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Sample records for distinct protease pathways

  1. Neuropeptidomics Mass Spectrometry Reveals Signaling Networks Generated by Distinct Protease Pathways in Human Systems

    NASA Astrophysics Data System (ADS)

    Hook, Vivian; Bandeira, Nuno

    2015-12-01

    Neuropeptides regulate intercellular signaling as neurotransmitters of the central and peripheral nervous systems, and as peptide hormones in the endocrine system. Diverse neuropeptides of distinct primary sequences of various lengths, often with post-translational modifications, coordinate and integrate regulation of physiological functions. Mass spectrometry-based analysis of the diverse neuropeptide structures in neuropeptidomics research is necessary to define the full complement of neuropeptide signaling molecules. Human neuropeptidomics has notable importance in defining normal and dysfunctional neuropeptide signaling in human health and disease. Neuropeptidomics has great potential for expansion in translational research opportunities for defining neuropeptide mechanisms of human diseases, providing novel neuropeptide drug targets for drug discovery, and monitoring neuropeptides as biomarkers of drug responses. In consideration of the high impact of human neuropeptidomics for health, an observed gap in this discipline is the few published articles in human neuropeptidomics compared with, for example, human proteomics and related mass spectrometry disciplines. Focus on human neuropeptidomics will advance new knowledge of the complex neuropeptide signaling networks participating in the fine control of neuroendocrine systems. This commentary review article discusses several human neuropeptidomics accomplishments that illustrate the rapidly expanding diversity of neuropeptides generated by protease processing of pro-neuropeptide precursors occurring within the secretory vesicle proteome. Of particular interest is the finding that human-specific cathepsin V participates in producing enkephalin and likely other neuropeptides, indicating unique proteolytic mechanisms for generating human neuropeptides. The field of human neuropeptidomics has great promise to solve new mechanisms in disease conditions, leading to new drug targets and therapeutic agents for human

  2. Neuropeptidomics Mass Spectrometry Reveals Signaling Networks Generated by Distinct Protease Pathways in Human Systems.

    PubMed

    Hook, Vivian; Bandeira, Nuno

    2015-12-01

    Neuropeptides regulate intercellular signaling as neurotransmitters of the central and peripheral nervous systems, and as peptide hormones in the endocrine system. Diverse neuropeptides of distinct primary sequences of various lengths, often with post-translational modifications, coordinate and integrate regulation of physiological functions. Mass spectrometry-based analysis of the diverse neuropeptide structures in neuropeptidomics research is necessary to define the full complement of neuropeptide signaling molecules. Human neuropeptidomics has notable importance in defining normal and dysfunctional neuropeptide signaling in human health and disease. Neuropeptidomics has great potential for expansion in translational research opportunities for defining neuropeptide mechanisms of human diseases, providing novel neuropeptide drug targets for drug discovery, and monitoring neuropeptides as biomarkers of drug responses. In consideration of the high impact of human neuropeptidomics for health, an observed gap in this discipline is the few published articles in human neuropeptidomics compared with, for example, human proteomics and related mass spectrometry disciplines. Focus on human neuropeptidomics will advance new knowledge of the complex neuropeptide signaling networks participating in the fine control of neuroendocrine systems. This commentary review article discusses several human neuropeptidomics accomplishments that illustrate the rapidly expanding diversity of neuropeptides generated by protease processing of pro-neuropeptide precursors occurring within the secretory vesicle proteome. Of particular interest is the finding that human-specific cathepsin V participates in producing enkephalin and likely other neuropeptides, indicating unique proteolytic mechanisms for generating human neuropeptides. The field of human neuropeptidomics has great promise to solve new mechanisms in disease conditions, leading to new drug targets and therapeutic agents for human

  3. Pathogen-secreted proteases activate a novel plant immune pathway.

    PubMed

    Cheng, Zhenyu; Li, Jian-Feng; Niu, Yajie; Zhang, Xue-Cheng; Woody, Owen Z; Xiong, Yan; Djonović, Slavica; Millet, Yves; Bush, Jenifer; McConkey, Brendan J; Sheen, Jen; Ausubel, Frederick M

    2015-05-14

    Mitogen-activated protein kinase (MAPK) cascades play central roles in innate immune signalling networks in plants and animals. In plants, however, the molecular mechanisms of how signal perception is transduced to MAPK activation remain elusive. Here we report that pathogen-secreted proteases activate a previously unknown signalling pathway in Arabidopsis thaliana involving the Gα, Gβ, and Gγ subunits of heterotrimeric G-protein complexes, which function upstream of an MAPK cascade. In this pathway, receptor for activated C kinase 1 (RACK1) functions as a novel scaffold that binds to the Gβ subunit as well as to all three tiers of the MAPK cascade, thereby linking upstream G-protein signalling to downstream activation of an MAPK cascade. The protease-G-protein-RACK1-MAPK cascade modules identified in these studies are distinct from previously described plant immune signalling pathways such as that elicited by bacterial flagellin, in which G proteins function downstream of or in parallel to an MAPK cascade without the involvement of the RACK1 scaffolding protein. The discovery of the new protease-mediated immune signalling pathway described here was facilitated by the use of the broad host range, opportunistic bacterial pathogen Pseudomonas aeruginosa. The ability of P. aeruginosa to infect both plants and animals makes it an excellent model to identify novel immunoregulatory strategies that account for its niche adaptation to diverse host tissues and immune systems.

  4. Targeting the AKT pathway: Repositioning HIV protease inhibitors as radiosensitizers.

    PubMed

    Goda, Jayant S; Pachpor, Tejaswini; Basu, Trinanjan; Chopra, Supriya; Gota, Vikram

    2016-02-01

    Cellular resistance in tumour cells to different therapeutic approaches has been a limiting factor in the curative treatment of cancer. Resistance to therapeutic radiation is a common phenomenon which significantly reduces treatment options and impacts survival. One of the mechanisms of acquiring resistance to ionizing radiation is the overexpression or activation of various oncogenes like the EGFR (epidermal growth factor receptor), RAS (rat sarcoma) oncogene or loss of PTEN (phosphatase and tensin homologue) which in turn activates the phosphatidyl inositol 3-kinase/protein kinase B (PI3-K)/AKT pathway responsible for radiation resistance in various tumours. Blocking the pathway enhances the radiation response both in vitro and in vivo. Due to the differential activation of this pathway (constitutively activated in tumour cells and not in the normal host cells), it is an excellent candidate target for molecular targeted therapy to enhance radiation sensitivity. In this regard, HIV protease inhibitors (HPIs) known to interfere with PI3-K/AKT signaling in tumour cells, have been shown to sensitize various tumour cells to radiation both in vitro and in vivo. As a result, HPIs are now being investigated as possible radiosensitizers along with various chemotherapeutic drugs. This review describes the mechanisms by which PI3-K/AKT pathway causes radioresistance and the role of HIV protease inhibitors especially nelfinavir as a potential candidate drug to target the AKT pathway for overcoming radioresistance and its use in various clinical trials for different malignancies.

  5. Targeting the AKT pathway: Repositioning HIV protease inhibitors as radiosensitizers

    PubMed Central

    Goda, Jayant S.; Pachpor, Tejaswini; Basu, Trinanjan; Chopra, Supriya; Gota, Vikram

    2016-01-01

    Cellular resistance in tumour cells to different therapeutic approaches has been a limiting factor in the curative treatment of cancer. Resistance to therapeutic radiation is a common phenomenon which significantly reduces treatment options and impacts survival. One of the mechanisms of acquiring resistance to ionizing radiation is the overexpression or activation of various oncogenes like the EGFR (epidermal growth factor receptor), RAS (rat sarcoma) oncogene or loss of PTEN (phosphatase and tensin homologue) which in turn activates the phosphatidyl inositol 3-kinase/protein kinase B (PI3-K)/AKT pathway responsible for radiation resistance in various tumours. Blocking the pathway enhances the radiation response both in vitro and in vivo. Due to the differential activation of this pathway (constitutively activated in tumour cells and not in the normal host cells), it is an excellent candidate target for molecular targeted therapy to enhance radiation sensitivity. In this regard, HIV protease inhibitors (HPIs) known to interfere with PI3-K/AKT signaling in tumour cells, have been shown to sensitize various tumour cells to radiation both in vitro and in vivo. As a result, HPIs are now being investigated as possible radiosensitizers along with various chemotherapeutic drugs. This review describes the mechanisms by which PI3-K/AKT pathway causes radioresistance and the role of HIV protease inhibitors especially nelfinavir as a potential candidate drug to target the AKT pathway for overcoming radioresistance and its use in various clinical trials for different malignancies. PMID:27121513

  6. Differential Disruption of Nucleocytoplasmic Trafficking Pathways by Rhinovirus 2A Proteases.

    PubMed

    Watters, Kelly; Inankur, Bahar; Gardiner, Jaye C; Warrick, Jay; Sherer, Nathan M; Yin, John; Palmenberg, Ann C

    2017-04-15

    The RNA rhinoviruses (RV) encode 2A proteases (2A(pro)) that contribute essential polyprotein processing and host cell shutoff functions during infection, including the cleavage of Phe/Gly-containing nucleoporin proteins (Nups) within nuclear pore complexes (NPC). Within the 3 RV species, multiple divergent genotypes encode diverse 2A(pro) sequences that act differentially on specific Nups. Since only subsets of Phe/Gly motifs, particularly those within Nup62, Nup98, and Nup153, are recognized by transport receptors (karyopherins) when trafficking large molecular cargos through the NPC, the processing preferences of individual 2A(pro) predict RV genotype-specific targeting of NPC pathways and cargos. To test this idea, transformed HeLa cell lines were created with fluorescent cargos (mCherry) for the importin α/β, transportin 1, and transportin 3 import pathways and the Crm1-mediated export pathway. Live-cell imaging of single cells expressing recombinant RV 2A(pro) (A16, A45, B04, B14, B52, C02, and C15) showed disruption of each pathway with measurably different efficiencies and reaction rates. The B04 and B52 proteases preferentially targeted Nups in the import pathways, while B04 and C15 proteases were more effective against the export pathway. Virus-type-specific trends were also observed during infection of cells with A16, B04, B14, and B52 viruses or their chimeras, as measured by NF-κB (p65/Rel) translocation into the nucleus and the rates of virus-associated cytopathic effects. This study provides new tools for evaluating the host cell response to RV infections in real time and suggests that differential 2A(pro) activities explain, in part, strain-dependent host responses and diverse RV disease phenotypes.IMPORTANCE Genetic variation among human rhinovirus types includes unexpected diversity in the genes encoding viral proteases (2A(pro)) that help these viruses achieve antihost responses. When the enzyme activities of 7 different 2A(pro) were measured

  7. Bacterial protease uses distinct thermodynamic signatures for substrate recognition.

    PubMed

    Bezerra, Gustavo Arruda; Ohara-Nemoto, Yuko; Cornaciu, Irina; Fedosyuk, Sofiya; Hoffmann, Guillaume; Round, Adam; Márquez, José A; Nemoto, Takayuki K; Djinović-Carugo, Kristina

    2017-06-06

    Porphyromonas gingivalis and Porphyromonas endodontalis are important bacteria related to periodontitis, the most common chronic inflammatory disease in humans worldwide. Its comorbidity with systemic diseases, such as type 2 diabetes, oral cancers and cardiovascular diseases, continues to generate considerable interest. Surprisingly, these two microorganisms do not ferment carbohydrates; rather they use proteinaceous substrates as carbon and energy sources. However, the underlying biochemical mechanisms of their energy metabolism remain unknown. Here, we show that dipeptidyl peptidase 11 (DPP11), a central metabolic enzyme in these bacteria, undergoes a conformational change upon peptide binding to distinguish substrates from end products. It binds substrates through an entropy-driven process and end products in an enthalpy-driven fashion. We show that increase in protein conformational entropy is the main-driving force for substrate binding via the unfolding of specific regions of the enzyme ("entropy reservoirs"). The relationship between our structural and thermodynamics data yields a distinct model for protein-protein interactions where protein conformational entropy modulates the binding free-energy. Further, our findings provide a framework for the structure-based design of specific DPP11 inhibitors.

  8. Distinct roles for Arabidopsis SUMO protease ESD4 and its closest homolog ELS1

    PubMed Central

    Hermkes, Rebecca; Fu, Yong-Fu; Nürrenberg, Kerstin; Budhiraja, Ruchika; Schmelzer, Elmon; Elrouby, Nabil; Dohmen, R. Jürgen; Coupland, George

    2010-01-01

    SUMO conjugation affects a broad range of processes in Arabidopsis thaliana, including flower initiation, pathogen defense, and responses to cold, drought and salt stress. We investigated two sequence-related SUMO-specific proteases that are both widely expressed and show that they differ significantly in their properties. The closest homolog of SUMO protease ESD4, ESD4-LIKE SUMO PROTEASE 1 (ELS1, alternatively called AtULP1a) has SUMO-specific proteolytic activity, but is functionally distinct from ESD4, as shown by intracellular localization, mutant phenotype and heterologous expression in yeast mutants. Furthermore, we show that the growth defects caused by loss of ESD4 function are not due to increased synthesis of the stress signal salicylic acid, as was previously shown for a SUMO ligase, indicating that impairment of the SUMO system affects plant growth in different ways. Our results demonstrate that two A. thaliana SUMO proteases showing close sequence similarity have distinct in vivo functions. Electronic supplementary material The online version of this article (doi:10.1007/s00425-010-1281-z) contains supplementary material, which is available to authorized users. PMID:20922545

  9. Proteases.

    PubMed

    Barrett, A J

    2001-05-01

    The processes of growth and remodeling of cells and tissues in multicellular organisms require the breakdown of old protein molecules, in concert with the synthesis of new ones. For example, many newly-synthesized molecules require proteolytic processing to convert them to biologically active forms. Proteolysis can terminate the activity of a protein--e.g., capsases mediate apoptosis, which is a vital step in the life cycle of the cell. Proteolysis contributes to defense systems too, as the recognition of peptide fragments of foreign proteins triggers the immune response. Proteases are the class of enzymes involved in these important reactions. This unit discusses the general categories of proteases, and sets the stage for addition of overview units on cysteine proteases, aspartic proteases, and metalloproteases, as well as protocol units featuring techniques for analyzing mammalian and yeast proteasomes and protease inhibitors, among other topics.

  10. Distinct and stage specific nuclear factors regulate the expression of falcipains, Plasmodium falciparum cysteine proteases

    PubMed Central

    Sunil, Sujatha; Chauhan, Virander S; Malhotra, Pawan

    2008-01-01

    Background Plasmodium falciparum cysteine proteases (falcipains) play indispensable roles in parasite infection and development, especially in the process of host erythrocyte rupture/invasion and hemoglobin degradation. No detailed molecular analysis of transcriptional regulation of parasite proteases especially cysteine proteases has yet been reported. In this study, using a combination of transient transfection assays and electrophoretic mobility shift assays (EMSA), we demonstrate the presence of stage specific nuclear factors that bind to unique sequence elements in the 5'upstream regions of the falcipains and probably modulate the expression of cysteine proteases. Results Falcipains differ in their timing of expression and exhibit ability to compensate each other's functions at asexual blood stages of the parasite. Present study was undertaken to study the transcriptional regulation of falcipains. Transient transfection assay employing firefly luciferase as a reporter revealed that a ~1 kb sequence upstream of translational start site is sufficient for the functional transcriptional activity of falcipain-1 gene, while falcipain-2, -2' and -3 genes that exist within 12 kb stretch on chromosome 11 require ~2 kb upstream sequences for the expression of reporter luciferase activity. EMSA analysis elucidated binding of distinct nuclear factors to specific sequences within the 5'upstream regions of falcipain genes. Analysis of falcipains' 5'upstream regulatory regions did not reveal the presence of sequences known to bind general eukaryotic factors. However, we did find parasite specific sequence elements such as poly(dA) poly(dT) tracts, CCAAT boxes and a single 7 bp-G rich sequence, (A/G)NGGGG(C/A) in the 5' upstream regulatory regions of these genes, thereby suggesting the role(s) of Plasmodium specific transcriptional factors in the regulation of falcipain genes. Conclusion Taken together, these results suggest that expression of Plasmodium cysteine proteases is

  11. Vestibular thalamus: Two distinct graviceptive pathways.

    PubMed

    Baier, Bernhard; Conrad, Julian; Stephan, Thomas; Kirsch, Valerie; Vogt, Thomas; Wilting, Janine; Müller-Forell, Wibke; Dieterich, Marianne

    2016-01-12

    To determine whether there are distinct thalamic regions statistically associated with either contraversive or ipsiversive disturbance of verticality perception measured by subjective visual vertical (SVV). We used modern statistical lesion behavior mapping on a sample of 37 stroke patients with isolated thalamic lesions to clarify which thalamic regions are involved in graviceptive otolith processing and whether there are distinct regions associated with contraversive or ipsiversive SVV deviation. We found 2 distinct systems of graviceptive processing within the thalamus. Contraversive tilt of SVV was associated with lesions to the nuclei dorsomedialis, intralamellaris, centrales thalami, posterior thalami, ventrooralis internus, ventrointermedii, ventrocaudales and superior parts of the nuclei parafascicularis thalami. The regions associated with ipsiversive tilt of SVV were located in more inferior regions, involving structures such as the nuclei endymalis thalami, inferior parts of the nuclei parafascicularis thalami, and also small parts of the junction zone of the nuclei ruber tegmenti and brachium conjunctivum. Our data indicate that there are 2 anatomically distinct graviceptive signal processing mechanisms within the vestibular network in humans that lead, when damaged, to a vestibular tone imbalance either to the contraversive or to the ipsiversive side. © 2015 American Academy of Neurology.

  12. Signaling pathways induced by serine proteases to increase intestinal epithelial barrier function

    PubMed Central

    Shang, Judie; Dion, Sébastien P.; Désilets, Antoine; Leduc, Richard

    2017-01-01

    Changes in barrier function of the gastrointestinal tract are thought to contribute to the inflammatory bowel diseases Crohn’s disease and ulcerative colitis. Previous work in our lab demonstrated that apical exposure of intestinal epithelial cell lines to serine proteases results in an increase in transepithelial electrical resistance (TER). However, the underlying mechanisms governing this response are unclear. We aimed to determine the requirement for proteolytic activity, epidermal growth factor receptor (EGFR) activation, and downstream intracellular signaling in initiating and maintaining enhanced barrier function following protease treatment using a canine intestinal epithelial cell line (SCBN). We also examined the role of phosphorylation of myosin regulatory light chain on the serine protease-induced increase in TER through. It was found that proteolytic activity of the serine proteases trypsin and matriptase is required to initiate and maintain the protease-mediated increase in TER. We also show that MMP-independent EGFR activation is essential to the sustained phase of the protease response, and that Src kinases may mediate EGFR transactivation. PI3-K and ERK1/2 signaling were important in reaching a maximal increase in TER following protease stimulation; however, their upstream activators are yet to be determined. CK2 inhibition prevented the increase in TER induced by serine proteases. The bradykinin B(2) receptor was not involved in the change in TER in response to serine proteases, and no change in phosphorylation of MLC was observed after trypsin or matriptase treatment. Taken together, our data show a requirement for ongoing proteolytic activity, EGFR transactivation, as well as downstream PI3-K, ERK1/2, and CK2 signaling in protease-mediated barrier enhancement of intestinal epithelial cells. The pathways mediating enhanced barrier function by proteases may be novel therapeutic targets for intestinal disorders characterized by disrupted

  13. Signaling pathways induced by serine proteases to increase intestinal epithelial barrier function.

    PubMed

    Lahey, Kelcie A; Ronaghan, Natalie J; Shang, Judie; Dion, Sébastien P; Désilets, Antoine; Leduc, Richard; MacNaughton, Wallace K

    2017-01-01

    Changes in barrier function of the gastrointestinal tract are thought to contribute to the inflammatory bowel diseases Crohn's disease and ulcerative colitis. Previous work in our lab demonstrated that apical exposure of intestinal epithelial cell lines to serine proteases results in an increase in transepithelial electrical resistance (TER). However, the underlying mechanisms governing this response are unclear. We aimed to determine the requirement for proteolytic activity, epidermal growth factor receptor (EGFR) activation, and downstream intracellular signaling in initiating and maintaining enhanced barrier function following protease treatment using a canine intestinal epithelial cell line (SCBN). We also examined the role of phosphorylation of myosin regulatory light chain on the serine protease-induced increase in TER through. It was found that proteolytic activity of the serine proteases trypsin and matriptase is required to initiate and maintain the protease-mediated increase in TER. We also show that MMP-independent EGFR activation is essential to the sustained phase of the protease response, and that Src kinases may mediate EGFR transactivation. PI3-K and ERK1/2 signaling were important in reaching a maximal increase in TER following protease stimulation; however, their upstream activators are yet to be determined. CK2 inhibition prevented the increase in TER induced by serine proteases. The bradykinin B(2) receptor was not involved in the change in TER in response to serine proteases, and no change in phosphorylation of MLC was observed after trypsin or matriptase treatment. Taken together, our data show a requirement for ongoing proteolytic activity, EGFR transactivation, as well as downstream PI3-K, ERK1/2, and CK2 signaling in protease-mediated barrier enhancement of intestinal epithelial cells. The pathways mediating enhanced barrier function by proteases may be novel therapeutic targets for intestinal disorders characterized by disrupted epithelial

  14. Regulation of distinct pools of amyloid β-protein by multiple cellular proteases

    PubMed Central

    2013-01-01

    Alzheimer’s disease (AD) is a progressive, age-related neurodegenerative disorder characterized by extracellular and intracellular deposition of the amyloid β-protein (Aβ). The study of rare, familial forms of AD has shown that sustained elevations in the production of Aβ (either all forms or specific pathogenic variants thereof) are sufficient to trigger the full spectrum of cognitive and histopathological features of the disease. Although the exact cause or causes remain unknown, emerging evidence suggests that impairments in the clearance of Aβ, after it is produced, may underlie the vast majority of sporadic AD cases. This review focuses on Aβ-degrading proteases (AβDPs), which have emerged as particularly important mediators of Aβ clearance. A wide variety of proteases that – by virtue of their particular regional and subcellular localization profiles – define distinct pools of Aβ have been identified. Different pools of Aβ, in turn, may contribute differentially to the pathogenesis of the disease. The study of individual AβDPs, therefore, promises to offer new insights into the mechanistic basis of AD pathogenesis and, ultimately, may facilitate the development of effective methods for its prevention or treatment or both. PMID:23953275

  15. Crystal structures of the viral protease Npro imply distinct roles for the catalytic water in catalysis.

    PubMed

    Zögg, Thomas; Sponring, Michael; Schindler, Sabrina; Koll, Maria; Schneider, Rainer; Brandstetter, Hans; Auer, Bernhard

    2013-06-04

    Npro is a key effector protein of pestiviruses such as bovine viral diarrhea virus and abolishes host cell antiviral defense mechanisms. Synthesized as the N-terminal part of the viral polyprotein, Npro releases itself via an autoproteolytic cleavage, triggering its immunological functions. However, the mechanisms of its proteolytic action and its immune escape were unclear. Here, we present the crystal structures of Npro to 1.25 Å resolution. Structures of pre- and postcleavage intermediates identify three catalytically relevant elements. The trapping of the putative catalytic water reveals its distinct roles as a base, acid, and nucleophile. The presentation of the substrate further explains the enigmatic latency of the protease, ensuring a single in cis cleavage. Additionally, we identified a zinc-free, disulfide-linked conformation of the TRASH motif, an interaction hub of immune factors. The structure opens additional opportunities in utilizing Npro as an autocleaving fusion protein and as a pharmaceutical target.

  16. Distinct contribution of Toxoplasma gondii rhomboid proteases 4 and 5 to micronemal protein protease 1 activity during invasion.

    PubMed

    Rugarabamu, George; Marq, Jean-Baptiste; Guérin, Amandine; Lebrun, Maryse; Soldati-Favre, Dominique

    2015-07-01

    Host cell entry by the Apicomplexa is associated with the sequential secretion of invasion factors from specialized apical organelles. Secretion of micronemal proteins (MICs) complexes by Toxoplasma gondii facilitates parasite gliding motility, host cell attachment and entry, as well as egress from infected cells. The shedding of MICs during these steps is mediated by micronemal protein proteases MPP1, MPP2 and MPP3. The constitutive activity of MPP1 leads to the cleavage of transmembrane MICs and is linked to the surface rhomboid protease 4 (ROM4) and possibly to rhomboid protease 5 (ROM5). To determine their importance and respective contribution to MPP1 activity, in this study ROM4 and ROM5 genes were abrogated using Cre-recombinase and CRISPR-Cas9 nuclease, respectively, and shown to be dispensable for parasite survival. Parasites lacking ROM4 predominantly engage in twirling motility and exhibit enhanced attachment and impaired invasion, whereas intracellular growth and egress is not affected. The substrates MIC2 and MIC6 are not cleaved in rom4-ko parasites, in contrast, intramembrane cleavage of AMA1 is reduced but not completely abolished. Shedding of MICs and invasion are not altered in the absence of ROM5; however, this protease responsible for the residual cleavage of AMA1 is able to cleave other AMA family members and exhibits a detectable contribution to invasion in the absence of ROM4.

  17. Cockroach induces inflammatory responses through protease-dependent pathways.

    PubMed

    Wada, Kota; Matsuwaki, Yoshinori; Moriyama, Hiroshi; Kita, Hirohito

    2011-01-01

    Exposure to cockroaches is a major risk factor for asthma. Products from cockroaches may contain proteases and ligands for pattern recognition receptors. These molecules may activate airway inflammatory cells, such as eosinophils, that are involved in asthma. Among inner-city children, cockroach allergens play an especially important role in increasing asthma morbidity. The molecular mechanism for this association between cockroach exposure and asthma is not fully understood. Enzymatic activities from cockroaches activate inflammatory cells in the airways and may also exacerbate certain human airway diseases, such as asthma. We recently reported that cockroach extracts contain pepstatin A-sensitive proteases that activate PAR-2 and induce activation and degranulation of human eosinophils. This review focuses on the effects of cockroach on various inflammatory cells, including eosinophils, epithelial cells, fibroblasts, dendritic cells, and T cells, in allergic reactions.

  18. Photostability and photodegradation pathways of distinctive pesticides.

    PubMed

    Kiss, Attila; Virág, Diána

    2009-01-01

    Transformation of pesticides in the environment is a highly complex process affected by different factors. Biological and physical-chemical factors may play a role in the degradation to variable extent. Photodecomposition might be regarded as one of the most crucial factors affecting the fate of pesticides. Therefore, our study focused on revealing specific details of the photolytic degradation of pesticides. The toxicity of the examined pesticides is well known; however, little information is available regarding their natural degradation processes. More detailed examinations are required to reveal the exact mechanism of the pesticide decomposition and the biological impacts of the degradates. Significance of this study is enhanced by the fact that decomposition of pesticides may result in the formation of toxic degradation products. The photolytic degradation of frequently applied pesticides (e.g., acetochlor, simazine, chlorpyrifos, and carbendazim) with different chemical structures was investigated. An immersible ultraviolet light source was applied to induce photodegradation. The degradation processes were followed by thin-layer chromatography and gas chromatography/mass spectrometry techniques. Electron ionization mass spectrometry was used to identify the degradation species. Detailed mechanisms of photolytic transformation were established by identification of each degradate. The photolytic degradation of pesticides of distinctive chemical character exhibited markedly different photodecomposition mechanisms. At least four degradation species were detected and identified in each case. Loss of alkyl, chloro, and hydroxyl groups as well as cleavage of alkyloxy, amide, amino-alkyl, and ester bonds might be regarded as typical decomposition patterns. Deamination and ring opening might be observed at the last stages of decomposition.

  19. AAA+ proteases and their role in distinct stages along the Vibrio cholerae lifecycle.

    PubMed

    Pressler, Katharina; Vorkapic, Dina; Lichtenegger, Sabine; Malli, Gerald; Barilich, Benjamin P; Cakar, Fatih; Zingl, Franz G; Reidl, Joachim; Schild, Stefan

    2016-09-01

    The facultative human pathogen Vibrio cholerae has to adapt to different environmental conditions along its lifecycle by means of transcriptional, translational and post-translational regulation. This study provides a first comprehensive analysis regarding the contribution of the cytoplasmic AAA+ proteases Lon, ClpP and HslV to distinct features of V. cholerae behaviour, including biofilm formation, motility, cholera toxin expression and colonization fitness in the mouse model. While absence of HslV did not yield to any altered phenotype compared to wildtype, absence of Lon or ClpP resulted in significantly reduced colonization in vivo. In addition, a Δlon deletion mutant showed altered biofilm formation and increased motility, which could be correlated with higher expression of V. cholerae flagella gene class IV. Concordantly, we could show by immunoblot analysis, that Lon is the main protease responsible for proteolytic control of FliA, which is required for class IV flagella gene transcription, but also downregulates virulence gene expression. FliA becomes highly sensitive to proteolytic degradation in absence of its anti-sigma factor FlgM, a scenario reported to occur during mucosal penetration due to FlgM secretion through the broken flagellum. Our results confirm that the high stability of FliA in the absence of Lon results in less cholera toxin and toxin corgulated pilus production under virulence gene inducing conditions and in the presence of a damaged flagellum. Thus, the data presented herein provide a molecular explanation on how V. cholerae can achieve full expression of virulence genes during early stages of colonization, despite FliA getting liberated from the anti-sigma factor FlgM. Copyright © 2016 Elsevier GmbH. All rights reserved.

  20. A plasma proteolysis pathway comprising blood coagulation proteases.

    PubMed

    Yang, Lu; Li, Yun; Bhattacharya, Arup; Zhang, Yuesheng

    2016-07-05

    Coagulation factors are essential for hemostasis. Here, we show that these factors also team up to degrade plasma proteins that are unrelated to hemostasis. Prolidase, SRC and amyloid β1-42 (Aβ1-42) are used as probes. Each probe, upon entering the blood circulation, binds and activates factor XII (FXII), triggering the intrinsic and common coagulation cascades, which in turn activate factor VII, a component of the extrinsic coagulation cascade. Activated factor VII (FVIIa) rapidly degrades the circulating probes. Therefore, FXII and FVIIa serve as the sensor/initiator and executioner, respectively, for the proteolysis pathway. Moreover, activation of this pathway by one probe leads to the degradation of all three probes. Significant activation of this pathway follows tissue injury and may also occur in other disorders, e.g., Alzheimer's disease, of which Aβ1-42 is a key driver. However, enoxaparin, a clinically used anticoagulant, inhibits the proteolysis pathway and elevates plasma levels of the probes. Enoxaparin may also mitigate potential impact of activators of the proteolysis pathway on coagulation. Our results suggest that the proteolysis pathway is important for maintaining low levels of various plasma proteins. Our finding that enoxaparin inhibits this pathway provides a means to control it. Inhibition of this pathway may facilitate the development of disease biomarkers and protein therapeutics, e.g., plasma Aβ1-42 as a biomarker of Alzheimer's disease or recombinant human prolidase as an antitumor agent.

  1. Proteases Inhibition Assessment on PC12 and NGF Treated Cells after Oxygen and Glucose Deprivation Reveals a Distinct Role for Aspartyl Proteases

    PubMed Central

    Kritis, Aristidis; Pourzitaki, Chryssa; Klagas, Ioannis; Chourdakis, Michael; Albani, Maria

    2011-01-01

    Hypoxia is a severe stressful condition and induces cell death leading to neuronal loss both to the developing and adult nervous system. Central theme to cellular death is the activation of different classes of proteases such as caspases calpains and cathepsins. In the present study we investigated the involvement of these proteases, in the hypoxia-induced PC12 cell death. Rat PC12 is a model cell line for experimentation relevant to the nervous system and several protocols have been developed for either lethal hypoxia (oxygen and glucose deprivation OGD) or ischemic preconditioning (IPS). Nerve Growth Factor (NGF) treated PC12 differentiate to a sympathetic phenotype, expressing neurites and excitability. Lethal hypoxia was established by exposing undifferentiated and NGF-treated PC12 cells to a mixture of N2/CO2 (93:5%) in DMEM depleted of glucose and sodium pyruvate for 16 h. The involvement of caspases, calpains and lysosomal cathepsins D and E to the cell death induced by lethal OGD was investigated employing protease specific inhibitors such as z-VAD-fmk for the caspases, MDL28170 for the calpains and pepstatin A for the cathepsins D and E. Our findings show that pepstatin A provides statistically significant protection from cell death of both naive and NGF treated PC12 cells exposed to lethal OGD. We propose that apart from the established processes of apoptosis and necrosis that are integral components of lethal OGD, the activation of cathepsins D and E launches additional cell death pathways in which these proteases are key partners. PMID:22028798

  2. Identification, functional expression and enzymic analysis of two distinct CaaX proteases from Caenorhabditis elegans.

    PubMed

    Cadiñanos, Juan; Schmidt, Walter K; Fueyo, Antonio; Varela, Ignacio; López-Otín, Carlos; Freije, José M P

    2003-03-15

    Post-translational processing of proteins such as the Ras GTPases, which contain a C-terminal CaaX motif (where C stands for cysteine, a for aliphatic and X is one of several amino acids), includes prenylation, proteolytic removal of the C-terminal tripeptide and carboxy-methylation of the isoprenyl-cysteine residue. In the present study, we report the presence of two distinct CaaX-proteolytic activities in membrane extracts from Caenorhabditis elegans, which are sensitive to EDTA and Tos-Phe-CH(2)Cl (tosylphenylalanylchloromethane; 'TPCK') respectively. A protein similar to the mammalian and yeast farnesylated-proteins converting enzyme-1 (FACE-1)/Ste24p CaaX metalloprotease, encoded by a hypothetical gene (CeFACE-1/C04F12.10) found in C. elegans chromosome I, probably accounts for the EDTA-sensitive activity. An orthologue of FACE-2/Rce1p, the enzyme responsible for the proteolytic maturation of Ras oncoproteins and other prenylated substrates, probably accounts for the Tos-Phe-CH(2)Cl-sensitive activity, even though the gene for FACE-2/Rce1 has not been previously identified in this model organism. We have identified a previously overlooked gene in C. elegans chromosome V, which codes for a 266-amino-acid protein (CeFACE-2) with 30% sequence identity to human FACE-2/Rce1. We show that both CeFACE-1 and CeFACE-2 have the ability to promote production of the farnesylated yeast pheromone a -factor in vivo and to cleave a farnesylated peptide in vitro. These results indicate that CeFACE-1 and CeFACE-2 are bona fide CaaX proteases and support the evolutionary conservation of this proteolytic system in eukaryotes.

  3. Identification, functional expression and enzymic analysis of two distinct CaaX proteases from Caenorhabditis elegans.

    PubMed Central

    Cadiñanos, Juan; Schmidt, Walter K; Fueyo, Antonio; Varela, Ignacio; López-Otín, Carlos; Freije, José M P

    2003-01-01

    Post-translational processing of proteins such as the Ras GTPases, which contain a C-terminal CaaX motif (where C stands for cysteine, a for aliphatic and X is one of several amino acids), includes prenylation, proteolytic removal of the C-terminal tripeptide and carboxy-methylation of the isoprenyl-cysteine residue. In the present study, we report the presence of two distinct CaaX-proteolytic activities in membrane extracts from Caenorhabditis elegans, which are sensitive to EDTA and Tos-Phe-CH(2)Cl (tosylphenylalanylchloromethane; 'TPCK') respectively. A protein similar to the mammalian and yeast farnesylated-proteins converting enzyme-1 (FACE-1)/Ste24p CaaX metalloprotease, encoded by a hypothetical gene (CeFACE-1/C04F12.10) found in C. elegans chromosome I, probably accounts for the EDTA-sensitive activity. An orthologue of FACE-2/Rce1p, the enzyme responsible for the proteolytic maturation of Ras oncoproteins and other prenylated substrates, probably accounts for the Tos-Phe-CH(2)Cl-sensitive activity, even though the gene for FACE-2/Rce1 has not been previously identified in this model organism. We have identified a previously overlooked gene in C. elegans chromosome V, which codes for a 266-amino-acid protein (CeFACE-2) with 30% sequence identity to human FACE-2/Rce1. We show that both CeFACE-1 and CeFACE-2 have the ability to promote production of the farnesylated yeast pheromone a -factor in vivo and to cleave a farnesylated peptide in vitro. These results indicate that CeFACE-1 and CeFACE-2 are bona fide CaaX proteases and support the evolutionary conservation of this proteolytic system in eukaryotes. PMID:12487630

  4. ADAM Proteases: Ligand Processing and Modulation of the Notch Pathway

    PubMed Central

    Zolkiewska, Anna

    2009-01-01

    ADAM metalloproteases play important roles in development and disease. One of the key functions of ADAMs is the proteolytic processing of Notch receptors and their ligands. ADAM-mediated cleavage of Notch represents the first step of the regulated intramembrane proteolysis of the receptor, leading to activation of the Notch pathway. Recent reports indicate that the transmembrane Notch ligands also undergo ADAM-mediated processing in cultured cells and in vivo. The proteolytic processing of Notch ligands modulates the strength and duration of Notch signals, leads to generation of soluble intracellular domains of the ligands, and may support a bi-directional signaling between cells. PMID:18344021

  5. Intraurethral stimulation evokes bladder responses via 2 distinct reflex pathways.

    PubMed

    Woock, John P; Yoo, Paul B; Grill, Warren M

    2009-07-01

    Recent animal studies have shown that selective activation of pudendal nerve branches can evoke bladder responses through 2 distinct reflex pathways. We examined intraurethral electrical stimulation as a minimally invasive means of selectively activating these pathways in the cat. Bladder responses evoked by intraurethral electrical stimulation were measured in alpha-chloralose anesthetized male cats at different stimulation frequencies, stimulation intensities and intraurethral locations. Intraurethral electrical stimulation evoked inhibitory and excitatory bladder reflexes depending on stimulation frequency and location. Stimulation in the penile urethra 0 to 3 cm from the urethral meatus at 33 Hz evoked bladder contraction and at 10 Hz it evoked bladder relaxation. These responses were abolished after bilateral transection of the dorsal penile nerves. Stimulation in the membranous urethra 5 to 7 cm from the urethral meatus at 2, 10 and 33 Hz evoked bladder contractions. These responses were abolished after bilateral transection of the cranial sensory nerves. Following acute spinal cord transection bladder contractions were still evoked by 33 Hz stimulation in the penile urethra but not by stimulation at any frequency in the membranous urethra. Intraurethral electrical stimulation selectively evoked bladder responses by activating 2 distinct pudendal afferent pathways. Responses depended on stimulation frequency and location. Intraurethral electrical stimulation is a valid means of determining the pathways involved in bladder responses evoked by pudendal nerve stimulation.

  6. Different sympathetic pathways control the metabolism of distinct bone envelopes.

    PubMed

    Bataille, Caroline; Mauprivez, Cédric; Haÿ, Eric; Baroukh, Brigitte; Brun, Adrian; Chaussain, Catherine; Marie, Pierre J; Saffar, Jean-Louis; Cherruau, Marc

    2012-05-01

    Bone remodeling, the mechanism that modulates bone mass adaptation, is controlled by the sympathetic nervous system through the catecholaminergic pathway. However, resorption in the mandible periosteum envelope is associated with cholinergic Vasoactive Intestinal Peptide (VIP)-positive nerve fibers sensitive to sympathetic neurotoxics, suggesting that different sympathetic pathways may control distinct bone envelopes. In this study, we assessed the role of distinct sympathetic pathways on rat femur and mandible envelopes. To this goal, adult male Wistar rats were chemically sympathectomized or treated with agonists/antagonists of the catecholaminergic and cholinergic pathways; femora and mandibles were sampled. Histomorphometric analysis showed that sympathectomy decreased the number of preosteoclasts and RANKL-expressing osteoblasts in mandible periosteum but had no effect on femur trabecular bone. In contrast, pharmacological stimulation or repression of the catecholaminergic cell receptors impacted the femur trabecular bone and mandible endosteal retromolar zone. VIP treatment of sympathectomized rats rescued the disturbances of the mandible periosteum and alveolar wall whereas the cholinergic pathway had no effect on the catecholaminergic-dependent envelopes. We also found that VIP receptor-1 was weakly expressed in periosteal osteoblasts in the mandible and was increased by VIP treatment, whereas osteoblasts of the retromolar envelope that was innervated only by tyrosine hydroxylase-immunoreactive fibers, constitutively expressed beta-2 adrenergic receptors. These data highlight the complexity of the sympathetic control of bone metabolism. Both the embryological origin of the bone (endochondral for the femur, membranous for the mandibular periosteum and the socket wall) and environmental factors specific to the innervated envelope may influence the phenotype of the sympathetic innervation. We suggest that an origin-dependent imprint of bone cells through

  7. The Effect of Clade-Specific Sequence Polymorphisms on HIV-1 Protease Activity and Inhibitor Resistance Pathways

    SciTech Connect

    Bandaranayake, Rajintha M.; Kolli, Madhavi; King, Nancy M.; Nalivaika, Ellen A.; Heroux, Annie; Kakizawa, Junko; Sugiura, Wataru; Schiffer, Celia A.

    2010-09-08

    The majority of HIV-1 infections around the world result from non-B clade HIV-1 strains. The CRF01{_}AE (AE) strain is seen principally in Southeast Asia. AE protease differs by {approx}10% in amino acid sequence from clade B protease and carries several naturally occurring polymorphisms that are associated with drug resistance in clade B. AE protease has been observed to develop resistance through a nonactive-site N88S mutation in response to nelfinavir (NFV) therapy, whereas clade B protease develops both the active-site mutation D30N and the nonactive-site mutation N88D. Structural and biochemical studies were carried out with wild-type and NFV-resistant clade B and AE protease variants. The relationship between clade-specific sequence variations and pathways to inhibitor resistance was also assessed. AE protease has a lower catalytic turnover rate than clade B protease, and it also has weaker affinity for both NFV and darunavir (DRV). This weaker affinity may lead to the nonactive-site N88S variant in AE, which exhibits significantly decreased affinity for both NFV and DRV. The D30N/N88D mutations in clade B resulted in a significant loss of affinity for NFV and, to a lesser extent, for DRV. A comparison of crystal structures of AE protease shows significant structural rearrangement in the flap hinge region compared with those of clade B protease and suggests insights into the alternative pathways to NFV resistance. In combination, our studies show that sequence polymorphisms within clades can alter protease activity and inhibitor binding and are capable of altering the pathway to inhibitor resistance.

  8. Distinct quaternary structures of the AAA+ Lon protease control substrate degradation

    PubMed Central

    Vieux, Ellen F.; Wohlever, Matthew L.; Chen, James Z.; Sauer, Robert T.; Baker, Tania A.

    2013-01-01

    Lon is an ATPase associated with cellular activities (AAA+) protease that controls cell division in response to stress and also degrades misfolded and damaged proteins. Subunits of Lon are known to assemble into ring-shaped homohexamers that enclose an internal degradation chamber. Here, we demonstrate that hexamers of Escherichia coli Lon also interact to form a dodecamer at physiological protein concentrations. Electron microscopy of this dodecamer reveals a prolate structure with the protease chambers at the distal ends and a matrix of N domains forming an equatorial hexamer–hexamer interface, with portals of ∼45 Å providing access to the enzyme lumen. Compared with hexamers, Lon dodecamers are much less active in degrading large substrates but equally active in degrading small substrates. Our results support a unique gating mechanism that allows the repertoire of Lon substrates to be tuned by its assembly state. PMID:23674680

  9. Engineered tobacco etch virus (TEV) protease active in the secretory pathway of mammalian cells.

    PubMed

    Cesaratto, Francesca; López-Requena, Alejandro; Burrone, Oscar R; Petris, Gianluca

    2015-10-20

    Tobacco etch virus protease (TEVp) is a unique endopeptidase with stringent substrate specificity. TEVp has been widely used as a purified protein for in vitro applications, but also as a biological tool directly expressing it in living cells. To adapt the protease to diverse applications, several TEVp mutants with different stability and enzymatic properties have been reported. Herein we describe the development of a novel engineered TEVp mutant designed to be active in the secretory pathway. While wild type TEVp targeted to the secretory pathway of mammalian cells is synthetized as an N-glycosylated and catalytically inactive enzyme, a TEVp mutant with selected mutations at two verified N-glycosylation sites and at an exposed cysteine was highly efficient. This mutant was very active in the endoplasmic reticulum (ER) of living cells and can be used as a biotechnological tool to cleave proteins within the secretory pathway. As an immediate practical application we report the expression of a complete functional monoclonal antibody expressed from a single polypeptide, which was cleaved by our TEVp mutant into the two antibody chains and secreted as an assembled and functional molecule. In addition, we show active TEVp mutants lacking auto-cleavage activity.

  10. Microtubule depolymerization induces traction force increase through two distinct pathways

    PubMed Central

    Rape, Andrew; Guo, Wei-hui; Wang, Yu-li

    2011-01-01

    Traction forces increase after microtubule depolymerization; however, the signaling mechanisms underlying this, in particular the dependence upon myosin II, remain unclear. We investigated the mechanism of traction force increase after nocodazole-induced microtubule depolymerization by applying traction force microscopy to cells cultured on micropatterned polyacrylamide hydrogels to obtain samples of homogeneous shape and size. Control cells and cells treated with a focal adhesion kinase (FAK) inhibitor showed similar increases in traction forces, indicating that the response is independent of FAK. Surprisingly, pharmacological inhibition of myosin II did not prevent the increase of residual traction forces upon nocodazole treatment. This increase was abolished upon pharmacological inhibition of FAK. These results suggest two distinct pathways for the regulation of traction forces. First, microtubule depolymerization activates a myosin-II-dependent mechanism through a FAK-independent pathway. Second, microtubule depolymerization also enhances traction forces through a myosin-II-independent, FAK-regulated pathway. Traction forces are therefore regulated by a complex network of complementary signals and force-generating mechanisms. PMID:22193960

  11. Microtubule depolymerization induces traction force increase through two distinct pathways.

    PubMed

    Rape, Andrew; Guo, Wei-hui; Wang, Yu-li

    2011-12-15

    Traction forces increase after microtubule depolymerization; however, the signaling mechanisms underlying this, in particular the dependence upon myosin II, remain unclear. We investigated the mechanism of traction force increase after nocodazole-induced microtubule depolymerization by applying traction force microscopy to cells cultured on micropatterned polyacrylamide hydrogels to obtain samples of homogeneous shape and size. Control cells and cells treated with a focal adhesion kinase (FAK) inhibitor showed similar increases in traction forces, indicating that the response is independent of FAK. Surprisingly, pharmacological inhibition of myosin II did not prevent the increase of residual traction forces upon nocodazole treatment. This increase was abolished upon pharmacological inhibition of FAK. These results suggest two distinct pathways for the regulation of traction forces. First, microtubule depolymerization activates a myosin-II-dependent mechanism through a FAK-independent pathway. Second, microtubule depolymerization also enhances traction forces through a myosin-II-independent, FAK-regulated pathway. Traction forces are therefore regulated by a complex network of complementary signals and force-generating mechanisms.

  12. In vitro protease cleavage and computer simulations reveal the HIV-1 capsid maturation pathway

    PubMed Central

    Ning, Jiying; Erdemci-Tandogan, Gonca; Yufenyuy, Ernest L.; Wagner, Jef; Himes, Benjamin A.; Zhao, Gongpu; Aiken, Christopher; Zandi, Roya; Zhang, Peijun

    2016-01-01

    HIV-1 virions assemble as immature particles containing Gag polyproteins that are processed by the viral protease into individual components, resulting in the formation of mature infectious particles. There are two competing models for the process of forming the mature HIV-1 core: the disassembly and de novo reassembly model and the non-diffusional displacive model. To study the maturation pathway, we simulate HIV-1 maturation in vitro by digesting immature particles and assembled virus-like particles with recombinant HIV-1 protease and monitor the process with biochemical assays and cryoEM structural analysis in parallel. Processing of Gag in vitro is accurate and efficient and results in both soluble capsid protein and conical or tubular capsid assemblies, seemingly converted from immature Gag particles. Computer simulations further reveal probable assembly pathways of HIV-1 capsid formation. Combining the experimental data and computer simulations, our results suggest a sequential combination of both displacive and disassembly/reassembly processes for HIV-1 maturation. PMID:27958264

  13. In vitro protease cleavage and computer simulations reveal the HIV-1 capsid maturation pathway.

    PubMed

    Ning, Jiying; Erdemci-Tandogan, Gonca; Yufenyuy, Ernest L; Wagner, Jef; Himes, Benjamin A; Zhao, Gongpu; Aiken, Christopher; Zandi, Roya; Zhang, Peijun

    2016-12-13

    HIV-1 virions assemble as immature particles containing Gag polyproteins that are processed by the viral protease into individual components, resulting in the formation of mature infectious particles. There are two competing models for the process of forming the mature HIV-1 core: the disassembly and de novo reassembly model and the non-diffusional displacive model. To study the maturation pathway, we simulate HIV-1 maturation in vitro by digesting immature particles and assembled virus-like particles with recombinant HIV-1 protease and monitor the process with biochemical assays and cryoEM structural analysis in parallel. Processing of Gag in vitro is accurate and efficient and results in both soluble capsid protein and conical or tubular capsid assemblies, seemingly converted from immature Gag particles. Computer simulations further reveal probable assembly pathways of HIV-1 capsid formation. Combining the experimental data and computer simulations, our results suggest a sequential combination of both displacive and disassembly/reassembly processes for HIV-1 maturation.

  14. Genomics of Chronic Obstructive Pulmonary Disease (COPD); Exploring the SNPs of Protease-Antiprotease Pathway

    PubMed Central

    Kumar, Manish; Phougat, Neetu; Ruhil, Sonam; Dhankhar, Sandeep; Balhara, Meenakshi; Chhillar, Anil Kumar

    2013-01-01

    The COPD has been an important respiratory condition that affects people worldwide and its incidence has been alarming. The increasing incidence of this disorder has been attributed to global industrialization and environmental pollution. Although the exposures to environmental pollutants and smoking have been important triggers, the genetic component of individuals has been shown to be important for development and progression of COPD. Recent literature reported that protease-antiprotease imbalance to be important in etiopathogenesis of COPD. The enzymes namely neutrophil elastase and matrix metalloprotienases are considered to be foremost proteolytic molecules released by neutrophils and macrophages during inflammatory events in COPD. Normally, the lungs remain protected from the destructive effect of these two antiproteases by α1-antitrypsin (α1AT) and tissue inhibitors of metalloproteinases (TIMPs) respectively. In this review, we are trying to highlight the work by various research groups in exploring the SNPs of various genes of inflammatory pathways and the protease-antiprotease pathway, which may have some degree of association with COPD. PMID:24179443

  15. In vitro protease cleavage and computer simulations reveal the HIV-1 capsid maturation pathway

    NASA Astrophysics Data System (ADS)

    Ning, Jiying; Erdemci-Tandogan, Gonca; Yufenyuy, Ernest L.; Wagner, Jef; Himes, Benjamin A.; Zhao, Gongpu; Aiken, Christopher; Zandi, Roya; Zhang, Peijun

    2016-12-01

    HIV-1 virions assemble as immature particles containing Gag polyproteins that are processed by the viral protease into individual components, resulting in the formation of mature infectious particles. There are two competing models for the process of forming the mature HIV-1 core: the disassembly and de novo reassembly model and the non-diffusional displacive model. To study the maturation pathway, we simulate HIV-1 maturation in vitro by digesting immature particles and assembled virus-like particles with recombinant HIV-1 protease and monitor the process with biochemical assays and cryoEM structural analysis in parallel. Processing of Gag in vitro is accurate and efficient and results in both soluble capsid protein and conical or tubular capsid assemblies, seemingly converted from immature Gag particles. Computer simulations further reveal probable assembly pathways of HIV-1 capsid formation. Combining the experimental data and computer simulations, our results suggest a sequential combination of both displacive and disassembly/reassembly processes for HIV-1 maturation.

  16. Distinct purinergic signaling pathways in prepubescent mouse spermatogonia

    PubMed Central

    Mundt, Nadine; Bruentgens, Felicitas; Geilenkirchen, Petra; Machado, Patricia A.; Veitinger, Thomas; Veitinger, Sophie; Lipartowski, Susanne M.; Engelhardt, Corinna H.; Oldiges, Marco; Spehr, Jennifer

    2016-01-01

    Spermatogenesis ranks among the most complex, yet least understood, developmental processes. The physiological principles that control male germ cell development in mammals are notoriously difficult to unravel, given the intricate anatomy and complex endo- and paracrinology of the testis. Accordingly, we lack a conceptual understanding of the basic signaling mechanisms within the testis, which control the seminiferous epithelial cycle and thus govern spermatogenesis. Here, we address paracrine signal transduction in undifferentiated male germ cells from an electrophysiological perspective. We identify distinct purinergic signaling pathways in prepubescent mouse spermatogonia, both in vitro and in situ. ATP—a dynamic, widespread, and evolutionary conserved mediator of cell to cell communication in various developmental contexts—activates at least two different spermatogonial purinoceptor isoforms. Both receptors operate within nonoverlapping stimulus concentration ranges, display distinct response kinetics and, in the juvenile seminiferous cord, are uniquely expressed in spermatogonia. We further find that spermatogonia express Ca2+-activated large-conductance K+ channels that appear to function as a safeguard against prolonged ATP-dependent depolarization. Quantitative purine measurements additionally suggest testicular ATP-induced ATP release, a mechanism that could increase the paracrine radius of initially localized signaling events. Moreover, we establish a novel seminiferous tubule slice preparation that allows targeted electrophysiological recordings from identified testicular cell types in an intact epithelial environment. This unique approach not only confirms our in vitro findings, but also supports the notion of purinergic signaling during the early stages of spermatogenesis. PMID:27574293

  17. Distinct Wnt signaling pathways have opposing roles in appendage regeneration.

    PubMed

    Stoick-Cooper, Cristi L; Weidinger, Gilbert; Riehle, Kimberly J; Hubbert, Charlotte; Major, Michael B; Fausto, Nelson; Moon, Randall T

    2007-02-01

    In contrast to mammals, lower vertebrates have a remarkable capacity to regenerate complex structures damaged by injury or disease. This process, termed epimorphic regeneration, involves progenitor cells created through the reprogramming of differentiated cells or through the activation of resident stem cells. Wnt/beta-catenin signaling regulates progenitor cell fate and proliferation during embryonic development and stem cell function in adults, but its functional involvement in epimorphic regeneration has not been addressed. Using transgenic fish lines, we show that Wnt/beta-catenin signaling is activated in the regenerating zebrafish tail fin and is required for formation and subsequent proliferation of the progenitor cells of the blastema. Wnt/beta-catenin signaling appears to act upstream of FGF signaling, which has recently been found to be essential for fin regeneration. Intriguingly, increased Wnt/beta-catenin signaling is sufficient to augment regeneration, as tail fins regenerate faster in fish heterozygous for a loss-of-function mutation in axin1, a negative regulator of the pathway. Likewise, activation of Wnt/beta-catenin signaling by overexpression of wnt8 increases proliferation of progenitor cells in the regenerating fin. By contrast, overexpression of wnt5b (pipetail) reduces expression of Wnt/beta-catenin target genes, impairs proliferation of progenitors and inhibits fin regeneration. Importantly, fin regeneration is accelerated in wnt5b mutant fish. These data suggest that Wnt/beta-catenin signaling promotes regeneration, whereas a distinct pathway activated by wnt5b acts in a negative-feedback loop to limit regeneration.

  18. Subtilisin-like autotransporter serves as maturation protease in a bacterial secretion pathway

    PubMed Central

    Coutte, Loic; Antoine, Rudy; Drobecq, Hervé; Locht, Camille; Jacob-Dubuisson, Françoise

    2001-01-01

    Proteins of Gram-negative bacteria destined to the extracellular milieu must cross the two cellular membranes and then fold at the appropriate time and place. The synthesis of a precursor may be a strategy to maintain secretion competence while preventing aggregation or premature folding (especially for large proteins). The secretion of 230 kDa filamentous haemagglutinin (FHA) of Bordetella pertussis requires the synthesis and the maturation of a 367 kDa precursor that undergoes the proteolytic removal of its ∼130 kDa C-terminal intramolecular chaperone domain. We have identified a specific protease, SphB1, responsible for the timely maturation of the precursor FhaB, which allows for extracellular release of FHA. SphB1 is a large exported protein with a subtilisin-like domain and a C-terminal domain typical of bacterial autotransporters. SphB1 is the first described subtilisin-like protein that serves as a specialized maturation protease in a secretion pathway of Gram-negative bacteria. This is reminiscent of pro-protein convertases of eukaryotic cells. PMID:11566869

  19. Crystal structure of TET protease reveals complementary protein degradation pathways in prokaryotes.

    PubMed

    Borissenko, Ljudmila; Groll, Michael

    2005-03-11

    Protein degradation is an essential and strictly controlled process with proteasome and functionally related proteases representing its central part. Tricorn protease (TRI) has been shown to act downstream of the proteasome, degrading produced peptides. Recently, a novel large prokaryotic aminopeptidase oligomeric complex, named TET, has been identified. This complex degrades peptides of different length in organisms where TRI is not present. We determined the crystal structure of TET from the thermophilic archaeon Pyrococcus horikoshii at 1.6 A resolution in native form and in complex with the inhibitor amastatin. We demonstrate that, beside the novel tetrahedral oligomerisation pattern, TET possesses a unique mechanism of substrate attraction and orientation. TET sequentially degrades peptides produced by the proteasome to single amino acids. Furthermore, we reconstituted in vitro the minimal protein degradation system from initial unfolding of labelled protein substrates, up to release of free amino acids. We propose that TET and TRI act as functional analogues in different organisms, with TET being more widely distributed. Thus, TET and TRI represent two evolutionarily diverged pathways of peptide degradation in prokaryotes.

  20. A major human immunodeficiency virus type 1-initiated killing pathway distinct from apoptosis.

    PubMed Central

    Kolesnitchenko, V; King, L; Riva, A; Tani, Y; Korsmeyer, S J; Cohen, D I

    1997-01-01

    We have investigated the relative contribution of apoptosis or programmed cell death (PCD) to cell killing during acute infection with T-cell-tropic, cytopathic human immunodeficiency virus type 1 (HIV-1), by employing diverse strategies to inhibit PCD or to detect its common end-stage sequelae. When Bcl-2-transfected cell lines were infected with HIV-1, their viability was only slightly higher than that of control infections. Although the adenovirus E1B 19-kDa protein has been reported to be a stronger competitor of apoptosis than Bcl-2, it did not inhibit HIV-mediated cell death better than Bcl-2 protein. Competition for Fas ligand or inactivation of the Fas pathway secondary to intracellular mutation (MOLT-4 T cells) also had modest effects on overall cell death during acute HIV infection. In contrast to these observations with HIV infection or with HIV envelope-initiated cell death, Tat-expressing cell lines were much more susceptible (200% enhancement) to Fas-induced apoptosis than controls and Bcl-2 overexpression strongly (75%) inhibited this apoptotic T-cell death. PCD associated with FasR ligation resulted in the cleavage of common interleukin-1beta-converting enzyme (ICE)-protease targets, poly(ADP-ribose) polymerase (PARP) and pro-ICE, whereas cleaved products were not readily detected during HIV infection of peripheral blood mononuclear cells or T-cell lines even during periods of extensive cell death. These results indicate that one important form of HIV-mediated cell killing proceeds by a pathway that lacks the characteristics of T-cell apoptosis. Our observations support the conclusion that at least two HIV genes (env and tat) can kill T cells by distinct pathways and that an envelope-initiated process of T-cell death can be discriminated from apoptosis by many of the properties most closely associated with apoptotic cell death. PMID:9371641

  1. Recycling and Endosomal Sorting of Protease-activated Receptor-1 Is Distinctly Regulated by Rab11A and Rab11B Proteins*

    PubMed Central

    Grimsey, Neil J.; Coronel, Luisa J.; Cordova, Isabel Canto; Trejo, JoAnn

    2016-01-01

    Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor that undergoes proteolytic irreversible activation by coagulant and anti-coagulant proteases. Given the irreversible activation of PAR1, signaling by the receptor is tightly regulated through desensitization and intracellular trafficking. PAR1 displays both constitutive and agonist-induced internalization. Constitutive internalization of PAR1 is important for generating an internal pool of naïve receptors that replenish the cell surface and facilitate resensitization, whereas agonist-induced internalization of PAR1 is critical for terminating G protein signaling. We showed that PAR1 constitutive internalization is mediated by the adaptor protein complex-2 (AP-2), whereas AP-2 and epsin control agonist-induced PAR1 internalization. However, the mechanisms that regulate PAR1 recycling are not known. In the present study we screened a siRNA library of 140 different membrane trafficking proteins to identify key regulators of PAR1 intracellular trafficking. In addition to known mediators of PAR1 endocytosis, we identified Rab11B as a critical regulator of PAR1 trafficking. We found that siRNA-mediated depletion of Rab11B and not Rab11A blocks PAR1 recycling, which enhanced receptor lysosomal degradation. Although Rab11A is not required for PAR1 recycling, depletion of Rab11A resulted in intracellular accumulation of PAR1 through disruption of basal lysosomal degradation of the receptor. Moreover, enhanced degradation of PAR1 observed in Rab11B-deficient cells is blocked by depletion of Rab11A and the autophagy related-5 protein, suggesting that PAR1 is shuttled to an autophagic degradation pathway in the absence of Rab11B recycling. Together these findings suggest that Rab11A and Rab11B differentially regulate intracellular trafficking of PAR1 through distinct endosomal sorting mechanisms. PMID:26635365

  2. Interference with nuclear factor kappaB signaling pathway by pathogen-encoded proteases: global and selective inhibition.

    PubMed

    Hodgson, Andrea; Wan, Fengyi

    2016-02-01

    Pathogens have evolved a myriad of ways to abrogate and manipulate the host response to infections. Of the various mechanisms involved, pathogen-encoded and sometimes host-encoded proteases are an important category of virulence factors that cause robust changes on the host response by targeting key proteins along signaling cascades. The nuclear factor kappaB (NF-κB) signaling pathway is a crucial regulatory mechanism for the cell, controlling the expression of survival, immune and proliferation genes. Proteases from pathogens of almost all types have been demonstrated to target and cleave members of the NF-κB signaling pathway at nearly every level. This review provides discussion of proteases targeting the most abundant NF-κB subunit, p65, and the impact of protease-mediated p65 cleavage on the immune responses and survival of the infected host cell. After examining various examples of protease interference, it becomes evident that the cleavage fragments produced by pathogen-driven proteolytic processing should be further characterized to determine whether they have novel and unique functions within the cell. The selective targeting of p65 and its effect on gene transcription reveals unique mechanisms by which pathogens acutely alter their microenvironment, and further research may open new opportunities for novel therapeutics to combat pathogens.

  3. The two main endoproteases present in dark-induced senescent wheat leaves are distinct subtilisin-like proteases.

    PubMed

    Roberts, Irma N; Passeron, Susana; Barneix, Atilio J

    2006-11-01

    We have previously reported the occurrence of two serine endoproteases (referred to as P1 and P2) in dark-induced senescent wheat (Triticum aestivum L.) leaves. P1 enzyme was already purified and identified as a subtilisin-like serine endoprotease (Roberts et al. in Physiol Plant 118:483-490, 2003). In this paper, we demonstrate by Western blot analysis of extracts obtained from dark-induced senescent leaves that an antiserum raised against P1 was able to recognise a second protein band of 78 kDa which corresponded to P2 activity. This result suggested that both enzymes must be structurally related. Therefore, we purified and characterised P2 activity. According to its biochemical and physical properties (inhibition by chymostatin and PMSF, broad pH range of activity, thermostability and ability to hydrolyse Suc-AAPF-pNA) P2 was classified as a serine protease with chymotrypsin-like activity. In addition, P2 was identified by mass spectrometry as a subtilisin-like protease distinct from P1. Western blot analysis demonstrated that P1 appeared in extracts from non-detached dark-induced senescent leaves but was undetectable in leaves senescing after nitrogen (N) deprivation. In contrast, P2 was already present in non-senescent leaves and showed increased levels in leaves senescing after N starvation or incubation in darkness. P1 signal was detected at late stages of ethephon or methyl jasmonate-induced senescence but was undetectable in senescent leaves from plants treated with abscisic acid. None of the three hormones have any effect on P2 protein levels. These results indicate that despite their biochemical and structural similarities, both enzymes are probably involved in different physiological roles.

  4. Distinct pathways leading to TDP-43-induced cellular dysfunctions.

    PubMed

    Yamashita, Makiko; Nonaka, Takashi; Hirai, Shinobu; Miwa, Akiko; Okado, Haruo; Arai, Tetsuaki; Hosokawa, Masato; Akiyama, Haruhiko; Hasegawa, Masato

    2014-08-15

    TAR DNA-binding protein of 43 kDa (TDP-43) is the major component protein of inclusions found in brains of patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). However, the molecular mechanisms by which TDP-43 causes neuronal dysfunction and death remain unknown. Here, we report distinct cytotoxic effects of full-length TDP-43 (FL-TDP) and its C-terminal fragment (CTF) in SH-SY5Y cells. When FL-TDP was overexpressed in the cells using a lentiviral system, exogenous TDP-43, like endogenous TDP-43, was expressed mainly in nuclei of cells without any intracellular inclusions. However, these cells showed striking cell death, caspase activation and growth arrest at G2/M phase, indicating that even simple overexpression of TDP-43 induces cellular dysfunctions leading to apoptosis. On the other hand, cells expressing TDP-43 CTF showed cytoplasmic aggregates but without significant cell death, compared with cells expressing FL-TDP. Confocal microscopic analyses revealed that RNA polymerase II (RNA pol II) and several transcription factors, such as specificity protein 1 and cAMP-response-element-binding protein, were co-localized with the aggregates of TDP-43 CTF, suggesting that sequestration of these factors into TDP-43 aggregates caused transcriptional dysregulation. Indeed, accumulation of RNA pol II at TDP-43 inclusions was detected in brains of patients with FTLD-TDP. Furthermore, apoptosis was not observed in affected neurons of FTLD-TDP brains containing phosphorylated and aggregated TDP-43 pathology. Our results suggest that different pathways of TDP-43-induced cellular dysfunction may contribute to the degeneration cascades involved in the onset of ALS and FTLD-TDP. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  5. An aspartyl protease defines a novel pathway for export of Toxoplasma proteins into the host cell

    PubMed Central

    Coffey, Michael J; Sleebs, Brad E; Uboldi, Alessandro D; Garnham, Alexandra; Franco, Magdalena; Marino, Nicole D; Panas, Michael W; Ferguson, David JP; Enciso, Marta; O'Neill, Matthew T; Lopaticki, Sash; Stewart, Rebecca J; Dewson, Grant; Smyth, Gordon K; Smith, Brian J; Masters, Seth L; Boothroyd, John C; Boddey, Justin A; Tonkin, Christopher J

    2015-01-01

    Infection by Toxoplasma gondii leads to massive changes to the host cell. Here, we identify a novel host cell effector export pathway that requires the Golgi-resident aspartyl protease 5 (ASP5). We demonstrate that ASP5 cleaves a highly constrained amino acid motif that has similarity to the PEXEL-motif of Plasmodium parasites. We show that ASP5 matures substrates at both the N- and C-terminal ends of proteins and also controls trafficking of effectors without this motif. Furthermore, ASP5 controls establishment of the nanotubular network and is required for the efficient recruitment of host mitochondria to the vacuole. Assessment of host gene expression reveals that the ASP5-dependent pathway influences thousands of the transcriptional changes that Toxoplasma imparts on its host cell. All these changes result in attenuation of virulence of Δasp5 tachyzoites in vivo. This work characterizes the first identified machinery required for export of Toxoplasma effectors into the infected host cell. DOI: http://dx.doi.org/10.7554/eLife.10809.001 PMID:26576949

  6. An aspartyl protease defines a novel pathway for export of Toxoplasma proteins into the host cell.

    PubMed

    Coffey, Michael J; Sleebs, Brad E; Uboldi, Alessandro D; Garnham, Alexandra; Franco, Magdalena; Marino, Nicole D; Panas, Michael W; Ferguson, David Jp; Enciso, Marta; O'Neill, Matthew T; Lopaticki, Sash; Stewart, Rebecca J; Dewson, Grant; Smyth, Gordon K; Smith, Brian J; Masters, Seth L; Boothroyd, John C; Boddey, Justin A; Tonkin, Christopher J

    2015-11-18

    Infection by Toxoplasma gondii leads to massive changes to the host cell. Here, we identify a novel host cell effector export pathway that requires the Golgi-resident aspartyl protease 5 (ASP5). We demonstrate that ASP5 cleaves a highly constrained amino acid motif that has similarity to the PEXEL-motif of Plasmodium parasites. We show that ASP5 matures substrates at both the N- and C-terminal ends of proteins and also controls trafficking of effectors without this motif. Furthermore, ASP5 controls establishment of the nanotubular network and is required for the efficient recruitment of host mitochondria to the vacuole. Assessment of host gene expression reveals that the ASP5-dependent pathway influences thousands of the transcriptional changes that Toxoplasma imparts on its host cell. All these changes result in attenuation of virulence of Δasp5 tachyzoites in vivo. This work characterizes the first identified machinery required for export of Toxoplasma effectors into the infected host cell.

  7. Mesenchymal stem cells from adipose and bone marrow promote angiogenesis via distinct cytokine and protease expression mechanisms

    PubMed Central

    Kachgal, Suraj; Putnam, Andrew J.

    2012-01-01

    Using a fibrin-based angiogenesis model, we have established that there is no canonical mechanism used by ECs to degrade the surrounding extracellular matrix (ECM), but rather the set of proteases used is dependent on the mural cells providing the angiogenic cues. Mesenchymal stem cells (MSCs) originating from different tissues, which are thought to be phenotypically similar, promote angiogenesis through distinct mechanisms. Specifically, adipose-derived stem cells (ASCs) promote utilization of the plasminogen activator-plasmin axis by ECs as the primary means of vessel invasion and elongation in fibrin. Matrix metalloproteinases (MMPs) serve a purpose in regulating capillary diameter and possibly in stabilizing the nascent vessels. These proteolytic mechanisms are more akin to those involved in fibroblast-mediated angiogenesis than to those in bone marrow-derived stem cell (BMSC)-mediated angiogenesis. In addition, expression patterns of angiogenic factors such as urokinase plasminogen activator (uPA), hepatocyte growth factor (HGF), and tumor necrosis factor alpha (TNFα) were similar for ASC and fibroblast-mediated angiogenesis, and in direct contrast to BMSC-mediated angiogenesis. The present study illustrates that the nature of the heterotypic interactions between mural cells and endothelial cells depend on the identity of the mural cell used. Even MSCs which are shown to behave phenotypically similar do not stimulate angiogenesis via the same mechanisms. PMID:21104120

  8. Distinct types of protease systems are involved in homeostasis regulation of mitochondrial morphology via balanced fusion and fission.

    PubMed

    Saita, Shotaro; Ishihara, Takaya; Maeda, Maki; Iemura, Shun-Ichiro; Natsume, Tohru; Mihara, Katsuyoshi; Ishihara, Naotada

    2016-05-01

    Mitochondrial morphology is dynamically regulated by fusion and fission. Several GTPase proteins control fusion and fission, and posttranslational modifications of these proteins are important for the regulation. However, it has not been clarified how the fusion and fission is balanced. Here, we report the molecular mechanism to regulate mitochondrial morphology in mammalian cells. Ablation of the mitochondrial fission, by repression of Drp1 or Mff, or by over-expression of MiD49 or MiD51, results in a reduction in the fusion GTPase mitofusins (Mfn1 and Mfn2) in outer membrane and long form of OPA1 (L-OPA1) in inner membrane. RNAi- or CRISPR-induced ablation of Drp1 in HeLa cells enhanced the degradation of Mfns via the ubiquitin-proteasome system (UPS). We further found that UPS-related protein BAT3/BAG6, here we identified as Mfn2-interacting protein, was implicated in the turnover of Mfns in the absence of mitochondrial fission. Ablation of the mitochondrial fission also enhanced the proteolytic cleavage of L-OPA1 to soluble S-OPA1, and the OPA1 processing was reversed by inhibition of the inner membrane protease OMA1 independent on the mitochondrial membrane potential. Our findings showed that the distinct degradation systems of the mitochondrial fusion proteins in different locations are enhanced in response to the mitochondrial morphology. © 2016 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  9. Signalling pathways induced by protease-activated receptors and integrins in T cells.

    PubMed

    Bar-Shavit, Rachel; Maoz, Miriam; Yongjun, Yin; Groysman, Maya; Dekel, Idit; Katzav, Shulamit

    2002-01-01

    Recent characterization of the thrombin receptor indicates that it plays a role in T-cell signalling pathways. However, little is known regarding the signalling events following stimulation of additional members of the protease-activated receptor (PAR) family, i.e. PAR2 and PAR3. Most of the postligand cascades are largely unknown. Here, we illustrate that in Jurkat T-leukaemic cells, activation of PAR1, PAR2 and PAR3 induce tyrosine phosphorylation of Vav1. This response was impaired in Jurkat T cells deficient in p56lck (JCaM1.6). Activation of PARs also led to an increase in tyrosine phosphorylation of ZAP-70 and SLP-76, two key proteins in T-cell receptor (TCR) signalling. We also demonstrated that p56lck is meaningful for integrin signalling. Thus, JCaM1.6 cells exhibited a marked reduction in their adherence to fibronectin-coated plates, as compared to the level of adherence of Jurkat T cells. While the phosphorylation of Vav1 in T cells is augmented following adhesion, no additional increase was noted following treatment of the adhered cells with PARs. Altogether, we have identified key components in the postligand-signalling cascade of PARs and integrins. Furthermore, we have identified Lck as a critical and possibly upstream component of PAR-induced Vav1 phosphorylation, as well as integrin activation, in Jurkat T cells.

  10. [Peptide hydrolases with catalytic dyad Ser-Lys. Similarity and distinctions of the active centers of ATP-dependent Lon proteases, LexA repressors, signal peptidases and C-terminal processing proteases].

    PubMed

    Rotanova, T V

    2002-01-01

    It is established that ATP-dependent protease Lon family belongs to the serine-lysine peptide hydrolases clan. Significant similarity of amino acid sequences of proteases Lon and repressors LexA in the regions including the catalytic serine and lysine residues is revealed by comparing primary structures of different families of the enzymes with Ser-Lys catalytic dyad. The both Lon and LexA families are shown to be divided into two subfamilies in accordance with the nature of amino acids in the catalytically active serine environment. Putative DNA binding sites are revealed in proteolytic domains of Lon A subfamily. Similarities and distinctions of the all families peptide hydrolases of the clan in the regions of their active centers are discussed.

  11. Distinct pathways of neural coupling for different basic emotions.

    PubMed

    Tettamanti, Marco; Rognoni, Elena; Cafiero, Riccardo; Costa, Tommaso; Galati, Dario; Perani, Daniela

    2012-01-16

    Emotions are complex events recruiting distributed cortical and subcortical cerebral structures, where the functional integration dynamics within the involved neural circuits in relation to the nature of the different emotions are still unknown. Using fMRI, we measured the neural responses elicited by films representing basic emotions (fear, disgust, sadness, happiness). The amygdala and the associative cortex were conjointly activated by all basic emotions. Furthermore, distinct arrays of cortical and subcortical brain regions were additionally activated by each emotion, with the exception of sadness. Such findings informed the definition of three effective connectivity models, testing for the functional integration of visual cortex and amygdala, as regions processing all emotions, with domain-specific regions, namely: i) for fear, the frontoparietal system involved in preparing adaptive motor responses; ii) for disgust, the somatosensory system, reflecting protective responses against contaminating stimuli; iii) for happiness: medial prefrontal and temporoparietal cortices involved in understanding joyful interactions. Consistently with these domain-specific models, the results of the effective connectivity analysis indicate that the amygdala is involved in distinct functional integration effects with cortical networks processing sensorimotor, somatosensory, or cognitive aspects of basic emotions. The resulting effective connectivity networks may serve to regulate motor and cognitive behavior based on the quality of the induced emotional experience. Copyright © 2011. Published by Elsevier Inc.

  12. Membrane proteases in the bacterial protein secretion and quality control pathway.

    PubMed

    Dalbey, Ross E; Wang, Peng; van Dijl, Jan Maarten

    2012-06-01

    Proteolytic cleavage of proteins that are permanently or transiently associated with the cytoplasmic membrane is crucially important for a wide range of essential processes in bacteria. This applies in particular to the secretion of proteins and to membrane protein quality control. Major progress has been made in elucidating the structure-function relationships of many of the responsible membrane proteases, including signal peptidases, signal peptide hydrolases, FtsH, the rhomboid protease GlpG, and the site 1 protease DegS. These enzymes employ very different mechanisms to cleave substrates at the cytoplasmic and extracytoplasmic membrane surfaces or within the plane of the membrane. This review highlights the different ways that bacterial membrane proteases degrade their substrates, with special emphasis on catalytic mechanisms and substrate delivery to the respective active sites.

  13. Maggot excretion products from the blowfly Lucilia sericata contain contact phase/intrinsic pathway-like proteases with procoagulant functions.

    PubMed

    Kahl, M; Gökçen, A; Fischer, S; Bäumer, M; Wiesner, J; Lochnit, G; Wygrecka, M; Vilcinskas, A; Preissner, K T

    2015-08-01

    For centuries, maggots have been used for the treatment of wounds by a variety of ancient cultures, as part of their traditional medicine. With increasing appearance of antimicrobial resistance and in association with diabetic ulcers, maggot therapy was revisited in the 1980s. Three mechanisms by which sterile maggots of the green bottle fly Lucilia sericata may improve healing of chronic wounds have been proposed: Biosurgical debridement, disinfecting properties, and stimulation of the wound healing process. However, the influence of maggot excretion products (MEP) on blood coagulation as part of the wound healing process has not been studied in detail. Here, we demonstrate that specific MEP-derived serine proteases from Lucilia sericata induce clotting of human plasma and whole blood, particularly by activating contact phase proteins factor XII and kininogen as well as factor IX, thereby providing kallikrein-bypassing and factor XIa-like activities, both in plasma and in isolated systems. In plasma samples deficient in contact phase proteins, MEP restored full clotting activity, whereas in plasma deficient in either factor VII, IX, X or II no effect was seen. The observed procoagulant/intrinsic pathway-like activity was mediated by (chymo-) trypsin-like proteases in total MEP, which were significantly blocked by C1-esterase inhibitor or other contact phase-specific protease inhibitors. No significant influence of MEP on platelet activation or fibrinolysis was noted. Together, MEP provides contact phase bypassing procoagulant activity and thereby induces blood clotting in the context of wound healing. Further characterisation of the active serine protease(s) may offer new perspectives for biosurgical treatment of chronic wounds.

  14. Human papillomaviruses: shared and distinct pathways for pathogenesis

    PubMed Central

    Galloway, Denise A.; Laimins, Laimonis A.

    2015-01-01

    Over 200 types of human papillomaviruses (HPV) have been identified that infect epithelial cells at different anatomic locations. HPVs are grouped into five genra with the alpha and beta viruses being the most commonly studied. Members of the alpha HPV genus infect genital epithelia and are the causative agents of many anogenital cancers. Beta HPVs infect cutaneous epithelia and have been suggested as co-factors in the development of non-melanoma skin cancers. Recent studies have shown that activation of DNA damage pathways is important for the productive life cycle of the alpha HPVs while the beta viruses suppress their activation. These differences likely contribute to the varying types of lesions and malignancies that are associated with these viruses. PMID:26398222

  15. Human papillomaviruses: shared and distinct pathways for pathogenesis.

    PubMed

    Galloway, Denise A; Laimins, Laimonis A

    2015-10-01

    Over 200 types of human papillomaviruses (HPV) have been identified that infect epithelial cells at different anatomic locations. HPVs are grouped into five genera with the alpha and beta viruses being the most commonly studied. Members of the alpha HPV genus infect genital epithelia and are the causative agents of many anogenital cancers. Beta HPVs infect cutaneous epithelia and have been suggested as co-factors in the development of non-melanoma skin cancers. Recent studies have shown that activation of DNA damage pathways is important for the productive life cycle of the alpha HPVs while the beta viruses suppress their activation. These differences likely contribute to the varying types of lesions and malignancies that are associated with these viruses. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Rhb1 regulates the expression of secreted aspartic protease 2 through the TOR signaling pathway in Candida albicans.

    PubMed

    Chen, Yu-Ting; Lin, Chia-Ying; Tsai, Pei-Wen; Yang, Cheng-Yao; Hsieh, Wen-Ping; Lan, Chung-Yu

    2012-02-01

    Candida albicans is a major fungal pathogen in humans. In C. albicans, secreted aspartyl protease 2 (Sap2) is the most highly expressed secreted aspartic protease in vitro and is a virulence factor. Recent research links the small GTPase Rhb1 to C. albicans target of rapamycin (TOR) signaling in response to nitrogen availability. The results of this study show that Rhb1 is related to cell growth through the control of SAP2 expression when protein is the major nitrogen source. This process involves various components of the TOR signaling pathway, including Tor1 kinase and its downstream effectors. TOR signaling not only controls SAP2 transcription but also affects Sap2 protein levels, possibly through general amino acid control. DNA microarray analysis identifies other target genes downstream of Rhb1 in addition to SAP2. These findings provide new insight into nutrients, Rhb1-TOR signaling, and expression of C. albicans virulence factor.

  17. Pharmacological and genetic reappraisals of protease and oxidative stress pathways in a mouse model of obstructive lung diseases

    PubMed Central

    Shuto, Tsuyoshi; Kamei, Shunsuke; Nohara, Hirofumi; Fujikawa, Haruka; Tasaki, Yukihiro; Sugahara, Takuya; Ono, Tomomi; Matsumoto, Chizuru; Sakaguchi, Yuki; Maruta, Kasumi; Nakashima, Ryunosuke; Kawakami, Taisei; Suico, Mary Ann; Kondo, Yoshitaka; Ishigami, Akihito; Takeo, Toru; Tanaka, Ken-ichiro; Watanabe, Hiroshi; Nakagata, Naomi; Uchimura, Kohei; Kitamura, Kenichiro; Li, Jian-Dong; Kai, Hirofumi

    2016-01-01

    Protease-antiprotease imbalance and oxidative stress are considered to be major pathophysiological hallmarks of severe obstructive lung diseases including chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF), but limited information is available on their direct roles in the regulation of pulmonary phenotypes. Here, we utilized βENaC-transgenic (Tg) mice, the previously established mouse model of severe obstructive lung diseases, to produce lower-mortality but pathophysiologically highly useful mouse model by backcrossing the original line with C57/BL6J mice. C57/BL6J-βENaC-Tg mice showed higher survival rates and key pulmonary abnormalities of COPD/CF, including mucous hypersecretion, inflammatory and emphysematous phenotypes and pulmonary dysfunction. DNA microarray analysis confirmed that protease- and oxidative stress-dependent pathways are activated in the lung tissue of C57/BL6J-βENaC-Tg mice. Treatments of C57/BL6J-βENaC-Tg mice with a serine protease inhibitor ONO-3403, a derivative of camostat methylate (CM), but not CM, and with an anti-oxidant N-acetylcystein significantly improved pulmonary emphysema and dysfunction. Moreover, depletion of a murine endogenous antioxidant vitamin C (VC), by genetic disruption of VC-synthesizing enzyme SMP30 in C57/BL6J-βENaC-Tg mice, exaggerated pulmonary phenotypes. Thus, these assessments clarified that protease-antiprotease imbalance and oxidative stress are critical pathways that exacerbate the pulmonary phenotypes of C57/BL6J-βENaC-Tg mice, consistent with the characteristics of human COPD/CF. PMID:27982104

  18. Pharmacological and genetic reappraisals of protease and oxidative stress pathways in a mouse model of obstructive lung diseases.

