Role for ADP ribosylation factor 1 in the regulation of hepatitis C virus replication

We hypothesized that ADP-ribosylation factor 1 (Arf1) plays an important role in the biogenesis and maintenance of infectious hepatitis C virus (HCV). Huh7.5 cells, in which HCV replicates and produces infectious viral particles, were exposed to brefeldin A or golgicide A, pharmacological inhibitors of Arf1 activation. Treatment with these agents caused a reduction in viral RNA levels, the accumulation of infectious particles within the cells, and a reduction in the levels of these particles in the extracellular medium. Fluorescence analyses showed that the viral nonstructural (NS) proteins NS5A and NS3, but not the viral structural protein core, shifted their localization from speckle-like structures in untreated cells to the rims of lipid droplets (LDs) in treated cells. Using pulldown assays, we showed that ectopic overexpression of NS5A in Huh7 cells reduces the levels of GTP-Arf1. Downregulation of Arf1 expression by small interfering RNA (siRNA) decreased both the levels of HCV RNA and the production of infectious viral particles and altered the localization of NS5A to the peripheries of LDs. Together, our data provide novel insights into the role of Arf1 in the regulation of viral RNA replication and the production of infectious HCV.     

Nonapoptotic role for Apaf-1 in the DNA damage checkpoint

Apaf-1 is an essential factor for cytochrome c-driven caspase activation during mitochondrial apoptosis but has also an apoptosis-unrelated function. Knockdown of Apaf-1 in human cells, knockout of apaf-1 in mice, and loss-of-function mutations in the Caenorhabditis elegans apaf-1 homolog ced-4 reveal the implication of Apaf-1/CED-4 in DNA damage-induced cell-cycle arrest. Apaf-1 loss compromised the DNA damage checkpoints elicited by ionizing irradiation or chemotherapy. Apaf-1 depletion reduced the activation of the checkpoint kinase Chk1 provoked by DNA damage, and knockdown of Chk1 abrogated the Apaf-1-mediated cell-cycle arrest. Nuclear translocation of Apaf-1, induced in vitro by exogenous DNA-damaging agents, correlated in non-small cell lung cancer (NSCLC) with the endogenous activation of Chk-1, suggesting that this pathway is clinically relevant. Hence, Apaf-1 exerts two distinct, phylogenetically conserved roles in response to mitochondrial membrane permeabilization and DNA damage. These data point to a role for Apaf-1 as a bona fide tumor suppressor.     

A mathematical model for apoptosome assembly: the optimal cytochrome c/Apaf-1 ratio

Apoptosis, a highly conserved form of cell suicide, is regulated by apoptotic signals and their transduction with caspases, a family of cystein proteases. Caspases are constantly expressed in the normal cells as inactive pro-enzymes. The activity of caspase is regulated by the proteolysis. Sequential proteolytic reactions of caspases are needed to execute apoptosis. Mitochondrial pathway is one of these apoptotic signal pathways, in which caspases are oligomerized into characteristic heptamer structure, called apoptosome, with caspase-9 that activate the effector caspases for apoptosis. To investigate the dynamics of signal transduction pathway regulated by oligomerization, we construct a mathematical model for Apaf-1 heptamer assembly process. The model first reveals that intermediate products can remain unconverted even after all assemble reactions are completed. The second result of the model is that the conversion efficiency of Apaf-1 heptamer assembly is maximized when the initial concentration of cytochrome c is equal to that of Apaf-1. When the concentration of cytochrome c is sufficiently larger or smaller than that of Apaf-1, the final Apaf-1 heptamer production is decreased, because intermediate Apaf-1 oligomers (tetramers and bigger oligomers), which themselves are unable to form active heptamer, accumulate too fast in the cells, choking a smooth production of Apaf-1 heptamer. Slow activation of Apaf-1 monomers and small oligomers increase the conversion efficiency. We also study the optimal number of subunits comprising an active oligomer that maximize the conversion efficiency in assembly process, and found that the tetramer is the optimum.     

A Critical Role for the Hippocampus in the Valuation of Imagined Outcomes

Many choice situations require imagining potential outcomes, a capacity that was shown to involve memory brain regions such as the hippocampus. We reasoned that the quality of hippocampus-mediated simulation might therefore condition the subjective value assigned to imagined outcomes. We developed a novel paradigm to assess the impact of hippocampus structure and function on the propensity to favor imagined outcomes in the context of intertemporal choices. The ecological condition opposed immediate options presented as pictures (hence directly observable) to delayed options presented as texts (hence requiring mental stimulation). To avoid confounding simulation process with delay discounting, we compared this ecological condition to control conditions using the same temporal labels while keeping constant the presentation mode. Behavioral data showed that participants who imagined future options with greater details rated them as more likeable. Functional MRI data confirmed that hippocampus activity could account for subjects assigning higher values to simulated options. Structural MRI data suggested that grey matter density was a significant predictor of hippocampus activation, and therefore of the propensity to favor simulated options. Conversely, patients with hippocampus atrophy due to Alzheimer''s disease, but not patients with Fronto-Temporal Dementia, were less inclined to favor options that required mental simulation. We conclude that hippocampus-mediated simulation plays a critical role in providing the motivation to pursue goals that are not present to our senses.     

