Sprouty2 Regulates PI(4,5)P2/Ca Signaling and HIV-1 Gag Release

We reported recently that activation of the inositol 1,4,5-triphosphate receptor (IP3R) is required for efficient HIV-1 Gag trafficking and viral particle release. IP3R activation requires phospholipase C (PLC)-catalyzed hydrolysis of PI(4,5)P₂ to IP3 and diacylglycerol. We show that Sprouty2 (Spry2), which binds PI(4,5)P₂ and PLCγ, interfered with PI(4,5)P₂ in a manner similar to that of U73122, an inhibitor of PI(4,5)P₂ hydrolysis, suggesting that Spry2 negatively regulates IP3R by preventing formation of its activating ligand, IP3. Mutation to Asp of R252, a crucial determinant of PI(4,5)P₂ binding in the C-terminal domain of Spry2, prevented the interference, indicating that binding to the phospholipid is required. By contrast, deletion of the PLCγ binding region or mutation of a critical Tyr residue in the region did not prevent the interference but Spry2-PI(4,5)P₂ colocalization was not detected, suggesting that PLC binding is required for their stable association. Like U73122, Spry2 over-expression inhibited wild type Gag release as virus-like particles. Disrupting either binding determinant relieved the inhibition. IP3R-mediated Ca²⁺signaling, in turn, was found to influence Spry2 subcellular distribution and ERK, a Spry2 regulator. Our findings suggest that Spry2 influences IP3R function through control of PI(4,5)P₂ and IP3R influences Spry2 function by controlling its distribution and ERK activation.     

Analysis of candidate susceptibility genes in canine diabetes

Canine diabetes is a complex genetic disease of unknown aetiology. It affects 0.005-1.5% of the canine population and shows a clear breed predisposition with the Samoyed being at high risk and the Boxer being at low risk of developing the disease. Canine diabetes is considered to be a disease homologue for human type 1 diabetes (T1D). It results in insulin deficiency as a consequence of autoimmune destruction of islet beta-cells in the pancreas and is believed to be mediated by Th1 cytokines (IFNgamma, TNFalpha, and IL-2). A number of genes have been associated with type 1 diabetes in humans, including the human leukocyte antigen region, the insulin variable number tandem repeat, PTPN22, CTLA4, IL-4, and IL-13. As yet, these genes have not been evaluated in canine diabetes. In this study, 483 cases of canine diabetes and 869 controls of known breed were analyzed for association with IFNgamma, IGF2, IL-10, IL-12beta, IL-6, insulin, PTPN22, RANTES, IL-4, IL-1alpha and TNFalpha. Minor allele frequencies were determined for these genes in each breed. These data were used for comparative analyses in a case-control study, and clear associations with diabetes were identified in some breeds with certain alleles of candidate genes. Some associations were with increased susceptibility to the disease (IFNgamma, IL-10, IL-12beta, IL-6, insulin, PTPN22, IL-4, and TNFalpha), whereas others were protective (IL-4, PTPN22, IL-6, insulin, IGF2, TNFalpha). This study demonstrates that a number of the candidate genes previously associated with human T1D also appear to be associated with canine diabetes and identifies an IL-10 haplotype which is associated with diabetes in the Cavalier King Charles Spaniel. This suggests that canine diabetes is an excellent comparative and spontaneously occurring disease model of human T1D.     

Post-translational modification of barley LTP1b: the lipid adduct lies in the hydrophobic cavity and alters the protein dynamics

NMR techniques have been used to characterise the effects of a lipid-like post-translational modification on barley lipid transfer protein (LTP1b). NMR chemical shift data indicate that the lipid-like molecule lies in the hydrophobic cavity of LTP1b, with Tyr 79 being displaced to accommodate the ligand in the cavity. The modified protein has a reduced level of backbone amide hydrogen exchange protection, presumably reflecting increased dynamics in the protein. This may result from a loosening of the protein structure and may explain the enhanced surface properties observed for LTP1b.     