    PubMed

    Shuto, Tsuyoshi; Kamei, Shunsuke; Nohara, Hirofumi; Fujikawa, Haruka; Tasaki, Yukihiro; Sugahara, Takuya; Ono, Tomomi; Matsumoto, Chizuru; Sakaguchi, Yuki; Maruta, Kasumi; Nakashima, Ryunosuke; Kawakami, Taisei; Suico, Mary Ann; Kondo, Yoshitaka; Ishigami, Akihito; Takeo, Toru; Tanaka, Ken-Ichiro; Watanabe, Hiroshi; Nakagata, Naomi; Uchimura, Kohei; Kitamura, Kenichiro; Li, Jian-Dong; Kai, Hirofumi

    2016-12-16

    Protease-antiprotease imbalance and oxidative stress are considered to be major pathophysiological hallmarks of severe obstructive lung diseases including chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF), but limited information is available on their direct roles in the regulation of pulmonary phenotypes. Here, we utilized βENaC-transgenic (Tg) mice, the previously established mouse model of severe obstructive lung diseases, to produce lower-mortality but pathophysiologically highly useful mouse model by backcrossing the original line with C57/BL6J mice. C57/BL6J-βENaC-Tg mice showed higher survival rates and key pulmonary abnormalities of COPD/CF, including mucous hypersecretion, inflammatory and emphysematous phenotypes and pulmonary dysfunction. DNA microarray analysis confirmed that protease- and oxidative stress-dependent pathways are activated in the lung tissue of C57/BL6J-βENaC-Tg mice. Treatments of C57/BL6J-βENaC-Tg mice with a serine protease inhibitor ONO-3403, a derivative of camostat methylate (CM), but not CM, and with an anti-oxidant N-acetylcystein significantly improved pulmonary emphysema and dysfunction. Moreover, depletion of a murine endogenous antioxidant vitamin C (VC), by genetic disruption of VC-synthesizing enzyme SMP30 in C57/BL6J-βENaC-Tg mice, exaggerated pulmonary phenotypes. Thus, these assessments clarified that protease-antiprotease imbalance and oxidative stress are critical pathways that exacerbate the pulmonary phenotypes of C57/BL6J-βENaC-Tg mice, consistent with the characteristics of human COPD/CF.

  19. A distinct pathway for tetrahymanol synthesis in bacteria

    PubMed Central

    Banta, Amy B.; Wei, Jeremy H.; Welander, Paula V.

    2015-01-01

    Tetrahymanol is a polycyclic triterpenoid lipid first discovered in the ciliate Tetrahymena pyriformis whose potential diagenetic product, gammacerane, is often used as a biomarker for water column stratification in ancient ecosystems. Bacteria are also a potential source of tetrahymanol, but neither the distribution of this lipid in extant bacteria nor the significance of bacterial tetrahymanol synthesis for interpreting gammacerane biosignatures is known. Here we couple comparative genomics with genetic and lipid analyses to link a protein of unknown function to tetrahymanol synthesis in bacteria. This tetrahymanol synthase (Ths) is found in a variety of bacterial genomes, including aerobic methanotrophs, nitrite-oxidizers, and sulfate-reducers, and in a subset of aquatic and terrestrial metagenomes. Thus, the potential to produce tetrahymanol is more widespread in the bacterial domain than previously thought. However, Ths is not encoded in any eukaryotic genomes, nor is it homologous to eukaryotic squalene-tetrahymanol cyclase, which catalyzes the cyclization of squalene directly to tetrahymanol. Rather, heterologous expression studies suggest that bacteria couple the cyclization of squalene to a hopene molecule by squalene-hopene cyclase with a subsequent Ths-dependent ring expansion to form tetrahymanol. Thus, bacteria and eukaryotes have evolved distinct biochemical mechanisms for producing tetrahymanol. PMID:26483502

  20. Uniform and Complementary Social Interaction: Distinct Pathways to Solidarity

    PubMed Central

    Koudenburg, Namkje; Postmes, Tom; Gordijn, Ernestine H.; van Mourik Broekman, Aafke

    2015-01-01

    We examine how different forms of co-action give rise to feelings of solidarity. We propose that (a) coordinated action elicits a sense of solidarity, and (b) the process through which such solidarity emerges differs for different forms of co-action. We suggest that whether solidarity within groups emerges from uniform action (e.g. synchronizing, as when people speak in unison) or from more complementary forms of action (e.g. alternating, when speaking in turns) has important consequences for the emergent position of individuals within the group. Uniform action relies on commonality, leaving little scope for individuality. In complementary action each individual makes a distinctive contribution to the group, thereby increasing a sense of personal value to the group, which should contribute to the emergence of solidarity. The predictions receive support from five studies, in which we study groups in laboratory and field settings. Results show that both complementary and uniform co-action increase a sense of solidarity compared to control conditions. However, in the complementary action condition, but not in the uniform action (or synchrony) condition, the effect on feelings of solidarity is mediated by a sense of personal value to the group. PMID:26047131

  1. Uniform and Complementary Social Interaction: Distinct Pathways to Solidarity.

    PubMed

    Koudenburg, Namkje; Postmes, Tom; Gordijn, Ernestine H; van Mourik Broekman, Aafke

    2015-01-01

    We examine how different forms of co-action give rise to feelings of solidarity. We propose that (a) coordinated action elicits a sense of solidarity, and (b) the process through which such solidarity emerges differs for different forms of co-action. We suggest that whether solidarity within groups emerges from uniform action (e.g. synchronizing, as when people speak in unison) or from more complementary forms of action (e.g. alternating, when speaking in turns) has important consequences for the emergent position of individuals within the group. Uniform action relies on commonality, leaving little scope for individuality. In complementary action each individual makes a distinctive contribution to the group, thereby increasing a sense of personal value to the group, which should contribute to the emergence of solidarity. The predictions receive support from five studies, in which we study groups in laboratory and field settings. Results show that both complementary and uniform co-action increase a sense of solidarity compared to control conditions. However, in the complementary action condition, but not in the uniform action (or synchrony) condition, the effect on feelings of solidarity is mediated by a sense of personal value to the group.

  2. A distinct pathway for tetrahymanol synthesis in bacteria

    NASA Astrophysics Data System (ADS)

    Banta, Amy B.; Wei, Jeremy H.; Welander, Paula V.

    2015-11-01

    Tetrahymanol is a polycyclic triterpenoid lipid first discovered in the ciliate Tetrahymena pyriformis whose potential diagenetic product, gammacerane, is often used as a biomarker for water column stratification in ancient ecosystems. Bacteria are also a potential source of tetrahymanol, but neither the distribution of this lipid in extant bacteria nor the significance of bacterial tetrahymanol synthesis for interpreting gammacerane biosignatures is known. Here we couple comparative genomics with genetic and lipid analyses to link a protein of unknown function to tetrahymanol synthesis in bacteria. This tetrahymanol synthase (Ths) is found in a variety of bacterial genomes, including aerobic methanotrophs, nitrite-oxidizers, and sulfate-reducers, and in a subset of aquatic and terrestrial metagenomes. Thus, the potential to produce tetrahymanol is more widespread in the bacterial domain than previously thought. However, Ths is not encoded in any eukaryotic genomes, nor is it homologous to eukaryotic squalene-tetrahymanol cyclase, which catalyzes the cyclization of squalene directly to tetrahymanol. Rather, heterologous expression studies suggest that bacteria couple the cyclization of squalene to a hopene molecule by squalene-hopene cyclase with a subsequent Ths-dependent ring expansion to form tetrahymanol. Thus, bacteria and eukaryotes have evolved distinct biochemical mechanisms for producing tetrahymanol.

  3. Distinct cortical pathways for processing tool versus animal sounds.

    PubMed

    Lewis, James W; Brefczynski, Julie A; Phinney, Raymond E; Janik, John J; DeYoe, Edgar A

    2005-05-25

    Human listeners can effortlessly categorize a wide range of environmental sounds. Whereas categorizing visual object classes (e.g., faces, tools, houses, etc.) preferentially activates different regions of visually sensitive cortex, it is not known whether the auditory system exhibits a similar organization for different types or categories of complex sounds outside of human speech. Using functional magnetic resonance imaging, we show that hearing and correctly or incorrectly categorizing animal vocalizations (as opposed to hand-manipulated tool sounds) preferentially activated middle portions of the left and right superior temporal gyri (mSTG). On average, the vocalization sounds had much greater harmonic and phase-coupling content (acoustically similar to human speech sounds), which may represent some of the signal attributes that preferentially activate the mSTG regions. In contrast, correctly categorized tool sounds (and even animal sounds that were miscategorized as being tool-related sounds) preferentially activated a widespread, predominantly left hemisphere cortical "mirror network." This network directly overlapped substantial portions of motor-related cortices that were independently activated when participants pantomimed tool manipulations with their right (dominant) hand. These data suggest that the recognition processing for some sounds involves a causal reasoning mechanism (a high-level auditory "how" pathway), automatically evoked when attending to hand-manipulated tool sounds, that effectively associates the dynamic motor actions likely to have produced the sound(s).

  4. Enzyme Substrate Specificity Conferred by Distinct Conformational Pathways.

    PubMed

    Rago, Florencia; Saltzberg, Daniel; Allen, Karen N; Tolan, Dean R

    2015-11-04

    Substrate recognition is one of the hallmarks of enzyme catalysis. Enzyme conformational changes have been linked to selectivity between substrates with little direct evidence. Aldolase, a glycolytic enzyme, must distinguish between two physiologically important substrates, fructose 1-phosphate and fructose 1,6-bisphosphate, and provides an excellent model system for the study of this question. Previous work has shown that isozyme specific residues (ISRs) distant from the active site are responsible for kinetic distinction between these substrates. Notably, most of the ISRs reside in a cluster of five surface α-helices, and the carboxyl-terminal region (CTR), and cooperative interactions among these helices have been demonstrated. To test the hypothesis that conformational changes are at the root of these changes, single surface-cysteine variants were created with the cysteine located on helices of the cluster and CTR. This allowed for site-specific labeling with an environmentally sensitive fluorophore, and subsequent monitoring of conformational changes by fluorescence emission spectrophotometry. These labeled variants revealed different spectra in the presence of saturating amounts of each substrate, which suggested the occurrence of different conformations. Emission spectra collected at various substrate concentrations showed a concentration dependence of the fluorescence spectra, consistent with binding events. Lastly, stopped-flow fluorescence spectrophotometry showed that the rate of these fluorescence changes was on the same time-scale as catalysis, thus suggesting a link between the different fluorescence changes and events during catalysis. On the basis of these results, we propose that different conformational changes may be a common mechanism for dictating substrate specificity in other enzymes with multiple substrates.

  5. Bacillus thuringiensis Crystal Protein Cry6Aa Triggers Caenorhabditis elegans Necrosis Pathway Mediated by Aspartic Protease (ASP-1)

    PubMed Central

    Zhang, Fengjuan; Peng, Donghai; Cheng, Chunsheng; Zhou, Wei; Ju, Shouyong; Wan, Danfeng; Yu, Ziquan; Shi, Jianwei; Deng, Yaoyao; Wang, Fenshan; Ye, Xiaobo; Hu, Zhenfei; Lin, Jian; Ruan, Lifang; Sun, Ming

    2016-01-01

    Cell death plays an important role in host-pathogen interactions. Crystal proteins (toxins) are essential components of Bacillus thuringiensis (Bt) biological pesticides because of their specific toxicity against insects and nematodes. However, the mode of action by which crystal toxins to induce cell death is not completely understood. Here we show that crystal toxin triggers cell death by necrosis signaling pathway using crystal toxin Cry6Aa-Caenorhabditis elegans toxin-host interaction system, which involves an increase in concentrations of cytoplasmic calcium, lysosomal lyses, uptake of propidium iodide, and burst of death fluorescence. We find that a deficiency in the necrosis pathway confers tolerance to Cry6Aa toxin. Intriguingly, the necrosis pathway is specifically triggered by Cry6Aa, not by Cry5Ba, whose amino acid sequence is different from that of Cry6Aa. Furthermore, Cry6Aa-induced necrosis pathway requires aspartic protease (ASP-1). In addition, ASP-1 protects Cry6Aa from over-degradation in C. elegans. This is the first demonstration that deficiency in necrosis pathway confers tolerance to Bt crystal protein, and that Cry6A triggers necrosis represents a newly added necrosis paradigm in the C. elegans. Understanding this model could lead to new strategies for nematode control. PMID:26795495

  6. Two Distinct Hepatitis C Virus Genotype 1a Clades Have Different Geographical Distribution and Association With Natural Resistance to NS3 Protease Inhibitors

    PubMed Central

    De Luca, Andrea; Di Giambenedetto, Simona; Lo Presti, Alessandra; Sierra, Saleta; Prosperi, Mattia; Cella, Eleonora; Giovanetti, Marta; Torti, Carlo; Caudai, Cinzia; Vicenti, Ilaria; Saladini, Francesco; Almi, Paolo; Grima, Pierfrancesco; Blanc, Pierluigi; Fabbiani, Massimiliano; Rossetti, Barbara; Gagliardini, Roberta; Kaiser, Rolf; Ciccozzi, Massimo; Zazzi, Maurizio

    2015-01-01

    Background. Hepatitis C virus (HCV) genotype 1 is the most prevalent worldwide. Subtype 1a, compared with 1b, shows lower response rates and higher propensity to select for drug resistance to NS3 and selected NS5A and nonnucleoside NS5B inhibitors. Two distinct clades of subtype 1a have been described. Methods. Using Bayesian methodology, we performed a time-scaled phylogeny reconstruction of clade separation and characterized the geographic distribution, phylodynamics, and association with natural resistance variants of NS3 sequences from 362 patients carrying subtype 1a HCV. Results. All sequences segregated in 2 clearly distinct clades. Clade I showed an earlier origin from the common ancestor compared with clade II. Clade I virus was more prevalent in non-European countries, represented mostly by United States, compared with European (75.7% vs 49.3%; P < .001). The prevalence of the natural NS3 variant Q80K, associated with resistance to the macrocyclic protease inhibitor simeprevir, was detected in 51.6% of clade I and 0% of clade II (P < .001); clade I showed a lower genetic barrier for Q80K, whereas no sign of selective pressure at any protease inhibitor resistance-associated codon was detected. Conclusions. Hepatitis C virus subtype 1a clades have a clearly different distribution in Europe and the United States, and the natural resistance mutation Q80K is exclusively associated with clade I. PMID:26213689

  7. Two Distinct Hepatitis C Virus Genotype 1a Clades Have Different Geographical Distribution and Association With Natural Resistance to NS3 Protease Inhibitors.

    PubMed

    De Luca, Andrea; Di Giambenedetto, Simona; Lo Presti, Alessandra; Sierra, Saleta; Prosperi, Mattia; Cella, Eleonora; Giovanetti, Marta; Torti, Carlo; Caudai, Cinzia; Vicenti, Ilaria; Saladini, Francesco; Almi, Paolo; Grima, Pierfrancesco; Blanc, Pierluigi; Fabbiani, Massimiliano; Rossetti, Barbara; Gagliardini, Roberta; Kaiser, Rolf; Ciccozzi, Massimo; Zazzi, Maurizio

    2015-04-01

    Background.  Hepatitis C virus (HCV) genotype 1 is the most prevalent worldwide. Subtype 1a, compared with 1b, shows lower response rates and higher propensity to select for drug resistance to NS3 and selected NS5A and nonnucleoside NS5B inhibitors. Two distinct clades of subtype 1a have been described. Methods.  Using Bayesian methodology, we performed a time-scaled phylogeny reconstruction of clade separation and characterized the geographic distribution, phylodynamics, and association with natural resistance variants of NS3 sequences from 362 patients carrying subtype 1a HCV. Results.  All sequences segregated in 2 clearly distinct clades. Clade I showed an earlier origin from the common ancestor compared with clade II. Clade I virus was more prevalent in non-European countries, represented mostly by United States, compared with European (75.7% vs 49.3%; P < .001). The prevalence of the natural NS3 variant Q80K, associated with resistance to the macrocyclic protease inhibitor simeprevir, was detected in 51.6% of clade I and 0% of clade II (P < .001); clade I showed a lower genetic barrier for Q80K, whereas no sign of selective pressure at any protease inhibitor resistance-associated codon was detected. Conclusions.  Hepatitis C virus subtype 1a clades have a clearly different distribution in Europe and the United States, and the natural resistance mutation Q80K is exclusively associated with clade I.

  8. Met-ase: Cloning and distinct chromosomal location of a serine protease preferentially expressed in human natural killer cells

    SciTech Connect

    Smyth, M.J.; Trapani, J.A. ); Sayers, T.J.; Wiltrout, T. ); Powers, J.C. )

    1993-12-01

    A cDNA clone encoding a human NK serine protease was obtained by screening a [lambda]-gt10 library from the Lopez NK leukemia with the rat natural killer Met-ase (RNK-Met-1) cDNA clone. In Northern blot analysis human Met-ase (Hu-Met-1) cDNA hybridized with a 0.9-kb mRNA in two human NK leukemia cell lines, unstimulated human PBMC, and untreated purified CD3[sup [minus

  9. Parallel prefrontal pathways reach distinct excitatory and inhibitory systems in memory-related rhinal cortices

    PubMed Central

    Bunce, Jamie G.; Zikopoulos, Basilis; Feinberg, Marcia; Barbas, Helen

    2013-01-01

    To investigate how prefrontal cortices impinge on medial temporal cortices we labeled pathways from the anterior cingulate cortex (ACC) and posterior orbitofrontal cortex (pOFC) in rhesus monkeys to compare their relationship with excitatory and inhibitory systems in rhinal cortices. The ACC pathway terminated mostly in areas 28 and 35 with a high proportion of large terminals, whereas the pOFC pathway terminated mostly through small terminals in area 36 and sparsely in areas 28 and 35. Both pathways terminated in all layers. Simultaneous labeling of pathways and distinct neurochemical classes of inhibitory neurons, followed by analyses of appositions of presynaptic and postsynaptic fluorescent signal, or synapses, showed overall predominant association with spines of putative excitatory neurons, but also significant interactions with presumed inhibitory neurons labeled for calretinin, calbindin or parvalbumin. In the upper layers of areas 28 and 35 the ACC pathway was associated with dendrites of neurons labeled with calretinin, which are thought to disinhibit neighboring excitatory neurons, suggesting facilitated hippocampal access. In contrast, in area 36 pOFC axons were associated with dendrites of calbindin neurons, which are poised to reduce noise and enhance signal. In the deep layers, both pathways innervated mostly dendrites of parvalbumin neurons, which strongly inhibit neighboring excitatory neurons, suggesting gating of hippocampal output to other cortices. These findings suggest that the ACC, associated with attention and context, and the pOFC, associated with emotional valuation, have distinct contributions to memory in rhinal cortices, in processes that are disrupted in psychiatric diseases. PMID:23839697

  10. Dysregulation of MS risk genes and pathways at distinct stages of disease

    PubMed Central

    Srinivasan, Sundararajan; Di Dario, Marco; Russo, Alessandra; Menon, Ramesh; Brini, Elena; Romeo, Marzia; Sangalli, Francesca; Costa, Gloria Dalla; Rodegher, Mariaemma; Radaelli, Marta; Moiola, Lucia; Cantarella, Daniela; Medico, Enzo; Martino, Gianvito; Furlan, Roberto; Martinelli, Vittorio; Comi, Giancarlo

    2017-01-01

    Objective: To perform systematic transcriptomic analysis of multiple sclerosis (MS) risk genes in peripheral blood mononuclear cells (PBMCs) of subjects with distinct MS stages and describe the pathways characterized by dysregulated gene expressions. Methods: We monitored gene expression levels in PBMCs from 3 independent cohorts for a total of 297 cases (including clinically isolated syndromes (CIS), relapsing-remitting MS, primary and secondary progressive MS) and 96 healthy controls by distinct microarray platforms and quantitative PCR. Differential expression and pathway analyses for distinct MS stages were defined and validated by literature mining. Results: Genes located in the vicinity of MS risk variants displayed altered expression in peripheral blood at distinct stages of MS compared with the healthy population. The frequency of dysregulation was significantly higher than expected in CIS and progressive forms of MS. Pathway analysis for each MS stage–specific gene list showed that dysregulated genes contributed to pathogenic processes with scientific evidence in MS. Conclusions: Systematic gene expression analysis in PBMCs highlighted selective dysregulation of MS susceptibility genes playing a role in novel and well-known pathogenic pathways. PMID:28349074

  11. Distinct 3D Architecture and Dynamics of the Human HtrA2(Omi) Protease and Its Mutated Variants

    PubMed Central

    Gieldon, Artur; Zurawa-Janicka, Dorota; Jarzab, Miroslaw; Wenta, Tomasz; Golik, Przemyslaw; Dubin, Grzegorz; Lipinska, Barbara; Ciarkowski, Jerzy

    2016-01-01

    HtrA2(Omi) protease controls protein quality in mitochondria and plays a major role in apoptosis. Its HtrA2S306A mutant (with the catalytic serine routinely disabled for an X-ray study to avoid self-degradation) is a homotrimer whose subunits contain the serine protease domain (PD) and the regulatory PDZ domain. In the inactive state, a tight interdomain interface limits penetration of both PDZ-activating ligands and PD substrates into their respective target sites. We successfully crystalized HtrA2V226K/S306A, whose active counterpart HtrA2V226K has had higher proteolytic activity, suggesting higher propensity to opening the PD-PDZ interface than that of the wild type HtrA2. Yet, the crystal structure revealed the HtrA2V226K/S306A architecture typical of the inactive protein. To get a consistent interpretation of crystallographic data in the light of kinetic results, we employed molecular dynamics (MD). V325D inactivating mutant was used as a reference. Our simulations demonstrated that upon binding of a specific peptide ligand NH2-GWTMFWV-COOH, the PDZ domains open more dynamically in the wild type protease compared to the V226K mutant, whereas the movement is not observed in the V325D mutant. The movement relies on a PDZ vs. PD rotation which opens the PD-PDZ interface in a lid-like (budding flower-like in trimer) fashion. The noncovalent hinges A and B are provided by two clusters of interfacing residues, harboring V325D and V226K in the C- and N-terminal PD barrels, respectively. The opening of the subunit interfaces progresses in a sequential manner during the 50 ns MD simulation. In the systems without the ligand only minor PDZ shifts relative to PD are observed, but the interface does not open. Further activation-associated events, e.g. PDZ-L3 positional swap seen in any active HtrA protein (vs. HtrA2), were not observed. In summary, this study provides hints on the mechanism of activation of wtHtrA2, the dynamics of the inactive HtrA2V325D, but does not

  12. Distinct folding pathways of two homologous disulfide proteins: bovine pancreatic trypsin inhibitor and tick anticoagulant peptide.

    PubMed

    Chang, Jui-Yoa

    2011-01-01

    The folding pathways of disulfide proteins vary substantially (Arolas et al., Trends Biochem Sci 31: 292-301, 2006). The diversity is mainly manifested by (a) the extent of heterogeneity of folding intermediates, (b) the extent of presence of native-like intermediates, and (c) the variation of folding kinetics. Even among structurally similar proteins, the difference can be enormous. This is demonstrated in this concise review with two structurally homologous kunitz-type protease inhibitors, bovine pancreatic trypsin inhibitor and tick anticoagulant peptide, as well as a group of cystine knot proteins. The diversity of their folding mechanisms is illustrated with two different folding techniques: (a) the conventional method of disulfide oxidation (oxidative folding), and (b) the novel method of disulfide scrambling (Chang, J Biol Chem 277: 120-126, 2002). This review also highlights the convergence of folding models concluded form the conventional conformational folding and those obtained by oxidative folding.

  13. Aspergillus protein degradation pathways with different secreted protease sets at neutral and acidic pH.

    PubMed

    Sriranganadane, Dev; Waridel, Patrice; Salamin, Karine; Reichard, Utz; Grouzmann, Eric; Neuhaus, Jean-Marc; Quadroni, Manfredo; Monod, Michel

    2010-07-02

    Aspergillus fumigatus grows well at neutral and acidic pH in a medium containing protein as the sole nitrogen source by secreting two different sets of proteases. Neutral pH favors the secretion of neutral and alkaline endoproteases, leucine aminopeptidases (Laps) which are nonspecific monoaminopeptidases, and an X-prolyl dipeptidase (DppIV). Acidic pH environment promotes the secretion of an aspartic endoprotease of pepsin family (Pep1) and tripeptidyl-peptidases of the sedolisin family (SedB and SedD). A novel prolyl peptidase, AfuS28, was found to be secreted in both alkaline and acidic conditions. In previous studies, Laps were shown to degrade peptides from their N-terminus until an X-Pro sequence acts as a stop signal. X-Pro sequences can be then removed by DppIV, which allows Laps access to the following residues. We have shown that at acidic pH Seds degrade large peptides from their N-terminus into tripeptides until Pro in P1 or P'1 position acts as a stop for these exopeptidases. However, X-X-Pro and X-X-X-Pro sequences can be removed by AfuS28 thus allowing Seds further sequential proteolysis. In conclusion, both alkaline and acidic sets of proteases contain exoprotease activity capable of cleaving after proline residues that cannot be removed during sequential digestion by nonspecific exopeptidases.

  14. Four distinct secretory pathways serve protein secretion, cell surface growth, and peroxisome biogenesis in the yeast Yarrowia lipolytica.

    PubMed Central

    Titorenko, V I; Ogrydziak, D M; Rachubinski, R A

    1997-01-01

    We have identified and characterized mutants of the yeast Yarrowia lipolytica that are deficient in protein secretion, in the ability to undergo dimorphic transition from the yeast to the mycelial form, and in peroxisome biogenesis. Mutations in the SEC238, SRP54, PEX1, PEX2, PEX6, and PEX9 genes affect protein secretion, prevent the exit of the precursor form of alkaline extracellular protease from the endoplasmic reticulum, and compromise peroxisome biogenesis. The mutants sec238A, srp54KO, pex2KO, pex6KO, and pex9KO are also deficient in the dimorphic transition from the yeast to the mycelial form and are affected in the export of only plasma membrane and cell wall-associated proteins specific for the mycelial form. Mutations in the SEC238, SRP54, PEX1, and PEX6 genes prevent or significantly delay the exit of two peroxisomal membrane proteins, Pex2p and Pex16p, from the endoplasmic reticulum en route to the peroxisomal membrane. Mutations in the PEX5, PEX16, and PEX17 genes, which have previously been shown to be essential for peroxisome biogenesis, affect the export of plasma membrane and cell wall-associated proteins specific for the mycelial form but do not impair exit from the endoplasmic reticulum of either Pex2p and Pex16p or of proteins destined for secretion. Biochemical analyses of these mutants provide evidence for the existence of four distinct secretory pathways that serve to deliver proteins for secretion, plasma membrane and cell wall synthesis during yeast and mycelial modes of growth, and peroxisome biogenesis. At least two of these secretory pathways, which are involved in the export of proteins to the external medium and in the delivery of proteins for assembly of the peroxisomal membrane, diverge at the level of the endoplasmic reticulum. PMID:9271399

  15. Distinct Molecular Evolutionary Mechanisms Underlie the Functional Diversification of the Wnt and TGFβ Signaling Pathways

    PubMed Central

    Konikoff, Charlotte E.; Wisotzkey, Robert G.; Stinchfield, Michael J.

    2010-01-01

    The canonical Wnt pathway is one of the oldest and most functionally diverse of animal intercellular signaling pathways. Though much is known about loss-of-function phenotypes for Wnt pathway components in several model organisms, the question of how this pathway achieved its current repertoire of functions has not been addressed. Our phylogenetic analyses of 11 multigene families from five species belonging to distinct phyla, as well as additional analyses employing the 12 Drosophila genomes, suggest frequent gene duplications affecting ligands and receptors as well as co-evolution of new ligand–receptor pairs likely facilitated the expansion of this pathway’s capabilities. Further, several examples of recent gene loss are visible in Drosophila when compared to family members in other phyla. By comparison the TGFβ signaling pathway is characterized by ancient gene duplications of ligands, receptors, and signal transducers with recent duplication events restricted to the vertebrate lineage. Overall, the data suggest that two distinct molecular evolutionary mechanisms can create a functionally diverse developmental signaling pathway. These are the recent dynamic generation of new genes and ligand–receptor interactions as seen in the Wnt pathway and the conservative adaptation of ancient pre-existing genes to new roles as seen in the TGFβ pathway. From a practical perspective, the former mechanism limits the investigator’s ability to transfer knowledge of specific pathway functions across species while the latter facilitates knowledge transfer. Electronic supplementary material The online version of this article (doi:10.1007/s00239-010-9337-z) contains supplementary material, which is available to authorized users. PMID:20339843

  16. The Plasmodium serine-type SERA proteases display distinct expression patterns and non-essential in vivo roles during life cycle progression of the malaria parasite

    PubMed Central

    Putrianti, Elyzana D; Schmidt-Christensen, Anja; Arnold, Iris; Heussler, Volker T; Matuschewski, Kai; Silvie, Olivier

    2010-01-01

    Parasite proteases play key roles in several fundamental steps of the Plasmodium life cycle, including haemoglobin degradation, host cell invasion and parasite egress. Plasmodium exit from infected host cells appears to be mediated by a class of papain-like cysteine proteases called ‘serine repeat antigens’ (SERAs). A SERA subfamily, represented by Plasmodium falciparum SERA5, contains an atypical active site serine residue instead of a catalytic cysteine. Members of this SERAser subfamily are abundantly expressed in asexual blood stages, rendering them attractive drug and vaccine targets. In this study, we show by antibody localization and in vivo fluorescent tagging with the red fluorescent protein mCherry that the two P. berghei serine-type family members, PbSERA1 and PbSERA2, display differential expression towards the final stages of merozoite formation. Via targeted gene replacement, we generated single and double gene knockouts of the P. berghei SERAser genes. These loss-of-function lines progressed normally through the parasite life cycle, suggesting a specialized, non-vital role for serine-type SERAs in vivo. Parasites lacking PbSERAser showed increased expression of the cysteine-type PbSERA3. Compensatory mechanisms between distinct SERA subfamilies may thus explain the absence of phenotypical defect in SERAser disruptants, and challenge the suitability to develop potent antimalarial drugs based on specific inhibitors of Plasmodium serine-type SERAs. PMID:20039882

  17. The Plasmodium serine-type SERA proteases display distinct expression patterns and non-essential in vivo roles during life cycle progression of the malaria parasite.

    PubMed

    Putrianti, Elyzana D; Schmidt-Christensen, Anja; Arnold, Iris; Heussler, Volker T; Matuschewski, Kai; Silvie, Olivier

    2010-06-01

    Parasite proteases play key roles in several fundamental steps of the Plasmodium life cycle, including haemoglobin degradation, host cell invasion and parasite egress. Plasmodium exit from infected host cells appears to be mediated by a class of papain-like cysteine proteases called 'serine repeat antigens' (SERAs). A SERA subfamily, represented by Plasmodium falciparum SERA5, contains an atypical active site serine residue instead of a catalytic cysteine. Members of this SERAser subfamily are abundantly expressed in asexual blood stages, rendering them attractive drug and vaccine targets. In this study, we show by antibody localization and in vivo fluorescent tagging with the red fluorescent protein mCherry that the two P. berghei serine-type family members, PbSERA1 and PbSERA2, display differential expression towards the final stages of merozoite formation. Via targeted gene replacement, we generated single and double gene knockouts of the P. berghei SERAser genes. These loss-of-function lines progressed normally through the parasite life cycle, suggesting a specialized, non-vital role for serine-type SERAs in vivo. Parasites lacking PbSERAser showed increased expression of the cysteine-type PbSERA3. Compensatory mechanisms between distinct SERA subfamilies may thus explain the absence of phenotypical defect in SERAser disruptants, and challenge the suitability to develop potent antimalarial drugs based on specific inhibitors of Plasmodium serine-type SERAs.

  18. Life and death of proteins after protease cleavage: protein degradation by the N-end rule pathway.

    PubMed

    Dissmeyer, Nico; Rivas, Susana; Graciet, Emmanuelle

    2017-06-05

    The N-end rule relates the stability of a protein to the identity of its N-terminal residue and some of its modifications. Since its discovery in the 1980s, the repertoire of N-terminal degradation signals has expanded, leading to a diversity of N-end rule pathways. Although some of these newly discovered N-end rule pathways remain largely unexplored in plants, recent discoveries have highlighted roles of N-end rule-mediated protein degradation in plant defense against pathogens and in cell proliferation during organ growth. Despite this progress, a bottleneck remains the proteome-wide identification of N-end rule substrates due to the prerequisite for endoproteolytic cleavage and technical limitations. Here, we discuss the recent diversification of N-end rule pathways and their newly discovered functions in plant defenses, stressing the role of proteases. We expect that novel proteomics techniques (N-terminomics) will be essential for substrate identification. We review these methods, their limitations and future developments. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  19. Toxin-Antitoxin Modules Are Pliable Switches Activated by Multiple Protease Pathways

    PubMed Central

    Muthuramalingam, Meenakumari; White, John C.; Bourne, Christina R.

    2016-01-01

    Toxin-antitoxin (TA) modules are bacterial regulatory switches that facilitate conflicting outcomes for cells by promoting a pro-survival phenotypic adaptation and/or by directly mediating cell death, all through the toxin activity upon degradation of antitoxin. Intensive study has revealed specific details of TA module functions, but significant gaps remain about the molecular details of activation via antitoxin degradation used by different bacteria and in different environments. This review summarizes the current state of knowledge about the interaction of antitoxins with cellular proteases Lon and ClpP to mediate TA module activation. An understanding of these processes can answer long-standing questions regarding stochastic versus specific activation of TA modules and provide insight into the potential for manipulation of TA modules to alter bacterial growth. PMID:27409636

  20. Wheat cysteine proteases triticain alpha, beta and gamma exhibit mutually distinct responses to gibberellin in germinating seeds.

    PubMed

    Kiyosaki, Toshihiro; Asakura, Tomiko; Matsumoto, Ichiro; Tamura, Tomoko; Terauchi, Kaede; Funaki, Junko; Kuroda, Masaharu; Misaka, Takumi; Abe, Keiko

    2009-01-01

    We cloned three novel papain-type cysteine proteases (CPs), triticain alpha, beta and gamma, from 1-d-germinating wheat seeds. Triticain alpha, beta and gamma were constituted with 461, 472 and 365 amino acid residues, respectively, and had Cys-His-Asn catalytic triads as well as signal and propeptide sequences. Triticain gamma contained a putative vacuole-sorting sequence. Phylogenetic analysis showed that these CPs were divided into mutually different clusters. Triticain alpha and gamma mRNAs were expressed in seeds at an early stage of maturation and at the stage of germination 2d after imbibition, while triticain beta mRNA appeared shortly after imbibition. The expression of mRNAs for triticain alpha and gamma was suppressed by uniconazol, a gibberellin synthesis inhibitor. All the three CP mRNAs were strongly expressed in both embryo and aleurone layers. These results suggest that triticain alpha, beta and gamma play differential roles in seed maturation as well as in digestion of storage proteins during germination.

  1. Three structurally similar odorants trigger distinct signaling pathways in a mouse olfactory neuron.

    PubMed

    Yu, Y; Boyer, N P; Zhang, C

    2014-09-05

    In the mammalian olfactory system, one olfactory sensory neuron (OSN) expresses a single olfactory receptor gene. By calcium imaging of individual OSNs in intact mouse olfactory turbinates, we observed that a subset of OSNs (Ho-OSNs) located in the most ventral olfactory receptor zone can mediate distinct signaling pathways when activated by structurally similar ligands. Calcium imaging showed that Ho-OSNs were highly sensitive to 2-heptanone, heptaldehyde and cis-4-heptenal. 2-heptanone-evoked intracellular calcium elevation was mediated by cAMP signaling while heptaldehyde triggered the diacylglycerol pathway. An increase of intracellular calcium evoked by cis-4-heptenal was due to a combination of activation mediated by the adenylate cyclase pathway and suppression generated by phospholipase C signaling. Pharmacological studies demonstrated that novel mechanisms were involved in the phospholipase C-mediated intracellular calcium changes. Binary-mixture studies and cross-adaptation data indicate that three odorants acted on the same olfactory receptor. The feature that an olfactory receptor mediates multiple signaling pathways was specific for Ho-OSNs and not established in another population of OSNs characterized. Our study suggests that distinct signaling pathways triggered by ligand-induced conformational changes of an olfactory receptor constitute a complex information process mechanism in olfactory transduction. This study has important implications beyond olfaction in that it provides insights of plasticity and complexity of G-protein-coupled receptor activation and signal transduction. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

  2. A Distinct and Parallel Pathway for the Nuclear Import of an mRNA-binding Protein

    PubMed Central

    Pemberton, Lucy F.; Rosenblum, Jonathan S.; Blobel, Günter

    1997-01-01

    Three independent pathways of nuclear import have so far been identified in yeast, each mediated by cognate nuclear transport factors, or karyopherins. Here we have characterized a new pathway to the nucleus, mediated by Mtr10p, a protein first identified in a screen for strains defective in polyadenylated RNA export. Mtr10p is shown to be responsible for the nuclear import of the shuttling mRNA-binding protein Npl3p. A complex of Mtr10p and Npl3p was detected in cytosol, and deletion of Mtr10p was shown to lead to the mislocalization of nuclear Npl3p to the cytoplasm, correlating with a block in import. Mtr10p bound peptide repeat-containing nucleoporins and Ran, suggesting that this import pathway involves a docking step at the nuclear pore complex and is Ran dependent. This pathway of Npl3p import is distinct and does not appear to overlap with another known import pathway for an mRNA-binding protein. Thus, at least two parallel pathways function in the import of mRNA-binding proteins, suggesting the need for the coordination of these pathways. PMID:9412460

  3. Evolutionary conservation and variation of protein folding pathways. Two protease inhibitor homologues from black mamba venom.

    PubMed

    Hollecker, M; Creighton, T E

    1983-08-05

    The pathways of unfolding and refolding of three homologous proteins are shown to be closely related. This implies that folding pathways, as well as the final folded conformation, have been largely conserved during the presumed evolutionary divergence of these proteins from a common ancestor. The pathways of the homologous proteins I and K from black mamba venom were determined here, using the disulphide interaction between their six cysteine residues to trap and identify the intermediate states, and are compared with those determined previously in the same way for the homologous bovine pancreatic trypsin inhibitor. The major one- and two-disulphide intermediates are the same with all three proteins; their kinetic roles are similar, although there are differences in the rates at which they are interconverted and in the minor intermediates that accumulate. As a consequence, different pathways may predominate with another homologous protein, even though the various most favourable pathways are the same. The energetics of the folding transitions and the stabilities of the folded states differ substantially for the three proteins. The differences in stabilities of the fully folded states are primarily reflected kinetically in the rate-determining rearrangements of the native-like conformation; the rates and equilibria of the other steps are not affected markedly. With the less stable proteins, the direct folding pathway of sequential formation of the three correct disulphide bonds becomes significant and is the most facile when considered on a solely intramolecular basis.

  4. Inhibitory properties of separate recombinant Kunitz-type-protease-inhibitor domains from tissue-factor-pathway inhibitor.

    PubMed

    Petersen, L C; Bjørn, S E; Olsen, O H; Nordfang, O; Norris, F; Norris, K

    1996-01-15

    Tissue-factor-pathway inhibitor (TFPI) is a multivalent inhibitor with three tandemly arranged Kunitz- type-protease-inhibitor (KPI) domains. Previous studies [Girard, Y. J., Warren, L. A., Novotny , W. F., Likert, K. M., Brown, S. G., Miletich, J. R & Broze, G. J. (1989) Nature 338, 518-520] by means of site-directed mutagenesis indicated that KPI domain 1 interacts with factor VIIa, that KPI domain 2 interacts with factor Xa, and that KPI domain 3 is apparently without inhibitory function. To elucidate the reaction mechanism of this complex inhibitor, we followed a different approach and studied the inhibitory properties of fragments of TFPI obtained by expression in yeast. Results obtained with TFPI-(1-161)-peptide and separate recombinant TFPI-KPI domains 1, 2 and 3 showed that KPI domain 1 inhibited factor VIIa/tissue factor (Ki = 250 nM), KPI domain 2 inhibited factor Xa (Ki = 90 nM), and that KPI domain 3 was without detectable inhibitory function. Studies with separate KPI domains also showed that KPI domain 2 was mainly responsible for inhibition of trypsin (Ki = 0.1 nM) and chymotrypsin (Ki = 0.75 nM), whereas KPI domain 1 inhibited plasmin (Ki = 26 nM) and cathepsin G (Ki = 200 nM). The structural basis for the interaction between serine proteases and KPI domains is discussed in terms of putative three-dimensional models of the proteins derived by comparative molecular-modelling methods. Studies of factor Xa inhibition by intact TFPI (Ki approximately 0.02 nM) suggested that regions other than the contact area of the KPI domain, interacted strongly with factor Xa. Secondary-site interactions were crucial for TFPI inhibition of factor Xa but was of little or no importance for its inhibition of trypsin.

  5. Distinct Membrane Disruption Pathways Are Induced by 40-Residue β-Amyloid Peptides.

    PubMed

    Delgado, Dennis A; Doherty, Katelynne; Cheng, Qinghui; Kim, Hyeongeun; Xu, Dawei; Dong, He; Grewer, Christof; Qiang, Wei

    2016-06-03

    Cellular membrane disruption induced by β-amyloid (Aβ) peptides has been considered one of the major pathological mechanisms for Alzheimer disease. Mechanistic studies of the membrane disruption process at a high-resolution level, on the other hand, are hindered by the co-existence of multiple possible pathways, even in simplified model systems such as the phospholipid liposome. Therefore, separation of these pathways is crucial to achieve an in-depth understanding of the Aβ-induced membrane disruption process. This study, which utilized a combination of multiple biophysical techniques, shows that the peptide-to-lipid (P:L) molar ratio is an important factor that regulates the selection of dominant membrane disruption pathways in the presence of 40-residue Aβ peptides in liposomes. Three distinct pathways (fibrillation with membrane content leakage, vesicle fusion, and lipid uptake through a temporarily stable ionic channel) become dominant in model liposome systems under specific conditions. These individual systems are characterized by both the initial states of Aβ peptides and the P:L molar ratio. Our results demonstrated the possibility to generate simplified Aβ-membrane model systems with a homogeneous membrane disruption pathway, which will benefit high-resolution mechanistic studies in the future. Fundamentally, the possibility of pathway selection controlled by P:L suggests that the driving forces for Aβ aggregation and Aβ-membrane interactions may be similar at the molecular level. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Formation of distinct inclusion bodies by inhibition of ubiquitin-proteasome and autophagy-lysosome pathways

    SciTech Connect

    Lee, Junho; Yang, Kyu-Hwan; Joe, Cheol O.; Kang, Seok-Seong

    2011-01-14

    Research highlights: {yields} Distinct inclusion bodies are developed by inhibition of UPP and ALP. {yields} The inclusion bodies differ in morphology, localization and formation process. {yields} The inclusion bodies are distinguishable by the localization of TSC2. {yields} Inhibition of both UPP and ALP simultaneously induces those inclusion bodies. -- Abstract: Accumulation of misfolded proteins is caused by the impairment of protein quality control systems, such as ubiquitin-proteasome pathway (UPP) and autophagy-lysosome pathway (ALP). In this study, the formation of inclusion bodies was examined after the blockade of UPP and/or ALP in A549 cells. UPP inhibition induced a single and large inclusion body localized in microtubule-organizing center. Interestingly, however, ALP inhibition generated dispersed small inclusion bodies in the cytoplasm. Tuberous sclerosis complex 2 was selectively accumulated in the inclusion bodies of UPP-inhibited cells, but not those of ALP-inhibited cells. Blockade of transcription and translation entirely inhibited the formation of inclusion body induced by UPP inhibition, but partially by ALP inhibition. Moreover, the simultaneous inhibition of two protein catabolic pathways independently developed two distinct inclusion bodies within a single cell. These findings clearly demonstrated that dysfunction of each catabolic pathway induced formation and accumulation of unique inclusion bodies on the basis of morphology, localization and formation process in A549 cells.

  7. A Distinct Pathway for Polar Exocytosis in Plant Cell Wall Formation1[OPEN

    PubMed Central

    Wang, Hao; Zhuang, Xiaohong; Wang, Xiangfeng; Law, Angus Ho Yin; Zhao, Teng; Du, Shengwang; Loy, Michael M.T.; Jiang, Liwen

    2016-01-01

    Post-Golgi protein sorting and trafficking to the plasma membrane (PM) is generally believed to occur via the trans-Golgi network (TGN). In this study using Nicotiana tabacum pectin methylesterase (NtPPME1) as a marker, we have identified a TGN-independent polar exocytosis pathway that mediates cell wall formation during cell expansion and cytokinesis. Confocal immunofluorescence and immunogold electron microscopy studies demonstrated that Golgi-derived secretory vesicles (GDSVs) labeled by NtPPME1-GFP are distinct from those organelles belonging to the conventional post-Golgi exocytosis pathway. In addition, pharmaceutical treatments, superresolution imaging, and dynamic studies suggest that NtPPME1 follows a polar exocytic process from Golgi-GDSV-PM/cell plate (CP), which is distinct from the conventional Golgi-TGN-PM/CP secretion pathway. Further studies show that ROP1 regulates this specific polar exocytic pathway. Taken together, we have demonstrated an alternative TGN-independent Golgi-to-PM polar exocytic route, which mediates secretion of NtPPME1 for cell wall formation during cell expansion and cytokinesis and is ROP1-dependent. PMID:27531442

  8. Azospirillum brasilense Chemotaxis Depends on Two Signaling Pathways Regulating Distinct Motility Parameters

    PubMed Central

    Mukherjee, Tanmoy; Kumar, Dhivya; Burriss, Nathan; Xie, Zhihong

    2016-01-01

    ABSTRACT The genomes of most motile bacteria encode two or more chemotaxis (Che) systems, but their functions have been characterized in only a few model systems. Azospirillum brasilense is a motile soil alphaproteobacterium able to colonize the rhizosphere of cereals. In response to an attractant, motile A. brasilense cells transiently increase swimming speed and suppress reversals. The Che1 chemotaxis pathway was previously shown to regulate changes in the swimming speed, but it has a minor role in chemotaxis and root surface colonization. Here, we show that a second chemotaxis system, named Che4, regulates the probability of swimming reversals and is the major signaling pathway for chemotaxis and wheat root surface colonization. Experimental evidence indicates that Che1 and Che4 are functionally linked to coordinate changes in the swimming motility pattern in response to attractants. The effect of Che1 on swimming speed is shown to enhance the aerotactic response of A. brasilense in gradients, likely providing the cells with a competitive advantage in the rhizosphere. Together, the results illustrate a novel mechanism by which motile bacteria utilize two chemotaxis pathways regulating distinct motility parameters to alter movement in gradients and enhance the chemotactic advantage. IMPORTANCE Chemotaxis provides motile bacteria with a competitive advantage in the colonization of diverse niches and is a function enriched in rhizosphere bacterial communities, with most species possessing at least two chemotaxis systems. Here, we identify the mechanism by which cells may derive a significant chemotactic advantage using two chemotaxis pathways that ultimately regulate distinct motility parameters. PMID:27068592

  9. Definition of Two Angiogenic Pathways by Distinct α_v Integrins

    NASA Astrophysics Data System (ADS)

    Friedlander, Martin; Brooks, Peter C.; Shaffer, Robert W.; Kincaid, Christine M.; Varner, Judith A.; Cheresh, David A.

    1995-12-01

    Angiogenesis depends on cytokines and vascular cell adhesion events. Two cytokine-dependent pathways of angiogenesis were shown to exist and were defined by their dependency on distinct vascular cell integrins. In vivo angiogenesis in corneal or chorioallantoic membrane models induced by basic fibroblast growth factor or by tumor necrosis factor-α depended on α_vβ_3, whereas angiogenesis initiated by vascular endothelial growth factor, transforming growth factor-α, or phorbol ester depended on α_vβ_5. Antibody to each integrin selectively blocked one of these pathways, and a cyclic peptide antagonist of both integrins blocked angiogenesis stimulated by each cytokine tested. These pathways are further distinguished by their sensitivity to calphostin C, an inhibitor of protein kinase C that blocked angiogenesis potentiated by α_vβ_5 but not by α_vβ_3.

  10. Distinct myeloid progenitor differentiation pathways identified through single cell RNA sequencing

    PubMed Central

    Drissen, Roy; Buza-Vidas, Natalija; Woll, Petter; Thongjuea, Supat; Gambardella, Adriana; Giustacchini, Alice; Mancini, Elena; Zriwil, Alya; Lutteropp, Michael; Grover, Amit; Mead, Adam; Sitnicka, Ewa

    2016-01-01

    According to current models for hematopoiesis, lymphoid-primed multi-potent progenitors (LMPPs; Lin−Sca-1+c-Kit+CD34+Flt3hi) and common myeloid progenitors (CMPs; Lin−Sca-1+c-Kit+CD34+CD41hi) establish an early branch point for separate lineage commitment pathways from hematopoietic stem cells, with the notable exception that both pathways are proposed to generate all myeloid innate immune cell types through the same myeloid-restricted pre-granulocyte-macrophage progenitor (pre-GM; Lin−Sca-1−c-Kit+CD41−FcγRII/III−CD150−CD105−). By single cell transcriptome profiling of pre-GMs we identify distinct myeloid differentiation pathways: a Gata1-expressing pathway generates mast cells, eosinophils, megakaryocytes and erythroid cells, and a Gata1-negative pathway that generates monocytes, neutrophils and lymphocytes. These results identify an early hematopoietic lineage bifurcation, separating the myeloid lineages prior to their segregation from other hematopoietic lineage potentials. PMID:27043410

  11. Steady and transient fluid shear stress stimulate NO release in osteoblasts through distinct biochemical pathways

    NASA Technical Reports Server (NTRS)

    McAllister, T. N.; Frangos, J. A.

    1999-01-01

    Fluid flow has been shown to be a potent stimulus in osteoblasts and osteocytes and may therefore play an important role in load-induced bone remodeling. The objective of this study was to investigate the characteristics of flow-activated pathways. Previously we reported that fluid flow stimulates rapid and continuous release of nitric oxide (NO) in primary rat calvarial osteoblasts. Here we demonstrate that flow-induced NO release is mediated by shear stress and that this response is distinctly biphasic. Transients in shear stress associated with the onset of flow stimulated a burst in NO production (8.2 nmol/mg of protein/h), while steady flow stimulated sustained NO production (2.2 nmol/mg of protein/h). Both G-protein inhibition and calcium chelation abolished the burst phase but had no effect on sustained production. Activation of G-proteins stimulated dose-dependent NO release in static cultures of both calvarial osteoblasts and UMR-106 osteoblast-like cells. Pertussis toxin had no effect on NO release. Calcium ionophore stimulated low levels of NO production within 15 minutes but had no effect on sustained production. Taken together, these data suggest that fluid shear stress stimulates NO release by two distinct pathways: a G-protein and calcium-dependent phase sensitive to flow transients, and a G-protein and calcium-independent pathway stimulated by sustained flow.

  12. Steady and transient fluid shear stress stimulate NO release in osteoblasts through distinct biochemical pathways

    NASA Technical Reports Server (NTRS)

    McAllister, T. N.; Frangos, J. A.

    1999-01-01

    Fluid flow has been shown to be a potent stimulus in osteoblasts and osteocytes and may therefore play an important role in load-induced bone remodeling. The objective of this study was to investigate the characteristics of flow-activated pathways. Previously we reported that fluid flow stimulates rapid and continuous release of nitric oxide (NO) in primary rat calvarial osteoblasts. Here we demonstrate that flow-induced NO release is mediated by shear stress and that this response is distinctly biphasic. Transients in shear stress associated with the onset of flow stimulated a burst in NO production (8.2 nmol/mg of protein/h), while steady flow stimulated sustained NO production (2.2 nmol/mg of protein/h). Both G-protein inhibition and calcium chelation abolished the burst phase but had no effect on sustained production. Activation of G-proteins stimulated dose-dependent NO release in static cultures of both calvarial osteoblasts and UMR-106 osteoblast-like cells. Pertussis toxin had no effect on NO release. Calcium ionophore stimulated low levels of NO production within 15 minutes but had no effect on sustained production. Taken together, these data suggest that fluid shear stress stimulates NO release by two distinct pathways: a G-protein and calcium-dependent phase sensitive to flow transients, and a G-protein and calcium-independent pathway stimulated by sustained flow.

  13. Magnetic resonance image features identify glioblastoma phenotypic subtypes with distinct molecular pathway activities

    PubMed Central

    Itakura, Haruka; Achrol, Achal S.; Mitchell, Lex A.; Loya, Joshua J.; Liu, Tiffany; Westbroek, Erick M.; Feroze, Abdullah H.; Rodriguez, Scott; Echegaray, Sebastian; Azad, Tej D.; Yeom, Kristen W.; Napel, Sandy; Rubin, Daniel L.; Chang, Steven D.; Harsh, Griffith R.; Gevaert, Olivier

    2015-01-01

    Glioblastoma (GBM) is the most common and highly lethal primary malignant brain tumor in adults. There is a dire need for easily accessible, noninvasive biomarkers that can delineate underlying molecular activities and predict response to therapy. To this end, we sought to identify subtypes of GBM, differentiated solely by quantitative MR imaging features, that could be used for better management of GBM patients. Quantitative image features capturing the shape, texture, and edge sharpness of each lesion were extracted from MR images of 121 patients with de novo, solitary, unilateral GBM. Three distinct phenotypic “clusters” emerged in the development cohort using consensus clustering with 10,000 iterations on these image features. These three clusters—pre-multifocal, spherical, and rim-enhancing, names reflecting their image features—were validated in an independent cohort consisting of 144 multi-institution patients with similar tumor characteristics from The Cancer Genome Atlas (TCGA). Each cluster mapped to a unique set of molecular signaling pathways using pathway activity estimates derived from analysis of TCGA tumor copy number and gene expression data with the PARADIGM algorithm. Distinct pathways, such as c-Kit and FOXA, were enriched in each cluster, indicating differential molecular activities as determined by image features. Each cluster also demonstrated differential probabilities of survival, indicating prognostic importance. Our imaging method offers a noninvasive approach to stratify GBM patients and also provides unique sets of molecular signatures to inform targeted therapy and personalized treatment of GBM. PMID:26333934

  14. A melanoma cell state distinction influences sensitivity to MAPK pathway inhibitors

    PubMed Central

    Konieczkowski, David J.; Johannessen, Cory M.; Abudayyeh, Omar; Kim, Jong Wook; Cooper, Zachary A.; Piris, Adriano; Frederick, Dennie T.; Barzily-Rokni, Michal; Straussman, Ravid; Haq, Rizwan; Fisher, David E.; Mesirov, Jill P.; Hahn, William C.; Flaherty, Keith T.; Wargo, Jennifer A.; Tamayo, Pablo; Garraway, Levi A.

    2014-01-01

    Most melanomas harbor oncogenic BRAFV600 mutations, which constitutively activate the MAP kinase (MAPK) pathway. Although MAPK pathway inhibitors show clinical benefit in BRAFV600-mutant melanoma, it remains incompletely understood why 10-20% of patients fail to respond. Here, we show that RAF inhibitor sensitive and resistant BRAFV600-mutant melanomas display distinct transcriptional profiles. Whereas most drug-sensitive cell lines and patient biopsies showed high expression and activity of the melanocytic lineage transcription factor MITF, intrinsically resistant cell lines and biopsies displayed low MITF expression but higher levels of NF-κB signaling and the receptor tyrosine kinase AXL. In vitro, these MITF-low/NF-κB-high melanomas were resistant to inhibition of RAF and MEK, singly or in combination, and ERK. Moreover, in cell lines, NF-κB activation antagonized MITF expression and induced both resistance marker genes and drug resistance. Thus, distinct cell states characterized by MITF or NF-κB activity may influence intrinsic resistance to MAPK pathway inhibitors in BRAFV600-mutant melanoma. PMID:24771846

  15. Proteases as therapeutics

    PubMed Central

    Craik, Charles S.; Page, Michael J.; Madison, Edwin L.

    2015-01-01

    Proteases are an expanding class of drugs that hold great promise. The U.S. FDA (Food and Drug Administration) has approved 12 protease therapies, and a number of next generation or completely new proteases are in clinical development. Although they are a well-recognized class of targets for inhibitors, proteases themselves have not typically been considered as a drug class despite their application in the clinic over the last several decades; initially as plasma fractions and later as purified products. Although the predominant use of proteases has been in treating cardiovascular disease, they are also emerging as useful agents in the treatment of sepsis, digestive disorders, inflammation, cystic fibrosis, retinal disorders, psoriasis and other diseases. In the present review, we outline the history of proteases as therapeutics, provide an overview of their current clinical application, and describe several approaches to improve and expand their clinical application. Undoubtedly, our ability to harness proteolysis for disease treatment will increase with our understanding of protease biology and the molecular mechanisms responsible. New technologies for rationally engineering proteases, as well as improved delivery options, will expand greatly the potential applications of these enzymes. The recognition that proteases are, in fact, an established class of safe and efficacious drugs will stimulate investigation of additional therapeutic applications for these enzymes. Proteases therefore have a bright future as a distinct therapeutic class with diverse clinical applications. PMID:21406063

  16. Distinct Cortical Pathways for Music and Speech Revealed by Hypothesis-Free Voxel Decomposition

    PubMed Central

    Norman-Haignere, Sam

    2015-01-01

    SUMMARY The organization of human auditory cortex remains unresolved, due in part to the small stimulus sets common to fMRI studies and the overlap of neural populations within voxels. To address these challenges, we measured fMRI responses to 165 natural sounds and inferred canonical response profiles (“components”) whose weighted combinations explained voxel responses throughout auditory cortex. This analysis revealed six components, each with interpretable response characteristics despite being unconstrained by prior functional hypotheses. Four components embodied selectivity for particular acoustic features (frequency, spectrotemporal modulation, pitch). Two others exhibited pronounced selectivity for music and speech, respectively, and were not explainable by standard acoustic features. Anatomically, music and speech selectivity concentrated in distinct regions of non-primary auditory cortex. However, music selectivity was weak in raw voxel responses, and its detection required a decomposition method. Voxel decomposition identifies primary dimensions of response variation across natural sounds, revealing distinct cortical pathways for music and speech. PMID:26687225

  17. Rac-1 and Raf-1 kinases, components of distinct signaling pathways, activate myotonic dystrophy protein kinase

    NASA Technical Reports Server (NTRS)

    Shimizu, M.; Wang, W.; Walch, E. T.; Dunne, P. W.; Epstein, H. F.

    2000-01-01

    Myotonic dystrophy protein kinase (DMPK) is a serine-threonine protein kinase encoded by the myotonic dystrophy (DM) locus on human chromosome 19q13.3. It is a close relative of other kinases that interact with members of the Rho family of small GTPases. We show here that the actin cytoskeleton-linked GTPase Rac-1 binds to DMPK, and coexpression of Rac-1 and DMPK activates its transphosphorylation activity in a GTP-sensitive manner. DMPK can also bind Raf-1 kinase, the Ras-activated molecule of the MAP kinase pathway. Purified Raf-1 kinase phosphorylates and activates DMPK. The interaction of DMPK with these distinct signals suggests that it may play a role as a nexus for cross-talk between their respective pathways and may partially explain the remarkable pleiotropy of DM.

  18. Azospirillum brasilense Chemotaxis Depends on Two Signaling Pathways Regulating Distinct Motility Parameters.

    PubMed

    Mukherjee, Tanmoy; Kumar, Dhivya; Burriss, Nathan; Xie, Zhihong; Alexandre, Gladys

    2016-06-15

    The genomes of most motile bacteria encode two or more chemotaxis (Che) systems, but their functions have been characterized in only a few model systems. Azospirillum brasilense is a motile soil alphaproteobacterium able to colonize the rhizosphere of cereals. In response to an attractant, motile A. brasilense cells transiently increase swimming speed and suppress reversals. The Che1 chemotaxis pathway was previously shown to regulate changes in the swimming speed, but it has a minor role in chemotaxis and root surface colonization. Here, we show that a second chemotaxis system, named Che4, regulates the probability of swimming reversals and is the major signaling pathway for chemotaxis and wheat root surface colonization. Experimental evidence indicates that Che1 and Che4 are functionally linked to coordinate changes in the swimming motility pattern in response to attractants. The effect of Che1 on swimming speed is shown to enhance the aerotactic response of A. brasilense in gradients, likely providing the cells with a competitive advantage in the rhizosphere. Together, the results illustrate a novel mechanism by which motile bacteria utilize two chemotaxis pathways regulating distinct motility parameters to alter movement in gradients and enhance the chemotactic advantage. Chemotaxis provides motile bacteria with a competitive advantage in the colonization of diverse niches and is a function enriched in rhizosphere bacterial communities, with most species possessing at least two chemotaxis systems. Here, we identify the mechanism by which cells may derive a significant chemotactic advantage using two chemotaxis pathways that ultimately regulate distinct motility parameters. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  19. Major histocompatibility complex class II expression distinguishes two distinct B cell developmental pathways during ontogeny

    PubMed Central

    1994-01-01

    All mature B cells coexpress major histocompatibility complex (MHC) class II molecules, I-A and I-E, which are restriction elements required for antigen presentation to CD4+ T cells. However, the expression of class II during the early stages of B cell development has been unclear. We demonstrate here that there is a difference in the expression of class II during murine B cell development in the fetal liver and adult bone marrow (BM). These differences define two distinct B cell developmental pathways. The Fetal-type (FT) pathway is characterized by pre-B and immature IgM+ B cells generated in the fetal liver which initially lack all class II expression. In contrast, the Adult-type (AT) pathway is typified by B cells developing in the adult BM which express class II molecules from the pre-B cell stage. In vitro stromal cell cultures of sorted fetal liver and adult BM pro-B cells indicated that the difference in I-A expression during B cell development is intrinsic to the progenitors. In addition, we show that FT B cell development is not restricted to the fetal liver but occurs in the peritoneal cavities, spleens, liver, and BM of young mice up to at least 1 mo of age. The AT B cell development begins to emerge after birth but is, however, restricted to the BM environment. These findings indicate that there are two distinct B cell developmental pathways during ontogeny, each of which could contribute differentially to the immune repertoire and thus the functions of B cell subsets and lineages. PMID:7913950

  20. Noise and interlocking signaling pathways promote distinct transcription factor dynamics in response to different stresses

    PubMed Central

    Petrenko, Natalia; Chereji, Raˇzvan V.; McClean, Megan N.; Morozov, Alexandre V.; Broach, James R.

    2013-01-01

    All cells perceive and respond to environmental stresses through elaborate stress-sensing networks. Yeast cells sense stress through diverse signaling pathways that converge on the transcription factors Msn2 and Msn4, which respond by initiating rapid, idiosyncratic cycles into and out of the nucleus. To understand the role of Msn2/4 nuclear localization dynamics, we combined time-lapse studies of Msn2-GFP localization in living cells with computational modeling of stress-sensing signaling networks. We find that several signaling pathways, including Ras/protein kinase A, AMP-activated kinase, the high-osmolarity response mitogen-activated protein kinase pathway, and protein phosphatase 1, regulate activation of Msn2 in distinct ways in response to different stresses. Moreover, we find that bursts of nuclear localization elicit a more robust transcriptional response than does sustained nuclear localization. Using stochastic modeling, we reproduce in silico the responses of Msn2 to different stresses, and demonstrate that bursts of localization arise from noise in the signaling pathways amplified by the small number of Msn2 molecules in the cell. This noise imparts diverse behaviors to genetically identical cells, allowing cell populations to “hedge their bets” in responding to an uncertain future, and to balance growth and survival in an unpredictable environment. PMID:23615444

  1. [Cognition-Emotion Interactions and Psychopathic Personality: Distinct Pathways to Antisocial and Violent Behavior].

    PubMed

    Verona, Edelyn

    Researchers have long acknowledged heterogeneity among persons who exhibit antisocial and violent behaviours. The study of psychopathic personality or psychopathy can help elucidate this heterogeneity through examination of the different facets that constitute this disorder. In particular, the distinct correlates of the interpersonal-affective traits (Factor 1) and the impulsive-antisocial traits (Factor 2) of psychopathy suggest at least two possible pathways to antisocial behaviours. Building on basic studies in cognitive and affective neuroscience, we provide a focused, non-comprehensive review of work identifying the biopsychological mechanisms involved in these two pathways, with special attention to studies using event-related potential (ERP) methods. In specific, a series of studies are discussed which examined affective and cognitive processes that may distinguish offenders high on psychopathic traits from other offenders, with emphasis on alterations in emotion-cognition interactions related to each factor of psychopathy. The set of findings reviewed highlight a central conclusion: Factor 1 represents a pathway involving reduced emotional responding, exacerbated by attentional abnormalities, that make for a more deliberate and emotionally insensitive offender profile. In contrast, Factor 2 characterizes a pathway marked by emotional and behavioural dysregulation and cognitive control dysfunctions, particularly in emotional contexts. Implications for identifying etiological processes and the further understanding of antisocial and violent behaviours are discussed.

  2. JAK2 V617F uses distinct signalling pathways to induce cell proliferation and neutrophil activation.

    PubMed

    Oku, Seido; Takenaka, Katsuto; Kuriyama, Takuro; Shide, Kotaro; Kumano, Takashi; Kikushige, Yoshikane; Urata, Shingo; Yamauchi, Takuji; Iwamoto, Chika; Shimoda, Haruko K; Miyamoto, Toshihiro; Nagafuji, Koji; Kishimoto, Junji; Shimoda, Kazuya; Akashi, Koichi

    2010-08-01

    The acquired JAK2 V617F mutation is observed in the majority of patients with BCR-ABL1 negative chronic myeloproliferative neoplasms (MPN). BCR-ABL1 negative MPN displays myeloproliferation with an elevated leucocyte alkaline phosphatase (LAP) activity, a neutrophil activation marker. We tried to separate the downstream signalling of JAK2 V617F to stimulate myeloproliferation and LAP activity. NB4, a myeloid lineage cell line, was transduced with Jak2 V617F mutation or wild-type Jak2. We found that Jak2 V617F mutation, but not wild-type Jak2 enhanced LAP expression in NB4-derived neutrophils and proliferation of NB4 cells. JAK2 V617F induces constitutive phosphorylation of STAT3 and STAT5, and uses signalling targets such as Ras/MEK/ERK and PI3K/Akt pathways. By using MEK1/2 inhibitor U0126, PI3K inhibitor LY294002, and STAT3 or STAT5 siRNAs, JAK2 V617F was found to specifically use the STAT3 pathway to enhance LAP expression, while STAT5, Ras/MEK/ERK and PI3K/Akt, but not STAT3 pathways, were able to stimulate cell proliferation. These data strongly suggest that JAK2 V617F uses distinct signalling pathways to induce typical pathological features of MPN, such as high LAP activity and enhanced cell proliferation.

  3. Noise and interlocking signaling pathways promote distinct transcription factor dynamics in response to different stresses.

    PubMed

    Petrenko, Natalia; Chereji, Razvan V; McClean, Megan N; Morozov, Alexandre V; Broach, James R

    2013-06-01

    All cells perceive and respond to environmental stresses through elaborate stress-sensing networks. Yeast cells sense stress through diverse signaling pathways that converge on the transcription factors Msn2 and Msn4, which respond by initiating rapid, idiosyncratic cycles into and out of the nucleus. To understand the role of Msn2/4 nuclear localization dynamics, we combined time-lapse studies of Msn2-GFP localization in living cells with computational modeling of stress-sensing signaling networks. We find that several signaling pathways, including Ras/protein kinase A, AMP-activated kinase, the high-osmolarity response mitogen-activated protein kinase pathway, and protein phosphatase 1, regulate activation of Msn2 in distinct ways in response to different stresses. Moreover, we find that bursts of nuclear localization elicit a more robust transcriptional response than does sustained nuclear localization. Using stochastic modeling, we reproduce in silico the responses of Msn2 to different stresses, and demonstrate that bursts of localization arise from noise in the signaling pathways amplified by the small number of Msn2 molecules in the cell. This noise imparts diverse behaviors to genetically identical cells, allowing cell populations to "hedge their bets" in responding to an uncertain future, and to balance growth and survival in an unpredictable environment.

  4. Formation of distinct inclusion bodies by inhibition of ubiquitin-proteasome and autophagy-lysosome pathways.

    PubMed

    Lee, Junho; Yang, Kyu-Hwan; Joe, Cheol O; Kang, Seok-Seong

    2011-01-14

    Accumulation of misfolded proteins is caused by the impairment of protein quality control systems, such as ubiquitin-proteasome pathway (UPP) and autophagy-lysosome pathway (ALP). In this study, the formation of inclusion bodies was examined after the blockade of UPP and/or ALP in A549 cells. UPP inhibition induced a single and large inclusion body localized in microtubule-organizing center. Interestingly, however, ALP inhibition generated dispersed small inclusion bodies in the cytoplasm. Tuberous sclerosis complex 2 was selectively accumulated in the inclusion bodies of UPP-inhibited cells, but not those of ALP-inhibited cells. Blockade of transcription and translation entirely inhibited the formation of inclusion body induced by UPP inhibition, but partially by ALP inhibition. Moreover, the simultaneous inhibition of two protein catabolic pathways independently developed two distinct inclusion bodies within a single cell. These findings clearly demonstrated that dysfunction of each catabolic pathway induced formation and accumulation of unique inclusion bodies on the basis of morphology, localization and formation process in A549 cells. Copyright © 2010 Elsevier Inc. All rights reserved.

  5. Distinct Annular Oligomers Captured along the Assembly and Disassembly Pathways of Transthyretin Amyloid Protofibrils

    PubMed Central

    Pires, Ricardo H.; Karsai, Árpád; Saraiva, Maria J.; Damas, Ana M.; Kellermayer, Miklós S. Z.

    2012-01-01

    Background Defects in protein folding may lead to severe degenerative diseases characterized by the appearance of amyloid fibril deposits. Cytotoxicity in amyloidoses has been linked to poration of the cell membrane that may involve interactions with amyloid intermediates of annular shape. Although annular oligomers have been detected in many amyloidogenic systems, their universality, function and molecular mechanisms of appearance are debated. Methodology/Principal Findings We investigated with high-resolution in situ atomic force microscopy the assembly and disassembly of transthyretin (TTR) amyloid protofibrils formed of the native protein by pH shift. Annular oligomers were the first morphologically distinct intermediates observed in the TTR aggregation pathway. Morphological analysis suggests that they can assemble into a double-stack of octameric rings with a 16±2 nm diameter, and displaying the tendency to form linear structures. According to light scattering data coupled to AFM imaging, annular oligomers appeared to undergo a collapse type of structural transition into spheroid oligomers containing 8–16 monomers. Disassembly of TTR amyloid protofibrils also resulted in the rapid appearance of annular oligomers but with a morphology quite distinct from that observed in the assembly pathway. Conclusions/Significance Our observations indicate that annular oligomers are key dynamic intermediates not only in the assembly but also in the disassembly of TTR protofibrils. The balance between annular and more compact forms of aggregation could be relevant for cytotoxicity in amyloidogenic disorders. PMID:22984597

  6. Distinct cellular pathways select germline-encoded and somatically mutated antibodies into immunological memory

    PubMed Central

    Kaji, Tomohiro; Ishige, Akiko; Hikida, Masaki; Taka, Junko; Hijikata, Atsushi; Kubo, Masato; Nagashima, Takeshi; Takahashi, Yoshimasa; Kurosaki, Tomohiro; Okada, Mariko; Ohara, Osamu

    2012-01-01

    One component of memory in the antibody system is long-lived memory B cells selected for the expression of somatically mutated, high-affinity antibodies in the T cell–dependent germinal center (GC) reaction. A puzzling observation has been that the memory B cell compartment also contains cells expressing unmutated, low-affinity antibodies. Using conditional Bcl6 ablation, we demonstrate that these cells are generated through proliferative expansion early after immunization in a T cell–dependent but GC-independent manner. They soon become resting and long-lived and display a novel distinct gene expression signature which distinguishes memory B cells from other classes of B cells. GC-independent memory B cells are later joined by somatically mutated GC descendants at roughly equal proportions and these two types of memory cells efficiently generate adoptive secondary antibody responses. Deletion of T follicular helper (Tfh) cells significantly reduces the generation of mutated, but not unmutated, memory cells early on in the response. Thus, B cell memory is generated along two fundamentally distinct cellular differentiation pathways. One pathway is dedicated to the generation of high-affinity somatic antibody mutants, whereas the other preserves germ line antibody specificities and may prepare the organism for rapid responses to antigenic variants of the invading pathogen. PMID:23027924

  7. Synonymous Mutations and Ribosome Stalling Can Lead to Altered Folding Pathways and Distinct Minima

    PubMed Central

    Tsai, Chung-Jung; Sauna, Zuben E.; Kimchi-Sarfaty, Chava; Ambudkar, Suresh V.; Gottesman, Michael M.; Nussinov, Ruth

    2008-01-01

    How can we understand a case where a given amino acid sequence folds into structurally and functionally distinct molecules? Synonymous single-nucleotide polymorphisms (SNPs) in the multidrug resistance 1 (MDR1 or ABCB1) gene involving frequent to rare codon substitutions lead to identical protein sequences. Remarkably these alternative sequences give a protein product with similar but different structures and functions. Here we propose that long-enough ribosomal pause time-scales may lead to alternate folding pathways and distinct minima on the folding free energy surface. While the conformational and functional differences between the native and alternate states may be minor, the MDR1 case illustrates that the barriers may nevertheless constitute sufficiently high hurdles in physiological time-scales, leading to kinetically trapped states with altered structures and functions. Different folding pathways leading to conformationally-similar trapped states may be due to swapping of (fairly symmetric) segments. Domain swapping is more likely in the no-pause case where the chain elongates and folds simulaneously; on the other hand, sufficiently long pause times between such segments may be expected to lessen the chances of swapping events. Here, we review the literature in this light. PMID:18722384

  8. ErbB2-dependent chemotaxis requires microtubule capture and stabilization coordinated by distinct signaling pathways.

    PubMed

    Benseddik, Khedidja; Sen Nkwe, Nadine; Daou, Pascale; Verdier-Pinard, Pascal; Badache, Ali

    2013-01-01

    Activation of the ErbB2 receptor tyrosine kinase stimulates breast cancer cell migration. Cell migration is a complex process that requires the synchronized reorganization of numerous subcellular structures including cell-to-matrix adhesions, the actin cytoskeleton and microtubules. How the multiple signaling pathways triggered by ErbB2 coordinate, in time and space, the various processes involved in cell motility, is poorly defined. We investigated the mechanism whereby ErbB2 controls microtubules and chemotaxis. We report that activation of ErbB2 increased both cell velocity and directed migration. Impairment of the Cdc42 and RhoA GTPases, but not of Rac1, prevented the chemotactic response. RhoA is a key component of the Memo/ACF7 pathway whereby ErbB2 controls microtubule capture at the leading edge. Upon Memo or ACF7 depletion, microtubules failed to reach the leading edge and cells lost their ability to follow the chemotactic gradient. Constitutive ACF7 targeting to the membrane in Memo-depleted cells reestablished directed migration. ErbB2-mediated activation of phospholipase C gamma (PLCγ) also contributed to cell guidance. We further showed that PLCγ signaling, via classical protein kinases C, and Memo signaling converged towards a single pathway controlling the microtubule capture complex. Finally, inhibiting the PI3K/Akt pathway did not affect microtubule capture, but disturbed microtubule stability, which also resulted in defective chemotaxis. PI3K/Akt-dependent stabilization of microtubules involved repression of GSK3 activity on the one hand and inhibition of the microtubule destabilizing protein, Stathmin, on the other hand. Thus, ErbB2 triggers distinct and complementary pathways that tightly coordinate microtubule capture and microtubule stability to control chemotaxis.

  9. Distinct Spatiotemporal Expression of Serine Proteases Prss23 and Prss35 in Periimplantation Mouse Uterus and Dispensable Function of Prss35 in Fertility

    PubMed Central

    Diao, Honglu; Xiao, Shuo; Li, Rong; Zhao, Fei; Ye, Xiaoqin

    2013-01-01

    PRSS23 and PRSS35 are homologous proteases originally identified in mouse ovaries. In the periimplantation mouse uterus, Prss23 was highly expressed in the preimplantation gestation day 3.5 (D3.5) uterine luminal epithelium (LE). It disappeared from the postimplantation LE and reappeared in the stromal compartment next to the myometrium on D6.5. It was undetectable in the embryo from D4.5 to D6.5 but highly expressed in the embryo on D7.5. Prss35 became detectable in the uterine stromal compartment surrounding the embryo on D4.5 and shifted towards the mesometrial side of the stromal compartment next to the embryo from D5.5 to D7.5. In the ovariectomized uterus, Prss23 was moderately and Prss35 was dramatically downregulated by progesterone and 17β-estradiol. Based on the expression of Prss35 in granulosa cells and corpus luteum of the ovary and the early pregnant uterus, we hypothesized that PRSS35 might play a role in female reproduction, especially in oocyte development, ovulation, implantation, and decidualization. This hypothesis was tested in Prss35(−/−) mice, which proved otherwise. Between wild type (WT) and Prss35(−/−) mice, superovulation of immature females produced comparable numbers of cumulus-oocyte complexes; there were comparable numbers of implantation sites detected on D4.5 and D7.5; there were no obvious differences in the expression of implantation and decidualization marker genes in D4.5 or D7.5 uteri. Comparable mRNA expression levels of a few known protease-related genes in the WT and Prss35(−/−) D4.5 uteri indicated no compensatory upregulation. Comparable litter sizes from WT × WT and Prss35(−/−)× Prss35(−/−) crosses suggested that Prss35 gene was unessential for fertility and embryo development. Prss35 gene has been linked to cleft lip/palate in humans. However, no obvious such defects were observed in Prss35(−/−) mice. This study demonstrates the distinct expression of Prss23 and Prss35 in the periimplantation

  10. Genetic approach identifies distinct asthma pathways in overweight vs normal weight children.

    PubMed

    Butsch Kovacic, M; Martin, L J; Biagini Myers, J M; He, H; Lindsey, M; Mersha, T B; Khurana Hershey, G K

    2015-08-01

    The pathogenesis of asthma in the context of excess body weight may be distinct from asthma that develops in normal weight children. The study's objective was to explore the biology of asthma in the context of obesity and normal weight status using genetic methodologies. Associations between asthma and SNPs in 49 genes were assessed, as well as, interactions between SNPs and overweight status in child participants of the Greater Cincinnati Pediatric Clinic Repository. Asthma was significantly associated with weight (OR = 1.38; P = 0.037). The number of genes and the magnitude of their associations with asthma were notably greater when considering overweight children alone vs normal weight and overweight children together. When considering weight, distinct sets of asthma-associated genes were observed, many times with opposing effects. We demonstrated that the underlying heterogeneity of asthma is likely due in part to distinct pathogenetic pathways that depend on preceding/comorbid overweight and/or allergy. It is therefore important to consider both obesity and asthma when conducting studies of asthma.

  11. Distinct microenvironmental cues stimulate divergent TLR4-mediated signaling pathways in macrophages

    PubMed Central

    Piccinini, Anna M.; Zuliani-Alvarez, Lorena; Lim, Jenny M. P.; Midwood, Kim S.

    2017-01-01

    Macrophages exhibit a phenotypic plasticity that enables them to orchestrate specific immune responses to distinct threats. The microbial product lipopolysaccharide (LPS) and the extracellular matrix glycoprotein tenascin-C are released during bacterial infection and tissue injury, respectively, and both activate Toll-like receptor 4 (TLR4). We found that these two TLR4 ligands stimulated distinct signaling pathways in macrophages, resulting in cells with divergent phenotypes. Although macrophages activated by LPS or tenascin-C displayed some common features, including activation of nuclear factor κB and mitogen-activated protein kinase signaling and cytokine synthesis, each ligand stimulated the production of different subsets of cytokines and generated different phosphoproteomic signatures. Moreover, tenascin-C promoted the generation of macrophages that exhibited increased synthesis and phosphorylation of extracellular matrix components, whereas LPS stimulated the production of macrophages that exhibited an enhanced capacity to degrade the matrix. These data reveal how the activation of one pattern recognition receptor by different microenvironmental cues generates macrophage with distinct phenotypes. PMID:27577261

  12. Comparative RNA-Seq transcriptome analyses reveal distinct metabolic pathways in diabetic nerve and kidney disease.

    PubMed

    Hinder, Lucy M; Park, Meeyoung; Rumora, Amy E; Hur, Junguk; Eichinger, Felix; Pennathur, Subramaniam; Kretzler, Matthias; Brosius, Frank C; Feldman, Eva L

    2017-03-08

    Treating insulin resistance with pioglitazone normalizes renal function and improves small nerve fibre function and architecture; however, it does not affect large myelinated nerve fibre function in mouse models of type 2 diabetes (T2DM), indicating that pioglitazone affects the body in a tissue-specific manner. To identify distinct molecular pathways regulating diabetic peripheral neuropathy (DPN) and nephropathy (DN), as well those affected by pioglitazone, we assessed DPN and DN gene transcript expression in control and diabetic mice with or without pioglitazone treatment. Differential expression analysis and self-organizing maps were then used in parallel to analyse transcriptome data. Differential expression analysis showed that gene expression promoting cell death and the inflammatory response was reversed in the kidney glomeruli but unchanged or exacerbated in sciatic nerve by pioglitazone. Self-organizing map analysis revealed that mitochondrial dysfunction was normalized in kidney and nerve by treatment; however, conserved pathways were opposite in their directionality of regulation. Collectively, our data suggest inflammation may drive large fibre dysfunction, while mitochondrial dysfunction may drive small fibre dysfunction in T2DM. Moreover, targeting both of these pathways is likely to improve DN. This study supports growing evidence that systemic metabolic changes in T2DM are associated with distinct tissue-specific metabolic reprogramming in kidney and nerve and that these changes play a critical role in DN and small fibre DPN pathogenesis. These data also highlight the potential dangers of a 'one size fits all' approach to T2DM therapeutics, as the same drug may simultaneously alleviate one complication while exacerbating another.