A Critical Role for the mTORC2 Pathway in Lung Fibrosis

A characteristic of dysregulated wound healing in IPF is fibroblastic-mediated damage to lung epithelial cells within fibroblastic foci. In these foci, TGF-β and other growth factors activate fibroblasts that secrete growth factors and matrix regulatory proteins, which activate a fibrotic cascade. Our studies and those of others have revealed that Akt is activated in IPF fibroblasts and it mediates the activation by TGF-β of pro-fibrotic pathways. Recent studies show that mTORC2, a component of the mTOR pathway, mediates the activation of Akt. In this study we set out to determine if blocking mTORC2 with MLN0128, an active site dual mTOR inhibitor, which blocks both mTORC1 and mTORC2, inhibits lung fibrosis. We examined the effect of MLN0128 on TGF-β-mediated induction of stromal proteins in IPF lung fibroblasts; also, we looked at its effect on TGF-β-mediated epithelial injury using a Transwell co-culture system. Additionally, we assessed MLN0128 in the murine bleomycin lung model. We found that TGF-β induces the Rictor component of mTORC2 in IPF lung fibroblasts, which led to Akt activation, and that MLN0128 exhibited potent anti-fibrotic activity in vitro and in vivo. Also, we observed that Rictor induction is Akt-mediated. MLN0128 displays multiple anti-fibrotic and lung epithelial-protective activities; it (1) inhibited the expression of pro-fibrotic matrix-regulatory proteins in TGF-β-stimulated IPF fibroblasts; (2) inhibited fibrosis in a murine bleomycin lung model; and (3) protected lung epithelial cells from injury caused by TGF-β-stimulated IPF fibroblasts. Our findings support a role for mTORC2 in the pathogenesis of lung fibrosis and for the potential of active site mTOR inhibitors in the treatment of IPF and other fibrotic lung diseases.     

A Critical Role of the C-terminal Segment for Allosteric Inhibitor-induced Aberrant Multimerization of HIV-1 Integrase

Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are a promising class of antiretroviral agents for clinical development. Although ALLINIs promote aberrant IN multimerization and inhibit IN interaction with its cellular cofactor LEDGF/p75 with comparable potencies in vitro, their primary mechanism of action in infected cells is through inducing aberrant multimerization of IN. Crystal structures have shown that ALLINIs bind at the IN catalytic core domain dimer interface and bridge two interacting subunits. However, how these interactions promote higher-order protein multimerization is not clear. Here, we used mass spectrometry-based protein footprinting to monitor surface topology changes in full-length WT and the drug-resistant A128T mutant INs in the presence of ALLINI-2. These experiments have identified protein-protein interactions that extend beyond the direct inhibitor binding site and which lead to aberrant multimerization of WT but not A128T IN. Specifically, we demonstrate that C-terminal residues Lys-264 and Lys-266 play an important role in the inhibitor induced aberrant multimerization of the WT protein. Our findings provide structural clues for exploiting IN multimerization as a new, attractive therapeutic target and are expected to facilitate development of improved inhibitors.     

A Novel Role for the Hypoxia Inducible Transcription Factor HIF-1alpha: Critical Regulation of Inflammatory Cell Function

No abstract available.     

To kill or to arrest: that is the new question for Apaf-1

In this issue of Molecular Cell, Zermati et al. (2007) report that Apaf-1 is essential for the DNA damage-induced intra-S phase checkpoint response. This new role for Apaf-1 is unrelated to its proapoptotic function but is evolutionarily conserved in Ced-4.     

DNA Interstrand Cross-Link Repair in the Cell Cycle: A Critical Role for Polymerase ζ in G1 Phase

DNA interstrand cross-links (ICLs) present a formidable challenge to the cellular repair apparatus, but to date ICL repair pathways have proved difficult to dissect genetically. It now appears that this is partly the result of a high degree of cell cycle phase selectivity in the choice of ICL pathway employed. Here we review recent results showing that Polymerase ζ, aspecialised translesion polymerase, plays an important role during ICL repair in G₁ phase yeast cells, and that PCNA modification by ubiquitin is a key regulator of its activity. Given that this reaction can occur outside the context of S-phase, these results imply a more general role for PCNA modification in the control of DNA repair pathways through the cell cycle, which is dependent on the type of damage or repair intermediate encountered.     

