Mapping of heparin/heparan sulfate binding sites on αvβ3 integrin by molecular docking

Heparin/heparan sulfate interact with growth factors, chemokines, extracellular proteins, and receptors. Integrins are αβ heterodimers that serve as receptors for extracellular proteins, regulate cell behavior, and participate in extracellular matrix assembly. Heparin binds to RGD‐dependent integrins (αIIbβ3, α5β1, αvβ3, and αvβ5) and to RGD‐independent integrins (α4β1, αXβ2, and αMβ2), but their binding sites have not been located on integrins. We report the mapping of heparin binding sites on the ectodomain of αvβ3 integrin by molecular modeling. The surface of the ectodomain was scanned with small rigid probes mimicking the sulfated domains of heparan sulfate. Docking results were clustered into binding spots. The best results were selected for further docking simulations with heparin hexasaccharide. Six potential binding spots containing lysine and/or arginine residues were identified on the ectodomain of αvβ3 integrin. Heparin would mostly bind to the top of the genu domain, the Calf‐I domain of the α subunit, and the top of the β subunit of RGD‐dependent integrins. Three spots were close enough from each other on the integrin surface to form an extended binding site that could interact with heparin/heparan sulfate chains. Because heparin does not bind to the same integrin site as protein ligands, no steric hindrance prevents the formation of ternary complexes comprising the integrin, its protein ligand, and heparin/heparan sulfate. The basic amino acid residues predicted to interact with heparin are conserved in the sequences of RGD‐dependent but not of RGD‐independent integrins suggesting that heparin/heparan sulfate could bind to different sites on these two integrin subfamilies. Copyright © 2013 John Wiley & Sons, Ltd.     

Erythrocytes anion transport and oxidative change in β‐thalassaemias

β‐Thalassaemia is characterized by a decrease in globin β‐chain synthesis and an excess in free α‐globin chains. This induces alterations in membrane lipids and proteins resulting from a reduction in spectrin/band 3 ratio, partial oxidation of band 4.1 and clustering of band 3. The membrane injury provokes hyperhaemolysis and bone marrow hyperplasia. The pathophysiology of thalassaemia is associated with iron overload that generates oxygen free radicals and oxidative tissue injury with ocular vessel alterations. The aim of this research is to investigate the influence of oxidative stress on band 3 efficiency, which is an integral membrane protein of RBCs (red blood cells). Band 3 protein, of which there are more than 1 million copies per cell, is the most abundant membrane protein in human RBCs. It mediates the anion exchange and acid–base equilibrium through the RBC membrane. Some experiments were performed on thalassaemic cells and β‐thalassaemia‐like cells and tested for sulfate uptake. To test the antioxidant effect of Mg²⁺, other experiments were performed using normal and pathological cells in the presence of Mg²⁺. The oxidant status in thalassaemic cells was verified by increased K⁺ efflux, by lower GSH levels and by increased G6PDH (glucose‐6‐phosphate dehydrogenase) activity. The rate constant of SO₄ ^2? uptake decreases in thalassaemic cells as well as in β‐thalassaemia‐like cells when compared with normal cells. It increases when both cells are incubated with Mg²⁺. Our data show that oxidative stress plays a relevant role in band 3 function of thalassaemic cells and that antioxidant treatment with Mg²⁺ could reduce oxidative damage to the RBC membrane and improve the anion transport efficiency regulated by band 3 protein.     

