Tranilast Inhibits Pulmonary Fibrosis by Suppressing TGFß/SMAD2 Pathway.

PURPOSE: Idiopathic pulmonary fibrosis (IPF) is characterized by the accumulation of extracellular matrix (ECM) protein in the lungs. Transforming growth factor (TGF) ß-induced ECM protein synthesis contributes to the development of IPF. Tranilast, an anti-allergy drug, suppresses TGFß expression and inhibits interstitial renal fibrosis in animal models. However, the beneficial effects of tranilast or its mechanism as a therapy for pulmonary fibrosis have not been clarified. METHODS: We investigated the in vitro effect of tranilast on ECM production and TGFß/SMAD2 pathway in TGFß2-stimulated A549 human alveolar epithelial cells, using quantitative polymerase chain reaction, Western blotting, and immunofluorescence. In vitro observations were validated in the lungs of a murine pulmonary fibrosis model, which we developed by intravenous injection of bleomycin. RESULTS: Treatment with tranilast suppressed the expression of ECM proteins, such as fibronectin and type IV collagen, and attenuated SMAD2 phosphorylation in TGFß2-stimulated A549 cells. In addition, based on a wound healing assay in these cells, tranilast significantly inhibited cell motility, with foci formation that comprised of ECM proteins. Histological analyses revealed that the administration of tranilast significantly attenuated lung fibrosis in mice. Furthermore, tranilast treatment significantly reduced levels of TGFß, collagen, fibronectin, and phosphorylated SMAD2 in pulmonary fibrotic tissues in mice. CONCLUSION: These findings suggest that tranilast inhibits pulmonary fibrosis by suppressing TGFß/SMAD2-mediated ECM protein production, presenting tranilast as a promising and novel anti-fibrotic agent for the treatment of IPF.

Amino-group carrier-protein-mediated secondary metabolite biosynthesis in Streptomyces.

Amino-group carrier proteins (AmCPs) mediate the biosynthesis of lysine and arginine in some bacteria and archaea. Here we demonstrate that an uncharacterized AmCP-mediated biosynthetic system functions to biosynthesize the previously uncharacterized and nonproteinogenic amino acid (2S,6R)-diamino-(5R,7)-dihydroxy-heptanoic acid (DADH) in Streptomyces sp. SANK 60404. DADH is incorporated into a novel peptide metabolite, vazabitide A, featuring an azabicyclo-ring structure, by nonribosomal peptide synthetases and successive modification enzymes in this bacterium. As the AmCP-mediated machinery for DADH biosynthesis is widely distributed in bacteria, further analysis of uncharacterized AmCP-containing gene clusters will lead to the discovery of novel bioactive compounds and novel biosynthetic enzymes.

Secreted tyrosine sulfated-eIF5A mediates oxidative stress-induced apoptosis.

Oxidative stress plays a critical role in ischemia/reperfusion-injury, atherosclerosis, and aging. It causes cell damage that leads to apoptosis via uncertain mechanisms. Because conditioned medium from cardiac myocytes subjected to hypoxia/reoxygenation induces extensive apoptosis of cardiac myocytes under normoxia, we hypothesized that a humoral factor released from the hypoxic/reoxygenated cardiac myocytes mediates apoptosis. We identified an apoptosis-inducing humoral factor in the hypoxia/reoxygenation-conditioned medium. Here, we found that eIF5A undergoes tyrosine sulfation in the trans-Golgi and is rapidly secreted from cardiac myocytes in response to hypoxia/reoxygenation; then, eIF5A induces apoptosis by acting as a pro-apoptotic ligand. The apoptosis of cardiac myocytes induced by hypoxia/reoxygenation or ultraviolet irradiation was suppressed by anti-eIF5A neutralizing monoclonal antibodies (mAbs) in vitro. Myocardial ischemia/reperfusion (but not ischemia alone) markedly increased the plasma levels of eIF5A, and treatment with anti-eIF5A neutralizing mAbs significantly reduced myocardial injury. These results identify an important, novel specific biomarker and a critical therapeutic target for oxidative stress-induced cell injury.

