Nuclear magnetic resonance structure of the nucleic acid-binding domain of severe acute respiratory syndrome coronavirus nonstructural protein 3

The nuclear magnetic resonance (NMR) structure of a globular domain of residues 1071 to 1178 within the previously annotated nucleic acid-binding region (NAB) of severe acute respiratory syndrome coronavirus nonstructural protein 3 (nsp3) has been determined, and N- and C-terminally adjoining polypeptide segments of 37 and 25 residues, respectively, have been shown to form flexibly extended linkers to the preceding globular domain and to the following, as yet uncharacterized domain. This extension of the structural coverage of nsp3 was obtained from NMR studies with an nsp3 construct comprising residues 1066 to 1181 [ nsp3(1066-1181)] and the constructs nsp3(1066-1203) and nsp3(1035-1181). A search of the protein structure database indicates that the globular domain of the NAB represents a new fold, with a parallel four-strand beta-sheet holding two alpha-helices of three and four turns that are oriented antiparallel to the beta-strands. Two antiparallel two-strand beta-sheets and two 3(10)-helices are anchored against the surface of this barrel-like molecular core. Chemical shift changes upon the addition of single-stranded RNAs (ssRNAs) identified a group of residues that form a positively charged patch on the protein surface as the binding site responsible for the previously reported affinity for nucleic acids. This binding site is similar to the ssRNA-binding site of the sterile alpha motif domain of the Saccharomyces cerevisiae Vts1p protein, although the two proteins do not share a common globular fold.

AtTRB1, a telomeric DNA-binding protein from Arabidopsis, is concentrated in the nucleolus and shows highly dynamic association with chromatin

AtTRB1, 2 and 3 are members of the SMH (single Myb histone) protein family, which comprises double-stranded DNA-binding proteins that are specific to higher plants. They are structurally conserved, containing a Myb domain at the N-terminus, a central H1/H5-like domain and a C-terminally located coiled-coil domain. AtTRB1, 2 and 3 interact through their Myb domain specifically with telomeric double-stranded DNA in vitro, while the central H1/H5-like domain interacts non-specifically with DNA sequences and mediates protein–protein interactions. Here we show that AtTRB1, 2 and 3 preferentially localize to the nucleus and nucleolus during interphase. Both the central H1/H5-like domain and the Myb domain from AtTRB1 can direct a GFP fusion protein to the nucleus and nucleolus. AtTRB1–GFP localization is cell cycle-regulated, as the level of nuclear-associated GFP diminishes during mitotic entry and GFP progressively re-associates with chromatin during anaphase/telophase. Using fluorescence recovery after photobleaching and fluorescence loss in photobleaching, we determined the dynamics of AtTRB1 interactions in vivo. The results reveal that AtTRB1 interaction with chromatin is regulated at two levels at least, one of which is coupled with cell-cycle progression, with the other involving rapid exchange.

A functional proteomic method for the enrichment of peripheral membrane proteins reveals the collagen binding protein Hsp47 is exposed on the surface of activated human platelets

Platelets are small blood cells vital for hemostasis. Following vascular damage, platelets adhere to collagens and activate, forming a thrombus that plugs the wound and prevents blood loss. Stimulation of the platelet collagen receptor glycoprotein VI (GPVI) allows recruitment of proteins to receptor-proximal signaling complexes on the inner-leaflet of the plasma membrane. These proteins are often present at low concentrations; therefore, signaling-complex characterization using mass spectrometry is limited due to high sample complexity. We describe a method that facilitates detection of signaling proteins concentrated on membranes. Peripheral membrane proteins (reversibly associated with membranes) were eluted from human platelets with alkaline sodium carbonate. Liquid-phase isoelectric focusing and gel electrophoresis were used to identify proteins that changed in levels on membranes from GPVI-stimulated platelets. Immunoblot analysis verified protein recruitment to platelet membranes and subsequent protein phosphorylation was preserved. Hsp47, a collagen binding protein, was among the proteins identified and found to be exposed on the surface of GPVI-activated platelets. Inhibition of Hsp47 abolished platelet aggregation in response to collagen, while only partially reducing aggregation in response to other platelet agonists. We propose that Hsp47 may therefore play a role in hemostasis and thrombosis.

