SMA-PAGE: A new method to examine complexes of membrane proteins using SMALP nano-encapsulation and native gel electrophoresis.

Most membrane proteins function through interactions with other proteins in the phospholipid bilayer, the cytosol or the extracellular milieu. Understanding the molecular basis of these interactions is key to understanding membrane protein function and dysfunction. Here we demonstrate for the first time how a nano-encapsulation method based on styrene maleic acid lipid particles (SMALPs) can be used in combination with native gel electrophoresis to separate membrane protein complexes in their native state. Using four model proteins, we show that this separation method provides an excellent measure of protein quaternary structure, and that the lipid environment surrounding the protein(s) can be probed using mass spectrometry. We also show that the method is complementary to immunoblotting. Finally we show that intact membrane protein-SMALPs extracted from a band on a gel could be visualised using electron microscopy (EM). Taken together these results provide a novel and elegant method for investigating membrane protein complexes in a native state.

Catalytic and non-catalytic functions of human IIA phospholipase A2.

Group IIA phospholipase A2 (PLA2) is a low-molecular-mass secreted PLA2 enzyme that has been identified as an acute phase protein with a role in the inflammatory response to infection and trauma. The protein is possibly unique in being highly cationic and having a global distribution of surface arginine and lysine residues. This structure supports two functions of the protein. (1) An anti-bacterial role where the enzyme is targeted to the anionic cell membrane of Gram-positive bacteria and phospholipid hydrolysis assists in bacterial killing. (2) A proposed non-catalytic role in which the protein forms supramolecular aggregates with anionic phospholipid vesicles or debris. These aggregates are then internalized via interactions with cell surface heparin sulphate proteoglycans and macropinocytosis for disposal by macrophages.

A catalytically independent physiological function for human acute phase protein group IIA phospholipase A2: cellular uptake facilitates cell debris removal.

Human group IIA phospholipase A2 (IIA PLA2) is an acute phase protein first identified at high concentrations in synovial fluid from patients with rheumatoid arthritis. Its physiological role has since been debated; the enzyme has a very high affinity for anionic phospholipid interfaces but expresses almost zero activity with zwitterionic phospholipid substrates, because of a lack of interfacial binding. We have prepared the cysteine-containing mutant (S74C) to allow the covalent attachment of fluorescent reporter groups. We show that fluorescently labeled IIA was taken up by phorbol 12-myristate 13-acetate-activated THP-1 cells in an energy-dependent process involving cell surface heparan sulfate proteoglycans. Uptake concurrently involved significant cell swelling, characteristic of macropinocytosis and the fluorescent enzyme localized to the nucleus. The endocytic process did not necessitate enzyme catalysis, ruling out membrane phospholipid hydrolysis as an essential requirement. The enzyme produced supramolecular aggregates with anionic phospholipid vesicles as a result of bridging between particles, a property that is unique to this globally cationic IIA PLA2. Uptake of such aggregates labeled with fluorescent anionic phospholipid was dramatically enhanced by the IIA protein, and uptake involved binding to heparan sulfate proteoglycans on activated THP-1 cells. A physiological role for this protein is proposed that involves the removal of anionic extracellular cell debris, including anionic microparticles generated as a result of trauma, infection, and the inflammatory response, and under such conditions serum levels of IIA PLA2 can increase approximately 1000-fold. A similar pathway may be significant in the uptake into cells of anionic vector DNA involving cationic lipid transfection protocols.

Immunolocalisation of the D. melanogaster Nramp homologue Malvolio to gut and Malpighian tubules provides evidence that Malvolio and Nramp2 are orthologous.

Nramp (Slc11a1) genes in mammals are associated with the transport of iron and other divalent cations; Nramp1 in macrophages involved in the innate immune response against intracellular pathogens, and Nramp2 with duodenal iron uptake and the transferrin-transferrin-receptor pathway of iron assimilation. The Drosophila melanogaster Nramp-related gene is known as Malvolio. The localisation of Malvolio protein was inferred from the enhancer trap line initially used to isolate Malvolio in a screen for mutants with defects in taste perception. Here we describe the generation of a Malvolio-reactive polyclonal antibody and apply it to evaluate Malvolio localisation during stages of D. melanogaster development, and compare the results with the localisation of the enhancer trap line identified with beta-galactosidase. All immunolocalisation studies have been confirmed to be specific with Malvolio-blocking peptides. Our results demonstrated expression within Malpighian tubules, testis, brain, the amnioserosa of embryos, the larval and adult alimentary canal. Expression within the gut was of significant interest, as mammalian Nramp2 in the gut plays a primary role in the acquisition of dietary iron. We confirm expression within the central nervous system and in cells of the haematopoietic system. By immunohistochemistry we showed that expression within cells was either punctuate, diffuse cytoplasmic or plasma membrane associated, or both. The staining within the gut indicates a degree of conservation of components for iron acquisition between flies and mammals, suggesting that a comparable mechanism has been retained during evolution.

