Histone deacetylase-2 controls IL-1ß production through the regulation of NLRP3 expression and activation in tuberculosis infection.

Histone deacetylases (HDACs) are critical immune regulators. However, their roles in interleukin-1ß (IL-1ß) production remain unclear. By screening 11 zinc-dependent HDACs with chemical inhibitors, we found that HDAC1 inhibitor, 4-(dimethylamino)-N-[6-(hydroxyamino)-6-oxohexyl]-benzamide (DHOB), enhanced IL-1ß production by macrophage and dendritic cells upon TLR4 stimulation or Mycobacterium tuberculosis infection through IL-1ß maturation via elevated NLRP3 expression, increased cleaved caspase-1, and enhanced ASC oligomerization. DHOB rescued defective IL-1ß production by dendritic cells infected with M. tuberculosis with ESAT-6 deletion, a virulence factor shown to activate NLRP3 inflammasome. DHOB increased IL-1ß production and NLRP3 expression in a tuberculosis mouse model. Although DHOB inhibited HDAC activities of both HDAC1 and HDAC2 by direct binding, knockdown of HDAC2, but not HDAC1, increased IL-1ß production and NLRP3 expression in M. tuberculosis-infected macrophages. These data suggest that HDAC2, but not HDAC1, controls IL-1ß production through NLRP3 inflammasome activation, a mechanism with a significance in chronic inflammatory diseases including tuberculosis.

Identification of amino acid residues in protein SRP72 required for binding to a kinked 5e motif of the human signal recognition particle RNA.

BACKGROUND: Human cells depend critically on the signal recognition particle (SRP) for the sorting and delivery of their proteins. The SRP is a ribonucleoprotein complex which binds to signal sequences of secretory polypeptides as they emerge from the ribosome. Among the six proteins of the eukaryotic SRP, the largest protein, SRP72, is essential for protein targeting and possesses a poorly characterized RNA binding domain. RESULTS: We delineated the minimal region of SRP72 capable of forming a stable complex with an SRP RNA fragment. The region encompassed residues 545 to 585 of the full-length human SRP72 and contained a lysine-rich cluster (KKKKKKKKGK) at postions 552 to 561 as well as a conserved Pfam motif with the sequence PDPXRWLPXXER at positions 572 to 583. We demonstrated by site-directed mutagenesis that both regions participated in the formation of a complex with the RNA. In agreement with biochemical data and results from chymotryptic digestion experiments, molecular modeling of SRP72 implied that the invariant W577 was located inside the predicted structure of an RNA binding domain. The 11-nucleotide 5e motif contained within the SRP RNA fragment was shown by comparative electrophoresis on native polyacrylamide gels to conform to an RNA kink-turn. The model of the complex suggested that the conserved A240 of the K-turn, previously identified as being essential for the binding to SRP72, could protrude into a groove of the SRP72 RNA binding domain, similar but not identical to how other K-turn recognizing proteins interact with RNA. CONCLUSIONS: The results from the presented experiments provided insights into the molecular details of a functionally important and structurally interesting RNA-protein interaction. A model for how a ligand binding pocket of SRP72 can accommodate a new RNA K-turn in the 5e region of the eukaryotic SRP RNA is proposed.

Mutagenesis studies toward understanding allostery in thrombin.

The binding of thrombomodulin (TM) to exosite-1 and the binding of Na(+) to 225-loop allosterically modulate the catalytic activity and substrate specificity of thrombin. To determine whether the conformation of these two cofactor-binding loops are energetically linked to each other and to the active site, we rationally designed two thrombin mutants in which either the 70-80 loop of exosite-1 or the 225-loop of the Na(+)-binding site was stabilized by an engineered disulfide bond. This was possible by replacing two residues, Arg-67 and Ile-82, in the first mutant and two residues, Glu-217 and Lys-224, in the second mutant with Cys residues. These mutants were expressed in mammalian cells as monomeric molecules, purified to homogeneity and characterized with respect to their ability to bind TM and Na(+) by kinetic and direct binding approaches. The Cys-67/Cys-82 mutant did not bind TM and exhibited a normal amidolytic activity, however, the activity of Cys-217/Cys-224 was dramatically impaired, though TM interacted with this mutant with >20-fold elevated K(D) to partially restore its activity. Both mutants exhibited approximately 2-3-fold higher K(D) for interaction with Na(+), and neither mutant clotted fibrinogen or activated protein C in the presence of TM. Both mutants interacted with heparin with a normal affinity. These results suggest that, while exosite-2 of thrombin is an independent cofactor binding-site, both Na(+)-binding and exosite-1 are energetically linked. Further studies with the fluorescein labeled Cys-195 mutant of thrombin revealed that the catalytic residue of thrombin is modulated by Na(+), but TM has no effect on the conformation of this residue.

