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Transforming growth factor-β (TGF-β) activation is involved in various pathogeneses, such as fibrosis and malignancy. We previously showed that TGF-β was activated by serine protease plasma kallikrein-dependent digestion of latency-associated peptides (LAPs) and developed a method to detect LAP degradation products (LAP-DPs) in the liver and blood using specific monoclonal antibodies. Clinical studies have revealed that blood LAP-DPs are formed in the early stages of liver fibrosis. This study aimed to identify the cell source of LAP-DP formation during liver fibrosis. The N-terminals of LAP-DPs ending at residue Arg58 (R58) were stained in liver sections of a bile duct-ligated liver fibrosis model at 3 and 13 days. R58 LAP-DPs were detected in quiescent hepatic stellate cells at day 3 and in macrophages on day 13 after ligation of the bile duct. We then performed a detailed analysis of the axial localization of R58 signals in a single macrophage, visualized the cell membrane with the anti-CLEC4F antibody, and found R58 LAP-DPs surrounded by the membrane in phagocytosed debris that appeared to be dead cells. These findings suggest that in the early stages of liver fibrosis, TGF-β is activated on the membrane of stellate cells, and then the cells are phagocytosed after cell death

Inhaled nebulized interferon (IFN)-α and IFN-β have been shown to be effective in the management of coronavirus disease 2019 (COVID-19). We aimed to construct a virus-free rapid detection system for high-throughput screening of IFN-like compounds that induce viral RNA degradation and suppress the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We prepared a SARS-CoV-2 subreplicon RNA expression vector which contained the SARS-CoV-2 5′-UTR, the partial sequence of ORF1a, luciferase, nucleocapsid, ORF10, and 3′-UTR under the control of the cytomegalovirus promoter. The expression vector was transfected into Calu-3 cells and treated with IFN-α and the IFNAR2 agonist CDM-3008 (RO8191) for 3 days. SARS-CoV-2 subreplicon RNA degradation was subsequently evaluated based on luciferase levels. IFN-α and CDM-3008 suppressed SARS-CoV-2 subreplicon RNA in a dose-dependent manner, with IC50 values of 193 IU/mL and 2.54 μM, respectively. HeLa cells stably expressing SARS-CoV-2 subreplicon RNA were prepared and treated with the IFN-α and pan-JAK inhibitor Pyridone 6 or siRNA-targeting ISG20. IFN-α activity was canceled with Pyridone 6. The knockdown of ISG20 partially canceled IFN-α activity. Collectively, we constructed a virus-free rapid detection system to measure SARS-CoV-2 RNA suppression. Our data suggest that the SARS-CoV-2 subreplicon RNA was degraded by IFN-α-induced ISG20 exonuclease activity.

Primary hepatic tumors mainly include hepatocellular carcinoma (HCC), which is one of the most frequent causes of cancer‐related deaths worldwide. Thus far, HCC prognosis has remained extremely poor given the lack of effective treatments. Numerous studies have described the roles played by microRNAs (miRNAs) in cancer progression and the potential of these small noncoding RNAs for diagnostic or therapeutic applications. The current consensus supports the idea that direct repression of a wide range of oncogenes by a single key miRNA could critically affect the malignant properties of cancer cells in a synergistic manner. In this study, we aimed to investigate the oncogenes controlled by miR‐493‐5p, a major tumor suppressor miRNA that inactivates miR‐483‐3p oncomir in hepatic cancer cells. Using global gene expression analysis, we highlighted a set of candidate genes potentially regulated by miR‐493‐5p. In particular, the canonical MYCN protooncogene (MYCN) appeared to be an attractive target of miR‐493‐5p given its significant inhibition through 3′‐UTR targeting in miR‐493‐5p‐rescued HCC cells. We showed that MYCN was overexpressed in liver cancer cell lines and clinical samples from HCC patients. Notably, MYCN expression levels were inversely correlated with miR‐493‐5p in tumor tissues. We confirmed that MYCN knockdown mimicked the anticancer effect of miR‐493‐5p by inhibiting HCC cell growth and invasion, whereas MYCN rescue hindered miR‐493‐5p activity. In summary, miR‐493‐5p is a pivotal miRNA that modulates various oncogenes after its reexpression in liver cancer cells, suggesting that tumor suppressor miRNAs with a large spectrum of action could provide valuable tools for miRNA replacement therapies., Our study highlighted the MYCN protooncogene as a critical target of microRNA (miR)‐493‐5p tumor suppressor. We found that MYCN was overexpressed in hepatic cancer cells and that miR‐493‐5p negatively repressed MYCN at the posttranscriptional level. We confirmed that MYCN silencing mimicked the anticancer activity of miR‐493‐5p by inhibiting hepatic tumor cell growth and invasion.

