Mayumi Shiota

Influenza virus RNA polymerase PA subunit is a novel serine protease with Ser624 at the active site

Influenza virus RNA polymerase is a multifunctional enzyme that catalyses both transcription and replication of the RNA genome. The function of the influenza virus RNA polymerase PA subunit in viral replication is poorly understood, although the enzyme is known to be required for cRNA → vRNA synthesis. The protease related activity of PA has been discussed ever since protease‐inducing activity was demonstrated in transfection experiments.

Identification of trypsin I as a candidate for influenza A virus and Sendai virus envelope glycoprotein processing protease in rat brain

Abstract Extracellular cleavage of virus envelope fusion glycoprotein hemagglutinin (HA0) by host trypsin-like proteases is a prerequisite for the infectivity and pathogenicity of human influenza A viruses and Sendai virus. The common epidemic influenza A viruses are pneumotropic, but occasionally cause encephalopathy or encephalitis, although the HA0 processing enzyme in the brain has not been identified. In searching for the brain processing proteases, we identified a processing enzyme in rat brain that was inducible by infection with these viruses. The purified enzyme exhibited an apparent molecular mass of approximately 22 kDa on SDS-PAGE and the N-terminal amino acid sequence was consistent with that of rat pancreatic trypsin I. Its substrate specificities and inhibition profiles were the same as those of pancreatic trypsin I. In situ hybridization and immunohistochemical studies on trypsin I distribution revealed heavy deposits in the brain capillaries, particularly in the allocortex, as well as in clustered neuronal cells of the hippocampus. The purified enzyme efficiently processed the HA0 of human influenza A virus and the fusion glycoprotein precursor of Sendai virus. Our results suggest that trypsin I in the brain potentiates virus multiplication in the pathogenesis and progression of influenza-associated encephalopathy or encephalitis.

The effect of topically applied secretory leukocyte protease inhibitor on the eosinophil response in the late phase of allergic conjunctivitis

Purpose. This study examined the effects of secretory leukocyte protease inhibitor (SLPI), a protease inhibitor in tears, in allergic conjunctivitis. Methods. Conjunctiva of male Hartley guinea pigs sensitized with ovalbumin were treated with SLPI or the vehicle 10min before antigen challenge or simultaneously. The animals were sacrificed after antigen challenges of 0-24 h duration, and the inhibition of eosinophil conjunctival migration and degranulation by SLPI was analyzed histochemically. The effects of SLPI on mast cell chymase and tryptase were also examined. Results. Treatment of sensitized guinea pigs with SLPI suppressed the conjunctival recruitment and degranulation of eosinophils after antigen challenge for 6 h, inhibiting the development of allergic conjunctivitis. The effects of SLPI were observed at concentrations =0.1µM, with a peak at 5µM. SLPI inhibited chymase in a dose-dependent manner, but had no effect on tryptase. Conclusion. The topical SLPI application may be therapeutic in allergic conjunctivitis.

Impaired long-chain fatty acid metabolism in mitochondria causes brain vascular invasion by a non-neurotropic epidemic influenza a virus in the newborn/suckling period: Implications for influenza-associated encephalopathy

The neuropathogenesis of influenza-associated encephalopathy in children and Reyes syndrome remains unclear. A surveillance effort conducted during 2000-2003 in South-West Japan reveals that almost all fatal and handicapped influenza-associated encephalopathy patients exhibit a disorder of mitochondrial β-oxidation with elevated serum acylcarnitine ratios (C16:0+C18:1)/C2. Here we show invasion by a non-neurotropic epidemic influenza A H3N2 virus in cerebral capillaries with progressive brain edema after intranasal infection of mice having impaired mitochondrial β-oxidation congenitally or posteriorly in the newborn/ suckling periods. Mice genetically lacking of carnitine transporter OCTN2, resulting in carnitine deficiency and impaired β-oxidation, exhibited significant higher virus-genome numbers in the brain, accumulation of virus antigen exclusively in the cerebral capillaries and increased brain vascular permeability compared to in wild type mice. Mini-plasmin, which proteolytically potentiates influenza virus multiplication in vivo and destroys the blood-brain barrier, accumulated with virus antigen in the brain capillaries of OCTN2-deficient mice but only a little in wild-type mice. These results suggest that the impaired mitochondrial β-oxidation changes the susceptibility to a non-neurotropic influenza A virus as to multiplication in the brain capillaries and to cause brain edema. These pathological findings in the brain of mice having impaired mitochondrial β-oxidation after influenza virus infection may have implications for human influenza-associated encephalopathy.

