The interaction of peripheral proteins and membranes studied with alpha-lactalbumin and phospholipid bilayers of various compositions

To characterize the interaction of peripheral proteins and membranes at the molecular level, we studied the reversible association of bovine alpha-lactalbumin (BLA) with lipid bilayers composed of different molecular forms of phosphatidylserine or equimolar mixtures of these phosphatidylserine forms and egg yolk phosphatidylcholine. At pH 4.5, almost all BLA (>90%) associates to negatively charged small unilamellar vesicles. The conformational changes that binding to these bilayers induced on the protein were characterized by circular dichroism and fluorescence spectroscopy. Because binding of BLA to negatively charged vesicles is reverted by adjusting the pH back to >6.0, we also investigated the conformation of the membrane-bound protein by NMR-monitored H-D exchange of the backbone amide protons. The conformation adopted by BLA bound to these bilayers resembles a molten globule-like state but the negative ellipticity at 222 nm and the apparent alpha-helix content of the bound protein senses the changes in the physical properties of the membrane. Binding to bilayers in the gel state appears to correlate with an increased amount of alpha-helical structure and with a lower extent of integration into the membrane, corresponding to the adsorbed protein, while the opposite is found for BLA bound to vesicles in the liquid-crystalline phase, corresponding to the embedded conformation. A common feature for the membrane-bound conformations of BLA is that the amphipathic helix C (residues 86 to 99) is an important determinant for the adsorption and further integration of the protein into the membrane.     

Platelet-derived growth factor (PDGF)-C,a PDGF family member with a vascular endothelial growth factor-like structure

Platelet-derived growth factor (PDGF)-C is a novel member of the PDGF family that binds to PDGF alphaalpha and alphabeta receptors. The growth factor domain of PDGF-C (GFD-PDGF-C) was expressed in high yields in Escherichia coli and was purified and refolded from inclusion bodies obtaining a biologically active growth factor with dimeric structure. The GFD-PDGF-C contains 12 cysteine residues, and Ellman assay analysis indicates that it contains three intramonomeric disulfide bonds, which is in accordance with GFD-PDGF-C being a member of the cystine knot superfamily of growth factors. The recombinant GFD-PDGF-C was characterized by CD, fluorescence, NMR, and infrared spectroscopy. Together, our data indicate that GFD-PDGF-C is a highly thermostable protein that contains mostly beta-sheet secondary structure and some (6%) alpha-helix structure. The structural model of PDGF-C, obtained by homology-based molecular modeling using the structural representatives of this family of growth factors, shows that GFD-PDGF-C has a higher structural homology to the vascular endothelial growth factor than to PDGF-B. The modeled structure can give further insights into the function and specificity of this molecule.     

Regulation of beta cell glucokinase by S-nitrosylation and association with nitric oxide synthase

Glucokinase (GK) activity plays a key role in glucose-stimulated insulin secretion from pancreatic beta cells. Insulin regulates GK activity by modulating its association with secretory granules, although little is known about the mechanisms involved in regulating this association. Using quantitative imaging of multicolor fluorescent proteins fused to GK, we found that the dynamic association of GK with secretory granules is modulated through nitric oxide (NO). Our results in cultured beta cells show that insulin stimulates NO production and leads to S-nitrosylation of GK. Furthermore, inhibition of NO synthase (NOS) activity blocks insulin-stimulated changes in both GK association with secretory granules and GK conformation. Mutation of cysteine 371 to serine blocks S-nitrosylation of GK and causes GK to remain tightly bound to secretory granules. GK was also found to interact stably with neuronal NOS as detected by coimmunoprecipitation and fluorescence resonance energy transfer. Finally, attachment of a nuclear localization signal sequence to NOS drives GK to the nucleus in addition to its normal cytoplasmic and granule targeting. Together, these data suggest that the regulation of GK localization and activity in pancreatic beta cells is directly related to NO production and that the association of GK with secretory granules occurs through its interaction with NOS.     

Estimation of population bottlenecks during systemic movement of tobacco mosaic virus in tobacco plants

More often than not, analyses of virus evolution have considered that virus populations are so large that evolution can be explained by purely deterministic models. However, virus populations could have much smaller effective numbers than the huge reported census numbers, and random genetic drift could be important in virus evolution. A reason for this would be population bottlenecks during the virus life cycle. Here we report a quantitative estimate of population bottlenecks during the systemic colonization of tobacco leaves by Tobacco mosaic virus (TMV). Our analysis is based on the experimental estimation of the frequency of different genotypes of TMV in the inoculated leaf, and in systemically infected leaves, of tobacco plants coinoculated with two TMV genotypes. A simple model, based on the probability that a leaf in coinoculated plants is infected by just one genotype and on the frequency of each genotype in the source, was used to estimate the effective number of founders for the populations in each leaf. Results from the analysis of three leaves per plant in plants inoculated with different combinations of three TMV genotypes yielded highly consistent estimates. Founder numbers for each leaf were small, in the order of units. This would result in effective population numbers much smaller than the census numbers and indicates that random effects due to genetic drift should be considered for understanding virus evolution within an infected plant.     

