Basic fibroblast growth factor is a β-rich protein

The conformation of the 153-residue form of human basic fibroblast growth factor (bFGF) was studied with circular dichroism (CD) and sequence prediction methods. The far-UV CD spectrum with a minimum at 202 nm resembled that of an unordered polypeptide/protein or a protein rich in distorted antiparallel β-sheets. Analysis of the CD spectrum by the least-squares method of Changet al. (1978) and the CONTIN program of Provencher and Glöckner (1981) suggested that about one half of the molecule consisted of β-sheet and there was no α-helix. These estimates agreed with the prediction by the sequence method of Garnieret al. (1978) using decision constants based on CD results. bFGF had an unusual CD band at 187 nm, which disappeared upon ionization of Tyr side chains atpH 11.7. It also had another unusual property of irreversibly converting the CD spectrum to a helix-like one with a double minimum at 205 and 215 and a maximum at 189 nm upon heating the solution to above 55°C. The helicity was also enhanced in trifluoroethanol and in sodium dodecyl sulfate. The mutant bFGF in which cysteines 76 and 94 were replaced by serine residues had essentially the same properties as the wild-type.     

A putative nuclear growth factor‐like globular nematode specific protein

Expressed sequence tags (ESTs) are an effective approach for discovery of novel genes. In the current study, approximately 250 ESTs of the cattle parasitic nematode Setaria digitata were examined and a cDNA clone identified whose coding sequence could not be functionally annotated by searching over publicly available genome, protein, EST and STS databases. Here, we report the extensive characterization of this ORF (UP) and its homologues using a bioinformatic approach. Uncharacterized protein (SDUP) of S. digitata consists of 204 amino acids with a predicted molecular weight and isoelectric point of 22.8KDa and 9.94, respectively. A search carried out using SDUP over nucleotide, EST and protein databases at NCBI, NEMBASE3 and Parasite Genome Database (PGD) identified homologous counterparts from the human parasitic nematodes Wuchereria bancrofti (WB), Brugia malayi (BM), Onchocerca volvulus (OV), the mouse filarial worm Litomosoides sigmodontis (LS), swine parasitic nematodes Ascaris suum (AS) and diverged counterparts from the plant parasitic nematode Meloidogyne hapla (MH) and free living nematodes Caenorhabditis elegans (CE) and Caenorhabditis briggsae (CB). Phylogenetic analyses revealed the UPs to be undergoing divergent evolution. A search of the ESTs at PGD showed that UP is expressed in all the stages of BM. Secondary structure analyses of multiply-aligned sequences of homologues using Jpred server indicated UPs to be rich in beta-pleated structures. TMMHH server and beta barrel finder programme indicated, UPs to be neither transmembrane or beta barrels proteins but are likely to be globular proteins. Further, the Motif discovery tool of MEME identified three novel potential motifs for UPS, of which only two are present in CE, CB & MH. Analyses of UPs using Signal IP, TargetP, Psort servers predicted this group of proteins to be devoid of signal peptide cleavage sites, are not mitochondrial targeting peptides but appear to be localized to the nucleus, respectively. Further analyses of the UPs using ScanProsite server for phosphorylation revealed potential sites for cAMP­ and cGMP­dependent protein kinase, Protein kinase C and Casein kinase II. Putative functional analysis using ProtFun 2.1 Server indicated UPs to be nonenzymatic, growth factor like protein. Finally, collating all the information derived from bioinformatic analyses, we conclude that the UPs of nematodes are most likely to be expressed at all stages in the life cycle, localized to the nucleus, regulated by phosphorylation, rich in beta­pleated strands and are growth factor like nematode specific proteins.     

