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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 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

  11. 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

  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. 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

  3. 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

  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. 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

  9. 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

  10. 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.

  11. 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

  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. 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

  11. 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

  12. 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

  13. 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

  14. 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

  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. 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

  17. 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

  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. 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

  20. 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

  1. 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

  2. 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

  3. 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

  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. 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

  7. 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

  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. 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

  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. 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

  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

    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 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

  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 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

  8. 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

  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. 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

  20. 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

  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. 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

  8. 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

  9. 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

  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 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

  14. 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

  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. 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

  9. 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

  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. 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

  19. 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

  20. 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

  1. 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

  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. 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

  4. 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

  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. Gene Expression Profile of Adult Human Olfactory Bulb and Embryonic Neural Stem Cell Suggests Distinct Signaling Pathways and Epigenetic Control

    PubMed Central

    Marei, Hany E. S.; Ahmed, Abd-Elmaksoud; Michetti, Fabrizio; Pescatori, Mario; Pallini, Roberto; Casalbore, Patricia; Cenciarelli, Carlo; Elhadidy, Mohamed

    2012-01-01

    Global gene expression profiling was performed using RNA from human embryonic neural stem cells (hENSC), and adult human olfactory bulb-derived neural stem cells (OBNSCs), to define a gene expression pattern and signaling pathways that are specific for each cell lineage. We have demonstrated large differences in the gene expression profile of human embryonic NSC, and adult human OBNSCs, but less variability between parallel cultures. Transcripts of genes involved in neural tube development and patterning (ALDH1A2, FOXA2), progenitor marker genes (LMX1a, ALDH1A1, SOX10), proliferation of neural progenitors (WNT1 and WNT3a), neuroplastin (NPTN), POU3F1 (OCT6), neuroligin (NLGN4X), MEIS2, and NPAS1 were up-regulated in both cell populations. By Gene Ontology, 325 out of 3875 investigated gene sets were scientifically different. 41 out of the 307 investigated Cellular Component (CC) categories, 45 out of the 620 investigated Molecular Function (MF) categories, and 239 out of the 2948 investigated Biological Process (BP) categories were significant. KEGG Pathway Class Comparison had revealed that 75 out of 171 investigated gene sets passed the 0.005 significance threshold. Levels of gene expression were explored in three signaling pathways, Notch, Wnt, and mTOR that are known to be involved in NS cell fates determination. The transcriptional signature also deciphers the role of genes involved in epigenetic modifications. SWI/SNF DNA chromatin remodeling complex family, including SMARCC1 and SMARCE1, were found specifically up-regulated in our OBNSC but not in hENSC. Differences in gene expression profile of transcripts controlling epigenetic modifications, and signaling pathways might indicate differences in the therapeutic potential of our examined two cell populations in relation to in cell survival, proliferation, migration, and differentiation following engraftments in different CNS insults. PMID:22485144

  8. 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

  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. 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

  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. 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

  14. 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

  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. 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

  20. 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

  1. 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

  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. 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.

  12. 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.

  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. 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

  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. 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

  5. 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

  6. 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

  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. Induction of tenascin-C by cyclic tensile strain versus growth factors: distinct contributions by Rho/ROCK and MAPK signaling pathways.

    PubMed

    Chiquet, Matthias; Sarasa-Renedo, Ana; Tunç-Civelek, Vildan

    2004-09-17

    Expression of the extracellular matrix (ECM) protein tenascin-C is induced in fibroblasts by growth factors as well as by tensile strain. Mechanical stress can act on gene regulation directly, or indirectly via the paracrine release of soluble factors by the stimulated cells. To distinguish between these possibilities for tenascin-C, we asked whether cyclic tensile strain and soluble factors, respectively, induced its mRNA via related or separate mechanisms. When cyclic strain was applied to chick embryo fibroblasts cultured on silicone membranes, tenascin-C mRNA and protein levels were increased twofold within 6 h compared to the resting control. Medium conditioned by strained cells did not stimulate tenascin-C mRNA in resting cells. Tenascin-C mRNA in resting cells was increased by serum; however, cyclic strain still caused an additional induction. Likewise, the effect of TGF-beta1 or PDGF-BB was additive to that of cyclic strain, whereas IL-4 or H2O2 (a reactive oxygen species, ROS) did not change tenascin-C mRNA levels. Antagonists for distinct mitogen-activated protein kinases (MAPK) inhibited tenascin-C induction by TGF-beta1 and PDGF-BB, but not by cyclic strain. Conversely, a specific inhibitor of Rho-dependent kinase strongly attenuated the response of tenascin-C mRNA to cyclic strain, but had limited effect on induction by growth factors. The data suggest that regulation of tenascin-C in fibroblasts by cyclic strain occurs independently from soluble mediators and MAPK pathways; however, it requires Rho/ROCK signaling. PMID:15363633

  11. 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

  12. Protease IV, a quorum sensing-dependent protease of Pseudomonas aeruginosa modulates insect innate immunity.

    PubMed

    Park, Su-Jin; Kim, Soo-Kyoung; So, Yong-In; Park, Ha-Young; Li, Xi-Hui; Yeom, Doo Hwan; Lee, Mi-Nan; Lee, Bok-Luel; Lee, Joon-Hee

    2014-12-01

    In Pseudomonas aeruginosa, quorum sensing (QS) plays an essential role in pathogenesis and the QS response controls many virulence factors. Using a mealworm, Tenebrio molitor as a host model, we found that Protease IV, a QS-regulated exoprotease of P. aeruginosa functions as a key virulence effector causing the melanization and death of T. molitor larvae. Protease IV was able to degrade zymogens of spätzle processing enzyme (SPE) and SPE-activating enzyme (SAE) without the activation of the antimicrobial peptide (AMP) production. Since SPE and SAE function to activate spätzle, a ligand of Toll receptor in the innate immune system of T. molitor, we suggest that Protease IV may interfere with the activation of the Toll signaling. Independently of the Toll pathway, the melanization response, another innate immunity was still generated, since Protease IV directly converted Tenebrio prophenoloxidase into active phenoloxidase. Protease IV also worked as an important factor in the virulence to brine shrimp and nematode. These results suggest that Protease IV provides P. aeruginosa with a sophisticated way to escape the immune attack of host by interfering with the production of AMPs. PMID:25315216

  13. Analysis of Δ12-fatty acid desaturase function revealed that two distinct pathways are active for the synthesis of PUFAs in T. aureum ATCC 34304

    PubMed Central

    Matsuda, Takanori; Sakaguchi, Keishi; Hamaguchi, Rie; Kobayashi, Takumi; Abe, Eriko; Hama, Yoichiro; Hayashi, Masahiro; Honda, Daiske; Okita, Yuji; Sugimoto, Shinichi; Okino, Nozomu; Ito, Makoto

    2012-01-01

    Thraustochytrids are known to synthesize PUFAs such as docosahexaenoic acid (DHA). Accumulating evidence suggests the presence of two synthetic pathways of PUFAs in thraustochytrids: the polyketide synthase-like (PUFA synthase) and desaturase/elongase (standard) pathways. It remains unclear whether the latter pathway functions in thraustochytrids. In this study, we report that the standard pathway produces PUFA in Thraustochytrium aureum ATCC 34304. We isolated a gene encoding a putative Δ12-fatty acid desaturase (TauΔ12des) from T. aureum. Yeasts transformed with the tauΔ12des converted endogenous oleic acid (OA) into linoleic acid (LA). The disruption of the tauΔ12des in T. aureum by homologous recombination resulted in the accumulation of OA and a decrease in the levels of LA and its downstream PUFAs. However, the DHA content was increased slightly in tauΔ12des-disruption mutants, suggesting that DHA is primarily produced in T. aureum via the PUFA synthase pathway. The transformation of the tauΔ12des-disruption mutants with a tauΔ12des expression cassette restored the wild-type fatty acid profiles. These data clearly indicate that TauΔ12des functions as Δ12-fatty acid desaturase in the standard pathway of T. aureum and demonstrate that this thraustochytrid produces PUFAs via both the PUFA synthase and the standard pathways. PMID:22368282

  14. Analysis of Δ12-fatty acid desaturase function revealed that two distinct pathways are active for the synthesis of PUFAs in T. aureum ATCC 34304.

    PubMed

    Matsuda, Takanori; Sakaguchi, Keishi; Hamaguchi, Rie; Kobayashi, Takumi; Abe, Eriko; Hama, Yoichiro; Hayashi, Masahiro; Honda, Daiske; Okita, Yuji; Sugimoto, Shinichi; Okino, Nozomu; Ito, Makoto

    2012-06-01

    Thraustochytrids are known to synthesize PUFAs such as docosahexaenoic acid (DHA). Accumulating evidence suggests the presence of two synthetic pathways of PUFAs in thraustochytrids: the polyketide synthase-like (PUFA synthase) and desaturase/elongase (standard) pathways. It remains unclear whether the latter pathway functions in thraustochytrids. In this study, we report that the standard pathway produces PUFA in Thraustochytrium aureum ATCC 34304. We isolated a gene encoding a putative Δ12-fatty acid desaturase (TauΔ12des) from T. aureum. Yeasts transformed with the tauΔ12des converted endogenous oleic acid (OA) into linoleic acid (LA). The disruption of the tauΔ12des in T. aureum by homologous recombination resulted in the accumulation of OA and a decrease in the levels of LA and its downstream PUFAs. However, the DHA content was increased slightly in tauΔ12des-disruption mutants, suggesting that DHA is primarily produced in T. aureum via the PUFA synthase pathway. The transformation of the tauΔ12des-disruption mutants with a tauΔ12des expression cassette restored the wild-type fatty acid profiles. These data clearly indicate that TauΔ12des functions as Δ12-fatty acid desaturase in the standard pathway of T. aureum and demonstrate that this thraustochytrid produces PUFAs via both the PUFA synthase and the standard pathways. PMID:22368282

  15. Protease nexin 1 induces apoptosis of prostate tumor cells through inhibition of X-chromosome-linked inhibitor of apoptosis protein

    PubMed Central

    Zhou, Jianfeng; Li, Chunrui; Huang, Liang; Xin, Xiangke; He, Jing; Allen, Joshua E.; El-Deiry, Wafik S.; Cao, Yunhong; Muschel, Ruth J.; Xu, Danmei

    2015-01-01

    Protease nexin 1 (PN1) is an endogenous serine protease inhibitor (SERPIN), expressed at high levels in the prostate, and capable of inhibiting the proliferation of prostate cancer cells. We previously showed that PN1-uPA complexes inhibited Sonic Hedgehog (SHH) signalling through engagement of the LRP receptor. Here, we describe an alternative anti-proliferative mechanism through which PN1 expression leads to apoptosis. In prostate cancer cells, increased expression of PN1 led to substantial reduction of XIAP levels and apoptosis mediated through the uPAR, but not the LRP receptor. The alterations in XIAP were effected in two ways 1) via alteration in the NF-κB pathway, a pathway known to signal XIAP transcription and 2) by promoting XIAP instability. The AKT pathway is known to phosphorylate XIAP at serine 87 leading to protein stability and PN1 expression is shown to interfere with this process. As a result of both mechanisms, programmed cell death is substantially increased. Consistent with these observations, reduced PN1 protein correlated with elevated p65/XIAP expression and with higher Gleason scores in human prostate tissue arrays. Thus, PN1 expression appears to differentially down-regulate distinct oncogenic pathways depending upon the cell surface receptor engaged by its complexes and demonstrates a novel molecular mechanism by which the protein can promote tumor cell apoptosis. PMID:25686839

  16. Protease production by Streptococcus sanguis associated with subacute bacterial endocarditis.

    PubMed Central

    Straus, D C

    1982-01-01

    A viridans streptococcus (Streptococcus sanguis biotype II) isolated from the blood of a patient with subacute bacterial endocarditis was examined for protease production. In broth culture, extracellular proteolytic enzymes were not produced by this organism until after the early exponential phase of growth, with maximal protease production occurring during the stationary phase. Four distinct proteases were isolated and purified from the supernatant fluids of stationary-phase cultures, employing a combination of ion-exchange column chromatography, gel filtration column chromatography, and polyacrylamide gel electrophoresis. All four proteases could be eluted from a diethylaminoethyl cellulose column at a sodium chloride gradient concentration of 0.25 M but were separable by gel filtration chromatography on a Sephadex G-100 column. They varied in molecular weights as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis from approximately 13,000 to 230,000. All four proteases had pH optima of between 8.0 and 9.0, and two of the proteases were active against casein, human serum albumin, and gelatin but were not active against elastin and collagen. The remaining two proteases were able to degrade only casein and gelatin. These results show that S. sanguis is able to excrete maximal levels of potentially destructive enzymes when the organisms are not actively multiplying. This finding may explain some of the damage caused in heart tissue by these organisms during subacute bacterial endocarditis. Images PMID:6759404

  17. Inhibitors of rhomboid proteases.

    PubMed

    Wolf, Eliane V; Verhelst, Steven H L

    2016-03-01

    Rhomboid proteases form one of the most widespread families of intramembrane proteases. They utilize a catalytic serine-histidine dyad located several Å below the surface of the membrane for substrate hydrolysis. Multiple studies have implicated rhomboid proteases in biologically and medically relevant processes. Several assays have been developed that are able to monitor rhomboid activity. With the aid of these assays, different types of inhibitors have been found, all based on electrophiles that covalently react with the active site machinery. Although the currently available inhibitors have limited selectivity and moderate potency, they can function as research tools and as starting point for the development of activity-based probes, which are reagents that can specifically detect active rhomboid species. Structural studies on complexes of inhibitors with the Escherichia coli rhomboid GlpG have provided insight into how substrate recognition may occur. Future synthetic efforts, aided by high-throughput screening or structure-based design, may lead to more potent and selective inhibitors for this interesting family of proteases. PMID:26166068

  18. Cysteine Protease Inhibitors as Chemotherapy: Lessons from a Parasite Target

    NASA Astrophysics Data System (ADS)

    Selzer, Paul M.; Pingel, Sabine; Hsieh, Ivy; Ugele, Bernhard; Chan, Victor J.; Engel, Juan C.; Bogyo, Matthew; Russell, David G.; Sakanari, Judy A.; McKerrow, James H.

    1999-09-01

    Papain family cysteine proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of cysteine protease inhibitors in cell culture or in vivo. We now report that specific cysteine protease inhibitors kill Leishmania parasites in vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition of Leishmania cysteine protease activity was accompanied by defects in the parasite's lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-protease antibodies with biotinylated surface proteins and accumulation of undigested debris and protease in the flagellar pocket of treated parasites were consistent with a pathway of protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stable in vivo to ameliorate the pathology associated with a mouse model of Leishmania infection.

  19. ADAM Proteases and Gastrointestinal Function.

    PubMed

    Jones, Jennifer C; Rustagi, Shelly; Dempsey, Peter J

    2016-01-01

    A disintegrin and metalloproteinases (ADAMs) are a family of cell surface proteases that regulate diverse cellular functions, including cell adhesion, migration, cellular signaling, and proteolysis. Proteolytically active ADAMs are responsible for ectodomain shedding of membrane-associated proteins. ADAMs rapidly modulate key cell signaling pathways in response to changes in the extracellular environment (e.g., inflammation) and play a central role in coordinating intercellular communication within the local microenvironment. ADAM10 and ADAM17 are the most studied members of the ADAM family in the gastrointestinal tract. ADAMs regulate many cellular processes associated with intestinal development, cell fate specification, and the maintenance of intestinal stem cell/progenitor populations. Several signaling pathway molecules that undergo ectodomain shedding by ADAMs [e.g., ligands and receptors from epidermal growth factor receptor (EGFR)/ErbB and tumor necrosis factor α (TNFα) receptor (TNFR) families] help drive and control intestinal inflammation and injury/repair responses. Dysregulation of these processes through aberrant ADAM expression or sustained ADAM activity is linked to chronic inflammation, inflammation-associated cancer, and tumorigenesis. PMID:26667078

  20. ADAM Proteases and Gastrointestinal Function

    PubMed Central

    Jones, Jennifer C.; Rustagi, Shelly; Dempsey, Peter J.

    2016-01-01

    A disintegrin and metalloproteinases (ADAMs) are a family of cell surface proteases that regulate diverse cellular functions, including cell adhesion, migration, cellular signaling, and proteolysis. Proteolytically active ADAMs are responsible for ectodomain shedding of membrane-associated proteins. ADAMs rapidly modulate key cell signaling pathways in response to changes in the extracellular environment (e.g., inflammation) and play a central role in coordinating intercellular communication within the local microenvironment. ADAM10 and ADAM17 are the most studied members of the ADAM family in the gastrointestinal tract. ADAMs regulate many cellular processes associated with intestinal development, cell fate specification, and the maintenance of intestinal stem cell/progenitor populations. Several signaling pathway molecules that undergo ectodomain shedding by ADAMs [e.g., ligands and receptors from epidermal growth factor receptor (EGFR)/ErbB and tumor necrosis factor α (TNFα) receptor (TNFR) families] help drive and control intestinal inflammation and injury/repair responses. Dysregulation of these processes through aberrant ADAM expression or sustained ADAM activity is linked to chronic inflammation, inflammation-associated cancer, and tumorigenesis. PMID:26667078

  1. Characterization of protease IV expression in Pseudomonas aeruginosa clinical isolates.

    PubMed

    Conibear, Tim C R; Willcox, Mark D P; Flanagan, Judith L; Zhu, Hua

    2012-02-01

    Expression of protease IV by Pseudomonas aeruginosa during ocular infections contributes significantly to tissue damage. However, several P. aeruginosa strains isolated from ocular infections or inflammatory events produce very low levels of protease IV. The aim of the present study was to characterize, genetically and phenotypically, the presence and expression of the protease IV gene in a group of clinical isolates that cause adverse ocular events of varying degrees, and to elucidate the possible control mechanisms of expression associated with this virulence factor. Protease IV gene sequences from seven clinical isolates of P. aeruginosa were determined and compared to P. aeruginosa strains PAO1 and PA103-29. Production and enzyme activity of protease IV were measured in test strains and compared to that of quorum-sensing gene (lasRI) mutants and the expression of other virulence factors. Protease IV gene sequence similarities between the isolates were 97.5-99.5 %. The strains were classified into two distinct phylogenetic groups that correlated with the presence of exo-enzymes from type three secretion systems (TTSS). Protease IV concentrations produced by PAOΔlasRI mutants and the two clinical isolates with a lasRI gene deficiency were restored to levels comparable to strain PAO1 following complementation of the quorum-sensing gene deficiencies. The protease IV gene is highly conserved in P. aeruginosa clinical isolates that cause a range of adverse ocular events. Observed variations within the gene sequence appear to correlate with presence of specific TTSS genes. Protease IV expression was shown to be regulated by the Las quorum-sensing system. PMID:21921113

  2. From proteases to proteomics

    PubMed Central

    Neurath, Hans

    2001-01-01

    This personal and professional autobiography covers the 50-yr period of 1950–2000 and includes the following topics: History of the University of Washington School of Medicine and its Department of Biochemistry (Mount Rainier and the University of Washington, recruiting faculty, biology, research programs); scientific editing (publication, Biochemistry, Protein Science, electronic publication); Europe revisited (Heidelberg, approaching retirement, the German Research Center, reunion in Vienna); and 50 yr of research on proteolytic enzymes (trypsin, carboxypeptidases, mast cell proteases, future developments). PMID:11274481

  3. PARP9-DTX3L ubiquitin ligase targets host histone H2BJ and viral 3C protease to enhance interferon signaling and control viral infection.

    PubMed

    Zhang, Yong; Mao, Dailing; Roswit, William T; Jin, Xiaohua; Patel, Anand C; Patel, Dhara A; Agapov, Eugene; Wang, Zhepeng; Tidwell, Rose M; Atkinson, Jeffrey J; Huang, Guangming; McCarthy, Ronald; Yu, Jinsheng; Yun, Nadezhda E; Paessler, Slobodan; Lawson, T Glen; Omattage, Natalie S; Brett, Tom J; Holtzman, Michael J

    2015-12-01

    Enhancing the response to interferon could offer an immunological advantage to the host. In support of this concept, we used a modified form of the transcription factor STAT1 to achieve hyper-responsiveness to interferon without toxicity and markedly improve antiviral function in transgenic mice and transduced human cells. We found that the improvement depended on expression of a PARP9-DTX3L complex with distinct domains for interaction with STAT1 and for activity as an E3 ubiquitin ligase that acted on host histone H2BJ to promote interferon-stimulated gene expression and on viral 3C proteases to degrade these proteases via the immunoproteasome. Thus, PARP9-DTX3L acted on host and pathogen to achieve a double layer of immunity within a safe reserve in the interferon signaling pathway. PMID:26479788

  4. Proteomic Profile Identifies Dysregulated Pathways in Cornelia de Lange Syndrome Cells With Distinct Mutations in SMC1A and SMC3 Genes

    PubMed Central

    Gimigliano, Anna; Mannini, Linda; Bianchi, Laura; Puglia, Michele; Deardorff, Matthew A.; Menga, Stefania; Krantz, Ian D; Musio, Antonio; Bini, Luca

    2012-01-01

    Mutations in cohesin genes have been identified in Cornelia de Lange syndrome (CdLS), but its etiopathogenetic mechanisms are still poorly understood. To define biochemical pathways that are affected in CdLS we analyzed the proteomic profile of CdLS cell lines carrying mutations in the core cohesin genes, SMC1A and SMC3. Dysregulated protein expression was found in CdLS probands compared to controls. The proteomics analysis was able to discriminate between probands harboring mutations in the different domains of the SMC proteins. In particular, proteins involved in the response to oxidative stress were specifically down-regulated in hinge mutated probands. In addition, the finding that CdLS cell lines show an increase in global oxidative stress argues that it could contribute to some CdLS phenotypic features such as premature physiological aging and genome instability. Finally, the c-MYC gene represents a convergent hub lying at the center of dysregulated pathways, and is down-regulated in CdLS. This study allowed us to highlight, for the first time, specific biochemical pathways that are affected in CdLS, providing plausible causal evidence for some of the phenotypic features seen in CdLS. PMID:23106691

  5. Human pre-B cell receptor signal transduction: evidence for distinct roles of PI3kinase and MAP-kinase signalling pathways

    PubMed Central

    Anbazhagan, Kolandaswamy; Rabbind Singh, Amrathlal; Isabelle, Piec; Stella, Ibata; Céline, Alleaume-De Martel; Bissac, Eliane; Bertrand, Brassart; Rémy, Nyga; Naomi, Taylor; Vincent, Fuentes; Rochette, Jacques; Lassoued, Kaïss

    2013-01-01

    Pre-BCR acts as a critical checkpoint in B cell development. However, its signalling cascade still remains indistinctly characterised in human. We investigated pre-BCR signalling pathway to examine its regulation in normal primary pre-B lymphocytes and pre-B cell lines. In cell lines, early signalling events occurring after pre-BCR stimulation include phosphorylation of Lyn, Blk and Syk together with ZAP70, Btk, Vav, PLC-γ2 and various adaptor proteins, such as BLNK, LAB, LAT and SLP-76. Further downstream, these molecules induced activation of the PI3K/AKT and MAP-kinase resulting in an augmentation of canonical NF-κB pathways and cFos/AP1 activation. PI3K and MAPK exerted opposing effects on the pre-BCR-induced activation of the canonical NF-κB and c-Fos/AP1 pathways. Immediate nuclear export of FoxO3A and delayed import of IRF4 were additional events observed after pre-BCR crosslinking in primary cells. Pre-BCR-induced down-regulation of Rag1, Rag2, E2A and Pax5 transcripts occurred in a PI3K-dependent manner. Finally we bring evidence that pre-BCR stimulation or co stimulation with CD19 enhances cell cycle signal. PMID:25400915

  6. Autocatalytic processing of m-AAA protease subunits in mitochondria.

    PubMed

    Koppen, Mirko; Bonn, Florian; Ehses, Sarah; Langer, Thomas

    2009-10-01

    m-AAA proteases are ATP-dependent proteolytic machines in the inner membrane of mitochondria which are crucial for the maintenance of mitochondrial activities. Conserved nuclear-encoded subunits, termed paraplegin, Afg3l1, and Afg3l2, form various isoenzymes differing in their subunit composition in mammalian mitochondria. Mutations in different m-AAA protease subunits are associated with distinct neuronal disorders in human. However, the biogenesis of m-AAA protease complexes or of individual subunits is only poorly understood. Here, we have examined the processing of nuclear-encoded m-AAA protease subunits upon import into mitochondria and demonstrate autocatalytic processing of Afg3l1 and Afg3l2. The mitochondrial processing peptidase MPP generates an intermediate form of Afg3l2 that is matured autocatalytically. Afg3l1 or Afg3l2 are also required for maturation of newly imported paraplegin subunits after their cleavage by MPP. Our results establish that mammalian m-AAA proteases can act as processing enzymes in vivo and reveal overlapping activities of Afg3l1 and Afg3l2. These findings might be of relevance for the pathogenesis of neurodegenerative disorders associated with mutations in different m-AAA protease subunits. PMID:19656850

  7. Distinct MAPK signaling pathways, p21 up-regulation and caspase-mediated p21 cleavage establishes the fate of U937 cells exposed to 3-hydrogenkwadaphnin: Differentiation versus apoptosis

    SciTech Connect

    Moosavi, Mohammad Amin; Yazdanparast, Razieh

    2008-07-01

    Despite the depth of knowledge concerning the pathogenesis of acute myeloblastic leukemia (AML), long-term survival remains unresolved. Therefore, new agents that act more selectively and more potently are required. In that line, we have recently characterized a novel diterpene ester, called 3-hydrogenkwadaphnin (3-HK), with capability to induce both differentiation and apoptosis in various leukemia cell lines. These effects of 3-HK were mediated through inhibition of inosine 5'-monophosphate dehydrogenase, a selective up-regulated enzyme in cancerous cells, especially leukemia. However, it remains elusive to understand how cells display different fates in response to 3-HK. Here, we report the distinct molecular signaling pathways involved in forcing of 3-HK-treated U937 cells to undergo differentiation and apoptosis. After 3-HK (15 nM) treatment, a portion of U937 cells adhered to the culture plates and showed macrophage criteria while others remained in suspension and underwent apoptosis. The differentiated cells arrested in G{sub 0}/G{sub 1} phase of cell cycle and showed early activation of ERK1/2 pathway (3 h) along with ERK-dependent p21{sup Cip/WAF1} (p21) up-regulation and expression of p27{sup Kip1} and Bcl-2. In contrast, the suspension cells underwent apoptosis through Fas/FasL and mitochondrial pathways. The occurrence of apoptosis in these cells were accompanied with caspase-8-mediated p21 cleavage and delayed activation (24 h) of JNK1/2 and p38 MAPK. Taken together, these results suggest that distinct signaling pathways play a pivotal role in fates of drug-treated leukemia cells, thus this may pave some novel therapeutical utilities.

  8. Activation of Plasmacytoid Dendritic Cells in Colon-Draining Lymph Nodes during Citrobacter rodentium Infection Involves Pathogen-Sensing and Inflammatory Pathways Distinct from Conventional Dendritic Cells.

    PubMed

    Toivonen, Raine; Kong, Lingjia; Rasool, Omid; Lund, Riikka J; Lahesmaa, Riitta; Hänninen, Arno

    2016-06-01

    Dendritic cells (DCs) bear the main responsibility for initiation of adaptive immune responses necessary for antimicrobial immunity. In the small intestine, afferent lymphatics convey Ags and microbial signals to mesenteric lymph nodes (LNs) to induce adaptive immune responses against microbes and food Ags derived from the small intestine. Whether the large intestine is covered by the same lymphatic system or represents its own lymphoid compartment has not been studied until very recently. We identified three small mesenteric LNs, distinct from small intestinal LNs, which drain lymph specifically from the colon, and studied DC responses to the attaching and effacing pathogen Citrobacter rodentium in these. Transcriptional profiling of conventional (CD11c(high)CD103(high)) DC and plasmacytoid (plasmacytoid DC Ag-1(high)B220(+)CD11c(int)) DC (pDC) populations during steady-state conditions revealed activity of distinct sets of genes in these two DC subsets, both in small intestinal and colon-draining LNs. C. rodentium activated DC especially in colon-draining LNs, and gene expression changed in pDC more profoundly than in conventional DC. Among the genes most upregulated in pDC were C-type lectin receptor CLEC4E, IL-1Rs (IL-1R1 and -2), proinflammatory cytokines (IL-1a and IL-6), and TLR6. Our results indicate that colon immune surveillance is distinct from that of the small intestine in terms of draining LNs, and identify pDC as active sentinels of colonic inflammation and/or microbial dysbiosis. PMID:27183629

  9. Proteases of human rhinovirus: role in infection.

    PubMed

    Jensen, Lora M; Walker, Erin J; Jans, David A; Ghildyal, Reena

    2015-01-01

    Human rhinoviruses (HRV) are the major etiological agents of the common cold and asthma exacerbations, with significant worldwide health and economic impact. Although large-scale population vaccination has proved successful in limiting or even eradicating many viruses, the more than 100 distinct serotypes mean that conventional vaccination is not a feasible strategy to combat HRV. An alternative strategy is to target conserved viral proteins such as the HRV proteases, 2A(pro) and 3C(pro), the focus of this review. Necessary for host cell shutoff, virus replication, and pathogenesis, 2A(pro) and 3C(pro) are clearly viable drug targets, and indeed, 3C(pro) has been successfully targeted for treating the common cold in experimental infection. 2A(pro) and 3C(pro) are crucial for virus replication due to their role in polyprotein processing as well as cleavage of key cellular proteins to inhibit cellular transcription and translation. Intriguingly, the action of the HRV proteases also disrupts nucleocytoplasmic trafficking, contributing to HRV cytopathic effects. Improved understanding of the protease-cell interactions should enable new therapeutic approaches to be identified for drug development. PMID:25261311

  10. Protease-mediated drug delivery

    NASA Astrophysics Data System (ADS)

    Dickson, Eva F.; Goyan, Rebecca L.; Kennedy, James C.; Mackay, M.; Mendes, M. A. K.; Pottier, Roy H.

    2003-12-01

    Drugs used in disease treatment can cause damage to both malignant and normal tissue. This toxicity limits the maximum therapeutic dose. Drug targeting is of high interest to increase the therapeutic efficacy of the drug without increasing systemic toxicity. Certain tissue abnormalities, disease processes, cancers, and infections are characterized by high levels of activity of specific extracellular and/or intracellular proteases. Abnormally high activity levels of specific proteases are present at sites of physical or chemical trauma, blood clots, malignant tumors, rheumatoid arthritis, inflammatory bowel disease, gingival disease, glomerulonerphritis, and acute pancreatitis. Abnormal protease activity is suspected in development of liver thrombosis, pulmonary emphysema, atherosclerosis, and muscular dystrophy. Inactiviating disease-associated proteases by the administration of appropriate protease inhibitors has had limited success. Instead, one could use such proteases to target drugs to treat the condition. Protease mediated drug delivery offers such a possibility. Solubilizing groups are attached to insoluble drugs via a polypeptide chain which is specifically cleavable by certian proteases. When the solubilized drug enounters the protease, the solubilizing moieties are cleaved, and the drug precipitates at the disease location. Thus, a smaller systemic dosage could result in a therapeutic drug concentration at the treatment site with less systemic toxicity.

  11. A group IIA-secreted phospholipase A2 from snake venom induces lipid body formation in macrophages: the roles of intracellular phospholipases A2 and distinct signaling pathways.

    PubMed

    Leiguez, Elbio; Zuliani, Juliana Pavan; Cianciarullo, Aurora Marques; Fernandes, Cristina Maria; Gutiérrez, José Maria; Teixeira, Catarina

    2011-07-01

    We investigated the ability of the sPLA(2), known as MT-III, isolated from the viperid snake Bothrops asper, to induce LB formation in macrophages and the major cellular signaling pathways involved in this process. The effects of MT-III on ADRP localization and expression and macrophage ultrastructure were assessed. Our results showed that this sPLA(2) induced a marked increase in LB numbers in macrophages, induced the recruitment of ADRP in macrophages, and up-regulated ADRP expression. Ultrastructural analysis showed the presence of weakly and strongly osmiophilic LBs in sPLA(2)-stimulated cells. Enlargement of the ER and Golgi cisterns was also observed. Pretreatment of cells with H7 or staurosporine (PKC inhibitors), LY294002 or wortmannin (PI3K inhibitors), SB202190 or PD98059 (p38(MAPK) and ERK1/2 inhibitors, respectively), or Pyr-2 or Bel (cPLA(2) and iPLA(2) inhibitors, respectively) significantly reduced sPLA(2)-induced LB formation. Herbimycin (a PTK inhibitor) and indomethacin or etoricoxib (COX inhibitors) failed to alter sPLA(2)-induced effects. In conclusion, our results show for the first time the ability of a venom sPLA(2) to induce the formation of LBs and the expression of ADRP in macrophages. Venom PLA(2)-induced LB formation is dependent on PKC, PI3K, p38(MAPK), ERK1/2, cPLA(2), and iPLA(2) signaling pathways but not on PTK, COX-1, or COX-2 pathways. Activation of the ER and Golgi complex may play an important role in the formation of LBs induced by this sPLA(2) in macrophages. PMID:21478270

  12. Distinct role and functional mode of TR3 and RARalpha in mediating ATRA-induced signalling pathway in breast and gastric cancer cells.