Concerted nitric oxide formation and release from the simultaneous reactions of nitrite with deoxy- and oxyhemoglobin

Recent studies reveal a novel role for hemoglobin as an allosterically regulated nitrite reductase that may mediate nitric oxide (NO)-dependent signaling along the physiological oxygen gradient. Nitrite reacts with deoxyhemoglobin in an allosteric reaction that generates NO and oxidizes deoxyhemoglobin to methemoglobin. NO then reacts at a nearly diffusion-limited rate with deoxyhemoglobin to form iron-nitrosyl-hemoglobin, which to date has been considered a highly stable adduct and, thus, not a source of bioavailable NO. However, under physiological conditions of partial oxygen saturation, nitrite will also react with oxyhemoglobin, and although this complex autocatalytic reaction has been studied for a century, the interaction of the oxy- and deoxy-reactions and the effects on NO disposition have never been explored. We have now characterized the kinetics of hemoglobin oxidation and NO generation at a range of oxygen partial pressures and found that the deoxy-reaction runs in parallel with and partially inhibits the oxy-reaction. In fact, intermediates in the oxy-reaction oxidize the heme iron of iron-nitrosyl-hemoglobin, a product of the deoxy-reaction, which releases NO from the iron-nitrosyl. This oxidative denitrosylation is particularly striking during cycles of hemoglobin deoxygenation and oxygenation in the presence of nitrite. These chemistries may contribute to the oxygen-dependent disposition of nitrite in red cells by limiting oxidative inactivation of nitrite by oxyhemoglobin, promoting nitrite reduction to NO by deoxyhemoglobin, and releasing free NO from iron-nitrosyl-hemoglobin.     

Prion protein genes in caribou from Alaska

Prion protein genes were sequenced in free-ranging Alaska caribou (Rangifer tarandus grantii). Caribou prion alleles are identical or nearly so to those of wapiti, white-tailed deer, and mule deer. Five single-nucleotide polymorphisms were detected with substitutions at residues 2 (V-->M), 129 (G-->S), 138 (S-->N), 146 (N-->N), and 169 (V-->M). The 138N codon had been previously reported only in prion pseudogenes of other cervids. In caribou, the 138S and 138N alleles are present at frequencies of approximately 0.7 and 0.3, respectively, and they are seen in both homozygotes and heterozygotes of three geographically separated herds, each a component of the continental metapopulation. Genetics seems to permit the spread of chronic wasting disease from middle-latitude deer to high-latitude caribou in North America.     

Greater uterine artery blood flow during pregnancy in multigenerational (Andean) than shorter-term (European) high-altitude residents

Multigenerational (Andean) compared with shorter-term (European) high-altitude residents exhibit less hypoxia-associated reductions in birth weight. Because differences in arterial O(2) content are not responsible, we asked whether greater pregnancy-associated increases in uterine artery (UA) blood flow and O(2) delivery were involved. Serial studies were conducted in 42 Andean and 26 European residents of La Paz, Bolivia (3600 m) at weeks 20, 30, 36 of pregnancy and 4 mo postpartum using Doppler ultrasound. There were no differences postpartum but Andean vs. European women had greater UA diameter (0.65 +/- 0.01 vs. 0.56 +/- 0.01 cm), cross-sectional area (33.1 +/- 0.97 vs. 24.7 +/- 1.18 mm(2)), and blood flow at week 36 (743 +/- 87 vs. 474 +/- 36 ml/min) (all P < 0.05) and thus 1.6-fold greater uteroplacental O(2) delivery near term (126.82 +/- 18.47 vs. 80.33 +/- 8.69 ml O(2).ml blood(-1).min(-1), P < 0.05). Andeans had greater common iliac (CI) flow and lower external iliac relative to CI flow (0.52 +/- 0.11 vs. 0.95 +/- 0.14, P < 0.05) than Europeans at week 36. After adjusting for gestational age, maternal height, and parity, Andean babies weighed 209 g more than the Europeans. Greater UA cross-sectional area at week 30 related positively to birth weight in Andeans (r = +0.39) but negatively in Europeans (r = -0.37) (both P < 0.01). We concluded that a greater pregnancy-associated increase in UA diameter raised UA blood flow and uteroplacental O(2) delivery in the Andeans and contributed to their ability to maintain normal fetal growth under conditions of high-altitude hypoxia. These data implicate the involvement of genetic factors in protecting multigenerational populations from hypoxia-associated reductions in fetal growth, but future studies are required for confirmation and identification of the specific genes involved.     

