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Sample records for adapter protein myd88

  1. The Brucella TIR-like protein TcpB interacts with the death domain of MyD88.

    PubMed

    Chaudhary, Anu; Ganguly, Kumkum; Cabantous, Stéphanie; Waldo, Geoffrey S; Micheva-Viteva, Sofiya N; Nag, Kamalika; Hlavacek, William S; Tung, Chang-Shung

    2012-01-06

    The pathogen Brucella melitensis secretes a Toll/interleukin-1 receptor (TIR) domain containing protein that abrogates host innate immune responses. In this study, we have characterized the biochemical interactions of Brucella TIR-like protein TcpB with host innate immune adaptor proteins. Using protein-fragment complementation assays based on Gaussia luciferase and green fluorescent protein, we find that TcpB interacts directly with MyD88 and that this interaction is significantly stronger than the interaction of TcpB with TIRAP, the only other adaptor protein that detectably interacts with TcpB. Surprisingly, the TcpB-MyD88 interaction depends on the death domain (DD) of MyD88, and TcpB does not interact with the isolated TIR domain of MyD88. TcpB disrupts MyD88(DD)-MyD88(DD), MyD88(DD)-MyD88(TIR) and MyD88(DD)-MyD88 interactions but not MyD88-MyD88 or MyD88(TIR)-MyD88(TIR) interactions. Structural models consistent with these results suggest how TcpB might inhibit TLR signaling by targeting MyD88 via a DD-TIR domain interface.

  2. Targeting the TLR9-MyD88 pathway in the regulation of adaptive immune responses

    PubMed Central

    Huang, Xiaopei; Yang, Yiping

    2010-01-01

    IMPORTANCE OF THE FIELD Toll-like receptors (TLRs) are innate immune receptors critical in the innate immune defense against invading pathogens. Recent advances also reveal a crucial role for TLRs in shaping adaptive immune responses, conferring a potential therapeutic value to their modulation in the treatment of diseases. AREAS COVERED IN THIS REVIEW The aim of this review is to discuss TLR9, the TLR9-MyD88 signaling pathway and its role in regulation of adaptive immune responses, as well as potential therapeutic implications by targeting this pathway. WHAT THE READER WILL GAIN This review shows that the TLR9-MyD88 signaling pathway plays a critical role in promoting adaptive immune responses and that modulation of this pathway may have enormous therapeutic potential in enhancing vaccine potency, controlling autoimmunity, as well as improving the outcome of viral vector-mediated gene therapy. TAKE HOME MESSAGE Although TLR9 agonists have been used as adjuvants for enhancing vaccine potency, further exploitation of the TLR9-MyD88 pathway and its dynamic interaction with the immune system in vivo is needed to provide more effective therapeutic inventions in the design of vaccines for infectious diseases, allergies and cancer, in the control of autoimmunity, as well as in the improvement of viral vector-mediated gene therapy. PMID:20560798

  3. Ultrastructural distribution of the death-domain-containing MyD88 protein in HeLa cells.

    PubMed

    Jaunin, F; Burns, K; Tschopp, J; Martin, T E; Fakan, S

    1998-08-25

    MyD88, a protein implicated in interleukin-1 signaling, was localized in HeLa cells transiently transfected with an epitope-tagged (flag) version of MyD88. Overexpression of MyD88 can induce apoptosis. We have analyzed the fine structural intracellular distribution of MyD88 using immunoelectron microscopy. MyD88 is localized to the nucleus and to the cytoplasm as revealed by immunofluorescence visualization. Ultrastructural immunocytochemistry shows that, in the cytoplasm, this protein is associated with fibrillar aggregates containing beta-actin. In the nucleus, MyD88 was found in fibrillar domains present only in cells not yet displaying morphological signs of apoptosis. These domains are not derived from nucleoli and do not constitute an accumulation site of splicing factors. We suggest that such structures could be involved in the formation of the apoptotic bodies and/or in the modification of the nuclear structure and of nucleocytoplasmic trafficking during apoptosis.

  4. The Adaptor Protein Myd88 Is a Key Signaling Molecule in the Pathogenesis of Irinotecan-Induced Intestinal Mucositis.

    PubMed

    Wong, Deysi V T; Lima-Júnior, Roberto C P; Carvalho, Cibele B M; Borges, Vanessa F; Wanderley, Carlos W S; Bem, Amanda X C; Leite, Caio A V G; Teixeira, Maraiza A; Batista, Gabriela L P; Silva, Rangel L; Cunha, Thiago M; Brito, Gerly A C; Almeida, Paulo R C; Cunha, Fernando Q; Ribeiro, Ronaldo A

    2015-01-01

    Intestinal mucositis is a common side effect of irinotecan-based anticancer regimens. Mucositis causes cell damage, bacterial/endotoxin translocation and production of cytokines including IL-1 and IL-18. These molecules and toll-like receptors (TLRs) activate a common signaling pathway that involves the Myeloid Differentiation adaptor protein, MyD88, whose role in intestinal mucositis is unknown. Then, we evaluated the involvement of TLRs and MyD88 in the pathogenesis of irinotecan-induced intestinal mucositis. MyD88-, TLR2- or TLR9-knockout mice and C57BL/6 (WT) mice were given either saline or irinotecan (75 mg/kg, i.p. for 4 days). On day 7, animal survival, diarrhea and bacteremia were assessed, and following euthanasia, samples of the ileum were obtained for morphometric analysis, myeloperoxidase (MPO) assay and measurement of pro-inflammatory markers. Irinotecan reduced the animal survival (50%) and induced a pronounced diarrhea, increased bacteremia, neutrophil accumulation in the intestinal tissue, intestinal damage and more than twofold increased expression of MyD88 (200%), TLR9 (400%), TRAF6 (236%), IL-1β (405%), IL-18 (365%), COX-2 (2,777%) and NF-κB (245%) in the WT animals when compared with saline-injected group (P<0.05). Genetic deletion of MyD88, TLR2 or TLR9 effectively controlled the signs of intestinal injury when compared with irinotecan-administered WT controls (P<0.05). In contrast to the MyD88-/- and TLR2-/- mice, the irinotecan-injected TLR9-/- mice showed a reduced survival, a marked diarrhea and an enhanced expression of IL-18 versus irinotecan-injected WT controls. Additionally, the expression of MyD88 was reduced in the TLR2-/- or TLR9-/- mice. This study shows a critical role of the MyD88-mediated TLR2 and TLR9 signaling in the pathogenesis of irinotecan-induced intestinal mucositis.

  5. The Adaptor Protein Myd88 Is a Key Signaling Molecule in the Pathogenesis of Irinotecan-Induced Intestinal Mucositis

    PubMed Central

    Wong, Deysi V. T.; Lima-Júnior, Roberto C. P.; Carvalho, Cibele B. M.; Borges, Vanessa F.; Wanderley, Carlos W. S.; Bem, Amanda X. C.; Leite, Caio A. V. G.; Teixeira, Maraiza A.; Batista, Gabriela L. P.; Silva, Rangel L.; Cunha, Thiago M.; Brito, Gerly A. C.; Almeida, Paulo R. C.; Cunha, Fernando Q.; Ribeiro, Ronaldo A.

    2015-01-01

    Intestinal mucositis is a common side effect of irinotecan-based anticancer regimens. Mucositis causes cell damage, bacterial/endotoxin translocation and production of cytokines including IL–1 and IL–18. These molecules and toll-like receptors (TLRs) activate a common signaling pathway that involves the Myeloid Differentiation adaptor protein, MyD88, whose role in intestinal mucositis is unknown. Then, we evaluated the involvement of TLRs and MyD88 in the pathogenesis of irinotecan-induced intestinal mucositis. MyD88-, TLR2- or TLR9-knockout mice and C57BL/6 (WT) mice were given either saline or irinotecan (75 mg/kg, i.p. for 4 days). On day 7, animal survival, diarrhea and bacteremia were assessed, and following euthanasia, samples of the ileum were obtained for morphometric analysis, myeloperoxidase (MPO) assay and measurement of pro-inflammatory markers. Irinotecan reduced the animal survival (50%) and induced a pronounced diarrhea, increased bacteremia, neutrophil accumulation in the intestinal tissue, intestinal damage and more than twofold increased expression of MyD88 (200%), TLR9 (400%), TRAF6 (236%), IL–1β (405%), IL–18 (365%), COX–2 (2,777%) and NF-κB (245%) in the WT animals when compared with saline-injected group (P<0.05). Genetic deletion of MyD88, TLR2 or TLR9 effectively controlled the signs of intestinal injury when compared with irinotecan-administered WT controls (P<0.05). In contrast to the MyD88-/- and TLR2-/- mice, the irinotecan-injected TLR9-/- mice showed a reduced survival, a marked diarrhea and an enhanced expression of IL–18 versus irinotecan-injected WT controls. Additionally, the expression of MyD88 was reduced in the TLR2-/- or TLR9-/- mice. This study shows a critical role of the MyD88-mediated TLR2 and TLR9 signaling in the pathogenesis of irinotecan-induced intestinal mucositis. PMID:26440613

  6. Contribution of TLR4 and MyD88 for adjuvant monophosphoryl lipid A (MPLA) activity in a DNA prime-protein boost HIV-1 vaccine.

    PubMed

    Pouliot, Kimberly; Buglione-Corbett, Rachel; Marty-Roix, Robyn; Montminy-Paquette, Sara; West, Kim; Wang, Shixia; Lu, Shan; Lien, Egil

    2014-09-03

    Recombinant protein vaccines are commonly formulated with an immune-stimulatory compound, or adjuvant, to boost immune responses to a particular antigen. Recent studies have shown that, through recognition of molecular motifs, receptors of the innate immune system are involved in the functions of adjuvants to generate and direct adaptive immune responses. However, it is not clear to which degree those receptors are also important when the adjuvant is used as part of a novel heterologous prime-boost immunization process in which the priming and boosting components are not the same type of vaccines. In the current study, we compared the immune responses elicited by a pentavalent HIV-1 DNA prime-protein boost vaccine in mice deficient in either Toll-like receptor 4 (TLR4) or myeloid differentiation primary response gene 88 (MyD88) to wildtype mice. HIV gp120 protein administered in the boost phase was formulated with either monophosphoryl lipid A (MPLA), QS-21, or Al(OH)3. Endpoint antibody titer, serum cytokine response and T-cell memory response were assessed. Neither TLR4 nor MyD88 deficiency had a significant effect on the immune response of mice given vaccine formulated with QS-21 or Al(OH)3. However, TLR4- and MyD88-deficiency decreased both the antibody and T-cell responses in mice administered HIV gp120 formulated with MPLA. These results further our understanding of the activation of TLR4 and MyD88 by MPLA in the context of a DNA prime/protein boost immunization strategy.

  7. Unique Roles of TLR9- and MyD88-Dependent and -Independent Pathways in Adaptive Immune Responses to AAV-Mediated Gene Transfer.

    PubMed

    Rogers, Geoffrey L; Suzuki, Masataka; Zolotukhin, Irene; Markusic, David M; Morel, Laurence M; Lee, Brendan; Ertl, Hildegund C J; Herzog, Roland W

    2015-01-01

    The immune system represents a significant barrier to successful gene therapy with adeno-associated viral (AAV) vectors. In particular, adaptive immune responses to the viral capsid or the transgene product are of concern. The sensing of AAV by toll-like receptors (TLRs) TLR2 and TLR9 has been suggested to play a role in innate immunity to the virus and may also shape subsequent adaptive immune responses. Here, we investigated the functions of TLR2, TLR9 and the downstream signaling adaptor MyD88 in antibody and CD8+ T-cell responses. Antibody formation against the transgene product occurred largely independently of TLR signaling following gene transfer with AAV1 or AAV2 vectors, whereas loss of signaling through the TLR9-MyD88 pathway substantially reduced CD8+ T-cell responses. In contrast, MyD88 (but neither of the TLRs) regulated antibody responses to capsid. B cell-intrinsic MyD88 was required for the formation of anti-capsid IgG2c independently of vector serotype or route of administration. However, MyD88(-/-) mice instead produced anti-capsid IgG1 that emerged with delayed kinetics but nonetheless completely prevented in vivo readministration. We conclude that there are distinct roles for TLR9 and MyD88 in promoting adaptive immune responses to AAV-mediated gene transfer and that there are redundant MyD88-dependent and MyD88-independent mechanisms that stimulate neutralizing antibody formation against AAV.

  8. Atg5 regulates formation of MyD88 condensed structures and MyD88-dependent signal transduction.

    PubMed

    Inomata, Megumi; Into, Takeshi; Niida, Shumpei; Murakami, Yukitaka

    2013-08-09

    MyD88 is known as an essential adaptor protein for Toll-like receptors (TLRs). Previous studies have shown that transfected MyD88 forms condensed structures in the cytoplasm. However, upon TLR stimulation, there is little formation of endogenous MyD88 condensed structures. Thus, the formation of MyD88 condensed structures is tightly suppressed, but the mechanism and significance of this suppression are currently unknown. Here we show that Atg5, a key regulatory protein of autophagy, inhibits the formation of MyD88 condensed structures. We found that endogenous MyD88 had already formed condensed structures in Atg5-deficient cells and that the formation of condensed structures was further enhanced by TLR stimulation. This suppressive effect of Atg5 may not be associated with autophagic processes because MyD88 itself was not degraded and because TLR stimulation did not induce LC3 punctate formation and LC3 conversion. Immunoprecipitation analysis revealed that Atg5 could interact with MyD88. Furthermore, Atg5 deficiency increased formation of the MyD88-TRAF6 signaling complex induced by TLR stimulation, and it enhanced activation of NF-κB signaling but not MAPKs and Akt. These findings indicate that Atg5 regulates the formation of MyD88 condensed structures through association with MyD88 and eventually exerts a modulatory effect on MyD88-dependent signaling.

  9. Inhibitory effect of miR-125b on hepatitis C virus core protein-induced TLR2/MyD88 signaling in THP-1 cells

    PubMed Central

    Peng, Cheng; Wang, Hua; Zhang, Wen-Jing; Jie, Sheng-Hua; Tong, Qiao-Xia; Lu, Meng-Ji; Yang, Dong-Liang

    2016-01-01

    AIM: To investigate the role of miR-125b in regulating monocyte immune responses induced by hepatitis C virus (HCV) core protein. METHODS: Monocytic THP-1 cells were treated with various concentrations of recombinant HCV core protein, and cytokines and miR-125b expression in these cells were analyzed. The requirement of Toll-like receptor 2 (TLR2) or MyD88 gene for HCV core protein-induced immune responses was determined by the transfection of THP-1 cells with gene knockdown vectors expressing either TLR2 siRNA or MyD88 siRNA. The effect of miR-125b overexpression on TLR2/MyD88 signaling was examined by transfecting THP-1 cells with miR-125b mimic RNA oligos. RESULTS: In response to HCV core protein stimulation, cytokine production was up-regulated and miR-125b expression was down-regulated in THP-1 cells. The modulatory effect of HCV core protein on cellular events was dose-dependent and required functional TLR2 or MyD88 gene. Forced miR-125b expression abolished the HCV core protein-induced enhancement of tumor necrosis factor-α, interleukin (IL)-6, and IL-10 expression by 66%, 54%, and 66%, respectively (P < 0.001), by inhibiting MyD88-mediated signaling, including phosphorylation of NF-κBp65, ERK, and P38. CONCLUSION: The inverse correlation between miR-125b and cytokine expression after HCV core challenge suggests that miR-125b may negatively regulate HCV-induced immune responses by targeting TLR2/MyD88 signaling in monocytes. PMID:27158204

  10. Dual Activation of TRIF and MyD88 Adaptor Proteins by Angiotensin II Evokes Opposing Effects on Pressure, Cardiac Hypertrophy and Inflammatory Gene Expression

    PubMed Central

    Singh, Madhu V.; Cicha, Michael Z.; Meyerholz, David K.; Chapleau, Mark W.; Abboud, François M.

    2015-01-01

    Hypertension is recognized as an immune disorder whereby immune cells play a defining role in the genesis and progression of the disease. The innate immune system and its component toll-like receptors (TLRs) are key determinants of the immunological outcome through their pro-inflammatory response. TLR activated signaling pathways utilize several adaptor proteins of which adaptor proteins MyD88 and TRIF define two major inflammatory pathways. In this study, we compared the contributions of MyD88 and TRIF adaptor proteins to angiotensin II (Ang II)-induced hypertension and cardiac hypertrophy in mice. Deletion of MyD88 did not prevent cardiac hypertrophy and the pressor response to Ang II tended to increase. Moreover, the increase in inflammatory gene expression (Tnfa, Nox4 and Agtr1a) was significantly greater in the heart and kidney of MyD88-deficient mice compared with wild type mice. Thus, pathways involving MyD88 may actually restrain the inflammatory responses. On the other hand, in mice with non-functional TRIF (Trifmut mice), Ang II induced hypertension and cardiac hypertrophy were abrogated, and pro-inflammatory gene expression in heart and kidneys was unchanged or decreased. Our results indicate that Ang II induces activation of a pro-inflammatory innate immune response, causing hypertension, and cardiac hypertrophy. These effects require functional adaptor protein TRIF-mediated pathways. However, the common MyD88 dependent signaling pathway, which is also activated simultaneously by Ang II, paradoxically exerts a negative regulatory influence on these responses. PMID:26195481

  11. Chlamydial heat shock protein 60 induces acute pulmonary inflammation in mice via the Toll-like receptor 4- and MyD88-dependent pathway.

    PubMed

    Bulut, Yonca; Shimada, Kenichi; Wong, Michelle H; Chen, Shuang; Gray, Pearl; Alsabeh, Randa; Doherty, Terence M; Crother, Timothy R; Arditi, Moshe

    2009-07-01

    Heat shock protein 60 derived from Chlamydia pneumoniae (cHSP60) activates Toll-like receptor 4 (TLR4) signaling through the MyD88 pathway in vitro, but it is not known how cHSP60 contributes to C. pneumoniae-induced lung inflammation. We treated wild-type (WT), TLR2(-/-), TLR4(-/-), or MyD88(-/-) mice intratracheally (i.t.) with recombinant cHSP60 (50 microg), UV-killed C. pneumoniae (UVCP; 5 x 10(6) inclusion-forming units/mouse), lipopolysaccharide (2 microg), or phosphate-buffered saline (PBS) and sacrificed mice 24 h later. Bronchoalveolar lavage (BAL) was obtained to measure cell counts and cytokine levels, lungs were analyzed for histopathology, and lung homogenate chemokine concentrations were determined. Bone marrow-derived dendritic cells (BMDDCs) were generated and stimulated with live C. pneumoniae (multiplicity of infection [MOI], 5), UVCP (MOI, 5), or cHSP60 for 24 h, and the expression of costimulatory molecules (CD80 and CD86) was measured by fluorescence-activated cell sorting. cHSP60 induced acute lung inflammation with the same intensity as that of UVCP-induced inflammation in WT mice but not in TLR4(-/-) or MyD88(-/-) mice. cHSP60- and UVCP-induced lung inflammation was associated with increased numbers of cells in BAL, increased neutrophil recruitment, and elevated BAL interleukin-6 (IL-6) levels. Both cHSP60 and UVCP induced IL-6 release and CD80 and CD86 expression in WT cells but not in MyD88(-/-) BMDDCs. cHSP60 stimulated DC activation in a TLR4- and MyD88-dependent manner with an intensity similar to that induced by UVCP. These data suggest that cHSP60 promotes lung inflammation and DC activation via TLR4 and MyD88 and therefore may play a significant role in the pathogenesis of C. pneumoniae-induced chronic inflammatory lung diseases.

  12. MyD88-mediated instructive signals in dendritic cells regulate pulmonary immune responses during respiratory virus infection.

    PubMed

    Rudd, Brian D; Schaller, Matthew A; Smit, Joost J; Kunkel, Steven L; Neupane, Rupak; Kelley, Lara; Berlin, Aaron A; Lukacs, Nicholas W

    2007-05-01

    Respiratory syncytial virus (RSV) is the leading cause of respiratory disease in infants worldwide. The induction of innate immunity and the establishment of adaptive immune responses are influenced by the recognition of pathogen-associated molecular patterns by TLRs. One of the primary pathways for TLR activation is by MyD88 adapter protein signaling. The present studies indicate that MyD88 deficiency profoundly impacts the pulmonary environment in RSV-infected mice characterized by the accumulation of eosinophils and augmented mucus production. Although there was little difference in CD4 T cell accumulation, there was also a significant decrease in conventional dendritic cells recruitment to the lungs of MyD88(-/-) mice. The exacerbation of RSV pathophysiology in MyD88(-/-) mice was associated with an enhanced Th2 cytokine profile that contributed to an inappropriate immune response. Furthermore, bone marrow-derived dendritic cells (BMDC) isolated from MyD88(-/-) mice were incapable of producing two important Th1 instructive signals, IL-12 and delta-like4, upon RSV infection. Although MyD88(-/-) BMDCs infected with RSV did up-regulate costimulatory molecules, they did not up-regulate class II as efficiently and stimulated less IFN-gamma from CD4(+) T cells in vitro compared with wild-type BMDCs. Finally, adoptive transfer of C57BL/6 BMDCs into MyD88(-/-) mice reconstituted Th1 immune responses in vivo, whereas transfer of MyD88(-/-) BMDCs into wild-type mice skewed the RSV responses toward a Th2 phenotype. Taken together, our data indicate that MyD88-mediated pathways are essential for the least pathogenic responses to this viral pathogen through the regulation of important Th1-associated instructive signals.

  13. Myd88 Initiates Early Innate Immune Responses and Promotes CD4 T Cells during Coronavirus Encephalomyelitis

    PubMed Central

    Butchi, Niranjan; Kapil, Parul; Puntambekar, Shweta; Stohlman, Stephen A.; Hinton, David R.

    2015-01-01

    ABSTRACT Myd88 signaling is critical to the control of numerous central nervous system (CNS) infections by promoting both innate and adaptive immune responses. Nevertheless, the extent to which Myd88 regulates type I interferon (IFN) versus proinflammatory factors and T cell function, as well as the anatomical site of action, varies extensively with the pathogen. CNS infection by neurotropic coronavirus with replication confined to the brain and spinal cord induces protective IFN-α/β via Myd88-independent activation of melanoma differentiation-associated gene 5 (MDA5). However, a contribution of Myd88-dependent signals to CNS pathogenesis has not been assessed. Infected Myd88−/− mice failed to control virus, exhibited enhanced clinical disease coincident with increased demyelination, and succumbed to infection within 3 weeks. The induction of IFN-α/β, as well as of proinflammatory cytokines and chemokines, was impaired early during infection. However, defects in both IFN-α/β and select proinflammatory factors were rapidly overcome prior to T cell recruitment. Myd88 deficiency also specifically blunted myeloid and CD4 T cell recruitment into the CNS without affecting CD8 T cells. Moreover, CD4 T cells but not CD8 T cells were impaired in IFN-γ production. Ineffective virus control indeed correlated most prominently with reduced antiviral IFN-γ in the CNS of Myd88−/− mice. The results demonstrate a crucial role for Myd88 both in early induction of innate immune responses during coronavirus-induced encephalomyelitis and in specifically promoting protective CD4 T cell activation. In the absence of these responses, functional CD8 T cells are insufficient to control viral spread within the CNS, resulting in severe demyelination. IMPORTANCE During central nervous system (CNS) infections, signaling through the adaptor protein Myd88 promotes both innate and adaptive immune responses. The extent to which Myd88 regulates antiviral type I IFN, proinflammatory

  14. Giardia lamblia binding immunoglobulin protein triggers maturation of dendritic cells via activation of TLR4-MyD88-p38 and ERK1/2 MAPKs.

    PubMed

    Lee, H-Y; Kim, J; Noh, H J; Kim, H-P; Park, S-J

    2014-12-01

    Much remains unknown about the mammalian immune response to Giardia lamblia, a protozoan pathogen that causes diarrhoeal outbreaks. We fractionated protein extracts of G. lamblia trophozoites by Viva-spin centrifugation, DEAE ion exchange and gel filtration chromatography. Resultant fractions were screened for antigenic molecules by western blots analysis using anti-G. lamblia antibodies (Abs), resulting in identification of G. lamblia binding immunoglobulin protein (GlBiP). Maturation of mouse dendritic cells (DCs) in response to recombinant GlBiP (rGlBiP) was detected by increased expression of surface molecules such as CD80, CD86 and MHC class II; these mature DCs, produced pro-inflammatory cytokines (TNF-α, IL-12 and IL-6). Especially, the truncated rGlBiP containing the heat-shock protein 70 domain-induced cytokine production from mouse DCs. rGlBiP-induced DC activation was initiated by TLR4 in a MyD88-dependent way and occurred through activation of p38 and ERK1/2 MAPKs as well as increased activity of NF-κB and AP-1. Moreover, CD4(+) T cells stimulated with rGlBiP-treated DCs produced high levels of IL-2 and IFN-γ. Together, our results suggest that GlBiP contributes to maturation of DCs via activation of TLR4-MyD88-p38, ERK1/2 MAPK, NF-κB and AP-1.

  15. Pathogenic and protective roles of MyD88 in leukocytes and epithelial cells in mouse models of inflammatory bowel disease

    PubMed Central

    Asquith, Mark J.; Boulard, Olivier; Powrie, Fiona; Maloy, Kevin J.

    2013-01-01

    Background & Aims Toll-Like Receptors (TLR) are innate immune receptors involved in recognition of the intestinal micro-flora; they are expressed by numerous cell types in the intestine, including epithelial cells, myeloid cells and lymphocytes. Little is known about the relative contributions of TLR signaling in distinct cellular compartments to intestinal homeostasis. We aimed to define the roles of TLR signals in distinct cell types in the induction and regulation of chronic intestinal inflammation. Methods We assessed the roles of the shared TLR signaling adaptor protein, MyD88, in several complementary mouse models of inflammatory bowel disease (IBD), mediated by either innate or adaptive immune activation. MyD88-deficient mice and bone marrow chimeras were used to selectively disrupt TLR signals in distinct cellular compartments in the intestine. Results MyD88-dependent activation of myeloid cells was required for development of chronic intestinal inflammation. By contrast, although epithelial cell MyD88 signals were required for host survival, they were insufficient to induce intestinal inflammation in the absence of a MyD88-competent myeloid compartment. MyD88 expression by T cells was not required for their pathogenic and regulatory functions in the intestine. Conclusions Cellular compartmentalization of MyD88 signals in the intestine allow the maintenance of host defense and prevent deleterious inflammatory responses. PMID:20433840

  16. MyD88 Is a Critical Regulator of Hematopoietic Cell-Mediated Neuroprotection Seen after Stroke

    PubMed Central

    Downes, Catherine E.; Wong, Connie H. Y.; Henley, Katya J.; Guio-Aguilar, Pedro L.; Zhang, Moses; Ates, Robert; Mansell, Ashley; Kile, Benjamin T.; Crack, Peter J.

    2013-01-01

    Neuroinflammation is critical in the neural cell death seen in stroke. It has been shown that CNS and peripheral responses drive this neuroinflammatory response in the brain. The Toll-like receptors (TLRs) are important regulators of inflammation in response to both exogenous and endogenous stressors. Taking advantage of a downstream adapter molecule that controls the majority of TLR signalling, this study investigated the role of the TLR adaptor protein myeloid differentiation factor 88 (MyD88) in the control of CNS and peripheral inflammation. Reversible middle-cerebral artery occlusion was used as the model of stroke in vivo; in vitro primary cultured neurons and glia were subject to four hours of oxygen and glucose deprivation (OGD). Both in vitro and in vivo Myd88−/− animals or cells were compared with wild type (WT). We found that after stroke Myd88−/− animals have a larger infarct volume compared to WT animals. Interestingly, in vitro there was no difference between the survival of Myd88−/− and WT cells following OGD, suggesting that peripheral responses were influencing stroke outcome. We therefore generated bone marrow chimeras and found that Myd88−/− animals have a smaller stroke infarct than their radiation naive counterparts if their hematopoietic cells are WT. Furthermore, WT animals have a larger stroke than their radiation naive counterparts if the hematopoietic cells are Myd88−/−. We have demonstrated that MyD88-dependent signalling in the hematopoietic cell lineage reduces infarct size following stroke and that infiltrating cells to the site of neuroinflammation are neuroprotective following stroke. PMID:23483951

  17. TLR signaling adaptor protein MyD88 in primary sensory neurons contributes to persistent inflammatory and neuropathic pain and neuroinflammation

    PubMed Central

    Liu, Xing-Jun; Liu, Tong; Chen, Gang; Wang, Bing; Yu, Xiao-Lu; Yin, Cui; Ji, Ru-Rong

    2016-01-01

    Increasing evidence suggests that neuro-immune and neuro-glial interactions are critically involved in chronic pain sensitization. It is well studied how immune/glial mediators sensitize pain, but how sensory neurons control neuroinflammation remains unclear. We employed Myd88 conditional knockout (CKO) mice, in which Myd88 was deleted in sodium channel subunit Nav1.8-expressing primary sensory neurons, to examine the unique role of neuronal MyD88 in regulating acute and chronic pain, and possible underlying mechanisms. We found that baseline pain and the formalin induced acute inflammatory pain were intact in CKO mice. However, the late phase inflammatory pain following complete Freund’s adjuvant injection and the late phase neuropathic pain following chronic constriction injury (CCI), were reduced in CKO mice. CCI induced up-regulation of MyD88 and chemokine C-C motif ligand 2 expression in DRG neurons and macrophage infiltration into DRGs, and microglia activation in spinal dorsal horns in wild-type mice, but all these changes were compromised in CKO mice. Finally, the pain hypersensitivity induced by intraplantar IL-1β was reduced in CKO mice. Our findings suggest that MyD88 in primary sensory neurons plays an active role in regulating IL-1β signaling and neuroinflammation in the peripheral and the central nervous systems, and contributes to the maintenance of persistent pain. PMID:27312666

  18. Elucidation of Novel Structural Scaffold in Rohu TLR2 and Its Binding Site Analysis with Peptidoglycan, Lipoteichoic Acid and Zymosan Ligands, and Downstream MyD88 Adaptor Protein

    PubMed Central

    Sahoo, Bikash Ranjan; Basu, Madhubanti; Swain, Banikalyan; Dikhit, Manas Ranjan; Jayasankar, Pallipuram; Samanta, Mrinal

    2013-01-01

    Toll-like receptors (TLRs) play key roles in sensing wide array of microbial signatures and induction of innate immunity. TLR2 in fish resembles higher eukaryotes by sensing peptidoglycan (PGN) and lipoteichoic acid (LTA) of bacterial cell wall and zymosan of yeasts. However, in fish TLR2, no study yet describes the ligand binding motifs in the leucine rich repeat regions (LRRs) of the extracellular domain (ECD) and important amino acids in TLR2-TIR (toll/interleukin-1 receptor) domain that could be engaged in transmitting downstream signaling. We predicted these in a commercially important freshwater fish species rohu (Labeo rohita) by constructing 3D models of TLR2-ECD, TLR2-TIR, and MyD88-TIR by comparative modeling followed by 40 ns (nanosecond) molecular dynamics simulation (MDS) for TLR2-ECD and 20 ns MDS for TLR2-TIR and MyD88-TIR. Protein (TLR2-ECD)–ligands (PGN, LTA, and zymosan) docking in rohu by AutoDock4.0, FlexX2.1, and GOLD4.1 anticipated LRR16–19, LRR12–14, and LRR20-CT as the most important ligand binding motifs. Protein (TLR2-TIR)—protein (MyD88-TIR) interaction by HADDOCK and ZDOCK predicted BB loop, αB-helix, αC-helix, and CD loop in TLR2-TIR and BB loop, αB-helix, and CD loop in MyD88-TIR as the critical binding domains. This study provides ligands recognition and downstream signaling. PMID:23956969

  19. Molecular cloning and functional characterization of a novel isoform of chicken myeloid differentiation factor 88 (MyD88).

    PubMed

    Qiu, Yafeng; Shen, Yang; Li, Xiangdong; Ding, Chan; Ma, Zhiyong

    2008-01-01

    Myeloid differentiation factor 88 (MyD88) is an adaptor protein involved in the interleukin-1 receptor- and Toll-like receptor-induced activation of nuclear factor-kappaB (NF-kappaB). A novel isoform of chicken MyD88, designated chicken MyD88-2, has been cloned and functionally characterized. Its open reading frame is of length 900bp, and it encodes a predicted 299 residue protein, similar in length to its mammalian orthologues, but, respectively, 77 and 69 amino acids shorter than the previously described chicken MyD88-1 and -3. The amino acid sequence of chicken MyD88-2 displays 96.9%, 96.9%, 70.4% and 70.2% identity with, respectively, chicken MyD88-1, -3, human and mouse MyD88. Chicken MyD88-2 expression was detected in a range of tissues tested, but no expression of either chicken MyD88-1 or -3 was observed. The over-expression of chicken MyD88-2 significantly induced the activation of NF-kappaB in vitro, suggesting that chicken MyD88-2 plays an important role in the innate immune responses of chicken.

  20. Chlamydia trachomatis induces expression of IFN-gamma-inducible protein 10 and IFN-beta independent of TLR2 and TLR4, but largely dependent on MyD88.

    PubMed

    Nagarajan, Uma M; Ojcius, David M; Stahl, Lynn; Rank, Roger G; Darville, Toni

    2005-07-01

    IFN-gamma-inducible protein 10 (IP-10) is a chemokine important in the attraction of T cells, which are essential for resolution of chlamydial genital tract infection. During infections with Gram-negative bacteria, the IP-10 response mediated through type I IFNs usually occurs as a result of TLR4 stimulation by bacterial LPS. However, we found that levels of IP-10 in genital tract secretions of Chlamydia trachomatis-infected female wild-type mice were similar to those of infected TLR2- and TLR4-deficient mice but significantly greater than those of infected MyD88-deficient mice. We investigated the mechanism of IP-10 and IFN-beta induction during chlamydial infection using mouse macrophages and fibroblasts infected ex vivo. The induction of IP-10 and IFN-beta was unchanged in Chlamydia-infected TLR2- and TLR4-deficient cells compared with wild-type cells. However, infection of MyD88-deficient cells resulted in significantly decreased responses. These results suggest a role for MyD88-dependent pathways in induction of IP-10 and IFN-beta during chlamydial infection. Furthermore, treatment of infected macrophages with an endosomal maturation inhibitor significantly reduced chlamydial-induced IFN-beta. Because endosomal maturation is required for MyD88-dependent intracellular pathogen recognition receptors to function, our data suggest a role for the intracellular pathogen recognition receptor(s) in induction of IFN-beta and IP-10 during chlamydial infection. Furthermore, the intracellular pathways that lead to chlamydial-induced IFN-beta function through TANK-binding kinase mediated phosphorylation and nuclear translocation of IFN regulatory factor-3.

