A new protein folding algorithm based on hydrophobic compactness: Rigid Unconnected Secondary Structure Iterative Assembly (RUSSIA). II: Applications

The RUSSIA procedure (Rigid Unconnected Secondary Structure Iterative Assembly) produces structural models of cores of small- and medium-sized proteins. Loops are omitted from this treatment and regular secondary structures are reduced to points, the centers of their hydrophobic faces. This methodology relies on the maximum compactness of the hydrophobic residues, as described in detail in Part I. Starting data are the sequence and the predicted limits and natures of regular secondary structures (alpha or beta). Helices are treated as rigid cylinders, whereas beta-strands are collectively taken into account within beta-sheets modeled by helicoid surfaces. Strands are allowed to shift along their mean axis to allow some flexibility and the alpha-helices can be placed on either side of beta-sheets. Numerous initial conformations are produced by discrete rotations of the helices and sheets around the direction going from the center of their hydrophobic face to the global center of the protein. Selection of proposed models is based upon a criterion lying on the minimization of distances separating hydrophobic residues belonging to different regular secondary structures. The procedure is rapid and appears to be robust relative to the quality of starting data (nature and length of regular secondary structures). This dependence of the quality of the model on secondary structure prediction and in particular the beta-sheet topology, is one of the limits of the present algorithm. We present here some results for a set of 12 proteins (alpha, beta and alpha/beta classes) of lengths 40-166 amino acids. The r.m.s. deviations for core models with respect to the native proteins are in the range 1.4-3.7 A.     

VCAM-1-mediated Rac signaling controls endothelial cell-cell contacts and leukocyte transmigration

Leukocyte adhesion is mediated totally and transendothelial migration partially by heterotypic interactions between the ₁- and ₂-integrins on the leukocytes and their ligands, Ig-like cell adhesion molecules (Ig-CAM), VCAM-1, and ICAM-1, on the endothelium. Both integrins and Ig-CAMs are known to have signaling capacities. In this study we analyzed the role of VCAM-1-mediated signaling in the control of endothelial cell-cell adhesion and leukocyte transendothelial migration. Antibody-mediated cross-linking of VCAM-1 on IL-1-activated primary human umbilical vein endothelial cells (pHUVEC) induced actin stress fiber formation, contractility, and intercellular gaps. The effects induced by VCAM-1 cross-linking were inhibited by C3 toxin, indicating that the small GTPase p21Rho is involved. In addition, the effects of VCAM-1 were accompanied by activation of Rac, which we recently showed induce intercellular gaps in pHUVEC in a Rho-dependent fashion. With the use of a cell-permeable peptide inhibitor, it was shown that Rac signaling is required for VCAM-1-mediated loss of cell-cell adhesion. Furthermore, VCAM-1-mediated signaling toward cell-cell junctions was accompanied by, and dependent on, Rac-mediated production of reactive oxygen species and activation of p38 MAPK. In addition, it was found that inhibition of Rac-mediated signaling blocks transendothelial migration of monocytic U937 cells. Together, these data indicate that VCAM-1-induced, Rac-dependent signaling plays a key role in the modulation of vascular-endothelial cadherin-mediated endothelial cell-cell adhesion and leukocyte extravasation. human umbilical vein endothelial cells; vascular-endothelial cadherin; F-actin; reactive oxygen species; p38 mitogen-activated protein kinase; vascular cell adhesion molecule     

Mode of action of a xylanase and its significance for the structural investigation of the branched l-arabino-d-glucurono-d-xylan from redwood (Sequoia sempervirens)

The substitution pattern of the water-soluble l-arabino-(4-O-methyl-d-glucurono)-d-xylan from redwood (Sequoia sempervirens) has been studied by enzymic degradation. Exhaustive hydrolysis by an endo-xylanase (EC 3.2.1.8) from a Basidiomycete Sporotrichum dimorphosporum left a residue accounting for 20% of the original d-xylan. In the dialyzable material, oligosaccharides having arabinose or 4-O-methylglucuronic acid residues attached to the non-reducing d-xylosyl end-group of xylobiose or xylotriose, respectively, were the smallest branched oligomers released. Action of the xylanase appears to involve a region of the polysaccharide backbone having three xylosyl residues. A mode of action is proposed that requires unsubstituted hydroxyl groups at C-2, C-3, and C-2′ of a xylobiosyl residue. The binding site seems to correspond to a shallow cavity. The composition and structure of the final residue of attack shows that the enzyme has no action when the xylosyl residues branched through O-2 are separated by only one, unsubstituted xylose residue. This pattern of action, the nature of the dialyzable products, and the production of a final residue in which the substituents are accumulated, suggest that the arabinosyl and glucosyl-uronic groups are irregularly distributed on the main chain of the xylan from redwood and that in some regions they are in close vicinity when not actually on adjacent xylosyl residues.     