  13. Human memory B cells originate from three distinct germinal center-dependent and -independent maturation pathways.

    PubMed

    Berkowska, Magdalena A; Driessen, Gertjan J A; Bikos, Vasilis; Grosserichter-Wagener, Christina; Stamatopoulos, Kostas; Cerutti, Andrea; He, Bing; Biermann, Katharina; Lange, Johan F; van der Burg, Mirjam; van Dongen, Jacques J M; van Zelm, Menno C

    2011-08-25

    Multiple distinct memory B-cell subsets have been identified in humans, but it remains unclear how their phenotypic diversity corresponds to the type of responses from which they originate. Especially, the contribution of germinal center-independent responses in humans remains controversial. We defined 6 memory B-cell subsets based on their antigen-experienced phenotype and differential expression of CD27 and IgH isotypes. Molecular characterization of their replication history, Ig somatic hypermutation, and class-switch profiles demonstrated their origin from 3 different pathways. CD27⁻IgG⁺ and CD27⁺IgM⁺ B cells are derived from primary germinal center reactions, and CD27⁺IgA⁺ and CD27⁺IgG⁺ B cells are from consecutive germinal center responses (pathway 1). In contrast, natural effector and CD27⁻IgA⁺ memory B cells have limited proliferation and are also present in CD40L-deficient patients, reflecting a germinal center-independent origin. Natural effector cells at least in part originate from systemic responses in the splenic marginal zone (pathway 2). CD27⁻IgA⁺ cells share low replication history and dominant Igλ and IgA2 use with gut lamina propria IgA+ B cells, suggesting their common origin from local germinal center-independent responses (pathway 3). Our findings shed light on human germinal center-dependent and -independent B-cell memory formation and provide new opportunities to study these processes in immunologic diseases.

  14. Spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome.

    PubMed

    Morselli, Eugenia; Mariño, Guillermo; Bennetzen, Martin V; Eisenberg, Tobias; Megalou, Evgenia; Schroeder, Sabrina; Cabrera, Sandra; Bénit, Paule; Rustin, Pierre; Criollo, Alfredo; Kepp, Oliver; Galluzzi, Lorenzo; Shen, Shensi; Malik, Shoaib Ahmad; Maiuri, Maria Chiara; Horio, Yoshiyuki; López-Otín, Carlos; Andersen, Jens S; Tavernarakis, Nektarios; Madeo, Frank; Kroemer, Guido

    2011-02-21

    Autophagy protects organelles, cells, and organisms against several stress conditions. Induction of autophagy by resveratrol requires the nicotinamide adenine dinucleotide-dependent deacetylase sirtuin 1 (SIRT1). In this paper, we show that the acetylase inhibitor spermidine stimulates autophagy independent of SIRT1 in human and yeast cells as well as in nematodes. Although resveratrol and spermidine ignite autophagy through distinct mechanisms, these compounds stimulate convergent pathways that culminate in concordant modifications of the acetylproteome. Both agents favor convergent deacetylation and acetylation reactions in the cytosol and in the nucleus, respectively. Both resveratrol and spermidine were able to induce autophagy in cytoplasts (enucleated cells). Moreover, a cytoplasm-restricted mutant of SIRT1 could stimulate autophagy, suggesting that cytoplasmic deacetylation reactions dictate the autophagic cascade. At doses at which neither resveratrol nor spermidine stimulated autophagy alone, these agents synergistically induced autophagy. Altogether, these data underscore the importance of an autophagy regulatory network of antagonistic deacetylases and acetylases that can be pharmacologically manipulated.

  15. Fear conditioning and extinction: emotional states encoded by distinct signaling pathways

    PubMed Central

    Tronson, Natalie C.; Corcoran, Kevin A.; Jovasevic, Vladimir; Radulovic, Jelena

    2011-01-01

    Conditioning and extinction of fear have traditionally been viewed as two independent learning processes for encoding representations of contexts or cues (conditioned stimuli, CS), aversive events (unconditioned stimuli, US), and their relationship. Based on the analysis of protein kinase signaling patterns in neurons of the fear circuit, we propose that fear and extinction are best conceptualized as emotional states triggered by a single CS representation with two opposing values: aversive and non-aversive. These values are conferred by the presence or absence of the US and encoded by distinct sets of kinase signaling pathways and their downstream targets. Modulating specific protein kinases thus has the potential to modify emotional states, and hence, may emerge as a promising treatment for anxiety disorders. PMID:22118930

  16. Receptor tyrosine kinases modulate distinct transcriptional programs by differential usage of intracellular pathways

    PubMed Central

    Vasudevan, Harish N; Mazot, Pierre; He, Fenglei; Soriano, Philippe

    2015-01-01

    Receptor tyrosine kinases (RTKs) signal through shared intracellular pathways yet mediate distinct outcomes across many cell types. To investigate the mechanisms underlying RTK specificity in craniofacial development, we performed RNA-seq to delineate the transcriptional response to platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) signaling in mouse embryonic palatal mesenchyme cells. While the early gene expression profile induced by both growth factors is qualitatively similar, the late response is divergent. Comparing the effect of MEK (Mitogen/Extracellular signal-regulated kinase) and PI3K (phosphoinositide-3-kinase) inhibition, we find the FGF response is MEK dependent, while the PDGF response is PI3K dependent. Furthermore, FGF promotes proliferation but PDGF favors differentiation. Finally, we demonstrate overlapping domains of PDGF-PI3K signaling and osteoblast differentiation in the palate and increased osteogenesis in FGF mutants, indicating this differentiation circuit is conserved in vivo. Our results identify distinct responses to PDGF and FGF and provide insight into the mechanisms encoding RTK specificity. DOI: http://dx.doi.org/10.7554/eLife.07186.001 PMID:25951516

  17. Ral-GTPases mediate a distinct downstream signaling pathway from Ras that facilitates cellular transformation.

    PubMed Central

    Urano, T; Emkey, R; Feig, L A

    1996-01-01

    Ral proteins (RalA and RalB) comprise a distinct family of Ras-related GTPases (Feig and Emkey, 1993). Recently, Ral-GDS, the exchange factor that activates Ral proteins, has been shown to bind specifically to the activated forms of RasH, R-Ras and Rap1A, in the yeast two-hybrid system. Here we demonstrate that although all three GTPases have the capacity to bind Ral-GDS in mammalian cells, only RasH activates Ral-GDS. Furthermore, although constitutively activated Ra1A does not induce oncogenic transformation on its own, its expression enhances the transforming activities of both RasH and Raf. Finally, a dominant inhibitory form of RalA suppresses the transforming activities of both RasH and Raf. These results demonstrate that activation of Ral-GDS and thus its target, Ral, constitutes a distinct downstream signaling pathway from RasH that potentiates oncogenic transformation. Images PMID:8631302

  18. Distinct Cortical Pathways for Music and Speech Revealed by Hypothesis-Free Voxel Decomposition.

    PubMed

    Norman-Haignere, Sam; Kanwisher, Nancy G; McDermott, Josh H

    2015-12-16

    The organization of human auditory cortex remains unresolved, due in part to the small stimulus sets common to fMRI studies and the overlap of neural populations within voxels. To address these challenges, we measured fMRI responses to 165 natural sounds and inferred canonical response profiles ("components") whose weighted combinations explained voxel responses throughout auditory cortex. This analysis revealed six components, each with interpretable response characteristics despite being unconstrained by prior functional hypotheses. Four components embodied selectivity for particular acoustic features (frequency, spectrotemporal modulation, pitch). Two others exhibited pronounced selectivity for music and speech, respectively, and were not explainable by standard acoustic features. Anatomically, music and speech selectivity concentrated in distinct regions of non-primary auditory cortex. However, music selectivity was weak in raw voxel responses, and its detection required a decomposition method. Voxel decomposition identifies primary dimensions of response variation across natural sounds, revealing distinct cortical pathways for music and speech. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Distinct Signaling Mechanisms in Multiple Developmental Pathways by the SCRAMBLED Receptor of Arabidopsis1[OPEN

    PubMed Central

    Kwak, Su-Hwan; Woo, Sooah; Lee, Myeong Min; Schiefelbein, John

    2014-01-01

    SCRAMBLED (SCM), a leucine-rich repeat receptor-like kinase in Arabidopsis (Arabidopsis thaliana), is required for positional signaling in the root epidermis and for tissue/organ development in the shoot. To further understand SCM action, we generated a series of kinase domain variants and analyzed their ability to complement scm mutant defects. We found that the SCM kinase domain, but not kinase activity, is required for its role in root epidermal patterning, supporting the view that SCM is an atypical receptor kinase. We also describe a previously uncharacterized role for SCM in fruit dehiscence, because mature siliques from scm mutants fail to open properly. Interestingly, the kinase domain of SCM appears to be dispensable for this developmental process. Furthermore, we found that most of the SCM kinase domain mutations dramatically inhibit inflorescence development. Because this process is not affected in scm null mutants, it is likely that SCM acts redundantly to regulate inflorescence size. The importance of distinct kinase residues for these three developmental processes provides an explanation for the maintenance of the conserved kinase domain in the SCM protein, and it may generally explain its conservation in other atypical kinases. Furthermore, these results indicate that individual leucine-rich repeat receptor-like kinases may participate in multiple pathways using distinct signaling mechanisms to mediate diverse cellular communication events. PMID:25136062

  20. Distinct signaling mechanisms in multiple developmental pathways by the SCRAMBLED receptor of Arabidopsis.

    PubMed

    Kwak, Su-Hwan; Woo, Sooah; Lee, Myeong Min; Schiefelbein, John

    2014-10-01

    SCRAMBLED (SCM), a leucine-rich repeat receptor-like kinase in Arabidopsis (Arabidopsis thaliana), is required for positional signaling in the root epidermis and for tissue/organ development in the shoot. To further understand SCM action, we generated a series of kinase domain variants and analyzed their ability to complement scm mutant defects. We found that the SCM kinase domain, but not kinase activity, is required for its role in root epidermal patterning, supporting the view that SCM is an atypical receptor kinase. We also describe a previously uncharacterized role for SCM in fruit dehiscence, because mature siliques from scm mutants fail to open properly. Interestingly, the kinase domain of SCM appears to be dispensable for this developmental process. Furthermore, we found that most of the SCM kinase domain mutations dramatically inhibit inflorescence development. Because this process is not affected in scm null mutants, it is likely that SCM acts redundantly to regulate inflorescence size. The importance of distinct kinase residues for these three developmental processes provides an explanation for the maintenance of the conserved kinase domain in the SCM protein, and it may generally explain its conservation in other atypical kinases. Furthermore, these results indicate that individual leucine-rich repeat receptor-like kinases may participate in multiple pathways using distinct signaling mechanisms to mediate diverse cellular communication events.

  1. Neurobeachin Regulates Glutamate- and GABA-Receptor Targeting to Synapses via Distinct Pathways.

    PubMed

    Farzana, F; Zalm, R; Chen, N; Li, K W; Grant, Seth G N; Smit, A B; Toonen, R F; Verhage, M

    2016-05-01

    Neurotransmission and synaptic strength depend on expression of post-synaptic receptors on the cell surface. Post-translational modification of receptors, trafficking to the synapse through the secretory pathway, and subsequent insertion into the synapse involves interaction of the receptor with A-kinase anchor proteins (AKAPs) and scaffolding proteins. Neurobeachin (Nbea), a brain specific AKAP, is required for synaptic surface expression of both glutamate and GABA receptors. Here, we investigated the role of Nbea-dependent targeting of postsynaptic receptors by studying Nbea interaction with synapse-associated protein 102 (SAP102/Dlg3) and protein kinase A subunit II (PKA II). A Nbea mutant lacking the PKA binding domain showed a similar distribution as wild-type Nbea in Nbea null neurons and partially restored GABA receptor surface expression. To understand the relevance of Nbea interaction with SAP102, we analysed SAP102 null mutant mice. Nbea levels were reduced by ~80% in SAP102 null mice, but glutamatergic receptor expression was normal. A single-point mutation in the pleckstrin homology domain of Nbea (E2218R) resulted in loss of binding with SAP102. When expressed in Nbea null neurons, this mutant fully restored GABA receptor surface expression, but not glutamate receptor expression. Our results suggest that the PKA-binding domain is not essential for Nbea's role in receptor targeting and that Nbea targets glutamate and GABA receptors to the synapse via distinct molecular pathways by interacting with specific effector proteins.

  2. Distinct outcomes of CRL–Nedd8 pathway inhibition reveal cancer cell plasticity

    PubMed Central

    Rulina, Anastasia V; Mittler, Frédérique; Obeid, Patricia; Gerbaud, Sophie; Guyon, Laurent; Sulpice, Eric; Kermarrec, Frédérique; Assard, Nicole; Dolega, Monika E; Gidrol, Xavier; Balakirev, Maxim Y

    2016-01-01

    Inhibition of protein degradation by blocking Cullin-RING E3 ligases (CRLs) is a new approach in cancer therapy though of unknown risk because CRL inhibition may stabilize both oncoproteins and tumor suppressors. Probing CRLs in prostate cancer cells revealed a remarkable plasticity of cells with TMPRSS2-ERG translocation. CRL suppression by chemical inhibition or knockdown of RING component RBX1 led to reversible G0/G1 cell cycle arrest that prevented cell apoptosis. Conversely, complete blocking of CRLs at a higher inhibitor dose-induced cytotoxicity that was amplified by knockdown of CRL regulator Cand1. We analyzed cell signaling to understand how varying degrees of CRL inhibition translated to distinct cell fates. Both tumor suppressor and oncogenic cell signaling pathways and transcriptional activities were affected, with pro-metastatic Wnt/β-catenin as the most upregulated. Suppression of the NF-κB pathway contributed to anti-apoptotic effect, and androgen receptor (AR) and ERG played decisive, though opposite, roles: AR was involved in protective quiescence, whereas ERG promoted apoptosis. These data define AR–ERG interaction as a key plasticity and survival determinant in prostate cancer and suggest supplementary treatments that may overcome drug resistance mechanisms regulated by AR–ERG interaction. PMID:27906189

  3. A distinct brain pathway links viral RNA exposure to sickness behavior

    PubMed Central

    Zhu, Xinxia; Levasseur, Pete R.; Michaelis, Katherine A.; Burfeind, Kevin G.; Marks, Daniel L.

    2016-01-01

    Sickness behaviors and metabolic responses to invading pathogens are common to nearly all types of infection. These responses evolved to provide short-term benefit to the host to ward off infection, but impact on quality of life, and when prolonged lead to neurodegeneration, depression, and cachexia. Among the major infectious agents, viruses most frequently enter the brain, resulting in profound neuroinflammation. We sought to define the unique features of the inflammatory response in the brain to these infections. We demonstrate that the molecular pathway defining the central response to dsRNA is distinct from that found in the periphery. The behavioral and physical response to the dsRNA mimetic poly I:C is dependent on signaling via MyD88 when it is delivered centrally, whereas this response is mediated via the TRIF pathway when delivered peripherally. We also define the likely cellular candidates for this MyD88-dependent step. These findings suggest that symptom management is possible without ameliorating protective antiviral immune responses. PMID:27435819

  4. Autism Spectrum Disorders and Drug Addiction: Common Pathways, Common Molecules, Distinct Disorders?

    PubMed Central

    Rothwell, Patrick E.

    2016-01-01

    Autism spectrum disorders (ASDs) and drug addiction do not share substantial comorbidity or obvious similarities in etiology or symptomatology. It is thus surprising that a number of recent studies implicate overlapping neural circuits and molecular signaling pathways in both disorders. The purpose of this review is to highlight this emerging intersection and consider implications for understanding the pathophysiology of these seemingly distinct disorders. One area of overlap involves neural circuits and neuromodulatory systems in the striatum and basal ganglia, which play an established role in addiction and reward but are increasingly implicated in clinical and preclinical studies of ASDs. A second area of overlap relates to molecules like Fragile X mental retardation protein (FMRP) and methyl CpG-binding protein-2 (MECP2), which are best known for their contribution to the pathogenesis of syndromic ASDs, but have recently been shown to regulate behavioral and neurobiological responses to addictive drug exposure. These shared pathways and molecules point to common dimensions of behavioral dysfunction, including the repetition of behavioral patterns and aberrant reward processing. The synthesis of knowledge gained through parallel investigations of ASDs and addiction may inspire the design of new therapeutic interventions to correct common elements of striatal dysfunction. PMID:26903789

  5. Pak1 regulates the orientation of apical polarization and lumen formation by distinct pathways.

    PubMed

    deLeon, Orlando; Puglise, Jason M; Liu, Fengming; Smits, Jos; ter Beest, Martin B; Zegers, Mirjam M

    2012-01-01

    The development of the basic architecture of branching tubules enclosing a central lumen that characterizes most epithelial organs crucially depends on the apico-basolateral polarization of epithelial cells. Signals from the extracellular matrix control the orientation of the apical surface, so that it faces the lumen interior, opposite to cell-matrix adhesion sites. This orientation of the apical surface is thought to be intrinsically linked to the formation of single lumens. We previously demonstrated in three-dimensional cyst cultures of Madin-Darby canine kidney (MDCK) cells that signaling by β1 integrins regulates the orientation of the apical surface, via a mechanism that depends on the activity of the small GTPase Rac1. Here, we investigated whether the Rac1 effector Pak1 is a downstream effector in this pathway. Expression of constitutive active Pak1 phenocopies the effect of β1 integrin inhibition in that it misorients the apical surface and induces a multilumen phenotype. The misorientation of apical surfaces depends on the interaction of active Pak1 with PIX proteins and is linked to defects in basement membrane assembly. In contrast, the multilumen phenotype was independent of PIX and the basement membrane. Therefore, Pak1 likely regulates apical polarization and lumen formation by two distinct pathways.

  6. Pak1 Regulates the Orientation of Apical Polarization and Lumen Formation by Distinct Pathways

    PubMed Central

    Smits, Jos; ter Beest, Martin B.; Zegers, Mirjam M.

    2012-01-01

    The development of the basic architecture of branching tubules enclosing a central lumen that characterizes most epithelial organs crucially depends on the apico-basolateral polarization of epithelial cells. Signals from the extracellular matrix control the orientation of the apical surface, so that it faces the lumen interior, opposite to cell-matrix adhesion sites. This orientation of the apical surface is thought to be intrinsically linked to the formation of single lumens. We previously demonstrated in three-dimensional cyst cultures of Madin-Darby canine kidney (MDCK) cells that signaling by β1 integrins regulates the orientation of the apical surface, via a mechanism that depends on the activity of the small GTPase Rac1. Here, we investigated whether the Rac1 effector Pak1 is a downstream effector in this pathway. Expression of constitutive active Pak1 phenocopies the effect of β1 integrin inhibition in that it misorients the apical surface and induces a multilumen phenotype. The misorientation of apical surfaces depends on the interaction of active Pak1 with PIX proteins and is linked to defects in basement membrane assembly. In contrast, the multilumen phenotype was independent of PIX and the basement membrane. Therefore, Pak1 likely regulates apical polarization and lumen formation by two distinct pathways. PMID:22815903

  7. Two distinct and competitive pathways confer the cellcidal actions of artemisinins

    PubMed Central

    Sun, Chen; Li, Jian; Cao, Yu; Long, Gongbo; Zhou, Bing

    2015-01-01

    The biological actions of artemisinin (ART), an antimalarial drug derived from Artemisia annua, remain poorly understood and controversial. Besides potent antimalarial activity, some of artemisinin derivatives (together with artemisinin, hereafter referred to as ARTs), in particular dihydroartemisinin (DHA), are also associated with anticancer and other antiparasitic activities. In this study, we used baker’s yeast Saccharomyces cerevisiae as cellular and genetic model to investigate the molecular and cellular properties of ARTs. Two clearly separable pathways exist. While all ARTs exhibit potent anti-mitochondrial actions as shown before, DHA exerts an additional strong heme-dependent, likely mitochondria-independent inhibitory action. More importantly, heme antagonizes the mitochondria-dependent cellcidal action. Indeed, when heme synthesis was inhibited, the mitochondria-dependent cellcidal action of ARTs could be dramatically strengthened, and significant yeast growth inhibition at as low as 100 nM ART, an increase of about 25 folds in sensitivity, was observed. We conclude that ARTs are endowed with two major and distinct types of properties: a potent and specific mitochondria-dependent reaction and a more general and less specific heme-mediated reaction. The competitive nature of these two actions could be explained by their shared source of the consumable ARTs, so that inhibition of the heme-mediated degradation pathway would enable more ARTs to be available for the mitochondrial action. These properties of ARTs can be used to interpret the divergent antimalarial and anticancer actions of ARTs. PMID:28357259

  8. Autism Spectrum Disorders and Drug Addiction: Common Pathways, Common Molecules, Distinct Disorders?

    PubMed

    Rothwell, Patrick E

    2016-01-01

    Autism spectrum disorders (ASDs) and drug addiction do not share substantial comorbidity or obvious similarities in etiology or symptomatology. It is thus surprising that a number of recent studies implicate overlapping neural circuits and molecular signaling pathways in both disorders. The purpose of this review is to highlight this emerging intersection and consider implications for understanding the pathophysiology of these seemingly distinct disorders. One area of overlap involves neural circuits and neuromodulatory systems in the striatum and basal ganglia, which play an established role in addiction and reward but are increasingly implicated in clinical and preclinical studies of ASDs. A second area of overlap relates to molecules like Fragile X mental retardation protein (FMRP) and methyl CpG-binding protein-2 (MECP2), which are best known for their contribution to the pathogenesis of syndromic ASDs, but have recently been shown to regulate behavioral and neurobiological responses to addictive drug exposure. These shared pathways and molecules point to common dimensions of behavioral dysfunction, including the repetition of behavioral patterns and aberrant reward processing. The synthesis of knowledge gained through parallel investigations of ASDs and addiction may inspire the design of new therapeutic interventions to correct common elements of striatal dysfunction.

  9. Associative Encoding and Retrieval Are Predicted by Functional Connectivity in Distinct Hippocampal Area CA1 Pathways

    PubMed Central

    Duncan, Katherine; Tompary, Alexa

    2014-01-01

    Determining how the hippocampus supports the unique demands of memory encoding and retrieval is fundamental for understanding the biological basis of episodic memory. One possibility proposed by theoretical models is that the distinct computational demands of encoding and retrieval are accommodated by shifts in the functional interaction between the hippocampal CA1 subregion and its input structures. However, empirical tests of this hypothesis are lacking. To test this in humans, we used high-resolution fMRI to measure functional connectivity between hippocampal area CA1 and regions of the medial temporal lobe and midbrain during extended blocks of associative encoding and retrieval tasks. We found evidence for a double dissociation between the pathways supporting successful encoding and retrieval. Specifically, during the associative encoding task, but not the retrieval task, functional connectivity only between area CA1 and the ventral tegmental area predicted associative long-term memory. In contrast, connectivity between area CA1 and DG/CA3 was greater, on average, during the retrieval task compared with the encoding task, and, importantly, the strength of this connectivity significantly correlated with retrieval success. Together, these findings serve as an important first step toward understanding how the demands of fundamental memory processes may be met by changes in the relative strength of connectivity within hippocampal pathways. PMID:25143600

  10. Distinct Structural Pathways Coordinate the Activation of AMPA Receptor-Auxiliary Subunit Complexes

    PubMed Central

    Dawe, G. Brent; Musgaard, Maria; Aurousseau, Mark R.P.; Nayeem, Naushaba; Green, Tim; Biggin, Philip C.; Bowie, Derek

    2016-01-01

    Summary Neurotransmitter-gated ion channels adopt different gating modes to fine-tune signaling at central synapses. At glutamatergic synapses, high and low activity of AMPA receptors (AMPARs) is observed when pore-forming subunits coassemble with or without auxiliary subunits, respectively. Whether a common structural pathway accounts for these different gating modes is unclear. Here, we identify two structural motifs that determine the time course of AMPAR channel activation. A network of electrostatic interactions at the apex of the AMPAR ligand-binding domain (LBD) is essential for gating by pore-forming subunits, whereas a conserved motif on the lower, D2 lobe of the LBD prolongs channel activity when auxiliary subunits are present. Accordingly, channel activity is almost entirely abolished by elimination of the electrostatic network but restored via auxiliary protein interactions at the D2 lobe. In summary, we propose that activation of native AMPAR complexes is coordinated by distinct structural pathways, favored by the association/dissociation of auxiliary subunits. PMID:26924438

  11. Molecular subtypes of diffuse large B-cell lymphoma arise by distinct genetic pathways

    PubMed Central

    Lenz, Georg; Wright, George W.; Emre, N. C. Tolga; Kohlhammer, Holger; Dave, Sandeep S.; Davis, R. Eric; Carty, Shannon; Lam, Lloyd T.; Shaffer, A. L.; Xiao, Wenming; Powell, John; Rosenwald, Andreas; Ott, German; Muller-Hermelink, Hans Konrad; Gascoyne, Randy D.; Connors, Joseph M.; Campo, Elias; Jaffe, Elaine S.; Delabie, Jan; Smeland, Erlend B.; Rimsza, Lisa M.; Fisher, Richard I.; Weisenburger, Dennis D.; Chan, Wing C.; Staudt, Louis M.

    2008-01-01

    Gene-expression profiling has been used to define 3 molecular subtypes of diffuse large B-cell lymphoma (DLBCL), termed germinal center B-cell-like (GCB) DLBCL, activated B-cell-like (ABC) DLBCL, and primary mediastinal B-cell lymphoma (PMBL). To investigate whether these DLBCL subtypes arise by distinct pathogenetic mechanisms, we analyzed 203 DLBCL biopsy samples by high-resolution, genome-wide copy number analysis coupled with gene-expression profiling. Of 272 recurrent chromosomal aberrations that were associated with gene-expression alterations, 30 were used differentially by the DLBCL subtypes (P < 0.006). An amplicon on chromosome 19 was detected in 26% of ABC DLBCLs but in only 3% of GCB DLBCLs and PMBLs. A highly up-regulated gene in this amplicon was SPIB, which encodes an ETS family transcription factor. Knockdown of SPIB by RNA interference was toxic to ABC DLBCL cell lines but not to GCB DLBCL, PMBL, or myeloma cell lines, strongly implicating SPIB as an oncogene involved in the pathogenesis of ABC DLBCL. Deletion of the INK4a/ARF tumor suppressor locus and trisomy 3 also occurred almost exclusively in ABC DLBCLs and was associated with inferior outcome within this subtype. FOXP1 emerged as a potential oncogene in ABC DLBCL that was up-regulated by trisomy 3 and by more focal high-level amplifications. In GCB DLBCL, amplification of the oncogenic mir-17–92 microRNA cluster and deletion of the tumor suppressor PTEN were recurrent, but these events did not occur in ABC DLBCL. Together, these data provide genetic evidence that the DLBCL subtypes are distinct diseases that use different oncogenic pathways. PMID:18765795

  12. Pediatric-type nodal follicular lymphoma: a biologically distinct lymphoma with frequent MAPK pathway mutations.

    PubMed

    Louissaint, Abner; Schafernak, Kristian T; Geyer, Julia T; Kovach, Alexandra E; Ghandi, Mahmoud; Gratzinger, Dita; Roth, Christine G; Paxton, Christian N; Kim, Sunhee; Namgyal, Chungdak; Morin, Ryan; Morgan, Elizabeth A; Neuberg, Donna S; South, Sarah T; Harris, Marian H; Hasserjian, Robert P; Hochberg, Ephraim P; Garraway, Levi A; Harris, Nancy Lee; Weinstock, David M

    2016-08-25

    Pediatric-type nodal follicular lymphoma (PTNFL) is a variant of follicular lymphoma (FL) characterized by limited-stage presentation and invariably benign behavior despite often high-grade histological appearance. It is important to distinguish PTNFL from typical FL in order to avoid unnecessary treatment; however, this distinction relies solely on clinical and pathological criteria, which may be variably applied. To define the genetic landscape of PTNFL, we performed copy number analysis and exome and/or targeted sequencing of 26 PTNFLs (16 pediatric and 10 adult). The most commonly mutated gene in PTNFL was MAP2K1, encoding MEK1, with a mutation frequency of 43%. All MAP2K1 mutations were activating missense mutations localized to exons 2 and 3, which encode negative regulatory and catalytic domains, respectively. Missense mutations in MAPK1 (2/22) and RRAS (1/22) were identified in cases that lacked MAP2K1 mutations. The second most commonly mutated gene in PTNFL was TNFRSF14, with a mutation frequency of 29%, similar to that seen in limited-stage typical FL (P = .35). PTNFL was otherwise genomically bland and specifically lacked recurrent mutations in epigenetic modifiers (eg, CREBBP, KMT2D). Copy number aberrations affected a mean of only 0.5% of PTNFL genomes, compared with 10% of limited-stage typical FL genomes (P < .02). Importantly, the mutational profiles of PTNFLs in children and adults were highly similar. Together, these findings define PTNFL as a biologically and clinically distinct indolent lymphoma of children and adults characterized by a high prevalence of MAPK pathway mutations and a near absence of mutations in epigenetic modifiers.

  13. Pediatric-type nodal follicular lymphoma: a biologically distinct lymphoma with frequent MAPK pathway mutations

    PubMed Central

    Schafernak, Kristian T.; Geyer, Julia T.; Kovach, Alexandra E.; Ghandi, Mahmoud; Gratzinger, Dita; Roth, Christine G.; Paxton, Christian N.; Kim, Sunhee; Namgyal, Chungdak; Morin, Ryan; Morgan, Elizabeth A.; Neuberg, Donna S.; South, Sarah T.; Harris, Marian H.; Hasserjian, Robert P.; Hochberg, Ephraim P.; Garraway, Levi A.; Harris, Nancy Lee; Weinstock, David M.

    2016-01-01

    Pediatric-type nodal follicular lymphoma (PTNFL) is a variant of follicular lymphoma (FL) characterized by limited-stage presentation and invariably benign behavior despite often high-grade histological appearance. It is important to distinguish PTNFL from typical FL in order to avoid unnecessary treatment; however, this distinction relies solely on clinical and pathological criteria, which may be variably applied. To define the genetic landscape of PTNFL, we performed copy number analysis and exome and/or targeted sequencing of 26 PTNFLs (16 pediatric and 10 adult). The most commonly mutated gene in PTNFL was MAP2K1, encoding MEK1, with a mutation frequency of 43%. All MAP2K1 mutations were activating missense mutations localized to exons 2 and 3, which encode negative regulatory and catalytic domains, respectively. Missense mutations in MAPK1 (2/22) and RRAS (1/22) were identified in cases that lacked MAP2K1 mutations. The second most commonly mutated gene in PTNFL was TNFRSF14, with a mutation frequency of 29%, similar to that seen in limited-stage typical FL (P = .35). PTNFL was otherwise genomically bland and specifically lacked recurrent mutations in epigenetic modifiers (eg, CREBBP, KMT2D). Copy number aberrations affected a mean of only 0.5% of PTNFL genomes, compared with 10% of limited-stage typical FL genomes (P < .02). Importantly, the mutational profiles of PTNFLs in children and adults were highly similar. Together, these findings define PTNFL as a biologically and clinically distinct indolent lymphoma of children and adults characterized by a high prevalence of MAPK pathway mutations and a near absence of mutations in epigenetic modifiers. PMID:27325104

  14. Cytochrome P450 3A Conjugation to Ubiquitin in a Process Distinct from Classical Ubiquitination Pathway

    SciTech Connect

    Zangar, Richard C. ); Kimzey, Amy L.; Okita, Janice R.; Wunschel, David S. ); Edwards, Robert J.; Kim, Hyesook; Okita, Richard T.

    2001-12-01

    We characterize a novel microsome system that forms high-molecular-mass (HMM) CYP3A, CYP2E1, and ubiquitin conjugates, but does not alter CYP4A or most other microsomal proteins. The formation of the HMM bands was observed in hepatic microsomes isolated from rats treated 1 week or more with high doses (50 mg/kg/day) of nicardipine, clotrimazole, or pregnenolone 16alpha-carbonitrile, but not microsomes from control, dexamethasone-, nifedipine-, or diltiazem-treated rats. Extensive washing of the microsomes to remove loosely attached proteins or cytosolic contaminants did not prevent the conjugation reaction. In contrast to prototypical ubiquitination pathways, this reaction did not require addition of ubiquitin, ATP, Mg(2+), or cytosol. Addition of cytosol did result in the degradation of the HMM CYP3A bands in a process that was not blocked by proteasome inhibitors. Immunoprecipitated CYP3A contained HMM ubiquitin. Even so, mass spectrometric analysis of tryptic peptides indicated that the HMM CYP3A was in molar excess to ubiquitin, suggesting that the formation of the HMM CYP3A may have resulted from conjugation to itself or a diffuse pool of ubiquitinated proteins already present in the microsomes. Addition of CYP3A substrates inhibited the formation of the HMM CYP3A and the cytosol-dependent degradation of HMM CYP3A. These results suggest that after extended periods of elevated CYP3A expression, microsomal factors are induced that catalyze the formation of HMM CYP3A conjugates that contain ubiquitin. This conjugation reaction, however, seems to be distinct from the classical ubiquitination pathway but may be related to the substrate-dependent stabilization of CYP3A observed in vivo.

  15. Pleiotropic action of CpG-ODN on endothelium and macrophages attenuates angiogenesis through distinct pathways

    PubMed Central

    Wu, Jiahui; Su, Wenru; Powner, Michael B.; Liu, Jian; Copland, David A.; Fruttiger, Marcus; Madeddu, Paolo; Dick, Andrew D.; Liu, Lei

    2016-01-01

    There is an integral relationship between vascular cells and leukocytes in supporting healthy tissue homeostasis. Furthermore, activation of these two cellular components is key for tissue repair following injury. Toll-like receptors (TLRs) play a role in innate immunity defending the organism against infection, but their contribution to angiogenesis remains unclear. Here we used synthetic TLR9 agonists, cytosine-phosphate-guanosine oligodeoxynucleotides (CpG-ODN), to investigate the role of TLR9 in vascular pathophysiology and identify potential therapeutic translation. We demonstrate that CpG-ODN stimulates inflammation yet inhibits angiogenesis. Regulation of angiogenesis by CpG-ODN is pervasive and tissue non-specific. Further, we noted that synthetic CpG-ODN requires backbone phosphorothioate but not TLR9 activation to render and maintain endothelial stalk cells quiescent. CpG-ODN pre-treated endothelial cells enhance macrophage migration but restrain pericyte mobilisation. CpG-ODN attenuation of angiogenesis, however, remains TLR9-dependent, as inhibition is lost in TLR9 deficient mice. Additionally, CpG-ODNs induce an M1 macrophage phenotype that restricts angiogenesis. The effects mediated by CpG-ODNs can therefore modulate both endothelial cells and macrophages through distinct pathways, providing potential therapeutic application in ocular vascular disease. PMID:27558877

  16. Iron isotopes reveal distinct dissolved iron sources and pathways in the intermediate versus deep Southern Ocean

    NASA Astrophysics Data System (ADS)

    Abadie, Cyril; Lacan, Francois; Radic, Amandine; Pradoux, Catherine; Poitrasson, Franck

    2017-01-01

    As an essential micronutrient, iron plays a key role in oceanic biogeochemistry. It is therefore linked to the global carbon cycle and climate. Here, we report a dissolved iron (DFe) isotope section in the South Atlantic and Southern Ocean. Throughout the section, a striking DFe isotope minimum (light iron) is observed at intermediate depths (200-1,300 m), contrasting with heavier isotopic composition in deep waters. This unambiguously demonstrates distinct DFe sources and processes dominating the iron cycle in the intermediate and deep layers, a feature impossible to see with only iron concentration data largely used thus far in chemical oceanography. At intermediate depths, the data suggest that the dominant DFe sources are linked to organic matter remineralization, either in the water column or at continental margins. In deeper layers, however, abiotic non-reductive release of Fe (desorption, dissolution) from particulate iron—notably lithogenic—likely dominates. These results go against the common but oversimplified view that remineralization of organic matter is the major pathway releasing DFe throughout the water column in the open ocean. They suggest that the oceanic iron cycle, and therefore oceanic primary production and climate, could be more sensitive than previously thought to continental erosion (providing lithogenic particles to the ocean), particle transport within the ocean, dissolved/particle interactions, and deep water upwelling. These processes could also impact the cycles of other elements, including nutrients.

  17. Acidic stimuli activates two distinct pathways in taste receptor cells from rat fungiform papillae.

    PubMed

    Liu, L; Simon, S A

    2001-12-27

    A sour taste sensation may be produced when acidic stimuli interact with taste receptor cells (TRCs) on the dorsal surface of the tongue. We have searched for pathways in TRCs that may be activated by acidic stimuli using RT-PCR and changes in intracellular calcium (Ca(2+)(I)) induced by acidic stimuli in rat fungiform papillae. RT-PCR revealed the presence of proton-gated subunits ASIC-beta and VR1. Ca(2+) imaging measurements of the TRCs revealed two distinct responses to acidic stimuli: Ca(2+)(i) was increased in 9% (28/308; Type I) and was decreased in 39% (121/308; Type II). Neither of these responses was affected by the removal of extracellular Ca(2+), indicating that the changes arise from the release and sequestration of Ca(2+) from intracellular stores. These responses were also not inhibited by the vanilloid receptor antagonist, capsazepine, suggesting they do not arise from the activation of vanilloid receptors. The Type I, but not the Type II response was inhibited by amiloride. Dose-response measurements for Types I and II responses yielded pH(50%) of 4.8 and 4.9, respectively. Type II responses were inhibited by pertussis toxin, suggesting G-protein involvement. TRCs that exhibit Type II responses could also be activated by quinine (which increased Ca(2+)(I)) thus suggesting a mechanism by which the addition of acid may be suppressive to other chemical stimuli.

  18. Pleiotropic action of CpG-ODN on endothelium and macrophages attenuates angiogenesis through distinct pathways.

    PubMed

    Wu, Jiahui; Su, Wenru; Powner, Michael B; Liu, Jian; Copland, David A; Fruttiger, Marcus; Madeddu, Paolo; Dick, Andrew D; Liu, Lei

    2016-08-25

    There is an integral relationship between vascular cells and leukocytes in supporting healthy tissue homeostasis. Furthermore, activation of these two cellular components is key for tissue repair following injury. Toll-like receptors (TLRs) play a role in innate immunity defending the organism against infection, but their contribution to angiogenesis remains unclear. Here we used synthetic TLR9 agonists, cytosine-phosphate-guanosine oligodeoxynucleotides (CpG-ODN), to investigate the role of TLR9 in vascular pathophysiology and identify potential therapeutic translation. We demonstrate that CpG-ODN stimulates inflammation yet inhibits angiogenesis. Regulation of angiogenesis by CpG-ODN is pervasive and tissue non-specific. Further, we noted that synthetic CpG-ODN requires backbone phosphorothioate but not TLR9 activation to render and maintain endothelial stalk cells quiescent. CpG-ODN pre-treated endothelial cells enhance macrophage migration but restrain pericyte mobilisation. CpG-ODN attenuation of angiogenesis, however, remains TLR9-dependent, as inhibition is lost in TLR9 deficient mice. Additionally, CpG-ODNs induce an M1 macrophage phenotype that restricts angiogenesis. The effects mediated by CpG-ODNs can therefore modulate both endothelial cells and macrophages through distinct pathways, providing potential therapeutic application in ocular vascular disease.

  19. Drosophila Abelson kinase mediates cell invasion and proliferation via two distinct MAPK pathways

    PubMed Central

    Singh, Jaskirat; Aaronson, Stuart A.; Mlodzik, Marek

    2010-01-01

    The Abelson (Abl) family of non-receptor tyrosine kinases plays important role in cell morphogenesis, motility and proliferation. Although the function of Abl has been extensively studied in leukemia, its role in epithelial cell invasion remains obscure. Using the Drosophila wing epithelium as an in-vivo model system, we demonstrate that overexpression (activation) of Drosophila Abl (dAbl) causes loss of epithelial apical/basal cell polarity and secretion of matrix metalloproteinases, resulting in a cellular invasion and apoptosis. Our in vivo data indicate that dAbl acts downstream of the Src kinases, which are known regulators of cell adhesion and invasion. Downstream of dAbl, Rac GTPases activate two distinct MAPK pathways: JNK signaling (required for cell invasion and apoptosis) and ERK signaling (inducing cell proliferation). Activated Abl also increases the activity of Src members through a positive feedback loop leading to signal amplification. Thus targeting Src-Abl, using available dual inhibitors, could be of therapeutic importance in tumor cell metastasis. PMID:20453880

  20. Iron isotopes reveal distinct dissolved iron sources and pathways in the intermediate versus deep Southern Ocean.

    PubMed

    Abadie, Cyril; Lacan, Francois; Radic, Amandine; Pradoux, Catherine; Poitrasson, Franck

    2017-01-31

    As an essential micronutrient, iron plays a key role in oceanic biogeochemistry. It is therefore linked to the global carbon cycle and climate. Here, we report a dissolved iron (DFe) isotope section in the South Atlantic and Southern Ocean. Throughout the section, a striking DFe isotope minimum (light iron) is observed at intermediate depths (200-1,300 m), contrasting with heavier isotopic composition in deep waters. This unambiguously demonstrates distinct DFe sources and processes dominating the iron cycle in the intermediate and deep layers, a feature impossible to see with only iron concentration data largely used thus far in chemical oceanography. At intermediate depths, the data suggest that the dominant DFe sources are linked to organic matter remineralization, either in the water column or at continental margins. In deeper layers, however, abiotic non-reductive release of Fe (desorption, dissolution) from particulate iron-notably lithogenic-likely dominates. These results go against the common but oversimplified view that remineralization of organic matter is the major pathway releasing DFe throughout the water column in the open ocean. They suggest that the oceanic iron cycle, and therefore oceanic primary production and climate, could be more sensitive than previously thought to continental erosion (providing lithogenic particles to the ocean), particle transport within the ocean, dissolved/particle interactions, and deep water upwelling. These processes could also impact the cycles of other elements, including nutrients.

  1. Selective removal of mitochondria via mitophagy: distinct pathways for different mitochondrial stresses.

    PubMed

    Wei, Huifang; Liu, Lei; Chen, Quan

    2015-10-01

    The efficient and selective elimination of damaged or excessive mitochondria in response to bioenergetic and environmental cues is critical for maintaining a healthy and appropriate population of mitochondria. Mitophagy is considered to be the central mechanism of mitochondrial quality and quantity control. Atg32, a mitophagy receptor in yeast, recruits mitochondria targeted for degradation into the isolation membrane via both direct and indirect interactions with Atg8. In mammals, different mitophagy effectors, including the mitophagy receptors NIX, BNIP3 and FUDNC1 and the PINK1/Parkin pathway, have been identified to participate in the selective clearance of mitochondria. One common feature of mitophagy receptors is that they harbor an LC3-interacting region (LIR) that interacts with LC3, thus promoting the sequestration of mitochondria into the isolation membrane. Additionally, both receptor- and Parkin/PINK1-mediated mitophagy have been found to be regulated by reversible phosphorylation. Here, we review the recent progress in the understanding of the molecular mechanisms involved in selective mitophagy at multiple levels. We also discuss different mitophagy receptors from an evolutionary perspective and highlight the specific functions of and possible cooperation between distinct mechanisms of mitophagy.

  2. Extensive cargo identification reveals distinct biological roles of the 12 importin pathways

    PubMed Central

    Kimura, Makoto; Morinaka, Yuriko; Imai, Kenichiro; Kose, Shingo; Horton, Paul; Imamoto, Naoko

    2017-01-01

    Vast numbers of proteins are transported into and out of the nuclei by approximately 20 species of importin-β family nucleocytoplasmic transport receptors. However, the significance of the multiple parallel transport pathways that the receptors constitute is poorly understood because only limited numbers of cargo proteins have been reported. Here, we identified cargo proteins specific to the 12 species of human import receptors with a high-throughput method that employs stable isotope labeling with amino acids in cell culture, an in vitro reconstituted transport system, and quantitative mass spectrometry. The identified cargoes illuminated the manner of cargo allocation to the receptors. The redundancies of the receptors vary widely depending on the cargo protein. Cargoes of the same receptor are functionally related to one another, and the predominant protein groups in the cargo cohorts differ among the receptors. Thus, the receptors are linked to distinct biological processes by the nature of their cargoes. DOI: http://dx.doi.org/10.7554/eLife.21184.001 PMID:28117667

  3. Iron isotopes reveal distinct dissolved iron sources and pathways in the intermediate versus deep Southern Ocean

    PubMed Central

    Abadie, Cyril; Lacan, Francois; Radic, Amandine; Pradoux, Catherine; Poitrasson, Franck

    2017-01-01

    As an essential micronutrient, iron plays a key role in oceanic biogeochemistry. It is therefore linked to the global carbon cycle and climate. Here, we report a dissolved iron (DFe) isotope section in the South Atlantic and Southern Ocean. Throughout the section, a striking DFe isotope minimum (light iron) is observed at intermediate depths (200–1,300 m), contrasting with heavier isotopic composition in deep waters. This unambiguously demonstrates distinct DFe sources and processes dominating the iron cycle in the intermediate and deep layers, a feature impossible to see with only iron concentration data largely used thus far in chemical oceanography. At intermediate depths, the data suggest that the dominant DFe sources are linked to organic matter remineralization, either in the water column or at continental margins. In deeper layers, however, abiotic non-reductive release of Fe (desorption, dissolution) from particulate iron—notably lithogenic—likely dominates. These results go against the common but oversimplified view that remineralization of organic matter is the major pathway releasing DFe throughout the water column in the open ocean. They suggest that the oceanic iron cycle, and therefore oceanic primary production and climate, could be more sensitive than previously thought to continental erosion (providing lithogenic particles to the ocean), particle transport within the ocean, dissolved/particle interactions, and deep water upwelling. These processes could also impact the cycles of other elements, including nutrients. PMID:28096366

  4. Differential submitochondrial localization of PINK1 as a molecular switch for mediating distinct mitochondrial signaling pathways.

    PubMed

    Fallaize, Dana; Chin, Lih-Shen; Li, Lian

    2015-12-01

    Mutations in mitochondrial kinase PINK1 cause Parkinson disease (PD), but the submitochondrial site(s) of PINK1 action remains unclear. Here, we report that three-dimensional structured illumination microscopy (3D-SIM) enables super-resolution imaging of protein submitochondrial localization. Dual-color 3D-SIM imaging analysis revealed that PINK1 resides in the cristae membrane and intracristae space but not on the outer mitochondrial membrane (OMM) of healthy mitochondria. Under normal physiological conditions, PINK1 colocalizes with its substrate TRAP1 in the cristae membrane and intracristae space. In response to mitochondrial depolarization, PINK1, but not TRAP1, translocates to the OMM. The PINK1 translocation to the OMM of depolarized mitochondria is independent of new protein synthesis and requires combined action of PINK1 transmembrane domain and C-terminal region. We found that mitochondrial depolarization-induced PINK1 OMM translocation is required for recruitment of parkin to the OMM of damaged mitochondria. Our findings suggest that differential submitochondrial localization of PINK1 serves as a molecular switch for mediating two distinct mitochondrial signaling pathways in maintenance of mitochondrial homeostasis. Furthermore, our study provides evidence for the involvement of deregulated PINK1 submitochondrial localization in PD pathogenesis.

  5. Spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome

    PubMed Central

    Morselli, Eugenia; Mariño, Guillermo; Bennetzen, Martin V.; Eisenberg, Tobias; Megalou, Evgenia; Schroeder, Sabrina; Cabrera, Sandra; Bénit, Paule; Rustin, Pierre; Criollo, Alfredo; Kepp, Oliver; Galluzzi, Lorenzo; Shen, Shensi; Malik, Shoaib Ahmad; Maiuri, Maria Chiara; Horio, Yoshiyuki; López-Otín, Carlos; Andersen, Jens S.; Tavernarakis, Nektarios

    2011-01-01

    Autophagy protects organelles, cells, and organisms against several stress conditions. Induction of autophagy by resveratrol requires the nicotinamide adenine dinucleotide–dependent deacetylase sirtuin 1 (SIRT1). In this paper, we show that the acetylase inhibitor spermidine stimulates autophagy independent of SIRT1 in human and yeast cells as well as in nematodes. Although resveratrol and spermidine ignite autophagy through distinct mechanisms, these compounds stimulate convergent pathways that culminate in concordant modifications of the acetylproteome. Both agents favor convergent deacetylation and acetylation reactions in the cytosol and in the nucleus, respectively. Both resveratrol and spermidine were able to induce autophagy in cytoplasts (enucleated cells). Moreover, a cytoplasm-restricted mutant of SIRT1 could stimulate autophagy, suggesting that cytoplasmic deacetylation reactions dictate the autophagic cascade. At doses at which neither resveratrol nor spermidine stimulated autophagy alone, these agents synergistically induced autophagy. Altogether, these data underscore the importance of an autophagy regulatory network of antagonistic deacetylases and acetylases that can be pharmacologically manipulated. PMID:21339330

  6. Epigenetic and genetic deregulation in cancer target distinct signaling pathway domains

    PubMed Central

    Gao, Yang; Teschendorff, Andrew E.

    2017-01-01

    Cancer is characterized by both genetic and epigenetic alterations. While cancer driver mutations and copy-number alterations have been studied at a systems-level, relatively little is known about the systems-level patterns exhibited by their epigenetic counterparts. Here we perform a pan-cancer wide systems-level analysis, mapping candidate cancer-driver DNA methylation (DNAm) alterations onto a human interactome. We demonstrate that functional DNAm alterations in cancer tend to map to nodes of lower connectivity and inter-connectivity, compared to the corresponding alterations at the genomic level. We find that epigenetic alterations are relatively over-represented in extracellular and transmembrane signaling domains, whereas cancer genes undergoing amplification or deletion tend to be enriched within the intracellular domain. A pan-cancer wide meta-analysis identifies WNT and chemokine signaling, as two key pathways where epigenetic deregulation preferentially targets extracellular components. We further pinpoint specific chemokine ligands/receptors whose epigenetic deregulation associates with key epigenetic enzymes, representing potential targets for epigenetic therapy. Our results suggest that epigenetic deregulation in cancer not only targets tissue-specific transcription factors, but also modulates signaling within the extra-cellular domain, providing novel system-level insight into the potential distinctive role of genetic and epigenetic alterations in cancer. PMID:27899617

  7. Two distinctive energy migration pathways of monolayer molecules on metal nanoparticle surfaces

    PubMed Central

    Li, Jiebo; Qian, Huifeng; Chen, Hailong; Zhao, Zhun; Yuan, Kaijun; Chen, Guangxu; Miranda, Andrea; Guo, Xunmin; Chen, Yajing; Zheng, Nanfeng; Wong, Michael S.; Zheng, Junrong

    2016-01-01

    Energy migrations at metal nanomaterial surfaces are fundamentally important to heterogeneous reactions. Here we report two distinctive energy migration pathways of monolayer adsorbate molecules on differently sized metal nanoparticle surfaces investigated with ultrafast vibrational spectroscopy. On a 5 nm platinum particle, within a few picoseconds the vibrational energy of a carbon monoxide adsorbate rapidly dissipates into the particle through electron/hole pair excitations, generating heat that quickly migrates on surface. In contrast, the lack of vibration-electron coupling on approximately 1 nm particles results in vibrational energy migration among adsorbates that occurs on a twenty times slower timescale. Further investigations reveal that the rapid carbon monoxide energy relaxation is also affected by the adsorption sites and the nature of the metal but to a lesser extent. These findings reflect the dependence of electron/vibration coupling on the metallic nature, size and surface site of nanoparticles and its significance in mediating energy relaxations and migrations on nanoparticle surfaces. PMID:26883665

  8. Distinct ECM mechanosensing pathways regulate microtubule dynamics to control endothelial cell branching morphogenesis

    PubMed Central

    Myers, Kenneth A.; Applegate, Kathryn T.

    2011-01-01

    During angiogenesis, cytoskeletal dynamics that mediate endothelial cell branching morphogenesis during vascular guidance are thought to be regulated by physical attributes of the extracellular matrix (ECM) in a process termed mechanosensing. Here, we tested the involvement of microtubules in linking mechanosensing to endothelial cell branching morphogenesis. We used a recently developed microtubule plus end–tracking program to show that specific parameters of microtubule assembly dynamics, growth speed and growth persistence, are globally and regionally modified by, and contribute to, ECM mechanosensing. We demonstrated that engagement of compliant two-dimensional or three-dimensional ECMs induces local differences in microtubule growth speed that require myosin II contractility. Finally, we found that microtubule growth persistence is modulated by myosin II–mediated compliance mechanosensing when cells are cultured on two-dimensional ECMs, whereas three-dimensional ECM engagement makes microtubule growth persistence insensitive to changes in ECM compliance. Thus, compliance and dimensionality ECM mechanosensing pathways independently regulate specific and distinct microtubule dynamics parameters in endothelial cells to guide branching morphogenesis in physically complex ECMs. PMID:21263030

  9. Diverse Developmental Disorders from The One Ring: Distinct Molecular Pathways Underlie the Cohesinopathies

    PubMed Central

    Horsfield, Julia A.; Print, Cristin G.; Mönnich, Maren

    2012-01-01

    The multi-subunit protein complex, cohesin, is responsible for sister chromatid cohesion during cell division. The interaction of cohesin with DNA is controlled by a number of additional regulatory proteins. Mutations in cohesin, or its regulators, cause a spectrum of human developmental syndromes known as the “cohesinopathies.” Cohesinopathy disorders include Cornelia de Lange Syndrome and Roberts Syndrome. The discovery of novel roles for chromatid cohesion proteins in regulating gene expression led to the idea that cohesinopathies are caused by dysregulation of multiple genes downstream of mutations in cohesion proteins. Consistent with this idea, Drosophila, mouse, and zebrafish cohesinopathy models all show altered expression of developmental genes. However, there appears to be incomplete overlap among dysregulated genes downstream of mutations in different components of the cohesion apparatus. This is surprising because mutations in all cohesion proteins would be predicted to affect cohesin’s roles in cell division and gene expression in similar ways. Here we review the differences and similarities between genetic pathways downstream of components of the cohesion apparatus, and discuss how such differences might arise, and contribute to the spectrum of cohesinopathy disorders. We propose that mutations in different elements of the cohesion apparatus have distinct developmental outcomes that can be explained by sometimes subtly different molecular effects. PMID:22988450

  10. Auditory agnosia and auditory spatial deficits following left hemispheric lesions: evidence for distinct processing pathways.

    PubMed

    Clarke, S; Bellmann, A; Meuli, R A; Assal, G; Steck, A J

    2000-01-01

    Auditory recognition and auditory spatial functions were studied in four patients with circumscribed left hemispheric lesions. Patient FD was severely deficient in recognition of environmental sounds but normal in auditory localisation and auditory motion perception. The lesion included the left superior, middle and inferior temporal gyri and lateral auditory areas (as identified in previous anatomical studies), but spared Heschl's gyrus, the acoustic radiation and the thalamus. Patient SD had the same profile as FD, with deficient recognition of environmental sounds but normal auditory localisation and motion perception. The lesion comprised the postero-inferior part of the frontal convexity and the anterior third of the temporal lobe; data from non-human primates indicate that the latter are interconnected with lateral auditory areas. Patient MA was deficient in recognition of environmental sounds, auditory localisation and auditory motion perception, confirming that auditory spatial functions can be disturbed by left unilateral damage; the lesion involved the supratemporal region as well as the temporal, postero-inferior frontal and antero-inferior parietal convexities. Patient CZ was severely deficient in auditory motion perception and partially deficient in auditory localisation, but normal in recognition of environmental sounds; the lesion involved large parts of the parieto-frontal convexity and the supratemporal region. We propose that auditory information is processed in the human auditory cortex along two distinct pathways, one lateral devoted to auditory recognition and one medial and posterior devoted to auditory spatial functions.

  11. Mutations in 2 distinct genetic pathways result in cerebral cavernous malformations in mice.

    PubMed

    Chan, Aubrey C; Drakos, Stavros G; Ruiz, Oscar E; Smith, Alexandra C H; Gibson, Christopher C; Ling, Jing; Passi, Samuel F; Stratman, Amber N; Sacharidou, Anastasia; Revelo, M Patricia; Grossmann, Allie H; Diakos, Nikolaos A; Davis, George E; Metzstein, Mark M; Whitehead, Kevin J; Li, Dean Y

    2011-05-01

    Cerebral cavernous malformations (CCMs) are a common type of vascular malformation in the brain that are a major cause of hemorrhagic stroke. This condition has been independently linked to 3 separate genes: Krev1 interaction trapped (KRIT1), Cerebral cavernous malformation 2 (CCM2), and Programmed cell death 10 (PDCD10). Despite the commonality in disease pathology caused by mutations in these 3 genes, we found that the loss of Pdcd10 results in significantly different developmental, cell biological, and signaling phenotypes from those seen in the absence of Ccm2 and Krit1. PDCD10 bound to germinal center kinase III (GCKIII) family members, a subset of serine-threonine kinases, and facilitated lumen formation by endothelial cells both in vivo and in vitro. These findings suggest that CCM may be a common tissue manifestation of distinct mechanistic pathways. Nevertheless, loss of heterozygosity (LOH) for either Pdcd10 or Ccm2 resulted in CCMs in mice. The murine phenotype induced by loss of either protein reproduced all of the key clinical features observed in human patients with CCM, as determined by direct comparison with genotype-specific human surgical specimens. These results suggest that CCM may be more effectively treated by directing therapies based on the underlying genetic mutation rather than treating the condition as a single clinical entity.

  12. Follistatin Targets Distinct Pathways To Promote Brown Adipocyte Characteristics in Brown and White Adipose Tissues.

    PubMed

    Singh, Rajan; Braga, Melissa; Reddy, Srinivasa T; Lee, Se-Jin; Parveen, Meher; Grijalva, Victor; Vergnes, Laurent; Pervin, Shehla

    2017-05-01

    We previously demonstrated that Fst expression is highest in brown adipose tissue (BAT) and skeletal muscle, but is also present at substantial levels in epididymal and subcutaneous white adipose tissues (WATs). Fst promotes mouse brown preadipocyte differentiation and promotes browning during differentiation of mouse embryonic fibroblasts. Fst-transgenic (Fst-Tg) mice show substantial increases in circulating Fst levels and increased brown adipose mass. BAT of Fst-Tg mice had increased expression of brown adipose-associated markers including uncoupling protein 1 (UCP1), PRDM16, PGC-1α, and Glut4. WATs from Fst-Tg mice show upregulation of brown/beige adipose markers and significantly increased levels of phosphorylated p38 MAPK/ERK1/2 proteins compared with the wild-type (WT) mice. Pharmacological inhibition of pp38 MAPK/pERK1/2 pathway of recombinant mouse Fst (rFst) treated differentiating 3T3-L1 cells led to significant blockade of Fst-induced UCP1 protein expression. On the other hand, BAT from Fst-Tg mice or differentiating mouse BAT cells treated with rFst show dramatic increase in Myf5 protein levels as well as upregulation of Zic1 and Lhx8 gene expression. Myf5 levels were significantly downregulated in Fst knock-out embryos and small inhibitory RNA-mediated inhibition of Myf5 led to significant inhibition of UCP1, Lhx8, and Zic1 gene expression and significant blockade of Fst-induced induction of UCP1 protein expression in mouse BAT cells. Both interscapular BAT and WAT tissues from Fst-Tg mice display enhanced response to CL316,243 treatment and decreased expression of pSmad3 compared with the WT mice. Therefore, our results indicate that Fst promotes brown adipocyte characteristics in both WAT and BAT depots in vivo through distinct mechanisms. Copyright © 2017 Endocrine Society.

  13. T-cell activation by transitory neo-antigens derived from distinct microbial pathways.

    PubMed

    Corbett, Alexandra J; Eckle, Sidonia B G; Birkinshaw, Richard W; Liu, Ligong; Patel, Onisha; Mahony, Jennifer; Chen, Zhenjun; Reantragoon, Rangsima; Meehan, Bronwyn; Cao, Hanwei; Williamson, Nicholas A; Strugnell, Richard A; Van Sinderen, Douwe; Mak, Jeffrey Y W; Fairlie, David P; Kjer-Nielsen, Lars; Rossjohn, Jamie; McCluskey, James

    2014-05-15

    T cells discriminate between foreign and host molecules by recognizing distinct microbial molecules, predominantly peptides and lipids. Riboflavin precursors found in many bacteria and yeast also selectively activate mucosal-associated invariant T (MAIT) cells, an abundant population of innate-like T cells in humans. However, the genesis of these small organic molecules and their mode of presentation to MAIT cells by the major histocompatibility complex (MHC)-related protein MR1 (ref. 8) are not well understood. Here we show that MAIT-cell activation requires key genes encoding enzymes that form 5-amino-6-d-ribitylaminouracil (5-A-RU), an early intermediate in bacterial riboflavin synthesis. Although 5-A-RU does not bind MR1 or activate MAIT cells directly, it does form potent MAIT-activating antigens via non-enzymatic reactions with small molecules, such as glyoxal and methylglyoxal, which are derived from other metabolic pathways. The MAIT antigens formed by the reactions between 5-A-RU and glyoxal/methylglyoxal were simple adducts, 5-(2-oxoethylideneamino)-6-D-ribitylaminouracil (5-OE-RU) and 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU), respectively, which bound to MR1 as shown by crystal structures of MAIT TCR ternary complexes. Although 5-OP-RU and 5-OE-RU are unstable intermediates, they became trapped by MR1 as reversible covalent Schiff base complexes. Mass spectra supported the capture by MR1 of 5-OP-RU and 5-OE-RU from bacterial cultures that activate MAIT cells, but not from non-activating bacteria, indicating that these MAIT antigens are present in a range of microbes. Thus, MR1 is able to capture, stabilize and present chemically unstable pyrimidine intermediates, which otherwise convert to lumazines, as potent antigens to MAIT cells. These pyrimidine adducts are microbial signatures for MAIT-cell immunosurveillance.

  14. Distinct Pathways of Humoral and Cellular Immunity Induced with the Mucosal Administration of a Nanoemulsion Adjuvant

    PubMed Central

    Makidon, Paul E.; Janczak, Katarzyna W.; Blanco, Luz P.; Swanson, Benjamin; Smith, Douglas M.; Pham, Tiffany; Szabo, Zsuzsanna; Kukowska-Latallo, Jolanta F.; Baker, James R.

    2014-01-01

    Nasal administration of an oil-in-water nanoemulsion (NE) adjuvant W805EC produces potent systemic and mucosal, Th-1– and Th-17–balanced cellular responses. However, its molecular mechanism of action has not been fully characterized and is of particular interest because NE does not contain specific ligands for innate immune receptors. In these studies, we demonstrate that W805EC NE adjuvant activates innate immunity, induces specific gene transcription, and modulates NF-κB activity via TLR2 and TLR4 by a mechanism that appears to be distinct from typical TLR agonists. Nasal immunization with NE-based vaccine showed that the TLR2, TLR4, and MyD88 pathways and IL-12 and IL-12Rβ1 expression are not required for an Ab response, but they are essential for the induction of balanced Th-1 polarization and Th-17 cellular immunity. NE adjuvant induces MHC class II, CD80, and CD86 costimulatory molecule expression and dendritic cell maturation. Further, upon immunization with NE, adjuvant mice deficient in the CD86 receptor had normal Ab responses but significantly reduced Th-1 cellular responses, whereas animals deficient in both CD80 and CD86 or lacking CD40 failed to produce either humoral or cellular immunity. Overall, our data show that intranasal administration of Ag with NE induces TLR2 and TLR4 activation along with a MyD88-independent Ab response and a MyD88-dependent Th-1 and Th-17 cell–mediated immune response. These findings suggest that the unique properties of NE adjuvant may offer novel opportunities for understanding previously unrecognized mechanisms of immune activation important for generating effective mucosal and systemic immune responses. PMID:24532579

  15. Murine Polyomavirus Cell Surface Receptors Activate Distinct Signaling Pathways Required for Infection

    PubMed Central

    O’Hara, Samantha D.

    2016-01-01

    ABSTRACT Virus binding to the cell surface triggers an array of host responses, including activation of specific signaling pathways that facilitate steps in virus entry. Using mouse polyomavirus (MuPyV), we identified host signaling pathways activated upon virus binding to mouse embryonic fibroblasts (MEFs). Pathways activated by MuPyV included the phosphatidylinositol 3-kinase (PI3K), FAK/SRC, and mitogen-activated protein kinase (MAPK) pathways. Gangliosides and α4-integrin are required receptors for MuPyV infection. MuPyV binding to both gangliosides and the α4-integrin receptors was required for activation of the PI3K pathway; however, either receptor interaction alone was sufficient for activation of the MAPK pathway. Using small-molecule inhibitors, we confirmed that the PI3K and FAK/SRC pathways were required for MuPyV infection, while the MAPK pathway was dispensable. Mechanistically, the PI3K pathway was required for MuPyV endocytosis, while the FAK/SRC pathway enabled trafficking of MuPyV along microtubules. Thus, MuPyV interactions with specific cell surface receptors facilitate activation of signaling pathways required for virus entry and trafficking. Understanding how different viruses manipulate cell signaling pathways through interactions with host receptors could lead to the identification of new therapeutic targets for viral infection. PMID:27803182

  16. Distinct Litter Stabilization Dynamics Pathways for Decomposition of Pine Needle and Fine Root Within Soil

    NASA Astrophysics Data System (ADS)

    Mambelli, S.; Filley, T. R.; Bird, J.; Dawson, T.; Torn, M. S.

    2008-12-01

    experiment while most lignin phenols for the humin from the root addition had greater 13C content than SFA. This indicates that molecular fragments of plant biopolymers can readily associate with both labile and stabilized SOM fractions. At the same time, these results suggest that distinct decomposition and stabilization pathways exist for litters, such as needles vs. roots, of different chemical quality.

  17. Protease signalling: the cutting edge

    PubMed Central

    Turk, Boris; Turk, Dus̆an; Turk, Vito

    2012-01-01

    Protease research has undergone a major expansion in the last decade, largely due to the extremely rapid development of new technologies, such as quantitative proteomics and in-vivo imaging, as well as an extensive use of in-vivo models. These have led to identification of physiological substrates and resulted in a paradigm shift from the concept of proteases as protein-degrading enzymes to proteases as key signalling molecules. However, we are still at the beginning of an understanding of protease signalling pathways. We have only identified a minor subset of true physiological substrates for a limited number of proteases, and their physiological regulation is still not well understood. Similarly, links with other signalling systems are not well established. Herein, we will highlight current challenges in protease research. PMID:22367392

  18. Protease signalling: the cutting edge.

    PubMed

    Turk, Boris; Turk, Dušan; Turk, Vito

    2012-04-04

    Protease research has undergone a major expansion in the last decade, largely due to the extremely rapid development of new technologies, such as quantitative proteomics and in-vivo imaging, as well as an extensive use of in-vivo models. These have led to identification of physiological substrates and resulted in a paradigm shift from the concept of proteases as protein-degrading enzymes to proteases as key signalling molecules. However, we are still at the beginning of an understanding of protease signalling pathways. We have only identified a minor subset of true physiological substrates for a limited number of proteases, and their physiological regulation is still not well understood. Similarly, links with other signalling systems are not well established. Herein, we will highlight current challenges in protease research.

  19. Transcriptional Gene Silencing Maintained by OTS1 SUMO Protease Requires a DNA-Dependent Polymerase V-Dependent Pathway1[OPEN

    PubMed Central

    Liu, Lei; Yan, Xiaojing; Zhao, Yiqiang

    2017-01-01

    The expression of genes with aberrant structure is prevented at both the transcriptional and posttranscriptional regulation levels. Aberrant gene silencing at the posttranscriptional level is well studied; however, it is not well understood how aberrant genes are silenced at the transcriptional level. In this study, through genetic screening a transgenic report line that harbors an aberrant gene (35S-LUC, lacking 3′-untranslated region [3′-UTR]) and lacks luciferase (LUC) activity, we identify that the small ubiquitin-like modifier (SUMO) protease OTS1 gene is required for maintaining the silence of the reporter 35S-LUC and an endogenous mutator-like element MULE-F19G14 at the transcriptional level, which requires DNA-dependent RNA polymerase (Pol) V and DDR complex, but not Pol IV. The increased transcripts in ots1 mutants are terminated by the 3′-UTRs of downstream genes. In addition to ots1 mutations, mutations in several known or putative SUMO proteases and two SUMO E3 ligases, SIZ1 and MMS21, have similar effects on this silencing regulation. Taken together, our results reveal that the enzymes involved in the SUMOylation process restrain aberrant gene transcription by using a downstream gene 3′-UTR, and this regulation requires a functional Pol V-dependent pathway in Arabidopsis (Arabidopsis thaliana). PMID:27852949

  20. Specific Hsp100 Chaperones Determine the Fate of the First Enzyme of the Plastidial Isoprenoid Pathway for Either Refolding or Degradation by the Stromal Clp Protease in Arabidopsis

    PubMed Central

    Pulido, Pablo; Llamas, Ernesto; Llorente, Briardo; Ventura, Salvador; Wright, Louwrance P.; Rodríguez-Concepción, Manuel

    2016-01-01

    The lifespan and activity of proteins depend on protein quality control systems formed by chaperones and proteases that ensure correct protein folding and prevent the formation of toxic aggregates. We previously found that the Arabidopsis thaliana J-protein J20 delivers inactive (misfolded) forms of the plastidial enzyme deoxyxylulose 5-phosphate synthase (DXS) to the Hsp70 chaperone for either proper folding or degradation. Here we show that the fate of Hsp70-bound DXS depends on pathways involving specific Hsp100 chaperones. Analysis of individual mutants for the four Hsp100 chaperones present in Arabidopsis chloroplasts showed increased levels of DXS proteins (but not transcripts) only in those defective in ClpC1 or ClpB3. However, the accumulated enzyme was active in the clpc1 mutant but inactive in clpb3 plants. Genetic evidence indicated that ClpC chaperones might be required for the unfolding of J20-delivered DXS protein coupled to degradation by the Clp protease. By contrast, biochemical and genetic approaches confirmed that Hsp70 and ClpB3 chaperones interact to collaborate in the refolding and activation of DXS. We conclude that specific J-proteins and Hsp100 chaperones act together with Hsp70 to recognize and deliver DXS to either reactivation (via ClpB3) or removal (via ClpC1) depending on the physiological status of the plastid. PMID:26815787

  1. PTS1 Peroxisomal Import Pathway Plays Shared and Distinct Roles to PTS2 Pathway in Development and Pathogenicity of Magnaporthe oryzae

    PubMed Central

    Wang, Jiaoyu; Zhang, Zhen; Wang, Yanli; Li, Ling; Chai, Rongyao; Mao, Xueqin; Jiang, Hua; Qiu, Haiping; Du, Xinfa; Lin, Fucheng; Sun, Guochang

    2013-01-01

    Peroxisomes participate in various important metabolisms and are required in pathogenicity of fungal plant pathogens. Peroxisomal matrix proteins are imported from cytoplasm into peroxisomes through peroxisomal targeting signal 1 (PTS1) or peroxisomal targeting signal 2 (PTS2) import pathway. PEX5 and PEX7 genes participate in the two pathways respectively. The involvement of PEX7 mediated PTS2 import pathway in fungal pathogenicity has been documented, while that of PTS1 remains unclear. Through null mutant analysis of MoPEX5, the PEX5 homolog in Magnaporthe oryzae, we report the crucial roles of PTS1 pathway in the development and host infection in the rice blast fungus, and compared with those of PTS2. We found that MoPEX5 disruption specifically blocked the PTS1 pathway. Δmopex5 was unable to use lipids as sole carbon source and lost pathogenicity completely. Similar as Δmopex7, Δmopex5 exhibited significant reduction in lipid utilization and mobilization, appressorial turgor genesis and H2O2 resistance. Additionally, Δmopex5 presented some distinct defects which were undetected in Δmopex7 in vegetative growth, conidial morphogenesis, appressorial morphogenesis and melanization. The results indicated that the PTS1 peroxisomal import pathway, in addition to PTS2, is required for fungal development and pathogenicity of the rice blast fungus, and also, as a main peroxisomal import pathway, played a more predominant role than PTS2. PMID:23405169

  2. MALT1 Protease Activity Is Required for Innate and Adaptive Immune Responses.

    PubMed

    Yu, Jong W; Hoffman, Sandy; Beal, Allison M; Dykon, Angela; Ringenberg, Michael A; Hughes, Anna C; Dare, Lauren; Anderson, Amber D; Finger, Joshua; Kasparcova, Viera; Rickard, David; Berger, Scott B; Ramanjulu, Joshi; Emery, John G; Gough, Peter J; Bertin, John; Foley, Kevin P

    2015-01-01

    CARMA-BCL10-MALT1 signalosomes play important roles in antigen receptor signaling and other pathways. Previous studies have suggested that as part of this complex, MALT1 functions as both a scaffolding protein to activate NF-κB through recruitment of ubiquitin ligases, and as a protease to cleave and inactivate downstream inhibitory signaling proteins. However, our understanding of the relative importance of these two distinct MALT1 activities has been hampered by a lack of selective MALT1 protease inhibitors with suitable pharmacologic properties. To fully investigate the role of MALT1 protease activity, we generated mice homozygous for a protease-dead mutation in MALT1. We found that some, but not all, MALT1 functions in immune cells were dependent upon its protease activity. Protease-dead mice had defects in the generation of splenic marginal zone and peritoneal B1 B cells. CD4+ and CD8+ T cells displayed decreased T cell receptor-stimulated proliferation and IL-2 production while B cell receptor-stimulated proliferation was partially dependent on protease activity. In dendritic cells, stimulation of cytokine production through the Dectin-1, Dectin-2, and Mincle C-type lectin receptors was also found to be partially dependent upon protease activity. In vivo, protease-dead mice had reduced basal immunoglobulin levels, and showed defective responses to immunization with T-dependent and T-independent antigens. Surprisingly, despite these decreased responses, MALT1 protease-dead mice, but not MALT1 null mice, developed mixed inflammatory cell infiltrates in multiple organs, suggesting MALT1 protease activity plays a role in immune homeostasis. These findings highlight the importance of MALT1 protease activity in multiple immune cell types, and in integrating immune responses in vivo.

  3. Distinct Corticostriatal and Intracortical Pathways Mediate Bilateral Sensory Responses in the Striatum

    PubMed Central

    Reig, Ramon; Silberberg, Gilad

    2016-01-01

    Individual striatal neurons integrate somatosensory information from both sides of the body, however, the afferent pathways mediating these bilateral responses are unclear. Whereas ipsilateral corticostriatal projections are prevalent throughout the neocortex, contralateral projections provide sparse input from primary sensory cortices, in contrast to the dense innervation from motor and frontal regions. There is, therefore, an apparent discrepancy between the observed anatomical pathways and the recorded striatal responses. We used simultaneous in vivo whole-cell and extracellular recordings combined with focal cortical silencing, to dissect the afferent pathways underlying bilateral sensory integration in the mouse striatum. We show that unlike direct corticostriatal projections mediating responses to contralateral whisker deflection, responses to ipsilateral stimuli are mediated mainly by intracortical projections from the contralateral somatosensory cortex (S1). The dominant pathway is the callosal projection from contralateral to ipsilateral S1. Our results suggest a functional difference between the cortico-basal ganglia pathways underlying bilateral sensory and motor processes. PMID:27664965

  4. Ubiquitin specific protease 21 upregulation in breast cancer promotes cell tumorigenic capability and is associated with the NOD-like receptor signaling pathway

    PubMed Central

    Peng, Liang; Hu, Yi; Chen, Demeng; Linghu, Ruixia; Wang, Yingzhe; Kou, Xiaoxue; Yang, Junlan; Jiao, Shunchang

    2016-01-01

    Ubiquitination and deubiquitination have emerged as critical regulators in cancer. In the present study, the expression pattern of 50 ubiquitin-specific proteases (USPs) was summarized in breast cancer using a bioinformatics approach, and USP21 was identified as the most altered gene in breast cancer. In particular, expression of USP21 in triple negative breast cancer (TNBC) cell lines was greater compared with other subtypes of breast cancer. Knockdown of USP21 in TNBC cells inhibited cell proliferation, migration and invasion. Microarray profiling of the USP21 knockdown cells revealed significant downregulation of multiple genes associated with the NOD-like receptor signaling pathway. The results of the present study suggest that USP21 has a significant role in TNBC progression, and therefore may represent a novel therapeutic target. PMID:28105162

  5. Cartography of Pathway Signal Perturbations Identifies Distinct Molecular Pathomechanisms in Malignant and Chronic Lung Diseases.

    PubMed

    Arakelyan, Arsen; Nersisyan, Lilit; Petrek, Martin; Löffler-Wirth, Henry; Binder, Hans

    2016-01-01

    Lung diseases are described by a wide variety of developmental mechanisms and clinical manifestations. Accurate classification and diagnosis of lung diseases are the bases for development of effective treatments. While extensive studies are conducted toward characterization of various lung diseases at molecular level, no systematic approach has been developed so far. Here we have applied a methodology for pathway-centered mining of high throughput gene expression data to describe a wide range of lung diseases in the light of shared and specific pathway activity profiles. We have applied an algorithm combining a Pathway Signal Flow (PSF) algorithm for estimation of pathway activity deregulation states in lung diseases and malignancies, and a Self Organizing Maps algorithm for classification and clustering of the pathway activity profiles. The analysis results allowed clearly distinguish between cancer and non-cancer lung diseases. Lung cancers were characterized by pathways implicated in cell proliferation, metabolism, while non-malignant lung diseases were characterized by deregulations in pathways involved in immune/inflammatory response and fibrotic tissue remodeling. In contrast to lung malignancies, chronic lung diseases had relatively heterogeneous pathway deregulation profiles. We identified three groups of interstitial lung diseases and showed that the development of characteristic pathological processes, such as fibrosis, can be initiated by deregulations in different signaling pathways. In conclusion, this paper describes the pathobiology of lung diseases from systems viewpoint using pathway centered high-dimensional data mining approach. Our results contribute largely to current understanding of pathological events in lung cancers and non-malignant lung diseases. Moreover, this paper provides new insight into molecular mechanisms of a number of interstitial lung diseases that have been studied to a lesser extent.

  6. Cartography of Pathway Signal Perturbations Identifies Distinct Molecular Pathomechanisms in Malignant and Chronic Lung Diseases

    PubMed Central

    Arakelyan, Arsen; Nersisyan, Lilit; Petrek, Martin; Löffler-Wirth, Henry; Binder, Hans

    2016-01-01

    Lung diseases are described by a wide variety of developmental mechanisms and clinical manifestations. Accurate classification and diagnosis of lung diseases are the bases for development of effective treatments. While extensive studies are conducted toward characterization of various lung diseases at molecular level, no systematic approach has been developed so far. Here we have applied a methodology for pathway-centered mining of high throughput gene expression data to describe a wide range of lung diseases in the light of shared and specific pathway activity profiles. We have applied an algorithm combining a Pathway Signal Flow (PSF) algorithm for estimation of pathway activity deregulation states in lung diseases and malignancies, and a Self Organizing Maps algorithm for classification and clustering of the pathway activity profiles. The analysis results allowed clearly distinguish between cancer and non-cancer lung diseases. Lung cancers were characterized by pathways implicated in cell proliferation, metabolism, while non-malignant lung diseases were characterized by deregulations in pathways involved in immune/inflammatory response and fibrotic tissue remodeling. In contrast to lung malignancies, chronic lung diseases had relatively heterogeneous pathway deregulation profiles. We identified three groups of interstitial lung diseases and showed that the development of characteristic pathological processes, such as fibrosis, can be initiated by deregulations in different signaling pathways. In conclusion, this paper describes the pathobiology of lung diseases from systems viewpoint using pathway centered high-dimensional data mining approach. Our results contribute largely to current understanding of pathological events in lung cancers and non-malignant lung diseases. Moreover, this paper provides new insight into molecular mechanisms of a number of interstitial lung diseases that have been studied to a lesser extent. PMID:27200087

  7. Mutation profiles of synchronous colorectal cancers from a patient with Lynch syndrome suggest distinct oncogenic pathways

    PubMed Central

    Shi, Chanjuan; Holt, Jonathan A.; Vnencak-Jones, Cindy L.

    2016-01-01

    Patients with Lynch syndrome often present with multiple synchronous or metachronous colorectal cancers (CRCs). The presence of multiple CRCs with distinct genetic profiles and driver mutations could complicate treatment as each cancer may respond differently to therapy. Studies of sporadic CRCs suggested that synchronous tumors have distinct etiologies, but could not rule out differences in genetic background. The presence of multiple cancers in a patient with a predisposing mutation provides an opportunity to profile synchronous cancers in the same genetic background. Here, we describe the case of a patient with Lynch syndrome that presented with six synchronous CRCs. Microsatellite instability (MSI) and genomic profiling indicated that each lesion had a unique pattern of instability and a distinct profile of affected genes. These findings support the idea that in Lynch syndrome, synchronous CRCs can develop in parallel with distinct mutation profiles and that these differences may inform treatment decisions. PMID:27284491

  8. Gut Symbionts from Distinct Hosts Exhibit Genotoxic Activity via Divergent Colibactin Biosynthesis Pathways

    PubMed Central

    Vizcaino, Maria I.; Crawford, Jason M.

    2014-01-01

    Secondary metabolites produced by nonribosomal peptide synthetase (NRPS) or polyketide synthase (PKS) pathways are chemical mediators of microbial interactions in diverse environments. However, little is known about their distribution, evolution, and functional roles in bacterial symbionts associated with animals. A prominent example is colibactin, a largely unknown family of secondary metabolites produced by Escherichia coli via a hybrid NRPS-PKS biosynthetic pathway that inflicts DNA damage upon eukaryotic cells and contributes to colorectal cancer and tumor formation in the mammalian gut. Thus far, homologs of this pathway have only been found in closely related Enterobacteriaceae, while a divergent variant of this gene cluster was recently discovered in a marine alphaproteobacterial Pseudovibrio strain. Herein, we sequenced the genome of Frischella perrara PEB0191, a bacterial gut symbiont of honey bees and identified a homologous colibactin biosynthetic pathway related to those found in Enterobacteriaceae. We show that the colibactin genomic island (GI) has conserved gene synteny and biosynthetic module architecture across F. perrara, Enterobacteriaceae, and the Pseudovibrio strain. Comparative metabolomics analyses of F. perrara and E. coli further reveal that these two bacteria produce related colibactin pathway-dependent metabolites. Finally, we demonstrate that F. perrara, like E. coli, causes DNA damage in eukaryotic cells in vitro in a colibactin pathway-dependent manner. Together, these results support that divergent variants of the colibactin biosynthetic pathway are widely distributed among bacterial symbionts, producing related secondary metabolites and likely endowing its producer with functional capabilities important for diverse symbiotic associations. PMID:25527542

  9. Pseudo-active sites of protease domains: HGF/Met and Sonic hedgehog signaling in cancer.

    PubMed

    Maun, Henry R; Kirchhofer, Daniel; Lazarus, Robert A

    2010-08-01

    Proteases represent a large class of enzymes with crucial biological functions. Although targeting various relevant proteases for therapeutic intervention has been widely investigated, structurally related proteins lacking proteolytic activity (pseudo-proteases) have received relatively little attention. Two distinct clinically relevant cancer pathways that contain signaling proteins with pseudo-protease domains include the Met and Hedgehog (Hh) pathways. The receptor tyrosine kinase Met pathway is driven by hepatocyte growth factor (HGF), a plasminogen-related ligand that binds Met and activates intracellular pathways resulting in cell proliferation, angiogenesis, motility and survival. HGF is a disulfide-linked alpha/beta-heterodimer having a trypsin serine protease-like beta-chain. The Hh pathway is driven by Sonic hedgehog (Shh), which has a Zn(2+) metalloprotease fold and binds Patched1 (Ptc1), which de-represses Smoothened and ultimately activates Gli-dependent transcription. Although HGF and Shh differ in structure and function, the pseudo-catalytic sites of both HGF and Shh are crucial for signal transduction. For HGF, this region binds the Met beta-propeller domain, which leads to Met dimerization and signaling. For Hh, this region binds to the antagonist receptor Hedgehog-interacting protein (Hhip) and most probably to Ptc1 as well. Thus, for both HGF and Hh pathways, targeting ligand pseudo-active sites represents a new strategy for regulation.