Possible mechanisms contributing to oxidative inactivation of activated protein C: molecular dynamics study.

Activated protein C (APC) is a serine protease, an effector enzyme of the natural anticoagulant pathway. APC is approved for treatment of severe sepsis characterized by the increased concentrations of H(2)O(2) and hypochlorite. We found that treatment of APC with these oxidants markedly inhibits the cleavage of the APC-specific chromogenic substrate, suggesting that oxidants can induce changes in the structure of the active site of APC. Resistance of oxidant-treated APC to chemical digestion with cyanogen bromide (CNBr) implies that methionine oxidation can at least in part be responsible for inhibition of APC. Since methionine residues, the main targets of oxidants in APC, are not included in the active site, we hypothesize that oxidation induces allosteric changes in the architecture of the catalytic triad of APC. Using molecular dynamics (MD) simulations we found that methionine oxidation alters the distance between cSer195Ogamma and cHis57Nepsilon2 atoms placing them in positions unfavorable for the catalysis. At the same time, neither distances between Calpha atoms of the catalytic triad cAsp102-cHis57-cSer195, nor the overall structure of APC changed significantly after oxidation of the methionine residues. Disruption of the H-bond between Ndelta1 of cHis57 and carboxyl group of cAsp102, which can take place during the hypochlorite-induced modification of cHis57, dramatically changed the architecture of the catalytic triad in oxidized APC. This mechanism could contribute to APC inactivation by hypochlorite concurrently with methionine oxidation. These are novel findings, which describe potentially pathophysiologically relevant changes in the functional stability of APC exposed to the oxidative stress.

Thrombomodulin-mediated catabolism of protein C by pleural mesothelial and vascular endothelial cells.

Pleural mesothelial and vascular endothelial cells express protein C (PC) pathway components including thrombomodulin (TM) and endothelial protein C receptor (EPCR) and activate PC by the thrombin-TM dependent mechanism. We used these cells as model systems to identify molecules involved in endocytosis and degradation of PC. We find that mesothelial and endothelial cells can bind, internalize and degrade PC. Addition of thrombin markedly induced degradation of PC by these cells in a TM-dependent fashion, implicating the involvement of the thrombin-TM complex in internalization and degradation of PC. This observation defines a novel function for the thrombin-TM complex as a degradation receptor for PC and suggests that PC is degraded concurrent with its activation. A PC Gla-domain mutant, which is unable to bind to the EPCR, was degraded by the cells to a lesser extent than wild-type PC, implicating the PC degradation concurrent with its activation. Consistent with the role of thrombin-TM complex as a degradation receptor, the catalytically inactive thrombin-S195A also induced PC degradation though to a lesser extent than wild-type thrombin. This suggests that generation of activated PC (APC) can contribute to accumulation of degradation products, but is not essential for the thrombin-induced degradation of PC. The thrombin-TM-mediated degradation of PC by both cell types suggest a previously unrecognized mechanism, which can contribute to PC consumption. This mechanism may be pathophysiologically relevant and can contribute to an acquired PC deficiency in conditions characterized by sustained thrombin generation.

Asbestos induces tissue factor in Beas-2B cells via PI3 kinase-PKC-mediated signaling.

