Hesperetin induces apoptosis in A549 cells via the Hsp70­mediated activation of Bax.

Hesperetin, a predominant flavonoid found in citrus fruits, has received considerable attention for its potential anticancer activity through the reduction of cell viability and the induction of apoptosis. Several effector mechanisms have been demonstrated underlying the antitumor properties of hesperetin but its specific mechanisms have not yet been fully elucidated. In the present study, how hesperetin affects the proliferation of A549 cells and the related cell proliferation regulatory mechanisms, were inevstigated. To elucidate the mechanisms underlying the effects of hesperetin on A549 cells, MTT assay, colony formation assay, flow cytometry, immunoblotting, reverse transcription­quantitative PCR and JC­1 staining were performed. The data revealed that hesperetin inhibited cell proliferation and induced apoptosis in these cells. Hesperetin also decreased the level of heat shock protein 70 (Hsp70), a negative regulator of the mitochondrial apoptosis pathway, often overexpressed in various cancer cells and suspected to contribute to tumor development. Hesperetin­induced Hsp70 suppression was associated with reduced cytosolic Bax and increased mitochondrial Bax levels, leading to the enhancement of the mitochondrial apoptotic cascade. The Hsp70 overexpression­induced reduction in the level of hesperetin­induced apoptosis provides evidence to hesperetin­induced apoptosis being mediated by affecting Hsp70. Furthermore, it was demonstrated that hesperetin reduced Hsp70 expression by inducing a proteasome­mediated degradation via the upregulation of E3­ligase, C­terminus of Hsp70­interacting protein (CHIP). The present study highlighted the importance of the Bax activation­triggered mitochondria­mediated pathway for hesperetin­induced apoptosis and demonstrated a novel mechanism of how Hsp70 played a critical role in the negative regulation of this apoptotic network in cancer cells.

Curcumin induces apoptosis in lung cancer cells by 14-3-3 protein-mediated activation of Bad.

The anticancer effects of curcumin are based on the induction of apoptosis, but the specific mechanisms have not yet been fully elucidated. To address this issue, we investigated the effects of curcumin on the intrinsic apoptosis pathway using mitochondria from A549 cells. Curcumin decreased the levels of 14-3-3 proteins, key molecules that inhibit the activation of proapoptotic factors known as BH3-only proteins (e.g. Bad). Curcumin-induced suppression of 14-3-3 protein levels was associated with reduced cytosolic Bad and elevation of mitochondrial Bad, leading to a drop in the mitochondrial membrane potential. 14-3-3 proteins generally interact with Bad phosphorylated by AKT, thus preventing its translocation to the mitochondria where it can promote cell death. Curcumin not only decreased the expression of 14-3-3 proteins but also promoted Bad dephosphorylation in an AKT-dependent fashion. Our results provide novel evidence for the induction of apoptosis by curcumin at multiple stages of the mitochondrial cascade.

Relationship between dietary patterns and brachial-ankle pulse wave velocity among middle-aged adults in Japan.

BACKGROUND AND OBJECTIVES: Arterial stiffness is a leading cause of cardiovascular disease (CVD), and it is considered to be affected by dietary intake. However, few studies have examined the relationship between major dietary patterns and brachial-ankle pulse wave velocity (baPWV) among Japanese middle-aged subjects. We studied whether major dietary patterns were associated with baPWV in this population. METHODS AND STUDY DESIGN: Between 2009 and 2012, 70 Japanese middle-aged subjects (39 men and 31 women) with no history of stroke, coronary heart disease, or cancer were studied. Dietary intake was documented using a validated food-frequency questionnaire, and dietary patterns were generated using factor analysis. Correlational analyses were performed between baPWV and identified dietary patterns and between baPWV and individual risk factors (total cholesterol, triglyceride, low-density lipoprotein cholesterol [LDL-C], high-density lipoprotein cholesterol [HDL-C], non- HDL-C, LDL/HDL ratio, and augmentation index). RESULTS: Two dietary patterns were identified: a rice-rich pattern (high in rice, breads, oils and fats, meats, and confectionery) and a vegetable-rich pattern (high in vegetables, fruit, fish, and seaweed). The rice-rich pattern was not associated with baPWVor other risk factors. A significant inverse correlation was found between baPWV and the vegetable-rich dietary pattern (p<0.05). CONCLUSIONS: Our findings suggest that a dietary pattern characterized by high consumption of vegetables, seafood, seaweed, fruit, and pulse is inversely associated with arterial stiffness measured by baPWV. This diet offers an additional nutritional approach to the prevention and treatment of arterial stiffness.