Recruitment of endothelial caveolae into mechanotransduction pathways by flow conditioning in vitro

The luminal surface of rat lung microvascular endothelial cells in situ is sensitive to changing hemodynamic parameters. Acute mechanosignaling events initiated in response to flow changes in perfused lung microvessels are localized within specialized invaginated microdomains called caveolae. Here we report that chronic exposure to shear stress alters caveolin expression and distribution, increases caveolae density, and leads to enhanced mechanosensitivity to subsequent changes in hemodynamic forces within cultured endothelial cells. Flow-preconditioned cells expressed a fivefold increase in caveolin (and other caveolar-residing proteins) at the luminal surface compared with no-flow controls. The density of morphologically identifiable caveolae was enhanced sixfold at the luminal cell surface of flow-conditioned cells. Laminar shear stress applied to static endothelial cultures (flow step of 5 dyn/cm2), enhanced the tyrosine phosphorylation of luminal surface proteins by 1.7-fold, including caveolin-1 by 1.3-fold, increased Ser1179 phosphorylation of endothelial nitric oxide synthase (eNOS) by 2.6-fold, and induced a 1.4-fold activation of mitogen-activated protein kinases (ERK1/2) over no-flow controls. The same shear step applied to endothelial cells preconditioned under 10 dyn/cm2 of laminar shear stress for 6 h and induced a sevenfold increase of total phosphotyrosine signal at the luminal endothelial cell surface enhanced caveolin-1 tyrosine phosphorylation 5.8-fold and eNOS phosphorylation by 3.3-fold over static control values. In addition, phosphorylated caveolin-1 and eNOS proteins were preferentially localized to caveolar microdomains. In contrast, ERK1/2 activation was not detected in conditioned cells after acute shear challenge. These data suggest that cultured endothelial cells respond to a sustained flow environment by directing caveolae to the cell surface where they serve to mediate, at least in part, mechanotransduction responses.     

Short-term storage of oocytes from the neotropical teleost fish Prochilodus marggravii

The loss of oocyte viability after ovulation is one of the limiting factors in controlled reproduction of several fish species. Experiments were performed with 15 feral Prochilodus marggravii female fish induced to spawn with crude carp pituitary extract to evaluate the viability of oocytes retained within the ovarian cavity (in situ storage) and outside of the ovarian cavity (ex situ storage). Because fertility rates rapidly declined after ovulation, simultaneously with an increase in the number of deformed larvae, P. marggravii oocytes could only be successfully stored for 1 h ex situ at room temperature ( approximately 26 degrees C). There was a highly negative correlation (r = -0.82) between fertilization and deformed larvae during in situ storage at approximately 26 degrees C. Ex situ cooling (18 degrees C) caused a drastic reduction in fertilization rates as compared with storage at approximately 26 degrees C. Oocyte structure was preserved during 2 h storage and the cortical reaction was induced before spawning. Since the micropylar apparatus remained open, it was not the primary cause for the loss of oocyte fertility. The cytoskeleton of the oocyte appeared to be affected since ooplasmic segregation was altered after 2 h storage.     

Probing cofactor specificity in phenylalanine hydroxylase by molecular dynamics simulations

Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin-dependent enzyme that catalyzes the hydroxylation of L-phenylalanine (L-Phe) to L-tyrosine using dioxygen as an additional substrate. The requirement of PAH for a cofactor is absolute, but several cofactor analogs are able to substitute the natural cofactor in catalysis. However, it is only the natural cofactor 6R-tetrahydrobiopterin (6R-BH(4)) that induces a negative regulatory effect on the enzyme. In order to get further insights on the molecular basis for this specificity, we studied the structure of the cofactor-enzyme complex and the conformational changes induced by cofactor binding by molecular dynamics simulations. Simulations were carried out on the enzyme alone and complexed with 6R-BH(4) and with two cofactor analogs, 6S-BH(4) and 6-methyl-tetrahydropterin (6M-PH(4)). In the resting unbound enzyme Tyr377 in the catalytic domain is hydrogen bonded to both Ser23 and Glu21 of the autoregulatory N-terminal sequence. This hydrogen bonding network is disturbed by the binding of BH(4), which interacts with Ser23. By doing so, 6R-BH(4) facilitates an interaction between Glu21 and the active site iron, further pulling the N-terminal into the active site of PAH and blocking the L-Phe binding site. Thus, in the 6R-BH(4) complexed enzyme, the N-terminal functions as an intrinsic amino acid regulatory sequence (IARS). Neither 6M-PH(4) nor 6S-BH(4) can interact favorably with Ser23, and do not induce an inhibitory effect on PAH. These simulations thus explain the previous findings that the two hydroxyl groups in the side chain of the 6R epimer of BH(4) are essential for the inhibitory regulatory effect on PAH.     

Safe and successful endoarterial infusion of liposomal amphotericin B in treatment of mucormycosis

Mucormycosis is a rare invasive mycotic infection treated by antifungini or amphotericin B. We describe the case of a patient with septic fever and a necrotic lesion, with phlegmon of medial left thigh. Surgery was performed to drain the abscess content and to remove the necrotic tissue; mucormycosis was diagnosized by histological and culture tests and treated by intravenous amphotericin B. Since the lesion worsened, liposomal amphotericin B was directly infused into the left common iliac artery, with progressive improvement, and treatment was continued until complete recovery. Therefore, the endoarterial infusion of liposomal amphotericin B was a safe and successful treatment of advanced lesions of mucormycosis. In such lesions, intravenous general antibiotic administration probably is not sufficient to reach the whole infected area.