Disuniting uniformity: a pied cladistic canvas of mtDNA haplogroup H in Eurasia

It has been often stated that the overall pattern of human maternal lineages in Europe is largely uniform. Yet this uniformity may also result from an insufficient depth and width of the phylogenetic analysis, in particular of the predominant western Eurasian haplogroup (Hg) H that comprises nearly a half of the European mitochondrial DNA (mtDNA) pool. Making use of the coding sequence information from 267 mtDNA Hg H sequences, we have analyzed 830 mtDNA genomes, from 11 European, Near and Middle Eastern, Central Asian, and Altaian populations. In addition to the seven previously specified subhaplogroups, we define fifteen novel subclades of Hg H present in the extant human populations of western Eurasia. The refinement of the phylogenetic resolution has allowed us to resolve a large number of homoplasies in phylogenetic trees of Hg H based on the first hypervariable segment (HVS-I) of mtDNA. As many as 50 out of 125 polymorphic positions in HVS-I were found to be mutated in more than one subcluster of Hg H. The phylogeographic analysis revealed that sub-Hgs H1*, H1b, H1f, H2a, H3, H6a, H6b, and H8 demonstrate distinct phylogeographic patterns. The monophyletic subhaplogroups of Hg H provide means for further progress in the understanding of the (pre)historic movements of women in Eurasia and for the understanding of the present-day genetic diversity of western Eurasians in general.     

The NOD2-RICK complex signals from the plasma membrane

NOD2 plays an important role in the innate immunity of the intestinal tract. By sensing the muramyl dipeptide (MDP), a bacterial wall component, NOD2 triggers the NF-kappaB signaling pathway and promotes the release of proinflammatory cytokines such as interleukin-8. Mutations in Nod2 (1007FS, R702W, G908R) impinge on NOD2 functions and are associated with the pathogenesis of Crohn disease, a chronic inflammatory bowel disease. Although NOD2 is usually described as a cytosolic receptor for MDP, the protein is also localized at the plasma membrane, and the 1007FS mutation delocalizes NOD2 to the cytoplasm (Barnich, N., Aguirre, J. E., Reinecker, H. C., Xavier, R., and Podolsky, D. K. (2005) J. Cell Biol. 170, 21-26; McDonald, C., Chen, F. F., Ollendorff, V., Ogura, Y., Marchetto, S., Lecine, P., Borg, J. P., and Nunez, G. (2005) J. Biol. Chem. 280, 40301-40309). In this study, we demonstrate that membrane-bound versions of NOD2 and Crohn disease-associated mutants R702W and G908R are capable of responding to MDP and activating the NF-kappaB pathway from this location. In contrast, the 1007FS mutant remains unable to respond to MDP from the plasma membrane. We also show that NOD2 promotes the membrane recruitment of RICK, a serine-threonine kinase involved in NF-kappaB activation downstream of NOD2. Furthermore, the artificial attachment of RICK at the plasma membrane provokes a constitutive and strong activation of the NF-kappaB pathway and secretion of interleukin-8 showing that optimal RICK activity depends upon its subcellular localization. Finally, we show that endogenous RICK localizes at the plasma membrane in the THP1 cell line. Thus, our data suggest that NOD2 is responsible for the membrane recruitment of RICK to induce a regulated NF-kappaB signaling and production of proinflammatory cytokines.     

Constitutive inhibition of plasma CETP by apolipoprotein C1 is blunted in dyslipidemic patients with coronary artery disease

Plasma cholesteryl ester transfer protein (CETP) promotes the cholesterol enrichment of apoB-containing lipoproteins (VLDL and LDL) at the expense of HDL. Recent studies demonstrated that apoC1 is a potent CETP inhibitor in plasma of healthy, normolipidemic subjects. Our goal was to establish whether the modulation of CETP activity by apoC1 is influenced by dyslipidemia in patients with documented coronary artery disease (CAD). In the total CAD population studied (n = 240), apoC1 levels correlated negatively with CETP activity, independently of apoE-epsilon, CETP-Taq1B, and apoC1-Hpa1 genotypes. In multivariate analysis, the negative relationship was observed only in normolipidemic patients, not in those with hypercholesterolemia, hypertriglyceridemia, or combined hyperlipidemia. In the normolipidemic subjects, apoC1 levels were positively associated with higher HDL- to LDL-cholesterol ratio (r = 0.359, P < 0.001). It is concluded that apoC1 as a CETP inhibitor no longer operates on cholesterol redistribution in high-risk patients with dyslipidemia, probably due to increasing amounts of VLDL-bound apoC1, which is inactive as a CETP inhibitor. Patients with dyslipidemia could experience major benefits from treatment with pharmacological CETP inhibitors, which might compensate for blunted endogenous inhibition.     

Spot position of rat liver ribosomal proteins by four different two-dimensional electrophoreses in polyacrylamide gel

Summary Separation of the proteins from rat liver 40S and 60S ribosomal subunits and polysomes was done in four different two-dimensional polyacrylamide gel electrophoresis systems. The first dimension was run at acidic or basic pH, the second dimension either with sodium dodecyl sulphate or at acidic pH in 18% acrylamide. The position of each individual protein of both subunits and polysomes was determined in each system. This identification resulted from a new method avoiding any previous purification of individual proteins. The new proposed uniform nomenclature for mammalian ribosomal proteins (McConkey et al. in press) was used for numbering the proteins in the four systems.