    PubMed

    Ye, Xiao feng; Wu, Qiao; Liu, Su; Lin, Xiao feng; Zhang, Bing; Wu, Jia fa; Cai, Jian huai; Zhang, Ming qing; Su, Wen jin

    2004-01-01

    All-trans retinoic acid (ATRA) affects cell proliferation, differentiation and apoptosis through its receptors, RARs and RXRs. Besides these, other receptors such as orphan receptor TR3, are also involved in the regulatory process of ATRA. However, how different receptors function in response to ATRA is still largely unknown. In the present study, we found that formation of TR3/RXRalpha heterodimers in the nucleus and their subsequent translocation into the cytoplasm, in association with regulation of apoptosis-related proteins Bcl-2, Bcl-xl and Bax, was critical for apoptosis induction by ATRA in breast cancer cells MCF-7. When such translocation was blocked by Leptomycin B (LMB), ATRA-induced apoptosis was consequently abolished. However, in ATRA-induced gastric cancer cells MGC80-3, RXRalpha heterodimerised with RARalpha but not with TR3, and remained in the nucleus exerting its effect on cell cycle regulation. When transfected with antisense-RARalpha, MGC80-3 cells changed from ATRA-sensitive to ATRA-resistant and most cells were arrested in the S phase, implying the importance of RARalpha in cell cycle regulation. Furthermore, we demonstrated that the effects of ATRA depend on the relative levels of TR3, RARalpha and RXRalpha expression in cancer cells. In ATRA-induced MCF-7 cells, highly expressed TR3 favours the formation of TR3/RXRalpha and promotes the TR3/RXRalpha signalling pathway causing apoptosis; while in ATRA-induced MGC80-3 cells, high expression of RARalpha favours the formation of RARalpha/RXRalpha and promotes the RXRalpha/RARalpha signalling pathway in mediating cell cycle regulation. In conclusion, these results reveal the novel mechanism that cellular expression and location of protein is associated with diverse signalling transduction pathways and the resultant physiological process. PMID:14592536

  13. Proteases in cardiometabolic diseases: Pathophysiology, molecular mechanisms and clinical applications.

    PubMed

    Hua, Yinan; Nair, Sreejayan

    2015-02-01

    Cardiovascular disease is the leading cause of death in the U.S. and other developed countries. Metabolic syndrome, including obesity, diabetes/insulin resistance, hypertension and dyslipidemia is a major threat for public health in the modern society. It is well established that metabolic syndrome contributes to the development of cardiovascular disease collective called as cardiometabolic disease. Despite documented studies in the research field of cardiometabolic disease, the underlying mechanisms are far from clear. Proteases are enzymes that break down proteins, many of which have been implicated in various diseases including cardiac disease. Matrix metalloproteinase (MMP), calpain, cathepsin and caspase are among the major proteases involved in cardiac remodeling. Recent studies have also implicated proteases in the pathogenesis of cardiometabolic disease. Elevated expression and activities of proteases in atherosclerosis, coronary heart disease, obesity/insulin-associated heart disease as well as hypertensive heart disease have been documented. Furthermore, transgenic animals that are deficient in or over-express proteases allow scientists to understand the causal relationship between proteases and cardiometabolic disease. Mechanistically, MMPs and cathepsins exert their effect on cardiometabolic diseases mainly through modifying the extracellular matrix. However, MMP and cathepsin are also reported to affect intracellular proteins, by which they contribute to the development of cardiometabolic diseases. On the other hand, activation of calpain and caspases has been shown to influence intracellular signaling cascade including the NF-κB and apoptosis pathways. Clinically, proteases are reported to function as biomarkers of cardiometabolic diseases. More importantly, the inhibitors of proteases are credited with beneficial cardiometabolic profile, although the exact molecular mechanisms underlying these salutary effects are still under investigation. A better

  14. Proteases in cardiometabolic diseases: Pathophysiology, molecular mechanisms and clinical applications

    PubMed Central

    Hua, Yinan; Nair, Sreejayan

    2014-01-01

    Cardiovascular disease is the leading cause of death in the U.S. and other developed country. Metabolic syndrome, including obesity, diabetes/insulin resistance, hypertension and dyslipidemia is major threat for public health in the modern society. It is well established that metabolic syndrome contributes to the development of cardiovascular disease collective called as cardiometabolic disease. Despite documented studies in the research field of cardiometabolic disease, the underlying mechanisms are far from clear. Proteases are enzymes that break down proteins, many of which have been implicated in various diseases including cardiac disease. Matrix metalloproteinase (MMP), calpain, cathepsin and caspase are among the major proteases involved in cardiac remodeling. Recent studies have also implicated proteases in the pathogenesis of cardiometabolic disease. Elevated expression and activities of proteases in atherosclerosis, coronary heart disease, obesity/insulin-associated heart disease as well as hypertensive heart disease have been documented. Furthermore, transgenic animals that are deficient in or overexpress proteases allow scientists to understand the causal relationship between proteases and cardiometabolic disease. Mechanistically, MMPs and cathepsins exert their effect on cardiometabolic diseases mainly through modifying the extracellular matrix. However, MMP and cathepsin are also reported to affect intracellular proteins, by which they contribute to the development of cardiometabolic diseases. On the other hand, activation of calpain and caspases has been shown to influence intracellular signaling cascade including the NF-κB and apoptosis pathways. Clinically, proteases are reported to function as biomarkers of cardiometabolic diseases. More importantly, the inhibitors of proteases are credited with beneficial cardiometabolic profile, although the exact molecular mechanisms underlying these salutary effects are still under investigation. A better

  15. Two mitochondrial matrix proteases act sequentially in the processing of mammalian matrix enzymes.

    PubMed

    Kalousek, F; Hendrick, J P; Rosenberg, L E

    1988-10-01

    The imported precursors of the mammalian matrix enzymes malate dehydrogenase [(S)-malate:NAD+ oxidoreductase, EC 1.1.1.37] and ornithine transcarbamylase (carbamoyl-phosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) are cleaved to their mature subunits in two steps, each catalyzed by matrix-localized processing proteases. The number and properties of these proteases are the subjects of this report. We have identified and characterized two distinct protease activities in a crude matrix fraction from rat liver: processing protease I, which cleaves these precursors to the corresponding intermediate form; and processing protease II, which cleaves the intermediate forms to mature subunits. Protease I is insensitive to chelation by EDTA and to inactivation with N-ethylmaleimide; protease II is inhibited by 5 mM EDTA and is inactivated by treatment with N-ethylmaleimide. We have prepared from mitochondrial matrix an 800-fold-enriched protease I fraction free of protease II activity by using the following steps: ion exchange, hydroxyapatite, molecular sieving, and hydrophobic chromatography. Using similar procedures, we also have prepared an approximately 2000-fold-enriched protease II fraction, which has a trace amount of contaminating protease I. This enriched protease II fraction has little or no cleavage activity toward mitochondrial precursors but rapidly and efficiently converts intermediate forms to mature size. Finally, we show that protease I alone is sufficient to cleave the precursor of a third nuclear-encoded mitochondrial protein subunit--the beta subunit of propionyl-CoA carboxylase [propanoyl-CoA:carbon dioxide ligase (ADP-forming), EC 6.4.1.3]--to its mature size. PMID:3050998

  16. The angiogenic responses induced by release of angiogenic proteins from tumor cell-activated platelets are regulated by distinct molecular pathways.

    PubMed

    Wu, Hongyan; Fan, Fangtian; Liu, Zhaoguo; Zhang, Feng; Liu, Yuping; Wei, Zhonghong; Shen, Cunsi; Cao, Yuzhu; Wang, Aiyun; Lu, Yin

    2015-08-01

    There is mounting evidence that tumor angiogenesis can be regulated by platelets (Plts), which serve as major sources and delivery vehicles of many proangiogenic cytokines including transforming growth factor-β and vascular endothelial growth factor. Although considerable progress has been made in understanding the role for Plt secretion in tumor angiogenesis, very little is known about the precise mechanisms underlying cancer cell induction of Plt granule release. Here, we demonstrated that nonsmall cell lung cancer (NSCLC) cells directly induced Plt secretion of several angiogenic regulatory cytokines that promoted angiogenesis in concert. Moreover, we discovered that these Plt-derived angiogenesis modulators were regulated by different molecular pathways and could be largely inhibited by combination of multiple signaling inhibitors. Our present studies indicated that manipulation of Plt secretion of angiogenic cytokines without compromising hemostatic functions could provide a novel option for management of tumor angiogenesis and metastasis in NSCLC patients with thrombocytosis. PMID:26283102

  17. The Golgi apparatus is a functionally distinct Ca2+ store regulated by PKA and Epac branches of the β1-adrenergic signaling pathway

    PubMed Central

    Yang, Zhaokang.; Kirton, Hannah M.; MacDougall, David A.; Boyle, John P.; Deuchars, James; Frater, Brenda; Ponnambalam, Sreenivasan; Hardy, Matthew E.; White, Edward; Calaghan, Sarah C.; Peers, Chris; Steele, Derek S.

    2016-01-01

    Ca2+ release from the Golgi apparatus regulates key functions of the organelle, including vesicle trafficking. However, the signaling pathways that control this form of Ca2+ release are poorly understood and evidence of discrete Golgi Ca2+ release events is lacking. Here, we identified the Golgi apparatus as the source of prolonged Ca2+ release events that originate from the nuclear ‘poles’ of primary cardiac cells. Once initiated, Golgi Ca2+ release was unaffected by global depletion of sarcoplasmic reticulum Ca2+, and disruption of the Golgi apparatus abolished Golgi Ca2+ release without affecting sarcoplasmic reticulum function, suggesting functional and anatomical independence of Golgi and sarcoplasmic reticulum Ca2+ stores. Maximal activation of β1-adrenoceptors had only a small stimulating effect on Golgi Ca2+ release. However, inhibition of phosphodiesterase (PDE) 3 or 4, or downregulation of PDE 3 and 4 in heart failure markedly potentiated β1-adrenergic stimulation of Golgi Ca2+ release, consistent with compartmentalization of cAMP signaling within the Golgi apparatus microenvironment. β1-adrenergic stimulation of Golgi Ca2+ release involved activation of both Epac and PKA signaling pathways and CaMKII. Interventions that stimulated Golgi Ca2+ release induced trafficking of vascular growth factor receptor-1 (VEGFR-1) from the Golgi apparatus to the surface membrane. These data establish the Golgi apparatus as a juxtanuclear focal point for Ca2+ and β1-adrenergic signaling, which functions independently from the sarcoplasmic reticulum and the global Ca2+ transients that underlie the primary contractile function of the cell. PMID:26462734

  18. Community Structure Analysis of Transcriptional Networks Reveals Distinct Molecular Pathways for Early- and Late-Onset Temporal Lobe Epilepsy with Childhood Febrile Seizures

    PubMed Central

    Moreira-Filho, Carlos Alberto; Bando, Silvia Yumi; Bertonha, Fernanda Bernardi; Iamashita, Priscila; Silva, Filipi Nascimento; Costa, Luciano da Fontoura; Silva, Alexandre Valotta; Castro, Luiz Henrique Martins; Wen, Hung-Tzu

    2015-01-01

    Age at epilepsy onset has a broad impact on brain plasticity and epilepsy pathomechanisms. Prolonged febrile seizures in early childhood (FS) constitute an initial precipitating insult (IPI) commonly associated with mesial temporal lobe epilepsy (MTLE). FS-MTLE patients may have early disease onset, i.e. just after the IPI, in early childhood, or late-onset, ranging from mid-adolescence to early adult life. The mechanisms governing early (E) or late (L) disease onset are largely unknown. In order to unveil the molecular pathways underlying E and L subtypes of FS-MTLE we investigated global gene expression in hippocampal CA3 explants of FS-MTLE patients submitted to hippocampectomy. Gene coexpression networks (GCNs) were obtained for the E and L patient groups. A network-based approach for GCN analysis was employed allowing: i) the visualization and analysis of differentially expressed (DE) and complete (CO) - all valid GO annotated transcripts - GCNs for the E and L groups; ii) the study of interactions between all the system’s constituents based on community detection and coarse-grained community structure methods. We found that the E-DE communities with strongest connection weights harbor highly connected genes mainly related to neural excitability and febrile seizures, whereas in L-DE communities these genes are not only involved in network excitability but also playing roles in other epilepsy-related processes. Inversely, in E-CO the strongly connected communities are related to compensatory pathways (seizure inhibition, neuronal survival and responses to stress conditions) while in L-CO these communities harbor several genes related to pro-epileptic effects, seizure-related mechanisms and vulnerability to epilepsy. These results fit the concept, based on fMRI and behavioral studies, that early onset epilepsies, although impacting more severely the hippocampus, are associated to compensatory mechanisms, while in late MTLE development the brain is less able to

  19. Protease degradable electrospun fibrous hydrogels

    PubMed Central

    Wade, Ryan J.; Bassin, Ethan J.; Rodell, Christopher B.; Burdick, Jason A.

    2015-01-01

    Electrospun nanofibers are promising in biomedical applications to replicate features of the natural extracellular matrix (ECM). However, nearly all electrospun scaffolds are either non-degradable or degrade hydrolytically, whereas natural ECM degrades proteolytically, often through matrix metalloproteinases (MMPs). Here, we synthesize reactive macromers that contain protease-cleavable and fluorescent peptides and are able to form both isotropic hydrogels and electrospun fibrous hydrogels through a photoinitiated polymerization. These biomimetic scaffolds are susceptible to protease-mediated cleavage in vitro in a protease dose dependent manner and in vivo in a subcutaneous mouse model using transdermal fluorescent imaging to monitor degradation. Importantly, materials containing an alternate and non-protease-cleavable peptide sequence are stable in both in vitro and in vivo settings. To illustrate the specificity in degradation, scaffolds with mixed fiber populations support selective fiber degradation based on individual fiber degradability. Overall, this represents a novel biomimetic approach to generate protease-sensitive fibrous scaffolds for biomedical applications. PMID:25799370

  20. Atg8 Transfer from Atg7 to Atg3: A Distinctive E1-E2 Architecture and Mechanism in the Autophagy Pathway

    SciTech Connect

    Taherbhoy, Asad M.; Tait, Stephen W.; Kaiser, Stephen E.; Williams, Allison H.; Deng, Alan; Nourse, Amanda; Hammel, Michal; Kurinov, Igor; Rock, Charles O.; Green, Douglas R.; Schulman, Brenda A.

    2012-07-11

    Atg7 is a noncanonical, homodimeric E1 enzyme that interacts with the noncanonical E2 enzyme, Atg3, to mediate conjugation of the ubiquitin-like protein (UBL) Atg8 during autophagy. Here we report that the unique N-terminal domain of Atg7 (Atg7{sup NTD}) recruits a unique 'flexible region' from Atg3 (Atg3{sup FR}). The structure of an Atg7{sup NTD}-Atg3{sup FR} complex reveals hydrophobic residues from Atg3 engaging a conserved groove in Atg7, important for Atg8 conjugation. We also report the structure of the homodimeric Atg7 C-terminal domain, which is homologous to canonical E1s and bacterial antecedents. The structures, SAXS, and crosslinking data allow modeling of a full-length, dimeric (Atg7 {approx} Atg8-Atg3){sub 2} complex. The model and biochemical data provide a rationale for Atg7 dimerization: Atg8 is transferred in trans from the catalytic cysteine of one Atg7 protomer to Atg3 bound to the N-terminal domain of the opposite Atg7 protomer within the homodimer. The studies reveal a distinctive E1 {approx} UBL-E2 architecture for enzymes mediating autophagy.

  1. Distinctive profiles of small RNA couple inverted repeat-induced post-transcriptional gene silencing with endogenous RNA silencing pathways in Arabidopsis

    PubMed Central

    Matvienko, Marta; Piskurewicz, Urszula; Xu, Huaqin; Martineau, Belinda; Wong, Joan; Govindarajulu, Manjula; Kozik, Alexander; Michelmore, Richard W.

    2014-01-01

    The experimental induction of RNA silencing in plants often involves expression of transgenes encoding inverted repeat (IR) sequences to produce abundant dsRNAs that are processed into small RNAs (sRNAs). These sRNAs are key mediators of post-transcriptional gene silencing (PTGS) and determine its specificity. Despite its application in agriculture and broad utility in plant research, the mechanism of IR-PTGS is incompletely understood. We generated four sets of 60 Arabidopsis plants, each containing IR transgenes expressing different configurations of uidA and CHALCONE SYNTHASE (At-CHS) gene fragments. Levels of PTGS were found to depend on the orientation and position of the fragment in the IR construct. Deep sequencing and mapping of sRNAs to corresponding transgene-derived and endogenous transcripts identified distinctive patterns of differential sRNA accumulation that revealed similarities among sRNAs associated with IR-PTGS and endogenous sRNAs linked to uncapped mRNA decay. Detailed analyses of poly-A cleavage products from At-CHS mRNA confirmed this hypothesis. We also found unexpected associations between sRNA accumulation and the presence of predicted open reading frames in the trigger sequence. In addition, strong IR-PTGS affected the prevalence of endogenous sRNAs, which has implications for the use of PTGS for experimental or applied purposes. PMID:25344399

  2. Single transcriptional and translational preQ1 riboswitches adopt similar pre-folded ensembles that follow distinct folding pathways into the same ligand-bound structure

    PubMed Central

    Suddala, Krishna C.; Rinaldi, Arlie J.; Feng, Jun; Mustoe, Anthony M.; Eichhorn, Catherine D.; Liberman, Joseph A.; Wedekind, Joseph E.; Al-Hashimi, Hashim M.; Brooks, Charles L.; Walter, Nils G.

    2013-01-01

    Riboswitches are structural elements in the 5′ untranslated regions of many bacterial messenger RNAs that regulate gene expression in response to changing metabolite concentrations by inhibition of either transcription or translation initiation. The preQ1 (7-aminomethyl-7-deazaguanine) riboswitch family comprises some of the smallest metabolite sensing RNAs found in nature. Once ligand-bound, the transcriptional Bacillus subtilis and translational Thermoanaerobacter tengcongensis preQ1 riboswitch aptamers are structurally similar RNA pseudoknots; yet, prior structural studies have characterized their ligand-free conformations as largely unfolded and folded, respectively. In contrast, through single molecule observation, we now show that, at near-physiological Mg2+ concentration and pH, both ligand-free aptamers adopt similar pre-folded state ensembles that differ in their ligand-mediated folding. Structure-based Gō-model simulations of the two aptamers suggest that the ligand binds late (Bacillus subtilis) and early (Thermoanaerobacter tengcongensis) relative to pseudoknot folding, leading to the proposal that the principal distinction between the two riboswitches lies in their relative tendencies to fold via mechanisms of conformational selection and induced fit, respectively. These mechanistic insights are put to the test by rationally designing a single nucleotide swap distal from the ligand binding pocket that we find to predictably control the aptamers′ pre-folded states and their ligand binding affinities. PMID:24003028

  3. The MAPKERK-1,2 pathway integrates distinct and antagonistic signals from TGF alpha and FGF7 in morphogenesis of mouse mammary epithelium

    SciTech Connect

    Fata, Jimmie E; Mori, Hidetoshi; Ewald, Andrew J; Zhang, Hui; Yao, Evelyn; Werb, Zena; Bissell, Mina J

    2006-10-03

    Transforming growth factor-{alpha} (TGF{alpha}) and fibroblast growth factor-7 (FGF7) exhibit distinct expression patterns in the mammary gland. Both factors signal through mitogen-activated kinase/extracellular regulated kinase-1,2 (MAPK{sup ERK1,2}); however, their unique and/or combined contributions to mammary morphogenesis have not been examined. In ex vivo mammary explants, we show that a sustained activation of MAPK{sup ERK1,2} for 1 h, induced by TGF{alpha}, was necessary and sufficient to initiate branching morphogenesis, whereas a transient activation (15 min) of MAPK{sup ERK1,2}, induced by FGF7, led to growth without branching. Unlike TGF{alpha}, FGF7 promoted sustained proliferation as well as ectopic localization of, and increase in, keratin-6 expressing cells. The response of the explants to FGF10 was similar to that to FGF7. Simultaneous stimulation by FGF7 and TGF{alpha} indicated that the FGF7-induced MAPK{sup ERK1,2} signaling and associated phenotypes were dominant: FGF7 may prevent branching by suppression of two necessary TGF{alpha}-induced morphogenetic effectors, matrix metalloproteinase-3 (MMP-3/stromelysin-1), and fibronectin. Our findings indicate that expression of morphogenetic effectors, proliferation, and cell-type decisions during mammary organoid morphogenesis are intimately dependent on the duration of activation of MAPK{sup ERK1,2} activation.

  4. Distinctive profiles of small RNA couple inverted repeat-induced post-transcriptional gene silencing with endogenous RNA silencing pathways in Arabidopsis.

    PubMed

    Wroblewski, Tadeusz; Matvienko, Marta; Piskurewicz, Urszula; Xu, Huaqin; Martineau, Belinda; Wong, Joan; Govindarajulu, Manjula; Kozik, Alexander; Michelmore, Richard W

    2014-12-01

    The experimental induction of RNA silencing in plants often involves expression of transgenes encoding inverted repeat (IR) sequences to produce abundant dsRNAs that are processed into small RNAs (sRNAs). These sRNAs are key mediators of post-transcriptional gene silencing (PTGS) and determine its specificity. Despite its application in agriculture and broad utility in plant research, the mechanism of IR-PTGS is incompletely understood. We generated four sets of 60 Arabidopsis plants, each containing IR transgenes expressing different configurations of uidA and CHALCONE Synthase (At-CHS) gene fragments. Levels of PTGS were found to depend on the orientation and position of the fragment in the IR construct. Deep sequencing and mapping of sRNAs to corresponding transgene-derived and endogenous transcripts identified distinctive patterns of differential sRNA accumulation that revealed similarities among sRNAs associated with IR-PTGS and endogenous sRNAs linked to uncapped mRNA decay. Detailed analyses of poly-A cleavage products from At-CHS mRNA confirmed this hypothesis. We also found unexpected associations between sRNA accumulation and the presence of predicted open reading frames in the trigger sequence. In addition, strong IR-PTGS affected the prevalence of endogenous sRNAs, which has implications for the use of PTGS for experimental or applied purposes. PMID:25344399

  5. Evolution under Drug Pressure Remodels the Folding Free-Energy Landscape of Mature HIV-1 Protease.

    PubMed

    Louis, John M; Roche, Julien

    2016-07-01

    Using high-pressure NMR spectroscopy and differential scanning calorimetry, we investigate the folding landscape of the mature HIV-1 protease homodimer. The cooperativity of unfolding was measured in the absence or presence of a symmetric active site inhibitor for the optimized wild type protease (PR), its inactive variant PRD25N, and an extremely multidrug-resistant mutant, PR20. The individual fit of the pressure denaturation profiles gives rise to first order, ∆GNMR, and second order, ∆VNMR (the derivative of ∆GNMR with pressure); apparent thermodynamic parameters for each amide proton considered. Heterogeneity in the apparent ∆VNMR values reflects departure from an ideal cooperative unfolding transition. The narrow to broad distribution of ∆VNMR spanning the extremes from inhibitor-free PR20D25N to PR-DMP323 complex, and distinctively for PRD25N-DMP323 complex, indicated large variations in folding cooperativity. Consistent with this data, the shape of thermal unfolding transitions varies from asymmetric for PR to nearly symmetric for PR20, as dimer-inhibitor ternary complexes. Lack of structural cooperativity was observed between regions located close to the active site, including the hinge and tip of the glycine-rich flaps, and the rest of the protein. These results strongly suggest that inhibitor binding drastically decreases the cooperativity of unfolding by trapping the closed flap conformation in a deep energy minimum. To evade this conformational trap, PR20 evolves exhibiting a smoother folding landscape with nearly an ideal two-state (cooperative) unfolding transition. This study highlights the malleability of retroviral protease folding pathways by illustrating how the selection of mutations under drug pressure remodels the free-energy landscape as a primary mechanism. PMID:27170547

  6. Distinct phospholipase C-gamma-dependent signaling pathways in the Drosophila eye and wing are revealed by a new small wing allele.

    PubMed Central

    Mankidy, Rishikesh; Hastings, Jeremy; Thackeray, Justin R

    2003-01-01

    The Drosophila genome contains a single phospholipase C-gamma (PLC-gamma) homolog, encoded by small wing (sl), that acts as an inhibitor of receptor tyrosine kinase (RTK) signaling during photoreceptor R7 development. Although the existing sl alleles behave genetically as nulls, they may still produce truncated Sl products that could in theory still provide limited PLC-gamma function. Both to identify a true null allele and to probe structure-function relationships in Sl, we carried out an F(1) screen for new sl mutations and identified seven new alleles. Flies homozygous for any of these alleles are viable, with the same short-wing phenotype described previously; however, two of the alleles differ from any of those previously isolated in the severity of the eye phenotype: sl(9) homozygotes have a slightly more extreme extra-R7 phenotype, whereas sl(7) homozygotes have an almost wild-type eye. We determined the mutant defect in all seven alleles, revealing that sl(9) is a molecular null due to a very early stop codon, while sl(7) has a missense mutation in the highly conserved Y catalytic domain. Together with in vitro mutagenesis of the residue affected by the sl(7) mutation, these results confirm the role of Sl in RTK signaling and provide evidence for two genetically separable PLC-gamma-dependent pathways affecting the development of the eye and the wing. PMID:12807776

  7. Nutritional Omega-3 Deficiency Alters Glucocorticoid Receptor-Signaling Pathway and Neuronal Morphology in Regionally Distinct Brain Structures Associated with Emotional Deficits.

    PubMed

    Larrieu, Thomas; Hilal, Muna L; De Smedt-Peyrusse, Véronique; Sans, Nathalie; Layé, Sophie

    2016-01-01

    Extensive evidence suggests that long term dietary n-3 polyunsaturated fatty acids (PUFAs) deficiency results in altered emotional behaviour. We have recently demonstrated that n-3 PUFAs deficiency induces emotional alterations through abnormal corticosterone secretion which leads to altered dendritic arborisation in the prefrontal cortex (PFC). Here we show that hypothalamic-pituitary-adrenal (HPA) axis feedback inhibition was not compromised in n-3 deficient mice. Rather, glucocorticoid receptor (GR) signaling pathway was inactivated in the PFC but not in the hippocampus of n-3 deficient mice. Consequently, only dendritic arborisation in PFC was affected by dietary n-3 PUFAs deficiency. In addition, occlusion experiment with GR blockade altered GR signaling in the PFC of control mice, with no further alterations in n-3 deficient mice. In conclusion, n-3 PUFAs deficiency compromised PFC, leading to dendritic atrophy, but did not change hippocampal GR function and dendritic arborisation. We argue that this GR sensitivity contributes to n-3 PUFAs deficiency-related emotional behaviour deficits. PMID:27057368

  8. Different exercise modalities have distinct effects on the integrin-linked kinase (ILK) and Ca2+ signaling pathways in the male rat bone

    PubMed Central

    Sontam, Dharani M; Firth, Elwyn C; Tsai, Peter; Vickers, Mark H; O’Sullivan, Justin M

    2015-01-01

    Mechanical loading is essential to maintain optimal skeletal health. Despite the fact that early-life exercise has positive, long-lasting effects on the musculo-skeletal system, the response of the musculo-skeletal system to spontaneous low-impact exercise has been poorly studied. Previously, we identified subtle morphological changes in the femoral diaphysis of exercised animals compared to nonexercised controls. We hypothesized that significant changes in gene expression of cells should precede significant measurable phenotypic changes in the tissues of which they are part. Here, we employed RNA-Seq to analyse the transcriptome of the cortical bone from the femoral mid-diaphysis of prepubertal male Sprague-Dawley rats that were assigned to control (CON); bipedal stance (BPS); or wheel exercise (WEX) groups for 15 days. We identified 808 and 324 differentially expressed transcripts in the BPS and WEX animals respectively. While a number of transcripts change their levels in an exercise-specific manner, we identified 191 transcripts that were differentially expressed in both BPS and WEX. Importantly, we observed that the exercise mode had diametrically opposite effects on transcripts for multiple genes within the integrin-linked kinase (ILK) and Ca2+ signaling pathways such that they were up-regulated in BPS and down-regulated in WEX. The findings are important for our understanding of possible ways in which different exercise regimens might affect bone when normal activities apply mechanical stimuli during postnatal growth and development. PMID:26471755

  9. Nutritional Omega-3 Deficiency Alters Glucocorticoid Receptor-Signaling Pathway and Neuronal Morphology in Regionally Distinct Brain Structures Associated with Emotional Deficits

    PubMed Central

    Larrieu, Thomas; Hilal, Muna L.; De Smedt-Peyrusse, Véronique; Sans, Nathalie; Layé, Sophie

    2016-01-01

    Extensive evidence suggests that long term dietary n-3 polyunsaturated fatty acids (PUFAs) deficiency results in altered emotional behaviour. We have recently demonstrated that n-3 PUFAs deficiency induces emotional alterations through abnormal corticosterone secretion which leads to altered dendritic arborisation in the prefrontal cortex (PFC). Here we show that hypothalamic-pituitary-adrenal (HPA) axis feedback inhibition was not compromised in n-3 deficient mice. Rather, glucocorticoid receptor (GR) signaling pathway was inactivated in the PFC but not in the hippocampus of n-3 deficient mice. Consequently, only dendritic arborisation in PFC was affected by dietary n-3 PUFAs deficiency. In addition, occlusion experiment with GR blockade altered GR signaling in the PFC of control mice, with no further alterations in n-3 deficient mice. In conclusion, n-3 PUFAs deficiency compromised PFC, leading to dendritic atrophy, but did not change hippocampal GR function and dendritic arborisation. We argue that this GR sensitivity contributes to n-3 PUFAs deficiency-related emotional behaviour deficits. PMID:27057368

  10. RNA sequencing supports distinct reactive oxygen species-mediated pathways of apoptosis by high and low size mass fractions of Bay leaf (Lauris nobilis) in HT-29 cells.

    PubMed

    Rodd, Annabelle L; Ververis, Katherine; Sayakkarage, Dheeshana; Khan, Abdul W; Rafehi, Haloom; Ziemann, Mark; Loveridge, Shanon J; Lazarus, Ross; Kerr, Caroline; Lockett, Trevor; El-Osta, Assam; Karagiannis, Tom C; Bennett, Louise E

    2015-08-01

    Anti-proliferative and pro-apoptotic effects of Bay leaf (Laurus nobilis) in mammalian cancer and HT-29 adenocarcinoma cells have been previously attributed to effects of polyphenolic and essential oil chemical species. Recently, we demonstrated differentiated growth-regulating effects of high (HFBL) versus low molecular mass (LFBL) aqueous fractions of bay leaf and now confirm by comparative effects on gene expression, that HFBL and LFBL suppress HT-29 growth by distinct mechanisms. Induction of intra-cellular lesions including DNA strand breakage by extra-cellular HFBL, invoked the hypothesis that iron-mediated reactive oxygen species with capacity to penetrate cell membrane, were responsible for HFBL-mediated effects, supported by equivalent effects of HFBL in combination with γ radiation. Activities of HFBL and LFBL were interpreted to reflect differentiated responses to iron-mediated reactive oxygen species (ROS), occurring either outside or inside cells. In the presence of LFBL, apoptotic death was relatively delayed compared with HFBL. ROS production by LFBL mediated p53-dependent apoptosis and recovery was suppressed by promoting G1/S phase arrest and failure of cellular tight junctions. In comparison, intra-cellular anti-oxidant protection exerted by LFBL was absent for extra-cellular HFBL (likely polysaccharide-rich), which potentiated more rapid apoptosis by producing DNA double strand breaks. Differentiated effects on expression of genes regulating ROS defense and chromatic condensation by LFBL versus HFBL, were observed. The results support ferrous iron in cell culture systems and potentially in vivo, can invoke different extra-cellular versus intra-cellular ROS-mediated chemistries, that may be regulated by exogenous, including dietary species. PMID:26114728

  11. Molecular Cloning and Functional Characterization of Three Distinct N-Methyltransferases Involved in the Caffeine Biosynthetic Pathway in Coffee Plants1

    PubMed Central

    Uefuji, Hirotaka; Ogita, Shinjiro; Yamaguchi, Yube; Koizumi, Nozomu; Sano, Hiroshi

    2003-01-01

    Caffeine is synthesized from xanthosine through N-methylation and ribose removal steps. In the present study, three types of cDNAs encoding N-methyltransferases were isolated from immature fruits of coffee (Coffea arabica) plants, and designated as CaXMT1, CaMXMT2, and CaDXMT1, respectively. The bacterially expressed encoded proteins were characterized for their catalytic properties. CaXMT1 catalyzed formation of 7-methylxanthosine from xanthosine with a Km value of 78 μm, CaMXMT2 catalyzed formation of 3,7-dimethylxanthine (theobromine) from 7-methylxanthine with a Km of 251 μm, and CaDXMT1 catalyzed formation of 1,3,7-trimethylxanthine (caffeine) from 3,7-dimethylxanthine with a Km of 1,222 μm. The crude extract of Escherichia coli was found to catalyze removal of the ribose moiety from 7-methylxanthosine, leading to the production of 7-methylxanthine. As a consequence, when all three recombinant proteins and E. coli extract were combined, xanthosine was successfully converted into caffeine in vitro. Transcripts for CaDXMT1 were predominantly found to accumulate in immature fruits, whereas those for CaXMT1 and CaMXMT2 were more broadly detected in sites encompassing the leaves, floral buds, and immature fruits. These results suggest that the presently identified three N-methyltransferases participate in caffeine biosynthesis in coffee plants and substantiate the proposed caffeine biosynthetic pathway: xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine → caffeine. PMID:12746542

  12. Speed of saccade execution and inhibition associated with fractional anisotropy in distinct fronto-frontal and fronto-striatal white matter pathways.

    PubMed

    Thakkar, Katharine N; van den Heiligenberg, Fiona M Z; Kahn, R S; Neggers, Sebastiaan F W

    2016-08-01

    Fast cancellation or switching of action plans is a critical cognitive function. Rapid signal transmission is key for quickly executing and inhibiting responses, and the structural integrity of connections between brain regions plays a crucial role in signal transmission speed. In this study, we used the search-step task, which has been used in nonhuman primates to measure dynamic alteration of saccade plans, in combination with functional and diffusion-weighted MRI. Functional MRI results were used to identify brain regions involved in the reactive control of gaze. Probabilistic tractography was used to identify white matter pathways connecting these structures, and the integrity of these connections, as indicated by fractional anisotropy (FA), was correlated with search-step task performance. Average FA from tracts between the right frontal eye field (FEF) and both right supplementary eye field (SEF) and the dorsal striatum were associated with faster saccade execution. Average FA of connections between the dorsal striatum and both right SEF and right inferior frontal cortex (IFC) as well as between SEF and IFC predicted the speed of inhibition. These relationships were largely behaviorally specific, despite the correlation between saccade execution and inhibition. Average FA of connections between the IFC and both SEF and the dorsal striatum specifically predicted the speed of inhibition, and connections between the FEF and SEF specifically predicted the speed of execution. In addition, these relationships were anatomically specific; correlations were observed after controlling for global FA. These data suggest that networks supporting saccade initiation and inhibition are at least partly dissociable. Hum Brain Mapp 37:2811-2822, 2016. © 2016 Wiley Periodicals, Inc. PMID:27091670

  13. CARRAGEENAN-INDUCED NFκB ACTIVATION DEPENDS ON DISTINCT PATHWAYS MEDIATED BY REACTIVE OXYGEN SPECIES AND HSP27 OR BY BCL10

    PubMed Central

    Bhattacharyya, Sumit; Dudeja, Pradeep K.; Tobacman, Joanne K.

    2013-01-01

    Carrageenans are highly sulfated polysaccharides that are widely used as food additives due to their ability to improve food texture. They are also widely recognized for their ability to induce inflammation in animal models of colitis. Recently, we reported that carrageenan (CGN) activated a pathway of innate immunity in human colonic epithelial cells mediated by Bcl10 (B-cell CLL/lymphoma 10). However, increases in phospho-IκBα and Interleukin-8 (IL-8) were not completely inhibited by silencing Bcl10, suggesting that CGN also influenced another mechanism, or mechanisms, of inflammation. In this report, we demonstrate that CGN increases production of reactive oxygen species (ROS) in human colonic epithelial cells. The combination of ROS quenching by the free radical scavenger Tempol and of Bcl10 silencing by siRNA completely inhibited the CGN-induced increases in nuclear NFκB (p65), phospho-IκBα, and secretion of IL-8. The CGN-induced increase in ROS was associated with declines in phosphorylation of MAPK 12 (p38γ), MAPK 13 (p38δ), and heat-shock protein (Hsp) 27. The CGN-induced decline in phospho-Hsp27 was reversed by co-administration of Tempol (100nM), but unaffected by silencing Bcl10. Since Hsp27 phosphorylation is inversely associated with phosphorylation of the IκBα kinase (IKK) signalosome, CGN exposure appears to affect the IKK signalosome by both the catalytic component, mediated by ROS-phospho-Hsp27, and the regulatory component, mediated by Bcl10 interaction with IKKγ (Nemo). Hence, the CGN-activated inflammatory cascades related to innate immunity and to generation of ROS may be integrated at the level of the IKK signalosome. PMID:18452717

  14. Poly(I:C) reduces expression of JAM-A and induces secretion of IL-8 and TNF-{alpha} via distinct NF-{kappa}B pathways in human nasal epithelial cells

    SciTech Connect

    Ohkuni, Tsuyoshi; Kojima, Takashi; Ogasawara, Noriko; Masaki, Tomoyuki; Fuchimoto, Jun; Kamekura, Ryuta; Koizumi, Jun-ichi; Ichimiya, Shingo; Murata, Masaki; Tanaka, Satoshi; Himi, Tetsuo; Sawada, Norimasa

    2011-01-01

    Human nasal epithelium is an important physical barrier and innate immune defense protecting against inhaled substances and pathogens. Toll-like receptor (TLR) signaling, which plays a key role in the innate immune response, has not been well characterized in human nasal epithelial cells (HNECs), including the epithelial tight junctional barrier. In the present study, mRNAs of TLR1-10 were detected in hTERT-transfected HNECs, which can be used as an indispensable and stable model of normal HNECs, similar to primary cultured HNECs. To investigate the changes of tight junction proteins and the signal transduction pathways via TLRs in HNECs in vitro, hTERT-transfected HNECs were treated with TLR2 ligand P{sub 3}CSK{sub 4}, TLR3 ligand poly(I:C), TLR4 ligand LPS, TLR7/8 ligand CL097, TLR8 ligand ssRNA40/LyoVec, and TLR9 ligand ODN2006. In hTERT-transfected HNECs, treatment with poly(I:C) significantly reduced expression of the tight junction protein JAM-A and induced secretion of proinflammatory cytokines IL-8 and TNF-{alpha}. Both the reduction of JAM-A expression and the induction of secretion of IL-8 and TNF-{alpha} after treatment with poly(I:C) were modulated by distinct signal transduction pathways via EGFR, PI3K, and p38 MAPK and finally regulated by a TLR3-mediated NF-{kappa}B pathway. The control of TLR3-mediated signaling pathways in HNECs may be important not only in infection by viral dsRNA but also in autoimmune diseases caused by endogenous dsRNA released from necrotic cells.