  1. Shrimp MyD88 responsive to bacteria and white spot syndrome virus.

    PubMed

    Wen, Rong; Li, Fuhua; Sun, Zheng; Li, Shihao; Xiang, Jianhai

    2013-02-01

    The myeloid differentiation factor 88 (MyD88) is an important adapter protein which links members of the toll-like receptor (TLR) to the downstream components to activate related signaling pathways. In the present study, a MyD88 homolog (FcMyD88) was cloned from penaeid shrimp Fenneropenaeus chinensis. The ORF of FcMyD88 consisted of 1434 bp encoding a polypeptide of 477 amino acids which contains a death domain (DD) and a typical TLR and interleukin-1 receptor (IL-1R)-related (TIR) domain. Homology analysis revealed that the predicted amino acid (aa) sequence of FcMyD88 shared high similarities with a variety of previously reported MyD88s. The time-dependent expression patterns of FcMyD88 in cephalothoraxes of shrimp injected with Vibrio anguillarum (Gram-negative bacteria, G(-)), Micrococcus lysodeikticu (Gram-positive bacteria, G(+)) and white syndrome spot virus (WSSV) were analyzed at transcription and protein level by real-time PCR and western blotting, respectively. The expression level of FcMyD88 mRNA was significantly up-regulated at one hour (h), 12 h and 24 h after stimulation with both V. anguillarum and M. lysodeikticu. The expression level of FcMyD88 protein was 2-fold up-regulated at 12 h post injection (hpi) of inactivated V. anguillarum while it didn't change after M. lysodeikticu injection during this period. After WSSV injection, the expression level of FcMyD88 mRNA remained relatively constant, while the FcMyD88 protein was significantly up-regulated at 12 and 24 hpi. These results suggested that the MyD88-dependent signaling pathway could be involved in the defense of both bacteria and WSSV infection.

  2. Pyogenic bacterial infections in humans with MyD88 deficiency.

    PubMed

    von Bernuth, Horst; Picard, Capucine; Jin, Zhongbo; Pankla, Rungnapa; Xiao, Hui; Ku, Cheng-Lung; Chrabieh, Maya; Mustapha, Imen Ben; Ghandil, Pegah; Camcioglu, Yildiz; Vasconcelos, Júlia; Sirvent, Nicolas; Guedes, Margarida; Vitor, Artur Bonito; Herrero-Mata, María José; Aróstegui, Juan Ignacio; Rodrigo, Carlos; Alsina, Laia; Ruiz-Ortiz, Estibaliz; Juan, Manel; Fortuny, Claudia; Yagüe, Jordi; Antón, Jordi; Pascal, Mariona; Chang, Huey-Hsuan; Janniere, Lucile; Rose, Yoann; Garty, Ben-Zion; Chapel, Helen; Issekutz, Andrew; Maródi, László; Rodriguez-Gallego, Carlos; Banchereau, Jacques; Abel, Laurent; Li, Xiaoxia; Chaussabel, Damien; Puel, Anne; Casanova, Jean-Laurent

    2008-08-01

    MyD88 is a key downstream adapter for most Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs). MyD88 deficiency in mice leads to susceptibility to a broad range of pathogens in experimental settings of infection. We describe a distinct situation in a natural setting of human infection. Nine children with autosomal recessive MyD88 deficiency suffered from life-threatening, often recurrent pyogenic bacterial infections, including invasive pneumococcal disease. However, these patients were otherwise healthy, with normal resistance to other microbes. Their clinical status improved with age, but not due to any cellular leakiness in MyD88 deficiency. The MyD88-dependent TLRs and IL-1Rs are therefore essential for protective immunity to a small number of pyogenic bacteria, but redundant for host defense to most natural infections.

  3. Dendritic cell specific targeting of MyD88 signalling pathways in vivo.

    PubMed

    Arnold-Schrauf, Catharina; Berod, Luciana; Sparwasser, Tim

    2015-01-01

    Dendritic cells (DCs) are key regulators of both innate and adaptive immunity. During infection, DCs recognise pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs) including the Toll-like receptor (TLR) family. TLRs mainly signal via the adaptor protein MyD88. This signalling pathway is required for immune protection during many infections, which are lethal in the absence of MyD88. However, the cell type specific importance of this pathway during both innate and adaptive immune responses against pathogens in vivo remains ill-defined. We discuss recent findings from conditional KO or gain-of-function mouse models targeting TLR/MyD88 signalling pathways in DCs and other myeloid cells during infection. While the general assumption that MyD88-dependent recognition by DCs is essential for inducing protective immunity holds true in some instances, the results surprisingly indicate a much more complex context-dependent requirement for this pathway in DCs and other myeloid or lymphoid cell-types in vivo. Furthermore, we highlight the advantages of Cre-mediated DC targeting approaches and their possible limitations. We also present future perspectives on the development of new genetic mouse models to target distinct DC subsets in vivo. Such models will serve to understand the functional heterogeneity of DCs in vivo.

  4. Myeloid-Specific Gene Deletion of Protein Phosphatase 2A Magnifies MyD88- and TRIF-Dependent Inflammation following Endotoxin Challenge.

    PubMed

    Sun, Lei; Pham, Tiffany T; Cornell, Timothy T; McDonough, Kelli L; McHugh, Walker M; Blatt, Neal B; Dahmer, Mary K; Shanley, Thomas P

    2017-01-01

    Protein phosphatase 2A (PP2A) is a member of the intracellular serine/threonine phosphatases. Innate immune cell activation triggered by pathogen-associated molecular patterns is mediated by various protein kinases, and PP2A plays a counter-regulatory role by deactivating these kinases. In this study, we generated a conditional knockout of the α isoform of the catalytic subunit of PP2A (PP2ACα). After crossing with myeloid-specific cre-expressing mice, effective gene knockout was achieved in various myeloid cells. The myeloid-specific knockout mice (lyM-PP2A(fl/fl)) showed higher mortality in response to endotoxin challenge and bacterial infection. Upon LPS challenge, serum levels of TNF-α, KC, IL-6, and IL-10 were significantly increased in lyM-PP2A(fl/fl) mice, and increased phosphorylation was observed in MAPK pathways (p38, ERK, JNK) and the NF-κB pathway (IKKα/β, NF-κB p65) in bone marrow-derived macrophages (BMDMs) from knockout mice. Heightened NF-κB activation was not associated with degradation of IκBα; instead, enhanced phosphorylation of the NF-κB p65 subunit and p38 phosphorylation-mediated TNF-α mRNA stabilization appear to contribute to the increased TNF-α expression. In addition, increased IL-10 expression appears to be due to PP2ACα-knockout-induced IKKα/β hyperactivation. Microarray experiments indicated that the Toll/IL-1R domain-containing adaptor inducing IFN-β/ TNFR-associated factor 3 pathway was highly upregulated in LPS-treated PP2ACα-knockout BMDMs, and knockout BMDMs had elevated IFN-α/β production compared with control BMDMs. Serum IFN-β levels from PP2ACα-knockout mice treated with LPS were also greater than those in controls. Thus, we demonstrate that PP2A plays an important role in regulating inflammation and survival in the setting of septic insult by targeting MyD88- and Toll/IL-1R domain-containing adaptor inducing IFN-β-dependent pathways.

  5. Panobinostat acts synergistically with ibrutinib in diffuse large B cell lymphoma cells with MyD88 L265 mutations

    PubMed Central

    Mondello, Patrizia; Brea, Elliott J.; De Stanchina, Elisa; Toska, Eneda; Chang, Aaron Y.; Fennell, Myles; Seshan, Venkatraman; Garippa, Ralph; Scheinberg, David A.; Baselga, José; Wendel, Hans-Guido

    2017-01-01

    Diffuse large B cell lymphoma (DLBCL) frequently harbors genetic alterations that activate the B cell receptor (BCR) and TLR pathways, which converge to activate NF-κB. While selective inhibition of BTK with ibrutinib causes clinical responses in relapsed DLBCL patients, most responses are partial and of a short duration. Here, we demonstrated that MyD88 silencing enhanced ibrutinib efficacy in DLBCL cells harboring MyD88 L265P mutations. Chemical downregulation of MyD88 expression with HDAC inhibitors also synergized with ibrutinib. We demonstrate that HDAC inhibitor regulation of MyD88 expression is mediated by STAT3. In turn, STAT3 silencing caused a decrease in MyD88 mRNA and protein levels, and enhanced the ibrutinib antilymphoma effect in MyD88 mutant DLBCL cells. Induced mutations in the STAT3 binding site in the MyD88 promotor region was associated with a decrease in MyD88 transcriptional activity. We also demonstrate that treatment with the HDAC inhibitor panobinostat decreased phosphorylated STAT3 binding to the MyD88 promotor. Accordingly, combined treatment with panobinostat and ibrutinib resulted in enhanced inhibition of NF-κB activity and caused regression of DLBCL xenografts. Our data provide a mechanistic rationale for combining HDAC inhibitors and ibrutinib for the treatment of DLBCL. PMID:28352655

  6. Burkholderia pseudomallei-induced expression of a negative regulator, sterile-alpha and Armadillo motif-containing protein, in mouse macrophages: a possible mechanism for suppression of the MyD88-independent pathway.

    PubMed

    Pudla, M; Limposuwan, K; Utaisincharoen, P

    2011-07-01

    Burkholderia pseudomallei, a causative agent of melioidosis, is a Gram-negative facultative intracellular bacterium that can survive and multiply in macrophages. Previously, we demonstrated that B. pseudomallei failed to activate gene expression downstream of the MyD88-independent pathway, particularly the expression of beta interferon (IFN-β) and inducible nitric oxide synthase (iNOS), leading to the inability of macrophages to kill this bacterium. In the present report, we extended our study to show that B. pseudomallei was able to activate sterile-α and Armadillo motif (SARM)-containing protein, a known negative regulator of the MyD88-independent pathway. Both live B. pseudomallei and heat-killed B. pseudomallei were able to upregulate SARM expression in a time-dependent manner in mouse macrophage cell line RAW 264.7. The expression of SARM required bacterial internalization, as it could be inhibited by cytochalasin D. In addition, the intracellular survival of B. pseudomallei was suppressed in SARM-deficient macrophages. Increased expression of IFN-β and iNOS and degradation of IκBα correlated with enhanced macrophage killing capability. These results demonstrated that B. pseudomallei modulated macrophage defense mechanisms by upregulating SARM, thus leading to the suppression of IFN-β and iNOS needed for bacterial elimination.

  7. A liquid crystal of ascorbyl palmitate, used as vaccine platform, provides sustained release of antigen and has intrinsic pro-inflammatory and adjuvant activities which are dependent on MyD88 adaptor protein.

    PubMed

    Sánchez Vallecillo, María F; Minguito de la Escalera, María M; Aguirre, María V; Ullio Gamboa, Gabriela V; Palma, Santiago D; González-Cintado, Leticia; Chiodetti, Ana L; Soldano, Germán; Morón, Gabriel; Allemandi, Daniel A; Ardavín, Carlos; Pistoresi-Palencia, María C; Maletto, Belkys A

    2015-09-28

    Modern subunit vaccines require the development of new adjuvant strategies. Recently, we showed that CpG-ODN formulated with a liquid crystal nanostructure formed by self-assembly of 6-O-ascorbyl palmitate (Coa-ASC16) is an attractive system for promoting an antigen-specific immune response to weak antigens. Here, we showed that after subcutaneous injection of mice with near-infrared fluorescent dye-labeled OVA antigen formulated with Coa-ASC16, the dye-OVA was retained at the injection site for a longer period than when soluble dye-OVA was administered. Coa-ASC16 alone elicited a local inflammation, but how this material triggers this response has not been described yet. Although it is known that some materials used as a platform are not immunologically inert, very few studies have directly focused on this topic. In this study, we explored the underlying mechanisms concerning the interaction between Coa-ASC16 and the immune system and we found that the whole inflammatory response elicited by Coa-ASC16 (leukocyte recruitment and IL-1β, IL-6 and IL-12 production) was dependent on the MyD88 protein. TLR2, TLR4, TLR7 and NLRP3-inflammasome signaling were not required for induction of this inflammatory response. Coa-ASC16 induced local release of self-DNA, and in TLR9-deficient mice IL-6 production was absent. In addition, Coa-ASC16 revealed an intrinsic adjuvant activity which was affected by MyD88 and IL-6 absence. Taken together these results indicate that Coa-ASC16 used as a vaccine platform is effective due to the combination of the controlled release of antigen and its intrinsic pro-inflammatory activity. Understanding how Coa-ASC16 works might have significant implications for rational vaccine design.

  8. CD4+ T cell expression of MyD88 is essential for normal resolution of Chlamydia muridarum genital tract infection1

    PubMed Central

    Frazer, Lauren C.; Sullivan, Jeanne E.; Zurenski, Matthew A.; Mintus, Margaret; Tomasak, Tammy E.; Prantner, Daniel; Nagarajan, Uma M.; Darville, Toni

    2013-01-01

    Resolution of Chlamydia genital tract infection is delayed in the absence of MyD88. In these studies, we first used bone marrow chimeras to demonstrate a requirement for MyD88 expression by hematopoietic cells in the presence of a wild-type epithelium. Using mixed bone marrow chimeras we then determined that MyD88 expression was specifically required in the adaptive immune compartment. Furthermore, adoptive transfer experiments revealed that CD4+ T cell expression of MyD88 was necessary for normal resolution of genital tract infection. This requirement was associated with a reduced ability of MyD88−/− CD4+ T cells to accumulate in the draining lymph nodes and genital tract when exposed to the same inflammatory milieu as wild-type CD4+ T cells. We also demonstrated that the impaired infection control we observed in the absence of MyD88 could not be recapitulated by deficiencies in TLR or IL-1R signaling. In vitro, we detected an increased frequency of apoptotic MyD88−/− CD4+ T cells upon activation in the absence of exogenous ligands for receptors upstream of MyD88. These data reveal an intrinsic requirement for MyD88 in CD4+ T cells during Chlamydia infection and indicate that the importance of MyD88 extends beyond innate immune responses by directly influencing adaptive immunity. PMID:24038087

  9. CD4+ T cell expression of MyD88 is essential for normal resolution of Chlamydia muridarum genital tract infection.

    PubMed

    Frazer, Lauren C; Sullivan, Jeanne E; Zurenski, Matthew A; Mintus, Margaret; Tomasak, Tammy E; Prantner, Daniel; Nagarajan, Uma M; Darville, Toni

    2013-10-15

    Resolution of Chlamydia genital tract infection is delayed in the absence of MyD88. In these studies, we first used bone marrow chimeras to demonstrate a requirement for MyD88 expression by hematopoietic cells in the presence of a wild-type epithelium. Using mixed bone marrow chimeras we then determined that MyD88 expression was specifically required in the adaptive immune compartment. Furthermore, adoptive transfer experiments revealed that CD4(+) T cell expression of MyD88 was necessary for normal resolution of genital tract infection. This requirement was associated with a reduced ability of MyD88(-/-)CD4(+) T cells to accumulate in the draining lymph nodes and genital tract when exposed to the same inflammatory milieu as wild-type CD4(+) T cells. We also demonstrated that the impaired infection control we observed in the absence of MyD88 could not be recapitulated by deficiencies in TLR or IL-1R signaling. In vitro, we detected an increased frequency of apoptotic MyD88(-/-)CD4(+) T cells upon activation in the absence of exogenous ligands for receptors upstream of MyD88. These data reveal an intrinsic requirement for MyD88 in CD4(+) T cells during Chlamydia infection and indicate that the importance of MyD88 extends beyond innate immune responses by directly influencing adaptive immunity.

  10. Effects of porcine MyD88 knockdown on the expression of TLR4 pathway-related genes and proinflammatory cytokines.

    PubMed

    Dai, Chaohui; Sun, Li; Yu, Lihuai; Zhu, Guoqiang; Wu, Shenglong; Bao, Wenbin

    2016-12-01

    As a critical adapter protein in Toll-like receptor (TLR)/Interleukin (IL)-1R signalling pathway, myeloid differentiation protein 88 (MyD88) plays an important role in immune responses and host defence against pathogens. The present study was designed to provide a foundation and an important reagent for the mechanistic study of MyD88 and its role TLR/IL-1R signalling pathways in porcine immunity. Lentivirus-mediated RNAi was used to generate a porcine PK15 cell line with a silenced MyD88 gene and quantitative real-time PCR (qPCR) and Western blotting were used to detect changes in the expression of critical genes in the Toll-like receptor 4 (TLR4) signalling pathway. ELISA was used to measure the levels of seven proinflammatory cytokines-interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α), IL-6, IL-8, IL-12, macrophage inflammatory protein (MIP)-1α and MIP-1β-in cell culture supernatants after MyD88 silencing. We successfully obtained a PK15 cell line with 61% MyD88 mRNA transcript down-regulated. In PK15 cells with MyD88 silencing, the transcript levels of TLR4 and IL-1β were significantly reduced, whereas there were no significant changes in the expression levels of cluster of differentiation antigen 14 (CD14), interferon-α (IFN-α) or TNF-α The ELISA results showed that the levels of most cytokines were not significantly changed apart from IL-8 without stimulation, which was significantly up-regulated. When cells were induced by lipopolysaccharide (LPS) (0.1 μg/ml) for 6 h, the global level of seven proinflammatory cytokines up-regulated and the level of IL-1β, TNF-α, IL-6, IL-8 and IL-12 of Blank and negative control (NC) group up-regulated more significantly than RNAi group (P<0.05), which revealed that the MyD88 silencing could reduce the TLR4 signal transduction which inhibited the release of proinflammatory cytokines and finally leaded to immunosuppression.

  11. HLA class I-restricted MYD88 L265P-derived peptides as specific targets for lymphoma immunotherapy

    PubMed Central

    Nelde, Annika; Walz, Juliane Sarah; Kowalewski, Daniel Johannes; Schuster, Heiko; Wolz, Olaf-Oliver; Peper, Janet Kerstin; Cardona Gloria, Yamel; Langerak, Anton W.; Muggen, Alice F.; Claus, Rainer; Bonzheim, Irina; Fend, Falko; Salih, Helmut Rainer; Kanz, Lothar; Rammensee, Hans-Georg; Stevanović, Stefan; Weber, Alexander N. R.

    2017-01-01

    ABSTRACT Genome sequencing has uncovered an array of recurring somatic mutations in different non-Hodgkin lymphoma (NHL) subtypes. If affecting protein-coding regions, such mutations may yield mutation-derived peptides that may be presented by HLA class I proteins and recognized by cytotoxic T cells. A recurring somatic and oncogenic driver mutation of the Toll-like receptor adaptor protein MYD88, Leu265Pro (L265P) was identified in up to 90% of different NHL subtype patients. We therefore screened the potential of MYD88L265P-derived peptides to elicit cytotoxic T cell responses as tumor-specific neoantigens. Based on in silico predictions, we identified potential MYD88L265P-containing HLA ligands for several HLA class I restrictions. A set of HLA class I MYD88L265P-derived ligands elicited specific cytotoxic T cell responses for HLA-B*07 and -B*15. These data highlight the potential of MYD88L265P mutation-specific peptide-based immunotherapy as a novel personalized treatment approach for patients with MYD88L265P+ NHLs that may complement pharmacological approaches targeting oncogenic MyD88 L265P signaling.

  12. The role of the MYD88-dependent pathway in MPTP-induced brain dopaminergic degeneration

    PubMed Central

    2011-01-01

    Background Mounting evidence supports a significant role of inflammation in Parkinson's disease (PD) pathophysiology, with several inflammatory pathways being suggested as playing a role in the dopaminergic degeneration seen in humans and animal models of the disease. These include tumor necrosis factor, prostaglandins and oxidative-related stress components. However, the role of innate immunity has not been established in PD. Methods Based on the fact that the myeloid differentiation primary response gene (88) (MyD88) is the most common adaptor protein implicated in toll-like receptor (TLR) signaling, critical in the innate immune response, we undertook a study to investigate the potential contribution of this specific pathway to MPTP-induced brain dopaminergic degeneration using MyD88 knock out mice (MyD88-/-), following our observations that the MyD88-dependent pathway was critical for MPTP dopaminergic toxicity in the enteric nervous system. Post-mortem analyses assessing nigrostriatal dopaminergic degeneration and inflammation were performed using HPLC, western blots, autoradiography and immunofluorescence. Results Our results demonstrate that MyD88-/- mice are as vulnerable to MPTP-induced dopamine and DOPAC striatal depletion as wild type mice. Furthermore, MyD88-/- mice show similar striatal dopamine transporter and tyrosine hydroxylase loss, as well as dopaminergic cell loss in the substantia nigra pars compacta in response to MPTP. To evaluate the extent of the inflammatory response created by the MPTP regimen utilized, we further performed bioluminescence imaging using TLR2-luc/gfp transgenic mice and microglial density analysis, which revealed a modest brain microglial response following MPTP. This was accompanied by a significant astrocytic reaction in the striatum, which was of similar magnitude both in wild type and MyD88-/- mice. Conclusions Our results suggest that subacute MPTP-induced dopaminergic degeneration observed in the central nervous

  13. The -938C>A Polymorphism in MYD88 Is Associated with Susceptibility to Tuberculosis: A Pilot Study

    PubMed Central

    Aggelou, Kalliopi; Siapati, Elena Konstantina; Gerogianni, Irini; Daniil, Zoe; Gourgoulianis, Konstantinos; Ntanos, Ioannis; Simantirakis, Emmanouel; Zintzaras, Elias; Mollaki, Vassiliki

    2016-01-01

    Introduction. Tuberculosis (TB) is a major disease worldwide, caused by Mycobacterium tuberculosis (MTB) infection. The Toll-Like Receptor (TLR) pathway plays a crucial role in the recognition of MTB. Aim. The present study aimed to investigate the involvement of myeloid differentiation primary response protein 88 (MYD88) gene polymorphisms in TB. Materials and Methods. A total of 103 TB cases and 92 control subjects were genotyped for the MYD88 -938C>A (rs4988453) and 1944C>G (rs4988457) polymorphisms. Results. The MYD88 -938CA and -938AA genotypes were associated with an increased risk for tuberculosis with odds ratio (OR) of 5.71 (95% confidence intervals [CIs] 2.89–11.28, p = 0.01). Conclusions. The MYD88 -938C>A genetic polymorphism is associated with increased susceptibility to TB and may serve as a marker to screen individuals who are at risk. PMID:28127112

  14. MyD88 Shapes Vaccine Immunity by Extrinsically Regulating Survival of CD4+ T Cells during the Contraction Phase

    PubMed Central

    Wang, Huafeng; Hung, Chiung Yu; Sinha, Meenal; Lee, Linda M.; Wiesner, Darin L.; LeBert, Vanessa; Lerksuthirat, Tassanee; Suresh, Marulasiddappa; DeFranco, Anthony L.; Lowell, Clifford A.; Klein, Bruce S.; Wüthrich, Marcel

    2016-01-01

    Soaring rates of systemic fungal infections worldwide underscore the need for vaccine prevention. An understanding of the elements that promote vaccine immunity is essential. We previously reported that Th17 cells are required for vaccine immunity to the systemic dimorphic fungi of North America, and that Card9 and MyD88 signaling are required for the development of protective Th17 cells. Herein, we investigated where, when and how MyD88 regulates T cell development. We uncovered a novel mechanism in which MyD88 extrinsically regulates the survival of activated T cells during the contraction phase and in the absence of inflammation, but is dispensable for the expansion and differentiation of the cells. The poor survival of activated T cells in Myd88-/- mice is linked to increased caspase3-mediated apoptosis, but not to Fas- or Bim-dependent apoptotic pathways, nor to reduced expression of the anti-apoptotic molecules Bcl-2 or Bcl-xL. Moreover, TLR3, 7, and/or 9, but not TLR2 or 4, also were required extrinsically for MyD88-dependent Th17 cell responses and vaccine immunity. Similar MyD88 requirements governed the survival of virus primed T cells. Our data identify unappreciated new requirements for eliciting adaptive immunity and have implications for designing vaccines. PMID:27542117

  15. Genetics Home Reference: MyD88 deficiency

    MedlinePlus

    ... 1 link) MYD88 DEFICIENCY Sources for This Page Maglione PJ, Simchoni N, Black S, Radigan L, Overbey JR, ... Picard C, von Bernuth H, Ghandil P, Chrabieh M, Levy O, Arkwright PD, McDonald D, Geha RS, ...

  16. Differential requirements of MyD88 and TRIF pathways in TLR4-mediated immune responses in murine B cells.

    PubMed

    Yanagibashi, Tsutomu; Nagai, Yoshinori; Watanabe, Yasuharu; Ikutani, Masashi; Hirai, Yoshikatsu; Takatsu, Kiyoshi

    2015-01-01

    LPS stimulates the TLR4/Myeloid differentiation protein-2 (MD-2) complex and promotes a variety of immune responses in B cells. TLR4 has two main signaling pathways, MyD88 and Toll/IL-1R (TIR)-domain-containing adaptor-inducing interferon-β (TRIF) pathways, but relatively few studies have examined these pathways in B cells. In this study, we investigated MyD88- or TRIF-dependent LPS responses in B cells by utilizing their knockout mice. Compared with wild-type (WT) B cells, MyD88(-/-) B cells were markedly impaired in up-regulation of CD86 and proliferation induced by lipid A moiety of LPS. TRIF(-/-) B cells were also impaired in these responses compared with WT B cells, but showed better responses than MyD88(-/-) B cells. Regarding class switch recombination (CSR) elicited by lipid A plus IL-4, MyD88(-/-) B cells showed similar patterns of CSR to WT B cells. However, TRIF(-/-) B cells showed the impaired in the CSR. Compared with WT and MyD88(-/-) B cells, TRIF(-/-) B cells exhibited reduced cell division, fewer IgG1(+) cells per division, and decreased activation-induced cytidine deaminase (Aicda) mRNA expression in response to lipid A plus IL-4. Finally, IgG1 production to trinitrophenyl (TNP)-LPS immunization was impaired in TRIF(-/-) mice, while MyD88(-/-) mice exhibited increased IgG1 production. Thus, MyD88 and TRIF pathways differently regulate TLR4-induced immune responses in B cells.

  17. MyD88-dependent pro-inflammatory cytokine response contributes to lethal toxicity of staphylococcal enterotoxin B in mice.

    PubMed

    Kissner, Teri L; Ruthel, Gordon; Cisney, Emily D; Ulrich, Robert G; Fernandez, Stefan; Saikh, Kamal U

    2011-10-01

    An elevated pro-inflammatory cytokine response is the primary cause of death by toxic shock after exposure to staphylococcal enterotoxin B (SEB). Identifying an intracellular signal mediator that predominantly controls the pro-inflammatory response is important for developing a therapeutic strategy. We examined the role of the signaling adaptor MyD88 in cell culture and in a mouse model of toxic shock. Our results indicated that elevated tumor necrosis factor-α, interferon-γ, interleukin (IL)-1α/β and IL-6 production from mouse spleen cells treated with SEB alone or in combination with lipopolysaccharide (LPS) was regulated by MyD88. Elevated levels of MyD88 protein in spleen cells, as well as in CD11c(+) or Mac3(+) cells, and activation of nuclear factor-κB in spleen cells were observed in mice treated with SEB. An SEB-dose dependent lethality was observed in LPS-potentiated and in D-galactosamine-sensitized mice. D-Galactosamine treatment of spleen cells had no effect in cytokine induction but rather increased the sensitivity to toxic shock in mice. Our results demonstrated an impaired pro-inflammatory cytokine production by spleen cells of MyD88(-/-) mice in response to SEB or SEB plus LPS. Most importantly, MyD88(-/-) mice were resistant to SEB-induced death. These results demonstrate that MyD88-dependent pro-inflammatory signaling is responsible for SEB intoxication. In addition, our studies also demonstrated that LPS potentiation, in comparison to D-galactosamine sensitization, contributes to a stronger SEB-induced lethality. This is due to the pro-inflammatory cytokine response elicited by MyD88 after exposure to SEB and LPS. These findings offer an important insight upon SEB intoxication and subsequent therapy targeting MyD88.

  18. The polysaccharide capsule of Streptococcus pneumonia partially impedes MyD88-mediated immunity during pneumonia in mice.

    PubMed

    de Vos, Alex F; Dessing, Mark C; Lammers, Adriana J J; de Porto, Alexander P N A; Florquin, Sandrine; de Boer, Onno J; de Beer, Regina; Terpstra, Sanne; Bootsma, Hester J; Hermans, Peter W; van 't Veer, Cornelis; van der Poll, Tom

    2015-01-01

    Toll-like receptors (TLR) and the downstream adaptor protein MyD88 are considered crucial for protective immunity during bacterial infections. Streptococcus (S.) pneumoniae is a human respiratory pathogen and a large majority of clinical pneumococcal isolates expresses an external polysaccharide capsule. We here sought to determine the role of pneumococcal capsule in MyD88-mediated antibacterial defense during S. pneumonia pneumonia. Wild type (WT) and Myd88(-/-) mice were inoculated intranasally with serotype 2 S. pneumoniae D39 or with an isogenic capsule locus deletion mutant (D39∆cps), and analysed for bacterial outgrowth and inflammatory responses in the lung. As compared to WT mice, Myd88(-/-) mice infected with D39 demonstrated a modestly impaired bacterial clearance accompanied by decreased inflammatory responses in the lung. Strikingly, while WT mice rapidly cleared D39∆cps, Myd88(-/-) mice showed 105-fold higher bacterial burdens in their lungs and dissemination to blood 24 hours after infection. These data suggest that the pneumococcal capsule impairs recognition of TLR ligands expressed by S. pneumoniae and thereby partially impedes MyD88-mediated antibacterial defense.

  19. MyD88-dependent Toll-like receptor 4 signal pathway in intervertebral disc degeneration

    PubMed Central

    Qin, Chuqiang; Zhang, Bo; Zhang, Liang; Zhang, Zhi; Wang, Le; Tang, Long; Li, Shuangqing; Yang, Yixi; Yang, Fuguo; Zhang, Ping; Yang, Bo

    2016-01-01

    Lower back pain (LBP) is a common and remitting problem. One of the primary causes of LBP is thought to be degeneration of the intervertebral disc (IVD). The aim of the present study was to investigate the role of the myeloid differentiation primary-response protein 88 (MyD88)-dependent Toll-like receptor 4 (TLR4) signal pathway in the mechanism of IVD degeneration. IVD nucleus pulposus cells isolated and cultured from the lumbar vertebrae of Wistar rats were stimulated by various doses of lipopolysaccharide (LPS; 0.1, 1, 10 and 100 µg/ml) to simulate IVD degeneration. Cells were rinsed and cultured in serum-free Dulbecco's modified Eagle's medium/F12. Reverse transcription-quantitative polymerase chain reaction was used to determine the levels of TLR4, MyD88, tumor necrosis factor α (TNFα), and interleukin-1β (IL-1β) mRNA expression after 1, 3, 6, 9 and 12 h of incubation. Additionally, western blot and enzyme-linked immunosorbent assay analyses were used to determine the levels of TLR4, MyD88, TNFα, and IL-1β protein expression after 24, 48 and 72 h of incubation. The levels of TLR4, MyD88, TNFα and IL-1β mRNA all increased in the cells stimulated by 10 µg/ml LPS at 3, 6 and 9 h (all P<0.001). Furthermore, the levels of TLR4, MyD88, TNFα and IL-1β protein all increased at 24, 48 and 72 h (all P<0.001). Additionally, the mRNA and protein levels of TLR4, MyD88, TNFα and IL-1β increased significantly in the cells stimulated by 1, 10 and 100 µg/ml LPS compared with the control group, and reached a peak in the 10 µg/ml LPS group (all P<0.001). These results suggest that the MyD88-dependent TLR4 signal pathway is a target pathway in IVD degeneration. This pathway is time phase- and dose-dependent, and when activated can lead to the release of inflammatory factors that participate in IVD degeneration. PMID:27446251

  20. Inhibition of Hepatitis B Virus Replication by MyD88 Involves Accelerated Degradation of Pregenomic RNA and Nuclear Retention of Pre-S/S RNAs▿

    PubMed Central

    Li, Jianhua; Lin, Shanshan; Chen, Qiying; Peng, Lu; Zhai, Jianwei; Liu, Yinghui; Yuan, Zhenghong

    2010-01-01

    Myeloid differentiation primary response protein 88 (MyD88), which can be induced by alpha interferon (IFN-α), has an antiviral activity against the hepatitis B virus (HBV). The mechanism of this antiviral activity remains poorly understood. Here, we report that MyD88 inhibited HBV replication in HepG2.2.15 cells and in a mouse model. The knockdown of MyD88 expression weakened the IFN-α-induced inhibition of HBV replication. Furthermore, MyD88 posttranscriptionally reduced the levels of viral RNA. Remarkably, MyD88 accelerated the decay of viral pregenomic RNA in the cytoplasm. Mapping analysis showed that the RNA sequence located in the 5′-proximal region of the pregenomic RNA was critical for the decay. In addition, MyD88 inhibited the nuclear export of pre-S/S RNAs via the posttranscriptional regulatory element (PRE). The retained pre-S/S RNAs were shown to degrade in the nucleus. Finally, we found that MyD88 inhibited the expression of polypyrimidine tract-binding protein (PTB), a key nuclear export factor for PRE-containing RNA. Taken together, our results define a novel antiviral mechanism against HBV mediated by MyD88. PMID:20410269

  1. Inflammation- and tumor-induced anorexia and weight loss require MyD88 in hematopoietic/myeloid cells but not in brain endothelial or neural cells.

    PubMed

    Ruud, Johan; Wilhelms, Daniel Björk; Nilsson, Anna; Eskilsson, Anna; Tang, Yan-Juan; Ströhle, Peter; Caesar, Robert; Schwaninger, Markus; Wunderlich, Thomas; Bäckhed, Fredrik; Engblom, David; Blomqvist, Anders

    2013-05-01

    Loss of appetite is a hallmark of inflammatory diseases. The underlying mechanisms remain undefined, but it is known that myeloid differentiation primary response gene 88 (MyD88), an adaptor protein critical for Toll-like and IL-1 receptor family signaling, is involved. Here we addressed the question of determining in which cells the MyD88 signaling that results in anorexia development occurs by using chimeric mice and animals with cell-specific deletions. We found that MyD88-knockout mice, which are resistant to bacterial lipopolysaccharide (LPS)-induced anorexia, displayed anorexia when transplanted with wild-type bone marrow cells. Furthermore, mice with a targeted deletion of MyD88 in hematopoietic or myeloid cells were largely protected against LPS-induced anorexia and displayed attenuated weight loss, whereas mice with MyD88 deletion in hepatocytes or in neural cells or the cerebrovascular endothelium developed anorexia and weight loss of similar magnitude as wild-type mice. Furthermore, in a model for cancer-induced anorexia-cachexia, deletion of MyD88 in hematopoietic cells attenuated the anorexia and protected against body weight loss. These findings demonstrate that MyD88-dependent signaling within the brain is not required for eliciting inflammation-induced anorexia. Instead, we identify MyD88 signaling in hematopoietic/myeloid cells as a critical component for acute inflammatory-driven anorexia, as well as for chronic anorexia and weight loss associated with malignant disease.