Syndecan-1 Signals Independently of β1 Integrins during Raji Cell Spreading

Syndecan-1-expressing Raji lymphoid cells (Raji-S1 cells) bind and spread rapidly when attaching to matrix ligands that contain heparan sulfate-binding domains. However, these ligands also contain binding sites for integrins, which are widely known to signal, raising the question of whether the proteoglycan core protein participates in generation of the signal for spreading. To address this question, the spreading of the Raji-S1 cells is examined on ligands specific for either β1 integrins, known to be present on the Raji cells, or the syndecan-1 core protein. The cells adhere and spread on invasin, a ligand that activates β1 integrins, the IIICS fragment of fibronectin, which is a specific ligand for the α4β1 integrin, or mAb281.2, an antibody specific for the syndecan-1 core protein. The signaling resulting from adhesion to the syndecan-specific antibody appears integrin independent as (i) the morphology of the cells spreading on the antibody is distinct from spreading initiated by the integrins alone; (ii) spreading on the syndecan or integrin ligands is affected differently by the kinase inhibitors tyrphostin 25, genistein, and staurosporine; and (iii) spreading on the syndecan-specific antibody is not disrupted by blocking β1 integrin activation with mAb13, a β1 inhibitory antibody. These data demonstrate that ligation of syndecan-1 initiates intracellular signaling and suggest that this signaling occurs when cells expressing syndecan-1 adhere to matrix ligands containing heparan sulfate-binding domains.     

Structural classification of αββ and ββα supersecondary structure units in proteins

We present a fully automatic structural classification of supersecondary structure units, consisting of two hydrogen-bonded β strands, preceded or followed by an α helix. The classification is performed on the spatial arrangement of the secondary structure elements, irrespective of the length and conformation of the intervening loops. The similarity of the arrangements is estimated by a structure alignment procedure that uses as similarity measure the root mean square deviation of superimposed backbone atoms. Applied to a set of 141 well-resolved nonhomologous protein structures, the classification yields 11 families of recurrent arrangements. In addition, fragments that are structurally intermediate between the families are found; they reveal the continuity of the classification. The analysis of the families shows that the α helix and β hairpin axes can adopt virtually all relative orientations, with, however, some preferable orientations; moreover, according to the orientation, preferences in the left/right handedness of the α–β connection are observed. These preferences can be explained by favorable side by side packing of the α helix and the β hairpin, local interactions in the region of the α–β connection or stabilizing environments in the parent protein. Furthermore, fold recognition procedures and structure prediction algorithms coupled to database-derived potentials suggest that the preferable nature of these arrangements does not imply their intrinsic stability. They usually accommodate a large number of sequences, of which only a subset is predicted to stabilize the motif. The motifs predicted as stable could correspond to nuclei formed at the very beginning of the folding process. Proteins 30:193–212, 1998. © 1998 Wiley-Liss, Inc.     

INTERACTIONS OF THE MIX‐LINKED β‐(1,3)/β‐(1,4)‐d‐XYLANS IN THE CELL WALLS OF PALMARIA PALMATA (RHODOPHYTA)1

Algal cell wall mechanical properties, crucial for biological functions and commercial applications, rely on interactions in macromolecular assemblies. In an effort to better understand the interactions of the matrix‐phase β‐(1,3)/(1,4)‐d ‐xylan in the edible seaweed Palmaria palmata ((L.) O. Kuntze, Rhodophyta, Palmariales), sequential extractions by saline, alkaline, and chaotropic solutions were done. The chemical composition and structure and the physicochemical properties of the isolated xylan revealed that it was partly acidic, probably due to the presence of sulfate (up to 5%) and phosphate groups (up to 4%). Although such acidity suggested ionic interactions of xylan in the cell walls, the high yields of polysaccharide extracted by alkali and particularly by 8 M urea and 4.5 M guanidium thiocyanate demonstrated that it was mainly hydrogen bonded in the cell wall. H‐bonds did not appear to be related to the mean proportions of β‐(1,3) and β‐(1,4)‐d ‐xylose linkages because these did not differ between extracts of increasing alkalinity. However, the decreasing molar weight and intrinsic viscosity of extracts obtained by alkaline solution containing a reducing agent used to prevent polysaccharide degradation suggested the presence of an alkali‐labile component in the xylan. These results are discussed with regard to the role of potential wall proteins as a means of control of these interactions.     