The guanine-nucleotide-exchange factor P-Rex1 is activated by protein phosphatase 1α

P-Rex1 is a GEF (guanine-nucleotide-exchange factor) for the small G-protein Rac that is activated by PIP3 (phosphatidylinositol 3,4,5-trisphosphate) and Gβγ subunits and inhibited by PKA (protein kinase A). In the present study we show that PP1α (protein phosphatase 1α) binds P-Rex1 through an RVxF-type docking motif. PP1α activates P-Rex1 directly in vitro, both independently of and additively to PIP3 and Gβγ. PP1α also substantially activates P-Rex1 in vivo, both in basal and PDGF (platelet-derived growth factor)- or LPA (lysophosphatidic acid)-stimulated cells. The phosphatase activity of PP1α is required for P-Rex1 activation. PP1β, a close homologue of PP1α, is also able to activate P-Rex1, but less effectively. PP1α stimulates P-Rex1-mediated Rac-dependent changes in endothelial cell morphology. MS analysis of wild-type P-Rex1 and a PP1α-binding-deficient mutant revealed that endogenous PP1α dephosphorylates P-Rex1 on at least three residues, Ser834, Ser1001 and Ser1165. Site-directed mutagenesis of Ser1165 to alanine caused activation of P-Rex1 to a similar degree as did PP1α, confirming Ser1165 as a dephosphorylation site important in regulating P-Rex1 Rac-GEF activity. In summary, we have identified a novel mechanism for direct activation of P-Rex1 through PP1α-dependent dephosphorylation.     

Liver-type fatty acid binding protein interacts with hepatocyte nuclear factor 4α

Hepatocyte nuclear factor 4α (HNF4α) regulates liver type fatty acid binding protein (L-FABP) gene expression. Conversely as shown herein, L-FABP structurally and functionally also interacts with HNF4α. Fluorescence resonance energy transfer (FRET) between Cy3-HNF4α (donor) and Cy5-L-FABP (acceptor) as well as FRET microscopy detected L-FABP in close proximity (∼80 Å) to HNF4α, binding with high affinity K_d ∼250–300 nM. Circular dichroism (CD) determined that the HNF4α/L-FABP interaction altered protein secondary structure. Finally, L-FABP potentiated transactivation of HNF4α in COS7 cells. Taken together, these data suggest that L-FABP provides a signaling path to HNF4α activation in the nucleus.     

Identifying protein–protein interaction sites in transient complexes with temperature factor, sequence profile and accessible surface area

Transient protein–protein interactions play a vital role in many biological processes, such as cell regulation and signal transduction. A nonredundant dataset of 130 protein chains extracted from transient complexes was used to analyze the features of transient interfaces. It was found that besides the two well-known features, sequence profile and accessible surface area (ASA), the temperature factor (B-factor) can also reflect the differences between interface and the rest of protein surface. These features were utilized to construct support vector machine (SVM) classifiers to identify interaction sites. The results of threefold cross-validation on the nonredundant dataset show that when B-factor was used as an additional feature, the prediction performance can be improved significantly. The sensitivity, specificity and correlation coefficient were raised from 54 to 62%, 41 to 45% and 0.20 to 0.29, respectively. To further illustrate the effectiveness of our method, the classifiers were tested with an independent set of 53 nonhomologous protein chains derived from benchmark 2.0. The sensitivity, specificity and correlation coefficient of the classifier based on the three features were 63%, 45% and 0.33, respectively. It is indicated that our classifiers are robust and can be applied to complement experimental techniques in studying transient protein–protein interactions.     

In vivo imaging of protein–protein and RNA–protein interactions using novel far-red fluorescence complementation systems

Imaging of protein–protein and RNA–protein interactions in vivo, especially in live animals, is still challenging. Here we developed far-red mNeptune-based bimolecular fluorescence complementation (BiFC) and trimolecular fluorescence complementation (TriFC) systems with excitation and emission above 600 nm in the ‘tissue optical window’ for imaging of protein–protein and RNA–protein interactions in live cells and mice. The far-red mNeptune BiFC was first built by selecting appropriate split mNeptune fragments, and then the mNeptune-TriFC system was built based on the mNeptune-BiFC system. The newly constructed mNeptune BiFC and TriFC systems were verified as useful tools for imaging protein–protein and mRNA–protein interactions, respectively, in live cells and mice. We then used the new mNeptune-TriFC system to investigate the interactions between human polypyrimidine-tract-binding protein (PTB) and HIV-1 mRNA elements as PTB may participate in HIV mRNA processing in HIV activation from latency. An interaction between PTB and the 3′long terminal repeat region of HIV-1 mRNAs was found and imaged in live cells and mice, implying a role for PTB in regulating HIV-1 mRNA processing. The study provides new tools for in vivo imaging of RNA–protein and protein–protein interactions, and adds new insight into the mechanism of HIV-1 mRNA processing.     