Sepsis results in lethal organ malfunction due to dysregulated host response to infection, which is a condition with increasing prevalence worldwide. Transglutaminase 2 (TG2) is a crosslinking enzyme that forms a covalent bond between lysine and glutamine. TG2 plays important roles in diverse cellular processes, including extracellular matrix stabilization, cytoskeletal function, cell motility, adhesion, signal transduction, apoptosis, and cell survival. We have shown that the co-culture of Candida albicans and hepatocytes activates and induces the translocation of TG2 into the nucleus. In addition, the expression and activation of TG2 in liver macrophages was dramatically induced in the lipopolysaccharide-injected and cecal ligation puncture-operated mouse models of sepsis. Based on these findings and recently published research, we have reviewed the current understanding of the relationship between TG2 and sepsis. Following the genetic and pharmacological inhibition of TG2, we also assessed the evidence regarding the use of TG2 as a potential marker and therapeutic target in inflammation and sepsis.

Hepatitis B, a viral infectious disease caused by hepatitis B virus (HBV), is a life-threatening disease that leads liver cirrhosis and liver cancer. Because the current treatments for HBV, such as an interferon (IFN) formulation or nucleoside/nucleotide analogues, are not sufficient, the development of a more effective agent for HBV is urgent required. CDM-3008 (1, 2-(2,4-bis(trifluoromethyl)imidazo[1,2-a][1,8]naphthyridin-8-yl)-1,3,4-oxadiazole) (RO8191)) is a small molecule with an imidazo[1,2-a][1,8]naphthyridine scaffold that shows anti-HCV activity with an IFN-like effect. Here, we report that 1 was also effective for HBV, although the solubility and metabolic stability were insufficient for clinical use. Through the structure-activity relationship (SAR), we discovered that CDM-3032 (11, N-(piperidine-4-yl)-2,4-bis(trifluoromethyl)imidazo[1,2-a][1,8]naphthyridine-8-carboxamide hydrochloride) was more soluble than 1 (>30 mg/mL for 11 versus 0.92 mg/mL for 1). In addition, the half-life period of 11 was dramatically improved in both mouse and human hepatic microsomes (T1/2, >120 min versus 58.2 min in mouse, and >120 min versus 34.1 min in human, for 11 and 1, respectively).

Hypoxia-inducible factor 1 (HIF-1) is a promising drug target for cancer chemotherapy. In our screening program aimed at identifying new HIF-1 inhibitors by using a hypoxia-responsive luciferase reporter gene assay, KUSC-5001 containing the 1-alkyl-1H-pyrazole-3-carboxamide moiety was found as a potential hit molecule. During an extensive structure–activity relationship (SAR) study, we developed a more effective HIF-1 inhibitor KUSC-5037 (IC50 = 1.2 μM). Under hypoxic conditions, KUSC-5037 suppressed the HIF-1α (a regulatory subunit of HIF-1) mRNA, causing decreases in the gene expression of HIF-1 target genes such as carbonic anhydrase 9 (CA9) and vascular endothelial growth factor (VEGF) genes. Furthermore, by applying our fluorescent and bifunctional probes, ATP5B, a catalytic β subunit of mitochondrial FoF1-ATP synthase, was identified as a target protein of KUSC-5037. These results indicate that the derivatives of KUSC-5037 containing the 1-alkyl-1H-pyrazole-3-carboxamide moiety are promising lead molecules for the inhibition of HIF-1 signaling via FoF1-ATP synthase suppression.

