GpIbα Interacts Exclusively with Exosite II of Thrombin

Activation of platelets by the serine protease thrombin is a critical event in haemostasis. This process involves the binding of thrombin to glycoprotein Ibα (GpIbα) and cleavage of protease-activated receptors (PARs). The N-terminal extracellular domain of GpIbα contains an acidic peptide stretch that has been identified as the main thrombin binding site, and both anion binding exosites of thrombin have been implicated in GpIbα binding, but it remains unclear how they are involved. This issue is of critical importance for the mechanism of platelet activation by thrombin. If both exosites bind to GpIbα, thrombin could potentially act as a platelet adhesion molecule or receptor dimerisation trigger. Alternatively, if only a single site is involved, GpIbα may serve as a cofactor for PAR-1 activation by thrombin. To determine the involvement of thrombin's two exosites in GpIbα binding, we employed the complementary methods of mutational analysis, binding studies, X-ray crystallography and NMR spectroscopy. Our results indicate that the peptide corresponding to the C-terminal portion of GpIbα and the entire extracellular domain bind exclusively to thrombin's exosite II. The interaction of thrombin with GpIbα thus serves to recruit thrombin activity to the platelet surface while leaving exosite I free for PAR-1 recognition.     

Type I Phosphatidylinositol Phosphate Kinase Beta Regulates Focal Adhesion Disassembly by Promoting β1 Integrin Endocytosis