  15. Regulation of Extracellular Protease Production in Bacillus cereus T: Characterization of Mutants Producing Altered Amounts of Protease

    PubMed Central

    Aronson, A. I.; Angelo, N.; Holt, S. C.

    1971-01-01

    Twenty-nine mutants of Bacillus cereus T were selected on casein agar for their inability to produce large amounts of extracellular protease. They all formed spores, and 27 were also auxotrophs for purines or pyrimidines. Upon reversion to prototrophy, a large fraction regained the capacity to produce protease. Conversely, reversion to normal protease production resulted in loss of the purine or pyrimidine requirement in a large fraction of the revertants. One spontaneous low-protease-producing pyrimidine auxotroph studied in detail grew as well as the wild type and produced spores which were identical to those produced by the wild type on the basis of heat resistance, dipicolinic acid content, density, and appearance in the electron microscope. The rate of protein turnover in the mutant was the same as the wild type. The mutant did grow poorly, however, when casein was the principal carbon source. A mutant excreting 5 to 10 times as much protease as the wild type was isolated as a secondary mutation from the hypoproducer discussed above. Loss of the pyrimidine requirement in this case did not alter the regulation of protease production. Although the secondary mutant grew somewhat faster in most media than the wild type, the final cell yield was lower. The spores of this mutant appeared to have excess coat on the basis of both electron microscopic and chemical studies. There appear to be closely related but distinct catabolic controls for both extracellular protease and spore formation. These controls can be dissociated as for the hypoproducers but can also appear integrated as for the hyperprotease producer. Images PMID:4104235

  16. A Low Dose of Fermented Soy Germ Alleviates Gut Barrier Injury, Hyperalgesia and Faecal Protease Activity in a Rat Model of Inflammatory Bowel Disease

    PubMed Central

    Moussa, Lara; Bézirard, Valérie; Salvador-Cartier, Christel; Bacquié, Valérie; Lencina, Corinne; Lévêque, Mathilde; Braniste, Viorica; Ménard, Sandrine

    2012-01-01

    Pro-inflammatory cytokines like macrophage migration inhibitory factor (MIF), IL-1β and TNF-α predominate in inflammatory bowel diseases (IBD) and TNBS colitis. Increased levels of serine proteases activating protease-activated receptor 2 (PAR-2) are found in the lumen and colonic tissue of IBD patients. PAR-2 activity and pro-inflammatory cytokines impair epithelial barrier, facilitating the uptake of luminal aggressors that perpetuate inflammation and visceral pain. Soy extracts contain phytoestrogens (isoflavones) and serine protease inhibitors namely Bowman-Birk Inhibitors (BBI). Since estrogens exhibit anti-inflammatory and epithelial barrier enhancing properties, and that a BBI concentrate improves ulcerative colitis, we aimed to evaluate if a fermented soy germ extract (FSG) with standardized isoflavone profile and stable BBI content exert cumulative or synergistic protection based on protease inhibition and estrogen receptor (ER)-ligand activity in colitic rats. Female rats received orally for 15 d either vehicle or FSG with or without an ER antagonist ICI 182.780 before TNBS intracolonic instillation. Macroscopic and microscopic damages, myeloperoxidase activity, cytokine levels, intestinal paracellular permeability, visceral sensitivity, faecal proteolytic activity and PAR-2 expression were assessed 24 h, 3 d and 5 d post-TNBS. FSG treatment improved the severity of colitis, by decreasing the TNBS-induced rise in gut permeability, visceral sensitivity, faecal proteolytic activity and PAR-2 expression at all post-TNBS points. All FSG effects were reversed by the ICI 182.780 except the decrease in faecal proteolytic activity and PAR-2 expression. In conclusion, the anti-inflammatory properties of FSG treatment result from two distinct but synergic pathways i.e an ER-ligand and a PAR-2 mediated pathway, providing rationale for potential use as adjuvant therapy in IBD. PMID:23166707

  17. Human B lymphoblastoid cells contain distinct patterns of cathepsin activity in endocytic compartments and regulate MHC class II transport in a cathepsin S-independent manner.

    PubMed

    Lautwein, Alfred; Kraus, Marianne; Reich, Michael; Burster, Timo; Brandenburg, J; Overkleeft, Herman S; Schwarz, Gerold; Kammer, Winfried; Weber, Ekkehard; Kalbacher, Hubert; Nordheim, Alfred; Driessen, Christoph

    2004-05-01

    Endocytic proteolysis represents a major functional component of the major histocompatibility complex class II antigen-presentation machinery. Although transport and assembly of class II molecules in the endocytic compartment are well characterized, we lack information about the pattern of endocytic protease activity along this pathway. Here, we used chemical tools that visualize endocytic proteases in an activity-dependent manner in combination with subcellular fractionation to dissect the subcellular distribution of the major cathepsins (Cat) CatS, CatB, CatH, CatD, CatC, and CatZ as well as the asparagine-specific endoprotease (AEP) in human B-lymphoblastoid cells (BLC). Endocytic proteases were distributed in two distinct patterns: CatB and CatZ were most prominent in early and late endosomes but absent from lysosomes, and CatH, CatS, CatD, CatC, and AEP distributed between late endosomes and lysosomes, suggesting that CatB and CatZ might be involved in the initial proteolytic attack on a given antigen. The entire spectrum of protease activity colocalized with human leukocyte antigen-DM and the C-terminal and N-terminal processing of invariant chain (Ii) in late endosomes. CatS was active in all endocytic compartments. Surprisingly and in contrast with results from dendritic cells, inhibition of CatS activity by leucine-homophenylalanine-vinylsulfone-phenol prevented N-terminal processing of Ii but did not alter the subcellular trafficking or surface delivery of class II complexes, as deferred from pulse-chase analysis in combination with subcellular fractionation and biotinylation of cell-surface protein. Thus, BLC contain distinct activity patterns of proteases in endocytic compartments and regulate the intracellular transport and surface-delivery of class II in a CatS-independent manner. PMID:14966190

  18. Serine proteases of parasitic helminths.

    PubMed

    Yang, Yong; Wen, Yun jun; Cai, Ya Nan; Vallée, Isabelle; Boireau, Pascal; Liu, Ming Yuan; Cheng, Shi Peng

    2015-02-01

    Serine proteases form one of the most important families of enzymes and perform significant functions in a broad range of biological processes, such as intra- and extracellular protein metabolism, digestion, blood coagulation, regulation of development, and fertilization. A number of serine proteases have been identified in parasitic helminths that have putative roles in parasite development and nutrition, host tissues and cell invasion, anticoagulation, and immune evasion. In this review, we described the serine proteases that have been identified in parasitic helminths, including nematodes (Trichinella spiralis, T. pseudospiralis, Trichuris muris, Anisakis simplex, Ascaris suum, Onchocerca volvulus, O. lienalis, Brugia malayi, Ancylostoma caninum, and Steinernema carpocapsae), cestodes (Spirometra mansoni, Echinococcus granulosus, and Schistocephalus solidus), and trematodes (Fasciola hepatica, F. gigantica, and Schistosoma mansoni). Moreover, the possible biological functions of these serine proteases in the endogenous biological phenomena of these parasites and in the host-parasite interaction were also discussed. PMID:25748703

  19. Serine Proteases of Parasitic Helminths

    PubMed Central

    Yang, Yong; Wen, Yun jun; Cai, Ya Nan; Vallée, Isabelle; Boireau, Pascal; Liu, Ming Yuan; Cheng, Shi Peng

    2015-01-01

    Serine proteases form one of the most important families of enzymes and perform significant functions in a broad range of biological processes, such as intra- and extracellular protein metabolism, digestion, blood coagulation, regulation of development, and fertilization. A number of serine proteases have been identified in parasitic helminths that have putative roles in parasite development and nutrition, host tissues and cell invasion, anticoagulation, and immune evasion. In this review, we described the serine proteases that have been identified in parasitic helminths, including nematodes (Trichinella spiralis, T. pseudospiralis, Trichuris muris, Anisakis simplex, Ascaris suum, Onchocerca volvulus, O. lienalis, Brugia malayi, Ancylostoma caninum, and Steinernema carpocapsae), cestodes (Spirometra mansoni, Echinococcus granulosus, and Schistocephalus solidus), and trematodes (Fasciola hepatica, F. gigantica, and Schistosoma mansoni). Moreover, the possible biological functions of these serine proteases in the endogenous biological phenomena of these parasites and in the host-parasite interaction were also discussed. PMID:25748703

  20. Reversible Unfolding of Rhomboid Intramembrane Proteases.

    PubMed

    Panigrahi, Rashmi; Arutyunova, Elena; Panwar, Pankaj; Gimpl, Katharina; Keller, Sandro; Lemieux, M Joanne

    2016-03-29

    Denaturant-induced unfolding of helical membrane proteins provides insights into their mechanism of folding and domain organization, which take place in the chemically heterogeneous, anisotropic environment of a lipid membrane. Rhomboid proteases are intramembrane proteases that play key roles in various diseases. Crystal structures have revealed a compact helical bundle with a buried active site, which requires conformational changes for the cleavage of transmembrane substrates. A dimeric form of the rhomboid protease has been shown to be important for activity. In this study, we examine the mechanism of refolding for two distinct rhomboids to gain insight into their secondary structure-activity relationships. Although helicity is largely abolished in the unfolded states of both proteins, unfolding is completely reversible for HiGlpG but only partially reversible for PsAarA. Refolding of both proteins results in reassociation of the dimer, with a 90% regain of catalytic activity for HiGlpG but only a 70% regain for PsAarA. For both proteins, a broad, gradual transition from the native, folded state to the denatured, partly unfolded state was revealed with the aid of circular dichroism spectroscopy as a function of denaturant concentration, thus arguing against a classical two-state model as found for many globular soluble proteins. Thermal denaturation has irreversible destabilizing effects on both proteins, yet reveals important functional details regarding substrate accessibility to the buried active site. This concerted biophysical and functional analysis demonstrates that HiGlpG, with a simple six-transmembrane-segment organization, is more robust than PsAarA, which has seven predicted transmembrane segments, thus rendering HiGlpG amenable to in vitro studies of membrane-protein folding. PMID:27028647

  1. Subclassification and Biochemical Analysis of Plant Papain-Like Cysteine Proteases Displays Subfamily-Specific Characteristics1[C][W

    PubMed Central

    Richau, Kerstin H.; Kaschani, Farnusch; Verdoes, Martijn; Pansuriya, Twinkal C.; Niessen, Sherry; Stüber, Kurt; Colby, Tom; Overkleeft, Hermen S.; Bogyo, Matthew; Van der Hoorn, Renier A.L.

    2012-01-01

    Papain-like cysteine proteases (PLCPs) are a large class of proteolytic enzymes associated with development, immunity, and senescence. Although many properties have been described for individual proteases, the distribution of these characteristics has not been studied collectively. Here, we analyzed 723 plant PLCPs and classify them into nine subfamilies that are present throughout the plant kingdom. Analysis of these subfamilies revealed previously unreported distinct subfamily-specific functional and structural characteristics. For example, the NPIR and KDEL localization signals are distinctive for subfamilies, and the carboxyl-terminal granulin domain occurs in two PLCP subfamilies, in which some individual members probably evolved by deletion of the granulin domains. We also discovered a conserved double cysteine in the catalytic site of SAG12-like proteases and two subfamily-specific disulfides in RD19A-like proteases. Protease activity profiling of representatives of the PLCP subfamilies using novel fluorescent probes revealed striking polymorphic labeling profiles and remarkably distinct pH dependency. Competition assays with peptide-epoxide scanning libraries revealed common and unique inhibitory fingerprints. Finally, we expand the detection of PLCPs by identifying common and organ-specific protease activities and identify previously undetected proteases upon labeling with cell-penetrating probes in vivo. This study provides the plant protease research community with tools for further functional annotation of plant PLCPs. PMID:22371507

  2. Allosteric antibody inhibition of human hepsin protease.

    PubMed

    Koschubs, Tobias; Dengl, Stefan; Dürr, Harald; Kaluza, Klaus; Georges, Guy; Hartl, Christiane; Jennewein, Stefan; Lanzendörfer, Martin; Auer, Johannes; Stern, Alvin; Huang, Kuo-Sen; Packman, Kathryn; Gubler, Ueli; Kostrewa, Dirk; Ries, Stefan; Hansen, Silke; Kohnert, Ulrich; Cramer, Patrick; Mundigl, Olaf

    2012-03-15

    Hepsin is a type II transmembrane serine protease that is expressed in several human tissues. Overexpression of hepsin has been found to correlate with tumour progression and metastasis, which is so far best studied for prostate cancer, where more than 90% of such tumours show this characteristic. To enable improved future patient treatment, we have developed a monoclonal humanized antibody that selectively inhibits human hepsin and does not inhibit other related proteases. We found that our antibody, hH35, potently inhibits hepsin enzymatic activity at nanomolar concentrations. Kinetic characterization revealed non-linear, slow, tight-binding inhibition. This correlates with the crystal structure we obtained for the human hepsin-hH35 antibody Fab fragment complex, which showed that the antibody binds hepsin around α3-helix, located far from the active centre. The unique allosteric mode of inhibition of hH35 is distinct from the recently described HGFA (hepatocyte growth factor activator) allosteric antibody inhibition. We further explain how a small change in the antibody design induces dramatic structural rearrangements in the hepsin antigen upon binding, leading to complete enzyme inactivation. PMID:22132769

  3. Mouse islet of Langerhans isolation using a combination of purified collagenase and neutral protease.

    PubMed

    Stull, Natalie D; Breite, Andrew; McCarthy, Robert; Tersey, Sarah A; Mirmira, Raghavendra G

    2012-01-01

    The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories(1-4), variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations(5, 6), but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh(7, 8), in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient(9), and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a(6) fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate(6). This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay(10). Utilizing these improved assays along with more

  4. Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

    PubMed Central

    Stull, Natalie D.; Breite, Andrew; McCarthy, Robert; Tersey, Sarah A.; Mirmira, Raghavendra G.

    2012-01-01

    The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional

  5. Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

    PubMed Central

    Klingler, Diana; Hardt, Markus

    2013-01-01

    Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic mechanisms of proteolytic enzymes1-3. With the advent of mass spectrometry-based proteomics, the enzymatically-catalyzed incorporation of 18O-atoms from stable isotopically enriched water has become a popular method to quantitatively compare protein expression levels (reviewed by Fenselau and Yao4, Miyagi and Rao5 and Ye et al.6). 18O-labeling constitutes a simple and low-cost alternative to chemical (e.g. iTRAQ, ICAT) and metabolic (e.g. SILAC) labeling techniques7. Depending on the protease utilized, 18O-labeling can result in the incorporation of up to two 18O-atoms in the C-terminal carboxyl group of the cleavage product3. The labeling reaction can be subdivided into two independent processes, the peptide bond cleavage and the carboxyl oxygen exchange reaction8. In our PALeO (protease-assisted labeling employing 18O-enriched water) adaptation of enzymatic 18O-labeling, we utilized 50% 18O-enriched water to yield distinctive isotope signatures. In combination with high-resolution matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), the characteristic isotope envelopes can be used to identify cleavage products with a high level of specificity. We previously have used the PALeO-methodology to detect and characterize endogenous proteases9 and monitor proteolytic reactions10-11. Since PALeO encodes the very essence of the proteolytic cleavage reaction, the experimental setup is simple and biochemical enrichment steps of cleavage products can be circumvented. The PALeO-method can easily be extended to (i) time course experiments that monitor the dynamics of proteolytic cleavage reactions and (ii) the analysis of proteolysis in complex biological samples that represent physiological conditions. PALeO-TimeCourse experiments help identifying rate-limiting processing

  6. Conditional Proteolysis of the Membrane Protein YfgM by the FtsH Protease Depends on a Novel N-terminal Degron.

    PubMed

    Bittner, Lisa-Marie; Westphal, Kai; Narberhaus, Franz

    2015-07-31

    Regulated proteolysis efficiently and rapidly adapts the bacterial proteome to changing environmental conditions. Many protease substrates contain recognition motifs, so-called degrons, that direct them to the appropriate protease. Here we describe an entirely new degron identified in the cytoplasmic N-terminal end of the membrane-anchored protein YfgM of Escherichia coli. YfgM is stable during exponential growth and degraded in stationary phase by the essential FtsH protease. The alarmone (p)ppGpp, but not the previously described YfgM interactors RcsB and PpiD, influence YfgM degradation. By scanning mutagenesis, we define individual amino acids responsible for turnover of YfgM and find that the degron does not at all comply with the known N-end rule pathway. The YfgM degron is a distinct module that facilitates FtsH-mediated degradation when fused to the N terminus of another monotopic membrane protein but not to that of a cytoplasmic protein. Several lines of evidence suggest that stress-induced degradation of YfgM relieves the response regulator RcsB and thereby permits cellular protection by the Rcs phosphorelay system. On the basis of these and other results in the literature, we propose a model for how the membrane-spanning YfgM protein serves as connector between the stress responses in the periplasm and cytoplasm. PMID:26092727

  7. Evidence for Reduced Drug Susceptibility without Emergence of Major Protease Mutations following Protease Inhibitor Monotherapy Failure in the SARA Trial

    PubMed Central

    Sutherland, Katherine A.; Parry, Chris M.; McCormick, Adele; Kapaata, Anne; Lyagoba, Fred; Kaleebu, Pontiano; Gilks, Charles F.; Goodall, Ruth; Spyer, Moira; Kityo, Cissy; Pillay, Deenan; Gupta, Ravindra K.

    2015-01-01

    Background Major protease mutations are rarely observed following failure with protease inhibitors (PI), and other viral determinants of failure to PI are poorly understood. We therefore characterized Gag-Protease phenotypic susceptibility in subtype A and D viruses circulating in East Africa following viral rebound on PIs. Methods Samples from baseline and treatment failure in patients enrolled in the second line LPV/r trial SARA underwent phenotypic susceptibility testing. Data were expressed as fold-change in susceptibility relative to a LPV-susceptible reference strain. Results We cloned 48 Gag-Protease containing sequences from seven individuals and performed drug resistance phenotyping from pre-PI and treatment failure timepoints in seven patients. For the six patients where major protease inhibitor resistance mutations did not emerge, mean fold-change EC50 to LPV was 4.07 fold (95% CI, 2.08–6.07) at the pre-PI timepoint. Following viral failure the mean fold-change in EC50 to LPV was 4.25 fold (95% CI, 1.39–7.11, p = 0.91). All viruses remained susceptible to DRV. In our assay system, the major PI resistance mutation I84V, which emerged in one individual, conferred a 10.5-fold reduction in LPV susceptibility. One of the six patients exhibited a significant reduction in susceptibility between pre-PI and failure timepoints (from 4.7 fold to 9.6 fold) in the absence of known major mutations in protease, but associated with changes in Gag: V7I, G49D, R69Q, A120D, Q127K, N375S and I462S. Phylogenetic analysis provided evidence of the emergence of genetically distinct viruses at the time of treatment failure, indicating ongoing viral evolution in Gag-protease under PI pressure. Conclusions Here we observe in one patient the development of significantly reduced susceptibility conferred by changes in Gag which may have contributed to treatment failure on a protease inhibitor containing regimen. Further phenotype-genotype studies are required to elucidate genetic

  8. Coupling of epithelial Na+ and Cl− channels by direct and indirect activation by serine proteases

    PubMed Central

    Gondzik, Veronika; Weber, Wolf Michael

    2012-01-01

    The mammalian collecting duct (CD) is continuously exposed to urinary proteases. The CD expresses an epithelial Na+ channel (ENaC) that is activated after cleavage by serine proteases. ENaC also exists at the plasma membrane in the uncleaved form, rendering activation by extracellular proteases an important mechanism for regulating Na+ transport. Many exogenous and a small number of endogenous extracellular serine proteases have been shown to activate the channel. Recently, kallikrein 1 (KLK1) was shown to increase γENaC cleavage in the native CD indicating a possible direct role of this endogenous protease in Na+ homeostasis. To explore this process, we examined the coordinated effect of this protease on Na+ and Cl− transport in a polarized renal epithelial cell line (Madin-Darby canine kidney). We also examined the role of native urinary proteases in this process. Short-circuit current (Isc) was used to measure transport of these ions. The Isc exhibited an ENaC-dependent Na+ component that was amiloride blockable and a cystic fibrosis transmembrane conductance regulator (CFTR)-dependent Cl− component that was blocked by inhibitor 172. Apical application of trypsin, an exogenous S1 serine protease, activated IENaC but was without effects on ICFTR. Subtilisin an exogenous S8 protease that mimics endogenous furin-type proteases activated both currents. A similar activation was also observed with KLK1 and native rat urinary proteases. Activation with urinary proteases occurred within minutes and at protease concentrations similar to those in the CD indicating physiological significance of this process. ENaC activation was irreversible and mediated by enhanced cleavage of γENaC. The activation of CFTR was indirect and likely dependent on activation of an endogenous apical membrane protease receptor. Collectively, these data demonstrate coordinated stimulation of separate Na+ and Cl− transport pathways in renal epithelia by extracellular luminal proteases. They

  9. The macromolecular assembly of pathogen-recognition receptors is impelled by serine proteases, via their complement control protein modules.

    PubMed

    Le Saux, Agnès; Ng, Patricia Miang Lon; Koh, Joanne Jing Yun; Low, Diana Hooi Ping; Leong, Geraldine E-Ling; Ho, Bow; Ding, Jeak Ling

    2008-03-28

    Although the innate immune response is triggered by the formation of a stable assembly of pathogen-recognition receptors (PRRs) onto the pathogens, the driving force that enables this PRR-PRR interaction is unknown. Here, we show that serine proteases, which are activated during infection, participate in associating with the PRRs. Inhibition of serine proteases gravely impairs the PRR assembly. Using yeast two-hybrid and pull-down methods, we found that two serine proteases in the horseshoe crab Carcinoscorpius rotundicauda are able to bind to the following three core members of PRRs: galactose-binding protein, Carcinolectin-5 and C-reactive protein. These two serine proteases are (1) Factor C, which activates the coagulation pathway, and (2) C2/Bf, a protein from the complement pathway. By systematic molecular dissection, we show that these serine proteases interact with the core "pathogen-recognition complex" via their complement control protein modules. PMID:18279891

  10. Microbial inhibitors of cysteine proteases.

    PubMed

    Kędzior, Mateusz; Seredyński, Rafał; Gutowicz, Jan

    2016-08-01

    Cysteine proteases are one of the major classes of proteolytic enzymes involved in a number of physiological and pathological processes in plants, animals and microorganisms. When their synthesis, activity and localization in mammalian cells are altered, they may contribute to the development of many diseases, including rheumatoid arthritis, osteoporosis and cancer. Therefore, cysteine proteases have become promising drug targets for the medical treatment of these disorders. Inhibitors of cysteine proteases are also produced by almost every group of living organisms, being responsible for the control of intracellular proteolytic activity. Microorganisms synthesize cysteine protease inhibitors not only to regulate the activity of endogenous, often virulent enzymes, but also to hinder the host's proteolytic defense system and evade its immune responses against infections. Present work describes known to date microbial inhibitors of cysteine proteases in terms of their structure, enzyme binding mechanism, specificity and pathophysiological roles. The overview of both proteinaceous and small-molecule inhibitors produced by all groups of microorganisms (bacteria, archaea, fungi, protists) and viruses is provided. Subsequently, possible applications of microbial inhibitors in science, medicine and biotechnology are also highlighted. PMID:27048482

  11. Bioinformatic analyses of male and female Amblyomma americanum tick expressed serine protease inhibitors (serpins)

    PubMed Central

    Porter, Lindsay; Radulovic, Zeljko; Kim, Tae; Braz, Gloria R. C.; Da Silva Vaz, Itabajara; Mulenga, Albert

    2014-01-01

    Serine protease inhibitors (serpins) are a diverse family of proteins that is conserved across taxa. The diversity of Amblyomma americanum serpins (AAS) is far more complex than previously thought as revealed by discovery of 57 and 33 AAS transcripts that are respectively expressed in male and female A. americanum ticks, with 30 found in both. While distinct reproductively, both male and female metastriate ticks, such as A. americanum, require a blood meal. Thus, 30 AAS sequences found in both male and female ticks could play important role(s) in regulating tick feeding and thus represent attractive candidates for anti-tick vaccine development. Of significant interest, 19 AAS sequences expressed in male and female ticks are also part of the 48 AAS sequences expressed in fed female tick salivary glands or midguts; two organs through which the tick interacts with host blood and immune response factors. Considered the most important domain for serpin function, the reactive center loop (RCL) is further characterized by a single ‘P1’ site amino acid residue, which is central to determining the protease regulated by the serpin. In this study, a diversity of 17 different P1 site amino acid residues were predicted, suggesting that A. americanum serpins potentially regulate a large number of proteolytic pathways. Our data also indicate that some serpins in this study could regulate target protease common to all tick species, in that more than 40% of AAS show 58–97% inter-species amino acid conservation. Of significance, 24% of AAS showed 62–100% inter-species conservation within the functional RCL domain, with 10 RCLs showing ≥90–100% conservation. In vertebrates, serpins with basic residues at the P1 site regulate key host defense pathways, which the tick must evade to feed successfully. Interestingly, we found that AAS sequences with basic or polar uncharged residues at the putative P1 site are more likely to be conserved across tick species. Another notable

  12. Distinct microRNA expression profile and targeted biological pathways in functional myeloid-derived suppressor cells induced by Δ9-tetrahydrocannabinol in vivo: regulation of CCAAT/enhancer-binding protein α by microRNA-690.

    PubMed

    Hegde, Venkatesh L; Tomar, Sunil; Jackson, Austin; Rao, Roshni; Yang, Xiaoming; Singh, Udai P; Singh, Narendra P; Nagarkatti, Prakash S; Nagarkatti, Mitzi

    2013-12-27

    Δ(9)-Tetrahydrocannabinol (THC), the major bioactive component of marijuana, has been shown to induce functional myeloid-derived suppressor cells (MDSCs) in vivo. Here, we studied the involvement of microRNA (miRNA) in this process. CD11b(+)Gr-1(+) MDSCs were purified from peritoneal exudates of mice administered with THC and used for genome-wide miRNA profiling. Expression of CD31 and Ki-67 confirmed that the THC-MDSCs were immature and proliferating. THC-induced MDSCs exhibited distinct miRNA expression signature relative to various myeloid cells and BM precursors. We identified 13 differentially expressed (>2-fold) miRNA in THC-MDSCs relative to control BM precursors. In silico target prediction for these miRNA and pathway analysis using multiple bioinformatics tools revealed significant overrepresentation of Gene Ontology clusters within hematopoiesis, myeloid cell differentiation, and regulation categories. Insulin-like growth factor 1 signaling involved in cell growth and proliferation, and myeloid differentiation pathways were among the most significantly enriched canonical pathways. Among the differentially expressed, miRNA-690 was highly overexpressed in THC-MDSCs (∼16-fold). Transcription factor CCAAT/enhancer-binding protein α (C/EBPα) was identified as a potential functional target of miR-690. Supporting this, C/EBPα expression was attenuated in THC-MDSCs as compared with BM precursors and exhibited an inverse relation with miR-690. miR-690 knockdown using peptide nucleic acid-antagomiR was able to unblock and significantly increase C/EBPα expression establishing the functional link. Further, CD11b(+)Ly6G(+)Ly6C(+) and CD11b(+)Ly6G(-)Ly6C(+) purified subtypes showed high levels of miR-690 with attenuated C/EBPα expression. Moreover, EL-4 tumor-elicited MDSCs showed increased miR-690 expression. In conclusion, miRNA are significantly altered during the generation of functional MDSC from BM. Select miRNA such as miR-690 targeting genes involved in

  13. Dengue Virus Infection Causes the Activation of Distinct NF-κB Pathways for Inducible Nitric Oxide Synthase and TNF-α Expression in RAW264.7 Cells.

    PubMed

    Cheng, Yi-Lin; Lin, Yee-Shin; Chen, Chia-Ling; Wan, Shu-Wen; Ou, Yi-Dan; Yu, Chia-Yi; Tsai, Tsung-Ting; Tseng, Po-Chun; Lin, Chiou-Feng

    2015-01-01

    Infection with dengue virus (DENV) causes an increase in proinflammatory responses, such as nitric oxide (NO) generation and TNF-α expression; however, the molecular mechanism underlying this inflammatory activation remains undefined, although the activation of the transcription factor NF-κB is generally involved. In addition to TNF-α production in DENV-infected murine macrophage RAW264.7 cells, inducible NO synthase was transcriptionally and posttranslationally elevated and accompanied by NO generation. NF-κB is known to be activated by DENV infection. Pharmacologically inhibiting NF-κB activation abolishes iNOS/NO biosynthesis and TNF-α production. With inhibition, the potential role of NF-κB in oxidative signaling regulation was prevented during DENV infection. Heat-inactivated DENV failed to cause the identified inflammatory responses. Pharmacological inhibition of TLR3 partly decreased NF-κB activation; however, it effectively abolished inducible iNOS/NO biosynthesis but did not inhibit TNF-α production. In contrast to TLR3, viral protein NS2B3 also independently contributed to NF-κB activation to regulate TNF-α production. These results show the distinct pathways for NF-κB activation caused by DENV infection individually for the regulation of iNOS/NO and TNF-α expression. PMID:26199460

  14. A Lys49 phospholipase A2, isolated from Bothrops asper snake venom, induces lipid droplet formation in macrophages which depends on distinct signaling pathways and the C-terminal region.

    PubMed

    Giannotti, Karina Cristina; Leiguez, Elbio; Moreira, Vanessa; Nascimento, Neide Galvão; Lomonte, Bruno; Gutiérrez, José Maria; Lopes de Melo, Robson; Teixeira, Catarina

    2013-01-01

    MT-II, a Lys49PLA2 homologue devoid of catalytic activity from B. asper venom, stimulates inflammatory events in macrophages. We investigated the ability of MT-II to induce formation of lipid droplets (LDs), key elements of inflammatory responses, in isolated macrophages and participation of protein kinases and intracellular PLA2s in this effect. Influence of MT-II on PLIN2 recruitment and expression was assessed, and the effects of some synthetic peptides on LD formation were further evaluated. At noncytotoxic concentrations, MT-II directly activated macrophages to form LDs. This effect was reproduced by a synthetic peptide corresponding to the C-terminal sequence 115-129 of MT-II, evidencing the critical role of C-terminus for MT-II-induced effect. Moreover, MT-II induced expression and recruitment of PLIN2. Pharmacological interventions with specific inhibitors showed that PKC, PI3K, ERK1/2, and iPLA2, but not P38(MAPK) or cPLA2, signaling pathways are involved in LD formation induced by MT-II. This sPLA2 homologue also induced synthesis of PGE2 that colocalized to LDs. In conclusion, MT-II is able to induce formation of LDs committed to PGE2 formation in a process dependent on C-terminal loop engagement and regulated by distinct protein kinases and iPLA2. LDs may constitute an important inflammatory mechanism triggered by MT-II in macrophages. PMID:23509782

  15. Dengue Virus Infection Causes the Activation of Distinct NF-κB Pathways for Inducible Nitric Oxide Synthase and TNF-α Expression in RAW264.7 Cells

    PubMed Central

    Cheng, Yi-Lin; Lin, Yee-Shin; Chen, Chia-Ling; Wan, Shu-Wen; Ou, Yi-Dan; Yu, Chia-Yi; Tsai, Tsung-Ting; Tseng, Po-Chun; Lin, Chiou-Feng

    2015-01-01

    Infection with dengue virus (DENV) causes an increase in proinflammatory responses, such as nitric oxide (NO) generation and TNF-α expression; however, the molecular mechanism underlying this inflammatory activation remains undefined, although the activation of the transcription factor NF-κB is generally involved. In addition to TNF-α production in DENV-infected murine macrophage RAW264.7 cells, inducible NO synthase was transcriptionally and posttranslationally elevated and accompanied by NO generation. NF-κB is known to be activated by DENV infection. Pharmacologically inhibiting NF-κB activation abolishes iNOS/NO biosynthesis and TNF-α production. With inhibition, the potential role of NF-κB in oxidative signaling regulation was prevented during DENV infection. Heat-inactivated DENV failed to cause the identified inflammatory responses. Pharmacological inhibition of TLR3 partly decreased NF-κB activation; however, it effectively abolished inducible iNOS/NO biosynthesis but did not inhibit TNF-α production. In contrast to TLR3, viral protein NS2B3 also independently contributed to NF-κB activation to regulate TNF-α production. These results show the distinct pathways for NF-κB activation caused by DENV infection individually for the regulation of iNOS/NO and TNF-α expression. PMID:26199460

  16. Hydrogen peroxide induces lysosomal protease alterations in PC12 cells.

    PubMed

    Lee, Daniel C; Mason, Ceceile W; Goodman, Carl B; Holder, Maurice S; Kirksey, Otis W; Womble, Tracy A; Severs, Walter B; Palm, Donald E

    2007-09-01

    Alterations in lysosomal proteases have been implicated in many neurodegenerative diseases. The current study demonstrates a concentration-dependent decrease in PC12 cell viability and transient changes in cystatin C (CYSC), cathepsin B (CATB), cathepsin D (CATD) and caspase-3 following exposure to H2O2. Furthermore, activation of CATD occurred following exposure to H2O2 and cysteine protease suppression, while inhibition of CATD with pepstatin A significantly improved cell viability. Additionally, significant PARP cleavage, suggestive of caspase-3-like activity, was observed following H2O2 exposure, while inhibition of caspase-3 significantly increased cell viability compared to H2O2 administration alone. Collectively, our data suggest that H2O2 induced cell death is regulated at least in part by caspase-3 and CATD. Furthermore, cysteine protease suppression increases CATD expression and activity. These studies provide insight for alternate pathways and potential therapeutic targets of cell death associated with oxidative stress and lysosomal protease alterations. PMID:17440810

  17. Serine proteases SP1 and SP13 mediate the melanization response of Asian corn borer, Ostrinia furnacalis, against entomopathogenic fungus Beauveria bassiana.

    PubMed

    Chu, Yuan; Liu, Yang; Shen, Dongxu; Hong, Fang; Wang, Guirong; An, Chunju

    2015-06-01

    Exposure to entomopathogenic fungi is one approach for insect pest control. Little is known about the immune interactions between fungus and its insect host. Melanization is a prominent immune response in insects in defending against pathogens such as bacteria and fungi. Clip domain serine proteases in insect plasma have been implicated in the activation of prophenoloxidase, a key enzyme in the melanization. The relationship between host melanization and the infection by a fungus needs to be established. We report here that the injection of entomopathogenic fungus Beauveria bassiana induced both melanin synthesis and phenoloxidase activity in its host insect, the Asian corn borer, Ostrinia furnacalis (Guenée). qRT-PCR analysis showed several distinct patterns of expression of 13 clip-domain serine proteases in response to the challenge of fungi, with seven increased, two decreased, and four unchanged. Of special interest among these clip-domain serine protease genes are SP1 and SP13, the orthologs of Manduca sexta HP6 and PAP1 which are involved in the prophenoloxidase activation pathway. Recombinant O. furnacalis SP1 was found to activate proSP13 and induce the phenoloxidase activity in corn borer plasma. Additionally, SP13 was determined to directly cleave prophenoloxidase and therefore act as the prophenoloxidase activating protease. Our work thus reveals a biochemical mechanism in the melanization in corn borer associated with the challenge by B. bassiana injection. These insights could provide valuable information for better understanding the immune responses of Asian corn borer against B. bassiana. PMID:25900291

  18. Structure-based design and functional studies of novel noroviral 3C protease chimaeras offer insights into substrate specificity

    PubMed Central

    Herod, Morgan R.; Prince, Cynthia A.; Skilton, Rachel J.; Ward, Vernon K.; Cooper, Jonathan B.; Clarke, Ian N.

