ATP7B expression in human breast epithelial cells is mediated by lactational hormones

A role for the copper transporter, ATP7B, in secretion of copper from the human breast into milk has previously not been reported, although it is known that the murine ortholog of ATP7B facilitates copper secretion in the mouse mammary gland. We show here that ATP7B is expressed in luminal epithelial cells in both the resting and lactating human breast, where it has a perinuclear localization in resting epithelial cells and a diffuse location in lactating tissue. ATP7B protein was present in a different subset of vesicles from those containing milk proteins and did not overlap with Menkes ATPase, ATP-7A, except in the perinuclear region of cells. In the cultured human mammary line, PMC42-LA, treatment with lactational hormones induced a redistribution of ATP7B from a perinuclear region to a region adjacent, but not coincident with, the apical plasma membrane. Trafficking of ATP7B was copper dependent, suggesting that the hormone-induced redistribution of ATP7A was mediated through an increase in intracellular copper. Radioactive copper (⁶⁴Cu) studies using polarized PMC42-LA cells that overexpressed mAtp7B protein showed that this transporter facilitates copper efflux from the apical surface of the cells. In summary, our results are consistent with an important function of ATP7B in the secretion of copper from the human mammary gland.

Copper-induced trafficking of the Cu-ATPase: a key mechanism for copper homeostasis

The Menkes protein (MNK) and Wilson protein (WND) are transmembrane, CPX-type Cu-ATPases with six metal binding sites (MBSs) in the N-terminal region containing the motif GMXCXXC. In cells cultured in low copper concentration MNK and WND localize to the transGolgi network but in high copper relocalize either to the plasma membrane (MNK) or a vesicular compartment (WND). In this paper we investigate the role of the MBSs in Cu-transport and trafficking. The copper transport activity of MBS mutants of MNK was determined by their ability to complement a strain of Saccharomyces cerevisiae deficient in CCC2 (Deltaccc2), the yeast MNK/WND homologue. Mutants (CXXC to SXXS) of MBS1, MBS6, and MBSs1-3 were able to complement Deltaccc2 while mutants of MBS4-6, MBS5-6 and all six MBS inactivated the protein. Each of the inactive mutants also failed to display Cu-induced trafficking suggesting a correlation between trafficking and transport activity. A similar correlation was found with mutants of MNK in which various MBSs were deleted, but two constructs with deletion of MBS5-6 were unable to traffic despite retaining 25% of copper transport activity. Chimeras in which the N-terminal MBSs of MNK were replaced with the corresponding MBSs of WND were used to investigate the region of the molecules that is responsible for the difference in Cu-trafficking of MNK and WND. The chimera which included the complete WND N-terminus localized to a vesicular compartment, similar to WND in elevated copper. Deletions of various MBSs of the WND N-terminus in the chimera indicate that a targeting signal in the region of MBS6 directs either WND/MNK or WND to a vesicular compartment of the cell.

Copper exposure induces trafficking of the Menkes protein in intestinal epithelium of ATP7A transgenic mice

The final steps in the absorption and excretion of copper at the molecular level are accomplished by 2 closely related proteins that catalyze the ATP-dependent transport of copper across the plasma membrane. These proteins, ATP7A and ATP7B, are encoded by the genes affected in human genetic copper-transport disorders, namely, Menkes and Wilson diseases. We studied the effect of copper perfusion of an isolated segment of the jejunum of ATP7A transgenic mice on the intracellular distribution of ATP7A by immunofluorescence of frozen sections. Our results indicate that ATP7A is retained in the trans-Golgi network under copper-limiting conditions, but relocalized to a vesicular compartment adjacent to the basolateral membrane in intestines perfused with copper. The findings support the hypothesis that the basolateral transport of copper from the enterocyte into the portal blood may involve ATP7A pumping copper into a vesicular compartment followed by exocytosis to release the copper, rather than direct pumping of copper across the basolateral membrane.