Dextran sulfate sodium inhibits amyloid‐β oligomer binding to cellular prion protein

Amyloid‐β peptide (Aβ), especially its oligomeric form, is believed to play an important role in the pathogenesis of Alzheimer's disease (AD). To this end, the binding of Aβ oligomer to cellular prion protein (PrP^C) plays an important role in synaptic dysfunction in a mouse model of AD. Here, we have screened for compounds that inhibit Aβ oligomer binding to PrP^C from medicines already used clinically (Mizushima Approved Medicine Library 1), and identified dextran sulfate sodium (DSS) as a candidate. In a cell‐free assay, DSS inhibited Aβ oligomer binding to PrP^C but not to ephrin receptor B2, another endogenous receptor for Aβ oligomers, suggesting that the drug's action is specific to the binding of Aβ oligomer to PrP^C. Dextran on the other hand did not affect this binding. DSS also suppressed Aβ oligomer binding to cells expressing PrP^C but not to control cells. Furthermore, while incubation of mouse hippocampal slices with Aβ oligomers inhibited the induction of long‐term potentiation, simultaneous treatment with DSS restored the long‐term potentiation. As DSS has already been approved for use in patients with hypertriglyceridemia, and its safety in humans has been confirmed, we propose further analysis of this drug as a candidate for AD treatment.

     

Engineered cystine knot peptides that bind αvβ3, αvβ5, and α5β1 integrins with low‐nanomolar affinity

There is a critical need for compounds that target cell surface integrin receptors for applications in cancer therapy and diagnosis. We used directed evolution to engineer the Ecballium elaterium trypsin inhibitor (EETI‐II), a knottin peptide from the squash family of protease inhibitors, as a new class of integrin‐binding agents. We generated yeast‐displayed libraries of EETI‐II by substituting its 6‐amino acid trypsin binding loop with 11‐amino acid loops containing the Arg‐Gly‐Asp integrin binding motif and randomized flanking residues. These libraries were screened in a high‐throughput manner by fluorescence‐activated cell sorting to identify mutants that bound to α_vβ₃ integrin. Select peptides were synthesized and were shown to compete for natural ligand binding to integrin receptors expressed on the surface of U87MG glioblastoma cells with half‐maximal inhibitory concentration values of 10–30 nM. Receptor specificity assays demonstrated that engineered knottin peptides bind to both α_vβ₃ and α_vβ₅ integrins with high affinity. Interestingly, we also discovered a peptide that binds with high affinity to α_vβ₃, α_vβ₅, and α₅β₁ integrins. This finding has important clinical implications because all three of these receptors can be coexpressed on tumors. In addition, we showed that engineered knottin peptides inhibit tumor cell adhesion to the extracellular matrix protein vitronectin, and in some cases fibronectin, depending on their integrin binding specificity. Collectively, these data validate EETI‐II as a scaffold for protein engineering, and highlight the development of unique integrin‐binding peptides with potential for translational applications in cancer. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Targeted inhibition of KCa3.1 attenuates TGF‐β‐induced reactive astrogliosis through the Smad2/3 signaling pathway

Reactive astrogliosis, characterized by cellular hypertrophy and various alterations in gene expression and proliferative phenotypes, is considered to contribute to brain injuries and diseases as diverse as trauma, neurodegeneration, and ischemia. KCa3.1 (intermediate‐conductance calcium‐activated potassium channel), a potassium channel protein, has been reported to be up‐regulated in reactive astrocytes after spinal cord injury in vivo. However, little is known regarding the exact role of KCa3.1 in reactive astrogliosis. To elucidate the role of KCa3.1 in regulating reactive astrogliosis, we investigated the effects of either blocking or knockout of KCa3.1 channels on the production of astrogliosis and astrocytic proliferation in response to transforming growth factor (TGF)‐β in primary cultures of mouse astrocytes. We found that TGF‐β increased KCa3.1 protein expression in astrocytes, with a concomitant marked increase in the expression of reactive astrogliosis, including glial fibrillary acidic protein and chondroitin sulfate proteoglycans. These changes were significantly attenuated by the KCa3.1 inhibitor 1‐((2‐chlorophenyl) (diphenyl)methyl)‐1H‐pyrazole (TRAM‐34). Similarly, the increase in glial fibrillary acidic protein and chondroitin sulfate proteoglycans in response to TGF‐β was attenuated in KCa3.1^?/? astrocytes. TRAM‐34 also suppressed astrocytic proliferation. In addition, the TGF‐β‐induced phosphorylation of Smad2 and Smad3 proteins was reduced with either inhibition of KCa3.1 with TRAM‐34 or in KCa3.1^?/? astrocytes. These findings highlight a novel role for the KCa3.1 channel in reactive astrogliosis phenotypic modulation and provide a potential target for therapeutic intervention for brain injuries.