Accumulation of mini-plasmin in the cerebral capillaries causes vascular invasion of the murine brain by a pneumotropic influenza A virus: implications for influenza-associated encephalopathy.

Abstract The infectivity and pathogenicity of influenza virus are primarily determined by host cellular trypsin-type processing proteases which cleave the viral membrane fusion glycoprotein hemagglutinin (HA). Therefore the distribution of the processing protease is a major determinant of the infectious organ tropism. The common epidemic human influenza A virus is pneumotropic and the HA processing proteases tryptase Clara, mini-plasmin, tryptase TC30 and ectopic anionic trypsin have all been isolated from mammalian airways. However, the pneumotropic influenza virus occasionally causes severe brain edema, particularly in children presenting with Reyes syndrome treated with aspirin, or in children with influenza associated encephalopathy without antipyretic treatment. We have observed that, after influenza virus infection, the accumulation of mini-plasmin in the cerebral capillaries in mice with a congenital or acquired abnormality of mitochondrial β-oxidation mimicking the pathological findings of Reyes syndrome, causes an invasion and multiplication of the pneumotropic influenza virus at these same locations. From these findings, we hypothesize that the accumulated mini-plasmin modifies the brain capillaries from a non-permissive to a permissive state, thereby allowing multiplication of pneumotropic influenza virus. In addition, mini-plasmin proteolytically destroys the bloodbrain barrier. These pathologic findings, consistent with encephalopathy in mice with a systemic impairment of β-oxidation, may have implications for human influenza encephalopathy.

A novel bioactive 31-amino acid ET-1 peptide stimulates eosinophil recruitment and increases the levels of eotaxin and IL-5

Abstract. Objective and design: Investigation of the role of a novel inflammatory mediator 31-amino acid endothelin-1 [ET-1(1-31)], a major ET derivative in granulocytes, in eosinophil recruitment after its subcutaneous administration to mice.¶Methods: Various ET-1 derivatives (100 pmol), with or without ET receptor antagonists (200 pmol), were administered subcutaneously to mice, and then the eosinophil migration into and chemokine levels in the injected loci were analyzed.¶Results: ET-1(1-31) and a 21-amino acid endothelin-1 (ET-1), but not big ET-1, induced eosinophil migration into the injected loci with a peak after administration for 12 h, and increased the levels of eotaxin and interleukin-5 with peaks at 6 and 24 h, respectively. These effects of ET-1(1-31) and ET-1 were significantly inhibited by an ETA receptor antagonist, BQ-123, but not by an ETB receptor antagonist, BQ-788.¶Conclusion: Novel bioactive ET-1(1-31) induces local eosinophil migration, and increases in eotaxin and interleukin-5 through an ETA or ETA-like receptor.

Gatekeeper role of 14-3-3τ protein in HIV-1 gp120-mediated apoptosis of human endothelial cells by inactivation of Bad

Objective:HIV-1-associated dementia (HAD) is a major neurological complication often observed in the advanced stages of AIDS. We have reported that 14-3-3 proteins in cerebrospinal fluid, reflecting neuronal cell destruction, is a real-time marker of HAD progression. This study was designed to examine the role of 14-3-3 proteins in HAD. Design:An in-vitro human umbilical vein endothelial cells (HUVEC) model of gp120 protein-induced apoptosis to study the protective role of 14-3-3 in HIV-1 gp120/CXCR4-mediated cell death. Methods:The α-chemokine receptor-mediated cell death by HIV-1 envelope protein, gp120, the critical event that causes neuron loss and endothelial cell injury, was evaluated in HUVEC undergoing gp120-induced apoptosis through the CXCR4 receptor. We studied the effects of siRNA for each 14-3-3 isoform on the death of HUVEC treated with CXCR4-preferring gp120 (IIIB). Results:Gp120 increased the expression of 14-3-3τ in HUVEC. The binding of Gp120 to CXCR4 induced apoptosis of HUVEC through decreased binding of 14-3-3τ to the pro-apoptotic molecule, Bad. Treatment of the cells with dsRNA against 14-3-3τ enhanced the gp120-mediated dephosphorylation of Bad and its association with Bcl-XL in mitochondria, accelerating the gp120-induced apoptosis, whereas suppression of Bad by RNAi rescued the cells from apoptosis triggered by gp120. Conclusions:The specific up-regulation of 14-3-3τ in HUVEC negatively regulated gp120/CXCR4-mediated cell death by protecting Bad dephosphorylation.