  2. MyD88 Mediates Instructive Signaling in Dendritic Cells and Protective Inflammatory Response during Rickettsial Infection

    PubMed Central

    Bechelli, Jeremy; Smalley, Claire; Zhao, Xuemei; Judy, Barbara; Valdes, Patricia; Walker, David H.

    2016-01-01

    Spotted fever group rickettsiae cause potentially life-threatening infections throughout the world. Several members of the Toll-like receptor (TLR) family are involved in host response to rickettsiae, and yet the mechanisms by which these TLRs mediate host immunity remain incompletely understood. In the present study, we found that host susceptibility of MyD88−/− mice to infection with Rickettsia conorii or Rickettsia australis was significantly greater than in wild-type (WT) mice, in association with severely impaired bacterial clearance in vivo. R. australis-infected MyD88−/− mice showed significantly lower expression levels of gamma interferon (IFN-γ), interleukin-6 (IL-6), and IL-1β, accompanied by significantly fewer inflammatory infiltrates of macrophages and neutrophils in infected tissues, than WT mice. The serum levels of IFN-γ, IL-12, IL-6, and granulocyte colony-stimulating factor were significantly reduced, while monocyte chemoattractant protein 1, macrophage inflammatory protein 1α, and RANTES were significantly increased in infected MyD88−/− mice compared to WT mice. Strikingly, R. australis infection was incapable of promoting increased expression of MHC-IIhigh and production of IL-12p40 in MyD88−/− bone marrow-derived dendritic cells (BMDCs) compared to WT BMDCs, although costimulatory molecules were upregulated in both types of BMDCs. Furthermore, the secretion levels of IL-1β by Rickettsia-infected BMDCs and in the sera of infected mice were significantly reduced in MyD88−/− mice compared to WT controls, suggesting that in vitro and in vivo production of IL-1β is MyD88 dependent. Taken together, our results suggest that MyD88 signaling mediates instructive signals in DCs and secretion of IL-1β and type 1 immune cytokines, which may account for the protective inflammatory response during rickettsial infection. PMID:26755162

  3. Transforming growth factor-beta differentially inhibits MyD88-dependent, but not TRAM- and TRIF-dependent, lipopolysaccharide-induced TLR4 signaling.

    PubMed

    Naiki, Yoshikazu; Michelsen, Kathrin S; Zhang, Wenxuang; Chen, Shuang; Doherty, Terence M; Arditi, Moshe

    2005-02-18

    Transforming growth factor-beta1 (TGF-beta1) is a multifunctional, potent anti-inflammatory cytokine produced by many cell types that regulates cell proliferation, apoptosis, and immune responses. Toll-like receptors (TLRs) recognize various pathogen-associated molecular patterns and are therefore a pivotal component of the innate immune system. In this study we show that TGF-beta1 blocks the NF-kappaB activation and cytokine release that is stimulated by ligands for TLRs 2, 4, and 5. We further show that TGF-beta1 can specifically interfere with TLR2, -4, or -5 ligand-induced responses involving the adaptor molecule MyD88 (myeloid differentiation factor 88) but not the TRAM/TRIF signaling pathway by decreasing MyD88 protein levels in a dose- and time-dependent manner without altering its mRNA expression. The proteasome inhibitor epoxomicin abolished the MyD88 degradation induced by TGF-beta1. Furthermore, TGF-beta1 resulted in ubiquitination of MyD88 protein, suggesting that TGF-beta1 facilitates ubiquitination and proteasomal degradation of MyD88 and thereby attenuates MyD88-dependent signaling by decreasing cellular levels of MyD88 protein. These findings importantly contribute to our understanding of molecular mechanisms mediating anti-inflammatory modulation of immune responses by TGF-beta1.

  4. Synergy between CD40 and MyD88 Does Not Influence Host Survival to Salmonella Infection.

    PubMed

    Wenzel, Ulf Alexander; Fernandez-Santoscoy, Maria; Tam, Miguel A; Tegtmeyer, Pia; Wick, Mary Jo

    2015-01-01

    Previous studies using purified toll-like receptor (TLR) ligands plus agonistic anti-CD40 antibodies showed that TLRs and CD40 can act synergistically on dendritic cells (DCs) to optimize T cell activation and Th1 differentiation. However, a synergistic effect of TLRs and CD40 during bacterial infection is not known. Here, we show that mice lacking the TLR adaptor MyD88 alone, or lacking both MyD88 and CD40 [double knockout (DKO) mice], are compromised in survival to Salmonella infection but have intact recruitment of neutrophils and inflammatory monocytes as well as unaltered abundance of DC subsets and DC activation in infected tissues. In contrast to infected wildtype and CD40(-/-) mice, both MyD88(-/-) mice and DKO mice lack detectable serum IFN-γ and have elevated IL-10. A synergistic effect of TLRs and CD40 was revealed in co-culture experiments where OT-II T cell proliferation was compromised when DKO DCs were pulsed with OVA protein and OVA323-339 peptide, but not with heat-killed Salmonella expressing OVA (HKSOVA), relative to MyD88(-/-) DCs. By contrast, MyD88(-/-) or DKO DCs pulsed with any of the antigens had a similar ability to induce IFN-γ that was lower than WT or CD40(-/-) DCs. DKO DCs pulsed with HKSOVA, but not with OVA or OVA323-339, had increased IL-10 relative to MyD88(-/-) DCs. Finally, HKSOVA-pulsed MyD88(-/-) and DKO DCs had similar and low induction of NFκB-dependent and -independent genes upon co-culture with OT-II cells. Overall, our data revealed that synergistic effects of CD40 and MyD88 do not influence host survival to Salmonella infection or serum levels of IFN-γ or IL-10. However, synergistic effects of MyD88 and CD40 may be apparent on some (IL-10 production) but not all (OT-II proliferation and IFN-γ production) DC functions and depend on the complexity of the antigen. Indeed, synergistic effects observed using purified ligands and well-defined antigens may not necessarily apply when complex antigens, such as live bacteria

  5. [The set-up of an in vitro model for stable knockdown of MyD88 by lentivirus-based RNAi in IEC-6 cell line and the study on its early apoptosis].

    PubMed

    Bao, Pingqian; Li, Yang; Chen, Keling; Zhou, Bin; Liu, Bin; Li, Yuan; Zhou, Zongguang

    2012-12-01

    Intestinal inflammatory disease is a kind of non-specific disease with morbidity increasing yearly. It has been proved that the Toll like receptor 4 (TLR4) signaling pathways are closely related to intestinal inflammatory diseases. Myeloid differentiation protein 88 (Myd88) is a critical adaptor protein of TLR4 signaling and critical for the study of intestinal inflammatory disease, but stable Myd88 knockdown in vitro models of cell line are still very few. In the present study, an HIV-1-based lentivirus three-plamid packaging system was used for the construction of a lentivirual vector mediating RNA interference (RNAi) against Myd88 in intestinal fossae epithelial cell line-6 (IEC-6). Real-time PCR and Western blot were used to detect Myd88 expression. Annexin V staining and flowcytometry (FLM) were applied to detect and evaluate the early apoptosis. The results showed that lentiviral vectors containing the shRNA expression cassette specifically targeting Myd88 were constructed and efficiently stably knocked down Myd88 expression in IEC-6 cell line. Early apoptosis was significantly decreased after Myd88 knockdown. This study successfully constructed a lentivirus-based RNAi for Myd88 and detailed the key technique combined with characteristics of the early apoptosis after the Myd88 knockdown, provided a novel, stable and repeatable in vitro model for the pathogenesis, targeting therapeutic study for the intestinal inflammatory diseases.

  6. MyD88 is essential for alveolar bone loss induced by Aggregatibacter actinomycetemcomitans lipopolysaccharide in mice.

    PubMed

    Madeira, M F M; Queiroz-Junior, C M; Cisalpino, D; Werneck, S M C; Kikuchi, H; Fujise, O; Ryffel, B; Silva, T A; Teixeira, M M; Souza, D G

    2013-12-01

    Aggregatibacter actinomycetemcomitans is a Gram-negative bacteria highly associated with localized aggressive periodontitis. The recognition of microbial factors, such as lipopolysaccharide from A. actinomycetemcomitans ((Aa)LPS), in the oral environment is made mainly by surface receptors known as Toll-like receptors (TLR). TLR4 is the major LPS receptor. This interaction leads to the production of inflammatory cytokines by myeloid differentiation primary-response protein 88 (MyD88) -dependent and -independent pathways, which may involve the adaptor Toll/interleukin-1 receptor-domain-containing adaptor inducing interferon-β (TRIF). The aim of this study was to assess the involvement of MyD88 in alveolar bone loss induced by (Aa)LPS in mice. C57BL6/J wild-type (WT) mice, MyD88, TRIF or TRIF/MyD88 knockout mice received 10 injections of Aa LPS strain FDC Y4 (5 μg in 3 μl), in the palatal gingival tissue of the right first molar, every 48 h. Phosphate-buffered saline was injected in the opposite side and used as control. Animals were sacrificed 24 h after the 10th injection and the maxillae were removed for macroscopic and biochemical analyses. The injections of Aa LPS induced significant alveolar bone loss in WT mice. In the absence of MyD88 or TRIF/MyD88 no bone loss induced by (Aa)LPS was observed. In contrast, responses in TRIF(-/-) mice were similar to those in WT mice. Diminished bone loss in the absence of MyD88 was associated with fewer TRAP-positive cells and increased expression of osteoblast markers, RUNX2 and osteopontin. There was also reduced tumor necrosis factor-α production in MyD88(-/-) mice. There was less osteoclast differentiation of hematopoietic bone marrow cells from MyD88(-/-) mice after (Aa)LPS stimulation. Hence, the signaling through MyD88 is pivotal for (Aa)LPS-induced osteoclast formation and alveolar bone loss.

  7. SARM modulates MyD88-mediated TLR activation through BB-loop dependent TIR-TIR interactions.

    PubMed

    Carlsson, Emil; Ding, Jeak Ling; Byrne, Bernadette

    2016-02-01

    Toll-like receptors (TLRs) recognise invading pathogens and initiate an innate immune response by recruiting intracellular adaptor proteins via heterotypic Toll/interleukin-1 receptor (TIR) domain interactions. Of the five TIR domain-containing adaptor proteins identified, Sterile α- and armadillo-motif-containing protein (SARM) is functionally unique; suppressing immune signalling instead of promoting it. Here we demonstrate that the recombinantly expressed and purified SARM TIR domain interacts with both the major human TLR adaptors, MyD88 and TRIF. A single glycine residue located in the BB-loop of the SARM TIR domain, G601, was identified as essential for interaction. A short peptide derived from this motif was also found to interact with MyD88 in vitro. SARM expression in HEK293 cells was found to significantly suppress lipopolysaccharide (LPS)-mediated upregulation of inflammatory cytokines, IL-8 and TNF-α, an effect lost in the G601A mutant. The same result was observed with cytokine activation initiated by MyD88 expression and stimulation of TLR2 with lipoteichoic acid (LTA), suggesting that SARM is capable of suppressing both TRIF- and MyD88- dependent TLR signalling. Our findings indicate that SARM acts on a broader set of target proteins than previously thought, and that the BB-loop motif is functionally important, giving further insight into the endogenous mechanisms used to suppress inflammation in immune cells.

  8. Sinomenine decreases MyD88 expression and improves inflammation-induced joint damage progression and symptoms in rat adjuvant-induced arthritis.

    PubMed

    Mu, Hui; Yao, Ru-Bing; Zhao, Ling-Jie; Shen, Si-Yu; Zhao, Zhi-Ming; Cai, Hui

    2013-10-01

    Sinomenine (SIN) is the active principle of the Chinese medical plant Sinomenium acutum which is widely used for the treatment of rheumatoid arthritis (RA) in China. Recently, several groups indicated that myeloid differentiation primary response protein 88 (MyD88) might be associated with disease progression of RA. Here, we observed the effect of SIN on MyD88 expression and showed its therapeutic role in RA. First, immunohistochemical staining in clinical specimens showed that MyD88 was mainly located in characteristic pathological structures of RA synovial tissues. Second, we found that MyD88 was overexpressed in the synovial tissues of the rats with adjuvant-induced arthritis (AIA). Treatment with SIN markedly decreased the expression of MyD88 in AIA rats. Finally, we provided evidences that SIN suppressed inflammation response and inflammation-induced joint destructive progression and arthritis symptoms in AIA rats. Therefore, SIN is an effective therapeutic agent for RA. Targeting MyD88 signaling may provide new methods for the treatment of RA.

  9. Toll-like receptor 4 signalling through MyD88 is essential to control Salmonella enterica serovar typhimurium infection, but not for the initiation of bacterial clearance.

    PubMed

    Talbot, Suzanne; Tötemeyer, Sabine; Yamamoto, Masahiro; Akira, Shizuo; Hughes, Katherine; Gray, David; Barr, Tom; Mastroeni, Pietro; Maskell, Duncan J; Bryant, Clare E

    2009-12-01

    Toll-like receptor-4 (TLR4) is important in protection against lethal Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. Control of the early stages of sublethal S. Typhimurium infection in mice depends on TLR4-dependent activation of macrophages and natural killer (NK) cells to drive an inflammatory response. TLR4 signals through the adapter proteins Mal/MyD88 and TRIF-related adaptor molecule (TRAM)/TIR-domain-containing adaptor-inducing interferon-b (TRIF). In the mouse typhoid model we showed that TLR4 and MyD88, but not Mal or TRIF, are essential for the control of exponential S. Typhimurium growth. TRIF(-/-) mice have a higher bacterial load in comparison with wild-type mice during a sublethal infection because TRIF is important for bacterial killing during the first day of systemic disease. Minimal pro-inflammatory responses were induced by S. Typhimurium infection of macrophages from TLR4(-/-), MyD88(-/-) and TRIF(-/-) mice in vitro. Pro-inflammatory responses from Mal(-/-) macrophages were similar to those from wild-type cells. The pro-inflammatory responses of TRIF(-/-) macrophages were partially restored by the addition of interferon-gamma (IFN-gamma), and TRIF(-/-) mice produced markedly enhanced IFN-gamma levels, in comparison to wild-type mice, probably explaining why bacterial growth can be controlled in these mice. TLR4(-/-), MyD88(-/-), TRIF(-/-) and Mal(-/-) mice all initiated clearance of S. Typhimurium, suggesting that TLR4 signalling is not important in driving bacterial clearance in comparison to its critical role in controlling early bacterial growth in mouse typhoid.

  10. Andrographolide alleviates imiquimod-induced psoriasis in mice via inducing autophagic proteolysis of MyD88.

    PubMed

    Shao, Fenli; Tan, Tao; Tan, Yang; Sun, Yang; Wu, Xingxin; Xu, Qiang

    2016-09-01

    Psoriasis is a chronic inflammatory skin disease with excessive activation of toll-like receptors (TLRs), which play important roles in developing psoriasis. Targeting TLR signaling remains a challenge for treating psoriasis. Here, we found that andrographolide (Andro), a small-molecule natural product, alleviated imiquimod- but not interleukin 23 (IL-23)-induced psoriasis in mice with reducing expressions of IL-23 and IL-1β in the skin. The improvement in imiquimod-induced psoriasis by Andro was not observed in microtubule-associated protein 1 light chain 3 beta (MAP1LC3B) knockout mice. Furthermore, Andro inhibited mRNA expressions of IL-23, IL-6 and IL-1β but not CD80 and CD86 in bone-marrow derived dendritic cells (BMDCs) treated with lipopolysaccharide (LPS) in a MAP1LC3B-dependent manner. In addition, Andro inhibited imiquimod-induced mRNA expressions of IL-23, IL-6, IL-1β, CD80 and CD86 in BMDCs from mice. Interestingly, Andro induced a degradation of myeloid differentiation factor 88 (MyD88) and blocked the recruitment of TNF receptor-associated factor 6 (TRAF6) to MyD88 upon LPS stimulation in BMDCs from mice. Blockade of autophagic proteolysis using NH4Cl or MAP1LC3B(-/-) BMDCs abolished the Andro-induced MyD88 degradation. In conclusion, Andro controls activation of MyD88-dependent cytokines and alleviates psoriasis in mice via inducing autophagic proteolysis of MyD88, which could be a novel strategy to treat psoriasis.

  11. MyD88-dependent IL-1 receptor signaling is essential for gouty inflammation stimulated by monosodium urate crystals

    PubMed Central

    Chen, Chun-Jen; Shi, Yan; Hearn, Arron; Fitzgerald, Kate; Golenbock, Douglas; Reed, George; Akira, Shizuo; Rock, Kenneth L.

    2006-01-01

    While it is known that monosodium urate (MSU) crystals cause the disease gout, the mechanism by which these crystals stimulate this inflammatory condition has not been clear. Here we find that the Toll/IL-1R (TIR) signal transduction adaptor myeloid differentiation primary response protein 88 (MyD88) is required for acute gouty inflammation. In contrast, other TIR adaptor molecules, TIRAP/Mal, TRIF, and TRAM, are not required for this process. The MyD88-dependent TLR1, -2, -4, -6, -7, -9, and -11 and IL-18 receptor (IL-18R) are not essential for MSU-induced inflammation. Moreover, MSU does not stimulate HEK cells expressing TLR1–11 to activate NF-κB. In contrast, mice deficient in the MyD88-dependent IL-1R showed reduced inflammatory responses, similar to those observed in MyD88-deficient mice. Similarly, mice treated with IL-1 neutralizing antibodies also showed reduced MSU-induced inflammation, demonstrating that IL-1 production and IL-1R activation play essential roles in MSU-triggered inflammation. IL-1R deficiency in bone marrow–derived cells did not affect the inflammatory response; however, it was required in non–bone marrow–derived cells. These results indicate that IL-1 is essential for the MSU-induced inflammatory response and that the requirement of MyD88 in this process is primarily through its function as an adaptor molecule in the IL-1R signaling pathway. PMID:16886064

  12. MyD88 deficiency leads to decreased NK cell gamma interferon production and T cell recruitment during Chlamydia muridarum genital tract infection, but a predominant Th1 response and enhanced monocytic inflammation are associated with infection resolution.

    PubMed

    Nagarajan, Uma M; Sikes, James; Prantner, Daniel; Andrews, Charles W; Frazer, Lauren; Goodwin, Anna; Snowden, Jessica N; Darville, Toni

    2011-01-01

    We have previously shown that MyD88 knockout (KO) mice exhibit delayed clearance of Chlamydia muridarum genital infection compared to wild-type (WT) mice. A blunted Th1 response and ineffective suppression of the Th2 response were also observed in MyD88 KO mice. The goal of the present study was to investigate specific mechanisms whereby absence of MyD88 leads to these effects and address the compensatory mechanisms in the genital tract that ultimately clear infection in the absence of MyD88. It was observed that NK cells recruited to the genital tract in MyD88 KO mice failed to produce gamma interferon (IFN-γ) mRNA and protein. This defect was associated with decreased local production of interleukin-17 (IL-17), IL-18, and tumor necrosis factor alpha (TNF-α) but normal levels of IL-12p70. Additionally, recruitment of CD4 T cells to the genital tract was reduced in MyD88 KO mice compared to that in WT mice. Although chronic infection in MyD88 KO mice resulted in oviduct pathology comparable to that of WT mice, increased histiocytic inflammation was observed in the uterine horns. This was associated with increased CCL2 levels and recruitment of macrophages as a potential compensatory mechanism. Further deletion of TLR4-TRIF signaling in MyD88 KO mice, using TLR4/MyD88 double-KO mice, did not further compromise host defense against chlamydiae, suggesting that compensatory mechanisms are Toll-like receptor (TLR) independent. Despite some polarization toward a Th2 response, a Th1 response remained predominant in the absence of MyD88, and it provided equivalent protection against a secondary infection as observed in WT mice.

  13. SARM inhibits both TRIF- and MyD88-mediated AP-1 activation.

    PubMed

    Peng, Jun; Yuan, Quan; Lin, Bin; Panneerselvam, Porkodi; Wang, Xiaowei; Luan, Xiao Lei; Lim, Soon Kok; Leung, Bernard P; Ho, Bow; Ding, Jeak Ling

    2010-06-01

    SARM (sterile alpha- and armadillo-motif-containing protein), the fifth identified TIR (Toll-interleukin 1 receptor (IL-1R)) domain-containing adaptors in humans, downregulates NF-kappaB and IRF3 (interferon-regulatory factor 3)-mediated TLR3 and TLR4 signaling. SARM was characterized as a negative regulator of the TRIF (TIR-domain-containing adaptor protein inducing IFN-beta)-dependent pathway via its interaction with TRIF. However, the precise mechanism of action of SARM remains unclear. Here, we demonstrate that SARM inhibits MAPK activation in human embryonic kidney 293 cells, and U937 cells. Both the TRIF- and MyD88-mediated, as well as basal MAPK activity, were repressed, indicating that SARM-mediated inhibition may not be exclusively directed at TRIF or MyD88, but that SARM may also directly inhibit MAPK phosphorylation. The MAPK inhibition effect was verified by RNAi, which increased the basal level of AP-1. Furthermore, LPS challenge upregulated SARM at both the mRNA and protein levels. Finally, we provide evidence to show that truncated SARM changes its subcellular localization, suggesting the importance of the N-terminal and sterile alpha motif domains in the autoregulation of SARM activity.

  14. Involvement of the MyD88-independent pathway in controlling the intracellular fate of Burkholderia pseudomallei infection in the mouse macrophage cell line RAW 264.7.

    PubMed

    Tangsudjai, S; Pudla, M; Limposuwan, K; Woods, D E; Sirisinha, S; Utaisincharoen, P

    2010-05-01

    Burkholderia pseudomallei is a facultative intracellular Gram-negative bacterium which is capable of surviving and multiplying inside macrophages. B. pseudomallei strain SRM117, a LPS mutant which lacks the O-antigenic polysaccharide moiety, is more susceptible to macrophage killing during the early phase of infection than is its parental wild type strain (1026b). In this study, it was shown that the wild type is able to induce expression of genes downstream of the MyD88-dependent (ikappabzeta, il-6 and tnf-alpha), but not of the MyD88-independent (inos, ifn-beta and irg-1), pathways in the mouse macrophage cell line RAW 264.7. In contrast, LPS mutant-infected macrophages were able to express genes downstream of both pathways. To elucidate the significance of activation of the MyD88-independent pathway in B. pseudomallei-infected macrophages, the expression of TBK1, an essential protein in the MyD88-independent pathway, was silenced prior to the infection. The results showed that silencing the tbk1 expression interferes with the gene expression profile in LPS mutant-infected macrophages and allows the bacteria to replicate intracellularly, thus suggesting that the MyD88-independent pathway plays an essential role in controlling intracellular survival of the LPS mutant. Moreover, exogenous IFN-gamma upregulated gene expression downstream of the MyD88-independent pathway, and interfered with intracellular survival in both wild type and tbk1-knockdown macrophages infected with either the wild type or the LPS mutant. These results suggest that gene expression downstream of the MyD88-independent pathway is essential in regulating the intracellular fate of B. pseudomallei, and that IFN-gamma regulates gene expression through the TBK1-independent pathway.

  15. Penta-O-galloyl-β-D-glucose ameliorates inflammation by inhibiting MyD88/NF-κB and MyD88/MAPK signalling pathways

    PubMed Central

    Jang, Se-Eun; Hyam, Supriya R; Jeong, Jin-Ju; Han, Myung Joo; Kim, Dong-Hyun

    2013-01-01

    Background and Purpose The gallnut of Rhus chinensis MILL and its main constituent penta-O-galloyl-β-D-glucose (PGG) inhibited NF-κB activation in LPS-stimulated peritoneal and colonic macrophages. Here we have investigated PGG mechanisms underlying anti-inflammatory effects of PGG in vitro and in vivo. Experimental Approach Male C57BL/6 mice (18–22 g, 6 weeks old) were used to prepare peritoneal and colonic macrophages and for the induction of colitis by intrarectal administration of 2,3,4-trinitrobenzene sulphonic acid (TNBS). A range of inflammatory markers and transcription factors were evaluated by elisa, immunoblotting, flow cytometry and confocal microscopy. Key Results Expression of Toll-like receptor (TLR)-4 or Lipopolysaccharide (LPS) binding to TLR-4 in LPS-stimulated peritoneal macrophages was not affected by PGG. However PGG inhibited binding of an anti-MyD88 antibody to peritoneal macrophages, but did not reduce binding of anti–IL-1 receptor-associated kinase (IRAK1) and IRAK4 antibodies to the macrophages with or without transfection with MyD88 siRNA. PGG potently reduced the activation of IRAK1, NF-κB, and MAPKs in LPS- or pepetidoglycan-stimulated peritoneal and colonic macrophages. PGG suppressed IL-1β, TNF-α and IL-6 in LPS-stimulated peritoneal macrophages, while increasing expression of the anti-inflammatorycytokine IL-10. Oral administration of PGG inhibited colon shortening and myeloperoxidase activity in mice with TNBS-induced colitis, along with reducing NF-κB activation and IL-1β, TNF-α, and IL-6 levels, whereas it increased IL-10. Conclusions and Implications PGG reduced activation of NF-κB and MAPK signalling pathways by directly interacting with the MyD88 adaptor protein. PGG may ameliorate inflammatory diseases such as colitis. PMID:23941302

  16. Nonredundant roles of TIRAP and MyD88 in airway response to endotoxin, independent of TRIF, IL-1 and IL-18 pathways.

    PubMed

    Togbe, Dieudonnée; Aurore, Gorse; Noulin, Nicolas; Quesniaux, Valérie F J; Schnyder-Candrian, Silvia; Schnyder, Bruno; Vasseur, Virginie; Akira, Shizuo; Hoebe, Kasper; Beutler, Bruce; Ryffel, Bernhard; Couillin, Isabelle

    2006-11-01

    Inhaled endotoxins induce an acute inflammatory response in the airways mediated through Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88). However, the relative roles of the TLR4 adaptor proteins TIRAP and TRIF and of the MyD88-dependent IL-1 and IL-18 receptor pathways in this response are unclear. Here, we demonstrate that endotoxin-induced acute bronchoconstriction, vascular damage resulting in protein leak, Th1 cytokine and chemokine secretion and neutrophil recruitment in the airways are abrogated in mice deficient for either TIRAP or MyD88, but not in TRIF deficient mice. The contribution of other TLR-independent, MyD88-dependent signaling pathways was investigated in IL-1R1, IL-18R and caspase-1 (ICE)-deficient mice, which displayed normal airway responses to endotoxin. In conclusion, the TLR4-mediated, bronchoconstriction and acute inflammatory lung pathology to inhaled endotoxin critically depend on the expression of both adaptor proteins, TIRAP and MyD88, suggesting cooperative roles, while TRIF, IL-1R1, IL-18R signaling pathways are dispensable.

  17. Baicalin inhibits TLR7/MYD88 signaling pathway activation to suppress lung inflammation in mice infected with influenza A virus

    PubMed Central

    WAN, QIAOFENG; WANG, HAO; HAN, XUEBO; LIN, YUAN; YANG, YANHUI; GU, LIGANG; ZHAO, JIAQING; WANG, LI; HUANG, LING; LI, YANBIN; YANG, YURONG

    2014-01-01

    The present study aimed to investigate the protective effects and underlying mechanisms of baicalin on imprinting control region mice infected with influenza A/FM/1/47 (H1N1) virus. Oral administration of baicalin into mice infected with H1N1 prevented death, increased the mean time to death and inhibited lung index and lung consolidation. Subsequently, fluorescence quantitative polymerase chain reaction was used to assess the mRNA expression of toll-like receptor 7 (TLR7) and myeloid differentiation primary response gene 88 (MYD88), and western blot analysis was used to determine the expression of phosphorylated nuclear factor κB (NF-κB)-P65 and c-jun/activator protein 1 (AP-1). An enzyme-linked immunosorbent assay was applied to test for the inflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-1β and IL-6, in the lung tissue. The findings indicated that baicalin downregulated the mRNA expression of TLR7 and MYD88, significantly downregulated the protein expression of NF-κB-P65 and AP-1 and also inhibited the secretion of TNF-α, IL-1β and IL-6. In conclusion, baicalin effectively reduced the pathological damage and inflammation of the lungs by downregulating the TLR7/MYD88-mediated signaling pathway. PMID:24748990

  18. Lipopolysaccharide regulation of intestinal tight junction permeability is mediated by TLR-4 signal transduction pathway activation of FAK and MyD88

    PubMed Central

    Guo, Shuhong; Nighot, Meghali; Al-Sadi, Rana; Alhmoud, Tarik; Nighot, Prashant; Ma, Thomas Y.

    2015-01-01

    Gut-derived bacterial lipopolysaccharides (LPS) play an essential role in inducing intestinal and systemic inflammatory responses and have been implicated as a pathogenic factor of necrotizing enterocolitis (NEC) and inflammatory bowel disease (IBD). The defective intestinal tight junction (TJ) barrier has been shown to be an important factor contributing to the development of intestinal inflammation. LPS, at physiological concentrations, cause an increase in intestinal tight junction permeability (TJP) via a TLR-4 dependent process; however the intracellular mechanisms that mediate LPS regulation of intestinal TJP remain unclear. The aim of this study was to investigate the adaptor proteins and the signaling interactions that mediate LPS modulation of intestinal TJ barrier using an in-vitro and in-vivo model system. LPS caused a TLR-4 dependent activation of membrane-associated adaptor protein FAK in Caco-2 monolayers. LPS caused an activation of both MyD88-dependent and –independent pathways. SiRNA silencing of MyD88 prevented LPS-induced increase in TJP. LPS caused a MyD88-dependent activation of IRAK4. TLR-4, FAK and MyD88 were co-localized. SiRNA silencing of TLR-4 inhibited TLR-4 associated FAK activation; and FAK knockdown prevented MyD88 activation. In-vivo studies also confirmed that LPS-induced increase in mouse intestinal permeability was associated with FAK and MyD88 activation; knockdown of intestinal epithelial FAK prevented LPS-induced increase in intestinal permeability. Additionally, high dose LPS-induced intestinal inflammation was also dependent on TLR-4/FAK/MyD88 signal-transduction axis. Our data show for the first time that LPS-induced increase in intestinal TJP and intestinal inflammation was regulated by TLR-4 dependent activation of FAK-MyD88-IRAK4 signaling pathway. PMID:26466961

  19. Multiple roles of Myd88 in the immune response to the plague F1-V vaccine and in protection against an aerosol challenge of Yersinia pestis CO92 in mice.

    PubMed

    Dankmeyer, Jennifer L; Fast, Randy L; Cote, Christopher K; Worsham, Patricia L; Fritz, David; Fisher, Diana; Kern, Steven J; Merkel, Tod; Kirschning, Carsten J; Amemiya, Kei

    2014-01-01

    The current candidate vaccine against Yersinia pestis infection consists of two subunit proteins: the capsule protein or F1 protein and the low calcium response V protein or V-antigen. Little is known of the recognition of the vaccine by the host's innate immune system and how it affects the acquired immune response to the vaccine. Thus, we vaccinated Toll-like receptor (Tlr) 2, 4, and 2/4-double deficient, as well as signal adaptor protein Myd88-deficient mice. We found that Tlr4 and Myd88 appeared to be required for an optimal immune response to the F1-V vaccine but not Tlr2 when compared to wild-type mice. However, there was a difference between the requirement for Tlr4 and MyD88 in vaccinated animals. When F1-V vaccinated Tlr4 mutant (lipopolysaccharide tolerant) and Myd88-deficient mice were challenged by aerosol with Y. pestis CO92, all but one Tlr4 mutant mice survived the challenge, but no vaccinated Myd88-deficient mice survived the challenge. Spleens from these latter nonsurviving mice showed that Y. pestis was not cleared from the infected mice. Our results suggest that MyD88 appears to be important for both an optimal immune response to F1-V and in protection against a lethal challenge of Y. pestis CO92 in F1-V vaccinated mice.

  20. Measurement of TLR-Induced Macrophage Spreading by Automated Image Analysis: Differential Role of Myd88 and MAPK in Early and Late Responses.

    PubMed

    Wenzel, Jens; Held, Christian; Palmisano, Ralf; Teufel, Stefan; David, Jean-Pierre; Wittenberg, Thomas; Lang, Roland

    2011-01-01

    Sensing of infectious danger by toll-like receptors (TLRs) on macrophages causes not only a reprogramming of the transcriptome but also changes in the cytoskeleton important for cell spreading and motility. Since manual determination of cell contact areas from fluorescence micrographs is very time-consuming and prone to bias, we have developed and tested algorithms for automated measurement of macrophage spreading. The two-step method combines identification of cells by nuclear staining with DAPI and cell surface staining of the integrin CD11b. Automated image analysis correlated very well with manual annotation in resting macrophages and early after stimulation, whereas at later time points the automated cell segmentation algorithm and manual annotation showed slightly larger variation. The method was applied to investigate the impact of genetic or pharmacological inhibition of known TLR signaling components. Deficiency in the adapter protein Myd88 strongly reduced spreading activity at the late time points, but had no impact early after LPS-stimulation. A similar effect was observed upon pharmacological inhibition of MEK1, the kinase activating the mitogen-activated protein kinases (MAPK) ERK1/2, indicating that ERK1/2 mediates Myd88-dependent macrophages spreading. In contrast, macrophages lacking the MAPK p38 were impaired in the initial spreading response but responded normally 8-24 h after stimulation. The dichotomy of p38 and ERK1/2 MAPK effects on early and late macrophage spreading raises the question which of the respective substrate proteins mediate(s) cytoskeletal remodeling and spreading. The automated measurement of cell spreading described here increases the objectivity and greatly reduces the time required for such investigations and is therefore expected to facilitate larger throughput analysis of macrophage spreading, e.g., in siRNA knockdown screens.

  1. Lactobacillus rhamnosus GR-1 Limits Escherichia coli-Induced Inflammatory Responses via Attenuating MyD88-Dependent and MyD88-Independent Pathway Activation in Bovine Endometrial Epithelial Cells.

