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Sample records for distinct protease pathways

  1. Distinct protease pathways control cell shape and apoptosis in v-src-transformed quail neuroretina cells

    SciTech Connect

    Neel, Benjamin D.; Gillet, Germain . E-mail: g.gillet@ibcp.fr

    2005-11-15

    Intracellular proteases play key roles in cell differentiation, proliferation and apoptosis. In nerve cells, little is known about their relative contribution to the pathways which control cell physiology, including cell death. Neoplastic transformation of avian neuroretina cells by p60 {sup v-src} tyrosine kinase results in dramatic morphological changes and deregulation of apoptosis. To identify the proteases involved in the cellular response to p60 {sup v-src}, we evaluated the effect of specific inhibitors of caspases, calpains and the proteasome on cell shape changes and apoptosis induced by p60 {sup v-src} inactivation in quail neuroretina cells transformed by tsNY68, a thermosensitive strain of Rous sarcoma virus. We found that the ubiquitin-proteasome pathway is recruited early after p60 {sup v-src} inactivation and is critical for morphological changes, whereas caspases are essential for cell death. This study provides evidence that distinct intracellular proteases are involved in the control of the morphology and fate of v-src-transformed cells.

  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. 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

  4. Pathogen-Secreted Proteases Activate a Novel Plant Immune Pathway

    PubMed Central

    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.

    2015-01-01

    Mitogen-Activated Protein Kinase (MAPK) cascades play central roles in innate immune signaling networks in plants and animals1,2. In plants, however, the molecular mechanisms of how signal perception is transduced to MAPK activation remain elusive1. We report that pathogen-secreted proteases activate a previously unknown signaling pathway in Arabidopsis thaliana involving the Gα, Gβ and Gγ subunits of heterotrimeric G-protein complexes, which function upstream of a 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 signaling to downstream activation of a MAPK cascade. The protease-G protein-RACK1-MAPK cascade modules identified in these studies are distinct from previously described plant immune signaling pathways such as the one elicited by bacterial flagellin, in which G proteins function downstream of or in parallel to a MAPK cascade without the involvement of the RACK1 scaffolding protein. The discovery of the novel protease-mediated immune signaling 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 types of immunoregulatory strategies that account for its niche adaptation to diverse host tissues and immune systems. PMID:25731164

  5. Signaling pathways activated by a protease allergen in basophils

    PubMed Central

    Rosenstein, Rachel K.; Bezbradica, Jelena S.; Yu, Shuang; Medzhitov, Ruslan

    2014-01-01

    Allergic diseases represent a significant burden in industrialized countries, but why and how the immune system responds to allergens remain largely unknown. Because many clinically significant allergens have proteolytic activity, and many helminths express proteases that are necessary for their life cycles, host mechanisms likely have evolved to detect the proteolytic activity of helminth proteases, which may be incidentally activated by protease allergens. A cysteine protease, papain, is a prototypic protease allergen that can directly activate basophils and mast cells, leading to the production of cytokines, including IL-4, characteristic of the type 2 immune response. The mechanism of papain’s immunogenic activity remains unknown. Here we have characterized the cellular response activated by papain in basophils. We find that papain-induced IL-4 production requires calcium flux and activation of PI3K and nuclear factor of activated T cells. Interestingly, papain-induced IL-4 production was dependent on the immunoreceptor tyrosine-based activation motif (ITAM) adaptor protein Fc receptor γ-chain, even though the canonical ITAM signaling was not activated by papain. Collectively, these data characterize the downstream signaling pathway activated by a protease allergen in basophils. PMID:25369937

  6. New soluble ATP-dependent protease, Ti, in Escherichia coli that is distinct from protease La

    SciTech Connect

    Chung, C.H.; Hwang, B.J.; Park, W.J.; Goldberg, A.L.

    1987-05-01

    E. coli must contain other ATP-requiring proteolytic systems in addition to protease La (the lon gene product). A new ATP-dependent protease was purified from lon cells which lack protease La, as shown by immuno-blotting. This enzyme hydrolyzes (TH)casein to acid-soluble products in the presence of ATP (or dATP) and MgS . Nonhydrolyzable ATP analogs, other nucleoside triphosphates and AMP can not replace ATP. Therefore, ATP hydrolysis appears necessary for proteolysis. The enzyme appears to be a serine protease, but also contains essential thiol residues. Unlike protease La, it is not inhibited by vanadate, heparin, or the defective R9 subunit of protease La. On gel filtration, this enzyme has an apparent Mr of 340,000 and is comprised of two components of 190,000D and 130,000D, which can be separated by phosphocellulose chromatography. By themselves, these components do not show ATP-dependent proteolysis, but when mixed, full activity is restored. These finding and similar ones of Maurizi and Gottesman indicate that E. coli contain two soluble ATP-dependent proteases, which function by different mechanisms. This new enzyme may contribute to the rapid breakdown of abnormal polypeptides or of normal proteins during starvation. The authors propose to name it protease Ti.

  7. Escherichia coli contains a soluble ATP-dependent protease (Ti) distinct from protease La

    SciTech Connect

    Hwang, B.J.; Park, W.J.; Chung, C.H.; Goldberg, A.L.

    1987-08-01

    The energy requirement for protein breakdown in Escherichia coli has generally been attributed to the ATP-dependence of protease La, the lon gene product. The authors have partially purified another ATP-dependent protease from lon/sup -/ cells that lack protease La (as shown by immunoblotting). This enzyme hydrolyzes (/sup 3/H)methyl-casein to acid-soluble products in the presence of ATP and Mg/sup 2 +/. ATP hydrolysis appears necessary for proteolytic activity. Since this enzyme is inhibited by diisopropyl fluorophosphate, it appears to be a serine protease, but it also contains essential thiol residues. They propose to name this enzyme protease Ti. It differs from protease La in nucleotide specificity, inhibitor sensitivity, and subunit composition. On gel filtration, protease Ti has an apparent molecular weight of 370,000. It can be fractionated by phosphocellulose chromatography or by DEAE chromatography into two components with apparent molecular weights of 260,000 and 140,000. When separated, they do not show preteolytic activity. One of these components, by itself, has ATPase activity and is labile in the absence of ATP. The other contains the diisopropyl fluorophosphate-sensitive proteolytic site. These results and the similar findings of Katayama-Fujimura et al. indicate that E. coli contains two ATP-hydrolyzing proteases, which differ in many biochemical features and probably in their physiological roles.

  8. 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

  9. Axon degeneration: context defines distinct pathways.

    PubMed

    Geden, Matthew J; Deshmukh, Mohanish

    2016-08-01

    Axon degeneration is an essential part of development, plasticity, and injury response and has been primarily studied in mammalian models in three contexts: 1) Axotomy-induced Wallerian degeneration, 2) Apoptosis-induced axon degeneration (axon apoptosis), and 3) Axon pruning. These three contexts dictate engagement of distinct pathways for axon degeneration. Recent advances have identified the importance of SARM1, NMNATs, NAD+ depletion, and MAPK signaling in axotomy-induced Wallerian degeneration. Interestingly, apoptosis-induced axon degeneration and axon pruning have many shared mechanisms both in signaling (e.g. DLK, JNKs, GSK3α/β) and execution (e.g. Puma, Bax, caspase-9, caspase-3). However, the specific mechanisms by which caspases are activated during apoptosis versus pruning appear distinct, with apoptosis requiring Apaf-1 but not caspase-6 while pruning requires caspase-6 but not Apaf-1. PMID:27197022

  10. Distinct properties of proteases and nucleases in the gut, salivary gland and saliva of southern green stink bug, Nezara viridula.

    PubMed

    Lomate, Purushottam R; Bonning, Bryony C

    2016-01-01

    Stink bugs negatively impact numerous plant species of agricultural and horticultural importance. While efforts to develop effective control measures are underway, the unique digestive physiology of these pests presents a significant hurdle for either protein- or nucleotide-based management options. Here we report the comparative biochemical and proteomic characterization of proteases and nucleases from the gut, salivary gland and saliva of the southern green stink bug, Nezara viridula. The pH optimum for protease activity was acidic (5 to 6) in the gut with the primary proteases being cysteine proteases, and alkaline (8 to 9) in the saliva and salivary gland with the primary proteases being serine proteases. The serine proteases in saliva differ biochemically from trypsin and chymotrypsin, and the cathepsins in the gut and saliva showed distinct properties in inhibitor assays. Nuclease activity (DNase, RNase, dsRNase) was concentrated in the salivary gland and saliva with negligible activity in the gut. The most abundant proteins of the gut (530) and salivary gland (631) identified by proteomic analysis included four gut proteases along with eight proteases and one nuclease from the salivary gland. Understanding of N. viridula digestive physiology will facilitate the design of new strategies for management of this significant pest. PMID:27282882

  11. Distinct properties of proteases and nucleases in the gut, salivary gland and saliva of southern green stink bug, Nezara viridula

    PubMed Central

    Lomate, Purushottam R.; Bonning, Bryony C.

    2016-01-01

    Stink bugs negatively impact numerous plant species of agricultural and horticultural importance. While efforts to develop effective control measures are underway, the unique digestive physiology of these pests presents a significant hurdle for either protein- or nucleotide-based management options. Here we report the comparative biochemical and proteomic characterization of proteases and nucleases from the gut, salivary gland and saliva of the southern green stink bug, Nezara viridula. The pH optimum for protease activity was acidic (5 to 6) in the gut with the primary proteases being cysteine proteases, and alkaline (8 to 9) in the saliva and salivary gland with the primary proteases being serine proteases. The serine proteases in saliva differ biochemically from trypsin and chymotrypsin, and the cathepsins in the gut and saliva showed distinct properties in inhibitor assays. Nuclease activity (DNase, RNase, dsRNase) was concentrated in the salivary gland and saliva with negligible activity in the gut. The most abundant proteins of the gut (530) and salivary gland (631) identified by proteomic analysis included four gut proteases along with eight proteases and one nuclease from the salivary gland. Understanding of N. viridula digestive physiology will facilitate the design of new strategies for management of this significant pest. PMID:27282882

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

    PubMed

    Leissring, Malcolm A; Turner, Anthony J

    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

  13. 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

  14. Crystal Structures of the Viral Protease Npro Imply Distinct Roles for the Catalytic Water in Catalysis

    PubMed Central

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

    2013-01-01

    Summary 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. PMID:23643950

  15. 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.

  16. Distinct purinergic signaling pathways in prepubescent mouse spermatogonia.

    PubMed

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

    2016-09-01

    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 Ca(2+)-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. Scabies mite inactive serine proteases are potent inhibitors of the human complement lectin pathway.

    PubMed

    Reynolds, Simone L; Pike, Robert N; Mika, Angela; Blom, Anna M; Hofmann, Andreas; Wijeyewickrema, Lakshmi C; Kemp, Dave; Fischer, Katja

    2014-05-01

    Scabies is an infectious skin disease caused by the mite Sarcoptes scabiei and has been classified as one of the six most prevalent epidermal parasitic skin diseases infecting populations living in poverty by the World Health Organisation. The role of the complement system, a pivotal component of human innate immunity, as an important defence against invading pathogens has been well documented and many parasites have an arsenal of anti-complement defences. We previously reported on a family of scabies mite proteolytically inactive serine protease paralogues (SMIPP-Ss) thought to be implicated in host defence evasion. We have since shown that two family members, SMIPP-S D1 and I1 have the ability to bind the human complement components C1q, mannose binding lectin (MBL) and properdin and are capable of inhibiting all three human complement pathways. This investigation focused on inhibition of the lectin pathway of complement activation as it is likely to be the primary pathway affecting scabies mites. Activation of the lectin pathway relies on the activation of MBL, and as SMIPP-S D1 and I1 have previously been shown to bind MBL, the nature of this interaction was examined using binding and mutagenesis studies. SMIPP-S D1 bound MBL in complex with MBL-associated serine proteases (MASPs) and released the MASP-2 enzyme from the complex. SMIPP-S I1 was also able to bind MBL in complex with MASPs, but MASP-1 and MASP-2 remained in the complex. Despite these differences in mechanism, both molecules inhibited activation of complement components downstream of MBL. Mutagenesis studies revealed that both SMIPP-Ss used an alternative site of the molecule from the residual active site region to inhibit the lectin pathway. We propose that SMIPP-Ss are potent lectin pathway inhibitors and that this mechanism represents an important tool in the immune evasion repertoire of the parasitic mite and a potential target for therapeutics. PMID:24854034

  18. Two distinct pathways for essential metabolic precursors for isoprenoid biosynthesis.

    PubMed

    Kuzuyama, Tomohisa; Seto, Haruo

    2012-01-01

    Isoprenoids are a diverse group of molecules found in all organisms, where they perform such important biological functions as hormone signaling (e.g., steroids) in mammals, antioxidation (e.g., carotenoids) in plants, electron transport (e.g., ubiquinone), and cell wall biosynthesis intermediates in bacteria. All isoprenoids are synthesized by the consecutive condensation of the five-carbon monomer isopentenyl diphosphate (IPP) to its isomer, dimethylallyl diphosphate (DMAPP). The biosynthetic pathway for the formation of IPP from acetyl-CoA (i.e., the mevalonate pathway) had been established mainly in mice and the budding yeast Saccharomyces cerevisiae. Curiously, most prokaryotic microorganisms lack homologs of the genes in the mevalonate pathway, even though IPP and DMAPP are essential for isoprenoid biosynthesis in bacteria. This observation provided an impetus to search for an alternative pathway to synthesize IPP and DMAPP, ultimately leading to the discovery of the mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate pathway. This review article focuses on our significant contributions to a comprehensive understanding of the biosynthesis of IPP and DMAPP. PMID:22450534

  19. Two distinct pathways for essential metabolic precursors for isoprenoid biosynthesis

    PubMed Central

    KUZUYAMA, Tomohisa; SETO, Haruo

    2012-01-01

    Isoprenoids are a diverse group of molecules found in all organisms, where they perform such important biological functions as hormone signaling (e.g., steroids) in mammals, antioxidation (e.g., carotenoids) in plants, electron transport (e.g., ubiquinone), and cell wall biosynthesis intermediates in bacteria. All isoprenoids are synthesized by the consecutive condensation of the five-carbon monomer isopentenyl diphosphate (IPP) to its isomer, dimethylallyl diphosphate (DMAPP). The biosynthetic pathway for the formation of IPP from acetyl-CoA (i.e., the mevalonate pathway) had been established mainly in mice and the budding yeast Saccharomyces cerevisiae. Curiously, most prokaryotic microorganisms lack homologs of the genes in the mevalonate pathway, even though IPP and DMAPP are essential for isoprenoid biosynthesis in bacteria. This observation provided an impetus to search for an alternative pathway to synthesize IPP and DMAPP, ultimately leading to the discovery of the mevalonate-independent 2-C-methyl-d-erythritol 4-phosphate pathway. This review article focuses on our significant contributions to a comprehensive understanding of the biosynthesis of IPP and DMAPP. PMID:22450534

  20. 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

  1. Tracing an allosteric pathway regulating the activity of the HslV protease.

    PubMed

    Shi, Lichi; Kay, Lewis E

    2014-02-11

    The HslU-HslV complex functions as a bacterial proteasome, degrading substrate polypeptides to preserve cellular homeostasis. Here, we use methyl-Transverse Relaxation-Optimized Spectroscopy (TROSY) and highly deuterated, methyl-protonated samples to study the 230 kDa dodecameric HslV protease component that is structurally homologous to the stacked pair of β7-rings of the proteasome. Chemical shift assignments for over 95% of the methyl groups are reported. From the pH dependence of methyl chemical shifts, a pKa of 7.7 is measured for the amine group of the catalytic residue T1, confirming that it can act as a proton acceptor during the initial step in substrate proteolysis. Analyses involving a series of single site mutants in HslV, localized to HslU binding sites or regions undergoing significant changes on HslU binding, have identified hot spots whose perturbation leads to an allosteric pathway of propagated changes in structure and ultimately, substrate proteolysis efficiency. HslV plasticity is explored through methyl-TROSY (13)C relaxation dispersion experiments that are sensitive to millisecond timescale dynamics. The data support a dynamic coupling between residues involved in both HslU and substrate binding and residues localized to the active sites of HslV that facilitate the allostery between these distal sites. An important role for dynamics has also been observed in the archaeal proteasome, suggesting a more generally conserved role of motion in the function of these barrel-like protease structures. PMID:24469799

  2. 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

  3. 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. PMID:26935475

  4. Structure-based discovery of small molecule hepsin and HGFA protease inhibitors: Evaluation of potency and selectivity derived from distinct binding pockets.

    PubMed

    Franco, Francisco M; Jones, Darin E; Harris, Peter K W; Han, Zhenfu; Wildman, Scott A; Jarvis, Cassie M; Janetka, James W

    2015-05-15

    Hepatocyte growth factor activator (HGFA), matriptase and hepsin are all S1 trypsin-like serine endopeptidases. HGFA is a plasma protease while hepsin and matriptase are type II transmembrane proteases (TTSPs). Upregulated expression and activity of all three proteases is associated with aberrant cancer cell signaling through c-MET and RON tyrosine kinase cell-signaling pathways in cancer. We modeled known benzamidine protease inhibitor scaffolds into the active sites of matriptase, hepsin and HGFA to design new non-peptide inhibitors of hepsin and HGFA. First, we used a docking model of the irreversible inhibitor, Nafamostat, bound to the active site of HGFA in order to explore structure activity relationships (SAR). Compounds were screened for inhibition of HGFA activity in a kinetic enzyme assay using a chromogenic substrate. Next, we designed matched pair compound libraries of 3-amidino and 4-amidino phenylalanine (benzamidine) arginine peptidomimetics based on the structure of matriptase inhibitor, CJ-672. Compounds were screened for inhibition of HGFA, matriptase, and hepsin enzyme activity using fluorogenic substrates. Using this strategy we have discovered the first reported non-peptide small molecule inhibitors of both HGFA and hepsin. These inhibitors have differential potency and selectivity towards all three proteases. A subset of piperazinyl ureas highlighted by 25a, have excellent potency and selectivity for hepsin over matriptase and HGFA. PMID:25882520

  5. 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. PMID:21888979

  6. Inhibition of M current in sensory neurons by exogenous proteases: a signaling pathway mediating inflammatory nociception.

    PubMed

    Linley, John E; Rose, Kirstin; Patil, Mayur; Robertson, Brian; Akopian, Armen N; Gamper, Nikita

    2008-10-29

    Inflammatory pain is thought to be mediated in part through the action of inflammatory mediators on membrane receptors of peripheral nerve terminals, however, the downstream signaling events which lead to pain are poorly understood. In this study we investigated the nociceptive pathways induced by activation of protease-activated receptor 2 (PAR-2) in damage-sensing (nociceptive) neurons from rat dorsal root ganglion (DRG). We found that activation of PAR-2 in these cells strongly inhibited M-type potassium currents (conducted by Kv7 potassium channels). Such inhibition caused depolarization of the neuronal resting membrane potential leading, ultimately, to nociception. Consistent with this mechanism, injection of the specific M channel blocker XE991 into rat paw induced nociception in a concentration-dependent manner. Injection of a PAR-2 agonist peptide also induced nociception but coinjection of XE991 and the PAR-2 agonist did not result in summation of nociception, suggesting that the action of both agents may share a similar mechanism. We also studied the signaling pathway of M current inhibition by PAR-2 using patch-clamp and fluorescence imaging of DRG neurons. These experiments revealed that the PAR-2 effect was mediated by phospholipase C (PLC). Further experiments demonstrated that M current inhibition required concurrent rises in cytosolic Ca(2+) concentration and depletion of membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)). We propose that PLC- and Ca(2+)/PIP(2)-mediated inhibition of M current in sensory neurons may represent one of the general mechanisms underlying pain produced by inflammatory mediators, and may therefore open up a new therapeutic window for treatment of this major clinical problem. PMID:18971466

  7. Evidence for the presence of a protease-activated receptor distinct from the thrombin receptor in human keratinocytes.

    PubMed Central

    Santulli, R J; Derian, C K; Darrow, A L; Tomko, K A; Eckardt, A J; Seiberg, M; Scarborough, R M; Andrade-Gordon, P

    1995-01-01

    Thrombin receptor activation was explored in human epidermal keratinocytes and human dermal fibroblasts, cells that are actively involved in skin tissue repair. The effects of thrombin, trypsin, and the receptor agonist peptides SFLLRN and TFRIFD were assessed in inositolphospholipid hydrolysis and calcium mobilization studies. Thrombin and SFLLRN stimulated fibroblasts in both assays to a similar extent, whereas TFRIFD was less potent. Trypsin demonstrated weak efficacy in these assays in comparison with thrombin. Results in fibroblasts were consistent with human platelet thrombin receptor activation. Keratinocytes, however, exhibited a distinct profile, with trypsin being a far better activator of inositolphospholipid hydrolysis and calcium mobilization than thrombin. Furthermore, SFLLRN was more efficacious than thrombin, whereas no response was observed with TFRIFD. Since our data indicated that keratinocytes possess a trypsin-sensitive receptor, we addressed the possibility that these cells express the human homologue of the newly described murine protease-activated receptor, PAR-2 [Nystedt, S., Emilsson, K., Wahlestedt, C. & Sundelin, J. (1994) Proc. Natl. Acad. Sci. USA 91, 9208-9212]. PAR-2 is activated by nanomolar concentrations of trypsin and possesses the tethered ligand sequence SLIGRL. SLIGRL was found to be equipotent with SFLLRN in activating keratinocyte inositolphospholipid hydrolysis and calcium mobilization. Desensitization studies indicated that SFLLRN, SLIGRL, and trypsin activate a common receptor, PAR-2. Northern blot analyses detected a transcript of PAR-2 in total RNA from keratinocytes but not fibroblasts. Levels of thrombin receptor message were equivalent in the two cell types. Our results indicate that human keratinocytes possess PAR-2, suggesting a potential role for this receptor in tissue repair and/or skin-related disorders. Images Fig. 6 PMID:7568091

  8. 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. PMID:26047131

  9. 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.

  10. 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

  11. A distinct pathway for tetrahymanol synthesis in bacteria.

    PubMed

    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. PMID:26483502

  12. 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

  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. PMID:25948398

  14. An alternative pathway for fibrinolysis. I. The cleavage of fibrinogen by leukocyte proteases at physiologic pH.

    PubMed Central

    Plow, E F; Edgington, T S

    1975-01-01

    An alternative fibrinolytic system, active at physiological pH, is present in peripheral blood leukocytes. The fibrinolytic proteases localized predominantly in the leukocyte granules are capable of degrading both fibrinogen and fibrin, and plasmin activity does not contribute significantly to this proteolytic event. The specificity of the alternative fibrinolytic proteases for fibrinogen and the characteristics of the derivative cleavage fragments are clearly distinguishable from the classical plasmin system. The high molecular weight derivatives of fibrinogen, generated by the alternative system, under physiological conditions, are larger than the plasmin-generated X fragment, exhibit immunoelectrophoretic mobility comparable to native fibrinogen, and are not coagulable by thrombin. Analysis of the constituent polypeptide chains of the fragments reveals cleavage of the Aalpha, Bbeta, and gamma chains of fibrinogen. The lower molecular weight derivatives of fibrinogen, generated by the alternative system, are structurally distinct from previously described fibrinogen degradation products and exhibit potent anticoagulant activity. This anticoagulant activity can be attributed to interference with normal fibrin polymerization. The proteases of the alternative fibrinolytic systems are actively secreted by leukocytes when stimulated to undergo a nonlytic release reaction. These results provide direct evidence for a fibrinolytic system resident in leukocyte granules that is associated with the leukocyte release reaction and is capable of generating unique fibrinogen cleavage fragments. Images PMID:237938

  15. Identifying Distinct Healthcare Pathways During Episodes of Chronic Obstructive Pulmonary Disease Exacerbations

    PubMed Central

    Kuwornu, John P.; Lix, Lisa M.; Quail, Jacqueline M.; Forget, Evelyn; Muthukumarana, Saman; Wang, Xiaoyun E.; Osman, Meric; Teare, Gary F.

