Identification of a new autoinhibitory domain of interferon-beta promoter stimulator-1 (IPS-1) for the tight regulation of oligomerization-driven signal activation.

Upon viral infection, retinoic acid-inducible gene-I (RIG-I)-like receptors detect viral foreign RNAs and transmit anti-viral signals via direct interaction with the downstream mitochondrial adaptor molecule, interferon (IFN)-ß promoter stimulator-1 (IPS-1), to inhibit viral replication. Although IPS-1 is known to form prion-like oligomers on mitochondria to activate signaling, the mechanisms that regulate oligomer formation remain unclear. Here, we identified an autoinhibitory domain (AD) at amino acids 180-349 to suppress oligomerization of IPS-1 in a resting state and regulate activation of downstream signaling. Size exclusion chromatography (SEC) analysis demonstrated that AD was required to suppress auto-oligomerization of the caspase recruitment domain (CARD) of IPS-1 via intramolecular interactions. This was supported by the observation that cleavage of a peptide bond between IPS-1 CARD and AD by Tobacco Etch virus (TEV) protease relieved autoinhibition. Conversely, deletion of this domain from IPS-1 enhanced signal activation in IFN-reporter assays, suggesting that IPS-1 AD played a critical role in the regulation of IPS-1-mediated anti-viral signal activation. These findings revealed novel molecular interactions involved in the tight regulation of innate anti-viral immunity.

Establishment of the BacMam system using silkworm baculovirus.

The BacMam system uses modified insect viruses (baculoviruses) as vehicles to efficiently deliver genes for expression in mammalian cells. The technique can be widely applied to large-scale recombinant protein production with appropriate modifications, high-throughput screening platforms for cell-based assays, and the delivery of large genes. The silkworm system is often employed as a rapid and cost-effective approach for recombinant baculovirus generation. Here we have developed the novel BacMam system using silkworm baculovirus, and shown the successful expression of EGFP in mammalian cells. The transduction to mammalian cells via the BacMam system was improved by adding phosphate-buffered saline and sodium butyrate to the culture medium and lowering the temperature after viral infection. This study provides an alternative gene delivery system for mammalian cells, which has various potential applications, including efficient native protein production and gene therapy.

Structures and interface mapping of the TIR domain-containing adaptor molecules involved in interferon signaling.

Homotypic and heterotypic interactions between Toll/interleukin-1 receptor (TIR) domains in Toll-like receptors (TLRs) and downstream adaptors are essential to evoke innate immune responses. However, such oligomerization properties present intrinsic difficulties in structural studies of TIR domains. Here, using BB-loop mutations that disrupt homotypic interactions, we determined the structures of the monomeric TIR domain-containing adaptor molecule (TICAM)-1 and TICAM-2 TIR domains. Docking of the monomeric structures, together with yeast two hybrid-based mutagenesis assays, reveals that the homotypic interaction between TICAM-2 TIR is indispensable to present a scaffold for recruiting the monomeric moiety of the TICAM-1 TIR dimer. This result proposes a unique idea that oligomerization of upstream TIR domains is crucial for binding of downstream TIR domains. Furthermore, the bivalent nature of each TIR domain dimer can generate a large signaling complex under the activated TLRs, which would recruit downstream signaling molecules efficiently. This model is consistent with previous reports that BB-loop mutants completely abrogate downstream signaling.

Selection of aptamers using ß-1,3-glucan recognition protein-tagged proteins and curdlan beads.

RNA aptamersare nucleic acids that are obtained using the systematic evolution of ligands by exponential enrichment (SELEX) method. When using conventional selection methods to immobilize target proteins on matrix beads using protein tags, sequences are obtained that bind not only to the target proteins but also to the protein tags and matrix beads. In this study, we performed SELEX using ß-1,3-glucan recognition protein (GRP)-tags and curdlan beads to immobilize the acute myeloid leukaemia 1 (AML1) Runt domain (RD) and analysed the enrichment of aptamers using high-throughput sequencing. Comparison of aptamer enrichment using the GRP-tag and His-tag suggested that aptamers were enriched using the GRP-tag as well as using the His-tag. Furthermore, surface plasmon resonance analysis revealed that the aptamer did not bind to the GRP-tag and that the conjugation of the GRP-tag to RD weakened the interaction between the aptamer and RD. The GRP-tag could have acted as a competitor to reduce weakly bound RNAs. Therefore, the affinity system of the GRP-tagged proteins and curdlan beads is suitable for obtaining specific aptamers using SELEX.