Amniotic fluid and umbilical cord plasma corticotropin-releasing factor (CRF), CRF-binding protein, adrenocorticotropin, and cortisol concentrations in intraamniotic infection and inflammation at term

Context: Pregnant tissues express corticotropin-releasing factor (CRF), a peptide modulating fetal and placental ACTH and cortisol secretion. These actions are modulated by the locally expressed CRF-binding protein (CRF-BP). Objective: The objective of the study was to determine whether CRF, CRF-BP, ACTH, and cortisol concentrations change in amniotic fluid and umbilical cord plasma in the presence of intraamniotic infection/inflammation (IAI) in women with spontaneous labor at term. Design: This was a cross-sectional study. Setting: The study was conducted at a tertiary referral center for obstetric care. Patients: Patients included women in active labor at term with (n = 39) and without (controls; n = 78) IAI. Main Outcome Measures: Amniotic fluid and umbilical cord plasma concentrations of CRF, CRF-BP, ACTH, and cortisol measured by RIA and immunoradiometric assays were measured. Results: In patients with IAI, amniotic fluid CRF (0.97 +/- 0.18 ng/ml) and CRF-BP (33.06 +/- 5.54 nmol/liter) concentrations were significantly (P < 0.001) higher than in controls (CRF: 0.32 +/- 0.04 ng/ml; CRF-BP: 14.69 +/- 2.79 ml). The umbilical cord plasma CRF and CRF-BP concentrations were significantly (P < 0.001 for all) higher in women with IAI than in controls (CRF: 2.96 +/- 0.35 ng/ml vs. 0.38 +/- 0.18 ng/ml; CRF-BP: 152.12 +/- 5.94 nmol/liter vs. 106.9 +/- 5.97 nmol/liter). In contrast, amniotic fluid and umbilical cord plasma ACTH and cortisol concentrations did not differ between groups. Conclusions: Amniotic fluid and umbilical cord plasma CRF and CRF-BP concentrations are increased in women with spontaneous labor at term and IAI. CRF-BP may modulate CRF actions on ACTH and cortisol secretion, playing a pivotal role in limiting the inflammatory process and thus avoiding an overactivation of the fetal/placental hypothalamus-pituitary-adrenal axis at birth.

Mapping the Sinorhizobium meliloti 1021 solute-binding protein-dependent transportome

The number of solute-binding protein-dependent transporters in rhizobia is dramatically increased compared with the majority of other bacteria so far sequenced. This increase may be due to the high affinity of solute-binding proteins for solutes, permitting the acquisition of a broad range of growth-limiting nutrients from soil and the rhizosphere. The transcriptional induction of these transporters was studied by creating a suite of plasmid and integrated fusions to nearly all ATP-binding cassette (ABC) and tripartite ATP-independent periplasmic (TRAP) transporters of Sinorhizobium meliloti. In total, specific inducers were identified for 76 transport systems, amounting to approximate to 47% of the ABC uptake systems and 53% of the TRAP transporters in S. meliloti. Of these transport systems, 64 are previously uncharacterized in Rhizobia and 24 were induced by solutes not known to be transported by ABC- or TRAP-uptake systems in any organism. This study provides a global expression map of one of the largest transporter families (transportome) and an invaluable tool to both understand their solute specificity and the relationships between members of large paralogous families.

The measurement of maternal plasma corticotropin-releasing factor (CRF) and CRF-binding protein improves the early prediction of preeclampsia