    PubMed

    Liu, Mingchao; Wu, Qiong; Wang, Mengling; Fu, Yunhe; Wang, Jiufeng

    2016-08-01

    Intrauterine Escherichia coli infection after calving reduces fertility and causes major economic losses in the dairy industry. We investigated the protective effect of the probiotic Lactobacillus rhamnosus GR-1 on E. coli-induced cell damage and inflammation in primary bovine endometrial epithelial cells (BEECs). L. rhamnosus GR-1 reduced ultrastructure alterations and the percentage of BEECs apoptosis after E. coli challenge. Increased messenger RNA (mRNA) expression of immune response indicators, including pattern recognition receptors (toll-like receptor [TLR]2, TLR4, nucleotide-binding oligomerization domain [NOD]1, and NOD2), inflammasome proteins (NOD-like receptor family member pyrin domain-containing protein 3, apoptosis-associated speck-like protein, and caspase-1), TLR4 downstream adaptor molecules (myeloid differentiation antigen 88 [MyD88], toll-like receptor adaptor molecule 2 [TICAM2]), nuclear transcription factor kB (NF-kB), and the inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-18, and interferon (IFN)-β, was observed following E. coli challenge. However, these increases were attenuated by L. rhamnosus GR-1 pretreatment. Our data indicate that L. rhamnosus GR-1 ameliorates the E. coli-induced disruption of cellular ultrastructure, subsequently reducing the percentage of BEECs apoptosis and limiting inflammatory responses, partly via attenuation of MyD88-dependent and MyD88-independent pathway activation. Certain probiotics could potentially prevent postpartum uterine diseases in dairy cows, ultimately reducing the use of antibiotics.

  2. Deficiency in MyD88 Signaling Results in Decreased Antibody Responses to an Adeno-Associated Virus Vector in Murine Pompe Disease

    PubMed Central

    Zhang, Ping; Luo, Xiaoyan; Bird, Andrew; Li, Songtao

    2012-01-01

    Abstract We have previously shown that antibody and T cell responses limit the efficacy of an adeno-associated virus (AAV) pseudotype 8 (2/8) vector containing the universally active cytomegalovirus enhancer/chicken β-actin regulatory cassette (AAV2/8-CBhGAA) in treating murine Pompe disease. However, the innate immune responses to AAV2/8-CBhGAA are largely unknown. In this study, we investigated acute immune responses to AAV2/8-CBhGAA and the role of MyD88/TRIF signaling pathway in shaping adaptive immune responses to this vector. We showed here that a small and transient increase in CXCL-1 and IL-1β expression in livers of acid-α-glucosidase knockout (GAAKO) mice 6 h following injection with AAV2/8-CBhGAA. There was a robust antibody response to GAA in wild-type mice injected with this vector. In contrast, the anti-GAA IgG1 response was diminished in MyD88KO mice, and showed a trend toward a decrease in TRIFKO mice. In addition, the vector genome and GAA activity were significantly higher in MyD88KO livers compared with wild-type livers, suggesting reduced cytotoxic T cell responses. Importantly, elevated CD4+ T cells were detected by immunohistochemistry in MyD88KO livers. When adoptively transferred to wild-type mice, these CD4+ T cells have an ability to suppress antibody responses against AAV2/8-CBhGAA and to prevent further immunization against rhGAA. Our study suggests that the MyD88 deficiency leads to the suppression of deleterious immune responses to AAV2/8-CBhGAA, which has implications for gene therapy in Pompe disease. PMID:23514839

  3. Deficiency in MyD88 Signaling Results in Decreased Antibody Responses to an Adeno-Associated Virus Vector in Murine Pompe Disease.

    PubMed

    Zhang, Ping; Luo, Xiaoyan; Bird, Andrew; Li, Songtao; Koeberl, Dwight D

    2012-06-01

    We have previously shown that antibody and T cell responses limit the efficacy of an adeno-associated virus (AAV) pseudotype 8 (2/8) vector containing the universally active cytomegalovirus enhancer/chicken β-actin regulatory cassette (AAV2/8-CBhGAA) in treating murine Pompe disease. However, the innate immune responses to AAV2/8-CBhGAA are largely unknown. In this study, we investigated acute immune responses to AAV2/8-CBhGAA and the role of MyD88/TRIF signaling pathway in shaping adaptive immune responses to this vector. We showed here that a small and transient increase in CXCL-1 and IL-1β expression in livers of acid-α-glucosidase knockout (GAAKO) mice 6 h following injection with AAV2/8-CBhGAA. There was a robust antibody response to GAA in wild-type mice injected with this vector. In contrast, the anti-GAA IgG1 response was diminished in MyD88KO mice, and showed a trend toward a decrease in TRIFKO mice. In addition, the vector genome and GAA activity were significantly higher in MyD88KO livers compared with wild-type livers, suggesting reduced cytotoxic T cell responses. Importantly, elevated CD4(+) T cells were detected by immunohistochemistry in MyD88KO livers. When adoptively transferred to wild-type mice, these CD4(+) T cells have an ability to suppress antibody responses against AAV2/8-CBhGAA and to prevent further immunization against rhGAA. Our study suggests that the MyD88 deficiency leads to the suppression of deleterious immune responses to AAV2/8-CBhGAA, which has implications for gene therapy in Pompe disease.

  4. Intrinsic MyD88-Akt1-mTOR Signaling Coordinates Disparate Tc17 and Tc1 Responses during Vaccine Immunity against Fungal Pneumonia.

    PubMed

    Nanjappa, Som Gowda; Hernández-Santos, Nydiaris; Galles, Kevin; Wüthrich, Marcel; Suresh, M; Klein, Bruce S

    2015-09-01

    Fungal infections have skyrocketed in immune-compromised patients lacking CD4+ T cells, underscoring the need for vaccine prevention. An understanding of the elements that promote vaccine immunity in this setting is essential. We previously demonstrated that vaccine-induced IL-17A+ CD8+ T cells (Tc17) are required for resistance against lethal fungal pneumonia in CD4+ T cell-deficient hosts, whereas the individual type I cytokines IFN-γ, TNF-α and GM-CSF, are dispensable. Here, we report that T cell-intrinsic MyD88 signals are crucial for these Tc17 cell responses and vaccine immunity against lethal fungal pneumonia in mice. In contrast, IFN-γ+ CD8+ cell (Tc1) responses are largely normal in the absence of intrinsic MyD88 signaling in CD8+ T cells. The poor accumulation of MyD88-deficient Tc17 cells was not linked to an early onset of contraction, nor to accelerated cell death or diminished expression of anti-apoptotic molecules Bcl-2 or Bcl-xL. Instead, intrinsic MyD88 was required to sustain the proliferation of Tc17 cells through the activation of mTOR via Akt1. Moreover, intrinsic IL-1R and TLR2, but not IL-18R, were required for MyD88 dependent Tc17 responses. Our data identify unappreciated targets for augmenting adaptive immunity against fungi. Our findings have implications for designing fungal vaccines and immune-based therapies in immune-compromised patients.

  5. Molecular cloning, structural modeling, and expression analysis of MyD88 and IRAK4 of golden pompano (Trachinotus ovatus).

    PubMed

    Qi, Zhitao; Sun, Baobao; Zhang, Qihuan; Meng, Fancui; Xu, Qiaoqing; Wei, Youchuan; Gao, Qian

    2017-04-10

    MyD88 and IRAK4 are important components of TLR signaling pathways. However, information about MyD88 and IRAK4 is vacant in golden pompano (Trachinotus ovatus), a marine teleost with great commercial value. Thus, in this study the full lengths of trMyD88 and trIRAK4 were cloned from golden pompano using RT-PCR and RACE-PCR methods. trMyD88 was 1213 bp in length, encoding a putative protein of 288 amino acids (aa), consisting of a 99 aa of death domain at its N-terminal and a 137 aa of the TIR domain at its C-terminal. trIRAK4 was 1606 bp in length, encoding a putative protein of 469 aa, including an N-terminal death domain and a central kinase domain, connected by a ProST domain. Other domains or aa residues needed for their functions were also identified in trMyD88 and trIRAK4. Physicochemical features and 3-D structures of trMyD88 and trIRAK4 were also analyzed. Quantitative real-time PCR revealed that the 2 genes were ubiquitously expressed in tissues from healthy pompano, especially highly in the spleen and head kidney, indicating their roles in the immune response. Further, trMyD88 and trIRAK4 were up-regulated at 12 h after the Vibrio alginilyticus and polyI:C challenge and continued to 48 h post challenge. Our results demonstrated that MyD88 and IRAK4 played important roles in the golden pompano innate immune system, providing the basis for further study of the signaling pathways that these 2 genes are involved in.

  6. Cloning, Characterization, and Expression Analysis of MyD88 in Rana dybowskii.

    PubMed

    Niu, Shudong; Shi, Xuecan; Zhang, Jingyu; Chai, Longhui; Xiao, Xianghong

    2016-05-01

    The myeloid differentiation factor 88 (MyD88) is the most common adaptor protein in toll-like receptor (TLR) signaling pathways and plays an important role in the innate immune system. In this report, we conducted rapid amplification of complementary DNA (cDNA) ends (RACE), multiple sequence alignment, conserved domain search, phylogenetic tree construction, and quantitative real-time PCR to obtain and analyze the full-length cDNA sequence, the amino acid sequential structures, and the expression patterns of Rana dybowskii (Rd) MyD88. The full-length cDNA of RdMyD88 is 1472 bp, with an open reading frame of 855 bp, encoding a protein of 285 amino acid residues. The RdMyD88 amino acid sequence contains a death domain (DD) and a Toll/interleukin-1 receptor (TIR) domain. RdMyD88 was calculated as a hydrophilic protein with predicted molecular mass and pI of 32.79 kDa and 6.00, respectively. Eighteen possible phosphorylation sites including eight serine residues, six tyrosine residues, and four threonine residues are predicted. Analysis of multiple sequence alignment and phylogenetic tree revealed that the predicted RdMyD88 protein is closest to its Xenopus counterparts. The PCR result showed that RdMyD88 is expressed in various tissues of R. dybowskii. Quantitative real-time PCR (qPCR) was used to examine the expression of RdMyD88 in the heart, liver, and kidney. After Rana grylio virus (RGV) exposure, the expression of RdMyD88 in the heart, liver, and kidney were significantly upregulated and reached peak levels at 48, 48, and 72 h post-infection (hpi), respectively. Meanwhile, in response to Aeromonas hydrophila (AH) infection, clear upregulation of RdMyD88 was observed in the heart, liver, and kidney and reached its peak at 48, 6, and 12 hpi, respectively. The highest levels of induction were found in the kidney after both RGV and AH infections. These findings indicate that RdMyD88 has a conserved structure and is probably an important component of the innate

  7. Mutations in TLR/MYD88 pathway identify a subset of young chronic lymphocytic leukemia patients with favorable outcome.

    PubMed

    Martínez-Trillos, Alejandra; Pinyol, Magda; Navarro, Alba; Aymerich, Marta; Jares, Pedro; Juan, Manel; Rozman, María; Colomer, Dolors; Delgado, Julio; Giné, Eva; González-Díaz, Marcos; Hernández-Rivas, Jesús M; Colado, Enrique; Rayón, Consolación; Payer, Angel R; Terol, Maria José; Navarro, Blanca; Quesada, Victor; Puente, Xosé S; Rozman, Ciril; López-Otín, Carlos; Campo, Elías; López-Guillermo, Armando; Villamor, Neus

    2014-06-12

    Mutations in Toll-like receptor (TLR) and myeloid differentiation primary response 88 (MYD88) genes have been found in chronic lymphocytic leukemia (CLL) at low frequency. We analyzed the incidence, clinicobiological characteristics, and outcome of patients with TLR/MYD88 mutations in 587 CLL patients. Twenty-three patients (3.9%) had mutations, 19 in MYD88 (one with concurrent IRAK1 mutation), 2 TLR2 (one with concomitant TLR6 mutation), 1 IRAK1, and 1 TLR5. No mutations were found in IRAK2 and IRAK4. TLR/MYD88-mutated CLL overexpressed genes of the nuclear factor κB pathway. Patients with TLR/MYD88 mutations were significantly younger (83% age ≤50 years) than those with no mutations. TLR/MYD88 mutations were the most frequent in young patients. Patients with mutated TLR/MYD88 CLL had a higher frequency of mutated IGHV and low expression of CD38 and ZAP-70. Overall survival (OS) was better in TLR/MYD88-mutated than unmutated patients in the whole series (10-year OS, 100% vs 62%; P = .002), and in the subset of patients age ≤50 years (100% vs 70%; P = .02). In addition, relative OS of TLR/MYD88-mutated patients was similar to that in the age- and gender-matched population. In summary, TLR/MYD88 mutations identify a population of young CLL patients with favorable outcome.

  8. Experimental and natural infections in MyD88- and IRAK-4-deficient mice and humans

    PubMed Central

    von Bernuth, Horst; Picard, Capucine; Puel, Anne; Casanova, Jean-Laurent

    2013-01-01

    Most Toll-like-receptors (TLRs) and interleukin-1 receptors (IL-1Rs) signal via myeloid differentiation primary response 88 (MyD88) and interleukin-1 receptor-associated kinase 4 (IRAK-4). The combined roles of these two receptor families in the course of experimental infections have been assessed in MyD88- and IRAK-4-deficient mice for almost fifteen years. These animals have been shown to be susceptible to 46 pathogens: 27 bacteria, 8 viruses, 7 parasites, and 4 fungi. Humans with inborn MyD88 or IRAK-4 deficiency were first identified in 2003. They suffer from naturally occurring life-threatening infections caused by a small number of bacterial species, although the incidence and severity of these infections decrease with age. Mouse TLR- and IL-1R-dependent immunity mediated by MyD88 and IRAK-4 seems to be vital to combat a wide array of experimentally administered pathogens at most ages. By contrast, human TLR- and IL-1R-dependent immunity mediated by MyD88 and IRAK-4 seems to be effective in the natural setting against only a few bacteria and is most important in infancy and early childhood. The roles of TLRs and IL-1Rs in protective immunity deduced from studies in mutant mice subjected to experimental infections should therefore be reconsidered in the light of findings for natural infections in humans carrying mutations as discussed in this review. PMID:23255009

  9. A Multifaceted Role for Myd88-Dependent Signaling in Progression of Murine Mammary Carcinoma

    PubMed Central

    Higgins, Mary J.; Serrano, Antonio; Boateng, Kofi Y.; Parsons, Victoria A.; Phuong, Tiffany; Seifert, Alyssa; Ricca, Jacob M.; Tucker, Kyle C.; Eidelman, Alec S.; Carey, Maureen A.; Kurt, Robert A.

    2016-01-01

    Previous data obtained in our laboratory suggested that there may be constitutive signaling through the myeloid differentiation primary response gene 88 (Myd88)-dependent signaling cascade in murine mammary carcinoma. Here, we extended these findings by showing that, in the absence of an added Toll-like receptor (TLR) agonist, the myddosome complex was preformed in 4T1 tumor cells, and that Myd88 influenced cytoplasmic extracellular signal–regulated kinase (Erk)1/Erk2 levels, nuclear levels of nuclear factor-kappaB (NFκB) and signal transducer and activator of transcription 5 (STAT5), tumor-derived chemokine (C–C motif) ligand 2 (CCL2) expression, and in vitro and in vivo tumor growth. In addition, RNA-sequencing revealed that Myd88-dependent signaling enhanced the expression of genes that could contribute to breast cancer progression and genes previously associated with poor outcome for patients with breast cancer, in addition to suppressing the expression of genes capable of inhibiting breast cancer progression. Yet, Myd88-dependent signaling in tumor cells also suppressed expression of genes that could contribute to tumor progression. Collectively, these data revealed a multifaceted role for Myd88-dependent signaling in murine mammary carcinoma. PMID:27812285

  10. Acute Brain Injury Triggers MyD88-Dependent, TLR2/4-Independent Inflammatory Responses

    PubMed Central

    Koedel, Uwe; Merbt, Ulrike Michaela; Schmidt, Caroline; Angele, Barbara; Popp, Bernadette; Wagner, Hermann; Pfister, Hans-Walter; Kirschning, Carsten J.

    2007-01-01

    Endogenous molecules released from disrupted cells and extracellular matrix degradation products activate Toll-like receptors (TLRs) and, thus, might contribute to immune activation after tissue injury. Here, we show that aseptic, cold-induced cortical injury triggered an acute immune response that involves increased production of multiple cytokines/chemokines accompanied by neutrophil recruitment to the lesion site. We observed selective reductions in injury-induced cytokine/chemokine expression as well as in neutrophil accumulation in mice lacking the common TLR signaling adaptor MyD88 compared with wild-type mice. Notably, attenuation of the immune response was paralleled by a reduction in lesion size. Neutrophil depletion of wild-type mice and transplantation of MyD88-deficient bone marrow into lethally irradiated wild-type recipients had no substantial impact on injury-induced expression of cytokines/chemokines and on lesion development. In contrast to MyD88 deficiency, double deficiency of TLR2 and TLR4—despite the two receptors being activated by specific endogenous molecules associated to danger and signal through MyD88—altered neither immune response nor extent of tissue lesion size on injury. Our data indicate modulation of the neuroinflammatory response and lesion development after aseptic cortical injury through MyD88-dependent but TLR2/4-independent signaling by central nervous system resident nonmyeloid cells. PMID:17591966

  11. Taurine Attenuates Hepatic Inflammation in Chronic Alcohol-Fed Rats Through Inhibition of TLR4/MyD88 Signaling

    PubMed Central

    Lin, Chao-Jen; Chiu, Chun-Ching; Chen, Yi-Chen; Chen, Mu-Lin

    2015-01-01

    Abstract Accumulating evidence indicates that overconsumption of ethanol contributes in many ways to the pathogenesis of hepatic injury. Although studies indicate that taurine decreases lipogenesis, oxidative stress, and inflammatory cytokines, the protective effect of taurine against alcohol-induced liver injury is still unclear. To clarify the precise signaling involved in the beneficial effect of taurine on alcohol-induced liver injury, rats were randomly divided into four treatment groups: (1) control (Ctl), (2) alcohol (Alc), (3) Alc+taurine (Tau), and (4) Alc+silymarin (Sil). The Tau and Sil groups had lower lymphocyte infiltration and significantly lower TLR-4/MyD88 and IκB/NFκB compared to the Alc group. The inducible nitric oxide synthase (iNOS), C-reactive protein (CRP), tumor necrosis factors (TNF)-α, interleukin (IL)-6, and IL-1β were also significantly lower in the Tau and Sil groups than in the Alc group. The experimental results indicated that hepatoprotection against alcohol-induced inflammation may be mediated by decreased TLR-4/MyD88 signaling. PMID:26090712

  12. VSV oncolytic virotherapy in the B16 model depends upon intact MyD88 signaling.

    PubMed

    Wongthida, Phonphimon; Diaz, Rosa M; Galivo, Feorillo; Kottke, Timothy; Thompson, Jill; Melcher, Alan; Vile, Richard

    2011-01-01

    We show here, for the first time to our knowledge, that the antitumor therapy of oncolytic vesicular stomatitis virus (VSV) in the B16ova model depends upon signaling through myeloid differentiation primary response gene 88 (MyD88) in host cells. VSV-mediated therapy of B16ova tumors was abolished in MyD88(-/-) mice despite generation of antigen-specific T cell responses similar to those in immune-competent mice. Mice defective in only toll-like receptor 4 (TLR4), TLR7, or interleukin 1 (IL-1) signaling retained VSV-induced therapy, suggesting that multiple, redundant pathways of innate immune activation by the virus contribute to antitumor immune reactivity. Lack of MyD88 signaling was associated with decreased expression of proinflammatory cytokines and neutrophil infiltration in response to intratumoral virus, as well as decreased infiltration of draining lymph nodes (LN) with plasmacytoid dendritic cells (pDCs) (CD11b(-)GR1(+)B220(+)) and myeloid-derived suppressor cells (CD11b(+)GR1(+)F4/80(+)). MyD88 signaling in response to VSV was also closely associated with a type I interferon (IFN) response. This inhibited virus replication within the tumor but also protected the host from viral dissemination from the tumor. Therefore, the innate immune response to oncolytic viruses can be, simultaneously, protherapeutic, antioncolytic, and systemically protective. These paradoxically conflicting roles need to be carefully considered in future strategies designed to improve the efficacy of oncolytic virotherapy.

  13. Signaling via MYD88 in the pancreatic tumor microenvironment: A double-edged sword.

    PubMed

    Zambirinis, Constantinos P; Miller, George

    2013-01-01

    We have recently shown that Toll-like receptor (TLR) signaling exacerbates pancreatic fibro-inflammation and promotes carcinogenesis in mice. Paradoxically, inhibition of the TLR-MYD88 signaling pathway is pro-tumorigenic owing to the dendritic cell-mediated TH2-polarization of CD4(+) T cells. TLR signaling appears to be central in pancreatic cancer-associated inflammation.

  14. Dissecting a Hub for Immune Response: Modeling the Structure of MyD88.

    PubMed

    Naro, Chiara; Sette, Claudio

    2016-03-01

    Immune cells sense foreign organisms through the evolutionarily conserved family of Toll-like receptors. Signaling from these receptors relies on oligomerization of adaptor molecules. In this issue of Structure, Vynke et al. (2016) shed light on the dynamical structure of the homo- and hetero-dimerization domain of MyD88, the main adaptor utilized by Toll-like receptors.

  15. Treg-mediated suppression of atherosclerosis requires MYD88 signaling in DCs

    PubMed Central

    Subramanian, Manikandan; Thorp, Edward; Hansson, Goran K.; Tabas, Ira

    2012-01-01

    TLR activation on CD11c+ DCs triggers DC maturation, which is critical for T cell activation. Given the expansion of CD11c+ DCs during the progression of atherosclerosis and the key role of T cell activation in atherogenesis, we sought to understand the role of TLR signaling in CD11c+ DCs in atherosclerosis. To this end, we used a mouse model in which a key TLR adaptor involved in DC maturation, MYD88, is deleted in CD11c+ DCs. We transplanted bone marrow containing Myd88-deficient CD11c+ DCs into Western diet–fed LDL receptor knockout mice and found that the transplanted mice had decreased activation of effector T cells in the periphery as well as decreased infiltration of both effector T cells and Tregs in atherosclerotic lesions. Surprisingly, the net effect was an increase in atherosclerotic lesion size due to an increase in the content of myeloid-derived inflammatory cells. The mechanism involves increased lesional monocyte recruitment associated with loss of Treg-mediated suppression of MCP-1. Thus, the dominant effect of MYD88 signaling in CD11c+ DCs in the setting of atherosclerosis is to promote the development of atheroprotective Tregs. In the absence of MYD88 signaling in CD11c+ DCs, the loss of this protective Treg response trumps the loss of proatherogenic T effector cell activation. PMID:23257360

  16. MyD88-dependent pro-inflammatory activity in Vi polysaccharide vaccine against typhoid promotes Ab switching to IgG.

    PubMed

    Garg, Rohini; Akhade, Ajay Suresh; Yadav, Jitender; Qadri, Ayub

    2015-10-01

    Vi capsular polysaccharide is currently in use as a vaccine against human typhoid caused by Salmonella Typhi. The vaccine efficacy correlates with IgG anti-Vi Abs. We have recently reported that Vi can generate inflammatory responses through activation of the TLR2/TLR1 complex. In the present study, we show that immunization with Vi produces IgM as well as IgG Abs in wild type mice. This ability is not compromised in mice deficient in T cells. However, immunization of mice lacking the TLR adaptor protein, MyD88, with Vi elicits only IgM Abs. These results suggest that MyD88-dependent pro-inflammatory ability of the Vi vaccine might be vital in generating IgG Abs with this T-independent Ag.

  17. TIFA upregulation after hypoxia-reoxygenation is TLR4- and MyD88-dependent and associated with HMGB1 upregulation and release.

    PubMed

    Ding, Ning; Zhang, Yong; Loughran, Patricia A; Wang, Qingde; Tsung, Allan; Billiar, Timothy R

    2013-10-01

    TRAF-interacting protein with a forkhead-associated domain (TIFA) is a tumor necrosis factor receptor-associated factor 6 (TRAF6) binding protein that mediates IL-1 signaling. We recently reported that TIFA mRNA is significantly upregulated early in the liver after trauma and hemorrhagic shock. In this study, we sought to characterize the upregulation of TIFA by hypoxia-reoxygenation and investigate its role in hypoxia-induced signaling. TIFA expression was detected by qRT-PCR and Western blotting in both mouse hemorrhagic shock with resuscitation (HS-R) and hepatocytes exposed to hypoxia-reoxygenation. Involvement of TLR4 and MyD88 was assessed using cells from TLR4(-/-) and MyD88(-/-) mice. The interaction of TIFA with TRAF6 and IRAK-1 was investigated using coimmunoprecipitation in vitro. RNAi was performed to knock down the endogenous expression of the TIFA gene in hepatocytes. High-mobility-group box 1 protein (HMGB1) expression was detected by Western blotting and ELISA, and the activation of NF-κB and MAPK was measured with EMSA and Western blotting. The results showed that TIFA expression was upregulated after HS-R in vivo and hypoxia-reoxygenation in vitro. Further analysis revealed that hypoxia-reoxygenation-induced upregulation of TIFA was TLR4- and MyD88-dependent. Moreover, TIFA was found to associate with TRAF6 constitutively, whereas its association with IRAK-1 was seen only after hypoxia-reoxygenation. Suppression of TIFA by siRNA reduced NF-κB activation and HMGB1 upregulation and release after hypoxia-reoxygenation. Taken together, these data suggest that TIFA is involved in the regulation of cell signaling in hypoxia-reoxygenation. The increase in TIFA level appears to be a feed-forward mechanism involved in TLR4/MyD88-dependent signaling, leading to NF-κB activation and HMGB1 release.

  18. Characterization of bbtTICAM from amphioxus suggests the emergence of a MyD88-independent pathway in basal chordates.

    PubMed

    Yang, Manyi; Yuan, Shaochun; Huang, Shengfeng; Li, Jun; Xu, Liqun; Huang, Huiqing; Tao, Xin; Peng, Jian; Xu, Anlong

    2011-10-01

    The MyD88-independent pathway, one of the two crucial TLR signaling routes, is thought to be a vertebrate innovation. However, a novel Toll/interleukin-1 receptor (TIR) adaptor, designated bbtTICAM, which was identified in the basal chordate amphioxus, links this pathway to invertebrates. The protein architecture of bbtTICAM is similar to that of vertebrate TICAM1 (TIR-containing adaptor molecule-1, also known as TRIF), while phylogenetic analysis based on the TIR domain indicated that bbtTICAM is the oldest ortholog of vertebrate TICAM1 and TICAM2 (TIR-containing adaptor molecule-2, also known as TRAM). Similar to human TICAM1, bbtTICAM activates NF-κB in a MyD88-independent manner by interacting with receptor interacting protein (RIP) via its RHIM motif. Such activation requires bbtTICAM to form homodimers in endosomes, and it may be negatively regulated by amphioxus SARM (sterile α and armadillo motif-containing protein) and TRAF2. However, bbtTICAM did not induce the production of type I interferon. Thus, our study not only presents the ancestral features of vertebrate TICAM1 and TICAM2, but also reveals the evolutionary origin of the MyD88-independent pathway from basal chordates, which will aid in understanding the development of the vertebrate TLR network.

  19. Characterization of bbtTICAM from amphioxus suggests the emergence of a MyD88-independent pathway in basal chordates

    PubMed Central

    Yang, Manyi; Yuan, Shaochun; Huang, Shengfeng; Li, Jun; Xu, Liqun; Huang, Huiqing; Tao, Xin; Peng, Jian; Xu, Anlong

    2011-01-01

    The MyD88-independent pathway, one of the two crucial TLR signaling routes, is thought to be a vertebrate innovation. However, a novel Toll/interleukin-1 receptor (TIR) adaptor, designated bbtTICAM, which was identified in the basal chordate amphioxus, links this pathway to invertebrates. The protein architecture of bbtTICAM is similar to that of vertebrate TICAM1 (TIR-containing adaptor molecule-1, also known as TRIF), while phylogenetic analysis based on the TIR domain indicated that bbtTICAM is the oldest ortholog of vertebrate TICAM1 and TICAM2 (TIR-containing adaptor molecule-2, also known as TRAM). Similar to human TICAM1, bbtTICAM activates NF-κB in a MyD88-independent manner by interacting with receptor interacting protein (RIP) via its RHIM motif. Such activation requires bbtTICAM to form homodimers in endosomes, and it may be negatively regulated by amphioxus SARM (sterile α and armadillo motif-containing protein) and TRAF2. However, bbtTICAM did not induce the production of type I interferon. Thus, our study not only presents the ancestral features of vertebrate TICAM1 and TICAM2, but also reveals the evolutionary origin of the MyD88-independent pathway from basal chordates, which will aid in understanding the development of the vertebrate TLR network. PMID:21931360

  20. S100A8/MYD88/NF-қB: a novel pathway involved in cardiomyocyte hypertrophy driven by thyroid hormone.

    PubMed

    Takano, Ana Paula Cremasco; Munhoz, Carolina Demarchi; Moriscot, Anselmo Sigari; Gupta, Sudhiranjan; Barreto-Chaves, Maria Luiza Morais

    2017-02-04

    Recent studies have evidenced the involvement of inflammation-related pathways to the development of cardiac hypertrophy and other consequences on the cardiovascular system, including the calcium-binding protein S100A8. However, this has never been investigated in the thyroid hormone (TH)-prompted cardiac hypertrophy. Thus, we aimed to test whether S100A8 and related signaling molecules, myeloid differentiation factor-88 (MyD88) and nuclear factor kappa B (NF-қB), could be associated with the cardiomyocyte hypertrophy induced by TH. Our results demonstrate that the S100A8/MyD88/NF-қB signaling pathway is activated in cardiomyocytes following TH stimulation. The knockdown of S100A8 and MyD88 indicates the contribution of those molecules to cardiomyocyte hypertrophy in response to TH, as evaluated by cell surface area, leucine incorporation assay, and gene expression. Furthermore, S100A8 and MyD88 are crucial mediators of NF-қB activation, which is also involved in the hypertrophic growth of TH-treated cardiomyocytes. Supporting the in vitro data, the contribution of NF-қB for TH-induced cardiac hypertrophy is confirmed in vivo, by using transgenic mice with cardiomyocyte-specific suppression of NF-қB. These data identify a novel pathway regulated by TH that mediates cardiomyocyte hypertrophy. However, the potential role of this new pathway in short and long-term cardiac effects of TH remains to be further investigated.

  1. MyD88-Dependent Immunity to a Natural Model of Vaccinia Virus Infection Does Not Involve Toll-Like Receptor 2

    PubMed Central

    Davies, Michael L.; Sei, Janet J.; Siciliano, Nicholas A.; Xu, Ren-Huan; Roscoe, Felicia; Sigal, Luis J.; Eisenlohr, Laurence C.

    2014-01-01

    ABSTRACT Although the pattern recognition receptor Toll-like receptor 2 (TLR2) is typically thought to recognize bacterial components, it has been described to alter the induction of both innate and adaptive immunity to a number of viruses, including vaccinia virus (VACV). However, many pathogens that reportedly encode TLR2 agonists may actually be artifactually contaminated during preparation, possibly with cellular debris or merely with molecules that sensitize cells to be activated by authentic TLR2 agonists. In both humans and mice, the most relevant natural route of infection with VACV is through intradermal infection of the skin. Therefore, we examined the requirement for TLR2 and its signaling adaptor MyD88 in protective immunity to VACV after intradermal infection. We find that although TLR2 may recognize virus preparations in vitro and have a minor role in preventing dissemination of VACV following systemic infection with large doses of virus, it is wholly disposable in both control of virus replication and induction of adaptive immunity following intradermal infection. In contrast, MyD88 is required for efficient induction of CD4 T cell and B cell responses and for local control of virus replication following intradermal infection. However, even MyD88 is not required to induce local inflammation, inflammatory cytokine production, or recruitment of cells that restrict virus from spreading systemically after peripheral infection. Thus, an effective antiviral response does require MyD88, but TLR2 is not required for control of a peripheral VACV infection. These findings emphasize the importance of studying relevant routes of infection when examining innate sensing mechanisms. IMPORTANCE Vaccinia virus (VACV) provides the backbone for some of the most widely used and successful viral vaccine vectors and is also related to the human pathogens Cantagalo virus and molluscum contagiosum virus that infect the skin of patients. Therefore, it is vital to understand

  2. Fish IRF6 is a positive regulator of IFN expression and involved in both of the MyD88 and TBK1 pathways.

    PubMed

    Li, Shun; Lu, Long-Feng; Wang, Zhao-Xi; Chen, Dan-Dan; Zhang, Yong-An

    2016-10-01

    Interferon (IFN) regulatory factors (IRF) are the crucial transcription factors for IFN expression, leading host cell response to viral infection. In mammals, only IRF6 is unaffected by IFN expression in the IRF family; however, in fish, a lower vertebrate, whether IRF6 is related to IFN regulation is unclear. In this study, we identified that zebrafish IRF6 was a positive regulator of IFN transcription and could be phosphorylated by both MyD88 and TBK1. First, the transcript level of cellular irf6 was upregulated by treatment with poly I:C (a mimic of viral RNAs), indicating IRF6 might be involved in the process of host cell response to viruses. Overexpression of IRF6 could upregulate IFN promoter activity significantly, meaning IRF6 is a positive regulator of IFN transcription. Subsequently, at the protein regulation level and in the interaction relationship, IRF6 was phosphorylated by and associated with both MyD88 and TBK1. In addition, overexpression of IRF6 activated the transcription of isg15, rig-i and mavs of host cells; meanwhile, the transcripts of p, m and n genes of SVCV were significantly declined in IRF6-overexpressing cells. Taken together, our data demonstrate that fish IRF6 is distinguished from the homolog of mammals by being a positive regulator of IFN transcription and phosphorylated by MyD88 and TBK1, suggesting that differences in the IRF6 regulation pattern exist between lower and higher vertebrates.

  3. MyD88 and TLR9 Dependent Immune Responses Mediate Resistance to Leishmania guyanensis Infections, Irrespective of Leishmania RNA Virus Burden

    PubMed Central

    Ives, Annette; Masina, Slavica; Castiglioni, Patrik; Prével, Florence; Revaz-Breton, Mélanie; Hartley, Mary-Anne; Launois, Pascal; Fasel, Nicolas; Ronet, Catherine

    2014-01-01

    Infections with Leishmania parasites of the Leishmania Viannia subgenus give rise to both localized cutaneous (CL), and metastatic leishmaniasis. Metastasizing disease forms including disseminated (DCL) and mutocutaneous (MCL) leishmaniasis result from parasitic dissemination and lesion formation at sites distal to infection and have increased inflammatory responses. The presence of Leishmania RNA virus (LRV) in L. guyanensis parasites contributes to the exacerbation of disease and impacts inflammatory responses via activation of TLR3 by the viral dsRNA. In this study we investigated other innate immune response adaptor protein modulators and demonstrated that both MyD88 and TLR9 played a crucial role in the development of Th1-dependent healing responses against L. guyanensis parasites regardless of their LRV status. The absence of MyD88- or TLR9-dependent signaling pathways resulted in increased Th2 associated cytokines (IL-4 and IL-13), which was correlated with low transcript levels of IL-12p40. The reliance of IL-12 was further confirmed in IL12AB−/− mice, which were completely susceptible to infection. Protection to L. guyanensis infection driven by MyD88- and TLR9-dependent immune responses arises independently to those induced due to high LRV burden within the parasites. PMID:24801628

  4. Natural killer cell intrinsic toll-like receptor MyD88 signaling contributes to IL-12-dependent IFN-γ production by mice during infection with Toxoplasma gondii.