Trypanosoma brucei 14-3-3I and II proteins predominantly form a heterodimer structure that acts as a potent cell cycle regulator in vivo.

Hetero- and homodimerization of 14-3-3 proteins demonstrate distinctive functions in mammals and plants. Trypanosoma brucei 14-3-3I and II (Tb14-3-3I and II) play pivotal roles in motility, cytokinesis and the cell cycle; however, the significance and the mechanism of Tb14-3-3 dimerization are remained to be elucidated. We found that ectopically expressed epitope-tagged Tb14-3-3I and II proteins formed hetero- and homodimers with endogenous Tb14-3-3I and II proteins. However, we also found the ability to form hetero- or homodimers between Tb14-3-3I and II proteins was clearly affected by the sequence and location of the epitope tag used. We found a blue native polyacrylamide gel electrophoresis system followed by western blotting may distinguish monomer from dimer structure, and stable from unstable conformation of Tb14-3-3. Combined with co-immunoprecipitation results, we revealed that Tb14-3-3 proteins mainly existed as heterodimeric form. Furthermore, co-overexpression of Tb14-3-3I and II proteins in T. brucei induced aberrant numbers of organelles in cells, but overexpression of either isoform alone rarely produced such morphology. These results suggest that heterodimers play more significant roles than homodimers not only in the maintenance of steady-state levels of the 14-3-3 proteins but also in the regulation of cytokinesis.

Induction and maintenance of anti-influenza antigen-specific nasal secretory IgA levels and serum IgG levels after influenza infection in adults.

OBJECTIVES: To determine the induction and changes in anti-influenza virus secretory IgA (s-IgA) levels in nasal washes and serum IgG levels in patients with influenza. METHODS: The study recruited 16 patients with influenza aged 35.6 ± 9.6 years in 2007/2008 and 2008/2009 seasons. Nasal washes and serum were obtained throughout the first year. Anti-viral s-IgA levels and neutralization activities in nasal washes, and serum anti-viral IgG levels and hemagglutination inhibition (HI) titers were measured. RESULTS: Anti-viral(H1N1) s-IgA to total IgA ratio and neutralizing antibody titer were low in nasal washes of all patients, whereas serum levels of anti-viral IgG and HI titers varied widely at day 1.4 ± 1.0 postinfection. Both nasal s-IgA and serum IgG levels later increased significantly, reaching peak levels at day 9.6 ± 3.3 postinfection. The induced nasal s-IgA then returned toward the initial levels within 300 days, although the levels at day 143 ± 70 were 3.03-fold of the initial. Individual serum IgG levels also returned toward the initial levels within 300 days, although the mean levels remained high probably because of re-infection in a subgroup of patients. Although influenza A (H3N2) was a minor epidemic subtype in both flu seasons, a significant rise in nasal anti-viral (H3N2) s-IgA levels and a slightly increase in serum IgG levels were noted. CONCLUSION: Low levels of nasal anti-viral s-IgA and neutralizing antibody were noted compared with a wide range of serum anti-viral IgG and HI titers at the onset of infection. Elevated s-IgA and IgG returned toward the initial levels within 300 days of infection with minor exceptions.

Role of host cellular proteases in the pathogenesis of influenza and influenza-induced multiple organ failure.

Influenza A virus (IAV) is one of the most common infectious pathogens in humans. Since the IVA genome does not have the processing protease for the viral hemagglutinin (HA) envelope glycoprotein precursors, entry of this virus into cells and infectious organ tropism of IAV are primarily determined by host cellular trypsin-type HA processing proteases. Several secretion-type HA processing proteases for seasonal IAV in the airway, and ubiquitously expressed furin and pro-protein convertases for highly pathogenic avian influenza (HPAI) virus, have been reported. Recently, other HA-processing proteases for seasonal IAV and HPAI have been identified in the membrane fraction. These proteases proteolytically activate viral multiplication at the time of viral entry and budding. In addition to the role of host cellular proteases in IAV pathogenicity, IAV infection results in marked upregulation of cellular trypsins and matrix metalloproteinase-9 in various organs and cells, particularly endothelial cells, through induced pro-inflammatory cytokines. These host cellular factors interact with each other as the influenza virus-cytokine-protease cycle, which is the major mechanism that induces vascular hyperpermeability and multiorgan failure in severe influenza. This mini-review discusses the roles of cellular proteases in the pathogenesis of IAV and highlights the molecular mechanisms of upregulation of trypsins as effective targets for the control of IAV infection. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.