Trafficking of the copper-ATPases, ATP7A and ATP7B: Role in copper homeostasis

Copper is essential for human health and copper imbalance is a key factor in the aetiology and pathology of several neurodegenerative diseases. The copper-transporting P-type ATPases, ATP7A and ATP7B are key molecules required for the regulation and maintenance of mammalian copper homeostasis. Their absence or malfunction leads to the genetically inherited disorders, Menkes and Wilson diseases, respectively. These proteins have a dual role in cells, namely to provide copper to essential cuproenzymes and to mediate the excretion of excess intracellular copper. A unique feature of ATP7A and ATP7B that is integral to these functions is their ability to sense and respond to intracellular copper levels, the latter manifested through their copper-regulated trafficking from the transGolgi network to the appropriate cellular membrane domain (basolateral or apical, respectively) to eliminate excess copper from the cell. Research over the last decade has yielded significant insight into the enzymatic properties and cell biology of the copper-ATPases. With recent advances in elucidating their localization and trafficking in human and animal tissues in response to physiological stimuli, we are progressing rapidly towards an integrated understanding of their physiological significance at the level of the whole animal. This knowledge in turn is helping to clarify the biochemical and cellular basis not only for the phenotypes conferred by individual Menkes and Wilson disease patient mutations, but also for the clinical variability of phenotypes associated with each of these diseases. Importantly, this information is also providing a rational basis for the applicability and appropriateness of certain diagnostic markers and therapeutic regimes. This overview will provide an update on the current state of our understanding of the localization and trafficking properties of the copper-ATPases in cells and tissues, the molecular signals and posttranslational interactions that govern their trafficking activities, and the cellular basis for the clinical phenotypes associated with disease-causing mutations.

Endosomal trafficking of the Menkes copper ATPase ATP7A is mediated by vesicles containing the Rab7 and Rab5 GTPase proteins

The Cu-ATPase ATP7A (MNK) is localized in the trans-Golgi network (TGN) and relocalizes in the plasma membrane via vesicle-mediated traffic following exposure of the cells to high concentrations of copper. Rab proteins are organelle-specific GTPases, markers of different endosomal compartments; their role has been recently reviewed (Trends Cell Biol. 11(2001) 487). In this article we analyze the endosomal pathway of trafficking of the MNK protein in stably transfected clones of CHO cells, expressing chimeric Rab5-myc or Rab7-myc proteins, markers of early or late endosome compartments, respectively. We demonstrate by immunofluorescence and confocal and electron microscopy techniques that the increase in the concentration of copper in the medium (189 μM) rapidly induces a redistribution of the MNK protein from early sorting endosomes, positive for Rab5-myc protein, to late endosomes, containing the Rab7-myc protein. Cell fractionation experiments confirm these results; i.e., the MNK protein is recruited to the endosomal fraction on copper stimulation and colocalizes with Rab5 and Rab7 proteins. These findings allow the first characterization of the vesicles involved in the intracellular routing of the MNK protein from the TGN to the plasma membrane, a key mechanism allowing appropriate efflux of copper in cells grown in high concentrations of the metal.

MSP119 miniproteins can serve as targets for invasion inhibitory antibodies in plasmodium falciparum provided they contain the correct domains for cell surface trafficking.

Antibodies from malaria-exposed individuals can agglutinate merozoites released from Plasmodium schizonts, thereby preventing them from invading new erythrocytes. Merozoite coat proteins attached to the plasma membrane are major targets for host antibodies and are therefore considered important malaria vaccine candidates. Prominent among these is the abundant glycosylphosphatidylinositol (GPI)-anchored merozoite surface protein 1 (MSP1) and particularly its C-terminal fragment (MSP1(19)) comprised of two epidermal growth factor (EGF)-like modules. In this paper, we revisit the role of agglutination and immunity using transgenic fluorescent marker proteins. We describe expression of heterologous MSP1(19)'miniproteins' on the surface of Plasmodium falciparum merozoites. To correctly express these proteins, we determined that GPI-anchoring and the presence of a signal sequence do not allow default export of proteins from the endoplasmic reticulum to merozoite surface and that extra sequence elements are required. The EGFs are insufficient for correct trafficking unless they are fused to additional residues that normally reside upstream of this fragment. Antibodies specifically targeting the surface-expressed miniprotein can inhibit erythrocyte invasion in vitro despite the presence of endogenous MSP1. Using a line expressing a green fluorescent protein-MSP1 fusion protein, we demonstrate that one mode of inhibition by antibodies targeting the MSP1(19) domain is the rapid agglutinating of merozoites prior to erythrocyte attachment.