Solution structure of the silkworm betaGRP/GNBP3 N-terminal domain reveals the mechanism for beta-1,3-glucan-specific recognition.

The beta-1,3-glucan recognition protein (betaGRP)/Gram-negative bacteria-binding protein 3 (GNBP3) is a crucial pattern-recognition receptor that specifically binds beta-1,3-glucan, a component of fungal cell walls. It evokes innate immunity against fungi through activation of the prophenoloxidase (proPO) cascade and Toll pathway in invertebrates. The betaGRP consists of an N-terminal beta-1,3-glucan-recognition domain and a C-terminal glucanase-like domain, with the former reported to be responsible for the proPO cascade activation. This report shows the solution structure of the N-terminal beta-1,3-glucan recognition domain of silkworm betaGRP. Although the N-terminal domain of betaGRP has a beta-sandwich fold, often seen in carbohydrate-binding modules, both NMR titration experiments and mutational analysis showed that betaGRP has a binding mechanism which is distinct from those observed in previously reported carbohydarate-binding domains. Our results suggest that betaGRP is a beta-1,3-glucan-recognition protein that specifically recognizes a triple-helical structure of beta-1,3-glucan.

A molecular mechanism for Toll-IL-1 receptor domain-containing adaptor molecule-1-mediated IRF-3 activation.

The Toll-IL-1 receptor (TIR) domain-containing adaptor molecule-1 (TICAM-1, also called TRIF) is a signaling adaptor for TLR3 and TLR4 that activates the transcription factors IRF-3, NF-kappaB, and AP-1, leading to induction of type I interferon and cytokines. The N-terminal region of TICAM-1 participates in IRF-3 activation, although the C-terminal region is involved in NF-kappaB activation. However, the mechanism by which TICAM-1 is activated and transmits signals is largely unknown. In this study, we identified Leu(194) as a critical amino acid for TICAM-1-mediated IRF-3 activation. When Leu(194) was substituted with Ala, the mutant TICAM-1 failed to recruit the IRF-3 kinase TBK1, resulting in lack of IRF-3 phosphorylation, although TRAF3 and NAP1 appeared to be recruited. The N-terminal 176 amino acids of TICAM-1 (N-terminal domain (NTD)) form a protease-resistant structural domain. A TICAM-1 mutant lacking the N-terminal 180 amino acids showed greater interferon-beta promoter activation than wild-type TICAM-1. Furthermore, immunoprecipitation and protein-protein interaction analysis revealed that the NTD interacted with the N terminus of TICAM-1-TIR. These results suggest that the NTD folds into the TIR domain structure to maintain the naive conformation of TICAM-1. Upon stimulation of TLR3/4, TICAM-1 oligomerizes through the TIR domain and the C-terminal region, which may break the intramolecular association and induce a conformational change that allows TBK1 access to TICAM-1.

Ser386 phosphorylation of transcription factor IRF-3 induces dimerization and association with CBP/p300 without overall conformational change.

The transcription factor IRF-3 is activated by microbial invasions and produces a variety of cytokines including type-I interferon. Upon microbial infection, IRF-3 is phosphorylated at its C-terminal regulatory domain, then oligomerized, translocated into the nucleus, and here it binds to CBP/p300. Although a number of studies have been reported investigating the activation mechanism of IRF-3, there are a number of unresolved issues, especially on the phosphorylation sites, the oligomerization process and the binding mechanism with CBP/p300. In this report, the phosphorylated IRF-3 regulatory domain (IRF-3 RD) was prepared using the kinase IKK-i, and the active form of phosphorylated IRF-3 RD was identified. The paper also reports the crystal structure of the active form of the phosphorylated IRF-3 RD. Furthermore, the phosphorylation of Ser386 was found to be essential for its dimerization and binding with CBP/p300 using mutational analysis and mass spectrometry. Thus, we conclude that the phosphorylation of Ser386 is essential for activation of IRF-3.