Vasohibin-2 (VASH2) is a homolog of VASH1, an endothelium-derived angiogenesis inhibitor. Vasohibin-2 is mainly expressed in cancer cells, and has been implicated in the progression of cancer by inducing angiogenesis and tumor growth. Although VASH2 has been recently reported to be involved in epithelial-mesenchymal transition (EMT), its precise roles are obscure. The aim of the present study was to clarify the role of VASH2 in the EMT of cancer cells in relation to transforming growth factor-β (TGF-β) signaling, which is a major stimulator of EMT. Decreased expression of VASH2 in ovarian cancer cells significantly repressed the expression of TGF-β type I receptor, namely activin receptor-like kinase 5. Transforming growth factor-β1-induced phosphorylation of Smad2 and Smad3 was markedly decreased in VASH2 knockdown cells while the expression of Smad2 and Smad3 was unchanged. Accordingly, the responses to TGF-β1 shown by promoter assay and plasminogen activator inhibitor type 1 expression were significantly attenuated in VASH2 knockdown cells. Furthermore, knockdown of VASH2 in cancer cells abrogated the TGF-β1-induced reduced expression of epithelial markers including E-cadherin, and the elevated expression of mesenchymal markers including fibronectin, ZEB2, and Snail2, suggesting that endogenous VASH2 is required for TGF-β1-induced EMT. In accordance with these results, the effects of TGF-β1 on cell morphology, migration, invasion, and MMP2 expression were also abrogated when VASH2 was knocked down. These results indicate that VASH2 played a significant role in the EMT by modulating the TGF-β signaling. We propose that VASH2 would be a novel molecular target for the prevention of EMT in cancers.

Sepsis is the leading cause of death in hospitalized patients and is characterized by a dysregulated inflammatory response to infection and multiple organ failure, including the liver. Transglutaminase 2 (TG2) is a multifunctional enzyme that exhibits transamidase, GTPase, and integrin-binding activities and has opposing roles in the regulation of cell growth, differentiation, and apoptosis. TG2 plays both pathogenic and protective roles in liver diseases, revealing the need to examine the activities of TG2. Here, we introduced an ex vivo imaging approach to examine the in vivo transamidase activity of TG2 based on the combination of intraperitoneal injection of 5-biotinamidopentylamine (5BAPA), a biotinylated substrate for TG2, and fluorescent streptavidin staining in frozen liver sections. Increased 5BAPA signals was observed in the livers of lipopolysaccharide (LPS) and cecal ligation and puncture (CLP)-induced sepsis mice. Pharmacological inhibition of TG2 activity ameliorated LPS-induced liver injury. 5BAPA signals were observed in TG2-expressing and F4/80-positive midzonal macrophages, providing direct evidence that activated macrophages are the major cellular source of active TG2 in the livers of sepsis mice. Further studies focusing on the activation of 5BAPA-stained midzonal macrophages may improve understanding of the molecular pathophysiology and the development of therapeutic strategies for sepsis.

Dendritic filopodia are thin and long protrusions based on the actin filament, and they extend and retract as if searching for a target axon. When the dendritic filopodia establish contact with a target axon, they begin maturing into spines, leading to the formation of a synapse. Telencephalin (TLCN) is abundantly localized in dendritic filopodia and is gradually excluded from spines. Overexpression of TLCN in cultured hippocampal neurons induces dendritic filopodia formation. We showed that telencephalin strongly binds to an extracellular matrix molecule, vitronectin. Vitronectin-coated microbeads induced phagocytic cup formation on neuronal dendrites. In the phagocytic cup, TLCN, TLCN-binding proteins such as phosphorylated Ezrin/Radixin/Moesin (phospho-ERM), and F-actin are accumulated, which suggests that components of the phagocytic cup are similar to those of dendritic filopodia. Thus, we developed a method for purifying the phagocytic cup instead of dendritic filopodia. Magnetic polystyrene beads were coated with vitronectin, which is abundantly present in the culture medium of hippocampal neurons and which induces phagocytic cup formation on neuronal dendrites. After 24 h of incubation, the phagocytic cups were mildly solubilized with detergent and collected using a magnet separator. After washing the beads, the binding proteins were eluted and analyzed by silver staining and Western blotting. In the binding fraction, TLCN and actin were abundantly present. In addition, many proteins identified from the fraction were localized to the dendritic filopodia; thus, we named the binding fraction as the dendritic filopodia-rich fraction. This article describes details regarding the purification method for the dendritic filopodia-rich fraction.