The Apaf-1 apoptosome: a large caspase-activating complex

It is increasingly recognized that many key biological processes, including apoptosis, are carried out within very large multi-protein complexes. Apoptosis can be initiated by activation of death receptors or perturbation of the mitochondria causing the release of apoptogenic proteins, which result in the activation of caspases which are responsible for most of the biochemical and morphological changes observed during apoptosis. Caspases are normally inactive and require proteolytic processing for activity and this is achieved by the formation of large protein complexes known as the DISC (death inducing signalling complex) and the apoptosome. In the case of the latter complex, the central scaffold protein is a mammalian CED-4 homologue known as Apaf-1. This is an approximately 130 kDa protein, which in the presence of cytochrome c and dATP oligomerizes to form a very large (approximately 700-1400 kDa) apoptosome complex. The apoptosome recruits and processes caspase-9 to form a holoenzyme complex, which in turn recruits and activates the effector caspases. The apoptosome has been described in cells undergoing apoptosis, in dATP activated cell lysates and in reconstitution studies with recombinant proteins. Recent studies show that formation and function of the apoptosome can be regulated by a variety of factors including intracellular levels of K(+), inhibitor of apoptosis proteins (IAPs), heat shock proteins and Smac/Diablo. These various factors thus ensure that the apoptosome complex is only fully assembled and functional when the cell is irrevocably destined to die.     

Structure of Acinetobacter strain ADP1 protocatechuate 3, 4-dioxygenase at 2.2 A resolution: implications for the mechanism of an intradiol dioxygenase

The crystal structures of protocatechuate 3,4-dioxygenase from the soil bacteria Acinetobacterstrain ADP1 (Ac 3,4-PCD) have been determined in space group I23 at pH 8.5 and 5.75. In addition, the structures of Ac 3,4-PCD complexed with its substrate 3, 4-dihydroxybenzoic acid (PCA), the inhibitor 4-nitrocatechol (4-NC), or cyanide (CN(-)) have been solved using native phases. The overall tertiary and quaternary structures of Ac 3,4-PCD are similar to those of the same enzyme from Pseudomonas putida[Ohlendorf et al. (1994) J. Mol. Biol. 244, 586-608]. At pH 8.5, the catalytic non-heme Fe(3+) is coordinated by two axial ligands, Tyr447(OH) (147beta) and His460(N)(epsilon)(2) (160beta), and three equatorial ligands, Tyr408(OH) (108beta), His462(N)(epsilon)(2) (162beta), and a hydroxide ion (d(Fe-OH) = 1.91 A) in a distorted bipyramidal geometry. At pH 5.75, difference maps suggest a sulfate binds to the Fe(3+) in an equatorial position and the hydroxide is shifted [d(Fe-OH) = 2.3 A] yielding octahedral geometry for the active site Fe(3+). This change in ligation geometry is concomitant with a shift in the optical absorbance spectrum of the enzyme from lambda(max) = 450 nm to lambda(max) = 520 nm. Binding of substrate or 4-NC to the Fe(3+) is bidentate with the axial ligand Tyr447(OH) (147beta) dissociating. The structure of the 4-NC complex supports the view that resonance delocalization of the positive character of the nitrogen prevents substrate activation. The cyanide complex confirms previous work that protocatechuate 3,4-dioxygenases have three coordination sites available for binding by exogenous substrates. A significant conformational change extending away from the active site is seen in all structures when compared to the native enzyme at pH 8.5. This conformational change is discussed in its relevance to enhancing catalysis in protocatechuate 3,4-dioxygenases.     

Trypanosoma cruzi: A Specific Surface Marker for the Amastigote Form1

ABSTRACT. Among the known life cycle stages of Trypanosoma cruzi only the amastigote form bound lactoferrin (LF), a glycoprotein produced by neutrophils. This capacity was readily demonstrable by indirect immunofluorescence in amastigotes derived from mice, a mammalian cell culture, or grown in an axenic medium. No LF binding was detectable on trypomastigotes from blood or mammalian cells, insect-derived metacyclics or epimastigotes, or on epimastigotes grown in Warren's medium. Serum levels of LF were increased in mice acutely infected with T. cruzi, and amastigotes from the spleens of these animals were found to have the glycoprotein on their surface. The amastigote LF receptor may have biological significance in parasite-host interaction since mononuclear phagocytes also express a LF receptor, and treatment of these cells with LF has been shown to increase their capacities to take up and kill T. cruzi amastigotes in vitro. The LF receptor is the first marker for T. cruzi amastigotes for which a naturally occurring ligand has been described.     