Structures of the tumor necrosis factor α inducing protein Tipα: A novel virulence factor from Helicobacter pylori

Helicobacter pylori secretes a unique virulence factor, Tipα, that enters gastric cells and both stimulates the production of the TNF-α and activates the NF-κB pathway. The structures of a truncated version of Tipα (TipαN34) in two crystal forms are presented here. Tipα adopts a novel β₁α₁α₂β₂β₃α₃α₄ topology that can be described as a combination of three domains. A first region consists in a short flexible extension, a second displays a dodecin-like fold and a third is a helical bundle domain similar to the sterile alpha motif (SAM). Analysis of the oligomerisation states of TipαN34 in the crystals and in solution suggests that the disulfide bridges could hold together Tipα monomers during their secretion in the gastric environment.

Structured summary

MINT-7033680:TIP alpha (uniprotkb:B2UTN0) and TIP alpha (uniprotkb:B2UTN0) bind (MI:0407) by cosedimentation (MI:0027)     

Conformational characterization of human eukaryotic initiation factor 2α: A single tryptophan protein

The α-subunit of the human eukaryotic initiation factor 2 (heIF2α), a GTP binding protein, plays a major role in the initiation of protein synthesis. During various cytoplasmic stresses, eIF2α gets phosphorylated by eIF2α-specific kinases resulting in inhibition of protein synthesis. The cloned and over expressed heIF2α, a protein with a single tryptophan (trp) residue was examined for its conformational characteristics using steady-state and time-resolved tryptophan fluorescence, circular dichroism (CD) and hydrophobic dye binding. The steady-state fluorescence spectrum, fluorescence lifetimes (τ₁ = 1.13 ns and τ₂ = 4.74 ns) and solute quenching studies revealed the presence of trp conformers in hydrophobic and differential polar environment at any given time. Estimation of the α-helix and β-sheet content showed: (i) more compact structure at pH 2.0, (ii) distorted α-helix and rearranged β-sheet in presence of 4 M guanidine hydrochloride and (iii) retention of more than 50% ordered structure at 95 °C. Hydrophobic dye binding to the protein with loosened tertiary structure was observed at pH 2.0 indicating the existence of a molten globule-like structure. These observations indicate the inherent structural stability of the protein under various denaturing conditions.     

Molecular insights into the interaction of the ribosomal stalk protein with elongation factor 1α

In all organisms, the large ribosomal subunit contains multiple copies of a flexible protein, the so-called ‘stalk’. The C-terminal domain (CTD) of the stalk interacts directly with the translational GTPase factors, and this interaction is required for factor-dependent activity on the ribosome. Here we have determined the structure of a complex of the CTD of the archaeal stalk protein aP1 and the GDP-bound archaeal elongation factor aEF1α at 2.3 Å resolution. The structure showed that the CTD of aP1 formed a long extended α-helix, which bound to a cleft between domains 1 and 3 of aEF1α, and bridged these domains. This binding between the CTD of aP1 and the aEF1α•GDP complex was formed mainly by hydrophobic interactions. The docking analysis showed that the CTD of aP1 can bind to aEF1α•GDP located on the ribosome. An additional biochemical assay demonstrated that the CTD of aP1 also bound to the aEF1α•GTP•aminoacyl-tRNA complex. These results suggest that the CTD of aP1 interacts with aEF1α at various stages in translation. Furthermore, phylogenetic perspectives and functional analyses suggested that the eukaryotic stalk protein also interacts directly with domains 1 and 3 of eEF1α, in a manner similar to the interaction of archaeal aP1 with aEF1α.     

A variant of the von Willebrand-Jürgens-syndrome with abnormalities of the factor VIII/von Willebrand factor protein (author's transl)]

A family is reported with a variant of von Willebrand's disease. The members of this family showed a qualitative defect of the factor VII/von Willebrand factor protein. The qualitative defect was characterized by an abnormal electrophoretical mobility of factor VIII-related antigen and an abnormal elution pattern as demonstrated by gelfiltration on Sepharose 4 B. Factor VIII-subunits in these patients were found to be normal by polyacrylamidgelelektrophoresis.     

