scholarly journals Nucleic acid recognition by Toll-like receptors is coupled to stepwise processing by cathepsins and asparagine endopeptidase

2011 ◽  
Vol 208 (4) ◽  
pp. 643-651 ◽  
Author(s):  
Sarah E. Ewald ◽  
Alex Engel ◽  
Jiyoun Lee ◽  
Miqi Wang ◽  
Matthew Bogyo ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Cell Types ◽  
Proteolytic Processing ◽  
Toll Like Receptor ◽  
The Core ◽  
Regulatory Strategy ◽  
Mouse Macrophages ◽  
Initial Cleavage ◽  
Analogous Manner ◽  
Multistep Process

Toll-like receptor (TLR) 9 requires proteolytic processing in the endolysosome to initiate signaling in response to DNA. However, recent studies conflict as to which proteases are required for receptor cleavage. We show that TLR9 proteolysis is a multistep process. The first step removes the majority of the ectodomain and can be performed by asparagine endopeptidase (AEP) or cathepsin family members. This initial cleavage event is followed by a trimming event that is solely cathepsin mediated and required for optimal receptor signaling. This dual requirement for AEP and cathepsins is observed in all cell types that we have analyzed, including mouse macrophages and dendritic cells. In addition, we show that TLR7 and TLR3 are processed in an analogous manner. These results define the core proteolytic steps required for TLR9 function and suggest that receptor proteolysis may represent a general regulatory strategy for all TLRs involved in nucleic acid recognition.

Imaging of ribosomal RNA-protein interactions by dark-field STEM

1989 ◽  
Vol 47 ◽  
pp. 250-251
Author(s):  
M. Boublik ◽  
V. Mandiyan ◽  
S. Tumminia ◽  
J.F. Hainfeld ◽  
J.S. Wall
Keyword(s):  
Nucleic Acid ◽  
16S Rrna ◽  
Dark Field ◽  
Radius Of Gyration ◽  
Central Domain ◽  
The Core ◽  
16S Rna ◽  
30S Subunit ◽  
Core Particles

Success in protein-free deposition of native nucleic acid molecules from solutions of selected ionic conditions prompted attempts for high resolution imaging of nucleic acid interactions with proteins, not attainable by conventional EM. Since the nucleic acid molecules can be visualized in the dark-field STEM mode without contrasting by heavy atoms, the established linearity between scattering cross-section and molecular weight can be applied to the determination of their molecular mass (M) linear density (M/L), mass distribution and radius of gyration (RG). Determination of these parameters promotes electron microscopic imaging of biological macromolecules by STEM to a quantitative analytical level. This technique is applied to study the mechanism of 16S rRNA folding during the assembly process of the 30S ribosomal subunit of E. coli. The sequential addition of protein S4 which binds to the 5'end of the 16S rRNA and S8 and S15 which bind to the central domain of the molecule leads to a corresponding increase of mass and increased coiling of the 16S rRNA in the core particles. This increased compactness is evident from the decrease in RG values from 114Å to 91Å (in “ribosomal” buffer consisting of 10 mM Hepes pH 7.6, 60 mM KCl, 2 m Mg(OAc)2, 1 mM DTT). The binding of S20, S17 and S7 which interact with the 5'domain, the central domain and the 3'domain, respectively, continues the trend of mass increase. However, the RG values of the core particles exhibit a reverse trend, an increase to 108Å. In addition, the binding of S7 leads to the formation of a globular mass cluster with a diameter of about 115Å and a mass of ∽300 kDa. The rest of the mass, about 330 kDa, remains loosely coiled giving the particle a “medusa-like” appearance. These results provide direct evidence that 16S RNA undergoes significant structural reorganization during the 30S subunit assembly and show that its interactions with the six primary binding proteins are not sufficient for 16S rRNA coiling into particles resembling the native 30S subunit, contrary to what has been reported in the literature.

scholarly journals Stomatal Development in the Context of Epidermal Tissues

2021 ◽  
Author(s):  
Keiko U Torii
Keyword(s):  
Arabidopsis Thaliana ◽  
Molecular Genetic ◽  
Cell Lineage ◽  
Optimal Growth ◽  
Cell Types ◽  
State Transitions ◽  
Stomatal Development ◽  
Water Control ◽  
Pavement Cells ◽  
The Core