Oral administration of nucleotide analogues and injection of interferon-α (IFNα) are used to achieve immediate suppression in replication of hepatitis B virus (HBV). Nucleotide analogs and IFNα inhibit viral polymerase activity and cause long-term eradication of the virus at least in part through removing covalently closed circular DNA (cccDNA) via induction of the APOBEC3 deaminases family of molecules, respectively. This study aimed to explore whether the orally administrable low molecular weight agent CDM-3008 (RO8191), which mimics IFNα through the binding to IFNα/β receptor 2 (IFNAR2) and the activation of the JAK/STAT pathway, can suppress HBV replication and reduce cccDNA levels. In primary cultured human hepatocytes, HBV DNA levels were decreased after CDM-3008-treatment in a dose-dependent manner with a half-maximal inhibitory concentration (IC50) value of 0.1 μM, and this was accompanied by significant reductions in cellular cccDNA levels, both HBeAg and HBsAg levels in the cell culture medium. Using a microarray we comprehensively analyzed and compared changes in gene (mRNA) expression in CDM-3008- and IFNα-treated primary cultured human hepatocytes. As reported previously, CDM-3008 mimicked the induction of genes that participate in the interferon signaling pathway. OAS1 and ISG20 mRNA expression was similarly enhanced by both CDM-3008 and IFNα. Thus, CDM-3008 could suppress pgRNA expression to show anti-HBV activity. APOBEC3F and 3G mRNA expression was also induced by CDM-3008 and IFNα treatments, suggesting that cccDNA could be degraded through induced APOBEC3 family proteins. We identified the genes whose expression was specifically enhanced in CDM-3008-treated cells compared to IFNα-treated cells. The expression of SOCS1, SOCS2, SOCS3, and CISH, which inhibit STAT activation, was enhanced in CDM-3008-treated cells suggesting that a feedback inhibition of the JAK/STAT pathway was enhanced in CDM-3008-treated cells compared to IFNα-treated cells. In addition, CDM-3008 showed an additive effect with a clinically-used nucleoside entecavir on inhibition of HBV replication. In summary, CDM-3008 showed anti-HBV activity through activation of the JAK/STAT pathway, inducing the expression of interferon-stimulated genes (ISGs), with greater feedback inhibition than IFNα., B型肝炎ウイルス抑制物質の作用機序解明 --新規抗B型肝炎治療薬の開発へ期待--. 京都大学プレスリリース. 2019-06-13.

Acute liver injury (ALI) is highly lethal acute liver failure caused by different etiologies. Transforming growth factor β (TGF-β) is a multifunctional cytokine and a well-recognized inducer of apoptotic and necrotic cell death in hepatocytes. Latent TGF-β is activated partly through proteolytic cleavage by a serine protease plasma kallikrein (PLK) between the R58 and L59 residues of its propeptide region. Recently, we developed a specific monoclonal antibody to detect the N-terminal side LAP degradation products ending at residue R58 (R58 LAP-DPs) that reflect PLK-dependent TGF-β activation. This study aimed to explore the potential roles of PLK-dependent TGF-β activation in the pathogenesis of ALI. We established a mouse ALI model via the injection of anti-Fas antibodies (Jo2) and observed increases in the TGF-β1 mRNA level, Smad3 phosphorylation, TUNEL-positive apoptotic hepatocytes and R58-positive cells in the liver tissues of Jo2-treated mice. The R58 LAP-DPs were observed in/around F4/80-positive macrophages, while macrophage depletion with clodronate liposomes partly alleviated the Jo2-induced liver injury. Blocking PLK-dependent TGF-β activation using either the serine proteinase inhibitor FOY305 or the selective PLK inhibitor PKSI-527 or blocking the TGF-β receptor-mediated signaling pathway using SB431542 significantly prevented Jo2-induced hepatic apoptosis and mortality. Furthermore, similar phenomena were observed in the mouse model of ALI with the administration of acetaminophen (APAP). In summary, R58 LAP-DPs reflecting PLK-dependent TGF-β activation may serve as a biomarker for ALI, and targeting PLK-dependent TGF-β activation has potential as a therapeutic strategy for ALI.