    PubMed

    Ge, Yiyue; Chen, Jinling; Qiu, Xiaoyan; Zhang, Jie; Cui, Lunbiao; Qi, Yuhua; Liu, Xinjian; Qiu, Jingfan; Shi, Zhiyang; Lun, Zhaorong; Shen, Jilong; Wang, Yong

    2014-06-01

    Myeloid differentiation factor 88 (MyD88)-dependent IL-12 secretion by dendritic cells is critical for natural killer cell-mediated IFN-γ production and innate resistance to Toxoplasma gondii. Although MyD88(-/-) mice challenged with T. gondii have defective IL-12 responses and succumb to infection, administration of IL-12 to MyD88(-/-) mice fails to prevent acute mortality, suggesting that MyD88 may mediate signals within natural killer cells important for IL-12-dependent IFN-γ production and innate resistance to this parasite. In this study, we found that T. gondii antigens and IL-12 could synergistically trigger IFN-γ secretion by natural killer cells, which was dependent on toll-like receptor-MyD88 signaling. Further analysis showed that p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, c-Jun N-terminal kinase and NF-κB multiple pathways downstream of MyD88 contributed to IFN-γ production by natural killer cells. Moreover, the well-established toll-like receptor agonists, T. gondii profilin (Tgprofilin) and T. gondii heat shock protein 70 (TgHSP70) could evoke a similar IFN-γ secretory response in natural killer cells to that evoked by T. gondii antigens. In vivo adoptive transfer experiments showed that, upon challenge with T. gondii, NOD/SCID-β2 microglobulin null (NOD/SCID-β2m(-/-)) mice injected i.v. with MyD88(-/-) natural killer cells had reduced serum IFN-γ levels and increased splenic tachyzoite burdens compared with those injected i.v. with wild-type natural killer cells. Taken together, these findings demonstrate a critical role for natural killer cell intrinsic toll-like receptor-MyD88 signaling in IL-12-dependent early IFN-γ production and innate resistance to T. gondii.

  5. Lymphoplasmacytic Lymphoma Presenting with Diarrhea and Joint Pain Which was Successfully Diagnosed by an MYD88 Mutation Analysis.

    PubMed

    Fukushima, Masaya; Okoshi, Yasushi; Fukazawa, Keiko; Koshino, Mayuko; Ishiguro, Shingo; Mitsuhashi, Shoichi; Saitoh, Hitoaki; Iijima, Tatsuo; Kojima, Hiroshi; Hori, Mitsuo

    2017-01-01

    A 55-year-old man presented to our department with diarrhea, weight loss, fatigability, and polyarthralgia. Blood tests revealed elevated soluble interleukin-2 receptor levels and IgG-type M protein positivity, without any findings that were suggestive of collagen disease. After computed tomography (CT) detected enlarged lymph nodes in the abdominal para-aortic region, lymphoma was suspected. CT-guided needle biopsy of the lymph node did not help to achieve a definitive diagnosis; however, a bone marrow test showed the pathological features of B-cell lymphoma. A genetic examination detected a MYD88 L265P mutation; the mutation analysis was valuable in diagnosing lymphoplasmacytic lymphoma in a IgM-type M protein-negative patient.

  6. Attenuation of Hepatic Graft-versus-host Disease in Allogeneic Recipients of MyD88-deficient Donor Bone Marrow.

    PubMed

    Lim, Ji-Young; Lee, Young-Kwan; Lee, Sung-Eun; Ju, Ji-Min; Park, Gyeongsin; Choi, Eun Young; Min, Chang-Ki

    2015-06-01

    Acute graft-versus-host-disease (GVHD) is characterized by selective damage to the liver, the skin, and the gastrointestinal tract. Following allogeneic hematopoietic stem cell transplantation, donor bone marrow (BM) cells repopulate the immune system of the recipient. We previously demonstrated that the acute intestinal GVHD (iGVHD) mortality rate was higher in MyD88-deficient BM recipients than that in the control BM recipients. In the present study, the role of MyD88 (expressed by donor BM) in the pathophysiology of hepatic GVHD (hGVHD) was examined. Unlike iGVHD, transplantation with MyD88-deficient T-cell depleted (TCD) BM attenuated hGVHD severity and was associated with low infiltration of T cells into the liver of the recipients. Moreover, GVHD hosts, transplanted with MyD88-deficient TCD BM, exhibited markedly reduced expansion of CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSC) in the liver. Adoptive injection of the MDSC from wild type mice, but not MyD88-deficient mice, enhanced hepatic T cell infiltration in the MyD88-deficient TCD BM recipients. Pre-treatment of BM donors with LPS increased MDSC levels in the liver of allogeneic wild type BM recipients. In conclusion, hGVHD and iGVHD may occur through various mechanisms based on the presence of MyD88 in the non-T cell compartment of the allograft.

  7. MyD88 is pivotal for immune recognition of Citrobacter koseri and astrocyte activation during CNS infection.

    PubMed

    Liu, Shuliang; Kielian, Tammy

    2011-04-16

    Citrobacter koseri (C. koseri) is a Gram-negative bacterium that can cause a highly aggressive form of neonatal meningitis, which often progresses to establish multi-focal brain abscesses. The roles of Toll-like receptor 4 (TLR4) and its signaling adaptor MyD88 during CNS C. koseri infection have not yet been examined, which is important since recent evidence indicates that innate immune responses are tailored towards specific pathogen classes. Here TLR4 WT (C3H/FeJ) and TLR4 mutant (C3H/HeJ) mice as well as MyD88 KO animals were infected intracerebrally with live C. koseri, resulting in meningitis and ventriculitis with accompanying brain abscess formation. MyD88 KO mice were exquisitely sensitive to C. koseri, demonstrating enhanced mortality rates and significantly elevated bacterial burdens compared to WT animals. Interestingly, although early proinflammatory mediator release (i.e. 12 h) was MyD88-dependent, a role for MyD88-independent signaling was evident at 24 h, revealing a compensatory response to CNS C. koseri infection. In contrast, TLR4 did not significantly impact bacterial burdens or proinflammatory mediator production in response to C. koseri. Similar findings were obtained with primary astrocytes, where MyD88-dependent pathways were essential for chemokine release in response to intact C. koseri, whereas TLR4 was dispensable; implicating the involvement of alternative TLRs since highly enriched astrocytes did not produce IL-1 upon bacterial exposure, which also signals via MyD88. Collectively, these findings demonstrate the importance of MyD88-dependent mechanisms in eliciting maximal proinflammatory responses, astrocyte activation, and bacterial containment during CNS C. koseri infection, as well as a late-phase MyD88-independent signaling pathway for cytokine/chemokine production.

  8. MyD88 in macrophages is critical for abscess resolution in staphylococcal skin infection.

    PubMed

    Feuerstein, Reinhild; Seidl, Maximilian; Prinz, Marco; Henneke, Philipp

    2015-03-15

    When Staphylococcus aureus penetrates the epidermis and reaches the dermis, polymorphonuclear leukocytes (PMLs) accumulate and an abscess is formed. However, the molecular mechanisms that orchestrate initiation and termination of inflammation in skin infection are incompletely understood. In human myeloid differentiation primary response gene 88 (MyD88) deficiency, staphylococcal skin and soft tissue infections are a leading and potentially life-threatening problem. In this study, we found that MyD88-dependent sensing of S. aureus by dermal macrophages (Mϕ) contributes to both timely escalation and termination of PML-mediated inflammation in a mouse model of staphylococcal skin infection. Mϕs were key to recruit PML within hours in response to staphylococci, irrespective of bacterial viability. In contrast with bone marrow-derived Mϕs, dermal Mϕs did not require UNC-93B or TLR2 for activation. Moreover, PMLs, once recruited, were highly activated in an MyD88-independent fashion, yet failed to clear the infection if Mϕs were missing or functionally impaired. In normal mice, clearance of the infection and contraction of the PML infiltrate were accompanied by expansion of resident Mϕs in a CCR2-dependent fashion. Thus, whereas monocytes were dispensable for the early immune response to staphylococci, they contributed to Mϕ renewal after the infection was overcome. Taken together, MyD88-dependent sensing of staphylococci by resident dermal Mϕs is key for a rapid and balanced immune response, and PMLs are dependent on intact Mϕ for full function. Renewal of resident Mϕs requires both local control of bacteria and inflammatory monocytes entering the skin.

  9. Susceptibility of prostate epithelial cells to Chlamydia muridarum infection and their role in innate immunity by recruitment of intracellular Toll-like receptors 4 and 2 and MyD88 to the inclusion.

    PubMed

    Mackern-Oberti, Juan Pablo; Maccioni, Mariana; Cuffini, Cecilia; Gatti, Gerardo; Rivero, Virginia E

    2006-12-01

    Although Chlamydia infections are widespread throughout the world, data about immunopathogenesis of genitourinary tract infections in males are very limited. In the present work we present an in vitro model of male genital tract-derived epithelial cells, more precisely prostate epithelial cells (PEC), to analyze if they are susceptible and able to respond to Chlamydia muridarum infection. Our results demonstrate that rat PEC are susceptible to C. muridarum infection and respond to this pathogen by up-regulating different proinflammatory cytokine and chemokine genes that could participate in the recruitment and local activation of immune cells, therefore influencing innate and adaptive immune responses during Chlamydia infection. Moreover, we analyzed the expression of Toll-like receptor 4 (TLR4), TLR2, and related molecules on PEC and the effect of C. muridarum infection on their expression. Our results demonstrate that PEC express significant levels of TLR4, CD14, TLR2, and the adaptor molecule MyD88 and up-regulate these proteins in response to C. muridarum infection. Indeed, TLR4, CD14, TLR2, and the adaptor MyD88 are specifically recruited to the vicinity of the bacterial inclusion, suggesting that these TLRs are actively engaged in signaling from this intracellular location in these cells. This is, to our knowledge, the first time that an in vitro model of infection with Chlamydia of male tract-derived epithelial cells has been achieved, and it provides the opportunity to determine how these cells respond and participate in modulating innate and adaptive immune response during Chlamydia infections.

  10. Lipid IVa incompletely activates MyD88-independent Toll-like receptor 4 signaling in mouse macrophage cell lines.

    PubMed

    Ogura, Norihiko; Muroi, Masashi; Sugiura, Yuka; Tanamoto, Ken-ichi

    2013-04-01

    We investigated the difference in the effect of synthetic lipid A compounds on MyD88-dependent and -independent Toll-like receptor 4 (TLR4) signaling in mouse macrophage cells. At higher concentrations, Escherichia coli-type hexa-acylated lipid A 506, Salmonella-type hepta-acylated lipid A 516, the lipid A precursor lipid IVa and monophosphoryl lipid A induced similar levels of production of the MyD88-dependent cytokine IL-1β although their potencies varied, whereas the maximum production of the MyD88-independent cytokine RANTES induced by lipid IVa was less than 50% that of other lipid A compounds. A maximum level of NF-κB activation, which is involved in IL-1β gene transcription, was also induced to a similar level by these four lipid A compounds, while the maximum level of IFN-β promoter activity induced during MyD88-independent signaling was also less than 50% for lipid IVa stimulation compared with other lipid A compounds. Early IκBα phosphorylation activated by MyD88-dependent signaling was similarly induced by 506 and lipid IVa, whereas lipid IVa barely stimulated the phosphorylation of IRF3, a MyD88-independent transcription factor, although efficient phosphorylation was observed with 506 stimulation. These results indicate that lipid IVa has limited activity toward MyD88-independent signaling of TLR4, in macrophage cell lines, despite having efficient activity in the MyD88-dependent pathway.

  11. MyD88 expression by CNS-resident cells is pivotal for eliciting protective immunity in brain abscesses.

    PubMed

    Garg, Sarita; Nichols, Jessica R; Esen, Nilufer; Liu, Shuliang; Phulwani, Nirmal K; Syed, Mohsin Md; Wood, William H; Zhang, Yongqing; Becker, Kevin G; Aldrich, Amy; Kielian, Tammy

    2009-05-05

    MyD88 KO (knockout) mice are exquisitely sensitive to CNS (central nervous system) infection with Staphylococcus aureus, a common aetiological agent of brain abscess, exhibiting global defects in innate immunity and exacerbated tissue damage. However, since brain abscesses are typified by the involvement of both activated CNS-resident and infiltrating immune cells, in our previous studies it has been impossible to determine the relative contribution of MyD88-dependent signalling in the CNS compared with the peripheral immune cell compartments. In the present study we addressed this by examining the course of S. aureus infection in MyD88 bone marrow chimaera mice. Interestingly, chimaeras where MyD88 was present in the CNS, but not bone marrow-derived cells, mounted pro-inflammatory mediator expression profiles and neutrophil recruitment equivalent to or exceeding that detected in WT (wild-type) mice. These results implicate CNS MyD88 as essential in eliciting the initial wave of inflammation during the acute response to parenchymal infection. Microarray analysis of infected MyD88 KO compared with WT mice revealed a preponderance of differentially regulated genes involved in apoptotic pathways, suggesting that the extensive tissue damage characteristic of brain abscesses from MyD88 KO mice could result from dysregulated apoptosis. Collectively, the findings of the present study highlight a novel mechanism for CNS-resident cells in initiating a protective innate immune response in the infected brain and, in the absence of MyD88 in this compartment, immunity is compromised.

  12. Helical assembly in the MyD88-IRAK4-IRAK2 complex in TLR/IL-1R signalling

    SciTech Connect

    Lin, Su-Chang; Lo, Yu-Chih; Wu, Hao

    2010-08-23

    MyD88, IRAK4 and IRAK2 are critical signalling mediators of the TLR/IL1-R superfamily. Here we report the crystal structure of the MyD88-IRAK4-IRAK2 death domain (DD) complex, which surprisingly reveals a left-handed helical oligomer that consists of 6 MyD88, 4 IRAK4 and 4 IRAK2 DDs. Assembly of this helical signalling tower is hierarchical, in which MyD88 recruits IRAK4 and the MyD88-IRAK4 complex recruits the IRAK4 substrates IRAK2 or the related IRAK1. Formation of these Myddosome complexes brings the kinase domains of IRAKs into proximity for phosphorylation and activation. Composite binding sites are required for recruitment of the individual DDs in the complex, which are confirmed by mutagenesis and previously identified signalling mutations. Specificities in Myddosome formation are dictated by both molecular complementarity and correspondence of surface electrostatics. The MyD88-IRAK4-IRAK2 complex provides a template for Toll signalling in Drosophila and an elegant mechanism for versatile assembly and regulation of DD complexes in signal transduction.

  13. The toll-like receptor signaling molecule Myd88 contributes to pancreatic beta-cell homeostasis in response to injury.

    PubMed

    Bollyky, Paul L; Bice, Jeffrey B; Sweet, Ian R; Falk, Ben A; Gebe, John A; Clark, April E; Gersuk, Vivian H; Aderem, Alan; Hawn, Thomas R; Nepom, Gerald T

    2009-01-01

    Commensal flora and pathogenic microbes influence the incidence of diabetes in animal models yet little is known about the mechanistic basis of these interactions. We hypothesized that Myd88, an adaptor molecule in the Toll-like-receptor (TLR) pathway, regulates pancreatic beta-cell function and homeostasis. We first examined beta-cells histologically and found that Myd88-/- mice have smaller islets in comparison to C57Bl/6 controls. Myd88-/- mice were nonetheless normoglycemic both at rest and after an intra-peritoneal glucose tolerance test (IPGTT). In contrast, after low-dose streptozotocin (STZ) challenge, Myd88-/-mice had an abnormal IPGTT relative to WT controls. Furthermore, Myd88-/- mice suffer enhanced beta-cell apoptosis and have enhanced hepatic damage with delayed recovery upon low-dose STZ treatment. Finally, we treated WT mice with broad-spectrum oral antibiotics to deplete their commensal flora. In WT mice, low dose oral lipopolysaccharide, but not lipotichoic acid or antibiotics alone, strongly promoted enhanced glycemic control. These data suggest that Myd88 signaling and certain TLR ligands mediate a homeostatic effect on beta-cells primarily in the setting of injury.

  14. The Toll-Like Receptor Signaling Molecule Myd88 Contributes to Pancreatic Beta-Cell Homeostasis in Response to Injury

    PubMed Central

    Bollyky, Paul L.; Bice, Jeffrey B.; Sweet, Ian R.; Falk, Ben A.; Gebe, John A.; Clark, April E.; Gersuk, Vivian H.; Aderem, Alan; Hawn, Thomas R.; Nepom, Gerald T.

    2009-01-01

    Commensal flora and pathogenic microbes influence the incidence of diabetes in animal models yet little is known about the mechanistic basis of these interactions. We hypothesized that Myd88, an adaptor molecule in the Toll-like-receptor (TLR) pathway, regulates pancreatic β-cell function and homeostasis. We first examined β-cells histologically and found that Myd88−/− mice have smaller islets in comparison to C57Bl/6 controls. Myd88−/− mice were nonetheless normoglycemic both at rest and after an intra-peritoneal glucose tolerance test (IPGTT). In contrast, after low-dose streptozotocin (STZ) challenge, Myd88−/−mice had an abnormal IPGTT relative to WT controls. Furthermore, Myd88−/− mice suffer enhanced β-cell apoptosis and have enhanced hepatic damage with delayed recovery upon low-dose STZ treatment. Finally, we treated WT mice with broad-spectrum oral antibiotics to deplete their commensal flora. In WT mice, low dose oral lipopolysaccharide, but not lipotichoic acid or antibiotics alone, strongly promoted enhanced glycemic control. These data suggest that Myd88 signaling and certain TLR ligands mediate a homeostatic effect on β-cells primarily in the setting of injury. PMID:19357791

  15. Lipopolysaccharide induced LOX-1 expression via TLR4/MyD88/ROS activated p38MAPK-NF-κB pathway.

    PubMed

    Zhao, Wenwen; Ma, Guixin; Chen, Xiuping

    2014-12-01

    Lectin-like receptor for oxidized low density lipoprotein (LOX-1) plays a key role in endothelial ox-LDL endocytosis, endothelial dysfunction and atherogenesis. In the present study, the effect of lipopolysaccharide (LPS) on LOX-1 expression and the underlying molecular pathways were investigated. Human umbilical vein endothelial cells (HUVECs) were treated with LPS and the protein expressions of LOX-1, TLR4, TLR2, MyD88, Nox4, Nox2, PI3K, p38MAPK, JNK, ERK, Nrf1, Nrf2 and p65 were examined by Western blotting. The intracellular reactive oxygen species (ROS) production was examined by flow cytometry with fluorescence probe DCFH2-DA. The role of TLR4, MyD88 and Nox4 were determined with specific siRNA. The endothelial ox-LDL uptake and the endothelial-monocyte adhesion were evaluated with DiI-ox-LDL and Hoechst 33342 respectively. The effect of LPS on LOX-1 expression in aorta tissue was also studied with male C57/BL6 mice by intraperitoneal injection of LPS. The results showed that LPS induced LOX-1 protein expression in a time- and concentration-dependent manner. The mRNA expression of LOX-1 was also upregulated. The protein expression of LOX-1 and phosphorylated p38MAPK, p65 was significantly enhanced by LPS both in vitro and in vivo. LPS induced LOX-1 expression was blocked by siRNA for TLR4, MyD88, and Nox4 and inhibitors for p38MAPK, NF-κB, cyclooxygenase-2, and NADPH oxidase. Both LPS induced ox-LDL uptake and endothelial-monocyte adhesion were significantly inhibited by anti-LOX-1 antibody. LPS dramatically induced LOX-1 protein expression in aorta tissues. In conclusion, our data suggested that LPS induces LOX-1 expression via TLR4/MyD88/ROS activated p38MAPK/NF-κB pathway in endothelial cells, which provides new regulatory mechanisms for LOX-1 expression.

  16. MyD88-Dependent Signaling Influences Fibrosis and Alternative Macrophage Activation during Staphylococcus aureus Biofilm Infection

    PubMed Central

    Hanke, Mark L.; Angle, Amanda; Kielian, Tammy

    2012-01-01

    Bacterial biofilms represent a significant therapeutic challenge based on their ability to evade host immune and antibiotic-mediated clearance. Recent studies have implicated IL-1β in biofilm containment, whereas Toll-like receptors (TLRs) had no effect. This is intriguing, since both the IL-1 receptor (IL-1R) and most TLRs impinge on MyD88-dependent signaling pathways, yet the role of this key adaptor in modulating the host response to biofilm growth is unknown. Therefore, we examined the course of S. aureus catheter-associated biofilm infection in MyD88 knockout (KO) mice. MyD88 KO animals displayed significantly increased bacterial burdens on catheters and surrounding tissues during early infection, which coincided with enhanced dissemination to the heart and kidney compared to wild type (WT) mice. The expression of several proinflammatory mediators, including IL-6, IFN-γ, and CXCL1 was significantly reduced in MyD88 KO mice, primarily at the later stages of infection. Interestingly, immunofluorescence staining of biofilm-infected tissues revealed increased fibrosis in MyD88 KO mice concomitant with enhanced recruitment of alternatively activated M2 macrophages. Taken in the context of previous studies with IL-1β, TLR2, and TLR9 KO mice, the current report reveals that MyD88 signaling is a major effector pathway regulating fibrosis and macrophage polarization during biofilm formation. Together these findings represent a novel example of the divergence between TLR and MyD88 action in the context of S. aureus biofilm infection. PMID:22879997

  17. TIFA Upregulation after Hypoxia-Reoxygenation is TLR4 and MyD88-Dependent and Associated with HMGB1 Upregulation and Release

    PubMed Central

    Ding, Ning; Zhang, Yong; Loughran, Patricia A.; Wang, Qingde; Billiar, Timothy R.

    2013-01-01

    TRAF-interacting protein with a forkhead-associated domain (TIFA) is a tumor necrosis factor receptor-associated factor 6 (TRAF6) binding protein that mediates IL-1 signaling. We recently reported that TIFA mRNA is significantly upregulated early in the liver following trauma and hemorrhagic shock. In the present study, we sought to characterize the upregulation of TIFA by hypoxia-reoxygenation and investigate its role in hypoxia-induced signaling. TIFA expression was detected by qRT-PCR and Western blotting in both mouse hemorrhagic shock with resuscitation (HS-R) and hepatocytes exposed to hypoxia-reoxygenation. Involvement of TLR4 and MyD88 was assessed using cells from TLR4−/− and MyD88−/− mice. The interaction of TIFA with TRAF6 and IRAK-1 was investigated using co-immunoprecipitation in vitro. RNAi was performed to knock down the endogenous expression of the TIFA gene in hepatocytes. High mobility group box 1 protein (HMGB1) expression was detected by Western blotting and ELISA, and the activation of NF-κB and MAPK was measured with EMSA and Western blotting. The results showed that TIFA expression was upregulated following HS-R in vivo and hypoxia-reoxygenation in vitro. Further analysis revealed that hypoxia-reoxygenation-induced upregulation of TIFA was TLR4 and MyD88-dependent. Moreover, TIFA was found to associate with TRAF6 constitutively, whereas its association with IRAK-1 was seen only after hypoxia-reoxygenation. Suppression of TIFA by siRNA reduced NF-κB activation and HMGB1 upregulation and release following hypoxia-reoxygenation. Taken together, these data suggest that TIFA is involved in the regulation of cell signaling in hypoxia-reoxygenation. The increase in TIFA level appears to be a feed-forward mechanism involved in TLR4/MyD88-dependent signaling, leading to NF-κB activation and HMGB1 release. PMID:23722163

  18. Positive Correlation between Enhanced Expression of TLR4/MyD88/NF-κB with Insulin Resistance in Placentae of Gestational Diabetes Mellitus.

    PubMed

    Feng, Hui; Su, Rina; Song, Yilin; Wang, Chen; Lin, Li; Ma, Jingmei; Yang, Huixia

    2016-01-01

    Insulin resistance (IR) is a critical factor of the pathophysiology of Gestational diabetes mellitus (GDM). Studies on key organs involved in IR, such as livers and adipose tissues, showed that Toll-like receptor 4 (TLR4) can regulate insulin sensitivity. As a maternal-fetal interface with multi-functions, placentae could contribute to the development of IR for GDM. Thus, we investigated the expressions of TLR4/Myeloid Differentiation factor 88 (MyD88)/Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-kB) in term placentae from 33 GDM women and 36 healthy pregnant women with normal glucose tolerance, evaluated local and systemic IR and furthermore identified the association between placental TLR4 and IR. TLR4 protein was expressed in various cells of term placenta, particularly in syncytiotrophoblast of villi. Compared with normal pregnancy, the expression of TLR4/MyD88/NF-kB pathway increased in the placenta of GDM (p<0.05), and these differences were more pronounced in the maternal section of the placenta and the syncytiotrophoblast of villi. In addition, more severe IR was observed in the placenta of GDM patients than the control group, evidenced with higher pIRS-1(ser312) (p<0.001) and lower IRS-1 (p<0.05) as well as pAkt proteins (p<0.01). The expression of TLR4 in placentae is positively correlated with local IR (pIRS-1: r = 0.76, p <0.001 and pAkt: r = -0.47, p <0.001) and maternal fasting (r = 0.42, p <0.01), one-hour (r = 0.52, p <0.01) and two-hour glucose (r = 0.54, p <0.01) at OGTT. We found an that enhanced expression of the TLR4-MyD88-NF-kB pathway occurs in GDM placentae, which positively correlates with heightened local IR in placentae and higher maternal hyperglycemia. The TLR4/MyD88/NF-kB pathway may play a potential role in the development of IR in placentae of GDM.

  19. Positive Correlation between Enhanced Expression of TLR4/MyD88/NF-κB with Insulin Resistance in Placentae of Gestational Diabetes Mellitus

    PubMed Central

    Feng, Hui; Wang, Chen; Lin, Li; Ma, Jingmei; Yang, Huixia

    2016-01-01

    Insulin resistance (IR) is a critical factor of the pathophysiology of Gestational diabetes mellitus (GDM). Studies on key organs involved in IR, such as livers and adipose tissues, showed that Toll-like receptor 4 (TLR4) can regulate insulin sensitivity. As a maternal-fetal interface with multi-functions, placentae could contribute to the development of IR for GDM. Thus, we investigated the expressions of TLR4/Myeloid Differentiation factor 88 (MyD88)/Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-kB) in term placentae from 33 GDM women and 36 healthy pregnant women with normal glucose tolerance, evaluated local and systemic IR and furthermore identified the association between placental TLR4 and IR. TLR4 protein was expressed in various cells of term placenta, particularly in syncytiotrophoblast of villi. Compared with normal pregnancy, the expression of TLR4/MyD88/NF-kB pathway increased in the placenta of GDM (p<0.05), and these differences were more pronounced in the maternal section of the placenta and the syncytiotrophoblast of villi. In addition, more severe IR was observed in the placenta of GDM patients than the control group, evidenced with higher pIRS-1(ser312) (p<0.001) and lower IRS-1 (p<0.05) as well as pAkt proteins (p<0.01). The expression of TLR4 in placentae is positively correlated with local IR (pIRS-1: r = 0.76, p <0.001 and pAkt: r = -0.47, p <0.001) and maternal fasting (r = 0.42, p <0.01), one-hour (r = 0.52, p <0.01) and two-hour glucose (r = 0.54, p <0.01) at OGTT. We found an that enhanced expression of the TLR4-MyD88-NF-kB pathway occurs in GDM placentae, which positively correlates with heightened local IR in placentae and higher maternal hyperglycemia. The TLR4/MyD88/NF-kB pathway may play a potential role in the development of IR in placentae of GDM. PMID:27340831

  20. IMQ-induced skin inflammation in mice is dependent on IL-1R1 and MyD88 signaling but independent of the NLRP3 inflammasome.

    PubMed

    Rabeony, Hanitriniaina; Pohin, Mathilde; Vasseur, Philippe; Petit-Paris, Isabelle; Jégou, Jean-François; Favot, Laure; Frouin, Eric; Boutet, Marie-Astrid; Blanchard, Frédéric; Togbe, Dieudonnée; Ryffel, Bernhard; Bernard, François-Xavier; Lecron, Jean-Claude; Morel, Franck

    2015-10-01

    The pathogenesis of inflammatory skin diseases such as psoriasis involves the release of numerous proinflammatory cytokines, including members of the IL-1 family. Here we report overexpression of IL-1α, IL-1β, and IL-1 receptor antagonist mRNA, associated to expression of IL-23p19, IL-17A, and IL-22 in skin cells, upon topical application of the TLR7 agonist imiquimod (IMQ) in C57BL/6J mice. IMQ-induced skin inflammation was partially reduced in mice deficient for both IL-1α/IL-1β or for IL-1 receptor type 1 (IL-1R1), but not in IL-1α- or IL-1β-deficient mice, demonstrating the redundant activity of IL-1α and IL-1β for skin inflammation. NLRP3 or apoptosis-associated Speck-like protein containing a Caspase recruitment domain-deficient mice had no significant reduction of skin inflammation in response to IMQ treatment, mainly due to the redundancy of IL-1α. However, IMQ-induced skin inflammation was abolished in the absence of MyD88, the adaptor protein shared by IL-1R and TLR signaling pathways. These results are consistent with the TLR7 dependence of IMQ-induced skin inflammation. Thus, IL-1R1 contributes to the IMQ-induced skin inflammation, and disruption of MyD88 signaling completely abrogates this response.

  1. Microglial activation by Citrobacter koseri is mediated by TLR4- and MyD88-dependent pathways.

    PubMed

    Liu, Shuliang; Kielian, Tammy

    2009-11-01

    Citrobacter koseri is a Gram-negative bacterium that can cause a highly aggressive form of neonatal meningitis, which often progresses to establish multifocal brain abscesses. Despite its tropism for the brain parenchyma, microglial responses to C. koseri have not yet been examined. Microglia use TLRs to recognize invading pathogens and elicit proinflammatory mediator expression important for infection containment. In this study, we investigated the importance of the LPS receptor TLR4 and MyD88, an adaptor molecule involved in the activation of the majority of TLRs in addition to the IL-1 and IL-18 receptors, for their roles in regulating microglial activation in response to C. koseri. Proinflammatory mediator release was significantly reduced in TLR4 mutant and MyD88 knockout microglia compared with wild-type cells following exposure to either live or heat-killed C. koseri, indicating a critical role for both TLR4- and MyD88-dependent pathways in microglial responses to this pathogen. However, residual proinflammatory mediator expression was still observed in TLR4 mutant and MyD88 KO microglia following C. koseri exposure, indicating a contribution of TLR4- and MyD88-independent pathway(s) for maximal pathogen recognition. Interestingly, C. koseri was capable of surviving intracellularly in both primary microglia and macrophages, suggesting that these cells may serve as a reservoir for the pathogen during CNS infections. These results demonstrate that microglia respond to C. koseri with the robust expression of proinflammatory molecules, which is dictated, in part, by TLR4- and MyD88-dependent signals.

  2. Chlamydial Lipoproteins Stimulate Toll-Like Receptors 1/2 Mediated Inflammatory Responses through MyD88-Dependent Pathway

    PubMed Central

    Wang, Yong; Liu, Qiong; Chen, Ding; Guan, Jie; Ma, Linghui; Zhong, Guangming; Shu, Hengping; Wu, Xiang

    2017-01-01

    Chlamydiae are very important pathogens which could cause several types of diseases in human, but little is known about its pathogenic mechanism. In order to elucidate host inflammatory response and the signal pathway induced by Chlamydial lipoproteins, the predicted lipoproteins of Chlamydia trachomatis were tested for their ability to induce the release of proinflammatory cytokines by mouse macrophages or human TLR (Toll-Like Receptor) expressing cell lines. The results showed that recombinant proteins of C. trachomatis D381, D541, D067, and D775 displayed a strong ability to induce the release of IL-8 in TLR expressing cell line. The signal pathways involved TLR1/2 and TLR2/CD14 but not TLR4. Moreover, except D067, the proinflammatory cytokine induction by D381, D541, and D775 required the thioacylation site (cysteine) for lipid modification and the induction was through MyD88-mediated pathway. Our data supported that lipoproteins played a vital role in pathogenesis of C. trachomatis-induced inflammatory responses via TLR pathway. It was the first study to characterize other chlamydial lipoproteins after identifying the role of MIP (D541) on pathogenesis of Chlamydial diseases. PMID:28184217

  3. Recurrent candidiasis and early-onset gastric cancer in a patient with a genetically defined partial MYD88 defect.

    PubMed

    Vogelaar, Ingrid P; Ligtenberg, Marjolijn J L; van der Post, Rachel S; de Voer, Richarda M; Kets, C Marleen; Jansen, Trees J G; Jacobs, Liesbeth; Schreibelt, Gerty; de Vries, I Jolanda M; Netea, Mihai G; Hoogerbrugge, Nicoline

    2016-04-01

    Gastric cancer is caused by both genetic and environmental factors. A woman who suffered from recurrent candidiasis throughout her life developed diffuse-type gastric cancer at the age of 23 years. Using whole-exome sequencing we identified a germline homozygous missense variant in MYD88. Immunological assays on peripheral blood mononuclear cells revealed an impaired immune response upon stimulation with Candida albicans, characterized by a defective production of the cytokine interleukin-17. Our data suggest that a genetic defect in MYD88 results in an impaired immune response and may increase gastric cancer risk.