Abstract Background Stomata are adjustable pores on the surface of plant shoots for efficient gas exchange and water control. The presence of stomata is essential for plant growth and survival, and the evolution of stomata is considered as a key developmental innovation of the land plants, allowing colonization on land from aquatic environments some 450 million years ago. In the past two decades, molecular genetic studies using the model plant Arabidopsis thaliana identified key genes and signalling modules that regulate stomatal development: master-regulatory transcription factors that orchestrate cell-state transitions and peptide-receptor signal transduction pathways, which, together, enforce proper patterning of stomata within the epidermis. Studies in diverse plant species, ranging from bryophytes to angiosperm grasses, have begun to unravel the conservation and uniqueness of the core modules in stomatal development. Scope Here, I review the mechanisms of stomatal development in the context of epidermal tissue patterning. First, I introduce the core regulatory mechanisms of stomatal patterning and differentiation in the model species Arabidopsis thaliana. Subsequently, experimental evidence is presented supporting the idea that different cell types within the leaf epidermis, namely stomata, hydathodes pores, pavement cells, and trichomes, either share developmental origins or mutually influence each other’s gene regulatory circuits during development. Emphasis is taken on extrinsic and intrinsic signals regulating the balance between stomata and pavement cells, specifically by controlling the fate of Stomatal-Lineage Ground Cells (SLGCs) to remain within the stomatal-cell lineage or differentiate into pavement cells. Finally, I discuss the influence of inter-tissue-layer communication between the epidermis and underlying mesophyll/vascular tissues on stomatal differentiation. Understanding the dynamic behaviors of stomatal precursor cells and their differentiation in the broader context of tissue and organ development may help design plants tailored for optimal growth and productivity in specific agricultural applications and a changing environment.

scholarly journals Differential Effects of Toll-Like Receptor Activation and Differential Mediation by MAP Kinases of Immune Responses in Microglial Cells

2021 ◽  
Author(s):  
Jaedeok Kwon ◽  
Christos Arsenis ◽  
Maria Suessmilch ◽  
Alison McColl ◽  
Jonathan Cavanagh ◽  
...  
Keyword(s):  
Map Kinase ◽  
Microglial Activation ◽  
Map Kinases ◽  
Mrna Level ◽  
Cell Types ◽  
Receptor Activation ◽  
Microglial Cells ◽  
Toll Like Receptor ◽  
Disease States ◽  
Nitrite Production

AbstractMicroglial activation is believed to play a role in many psychiatric and neurodegenerative diseases. Based largely on evidence from other cell types, it is widely thought that MAP kinase (ERK, JNK and p38) signalling pathways contribute strongly to microglial activation following immune stimuli acting on toll-like receptor (TLR) 3 or TLR4. We report here that exposure of SimA9 mouse microglial cell line to immune mimetics stimulating TLR4 (lipopolysaccharide—LPS) or TLR7/8 (resiquimod/R848), results in marked MAP kinase activation, followed by induction of nitric oxide synthase, and various cytokines/chemokines. However, in contrast to TLR4 or TLR7/8 stimulation, very few effects of TLR3 stimulation by poly-inosine/cytidine (polyI:C) were detected. Induction of chemokines/cytokines at the mRNA level by LPS and resiquimod were, in general, only marginally affected by MAP kinase inhibition, and expression of TNF, Ccl2 and Ccl5 mRNAs, along with nitrite production, were enhanced by p38 inhibition in a stimulus-specific manner. Selective JNK inhibition enhanced Ccl2 and Ccl5 release. Many distinct responses to stimulation of TLR4 and TLR7 were observed, with JNK mediating TNF protein induction by the latter but not the former, and suppressing Ccl5 release by the former but not the latter. These data reveal complex modulation by MAP kinases of microglial responses to immune challenge, including a dampening of some responses. They demonstrate that abnormal levels of JNK or p38 signalling in microglial cells will perturb their profile of cytokine and chemokine release, potentially contributing to abnormal inflammatory patterns in CNS disease states.

scholarly journals Cell-substratum interaction of cultured avian osteoclasts is mediated by specific adhesion structures.

1984 ◽  
Vol 99 (5) ◽  
pp. 1696-1705 ◽  
Author(s):  
P C Marchisio ◽  
D Cirillo ◽  
L Naldini ◽  
M V Primavera ◽  
A Teti ◽  
...  
Keyword(s):  
Electron Microscope ◽  
Intermediate Filaments ◽  
Immunofluorescence Microscopy ◽  
Laying Hens ◽  
Cell Types ◽  
Medullary Bone ◽  
Transformed Cell ◽  
The Core ◽  
Low Calcium Diet