Liver fibrosis is linked to VEGF-induced angiogenesis. Overexpression of exogenous vasohibin-1, a feedback inhibitor of angiogenesis, has been reported to reduce liver fibrosis after bile duct ligation (BDL). To uncover the function of endogenous vasohibin-1, we performed BDL using vasohibin-1-deficient mice and analyzed liver fibrosis, injury, and angiogenesis. Liver fibrosis was induced by 14-days of BDL in both wild-type and vasohibin-1-deficient mice. The liver sections were stained with anti-CD31 to visualize endothelial cells and with Sirius red to observe fibrotic regions. Total RNAs were purified from the livers and expression of collagen I α1 mRNA was measured by quantitative PCR. Plasma ALT activity was determined to assess liver injury. Surprisingly, the same extents of increases were seen in anti-CD31 and Sirius red stainings, collagen I α1 mRNA expressions, hepatic hydroxyproline contents, and ALT activity after 14-days of BDL in both wild-type and vasohibin-1-deficient mice. There was unexpectedly no difference between these mice, suggesting that anti-fibrogenic and angiogenic activities of the endogenous vasohibin-1 might be masked in the normal liver at early stage of hepatic fibrosis in mice.

Dendritic filopodia are thin, long, and highly mobile protrusions functioning as spine precursors. By contrast with a wealth of knowledge on molecular profiles in spines, little is known about structural and functional proteins present in dendritic filopodia. To reveal the molecular constituents of dendritic filopodia, we developed a new method for biochemical preparation of proteins enriched in dendritic filopodia, by taking advantage of specific and strong binding between a dendritic filopodial membrane protein, telencephalin, and its extracellular matrix ligand, vitronectin. When vitronectin-coated magnetic microbeads were added onto cultured hippocampal neurons, phagocytic cup-like membrane protrusions were formed on dendrites through the binding to telencephalin. Magnetically purified membrane protrusion fraction was subjected to comprehensive mass spectrometric analysis and 319 proteins were identified, many of which were confirmed to be localized to dendritic filopodia. Thus, this study provides a useful resource for studying molecular mechanisms underlying dendritic development, synapse formation, and plasticity.

Transglutaminase 2 (TG2) localizes to the nucleus and induces apoptosis through a crosslinking inactivation of Sp1 in JHH-7 cells treated with acyclic retinoid. We screened an inhibitor suppressing transamidase activity in the nucleus without affecting transamidase activity itself. Phenosafranin was found to inhibit nuclear localization of EGFP-tagged TG2 and dose-dependently reduce nuclear transamidase activity without affecting the activity in a tube. We concluded that phenosafranin was a novel TG2 inhibitor capable of suppressing its nuclear localization.

Vitronectin (VN) is an extracellular matrix protein abundantly present in blood and a wide variety of tissues and plays important roles in a number of biological phenomena mainly through its binding to αV integrins. However, its definite function in the brain remains largely unknown. Here we report the identification of telencephalin (TLCN/ICAM-5) as a novel VN receptor on neuronal dendrites. VN strongly binds to TLCN, a unique neuronal member of the ICAM family, which is specifically expressed on dendrites of spiny neurons in the mammalian telencephalon. VN-coated microbeads induce the formation of phagocytic cup-like plasma membrane protrusions on dendrites of cultured hippocampal neurons and trigger the activation of TLCN-dependent intracellular signaling cascade including the phosphorylation of ezrin/radixin/moesin actin-binding proteins and recruitment of F-actin and phosphatidylinositol 4,5-bisphosphate for morphological transformation of the dendritic protrusions. These results suggest that the extracellular matrix molecule VN and its neuronal receptor TLCN play a pivotal role in the phosphorylation of ezrin/radixin/moesin proteins and the formation of phagocytic cup-like structures on neuronal dendrites.

Neurons are compartmentalized into two morphologically, molecularly, and functionally distinct domains: axons and dendrites, and precise targeting and localization of proteins within these domains are critical for proper neuronal functions. It has been reported that several members of the Rab family small GTPases that are key mediators of membrane trafficking, regulate axon-specific trafficking events, but little has been elucidated regarding the molecular mechanisms that underlie dendrite-specific membrane trafficking. Here we show that Rab17 regulates dendritic morphogenesis and postsynaptic development in mouse hippocampal neurons. Rab17 is localized at dendritic growth cones, shafts, filopodia, and mature spines, but it is mostly absent in axons. We also found that Rab17 mediates dendrite growth and branching and that it does not regulate axon growth or branching. Moreover, shRNA-mediated knockdown of Rab17 expression resulted in a dramatically reduced number of dendritic spines, probably because of impaired filopodia formation. These findings have revealed the first molecular link between membrane trafficking and dendritogenesis.