  10. Homology-directed repair of DNA nicks via pathways distinct from canonical double-strand break repair.

    PubMed

    Davis, Luther; Maizels, Nancy

    2014-03-11

    DNA nicks are the most common form of DNA damage, and if unrepaired can give rise to genomic instability. In human cells, nicks are efficiently repaired via the single-strand break repair pathway, but relatively little is known about the fate of nicks not processed by that pathway. Here we show that homology-directed repair (HDR) at nicks occurs via a mechanism distinct from HDR at double-strand breaks (DSBs). HDR at nicks, but not DSBs, is associated with transcription and is eightfold more efficient at a nick on the transcribed strand than at a nick on the nontranscribed strand. HDR at nicks can proceed by a pathway dependent upon canonical HDR factors RAD51 and BRCA2; or by an efficient alternative pathway that uses either ssDNA or nicked dsDNA donors and that is strongly inhibited by RAD51 and BRCA2. Nicks generated by either I-AniI or the CRISPR/Cas9(D10A) nickase are repaired by the alternative HDR pathway with little accompanying mutagenic end-joining, so this pathway may be usefully applied to genome engineering. These results suggest that alternative HDR at nicks may be stimulated in physiological contexts in which canonical RAD51/BRCA2-dependent HDR is compromised or down-regulated, which occurs frequently in tumors.

  11. Homology-directed repair of DNA nicks via pathways distinct from canonical double-strand break repair

    PubMed Central

    Davis, Luther; Maizels, Nancy

    2014-01-01

    DNA nicks are the most common form of DNA damage, and if unrepaired can give rise to genomic instability. In human cells, nicks are efficiently repaired via the single-strand break repair pathway, but relatively little is known about the fate of nicks not processed by that pathway. Here we show that homology-directed repair (HDR) at nicks occurs via a mechanism distinct from HDR at double-strand breaks (DSBs). HDR at nicks, but not DSBs, is associated with transcription and is eightfold more efficient at a nick on the transcribed strand than at a nick on the nontranscribed strand. HDR at nicks can proceed by a pathway dependent upon canonical HDR factors RAD51 and BRCA2; or by an efficient alternative pathway that uses either ssDNA or nicked dsDNA donors and that is strongly inhibited by RAD51 and BRCA2. Nicks generated by either I-AniI or the CRISPR/Cas9D10A nickase are repaired by the alternative HDR pathway with little accompanying mutagenic end-joining, so this pathway may be usefully applied to genome engineering. These results suggest that alternative HDR at nicks may be stimulated in physiological contexts in which canonical RAD51/BRCA2-dependent HDR is compromised or down-regulated, which occurs frequently in tumors. PMID:24556991

  12. Acetylcholinesterase staining differentiates functionally distinct auditory pathways in the barn owl.

    PubMed

    Adolphs, R

    1993-03-15

    The aim of this study was to examine how the functional specialization of the barn owl's auditory brainstem might correlate with histochemical compartmentalization. The barn owl uses interaural intensity and time differences to encode, respectively, the vertical and azimuthal positions of sound sources in space. These two auditory cues are processed in parallel ascending pathways that separate from each other at the level of the cochlear nuclei. Sections through the auditory brainstem were stained for acetylcholinesterase (AChE) to examine whether nuclei that process different auditory cues stain differentially for this enzyme. Of the two cochlear nuclei, angularis showed more intense staining than nucleus magnocellularis. Nucleus angularis projects to all of the nuclei and subdivisions of nuclei that belong to the intensity processing pathway. Acetylcholinesterase stained all regions that contain terminal fields of nucleus angularis and thus provided discrimination between the time and intensity pathways. Moreover, staining patterns with acetylcholinesterase were complementary to those previously reported with an anti-calbindin antibody, which stains terminal fields of nucleus laminaris, and thus stains all the nuclei and subdivisions of nuclei that belong to the time pathway. Some of the gross staining patterns observed with AChE were similar to those reported with antibodies to glutamate decarboxylase. However, AChE is a more convenient and definitive marker in discriminating between these pathways than is calbindin or glutamate decarboxylase. Acetylcholinesterase staining of the intensity pathway in the owl may be related to encoding of sound intensity by spike rate over large dynamic ranges.

  13. Overlapping and distinct pRb pathways in the mammalian auditory and vestibular organs

    PubMed Central

    Huang, Mingqian; Sage, Cyrille; Tang, Yong; Lee, Sang Goo; Petrillo, Marco; Hinds, Philip W

    2011-01-01

    Retinoblastoma gene (Rb1) is required for proper cell cycle exit in the developing mouse inner ear and its deletion in the embryo leads to proliferation of sensory progenitor cells that differentiate into hair cells and supporting cells. In a conditional hair cell Rb1 knockout mouse, Pou4f3-Cre-pRb™/™, pRb™/™ utricular hair cells differentiate and survive into adulthood whereas differentiation and survival of pRb™/™ cochlear hair cells are impaired. To comprehensively survey the pRb pathway in the mammalian inner ear, we performed microarray analysis of pRb™/™ cochlea and utricle. The comparative analysis shows that the core pathway shared between pRb™/™ cochlea and utricle is centered on e2F, the key pathway that mediates pRb function. A majority of differentially expressed genes and enriched pathways are not shared but uniquely associated with pRb™/™ cochlea or utricle. In pRb™/™ cochlea, pathways involved in early inner ear development such as Wnt/β-catenin and Notch were enriched, whereas pathways involved in proliferation and survival are enriched in pRb™/™ utricle. Clustering analysis showed that the pRb™/™ inner ear has characteristics of a younger control inner ear, an indication of delayed differentiation. We created a transgenic mouse model (ER-Cre-pRbflox/flox) in which Rb1 can be acutely deleted postnatally. Acute Rb1 deletion in the adult mouse fails to induce proliferation or cell death in inner ear, strongly indicating that Rb1 loss in these postmitotic tissues can be effectively compensated for, or that pRb-mediated changes in the postmitotic compartment result in events that are functionally irreversible once enacted. This study thus supports the concept that pRb-regulated pathways relevant to hair cell development, encompassing proliferation, differentiation and survival, act predominantly during early development. PMID:21239885

  14. Distinct roles of GABAergic interneurons in the regulation of striatal output pathways

    PubMed Central

    Gittis, Aryn H.; Nelson, Alexandra B.; Thwin, Myo T.; Palop, Jorge J.; Kreitzer, Anatol C.

    2010-01-01

    Striatal GABAergic microcircuits are critical for motor function, yet their properties remain enigmatic due to difficulties in targeting striatal interneurons for electrophysiological analysis. Here, we utilize Lhx6-GFP transgenic mice to identify GABAergic interneurons and investigate their regulation of striatal direct- and indirect-pathway medium spiny neurons (MSNs). We find that the two major interneuron populations, persistent low-threshold spiking (PLTS) and fast spiking (FS) interneurons, differ substantially in their excitatory inputs and inhibitory outputs. Excitatory synaptic currents recorded from PLTS interneurons are characterized by a small, non-rectifying AMPA receptor-mediated component and a NMDA receptor-mediated component. In contrast, glutamatergic synaptic currents in FS interneurons have a large, strongly-rectifying AMPA receptor-mediated component, but no detectable NMDA receptor-mediated responses. Consistent with their axonal morphology, the output of individual PLTS interneurons is relatively weak and sparse, whereas FS interneurons are robustly connected to MSNs and other FS interneurons and appear to mediate the bulk of feedforward inhibition. Synaptic depression of FS outputs is relatively insensitive to firing frequency, and dynamic-clamp experiments reveal that these short-term dynamics enable feedforward inhibition to remain efficacious across a broad frequency range. Surprisingly, we find that FS interneurons preferentially target direct-pathway MSNs over indirect-pathway MSNs, suggesting a potential mechanism for rapid pathway-specific regulation of striatal output pathways. PMID:20147549

  15. Parsing the construct of maternal insensitivity: distinct longitudinal pathways associated with early maternal withdrawal.

    PubMed

    Lyons-Ruth, Karlen; Bureau, Jean-Francois; Easterbrooks, M Ann; Obsuth, Ingrid; Hennighausen, Kate; Vulliez-Coady, Lauriane

    2013-01-01

    The current paper expands on Ainsworth's seminal construct of maternal sensitivity by exploring the developmental pathways associated with one particular form of insensitivity: maternal withdrawal. Drawing on longitudinal data from infancy to age 20 in a high-risk cohort, we highlight how maternal withdrawal over the first eight years of life is associated with child caregiving behavior and with maternal role confusion, as well as with features of borderline and antisocial personality disorders. We also present evidence for the specificity of this pathway in relation to other aspects of maternal insensitivity and other aspects of child adaptation. To illuminate these pathways we both review recent published work and report new findings on the middle childhood and adolescent components of these trajectories. Finally, we consider the implications for assessment of maternal behavior in high-risk samples and indicate directions for productive future work.

  16. Supermarket Proteases.

    ERIC Educational Resources Information Center

    Hagar, William G.; Bullerwell, Lornie D.

    2003-01-01

    Presents a laboratory activity on enzymes. Uses common items found in the supermarket that contain protease enzymes, such as contact lens cleaner and meat tenderizer. Demonstrates the digestion of gelatin proteins as part of enzymatic reactions. (Author/SOE)

  17. Supermarket Proteases.

    ERIC Educational Resources Information Center

    Hagar, William G.; Bullerwell, Lornie D.

    2003-01-01

    Presents a laboratory activity on enzymes. Uses common items found in the supermarket that contain protease enzymes, such as contact lens cleaner and meat tenderizer. Demonstrates the digestion of gelatin proteins as part of enzymatic reactions. (Author/SOE)

  18. Two pathways of activation of the superoxide-generating NADPH oxidase of phagocytes in vitro--distinctive effects of inhibitors.

    PubMed

    Sigal, Natalia; Gorzalczany, Yara; Pick, Edgar

    2003-06-01

    The NADPH oxidase complex of phagocytes comprises a membrane-associated flavocytochrome b559, and 4 cytosolic components: p47phox, p67phox, p40phox, and the small GTPase Rac. Activation of the oxidase in vivo is the result of assembly of the cytosolic components with cytochrome b559 and is mimicked in vitro by a cell-free system consisting of membranes, p47phox, p67phox, nonprenylated or prenylated Rac, and an anionic amphiphile as activator (defined as "p47phox and amphiphile-dependent" or canonical pathway). We reported that prenylated Rac1 is capable of activating the NADPH oxidase in vitro in the absence of p47phox and amphiphile (defined as "p47phox and amphiphile-independent" pathway). We now demonstrate that the 2 pathways exhibit distinctive susceptibilities to inhibitors: 1) The anionic amphiphile lithium dodecyl sulfate, an activator of the canonical pathway, has the opposite effect (inhibition) on oxidase activation by prenylated Rac and p67phox; 2) GDP and, paradoxically, GTP (but not GMP, ATP, ADP, and AMP) prevent oxidase activation by the p47phox and amphiphile-independent pathway but do not affect activation by the canonical pathway; 3) The Rac-binding domain of p21-activated kinase is a potent inhibitor of activation by the p47phox and amphiphile-independent pathway while exerting a milder inhibitory effect on the canonical pathway; 4) The C-terminal polybasic Rac1 peptide 177-191 and the cationic antibiotic neomycin sulfate inhibit activation by the canonical pathway but do not affect activation by the p47phox and amphiphile-independent pathway; 5) Binding of prenylated Rac1 to membrane-mimicking phospholipid vesicles is, nevertheless, enhanced when these contain negatively charged lipids. It is proposed that preferential inhibition of oxidase activation, via the p47phox and amphiphile-independent pathway, is a reflection of interference by the inhibitors with Rac-dependent recruitment of p67phox to the membrane.

  19. Distinct Pathways Regulated by RET and Estrogen Receptor in Luminal Breast Cancer Demonstrate the Biological Basis for Combination Therapy

    PubMed Central

    Spanheimer, Philip M.; Cyr, Anthony R.; Gillum, Matthew P.; Woodfield, George W.; Askeland, Ryan W.; Weigel, Ronald J.

    2014-01-01

    Objectives We investigated directed therapy based on TFAP2C-regulated pathways to inform new therapeutic approaches for treatment of luminal breast cancer. Background TFAP2C regulates the expression of genes characterizing the luminal phenotype including ESR1 and RET, but pathway cross talk and potential for distinct elements have not been characterized. Methods Activation of extracellular signal-regulated kinases (ERK) and AKT was assessed using phosphorylation-specific Western blot. Cell proliferation was measured with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] after siRNA (small interfering RNA) gene knockdown or drug treatment. Cell cycle, Ki-67, and cleaved caspase 3 were measured by fluorescence-activated cell sorting. Tumorigenesis was assessed in mice xenografts. Results Knockdown of TFAP2C or RET inhibited GDNF (glial cell line–derived neurotrophic factor)–mediated activation of ERK and AKT in MCF-7 cells. Similarly, sunitinib, a small-molecule inhibitor of RET, blocked GDNF-mediated activation of ERK and AKT. Inhibition of RET either by gene knockdown or by treatment with sunitinib or vandetanib reduced RET-dependent growth of luminal breast cancer cells. Interestingly, knockdown of TFAP2C, which controls both ER (estrogen receptor) and RET, demonstrated a greater effect on cell growth than either RET or ER alone. Parallel experiments using treatment with tamoxifen and sunitinib confirmed the increased effectiveness of dual inhibition of the ER and RET pathways in regulating cell growth. Whereas targeting the ER pathway altered cell proliferation, as measured by Ki-67 and S-phase, anti-RET primarily increased apoptosis, as demonstrated by cleaved caspase 3 and increased TUNEL (terminal deoxyneucleotidyl transferase dUTP nick end labeling) expression in xenografts. Conclusions ER and RET primarily function through distinct pathways regulating proliferation and cell survival, respectively. The findings inform a therapeutic

  20. Distinct pathways regulated by RET and estrogen receptor in luminal breast cancer demonstrate the biological basis for combination therapy.

    PubMed

    Spanheimer, Philip M; Cyr, Anthony R; Gillum, Matthew P; Woodfield, George W; Askeland, Ryan W; Weigel, Ronald J

    2014-04-01

    We investigated directed therapy based on TFAP2C-regulated pathways to inform new therapeutic approaches for treatment of luminal breast cancer. TFAP2C regulates the expression of genes characterizing the luminal phenotype including ESR1 and RET, but pathway cross talk and potential for distinct elements have not been characterized. Activation of extracellular signal-regulated kinases (ERK) and AKT was assessed using phosphorylation-specific Western blot. Cell proliferation was measured with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] after siRNA (small interfering RNA) gene knockdown or drug treatment. Cell cycle, Ki-67, and cleaved caspase 3 were measured by fluorescence-activated cell sorting. Tumorigenesis was assessed in mice xenografts. Knockdown of TFAP2C or RET inhibited GDNF (glial cell line-derived neurotrophic factor)-mediated activation of ERK and AKT in MCF-7 cells. Similarly, sunitinib, a small-molecule inhibitor of RET, blocked GDNF-mediated activation of ERK and AKT. Inhibition of RET either by gene knockdown or by treatment with sunitinib or vandetanib reduced RET-dependent growth of luminal breast cancer cells. Interestingly, knockdown of TFAP2C, which controls both ER (estrogen receptor) and RET, demonstrated a greater effect on cell growth than either RET or ER alone. Parallel experiments using treatment with tamoxifen and sunitinib confirmed the increased effectiveness of dual inhibition of the ER and RET pathways in regulating cell growth. Whereas targeting the ER pathway altered cell proliferation, as measured by Ki-67 and S-phase, anti-RET primarily increased apoptosis, as demonstrated by cleaved caspase 3 and increased TUNEL (terminal deoxyneucleotidyl transferase dUTP nick end labeling) expression in xenografts. ER and RET primarily function through distinct pathways regulating proliferation and cell survival, respectively. The findings inform a therapeutic approach based on combination therapy with antiestrogen and

  1. Histone Deacetylase Inhibitors Antagonize Distinct Pathways to Suppress Tumorigenesis of Embryonal Rhabdomyosarcoma

    PubMed Central

    Vleeshouwer-Neumann, Terra; Phelps, Michael; Bammler, Theo K.; MacDonald, James W.; Jenkins, Isaac; Chen, Eleanor Y.

    2015-01-01

    Embryonal rhabdomyosarcoma (ERMS) is the most common soft tissue cancer in children. The prognosis of patients with relapsed or metastatic disease remains poor. ERMS genomes show few recurrent mutations, suggesting that other molecular mechanisms such as epigenetic regulation might play a major role in driving ERMS tumor biology. In this study, we have demonstrated the diverse roles of histone deacetylases (HDACs) in the pathogenesis of ERMS by characterizing effects of HDAC inhibitors, trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA; also known as vorinostat) in vitro and in vivo. TSA and SAHA suppress ERMS tumor growth and progression by inducing myogenic differentiation as well as reducing the self-renewal and migratory capacity of ERMS cells. Differential expression profiling and pathway analysis revealed downregulation of key oncogenic pathways upon HDAC inhibitor treatment. By gain-of-function, loss-of-function, and chromatin immunoprecipitation (ChIP) studies, we show that Notch1- and EphrinB1-mediated pathways are regulated by HDACs to inhibit differentiation and enhance migratory capacity of ERMS cells, respectively. Our study demonstrates that aberrant HDAC activity plays a major role in ERMS pathogenesis. Druggable targets in the molecular pathways affected by HDAC inhibitors represent novel therapeutic options for ERMS patients. PMID:26636678

  2. Two distinct, calcium-mediated, signal transduction pathways can trigger deflagellation in Chlamydomonas reinhardtii

    PubMed Central

    1994-01-01

    The molecular machinery of deflagellation can be activated in detergent permeabilized Chlamydomonas reinhardtii by the addition of Ca2+ (Sanders, M. A., and J. L. Salisbury, 1989. J. Cell Biol. 108:1751- 1760). This suggests that stimuli which induce deflagellation in living cells cause an increase in the intracellular concentration of Ca2+, but this has never been demonstrated. In this paper we report that the wasp venom peptide, mastoparan, and the permeant organic acid, benzoate, activate two different signalling pathways to trigger deflagellation. We have characterized each pathway with respect to: (a) the requirement for extracellular Ca2+; (b) sensitivity to Ca2+ channel blockers; and (c) 45Ca influx. We also report that a new mutant strain of C. reinhardtii, adf-1, is specifically defective in the acid-activated signalling pathway. Both signalling pathways appear normal in another mutant, fa-1, that is defective in the machinery of deflagellation (Lewin, R. and C. Burrascano. 1983. Experientia. 39:1397-1398; Sanders, M. A., and J. L. Salisbury. 1989. J. Cell Biol. 108:1751-1760). We conclude that mastoparan induces the release of an intracellular pool of Ca2+ whereas acid induces an influx of extracellular Ca2+ to activate the machinery of deflagellation. PMID:8120101

  3. Two distinct pathways supply anthranilate as a precursor of the Pseudomonas quinolone signal.

    PubMed

    Farrow, John M; Pesci, Everett C

    2007-05-01

    Pseudomonas aeruginosa is an opportunistic pathogen that causes serious infections in immunocompromised patients and those with cystic fibrosis (CF). This gram-negative bacterium uses multiple cell-to-cell signals to control numerous cellular functions and virulence. One of these signals is 2-heptyl-3-hydroxy-4-quinolone, which is referred to as the Pseudomonas quinolone signal (PQS). This signal functions as a coinducer for a transcriptional regulator (PqsR) to positively control multiple virulence genes and its own synthesis. PQS production is required for virulence in multiple models of infection, and it has been shown to be produced in the lungs of CF patients infected by P. aeruginosa. One of the precursor compounds from which PQS is synthesized is the metabolite anthranilate. This compound can be derived from the conversion of chorismate to anthranilate by an anthranilate synthase or through the degradation of tryptophan via the anthranilate branch of the kynurenine pathway. In this study, we present data which help to define the kynurenine pathway in P. aeruginosa and show that the kynurenine pathway serves as a critical source of anthranilate for PQS synthesis. We also show that the kyn pathway genes are induced during growth with tryptophan and that they are autoregulated by kynurenine. This study provides solid foundations for the understanding of how P. aeruginosa produces the anthranilate that serves as a precursor to PQS and other 4-quinolones.

  4. Coagulation factor VIIa-mediated protease-activated receptor 2 activation leads to β-catenin accumulation via the AKT/GSK3β pathway and contributes to breast cancer progression.

    PubMed

    Roy, Abhishek; Ansari, Shabbir A; Das, Kaushik; Prasad, Ramesh; Bhattacharya, Anindita; Mallik, Suman; Mukherjee, Ashis; Sen, Prosenjit

    2017-08-18

    Cell migration and invasion are very characteristic features of cancer cells that promote metastasis, which is one of the most common causes of mortality among cancer patients. Emerging evidence has shown that coagulation factors can directly mediate cancer-associated complications either by enhancing thrombus formation or by initiating various signaling events leading to metastatic cancer progression. It is well established that, apart from its distinct role in blood coagulation, coagulation factor FVIIa enhances aggressive behaviors of breast cancer cells, but the underlying signaling mechanisms still remain elusive. To this end, we investigated FVIIa's role in the migration and invasiveness of the breast cancer cell line MDA-MB-231. Consistent with previous observations, we observed that FVIIa increased the migratory and invasive potential of these cells. We also provide molecular evidence that protease-activated receptor 2 activation followed by PI3K-AKT activation and GSK3β inactivation is involved in these processes and that β-catenin, a well known tumor-regulatory protein, contributes to this signaling pathway. The pivotal role of β-catenin was further indicated by the up-regulation of its downstream targets cyclin D1, c-Myc, COX-2, MMP-7, MMP-14, and Claudin-1. β-Catenin knockdown almost completely attenuated the FVIIa-induced enhancement of breast cancer migration and invasion. These findings provide a new perspective to counteract the invasive behavior of breast cancer, indicating that blocking PI3K-AKT pathway-dependent β-catenin accumulation may represent a potential therapeutic approach to control breast cancer. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Direct neural pathways convey distinct visual information to Drosophila mushroom bodies

    PubMed Central

    Vogt, Katrin; Aso, Yoshinori; Hige, Toshihide; Knapek, Stephan; Ichinose, Toshiharu; Friedrich, Anja B; Turner, Glenn C; Rubin, Gerald M; Tanimoto, Hiromu

    2016-01-01

    Previously, we demonstrated that visual and olfactory associative memories of Drosophila share mushroom body (MB) circuits (Vogt et al., 2014). Unlike for odor representation, the MB circuit for visual information has not been characterized. Here, we show that a small subset of MB Kenyon cells (KCs) selectively responds to visual but not olfactory stimulation. The dendrites of these atypical KCs form a ventral accessory calyx (vAC), distinct from the main calyx that receives olfactory input. We identified two types of visual projection neurons (VPNs) directly connecting the optic lobes and the vAC. Strikingly, these VPNs are differentially required for visual memories of color and brightness. The segregation of visual and olfactory domains in the MB allows independent processing of distinct sensory memories and may be a conserved form of sensory representations among insects. DOI: http://dx.doi.org/10.7554/eLife.14009.001 PMID:27083044

  6. Distinct visual pathways mediate Drosophila larval light avoidance and circadian clock entrainment.

    PubMed

    Keene, Alex C; Mazzoni, Esteban O; Zhen, Jamie; Younger, Meg A; Yamaguchi, Satoko; Blau, Justin; Desplan, Claude; Sprecher, Simon G

    2011-04-27

    Visual organs perceive environmental stimuli required for rapid initiation of behaviors and can also entrain the circadian clock. The larval eye of Drosophila is capable of both functions. Each eye contains only 12 photoreceptors (PRs), which can be subdivided into two subtypes. Four PRs express blue-sensitive rhodopsin5 (rh5) and eight express green-sensitive rhodopsin6 (rh6). We found that either PR-subtype is sufficient to entrain the molecular clock by light, while only the Rh5-PR subtype is essential for light avoidance. Acetylcholine released from PRs confers both functions. Both subtypes of larval PRs innervate the main circadian pacemaker neurons of the larva, the neuropeptide PDF (pigment-dispersing factor)-expressing lateral neurons (LNs), providing sensory input to control circadian rhythms. However, we show that PDF-expressing LNs are dispensable for light avoidance, and a distinct set of three clock neurons is required. Thus we have identified distinct sensory and central circuitry regulating light avoidance behavior and clock entrainment. Our findings provide insights into the coding of sensory information for distinct behavioral functions and the underlying molecular and neuronal circuitry.

  7. Distinct visual pathways mediate Drosophila larval light avoidance and circadian clock entrainment

    PubMed Central

    Keene, Alex C.; Mazzoni, Esteban O.; Zhen, Jamie; Younger, Meg A.; Yamaguchi, Satoko; Blau, Justin; Desplan, Claude; Sprecher, Simon G.

    2011-01-01

    Visual organs perceive environmental stimuli required for rapid initiation of behaviors and can also entrain the circadian clock. The larval eye of Drosophila is capable of both functions. Each eye contains only 12 photoreceptors (PRs), which can be subdivided into two subtypes. Four PRs express blue-sensitive rhodopsin5 (rh5) and eight express green-sensitive rhodopsin6 (rh6). We found that either PR-subtype is sufficient to entrain the molecular clock by light, while only the Rh5-PR subtype is essential for light avoidance. Acetylcholine (ACh) released from PRs confers both functions. Both subtypes of larval PRs innervate the main circadian pacemaker neurons of the larva, the PDF-expressing lateral neurons (LNs), providing sensory input to control circadian rhythms. However, we show that PDF-expressing LNs are dispensable for light avoidance, and a distinct set of three clock neurons is required. Thus we have identified distinct sensory and central circuitry regulating light avoidance behavior and clock entrainment. Our findings provide insights into the coding of sensory information for distinct behavioral functions and the underlying molecular and neuronal circuitry. PMID:21525293

  8. Activation of Type I Interferon Pathway in Systemic Lupus Erythematosus: Association with Distinct Clinical Phenotypes

    PubMed Central

    Karageorgas, Theophanis P.; Tseronis, Dimitrios D.; Mavragani, Clio P.

    2011-01-01

    Growing evidence over the last few years suggests a central role of type I IFN pathway in the pathogenesis of systemic autoimmune disorders. Data from clinical and genetic studies in patients with systemic lupus erythematosus (SLE) and lupus-prone mouse models, indicates that the type I interferon system may play a pivotal role in the pathogenesis of several lupus and associated clinical features, such as nephritis, neuropsychiatric and cutaneous lupus, premature atherosclerosis as well as lupus-specific autoantibodies particularly against ribonucleoproteins. In the current paper, our aim is to summarize the latest findings supporting the association of type I IFN pathway with specific clinical manifestations in the setting of SLE providing insights on the potential use of type I IFN as a therapeutic target. PMID:22162633

  9. Distinct routes to metastasis: plasticity-dependent and plasticity-independent pathways

    PubMed Central

    Somarelli, J A; Schaeffer, D; Marengo, M S; Bepler, T; Rouse, D; Ware, K E; Hish, A J; Zhao, Y; Buckley, A F; Epstein, J I; Armstrong, A J; Virshup, D M; Garcia-Blanco, M A

    2016-01-01

    The cascade that culminates in macrometastases is thought to be mediated by phenotypic plasticity, including epithelial–mesenchymal and mesenchymal–epithelial transitions (EMT and MET). Although there is substantial support for the role of EMT in driving cancer cell invasion and dissemination, much less is known about the importance of MET in the later steps of metastatic colonization. We created novel reporters, which integrate transcriptional and post-transcriptional regulation, to test whether MET is required for metastasis in multiple in vivo cancer models. In a model of carcinosarcoma, metastasis occurred via an MET-dependent pathway; however, in two prostate carcinoma models, metastatic colonization was MET independent. Our results provide evidence for both MET-dependent and MET-independent metastatic pathways. PMID:26751776

  10. Repeated LPS Injection Induces Distinct Changes in the Kynurenine Pathway in Mice.

    PubMed

    Larsson, M K; Faka, A; Bhat, M; Imbeault, S; Goiny, M; Orhan, F; Oliveros, A; Ståhl, S; Liu, X C; Choi, D S; Sandberg, K; Engberg, G; Schwieler, L; Erhardt, S

    2016-09-01

    The immune system has been recognized as a potential contributor to psychiatric disorders. In animals, lipopolysaccharide (LPS) is used to induce inflammation and behaviors analogous to some of the symptoms in these disorders. Recent data indicate that the kynurenine pathway contributes to LPS-induced aberrant behaviors. However, data are inconclusive regarding optimal LPS dose and treatment strategy. Here, we therefore aimed to evaluate the effects of single versus repeated administration of LPS on the kynurenine pathway. Adult C57BL6 mice were given 0.83 mg/kg LPS as a single or a repeated injection (LPS + LPS) and sacrificed after 24, 48, 72, or 120 h. Mice receiving LPS + LPS had significantly elevated brain kynurenine levels at 24 and 48 h, and elevated serum kynurenine at 24, 48 and 72 h. Brain kynurenic acid and quinolinic acid were significantly increased at 24 and 48 h in mice receiving LPS + LPS, whereas serum kynurenic acid levels were significantly decreased at 24 h. The increase of brain kynurenic acid by LPS + LPS was likely unrelated to the higher total dose as a separate group of mice receiving 1.66 mg/kg LPS as single injection 24 h prior to sacrifice did not show increased brain kynurenic acid. Serum quinolinic acid levels were not affected by LPS + LPS compared to vehicle. Animals given repeated injections of LPS showed a more robust induction of the kynurenine pathway in contrast to animals receiving a single injection. These results may be valuable in light of data showing the importance of the kynurenine pathway in psychiatric disorders.

  11. Carbon black and titanium dioxide nanoparticles elicit distinct apoptotic pathways in bronchial epithelial cells

    PubMed Central

    2010-01-01

    Background Increasing environmental and occupational exposures to nanoparticles (NPs) warrant deeper insight into the toxicological mechanisms induced by these materials. The present study was designed to characterize the cell death induced by carbon black (CB) and titanium dioxide (TiO2) NPs in bronchial epithelial cells (16HBE14o- cell line and primary cells) and to investigate the implicated molecular pathways. Results Detailed time course studies revealed that both CB (13 nm) and TiO2(15 nm) NP exposed cells exhibit typical morphological (decreased cell size, membrane blebbing, peripheral chromatin condensation, apoptotic body formation) and biochemical (caspase activation and DNA fragmentation) features of apoptotic cell death. A decrease in mitochondrial membrane potential, activation of Bax and release of cytochrome c from mitochondria were only observed in case of CB NPs whereas lipid peroxidation, lysosomal membrane destabilization and cathepsin B release were observed during the apoptotic process induced by TiO2 NPs. Furthermore, ROS production was observed after exposure to CB and TiO2 but hydrogen peroxide (H2O2) production was only involved in apoptosis induction by CB NPs. Conclusions Both CB and TiO2 NPs induce apoptotic cell death in bronchial epithelial cells. CB NPs induce apoptosis by a ROS dependent mitochondrial pathway whereas TiO2 NPs induce cell death through lysosomal membrane destabilization and lipid peroxidation. Although the final outcome is similar (apoptosis), the molecular pathways activated by NPs differ depending upon the chemical nature of the NPs. PMID:20398356

  12. Distinct amino acid-sensing mTOR pathways regulate skeletal myogenesis.

    PubMed

    Yoon, Mee-Sup; Chen, Jie

    2013-12-01

    Signaling through the mammalian target of rapamycin (mTOR) in response to amino acid availability controls many cellular and developmental processes. mTOR is a master regulator of myogenic differentiation, but the pathways mediating amino acid signals in this process are not known. Here we examine the Rag GTPases and the class III phosphoinositide 3-kinase (PI3K) Vps34, two mediators of amino acid signals upstream of mTOR complex 1 (mTORC1) in cell growth regulation, for their potential involvement in myogenesis. We find that, although both Rag and Vps34 mediate amino acid activation of mTORC1 in C2C12 myoblasts, they have opposing functions in myogenic differentiation. Knockdown of RagA/B enhances, whereas overexpression of active RagB/C mutants impairs, differentiation, and this inhibitory function of Rag is mediated by mTORC1 suppression of the IRS1-PI3K-Akt pathway. On the other hand, Vps34 is required for myogenic differentiation. Amino acids activate a Vps34-phospholipase D1 (PLD1) pathway that controls the production of insulin-like growth factor II, an autocrine inducer of differentiation, through the Igf2 muscle enhancer. The product of PLD, phosphatidic acid, activates the enhancer in a rapamycin-sensitive but mTOR kinase-independent manner. Our results uncover amino acid-sensing mechanisms controlling the homeostasis of myogenesis and underline the versatility and context dependence of mTOR signaling.

  13. Caenorhabditis elegans POLQ-1 and HEL-308 function in two distinct DNA interstrand cross-link repair pathways.

    PubMed

    Muzzini, Diego M; Plevani, Paolo; Boulton, Simon J; Cassata, Giuseppe; Marini, Federica

    2008-06-01

    DNA interstrand cross-links (ICLs) are highly cytotoxic DNA lesions hindering DNA replication and transcription. Whereas in bacteria and yeast the molecular mechanisms involved in ICL repair are genetically well dissected, the scenario in multicellular organisms remains unclear. Here, we report that the two new mus308 genes, polq-1 and hel-308 are involved in ICL repair in Caenorhabditis elegans. After treatment with ICL agents, a decrease in survival and an increase in checkpoint-induced cell-cycle arrest and apoptosis of germ cells is observed in mutants of both genes. Although sensitive to ICL agents and to a minor extent to IR, cytological and epistatic analyses suggest that polq-1 and hel-308 are involved in different DNA repair pathways. While hel-308 functions in a Fanconi anemia-dependent pathway, polq-1 has a role in a novel distinct and brc-1 (CeBRCA1)-dependent ICL repair process in metazoans.

  14. SARS coronavirus papain-like protease inhibits the type I interferon signaling pathway through interaction with the STING-TRAF3-TBK1 complex.

    PubMed

    Chen, Xiaojuan; Yang, Xingxing; Zheng, Yang; Yang, Yudong; Xing, Yaling; Chen, Zhongbin

    2014-05-01

    SARS coronavirus (SARS-CoV) develops an antagonistic mechanism by which to evade the antiviral activities of interferon (IFN). Previous studies suggested that SARS-CoV papain-like protease (PLpro) inhibits activation of the IRF3 pathway, which would normally elicit a robust IFN response, but the mechanism(s) used by SARS PLpro to inhibit activation of the IRF3 pathway is not fully known. In this study, we uncovered a novel mechanism that may explain how SARS PLpro efficiently inhibits activation of the IRF3 pathway. We found that expression of the membrane-anchored PLpro domain (PLpro-TM) from SARS-CoV inhibits STING/TBK1/IKKε-mediated activation of type I IFNs and disrupts the phosphorylation and dimerization of IRF3, which are activated by STING and TBK1. Meanwhile, we showed that PLpro-TM physically interacts with TRAF3, TBK1, IKKε, STING, and IRF3, the key components that assemble the STING-TRAF3-TBK1 complex for activation of IFN expression. However, the interaction between the components in STING-TRAF3-TBK1 complex is disrupted by PLpro-TM. Furthermore, SARS PLpro-TM reduces the levels of ubiquitinated forms of RIG-I, STING, TRAF3, TBK1, and IRF3 in the STING-TRAF3-TBK1 complex. These results collectively point to a new mechanism used by SARS-CoV through which PLpro negatively regulates IRF3 activation by interaction with STING-TRAF3-TBK1 complex, yielding a SARS-CoV countermeasure against host innate immunity.

  15. Distinct pathways for rule-based retrieval and spatial mapping of memory representations in hippocampal neurons

    PubMed Central

    Navawongse, Rapeechai; Eichenbaum, Howard

    2013-01-01

    Hippocampal neurons encode events within the context in which they occurred, a fundamental feature of episodic memory. Here we explored the sources of event and context information represented by hippocampal neurons during the retrieval of object associations in rats. Temporary inactivation of the medial prefrontal cortex differentially reduced the selectivity of rule-based object associations represented by hippocampal neuronal firing patterns but did not affect spatial firing patterns. By contrast, inactivation of the medial entorhinal cortex resulted in a pervasive reorganization of hippocampal mappings of spatial context and events. These results suggest distinct and cooperative prefrontal and medial temporal mechanisms in memory representation. PMID:23325238

  16. Function and Regulation of SUMO Proteases

    PubMed Central

    Hickey, Christopher M.; Wilson, Nicole R.; Hochstrasser, Mark

    2013-01-01

    Covalent attachment of small ubiquitin-like modifier (SUMO) to proteins is highly dynamic, and both SUMO-protein conjugation and cleavage can be regulated. Protein desumoylation is performed by SUMO proteases, which control cellular mechanisms ranging from transcription and cell division to ribosome biogenesis. Recent advances include the discovery of two novel classes of SUMO proteases, insights regarding SUMO protease specificity, and revelations of previously unappreciated SUMO protease functions in several key cellular pathways. These developments, together with new connections between SUMO proteases and the recently discovered SUMO-targeted ubiquitin ligases (STUbLs), make this an exciting period for the study of these enzymes. PMID:23175280

  17. SPC4, SPC6, and the novel protease SPC7 are coexpressed with bone morphogenetic proteins at distinct sites during embryogenesis

    PubMed Central

    1996-01-01

    In the present study, we screened for subtilisin-like proprotein convertases (SPCs) that potentially regulate the activation of known growth factors during embryonic development. We isolated a novel protease, SPC7, as well as several known SPCs. SPC7, like SPC1, is expressed ubiquitously throughout development. In contrast, SPC4 and SPC6 exhibit dynamic expression patterns. SPC4 transcripts were initially detected in the granulosa cells of secondary follicles. Shortly after implantation, SPC4 transcripts are localized to extraembryonic cell populations, and at later stages are detected in discrete tissues including the primitive gut, heart, neural tube, and limb buds. Within the limb buds, SPC4 mRNA is most abundant in the apical ectodermal ridge (AER). At later stages of limb development, SPC4 mRNA is strongly expressed in cartilage and in the interdigital mesenchyme. In contrast, high SPC6 mRNA levels are detected in somites, the dorsal surface ectoderm, and in vertebral cartilage primordia. In limb buds, SPC6 is strongly expressed in the AER, and at later stages in dorsal mesenchyme. A comparison of these expression patterns with those of several bone morphogenetic proteins (BMPs) indicates that processing of these growth factors may be limited by the local availability of SPCs. PMID:8698813

  18. Distinct amino acid–sensing mTOR pathways regulate skeletal myogenesis

    PubMed Central

    Yoon, Mee-Sup; Chen, Jie

    2013-01-01

    Signaling through the mammalian target of rapamycin (mTOR) in response to amino acid availability controls many cellular and developmental processes. mTOR is a master regulator of myogenic differentiation, but the pathways mediating amino acid signals in this process are not known. Here we examine the Rag GTPases and the class III phosphoinositide 3-kinase (PI3K) Vps34, two mediators of amino acid signals upstream of mTOR complex 1 (mTORC1) in cell growth regulation, for their potential involvement in myogenesis. We find that, although both Rag and Vps34 mediate amino acid activation of mTORC1 in C2C12 myoblasts, they have opposing functions in myogenic differentiation. Knockdown of RagA/B enhances, whereas overexpression of active RagB/C mutants impairs, differentiation, and this inhibitory function of Rag is mediated by mTORC1 suppression of the IRS1-PI3K-Akt pathway. On the other hand, Vps34 is required for myogenic differentiation. Amino acids activate a Vps34-phospholipase D1 (PLD1) pathway that controls the production of insulin-like growth factor II, an autocrine inducer of differentiation, through the Igf2 muscle enhancer. The product of PLD, phosphatidic acid, activates the enhancer in a rapamycin-sensitive but mTOR kinase–independent manner. Our results uncover amino acid–sensing mechanisms controlling the homeostasis of myogenesis and underline the versatility and context dependence of mTOR signaling. PMID:24068326

  19. Non-proteolytic functions of microbial proteases increase pathological complexity.

    PubMed

    Jarocki, Veronica M; Tacchi, Jessica L; Djordjevic, Steven P

    2015-03-01

    Proteases are enzymes that catalyse hydrolysis of peptide bonds thereby controlling the shape, size, function, composition, turnover and degradation of other proteins. In microbes, proteases are often identified as important virulence factors and as such have been targets for novel drug design. It is emerging that some proteases possess additional non-proteolytic functions that play important roles in host epithelia adhesion, tissue invasion and in modulating immune responses. These additional "moonlighting" functions have the potential to obfuscate data interpretation and have implications for therapeutic design. Moonlighting enzymes comprise a subcategory of multifunctional proteins that possess at least two distinct biological functions on a single polypeptide chain. Presently, identifying moonlighting proteins relies heavily on serendipitous empirical data with clues arising from proteins lacking signal peptides that are localised to the cell surface. Here, we describe examples of microbial proteases with additional non-proteolytic functions, including streptococcal pyrogenic exotoxin B, PepO and C5a peptidases, mycoplasmal aminopeptidases, mycobacterial chaperones and viral papain-like proteases. We explore how these non-proteolytic functions contribute to host cell adhesion, modulate the coagulation pathway, assist in non-covalent folding of proteins, participate in cell signalling, and increase substrate repertoire. We conclude by describing how proteomics has aided in moonlighting protein discovery, focusing attention on potential moonlighters in microbial exoproteomes.

  20. Two Distinct Pathways for Trehalose Assimilation in the Yeast Saccharomyces cerevisiae

    PubMed Central

    Jules, Matthieu; Guillou, Vincent; François, Jean; Parrou, Jean-Luc

    2004-01-01

    The yeast Saccharomyces cerevisiae can synthesize trehalose and also use this disaccharide as a carbon source for growth. However, the molecular mechanism by which extracellular trehalose can be transported to the vacuole and degraded by the acid trehalase Ath1p is not clear. By using an adaptation of the assay of invertase on whole cells with NaF, we showed that more than 90% of the activity of Ath1p is extracellular, splitting of the disaccharide into glucose. We also found that Agt1p-mediated trehalose transport and the hydrolysis of the disaccharide by the cytosolic neutral trehalase Nth1p are coupled and represent a second, independent pathway, although there are several constraints on this alternative route. First, the AGT1/MAL11 gene is controlled by the MAL system, and Agt1p was active in neither non-maltose-fermenting nor maltose-inducible strains. Second, Agt1p rapidly lost activity during growth on trehalose, by a mechanism similar to the sugar-induced inactivation of the maltose permease. Finally, both pathways are highly pH sensitive and effective growth on trehalose occurred only when the medium was buffered at around pH 5.0. The catabolism of trehalose was purely oxidative, and since levels of Ath1p limit the glucose flux in the cells, batch cultures on trehalose may provide a useful alternative to glucose-limited chemostat cultures for investigation of metabolic responses in yeast. PMID:15128531

  1. Distinct Effects of p19 RNA Silencing Suppressor on Small RNA Mediated Pathways in Plants

    PubMed Central

    Kontra, Levente; Tavazza, Mario; Lucioli, Alessandra; Tavazza, Raffaela; Moxon, Simon; Medzihradszky, Anna; Burgyán, József

    2016-01-01

    RNA silencing is one of the main defense mechanisms employed by plants to fight viruses. In change, viruses have evolved silencing suppressor proteins to neutralize antiviral silencing. Since the endogenous and antiviral functions of RNA silencing pathway rely on common components, it was suggested that viral suppressors interfere with endogenous silencing pathway contributing to viral symptom development. In this work, we aimed to understand the effects of the tombusviral p19 suppressor on endogenous and antiviral silencing during genuine virus infection. We showed that ectopically expressed p19 sequesters endogenous small RNAs (sRNAs) in the absence, but not in the presence of virus infection. Our presented data question the generalized model in which the sequestration of endogenous sRNAs by the viral suppressor contributes to the viral symptom development. We further showed that p19 preferentially binds the perfectly paired ds-viral small interfering RNAs (vsiRNAs) but does not select based on their sequence or the type of the 5’ nucleotide. Finally, co-immunoprecipitation of sRNAs with AGO1 or AGO2 from virus-infected plants revealed that p19 specifically impairs vsiRNA loading into AGO1 but not AGO2. Our findings, coupled with the fact that p19-expressing wild type Cymbidium ringspot virus (CymRSV) overcomes the Nicotiana benthamiana silencing based defense killing the host, suggest that AGO1 is the main effector of antiviral silencing in this host-virus combination. PMID:27711201

  2. Distinct Signaling Pathways and Transcriptome Response Signatures Differentiate Ammonium- and Nitrate-supplied Plants

    PubMed Central

    Patterson, Kurt; Cakmak, Turgay; Cooper, Andrew; Lager, Ida; Rasmusson, Allan G.; Escobar, Matthew A.

    2010-01-01

    Nitrogen is the only macronutrient that is commonly available to plants in both oxidized and reduced forms, mainly nitrate and ammonium. The physiological and molecular effects of nitrate supply have been well studied, but comparatively little is known about ammonium nutrition and its differential effects on cell function and gene expression. We have used a physiologically realistic hydroponic growth system to compare the transcriptomes and redox status of the roots of ammonium- and nitrate-supplied Arabidopsis thaliana plants. While ~60% of nitrogen-regulated genes displayed common responses to both ammonium and nitrate, significant “nitrate-specific” and “ammonium-specific” gene sets were identified. Pathways involved in cytokinin response and reductant generation/distribution were specifically altered by nitrate, while a complex biotic stress response and changes in nodulin gene expression were characteristic of ammonium-supplied plants. Nitrate supply was associated with a rapid decrease in H2O2 production, potentially due to an increased export of reductant from the mitochondrial matrix. The underlying basis of the nitrate- and ammonium-specific patterns of gene expression appears to be different signals elaborated from each nitrogen source, including alterations in extracellular pH that are associated with ammonium uptake, downstream metabolites in the ammonium assimilation pathway, and the presence or absence of the nitrate ion. PMID:20444219

  3. Redistribution of cholesterol in oligodendrocyte membrane sheets after activation of distinct signal transduction pathways.

    PubMed

    Lintner, R N; Dyer, C A

    2000-05-15

    Cultured oligodendrocytes produce extensive membrane sheets that contain an internal lacy network of vein-like structures composed of microtubules, actin filaments, and 2'3'-cyclic nucleotide 3'-phosphohydrolase (CNPase). These cytoplasmic vein-like structures surround domains of myelin basic protein (MBP). Using the antibiotic filipin, that binds to cholesterol, the relationship between plasma membrane cholesterol and cytoskeleton in membrane sheets was examined. Our results show that cholesterol was relatively uniformly distributed within the plasma membranes of prefixed control oligodendrocyte membrane sheets. When live cultures were extracted with Triton X-100, however, a subpopulation of cholesterol molecules remained colocalized with cytoskeleton in the membrane sheets. Activation of two well-characterized signaling pathways that differentially affect microtubule and actin filament stability in membrane sheets resulted in an apparent massive lateral movement of cholesterol molecules away from membrane regions overlying internal MBP domains to membrane tracts directly overlying cytoplasmic cytoskeletal veins. Depolymerization of microtubules by colchicine resulted in redistribution of cholesterol directly over actin filaments, whereas depolymerization of actin filaments by cytochalasin B resulted in redistribution of cholesterol directly over CNPase/microtubular veins. These data suggest that cholesterol forms an association with cytoskeletal components or proteins associated with cytoskeleton. These data also suggest that cholesterol, via interactions with cytoskeleton, plays a role in signaling pathways in oligodendrocyte membrane sheets. Copyright 2000 Wiley-Liss, Inc.

  4. Two distinct pathways for trehalose assimilation in the yeast Saccharomyces cerevisiae.

    PubMed

    Jules, Matthieu; Guillou, Vincent; François, Jean; Parrou, Jean-Luc

    2004-05-01

    The yeast Saccharomyces cerevisiae can synthesize trehalose and also use this disaccharide as a carbon source for growth. However, the molecular mechanism by which extracellular trehalose can be transported to the vacuole and degraded by the acid trehalase Ath1p is not clear. By using an adaptation of the assay of invertase on whole cells with NaF, we showed that more than 90% of the activity of Ath1p is extracellular, splitting of the disaccharide into glucose. We also found that Agt1p-mediated trehalose transport and the hydrolysis of the disaccharide by the cytosolic neutral trehalase Nth1p are coupled and represent a second, independent pathway, although there are several constraints on this alternative route. First, the AGT1/MAL11 gene is controlled by the MAL system, and Agt1p was active in neither non-maltose-fermenting nor maltose-inducible strains. Second, Agt1p rapidly lost activity during growth on trehalose, by a mechanism similar to the sugar-induced inactivation of the maltose permease. Finally, both pathways are highly pH sensitive and effective growth on trehalose occurred only when the medium was buffered at around pH 5.0. The catabolism of trehalose was purely oxidative, and since levels of Ath1p limit the glucose flux in the cells, batch cultures on trehalose may provide a useful alternative to glucose-limited chemostat cultures for investigation of metabolic responses in yeast.

  5. Distinct Fragmentation Pathways of Anticancer Drugs Induced by Charge-Carrying Cations in the Gas Phase

    NASA Astrophysics Data System (ADS)

    Hong, Areum; Lee, Hong Hee; Heo, Chae Eun; Cho, Yunju; Kim, Sunghwan; Kang, Dukjin; Kim, Hugh I.

    2017-04-01

    With the growth of the pharmaceutical industry, structural elucidation of drugs and derivatives using tandem mass spectrometry (MS2) has become essential for drug development and pharmacokinetics studies because of its high sensitivity and low sample requirement. Thus, research seeking to understand fundamental relationships between fragmentation patterns and precursor ion structures in the gas phase has gained attention. In this study, we investigate the fragmentation of the widely used anticancer drugs, doxorubicin (DOX), vinblastine (VBL), and vinorelbine (VRL), complexed by a singly charged proton or alkali metal ion (Li+, Na+, K+) in the gas phase. The drug-cation complexes exhibit distinct fragmentation patterns in tandem mass spectra as a function of cation size. The trends in fragmentation patterns are explicable in terms of structures derived from ion mobility mass spectrometry (IM-MS) and theoretical calculations.

  6. Distinct Fragmentation Pathways of Anticancer Drugs Induced by Charge-Carrying Cations in the Gas Phase

    NASA Astrophysics Data System (ADS)

    Hong, Areum; Lee, Hong Hee; Heo, Chae Eun; Cho, Yunju; Kim, Sunghwan; Kang, Dukjin; Kim, Hugh I.

    2016-12-01

    With the growth of the pharmaceutical industry, structural elucidation of drugs and derivatives using tandem mass spectrometry (MS2) has become essential for drug development and pharmacokinetics studies because of its high sensitivity and low sample requirement. Thus, research seeking to understand fundamental relationships between fragmentation patterns and precursor ion structures in the gas phase has gained attention. In this study, we investigate the fragmentation of the widely used anticancer drugs, doxorubicin (DOX), vinblastine (VBL), and vinorelbine (VRL), complexed by a singly charged proton or alkali metal ion (Li+, Na+, K+) in the gas phase. The drug-cation complexes exhibit distinct fragmentation patterns in tandem mass spectra as a function of cation size. The trends in fragmentation patterns are explicable in terms of structures derived from ion mobility mass spectrometry (IM-MS) and theoretical calculations.

  7. Size evolution of highly amphiphilic macromolecular solution assemblies via a distinct bimodal pathway

    PubMed Central

    Kelley, Elizabeth G.; Murphy, Ryan P.; Seppala, Jonathan E.; Smart, Thomas P.; Hann, Sarah D.

    2014-01-01

    The solution self-assembly of macromolecular amphiphiles offers an efficient, bottom-up strategy for producing well--defined nanocarriers, with applications ranging from drug delivery to nanoreactors. Typically, the generation of uniform nanocarrier architecturesis controlled by processing methods that rely upon cosolvent mixtures. These preparation strategies hinge on the assumption that macromolecular solution nanostructures are kinetically stable following transfer from an organic/aqueous cosolvent into aqueous solution. Herein we demonstrate that unequivocal step-change shifts in micelle populations occur over several weeks following transfer into a highly selective solvent. The unexpected micelle growth evolves through a distinct bimodal distribution separated by multiple fusion events and critically depends on solution agitation. Notably, these results underscore fundamental similarities between assembly processes in amphiphilic polymer, small molecule, and protein systems. Moreover, the non-equilibrium micelle size increase can have a major impact on the assumed stability of solution assemblies, for which performance is dictated by nanocarrier size and structure. PMID:24710204

  8. Network Analyses Reveal Pervasive Functional Regulation Between Proteases in the Human Protease Web

    PubMed Central

    Fortelny, Nikolaus; Cox, Jennifer H.; Kappelhoff, Reinhild; Starr, Amanda E.; Lange, Philipp F.; Pavlidis, Paul; Overall, Christopher M.

    2014-01-01

    Proteolytic processing is an irreversible posttranslational modification affecting a large portion of the proteome. Protease-cleaved mediators frequently exhibit altered activity, and biological pathways are often regulated by proteolytic processing. Many of these mechanisms have not been appreciated as being protease-dependent, and the potential in unraveling a complex new dimension of biological control is increasingly recognized. Proteases are currently believed to act individually or in isolated cascades. However, conclusive but scattered biochemical evidence indicates broader regulation of proteases by protease and inhibitor interactions. Therefore, to systematically study such interactions, we assembled curated protease cleavage and inhibition data into a global, computational representation, termed the protease web. This revealed that proteases pervasively influence the activity of other proteases directly or by cleaving intermediate proteases or protease inhibitors. The protease web spans four classes of proteases and inhibitors and so links both recently and classically described protease groups and cascades, which can no longer be viewed as operating in isolation in vivo. We demonstrated that this observation, termed reachability, is robust to alterations in the data and will only increase in the future as additional data are added. We further show how subnetworks of the web are operational in 23 different tissues reflecting different phenotypes. We applied our network to develop novel insights into biologically relevant protease interactions using cell-specific proteases of the polymorphonuclear leukocyte as a system. Predictions from the protease web on the activity of matrix metalloproteinase 8 (MMP8) and neutrophil elastase being linked by an inactivating cleavage of serpinA1 by MMP8 were validated and explain perplexing Mmp8 −/− versus wild-type polymorphonuclear chemokine cleavages in vivo. Our findings supply systematically derived and

  9. Functional genomics identifies five distinct molecular subtypes with clinical relevance and pathways for growth control in epithelial ovarian cancer

    PubMed Central

    Tan, Tuan Zea; Miow, Qing Hao; Huang, Ruby Yun-Ju; Wong, Meng Kang; Ye, Jieru; Lau, Jieying Amelia; Wu, Meng Chu; Bin Abdul Hadi, Luqman Hakim; Soong, Richie; Choolani, Mahesh; Davidson, Ben; Nesland, Jahn M; Wang, Ling-Zhi; Matsumura, Noriomi; Mandai, Masaki; Konishi, Ikuo; Goh, Boon-Cher; Chang, Jeffrey T; Thiery, Jean Paul; Mori, Seiichi

    2013-01-01

    Epithelial ovarian cancer (EOC) is hallmarked by a high degree of heterogeneity. To address this heterogeneity, a classification scheme was developed based on gene expression patterns of 1538 tumours. Five, biologically distinct subgroups — Epi-A, Epi-B, Mes, Stem-A and Stem-B — exhibited significantly distinct clinicopathological characteristics, deregulated pathways and patient prognoses, and were validated using independent datasets. To identify subtype-specific molecular targets, ovarian cancer cell lines representing these molecular subtypes were screened against a genome-wide shRNA library. Focusing on the poor-prognosis Stem-A subtype, we found that two genes involved in tubulin processing, TUBGCP4 and NAT10, were essential for cell growth, an observation supported by a pathway analysis that also predicted involvement of microtubule-related processes. Furthermore, we observed that Stem-A cell lines were indeed more sensitive to inhibitors of tubulin polymerization, vincristine and vinorelbine, than the other subtypes. This subtyping offers new insights into the development of novel diagnostic and personalized treatment for EOC patients. PMID:23666744

  10. Puma and Trail/Dr5 pathways control radiation-induced apoptosis in distinct populations of testicular progenitors.

    PubMed

    Coureuil, Mathieu; Ugolin, Nicolas; Tavernier, Marie; Chevillard, Sylvie; Barroca, Vilma; Fouchet, Pierre; Allemand, Isabelle

    2010-08-12

    Spermatogonia- stem cells and progenitors of adult spermatogenesis- are killed through a p53-regulated apoptotic process after gamma-irradiation but the death effectors are still poorly characterized. Our data demonstrate that both intrinsic and extrinsic apoptotic pathways are involved, and especially that spermatogonia can be split into two main populations, according to apoptotic effectors. Following irradiation both Dr5 and Puma genes are upregulated in the alpha6-integrin-positive Side Population (SP) fraction, which is highly enriched in spermatogonia. Flow cytometric analysis confirms an increased number of Dr5-expressing SP cells, and Puma-beta isoform accumulates in alpha6-integrin positive cellular extracts, enriched in spermatogonia. Trail-/- or Puma-/- spermatogonia display a reduced sensitivity to radiation-induced apoptosis. The TUNEL kinetics strongly suggest that the extrinsic and intrinsic pathways, via Trail/Dr5 and Puma respectively, could be engaged in distinct subpopulations of spermatogonia. Indeed flow cytometric studies show that Dr5 receptor is constitutively present on more than half of the undifferentiated progenitors (Kit- alpha6+ SP) and half of the differentiated ones (Kit+ alpha6+ SP). In addition after irradiation, Puma is not detected in the Dr5-positive cellular fraction isolated by immunomagnetic purification, while Puma is present in the Dr5-negative cell extracts. In conclusion, adult testicular progenitors are divided into distinct sub-populations by apoptotic effectors, independently of progenitor types (immature Kit-negative versus mature Kit-positive), underscoring differential radiosensitivities characterizing the stem cell/progenitors compartment.

  11. Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways

    PubMed Central

    Naqvi, Shaista; Zhu, Changfu; Farre, Gemma; Ramessar, Koreen; Bassie, Ludovic; Breitenbach, Jürgen; Perez Conesa, Dario; Ros, Gaspar; Sandmann, Gerhard; Capell, Teresa; Christou, Paul

    2009-01-01

    Vitamin deficiency affects up to 50% of the world's population, disproportionately impacting on developing countries where populations endure monotonous, cereal-rich diets. Transgenic plants offer an effective way to increase the vitamin content of staple crops, but thus far it has only been possible to enhance individual vitamins. We created elite inbred South African transgenic corn plants in which the levels of 3 vitamins were increased specifically in the endosperm through the simultaneous modification of 3 separate metabolic pathways. The transgenic kernels contained 169-fold the normal amount of β-carotene, 6-fold the normal amount of ascorbate, and double the normal amount of folate. Levels of engineered vitamins remained stable at least through to the T3 homozygous generation. This achievement, which vastly exceeds any realized thus far by conventional breeding alone, opens the way for the development of nutritionally complete cereals to benefit the world's poorest people. PMID:19416835

  12. Distinct silicon and germanium pathways in the soil-plant system: Evidence from banana and horsetail

    NASA Astrophysics Data System (ADS)

    Delvigne, C.; Opfergelt, S.; Cardinal, D.; Delvaux, B.; André, L.

    2009-06-01

    Plants strongly impact the continental silicon cycle by taking up Si and precipitating opal phytoliths which are recycled into the soil. Studying Ge incorporation, a chemical analog of Si, relative to Si may provide a useful tracer of Si pathways. However, Ge uptake and transport through plants and the impact on Ge/Si of phytoliths remain poorly understood. Here, we report Ge uptake and accumulation and Ge/Si fractionation in all plant parts and solutions from: (1) hydroponic banana, (2) in situ sampled banana, and (3) horsetails. We further combine these data with δ29Si from banana plants. Our data reconcile opposite conclusions drawn from previous studies on Ge uptake and pathways. No discrimination of Ge occurred at the root-solution interface. Banana and horsetails were shown to accumulate Ge in roots: a previous study provided evidence of low Ge/Si ratios in root phytoliths which contrasts with high bulk Ge/Si ratios in roots we report here. This suggests that Ge is organically trapped in roots. Consequently, shoots display lower Ge/Si ratios, without fractionation between shoot parts since Ge would follow transpiration stream as silicon, and is not discriminated between shoot parts. This contrasts with the two-step discrimination against heavy Si isotopes, at the root-solution interface, and then within the shoots. The soil composition (clays versus Fe oxides) has a leading role on the Ge/Si signatures of plants which may in turn impact on the Si and Ge fluxes to the global ocean.

  13. The non-phagocytic route of collagen uptake: a distinct degradation pathway.

    PubMed

    Madsen, Daniel H; Ingvarsen, Signe; Jürgensen, Henrik J; Melander, Maria C; Kjøller, Lars; Moyer, Amanda; Honoré, Christian; Madsen, Charlotte A; Garred, Peter; Burgdorf, Sven; Bugge, Thomas H; Behrendt, Niels; Engelholm, Lars H

    2011-07-29

    The degradation of collagens, the most abundant proteins of the extracellular matrix, is involved in numerous physiological and pathological conditions including cancer invasion. An important turnover pathway involves cellular internalization and degradation of large, soluble collagen fragments, generated by initial cleavage of the insoluble collagen fibers. We have previously observed that in primary mouse fibroblasts, this endocytosis of collagen fragments is dependent on the receptor urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180. Others have identified additional mechanisms of collagen uptake, with different associated receptors, in other cell types. These receptors include β1-integrins, being responsible for collagen phagocytosis, and the mannose receptor. We have now utilized a newly developed monoclonal antibody against uPARAP/Endo180, which down-regulates the receptor protein level on treated cells, to examine the role of uPARAP/Endo180 as a mediator of collagen internalization by a wide range of cultured cell types. With the exception of macrophages, all cells that proved capable of efficient collagen internalization were of mesenchymal origin and all of these utilized uPARAP/Endo180 for their collagen uptake process. Macrophages internalized collagen in a process mediated by the mannose receptor, a protein belonging to the same protein family as uPARAP/Endo180. β1-Integrins were found not to be involved in the endocytosis of soluble collagen, irrespectively of whether this was mediated by uPARAP/Endo180 or the mannose receptor. This further distinguishes these pathways from the phagocytic uptake of particulate collagen.

  14. Deregulation of Listeria monocytogenes virulence gene expression by two distinct and semi-independent pathways.

    PubMed

    Milenbachs Lukowiak, Andrea; Mueller, Kimberly J; Freitag, Nancy E; Youngman, Philip

    2004-02-01

    Expression of the major virulence cluster in Listeria monocytogenes is positively regulated by the transcription factor PrfA and is influenced by several environmental factors, including the presence of readily metabolized carbohydrates such as cellobiose and glucose. Although little is understood about the mechanisms through which environmental factors influence expression of the PrfA regulon, evidence for structural and functional similarities of PrfA to the CRP-FNR family of regulatory proteins suggests the possibility that PrfA activity could be modulated by a small molecule ligand. The identity of components of the PrfA-associated regulatory pathway was sought through the isolation of mutants that exhibit high levels of PrfA-controlled gene expression in the presence of cellobiose or glucose. Here are described the properties and preliminary genetic analysis in two different genetic loci, gcr and csr, both unlinked by general transduction to the major virulence cluster. A mutation in gcr deregulates the expression of PrfA-controlled genes in the presence of several repressing sugars and other environmental conditions, a phenotype similar to that of a G145S substitution in PrfA itself. A mutation in the csr locus, within csrA, results in a cellobiose-specific defect in virulence gene regulation. Gene products encoded by the csr locus share homology with proteins involved in the sensing and transport of beta-glucosides in other bacteria. Mutations in both gcr and csr are required for full relief of cellobiose-mediated repression of the PrfA regulon. These results suggest the existence of two semi-independent pathways for cellobiose-mediated repression and further reconcile conflicting reports in previous literature concerning the repressive effects of carbohydrates on virulence gene expression in L. monocytogenes.

  15. Proteases in blood clotting.

    PubMed

    Walsh, Peter N; Ahmad, Syed S

    2002-01-01

    The serine proteases, cofactors and cell-receptor molecules that comprise the haemostatic mechanism are highly conserved modular proteins that have evolved to participate in biochemical reactions in blood coagulation, anticoagulation and fibrinolysis. Blood coagulation is initiated by exposure of tissue factor, which forms a complex with factor VIIa and factor X, which results in the generation of small quantities of thrombin and is rapidly shutdown by the tissue factor pathway inhibitor. The generation of these small quantities of thrombin then activates factor XI, resulting in a sequence of events that lead to the activation of factor IX, factor X and prothrombin. Sufficient thrombin is generated to effect normal haemostasis by converting fibrinogen into fibrin. The anticoagulant pathways that regulate blood coagulation include the protein C anticoagulant mechanism, the serine protease inhibitors in plasma, and the Kunitz-like inhibitors, tissue factor pathway inhibitor and protease nexin 2. Finally, the fibrinolytic mechanism that comprises the activation of plasminogen into plasmin prevents excessive fibrin accumulation by promoting local dissolution of thrombi and promoting wound healing by reestablishment of blood flow.

  16. Hemagglutinin protease secreted by V. cholerae induced apoptosis in breast cancer cells by ROS mediated intrinsic pathway and regresses tumor growth in mice model.

    PubMed

    Ray, Tanusree; Chakrabarti, Monoj Kumar; Pal, Amit

    2016-02-01

    Conventional anticancer therapies are effective but have side effects, so alternative targets are being developed. Bacterial toxins that can kill cells or alter the cellular processes like proliferation, apoptosis and differentiation have been reported for cancer treatment. In this study we have shown antitumor activity of hemagglutinin protease (HAP) secreted by Vibrio cholerae. One µg of HAP showed potent antitumor activity when injected into Ehrlich ascites carcinoma (EAC) tumors in Swiss albino mice. Weekly administration of this dose is able to significantly diminish a large tumor volume within 3 weeks and increases the survival rates of cancerous mice. HAP showed apoptotic activity on EAC and other malignant cells. Increased level of pro-apoptotic p53 with increased ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2 signify that HAP induced apoptogenic signals lead to death of the tumor cells. In vivo and ex vivo studies suggest that mitochondrial dependent intrinsic pathway is responsible for this apoptosis. The level of ROS in malignant cells is reported to be higher than the normal healthy cells. HAP induces oxidative stress and increases the level of ROS in malignant cells which is significantly higher than the normal healthy cells. As a result the malignant cells cross the threshold level of ROS for cell survival faster than normal healthy cells. This mechanism causes HAP mediated apoptosis in malignant cells, but normal cells remain unaltered in the same environment. Our study suggests that HAP may be used as a new candidate drug for cancer therapy.

  17. SARS Coronavirus Papain-Like Protease Inhibits the TLR7 Signaling Pathway through Removing Lys63-Linked Polyubiquitination of TRAF3 and TRAF6.

    PubMed

    Li, Shih-Wen; Wang, Ching-Ying; Jou, Yu-Jen; Huang, Su-Hua; Hsiao, Li-Hsin; Wan, Lei; Lin, Ying-Ju; Kung, Szu-Hao; Lin, Cheng-Wen

    2016-05-05

    Severe acute respiratory syndrome coronavirus (SARS-CoV) papain-like protease (PLPro) reportedly inhibits the production of type I interferons (IFNs) and pro-inflammatory cytokines in Toll-like receptor 3 (TLR3) and retinoic acid-inducible gene 1 (RIG-I) pathways. The study investigated the inhibitory effect and its antagonistic mechanism of SARS-CoV PLPro on TLR7-mediated cytokine production. TLR7 agonist (imiquimod (IMQ)) concentration-dependently induced activation of ISRE-, NF-κB- and AP-1-luciferase reporters, as well as the production of IFN-α, IFN-β, TNF-α, IL-6 and IL-8 in human promonocyte cells. However, SARS-CoV PLPro significantly inhibited IMQ-induced cytokine production through suppressing the activation of transcription factors IRF-3, NF-κB and AP-1. Western blot analysis with anti-Lys48 and anti-Lys63 ubiquitin antibodies indicated the SARS-CoV PLPro removed Lys63-linked ubiquitin chains of TRAF3 and TRAF6, but not Lys48-linked ubiquitin chains in un-treated and treated cells. The decrease in the activated state of TRAF3 and TRAF6 correlated with the inactivation of TBK1 in response to IMQ by PLPro. The results revealed that the antagonism of SARS-CoV PLPro on TLR7-mediated innate immunity was associated with the negative regulation of TRAF3/6-TBK1-IRF3/NF-κB/AP1 signals.

  18. Genome-Wide Survey of Pseudomonas aeruginosa PA14 Reveals a Role for the Glyoxylate Pathway and Extracellular Proteases in the Utilization of Mucin.

    PubMed

    Flynn, Jeffrey M; Phan, Chi; Hunter, Ryan C

    2017-08-01

    Chronic airway infections by the opportunistic pathogen Pseudomonas aeruginosa are a major cause of mortality in cystic fibrosis (CF) patients. Although this bacterium has been extensively studied for its virulence determinants, biofilm growth, and immune evasion mechanisms, comparatively little is known about the nutrient sources that sustain its growth in vivo Respiratory mucins represent a potentially abundant bioavailable nutrient source, although we have recently shown that canonical pathogens inefficiently use these host glycoproteins as a growth substrate. However, given that P. aeruginosa, particularly in its biofilm mode of growth, is thought to grow slowly in vivo, the inefficient use of mucin glycoproteins may be relevant to its persistence within the CF airways. To this end, we used whole-genome fitness analysis, combining transposon mutagenesis with high-throughput sequencing, to identify genetic determinants required for P. aeruginosa growth using intact purified mucins as a sole carbon source. Our analysis reveals a biphasic growth phenotype, during which the glyoxylate pathway and amino acid biosynthetic machinery are required for mucin utilization. Secondary analyses confirmed the simultaneous liberation and consumption of acetate during mucin degradation and revealed a central role for the extracellular proteases LasB and AprA. Together, these studies describe a molecular basis for mucin-based nutrient acquisition by P. aeruginosa and reveal a host-pathogen dynamic that may contribute to its persistence within the CF airways. Copyright © 2017 American Society for Microbiology.

  19. ZHOUPI controls embryonic cuticle formation via a signalling pathway involving the subtilisin protease ABNORMAL LEAF-SHAPE1 and the receptor kinases GASSHO1 and GASSHO2.

    PubMed

    Xing, Qian; Creff, Audrey; Waters, Andrew; Tanaka, Hirokazu; Goodrich, Justin; Ingram, Gwyneth C

    2013-02-01

    Seed production in angiosperms requires tight coordination of the development of the embryo and the endosperm. The endosperm-specific transcription factor ZHOUPI has previously been shown to play a key role in this process, by regulating both endosperm breakdown and the formation of the embryonic cuticle. To what extent these processes are functionally linked is, however, unclear. In order to address this issue we have concentrated on the subtilisin-like serine protease encoding gene ABNORMAL LEAF-SHAPE1. Expression of ABNORMAL LEAF-SHAPE1 is endosperm specific, and dramatically decreased in zhoupi mutants. We show that, although ABNORMAL LEAF-SHAPE1 is required for normal embryonic cuticle formation, it plays no role in regulating endosperm breakdown. Furthermore, we show that re-introducing ABNORMAL LEAF-SHAPE1 expression in the endosperm of zhoupi mutants partially rescues embryonic cuticle formation without rescuing their persistent endosperm phenotype. Thus, we conclude that ALE1 can normalize cuticle formation in the absence of endosperm breakdown, and that ZHOUPI thus controls two genetically separable developmental processes. Finally, our genetic study shows that ZHOUPI and ABNORMAL LEAF-SHAPE1 promotes formation of embryonic cuticle via a pathway involving embryonically expressed receptor kinases GASSHO1 and GASSHO2. We therefore provide a molecular framework of inter-tissue communication for embryo-specific cuticle formation during embryogenesis.

  20. Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways

    SciTech Connect

    Lund, Leif R; Romer, John; Thomasset, Nicole; Solberg, Helene; Pyke, Charles; Bissell, Mina J; Dano, Keld; Werb, Zena

    1996-01-01

    Postlactational involution of the mammary gland is characterized by two distinct physiological events: apoptosis of the secretory, epithelial cells undergoing programmed cell death, and proteolytic degradation of the mammary gland basement membrane. We examined the spatial and temporal patterns of apoptotic cells in relation to those of proteinases during involution of the BALB/c mouse mammary gland. Apoptosis was almost absent during lactation but became evident at day 2 of involution, when {beta}-casein gene expression was still high. Apoptotic cells were then seen at least up to day 8 of involution, when {beta}-casein gene expression was being extinguished. Expression of sulfated glycoprotein-2 (SGP-2), interleukin-1{beta} converting enzyme (ICE) and tissue inhibitor of metalloproteinases-1 was upregulated at day 2, when apoptotic cells were seen initially. Expression of the matrix metalloproteinases gelatinase A and stromelysin-1 and the serine proteinase urokinase-type plasminogen activator, which was low during lactation, was strongly upregulated in parallel starting at day 4 after weaning, coinciding with start of the collapse of the lobulo-alveolar structures and the intensive tissue remodeling in involution. The major sites of mRNA synthesis for these proteinases were fibroblast-like cells in the periductal stroma and stromal cells surrounding the collapsed alveoli, suggesting that the degradative phase of involution is due to a specialized mesenchymal-epithelial interaction. To elucidate the functional role of these proteinases during involution, at the onset of weaning we treated mice systemically with the glucocorticoid hydrocortisone, which is known to inhibit mammary gland involution. Although the initial wave of apoptotic cells appeared in the lumina of the gland, the dramatic regression and tissue remodeling usually evident by day 5 was substantially inhibited by systemic treatment with hydrocortisone. mRNA and protein for gelatinase A, stromelysin

  1. A chimeric prokaryotic pentameric ligand–gated channel reveals distinct pathways of activation

    SciTech Connect

    Schmandt, Nicolaus; Velisetty, Phanindra; Chalamalasetti, Sreevatsa V.; Stein, Richard A.; Bonner, Ross; Talley, Lauren; Parker, Mark D.; Mchaourab, Hassane S.; Yee, Vivien C.; Lodowski, David T.; Chakrapani, Sudha

    2015-09-28

    Recent high resolution structures of several pentameric ligand–gated ion channels have provided unprecedented details of their molecular architecture. However, the conformational dynamics and structural rearrangements that underlie gating and allosteric modulation remain poorly understood. We used a combination of electrophysiology, double electron–electron resonance (DEER) spectroscopy, and x-ray crystallography to investigate activation mechanisms in a novel functional chimera with the extracellular domain (ECD) of amine-gated Erwinia chrysanthemi ligand–gated ion channel, which is activated by primary amines, and the transmembrane domain of Gloeobacter violaceus ligand–gated ion channel, which is activated by protons. We found that the chimera was independently gated by primary amines and by protons. The crystal structure of the chimera in its resting state, at pH 7.0 and in the absence of primary amines, revealed a closed-pore conformation and an ECD that is twisted with respect to the transmembrane region. Amine- and pH-induced conformational changes measured by DEER spectroscopy showed that the chimera exhibits a dual mode of gating that preserves the distinct conformational changes of the parent channels. Collectively, our findings shed light on both conserved and divergent features of gating mechanisms in this class of channels, and will facilitate the design of better allosteric modulators.

  2. A chimeric prokaryotic pentameric ligand–gated channel reveals distinct pathways of activation

    DOE PAGES

    Schmandt, Nicolaus; Velisetty, Phanindra; Chalamalasetti, Sreevatsa V.; ...

    2015-09-28

    Recent high resolution structures of several pentameric ligand–gated ion channels have provided unprecedented details of their molecular architecture. However, the conformational dynamics and structural rearrangements that underlie gating and allosteric modulation remain poorly understood. We used a combination of electrophysiology, double electron–electron resonance (DEER) spectroscopy, and x-ray crystallography to investigate activation mechanisms in a novel functional chimera with the extracellular domain (ECD) of amine-gated Erwinia chrysanthemi ligand–gated ion channel, which is activated by primary amines, and the transmembrane domain of Gloeobacter violaceus ligand–gated ion channel, which is activated by protons. We found that the chimera was independently gated by primarymore » amines and by protons. The crystal structure of the chimera in its resting state, at pH 7.0 and in the absence of primary amines, revealed a closed-pore conformation and an ECD that is twisted with respect to the transmembrane region. Amine- and pH-induced conformational changes measured by DEER spectroscopy showed that the chimera exhibits a dual mode of gating that preserves the distinct conformational changes of the parent channels. Collectively, our findings shed light on both conserved and divergent features of gating mechanisms in this class of channels, and will facilitate the design of better allosteric modulators.« less

  3. A chimeric prokaryotic pentameric ligand–gated channel reveals distinct pathways of activation

    PubMed Central

    Schmandt, Nicolaus; Velisetty, Phanindra; Chalamalasetti, Sreevatsa V.; Stein, Richard A.; Bonner, Ross; Talley, Lauren; Parker, Mark D.; Mchaourab, Hassane S.; Yee, Vivien C.; Lodowski, David T.

    2015-01-01

    Recent high resolution structures of several pentameric ligand–gated ion channels have provided unprecedented details of their molecular architecture. However, the conformational dynamics and structural rearrangements that underlie gating and allosteric modulation remain poorly understood. We used a combination of electrophysiology, double electron–electron resonance (DEER) spectroscopy, and x-ray crystallography to investigate activation mechanisms in a novel functional chimera with the extracellular domain (ECD) of amine-gated Erwinia chrysanthemi ligand–gated ion channel, which is activated by primary amines, and the transmembrane domain of Gloeobacter violaceus ligand–gated ion channel, which is activated by protons. We found that the chimera was independently gated by primary amines and by protons. The crystal structure of the chimera in its resting state, at pH 7.0 and in the absence of primary amines, revealed a closed-pore conformation and an ECD that is twisted with respect to the transmembrane region. Amine- and pH-induced conformational changes measured by DEER spectroscopy showed that the chimera exhibits a dual mode of gating that preserves the distinct conformational changes of the parent channels. Collectively, our findings shed light on both conserved and divergent features of gating mechanisms in this class of channels, and will facilitate the design of better allosteric modulators. PMID:26415570

  4. LIM Domain Only-2 (LMO2) Induces T-Cell Leukemia by Two Distinct Pathways

    PubMed Central

    Goodings, Charnise; Cleveland, Susan; Mathias, Elizabeth; Hardaway, J. Andrew; Elliott, Natalina; Yi, Yajun; Chen, Xi; Downing, James; Mullighan, Charles; Swing, Deborah A.; Tessarollo, Lino; Li, Liqi; Love, Paul; Jenkins, Nancy A.; Copeland, Neal G.; Thompson, Mary Ann; Du, Yang; Davé, Utpal P.

    2014-01-01

    The LMO2 oncogene is deregulated in the majority of human T-cell leukemia cases and in most gene therapy-induced T-cell leukemias. We made transgenic mice with enforced expression of Lmo2 in T-cells by the CD2 promoter/enhancer. These transgenic mice developed highly penetrant T-ALL by two distinct patterns of gene expression: one in which there was concordant activation of Lyl1, Hhex, and Mycn or alternatively, with Notch1 target gene activation. Most strikingly, this gene expression clustering was conserved in human Early T-cell Precursor ALL (ETP-ALL), where LMO2, HHEX, LYL1, and MYCN were most highly expressed. We discovered that HHEX is a direct transcriptional target of LMO2 consistent with its concordant gene expression. Furthermore, conditional inactivation of Hhex in CD2-Lmo2 transgenic mice markedly attenuated T-ALL development, demonstrating that Hhex is a crucial mediator of Lmo2's oncogenic function. The CD2-Lmo2 transgenic mice offer mechanistic insight into concordant oncogene expression and provide a model for the highly treatment-resistant ETP-ALL subtype. PMID:24465765

  5. Amyloid -protein (A) assembly: A40 and A42 oligomerize through distinct pathways

    NASA Astrophysics Data System (ADS)

    Bitan, Gal; Kirkitadze, Marina D.; Lomakin, Aleksey; Vollers, Sabrina S.; Benedek, George B.; Teplow, David B.

    2003-01-01

    Amyloid -protein (A) is linked to neuronal injury and death in Alzheimer's disease (AD). Of particular relevance for elucidating the role of A in AD is new evidence that oligomeric forms of A are potent neurotoxins that play a major role in neurodegeneration and the strong association of the 42-residue form of A, A42, with the disease. Detailed knowledge of the structure and assembly dynamics of A thus is important for the development of properly targeted AD therapeutics. Recently, we have shown that A oligomers can be cross-linked efficiently, and their relative abundances quantified, by using the technique of photo-induced cross-linking of unmodified proteins (PICUP). Here, PICUP, size-exclusion chromatography, dynamic light scattering, circular dichroism spectroscopy, and electron microscopy have been combined to elucidate fundamental features of the early assembly of A40 and A42. Carefully prepared aggregate-free A40 existed as monomers, dimers, trimers, and tetramers, in rapid equilibrium. In contrast, A42 preferentially formed pentamer/hexamer units (paranuclei) that assembled further to form beaded superstructures similar to early protofibrils. Addition of Ile-41 to A40 was sufficient to induce formation of paranuclei, but the presence of Ala-42 was required for their further association. These data demonstrate that A42 assembly involves formation of several distinct transient structures that gradually rearrange into protofibrils. The strong etiologic association of A42 with AD may thus be a result of assemblies formed at the earliest stages of peptide oligomerization.

  6. Inflammation-Induced CCR7 Oligomers Form Scaffolds to Integrate Distinct Signaling Pathways for Efficient Cell Migration.