Cell migration requires the regulated disassembly of focal adhesions, but the underlying mechanisms remain poorly defined. We have previously shown that focal adhesion disassembly requires the dynamin 2- and clathrin-dependent endocytosis of ligand-activated β1 integrins. Here, we identify type I phosphatidylinositol phosphate kinase beta (PIPKIβ), an enzyme that generates phosphatidylinositol-4,5-bisphosphate (PI4,5P₂), as a key regulator of this process. We found that knockdown of PIPKIβ by RNA interference blocks the internalization of active β1 integrins and impairs focal adhesion turnover and cell migration. These defects are caused by the failure to target the endocytic machinery, including clathrin adaptors and dynamin 2, to focal adhesion sites. As a consequence, depletion of PIPKIβ blocks clathrin assembly at adhesion plaques and prevents complex formation between dynamin 2 and focal adhesion kinase (FAK), a critical step in focal adhesion turnover. Together, our findings identify PIPKIβ as a novel regulator of focal adhesion disassembly and suggest that PIPKIβ spatially regulates integrin endocytosis at adhesion sites to control cell migration.Cell migration is a highly dynamic process that depends on the ability of a cell to adhere to and deadhere from the extracellular matrix in a coordinated manner. Adhesion is mediated through focal adhesion sites, which assemble in response to activation and clustering of integrin receptors and comprise signaling and scaffolding proteins, such as focal adhesion kinase (FAK), talin, vinculin, paxillin, and zyxin (9, 55). These complexes anchor the extracellular matrix to the actin cytoskeleton and also serve as signaling platforms (9, 55). The formation of adhesive complexes is essential for the stabilization of membrane protrusions and to provide the tensile forces for migration (9, 55). However, rapid cell movement requires that focal adhesions not only be continuously formed, but also disassembled (9, 56). The coordinated control of cell adhesion, and release thereof, is therefore a critical regulatory function for migrating cells. However, while much has been learned about the mechanisms underlying focal adhesion assembly, comparatively little is known about how the turnover of adhesion sites is regulated, despite the importance of this process for cell migration.Recently, the protease calpain, FAK, and phosphatases and kinases that control the activity of FAK, as well as microtubules and the large GTPase dynamin 2, have been identified as regulators of focal adhesion disassembly (9, 10, 21, 22). In particular, a pathway has been defined in which microtubule targeting of focal adhesions leads to their disassembly (21). A critical step in this process is the formation of a protein complex between FAK and dynamin 2, a key regulator of endocytosis (21). Dynamin 2, together with components of the clathrin machinery, then mediates the turnover of focal adhesions by promoting the internalization of β1 integrins (14, 41). Notably, dynamin 2 and clathrin adaptors become enriched at focal adhesion sites prior to their disassembly (14, 21). Therefore, mechanisms that control the recruitment of the endocytic machinery to focal adhesion sites must exist. However, how this process is regulated during focal adhesion turnover remains unknown.Phosphatidylinositol-4,5-bisphosphate (PI4,5P₂) has recently emerged as an important regulator of focal adhesion dynamics (38, 47, 51, 57). In addition to serving as the precursor to other second messengers, PI4,5P₂ directly binds and modulates many focal adhesion components, including talin, vinculin, and α-actinin, that regulate adhesion assembly and their linkage to the actin cytoskeleton (38, 47, 51, 57). Adhesion to the extracellular matrix stimulates the synthesis of PI4,5P₂, and the general paradigm has been that the resulting local increase in PI4,5P₂ levels promotes focal adhesion assembly (23, 38, 40). Intriguingly, emerging evidence suggests that PI4,5P₂ also promotes the disassembly of focal adhesions (13, 46). This finding implies that PI4,5P₂ levels at adhesion sites must be tightly regulated, both spatially and temporally, to elicit its specific, yet inverse, effects on focal adhesion dynamics.The generation of PI4,5P₂ at specific subcellular sites is modulated in part by the selective targeting and activation of specific type I phosphatidylinositol phosphate kinases (PIPKI), which synthesize PI4,5P₂ (38). Three related PIPKI isoforms, designated PIPKIα, PIPKIβ, and PIPKIγ, and multiple splice variants are present in mammalian cells (27, 28, 39, 49). Recent studies have shown that the increase of PI4,5P₂ synthesis leading to focal adhesion assembly is mediated through the specific recruitment of PIPKIγ661, a splice variant of PIPKIγ, to focal adhesions (18, 37). However, whether PIPKIγ661 or another member of the PIPKI family is responsible for synthesizing the PI4,5P₂ pool regulating focal adhesion disassembly is currently unknown, and the molecular mechanisms whereby PI4,5P₂ regulates this process are not well defined.Coincidently, PI4,5P₂ is also an important organizer of clathrin assembly at the plasma membrane (17). In this study, we therefore set out to determine whether PI4,5P₂ promotes focal adhesion disassembly through its effects on endocytosis and to identify the PIPKI isoform involved in generating this pool of PI4,5P₂. We show that knockdown of one specific PIPKI isoform, PIPKIβ, blocks adhesion turnover leading to the inhibition of cell migration. We further show that PIPKIβ is necessary for the uptake of activated β1 integrins and provide evidence that PI4,5P₂ produced by PIPKIβ orchestrates the recruitment of components of the endocytic machinery to adhesion sites. Together, these studies define the role of PI4,5P₂ in the regulation of focal adhesion disassembly and identify PIPKIβ as the enzyme synthesizing this pool of PI4,5P₂.     

Protein Phosphatase 6 Interacts with the DNA-Dependent Protein Kinase Catalytic Subunit and Dephosphorylates γ-H2AX