     

Studies on the 5α-androstane-3β, 17β-diol-6α-hydroxylase and on the 5α-androstane-3β, 17β-diol-7α-hydroxylase in anterior hypophysis of male rats.

In anterior pituitaries from male rats, it appeared that 5α-androstane-3β, 17β-diol was quickly metabolized into 5α-androstane-3β,6α-17β-triol and 5α-androstane-3β,7α, 17β-triol by action of 6α- and 7α-hydroxylases. Hydroxysteroid hydroxylases were located in endoplasmic reticulum and were dependent on NADPH⁺. Their optimum pH was 8.0, optima temperature, 37°C, and their apparent Km was 2.7 μM. Hydroxylative reactions were not reversible and not modified by gonadectomy. Hydroxylation seemed an efficient control of the pituitary level of 5α-andros-tane-3β, 17β-diol.     

Modulation of α5β1 and αVβ3 integrins on the cell surface during mitosis

One of the hallmarks of cells undergoing mitotic division is their rounded morphology and reduced adhesion to the substratum. We have studied and compared the attachment of interphase and mitotic cells to substrata coated with fibronectin and vitronectin. We have found that adhesion of mitotic cells, as compared to interphase cells, is significantly reduced to fibronectin, but is higher to vitronectin. These results correlate well with the expression of α5β1 and αVβ3 integrins, the respective receptors for fibronectin and vitronectin, on the cell surface. Mitotic cells show higher levels of αVβ3 and very low levels of α5β1 proteins on the cell surface as compared to interphase cells. This difference in the levels of these integrins also reflects in the total amounts of fibronectin and vitronectin present on the cell surface of these cells. We have further shown, by flow cytometry, that binding of vitronectin, or the synthetic peptide-GRGDSP-, causes an increase in the intracellular levels of Ca²⁻ in mitotic cells, but no change is seen in the interphase cells. Binding of fibronectin to either of these cells fails to elicit any response. One interesting feature of our results is that the levels of total, i.e., cytoplasmic plus membrane bound, α5β1 and αVβ3 integrins of mitotic and interphase cells remain the same, thus implying an alteration in the distribution of integrin chains between the plasma membrane and the cytoplasm during the conversion of interphase cells into the mitotic phase. © 1996 Wiley-Liss, Inc.     

Integrin αvβ3 enhances the suppressive effect of interferon‐γ on hematopoietic stem cells

Hematopoietic homeostasis depends on the maintenance of hematopoietic stem cells (HSCs), which are regulated within a specialized bone marrow (BM) niche. When HSC sense external stimuli, their adhesion status may be critical for determining HSC cell fate. The cell surface molecule, integrin αvβ3, is activated through HSC adhesion to extracellular matrix and niche cells. Integrin β3 signaling maintains HSCs within the niche. Here, we showed the synergistic negative regulation of the pro‐inflammatory cytokine interferon‐γ (IFNγ) and β3 integrin signaling in murine HSC function by a novel definitive phenotyping of HSCs. Integrin αvβ3 suppressed HSC function in the presence of IFNγ and impaired integrin β3 signaling mitigated IFNγ‐dependent negative action on HSCs. During IFNγ stimulation, integrin β3 signaling enhanced STAT1‐mediated gene expression via serine phosphorylation. These findings show that integrin β3 signaling intensifies the suppressive effect of IFNγ on HSCs, which indicates that cell adhesion via integrin αvβ3 within the BM niche acts as a context‐dependent signal modulator to regulate the HSC function under both steady‐state and inflammatory conditions.     

