HMGB1 interacts differentially with members of the Rel family of transcription factors

HMGB1 is an architectural factor that enhances the DNA binding affinity of several proteins. We have investigated the influence of HMGB1 on DNA binding by members of the Rel family. HMGB1 enhances DNA binding by p65/p50 and p50/p50, but reduces binding by p65/p65, c-Rel/c-Rel, p65/c-Rel, and p50/c-Rel. In pull-down assays, HMGB1 interacts directly with the p50 subunit via its HMG boxes and this interaction is weakened by the presence of the acidic tail. Functionally, HMGB1 is required for the NF-kappaB-dependent expression of the adhesion molecule VCAM-1.     

Interaction with membrane lipids and heme ligand binding properties of Escherichia coli flavohemoglobin

Escherichia coli flavohemoglobin has been shown to be able to bind specifically unsaturated and/or cyclopropanated fatty acids with very high affinity. Unsaturated or cyclopropanated fatty acid binding results in a modification of the visible absorption spectrum of the ferric heme, corresponding to a transition from a pentacoordinated (typical of the ligand free protein) to a hexacoordinated, high spin, heme iron. In contrast, no detectable interaction has been observed with saturated fatty acid, saturated phospholipids, linear, cyclic, and aromatic hydrocarbons pointing out that the protein recognizes specifically double bonds in cis conformation within the hydrocarbon chain of the fatty acid molecule. Accordingly, as demonstrated in gel filtration experiments, flavohemoglobin is able to bind liposomes obtained from lipid extracts of E. coli membranes and eventually abstract phospholipids containing cis double bonds and/or cyclopropane rings along the acyl chains. The presence of a protein bound lipid strongly affects the thermodynamic and kinetic properties of imidazole binding to the ferric protein and brings about significant modifications in the reactivity of the ferrous protein with oxygen and carbon monoxide. The effect of the bound lipid has been accounted for by a reaction scheme that involves the presence of two sites for the lipid/ligand recognition, namely, the heme iron and a non-heme site located in a loop region above the heme pocket.     

Ni2+ enhances Fe2+/peroxide-induced oxidation of arachidonic acid and formation of geno/cytotoxic 4-hydroxynonenal: a possible contributory mechanism in nickel toxicity and allergenicity

Ni(2+), a toxic, carcinogenic and allergenic agent, affected both the kinetic and chemical courses of the Fe(2+)-induced oxidation of arachidonic acid (AA) in 0.05 M phosphate buffer (pH 7.4) and at 37 degrees C. At 10 microM concentration, Ni(2+) decreased the rate of oxidation of peroxide-free AA (200 microM) promoted by 50 microM Fe(2+), as determined by measurement of thiobarbituric acid reactive species (TBARS) and 1H NMR analysis. However, in the presence of low levels of peroxides (e.g. 2%), Ni(2+) exerted a significant stimulatory effect on Fe(2+)-induced AA oxidation and TBARS formation. 1H NMR analysis showed that Ni(2+) (10 microM) enhanced formation of genotoxic alkenals including 4-hydroxy-2-nonenal (4-HNE, GC/MS evidence) by Fe(2+)-promoted degradation of both AA and 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) methyl esters. The observed stimulatory effects of Ni(2+) on peroxide breakdown and cytotoxic aldehyde formation provide an attractive explanation to the enhanced sensitization capacity of nickel in inflammatory states compared to normal states.     

T cell receptor can be recruited to a subset of plasma membrane rafts,independently of cell signaling and attendantly to raft clustering

The constitutive/inducible association of the T cell receptor (TCR) with isolated detergent-resistant, lipid raft-derived membranes has been studied in Jurkat T lymphocytes. Membranes resistant to 1% Triton X-100 contained virtually no CD3epsilon, part of the TCR complex, irrespective of cell stimulation. On the other hand, membranes resistant either to a lower Triton X-100 concentration (i.e. 0.2%) or to the less hydrophobic detergent Brij 58 (1%) contained (i) a low CD3epsilon amount (approximate 2.7% of total) in resting cells and (ii) a several times higher amount of the TCR component, after T cell stimulation with either antigen-presenting cells or with phytohemagglutinin. It appeared that CD3/TCR was constitutively associated with and recruited to a raft-derived membrane subset because (i) all three membrane preparations contained a similar amount of the raft marker tyrosine kinase Lck but no detectable amounts of the conventional membrane markers, CD45 phosphatase and transferrin receptor; (ii) a larger amount of particulate membranes were resistant to solubilization with 0.2% Triton X-100 and Brij 58 than to solubilization with 1% Triton X-100; and (iii) higher cholesterol levels were present in membranes resistant to either the lower Triton X-100 concentration or to Brij 58, as compared with those resistant to 1% Triton X-100. The recruitment of CD3 to the raft-derived membrane subset appeared (i) to occur independently of cell signaling events, such as protein-tyrosine phosphorylation and Ca(2+) mobilization/influx, and (ii) to be associated with clustering of plasma membrane rafts induced by multiple cross-linking of either TCR or the raft component, ganglioside GM(1). We suggest that during T cell stimulation a lateral reorganization of rafts into polarized larger domains can determine the recruitment of TCR into these domains, which favors a polarization of the signaling cascade.     