    2016-01-01

    Abstract Healthcare pathways are important to measure because they are expected to affect outcomes. However, they are challenging to define because patients exhibit heterogeneity in their use of healthcare services. The objective of this study was to identify and describe healthcare pathways during episodes of chronic obstructive pulmonary disease (COPD) exacerbations. Linked administrative databases from Saskatchewan, Canada were used to identify a cohort of newly diagnosed COPD patients and their episodes of healthcare use for disease exacerbations. Latent class analysis (LCA) was used to classify the cohort into homogeneous pathways using indicators of respiratory-related hospitalizations, emergency department (ED) visits, general and specialist physician visits, and outpatient prescription drug dispensations. Multinomial logistic regression models tested patients’ demographic and disease characteristics associated with pathway group membership. The most frequent healthcare contact sequences in each pathway were described. Tests of mean costs across groups were conducted using a model-based approach with χ2 statistics. LCA identified 3 distinct pathways for patients with hospital- (n = 963) and ED-initiated (n = 364) episodes. For the former, pathway group 1 members followed complex pathways in which multiple healthcare services were repeatedly used and incurred substantially higher costs than patients in the other pathway groups. For patients with an ED-initiated episode, pathway group 1 members also had higher costs than other groups. Pathway groups differed with respect to patient demographic and disease characteristics. A minority of patients were discharged from ED or hospital, but did not have any follow-up care during the remainder of their episode. Patients who followed complex pathways could benefit from case management interventions to streamline their journeys through the healthcare system. The minority of patients whose pathways were not

  16. Mycobacteria exploit three genetically distinct DNA double-strand break repair pathways

    PubMed Central

    Gupta, Richa; Barkan, Daniel; Redelman-Sidi, Gil; Shuman, Stewart; Glickman, Michael S.

    2013-01-01

    Bacterial pathogens rely on their DNA repair pathways to resist genomic damage inflicted by the host. DNA double-strand breaks (DSBs) are especially threatening to bacterial viability. DSB repair by homologous recombination (HR) requires nucleases that resect DSB ends and a strand exchange protein that facilitates homology search. RecBCD and RecA perform these functions in E. coli and constitute the major pathway of error free DSB repair. Mycobacteria, including the human pathogen M. tuberculosis, elaborate an additional error-prone pathway of DSB repair via nonhomologous end-joining (NHEJ) catalyzed by Ku and DNA ligase D (LigD). Little is known about the relative contributions of HR and NHEJ to mycobacterial chromosome repair, the factors that dictate pathway choice, or the existence of additional DSB repair pathways. Here we demonstrate that Mycobacterium smegmatis has three DSB repair pathway options: HR, NHEJ, and a novel mechanism of single-strand annealing (SSA). Inactivation of NHEJ or SSA is compensated by elevated HR. We find that mycobacterial RecBCD does not participate in HR or confer resistance to ionizing radiation (IR), but is required for the RecA-independent SSA pathway. In contrast, the mycobacterial helicase-nuclease AdnAB participates in the RecA-dependent HR pathway, and is a major determinant of resistance to IR and oxidative DNA damage. These findings reveal distinctive features of mycobacterial DSB repair, most notably the dedication of the RecBCD and AdnAB helicase-nuclease machines to distinct repair pathways. PMID:21219454

  17. 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

  18. 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. PMID:26213689

  19. 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

  20. 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

  1. [Distinct roles of the direct and indirect pathways in the basal ganglia circuit mechanism].

    PubMed

    Morita, Makiko; Hikida, Takatoshi

    2015-11-01

    The basal ganglia are key neural substrates that control not only motor balance but also emotion, motivation, cognition, learning, and decision-making. Dysfunction of the basal ganglia leads to neurodegenerative diseases (e.g. Parkinson's disease and Huntington's disease) and psychiatric disorders (e.g. drug addiction, schizophrenia, and depression). In the basal ganglia circuit, there are two important pathways: the direct and indirect striatal pathways. Recently, new molecular techniques that activate or inactive selectively the direct or indirect pathway neurons have revealed the function of each pathway. Here we review the distinct roles of the direct and indirect striatal pathways in brain function and drug addiction. We have developed a reversible neurotransmission blocking technique, in which transmission of each pathway is selectively blocked by specific expression of transmission-blocking tetanus toxin, and revealed that the activation of D1 receptors in the direct pathway is critical for reward learning/cocaine addiction, and that the inactivation of D2 receptors is critical for aversive learning/learning flexibility. We propose a new circuit mechanism by which the dopaminergic input from the ventral tegmental area can switch the direct and indirect pathways in the nucleus accumbens. These basal ganglia circuit mechanisms will give us insights into the pathophysiology of mental diseases. PMID:26785520

  2. 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

  3. Fas activation of the p38 mitogen-activated protein kinase signalling pathway requires ICE/CED-3 family proteases.

    PubMed Central

    Juo, P; Kuo, C J; Reynolds, S E; Konz, R F; Raingeaud, J; Davis, R J; Biemann, H P; Blenis, J

    1997-01-01

    The Fas receptor mediates a signalling cascade resulting in programmed cell death (apoptosis) within hours of receptor cross-linking. In this study Fas activated the stress-responsive mitogen-activated protein kinases, p38 and JNK, within 2 h in Jurkat T lymphocytes but not the mitogen-responsive kinase ERK1 or pp70S6k. Fas activation of p38 correlated temporally with the onset of apoptosis, and transfection of constitutively active MKK3 (glu), an upstream regulator of p38, potentiated Fas-induced cell death, suggesting a potential involvement of the MKK3/p38 activation pathway in Fas-mediated apoptosis. Fas has been shown to require ICE (interleukin-1 beta-converting enzyme) family proteases to induce apoptosis from studies utilizing the cowpox ICE inhibitor protein CrmA, the synthetic tetrapeptide ICE inhibitor YVAD-CMK, and the tripeptide pan-ICE inhibitor Z-VAD-FMK. In this study, crmA antagonized, and YVAD-CMK and Z-VAD-FMK completely inhibited, Fas activation of p38 kinase activity, demonstrating that Fas-dependent activation of p38 requires ICE/CED-3 family members and conversely that the MKK3/p38 activation cascade represents a downstream target for the ICE/CED-3 family proteases. Intriguingly, p38 activation by sorbitol and etoposide was resistant to YVAD-CMK and Z-VAD-FMK, suggesting the existence of an additional mechanism(s) of p38 regulation. The ICE/CED-3 family-p38 regulatory relationship described in the current work indicates that in addition to the previously described destructive cleavage of substrates such as poly(ADP ribose) polymerase, lamins, and topoisomerase, the apoptotic cysteine proteases also function to regulate stress kinase signalling cascades. PMID:8972182

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

    PubMed

    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

  5. 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

  6. Per a 10 protease activity modulates CD40 expression on dendritic cell surface by nuclear factor-kappaB pathway

    PubMed Central

    Goel, C; Kalra, N; Dwarakanath, B S; Gaur, S N; Arora, N

    2015-01-01

    Serine protease activity of Per a 10 from Periplaneta americana modulates dendritic cell (DC) functions by a mechanism(s) that remains unclear. In the present study, Per a 10 protease activity on CD40 expression and downstream signalling was evaluated in DCs. Monocyte-derived DCs from cockroach-allergic patients were treated with proteolytically active/heat-inactivated Per a 10. Stimulation with active Per a 10 demonstrated low CD40 expression on DCs surface (P < 0·05), while enhanced soluble CD40 level in the culture supernatant (P < 0·05) compared to the heat-inactivated Per a 10, suggesting cleavage of CD40. Per a 10 activity reduced the interleukin (IL)-12 and interferon (IFN)-γ secretion by DCs (P < 0·05) compared to heat-inactivated Per a 10, indicating that low CD40 expression is associated with low levels of IL-12 secretion. Active Per a 10 stimulation caused low nuclear factor-kappa B (NF-κB) activation in DCs compared to heat-inactivated Per a 10. Inhibition of the NF-κB pathway suppressed the CD40 expression and IL-12 secretion by DCs, further indicating that NF-κB is required for CD40 up-regulation. CD40 expression activated the tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6), thereby suggesting its involvement in NF-κB activation. Protease activity of Per a 10 induced p38 mitogen-activated protein kinase (MAPK) activation that showed no significant effect on CD40 expression by DCs. However, inhibiting p38 MAPK or NF-κB suppressed the secretion of IL-12, IFN-γ, IL-6 and TNF-α by DCs. Such DCs further reduced the secretion of IL-4, IL-6, IL-12 and TNF-α by CD4+ T cells. In conclusion, protease activity of Per a 10 reduces CD40 expression on DCs. CD40 down-regulation leads to low NF-κB levels, thereby modulating DC-mediated immune responses. PMID:25492061

  7. 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

    2016-06-01

    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. PMID:27056326

  8. 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.

  9. 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. PMID:20039882

  10. Novel proteases: common themes and surprising features.

    PubMed

    Vandeputte-Rutten, Lucy; Gros, Piet

    2002-12-01

    Proteases perform a wide variety of functions, inside and outside cells, regulating many biological processes. Recent years have witnessed a number of significant advances in the structural biology of proteases, including aspects of intracellular protein and peptide degradation by self-compartmentalizing proteases, activation of proteases in proteolytic cascades of regulatory pathways, and mechanisms of microbial proteases in pathogenicity. PMID:12504673

  11. 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

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

    PubMed

    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; Jacobsen, Sten Eirik W; Nerlov, Claus

    2016-06-01

    According to current models of hematopoiesis, lymphoid-primed multi-potent progenitors (LMPPs) (Lin(-)Sca-1(+)c-Kit(+)CD34(+)Flt3(hi)) and common myeloid progenitors (CMPs) (Lin(-)Sca-1(+)c-Kit(+)CD34(+)CD41(hi)) 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 identified distinct myeloid differentiation pathways: a pathway expressing the gene encoding the transcription factor GATA-1 generated mast cells, eosinophils, megakaryocytes and erythroid cells, and a pathway lacking expression of that gene generated monocytes, neutrophils and lymphocytes. These results identify an early hematopoietic-lineage bifurcation that separates the myeloid lineages before their segregation from other hematopoietic-lineage potential. PMID:27043410

  13. Vestibular function in the temporal and parietal cortex: distinct velocity and inertial processing pathways

    PubMed Central

    Ventre-Dominey, Jocelyne

    2014-01-01

    A number of behavioral and neuroimaging studies have reported converging data in favor of a cortical network for vestibular function, distributed between the temporo-parietal cortex and the prefrontal cortex in the primate. In this review, we focus on the role of the cerebral cortex in visuo-vestibular integration including the motion sensitive temporo-occipital areas i.e., the middle superior temporal area (MST) and the parietal cortex. Indeed, these two neighboring cortical regions, though they both receive combined vestibular and visual information, have distinct implications in vestibular function. In sum, this review of the literature leads to the idea of two separate cortical vestibular sub-systems forming (1) a velocity pathway including MST and direct descending pathways on vestibular nuclei. As it receives well-defined visual and vestibular velocity signals, this pathway is likely involved in heading perception and rapid top-down regulation of eye/head coordination and (2) an inertial processing pathway involving the parietal cortex in connection with the subcortical vestibular nuclei complex responsible for velocity storage integration. This vestibular cortical pathway would be implicated in high-order multimodal integration and cognitive functions, including world space and self-referential processing. PMID:25071481

  14. Two novel regulators of N-acetyl-galactosamine utilization pathway and distinct roles in bacterial infections.

    PubMed

    Zhang, Huimin; Ravcheev, Dmitry A; Hu, Dan; Zhang, Fengyu; Gong, Xiufang; Hao, Lina; Cao, Min; Rodionov, Dmitry A; Wang, Changjun; Feng, Youjun

    2015-12-01

    Bacterial pathogens can exploit metabolic pathways to facilitate their successful infection cycles, but little is known about roles of d-galactosamine (GalN)/N-acetyl-d-galactosamine (GalNAc) catabolism pathway in bacterial pathogenesis. Here, we report the genomic reconstruction of GalN/GalNAc utilization pathway in Streptococci and the diversified aga regulons. We delineated two new paralogous AgaR regulators for the GalN/GalNAc catabolism pathway. The electrophoretic mobility shift assays experiment demonstrated that AgaR2 (AgaR1) binds the predicted palindromes, and the combined in vivo data from reverse transcription quantitative polymerase chain reaction and RNA-seq suggested that AgaR2 (not AgaR1) can effectively repress the transcription of the target genes. Removal of agaR2 (not agaR1) from Streptococcus suis 05ZYH33 augments significantly the abilities of both adherence to Hep-2 cells and anti-phagocytosis against RAW264.7 macrophage. As anticipated, the dysfunction in AgaR2-mediated regulation of S. suis impairs its pathogenicity in experimental models of both mice and piglets. Our finding discovered two novel regulators specific for GalN/GalNAc catabolism and assigned them distinct roles into bacterial infections. To the best of our knowledge, it might represent a first paradigm that links the GalN/GalNAc catabolism pathway to bacterial pathogenesis. PMID:26540018

  15. Nicotiana benthamiana cathepsin B displays distinct enzymatic features which differ from its human relative and aleurain-like protease.

    PubMed

    Niemer, Melanie; Mehofer, Ulrich; Verdianz, Maria; Porodko, Andreas; Schähs, Philipp; Kracher, Daniel; Lenarcic, Brigita; Novinec, Marko; Mach, Lukas

    2016-03-01

    The tobacco-related plant species Nicotiana benthamiana has recently emerged as a versatile expression platform for the rapid generation of recombinant biopharmaceuticals, but product yield and quality frequently suffer from unintended proteolysis. Previous studies have highlighted that recombinant protein fragmentation in plants involves papain-like cysteine proteinases (PLCPs). For this reason, we have now characterized two major N. benthamiana PLCPs in detail: aleurain-like protease (NbALP) and cathepsin B (NbCathB). As typical for PLCPs, the precursor of NbCathB readily undergoes autocatalytic activation when incubated at low pH. On the contrary, maturation of NbALP requires the presence of a cathepsin L-like PLCP as processing enzyme. While the catalytic features of NbALP closely resemble those of its mammalian homologue cathepsin H, NbCathB displays remarkable differences to human cathepsin B. In particular, NbCathB appears to be a far less efficient peptidyldipeptidase (removing C-terminal dipeptides) than its human counterpart, suggesting that it functions primarily as an endopeptidase. Importantly, NbCathB was far more efficient than NbALP in processing the human anti-HIV-1 antibody 2F5 into fragments observed during its production in N. benthamiana. This suggests that targeted down-regulation of NbCathB could improve the performance of this plant-based expression platform. PMID:26166069

  16. Toxic and chemopreventive ligands preferentially activate distinct aryl hydrocarbon receptor pathways: implications for cancer prevention.

    PubMed

    Okino, Steven T; Pookot, Deepa; Basak, Shashwati; Dahiya, Rajvir

    2009-03-01

    The aryl hydrocarbon receptor (AhR) is a ligand-activated regulatory protein that controls estrogen action through two distinct pathways. In one pathway, AhR acts as a transcription factor that induces the expression of the CYP1 family of estrogen-metabolizing genes; in the other pathway, AhR initiates the degradation of the estrogen receptor and suppresses estrogen signaling. The AhR ligand 3,3'-diindolylmethane (DIM) is a beneficial dietary constituent that prevents breast tumors in rodents and is associated with decreased breast cancer risk in humans. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a toxic AhR ligand that is implicated in birth defects, infertility, and cancer. We analyzed MCF-7 cells to gain insight into how two AhR ligands can exert such fundamentally different health effects. We find that DIM and TCDD have differing abilities to activate the distinct AhR-controlled pathways. TCDD strongly induces AhR-dependent CYP1 gene expression, whereas DIM is a relatively weak CYP1 inducer. DIM strongly inhibits estrogen receptor-alpha expression and estrogen signaling, whereas TCDD has a notably weaker effect on these processes. Small interfering RNA knockdown of AhR confirms that the effects of DIM and TCDD are indeed AhR dependent. Our findings reveal that DIM and TCDD each elicit a unique pattern of change in pathways that control estrogen action; such patterns may determine if an AhR ligand has beneficial or adverse health effects. PMID:19223575

  17. Coordinate Control of Muscle Cell Survival by Distinct Insulin-like Growth Factor Activated Signaling Pathways

    PubMed Central

    Lawlor, Margaret A.; Rotwein, Peter

    2000-01-01

    Peptide growth factors control diverse cellular functions by regulating distinct signal transduction pathways. In cultured myoblasts, insulin-like growth factors (IGFs) stimulate differentiation and promote hypertrophy. IGFs also maintain muscle cell viability. We previously described C2 skeletal muscle lines lacking expression of IGF-II. These cells did not differentiate, but underwent progressive apoptotic death when incubated in differentiation medium. Viability could be sustained and differentiation enabled by IGF analogues that activated the IGF-I receptor; survival was dependent on stimulation of phosphatidylinositol 3-kinase (PI3-kinase). We now find that IGF action promotes myoblast survival through two distinguishable PI3-kinase–regulated pathways that culminate in expression of the cyclin-dependent kinase inhibitor, p21. Incubation with IGF-I or transfection with active PI3-kinase led to rapid induction of MyoD and p21, and forced expression of either protein maintained viability in the absence of growth factors. Ectopic expression of MyoD induced p21, and inhibition of p21 blocked MyoD-mediated survival, thus defining one PI3-kinase–dependent pathway as leading first to MyoD, and then to p21 and survival. Unexpectedly, loss of MyoD expression did not impede IGF-mediated survival, revealing a second pathway involving activation by PI3-kinase of Akt, and subsequent induction of p21. Since inhibition of p21 caused death even in the presence of IGF-I, these results establish a central role for p21 as a survival factor for muscle cells. Our observations also define a MyoD-independent pathway for regulating p21 in muscle, and demonstrate that distinct mechanisms help ensure appropriate expression of this key protein during differentiation. PMID:11121430

  18. 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.

  19. 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.

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

    PubMed

    Verona, Edelyn

    2016-01-01

    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. PMID:27570952

  1. Three Distinct Blue-Green Color Pathways in a Mammalian Retina

    PubMed Central

    Tian, Lian-Ming; Hoshi, Hideo; Whitaker, Christopher M.; Massey, Stephen C.

    2014-01-01

    In mammalian retinae, the first steps in the process of discrimination of color are mediated by color-opponent neurons that respond with opposite polarity to signals from short (S, blue) and longer wavelength (M, green or L, red) cones. Primates also contain a second system that is different from M and L cones. Although pathways responding to the onset of S-cone stimulation (S-ON) are well known, the existence of bipolar cells and retinal ganglion cells that respond to the offset of S-cone stimulation (S-OFF) has been controversial. We have recorded from and stained three different types of S/M color-opponent ganglion cells in the rabbit retina that are distinguished by the polarity of their responses to S-cone stimulation, the stratification pattern of their dendrites, and the distinct mechanisms underlying their color-opponent responses. We describe an S-ON and an S-OFF pathway formed by amacrine cells inverting the S-ON signal. Most importantly, we also provide both anatomical and physiological evidence for a direct S-OFF pathway dependent on an S-OFF cone bipolar cell. The results indicate a greater diversity of pathways for processing of signals from S-cones than previously suspected. PMID:24478358

  2. 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

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

    PubMed

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

    2016-01-01

    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

  4. Vesicular Monoamine and Glutamate Transporters Select Distinct Synaptic Vesicle Recycling Pathways

    PubMed Central

    Onoa, Bibiana; Li, Haiyan; Gagnon-Bartsch, Johann A.; Elias, Laura A. B.; Edwards, Robert H.

    2011-01-01

    Previous work has characterized the properties of neurotransmitter release at excitatory and inhibitory synapses, but we know remarkably little about the properties of monoamine release because these neuromodulators do not generally produce a fast ionotropic response. Since dopamine and serotonin neurons can also release glutamate in vitro and in vivo, we have used the vesicular monoamine transporter VMAT2 and the vesicular glutamate transporter VGLUT1 to compare the localization and recycling of synaptic vesicles that store, respectively, monoamines and glutamate. First, VMAT2 segregates partially from VGLUT1 in the boutons of midbrain dopamine neurons, indicating the potential for distinct release sites. Second, endocytosis after stimulation is slower for VMAT2 than VGLUT1. During the stimulus, however, the endocytosis of VMAT2 (but not VGLUT1) accelerates dramatically in midbrain dopamine but not hippocampal neurons, indicating a novel, cell-specific mechanism to sustain high rates of release. On the other hand, we find that in both midbrain dopamine and hippocampal neurons, a substantially smaller proportion of VMAT2 than VGLUT1 is available for evoked release, and VMAT2 shows considerably more dispersion along the axon after exocytosis than VGLUT1. Even when expressed in the same neuron, the two vesicular transporters thus target to distinct populations of synaptic vesicles, presumably due to their selection of distinct recycling pathways. PMID:20534840

  5. Distinct molecular pathways mediate glial activation and engulfment of axonal debris after axotomy

    PubMed Central

    Ziegenfuss, Jennifer S.; Doherty, Johnna; Freeman, Marc R.

    2016-01-01

    Glial cells efficiently recognize and clear cellular debris after nervous system injury to maintain brain homeostasis, but pathways governing glial responses to neural injury remain poorly defined. We identify the Drosophila guanine-nucleotide exchange factor (GEF) complex Crk/Mbc/dCed-12, and the small GTPase Rac1 as novel modulators of glial clearance of axonal debris. We show Crk/Mbc/dCed-12 and Rac1 function in a non-redundant fashion with the Draper pathway—loss of either pathway fully suppresses clearance of axonal debris. Draper signaling is required early during glial responses, promoting glial activation, which includes increased Draper and dCed-6 expression and extension of glial membranes to degenerating axons. In contrast, the Crk/Mbc/dCed-12 complex functions at later phases promoting glial phagocytosis of axonal debris. Our work identifies new components of the glial engulfment machinery and shows that glial activation, phagocytosis of axonal debris, and termination of responses to injury are genetically separable events mediated by distinct signaling pathways. PMID:22706267

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

    PubMed Central

    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.

    2011-01-01

    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. PMID:21690558

  7. Dual Control of Muscle Cell Survival by Distinct Growth Factor-Regulated Signaling Pathways

    PubMed Central

    Lawlor, Margaret A.; Feng, Xiuhong; Everding, Daniel R.; Sieger, Kerry; Stewart, Claire E. H.; Rotwein, Peter

    2000-01-01

    In addition to their ability to stimulate cell proliferation, polypeptide growth factors are able to maintain cell survival under conditions that otherwise lead to apoptotic death. Growth factors control cell viability through regulation of critical intracellular signal transduction pathways. We previously characterized C2 muscle cell lines that lacked endogenous expression of insulin-like growth factor II (IGF-II). These cells did not differentiate but underwent apoptotic death in low-serum differentiation medium. Death could be prevented by IGF analogues that activated the IGF-I receptor or by unrelated growth factors such as platelet-derived growth factor BB (PDGF-BB). Here we analyze the signaling pathways involved in growth factor-mediated myoblast survival. PDGF treatment caused sustained activation of extracellular-regulated kinases 1 and 2 (ERK1 and -2), while IGF-I only transiently induced these enzymes. Transient transfection of a constitutively active Mek1, a specific upstream activator of ERKs, maintained myoblast viability in the absence of growth factors, while inhibition of Mek1 by the drug UO126 blocked PDGF-mediated but not IGF-stimulated survival. Although both growth factors activated phosphatidylinositol 3-kinase (PI3-kinase) to similar extents, only IGF-I treatment led to sustained stimulation of its downstream kinase, Akt. Transient transfection of a constitutively active PI3-kinase or an inducible Akt promoted myoblast viability in the absence of growth factors, while inhibition of PI3-kinase activity by the drug LY294002 selectively blocked IGF- but not PDGF-mediated muscle cell survival. In aggregate, these observations demonstrate that distinct growth factor-regulated signaling pathways independently control myoblast survival. Since IGF action also stimulates muscle differentiation, these results suggest a means to regulate myogenesis through selective manipulation of different signal transduction pathways. PMID:10757809

  8. Distinct Pathways Mediate the Sorting of Tail-Anchored Proteins to the Plastid Outer Envelope

    PubMed Central

    Dhanoa, Preetinder K.; Richardson, Lynn G. L.; Smith, Matthew D.; Gidda, Satinder K.; Henderson, Matthew P. A.; Andrews, David W.; Mullen, Robert T.

    2010-01-01

    Background Tail-anchored (TA) proteins are a distinct class of membrane proteins that are sorted post-translationally to various organelles and function in a number of important cellular processes, including redox reactions, vesicular trafficking and protein translocation. While the molecular targeting signals and pathways responsible for sorting TA proteins to their correct intracellular destinations in yeasts and mammals have begun to be characterized, relatively little is known about TA protein biogenesis in plant cells, especially for those sorted to the plastid outer envelope. Methodology/Principal Findings Here we investigated the biogenesis of three plastid TA proteins, including the 33-kDa and 34-kDa GTPases of the translocon at the outer envelope of chloroplasts (Toc33 and Toc34) and a novel 9-kDa protein of unknown function that we define here as an outer envelope TA protein (OEP9). Using a combination of in vivo and in vitro assays we show that OEP9 utilizes a different sorting pathway than that used by Toc33 and Toc34. For instance, while all three TA proteins interact with the cytosolic OEP chaperone/receptor, AKR2A, the plastid targeting information within OEP9 is distinct from that within Toc33 and Toc34. Toc33 and Toc34 also appear to differ from OEP9 in that their insertion is dependent on themselves and the unique lipid composition of the plastid outer envelope. By contrast, the insertion of OEP9 into the plastid outer envelope occurs in a proteinaceous-dependent, but Toc33/34-independent manner and membrane lipids appear to serve primarily to facilitate normal thermodynamic integration of this TA protein. Conclusions/Significance Collectively, the results provide evidence in support of at least two sorting pathways for plastid TA outer envelope proteins and shed light on not only the complex diversity of pathways involved in the targeting and insertion of proteins into plastids, but also the molecular mechanisms that underlie the delivery of TA

  9. 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

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

    PubMed

    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

  11. 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

  12. 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

  13. 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

  14. 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

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

    PubMed

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

    2016-03-16

    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

  16. 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. PMID:27325104

  17. 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

  18. 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.

