Structure of the house dust mite allergen Der f 2: implications for function and molecular basis of IgE cross-reactivity

The X-ray structure of the group 2 major allergen from Dermatophagoides farinae (Der f 2) was determined to 1.83 A resolution. The overall Der f 2 structure comprises a single domain of immunoglobulin fold with two anti-parallel beta-sheets. A large hydrophobic cavity is formed in the interior of Der f 2. Structural comparisons to distantly related proteins suggest a role in lipid binding. Immunoglobulin E (IgE) cross-reactivity between group 2 house dust mite major allergens can be explained by conserved surface areas representing IgE binding epitopes.     

Proteolytic processing of the ovine prion protein in cell cultures

The cellular compartment and purpose of the proteolytic processing of the prion protein (PrP) are still under debate. We have studied ovine PrP constructs expressed in four cell lines; murine neuroblastoma cells (N2a), human neuroblastoma cells (SH-SY5Y), dog kidney epithelial cells (MDCK), and human furin-deficient colon cancer cells (LoVo). Cleavage of PrP in LoVo cells indicates that the processing is furin independent. Neither is it reduced by some inhibitors of lysosomal proteinases, proteasomes or zinc-metalloproteinases, but incubation with bafilomycin A1, an inhibitor of vacuolar H+/ATPases, increases the amount of uncleaved PrP in the apical medium of MDCK cells. Mutations affecting the putative cleavage site near amino acid 113 reveal that the cleavage is independent of primary structure at this site. Absence of glycosylphosphatidylinositol anchor and glycan modifications does not influence the proteolytic processing of PrP. Our data indicate that PrP is cleaved during transit to the cell membrane.     

The crystal structure of recombinant proDer p 1, a major house dust mite proteolytic allergen

Allergy to house dust mite is among the most prevalent allergic diseases worldwide. Most house dust mite allergic patients react to Der p 1 from Dermatophagoides pteronyssinus, which is a cysteine protease. To avoid heterogeneity in the sample used for crystallization, a modified recombinant molecule was produced. The sequence of the proDer p 1 allergen was modified to reduce glycosylation and to abolish enzymatic activity. The resulting rproDer p 1 preparation was homogenous and stable and yielded crystals diffracting to a resolution of 1.61 A. The active site is located in a large cleft on the surface of the molecule. The 80-aa pro-peptide adopts a unique fold that interacts with the active site cleft and a substantial adjacent area on the mature region, excluding access to the cleft and the active site. Studies performed using crossed-line immunoelectrophoresis and IgE inhibition experiments indicated that several epitopes are covered by the pro-peptide and that the epitopes on the recombinant mature molecule are indistinguishable from those on the natural one. The structure confirms previous results suggesting a preference for aliphatic residues in the important P2 position in substrates. Sequence variations in related species are concentrated on the surface, which explains the existence of cross-reacting and species-specific antibodies. This study describes the first crystal structure of one of the clinically most important house dust mite allergens, the cysteine protease Der p 1.     

Importance of transmembrane segment M1 of the sarcoplasmic reticulum Ca2+-ATPase in Ca2+ occlusion and phosphoenzyme processing

The functional consequences of a series of point mutations in transmembrane segment M1 of sarcoplasmic reticulum Ca2+-ATPase were analyzed in steady-state and transient kinetic experiments examining the partial reaction steps involved in Ca2+ interaction and phosphoenzyme turnover. Arginine or leucine substitution of Glu51, Glu55, or Glu58, located in the N-terminal third of M1, did not affect these functions. Arginine or leucine substitution of Asp59, located right at the bend of M1 seen in the crystal structure of the thapsigargin-bound form, caused a 10-fold increase of the rate of Ca2+ dissociation toward the cytoplasmic side. Mutation of Leu60 to alanine or proline and of Val62 to alanine also enhanced Ca2+ dissociation, whereas an 11-fold reduction of the rate of Ca2+ dissociation was observed upon alanine substitution of Leu65, thus providing evidence for a relation of the middle part of M1 to a gating mechanism controlling the dissociation of occluded Ca2+ from its membranous binding sites. Moreover, phosphoenzyme processing was affected by some of the latter mutations, in particular leucine substitution of Asp59, and alanine substitution of Leu65 accelerated the transition to ADP-insensitive phosphoenzyme and blocked its dephosphorylation, thus demonstrating that this part of M1, besides being important in Ca2+ interaction, furthermore, is a critical element in the long range signaling between the transmembrane domain and the cytoplasmic catalytic site.     

Equol is a novel anti-androgen that inhibits prostate growth and hormone feedback

Equol (7-hydroxy-3[4'hydroxyphenyl]-chroman) is the major metabolite of the phytoestrogen daidzein, one of the main isoflavones found abundantly in soybeans and soy foods. Equol may be an important biologically active molecule based on recent studies demonstrating that equol can modulate reproductive function. In this study, we examined the effects of equol on prostate growth and LH secretion and determined some of the mechanisms by which it might act. Administration of equol to intact male rats for 4-7 days reduced ventral prostate and epididymal weight and increased circulating LH levels. Using binding assays, we determined that equol specifically binds 5alpha-dihydrotestosterone (DHT), but not testosterone, dehydroepiandrosterone, or estrogen with high affinity. Equol does not bind the prostatic androgen receptor, and has a modest affinity for recombinant estrogen receptor (ER) beta, and no affinity for ERalpha. In castrated male rats treated with DHT, concomitant treatment with equol blocked DHT's trophic effects on the ventral prostate gland growth and inhibitory feedback effects on plasma LH levels without changes in circulating DHT. Therefore, equol can bind circulating DHT and sequester it from the androgen receptor, thus altering growth and physiological hormone responses that are regulated by androgens. These data suggest a novel model to explain equol's biological properties. The significance of equol's ability to specifically bind and sequester DHT from the androgen receptor have important ramifications in health and disease and may indicate a broad and important usage for equol in the treatment of androgen-mediated pathologies.     