Nramp1-mediated innate resistance to intraphagosomal pathogens is regulated by IRF-8, PU.1, and Miz-1.

Natural resistance-associated macrophage protein 1 (Nramp1) is a proton/divalent cation antiporter exclusively expressed in monocyte/macrophage cells with a unique role in innate resistance to intraphagosomal pathogens. In humans, it is linked to several infectious diseases, including leprosy, pulmonary tuberculosis, visceral leishmaniasis, meningococcal meningitis, and human immunodeficiency virus as well as to autoimmune diseases such as rheumatoid arthritis and Crohn's disease. Here we demonstrate that the restricted expression of Nramp1 is mediated by the macrophage-specific transcription factor IRF-8. This factor exerts its activity via protein-protein interaction, which facilitates its binding to target DNA. Using yeast two-hybrid screen we identified Myc Interacting Zinc finger protein 1 (Miz-1) as new interacting partner. This interaction is restricted to immune cells and takes place on the promoter Nramp1 in association with PU.1, a transcription factor essential for myelopoiesis. Consistent with these data, IRF-8 knockout mice are sensitive to a repertoire of intracellular pathogens. Accordingly, IRF-8-/- mice express low levels of Nramp1 that can not be induced any further. Thus, our results explain in molecular terms the role of IRF-8 in conferring innate resistance to intracellular pathogens and point to its possible involvement in autoimmune diseases.

Characterization of the murine Nramp1 promoter: requirements for transactivation by Miz-1.

Murine Nramp1 encodes a divalent cation transporter that is expressed in late endosomes/lysosomes of macrophages, and the transported cations facilitate intracellular pathogen growth control. The Nramp1 promoter is TATA box-deficient, has two initiator elements, and is repressed by c-Myc, in accordance with the notion that genes that deplete the iron content of the cell cytosol antagonize cell growth. Repression via c-Myc occurs at the initiator elements, whereas a c-Myc-interacting protein (Miz-1) stimulates transcription. Here we demonstrate that a non-canonical E box (CAACTG) inhibits basal promoter activity and activation by Miz-1. A consensus Sp1-binding site or GC box is also necessary for Miz-1-dependent transactivation, but not repression. Repression occurs by c-Myc competing with p300/CBP for binding Miz-1. Our results show that an Sp1 site mutant inhibits coactivation by p300 and that the murine Nramp1 promoter is preferentially expressed within macrophages (relative to a beta-actin control) compared with non-macrophage cells. The effect of the Sp1 site mutation on promoter function shows cell-type specificity: stimulation in COS-1 and inhibition in RAW264.7 cells. Miz-1-directed RNA interference confirms a stimulatory role for Miz-1 in Nramp1 promoter function. c-Myc, Miz-1, and Sp1 were identified as binding to the Nramp1 core promoter in control cells and following acute stimulation with interferon-gamma and lipopolysaccharide. These results provide a description of sites that modulate the activity of the initiator-binding protein Miz-1 and indicate a stimulatory role for GC box-binding factors in macrophages and a inhibitory role for E box elements in proliferating cells.

c-Myc represses and Miz-1 activates the murine natural resistance-associated protein 1 promoter.

Iron is essential for growth, and impaired iron homoeostasis through a non-conserved mutation within murine Nramp1, also termed Slc11a1, contributes to susceptibility to infection. Nramp1 depletes the macrophage cytosol of iron, with effects on iron-regulated gene expression and iron-dependent processes. Wu and colleagues (Wu, K.-J., Polack, A., and Dalla-Favera, R. (1999) Science 283, 676-679) showed converse control of iron regulatory protein expression (IRP2) and H-ferritin by c-Myc, suggesting a role for c-Myc in enhancing cytoplasmic iron levels for growth. We investigated if c-Myc also regulates Nramp1 expression. We show an inverse correlation with cell growth, and in co-transfection experiments c-Myc represses the Nramp1 promoter. Within the Nramp1 promoter we identified six non-canonical E boxes, which are not important for c-Myc repression. By deletion analysis the repressor site maps to one or more initiator elements flanking the transcriptional initiation site. Co-transfections with the c-Myc interacting zinc finger protein (Miz-1) show that Miz-1 can overcome c-Myc repression of Nramp1, and, from a deletion construct lacking E box sites, Miz-1 activates the Nramp1 promoter. These studies reinforce the link between c-Myc and iron regulation and provide further evidence that c-Myc negatively regulates genes that decrease the iron content of the cytosol. The results provide further support for a divalent cation antiporter function for Nramp1.