  19. 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

  20. 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

  1. Viral Reorganization of the Secretory Pathway Generates Distinct Organelles for RNA Replication

    PubMed Central

    Hsu, Nai-Yun; Ilnytska, Olha; Belov, Georgiy; Santiana, Marianita; Chen, Ying-Han; Takvorian, Peter M.; Pau, Cyrilla; van der Schaar, Hilde; Kaushik-Basu, Neerja; Balla, Tamas; Cameron, Craig E.; Ehrenfeld, Ellie; van Kuppeveld, Frank J.M.; Altan-Bonnet, Nihal

    2010-01-01

    SUMMARY Many RNA viruses remodel intracellular membranes to generate specialized sites for RNA replication. How membranes are remodeled and what properties make them conducive for replication are unknown. Here we show how RNA viruses can manipulate multiple components of the cellular secretory pathway to generate organelles specialized for replication that are distinct in protein and lipid composition from the host cell. Specific viral proteins modulate effector recruitment by Arf1 GTPase and its guanine nucleotide exchange factor GBF1, promoting preferential recruitment of phosphatidylinositol-4-kinase IIIβ (PI4KIIIβ) to membranes over coat proteins, yielding uncoated phosphatidylinositol-4-phosphate (PI4P) lipid-enriched organelles. The PI4P-rich lipid micro-environment is essential for both enteroviral and flaviviral RNA replication; PI4KIIIβ inhibition interferes with this process; and enteroviral RNA polymerases specifically bind PI4P. These findings reveal how RNA viruses can selectively exploit specific elements of the host to form specialized organelles where cellular phosphoinositide lipids are key to regulating viral RNA replication. PMID:20510927

  2. 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

  3. 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

  4. Erythropoiesis and Blood Pressure Are Regulated via AT1 Receptor by Distinctive Pathways

    PubMed Central

    Kato, Hideki; Ishida, Junji; Matsusaka, Taiji; Ishimaru, Tomohiro; Tanimoto, Keiji; Sugiyama, Fumihiro; Yagami, Ken-ichi; Nangaku, Masaomi; Fukamizu, Akiyoshi

    2015-01-01

    The renin–angiotensin system (RAS) plays a central role in blood pressure regulation. Although clinical and experimental studies have suggested that inhibition of RAS is associated with progression of anemia, little evidence is available to support this claim. Here we report that knockout mice that lack angiotensin II, including angiotensinogen and renin knockout mice, exhibit anemia. The anemia of angiotensinogen knockout mice was rescued by angiotensin II infusion, and rescue was completely blocked by simultaneous administration of AT1 receptor blocker. To genetically determine the responsible receptor subtype, we examined AT1a, AT1b, and AT2 knockout mice, but did not observe anemia in any of them. To investigate whether pharmacological AT1 receptor inhibition recapitulates the anemic phenotype, we administered AT1 receptor antagonist in hypotensive AT1a receptor knockout mice to inhibit the remaining AT1b receptor. In these animals, hematocrit levels barely decreased, but blood pressure further decreased to the level observed in angiotensinogen knockout mice. We then generated AT1a and AT1b double-knockout mice to completely ablate the AT1 receptors; the mice finally exhibited the anemic phenotype. These results provide clear evidence that although erythropoiesis and blood pressure are negatively controlled through the AT1 receptor inhibition in vivo, the pathways involved are complex and distinct, because erythropoiesis is more resistant to AT1 receptor inhibition than blood pressure control. PMID:26107632

  5. Integrated data analysis reveals uterine leiomyoma subtypes with distinct driver pathways and biomarkers

    PubMed Central

    Mehine, Miika; Kaasinen, Eevi; Heinonen, Hanna-Riikka; Mäkinen, Netta; Kämpjärvi, Kati; Sarvilinna, Nanna; Aavikko, Mervi; Vähärautio, Anna; Pasanen, Annukka; Bützow, Ralf; Heikinheimo, Oskari; Sjöberg, Jari; Pitkänen, Esa; Vahteristo, Pia; Aaltonen, Lauri A.

    2016-01-01

    Uterine leiomyomas are common benign smooth muscle tumors that impose a major burden on women’s health. Recent sequencing studies have revealed recurrent and mutually exclusive mutations in leiomyomas, suggesting the involvement of molecularly distinct pathways. In this study, we explored transcriptional differences among leiomyomas harboring different genetic drivers, including high mobility group AT-hook 2 (HMGA2) rearrangements, mediator complex subunit 12 (MED12) mutations, biallelic inactivation of fumarate hydratase (FH), and collagen, type IV, alpha 5 and collagen, type IV, alpha 6 (COL4A5-COL4A6) deletions. We also explored the transcriptional consequences of 7q22, 22q, and 1p deletions, aiming to identify possible target genes. We investigated 94 leiomyomas and 60 corresponding myometrial tissues using exon arrays, whole genome sequencing, and SNP arrays. This integrative approach revealed subtype-specific expression changes in key driver pathways, including Wnt/β-catenin, Prolactin, and insulin-like growth factor (IGF)1 signaling. Leiomyomas with HMGA2 aberrations displayed highly significant up-regulation of the proto-oncogene pleomorphic adenoma gene 1 (PLAG1), suggesting that HMGA2 promotes tumorigenesis through PLAG1 activation. This was supported by the identification of genetic PLAG1 alterations resulting in expression signatures as seen in leiomyomas with HMGA2 aberrations. RAD51 paralog B (RAD51B), the preferential translocation partner of HMGA2, was up-regulated in MED12 mutant lesions, suggesting a role for this gene in the genesis of leiomyomas. FH-deficient leiomyomas were uniquely characterized by activation of nuclear factor erythroid 2-related factor 2 (NRF2) target genes, supporting the hypothesis that accumulation of fumarate leads to activation of the oncogenic transcription factor NRF2. This study emphasizes the need for molecular stratification in leiomyoma research and possibly in clinical practice as well. Further research is

  6. Genetically distinct pathways guide effector export through the type VI secretion system

    PubMed Central

    Whitney, John C.; Beck, Christina M.; Goo, Young Ah; Russell, Alistair B.; Harding, Brittany; De Leon, Justin A.; Cunningham, David A.; Tran, Bao Q.; Low, David A.; Goodlett, David R.; Hayes, Christopher S.; Mougous, Joseph D.

    2014-01-01

    Summary Bacterial secretion systems often employ molecular chaperones to recognize and facilitate export of their substrates. Recent work demonstrated that a secreted component of the type VI secretion system (T6SS), hemolysin co-regulated protein (Hcp), binds directly to effectors, enhancing their stability in the bacterial cytoplasm. Herein, we describe a quantitative cellular proteomics screen for T6S substrates that exploits this chaperone-like quality of Hcp. Application of this approach to the Hcp secretion island I-encoded T6SS (H1-T6SS) of Pseudomonas aeruginosa led to the identification of a novel effector protein, termed Tse4 (type VI secretion exported 4), subsequently shown to act as a potent intra-specific H1-T6SS-delivered antibacterial toxin. Interestingly, our screen failed to identify two predicted H1-T6SS effectors, Tse5 and Tse6, which differ from Hcp-stabilized substrates by the presence of toxin-associated PAAR-repeat motifs and genetic linkage to members of the valine-glycine repeat protein G (vgrG) genes. Genetic studies further distinguished these two groups of effectors: Hcp-stabilized effectors were found to display redundancy in interbacterial competition with respect to the requirement for the two H1-T6SS-exported VgrG proteins, whereas Tse5 and Tse6 delivery strictly required a cognate VgrG. Together, we propose that interaction with either VgrG or Hcp defines distinct pathways for T6S effector export. PMID:24589350

  7. 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

  8. Subcutaneous Allergic Sensitization to Protease Allergen Is Dependent on Mast Cells but Not IL-33: Distinct Mechanisms between Subcutaneous and Intranasal Routes.

    PubMed

    Kamijo, Seiji; Suzuki, Mayu; Hara, Mutsuko; Shimura, Sakiko; Ochi, Hirono; Maruyama, Natsuko; Matsuda, Akira; Saito, Hirohisa; Nakae, Susumu; Suto, Hajime; Ichikawa, Saori; Ikeda, Shigaku; Ogawa, Hideoki; Okumura, Ko; Takai, Toshiro

    2016-05-01

    Protease activity of papain, a plant-derived occupational allergen homologous to mite major allergens, is essential to IgE/IgG1 production and lung eosinophilia induced by intranasal papain administration in mice, and IL-33 contributes to these responses. In this work, we investigate skin and Ab responses induced by s.c. papain administration into ear lobes and responses induced by subsequent airway challenge with papain. Subcutaneous papain injection induced swelling associated with increased epidermal thickness, dermal inflammation, serum IgE/IgG1 responses, and Th2 cytokine production in draining lymph node cells restimulated in vitro. These responses were markedly less upon s.c. administration of protease inhibitor-treated papain. Results obtained by using mast cell-deficient mice and reconstitution of tissue mast cells suggested the contribution of mast cells to papain-specific IgE/IgG1 responses and eosinophil infiltration. The responses were equivalent between wild-type and IL-33(-/-) mice. After the subsequent airway challenge, the s.c. presensitized wild-type mice showed more severe lung eosinophilia than those without the presensitization. The presensitized IL-33(-/-) mice showed modest lung eosinophilia, which was absent without the presensitization, but its severity and IgE boost by the airway challenge were markedly less than the presensitized wild-type mice, in which protease activity of inhaled papain contributed to the responses. The results suggest that mechanisms for the protease-dependent sensitization differ between skin and airway and that cooperation of mast cell-dependent, IL-33-independent initial sensitization via skin and protease-induced, IL-33-mediated mechanism in re-exposure via airway to protease allergens maximizes the magnitude of the transition from skin inflammation to asthma in natural history of progression of allergic diseases. PMID:27001956

  9. The PAF Complex and Prf1/Rtf1 Delineate Distinct Cdk9-Dependent Pathways Regulating Transcription Elongation in Fission Yeast

    PubMed Central

    Mbogning, Jean; Nagy, Stephen; Pagé, Viviane; Schwer, Beate; Shuman, Stewart; Fisher, Robert P.; Tanny, Jason C.

    2013-01-01

    Cyclin-dependent kinase 9 (Cdk9) promotes elongation by RNA polymerase II (RNAPII), mRNA processing, and co-transcriptional histone modification. Cdk9 phosphorylates multiple targets, including the conserved RNAPII elongation factor Spt5 and RNAPII itself, but how these different modifications mediate Cdk9 functions is not known. Here we describe two Cdk9-dependent pathways in the fission yeast Schizosaccharomyces pombe that involve distinct targets and elicit distinct biological outcomes. Phosphorylation of Spt5 by Cdk9 creates a direct binding site for Prf1/Rtf1, a transcription regulator with functional and physical links to the Polymerase Associated Factor (PAF) complex. PAF association with chromatin is also dependent on Cdk9 but involves alternate phosphoacceptor targets. Prf1 and PAF are biochemically separate in cell extracts, and genetic analyses show that Prf1 and PAF are functionally distinct and exert opposing effects on the RNAPII elongation complex. We propose that this opposition constitutes a Cdk9 auto-regulatory mechanism, such that a positive effect on elongation, driven by the PAF pathway, is kept in check by a negative effect of Prf1/Rtf1 and downstream mono-ubiquitylation of histone H2B. Thus, optimal RNAPII elongation may require balanced action of functionally distinct Cdk9 pathways. PMID:24385927

  10. Influenza A induces the major secreted airway mucin MUC5AC in a protease-EGFR-extracellular regulated kinase-Sp1-dependent pathway.

    PubMed

    Barbier, Diane; Garcia-Verdugo, Ignacio; Pothlichet, Julien; Khazen, Roxana; Descamps, Delphyne; Rousseau, Karine; Thornton, David; Si-Tahar, Mustapha; Touqui, Lhousseine; Chignard, Michel; Sallenave, Jean-Michel

    2012-08-01

    Mucins, the main glycoproteins present within mucus, modulate the rheologic properties of airways and participate in lung defense. They are thought to be able to trap and eliminate microorganisms from the lung. Among the mucins secreted in the lung, MUC5AC is the most prominent factor secreted by surface epithelial cells. Although much is known about the signaling pathways involved in the regulation of MUC5AC by host factors such as cytokines or proteases, less is known about the pathways triggered by microorganisms and, specifically, by influenza A virus (IAV). We therefore set up experiments to dissect the molecular mechanisms responsible for the potential modulation of MUC5AC by IAV. Using epithelial cells, C57/Bl6 mice, and IAV strains, we measured MUC5AC expression at the RNA and protein levels, specificity protein 1 (Sp1) activation, and protease activity. Intermediate molecular partners were confirmed using pharmacological inhibitors, blocking antibodies, and small interfering (si)RNAs. We showed in vitro and in vivo that IAV up-regulates epithelial cell-derived MUC5AC and Muc5ac expression in mice, both at transcriptional (through the induction of Sp1) and translational levels. In addition, we determined that this induction was dependent on a protease-epithelial growth factor receptor-extracellular regulated kinase-Sp1 signaling cascade, involving in particular the human airway trypsin. Our data point to MUC5AC as a potential modulatory mechanism by which the lung epithelia respond to IAV infection, and we dissect, for the first time to the best of our knowledge, the molecular partners involved. Future experiments using MUC5AC-targeted strategies should help further unravel the pathophysiological consequences of IAV-induced MUC5AC expression for lung homeostasis. PMID:22383584

  11. The papain-like protease of porcine epidemic diarrhea virus negatively regulates type I interferon pathway by acting as a viral deubiquitinase

    PubMed Central

    Xing, Yaling; Chen, Jianfei; Tu, Jian; Zhang, Bailing; Chen, Xiaojuan; Shi, Hongyan; Baker, Susan C.

    2013-01-01

    Porcine epidemic diarrhea virus (PEDV) is the cause of an economically important swine disease. Previous studies suggested that PEDV does not elicit a robust IFN response, but the mechanism(s) used to evade or block this innate immune response was not known. In this study, we found that PEDV infection blocked synthetic dsRNA-induced IFN-β production by interfering with the activation of interferon regulatory factor 3 (IRF3). We identified PEDV replicase encoded papain-like protease 2 (PLP2) as an IFN antagonist that depends on catalytic activity for its function. We show that levels of ubiquitinated proteins are reduced during PEDV infection and that PEDV PLP2 has deubiquitinase (DUB) activity that recognizes and processes both K-48 and K-63 linked polyubiquitin chains. Furthermore, we found that PEDV PLP2 strongly inhibits RIG-I- and STING-activated IFN expression and that PEDV PLP2 can be co-immunoprecipitated with and deubiquitinates RIG-I and STING, the key components of the signalling pathway for IFN expression. These results show that PEDV infection suppresses production of IFN-β and provides evidence indicating that the PEDV papain-like protease 2 acts as a viral DUB to interfere with the RIG-I- and STING-mediated signalling pathway. PMID:23596270

  12. 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

  13. 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. PMID:27200087

  14. 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

  15. Distinct localization of two closely related Ypt3/Rab11 proteins on the trafficking pathway in higher plants.

    PubMed

    Inaba, Takehito; Nagano, Yukio; Nagasaki, Takeshi; Sasaki, Yukiko

    2002-03-15

    Ypt/Rab proteins are Ras-related small GTPases that act on the intracellular membrane through the trafficking pathway, and their function depends on their localization. Approximately 25 genes encoding Ypt3/Rab11-related proteins exist in Arabidopsis, but the reason for the presence of many genes in plants remains unclear. Pea Pra2 and Pra3, members of Ypt3/Rab11, are closely related proteins. Because possible orthologs are conserved among dicots, they can be studied to determine their possible localization. Biochemical analysis revealed that these proteins were localized on distinct membranes in pea. Furthermore, using green fluorescent protein-Pra2 and green fluorescent protein-Pra3 fusion proteins, we demonstrated that these proteins are distinctively localized on the trafficking pathway in tobacco Bright Yellow 2 cells. Pra2 was predominantly localized on Golgi stacks and endosomes, which did not support the localization of Pra2 on the endoplasmic reticulum (Kang, J. G., Yun, J., Kim, D. H., Chung, K. S., Fujioka, S., Kim, J. I., Dae, H. W., Yoshida, S., Takatsuto, S., Song, P. S., and Park, C. M. (2001) Cell 105, 625--636). In contrast, Pra3 was likely to be localized on the trans-Golgi network and/or the prevacuolar compartment. We concluded that Pra2 and Pra3 proteins are distinctively localized on the trafficking pathway. This finding suggests that functional diversification takes place in the plant Ypt3/Rab11 family. PMID:11756458

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

    PubMed

    Wheeler, Scott R; Shi, Chanjuan; Holt, Jonathan A; Vnencak-Jones, Cindy L

    2016-06-01

    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

  17. 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

  18. Two distinct signaling pathways participate in auxin-induced swelling of pea epidermal protoplasts.

    PubMed

    Yamagami, Mutsumi; Haga, Ken; Napier, Richard M; Iino, Moritoshi

    2004-02-01

    Protoplast swelling was used to investigate auxin signaling in the growth-limiting stem epidermis. The protoplasts of epidermal cells were isolated from elongating internodes of pea (Pisum sativum). These protoplasts swelled in response to auxin, providing the clearest evidence that the epidermis can directly perceive auxin. The swelling response to the natural auxin IAA showed a biphasic dose response curve but that to the synthetic auxin 1-naphthalene acetic acid (NAA) showed a simple bell-shaped dose response curve. The responses to IAA and NAA were further analyzed using antibodies raised against ABP1 (auxin-binding protein 1), and their dependency on extracellular ions was investigated. Two signaling pathways were resolved for IAA, an ABP1-dependent pathway and an ABP1-independent pathway that is much more sensitive to IAA than the former. The response by the ABP1 pathway was eliminated by anti-ABP1 antibodies, had a higher sensitivity to NAA, and did not depend on extracellular Ca(2+). In contrast, the response by the non-ABP1 pathway was not affected by anti-ABP1 antibodies, had no sensitivity to NAA, and depended on extracellular Ca(2+). The swelling by either pathway required extracellular K(+) and Cl(-). The auxin-induced growth of pea internode segments showed similar response patterns, including the occurrence of two peaks in the dose response curve for IAA and the difference in Ca(2+) requirements. It is suggested that two signaling pathways participate in auxin-induced internode growth and that the non-ABP1 pathway is more likely to be involved in the control of growth by constitutive concentrations of endogenous auxin. PMID:14764902

  19. 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. PMID:7681456

  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. 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

    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

  2. PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways.

    PubMed

    Wang, Minli; Wu, Weizhong; Wu, Wenqi; Rosidi, Bustanur; Zhang, Lihua; Wang, Huichen; Iliakis, George

    2006-01-01

    Poly(ADP-ribose)polymerase 1 (PARP-1) recognizes DNA strand interruptions in vivo and triggers its own modification as well as that of other proteins by the sequential addition of ADP-ribose to form polymers. This modification causes a release of PARP-1 from DNA ends and initiates a variety of responses including DNA repair. While PARP-1 has been firmly implicated in base excision and single strand break repair, its role in the repair of DNA double strand breaks (DSBs) remains unclear. Here, we show that PARP-1, probably together with DNA ligase III, operates in an alternative pathway of non-homologous end joining (NHEJ) that functions as backup to the classical pathway of NHEJ that utilizes DNA-PKcs, Ku, DNA ligase IV, XRCC4, XLF/Cernunnos and Artemis. PARP-1 binds to DNA ends in direct competition with Ku. However, in irradiated cells the higher affinity of Ku for DSBs and an excessive number of other forms of competing DNA lesions limit its contribution to DSB repair. When essential components of the classical pathway of NHEJ are absent, PARP-1 is recruited for DSB repair, particularly in the absence of Ku and non-DSB lesions. This form of DSB repair is sensitive to PARP-1 inhibitors. The results define the function of PARP-1 in DSB repair and characterize a candidate pathway responsible for joining errors causing genomic instability and cancer. PMID:17088286

  3. PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways

    PubMed Central

    Wang, Minli; Wu, Weizhong; Wu, Wenqi; Rosidi, Bustanur; Zhang, Lihua; Wang, Huichen; Iliakis, George

    2006-01-01

    Poly(ADP-ribose)polymerase 1 (PARP-1) recognizes DNA strand interruptions in vivo and triggers its own modification as well as that of other proteins by the sequential addition of ADP-ribose to form polymers. This modification causes a release of PARP-1 from DNA ends and initiates a variety of responses including DNA repair. While PARP-1 has been firmly implicated in base excision and single strand break repair, its role in the repair of DNA double strand breaks (DSBs) remains unclear. Here, we show that PARP-1, probably together with DNA ligase III, operates in an alternative pathway of non-homologous end joining (NHEJ) that functions as backup to the classical pathway of NHEJ that utilizes DNA-PKcs, Ku, DNA ligase IV, XRCC4, XLF/Cernunnos and Artemis. PARP-1 binds to DNA ends in direct competition with Ku. However, in irradiated cells the higher affinity of Ku for DSBs and an excessive number of other forms of competing DNA lesions limit its contribution to DSB repair. When essential components of the classical pathway of NHEJ are absent, PARP-1 is recruited for DSB repair, particularly in the absence of Ku and non-DSB lesions. This form of DSB repair is sensitive to PARP-1 inhibitors. The results define the function of PARP-1 in DSB repair and characterize a candidate pathway responsible for joining errors causing genomic instability and cancer. PMID:17088286

  4. Distinctive microRNA signature associated of neoplasms with the Wnt/β-catenin signaling pathway.

    PubMed

    Sun, Xu; He, Yong; Huang, Cheng; Ma, Tao-Tao; Li, Jun

    2013-12-01

    As the crucial biological regulators, microRNAs that act by suppressing their target genes are involved in a variety of pathophysiological processes. It is generally accepted that microRNAs are often dysregulated in many types of neoplasm and other human diseases. In neoplasm, microRNAs may function as oncogenes or tumor suppressors. As constitutive activation of the Wnt signaling pathway is a common feature of neoplasm and contributes to its development, progression and metastasis in various cancers, numerous studies have revealed that microRNA-mediated gene regulation are interconnected with the Wnt/β-catenin signaling pathway, forming a Wnt/β-catenin-microRNA regulatory network, which is critical to successful targeting of the Wnt/β-catenin pathway for oncotherapy. In this review, we aim to accumulate recent advances on microRNAs that work in tandem with Wnt/β-catenin signaling in tumorigenesis, with particular focus on how microRNAs affect Wnt/β-catenin activity as well as how microRNAs are regulated through the Wnt/β-catenin pathway. PMID:24041653

  5. 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

  6. Blockade of the granzyme B/perforin pathway through overexpression of the serine protease inhibitor PI-9/SPI-6 constitutes a mechanism for immune escape by tumors

    PubMed Central

    Medema, J. P.; de Jong, J.; Peltenburg, L. T. C.; Verdegaal, E. M. E.; Gorter, A.; Bres, S. A.; Franken, K. L. M. C.; Hahne, M.; Albar, J. P.; Melief, C. J. M.; Offringa, R.

    2001-01-01

    The concept for cellular immunotherapy of solid tumors relies heavily on the capacity of class I MHC-restricted cytotoxic T lymphocytes (CTLs) to eliminate tumor cells. However, tumors often have managed to escape from the cytolytic machinery of these effector cells. Therefore, it is very important to chart the mechanisms through which this escape can occur. Target-cell killing by CTLs involves the induction of apoptosis by two major mechanisms: through death receptors and the perforin/granzyme B (GrB) pathway. Whereas tumors previously were shown to exhibit mechanisms for blocking the death receptor pathway, we now demonstrate that they also can resist CTL-mediated killing through interference with the perforin/GrB pathway. This escape mechanism involves expression of the serine protease inhibitor PI-9/SPI-6, which inactivates the apoptotic effector molecule GrB. Expression of PI-9 was observed in a variety of human and murine tumors. Moreover, we show that, indeed, expression results in the resistance of tumor cells to CTL-mediated killing both in vitro and in vivo. Our data reveal that PI-9/SPI-6 is an important parameter determining the success of T cell-based immunotherapeutic modalities against cancer. PMID:11562487

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

    PubMed

    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. PMID:27083044

  8. 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

  9. 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. PMID:21525293

  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. PMID:27165635

  11. Regulator of G protein signaling 8 inhibits protease-activated receptor 1/Gi/o signaling by forming a distinct G protein-dependent complex in live cells.

