Structural characterization and oligomerization of PB1-F2, a proapoptotic influenza A virus protein

Recently, a novel 87-amino acid influenza A virus protein with proapoptotic properties, PB1-F2, has been reported that originates from an alternative reading frame in the PB1 polymerase gene and is encoded in most known human influenza A virus isolates. Here we characterize the molecular structure of a biologically active synthetic version of the protein (sPB1-F2). Western blot analysis, chemical cross-linking, and NMR spectroscopy afforded direct evidence of the inherent tendency of sPB1-F2 to undergo oligomerization mediated by two distinct domains located in the N and C termini, respectively. CD and (1)H NMR spectroscopic analyses indicate that the stability of structured regions in the molecule clearly depends upon the hydrophobicity of the solvent. In aqueous solutions, the behavior of sPB1-F2 is typical of a largely random coil peptide that, however, adopts alpha-helical structure upon the addition of membrane mimetics. (1)H NMR analysis of three overlapping peptides afforded, for the first time, direct experimental evidence of the presence of a C-terminal region with strong alpha-helical propensity comprising amino acid residues Ile(55)-Lys(85) connected via an essentially random coil structure to a much weaker helix-like region, located in the N terminus between residues Trp(9) and Lys(20). The C-terminal helix is not a true amphipathic helix and is more compact than previously predicted. It corresponds to a positively charged region previously shown to include the mitochondrial targeting sequence of PB1-F2. The consequences of the strong oligomerization and helical propensities of the molecule are discussed and used to formulate a hypothetical model of its interaction with the mitochondrial membrane.     

Anthocyanin from strawberry (Fragaria ananassa) with the novel aglycone, 5-carboxypyranopelargonidin

An anthocyanin, 1, with the novel 4-substituted aglycone, 5-carboxypyranopelargonidin, was isolated in small amounts from the acidified, methanolic extract of strawberries, Fragaria ananassa Duch., by preparative HPLC after purification by partition against ethyl acetate, Amberlite XAD-7 and Sephadex LH-20 column chromatography. It was identified mainly by 2D NMR spectroscopy and electrospray LC-MS as the 3-O-beta-glucopyranoside of 5-carboxy-2-(4-hydroxyphenyl)-3,8-dihydroxy-pyrano[4,3,2-de]-1-benzopyrylium, an anthocyanidin which is homologous to 5-carboxypyranomalvidin (vitisidin A) reported in red wines and 5-carboxypyranocyanidin recently isolated from red onions. By comparison of UV-Vis absorption spectra, 1 showed in contrast to 2, pelargonidin 3-O-beta-glucopyranoside, a local absorption peak around 360 nm, a hypsochromic shift (8 nm) of the visible absorption maximum, and lack of a distinct UV absorption peak around 280 nm. The similarities between the absorption spectra of 1 in various acidic and neutral buffer solutions implied restricted formation of the instable colourless equilibrium forms, which are typical for most anthocyanins in weakly acidic solutions. The molar absorptivity (epsilon) of 1 varied little with pH contrary to similar values of for instance the major anthocyanin in strawberry, 2. However, 2 revealed higher epsilon-values than 1 at all pH values except 5.1. At pH 5.1, the epsilon-value of 1 (6250) was nearly four times the corresponding value of 2 (1720), which showed the potential of 5-carboxypyranopelargonidin derivatives as colorants in solutions with pH around 5. The colours of 1 and 2 in buffered solutions with pH 1.1 and pH 6.9 have been described by the CIELAB coordinates h(ab) (hue angle), C* (chroma), and L* (lightness).     