Cysteine protease antigens cleave CD123, the α subunit of murine IL-3 receptor, on basophils and suppress IL-3-mediated basophil expansion.

Th2 type immune responses are essential for protective immunity against parasites and play crucial roles in allergic disorders. Helminth parasites secrete a variety of proteases for their infectious cycles including for host entry, tissue migration, and suppression of host immune effector cell function. Furthermore, a number of pathogen-derived antigens, as well as allergens such as papain, belong to the family of cysteine proteases. Although the link between protease activity and Th2 type immunity is well documented, the mechanisms by which proteases regulate host immune responses are largely unknown. Here, we demonstrate that the cysteine proteases papain and bromelain selectively cleave the α subunit of the IL-3 receptor (IL-3Rα/CD123) on the surface of murine basophils. The decrease in CD123 expression on the cell surface, and the degradation of the extracellular domain of recombinant CD123 were dependent on the protease activity of papain and bromelain. Pre-treatment of murine basophils with papain resulted in inhibition of IL-3-IL-3R signaling and suppressed IL-3- but not thymic stromal lymphopoietin-induced expansion of basophils in vitro. Our unexpected findings illuminate a novel mechanism for the regulation of basophil functions by protease antigens. Because IL-3 plays pivotal roles in the activation and proliferation of basophils and in protective immunity against helminth parasites, pathogen-derived proteases might contribute to the pathogenesis of infections by regulating IL-3-mediated functions in basophils.

A treadmill exercise reactivates the signaling of the mammalian target of rapamycin (mTor) in the skeletal muscles of starved mice.

It has been well established that a starvation-induced decrease in insulin/IGF-I and serum amino acids effectively suppresses the mammalian target of rapamycin (mTor) signaling to induce autophagy, which is a major degradative cellular pathway in skeletal muscles. In this study, we investigated the systematic effects of exercise on the mTor signaling of skeletal muscles. Wild type C57BL/6J mice were starved for 24h under synchronous autophagy induction conditions. Under these conditions, endogenous LC3-II increased, while both S6-kinse and S6 ribosomal protein were dephosphorylated in the skeletal muscles, which indicated mTor inactivation. Using GFP-LC3 transgenic mice, it was also confirmed that fluorescent GFP-LC3 dots in the skeletal muscles increased, including soleus, plantaris, and gastrocnemius, which clearly showed autophagosomal induction. These starved mice were then subjected to a single bout of running on a treadmill (12m/min, 2h, with a lean of 10 degrees). Surprisingly, biochemical analyses revealed that the exercise elicited a decrease in the LC3-II/LC3-I ratio as well as an inversion from the dephosphorylated state to the rephosphorylated state of S6-kinase and ribosomal S6 in these skeletal muscles. Consistently, the GFP-LC3 dots of the skeletal muscles were diminished immediately after the exercise. These results indicated that exercise suppressed starvation-induced autophagy through a reactivation of mTor signaling in the skeletal muscles of these starved mice.

Galectin-4, a novel predictor for lymph node metastasis in lung adenocarcinoma.

Metastasis is still a major issue in cancer, and the discovery of biomarkers predicting metastatic capacity is essential for the development of better therapeutic strategies for treating lung adenocarcinoma. By using a proteomic approach, we aimed to identify novel predictors for lymph node metastasis in lung adenocarcinoma. Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis showed 6 spots differentially expressed between lymph node metastasis-positive and lymph node metastasis-negative groups in a discovery set. Subsequent mass spectrometry showed that 2 of these spots were derived from galectin-4, and western blot analysis confirmed the overexpression of galectin-4 in metastatic samples. The predictive value of galectin-4 was confirmed by immunohistochemical analysis for a validation set consisting of 707 surgically resected specimens of lung adenocarcinomas (stages I to IV). We observed that 148 lung adenocarcinomas (20.9%) expressed galectin-4, which was significantly associated with variables of disease progression such as tumor size (p<0.0001), pleural invasion (p = 0.0071), venous invasion (p = 0.0178), nodal status (p = 0.0007), and TNM stage (p<0.0001). By the multivariate analysis, Galectin-4 expression was revealed as one of the independent predictor for lymph node metastasis, together with solid predominant and micropapillary histologic pattern. Furthermore, galectin-4 expression was revealed to be an independent predictor for lymph node metastasis and an adverse survival factor in patients with lung adenocarcinoma of acinar predominant type. Galectin-4 plays an important role in metastatic process of lung adenocarcinoma. Immunohistochemical testing for galectin-4 expression may be useful together with the detection of specific histology to predict the metastatic potential of lung adenocarcinoma.