    PubMed

    Lee, Jinyong; Ghil, Sungho

    2016-05-01

    Activation of seven-transmembrane-domain-possessing G protein-coupled receptors (GPCRs) by extracellular stimuli elicits intracellular responses. One class of GPCRs-protease-activated receptors (PARs)-is activated by endogenous proteases, such as thrombin and trypsin. Members of the regulator of G protein signaling (RGS) family stimulate GTP hydrolysis of G protein alpha (Gα) subunits, thereby inhibiting GPCR/Gα-mediated signaling. We previously reported that RGS2 and RGS4 inhibit PAR1/Gα-mediated signaling by interacting with PAR1 in a Gα-dependent manner. Here, employing the bioluminescence resonance energy transfer (BRET) technique, we identified RGS8 as a novel PAR1-interacting protein. Very little BRET activity was observed between PAR1-Venus (PAR1-Ven) and RGS8-Luciferase (RGS8-Luc) in the absence of Gα. However, in the presence of Gαo, BRET activity was specifically and significantly increased. This interaction was confirmed by biochemical and immunofluorescence assays. Notably, RGS8 inhibited PAR1/Gαi/o-mediated adenylyl cyclase and ERK activation, and prevented Gαo-induced neurite outgrowth and activation of Necdin protein, a downstream target of Gαo. Our findings suggest a novel function of RGS8 and reveal cellular mechanisms by which RGS8 mediates PAR1 inhibition. PMID:26829215

  12. Distinct patterns of the histone marks associated with recruitment of the methionine chain-elongation pathway from leucine biosynthesis.

    PubMed

    Xue, Ming; Long, Jingcheng; Jiang, Qinlong; Wang, Minghui; Chen, Sixue; Pang, Qiuying; He, Yan

    2015-02-01

    Aliphatic glucosinolates (GLSs) are derived from chain-elongated methionine produced by an iterative three-step process, known to be evolutionarily recruited from leucine biosynthesis. The divergence of homologous genes between two pathways is mainly linked to the alterations in biochemical features. In this study, it was discovered that a distinct pattern of histone modifications is associated with and/or contributes to the divergence of the two pathways. In general, genes involved in leucine biosynthesis were robustly associated with H3k4me2 and H3K4me3. In contrast, despite the considerable abundances of H3K4me2 observed in some of genes involved in methionine chain elongation, H3K4me3 was completely missing. This H3K4m3-depleted pattern had no effect on gene transcription, whereas it seemingly co-evolved with the entire pathway of aliphatic GLS biosynthesis. The results reveal a novel association of the epigenetic marks with plant secondary metabolism, and may help to understand the recruitment of the methionine chain-elongation pathway from leucine biosynthesis. PMID:25428994

  13. 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

  14. 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. PMID:24068326

  15. Interleukin-13 Activates Distinct Cellular Pathways Leading to Ductular Reaction, Steatosis, and Fibrosis.

    PubMed

    Gieseck, Richard L; Ramalingam, Thirumalai R; Hart, Kevin M; Vannella, Kevin M; Cantu, David A; Lu, Wei-Yu; Ferreira-González, Sofía; Forbes, Stuart J; Vallier, Ludovic; Wynn, Thomas A

    2016-07-19

    Fibroproliferative diseases are driven by dysregulated tissue repair responses and are a major cause of morbidity and mortality because they affect nearly every organ system. Type 2 cytokine responses are critically involved in tissue repair; however, the mechanisms that regulate beneficial regeneration versus pathological fibrosis are not well understood. Here, we have shown that the type 2 effector cytokine interleukin-13 simultaneously, yet independently, directed hepatic fibrosis and the compensatory proliferation of hepatocytes and biliary cells in progressive models of liver disease induced by interleukin-13 overexpression or after infection with Schistosoma mansoni. Using transgenic mice with interleukin-13 signaling genetically disrupted in hepatocytes, cholangiocytes, or resident tissue fibroblasts, we have revealed direct and distinct roles for interleukin-13 in fibrosis, steatosis, cholestasis, and ductular reaction. Together, these studies show that these mechanisms are simultaneously controlled but distinctly regulated by interleukin-13 signaling. Thus, it may be possible to promote interleukin-13-dependent hepatobiliary expansion without generating pathological fibrosis. VIDEO ABSTRACT. PMID:27421703

  16. Blue Light Induces a Distinct Starch Degradation Pathway in Guard Cells for Stomatal Opening.

    PubMed

    Horrer, Daniel; Flütsch, Sabrina; Pazmino, Diana; Matthews, Jack S A; Thalmann, Matthias; Nigro, Arianna; Leonhardt, Nathalie; Lawson, Tracy; Santelia, Diana

    2016-02-01

    Stomatal pores form a crucial interface between the leaf mesophyll and the atmosphere, controlling water and carbon balance in plants [1]. Major advances have been made in understanding the regulatory networks and ion fluxes in the guard cells surrounding the stomatal pore [2]. However, our knowledge on the role of carbon metabolism in these cells is still fragmentary [3-5]. In particular, the contribution of starch in stomatal opening remains elusive [6]. Here, we used Arabidopsis thaliana as a model plant to provide the first quantitative analysis of starch turnover in guard cells of intact leaves during the diurnal cycle. Starch is present in guard cells at the end of night, unlike in the rest of the leaf, but is rapidly degraded within 30 min of light. This process is critical for the rapidity of stomatal opening and biomass production. We exploited Arabidopsis molecular genetics to define the mechanism and regulation of guard cell starch metabolism, showing it to be mediated by a previously uncharacterized pathway. This involves the synergistic action of β-amylase 1 (BAM1) and α-amylase 3 (AMY3)-enzymes that are normally not required for nighttime starch degradation in other leaf tissues. This pathway is under the control of the phototropin-dependent blue-light signaling cascade and correlated with the activity of the plasma membrane H(+)-ATPase. Our results show that guard cell starch degradation has an important role in plant growth by driving stomatal responses to light. PMID:26774787

  17. 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

  18. 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

  19. Distinct Gene Regulatory Pathways for Human Innate versus Adaptive Lymphoid Cells.

    PubMed

    Koues, Olivia I; Collins, Patrick L; Cella, Marina; Robinette, Michelle L; Porter, Sofia I; Pyfrom, Sarah C; Payton, Jacqueline E; Colonna, Marco; Oltz, Eugene M

    2016-05-19

    Innate lymphoid cells (ILCs) serve as sentinels in mucosal tissues, sensing release of soluble inflammatory mediators, rapidly communicating danger via cytokine secretion, and functioning as guardians of tissue homeostasis. Although ILCs have been extensively studied in model organisms, little is known about these "first responders" in humans, especially their lineage and functional kinships to cytokine-secreting T helper (Th) cell counterparts. Here, we report gene regulatory circuitries for four human ILC-Th counterparts derived from mucosal environments, revealing that each ILC subset diverges as a distinct lineage from Th and circulating natural killer cells but shares circuitry devoted to functional polarization with their Th counterparts. Super-enhancers demarcate cohorts of cell-identity genes in each lineage, uncovering new modes of regulation for signature cytokines, new molecules that likely impart important functions to ILCs, and potential mechanisms for autoimmune disease SNP associations within ILC-Th subsets. PMID:27156452

  20. 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

  1. REDOX regulation of IL-13 signaling in intestinal epithelial cells: usage of alternate pathways mediates distinct gene expression patterns

    PubMed Central

    Mandal, Debasmita; Fu, Pingfu; Levine, Alan D.

    2010-01-01

    In the classic view interleukin-13 (IL-13) binds to a heterodimer protein complex of the IL-13Rα1 and IL-4Rα chains and signals through a janus kinase 1 (JAK1)-signal transducer and activator of transcription 6 (STAT6) mechanism. We recently reported that IL-13 also signals through the IL-13Rα2 chain initiating all three mitogen activated protein kinase (MAPK) pathways, and the relative expression of IL-13Rα1 and IL-13Rα2 modulates one another’s transduction pathway. Therefore we investigated whether generation of reactive oxygen species (ROS) as second messengers may serve as a common nexus between these two pathways emanating from the individual IL-13 receptor chains in intestinal epithelial cells (IEC). IL-13 stimulates intracellular ROS synthesis within 5 min via IL-13Rα1-JAK1-STAT6- and IL-13Rα2-MEK1/2-ERK1/2-dependent activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-1 (NOX-1). IL-13-induced ROS generation in turn positively regulates phosphorylation of ERK1/2 and STAT6, yielding a feed forward amplification loop. IL-13 also stimulates the stable, long-term gene expression of two other NADPH oxidases, NOX-4 and DUOX-2, which along with constitutive NOX-1, might facilitate elevated, continuous production of ROS in IL-13-activated IEC. The contribution of each signal transduction pathway initiated by IL-13 engagement to such biological functions as wound healing, inflammation, and apoptosis was mapped for representative, responsive genes. Distinct usage patterns were observed, demonstrating that not only is IL-13 signal transduction through STAT6, MAPK, and ROS regulated in both an antagonistic and cyclic fashion, but each pathway also plays a specific role in modulating the wound healing and anti-apoptotic capabilities of the intestinal epithelium. PMID:20570727

  2. 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

  3. 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.

  4. Mouse Ficolin B Has an Ability to Form Complexes with Mannose-Binding Lectin-Associated Serine Proteases and Activate Complement through the Lectin Pathway

    PubMed Central

    Endo, Yuichi; Iwaki, Daisuke; Ishida, Yumi; Takahashi, Minoru; Matsushita, Misao; Fujita, Teizo

    2012-01-01

    Ficolins are thought to be pathogen-associated-molecular-pattern-(PAMP-) recognition molecules that function to support innate immunity. Like mannose-binding lectins (MBLs), most mammalian ficolins form complexes with MBL-associated serine proteases (MASPs), leading to complement activation via the lectin pathway. However, the ability of murine ficolin B, a homologue of human M-ficolin, to perform this function is still controversial. The results of the present study show that ficolin B in mouse bone marrow is an oligomeric protein. Ficolin B, pulled down using GlcNAc-agarose, contained very low, but detectable, amounts of MASP-2 and small MBL-associated protein (sMAP) and showed detectable C4-deposition activity on immobilized N-acetylglucosamine. These biochemical features of ficolin B were confirmed using recombinant mouse ficolin B produced in CHO cells. Taken together, these results suggest that like other mammalian homologues, murine ficolin B has an ability to exert its function via the lectin pathway. PMID:22523468

  5. 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

  6. Two Distinct N-Glycosylation Pathways Process the Haloferax volcanii S-Layer Glycoprotein upon Changes in Environmental Salinity

    PubMed Central

    Kaminski, Lina; Guan, Ziqiang; Yurist-Doutsch, Sophie; Eichler, Jerry

    2013-01-01

    ABSTRACT N-glycosylation in Archaea presents aspects of this posttranslational modification not seen in either Eukarya or Bacteria. In the haloarchaeon Haloferax volcanii, the surface (S)-layer glycoprotein can be simultaneously modified by two different N-glycans. Asn-13 and Asn-83 are modified by a pentasaccharide, whereas Asn-498 is modified by a tetrasaccharide of distinct composition, with N-glycosylation at this position being related to environmental conditions. Specifically, N-glycosylation of Asn-498 is detected when cells are grown in the presence of 1.75 but not 3.4 M NaCl. While deletion of genes encoding components of the pentasaccharide assembly pathway had no effect on the biosynthesis of the tetrasaccharide bound to Asn-498, deletion of genes within the cluster spanning HVO_2046 to HVO_2061 interfered with the assembly and attachment of the Asn-498-linked tetrasaccharide. Transfer of the “low-salt” tetrasaccharide from the dolichol phosphate carrier upon which it is assembled to S-layer glycoprotein Asn-498 did not require AglB, the oligosaccharyltransferase responsible for pentasaccharide attachment to Asn-13 and Asn-83. Finally, although biogenesis of the low-salt tetrasaccharide is barely discernible upon growth at the elevated salinity, this glycan was readily detected under such conditions in strains deleted of pentasaccharide biosynthesis pathway genes, indicative of cross talk between the two N-glycosylation pathways. PMID:24194539

  7. LIM domain only-2 (LMO2) induces T-cell leukemia by two distinct pathways.

    PubMed

    Smith, Stephen; Tripathi, Rati; 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

  8. Interleukin 2 and erythropoietin activate STAT5/MGF via distinct pathways.

    PubMed Central

    Wakao, H; Harada, N; Kitamura, T; Mui, A L; Miyajima, A

    1995-01-01

    Signal transducers and activators of transcription (STAT) proteins play an important role in cytokine signal transduction in conjunction with Janus kinases (JAKs). MGF/STAT5 is known as prolactin regulated STAT. Here we demonstrate that interleukin 2 (IL-2) as well as erythropoietin (EPO) stimulate STAT5 and induce tyrosine phosphorylation of STAT5. These IL-2- and EPO-induced STATs have an identical DNA binding specificity and immunoreactivity. We also show that IL-4 induces a DNA binding factor which possesses similar, but distinct, DNA binding specificity from that of STAT5 and is immunologically different from STAT5. Analysis of two EPO receptor (EPOR) transfected CTLL-2 cell lines discloses that IL-2 activates JAK1 and JAK3 as well as STAT5, while EPO stimulates STAT5 and JAK2 in EPO-responsive CTLL-2 cells (ERT/E2). On the contrary, EPO activates neither JAK2 nor STAT5 in other cell lines that failed to respond to EPO (ERT cells). EPOR and JAK2 associate with each other regardless of EPO presence in ERT/E2 cells, however, such an interaction is not present in ERT cells. Thus, EPOR and JAK2 association seems to be important for EPO responsiveness in CTLL-2 cells. Images PMID:7781605

  9. 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

  10. Distinct core thalamocortical pathways to central and dorsal primary auditory cortex.

    PubMed

    Read, Heather L; Nauen, David W; Escabí, Monty A; Miller, Lee M; Schreiner, Christoph E; Winer, Jeffery A

    2011-04-01

    The cat primary auditory cortex (AI) is usually assumed to form one continuous functional region. However, the dorsal and central parts of the AI iso-frequency domain contain neurons that have distinct response properties to acoustic stimuli. In this study, we asked whether neurons projecting to dorsal versus central regions of AI originate in different parts of the medial geniculate body (MGB). Spike rate responses to variations in the sound level and frequency of pure tones were used to measure characteristic frequency (CF) and frequency resolution. These were mapped with high spatial density in order to place retrograde tracers into matching frequency regions of the central narrow-band region (cNB) and dorsal AI. Labeled neurons projecting to these two parts of AI were concentrated in the middle and rostral thirds of the MGB, respectively. There was little evidence that differences in dorsal and central AI function could be due to convergent input from cells outside the ventral division of the MGB (MGBv). Instead, inputs arising from different locations along the caudal-to-rostral dimension of MGBv represent potential sources of response differences between central and dorsal sub-regions of AI. PMID:21145383

  11. Endocytosis of lutropin by Leydig cells through a pathway distinct from the high-affinity receptor.

    PubMed

    Bozon, V; Pajot-Augy, E; Vignon, X; Salesse, R

    1998-08-25

    In porcine Leydig cells in primary culture, 95% of the internalization of [125I]porcine lutropin ([125I]pLH, which bears sulfated GalNAc) could not be ascribed to the high-affinity LH receptor (LHR). In contrast, >40% of [125I]human choriogonadotropin (hCG, with sialylated sugar chains) uptake was performed by the LHR itself. When the LHR was down-regulated by excess unlabeled hormone, the LHR-independent incorporation of [125I]pLH could be inhibited in a dose-dependent fashion by sulfated polysaccharides such as fucoidan or chondroitin-(4 or 6)-sulfate, but not by other polyanionic compounds, nor by sulfated chondroitin disaccharides. Endocytosis occurred through a clathrin-dependent pathway and was inhibited by low temperature, endocytosis inhibitors, increased ionic strength, or by EDTA and dithiothreitol. Taken together, these results suggest that a Leydig cell membrane protein (possibly a lectin, or a glycosaminoglycan receptor) could perform specific LH clearance in the testis via recognition of its sulfated sugars. PMID:9806348

  12. Metformin and rapamycin have distinct effects on the AKT pathway and proliferation in breast cancer cells.

    PubMed

    Zakikhani, Mahvash; Blouin, Marie-José; Piura, Esther; Pollak, Michael N

    2010-08-01

    Rapamycin and its analogues inhibit mTOR, which leads to decreased protein synthesis and decreased cancer cell proliferation in many experimental systems. Adenosine 5'- monophosphate-activated protein kinase (AMPK) activators such as metformin have similar actions, in keeping with the TSC2/1 pathway linking activation of AMPK to inhibition of mTOR. As mTOR inhibition by rapamycin is associated with attenuation of negative feedback to IRS-1, rapamycin is known to increase activation of AKT, which may reduce its anti-neoplastic activity. We observed that metformin exposure decreases AKT activation, an action opposite to that of rapamycin. We show that metformin (but not rapamycin) exposure leads to increased phosphorylation of IRS-1 at Ser(789), a site previously reported to inhibit downstream signaling and to be an AMPK substrate phosphorylated under conditions of cellular energy depletion. siRNA methods confirmed that reduction of AMPK levels attenuates both the IRS-1 Ser(789) phosphorylation and the inhibition of AKT activation associated with metformin exposure. Although both rapamycin and metformin inhibit mTOR (the former directly and the latter through AMPK signaling), our results demonstrate previously unrecognized differences between these agents. The data are consistent with the observation that maximal induction of apoptosis and inhibition of proliferation are greater for metformin than rapamycin. PMID:20135346

  13. Distinct pathways regulate proapoptotic Nix and BNip3 in cardiac stress.

    PubMed

    Gálvez, Anita S; Brunskill, Eric W; Marreez, Yehia; Benner, Bonnie J; Regula, Kelly M; Kirschenbaum, Lorrie A; Dorn, Gerald W

    2006-01-20

    Up-regulation of myocardial Nix and BNip3 is associated with apoptosis in cardiac hypertrophy and ischemia, respectively. To identify mechanisms of gene regulation for these critical cardiac apoptosis effectors, the determinants of Nix and BNip3 promoter activation were elucidated by luciferase reporter gene expression in neonatal rat cardiac myocytes. BNip3 transcription was increased by hypoxia but not by phenylephrine (10 microM), angiotensin II (100 nM), or isoproterenol (10 microM). In contrast, Nix transcription was increased by phenylephrine but not by isoproterenol, angiotensin II, or hypoxia. Since phenylephrine stimulates cardiomyocyte hypertrophy via protein kinase C (PKC), the effects of phorbol myristate acetate (PMA, 10 nM for 24 h) and adenoviral PKC expression were assessed. PMA and PKC alpha, but not PKC epsilon or dominant negative PKC alpha, increased Nix transcription. Multiple Nix promoter GC boxes bound transcription factor Sp-1, and basal and PMA- or PKC alpha-stimulated Nix promoter activity was suppressed by mithramycin inhibition of Sp1-DNA interactions. In vivo determinants of Nix expression were evaluated in Nix promoter-luciferase (NixP) transgenic mice that underwent ischemia-reperfusion (1 h/24 h), transverse aortic coarctation (TAC), or cross-breeding with the G(q) overexpression model of hypertrophy. Luciferase activity increased in G alpha(q)-NixP hearts 3.2 +/- 0.4-fold and in TAC hearts 2.8 +/- 0.4-fold but did not increase with infarction-reperfusion. NixP activity was proportional to the extent of TAC hypertrophy and was inhibited by mithramycin. These studies revealed distinct mechanisms of transcriptional regulation for cardiac Nix and BNip3. BNip3 is hypoxia-inducible, whereas Nix expression was induced by G alpha(q)-mediated hypertrophic stimuli. PKC alpha, a G(q) effector, transduced Nix transcriptional induction via Sp1. PMID:16291751

  14. 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

  15. 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

  16. 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. PMID:24622840

  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. Distinct roles of secreted HtrA proteases from gram-negative pathogens in cleaving the junctional protein and tumor suppressor E-cadherin.

    PubMed

    Hoy, Benjamin; Geppert, Tim; Boehm, Manja; Reisen, Felix; Plattner, Patrick; Gadermaier, Gabriele; Sewald, Norbert; Ferreira, Fatima; Briza, Peter; Schneider, Gisbert; Backert, Steffen; Wessler, Silja

    2012-03-23

    The periplasmic chaperone and serine protease HtrA is important for bacterial stress responses and protein quality control. Recently, we discovered that HtrA from Helicobacter pylori is secreted and cleaves E-cadherin to disrupt the epithelial barrier, but it remained unknown whether this maybe a general virulence mechanism. Here, we show that important other pathogens including enteropathogenic Escherichia coli, Shigella flexneri, and Campylobacter jejuni, but not Neisseria gonorrhoeae, cleaved E-cadherin on host cells. HtrA deletion in C. jejuni led to severe defects in E-cadherin cleavage, loss of cell adherence, paracellular transmigration, and basolateral invasion. Computational modeling of HtrAs revealed a conserved pocket in the active center exhibiting pronounced proteolytic activity. Differential E-cadherin cleavage was determined by an alanine-to-glutamine exchange in the active center of neisserial HtrA. These data suggest that HtrA-mediated E-cadherin cleavage is a prevalent pathogenic mechanism of multiple gram-negative bacteria representing an attractive novel target for therapeutic intervention to combat bacterial infections. PMID:22337879

  19. 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

  20. 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.

  1. The ARG1-LIKE2 Gene of Arabidopsis Functions in a Gravity Signal Transduction Pathway That Is Genetically Distinct from the PGM Pathway1

    PubMed Central

    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. PMID:12970478

  2. The regulation of p53 by phosphorylation: a model for how distinct signals integrate into the p53 pathway.

    PubMed

    Maclaine, Nicola J; Hupp, Ted R

    2009-05-01

    The tumour suppressor p53 is a transcription factor that has evolved the ability to integrate distinct environmental signals including DNA damage, virus infection, and cytokine signaling into a common biological outcome that maintains normal cellular control. Mutations in p53 switch the cellular transcription program resulting in deregulation of the stress responses that normally maintain cell and tissue integrity. Transgenic studies in mice have indicated that changes in the specific activity of p53 can have profound effects not only on cancer development, but also on organism aging. As the specific activity of p53 is regulated at a post-translational level by sets of enzymes that mediate phosphorylation, acetylation, methylation, and ubiquitin-like modifications, it is likely that physiological modifiers of the aging function of p53 would be enzymes that catalyze such covalent modifications. We demonstrate that distinct stress-activated kinases, including ataxia telangiectasia mutated (ATM), casein kinase 1 (CK1) and AMP-activated protein kinase (AMPK), mediate phosphorylation of a key phospho-acceptor site in the p53 transactivation domain in response to diverse stresses including ionizing radiation, DNA virus infection, and elevation in the intracellular AMP/ATP ratio. As diseases linked to aging can involve activation of p53-dependent changes in cellular protective pathways, the development of specific physiological models might further shed light on the role of p53 kinases in modifying age-related diseases. PMID:20157532

  3. 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

  4. Distinct Argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline

    PubMed Central

    Gu, Weifeng; Shirayama, Masaki; Conte, Darryl; Vasale, Jessica; Batista, Pedro J.; Claycomb, Julie M.; Moresco, James J.; Youngman, Elaine; Keys, Jennifer; Stoltz, Matthew J.; Chen, Chun-Chieh G.; Chaves, Daniel A.; Duan, Shenghua E.; Kasschau, Kristen D.; Falgren, Noah; Yates, John R.; Mitani, Shohei; Carrington, James C.; Mello, Craig C.