    PubMed

    Hauser, Mark A; Schaeuble, Karin; Kindinger, Ilona; Impellizzieri, Daniela; Krueger, Wolfgang A; Hauck, Christof R; Boyman, Onur; Legler, Daniel F

    2016-01-19

    Host defense depends on orchestrated cell migration guided by chemokines that elicit selective but biased signaling pathways to control chemotaxis. Here, we showed that different inflammatory stimuli provoked oligomerization of the chemokine receptor CCR7, enabling human dendritic cells and T cell subpopulations to process guidance cues not only through classical G protein-dependent signaling but also by integrating an oligomer-dependent Src kinase signaling pathway. Efficient CCR7-driven migration depends on a hydrophobic oligomerization interface near the conserved NPXXY motif of G protein-coupled receptors as shown by mutagenesis screen and a CCR7-SNP demonstrating super-oligomer characteristics leading to enhanced Src activity and superior chemotaxis. Furthermore, Src phosphorylates oligomeric CCR7, thereby creating a docking site for SH2-domain-bearing signaling molecules. Finally, we identified CCL21-biased signaling that involved the phosphatase SHP2 to control efficient cell migration. Collectively, our data showed that CCR7 oligomers serve as molecular hubs regulating distinct signaling pathways.

  7. Pentraxin 3, ficolin-2 and lectin pathway associated serine protease MASP-3 as early predictors of myocardial infarction - the HUNT2 study

    PubMed Central

    Vengen, Inga Thorsen; Enger, Tone Bull; Videm, Vibeke; Garred, Peter

    2017-01-01

    The lectin complement pathway is suggested to play a role in atherogenesis. Pentraxin-3 (PTX3), ficolin-1, ficolin-2, ficolin-3, MBL/ficolin/collectin-associated serine protease-3 (MASP-3) and MBL/ficolin/collectin-associated protein-1 (MAP-1) are molecules related to activation of the lectin complement pathway. We hypothesized that serum levels of these molecules may be associated with the incidence of myocardial infarction (MI). In a Norwegian population-based cohort (HUNT2) where young to middle-aged relatively healthy Caucasians were followed up for a first-time MI from 1995–1997 through 2008, the 370 youngest MI patients were matched by age (range 29–62 years) and gender to 370 controls. After adjustments for traditional risk factors, the two highest tertiles of PTX3 and the highest tertiles of ficolin-2 and MASP-3 were associated with MI, with odds ratios (95% confidence interval) of 1.65 (1.10–2.47) and 2.79 (1.83–4.24) for PTX3, 1.55 (1.04–2.30) for ficolin-2, and 0.63 (0.043–0.94) for MASP-3. Ficolin-1, ficolin-3 and MAP-1 were not associated with MI. In a multimarker analysis of all associated biomarkers, only PTX3 and MASP-3 remained significant. PTX-3 and MASP-3 enhanced prediction of MI compared to the traditional Framingham risk score alone (AUC increased from 0.64 to 0.68, p = 0.006). These results support the role of complement-dependent inflammation in the pathophysiology of cardiovascular disease. PMID:28216633

  8. SARS coronavirus papain-like protease induces Egr-1-dependent up-regulation of TGF-β1 via ROS/p38 MAPK/STAT3 pathway.

    PubMed

    Li, Shih-Wein; Wang, Ching-Ying; Jou, Yu-Jen; Yang, Tsuey-Ching; Huang, Su-Hua; Wan, Lei; Lin, Ying-Ju; Lin, Cheng-Wen

    2016-05-13

    SARS coronavirus (SARS-CoV) papain-like protease (PLpro) has been identified in TGF-β1 up-regulation in human promonocytes (Proteomics 2012, 12: 3193-205). This study investigates the mechanisms of SARS-CoV PLpro-induced TGF-β1 promoter activation in human lung epithelial cells and mouse models. SARS-CoV PLpro dose- and time-dependently up-regulates TGF-β1 and vimentin in A549 cells. Dual luciferase reporter assays with TGF-β1 promoter plasmids indicated that TGF-β1 promoter region between -175 to -60, the Egr-1 binding site, was responsible for TGF-β1 promoter activation induced by SARS-CoV PLpro. Subcellular localization analysis of transcription factors showed PLpro triggering nuclear translocation of Egr-1, but not NF-κB and Sp-1. Meanwhile, Egr-1 silencing by siRNA significantly reduced PLpro-induced up-regulation of TGF-β1, TSP-1 and pro-fibrotic genes. Furthermore, the inhibitors for ROS (YCG063), p38 MAPK (SB203580), and STAT3 (Stattic) revealed ROS/p38 MAPK/STAT3 pathway involving in Egr-1 dependent activation of TGF-β1 promoter induced by PLpro. In a mouse model with a direct pulmonary injection, PLpro stimulated macrophage infiltration into lung, up-regulating Egr-1, TSP-1, TGF-β1 and vimentin expression in lung tissues. The results revealed that SARS-CoV PLpro significantly triggered Egr-1 dependent activation of TGF-β1 promoter via ROS/p38 MAPK/STAT3 pathway, correlating with up-regulation of pro-fibrotic responses in vitro and in vivo.

  9. ShRNA-mediated silencing of the ubiquitin-specific protease 22 gene restrained cell progression and affected the Akt pathway in nasopharyngeal carcinoma

    PubMed Central

    Zhuang, Ya-Jing; Liao, Zhi-Wei; Yu, Hong-wei; Song, Xian-Lu; Liu, Yuan; Shi, Xing-Yuan; Lin, Xiao-dan; Zhou, Tong-Chong

    2015-01-01

    Ubiquitin-specific protease 22 (USP22) is closely related with poor prognosis of cancer patients. However, the role of USP22 expression in nasopharyngeal carcinoma (NPC) has not been determined. The main aim of this study was to determine the role of USP22 in the pathologic processes of NPC. Immunohistochemistry (IHC), western blot (WB), and real-time polymerase chain reaction (RT-PCR) were used to measure the expression of USP22 in cell lines and tissues of NPC in comparison with expression in non-cancerous cells and tissues. USP22-specific short hairpin RNA (shRNA) was used to knock down USP22 expression in the NPC cell line CNE-1 and CNE-2. Furthermore, the impact of USP22 in cellular proliferation, growth, and cell cycle were detected respectively. WB was used to determine the role of USP22 in the AKT/GSK-3/Cyclin signaling pathway. The expression levels of USP22 were remarkably higher in NPC cell lines and tissues. With cell counting and the MTS assay, cellular growth and proliferation progression of USP22 knockdown cell line was shown to be effectively restrained. The USP22 silencing both in CNE-1 and CNE-2 cells caused them to accumulate in the G0/G1 phase of the cell cycle. USP22 knockdown was also found to modulate the AKT/GSK-3/Cyclin pathway, resulting in downregulation of p-AKT, p-GSK-3β, and cyclinD1. This study suggests that USP22 plays a critical regulatory role in the pathologic processes of NPC, and that it may be a potential biological treatment target in the future. PMID:25482932

  10. SARS coronavirus papain-like protease induces Egr-1-dependent up-regulation of TGF-β1 via ROS/p38 MAPK/STAT3 pathway

    PubMed Central

    Li, Shih-Wein; Wang, Ching-Ying; Jou, Yu-Jen; Yang, Tsuey-Ching; Huang, Su-Hua; Wan, Lei; Lin, Ying-Ju; Lin, Cheng-Wen

    2016-01-01

    SARS coronavirus (SARS-CoV) papain-like protease (PLpro) has been identified in TGF-β1 up-regulation in human promonocytes (Proteomics 2012, 12: 3193-205). This study investigates the mechanisms of SARS-CoV PLpro-induced TGF-β1 promoter activation in human lung epithelial cells and mouse models. SARS-CoV PLpro dose- and time-dependently up-regulates TGF-β1 and vimentin in A549 cells. Dual luciferase reporter assays with TGF-β1 promoter plasmids indicated that TGF-β1 promoter region between −175 to −60, the Egr-1 binding site, was responsible for TGF-β1 promoter activation induced by SARS-CoV PLpro. Subcellular localization analysis of transcription factors showed PLpro triggering nuclear translocation of Egr-1, but not NF-κB and Sp-1. Meanwhile, Egr-1 silencing by siRNA significantly reduced PLpro-induced up-regulation of TGF-β1, TSP-1 and pro-fibrotic genes. Furthermore, the inhibitors for ROS (YCG063), p38 MAPK (SB203580), and STAT3 (Stattic) revealed ROS/p38 MAPK/STAT3 pathway involving in Egr-1 dependent activation of TGF-β1 promoter induced by PLpro. In a mouse model with a direct pulmonary injection, PLpro stimulated macrophage infiltration into lung, up-regulating Egr-1, TSP-1, TGF-β1 and vimentin expression in lung tissues. The results revealed that SARS-CoV PLpro significantly triggered Egr-1 dependent activation of TGF-β1 promoter via ROS/p38 MAPK/STAT3 pathway, correlating with up-regulation of pro-fibrotic responses in vitro and in vivo. PMID:27173006

  11. Two distinct domains of Bicoid mediate its transcriptional downregulation by the Torso pathway.

    PubMed

    Janody, F; Sturny, R; Schaeffer, V; Azou, Y; Dostatni, N

    2001-06-01

    The transcriptional activity of the Bicoid morphogen is directly downregulated by the Torso signal transduction cascade at the anterior pole of the Drosophila embryo. This regulation does not involve the homeodomain or direct phosphorylation of Bicoid. We analyse the transcriptional regulation of Bicoid in response to the Torso pathway, using Bicoid variants and fusion proteins between the Bicoid domains and the Gal4 DNA-binding domain. We show that Bicoid possesses three autonomous activation domains. Two of these domains, the serine/threonine-rich and the acidic domains, are downregulated by Torso, whereas the third activation domain, which is rich in glutamine, is not. The alanine-rich domain, previously described as an activation domain in vitro, has a repressive activity that is independent of Torso. Thus, Bicoid downregulation by Torso results from a competition between the glutamine-rich domain that is insensitive to Torso and the serine/threonine-rich and acidic activation domains downregulated by Torso. The alanine-rich domain contributes to this process indirectly by reducing the global activity of the protein and in particular the activity of the glutamine-rich domain that might otherwise prevent downregulation by Torso.

  12. Distinct surveillance pathway for immunopathology during acute infection via autophagy and SR-BI

    PubMed Central

    Pfeiler, Susanne; Khandagale, Avinash B.; Magenau, Astrid; Nichols, Maryana; Heijnen, Harry F. G.; Rinninger, Franz; Ziegler, Tilman; Seveau, Stephanie; Schubert, Sören; Zahler, Stefan; Verschoor, Admar; Latz, Eicke; Massberg, Steffen; Gaus, Katharina; Engelmann, Bernd

    2016-01-01

    The mechanisms protecting from immunopathology during acute bacterial infections are incompletely known. We found that in response to apoptotic immune cells and live or dead Listeria monocytogenes scavenger receptor BI (SR-BI), an anti-atherogenic lipid exchange mediator, activated internalization mechanisms with characteristics of macropinocytosis and, assisted by Golgi fragmentation, initiated autophagic responses. This was supported by scavenger receptor-induced local increases in membrane cholesterol concentrations which generated lipid domains particularly in cell extensions and the Golgi. SR-BI was a key driver of beclin-1-dependent autophagy during acute bacterial infection of the liver and spleen. Autophagy regulated tissue infiltration of neutrophils, suppressed accumulation of Ly6C+ (inflammatory) macrophages, and prevented hepatocyte necrosis in the core of infectious foci. Perifocal levels of Ly6C+ macrophages and Ly6C− macrophages were unaffected, indicating predominant regulation of the focus core. SR-BI-triggered autophagy promoted co-elimination of apoptotic immune cells and dead bacteria but barely influenced bacterial sequestration and survival or inflammasome activation, thus exclusively counteracting damage inflicted by immune responses. Hence, SR-BI- and autophagy promote a surveillance pathway that partially responds to products of antimicrobial defenses and selectively prevents immunity-induced damage during acute infection. Our findings suggest that control of infection-associated immunopathology can be based on a unified defense operation. PMID:27694929

  13. Distinct TCR signaling pathways drive proliferation and cytokine production in T cells.

    PubMed

    Guy, Clifford S; Vignali, Kate M; Temirov, Jamshid; Bettini, Matthew L; Overacre, Abigail E; Smeltzer, Matthew; Zhang, Hui; Huppa, Johannes B; Tsai, Yu-Hwai; Lobry, Camille; Xie, Jianming; Dempsey, Peter J; Crawford, Howard C; Aifantis, Iannis; Davis, Mark M; Vignali, Dario A A

    2013-03-01

    The physiological basis and mechanistic requirements for a large number of functional immunoreceptor tyrosine-based activation motifs (ITAMs; high ITAM multiplicity) in the complex of the T cell antigen receptor (TCR) and the invariant signaling protein CD3 remain obscure. Here we found that whereas a low multiplicity of TCR-CD3 ITAMs was sufficient to engage canonical TCR-induced signaling events that led to cytokine secretion, a high multiplicity of TCR-CD3 ITAMs was required for TCR-driven proliferation. This was dependent on the formation of compact immunological synapses, interaction of the adaptor Vav1 with phosphorylated CD3 ITAMs to mediate the recruitment and activation of the oncogenic transcription factor Notch1 and, ultimately, proliferation induced by the cell-cycle regulator c-Myc. Analogous mechanistic events were also needed to drive proliferation in response to weak peptide agonists. Thus, the TCR-driven pathways that initiate cytokine secretion and proliferation are separable and are coordinated by the multiplicity of phosphorylated ITAMs in TCR-CD3.

  14. Reading for meaning in dyslexic and young children: distinct neural pathways but common endpoints.

    PubMed

    Schulz, Enrico; Maurer, Urs; van der Mark, Sanne; Bucher, Kerstin; Brem, Silvia; Martin, Ernst; Brandeis, Daniel

    2009-10-01

    Developmental dyslexia is a highly prevalent and specific disorder of reading acquisition characterised by impaired reading fluency and comprehension. We have previously identified fMRI- and ERP-based neural markers of impaired sentence reading in dyslexia that indicated both deviant basic word processing and deviant semantic incongruency processing. However, it remained unclear how specific these impairments are for dyslexia, as they occurred when children with dyslexia (DYS) were compared to chronological age-matched controls (CA) who also differ in the amount of reading experience. Adding a younger control group at a similar reading level (RL) as the dyslexic group, we examined here which of these markers would be specific for dyslexia despite matched performance, and which would resemble a developmental delay. Both the RL group and the DYS group showed a similar reversal of incongruency effects in the inferior parietal region (fMRI data) and similarly reduced incongruency effects around 400 ms (ERP data) compared to the CA group, suggesting that the semantic impairment in dyslexia resembles a developmental delay. Furthermore, the DYS group showed reduced sentence reading-related activation in the inferior parietal cortex in the fMRI data, and at around 100 ms (trend) and 400 ms in the ERP data when compared to both CA and RL groups, suggesting dyslexia-specific deficits in basic word processing during sentence reading. Low reading skills due to young age and due to dyslexia-specific word processing deficits thus reflect different pathways which impair semantic processing in similar ways.

  15. TLR ligands and butyrate increase Pyy expression through two distinct but inter-regulated pathways.

    PubMed

    Larraufie, Pierre; Doré, Joël; Lapaque, Nicolas; Blottière, Hervé M

    2017-02-01

    The intestinal epithelium is an active barrier separating the host from its microbiota. It senses microbial compounds through expression of a wide range of receptors including the Toll-like receptors (TLRs). TLRs have been shown to regulate epithelium permeability or secretion of defensin by Paneth cells. However, the expression and function of TLRs in enteroendocrine L-cells, a specific subtype of intestinal cells secreting PYY and GLP-1, have not yet been assessed. PYY and GLP-1 are implicated in regulation of gut motility, food intake and insulin secretion, and are of great interest regarding obesity and type 2 diabetes. Using a cellular model of human L-cells and a reporter system for NF-κB activation pathway, we reported functional expression of TLRs in these cells. Stimulation with specific TLR-agonists increased expression of Pyy but not Proglucagon in an NF-κB-dependent manner. Moreover, the effect of TLR stimulation was additive to butyrate, a product of bacterial fermentation, on Pyy expression. Additionally, butyrate also increased Tlr expression, including Tlr4, and the NF-κB response to TLR stimulation. Altogether, our results demonstrated a role of TLRs in the modulation of Pyy expression and the importance of butyrate, a product of bacterial fermentation in regulation of microbial TLR-dependent sensing.

  16. Brg1 governs distinct pathways to direct multiple aspects of mammalian neural crest cell development.

    PubMed

    Li, Wei; Xiong, Yiqin; Shang, Ching; Twu, Karen Y; Hang, Calvin T; Yang, Jin; Han, Pei; Lin, Chieh-Yu; Lin, Chien-Jung; Tsai, Feng-Chiao; Stankunas, Kryn; Meyer, Tobias; Bernstein, Daniel; Pan, Minggui; Chang, Ching-Pin

    2013-01-29

    Development of the cerebral vessels, pharyngeal arch arteries (PAAs). and cardiac outflow tract (OFT) requires multipotent neural crest cells (NCCs) that migrate from the neural tube to target tissue destinations. Little is known about how mammalian NCC development is orchestrated by gene programming at the chromatin level, however. Here we show that Brahma-related gene 1 (Brg1), an ATPase subunit of the Brg1/Brahma-associated factor (BAF) chromatin-remodeling complex, is required in NCCs to direct cardiovascular development. Mouse embryos lacking Brg1 in NCCs display immature cerebral vessels, aberrant PAA patterning, and shortened OFT. Brg1 suppresses an apoptosis factor, Apoptosis signal-regulating kinase 1 (Ask1), and a cell cycle inhibitor, p21(cip1), to inhibit apoptosis and promote proliferation of NCCs, thereby maintaining a multipotent cell reservoir at the neural crest. Brg1 also supports Myosin heavy chain 11 (Myh11) expression to allow NCCs to develop into mature vascular smooth muscle cells of cerebral vessels. Within NCCs, Brg1 partners with chromatin remodeler Chromodomain-helicase-DNA-binding protein 7 (Chd7) on the PlexinA2 promoter to activate PlexinA2, which encodes a receptor for semaphorin to guide NCCs into the OFT. Our findings reveal an important role for Brg1 and its downstream pathways in the survival, differentiation, and migration of the multipotent NCCs critical for mammalian cardiovascular development.

  17. Dopamine elevates and lowers astroglial Ca2+ through distinct pathways depending on local synaptic circuitry

    PubMed Central

    Jennings, Alistair; Tyurikova, Olga; Bard, Lucie; Zheng, Kaiyu; Semyanov, Alexey; Henneberger, Christian

    2016-01-01

    Whilst astrocytes in culture invariably respond to dopamine with cytosolic Ca2+ rises, the dopamine sensitivity of astroglia in situ and its physiological roles remain unknown. To minimize effects of experimental manipulations on astroglial physiology, here we monitored Ca2+ in cells connected via gap junctions to astrocytes loaded whole‐cell with cytosolic indicators in area CA1 of acute hippocampal slices. Aiming at high sensitivity of [Ca2+] measurements, we also employed life‐time imaging of the Ca2+ indicator Oregon Green BAPTA‐1. We found that dopamine triggered a dose‐dependent, bidirectional Ca2+ response in stratum radiatum astroglia, a jagged elevation accompanied and followed by below‐baseline decreases. The elevation depended on D1/D2 receptors and engaged intracellular Ca2+ storage and removal whereas the dopamine‐induced [Ca2+] decrease involved D2 receptors only and was sensitive to Ca2+ channel blockade. In contrast, the stratum lacunosum moleculare astroglia generated higher‐threshold dopamine‐induced Ca2+ responses which did not depend on dopamine receptors and were uncoupled from the prominent inhibitory action of dopamine on local perforant path synapses. Our findings thus suggest that a single neurotransmitter—dopamine—could either elevate or decrease astrocyte [Ca2+] depending on the receptors involved, that such actions are specific to the regional neural circuitry and that they may be causally uncoupled from dopamine actions on local synapses. The results also indicate that [Ca2+] elevations commonly detected in astroglia can represent the variety of distinct mechanisms acting on the microscopic scale. GLIA 2017;65:447–459 PMID:27896839

  18. Distinctive neuronal networks and biochemical pathways for appetitive and aversive memory in Drosophila larvae.

    PubMed

    Honjo, Ken; Furukubo-Tokunaga, Katsuo

    2009-01-21

    Associative strength between conditioned stimulus (CS) and unconditioned stimulus (US) is thought to determine learning efficacy in classical conditioning. Elucidation of the neuronal mechanism that underlies the association between CS and US in the brain is thus critical to understand the principle of memory formation. With a simple brain organization, the Drosophila larva provides an attractive model system to investigate learning at the neurocircuitry level. Previously, we described a single-odor paradigm for larval associative learning using sucrose as a reward, and showed that larval appetitive memory lasts longer than 2 h. In this work, we describe behavioral and genetic characterization of larval aversive olfactory memory formed in our paradigm, and compare its stability and neurocircuitry with those of appetitive memory. Despite identical training paradigms, larval olfactory memory formed with quinine or NaCl is short-lived to be lost in 20 min. As with appetitive memory, larval aversive memory produced in this paradigm depends on intact cAMP signaling, but neither mutation of amnesiac nor suppression of CREB activity affects its kinetics. Neurocircuitry analyses suggest that aversive memory is stored before the presynaptic termini of the larval mushroom body neurons as is the case with appetitive memory. However, synaptic output of octopaminergic and dopaminergic neurons, which exhibit distinctive innervation patterns on the larval mushroom body and antennal lobe, is differentially required for the acquisition of appetitive and aversive memory, respectively. These results as a whole suggest that the genetically programmed memory circuitries might provide predisposition in the efficacy of inducing longer-lived memory components in associative learning.

  19. Distinct reaction pathway promoted by non-divalent-metal cations in a tertiary stabilized hammerhead ribozyme

    PubMed Central

    Roychowdhury-Saha, Manami; Burke, Donald H.

    2007-01-01

    Divalent ion sensitivity of hammerhead ribozymes is significantly reduced when the RNA structure includes appropriate tertiary stabilization. Therefore, we investigated the activity of the tertiary stabilized “RzB” hammerhead ribozyme in several nondivalent ions. Ribozyme RzB is active in spermidine and Na+ alone, although the cleavage rates are reduced by more than 1,000-fold relative to the rates observed in Mg2+ and in transition metal ions. The trivalent cobalt hexammine (CoHex) ion is often used as an exchange-inert analog of hydrated magnesium ion. Trans-cleavage rates exceeded 8 min−1 in 20 mM CoHex, which promoted cleavage through outersphere interactions. The stimulation of catalysis afforded by the tertiary structural interactions within RzB does not require Mg2+, unlike other extended hammerhead ribozymes. Site-specific interaction with at least one Mg2+ ion is suggested by CoHex competition experiments. In the presence of a constant, low concentration of Mg2+, low concentrations of CoHex decreased the rate by two to three orders of magnitude relative to the rate in Mg2+ alone. Cleavage rates increased as CoHex concentrations were raised further, but the final fraction cleaved was lower than what was observed in CoHex or Mg2+ alone. These observations suggest that Mg2+ and CoHex compete for binding and that they cause misfolded structures when they are together. The results of this study support the existence of an alternate catalytic mechanism used by nondivalent ions (especially CoHex) that is distinct from the one promoted by divalent metal ions, and they imply that divalent metals influence catalysis through a specific nonstructural role. PMID:17456566

  20. Abl2/Arg controls dendritic spine and dendrite arbor stability via distinct cytoskeletal control pathways.

    PubMed

    Lin, Yu-Chih; Yeckel, Mark F; Koleske, Anthony J

    2013-01-30

    Rho family GTPases coordinate cytoskeletal rearrangements in neurons, and mutations in their regulators are associated with mental retardation and other neurodevelopmental disorders (Billuart et al., 1998; Kutsche et al., 2000; Newey et al., 2005; Benarroch, 2007). Chromosomal microdeletions encompassing p190RhoGAP or its upstream regulator, the Abl2/Arg tyrosine kinase, have been observed in cases of mental retardation associated with developmental defects (Scarbrough et al., 1988; James et al., 1996; Takano et al., 1997; Chaabouni et al., 2006; Leal et al., 2009). Genetic knock-out of Arg in mice leads to synapse, dendritic spine, and dendrite arbor loss accompanied by behavioral deficits (Moresco et al., 2005; Sfakianos et al., 2007). To elucidate the cell-autonomous mechanisms by which Arg regulates neuronal stability, we knocked down Arg in mouse hippocampal neuronal cultures. We find that Arg knockdown significantly destabilizes dendrite arbors and reduces dendritic spine density by compromising dendritic spine stability. Inhibiting RhoA prevents dendrite arbor loss following Arg knockdown in neurons, but does not block spine loss. Interestingly, Arg-deficient neurons exhibit increased miniature EPSC amplitudes, and their remaining spines exhibit larger heads deficient in the actin stabilizing protein cortactin. Spine destabilization in Arg knockdown neurons is prevented by blocking NMDA receptor-dependent relocalization of cortactin from spines, or by forcing cortactin into spines via fusion to an actin-binding region of Arg. Thus, Arg employs distinct mechanisms to selectively regulate spine and dendrite stability: Arg dampens activity-dependent disruption of cortactin localization to stabilize spines and attenuates Rho activity to stabilize dendrite arbors.

  1. Amiloride-sensitive sodium transport in lamprey red blood cells: evidence for two distinct transport pathways.

    PubMed

    Gusev, G P; Ivanova, T I

    2004-12-01

    To determine Na+/H+ exchange in lamprey erythrocyte membranes, the cells were acidified to pH(i) 6.0 using the K+/H+ ionophore nigericin. Incubation of acidified erythrocytes in a NaCl medium at pH 8.0 caused a considerable rise in 22Na+ influx and H+ efflux during the first 1 min of exposure. In addition, exposure of acidified red cells to NaCl medium was associated with rapid elevation of intracellular Na+ content. The acid-induced changes in Na+ influx and H+ efflux were almost completely inhibited by amiloride and dimethylamiloride. In native lamprey erythrocytes, amiloride-sensitive Na+ influx progressively increased as the osmolality of incubation medium was increased by addition of 100, 200, or 300 mmol/l sucrose. Unexpectedly, the hypertonic stress induced a small, yet statistically significant decrease in intracellular Na+ content in these cells. The reduction in the cellular Na+ content increased with hypertonicity of the medium. The acid- and shrinkage-induced Na+ influxes were inhibited by both amiloride and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) in a dose-dependent manner. For both blockers, the half-maximal inhibitory values (IC50) were much greater for the shrinkage-induced (44 and 15 micromol/l for amiloride and EIPA, respectively) than for the acid-induced Na+ influx (5.1 and 3.3 micromol/l, respectively). The data obtained are the first demonstration of the presence of a Na+/H+ exchanger with high activity in acidified (pH(i) 6.0) lamprey red blood cells (on average, 512 +/- 56 mmol/l cells/h, n = 13). The amiloride-sensitive Na+ influxes produced by hypertonic cell shrinkage and acid load are likely to be mediated by distinct ion transporters in these cells.

  2. Perfluorooctanoic acid (PFOA) affects distinct molecular signalling pathways in human primary hepatocytes.

    PubMed

    Buhrke, Thorsten; Krüger, Eileen; Pevny, Sophie; Rößler, Manuela; Bitter, Katja; Lampen, Alfonso

    2015-07-03

    Perfluorooctanoic acid (PFOA) was shown to damage the liver of rodents and to impair embryonic development. At the molecular level, the hepatotoxic effects were attributed to the PFOA-mediated activation of peroxisome proliferator-activated receptor alpha (PPARα). In general, PPARα-dependent effects are less pronounced in humans than in rodents, and the hazard potential of PFOA for humans is controversially discussed. To analyse the effects of PFOA in human hepatocytes, a microarray analysis was conducted to screen for PFOA-mediated alterations in the transcriptome of human primary hepatocytes. A subsequent network analysis revealed that PFOA had an impact on several signalling pathways in addition to the well-known activation of PPARα. The microarray data confirmed earlier findings that PFOA: (i) affects the estrogen receptor ERα, (ii) activates the peroxisome proliferator-activated receptor gamma (PPARγ), and (iii) inhibits the function of the hepatocyte nuclear factor 4α (HNF4α) which is an essential factor for liver development and embryogenesis. Finally, as a novel finding, PFOA was shown to stimulate gene expression of the proto-oncogenes c-Jun and c-Fos. This was confirmed by using the HepG2 cell line as a model for human hepatocytes. PFOA stimulated cellular proliferation and the metabolic activity of the cells, and upregulated the expression of various cyclins which have a central function in the regulation of cell cycle control. Functional studies, however, indicated that PFOA had no impact on c-Jun and c-Fos phosphorylation and on AP-1-dependent gene transcription, thus demonstrating that PFOA-induced proliferation occurs largely independent of c-Jun and c-Fos. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  3. Brassinosteroids Regulate Plant Growth through Distinct Signaling Pathways in Selaginella and Arabidopsis

    PubMed Central

    Cheon, Jinyeong; Fujioka, Shozo; Dilkes, Brian P.; Choe, Sunghwa

    2013-01-01

    Brassinosteroids (BRs) are growth-promoting steroid hormones that regulate diverse physiological processes in plants. Most BR biosynthetic enzymes belong to the cytochrome P450 (CYP) family. The gene encoding the ultimate step of BR biosynthesis in Arabidopsis likely evolved by gene duplication followed by functional specialization in a dicotyledonous plant-specific manner. To gain insight into the evolution of BRs, we performed a genomic reconstitution of Arabidopsis BR biosynthetic genes in an ancestral vascular plant, the lycophyte Selaginella moellendorffii. Selaginella contains four members of the CYP90 family that cluster together in the CYP85 clan. Similar to known BR biosynthetic genes, the Selaginella CYP90s exhibit eight or ten exons and Selaginella produces a putative BR biosynthetic intermediate. Therefore, we hypothesized that Selaginella CYP90 genes encode BR biosynthetic enzymes. In contrast to typical CYPs in Arabidopsis, Selaginella CYP90E2 and CYP90F1 do not possess amino-terminal signal peptides, suggesting that they do not localize to the endoplasmic reticulum. In addition, one of the three putative CYP reductases (CPRs) that is required for CYP enzyme function co-localized with CYP90E2 and CYP90F1. Treatments with a BR biosynthetic inhibitor, propiconazole, and epi-brassinolide resulted in greatly retarded and increased growth, respectively. This suggests that BRs promote growth in Selaginella, as they do in Arabidopsis. However, BR signaling occurs through different pathways than in Arabidopsis. A sequence homologous to the Arabidopsis BR receptor BRI1 was absent in Selaginella, but downstream components, including BIN2, BSU1, and BZR1, were present. Thus, the mechanism that initiates BR signaling in Selaginella seems to differ from that in Arabidopsis. Our findings suggest that the basic physiological roles of BRs as growth-promoting hormones are conserved in both lycophytes and Arabidopsis; however, different BR molecules and BRI1-based

  4. Brassinosteroids regulate plant growth through distinct signaling pathways in Selaginella and Arabidopsis.

    PubMed

    Cheon, Jinyeong; Fujioka, Shozo; Dilkes, Brian P; Choe, Sunghwa

    2013-01-01

    Brassinosteroids (BRs) are growth-promoting steroid hormones that regulate diverse physiological processes in plants. Most BR biosynthetic enzymes belong to the cytochrome P450 (CYP) family. The gene encoding the ultimate step of BR biosynthesis in Arabidopsis likely evolved by gene duplication followed by functional specialization in a dicotyledonous plant-specific manner. To gain insight into the evolution of BRs, we performed a genomic reconstitution of Arabidopsis BR biosynthetic genes in an ancestral vascular plant, the lycophyte Selaginella moellendorffii. Selaginella contains four members of the CYP90 family that cluster together in the CYP85 clan. Similar to known BR biosynthetic genes, the Selaginella CYP90s exhibit eight or ten exons and Selaginella produces a putative BR biosynthetic intermediate. Therefore, we hypothesized that Selaginella CYP90 genes encode BR biosynthetic enzymes. In contrast to typical CYPs in Arabidopsis, Selaginella CYP90E2 and CYP90F1 do not possess amino-terminal signal peptides, suggesting that they do not localize to the endoplasmic reticulum. In addition, one of the three putative CYP reductases (CPRs) that is required for CYP enzyme function co-localized with CYP90E2 and CYP90F1. Treatments with a BR biosynthetic inhibitor, propiconazole, and epi-brassinolide resulted in greatly retarded and increased growth, respectively. This suggests that BRs promote growth in Selaginella, as they do in Arabidopsis. However, BR signaling occurs through different pathways than in Arabidopsis. A sequence homologous to the Arabidopsis BR receptor BRI1 was absent in Selaginella, but downstream components, including BIN2, BSU1, and BZR1, were present. Thus, the mechanism that initiates BR signaling in Selaginella seems to differ from that in Arabidopsis. Our findings suggest that the basic physiological roles of BRs as growth-promoting hormones are conserved in both lycophytes and Arabidopsis; however, different BR molecules and BRI1-based

  5. Two Distinct Ca2+ Signaling Pathways Modulate Sperm Flagellar Beating Patterns in Mice1

    PubMed Central

    Chang, Haixin; Suarez, Susan S.

    2011-01-01

    Hyperactivation, a swimming pattern of mammalian sperm in the oviduct, is essential for fertilization. It is characterized by asymmetrical flagellar beating and an increase of cytoplasmic Ca2+. We observed that some mouse sperm swimming in the oviduct produce high-amplitude pro-hook bends (bends in the direction of the hook on the head), whereas other sperm produce high-amplitude anti-hook bends. Switching direction of the major bends could serve to redirect sperm toward oocytes. We hypothesized that different Ca2+ signaling pathways produce high-amplitude pro-hook and anti-hook bends. In vitro, sperm that hyperactivated during capacitation (because of activation of CATSPER plasma membrane Ca2+ channels) developed high-amplitude pro-hook bends. The CATSPER activators procaine and 4-aminopyridine (4-AP) also induced high-amplitude pro-hook bends. Thimerosal, which triggers a Ca2+ release from internal stores, induced high-amplitude anti-hook bends. Activation of CATSPER channels is facilitated by a pH rise, so both Ca2+ and pH responses to treatments with 4-AP and thimerosal were monitored. Thimerosal triggered a Ca2+ increase that initiated at the base of the flagellum, whereas 4-AP initiated a rise in the proximal principal piece. Only 4-AP triggered a flagellar pH rise. Proteins were extracted from sperm for examination of phosphorylation patterns induced by Ca2+ signaling. Procaine and 4-AP induced phosphorylation of proteins on threonine and serine, whereas thimerosal primarily induced dephosphorylation of proteins. Tyrosine phosphorylation was unaffected. We concluded that hyperactivation, which is associated with capacitation, can be modulated by release of Ca2+ from intracellular stores to reverse the direction of the dominant flagellar bend and, thus, redirect sperm. PMID:21389347

  6. Chronic stress and peripheral pain: Evidence for distinct, region-specific changes in visceral and somatosensory pain regulatory pathways.

    PubMed

    Zheng, Gen; Hong, Shuangsong; Hayes, John M; Wiley, John W

    2015-11-01

    Chronic stress alters the hypothalamic-pituitary-adrenal (HPA) axis and enhances visceral and somatosensory pain perception. It is unresolved whether chronic stress has distinct effects on visceral and somatosensory pain regulatory pathways. Previous studies reported that stress-induced visceral hyperalgesia is associated with reciprocal alterations of endovanilloid and endocannabinoid pain pathways in DRG neurons innervating the pelvic viscera. In this study, we compared somatosensory and visceral hyperalgesia with respect to differential responses of peripheral pain regulatory pathways in a rat model of chronic, intermittent stress. We found that chronic stress induced reciprocal changes in the endocannabinoid 2-AG (increased) and endocannabinoid degradation enzymes COX-2 and FAAH (decreased), associated with down-regulation of CB1 and up-regulation of TRPV1 receptors in L6-S2 DRG but not L4-L5 DRG neurons. In contrast, sodium channels Nav1.7 and Nav1.8 were up-regulated in L4-L5 but not L6-S2 DRGs in stressed rats, which was reproduced in control DRGs treated with corticosterone in vitro. The reciprocal changes of CB1, TRPV1 and sodium channels were cell-specific and observed in the sub-population of nociceptive neurons. Behavioral assessment showed that visceral hyperalgesia persisted, whereas somatosensory hyperalgesia and enhanced expression of Nav1.7 and Nav1.8 sodium channels in L4-L5 DRGs normalized 3 days after completion of the stress phase. These data indicate that chronic stress induces visceral and somatosensory hyperalgesia that involves differential changes in endovanilloid and endocannabinoid pathways, and sodium channels in DRGs innervating the pelvic viscera and lower extremities. These results suggest that chronic stress-induced visceral and lower extremity somatosensory hyperalgesia can be treated selectively at different levels of the spinal cord. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Chronic Stress and Peripheral Pain: Evidence for Distinct, Region-specific Changes in Visceral and Somatosensory Pain Regulatory Pathways

    PubMed Central

    Zheng, Gen; Hong, Shuangsong; Hayes, John M; Wiley, John W

    2015-01-01

    Chronic stress alters the hypothalamic-pituitary-adrenal (HPA) axis and enhances visceral and somatosensory pain perception. It is unresolved whether chronic stress has distinct effects on visceral and somatosensory pain regulatory pathways. Previous studies reported that stress-induced visceral hyperalgesia is associated with reciprocal alterations of endovanilloid and endocannabinoid pain pathways in DRG neurons innervating the pelvic viscera. In this study, we compared somatosensory and visceral hyperalgesia with respect to differential responses of peripheral pain regulatory pathways in a rat model of chronic, intermittent stress. We found that chronic stress induced reciprocal changes in the endocannabinoid 2-AG (increased) and endocannabinoid degradation enzymes COX-2 and FAAH (decreased), associated with down-regulation of CB1 and up-regulation of TRPV1 receptors in L6-S2 DRG but not L4-L5 DRG neurons. In contrast, sodium channels Nav1.7 and Nav1.8 were up-regulated in L4-L5 but not L6-S2 DRGs in stressed rats, which was reproduced in control L4-L5 DRGs treated with corticosterone in vitro. The reciprocal changes of CB1, TRPV1 and sodium channels were cell-specific and observed in the sub-population of nociceptive neurons. Behavioral assessment showed that visceral hyperalgesia persisted, whereas somatosensory hyperalgesia and enhanced expression of Nav1.7 and Nav1.8 sodium channels in L4-L5 DRGs normalized 3 days after completion of the stress phase. These data indicate that chronic stress induces visceral and somatosensory hyperalgesia that involves differential changes in endovanilloid and endocannabinoid pathways, and sodium channels in DRGs innervating the pelvic viscera and lower extremities. These results suggest that chronic stress-induced visceral and lower extremity somatosensory hyperalgesia can be treated selectively at different levels of the spinal cord. PMID:26408049

  8. Rotavirus Controls Activation of the 2'-5'-Oligoadenylate Synthetase/RNase L Pathway Using at Least Two Distinct Mechanisms.

    PubMed

    Sánchez-Tacuba, Liliana; Rojas, Margarito; Arias, Carlos F; López, Susana

    2015-12-01

    The innate immune response is the first line of defense of the host cell against a viral infection. In turn, viruses have evolved a wide variety of strategies to hide from, and to directly antagonize, the host innate immune pathways. One of these pathways is the 2'-5'-oligoadenylate synthetase (OAS)/RNase L pathway. OAS is activated by double-stranded RNA (dsRNA) to produce 2'-5' oligoadenylates, which are the activators of RNase L; this enzyme degrades viral and cellular RNAs, restricting viral infection. It has been recently found that the carboxy-terminal domain (CTD) of rotavirus VP3 has a 2'-5'-phosphodiesterase (PDE) activity that is able to functionally substitute for the PDE activity of the mouse hepatitis virus ns2 protein. This particular phosphodiesterase cleaves the 2'-5'-phosphodiester bond of the oligoadenylates, antagonizing the OAS/RNase L pathway. However, whether this activity of VP3 is relevant during the replication cycle of rotavirus is not known. Here, we demonstrate that after rotavirus infection the OAS/RNase L complex becomes activated; however, the virus is able to control its activity using at least two distinct mechanisms. A virus-cell interaction that occurs during or before rotavirus endocytosis triggers a signal that prevents the early activation of RNase L, while later on the control is taken by the newly synthesized VP3. Cosilencing the expression of VP3 and RNase L in infected cells yields viral infectious particles at levels similar to those obtained in control infected cells, where no genes were silenced, suggesting that the capping activity of VP3 is not essential for the formation of infectious viral particles. Rotaviruses represent an important cause of severe gastroenteritis in the young of many animal species, including humans. In this work, we have found that the OAS/RNase L pathway is activated during rotavirus infection, but the virus uses two different strategies to prevent the deleterious effects of this innate immune

  9. A Coronavirus E Protein Is Present in Two Distinct Pools with Different Effects on Assembly and the Secretory Pathway

    PubMed Central

    Westerbeck, Jason W.

    2015-01-01

    ABSTRACT Coronaviruses (CoVs) assemble by budding into the lumen of the early Golgi complex prior to exocytosis. The small CoV envelope (E) protein plays roles in assembly, virion release, and pathogenesis. CoV E has a single hydrophobic domain (HD), is targeted to Golgi complex membranes, and has cation channel activity in vitro. However, the precise functions of the CoV E protein during infection are still enigmatic. Structural data for the severe acute respiratory syndrome (SARS)-CoV E protein suggest that it assembles into a homopentamer. Specific residues in the HD regulate the ion-conducting pore formed by SARS-CoV E in artificial bilayers and the pathogenicity of the virus during infection. The E protein from the avian infectious bronchitis virus (IBV) has dramatic effects on the secretory system which require residues in the HD. Here, we use the known structural data from SARS-CoV E to infer the residues important for ion channel activity and the oligomerization of IBV E. We present biochemical data for the formation of two distinct oligomeric pools of IBV E in transfected and infected cells and the residues required for their formation. A high-order oligomer of IBV E is required for the production of virus-like particles (VLPs), implicating this form of the protein in virion assembly. Additionally, disruption of the secretory pathway by IBV E correlates with a form that is likely monomeric, suggesting that the effects on the secretory pathway are independent of E ion channel activity. IMPORTANCE CoVs are important human pathogens with significant zoonotic potential, as demonstrated by the emergence of SARS-CoV and Middle East respiratory syndrome (MERS)-CoV. Progress has been made toward identifying potential vaccine candidates in mouse models of CoV infection, including the use of attenuated viruses that lack the CoV E protein or express E-protein mutants. However, no approved vaccines or antiviral therapeutics exist. We previously reported that the

  10. The HIV-1 protease inhibitor nelfinavir activates PP2 and inhibits MAPK signaling in macrophages: a pathway to reduce inflammation.

    PubMed

    Wallet, Mark A; Reist, Caroline M; Williams, Julie C; Appelberg, Sofia; Guiulfo, Giorgio L; Gardner, Brent; Sleasman, John W; Goodenow, Maureen M

    2012-10-01

    The HIV-1 PI NFV has off-target effects upon host enzymes, including inhibition of the 20S proteasome, resulting in activation of PP1. HIV-1-associated monocyte/macrophage activation, in part a result of systemically elevated levels of microbial products including LPS, is associated with risk of mortality, independent of viremia or CD4 T cell loss. This study tested the hypothesis that activation of protein phosphatases by NFV would reduce activation of monocytes/macrophages through dephosphorylation of signal transduction proteins. NFV uniquely blocked LPS-induced production by human monocyte-derived macrophages of the inflammatory cytokines TNF and IL-6, as well as sCD14. Although NFV failed to modulate NF-κB, NFV treatment reduced phosphorylation of AKT and MAPKs. Inhibition of PP2 with okadaic acid blocked the anti-inflammatory effect of NFV, whereas the PP1 inhibitor calyculin A failed to counter the anti-inflammatory effects of NFV. For in vivo studies, plasma sCD14 and LPS were monitored in a cohort of 31 pediatric HIV-1 patients for over 2 years of therapy. Therapy, including NFV, reduced sCD14 levels significantly compared with IDV or RTV, independent of ΔLPS levels, VL, CD4 T cell frequency, or age. The hypothesis was supported as NFV induced activation of PP2 in macrophages, resulting in disruption of inflammatory cell signaling pathways. In vivo evidence supports that NFV may offer beneficial effects independent of antiviral activity by reducing severity of chronic innate immune activation in HIV-1 infection.

  11. The HIV-1 protease inhibitor nelfinavir activates PP2 and inhibits MAPK signaling in macrophages: a pathway to reduce inflammation

    PubMed Central

    Wallet, Mark A.; Reist, Caroline M.; Williams, Julie C.; Appelberg, Sofia; Guiulfo, Giorgio L.; Gardner, Brent; Sleasman, John W.; Goodenow, Maureen M.

    2012-01-01

    The HIV-1 PI NFV has off-target effects upon host enzymes, including inhibition of the 20S proteasome, resulting in activation of PP1. HIV-1-associated monocyte/macrophage activation, in part a result of systemically elevated levels of microbial products including LPS, is associated with risk of mortality, independent of viremia or CD4 T cell loss. This study tested the hypothesis that activation of protein phosphatases by NFV would reduce activation of monocytes/macrophages through dephosphorylation of signal transduction proteins. NFV uniquely blocked LPS-induced production by human monocyte-derived macrophages of the inflammatory cytokines TNF and IL-6, as well as sCD14. Although NFV failed to modulate NF-κB, NFV treatment reduced phosphorylation of AKT and MAPKs. Inhibition of PP2 with okadaic acid blocked the anti-inflammatory effect of NFV, whereas the PP1 inhibitor calyculin A failed to counter the anti-inflammatory effects of NFV. For in vivo studies, plasma sCD14 and LPS were monitored in a cohort of 31 pediatric HIV-1 patients for over 2 years of therapy. Therapy, including NFV, reduced sCD14 levels significantly compared with IDV or RTV, independent of ΔLPS levels, VL, CD4 T cell frequency, or age. The hypothesis was supported as NFV induced activation of PP2 in macrophages, resulting in disruption of inflammatory cell signaling pathways. In vivo evidence supports that NFV may offer beneficial effects independent of antiviral activity by reducing severity of chronic innate immune activation in HIV-1 infection. PMID:22786868

  12. Protease-activated receptor-2 promotes kidney tubular epithelial inflammation by inhibiting autophagy via the PI3K/Akt/mTOR signalling pathway.

    PubMed

    Du, Chunyang; Zhang, Tao; Xiao, Xia; Shi, Yonghong; Duan, Huijun; Ren, Yunzhuo

    2017-08-02

    Protease-activated receptor-2 (PAR2), which belongs to a specific class of the G-protein-coupled receptors, is central to several inflammation processes. However, the precise molecular mechanism involved remains undefined. Autophagy has been previously shown to affect inflammation. In the present study, we examine the effect of PAR2 on kidney tubular epithelial autophagy and on autophagy-related inflammation and reveal the underlying mechanism involved. Autophagic activity and levels of autophagic marker LC3 were examined in human kidney tubular epithelial cells with PAR2 knockdown or overexpression. We administered the mammalian target of rapamycin (mTOR) inhibitor (rapamycin) or activator (MHY1485) to investigate the function of the phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway. We also used transforming growth factor-β1 (TGF-β1)-induced HK-2 cell inflammation models to investigate the role of PAR2-associated autophagy in kidney tubular epithelial inflammation. PAR2 antagonist and rapamycin were administered to mice after unilateral ureteral obstruction to detect the correlations between PAR2, autophagy, and inflammation. Our results show that PAR2 overexpression in HK-2 cells led to a greater reduction in autophagy via the PI3K/Akt/mTOR pathway activation and induces autophagy-related inflammation. Meanwhile, a knockdown of PAR2 via PAR2 RNAi transfection greatly increased autophagy and alleviated autophagy-associated inflammation. In unilateral ureteral obstruction (UUO) kidneys, PAR2 antagonist treatment greatly attenuated renal inflammation and interstitial injury by enhancing autophagy. Moreover, inhibition of mTOR, rapa, markedly increased autophagy and inhibited the UUO-induced inflammation. We conclude that PAR2 induces kidney tubular epithelial inflammation by inhibiting autophagy via the PI3K/Akt/mTOR signalling pathway. Our results are suggestive that PAR2 inhibition may play a role in the treatment of diseases with increased inflammatory

  13. Puma and Trail/Dr5 Pathways Control Radiation-Induced Apoptosis in Distinct Populations of Testicular Progenitors

    PubMed Central

    Coureuil, Mathieu; Ugolin, Nicolas; Tavernier, Marie; Chevillard, Sylvie; Barroca, Vilma; Fouchet, Pierre; Allemand, Isabelle

    2010-01-01

    Spermatogonia- stem cells and progenitors of adult spermatogenesis- are killed through a p53-regulated apoptotic process after γ-irradiation but the death effectors are still poorly characterized. Our data demonstrate that both intrinsic and extrinsic apoptotic pathways are involved, and especially that spermatogonia can be split into two main populations, according to apoptotic effectors. Following irradiation both Dr5 and Puma genes are upregulated in the α6-integrin-positive Side Population (SP) fraction, which is highly enriched in spermatogonia. Flow cytometric analysis confirms an increased number of Dr5-expressing SP cells, and Puma-β isoform accumulates in α6-integrin positive cellular extracts, enriched in spermatogonia. Trail−/− or Puma−/− spermatogonia display a reduced sensitivity to radiation-induced apoptosis. The TUNEL kinetics strongly suggest that the extrinsic and intrinsic pathways, via Trail/Dr5 and Puma respectively, could be engaged in distinct subpopulations of spermatogonia. Indeed flow cytometric studies show that Dr5 receptor is constitutively present on more than half of the undifferentiated progenitors (Kit− α6+ SP) and half of the differentiated ones (Kit+ α6+ SP). In addition after irradiation, Puma is not detected in the Dr5-positive cellular fraction isolated by immunomagnetic purification, while Puma is present in the Dr5-negative cell extracts. In conclusion, adult testicular progenitors are divided into distinct sub-populations by apoptotic effectors, independently of progenitor types (immature Kit-negative versus mature Kit-positive), underscoring differential radiosensitivities characterizing the stem cell/progenitors compartment. PMID:20711434

  14. The ARG1-LIKE2 gene of Arabidopsis functions in a gravity signal transduction pathway that is genetically distinct from the PGM pathway

    NASA Technical Reports Server (NTRS)

    Guan, Changhui; Rosen, Elizabeth S.; Boonsirichai, Kanokporn; Poff, Kenneth L.; Masson, Patrick H.

    2003-01-01

    The arl2 mutants of Arabidopsis display altered root and hypocotyl gravitropism, whereas their inflorescence stems are fully gravitropic. Interestingly, mutant roots respond like the wild type to phytohormones and an inhibitor of polar auxin transport. Also, their cap columella cells accumulate starch similarly to wild-type cells, and mutant hypocotyls display strong phototropic responses to lateral light stimulation. The ARL2 gene encodes a DnaJ-like protein similar to ARG1, another protein previously implicated in gravity signal transduction in Arabidopsis seedlings. ARL2 is expressed at low levels in all organs of seedlings and plants. arl2-1 arg1-2 double mutant roots display kinetics of gravitropism similar to those of single mutants. However, double mutants carrying both arl2-1 and pgm-1 (a mutation in the starch-biosynthetic gene PHOSPHOGLUCOMUTASE) at the homozygous state display a more pronounced root gravitropic defect than the single mutants. On the other hand, seedlings with a null mutation in ARL1, a paralog of ARG1 and ARL2, behave similarly to the wild type in gravitropism and other related assays. Taken together, the results suggest that ARG1 and ARL2 function in the same gravity signal transduction pathway in the hypocotyl and root of Arabidopsis seedlings, distinct from the pathway involving PGM.

  15. The ARG1-LIKE2 gene of Arabidopsis functions in a gravity signal transduction pathway that is genetically distinct from the PGM pathway.

    PubMed

    Guan, Changhui; Rosen, Elizabeth S; Boonsirichai, Kanokporn; Poff, Kenneth L; Masson, Patrick H

    2003-09-01

    The arl2 mutants of Arabidopsis display altered root and hypocotyl gravitropism, whereas their inflorescence stems are fully gravitropic. Interestingly, mutant roots respond like the wild type to phytohormones and an inhibitor of polar auxin transport. Also, their cap columella cells accumulate starch similarly to wild-type cells, and mutant hypocotyls display strong phototropic responses to lateral light stimulation. The ARL2 gene encodes a DnaJ-like protein similar to ARG1, another protein previously implicated in gravity signal transduction in Arabidopsis seedlings. ARL2 is expressed at low levels in all organs of seedlings and plants. arl2-1 arg1-2 double mutant roots display kinetics of gravitropism similar to those of single mutants. However, double mutants carrying both arl2-1 and pgm-1 (a mutation in the starch-biosynthetic gene PHOSPHOGLUCOMUTASE) at the homozygous state display a more pronounced root gravitropic defect than the single mutants. On the other hand, seedlings with a null mutation in ARL1, a paralog of ARG1 and ARL2, behave similarly to the wild type in gravitropism and other related assays. Taken together, the results suggest that ARG1 and ARL2 function in the same gravity signal transduction pathway in the hypocotyl and root of Arabidopsis seedlings, distinct from the pathway involving PGM.

  16. The ARG1-LIKE2 gene of Arabidopsis functions in a gravity signal transduction pathway that is genetically distinct from the PGM pathway

    NASA Technical Reports Server (NTRS)

    Guan, Changhui; Rosen, Elizabeth S.; Boonsirichai, Kanokporn; Poff, Kenneth L.; Masson, Patrick H.

    2003-01-01

    The arl2 mutants of Arabidopsis display altered root and hypocotyl gravitropism, whereas their inflorescence stems are fully gravitropic. Interestingly, mutant roots respond like the wild type to phytohormones and an inhibitor of polar auxin transport. Also, their cap columella cells accumulate starch similarly to wild-type cells, and mutant hypocotyls display strong phototropic responses to lateral light stimulation. The ARL2 gene encodes a DnaJ-like protein similar to ARG1, another protein previously implicated in gravity signal transduction in Arabidopsis seedlings. ARL2 is expressed at low levels in all organs of seedlings and plants. arl2-1 arg1-2 double mutant roots display kinetics of gravitropism similar to those of single mutants. However, double mutants carrying both arl2-1 and pgm-1 (a mutation in the starch-biosynthetic gene PHOSPHOGLUCOMUTASE) at the homozygous state display a more pronounced root gravitropic defect than the single mutants. On the other hand, seedlings with a null mutation in ARL1, a paralog of ARG1 and ARL2, behave similarly to the wild type in gravitropism and other related assays. Taken together, the results suggest that ARG1 and ARL2 function in the same gravity signal transduction pathway in the hypocotyl and root of Arabidopsis seedlings, distinct from the pathway involving PGM.

  17. Distinct Signal Transduction Pathways Downstream of the (P)RR Revealed by Microarray and ChIP-chip Analyses

    PubMed Central

    Zaade, Daniela; Schmitz, Jennifer; Benke, Eileen; Klare, Sabrina; Seidel, Kerstin; Kirsch, Sebastian; Goldin-Lang, Petra; Zollmann, Frank S.; Unger, Thomas; Funke-Kaiser, Heiko

    2013-01-01

    The (pro)renin receptor ((P)RR) signaling is involved in different pathophysiologies ranging from cardiorenal end-organ damage via diabetic retinopathy to tumorigenesis. We have previously shown that the transcription factor promyelocytic leukemia zinc finger (PLZF) is an adaptor protein of the (P)RR. Furthermore, recent publications suggest that major functions of the (P)RR are mediated ligand-independently by its transmembrane and intracellular part, which acts as an accessory protein of V-ATPases. The transcriptome and recruitmentome downstream of the V-ATPase function and PLZF in the context of the (P)RR are currently unknown. Therefore, we performed a set of microarray and chromatin-immunoprecipitation (ChIP)-chip experiments using siRNA against the (P)RR, stable overexpression of PLZF, the PLZF translocation inhibitor genistein and the specific V-ATPase inhibitor bafilomycin to dissect transcriptional pathways downstream of the (P)RR. We were able to identify distinct and overlapping genetic signatures as well as novel real-time PCR-validated target genes of the different molecular functions of the (P)RR. Moreover, bioinformatic analyses of our data confirm the role of (P)RŔs signal transduction pathways in cardiovascular disease and tumorigenesis. PMID:23469216

  18. A pathway switch directs BAFF signaling to distinct NFκB transcription factors in maturing and proliferating B cells.

    PubMed

    Almaden, Jonathan V; Tsui, Rachel; Liu, Yi C; Birnbaum, Harry; Shokhirev, Maxim N; Ngo, Kim A; Davis-Turak, Jeremy C; Otero, Dennis; Basak, Soumen; Rickert, Robert C; Hoffmann, Alexander

    2014-12-24

    BAFF, an activator of the noncanonical NFκB pathway, provides critical survival signals during B cell maturation and contributes to B cell proliferation. We found that the NFκB family member RelB is required ex vivo for B cell maturation, but cRel is required for proliferation. Combined molecular network modeling and experimentation revealed Nfkb2 p100 as a pathway switch; at moderate p100 synthesis rates in maturing B cells, BAFF fully utilizes p100 to generate the RelB:p52 dimer, whereas at high synthesis rates, p100 assembles into multimeric IκBsome complexes, which BAFF neutralizes in order to potentiate cRel activity and B cell expansion. Indeed, moderation of p100 expression or disruption of IκBsome assembly circumvented the BAFF requirement for full B cell expansion. Our studies emphasize the importance of p100 in determining distinct NFκB network states during B cell biology, which causes BAFF to have context-dependent functional consequences.

  19. Immune Exhaustion and Immune Senescence – Two Distinct Pathways for HBV Vaccine Failure during HCV and/or HIV Infection

    PubMed Central

    Yao, Zhi Q.; Moorman, Jonathan P.

    2013-01-01

    Given the shared risk factors for transmission, co-infection of hepatitis B virus (HBV) with hepatitis C virus (HCV) and/or human immunodeficiency virus (HIV) is quite common, and may lead to increases in morbidity and mortality. As such, HBV vaccine is recommended as the primary means to prevent HBV super-infection in HCV- and/or HIV-infected individuals. However, vaccine response (sero-conversion with a hepatitis B surface antibody titer >10 IU/L) in this setting is often blunted, with poor response rates to standard HBV vaccinations in virally infected individuals when compared to the healthy subjects. This phenomenon also occurs to other vaccines in adults, such as pneumococcal and influenza vaccines, in other immunocompromised hosts who are really at risk for opportunistic infections, such as individuals with hemodialysis, transplant, and malignancy. In this review, we summarize the underlying mechanisms involving vaccine failure in these conditions, focusing on immune exhaustion and immune senescence - two distinct signaling pathways regulating cell function and fate. We raise the possibility that blocking these negative signaling pathways might improve success rates of immunizations in the setting of chronic viral infection. PMID:23400275

  20. Distinct signaling pathways for induction of type II NOS by IFNgamma and LPS in BV-2 microglial cells.

    PubMed

    Shen, Siming; Yu, Sue; Binek, Joshua; Chalimoniuk, Malgorzata; Zhang, Xiaolin; Lo, Shih-Ching; Hannink, Mark; Wu, Jinmei; Fritsche, Kevin; Donato, Rosario; Sun, Grace Y

    2005-09-01

    Nitric oxide (NO) release upon microglial cell activation has been implicated in the tissue injury and cell death in many neurodegenerative diseases. Recent studies have indicated the ability of interferon-gamma (IFNgamma) and lipopolysaccharides (LPS) to independently induce type II nitric oxide synthase (iNOS) expression and NO production in BV-2 microglial cells. However, a detailed comparison between the signaling pathways activating iNOS by these two agents has not been accomplished. Analysis of PKC isoforms revealed mainly the presence of PKCdelta, iota and lambda in BV-2 cells. Although both IFNgamma and LPS could specifically enhance the tyrosine phosphorylation of PKCdelta, treatment with IFNgamma induced a steady increase of phospho-PKCdelta for up to 1h, whereas treatment with LPS elevated phospho-PKCdelta levels only transiently, with peak activity at 5 min. Rottlerin, a specific inhibitor for PKCdelta, dose-dependently inhibited IFNgamma- and LPS-induced NO production. Despite the common involvement of PKCdelta, IFNgamma- but not LPS-induced NO production involved extracellular signal-regulated kinases (ERK1/2) cascade and IFNgamma-induced phosphorylation of ERK1/2 was mediated through PKC. On the other hand, LPS- but not IFNgamma-induced NO production was through stimulation of NF-kappaB activation and nuclear translocation to interact with DNA. These results demonstrated distinct signaling pathways for induction of iNOS by IFNgamma and LPS in BV-2 microglial cells.

  1. Human T-cell leukemia virus type 1 Tax protein transforms rat fibroblasts via two distinct pathways.

    PubMed Central

    Matsumoto, K; Shibata, H; Fujisawa, J I; Inoue, H; Hakura, A; Tsukahara, T; Fujii, M

    1997-01-01

    The human T-cell leukemia virus type 1 (HTLV-1) Tax protein activates the transcription of several cellular genes. This function is thought to play a critical role in the Tax-dependent transformation step in HTLV-1 leukemogenesis. Tax activates transcription via three enhancers: the cyclic AMP response element (CRE)-like sequence, the kappaB element, and the CArG box. Their involvement in the transformation of rat fibroblasts by Tax was examined by colony formation of Rat-1 cells in soft agar and Ras cooperative focus formation of rat embryo fibroblasts (REF). Among Tax mutants, those retaining activity for the CArG box transformed REF like wild-type Tax, while those inactive for the CArG box did not. Thus, the activation of the CArG box pathway is essential for the transformation of REF by Tax. In contrast, activation of the kappaB element correlated with the transformation of Rat-1 by Tax. These results show that Tax transforms rat fibroblasts via two distinct pathways. PMID:9151835

  2. Both overlapping and distinct signaling pathways for somatostatin receptor subtypes SSTR1 and SSTR2 in pituitary cells.

    PubMed

    Chen, L; Fitzpatrick, V D; Vandlen, R L; Tashjian, A H

    1997-07-25

    To elucidate the signaling events mediated by specific somatostatin receptor (SSTR) subtypes, we expressed SSTR1 and SSTR2 individually in rat pituitary GH12C1 and F4C1 cells, which lack endogenous somatostatin receptors. In transfected GH12C1 cells, both SSTR1 and SSTR2 coupled to inhibition of Ca2+ influx and hyperpolarization of membrane potential via a pertussis toxin (PTx)-sensitive mechanism. These effects reflected modulation of ion channel activities which are important for regulation of hormone secretion. Somatostatin analogs MK678 and CH275 acted as subtype selective agonists as expected. In transfected F4C1 cells, both SSTR1 and SSTR2 mediated somatostatin-induced inhibition of adenylyl cyclase via a PTx-sensitive pathway. In addition, activation of SSTR2 in F4C1 cells, but not SSTR1, stimulated phospholipase C (PLC) activity and an increase in [Ca2+]i due to release of Ca2+ from intracellular stores. Unlike adenylyl cyclase inhibition, the PLC-mediated response was only partially sensitive to PTx. To determine the structural determinants in SSTR2 necessary for activation of PLC, we constructed chimeric receptors in which domains of SSTR2 were introduced into SSTR1. Chimeric receptors containing only the third intracellular loop, or all three intracellular loops from SSTR2, mediated inhibition of adenylyl cyclase, but failed to stimulate PLC activity as did wild-type SSTR2. Furthermore, the C-terminal tail of SSTR2 was not required for coupling to PLC. Thus, by expressing individual somatostatin receptor subtypes in pituitary cells, we have identified both overlapping and distinct signaling pathways for SSTR1 and SSTR2, and have shown that sequences other than simply the intracellular domains are required for SSTR2 to couple to the PLC signaling pathway.

  3. Distinct sensory pathways in Vibrio cholerae El Tor and classical biotypes modulate cyclic dimeric GMP levels to control biofilm formation.

    PubMed

    Hammer, Brian K; Bassler, Bonnie L

    2009-01-01

    Quorum sensing (QS), or cell-cell communication in bacteria, is achieved through the production and subsequent response to the accumulation of extracellular signal molecules called autoinducers (AIs). To identify AI-regulated target genes in Vibrio cholerae El Tor (V. cholerae(El)), the strain responsible for the current cholera pandemic, luciferase expression was assayed in an AI(-) strain carrying a random lux transcriptional reporter library in the presence and absence of exogenously added AIs. Twenty-three genes were identified and shown to require the QS transcription factor, HapR, for their regulation. Several of the QS-dependent target genes, annotated as encoding hypothetical proteins, in fact encode HD-GYP proteins, phosphodiesterases that degrade the intracellular second messenger cyclic dimeric GMP (c-di-GMP), which is important for controlling biofilm formation. Indeed, overexpression of a representative QS-activated HD-GYP protein in V. cholerae(El) reduced the intracellular concentration of c-di-GMP, which in turn decreased exopolysaccharide production and biofilm formation. The V. cholerae classical biotype (V. cholerae(Cl)), which caused previous cholera pandemics and is HapR(-), controls c-di-GMP levels and biofilm formation by the VieA signaling pathway. We show that the VieA pathway is dispensable for biofilm formation in V. cholerae(El) but that restoring HapR in V. cholerae(Cl) reestablishes QS-dependent repression of exopolysaccharide production. Thus, different pandemic strains of V. cholerae modulate c-di-GMP levels and control biofilm formation in response to distinct sensory pathways.

  4. Functional overlap among distinct G1/S inhibitory pathways allows robust G1 arrest by yeast mating pheromones.

    PubMed

    Pope, Patricia A; Pryciak, Peter M

    2013-12-01

    In budding yeast, mating pheromones arrest the cell cycle in G1 phase via a pheromone-activated Cdk-inhibitor (CKI) protein, Far1. Alternate pathways must also exist, however, because deleting the cyclin CLN2 restores pheromone arrest to far1 cells. Here we probe whether these alternate pathways require the G1/S transcriptional repressors Whi5 and Stb1 or the CKI protein Sic1, whose metazoan analogues (Rb or p27) antagonize cell cycle entry. Removing Whi5 and Stb1 allows partial escape from G1 arrest in far1 cln2 cells, along with partial derepression of G1/S genes, which implies a repressor-independent route for inhibiting G1/S transcription. This route likely involves pheromone-induced degradation of Tec1, a transcriptional activator of the cyclin CLN1, because Tec1 stabilization also causes partial G1 escape in far1 cln2 cells, and this is additive with Whi5/Stb1 removal. Deleting SIC1 alone strongly disrupts Far1-independent G1 arrest, revealing that inhibition of B-type cyclin-Cdk activity can empower weak arrest pathways. Of interest, although far1 cln2 sic1 cells escaped G1 arrest, they lost viability during pheromone exposure, indicating that G1 exit is deleterious if the arrest signal remains active. Overall our findings illustrate how multiple distinct G1/S-braking mechanisms help to prevent premature cell cycle commitment and ensure a robust signal-induced G1 arrest.

  5. Honokiol traverses the blood-brain barrier and induces apoptosis of neuroblastoma cells via an intrinsic bax-mitochondrion-cytochrome c-caspase protease pathway

    PubMed Central

    Lin, Jia-Wei; Chen, Juei-Tai; Hong, Chung-Ye; Lin, Yi-Ling; Wang, Kuan-Ting; Yao, Chih-Jung; Lai, Gi-Ming; Chen, Ruei-Ming

    2012-01-01

    Neuroblastomas, an embryonic cancer of the sympathetic nervous system, often occur in young children. Honokiol, a small-molecule polyphenol, has multiple therapeutic effects and pharmacological activities. This study was designed to evaluate whether honokiol could pass through the blood-brain barrier (BBB) and induce death of neuroblastoma cells and its possible mechanisms. Primary cerebral endothelial cells (CECs) prepared from mouse brain capillaries were cultured at a high density for 4 days, and these cells formed compact morphologies and expressed the ZO-1 tight-junction protein. A permeability assay showed that the CEC-constructed barrier obstructed the passing of FITC-dextran. Analyses by high-performance liquid chromatography and the UV spectrum revealed that honokiol could traverse the CEC-built junction barrier and the BBB of ICR mice. Exposure of neuroblastoma neuro-2a cells and NB41A3 cells to honokiolinduced cell shrinkage and decreased cell viability. In parallel, honokiol selectively induced DNA fragmentation and cell apoptosis rather than cell necrosis. Sequential treatment of neuro-2a cells with honokiol increased the expression of the proapoptotic Bax protein and its translocation from the cytoplasm to mitochondria. Honokiol successively decreased the mitochondrial membrane potential but increased the release of cytochrome c from mitochondria. Consequently, honokiol induced cascade activation of caspases-9, -3, and -6. In comparison, reducing caspase-6 activity by Z-VEID-FMK, an inhibitor of caspase-6, simultaneously attenuated honokiol-induced DNA fragmentation and cell apoptosis. Taken together, this study showed that honokiol can pass through the BBB and induce apoptotic insults to neuroblastoma cells through a Bax-mitochondrion-cytochrome c-caspase protease pathway. Therefore, honokiol may be a potential candidate drug for treating brain tumors. PMID:22259050

  6. Honokiol traverses the blood-brain barrier and induces apoptosis of neuroblastoma cells via an intrinsic bax-mitochondrion-cytochrome c-caspase protease pathway.

    PubMed

    Lin, Jia-Wei; Chen, Juei-Tai; Hong, Chung-Ye; Lin, Yi-Ling; Wang, Kuan-Ting; Yao, Chih-Jung; Lai, Gi-Ming; Chen, Ruei-Ming

    2012-03-01

    Neuroblastomas, an embryonic cancer of the sympathetic nervous system, often occur in young children. Honokiol, a small-molecule polyphenol, has multiple therapeutic effects and pharmacological activities. This study was designed to evaluate whether honokiol could pass through the blood-brain barrier (BBB) and induce death of neuroblastoma cells and its possible mechanisms. Primary cerebral endothelial cells (CECs) prepared from mouse brain capillaries were cultured at a high density for 4 days, and these cells formed compact morphologies and expressed the ZO-1 tight-junction protein. A permeability assay showed that the CEC-constructed barrier obstructed the passing of FITC-dextran. Analyses by high-performance liquid chromatography and the UV spectrum revealed that honokiol could traverse the CEC-built junction barrier and the BBB of ICR mice. Exposure of neuroblastoma neuro-2a cells and NB41A3 cells to honokiolinduced cell shrinkage and decreased cell viability. In parallel, honokiol selectively induced DNA fragmentation and cell apoptosis rather than cell necrosis. Sequential treatment of neuro-2a cells with honokiol increased the expression of the proapoptotic Bax protein and its translocation from the cytoplasm to mitochondria. Honokiol successively decreased the mitochondrial membrane potential but increased the release of cytochrome c from mitochondria. Consequently, honokiol induced cascade activation of caspases-9, -3, and -6. In comparison, reducing caspase-6 activity by Z-VEID-FMK, an inhibitor of caspase-6, simultaneously attenuated honokiol-induced DNA fragmentation and cell apoptosis. Taken together, this study showed that honokiol can pass through the BBB and induce apoptotic insults to neuroblastoma cells through a Bax-mitochondrion-cytochrome c-caspase protease pathway. Therefore, honokiol may be a potential candidate drug for treating brain tumors.

  7. Target-mediated clearance and bio-distribution of a monoclonal antibody against the Kunitz-type protease inhibitor 2 domain of Tissue Factor Pathway Inhibitor.

    PubMed

    Hansen, Lene; Petersen, Lars Christian; Lauritzen, Brian; Clausen, Jes Thorn; Grell, Susanne Nedergaard; Agersø, Henrik; Sørensen, Brit Binow; Hilden, Ida; Almholt, Kasper

    2014-03-01

    A humanised monoclonal antibody, concizumab, that binds with high affinity to the Kunitz-type protease inhibitor (KPI) 2 domain of human tissue factor pathway inhibitor (TFPI) is in clinical development. It promotes coagulation by neutralising the inhibitory function of TFPI and may provide a subcutaneous prophylaxis option for patients with haemophilia. We aimed to study biodistribution and pharmacokinetics (PK) of concizumab. Blockage of cellular TFPI by concizumab was measured by tissue factor/Factor VIIa-mediated Factor X activation on human EA.hy926 cells. Biodistribution of concizumab was analysed in rabbits by immunohistology, and the PK was measured in rabbits and rats. Concizumab bound to cell surface TFPI on EA.hy926 cells and neutralised TFPI inhibition of Factor X activation. The antibody cross-reacted with rabbit TFPI, but not with rat TFPI, allowing for comparative PK studies. PK data in rats described a log-linear profile typical for a non-binding antibody, whereas PK data in rabbits revealed a non-linear, dose-dependent profile, consistent with a target-mediated clearance mechanism. Immunohistology in rabbits during target-saturation showed localisation of the antibody on the endothelium of the microvasculature in several organs. We observed a marked co-localisation with endogenous rabbit TFPI, but a negligible sub-endothelial build-up. Concizumab binds and neutralises the inhibitory effect of cell surface-bound TFPI. The PK profile observed in rabbits is consistent with a TFPI-mediated drug disposition. Double immunofluorescence shows co-localisation of the antibody with TFPI on the endothelium of the microvasculature and points to this TFPI as a putative target involved in the clearance mechanism. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Lectin pathway effector enzyme mannan-binding lectin-associated serine protease-2 can activate native complement C3 in absence of C4 and/or C2.

    PubMed

    Yaseen, Sadam; Demopulos, Gregory; Dudler, Thomas; Yabuki, Munehisa; Wood, Christi L; Cummings, W Jason; Tjoelker, Larry W; Fujita, Teizo; Sacks, Steven; Garred, Peter; Andrew, Peter; Sim, Robert B; Lachmann, Peter J; Wallis, Russell; Lynch, Nicholas; Schwaeble, Wilhelm J

    2017-05-01

    All 3 activation pathways of complement-the classic pathway (CP), the alternative pathway, and the lectin pathway (LP)- converge into a common central event: the cleavage and activation of the abundant third complement component, C3, via formation of C3-activating enzymes (C3 convertases). The fourth complement component, C4, and the second component, C2, are indispensable constituents of the C3 convertase complex, C4bC2a, which is formed by both the CP and the LP. Whereas in the absence of C4, CP can no longer activate C3, LP retains a residual but physiologically critical capacity to convert native C3 into its activation fragments, C3a and C3b. This residual C4 and/or C2 bypass route is dependent on LP-specific mannan-binding lectin-associated serine protease-2. By using various serum sources with defined complement deficiencies, we demonstrate that, under physiologic conditions LP-specific C4 and/or C2 bypass activation of C3 is mediated by direct cleavage of native C3 by mannan-binding lectin-associated serine protease-2 bound to LP-activation complexes captured on ligand-coated surfaces.-Yaseen, S., Demopulos, G., Dudler, T., Yabuki, M., Wood, C. L., Cummings, W. J., Tjoelker, L. W., Fujita, T., Sacks, S., Garred, P., Andrew, P., Sim, R. B., Lachmann, P. J., Wallis, R., Lynch, N., Schwaeble, W. J. Lectin pathway effector enzyme mannan-binding lectin-associated serine protease-2 can activate native complement C3 in absence of C4 and/or C2. © FASEB.

  9. Cross-talk between non-genomic and genomic signalling pathways - Distinct effect profiles of environmental estrogens

    SciTech Connect

    Silva, Elisabete; Kabil, Alena; Kortenkamp, Andreas

    2010-06-01

    Estrogen receptor (ER) transcriptional cross-talk after activation by 17{beta}-estradiol (E2) has been studied in considerable detail, but comparatively little is known about the ways in which synthetic estrogen-like chemicals, so-called xenoestrogens, interfere with these signalling pathways. E2 can stimulate rapid, non-genomic signalling events, such as activation of the Src/Ras/Erk signalling pathway. We investigated how activation of this pathway by E2, the estrogenic environmental contaminants o,p'-DDT, {beta}-HCH and p,p'-DDE, and epidermal growth factor (EGF) influences the expression of ER target genes, such as TFF1, ER, PR, BRCA1 and CCND1, and the proliferation of breast cancer cells. Despite commonalities in their estrogenicity as judged by cell proliferation assays, the environmental contaminants exhibited striking differences in their non-genomic and genomic signalling. The gene expression profiles of o,p'-DDT and {beta}-HCH resembled the effects observed with E2. In the case of {beta}-HCH this is surprising, considering its reported lack of affinity to the 'classical' ER. The expression profiles seen with p,p'-DDE showed some similarities with E2, but overall, p,p'-DDE was a fairly weak transcriptional inducer of TFF1, ER, PR, BRCA1 and CCND1. We observed distinct differences in the non-genomic signalling of the tested compounds. p,p'-DDE was unable to stimulate Src and Erk1/Erk2 activations. The effects of E2 on Src and Erk1/Erk2 phosphorylation were transient and weak when compared to EGF, but {beta}-HCH induced strong and sustained activation of all tested kinases. Transcription of TFF1, ER, PR and BRCA1 by E2, o,p'-DDT and {beta}-HCH could be suppressed partially by inhibiting the Src/Ras/Erk pathway with PD 98059. However, this was not seen with p,p'-DDE. Our investigations show that the cellular activities of estrogens and xenoestrogens are the result of a combination of extranuclear (non-genomic) and nuclear (genomic) events and highlight the

  10. Semaphorin 3A inhibits growth of adult sympathetic and parasympathetic neurones via distinct cyclic nucleotide signalling pathways

    PubMed Central

    Nangle, MR; Keast, JR

    2011-01-01

    BACKGROUND AND PURPOSE Semaphorin 3A (Sema3A) is an important secreted repulsive guidance factor for many developing neurones. Sema3A continues to be expressed in adulthood, and expression of its receptor, neuropilin-1 (Nrp-1), can be altered by nerve injury. Autonomic neurones innervating the pelvic viscera are particularly susceptible to damage during pelvic surgical procedures, and failure to regenerate or aberrant growth of sympathetic and parasympathetic nerves lead to organ dysfunction. However, it is not known if adult pelvic neurones are potential targets for Sema3A. EXPERIMENTAL APPROACH The effects of Sema3A and activation or inhibition of cyclic nucleotide signalling were assessed in adult rat pelvic ganglion neurones in culture using a growth cone collapse assay. KEY RESULTS Sema3A caused growth cone collapse in both parasympathetic and sympathetic neurones expressing Nrp-1. However, the effect of Sema3A was mediated by distinct cyclic nucleotide signalling pathways in each neurone type. In parasympathetic neurones, cAMP and downstream activation of protein kinase A were required for growth cone collapse. In sympathetic neurones, cGMP was required for Sema3A-induced collapse; cAMP can also cause collapse but was not required. Sema3A-mediated, cGMP-dependent collapse in sympathetic neurones may require activation of cyclic nucleotide-gated ion channels (CNGCs). CONCLUSIONS AND IMPLICATIONS We propose that Sema3A is an important guidance factor for adult pelvic autonomic neurones, and that manipulation of their distinct signalling mechanisms could potentially promote functional selective regeneration or attenuate aberrant growth. To our knowledge, this is also the first study to implicate CNGCs in regulating growth cone dynamics of adult neurones. PMID:21054346

  11. Dual-tagged amyloid-β precursor protein reveals distinct transport pathways of its N- and C-terminal fragments.

    PubMed

    Villegas, Christine; Muresan, Virgil; Ladescu Muresan, Zoia

    2014-03-15

    The amyloid-β precursor protein (APP), a type I transmembrane protein genetically associated with Alzheimer's disease, has a complex biology that includes proteolytic processing into potentially toxic fragments, extensive trafficking and multiple, yet poorly-defined functions. We recently proposed that a significant fraction of APP is proteolytically cleaved in the neuronal soma into N- and C-terminal fragments (NTFs and CTFs), which then target independently of each other to separate destinations in the cell. Here, we prove this concept with live imaging and immunolocalization of two dual, N- and C-termini-tagged APP constructs: CFP-APP-YFP [containing the fluorescent tags, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP)] and FLAG-APP-Myc. When expressed at low levels in neuronal cells, these constructs are processed into differently tagged NTFs and CTFs that reveal distinct distributions and characteristics of transport. Like the endogenous N- and C-terminal epitopes of APP, the FLAG-tagged NTFs are present in trains of vesicles and tubules that localize to short filaments, which often immunostain for acetylated tubulin, whereas the Myc-tagged CTFs are detected on randomly distributed vesicle-like structures. The experimental treatments that selectively destabilize the acetylated microtubules abrogate the distribution of NTFs along filaments, without altering the random distribution of CTFs. These results indicate that the NTFs and CTFs are recruited to distinct transport pathways and reach separate destinations in neurons, where they likely accomplish functions independent of the parental, full-length APP. They also point to a compartment associated with acetylated microtubules in the neuronal soma--not the neurite terminals--as a major site of APP cleavage, and segregation of NTFs from CTFs.

  12. Semaphorin 3A inhibits growth of adult sympathetic and parasympathetic neurones via distinct cyclic nucleotide signalling pathways.

    PubMed

    Nangle, M R; Keast, J R

    2011-03-01

    Semaphorin 3A (Sema3A) is an important secreted repulsive guidance factor for many developing neurones. Sema3A continues to be expressed in adulthood, and expression of its receptor, neuropilin-1 (Nrp-1), can be altered by nerve injury. Autonomic neurones innervating the pelvic viscera are particularly susceptible to damage during pelvic surgical procedures, and failure to regenerate or aberrant growth of sympathetic and parasympathetic nerves lead to organ dysfunction. However, it is not known if adult pelvic neurones are potential targets for Sema3A. The effects of Sema3A and activation or inhibition of cyclic nucleotide signalling were assessed in adult rat pelvic ganglion neurones in culture using a growth cone collapse assay. Sema3A caused growth cone collapse in both parasympathetic and sympathetic neurones expressing Nrp-1. However, the effect of Sema3A was mediated by distinct cyclic nucleotide signalling pathways in each neurone type. In parasympathetic neurones, cAMP and downstream activation of protein kinase A were required for growth cone collapse. In sympathetic neurones, cGMP was required for Sema3A-induced collapse; cAMP can also cause collapse but was not required. Sema3A-mediated, cGMP-dependent collapse in sympathetic neurones may require activation of cyclic nucleotide-gated ion channels (CNGCs). We propose that Sema3A is an important guidance factor for adult pelvic autonomic neurones, and that manipulation of their distinct signalling mechanisms could potentially promote functional selective regeneration or attenuate aberrant growth. To our knowledge, this is also the first study to implicate CNGCs in regulating growth cone dynamics of adult neurones. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  13. Dual-tagged amyloid-β precursor protein reveals distinct transport pathways of its N- and C-terminal fragments

    PubMed Central

    Villegas, Christine; Muresan, Virgil; Ladescu Muresan, Zoia

    2014-01-01

    The amyloid-β precursor protein (APP), a type I transmembrane protein genetically associated with Alzheimer's disease, has a complex biology that includes proteolytic processing into potentially toxic fragments, extensive trafficking and multiple, yet poorly-defined functions. We recently proposed that a significant fraction of APP is proteolytically cleaved in the neuronal soma into N- and C-terminal fragments (NTFs and CTFs), which then target independently of each other to separate destinations in the cell. Here, we prove this concept with live imaging and immunolocalization of two dual, N- and C-termini-tagged APP constructs: CFP-APP-YFP [containing the fluorescent tags, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP)] and FLAG-APP-Myc. When expressed at low levels in neuronal cells, these constructs are processed into differently tagged NTFs and CTFs that reveal distinct distributions and characteristics of transport. Like the endogenous N- and C-terminal epitopes of APP, the FLAG-tagged NTFs are present in trains of vesicles and tubules that localize to short filaments, which often immunostain for acetylated tubulin, whereas the Myc-tagged CTFs are detected on randomly distributed vesicle-like structures. The experimental treatments that selectively destabilize the acetylated microtubules abrogate the distribution of NTFs along filaments, without altering the random distribution of CTFs. These results indicate that the NTFs and CTFs are recruited to distinct transport pathways and reach separate destinations in neurons, where they likely accomplish functions independent of the parental, full-length APP. They also point to a compartment associated with acetylated microtubules in the neuronal soma—not the neurite terminals—as a major site of APP cleavage, and segregation of NTFs from CTFs. PMID:24203698

  14. The Human Coparental Bond Implicates Distinct Corticostriatal Pathways: Longitudinal Impact on Family Formation and Child Well-Being.