Bcl-xL does not inhibit the function of Apaf-1

Bcl-2 and its relative, Bcl-xL, inhibit apoptotic cell death primarily by controlling the activation of caspase proteases. Previous reports have suggested at least two distinct mechanisms: Bcl-2 and Bcl-xL may inhibit either the formation of the cytochrome c/Apaf-1/caspase-9 apoptosome complex (by preventing cytochrome c release from mitochondria) or the function of this apoptosome (through a direct interaction of Bcl-2 or Bcl-xL with Apaf-1). To evaluate this latter possibility, we added recombinant Bcl-xL protein to cell-free apoptotic systems derived from Jurkat cells and Xenopus eggs. At low concentrations (50 nM), Bcl-xL was able to block the release of cytochrome c from mitochondria. However, although Bcl-xL did associate with Apaf-1, it was unable to inhibit caspase activation induced by the addition of cytochrome c, even at much higher concentrations (1-5 microM). These observations, together with previous results obtained with Bcl-2, argue that Bcl-xL and Bcl-2 cannot block the apoptosome-mediated activation of caspase-9.     

A novel Apaf-1-independent putative caspase-2 activation complex

Caspase activation is a key event in apoptosis execution. In stress-induced apoptosis, the mitochondrial pathway of caspase activation is believed to be of central importance. In this pathway, cytochrome c released from mitochondria facilitates the formation of an Apaf-1 apoptosome that recruits and activates caspase-9. Recent data indicate that in some cells caspase-9 may not be the initiator caspase in stress-mediated apoptosis because caspase-2 is required upstream of mitochondria for the release of cytochrome c and other apoptogenic factors. To determine how caspase-2 is activated, we have studied the formation of a complex that mediates caspase-2 activation. Using gel filtration analysis of cell lysates, we show that caspase-2 is spontaneously recruited to a large protein complex independent of cytochrome c and Apaf-1 and that recruitment of caspase-2 to this complex is sufficient to mediate its activation. Using substrate-binding assays, we also provide the first evidence that caspase-2 activation may occur without processing of the precursor molecule. Our data are consistent with a model where caspase-2 activation occurs by oligomerization, independent of the Apaf-1 apoptosome.     

A Nanoconjugate Apaf-1 Inhibitor Protects Mesothelial Cells from Cytokine-Induced Injury

Background

Inflammation may lead to tissue injury. We have studied the modulation of inflammatory milieu-induced tissue injury, as exemplified by the mesothelium. Peritoneal dialysis is complicated by peritonitis episodes that cause loss of mesothelium. Proinflammatory cytokines are increased in the peritoneal cavity during peritonitis episodes. However there is scarce information on the modulation of cell death by combinations of cytokines and on the therapeutic targets to prevent desmesothelization.

Methodology

Human mesothelial cells were cultured from effluents of stable peritoneal dialysis patients and from omentum of non-dialysis patients. Mesothelial cell death was studied in mice with S. aureus peritonitis and in mice injected with tumor necrosis factor alpha and interferon gamma.Tumor necrosis factor alpha and interferon gamma alone do not induce apoptosis in cultured mesothelial cells. By contrast, the cytokine combination increased the rate of apoptosis 2 to 3-fold over control. Cell death was associated with the activation of caspases and a pancaspase inhibitor prevented apoptosis. Specific caspase-8 and caspase-3 inhibitors were similarly effective. Co-incubation with both cytokines also impaired mesothelial wound healing in an in vitro model. However, inhibition of caspases did not improve wound healing and even impaired the long-term recovery from injury. By contrast, a polymeric nanoconjugate Apaf-1 inhibitor protected from apoptosis and allowed wound healing and long-term recovery. The Apaf-1 inhibitor also protected mesothelial cells from inflammation-induced injury in vivo in mice.

Conclusion

Cooperation between tumor necrosis factor alpha and interferon gamma contributes to mesothelial injury and impairs the regenerative capacity of the monolayer. Caspase inhibition attenuates mesothelial cell apoptosis but does not facilitate regeneration. A drug targeting Apaf-1 allows protection from apoptosis as well as regeneration in the course of inflammation-induced tissue injury.     

A Role for LHC1 in Higher Order Structure and Complement Binding of the Cryptococcus neoformans Capsule

Polysaccharide capsules are important virulence factors for many microbial pathogens including the opportunistic fungus Cryptococcus neoformans. In the present study, we demonstrate an unusual role for a secreted lactonohydrolase of C. neoformans, LHC1 in capsular higher order structure. Analysis of extracted capsular polysaccharide from wild-type and lhc1Δ strains by dynamic and static light scattering suggested a role for the LHC1 locus in altering the capsular polysaccharide, both reducing dimensions and altering its branching, density and solvation. These changes in the capsular structure resulted in LHC1-dependent alterations of antibody binding patterns, reductions in human and mouse complement binding and phagocytosis by the macrophage-like cell line J774, as well as increased virulence in mice. These findings identify a unique molecular mechanism for tertiary structural changes in a microbial capsule, facilitating immune evasion and virulence of a fungal pathogen.