Effect of milk- and TRIS-based extenders on the fertility of sheep inseminated vaginally once or twice with liquid semen

We studied the influence of two different extenders, a milk-based versus a TRIS-based extender, using a split-sample technique, on fertility after single and double vaginal inseminations in natural estrous in Norwegian Crossbred ewes. Semen from 21 Norwegian Crossbred rams, all aged approximately 0.5 years, was used for AI of totally 561 Norwegian Crossbred ewes housed at 37 different farms. The farmers performed the inseminations themselves. The ewes were allocated to four parallel groups based on the two extenders and single or double inseminations (2 x 2). The farmers were recommended to inseminate the ewes between 12 and 24 h after detection of natural standing estrous. Vaginal insemination with cooled liquid semen diluted in the milk-based extender resulted in a statistically significant (P<0.01) better fertility of about 10% units both as 25-day NR (non return rate)-and lambing rates, compared with semen diluted in the TRIS-based extender. Double inseminations gave significantly higher (P=0.03) fertility results for both extenders expressed as 25-day NR results, but was not quite statistically significant when expressed as lambing rates (P=0.06) compared with single insemination. The overall 25-day NR results for the milk-based extender (66.4%) after single inseminations is in accordance with both the national results (67.1%) based on vaginal inseminations of 11,377 ewes, as well as with the results from a previous study in the same region achieving a 25-day NR results of 63.3%. In conclusion, liquid ram semen diluted in a milk-based extender and vaginally inseminated once in natural heat, with a semen dose of 150 x 10(6) spermatozoa, gave acceptable fertility results and is to be recommended as the method of choice in Norway.     

Anthocyanin 3-galactosides from Cornus alba 'Sibirica' with glucosidation of the B-ring

The three anthocyanins, delphinidin 3-O-beta-galactopyranoside-3',5'-di-O-beta-glucopyranoside (1), delphinidin 3-O-beta-galactopyranoside-3'-O-beta-glucopyranoside (2) and cyanidin 3-O-beta-galactopyranoside-3'-O-beta-glucopyranoside (3), and the 3-O-beta-galactopyranosides of delphinidin (4) and cyanidin (5) were isolated from the bluish white berries and compound umbel of Siberian dogwood, Cornus alba 'Sibirica'. The ornamental autumn leaves and the characteristic purplish red bark of this variety were found to contain only pigment 5.     

Synthesis of full length PB1-F2 influenza A virus proteins from 'Spanish flu' and 'bird flu'.

The proapoptotic influenza A virus PB1-F2 protein contributes to viral pathogenicity and is present in most human and avian isolates. Previous synthetic protocols have been improved to provide a synthetic full length H1N1 type PB1-F2 protein that is encoded by the 'Spanish flu' isolate and an equivalent protein from an avian host that is representative of a highly pathogenic H5N1 'bird flu' isolate, termed SF2 and BF2, respectively. Full length SF2, different mutants of BF2 and a number of fragments of these peptides have been synthesized by either the standard solid-phase peptide synthesis method or by native chemical ligation of unprotected N- and C-terminal peptide fragments. For SF2 chemical ligation made use of the histidine and the cysteine residues located in positions 41 and 42 of the native sequence, respectively, to afford a highly efficient synthesis of SF2 compared to the standard SPPS elongation method. By-product formation at the aspartic acid residue in position 23 was prevented by specific modifications of the SPPS protocol. As the native sequence of BF2 does not contain a cysteine residue two different mutants of BF2 (Y42C) and BF2 (S47C) with appropriate cysteine exchanges were produced. In addition to the full length molecules, fragments of the native sequences were synthesized for comparison of their physical characteristics with those from the H1N1 human isolate A/Puerto Rico/8/34 (H1N1). All peptides were analyzed by mass spectrometry, (1)H NMR spectroscopy, and SDS-PAGE. The protocols allow the synthesis of significant amounts of PB1-F2 and its related peptides. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Paired opposing leukocyte receptors recognizing rapidly evolving ligands are subject to homogenization of their ligand binding domains

Some leukocyte receptors come in groups of two or more where the partners share ligand(s) but transmit opposite signals. Some of the ligands, such as MHC class I, are fast evolving, raising the problem of how paired opposing receptors manage to change in step with respect to ligand binding properties and at the same time conserve opposite signaling functions. An example is the KLRC (NKG2) family, where opposing variants have been conserved in both rodents and primates. Phylogenetic analyses of the KLRC receptors within and between the two orders show that the opposing partners have been subject to post-speciation gene homogenization restricted mainly to the parts of the genes that encode the ligand binding domains. Concerted evolution similarly restricted is demonstrated also for the KLRI, KLRB (NKR-P1), KLRA (Ly49), and PIR receptor families. We propose the term merohomogenization for this phenomenon and discuss its significance for the evolution of immune receptors.     