    PubMed

    Abraham, Eyal; Gilam, Gadi; Kanat-Maymon, Yaniv; Jacob, Yael; Zagoory-Sharon, Orna; Hendler, Talma; Feldman, Ruth

    2017-11-01

    Alloparental care, the cooperative care of offspring by group members other than the biological mother, has been widely practiced since early hominin evolution to increase infant survival and thriving. The coparental bond-a relationship of solidarity and commitment between two adults who join their effort to care for children-is a central contributor to children's well-being and sociality; yet, the neural basis of coparenting has not been studied in humans. Here, we followed 84 first-time co-parents (42 couples) across the first 6 years of family formation, including opposite-sex and same-sex couples, measured brain response to coparental stimuli, observed collaborative and undermining coparental behaviors in infancy and preschool, assayed oxytocin (OT) and vasopressin (AVP), and measured coparenting and child behavior problems at 6 years. Across family types, coparental stimuli activated the striatum, specifically the ventral striatum and caudate, striatal nodes implicated in motivational goal-directed social behavior. Psychophysiological interaction analysis indicated that both nodes were functionally coupled with the vmPFC in support of the human coparental bond and this connectivity was stronger as collaborative coparental behavior increased. Furthermore, caudate functional connectivity patterns differentiated distinct corticostriatal pathways associated with two stable coparental behavioral styles; stronger caudate-vmPFC connectivity was associated with more collaborative coparenting and was linked to OT, whereas a stronger caudate-dACC connectivity was associated with increase in undermining coparenting and was related to AVP. Finally, dyadic path-analysis model indicated that the parental caudate-vmPFC connectivity in infancy predicted lower child externalizing symptoms at 6 years as mediated by collaborative coparenting in preschool. Findings indicate that the coparental bond is underpinned by striatal activations and corticostriatal connectivity similar to

  15. Distinct Signaling Pathways After Higher or Lower Doses of Radiation in Three Closely Related Human Lymphoblast Cell Lines

    SciTech Connect

    Lu, T.-P.; Lai, L.-C.; Lin, B.-I.; Chen, L.-H.; Hsiao, T.-H.; Liber, Howard L.; Cook, John A.; Mitchell, James B.; Tsai, M.-H.; Chuang, Eric Y.

    2010-01-15

    Purpose: The tumor suppressor p53 plays an essential role in cellular responses to DNA damage caused by ionizing radiation; therefore, this study aims to further explore the role that p53 plays at different doses of radiation. Materials and Methods: The global cellular responses to higher-dose (10 Gy) and lower dose (iso-survival dose, i.e., the respective D0 levels) radiation were analyzed using microarrays in three human lymphoblast cell lines with different p53 status: TK6 (wild-type p53), NH32 (p53-null), and WTK1 (mutant p53). Total RNAs were extracted from cells harvested at 0, 1, 3, 6, 9, and 24 h after higher and lower dose radiation exposures. Template-based clustering, hierarchical clustering, and principle component analysis were applied to examine the transcriptional profiles. Results: Differential expression profiles between 10 Gy and iso-survival radiation in cells with different p53 status were observed. Moreover, distinct gene expression patterns were exhibited among these three cells after 10 Gy radiation treatment, but similar transcriptional responses were observed in TK6 and NH32 cells treated with iso-survival radiation. Conclusions: After 10 Gy radiation exposure, the p53 signaling pathway played an important role in TK6, whereas the NFkB signaling pathway appeared to replace the role of p53 in WTK1. In contrast, after iso-survival radiation treatment, E2F4 seemed to play a dominant role independent of p53 status. This study dissected the impacts of p53, NFkB and E2F4 in response to higher or lower doses of gamma-irradiation.

  16. Melatonin Regulates Somatotrope and Lactotrope Function Through Common and Distinct Signaling Pathways in Cultured Primary Pituitary Cells From Female Primates

    PubMed Central

    Ibáñez-Costa, Alejandro; Córdoba-Chacón, José; Gahete, Manuel D.; Kineman, Rhonda D.; Castaño, Justo P.

    2015-01-01

    Melatonin (MT) is secreted by the pineal gland and exhibits a striking circadian rhythm in its release. Depending on the species studied, some pituitary hormones also display marked circadian/seasonal patterns and rhythms of secretion. However, the precise relationship between MT and pituitary function remains controversial, and studies focusing on the direct role of MT in normal pituitary cells are limited to nonprimate species. Here, adult normal primate (baboons) primary pituitary cell cultures were used to determine the direct impact of MT on the functioning of all pituitary cell types from the pars distalis. MT increased GH and prolactin (PRL) expression/release in a dose- and time-dependent fashion, a response that was blocked by somatostatin. However, MT did not significantly affect ACTH, FSH, LH, or TSH expression/release. MT did not alter GHRH- or ghrelin-induced GH and/or PRL secretions, suggesting that MT may activate similar signaling pathways as ghrelin/GHRH. The effects of MT on GH/PRL release, which are likely mediated through MT1 receptor, involve both common (adenylyl cyclase/protein kinase A/extracellular calcium-channels) and distinct (phospholipase C/intracellular calcium-channels) signaling pathways. Actions of MT on pituitary cells also included regulation of the expression of other key components for the control of somatotrope/lactotrope function (GHRH, ghrelin, and somatostatin receptors). These results show, for the first time in a primate model, that MT directly regulates somatotrope/lactotrope function, thereby lending support to the notion that the actions of MT on these cells might substantially contribute to the define daily patterns of GH and PRL observed in primates and perhaps in humans. PMID:25545385

  17. Distinct activation of monoaminergic pathways in chick brain in relation to auditory imprinting and stressful situations: a microdialysis study.

    PubMed

    Gruss, M; Braun, K

    1997-02-01

    /hyperstriatum ventrale related to different emotional conditions after acoustic imprinting and during stressful situations is, at least in part, mediated by dopaminergic and/or serotonergic pathways. Furthermore, the results from the present study indicate a distinct activation of dopaminergic and serotonergic pathways in relation to the behavioural situation and the associated changes of emotional status.

  18. Multifunctional Mitochondrial AAA Proteases

    PubMed Central

    Glynn, Steven E.

    2017-01-01

    Mitochondria perform numerous functions necessary for the survival of eukaryotic cells. These activities are coordinated by a diverse complement of proteins encoded in both the nuclear and mitochondrial genomes that must be properly organized and maintained. Misregulation of mitochondrial proteostasis impairs organellar function and can result in the development of severe human diseases. ATP-driven AAA+ proteins play crucial roles in preserving mitochondrial activity by removing and remodeling protein molecules in accordance with the needs of the cell. Two mitochondrial AAA proteases, i-AAA and m-AAA, are anchored to either face of the mitochondrial inner membrane, where they engage and process an array of substrates to impact protein biogenesis, quality control, and the regulation of key metabolic pathways. The functionality of these proteases is extended through multiple substrate-dependent modes of action, including complete degradation, partial processing, or dislocation from the membrane without proteolysis. This review discusses recent advances made toward elucidating the mechanisms of substrate recognition, handling, and degradation that allow these versatile proteases to control diverse activities in this multifunctional organelle. PMID:28589125

  19. Multiple motifs regulate the trafficking of GABA(B) receptors at distinct checkpoints within the secretory pathway.

    PubMed

    Restituito, Sophie; Couve, Andrés; Bawagan, Hinayana; Jourdain, Sabine; Pangalos, Menelas N; Calver, Andrew R; Freeman, Katie B; Moss, Stephen J

    2005-04-01

    gamma-Aminobutyric acid type B receptors (GABA(B)) are G-protein-coupled receptors that mediate GABAergic inhibition in the brain. Their functional expression is dependent upon the formation of heterodimers between GABA(B)R1 and GABA(B)R2 subunits, a process that occurs within the endoplasmic reticulum (ER). However, the mechanisms that regulate receptor surface expression remain largely unknown. Here, we demonstrate that access to the cell surface for GABA(B)R1 is sequentially controlled by an RSR(R) motif and a LL motif within its cytoplasmic domain. In addition, we reveal that msec7-1, a guanine-nucleotide-exchange factor (GEF) for the ADP-ribosylation factor (ARF) family of GTPases, critical regulators of vesicular membrane trafficking, interacts with GABA(B)R1 via the LL motif in this subunit. Finally, we establish that msec7-1 modulates the cell surface expression of GABA(B) receptors, a process that is dependent upon the integrity of the LL motif in GABA(B)R1. Together, our results demonstrate that the cell surface expression of the GABA(B)R1 subunit is regulated by multiple motifs, which act at distinct checkpoints in the secretory pathway, and also suggest a novel role for msec7-1 in regulating the membrane trafficking of GABA(B)R1 subunits.

  20. Immune cell-specific transcriptional profiling highlights distinct molecular pathways controlled by Tob1 upon experimental autoimmune encephalomyelitis

    PubMed Central

    Didonna, Alessandro; Cekanaviciute, Egle; Oksenberg, Jorge R.; Baranzini, Sergio E.

    2016-01-01

    Multiple sclerosis (MS) is an autoimmune disease of the central nervous system characterized by focal lymphocytic infiltration, demyelination and neurodegeneration. Despite the recent advances in understanding MS molecular basis, no reliable biomarkers have been identified yet to monitor disease progression. Our group has previously reported that low levels of TOB1 in CD4+ T cells are strongly associated with a higher risk of MS conversion in individuals experiencing an initial demyelinating event. Consistently, Tob1 ablation in mice exacerbates the clinical phenotype of the MS model experimental autoimmune encephalomyelitis (EAE). To shed light on Tob1 molecular functions in the immune system, we have conducted the first cell-based transcriptomic analysis in Tob1−/− and wildtype mice upon EAE. Next-generation sequencing was employed to characterize the changes in gene expression in T and B cells at pre- and post-symptomatic EAE stages. Remarkably, we found only modest overlap among the different genetic signatures, suggesting that Tob1 may control distinct genetic programs in the different cytotypes. This hypothesis was corroborated by gene ontology and global interactome analyses, which highlighted specific cellular pathways in each cellular subset before and after EAE induction. In summary, our work pinpoints a multifaceted activity of Tob1 in both homeostasis and disease progression. PMID:27546286

  1. Brevetoxin-induced phosphorylation of Pyk2 and Src in murine neocortical neurons involves distinct signaling pathways

    PubMed Central

    Cao, Zhengyu; George, Joju; Baden, Daniel G.; Murray, Thomas F.

    2009-01-01

    Brevetoxins (PbTx-1 to PbTx-10) are potent lipid soluble polyether neurotoxins produced by the marine dinoflagellate Karenia brevis. Brevetoxins bind to site 5 of the α-subunit of voltage-gated sodium channels (VGSCs) and augment Na+ influx. In neocortical neurons brevetoxins elevate intracellular Ca2+ and augment NMDA receptor signaling. In this study, we explored the effects of PbTx-2 on Pyk2 and Src activation in neocortical neurons. We found that both Pyk2 and Src were activated following PbTx-2 exposure. PbTx-2-induced Pyk2 Tyr402 phosphorylation was dependent on elevation of Ca2+ influx through NMDA receptors. Moreover, Pyk2 Tyr402 phosphorylation was also found to require PKC activation inasmuch as RO-31-8425 and GF 109203x both attenuated the response. In contrast, PbTx-2-induced Src Tyr416 phosphorylation involved a Gq-coupled receptor inasmuch as U73122, a specific PLC inhibitor, abolished the response. This Gq-coupled receptor appears to be mGluR 5. The PKCδ inhibitor rottlerin abolished PbTx-2-induced Src activation demonstrating that this isoform of PKC is involved in the activation of Src by PbTx-2. Considered together these data suggest that although activation of neuronal Pyk2 and Src result from PbTx-2 stimulation of VGSC, engagement of these two non-receptor tyrosine kinases involves distinct signaling pathways. PMID:17963734

  2. Involvement of Net and Hif1α in Distinct yet Intricately Linked Hypoxia-induced Signaling Pathways*

    PubMed Central

    Serchov, Tsvetan; Dubois-Pot-Schneider, Helene; Charlot, Celine; Rösl, Frank; Wasylyk, Bohdan

    2010-01-01

    The present study compares negative Ets transcription factor (Net) and hypoxia-inducible factor 1α (HIF1α) regulation by hypoxia. Their protein stabilities are differently regulated by hypoxia, defining three periods in the kinetics: normoxia (high Net levels and low HIF1α levels), early hypoxia (high levels of Net and HIF1α), and late hypoxia (degradation of Net and HIF1α). Modulators of prolyl hydroxylase domain protein (PHD) activity induce a mobility shift of Net, similar to HIF1α, suggesting that post-translational modifications of both factors depend on PHD activity. The three PHDs have different roles in the regulation of Net protein levels; PHD1 and PHD3 are involved in the stabilization of Net, whereas PHD2 controls its degradation in late hypoxia. Net physically interacts with PHD2 in hypoxia, whereas PHD1 and PHD3 bind to Net in normoxia and hypoxia. Under the same conditions, PHD2 and PHD3 regulate both HIF1α stabilization in early hypoxia and its degradation at late hypoxia, whereas PHD1 is involved in HIF1α degradation in late hypoxia. We describe interconnections between the regulation of both Net and HIF1α at the protein level. Evidence is provided for a direct physical interaction between Net and HIF1α and indirect transcriptional regulation loops that involve the PHDs. Taken together our results indicate that Net and HIF1α are components of distinct signaling pathways that are intricately linked. PMID:20427288

  3. Specific residues within the alpha 2 integrin subunit cytoplasmic domain regulate migration and cell cycle progression via distinct MAPK pathways.

    PubMed

    Klekotka, P A; Santoro, S A; Wang, H; Zutter, M M

    2001-08-24

    The alpha(2) integrin subunit cytoplasmic domain is necessary for epidermal growth factor (EGF)-stimulated chemotactic migration and insulin-dependent entry into S-phase of mammary epithelial cells adherent to type I collagen. Truncation mutants revealed that the seven amino acids, KYEKMTK, in addition to the GFFKR motif were sufficient for these functions. Mutation of tyrosine 1134 to alanine inhibited the ability of the cells to phosphorylate p38 MAPK and to migrate in response to EGF but had only a modest effect on the ability of the cells to induce sustained phosphorylation of the ERK MAPK, to up-regulate cyclin E and cdk2 expression, and to enter S-phase when adherent to type I collagen. Conversely, mutation of the lysine 1136 inhibited the ability of the cells to increase cyclin E and cdk2 expression, to maintain long term phosphorylation of the ERK MAPK, and to enter S-phase but had no effect on the ability of the cells to phosphorylate the p38 MAPK or to migrate on type I collagen in response to EGF. Methionine 1137 was essential for both migration and entry into S-phase. Thus, distinctly different structural elements of the alpha(2) integrin cytoplasmic domain are required to engage the signaling pathways leading to cell migration or cell cycle progression.

  4. Proteases as Insecticidal Agents

    PubMed Central

    Harrison, Robert L.; Bonning, Bryony C.

    2010-01-01

    Proteases from a variety of sources (viruses, bacteria, fungi, plants, and insects) have toxicity towards insects. Some of these insecticidal proteases evolved as venom components, herbivore resistance factors, or microbial pathogenicity factors, while other proteases play roles in insect development or digestion, but exert an insecticidal effect when over-expressed from genetically engineered plants or microbial pathogens. Many of these proteases are cysteine proteases, although insect-toxic metalloproteases and serine proteases have also been examined. The sites of protease toxic activity range from the insect midgut to the hemocoel (body cavity) to the cuticle. This review discusses these insecticidal proteases along with their evaluation and use as potential pesticides. PMID:22069618

  5. Oxidized low-density lipoprotein induces apoptotic insults to mouse cerebral endothelial cells via a Bax-mitochondria-caspase protease pathway

    SciTech Connect

    Chen, T.-G.; Chen, T.-L.; Chang, H.-C.; Tai, Y.-T.; Cherng, Y.-G.; Chang, Y.-T.; Chen, R.-M. . E-mail: rmchen@tmu.edu.tw

    2007-02-15

    LDL can damage the blood-brain barrier through induction of CEC apoptosis via a Bax-mitochondria-caspase protease pathway.

  6. Oxidized low-density lipoprotein induces apoptotic insults to mouse cerebral endothelial cells via a Bax-mitochondria-caspase protease pathway.

    PubMed

    Chen, Tyng-Guey; Chen, Ta-Liang; Chang, Huai-Chia; Tai, Yu-Ting; Cherng, Yih-Giun; Chang, Ya-Ting; Chen, Ruei-Ming

    2007-02-15

    LDL can damage the blood-brain barrier through induction of CEC apoptosis via a Bax-mitochondria-caspase protease pathway.

  7. Yeast extracellular proteases.

    PubMed

    Ogrydziak, D M

    1993-01-01

    Many species of yeast secrete significant amounts of protease(s). In this article, results of numerous surveys of yeast extracellular protease production have been compiled and inconsistencies in the data and limitations of the methodology have been examined. Regulation, purification, characterization, and processing of yeast extracellular proteases are reviewed. Results obtained from the sequences of cloned genes, especially the Saccharomyces cerevisiae Bar protease, the Candida albicans acid protease, and the Yarrowia lipolytica alkaline protease, have been emphasized. Biotechnological applications and the medical relevance of yeast extracellular proteases are covered. Yeast extracellular proteases have potential in beer and wine stabilization, and they probably contribute to pathogenicity of Candida spp. Yeast extracellular protease genes also provide secretion and processing signals for yeast expression systems designed for secretion of heterologous proteins. Coverage of the secretion of foreign proteases such as prochymosin, urokinase, and tissue plasminogen activator by yeast in included.

  8. Genomic and phenotypic characterization of myxoma virus from Great Britain reveals multiple evolutionary pathways distinct from those in Australia.

    PubMed

    Kerr, Peter J; Cattadori, Isabella M; Rogers, Matthew B; Fitch, Adam; Geber, Adam; Liu, June; Sim, Derek G; Boag, Brian; Eden, John-Sebastian; Ghedin, Elodie; Read, Andrew F; Holmes, Edward C

    2017-03-01

    The co-evolution of myxoma virus (MYXV) and the European rabbit occurred independently in Australia and Europe from different progenitor viruses. Although this is the canonical study of the evolution of virulence, whether the genomic and phenotypic outcomes of MYXV evolution in Europe mirror those observed in Australia is unknown. We addressed this question using viruses isolated in the United Kingdom early in the MYXV epizootic (1954-1955) and between 2008-2013. The later UK viruses fell into three distinct lineages indicative of a long period of separation and independent evolution. Although rates of evolutionary change were almost identical to those previously described for MYXV in Australia and strongly clock-like, genome evolution in the UK and Australia showed little convergence. The phenotypes of eight UK viruses from three lineages were characterized in laboratory rabbits and compared to the progenitor (release) Lausanne strain. Inferred virulence ranged from highly virulent (grade 1) to highly attenuated (grade 5). Two broad disease types were seen: cutaneous nodular myxomatosis characterized by multiple raised secondary cutaneous lesions, or an amyxomatous phenotype with few or no secondary lesions. A novel clinical outcome was acute death with pulmonary oedema and haemorrhage, often associated with bacteria in many tissues but an absence of inflammatory cells. Notably, reading frame disruptions in genes defined as essential for virulence in the progenitor Lausanne strain were compatible with the acquisition of high virulence. Combined, these data support a model of ongoing host-pathogen co-evolution in which multiple genetic pathways can produce successful outcomes in the field that involve both different virulence grades and disease phenotypes, with alterations in tissue tropism and disease mechanisms.

  9. Targeting of apoptotic pathways by SMAC or BH3 mimetics distinctly sensitizes paclitaxel-resistant triple negative breast cancer cells.

    PubMed

    Panayotopoulou, Effrosini G; Müller, Anna-Katharina; Börries, Melanie; Busch, Hauke; Hu, Guohong; Lev, Sima

    2017-02-06

    Standard chemotherapy is the only systemic treatment for triple-negative breast cancer (TNBC), and despite the good initial response, resistance remains a major therapeutic obstacle. Here, we employed a High-Throughput Screen to identify targeted therapies that overcome chemoresistance in TNBC. We applied short-term paclitaxel treatment and screened 320 small-molecule inhibitors of known targets to identify drugs that preferentially and efficiently target paclitaxel-treated TNBC cells. Among these compounds the SMAC mimetics (BV6, Birinapant) and BH3-mimetics (ABT-737/263) were recognized as potent targeted therapy for multiple paclitaxel-residual TNBC cell lines. However, acquired paclitaxel resistance through repeated paclitaxel pulses result in desensitization to BV6, but not to ABT-263, suggesting that short- and long-term paclitaxel resistance are mediated by distinct mechanisms. Gene expression profiling of paclitaxel-residual, -resistant and naïve MDA-MB-231 cells demonstrated that paclitaxel-residual, as opposed to -resistant cells, were characterized by an apoptotic signature, with downregulation of anti-apoptotic genes (BCL2, BIRC5), induction of apoptosis inducers (IL24, PDCD4), and enrichment of TNFα/NF-κB pathway, including upregulation of TNFSF15, coupled with cell-cycle arrest. BIRC5 and FOXM1 downregulation and IL24 induction was also evident in breast cancer patient datasets following taxane treatment. Exposure of naïve or paclitaxel-resistant cells to supernatants of paclitaxel-residual cells sensitized them to BV6, and treatment with TNFα enhanced BV6 potency, suggesting that sensitization to BV6 is mediated, at least partially, by secreted factor(s). Our results suggest that administration of SMAC or BH3 mimetics following short-term paclitaxel treatment could be an effective therapeutic strategy for TNBC, while only BH3-mimetics could effectively overcome long-term paclitaxel resistance.

  10. Membrane lipid domains distinct from cholesterol/sphingomyelin-rich rafts are involved in the ABCA1-mediated lipid secretory pathway.

    PubMed

    Mendez, A J; Lin, G; Wade, D P; Lawn, R M; Oram, J F

    2001-02-02

    Efflux of excess cellular cholesterol mediated by lipid-poor apolipoproteins occurs by an active mechanism distinct from passive diffusion and is controlled by the ATP-binding cassette transporter ABCA1. Here we examined whether ABCA1-mediated lipid efflux involves the selective removal of lipids associated with membrane rafts, plasma membrane domains enriched in cholesterol and sphingomyelin. ABCA1 was not associated with cholesterol and sphingolipid-rich membrane raft domains based on detergent solubility and lack of colocalization with marker proteins associated with raft domains. Lipid efflux to apoA-I was accounted for by decreases in cellular lipids not associated with cholesterol/sphingomyelin-rich membranes. Treating cells with filipin, to disrupt raft structure, or with sphingomyelinase, to digest plasma membrane sphingomyelin, did not impair apoA-I-mediated cholesterol or phosphatidylcholine efflux. In contrast, efflux of cholesterol to high density lipoproteins (HDL) or plasma was partially accounted for by depletion of cholesterol from membrane rafts. Additionally, HDL-mediated cholesterol efflux was partially inhibited by filipin and sphingomyelinase treatment. Apo-A-I-mediated cholesterol efflux was absent from fibroblasts with nonfunctional ABCA1 (Tangier disease cells), despite near normal amounts of cholesterol associated with raft domains and normal abilities of plasma and HDL to deplete cholesterol from these domains. Thus, the involvement of membrane rafts in cholesterol efflux applies to lipidated HDL particles but not to lipid-free apoA-I. We conclude that cholesterol and sphingomyelin-rich membrane rafts do not provide lipid for efflux promoted by apolipoproteins through the ABCA1-mediated lipid secretory pathway and that ABCA1 is not associated with these domains.

  11. Genomic and phenotypic characterization of myxoma virus from Great Britain reveals multiple evolutionary pathways distinct from those in Australia

    PubMed Central

    Kerr, Peter J.; Cattadori, Isabella M.; Fitch, Adam; Geber, Adam; Liu, June; Sim, Derek G.; Boag, Brian; Ghedin, Elodie

    2017-01-01

    The co-evolution of myxoma virus (MYXV) and the European rabbit occurred independently in Australia and Europe from different progenitor viruses. Although this is the canonical study of the evolution of virulence, whether the genomic and phenotypic outcomes of MYXV evolution in Europe mirror those observed in Australia is unknown. We addressed this question using viruses isolated in the United Kingdom early in the MYXV epizootic (1954–1955) and between 2008–2013. The later UK viruses fell into three distinct lineages indicative of a long period of separation and independent evolution. Although rates of evolutionary change were almost identical to those previously described for MYXV in Australia and strongly clock-like, genome evolution in the UK and Australia showed little convergence. The phenotypes of eight UK viruses from three lineages were characterized in laboratory rabbits and compared to the progenitor (release) Lausanne strain. Inferred virulence ranged from highly virulent (grade 1) to highly attenuated (grade 5). Two broad disease types were seen: cutaneous nodular myxomatosis characterized by multiple raised secondary cutaneous lesions, or an amyxomatous phenotype with few or no secondary lesions. A novel clinical outcome was acute death with pulmonary oedema and haemorrhage, often associated with bacteria in many tissues but an absence of inflammatory cells. Notably, reading frame disruptions in genes defined as essential for virulence in the progenitor Lausanne strain were compatible with the acquisition of high virulence. Combined, these data support a model of ongoing host-pathogen co-evolution in which multiple genetic pathways can produce successful outcomes in the field that involve both different virulence grades and disease phenotypes, with alterations in tissue tropism and disease mechanisms. PMID:28253375

  12. Quantitative Characterization of the Activation Steps of Mannan-binding Lectin (MBL)-associated Serine Proteases (MASPs) Points to the Central Role of MASP-1 in the Initiation of the Complement Lectin Pathway*

    PubMed Central

    Megyeri, Márton; Harmat, Veronika; Major, Balázs; Végh, Ádám; Balczer, Júlia; Héja, Dávid; Szilágyi, Katalin; Datz, Dániel; Pál, Gábor; Závodszky, Péter; Gál, Péter; Dobó, József

    2013-01-01

    Mannan-binding lectin (MBL)-associated serine proteases, MASP-1 and MASP-2, have been thought to autoactivate when MBL/ficolin·MASP complexes bind to pathogens triggering the complement lectin pathway. Autoactivation of MASPs occurs in two steps: 1) zymogen autoactivation, when one proenzyme cleaves another proenzyme molecule of the same protease, and 2) autocatalytic activation, when the activated protease cleaves its own zymogen. Using recombinant catalytic fragments, we demonstrated that a stable proenzyme MASP-1 variant (R448Q) cleaved the inactive, catalytic site Ser-to-Ala variant (S646A). The autoactivation steps of MASP-1 were separately quantified using these mutants and the wild type enzyme. Analogous mutants were made for MASP-2, and rate constants of the autoactivation steps as well as the possible cross-activation steps between MASP-1 and MASP-2 were determined. Based on the rate constants, a kinetic model of lectin pathway activation was outlined. The zymogen autoactivation rate of MASP-1 is ∼3000-fold higher, and the autocatalytic activation of MASP-1 is about 140-fold faster than those of MASP-2. Moreover, both activated and proenzyme MASP-1 can effectively cleave proenzyme MASP-2. MASP-3, which does not autoactivate, is also cleaved by MASP-1 quite efficiently. The structure of the catalytic region of proenzyme MASP-1 R448Q was solved at 2.5 Å. Proenzyme MASP-1 R448Q readily cleaves synthetic substrates, and it is inhibited by a specific canonical inhibitor developed against active MASP-1, indicating that zymogen MASP-1 fluctuates between an inactive and an active-like conformation. The determined structure provides a feasible explanation for this phenomenon. In summary, autoactivation of MASP-1 is crucial for the activation of MBL/ficolin·MASP complexes, and in the proenzymic phase zymogen MASP-1 controls the process. PMID:23386610

  13. Electrophysiological evidence for distinct vagal pathways mediating CCK-evoked motor effects in the proximal versus distal stomach

    PubMed Central

    Okano-Matsumoto, Shiho; McRoberts, James A; Taché, Yvette; Adelson, David W

    2011-01-01

    Intravenous cholecystokinin octapeptide (CCK-8) elicits vago-vagal reflexes that inhibit phasic gastric contractions and reduce gastric tone in urethane-anaesthetized rats. A discrete proximal subdivision of the ventral gastric vagus nerve (pVGV) innervates the proximal stomach, but the fibre populations within it have not been characterized previously. We hypothesized that i.v. CCK-8 injection would excite inhibitory efferent outflow in the pVGV, in contrast to its inhibitory effect on excitatory efferent outflow in the distal subdivision (dVGV), which supplies the distal stomach. In each VGV subdivision, a dual-recording technique was used to record afferent and efferent activity simultaneously, while also monitoring intragastric pressure (IGP). CCK-8 dose dependently (100–1000 pmol kg−1, i.v.) reduced gastric tone, gastric contractile activity and multi-unit dVGV efferent discharge, but increased pVGV efferent firing. Single-unit analysis revealed a minority of efferent fibres in each branch whose response differed in direction from the bulk response. Unexpectedly, efferent excitation in the pVGV was significantly shorter lived and had a significantly shorter decay half-time than did efferent inhibition in the dVGV, indicating that distinct pathways drive CCK-evoked outflow to the proximal vs. the distal stomach. Efferent inhibition in the dVGV began several seconds before, and persisted significantly longer than, simultaneously recorded dVGV afferent excitation. Thus, dVGV afferent excitation could not account for the pattern of dVGV efferent inhibition. However, the time course of dVGV afferent excitation paralleled that of pVGV efferent excitation. Similarly, the duration of CCK-8-evoked afferent responses recorded in the accessory celiac branch of the vagus (ACV) matched the duration of dVGV efferent responses. The observed temporal relationships suggest that postprandial effects on gastric complicance of CCK released from intestinal endocrine cells may

  14. Ca(2+) and H+ homeostasis in fission yeast: a role of Ca(2+)/H+ exchange and distinct V-H+-ATPases of the secretory pathway organelles.

    PubMed

    Okorokov, L A; Silva, F E; Okorokova Façanha, A L

    2001-09-14

    We determined the H+ and Ca(2+) uptake by fission yeast membranes separated on sucrose gradient and found that (i) Ca(2+) sequestering is due to Ca(2+)/H+ antiporter(s) localized to secretory pathway organelles while Ca(2+)-ATPase activity is not detectable in their membranes; (ii) immunochemically distinct V-H+-ATPases acidify the lumen of the secretory pathway organelles. The data indicate that the endoplasmic reticulum, Golgi and vacuole form a network of Ca(2+) and H+ stores in the single cell, providing favorable conditions for such key processes as protein folding/sorting, membrane fusion, ion homeostasis and Ca(2+) signaling in a differential and local manner.

  15. Identification of a mutant locus that bypasses the BsgA protease requirement for social development in Myxococcus xanthus.

    PubMed

    Cusick, John K; Hager, Elizabeth; Gill, Ronald E

    2015-01-01

    The BsgA protease is required for the earliest morphological changes observed in Myxococcus xanthus development. We hypothesize that the BsgA protease is required to cleave an inhibitor of the developmental program, and isolation of genetic bypass suppressors of a bsgA mutant was used to identify signaling components controlling development downstream of the BsgA protease. Strain M955 was created by transposon mutagenesis of a bsgA mutant followed by screening for strains that could develop despite the absence of the BsgA protease. Strain M955 was able to aggregate, form fruiting bodies, and partially restored the production of viable spores in comparison to the parental bsgA mutant. The bsgA Tn5Ω955 strain partially restored developmental expression to a subset of genes normally induced during development, and expressed one developmentally induced fusion at higher amounts during vegetative growth in comparison to wild-type cells. The transposon in strain M955 was localized to a Ribonuclease D homolog that appears to exist in an operon with a downstream aminopeptidase-encoding gene. The identification of a third distinct bypass suppressor of the BsgA protease suggests that the BsgA protease may regulate a potentially complex pathway during the initiation of the M. xanthus developmental program.

  16. Investigations with Protease.

    ERIC Educational Resources Information Center

    Yip, Din Yan

    1997-01-01

    Presents two simple and reliable ways for measuring protease activity that can be used for a variety of investigations in a range of biology class levels. The investigations use protease from a variety of sources. (DDR)

  17. Investigations with Protease.

    ERIC Educational Resources Information Center

    Yip, Din Yan

    1997-01-01

    Presents two simple and reliable ways for measuring protease activity that can be used for a variety of investigations in a range of biology class levels. The investigations use protease from a variety of sources. (DDR)

  18. Distinct growth factor-induced dynamic mass redistribution (DMR) profiles for monitoring oncogenic signaling pathways in various cancer cells.

    PubMed

    Du, Yuhong; Li, Zijian; Li, Lian; Chen, Zhuo Georgia; Sun, Shi-Yong; Chen, Peifang; Shin, Dong M; Khuri, Fadlo R; Fu, Haian

    2009-01-01

    Targeting dysregulated signaling pathways in tumors has led to the development of a novel class of signal transduction inhibitors, including inhibitors of the epidermal growth factor (EGF) receptor (EGFR). To dissect oncogenic pathways, identify key pathway determinants, and evaluate the efficacy of targeted agents, it is vital to develop technologies that allow the detection of temporal signaling events under physiological conditions. Here we report the application of a label-free optical biosensor to reveal the rapid response of cancer cells to EGF, expressed as a dynamic mass redistribution (DMR) signal. In response to EGF, squamous cell carcinoma of the head and neck cells exhibited a rapid rise in DMR signal, whereas lung adenocarcinoma cells showed a biphasic DMR profile, suggesting a cell type-dependent DMR response. Pharmacological studies suggested the importance of EGFR and the phosphatidylinositol-3 kinase pathway in mediating the EGF-induced DMR response. The defined DMR signatures offer a simple yet sensitive tool for evaluating EGFR-targeted agents, as shown with gefitinib and erlotinib. The assay can also be used for cell-based high-throughput screening of EGF pathway inhibitors, as demonstrated by its robust performance in a 384-well plate format (Z' > 0.5). This technology is applicable to other oncogenic pathways for the discovery of novel therapeutic agents for the treatment of various cancers.

  19. Protease and protease inhibitory activity in pregnant and postpartum involuting uterus

    SciTech Connect

    Milwidsky, A.; Beller, U.; Palti, Z.; Mayer, M.

    1982-08-15

    The presence of two distinct proteolytic activities in the rat uterus was confirmed with /sup 14/C-labeled globin used as a sensitive protein substrate and following release of label into the trichloroacetic acid-soluble supernatant fraction. Protease I is a cytoplasmic acid protease while protease II is associated with the pellet fraction, can be extracted by 0.6 M sodium chloride, and is active at pH 7.0. Protease I activity is low during pregnancy and markedly increases at term achieving maximal activity at day 3 post partum with a subsequent decline to preterm activity values. Lactation did not affect the uterine protease I activity. Protease II activity is not significantly different during pregnancy, at term, and post partum. The presence of an inhibitor of protease I was suggested by a decrease in enzyme activity with an increased cytosolic protein concentration. The inhibitor also lessened bovine trypsin activity but had no effect on protease II. Although its inhibitory potency on trypsin fluctuated during the various uterine physiologic stages, these changes appeared to be statistically insignificant. Human uterine samples were also found to contain the two protease activities with similar changes in protease I post partum. It is suggested that, both in the rat and in man, uterine involution post partum is associated with a marked increase in activity of acid cytosolic protease, while a particulate neutral protease and a soluble inhibitor of trypsin, which are also present in uterine cells, do not appear to play a significant role in the dissolution of uterine tissues after parturition.

  20. Experimentally-Derived Fibroblast Gene Signatures Identify Molecular Pathways Associated with Distinct Subsets of Systemic Sclerosis Patients in Three Independent Cohorts

    PubMed Central

    Johnson, Michael E.; Mahoney, J. Matthew; Taroni, Jaclyn; Sargent, Jennifer L.; Marmarelis, Eleni; Wu, Ming-Ru; Varga, John; Hinchcliff, Monique E.; Whitfield, Michael L.

    2015-01-01

    Genome-wide expression profiling in systemic sclerosis (SSc) has identified four ‘intrinsic’ subsets of disease (fibroproliferative, inflammatory, limited, and normal-like), each of which shows deregulation of distinct signaling pathways; however, the full set of pathways contributing to this differential gene expression has not been fully elucidated. Here we examine experimentally derived gene expression signatures in dermal fibroblasts for thirteen different signaling pathways implicated in SSc pathogenesis. These data show distinct and overlapping sets of genes induced by each pathway, allowing for a better understanding of the molecular relationship between profibrotic and immune signaling networks. Pathway-specific gene signatures were analyzed across a compendium of microarray datasets consisting of skin biopsies from three independent cohorts representing 80 SSc patients, 4 morphea, and 26 controls. IFNα signaling showed a strong association with early disease, while TGFβ signaling spanned the fibroproliferative and inflammatory subsets, was associated with worse MRSS, and was higher in lesional than non-lesional skin. The fibroproliferative subset was most strongly associated with PDGF signaling, while the inflammatory subset demonstrated strong activation of innate immune pathways including TLR signaling upstream of NF-κB. The limited and normal-like subsets did not show associations with fibrotic and inflammatory mediators such as TGFβ and TNFα. The normal-like subset showed high expression of genes associated with lipid signaling, which was absent in the inflammatory and limited subsets. Together, these data suggest a model by which IFNα is involved in early disease pathology, and disease severity is associated with active TGFβ signaling. PMID:25607805

  1. A Multifunctional Protease Inhibitor To Regulate Endolysosomal Function

    PubMed Central

    2011-01-01

    Proteases constitute a major class of drug targets. Endosomal compartments harbor several protease families whose attenuation may be beneficial to a number of biological processes, including inflammation, cancer metastasis, antigen presentation, and parasite clearance. As a step toward the goal of generalized but targeted protease inhibition in the endocytic pathway, we describe here the synthesis, characterization, and cellular application of a novel multifunctional protease inhibitor. We show that pepstatin A, a potent but virtually insoluble inhibitor of cathepsins D and E, can be conjugated to a single site on cystatin C, a potent inhibitor of the papain-like cysteine proteases (PLCP) and of asparagine endopeptidease (AEP), to create a highly soluble compound capable of suppressing the activity of all 3 principal protease families found in endosomes and lysosomes. We demonstrate that this cystatin–pepstatin inhibitor (CPI) can be taken up by cells to modulate protease activity and affect biological responses. PMID:21910425

  2. A multifunctional protease inhibitor to regulate endolysosomal function.

    PubMed

    van Kasteren, Sander I; Berlin, Ilana; Colbert, Jeff D; Keane, Doreen; Ovaa, Huib; Watts, Colin

    2011-11-18

    Proteases constitute a major class of drug targets. Endosomal compartments harbor several protease families whose attenuation may be beneficial to a number of biological processes, including inflammation, cancer metastasis, antigen presentation, and parasite clearance. As a step toward the goal of generalized but targeted protease inhibition in the endocytic pathway, we describe here the synthesis, characterization, and cellular application of a novel multifunctional protease inhibitor. We show that pepstatin A, a potent but virtually insoluble inhibitor of cathepsins D and E, can be conjugated to a single site on cystatin C, a potent inhibitor of the papain-like cysteine proteases (PLCP) and of asparagine endopeptidease (AEP), to create a highly soluble compound capable of suppressing the activity of all 3 principal protease families found in endosomes and lysosomes. We demonstrate that this cystatin-pepstatin inhibitor (CPI) can be taken up by cells to modulate protease activity and affect biological responses.

  3. A preliminary neutron diffraction analysis of Achromobacter protease I

    NASA Astrophysics Data System (ADS)

    Ohnishi, Yuki; Masaki, Takeharu; Yamada, Taro; Kurihara, Kazuo; Tanaka, Ichiro; Niimura, Nobuo

    2010-11-01

    Achromobacter protease I (API, E.C. 3.4.21.50) is one of the serine proteases produced by Achromobacter lyticus M497-1. API is distinct from the other tripsin type protease in its lysine specificity. The neutron structure analysis of catalytic triad with Trp169 and His210 was presented. His57 was double protonated and formed hydrogen bonds to Ser194Oγ and Asp113Oδ1, Oδ2.

  4. A minimal dose of electrically induced muscle activity regulates distinct gene signaling pathways in humans with spinal cord injury.

    PubMed

    Petrie, Michael A; Suneja, Manish; Faidley, Elizabeth; Shields, Richard K

    2014-01-01

    Paralysis after a spinal cord injury (SCI) induces physiological adaptations that compromise the musculoskeletal and metabolic systems. Unlike non-SCI individuals, people with spinal cord injury experience minimal muscle activity which compromises optimal glucose utilization and metabolic control. Acute or chronic muscle activity, induced through electrical stimulation, may regulate key genes that enhance oxidative metabolism in paralyzed muscle. We investigated the short and long term effects of electrically induced exercise on mRNA expression of human paralyzed muscle. We developed an exercise dose that activated the muscle for only 0.6% of the day. The short term effects were assessed 3 hours after a single dose of exercise, while the long term effects were assessed after training 5 days per week for at least one year (adherence 81%). We found a single dose of exercise regulated 117 biological pathways as compared to 35 pathways after one year of training. A single dose of electrical stimulation increased the mRNA expression of transcriptional, translational, and enzyme regulators of metabolism important to shift muscle toward an oxidative phenotype (PGC-1α, NR4A3, IFRD1, ABRA, PDK4). However, chronic training increased the mRNA expression of specific metabolic pathway genes (BRP44, BRP44L, SDHB, ACADVL), mitochondrial fission and fusion genes (MFF, MFN1, MFN2), and slow muscle fiber genes (MYH6, MYH7, MYL3, MYL2). These findings support that a dose of electrical stimulation (∼10 minutes/day) regulates metabolic gene signaling pathways in human paralyzed muscle. Regulating these pathways early after SCI may contribute to reducing diabetes in people with longstanding paralysis from SCI.

  5. A Minimal Dose of Electrically Induced Muscle Activity Regulates Distinct Gene Signaling Pathways in Humans with Spinal Cord Injury

    PubMed Central

    Petrie, Michael A.; Suneja, Manish; Faidley, Elizabeth; Shields, Richard K.

    2014-01-01

    Paralysis after a spinal cord injury (SCI) induces physiological adaptations that compromise the musculoskeletal and metabolic systems. Unlike non-SCI individuals, people with spinal cord injury experience minimal muscle activity which compromises optimal glucose utilization and metabolic control. Acute or chronic muscle activity, induced through electrical stimulation, may regulate key genes that enhance oxidative metabolism in paralyzed muscle. We investigated the short and long term effects of electrically induced exercise on mRNA expression of human paralyzed muscle. We developed an exercise dose that activated the muscle for only 0.6% of the day. The short term effects were assessed 3 hours after a single dose of exercise, while the long term effects were assessed after training 5 days per week for at least one year (adherence 81%). We found a single dose of exercise regulated 117 biological pathways as compared to 35 pathways after one year of training. A single dose of electrical stimulation increased the mRNA expression of transcriptional, translational, and enzyme regulators of metabolism important to shift muscle toward an oxidative phenotype (PGC-1α, NR4A3, IFRD1, ABRA, PDK4). However, chronic training increased the mRNA expression of specific metabolic pathway genes (BRP44, BRP44L, SDHB, ACADVL), mitochondrial fission and fusion genes (MFF, MFN1, MFN2), and slow muscle fiber genes (MYH6, MYH7, MYL3, MYL2). These findings support that a dose of electrical stimulation (∼10 minutes/day) regulates metabolic gene signaling pathways in human paralyzed muscle. Regulating these pathways early after SCI may contribute to reducing diabetes in people with longstanding paralysis from SCI. PMID:25531450

  6. Distinct development of peripheral trigeminal pathways in the platypus (Ornithorhynchus anatinus) and short-beaked echidna (Tachyglossus aculeatus).

    PubMed

    Ashwell, Ken W S; Hardman, Craig D; Giere, Peter

    2012-01-01

    The extant monotremes (platypus and echidnas) are believed to all be capable of electroreception in the trigeminal pathways, although they differ significantly in the number and distribution of electroreceptors. It has been argued by some authors that electroreception was first developed in an aquatic environment and that echidnas are descended from a platypus-like ancestor that invaded an available terrestrial habitat. If this were the case, one would expect the developmental trajectories of the trigeminal pathways to be similar in the early stages of platypus and short-beaked echidna development, with structural divergence occurring later. We examined the development of the peripheral trigeminal pathway from snout skin to trigeminal ganglion in sectioned material in the Hill and Hubrecht collections to test for similarities and differences between the two during the development from egg to adulthood. Each monotreme showed a characteristic and different pattern of distribution of developing epidermal sensory gland specializations (electroreceptor primordia) from the time of hatching. The cross-sectional areas of the trigeminal divisions and the volume of the trigeminal ganglion itself were also very different between the two species at embryonic ages, and remained consistently different throughout post-hatching development. Our findings indicate that the trigeminal pathways in the short-beaked echidna and the platypus follow very different developmental trajectories from the earliest ages. These findings are more consistent with the notion that the platypus and echidna have both diverged from an ancestor with rudimentary electroreception and/or trigeminal specialization, rather than the contention that the echidna is derived from a platypus-like ancestor. Copyright © 2011 S. Karger AG, Basel.

  7. Gene Expression Profile of Adult Human Olfactory Bulb and Embryonic Neural Stem Cell Suggests Distinct Signaling Pathways and Epigenetic Control

    PubMed Central

    Marei, Hany E. S.; Ahmed, Abd-Elmaksoud; Michetti, Fabrizio; Pescatori, Mario; Pallini, Roberto; Casalbore, Patricia; Cenciarelli, Carlo; Elhadidy, Mohamed

    2012-01-01

    Global gene expression profiling was performed using RNA from human embryonic neural stem cells (hENSC), and adult human olfactory bulb-derived neural stem cells (OBNSCs), to define a gene expression pattern and signaling pathways that are specific for each cell lineage. We have demonstrated large differences in the gene expression profile of human embryonic NSC, and adult human OBNSCs, but less variability between parallel cultures. Transcripts of genes involved in neural tube development and patterning (ALDH1A2, FOXA2), progenitor marker genes (LMX1a, ALDH1A1, SOX10), proliferation of neural progenitors (WNT1 and WNT3a), neuroplastin (NPTN), POU3F1 (OCT6), neuroligin (NLGN4X), MEIS2, and NPAS1 were up-regulated in both cell populations. By Gene Ontology, 325 out of 3875 investigated gene sets were scientifically different. 41 out of the 307 investigated Cellular Component (CC) categories, 45 out of the 620 investigated Molecular Function (MF) categories, and 239 out of the 2948 investigated Biological Process (BP) categories were significant. KEGG Pathway Class Comparison had revealed that 75 out of 171 investigated gene sets passed the 0.005 significance threshold. Levels of gene expression were explored in three signaling pathways, Notch, Wnt, and mTOR that are known to be involved in NS cell fates determination. The transcriptional signature also deciphers the role of genes involved in epigenetic modifications. SWI/SNF DNA chromatin remodeling complex family, including SMARCC1 and SMARCE1, were found specifically up-regulated in our OBNSC but not in hENSC. Differences in gene expression profile of transcripts controlling epigenetic modifications, and signaling pathways might indicate differences in the therapeutic potential of our examined two cell populations in relation to in cell survival, proliferation, migration, and differentiation following engraftments in different CNS insults. PMID:22485144

  8. p53 and ATF4 mediate distinct and additive pathways to skeletal muscle atrophy during limb immobilization

    PubMed Central

    Fox, Daniel K.; Ebert, Scott M.; Bongers, Kale S.; Dyle, Michael C.; Bullard, Steven A.; Dierdorff, Jason M.; Kunkel, Steven D.

    2014-01-01

    Immobilization causes skeletal muscle atrophy via complex signaling pathways that are not well understood. To better understand these pathways, we investigated the roles of p53 and ATF4, two transcription factors that mediate adaptations to a variety of cellular stresses. Using mouse models, we demonstrate that 3 days of muscle immobilization induces muscle atrophy and increases expression of p53 and ATF4. Furthermore, muscle fibers lacking p53 or ATF4 are partially resistant to immobilization-induced muscle atrophy, and forced expression of p53 or ATF4 induces muscle fiber atrophy in the absence of immobilization. Importantly, however, p53 and ATF4 do not require each other to promote atrophy, and coexpression of p53 and ATF4 induces more atrophy than either transcription factor alone. Moreover, muscle fibers lacking both p53 and ATF4 are more resistant to immobilization-induced atrophy than fibers lacking only p53 or ATF4. Interestingly, the independent and additive nature of the p53 and ATF4 pathways allows for combinatorial control of at least one downstream effector, p21. Using genome-wide mRNA expression arrays, we identified p21 mRNA as a skeletal muscle transcript that is highly induced in immobilized muscle via the combined actions of p53 and ATF4. Additionally, in mouse muscle, p21 induces atrophy in a manner that does not require immobilization, p53 or ATF4, and p21 is required for atrophy induced by immobilization, p53, and ATF4. Collectively, these results identify p53 and ATF4 as essential and complementary mediators of immobilization-induced muscle atrophy and discover p21 as a critical downstream effector of the p53 and ATF4 pathways. PMID:24895282

  9. Unique and shared signaling pathways cooperate to regulate the differentiation of human CD4+ T cells into distinct effector subsets.

    PubMed

    Ma, Cindy S; Wong, Natalie; Rao, Geetha; Nguyen, Akira; Avery, Danielle T; Payne, Kathryn; Torpy, James; O'Young, Patrick; Deenick, Elissa; Bustamante, Jacinta; Puel, Anne; Okada, Satoshi; Kobayashi, Masao; Martinez-Barricarte, Ruben; Elliott, Michael; Sebnem Kilic, Sara; El Baghdadi, Jamila; Minegishi, Yoshiyuki; Bousfiha, Aziz; Robertson, Nic; Hambleton, Sophie; Arkwright, Peter D; French, Martyn; Blincoe, Annaliesse K; Hsu, Peter; Campbell, Dianne E; Stormon, Michael O; Wong, Melanie; Adelstein, Stephen; Fulcher, David A; Cook, Matthew C; Stepensky, Polina; Boztug, Kaan; Beier, Rita; Ikincioğullari, Aydan; Ziegler, John B; Gray, Paul; Picard, Capucine; Boisson-Dupuis, Stéphanie; Phan, Tri Giang; Grimbacher, Bodo; Warnatz, Klaus; Holland, Steven M; Uzel, Gulbu; Casanova, Jean-Laurent; Tangye, Stuart G

    2016-07-25

    Naive CD4(+) T cells differentiate into specific effector subsets-Th1, Th2, Th17, and T follicular helper (Tfh)-that provide immunity against pathogen infection. The signaling pathways involved in generating these effector cells are partially known. However, the effects of mutations underlying human primary immunodeficiencies on these processes, and how they compromise specific immune responses, remain unresolved. By studying individuals with mutations in key signaling pathways, we identified nonredundant pathways regulating human CD4(+) T cell differentiation in vitro. IL12Rβ1/TYK2 and IFN-γR/STAT1 function in a feed-forward loop to induce Th1 cells, whereas IL-21/IL-21R/STAT3 signaling is required for Th17, Tfh, and IL-10-secreting cells. IL12Rβ1/TYK2 and NEMO are also required for Th17 induction. Strikingly, gain-of-function STAT1 mutations recapitulated the impact of dominant-negative STAT3 mutations on Tfh and Th17 cells, revealing a putative inhibitory effect of hypermorphic STAT1 over STAT3. These findings provide mechanistic insight into the requirements for human T cell effector function, and explain clinical manifestations of these immunodeficient conditions. Furthermore, they identify molecules that could be targeted to modulate CD4(+) T cell effector function in the settings of infection, vaccination, or immune dysregulation. © 2016 Ma et al.

  10. Distinct spatial activation of intrinsic and extrinsic apoptosis pathways in natural scrapie: association with prion-related lesions

    PubMed Central

    Serrano, Carmen; Lyahyai, Jaber; Bolea, Rosa; Varona, Luis; Monleón, Eva; Badiola, Juan J.; Zaragoza, Pilar; Martín-Burriel, Inmaculada

    2009-01-01

    Neurodegeneration and gliosis are the main neuropathological features of prion diseases. However, the molecular mechanisms involved in these processes remain unclear. Several studies have demonstrated changes in the expression of apoptotic factors and inflammatory cytokines in animals with experimental infection. Here we present the expression profiles of 15 genes implicated in the intrinsic and extrinsic apoptotic pathways in the central nervous systems of sheep naturally infected with scrapie. Expression changes obtained by real-time RT-PCR were also compared with the extent of classical scrapie lesions, such as prion deposition, neuronal vacuolisation, spongiosis, and astrogliosis as well as with the activation of caspase-3, using a stepwise regression. The results suggest that the factors assessed participate in apoptotic or inflammatory functions, depending on the affected area. The mitochondrial apoptosis pathway was associated with prion deposition in the prefrontal cortex (the less affected area), and with activation of caspase-3-mediated cell death via over-expression of BAK. In addition to its known association with astroglial activation, the extrinsic apoptosis pathway was also related to cell death and neuronal vacuolisation. PMID:19401142

  11. Distinct photoproducts of hydroxylated polybromodiphenyl ethers from different photodegradation pathways: a case study of 2'-HO-BDE-68.

    PubMed

    Xie, Qing; Chen, Jingwen; Zhao, Hongxia; Wang, Xingbao; Xie, Hong-Bin

    2015-02-01

    Hydroxylated polyhalodiphenyl ethers (HO-PXDEs) are emerging aquatic pollutants. Previous studies have shown that HO-PXDEs can photogenerate dioxins and phenolic compounds. However, it is unclear which photochemical pathways are responsible for the various photoproducts. This study investigates the direct photolysis and photooxidation initiated by (1)O2 and ˙OH that can be formed by photosensitization, taking 2'-HO-2,3',4,5'-tetrabromodiphenyl ether (2'-HO-BDE-68) as a case study. The results show that 1,3,8-tribromodibenzo-p-dioxin can only be produced during direct photolysis. By mass spectrum analysis, four dihydroxylated polybromodiphenyl ethers, generated from both direct and indirect photodegradation were confirmed. Among them, di-HO-tribromodiphenyl ether (di-HO-TBDE) was the main product generated from direct photohydrolysis. Most probably, the di-HO-TBDE is 2',5'-HO-2,3',4-tribromodiphenyl ether, as was suggested by density functional theory calculations. Ether bond cleavage is a dominant pathway for the direct photolysis and photooxidation reactions leading to 2,4-dibromophenol as the dominant product. The yields of the products, which are irrespective of reaction time and can be employed to compare the ability of different HO-PXDEs to photogenerate a given product, were reported. This study indicates that for accurate ecological risk assessment of HO-PXDEs, their different photodegradation pathways that may lead to different photoproducts should be considered.

  12. Adaptin-like protein TPLATE and clathrin recruitment during plant somatic cytokinesis occurs via two distinct pathways

    PubMed Central

    Van Damme, Daniël; Gadeyne, Astrid; Vanstraelen, Marleen; Inzé, Dirk; Van Montagu, Marc C. E.; De Jaeger, Geert; Russinova, Eugenia; Geelen, Danny

    2011-01-01

    Plant cytokinesis deploys a transport system that centers cell plate-forming vesicles and fuses them to form a cell plate. Here we show that the adaptin-like protein TPLATE and clathrin light chain 2 (CLC2) are targeted to the expanding cell plate and to the equatorial subregion of the plasma membrane referred to as the cortical division zone (CDZ). Bimolecular fluorescence complementation and immunodetection indicates that TPLATE interacts with clathrin. Pharmacological tools as well as analysis of protein targeting in a mutant background affecting cell plate formation allowed to discriminate two recruitment pathways for TPLATE and CLC2. The cell plate recruitment pathway is dependent on phragmoplast microtubule organization and the formation and transport of secretory vesicles. The CDZ recruitment pathway, on the other hand, is activated at the end of cytokinesis and independent of trans-Golgi–derived vesicle trafficking. TPLATE and CLC2 do not accumulate at a narrow zone central of the CDZ. We have dubbed this subdomain the cortical division site and show that it corresponds precisely with the position where the cell plate merges with the parental wall. These data provide evidence that the plasma membrane is subject to localized endocytosis or membrane remodeling processes that are required for the fusion of the cell plate with a predefined region of the plasma membrane. PMID:21187379

  13. Unique and shared signaling pathways cooperate to regulate the differentiation of human CD4+ T cells into distinct effector subsets

    PubMed Central

    Wong, Natalie; Rao, Geetha; Nguyen, Akira; Avery, Danielle T.; Payne, Kathryn; Torpy, James; O’Young, Patrick; Deenick, Elissa; Bustamante, Jacinta; Puel, Anne; Okada, Satoshi; Kobayashi, Masao; Martinez-Barricarte, Ruben; Elliott, Michael; Sebnem Kilic, Sara; El Baghdadi, Jamila; Minegishi, Yoshiyuki; Bousfiha, Aziz; Robertson, Nic; Hambleton, Sophie; Arkwright, Peter D.; French, Martyn; Blincoe, Annaliesse K.; Hsu, Peter; Campbell, Dianne E.; Stormon, Michael O.; Wong, Melanie; Adelstein, Stephen; Fulcher, David A.; Cook, Matthew C.; Stepensky, Polina; Boztug, Kaan; Beier, Rita; Ikincioğullari, Aydan; Ziegler, John B.; Gray, Paul; Picard, Capucine; Boisson-Dupuis, Stéphanie; Phan, Tri Giang; Grimbacher, Bodo; Warnatz, Klaus; Holland, Steven M.; Uzel, Gulbu; Casanova, Jean-Laurent; Tangye, Stuart G.

    2016-01-01

    Naive CD4+ T cells differentiate into specific effector subsets—Th1, Th2, Th17, and T follicular helper (Tfh)—that provide immunity against pathogen infection. The signaling pathways involved in generating these effector cells are partially known. However, the effects of mutations underlying human primary immunodeficiencies on these processes, and how they compromise specific immune responses, remain unresolved. By studying individuals with mutations in key signaling pathways, we identified nonredundant pathways regulating human CD4+ T cell differentiation in vitro. IL12Rβ1/TYK2 and IFN-γR/STAT1 function in a feed-forward loop to induce Th1 cells, whereas IL-21/IL-21R/STAT3 signaling is required for Th17, Tfh, and IL-10–secreting cells. IL12Rβ1/TYK2 and NEMO are also required for Th17 induction. Strikingly, gain-of-function STAT1 mutations recapitulated the impact of dominant-negative STAT3 mutations on Tfh and Th17 cells, revealing a putative inhibitory effect of hypermorphic STAT1 over STAT3. These findings provide mechanistic insight into the requirements for human T cell effector function, and explain clinical manifestations of these immunodeficient conditions. Furthermore, they identify molecules that could be targeted to modulate CD4+ T cell effector function in the settings of infection, vaccination, or immune dysregulation. PMID:27401342

  14. Identification of Distinct Conformations of the Angiotensin-II Type 1 Receptor Associated with the Gq/11 Protein Pathway and the β-Arrestin Pathway Using Molecular Dynamics Simulations*

    PubMed Central

    Cabana, Jérôme; Holleran, Brian; Leduc, Richard; Escher, Emanuel; Guillemette, Gaétan; Lavigne, Pierre

    2015-01-01

    Biased signaling represents the ability of G protein-coupled receptors to engage distinct pathways with various efficacies depending on the ligand used or on mutations in the receptor. The angiotensin-II type 1 (AT1) receptor, a prototypical class A G protein-coupled receptor, can activate various effectors upon stimulation with the endogenous ligand angiotensin-II (AngII), including the Gq/11 protein and β-arrestins. It is believed that the activation of those two pathways can be associated with distinct conformations of the AT1 receptor. To verify this hypothesis, microseconds of molecular dynamics simulations were computed to explore the conformational landscape sampled by the WT-AT1 receptor, the N111G-AT1 receptor (constitutively active and biased for the Gq/11 pathway), and the D74N-AT1 receptor (biased for the β-arrestin1 and -2 pathways) in their apo-forms and in complex with AngII. The molecular dynamics simulations of the AngII-WT-AT1, N111G-AT1, and AngII-N111G-AT1 receptors revealed specific structural rearrangements compared with the initial and ground state of the receptor. Simulations of the D74N-AT1 receptor revealed that the mutation stabilizes the receptor in the initial ground state. The presence of AngII further stabilized the ground state of the D74N-AT1 receptor. The biased agonist [Sar1,Ile8]AngII also showed a preference for the ground state of the WT-AT1 receptor compared with AngII. These results suggest that activation of the Gq/11 pathway is associated with a specific conformational transition stabilized by the agonist, whereas the activation of the β-arrestin pathway is linked to the stabilization of the ground state of the receptor. PMID:25934394

  15. Decreasing electron flux through the cytochrome and/or alternative respiratory pathways triggers common and distinct cellular responses dependent on growth conditions.

    PubMed

    Kühn, Kristina; Yin, Guangkun; Duncan, Owen; Law, Simon R; Kubiszewski-Jakubiak, Szymon; Kaur, Parwinder; Meyer, Etienne; Wang, Yan; Small, Catherine Colas des Francs; Giraud, Estelle; Narsai, Reena; Whelan, James

    2015-01-01

    Diverse signaling pathways are activated by perturbation of mitochondrial function under different growth conditions.Mitochondria have emerged as an important organelle for sensing and coping with stress in addition to being the sites of important metabolic pathways. Here, responses to moderate light and drought stress were examined in different Arabidopsis (Arabidopsis thaliana) mutant plants lacking a functional alternative oxidase (alternative oxidase1a [aox1a]), those with reduced cytochrome electron transport chain capacity (T3/T7 bacteriophage-type RNA polymerase, mitochondrial, and plastidial [rpoTmp]), and double mutants impaired in both pathways (aox1a:rpoTmp). Under conditions considered optimal for growth, transcriptomes of aox1a and rpoTmp were distinct. Under adverse growth conditions, however, transcriptome changes in aox1a and rpoTmp displayed a highly significant overlap and were indicative of a common mitochondrial stress response and down-regulation of photosynthesis. This suggests that the role of mitochondria to support photosynthesis is provided through either the alternative pathway or the cytochrome pathway, and when either pathway is inhibited, such as under environmental stress, a common, dramatic, and succinct mitochondrial signal is activated to alter energy metabolism in both organelles. aox1a:rpoTmp double mutants grown under optimal conditions showed dramatic reductions in biomass production compared with aox1a and rpoTmp and a transcriptome that was distinct from aox1a or rpoTmp. Transcript data indicating activation of mitochondrial biogenesis in aox1a:rpoTmp were supported by a proteomic analysis of over 200 proteins. Under optimal conditions, aox1a:rpoTmp plants seemed to switch on many of the typical mitochondrial stress regulators. Under adverse conditions, aox1a:rpoTmp turned off these responses and displayed a biotic stress response. Taken together, these results highlight the diverse signaling pathways activated by the

  16. Distinct and Atypical Intrinsic and Extrinsic Cell Death Pathways between Photoreceptor Cell Types upon Specific Ablation of Ranbp2 in Cone Photoreceptors

    PubMed Central

    Cho, Kyoung-in; Yu, Minzhong; Hao, Ying; Qiu, Sunny; Pillai, Indulekha C. L.; Peachey, Neal S.; Ferreira, Paulo A.

    2013-01-01

    Non-autonomous cell-death is a cardinal feature of the disintegration of neural networks in neurodegenerative diseases, but the molecular bases of this process are poorly understood. The neural retina comprises a mosaic of rod and cone photoreceptors. Cone and rod photoreceptors degenerate upon rod-specific expression of heterogeneous mutations in functionally distinct genes, whereas cone-specific mutations are thought to cause only cone demise. Here we show that conditional ablation in cone photoreceptors of Ran-binding protein-2 (Ranbp2), a cell context-dependent pleiotropic protein linked to neuroprotection, familial necrotic encephalopathies, acute transverse myelitis and tumor-suppression, promotes early electrophysiological deficits, subcellular erosive destruction and non-apoptotic death of cones, whereas rod photoreceptors undergo cone-dependent non-autonomous apoptosis. Cone-specific Ranbp2 ablation causes the temporal activation of a cone-intrinsic molecular cascade highlighted by the early activation of metalloproteinase 11/stromelysin-3 and up-regulation of Crx and CoREST, followed by the down-modulation of cone-specific phototransduction genes, transient up-regulation of regulatory/survival genes and activation of caspase-7 without apoptosis. Conversely, PARP1+-apoptotic rods develop upon sequential activation of caspase-9 and caspase-3 and loss of membrane permeability. Rod photoreceptor demise ceases upon cone degeneration. These findings reveal novel roles of Ranbp2 in the modulation of intrinsic and extrinsic cell death mechanisms and pathways. They also unveil a novel spatiotemporal paradigm of progression of neurodegeneration upon cell-specific genetic damage whereby a cone to rod non-autonomous death pathway with intrinsically distinct cell-type death manifestations is triggered by cell-specific loss of Ranbp2. Finally, this study casts new light onto cell-death mechanisms that may be shared by human dystrophies with distinct retinal spatial

  17. Distinct and atypical intrinsic and extrinsic cell death pathways between photoreceptor cell types upon specific ablation of Ranbp2 in cone photoreceptors.

    PubMed

    Cho, Kyoung-In; Haque, Mdemdadul; Wang, Jessica; Yu, Minzhong; Hao, Ying; Qiu, Sunny; Pillai, Indulekha C L; Peachey, Neal S; Ferreira, Paulo A

    2013-06-01

    Non-autonomous cell-death is a cardinal feature of the disintegration of neural networks in neurodegenerative diseases, but the molecular bases of this process are poorly understood. The neural retina comprises a mosaic of rod and cone photoreceptors. Cone and rod photoreceptors degenerate upon rod-specific expression of heterogeneous mutations in functionally distinct genes, whereas cone-specific mutations are thought to cause only cone demise. Here we show that conditional ablation in cone photoreceptors of Ran-binding protein-2 (Ranbp2), a cell context-dependent pleiotropic protein linked to neuroprotection, familial necrotic encephalopathies, acute transverse myelitis and tumor-suppression, promotes early electrophysiological deficits, subcellular erosive destruction and non-apoptotic death of cones, whereas rod photoreceptors undergo cone-dependent non-autonomous apoptosis. Cone-specific Ranbp2 ablation causes the temporal activation of a cone-intrinsic molecular cascade highlighted by the early activation of metalloproteinase 11/stromelysin-3 and up-regulation of Crx and CoREST, followed by the down-modulation of cone-specific phototransduction genes, transient up-regulation of regulatory/survival genes and activation of caspase-7 without apoptosis. Conversely, PARP1+ -apoptotic rods develop upon sequential activation of caspase-9 and caspase-3 and loss of membrane permeability. Rod photoreceptor demise ceases upon cone degeneration. These findings reveal novel roles of Ranbp2 in the modulation of intrinsic and extrinsic cell death mechanisms and pathways. They also unveil a novel spatiotemporal paradigm of progression of neurodegeneration upon cell-specific genetic damage whereby a cone to rod non-autonomous death pathway with intrinsically distinct cell-type death manifestations is triggered by cell-specific loss of Ranbp2. Finally, this study casts new light onto cell-death mechanisms that may be shared by human dystrophies with distinct retinal spatial

  18. Unified Approach to (Thio)chromenones via One-Pot Friedel-Crafts Acylation/Cyclization: Distinctive Mechanistic Pathways of β-Chlorovinyl Ketones.

    PubMed

    Kim, Hun Young; Song, Eunsun; Oh, Kyungsoo

    2017-01-20

    A facile synthetic method to chromenones and thiochromenones has been developed using a one-pot Friedel-Crafts acylation of alkynes with suitably substituted benzoyl chlorides. This unified approach to (thio)chromenones is readily applicable to aryl- and alkylalkynes where the stereochemically well-defined β-chlorovinyl ketone intermediates undergo distinctively different cyclization pathways. The ready availability of both starting materials, alkynes and benzoyl chlorides, coupled with the experimental simplicity makes the current synthetic method to (thio)chromenones fast, efficient, and practical.

  19. Interleukin-27 and interleukin-12 augment activation of distinct cord blood natural killer cells responses via STAT3 pathways.

    PubMed

    Chen, Juei-Chang; Huang, Ai-Ju; Chen, Shih-Chang; Wu, Jia-Long; Wu, Wen-Mein; Chiang, Han-Sun; Chan, Chia-Hao; Lin, Chih-Ming; Huang, Yu-Tzu

    2012-05-01

    Umbilical cord blood is rich in primitive natural killer (NK) cells, which are activated by interleukin (IL)-12. It was previously reported that a novel IL-12 family cytokine, IL-27 comprised of EBI3 and p28, was elevated in maternal serum during normal pregnancy. Thus, we compared the immune regulatory functions of IL-27 and IL-12 on mononuclear cells derived from cord blood and adult peripheral blood. After stimulation with IL-27, IL-12, and IL-27 combined with IL-12, the cytotoxicity against BJAB lymphoma cells by blood mononuclear cells was performed. Then immunofluorescence staining, reverse transcriptase-polymerase chain reaction and Western blotting were used to detect the effects of IL-27 and IL-12 in isolated NK cells. IL-27, IL-12, and IL-27 combined with IL-12 enhanced the cytotoxicity of adult peripheral blood cells and cord blood cells, but the proliferation of distinct subpopulations of cells was not evident. Similar results were also obtained with purified cord blood NK cells. Interestingly, distinct from IL-12, IL-27 could induce aggregation and morphological changes of umbilical cord blood cells. Finally, IL-27 combined with IL-12 could stimulate increased IL-27 receptor (gp130 and WSX-1) transcripts in purified cord blood NK cells. However, the activation of signal transducer and activator of transcription 3 (STAT3) in NK cells was only detected in the presence of IL-27, but not IL-12 alone. From previous results, we summarize our current understanding of the augmentation of distinct regulation of NK cells by IL-27 and IL-12. Copyright © 2012. Published by Elsevier B.V.

  20. Botulinum neurotoxin type B uses a distinct entry pathway mediated by CDC42 into intestinal cells versus neuronal cells.

    PubMed

    Connan, Chloé; Voillequin, Marie; Chavez, Carolina Varela; Mazuet, Christelle; Leveque, Christian; Vitry, Sandrine; Vandewalle, Alain; Popoff, Michel R

    2017-08-01

    Botulinum neurotoxins (BoNTs) are responsible for severe flaccid paralysis by inhibiting the release of acetylcholine at the neuromuscular junctions. BoNT type B (BoNT/B) most often induces mild forms of botulism with predominant dysautonomic symptoms. In food borne botulism and botulism by intestinal colonisation such as infant botulism, which are the most frequent naturally acquired forms of botulism, the digestive tract is the main entry route of BoNTs into the organism. We previously showed that BoNT/B translocates through mouse intestinal barrier by an endocytosis-dependent mechanism and subsequently targets neuronal cells, mainly cholinergic neurons, in the intestinal mucosa and musculosa. Here, we investigated the entry pathway of BoNT/B using fluorescent C-terminal domain of the heavy chain (HcB), which is involved in the binding to specific receptor(s) and entry process into target cells. While the combination of gangliosides GD1a /GD1b /GT1b and synaptotagmin I and to a greater extent synaptotagmin II constitutes the functional HcB receptor on NG108-15 neuronal cells, HcB only uses the gangliosides GD1a /GD1b /GT1b to efficiently bind to m-ICcl2 intestinal cells. HcB enters both cell types by a dynamin-dependent endocytosis, which is efficiently prevented by Dynasore, a dynamin inhibitor, and reaches a common early endosomal compartment labeled by early endosome antigen (EEA1). In contrast to neuronal cells, HcB uses a Cdc42-dependent pathway to enter intestinal cells. Then, HcB is transported to late endosomes in neuronal cells, whereas it exploits a nonacidified pathway from apical to basal lateral side of m-ICcl2 cells supporting a transcytotic route in epithelial intestinal cells. © 2017 John Wiley & Sons Ltd.

  1. Distinct Cell Guidance Pathways Controlled by the Rac and Rho GEF Domains of UNC-73/TRIO in Caenorhabditis elegans

    PubMed Central

    Marcus-Gueret, Nancy; Schmidt, Kristopher L.; Stringham, Eve G.

    2012-01-01

    The cytoskeleton regulator UNC-53/NAV2 is required for both the anterior and posterior outgrowth of several neurons as well as that of the excretory cell while the kinesin-like motor VAB-8 is essential for most posteriorly directed migrations in Caenorhabditis elegans. Null mutations in either unc-53 or vab-8 result in reduced posterior excretory canal outgrowth, while double null mutants display an enhanced canal extension defect, suggesting the genes act in separate pathways to control this posteriorly directed outgrowth. Genetic analysis of putative interactors of UNC-53 or VAB-8, and cell-specific rescue experiments suggest that VAB-8, SAX-3/ROBO, SLT-1/Slit, and EVA-1 are functioning together in the outgrowth of the excretory canals, while UNC-53 appears to function in a parallel pathway with UNC-71/ADAM. The known VAB-8 interactor, the Rac/Rho GEF UNC-73/TRIO operates in both pathways, as isoform specific alleles exhibit enhancement of the phenotype in double-mutant combination with either unc-53 or vab-8. On the basis of these results, we propose a bipartite model for UNC-73/TRIO activity in excretory canal extension: a cell autonomous function that is mediated by the Rho-specific GEF domain of the UNC-73E isoform in conjunction with UNC-53 and UNC-71 and a cell nonautonomous function that is mediated by the Rac-specific GEF domain of the UNC-73B isoform, through partnering with VAB-8 and the receptors SAX-3 and EVA-1. PMID:21996675

  2. Profiling dose-dependent activation of p53-mediated signaling pathways by chemicals with distinct mechanisms of DNA damage.

    PubMed

    Clewell, Rebecca A; Sun, Bin; Adeleye, Yeyejide; Carmichael, Paul; Efremenko, Alina; McMullen, Patrick D; Pendse, Salil; Trask, O J; White, Andy; Andersen, Melvin E

    2014-11-01

    As part of a larger effort to provide proof-of-concept in vitro-only risk assessments, we have developed a suite of high-throughput assays for key readouts in the p53 DNA damage response toxicity pathway: double-strand break DNA damage (p-H2AX), permanent chromosomal damage (micronuclei), p53 activation, p53 transcriptional activity, and cell fate (cell cycle arrest, apoptosis, micronuclei). Dose-response studies were performed with these protein and cell fate assays, together with whole genome transcriptomics, for three prototype chemicals: etoposide, quercetin, and methyl methanesulfonate. Data were collected in a human cell line expressing wild-type p53 (HT1080) and results were confirmed in a second p53 competent cell line (HCT 116). At chemical concentrations causing similar increases in p53 protein expression, p53-mediated protein expression and cellular processes showed substantial chemical-specific differences. These chemical-specific differences in the p53 transcriptional response appear to be determined by augmentation of the p53 response by co-regulators. More importantly, dose-response data for each of the chemicals indicate that the p53 transcriptional response does not prevent micronuclei induction at low concentrations. In fact, the no observed effect levels and benchmark doses for micronuclei induction were less than or equal to those for p53-mediated gene transcription regardless of the test chemical, indicating that p53's post-translational responses may be more important than transcriptional activation in the response to low dose DNA damage. This effort demonstrates the process of defining key assays required for a pathway-based, in vitro-only risk assessment, using the p53-mediated DNA damage response pathway as a prototype.

  3. Transgenic Mouse Lines Subdivide External Segment of the Globus Pallidus (GPe) Neurons and Reveal Distinct GPe Output Pathways

    PubMed Central

    Mastro, Kevin J.; Bouchard, Rachel S.; Holt, Hiromi A. K.

    2014-01-01

    Cell-type diversity in the brain enables the assembly of complex neural circuits, whose organization and patterns of activity give rise to brain function. However, the identification of distinct neuronal populations within a given brain region is often complicated by a lack of objective criteria to distinguish one neuronal population from another. In the external segment of the globus pallidus (GPe), neuronal populations have been defined using molecular, anatomical, and electrophysiological criteria, but these classification schemes are often not generalizable across preparations and lack consistency even within the same preparation. Here, we present a novel use of existing transgenic mouse lines, Lim homeobox 6 (Lhx6)–Cre and parvalbumin (PV)–Cre, to define genetically distinct cell populations in the GPe that differ molecularly, anatomically, and electrophysiologically. Lhx6–GPe neurons, which do not express PV, are concentrated in the medial portion of the GPe. They have lower spontaneous firing rates, narrower dynamic ranges, and make stronger projections to the striatum and substantia nigra pars compacta compared with PV–GPe neurons. In contrast, PV–GPe neurons are more concentrated in the lateral portions of the GPe. They have narrower action potentials, deeper afterhyperpolarizations, and make stronger projections to the subthalamic nucleus and parafascicular nucleus of the thalamus. These electrophysiological and anatomical differences suggest that Lhx6–GPe and PV–GPe neurons participate in different circuits with the potential to contribute to different aspects of motor function and dysfunction in disease. PMID:24501350

  4. A facile analytical method for the identification of protease gene profiles from Bacillus thuringiensis strains.

    PubMed

    Chen, Fu-Chu; Shen, Li-Fen; Chak, Kin-Fu

    2004-01-01

    Five pairs of degenerate universal primers have been designed to identify the general protease gene profiles from some distinct Bacillus thuringiensis strains. Based on the PCR amplification patterns and DNA sequences of the cloned fragments, it was noted that the protease gene profiles of the three distinct strains of B. thuringiensis subsp. kurstaki HD73, tenebrionis and israelensis T14001 are varied. Seven protease genes, neutral protease B (nprB), intracellular serine protease A (ispA), extracellular serine protease (vpr), envelope-associated protease (prtH), neutral protease F (nprF), thermostable alkaline serine protease and alkaline serine protease (aprS), with known functions were identified from three distinct B. thuringiensis strains. In addition, five DNA sequences with unknown functions were also identified by this facile analytical method. However, based on the alignment of the derived protein sequences with the protein domain database, it suggested that at least one of these unknown genes, yunA, might be highly protease-related. Thus, the proposed PCR-mediated amplification design could be a facile method for identifying the protease gene profiles as well as for detecting novel protease genes of the B. thuringiensis strains.

  5. Evidence for distinct L-methionine catabolic pathways in the yeast Geotrichum candidum and the bacterium Brevibacterium linens.

    PubMed

    Arfi, Kenza; Landaud, Sophie; Bonnarme, Pascal

    2006-03-01

    Tracing experiments were carried out to identify volatile and nonvolatile L-methionine degradation intermediates and end products in the yeast Geotrichum candidum and in the bacterium Brevibacterium linens, both of which are present in the surface flora of certain soft cheeses and contribute to the ripening reactions. Since the acid-sensitive bacterium B. linens is known to produce larger amounts and a greater variety of volatile sulfur compounds (VSCs) than the yeast G. candidum produces, we examined whether the L-methionine degradation routes of these microorganisms differ. In both microorganisms, methanethiol and alpha-ketobutyrate are generated; the former compound is the precursor of other VSCs, and the latter is subsequently degraded to 2,3-pentanedione, which has not been described previously as an end product of L-methionine catabolism. However, the L-methionine degradation pathways differ in the first steps of L-methionine degradation. L-Methionine degradation is initiated by a one-step degradation process in the bacterium B. linens, whereas a two-step degradation pathway with 4-methylthio-2-oxobutyric acid (MOBA) and 4-methylthio-2-hydroxybutyric acid (MHBA) as intermediates is used in the yeast G. candidum. Since G. candidum develops earlier than B. linens during the ripening process, MOBA and MHBA generated by G.candidum could also be used as precursors for VSC production by B. linens.

  6. Distinct signal transduction pathways are utilized during the tube formation and survival phases of in vitro angiogenesis.

    PubMed

    Ilan, N; Mahooti, S; Madri, J A

    1998-12-18

    Angiogenesis, the formation of new blood vessels from pre-existing ones, occurs during development, wound healing and cancer and involves stages that orchestrate a network of cooperative interactions. Peptide growth factors and extracellular matrix (ECM) components are two major groups of angiogenesis mediators. Among the different ECM proteins, collagens have been well-associated with in vivo angiogenesis. Using human umbilical vein endothelial cells (HUVEC) grown in 3-D collagen gels we show that: (1) HUVEC do not survive well in 3-D collagen gels due to rapid induction of apoptosis. (2) VEGF, a potent in vivo angiogenic factor, fails to induce tube formation. (3) PMA was effective in inducing tube formation and survival in HUVEC dispersed in 3-D collagen gels, activating MAP kinase, phosphoinositide 3-OH kinase (PI-3-kinase) and Akt/PKB (protein kinase B) pathways. (4) VEGF was effective in preventing PMA-induced tube-like structure regression after PMA-withdrawal by (5) activating the mitogen activated protein kinase (MAPK), rather than the Akt/PKB, signaling pathway.

