not for copper efflux. They suggested that this dileucine motif is a putative endocytic targeting motif necessary for the retrieval of MNK from the plasma membrane to the TGN. Qian et al. (1998) and Francis et al. (1998) demonstrated that the third transmembrane region of the MNK protein functions as a TGN targeting signal; Petris et al. (1998) suggested that MNK localization to the TGN may be a 2-step process involving TGN retention by the transmembrane region, and recycling to this compartment from the plasma membrane via the L1487L1488 motif.

Petris et al. (2000) investigated whether the ATP7A protein is required for the activity of tyrosinase (606933), a copper-dependent enzyme involved in melanogenesis that is synthesized within the secretory pathway. Recombinant tyrosinase expressed in immortalized Menkes fibroblast cell lines was inactive, whereas in normal fibroblasts known to express ATP7A there was substantial tyrosinase activity. Coexpression of ATP7A and tyrosinase from plasmid constructs in Menkes fibroblasts led to the activation of tyrosinase and melanogenesis. This ATP7A-dependent activation of tyrosinase was impaired by the chelation of copper in the medium of cells and after mutation of the invariant phosphorylation site at aspartic acid residue 1044 of ATP7A. The authors proposed that ATP7A transports copper into the secretory pathway of mammalian cells to activate copper-dependent enzymes.

Cobbold et al. (2002) showed that endogenous ATP7A in cultured cell lines was localized to the distal Golgi apparatus and translocated to the plasma membrane in response to exogenous copper ions. This transport event was not blocked by expression of a dominant-negative mutant protein kinase D (PRKCM; 605435), an enzyme implicated in regulating constitutive trafficking from the TGN to the plasma membrane, whereas constitutive transport of CD4 (186940) was inhibited. In contrast, protein kinase A inhibitors blocked copper-stimulated ATP7A delivery to the plasma membrane. Expression of constitutively active Rho GTPases such as CDC42 (116952), RAC1 (602048), and RhoA (ARHA; 165390) revealed a requirement for CDC42 in the trafficking of ATP7A to the cell surface. Furthermore, overexpression of WASP (300392) inhibited anterograde transport of ATP7A, further supporting regulation by the CDC42 GTPase.

Cobbold et al. (2003) showed that ATP7A is internalized by a novel pathway that is independent of clathrin (see 118960)-mediated endocytosis. Expression of dominant-negative mutants of the dynamin-1 (DNM1; 602377), dynamin-2 (DNM2; 602378), and EPS15 (600051) proteins that block clathrin-dependent endocytosis of the transferrin receptor did not inhibit internalization of endogenous ATP7A or an ATP7A reporter molecule (CD8-MCF1). Similarly, inhibitors of caveolae (see 601047)-mediated uptake did not affect ATP7A internalization and prevented uptake of BODIPY-ganglioside GM1, a caveolae marker. In contrast, expression of a constitutively active mutant of the RAC1 GTPase inhibited plasma membrane internalization of both the ATP7A and transferrin receptor transmembrane proteins. Cobbold et al. (2003) concluded that their findings defined a novel route required for ATP7A internalization and delivery to endosomes.

Schlief et al. (2006) stated that ATP7A is required for production of an NMDA receptor (see GRIN1; 138249)-dependent releasable copper pool within hippocampal neurons, suggesting a role for copper in activity-dependent modulation of synaptic activity. In support of this hypothesis, they found that copper chelation exacerbated NMDA-mediated excitotoxic cell death in rat primary hippocampal neurons, whereas addition of copper was protective and significantly decreased cytoplasmic calcium levels after NMDA receptor activation. The protective effect of copper in hippocampal neurons depended on endogenous nitric oxide production, demonstrating an in vivo link between neuroprotection, copper metabolism, and nitrosylation. Using 'brindled' mice, a model of Menkes disease (see ANIMAL MODEL), Schlief et al. (2006) showed that ATP7A was required for these copper-dependent effects. Hippocampal neurons isolated from newborn brindled mice showed marked sensitivity to endogenous glutamate-mediated NMDA receptor-dependent excitotoxicity in vitro, and mild hypoxic/ischemic insult to these mice in vivo resulted in significantly increased caspase-3 (CASP3; 600636) activation and neuronal injury.

praspowt said...

Tanzi et al. (1993) noted that the protein encoded by the ATP7B gene had the characteristics of a copper-transporting ATPase. They suggested that it may serve a function in the export of copper from cells, whereas the Menkes gene product has a role in the import of copper. Dijkstra et al. (1995) studied copper transport in rat liver plasma membranes and suggested that the ATP7B protein functions to transport copper across these membranes in the presence of ATP. Harris (2000) reviewed cellular copper transport and metabolism and stated that the ATP7B protein resides within internal compartments of the cell, where it may function to incorporate copper into apo-ceruloplasmin, and to release copper into bile.

Variant Proteins
Forbes and Cox (2000) analyzed the intracellular localization of ATP7B variant proteins using transient transfection and triple-label immunofluorescence microscopy. Two human WND ATP7B variants, asp765 to asn (606882.0012) and leu776 to val, which have normal copper transport activity in yeast, retained partial normal Golgi network localization, but were predominantly mislocalized throughout the cell and were capable of only partial copper-dependent redistribution. Variant protein arg778 to leu (606882.0009), which has defective function in yeast, was extensively mislocalized, presumably to the endoplasmic reticulum. Variant proteins gly943 to ser (606882.0013), which has nearly normal function in yeast, and cys-pro-cys/ser (mutation of the conserved cys-pro-cys motif to ser-pro-ser), which is inactive in yeast, were localized normally but were unable to redistribute in response to copper. The authors hypothesized that mislocalization and/or deficient copper transport are defects seen in some mutant proteins, and that the nature of the malfunction(s) may explain, in part, the variable biochemical features of WND, in particular the normal holoceruloplasmin levels observed in some patients.

La Fontaine et al. (2001) generated cDNA constructs encoding the wildtype (Wnd-wt) and mutant (Wnd-tx) Wilson proteins (Wnd) and expressed them in Chinese hamster ovary (CHO) cells. The tx mutation disrupted the copper-induced relocalization of Wnd in CHO cells and abrogated Wnd-mediated copper resistance of transfected CHO cells. In addition, colocalization experiments demonstrated that while Wnd and MNK (309400) are located in the trans-Golgi network in basal copper conditions, with elevated copper, these proteins are sorted to different destinations within the same cell. Ultrastructural studies showed that with elevated copper levels, Wnd accumulated in large multivesicular structures resembling late endosomes that may represent a novel compartment for copper transport.