  4. MyD88 is a key mediator of anorexia, but not weight loss, induced by lipopolysaccharide and interleukin-1 beta.

    PubMed

    Ogimoto, Kayoko; Harris, Marvin K; Wisse, Brent E

    2006-09-01

    Systemic inflammatory signals can disrupt the physiological regulation of energy balance, causing anorexia and weight loss. In the current studies, we investigated whether MyD88, the primary, but not exclusive, intracellular signal transduction pathway for Toll-like receptor 4 and IL-1 receptor I, is necessary for anorexia and weight loss to occur in response to stimuli that activate these key innate immune receptors. Our findings demonstrate that the absence of MyD88 signaling confers complete protection against anorexia induced by either lipopolysaccharide (LPS) (20 h food intake in MyD88-/- mice 5.4 +/- 0.3 vs. 3.3 +/- 0.4 g in MyD88+/+ control mice, P < 0.001) or IL-1 beta (20 h food intake in MyD88-/- mice 4.9 +/- 0.5 vs. 4.0 +/- 0.3 g in MyD88+/+ control mice, P < 0.001). However, absent MyD88 signaling does not prevent these inflammatory mediators from causing weight loss (LPS, -0.4 +/- 0.1 g; IL1 beta, -0.1 +/- 0.1 g, both P < 0.01 vs. vehicle-injected MyD88-/- mice, +0.4 +/- 0.2 g). Furthermore, LPS-induced weight loss occurs in the absence of adipsia, fever, or hypothalamus-pituitary-adrenal axis activation in MyD88-deficient mice. In addition, the peripheral inflammatory response to LPS is surprisingly intact in mice lacking MyD88. Together, these observations indicate that LPS reduces food intake via a mechanism that is dissociated from its effect on peripheral cytokine production, and whereas the presence of circulating proinflammatory cytokines per se is insufficient to cause anorexia in the absence of MyD88 signaling, it may contribute to LPS-induced weight loss.

  5. MyD88 regulates physical inactivity-induced skeletal muscle inflammation, ceramide biosynthesis signaling, and glucose intolerance.

    PubMed

    Kwon, Oh Sung; Tanner, Ruth E; Barrows, Katherine M; Runtsch, Marah; Symons, J David; Jalili, Thunder; Bikman, Benjamin T; McClain, Donald A; O'Connell, Ryan M; Drummond, Micah J

    2015-07-01

    Physical inactivity in older adults is a risk factor for developing glucose intolerance and impaired skeletal muscle function. Elevated inflammation and ceramide biosynthesis have been implicated in metabolic disruption and are linked to Toll-like receptor (TLR)/myeloid differentiation primary response 88 (MyD88) signaling. We hypothesize that a physical inactivity stimulus, capable of inducing glucose intolerance, would increase skeletal muscle inflammation and ceramide biosynthesis signaling and that this response would be regulated by the TLR/MyD88 pathway. Therefore, we subjected wild-type (WT) and MyD88(-/-) mice to hindlimb unloading (HU) for 14 days or an ambulatory control period. We observed impaired glucose uptake, muscle insulin signaling (p-Akt), and increased markers of NF-κB signaling (p-IκBα), inflammation (p-JNK, IL-6), TLR4, and the rate-limiting enzyme of ceramide biosynthesis, SPT2, with HU WT (P < 0.05), but not in HU MyD88(-/-) mice. Concurrently, we found that 5 days of bed rest in older adults resulted in whole body glucose dysregulation, impaired skeletal muscle insulin signaling, and upregulation of muscle IL-6 and SPT2 (P < 0.05). Post-bed rest TLR4 abundance was tightly correlated with impaired postprandial insulin and glucose levels. In conclusion, MyD88 signaling is necessary for the increased inflammation, ceramide biosynthesis signaling, and compromised metabolic function that accompanies physical inactivity.

  6. MyD88 regulates physical inactivity-induced skeletal muscle inflammation, ceramide biosynthesis signaling, and glucose intolerance

    PubMed Central

    Kwon, Oh Sung; Tanner, Ruth E.; Barrows, Katherine M.; Runtsch, Marah; Symons, J. David; Jalili, Thunder; Bikman, Benjamin T.; McClain, Donald A.; O'Connell, Ryan M.

    2015-01-01

    Physical inactivity in older adults is a risk factor for developing glucose intolerance and impaired skeletal muscle function. Elevated inflammation and ceramide biosynthesis have been implicated in metabolic disruption and are linked to Toll-like receptor (TLR)/myeloid differentiation primary response 88 (MyD88) signaling. We hypothesize that a physical inactivity stimulus, capable of inducing glucose intolerance, would increase skeletal muscle inflammation and ceramide biosynthesis signaling and that this response would be regulated by the TLR/MyD88 pathway. Therefore, we subjected wild-type (WT) and MyD88−/− mice to hindlimb unloading (HU) for 14 days or an ambulatory control period. We observed impaired glucose uptake, muscle insulin signaling (p-Akt), and increased markers of NF-κB signaling (p-IκBα), inflammation (p-JNK, IL-6), TLR4, and the rate-limiting enzyme of ceramide biosynthesis, SPT2, with HU WT (P < 0.05), but not in HU MyD88−/− mice. Concurrently, we found that 5 days of bed rest in older adults resulted in whole body glucose dysregulation, impaired skeletal muscle insulin signaling, and upregulation of muscle IL-6 and SPT2 (P < 0.05). Post-bed rest TLR4 abundance was tightly correlated with impaired postprandial insulin and glucose levels. In conclusion, MyD88 signaling is necessary for the increased inflammation, ceramide biosynthesis signaling, and compromised metabolic function that accompanies physical inactivity. PMID:25968578

  7. Lung epithelial MyD88 drives early pulmonary clearance of Pseudomonas aeruginosa by a flagellin dependent mechanism.

    PubMed

    Anas, Adam A; van Lieshout, Miriam H P; Claushuis, Theodora A M; de Vos, Alex F; Florquin, Sandrine; de Boer, Onno J; Hou, Baidong; Van't Veer, Cornelis; van der Poll, Tom

    2016-08-01

    Pseudomonas aeruginosa is a flagellated pathogen frequently causing pneumonia in hospitalized patients and sufferers of chronic lung disease. Here we investigated the role of the common Toll-like receptor (TLR) adaptor myeloid differentiation factor (MyD)88 in myeloid vs. lung epithelial cells in clearance of P. aeruginosa from the airways. Mice deficient for MyD88 in lung epithelial cells (Sftpccre-MyD88-lox mice) or myeloid cells (LysMcre-MyD88-lox mice) and bone marrow chimeric mice deficient for TLR5 (the receptor recognizing Pseudomonas flagellin) in either parenchymal or hematopoietic cells were infected with P. aeruginosa via the airways. Sftpccre-MyD88-lox mice demonstrated a reduced influx of neutrophils into the bronchoalveolar space and an impaired early antibacterial defense after infection with P. aeruginosa, whereas the response of LysMcre-MyD88-lox mice did not differ from control mice. The immune-enhancing role of epithelial MyD88 was dependent on recognition of pathogen-derived flagellin by epithelial TLR5, as demonstrated by an unaltered clearance of mutant P. aeruginosa lacking flagellin from the lungs of Sftpccre-MyD88-lox mice and an impaired bacterial clearance in bone marrow chimeric mice lacking TLR5 in parenchymal cells. These data indicate that early clearance of P. aeruginosa from the airways is dependent on flagellin-TLR5-MyD88-dependent signaling in respiratory epithelial cells.

  8. Goose Toll-like receptor 7 (TLR7), myeloid differentiation factor 88 (MyD88) and antiviral molecules involved in anti-H5N1 highly pathogenic avian influenza virus response.

    PubMed

    Wei, Liangmeng; Jiao, Peirong; Yuan, Runyu; Song, Yafen; Cui, Pengfei; Guo, Xuchen; Zheng, Bofang; Jia, Weixin; Qi, Wenbao; Ren, Tao; Liao, Ming

    2013-05-15

    In mammals, Toll-like receptor 7 (TLR7) is an important membrane-bound receptor triggered by antiviral compounds and single-stranded RNA. It is implicated in the immune response to viruses such as influenza virus. It was not known whether geese, a natural host for avian influenza viruses, possess a homologue of mammalian TLR7 for recognizing avian influenza virus. In this study, we cloned the full-length of goose TLR7 and partial sequences of its adaptor protein, myeloid differentiation factor 88 (MyD88), some antiviral molecules such as RNA-dependent protein kinase (PKR) and 2',5'-oligoadenylate synthetase (OAS). Goose TLR7 has a protein secondary structure identical to that of mammals, consisting of several leucine-rich domains, a transmembrane domain, and Toll/interleukin-1 receptor domain. To further understand whether the MyD88-dependent pathway of TLR7 is involved in the antiviral innate immune response against highly pathogenic avian influenza virus (HPAIV) infection in geese, we inoculated geese with an H5N1 HPAIV isolated from ducks in 2004. The virus, A/Duck/Guangdong/212/2004, replicated in various tissues resulting in 40% mortality. Quantitative real-time PCR analysis showed upregulation of mRNA transcripts for TLR7, MyD88, PKR and OAS in the lungs of geese at 1, 2 and 3 days post-inoculation. Therefore, the MyD88-dependent pathway of TLR7 was involved in the early stage of antiviral innate immune response in geese during H5N1 HPAIV infection.

  9. Utility of MYD88 in the Differential Diagnosis and Choice of Second-Line Therapy in a Case of Nonsecretory Lymphoplasmacytic Lymphoma versus Free Light Chain Waldenstrom's Macroglobulinemia

    PubMed Central

    Kazmierski, D.; Palomba, M. L.

    2017-01-01

    The MYD88 L265P somatic variant (MYD88) has a high prevalence in Waldenstrom's Macroglobulinemia (WM), a form of lymphoplasmacytic lymphoma (LPL) associated with monoclonal IgM. Although the role of MYD88 in WM was initially reported in 2012, it was not until 2016 that MYD88 testing was included in the National Cancer Care Network (NCCN) Guidelines. We present a case illustrating the utility of MYD88 status in distinguishing atypical forms of WM from marginal zone lymphoma (MZL) and in selecting second-line therapy with ibrutinib. In 2012, a 64-year-old male presented with dyspnea on exertion, a hemoglobin of 5.6 g/dL, a platelet count of 86,000, and monoclonal IgM kappa on serum immunofixation but no detectable M-spike. Bone marrow biopsy revealed 95% monoclonal B-lymphocytes with lymphoplasmacytic differentiation favoring a diagnosis of LPL/WM over MZL, with a favorable response to chemotherapy. This diagnosis was called into question 3 years later following relapse, and MZL was favored based on the lack of MYD88 mutation. One year later, however, repeat bone marrow biopsy detected the MYD88 mutation and therapy with ibrutinib yielded a favorable response. The distinction between certain lymphomas can be problematic and in this case MYD88 was helpful in clarifying a diagnosis of atypical LPL/WM from MZL and in selecting effective second-line therapy. PMID:28286680

  10. Deletion of the gene encoding MyD88 protects from anorexia in a mouse tumor model.

    PubMed

    Ruud, Johan; Bäckhed, Fredrik; Engblom, David; Blomqvist, Anders

    2010-05-01

    The anorexia-cachexia syndrome, characterized by a rise in energy expenditure and loss of body weight that paradoxically are associated with loss of appetite and decreased food intake, contributes significantly to the morbidity and mortality in cancer. While the pathophysiology of cancer anorexia-cachexia is poorly understood, evidence indicates that pro-inflammatory cytokines are key mediators of this response. Although inflammation hence is recognized as an important component of cancer anorexia-cachexia, the molecular pathways involved are largely unknown. We addressed this issue in mice carrying a deletion of the gene encoding MyD88, the key intracellular adaptor molecule in Toll-like and interleukin-1 family receptor signaling. Wild-type and MyD88-deficient mice were transplanted subcutaneously with a syngenic methylcholanthrene-induced tumor (MCG 101) and daily food intake and body weight were recorded. Wild-type mice showed progressively reduced food intake from about 5days after tumor transplantation and displayed a slight body weight loss after 10days when the experiment was terminated. In contrast, MyD88-deficient mice did not develop anorexia, and displayed a positive body weight development during the observation period. While the MyD88-deficient mice on average developed somewhat smaller tumors than wild-type mice, this did not explain the absence of anorexia, because anorexia was seen in wild-type mice with similar tumor mass as non-anorexic knock-out mice. These data suggest that MyD88-dependent mechanisms are involved in the metabolic derangement during cancer anorexia-cachexia and that innate immune signaling is important for the development of this syndrome.

  11. Absence of MyD88 results in enhanced TLR3-dependent phosphorylation of IRF3 and increased IFN-β and RANTES production.

    PubMed

    Siednienko, Jakub; Gajanayake, Thusitha; Fitzgerald, Katherine A; Moynagh, Paul; Miggin, Sinéad M

    2011-02-15

    Toll-like receptors are a group of pattern-recognition receptors that play a crucial role in "danger" recognition and induction of the innate immune response against bacterial and viral infections. TLR3 has emerged as a key sensor of viral dsRNA, resulting in the induction of the anti-viral molecule, IFN-β. Thus, a clearer understanding of the biological processes that modulate TLR3 signaling is essential. Previous studies have shown that the TLR adaptor, Mal/TIRAP, an activator of TLR4, inhibits TLR3-mediated IFN-β induction through a mechanism involving IRF7. In this study, we sought to investigate whether the TLR adaptor, MyD88, an activator of all TLRs except TLR3, has the ability to modulate TLR3 signaling. Although MyD88 does not significantly affect TLR3 ligand-induced TNF-α induction, MyD88 negatively regulates TLR3-, but not TLR4-, mediated IFN-β and RANTES production; this process is mechanistically distinct from that employed by Mal/TIRAP. We show that MyD88 inhibits IKKε-, but not TBK1-, induced activation of IRF3. In doing so, MyD88 curtails TLR3 ligand-induced IFN-β induction. The present study shows that while MyD88 activates all TLRs except TLR3, MyD88 also functions as a negative regulator of TLR3. Thus, MyD88 is essential in restricting TLR3 signaling, thereby protecting the host from unwanted immunopathologies associated with the excessive production of IFN-β. Our study offers a new role for MyD88 in restricting TLR3 signaling through a hitherto unknown mechanism whereby MyD88 specifically impairs IKKε-mediated induction of IRF3 and concomitant IFN-β and RANTES production.

  12. Telmisartan mediates anti-inflammatory and not cognitive function through PPAR-γ agonism via SARM and MyD88 signaling.

    PubMed

    Prathab Balaji, S; Vijay Chand, C; Justin, A; Ramanathan, M

    2015-10-01

    Telmisartan (TM), an angiotensin II receptor I (AT1) blocker, has been reported to have agonist property with respect to PPAR-γ. Activation of PPAR-γ receptor by TM attenuated the lipopolysaccharide (LPS) mediated TLR4 central downstream inflammatory responses. However, the missing link between PPAR-γ and TLR4 signaling with TM stimulation has not been clarified. Hence, the present study has been designed to evaluate the molecular mechanism involving PPARγ-TLR4 signaling with TM stimulation in LPS induced inflammatory model. LPS was administered in rats through ICV and the rats were treated with either PPAR-γ antagonist GW9662 (GW) or TM or both. After 14days of LPS administration, the rats were subjected to behavioral tests and their brains were isolated for blotting techniques. The protein study includes NF-κB, PPAR-γ receptors, and their downstream proteins (MyD88 & SARM). The pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) levels were measured by ELISA and cresyl violet staining in the hippocampus region to measure the neuroprotective activity. Results have shown that TM significantly increased the motor co-ordination, cognitive functions, and activated SARM and PPAR-γ protein levels. Also, TM treatment decreased the NF-κB, MyD88 activation, and cytokines release in LPS rats. The co-administration of GW attenuated the TM responses in the parameters studied except cognitive functions. TM (10mg/kg) has significantly reduced the LPS mediated inflammatory responses. This resulted in effective regeneration of hippocampal neurons as observed by cresyl violet staining. It can be concluded that the activation of PPAR-γ receptors may increase the SARM and decrease the MyD88 and NF-κB expression. This negative regulation of SARM dependent inflammation control could be a possible mechanism for TM anti-neuroinflammatory activity. This study of TM in neuro-inflammatory model may further confirm the dual activities of TM that controls hypertension and cognition

  13. Protective role of MyD88-independent innate immune responses against prion infection

    PubMed Central

    Ishibashi, Daisuke; Atarashi, Ryuichiro; Nishida, Noriyuki

    2012-01-01

    Despite recent progress in the understanding of prion diseases, little is known about the host-defense mechanisms against prion. Although it has long been thought that type I interferon (IFN-I) has no protective effect on prion infection, certain key molecules in innate immunity such as toll-like receptor (TLR) 4 seemed to be involved in the host response. For this reason we decided to focus on TLRs and investigate the role of a transcription factor, interferon regulatory factor 3 (IRF3), because the absence of MyD88, a major adaptor signaling molecule of TLRs, has no effect on the survival of prion infected mice. Intriguingly, survival periods of prion inoculated IRF3-knockout mice became significantly shorter than those of wild-type mice. In addition, IRF3 stimulation inhibited PrPSc replication in prion persistently-infected cells, and a de novo prion infection assay revealed that IRF3-overexpression could make host cells resistant to prion infection. Our work suggests that IRF3 may play a key role in innate immune responses against invasion of prion pathogens. Activated IRF3 could upregulate several anti-pathogen factors, including IFN-I, and induce sequential responses. Although the mechanism for the anti-prion effects mediated by IRF3 has yet to be clarified, certain interferon responsive genes might be involved in the anti-prion host-defense mechanism. PMID:23093799

  14. Salmonella inhibits monocyte differentiation into CD11c hi MHC-II hi cells in a MyD88-dependent fashion.

    PubMed

    Rydström, Anna; Wick, Mary Jo

    2010-05-01

    Monocytes and DCs originate from a shared precursor in the bone marrow, and steady-state DCs in lymphoid organs develop directly from the precursor rather than via a monocyte intermediate. However, monocytes can differentiate into DCs in tissues such as the lung and gut mucosa and into macrophages in most tissues. As Ly6C hi monocytes accumulate in lymphoid organs during oral Salmonella infection, we investigated their ability to develop into potential DCs, identified as CD11c hi MHC-II hi cells, in infected hosts. Ly6C hi monocytes, isolated from the blood of Salmonella-infected mice, developed into CD11c hi MHC-II hi cells after culture with GM-CSF or Flt3L. In contrast, the same monocytes cultured in the presence of GM-CSF and heat-killed Salmonella did not differentiate into CD11c hi MHC-II hi cells. The bacteria-induced differentiation block was dependent on TLRs, as monocytes from MyD88-/- mice converted into CD11c hi MHC-II hi cells even in the presence of bacteria. We hypothesized that Salmonella-activated wild-type monocytes secreted mediators that inhibited differentiation of MyD88-/--derived monocytes. However, IL-6, IL-10, TNF-alpha, or IL-12p70 did not account for the inhibition. Finally, monocyte-derived CD11c hi MHC-II hi cells pulsed with OVA peptide or protein did not induce proliferation of antigen-specific CD4+ T cells but rather, suppressed the ability of DCs to activate CD4+ T cells. Overall, the data show that Ly6C hi monocytes from Salmonella-infected mice develop into CD11c hi MHC-II hi cells with poor antigen-presentation capacity when cultured ex vivo, and that monocyte exposure to Salmonella inhibits their differentiation into CD11c hi MHC-II hi cells in a MyD88-dependent fashion.

  15. HCV-Induced miR-21 Contributes to Evasion of Host Immune System by Targeting MyD88 and IRAK1

    PubMed Central

    Chen, Yanni; Chen, Junbo; Wang, Hui; Shi, Jingjing; Wu, Kailang; Liu, Shi; Liu, Yingle; Wu, Jianguo

    2013-01-01

    Upon recognition of viral components by pattern recognition receptors, such as the toll-like receptors (TLRs) and retinoic acid-inducible gene I (RIG-I)-like helicases, cells are activated to produce type I interferon (IFN) and proinflammatory cytokines. These pathways are tightly regulated by the host to prevent an inappropriate cellular response, but viruses can modulate these pathways to proliferate and spread. In this study, we revealed a novel mechanism in which hepatitis C virus (HCV) evades the immune surveillance system to proliferate by activating microRNA-21 (miR-21). We demonstrated that HCV infection upregulates miR-21, which in turn suppresses HCV-triggered type I IFN production, thus promoting HCV replication. Furthermore, we demonstrated that miR-21 targets two important factors in the TLR signaling pathway, myeloid differentiation factor 88 (MyD88) and interleukin-1 receptor-associated kinase 1 (IRAK1), which are involved in HCV-induced type I IFN production. HCV-mediated activation of miR-21 expression requires viral proteins and several signaling components. Moreover, we identified a transcription factor, activating protein-1 (AP-1), which is partly responsible for miR-21 induction in response to HCV infection through PKCε/JNK/c-Jun and PKCα/ERK/c-Fos cascades. Taken together, our results indicate that miR-21 is upregulated during HCV infection and negatively regulates IFN-α signaling through MyD88 and IRAK1 and may be a potential therapeutic target for antiviral intervention. PMID:23633945

  16. IRAK-4 and MyD88 deficiencies impair IgM responses against T-independent bacterial antigens

    PubMed Central

    Maglione, Paul J.; Simchoni, Noa; Black, Samuel; Radigan, Lin; Overbey, Jessica R.; Bagiella, Emilia; Bussel, James B.; Bossuyt, Xavier; Casanova, Jean-Laurent; Meyts, Isabelle; Cerutti, Andrea; Picard, Capucine

    2014-01-01

    IRAK-4 and MyD88 deficiencies impair interleukin 1 receptor and Toll-like receptor (TLR) signaling and lead to heightened susceptibility to invasive bacterial infections. Individuals with these primary immunodeficiencies have fewer immunoglobulin M (IgM)+IgD+CD27+ B cells, a population that resembles murine splenic marginal zone B cells that mount T-independent antibody responses against bacterial antigens. However, the significance of this B-cell subset in humans is poorly understood. Using both a 610 carbohydrate array and enzyme-linked immunosorbent assay, we found that patients with IRAK-4 and MyD88 deficiencies have reduced serum IgM, but not IgG antibody, recognizing T-independent bacterial antigens. Moreover, the quantity of specific IgM correlated with IgM+IgD+CD27+ B-cell frequencies. As with mouse marginal zone B cells, human IgM+CD27+ B cells activated by TLR7 or TLR9 agonists produced phosphorylcholine-specific IgM. Further linking splenic IgM+IgD+CD27+ B cells with production of T-independent IgM, serum from splenectomized subjects, who also have few IgM+IgD+CD27+ B cells, had reduced antibacterial IgM. IRAK-4 and MyD88 deficiencies impaired TLR-induced proliferation of this B-cell subset, suggesting a means by which loss of this activation pathway leads to reduced cell numbers. Thus, by bolstering the IgM+IgD+CD27+ B-cell subset, IRAK-4 and MyD88 promote optimal T-independent IgM antibody responses against bacteria in humans. PMID:25320238

  17. IRAK-4 and MyD88 deficiencies impair IgM responses against T-independent bacterial antigens.

    PubMed

    Maglione, Paul J; Simchoni, Noa; Black, Samuel; Radigan, Lin; Overbey, Jessica R; Bagiella, Emilia; Bussel, James B; Bossuyt, Xavier; Casanova, Jean-Laurent; Meyts, Isabelle; Cerutti, Andrea; Picard, Capucine; Cunningham-Rundles, Charlotte

    2014-12-04

    IRAK-4 and MyD88 deficiencies impair interleukin 1 receptor and Toll-like receptor (TLR) signaling and lead to heightened susceptibility to invasive bacterial infections. Individuals with these primary immunodeficiencies have fewer immunoglobulin M (IgM)(+)IgD(+)CD27(+) B cells, a population that resembles murine splenic marginal zone B cells that mount T-independent antibody responses against bacterial antigens. However, the significance of this B-cell subset in humans is poorly understood. Using both a 610 carbohydrate array and enzyme-linked immunosorbent assay, we found that patients with IRAK-4 and MyD88 deficiencies have reduced serum IgM, but not IgG antibody, recognizing T-independent bacterial antigens. Moreover, the quantity of specific IgM correlated with IgM(+)IgD(+)CD27(+) B-cell frequencies. As with mouse marginal zone B cells, human IgM(+)CD27(+) B cells activated by TLR7 or TLR9 agonists produced phosphorylcholine-specific IgM. Further linking splenic IgM(+)IgD(+)CD27(+) B cells with production of T-independent IgM, serum from splenectomized subjects, who also have few IgM(+)IgD(+)CD27(+) B cells, had reduced antibacterial IgM. IRAK-4 and MyD88 deficiencies impaired TLR-induced proliferation of this B-cell subset, suggesting a means by which loss of this activation pathway leads to reduced cell numbers. Thus, by bolstering the IgM(+)IgD(+)CD27(+) B-cell subset, IRAK-4 and MyD88 promote optimal T-independent IgM antibody responses against bacteria in humans.

  18. MyD88 Adaptor-Dependent Microbial Sensing by Regulatory T Cells Promotes Mucosal Tolerance and Enforces Commensalism.

    PubMed

    Wang, Sen; Charbonnier, Louis-Marie; Noval Rivas, Magali; Georgiev, Peter; Li, Ning; Gerber, Georg; Bry, Lynn; Chatila, Talal A

    2015-08-18

    Commensal microbiota promote mucosal tolerance in part by engaging regulatory T (Treg) cells via Toll-like receptors (TLRs). We report that Treg-cell-specific deletion of the TLR adaptor MyD88 resulted in deficiency of intestinal Treg cells, a reciprocal increase in T helper 17 (Th17) cells and heightened interleukin-17 (IL-17)-dependent inflammation in experimental colitis. It also precipitated dysbiosis with overgrowth of segmented filamentous bacteria (SFB) and increased microbial loads in deep tissues. The Th17 cell dysregulation and bacterial dysbiosis were linked to impaired anti-microbial intestinal IgA responses, related to defective MyD88 adaptor- and Stat3 transcription factor-dependent T follicular regulatory and helper cell differentiation in the Peyer's patches. These findings establish an essential role for MyD88-dependent microbial sensing by Treg cells in enforcing mucosal tolerance and maintaining commensalism by promoting intestinal Treg cell formation and anti-commensal IgA responses.

  19. Inhibition of IL-1R1/MyD88 signalling promotes mesenchymal stem cell-driven tissue regeneration.

    PubMed

    Martino, Mikaël M; Maruyama, Kenta; Kuhn, Gisela A; Satoh, Takashi; Takeuchi, Osamu; Müller, Ralph; Akira, Shizuo

    2016-03-22

    Tissue injury and the healing response lead to the release of endogenous danger signals including Toll-like receptor (TLR) and interleukin-1 receptor, type 1 (IL-1R1) ligands, which modulate the immune microenvironment. Because TLRs and IL-1R1 have been shown to influence the repair process of various tissues, we explored their role during bone regeneration, seeking to design regenerative strategies integrating a control of their signalling. Here we show that IL-1R1/MyD88 signalling negatively regulates bone regeneration, in the mouse. Furthermore, IL-1β which is released at the bone injury site, inhibits the regenerative capacities of mesenchymal stem cells (MSCs). Mechanistically, IL-1R1/MyD88 signalling impairs MSC proliferation, migration and differentiation by inhibiting the Akt/GSK-3β/β-catenin pathway. Lastly, as a proof of concept, we engineer a MSC delivery system integrating inhibitors of IL-1R1/MyD88 signalling. Using this strategy, we considerably improve MSC-based bone regeneration in the mouse, demonstrating that this approach may be useful in regenerative medicine applications.

  20. Overexpression of FOXO3, MYD88, and GAPDH Identified by Suppression Subtractive Hybridization in Esophageal Cancer Is Associated with Autophagy.

    PubMed

    Soltany-Rezaee-Rad, Mohammad; Mottaghi-Dastjerdi, Negar; Setayesh, Neda; Roshandel, Gholamreza; Ebrahimifard, Farzaneh; Sepehrizadeh, Zargham

    2014-01-01

    To find genes involved in tumorigenesis and the development of esophageal cancer, the suppression subtractive hybridization (SSH) method was used to identify genes that are overexpressed in esophageal cancer tissues compared to normal esophageal tissues. In our SSH library, the forkhead box O3 (FOXO3), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and myeloid differentiation primary response 88 (MYD88) genes were the most highly upregulated genes, and they were selected for further studies because of their potential role in the induction of autophagy. Upregulation of these genes was also observed in clinical samples using qRT-PCR. In addition, coexpression analysis of the autophagy-related genes Beclin1, ATG12, Gabarapl, PIK3C3, and LC3 demonstrated a significant correlation between the differentially overexpressed genes and autophagy. Autophagy is an important mechanism in tumorigenesis and the development of chemoresistance in cancer cells. The upregulation of FOXO3, GAPDH, and MYD88 variants in esophageal cancer suggests a role for autophagy and provides new insight into the biology of esophageal cancer. We propose that FOXO3, GAPDH, and MYD88 are novel targets for combating autophagy in esophageal cancer.

  1. PPAR-γ/IL-10 axis inhibits MyD88 expression and ameliorates murine polymicrobial sepsis.

    PubMed

    Ferreira, Ana Elisa; Sisti, Flavia; Sônego, Fabiane; Wang, Suojuan; Filgueiras, Luciano Ribeiro; Brandt, Stephanie; Serezani, Ana Paula Moreira; Du, Hong; Cunha, Fernando Q; Alves-Filho, Jose Carlos; Serezani, Carlos Henrique

    2014-03-01

    Polymicrobial sepsis induces organ failure and is accompanied by overwhelming inflammatory response and impairment of microbial killing. Peroxisome proliferator-activated receptor (PPAR)-γ is a nuclear receptor with pleiotropic effects on lipid metabolism, inflammation, and cell proliferation. The insulin-sensitizing drugs thiazolidinediones (TZDs) are specific PPAR-γ agonists. TZDs exert anti-inflammatory actions in different disease models, including polymicrobial sepsis. The TZD pioglitazone, which has been approved by the U.S. Food and Drug Administration, improves sepsis outcome; however, the molecular programs that mediate its effect have not been determined. In a murine model of sepsis, we now show that pioglitazone treatment improves microbial clearance and enhances neutrophil recruitment to the site of infection. We also observed reduced proinflammatory cytokine production and high IL-10 levels in pioglitazone-treated mice. These effects were associated with a decrease in STAT-1-dependent expression of MyD88 in vivo and in vitro. IL-10R blockage abolished PPAR-γ-mediated inhibition of MyD88 expression. These data demonstrate that the primary mechanism by which pioglitazone protects against polymicrobial sepsis is through the impairment of MyD88 responses. This appears to represent a novel regulatory program. In this regard, pioglitazone provides advantages as a therapeutic tool, because it improves different aspects of host defense during sepsis, ultimately enhancing survival.

  2. Interleukin 1 receptor-driven neutrophil recruitment accounts to MyD88-dependent pulmonary clearance of legionella pneumophila infection in vivo.

    PubMed

    Mascarenhas, Danielle P A; Pereira, Marcelo S F; Manin, Graziele Z; Hori, Juliana I; Zamboni, Dario S

    2015-01-15

    Legionella pneumophila, the etiological agent of Legionnaires' disease, triggers activation of multiple innate immune pathways that lead to the restriction of bacterial replication in vivo. Despite the critical role for MyD88 in infection clearance, the receptors and mechanisms responsible for MyD88-mediated pulmonary bacterial clearance are still unclear. Here, we used flagellin mutants of L. pneumophila, which bypass the NAIP5/NLRC4-mediated restriction of bacterial replication, to assess the receptors involved in MyD88-mediated pulmonary bacterial clearance. By systematically comparing pulmonary clearance of L. pneumophila in C57BL/6 MyD88(-/-), TLR2(-/-), TLR3(-/-), TLR4(-/-), TLR9(-/-), IL-1R(-/-), and IL-18(-/-) mice, we found that, while the knockout of a single Toll-like receptor or interleukin 18 resulted only in minor impairment of bacterial clearance, deficiency in the interleukin 1 (IL-1) receptor led to a significant impairment. IL-1/MyD88-mediated pulmonary bacterial clearance occurs via processes involving the recruitment of neutrophils. Collectively, our data contribute to the understanding of the effector mechanisms involved in MyD88-mediated pulmonary bacterial clearance.

  3. MyD88 deficiency markedly worsens tissue inflammation and bacterial clearance in mice infected with Treponema pallidum, the agent of syphilis.

    PubMed

    Silver, Adam C; Dunne, Dana W; Zeiss, Caroline J; Bockenstedt, Linda K; Radolf, Justin D; Salazar, Juan C; Fikrig, Erol

    2013-01-01

    Research on syphilis, a sexually transmitted infection caused by the non-cultivatable spirochete Treponema pallidum, has been hampered by the lack of an inbred animal model. We hypothesized that Toll-like receptor (TLR)-dependent responses are essential for clearance of T. pallidum and, consequently, compared infection in wild-type (WT) mice and animals lacking MyD88, the adaptor molecule required for signaling by most TLRs. MyD88-deficient mice had significantly higher pathogen burdens and more extensive inflammation than control animals. Whereas tissue infiltrates in WT mice consisted of mixed mononuclear and plasma cells, infiltrates in MyD88-deficient animals were predominantly neutrophilic. Although both WT and MyD88-deficient mice produced antibodies that promoted uptake of treponemes by WT macrophages, MyD88-deficient macrophages were deficient in opsonophagocytosis of treponemes. Our results demonstrate that TLR-mediated responses are major contributors to the resistance of mice to syphilitic disease and that MyD88 signaling and FcR-mediated opsonophagocytosis are linked to the macrophage-mediated clearance of treponemes.

  4. MyD88 is pivotal for the early inflammatory response and subsequent bacterial clearance and survival in a mouse model of Chlamydia pneumoniae pneumonia.

    PubMed

    Naiki, Yoshikazu; Michelsen, Kathrin S; Schröder, Nicolas W J; Alsabeh, Randa; Slepenkin, Anatoly; Zhang, Wenxuan; Chen, Shuang; Wei, Bo; Bulut, Yonca; Wong, Michelle H; Peterson, Ellena M; Arditi, Moshe

    2005-08-12

    Chlamydia pneumoniae is the causative agent of respiratory tract infections and a number of chronic diseases. Here we investigated the involvement of the common TLR adaptor molecule MyD88 in host responses to C. pneumoniae-induced pneumonia in mice. MyD88-deficient mice were severely impaired in their ability to mount an acute early inflammatory response toward C. pneumoniae. Although the bacterial burden in the lungs was comparable 5 days after infection, MyD88-deficient mice exhibited only minor signs of pneumonia and reduced expression of inflammatory mediators. MyD88-deficient mice were unable to up-regulate proinflammatory cytokines and chemokines, demonstrated delayed recruitment of CD8+ and CD4+ T cells to the lungs, and were unable to clear the pathogen from their lungs at day 14. At day 14 the MyD88-deficent mice developed a severe, chronic lung inflammation with elevated IL-1beta and IFN-gamma leading to increased mortality, whereas wild-type mice as well as TLR2- or TLR4-deficient mice recovered from acute pneumonia and did not show delayed bacterial clearance. Thus, MyD88 is essential to recognize C. pneumoniae infection and initiate a prompt and effective immune host response against this organism leading to clearance of bacteria from infected lungs.