The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) plays a major role in the repair of DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ). We have previously shown that DNA-PKcs is autophosphorylated in response to ionizing radiation (IR) and that dephosphorylation by a protein phosphatase 2A (PP2A)-like protein phosphatase (PP2A, PP4, or PP6) regulates the protein kinase activity of DNA-PKcs. Here we report that DNA-PKcs interacts with the catalytic subunits of PP6 (PP6c) and PP2A (PP2Ac), as well as with the PP6 regulatory subunits PP6R1, PP6R2, and PP6R3. Consistent with a role in the DNA damage response, silencing of PP6c by small interfering RNA (siRNA) induced sensitivity to IR and delayed release from the G₂/M checkpoint. Furthermore, siRNA silencing of either PP6c or PP6R1 led to sustained phosphorylation of histone H2AX on serine 139 (γ-H2AX) after IR. In contrast, silencing of PP6c did not affect the autophosphorylation of DNA-PKcs on serine 2056 or that of the ataxia-telangiectasia mutated (ATM) protein on serine 1981. We propose that a novel function of DNA-PKcs is to recruit PP6 to sites of DNA damage and that PP6 contributes to the dephosphorylation of γ-H2AX, the dissolution of IR-induced foci, and release from the G₂/M checkpoint in vivo.DNA double-strand breaks (DSBs) are the most cytotoxic form of DNA damage. In human cells there are two main pathways for the repair of DSBs, namely, nonhomologous end joining (NHEJ) and homologous recombination (HR) (reviewed in reference 26). In the initial phase of NHEJ, DSBs are detected by the Ku70/80 heterodimer, which leads to recruitment of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and stimulation of its serine/threonine protein kinase activity. Upon autophosphorylation, DNA-PKcs undergoes a conformational change and dissociates from the DSB (25), providing other DNA repair proteins with access to the damage site (reviewed in reference 33). Another physiological substrate of DNA-PK is a histone H2A variant, H2AX. DNA-PKcs and the related protein kinase ATM (ataxia-telangiectasia mutated) both contribute to DNA damage-induced phosphorylation of H2AX on serine 139 to form γ-H2AX (51), which acts as a recruitment platform for MDC1, 53BP1, and other proteins involved in the DNA damage response and cell cycle checkpoint activation (7, 52).While the effects of phosphorylation on the repair process have been well documented, comparatively little is known about the role of serine/threonine phosphoprotein phosphatases (PPPs) in the DNA damage response. Within the PPP family, the catalytic subunits of PP2A (PP2Ac), PP4 (PP4c), and PP6 (PP6c) are most closely related and form a subgroup referred to as the PP2A-like protein phosphatases (reviewed in reference 40). In vitro, the PP2A-like enzymes display similar sensitivities to small-molecule inhibitors such as okadaic acid and microcystin (27, 45, 53). The specificity of PP2Ac, PP4c, and PP6c function in vivo is derived from a group of regulatory subunits that, with the exception of α4/TAP42 and TIP41, are unique to each enzyme (12, 13, 27, 45, 49). PP2Ac associates with a scaffolding A-α or A-β subunit and additional B-type subunits, while four direct binding partners and several other complex partners unique to PP4c have been characterized (12). The Saccharomyces cerevisiae homologue of PP6c, known as Sit4, interacts with three related proteins: the Sit4-associated proteins SAP155, SAP185, and SAP190, each of which contains a conserved domain known as the SAPs domain (32, 50). The SAPs domain is present in three human orthologues designated PP6R1, PP6R2, and PP6R3, which are therefore considered PP6c regulatory subunits, and each has been shown to bind independently to PP6c (48). More recently, three ankyrin repeat-containing proteins (ARS-A, ARS-B, and ARS-C) were identified as PP6R1 binding partners. One of these, ARS-A, has been shown to dock all three SAPs domain proteins (50), suggesting that, like PP2Ac, PP6c forms stable heterotrimers in vivo and that together these subunits define PP6 function.We have previously shown that inhibition of PP2A-like protein phosphatase activity by okadaic acid increases the phosphorylation status of DNA-PKcs and decreases its protein kinase activity (20), thus implicating PP2A-like phosphatases in the regulation of DNA-PK activity in vivo. More recently, both PP4 and PP2A have been shown to play roles in the DNA damage response by dephosphorylating γ-H2AX (14, 15, 28, 42). However, the potential role of PP6 in γ-H2AX dephosphorylation has not been addressed.Here we show that DNA-PKcs interacts with PP2Ac and PP6c, as well as with the PP6c regulatory subunits, PP6R1, PP6R2, and PP6R3. Depletion of PP6c by small interfering RNA (siRNA) induces sensitivity to ionizing radiation (IR) and delayed release from the G₂/M checkpoint. Furthermore, siRNA silencing of either PP6c or PP6R1 leads to sustained phosphorylation of γ-H2AX after DNA damage. Together, our studies reveal that a novel and previously unrecognized function of DNA-PKcs may be to recruit PP6 to sites of DNA damage and that PP6 regulates the phosphorylation status of γ-H2AX, the dissolution of IR-induced foci, and release from the G₂/M checkpoint.     