Specific inhibition of FGF5-induced cell proliferation by RNA aptamers.

Fibroblast growth factor 5 (FGF5) is a crucial regulator of hair growth and an oncogenic factor in several human cancers. To generate FGF5 inhibitors, we performed Systematic Evolution of Ligands by EXponential enrichment and obtained novel RNA aptamers that have high affinity to human FGF5. These aptamers inhibited FGF5-induced cell proliferation, but did not inhibit FGF2-induced cell proliferation. Surface plasmon resonance demonstrated that one of the aptamers, F5f1, binds to FGF5 tightly (Kd = 0.7 ± 0.2 nM), but did not fully to FGF1, FGF2, FGF4, FGF6, or FGFR1. Based on sequence and secondary structure similarities of the aptamers, we generated the truncated aptamer, F5f1_56, which has higher affinity (Kd = 0.118 ± 0.003 nM) than the original F5f1. Since the aptamers have high affinity and specificity to FGF5 and inhibit FGF5-induced cell proliferation, they may be candidates for therapeutic use with FGF5-related diseases or hair disorders.

Examination of stent deformation and gap formation after complex stenting of left main coronary artery bifurcations using microfocus computed tomography.

BACKGROUND: Fluoroscopy and intravascular ultrasound (IVUS) lack sufficient resolution for assessing the results of complex stenting in true bifurcation lesions. OBJECTIVES: After diverse bifurcation stenting at the left main coronary artery (LM) bifurcation model, the results were examined using microfocus computed tomography (MFCT). METHODS: The strut distribution of three kinds of stents deployed on a straight vessel segment was investigated. Classical crush, double kissing (DK)-double crush, and culotte stenting were performed on a three-dimensional (3D) LM model. The results were assessed using cross-sectional, longitudinal, and 3D reconstruction views of MFCT. RESULTS: Nonuniform strut distribution was observed in a corrugated stent design deployed on a straight vessel segment. Following classical crush stenting, a relatively large gap at the nonmyocardial site was observed in the corrugated stents. When the guidewire recrossed outside the ostium of the crushed side branch stent, kissing balloon inflation caused further crushing of the stent at the more distal segment. The dilated strut rose up from the main vessel bed after the first kissing balloon inflation in DK crush stenting; the advantage of DK would be cancelled after main vessel stenting due to recrushing the raised strut. The culotte stenting with closed-cell stents showed the restriction of the expansion at the branch ostium when it was dilated with a 3.5-mm balloon. The culotte stenting with open-cell-based stents showed a good stent apposition except for a tiny gap and small metallic carina at the distal bifurcation. CONCLUSION: MFCT analysis in the 3D phantom model is useful to assess the structural deformation of the stents and gap on vessel wall coverage after complex stenting at the LM bifurcation.

Crystallization and preliminary crystallographic analysis of the Tob-hCaf1 complex.

The Tob/BTG family is a group of antiproliferative proteins that contain two highly homologous regions named Box A and Box B. These proteins all associate with CCR4-associated factor 1 (Caf1), which belongs to the ribonuclease D family of deadenylases. The antiproliferative region of human Tob (residues 1-138) and intact hCaf1 were co-expressed in Escherichia coli, purified and successfully cocrystallized. The crystal belongs to the tetragonal space group I422, with unit-cell parameters a = b = 150.9, c = 113.9 A, and is estimated to contain one heterodimer per asymmetric unit. The crystal diffracted to around 2.6 A resolution.

Long-term treatment with N(omega)-nitro-L-arginine methyl ester causes arteriosclerotic coronary lesions in endothelial nitric oxide synthase-deficient mice.