PLCγ1–PKCγ Signaling‐Mediated Hsp90α Plasma Membrane Translocation Facilitates Tumor Metastasis

The 90‐kDa heat shock protein (Hsp90α) has been identified on the surface of cancer cells, and is implicated in tumor invasion and metastasis, suggesting that it is a potentially important target for tumor therapy. However, the regulatory mechanism of Hsp90α plasma membrane translocation during tumor invasion remains poorly understood. Here, we show that Hsp90α plasma membrane expression is selectively upregulated upon epidermal growth factor (EGF) stimulation, which is a process independent of the extracellular matrix. Abrogation of EGF‐mediated activation of phospholipase (PLCγ1) by its siRNA or inhibitor prevents the accumulation of Hsp90α at cell protrusions. Inhibition of the downstream effectors of PLCγ1, including Ca²⁺ and protein kinase C (PKCγ), also blocks the membrane translocation of Hsp90α, while activation of PKCγ leads to increased levels of cell‐surface Hsp90α. Moreover, overexpression of PKCγ increases extracellular vesicle release, on which Hsp90α is present. Furthermore, activation or overexpression of PKCγ promotes tumor cell motility in vitro and tumor metastasis in vivo, whereas a specific neutralizing monoclonal antibody against Hsp90α inhibits such effects, demonstrating that PKCγ‐induced Hsp90α translocation is required for tumor metastasis. Taken together, our study provides a mechanistic basis for the role for the PLCγ1–PKCγ pathway in regulating Hsp90α plasma membrane translocation, which facilitates tumor cell motility and promotes tumor metastasis.     

Anti-inflammatory properties of α- and γ-tocopherol

Natural vitamin E consists of four different tocopherol and four different tocotrienol homologues (α, β, γ, δ) that all have antioxidant activity. However, recent data indicate that the different vitamin E homologues also have biological activity unrelated to their antioxidant activity. In this review, we discuss the anti-inflammatory properties of the two major forms of vitamin E, α-tocopherol (αT) and γ-tocopherol (γT), and discuss the potential molecular mechanisms involved in these effects. While both tocopherols exhibit anti-inflammatory activity in vitro and in vivo, supplementation with mixed (γT-enriched) tocopherols seems to be more potent than supplementation with αT alone. This may explain the mostly negative outcomes of the recent large-scale interventional chronic disease prevention trials with αT only and thus warrants further investigation.     

Modeling the αIIbβ3 integrin solution conformation

The α_IIbβ₃ platelet integrin is the prototypical member of a widely distributed class of transmembrane receptors formed by the noncovalent association of α and β subunits. Electron microscopic (EM) images of the α_IIbβ₃ complex show an asymmetric particle with a globular domain from which two extended regions protrude to contact the lipid bilayer. Distance constraints provided by disulfide bond patterns, epitope mapping, and ligand mimetic cross-linking studies rather suggest a somewhat more compact conformation for the α_IIbβ₃ complex. We have studied the shape of detergent-solubilized α_IIbβ₃ by employing a low-resolution modeling procedure in which each polypeptide has been represented as an array of interconnected, nonoverlapping spheres (beads) of various sizes. The number, size, and three-dimensional relationships among the beads were defined either solely by dimensions obtained from published EM images of integrin receptors (EM models, 21 beads), or solely by interdomain constraints derived from published biochemical data (biochemical model, 37 beads). Interestingly, although no EM data were employed in its construction, the resulting overall shape of the biochemical model was still compatible with the EM data. Both kinds of models were then evaluated for their calculated solution properties. The more elongated EM models have diffusion and sedimentation coefficients that differ, at best, by +2% and -18% from the experimental values, determined, respectively, in octyl glucoside and Triton X-100. On the other hand, the parameters calculated for the more compact biochemical model showed a more consistent agreement with experimental values, differing by -7% (octyl glucoside) to -6% (Triton X-100). Thus, it appears that using the biochemical constraints as a starting point has resulted in not only a more detailed model of the detergent-solubilized α_IIbβ₃ complex, where the relative spatial location of specific domains the size of 5–10 kDa can be tentatively mapped, but in a model that can also reconcile the electron microscopy with the biochemical and the solution data.     