Residues in Conserved Loops of Intramembrane Metalloprotease SpoIVFB Interact with Residues near the Cleavage Site in Pro-σK

Intramembrane metalloproteases (IMMPs) control critical biological processes by cleaving membrane-associated proteins within a transmembrane segment or at a site near the membrane surface. Phylogenetic analysis divides IMMPs into four groups. SpoIVFB is a group III IMMP that regulates Bacillus subtilis endospore formation by cleaving Pro-σ^K and releasing the active sigma factor from a membrane. To elucidate the enzyme-substrate interaction, single-cysteine versions of catalytically inactive SpoIVFB and C-terminally truncated Pro-σ^K(1-126) (which can be cleaved by active SpoIVFB) were coexpressed in Escherichia coli, and proximity was tested by disulfide cross-linking in vivo. As expected, the results provided evidence that catalytic residue Glu-44 of SpoIVFB is near the cleavage site in the substrate. Also near the cleavage site were two residues of SpoIVFB in predicted conserved loops; Pro-135 in a short loop and Val-70 in a longer loop. Pro-135 corresponds to Pro-399 of RseP, a group I IMMP, and Pro-399 was reported previously to interact with substrate near the cleavage site, suggesting a conserved interaction across IMMP subfamilies. Val-70 follows a newly recognized conserved motif, PXGG (X is a large hydrophobic residue), which is in a hydrophobic region predicted to be a membrane reentrant loop. Following the hydrophobic region is a negatively charged region that is conserved in IMMPs of groups I and III. At least two residues with a negatively charged side chain are required in this region for activity of SpoIVFB. The region exhibits other features in IMMPs of groups II and IV. Its possible roles, as well as that of the short loop, are discussed. New insights into IMMP-substrate interaction build toward understanding how IMMPs function and may facilitate manipulation of their activity.     

Expression and Localization of Type II Diacylglycerol Kinase Isozymes δ and η in the Developing Mouse Brain

The functions of type II diacylglycerol kinase (DGK) δ and -η in the brain are still unclear. As a first step, we investigated the spatial and temporal expression of DGKδ and -η in the brains of mice. DGKδ2, but not DGKδ1, was highly expressed in layers II–VI of the cerebral cortex; CA–CA3 regions and dentate gyrus of hippocampus; mitral cell, glomerular and granule cell layers of the olfactory bulb; and the granule cell layer in the cerebellum in 1- to 32-week-old mice. DGKδ2 was expressed just after birth, and its expression levels dramatically increased from weeks 1 to 4. A substantial amount of DGKη (η1/η2) was detected in layers II–VI of the cerebral cortex, CA1 and CA2 regions and dentate gyrus of the hippocampus, mitral cell and glomerular layers of the olfactory bulb, and Purkinje cells in the cerebellum of 1- to 32-week-old mice. DGKη2 expression reached maximum levels at P5 and decreased by 4 weeks, whereas DGKη1 increased over the same time frame. These results indicate that the expression patterns of DGK isozymes differ from each other and also from other isozymes, and this suggests that DGKδ and -η play distinct and specific roles in the brain.     

Phosphatidylinositol Phosphate 5-Kinase Iγi2 in Association with Src Controls Anchorage-independent Growth of Tumor Cells

A fundamental property of tumor cells is to defy anoikis, cell death caused by a lack of cell-matrix interaction, and grow in an anchorage-independent manner. How tumor cells organize signaling molecules at the plasma membrane to sustain oncogenic signals in the absence of cell-matrix interactions remains poorly understood. Here, we describe a role for phosphatidylinositol 4-phosphate 5-kinase (PIPK) Iγi2 in controlling anchorage-independent growth of tumor cells in coordination with the proto-oncogene Src. PIPKIγi2 regulated Src activation downstream of growth factor receptors and integrins. PIPKIγi2 directly interacted with the C-terminal tail of Src and regulated its subcellular localization in concert with talin, a cytoskeletal protein targeted to focal adhesions. Co-expression of PIPKIγi2 and Src synergistically induced the anchorage-independent growth of nonmalignant cells. This study uncovers a novel mechanism where a phosphoinositide-synthesizing enzyme, PIPKIγi2, functions with the proto-oncogene Src, to regulate oncogenic signaling.     