Surfactant protein C is an essential constituent for mucosal adjuvanticity of Surfacten, acting as an antigen delivery vehicle and inducing both local and systemic immunity.

We have reported that Surfacten(®) (St), a bovine pulmonary surfactant free of antigenic c-type lectins, is a useful mucosal adjuvant for nasal vaccination. To prepare ample supplies a synthetic adjuvant that mimics St, we analyzed essential constituents of St for mucosal adjuvanticity. Intranasal inoculation of influenza virus hemagglutinin (HA) vaccine combined with St free of surfactant protein (SP)-C resulted in failure of HA vaccine delivery to dendritic cells and loss of local and systemic immune responses. Naïve bovine SP-C, synthetic human or bovine SP-C peptide reconstituted with three major St lipids restored delivery activity and local and systemic immune responses to levels similar to those of St and provided almost complete protection against lethal doses of influenza virus challenge in mice. The delivery of fluoresceinated HA vaccine to cultured dendritic cells was significantly enhanced by co-administration of St or synthetic adjuvant, and moderately stimulated the expression of MHC class II and CD86. In addition, both St and synthetic adjuvant markedly sustained HA vaccine and achieved a wide antigen distribution in murine nasal cavity. These results suggest that synthetic mucosal adjuvant reconstituted with SP-C peptide and major St lipids is useful for ample supply of the potent mucosal adjuvant as an antigen delivery vehicle for intranasal vaccination.

Effects of inhibitors of Toll-like receptors, protease-activated receptor-2 signalings and trypsin on influenza A virus replication and upregulation of cellular factors in cardiomyocytes.

Severe influenza sometimes causes myocarditis. We recently found that influenza A virus (IAV) infection induces various cellular factors, such as proinflammatory cytokines IL-6, IL-1ß and TNF-α, matrix metalloproteinases (MMPs) and ectopic trypsin in mice hearts and in H9c2 cardiomyocytes. The induction of these cellular factors in turn promotes viral replication, myocardial inflammation and cellular damage through their intracellular signal transductions in cooperation with the IAV-induced Toll-like receptors (TLRs) and proteinase-activated receptor-2 (PAR-2) signalings, although the precise nature of these interactions remain obscure. By using specific inhibitors of TLRs and PAR-2 signalings and trypsin inhibitor aprotinin, we analyzed the role of TLR signaling and PAR-2 signaling in the IAV-induced pathological changes in cardiomyocytes. Inhibitors of TLR7/8-Myeloid Differentiation factor 88-nuclear factor-κB signaling and aprotinin effectively suppressed IAV-induced upregulation of proinflammatory cytokines, MMPs, trypsinogen and viral replication. Inhibitor of TLR3-Toll/interleukin-1 receptor domain-containing adaptor inducing interferons-dependent signaling predominantly suppressed the upregulation of interferon-ß, a key intracellular host immune response factor. In contrast to the suppressive effect of trypsin inhibitor aprotinin on IAV replication, PAR-2 inhibitor FSY-NH(2), induced marginal upregulation of trypsinogen and subsequent stimulation of IAV replication.

Up-regulation of ectopic trypsins in the myocardium by influenza A virus infection triggers acute myocarditis.