The cell-substratum interaction was studied in cultures of osteoclasts isolated from the medullary bone of laying hens kept on low calcium diet. In fully spread osteoclasts, cell-substratum adhesion mostly occurred within a continuous paramarginal area that corresponded also to the location of a thick network of intermediate filaments of the vimentin type. In this area, regular rows of short protrusions contacting the substratum and often forming a cup-shaped adhesion area were observed in the electron microscope. These short protrusions showed a core of F-actin-containing material presumably organized as a network of microfilaments and surrounded by a rosette-like structure in which vinculin and alpha-actinin were found by immunofluorescence microscopy. Rosettes were superposable to dark circles in interference-reflection microscopy and thus represented circular forms of close cell-substratum contact. The core of ventral protrusions also contained, beside F-actin, fimbrin and alpha-actinin. Villin was absent. This form of cell-substratum contact occurring at the tip of a short ventral protrusion differed from other forms of cell-substratum contact and represented an osteoclast-specific adhesion device that might also be present in in vivo osteoclasts as well as in other normal and transformed cell types.

scholarly journals Differential Requirement for TANK-binding Kinase-1 in Type I Interferon Responses to Toll-like Receptor Activation and Viral Infection

2004 ◽  
Vol 199 (12) ◽  
pp. 1651-1658 ◽  
Author(s):  
Andrea K. Perry ◽  
Edward K. Chow ◽  
Julia B. Goodnough ◽  
Wen-Chen Yeh ◽  
Genhong Cheng
Keyword(s):  
Viral Infection ◽  
Nuclear Translocation ◽  
Sendai Virus ◽  
Cell Types ◽  
Receptor Activation ◽  
Northern Blotting ◽  
Type I ◽  
Toll Like Receptor ◽  
Embryonic Fibroblasts ◽  
Iκb Kinase

TANK-binding kinase-1 (TBK1) and the inducible IκB kinase (IKK-i) have been shown recently to activate interferon (IFN) regulatory factor-3 (IRF3), the primary transcription factor regulating induction of type I IFNs. Here, we have compared the role and specificity of TBK1 in the type I IFN response to lipopolysaccharide (LPS), polyI:C, and viral challenge by examining IRF3 nuclear translocation, signal transducer and activator of transcription 1 phosphorylation, and induction of IFN-regulated genes. The LPS and polyI:C-induced IFN responses were abolished and delayed, respectively, in macrophages from mice with a targeted disruption of the TBK1 gene. When challenged with Sendai virus, the IFN response was normal in TBK1−/− macrophages, but defective in TBK1−/− embryonic fibroblasts. Although both TBK1 and IKK-i are expressed in macrophages, only TBK1 but not IKK-i was detected in embryonic fibroblasts by Northern blotting analysis. Furthermore, the IFN response in TBK1−/− embryonic fibroblasts can be restored by reconstitution with wild-type IKK-i but not a mutant IKK-i lacking kinase activity. Thus, our studies suggest that TBK1 plays an important role in the Toll-like receptor–mediated IFN response and is redundant with IKK-i in the response of certain cell types to viral infection.

scholarly journals Toll-Like Receptor 2 Controls the Gamma Interferon Response to Francisella tularensis by Mouse Liver Lymphocytes

2007 ◽  
Vol 75 (11) ◽  
pp. 5338-5345 ◽  
Author(s):  
Kee-Jong Hong ◽  
Jason R. Wickstrum ◽  
Hung-Wen Yeh ◽  
Michael J. Parmely
Keyword(s):  
Francisella Tularensis ◽  
Cell Types ◽  
Gamma Interferon ◽  
Interferon Response ◽  
Interleukin 12 ◽  
Toll Like Receptor ◽  
Wild Type ◽  
Ifn Γ ◽  
Deficient Mice

ABSTRACT The production of gamma interferon (IFN-γ) is a key step in the protective innate immune response to Francisella tularensis. Natural killer cells and T cells in the liver are important sources of this cytokine during primary F. tularensis infections, and interleukin-12 (IL-12) appears to be an essential coactivating cytokine for hepatic IFN-γ expression. The present study was undertaken to determine whether or not macrophages (Mφ) or dendritic cells (DC) provide coactivating signals for the liver IFN-γ response in vitro, whether IL-12 mediates these effects, and whether Toll-like receptor (TLR) signaling is essential to induce this costimulatory activity. Both bone marrow-derived Mφ and DC significantly augmented the IFN-γ response of F. tularensis-challenged liver lymphocytes in vitro. While both cell types produced IL-12p40 in response to F. tularensis challenge, only DC secreted large quantities of IL-12p70. DC from both IL-12p35-deficient and TLR2-deficient mice failed to produce IL-12p70 and did not costimulate liver lymphocytes for IFN-γ production in response to viable F. tularensis organisms. Conversely, liver lymphocytes from TLR2-deficient mice cocultured with wild-type accessory cells produced IFN-γ at levels comparable to those for wild-type hepatic lymphocytes. These findings indicate that TLR2 controls hepatic lymphocyte IFN-γ responses to F. tularensis by regulating DC IL-12 production. While Mφ also coinduced hepatic IFN-γ production in response to F. tularensis, they did so in a fashion less dependent on TLR2.