Type II and VI collagen in nasal and articular cartilage and the effect of IL-1α on the distribution of these collagens

The distribution of type II and VI collagen was immunocytochemically investigated in bovine articular and nasal cartilage. Cartilage explants were used either fresh or cultured for up to 4 weeks with or without interleukin 1α (IL-1α). Sections of the explants were incubated with antibodies for both types of collagen. Microscopic analyses revealed that type II collagen was preferentially localized in the interchondron matrix whereas type VI collagen was primarily found in the direct vicinity of the chondrocytes. Treatment of the sections with hyaluronidase greatly enhanced the signal for both types of collagen. Also in sections of explants cultured with IL-1α a higher level of labeling of the collagens was found. This was apparent without any pre-treatment with hyaluronidase. Under the influence of IL-1α the area positive for type VI collagen that surrounded the chondrocytes broadened. Although the two collagens in both types of cartilage were distributed similarly, a remarkable difference was the higher degree of staining of type VI collagen in articular cartilage. Concomitantly we noted that digestion of this type of cartilage hardly occurred in the presence of IL-1α whereas nasal cartilage was almost completely degraded within 18 days of culture. Since type VI collagen is known to be relatively resistant to proteolysis we speculate that the higher level of type VI collagen in articular cartilage is important in protecting cartilage from digestion.     

Comparison of the Regulation of β-Catenin Signaling by Type I,Type II and Type III Interferons in Hepatocellular Carcinoma Cells

Background/Objective

IFNs are a group of cytokines that possess potent antiviral and antitumor activities, while β-catenin pathway is a proliferative pathway involved in carcinogenesis. Interaction between these two pathways has not been well elaborated in hepatocellular carcinoma (HCC).

Methods

HCC cell lines, HepG2 and Huh7, were used in this study. β-catenin protein levels and corresponding signaling activities were observed by flow cytometry and luciferase assay, respectively. Cell proliferation was quantified by counting viable cells under microscope, and apoptosis by TUNEL assay. DKK1 and GSK3β levels were determined by flow cytometry. Secreted DKK1 was tested by ELISA. FLUD, S3I and aDKK1 were used to inhibit STAT1, STAT3 and DKK1 activities, respectively.

Results

Our findings show that all three types of IFNs, IFNα, IFNγ and IFNλ, are capable of inhibiting β-catenin signaling activity in HepG2 and Huh7 cells, where IFNγ was the strongest (p<0.05). They expressed suppression of cellular proliferation and induced apoptosis. IFNγ expressed greater induction ability when compared to IFNα and IFNλ (p<0.05). All tested IFNs could induce DKK1 activation but not GSK3β in HepG2 and Huh7 cells. IFNs induced STAT1 and STAT3 activation but by using specific inhibitors, we found that only STAT3 is vital for IFN-induced DKK1 activation and apoptosis. In addition, DKK1 inhibitor blocked IFN-induced apoptosis. The pattern of STAT3 activation by different IFNs is found consistent with the levels of apoptosis with the corresponding IFNs (p<0.05).

Conclusions

In hepatocellular carcinoma, all three types of IFNs are found to induce apoptosis by inhibiting β-catenin signaling pathway via a STAT3- and DKK1-dependent pathway. This finding points to a cross-talk between different IFN types and β-catenin signaling pathways which might be carrying a biological effect not only on HCC, but also on processes where the two pathways bridge.     