    2009-01-01

    Summary Endogenous small RNAs (endo-siRNAs) interact with Argonaute (AGO) proteins to mediate sequence-specific regulation of diverse biological processes. Here, we combine deep-sequencing and genetic approaches to explore the biogenesis and function of endo-siRNAs in C. elegans. We describe conditional alleles of the dicer-related helicase, drh-3, that abrogate both RNA interference and the biogenesis of endo-siRNAs, called 22G-RNAs. DRH-3 is a core component of RNA-dependent RNA polymerase (RdRP) complexes essential for several distinct 22G-RNA systems. We show that in the germ-line, one system is dependent on worm-specific AGOs, including WAGO-1, which localizes to germ-line nuage structures called P-granules. WAGO-1 silences certain genes, transposons, pseudogenes and cryptic loci. Finally, we demonstrate that components of the nonsense-mediated decay pathway function in at least one WAGO-mediated surveillance pathway. These findings broaden our understanding of the biogenesis and diversity of 22G-RNAs and suggest novel regulatory functions for small RNAs. PMID:19800275

  5. 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

  6. Tuning of AKT-pathway by Nef and its blockade by protease inhibitors results in limited recovery in latently HIV infected T-cell line

    PubMed Central

    Kumar, Amit; Abbas, Wasim; Colin, Laurence; Khan, Kashif Aziz; Bouchat, Sophie; Varin, Audrey; Larbi, Anis; Gatot, Jean-Stéphane; Kabeya, Kabamba; Vanhulle, Caroline; Delacourt, Nadège; Pasquereau, Sébastien; Coquard, Laurie; Borch, Alexandra; König, Renate; Clumeck, Nathan; De Wit, Stephane; Rohr, Olivier; Rouzioux, Christine; Fulop, Tamas; Van Lint, Carine; Herbein, Georges

    2016-01-01

    Akt signaling plays a central role in many biological processes, which are key players in human immunodeficiency virus 1 (HIV-1) pathogenesis. We found that Akt interacts with HIV-1 Nef protein. In primary T cells treated with exogenous Nef or acutely infected with Nef-expressing HIV-1 in vitro, Akt became phosphorylated on serine473 and threonine308. In vitro, Akt activation mediated by Nef in T-cells was blocked by HIV protease inhibitors (PI), but not by reverse transcriptase inhibitors (RTI). Ex vivo, we found that the Akt pathway is hyperactivated in peripheral blood lymphocytes (PBLs) from cART naïve HIV-1-infected patients. PBLs isolated from PI-treated patients, but not from RTI-treated patients, exhibited decreased Akt activation, T-cell proliferation and IL-2 production. We found that PI but not RTI can block HIV-1 reactivation in latently infected J-Lat lymphoid cells stimulated with various stimuli. Using luciferase measurement, we further confirmed that Nef-mediated reactivation of HIV-1 from latency in 1G5 cells was blocked by PI parallel to decreased Akt activation. Our results indicate that PI-mediated blockade of Akt activation could impact the HIV-1 reservoir and support the need to further assess the therapeutic use of HIV-1 PI in order to curtail latently infected cells in HIV-1-infected patients. PMID:27076174

  7. Tuning of AKT-pathway by Nef and its blockade by protease inhibitors results in limited recovery in latently HIV infected T-cell line.

    PubMed

    Kumar, Amit; Abbas, Wasim; Colin, Laurence; Khan, Kashif Aziz; Bouchat, Sophie; Varin, Audrey; Larbi, Anis; Gatot, Jean-Stéphane; Kabeya, Kabamba; Vanhulle, Caroline; Delacourt, Nadège; Pasquereau, Sébastien; Coquard, Laurie; Borch, Alexandra; König, Renate; Clumeck, Nathan; De Wit, Stephane; Rohr, Olivier; Rouzioux, Christine; Fulop, Tamas; Van Lint, Carine; Herbein, Georges

    2016-01-01

    Akt signaling plays a central role in many biological processes, which are key players in human immunodeficiency virus 1 (HIV-1) pathogenesis. We found that Akt interacts with HIV-1 Nef protein. In primary T cells treated with exogenous Nef or acutely infected with Nef-expressing HIV-1 in vitro, Akt became phosphorylated on serine(473) and threonine(308). In vitro, Akt activation mediated by Nef in T-cells was blocked by HIV protease inhibitors (PI), but not by reverse transcriptase inhibitors (RTI). Ex vivo, we found that the Akt pathway is hyperactivated in peripheral blood lymphocytes (PBLs) from cART naïve HIV-1-infected patients. PBLs isolated from PI-treated patients, but not from RTI-treated patients, exhibited decreased Akt activation, T-cell proliferation and IL-2 production. We found that PI but not RTI can block HIV-1 reactivation in latently infected J-Lat lymphoid cells stimulated with various stimuli. Using luciferase measurement, we further confirmed that Nef-mediated reactivation of HIV-1 from latency in 1G5 cells was blocked by PI parallel to decreased Akt activation. Our results indicate that PI-mediated blockade of Akt activation could impact the HIV-1 reservoir and support the need to further assess the therapeutic use of HIV-1 PI in order to curtail latently infected cells in HIV-1-infected patients. PMID:27076174

  8. The glycoinositol phospholipids of Leishmania mexicana promastigotes. Evidence for the presence of three distinct pathways of glycolipid biosynthesis.

    PubMed

    McConville, M J; Collidge, T A; Ferguson, M A; Schneider, P

    1993-07-25

    was highly enriched for C24:0 or C26:0 alkyl chains. These data suggest that L. mexicana promastigotes contain three distinct pathways of GPI biosynthesis. The possibility that the distinct alkyl chain compositions of the different GPI glycolipids reflects the subcellular compartmentalization of different GPI biosynthetic pathways is discussed. PMID:8340385

  9. Distinct Splice Variants and Pathway Enrichment in the Cell Line Models of Aggressive Human Breast Cancer Subtypes

    PubMed Central

    Menon, Rajasree; Im, Hogune; Zhang, Emma (Yue); Wu, Shiaw-Lin; Chen, Rui; Snyder, Michael; Hancock, William S.; Omenn, Gilbert S.

    2013-01-01

    This study was conducted as a part of the Chromosome-Centric Human Proteome Project (C-HPP) of the Human Proteome Organization. The United States team of C-HPP is focused on characterizing the protein-coding genes in chromosome 17. Despite its small size, chromosome 17 is rich in protein-coding genes, it contains many cancer-associated genes, including BRCA1, ERBB2 (Her2/neu), and TP53. The goal of this study was to examine the splice variants expressed in three ERBB2 expressed breast cancer cell line models of hormone receptor negative breast cancers by integrating RNA-Seq and proteomic mass spectrometry data. The cell-lines represent distinct phenotypic variations subtype: SKBR3 (ERBB2+ (over-expression)/ ER−/PR−; adenocarcinoma), SUM190 (ERBB2+ (over-expression)/ER−/PR−; inflammatory breast cancer) and SUM149 (ERBB2 (low expression) ER−/PR −; inflammatory breast cancer). We identified more than one splice variant for 1167 genes expressed in at least one of the three cancer cell lines. We found multiple variants of genes that are in the signaling pathways downstream of ERBB2 along with variants specific to one cancer cell line compared to the other two cancer cell lines and to normal mammary cells. The overall transcript profiles based on read counts indicated more similarities between SKBR3 and SUM190. The top-ranking Gene Ontology and BioCarta pathways for the cell-line specific variants pointed to distinct key mechanisms including: amino sugar metabolism, caspase activity, and endocytosis in SKBR3; different aspects of metabolism, especially of lipids in SUM190; cell- to-cell adhesion, integrin and ERK1/ERK2 signaling, and translational control in SUM149. The analyses indicated an enrichment in the electron transport chain processes in the ERBB2 over-expressed cell line models; and an association of nucleotide binding, RNA splicing and translation processes with the IBC models, SUM190 and SUM149. Detailed experimental studies on the distinct

  10. 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

    2016-01-01

    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. 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

  12. 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

  13. 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. PMID:24203698

  14. The Candida albicans Histone Acetyltransferase Hat1 Regulates Stress Resistance and Virulence via Distinct Chromatin Assembly Pathways

    PubMed Central

    Tscherner, Michael; Zwolanek, Florian; Jenull, Sabrina; Sedlazeck, Fritz J.; Petryshyn, Andriy; Frohner, Ingrid E.; Mavrianos, John; Chauhan, Neeraj; von Haeseler, Arndt; Kuchler, Karl

    2015-01-01

    Human fungal pathogens like Candida albicans respond to host immune surveillance by rapidly adapting their transcriptional programs. Chromatin assembly factors are involved in the regulation of stress genes by modulating the histone density at these loci. Here, we report a novel role for the chromatin assembly-associated histone acetyltransferase complex NuB4 in regulating oxidative stress resistance, antifungal drug tolerance and virulence in C. albicans. Strikingly, depletion of the NuB4 catalytic subunit, the histone acetyltransferase Hat1, markedly increases resistance to oxidative stress and tolerance to azole antifungals. Hydrogen peroxide resistance in cells lacking Hat1 results from higher induction rates of oxidative stress gene expression, accompanied by reduced histone density as well as subsequent increased RNA polymerase recruitment. Furthermore, hat1Δ/Δ cells, despite showing growth defects in vitro, display reduced susceptibility to reactive oxygen-mediated killing by innate immune cells. Thus, clearance from infected mice is delayed although cells lacking Hat1 are severely compromised in killing the host. Interestingly, increased oxidative stress resistance and azole tolerance are phenocopied by the loss of histone chaperone complexes CAF-1 and HIR, respectively, suggesting a central role for NuB4 in the delivery of histones destined for chromatin assembly via distinct pathways. Remarkably, the oxidative stress phenotype of hat1Δ/Δ cells is a species-specific trait only found in C. albicans and members of the CTG clade. The reduced azole susceptibility appears to be conserved in a wider range of fungi. Thus, our work demonstrates how highly conserved chromatin assembly pathways can acquire new functions in pathogenic fungi during coevolution with the host. PMID:26473952

  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. Monospecific inhibitors show that both mannan-binding lectin-associated serine protease-1 (MASP-1) and -2 Are essential for lectin pathway activation and reveal structural plasticity of MASP-2.

    PubMed

    Héja, Dávid; Harmat, Veronika; Fodor, Krisztián; Wilmanns, Matthias; Dobó, József; Kékesi, Katalin A; Závodszky, Péter; Gál, Péter; Pál, Gábor

    2012-06-01

    The lectin pathway is an antibody-independent activation route of the complement system. It provides immediate defense against pathogens and altered self-cells, but it also causes severe tissue damage after stroke, heart attack, and other ischemia reperfusion injuries. The pathway is triggered by target binding of pattern recognition molecules leading to the activation of zymogen mannan-binding lectin-associated serine proteases (MASPs). MASP-2 is considered as the autonomous pathway-activator, while MASP-1 is considered as an auxiliary component. We evolved a pair of monospecific MASP inhibitors. In accordance with the key role of MASP-2, the MASP-2 inhibitor completely blocks the lectin pathway activation. Importantly, the MASP-1 inhibitor does the same, demonstrating that MASP-1 is not an auxiliary but an essential pathway component. We report the first Michaelis-like complex structures of MASP-1 and MASP-2 formed with substrate-like inhibitors. The 1.28 Å resolution MASP-2 structure reveals significant plasticity of the protease, suggesting that either an induced fit or a conformational selection mechanism should contribute to the extreme specificity of the enzyme. PMID:22511776

  17. Monospecific Inhibitors Show That Both Mannan-binding Lectin-associated Serine Protease-1 (MASP-1) and -2 Are Essential for Lectin Pathway Activation and Reveal Structural Plasticity of MASP-2*

    PubMed Central

    Héja, Dávid; Harmat, Veronika; Fodor, Krisztián; Wilmanns, Matthias; Dobó, József; Kékesi, Katalin A.; Závodszky, Péter; Gál, Péter; Pál, Gábor

    2012-01-01

    The lectin pathway is an antibody-independent activation route of the complement system. It provides immediate defense against pathogens and altered self-cells, but it also causes severe tissue damage after stroke, heart attack, and other ischemia reperfusion injuries. The pathway is triggered by target binding of pattern recognition molecules leading to the activation of zymogen mannan-binding lectin-associated serine proteases (MASPs). MASP-2 is considered as the autonomous pathway-activator, while MASP-1 is considered as an auxiliary component. We evolved a pair of monospecific MASP inhibitors. In accordance with the key role of MASP-2, the MASP-2 inhibitor completely blocks the lectin pathway activation. Importantly, the MASP-1 inhibitor does the same, demonstrating that MASP-1 is not an auxiliary but an essential pathway component. We report the first Michaelis-like complex structures of MASP-1 and MASP-2 formed with substrate-like inhibitors. The 1.28 Å resolution MASP-2 structure reveals significant plasticity of the protease, suggesting that either an induced fit or a conformational selection mechanism should contribute to the extreme specificity of the enzyme. PMID:22511776

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

    PubMed

    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

  19. 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

  20. Involvement of net and Hif1alpha in distinct yet intricately linked hypoxia-induced signaling pathways.

    PubMed

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

    2010-07-01

    The present study compares negative Ets transcription factor (Net) and hypoxia-inducible factor 1alpha (HIF1alpha) regulation by hypoxia. Their protein stabilities are differently regulated by hypoxia, defining three periods in the kinetics: normoxia (high Net levels and low HIF1alpha levels), early hypoxia (high levels of Net and HIF1alpha), and late hypoxia (degradation of Net and HIF1alpha). Modulators of prolyl hydroxylase domain protein (PHD) activity induce a mobility shift of Net, similar to HIF1alpha, 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 HIF1alpha stabilization in early hypoxia and its degradation at late hypoxia, whereas PHD1 is involved in HIF1alpha degradation in late hypoxia. We describe interconnections between the regulation of both Net and HIF1alpha at the protein level. Evidence is provided for a direct physical interaction between Net and HIF1alpha and indirect transcriptional regulation loops that involve the PHDs. Taken together our results indicate that Net and HIF1alpha are components of distinct signaling pathways that are intricately linked. PMID:20427288

  1. Dietary protein quality differentially regulates trypsin enzymes at the secretion and transcription level in Panulirus argus by distinct signaling pathways.

    PubMed

    Perera, Erick; Rodríguez-Viera, Leandro; Rodríguez-Casariego, Javier; Fraga, Iliana; Carrillo, Olimpia; Martínez-Rodríguez, Gonzalo; Mancera, Juan M

    2012-03-01

    The effects of pelleted diets with different protein composition (fish, squid or soybean meals as main protein sources) on trypsin secretion and expression were studied in the lobster Panulirus argus. Trypsin secretion was shown to be maximal 4 h after ingestion. At this time, fish- and squid-based diets induced trypsin secretion, as well as up-regulation of the major trypsin isoform at the transcription level. While fish- and squid-based diets elicited a prandial response, soybean-based diet failed to stimulate the digestive gland to secrete trypsin into the gastric fluid or induce trypsin expression above the levels observed in fasting lobsters. In vitro assays showed that intact proteins rather than protein hydrolysates stimulate trypsin secretion in the lobster. However, the signal for trypsin transcription appears to be different to that for secretion and is probably mediated by the appearance of free amino acids in the digestive gland, suggesting a stepwise regulation of trypsin enzymes during digestion. We conclude that trypsin enzymes in P. argus are regulated at the transcription and secretion level by the quality of dietary proteins through two distinct signaling pathways. Our results indicate that protein digestion efficiency in spiny lobsters can be improved by selecting appropriated protein sources. However, other factors like the poor solubility of dietary proteins in dry diets could hamper further enhancement of digestion efficiency. PMID:22323208

  2. Transformation processes, pathways, and possible sources of distinctive polychlorinated dibenzo-p-dioxin signatures in sink environments.

    PubMed

    Gaus, Caroline; Brunskill, Gregg J; Connell, W; Prange, Joelle; Müller, Jochen F; Päpke, Olaf; Weber, Roland

    2002-08-15

    In recent years, studies on environmental samples with unusual dibenzo-p-dioxin (PCDD) congener profiles were reported from a range of countries. These profiles, characterized by a dominance of octachlorinated dibenzodioxin (OCDD) and relatively low in dibenzofuran (PCDF) concentrations, could not be attributed to known sources or formation processes. In the present study, the processes that result in these unusual profiles were assessed using the concentrations and isomer signatures of PCDDs from dated estuarine sediment cores in Queensland, Australia. Increases in relative concentrations of lower chlorinated PCDDs and a relative decrease of OCDD were correlated with time of sediment deposition. Preferred lateral, anaerobic dechlorination of OCDD represents a likely pathway for these changes. In Queensland sediments, these transformations result in a distinct dominance of isomers fully chlorinated in the 1,4,6,9-positions (1,4-patterns), and similar 1,4-patterns were observed in sediments from elsewhere. Consequently, these environmental samples may not reflect the signatures of the original source, and a reevaluation of source inputs was undertaken. Natural formation of PCDDs, which has previously been suggested, is discussed; however, based on the present results and literature comparisons, we propose an alternative scenario. This scenario hypothesizes that an anthropogenic PCDD precursor input (e.g. pentachlorophenol) results in the contamination. These results and hypothesis imply further investigations are warranted into possible anthropogenic sources in areas where natural PCDD formation has been suggested. PMID:12214647

  3. 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

  4. Distinct pathways of insulin-regulated versus diabetes-regulated gene expression: An in vivo analysis in MIRKO mice

    PubMed Central

    Yechoor, Vijay K.; Patti, Mary-Elizabeth; Ueki, Kohjiro; Laustsen, Palle G.; Saccone, Robert; Rauniyar, Ravi; Kahn, C. Ronald

    2004-01-01

    Diabetes mellitus is a complex metabolic disorder accompanied by alterations in cellular physiology, metabolism, and gene expression. These alterations can be primary (due to loss of direct insulin action) or secondary (due to the metabolic perturbations associated with the disease). To dissect and quantitate these two separate effects, we compared the skeletal muscle gene-expression profiles of muscle insulin receptor knockout (MIRKO) mice and their Lox controls in the basal, streptozotocin-induced diabetic, and insulin-treated diabetic states. Pure deficiency of insulin action as present in the MIRKO mouse results in regulation of 130 genes, with down-regulation of NSF (N-ethylmaleimide-sensitive fusion protein) and VAMP-2 (vesicle-associated membrane protein 2), stearoyl CoA desaturase 1, and cAMP-specific phosphodiesterase 4B, as well as up-regulation of some signaling-related genes, such as Akt2, and the fatty-acid transporter CD36. In diabetes, additional transcriptional mechanisms are activated, resulting in alterations in expression of ≈500 genes, including a highly coordinated down-regulation of genes of the mitochondrial electron-transport chain and one of the mammalian homologues of the histone deacetylase Sir2, which has been implicated in the link between nutrition and longevity. These distinct pathways of direct and indirect regulation of gene expression provide insights into the complex mechanisms of transcriptional control in diabetes and areas of potential therapeutic targeting. PMID:15546994

  5. 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

    2016-01-01

    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. PMID:27164085

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

    PubMed Central

    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

    2016-01-01

    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. PMID:27164085

  7. The Levels of the Lectin Pathway Serine Protease MASP-1 and Its Complex Formation with C1 Inhibitor Are Linked to the Severity of Hereditary Angioedema.

    PubMed

    Hansen, Cecilie Bo; Csuka, Dorottya; Munthe-Fog, Lea; Varga, Lilian; Farkas, Henriette; Hansen, Karin Møller; Koch, Claus; Skjødt, Karsten; Garred, Peter; Skjoedt, Mikkel-Ole

    2015-10-15

    C1 inhibitor (C1-INH) is known to form complexes with the lectin complement pathway serine proteases MASP-1 and MASP-2. Deficiency of C1-INH is associated with hereditary angioedema (HAE), an autosomal inherited disease characterized by swelling attacks caused by elevated levels of bradykinin. MASP-1 was shown to cleave high m.w. kininogen into bradykinin; therefore, we hypothesized that MASP-1 levels and the quantity of MASP-1/C1-INH complexes might be associated with different paraclinical and clinical outcomes of HAE. We measured MASP-1 serum concentrations and endogenous MASP-1/C1-INH complex levels in 128 HAE patients and 100 controls. Relatively high levels of pre-existing MASP-1/C1-INH complexes were observed in normal serum, and we found that both the serum levels of MASP-1 and the complex formation between MASP-1 and C1-INH were significantly reduced in HAE patients compared with matched controls (p < 0.0001). The level of MASP-1 and MASP-1/C1-INH complexes in HE patients correlated with the level of C1-INH (p = 0.0009 and p = 0.0047, respectively), the level of C4 (p = 0.0084 and p < 0.0001, respectively), and the number of attacks in the year of blood sampling (p = 0.0075 and p = 0.0058, respectively). In conclusion, we show that MASP-1/C1-INH complexes circulate in normal human blood. The levels of MASP-1 and MASP-1/C1-INH complexes are reduced in HAE patients compared with controls. Both MASP-1 and MASP-1/C1-INH complexes are related to the degree of complement C4 consumption, as well as the severity of disease. These results suggest that MASP-1 may exert a previously unrecognized role in the pathophysiology of HAE. PMID:26371246

  8. 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

  9. Distinctive G Protein-Dependent Signaling by Protease-Activated Receptor 2 (PAR2) in Smooth Muscle: Feedback Inhibition of RhoA by cAMP-Independent PKA

    PubMed Central

    Sriwai, Wimolpak; Mahavadi, Sunila; Al-Shboul, Othman; Grider, John R.; Murthy, Karnam S.

    2013-01-01

    We examined expression of protease-activated receptors 2 (PAR2) and characterized their signaling pathways in rabbit gastric muscle cells. The PAR2 activating peptide SLIGRL (PAR2-AP) stimulated Gq, G13, Gi1, PI hydrolysis, and Rho kinase activity, and inhibited cAMP formation. Stimulation of PI hydrolysis was partly inhibited in cells expressing PAR2 siRNA, Gaq or Gai minigene and in cells treated with pertussis toxin, and augmented by expression of dominant negative regulator of G protein signaling (RGS4(N88S)). Stimulation of Rho kinase activity was abolished by PAR-2 or Ga13 siRNA, and by Ga13 minigene. PAR2-AP induced a biphasic contraction; initial contraction was selectively blocked by the inhibitor of PI hydrolysis (U73122) or MLC kinase (ML-9), whereas sustained contraction was selectively blocked by the Rho kinase inhibitor (Y27632). PAR2-AP induced phosphorylation of MLC20, MYPT1 but not CPI-17. PAR2-AP also caused a decrease in the association of NF-kB and PKA catalytic subunit: the effect of PAR2-AP was blocked by PAR2 siRNA or phosphorylation-deficient RhoA (RhoA(S188A)). PAR2-AP-induced degradation of IkBa and activation of NF-kB were abolished by the blockade of RhoA activity by Clostridium botulinum C3 exoenzyme suggesting RhoA-dependent activation of NF-kB. PAR2-AP-stimulated Rho kinase activity was significantly augmented by the inhibitors of PKA (myristoylated PKI), IKK2 (IKKIV) or NF-kB (MG132), and in cells expressing dominant negative mutants of IKK (IKK(K44A), IkBa (IkBa (S32A/S36A)) or RhoA(S188A), suggesting feedback inhibition of Rho kinase activity via PKA derived from NF-kB pathway. PAR2-AP induced phosphorylation of RhoA and the phosphorylation was attenuated in cells expressing phosphorylation-deficient RhoA(S188A). Our results identified signaling pathways activated by PAR2 to mediate smooth muscle contraction and a novel pathway for feedback inhibition of PAR2-stimulated RhoA. The pathway involves activation of the NF-kB to release

  10. Two distinct pathways responsible for the loading of CENP-A to centromeres in the fission yeast cell cycle.

    PubMed

    Takahashi, Kohta; Takayama, Yuko; Masuda, Fumie; Kobayashi, Yasuyo; Saitoh, Shigeaki

    2005-03-29

    CENP-A is a centromere-specific histone H3 variant that is- essential for faithful chromosome segregation in all eukaryotes thus far investigated. We genetically identified two factors, Ams2 and Mis6, each of which is required for the correct centromere localization of SpCENP-A (Cnp1), the fission yeast homologue of CENP-A. Ams2 is a cell-cycle-regulated GATA factor that localizes on the nuclear chromatin, including on centromeres, during the S phase. Ams2 may be responsible for the replication-coupled loading of SpCENP-A by facilitating nucleosomal formation during the S phase. Consistently, overproduction of histone H4, but not that of H3, suppressed the defect of SpCENP-A localization in Ams2-deficient cells. We demonstrated the existence of at least two distinct phases for SpCENP-A loading during the cell cycle: the S phase and the late-G2 phase. Ectopically induced SpCENP-A was efficiently loaded onto the centromeres in G2-arrested cells, indicating that SpCENP-A probably undergoes replication-uncoupled loading after the completion of S phase. This G2 loading pathway of SpCENP-A may require Mis6, a constitutive centromere-binding protein that is also implicated in the Mad2-dependent spindle attachment checkpoint response. Here, we discuss the functional relationship between the flexible loading mechanism of CENP-A and the plasticity of centromere chromatin formation in fission yeast. PMID:15897182

  11. The Dopamine D1–D2 Receptor Heteromer in Striatal Medium Spiny Neurons: Evidence for a Third Distinct Neuronal Pathway in Basal Ganglia

    PubMed Central

    Perreault, Melissa L.; Hasbi, Ahmed; O’Dowd, Brian F.; George, Susan R.