T cell receptor engagement leads to the recruitment of IBP,a novel guanine nucleotide exchange factor,to the immunological synapse

Reorganization of the actin cytoskeleton is crucial to the formation and function of the immunological synapse. Rho GTPases are critical mediators of cytoskeletal reorganization, and their activity at the synapse is likely to be stringently regulated. Guanine nucleotide exchange factors (GEFs) belonging to the Dbl family of proteins represent one major class of proteins that regulate the activity of Rho GTPases. Here we demonstrate that IBP, a homologue of SWAP-70, is a novel GEF for Rac1 and Cdc42 in T lymphocytes, which is recruited to the immunological synapse upon engagement of the antigen receptor. Mutational analysis supports a model whereby IBP is inactive in unstimulated cells. Upon engagement of the T cell receptor, its GEF activity is enhanced by tyrosine phosphorylation, as well as by binding newly generated phosphatidylinositol 3,4,5-trisphosphate. Although it is known that T cell receptor engagement leads to the recruitment of Vav to the immunological synapse, these findings indicate that other GEFs, such as IBP, also relocalize to this intercellular region. The recruitment and activation of distinct classes of GEFs may allow for precise control of Rho GTPase function at the crucial interface between T cells and antigen presenting cells.     

Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-B1 scavenger receptor

Several cell surface molecules have been proposed as receptor candidates, mediating cell entry of hepatitis C virus (HCV) on the basis of their physical association with virions or with soluble HCV E2 glycoproteins. However, due to the lack of infectious HCV particles, evidence that these receptor candidates support infection was missing. Using our recently described infectious HCV pseudotype particles (HCVpp) that display functional E1E2 glycoprotein complexes, here we show that HCV is a pH-dependent virus, implying that its receptor component(s) mediate virion internalization by endocytosis. Expression of the CD81 tetraspanin in non-permissive CD81-negative hepato-carcinoma cells was sufficient to restore susceptibility to HCVpp infection, confirming its critical role as a cell attachment factor. As a cell surface molecule likely to mediate endosomal trafficking, we demonstrate that the human scavenger receptor class B type 1 (SR-B1), a high-density lipoprotein-internalization molecule that we previously proposed as a novel HCV receptor candidate due to its affinity with E2 glycoproteins, is required for infection of CD81-expressing hepatic cells. By receptor competition assays, we found that SR-B1 antibodies that blocked binding of soluble E2 could prevent HCVpp infectivity. Furthermore, we establish that the hyper-variable region 1 of the HCV E2 glycoprotein is a critical determinant mediating entry in SR-B1-positive cells. Finally, by correlating expression of HCV receptors and infectivity, we suggest that, besides CD81 and SR-B1, additional hepatocyte-specific co-factor(s) are necessary for HCV entry.     

Escherichia coli flavohemoglobin is an efficient alkylhydroperoxide reductase

Escherichia coli flavohemoglobin (HMP) is shown to be capable of catalyzing the reduction of several alkylhydroperoxide substrates into their corresponding alcohols using NADH as an electron donor. In particular, HMP possesses a high catalytic activity and a low Km toward cumyl, linoleic acid, and tert-butyl hydroperoxides, whereas it is a less efficient hydrogen peroxide scavenger. An analysis of UV-visible spectra during the stationary state reveals that at variance with classical peroxidases, HMP turns over in the ferrous state. In particular, an iron oxygen adduct intermediate whose spectrum is similar to that reported for the oxo-ferryl derivative in peroxidases (Compound II), has been identified during the catalysis of hydrogen peroxide reduction. This finding suggests that hydroperoxide cleavage occurs upon direct binding of a peroxide oxygen atom to the ferrous heme iron. Competitive inhibition of the alkylhydroperoxide reductase activity by carbon monoxide has also been observed, thus confirming that heme iron is directly involved in the catalytic mechanism of hydroperoxide reduction. The alkylhydroperoxide reductase activity taken together with the unique lipid binding properties of HMP suggests that this protein is most likely involved in the repair of the lipid membrane oxidative damage generated during oxidative/nitrosative stress.     