  5. Natural Killer Cell Sensing of Infected Cells Compensates for MyD88 Deficiency but Not IFN-I Activity in Resistance to Mouse Cytomegalovirus.

    PubMed

    Cocita, Clément; Guiton, Rachel; Bessou, Gilles; Chasson, Lionel; Boyron, Marilyn; Crozat, Karine; Dalod, Marc

    2015-05-01

    In mice, plasmacytoid dendritic cells (pDC) and natural killer (NK) cells both contribute to resistance to systemic infections with herpes viruses including mouse Cytomegalovirus (MCMV). pDCs are the major source of type I IFN (IFN-I) during MCMV infection. This response requires pDC-intrinsic MyD88-dependent signaling by Toll-Like Receptors 7 and 9. Provided that they express appropriate recognition receptors such as Ly49H, NK cells can directly sense and kill MCMV-infected cells. The loss of any one of these responses increases susceptibility to infection. However, the relative importance of these antiviral immune responses and how they are related remain unclear. In humans, while IFN-I responses are essential, MyD88 is dispensable for antiviral immunity. Hence, a higher redundancy has been proposed in the mechanisms promoting protective immune responses against systemic infections by herpes viruses during natural infections in humans. It has been assumed, but not proven, that mice fail to mount protective MyD88-independent IFN-I responses. In humans, the mechanism that compensates MyD88 deficiency has not been elucidated. To address these issues, we compared resistance to MCMV infection and immune responses between mouse strains deficient for MyD88, the IFN-I receptor and/or Ly49H. We show that selective depletion of pDC or genetic deficiencies for MyD88 or TLR9 drastically decreased production of IFN-I, but not the protective antiviral responses. Moreover, MyD88, but not IFN-I receptor, deficiency could largely be compensated by Ly49H-mediated antiviral NK cell responses. Thus, contrary to the current dogma but consistent with the situation in humans, we conclude that, in mice, in our experimental settings, MyD88 is redundant for IFN-I responses and overall defense against a systemic herpes virus infection. Moreover, we identified direct NK cell sensing of infected cells as one mechanism able to compensate for MyD88 deficiency in mice. Similar mechanisms likely contribute to protect MyD88- or IRAK4-deficient patients from viral infections.

  6. Detection of MYD88 L265P in peripheral blood of patients with Waldenström's Macroglobulinemia and IgM monoclonal gammopathy of undetermined significance.

    PubMed

    Xu, L; Hunter, Z R; Yang, G; Cao, Y; Liu, X; Manning, R; Tripsas, C; Chen, J; Patterson, C J; Kluk, M; Kanan, S; Castillo, J; Lindeman, N; Treon, S P

    2014-08-01

    MYD88 L265P is highly prevalent in Waldenstrom's Macroglobulinemia (WM) and IgM monoclonal gammopathy of unknown significance (MGUS). We investigated whether MYD88 L265P could be identified by peripheral blood (PB) allele-specific PCR. MYD88 L265P was detected in untreated WM (114/118; 96.6%); previously treated WM (63/102; 61.8%); and IgM MGUS (5/12; 41.7%) but in none of 3 hyper-IgM or 40 healthy individuals. Median PB MYD88 L265P ΔCt was 3.77, 7.24, 10.89, 12.33 and 14.07 for untreated WM, previously treated WM, IgM MGUS, hyper-IgM and healthy individuals, respectively (P<0.0001). For the 232 IgM MGUS and WM patients, PB MYD88 L265P ΔCt moderately correlated to bone marrow (BM) disease (r=-0.3553; P<0.0001), serum IgM (r=-0.3262; P<0.0001) and hemoglobin (r=0.3005; P<0.0001) levels. PB MYD88 L265P ΔCt and serum IgM correlated similarly with BM disease burden. For positive patients, PB MYD88 L265P ΔCt was <6.5 in 100/114 (88%) untreated WM, and >6.5 in 4/5 (80%) IgM MGUS patients (P=0.0034). Attainment of a negative PB MYD88 L265P mutation status was associated with lower BM disease (P=0.001), serum IgM (P=0.019) and higher hemoglobin (P=0.004) levels in treated patients. These studies show the feasibility for detecting MYD88 L265P by PB examination, and the potential for PB MYD88 L265P ΔCt use in the diagnosis and management of WM patients.

  7. Participation of MyD88 and Interleukin-33 as Innate Drivers of Th2 Immunity to Trichinella spiralis

    PubMed Central

    Scalfone, Lisa K.; Nel, Hendrik J.; Gagliardo, Lucille F.; Cameron, Jody L.; Al-Shokri, Shaikha; Leifer, Cynthia A.; Fallon, Padraic G.

    2013-01-01

    Trichinella spiralis is a highly destructive parasitic nematode that invades and destroys intestinal epithelial cells, injures many different tissues during its migratory phase, and occupies and transforms myotubes during the final phase of its life cycle. We set out to investigate the role in immunity of innate receptors for potential pathogen- or danger-associated molecular patterns (PAMPs or DAMPs). Focusing on the MyD88-dependent receptors, which include Toll-like receptors (TLRs) and interleukin-1 (IL-1) family members, we found that MyD88-deficient mice expelled worms normally, while TLR2/4-deficient mice showed accelerated worm expulsion, suggesting that MyD88 was active in signaling pathways for more than one receptor during intestinal immunity. A direct role for PAMPs in TLR activation was not supported in a transactivation assay involving a panel of murine and human TLRs. Mice deficient in the IL-1 family receptor for the DAMP, IL-33 (called ST2), displayed reduced intestinal Th2 responses and impaired mast cell activation. IL-33 was constitutively expressed in intestinal epithelial cells, where it became concentrated in nuclei within 2 days of infection. Nuclear localization was an innate response to infection that occurred in intestinal regions where worms were actively migrating. Th2 responses were also compromised in the lymph nodes draining the skeletal muscles of ST2-deficient mice, and this correlated with increased larval burdens in muscle. Our results support a mechanism in which the immune system recognizes and responds to tissue injury in a way that promotes Th2 responses. PMID:23403558

  8. CpG-ODN Shapes Alum Adjuvant Activity Signaling via MyD88 and IL-10.

    PubMed

    Mirotti, Luciana; Alberca Custódio, Ricardo Wesley; Gomes, Eliane; Rammauro, Florencia; de Araujo, Eliseu Frank; Garcia Calich, Vera Lucia; Russo, Momtchilo

    2017-01-01

    Aluminum-containing adjuvants usually referred as Alum are considered as T helper type-2 (Th2) adjuvants, while agonists of toll-like receptors (TLRs) are viewed as adjuvants that favor Th1/Th17 immunity. Alum has been used in numerous vaccine formulations; however, its undesired pro-Th2 adjuvant activity constitutes a caveat for Alum-based vaccines. Combining Alum with TLR-dependent, pro-Th1/Th17 adjuvants might dampen the pro-Th2 activity and improve the effectiveness of vaccine formulations. Here, using the ovalbumin (OVA) model of allergic lung inflammation, we found that sensitization with the synthetic TLR9 agonist, which is composed of oligodeoxynucleotides containing CpG motifs adsorbed to Alum, inhibited the development of OVA-induced lung allergic Th2 responses without shifting toward a Th1 pattern. The conversion of T cell immunity from the polarized allergic Th2 response to a non-polarized form by sensitization with OVA/Alum/CpG was dependent on MyD88 signaling in myeloid cells. Notably, sensitization of IL-10-deficient mice with OVA/Alum/CpG resulted in the development of neutrophilic lung inflammation associated with IFNγ production. However, in IL-10/IL-12-deficient mice, it resulted in neutrophilic inflammation dominated by IL-17 production. We conclude that OVA/Alum/CpG sensitization signaling via MyD88 and IL-10 molecules results in non-polarized immunity. Conversely, OVA/Alum/CpG sensitization in presence of MyD88 but absence of IL-10 or IL-10/IL-12 molecules results, respectively, in neutrophilic inflammation associated with IFNγ or IL-17 production. Our work provides novel OVA models of lung inflammation and suggests that Alum/CpG-based formulations might be of potential use in anti-allergic or anti-infectious processes.

  9. MYD88 and functionally related genes are associated with multiple infections in a model population of Kenyan village dogs.

    PubMed

    Necesankova, Michaela; Vychodilova, Leona; Albrechtova, Katerina; Kennedy, Lorna J; Hlavac, Jan; Sedlak, Kamil; Modry, David; Janova, Eva; Vyskocil, Mirko; Horin, Petr

    2016-12-01

    The purpose of this study was to seek associations between immunity-related molecular markers and endemic infections in a model population of African village dogs from Northern Kenya with no veterinary care and no selective breeding. A population of village dogs from Northern Kenya composed of three sub-populations from three different areas (84, 50 and 55 dogs) was studied. Canine distemper virus (CDV), Hepatozoon canis, Microfilariae (Acantocheilonema dracunculoides, Acantocheilonema reconditum) and Neospora caninum were the pathogens studied. The presence of antibodies (CDV, Neospora), light microscopy (Hepatozoon) and diagnostic PCR (Microfilariae) were the methods used for diagnosing infection. Genes involved in innate immune mechanisms, NOS3, IL6, TLR1, TLR2, TLR4, TLR7, TLR9, LY96, MYD88, and three major histocompatibility genes class II genes were selected as candidates. Single nucleotide polymorphism (SNP) markers were detected by Sanger sequencing, next generation sequencing and PCR-RFLP. The Fisher´s exact test for additive and non-additive models was used for association analyses. Three SNPs within the MYD88 gene and one TLR4 SNP marker were associated with more than one infection. Combined genotypes and further markers identified by next generation sequencing confirmed associations observed for individual genes. The genes associated with infection and their combinations in specific genotypes match well our knowledge on their biological role and on the role of the relevant biological pathways, respectively. Associations with multiple infections observed between the MYD88 and TLR4 genes suggest their involvement in the mechanisms of anti-infectious defenses in dogs.

  10. CpG-ODN Shapes Alum Adjuvant Activity Signaling via MyD88 and IL-10

    PubMed Central

    Mirotti, Luciana; Alberca Custódio, Ricardo Wesley; Gomes, Eliane; Rammauro, Florencia; de Araujo, Eliseu Frank; Garcia Calich, Vera Lucia; Russo, Momtchilo

    2017-01-01

    Aluminum-containing adjuvants usually referred as Alum are considered as T helper type-2 (Th2) adjuvants, while agonists of toll-like receptors (TLRs) are viewed as adjuvants that favor Th1/Th17 immunity. Alum has been used in numerous vaccine formulations; however, its undesired pro-Th2 adjuvant activity constitutes a caveat for Alum-based vaccines. Combining Alum with TLR-dependent, pro-Th1/Th17 adjuvants might dampen the pro-Th2 activity and improve the effectiveness of vaccine formulations. Here, using the ovalbumin (OVA) model of allergic lung inflammation, we found that sensitization with the synthetic TLR9 agonist, which is composed of oligodeoxynucleotides containing CpG motifs adsorbed to Alum, inhibited the development of OVA-induced lung allergic Th2 responses without shifting toward a Th1 pattern. The conversion of T cell immunity from the polarized allergic Th2 response to a non-polarized form by sensitization with OVA/Alum/CpG was dependent on MyD88 signaling in myeloid cells. Notably, sensitization of IL-10-deficient mice with OVA/Alum/CpG resulted in the development of neutrophilic lung inflammation associated with IFNγ production. However, in IL-10/IL-12-deficient mice, it resulted in neutrophilic inflammation dominated by IL-17 production. We conclude that OVA/Alum/CpG sensitization signaling via MyD88 and IL-10 molecules results in non-polarized immunity. Conversely, OVA/Alum/CpG sensitization in presence of MyD88 but absence of IL-10 or IL-10/IL-12 molecules results, respectively, in neutrophilic inflammation associated with IFNγ or IL-17 production. Our work provides novel OVA models of lung inflammation and suggests that Alum/CpG-based formulations might be of potential use in anti-allergic or anti-infectious processes. PMID:28220116

  11. Early alpha/beta interferon production by myeloid dendritic cells in response to UV-inactivated virus requires viral entry and interferon regulatory factor 3 but not MyD88.

    PubMed

    Hidmark, Asa S; McInerney, Gerald M; Nordström, Eva K L; Douagi, Iyadh; Werner, Kristen M; Liljeström, Peter; Karlsson Hedestam, Gunilla B

    2005-08-01

    Alpha/beta interferons (IFN-alpha/beta) are key mediators of innate immunity and important modulators of adaptive immunity. The mechanisms by which IFN-alpha/beta are induced are becoming increasingly well understood. Recent studies showed that Toll-like receptors 7 and 8 expressed by plasmacytoid dendritic cells (pDCs) mediate the endosomal recognition of incoming viral RNA genomes, a process which requires myeloid differentiation factor 88 (MyD88). Here we investigate the requirements for virus-induced IFN-alpha/beta production in cultures of bone marrow-derived murine myeloid DCs (mDCs). Using recombinant Semliki Forest virus blocked at different steps in the viral life cycle, we show that replication-defective virus induced IFN-alpha/beta in mDCs while fusion-defective virus did not induce IFN-alpha/beta. The response to replication-defective virus was largely intact in MyD88-/- mDC cultures but was severely reduced in mDC cultures from mice lacking IFN regulatory factor 3. Our observations suggest that mDCs respond to incoming virus via a pathway that differs from the fusion-independent, MyD88-mediated endosomal pathway described for the induction of IFN-alpha/beta in pDCs. We propose that events during or downstream of viral fusion, but prior to replication, can activate IFN-alpha/beta in mDCs. Thus, mDCs may contribute to the antiviral response activated by the immune system at early time points after infection.

  12. Rifaximin Improves Clostridium difficile Toxin A-Induced Toxicity in Caco-2 Cells by the PXR-Dependent TLR4/MyD88/NF-κB Pathway

    PubMed Central

    Esposito, Giuseppe; Nobile, Nicola; Gigli, Stefano; Seguella, Luisa; Pesce, Marcella; d’Alessandro, Alessandra; Bruzzese, Eugenia; Capoccia, Elena; Steardo, Luca; Cuomo, Rosario; Sarnelli, Giovanni

    2016-01-01

    Background: Clostridium difficile infections (CDIs) caused by Clostridium difficile toxin A (TcdA) lead to severe ulceration, inflammation and bleeding of the colon, and are difficult to treat. Aim: The study aimed to evaluate the effect of rifaximin on TcdA-induced apoptosis in intestinal epithelial cells and investigate the role of PXR in its mechanism of action. Methods: Caco-2 cells were incubated with TcdA and treated with rifaximin (0.1-10 μM) with or without ketoconazole (10 μM). The transepithelial electrical resistance (TEER) and viability of the treated cells was determined. Also, the expression of zona occludens-1 (ZO-1), toll-like receptor 4 (TLR4), Bcl-2-associated X protein (Bax), transforming growth factor-β-activated kinase-1 (TAK1), myeloid differentiation factor 88 (MyD88), and nuclear factor-kappaB (NF-κB) was determined. Results: Rifaximin treatment (0.1, 1.0, and 10 μM) caused a significant and concentration-dependent increase in the TEER of Caco-2 cells (360, 480, and 680% vs. TcdA treatment) 24 h after the treatment and improved their viability (61, 79, and 105%). Treatment also concentration-dependently decreased the expression of Bax protein (-29, -65, and -77%) and increased the expression of ZO-1 (25, 54, and 87%) and occludin (71, 114, and 262%) versus TcdA treatment. The expression of TLR4 (-33, -50, and -75%), MyD88 (-29, -60, and -81%) and TAK1 (-37, -63, and -79%) were also reduced with rifaximin versus TcdA treatment. Ketoconazole treatment inhibited these effects. Conclusion: Rifaximin improved TcdA-induced toxicity in Caco-2 cells by the PXR-dependent TLR4/MyD88/NF-κB pathway mechanism, and may be useful in the treatment of CDIs. PMID:27242527

  13. Staphylococcus aureus recognition by hematopoietic stem and progenitor cells via TLR2/MyD88/PGE2 stimulates granulopoiesis in wounds.

    PubMed

    Granick, Jennifer L; Falahee, Patrick C; Dahmubed, Delsheen; Borjesson, Dori L; Miller, Lloyd S; Simon, Scott I

    2013-09-05

    During bacterial infection, hematopoietic stem and progenitor cells (HSPCs) differentiate into polymorphonuclear leukocytes (PMNs) in the bone marrow. We reported that HSPCs recruited to Staphylococcus aureus-infected skin wounds in mice undergo granulopoiesis, whereas other authors have demonstrated their differentiation in vitro after Toll-like receptor 2 (TLR2)/MyD88 stimulation. Here, we examined this pathway in HSPC trafficking and granulopoiesis within S aureus-infected wounds. Lineage- HSPCs from TLR2- or MyD88-deficient mice injected into infected wounds of wild-type (WT) mice exhibited impaired granulopoiesis. However, HSPCs from WT mice produced similar numbers of PMNs whether transferred into wounds of TLR2-, MyD88-deficient, or WT mice. Prostaglandin E2 (PGE2), which stimulates HSPC survival and proliferation, was produced by HSPCs after TLR2 stimulation, suggesting that TLR2/MyD88 activation promotes granulopoiesis in part by production and autocrine activity of PGE2. Pretreatment of TLR2- or MyD88-deficient HSPCs with PGE2 rescued granulocytic differentiation in vivo. Finally, we demonstrate that bone marrow-derived lin-/Sca-1+/c-kit+ cells produced PGE2 and underwent granulopoiesis after TLR2 stimulation. lin-/Sca-1+/c-kit+ cells deficient in TLR2 or MyD88 produced PMNs after PGE2 treatment when transferred into uninfected wounds. We conclude that granulopoiesis in S aureus-infected wounds is induced by TLR2/MyD88 activation of HSPCs through a mechanism that involves autocrine production and activity of PGE2.

  14. Astragalus polysaccharides exerts immunomodulatory effects via TLR4-mediated MyD88-dependent signaling pathway in vitro and in vivo

    PubMed Central

    Zhou, Lijing; Liu, Zijing; Wang, Zhixue; Yu, Shuang; Long, Tingting; Zhou, Xing; Bao, Yixi

    2017-01-01

    Astragalus polysaccharides (APS), which is widely used as a remedy to promote immunity of breast cancer patients, can enhance immune responses and exert anti-tumor effects. In this study, we investigated the effects and mechanisms of APS on macrophage RAW 264.7 and EAC tumor-bearing mice. Griess reaction and ELISA assays revealed that the concentrations of nitric oxide, TNF-α, IL-1β and IL-6 were increased by APS. However, this effect was diminished in the presence of TAK-242 (TLR4 inhibitor) or ST-2825(MyD88 inhibitor). In C57BL/10J (TLR4+/+wild-type) and C57BL/6J (MyD88+/+wild-type) tumor-bearing mice, the tumor apoptosis rate, immune organ indexes and the levels of TNF-α, IL-1β and IL-6 in blood increased and the tumor weight decreased by oral administration of APS for 25 days. APS had no obvious effects on IL-12p70. However, these effects were not significant in C57BL/10ScNJ (TLR4-deficient) and C57BL/B6.129P2(SJL)-Myd88m1.1Defr/J (MyD88-deficient) tumor-bearing mice. qRT-PCR and Western blot indicated that APS stimulated the key nodes in the TLR4-MyD88 dependent signaling pathway, including TLR4, MyD88, TRAF-6, NF-κB and AP-1, both in vitro and in vivo. However, TRAM was an exception. Moreover, TRAF-6 and NF-κB were not triggered by APS in gene-deficient tumor-bearing mice. Therefore, APS may modulate immunity of host organism through activation of TLR4-mediated MyD88-dependent signaling pathway. PMID:28303957

  15. Resveratrol inhibits the IL-1β-induced expression of MMP-13 and IL-6 in human articular chondrocytes via TLR4/MyD88-dependent and -independent signaling cascades.

    PubMed

    Gu, Hailun; Jiao, Yongliang; Yu, Xiaolu; Li, Xingyao; Wang, Wei; Ding, Lifeng; Liu, Li

    2017-03-01

    The natural polyphenolic compound, resveratrol, has been shown to exhibit anti-osteoarthritic activity. Therefore it is hypothesized that resveratrol may serve as a nutritional supplement to counteract osteoarthritis (OA). However, the mechanisms responsible for these anti-osteoarthritic effects have not yet been fully elucidated. The aim of this study was to determine whether the biological effects of resveratrol against interleukin (IL)-1β‑induced inflammation in human articular chondrocytes involved both Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)-dependent and -independent signaling pathways. Human articular chondrocytes derived from patients with OA were stimulated with IL-1β, and then co-treated with resveratrol. Cell viability was subsequently evaluated by MTS assays, and the concentrations of matrix metalloproteinase (MMP)-13 and the pro-inflammatory factor, IL-6, were detected in culture supernatants using ELISA. The mRNA and protein levels of downstream mediators of TLR4/MyD88-dependent and -independent signaling pathways were also assayed by RT-qPCR and western blot analysis, respectively. Our results revealed that resveratrol prevented the IL-1β-induced reduction in cell viability. Furthermore, stimulation of the chondrocytes with IL-1β resulted in a significant upregulation of TLR4 and downstream targets of both TLR4/MyD88-dependent and -independent signaling pathways that are associated with the synthesis of MMP-13 and IL-6. Correspondingly, IL-1β-induced catabolic and inflammatory responses were effectively reversed by resveratrol. Taken together, these data suggest that resveratrol exerted protective effects against matrix degradation and inflammation in OA-affected chondrocytes by inhibiting both TLR4/MyD88-dependent and -independent signaling pathways. Thus, resveratrol represents a potential treatment for OA and warrants further investigation.

  16. Molecular characterization of Pacific oyster (Crassostrea gigas) IRAK4 gene and its role in MyD88-dependent pathway.

    PubMed

    Tang, Xueying; Huang, Baoyu; Zhang, Linlin; Li, Li; Zhang, Guofan

    2017-02-13

    Interleukin-1 receptor-associated kinases (IRAKs) play important roles in MyD88-dependent TLR signaling, the crucial innate immune pathway in molluscs. In this study, we examined the full-length IRAK4 genetic sequence in the Pacific oyster (Crassostrea gigas) by molecular cloning. Phylogenetic analysis revealed that CgIRAK4 is most closely related to Mytilus edulis, and forms a clade with other molluscs. CgIRAK4 transcripts are widely expressed in all tissues, with the highest expression observed in the hemocytes and gill. Moreover, CgIRAK4 is significantly upregulated after Oyster herpesvirus-1 microvariant (OsHV-1 μvar), Vibrio alginolyticus, and poly I:C challenge. Yeast two-hybrid and co-immunoprecipitation assays reveal that the CgIRAK4 death domain is necessary to mediate interaction between CgIRAK4 and two CgMyD88 isoforms. In addition, CgIRAK4 overexpression cannot induce NF-κB transcriptional activity, but blocks that induced by CgMyD88 in HEK293T cells. These findings elucidate the mechanisms of MyD88-dependent TLR signaling in molluscs, and the differences in IRAK-mediated pathway activation between invertebrates and vertebrates.

  17. Resveratrol suppresses NTHi-induced inflammation via up-regulation of the negative regulator MyD88 short

    PubMed Central

    Andrews, Carla S.; Matsuyama, Shingo; Lee, Byung-Cheol; Li, Jian-Dong

    2016-01-01

    Upper respiratory tract inflammatory diseases such as asthma and chronic obstructive pulmonary diseases (COPD) affect more than one-half billion people globally and are characterized by chronic inflammation that is often exacerbated by respiratory pathogens such as nontypeable Haemophilus influenzae (NTHi). The increasing numbers of antibiotic-resistant bacterial strains and the limited success of currently available pharmaceuticals used to manage the symptoms of these diseases present an urgent need for the development of novel anti-inflammatory therapeutic agents. Resveratrol has long been thought as an interesting therapeutic agent for various diseases including inflammatory diseases. However, the molecular mechanisms underlying its anti-inflammatory properties remain largely unknown. Here we show for the first time that resveratrol decreases expression of pro-inflammatory mediators in airway epithelial cells and in the lung of mice by enhancing NTHi-induced MyD88 short, a negative regulator of inflammation, via inhibition of ERK1/2 activation. Furthermore, resveratrol inhibits NTHi-induced ERK1/2 phosphorylation by increasing MKP-1 expression via a cAMP-PKA-dependent signaling pathway. Finally, we show that resveratrol has anti-inflammatory effects post NTHi infection, thereby demonstrating its therapeutic potential. Together these data reveal a novel mechanism by which resveratrol alleviates NTHi-induced inflammation in airway disease by up-regulating the negative regulator of inflammation MyD88s. PMID:27677845

  18. Trichinella spiralis excretory-secretory products protect against polymicrobial sepsis by suppressing MyD88 via mannose receptor.

    PubMed

    Du, Linlin; Liu, Lihua; Yu, Yang; Shan, Hui; Li, Leiqing

    2014-01-01

    Trichinella spiralis (T. spiralis) or its excretory-secretory products (TsES) protect hosts from autoimmune diseases, which depend on inducing host T helper (Th) 2 immune response and inhibiting inflammatory factors. Sepsis is a systemic inflammatory response syndrome (SIRS) evoked by infection. Little is known about the effects of helminths or their excretory-secretory products on sepsis. Here, we investigated the effects of TsES in a mice model of polymicrobial sepsis. TsES improved survival, reduced organ injury, and enhanced bacterial clearance in septic mice. To investigate the molecular mechanism, macrophages from septic patients or the control group were incubated with TsES. TsES reduced sepsis-inducing inflammatory cytokines mediated by Toll-like receptors (TLR) in vitro by suppressing TLR adaptor-transducer myeloid differentiation factor 88 (MyD88) and nuclear factor- (NF-)-κB. Furthermore, TsES upregulated mannose receptor (MR) expression during sepsis. MR blocking attenuated the effects of TsES on MyD88 and NF-κB expression. In vivo, MR RNAi reduced the survival rate of septic mice treated with TsES, suggesting that TsES-mediated protection against polymicrobial sepsis is dependent on MR. Thus, TsES administration might be a potential therapeutic strategy for treating sepsis.

  19. The frustrated host response to Legionella pneumophila is bypassed by MyD88-dependent translation of pro-inflammatory cytokines.

    PubMed

    Asrat, Seblewongel; Dugan, Aisling S; Isberg, Ralph R

    2014-07-01

    Many pathogens, particularly those that require their host for survival, have devised mechanisms to subvert the host immune response in order to survive and replicate intracellularly. Legionella pneumophila, the causative agent of Legionnaires' disease, promotes intracellular growth by translocating proteins into its host cytosol through its type IV protein secretion machinery. At least 5 of the bacterial translocated effectors interfere with the function of host cell elongation factors, blocking translation and causing the induction of a unique host cell transcriptional profile. In addition, L. pneumophila also interferes with translation initiation, by preventing cap-dependent translation in host cells. We demonstrate here that protein translation inhibition by L. pneumophila leads to a frustrated host MAP kinase response, where genes involved in the pathway are transcribed but fail to be translated due to the bacterium-induced protein synthesis inhibition. Surprisingly, few pro-inflammatory cytokines, such as IL-1α and IL-1β, bypass this inhibition and get synthesized in the presence of Legionella effectors. We show that the selective synthesis of these genes requires MyD88 signaling and takes place in both infected cells that harbor bacteria and neighboring bystander cells. Our findings offer a perspective of how host cells are able to cope with pathogen-encoded activities that disrupt normal cellular process and initiate a successful inflammatory response.

  20. Rare Circulating Cells in Familial Waldenström Macroglobulinemia Displaying the MYD88 L265P Mutation Are Enriched by Epstein-Barr Virus Immortalization

    PubMed Central

    Pertesi, Maroulio; Galia, Perrine; Nazaret, Nicolas; Vallée, Maxime; Garderet, Laurent; Leleu, Xavier; Avet-Loiseau, Hervé; Foll, Matthieu; Byrnes, Graham; Lachuer, Joel; McKay, James D.; Dumontet, Charles

    2015-01-01

    The MYD88 L265P is a recurrent somatic mutation in neoplastic cells from patients with Waldenström Macroglobulinemia (WM). We identified the MYD88 L265P mutation in three individuals from unrelated families, but its presence did not explain the disease segregation within these WM pedigrees. We observed the mutation in these three individuals at high allele fractions in DNA extracted from EBV-immortalized Lymphoblastoid cell lines established from peripheral blood (LCL), but at much lower allele fractions in DNA extracted directly from peripheral blood, suggesting that this mutation is present in a clonal cell subpopulation rather than of germ-line origin. Furthermore, we observed that the MYD88 L265P mutation is enriched in WM families, detected in 40.5% of patients with familial WM or MGUS (10/22 WM, 5/15 MGUS), compared to 3.5% of patients with familial MM or MGUS (0/72 MM, 4/41 MGUS) (p = 10−7). The mutant allele frequency increased with passages in vitro after immortalization with Epstein-Barr virus (EBV) consistent with the MYD88 L265P described gain-of-function proposed for this mutation. The MYD88 L265P mutation appears to be frequently present in circulating cells in patients with WM, and MGUS, and these cells are amenable to immortalization by EBV. PMID:26352266

  1. MyD88 signaling in the CNS is required for development of fatty acid induced leptin resistance and diet-induced obesity

    PubMed Central

    Kleinridders, André; Schenten, Dominik; Könner, A. Christine; Belgardt, Bengt F.; Mauer, Jan; Okamura, Tomoo; Wunderlich, F. Thomas; Medzhitov, Ruslan; Brüning, Jens C.

    2014-01-01

    Summary Obesity-associated activation of inflammatory pathways represents a key step in the development of insulin resistance in peripheral organs, partially via activation of TLR-4 signaling by fatty acids. Here we demonstrate that palmitate acting in the central nervous system (CNS) inhibits leptin-induced anorexia and Stat-3 activation. To determine the functional significance of TLR signaling in the CNS in the development of leptin resistance and diet-induced obesity in vivo, we have characterized mice deficient for the TLR adaptor molecule MyD88 in the CNS (MyD88ΔCNS). Compared to control mice, MyD88ΔCNS mice are protected from high-fat diet (HFD)-induced weight gain, from the development of HFD-induced leptin resistance and from the induction of leptin resistance by acute central application of palmitate. Moreover, CNS-restricted MyD88 deletion protects from HFD- and icv palmitate-induced impairment of peripheral glucose metabolism. Thus, we define neuronal MyD88-dependent signaling as a key regulator of diet-induced leptin and insulin resistance in vivo. PMID:19808018

  2. Detection of MYD88 L265P in patients with lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia and other B-cell non-Hodgkin lymphomas

    PubMed Central

    Shin, Sang-Yong; Kim, Hyun-Young; Park, Chang-Hun; Kim, Hee-Jin; Kim, Jong-Won; Kim, Seok Jin; Kim, Won Seog

    2016-01-01

    Background Recent studies have identified a high prevalence of the MYD88 L265P mutation in lymphoplasmacytic lymphoma (LPL)/Waldenstrom macroglobulinemia (WM) cases, whereas low frequencies have been observed in other B cell non-Hodgkin lymphomas (NHLs). Methods We evaluated the sensitivity of the mutant enrichment 3'-modified oligonucleotide (MEMO)-PCR technique, a new detection method. We examined the MYD88 L265P mutation in a series of Korean patients with LPL/WM and other B cell NHLs in bone marrow aspirates, using the MEMO-PCR technique. Results The sensitivity of MEMO-PCR was estimated to be approximately 10-16.7%. MYD88 L265P was detected in 21 of 28 LPL cases (75%) and only three of 69 B cell NHL cases (4.3%). Conclusion Although MEMO-PCR had relatively low sensitivity, we confirmed the high prevalence of the MYD88 L265P mutation in Korean LPL patients. Our study suggests the diagnostic value of MYD88 L265P for differentiating B-cell NHLs. PMID:27722129

  3. Gene expression profiles identify both MyD88-independent and MyD88-dependent pathways involved in the maturation of dendritic cells mediated by heparan sulfate: A novel adjuvant

    PubMed Central

    Wu, Meini; Wang, Haixuan; Shi, Jiandong; Sun, Jing; Duan, Zhiqing; Li, Yanhan; Li, Jianfang; Hu, Ningzhu; Wei, Yiju; Chen, Yang; Hu, Yunzhang

    2015-01-01

    The traditional vaccine adjuvant research is mainly based on the trial and error method, and the mechanisms underlying the immune system stimulation remaining largely unknown. We previously demonstrated that heparan sulfate (HS), a TLR-4 ligand and endogenous danger signal, effectively enhanced humoral and cellular immune responses in mice immunized by HBsAg. This study aimed to evaluate whether HS induces better humoral immune responses against inactivated Hepatitis A or Rabies Vaccines, respectively, compared with traditional adjuvants (e.g. Alum and complete Freund's adjuvant). In order to investigate the molecular mechanisms of its adjuvanticity, the gene expression pattern of peripheral blood monocytes derived DCs (dendritic cells) stimulated with HS was analyzed at different times points. Total RNA was hybridized to Agilent SurePrint G3 Human Gene Expression 8 × 60 K one-color oligo-microarray. Through intersection analysis of the microarray results, we found that the Toll-like receptor signaling pathway was significantly activated, and NF-kB, TRAF3 and IRF7 were activated as early as 12 h, and MyD88 was activated at 48 h post-stimulation. Furthermore, the expression of the surface marker CD83 and the co-stimulatory molecules CD80 and CD86 was up-regulated as early as 24 h. Therefore, we speculated that HS-induced human monocyte-derived DC maturation may occur through both MyD88-independent and dependent pathways, but primarily through the former (TRIF pathway). These data provide an important basis for understanding the mechanisms underlying HS enhancement of the immune response. PMID:25668674

  4. Case-control study and mRNA expression analysis reveal the MyD88 gene is associated with digestive disorders in rabbit.