    2014-01-01

    The norovirus NS6 protease is a key target for anti-viral drug development. Noroviruses encode a 2200 amino acid polyprotein which is cleaved by this critical protease at five defined boundary substrates into six mature non-structural (NS) proteins. Studies of the human norovirus (HNV) NS6 protease, in the context of a full ORF1 polyprotein, have been severely hampered because HNVs are not culturable. Thus, investigations into the HNV NS6 protease have been largely restricted to in vitro assays using Escherichia coli-expressed, purified enzyme. The NS6 protease is formed of two distinct domains joined by a linking loop. Structural data suggest that domain 2 of the protease possesses substantial substrate binding pockets which form the bulk of the interactions with the NS boundaries and largely dictate boundary specificity and cleavage. We have constructed chimaeric murine norovirus (MNV) genomes carrying individual domains from the HNV protease and demonstrated by cell transfection that chimaeric HNV proteases have functional activity in the context of the full-length ORF1 polyprotein. Although domain 2 primarily confers boundary specificity, our data suggest that an inter-domain interaction exists within HNV NS6 protease which influences cleavage of specific substrates. The present study also shows that chimaeric MNVs provide improved models for studying HNV protein function in the context of a full ORF1 polyprotein. PMID:25275273

  19. Structure-based design and functional studies of novel noroviral 3C protease chimaeras offer insights into substrate specificity.

    PubMed

    Herod, Morgan R; Prince, Cynthia A; Skilton, Rachel J; Ward, Vernon K; Cooper, Jonathan B; Clarke, Ian N

    2014-12-15

    The norovirus NS6 protease is a key target for anti-viral drug development. Noroviruses encode a 2200 amino acid polyprotein which is cleaved by this critical protease at five defined boundary substrates into six mature non-structural (NS) proteins. Studies of the human norovirus (HNV) NS6 protease, in the context of a full ORF1 polyprotein, have been severely hampered because HNVs are not culturable. Thus, investigations into the HNV NS6 protease have been largely restricted to in vitro assays using Escherichia coli-expressed, purified enzyme. The NS6 protease is formed of two distinct domains joined by a linking loop. Structural data suggest that domain 2 of the protease possesses substantial substrate binding pockets which form the bulk of the interactions with the NS boundaries and largely dictate boundary specificity and cleavage. We have constructed chimaeric murine norovirus (MNV) genomes carrying individual domains from the HNV protease and demonstrated by cell transfection that chimaeric HNV proteases have functional activity in the context of the full-length ORF1 polyprotein. Although domain 2 primarily confers boundary specificity, our data suggest that an inter-domain interaction exists within HNV NS6 protease which influences cleavage of specific substrates. The present study also shows that chimaeric MNVs provide improved models for studying HNV protein function in the context of a full ORF1 polyprotein. PMID:25275273

  20. Treatment of chronic hepatitis C: anticipated impact of resistance in patients treated with protease inhibitors.

    PubMed

    Kronenberger, Bernd; Zeuzem, Stefan

    2009-02-01

    A main target of specifically targeted antiviral therapy for hepatitis C (STAT-C) is the NS3-protease, which has key functions in the hepatitis C virus (HCV) replication cycle. HCV/NS3-protease inhibitors have shown high antiviral activity in vitro and in patients with chronic hepatitis C. Protease-resistant HCV variants occurred rapidly in patients receiving protease-inhibitor monotherapy. The development of resistance can be best explained by selection of preexisting resistant variants, which grow out under selective pressure. Numerous mutations associated with resistance were identified. Clinical trials showed that protease-resistant strains are sensitive to interferon and that a triple combination of protease inhibitors, peginterferon, and ribavirin may improve the sustained virologic response rate compared with standard peginterferon/ribavirin combination therapy. Overall, it can be anticipated that successful treatment with protease inhibitors will require either combination therapy with peginterferon/ribavirin or a combination of STAT-C compounds with distinct modes of action and resistance patterns. PMID:19166654

  1. Exogenous proteases for meat tenderization.

    PubMed

    Bekhit, Alaa A; Hopkins, David L; Geesink, Geert; Bekhit, Adnan A; Franks, Philip

    2014-01-01

    The use of exogenous proteases to improve meat tenderness has attracted much interest recently, with a view to consistent production of tender meat and added value to lower grade meat cuts. This review discusses the sources, characteristics, and use of exogenous proteases in meat tenderization to highlight the specificity of the proteases toward meat proteins and their impact on meat quality. Plant enzymes (such as papain, bromelain, and ficin) have been extensively investigated as meat tenderizers. New plant proteases (actinidin and zingibain) and microbial enzyme preparations have been of recent interest due to controlled meat tenderization and other advantages. Successful use of these enzymes in fresh meat requires their enzymatic kinetics and characteristics to be determined, together with an understanding of the impact of the surrounding environmental conditions of the meat (pH, temperature) on enzyme function. This enables the optimal conditions for tenderizing fresh meat to be established, and the elimination or reduction of any negative impacts on other quality attributes. PMID:24499119

  2. Evaluation on Potential Contributions of Protease Activated Receptors Related Mediators in Allergic Inflammation

    PubMed Central

    Zhang, Huiyun; Zeng, Xiaoning; He, Shaoheng

    2014-01-01

    Protease activated receptors (PARs) have been recognized as a distinctive four-member family of seven transmembrane G protein-coupled receptors (GPCRs) that can be cleaved by certain serine proteases. In recent years, there has been considerable interest in the role of PARs in allergic inflammation, the fundamental pathologic changes of allergy, but the potential roles of PARs in allergy remain obscure. Since many of these proteases are produced and actively involved in the pathologic process of inflammation including exudation of plasma components, inflammatory cell infiltration, and tissue damage and repair, PARs appear to make important contribution to allergy. The aim of the present review is to summarize the expression of PARs in inflammatory and structural cells, the influence of agonists or antagonists of PARs on cell behavior, and the involvement of PARs in allergic disorders, which will help us to better understand the roles of serine proteases and PARs in allergy. PMID:24876677

  3. Activation mechanism of thiol protease precursor from broiler chicken specific Staphylococcus aureus strain CH-91.

    PubMed

    Wladyka, Benedykt; Dubin, Grzegorz; Dubin, Adam

    2011-01-10

    Staphylococcus aureus strain CH-91 isolated from chicken dermatitis lesions produces large quantities of thiol protease implicated in disease formation. Observed overproduction requires efficient activation of the protease precursor which mechanism is studied here in detail. Wild type and mutant precursor forms are expressed in E. coli to test different hypotheses on the activation process. It is demonstrated that wild type precursor undergoes rapid autocatalytic processing whereas proteolytically inactive catalytic triad cysteine mutant (C(249)A) of the precursor is stable, but can be processed by minute quantities of active protease. It is concluded that limited intramolecular proteolysis is mainly responsible for efficient activation but, a positive feedback loop also contributes to the process. Both activation pathways allow efficient production of mature extracellular thiol protease, a putative virulence factor specific for avian strains of S. aureus. PMID:20598816

  4. Intestinal proteases of free-living and parasitic astigmatid mites.

    PubMed

    Holt, Deborah C; Burgess, Stewart T G; Reynolds, Simone L; Mahmood, Wajahat; Fischer, Katja

    2013-02-01

    Among arthropod pests, mites are responsible for considerable damage to crops, humans and other animals. However, detailed physiological data on these organisms remain sparse, mainly because of their small size but possibly also because of their extreme diversity. Focusing on intestinal proteases, we draw together information from three distinct mite species that all feed on skin but have separately adapted to a free-living, a strictly ecto-parasitic and a parasitic lifestyle. A wide range of studies involving immunohistology, molecular biology, X-ray crystallography and enzyme biochemistry of mite gut proteases suggests that these creatures have diverged considerably as house dust mites, sheep scab mites and scabies mites. Each species has evolved a particular variation of a presumably ancestral repertoire of digestive enzymes that have become specifically adapted to their individual environmental requirements. PMID:22427061

  5. A computational module assembled from different protease family motifs identifies PI PLC from Bacillus cereus as a putative prolyl peptidase with a serine protease scaffold.

    PubMed

    Rendón-Ramírez, Adela; Shukla, Manish; Oda, Masataka; Chakraborty, Sandeep; Minda, Renu; Dandekar, Abhaya M; Ásgeirsson, Bjarni; Goñi, Félix M; Rao, Basuthkar J

    2013-01-01

    Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP). CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP) and a scaffold recognizing a β-lactam (imipenem) in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV), we sought to query other OMV proteins, like phospholipase C (PLC), using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM). Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues. PMID:23940667

  6. Biotechnology of Cold-Active Proteases

    PubMed Central

    Joshi, Swati; Satyanarayana, Tulasi

    2013-01-01

    The bulk of Earth’s biosphere is cold (<5 °C) and inhabited by psychrophiles. Biocatalysts from psychrophilic organisms (psychrozymes) have attracted attention because of their application in the ongoing efforts to decrease energy consumption. Proteinases as a class represent the largest category of industrial enzymes. There has been an emphasis on employing cold-active proteases in detergents because this allows laundry operations at ambient temperatures. Proteases have been used in environmental bioremediation, food industry and molecular biology. In view of the present limited understanding and availability of cold-active proteases with diverse characteristics, it is essential to explore Earth’s surface more in search of an ideal cold-active protease. The understanding of molecular and mechanistic details of these proteases will open up new avenues to tailor proteases with the desired properties. A detailed account of the developments in the production and applications of cold-active proteases is presented in this review. PMID:24832807

  7. Retrovirus Entry by Endocytosis and Cathepsin Proteases

    PubMed Central

    Kubo, Yoshinao; Hayashi, Hideki; Matsuyama, Toshifumi; Sato, Hironori; Yamamoto, Naoki

    2012-01-01

    Retroviruses include infectious agents inducing severe diseases in humans and animals. In addition, retroviruses are widely used as tools to transfer genes of interest to target cells. Understanding the entry mechanism of retroviruses contributes to developments of novel therapeutic approaches against retrovirus-induced diseases and efficient exploitation of retroviral vectors. Entry of enveloped viruses into host cell cytoplasm is achieved by fusion between the viral envelope and host cell membranes at either the cell surface or intracellular vesicles. Many animal retroviruses enter host cells through endosomes and require endosome acidification. Ecotropic murine leukemia virus entry requires cathepsin proteases activated by the endosome acidification. CD4-dependent human immunodeficiency virus (HIV) infection is thought to occur via endosomes, but endosome acidification is not necessary for the entry whereas entry of CD4-independent HIVs, which are thought to be prototypes of CD4-dependent viruses, is low pH dependent. There are several controversial results on the retroviral entry pathways. Because endocytosis and endosome acidification are complicatedly controlled by cellular mechanisms, the retrovirus entry pathways may be different in different cell lines. PMID:23304142

  8. Proteases in agricultural dust induce lung inflammation through PAR-1 and PAR-2 activation.

    PubMed

    Romberger, Debra J; Heires, Art J; Nordgren, Tara M; Souder, Chelsea P; West, William; Liu, Xiang-de; Poole, Jill A; Toews, Myron L; Wyatt, Todd A

    2015-08-15

    Workers exposed to aerosolized dust present in concentrated animal feeding operations (CAFOs) are susceptible to inflammatory lung diseases, such as chronic obstructive pulmonary disease. Extracts of dust collected from hog CAFOs [hog dust extract (HDE)] are potent stimulators of lung inflammatory responses in several model systems. The observation that HDE contains active proteases prompted the present study, which evaluated the role of CAFO dust proteases in lung inflammatory processes and tested whether protease-activated receptors (PARs) are involved in the signaling pathway for these events. We hypothesized that the damaging proinflammatory effect of HDE is due, in part, to the proteolytic activation of PARs, and inhibiting the proteases in HDE or disrupting PAR activation would attenuate HDE-mediated inflammatory indexes in bronchial epithelial cells (BECs), in mouse lung slices in vitro, and in a murine in vivo exposure model. Human BECs and mouse lung slice cultures stimulated with 5% HDE released significantly more of each of the cytokines measured (IL-6, IL-8, TNF-α, keratinocyte-derived chemokine/CXC chemokine ligand 1, and macrophage inflammatory protein-2/CXC chemokine ligand 2) than controls, and these effects were markedly diminished by protease inhibition. Inhibition of PARs also blunted the HDE-induced cytokine release from BECs. In addition, protease depletion inhibited HDE-induced BEC intracellular PKCα and PKCε activation. C57BL/6J mice administered 12.5% HDE intranasally, either once or daily for 3 wk, exhibited increased total cellular and neutrophil influx, bronchial alveolar fluid inflammatory cytokines, lung histopathology, and inflammatory scores compared with mice receiving protease-depleted HDE. These data suggest that proteases in dust from CAFOs are important mediators of lung inflammation, and these proteases and their receptors may provide novel targets for therapeutic intervention in CAFO dust-induced airways disease. PMID

  9. Rubus idaeus L. reverses epithelial-to-mesenchymal transition and suppresses cell invasion and protease activities by targeting ERK1/2 and FAK pathways in human lung cancer cells.

    PubMed

    Hsieh, Yih-Shou; Chu, Shu-Chen; Hsu, Li-Sung; Chen, Kuo-Shuen; Lai, Ming-Tsung; Yeh, Chia-Heng; Chen, Pei-Ni

    2013-12-01

    Epithelial to mesenchymal transition (EMT) has been considered essential for cancer metastasis, a multistep complicated process including local invasion, intravasation, extravasation, and proliferation at distant sites. Herein we provided molecular evidence associated with the antimetastatic effect of Rubus idaeus L. extracts (RIE) by showing a nearly complete inhibition on the invasion (p<0.001) of highly metastatic A549 cells via reduced activities of matrix metalloproteinase-2 (MMP-2) and urokinasetype plasminogen activator (u-PA). We performed Western blot to find that RIE could induce up-regulation of epithelial marker such as E-cadherin and α-catenin and inhibit the mesenchymal markers such as N-cadherin, fibronectin, snail-1, and vimentin. Selective snail-1 inhibition by snail-1-specific-siRNA also showed increased E-cadherin expression in A549 cells suggesting a possible involvement of snail-1 inhibition in RIE-caused increase in E-cadherin level. RIE also inhibited p-FAK, p-paxillin and AP-1 by Western blot analysis, indicating the anti-EMT effect of RIE in human lung carcinoma. Importantly, an in vivo BALB/c nude mice xenograft model showed that RIE treatment reduced tumor growth by oral gavage, and RIE represent promising candidates for future phytochemical-based mechanistic pathway-targeted cancer prevention strategies. PMID:24161487

  10. Secretory Leukocyte Protease Inhibitor Suppresses the Inflammation and Joint Damage of Bacterial Cell Wall–Induced Arthritis

    PubMed Central

    Song, Xiao-yu; Zeng, Li; Jin, Wenwen; Thompson, John; Mizel, Diane E.; Lei, Ke-jian; Billinghurst, R.C.; Poole, A. Robin; Wahl, Sharon M.

    1999-01-01

    Disruption of the balance between proteases and protease inhibitors is often associated with pathologic tissue destruction. To explore the therapeutic potential of secretory leukocyte protease inhibitor (SLPI) in erosive joint diseases, we cloned, sequenced, and expressed active rat SLPI, which shares the protease-reactive site found in human SLPI. In a rat streptococcal cell wall (SCW)-induced model of inflammatory erosive polyarthritis, endogenous SLPI was unexpectedly upregulated at both mRNA and protein levels in inflamed joint tissues. Systemic delivery of purified recombinant rat SLPI inhibited joint inflammation and cartilage and bone destruction. Inflammatory pathways as reflected by circulating tumor necrosis factor α and nuclear factor κB activation and cartilage resorption detected by circulating levels of type II collagen collagenase-generated cleavage products were all diminished by SLPI treatment in acute and chronic arthritis, indicating that the action of SLPI may extend beyond inhibition of serine proteases. PMID:10449524

  11. ATP-dependent incorporation of 20S protease into the 26S complex that degrades proteins conjugated to ubiquitin.

    PubMed Central

    Eytan, E; Ganoth, D; Armon, T; Hershko, A

    1989-01-01

    Previous studies have indicated that the ATP-dependent 26S protease complex that degrades proteins conjugated to ubiquitin is formed by the assembly of three factors in an ATP-requiring process. We now identify one of the factors as the 20S "multicatalytic" protease, a complex of low molecular weight subunits widely distributed in eukaryotic cells. Comparison of the subunit compositions of purified 20S and 26S complexes indicates that the former is an integral part of the latter. By the use of detergent treatment to activate latent protease activity, we show that the 20S protease becomes incorporated into the 26S complex in the ATP-dependent assembly process. It thus seems that the 20S protease is the "catalytic core" of the 26S complex of the ubiquitin proteolytic pathway. Images PMID:2554287

  12. Anti-Severe Acute Respiratory Syndrome Coronavirus Spike Antibodies Trigger Infection of Human Immune Cells via a pH- and Cysteine Protease-Independent FcγR Pathway

    PubMed Central

    Jaume, Martial; Yip, Ming S.; Cheung, Chung Y.; Leung, Hiu L.; Li, Ping H.; Kien, Francois; Dutry, Isabelle; Callendret, Benoît; Escriou, Nicolas; Altmeyer, Ralf; Nal, Beatrice; Daëron, Marc; Bruzzone, Roberto; Peiris, J. S. Malik

    2011-01-01

    Public health measures successfully contained outbreaks of the severe acute respiratory syndrome coronavirus (SARS-CoV) infection. However, the precursor of the SARS-CoV remains in its natural bat reservoir, and reemergence of a human-adapted SARS-like coronavirus remains a plausible public health concern. Vaccination is a major strategy for containing resurgence of SARS in humans, and a number of vaccine candidates have been tested in experimental animal models. We previously reported that antibody elicited by a SARS-CoV vaccine candidate based on recombinant full-length Spike-protein trimers potentiated infection of human B cell lines despite eliciting in vivo a neutralizing and protective immune response in rodents. These observations prompted us to investigate the mechanisms underlying antibody-dependent enhancement (ADE) of SARS-CoV infection in vitro. We demonstrate here that anti-Spike immune serum, while inhibiting viral entry in a permissive cell line, potentiated infection of immune cells by SARS-CoV Spike-pseudotyped lentiviral particles, as well as replication-competent SARS coronavirus. Antibody-mediated infection was dependent on Fcγ receptor II but did not use the endosomal/lysosomal pathway utilized by angiotensin I converting enzyme 2 (ACE2), the accepted receptor for SARS-CoV. This suggests that ADE of SARS-CoV utilizes a novel cell entry mechanism into immune cells. Different SARS vaccine candidates elicit sera that differ in their capacity to induce ADE in immune cells despite their comparable potency to neutralize infection in ACE2-bearing cells. Our results suggest a novel mechanism by which SARS-CoV can enter target cells and illustrate the potential pitfalls associated with immunization against it. These findings should prompt further investigations into SARS pathogenesis. PMID:21775467

  13. Molecular Imaging of Proteases in Cancer

    PubMed Central

    Yang, Yunan; Hong, Hao; Zhang, Yin; Cai, Weibo

    2010-01-01

    Proteases play important roles during tumor angiogenesis, invasion, and metastasis. Various molecular imaging techniques have been employed for protease imaging: optical (both fluorescence and bioluminescence), magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), and positron emission tomography (PET). In this review, we will summarize the current status of imaging proteases in cancer with these techniques. Optical imaging of proteases, in particular with fluorescence, is the most intensively validated and many of the imaging probes are already commercially available. It is generally agreed that the use of activatable probes is the most accurate and appropriate means for measuring protease activity. Molecular imaging of proteases with other techniques (i.e. MRI, SPECT, and PET) has not been well-documented in the literature which certainly deserves much future effort. Optical imaging and molecular MRI of protease activity has very limited potential for clinical investigation. PET/SPECT imaging is suitable for clinical investigation; however the optimal probes for PET/SPECT imaging of proteases in cancer have yet to be developed. Successful development of protease imaging probes with optimal in vivo stability, tumor targeting efficacy, and desirable pharmacokinetics for clinical translation will eventually improve cancer patient management. Not limited to cancer, these protease-targeted imaging probes will also have broad applications in other diseases such as arthritis, atherosclerosis, and myocardial infarction. PMID:20234801

  14. The effect of environmental conditions on expression of Bacteroides fragilis and Bacteroides thetaiotaomicron C10 protease genes

    PubMed Central

    2012-01-01

    Background Bacteroides fragilis and Bacteroides thetaiotaomicron are members of the normal human intestinal microbiota. However, both organisms are capable of causing opportunistic infections, during which the environmental conditions to which the bacteria are exposed change dramatically. To further explore their potential for contributing to infection, we have characterized the expression in B. thetaiotaomicron of four homologues of the gene encoding the C10 cysteine protease SpeB, a potent extracellular virulence factor produced by Streptococcus pyogenes. Results We identified a paralogous set of genes (btp genes) in the B. thetaiotaomicron genome, that were related to C10 protease genes we recently identified in B. fragilis. Similar to C10 proteases found in B. fragilis, three of the B. thetaiotaomicron homologues were transcriptionally coupled to genes encoding small proteins that are similar in structural architecture to Staphostatins, protease inhibitors associated with Staphopains in Staphylococcus aureus. The expression of genes for these C10 proteases in both B. fragilis and B. thetaiotaomicron was found to be regulated by environmental stimuli, in particular by exposure to oxygen, which may be important for their contribution to the development of opportunistic infections. Conclusions Genes encoding C10 proteases are increasingly identified in operons which also contain genes encoding proteins homologous to protease inhibitors. The Bacteroides C10 protease gene expression levels are responsive to different environmental stimuli suggesting they may have distinct roles in the bacterial-host interaction. PMID:22943521

  15. Advances in protease engineering for laundry detergents.

    PubMed

    Vojcic, Ljubica; Pitzler, Christian; Körfer, Georgette; Jakob, Felix; Ronny Martinez; Maurer, Karl-Heinz; Schwaneberg, Ulrich

    2015-12-25

    Proteases are essential ingredients in modern laundry detergents. Over the past 30 years, subtilisin proteases employed in the laundry detergent industry have been engineered by directed evolution and rational design to tailor their properties towards industrial demands. This comprehensive review discusses recent success stories in subtilisin protease engineering. Advances in protease engineering for laundry detergents comprise simultaneous improvement of thermal resistance and activity at low temperatures, a rational strategy to modulate pH profiles, and a general hypothesis for how to increase promiscuous activity towards the production of peroxycarboxylic acids as mild bleaching agents. The three protease engineering campaigns presented provide in-depth analysis of protease properties and have identified principles that can be applied to improve or generate enzyme variants for industrial applications beyond laundry detergents. PMID:25579194

  16. Extracellular proteases as targets for drug development.

    PubMed

    Cudic, Mare; Fields, Gregg B

    2009-08-01

    Proteases constitute one of the primary targets in drug discovery. In the present review, we focus on extracellular proteases (ECPs) because of their differential expression in many pathophysiological processes, including cancer, cardiovascular conditions, and inflammatory, pulmonary, and periodontal diseases. Many new ECP inhibitors are currently under clinical investigation and a significant increase in new therapies based on protease inhibition can be expected in the coming years. In addition to directly blocking the activity of a targeted protease, one can take advantage of differential expression in disease states to selectively deliver therapeutic or imaging agents. Recent studies in targeted drug development for the metalloproteases (matrix metalloproteinases, adamalysins, pappalysins, neprilysin, angiotensin-converting enzyme, metallocarboxypeptidases, and glutamate carboxypeptidase II), serine proteases (elastase, coagulation factors, tissue/urokinase plasminogen activator system, kallikreins, tryptase, dipeptidyl peptidase IV) and cysteine proteases (cathepsin B) are discussed herein. PMID:19689354

  17. Type-I Prenyl Protease Function Is Required in the Male Germline of Drosophila melanogaster

    PubMed Central

    Adolphsen, Katie; Amell, Amanda; Havko, Nathan; Kevorkian, Sara; Mears, Kyle; Neher, Hayley; Schwarz, Dietmar; Schulze, Sandra R.

    2012-01-01

    Many proteins require the addition of a hydrophobic prenyl anchor (prenylation) for proper trafficking and localization in the cell. Prenyl proteases play critical roles in modifying proteins for membrane anchorage. The type I prenyl protease has a defined function in yeast (Ste24p/Afc1p) where it modifies a mating pheromone, and in humans (Zmpste24) where it has been implicated in a disease of premature aging. Despite these apparently very different biological processes, the type I prenyl protease gene is highly conserved, encoded by a single gene in a wide range of animal and plant groups. A notable exception is Drosophila melanogaster, where the gene encoding the type I prenyl protease has undergone an unprecedented series of duplications in the genome, resulting in five distinct paralogs, three of which are organized in a tandem array, and demonstrate high conservation, particularly in the vicinity of the active site of the enzyme. We have undertaken targeted deletion to remove the three tandem paralogs from the genome. The result is a male fertility defect, manifesting late in spermatogenesis. Our results also show that the ancestral type I prenyl protease gene in Drosophila is under strong purifying selection, while the more recent replicates are evolving rapidly. Our rescue data support a role for the rapidly evolving tandem paralogs in the male germline. We propose that potential targets for the male-specific type I prenyl proteases include proteins involved in the very dramatic cytoskeletal remodeling events required for spermatid maturation. PMID:22690372

  18. Proteases in biological control and biotechnology

    SciTech Connect

    Cunningham, D.D.; Long, G.L.

    1987-01-01

    This book explores the role of proteases in biological control systems and diseases, examines their structures and evolution, and reviews the methods by which proteases and protease inhibitors are engineered. In addition, the use of recombinant DNA technology is explained throughout the volume. Specific topics examined include: the versatility of proteolytic enzymes, the intricate proteolytic control mechanisms in hemostasis and their application to thrombolytic therapy, the evolution of proteolytic enzymes, and the role of limited proteolytic processing in several biological control processes.

  19. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, David B.; Lao, Guifang

    1998-01-01

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium.

  20. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, D.B.; Lao, G.

    1998-01-06

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium. 3 figs.

  1. PARP9-DTX3L ubiquitin ligase targets host histone H2BJ and viral 3C protease to enhance interferon signaling and control viral infection

    PubMed Central

    Zhang, Yong; Mao, Dailing; Roswit, William T.; Jin, Xiaohua; Patel, Anand C.; Patel, Dhara A.; Agapov, Eugene; Wang, Zhepeng; Tidwell, Rose M.; Atkinson, Jeffrey J.; Huang, Guangming; McCarthy, Ronald; Yu, Jinsheng; Yun, Nadezhda E.; Paessler, Slobodan; Lawson, T. Glen; Omattage, Natalie S.; Brett, Tom J.; Holtzman, Michael J.

    2015-01-01

    Enhancing the response to interferon could offer an immunological advantage to the host. In support of this concept, we used a modified form of the transcription factor STAT1 to achieve interferon hyperresponsiveness without toxicity and markedly improve antiviral function in transgenic mice and transduced human cells. We found that the improvement depends on expression of a PARP9-DTX3L complex with distinct domains for interaction with STAT1 and for activity as an E3 ubiquitin ligase that acts on host histone H2BJ to promote interferon-stimulated gene expression and on viral 3C proteases to initiate their degradation via the immunoproteasome. Together, PARP9-DTX3L acts on host and pathogen to achieve a double layer of immunity within a safe reserve in the interferon signaling pathway. PMID:26479788

  2. Proteolytic crosstalk in multi-protease networks

    NASA Astrophysics Data System (ADS)

    Ogle, Curtis T.; Mather, William H.

    2016-04-01

    Processive proteases, such as ClpXP in E. coli, are conserved enzyme assemblies that can recognize and rapidly degrade proteins. These proteases are used for a number of purposes, including degrading mistranslated proteins and controlling cellular stress response. However, proteolytic machinery within the cell is limited in capacity and can lead to a bottleneck in protein degradation, whereby many proteins compete (‘queue’) for proteolytic resources. Previous work has demonstrated that such queueing can lead to pronounced statistical relationships between different protein counts when proteins compete for a single common protease. However, real cells contain many different proteases, e.g. ClpXP, ClpAP, and Lon in E. coli, and it is not clear how competition between proteins for multiple classes of protease would influence the dynamics of cellular networks. In the present work, we theoretically demonstrate that a multi-protease proteolytic bottleneck can substantially couple the dynamics for both simple and complex (oscillatory) networks, even between substrates with substantially different affinities for protease. For these networks, queueing often leads to strong positive correlations between protein counts, and these correlations are strongest near the queueing theoretic point of balance. Furthermore, we find that the qualitative behavior of these networks depends on the relative size of the absolute affinity of substrate to protease compared to the cross affinity of substrate to protease, leading in certain regimes to priority queue statistics.

  3. Proteolytic crosstalk in multi-protease networks.

    PubMed

    Ogle, Curtis T; Mather, William H

    2016-01-01

    Processive proteases, such as ClpXP in E. coli, are conserved enzyme assemblies that can recognize and rapidly degrade proteins. These proteases are used for a number of purposes, including degrading mistranslated proteins and controlling cellular stress response. However, proteolytic machinery within the cell is limited in capacity and can lead to a bottleneck in protein degradation, whereby many proteins compete ('queue') for proteolytic resources. Previous work has demonstrated that such queueing can lead to pronounced statistical relationships between different protein counts when proteins compete for a single common protease. However, real cells contain many different proteases, e.g. ClpXP, ClpAP, and Lon in E. coli, and it is not clear how competition between proteins for multiple classes of protease would influence the dynamics of cellular networks. In the present work, we theoretically demonstrate that a multi-protease proteolytic bottleneck can substantially couple the dynamics for both simple and complex (oscillatory) networks, even between substrates with substantially different affinities for protease. For these networks, queueing often leads to strong positive correlations between protein counts, and these correlations are strongest near the queueing theoretic point of balance. Furthermore, we find that the qualitative behavior of these networks depends on the relative size of the absolute affinity of substrate to protease compared to the cross affinity of substrate to protease, leading in certain regimes to priority queue statistics. PMID:27042892

  4. Proteases from Canavalia ensiformis: Active and Thermostable Enzymes with Potential of Application in Biotechnology

    PubMed Central

    Gonçalves, Rayane Natshe; Gozzini Barbosa, Suellen Duarte

    2016-01-01

    Extracts of leaves, seeds, roots, and stem from a tropical legume, C. ensiformis, were prepared employing buffers and detergent in aqueous solution. Leaf extracts had the highest protein content and the most pronounced peptidase activity with optimal pH in the neutral to alkaline range. All extracts exhibited peaks of activity at various pH values, suggesting the presence of distinctive classes of proteases. N-α-Tosyl-L-arginine methyl ester hydrolysis was maximal at 30°C to 60°C and peptidase activity from all extracts presented very good thermal stability after 24 h incubation at 70°C. C. ensiformis proteases exhibited molecular masses of about 200–57, 40–37, and 20–15 kDa by SDS-PAGE analysis. These enzymes cleaved hemoglobin, bovine serum albumin, casein, and gelatin at different levels. Serine and metalloproteases are the major proteases in C. ensiformis extracts, modulated by divalent cations, stable at 1% of surfactant Triton X-100 and at different concentrations of the reducing agent β-mercaptoethanol. Thus, C. ensiformis expresses a particular set of proteases in distinctive organs with high activity and stability, making this legume an important source of proteases with biotechnological potential.

  5. Phytaspase, a relocalisable cell death promoting plant protease with caspase specificity

    PubMed Central

    Chichkova, Nina V; Shaw, Jane; Galiullina, Raisa A; Drury, Georgina E; Tuzhikov, Alexander I; Kim, Sang Hyon; Kalkum, Markus; Hong, Teresa B; Gorshkova, Elena N; Torrance, Lesley; Vartapetian, Andrey B; Taliansky, Michael

    2010-01-01

    Caspases are cysteine-dependent proteases and are important components of animal apoptosis. They introduce specific breaks after aspartate residues in a number of cellular proteins mediating programmed cell death (PCD). Plants encode only distant homologues of caspases, the metacaspases that are involved in PCD, but do not possess caspase-specific proteolytic activity. Nevertheless, plants do display caspase-like activities indicating that enzymes structurally distinct from classical caspases may operate as caspase-like proteases. Here, we report the identification and characterisation of a novel PCD-related subtilisin-like protease from tobacco and rice named phytaspase (plant aspartate-specific protease) that possesses caspase specificity distinct from that of other known caspase-like proteases. We provide evidence that phytaspase is synthesised as a proenzyme, which is autocatalytically processed to generate the mature enzyme. Overexpression and silencing of the phytaspase gene showed that phytaspase is essential for PCD-related responses to tobacco mosaic virus and abiotic stresses. Phytaspase is constitutively secreted into the apoplast before PCD, but unexpectedly is re-imported into the cell during PCD providing insights into how phytaspase operates. PMID:20111004

  6. Structural Evidence for Regulation and Specificity of Flaviviral Proteases and Evolution of the Flaviviridae Fold

    SciTech Connect

    Aleshin,A.; Shiryaev, S.; Strongin, A.; Liddington, R.

    2007-01-01

    Pathogenic members of the flavivirus family, including West Nile Virus (WNV) and Dengue Virus (DV), are growing global threats for which there are no specific treatments. The two-component flaviviral enzyme NS2B-NS3 cleaves the viral polyprotein precursor within the host cell, a process that is required for viral replication. Here, we report the crystal structure of WNV NS2B-NS3pro both in a substrate-free form and in complex with the trypsin inhibitor aprotinin/BPTI. We show that aprotinin binds in a substrate-mimetic fashion in which the productive conformation of the protease is fully formed, providing evidence for an 'induced fit' mechanism of catalysis and allowing us to rationalize the distinct substrate specificities of WNV and DV proteases. We also show that the NS2B cofactor of WNV can adopt two very distinct conformations and that this is likely to be a general feature of flaviviral proteases, providing further opportunities for regulation. Finally, by comparing the flaviviral proteases with the more distantly related Hepatitis C virus, we provide insights into the evolution of the Flaviviridae fold. Our work should expedite the design of protease inhibitors to treat a range of flaviviral infections.