Mechanism of enhanced cardiac function in mice with hypertrophy induced by overexpressed Akt

Transgenic mice with cardiac-specific overexpression of active Akt (TG) not only exhibit hypertrophy but also show enhanced left ventricular (LV) function. In 3-4-month-old TG, heart/body weight was increased by 60% and LV ejection fraction was elevated (84 +/- 2%, p < 0.01) compared with nontransgenic littermates (wild type (WT)) (73 +/- 1%). An increase in isolated ventricular myocyte contractile function (% contraction) in TG compared with WT (6.1 +/- 0.2 versus 3.5 +/- 0.2%, p < 0.01) was associated with increased Fura-2 Ca2+ transients (396 +/- 50 versus 250 +/- 24 nmol/liter, p < 0.05). The rate of relaxation (+dL/dt) was also enhanced in TG (214 +/- 15 versus 98 +/- 18 microm/s, p < 0.01). L-type Ca2+ current (ICa) density was increased in TG compared with WT (-9.0 +/- 0.3 versus 7.2 +/- 0.3 pA/pF, p < 0.01). Sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) protein levels were increased (p < 0.05) by 6.6-fold in TG, which could be recapitulated in vitro by adenovirus-mediated overexpression of Akt in cultured adult ventricular myocytes. Conversely, inhibiting SERCA with either ryanodine or thapsigargin affected myocyte contraction and relaxation and Ca2+ channel kinetics more in TG than in WT. Thus, myocytes from mice with overexpressed Akt demonstrated enhanced contractility and relaxation, Fura-2 Ca2+ transients, and Ca2+ channel currents. Furthermore, increased protein expression of SERCA2a plays an important role in mediating enhanced LV function by Akt. Up-regulation of SERCA2a expression and enhanced LV myocyte contraction and relaxation in Akt-induced hypertrophy is opposite to the down-regulation of SERCA2a and reduced contractile function observed in many other forms of LV hypertrophy.     

The mitochondrial barriers segregate agonist-induced calcium-dependent functions in human airway epithelia

In airway epithelia, purinergic receptor (P2Y2-R) stimulation of intracellular calcium (Ca2+i)-regulated ion transport is restricted to the membrane domain ipsilateral to receptor activation, implying compartmentalization of Ca2+i signaling. Because mitochondria can spatially restrict cellular Ca2+i signals, immunocytochemical, electron microscopic, and fluorescent studies of mitochondria localization were performed in human airway epithelia. Although concentrated at the apical domain, mitochondria were found distributed at both the apical and the basolateral poles and in close association with the endoplasmic reticulum. The role of mitochondria in locally restricting P2Y2-R-induced Ca2+i signals was investigated by measuring changes in mitochondrial Ca2+ (Ca2+m) in human airway epithelial monolayers. P2Y2-R activation induced Ca2+m accumulation in mitochondria confined to the domain ipsilateral to P2Y2-R stimulation, which was blocked by mitochondrial uncoupling with 1 microM CCCP and 2.5 microg/ml oligomycin. The role of mitochondria in restricting the cellular cross-talk between basolateral P2Y2-R-dependent Ca2+i mobilization and apical membrane Ca2+-activated Cl- secretion was investigated in studies simultaneously measuring Ca2+i and Cl- secretion in cystic fibrosis human airway epithelial monolayers. Activation of basolateral P2Y2-Rs produced similar increases in Ca2+i in monolayers without and with pretreatment with uncouplers, whereas Ca2+i-activated Cl- secretion was only efficiently triggered in mitochondria-uncoupled conditions. We conclude that (a) mitochondria function as a Ca2+i-buffering system in airway epithelia, compartmentalizing Ca2+i-dependent functions to the membrane ipsilateral to receptor stimulation; and (b) the mitochondria provide structural barriers that protect the airway epithelia against nonspecific activation of Ca2+i-modulated functions associated with Ca2+i signals emanating from the apical or the basolateral membrane domains.     

Cutting edge: histamine inhibits IFN-alpha release from plasmacytoid dendritic cells

Plasmacytoid dendritic cells (DC) are professional APC and a major source of type I IFN following viral infection. We previously showed that histamine alters the cytokine profiles of maturing monocyte-derived DC resulting in a change from Th1 to Th2 in their T cell polarizing function. In this study, we show that human plasmacytoid DC, activated by either CpG oligodeoxynucleotides or viral infection, also respond to histamine through H2 receptors, leading to a marked down-regulation of IFN-alpha and TNF-alpha and a moderate switch in their capacity to polarize naive T cells. Our findings provide an explanation for low levels of type I IFN frequently observed in atopic individuals.