  7. Differential protein-protein interactions of LRRK1 and LRRK2 indicate roles in distinct cellular signaling pathways

    PubMed Central

    Reyniers, Lauran; Del Giudice, Maria Grazia; Civiero, Laura; Belluzzi, Elisa; Lobbestael, Evy; Beilina, Alexandra; Arrigoni, Giorgio; Derua, Rita; Waelkens, Etienne; Li, Yan; Crosio, Claudia; Iaccarino, Ciro; Cookson, Mark R.; Baekelandt, Veerle; Greggio, Elisa; Taymans, Jean-Marc

    2014-01-01

    Genetic studies show that LRRK2, and not its closest paralogue LRRK1, is linked to Parkinson’s disease. To gain insight into the molecular and cellular basis of this discrepancy, we searched for LRRK1- and LRRK2-specific cellular processes by identifying their distinct interacting proteins. A protein microarray-based interaction screen was performed with recombinant 3xFlag-LRRK1 and 3xFlag-LRRK2 and, in parallel, co-immunoprecipitation followed by mass spectrometry was performed from SH-SY5Y neuroblastoma cell lines stably expressing 3xFlag-LRRK1 or 3xFlag-LRRK2. We identified a set of LRRK1- and LRRK2-specific as well as common interactors. One of our most prominent findings was that both screens pointed to epidermal growth factor receptor (EGF-R) as a LRRK1-specific interactor, while 14-3-3 proteins were LRRK2-specific. This is consistent with phosphosite mapping of LRRK1, revealing phosphosites outside of 14-3-3 consensus binding motifs. To assess the functional relevance of these interactions, SH-SY5Y-LRRK1 and -LRRK2 cell lines were treated with LRRK2 kinase inhibitors that disrupt 14-3-3 binding, or with EGF, an EGF-R agonist. Redistribution of LRRK2, not LRRK1, from diffuse cytoplasmic to filamentous aggregates was observed after inhibitor treatment. Similarly, EGF induced translocation of LRRK1, but not of LRRK2, to endosomes. Our study confirms that LRRK1 and LRRK2 can carry out distinct functions by interacting with different cellular proteins. PMID:24947832

  8. Yeast heat shock mRNAs are exported through a distinct pathway defined by Rip1p

    PubMed Central

    Saavedra, Claudio A.; Hammell, Christopher M.; Heath, Catherine V.; Cole, Charles N.

    1997-01-01

    We reported previously that heat or ethanol shock in Saccharomyces cerevisiae leads to nuclear retention of most poly(A)+ RNA but heat shock mRNAs (encoding Hsp70 proteins Ssa1p and Ssa4p) are efficiently exported in a process that is independent of the small GTPase Ran/Gsp1p, which is essential for most nucleocytoplasmic transport. To gain further insights into proteins essential or nonessential for export of heat shock mRNAs, in situ hybridization analyses to detect mRNA and pulse-labeling of proteins were used to examine several yeast mutant strains for their ability to export heat shock mRNAs following stress. Rip1p is a 42-kD protein associated with nuclear pore complexes and contains nucleoporin-like repeat sequences. It is dispensable for growth of yeast cells under normal conditions, but we report that it is essential for the export of heat shock mRNAs following stress. When SSA4 mRNA was induced from a GAL promoter in the absence of stress, it was efficiently exported in a strain lacking RIP1, indicating that Rip1p is required for export of heat shock mRNAs only following stress. Npl3p, a key mediator of export of poly(A)+ RNA, was not required for heat shock mRNA export, whereas Rss1p/Gle1p, a NES-containing factor essential for poly(A)+ RNA export, was also required for export of heat shock mRNAs after stress. High-level expression of the HIV-1 Rev protein, but not of Rev mutants, led to a partial block in export of heat shock mRNAs following stress. The data suggest a model wherein the requirement for Npl3p defines the mRNA export pathway, the requirement for Rip1p defines a pathway used for export of heat shock mRNAs after stress, and additional factors, including Rss1p/Gle1p and several nucleoporins (Rat7p/Nup159p, Rat2p/Nup120p, and Nup145p/Rat10p), are required in both pathways. PMID:9353254

  9. Toll-like receptor ligands synergize through distinct dendritic cell pathways to induce T cell responses: Implications for vaccines

    PubMed Central

    Zhu, Qing; Egelston, Colt; Vivekanandhan, Aravindhan; Uematsu, Satoshi; Akira, Shizuo; Klinman, Dennis M.; Belyakov, Igor M.; Berzofsky, Jay A.

    2008-01-01

    Toll-like receptors (TLRs) may need to cooperate with each other to be effective in detecting imminent infection and trigger immune responses. Understanding is still limited about the intracellular mechanism of this cooperation. We found that when certain TLRs are involved, dendritic cells (DCs) establish unidirectional intracellular cross-talk, in which the MyD88-independent TRIF-dependent pathway amplifies the MyD88-dependent DC function through a JNK-dependent mechanism. The amplified MyD88-dependent DC function determines the induction of the T cell response to a given vaccine in vivo. Therefore, our study revealed an underlying TLR mechanism governing the functional, nonrandom interplay among TLRs for recognition of combinatorial ligands that may be dangerous to the host, providing important guidance for design of novel synergistic molecular vaccine adjuvants. PMID:18845682

  10. A Distinct Replication Fork Protection Pathway Connects Fanconi Anemia Tumor Suppressors to RAD51-BRCA1/2

    PubMed Central

    Schlacher, Katharina; Wu, Hong; Jasin, Maria

    2014-01-01

    SUMMARY Genes mutated in patients with Fanconi anemia (FA) interact with the DNA repair genes BRCA1 and BRCA2/FANCD1 to suppress tumorigenesis, but the molecular functions ascribed to them cannot fully explain all of their cellular roles. Here, we show a repair-independent requirement for FA genes, including FANCD2, and BRCA1 in protecting stalled replication forks from degradation. Fork protection is surprisingly rescued in FANCD2-deficient cells by elevated RAD51 levels or stabilized RAD51 filaments. Moreover, FANCD2-mediated fork protection is epistatic with RAD51 functions, revealing an unanticipated fork protection pathway that connects FA genes to RAD51 and the BRCA1/2 breast cancer suppressors. Collective results imply a unified molecular mechanism for repair-independent functions of FA, RAD51, and BRCA1/2 proteins in preventing genomic instability and suppressing tumorigenesis. PMID:22789542

  11. A distinct replication fork protection pathway connects Fanconi anemia tumor suppressors to RAD51-BRCA1/2.

    PubMed

    Schlacher, Katharina; Wu, Hong; Jasin, Maria

    2012-07-10

    Genes mutated in patients with Fanconi anemia (FA) interact with the DNA repair genes BRCA1 and BRCA2/FANCD1 to suppress tumorigenesis, but the molecular functions ascribed to them cannot fully explain all of their cellular roles. Here, we show a repair-independent requirement for FA genes, including FANCD2, and BRCA1 in protecting stalled replication forks from degradation. Fork protection is surprisingly rescued in FANCD2-deficient cells by elevated RAD51 levels or stabilized RAD51 filaments. Moreover, FANCD2-mediated fork protection is epistatic with RAD51 functions, revealing an unanticipated fork protection pathway that connects FA genes to RAD51 and the BRCA1/2 breast cancer suppressors. Collective results imply a unified molecular mechanism for repair-independent functions of FA, RAD51, and BRCA1/2 proteins in preventing genomic instability and suppressing tumorigenesis.

  12. The endocannabinoid system in renal cells: regulation of Na+ transport by CB1 receptors through distinct cell signalling pathways

    PubMed Central

    Sampaio, L S; Taveira Da Silva, R; Lima, D; Sampaio, C L C; Iannotti, F A; Mazzarella, E; Di Marzo, V; Vieyra, A; Reis, R A M; Einicker-Lamas, M

    2015-01-01

    Background and Purpose The function of the endocannabinoid system (ECS) in renal tissue is not completely understood. Kidney function is closely related to ion reabsorption in the proximal tubule, the nephron segment responsible for the re-absorption of 70–80% of the filtrate. We studied the effect of compounds modulating the activity of cannabinoid (CB) receptors on the active re-absorption of Na+ in LLC-PK1 cells. Experimental Approach Changes in Na+/K+-ATPase activity were assessed after treatment with WIN55,212-2 (WIN), a non-selective lipid agonist, and haemopressin (HP), an inverse peptide agonist at CB1 receptors. Pharmacological tools were used to investigate the signalling pathways involved in the modulation of Na+ transport. Key Results In addition to CB1 and CB2 receptors and TRPV1 channels, the mRNAs encoding for enzymes of the ECS were also expressed in LLC-PK1. WIN (10−7 M) and HP (10−6 M) altered Na+ re-absorption in LLC-PK1 in a dual manner. They both acutely (after 1 min) increased Na+/K+-ATPase activity in a TRPV1 antagonist-sensitive way. WIN's stimulating effect persisted for 30 min, and this effect was partially blocked by a CB1 antagonist or a PKC inhibitor. In contrast, HP inhibited Na+/K+-ATPase after 30 min incubation, and this effect was attenuated by a CB1 antagonist or a PKA inhibitor. Conclusion and Implications The ECS is expressed in LLC-PK1 cells. Both CB1 receptors and TRPV1 channels regulate Na+/K+-ATPase activity in these cells, and are modulated by lipid and peptide CB1 receptor ligands, which act via different signalling pathways. PMID:25537261

  13. Erythropoietin activates two distinct signaling pathways required for the initiation and the elongation of c-myc

    NASA Technical Reports Server (NTRS)

    Chen, C.; Sytkowski, A. J.

    2001-01-01

    Erythropoietin (Epo) stimulation of erythroid cells results in the activation of several kinases and a rapid induction of c-myc expression. Protein kinase C is necessary for Epo up-regulation of c-myc by promoting elongation at the 3'-end of exon 1. PKCepsilon mediates this signal. We now show that Epo triggers two signaling pathways to c-myc. Epo rapidly up-regulated Myc protein in BaF3-EpoR cells. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 blocked Myc up-regulation in a concentration-dependent manner but had no effect on the Epo-induced phosphorylation of ERK1 and ERK2. LY294002 also had no effect on Epo up-regulation of c-fos. MEK1 inhibitor PD98059 blocked both the c-myc and the c-fos responses to Epo. PD98059 and the PKC inhibitor H7 also blocked the phosphorylation of ERK1 and ERK2. PD98059 but not LY294002 inhibited Epo induction of ERK1 and ERK2 phosphorylation in normal erythroid cells. LY294002 blocked transcription of c-myc at exon 1. PD98059 had no effect on transcription from exon 1 but, rather, blocked Epo-induced c-myc elongation at the 3'-end of exon 1. These results identify two Epo signaling pathways to c-myc, one of which is PI3K-dependent operating on transcriptional initiation, whereas the other is mitogen-activated protein kinase-dependent operating on elongation.

  14. Erythropoietin activates two distinct signaling pathways required for the initiation and the elongation of c-myc

    NASA Technical Reports Server (NTRS)

    Chen, C.; Sytkowski, A. J.

    2001-01-01

    Erythropoietin (Epo) stimulation of erythroid cells results in the activation of several kinases and a rapid induction of c-myc expression. Protein kinase C is necessary for Epo up-regulation of c-myc by promoting elongation at the 3'-end of exon 1. PKCepsilon mediates this signal. We now show that Epo triggers two signaling pathways to c-myc. Epo rapidly up-regulated Myc protein in BaF3-EpoR cells. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 blocked Myc up-regulation in a concentration-dependent manner but had no effect on the Epo-induced phosphorylation of ERK1 and ERK2. LY294002 also had no effect on Epo up-regulation of c-fos. MEK1 inhibitor PD98059 blocked both the c-myc and the c-fos responses to Epo. PD98059 and the PKC inhibitor H7 also blocked the phosphorylation of ERK1 and ERK2. PD98059 but not LY294002 inhibited Epo induction of ERK1 and ERK2 phosphorylation in normal erythroid cells. LY294002 blocked transcription of c-myc at exon 1. PD98059 had no effect on transcription from exon 1 but, rather, blocked Epo-induced c-myc elongation at the 3'-end of exon 1. These results identify two Epo signaling pathways to c-myc, one of which is PI3K-dependent operating on transcriptional initiation, whereas the other is mitogen-activated protein kinase-dependent operating on elongation.

  15. Bub1, Sgo1, and Mps1 mediate a distinct pathway for chromosome biorientation in budding yeast

    PubMed Central

    Storchová, Zuzana; Becker, Justin S.; Talarek, Nicolas; Kögelsberger, Sandra; Pellman, David

    2011-01-01

    The conserved mitotic kinase Bub1 performs multiple functions that are only partially characterized. Besides its role in the spindle assembly checkpoint and chromosome alignment, Bub1 is crucial for the kinetochore recruitment of multiple proteins, among them Sgo1. Both Bub1 and Sgo1 are dispensable for growth of haploid and diploid budding yeast, but they become essential in cells with higher ploidy. We find that overexpression of SGO1 partially corrects the chromosome segregation defect of bub1Δ haploid cells and restores viability to bub1Δ tetraploid cells. Using an unbiased high-copy suppressor screen, we identified two members of the chromosomal passenger complex (CPC), BIR1 (survivin) and SLI15 (INCENP, inner centromere protein), as suppressors of the growth defect of both bub1Δ and sgo1Δ tetraploids, suggesting that these mutants die due to defects in chromosome biorientation. Overexpression of BIR1 or SLI15 also complements the benomyl sensitivity of haploid bub1Δ and sgo1Δ cells. Mutants lacking SGO1 fail to biorient sister chromatids attached to the same spindle pole (syntelic attachment) after nocodazole treatment. Moreover, the sgo1Δ cells accumulate syntelic attachments in unperturbed mitoses, a defect that is partially corrected by BIR1 or SLI15 overexpression. We show that in budding yeast neither Bub1 nor Sgo1 is required for CPC localization or affects Aurora B activity. Instead we identify Sgo1 as a possible partner of Mps1, a mitotic kinase suggested to have an Aurora B–independent function in establishment of biorientation. We found that Sgo1 overexpression rescues defects caused by metaphase inactivation of Mps1 and that Mps1 is required for Sgo1 localization to the kinetochore. We propose that Bub1, Sgo1, and Mps1 facilitate chromosome biorientation independently of the Aurora B–mediated pathway at the budding yeast kinetochore and that both pathways are required for the efficient turnover of syntelic attachments. PMID:21389114

  16. CtpB assembles a gated protease tunnel regulating cell-cell signaling during spore formation in Bacillus subtilis.

    PubMed

    Mastny, Markus; Heuck, Alexander; Kurzbauer, Robert; Heiduk, Anja; Boisguerin, Prisca; Volkmer, Rudolf; Ehrmann, Michael; Rodrigues, Christopher D A; Rudner, David Z; Clausen, Tim

    2013-10-24

    Spore formation in Bacillus subtilis relies on a regulated intramembrane proteolysis (RIP) pathway that synchronizes mother-cell and forespore development. To address the molecular basis of this SpoIV transmembrane signaling, we carried out a structure-function analysis of the activating protease CtpB. Crystal structures reflecting distinct functional states show that CtpB constitutes a ring-like protein scaffold penetrated by two narrow tunnels. Access to the proteolytic sites sequestered within these tunnels is controlled by PDZ domains that rearrange upon substrate binding. Accordingly, CtpB resembles a minimal version of a self-compartmentalizing protease regulated by a unique allosteric mechanism. Moreover, biochemical analysis of the PDZ-gated channel combined with sporulation assays reveal that activation of the SpoIV RIP pathway is induced by the concerted activity of CtpB and a second signaling protease, SpoIVB. This proteolytic mechanism is of broad relevance for cell-cell communication, illustrating how distinct signaling pathways can be integrated into a single RIP module.

  17. CtpB Assembles a Gated Protease Tunnel Regulating Cell-Cell Signaling during Spore Formation in Bacillus subtilis

    PubMed Central

    Mastny, Markus; Heuck, Alexander; Kurzbauer, Robert; Heiduk, Anja; Boisguerin, Prisca; Volkmer, Rudolf; Ehrmann, Michael; Rodrigues, Christopher D.A.; Rudner, David Z.; Clausen, Tim

    2013-01-01

    Summary Spore formation in Bacillus subtilis relies on a regulated intramembrane proteolysis (RIP) pathway that synchronizes mother-cell and forespore development. To address the molecular basis of this SpoIV transmembrane signaling, we carried out a structure-function analysis of the activating protease CtpB. Crystal structures reflecting distinct functional states show that CtpB constitutes a ring-like protein scaffold penetrated by two narrow tunnels. Access to the proteolytic sites sequestered within these tunnels is controlled by PDZ domains that rearrange upon substrate binding. Accordingly, CtpB resembles a minimal version of a self-compartmentalizing protease regulated by a unique allosteric mechanism. Moreover, biochemical analysis of the PDZ-gated channel combined with sporulation assays reveal that activation of the SpoIV RIP pathway is induced by the concerted activity of CtpB and a second signaling protease, SpoIVB. This proteolytic mechanism is of broad relevance for cell-cell communication, illustrating how distinct signaling pathways can be integrated into a single RIP module. PMID:24243021

  18. Mechanosensitive transient receptor potential vanilloid 4 regulates Dermatophagoides farinae-induced airway remodeling via 2 distinct pathways modulating matrix synthesis and degradation.

    PubMed

    Gombedza, Farai; Kondeti, Vinay; Al-Azzam, Nosayba; Koppes, Stephanie; Duah, Ernest; Patil, Prachi; Hexter, Madison; Phillips, Daniel; Thodeti, Charles K; Paruchuri, Sailaja

    2017-04-01

    Contributions of mechanical signals to airway remodeling during asthma are poorly understood. Transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive ion channel, has been implicated in cardiac and pulmonary fibrosis; however, its role in asthma remains elusive. Employing a Dermatophagoides farinae-induced asthma model, we report here that TRPV4-knockout mice were protected from D. farinae-induced airway remodeling. Furthermore, lung fibroblasts that were isolated from TRPV4-knockout mice showed diminished differentiation potential compared with wild-type mice. Fibroblasts from asthmatic lung exhibited increased TRPV4 activity and enhanced differentiation potential compared with normal human lung fibroblasts. Of interest, TGF-β1 treatment enhanced TRPV4 activation in a PI3K-dependent manner in normal human lung fibroblasts in vitro Mechanistically, TRPV4 modulated matrix remodeling in the lung via 2 distinct but dependent pathways: one enhances matrix deposition by fibrotic gene activation, whereas the other slows down matrix degradation by increased plasminogen activator inhibitor 1. Of importance, both pathways are regulated by Rho/myocardin-related transcription factor-A and contribute to fibroblast differentiation and matrix remodeling in the lung. Thus, our results support a unique role for TRPV4 in D. farinae-induced airway remodeling and warrant further studies in humans for it to be used as a novel therapeutic target in the treatment of asthma.-Gombedza, F., Kondeti, V., Al-Azzam, N., Koppes, S., Duah, E., Patil, P., Hexter, M., Phillips, D., Thodeti, C. K., Paruchuri, S. Mechanosensitive transient receptor potential vanilloid 4 regulates Dermatophagoides farinae-induced airway remodeling via 2 distinct pathways modulating matrix synthesis and degradation. © FASEB.

  19. TGF-beta and vitamin D3 utilize distinct pathways to suppress IL-12 production and modulate rapid differentiation of human monocytes into CD83+ dendritic cells.

    PubMed

    Lyakh, Lyudmila A; Sanford, Michael; Chekol, Sebel; Young, Howard A; Roberts, Anita B

    2005-02-15

    We previously demonstrated that agents known to signal infection or inflammation can rapidly and directly drive differentiation of human CD14+ monocytes into CD83+ dendritic cells (DCs) when introduced to cells under serum-free conditions. In this study, we evaluated the effects of TGF-beta and vitamin D3 (VitD3) on the proportion and function of monocytes that adopt DC characteristics. TGF-beta significantly decreased the proportion of cells that rapidly adopted stable DC characteristics in response to LPS, but had little or no effect on calcium ionophore-induced differentiation. In contrast, VitD3 showed no such pathway specificity and dramatically suppressed differentiation of monocytes into DCs in response to these agents. Both TGF-beta and VitD3 altered cytokine and chemokine production in LPS-treated monocytes, inhibited IL-12 and IL-10 secretion, and decreased the functional capacity of DCs. Despite the similar effects of TGF-beta and VitD3, there are significant differences in the signaling pathways used by these agents, as evidenced by their distinct effects on LPS- and calcium ionophore-induced DC differentiation, on LPS-induced secretion of IL-10, and on two members of the NF-kappaB family of transcription factors, RelB and cRel. These studies identify TGF-beta and VitD3 as potent regulatory factors that use distinct pathways to suppress both the differentiation of DCs as well as their capacity to secrete the Th1-polarizing cytokine IL-12. Because these agents are present in serum and negatively affect DC differentiation at physiological concentrations, our findings are likely to have significance regarding the in vivo role of TGF-beta and VitD3 in determining the type of immune responses.

  20. Distinct effect of CacyBP/SIP on the ERK1/2-CREB-BDNF pathway in undifferentiated and differentiated neuroblastoma NB2a cells.

    PubMed

    Rosińska, Sara; Leśniak, Wiesława; Filipek, Anna

    2016-07-01

    CacyBP/SIP, a protein expressed to high extent in the brain, has been shown to act as ERK1/2 phosphatase in vitro and in cultured cells. It has been demonstrated recently that CacyBP/SIP can modulate the activity of some transcription factors in neurons and glioma cells. In the present work we have examined the effect of CacyBP/SIP overexpression and silencing on the phosphorylation/activity of ERK1/2 (pERK1/2) and CREB (pCREB) and on the level of BDNF mRNA in differentiated and undifferentiated neuroblastoma NB2a cells. We have shown that in undifferentiated cells the amount of pERK1/2 decreased upon CacyBP/SIP overexpression. Further studies have shown that the activity of CREB and the level of BDNF mRNA, downstream effectors of the ERK1/2 signaling pathway, also depended on the CacyBP/SIP level and strictly matched the level of pERK1/2. Interestingly, in differentiated NB2a cells, overexpression of CacyBP/SIP appeared to have a distinct effect on the pERK1/2 level from that observed in undifferentiated cells. Subsequent studies have revealed that distinct function of CacyBP/SIP in undifferentiated and differentiated NB2a cells might be due to changes in its posttranslational modifications and protein ligands. Altogether, our studies suggest that CacyBP/SIP is involved in the ERK1/2-CREB-BDNF pathway and that it might regulate this pathway depending on the stage of NB2a cell differentiation.

  1. Mutant Allele-Specific Uncoupling of PENETRATION3 Functions Reveals Engagement of the ATP-Binding Cassette Transporter in Distinct Tryptophan Metabolic Pathways1[OPEN

    PubMed Central

    Lu, Xunli; Dittgen, Jan; Piślewska-Bednarek, Mariola; Molina, Antonio; Schneider, Bernd; Doubský, Jan; Schneeberger, Korbinian; Schulze-Lefert, Paul

    2015-01-01

    Arabidopsis (Arabidopsis thaliana) PENETRATION (PEN) genes quantitatively contribute to the execution of different forms of plant immunity upon challenge with diverse leaf pathogens. PEN3 encodes a plasma membrane-resident pleiotropic drug resistance-type ATP-binding cassette transporter and is thought to act in a pathogen-inducible and PEN2 myrosinase-dependent metabolic pathway in extracellular defense. This metabolic pathway directs the intracellular biosynthesis and activation of tryptophan-derived indole glucosinolates for subsequent PEN3-mediated efflux across the plasma membrane at pathogen contact sites. However, PEN3 also functions in abiotic stress responses to cadmium and indole-3-butyric acid (IBA)-mediated auxin homeostasis in roots, raising the possibility that PEN3 exports multiple functionally unrelated substrates. Here, we describe the isolation of a pen3 allele, designated pen3-5, that encodes a dysfunctional protein that accumulates in planta like wild-type PEN3. The specific mutation in pen3-5 uncouples PEN3 functions in IBA-stimulated root growth modulation, callose deposition induced with a conserved peptide epitope of bacterial flagellin (flg22), and pathogen-inducible salicylic acid accumulation from PEN3 activity in extracellular defense, indicating the engagement of multiple PEN3 substrates in different PEN3-dependent biological processes. We identified 4-O-β-d-glucosyl-indol-3-yl formamide (4OGlcI3F) as a pathogen-inducible, tryptophan-derived compound that overaccumulates in pen3 leaf tissue and has biosynthesis that is dependent on an intact PEN2 metabolic pathway. We propose that a precursor of 4OGlcI3F is the PEN3 substrate in extracellular pathogen defense. These precursors, the shared indole core present in IBA and 4OGlcI3F, and allele-specific uncoupling of a subset of PEN3 functions suggest that PEN3 transports distinct indole-type metabolites in distinct biological processes. PMID:26023163

  2. Dual origin of gut proteases in Formosan subterranean termites (Coptotermes formosanus Shiraki) (Isoptera: Rhinotermitidae).

    PubMed

    Sethi, Amit; Xue, Qing-Gang; La Peyre, Jerome F; Delatte, Jennifer; Husseneder, Claudia

    2011-07-01

    Cellulose digestion in lower termites, mediated by carbohydrases originating from both termite and endosymbionts, is well characterized. In contrast, limited information exists on gut proteases of lower termites, their origins and roles in termite nutrition. The objective of this study was to characterize gut proteases of the Formosan subterranean termite (Coptotermes formosanus Shiraki) (Isoptera: Rhinotermitidae). The protease activity of extracts from gut tissues (fore-, mid- and hindgut) and protozoa isolated from hindguts of termite workers was quantified using hide powder azure as a substrate and further characterized by zymography with gelatin SDS-PAGE. Midgut extracts showed the highest protease activity followed by the protozoa extracts. High level of protease activity was also detected in protozoa culture supernatants after 24 h incubation. Incubation of gut and protozoa extracts with class-specific protease inhibitors revealed that most of the proteases were serine proteases. All proteolytic bands identified after gelatin SDS-PAGE were also inhibited by serine protease inhibitors. Finally, incubation with chromogenic substrates indicated that extracts from fore- and hindgut tissues possessed proteases with almost exclusively trypsin-like activity while both midgut and protozoa extracts possessed proteases with trypsin-like and subtilisin/chymotrypsin-like activities. However, protozoa proteases were distinct from midgut proteases (with different molecular mass). Our results suggest that the Formosan subterranean termite not only produces endogenous proteases in its gut tissues, but also possesses proteases originating from its protozoan symbionts.

  3. Two functionally distinct domains generated by in vivo cleavage of Nup145p: a novel biogenesis pathway for nucleoporins.

    PubMed Central

    Teixeira, M T; Siniossoglou, S; Podtelejnikov, S; Bénichou, J C; Mann, M; Dujon, B; Hurt, E; Fabre, E

    1997-01-01

    Nup145p is an essential yeast nucleoporin involved in nuclear export of polyadenylated RNAs. We demonstrate here that Nup145p is cleaved in vivo to yield two functionally distinct domains: a carboxy-terminal domain (C-Nup145p) which is located at the nuclear pore complex (NPC) and assembles into the Nup84p complex, and a GLFG-containing amino-terminal domain (N-Nup145p) which is not part of this complex. Whereas the essential C-Nup145p accomplishes the functions required for efficient mRNA export and normal NPC distribution, N-Nup145p, which is homologous to the GLFG-containing nucleoporins Nup100p and Nup116p, is not necessary for cell growth. However, the N-Nup145p becomes essential in a nup188 mutant background. Strikingly, generation of a free N-domain is a prerequisite for complementation of this peculiar synthetic lethal mutant. These data suggest that N- and C-domains of Nup145p perform independent functions, and that the in vivo cleavage observed is of functional importance. PMID:9305650

  4. β-Adrenergic Receptors Regulate the Acquisition and Consolidation Phases of Aversive Memory Formation Through Distinct, Temporally Regulated Signaling Pathways.

    PubMed

    Schiff, Hillary C; Johansen, Joshua P; Hou, Mian; Bush, David E A; Smith, Emily K; Klein, JoAnna E; LeDoux, Joseph E; Sears, Robert M

    2017-03-01

    Memory formation requires the temporal coordination of molecular events and cellular processes following a learned event. During Pavlovian threat (fear) conditioning (PTC), sensory and neuromodulatory inputs converge on post-synaptic neurons within the lateral nucleus of the amygdala (LA). By activating an intracellular cascade of signaling molecules, these G-protein-coupled neuromodulatory receptors are capable of recruiting a diverse profile of plasticity-related proteins. Here we report that norepinephrine, through its actions on β-adrenergic receptors (βARs), modulates aversive memory formation following PTC through two molecularly and temporally distinct signaling mechanisms. Specifically, using behavioral pharmacology and biochemistry in adult rats, we determined that βAR activity during, but not after PTC training initiates the activation of two plasticity-related targets: AMPA receptors (AMPARs) for memory acquisition and short-term memory and extracellular regulated kinase (ERK) for consolidating the learned association into a long-term memory. These findings reveal that βAR activity during, but not following PTC sets in motion cascading molecular events for the acquisition (AMPARs) and subsequent consolidation (ERK) of learned associations.

  5. Distinct Abscisic Acid Signaling Pathways for Modulation of Guard Cell versus Mesophyll Cell Potassium Channels Revealed by Expression Studies in Xenopus laevis Oocytes1

    PubMed Central

    Sutton, Fedora; Paul, Sunil S.; Wang, Xi-Qing; Assmann, Sarah M.

    2000-01-01

    Regulation of guard cell ion transport by abscisic acid (ABA) and in particular ABA inhibition of a guard cell inward K+ current (IKin) is well documented. However, little is known concerning ABA effects on ion transport in other plant cell types. Here we applied patch clamp techniques to mesophyll cell protoplasts of fava bean (Vicia faba cv Long Pod) plants and demonstrated ABA inhibition of an outward K+ current (IKout). When mesophyll cell protoplast mRNA (mesophyll mRNA) was expressed in Xenopus laevis oocytes, IKout was generated that displayed similar properties to IKout observed from direct analysis of mesophyll cell protoplasts. IKout expressed by mesophyll mRNA-injected oocytes was inhibited by ABA, indicating that the ABA signal transduction pathway observed in mesophyll cells was preserved in the frog oocytes. Co-injection of oocytes with guard cell protoplast mRNA and cRNA for KAT1, an inward K+ channel expressed in guard cells, resulted in IKin that was similarly inhibited by ABA. However, oocytes co-injected with mesophyll mRNA and KAT1 cRNA produced IKin that was not inhibited by ABA. These results demonstrate that the mesophyll-encoded signaling mechanism could not substitute for the guard cell pathway. These findings indicate that mesophyll cells and guard cells use distinct and different receptor types and/or signal transduction pathways in ABA regulation of K+ channels. PMID:10982437

  6. Gadd45a and Gadd45b protect hematopoietic cells from UV-induced apoptosis via distinct signaling pathways, including p38 activation and JNK inhibition.

    PubMed

    Gupta, Mamta; Gupta, Shiv Kumar; Hoffman, Barbara; Liebermann, Dan A

    2006-06-30

    Gadd45a, Gadd45b, and Gadd45g (Gadd45/MyD118/CR6) are genes that are rapidly induced by genotoxic stress and have been implicated in genotoxic stress-induced responses, notably in apoptosis. Recently, using myeloid-enriched bone marrow (BM) cells obtained from wild-type (WT), Gadd45a-deficient, and Gadd45b-deficient mice, we have shown that in hematopoietic cells Gadd45a and Gadd45b play a survival function to protect hematopoietic cells from DNA-damaging agents, including ultra violet (UV)-induced apoptosis. The present study was undertaken to decipher the molecular paths that mediate the survival functions of Gadd45a and Gadd45b against genotoxic stress induced by UV radiation. It is shown that in hematopoietic cells exposed to UV radiation Gaddd45a and Gadd45b cooperate to promote cell survival via two distinct signaling pathways involving activation of the GADD45a-p38-NF-kappaB-mediated survival pathway and GADD45b-mediated inhibition of the stress response MKK4-JNK pathway.

  7. Co-expression modules of NF1, PTEN and sprouty enable distinction of adult diffuse gliomas according to pathway activities of receptor tyrosine kinases

    PubMed Central

    Xue, Yang; Wu, Chenxing; Yao, Kun; Zhang, Chuanbao; Jin, Qiang; Huang, Rong; Li, Jiuyi; Sun, Yingyu; Su, Xiaodong; Jiang, Tao; Fan, Xiaolong

    2016-01-01

    Inter-individual variability causing elevated signaling of receptor tyrosine kinases (RTK) may have hampered the efficacy of targeted therapies. We developed a molecular signature for clustering adult diffuse gliomas based on the extent of RTK pathway activities. Glioma gene modules co-expressed with NF1 (NF1-M), Sprouty (SPRY-M) and PTEN (PTEN-M) were identified, their signatures enabled robust clustering of adult diffuse gliomas of WHO grades II-IV from five independent data sets into two subtypes with distinct activities of RAS-RAF-MEK-MAPK cascade and PI3K-AKT pathway (named RMPAhigh and RMPAlow subtypes) in a morphology-independent manner. The RMPAhigh gliomas were associated with poor prognosis compared to the RMPAlow gliomas. The RMPAhigh and RMPAlow glioma subtypes harbored unique sets of genomic alterations in the RTK signaling-related genes. The RMPAhigh gliomas were enriched in immature vessel cells and tumor associated macrophages, and both cell types expressed high levels of pro-angiogenic RTKs including MET, VEGFR1, KDR, EPHB4 and NRP1. In gliomas with major genomic lesions unrelated to RTK pathway, high RMPA signature was associated with short survival. Thus, the RMPA signatures capture RTK activities in both glioma cells and glioma microenvironment, and RTK signaling in the glioma microenvironment contributes to glioma progression. PMID:27385209

  8. Distinct abscisic acid signaling pathways for modulation of guard cell versus mesophyll cell potassium channels revealed by expression studies in Xenopus laevis oocytes

    NASA Technical Reports Server (NTRS)

    Sutton, F.; Paul, S. S.; Wang, X. Q.; Assmann, S. M.; Evans, M. L. (Principal Investigator)

    2000-01-01

    Regulation of guard cell ion transport by abscisic acid (ABA) and in particular ABA inhibition of a guard cell inward K(+) current (I(Kin)) is well documented. However, little is known concerning ABA effects on ion transport in other plant cell types. Here we applied patch clamp techniques to mesophyll cell protoplasts of fava bean (Vicia faba cv Long Pod) plants and demonstrated ABA inhibition of an outward K(+) current (I(Kout)). When mesophyll cell protoplast mRNA (mesophyll mRNA) was expressed in Xenopus laevis oocytes, I(Kout) was generated that displayed similar properties to I(Kout) observed from direct analysis of mesophyll cell protoplasts. I(Kout) expressed by mesophyll mRNA-injected oocytes was inhibited by ABA, indicating that the ABA signal transduction pathway observed in mesophyll cells was preserved in the frog oocytes. Co-injection of oocytes with guard cell protoplast mRNA and cRNA for KAT1, an inward K(+) channel expressed in guard cells, resulted in I(Kin) that was similarly inhibited by ABA. However, oocytes co-injected with mesophyll mRNA and KAT1 cRNA produced I(Kin) that was not inhibited by ABA. These results demonstrate that the mesophyll-encoded signaling mechanism could not substitute for the guard cell pathway. These findings indicate that mesophyll cells and guard cells use distinct and different receptor types and/or signal transduction pathways in ABA regulation of K(+) channels.

  9. Distinct abscisic acid signaling pathways for modulation of guard cell versus mesophyll cell potassium channels revealed by expression studies in Xenopus laevis oocytes

    NASA Technical Reports Server (NTRS)

    Sutton, F.; Paul, S. S.; Wang, X. Q.; Assmann, S. M.; Evans, M. L. (Principal Investigator)

    2000-01-01

    Regulation of guard cell ion transport by abscisic acid (ABA) and in particular ABA inhibition of a guard cell inward K(+) current (I(Kin)) is well documented. However, little is known concerning ABA effects on ion transport in other plant cell types. Here we applied patch clamp techniques to mesophyll cell protoplasts of fava bean (Vicia faba cv Long Pod) plants and demonstrated ABA inhibition of an outward K(+) current (I(Kout)). When mesophyll cell protoplast mRNA (mesophyll mRNA) was expressed in Xenopus laevis oocytes, I(Kout) was generated that displayed similar properties to I(Kout) observed from direct analysis of mesophyll cell protoplasts. I(Kout) expressed by mesophyll mRNA-injected oocytes was inhibited by ABA, indicating that the ABA signal transduction pathway observed in mesophyll cells was preserved in the frog oocytes. Co-injection of oocytes with guard cell protoplast mRNA and cRNA for KAT1, an inward K(+) channel expressed in guard cells, resulted in I(Kin) that was similarly inhibited by ABA. However, oocytes co-injected with mesophyll mRNA and KAT1 cRNA produced I(Kin) that was not inhibited by ABA. These results demonstrate that the mesophyll-encoded signaling mechanism could not substitute for the guard cell pathway. These findings indicate that mesophyll cells and guard cells use distinct and different receptor types and/or signal transduction pathways in ABA regulation of K(+) channels.

  10. Distinct Pathways Regulate Syk Protein Activation Downstream of Immune Tyrosine Activation Motif (ITAM) and hemITAM Receptors in Platelets*

    PubMed Central

    Manne, Bhanu Kanth; Badolia, Rachit; Dangelmaier, Carol; Eble, Johannes A.; Ellmeier, Wilfried; Kahn, Mark; Kunapuli, Satya P.

    2015-01-01

    Tyrosine kinase pathways are known to play an important role in the activation of platelets. In particular, the GPVI and CLEC-2 receptors are known to activate Syk upon tyrosine phosphorylation of an immune tyrosine activation motif (ITAM) and hemITAM, respectively. However, unlike GPVI, the CLEC-2 receptor contains only one tyrosine motif in the intracellular domain. The mechanisms by which this receptor activates Syk are not completely understood. In this study, we identified a novel signaling mechanism in CLEC-2-mediated Syk activation. CLEC-2-mediated, but not GPVI-mediated, platelet activation and Syk phosphorylation were abolished by inhibition of PI3K, which demonstrates that PI3K regulates Syk downstream of CLEC-2. Ibrutinib, a Tec family kinase inhibitor, also completely abolished CLEC-2-mediated aggregation and Syk phosphorylation in human and murine platelets. Furthermore, embryos lacking both Btk and Tec exhibited cutaneous edema associated with blood-filled vessels in a typical lymphatic pattern similar to CLEC-2 or Syk-deficient embryos. Thus, our data show, for the first time, that PI3K and Tec family kinases play a crucial role in the regulation of platelet activation and Syk phosphorylation downstream of the CLEC-2 receptor. PMID:25767114

  11. Expression of three topologically distinct membrane proteins elicits unique stress response pathways in the yeast Saccharomyces cerevisiae

    PubMed Central

    Buck, Teresa M.; Jordan, Rick; Lyons-Weiler, James; Adelman, Joshua L.; Needham, Patrick G.; Kleyman, Thomas R.

    2015-01-01

    Misfolded membrane proteins are retained in the endoplasmic reticulum (ER) and are subject to ER-associated degradation, which clears the secretory pathway of potentially toxic species. While the transcriptional response to environmental stressors has been extensively studied, limited data exist describing the cellular response to misfolded membrane proteins. To this end, we expressed and then compared the transcriptional profiles elicited by the synthesis of three ER retained, misfolded ion channels: The α-subunit of the epithelial sodium channel, ENaC, the cystic fibrosis transmembrane conductance regulator, CFTR, and an inwardly rectifying potassium channel, Kir2.1, which vary in their mass, membrane topologies, and quaternary structures. To examine transcriptional profiles in a null background, the proteins were expressed in yeast, which was previously used to examine the degradation requirements for each substrate. Surprisingly, the proteins failed to induce a canonical unfolded protein response or heat shock response, although messages encoding several cytosolic and ER lumenal protein folding factors rose when αENaC or CFTR was expressed. In contrast, the levels of these genes were unaltered by Kir2.1 expression; instead, the yeast iron regulon was activated. Nevertheless, a significant number of genes that respond to various environmental stressors were upregulated by all three substrates, and compared with previous microarray data we deduced the existence of a group of genes that reflect a novel misfolded membrane protein response. These data indicate that aberrant proteins in the ER elicit profound yet unique cellular responses. PMID:25759377

  12. SLY1 and Syntaxin 18 specify a distinct pathway for procollagen VII export from the endoplasmic reticulum

    PubMed Central

    Nogueira, Cristina; Erlmann, Patrik; Villeneuve, Julien; Santos, António JM; Martínez-Alonso, Emma; Martínez-Menárguez, José Ángel; Malhotra, Vivek

    2014-01-01

    TANGO1 binds and exports Procollagen VII from the endoplasmic reticulum (ER). In this study, we report a connection between the cytoplasmic domain of TANGO1 and SLY1, a protein that is required for membrane fusion. Knockdown of SLY1 by siRNA arrested Procollagen VII in the ER without affecting the recruitment of COPII components, general protein secretion, and retrograde transport of the KDEL-containing protein BIP, and ERGIC53. SLY1 is known to interact with the ER-specific SNARE proteins Syntaxin 17 and 18, however only Syntaxin 18 was required for Procollagen VII export. Neither SLY1 nor Syntaxin 18 was required for the export of the equally bulky Procollagen I from the ER. Altogether, these findings reveal the sorting of bulky collagen family members by TANGO1 at the ER and highlight the existence of different export pathways for secretory cargoes one of which is mediated by the specific SNARE complex containing SLY1 and Syntaxin 18. DOI: http://dx.doi.org/10.7554/eLife.02784.001 PMID:24842878

  13. The ethanol metabolite acetaldehyde induces water and salt intake via two distinct pathways in the central nervous system of rats.

    PubMed

    Ujihara, Izumi; Hitomi, Suzuro; Ono, Kentaro; Kakinoki, Yasuaki; Hashimoto, Hirofumi; Ueta, Yoichi; Inenaga, Kiyotoshi

    2015-12-01

    The sensation of thirst experienced after heavy alcohol drinking is widely regarded as a consequence of ethanol (EtOH)-induced diuresis, but EtOH in high doses actually induces anti-diuresis. The present study was designed to investigate the introduction mechanism of water and salt intake after heavy alcohol drinking, focusing on action of acetaldehyde, a metabolite of EtOH and a toxic substance, using rats. The aldehyde dehydrogenase (ALDH) inhibitor cyanamide was used to mimic the effect of prolonged acetaldehyde exposure because acetaldehyde is quickly degraded by ALDH. Systemic administration of a high-dose of EtOH at 2.5 g/kg induced water and salt intake with anti-diuresis. Cyanamide enhanced the fluid intake following EtOH and acetaldehyde administration. Systemic administration of acetaldehyde with cyanamide suppressed blood pressure and increased plasma renin activity. Blockade of central angiotensin receptor AT1R suppressed the acetaldehyde-induced fluid intake and c-Fos expression in the circumventricular organs (CVOs), which form part of dipsogenic mechanism in the brain. In addition, central administration of acetaldehyde together with cyanamide selectively induced water but not salt intake without changes in blood pressure. In electrophysiological recordings from slice preparations, acetaldehyde specifically excited angiotensin-sensitive neurons in the CVO. These results suggest that acetaldehyde evokes the thirst sensation following heavy alcohol drinking, by two distinct and previously unsuspected mechanisms, independent of diuresis. First acetaldehyde indirectly activates AT1R in the dipsogenic centers via the peripheral renin-angiotensin system following the depressor response and induces both water and salt intake. Secondly acetaldehyde directly activates neurons in the dipsogenic centers and induces only water intake.

  14. Distinct Severe Acute Respiratory Syndrome Coronavirus-Induced Acute Lung Injury Pathways in Two Different Nonhuman Primate Species▿†

    PubMed Central

    Smits, Saskia L.; van den Brand, Judith M. A.; de Lang, Anna; Leijten, Lonneke M. E.; van IJcken, Wilfred F.; van Amerongen, Geert; Osterhaus, Albert D. M. E.; Andeweg, Arno C.; Haagmans, Bart L.

    2011-01-01

    Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), caused by influenza A virus H5N1 and severe acute respiratory syndrome coronavirus (SARS-CoV), supposedly depend on activation of the oxidative-stress machinery that is coupled with innate immunity, resulting in a strong proinflammatory host response. Inflammatory cytokines, such as interleukin 1β (IL-1β), IL-8, and IL-6, play a major role in mediating and amplifying ALI/ARDS by stimulating chemotaxis and activation of neutrophils. To obtain further insight into the pathogenesis of SARS-CoV-associated ALI, we compared SARS-CoV infections in two different nonhuman primate species, cynomolgus macaques and African green monkeys. Viral titers in the upper and lower respiratory tract were not significantly different in SARS-CoV-infected macaques and African green monkeys. Inflammatory cytokines that play a major role in mediating and amplifying ALI/ARDS or have neutrophil chemoattractant activity, such as IL-6, IL-8, CXCL1, and CXCL2, were, however, induced only in macaques. In contrast, other proinflammatory cytokines and chemokines, including osteopontin and CCL3, were upregulated in the lungs of African green monkeys to a significantly greater extent than in macaques. Because African green monkeys developed more severe ALI than macaques, with hyaline membrane formation, some of these differentially expressed proinflammatory genes may be critically involved in development of the observed pathological changes. Induction of distinct proinflammatory genes after SARS-CoV infection in different nonhuman primate species needs to be taken into account when analyzing outcomes of intervention strategies in these species. PMID:21325418

  15. Engagement of signaling pathways of protease-activated receptor 2 and μ-opioid receptor in bone cancer pain and morphine tolerance.

    PubMed

    Bao, Yanju; Gao, Yebo; Hou, Wei; Yang, Liping; Kong, Xiangying; Zheng, Honggang; Li, Conghuang; Hua, Baojin

    2015-09-15

    Pain is one of the most common and distressing symptoms suffered by patients with progression of cancer. Using a rat model of bone cancer, recent findings suggest that proteinase-activated receptor 2 (PAR2) signaling pathways contribute to neuropathic pain and blocking PAR2 amplifies antinociceptive effects of systemic morphine. The purpose of our study was to examine the underlying mechanisms responsible for the role of PAR2 in regulating bone cancer-evoked pain and the tolerance of systemic morphine. Breast sarcocarcinoma Walker 256 cells were implanted into the tibia bone cavity of rats and this evoked significant mechanical and thermal hyperalgesia. Our results showed that the protein expression of PAR2 and its downstream pathways (protein kinases namely, PKCε and PKA) and transient receptor potential vanilloid 1 (TRPV1) were amplified in the dorsal horn of the spinal cord of bone cancer rats compared to control rats. Blocking spinal PAR2 by using FSLLRY-NH2 significantly attenuated the activities of PKCε/PKA signaling pathways and TRPV1 expression as well as mechanical and thermal hyperalgesia. Also, inhibition of PKCε/PKA and TRPV1 significantly diminished the hyperalgesia observed in bone cancer rats. Additionally, blocking PAR2 enhanced the attenuations of PKCε/PKA and cyclic adenosine monophosphate induced by morphine and further extended analgesia of morphine via μ-opioid receptor (MOR). Our data revealed specific signaling pathways, leading to bone cancer pain, including the activation of PAR2, downstream PKCε/PKA, TRPV1 and resultant sensitization of MOR. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of bone cancer pain often observed in clinics.

  16. Distinct muscle apoptotic pathways are activated in muscles with different fiber types in a rat model of critical illness myopathy.

    PubMed

    Barnes, Benjamin T; Confides, Amy L; Rich, Mark M; Dupont-Versteegden, Esther E

    2015-06-01

    Critical illness myopathy (CIM) is associated with severe muscle atrophy and fatigue in affected patients. Apoptotic signaling is involved in atrophy and is elevated in muscles from patients with CIM. In this study we investigated underlying mechanisms of apoptosis-related pathways in muscles with different fiber type composition in a rat model of CIM using denervation and glucocorticoid administration (denervation and steroid-induced myopathy, DSIM). Soleus and tibialis anterior (TA) muscles showed severe muscle atrophy (40-60% of control muscle weight) and significant apoptosis in interstitial as well as myofiber nuclei that was similar between the two muscles with DSIM. Caspase-3 and -8 activities, but not caspase-9 and -12, were elevated in TA and not in soleus muscle, while the caspase-independent proteins endonuclease G (EndoG) and apoptosis inducing factor (AIF) were not changed in abundance nor differentially localized in either muscle. Anti-apoptotic proteins HSP70, -27, and apoptosis repressor with a caspase recruitment domain (ARC) were elevated in soleus compared to TA muscle and ARC was significantly decreased with induction of DSIM in soleus. Results indicate that apoptosis is a significant process associated with DSIM in both soleus and TA muscles, and that apoptosis-associated processes are differentially regulated in muscles of different function and fiber type undergoing atrophy due to DSIM. We conclude that interventions combating apoptosis with CIM may need to be directed towards inhibiting caspase-dependent as well as -independent mechanisms to be able to affect muscles of all fiber types.

  17. Distinct Patterns of Tryptophan Maintenance in Tissues during Kynurenine Pathway Activation in Simian Immunodeficiency Virus-Infected Macaques

    PubMed Central

    Drewes, Julia L.; Croteau, Joshua D.; Shirk, Erin N.; Engle, Elizabeth L.; Zink, M. C.; Graham, David R.

    2016-01-01

    Induction of the kynurenine pathway (KP) of tryptophan (TRP) catabolism has been proposed to contribute to T cell dysfunction during human/simian immunodeficiency virus (SIV) infection via depletion of local TRP levels and production of immunomodulatory KP metabolites. However, while changes in TRP and KP metabolites have been observed in plasma, their levels in lymphoid tissues and levels of enzymes downstream of indoleamine 2,3-dioxygenase (IDO1) have been relatively unexplored. We used our SIV-infected pigtailed macaque model to analyze longitudinal changes in KP metabolites and enzymes by gas chromatography/mass spectrometry and NanoString nCounter gene expression analysis, respectively, in spleen and blood compared to changes previously established in brain and CSF. We found that TRP levels were remarkably stable in tissue sites despite robust depletion in the circulating plasma and CSF. We also demonstrated that intracellular TRP reserves were maintained in cultured cells even in the presence of depleted extracellular TRP levels. Kynurenine (KYN), 3-hydroxykynurenine, quinolinic acid, and the KP enzymes all displayed highly divergent patterns in the sites examined, though IDO1 expression always correlated with local KYN/TRP ratios. Finally, we demonstrated by fluorescence-activated cell sorting that myeloid dendritic cells and cells of monocytic lineage were the highest producers of IDO1 in chronically infected spleens. Overall, our study reveals insights into the tissue-specific regulation of KP enzymes and metabolites and, in particular, highlights the multiple mechanisms by which cells and tissues seek to prevent TRP starvation during inflammation. PMID:28066416

  18. Turnover of Phosphatidic Acid through Distinct Signaling Pathways Affects Multiple Aspects of Pollen Tube Growth in Tobacco.

    PubMed

    Pleskot, Roman; Pejchar, Přemysl; Bezvoda, Radek; Lichtscheidl, Irene K; Wolters-Arts, Mieke; Marc, Jan; Zárský, Viktor; Potocký, Martin

    2012-01-01

    Phosphatidic acid (PA) is an important intermediate in membrane lipid metabolism that acts as a key component of signaling networks, regulating the spatio-temporal dynamics of the endomembrane system and the cytoskeleton. Using tobacco pollen tubes as a model, we addressed the signaling effects of PA by probing the functions of three most relevant enzymes that regulate the production and degradation of PA, namely, phospholipases D (PLD), diacylglycerol kinases (DGKs), and lipid phosphate phosphatases (LPPs). Phylogenetic analysis indicated a highly dynamic evolution of all three lipid-modifying enzymes in land plants, with many clade-specific duplications or losses and massive diversification of the C2-PLD family. In silico transcriptomic survey revealed increased levels of expression of all three PA-regulatory genes in pollen development (particularly the DGKs). Using specific inhibitors we were able to distinguish the contributions of PLDs, DGKs, and LPPs into PA-regulated processes. Thus, suppressing PA production by inhibiting either PLD or DGK activity compromised membrane trafficking except early endocytosis, disrupted tip-localized deposition of cell wall material, especially pectins, and inhibited pollen tube growth. Conversely, suppressing PA degradation by inhibiting LPP activity using any of three different inhibitors significantly stimulated pollen tube growth, and similar effect was achieved by suppressing the expression of tobacco pollen LPP4 using antisense knock-down. Interestingly, inhibiting specifically DGK changed vacuolar dynamics and the morphology of pollen tubes, whereas inhibiting specifically PLD disrupted the actin cytoskeleton. Overall, our results demonstrate the critical importance of all three types of enzymes involved in PA production and degradation, with strikingly different roles of PA produced by the PLD and DGK pathways, in pollen tube growth.

  19. Distinct muscle apoptotic pathways are activated in muscles with different fiber types a rat model of critical illness myopathy

    PubMed Central

    Barnes, Benjamin T.; Confides, Amy L.; Rich, Mark M.; Dupont-Versteegden, Esther E.

    2015-01-01

    Critical illness myopathy (CIM) is associated with severe muscle atrophy and fatigue in affected patients. Apoptotic signaling is involved in atrophy and is elevated in muscles from patients with CIM. In this study we investigated underlying mechanisms of apoptosis-related pathways in muscles with different fiber type composition in a rat model of CIM using denervation and glucocorticoid administration (denervation and steroid-induced myopathy, DSIM). Soleus and tibialis anterior (TA) muscles showed severe muscle atrophy (40–60% of control muscle weight) and significant apoptosis in interstitial as well as myofiber nuclei that was similar between the two muscles with DSIM. Caspase-3 and −8 activities, but not caspase-9 and −12, were elevated in TA and not in soleus muscle, while the caspase-independent proteins endonuclease G (EndoG) and apoptosis inducing factor (AIF) were not changed in abundance nor differentially localized in either muscle. Anti-apoptotic proteins HSP70, −27, and apoptosis repressor with a caspase recruitment domain (ARC) were elevated in soleus compared to TA muscle and ARC was significantly decreased with induction of DSIM in soleus. Results indicate that apoptosis is a significant process associated with DSIM in both soleus and TA muscles, and that apoptosis-associated processes are differentially regulated in muscles of different function and fiber type undergoing atrophy due to DSIM. We conclude that interventions combating apoptosis with CIM may need to be directed towards inhibiting caspase-dependent as well as -independent mechanisms to be able to affect muscles of all fiber types. PMID:25740800

  20. Turnover of Phosphatidic Acid through Distinct Signaling Pathways Affects Multiple Aspects of Pollen Tube Growth in Tobacco

    PubMed Central

    Pleskot, Roman; Pejchar, Přemysl; Bezvoda, Radek; Lichtscheidl, Irene K.; Wolters-Arts, Mieke; Marc, Jan; Žárský, Viktor; Potocký, Martin

    2012-01-01

    Phosphatidic acid (PA) is an important intermediate in membrane lipid metabolism that acts as a key component of signaling networks, regulating the spatio-temporal dynamics of the endomembrane system and the cytoskeleton. Using tobacco pollen tubes as a model, we addressed the signaling effects of PA by probing the functions of three most relevant enzymes that regulate the production and degradation of PA, namely, phospholipases D (PLD), diacylglycerol kinases (DGKs), and lipid phosphate phosphatases (LPPs). Phylogenetic analysis indicated a highly dynamic evolution of all three lipid-modifying enzymes in land plants, with many clade-specific duplications or losses and massive diversification of the C2-PLD family. In silico transcriptomic survey revealed increased levels of expression of all three PA-regulatory genes in pollen development (particularly the DGKs). Using specific inhibitors we were able to distinguish the contributions of PLDs, DGKs, and LPPs into PA-regulated processes. Thus, suppressing PA production by inhibiting either PLD or DGK activity compromised membrane trafficking except early endocytosis, disrupted tip-localized deposition of cell wall material, especially pectins, and inhibited pollen tube growth. Conversely, suppressing PA degradation by inhibiting LPP activity using any of three different inhibitors significantly stimulated pollen tube growth, and similar effect was achieved by suppressing the expression of tobacco pollen LPP4 using antisense knock-down. Interestingly, inhibiting specifically DGK changed vacuolar dynamics and the morphology of pollen tubes, whereas inhibiting specifically PLD disrupted the actin cytoskeleton. Overall, our results demonstrate the critical importance of all three types of enzymes involved in PA production and degradation, with strikingly different roles of PA produced by the PLD and DGK pathways, in pollen tube growth. PMID:22639652

  1. Calcium and SOL Protease Mediate Temperature Resetting of Circadian Clocks.

    PubMed

    Tataroglu, Ozgur; Zhao, Xiaohu; Busza, Ania; Ling, Jinli; O'Neill, John S; Emery, Patrick

    2015-11-19

    Circadian clocks integrate light and temperature input to remain synchronized with the day/night cycle. Although light input to the clock is well studied, the molecular mechanisms by which circadian clocks respond to temperature remain poorly understood. We found that temperature phase shifts Drosophila circadian clocks through degradation of the pacemaker protein TIM. This degradation is mechanistically distinct from photic CRY-dependent TIM degradation. Thermal TIM degradation is triggered by cytosolic calcium increase and CALMODULIN binding to TIM and is mediated by the atypical calpain protease SOL. This thermal input pathway and CRY-dependent light input thus converge on TIM, providing a molecular mechanism for the integration of circadian light and temperature inputs. Mammals use body temperature cycles to keep peripheral clocks synchronized with their brain pacemaker. Interestingly, downregulating the mammalian SOL homolog SOLH blocks thermal mPER2 degradation and phase shifts. Thus, we propose that circadian thermosensation in insects and mammals share common principles.

  2. Serine proteases inhibiting cyanopeptides.

    PubMed

    Radau, G

    2000-08-01

    There are many compounds inhibiting serine proteases which play an important role in the human organism. This article reviews publications on the low-molecular weight, serine protease inhibitory cyanopeptides and reports on new developments in establishing structure-activity relationships.

  3. Structural determinants of tobacco vein mottling virus protease substrate specificity

    SciTech Connect

    Sun, Ping; Austin, Brian P.; Tozer, Jozsef; Waugh, David

    2010-10-28

    Tobacco vein mottling virus (TVMV) is a member of the Potyviridae, one of the largest families of plant viruses. The TVMV genome is translated into a single large polyprotein that is subsequently processed by three virally encoded proteases. Seven of the nine cleavage events are carried out by the NIa protease. Its homolog from the tobacco etch virus (TEV) is a widely used reagent for the removal of affinity tags from recombinant proteins. Although TVMV protease is a close relative of TEV protease, they exhibit distinct sequence specificities. We report here the crystal structure of a catalytically inactive mutant TVMV protease (K65A/K67A/C151A) in complex with a canonical peptide substrate (Ac-RETVRFQSD) at 1.7-{angstrom} resolution. As observed in several crystal structures of TEV protease, the C-terminus ({approx}20 residues) of TVMV protease is disordered. Unexpectedly, although deleting the disordered residues from TEV protease reduces its catalytic activity by {approx}10-fold, an analogous truncation mutant of TVMV protease is significantly more active. Comparison of the structures of TEV and TVMV protease in complex with their respective canonical substrate peptides reveals that the S3 and S4 pockets are mainly responsible for the differing substrate specificities. The structure of TVMV protease suggests that it is less tolerant of variation at the P1{prime} position than TEV protease. This conjecture was confirmed experimentally by determining kinetic parameters k{sub cat} and K{sub m} for a series of oligopeptide substrates. Also, as predicted by the cocrystal structure, we confirm that substitutions in the P6 position are more readily tolerated by TVMV than TEV protease.

  4. Structural determinants of tobacco vein mottling virus protease substrate specificity.

    PubMed

    Sun, Ping; Austin, Brian P; Tözsér, József; Waugh, David S

    2010-11-01

    Tobacco vein mottling virus (TVMV) is a member of the Potyviridae, one of the largest families of plant viruses. The TVMV genome is translated into a single large polyprotein that is subsequently processed by three virally encoded proteases. Seven of the nine cleavage events are carried out by the NIa protease. Its homolog from the tobacco etch virus (TEV) is a widely used reagent for the removal of affinity tags from recombinant proteins. Although TVMV protease is a close relative of TEV protease, they exhibit distinct sequence specificities. We report here the crystal structure of a catalytically inactive mutant TVMV protease (K65A/K67A/C151A) in complex with a canonical peptide substrate (Ac-RETVRFQSD) at 1.7-Å resolution. As observed in several crystal structures of TEV protease, the C-terminus (∼20 residues) of TVMV protease is disordered. Unexpectedly, although deleting the disordered residues from TEV protease reduces its catalytic activity by ∼10-fold, an analogous truncation mutant of TVMV protease is significantly more active. Comparison of the structures of TEV and TVMV protease in complex with their respective canonical substrate peptides reveals that the S3 and S4 pockets are mainly responsible for the differing substrate specificities. The structure of TVMV protease suggests that it is less tolerant of variation at the P1' position than TEV protease. This conjecture was confirmed experimentally by determining kinetic parameters k(cat) and K(m) for a series of oligopeptide substrates. Also, as predicted by the cocrystal structure, we confirm that substitutions in the P6 position are more readily tolerated by TVMV than TEV protease.

  5. Structural determinants of tobacco vein mottling virus protease substrate specificity

    PubMed Central

    Sun, Ping; Austin, Brian P; Tözsér, József; Waugh, David S

    2010-01-01

    Tobacco vein mottling virus (TVMV) is a member of the Potyviridae, one of the largest families of plant viruses. The TVMV genome is translated into a single large polyprotein that is subsequently processed by three virally encoded proteases. Seven of the nine cleavage events are carried out by the NIa protease. Its homolog from the tobacco etch virus (TEV) is a widely used reagent for the removal of affinity tags from recombinant proteins. Although TVMV protease is a close relative of TEV protease, they exhibit distinct sequence specificities. We report here the crystal structure of a catalytically inactive mutant TVMV protease (K65A/K67A/C151A) in complex with a canonical peptide substrate (Ac-RETVRFQSD) at 1.7-Å resolution. As observed in several crystal structures of TEV protease, the C-terminus (∼20 residues) of TVMV protease is disordered. Unexpectedly, although deleting the disordered residues from TEV protease reduces its catalytic activity by ∼10-fold, an analogous truncation mutant of TVMV protease is significantly more active. Comparison of the structures of TEV and TVMV protease in complex with their respective canonical substrate peptides reveals that the S3 and S4 pockets are mainly responsible for the differing substrate specificities. The structure of TVMV protease suggests that it is less tolerant of variation at the P1′ position than TEV protease. This conjecture was confirmed experimentally by determining kinetic parameters kcat and Km for a series of oligopeptide substrates. Also, as predicted by the cocrystal structure, we confirm that substitutions in the P6 position are more readily tolerated by TVMV than TEV protease. PMID:20862670

  6. Non-esterified fatty acids generate distinct low-molecular weight amyloid-β (Aβ42) oligomers along pathway different from fibril formation.

    PubMed

    Kumar, Amit; Bullard, Rebekah L; Patel, Pritesh; Paslay, Lea C; Singh, Dipti; Bienkiewicz, Ewa A; Morgan, Sarah E; Rangachari, Vijayaraghavan

    2011-04-19

    Amyloid-β (Aβ) peptide aggregation is known to play a central role in the etiology of Alzheimer's disease (AD). Among various aggregates, low-molecular weight soluble oligomers of Aβ are increasingly believed to be the primary neurotoxic agents responsible for memory impairment. Anionic interfaces are known to influence the Aβ aggregation process significantly. Here, we report the effects of interfaces formed by medium-chain (C9-C12), saturated non-esterified fatty acids (NEFAs) on Aβ42 aggregation. NEFAs uniquely affected Aβ42 aggregation rates that depended on both the ratio of Aβ:NEFA as well the critical micelle concentration (CMC) of the NEFAs. More importantly, irrespective of the kind of NEFA used, we observed that two distinct oligomers, 12-18 mers and 4-5 mers were formed via different pathway of aggregation under specific experimental conditions: (i) 12-18 mers were generated near the CMC in which NEFAs augment the rate of Aβ42 aggregation towards fibril formation, and, (ii) 4-5 mers were formed above the CMC, where NEFAs inhibit fibril formation. The data indicated that both 12-18 mers and 4-5 mers are formed along an alternate pathway called 'off-pathway' that did not result in fibril formation and yet have subtle structural and morphological differences that distinguish their bulk molecular behavior. These observations, (i) reflect the possible mechanism of Aβ aggregation in physiological lipid-rich environments, and (ii) reiterate the fact that all oligomeric forms of Aβ need not be obligatory intermediates of the fibril formation pathway.

  7. Alterations of the p53 and PIK3CA/AKT/mTOR pathways in angiosarcomas: a pattern distinct from other sarcomas with complex genomics.

    PubMed

    Italiano, Antoine; Chen, Chun-Liang; Thomas, Rachael; Breen, Matthew; Bonnet, Françoise; Sevenet, Nicolas; Longy, Michel; Maki, Robert G; Coindre, Jean-Michel; Antonescu, Cristina R

    2012-12-01

    The p53 and phosphoinositide-3-kinase, catalytic, alpha polypeptide/v-akt murine thymoma viral oncogene homolog/mechanistic target of rapamycin (PIK3CA/AKT/mTOR) pathways frequently are altered in sarcoma with complex genomics, such as leiomyosarcoma (LMS) or undifferentiated pleomorphic sarcoma (UPS). The scale of genetic abnormalities in these pathways remains unknown in angiosarcoma (AS). The authors investigated the status of critical genes involved in the p53 and PIK3CA/AKT/mTOR pathways in a series of 62 AS. The mutation and deletion rates of tumor protein 53 (TP53) were 4% and 0%, respectively. Overexpression of p53 was detected by immunohistochemistry in 49% of patients and was associated with inferior disease-free survival. Although p14 inactivation or overexpression of the human murine double minute homolog (HDM2) were frequent in LMS and UPS and could substitute for TP53 mutation or deletion, such alterations were rare in angiosarcomas. Phosphorylated ribosomal protein S6 kinase (p-S6K) and/or phosphorylated eukaryotic translation initiation factor 4E binding protein 1 (p-4eBP1) overexpression was observed in 42% of patients, suggesting frequent activation of the PIK3CA/AKT/mTOR pathway in angiosarcomas. Activation was not related to intragenic deletion of phosphatase and tensin homolog (PTEN), an aberration that is frequent in LMS and UPS but absent in angiosarcomas. The current results indicated that angiosarcomas constitute a distinct subgroup among sarcomas with complex genomics. Although TP53 mutation and PTEN deletion are frequent in LMS and UPS, these aberrations are rarely involved in the pathogenesis of angiosarcoma. Copyright © 2012 American Cancer Society.

  8. Distinct effects of folate pathway genes MTHFR and MTHFD1L on ruminative response style: a potential risk mechanism for depression

    PubMed Central

    Eszlari, N; Kovacs, D; Petschner, P; Pap, D; Gonda, X; Elliott, R; Anderson, I M; Deakin, J F W; Bagdy, G; Juhasz, G

    2016-01-01

    Alterations in the folate pathway have been related to both major depression and cognitive inflexibility; however, they have not been investigated in the genetic background of ruminative response style, which is a form of perseverative cognition and a risk factor for depression. In the present study, we explored the association of rumination (measured by the Ruminative Responses Scale) with polymorphisms of two distinct folate pathway genes, MTHFR rs1801133 (C677T) and MTHFD1L rs11754661, in a combined European white sample from Budapest, Hungary (n=895) and Manchester, United Kingdom (n=1309). Post hoc analysis investigated whether the association could be replicated in each of the two samples, and the relationship between folate pathway genes, rumination, lifetime depression and Brief Symptom Inventory depression score. Despite its functional effect on folate metabolism, the MTHFR rs1801133 showed no effect on rumination. However, the A allele of MTHFD1L rs11754661 was significantly associated with greater rumination, and this effect was replicated in both the Budapest and Manchester samples. In addition, rumination completely mediated the effects of MTHFD1L rs11754661 on depression phenotypes. These findings suggest that the MTHFD1L gene, and thus the C1-THF synthase enzyme of the folate pathway localized in mitochondria, has an important effect on the pathophysiology of depression through rumination, and maybe via this cognitive intermediate phenotype on other mental and physical disorders. Further research should unravel whether the reversible metabolic effect of MTHFD1L is responsible for increased rumination or other long-term effects on brain development. PMID:26926881

  9. Formation of Thiolated Nucleosides Present in tRNA from Salmonella enterica serovar Typhimurium Occurs in Two Principally Distinct Pathways

    PubMed Central

    Leipuviene, Ramune; Qian, Qiang; Björk, Glenn R.

    2004-01-01

    tRNA from Salmonella enterica serovar Typhimurium contains five thiolated nucleosides, 2-thiocytidine (s2C), 4-thiouridine (s4U), 5-methylaminomethyl-2-thiouridine (mnm5s2U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), and N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A). The levels of all of them are significantly reduced in cells with a mutated iscS gene, which encodes the cysteine desulfurase IscS, a member of the ISC machinery that is responsible for [Fe-S] cluster formation in proteins. A mutant (iscU52) was isolated that carried an amino acid substitution (S107T) in the IscU protein, which functions as a major scaffold in the formation of [Fe-S] clusters. In contrast to the iscS mutant, the iscU52 mutant showed reduced levels of only two of the thiolated nucleosides, ms2io6A (10-fold) and s2C (more than 2-fold). Deletions of the iscU, hscA, or fdx genes from the isc operon lead to a similar tRNA thiolation pattern to that seen for the iscU52 mutant. Unexpectedly, deletion of the iscA gene, coding for an alternative scaffold protein for the [Fe-S] clusters, showed a novel tRNA thiolation pattern, where the synthesis of only one thiolated nucleoside, ms2io6A, was decreased twofold. Based on our results, we suggest two principal distinct routes for thiolation of tRNA: (i) a direct sulfur transfer from IscS to the tRNA modifying enzymes ThiI and MnmA, which form s4U and the s2U moiety of (c)mnm5s2U, respectively; and (ii) an involvement of [Fe-S] proteins (an unidentified enzyme in the synthesis of s2C and MiaB in the synthesis of ms2io6A) in the transfer of sulfur to the tRNA. PMID:14729702

  10. Bifidobacterium breve UCC2003 metabolises the human milk oligosaccharides lacto-N-tetraose and lacto-N-neo-tetraose through overlapping, yet distinct pathways

    PubMed Central

    James, Kieran; Motherway, Mary O’Connell; Bottacini, Francesca; van Sinderen, Douwe

    2016-01-01

    In this study, we demonstrate that the prototype B. breve strain UCC2003 possesses specific metabolic pathways for the utilisation of lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT), which represent the central moieties of Type I and Type II human milk oligosaccharides (HMOs), respectively. Using a combination of experimental approaches, the enzymatic machinery involved in the metabolism of LNT and LNnT was identified and characterised. Homologs of the key genetic loci involved in the utilisation of these HMO substrates were identified in B. breve, B. bifidum, B. longum subsp. infantis and B. longum subsp. longum using bioinformatic analyses, and were shown to be variably present among other members of the Bifidobacterium genus, with a distinct pattern of conservation among human-associated bifidobacterial species. PMID:27929046

  11. Interference with Protease-activated Receptor 1 Alleviates Neuronal Cell Death Induced by Lipopolysaccharide-Stimulated Microglial Cells through the PI3K/Akt Pathway

    PubMed Central

    Li, Yuxin; Yang, Wuyang; Quinones-Hinojosa, Alfredo; Wang, Baocheng; Xu, Shujun; Zhu, Weijie; Yu, Feng; Yuan, Shaoji; Lu, Peigang

    2016-01-01

    Excessive microglial cells activation in response to inflammatory stimuli leads to synaptic loss, dysfunction, and neuronal cell death. Activated microglia are involved in the pathogenesis of neurological conditions and frequently contribute to several complications. Accumulating evidence suggests that signaling through PAR-1 is involved in inflammation, however, its function has yet to be fully elucidated. Here, we have demonstrated that the suppression of PAR-1 leads to down-regulation of inflammatory factors including IL-1β, IL-6, TNF-α, NO, as well as the prevention of activation of NF-κB in BV2 cells. In addition, we found that a PAR-1 antagonist, SCH, prevented LPS-induced excessive microglial activation in a dose-dependent manner. As a result of SCH treatment, neuronal cell death via up-regulation of Akt-mediated pathways was reduced. Our results demonstrate that the beneficial effects of SCH are linked to its ability to block an inflammatory response. Further, we found that SCH inhibited the death of PC12 neurons from the cytotoxicity of activated BV2 cells via activation of the PI3K/Akt pathway. These neuro-protective effects appear to be related to inhibition of PAR-1, and represents a novel neuroprotective strategy that could has potential for use in therapeutic interventions of neuroinflammatory disease. PMID:27910893

  12. Finding novel distinctions between the sAPPα-mediated anabolic biochemical pathways in Autism Spectrum Disorder and Fragile X Syndrome plasma and brain tissue

    PubMed Central

    Ray, Balmiki; Sokol, Deborah K.; Maloney, Bryan; Lahiri, Debomoy K.

    2016-01-01

    Autism spectrum disorder (ASD) and Fragile X syndrome (FXS) are developmental disorders. No validated blood-based biomarkers exist for either, which impedes bench-to-bedside approaches. Amyloid-β (Aβ) precursor protein (APP) and metabolites are usually associated with Alzheimer’s disease (AD). APP cleavage by α-secretase produces potentially neurotrophic secreted APPα (sAPPα) and the P3 peptide fragment. β-site APP cleaving enzyme (BACE1) cleavage produces secreted APPβ (sAPPβ) and intact Aβ. Excess Aβ is potentially neurotoxic and can lead to atrophy of brain regions such as amygdala in AD. By contrast, amygdala is enlarged in ASD but not FXS. We previously reported elevated levels of sAPPα in ASD and FXS vs. controls. We now report elevated plasma Aβ and total APP levels in FXS compared to both ASD and typically developing controls, and elevated levels of sAPPα in ASD and FXS vs. controls. By contrast, plasma and brain sAPPβ and Aβ were lower in ASD vs. controls but elevated in FXS plasma vs. controls. We also detected age-dependent increase in an α-secretase in ASD brains. We report a novel mechanistic difference in APP pathways between ASD (processing) and FXS (expression) leading to distinct APP metabolite profiles in these two disorders. These novel, distinctive biochemical differences between ASD and FXS pave the way for blood-based biomarkers for ASD and FXS. PMID:27212113

  13. Bacterial proteases and virulence.

    PubMed

    Frees, Dorte; Brøndsted, Lone; Ingmer, Hanne

    2013-01-01

    Bacterial pathogens rely on proteolysis for variety of purposes during the infection process. In the cytosol, the main proteolytic players are the conserved Clp and Lon proteases that directly contribute to virulence through the timely degradation of virulence regulators and indirectly by providing tolerance to adverse conditions such as those experienced in the host. In the membrane, HtrA performs similar functions whereas the extracellular proteases, in close contact with host components, pave the way for spreading infections by degrading host matrix components or interfering with host cell signalling to short-circuit host cell processes. Common to both intra- and extracellular proteases is the tight control of their proteolytic activities. In general, substrate recognition by the intracellular proteases is highly selective which is, in part, attributed to the chaperone activity associated with the proteases either encoded within the same polypeptide or on separate subunits. In contrast, substrate recognition by extracellular proteases is less selective and therefore these enzymes are generally expressed as zymogens to prevent premature proteolytic activity that would be detrimental to the cell. These extracellular proteases are activated in complex cascades involving auto-processing and proteolytic maturation. Thus, proteolysis has been adopted by bacterial pathogens at multiple levels to ensure the success of the pathogen in contact with the human host.

  14. AtNUDT7, a negative regulator of basal immunity in Arabidopsis, modulates two distinct defense response pathways and is involved in maintaining redox homeostasis.

    PubMed

    Ge, Xiaochun; Li, Guo-Jing; Wang, Sheng-Bing; Zhu, Huifen; Zhu, Tong; Wang, Xun; Xia, Yiji

    2007-09-01

    Plants have evolved complicated regulatory systems to control immune responses. Both positive and negative signaling pathways interplay to coordinate development of a resistance response with the appropriate amplitude and duration. AtNUDT7, a Nudix domain-containing protein in Arabidopsis (Arabidopsis thaliana) that hydrolyzes nucleotide derivatives, was found to be a negative regulator of the basal defense response, and its loss-of-function mutation results in enhanced resistance to infection by Pseudomonas syringae. The nudt7 mutation does not cause a strong constitutive disease resistance phenotype, but it leads to a heightened defense response, including accelerated activation of defense-related genes that can be triggered by pathogenic and nonpathogenic microorganisms. The nudt7 mutation enhances two distinct defense response pathways: one independent of and the other dependent on NPR1 and salicylic acid accumulation. In vitro enzymatic assays revealed that ADP-ribose and NADH are preferred substrates of NUDT7, and the hydrolysis activity of NUDT7 is essential for its biological function and is sensitive to inhibition by Ca(2+). Further analyses indicate that ADP-ribose is not likely the physiological substrate of NUDT7. However, the nudt7 mutation leads to perturbation of cellular redox homeostasis and a higher level of NADH in pathogen-challenged leaves. The study suggests that the alteration in cellular antioxidant status caused by the nudt7 mutation primes the cells for the amplified defense response and NUDT7 functions to modulate the defense response to prevent excessive stimulation.

  15. Extracellular Bacterial Proteases in Chronic Wounds: A Potential Therapeutic Target?

    PubMed

    Suleman, Louise

    2016-10-01

    Significance: Bacterial biofilms are considered to be responsible for over 80% of persistent infections, including chronic lung infections, osteomyelitis, periodontitis, endocarditis, and chronic wounds. Over 60% of chronic wounds are colonized with bacteria that reside within a biofilm. The exaggerated proteolytic environment of chronic wounds, more specifically elevated matrix metalloproteinases, is thought to be one of the possible reasons as to why chronic wounds fail to heal. However, the role of bacterial proteases within chronic wounds is not fully understood. Recent Advances: Recent research has shown that bacterial proteases can enable colonization and facilitate bacterial immune evasion. The inhibition of bacterial proteases such as Pseudomonas aeruginosa elastase B (LasB) has resulted in the disruption of the bacterial biofilm in vitro. P. aeruginosa is thought to be a key pathogen in chronic wound infection, and therefore, the disruption of these biofilms, potentially through the targeting of P. aeruginosa bacterial proteases, is an attractive therapeutic endeavor. Critical Issues: Disrupting biofilm formation through the inhibition of bacterial proteases may lead to the dissemination of bacteria from the biofilm, allowing planktonic cells to colonize new sites within the wound. Future Directions: Despite a plethora of evidence supporting the role of bacterial proteases as virulence factors in infection, there remains a distinct lack of research into the effect of bacterial proteases in chronic wounds. To assess the viability of targeting bacterial proteases, future research should aim to understand the role of these proteases in a variety of chronic wound subtypes.

  16. Two distinct amyloid beta-protein (Abeta) assembly pathways leading to oligomers and fibrils identified by combined fluorescence correlation spectroscopy, morphology, and toxicity analyses.

    PubMed

    Matsumura, Satoko; Shinoda, Keiko; Yamada, Mayumi; Yokojima, Satoshi; Inoue, Masafumi; Ohnishi, Takayuki; Shimada, Tetsuya; Kikuchi, Kazuya; Masui, Dai; Hashimoto, Shigeki; Sato, Michio; Ito, Akane; Akioka, Manami; Takagi, Shinsuke; Nakamura, Yoshihiro; Nemoto, Kiyokazu; Hasegawa, Yutaka; Takamoto, Hisayoshi; Inoue, Haruo; Nakamura, Shinichiro; Nabeshima, Yo-ichi; Teplow, David B; Kinjo, Masataka; Hoshi, Minako

    2011-04-01

    Nonfibrillar assemblies of amyloid β-protein (Aβ) are considered to play primary roles in Alzheimer disease (AD). Elucidating the assembly pathways of these specific aggregates is essential for understanding disease pathogenesis and developing knowledge-based therapies. However, these assemblies cannot be monitored in vivo, and there has been no reliable in vitro monitoring method at low protein concentration. We have developed a highly sensitive in vitro monitoring method using fluorescence correlation spectroscopy (FCS) combined with transmission electron microscopy (TEM) and toxicity assays. Using Aβ labeled at the N terminus or Lys(16), we uncovered two distinct assembly pathways. One leads to highly toxic 10-15-nm spherical Aβ assemblies, termed amylospheroids (ASPDs). The other leads to fibrils. The first step in ASPD formation is trimerization. ASPDs of ∼330 kDa in mass form from these trimers after 5 h of slow rotation. Up to at least 24 h, ASPDs remain the dominant structures in assembly reactions. Neurotoxicity studies reveal that the most toxic ASPDs are ∼128 kDa (∼32-mers). In contrast, fibrillogenesis begins with dimer formation and then proceeds to formation of 15-40-nm spherical intermediates, from which fibrils originate after 15 h. Unlike ASPD formation, the Lys(16)-labeled peptide disturbed fibril formation because the Aβ(16-20) region is critical for this final step. These differences in the assembly pathways clearly indicated that ASPDs are not fibril precursors. The method we have developed should facilitate identifying Aβ assembly steps at which inhibition may be beneficial.

  17. Distinct and concurrent pathways of Pol II- and Pol IV-dependent siRNA biogenesis at a repetitive trans-silencer locus in Arabidopsis thaliana.

    PubMed

    Sasaki, Taku; Lee, Tzuu-fen; Liao, Wen-Wei; Naumann, Ulf; Liao, Jo-Ling; Eun, Changho; Huang, Ya-Yi; Fu, Jason L; Chen, Pao-Yang; Meyers, Blake C; Matzke, Antonius J M; Matzke, Marjori

    2014-07-01

    Short interfering RNAs (siRNAs) homologous to transcriptional regulatory regions can induce RNA-directed DNA methylation (RdDM) and transcriptional gene silencing (TGS) of target genes. In our system, siRNAs are produced by transcribing an inverted DNA repeat (IR) of enhancer sequences, yielding a hairpin RNA that is processed by several Dicer activities into siRNAs of 21-24 nt. Primarily 24-nt siRNAs trigger RdDM of the target enhancer in trans and TGS of a downstream GFP reporter gene. We analyzed siRNA accumulation from two different structural forms of a trans-silencer locus in which tandem repeats are embedded in the enhancer IR and distinguished distinct RNA polymerase II (Pol II)- and Pol IV-dependent pathways of siRNA biogenesis. At the original silencer locus, Pol-II transcription of the IR from a 35S promoter produces a hairpin RNA that is diced into abundant siRNAs of 21-24 nt. A silencer variant lacking the 35S promoter revealed a normally masked Pol IV-dependent pathway that produces low levels of 24-nt siRNAs from the tandem repeats. Both pathways operate concurrently at the original silencer locus. siRNAs accrue only from specific regions of the enhancer and embedded tandem repeat. Analysis of these sequences and endogenous tandem repeats producing siRNAs revealed the preferential accumulation of siRNAs at GC-rich regions containing methylated CG dinucleotides. In addition to supporting a correlation between base composition, DNA methylation and siRNA accumulation, our results highlight the complexity of siRNA biogenesis at repetitive loci and show that Pol II and Pol IV use different promoters to transcribe the same template.

  18. High-grade Müllerian Adenosarcoma: Genomic and Clinicopathologic Characterization of a Distinct Neoplasm With Prevalent TP53 Pathway Alterations and Aggressive Behavior.