scholarly journals Core-shell bioprinting as a strategy to apply differentiation factors in a spatially defined manner inside osteochondral tissue substitutes

2021 ◽  
Author(s):  
David Kilian ◽  
Silvia Cometta ◽  
Anne Bernhardt ◽  
Rania Taymour ◽  
Jonas Golde ◽  
...  
Keyword(s):  
Binding Capacity ◽  
Close Contact ◽  
Release Kinetics ◽  
Cell Types ◽  
Core Shell ◽  
The Core ◽  
Differentiation Factors ◽  
Local Supply ◽  
Osteochondral Tissue ◽  
Different Cell Types

Abstract One of the key challenges in osteochondral tissue engineering is to define specified zones with varying material properties, cell types and biochemical factors supporting locally adjusted differentiation into the osteogenic and chondrogenic lineage, respectively. Herein, extrusion-based core-shell bioprinting is introduced as a potent tool allowing a spatially defined delivery of cell types and differentiation factors TGF-β3 and BMP-2 in separated compartments of hydrogel strands, and, therefore, a local supply of matching factors for chondrocytes and osteoblasts. Ink development was based on blends of alginate and methylcellulose, in combination with varying concentrations of the nanoclay Laponite whose high affinity binding capacity for various molecules was exploited. Release kinetics of model molecules was successfully tuned by Laponite addition. Core-shell bioprinting was proven to generate well-oriented compartments within one strand as monitored by optical coherence tomography in a non-invasive manner. Chondrocytes and osteoblasts were applied each in the shell while the respective differentiation factors (TGF-β3, BMP-2) were provided by a Laponite-supported core serving as central factor depot within the strand, allowing directed differentiation of cells in close contact to the core. Experiments with bi-zonal constructs, comprising an osteogenic and a chondrogenic zone, revealed that the local delivery of the factors from the core reduces effects of these factors on the cells in the other scaffold zone. These observations prove the general suitability of the suggested system for co-differentiation of different cell types within a zonal construct.

scholarly journals B cell survival, surface BCR and BAFFR expression, CD74 metabolism, and CD8− dendritic cells require the intramembrane endopeptidase SPPL2A

2012 ◽  
Vol 210 (1) ◽  
pp. 31-40 ◽  
Author(s):  
Hannes Bergmann ◽  
Mehmet Yabas ◽  
Alanna Short ◽  
Lisa Miosge ◽  
Nadine Barthel ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
B Cell ◽  
B Lymphocyte ◽  
B Cell Lymphoma ◽  
Cell Types ◽  
Proteolytic Processing ◽  
Mhc Ii ◽  
Humoral Immunodeficiency ◽  
B Cell Subsets ◽  
New Treatments

Druggable proteins required for B lymphocyte survival and immune responses are an emerging source of new treatments for autoimmunity and lymphoid malignancy. In this study, we show that mice with an inactivating mutation in the intramembrane protease signal peptide peptidase–like 2A (SPPL2A) unexpectedly exhibit profound humoral immunodeficiency and lack mature B cell subsets, mirroring deficiency of the cytokine B cell–activating factor (BAFF). Accumulation of Sppl2a-deficient B cells was rescued by overexpression of the BAFF-induced survival protein B cell lymphoma 2 (BCL2) but not BAFF and was distinguished by low surface BAFF receptor and IgM and IgD B cell receptors. CD8-negative dendritic cells were also greatly decreased. SPPL2A deficiency blocked the proteolytic processing of CD74 MHC II invariant chain in both cell types, causing dramatic build-up of the p8 product of Cathepsin S and interfering with earlier steps in CD74 endosomal retention and processing. The findings illuminate an important role for the final step in the CD74–MHC II pathway and a new target for protease inhibitor treatment of B cell diseases.

scholarly journals Nucleic acid agonists for Toll-like receptor 7 are defined by the presence of uridine ribonucleotides

2006 ◽  
Vol 36 (12) ◽  
pp. 3256-3267 ◽  
Author(s):  
Sandra S. Diebold ◽  
Catherine Massacrier ◽  
Shizuo Akira ◽  
Carine Paturel ◽  
Yannis Morel ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Toll Like Receptor
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