    2011-01-01

    Dopaminergic signaling within the basal ganglia has classically been thought to occur within two distinct neuronal pathways; the direct striatonigral pathway which contains the dopamine D1 receptor and the neuropeptides dynorphin (DYN) and substance P, and the indirect striatopallidal pathway which expresses the dopamine D2 receptor and enkephalin (ENK). A number of studies have also shown, however, that D1 and D2 receptors can co-exist within the same medium spiny neuron and emerging evidence indicates that these D1/D2-coexpressing neurons, which also express DYN and ENK, may comprise a third neuronal pathway, with representation in both the striatonigral and striatopallidal projections of the basal ganglia. Furthermore, within these coexpressing neurons it has been shown that the dopamine D1 and D2 receptor can form a novel and pharmacologically distinct receptor complex, the dopamine D1–D2 receptor heteromer, with unique signaling properties. This is indicative of a functionally unique role for these neurons in brain. The aim of this review is to discuss the evidence in support of a novel third pathway coexpressing the D1 and D2 receptor, to discuss the potential relevance of this pathway to basal ganglia signaling, and to address its potential value, and that of the dopamine D1–D2 receptor heteromer, in the search for new therapeutic strategies for disorders involving dopamine neurotransmission. PMID:21747759

  12. 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.

  13. 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. PMID:17239413

  14. Protease proteomics: revealing protease in vivo functions using systems biology approaches.

    PubMed

    Doucet, Alain; Overall, Christopher M

    2008-10-01

    Proteases irreversibly modify proteins by cleaving their amide bonds and are implicated in virtually every important biological process such as immunity, development and tissue repair. Accordingly, it is easy to see that deregulated proteolysis is a pathognomic feature of many diseases. Most of the current information available on proteases was acquired using in vitro methods, which reveals molecular structure, enzyme kinetics and active-site specificity. However, considerably less is known about the relevant biological functions and combined roles of proteases in moulding the proteome. Although models using genetically modified animals are powerful, they are slow to develop, they can be difficult to interpret, and while useful, they remain only models of human disease. Therefore, to understand how proteases accomplish their tasks in organisms and how they participate in pathology, we need to elucidate the protease degradome-the repertoire of proteases expressed by a cell, a tissue or an organism at a particular time-their expression level, activation state, their biological substrates, also known as the substrate degradome-the repertoire of substrates for each protease-and the effect of the activity of each protease on the pathways of the system under study. Achieving this goal is challenging because several proteases might cleave the same protein, and proteases also form pathways and interact to form the protease web [Overall, C.M., Kleifeld, O., 2006. Tumour microenvironment - opinion: validating matrix metalloproteinases as drug targets and anti-targets for cancer therapy. Nat. Rev. Cancer 6 (3), 227-239]. Hence, the net proteolytic potential of the degradome at a particular time on a substrate and pathway must also be understood. Proteomics offers one of the few routes to the understanding of proteolysis in complex in vivo systems and especially in man where genetic manipulations are impossible. The aim of this chapter is to review methods and tools that allow

  15. 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

  16. Toxins that are activated by HIV type-1 protease through removal of a signal for degradation by the N-end-rule pathway.

    PubMed Central

    Falnes, P O; Welker, R; Kräusslich, H G; Olsnes, S

    1999-01-01

    Diphtheria toxin enters the cytosol of mammalian cells where it inhibits cellular protein synthesis, leading to cell death. Recently we found that the addition of a signal for N-end-rule-mediated protein degradation to diphtheria toxin substantially reduced its intracellular stability and toxicity. These results prompted us to construct a toxin containing a degradation signal that is removable through the action of a viral protease. In principle, such a toxin would be preferentially stabilized, and thus activated, in cells expressing the viral protease in the cytosol, i.e. virus-infected cells, thereby providing a specific eradication of these cells. In the present work we describe the construction of toxins that contain a signal for N-end-rule-mediated degradation just upstream of a cleavage site for the protease from HIV type 1 (HIV-1 PR). We show that the toxins are cleaved by HIV-1 PR exclusively at the introduced sites, and thereby are converted from unstable to stable proteins. Furthermore, this cleavage substantially increased the ability of the toxins to inhibit cellular protein synthesis. However, the toxins were unable to selectively eradicate HIV-1-infected cells, apparently due to low cytosolic HIV-1 PR activity, since we could not detect cleavage of the toxins by HIV-1 PR in infected cells. Alternative strategies for the construction of toxins that can specifically be activated by viral proteases are discussed. PMID:10493930

  17. 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

  18. 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

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

    2013-03-29

    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

  19. 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

  20. Immune challenge and satiety-related activation of both distinct and overlapping neuronal populations in the brainstem indicate parallel pathways for viscerosensory signaling.

    PubMed

    Gaykema, Ronald P A; Daniels, Teresa E; Shapiro, Nathan J; Thacker, Gregory C; Park, Su-Mi; Goehler, Lisa E

    2009-10-19

    Caudal brainstem viscerosensory nuclei convey information about the body's internal state to forebrain regions implicated in feeding behavior and responses to immune challenge, and may modulate ingestive behavior following immune activation. Illness-induced appetite loss might be attributed to accentuated "satiety" pathways, activation of a distinct "danger channel" separate from satiety pathways, or both. To evaluate neural substrates that could mediate the effects of illness on ingestive behavior, we analyzed the pattern and phenotypes of medullary neurons responsive to consumption of a preferred food, sweetened milk, and to intraperitoneal lipopolysaccharide challenge that reduced sweetened milk intake. Brainstem sections were stained for c-Fos, dopamine beta-hydroxylase, phenylethanolamine-N-methyltransferase, and glucagon-like peptide-1 (GLP-1) immunoreactivity. Sweetened milk intake activated many neurons throughout the nucleus of the solitary tract (NTS), including A2 noradrenergic neurons in the caudal half of the NTS. LPS challenge activated a similar population of neurons in the NTS, in addition to rostral C2 adrenergic and mid-level A2 noradrenergic neurons in the NTS, many C1 and A1 neurons in the ventrolateral medulla, and in GLP-1 neurons in the dorsal medullary reticular nucleus. Increased numbers of activated GLP-1 neurons in the NTS were only associated with sweetened milk ingestion. Evidence for parallel processing was reflected in the parabrachial nucleus, where sweetened milk intake resulted in activation of the inner external lateral, ventrolateral and central medial portions, whereas LPS challenge induced c-Fos expression in the outer external lateral portions. Thus, signals generated in response to potentially dangerous physiological conditions seem to be propagated via specific populations of catecholaminergic neurons in the NTS and VLM, and likely include a pathway through the external lateral PBN. The data indicate that immune challenge

  1. Serine protease autotransporters of enterobacteriaceae (SPATEs): biogenesis and function.

    PubMed

    Dautin, Nathalie

    2010-06-01

    Serine Protease Autotransporters of Enterobacteriaceae (SPATEs) constitute a large family of proteases secreted by Escherichia coli and Shigella. SPATEs exhibit two distinct proteolytic activities. First, a C-terminal catalytic site triggers an intra-molecular cleavage that releases the N-terminal portion of these proteins in the extracellular medium. Second, the secreted N-terminal domains of SPATEs are themselves proteases; each contains a canonical serine-protease catalytic site. Some of these secreted proteases are toxins, eliciting various effects on mammalian cells. Here, we discuss the biogenesis of SPATEs and their function as toxins. PMID:22069633

  2. Transport through the yeast endocytic pathway occurs through morphologically distinct compartments and requires an active secretory pathway and Sec18p/N-ethylmaleimide-sensitive fusion protein.

    PubMed Central

    Hicke, L; Zanolari, B; Pypaert, M; Rohrer, J; Riezman, H

    1997-01-01

    Molecules travel through the yeast endocytic pathway from the cell surface to the lysosome-like vacuole by passing through two sequential intermediates. Immunofluorescent detection of an endocytosed pheromone receptor was used to morphologically identify these intermediates, the early and late endosomes. The early endosome is a peripheral organelle that is heterogeneous in appearance, whereas the late endosome is a large perivacuolar compartment that corresponds to the prevacuolar compartment previously shown to be an endocytic intermediate. We demonstrate that inhibiting transport through the early secretory pathway in sec mutants quickly impedes transport from the early endosome. Treatment of sensitive cells with brefeldin A also blocks transport from this compartment. We provide evidence that Sec18p/N-ethylmaleimide-sensitive fusion protein, a protein required for membrane fusion, is directly required in vivo for forward transport early in the endocytic pathway. Inhibiting protein synthesis does not affect transport from the early endosome but causes endocytosed proteins to accumulate in the late endosome. As newly synthesized proteins and the late steps of secretion are not required for early to late endosome transport, but endoplasmic reticulum through Golgi traffic is, we propose that efficient forward transport in the early endocytic pathway requires delivery of lipid from secretory organelles to endosomes. Images PMID:9017592

  3. Differential modulation of the glutamate-nitric oxide-cyclic GMP pathway by distinct neurosteroids in cerebellum in vivo.

    PubMed

    Cauli, O; González-Usano, A; Agustí, A; Felipo, V

    2011-09-01

    The glutamate-nitric oxide (NO)-cGMP pathway mediates many responses to activation of N-methyl-d-aspartate (NMDA) receptors, including modulation of some types of learning and memory. The glutamate-NO-cGMP pathway is modulated by GABAergic neurotransmission. Activation of GABA(A) receptors reduces the function of the pathway. Several neurosteroids modulate the activity of GABA(A) and/or NMDA receptors, suggesting that they could modulate the function of the glutamate-NO-cGMP pathway. The aim of this work was to assess, by in vivo microdialysis, the effects of several neurosteroids with different effects on GABA(A) and NMDA receptors on the function of the glutamate-NO-cGMP pathway in cerebellum in vivo. To assess the effects of the neurosteroids on the glutamate-NO-cGMP pathway, they were administered through the microdialysis probe before administration of NMDA and the effects on NMDA-induced increase in extracellular cGMP were analyzed. We also assessed the effects of the neurosteroids on basal levels of extracellular cGMP. To assess the effects of neurosteroids on nitric oxide synthase (NOS) activity and on NMDA-induced activation of NOS, we also measured the effects of the neurosteroids on extracellular citrulline. Pregnanolone and tetrahydrodeoxy-corticosterone (THDOC) behave as agonists of GABA(A) receptors and completely block NMDA-induced increase in cGMP. Pregnanolone but not THDOC also reduced basal levels of extracellular cGMP. Pregnenolone did not affect extracellular cGMP or its increase by NMDA administration. Pregnenolone sulfate increased basal extracellular cGMP and potentiated NMDA-induced increase in cGMP, behaving as an enhancer of NMDA receptors activation. Allopregnanolone and dehydroepiandrosterone sulphate behave as antagonists of NMDA receptors, increasing basal cGMP and blocking completely NMDA-induced increase in cGMP. Dehydroepiandrosterone sulphate seems to do this by activating sigma receptors. These data support the concept that, at

  4. The striatal balancing act in drug addiction: distinct roles of direct and indirect pathway medium spiny neurons.

    PubMed

    Lobo, Mary Kay; Nestler, Eric J

    2011-01-01

    The striatum plays a key role in mediating the acute and chronic effects of addictive drugs, with drugs of abuse causing long-lasting molecular and cellular alterations in both dorsal striatum and nucleus accumbens (ventral striatum). Despite the wealth of research on the biological actions of abused drugs in striatum, until recently, the distinct roles of the striatum's two major subtypes of medium spiny neurons (MSNs) in drug addiction remained elusive. Recent advances in cell-type-specific technologies, including fluorescent reporter mice, transgenic, or knockout mice, and viral-mediated gene transfer, have advanced the field toward a more comprehensive understanding of the two MSN subtypes in the long-term actions of drugs of abuse. Here we review progress in defining the distinct molecular and functional contributions of the two MSN subtypes in mediating addiction. PMID:21811439

  5. The Striatal Balancing Act in Drug Addiction: Distinct Roles of Direct and Indirect Pathway Medium Spiny Neurons

    PubMed Central

    Lobo, Mary Kay; Nestler, Eric J.

    2011-01-01

    The striatum plays a key role in mediating the acute and chronic effects of addictive drugs, with drugs of abuse causing long-lasting molecular and cellular alterations in both dorsal striatum and nucleus accumbens (ventral striatum). Despite the wealth of research on the biological actions of abused drugs in striatum, until recently, the distinct roles of the striatum’s two major subtypes of medium spiny neurons (MSNs) in drug addiction remained elusive. Recent advances in cell-type-specific technologies, including fluorescent reporter mice, transgenic, or knockout mice, and viral-mediated gene transfer, have advanced the field toward a more comprehensive understanding of the two MSN subtypes in the long-term actions of drugs of abuse. Here we review progress in defining the distinct molecular and functional contributions of the two MSN subtypes in mediating addiction. PMID:21811439

  6. 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

  7. 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. PMID:22179203

  8. 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

  9. 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

  10. SIKVAV, a Laminin α1-Derived Peptide, Interacts with Integrins and Increases Protease Activity of a Human Salivary Gland Adenoid Cystic Carcinoma Cell Line through the ERK 1/2 Signaling Pathway

    PubMed Central

    Freitas, Vanessa M.; Vilas-Boas, Vanessa F.; Pimenta, Daniel C.; Loureiro, Vania; Juliano, Maria A.; Carvalho, Márcia R.; Pinheiro, João J.V.; Camargo, Antonio C.M.; Moriscot, Anselmo S.; Hoffman, Matthew P.; Jaeger, Ruy G.

    2007-01-01

    Adenoid cystic carcinoma is a frequently occurring malignant salivary gland neoplasm. We studied the induction of protease activity by the laminin-derived peptide, SIKVAV, in cells (CAC2) derived from this neoplasm. Laminin α1 and matrix metalloproteinases (MMPs) 2 and 9 were immunolocalized in adenoid cystic carcinoma cells in vivo and in vitro. CAC2 cells cultured on SIKVAV showed a dose-dependent increase of MMP9 as detected by zymography and colocalization of α3 and α6 integrins. Small interfering RNA (siRNA) knockdown of integrin expression in CAC2 cells resulted in decreased adhesion to the peptide. SIKVAV affinity chromatography and immunoblot analysis showed that α3, α6, and β1 integrins were eluted from the SIKVAV column, which was confirmed by mass spectrometry and a solid-phase binding assay. Small interfering RNA experiments also showed that these integrins, through extracellular signal-regulated kinase (ERK) 1/2 signaling, regulate MMP secretion induced by SIKVAV in CAC2 cells. We propose that SIKVAV increases protease activity of a human salivary gland adenoid cystic carcinoma cell line through α3β1 and α6β1 integrins and the ERK 1/2 signaling pathway. PMID:17591960

  11. Fear conditioning and early life vulnerabilities: two distinct pathways of emotional dysregulation and brain dysfunction in PTSD

    PubMed Central

    Lanius, Ruth A.; Frewen, Paul A.; Vermetten, Eric; Yehuda, Rachel

    2010-01-01

    The newly proposed criteria for posttraumatic stress disorder (PTSD) in the Diagnostic and Statistical Manual (DSM-V) include dysregulation of a variety of emotional states including fear, anger, guilt, and shame, in addition to dissociation and numbing. Consistent with these revisions, we postulate two models of emotion dysregulation in PTSD in which fear is not the prevailing emotion but is only one of several components implicated in a dysregulated emotional system that also mediates problems regulating anger, guilt, shame, dissociation, and numbing. We discuss whether there is a relationship between fear and other emotion regulation systems that may help further our understanding of PTSD and its underlying neurocircuitry. Two pathways describing the relationship between fear and other emotion regulation systems in PTSD are proposed. The first pathway describes emotion dysregulation as an outcome of fear conditioning through stress sensitization and kindling. The second pathway views emotion dysregulation as a distal vulnerability factor and hypothesizes a further exacerbation of fear and other emotion regulatory problems, including the development of PTSD after exposure to one or several traumatic event(s) later in life. Future research and treatment implications are discussed. PMID:22893793

  12. 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. PMID:25569145

  13. 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

  14. 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. PMID:27401342

  15. Distinct UV-B and UV-A/blue light signal transduction pathways induce chalcone synthase gene expression in Arabidopsis cells.

    PubMed Central

    Christie, J M; Jenkins, G I

    1996-01-01

    UV and blue light control the expression of flavonoid biosynthesis genes in a range of higher plants. To investigate the signal transduction processes involved in the induction of chalcone synthase (CHS) gene expression by UV-B and UV-A/blue light, we examined the effects of specific agonists and inhibitors of known signaling components in mammalian systems in a photomixotrophic Arabidopsis cell suspension culture. CHS expression is induced specifically by these wavelengths in the cell culture, in a manner similar to that in mature Arabidopsis leaf tissue. Both the UV-B and UV-A/blue phototransduction processes involve calcium, although the elevation of cytosolic calcium is insufficient on its own to stimulate CHS expression. The UV-A/blue light induction of CHS expression does not appear to involve calmodulin, whereas the UV-B response does; this difference indicates that the signal transduction pathways are, at least in part, distinct. We provide evidence that both pathways involve reversible protein phosphorylation and require protein synthesis. The UV-B and UV-A/blue light signaling pathways are therefore different from the phytochrome signal transduction pathway regulating CHS expression in other species. PMID:8837509

  16. α-Ketobenzothiazole Serine Protease Inhibitors of Aberrant HGF/c-MET and MSP/RON Kinase Pathway Signaling in Cancer.

    PubMed

    Han, Zhenfu; Harris, Peter K W; Karmakar, Partha; Kim, Tommy; Owusu, Ben Y; Wildman, Scott A; Klampfer, Lidija; Janetka, James W

    2016-03-17

    Upregulation of the HGF and MSP growth-factor processing serine endopeptidases HGFA, matriptase and hepsin is correlated with increased metastasis in multiple tumor types driven by c-MET or RON kinase signaling. We rationally designed P1' α-ketobenzothiazole mechanism-based inhibitors of these proteases. Structure-activity studies are presented, which resulted in the identification of potent inhibitors with differential selectivity. The tetrapeptide inhibitors span the P1-P1' substrate cleavage site via a P1' amide linker off the benzothiazole, occupying the S3' pocket. Optimized inhibitors display sub-nanomolar enzyme inhibition against one, two, or all three of HGFA, matriptase, and hepsin. Several compounds also have good selectivity against the related trypsin-like proteases, thrombin and Factor Xa. Finally, we show that inhibitors block the fibroblast (HGF)-mediated migration of invasive DU145 prostate cancer cells. In addition to prostate cancer, breast, colon, lung, pancreas, gliomas, and multiple myeloma tumors all depend on HGF and MSP for tumor survival and progression. Therefore, these unique inhibitors have potential as new therapeutics for a diverse set of tumor types. PMID:26889658

  17. Profiling Dose-Dependent Activation of p53-Mediated Signaling Pathways by Chemicals with Distinct Mechanisms of DNA Damage

    PubMed Central

    Clewell, Rebecca A.; Sun, Bin; Adeleye, Yeyejide; Carmichael, Paul; Efremenko, Alina; McMullen, Patrick D.; Pendse, Salil; Trask, O. J.; White, Andy; Andersen, Melvin E.

    2014-01-01

    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. PMID:25078064

  18. Distinct populations within Isl1 lineages contribute to appendicular and facial skeletogenesis through the β-catenin pathway.

    PubMed

    Akiyama, Ryutaro; Kawakami, Hiroko; Taketo, M Mark; Evans, Sylvia M; Wada, Naoyuki; Petryk, Anna; Kawakami, Yasuhiko

    2014-03-01

    Isl1 expression marks progenitor populations in developing embryos. In this study, we investigated the contribution of Isl1-expressing cells that utilize the β-catenin pathway to skeletal development. Inactivation of β-catenin in Isl1-expressing cells caused agenesis of the hindlimb skeleton and absence of the lower jaw (agnathia). In the hindlimb, Isl1-lineages broadly contributed to the mesenchyme; however, deletion of β-catenin in the Isl1-lineage caused cell death only in a discrete posterior domain of nascent hindlimb bud mesenchyme. We found that the loss of posterior mesenchyme, which gives rise to Shh-expressing posterior organizer tissue, caused loss of posterior gene expression and failure to expand chondrogenic precursor cells, leading to severe truncation of the hindlimb. In facial tissues, Isl1-expressing cells broadly contributed to facial epithelium. We found reduced nuclear β-catenin accumulation and loss of Fgf8 expression in mandibular epithelium of Isl1(-/-) embryos. Inactivating β-catenin in Isl1-expressing epithelium caused both loss of epithelial Fgf8 expression and death of mesenchymal cells in the mandibular arch without affecting epithelial proliferation and survival. These results suggest a Isl1→β-catenin→Fgf8 pathway that regulates mesenchymal survival and development of the lower jaw in the mandibular epithelium. By contrast, activating β-catenin signaling in Isl1-lineages caused activation of Fgf8 broadly in facial epithelium. Our results provide evidence that, despite its broad contribution to hindlimb mesenchyme and facial epithelium, the Isl1-β-catenin pathway regulates skeletal development of the hindlimb and lower jaw through discrete populations of cells that give rise to Shh-expressing posterior hindlimb mesenchyme and Fgf8-expressing mandibular epithelium. PMID:24424161

  19. Distinctive effects of eicosapentaenoic and docosahexaenoic acids in regulating neural stem cell fate are mediated via endocannabinoid signalling pathways.

    PubMed

    Dyall, S C; Mandhair, H K; Fincham, R E A; Kerr, D M; Roche, M; Molina-Holgado, F

    2016-08-01

    Emerging evidence suggests a complex interplay between the endocannabinoid system, omega-3 fatty acids and the immune system in the promotion of brain self-repair. However, it is unknown if all omega-3 fatty acids elicit similar effects on adult neurogenesis and if such effects are mediated or regulated by interactions with the endocannabinoid system. This study investigated the effects of DHA and EPA on neural stem cell (NSC) fate and the role of the endocannabinoid signalling pathways in these effects. EPA, but not DHA, significantly increased proliferation of NSCs compared to controls, an effect associated with enhanced levels of the endocannabinoid 2-arachidonylglycerol (2-AG) and p-p38 MAPK, effects attenuated by pre-treatment with CB1 (AM251) or CB2 (AM630) receptor antagonists. Furthermore, in NSCs derived from IL-1β deficient mice, EPA significantly decreased proliferation and p-p38 MAPK levels compared to controls, suggesting a key role for IL-1β signalling in the effects observed. Although DHA similarly increased 2-AG levels in wild-type NSCs, there was no concomitant increase in proliferation or p-p38 MAPK activity. In addition, in NSCs from IL-1β deficient mice, DHA significantly increased proliferation without effects on p-P38 MAPK, suggesting effects of DHA are mediated via alternative signalling pathways. These results provide crucial new insights into the divergent effects of EPA and DHA in regulating NSC proliferation and the pathways involved, and highlight the therapeutic potential of their interplay with endocannabinoid signalling in brain repair. PMID:27044662

  20. Distinct populations within Isl1 lineages contribute to appendicular and facial skeletogenesis through the β-catenin pathway

    PubMed Central

    Akiyama, Ryutaro; Kawakami, Hiroko; Taketo, M. Mark; Evans, Sylvia M.; Wada, Naoyuki; Petryk, Anna; Kawakami, Yasuhiko

    2014-01-01

    Isl1 expression marks progenitor populations in developing embryos. In this study, we investigated the contribution of Isl1-expressing cells that utilize the β-catenin pathway to skeletal development. Inactivation of β-catenin in Isl1-expressing cells caused agenesis of the hindlimb skeleton and absence of the lower jaw (agnathia). In the hindlimb, Isl1-lineages broadly contributed to the mesenchyme, however, deletion of β-catenin in the Isl1-lineage caused cell death only in a discrete posterior domain of nascent hindlimb bud mesenchyme. We found that the loss of posterior mesenchyme, which gives rise to Shh-expressing posterior organizer tissue, caused loss of posterior gene expression and failure to expand chondrogenic precursor cells, leading to severe truncation of the hindlimb. In facial tissues, Isl1-expressing cells broadly contributed to facial epithelium. We found reduced nuclear β-catenin accumulation and loss of Fgf8 expression in mandibular epithelium of Isl1−/− embryos. Inactivating β-catenin in Isl1-expressing epithelium caused both loss of epithelial Fgf8 expression and death of mesenchymal cells in the mandibular arch without affecting epithelial proliferation and survival. These results suggest a Isl1-> β-catenin-> Fgf8 pathway that regulates mesenchymal survival and development of the lower jaw in the mandibular epithelium. By contrast, activating β-catenin signaling in Isl1-lineages caused activation of Fgf8 broadly in facial epithelium. Our results provide evidence that, despite its broad contribution to hindlimb mesenchyme and facial epithelium, the Isl1-β-catenin pathway regulates skeletal development of the hindlimb and lower jaw through discrete populations of cells that give rise to Shh-expressing posterior hindlimb mesenchyme and Fgf8-expressing mandibular epithelium. PMID:24424161

  1. Distinct Patterns of Wnt3a and Wnt5a Signaling Pathway in the Lung from Rats with Endotoxic Shock.

    PubMed

    Hii, Hiong-Ping; Liao, Mei-Hui; Chen, Shiu-Jen; Wu, Chin-Chen; Shih, Chih-Chin

    2015-01-01

    Septic shock is a syndrome with severe hypotension and multiple organ dysfunction caused by an imbalance between pro-inflammatory and anti-inflammatory response. The most common risk factor of acute lung injury is severe sepsis. Patients with sepsis-related acute respiratory distress syndrome have higher mortality. Recent studies reveal regulatory roles of Wnt3a and Wnt5a signaling in inflammatory processes. Wnt3a signaling has been implicated in anti-inflammatory effects, whereas Wnt5a signaling has been postulated to have pro-inflammatory properties. However, the balance between Wnt3a and Wnt5a signaling pathway in the lung of rats with endotoxic shock has not been determined. Thus, we investigated the major components of Wnt3a and Wnt5a signaling pathway in the lung of endotoxemic rats. Male Wistar rats were intravenously infused with saline or lipopolysaccharide (LPS, 10 mg/kg). The changes of hemodynamics, biochemical variables, and arterial blood gas were examined during the experimental period. At 6 h after saline or LPS, animals were sacrificed, and lungs were obtained for analyzing superoxide production, water accumulation, histologic assessment, and protein expressions of Wnt3a and Wnt5a signaling pathway. Animals that received LPS showed circulatory failure, multiple organ dysfunction, metabolic acidosis, hyperventilation, lung edema, and high mortality. The lung from rats with endotoxic shock exhibited significant decreases in the levels of Wnt3a, Fzd1, Dsh1, phosphorylated GSK-3β at Ser9, and β-catenin. In contrast, the expressions of Wnt5a, Fzd5, and CaMKII were up-regulated in the lung of endotoxemic rats. These findings indicate the major components of Wnt3a and Wnt5a signaling in the lung are disturbed under endotoxic insult. PMID:26218875

  2. 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.