Inhibition of the protease activity of influenza virus RNA polymerase PA subunit by viral matrix protein.

Influenza virus PA is a subunit of RNA‐dependent RNA polymerase. We demonstrated that PA has a unique chymotrypsin‐like serine protease activity with Ser624 as an active site. To obtain further insight into the role of the protease activity of PA in viral proliferation, we examined the interaction between PA and matrix protein (M1). Both M1 purified from virion and hexa‐histidine‐tagged M1 expressed in Escherichia coli bound to PA. Hexa‐histidine‐tagged M1 pulled down PA. The interaction of PA with M1 was sensitive to ionic strength, suggesting that the interaction is formed by electrostatic force. Using Suc‐Leu‐Leu‐Val‐Tyr‐MCA, a specific substrate for PA protease, M1 was demonstrated to inhibit the amidolytic activity of PA, whereas M1 did not inhibit that of chymotrypsin or trypsin at all. These results suggest that M1 binds to and inhibits the amidolytic activity of PA.

Cloning and Characterization of a Transmembrane-Type Serine Protease from Rat Kidney, a New Sodium Channel Activator

Abstract We have cloned the gene of a new transmembranetype serine protease from rat kidney, which activates sodium channels. The amino acid sequence deduced from a full-length cDNA revealed that transmembrane serine protease-1 (TMSP-1) is a member of the clan SA/family S1 of serine proteases, comprising a 30 amino acid prepropeptide, a mature form sequence of 274 amino acids starting with the Ile-Val-Gly-Gly-Gln motif, and a common catalytic triad of serine proteases. The hydrophobic amino acid sequence in the carboxyterminus of this enzyme suggests that it is a glycosylphosphatidylinositol-anchored protein. As revealed by quantitative reverse transcription-polymerase chain reaction analysis, it is highly expressed in kidney, small intestine, and stomach, and moderately expressed in lung, thymus, spleen and skin. The recombinant protease had an optimal pH at 9.0, selectively cleaved synthetic peptide substrates of trypsin, and was inhibited by aprotinin, leupeptin and benzamidine. Immunohistochemical studies revealed that this protease is predominantly expressed in cells from collecting ducts of the renal medulla. We also demonstrate that a C-terminally truncated variant of TMSP-1 significantly activates the epithelial sodium channel, and that its mRNA levels are upregulated by aldosterone. These observations suggest that it is a new member of the trypsin-type transmembrane proteases, which regulate sodium balance by activating the epithelial sodium channel.

Human Mast Cell Chymase and 31 Amino Acid Endothelin-1

Human mast cell chymase, which is predominantly distributed in the perivascular tissues and heart, selectively, and neutrophil cathepsin G transiently, cleaved the Tyr31-Gly32 bond of three forms of big endothelin (ET)-1, -2 and -3, and generated new bioactive 31-arnino acid ETs, Ets( 1-31). Unlike the distribution of 21-arnino acid ET-1 in cardiovascular endothelial cells and bronchiolar epithelial cells, ET-1(1-31) was distributed predominantly in neutrophils, eosinophils and mast cells in addition to these tissues. Besides the biological activities of ET-l(1-31) on smooth muscle constriction and stimulation of mitosis, it induced local eotaxin and interleukin-5, resulting in eosinophil recruitment in mice, and also exhibited chemotactism for human neutrophils and monocytes. Concentrations of ET1(1-31) were markedly increased at sites of inflammation in lungs, while the increase in ET-1 (1-21) concentrations was not significant, suggesting that ET-1 (1-31) is a sensitive inflammatory mediator. In addition, human chymase itself was a potent chemoattractant for neutrophils and monocytes. Overall, these findings indicate that chymase, cathepsin G and ET derivatives in the perivascular tissues and heart represent a network of inflammatory mediators, and play pathophysiologic roles in cardiovascular diseases.