  7. Identification of novel secreted proteases during extracellular proteolysis by dermatophytes at acidic pH.

    PubMed

    Sriranganadane, Dev; Waridel, Patrice; Salamin, Karine; Feuermann, Marc; Mignon, Bernard; Staib, Peter; Neuhaus, Jean-Marc; Quadroni, Manfredo; Monod, Michel

    2011-11-01

    The dermatophytes are a group of closely related fungi which are responsible for the great majority of superficial mycoses in humans and animals. Among various potential virulence factors, their secreted proteolytic activity attracts a lot of attention. Most dermatophyte-secreted proteases which have so far been isolated in vitro are neutral or alkaline enzymes. However, inspection of the recently decoded dermatophyte genomes revealed many other hypothetical secreted proteases, in particular acidic proteases similar to those characterized in Aspergillus spp. The validation of such genome predictions instigated the present study on two dermatophyte species, Microsporum canis and Arthroderma benhamiae. Both fungi were found to grow well in a protein medium at acidic pH, accompanied by extracellular proteolysis. Shotgun MS analysis of secreted protein revealed fundamentally different protease profiles during fungal growth in acidic versus neutral pH conditions. Most notably, novel dermatophyte-secreted proteases were identified at acidic pH such as pepsins, sedolisins and acidic carboxypeptidases. Therefore, our results not only support genome predictions, but demonstrate for the first time the secretion of acidic proteases by dermatophytes. Our findings also suggest the existence of different pathways of protein degradation into amino acids and short peptides in these highly specialized pathogenic fungi. PMID:21919205

  8. Distinct cathepsins control necrotic cell death mediated by pyroptosis inducers and lysosome-destabilizing agents.

    PubMed

    Brojatsch, Jürgen; Lima, Heriberto; Palliser, Deborah; Jacobson, Lee S; Muehlbauer, Stefan M; Furtado, Raquel; Goldman, David L; Lisanti, Michael P; Chandran, Kartik

    2015-01-01

    Necrotic cell death triggers a range of biological responses including a strong adaptive immune response, yet we know little about the cellular pathways that control necrotic cell death. Inhibitor studies suggest that proteases, and in particular cathepsins, drive necrotic cell death. The cathepsin B-selective inhibitor CA-074-Me blocks all forms of programmed necrosis by an unknown mechanism. We found that cathepsin B deficiency does not prevent induction of pyroptosis and lysosome-mediated necrosis suggesting that CA-074-Me blocks necrotic cell death by targeting cathepsins other than cathepsin B. A single cathepsin, cathepsin C, drives necrotic cell death mediated by the lysosome-destabilizing agent Leu-Leu-OMe (LLOMe). Here we present evidence that cathepsin C-deficiency and CA-074-Me block LLOMe killing in a distinct and cell type-specific fashion. Cathepsin C-deficiency and CA-074-Me block LLOMe killing of all myeloid cells, except for neutrophils. Cathepsin C-deficiency, but not CA-074-Me, blocks LLOMe killing of neutrophils suggesting that CA-074-Me does not target cathepsin C directly, consistent with inhibitor studies using recombinant cathepsin C. Unlike other cathepsins, cathepsin C lacks endoproteolytic activity, and requires activation by other lysosomal proteases, such as cathepsin D. Consistent with this theory, we found that lysosomotropic agents and cathepsin D downregulation by siRNA block LLOMe-mediated necrosis. Our findings indicate that a proteolytic cascade, involving cathepsins C and D, controls LLOMe-mediated necrosis. In contrast, cathepsins C and D were not required for pyroptotic cell death suggesting that distinct cathepsins control pyroptosis and lysosome-mediated necrosis. PMID:25830414

  9. Organ Length Control by an ADAMTS Extracellular Protease in Caenorhabditis elegans.

    PubMed

    Shibata, Yukimasa; Kawakado, Yuri; Hori, Noriyoshi; Tanaka, Kota; Inoue, Ryo; Takano, Tomomi; Kubota, Yukihiko; Nishiwaki, Kiyoji

    2016-01-01

    MIG-17, a secreted protease of the ADAMTS family, acts in the directed migration of gonadal distal tip cells (DTCs) through regulation of the gonadal basement membrane in Caenorhabditis elegans Here, we show that MIG-17 is also required for the control of pharynx elongation during animal growth. We found that the pharynx was elongated in mig-17 mutants compared with wild type. MIG-17 localized to the pharyngeal basement membrane as well as to the gonadal basement membrane. The number of nuclei in the pharynx, and the pumping rate of the pharynx, were not affected in mig-17 mutants, suggesting that cells constituting the pharynx are elongated, although the pharynx functions normally in these mutants. In contrast to the control of DTC migration, MIG-18, a secreted cofactor of MIG-17, was not essential for pharynx length regulation. In addition, the downstream pathways of MIG-17 involving LET-2/type IV collagen, FBL-1/fibulin-1, and NID-1/nidogen, partly diverged from those in gonad development. These results indicate that basement membrane remodeling is important for organ length regulation, and suggest that MIG-17/ADAMTS functions in similar but distinct molecular machineries in pharyngeal and gonadal basement membranes. PMID:26994289

  10. Organ Length Control by an ADAMTS Extracellular Protease in Caenorhabditis elegans

    PubMed Central

    Shibata, Yukimasa; Kawakado, Yuri; Hori, Noriyoshi; Tanaka, Kota; Inoue, Ryo; Takano, Tomomi; Kubota, Yukihiko; Nishiwaki, Kiyoji

    2016-01-01

    MIG-17, a secreted protease of the ADAMTS family, acts in the directed migration of gonadal distal tip cells (DTCs) through regulation of the gonadal basement membrane in Caenorhabditis elegans. Here, we show that MIG-17 is also required for the control of pharynx elongation during animal growth. We found that the pharynx was elongated in mig-17 mutants compared with wild type. MIG-17 localized to the pharyngeal basement membrane as well as to the gonadal basement membrane. The number of nuclei in the pharynx, and the pumping rate of the pharynx, were not affected in mig-17 mutants, suggesting that cells constituting the pharynx are elongated, although the pharynx functions normally in these mutants. In contrast to the control of DTC migration, MIG-18, a secreted cofactor of MIG-17, was not essential for pharynx length regulation. In addition, the downstream pathways of MIG-17 involving LET-2/type IV collagen, FBL-1/fibulin-1, and NID-1/nidogen, partly diverged from those in gonad development. These results indicate that basement membrane remodeling is important for organ length regulation, and suggest that MIG-17/ADAMTS functions in similar but distinct molecular machineries in pharyngeal and gonadal basement membranes. PMID:26994289

  11. Tissue dissociation enzyme neutral protease assessment.

    PubMed

    Breite, A G; Dwulet, F E; McCarthy, R C

    2010-01-01

    Neutral proteases, essential components of purified tissue dissociation enzymes required for successful human islet isolation, show variable activities and effects of substrate on their activities. Initially we used a spectrophotometric endpoint assay with azocasein substrate to measure neutral protease activity. After critical review of the results, we observed these data to be inconsistent and not correlating expected differences in specific activities between thermolysin and Bacillus polymyxa proteases. This observation led to the development of a fluorescent microplate assay using fluorescein isothyocyanate-conjugated bovine serum albumin (FITC-BSA) as the substrate. This simpler, more flexible method offered a homogeneous, kinetic enzyme assay allowing determination of steady state reaction rates of sample replicates at various dilutions. The assay had a linear range of 4- to 8-fold and interassay coefficients of variation for B polymyxa protease and thermolysin of <9% and <15%, respectively, which were lower than those using the spectrophotometric endpoint assay, namely, 54% and 36%, respectively. This format allowed for incorporation of enzyme inhibitors, as illustrated by addition of sulfhydryl protease inhibitors, which, consistent with earlier reports, strongly indicated that the main contaminant in purified collagenase preparations was clostripain. Determination of the specific activities for several purified neutral proteases showed that the B polymyxa and Clostridium histolyticum proteases had approximately 40% and 15% specific activities, respectively, of those obtained with purified thermolysin, indicating the different characteristics of neutral protease enzymes for cell isolation procedures. PMID:20692405

  12. The serine protease inhibitor protease nexin-1 controls mammary cancer metastasis through LRP-1-mediated MMP-9 expression.

    PubMed

    Fayard, Bérengère; Bianchi, Fabrizio; Dey, Julien; Moreno, Eliza; Djaffer, Sabrina; Hynes, Nancy E; Monard, Denis

    2009-07-15

    Through their ability to degrade the extracellular matrix, proteases mediate cancer cell invasion and metastasis. Paradoxically, some serine protease inhibitors (serpins) are often overexpressed in human tumors. Using computational analysis, we found that the RNA level of protease nexin-1 (PN-1), a serpin that blocks numerous proteases activity, is significantly elevated in estrogen receptor-alpha-negative and in high-grade breast cancer. The in silico approach was complemented by mechanistic studies on two mammary cancer cell lines, the PN-1-negative 168FARN cells and the PN-1-positive 4T1 cells, both of which form primary mammary tumors, but only 4T1 tumors are able to metastasize to the lungs. We show that treatment of 168FARN cells with PN-1 stimulates extracellular signal-regulated kinase activation via low-density lipoprotein receptor-related protein-1 (LRP-1) binding, resulting in increased matrix metalloproteinase (MMP)-9 RNA, protein, and secreted activity. PN-1-silenced 4T1 cells express low MMP-9 levels. Moreover, injection of PN-1-silenced cells into mice did not affect 4T1 primary mammary tumor outgrowth; however, the tumors had impaired metastatic potential, which could be restored by reexpressing soluble MMP-9 in the PN-1-silenced 4T1 cells. Thus, using mammary tumor models, we describe a novel pathway whereby the serpin PN-1 by binding LRP-1 stimulates extracellular signal-regulated kinase signaling, MMP-9 expression, and metastatic spread of mammary tumors. Importantly, an analysis of 126 breast cancer patients revealed that those whose breast tumors had elevated PN-1 levels had a significantly higher probability to develop lung metastasis, but not metastasis to other sites, on relapse. These results suggest that PN-1 might become a prognostic marker in breast cancer. PMID:19584287

  13. Protease-degradable electrospun fibrous hydrogels

    NASA Astrophysics Data System (ADS)

    Wade, Ryan J.; Bassin, Ethan J.; Rodell, Christopher B.; Burdick, Jason A.

    2015-03-01

    Electrospun nanofibres are promising in biomedical applications to replicate features of the natural extracellular matrix (ECM). However, nearly all electrospun scaffolds are either non-degradable or degrade hydrolytically, whereas natural ECM degrades proteolytically, often through matrix metalloproteinases. Here we synthesize reactive macromers that contain protease-cleavable and fluorescent peptides and are able to form both isotropic hydrogels and electrospun fibrous hydrogels through a photoinitiated polymerization. These biomimetic scaffolds are susceptible to protease-mediated cleavage in vitro in a protease dose-dependent manner and in vivo in a subcutaneous mouse model using transdermal fluorescent imaging to monitor degradation. Importantly, materials containing an alternate and non-protease-cleavable peptide sequence are stable in both in vitro and in vivo settings. To illustrate the specificity in degradation, scaffolds with mixed fibre populations support selective fibre degradation based on individual fibre degradability. Overall, this represents a novel biomimetic approach to generate protease-sensitive fibrous scaffolds for biomedical applications.

  14. Progress and prospects on DENV protease inhibitors.

    PubMed

    Timiri, Ajay Kumar; Sinha, Barij Nayan; Jayaprakash, Venkatesan

    2016-07-19

    New treatments are desperately required to combat increasing rate of dengue fever cases reported in tropical and sub-tropical parts of the world. Among the ten proteins (structural and non-structural) encoded by dengue viral genome, NS2B-NS3 protease is an ideal target for drug discovery. It is responsible for the processing of poly protein that is required for genome replication of the virus. Moreover, inhibitors designed against proteases were found successful in Human Immuno-deficiency Virus (HIV) and Hepatitis C Virus (HCV). Complete molecular mechanism and a survey of inhibitors reported against dengue protease will be helpful in designing effective and potent inhibitors. This review provides an insight on molecular mechanism of dengue virus protease and covers up-to-date information on different inhibitors reported against dengue proteases with medicinal chemistry perspective. PMID:27092412

  15. HIV-1 protease mutations and protease inhibitor cross-resistance.

    PubMed

    Rhee, Soo-Yon; Taylor, Jonathan; Fessel, W Jeffrey; Kaufman, David; Towner, William; Troia, Paolo; Ruane, Peter; Hellinger, James; Shirvani, Vivian; Zolopa, Andrew; Shafer, Robert W

    2010-10-01

    The effects of many protease inhibitor (PI)-selected mutations on the susceptibility to individual PIs are unknown. We analyzed in vitro susceptibility test results on 2,725 HIV-1 protease isolates. More than 2,400 isolates had been tested for susceptibility to fosamprenavir, indinavir, nelfinavir, and saquinavir; 2,130 isolates had been tested for susceptibility to lopinavir; 1,644 isolates had been tested for susceptibility to atazanavir; 1,265 isolates had been tested for susceptibility to tipranavir; and 642 isolates had been tested for susceptibility to darunavir. We applied least-angle regression (LARS) to the 200 most common mutations in the data set and identified a set of 46 mutations associated with decreased PI susceptibility of which 40 were not polymorphic in the eight most common HIV-1 group M subtypes. We then used least-squares regression to ascertain the relative contribution of each of these 46 mutations. The median number of mutations associated with decreased susceptibility to each PI was 28 (range, 19 to 32), and the median number of mutations associated with increased susceptibility to each PI was 2.5 (range, 1 to 8). Of the mutations with the greatest effect on PI susceptibility, I84AV was associated with decreased susceptibility to eight PIs; V32I, G48V, I54ALMSTV, V82F, and L90M were associated with decreased susceptibility to six to seven PIs; I47A, G48M, I50V, L76V, V82ST, and N88S were associated with decreased susceptibility to four to five PIs; and D30N, I50L, and V82AL were associated with decreased susceptibility to fewer than four PIs. This study underscores the greater impact of nonpolymorphic mutations compared with polymorphic mutations on decreased PI susceptibility and provides a comprehensive quantitative assessment of the effects of individual mutations on susceptibility to the eight clinically available PIs. PMID:20660676

  16. Characterization of human liver enzymes involved in the biotransformation of boceprevir, a hepatitis C virus protease inhibitor.

    PubMed

    Ghosal, Anima; Yuan, Yuan; Tong, Wei; Su, Ai-Duen; Gu, Chunyan; Chowdhury, Swapan K; Kishnani, Narendra S; Alton, Kevin B

    2011-03-01

    Boceprevir (SCH 503034), a protease inhibitor, is under clinical development for the treatment of human hepatitis C virus infections. In human liver microsomes, formation of oxidative metabolites after incubations with [(14)C]boceprevir was catalyzed by CYP3A4 and CYP3A5. In addition, the highest turnover was observed in recombinant CYP3A4 and CYP3A5. After a single radiolabeled dose to human, boceprevir was subjected to two distinct pathways, namely cytochrome P450-mediated oxidation and ketone reduction. Therefore, attempts were made to identify the enzymes responsible for the formation of carbonyl-reduced metabolites. Human liver S9 and cytosol converted ∼ 28 and ∼ 68% of boceprevir to M28, respectively, in the presence of an NADPH-generating system. Screening of boceprevir with recombinant human aldo-keto reductases (AKRs) revealed that AKR1C2 and AKR1C3 exhibited catalytic activity with respect to the formation of M+2 metabolites (M28 and M31). The formation of M28 was inhibited by 100 μM flufenamic acid (80.3%), 200 μM mefenamic acid (83.7%), and 100 μM phenolphthalein (86.1%), known inhibitors of AKRs, suggesting its formation through carbonyl reduction pathway. Formation of M28 was also inhibited by 100 μM diazepam (75.1%), 1 mM ibuprofen (70%), and 200 μM diflunisal (89.4%). These data demonstrated that CYP3A4 and CYP3A5 are primarily responsible for the formation of oxidative metabolites and the formation of M28 and M31, the keto-reduced metabolites, are most likely mediated by AKR1C2 and AKR1C3. Because the biotransformation and clearance of boceprevir involves two different enzymatic pathways, boceprevir is less likely to be a victim of significant drug-drug interaction with concomitant medication affecting either of these pathways. PMID:21123164

  17. Drought and flooding have distinct effects on herbivore-induced responses and resistance in Solanum dulcamara.

    PubMed

    Nguyen, Duy; D'Agostino, Nunzio; Tytgat, Tom O G; Sun, Pulu; Lortzing, Tobias; Visser, Eric J W; Cristescu, Simona M; Steppuhn, Anke; Mariani, Celestina; van Dam, Nicole M; Rieu, Ivo

    2016-07-01

    In the field, biotic and abiotic stresses frequently co-occur. As a consequence, common molecular signalling pathways governing adaptive responses to individual stresses can interact, resulting in compromised phenotypes. How plant signalling pathways interact under combined stresses is poorly understood. To assess this, we studied the consequence of drought and soil flooding on resistance of Solanum dulcamara to Spodoptera exigua and their effects on hormonal and transcriptomic profiles. The results showed that S. exigua larvae performed less well on drought-stressed plants than on well-watered and flooded plants. Both drought and insect feeding increased abscisic acid and jasmonic acid (JA) levels, whereas flooding did not induce JA accumulation. RNA sequencing analyses corroborated this pattern: drought and herbivory induced many biological processes that were repressed by flooding. When applied in combination, drought and herbivory had an additive effect on specific processes involved in secondary metabolism and defence responses, including protease inhibitor activity. In conclusion, drought and flooding have distinct effects on herbivore-induced responses and resistance. Especially, the interaction between abscisic acid and JA signalling may be important to optimize plant responses to combined drought and insect herbivory, making drought-stressed plants more resistant to insects than well-watered and flooded plants. PMID:26759219

  18. Extracellular proteases of Trichoderma species. A review.

    PubMed

    Kredics, L; Antal, Zsuzsanna; Szekeres, A; Hatvani, L; Manczinger, L; Vágvölgyi, Cs; Nagy, Erzsébet

    2005-01-01

    Cellulolytic, xylanolytic, chitinolytic and beta-1,3-glucanolytic enzyme systems of species belonging to the filamentous fungal genus Trichoderma have been investigated in details and are well characterised. The ability of Trichoderma strains to produce extracellular proteases has also been known for a long time, however, the proteolytic enzyme system is relatively unknown in this genus. Fortunately, in the recent years more and more attention is focused on the research in this field. The role of Trichoderma proteases in the biological control of plant pathogenic fungi and nematodes has been demonstrated, and it is also suspected that they may be important for the competitive saprophytic ability of green mould isolates and may represent potential virulence factors of Trichoderma strains as emerging fungal pathogens of clinical importance. The aim of this review is to summarize the information available about the extracellular proteases of Trichoderma. Numerous studies are available about the extracellular proteolytic enzyme profiles of Trichoderma strains and about the effect of abiotic environmental factors on protease activities. A number of protease enzymes have been purified to homogeneity and some protease encoding genes have been cloned and characterized. These results will be reviewed and the role of Trichoderma proteases in biological control as well as their advantages and disadvantages in biotechnology will be discussed. PMID:16003937

  19. Regulation of protease production in Clostridium sporogenes.

    PubMed Central

    Allison, C; Macfarlane, G T

    1990-01-01

    The physiological and nutritional factors that regulate protease synthesis in Clostridium sporogenes C25 were studied in batch and continuous cultures. Formation of extracellular proteases occurred at the end of active growth and during the stationary phase in batch cultures. Protease production was inversely related to growth rate in glucose-excess and glucose-limited chemostats over the range D = 0.05 to 0.70 h-1. In pulse experiments, glucose, ammonia, phosphate, and some amino acids (tryptophan, proline, tyrosine, and isoleucine) strongly repressed protease synthesis. This repression was not relieved by addition of 4 mM cyclic AMP, cyclic GMP, or dibutyryl cyclic AMP. Protease formation was markedly inhibited by 4 mM ATP and ADP, but GTP and GDP had little effect on the process. It is concluded that protease production by C. sporogenes is strongly influenced by the amount of energy available to the cells, with the highest levels of protease synthesis occurring under energy-limiting conditions. PMID:2268158

  20. A biotechnology perspective of fungal proteases

    PubMed Central

    de Souza, Paula Monteiro; Bittencourt, Mona Lisa de Assis; Caprara, Carolina Canielles; de Freitas, Marcela; de Almeida, Renata Paula Coppini; Silveira, Dâmaris; Fonseca, Yris Maria; Ferreira, Edivaldo Ximenes; Pessoa, Adalberto; Magalhães, Pérola Oliveira

    2015-01-01

    Proteases hydrolyze the peptide bonds of proteins into peptides and amino acids, being found in all living organisms, and are essential for cell growth and differentiation. Proteolytic enzymes have potential application in a wide number of industrial processes such as food, laundry detergent and pharmaceutical. Proteases from microbial sources have dominated applications in industrial sectors. Fungal proteases are used for hydrolyzing protein and other components of soy beans and wheat in soy sauce production. Proteases can be produced in large quantities in a short time by established methods of fermentation. The parameters such as variation in C/N ratio, presence of some sugars, besides several other physical factors are important in the development of fermentation process. Proteases of fungal origin can be produced cost effectively, have an advantage faster production, the ease with which the enzymes can be modified and mycelium can be easily removed by filtration. The production of proteases has been carried out using submerged fermentation, but conditions in solid state fermentation lead to several potential advantages for the production of fungal enzymes. This review focuses on the production of fungal proteases, their distribution, structural-functional aspects, physical and chemical parameters, and the use of these enzymes in industrial applications. PMID:26273247

  1. A biotechnology perspective of fungal proteases.

    PubMed

    de Souza, Paula Monteiro; Bittencourt, Mona Lisa de Assis; Caprara, Carolina Canielles; de Freitas, Marcela; de Almeida, Renata Paula Coppini; Silveira, Dâmaris; Fonseca, Yris Maria; Ferreira Filho, Edivaldo Ximenes; Pessoa Junior, Adalberto; Magalhães, Pérola Oliveira

    2015-06-01

    Proteases hydrolyze the peptide bonds of proteins into peptides and amino acids, being found in all living organisms, and are essential for cell growth and differentiation. Proteolytic enzymes have potential application in a wide number of industrial processes such as food, laundry detergent and pharmaceutical. Proteases from microbial sources have dominated applications in industrial sectors. Fungal proteases are used for hydrolyzing protein and other components of soy beans and wheat in soy sauce production. Proteases can be produced in large quantities in a short time by established methods of fermentation. The parameters such as variation in C/N ratio, presence of some sugars, besides several other physical factors are important in the development of fermentation process. Proteases of fungal origin can be produced cost effectively, have an advantage faster production, the ease with which the enzymes can be modified and mycelium can be easily removed by filtration. The production of proteases has been carried out using submerged fermentation, but conditions in solid state fermentation lead to several potential advantages for the production of fungal enzymes. This review focuses on the production of fungal proteases, their distribution, structural-functional aspects, physical and chemical parameters, and the use of these enzymes in industrial applications. PMID:26273247

  2. Quantitative Correlation of Conformational Binding Enthalpy with Substrate Specificity of Serine Proteases

    PubMed Central

    2015-01-01

    Members of the same protease family show different substrate specificity, even if they share identical folds, depending on the physiological processes they are part of. Here, we investigate the key factors for subpocket and global specificity of factor Xa, elastase, and granzyme B which despite all being serine proteases and sharing the chymotrypsin-fold show distinct substrate specificity profiles. We determined subpocket interaction potentials with GRID for static X-ray structures and an in silico generated ensemble of conformations. Subpocket interaction potentials determined for static X-ray structures turned out to be insufficient to explain serine protease specificity for all subpockets. Therefore, we generated conformational ensembles using molecular dynamics simulations. We identified representative binding site conformations using distance-based hierarchical agglomerative clustering and determined subpocket interaction potentials for each representative conformation of the binding site. Considering the differences in subpocket interaction potentials for these representative conformations as well as their abundance allowed us to quantitatively explain subpocket specificity for the nonprime side for all three example proteases on a molecular level. The methods to identify key regions determining subpocket specificity introduced in this study are directly applicable to other serine proteases, and the results provide starting points for new strategies in rational drug design. PMID:26709959

  3. Affinity-Tagged Miniprion Derivatives Spontaneously Adopt Protease-Resistant Conformations

    PubMed Central

    Supattapone, Surachai; Nguyen, Hoang-Oanh B.; Muramoto, Tamaki; Cohen, Fred E.; DeArmond, Stephen J.; Prusiner, Stanley B.; Scott, Michael

    2000-01-01

    An abridged PrP molecule of 106 amino acids designated PrP106 can form infectious miniprions in transgenic (Tg) mice (29). Addition of six-histidine (His6) affinity tags to selective sites within PrP106 resulted unexpectedly in new PrP proteins that spontaneously adopted protease-resistant conformations when expressed in neuroblastoma cells and Tg mice. Acquisition of protease resistance depended on the length, charge, and placement of the affinity tag. Introduction of the disease-linked mutation E200K into the sequence of PrP106(140/6His) increased the recovery of protease-resistant PrP fivefold, whereas introduction of the mutations C213A and Δ214–220 did not affect the recovery of protease-resistant PrP. Treatment of cultured cells expressing affinity-tagged PrP106 mutants with polypropyleneimine dendrimer rendered these proteins sensitive to protease digestion in a manner similar to wild-type PrPSc. We conclude that certain affinity-tagged PrP106 proteins spontaneously fold into conformations partially resembling, yet distinct from, wild-type PrPSc. These proteins might be useful tools in the identification of new disease-causing mutations as well as for screening compounds for therapeutic efficacy. PMID:11090193

  4. HIV-1 Protease: Structure, Dynamics and Inhibition

    SciTech Connect

    Louis, John M.; Ishima, R.; Torchia, D.A.; Weber, Irene T.

    2008-06-03

    The HIV-1 protease is synthesized as part of a large Gag-Pol precursor protein. It is responsible for its own release from the precursor and the processing of the Gag and Gag-Pol polyproteins into the mature structural and functional proteins required for virus maturation. Because of its indispensable role, the mature HIV-1 protease dimer has proven to be a successful target for the development of antiviral agents. In the last 5 years, a major emphasis in protease research has been to improve inhibitor design and treatment regimens.

  5. Role of NADPH Oxidase versus Neutrophil Proteases in Antimicrobial Host Defense

    PubMed Central

    Grimm, Melissa J.; Lewandowski, David C.; Pham, Christine T. N.; Blackwell, Timothy S.; Petraitiene, Ruta; Petraitis, Vidmantas; Walsh, Thomas J.; Urban, Constantin F.; Segal, Brahm H.

    2011-01-01

    NADPH oxidase is a crucial enzyme in mediating antimicrobial host defense and in regulating inflammation. Patients with chronic granulomatous disease, an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates (ROIs), suffer from life-threatening bacterial and fungal infections. The mechanisms by which NADPH oxidase mediate host defense are unclear. In addition to ROI generation, neutrophil NADPH oxidase activation is linked to the release of sequestered proteases that are posited to be critical effectors of host defense. To definitively determine the contribution of NADPH oxidase versus neutrophil serine proteases, we evaluated susceptibility to fungal and bacterial infection in mice with engineered disruptions of these pathways. NADPH oxidase-deficient mice (p47phox−/−) were highly susceptible to pulmonary infection with Aspergillus fumigatus. In contrast, double knockout neutrophil elastase (NE)−/−×cathepsin G (CG)−/− mice and lysosomal cysteine protease cathepsin C/dipeptidyl peptidase I (DPPI)-deficient mice that are defective in neutrophil serine protease activation demonstrated no impairment in antifungal host defense. In separate studies of systemic Burkholderia cepacia infection, uniform fatality occurred in p47phox−/− mice, whereas NE−/−×CG−/− mice cleared infection. Together, these results show a critical role for NADPH oxidase in antimicrobial host defense against A. fumigatus and B. cepacia, whereas the proteases we evaluated were dispensable. Our results indicate that NADPH oxidase dependent pathways separate from neutrophil serine protease activation are required for host defense against specific pathogens. PMID:22163282

  6. Cleaving for growth: threonine aspartase 1-a protease relevant for development and disease.

    PubMed

    Stauber, Roland H; Hahlbrock, Angelina; Knauer, Shirley K; Wünsch, Désirée

    2016-03-01

    From the beginning of life, proteases are key to organismal development comprising morphogenesis, cellular differentiation, and cell growth. Regulated proteolytic activity is essential for the orchestration of multiple developmental pathways, and defects in protease activity can account for multiple disease patterns. The highly conserved protease threonine aspartase 1 is a member of such developmental proteases and critically involved in the regulation of complex processes, including segmental identity, head morphogenesis, spermatogenesis, and proliferation. Additionally, threonine aspartase 1 is overexpressed in numerous liquid as well as in solid malignancies. Although threonine aspartase 1 is able to cleave the master regulator mixed lineage leukemia protein as well as other regulatory proteins in humans, our knowledge of its detailed pathobiological function and the underlying molecular mechanisms contributing to development and disease is still incomplete. Moreover, neither effective genetic nor chemical inhibitors for this enzyme are available so far precluding the detailed dissection of the pathobiological functions of threonine aspartase 1. Here, we review the current knowledge of the structure-function relationship of threonine aspartase 1 and its mechanistic impact on substrate-mediated coordination of the cell cycle and development. We discuss threonine aspartase 1-mediated effects on cellular transformation and conclude by presenting a short overview of recent interference strategies.-Stauber, R. H., Hahlbrock, A., Knauer, S. K., Wünsch, D. Cleaving for growth: threonine aspartase 1-a protease relevant for development and disease. PMID:26578689

  7. Differential Signaling by Protease-Activated Receptors: Implications for Therapeutic Targeting

    PubMed Central

    Sidhu, Tejminder S.; French, Shauna L.; Hamilton, Justin R.

    2014-01-01

    Protease-activated receptors (PARs) are a family of four G protein-coupled receptors that exhibit increasingly appreciated differences in signaling and regulation both within and between the receptor class. By nature of their proteolytic self-activation mechanism, PARs have unique processes of receptor activation, “ligand” binding, and desensitization/resensitization. These distinctive aspects have presented both challenges and opportunities in the targeting of PARs for therapeutic benefit—the most notable example of which is inhibition of PAR1 on platelets for the prevention of arterial thrombosis. However, more recent studies have uncovered further distinguishing features of PAR-mediated signaling, revealing mechanisms by which identical proteases elicit distinct effects in the same cell, as well as how distinct proteases produce different cellular consequences via the same receptor. Here we review this differential signaling by PARs, highlight how important distinctions between PAR1 and PAR4 are impacting on the progress of a new class of anti-thrombotic drugs, and discuss how these more recent insights into PAR signaling may present further opportunities for manipulating PAR activation and signaling in the development of novel therapies. PMID:24733067

  8. Protease nexin-1 regulates retinal vascular development.

    PubMed

    Selbonne, Sonia; Francois, Deborah; Raoul, William; Boulaftali, Yacine; Sennlaub, Florian; Jandrot-Perrus, Martine; Bouton, Marie-Christine; Arocas, Véronique

    2015-10-01

    We recently identified protease nexin-1 (PN-1) or serpinE2, as a possibly underestimated player in maintaining angiogenic balance. Here, we used the well-characterized postnatal vascular development of newborn mouse retina to further investigate the role and the mechanism of action of PN-1 in physiological angiogenesis. The development of retinal vasculature was analysed by endothelial cell staining with isolectin B4. PN-1-deficient (PN-1(-/-)) retina displayed increased vascularization in the postnatal period, with elevated capillary thickness and density, compared to their wild-type littermate (WT). Moreover, PN-1(-/-) retina presented more veins/arteries than WT retina. The kinetics of retinal vasculature development, retinal VEGF expression and overall retinal structure were similar in WT and PN-1(-/-) mice, but we observed a hyperproliferation of vascular cells in PN-1(-/-) retina. Expression of PN-1 was analysed by immunoblotting and X-Gal staining of retinas from mice expressing beta-galactosidase under a PN-1 promoter. PN-1 was highly expressed in the first week following birth and then progressively decreased to a low level in adult retina where it localized on the retinal arteries. PCR arrays performed on mouse retinal RNA identified two angiogenesis-related factors, midkine and Smad5, that were overexpressed in PN-1(-/-) newborn mice and this was confirmed by RT-PCR. Both the higher vascularization and the overexpression of midkine and Smad5 mRNA were also observed in gastrocnemius muscle of PN-1(-/-) mice, suggesting that PN-1 interferes with these pathways. Together, our results demonstrate that PN-1 strongly limits physiological angiogenesis and suggest that modulation of PN-1 expression could represent a new way to regulate angiogenesis. PMID:26109427

  9. HIV-1 protease-induced apoptosis

    PubMed Central

    2014-01-01

    Background Apoptosis is one of the presumptive causes of CD4+ T cell depletion during HIV infection and progression to AIDS. However, the precise role of HIV-1 in this process remains unexplained. HIV-1 protease (PR) has been suggested as a possible factor, but a direct link between HIV-1 PR enzymatic activity and apoptosis has not been established. Results Here, we show that expression of active HIV-1 PR induces death in HeLa and HEK-293 cells via the mitochondrial apoptotic pathway. This conclusion is based on in vivo observations of the direct localization of HIV-1 PR in mitochondria, a key player in triggering apoptosis. Moreover, we observed an HIV-1 PR concentration-dependent decrease in mitochondrial membrane potential and the role of HIV-1 PR in activation of caspase 9, PARP cleavage and DNA fragmentation. In addition, in vitro data demonstrated that HIV-1 PR mediates cleavage of mitochondrial proteins Tom22, VDAC and ANT, leading to release of AIF and Hsp60 proteins. By using yeast two-hybrid screening, we also identified a new HIV-1 PR interaction partner, breast carcinoma-associated protein 3 (BCA3). We found that BCA3 accelerates p53 transcriptional activity on the bax promoter, thus elevating the cellular level of pro-apoptotic Bax protein. Conclusion In summary, our results describe the involvement of HIV-1 PR in apoptosis, which is caused either by a direct effect of HIV-1 PR on mitochondrial membrane integrity or by its interaction with cellular protein BCA3. PMID:24886575

  10. Cleavage of fibrinogen by the human neutrophil neutral peptide-generating protease.

    PubMed Central

    Wintroub, B U; Coblyn, J S; Kaempfer, C E; Austen, K F

    1980-01-01

    The human neutrophil peptide-generating protease, which generates a low molecular weight vasoactive peptide from a plasma protein substrate, is directly fibrinolytic and cleaves human fibrinogen in a manner distinct from plasmin. Fibrinogen was reduced from 340,000 Mr to derivatives of 270,000-325,000 Mr during interaction with the protease at enzyme-to-substrate ratios of 0.3 or 1.0 microgram/1.0 mg. The 310,000-325,000 Mr cleavage fragments exhibited prolonged thrombin-induced clotting activity but were able to be coagulated, whereas the 270,000-290,000 Mr fragments were not able to be coagulated. Anticoagulants were not generated at either enzyme dose. As analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis in 4-30% gradient gels and 10% gels stained for protein and carbohydrate, the diminution to 310,000-325,000 Mr and the prolongation of thrombin-induced clotting time resulted from cleavage of the fibrinogen A alpha chain. The further decrease in size to 270,000-290,000 Mr was associated with B beta-chain and gamma-chain cleavage and an inability to form gamma-gamma dimers. The neutral peptide-generating protease, a distinct human neutrophil neutral protease with fibrinolytic and fibrinogenolytic activities comparable to those of plasmin on a weight basis, cleaves fibrinogen in a manner that is distinct from the action of plasmin, leukocyte elastase, and leukocyte granule extracts. It may be that the concerted action of this neutrophil protease to generate a vasoactive peptide and to digest fibrinogen and fibrin facilitates neutrophil movement through vascular and extravascular sites. Images PMID:7001479

  11. Lung protease/anti-protease network and modulation of mucus production and surfactant activity.

    PubMed

    Garcia-Verdugo, Ignacio; Descamps, Delphyne; Chignard, Michel; Touqui, Lhousseine; Sallenave, Jean-Michel

    2010-11-01

    Lung epithelium guarantees gas-exchange (performed in the alveoli) and protects from external insults (pathogens, pollutants…) present within inhaled air. Both functions are facilitated by secretions lining airway surface liquid, mucus (in the upper airways) and pulmonary surfactant (in the alveoli). Mucins, the main glycoproteins present within the mucus, are responsible for its rheologic properties and participate in lung defense mechanisms. In parallel, lung collectins are pattern recognition molecules present in pulmonary surfactant that also modulate lung defense. During chronic airways diseases, excessive protease activity can promote mucus hypersecretion and degradation of lung collectins and therefore contribute to the pathophysiology of these diseases. Importantly, secretion of local and systemic anti-proteases might be crucial to equilibrate the protease/anti-protease unbalance and therefore preserve the function of lung host defense compounds and airway surface liquid homeostasis. In this review we will present information relative to proteases able to modulate mucin production and lung collectin integrity, two important compounds of innate immune defense. One strategy to preserve physiological mucus production and collectin integrity during chronic airways diseases might be the over-expression of local 'alarm' anti-proteases such as SLPI and elafin. Interestingly, a cross-talk between lung collectins and anti-protease activity has recently been described, implicating the presence within the lung of a complex network between proteases, anti-proteases and pattern recognition molecules, which aims to keep or restore homeostasis in resting or inflamed lungs. PMID:20493919

  12. Synthesis of amino heterocycle aspartyl protease inhibitors.

    PubMed

    Chambers, Rachel K; Khan, Tanweer A; Olsen, David B; Sleebs, Brad E

    2016-06-14

    Aspartyl proteases are important pharmacological targets. Historically aspartyl proteases have been commonly targeted with transition state derived peptidomimetics. The strategy to develop aspartyl protease inhibitors has undertaken a dramatic paradigm shift in the last 10 years. The pharmaceutical industry in 2005 disclosed several scaffolds or "head groups" that prompted the field to move beyond peptidomimetic derived inhibitors. Since the discovery of the first amino heterocycle aspartyl protease inhibitor, the amino hydantoin, industry and academia have positioned themselves for a foothold on the new molecular space, designing a variety of related "head groups". Both the design and synthetic efforts involved in constructing these scaffolds are varied and complex. Here we highlight the synthetic strategies used to access these amino heterocycle scaffolds. PMID:27143279

  13. Four proteolytic processes of myocardium, one insensitive to thiol reactive agents and thiol protease inhibitor.

    PubMed

    Thorne, D P; Lockwood, T D

    1993-07-01

    Four distinct processes mediating protein degradation were identified in the Langendorff perfused rat heart. Hearts were biosynthetically labeled in vitro with [3H]leucine for 10 min. The subsequent release of [3H]leucine at 1.5-min intervals (2 mM nonradioactive leucine) was determined from 20 min to 8 h after labeling in rhythmically contracting hearts. Rapid turnover proteins were eliminated during the first 3 h; this degradation was not inhibited by insulin (5 nM) or isoproterenol (0.5 microM). However, the nontoxic thiol reactive agent diamide (100 microM) caused a complete inhibition of the [3H]leucine release from rapidly degraded proteins. After the elimination of rapidly degraded proteins at 3 h, the release of [3H]leucine was inhibited 35-40% by insulin (5 nM) or the lysosomal inhibitor chloroquine (30 microM), thereby defining a second vesicular process. The beta-agonist isoproterenol (0.5 microM) or the nonselective alpha-agonist naphazoline (100 microM) caused 30-35% proteolytic inhibitions, defining a third adrenergic-responsive process. The inhibitory effects of simultaneously combined insulin and chloroquine did not exceed the effect of either agent alone. However, the combined effects of insulin and isoproterenol were additive, inhibiting two-thirds of basal degradation. Beginning at 3 h after labeling a 75% proteolytic inhibition resulted from the thiol reactive agents diamide (100 microM) or N-ethylmaleimide (10 microM); the thiol protease active site inhibitor trans-epoxysuccinly-L-leucylamino-(4-quinidino)butane (50 microM) caused 65% inhibition. The 75% inhibition caused by diamide includes both the insulin-responsive and beta-adrenergic-responsive pathways. A novel fourth proteolytic process (25% of proteolysis) was thereby distinguished from the above three by its resistance to inhibition by insulin, adrenergic agonists, thiol reactive agents, or thiol protease inhibitor. Only the adrenergic-responsive process was correlated with changes in

  14. A radiometric assay for HIV-1 protease

    SciTech Connect

    Hyland, L.J.; Dayton, B.D.; Moore, M.L.; Shu, A.Y.; Heys, J.R.; Meek, T.D. )

    1990-08-01

    A rapid, high-throughput radiometric assay for HIV-1 protease has been developed using ion-exchange chromatography performed in 96-well filtration plates. The assay monitors the activity of the HIV-1 protease on the radiolabeled form of a heptapeptide substrate, (tyrosyl-3,5-3H)Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, which is based on the p17-p24 cleavage site found in the viral polyprotein substrate Pr55gag. Specific cleavage of this uncharged heptapeptide substrate by HIV-1 protease releases the anionic product (tyrosyl-3,5-3H)Ac-Ser-Gln-Asn-Tyr, which is retained upon minicolumns of the anion-exchange resin AG1-X8. Protease activity is determined from the recovery of this radiolabeled product following elution with formic acid. This facile and highly sensitive assay may be utilized for steady-state kinetic analysis of the protease, for measurements of enzyme activity during its purification, and as a routine assay for the evaluation of protease inhibitors from natural product or synthetic sources.