International audience; An extracellular form of the Ca2+-dependent protein-cross-linking enzyme transglutaminase (TGase) was demonstrated to be involved in the apical growth of Malus domestica pollen tube. Apple pollen transglutaminase and its substrates were co-localized within aggregates on the pollen tube surface, as determined by indirect immuno-fluorescence staining and the in situ cross-linking of fluorescently labeled substrates. Transglutaminase-specific inhibitors and an anti-TGase monoclonal antibody blocked pollen tube growth, whereas incorporation of a recombinant fluorescent mammalian TGase substrate (histidine-tagged green fluorescent protein: His6-Xpr-GFP) into the growing tube wall enhanced tube length and germination, consistent with a role of TGase as modulator of cell wall building and strengthening. The secreted pollen TGase catalyzed the cross-linking of both polyamines (PAs) into proteins (released by the pollen tube) and His6-Xpr-GFP into endogenous or exogenously added substrates. A similar distribution of TGase activity was observed in planta on pollen tubes germinating inside the style, consistent with a possible additional role for TGase in the interaction between the pollen tube and the style during fertilization.

We screened more than 60000 RIKEN mouse cDNAs for novel ECM (extracellular matrix) proteins by extensive computational screening followed by recombinant expression and immunohistochemical characterization. We identified two novel olfactomedin-family proteins characterized by the presence of tandem CXCXCX9C motifs in the N-terminal region, a coiled-coil domain and an olfactomedin domain in the C-terminal region. These proteins, named photomedin-1 and photomedin-2, were secreted as disulphide-bonded dimers (photomedin-1) or oligomers/multimers (photomedin-2) with O-linked carbohydrate chains, although photomedin-1 was proteolytically processed in the middle of the molecule after secretion. In the retina, photomedin-1 was selectively expressed in the outer segment of photoreceptor cells and photomedin-2 was expressed in all retinal neurons. Among a panel of ECM components, including glycosaminoglycans, photomedins preferentially bound to chondroitin sulphate-E and heparin. These results, together, indicate that photomedins are novel olfactomedin-domain-containing extracellular proteins capable of binding to proteoglycans containing these glycosaminoglycan chains.

Trappins are found in human, bovine, hippopotamus, and members of the pig family, but not in rat and mouse. To clarify the evolution of the trappin genes and the functional significance of their products, we isolated the trappin gene in guinea pig, a species belonging to a rodent family distinct from rat and mouse. Guinea pig trappin was confirmed to encode the same domain structure as trappin, consisting of a signal sequence, an extra large transglutaminase substrate domain, and a whey acidic protein motif. Northern blot analysis and in situ hybridization histochemistry as well as immunohistochemistry demonstrated that guinea pig trappin is expressed solely in the secretory epithelium of the seminal vesicle and that its expression is androgen-dependent. We confirmed that guinea pig trappin is cross-linked by prostate transglutaminase and that the whey acidic protein motif derived from guinea pig trappin has an inhibitory activity against leukocyte elastase. Genome sequence analysis showed that guinea pig trappin belongs to the family of REST (rapidly evolving seminal vesicle transcribed) genes.

We determined the cDNA and gene structures of guinea pig caltrin II, a unique member of the calcium transporter inhibitors containing a whey acidic protein (WAP) motif, and we established that it is a secretory protein with a potential 21-amino acid signal peptide in its N-terminus. Northern blot analysis and in situ hybridization histochemistry indicated that the expression of caltrin II is restricted to luminal epithelial cells in the seminal vesicles. Its message levels markedly decreased either after castration (and were restored by simultaneous administration of testosterone) or after treatment of the animals with estradiol, suggesting that the expression of caltrin II is androgen-dependent. Recombinant caltrin II had an elastase-inhibitor activity. Comparison of sequence between the caltrin II and related genes and their molecular evolutionary analyses revealed that caltrin II and seminal vesicle secretory proteins (SVPs) appear to be evolved from a common ancestor gene that is made by the fusion of semenogelin and trappin genes. Caltrin II and SVPs lost the transglutaminase substrate domain and the WAP motif, respectively, within a single exon, resulting in the exertion of different functions.