    PubMed

    Hodgson, Anjelica; Amemiya, Yutaka; Seth, Arun; Djordjevic, Bojana; Parra-Herran, Carlos

    2017-08-22

    Müllerian adenosarcoma harbors low malignant potential, except in cases with myometrial invasion or sarcomatous overgrowth. The presence of a high-grade stromal component has been proposed as an important pathologic predictor of outcome. We hypothesized that high-grade adenosarcoma has distinct clinical and molecular features, distinct from low-grade adenosarcoma. We analyzed the clinicopathologic features and follow-up of 9 high-grade adenosarcomas and a control group of 9 low-grade adenosarcomas. Comprehensive genomic analysis of the high-grade group was performed targeting exons of 409 oncogenes and tumor suppressor genes. In 1 case, the high-grade and low-grade components were separately sequenced. High-grade and low-grade adenosarcomas were comparable in patient age, myometrial invasion, and stage at presentation. Sarcomatous overgrowth was observed in 2/9 (22%) low-grade and 8/9 (89%) high-grade adenosarcomas. Six of 9 (67%) patients with high-grade adenosarcoma developed rapid recurrence; 1 died of her disease. Conversely, no low-grade tumors recurred or metastasized. Sequencing of high-grade adenosarcomas revealed frequent TP53 pathway alterations, identified in 7/9 (78%) cases. p53 expression by immunohistochemistry highly correlated with mutation status. Copy number variations occurred at a mean of 28.8 per tumor; most frequently involved genes included CDK4, MDM2, GNAS, SGK1, and DICER1. High-grade adenosarcoma is an aggressive neoplasm with propensity for short-interval recurrence and metastasis. The proportion of copy number alterations is similar to that reported for adenosarcoma with sarcomatous overgrowth. However, the high frequency of TP53 abnormalities is a novel finding, indicating that high-grade adenosarcoma is a distinct subset with driver TP53 pathway alterations. p53 immunohistochemistry can be used to confirm the presence of a high-grade component. Given its aggressive potential, the presence of any high-grade component in an adenosarcoma

  19. Proteases and Protease Inhibitors of Urinary Extracellular Vesicles in Diabetic Nephropathy

    PubMed Central

    Tataruch, Dorota; Gu, Dongfeng; Liu, Xinyu; Forsblom, Carol; Groop, Per-Henrik; Holthofer, Harry

    2015-01-01

    Diabetic nephropathy (DN) is one of the major complications of diabetes mellitus (DM), leads to chronic kidney disease (CKD), and, ultimately, is the main cause for end-stage kidney disease (ESKD). Beyond urinary albumin, no reliable biomarkers are available for accurate early diagnostics. Urinary extracellular vesicles (UEVs) have recently emerged as an interesting source of diagnostic and prognostic disease biomarkers. Here we used a protease and respective protease inhibitor array to profile urines of type 1 diabetes patients at different stages of kidney involvement. Urine samples were divided into groups based on the level of albuminuria and UEVs isolated by hydrostatic dialysis and screened for relative changes of 34 different proteases and 32 protease inhibitors, respectively. Interestingly, myeloblastin and its natural inhibitor elafin showed an increase in the normo- and microalbuminuric groups. Similarly, a characteristic pattern was observed in the array of protease inhibitors, with a marked increase of cystatin B, natural inhibitor of cathepsins L, H, and B as well as of neutrophil gelatinase-associated Lipocalin (NGAL) in the normoalbuminuric group. This study shows for the first time the distinctive alterations in comprehensive protease profiles of UEVs in diabetic nephropathy and uncovers intriguing mechanistic, prognostic, and diagnostic features of kidney damage in diabetes. PMID:25874235

  20. PSM/SH2-B distributes selected mitogenic receptor signals to distinct components in the PI3-kinase and MAP kinase signaling pathways.

    PubMed

    Deng, Youping; Xu, Hu; Riedel, Heimo

    2007-02-15

    The Pro-rich, PH, and SH2 domain containing mitogenic signaling adapter PSM/SH2-B has been implicated as a cellular partner of various mitogenic receptor tyrosine kinases and related signaling mechanisms. Here, we report in a direct comparison of three peptide hormones, that PSM participates in the assembly of distinct mitogenic signaling complexes in response to insulin or IGF-I when compared to PDGF in cultured normal fibroblasts. The complex formed in response to insulin or IGF-I involves the respective peptide hormone receptor and presumably the established components leading to MAP kinase activation. However, our data suggest an alternative link from the PDGF receptor via PSM directly to MEK1/2 and consequently also to p44/42 activation, possibly through a scaffold protein. At least two PSM domains participate, the SH2 domain anticipated to link PSM to the respective receptor and the Pro-rich region in an association with an unidentified downstream component resulting in direct MEK1/2 and p44/42 regulation. The PDGF receptor signaling complex formed in response to PDGF involves PI 3-kinase in addition to the same components and interactions as described for insulin or IGF-I. PSM associates with PI 3-kinase via p85 and in addition the PSM PH domain participates in the regulation of PI 3-kinase activity, presumably through membrane interaction. In contrast, the PSM Pro-rich region appears to participate only in the MAP kinase signal. Both pathways contribute to the mitogenic response as shown by cell proliferation, survival, and focus formation. PSM regulates p38 MAP kinase activity in a pathway unrelated to the mitogenic response.

  1. Gene Expression Patterns of Hemizygous and Heterozygous KIT Mutations Suggest Distinct Oncogenic Pathways: A Study in NIH3T3 Cell Lines and GIST Samples

    PubMed Central

    Dessaux, Sophie; Besse, Anthony; Brahimi-Adouane, Sabrina; Emile, Jean-François; Blay, Jean-Yves; Alberti, Laurent

    2013-01-01

    Objective Most gain of function mutations of tyrosine kinase receptors in human tumours are hemizygous. Gastrointestinal stromal tumours (GIST) with homozygous mutations have a worse prognosis. We aimed to identify genes differentially regulated by hemizygous and heterozygous KIT mutations. Materials and Methods Expression of 94 genes and 384 miRNA was analysed with low density arrays in five NIH3T3 cell lines expressing the full-length human KIT cDNA wild-type (WT), hemizygous KIT mutation with del557-558 (D6) or del564-581 (D54) and heterozygous WT/D6 or WT/D54. Expression of 5 of these genes and 384 miRNA was then analysed in GISTs samples. Results Unsupervised and supervised hierarchical clustering of the mRNA and miRNA profiles showed that heterozygous mutants clustered with KIT WT expressing cells while hemizygous mutants were distinct. Among hemizygous cells, D6 and D54 expressing cells clustered separately. Most deregulated genes have been reported as potentially implicated in cancer and severals, as ANXA8 and FBN1, are highlighted by both, mRNA and miRNA analyses. MiRNA and mRNA analyses in GISTs samples confirmed that their expressions varied according to the mutation of the alleles. Interestingly, RGS16, a membrane protein of the regulator of G protein family, correlate with the subcellular localization of KIT mutants and might be responsible for regulation of the PI3K/AKT signalling pathway. Conclusion Patterns of mRNA and miRNA expression in cells and tumours depend on heterozygous/hemizygous status of KIT mutations, and deletion/presence of TYR568 & TYR570 residues. Thus each mutation of KIT may drive specific oncogenic pathways. PMID:23593401

  2. The Drosophila TRPP Cation Channel, PKD2 and Dmel/Ced-12 Act in Genetically Distinct Pathways during Apoptotic Cell Clearance

    PubMed Central

    Van Goethem, Emeline; Silva, Elizabeth A.; Xiao, Hui; Franc, Nathalie C.

    2012-01-01

    Apoptosis, a genetically programmed cell death, allows for homeostasis and tissue remodelling during development of all multi-cellular organisms. Phagocytes swiftly recognize, engulf and digest apoptotic cells. Yet, to date the molecular mechanisms underlying this phagocytic process are still poorly understood. To delineate the molecular mechanisms of apoptotic cell clearance in Drosophila, we have carried out a deficiency screen and have identified three overlapping phagocytosis-defective mutants, which all delete the fly homologue of the ced-12 gene, known as Dmel\\ced12. As anticipated, we have found that Dmel\\ced-12 is required for apoptotic cell clearance, as for its C. elegans and mammalian homologues, ced-12 and elmo, respectively. However, the loss of Dmel\\ced-12 did not solely account for the phenotypes of all three deficiencies, as zygotic mutations and germ line clones of Dmel\\ced-12 exhibited weaker phenotypes. Using a nearby genetically interacting deficiency, we have found that the polycystic kidney disease 2 gene, pkd2, which encodes a member of the TRPP channel family, is also required for phagocytosis of apoptotic cells, thereby demonstrating a novel role for PKD2 in this process. We have also observed genetic interactions between pkd2, simu, drpr, rya-r44F, and retinophilin (rtp), also known as undertaker (uta), a gene encoding a MORN-repeat containing molecule, which we have recently found to be implicated in calcium homeostasis during phagocytosis. However, we have not found any genetic interaction between Dmel\\ced-12 and simu. Based on these genetic interactions and recent reports demonstrating a role for the mammalian pkd-2 gene product in ER calcium release during store-operated calcium entry, we propose that PKD2 functions in the DRPR/RTP pathway to regulate calcium homeostasis during this process. Similarly to its C. elegans homologue, Dmel\\Ced-12 appears to function in a genetically distinct pathway. PMID:22347485

  3. Putting one step before the other: distinct activation pathways for Cdk1 and Cdk2 bring order to the mammalian cell cycle.

    PubMed

    Merrick, Karl A; Fisher, Robert P

    2010-02-15

    Eukaryotic cell division is controlled by the activity of cyclin-dependent kinases (CDKs). Cdk1 and Cdk2, which function at different stages of the mammalian cell cycle, both require cyclin-binding and phosphorylation of the activation (T-) loop for full activity, but differ with respect to the order in which the two steps occur in vivo. To form stable complexes with either of its partners-cyclins A and B-Cdk1 must be phosphorylated on its T-loop, but that phosphorylation in turn depends on the presence of cyclin. Cdk2 can follow a kinetically distinct path to activation in which T-loop phosphorylation precedes cyclin-binding, and thereby out-compete the more abundant Cdk1 for limiting amounts of cyclin A. Mathematical modeling suggests this could be a principal basis for the temporal ordering of CDK activation during S phase, which may dictate the sequence in which replication origins fire. Still to be determined are how: (1) the activation machinery discriminates between closely related CDKs, and (2) coordination of the cell cycle is affected when this mechanism of pathway insulation breaks down.

  4. Excitation-transcription coupling in parvalbumin-positive interneurons employs a novel CaM Kinase-dependent pathway distinct from excitatory neurons

    PubMed Central

    Cohen, Samuel M.; Ma, Huan; Kuchibhotla, Kishore V.; Watson, Brendon O.; Buzsáki, György; Froemke, Robert C.; Tsien, Richard W.

    2016-01-01

    Properly functional CNS circuits depend on inhibitory interneurons that in turn rely upon activity-dependent gene expression for morphological development, connectivity and excitatory-inhibitory coordination. Despite its importance, excitation-transcription coupling in inhibitory interneurons is poorly understood. Here, we report that PV+ interneurons employ a novel CaMK-dependent pathway to trigger CREB phosphorylation and gene expression. As in excitatory neurons, voltage-gated Ca2+ influx through CaV1 channels triggers CaM nuclear translocation via local Ca2+ signaling. However, PV+ interneurons are distinct in that nuclear signaling is mediated by γCaMKI, not γCaMKII. CREB phosphorylation also proceeds with slow, sigmoid kinetics, rate-limited by paucity of CaMKIV, protecting against saturation of phospho-CREB in the face of higher firing rates and bigger Ca2+ transients. Our findings support the generality of CaM shuttling to drive nuclear CaMK activity, and are relevant to disease pathophysiology, insofar as dysfunction of PV+ interneurons and molecules underpinning their excitation-transcription coupling both relate to neuropsychiatric disease. PMID:27041500

  5. [Chloroplast Deg proteases].

    PubMed

    Grabsztunowicz, Magda; Luciński, Robert; Baranek, Małgorzata; Sikora, Bogna; Jackowski, Grzegorz

    2011-01-01

    For some chloroplast proteases ATP binding and hydrolysis is not necessary for their catalytic activity, most probably because even strongly unfolded substrates may penetrate their catalytic chamber. Deg1, 2, 5 and 8 are the best known of Arabidopsis thaliana ATP- independent chloroplast proteases, encoded by orthologues of genes coding for DegP, DegQ and DegS proteases of Escherichia coli. Current awareness in the area of structure and functions of chloroplast Degs is much more limited vs the one about their bacterial counterparts. Deg5 and Deg8 form a catalytic heterododecamer which is loosely attached to luminal side of thylakoid membrane. The complex catalyses--supported by Deg1 and one of FtsH proteases--the degradation of PsbA damaged due to plant exposition to elevated irradiance and thus these protease are of key importance for the plants' sensitivity to photoinhibition. Deg2 role in the disposal of damaged PsbA has not been elucidated. Recombinant Deg1 may degrade PsbO and plastocyanin in vitro but it is not clear whether this reaction is performed in vivo as well.

  6. Protease inhibitor studies enrolling.

    PubMed

    1995-01-01

    The protease enzyme is essential for HIV to make copies of itself. So far, research has failed to find a protease inhibitor that works against HIV. It is believed that, regardless of what type of protease inhibitor someone takes, it will need to be supplemented with other anti-HIV drugs. Three protease inhibitors have thus far been found to be safe, although long-term effects are unknown. These drugs are saquinavir, ABT-538, and L-735,524 produced by Hoffman-LaRoche, Abbott, and Merck respectively. Clinical trials of saquinavir are promising but it has not been shown to be the knock-out drug needed. ABT-538 has high bioavailability, but studies are showing it can cause liver and eye damage. L-735,524 studies are showing that resistance develops quite quickly. Future studies at higher doses are expected. To obtain information on protease studies currently looking for participants, contact The Network. Information on other approved, alternative, and experimental drugs is also available.

  7. Expression and functions of proteases in vascular tissues.

    PubMed

    Petzold, H Earl; Zhao, Mingzhe; Beers, Eric P

    2012-05-01

    With the emergence of new models for wood formation and the increasing emphasis on improving the efficiency of cellulosic biofuel production, research on vascular tissue biology has intensified in recent years. Some of the most active areas of research focus on manipulating activity of enzymes in the cellulose, hemicellulose, pectin and lignin pathways. In addition, great strides have been made in the characterization of transcriptional networks controlling genes that affect differentiation, secondary cell wall synthesis and programmed cell death in xylem. Less attention has been devoted to the characterization of proteases that may be important regulators of post-translational events that affect vascular cell differentiation and function and cell wall composition. Several genes for proteases and components of the ubiquitin/26S proteasome pathway are upregulated in xylem and phloem and in cell culture systems for studying the differentiation of xylem tracheary elements (TEs). Although small molecule protease inhibitors have been used to explore the roles of proteases during the differentiation of cultured TEs, only a small number of vascular tissue-associated protease genes have been directly tested to determine whether they play roles in vascular tissue biology. In this report, we review roles for proteases in vascular cell differentiation and function as determined through the use of protease inhibitors and genetic analyses and conclude by identifying opportunities for future research in this area. Copyright © Physiologia Plantarum 2011.

  8. Spatially dependent activation of the patterning protease, Easter.

    PubMed

    LeMosy, Ellen K

    2006-04-17

    The dorsoventral axis of the Drosophila embryo is established by the activating cleavage of a signaling ligand by a serine protease, Easter, only on the ventral side of the embryo. Easter is the final protease in a serine protease cascade in which initial reaction steps appear not to be ventrally restricted, but where Easter activity is promoted ventrally through the action of a spatial cue at an unknown step in the pathway. Here, biochemical studies demonstrate that this spatial control occurs at or above the level of Easter zymogen activation, rather than through direct promotion of Easter's catalytic activity against the signaling ligand.

  9. The site-2 protease.

    PubMed

    Rawson, Robert B

    2013-12-01

    The site-2 protease (S2P) is an unusually-hydrophobic integral membrane protease. It cleaves its substrates, which are membrane-bound transcription factors, within membrane-spanning helices. Although structural information for S2P from animals is lacking, the available data suggest that cleavage may occur at or within the lipid bilayer. In mammalian cells, S2P is essential owing to its activation of the sterol regulatory element binding proteins (SREBPs); in the absence of exogenous lipid, cells lacking S2P cannot survive. S2P is also important in the endoplasmic reticulum (ER) stress response, activating several different membrane-bound transcription factors. Human patients harboring reduction-of-function mutations in S2P exhibit an array of pathologies ranging from skin defects to neurological abnormalities. Surprisingly, Drosophila melanogaster lacking S2P are viable and fertile. This article is part of a Special Issue entitled: Intramembrane Proteases.

  10. A Genomic Analysis of Rat Proteases and Protease Inhibitors

    PubMed Central

    Puente, Xose S.; López-Otín, Carlos

    2004-01-01

    Proteases perform important roles in multiple biological and pathological processes. The availability of the rat genome sequence has facilitated the analysis of the complete protease repertoire or degradome of this model organism. The rat degradome consists of at least 626 proteases and homologs, which are distributed into 24 aspartic, 160 cysteine, 192 metallo, 221 serine, and 29 threonine proteases. This distribution is similar to that of the mouse degradome but is more complex than that of the human degradome composed of 561 proteases and homologs. This increased complexity of rat proteases mainly derives from the expansion of several families, including placental cathepsins, testases, kallikreins, and hematopoietic serine proteases, involved in reproductive or immunological functions. These protease families have also evolved differently in rat and mouse and may contribute to explain some functional differences between these closely related species. Likewise, genomic analysis of rat protease inhibitors has shown some differences with mouse protease inhibitors and the expansion of families of cysteine and serine protease inhibitors in rodents with respect to human. These comparative analyses may provide new views on the functional diversity of proteases and inhibitors and contribute to the development of innovative strategies for treating proteolysis diseases. PMID:15060002

  11. Distinct patterns of mucosal apoptosis in H pylori-associated gastric ulcer are associated with altered FasL and perforin cytotoxic pathways

    PubMed Central

    Souza, Heitor SP; Neves, Marcelo S; Elia, Celeste CS; Tortori, Claudio JA; Dines, Ilana; Martinusso, Cesonia A; Madi, Kalil; Andrade, Leonardo; Castelo-Branco, Morgana TL

    2006-01-01

    AIM: To analyze the level of apoptosis in different mucosal compartments and the differential expression of Fas/Fas-ligand and perforin in H pylori-associated gastric ulcer. METHODS: Antral specimens from patients with H pylori-related active gastric ulcer (GU), H pylori-related gastritis, and non-infected controls were analysed for densities and distribution of apoptotic cells determined by the TdT-mediated dUDP-biotin nick-end-labelling method. GU patients were submitted to eradication therapy with follow-up biopsy after 60 d. Fas, FasL, and perforin-expressing cells were assessed by immunoperoxidase, and with anti-CD3, anti-CD20 and anti-CD68 by double immunofluorescence and confocal microscopy. Quantitative analysis was performed using a computer-assisted image analyser. RESULTS: H pylori-infected antrum showed greater surface epithelial apoptosis which decreased after eradication therapy. In the lamina propria, higher rates of mononuclear cell apoptosis were observed in H pylori-gastritis. Co-expression of Fas with T-cell and macrophage markers was reduced in GU. FasL- and perforin-expressing cells were increased in H pylori-infection and correlated with epithelial apoptosis. Perforin-expressing cells were also increased in GU compared with H pylori-gastritis. CONCLUSION: Epithelial apoptosis is increased in H pylori-infection and correlates to FasL- and perforin-expression by T cells. Expression of perforin is correlated with the tissue damage, and may represent the enhancement of a distinct cytotoxic pathway in GU. Increased expression of FasL not paralleled by Fas on T-cells and macrophages may indicate a reduced susceptibility to the Fas/FasL-mediated apoptosis of lymphoid cells in H pylori-infection. PMID:17036384

  12. Characterization of New Otic Enhancers of the Pou3f4 Gene Reveal Distinct Signaling Pathway Regulation and Spatio-Temporal Patterns

    PubMed Central

    Robert-Moreno, Àlex; Naranjo, Silvia; de la Calle-Mustienes, Elisa; Gómez-Skarmeta, José Luis; Alsina, Berta

    2010-01-01

    POU3F4 is a member of the POU-homedomain transcription factor family with a prominent role in inner ear development. Mutations in the human POU3F4 coding unit leads to X-linked deafness type 3 (DFN3), characterized by conductive hearing loss and progressive sensorineural deafness. Microdeletions found 1 Mb 5′ upstream of the coding region also displayed the same phenotype, suggesting that cis-regulatory elements might be present in that region. Indeed, we and others have recently identified several enhancers at the 1 Mb 5′ upstream interval of the pou3f4 locus. Here we characterize the spatio-temporal patterns of these regulatory elements in zebrafish transgenic lines. We show that the most distal enhancer (HCNR 81675) is activated earlier and drives GFP reporter expression initially to a broad ear domain to progressively restrict to the sensory patches. The proximal enhancer (HCNR 82478) is switched later during development and promotes expression, among in other tissues, in sensory patches from its onset. The third enhancer (HCNR 81728) is also active at later stages in the otic mesenchyme and in the otic epithelium. We also characterize the signaling pathways regulating these enhancers. While HCNR 81675 is regulated by very early signals of retinoic acid, HCNR 82478 is regulated by Fgf activity at a later stage and the HCNR 81728 enhancer is under the control of Hh signaling. Finally, we show that Sox2 and Pax2 transcription factors are bound to HCNR 81675 genomic region during otic development and specific mutations to these transcription factor binding sites abrogates HCNR 81675 enhancer activity. Altogether, our results suggest that pou3f4 expression in inner ear might be under the control of distinct regulatory elements that fine-tune the spatio-temporal activity of this gene and provides novel data on the signaling mechanisms controlling pou3f4 function. PMID:21209840

  13. Proteases in bacterial pathogenesis.

    PubMed

    Ingmer, Hanne; Brøndsted, Lone

    2009-11-01

    Bacterial pathogens rely on proteolysis for protein quality control under adverse conditions experienced in the host, as well as for the timely degradation of central virulence regulators. We have focused on the contribution of the conserved Lon, Clp, HtrA and FtsH proteases to pathogenesis and have highlighted common biological processes for which their activities are important for virulence.

  14. Rapid qualitative protease microassay (RPM).

    PubMed

    Mohan, S; Ma, P W K; Luthe, D S

    2005-09-30

    A rapid qualitative protease microassay (RPM) was developed as an alternative to conventional assays of cysteine protease activity in HPLC fractions. Using this technique protease activity in samples could be visually determined within 5 min. The method was sensitive to 3.3x10(-7) U/mL of papain and detected cysteine protease activity in dilute HPLC fractions with activity of 5.4x10(-5) U/mL. Because the method monitors the decolorization of Coomassie Brilliant Blue stained substrate, it can be modified to detect other classes of proteases.

  15. Cold-adapted proteases as an emerging class of therapeutics.

    PubMed

    Fornbacke, Marcus; Clarsund, Mats

    2013-06-01

    Proteases have been used in medicine for several decades and are an established and well tolerated class of therapeutic agent. These proteases were sourced from mammals or bacteria that exist or have adapted to moderate temperatures (mesophilic organisms); however, proteases derived from organisms from cold environments-cold-adapted or psychrophilic proteases-generally have high specific activity, low substrate affinity, and high catalytic rates at low and moderate temperatures. Made possible by greater flexibility, psychrophilic enzymes interact with and transform the substrate at lower energy costs. Cold-adapted proteases have been used in a wide range of applications, including industrial functions, textiles, cleaning/hygiene products, molecular biology, environmental bioremediations, consumer food products, cosmetics, and pharmaceutical production. In addition to these applications, they have also shown promise as therapeutic modalities for cosmeceutical applications (by reducing glabellar [frown] lines) and a number of disease conditions, including bacterial infections (by disrupting biofilms to prevent bacterial infection), topical wound management (when used as a debridement agent to remove necrotic tissue and fibrin clots), oral/dental health management (by removing plaque and preventing periodontal disease), and in viral infections (by reducing the infectivity of viruses, such as human rhinovirus 16 and herpes simplex virus). Psychrophilic proteases with greater activity and stability (than the original organism-derived variant) have been developed; this coupled with available manufacturing recombinant production techniques suggests that cold-adapted proteases have a promising future as a distinct therapeutic class with diverse clinical applications.

  16. A novel class of cysteine protease inhibitors: solution structure of staphostatin A from Staphylococcus aureus.

    PubMed

    Dubin, Grzegorz; Krajewski, Marcin; Popowicz, Grzegorz; Stec-Niemczyk, Justyna; Bochtler, Matthias; Potempa, Jan; Dubin, Adam; Holak, Tad A

    2003-11-25

    A series of secreted proteases are included among the virulence factors documented for Staphylococcus aureus. In light of increasing antibiotic resistance of this dangerous human pathogen, these proteases are considered as suitable targets for the development of novel therapeutic strategies. The recent discovery of staphostatins, endogenous, highly specific, staphylococcal cysteine protease inhibitors, opened a possibility for structure-based design of low molecular weight analogues. Moreover, the crystal structure of staphostatin B revealed a distinct folding pattern and an unexpected, substrate-like binding mode. The solution structure of staphostatin A reported here confirms that staphostatins constitute a novel, distinct class of cysteine protease inhibitors. In addition, the structure knowledge-based mutagenesis studies shed light on individual structural features of staphostatin A, the inhibition mechanism, and the determinants of distinct specificity of staphostatins toward their target proteases.

  17. Staphylococcal proteases aid in evasion of the human complement system

    PubMed Central

    Jusko, Monika; Potempa, Jan; Kantyka, Tomasz; Bielecka, Ewa; Miller, Halie K.; Kalinska, Magdalena; Dubin, Grzegorz; Garred, Peter; Shaw, Lindsey N.; Blom, Anna M.

    2014-01-01

    Staphylococcus aureus is an opportunistic pathogen that presents severe healthcare concerns due to the prevalence of multiple antibiotic resistant strains. New treatment strategies are urgently needed, which requires an understanding of disease causation mechanisms. Complement is one of the first lines of defense against bacterial pathogens, and S. aureus expresses several specific complement inhibitors. The effect of extracellular proteases from this bacterium on complement, however, has been the subject of limited investigation, except for a recent report regarding cleavage of the C3 component by aureolysin. We demonstrate here that four major extracellular proteases of S. aureus are potent complement inhibitors. Incubation of human serum with the cysteine proteases staphopain A and staphopain B, the serine protease V8, and the metalloproteinase aureolysin resulted in a drastic decrease in the haemolytic activity of serum; whereas two serine-protease like enzymes, SplD and SplE, had no effect. These four proteases were found to inhibit all pathways of complement due to the efficient degradation of several crucial components. Furthermore, S. aureus mutants lacking proteolytic enzymes were found to be more efficiently killed in human blood. Taken together, the major proteases of S. aureus appear to be important for pathogen-mediated evasion of the human complement system. PMID:23838186

  18. Cysteine Protease Inhibitors as Chemotherapy: Lessons from a Parasite Target

    NASA Astrophysics Data System (ADS)

    Selzer, Paul M.; Pingel, Sabine; Hsieh, Ivy; Ugele, Bernhard; Chan, Victor J.; Engel, Juan C.; Bogyo, Matthew; Russell, David G.; Sakanari, Judy A.; McKerrow, James H.

    1999-09-01

    Papain family cysteine proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of cysteine protease inhibitors in cell culture or in vivo. We now report that specific cysteine protease inhibitors kill Leishmania parasites in vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition of Leishmania cysteine protease activity was accompanied by defects in the parasite's lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-protease antibodies with biotinylated surface proteins and accumulation of undigested debris and protease in the flagellar pocket of treated parasites were consistent with a pathway of protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stable in vivo to ameliorate the pathology associated with a mouse model of Leishmania infection.

  19. Distinct effects of methylseleninic acid versus selenite on apoptosis, cell cycle, and protein kinase pathways in DU145 human prostate cancer cells.

    PubMed

    Jiang, Cheng; Wang, Zaisen; Ganther, Howard; Lü, Junxuan

    2002-10-01

    /2 de-phosphorylation changes before the onset of PARP cleavage in MSeA-exposed cells, experiments with phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 did not show an enhancing effect of specific blocking of AKT on MSeA-induction of PARP cleavage. Taken together, exposure of DU145 cells to MSeA versus selenite induced differential patterns of cell cycle arrest and apoptosis execution as well as distinct patterns of effects on AKT, ERK1/2, JNK1/2, and p38MAPK phosphorylation and p27kip1 and p21cip1 expression. Multiple molecular pathways are likely differentially targeted by selenium metabolite pools to mediate cancer chemoprevention.

  20. Different Doublecortin (DCX) Patient Alleles Show Distinct Phenotypes in Cultured Neurons: EVIDENCE FOR DIVERGENT LOSS-OF-FUNCTION AND "OFF-PATHWAY" CELLULAR MECHANISMS.

    PubMed

    Yap, Chan Choo; Digilio, Laura; McMahon, Lloyd; Roszkowska, Matylda; Bott, Christopher J; Kruczek, Kamil; Winckler, Bettina

    2016-12-23

    Doublecortin on the X-chromosome (DCX) is a neuronal microtubule-binding protein with a multitude of roles in neurodevelopment. In humans, DCX is a major genetic locus for X-linked lissencephaly. The best studied defects are in neuronal migration during corticogenesis and in the hippocampus, as well as axon and dendrite growth defects. Much effort has been directed at understanding the molecular and cellular bases of DCX-linked lissencephaly. The focus has been in particular on defects in microtubule assembly and bundling, using knock-out mice and expression of WT and mutant Dcx in non-neuronal cells. Dcx also binds other proteins besides microtubules, such as spinophilin (abbreviated spn; gene name Ppp1r9b protein phosphatase 1 regulatory subunit 9b) and the clathrin adaptors AP-1 and AP-2. Even though many non-sense and missense mutations of Dcx are known, their molecular and cellular defects are still only incompletely understood. It is also largely unknown how neurons are affected by expression of DCX patient alleles. We have now characterized several patient DCX alleles (DCX-R89G, DCX-R59H, DCX-246X, DCX-272X, and DCX-303X) using a gain-of-function dendrite growth assay in cultured rat neurons in combination with the determination of molecular binding activities and subcellular localization in non-neuronal and neuronal cells. First, we find that several mutants (Dcx-R89G and Dcx-272X) were loss-of-function alleles (as had been postulated) but surprisingly acted via different cellular mechanisms. Second, one allele (Dcx-R59H) formed cytoplasmic aggregates, which contained Hspa1B (heat shock protein 1B hsp70) and ubiquitinated proteins, trapped other cytoskeletal proteins, including spinophilin, and led to increased autophagy. This allele could thus be categorized as "off-pathway"/possibly neomorph. Our findings thus suggested that distinct DCX alleles caused dysfunction by different mechanisms. © 2016 by The American Society for Biochemistry and Molecular

  1. Functional MRI evidence for distinctive binding and consolidation pathways for face-name associations: analysis of activation maps and BOLD response amplitudes.

    PubMed

    Robinson-Long, Melissa; Eslinger, Paul J; Wang, Jianli; Meadowcroft, Mark; Yang, Qing X

    2009-10-01

    bilaterally, where multimodal visual association, language, memory, and decision-making systems interact. Higher BOLD response amplitudes in the right fusiform gyrus and the right hippocampus during face-name encoding suggest a potentially specific binding pathway where disparate information might be neurally linked. In contrast, the increased BOLD response in the lingual gyrus during recognition memory may indicate a key neural substrate for memory consolidation and long-term knowledge of what is learned. Whole-brain activation maps revealed task-specific differences in areas of the prefrontal, temporal, and occipital-parietal-temporal junctions as well. Findings suggest that there are distinctive anatomical and physiological nodes for face-name learning and memory within large-scale cortical-subcortical networks. Hence, lesions in fairly widespread cerebral regions may potentially disrupt specific binding and/or memory consolidation processes.

  2. From proteases to proteomics

    PubMed Central

    Neurath, Hans

    2001-01-01

    This personal and professional autobiography covers the 50-yr period of 1950–2000 and includes the following topics: History of the University of Washington School of Medicine and its Department of Biochemistry (Mount Rainier and the University of Washington, recruiting faculty, biology, research programs); scientific editing (publication, Biochemistry, Protein Science, electronic publication); Europe revisited (Heidelberg, approaching retirement, the German Research Center, reunion in Vienna); and 50 yr of research on proteolytic enzymes (trypsin, carboxypeptidases, mast cell proteases, future developments). PMID:11274481

  3. From proteases to proteomics.

    PubMed

    Neurath, H

    2001-04-01

    This personal and professional autobiography covers the 50-yr period of 1950-2000 and includes the following topics: History of the University of Washington School of Medicine and its Department of Biochemistry (Mount Rainier and the University of Washington, recruiting faculty, biology, research programs); scientific editing (publication, Biochemistry, Protein Science, electronic publication); Europe revisited (Heidelberg, approaching retirement, the German Research Center, reunion in Vienna); and 50 yr of research on proteolytic enzymes (trypsin, carboxypeptidases, mast cell proteases, future developments).

  4. Serine protease inhibitors suppress pancreatic endogenous proteases and modulate bacterial neutral proteases.

    PubMed

    Nduaguibe, Chikodili C; Bentsi-Barnes, Kwamina; Mullen, Yoko; Kandeel, Fouad; Al-Abdullah, Ismail

    2010-01-01

    Pefabloc, Trasylol and Urinary Trypsin Inhibitor (UTI) have been reported to be effective serine protease inhibitors that impair pancreatic endogenous proteases resulting in improved islet yield. Here we evaluated the effect of these inhibitors on endogenous proteases (trypsin, chymotrypsin and elastase), bacterial neutral proteases (thermolysin and neutral protease) and islet isolation digestion samples. Protease activity was measured using a fluorimetric assay and islet function was assessed by dynamic perifusion. Trypsin, chymotrypsin and elastase were significantly inhibited by Pefabloc and UTI. Trasylol showed strong inhibitory effects on trypsin and chymotrypsin but also decreased thermolysin activity. UTI was found to inhibit the activity of endogenous proteases and increase the activity of bacterial neutral proteases. Human islets exposed to Pefabloc had reduced insulin response, unlike Trasylol or UTI, which had no detrimental effect on insulin secretion. Although Trasylol was an effective inhibitor of endogenous proteases, FDA regulatory issues preclude its use in clinical application and thus in the isolation process. UTI has the greatest potential because it impairs endogenous pancreatic proteases and enhances digestion enzymes.

  5. Protease-mediated drug delivery

    NASA Astrophysics Data System (ADS)

    Dickson, Eva F.; Goyan, Rebecca L.; Kennedy, James C.; Mackay, M.; Mendes, M. A. K.; Pottier, Roy H.

    2003-12-01

    Drugs used in disease treatment can cause damage to both malignant and normal tissue. This toxicity limits the maximum therapeutic dose. Drug targeting is of high interest to increase the therapeutic efficacy of the drug without increasing systemic toxicity. Certain tissue abnormalities, disease processes, cancers, and infections are characterized by high levels of activity of specific extracellular and/or intracellular proteases. Abnormally high activity levels of specific proteases are present at sites of physical or chemical trauma, blood clots, malignant tumors, rheumatoid arthritis, inflammatory bowel disease, gingival disease, glomerulonerphritis, and acute pancreatitis. Abnormal protease activity is suspected in development of liver thrombosis, pulmonary emphysema, atherosclerosis, and muscular dystrophy. Inactiviating disease-associated proteases by the administration of appropriate protease inhibitors has had limited success. Instead, one could use such proteases to target drugs to treat the condition. Protease mediated drug delivery offers such a possibility. Solubilizing groups are attached to insoluble drugs via a polypeptide chain which is specifically cleavable by certian proteases. When the solubilized drug enounters the protease, the solubilizing moieties are cleaved, and the drug precipitates at the disease location. Thus, a smaller systemic dosage could result in a therapeutic drug concentration at the treatment site with less systemic toxicity.

  6. SK-N-MC cell death occurs by distinct molecular mechanisms in response to hydrogen peroxide and superoxide anions: involvements of JAK2-STAT3, JNK, and p38 MAP kinases pathways.

    PubMed

    Moslehi, Maryam; Yazdanparast, Razieh

    2013-07-01

    Oxidative stress plays a vital role in the pathogenesis of neurodegenerative diseases. Nerve cells are incessantly exposed to environmental stresses leading to overproduction of some harmful species like reactive oxygen species (ROS). ROS including hydrogen peroxide and superoxide anion are potent inducers of various signaling pathways encompassing MAPKs and JAK-STAT pathways. In the current study, we scrutinized the effects of hydrogen peroxide and/or menadione (superoxide anion generator) on JNK/p38-MAPKs and JAK2-STAT3 pathways to elucidate the mechanism(s) by which each oxidant modulated the above-mentioned pathways leading to SK-N-MC cell death. Our results delineated that hydrogen peroxide and superoxide anion radical induced distinct responses as we showed that STAT3 and p38 were activated in response to hydrogen peroxide, but not superoxide anion radicals indicating the specificity in ROS-induced signaling pathways activations and behaviors. We also observed that menadione induced JNK-dependent p53 expression and apoptotic death in SK-N-MC cells while H2O2-induced JNK activation was p53 independent. Thus, we declare that ROS type has a key role in selective instigation of JNK/p38-MAPKs and JAK2-STAT3 pathways in SK-N-MC cells. Identifying these differential behaviors and mechanisms of hydrogen peroxide and superoxide anion functions illuminates the possible therapeutic targets in the prevention or treatment of ROS-induced neurodegenerative diseases such as Alzheimer's disease.

  7. Dose-dependent insulin regulation of insulin-like growth factor binding protein-1 in human endometrial stromal cells is mediated by distinct signaling pathways.

    PubMed

    Lathi, R B; Hess, A P; Tulac, S; Nayak, N R; Conti, M; Giudice, L C

    2005-03-01

    IGF binding protein-1 (IGFBP-1) is a major product of decidualized human endometrial stromal cells and decidua, and as a modulator of IGF action and/or by independent mechanisms, it regulates cell growth and differentiation and embryonic implantation in these tissues. IGFBP-1 secretion is primarily stimulated by progesterone and cAMP and is inhibited by insulin and IGFs. The signaling pathways mediating the latter are not well defined, and the current study was conducted to determine which pathways mediate the effects of insulin on IGFBP-1 mRNA and protein expression by human endometrial stromal cells decidualized in vitro by progesterone. Cells were cultured and treated with different combinations of insulin; wortmannin, an inhibitor of the phosphatidylinositide-3-kinase (PI3-kinase) pathway; and PD98059, an inhibitor of the MAPK pathway. IGFBP-1 mRNA was determined by real-time PCR, and protein secretion in the conditioned medium was measured by ELISA. Activation of the PI3-kinase and the MAPK pathways was assessed by the detection of phosphorylated AKT and ERK in Western blots, respectively. Insulin inhibited IGFBP-1 mRNA and protein secretion in a dose-dependent fashion, with an ED(50) for the latter 0.127 ng/ml (21.6 pm). Inhibitor studies revealed that at low doses, insulin acts through the PI3-kinase pathway, whereas at higher levels it also activates the MAPK pathway in the inhibition of IGFBP-1. The data demonstrate that human endometrium is a target for insulin action in the regulation of IGFBP-1. At physiological levels insulin likely plays a homeostatic role for energy metabolism in the endometrium, and in hyperinsulinemic states, insulin action on the endometrium may activate cellular mitosis via the MAPK pathway and perhaps predispose this tissue to hyperplasia and/or cancer.

  8. Distinct functions for ERKs?

    PubMed Central

    Lloyd, Alison C

    2006-01-01

    The Ras/Raf/MEK/ERK signaling pathway is one of the best understood signal routes in cells. Recent studies add complexity to this cascade by indicating that the two ERK kinases, ERK1 (p44ERK1) and ERK2 (p42ERK2), may have distinct functions. PMID:16879721

  9. Enteric bacterial proteases in inflammatory bowel disease- pathophysiology and clinical implications.

    PubMed

    Carroll, Ian M; Maharshak, Nitsan

    2013-01-01

    Numerous reports have identified a dysbiosis in the intestinal microbiota in patients suffering from inflammatory bowel diseases (IBD), yet the mechanism(s) in which this complex microbial community initiates or perpetuates inflammation remains unclear. The purpose of this review is to present evidence for one such mechanism that implicates enteric microbial derived proteases in the pathogenesis of IBD. We highlight and discuss studies demonstrating that proteases and protease receptors are abundant in the digestive system. Additionally, we investigate studies demonstrating an association between increased luminal protease activity and activation of protease receptors, ultimately resulting in increased intestinal permeability and exacerbation of colitis in animal models as well as in human IBD. Proteases are essential for the normal functioning of bacteria and in some cases can serve as virulence factors for pathogenic bacteria. Although not classified as traditional virulence factors, proteases originating from commensal enteric bacteria also have a potential association with intestinal inflammation via increased enteric permeability. Reports of increased protease activity in stools from IBD patients support a possible mechanism for a dysbiotic enteric microbiota in IBD. A better understanding of these pathways and characterization of the enteric bacteria involved, their proteases, and protease receptors may pave the way for new therapeutic approaches for these diseases.

  10. Proteases in cardiometabolic diseases: Pathophysiology, molecular mechanisms and clinical applications

    PubMed Central

    Hua, Yinan; Nair, Sreejayan

    2014-01-01

    Cardiovascular disease is the leading cause of death in the U.S. and other developed country. Metabolic syndrome, including obesity, diabetes/insulin resistance, hypertension and dyslipidemia is major threat for public health in the modern society. It is well established that metabolic syndrome contributes to the development of cardiovascular disease collective called as cardiometabolic disease. Despite documented studies in the research field of cardiometabolic disease, the underlying mechanisms are far from clear. Proteases are enzymes that break down proteins, many of which have been implicated in various diseases including cardiac disease. Matrix metalloproteinase (MMP), calpain, cathepsin and caspase are among the major proteases involved in cardiac remodeling. Recent studies have also implicated proteases in the pathogenesis of cardiometabolic disease. Elevated expression and activities of proteases in atherosclerosis, coronary heart disease, obesity/insulin-associated heart disease as well as hypertensive heart disease have been documented. Furthermore, transgenic animals that are deficient in or overexpress proteases allow scientists to understand the causal relationship between proteases and cardiometabolic disease. Mechanistically, MMPs and cathepsins exert their effect on cardiometabolic diseases mainly through modifying the extracellular matrix. However, MMP and cathepsin are also reported to affect intracellular proteins, by which they contribute to the development of cardiometabolic diseases. On the other hand, activation of calpain and caspases has been shown to influence intracellular signaling cascade including the NF-κB and apoptosis pathways. Clinically, proteases are reported to function as biomarkers of cardiometabolic diseases. More importantly, the inhibitors of proteases are credited with beneficial cardiometabolic profile, although the exact molecular mechanisms underlying these salutary effects are still under investigation. A better

  11. Protease signaling in animal and plant-regulated cell death.

    PubMed

    Salvesen, Guy S; Hempel, Anne; Coll, Nuria S

    2016-07-01

    This review aims to highlight the proteases required for regulated cell death mechanisms in animals and plants. The aim is to be incisive, and not inclusive of all the animal proteases that have been implicated in various publications. The review also aims to focus on instances when several publications from disparate groups have demonstrated the involvement of an animal protease, and also when there is substantial biochemical, mechanistic and genetic evidence. In doing so, the literature can be culled to a handful of proteases, covering most of the known regulated cell death mechanisms: apoptosis, regulated necrosis, necroptosis, pyroptosis and NETosis in animals. In plants, the literature is younger and not as extensive as for mammals, although the molecular drivers of vacuolar death, necrosis and the hypersensitive response in plants are becoming clearer. Each of these death mechanisms has at least one proteolytic component that plays a major role in controlling the pathway, and sometimes they combine in networks to regulate cell death/survival decision nodes. Some similarities are found among animal and plant cell death proteases but, overall, the pathways that they govern are kingdom-specific with very little overlap. © 2015 FEBS.

  12. Hippocampal Proteomic Analysis Reveals Distinct Pathway Deregulation Profiles at Early and Late Stages in a Rat Model of Alzheimer's-Like Amyloid Pathology.

    PubMed

    Do Carmo, Sonia; Crynen, Gogce; Paradis, Tiffany; Reed, Jon; Iulita, M Florencia; Ducatenzeiler, Adriana; Crawford, Fiona; Cuello, A Claudio

    2017-05-13

    The cerebral accumulation and cytotoxicity of amyloid beta (Aβ) is central to Alzheimer's pathogenesis. However, little is known about how the amyloid pathology affects the global expression of brain proteins at different disease stages. In order to identify genotype and time-dependent significant changes in protein expression, we employed quantitative proteomics analysis of hippocampal tissue from the McGill-R-Thy1-APP rat model of Alzheimer-like amyloid pathology. McGill transgenic rats were compared to wild-type rats at early and late pathology stages, i.e., when intraneuronal Aβ amyloid burden is conspicuous and when extracellular amyloid plaques are abundant with more pronounced cognitive deficits. After correction for multiple testing, the expression levels of 64 proteins were found to be considerably different in transgenic versus wild-type rats at the pre-plaque stage (3 months), and 86 proteins in the post-plaque group (12 months), with only 9 differentially regulated proteins common to the 2 time-points. This minimal overlap supports the hypothesis that different molecular pathways are affected in the hippocampus at early and late stages of the amyloid pathology throughout its continuum. At early stages, disturbances in pathways related to cellular responses to stress, protein homeostasis, and neuronal structure are predominant, while disturbances in metabolic energy generation dominate at later stages. These results shed new light on the molecular pathways affected by the early accumulation of Aβ and how the evolving amyloid pathology impacts other complex metabolic pathways.

  13. Increasing maternal body mass index is associated with systemic inflammation in the mother and the activation of distinct placental inflammatory pathways.

    PubMed

    Aye, Irving L M H; Lager, Susanne; Ramirez, Vanessa I; Gaccioli, Francesca; Dudley, Donald J; Jansson, Thomas; Powell, Theresa L

    2014-06-01

    Obese pregnant women have increased levels of proinflammatory cytokines in maternal circulation and placental tissues. However, the pathways contributing to placental inflammation in obesity are largely unknown. We tested the hypothesis that maternal body mass index (BMI) was associated with elevated proinflammatory cytokines in maternal and fetal circulations and increased activation of placental inflammatory pathways. A total of 60 women of varying pre-/early pregnancy BMI, undergoing delivery by Cesarean section at term, were studied. Maternal and fetal (cord) plasma were collected for analysis of insulin, leptin, IL-1beta, IL-6, IL-8, monocyte chemoattractant protein (MCP) 1, and TNFalpha by multiplex ELISA. Activation of the inflammatory pathways in the placenta was investigated by measuring the phosphorylated and total protein expression of p38-mitogen-activated protein kinase (MAPK), c-Jun-N-terminal kinase (JNK)-MAPK, signal transducer-activated transcription factor (STAT) 3, caspase-1, IL-1beta, IkappaB-alpha protein, and p65 DNA-binding activity. To determine the link between activated placental inflammatory pathways and elevated maternal cytokines, cultured primary human trophoblast (PHT) cells were treated with physiological concentrations of insulin, MCP-1, and TNFalpha, and inflammatory signaling analyzed by Western blot. Maternal BMI was positively correlated with maternal insulin, leptin, MCP-1, and TNFalpha, whereas only fetal leptin was increased with BMI. Placental phosphorylation of p38-MAPK and STAT3, and the expression of IL-1beta protein, were increased with maternal BMI; phosphorylation of p38-MAPK was also correlated with birth weight. In contrast, placental NFkappaB, JNK and caspase-1 signaling, and fetal cytokine levels were unaffected by maternal BMI. In PHT cells, p38-MAPK was activated by MCP-1 and TNFalpha, whereas STAT3 phosphorylation was increased following TNFalpha treatment. Maternal BMI is associated with elevated maternal

  14. Regulation by proteolysis: energy-dependent proteases and their targets.

    PubMed Central

    Gottesman, S; Maurizi, M R

    1992-01-01

    A number of critical regulatory proteins in both prokaryotic and eukaryotic cells are subject to rapid, energy-dependent proteolysis. Rapid degradation combined with control over biosynthesis provides a mechanism by which the availability of a protein can be limited both temporally and spatially. Highly unstable regulatory proteins are involved in numerous biological functions, particularly at the commitment steps in developmental pathways and in emergency responses. The proteases involved in energy-dependent proteolysis are large proteins with the ability to use ATP to scan for appropriate targets and degrade complete proteins in a processive manner. These cytoplasmic proteases are also able to degrade many abnormal proteins in the cell. PMID:1480111

  15. Retrograde lipid traffic in yeast: identification of two distinct pathways for internalization of fluorescent-labeled phosphatidylcholine from the plasma membrane

    PubMed Central

    1993-01-01

    Digital, video-enhanced fluorescence microscopy and spectrofluorometry were used to follow the internalization into the yeast Saccharomyces cerevisiae of phosphatidylcholine molecules labeled on one acyl chain with the fluorescent probe 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD). Two pathways were found: (1) transport by endocytosis to the vacuole and (2) transport by a non-endocytic pathway to the nuclear envelope and mitochondria. The endocytic pathway was inhibited at low temperature (< 2 degrees C) and by ATP depletion. Mutations in secretory (SEC) genes that are necessary for membrane traffic through the secretory pathway (including SEC1, SEC2, SEC4, SEC6, SEC7, SEC12, SEC14, SEC17, SEC18, and SEC21) almost completely blocked endocytic uptake. In contrast, mutations in the SEC63, SEC65, or SEC11 genes, required for translocation of nascent secretory polypeptides into the ER or signal peptide processing in the ER, only slightly reduced endocytic uptake. Phospholipid endocytosis was also independent of the gene encoding the clathrin heavy chain, CHC1. The correlation of biochemical analysis with fluorescence microscopy indicated that the fluorescent phosphatidylcholine was degraded in the vacuole and that degradation was, at least in part, dependent on the vacuolar proteolytic cascade. The non-endocytic route functioned with a lower cellular energy charge (ATP levels 80% reduced) and was largely independent of the SEC genes. Non-endocytic transport of NBD-phosphatidylcholine to the nuclear envelope and mitochondria was inhibited by pretreatment of cells with the sulfhydryl reagents N-ethylmaleimide and p- chloromercuribenzenesulfonic acid, suggesting the existence of protein- mediated transmembrane transfer (flip-flop) of phosphatidylcholine across the yeast plasma membrane. These data establish a link between lipid movement during secretion and endocytosis in yeast and suggest that phospholipids may also gain access to intracellular organelles through non

  16. A dominant-negative clathrin mutant differentially affects trafficking of molecules with distinct sorting motifs in the class II major histocompatibility complex (MHC) pathway.

    PubMed

    Liu, S H; Marks, M S; Brodsky, F M

    1998-03-09

    The role of clathrin in intracellular sorting was investigated by expression of a dominant-negative mutant form of clathrin, termed the hub fragment. Hub inhibition of clathrin-mediated membrane transport was established by demonstrating a block of transferrin internalization and an alteration in the intracellular distribution of the cation-independent mannose-6-phosphate receptor. Hubs had no effect on uptake of FITC-dextran, adaptor distribution, organelle integrity in the secretory pathway, or cell surface expression of constitutively secreted molecules. Hub expression blocked lysosomal delivery of chimeric molecules containing either the tyrosine-based sorting signal of H2M or the dileucine-based sorting signal of CD3gamma, confirming a role for clathrin-coated vesicles (CCVs) in recognizing these signals and sorting them to the endocytic pathway. Hub expression was then used to probe the role of CCVs in targeting native molecules bearing these sorting signals in the context of HLA-DM and the invariant chain (I chain) complexed to HLA-DR. The distribution of these molecules was differentially affected. Accumulation of hubs before expression of the DM dimer blocked DM export from the TGN, whereas hubs had no effect on direct targeting of the DR-I chain complex from the TGN to the endocytic pathway. However, concurrent expression of hubs, such that hubs were building to inhibitory concentrations during DM or DR-I chain expression, caused cell surface accumulation of both complexes. These observations suggest that both DM and DR-I chain are directly transported to the endocytic pathway from the TGN, DM in CCVs, and DR-I chain independent of CCVs. Subsequently, both complexes can appear at the cell surface from where they are both internalized by CCVs. Differential packaging in CCVs in the TGN, mediated by tyrosine- and dileucine-based sorting signals, could be a mechanism for functional segregation of DM from DR-I chain until their intended rendezvous in late

  17. A Distinct Profile of Tryptophan Metabolism along the Kynurenine Pathway Downstream of Toll-Like Receptor Activation in Irritable Bowel Syndrome

    PubMed Central

    Clarke, Gerard; McKernan, Declan P.; Gaszner, Gabor; Quigley, Eamonn M.; Cryan, John F.; Dinan, Timothy G.

    2012-01-01

    Irritable bowel syndrome (IBS), a disorder of the brain-gut axis, is characterised by the absence of reliable biological markers. Tryptophan is an essential amino acid that serves as a precursor to serotonin but which can alternatively be metabolised along the kynurenine pathway leading to the production of other neuroactive agents. We previously reported an increased degradation of tryptophan along this immunoresponsive pathway in IBS. Recently, altered cytokine production following activation of specific members of the toll-like receptor (TLR) family (TLR1-9) has also been demonstrated in IBS. However, the relationship between TLR activation and kynurenine pathway activity in IBS is unknown. In this study, we investigated whether activation of specific TLRs elicits exaggerated kynurenine production in IBS patients compared to controls. Whole blood from IBS patients and healthy controls was cultured with a panel of nine different TLR agonists for 24 h. Cell culture supernatants were then analyzed for both tryptophan and kynurenine concentrations, as were plasma samples from both cohorts. IBS subjects had an elevated plasma kynurenine:tryptophan ratio compared to healthy controls. Furthermore, we demonstrated a differential downstream profile of kynurenine production subsequent to TLR activation in IBS patients compared to healthy controls. This profile included alterations at TLR1/2, TLR2, TLR3, TLR5, TLR7, and TLR8. Our data expands on our previous understanding of altered tryptophan metabolism in IBS and suggests that measurement of tryptophan metabolites downstream of TLR activation may ultimately find utility as components of a biomarker panel to aid gastroenterologists in the diagnosis of IBS. Furthermore, these studies implicate the modulation of TLRs as means through which aberrant tryptophan metabolism along the kynurenine pathway can be controlled, a novel potential therapeutic strategy in this and other disorders. PMID:22661947

  18. Human pre-B cell receptor signal transduction: evidence for distinct roles of PI3kinase and MAP-kinase signalling pathways

    PubMed Central

    Anbazhagan, Kolandaswamy; Rabbind Singh, Amrathlal; Isabelle, Piec; Stella, Ibata; Céline, Alleaume-De Martel; Bissac, Eliane; Bertrand, Brassart; Rémy, Nyga; Naomi, Taylor; Vincent, Fuentes; Rochette, Jacques; Lassoued, Kaïss

    2013-01-01

    Pre-BCR acts as a critical checkpoint in B cell development. However, its signalling cascade still remains indistinctly characterised in human. We investigated pre-BCR signalling pathway to examine its regulation in normal primary pre-B lymphocytes and pre-B cell lines. In cell lines, early signalling events occurring after pre-BCR stimulation include phosphorylation of Lyn, Blk and Syk together with ZAP70, Btk, Vav, PLC-γ2 and various adaptor proteins, such as BLNK, LAB, LAT and SLP-76. Further downstream, these molecules induced activation of the PI3K/AKT and MAP-kinase resulting in an augmentation of canonical NF-κB pathways and cFos/AP1 activation. PI3K and MAPK exerted opposing effects on the pre-BCR-induced activation of the canonical NF-κB and c-Fos/AP1 pathways. Immediate nuclear export of FoxO3A and delayed import of IRF4 were additional events observed after pre-BCR crosslinking in primary cells. Pre-BCR-induced down-regulation of Rag1, Rag2, E2A and Pax5 transcripts occurred in a PI3K-dependent manner. Finally we bring evidence that pre-BCR stimulation or co stimulation with CD19 enhances cell cycle signal. PMID:25400915

  19. Human pre-B cell receptor signal transduction: evidence for distinct roles of PI3kinase and MAP-kinase signalling pathways.

    PubMed

    Anbazhagan, Kolandaswamy; Rabbind Singh, Amrathlal; Isabelle, Piec; Stella, Ibata; Céline, Alleaume-De Martel; Bissac, Eliane; Bertrand, Brassart; Rémy, Nyga; Naomi, Taylor; Vincent, Fuentes; Rochette, Jacques; Lassoued, Kaïss

    2013-10-01

    Pre-BCR acts as a critical checkpoint in B cell development. However, its signalling cascade still remains indistinctly characterised in human. We investigated pre-BCR signalling pathway to examine its regulation in normal primary pre-B lymphocytes and pre-B cell lines. In cell lines, early signalling events occurring after pre-BCR stimulation include phosphorylation of Lyn, Blk and Syk together with ZAP70, Btk, Vav, PLC-γ2 and various adaptor proteins, such as BLNK, LAB, LAT and SLP-76. Further downstream, these molecules induced activation of the PI3K/AKT and MAP-kinase resulting in an augmentation of canonical NF-κB pathways and cFos/AP1 activation. PI3K and MAPK exerted opposing effects on the pre-BCR-induced activation of the canonical NF-κB and c-Fos/AP1 pathways. Immediate nuclear export of FoxO3A and delayed import of IRF4 were additional events observed after pre-BCR crosslinking in primary cells. Pre-BCR-induced down-regulation of Rag1, Rag2, E2A and Pax5 transcripts occurred in a PI3K-dependent manner. Finally we bring evidence that pre-BCR stimulation or co stimulation with CD19 enhances cell cycle signal.

  20. Proteases from psychrotrophs: an overview.

    PubMed

    Kasana, Ramesh Chand

    2010-05-01

    Proteases are hydrolytic enzymes which catalyze the total hydrolysis of proteins in to amino acids. Although proteolytic enzymes can be obtained from animals and plants but microorganisms are the preferred source for industrial applications in view of scientific and economical advantage. Among various groups of microbes, psychrotrophs are ideal candidates for enzymes production keeping in mind that enzymes active at low temperature and stable under alkaline condition, in presence of oxidants and detergents are in large demand as laundry additive. The proteases from psychrotrophs also find application in environmental bioremediation, food and molecular biology. During the previous two decades, proteases from psychrotrophs have received increased attention because of their wide range of applications, but the full potential of psychrotrophic proteases has not been exploited. This review focuses attention on the present status of knowledge on the production, optimization, molecular characteristics, applications, substrate specificity, and crystal structure of psychrotrophic proteases. The review will help in making strategies for exploitation of psychrotrophic protease resources and improvement of enzymes to obtain more robust proteases of industrial and biotechnological significance.

  1. Proteases in gastrointestinal neoplastic diseases.

    PubMed

    Herszényi, L; Plebani, M; Carraro, P; De Paoli, M; Roveroni, G; Cardin, R; Foschia, F; Tulassay, Z; Naccarato, R; Farinati, F

    2000-02-15

    Cysteine and serine proteases are involved in cancer invasion and metastasis. In the past few years we investigated the tissue levels of these proteases in gastric cancer (GC), gastric precancerous changes (CAG), colorectal cancer (CRC) and the plasma and serum levels of proteases in several gastrointestinal tumours, using ELISA methods. Significantly higher antigen levels were found not only in GC tissue but also in CAG with respect to the levels found normal tissue; with respect to CAG, patients with dysplasia had higher levels than patients without dysplasia. The same findings were obtained in CRC. In general protease levels correlated with the major histomorphological parameters, such as grading and histotype in GC as well as in CRC. Tissue protease levels had a strong prognostic impact in GC, in which UPA was singled out by multivariate analysis as the major prognostic factor, and CRC. The plasma levels of urokinase-type plasminogen activator (UPA) and the serum levels of cathepsin B were significantly increased in patients with gastrointestinal tumours. In conclusions, cysteine and serine proteases may have a part not only in GC and CRC invasion and metastasis, but also in the progression of gastric precancerous changes into cancer. They are strong prognostic factors in GC and CRC. These proteases may also have a role as tumour markers in the early diagnosis of gastrointestinal tract tumours.

  2. Pseudomonas aeruginosa protease IV degrades surfactant proteins and inhibits surfactant host defense and biophysical functions.

    PubMed

    Malloy, Jaret L; Veldhuizen, Ruud A W; Thibodeaux, Brett A; O'Callaghan, Richard J; Wright, Jo Rae

    2005-02-01

    Pulmonary surfactant has two distinct functions within the lung: reduction of surface tension at the air-liquid interface and participation in innate host defense. Both functions are dependent on surfactant-associated proteins. Pseudomonas aeruginosa is primarily responsible for respiratory dysfunction and death in cystic fibrosis patients and is also a leading pathogen in nosocomial pneumonia. P. aeruginosa secretes a number of proteases that contribute to its virulence. We hypothesized that P. aeruginosa protease IV degrades surfactant proteins and results in a reduction in pulmonary surfactant host defense and biophysical functions. Protease IV was isolated from cultured supernatant of P. aeruginosa by gel chromatography. Incubation of cell-free bronchoalveolar lavage fluid with protease IV resulted in degradation of surfactant proteins (SP)-A, -D, and -B. SPs were degraded in a time- and dose-dependent fashion by protease IV, and degradation was inhibited by the trypsin-like serine protease inhibitor Nalpha-p-tosyl-L-lysine-chloromethyl ketone (TLCK). Degradation by protease IV inhibited SP-A- and SP-D-mediated bacterial aggregation and uptake by macrophages. Surfactant treated with protease IV was unable to reduce surface tension as effectively as untreated surfactant, and this effect was inhibited by TLCK. We speculate that protease IV may be an important contributing factor to the development and propagation of acute lung injury associated with P. aeruginosa via loss of surfactant function within the lung.

  3. Cathepsin proteases in Toxoplasma gondii

    PubMed Central

    Dou, Zhicheng; Carruthers, Vern B.

    2014-01-01

    Cysteine proteases are important for the growth and survival of apicomplexan parasites that infect humans. The apicomplexan Toxoplasma gondii expresses five members of the C1 family of cysteine proteases, including one cathepsin L-like (TgCPL), one cathepsin B-like (TgCPB), and three cathepsin C-like (TgCPC1, 2 and 3) proteases. Recent genetic, biochemical and structural studies reveal that cathepsins function in microneme and rhoptry protein maturation, host cell invasion, replication, and nutrient acquisition.. Here, we review the key features and roles of T. gondii cathepsins and discuss the therapeutic potential for specific inhibitor development. PMID:21660658

  4. Distinct MAPK signaling pathways, p21 up-regulation and caspase-mediated p21 cleavage establishes the fate of U937 cells exposed to 3-hydrogenkwadaphnin: Differentiation versus apoptosis

    SciTech Connect

    Moosavi, Mohammad Amin; Yazdanparast, Razieh

    2008-07-01

    Despite the depth of knowledge concerning the pathogenesis of acute myeloblastic leukemia (AML), long-term survival remains unresolved. Therefore, new agents that act more selectively and more potently are required. In that line, we have recently characterized a novel diterpene ester, called 3-hydrogenkwadaphnin (3-HK), with capability to induce both differentiation and apoptosis in various leukemia cell lines. These effects of 3-HK were mediated through inhibition of inosine 5'-monophosphate dehydrogenase, a selective up-regulated enzyme in cancerous cells, especially leukemia. However, it remains elusive to understand how cells display different fates in response to 3-HK. Here, we report the distinct molecular signaling pathways involved in forcing of 3-HK-treated U937 cells to undergo differentiation and apoptosis. After 3-HK (15 nM) treatment, a portion of U937 cells adhered to the culture plates and showed macrophage criteria while others remained in suspension and underwent apoptosis. The differentiated cells arrested in G{sub 0}/G{sub 1} phase of cell cycle and showed early activation of ERK1/2 pathway (3 h) along with ERK-dependent p21{sup Cip/WAF1} (p21) up-regulation and expression of p27{sup Kip1} and Bcl-2. In contrast, the suspension cells underwent apoptosis through Fas/FasL and mitochondrial pathways. The occurrence of apoptosis in these cells were accompanied with caspase-8-mediated p21 cleavage and delayed activation (24 h) of JNK1/2 and p38 MAPK. Taken together, these results suggest that distinct signaling pathways play a pivotal role in fates of drug-treated leukemia cells, thus this may pave some novel therapeutical utilities.

  5. Distinct Nongenomic Signal Transduction Pathways Controlled by 17β-Estradiol Regulate DNA Synthesis and Cyclin D1 Gene Transcription in HepG2 Cells

    PubMed Central

    Marino, Maria; Acconcia, Filippo; Bresciani, Francesco; Weisz, Alessandro; Trentalance, Anna

    2002-01-01

    Estrogens induce cell proliferation in target tissues by stimulating progression through the G1 phase of the cell cycle. Activation of cyclin D1 gene expression is a critical feature of this hormonal action. The existence of rapid/nongenomic estradiol-regulated protein kinase C (PKC-α) and extracellular signal-regulated kinase (ERK) signal transduction pathways, their cross talk, and role played in DNA synthesis and cyclin D1 gene transcription have been studied herein in human hepatoma HepG2 cells. 17β-Estradiol was found to rapidly activate PKC-α translocation and ERK-2/mitogen-activated protein kinase phosphorylation in this cell line. These actions were independent of each other, preceding the increase of thymidine incorporation into DNA and cyclin D1 expression, and did not involve DNA binding by estrogen receptor. The results obtained with specific inhibitors indicated that PKC-α pathway is necessary to mediate the estradiol-induced G1-S progression of HepG2 cells, but it does not exert any effect(s) on cyclin D1 gene expression. On the contrary, ERK-2 cascade was strongly involved in both G1-S progression and cyclin D1 gene transcription. Deletion of its activating protein-1 responsive element motif resulted in attenuation of cyclin D1 promoter responsiveness to estrogen. These results indicate that estrogen-induced cyclin D1 transcription can occur in HepG2 cells independently of the transcriptional activity of estrogen receptor, sustaining the pivotal role played by nongenomic pathways of estrogen action in hormone-induced proliferation. PMID:12388769

  6. Microarray and Pathway Analysis Reveal Distinct Mechanisms Underlying Cannabinoid-Mediated Modulation of LPS-Induced Activation of BV-2 Microglial Cells

    PubMed Central

    Juknat, Ana; Kozela, Ewa; Rimmerman, Neta; Levy, Rivka; Gao, Fuying; Coppola, Giovanni; Geschwind, Daniel; Vogel, Zvi

    2013-01-01

    Cannabinoids are known to exert immunosuppressive activities. However, the mechanisms which contribute to these effects are unknown. Using lipopolysaccharide (LPS) to activate BV-2 microglial cells, we examined how Δ9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, and cannabidiol (CBD) the non-psychoactive component, modulate the inflammatory response. Microarray analysis of genome-wide mRNA levels was performed using Illumina platform and the resulting expression patterns analyzed using the Ingenuity Pathway Analysis to identify functional subsets of genes, and the Ingenuity System Database to denote the gene networks regulated by CBD and THC. From the 5338 transcripts that were differentially expressed across treatments, 400 transcripts were found to be upregulated by LPS, 502 by CBD+LPS and 424 by THC+LPS, while 145 were downregulated by LPS, 297 by CBD+LPS and 149 by THC+LPS, by 2-fold or more (p≤0.005). Results clearly link the effects of CBD and THC to inflammatory signaling pathways and identify new cannabinoid targets in the MAPK pathway (Dusp1, Dusp8, Dusp2), cell cycle related (Cdkn2b, Gadd45a) as well as JAK/STAT regulatory molecules (Socs3, Cish, Stat1). The impact of CBD on LPS-stimulated gene expression was greater than that of THC. We attribute this difference to the fact that CBD highly upregulated several genes encoding negative regulators of both NFκB and AP-1 transcriptional activities, such as Trib3 and Dusp1 known to be modulated through Nrf2 activation. The CBD-specific expression profile reflected changes associated with oxidative stress and glutathione depletion via Trib3 and expression of ATF4 target genes. Furthermore, the CBD affected genes were shown to be controlled by nuclear factors usually involved in regulation of stress response and inflammation, mainly via Nrf2/Hmox1 axis and the Nrf2/ATF4-Trib3 pathway. These observations indicate that CBD, and less so THC, induce a cellular stress response and

  7. Large-conductance channel formation mediated by P2X7 receptor activation is regulated through distinct intracellular signaling pathways in peritoneal macrophages and 2BH4 cells.

    PubMed

    Faria, R X; Cascabulho, C M; Reis, R A M; Alves, Luiz Anastácio

    2010-07-01

    The P2X(7) receptor (P2X7R) is a ligand-gated ATP receptor that acts as a low- and large-conductance channel (pore) and is known to be coupled to several downstream effectors. Recently, we demonstrated that the formation of a large-conductance channel associated with the P2X(7) receptor is induced by increasing the intracellular Ca(2+) concentration (Faria et al., Am J Physiol Cell Physiol 297:C28-C42, 2005). Here, we investigated the intracellular signaling pathways associated with P2X(7) large-conductance channel formation using the patch clamp technique in conjunction with fluorescent imaging and flow cytometry assays in 2BH4 cells and peritoneal macrophages. Different antagonists were applied to investigate the following pathways: Ca(2+)-calmodulin, phospholipase A, phospholipase D, phospholipase C, protein kinase C (PKC), mitogen-activated protein kinase (MAPK), MAPK/extracellular signal-regulated kinase, phosphoinositide 3-kinase (PI3K), and cytoskeletal proteins. Macroscopic ionic currents induced by 1 mM ATP were reduced by 85% in the presence of PKC antagonists. The addition of antagonists for MAPK, PI3K, and the cytoskeleton (actin, intermediary filament, and microtubule) blocked 92%, 83%, and 95% of the ionic currents induced by 1 mM ATP, respectively. Our results show that PKC, MAPK, PI3K, and cytoskeletal components are involved in P2X(7) receptor large-channel formation in 2BH4 cells and peritoneal macrophages.

  8. Exogenous proteases for meat tenderization.

    PubMed

    Bekhit, Alaa A; Hopkins, David L; Geesink, Geert; Bekhit, Adnan A; Franks, Philip

    2014-01-01

    The use of exogenous proteases to improve meat tenderness has attracted much interest recently, with a view to consistent production of tender meat and added value to lower grade meat cuts. This review discusses the sources, characteristics, and use of exogenous proteases in meat tenderization to highlight the specificity of the proteases toward meat proteins and their impact on meat quality. Plant enzymes (such as papain, bromelain, and ficin) have been extensively investigated as meat tenderizers. New plant proteases (actinidin and zingibain) and microbial enzyme preparations have been of recent interest due to controlled meat tenderization and other advantages. Successful use of these enzymes in fresh meat requires their enzymatic kinetics and characteristics to be determined, together with an understanding of the impact of the surrounding environmental conditions of the meat (pH, temperature) on enzyme function. This enables the optimal conditions for tenderizing fresh meat to be established, and the elimination or reduction of any negative impacts on other quality attributes.

  9. Serine Proteases of Parasitic Helminths

    PubMed Central

    Yang, Yong; Wen, Yun jun; Cai, Ya Nan; Vallée, Isabelle; Boireau, Pascal; Liu, Ming Yuan; Cheng, Shi Peng

    2015-01-01

    Serine proteases form one of the most important families of enzymes and perform significant functions in a broad range of biological processes, such as intra- and extracellular protein metabolism, digestion, blood coagulation, regulation of development, and fertilization. A number of serine proteases have been identified in parasitic helminths that have putative roles in parasite development and nutrition, host tissues and cell invasion, anticoagulation, and immune evasion. In this review, we described the serine proteases that have been identified in parasitic helminths, including nematodes (Trichinella spiralis, T. pseudospiralis, Trichuris muris, Anisakis simplex, Ascaris suum, Onchocerca volvulus, O. lienalis, Brugia malayi, Ancylostoma caninum, and Steinernema carpocapsae), cestodes (Spirometra mansoni, Echinococcus granulosus, and Schistocephalus solidus), and trematodes (Fasciola hepatica, F. gigantica, and Schistosoma mansoni). Moreover, the possible biological functions of these serine proteases in the endogenous biological phenomena of these parasites and in the host-parasite interaction were also discussed. PMID:25748703

  10. Application of Protease Technology in Dermatology

    PubMed Central

    Del Rosso, James Q.

    2013-01-01

    This article reviews background on proteases and their functions, their physiological significance in skin, and the potential implications of incorporating specific proteases and protease blends into dermatological products, including skin care formulations. The history of protease blend formulations used in wound model studies and for other disorders is reviewed. In vitro data with use of a specific 3-protease blend with evaluation of the impact on various skin proteins and peptides is also discussed in this article. PMID:23882305

  11. The chloroplast ATP-dependent Clp protease in vascular plants - new dimensions and future challenges.

    PubMed

    Clarke, Adrian K

    2012-05-01

    The ATP-dependent Clp protease is by far the most intricate protease in chloroplasts of vascular plants. Structurally, it is particularly complex with a proteolytic core complex containing 11 distinct subunits along with three potential chaperone partners. The Clp protease is also essential for chloroplast development and overall plant viability. Over the past decade, many of the important characteristics of this crucial protease have been revealed in the model plant species Arabidopsis thaliana. Despite this, challenges still remain in fully resolving certain key features, in particular, how the assembly of this multisubunit protease is regulated, the full range of native protein substrates and how they are targeted for degradation and how this complicated enzyme might have developed from simpler bacterial forms. This article focuses upon the recent advances in revealing the details underlying these important features. It also take the opportunity to speculate upon many of these findings in the hope of stimulating further investigation.

  12. NFAT activation by membrane potential follows a calcium pathway distinct from other activity-related transcription factors in skeletal muscle cells.

    PubMed

    Valdés, Juan Antonio; Gaggero, Eduardo; Hidalgo, Jorge; Leal, Nancy; Jaimovich, Enrique; Carrasco, M Angélica

    2008-03-01

    Depolarization of skeletal muscle cells triggers intracellular Ca2+ signals mediated by ryanodine and inositol 1,4,5-trisphosphate (IP3) receptors. Previously, we have reported that K+-induced depolarization activates transcriptional regulators ERK, cAMP response element-binding protein, c-fos, c-jun, and egr-1 through IP3-dependent Ca2+ release, whereas NF-kappa B activation is elicited by both ryanodine and IP3 receptor-mediated Ca2+ signals. We have further shown that field stimulation with electrical pulses results in an NF-kappa B activation increase dependent of the amount of pulses and independent of their frequency. In this work, we report the results obtained for nuclear factor of activated T cells (NFAT)-mediated transcription and translocation generated by both K+ and electrical stimulation protocols in primary skeletal muscle cells and C2C12 cells. The Ca2+ source for NFAT activation is through release by ryanodine receptors and extracellular Ca2+ entry. We found this activation to be independent of the number of pulses within a physiological range of stimulus frequency and enhanced by long-lasting low-frequency stimulation. Therefore, activation of the NFAT signaling pathway differs from that of NF-kappa B and other transcription factors. Calcineurin enzyme activity correlated well with the relative activation of NFAT translocation and transcription using different stimulation protocols. Furthermore, both K+-induced depolarization and electrical stimulation increased mRNA levels of the type 1 IP3 receptor mediated by calcineurin activity, which suggests that depolarization may regulate IP3 receptor transcription. These results confirm the presence of at least two independent pathways for excitation-transcription coupling in skeletal muscle cells, both dependent on Ca2+ release and triggered by the same voltage sensor but activating different intracellular release channels.

  13. Suppression of Canine Dendritic Cell Activation/Maturation and Inflammatory Cytokine Release by Mesenchymal Stem Cells Occurs Through Multiple Distinct Biochemical Pathways.

    PubMed

    Wheat, William H; Chow, Lyndah; Kurihara, Jade N; Regan, Daniel P; Coy, Jonathan W; Webb, Tracy L; Dow, Steven W

    2017-02-15

    Mesenchymal stem cells (MSC) represent a readily accessible source of cells with potent immune modulatory activity. MSC can suppress ongoing inflammatory responses by suppressing T cell function, while fewer studies have examined the impact of MSC on dendritic cell (DC) function. The dog spontaneous disease model represents an important animal model with which to evaluate the safety and effectiveness of cellular therapy with MSC. This study evaluated the effects of canine MSC on the activation and maturation of canine monocyte-derived DC, as well as mechanisms underlying these effects. Adipose-derived canine MSC were cocultured with canine DC, and the MSC effects on DC maturation and activation were assessed by flow cytometry, cytokine ELISA, and confocal microscopy. We found that canine MSC significantly suppressed lipopolysaccharide (LPS)-stimulated upregulation of DC activation markers such as major histocompatibility class II (MHCII), CD86, and CD40. Furthermore, pretreatment of MSC with interferon gamma (IFNγ) augmented this suppressive activity. IFNγ-activated MSC also significantly reduced LPS-elicited DC secretion of tumor necrosis factor alpha without reducing secretion of interleukin-10. The suppressive effect of IFNγ-treated MSC on LPS-induced DC activation was mediated by soluble factors secreted by both MSC and DC. Pathways of DC functional suppression included programmed death ligand-1 expression and secretion of nitrous oxide, prostaglandin E2, and adenosine by activated MSC. Coculture of DC with IFNγ-treated MSC maintained DC in an immature state and prolonged DC antigen uptake during LPS maturation stimulus. Taken together, canine MSC are capable of potently suppressing DC function in a potentially inflammatory microenvironment through several separate immunological pathways and confirm the potential for immune therapy with MSC in canine immune-mediated disease models.

  14. Community structure analysis of transcriptional networks reveals distinct molecular pathways for early- and late-onset temporal lobe epilepsy with childhood febrile seizures.

    PubMed

    Moreira-Filho, Carlos Alberto; Bando, Silvia Yumi; Bertonha, Fernanda Bernardi; Iamashita, Priscila; Silva, Filipi Nascimento; Costa, Luciano da Fontoura; Silva, Alexandre Valotta; Castro, Luiz Henrique Martins; Wen, Hung-Tzu

    2015-01-01

    Age at epilepsy onset has a broad impact on brain plasticity and epilepsy pathomechanisms. Prolonged febrile seizures in early childhood (FS) constitute an initial precipitating insult (IPI) commonly associated with mesial temporal lobe epilepsy (MTLE). FS-MTLE patients may have early disease onset, i.e. just after the IPI, in early childhood, or late-onset, ranging from mid-adolescence to early adult life. The mechanisms governing early (E) or late (L) disease onset are largely unknown. In order to unveil the molecular pathways underlying E and L subtypes of FS-MTLE we investigated global gene expression in hippocampal CA3 explants of FS-MTLE patients submitted to hippocampectomy. Gene coexpression networks (GCNs) were obtained for the E and L patient groups. A network-based approach for GCN analysis was employed allowing: i) the visualization and analysis of differentially expressed (DE) and complete (CO) - all valid GO annotated transcripts - GCNs for the E and L groups; ii) the study of interactions between all the system's constituents based on community detection and coarse-grained community structure methods. We found that the E-DE communities with strongest connection weights harbor highly connected genes mainly related to neural excitability and febrile seizures, whereas in L-DE communities these genes are not only involved in network excitability but also playing roles in other epilepsy-related processes. Inversely, in E-CO the strongly connected communities are related to compensatory pathways (seizure inhibition, neuronal survival and responses to stress conditions) while in L-CO these communities harbor several genes related to pro-epileptic effects, seizure-related mechanisms and vulnerability to epilepsy. These results fit the concept, based on fMRI and behavioral studies, that early onset epilepsies, although impacting more severely the hippocampus, are associated to compensatory mechanisms, while in late MTLE development the brain is less able to

  15. The Golgi apparatus is a functionally distinct Ca2+ store regulated by PKA and Epac branches of the β1-adrenergic signaling pathway

    PubMed Central

    Yang, Zhaokang.; Kirton, Hannah M.; MacDougall, David A.; Boyle, John P.; Deuchars, James; Frater, Brenda; Ponnambalam, Sreenivasan; Hardy, Matthew E.; White, Edward; Calaghan, Sarah C.; Peers, Chris; Steele, Derek S.

    2016-01-01

    Ca2+ release from the Golgi apparatus regulates key functions of the organelle, including vesicle trafficking. However, the signaling pathways that control this form of Ca2+ release are poorly understood and evidence of discrete Golgi Ca2+ release events is lacking. Here, we identified the Golgi apparatus as the source of prolonged Ca2+ release events that originate from the nuclear ‘poles’ of primary cardiac cells. Once initiated, Golgi Ca2+ release was unaffected by global depletion of sarcoplasmic reticulum Ca2+, and disruption of the Golgi apparatus abolished Golgi Ca2+ release without affecting sarcoplasmic reticulum function, suggesting functional and anatomical independence of Golgi and sarcoplasmic reticulum Ca2+ stores. Maximal activation of β1-adrenoceptors had only a small stimulating effect on Golgi Ca2+ release. However, inhibition of phosphodiesterase (PDE) 3 or 4, or downregulation of PDE 3 and 4 in heart failure markedly potentiated β1-adrenergic stimulation of Golgi Ca2+ release, consistent with compartmentalization of cAMP signaling within the Golgi apparatus microenvironment. β1-adrenergic stimulation of Golgi Ca2+ release involved activation of both Epac and PKA signaling pathways and CaMKII. Interventions that stimulated Golgi Ca2+ release induced trafficking of vascular growth factor receptor-1 (VEGFR-1) from the Golgi apparatus to the surface membrane. These data establish the Golgi apparatus as a juxtanuclear focal point for Ca2+ and β1-adrenergic signaling, which functions independently from the sarcoplasmic reticulum and the global Ca2+ transients that underlie the primary contractile function of the cell. PMID:26462734

  16. Community Structure Analysis of Transcriptional Networks Reveals Distinct Molecular Pathways for Early- and Late-Onset Temporal Lobe Epilepsy with Childhood Febrile Seizures

    PubMed Central

    Moreira-Filho, Carlos Alberto; Bando, Silvia Yumi; Bertonha, Fernanda Bernardi; Iamashita, Priscila; Silva, Filipi Nascimento; Costa, Luciano da Fontoura; Silva, Alexandre Valotta; Castro, Luiz Henrique Martins; Wen, Hung-Tzu

    2015-01-01

    Age at epilepsy onset has a broad impact on brain plasticity and epilepsy pathomechanisms. Prolonged febrile seizures in early childhood (FS) constitute an initial precipitating insult (IPI) commonly associated with mesial temporal lobe epilepsy (MTLE). FS-MTLE patients may have early disease onset, i.e. just after the IPI, in early childhood, or late-onset, ranging from mid-adolescence to early adult life. The mechanisms governing early (E) or late (L) disease onset are largely unknown. In order to unveil the molecular pathways underlying E and L subtypes of FS-MTLE we investigated global gene expression in hippocampal CA3 explants of FS-MTLE patients submitted to hippocampectomy. Gene coexpression networks (GCNs) were obtaine