Tumor necrosis factor α-induced adipose-related protein expression in experimental arthritis and in rheumatoid arthritis

Introduction  

Tumor necrosis factor-alpha (TNFα) plays a pivotal role in rheumatoid arthritis (RA); however, the mechanism of action of TNFα antagonists in RA is poorly defined. Immunization of DBA/1 mice with glucose-6-phosphate isomerase (GPI) induces severe acute arthritis. This arthritis can be controlled by TNFα antagonists, suggesting similar etiology to RA. In this study, we explored TNFα-related mechanisms of arthritis.     

Tumor necrosis factor α-induced adipose-related protein expression in experimental arthritis and in rheumatoid arthritis

Introduction

Broad-range rDNA PCR provides an alternative, cultivation-independent approach for identifying bacterial DNA in reactive and other form of arthritis. The aim of this study was to use broad-range rDNA PCR targeting the 16S rRNA gene in patients with reactive and other forms of arthritis and to screen for the presence of DNA from any given bacterial species in synovial fluid (SF) samples.

Methods

We examined the SF samples from a total of 27 patients consisting of patients with reactive arthritis (ReA) (n = 5), undifferentiated arthritis (UA) (n = 9), rheumatoid arthritis (n = 7), and osteoarthritis (n = 6) of which the latter two were used as controls. Using broad-range bacterial PCR amplifying a 1400 bp fragment from the 16S rRNA gene, we identified and sequenced at least 24 clones from each SF sample. To identify the corresponding bacteria, DNA sequences were compared to the EMBL (European Molecular Biology Laboratory) database.

Results

Bacterial DNA was identified in 20 of the 27 SF samples (74, 10%). Analysis of a large number of sequences revealed the presence of DNA from more than one single bacterial species in the SF of all patients studied. The nearly complete sequences of the 1400 bp were obtained for most of the detected species. DNA of bacterial species including Shigella species, Escherichia species, and other coli-form bacteria as well as opportunistic pathogens such as Stenotrophomonas maltophilia and Achromobacter xylosoxidans were shared in all arthritis patients. Among pathogens described to trigger ReA, DNA from Shigella sonnei was found in ReA and UA patients. We also detected DNA from rarely occurring human pathogens such as Aranicola species and Pantoea ananatis. We also found DNA from bacteria so far not described in human infections such as Bacillus niacini, Paenibacillus humicus, Diaphorobacter species and uncultured bacterium genera incertae sedis OP10.

Conclusions

Broad-range PCR followed by cloning and sequencing the entire 16S rDNA, allowed the identification of the bacterial DNA environment in the SF samples of arthritic patients. We found a wide spectrum of bacteria including those known to be involved in ReA and others not previously associated with arthritis.     

On the mechanism of activation of protein kinase FA (an activating factor of ATP·Mg-dependent protein phosphatase) in brain myelin

Protein kinase F_A (an activating factor of ATP·Mg-dependent protein phosphatase) has been characterized to exist in two forms in the purified brain myelin. One form of kinase F_A is spontaneously active and trypsin-labile, whereas the other form of kinase F_A is inactive and trypsin-resistant, suggesting a different membrane topography with active F_A exposed on the outer face of the myelin membrane and inactivu F_Q buried within the myelin membrane. When myelin was solubilized in 1% Triton X-100, all kinase F_A became active and trypsin-labile. Phospholipid reconstitution studies further indicated that when kinase F_A was reconstituted in acidic phospholipids, such as phosphatidylinositol and phosphatidylserine, the enzyme activity was inhibited in a dose-dependent manner, suggesting that kinase F_A interacts with acidic phospholipids which inhibit its activity. Furthermore, when myelin was incubated with exogenous phospholipase C, the inactive/trypsin-resistant F_A could be converted to the active/trypsin-labile F_A in a time- and dose-dependent manner. Taken together, it is concluded that membrane phospholipids play an important role in modulating the activity of kinase F_A in the brain myelin. It is suggested that phospholipase C may mediate the activation-sequestration of inactive/trypsin-resistant kinase F_A in the brain myelin through the phospholipase C-katalyzed degradation of acidic membrane phospholipids. The activation-sequestration of protein Kinase F_A may represent one mode of control modulating the activity of kinase F_A in the central nervous system myelin.