BACKGROUND: N(omega)-nitro-L-arginine methyl ester (l-NAME) is widely used to inhibit endothelial synthesis of NO in vivo. However, it is controversial whether the long-term vascular effects of l-NAME are mediated primarily by inhibition of endothelial NO synthesis. We addressed this point in mice that are deficient in the endothelial NO synthase gene (eNOS-KO mice). METHODS AND RESULTS: Wild-type and eNOS-KO mice received l-NAME in drinking water for 8 weeks. In wild-type mice, long-term treatment with l-NAME caused significant medial thickening and perivascular fibrosis in coronary microvessels but not in large coronary arteries. Importantly, in eNOS-KO mice, treatment with l-NAME also caused an extent of medial thickening and perivascular fibrosis in coronary microvessels that was comparable to that in wild-type mice and that was not prevented by supplementation of L-arginine. Vascular NO and cGMP levels were not significantly reduced by l-NAME treatment, and no expression of inducible or neuronal NO synthase was noted in microvessels of eNOS-KO mice, suggesting an involvement of NO-independent mechanisms. Treatment with l-NAME caused an upregulation of vascular ACE and an increase in cardiac lucigenin chemiluminescence that were comparable in both strains and that were abolished by simultaneous treatment with temocapril (ACE inhibitor) or CS866 (angiotensin II type 1 receptor antagonist) along with the suppression of vascular lesion formation. CONCLUSIONS: These results provide the first direct evidence that the long-term vascular effects of l-NAME are not mediated by simple inhibition of endothelial NO synthesis. Direct upregulation of local ACE and increased oxidative stress appear to be involved in the long-term vascular effects of l-NAME in vivo.

Vasculoprotective roles of neuronal nitric oxide synthase.

Nitric oxide (NO) has multiple important actions that contribute to the maintenance of vascular homeostasis. NO is synthesized by three different isoforms of NO synthase (NOS), all of which have been reported to be expressed in human atherosclerotic vascular lesions. Although the regulatory roles of endothelial NOS (eNOS) and inducible NOS (iNOS) on the development of atherosclerosis have been described, little is known about the role of neuronal NOS (nNOS). Here, we show that nNOS also exerts important vasculoprotective effects in vivo. In a carotid artery ligation model, nNOS gene-deficient (nNOS-KO) mice exhibited accelerated neointimal formation and constrictive vascular remodeling caused by blood flow disruption. In a rat balloon injury model, the selective inhibition of nNOS activity potently enhanced vasoconstrictor responses to a variety of calcium-mobilizing stimuli, suppressed tissue cGMP concentrations, a marker of vascular NO production, and exacerbated neointimal formation. In both models, nNOS was absent before injury and was up-regulated only after the injury, and was predominantly expressed in the neointima and medial smooth muscle cells. These results provide the first direct evidence that nNOS plays important roles in suppressing arteriosclerotic vascular lesion formation in vivo.

Upregulation of vascular extracellular superoxide dismutase in patients with acute coronary syndromes.

OBJECTIVE: We examined the vascular expression levels of extracellular superoxide dismutase (EC-SOD), a major antioxidant enzyme in the cardiovascular system, in patients with acute coronary syndromes. METHODS AND RESULTS: Twenty-one consecutive patients with acute myocardial infarction (AMI), 14 patients with unstable angina, 11 patients with stable angina, and 20 control subjects were studied. The levels of vascular EC-SOD expression were assessed by the difference in plasma EC-SOD concentrations before and after intravenous heparan injection. In the patients with AMI, vascular EC-SOD expression (ng/mL) was significantly higher on day 1 after the onset of AMI (148+/-10) as compared with the control subjects (116+/-6, P<0.05). The vascular EC-SOD expression returned to the normal range on day 7 (104+/-8), and that level persisted thereafter. The vascular EC-SOD expression was also significantly higher in the patients with unstable angina (160+/-13) than in those with stable angina (122+/-10) or in the controls (116+/-6) (P<0.05 each). Moreover, in the patients with AMI, higher levels of vascular EC-SOD expression on day 1 were significantly associated with smaller myocardial infarct size (P<0.05). CONCLUSIONS: This is the first clinical demonstration showing that vascular EC-SOD may be upregulated in acute coronary syndromes in humans in vivo. EC-SOD may play an important protective role against increased oxidative stress during acute ischemic coronary events.

Experimental hyperhomocysteinemia impairs coronary flow velocity reserve.