The intriguing Cyclophilin A-HIV-1 Vpr interaction: prolyl cis/trans isomerisation catalysis and specific binding

Background

Cyclophilin A (CypA) represents a potential target for antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication, although the mechanism through which CypA modulates HIV-1 infectivity still remains unclear. The interaction of HIV-1 viral protein R (Vpr) with the human peptidyl prolyl isomerase CypA is known to occur in vitro and in vivo. However, the nature of the interaction of CypA with Pro-35 of N-terminal Vpr has remained undefined.

Results

Characterization of the interactions of human CypA with N-terminal peptides of HIV-1 Vpr has been achieved using a combination of nuclear magnetic resonace (NMR) exchange spectroscopy and surface plasmon resonance spectroscopy (SPR). NMR data at atomic resolution indicate prolyl cis/trans isomerisation of the highly conserved proline residues Pro-5, -10, -14 and -35 of Vpr are catalyzed by human CypA and require only very low concentrations of the isomerase relative to that of the peptide substrates. Of the N-terminal peptides of Vpr only those containing Pro-35 bind to CypA in a biosensor assay. SPR studies of specific N-terminal peptides with decreasing numbers of residues revealed that a seven-residue motif centred at Pro-35 consisting of RHFPRIW, which under membrane-like solution conditions comprises the loop region connecting helix 1 and 2 of Vpr and the two terminal residues of helix 1, is sufficient to maintain strong specific binding.

Conclusions

Only N-terminal peptides of Vpr containing Pro-35, which appears to be vital for manifold functions of Vpr, bind to CypA in a biosensor assay. This indicates that Pro-35 is essential for a specific CypA-Vpr binding interaction, in contrast to the general prolyl cis/trans isomerisation observed for all proline residues of Vpr, which only involve transient enzyme-substrate interactions. Previously suggested models depicting CypA as a chaperone that plays a role in HIV-1 virulence are now supported by our data. In detail the SPR data of this interaction were compatible with a two-state binding interaction model that involves a conformational change during binding. This is in accord with the structural changes observed by NMR suggesting CypA catalyzes the prolyl cis/trans interconversion during binding to the RHFP³⁵RIW motif of N-terminal Vpr.     

Solution structure of the human immunodeficiency virus type 1 p6 protein

The human immunodeficiency virus type 1 p6 protein represents a docking site for several cellular and viral binding factors and fulfills major roles in the formation of infectious viruses. To date, however, the structure of this 52-amino acid protein, by far the smallest lentiviral protein known, either in its mature form as free p6 or as the C-terminal part of the Pr55 Gag polyprotein has not been unraveled. We have explored the high resolution structure and folding of p6 by CD and NMR spectroscopy. Under membranous solution conditions, p6 can adopt a helix-flexible helix structure; a short helix-1 (amino acids 14-18) is connected to a pronounced helix-2 (amino acids 33-44) by a flexible hinge region. Thus, p6 can be subdivided into two distinct structural and functional domains; helix-2 perfectly defines the region that binds to the virus budding factor AIP-1/ALIX, indicating that this structure is required for interaction with the endosomal sorting complex required for transport. The PTAP motif at the N terminus, comprising the primary late assembly domain, which is crucial for interaction with another cellular budding factor, Tsg101, does not exhibit secondary structure. However, the adjacent helix-1 may play an indirect role in the specific complex formation between p6 and the binding groove in Tsg101. Moreover, binding studies by NMR demonstrate that helix-2, which also comprises the LXXLF motif required for incorporation of the human immunodeficiency virus type 1 accessory protein Vpr into budding virions, specifically interacts with the Vpr binding region, indicating that under the specific solution conditions used for structure analysis, p6 adopted a functional conformation.     

Growing with dinosaurs: natural products from the Cretaceous relict Metasequoia glyptostroboides Hu & Cheng—a molecular reservoir from the ancient world with potential in modern medicine

Phytochemistry Reviews - After the sensational rediscovery of living exemplars of the Cretaceous relict Metasequoia glyptostroboides—a tree previously known exclusively from fossils from...