In the present study we measured maternal plasma concentrations of two placental neurohormones, corticotropin-releasing factor (CRF) and CRF-binding protein (CRF-BP), in 58 at-risk pregnant women consecutively enrolled between 28 and 29 wk of pregnancy to evaluate whether their evaluation may predict third trimester-onset preeclampsia ( PE). The statistical significance was assessed by t test. The cut-off points for defining altered CRF and CRF-BP levels for prediction of PE were chosen by receiving operator characteristics curve analysis, and the probability of developing PE was calculated for several combinations of hormone testing results. CRF and CRF-BP levels were significantly ( both P < 0.0001) higher and lower, respectively, in the patients (n = 20) who later developed PE than in those who did not present PE at follow-up. CRF at the cut-off 425.95 pmol/liter achieved a sensitivity of 94.8% and a specificity of 96.9%, whereas CRF-BP at the cut-off 125.8 nmol/liter combined a sensitivity of 92.5% and a specificity of 82.5% as single markers for prediction of PE. The probability of PE was 34.5% in the whole study population, 93.75% when both CRF and CRF-BP levels were changed, and 0% if both hormone markers were unaltered. The measurement of CRF and CRF-BP levels may add significant prognostic information for predicting PE in at-risk pregnant women.

Platelet endothelial cell adhesion molecule-1 regulates collagen-stimulated platelet function by modulating the association of phosphatidylinositol 3-kinase with Grb-2-associated binding protein-1 and linker for activation of T cells

Background: Platelet activation by collagen depends on signals transduced by the glycoprotein (GP)VI–Fc receptor (FcR)-chain collagen receptor complex, which involves recruitment of phosphatidylinositol 3-kinase (PI3K) to phosphorylated tyrosines in the linker for activation of T cells (LAT). An interaction between the p85 regulatory subunit of PI3K and the scaffolding molecule Grb-2-associated binding protein-1 (Gab1), which is regulated by binding of the Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) to Gab1, has been shown in other cell types to sustain PI3K activity to elicit cellular responses. Platelet endothelial cell adhesion molecule-1 (PECAM-1) functions as a negative regulator of platelet reactivity and thrombosis, at least in part by inhibiting GPVI–FcR-chain signaling via recruitment of SHP-2 to phosphorylated immunoreceptor tyrosine-based inhibitory motifs in PECAM-1. Objective: To investigate the possibility that PECAM-1 regulates the formation of the Gab1–p85 signaling complexes, and the potential effect of such interactions on GPVI-mediated platelet activation in platelets. Methods: The ability of PECAM-1 signaling to modulate the LAT signalosome was investigated with immunoblotting assays on human platelets and knockout mouse platelets. Results: PECAM-1-associated SHP-2 in collagen-stimulated platelets binds to p85, which results in diminished levels of association with both Gab1 and LAT and reduced collagen-stimulated PI3K signaling. We therefore propose that PECAM-1-mediated inhibition of GPVI-dependent platelet responses result, at least in part, from recruitment of SHP-2–p85 complexes to tyrosine-phosphorylated PECAM-1, which diminishes the association of PI3K with activatory signaling molecules, such as Gab1 and LAT.

Puroindoline-a, a lipid binding protein from common wheat, spontaneously forms prolate protein micelles in solution

The self-assembly in solution of puroindoline-a (Pin-a), an amphiphilic lipid binding protein from common wheat, was investigated by small angle neutron scattering, dynamic light scattering and size exclusion chromatography. Pin-a was found to form monodisperse prolate ellipsoidal micelles with a major axial radius of 112 +/- 4.5 A ˚ and minor axial radius of 40.4 +/- 0.18 A ˚ . These protein micelles were formed by the spontaneous self-assembly of 38 Pin-a molecules in solution and were stable over a wide pH range (3.5–11) and at elevated temperatures (20–65 degC). Pin-a micelles could be disrupted upon addition of the non-ionic surfactant dodecyl-b-maltoside, suggesting that the protein self-assembly is driven by hydrophobic forces, consisting of intermolecular interactions between Trp residues located within a well-defined Trp-rich domain of Pin-a.

