Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The role of Toll-like receptors (TLRs) in innate immunity to Legionella pneumophila, a gram-negative facultative intracellular bacterium, was studied by using bone marrow-derived macrophages and dendritic cells from TLR2-deficient (TLR2(-/-)), TLR4(-/-), and wild-type (WT) littermate (C57BL/6 x 129Sv) mice. Intracellular growth of L. pneumophila was enhanced within TLR2(-/-) macrophages compared to WT and TLR4(-/-) macrophages. There was no difference in the bacterial growth within dendritic cells from WT and TLR-deficient mice. Production of interleukin-12p40 (IL-12p40) and IL-10 after infection with L. pneumophila was attenuated in TLR2(-/-) macrophages compared to WT and TLR4(-/-) macrophages. Induction of IL-12p40, IL-10, and tumor necrosis factor alpha secretion from macrophages by the L. pneumophila dotO mutant, which cannot multiply within macrophages, and heat-killed bacteria, was similar to that caused by a viable virulent strain. There was no difference between the WT and its mutants in susceptibility to the cytopathic effect of bacteria. An L. pneumophila sonicated lysate induced IL-12p40 production by macrophages, but that of TLR2(-/-) macrophages was significantly lower than those of WT and TLR4(-/-) macrophages. Treatment of L. pneumophila sonicated lysate with proteinase K and heating did not abolish TLR2-dependent IL-12p40 production. Our results show that TLR2, but not TLR4, is involved in murine innate immunity against L. pneumophila, although other TLRs may also contribute to innate immunity against this organism.
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PMID:Differential roles of Toll-like receptors 2 and 4 in in vitro responses of macrophages to Legionella pneumophila. 1561 72

Mycoplasma arthritidis induces toxicity, arthritis, and dermal necrosis in mice. Virulence factors include a superantigen and membrane adhesins and possibly also a bacteriophage component. Here we compare the biological properties of Triton X-114 extracts derived from avirulent and virulent M. arthritidis strains. Macrophage cell lines and resident peritoneal macrophages were used to assess inflammatory potential as indicated by production of tumor necrosis factor alpha, interleukin-6, and/or nitric oxide. The activity resided exclusively within the hydrophobic detergent phase, was unaffected by heat treatment at 100 degrees C for 30 min, and was resistant to proteinase K digestion, suggesting involvement of a lipopeptide. Contamination of extracts with endotoxin or superantigen was excluded. Extracts of the more virulent strain had higher activity than did those of the avirulent strain. Using CHO cells expressing Toll-like receptor 2 (TLR2) or TLR4, both with transfected CD14, we showed that extracts activated these cells via TLR2 but not by TLR4. Also, macrophages from C57BL/6 TLR2(-/-) mice failed to respond to the extracts, whereas those from TLR2(+/+) cells did respond. The preparations from the virulent strain of M. arthritidis were also more potent in activating dendritic cells, as evidenced by up-regulation of major histocompatibility complex class II, CD40, B7-1, and B7-2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent elution of gel slices revealed the presence of three active moieties which corresponded to molecular masses of approximately 24, 28, and 40 kDa. Three active components were also found by reverse-phase chromatography. We suggest that macrophage activation by M. arthritidis could play a significant role in the inflammatory response induced in the host by this organism.
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PMID:Isolation and partial purification of macrophage- and dendritic cell-activating components from Mycoplasma arthritidis: association with organism virulence and involvement with Toll-like receptor 2. 1611 24