AIMS: Influenza A virus (IAV) infection markedly up-regulates ectopic trypsins in various organs, viral envelope glycoprotein processing proteases, which are pre-requisites for virus entry and multiplication. We investigated the pathological roles of trypsin up-regulation in the progression of IAV-induced myocarditis, cytokine induction, and viral replication in the hearts, and also investigated the protective effects of trypsin inhibitor on cardiac dysfunction in vivo and selective knockdown of trypsin on IAV-induced cellular damage in cardiomyoblasts. METHODS AND RESULTS: The relationship of the expression among IAV RNA, trypsins, matrix metalloproteinase (MMP)-9, MMP-2, pro-inflammatory cytokines interleukin (IL)-6, IL-1ß, and tumour necrosis factor-α was analysed in mice hearts and cardiomyoblasts after IAV infection. The severity of myocarditis was most noticeable during Day 6-9 post-infection, along with peak expression of viral RNA, trypsins, particularly trypsin2, MMPs, and cytokines. Cardiac ATP levels were the lowest at Day 9. Up-regulated trypsins, viral protein, and tissue-injured loci in the myocardium were closely localized. Trypsin inhibitor aprotinin treatment in vivo and selective trypsin1- and trypsin2-knockdown, particularly the latter, in H9c2 cardiomyoblasts significantly suppressed viral replication, up-regulation of MMPs, and production of active MMP-9 and cytokines, resulting in marked protection against cellular damage, ATP depletion, and apoptosis. IAV infection-induced cardiac dysfunction monitored by echocardiography was improved significantly by aprotinin treatment. CONCLUSIONS: IAV-induced trypsins, particularly trypsin2, in the myocardium trigger acute viral myocarditis through stimulation of IAV replication, proMMP-9 activation, and cytokine induction. These results suggest that up-regulation of trypsins is one of the key host pathological findings in IAV-induced myocarditis.

Influenza virus-cytokine-protease cycle in the pathogenesis of vascular hyperpermeability in severe influenza.

BACKGROUND: Severe influenza is characterized by cytokine storm and multiorgan failure with edema. The aim of this study was to define the impact of the cytokine storm on the pathogenesis of vascular hyperpermeability in severe influenza. METHODS: Weanling mice were infected with influenza A WSN/33(H1N1) virus. The levels of proinflammatory cytokines, tumor necrosis factor (TNF) alpha, interleukin (IL) 6, IL-1beta, and trypsin were analyzed in the lung, brain, heart, and cultured human umbilical vein endothelial cells. The effects of transcriptional inhibitors on cytokine and trypsin expressions and viral replication were determined. RESULTS: Influenza A virus infection resulted in significant increases in TNF-alpha, IL-6, IL-1beta, viral hemagglutinin-processing protease trypsin levels, and viral replication with vascular hyperpermeability in lung and brain in the first 6 days of infection. Trypsin upregulation was suppressed by transcriptional inhibition of cytokines in vivo and by anti-cytokine antibodies in endothelial cells. Calcium mobilization and loss of tight junction constituent, zonula occludens-1, associated with cytokine- and trypsin-induced endothelial hyperpermeability were inhibited by a protease-activated receptor-2 antagonist and a trypsin inhibitor. CONCLUSIONS: The influenza virus-cytokine-protease cycle is one of the key mechanisms of vascular hyperpermeability in severe influenza.

Mechanisms of matrix metalloproteinase-9 upregulation and tissue destruction in various organs in influenza A virus infection.

Severe influenza is characterized clinicopathologically by multiple organ failure, although the relationship amongst virus and host factors that influence this morbid outcome and the underlying mechanisms of action remain unclear. The present study identified marked upregulation of matrix metalloproteinase (MMP)-9 and pro-inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) in various organs after intranasal infection of influenza A WSN virus. MMP-9 and TNF-alpha were upregulated in the lung, the site of initial infection, as well as in the brain and heart. The infection-induced MMP-9 upregulation was inhibited by anti-TNF-alpha antibodies and by anti-oxidative reagents pyrrolidine dithiocarbamate and N-acetyl-L-cysteine, which inhibit activation of nuclear factor kappa B (NF-kappaB), as well as by nordihydroguaiaretic acid, which inhibits activation of activator protein 1 (AP-1). In addition, MMP-9 upregulation via TNF-alpha was also suppressed by inhibitors of mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase 1/2 and p38, and partly by a c-Jun N-terminal kinase inhibitor. These results indicated that the influenza-induced MMP-9 upregulation in various organs is mediated through MAPK-NF-kappaB- and/or AP-1-dependent mechanisms. Strategies that neutralize TNF-alpha as well as inhibitors of MAPK-NF-kappa B- and/or AP-1-dependent pathways may be useful for suppressing the MMP-9 effect and thus preventing multiple organ failure in severe influenza.

Novel type II transmembrane serine proteases, MSPL and TMPRSS13, Proteolytically activate membrane fusion activity of the hemagglutinin of highly pathogenic avian influenza viruses and induce their multicycle replication.