Lysine and arginine biosyntheses mediated by a common carrier protein in Sulfolobus.

LysW has been identified as a carrier protein in the lysine biosynthetic pathway that is active through the conversion of α-aminoadipate (AAA) to lysine. In this study, we found that the hyperthermophilic archaeon, Sulfolobus acidocaldarius, not only biosynthesizes lysine through LysW-mediated protection of AAA but also uses LysW to protect the amino group of glutamate in arginine biosynthesis. In this archaeon, after LysW modification, AAA and glutamate are converted to lysine and ornithine, respectively, by a single set of enzymes with dual functions. The crystal structure of ArgX, the enzyme responsible for modification and protection of the amino moiety of glutamate with LysW, was determined in complex with LysW. Structural comparison and enzymatic characterization using Sulfolobus LysX, Sulfolobus ArgX and Thermus LysX identify the amino acid motif responsible for substrate discrimination between AAA and glutamate. Phylogenetic analysis reveals that gene duplication events at different stages of evolution led to ArgX and LysX.

NP-1250, an ABCG2 inhibitor, induces apoptotic cell death in mitoxantrone-resistant breast carcinoma MCF7 cells via a caspase-independent pathway.

Chemoresistance is one of the main obstacles to successful cancer therapy and is frequently associated with multidrug resistance (MDR). One of the most studied mechanisms of MDR is the high expression of ATP-binding cassette (ABC) transporters. Here, we demonstrated that NP-1250, an ABCG2 inhibitor, induced apoptotic cell death in ABCG2-overexpressing multidrug-resistant MCF7/mitoxantrone-resistant (MX) human breast carcinoma cells via a caspase-independent pathway. Incubation of MCF7/MX cells with NP-1250 significantly reduced cell viability, while NP-1250 had little effect on the viability of drug-sensitive MCF7/wild-type cells. Although the target molecules of NP-1250 in cell death remain unknown, investigation of NP-1250 will aid in the elucidation of the molecular mechanism of drug resistance and NP-1250 may become a new therapy for MDR cancers.

Molecular interaction of the first 3 enzymes of the de novo pyrimidine biosynthetic pathway of Trypanosoma cruzi.

The first 3 reaction steps of the de novo pyrimidine biosynthetic pathway are catalyzed by carbamoyl-phosphate synthetase II (CPSII), aspartate transcarbamoylase (ATC), and dihydroorotase (DHO), respectively. In eukaryotes, these enzymes are structurally classified into 2 types: (1) a CPSII-DHO-ATC fusion enzyme (CAD) found in animals, fungi, and amoebozoa, and (2) stand-alone enzymes found in plants and the protist groups. In the present study, we demonstrate direct intermolecular interactions between CPSII, ATC, and DHO of the parasitic protist Trypanosoma cruzi, which is the causative agent of Chagas disease. The 3 enzymes were expressed in a bacterial expression system and their interactions were examined. Immunoprecipitation using an antibody specific for each enzyme coupled with Western blotting-based detection using antibodies for the counterpart enzymes showed co-precipitation of all 3 enzymes. From an evolutionary viewpoint, the formation of a functional tri-enzyme complex may have preceded-and led to-gene fusion to produce the CAD protein. This is the first report to demonstrate the structural basis of these 3 enzymes as a model of CAD. Moreover, in conjunction with the essentiality of de novo pyrimidine biosynthesis in the parasite, our findings provide a rationale for new strategies for developing drugs for Chagas disease, which target the intermolecular interactions of these 3 enzymes.