Granulosal and thecal expression of bone morphogenetic protein- and activin-binding protein mRNA transcripts during bovine follicle development and factors modulating their expression in vitro

Evidence supports local roles for TGFβ superfamily members including activins and bone morphogenetic proteins (BMP) in follicle development. Access of these ligands to signaling receptors is likely modulated by extracellular binding proteins (BP). In this study we compared expression of four BPs (chordin, gremlin, noggin, follistatin) in granulosal (GC) and theca interna (TC) compartments of developing bovine antral follicles (1-18mm). Effects of FSH and IGF on BMP and BP expression by cultured GC, and effects of LH and BMPs on BP expression by cultured TC were also examined. Follicular expression of all four BP transcripts was higher in GC than TC compartments (P<0.001) a finding confirmed by immunohistochemistry. Follicle category affected (P<0.01) gremlin and follistatin mRNA abundance, with a significant cell-type x follicle category interaction for chordin, follistatin and noggin. Noggin transcript abundance was lower (P<0.05) in GC of large 'E-active' than 'E-inactive' follicles while follistatin mRNA level was higher (P<0.01). FSH enhanced CYP19, FSHR, INHBA and follistatin by GC without affecting BMP or BMP-BP expression. IGF increased CYP19 and follistatin, reduced BMP4, noggin and gremlin but did not affect chordin or FSHR mRNA levels. LH increased TC androgen secretion but had no effect on BMP or BP expression. BMPs uniformly suppressed TC androgen production whilst increasing chordin, noggin, and gremlin mRNA levels up to 20-fold (P<0.01). These findings support the hypothesis that extracellular BP, mostly from GC, contribute to the regulation of intrafollicular BMP/activin signaling. Enhancement of thecal BP expression by BMP implies an autoregulatory feedback role to prevent excessive signaling.

Elevated levels of corticotrophin-releasing factor binding protein in the blood of patients suffering from arthritis and septicaemia and the presence of novel ligands in synovial fluid.

In view of the reported inflammatory effects of corticotrophin-releasing factor (CRF) and the associated regulatory elements in the gene of its binding protein (BP), we postulate that both BP as well as novel BP-ligands other than CRF may be involved in inflammatory disease. We have investigated BP in the blood of patients with arthritis and septicaemia and have attempted to identify CRF and other BP-ligands in synovial fluid. The BP was found to be significantly elevated in the blood of patients with rheumatoid arthritis and septicaemia. There was less BP-ligand and CRF in synovial fluid from patients with rheumatoid arthritis that from those with osteo- or psoriatic arthritis. There was at least 10-fold more BP-ligand than CRF in the fluid of all three groups of patients. A small amount of immunoreactive human (h)CRF, eluting in the expected position of CRF-41, was detected after high-pressure liquid chromatography of arthritic synovial fluid; however, the bulk of material with BP-ligand binding activity eluted earlier, suggesting that synovial fluid contained novel peptides that interacted with the BP. These results would suggest that the BP and its ligands could play an endocrine immunomodulatory role in inflammatory disease.

Corticotropin-releasing factor binding protein dimerizes after association with ligand.

We have suggested recently that the fall in plasma CRF-binding protein (BP) during the last few weeks of pregnancy is a direct effect of association with its ligand because of the rapid decrease in plasma BP concentration seen in normal males reaching a nadir some 15 min after a bolus injection of synthetic CRF. In the present study, we have investigated the physicochemical properties of both natural and recombinant BP by gel filtration under physiological conditions and have shown that association of human CRF to this BP results in an increase in molecular weight consistent with the formation of a dimer form of the BP ligand complex. The dimer is more stable when the interaction occurs in the presence of serum or if a peptide with a higher affinity for the BP is substituted as ligand. Experimental evidence would also suggest that the dimer BP has a higher affinity for ligand than the monomeric form. We suggest that this dimerization occurs in vivo when CRF is released into the bloodstream and provides the trigger that causes the uptake of the complex at specific receptor sites.