  3. The Acute Phase Protein Serum Amyloid A Induces Lipolysis and Inflammation in Human Adipocytes through Distinct Pathways

    PubMed Central

    Faty, Aurélie; Ferré, Pascal; Commans, Stéphane

    2012-01-01

    Background The acute phase response (APR) is characterized by alterations in lipid and glucose metabolism leading to an increased delivery of energy substrates. In adipocytes, there is a coordinated decrease in Free Fatty acids (FFAs) and glucose storage, in addition to an increase in FFAs mobilization. Serum Amyloid A (SAA) is an acute phase protein mainly associated with High Density Lipoproteins (HDL). We hypothesized that enrichment of HDL with SAA, during the APR, could be implicated in the metabolic changes occurring in adipocytes. Methodology/Principal Findings In vitro differentiated human adipocytes (hMADS) were treated with SAA enriched HDL or recombinant SAA and the metabolic phenotype of the cells analyzed. In hMADS, SAA induces an increased lipolysis through an ERK dependent pathway. At the molecular level, SAA represses PPARγ2, C/EBPα and SREBP-1c gene expression, three transcription factors involved in adipocyte differentiation or lipid synthesis. In addition, the activation of the NF-κB pathway by SAA leads to the induction of pro-inflammatory cytokines and chemokines, as in the case of immune cells. These latter findings were replicated in freshly isolated mature human adipocytes. Conclusions/Significance Besides its well-characterized role in cholesterol metabolism, SAA has direct metabolic effects on human adipocytes. These metabolic changes could be at least partly responsible for alterations of adipocyte metabolism observed during the APR as well as during pathophysiological conditions such as obesity and conditions leading to insulin resistant states. PMID:22532826

  4. Shibire mutations reveal distinct dynamin-independent and -dependent endocytic pathways in primary cultures of Drosophila hemocytes.

    PubMed

    Guha, A; Sriram, V; Krishnan, K S; Mayor, S

    2003-08-15

    We have developed a primary cell culture system derived from embryonic and larval stages of Drosophila. This allows for high-resolution imaging and genetic analyses of endocytic processes. Here, we have investigated endocytic pathways of three types of molecules: an endogenous receptor that binds anionic ligands (ALs), glycosylphosphatidylinositol (GPI)-anchored protein (GPI-AP), and markers of the fluid phase in primary hemocytes. We find that the endogenous AL-binding receptor (ALBR) is internalized into Rab5-positive endosomes, whereas the major portion of the fluid phase is taken up into Rab5-negative endosomes; GPI-APs are endocytosed into both classes of endosomes. ALBR and fluid-phase-containing early endosomes subsequently fuse to yield a population of Rab7-positive late endosomes. In primary culture, the endocytic phenotype of ALBR internalization in cells carrying mutations in Drosophila Dynamin (dDyn) at the shibire locus (shits) parallels the temperature-sensitive behavior of shits animals. At the restrictive temperature in shits cells, receptor-bound ALs remain completely surface accessible, localized to clathrin and alpha-adaptin-positive structures. On lowering the temperature, ALs are rapidly sequestered, suggesting a reversible block at a late step in dDyn-dependent endocytosis. By contrast, GPI-AP and fluid-phase endocytosis are quantitatively unaffected at the restrictive temperature in shits hemocytes, demonstrating a constitutive dDyn and Rab5-independent endocytic pathway in Drosophila. PMID:12857788

  5. Cancer Stem-like Cells Act via Distinct Signaling Pathways in Promoting Late Stages of Malignant Progression.

    PubMed

    da Silva-Diz, Victoria; Simón-Extremera, Pilar; Bernat-Peguera, Adrià; de Sostoa, Jana; Urpí, Maria; Penín, Rosa M; Sidelnikova, Diana Pérez; Bermejo, Oriol; Viñals, Joan Maria; Rodolosse, Annie; González-Suárez, Eva; Moruno, Antonio Gómez; Pujana, Miguel Ángel; Esteller, Manel; Villanueva, Alberto; Viñals, Francesc; Muñoz, Purificación

    2016-03-01

    Cancer stem-like cells (CSC) play key roles in long-term tumor propagation and metastasis, but their dynamics during disease progression are not understood. Tumor relapse in patients with initially excised skin squamous cell carcinomas (SCC) is characterized by increased metastatic potential, and SCC progression is associated with an expansion of CSC. Here, we used genetically and chemically-induced mouse models of skin SCC to investigate the signaling pathways contributing to CSC function during disease progression. We found that CSC regulatory mechanisms change in advanced SCC, correlating with aggressive tumor growth and enhanced metastasis. β-Catenin and EGFR signaling, induced in early SCC CSC, were downregulated in advanced SCC. Instead, autocrine FGFR1 and PDGFRα signaling, which have not been previously associated with skin SCC CSC, were upregulated in late CSC and promoted tumor growth and metastasis, respectively. Finally, high-grade and recurrent human skin SCC recapitulated the signaling changes observed in advanced mouse SCC. Collectively, our findings suggest a stage-specific switch in CSC regulation during disease progression that could be therapeutically exploited by targeting the PDGFR and FGFR1 pathways to block relapse and metastasis of advanced human skin SCC. PMID:26719534

  6. 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)

  7. Real-Time Monitoring of Calcineurin Activity in Living Cells: Evidence for Two Distinct Ca2+-dependent Pathways in Fission Yeast

    PubMed Central

    Deng, Lu; Sugiura, Reiko; Takeuchi, Mai; Suzuki, Masahiro; Ebina, Hidemine; Takami, Tomonori; Koike, Atsushi; Iba, Shiori

    2006-01-01

    In fission yeast, calcineurin dephosphorylates and activates the Prz1 transcription factor. Here, we identified the calcineurin-dependent response element (CDRE) in the promoter region of prz1+ gene and monitored the calcineurin activity in living cells using a destabilized luciferase reporter gene fused to three tandem repeats of CDRE. Elevated extracellular CaCl2 caused an increase in calcineurin activity with an initial peak and then approached a sustained constant level in a concentration-dependent manner. In CaCl2-sensitive mutants such as Δpmc1, the response was markedly enhanced, reflecting its high intracellular Ca2+. Agents expected to induce Ca2+ influx showed distinct patterns of the CDRE-reporter activity, suggesting different mechanisms of calcineurin activation. Knockout of yam8+ or cch1+ encoding putative subunits of a Ca2+ channel abolished the activation of calcineurin upon exposure to various stimuli, including high extracellular NaCl and cell wall–damaging agents. However, knockout of yam8+ or cch1+ did not affect the activation of calcineurin upon stimulation by elevated extracellular Ca2+. The Pck2 protein kinase C-Pmk1 mitogen-activate protein kinase pathway was required for the stimulation of calcineurin via Yam8/Cch1-mediated Ca2+ influx, but it was not required for the stimulation by elevated extracellular Ca2+, suggesting two distinct pathways for calcineurin activation. PMID:16928959

  8. Neural activity selects myosin IIB and VI with a specific time window in distinct dynamin isoform-mediated synaptic vesicle reuse pathways.

    PubMed

    Hayashida, Michikata; Tanifuji, Shota; Ma, Huan; Murakami, Noriko; Mochida, Sumiko

    2015-06-10

    Presynaptic nerve terminals must maintain stable neurotransmissions via synaptic vesicle (SV) resupply despite encountering wide fluctuations in the number and frequency of incoming action potentials (APs). However, the molecular mechanism linking variation in neural activity to SV resupply is unknown. Myosins II and VI are actin-based cytoskeletal motors that drive dendritic actin dynamics and membrane transport, respectively, at brain synapses. Here we combined genetic knockdown or molecular dysfunction and direct physiological measurement of fast synaptic transmission from paired rat superior cervical ganglion neurons in culture to show that myosins IIB and VI work individually in SV reuse pathways, having distinct dependency and time constants with physiological AP frequency. Myosin VI resupplied the readily releasable pool (RRP) with slow kinetics independently of firing rates but acted quickly within 50 ms after AP. Under high-frequency AP firing, myosin IIB resupplied the RRP with fast kinetics in a slower time window of 200 ms. Knockdown of both myosin and dynamin isoforms by mixed siRNA microinjection revealed that myosin IIB-mediated SV resupply follows amphiphysin/dynamin-1-mediated endocytosis, while myosin VI-mediated SV resupply follows dynamin-3-mediated endocytosis. Collectively, our findings show how distinct myosin isoforms work as vesicle motors in appropriate SV reuse pathways associated with specific firing patterns. PMID:26063922

  9. 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

  10. Comparison of hepatic transcription profiles of locked ribonucleic acid antisense oligonucleotides: evidence of distinct pathways contributing to non-target mediated toxicity in mice.

    PubMed

    Kakiuchi-Kiyota, Satoko; Koza-Taylor, Petra H; Mantena, Srinivasa R; Nelms, Linda F; Enayetallah, Ahmed E; Hollingshead, Brett D; Burdick, Andrew D; Reed, Lori A; Warneke, James A; Whiteley, Lawrence O; Ryan, Anne M; Mathialagan, Nagappan

    2014-03-01

    Development of LNA gapmers, antisense oligonucleotides used for efficient inhibition of target RNA expression, is limited by non-target mediated hepatotoxicity issues. In the present study, we investigated hepatic transcription profiles of mice administered non-toxic and toxic LNA gapmers. After repeated administration, a toxic LNA gapmer (TS-2), but not a non-toxic LNA gapmer (NTS-1), caused hepatocyte necrosis and increased serum alanine aminotransferase levels. Microarray data revealed that, in addition to gene expression patterns consistent with hepatotoxicity, 17 genes in the clathrin-mediated endocytosis (CME) pathway were altered in the TS-2 group. TS-2 significantly down-regulated myosin 1E (Myo1E), which is involved in release of clathrin-coated pits from plasma membranes. To map the earliest transcription changes associated with LNA gapmer-induced hepatotoxicity, a second microarray analysis was performed using NTS-1, TS-2, and a severely toxic LNA gapmer (HTS-3) at 8, 16, and 72 h following a single administration in mice. The only histopathological change observed was minor hepatic hypertrophy in all LNA groups across time points. NTS-1, but not 2 toxic LNA gapmers, increased immune response genes at 8 and 16 h but not at 72 h. TS-2 significantly perturbed the CME pathway only at 72 h, while Myo1E levels were decreased at all time points. In contrast, HTS-3 modulated DNA damage pathway genes at 8 and 16 h and also modulated the CME pathway genes (but not Myo1E) at 16 h. Our results may suggest that different LNAs modulate distinct transcriptional genes and pathways contributing to non-target mediated hepatotoxicity in mice. PMID:24336348

  11. Ionic transport in mixed-alkali glasses: hop through the distinctly different conduction pathways of low dimensionality

    NASA Astrophysics Data System (ADS)

    Rim, Young-Hoon; Kim, Mac; Kim, Jeong Eun; Yang, Yong Suk

    2013-02-01

    A feature common to various solids, including single-alkali (SA) and mixed-alkali (MA) glasses, is a frequency-dependent ionic conductivity that shows the power law and the linear behavior with frequency. In spite of the advances made, the origin of this behavior continues to be controversial. We report our measurements of the conductivity of a series of MA borate glasses (Li1-xAx)2B4O7 (A = Na, K, Rb, Cs; 0 ⩽ x ⩽ 1.0) in the frequency range of 100 Hz-15 MHz and in the temperature range from 300 K to less than the glass transition temperature Tg . Using a self-similar spatial structure model, we show that the real process of ionic transport in the SA and the MA glass systems can be described by the fractional kinetic equations containing non-integer integration/differentiation operators. In the procedure of a systematic deduction of the ionic transport in glass systems, we obtained two important insights. Firstly, the time-dependent conductivity σ(t) ˜ exp (t/τ c)α reproduces the empirical expression of mean square displacement of the mobile ions ˜tα as a first approximation of ions moving through the fractal pathway and leads to the universal power-law behavior at frequency scales. Secondly, the modified fractional Rayleigh equation with a repulsive interaction provides a quantitative explanation for experimental findings on the SA and the MA glasses. Investigations on the power-law exponent β in the SA and the MA borate glasses indicate that the ions move through the different branches of the fractal structured conduction pathways due to the structural character, associated with a site mismatch effect, and Coulomb blockade by the randomly distributed ions.

  12. 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.

  13. 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

  14. 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. PMID:27180052

  15. Distinct abscisic acid signaling pathways for modulation of guard cell versus mesophyll cell potassium channels revealed by expression studies in Xenopus laevis oocytes.

    PubMed

    Sutton, F; Paul, S S; Wang, X Q; Assmann, S M

    2000-09-01

    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. PMID:10982437

  16. 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.

  17. 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

  18. Cytokeratin and protein expression patterns in squamous cell carcinoma of the oral cavity provide evidence for two distinct pathogenetic pathways

    PubMed Central

    FROHWITTER, GESCHE; BUERGER, HORST; VAN DIEST, PAUL J.; KORSCHING, EBERHARD; KLEINHEINZ, JOHANNES; FILLIES, THOMAS

    2016-01-01

    Squamous cell carcinoma (SCC) of the oral cavity is a morphological heterogeneous disease. Various cytokeratin (CK) expression patterns with different prognostic values have been described, but little is known concerning the underlying biological cell mechanisms. Therefore, the present study investigated 193 cases of oral SCCs using immunohistochemistry for α/β/γ-catenin, glucose transporter 1, caspase-3, X-linked inhibitor of apoptosis protein, hypoxia inducible factor-1α, carbonic anhydrase 9, heat shock protein (hsp) 70, mast/stem cell growth factor receptor, p21, p27, p16, p53, B-cell lymphoma 6, epidermal growth factor receptor, cyclin D1 and CK1, 5/6, 8/18, 10, 14 and 19. Expression patterns were analyzed with biomathematical permutation analysis. The present results revealed a significant association between the expression of low-molecular weight CK8/18 and 19 and a high-tumor grade, β and γ-catenin expression, deregulated cell cycle proteins and a predominant localization of the tumor on the floor of the mouth. By contrast, expression of high-molecular weight CK1, 5/6, 10 and 14 was significantly associated with the expression of p21 and hsp70. In conclusion, the current study presents evidence for the existence of two parallel pathogenetic pathways in oral SCCs, characterized by the expression of low- and high-molecular weight CKs. Additional studies are required to demonstrate the extent that these results may be used to improve therapeutic regimens. PMID:27347109

  19. 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

  20. 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

  1. Distinct GAB2 signaling pathways are essential for myeloid and lymphoid transformation and leukemogenesis by BCR-ABL1.

    PubMed

    Gu, Shengqing; Chan, Wayne W; Mohi, Golam; Rosenbaum, Joel; Sayad, Azin; Lu, Zhibin; Virtanen, Carl; Li, Shaoguang; Neel, Benjamin G; Van Etten, Richard A

    2016-04-01

    Tyrosine kinase inhibitors (TKIs) directed against BCR-ABL1, the product of the Philadelphia (Ph) chromosome, have revolutionized treatment of patients with chronic myeloid leukemia (CML). However, acquired resistance to TKIs is a significant clinical problem in CML, and TKI therapy is much less effective against Ph(+)B-cell acute lymphoblastic leukemia (B-ALL). BCR-ABL1, via phosphorylated Tyr177, recruits the adapter GRB2-associated binding protein 2 (GAB2) as part of a GRB2/GAB2 complex. We showed previously that GAB2 is essential for BCR-ABL1-evoked myeloid transformation in vitro. Using a genetic strategy and mouse models of CML and B-ALL, we show here that GAB2 is essential for myeloid and lymphoid leukemogenesis by BCR-ABL1. In the mouse model, recipients of BCR-ABL1-transducedGab2(-/-)bone marrow failed to develop CML-like myeloproliferative neoplasia. Leukemogenesis was restored by expression of GAB2 but not by GAB2 mutants lacking binding sites for its effectors phosphatidylinositol 3-kinase (PI3K) or SRC homology 2-containing phosphotyrosine phosphatase 2 (SHP2). GAB2 deficiency also attenuated BCR-ABL1-induced B-ALL, but only the SHP2 binding site was required. The SHP2 and PI3K binding sites were differentially required for signaling downstream of GAB2. Hence, GAB2 transmits critical transforming signals from Tyr177 to PI3K and SHP2 for CML pathogenesis, whereas only the GAB2-SHP2 pathway is essential for lymphoid leukemogenesis. Given that GAB2 is dispensable for normal hematopoiesis, GAB2 and its effectors PI3K and SHP2 represent promising targets for therapy in Ph(+)hematologic neoplasms. PMID:26773044

  2. Endothelial and Neuronal Nitric Oxide Activate Distinct Pathways on Sympathetic Neurotransmission in Rat Tail and Mesenteric Arteries.

    PubMed

    Sousa, Joana Beatriz; Vieira-Rocha, Maria Sofia; Arribas, Silvia M; González, Maria Carmen; Fresco, Paula; Diniz, Carmen

    2015-01-01

    Nitric oxide (NO) seems to contribute to vascular homeostasis regulating neurotransmission. This work aimed at assessing the influence of NO from different sources and respective intracellular pathways on sympathetic neurotransmission, in two vascular beds. Electrically-evoked [3H]-noradrenaline release was assessed in rat mesenteric and tail arteries in the presence of NO donors or endothelial/neuronal nitric oxide synthase (NOS) inhibitors. The influence of NO on adenosine-mediated effects was also studied using selective antagonists for adenosine receptors subtypes. Location of neuronal NOS (nNOS) was investigated by immunohistochemistry (with specific antibodies for nNOS and for Schwann cells) and Confocal Microscopy. Results indicated that: 1) in mesenteric arteries, noradrenaline release was reduced by NO donors and it was increased by nNOS inhibitors; the effect of NO donors was only abolished by the adenosine A1 receptors antagonist; 2) in tail arteries, noradrenaline release was increased by NO donors and it was reduced by eNOS inhibitors; adenosine receptors antagonists were devoid of effect; 3) confocal microscopy showed nNOS staining in adventitial cells, some co-localized with Schwann cells. nNOS staining and its co-localization with Schwann cells were significantly lower in tail compared to mesenteric arteries. In conclusion, in mesenteric arteries, nNOS, mainly located in Schwann cells, seems to be the main source of NO influencing perivascular sympathetic neurotransmission with an inhibitory effect, mediated by adenosine A1 receptors activation. Instead, in tail arteries endothelial NO seems to play a more relevant role and has a facilitatory effect, independent of adenosine receptors activation. PMID:26075386

  3. Endothelial and Neuronal Nitric Oxide Activate Distinct Pathways on Sympathetic Neurotransmission in Rat Tail and Mesenteric Arteries

    PubMed Central

    Sousa, Joana Beatriz; Vieira-Rocha, Maria Sofia; Arribas, Silvia M.; González, Maria Carmen; Fresco, Paula; Diniz, Carmen

    2015-01-01

    Nitric oxide (NO) seems to contribute to vascular homeostasis regulating neurotransmission. This work aimed at assessing the influence of NO from different sources and respective intracellular pathways on sympathetic neurotransmission, in two vascular beds. Electrically-evoked [3H]-noradrenaline release was assessed in rat mesenteric and tail arteries in the presence of NO donors or endothelial/neuronal nitric oxide synthase (NOS) inhibitors. The influence of NO on adenosine-mediated effects was also studied using selective antagonists for adenosine receptors subtypes. Location of neuronal NOS (nNOS) was investigated by immunohistochemistry (with specific antibodies for nNOS and for Schwann cells) and Confocal Microscopy. Results indicated that: 1) in mesenteric arteries, noradrenaline release was reduced by NO donors and it was increased by nNOS inhibitors; the effect of NO donors was only abolished by the adenosine A1 receptors antagonist; 2) in tail arteries, noradrenaline release was increased by NO donors and it was reduced by eNOS inhibitors; adenosine receptors antagonists were devoid of effect; 3) confocal microscopy showed nNOS staining in adventitial cells, some co-localized with Schwann cells. nNOS staining and its co-localization with Schwann cells were significantly lower in tail compared to mesenteric arteries. In conclusion, in mesenteric arteries, nNOS, mainly located in Schwann cells, seems to be the main source of NO influencing perivascular sympathetic neurotransmission with an inhibitory effect, mediated by adenosine A1 receptors activation. Instead, in tail arteries endothelial NO seems to play a more relevant role and has a facilitatory effect, independent of adenosine receptors activation. PMID:26075386

  4. 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

  5. Distinct effects of folate pathway genes MTHFR and MTHFD1L on ruminative response style: a potential risk mechanism for depression.

    PubMed

    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

  6. 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

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

    PubMed Central

    Sánchez-Tacuba, Liliana; Rojas, Margarito; Arias, Carlos F.

    2015-01-01

    ABSTRACT 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. IMPORTANCE 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

  8. 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. PMID:24243021

  9. AAA proteases in mitochondria: diverse functions of membrane-bound proteolytic machines.

    PubMed

    Tatsuta, Takashi; Langer, Thomas

    2009-11-01

    FtsH/AAA proteases comprise a distinct family of membrane-bound, ATP-dependent proteases present in eubacteria and eukaryotic cells, where they are confined to mitochondria and chloroplasts. Here, we will summarize versatile functions of AAA proteases within mitochondria, which ensure mitochondrial integrity and cell survival, acting both as quality control and processing enzymes. PMID:19781639

  10. 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

  11. Confocal Microscopy Studies of Cationic Lipid/dna Complexes Reveal Distinct Pathways of Gene Delivery in Cells as a Function of Structure

    NASA Astrophysics Data System (ADS)

    Lin, Alison J.; Slack, Nelle L.; Ahmad, Ayesha; Evans, Heather M.; George, Cyril X.; Samuel, Charles E.; Safinya, Cyrus R.

    2000-03-01

    We have identified multiple pathways of gene delivery in mouse L cells using cationic lipids as carriers of DNA. Transfection, the process of delivering foreign DNA into cells, using cationic lipid/DNA (CL-DNA) complexes was monitored by laser confocal microscopy. By following the progress of fluorescently labelled lipid and DNA, we have determined distinct pathways of entry of complexes into cells and the subsequent DNA release from the complexes. We have correlated the microscopy results with the x-ray diffraction data on complex structures and the corresponding transfection efficiencies. X-ray diffraction results elucidated the structures of CL-DNA complexes as a function of the membrane charge density of the system. Luciferase protein assays disclosed novel trends of transfection efficiencies along the structural phase diagram. Similar results were obtained with GFP plasmids. Funded by NIH R01-GM59288-01, R37-AI12520-24, NSF-DMR 9972246, UC-Biotechnology Research and Education Program (97-02).