  15. β-Lactam Resistance in Methicillin-Resistant Staphylococcus aureus USA300 Is Increased by Inactivation of the ClpXP Protease

    PubMed Central

    Bæk, Kristoffer T.; Gründling, Angelika; Mogensen, René G.; Thøgersen, Louise; Petersen, Andreas; Paulander, Wilhelm

    2014-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) has acquired the mecA gene encoding a peptidoglycan transpeptidase, penicillin binding protein 2a (PBP2a), which has decreased affinity for β-lactams. Quickly spreading and highly virulent community-acquired (CA) MRSA strains recently emerged as a frequent cause of infection in individuals without exposure to the health care system. In this study, we found that the inactivation of the components of the ClpXP protease substantially increased the β-lactam resistance level of a CA-MRSA USA300 strain, suggesting that the proteolytic activity of ClpXP controls one or more pathways modulating β-lactam resistance. These pathways do not involve the control of mecA expression, as the cellular levels of PBP2a were unaltered in the clp mutants. An analysis of the cell envelope properties of the clpX and clpP mutants revealed a number of distinct phenotypes that may contribute to the enhanced β-lactam tolerance. Both mutants displayed significantly thicker cell walls, increased peptidoglycan cross-linking, and altered composition of monomeric muropeptide species compared to those of the wild types. Moreover, changes in Sle1-mediated peptidoglycan hydrolysis and altered processing of the major autolysin Atl were observed in the clp mutants. In conclusion, the results presented here point to an important role for the ClpXP protease in controlling cell wall metabolism and add novel insights into the molecular factors that determine strain-dependent β-lactam resistance. PMID:24867990

  16. Neutral serine proteases of neutrophils.

    PubMed

    Kettritz, Ralph

    2016-09-01

    Neutrophil serine proteases (NSPs) exercise tissue-degrading and microbial-killing effects. The spectrum of NSP-mediated functions grows continuously, not least because of methodological progress. Sensitive and specific FRET substrates were developed to study the proteolytic activity of each NSP member. Advanced biochemical methods are beginning to characterize common and specific NSP substrates. The resulting novel information indicates that NSPs contribute not only to genuine inflammatory neutrophil functions but also to autoimmunity, metabolic conditions, and cancer. Tight regulatory mechanisms control the proteolytic potential of NSPs. However, not all NSP functions depend on their enzymatic activity. Proteinase-3 (PR3) is somewhat unique among the NSPs for PR3 functions as an autoantigen. Patients with small-vessel vasculitis develop autoantibodies to PR3 that bind their target antigens on the neutrophil surface and trigger neutrophil activation. These activated cells subsequently contribute to vascular necrosis with life-threatening multiorgan failure. This article discusses various aspects of NSP biology and highlights translational aspects with strong clinical implications. PMID:27558338

  17. Synergistic Caseinolytic Activity and Differential Fibrinogenolytic Action of Multiple Proteases of Maclura spinosa (Roxb. ex Willd.) latex

    PubMed Central

    Venkatesh, B. K.; Achar, Raghu Ram; Sharanappa, P.; Priya, B. S.; Swamy, S. Nanjunda

    2015-01-01

    Background: Kollamalayaali tribes of South India use latex of Maclura spinosa for milk curdling. This action is implicated to proteases which exhibit strong pharmacological potential in retardation of blood flow and acceleration of wound healing. Objective: To validate the presence of a proteolytic enzyme(s) in Maclura spinosa latex (MSL), and to investigate their probable role in hemostasis. Materials and Methods: Processed latex was examined for proteolytic and hemostatic activity using casein and human fibrinogen as substrates, respectively. Caseinoltyic activity was compared with two standard proteases viz., trypsin I and trypsin II. Effect of various standard protease inhibitors viz., iodoacetic acid (IAA), phenylmethylsulfonyl fluoride (PMSF), ethylene glycol tetraacetic acid, and ethylenediaminetetraacetic acid on both caseinolytic and fibrinogenolytic activities were examined. Electrophoretogram of fibrinogenolytic assays were subjected to densitometric analysis. Results: Proteolytic action of MSL was found to be highly efficient over trypsin I and trypsin II in dose-dependent caseinolytic activity (P < 0.05; specific activity of 1,080 units/mg protein). The Aα and Bβ bands of human fibrinogen were readily cleaved by MSL (for 1 μg crude protein and 30 min of incubation time). Furthermore, MSL cleaved γ subunit in dose- and time-dependent manner. Quantitative correlation of these results was obtained by densitometric analysis. The caseinolytic activity of MSL was inhibited by IAA, PMSF. While, only PMSF inhibited fibrinogenolytic activity. Conclusions: MSL contains proteolytic enzymes belonging to two distinct superfamilies viz., serine protease and cysteine proteases. The fibrinogenolytic activity of MSL is restricted to serine proteases only. The study extrapolates the use of M. spinosa latex from milk curdling to hemostasis. SUMMARY Proteolytic enzymes present in latex of Maclura spinosa can be assigned to two different protease superfamilies viz

  18. Carbohydrate protease conjugates: Stabilized proteases for peptide synthesis

    SciTech Connect

    Wartchow, C.A.; Wang, Peng; Bednarski, M.D.; Callstrom, M.R. |

    1995-12-31

    The synthesis of oligopeptides using stable carbohydrate protease conjugates (CPCs) was examined in acetonitrile solvent systems. CPC[{alpha}-chymotrypsin] was used for the preparation of peptides containing histidine, phenylalanine, tryptophan in the P{sub 1} position in 60-93% yield. The CPC[{alpha}-chymotrypsin]-catalyzed synthesis of octamer Z-Gly-Gly-Phe-Gly-Gly-Phe-Gly-Gly-OEt from Z-Gly-Gly-Phe-Gly-Gly-Phe-OMe was achieved in 71% yield demonstrating that synthesis peptides containing both hydrophylic and hydrophobic amino acids. The P{sub 2} specificity of papain for aromatic residues was utilized for the 2 + 3 coupling of Z-Tyr-Gly-OMe to H{sub 2}N-Gly-Phe-Leu-OH to generate the leucine enkephalin derivative in 79% yield. Although papain is nonspecific for the hydrolysis of N-benzyloxycarbonyl amino acid methyl esters in aqueous solution, the rates of synthesis for these derivitives with nucleophile leucine tert-butyl ester differed by nearly 2 orders of magnitude. CPC[thermolysin] was used to prepare the aspartame precursor Z-Asp-Phe-OMe in 90% yield. The increased stability of CPCs prepared from periodate-modified poly(2-methacryl- amido-2-deoxy-D-glucose), poly(2-methacrylamido-2-deoxy-D-galactose), and poly(5-methacryl-amido-5-deoxy-D-ribose), carbohydrate materials designed to increase the aldehyde concentration in aqueous solution, suggests that the stability of CPCs is directly related to the aldehyde concentration of the carbohydrate material. Periodate oxidation of poly(2-methacrylamido-2-deoxy-D-glucose) followed by covalent attachment to {alpha}-chymotrypsin gave a CPC with catalytic activity in potassium phosphate buffer at 90{degrees}C for 2 h. 1 fig., 1 tab., 40 refs.

  19. The spectrum of low molecular weight alpha-amylase/protease inhibitor genes expressed in the US bread wheat Butte 86

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The complement of genes encoding alpha-amylase/protease inhibitors expressed in Triticum aestivum cv. Butte 86 was characterized by transcript and proteomic analysis. Coding sequences for 18 distinct proteins were identified among a collection of expressed sequence tags (ESTs) from Butte 86 developi...

  20. Protease inhibitors targeting coronavirus and filovirus entry.

    PubMed

    Zhou, Yanchen; Vedantham, Punitha; Lu, Kai; Agudelo, Juliet; Carrion, Ricardo; Nunneley, Jerritt W; Barnard, Dale; Pöhlmann, Stefan; McKerrow, James H; Renslo, Adam R; Simmons, Graham

    2015-04-01

    In order to gain entry into cells, diverse viruses, including Ebola virus, SARS-coronavirus and the emerging MERS-coronavirus, depend on activation of their envelope glycoproteins by host cell proteases. The respective enzymes are thus excellent targets for antiviral intervention. In cell culture, activation of Ebola virus, as well as SARS- and MERS-coronavirus can be accomplished by the endosomal cysteine proteases, cathepsin L (CTSL) and cathepsin B (CTSB). In addition, SARS- and MERS-coronavirus can use serine proteases localized at the cell surface, for their activation. However, it is currently unclear which protease(s) facilitate viral spread in the infected host. We report here that the cysteine protease inhibitor K11777, ((2S)-N-[(1E,3S)-1-(benzenesulfonyl)-5-phenylpent-1-en-3-yl]-2-{[(E)-4-methylpiperazine-1-carbonyl]amino}-3-phenylpropanamide) and closely-related vinylsulfones act as broad-spectrum antivirals by targeting cathepsin-mediated cell entry. K11777 is already in advanced stages of development for a number of parasitic diseases, such as Chagas disease, and has proven to be safe and effective in a range of animal models. K11777 inhibition of SARS-CoV and Ebola virus entry was observed in the sub-nanomolar range. In order to assess whether cysteine or serine proteases promote viral spread in the host, we compared the antiviral activity of an optimized K11777-derivative with that of camostat, an inhibitor of TMPRSS2 and related serine proteases. Employing a pathogenic animal model of SARS-CoV infection, we demonstrated that viral spread and pathogenesis of SARS-CoV is driven by serine rather than cysteine proteases and can be effectively prevented by camostat. Camostat has been clinically used to treat chronic pancreatitis, and thus represents an exciting potential therapeutic for respiratory coronavirus infections. Our results indicate that camostat, or similar serine protease inhibitors, might be an effective option for treatment of SARS and

  1. Dephosphorylation of the linker regions of Smad1 and Smad2/3 by small C-terminal domain phosphatases has distinct outcomes for bone morphogenetic protein and transforming growth factor-beta pathways.

    PubMed

    Sapkota, Gopal; Knockaert, Marie; Alarcón, Claudio; Montalvo, Ermelinda; Brivanlou, Ali H; Massagué, Joan

    2006-12-29

    Smad proteins transduce bone morphogenetic protein (BMP) and transforming growth factor-beta (TGFbeta) signals upon phosphorylation of their C-terminal SXS motif by receptor kinases. The activity of Smad1 in the BMP pathway and Smad2/3 in the TGFbeta pathway is restricted by pathway cross-talk and feedback through protein kinases, including MAPK, CDK2/4, p38MAPK, JNK, and others. These kinases phosphorylate Smads 1-3 at the region that links the N-terminal DNA-binding domain and the C-terminal transcriptional domain. Phosphatases that dephosphorylate the linker region are therefore likely to play an integral part in the regulation of Smad activity. We reported previously that small C-terminal domain phosphatases 1, 2, and 3 (SCP1-3) dephosphorylate Smad1 C-terminal tail, thereby attenuating BMP signaling. Here we provide evidence that SCP1-3 also dephosphorylate the linker regions of Smad1 and Smad2/3 in vitro, in mammalian cells and in Xenopus embryos. Overexpression of SCP 1, 2, or 3 decreased linker phosphorylation of Smads 1, 2 and 3. Moreover, RNA interference-mediated knockdown of SCP1/2 increased the BMP-dependent phosphorylation of the Smad1 linker region as well as the C terminus. In contrast, SCP1/2 knockdown increased the TGFbeta-dependent linker phosphorylation of Smad2/3 but not the C-terminal phosphorylation. Consequently, SCP1/2 knockdown inhibited TGFbeta transcriptional responses, but it enhanced BMP transcriptional responses. Thus, by dephosphorylating Smad2/3 at the linker (inhibitory) but not the C-terminal (activating) site, the SCPs enhance TGFbeta signaling, and by dephosphorylating Smad1 at both sites, the SCPs reset Smad1 to the basal unphosphorylated state. PMID:17085434

  2. Protease-activated receptors mediate crosstalk between coagulation and fibrinolysis.

    PubMed

    McEachron, Troy A; Pawlinski, Rafal; Richards, Kristy L; Church, Frank C; Mackman, Nigel

    2010-12-01

    The coagulation and fibrinolytic systems contribute to malignancy by increasing angiogenesis, tumor growth, tumor invasion, and tumor metastasis. Oncogenic transformation increases the expression of tissue factor (TF) that results in local generation of coagulation proteases and activation of protease-activated receptor (PAR)-1 and PAR-2. We compared the PAR-dependent expression of urokinase plasminogen activator (uPA) and plasminogen activator inhibitor (PAI)-1 in 2 murine mammary adencocarcinoma cell lines: metastatic 4T1 cells and nonmetastatic 67NR cells. 4T1 cells expressed TF, PAR-1 and PAR-2 whereas 67NR cells expressed TF and PAR-1. We also silenced PAR-1 or PAR-2 expression in the 4T1 cells. We discovered 2 distinct mechanisms for PAR-dependent expression of uPA and PAI-1. First, we found that factor Xa or thrombin activation of PAR-1 led to a rapid release of stored intracellular uPA into the culture supernatant. Second, thrombin transactivation of a PAR-1/PAR-2 complex resulted in increases in PAI-1 mRNA and protein expression. Cells lacking PAR-2 failed to express PAI-1 in response to thrombin and factor Xa did not activate the PAR-1/PAR-2 complex. Our results reveal how PAR-1 and PAR-2 on tumor cells mediate crosstalk between coagulation and fibrinolysis. PMID:20736455

  3. Protease-activated receptors mediate crosstalk between coagulation and fibrinolysis

    PubMed Central

    McEachron, Troy A.; Pawlinski, Rafal; Richards, Kristy L.; Church, Frank C.

    2010-01-01

    The coagulation and fibrinolytic systems contribute to malignancy by increasing angiogenesis, tumor growth, tumor invasion, and tumor metastasis. Oncogenic transformation increases the expression of tissue factor (TF) that results in local generation of coagulation proteases and activation of protease-activated receptor (PAR)-1 and PAR-2. We compared the PAR-dependent expression of urokinase plasminogen activator (uPA) and plasminogen activator inhibitor (PAI)-1 in 2 murine mammary adencocarcinoma cell lines: metastatic 4T1 cells and nonmetastatic 67NR cells. 4T1 cells expressed TF, PAR-1 and PAR-2 whereas 67NR cells expressed TF and PAR-1. We also silenced PAR-1 or PAR-2 expression in the 4T1 cells. We discovered 2 distinct mechanisms for PAR-dependent expression of uPA and PAI-1. First, we found that factor Xa or thrombin activation of PAR-1 led to a rapid release of stored intracellular uPA into the culture supernatant. Second, thrombin transactivation of a PAR-1/PAR-2 complex resulted in increases in PAI-1 mRNA and protein expression. Cells lacking PAR-2 failed to express PAI-1 in response to thrombin and factor Xa did not activate the PAR-1/PAR-2 complex. Our results reveal how PAR-1 and PAR-2 on tumor cells mediate crosstalk between coagulation and fibrinolysis. PMID:20736455

  4. ADAMTS: a novel family of extracellular matrix proteases.

    PubMed

    Tang, B L

    2001-01-01

    ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) is a novel family of extracellular proteases found in both mammals and invertebrates. Members of the family may be distinguished from the ADAM (a disintegrin and metalloprotease) family members based on the multiple copies of thrombospondin 1-like repeats they carry. With at least nine members in mammals alone, the ADAMTS family members are predicted by their structural domains to be extracellular matrix (ECM) proteins with a wide range of activities and functions distinct from members of the ADAM family that are largely anchored on the cell surface. ADAMTS2 is a procollagen N-proteinase, and the mutations of its gene are responsible for Human Ehlers-Danlos syndrome type VII C and bovine dermatosparaxis. ADAMTS4 and ADAMTS5 are aggrecanases implicated in the degradation of cartilage aggrecan in arthritic diseases. Other members of the ADAMTS family have also been implicated in roles during embryonic development and angiogenesis. Current and future studies on this emerging group of ECM proteases may provide important insights into developmental or pathological processes involving ECM remodeling. PMID:11167130

  5. Structurally Similar Triphenylphosphine-Stabilized Undecagolds, Au11(PPh3)7Cl3 and [Au11(PPh3)8Cl2]Cl, Exhibit Distinct Ligand Exchange Pathways with Glutathione

    PubMed Central

    2015-01-01

    Ligand exchange is frequently used to introduce new functional groups on the surface of inorganic nanoparticles or clusters while preserving the core size. For one of the smallest clusters, triphenylphosphine (TPP)-stabilized undecagold, there are conflicting reports in the literature regarding whether core size is retained or significant growth occurs during exchange with thiol ligands. During an investigation of these differences in reactivity, two distinct forms of undecagold were isolated. The X-ray structures of the two forms, Au11(PPh3)7Cl3 and [Au11(PPh3)8Cl2]Cl, differ only in the number of TPP ligands bound to the core. Syntheses were developed to produce each of the two forms, and their spectroscopic features correlated with the structures. Ligand exchange on [Au11(PPh3)8Cl2]Cl yields only small clusters, whereas exchange on Au11(PPh3)7Cl3 (or mixtures of the two forms) yields the larger Au25 cluster. The distinctive features in the optical spectra of the two forms made it possible to evaluate which of the cluster forms were used in the previously published papers and clarify the origin of the differences in reactivity that had been reported. The results confirm that reactions of clusters and nanoparticles may be influenced by small variations in the arrangement of ligands and suggest that the role of the ligand shell in stabilizing intermediates during ligand exchange may be essential to preventing particle growth or coalescence. PMID:25171178

  6. Isolation of the human PC6 gene encoding the putative host protease for HIV-1 gp160 processing in CD4+ T lymphocytes.

    PubMed Central

    Miranda, L; Wolf, J; Pichuantes, S; Duke, R; Franzusoff, A

    1996-01-01

    Production of infectious HIV-1 virions is dependent on the processing of envelope glycoprotein gp160 by a host cell protease. The protease in human CD4+ T lymphocytes has not been unequivocally identified, yet members of the family of mammalian subtilisin-like protein convertases (SPCs), which are soluble or membrane-bound proteases of the secretory pathway, best fulfill the criteria. These proteases are required for proprotein maturation and cleave at paired basic amino acid motifs in numerous cellular and viral glycoprotein precursors, both in vivo and in vitro. To identify the gp160 processing protease, we have used reverse transcription-PCR and Northern blot analyses to ascertain the spectrum of SPC proteases in human CD4+ T cells. We have cloned novel members of the SPC family, known as the human PC6 genes. Two isoforms of the hPC6 protease are expressed in human T cells, hPC6A and the larger hPC6B. The patterns of SPC gene expression in human T cells has been compared with the furin-defective LoVo cell line, both of which are competent in the production of infectious HIV virions. This comparison led to the conclusion that the hPC6 gene products are the most likely candidates for the host cell protease responsible for HIV-1 gp160 processing in human CD4+ T cells. Images Fig. 1 Fig. 3 PMID:8755538

  7. A Novel Bioluminescent Protease Assay Using Engineered Firefly Luciferase

    PubMed Central

    Wigdal, Susan S; Anderson, Jessica L; Vidugiris, Gediminas J; Shultz, John; Wood, Keith V; Fan, Frank

    2008-01-01

    Proteases play important roles in a variety of disease processes. Understanding their biological functions underpins the efforts of drug discovery. We have developed a bioluminescent protease assay using a circularly permuted form of firefly luciferase, wherein the native enzyme termini were joined by a peptide containing a protease site of interest. Protease cleavage of these mutant luciferases greatly activates the enzyme, typically over 100 fold. The mutant luciferase substrates are easily generated by molecular cloning and cell-free translation reactions and thus the protease substrates do not need to be chemically synthesized or purchased. The assay has broad applicability using a variety of proteases and their cognate sites and can sensitively detect protease activity. In this report we further demonstrate its utility for the evaluation of protease recognition sequence specificity and subsequent establishment of an optimized assay for the identification and characterization of protease inhibitors using high throughput screening. PMID:20161840

  8. Function of site-2 proteases in bacteria and bacterial pathogens

    PubMed Central

    Schneider, Jessica S.; Glickman, Michael S.

    2014-01-01

    Site-2 Proteases (S2Ps) are a class of intramembrane metalloproteases named after the founding member of this protein family, human S2P, which cleaves Sterol Regulatory Element Binding Proteins which control cholesterol and fatty acid biosynthesis. S2Ps are widely distributed in bacteria and participate in diverse pathways that control such diverse functions as membrane integrity, sporulation, lipid biosynthesis, pheromone production, virulence, and others. The most common signaling mechanism mediated by S2Ps is the coupled degradation of transmembrane anti-Sigma factors to activate ECF Sigma factor regulons. However, additional signaling mechanisms continue to emerge as more prokaryotic S2Ps are characterized, including direct proteolysis of membrane embedded transcription factors and proteolysis of non-transcriptional membrane proteins or membrane protein remnants. In this review we seek to comprehensively review the functions of S2Ps in bacteria and bacterial pathogens and attempt to organize these proteases into conceptual groups that will spur further study. PMID:24099002

  9. The Spn4 gene from Drosophila melanogaster is a multipurpose defence tool directed against proteases from three different peptidase families

    PubMed Central

    Brüning, Mareke; Lummer, Martina; Bentele, Caterina; Smolenaars, Marcel M. W.; Rodenburg, Kees W.; Ragg, Hermann

    2006-01-01

    By alternative use of four RSL (reactive site loop) coding exon cassettes, the serpin (serine protease inhibitor) gene Spn4 from Drosophila melanogaster was proposed to enable the synthesis of multiple protease inhibitor isoforms, one of which has been shown to be a potent inhibitor of human furin. Here, we have investigated the inhibitory spectrum of all Spn4 RSL variants. The analyses indicate that the Spn4 gene encodes inhibitors that may inhibit serine proteases of the subtilase family (S8), the chymotrypsin family (S1), and the papain-like cysteine protease family (C1), most of them at high rates. Thus a cohort of different protease inhibitors is generated simply by grafting enzyme-adapted RSL sequences on to a single serpin scaffold, even though the target proteases contain different types and/or a varying order of catalytic residues and are descendents of different phylogenetic lineages. Since all of the Spn4 RSL isoforms are produced as intracellular residents and additionally as variants destined for export or associated with the secretory pathway, the Spn4 gene represents a versatile defence tool kit that may provide multiple antiproteolytic functions. PMID:16989645

  10. Development of potent inhibitors of the coxsackievirus 3C protease

    SciTech Connect

    Lee, Eui Seung; Lee, Won Gil; Yun, Soo-Hyeon; Rho, Seong Hwan; Im, Isak; Yang, Sung Tae; Sellamuthu, Saravanan; Lee, Yong Jae; Kwon, Sun Jae; Park, Ohkmae K.; Jeon, Eun-Seok; Park, Woo Jin . E-mail: wjpark@gist.ac.kr; Kim, Yong-Chul . E-mail: yongchul@gist.ac.kr

    2007-06-22

    Coxsackievirus B3 (CVB3) 3C protease (3CP) plays essential roles in the viral replication cycle, and therefore, provides an attractive therapeutic target for treatment of human diseases caused by CVB3 infection. CVB3 3CP and human rhinovirus (HRV) 3CP have a high degree of amino acid sequence similarity. Comparative modeling of these two 3CPs revealed one prominent distinction; an Asn residue delineating the S2' pocket in HRV 3CP is replaced by a Tyr residue in CVB3 3CP. AG7088, a potent inhibitor of HRV 3CP, was modified by substitution of the ethyl group at the P2' position with various hydrophobic aromatic rings that are predicted to interact preferentially with the Tyr residue in the S2' pocket of CVB3 3CP. The resulting derivatives showed dramatically increased inhibitory activities against CVB3 3CP. In addition, one of the derivatives effectively inhibited the CVB3 proliferation in vitro.

  11. Identification of covalent active site inhibitors of dengue virus protease

    PubMed Central

    Koh-Stenta, Xiaoying; Joy, Joma; Wang, Si Fang; Kwek, Perlyn Zekui; Wee, John Liang Kuan; Wan, Kah Fei; Gayen, Shovanlal; Chen, Angela Shuyi; Kang, CongBao; Lee, May Ann; Poulsen, Anders; Vasudevan, Subhash G; Hill, Jeffrey; Nacro, Kassoum

    2015-01-01

    Dengue virus (DENV) protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described. PMID:26677315

  12. Integrated innate mechanisms involved in airway allergic inflammation to the serine protease subtilisin.

    PubMed

    Florsheim, Esther; Yu, Shuang; Bragatto, Ivan; Faustino, Lucas; Gomes, Eliane; Ramos, Rodrigo N; Barbuto, José Alexandre M; Medzhitov, Ruslan; Russo, Momtchilo

    2015-05-15

    Proteases are recognized environmental allergens, but little is known about the mechanisms responsible for sensing enzyme activity and initiating the development of allergic inflammation. Because usage of the serine protease subtilisin in the detergent industry resulted in an outbreak of occupational asthma in workers, we sought to develop an experimental model of allergic lung inflammation to subtilisin and to determine the immunological mechanisms involved in type 2 responses. By using a mouse model of allergic airway disease, we have defined in this study that s.c. or intranasal sensitization followed by airway challenge to subtilisin induces prototypic allergic lung inflammation, characterized by airway eosinophilia, type 2 cytokine release, mucus production, high levels of serum IgE, and airway reactivity. These allergic responses were dependent on subtilisin protease activity, protease-activated receptor-2, IL-33R ST2, and MyD88 signaling. Also, subtilisin stimulated the expression of the proallergic cytokines IL-1α, IL-33, thymic stromal lymphopoietin, and the growth factor amphiregulin in a human bronchial epithelial cell line. Notably, acute administration of subtilisin into the airways increased lung IL-5-producing type 2 innate lymphoid cells, which required protease-activated receptor-2 expression. Finally, subtilisin activity acted as a Th2 adjuvant to an unrelated airborne Ag-promoting allergic inflammation to inhaled OVA. Therefore, we established a murine model of occupational asthma to a serine protease and characterized the main molecular pathways involved in allergic sensitization to subtilisin that potentially contribute to initiate allergic airway disease. PMID:25876764

  13. Non-Secreted Clusterin Isoforms Are Translated in Rare Amounts from Distinct Human mRNA Variants and Do Not Affect Bax-Mediated Apoptosis or the NF-κB Signaling Pathway

    PubMed Central

    Prochnow, Hans; Gollan, Rene; Rohne, Philipp; Hassemer, Matthias; Koch-Brandt, Claudia; Baiersdörfer, Markus

    2013-01-01

    Clusterin, also known as apolipoprotein J, is expressed from a variety of tissues and implicated in pathological disorders such as neurodegenerative diseases, ischemia and cancer. In contrast to secretory clusterin (sCLU), which acts as an extracellular chaperone, the synthesis, subcellular localization and function(s) of intracellular CLU isoforms is currently a matter of intense discussion. By investigating human CLU mRNAs we here unravel mechanisms leading to the synthesis of distinct CLU protein isoforms and analyze their subcellular localization and their impact on apoptosis and on NF-κB-activity. Quantitative PCR-analyses revealed the expression of four different stress-inducible CLU mRNA variants in non-cancer and cancer cell lines. In all cell lines variant 1 represents the most abundant mRNA, whereas all other variants collectively account for no more than 0.34% of total CLU mRNA, even under stressed conditions. Overexpression of CLU cDNAs combined with in vitro mutagenesis revealed distinct translational start sites including a so far uncharacterized non-canonical CUG start codon. We show that all exon 2-containing mRNAs encode sCLU and at least three non-glycosylated intracellular isoforms, CLU1‑449, CLU21‑449 and CLU34‑449, which all reside in the cytosol of unstressed and stressed HEK‑293 cells. The latter is the only form expressed from an alternatively spliced mRNA variant lacking exon 2. Functional analysis revealed that none of these cytosolic CLU forms modulate caspase-mediated intrinsic apoptosis or significantly affects TNF-α-induced NF-κB-activity. Therefore our data challenge some of the current ideas regarding the physiological functions of CLU isoforms in pathologies. PMID:24073260

  14. Heme oxygenase-1-derived bilirubin counteracts HIV protease inhibitor-mediated endothelial cell dysfunction.

    PubMed

    Liu, Xiao-Ming; Durante, Zane E; Peyton, Kelly J; Durante, William

    2016-05-01

    The use of HIV protease inhibitors (PIs) has extended the duration and quality of life for HIV-positive individuals. However there is increasing concern that this antiviral therapy may promote premature cardiovascular disease by impairing endothelial cell (EC) function. In the present study, we investigated the effect of HIV PIs on EC function and determined if the enzyme heme oxygenase (HO-1) influences the biological action of these drugs. We found that three distinct PIs, including ritonavir, atazanavir, and lopinavir, stimulated the expression of HO-1 protein and mRNA. The induction of HO-1 was associated with an increase in NF-E2-related factor-2 (Nrf2) activity and reactive oxygen species (ROS). PIs also stimulated HO-1 promoter activity and this was prevented by mutating the antioxidant responsive element or by overexpressing dominant-negative Nrf2. In addition, the PI-mediated induction of HO-1 was abolished by N-acetyl-l-cysteine and rotenone. Furthermore, PIs blocked EC proliferation and migration and stimulated the expression of intercellular adhesion molecule-1 and the adhesion of monocytes on ECs. Inhibition of HO-1 activity or expression potentiated the anti-proliferative and inflammatory actions of PIs which was reversed by bilirubin but not carbon monoxide. Alternatively, adenovirus-mediated overexpression of HO-1 attenuated the growth-inhibitory and inflammatory effect of PIs. In contrast, blocking HO-1 activity failed to modify the anti-migratory effect of the PIs. Thus, induction of HO-1 via the ROS-Nrf2 pathway in human ECs counteracts the anti-proliferative and inflammatory actions of PIs by generating bilirubin. Therapeutic approaches targeting HO-1 may provide a novel approach in preventing EC dysfunction and vascular disease in HIV-infected patients undergoing antiretroviral therapy. PMID:26968795

  15. Production of alkaline protease from Cellulosimicrobium cellulans

    PubMed Central

    Ferracini-Santos, Luciana; Sato, Hélia H

    2009-01-01

    Cellulosimicrobium cellulans is one of the microorganisms that produces a wide variety of yeast cell wall-degrading enzymes, β-1,3-glucanase, protease and chitinase. Dried cells of Saccharomyces cerevisiae were used as carbon and nitrogen source for cell growth and protease production. The medium components KH2PO4, KOH and dried yeast cells showed a significant effect (p<0.05) on the factorial fractional design. A second design was prepared using two factors: pH and percentage of dried yeast cells. The results showed that the culture medium for the maximum production of protease was 0.2 g/l of MgSO4.7H2O, 2.0 g/l of (NH4)2SO4 and 8% of dried yeast cells in 0.15M phosphate buffer at pH 8.0. The maximum alkaline protease production was 7.0 ± 0.27 U/ml over the center point. Crude protease showed best activity at 50ºC and pH 7.0-8.0, and was stable at 50ºC. PMID:24031317

  16. Subtilisin-like proteases in nematodes.

    PubMed

    Poole, Catherine B; Jin, Jingmin; McReynolds, Larry A

    2007-09-01

    Cleavage by subtilisin-like proteases (subtilases) is an essential step in post-translational processing of proteins found in organisms ranging from yeast to mammals. Our knowledge of the diversity of this protease family in nematodes is aided by the rapid increase in sequence information, especially from the Brugia malayi genome project. Genetic studies of the subtilases in Caenorhabitis elegans give valuable insight into the biological function of these proteases in other nematode species. In this review, we focus on the subtilases in filarial nematodes as well as other parasitic and free-living nematodes in comparison to what is known in C. elegans. Topics to be addressed include expansion and diversity of the subtilase gene family during evolution, enhanced complexity created by alternative RNA splicing, molecular and biochemical characterization of the different subtilases and the challenges of designing subtilase-specific inhibitors for parasitic nematodes. PMID:17570539

  17. Serine protease inhibitors of parasitic helminths.

    PubMed

    Molehin, Adebayo J; Gobert, Geoffrey N; McManus, Donald P

    2012-05-01

    Serine protease inhibitors (serpins) are a superfamily of structurally conserved proteins that inhibit serine proteases and play key physiological roles in numerous biological systems such as blood coagulation, complement activation and inflammation. A number of serpins have now been identified in parasitic helminths with putative involvement in immune regulation and in parasite survival through interference with the host immune response. This review describes the serpins and smapins (small serine protease inhibitors) that have been identified in Ascaris spp., Brugia malayi, Ancylostoma caninum Onchocerca volvulus, Haemonchus contortus, Trichinella spiralis, Trichostrongylus vitrinus, Anisakis simplex, Trichuris suis, Schistosoma spp., Clonorchis sinensis, Paragonimus westermani and Echinococcus spp. and discusses their possible biological functions, including roles in host-parasite interplay and their evolutionary relationships. PMID:22310379

  18. Dataset of cocoa aspartic protease cleavage sites.

    PubMed

    Janek, Katharina; Niewienda, Agathe; Wöstemeyer, Johannes; Voigt, Jürgen

    2016-09-01

    The data provide information in support of the research article, "The cleavage specificity of the aspartic protease of cocoa beans involved in the generation of the cocoa-specific aroma precursors" (Janek et al., 2016) [1]. Three different protein substrates were partially digested with the aspartic protease isolated from cocoa beans and commercial pepsin, respectively. The obtained peptide fragments were analyzed by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS/MS) and identified using the MASCOT server. The N- and C-terminal ends of the peptide fragments were used to identify the corresponding in-vitro cleavage sites by comparison with the amino acid sequences of the substrate proteins. The same procedure was applied to identify the cleavage sites used by the cocoa aspartic protease during cocoa fermentation starting from the published amino acid sequences of oligopeptides isolated from fermented cocoa beans. PMID:27508221

  19. Coagulation, Protease Activated Receptors and Viral Myocarditis

    PubMed Central

    Antoniak, Silvio; Mackman, Nigel

    2013-01-01

    The coagulation protease cascade plays an essential role in hemostasis. In addition, a clot contributes to host defense by limiting the spread of pathogens. Coagulation proteases induce intracellular signaling by cleavage of cell surface receptors called protease-activated receptors (PARs). These receptors allow cells to sense changes in the extracellular environment, such as infection. Viruses activate the coagulation cascade by inducing tissue factor expression and by disrupting the endothelium. Virus infection of the heart can cause myocarditis, cardiac remodeling and heart failure. Recent studies using a mouse model have shown that tissue factor, thrombin and PAR-1 signaling all positively regulate the innate immune during viral myocarditis. In contrast, PAR-2 signaling was found to inhibit interferon-β expression and the innate immune response. These observations suggest that anticoagulants may impair the innate immune response to viral infection and that inhibition of PAR-2 may be a new target to reduce viral myocarditis.. PMID:24203054

  20. The Structure of the Cell-Wall Protease from Streptococci that Inactivates the Human Complement Factor 5A

    SciTech Connect

    Brown,C.; Gu, Z.; Matsuka, Y.; Olmsted, S.; Cleary, P.; Ohlendorf, D.; Earhart, C.