BACKGROUND: Hyperhomocysteinemia has been identified as an independent risk factor for coronary artery disease. One mechanism is considered to be deteriorated endothelial function that is recovered by vitamin C. However, its direct action on coronary circulation has yet to be examined. This study was designed to test the hypothesis that experimental acute hyperhomocysteinemia would impair coronary flow velocity reserve (CFR) by increasing oxidative stress. METHODS: Eleven healthy male volunteers (aged 23.3+/-0.9 years) were enrolled. CFR induced by intravenous 5'-adenosine triphosphate infusion was measured by transthoracic-Doppler echocardiography. Measurements were taken before and 4 h after administration of a placebo, oral methionine (L-methionine 0.1 g/kg) or oral methionine plus vitamin C (2 g) on 3 separate days. RESULTS: The baseline average diastolic peak velocity (APV) was similar in all 3 groups. In the methionine group, plasma homocysteine increased (12.9+/-7.0 to 32.1+/-9.4 nmol/ml, p<0.0001), while APV under hyperemic conditions (APV-hyp) and CFR significantly decreased (87.2+/-11.4 cm/sec and 4.02+/-0.70 to 73.2+/-10.2 cm/sec and 3.35+/-0.52, p=0.0022 and 0.0030, respectively). Moreover, there was a significant inverse correlation between the plasma homocysteine and CFR (r=-0.620, p=0.0021). However, upon simultaneous administration of vitamin C, APV-hyp and CVR did not decrease despite an elevation in plasma homocysteine. CONCLUSIONS: Experimentally induced acute hyperhomocysteinemia significantly decreased CFR, and this decrease was significantly reversed by vitamin C administration. Oxidative stress is suggested to play a major role in the deleterious effects of homocysteine on the coronary microcirculation.

Safety and feasibility of high-dose administration of nicorandil before reperfusion therapy in acute myocardial infarction.

The efficacy and safety of high-dose nicorandil therapy in acute myocardial infarction (AMI) have not yet been clarified. This is a prospective study including 30 patients who received nicorandil at 0.06 mg/kg/h [standard dose nicorandil (SDN) group] and 32 patients who received a bolus injection of nicorandil 0.2 mg/kg followed by a continuous infusion at 0.2 mg/kg/h [high-dose nicorandil (HDN) group]. The benefits and adverse events were assessed during acute phase and 12-month follow-up period. There were no significant differences between the groups in blood pressure, heart rate or urine volume 2, 6 and 24 h after drug administration, although blood pressure decreased during acute phase. The percentages of patients who required dose reduction or discontinuation of nicorandil were 34.4 and 16.7 % in HDN and SDN groups, respectively (p = 0.11). In HDN group, subgroup analysis revealed that the TIMI frame count (TFC) was significantly lower in patients in whom the treatment was started within 12 h compared to those more than 12 h (17.0 vs. 21.0, p = 0.017) and in patients with baseline WBC elevation compared to those without it (16.5 vs. 22.0, p = 0.029). A TFC of >20 was significantly associated with being in HDN group [odds ratio (OR) 0.27; 95 % confidence interval, CI 0.07-0.89], onset-to-balloon time (OR 1.06; 95 % CI 1.01-1.16), and ∑creatine kinase (OR 7.27; 95 % CI 1.40-57.83). There were no significant differences in incidences of cardiovascular death, rehospitalization, and target lesion revascularization between the groups. HDN therapy may improve coronary microcirculation in patients with AMI.

SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation.

Chromophore-assisted light inactivation (CALI) is a powerful technique for acute perturbation of biomolecules in a spatio-temporally defined manner in living specimen with reactive oxygen species (ROS). Whereas a chemical photosensitizer including fluorescein must be added to specimens exogenously and cannot be restricted to particular cells or sub-cellular compartments, a genetically-encoded photosensitizer, KillerRed, can be controlled in its expression by tissue specific promoters or subcellular localization tags. Despite of this superiority, KillerRed hasn't yet become a versatile tool because its dimerization tendency prevents fusion with proteins of interest. Here, we report the development of monomeric variant of KillerRed (SuperNova) by direct evolution using random mutagenesis. In contrast to KillerRed, SuperNova in fusion with target proteins shows proper localization. Furthermore, unlike KillerRed, SuperNova expression alone doesn't perturb mitotic cell division. Supernova retains the ability to generate ROS, and hence promote CALI-based functional analysis of target proteins overcoming the major drawbacks of KillerRed.