Long-distance delivery of bacterial virulence factors by Pseudomonas aeruginosa outer membrane vesicles

Bacteria use a variety of secreted virulence factors to manipulate host cells, thereby causing significant morbidity and mortality. We report a mechanism for the long-distance delivery of multiple bacterial virulence factors, simultaneously and directly into the host cell cytoplasm, thus obviating the need for direct interaction of the pathogen with the host cell to cause cytotoxicity. We show that outer membrane–derived vesicles (OMV) secreted by the opportunistic human pathogen Pseudomonas aeruginosa deliver multiple virulence factors, including b-lactamase, alkaline phosphatase, hemolytic phospholipase C, and Cif, directly into the host cytoplasm via fusion of OMV with lipid rafts in the host plasma membrane. These virulence factors enter the cytoplasm of the host cell via N-WASP–mediated actin trafficking, where they rapidly distribute to specific subcellular locations to affect host cell biology. We propose that secreted virulence factors are not released individually as naked proteins into the surrounding milieu where they may randomly contact the surface of the host cell, but instead bacterial derived OMV deliver multiple virulence factors simultaneously and directly into the host cell cytoplasm in a coordinated manner.

Host cell remodelling and protein trafficking

Inside their respective vertebrate hosts, Plasmodium spp spend most of their life residing within hepatocytes and erythrocytes, with large-scale infection of the latter responsible for the clinical symptoms associated with malaria. These parasites extensively remodel these host cells for a variety of purposes relating to both pathogenesis and maintaining growth. Remodelling of the erythrocytic stage has been most intensively studied in P. falciparum and is the subject of this chapter. To help remodel their hosts these parasites export hundreds of proteins into the erythrocytic compartment. This principally alters the architecture of the erythrocyte, rendering the host membrane more permeable to solutes and nutrients, and also increasing the rigidity and adhesiveness of the infected erythrocyte. Moreover, because erythrocytes lack a secretory apparatus, the parasite must also export many additional proteins to help traffic other proteins to their correct destination within the host cell. The functions of some of these exported proteins will be discussed as will recent progress that has been made in unravelling how exported proteins gain access to the host compartment.

The deleterious effects of oxidative and nitrosative stress on palmitoylation, membrane lipid rafts and lipid-based cellular signalling : new drug targets in neuroimmune disorders

Oxidative and nitrosative stress (O&NS) is causatively implicated in the pathogenesis of Alzheimer’s and Parkinson’s disease, multiple sclerosis, chronic fatigue syndrome, schizophrenia and depression. Many of the consequences stemming from O&NS, including damage to proteins, lipids and DNA, are well known, whereas the effects of O&NS on lipoprotein-based cellular signalling involving palmitoylation and plasma membrane lipid rafts are less well documented. The aim of this narrative review is to discuss the mechanisms involved in lipid-based signalling, including palmitoylation, membrane/lipid raft (MLR) and n-3 polyunsaturated fatty acid (PUFA) functions, the effects of O&NS processes on these processes and their role in the abovementioned diseases. S-palmitoylation is a post-translational modification, which regulates protein trafficking and association with the plasma membrane, protein subcellular location and functions. Palmitoylation and MRLs play a key role in neuronal functions, including glutamatergic neurotransmission, and immune-inflammatory responses. Palmitoylation, MLRs and n-3 PUFAs are vulnerable to the corruptive effects of O&NS. Chronic O&NS inhibits palmitoylation and causes profound changes in lipid membrane composition, e.g. n-3 PUFA depletion, increased membrane permeability and reduced fluidity, which together lead to disorders in intracellular signal transduction, receptor dysfunction and increased neurotoxicity. Disruption of lipid-based signalling is a source of the neuroimmune disorders involved in the pathophysiology of the abovementioned diseases. n-3 PUFA supplementation is a rational therapeutic approach targeting disruptions in lipid-based signalling.