    2006-01-01

    The structure of a 949-residue fragment of complement factor 5a peptidase (SCP) was determined to 1.9 Angstroms resolution. The molecule is made of five distinct domains in an elongated head-stalk structure. The structure suggests that activity of SCP can be modulated through binding of integrins to 2 RGD sequences. This structure is the first of an enzyme that is covalently attached to the cell wall of a Gram-positive bacteria. SCP is also the first functional protease containing a protease-associated domain to have its structure elucidated.

  1. Enteroviral proteases: structure, host interactions and pathogenicity.

    PubMed

    Laitinen, Olli H; Svedin, Emma; Kapell, Sebastian; Nurminen, Anssi; Hytönen, Vesa P; Flodström-Tullberg, Malin

    2016-07-01

    Enteroviruses are common human pathogens, and infections are particularly frequent in children. Severe infections can lead to a variety of diseases, including poliomyelitis, aseptic meningitis, myocarditis and neonatal sepsis. Enterovirus infections have also been implicated in asthmatic exacerbations and type 1 diabetes. The large disease spectrum of the closely related enteroviruses may be partially, but not fully, explained by differences in tissue tropism. The molecular mechanisms by which enteroviruses cause disease are poorly understood, but there is increasing evidence that the two enteroviral proteases, 2A(pro) and 3C(pro) , are important mediators of pathology. These proteases perform the post-translational proteolytic processing of the viral polyprotein, but they also cleave several host-cell proteins in order to promote the production of new virus particles, as well as to evade the cellular antiviral immune responses. Enterovirus-associated processing of cellular proteins may also contribute to pathology, as elegantly demonstrated by the 2A(pro) -mediated cleavage of dystrophin in cardiomyocytes contributing to Coxsackievirus-induced cardiomyopathy. It is likely that improved tools to identify targets for these proteases will reveal additional host protein substrates that can be linked to specific enterovirus-associated diseases. Here, we discuss the function of the enteroviral proteases in the virus replication cycle and review the current knowledge regarding how these proteases modulate the infected cell in order to favour virus replication, including ways to avoid detection by the immune system. We also highlight new possibilities for the identification of protease-specific cellular targets and thereby a way to discover novel mechanisms contributing to disease. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27145174

  2. German cockroach proteases and protease-activated receptor-2 regulate chemokine production and dendritic cell recruitment.

    PubMed

    Day, Scottie B; Ledford, John R; Zhou, Ping; Lewkowich, Ian P; Page, Kristen

    2012-01-01

    We recently showed that serine proteases in German cockroach (GC) feces (frass) decreased experimental asthma through the activation of protease-activated receptor (PAR)-2. Since dendritic cells (DCs) play an important role in the initiation of asthma, we queried the role of GC frass proteases in modulating CCL20 (chemokine C-C motif ligand 20) and granulocyte macrophage colony-stimulating factor (GM-CSF) production, factors that regulate pulmonary DCs. A single exposure to GC frass resulted in a rapid, but transient, increase in GM-CSF and a steady increase in CCL20 in the airways of mice. Instillation of protease-depleted GC frass or instillation of GC frass in PAR-2-deficient mice significantly decreased chemokine release. A specific PAR-2-activating peptide was also sufficient to induce CCL20 production. To directly assess the role of the GC frass protease in chemokine release, we enriched the protease from GC frass and confirmed that the protease was sufficient to induce both GM-CSF and CCL20 production in vivo. Primary airway epithelial cells produced both GM-CSF and CCL20 in a protease- and PAR-2-dependent manner. Finally, we show a decreased percentage of myeloid DCs in the lung following allergen exposure in PAR-2-deficient mice compared to wild-type mice. However, there was no difference in GC frass uptake. Our data indicate that, through the activation of PAR-2, allergen-derived proteases are sufficient to induce CCL20 and GM-CSF production in the airways. This leads to increased recruitment and/or differentiation of myeloid DC populations in the lungs and likely plays an important role in the initiation of allergic airway responses. PMID:21876326

  3. Crystal structure of a novel cysteinless plant Kunitz-type protease inhibitor

    SciTech Connect

    Hansen, Daiane; Macedo-Ribeiro, Sandra; Verissimo, Paula; Yoo Im, Sonia; Sampaio, Misako Uemura; Oliva, Maria Luiza Vilela . E-mail: olivaml.bioq@epm.br

    2007-09-07

    Bauhinia bauhinioides Cruzipain Inhibitor (BbCI) is a cysteine protease inhibitor highly homologous to plant Kunitz-type inhibitors. However, in contrast to classical Kunitz family inhibitors it lacks cysteine residues and therefore disulfide bridges. BbCI is also distinct in the ability to inactivate enzymes belonging to two different classes, cysteine and serine proteases. Besides inhibiting the cysteine protease cruzipain, BbCI also inhibits cathepsin L and the serine proteases HNE (human neutrophil elastase) and PPE (porcine pancreatic elastase). Monoclinic crystals of the recombinant inhibitor that diffract to 1.7 A resolution were obtained using hanging drop method by vapor diffusion at 18 {sup o}C. The refined structure shows the conservative {beta}-trefoil fold features of the Kunitz inhibitors. In BbCI, one of the two characteristic S-S bonds is replaced by the water-mediated interaction between Tyr125 and Gly132. In this work we explore the structural differences between Kunitz-type inhibitors and analyze the essential interactions that maintain the protein structural stability preserving its biological function.

  4. Broad Spectrum Activity of a Lectin-Like Bacterial Serine Protease Family on Human Leukocytes

    PubMed Central

    Ayala-Lujan, Jorge Luis; Vijayakumar, Vidhya; Gong, Mei; Smith, Rachel; Santiago, Araceli E.; Ruiz-Perez, Fernando

    2014-01-01

    The serine protease autotransporter from Enterobacteriaceae (SPATE) family, which number more than 25 proteases with apparent diverse functions, have been phylogenetically divided into two distinct classes, designated 1 and 2. We recently demonstrated that Pic and Tsh, two members of the class-2 SPATE family produced by intestinal and extraintestinal pathogenic E. coli, were able to cleave a number of O-glycosylated proteins on neutrophils and lymphocytes resulting in impaired leukocyte functions. Here we show that most members of the class-2 SPATE family have lectin-like properties and exhibit differential protease activity reliant on glycoprotein type and cell lineage. Protease activity was seen in virtually all tested O-glycosylated proteins including CD34, CD55, CD164, TIM1, TIM3, TIM4 and C1-INH. We also show that although SPATE proteins bound and cleaved glycoproteins more efficiently on granulocytes and monocytes, they also targeted glycoproteins on B, T and natural killer lymphocytes. Finally, we found that the characteristic domain-2 of class-2 SPATEs is not required for glycoprotease activity, but single amino acid mutations in Pic domain-1 to those residues naturally occurring in domain-1 of SepA, were sufficient to hamper Pic glycoprotease activity. This study shows that most class-2 SPATEs have redundant activities and suggest that they may function as immunomodulators at several levels of the immune system. PMID:25251283

  5. Broad spectrum activity of a lectin-like bacterial serine protease family on human leukocytes.

    PubMed

    Ayala-Lujan, Jorge Luis; Vijayakumar, Vidhya; Gong, Mei; Smith, Rachel; Santiago, Araceli E; Ruiz-Perez, Fernando

    2014-01-01

    The serine protease autotransporter from Enterobacteriaceae (SPATE) family, which number more than 25 proteases with apparent diverse functions, have been phylogenetically divided into two distinct classes, designated 1 and 2. We recently demonstrated that Pic and Tsh, two members of the class-2 SPATE family produced by intestinal and extraintestinal pathogenic E. coli, were able to cleave a number of O-glycosylated proteins on neutrophils and lymphocytes resulting in impaired leukocyte functions. Here we show that most members of the class-2 SPATE family have lectin-like properties and exhibit differential protease activity reliant on glycoprotein type and cell lineage. Protease activity was seen in virtually all tested O-glycosylated proteins including CD34, CD55, CD164, TIM1, TIM3, TIM4 and C1-INH. We also show that although SPATE proteins bound and cleaved glycoproteins more efficiently on granulocytes and monocytes, they also targeted glycoproteins on B, T and natural killer lymphocytes. Finally, we found that the characteristic domain-2 of class-2 SPATEs is not required for glycoprotease activity, but single amino acid mutations in Pic domain-1 to those residues naturally occurring in domain-1 of SepA, were sufficient to hamper Pic glycoprotease activity. This study shows that most class-2 SPATEs have redundant activities and suggest that they may function as immunomodulators at several levels of the immune system. PMID:25251283

  6. Thermostable alkaline halophilic-protease production by Natronolimnobius innermongolicus WN18.

    PubMed

    Selim, Samy; Hagagy, Nashwa; Abdel Aziz, Mohamed; El-Meleigy, El Syaed; Pessione, Enrica

    2014-01-01

    This study reports the production and biochemical characterisation of a thermostable alkaline halophilic protease from Natronolimnobius innermongolicus WN18 (HQ658997), isolated from soda Lake of Wadi An-Natrun, Egypt. The enzyme was concentrated by spinning through a centriplus, centrifugal ultrafiltration Millipore membrane with a total yield of 25%. The relative molecular mass of this protease determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis ranged from 67 to 43 kDa. The extracellular protease of N. innermongolicus WN18 was dependent on high salt concentrations for activity and stability, and it had an optimum temperature of 60°C in the presence of 2.5 M NaCl. This enzyme was stable in a broad pH range (6-12) with an optimum pH of 9-10 for azocasein hydrolysis. This extracellular protease, therefore, could be defined as thermostable and haloalkaliphilic with distinct properties that make the enzyme applicable for different industrial purposes. PMID:24716669

  7. Using specificity to strategically target proteases

    PubMed Central

    Lim, Mark D.; Craik, Charles S.

    2009-01-01

    Proteases are a family of naturally occurring enzymes in the body whose dysregulation has been implicated in numerous diseases and cancers. Their ability to selectively and catalytically turnover substrate adds both signal amplification and functionality as parameters for the detection of disease. This review will focus on the development of activity-based methodologies to characterize proteases, and in particular, the use of positional scanning, synthetic combinatorial libraries (PS-SCL’s), and substrate activity screening (SAS) assays. The use of these approaches to better understand a protease’s natural substrate will be discussed as well as the technologies that emerged. PMID:18434168

  8. Detection of protease and protease activity using a single nanoscrescent SERS probe

    DOEpatents

    Liu, Gang L.; Ellman, Jonathan A.; Lee, Luke P.; Chen, Fanqing Frank

    2013-01-29

    This invention pertains to the in vitro detection of proteases using a single peptide-conjugate nanocrescent surface enhanced Raman scattering (SERS) probes with at least nanomolar sensitivity. The probe enables detection of proteolytic activity in extremely small volume and at low concentration. In certain embodiments the probes comprise an indicator for the detection of an active protease, where the indicator comprises a nanocrescent attached to a peptide, where said peptide comprises a recognition site for the protease and a Raman tag attached to the peptide.

  9. Nepenthesin protease activity indicates digestive fluid dynamics in carnivorous nepenthes plants.

    PubMed

    Buch, Franziska; Kaman, Wendy E; Bikker, Floris J; Yilamujiang, Ayufu; Mithöfer, Axel

    2015-01-01

    Carnivorous plants use different morphological features to attract, trap and digest prey, mainly insects. Plants from the genus Nepenthes possess specialized leaves called pitchers that function as pitfall-traps. These pitchers are filled with a digestive fluid that is generated by the plants themselves. In order to digest caught prey in their pitchers, Nepenthes plants produce various hydrolytic enzymes including aspartic proteases, nepenthesins (Nep). Knowledge about the generation and induction of these proteases is limited. Here, by employing a FRET (fluorescent resonance energy transfer)-based technique that uses a synthetic fluorescent substrate an easy and rapid detection of protease activities in the digestive fluids of various Nepenthes species was feasible. Biochemical studies and the heterologously expressed Nep II from Nepenthes mirabilis proved that the proteolytic activity relied on aspartic proteases, however an acid-mediated auto-activation mechanism was necessary. Employing the FRET-based approach, the induction and dynamics of nepenthesin in the digestive pitcher fluid of various Nepenthes plants could be studied directly with insect (Drosophila melanogaster) prey or plant material. Moreover, we observed that proteolytic activity was induced by the phytohormone jasmonic acid but not by salicylic acid suggesting that jasmonate-dependent signaling pathways are involved in plant carnivory. PMID:25750992

  10. The Clp Chaperones and Proteases of the Human Malaria Parasite Plasmodium falciparum

    SciTech Connect

    M El Bakkouri; A Pow; A Mulichak; K Cheung; J Artz; M Amani; S Fell; T de Koning-Ward; C Goodman; et al.

    2011-12-31

    The Clpchaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clpchaperones and proteases in the humanmalariaparasitePlasmodiumfalciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clpchaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.

  11. The Clp Chaperones and Proteases of the Human Malaria Parasite Plasmodium falciparum

    SciTech Connect

    Bakkouri, Majida El; Pow, Andre; Mulichak, Anne; Cheung, Kevin L.Y.; Artz, Jennifer D.; Amani, Mehrnaz; Fell, Stuart; de Koning-Ward, Tania F.; Goodman, C. Dean; McFadden, Geoffrey I.; Ortega, Joaquin; Hui, Raymond; Houry, Walid A.

    2015-02-09

    The Clp chaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clp chaperones and proteases in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clp chaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.

  12. Structure of granzyme C reveals an unusual mechanism of protease autoinhibition

    SciTech Connect

    Kaiserman, Dion; Buckle, Ashley M.; Van Damme, Petra; Irving, James A.; Law, Ruby H.P.; Matthews, Antony Y.; Bashtannyk-Puhalovich, Tanya; Langendorf, Chris; Thompson, Philip; Vandekerckhove, Joël; Gevaert, Kris; Whisstock, James C.; Bird, Phillip I.

    2009-05-21

    Proteases act in important homeostatic pathways and are tightly regulated. Here, we report an unusual structural mechanism of regulation observed by the 2.5-{angstrom} X-ray crystal structure of the serine protease, granzyme C. Although the active-site triad residues adopt canonical conformations, the oxyanion hole is improperly formed, and access to the primary specificity (S1) pocket is blocked through a reversible rearrangement involving Phe-191. Specifically, a register shift in the 190-strand preceding the active-site serine leads to Phe-191 filling the S1 pocket. Mutation of a unique Glu-Glu motif at positions 192-193 unlocks the enzyme, which displays chymase activity, and proteomic analysis confirms that activity of the wild-type protease can be released through interactions with an appropriate substrate. The 2.5-{angstrom} structure of the unlocked enzyme reveals unprecedented flexibility in the 190-strand preceding the active-site serine that results in Phe-191 vacating the S1 pocket. Overall, these observations describe a broadly applicable mechanism of protease regulation that cannot be predicted by template-based modeling or bioinformatic approaches alone.

  13. Nepenthesin Protease Activity Indicates Digestive Fluid Dynamics in Carnivorous Nepenthes Plants

    PubMed Central

    Buch, Franziska; Kaman, Wendy E.; Bikker, Floris J.; Yilamujiang, Ayufu; Mithöfer, Axel

    2015-01-01

    Carnivorous plants use different morphological features to attract, trap and digest prey, mainly insects. Plants from the genus Nepenthes possess specialized leaves called pitchers that function as pitfall-traps. These pitchers are filled with a digestive fluid that is generated by the plants themselves. In order to digest caught prey in their pitchers, Nepenthes plants produce various hydrolytic enzymes including aspartic proteases, nepenthesins (Nep). Knowledge about the generation and induction of these proteases is limited. Here, by employing a FRET (fluorescent resonance energy transfer)-based technique that uses a synthetic fluorescent substrate an easy and rapid detection of protease activities in the digestive fluids of various Nepenthes species was feasible. Biochemical studies and the heterologously expressed Nep II from Nepenthes mirabilis proved that the proteolytic activity relied on aspartic proteases, however an acid-mediated auto-activation mechanism was necessary. Employing the FRET-based approach, the induction and dynamics of nepenthesin in the digestive pitcher fluid of various Nepenthes plants could be studied directly with insect (Drosophila melanogaster) prey or plant material. Moreover, we observed that proteolytic activity was induced by the phytohormone jasmonic acid but not by salicylic acid suggesting that jasmonate-dependent signaling pathways are involved in plant carnivory. PMID:25750992

  14. The Flavodiiron Protein Flv3 Functions as a Homo-Oligomer During Stress Acclimation and is Distinct from the Flv1/Flv3 Hetero-Oligomer Specific to the O2 Photoreduction Pathway

    PubMed Central

    Mustila, Henna; Paananen, Pasi; Battchikova, Natalia; Santana-Sánchez, Anita; Muth-Pawlak, Dorota; Hagemann, Martin; Aro, Eva-Mari; Allahverdiyeva, Yagut

    2016-01-01

    The flavodiiron proteins (FDPs) Flv1 and Flv3 in cyanobacteria function in photoreduction of O2 to H2O, without concomitant formation of reactive oxygen species, known as the Mehler-like reaction. Both Flv1 and Flv3 are essential for growth under fluctuating light (FL) intensities, providing protection for PSI. Here we compared the global transcript profiles of the wild type (WT), Δflv1 and Δflv1/Δflv3 grown under constant light (GL) and FL. In the WT, FL induced the largest down-regulation in transcripts involved in carbon-concentrating mechanisms (CCMs), while those of the nitrogen assimilation pathways increased as compared with GL. Already under GL the Δflv1/Δflv3 double mutant demonstrated a partial down-regulation of transcripts for CCM and nitrogen metabolism, while in FL conditions the transcripts for nitrogen assimilation were strongly down-regulated. Many alterations were specific only for Δflv1/Δflv3, and not detected in Δflv1, suggesting that certain transcripts are affected primarily because of the lack of flv3. By constructing the strains overproducing solely either Flv1 or Flv3, we demonstrate that the homo-oligomers of these proteins also function in acclimation of cells to FL, by catalyzing reactions with as yet unidentified components, while the presence of both Flv1 and Flv3 is a prerequisite for the Mehler-like reaction and thus the electron transfer to O2. Considering the low expression of flv1, it is unlikely that the Flv1 homo-oligomer is present in the WT. PMID:26936793

  15. Activation of human monocytic cells by Treponema pallidum and Borrelia burgdorferi lipoproteins and synthetic lipopeptides proceeds via a pathway distinct from that of lipopolysaccharide but involves the transcriptional activator NF-kappa B.

    PubMed Central

    Norgard, M V; Arndt, L L; Akins, D R; Curetty, L L; Harrich, D A; Radolf, J D

    1996-01-01

    There is increasing evidence that lipoproteins of Treponema pallidum and Borrelia burgdorferi are key inflammatory mediators during syphilis and Lyme disease. A principal objective of the present study was to identify more precisely similarities and divergences among lipopolysaccharide (LPS)- and lipoprotein-lipopeptide-induced immune cell signaling events. Like LPS, purified native B. burgdorferi OspA and synthetic analogs of OspA, OspB, and two T. pallidum lipoproteins (Tpp47 and Tpp17) all induced NF-kappa B translocation in THP-1 human monocytoid cells. Acylation of OspA and the synthetic peptides was requisite for cell activation. Polymyxin B abrogated only the response to LPS. By using 70Z/3-derived pre-B-cell lines either lacking or expressing human CD14 (the LPS receptor), it was observed that expression of human CD14 imparted responsiveness to LPS but not to OspA or spirochetal lipopeptides (assessed by induction of NF-kappa B and expression of surface immunoglobulin M). Finally, the biological relevance of the observation that T. pallidum lipoproteins-lipopeptides induce both NF-kappa B and cytokine production in monocytes was supported by the ability of the synthetic analogs to promote human immunodeficiency virus replication in chronically infected U1 monocytoid cells; these observations also suggest a potential mechanism whereby a syphilitic chancre can serve as a cofactor for human immunodeficiency virus transmission. The combined data lend additional support to the proposal that spirochetal lipoproteins and LPS initiate monocyte activation via different cell surface events but that the signaling pathways ultimately converge to produce qualitatively similar cellular responses. PMID:8751937

  16. Leader Peptide-Free In Vitro Reconstitution of Microviridin Biosynthesis Enables Design of Synthetic Protease-Targeted Libraries.

    PubMed

    Reyna-González, Emmanuel; Schmid, Bianca; Petras, Daniel; Süssmuth, Roderich D; Dittmann, Elke

    2016-08-01

    Microviridins are a family of ribosomally synthesized and post-translationally modified peptides with a highly unusual architecture featuring non-canonical lactone as well as lactam rings. Individual variants specifically inhibit different types of serine proteases. Here we have established an efficient in vitro reconstitution approach based on two ATP-grasp ligases that were constitutively activated using covalently attached leader peptides and a GNAT-type N-acetyltransferase. The method facilitates the efficient in vitro one-pot transformation of microviridin core peptides to mature microviridins. The engineering potential of the chemo-enzymatic technology was demonstrated for two synthetic peptide libraries that were used to screen and optimize microviridin variants targeting the serine proteases trypsin and subtilisin. Successive analysis of intermediates revealed distinct structure-activity relationships for respective target proteases. PMID:27336908

  17. Proteases decode the extracellular matrix cryptome.

    PubMed

    Ricard-Blum, Sylvie; Vallet, Sylvain D

    2016-03-01

    The extracellular matrix is comprised of 1100 core-matrisome and matrisome-associated proteins and of glycosaminoglycans. This structural scaffold contributes to the organization and mechanical properties of tissues and modulates cell behavior. The extracellular matrix is dynamic and undergoes constant remodeling, which leads to diseases if uncontrolled. Bioactive fragments, called matricryptins, are released from the extracellular proteins by limited proteolysis and have biological activities on their own. They regulate numerous physiological and pathological processes such as angiogenesis, cancer, diabetes, wound healing, fibrosis and infectious diseases and either improve or worsen the course of diseases depending on the matricryptins and on the molecular and biological contexts. Several protease families release matricryptins from core-matrisome and matrisome-associated proteins both in vitro and in vivo. The major proteases, which decrypt the extracellular matrix, are zinc metalloproteinases of the metzincin superfamily (matrixins, adamalysins and astacins), cysteine proteinases and serine proteases. Some matricryptins act as enzyme inhibitors, further connecting protease and matricryptin fates and providing intricate regulation of major physiopathological processes such as angiogenesis and tumorigenesis. They strengthen the role of the extracellular matrix as a key player in tissue failure and core-matrisome and matrisome-associated proteins as important therapeutic targets. PMID:26382969

  18. Transient ECM protease activity promotes synaptic plasticity.

    PubMed

    Magnowska, Marta; Gorkiewicz, Tomasz; Suska, Anna; Wawrzyniak, Marcin; Rutkowska-Wlodarczyk, Izabela; Kaczmarek, Leszek; Wlodarczyk, Jakub

    2016-01-01

    Activity-dependent proteolysis at a synapse has been recognized as a pivotal factor in controlling dynamic changes in dendritic spine shape and function; however, excessive proteolytic activity is detrimental to the cells. The exact mechanism of control of these seemingly contradictory outcomes of protease activity remains unknown. Here, we reveal that dendritic spine maturation is strictly controlled by the proteolytic activity, and its inhibition by the endogenous inhibitor (Tissue inhibitor of matrix metalloproteinases-1 - TIMP-1). Excessive proteolytic activity impairs long-term potentiation of the synaptic efficacy (LTP), and this impairment could be rescued by inhibition of protease activity. Moreover LTP is altered persistently when the ability of TIMP-1 to inhibit protease activity is abrogated, further demonstrating the role of such inhibition in the promotion of synaptic plasticity under well-defined conditions. We also show that dendritic spine maturation involves an intermediate formation of elongated spines, followed by their conversion into mushroom shape. The formation of mushroom-shaped spines is accompanied by increase in AMPA/NMDA ratio of glutamate receptors. Altogether, our results identify inhibition of protease activity as a critical regulatory mechanism for dendritic spines maturation. PMID:27282248

  19. Proteases and Peptidases of Castor Bean Endosperm

    PubMed Central

    Tully, Raymond E.; Beevers, Harry

    1978-01-01

    The endosperm of castor bean seeds (Ricinus communis L.) contains two —SH-dependent aminopeptidases, one hydrolyzing l-leucine-β-naphthylamide optimally at pH 7.0, and the other hydrolyzing l-proline-β-naphthylamide optimally at pH 7.5. After germination the endosperm contains in addition an —SH-dependent hemoglobin protease, a serine-dependent carboxypeptidase, and at least two —SH-dependent enzymes hydrolyzing the model substrate α-N-benzoyl-dl-arginine-β-naphthylamide (BANA). The carboxypeptidase is active on a variety of N-carbobenzoxy dipeptides, especially N-carbobenzoxy-L-phenylalanine-l-alanine and N-carbobenzoxy-l-tyrosine-l-leucine. The pH optima for the protease, carboxypeptidase, and BANAase acivities are 3.5 to 4.0, 5.0 to 5.5, and 6 to 8, respectively. The two aminopeptidases increased about 4-fold in activity during the first 4 days of growth, concurrent with the period of rapid depletion of storage protein. Activities then declined as the endosperm senesced, but were still evident after 6 days. Senescence was complete by day 7 to 8. Hemoglobin protease, carboxypeptidase, and BANAase activities appeared in the endosperm at day 2 to 3, and reached peak activity at day 5 to 6. The data indicate that the aminopeptidases are involved in the early mobilization of endosperm storage protein, whereas protease, carboxypeptidase, and BANAase may take part in later turnover and/or senescence. PMID:16660598

  20. Transient ECM protease activity promotes synaptic plasticity

    PubMed Central

    Magnowska, Marta; Gorkiewicz, Tomasz; Suska, Anna; Wawrzyniak, Marcin; Rutkowska-Wlodarczyk, Izabela; Kaczmarek, Leszek; Wlodarczyk, Jakub

    2016-01-01

    Activity-dependent proteolysis at a synapse has been recognized as a pivotal factor in controlling dynamic changes in dendritic spine shape and function; however, excessive proteolytic activity is detrimental to the cells. The exact mechanism of control of these seemingly contradictory outcomes of protease activity remains unknown. Here, we reveal that dendritic spine maturation is strictly controlled by the proteolytic activity, and its inhibition by the endogenous inhibitor (Tissue inhibitor of matrix metalloproteinases-1 – TIMP-1). Excessive proteolytic activity impairs long-term potentiation of the synaptic efficacy (LTP), and this impairment could be rescued by inhibition of protease activity. Moreover LTP is altered persistently when the ability of TIMP-1 to inhibit protease activity is abrogated, further demonstrating the role of such inhibition in the promotion of synaptic plasticity under well-defined conditions. We also show that dendritic spine maturation involves an intermediate formation of elongated spines, followed by their conversion into mushroom shape. The formation of mushroom-shaped spines is accompanied by increase in AMPA/NMDA ratio of glutamate receptors. Altogether, our results identify inhibition of protease activity as a critical regulatory mechanism for dendritic spines maturation. PMID:27282248

  1. Emergence of Resistance to Protease Inhibitor Amprenavir in Human Immunodeficiency Virus Type 1-Infected Patients: Selection of Four Alternative Viral Protease Genotypes and Influence of Viral Susceptibility to Coadministered Reverse Transcriptase Nucleoside Inhibitors

    PubMed Central

    Maguire, Michael; Shortino, Denise; Klein, Astrid; Harris, Wendy; Manohitharajah, Varsha; Tisdale, Margaret; Elston, Robert; Yeo, Jane; Randall, Sharon; Xu, Fan; Parker, Hayley; May, Jackie; Snowden, Wendy

    2002-01-01

    Previous data have indicated that the development of resistance to amprenavir, an inhibitor of the human immunodeficiency virus type 1 protease, is associated with the substitution of valine for isoleucine at residue 50 (I50V) in the viral protease. We present further findings from retrospective genotypic and phenotypic analyses of plasma samples from protease inhibitor-naïve and nucleoside reverse transcriptase inhibitor (NRTI)-experienced patients who experienced virological failure while participating in a clinical trial where they had been randomized to receive either amprenavir or indinavir in combination with NRTIs. Paired baseline and on-therapy isolates from 31 of 48 (65%) amprenavir-treated patients analyzed demonstrated the selection of protease mutations. These mutations fell into four distinct categories, characterized by the presence of either I50V, I54L/I54M, I84V, or V32I+I47V and often included accessory mutations, commonly M46I/L. The I50V and I84V genotypes displayed the greatest reductions in susceptibility to amprenavir, although each of the amprenavir-selected genotypes conferred little or no cross-resistance to other protease inhibitors. There was a significant association, for both amprenavir and indinavir, between preexisting baseline resistance to NRTIs subsequently received during the study and development of protease mutations (P = 0.014 and P = 0.031, respectively). Our data provide a comprehensive analysis of the mechanisms by which amprenavir resistance develops during clinical use and present evidence that resistance to concomitant agents in the treatment regimen predisposes to the development of mutations associated with protease inhibitor resistance and treatment failure. PMID:11850255

  2. [Physiology of protease-activated receptors (PARs): involvement of PARs in digestive functions].

    PubMed

    Kawabata, A; Kuroda, R; Hollenberg, M D

    1999-10-01

    The protease-activated receptor (PAR), a G protein-coupled receptor present on cell surface, mediates cellular actions of extracellular proteases. Proteases cleave the extracellular N-terminal of PAR molecules at a specific site, unmasking and exposing a novel N-terminal, a tethered ligand, that binds to the body of receptor molecules resulting in receptor activation. Amongst four distinct PARs that have been cloned, PARs 1, 3 and 4 are activated by thrombin, but PAR-2 is activated by trypsin or mast cell tryptase. Human platelets express two distinct thrombin receptors, PAR-1 and PAR-4, while murine platelets express PAR-3 and PAR-4. Apart from roles of PARs in platelet activation, PARs are distributed to a number of organs in various species, predicting their physiological importance. We have been evaluating agonists specific for each PAR, using multiple procedures including a HEK cell calcium signal receptor desensitization assay. Using specific agonists that we developed, we found the following: 1) the salivary glands express PAR-2 mRNA and secret saliva in response to PAR-2 activation; 2) pancreatic juice secretion occurs following in vivo PAR-2 activation; 3) PAR-1 and PAR-2 modulate duodenal motility. Collectively, PAR plays various physiological and/or pathophysiological roles, especially in the digestive systems, and could be a novel target for drug development. PMID:10629876

  3. A novel protease activity assay using a protease-responsive chaperone protein

    SciTech Connect

    Sao, Kentaro; Murata, Masaharu; Fujisaki, Yuri; Umezaki, Kaori; Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki; Hashizume, Makoto

    2009-06-05

    Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.

  4. Coagulation factor XII protease domain crystal structure

    PubMed Central

    Pathak, M; Wilmann, P; Awford, J; Li, C; Hamad, BK; Fischer, PM; Dreveny, I; Dekker, LV; Emsley, J

    2015-01-01

    Background Coagulation factor XII is a serine protease that is important for kinin generation and blood coagulation, cleaving the substrates plasma kallikrein and FXI. Objective To investigate FXII zymogen activation and substrate recognition by determining the crystal structure of the FXII protease domain. Methods and results A series of recombinant FXII protease constructs were characterized by measurement of cleavage of chromogenic peptide and plasma kallikrein protein substrates. This revealed that the FXII protease construct spanning the light chain has unexpectedly weak proteolytic activity compared to β-FXIIa, which has an additional nine amino acid remnant of the heavy chain present. Consistent with these data, the crystal structure of the light chain protease reveals a zymogen conformation for active site residues Gly193 and Ser195, where the oxyanion hole is absent. The Asp194 side chain salt bridge to Arg73 constitutes an atypical conformation of the 70-loop. In one crystal form, the S1 pocket loops are partially flexible, which is typical of a zymogen. In a second crystal form of the deglycosylated light chain, the S1 pocket loops are ordered, and a short α-helix in the 180-loop of the structure results in an enlarged and distorted S1 pocket with a buried conformation of Asp189, which is critical for P1 Arg substrate recognition. The FXII structures define patches of negative charge surrounding the active site cleft that may be critical for interactions with inhibitors and substrates. Conclusions These data provide the first structural basis for understanding FXII substrate recognition and zymogen activation. PMID:25604127

  5. Expression and characterization of Drosophila signal peptide peptidase-like (sppL), a gene that encodes an intramembrane protease.

    PubMed

    Casso, David J; Liu, Songmei; Biehs, Brian; Kornberg, Thomas B

    2012-01-01

    Intramembrane proteases of the Signal Peptide Peptidase (SPP) family play important roles in developmental, metabolic and signaling pathways. Although vertebrates have one SPP and four SPP-like (SPPL) genes, we found that insect genomes encode one Spp and one SppL. Characterization of the Drosophila sppL gene revealed that the predicted SppL protein is a highly conserved structural homolog of the vertebrate SPPL3 proteases, with a predicted nine-transmembrane topology, an active site containing aspartyl residues within a transmembrane region, and a carboxy-terminal PAL domain. SppL protein localized to both the Golgi and ER. Whereas spp is an essential gene that is required during early larval stages and whereas spp loss-of-function reduced the unfolded protein response (UPR), sppL loss of function had no apparent phenotype. This was unexpected given that genetic knockdown phenotypes in other organisms suggested significant roles for Spp-related proteases. PMID:22439002

  6. Rhomboid intramembrane protease RHBDL4 triggers ER-export and non-canonical secretion of membrane-anchored TGFα

    PubMed Central

    Wunderle, Lina; Knopf, Julia D.; Kühnle, Nathalie; Morlé, Aymeric; Hehn, Beate; Adrain, Colin; Strisovsky, Kvido; Freeman, Matthew; Lemberg, Marius K.