There is increasing epidemiologic evidence implying a role for chronic infection in atherosclerosis and that microbial TLR agonists may contribute to this disease. Mycoplasma arthritidis is an agent of acute and chronic inflammatory disease in rodents, and has been used extensively as a model for defining the mechanisms involved in arthritis and other inflammatory diseases. We have purified a 28-kDa, apolipoprotein A-1 (apoA-1)-like TLR2-dependent macrophage-activating moiety from a culture of a virulent strain of M. arthritidis. ApoA-1 similarly isolated from uninoculated mycoplasma medium was without bioactivity. The activity of the mycoplasma-derived molecule was resistant to heat and to digestion with proteinase K, but was susceptible to alkaline hydrolysis and H(2)O(2) oxidation. Infrared profiles of normal apoA-1 and that derived from mycoplasma were distinct. Unlike the activity of other mycoplasmal TLR2 agonists such as macrophage-activating lipopeptide-2, activity of the M. arthritidis-derived 28-kDa component was dependent upon CD14, a coreceptor for LPS. Finally, we showed that bioactive lipopeptides prepared from M. arthritidis grown in serum-free medium and also from a 41-kDa known bioactive lipoprotein of M. arthritidis, avidly bound to purified apoA-1 that separated out by SDS-PAGE, induced TNF-alpha and IL-12p40 both in vitro and in vivo. ApoA-1 is a key functional component of the high-density lipoprotein cholesterol complex by scavenging and removing unwanted lipids. Our finding that this molecule can acquire macrophage-activating properties from microbial TLR2-dependent agonists suggests a novel mechanism whereby some microbial agents might reverse the protective role of apoA-1, thus contributing to the genesis of atherosclerosis.
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PMID:A microbial TLR2 agonist imparts macrophage-activating ability to apolipoprotein A-1. 1698 24

Mycoplasma arthritidis is a naturally occurring murine pathogen, and the disease model has been used extensively to understand inflammatory mechanisms. Recently, Triton X-114 extracts of a virulent strain of M. arthritidis were found to be more potent in activating macrophages than were those from an avirulent strain, suggesting a role in disease. Here, octyl glucoside extraction of cells was used to identify four distinct bioactive moieties, with molecular masses of approximately 41, 37, 34, and 17 kDa. Their bioactivities were resistant to proteinase K but were destroyed by alkaline hydrolysis and oxidation. As for MALP-2, all were dependent upon Toll-like receptor 2, but unlike MALP-2, they were also dependent upon CD14. The M. arthritidis lipoproteins exhibited infrared absorbances at 2,900 cm(-1) and 1,662 cm(-1), similar to those seen in Pam(3)-Cys-Ser-(Lys)(4). Edman degradation failed to reveal N-terminal sequences, suggesting that they were blocked and therefore might be triacylated. However, mass spectrometry of fragments revealed that the 41-kDa moiety, which binds to serum apolipoprotein A-1, had similarity with the recently described MlpD lipoprotein of M. arthritidis.
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PMID:Inflammatory lipoproteins purified from a toxigenic and arthritogenic strain of Mycoplasma arthritidis are dependent on Toll-like receptor 2 and CD14. 1728 6

Chlamydiae components and signaling pathway(s) responsible for the production of proinflammatory cytokines by human monocytes/macrophages are not clearly identified. To this aim, Chlamydia trachomatis-inactivated elementary bodies (EB) as well as the following seven individual Ags were tested for their ability to induce the production of proinflammatory cytokines by human monocytes/macrophages and THP-1 cells: purified LPS, recombinant heat shock protein (rhsp)70, rhsp60, rhsp10, recombinant polypeptide encoded by open reading frame 3 of the plasmid (rpgp3), recombinant macrophage infectivity potentiator (rMip), and recombinant outer membrane protein 2 (rOmp2). Aside from EB, rMip displayed the highest ability to induce release of IL-1beta, TNF-alpha, IL-6, and IL-8. rMip proinflammatory activity could not be attributed to Escherichia coli LPS contamination as determined by the Limulus Amoebocyte lysate assay, insensitivity to polymyxin B (50 microg/ml), and different serum requirement. We have recently demonstrated that Mip is a "classical" bacterial lipoprotein, exposed at the surface of EB. The proinflammatory activity of EB was significantly attenuated in the presence of polyclonal Ab to rMip. Native Mip was able to induce TNF-alpha and IL-8 secretion, whereas a nonlipidated C20A rMip variant was not. Proinflammatory activity of rMip was unaffected by heat or proteinase K treatments but was greatly reduced by treatment with lipases, supporting a role of lipid modification in this process. Stimulating pathways appeared to involve TLR2/TLR1/TLR6 with the help of CD14 but not TLR4. These data support a role of Mip lipoprotein in pathogenesis of C. trachomatis-induced inflammatory responses.
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PMID:The proinflammatory cytokine response to Chlamydia trachomatis elementary bodies in human macrophages is partly mediated by a lipoprotein, the macrophage infectivity potentiator, through TLR2/TLR1/TLR6 and CD14. 1817 56