Host cellular proteases induce influenza virus entry into cells by cleaving the viral surface envelope glycoprotein hemagglutinin (HA). However, details on the cellular proteases involved in this event are not fully available. We report here that ubiquitous type II transmembrane serine proteases, MSPL and its splice variant TMPRSS13, are novel candidates for proteases processing HA proteins of highly pathogenic avian influenza (HPAI) viruses, apart from the previously identified furin and proprotein convertases 5 and 6. HAs from all HPAI virus H5 and H7 strains have one of two cleavage site motifs, the R-X-K/R-R motif with R at position P4 and the K-K/R-K/T-R motif with K at position P4. In studies of synthetic 14-residue HPAI virus HA peptides with these cleavage site motifs, furin preferentially cleaved only HA peptides with the R-K-K-R motif in the presence of calcium and not peptides with the other motif, whereas MSPL and TMPRSS13 cleaved both types of HA peptides (those with the R/K-K-K-R motif) efficiently in the absence of calcium. Full-length recombinant HPAI virus HA with the K-K-K-R cleavage motif exhibited poor susceptibility to cleavage in the absence of MSPL or TMPRSS13 and the presence of furin in infected cells, but it was converted to mature HA subunits in transfected cells expressing MSPL or TMPRSS13, with membrane-fused giant-cell formation. This conversion and membrane fusion were suppressed by inhibitors of MSPL and TMPRSS13. Furthermore, infection with and multiplication of genetically modified live HPAI virus A/Crow/Kyoto/53/2004 (H5N1) with the K-K-K-R cleavage site motif were detected only in MSPL- and TMPRSS13-expressing cells.

Human immunodeficiency virus type 1 gp120-mediated disruption of tight junction proteins by induction of proteasome-mediated degradation of zonula occludens-1 and -2 in human brain microvascular endothelial cells.

The infiltration of human immunodeficiency virus (HIV)-1, such as by HIV-infected leukocytes, across an injured blood-brain barrier (BBB) is a characteristic pathologic manifestation of HIV-1-associated dementia. HIV-1 gp120 has been implicated as a cause of breakdown of tight junctions between endothelial cells of the BBB, though the disrupting molecular mechanisms are unexplained. This study offers a new explanation for the increased BBB microvascular permeability, due to the degradation of tight junction proteins by the proteasome induced by gp120, and the negative regulation of this process by the scaffold protein, 14-3-3tau. gp120 reduced the amount of zonula occludens (ZO)-1 and ZO-2 in human brain microvascular endothelial cells (HBMECs). The treatment of HBMECs with the proteasome inhibitor, lactacystin, blocked the degradation of ZO-1 and ZO-2, suggesting that these proteins were targeted by gp120 for degradation by the proteasome. gp120 also specifically increased the expression of 14-3-3tau in HBMECs, and its down-regulation by RNAi facilitated the breakdown of tight junction proteins induced by gp120. Our results demonstrate the novel molecular mechanisms of the BBB breakdown by gp120.

Host envelope glycoprotein processing proteases are indispensable for entry into human cells by seasonal and highly pathogenic avian influenza viruses.

Influenza A virus (IAV) is one of the most common infectious pathogens in humans and causes considerable morbidity and mortality. The recent spread of highly-pathogenic avian IAV H5N1 viruses has reinforced the importance of pandemic preparedness. In the pathogenesis of IAV infection, cellular proteases play critical roles in the process of viral entry into cells that subsequently leads to tissue damage in the infected organs. Since there are no processing protease for the viral membrane fusion glycoprotein hemagglutinin precursor (HA(0)) in IAV, entry of the virus into cells is determined primarily by the host cellular HA(0) processing proteases that proteolytically activate membrane fusion activity. HA(0) of seasonal human IAV has the consensus cleavage site motif Q(E)-T/X-R and is selectively processed by at least seven different trypsin-type processing proteases identified to-date in animal model experiments using mouse-adapted IAV or gene expression system in MDCK cells. As is the case for the highly pathogenic avian influenza (HPAI) A virus, endoproteolytic processing of the HA(0) occurs through ubiquitous cellular processing proteases, which selectively recognize the multi-basic consensus cleavage site motifs, such as R-X-K/R-R, and K-X-K/R-R. The cleavage enzymes for the R-X-K/R-R motif, but not K-X-K/R-R motif, have been reported to be furin and pro-protein convertase (PC)5/6 in the trans-Golgi network. Here we report new members of type II transmembrane serine proteases of the cell membrane, mosaic serine protease large form (MSPL) and its splice variant TMPRSS13, which recognize and cleave both R-X-K/R-R and K-X-K/R-R motifs without calcium. Furthermore, IAV infection significantly up-regulates a latent ectopic pancreatic trypsin, one of the potent HA processing proteases, and pro-matrix metalloprotease-9, in various organs. These proteases may synergistically damage the blood-brain barrier in the brain and basement membrane of blood vessels in various organs, resulting in severe edema and multiple organ failure. In this review, we discuss these proteases as new drug target molecules for IAV treatment acting by inhibition of IAV multiplication and prevention of multiple organ failure, other than anti-viral agents, viral neuraminidase inhibitors.