Glyceraldehyde-3-phosphate dehydrogenase interacts with phosphorylated Akt resulting from increased blood glucose in rat cardiac muscle.

Here we describe the interaction of phosphorylated approximately 40 kDa protein with phosphorylated Akt which is a serine/threonine kinase resulting from increased blood glucose in rat cardiac muscle. Mass spectrometry analysis revealed that this protein was glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Furthermore, increase in Akt and GAPDH phosporylation and induction of their association were both observed after insulin stimulation in the H9c2 cell line derived from embryonic rat ventricle. Moreover, the activation of GAPDH was upregulated when the GAPDH phosphorylation was increased. Our data suggest that GAPDH phosphorylation and association with Akt by insulin treatment have some bearing on the enhancement of GAPDH activity.

Serine 62 is a phosphorylation site in folliculin, the Birt-Hogg-Dubé gene product.

Recently, it was reported that the product of Birt-Hogg-Dubé syndrome gene (folliculin, FLCN) is directly phosphorylated by 5'-AMP-activated protein kinase (AMPK). In this study, we identified serine 62 (Ser62) as a phosphorylation site in FLCN and generated an anti-phospho-Ser62-FLCN antibody. Our analysis suggests that Ser62 phosphorylation is indirectly up-regulated by AMPK and that another residue is directly phosphorylated by AMPK. By binding with FLCN-interacting proteins (FNIP1 and FNIP2/FNIPL), Ser62 phosphorylation is increased. A phospho-mimic mutation at Ser62 enhanced the formation of the FLCN-AMPK complex. These results suggest that function(s) of FLCN-AMPK-FNIP complex is regulated by Ser62 phosphorylation.

Discovery of proteinaceous N-modification in lysine biosynthesis of Thermus thermophilus.

Although the latter portion of lysine biosynthesis, the conversion of alpha-aminoadipate (AAA) to lysine, in Thermus thermophilus is similar to the latter portion of arginine biosynthesis, enzymes homologous to ArgA and ArgJ are absent from the lysine pathway. Because ArgA and ArgJ are known to modify the amino group of glutamate to avoid intramolecular cyclization of intermediates, their absence suggests that the pathway includes an alternative N-modification system. We reconstituted the conversion of AAA to lysine and found that the amino group of AAA is modified by attachment to the gamma-carboxyl group of the C-terminal Glu54 of a small protein, LysW; that the side chain of AAA is converted to the lysyl side chain while still attached to LysW; and that lysine is subsequently liberated from the LysW-lysine fusion. The fact that biosynthetic enzymes recognize the acidic globular domain of LysW indicates that LysW acts as a carrier protein or protein scaffold for the biosynthetic enzymes. This study thus reveals the previously unknown function of a small protein in primary metabolism.

Novel mitochondrial complex II isolated from Trypanosoma cruzi is composed of 12 peptides including a heterodimeric Ip subunit.

Mitochondrial respiratory enzymes play a central role in energy production in aerobic organisms. They differentiated from the alpha-proteobacteria-derived ancestors by adding noncatalytic subunits. An exception is Complex II (succinate: ubiquinone reductase), which is composed of four alpha-proteobacteria-derived catalytic subunits (SDH1-SDH4). Complex II often plays a pivotal role in adaptation of parasites in host organisms and would be a potential target for new drugs. We purified Complex II from the parasitic protist Trypanosoma cruzi and obtained the unexpected result that it consists of six hydrophilic (SDH1, SDH2N, SDH2C, and SDH5-SDH7) and six hydrophobic (SDH3, SDH4, and SDH8-SDH11) nucleus-encoded subunits. Orthologous genes for each subunit were identified in Trypanosoma brucei and Leishmania major. Notably, the iron-sulfur subunit was heterodimeric; SDH2N and SDH2C contain the plant-type ferredoxin domain in the N-terminal half and the bacterial ferredoxin domain in the C-terminal half, respectively. Catalytic subunits (SDH1, SDH2N plus SDH2C, SDH3, and SDH4) contain all key residues for binding of dicarboxylates and quinones, but the enzyme showed the lower affinity for both substrates and inhibitors than mammalian enzymes. In addition, the enzyme binds protoheme IX, but SDH3 lacks a ligand histidine. These unusual features are unique in the Trypanosomatida and make their Complex II a target for new chemotherapeutic agents.