A mathematical model of the sterol regulatory element binding protein 2 cholesterol biosynthesis pathway

Cholesterol is one of the key constituents for maintaining the cellular membrane and thus the integrity of the cell itself. In contrast high levels of cholesterol in the blood are known to be a major risk factor in the development of cardiovascular disease. We formulate a deterministic nonlinear ordinary differential equation model of the sterol regulatory element binding protein 2 (SREBP-2) cholesterol genetic regulatory pathway in an hepatocyte. The mathematical model includes a description of genetic transcription by SREBP-2 which is subsequently translated to mRNA leading to the formation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), a main precursor of cholesterol synthesis. Cholesterol synthesis subsequently leads to the regulation of SREBP-2 via a negative feedback formulation. Parameterised with data from the literature, the model is used to understand how SREBP-2 transcription and regulation affects cellular cholesterol concentration. Model stability analysis shows that the only positive steady-state of the system exhibits purely oscillatory, damped oscillatory or monotic behaviour under certain parameter conditions. In light of our findings we postulate how cholesterol homestasis is maintained within the cell and the advantages of our model formulation are discussed with respect to other models of genetic regulation within the literature.

Activation of the small GTP-binding protein Ras in the heart by hypertrophic agonists.

The small (21-kDa) guanine nucleotide-binding protein Ras plays a central role in the regulation of cell growth and division. In the cardiac myocyte, it has been implicated in the hypertrophic adaptation. We have recently examined the ability of hypertrophic agonists such as endothelin-1, phenylephrine and phorbol esters to increase the "activity" (GTP loading) of Ras. We have also studied the signaling events that lead to activation of Ras and the processes that respond to Ras activation. In this brief review, we describe these studies and set them within the context of the hypertrophic response.

Influence of birth weight on gene regulators of lipid metabolism and utilization in subcutaneous adipose tissue and skeletal muscle of neonatal pigs.

Epidemiological studies suggest that low-birth weight infants show poor neonatal growth and increased susceptibility to metabolic syndrome, in particular, obesity and diabetes. Adipose tissue development is regulated by many genes, including members of the peroxisome proliferator-activated receptor (PPAR) and the fatty acid-binding protein (FABP) families. The aim of this study was to determine the influence of birth weight on key adipose and skeletal muscle tissue regulating genes. Piglets from 11 litters were ranked according to birth weight and 3 from each litter assigned to small, normal, or large-birth weight groups. Tissue samples were collected on day 7 or 14. Plasma metabolite concentrations and the expression of PPARG2, PPARA, FABP3, and FABP4 genes were determined in subcutaneous adipose tissue and skeletal muscle. Adipocyte number and area were determined histologically. Expression of FABP3 and 4 was significantly reduced in small and large, compared with normal, piglets in adipose tissue on day 7 and in skeletal muscle on day 14. On day 7, PPARA and PPARG2 were significantly reduced in adipose tissue from small and large piglets. Adipose tissue from small piglets contained more adipocytes than normal or large piglets. Birth weight had no effect on adipose tissue and skeletal muscle lipid content. Low-birth weight is associated with tissue-specific and time-dependent effects on lipid-regulating genes as well as morphological changes in adipose tissue. It remains to be seen whether these developmental changes alter an individual's susceptibility to metabolic syndrome.

Intestinal injury and endotoxemia in children undergoing surgery for congenital heart disease

RATIONALE: Children with congenital heart disease are at risk of gut barrier dysfunction and translocation of gut bacterial antigens into the bloodstream. This may contribute to inflammatory activation and organ dysfunction postoperatively. OBJECTIVES: To investigate the role of intestinal injury and endotoxemia in the pathogenesis of organ dysfunction after surgery for congenital heart disease. METHODS: We analyzed blood levels of intestinal fatty acid binding protein and endotoxin (endotoxin activity assay) alongside global transcriptomic profiling and assays of monocyte endotoxin receptor expression in children undergoing surgery for congenital heart disease. MEASUREMENTS AND MAIN RESULTS: Levels of intestinal fatty acid binding protein and endotoxin were greater in children with duct-dependent cardiac lesions. Endotoxemia was associated with severity of vital organ dysfunction and intensive care stay. We identified activation of pathogen-sensing, antigen-processing, and immune-suppressing pathways at the genomic level postoperatively and down-regulation of pathogen-sensing receptors on circulating immune cells. CONCLUSIONS: Children undergoing surgery for congenital heart disease are at increased risk of intestinal mucosal injury and endotoxemia. Endotoxin activity correlates with a number of outcome variables in this population, and may be used to guide the use of gut-protective strategies.