Treatment of Beas-2B airway epithelial cells with crocidolite asbestos induced tissue factor (TF) mRNA and TF-dependent procoagulant activity. The mitogen-activated protein kinase (MAPK) inhibitors UO126 and SB203850 decreased TF expression in both naive and crocidolite-treated Beas-2B cells to the same extent. Calphostin, an inhibitor of classical and novel protein kinase C (PKC) isotypes, reduced TF mRNA in both intact and crocidolite-treated Beas-2B cells by about 50%. Conversely, the phosphatidylinositol 3-kinase (PI3 kinase) inhibitor LY294002 and a selective PKCzeta inhibitory peptide decreased TF mRNA expression in asbestos-treated cells to a greater extent than in naive cells, suggesting that signaling via this pathway contributes to asbestos-induced TF expression. These results demonstrate that crocidolite asbestos induces TF expression by Beas-2B cells and suggest that the process involves the PI3 kinase-PKCzeta signaling pathway, representing a newly recognized potential mechanism by which asbestos may contribute to lung remodeling.

Mapping the intramolecular signal propagation pathways in protein using Bayesian change point analysis of atomic motions.

We propose to use change points of atomic positions in the molecular dynamics trajectory as indicators of the propagating signals in protein. We designate these changes as signals because they can propagate within the molecule in the form of "perturbation wave", transmit energy or information between different parts of protein, and serve as allosteric signals. We found that change points can distinguish between thermal fluctuations of atoms (noise) and signals in a protein despite the differences in the motility of amino acid residues. Clustering of the spatially close residues that were experiencing change points close in time, allowed us to map pathways of signal propagation in a protein at the atomic level of resolution. We propose a potential mechanism for the origin of the signal and its propagation that relies on the autonomic coherence resonance in atomic fluctuations. According to this mechanism, random synchronization of fluctuations of neighboring atoms results in a resonance, which increases amplitude of vibration of these atoms. This increase can be transmitted to the atoms colliding with the resonant atoms, leading to the propagating signal. The wavelet-based coherence analysis of the inter-atomic distances between carbon-alpha atoms and surrounding atoms for the residue pairs that belong to the same communication pathway allowed us to find time periods with temporarily locked phases, confirming the occurrence of conditions for resonance. Analysis of the mapped pathways demonstrated that they form a network that connects different regions of the protein.

Graph-theoretical comparison of protein surfaces reveals potential determinants of cross-reactivity and the molecular mimicry.

Different proteins, even without sequence similarity, still can contain similar surface regions involved in protein-protein interactions with common target. These regions can serve as structural determinants of cross-reactivity and molecular mimicry. Molecular mimicry, defined as the process in which structural properties of one molecule are simulated by the dissimilar molecules, is implicated in several biologically important processes, including autoimmune and allergic reactions, binding of some ligands to common receptor, and interactions in cell signaling. The problem of identification of the determinants of molecular mimicry is not completely solved at this time. We hypothesize that identification of structurally and chemically similar surface regions of two protein molecules capable of binding to the same target will allow us to identify sites involved in cross-reactivity including determinants of the molecular mimicry. We used a graph-theoretical approach in order to determine highly similar surface regions of two proteins with known three-dimensional structures. This approach uses a variation of Maximal Common Subgraph (MCS) isomorphism, where an association graph is constructed based on the surface-exposed residues of the two molecules and the matching regions are found based on the maximum cliques in the association graph. Testing the proposed method on the targets of autoantibody involved in antiphopholipid syndrome (APS)--beta2-GPI, PC, thrombin, factor IX, factor X, and plasmin allowed identifying potential epitopes for antibody that can inhibit coagulation proteases. Application of this method to the Activated Protein C and factor VII Gla-domains revealed surface regions involved in EPCR and plasma membrane binding, consistent with known experimental results. Analysis of major pollen allergen that can cause food allergies through cross-reactivity found known epitopes involved in cross-reactivity and also revealed additional surface regions that can complement the list of epitopes. Taken together, our results suggest that the proposed graph-theoretical approach can identify determinants of cross-reactivity and molecular mimicry.

Activation and degradation of protein C by primary rabbit pleural mesothelial cells.