Multiple molecular interactions implicate the connectin/titin N2A region as a modulating scaffold for p94/calpain 3 activity in skeletal muscle.

p94/calpain 3 is a skeletal muscle-specific Ca(2+)-regulated cysteine protease (calpain), and genetic loss of p94 protease activity causes muscular dystrophy (calpainopathy). In addition, a small in-frame deletion in the N2A region of connectin/titin that impairs p94-connectin interaction causes a severe muscular dystrophy (mdm) in mice. Since p94 via its interaction with the N2A and M-line regions of connectin becomes part of the connectin filament system that serves as a molecular scaffold for the myofibril, it has been proposed that structural and functional integrity of the p94-connectin complex is essential for health and maintenance of myocytes. In this study, we have surveyed the interactions made by p94 and connectin N2A inside COS7 cells. This revealed that p94 binds to connectin at multiple sites, including newly identified loci in the N2A and PEVK regions of connectin. Functionally, p94-N2A interactions suppress p94 autolysis and protected connectin from proteolysis. The connectin N2A region also contains a binding site for the muscle ankyrin repeat proteins (MARPs), a protein family involved in the cellular stress responses. MARP2/Ankrd2 competed with p94 for binding to connectin and was also proteolyzed by p94. Intriguingly, a connectin N2A fragment with the mdm deletion possessed enhanced resistance to proteases, including p94, and its interaction with MARPs was weakened. Our data support a model in which MARP2-p94 signaling converges within the N2A connectin segment and the mdm deletion disrupts their coordination. These results also implicate the dynamic nature of connectin molecule as a regulatory scaffold of p94 functions.

Comprehensive proteomics analysis of autophagy-deficient mouse liver.

Autophagy is a bulk protein degradation system for the entire organelles and cytoplasmic proteins. Previously, we have shown the liver dysfunction by autophagy deficiency. To examine the pathological effect of autophagy deficiency, we examined protein composition and their levels in autophagy-deficient liver by the proteomic analysis. While impaired autophagy led to an increase in total protein mass, the protein composition was largely unchanged, consistent with non-selective proteins/organelles degradation of autophagy. However, a series of oxidative stress-inducible proteins, including glutathione S-transferase families, protein disulfide isomerase and glucose-regulated proteins were specifically increased in autophagy-deficient liver, probably due to enhanced gene expression, which is induced by accumulation of Nrf2 in the nuclei of mutant hepatocytes. Our results suggest that autophagy deficiency causes oxidative stress, and such stress might be the main cause of liver injury in autophagy-deficient liver.

A case of neonatal lupus erythematosus presenting delayed dilated cardiomyopathy with circulating autoantibody to annexin A6.

Patients with neonatal lupus erythematosus (NLE) often have congenital heart block with or without heart failure and are born to mothers who have anti-SS-A and/or anti-SS-B antibodies. NLE has been considered to result from the placental transmission of maternal autoantibodies into the fetal circulation causing myocardial damage. We report a case of NLE with congenital heart block who had undergone pacemaker implantation at the age of 17, and then developed dilated cardiomyopathy (DCM) at the age of 19, which is much later than in most other cases. The patient's mother was positive for anti-SS-A and anti-SS-B antibodies, whereas the patient was negative for both anti-SS-A and anti-SS-B antibodies. There were some autoantibodies against cell surface antigens of cardiac myocytes in the serum from the patient, and annexin A6 was identified as one of the autoantigens. This is the first report demonstrating that annexin A6 is involved in the myocardial injury in patients with NLE. The results indicate that inhibition of annexin A6 function may prevent autoantibody-mediated myocardial injury in at least some cases of DCM.

Dihydroorotate dehydrogenase arises from novel fused gene product with aspartate carbamoyltransferase in Bodo saliens.