Ecosystem stability in space: α, β and γ variability

The past two decades have seen great progress in understanding the mechanisms of ecosystem stability in local ecological systems. There is, however, an urgent need to extend existing knowledge to larger spatial scales to match the scale of management and conservation. Here, we develop a general theoretical framework to study the stability and variability of ecosystems at multiple scales. Analogously to the partitioning of biodiversity, we propose the concepts of alpha, beta and gamma variability. Gamma variability at regional (metacommunity) scale can be partitioned into local alpha variability and spatial beta variability, either multiplicatively or additively. On average, variability decreases from local to regional scales, which creates a negative variability–area relationship. Our partitioning framework suggests that mechanisms of regional ecosystem stability can be understood by investigating the influence of ecological factors on alpha and beta variability. Diversity can provide insurance effects at the various levels of variability, thus generating alpha, beta and gamma diversity–stability relationships. As a consequence, the loss of biodiversity and habitat impairs ecosystem stability at the regional scale. Overall, our framework enables a synthetic understanding of ecosystem stability at multiple scales and has practical implications for landscape management.     

Divalent cations regulate the organization of integrins αvβ3 and αvβ5 on the cell surface

Extracellular divalent cations are important regulators of integrin ligand binding activity. In this study we evaluated how divalent cations affect the organization of integrins into focal adhesion sites. Integrins αvβ3 and αvβ5 were compared because they share a high degree of structural homology and because both integrins mediate cell adhesion to vitronectin. On MG-63 osteosarcoma cells, we found that both the extent and pattern of integrin organization was regulated by the type of extracellular divalent ion. Integrin αvβ3 organized in focal contacts when Mn²⁺ or Mg²⁺ was present, but not in Ca²⁺. In contrast, αvβ5 organized in focal contacts only when Ca²⁺ or Mg²⁺ was present. Integrin αvβ5 clustered in a centrally located punctate field on the ventral surface of the cell in the presence of Mn²⁺. These observations reveal a previously unappreciated role for divalent ions in regulating the organization of integrins into focal adhesion sites. © 1996 Wiley-Liss, Inc.     

LYMPHOTOXIN-α (TNF-β) DURING SEPSIS

T cell release of lymphotoxin-α (LT-α, or TNF-β) is stimulated by pyrogenic exotoxins of Gram-positive bacteria and mitogens. In contrast to TNF-α, it is unknown whether LT-α plays any role in the pathogenesis of sepsis and, in particular, the pathogenesis of Gram-positive sepsis. Sera from patients with sepsis were examined for LT-α and compared with normal volunteers and pregnant women. LT-α was detected in 33% of sepsis sera (mean 608.4 pg/ml SE 306), 16% of normal sera (mean 167 pg/ml SE 87) and 23% of sera from pregnant women (mean 714 pg/ml SE 191). These differences were not significant and there were no differences within species sera when grouped by the type of causative organism, or disease severity. LT-α detected by immunoassay in serum was not bioactive, in contrast to that produced in cell culture. Recombinant soluble TNF receptors (rSTNFR) neutralized the bioactivity of recombinant LT-α at rSTNFR concentrations which did not interfere with immunoreactivity and which are known to prevailin vivo. Hence, LT-α is unlikely to have a critical role in the pathogenesis of sepsis. Much of the potential bioactivity of this lymphokine may be abrogated by TNFR in serum.