    2016-01-01

    Rhomboid intramembrane proteases are the enzymes that release active epidermal growth factor receptor (EGFR) ligands in Drosophila and C. elegans, but little is known about their functions in mammals. Here we show that the mammalian rhomboid protease RHBDL4 (also known as Rhbdd1) promotes trafficking of several membrane proteins, including the EGFR ligand TGFα, from the endoplasmic reticulum (ER) to the Golgi apparatus, thereby triggering their secretion by extracellular microvesicles. Our data also demonstrate that RHBDL4-dependent trafficking control is regulated by G-protein coupled receptors, suggesting a role for this rhomboid protease in pathological conditions, including EGFR signaling. We propose that RHBDL4 reorganizes trafficking events within the early secretory pathway in response to GPCR signaling. Our work identifies RHBDL4 as a rheostat that tunes secretion dynamics and abundance of specific membrane protein cargoes. PMID:27264103

  7. Rhomboid intramembrane protease RHBDL4 triggers ER-export and non-canonical secretion of membrane-anchored TGFα.

    PubMed

    Wunderle, Lina; Knopf, Julia D; Kühnle, Nathalie; Morlé, Aymeric; Hehn, Beate; Adrain, Colin; Strisovsky, Kvido; Freeman, Matthew; Lemberg, Marius K

    2016-01-01

    Rhomboid intramembrane proteases are the enzymes that release active epidermal growth factor receptor (EGFR) ligands in Drosophila and C. elegans, but little is known about their functions in mammals. Here we show that the mammalian rhomboid protease RHBDL4 (also known as Rhbdd1) promotes trafficking of several membrane proteins, including the EGFR ligand TGFα, from the endoplasmic reticulum (ER) to the Golgi apparatus, thereby triggering their secretion by extracellular microvesicles. Our data also demonstrate that RHBDL4-dependent trafficking control is regulated by G-protein coupled receptors, suggesting a role for this rhomboid protease in pathological conditions, including EGFR signaling. We propose that RHBDL4 reorganizes trafficking events within the early secretory pathway in response to GPCR signaling. Our work identifies RHBDL4 as a rheostat that tunes secretion dynamics and abundance of specific membrane protein cargoes. PMID:27264103

  8. A subset of membrane-altering agents and γ-secretase modulators provoke non-substrate cleavage by rhomboid proteases

    PubMed Central

    Urban, Siniša; Moin, Syed M.

    2015-01-01

    SUMMARY Rhomboid proteases are integral membrane enzymes that regulate cell signaling, adhesion, and organelle homeostasis pathways, making substrate specificity a key feature of their function. Interestingly, we found that perturbing the membrane pharmacologically in living cells had little effect on substrate processing, but induced inappropriate cleavage of non-substrates by rhomboid proteases. A subclass of drugs known to modulate γ-secretase activity acted on the membrane directly and induced non-substrate cleavage by rhomboid proteases, but left true substrate cleavage sites unaltered. These observations highlight an active role for the membrane in guiding rhomboid selectivity, and caution that membrane-targeted drugs should be evaluated for cross-activity against membrane-resident enzymes that are otherwise unrelated to the intended drug target. Further, some γ-secretase-modulating activity or toxicity could partly result from global membrane effects. PMID:25159145

  9. Global Substrate Profiling of Proteases in Human Neutrophil Extracellular Traps Reveals Consensus Motif Predominantly Contributed by Elastase

    PubMed Central

    Knudsen, Giselle M.; Perera, Natascha C.; Jenne, Dieter E.; Murphy, John E.; Craik, Charles S.; Hermiston, Terry W.

    2013-01-01

    Neutrophil extracellular traps (NETs) consist of antimicrobial molecules embedded in a web of extracellular DNA. Formation of NETs is considered to be a defense mechanism utilized by neutrophils to ensnare and kill invading pathogens, and has been recently termed NETosis. Neutrophils can be stimulated to undergo NETosis ex vivo, and are predicted to contain high levels of serine proteases, such as neutrophil elastase (NE), cathepsin G (CG) and proteinase 3 (PR3). Serine proteases are important effectors of neutrophil-mediated immunity, which function directly by degrading pathogenic virulent factors and indirectly via proteolytic activation or deactivation of cytokines, chemokines and receptors. In this study, we utilized a diverse and unbiased peptide library to detect and profile protease activity associated with NETs induced by phorbol-12-myristate-13-acetate (PMA). We obtained a “proteolytic signature” from NETs derived from healthy donor neutrophils and used proteomics to assist in the identification of the source of this proteolytic activity. In addition, we profiled each neutrophil serine protease and included the newly identified enzyme, neutrophil serine protease 4 (NSP4). Each enzyme had overlapping yet distinct endopeptidase activities and often cleaved at unique sites within the same peptide substrate. The dominant proteolytic activity in NETs was attributed to NE; however, cleavage sites corresponding to CG and PR3 activity were evident. When NE was immunodepleted, the remaining activity was attributed to CG and to a lesser extent PR3 and NSP4. Our results suggest that blocking NE activity would abrogate the major protease activity associated with NETs. In addition, the newly identified substrate specificity signatures will guide the design of more specific probes and inhibitors that target NET-associated proteases. PMID:24073241

  10. In Vivo Assessment of Protease Dynamics in Cutaneous Wound Healing by Degradomics Analysis of Porcine Wound Exudates*

    PubMed Central

    Sabino, Fabio; Hermes, Olivia; Egli, Fabian E.; Kockmann, Tobias; Schlage, Pascal; Croizat, Pierre; Kizhakkedathu, Jayachandran N.; Smola, Hans; auf dem Keller, Ulrich

    2015-01-01

    Proteases control complex tissue responses by modulating inflammation, cell proliferation and migration, and matrix remodeling. All these processes are orchestrated in cutaneous wound healing to restore the skin's barrier function upon injury. Altered protease activity has been implicated in the pathogenesis of healing impairments, and proteases are important targets in diagnosis and therapy of this pathology. Global assessment of proteolysis at critical turning points after injury will define crucial events in acute healing that might be disturbed in healing disorders. As optimal biospecimens, wound exudates contain an ideal proteome to detect extracellular proteolytic events, are noninvasively accessible, and can be collected at multiple time points along the healing process from the same wound in the clinics. In this study, we applied multiplexed Terminal Amine Isotopic Labeling of Substrates (TAILS) to globally assess proteolysis in early phases of cutaneous wound healing. By quantitative analysis of proteins and protein N termini in wound fluids from a clinically relevant pig wound model, we identified more than 650 proteins and discerned major healing phases through distinctive abundance clustering of markers of inflammation, granulation tissue formation, and re-epithelialization. TAILS revealed a high degree of proteolysis at all time points after injury by detecting almost 1300 N-terminal peptides in ∼450 proteins. Quantitative positional proteomics mapped pivotal interdependent processing events in the blood coagulation and complement cascades, temporally discerned clotting and fibrinolysis during the healing process, and detected processing of complement C3 at distinct time points after wounding and by different proteases. Exploiting data on primary cleavage specificities, we related candidate proteases to cleavage events and revealed processing of the integrin adapter protein kindlin-3 by caspase-3, generating new hypotheses for protease

  11. In vivo assessment of protease dynamics in cutaneous wound healing by degradomics analysis of porcine wound exudates.

    PubMed

    Sabino, Fabio; Hermes, Olivia; Egli, Fabian E; Kockmann, Tobias; Schlage, Pascal; Croizat, Pierre; Kizhakkedathu, Jayachandran N; Smola, Hans; auf dem Keller, Ulrich

    2015-02-01

    Proteases control complex tissue responses by modulating inflammation, cell proliferation and migration, and matrix remodeling. All these processes are orchestrated in cutaneous wound healing to restore the skin's barrier function upon injury. Altered protease activity has been implicated in the pathogenesis of healing impairments, and proteases are important targets in diagnosis and therapy of this pathology. Global assessment of proteolysis at critical turning points after injury will define crucial events in acute healing that might be disturbed in healing disorders. As optimal biospecimens, wound exudates contain an ideal proteome to detect extracellular proteolytic events, are noninvasively accessible, and can be collected at multiple time points along the healing process from the same wound in the clinics. In this study, we applied multiplexed Terminal Amine Isotopic Labeling of Substrates (TAILS) to globally assess proteolysis in early phases of cutaneous wound healing. By quantitative analysis of proteins and protein N termini in wound fluids from a clinically relevant pig wound model, we identified more than 650 proteins and discerned major healing phases through distinctive abundance clustering of markers of inflammation, granulation tissue formation, and re-epithelialization. TAILS revealed a high degree of proteolysis at all time points after injury by detecting almost 1300 N-terminal peptides in ∼450 proteins. Quantitative positional proteomics mapped pivotal interdependent processing events in the blood coagulation and complement cascades, temporally discerned clotting and fibrinolysis during the healing process, and detected processing of complement C3 at distinct time points after wounding and by different proteases. Exploiting data on primary cleavage specificities, we related candidate proteases to cleavage events and revealed processing of the integrin adapter protein kindlin-3 by caspase-3, generating new hypotheses for protease

  12. p38 MAPK regulates PKAα and CUB-serine protease in Amphibalanus amphitrite cyprids.

    PubMed

    Zhang, Gen; He, Li-Sheng; Him Wong, Yue; Xu, Ying; Zhang, Yu; Qian, Pei-Yuan

    2015-01-01

    The MKK3-p38 MAPK pathway has been reported to mediate larval settlement in Amphibalanus (=Balanus) amphitrite. To clarify the underlying molecular mechanism, we applied label-free proteomics to analyze changes in the proteome of cyprids treated with a p38 MAPK inhibitor. The results showed that the expression levels of 80 proteins were significantly modified (p < 0.05). These differentially expressed proteins were assigned to 15 functional groups according to the KOG database and 9 pathways were significantly enriched. Further analysis revealed that p38 MAPK might regulate the energy supply and metamorphosis. Two potential regulatory proteins, CUB-serine protease and PKAα, were both down-regulated in expression. CUB-serine protease localized to postaxial seta 2 and 3, as well as the 4 subterminal sensilla in the antennule. Importantly, it was co-localized with the neuron transmitter serotonin in the sections, suggesting that the CUB-serine protease was present in the neural system. PKAα was highly expressed during the cyprid and juvenile stages, and it was co-localized with phospho-p38 MAPK (pp38 MAPK) to the cement gland, suggesting that PKAα might have some functions in cement glands. Overall, p38 MAPK might regulate multiple functions in A. amphitrite cyprids, including the energy supply, metamorphosis, neural system and cement glands. PMID:26434953

  13. p38 MAPK regulates PKAα and CUB-serine protease in Amphibalanus amphitrite cyprids

    PubMed Central

    Zhang, Gen; He, Li-Sheng; Him Wong, Yue; Xu, Ying; Zhang, Yu; Qian, Pei-Yuan

    2015-01-01

    The MKK3-p38 MAPK pathway has been reported to mediate larval settlement in Amphibalanus (=Balanus) amphitrite. To clarify the underlying molecular mechanism, we applied label-free proteomics to analyze changes in the proteome of cyprids treated with a p38 MAPK inhibitor. The results showed that the expression levels of 80 proteins were significantly modified (p < 0.05). These differentially expressed proteins were assigned to 15 functional groups according to the KOG database and 9 pathways were significantly enriched. Further analysis revealed that p38 MAPK might regulate the energy supply and metamorphosis. Two potential regulatory proteins, CUB-serine protease and PKAα, were both down-regulated in expression. CUB-serine protease localized to postaxial seta 2 and 3, as well as the 4 subterminal sensilla in the antennule. Importantly, it was co-localized with the neuron transmitter serotonin in the sections, suggesting that the CUB-serine protease was present in the neural system. PKAα was highly expressed during the cyprid and juvenile stages, and it was co-localized with phospho-p38 MAPK (pp38 MAPK) to the cement gland, suggesting that PKAα might have some functions in cement glands. Overall, p38 MAPK might regulate multiple functions in A. amphitrite cyprids, including the energy supply, metamorphosis, neural system and cement glands. PMID:26434953

  14. Deficiency for the cysteine protease cathepsin L promotes tumor progression in mouse epidermis

    PubMed Central

    Dennemärker, J; Lohmüller, T; Mayerle, J; Tacke, M; Lerch, MM; Coussens, LM; Peters, C; Reinheckel, T

    2011-01-01

    To define a functional role for the endosomal/lysosomal cysteine protease cathepsin L (Ctsl) during squamous carcinogenesis, we generated mice harboring a constitutive Ctsl deficiency in addition to epithelial expression of the human papillomavirus type 16 oncogenes (human cytokeratin 14 (K14)–HPV16).We found enhanced tumor progression and metastasis in the absence of Ctsl. As tumor progression in K14–HPV16 mice is dependent on inflammation and angiogenesis, we examined immune cell infiltration and vascularization without finding any effect of the Ctsl genotype. In contrast, keratinocyte-specific transgenic expression of cathepsin V, the human orthologue of mouse Ctsl, in otherwise Ctsl-deficient K14–HPV16 mice restored the phenotype observed in the control HPV16 skin. To better understand this phenotype at the molecular level, we measured several oncogenic signal transduction pathways in primary keratinocytes on stimulation with keratinocyte-conditioned cell culture medium. We found increased activation of protein kinase B/Akt and mitogen-activated protein kinase pathways in protease-deficient cells, especially if treated with media conditioned by Ctsl-deficient keratinocytes. Similarly, the level of active GTP-Ras was increased in Ctsl-deficient epidermis. We conclude that Ctsl is critical for the termination of growth factor signaling in the endosomal/lysosomal compartment of keratinocytes and, therefore, functions as an anti-tumor protease. PMID:20023699

  15. Cloning and nucleotide sequence of the Vibrio cholerae hemagglutinin/protease (HA/protease) gene and construction of an HA/protease-negative strain.

    PubMed Central

    Häse, C C; Finkelstein, R A

    1991-01-01

    The structural gene hap for the extracellular hemagglutinin/protease (HA/protease) of Vibrio cholerae was cloned and sequenced. The cloned DNA fragment contained a 1,827-bp open reading frame potentially encoding a 609-amino-acid polypeptide. The deduced protein contains a putative signal sequence followed by a large propeptide. The extracellular HA/protease consists of 414 amino acids with a computed molecular weight of 46,700. In the absence of protease inhibitors, this is processed to the 32-kDa form which is usually isolated. The deduced amino acid sequence of the mature HA/protease showed 61.5% identity with the Pseudomonas aeruginosa elastase. The cloned hap gene was inactivated and introduced into the chromosome of V. cholerae by recombination to construct the HA/protease-negative strain HAP-1. The cloned fragment containing the hap gene was then shown to complement the mutant strain. Images PMID:2045361

  16. Isolation of a thiol-dependent serine protease in peanut and investigation of its role in the complement and the allergic reaction.

    PubMed

    Javaux, Cédric; Stordeur, Patrick; Azarkan, Mohamed; Mascart, Françoise; Baeyens-Volant, Danielle

    2016-07-01

    A serine protease activity was detected in aqueous peanuts seeds extracts, partially purified and characterized as a thiol-dependent serine protease. The potential role of this proteolytic activity on allergic reaction to peanuts was prospected through complement activation studies in human plasma and serum, and MDCK cells to investigate a possible occludin degradation in tight junctions. The peanut protease activity induced the production of anaphylatoxins C3a and C5a, and of the terminal membrane attack complex SC5b-9 whatever the complement activation pathway. The protease activity was also involved in the partial digestion of occludin within tight junctions, with for result, an increase of the epithelial permeability to antigen absorption. PMID:27280846

  17. Proteases from the Regenerating Gut of the Holothurian Eupentacta fraudatrix

    PubMed Central

    Lamash, Nina E.; Dolmatov, Igor Yu

    2013-01-01

    Four proteases with molecular masses of 132, 58, 53, and 47 kDa were detected in the digestive system of the holothurian Eupentacta fraudatrix. These proteases displayed the gelatinase activity and characteristics of zinc metalloproteinases. The 58 kDa protease had similar protease inhibitor sensitivity to that of mammalian matrix metalloproteinases. Zymographic assay revealed different lytic activities of all four proteases during intestine regeneration in the holothurian. The 132 kDa protease showed the highest activity at the first stage. During morphogenesis (stages 2–4 of regeneration), the highest activity was measured for the 53 and 58 kDa proteases. Inhibition of protease activity exerts a marked effect on regeneration, which was dependent on the time when 1,10-phenanthroline injections commenced. When metalloproteinases were inhibited at the second stage of regeneration, the restoration rates were decreased. However, such an effect proved to be reversible, and when inhibition ceased, the previous rate of regeneration was recovered. When protease activity is inhibited at the first stage, regeneration is completely abolished, and the animals die, suggesting that early activation of the proteases is crucial for triggering the regenerative process in holothurians. The role of the detected proteases in the regeneration processes of holothurians is discussed. PMID:23505505

  18. Initiating protease with modular domains interacts with β-glucan recognition protein to trigger innate immune response in insects

    PubMed Central

    Takahashi, Daisuke; Garcia, Brandon L.; Kanost, Michael R.

    2015-01-01

    The autoactivation of an initiating serine protease upon binding of pattern recognition proteins to pathogen surfaces is a crucial step in eliciting insect immune responses such as the activation of Toll and prophenoloxidase pathways. However, the molecular mechanisms responsible for autoactivation of the initiating protease remains poorly understood. Here, we investigated the molecular basis for the autoactivation of hemolymph protease 14 (HP14), an initiating protease in hemolymph of Manduca sexta, upon the binding of β-1,3-glucan by its recognition protein, βGRP2. Biochemical analysis using HP14 zymogen (proHP14), βGRP2, and the recombinant proteins as truncated forms showed that the amino-terminal modular low-density lipoprotein receptor class A (LA) domains within HP14 are required for proHP14 autoactivation that is stimulated by its interaction with βGRP2. Consistent with this result, recombinant LA domains inhibit the activation of proHP14 and prophenoloxidase, likely by competing with the interaction between βGRP2 and LA domains within proHP14. Using surface plasmon resonance, we demonstrated that immobilized LA domains directly interact with βGRP2 in a calcium-dependent manner and that high-affinity interaction requires the C-terminal glucanase-like domain of βGRP2. Importantly, the affinity of LA domains for βGRP2 increases nearly 100-fold in the presence of β-1,3-glucan. Taken together, these results present the first experimental evidence to our knowledge that LA domains of an insect modular protease and glucanase-like domains of a βGRP mediate their interaction, and that this binding is essential for the protease autoactivation. Thus, our study provides important insight into the molecular basis underlying the initiation of protease cascade in insect immune responses. PMID:26504233

  19. RC1339/APRc from Rickettsia conorii is a novel aspartic protease with properties of retropepsin-like enzymes.

    PubMed

    Cruz, Rui; Huesgen, Pitter; Riley, Sean P; Wlodawer, Alexander; Faro, Carlos; Overall, Christopher M; Martinez, Juan J; Simões, Isaura

    2014-08-01

    Members of the species Rickettsia are obligate intracellular, gram-negative, arthropod-borne pathogens of humans and other mammals. The life-threatening character of diseases caused by many Rickettsia species and the lack of reliable protective vaccine against rickettsioses strengthens the importance of identifying new protein factors for the potential development of innovative therapeutic tools. Herein, we report the identification and characterization of a novel membrane-embedded retropepsin-like homologue, highly conserved in 55 Rickettsia genomes. Using R. conorii gene homologue RC1339 as our working model, we demonstrate that, despite the low overall sequence similarity to retropepsins, the gene product of rc1339 APRc (for Aspartic Protease from Rickettsia conorii) is an active enzyme with features highly reminiscent of this family of aspartic proteases, such as autolytic activity impaired by mutation of the catalytic aspartate, accumulation in the dimeric form, optimal activity at pH 6, and inhibition by specific HIV-1 protease inhibitors. Moreover, specificity preferences determined by a high-throughput profiling approach confirmed common preferences between this novel rickettsial enzyme and other aspartic proteases, both retropepsins and pepsin-like. This is the first report on a retropepsin-like protease in gram-negative intracellular bacteria such as Rickettsia, contributing to the analysis of the evolutionary relationships between the two types of aspartic proteases. Additionally, we have also shown that APRc is transcribed and translated in R. conorii and R. rickettsii and is integrated into the outer membrane of both species. Finally, we demonstrated that APRc is sufficient to catalyze the in vitro processing of two conserved high molecular weight autotransporter adhesin/invasion proteins, Sca5/OmpB and Sca0/OmpA, thereby suggesting the participation of this enzyme in a relevant proteolytic pathway in rickettsial life-cycle. As a novel bona fide member

  20. RC1339/APRc from Rickettsia conorii Is a Novel Aspartic Protease with Properties of Retropepsin-Like Enzymes

    PubMed Central

    Cruz, Rui; Huesgen, Pitter; Riley, Sean P.; Wlodawer, Alexander; Faro, Carlos; Overall, Christopher M.; Martinez, Juan J.; Simões, Isaura

    2014-01-01

    Members of the species Rickettsia are obligate intracellular, gram-negative, arthropod-borne pathogens of humans and other mammals. The life-threatening character of diseases caused by many Rickettsia species and the lack of reliable protective vaccine against rickettsioses strengthens the importance of identifying new protein factors for the potential development of innovative therapeutic tools. Herein, we report the identification and characterization of a novel membrane-embedded retropepsin-like homologue, highly conserved in 55 Rickettsia genomes. Using R. conorii gene homologue RC1339 as our working model, we demonstrate that, despite the low overall sequence similarity to retropepsins, the gene product of rc1339 APRc (for Aspartic Protease from Rickettsia conorii) is an active enzyme with features highly reminiscent of this family of aspartic proteases, such as autolytic activity impaired by mutation of the catalytic aspartate, accumulation in the dimeric form, optimal activity at pH 6, and inhibition by specific HIV-1 protease inhibitors. Moreover, specificity preferences determined by a high-throughput profiling approach confirmed common preferences between this novel rickettsial enzyme and other aspartic proteases, both retropepsins and pepsin-like. This is the first report on a retropepsin-like protease in gram-negative intracellular bacteria such as Rickettsia, contributing to the analysis of the evolutionary relationships between the two types of aspartic proteases. Additionally, we have also shown that APRc is transcribed and translated in R. conorii and R. rickettsii and is integrated into the outer membrane of both species. Finally, we demonstrated that APRc is sufficient to catalyze the in vitro processing of two conserved high molecular weight autotransporter adhesin/invasion proteins, Sca5/OmpB and Sca0/OmpA, thereby suggesting the participation of this enzyme in a relevant proteolytic pathway in rickettsial life-cycle. As a novel bona fide member

  1. Cleavage and activation of a Toll-like receptor by microbial proteases

    PubMed Central

    de Zoete, Marcel R.; Bouwman, Lieneke I.; Keestra, A. Marijke; van Putten, Jos P. M.

    2011-01-01

    Toll-like receptors (TLRs) are innate receptors that show high conservation throughout the animal kingdom. Most TLRs can be clustered into phylogenetic groups that respond to similar types of ligands. One exception is avian TLR15. This receptor does not categorize into one of the existing groups of TLRs and its ligand is still unknown. Here we report that TLR15 is a sensor for secreted virulence-associated fungal and bacterial proteases. Activation of TLR15 involves proteolytic cleavage of the receptor ectodomain and stimulation of NF-κB–dependent gene transcription. Receptor activation can be mimicked by the expression of a truncated TLR15 of which the entire ectodomain is removed, suggesting that receptor cleavage alleviates receptor inhibition by the leucine-rich repeat domain. Our results indicate TLR15 as a unique type of innate immune receptor that combines TLR characteristics with an activation mechanism typical for the evolutionary distinct protease-activated receptors. PMID:21383168

  2. Cleavage and activation of a Toll-like receptor by microbial proteases.

    PubMed

    de Zoete, Marcel R; Bouwman, Lieneke I; Keestra, A Marijke; van Putten, Jos P M

    2011-03-22

    Toll-like receptors (TLRs) are innate receptors that show high conservation throughout the animal kingdom. Most TLRs can be clustered into phylogenetic groups that respond to similar types of ligands. One exception is avian TLR15. This receptor does not categorize into one of the existing groups of TLRs and its ligand is still unknown. Here we report that TLR15 is a sensor for secreted virulence-associated fungal and bacterial proteases. Activation of TLR15 involves proteolytic cleavage of the receptor ectodomain and stimulation of NF-κB-dependent gene transcription. Receptor activation can be mimicked by the expression of a truncated TLR15 of which the entire ectodomain is removed, suggesting that receptor cleavage alleviates receptor inhibition by the leucine-rich repeat domain. Our results indicate TLR15 as a unique type of innate immune receptor that combines TLR characteristics with an activation mechanism typical for the evolutionary distinct protease-activated receptors. PMID:21383168

  3. Cloning and analysis of a Trichinella pseudospiralis muscle larva secreted serine protease gene

    PubMed Central

    Cwiklinski, Krystyna; Meskill, Diana; Robinson, Mark W.; Pozio, E.; Appleton, Judith A.; Connolly, Bernadette

    2009-01-01

    Nematode parasites of the genus Trichinella are intracellular and distinct life cycle stages invade intestinal epithelial and skeletal muscle cells. Within the genus, Trichinella spiralis and Trichinella pseudospiralis exhibit species-specific differences with respect to host-parasite complex formation and host immune modulation. Parasite excretory-secretory (ES) proteins play important roles at the host-parasite interface and are thought to underpin these differences in biology. Serine proteases are among the most abundant group of T. spiralis ES proteins and multiple isoforms of the muscle larvae-specific TspSP-1 serine protease have been identified. Recently, a similar protein (TppSP-1) in T. pseudospiralis muscle larvae was identified. Here we report the cloning and characterisation of the full-length transcript of TppSP-1 and present comparative data between TspSP-1 and TppSP-1. PMID:19054614

  4. Cloning and analysis of a Trichinella pseudospiralis muscle larva secreted serine protease gene.

    PubMed

    Cwiklinski, Krystyna; Meskill, Diana; Robinson, Mark W; Pozio, Eduardo; Appleton, Judith A; Connolly, Bernadette

    2009-02-23

    Nematode parasites of the genus Trichinella are intracellular and distinct life cycle stages invade intestinal epithelial and skeletal muscle cells. Within the genus, Trichinella spiralis and Trichinella pseudospiralis exhibit species-specific differences with respect to host-parasite complex formation and host immune modulation. Parasite excretory-secretory (ES) proteins play important roles at the host-parasite interface and are thought to underpin these differences in biology. Serine proteases are among the most abundant group of T. spiralis ES proteins and multiple isoforms of the muscle larvae-specific TspSP-1 serine protease have been identified. Recently, a similar protein (TppSP-1) in T. pseudospiralis muscle larvae was identified. Here we report the cloning and characterisation of the full-length transcript of TppSP-1 and present comparative data between TspSP-1 and TppSP-1. PMID:19054614

  5. Inhibition of kallikrein-related peptidases by the serine protease inhibitor of Kazal-type 6.

    PubMed

    Kantyka, Tomasz; Fischer, Jan; Wu, Zhihong; Declercq, Wim; Reiss, Karina; Schröder, Jens-Michael; Meyer-Hoffert, Ulf

    2011-06-01

    Kallikrein-related peptidases (KLKs) are a group of serine proteases, expressed in several tissues. Their activity is regulated by inhibitors including members of the serine protease of Kazal-type (SPINK) family. Recently, we discovered that SPINK6 is expressed in human skin and inhibits KLK5, KLK7, KLK14 but not KLK8. In this study we tested whether SPINK6 inhibits other members of the KLK family and caspase-14. Using chromogenic substrates, SPINK6 exhibited inhibitory activity against KLK12 and KLK13 with K(i) around 1nM, KLK4 with K(i)=27.3nM, KLK6 with K(i)=140nM, caspase-14 with a K(i) approximating 1μM and no activity against KLK1, KLK3 and KLK11. Taken together, SPINK6 is a potent inhibitor of distinct KLKs members. PMID:21439340

  6. Enzymatic characterization of germination-specific cysteine protease-1 expressed transiently in cotyledons during the early phase of germination.

    PubMed

    Tsuji, Akihiko; Tsukamoto, Kana; Iwamoto, Keiko; Ito, Yuka; Yuasa, Keizo

    2013-01-01

    Papain-like cysteine protease activity that shows a unique transient expression profile in cotyledons of daikon radish during germination was detected. The enzyme showed a distinct elution pattern on DEAE-cellulose compared with cathepsin B-like and Responsive to dessication-21 cysteine protease. Although this activity was not detected in seed prior to imbibition, the activity increased markedly and reached a maximum at 2 days after imbibition and then decreased rapidly and completely disappeared after 5 days. Using cystatin-Sepharose, the 26 kDa cysteine protease (DRCP26) was isolated from cotyledons at 2 days after imbibition. The deduced amino acid sequence from the cDNA nucleotide sequence indicated that DRCP26 is an orthologue of Arabidopsis unidentified protein, germination-specific cysteine protease-1, belonging to the C1 family of cysteine protease predicted from genetic information. In an effort to characterize the enzymatic properties of DRCP26, the enzyme was purified to homogeneity from cotyledons at 48 h after imbibition. The best synthetic substrate for the enzyme was carbobenzoxy-Phe-Arg-4-methylcoumaryl-7-amide. All model peptides were digested to small peptides by the enzyme, suggesting that DRCP26 possesses broad cleavage specificity. These results indicated that DRCP26 plays a role in the mobilization of storage proteins in the early phase of seed germination. PMID:23112094

  7. Expression profiling and comparative analyses of seven midgut serine proteases from the yellow fever mosquito, Aedes aegypti

    PubMed Central

    Brackney, Doug E.; Isoe, Jun; Black, W.C.; Zamora, Jorge; Foy, Brian D.; Miesfeld, Roger L.; Olson, Ken E.

    2010-01-01

    Aedes aegypti utilizes blood for energy production, egg maturation and replenishment of maternal reserves. The principle midgut enzymes responsible for bloodmeal digestion are endoproteolytic serine-type proteases within the S1.A subfamily. While there are hundreds of serine protease-like genes in the A. aegypti genome, only five are known to be expressed in the midgut. We describe the cloning, sequencing and expression profiling of seven additional serine proteases and provide a genomic and phylogenetic assessment of these findings. Of the seven genes, four are constitutively expressed and three are transcriptionally induced upon blood feeding. The amount of transcriptional induction is strongly correlated among these genes. Alignments reveal that, in general, the conserved catalytic triad, active site and accessory catalytic residues are maintained in these genes and phylogenetic analysis shows that these genes fall within three distinct clades; trypsins, chymotrypsins and serine collagenases. Interestingly, a previously described trypsin consistently arose with other serine collagenases in phylogenetic analyses. These results suggest that multiple gene duplications have arisen within the S1.A subfamily of midgut serine proteases and/or that A. aegypti has evolved an array of proteases with a broad range of substrate specificities for rapid, efficient digestion of bloodmeals. PMID:20100490

  8. Human immunodeficiency virus type 1 viral background plays a major role in development of resistance to protease inhibitors.

    PubMed Central

    Rose, R E; Gong, Y F; Greytok, J A; Bechtold, C M; Terry, B J; Robinson, B S; Alam, M; Colonno, R J; Lin, P F

    1996-01-01

    The observed in vitro and in vivo benefit of combination treatment with anti-human immunodeficiency virus (HIV) agents prompted us to examine the potential of resistance development when two protease inhibitors are used concurrently. Recombinant HIV-1 (NL4-3) proteases containing combined resistance mutations associated with BMS-186318 and A-77003 (or saquinavir) were either inactive or had impaired enzyme activity. Subsequent construction of HIV-1 (NL4-3) proviral clones containing the same mutations yielded viruses that were severely impaired in growth or nonviable, confirming that combination therapy may be advantageous. However, passage of BMS-186318-resistant HIV-1 (RF) in the presence of either saquinavir or SC52151, which represented sequential drug treatment, produced viable viruses resistant to both BMS-186318 and the second compound. The predominant breakthrough virus contained the G48V/A71T/V82A protease mutations. The clone-purified RF (G48V/A71T/V82A) virus, unlike the corresponding defective NL4-3 triple mutant, grew well and displayed cross-resistance to four distinct protease inhibitors. Chimeric virus and in vitro mutagenesis studies indicated that the RF-specific protease sequence, specifically the Ile at residue 10, enabled the NL4-3 strain with the triple mutant to grow. Our results clearly indicate that viral genetic background will play a key role in determining whether cross-resistance variants will arise. Images Fig. 1 PMID:8643685

  9. Comparative study on the protease inhibitors from fish eggs

    NASA Astrophysics Data System (ADS)

    Ustadi; Kim, K. Y.; Kim, S. M.

    2005-07-01

    The protease inhibitor was purified from five different fish eggs. The molecular weights of Pacific herring, chum salmon, pond smelt, glassfish, and Alaska pollock egg protease inhibitors were 120, 89, 84.5, 17, and l6.8kDa, respectively. The specific inhibitory activity of glassfish egg protease inhibitor was the highest followed by those of Pacific herring and Alaska pollock in order. The specific inhibitory activity and purity of glassfish egg protease inhibitor were 19.70 Umg-1 protein and 164.70 folds of purification, respectively. Glassfish egg protease inhibitor was reasonably stable at 50-65°C and pH 8, which was more stable at high temperature and pH than protease inhibitors from the other fish species. Glassfish egg protease inhibitor was noncompetitive with inhibitor constant ( K i) of 4.44 nmolL-1.

  10. New directions for protease inhibitors directed drug discovery.

    PubMed

    Hamada, Yoshio; Kiso, Yoshiaki

    2016-11-01

    Proteases play crucial roles in various biological processes, and their activities are essential for all living organisms-from viruses to humans. Since their functions are closely associated with many pathogenic mechanisms, their inhibitors or activators are important molecular targets for developing treatments for various diseases. Here, we describe drugs/drug candidates that target proteases, such as malarial plasmepsins, β-secretase, virus proteases, and dipeptidyl peptidase-4. Previously, we reported inhibitors of aspartic proteases, such as renin, human immunodeficiency virus type 1 protease, human T-lymphotropic virus type I protease, plasmepsins, and β-secretase, as drug candidates for hypertension, adult T-cell leukaemia, human T-lymphotropic virus type I-associated myelopathy, malaria, and Alzheimer's disease. Our inhibitors are also described in this review article as examples of drugs that target proteases. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 563-579, 2016. PMID:26584340

  11. Two Proteases, Trypsin Domain-containing 1 (Tysnd1) and Peroxisomal Lon Protease (PsLon), Cooperatively Regulate Fatty Acid β-Oxidation in Peroxisomal Matrix*

    PubMed Central

    Okumoto, Kanji; Kametani, Yukari; Fujiki, Yukio

    2011-01-01

    The molecular mechanisms underlying protein turnover and enzyme regulation in the peroxisomal matrix remain largely unknown. Trypsin domain-containing 1 (Tysnd1) and peroxisomal Lon protease (PsLon) are newly identified peroxisomal matrix proteins that harbor both a serine protease-like domain and a peroxisome-targeting signal 1 (PTS1) sequence. Tysnd1 processes several PTS1-containing proteins and cleaves N-terminal presequences from PTS2-containing protein precursors. Here we report that knockdown of Tysnd1, but not PsLon, resulted in accumulation of endogenous β-oxidation enzymes in their premature form. The protease activity of Tysnd1 was inactivated by intermolecular self-conversion of the 60-kDa form to 15- and 45-kDa chains, which were preferentially degraded by PsLon. Peroxisomal β-oxidation of a very long fatty acid was significantly decreased by knockdown of Tysnd1 and partially lowered by PsLon knockdown. Taken together, these data suggest that Tysnd1 is a key regulator of the peroxisomal β-oxidation pathway via proteolytic processing of β-oxidation enzymes. The proteolytic activity of oligomeric Tysnd1 is in turn controlled by self-cleavage of Tysnd1 and degradation of Tysnd1 cleavage products by PsLon. PMID:22002062

  12. Characterization of cysteine proteases in Malian medicinal plants.

    PubMed

    Bah, Sékou; Paulsen, Berit S; Diallo, Drissa; Johansen, Harald T

    2006-09-19

    Extracts form 10 different Malian medicinal plants with a traditional use against schistosomiasis were investigated for their possible content of proteolytic activity. The proteolytic activity was studied by measuring the hydrolysis of two synthetic peptide substrates Z-Ala-Ala-Asn-NHMec and Z-Phe-Arg-NHMec. Legumain- and papain-like activities were found in all tested crude extracts except those from Entada africana, with the papain-like activity being the strongest. Cissus quadrangularis, Securidaca longepedunculata and Stylosanthes erecta extracts showed high proteolytic activities towards both substrates. After gel filtration the proteolytic activity towards the substrate Z-Ala-Ala-Asn-NHMec in root extract of Securidaca longepedunculata appeared to have Mr of 30 and 97kDa, while the activity in extracts from Cissus qu