The protein C (PC) anticoagulant pathway is the major mechanism that controls thrombin generation in vivo and may thereby influence pathophysiologic fibrin turnover associated with intrapleural inflammation. We hypothesized that pleural mesothelial cells could regulate local expression of PC in evolving pleurodesis where inflammation and thrombosis play an important role. To test this hypothesis, we determined the ability of rabbit pleural mesothelial cells (RPMC) to support the activation of PC as well as its binding, internalization, and degradation. Lung fibroblasts were also assessed to test the specificity of the responses. We found that both cell types could support thrombin-dependent activation of PC in vitro. Both cell types were capable of binding, internalizing, and degrading 125I-PC. Degradation of 125I-PC by these cells was prevented by the lysosomal inhibitor chloroquine but not the proteasomal inhibitor lactacystin, supporting involvement of a lysosomal mechanism of PC degradation. During evolving tetracycline (TCN)-induced pleural injury in rabbits, PC levels in pleural fluids were sustained, exhibited a trend toward progressive decline, and were temporally correlated with pleural adhesion formation in vivo. These observations indicate that sustained expression of PC during evolving pleurodesis induced by TCN is subject to regulation by resident pleural cells: both RPMC and lung fibroblasts. Both cell types support local generation of APC. Internalization and degradation of PC by RPMC and fibroblasts may regulate its intrapleural expression and influence remodeling of extravascular fibrin in the setting of evolving pleurodesis induced by TCN.

Acute cholesterol depletion impairs functional expression of tissue factor in fibroblasts: modulation of tissue factor activity by membrane cholesterol.

Cholesterol, in addition to providing rigidity to the fluid membrane, plays a critical role in receptor function, endocytosis, recycling, and signal transduction. In the present study, we examined the effect of membrane cholesterol on functional expression of tissue factor (TF), a cellular receptor for clotting factor VIIa. Depletion of cholesterol in human fibroblasts (WI-38) with methyl-beta-cyclodextrin-reduced TF activity at the cell surface. Binding studies with radiolabeled VIIa and TF monoclonal antibody (mAB) revealed that reduced TF activity in cholesterol-depleted cells stems from the impairment of VIIa interaction with TF rather than the loss of TF receptors at the cell surface. Repletion of cholesterol-depleted cells with cholesterol restored TF function. Loss of caveolar structure on cholesterol removal is not responsible for reduced TF activity. Solubilization of cellular TF in different detergents indicated that a substantial portion of TF in fibroblasts is associated with noncaveolar lipid rafts. Cholesterol depletion studies showed that the TF association with these rafts is cholesterol dependent. Overall, the data presented herein suggest that membrane cholesterol functions as a positive regulator of TF function by maintaining TF receptors, probably in noncaveolar lipid rafts, in a high-affinity state for VIIa binding.

Asbestos induces tissue factor in Beas-2B human lung bronchial epithelial cells in vitro.

Asbestos has been implicated in the pathogenesis of interstitial lung diseases including asbestosis. Tissue factor (TF) initiates blood coagulation in vivo contributing to inflammation and tissue remodeling via extravascular fibrin deposition and signaling for profibrogenic mediators. We hypothesized that asbestos could induce TF expression by lung epithelial cells. We found that TF mRNA and TF-dependent procoagulant activity were induced in asbestos-treated Beas-2B human airway epithelial cells, which we used as a model system. The effect was increased by crocidolite and chrysotile versus control particulates, including titanium dioxide (TiO2) and Wollastonite (W). Transcription factors that bind the TF gene promoter, including NF-kappaB, AP1 and Sp1, were induced by asbestos while TF mRNA was unstable. TF mRNA was inhibited by mithramycin in asbestos-treated as well as control cells suggesting that Sp1 contributes to TF maintenance in Beas-2B cells. Sp1 knockdown with specific siRNA decreased TF antigen, which is consistent with Sp1-mediated control of TF in Beas-2B cells. The results demonstrate that asbestos induces TF expression in lung epithelial cells in vitro, representing a newly recognized potential mechanism by which asbestos may modulate epithelial cell responses germane to lung remodeling. The mechanism involves alterations in steady-state TF mRNA that do not involve posttranscriptional regulation, implicating control of TF gene expression at the transcriptional level through Sp1 or other transcription factors.