One of the unique characteristics of the transglutaminase (TG) family enzymes is multiple distribution of the members. It is known that TGs exist in the cytosol, plasma membrane, nucleus, and mitochondria, as well as outside cells, being associated with the extracellular matrix and being secreted into tissue fluids or blood circulation. Depending on where TGs locate, their molecular conformation is altered, and they exert multiple biological activities. Furthermore, their localization and thus their activities are changed in pathological situations, which is highly associated with progression of many diseases, including thrombotic disease, immune diseases including celiac disease, skin diseases, neurodegenerative diseases, hepatic diseases, fibrotic diseases and cancers.

Transglutaminase 2 (TG2) is primarily known as the most ubiquitously expressed member of the transglutaminase family with Ca2+-dependent protein crosslinking activity; however, this enzyme exhibits multiple additional functions through GTPase, cell adhesion, protein disulfide isomerase, kinase, and scaffold activities and is associated with cell growth, differentiation, and apoptosis. TG2 is found in the extracellular matrix, plasma membrane, cytosol, mitochondria, recycling endosomes, and nucleus, and its subcellular localization is an important determinant of its function. Depending upon the cell type and stimuli, TG2 changes its subcellular localization and biological activities, playing both anti- and pro-apoptotic roles. Increasing evidence indicates that the GTP-bound form of the enzyme (in its closed form) protects cells from apoptosis but that the transamidation activity of TG2 (in its open form) participates in both facilitating and inhibiting apoptosis. A difficulty in the study and understanding of this enigmatic protein is that opposing effects have been reported regarding its roles in the same physiological and/or pathological systems. These include neuroprotective or neurodegenerative effects, hepatic cell growth-promoting or hepatic cell death-inducing effects, exacerbating or having no effect on liver fibrosis, and anti- and pro-apoptotic effects on cancer cells. The reasons for these discrepancies have been ascribed to TG2’s multifunctional activities, genetic variants, conformational changes induced by the immediate environment, and differences in the genetic background of the mice used in each of the experiments. In this article, we first report that TG2 has opposing roles like the protagonist in the novel Dr. Jekyll and Mr. Hyde, followed by a summary of the controversies reported, and finally discuss the possible reasons for these discrepancies.

The C1q family is characterized by the C-terminally conserved globular C1q (gC1q) domain. Although more than 30 C1q family proteins have been identified in mammals, many of them remain ill-defined with respect to their molecular and biological properties. Here, we report on a novel C1q family protein specifically expressed in the central nervous system (CNS), which we designated neural C1q-like protein (nCLP) 2. nCLP2 was secreted as disulphide-bonded multimers comprising trimeric units. The multimers were stabilized by interchain disulphide bonds involving the cysteine residues in the N-terminal variable region and the C-terminal gC1q domain. The expression of nCLP2 was restricted to several brain regions and retina, including regions associated with memory formation (i.e. hippocampus, entorhinal cortex, anterodorsal thalamic nucleus). Immunoelectron microscopy revealed that nCLP2 was localized in the mossy fibre axons of hippocampal granule cells and their synaptic boutons and clefts, implying that nCLP2 was anterogradely transported in mossy fibres and secreted from the presynaptic termini. These results suggest that nCLP2 plays roles in synaptic function and maintenance in the CNS.

Extracellular matrix (ECM), which provides critical scaffolds for all adhesive cells, regulates proliferation, differentiation, and apoptosis. Different cell types employ customized ECMs, which are thought to play important roles in the generation of so-called niches that contribute to cell-specific functions. The molecular entities of these customized ECMs, however, have not been elucidated. Here, we describe a strategy for transcriptome-wide identification of ECM proteins based on computational screening of >60,000 full-length mouse cDNAs for secreted proteins, followed by in vitro functional assays. These assays screened the candidate proteins for ECM-assembling activities, interactions with other ECM molecules, modifications with glycosaminoglycans, and cell-adhesive activities, and were then complemented with immunohistochemical analysis. We identified 16 ECM proteins, of which seven were localized in basement membrane (BM) zones. The identification of these previously unknown BM proteins allowed us to construct a body map of BM proteins, which represents the comprehensive immunohistochemistry-based expression profiles of the tissue-specific customization of BMs.