Gatekeeper role of 14-3-3tau 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 alpha-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 14-3-3tau in HUVEC. The binding of Gp120 to CXCR4 induced apoptosis of HUVEC through decreased binding of 14-3-3tau to the pro-apoptotic molecule, Bad. Treatment of the cells with dsRNA against 14-3-3tau 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 14-3-3tau in HUVEC negatively regulated gp120/CXCR4-mediated cell death by protecting Bad dephosphorylation.

Proteases essential for human influenza virus entry into cells and their inhibitors as potential therapeutic agents.

Influenza A virus (IAV) is one of the most common infectious pathogens in humans. Since IVA genome does not have the processing protease for the viral membrane fusion glycoprotein precursors, entry of this virus into cells is determined primarily by host cellular, trypsin-type, processing proteases that proteolytically activate the fusion glycoprotein precursors of IAV. At least five different processing proteases have been identified in the airways of animals and humans. These proteases determine the infectious organ tropism of IAV infection as well as the efficiency of viral multiplication in the airway, and sometimes in the brain. Proteases in the upper respiratory tract are suppressed by secretory leukoprotease inhibitor, and those in the lower respiratory tract are suppressed by pulmonary surfactant which, by adsorption, inhibits the interaction between the proteases and viral membrane proteins. Since protease activities predominate over those of endogenous inhibitory compounds under normal airway conditions, administration of protease inhibitors in the early-stage of infection significantly suppresses viral entry and viral multiplication. Several viral neuraminidase inhibitors are used clinically as anti-influenza virus agents, based on their inhibitory action on viral release from infected cells. Furthermore, protease inhibitors of viral entry could be potentially useful against influenza virus as well as neuraminidase inhibitor-resistant viruses. We also found that ambroxol, a mucolytic and anti-oxidant agent, up-regulates the levels of endogenous protease inhibitory compounds in the airway fluids in early-phase infection, and that clarithromycin, a macrolide antibiotic, increases IgA levels and mucosal immunity through augmentation of interleukin-12 levels in the airway. The combination of neuraminidase inhibitors and protease inhibitors, clarithromycin or ambroxol, could be potentially used as a potent anti-influenza therapy to minimize the emergence of drug-resistant mutant viruses.

A novel function of 14-3-3 protein: 14-3-3zeta is a heat-shock-related molecular chaperone that dissolves thermal-aggregated proteins.

The 14-3-3 proteins are highly conserved molecules that function as intracellular adaptors in a variety of biological processes, such as signal transduction, cell cycle control, and apoptosis. Here, we show that a 14-3-3 protein is a heat-shock protein (Hsp) that protects cells against physiological stress as its new cellular function. We have observed that, in Drosophila cells, the 14-3-3zeta is up-regulated under heat stress conditions, a process mediated by a heat shock transcription factor. As the biological action linked to heat stress, 14-3-3zeta interacted with apocytochrome c, a mitochondrial precursor protein of cytochrome c, in heat-treated cells, and the suppression of 14-3-3zeta expression by RNA interference resulted in the formation of significant amounts of aggregated apocytochrome c in the cytosol. The aggregated apocytochrome c was converted to a soluble form by the addition of 14-3-3zeta protein and ATP in vitro. 14-3-3zeta also resolubilized heat-aggregated citrate synthase and facilitated its reactivation in cooperation with Hsp70/Hsp40 in vitro. Our observations provide the first direct evidence that a 14-3-3 protein functions as a stress-induced molecular chaperone that dissolves and renaturalizes thermal-aggregated proteins.

Serase-1B, a new splice variant of polyserase-1/TMPRSS9, activates urokinase-type plasminogen activator and the proteolytic activation is negatively regulated by glycosaminoglycans.