A low-cost affinity purification system using ß-1,3-glucan recognition protein and curdlan beads.

Silkworm ß-1,3-glucan recognition protein (ßGRP) tightly and specifically associates with ß-1,3-glucan. We report here an affinity purification system named the 'GRP system', which uses the association between the ß-1,3-glucan recognition domain of ßGRP (GRP-tag), as an affinity tag, and curdlan beads. Curdlan is a water-insoluble ß-1,3-glucan reagent, the low cost of which (about 100 JPY/g) allows the economical preparation of beads. Curdlan beads can be readily prepared by solubilization in an alkaline solution, followed by neutralization, sonication and centrifugation. We applied the GRP system to preparation of several proteins and revealed that the expression levels of the GRP-tagged proteins in soluble fractions were two or three times higher than those of the glutathione S-transferase (GST)-tagged proteins. The purity of the GRP-tagged proteins on the curdlan beads was comparable to that of the GST-tagged proteins on glutathione beads. The chemical stability of the GRP system was more robust than conventional affinity systems under various conditions, including low pH (4-6). Biochemical and structural analyses revealed that proteins produced using the GRP system were structurally and functionally active. Thus, the GRP system is suitable for both the large- and small-scale preparation of recombinant proteins for functional and structural analyses.

Solution structures of cytosolic RNA sensor MDA5 and LGP2 C-terminal domains: identification of the RNA recognition loop in RIG-I-like receptors.

The RIG-I like receptor (RLR) comprises three homologues: RIG-I (retinoic acid-inducible gene I), MDA5 (melanoma differentiation-associated gene 5), and LGP2 (laboratory of genetics and physiology 2). Each RLR senses different viral infections by recognizing replicating viral RNA in the cytoplasm. The RLR contains a conserved C-terminal domain (CTD), which is responsible for the binding specificity to the viral RNAs, including double-stranded RNA (dsRNA) and 5'-triphosphated single-stranded RNA (5'ppp-ssRNA). Here, the solution structures of the MDA5 and LGP2 CTD domains were solved by NMR and compared with those of RIG-I CTD. The CTD domains each have a similar fold and a similar basic surface but there is the distinct structural feature of a RNA binding loop; The LGP2 and RIG-I CTD domains have a large basic surface, one bank of which is formed by the RNA binding loop. MDA5 also has a large basic surface that is extensively flat due to open conformation of the RNA binding loop. The NMR chemical shift perturbation study showed that dsRNA and 5'ppp-ssRNA are bound to the basic surface of LGP2 CTD, whereas dsRNA is bound to the basic surface of MDA5 CTD but much more weakly, indicating that the conformation of the RNA binding loop is responsible for the sensitivity to dsRNA and 5'ppp-ssRNA. Mutation study of the basic surface and the RNA binding loop supports the conclusion from the structure studies. Thus, the CTD is responsible for the binding affinity to the viral RNAs.

Structural basis for the antiproliferative activity of the Tob-hCaf1 complex.

The Tob/BTG family is a group of antiproliferative proteins containing two highly homologous regions, Box A and Box B. These proteins all associate with CCR4-associated factor 1 (Caf1), which belongs to the ribonuclease D (RNase D) family of deadenylases and is a component of the CCR4-Not deadenylase complex. Here we determined the crystal structure of the complex of the N-terminal region of Tob and human Caf1 (hCaf1). Tob exhibited a novel fold, whereas hCaf1 most closely resembled the catalytic domain of yeast Pop2 and human poly(A)-specific ribonuclease. Interestingly, the association of hCaf1 was mediated by both Box A and Box B of Tob. Cell growth assays using both wild-type and mutant proteins revealed that deadenylase activity of Caf1 is not critical but complex formation is crucial to cell growth inhibition. Caf1 tethers Tob to the CCR4-Not deadenylase complex, and thereby Tob gathers several factors at its C-terminal region, such as poly(A)-binding proteins, to exert antiproliferative activity.