  12. Two Distinct Amyloid β-Protein (Aβ) Assembly Pathways Leading to Oligomers and Fibrils Identified by Combined Fluorescence Correlation Spectroscopy, Morphology, and Toxicity Analyses*

    PubMed Central

    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

    2011-01-01

    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 Lys16, 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 Lys16-labeled peptide disturbed fibril formation because the Aβ16–20 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. PMID:21292768

  13. High Throughput Substrate Phage Display for Protease Profiling

    PubMed Central

    Ratnikov, Boris; Cieplak, Piotr; Smith, Jeffrey W.

    2012-01-01

    Summary The interplay between a protease and its substrates is controlled at many different levels, including coexpression, colocalization, binding driven by ancillary contacts, and the presence of natural inhibitors. Here we focus on the most basic parameter that guides substrate recognition by a protease, the recognition specificity at the catalytic cleft. An understanding of this substrate specificity can be used to predict the putative substrates of a protease, to design protease activated imaging agents, and to initiate the design of active site inhibitors. Our group has characterized protease specificities of several matrix metalloproteinases using substrate phage display. Recently, we have adapted this method to a semiautomated platform that includes several high-throughput steps. The semiautomated platform allows one to obtain an order of magnitude more data, thus permitting precise comparisons among related proteases to define their functional distinctions. PMID:19377968

  14. High throughput substrate phage display for protease profiling.

    PubMed

    Ratnikov, Boris; Cieplak, Piotr; Smith, Jeffrey W

    2009-01-01

    The interplay between a protease and its substrates is controlled at many different levels, including coexpression, colocalization, binding driven by ancillary contacts, and the presence of natural inhibitors. Here we focus on the most basic parameter that guides substrate recognition by a protease, the recognition specificity at the catalytic cleft. An understanding of this substrate specificity can be used to predict the putative substrates of a protease, to design protease activated imaging agents, and to initiate the design of active site inhibitors. Our group has characterized protease specificities of several matrix metalloproteinases using substrate phage display. Recently, we have adapted this method to a semiautomated platform that includes several high-throughput steps. The semiautomated platform allows one to obtain an order of magnitude more data, thus permitting precise comparisons among related proteases to define their functional distinctions. PMID:19377968

  15. The outer membrane protease PgtE of Salmonella enterica interferes with the alternative complement pathway by cleaving factors B and H.

    PubMed

    Riva, Rauna; Korhonen, Timo K; Meri, Seppo

    2015-01-01

    The virulence factor PgtE is an outer membrane protease (omptin) of the zoonotic pathogen Salmonella enterica that causes diseases ranging from gastroenteritis to severe enteric fever. It is surface exposed in bacteria that have a short-chain, i.e., rough LPS, as observed e.g., in bacteria residing inside macrophages or just emerging from them. We investigated whether PgtE cleaves the complement factors B (B) and H (H), key proteins controlling formation and inactivation of the complement protein C3b and thereby the activity of the complement system. S. enterica serovar Typhimurium or omptin-expressing recombinant E. coli bacteria were incubated with purified human complement proteins or recombinant H fragments. PgtE cleaved both B and H, whereas its close homolog Pla of Yersinia pestis cleaved only H. H was cleaved at both N- and C-termini, while the central region resisted proteolysis. Because of multiple effects of PgtE on complement components (cleavage of C3, C3b, B, and H) we assessed its effect on the opsonophagocytosis of Salmonella. In human serum, C3 cleavage was dependent on proteolytically active PgtE. Human neutrophils interacted less with serum-opsonized FITC-stained S. enterica 14028R than with the isogenic ΔpgtE strain, as analyzed by flow cytometry. In conclusion, cleavage of B and H by PgtE, together with C3 cleavage, affects the C3-mediated recognition of S. enterica by human neutrophils, thus thwarting the immune protection against Salmonella. PMID:25705210

  16. The outer membrane protease PgtE of Salmonella enterica interferes with the alternative complement pathway by cleaving factors B and H

    PubMed Central

    Riva, Rauna; Korhonen, Timo K.; Meri, Seppo

    2015-01-01

    The virulence factor PgtE is an outer membrane protease (omptin) of the zoonotic pathogen Salmonella enterica that causes diseases ranging from gastroenteritis to severe enteric fever. It is surface exposed in bacteria that have a short-chain, i.e., rough LPS, as observed e.g., in bacteria residing inside macrophages or just emerging from them. We investigated whether PgtE cleaves the complement factors B (B) and H (H), key proteins controlling formation and inactivation of the complement protein C3b and thereby the activity of the complement system. S. enterica serovar Typhimurium or omptin-expressing recombinant E. coli bacteria were incubated with purified human complement proteins or recombinant H fragments. PgtE cleaved both B and H, whereas its close homolog Pla of Yersinia pestis cleaved only H. H was cleaved at both N- and C-termini, while the central region resisted proteolysis. Because of multiple effects of PgtE on complement components (cleavage of C3, C3b, B, and H) we assessed its effect on the opsonophagocytosis of Salmonella. In human serum, C3 cleavage was dependent on proteolytically active PgtE. Human neutrophils interacted less with serum-opsonized FITC-stained S. enterica 14028R than with the isogenic ΔpgtE strain, as analyzed by flow cytometry. In conclusion, cleavage of B and H by PgtE, together with C3 cleavage, affects the C3-mediated recognition of S. enterica by human neutrophils, thus thwarting the immune protection against Salmonella. PMID:25705210

  17. Distinct Escape Pathway by Hepatitis C Virus Genotype 1a from a Dominant CD8+ T Cell Response by Selection of Altered Epitope Processing

    PubMed Central

    Walker, Andreas; Skibbe, Kathrin; Steinmann, Eike; Pfaender, Stephanie; Kuntzen, Thomas; Megger, Dominik A.; Groten, Svenja; Sitek, Barbara; Lauer, Georg M.; Kim, Arthur Y.; Pietschmann, Thomas; Allen, Todd M.

    2015-01-01

    ABSTRACT Antiviral CD8+ T cells are a key component of the adaptive immune response against HCV, but their impact on viral control is influenced by preexisting viral variants in important target epitopes and the development of viral escape mutations. Immunodominant epitopes highly conserved across genotypes therefore are attractive for T cell based prophylactic vaccines. Here, we characterized the CD8+ T cell response against the highly conserved HLA-B*51-restricted epitope IPFYGKAI1373–1380 located in the helicase domain of NS3 in people who inject drugs (PWID) exposed predominantly to HCV genotypes 1a and 3a. Despite this epitope being conserved in both genotypes, the corresponding CD8+ T cell response was detected only in PWID infected with genotype 3a and HCV-RNA negative PWID, but not in PWID infected with genotype 1a. In genotype 3a, the detection of strong CD8+ T cell responses was associated with epitope variants in the autologous virus consistent with immune escape. Analysis of viral sequences from multiple cohorts confirmed HLA-B*51-associated escape mutations inside the epitope in genotype 3a, but not in genotype 1a. Here, a distinct substitution in the N-terminal flanking region located 5 residues upstream of the epitope (S1368P; P = 0.00002) was selected in HLA-B*51-positive individuals. Functional assays revealed that the S1368P substitution impaired recognition of target cells presenting the endogenously processed epitope. The results highlight that, despite an epitope being highly conserved between two genotypes, there are major differences in the selected viral escape pathways and the corresponding T cell responses. IMPORTANCE HCV is able to evolutionary adapt to CD8+ T cell immune pressure in multiple ways. Beyond selection of mutations inside targeted epitopes, this study demonstrates that HCV inhibits epitope processing by modification of the epitope flanking region under T cell immune pressure. Selection of a substitution five amino acids

  18. Calcium and SOL Protease Mediate Temperature Resetting of Circadian Clocks

    PubMed Central

    Tataroglu, Ozgur; Zhao, Xiaohu; Busza, Ania; Ling, Jinli; O’Neill, John S.; Emery, Patrick

    2015-01-01

    Summary 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. PMID:26590423

  19. Mass balance, bedload extraction, surface morphology, and Holocene stratigraphic architecture along distinct sediment transport pathways of the Brahmaputra River in Bengal Basin

    NASA Astrophysics Data System (ADS)

    Sincavage, R.; Goodbred, S. L., Jr.; Wilson, C.; Pickering, J.; Paola, C.

    2015-12-01

    The Brahmaputra River has followed three preferred pathways within the Ganges-Brahmaputra-Meghna delta during the Holocene: the principal course along the modern Jamuna braidbelt and two subsidiary routes through the tectonically deforming Sylhet Basin. The Jamuna braidbelt follows a relatively straight and tectonically stable (subsidence rates generally <2 mm/yr) route along much of its reach. In contrast, pathways through Sylhet Basin traverse an actively subsiding basin (typically 3-4 mm/yr, with rates as high as 7 mm/year near the adjacent Dauki foredeep). In spite of differential subsidence, the Brahmaputra has only episodically occupied Sylhet Basin for brief periods during most of the Holocene. We compare downstream rates of mass extraction via selective deposition along multiple sediment delivery pathways using quantitative grain size data from a network of over 400 closely-spaced (3-5 km) boreholes (up to 90 m deep). An exponential function to describe downstream fining was fitted to the data, with the exponent alpha taken to be the rate of downstream fining. Our data indicate 1.) the upper and lower reaches (~100 km) of all pathways exhibit limited to no fining, 2.) once fining begins it proceeds rapidly in a downstream direction until the coarse fraction has been extracted, and 3.) the fining rate in Sylhet Basin (α = 0.005) is considerably higher than that for the modern braidbelt (α = 0.001), likely due to a steeper slope down the fan delta. When converted to a scale-independent chi space, preserved stratigraphy along both courses show declining sand:mud ratios and sand body thickness at a chi distance of about 0.7, consistent with previous studies in other depositional settings. This downstream distance also generally coincides with a decrease in slope from the fan delta to the lower fluvial-tidal delta, as well as the cessation of downstream fining. Furthermore, this transition corresponds with the backwater length as calculated by a number of

  20. 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.

  1. The erbB3- and IGF-1 receptor-initiated signaling pathways exhibit distinct effects on lapatinib sensitivity against trastuzumab-resistant breast cancer cells.

    PubMed

    Lyu, Hui; Yang, Xiao He; Edgerton, Susan M; Thor, Ann D; Wu, Xiaoying; He, Zhimin; Liu, Bolin

    2016-01-19

    Both erbB3 and IGF-1 receptor (IGF-1R) have been shown to play an important role in trastuzumab resistance. However, it remains unclear whether erbB3- and IGF-1R-initiated signaling pathways possess distinct effects on the sensitivity of lapatinib, a dual tyrosine kinase inhibitor against both EGFR and erbB2, in trastuzumab-resistant breast cancer. Here, we show that the trastuzumab-resistant SKBR3-pool2 and BT474-HR20 breast cancer sublines, as compared the parental SKBR3 and BT474 cells, respectively, exhibit refractoriness to lapatinib. Knockdown of erbB3 inhibited Akt in SKBR3-pool2 and BT474-HR20 cells, significantly increased lapatinib efficacy, and dramatically re-sensitized the cells to lapatinib-induced apoptosis. In contrast, specific knockdown of IGF-1R did not alter the cells' responsiveness to lapatinib. While the levels of phosphorylated Src (P-Src) were reduced upon IGF-1R downregulation, the P-Akt levels remained unchanged. Furthermore, a specific inhibitor of Akt, but not Src, significantly enhanced lapatinib-mediated anti-proliferative/anti-survival effects on SKBR3-pool2 and BT474-HR20 cells. These data indicate that erbB3 signaling is critical for both trastuzumab and lapatinib resistances mainly through the PI-3K/Akt pathway, whereas IGF-1R-initiated Src activation results in trastuzumab resistance without affecting lapatinib sensitivity. Our findings may facilitate the development of precision therapeutic regimens for erbB2-positive breast cancer patients who become resistant to erbB2-targeted therapy. PMID:26621843

  2. Nuclear Export of African Swine Fever Virus p37 Protein Occurs through Two Distinct Pathways and Is Mediated by Three Independent Signals

    PubMed Central

    Eulálio, Ana; Nunes-Correia, Isabel; Carvalho, Ana Luísa; Faro, Carlos; Citovsky, Vitaly; Salas, José; Salas, Maria L.; Simões, Sérgio; de Lima, Maria C. Pedroso

    2006-01-01

    Nucleocytoplasmic shuttling activity of the African swine fever virus p37 protein, a major structural protein of this highly complex virus, has been recently reported. The systematic characterization of the nuclear export ability of this protein constituted the major purpose of the present study. We report that both the N- and C-terminal regions of p37 protein are actively exported from the nucleus to the cytoplasm of yeast and mammalian cells. Moreover, experiments using leptomycin B and small interfering RNAs targeting the CRM1 receptor have demonstrated that the export of p37 protein is mediated by both the CRM1-dependent and CRM1-independent nuclear export pathways. Two signals responsible for the CRM1-mediated nuclear export of p37 protein were identified at the N terminus of the protein, and an additional signal was identified at the C-terminal region, which mediates the CRM1-independent nuclear export. Interestingly, site-directed mutagenesis revealed that hydrophobic amino acids are critical to the function of these three nuclear export signals. Overall, our results demonstrate that two distinct pathways contribute to the strong nuclear export of full-length p37 protein, which is mediated by three independent nuclear export signals. The existence of overlapping nuclear export mechanisms, together with our observation that p37 protein is localized in the nucleus at early stages of infection and exclusively in the cytoplasm at later stages, suggests that the nuclear transport ability of this protein may be critical to the African swine fever virus replication cycle. PMID:16415017

  3. 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

  4. Hematopoietic Tissue Factor–Protease-Activated Receptor 2 Signaling Promotes Hepatic Inflammation and Contributes to Pathways of Gluconeogenesis and Steatosis in Obese Mice

    PubMed Central

    Wang, Jing; Chakrabarty, Sagarika; Bui, Quyen; Ruf, Wolfram; Samad, Fahumiya

    2016-01-01

    Failure to inhibit hepatic gluconeogenesis is a major mechanism contributing to fasting hyperglycemia in type 2 diabetes and, along with steatosis, is the hallmark of hepatic insulin resistance. Obesity is associated with chronic inflammation in multiple tissues, and hepatic inflammation is mechanistically linked to both steatosis and hepatic insulin resistance. Here, we delineate a role for coagulation signaling via tissue factor (TF) and proteinase-activated receptor 2 (PAR2) in obesity-mediated hepatic inflammation, steatosis, and gluconeogenesis. In diet-induced obese mice, TF tail signaling independent of PAR2 drives CD11b+CD11c+ hepatic macrophage recruitment, and TF–PAR2 signaling contributes to the accumulation of hepatic CD8+ T cells. Transcripts of key pathways of gluconeogenesis, lipogenesis, and inflammatory cytokines were reduced in high-fat diet–fed mice that lack the cytoplasmic domain of TF (F3) (TFΔCT) or that are deficient in PAR2 (F2rl1), as well as by pharmacological inhibition of TF–PAR2 signaling in diet-induced obese mice. These gluconeogenic, lipogenic, and inflammatory pathway transcripts were similarly reduced in response to genetic ablation or pharmacological inhibition of TF–PAR2 signaling in hematopoietic cells and were mechanistically associated with activation of AMP-activated protein kinase (AMPK). These findings indicate that hematopoietic TF–PAR2 signaling plays a pivotal role in the hepatic inflammatory responses, steatosis, and hepatic insulin resistance that lead to systemic insulin resistance and type 2 diabetes in obesity. PMID:25476527

  5. Cu(I)-catalyzed diamination of conjugated dienes. Complementary regioselectivity from two distinct mechanistic pathways involving Cu(II) and Cu(III) species.

    PubMed

    Zhao, Baoguo; Peng, Xingao; Zhu, Yingguang; Ramirez, Thomas A; Cornwall, Richard G; Shi, Yian

    2011-12-28

    Conjugated dienes can be diaminated at the internal and/or terminal double bonds using Cu(I) as catalyst and N,N-di-t-butyldiaziridinone (1) as nitrogen source. The regioselectivity is highly dependent upon the choice of Cu(I) catalyst and the substituents on diene substrates. The diamination likely proceeds via two mechanistically distinct pathways. The N-N bond of N,N-di-t-butyldiaziridinone (1) is first homolytically cleaved by the Cu(I) catalyst to form four-membered Cu(III) species A and Cu(II) radical species B, which are in rapid equilibrium. The internal diamination likely proceeds in a concerted manner via Cu(III) species A, and the terminal diamination likely involves Cu(II) radical species B. Kinetic studies have shown that the diamination is first-order in N,N-di-t-butyldiaziridinone (1), zero-order in olefin, and first-order in total Cu(I) catalyst, and the cleavage of the N-N bond of 1 by the Cu(I) catalyst is the rate-determining step. The internal diamination is favored by use of CuBr without ligand and electron-rich dienes. The terminal diamination is favored when using CuCl-L and dienes with radical-stabilizing groups. PMID:22081888

  6. Cu(I)-Catalyzed Diamination of Conjugated Dienes. Complementary Regioselectivity from Two Distinct Mechanistic Pathways Involving Cu(II) and Cu(III) Species

    PubMed Central

    Zhao, Baoguo; Peng, Xingao; Zhu, Yingguang; Ramirez, Thomas A.; Cornwall, Richard G.

    2011-01-01

    Conjugated dienes can be diaminated at the internal and/or terminal double bonds using Cu(I) as catalyst and N,N-di-t-butyldiaziridinone (1) as nitrogen source. The regioselectivity is highly dependent upon the choice of Cu(I) catalyst and the substituents on diene substrates. The diamination likely proceeds via two mechanistically distinct pathways. The N-N bond of N,N-di-t-butyldiaziridinone (1) is first homolytically cleaved by the Cu(I) catalyst to form four-membered Cu(III) species A and Cu(II) radical species B, which are in rapid equilibrium. The internal diamination likely proceeds in a concerted manner via Cu(III) species A and the terminal diamination likely involves Cu(II) radical species B. Kinetic studies have shown that the diamination is first-order in N,N-di-t-butyldiaziridinone (1), zero-order in olefin, first-order in total Cu(I) catalyst, and the cleavage of the N-N bond of 1 by the Cu(I) catalyst is the rate-determining step. The internal diamination is favored by use of CuBr without ligand and electron-rich dienes. The terminal diamination is favored when using CuCl-L and dienes with radical-stabilizing groups. PMID:22081888

  7. Extracellular acid proteases from Neurospora crassa.

    PubMed Central

    Lindberg, R A; Rhodes, W G; Eirich, L D; Drucker, H

    1982-01-01

    Three electrophoretically distinct acid proteases appear in culture filtrates of Neurospora crassa. Like the previously investigated alkaline and neutral proteases, these enzymes require induction by an exogenous protein. But in contrast to alkaline and neutral proteases, which are synthesized and secreted in response to limitation of any one of three nutrilites (carbon, nitrogen or sulfur), extracellular elaboration of the acidic proteases is more specifically a function of the missing nutrilite. AcP, a pepstatin-inhibitable enzyme similar to other fungal carboxyl proteases, was secreted in large amounts when protein was the sole source of sulfur. Only trace amounts were secreted when nitrogen was the limiting nutrilite, and it was undetectable under carbon limitation. M-1, a chelator-sensitive protease, was secreted when nitrogen or carbon was limiting. M-2, also chelator sensitive, was present only when nitrogen or sulfur was limiting. The evidence presented suggests that the differential regulation of the acidic proteases with respect to nutrilite deprivation may not occur at the level of transcription. AcP and M-2 were partially purified from nitrogen-derepressed cultures by ultrafiltration, cation-exchange chromatography, and gel filtration. AcP has a molecular weight of 66,000, is stable from pH 3.0 to 6.0, and is optimally active toward bovine serum albumin at pH 4.0. M-2 has a molecular weight of 18,000, is stable from pH 1.6 to 5.5, and has optimal activity at pH 4.5. Images PMID:6210687

  8. Extracellular acid proteases from Neurospora crassa.

    PubMed

    Lindberg, R A; Rhodes, W G; Eirich, L D; Drucker, H

    1982-06-01

    Three electrophoretically distinct acid proteases appear in culture filtrates of Neurospora crassa. Like the previously investigated alkaline and neutral proteases, these enzymes require induction by an exogenous protein. But in contrast to alkaline and neutral proteases, which are synthesized and secreted in response to limitation of any one of three nutrilites (carbon, nitrogen or sulfur), extracellular elaboration of the acidic proteases is more specifically a function of the missing nutrilite. AcP, a pepstatin-inhibitable enzyme similar to other fungal carboxyl proteases, was secreted in large amounts when protein was the sole source of sulfur. Only trace amounts were secreted when nitrogen was the limiting nutrilite, and it was undetectable under carbon limitation. M-1, a chelator-sensitive protease, was secreted when nitrogen or carbon was limiting. M-2, also chelator sensitive, was present only when nitrogen or sulfur was limiting. The evidence presented suggests that the differential regulation of the acidic proteases with respect to nutrilite deprivation may not occur at the level of transcription. AcP and M-2 were partially purified from nitrogen-derepressed cultures by ultrafiltration, cation-exchange chromatography, and gel filtration. AcP has a molecular weight of 66,000, is stable from pH 3.0 to 6.0, and is optimally active toward bovine serum albumin at pH 4.0. M-2 has a molecular weight of 18,000, is stable from pH 1.6 to 5.5, and has optimal activity at pH 4.5. PMID:6210687

  9. Extracellular acid proteases from Neurospora crassa

    SciTech Connect

    Lindberg, R.A.; Rhodes, W.G.; Eirich, L.D.; Drucker, H.

    1982-06-01

    Three electrophoretically distinct acid proteases appear in culture filtrates of Neurospora crassa. Like the previously investigated alkaline and neutral proteases, these enzymes require induction by an exogenous protein. But in contrast to alkaline and neutral proteases, which are synthesized and secreted in response to limitation of any one of three nutrilites (carbon, nitrogen or sulfur), extracellular elaboration of the acidic proteases is more specifically a function of the missing nutrilite. AcP, a pepstatin-inhibitable enzyme similar to other fungal carboxyl proteases, was secreted in large amounts when protein was the sole source of sulfur. Only trace amounts were secreted when nitrogen was the limiting nutrilite, and it was undetectable under carbon limitation. M-1, a chelator-sensitive protease, was secreted when nitrogen or carbon was limiting. M-2, also chelator sensitive, was present only when nitrogen or sulfur was limiting. The evidence presented suggests that the differential regulation of the acidic proteases with respect to nutrilite deprivation may not occur at the level of transcription. AcP and M-2 were partially purified from nitrogen-derepressed cultures by ultrafiltration, cation-exchange chromatography, and gel filtration. AcP has a molecular weight of 66,000, is stable from pH 3.0 to 6.0, and is optimally active toward bovine serum albumin at pH 4.0. M-2 has a molecular weight of 18,000, is stable from pH 1.6 to 5.5, and has optimal activity at pH 4.5.

  10. Distinctive features of the biological catch bond in the jump-ramp force regime predicted by the two-pathway model.

    PubMed

    Pereverzev, Yuriy V; Prezhdo, Oleg V; Thomas, Wendy E; Sokurenko, Evgeni V

    2005-07-01

    The receptor-ligand unbinding in the biological catch bond is analyzed within a simple model that comprises a single bound state and two unbinding pathways. This model is investigated in detail for the jump-ramp force regime, where the pulling force quickly jumps to a finite value and then is ramped linearly with time. Two qualitative criteria are identified that distinguish the catch bond from the slip bond. First, the rupture force probability density of the catch-bond exhibits a maximum-minimum pair, which develops at finite forces. In contrast, the slip bond produces a maximum that first appears at zero force. Second, the catch bond can be identified over a wide range of ramp rates by high rupture probabilities at low forces relative to the probability at the maximum, in contrast to the slip bond, where the probability at the maximum always corresponds to the most likely rupture force. Both distinctive features of the catch bond are masked by large jump forces, indicating that the catch bond is best identified in experiments with moderate loading rates and small jump forces. The catch-bond lifetime in the constant force regime is related to the probability density in the jump-ramp regime, allowing one to determine the bond lifetime for a constant force by measuring the initial probability density in the jump-ramp experiments with different jump forces and a fixed ramp rate. The key analytic results are illustrated with the P -selectin/P-selectin glucoprotein ligand-1 bond. PMID:16089930