Apolipoprotein A-I-stimulated apolipoprotein E secretion from human macrophages is independent of cholesterol efflux

Apolipoprotein A-I (apoA-I)-mediated cholesterol efflux involves the binding of apoA-I to the plasma membrane via its C terminus and requires cellular ATP-binding cassette transporter (ABCA1) activity. ApoA-I also stimulates secretion of apolipoprotein E (apoE) from macrophage foam cells, although the mechanism of this process is not understood. In this study, we demonstrate that apoA-I stimulates secretion of apoE independently of both ABCA1-mediated cholesterol efflux and of lipid binding by its C terminus. Pulse-chase experiments using (35)S-labeled cellular apoE demonstrate that macrophage apoE exists in both relatively mobile (E(m)) and stable (E(s)) pools, that apoA-I diverts apoE from degradation to secretion, and that only a small proportion of apoA-I-mobilized apoE is derived from the cell surface. The structural requirements for induction of apoE secretion and cholesterol efflux are clearly dissociated, as C-terminal deletions in recombinant apoA-I reduce cholesterol efflux but increase apoE secretion, and deletion of central helices 5 and 6 decreases apoE secretion without perturbing cholesterol efflux. Moreover, a range of 11- and 22-mer alpha-helical peptides representing amphipathic alpha-helical segments of apoA-I stimulate apoE secretion whereas only the C-terminal alpha-helix (domains 220-241) stimulates cholesterol efflux. Other alpha-helix-containing apolipoproteins (apoA-II, apoA-IV, apoE2, apoE3, apoE4) also stimulate apoE secretion, implying a positive feedback autocrine loop for apoE secretion, although apoE4 is less effective. Finally, apoA-I stimulates apoE secretion normally from macrophages of two unrelated subjects with genetically confirmed Tangier Disease (mutations C733R and c.5220-5222delTCT; and mutations A1046D and c.4629-4630insA), despite severely inhibited cholesterol efflux. We conclude that apoA-I stimulates secretion of apoE independently of cholesterol efflux, and that this represents a novel, ABCA-1-independent, positive feedback pathway for stimulation of potentially anti-atherogenic apoE secretion by alpha-helix-containing molecules including apoA-I and apoE.     

AICAR stimulates adiponectin and inhibits cytokines in adipose tissue

5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) can be used as an experimental tool to activate 5'-AMP-activated protein kinase (AMPK) and has been shown to improve insulin sensitivity. In parallel adiponectin also seems to activate AMPK and to improve insulin sensitivity. We have investigated the effects of AICAR on the gene expression of adiponectin and on gene expression and release of cytokines in human adipose tissue in vitro. AICAR stimulated AMPK alpha1 activity 3-4-fold (p<0.001), and dose-dependently increased adiponectin mRNA levels with significant stimulation (2-4-fold) at AICAR concentrations of 0.5-2mM (p<0.05). The adipose tissue protein release of tumor necrosis factor-alpha (TNF- alpha) and interleukin-6 (IL-6) was decreased by AICAR (p<0.05). In conclusion, AICAR stimulated adipose tissue AMPK alpha1 activity and adiponectin gene expression, while attenuating the release of TNF-alpha and IL-6. Reduced concentrations of these cytokines and increased levels of adiponectin might play a role for the insulin sensitizing effects of AICAR.     

Exercise normalises overexpression of TNF-alpha in knockout mice

TNF-alpha is linked with insulin resistance, as greater amounts of TNF are detected in muscle and adipose tissue in glycemically challenged people and TNF-alpha inhibits insulin receptor signalling. However, what modulates this overexpression of TNF-alpha is currently unknown. We examined the effect of 1 h exercise on overexpression of the TNF-alpha gene in TNF receptor 1 and 2 knockout mice. IL-6 knockout mice were included to elucidate the importance of IL-6 in regulating TNF-alpha in response to exercise. TNF-alpha gene expression was over-expressed in muscle in both TNFR knockout models. TNF-alpha overexpression returned to normal levels after exercise in the TNF-alpha receptor knockout models. In IL-6 knockout mice, a modest decrease in TNF-alpha was also observed. These data suggest that TNF-alpha-induced insulin resistance can be regulated by a single exercise bout by normalising TNF-alpha expression. This exercise effect can be mediated via IL-6, but also an IL-6 independent mechanism seems to exist.     

Molecular phylogeography and hybridization in members of the circumpolar Potentilla sect. Niveae (Rosaceae)

Glacial events and the formation of ice-free areas serving as refugia for plants and animals are important in shaping present patterns of genetic diversity in arctic areas. Beringia, situated in northeastern Russia and Alaska, has been pointed out as a major refugium. This study focuses on the historical biogeography of the circumpolar taxon Potentilla sect. Niveae. The taxonomy of the group is complex, most likely highly influenced by hybridization and apomixis. cpDNA microsatellites together with AFLP fragments were used to map the genetic variability in the section, from Beringia across the Canadian Arctic to Greenland. The data support the hypothesis that Beringia, as well as parts of adjacent arctic Canada, served as refugia during the Wisconsinan glaciation, and there is some evidence for a northern and a southern migration route out of Beringia. The hair type groups within sect. Niveae are more or less genetically distinct, and hybridization, especially with sect. Multifida, takes place. Haplotype diversity as well as frequency is at its maximum close to the Last Glacial Maximum ice cap edge. This pattern can be explained by merging of previously isolated refugia, by repeated extinction/colonization events close to the ice edge, and by hybridization among sympatric taxonomical lineages.