In this study, 2-Cys Plasmodium berghei ANKA (PbA) peroxiredoxin (Prx) was identified as an antigenic protein recognized by an anti-PbA IgE antibody using two-dimensional polyacrylamide gel electrophoresis and proteomic analysis. Innate immune responses to PbAPrx were examined using cells from mice deficient in Toll-like receptors (TLR) or related molecules, and it was demonstrated that responses were severely impaired in TLR4(-/-), MyD88(-/-) and MD-2(-/-) mice, but not in Toll/IL-1 receptor domain-containing adaptor inducing IFN-gamma (TRIF)(-/-), TLR2(-/-) or radioprotective 105 (RP105)(-/-) mice. An association between PbAPrx and TLR4 was observed following immunoprecipitation and immunoblotting, suggesting that PbAPrx was associated with TLR4/MD-2. Interactions between Prx and TLR4/MD-2 were also examined by flow cytometry using TLR4/MD-2- or TLR2-expressing cells. NFkappaB/GFP activity was observed in TLR4/MD-2- but not in TLR2-expressing cells following stimulation with Prx. However, this effect was not observed after treatment with proteinase K, suggesting that PbAPrx is a protein ligand for TLR4 and that the PbAPrx activity observed in this study is not due to contamination with LPS. These findings indicate that malarial Prx induces IgE-mediated protection through FcepsilonRI on mast cells and innate immunity through TLR4 with MyD88 and MD-2, suggesting a novel function for malarial Prx in innate and acquired immune responses in malaria.
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PMID:Mast cell-mediated immune responses through IgE antibody and Toll-like receptor 4 by malarial peroxiredoxin. 1839 34

The acute-phase protein serum amyloid A (SAA) is commonly considered a marker for inflammatory diseases; however, its precise role in inflammation and infection, which often result in neutrophilia, remains ambiguous. In this study, we demonstrate that SAA is a potent endogenous stimulator of granulocyte colony-stimulated factor (G-CSF), a principal cytokine-regulating granulocytosis. This effect of SAA is dependent on Toll-like receptor 2 (TLR2). Our data demonstrate that, in mouse macrophages, both G-CSF mRNA and protein were significantly increased after SAA stimulation. The induction of G-CSF was blocked by an anti-TLR2 antibody and markedly decreased in the TLR2-deficient macrophages. SAA stimulation results in the activation of nuclear factor-kappaB and binding activity to the CK-1 element of the G-CSF promoter region. In vitro reconstitution experiments also support that TLR2 mediates SAA-induced G-CSF expression. In addition, SAA-induced secretion of G-CSF was sensitive to heat and proteinase K treatment, yet insensitive to polymyxin B treatment, indicating that the induction is a direct effect of SAA. Finally, our in vivo studies confirmed that SAA treatment results in a significant increase in plasma G-CSF and neutrophilia, whereas these responses are ablated in G-CSF- or TLR2-deficient mice.
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PMID:Serum amyloid A induces G-CSF expression and neutrophilia via Toll-like receptor 2. 1895 97