Dendritic filopodia are long, thin, actin-rich, and dynamic protrusions that are thought to play a critical role as a precursor of spines during neural development. We reported previously that a telencephalon-specific cell adhesion molecule, telencephalin (TLCN) [intercellular adhesion molecule-5 (ICAM-5)], is highly expressed in dendritic filopodia, facilitates the filopodia formation, and slows spine maturation. Here we demonstrate that TLCN cytoplasmic region binds ERM (ezrin/radixin/moesin) family proteins that link membrane proteins to actin cytoskeleton. In cultured hippocampal neurons, phosphorylated active forms of ERM proteins are colocalized with TLCN in dendritic filopodia, whereas α-actinin, another binding partner of TLCN, is colocalized with TLCN at surface membranes of soma and dendritic shafts. Expression of constitutively active ezrin induces dendritic filopodia formation, whereas small interference RNA-mediated knockdown of ERM proteins decreases filopodia density and accelerates spine maturation. These results indicate the important role of TLCN–ERM interaction in the formation of dendritic filopodia, which leads to subsequent synaptogenesis and establishment of functional neural circuitry in the developing brain.

SUMMARY Histidine-tagged green fluorescent protein (His6-Xpress-GFP), a widely used fluorescent probe, was found to be a good substrate for transglutaminase, an enzyme that catalyzes covalent crosslinking of proteins. GFP alone did not serve as a substrate but its derivative His6-Xpress-GFP was readily crosslinked through the Gln and Lys residues present in the short N-terminal extension (His6-Xpress). His6-Xpress-GFP was sensitive enough to detect the transglutaminase activity in guinea pig liver homogenates. The fluorescent substrate could also be used for activity staining of transglutaminase on histological tissue sections, and such applications revealed a surprisingly wide distribution of transglutaminase in the body, especially in the extracellular matrices of various tissues, suggesting an important role for transglutaminase in maintaining the integrity of the extracellular matrix and connective tissues by crosslinking its constituent proteins.(J Histochem Cytochem 49:247–258, 2001)

Background Trappin is a multifunctional host-defense peptide that has antiproteolytic, antiinflammatory, and antimicrobial activities. The numbers and compositions of trappin paralogs vary among mammalian species: human and sheep have a single trappin-2 gene; mouse and rat have no trappin gene; pig and cow have multiple trappin genes; and guinea pig has a trappin gene and two other derivativegenes. Independent duplications of trappin genes in pig and cow were observed recently after the species were separated. To determine whether these trappin gene duplications are restricted only to certain mammalian lineages, we analyzed recently-developed genome databases for the presence of duplicate trappin genes. Results The database analyses revealed that: 1) duplicated trappin multigenes were found recently in the nine-banded armadillo; 2) duplicated two trappin genes had been found in the Afrotherian species (elephant, tenrec, and hyrax) since ancient days; 3) a single trappin-2 gene was found in various eutherians species; and 4) no typical trappin gene has been found in chicken, zebra finch, and opossum. Bayesian analysis estimated the date of the duplication of trappin genes in the Afrotheria, guinea pig, armadillo, cow, and pig to be 244, 35, 11, 13, and 3 million-years ago, respectively. The coding regions of trappin multigenes of almadillo, bovine, and pig evolved much faster than the noncoding exons, introns, and the flanking regions, showing that these genes have undergone accelerated evolution, and positive Darwinian selection was observed in pig-specific trappin paralogs. Conclusion These results suggest that trappin is an eutherian-specific molecule and eutherian genomes have the potential to form trappin multigenes.

The force curve measurement mode of an atomic force microscope was used to record the force required to stretch a protein molecule that was covalently sandwiched through gold-thiol bonds between a mica substrate and a silicon nitride tip, both coated with gold. In one experiment, 8 ±1 out of 20 lysyl residues of bovine carbonic anhydrase B were randomly derivatized to give free thiols, and grafted on an atomically flat gold (111) surface on mica. Force curves taken on the surface covered with protein molecules using a gold coated tip occasionally showed a large downward deflection indicating trapping and subsequent stretching of protein molecules between the tip and the substrate. In another experiment, the same protein was genetically engineered so that cysteine residues were introduced at both the amino and the carboxyl terminus. Force curves taken in a similar manner as in the first experiment indicated almost complete extension of a linear polypeptide chain. The result was explained in terms of extension of a pseudo-three-dimensional gel in both cases, with additional stretching of a linear chain in the second case.