The ACT-DHOD gene in the kinetoplastid Bodo saliens encodes aspartate carbamoyltransferase and dihydroorotate dehydrogenase, the second and fourth enzymes of pyrimidine biosynthesis. Although the single mRNA species yielded a 70-kDa ACT-DHOD protein, Western blotting with anti-DHOD-peptide antibody showed a major band of 35-kDa and minor bands. In-gel digestion and liquid chromatography-tandem mass (MS/MS) spectrometry showed that the 35-kDa band contained DHOD-specific polypeptides and an ACT-specific polypeptide, suggesting the occurrence of independent DHOD and ACT. Immunoprecipitation and MS/MS analysis identified a 70-kDa ACT-DHOD and a 35-kDa DHOD independently, and the N-terminal amino acid of 35-kDa DHOD was blocked. In vitro processing assay showed that recombinant ACT-DHOD was decreased by the B. saliens lysate, accompanying the appearance of 35-kDa DHOD and 35-kDa ACT. These results indicate that fused ACT-DHOD is the precursor to mature DHOD. Large amount of 35-kDa DHOD in B. saliens is discussed from a viewpoint of its physiological roles.

Detection of 6-nitrotryptophan in proteins by Western blot analysis and its application for peroxynitrite-treated PC12 cells.

We have previously reported on the formation of 6-nitrotryptophan by the reaction of reactive nitrogen species with a tryptophan residue in human Cu, Zn-superoxide dismutase (SOD) (F. Yamakura et al., J. Biochem. 138 (2005) 57-69). Here, we report on the preparation of anti-6-nitrotryptophan antiserum by using synthesized 6-nitrotryptophan-conjugated keyhole limpet hemocyanin as an antigen and the purification of the antibody by using a 6-nitrotryptophan-conjugated affinity column. The purified antibody was immunoreactive with 6-nitrotryptophan residue containing Cu, Zn-SOD but not immunoreactive with Cu, Zn-SOD, Mn-SOD, bovine serum albumin, and 3-nitrotyrosine residue containing Mn-SOD. Nitro group of 6-nitrotryptophan was reduced by sodium hydrosulfite to form 6-aminotryptophan as a major product. The reduced 6-nitrotryptophan residues lost its immunoreactivity with the antibody. We detected different immunoreactive bands between using antibody for 6-nitrotryptophan residues and that for 3-nitrotyrosine residues in crude extracts of neuron-like PC12 cells treated with peroxynitrite by a Western blot analysis. Western blot analysis for two-dimensional gel electrophoresis showed nine intensively stained immunoreactive spots for 6-nitrotryptophan residues in the peroxynitrite-treated PC12 cells, which were subjected to trypsin digestion and LC-ESI-MS/MS analysis. We identified M2 pyruvate kinase, elongation factor 2, mitochondrial aconitase, pyruvate carboxylase, and heat shock protein HSP90alpha as candidates for 6-nitrotryptophan residues containing proteins, with peptide coverage over 10%, in crude extracts of peroxynitrite-treated PC12 cells.

Proteome analysis of a rat liver nuclear insoluble protein fraction and localization of a novel protein, ISP36, to compartments in the interchromatin space.

A rat liver nuclear insoluble protein fraction was analyzed to investigate candidate proteins participating in nuclear architecture formation. Proteins were subjected to two-dimensional separation by reversed-phase HPLC in 60% formic acid and SDS/PAGE. The method produced good resolution of insoluble proteins. One hundred and thirty-eight proteins were separated, and 28 of these were identified. The identified proteins included one novel protein, seven known nuclear proteins and 12 known nuclear matrix proteins. The novel 36 kDa protein was further investigated for its subnuclear localization. The human ortholog of the protein was expressed in Escherichia coli and antibodies were raised against the recombinant protein. Exclusive localization of the protein to the nuclear insoluble protein fraction was confirmed by cell fractionation followed by immunoblotting. Immunostaining of mouse C3H cells suggested that the 36 kDa protein was a constituent of an insoluble macromolecular complex spread throughout the interchromatin space of the nucleus. The protein was designated 'interchromatin space protein of 36 kDa', ISP36.