Mycoplasma genitalium has been implicated in several important reproductive tract syndromes in women, including pelvic inflammatory disease, cervicitis, and tubal factor infertility. The mechanisms of immune activation are unclear, and we sought to determine whether M. genitalium was capable of activating innate immune responses through ligation of highly expressed Toll-like receptors (TLR) of the genital tract. Using HEK293 cells expressing specific human TLR, viable M. genitalium and the recombinant C-terminal portion of the immunogenic protein MG309 (rMG309c) were shown to activate NF-kappaB via TLR2/6. These data provided a putative mechanism for activation of the innate response in genital tissues. Genital epithelial cells (EC) are the first responders to sexually transmitted pathogens and express high levels of TLR2 and -6. Following exposure to purified rMG309c, vaginal and ecto- and endocervical EC secreted proinflammatory cytokines, including interleukin-6 (IL-6) and IL-8. Vaginal EC were less responsive than cervical EC. The capacity of rMG309c to bind TLR2/6 and elicit inflammation was sensitive to proteinase K digestion and independent of traditional N-terminal lipoylation. Furthermore, the immunostimulatory capacity of rMG309c was localized specifically to a 91-amino-acid subfragment of the recombinant protein, suggesting that TLR activation is likely amino acid based. Together, these data indicated that human vaginal and cervical EC are immunologically responsive to M. genitalium and to purified rMG309c via highly expressed TLR of the genital tract. These findings provide valuable insights into the mechanisms for activation of acute-phase inflammatory responses and suggest that M. genitalium colonization of reproductive tract tissues may result in inflammatory sequelae.
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PMID:Mycoplasma genitalium-encoded MG309 activates NF-kappaB via Toll-like receptors 2 and 6 to elicit proinflammatory cytokine secretion from human genital epithelial cells. 1910 62

Streptococcus suis causes invasive infections in pigs and occasionally in humans. Worldwide, S. suis serotype 2 is most frequently isolated from diseased piglets, but the less virulent serotype 9 is emerging, at least in Europe. We compared the activation of human Toll-like receptors (hTLRs) by S. suis serotype 2 and 9 strains to better understand the role of the innate immune response in fighting S. suis infections. Neither live nor heat-killed log phase grown S. suis activated the hTLR1/2, hTLR2/6 and hTLR4/MD-2 complexes. However, the hTLR2/6 complex was specifically activated by both serotypes after disruption of the cell wall synthesis using penicillin. Activation levels of the hTLR2/6 complex were higher for serotype 9 strains compared to serotype 2 strains suggesting intrinsic differences in cell wall composition between both serotypes. The hTLR2/6 activating fractions decreased in molecular size after digestion with proteinase K and were sensitive for lipoprotein lipase digestion and NaOH hydrolysis, indicating lipoprotein(s) as active component(s). Overall, our results indicate that S. suis lipoproteins activate TLR2/6 but not TLR1/2 and that the clinically different serotypes 2 and 9 display differential release of TLR ligand when cell wall integrity is compromised.
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PMID:Differential activation of the Toll-like receptor 2/6 complex by lipoproteins of Streptococcus suis serotypes 2 and 9. 2004 19

Bacterial fimbriae are an important pathogenic factor. It has been demonstrated that fimbrial protein encoded by fimA gene (FimA fimbriae) of Porphyromonas gingivalis not only contributes to the abilities of bacterial adhesion and invasion to host cells, but also strongly stimulates host innate immune responses. However, FimA fimbriae separated from P. gingivalis ATCC 33277 using a gentle procedure showed very weak proinflammatory activity compared with previous reports. Therefore, in the present study, biological characteristics of FimA fimbriae were further analyzed in terms of proinflammatory activity in macrophages. Macrophages differentiated from THP-1 cells were stimulated with native, heat-denatured, or either proteinase- or lipoprotein lipase-treated FimA fimbriae of P. gingivalis ATCC 33277. Stimulating activities of these FimA fimbriae were evaluated by TNF-alpha-inducing activity in the macrophages. To clarify the mode of action of FimA fimbriae, anti-Toll-like receptor (TLR) 2 blocking antibody was added prior to stimulation. Weak stimulatory activity of native FimA fimbriae was enhanced by heat treatment and low-dose proteinase K treatment. Higher dose of proteinase K treatment abrogated this up-regulation. The activity of treated FimA fimbriae was suppressed by anti-TLR2 antibody, and more substantially by lipoprotein lipase treatment. These results suggest that lipoproteins or lipopeptides associated with FimA fimbriae could at least in part account for signaling via TLR2 and subsequent TNF-alpha production in macrophages.
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PMID:Analysis of immunostimulatory activity of Porphyromonas gingivalis fimbriae conferred by Toll-like receptor 2. 2055 41


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