Chromatin Protein HP1α Interacts with the Mitotic Regulator Borealin Protein and Specifies the Centromere Localization of the Chromosomal Passenger Complex

Accurate mitosis requires the chromosomal passenger protein complex (CPC) containing Aurora B kinase, borealin, INCENP, and survivin, which orchestrates chromosome dynamics. However, the chromatin factors that specify the CPC to the centromere remain elusive. Here we show that borealin interacts directly with heterochromatin protein 1α (HP1α) and that this interaction is mediated by an evolutionarily conserved PXVXL motif in the C-terminal borealin with the chromo shadow domain of HP1α. This borealin-HP1α interaction recruits the CPC to the centromere and governs an activation of Aurora B kinase judged by phosphorylation of Ser-7 in CENP-A, a substrate of Aurora B. Consistently, modulation of the motif PXVXL leads to defects in CPC centromere targeting and aberrant Aurora B activity. On the other hand, the localization of the CPC in the midzone is independent of the borealin-HP1α interaction, demonstrating the spatial requirement of HP1α in CPC localization to the centromere. These findings reveal a previously unrecognized but direct link between HP1α and CPC localization in the centromere and illustrate the critical role of borealin-HP1α interaction in orchestrating an accurate cell division.     

Characterization of the Catalytic and Nucleotide Binding Properties of the α-Kinase Domain of Dictyostelium Myosin-II Heavy Chain Kinase A

The α-kinases are a widely expressed family of serine/threonine protein kinases that exhibit no sequence identity with conventional eukaryotic protein kinases. In this report, we provide new information on the catalytic properties of the α-kinase domain of Dictyostelium myosin-II heavy chain kinase-A (termed A-CAT). Crystallization of A-CAT in the presence of MgATP yielded structures with AMP or adenosine in the catalytic cleft together with a phosphorylated Asp-766 residue. The results show that the β- and α-phosphoryl groups are transferred either directly or indirectly to the catalytically essential Asp-766. Biochemical assays confirmed that A-CAT hydrolyzed ATP, ADP, and AMP with k_cat values of 1.9, 0.6, and 0.32 min⁻¹, respectively, and showed that A-CAT can use ADP to phosphorylate peptides and proteins. Binding assays using fluorescent 2′/3′-O-(N-methylanthraniloyl) analogs of ATP and ADP yielded K_d values for ATP, ADP, AMP, and adenosine of 20 ± 3, 60 ± 20, 160 ± 60, and 45 ± 15 μm, respectively. Site-directed mutagenesis showed that Glu-713, Leu-716, and Lys-645, all of which interact with the adenine base, were critical for nucleotide binding. Mutation of the highly conserved Gln-758, which chelates a nucleotide-associated Mg²⁺ ion, eliminated catalytic activity, whereas loss of the highly conserved Lys-722 and Arg-592 decreased k_cat values for kinase and ATPase activities by 3–6-fold. Mutation of Asp-663 impaired kinase activity to a much greater extent than ATPase, indicating a specific role in peptide substrate binding, whereas mutation of Gln-768 doubled ATPase activity, suggesting that it may act to exclude water from the active site.     

TNFα Inhibits IGFBP-3 through Activation of p38α and Casein Kinase 2 in Human Retinal Endothelial Cells

We recently reported a reciprocal relationship between tumor necrosis factor alpha (TNFα) and insulin-like receptor growth factor binding protein 3 (IGFBP-3) in whole retina of normal and IGFBP-3 knockout mice. A similar relationship was also observed in cultured retinal endothelial cells (REC). We found that TNFα significantly reduced IGFBP-3 levels and vice-versa, IGFBP-3 can lower TNFα and TNFα receptor expression. Since IGFBP-3 is protective to the diabetic retina and TNFα is causative in the development of diabetic retinopathy, we wanted to better understand the cellular mechanisms by which TNFα can reduce IGFBP-3 levels. For these studies, primary human retinal endothelial cells (REC) were used since these cells undergo TNFα-mediated apoptosis under conditions of high glucose conditions and contribute to diabetic retinopathy. We first cultured REC in normal or high glucose, treated with exogenous TNFα, then measured changes in potential signaling pathways, with a focus on P38 mitogen-activated protein kinase alpha (P38α) and casein kinase 2 (CK2) as these pathways have been linked to both TNFα and IGFBP-3. We found that TNFα significantly increased phosphorylation of P38α and CK2. Furthermore, specific inhibitors of P38α or CK2 blocked TNFα inhibition of IGFBP-3 expression, demonstrating that TNFα reduces IGFBP-3 through activation of P38α and CK2. Since TNFα and IGFBP-3 are key mediators of retinal damage and protection respectively in diabetic retinopathy, increased understanding of the relationship between these two proteins will offer new therapeutic options for treatment.     