Polyserase-1 (polyserine protease-1)/TMPRSS9 (transmembrane serine protease 9) is a type II transmembrane serine protease (TTSP) that possesses unique three tandem serine protease domains. However, the physiological function of each protease domain remains poorly understood. We discovered a new splice variant of polyserase-1, termed Serase-1B, which contains 34 extra amino acids consisting a SEA module (a domain found in sea urchin sperm protein, enterokinase and agrin) adjacent to the transmembrane domain and the first protease domain with a mucin-like box at the C-terminus. The tissue distribution of this enzyme by RT (reverse transcription)-PCR analysis revealed high expression in the liver, small intestine, pancreas, testis and peripheral blood CD14+ and CD8+ cells. To investigate the role of Serase-1B, a full-length form recombinant protein was produced. Interestingly, recombinant Serase-1B was partly secreted as a soluble inactive precursor and it was also activated by trypsin. This activated enzyme selectively cleaved synthetic peptides for trypsin and activated protein C, and it was inhibited by several natural serine protease inhibitors, such as aprotinin, alpha2-antiplasmin and plasminogen activator inhibitor 1. In addition, Serase-1B efficiently converted pro-uPA (urokinase-type plasminogen activator) into active uPA and this activation was strongly inhibited by these natural inhibitors. Furthermore, this activation was also negatively regulated by glycosaminoglycans. Our results indicate that Serase-1B is a novel member of TTSPs that might be involved in uPA/plasmin-mediated proteolysis and possibly implicated in biological events such as fibrinolysis and tumour progression.

Chaperone activities of the 26S and 20S proteasome.

The accumulation of misfolded or damaged proteins causes the failure of normal cell structure and functions necessary for growth and viability. To abort this adverse development, defective proteins must be rapidly repaired by molecular chaperones or destroyed by energy-dependent cytoplasmic proteases. A balance among these processes ultimately maintains cellular homeostasis. In eukaryotes, the 26S proteasome, a protease/chaperone complex, is a central component in the protein triage decision process. The 26S proteasome generally acts as a ubiquitination system, though it also selectively degrades structurally abnormal proteins in an ubiquitin-independent manner. In either case, all substrate proteins must undergo structural changes and stabilization necessary for their rapid degradation. It has, therefore, often been suggested that several chaperone functions are closely related to the stimulation of proteasomal degradation. This review summarizes recent discoveries pertaining to chaperone activities in the proteasomal degradation pathway, and to their regulation of protein breakdown mediated by the proteasome.

Secretory leukoprotease inhibitor and pulmonary surfactant serve as principal defenses against influenza A virus infection in the airway and chemical agents up-regulating their levels may have therapeutic potential.

Influenza A virus (IAV) is one of the most common infectious pathogens in humans. Entry of this virus into cells is primarily determined by host cellular trypsin-type processing proteases, which proteolytically activate viral membrane fusion glycoprotein precursors. Human IAV and murine parainfluenza virus type 1 Sendai virus are exclusively pneumotropic, and the infectious organ tropism of these viruses is determined by the susceptibility of the viral envelope glycoprotein to cleavage by proteases in the airway. Proteases in the upper respiratory tract are suppressed by secretory leukoprotease inhibitor, and those in the lower respiratory tract are suppressed by pulmonary surfactant, which by adsorption inhibits the interaction between the proteases and viral membrane proteins. Although the protease activities are predominant over the activities of inhibitory compounds under normal airway conditions, intranasal administration of inhibitors was able to significantly suppress multi-cycles of viral replication in the airway. In addition, we identified chemical agents that could act as defensive factors by up-regulating the levels of the natural inhibitors and immunoglobulin A (IgA) in airway fluids. One of these compounds, ambroxol, is a mucolytic and anti-oxidant agent that stimulates the release of secretory leukoprotease inhibitor and pulmonary surfactant in the early phase, and IgA in the late phase of infection at an optimal dose, i.e. a dose sufficient to inhibit virus proliferation and increase the survival rate of animals after treatment with a lethal dose of IAV. Another agent, clarithromycin, is a macrolide antibiotic that increases IgA levels through augmentation of interleukin-12 levels and mucosal immunization in the airway. In addition to the sialidase inhibitors, which prevent the release of IAV from infected cells, inhibitors of the processing proteases and chemical agents that augment mucosal immunity and/or levels of the relevant defensive compounds may also ultimately prove to be useful as new anti-influenza agents.