BI-D1870 is a specific inhibitor of the p90 RSK (ribosomal S6 kinase) isoforms in vitro and in vivo

Lipocalins are extracellular proteins (17-25 kDa) that bind and transport small lipophilic molecules. The three-dimensional structure of the first lipocalin from a metatherian has been determined at different values of pH both with and without bound ligands. Trichosurin, a protein from the milk whey of the common brushtail possum, Trichosurus vulpecula, has been recombinantly expressed in Escherichia coli, refolded from inclusion bodies, purified and crystallized at two different pH values. The three-dimensional structure of trichosurin was solved by X-ray crystallography in two different crystal forms to 1.9 A (1 A=0.1 nm) and 2.6 A resolution, from crystals grown at low and high pH values respectively. Trichosurin has the typical lipocalin fold, an eight-stranded anti-parallel beta-barrel but dimerizes in an orientation that has not been seen previously. The putative binding pocket in the centre of the beta-barrel is well-defined in both high and low pH structures and is occupied by water molecules along with isopropanol molecules from the crystallization medium. Trichosurin was also co-crystallized with a number of small molecule ligands and structures were determined with 2-naphthol and 4-ethylphenol bound in the centre of the beta-barrel. The binding of phenolic compounds by trichosurin provides clues to the function of this important marsupial milk protein, which is highly conserved across metatherians.     

Heat treatment of bovine alpha-lactalbumin results in partially folded, disulfide bond shuffled states with enhanced surface activity

Prolonged heating of holo bovine alpha-lactalbumin (BLA) at 80 degrees C in pH 7 phosphate buffer in the absence of a thiol initiator improves the surface activity of the protein at the air:water interface, as determined by surface tension measurements. Samples after 30, 60, and 120 min of heating were analyzed on cooling to room temperature. Size-exclusion chromatography shows sample heterogeneity that increases with the length of heating. After 120 min of heating monomeric, dimeric, and oligomeric forms of BLA are present, with aggregates formed from disulfide bond linked hydrolyzed protein fragments. NMR characterization at pH 7 in the presence of Ca2+ of the monomer species isolated from the sample heated for 120 min showed that it consisted of a mixture of refolded native protein and partially folded protein and that the partially folded protein species had spectral characteristics similar to those of the pH 2 molten globule state of the protein. Circular dichroism spectroscopy showed that the non-native species had approximately 40% of the alpha-helical content of the native state, but lacked persistent tertiary interactions. Proteomic analysis using thermolysin digestion of three predominant non-native monomeric forms isolated by high-pressure liquid chromatography indicated the presence of disulfide shuffled isomers, containing the non-native 61-73 disulfide bond. These partially folded, disulfide shuffled species are largely responsible for the pronounced improvement in surface activity of the protein on heating.     

Roles of basic residues and salt-bridge interaction in a vacuolar H+-pumping pyrophosphatase (AVP1) from Arabidopsis thaliana

To investigate the possible role of basic residues in H+ translocation through vacuolar-type H+-pumping pyrophosphatases (V-PPases), conserved arginine and lysine residues predicted to reside within or close to transmembrane domains of an Arabidopsis thaliana V-PPase (AVP1) were subjected to site-directed mutagenesis. One of these mutants (K461A) exhibited a "decoupled" phenotype in which proton-pumping but not hydrolysis was inhibited. Similar results were reported previously for an E427Q mutant, resulting in the proposal that E427 might be involved in proton translocation. However, the double mutant E427K/K461E has a wild type phenotype, suggesting that E427 and K461 form a stabilising salt bridge, but that neither residue plays a critical role in proton translocation.