Acute Systemic Infection with Dengue Virus Leads to Vascular Leakage and Death through Tumor Necrosis Factor-α and Tie2/Angiopoietin Signaling in Mice Lacking Type I and II Interferon Receptors

Severe dengue is caused by host responses to viral infection, but the pathogenesis remains unknown. This is, in part, due to the lack of suitable animal models. Here, we report a non-mouse-adapted low-passage DENV-3 clinical isolate, DV3P12/08, derived from recently infected patients. DV3P12/08 caused a lethal systemic infection in type I and II IFN receptor KO mice (IFN-α/β/γR KO mice), which have the C57/BL6 background. Infection with DV3P12/08 induced a cytokine storm, resulting in severe vascular leakage (mainly in the liver, kidney and intestine) and organ damage, leading to extensive hemorrhage and rapid death. DV3P12/08 infection triggered the release of large amounts of TNF-α, IL-6, and MCP-1. Treatment with a neutralizing anti-TNF-α antibody (Ab) extended survival and reduced liver damage without affecting virus production. Anti-IL-6 neutralizing Ab partly prolonged mouse survival. The anti-TNF-α Ab suppressed IL-6, MCP-1, and IFN-γ levels, suggesting that the severe response to infection was triggered by TNF-α. High levels of TNF-α mRNA were expressed in the liver and kidneys, but not in the small intestine, of infected mice. Conversely, high levels of IL-6 mRNA were expressed in the intestine. Importantly, treatment with Angiopoietin-1, which is known to stabilize blood vessels, prolonged the survival of DV3P12/08-infected mice. Taken together, the results suggest that an increased level of TNF-α together with concomitant upregulation of Tie2/Angiopoietin signaling have critical roles in severe dengue infection.     

Cytosolic 5’-Nucleotidase II Interacts with the Leucin Rich Repeat of NLR Family Member Ipaf

IMP/GMP preferring cytosolic 5''-nucleotidase II (cN-II) is a bifunctional enzyme whose activities and expression play crucial roles in nucleotide pool maintenance, nucleotide-dependent pathways and programmed cell death. Alignment of primary amino acid sequences of cN-II from human and other organisms show a strong conservation throughout the entire vertebrata taxon suggesting a fundamental role in eukaryotic cells. With the aim to investigate the potential role of this homology in protein-protein interactions, a two hybrid system screening of cN-II interactors was performed in S. cerevisiae. Among the X positive hits, the Leucin Rich Repeat (LRR) domain of Ipaf was found to interact with cN-II. Recombinant Ipaf isoform B (lacking the Nucleotide Binding Domain) was used in an in vitro affinity chromatography assay confirming the interaction obtained in the screening. Moreover, co-immunoprecipitation with proteins from wild type Human Embryonic Kidney 293 T cells demonstrated that endogenous cN-II co-immunoprecipitated both with wild type Ipaf and its LRR domain after transfection with corresponding expression vectors, but not with Ipaf lacking the LRR domain. These results suggest that the interaction takes place through the LRR domain of Ipaf. In addition, a proximity ligation assay was performed in A549 lung carcinoma cells and in MDA-MB-231 breast cancer cells and showed a positive cytosolic signal, confirming that this interaction occurs in human cells. This is the first report of a protein-protein interaction involving cN-II, suggesting either novel functions or an additional level of regulation of this complex enzyme.