    PubMed

    Chen, Shi-Yi; Zhang, Wen-Xiu; Zhang, Gong-Wei; Peng, Jin; Zhao, Xiao-Bing; Lai, Song-Jia

    2013-12-01

    As in humans, significant associations between Toll-like receptor 4 (TLR4) and digestive disorders have been identified in rabbit and dog. However, as an essential adaptor downstream of TLR4, the genetic variation of myeloid differentiating factor 88 (MyD88) and its association with digestive disorders have remained unknown. In this study, we detected 10 single nucleotide polymorphisms (SNPs) in the entire genomic region of rabbit MyD88. The genetic variation in susceptibility to digestive disorders for the only coding SNP (synonymous c.699T>C) was studied in Yaan (183 cases and 142 controls) and Chengdu populations (145 cases and 140 controls). A case-control association study revealed that individuals with the C allele had significant protection against digestive disorders in the Yaan population (OR = 0.71; 95% CI, 0.51-0.99; P < 0.05), the Chengdu population (OR = 0.55; 95% CI, 0.39-0.78; P < 0.01) and for joint analysis (OR = 0.62; 95% CI, 0.49-0.79; P < 0.01). We also experimentally induced digestive disorders by feeding a fiber-deficient diet and found that increased susceptibility was significantly associated with higher MyD88 mRNA expression (P < 0.05). The lowest MyD88 mRNA expression was observed in individuals carrying the protective CC genotype. These results suggest that MyD88 is one of the most plausible candidate genes in relation to digestive disorders in rabbit. Further studies are required to explore the biological implications of MyD88 in the pathogenesis of digestive disorders.

  5. Interleukin-1 receptor but not Toll-like receptor 2 is essential for MyD88-dependent Th17 immunity to Coccidioides infection.

    PubMed

    Hung, Chiung-Yu; Jiménez-Alzate, María del Pilar; Gonzalez, Angel; Wüthrich, Marcel; Klein, Bruce S; Cole, Garry T

    2014-05-01

    Interleukin-17A (IL-17A)-producing CD4(+) T helper (Th17) cells have been shown to be essential for defense against pulmonary infection with Coccidioides species. However, we have just begun to identify the required pattern recognition receptors and understand the signal pathways that lead to Th17 cell activation after fungal infection. We previously reported that Card9(-/-) mice vaccinated with formalin-killed spherules failed to acquire resistance to Coccidioides infection. Here, we report that both MyD88(-/-) and Card9(-/-) mice immunized with a live, attenuated vaccine also fail to acquire protective immunity to this respiratory disease. Like Card9(-/-) mice, vaccinated MyD88(-/-) mice revealed a significant reduction in numbers of both Th17 and Th1 cells in their lungs after Coccidioides infection. Both Toll-like receptor 2 (TLR2) and IL-1 receptor type 1 (IL-1r1) upstream of MyD88 have been implicated in Th17 cell differentiation. Surprisingly, vaccinated TLR2(-/-) and wild-type (WT) mice showed similar outcomes after pulmonary infection with Coccidioides, while vaccinated IL-1r1(-/-) mice revealed a significant reduction in the number of Th17 cells in their infected lungs compared to WT mice. Thus, activation of both IL-1r1/MyD88- and Card9-mediated Th17 immunity is essential for protection against Coccidioides infection. Our data also reveal that the numbers of Th17 cells were reduced in IL-1r1(-/-) mice to a lesser extent than in MyD88(-/-) mice, raising the possibility that other TLRs are involved in MyD88-dependent Th17 immunity to coccidioidomycosis. An antimicrobial action of Th17 cells is to promote early recruitment of neutrophils to infection sites. Our data revealed that neutrophils are required for vaccine immunity to this respiratory disease.

  6. Bifidobacterium breve reduces apoptotic epithelial cell shedding in an exopolysaccharide and MyD88-dependent manner

    PubMed Central

    Hughes, K. R.; Harnisch, L. C.; Alcon-Giner, C.; Mitra, S.; Wright, C. J.; Ketskemety, J.

    2017-01-01

    Certain members of the microbiota genus Bifidobacterium are known to positively influence host well-being. Importantly, reduced bifidobacterial levels are associated with inflammatory bowel disease (IBD) patients, who also have impaired epithelial barrier function, including elevated rates of apoptotic extrusion of small intestinal epithelial cells (IECs) from villi—a process termed ‘cell shedding’. Using a mouse model of pathological cell shedding, we show that mice receiving Bifidobacterium breve UCC2003 exhibit significantly reduced rates of small IEC shedding. Bifidobacterial-induced protection appears to be mediated by a specific bifidobacterial surface exopolysaccharide and interactions with host MyD88 resulting in downregulation of intrinsic and extrinsic apoptotic responses to protect epithelial cells under highly inflammatory conditions. Our results reveal an important and previously undescribed role for B. breve, in positively modulating epithelial cell shedding outcomes via bacterial- and host-dependent factors, supporting the notion that manipulation of the microbiota affects intestinal disease outcomes. PMID:28123052

  7. Bifidobacterium breve reduces apoptotic epithelial cell shedding in an exopolysaccharide and MyD88-dependent manner.

    PubMed

    Hughes, K R; Harnisch, L C; Alcon-Giner, C; Mitra, S; Wright, C J; Ketskemety, J; van Sinderen, D; Watson, A J M; Hall, L J

    2017-01-01

    Certain members of the microbiota genus Bifidobacterium are known to positively influence host well-being. Importantly, reduced bifidobacterial levels are associated with inflammatory bowel disease (IBD) patients, who also have impaired epithelial barrier function, including elevated rates of apoptotic extrusion of small intestinal epithelial cells (IECs) from villi-a process termed 'cell shedding'. Using a mouse model of pathological cell shedding, we show that mice receiving Bifidobacterium breve UCC2003 exhibit significantly reduced rates of small IEC shedding. Bifidobacterial-induced protection appears to be mediated by a specific bifidobacterial surface exopolysaccharide and interactions with host MyD88 resulting in downregulation of intrinsic and extrinsic apoptotic responses to protect epithelial cells under highly inflammatory conditions. Our results reveal an important and previously undescribed role for B. breve, in positively modulating epithelial cell shedding outcomes via bacterial- and host-dependent factors, supporting the notion that manipulation of the microbiota affects intestinal disease outcomes.

  8. Monoclonal antibody against Toll-like receptor 4 attenuates ventilator-induced lung injury in rats by inhibiting MyD88- and NF-κB-dependent signaling.

    PubMed

    Huang, Cuiyuan; Pan, Linghui; Lin, Fei; Dai, Huijun; Fu, Ruili

    2017-03-01

    The mechanisms through which mechanical ventilation causes non-infectious inflammatory diseases and lung injury are poorly understood. Animals models of this type of injury suggest that it involves signaling mediated by Toll‑like receptor (TLR)4 and 9. In this study, in order to gain further insight into the involvement of TLR4 in this type of injury, we performed in vivo and in vitro experiments to determine the mechanisms through which TLR4 triggers inflammation. We also examined whether the use of TLR4 monoclonal antibody (mAb) can alleviate this type of injury. For this purpose, rats were tracheotomized and administered intratracheal injections of anti‑TLR4 mAb or saline, and then ventilated for 4 h at a high tidal volume (HTV) of 40 ml/ kg or allowed to breathe spontaneously for the same period of time (controls). Alveolar macrophages (AMs) were isolated from the bronchoalveolar lavage fluid (BALF) of the rats and stimulated for 16 h with tumor necrosis factor (TNF)‑α in the presence or absence of anti‑TLR4 mAb. Lung injury was assessed by examining lung histopathology, lung wet/dry weight ratio, BALF total protein and cytokine levels in BALF and plasma. The mRNA and protein expression levels of TLR4, TLR9, myeloid differentiation factor 88 (Myd88) and nuclear factor (NF)‑κB were measured in cultured macrophages. Compared to the controls (spontaneous breathing), the ventilated rats exhibited greater pulmonary permeability, more severe inflammatory cell infiltration/lung edema, and higher levels of interleukin (IL)‑1β, IL‑6 and TNF‑α in BALF and plasma. The AMs from the ventilated rats expressed higher mRNA and protein levels of TLR4, TLR9, Myd88 and NF‑κB compared with the macrophages from the spontaneously breathing rats. The ventilated rats pre‑treated with anti‑TLR4 mAb exhibited markedly attenuated signs of ventilation‑induced injury, such as less lung inflammation and pulmonary edema, fewer cells in BALF, and

  9. Toll-like receptor 5 agonist inhibition of growth of A549 lung cancer cells in vivo in a Myd88 dependent manner.

    PubMed

    Zhou, Shi-Xiang; Li, Feng-Sheng; Qiao, Yu-Lei; Zhang, Xue-Qing; Wang, Zhi-Dong

    2012-01-01

    The purpose of this study was to examine the effect of a Toll-like receptor 5 (TLR5) agonist, CBLB502, on the growth and radiosensitivity of A549 lung cancer cells in vivo. Expression of myeloid differentiation factor 88 (MyD88) or TLR5 was stably knocked down in human lung cancer cells (A549) using lentivirus expressing short hairpin RNA targeting human MyD88 or TLR5. Lack of MyD88 or TLR5 expression enhanced tumor growth in mouse xenografts of A549 lung cancer cells. CBLB502 inhibited the growth of A549 lung cancer cells, not A549-MyD88-KD cells in vivo in the murine xenograft model. Our results showed that the inhibition of A549 by CBLB502 in vivo was realized through regulating the expression of neutrophil recruiting cytokines and neutrophil infiltration. Finally, we found that activation of TLR5 signaling did not affect the radiosensitivity of tumors in vivo.

  10. The Type II Secretion System of Legionella pneumophila Dampens the MyD88 and Toll-Like Receptor 2 Signaling Pathway in Infected Human Macrophages.

    PubMed

    Mallama, Celeste A; McCoy-Simandle, Kessler; Cianciotto, Nicholas P

    2017-04-01

    Previously, we reported that mutants of Legionella pneumophila lacking a type II secretion (T2S) system elicit higher levels of cytokines (e.g., interleukin-6 [IL-6]) following infection of U937 cells, a human macrophage-like cell line. We now show that this effect of T2S is also manifest upon infection of human THP-1 macrophages and peripheral blood monocytes but does not occur during infection of murine macrophages. Supporting the hypothesis that T2S acts to dampen the triggering of an innate immune response, we observed that the mitogen-activated protein kinase (MAPK) and nuclear transcription factor kappa B (NF-κB) pathways are more highly stimulated upon infection with the T2S mutant than upon infection with the wild type. By using short hairpin RNA to deplete proteins involved in specific pathogen-associated molecular pattern (PAMP) recognition pathways, we determined that the dampening effect of the T2S system was not dependent on nucleotide binding oligomerization domain (NOD)-like receptors (NLRs), retinoic acid-inducible protein I (RIG-I)-like receptors (RLRs), double-stranded RNA (dsRNA)-dependent protein kinase receptor (PKR), or TIR domain-containing adaptor inducing interferon beta (TRIF) signaling or an apoptosis-associated speck-like protein containing a CARD (ASC)- or caspase-4-dependent inflammasome. However, the dampening effect of T2S on IL-6 production was significantly reduced upon gene knockdown of myeloid differentiation primary response 88 (MyD88), TANK binding kinase 1 (TBK1), or Toll-like receptor 2 (TLR2). These data indicate that the L. pneumophila T2S system dampens the signaling of the TLR2 pathway in infected human macrophages. We also document the importance of PKR, TRIF, and TBK1 in cytokine secretion during L. pneumophila infection of macrophages.

  11. MyD88/CD40 Genetic Adjuvant Function in Cutaneous Atypical Antigen-Presenting Cells Contributes to DNA Vaccine Immunogenicity

    PubMed Central

    Slawin, Kevin M.; Levitt, Jonathan M.; Spencer, David M.

    2016-01-01

    Therapeutic DNA-based vaccines aim to prime an adaptive host immune response against tumor-associated antigens, eliminating cancer cells primarily through CD8+ cytotoxic T cell-mediated destruction. To be optimally effective, immunological adjuvants are required for the activation of tumor-specific CD8+ T cells responses by DNA vaccination. Here, we describe enhanced anti-tumor efficacy of an in vivo electroporation-delivered DNA vaccine by inclusion of a genetically encoded chimeric MyD88/CD40 (MC) adjuvant, which integrates both innate and adaptive immune signaling pathways. When incorporated into a DNA vaccine, signaling by the MC adjuvant increased antigen-specific CD8+ T cells and promoted elimination of pre-established tumors. Interestingly, MC-enhanced vaccine efficacy did not require direct-expression of either antigen or adjuvant by local antigen-presenting cells, but rather our data supports a key role for MC function in “atypical” antigen-presenting cells of skin. In particular, MC adjuvant-modified keratinocytes increased inflammatory cytokine secretion, upregulated surface MHC class I, and were able to increase in vitro and in vivo priming of antigen-specific CD8+ T cells. Furthermore, in the absence of critical CD8α+/CD103+ cross-priming dendritic cells, MC was still able to promote immune priming in vivo, albeit at a reduced level. Altogether, our data support a mechanism by which MC signaling activates an inflammatory phenotype in atypical antigen-presenting cells within the cutaneous vaccination site, leading to an enhanced CD8+ T cell response against DNA vaccine-encoded antigens, through both CD8α+/CD103+ dendritic cell-dependent and independent pathways. PMID:27741278

  12. Invasive group A Streptococcus disease in French-Canadian children is not associated with a defect in MyD88/IRAK4-pathway

    PubMed Central

    2014-01-01

    Background Beta-hemolytic Group A Streptococcus invasive disease (iGASd) has been subject to intense research since its re-emergence in the late 1980s. In Quebec, an increase in the number of severe iGASd cases has recently been observed. Because of the inter-individual variability in the severity of iGASd, a hereditary predisposition to invasive disease can be suspected. Given that iGASd occurs in MyD88- and IRAK4-deficient patients, although rarely, the increasing frequency of iGASd in the population of French-Canadian children may be associated with a deficiency in the host’s innate immune response. Methods In this report, we assessed the influence of: (i) bacterial genotype and virulence factors, (ii) immune-cellular features, and (iii) Myd88/IRAK4-dependent response to GAS in vitro on the susceptibility to iGASd in a paediatric cohort of 16 children: 11 French-Canadian and 5 from diverse origin. Findings GAS virulence factors and genotype are not implicated in the susceptibility toward iGASd, and cellular and MyD88/IRAK4 deficiencies are excluded in our patients. Conclusions Although it has been shown that the MyD88/IRAK4-dependent signal is involved in the response to invasive GAS, our data indicates that a MyD88/IRAK4-mediated signalling defect is not the main factor responsible for the susceptibility to severe iGASd in a paediatric population from the province of Quebec. PMID:24499202

  13. Chlamydia pneumoniae-induced foam cell formation requires MyD88-dependent and -independent signaling and is reciprocally modulated by liver X receptor activation.

    PubMed

    Chen, Shuang; Sorrentino, Rosalinda; Shimada, Kenichi; Bulut, Yonca; Doherty, Terence M; Crother, Timothy R; Arditi, Moshe

    2008-11-15

    Chlamydia pneumoniae is detected by macrophages and other APCs via TLRs and can exacerbate developing atherosclerotic lesions, but how that occurs is not known. Liver X receptors (LXRs) centrally control reverse cholesterol transport, but also negatively modulate TLR-mediated inflammatory pathways. We isolated peritoneal macrophages from wild-type, TLR2, TLR3, TLR4, TLR2/4, MyD88, TRIF, MyD88/TRIF, and IFN regulatory factor 3 (IRF3) KO mice, treated them with live or UV-killed C. pneumoniae in the presence or absence of oxidized LDL, then measured foam cell formation. In some experiments, the synthetic LXR agonist GW3965 was added to macrophages infected with C. pneumoniae in the presence of oxidized LDL. Both live and UV-killed C. pneumoniae induced IRF3 activation and promoted foam cell formation in wild-type macrophages, whereas the genetic absence of TLR2, TLR4, MyD88, TRIF, or IRF3, but not TLR3, significantly reduced foam cell formation. C. pneumoniae-induced foam cell formation was significantly reduced by the LXR agonist GW3965, which in turn inhibited C. pneumoniae-induced IRF3 activation, suggesting a bidirectional cross-talk. We conclude that C. pneumoniae facilitates foam cell formation via activation of both MyD88-dependent and MyD88-independent (i.e., TRIF-dependent and IRF3-dependent) pathways downstream of TLR2 and TLR4 signaling and that TLR3 is not involved in this process. This mechanism could at least partly explain why infection with C. pneumoniae accelerates the development of atherosclerotic plaque and lends support to the proposal that LXR agonists might prove clinically useful in suppressing atherogenesis.

  14. Toll-Like Receptor Ligand-Based Vaccine Adjuvants Require Intact MyD88 Signaling in Antigen-Presenting Cells for Germinal Center Formation and Antibody Production

    PubMed Central

    Mosaheb, Munir M.; Reiser, Michael L.; Wetzler, Lee M.

    2017-01-01

    Vaccines are critical in the fight against infectious diseases, and immune-stimulating adjuvants are essential for enhancing vaccine efficacy. However, the precise mechanisms of action of most adjuvants are unknown. There is an urgent need for customized and adjuvant formulated vaccines against immune evading pathogens that remain a risk today. Understanding the specific role of various cell types in adjuvant-induced protective immune responses is vital for an effective vaccine design. We have investigated the role of cell-specific MyD88 signaling in vaccine adjuvant activity in vivo, using Neisserial porin B (PorB), a TLR2 ligand-based adjuvant, compared with an endosomal TLR9 ligand (CpG) and toll-like receptor (TLR)-independent (alum, MF59) adjuvants. We found that intact MyD88 signaling is essential, separately, in all three antigen-presenting cell types [B cells, macrophages, and dendritic cells (DCs)] for optimal TLR ligand-based adjuvant activity. The role of MyD88 signaling in B cell and DC in vaccine adjuvant has been previously investigated. In this study, we now demonstrate that the immune response was also reduced in mice with macrophage-specific MyD88 deletion (Mac-MyD88−/−). We demonstrate that TLR-dependent adjuvants are potent inducers of germinal center (GC) responses, but GCs are nearly absent in Mac-MyD88−/− mice following immunization with TLR-dependent adjuvants PorB or CpG, but not with TLR-independent adjuvants MF59 or alum. Our findings reveal a unique and here-to-for unrecognized importance of intact MyD88 signaling in macrophages, to allow for a robust vaccine-induced immune responses when TLR ligand-based adjuvants are used. PMID:28316602

  15. The effects of MyD88 deficiency on exploratory activity, anxiety, motor coordination, and spatial learning in C57BL/6 and APPswe/PS1dE9 mice.

    PubMed

    Lim, Jeong-Eun; Song, Min; Jin, Jingji; Kou, Jinghong; Pattanayak, Abhinandan; Lalonde, Robert; Fukuchi, Ken-Ichiro

    2012-02-01

    Toll-like receptors (TLRs) are a family of pattern-recognition receptors in innate immunity and provide a first line defense against pathogens and tissue injuries. In addition to important roles in infection, inflammation, and immune diseases, recent studies show that TLR signaling is involved in modulation of learning, memory, mood, and neurogenesis. Because MyD88 is essential for the downstream signaling of all TLRs, except TLR3, we investigated the effects of MyD88 deficiency (MyD88-/-) on behavioral functions in mice. Additionally, we recently demonstrated that a mouse model of Alzheimer's disease (AD) deficient for MyD88 had decreases in Aβ deposits and soluble Aβ in the brain as compared with MyD88 sufficient AD mouse models. Because accumulation of Aβ in the brain is postulated to be a causal event leading to cognitive deficits in AD, we investigated the effects of MyD88 deficiency on behavioral functions in the AD mouse model at 10 months of age. MyD88 deficient mice showed more anxiety in the elevated plus-maze. In the motor coordination tests, MyD88 deficient mice remained on a beam and a bar for a longer time, but with slower initial movement on the bar. In the Morris water maze test, MyD88 deficiency appeared to improve spatial learning irrespective of the transgene. Our findings suggest that the MyD88-dependent pathway contributes to behavioral functions in an AD mouse model and its control group.

  16. Anti-Inflammatory Activity of Bee Venom in BV2 Microglial Cells: Mediation of MyD88-Dependent NF-κB Signaling Pathway

    PubMed Central

    Kim, Su Jung; Hong, Seung Bok; Park, Jin-Kyu

    2016-01-01

    Bee venom has long been used as a traditional folk medicine in Korea. It has been reportedly used for the treatment of arthritis, cancer, and inflammation. Although its anti-inflammatory activity in lipopolysaccharide- (LPS-) stimulated inflammatory cells has been reported, the exact mechanism of its anti-inflammatory action has not been fully elucidated. Therefore, the aim of this study was to investigate the anti-inflammatory mechanism of bee venom in BV2 microglial cells. We first investigated whether NO production in LPS-activated BV2 cells was inhibited by bee venom, and further iNOS mRNA and protein expressions were determined. The mRNA and protein levels of proinflammatory cytokines were examined using semiquantitative RT-PCR and immunoblotting, respectively. Moreover, modulation of the transcription factor NF-κB by bee venom was also investigated using a luciferase assay. LPS-induced NO production in BV2 microglial cells was significantly inhibited in a concentration-dependent manner upon pretreatment with bee venom. Bee venom markedly reduced the mRNA expression of COX-2, TNF-α, IL-1β, and IL-6 and suppressed LPS-induced activation of MyD88 and IRAK1 and phosphorylation of TAK1. Moreover, NF-κB translocation by IKKα/β phosphorylation and subsequent IκB-α degradation were also attenuated. Thus, collectively, these results indicate that bee venom exerts its anti-inflammatory activity via the IRAK1/TAK1/NF-κB signaling pathway. PMID:27563334

  17. Detection of MYD88 L265P and WHIM-like CXCR4 mutation in patients with IgM monoclonal gammopathy related disease.

    PubMed

    Cao, Xin-Xin; Meng, Qi; Cai, Hao; He, Tian-Hua; Zhang, Cong-Li; Su, Wei; Sun, Jian; Li, Yue; Xu, Wei; Zhou, Dao-Bin; Li, Jian

    2017-03-09

    A broad spectrum of diseases are associated with IgM monoclonal gammopathy, including Waldenstrom macroglobulinemia (WM), various types of B cell non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), primary amyloidosis (AL), and monoclonal gammopathy of undetermined significance (MGUS); these are called IgM monoclonal gammopathy related diseases (IgM-RD). We investigated MYD88 L265P and WHIM-like CXCR4 mutations in various IgM-RD. Patients with serum immunofixation electrophoresis confirmed IgM monoclonal gammopathy who had enough material for DNA extraction and presented between January 2008 and October 2016 at Peking Union Medical College Hospital were enrolled in this cohort. We performed real-time allele-specific-polymerase chain reaction and Sanger sequencing to explore the presence of MYD88 L265P and WHIM-like CXCR4 mutations. One hundred and twelve patients (64 male and 48 female patients) were included in this retrospective study. The median age at diagnosis was 62 years (range, 30-84 years). In total, 64 patients (57.1%) carried the MYD88 L265P mutation and 14 patients (12.5%) carried the CXCR4 WHIM-like mutation. We identified the MYD88 L265P somatic variant in cases with WM (39/42), MGUS (8/18), NHL (14/41, including 4/13 diffuse large B cell lymphoma (DLBCL), 1/8 mucosa-associated lymphoid tissue, 3/6 splenic marginal zone lymphoma (SMZL), 1/4 chronic lymphocytic leukemia, 2/3 nodal marginal zone lymphoma (NMZL), 1/2 mantle cell lymphoma, 1 Burkitt lymphoma, and 1 B cell NHL that could not be classified), primary AL (2/2), and IgM-PN (1/1). The mutation was absent in five patients with Cryoglobulinemia, two with primary cold agglutinin disease and one with MM. The CXCR4 WHIM-like mutation was present in 10/42 patients with WM, 3/41 with NHL (1 DLBCL, 1 SMZL, and 1 NMZL), and 1/18 patients with IgM MGUS. Among the patients with NHL, those with the mutated MYD88 L265P genotype were younger and had lower level of IgG and IgA than the patients with the

  18. CARMA1- and MyD88-dependent activation of Jun/ATF-type AP-1 complexes is a hallmark of ABC diffuse large B-cell lymphomas.

    PubMed

    Juilland, Mélanie; Gonzalez, Montserrat; Erdmann, Tabea; Banz, Yara; Jevnikar, Zala; Hailfinger, Stephan; Tzankov, Alexandar; Grau, Michael; Lenz, Georg; Novak, Urban; Thome, Margot

    2016-04-07

    A hallmark of the diffuse large B-cell lymphoma (DLBCL) of the activated B-cell (ABC) type, a molecular subtype characterized by adverse outcome, is constitutive activation of the transcription factor nuclear factor-κB (NF-κB), which controls expression of genes promoting cellular survival and proliferation. Much less, however, is known about the role of the transcription factor activator protein-1 (AP-1) in ABC DLBCL. Here, we show that AP-1, like NF-κB, was controlled by constitutive activation of the B-cell receptor signaling component caspase recruitment domain-containing membrane-associated guanylate kinase 1 (CARMA1) and/or the Toll-like receptor signaling component myeloid differentiation primary response gene 88 (MyD88) in ABC DLBCL cell lines. In contrast to germinal center (GC) B-cell (GCB) DLBCL, ABC DLBCL cell lines expressed high levels of the AP-1 family members c-Jun, JunB, and JunD, which formed heterodimeric complexes with the AP-1 family members activating transcription factor (ATF) 2, ATF3, and ATF7. Inhibition of these complexes by a dominant-negative approach led to impaired growth of a majority of ABC DLBCL cell lines. Individual silencing of c-Jun, ATF2, or ATF3 decreased cellular survival and revealed c-Jun/ATF2-dependent control of ATF3 expression. As a consequence, ATF3 expression was much higher in ABC vs GCB DLBCL cell lines. Samples derived from DLBCL patients showed a clear trend toward high and nuclear ATF3 expression in nodal DLBCL of the non-GC or ABC subtype. These findings identify the activation of AP-1 complexes of the Jun/ATF-type as an important element controlling the growth of ABC DLBCL.

  19. CARMA1- and MyD88-dependent activation of Jun/ATF-type AP-1 complexes is a hallmark of ABC diffuse large B-cell lymphomas

    PubMed Central

    Juilland, Mélanie; Gonzalez, Montserrat; Erdmann, Tabea; Banz, Yara; Jevnikar, Zala; Hailfinger, Stephan; Tzankov, Alexandar; Grau, Michael; Lenz, Georg; Novak, Urban

    2016-01-01

    A hallmark of the diffuse large B-cell lymphoma (DLBCL) of the activated B-cell (ABC) type, a molecular subtype characterized by adverse outcome, is constitutive activation of the transcription factor nuclear factor–κB (NF-κB), which controls expression of genes promoting cellular survival and proliferation. Much less, however, is known about the role of the transcription factor activator protein-1 (AP-1) in ABC DLBCL. Here, we show that AP-1, like NF-κB, was controlled by constitutive activation of the B-cell receptor signaling component caspase recruitment domain-containing membrane-associated guanylate kinase 1 (CARMA1) and/or the Toll-like receptor signaling component myeloid differentiation primary response gene 88 (MyD88) in ABC DLBCL cell lines. In contrast to germinal center (GC) B-cell (GCB) DLBCL, ABC DLBCL cell lines expressed high levels of the AP-1 family members c-Jun, JunB, and JunD, which formed heterodimeric complexes with the AP-1 family members activating transcription factor (ATF) 2, ATF3, and ATF7. Inhibition of these complexes by a dominant-negative approach led to impaired growth of a majority of ABC DLBCL cell lines. Individual silencing of c-Jun, ATF2, or ATF3 decreased cellular survival and revealed c-Jun/ATF2-dependent control of ATF3 expression. As a consequence, ATF3 expression was much higher in ABC vs GCB DLBCL cell lines. Samples derived from DLBCL patients showed a clear trend toward high and nuclear ATF3 expression in nodal DLBCL of the non-GC or ABC subtype. These findings identify the activation of AP-1 complexes of the Jun/ATF-type as an important element controlling the growth of ABC DLBCL. PMID:26747248

  20. Immunological basis of M13 phage vaccine: Regulation under MyD88 and TLR9 signaling.

    PubMed

    Hashiguchi, Shuhei; Yamaguchi, Yuya; Takeuchi, Osamu; Akira, Shizuo; Sugimura, Kazuhisa

    2010-11-05

    Peptide-displaying bacteriophages induce mimotope-specific antibody responses, suggesting a novel application of phage-display library as bacteriophage vaccine. We examined the antibody response against M13 phage in mice induced by an i.p. administration of M13 phage in phosphate-buffered saline. We showed here that firstly, mice showed strong IgG antibody responses, particularly, in IgG2b, IgG2c, and IgG3 subclasses even in primary responses. Secondly, IgG production in primary response is totally dependent on MyD88 signaling. These responses were almost comparable, but slightly weaker, in TLR2-, TLR4- and TLR7-deficient mice relative to wild-type mice, suggesting that this enhancing effect is not due to plausible LPS contamination. Thirdly, although primary IgG1 response was not detected in wild-type mice, remarkable IgG1 response was induced in TLR9-deficient mice, suggesting that TLR9 pathway functions as regulatory, but not a simple augmenting signaling cascade, and furthermore, the enhanced IgG1 response was not due to adjuvant effect of single-stranded DNA derived from M13 phage. Thus, innate immunity including TLR regulation is crucial for M13 phage vaccine design.

  1. SARM1, not MyD88, mediates TLR7/TLR9-induced apoptosis in neurons1

    PubMed Central

    Mukherjee, Piyali; Winkler, Clayton W.; Taylor, Katherine G.; Woods, Tyson A.; Nair, Vinod; Khan, Burhan A.; Peterson, Karin E.

    2015-01-01

    Neuronal apoptosis is a key aspect of many different neurological diseases, but the mechanisms remain unresolved. Recent studies have suggested a mechanism of innate immune-induced neuronal apoptosis that may act through the stimulation of toll-like receptors (TLR) in neurons. TLRs are stimulated both by pathogen associated molecular patterns (PAMPs) as well as by damage-associated molecular patterns (DAMPs), including micro-RNAs released by damaged neurons. In the current study, we identified the mechanism responsible for TLR7/TLR9-mediated neuronal apoptosis. TLR-induced apoptosis required endosomal localization of TLRs but was independent of MyD88 signaling. Instead, apoptosis required the TLR adaptor molecule, sterile alpha armadillo motif (SARM1), which localized to the mitochondria following TLR activation and was associated with mitochondrial accumulation in neurites. Deficiency in SARM1 inhibited both mitochondrial accumulation in neurites and TLR-induced apoptosis. These studies identify a non-MyD88 pathway of TLR7/TLR9 signaling in neurons and provide a mechanism for how innate immune responses in the CNS directly induce neuronal damage. PMID:26423149

  2. Heart-resident CCR2(+) macrophages promote neutrophil extravasation through TLR9/MyD88/CXCL5 signaling.

    PubMed

    Li, Wenjun; Hsiao, Hsi-Min; Higashikubo, Ryuji; Saunders, Brian T; Bharat, Ankit; Goldstein, Daniel R; Krupnick, Alexander S; Gelman, Andrew E; Lavine, Kory J; Kreisel, Daniel

    2016-08-04

    It is well established that maladaptive innate immune responses to sterile tissue injury represent a fundamental mechanism of disease pathogenesis. In the context of cardiac ischemia reperfusion injury, neutrophils enter inflamed heart tissue, where they play an important role in potentiating tissue damage and contributing to contractile dysfunction. The precise mechanisms that govern how neutrophils are recruited to and enter the injured heart are incompletely understood. Using a model of cardiac transplant-mediated ischemia reperfusion injury and intravital 2-photon imaging of beating mouse hearts, we determined that tissue-resident CCR2(+) monocyte-derived macrophages are essential mediators of neutrophil recruitment into ischemic myocardial tissue. Our studies revealed that neutrophil extravasation is mediated by a TLR9/MyD88/CXCL5 pathway. Intravital 2-photon imaging demonstrated that CXCL2 and CXCL5 play critical and nonredundant roles in guiding neutrophil adhesion and crawling, respectively. Together, these findings uncover a specific role for a tissue-resident monocyte-derived macrophage subset in sterile tissue inflammation and support the evolving concept that macrophage ontogeny is an important determinant of function. Furthermore, our results provide the framework for targeting of cell-specific signaling pathways in myocardial ischemia reperfusion injury.

  3. Heart-resident CCR2+ macrophages promote neutrophil extravasation through TLR9/MyD88/CXCL5 signaling

    PubMed Central

    Li, Wenjun; Higashikubo, Ryuji; Saunders, Brian T.; Bharat, Ankit; Goldstein, Daniel R.; Krupnick, Alexander S.; Gelman, Andrew E.; Lavine, Kory J.

    2016-01-01

    It is well established that maladaptive innate immune responses to sterile tissue injury represent a fundamental mechanism of disease pathogenesis. In the context of cardiac ischemia reperfusion injury, neutrophils enter inflamed heart tissue, where they play an important role in potentiating tissue damage and contributing to contractile dysfunction. The precise mechanisms that govern how neutrophils are recruited to and enter the injured heart are incompletely understood. Using a model of cardiac transplant–mediated ischemia reperfusion injury and intravital 2-photon imaging of beating mouse hearts, we determined that tissue-resident CCR2+ monocyte–derived macrophages are essential mediators of neutrophil recruitment into ischemic myocardial tissue. Our studies revealed that neutrophil extravasation is mediated by a TLR9/MyD88/CXCL5 pathway. Intravital 2-photon imaging demonstrated that CXCL2 and CXCL5 play critical and nonredundant roles in guiding neutrophil adhesion and crawling, respectively. Together, these findings uncover a specific role for a tissue-resident monocyte-derived macrophage subset in sterile tissue inflammation and support the evolving concept that macrophage ontogeny is an important determinant of function. Furthermore, our results provide the framework for targeting of cell-specific signaling pathways in myocardial ischemia reperfusion injury. PMID:27536731

  4. Salvianolic Acid B Ameliorates Cerebral Ischemia/Reperfusion Injury Through Inhibiting TLR4/MyD88 Signaling Pathway.

    PubMed

    Wang, Yujue; Chen, Guang; Yu, Xiangdong; Li, Yunchao; Zhang, Li; He, Zongze; Zhang, Nannan; Yang, Xiuping; Zhao, Yansheng; Li, Na; Qiu, Hong

    2016-08-01

    Ischemic stroke can activate multiple transcription factors and cause inflammatory reactions, which involve pattern recognition receptors with immunostimulatory effects. Toll-like receptor 4 (TLR4) is one of the receptors related to innate immunity and several inflammatory reactions. The promising anti- inflammatory activity of salvianolic acid B (SAB) had been previously reported, but its effect on ischemic stroke remains unknown. An oxygen-glucose deprivation and reoxygenation (OGD/R) model in vitro and a middle cerebral artery occlusion (MCAO) model in vivo were used in this paper, and the results showned that SAB remarkably increased the viabilities of PC12 cells and primary cortical neurons after OGD/R injury and notably prevented cerebral ischemia/reperfusion (I/R) injury. SAB also significantly ameliorated NeuN release from primary cortical neurons. Further research indicated that the neuroprotection of SAB was completed through inhibiting the TLR4/MyD88/TRAF6 signaling pathway. The blocking of TLR4 by SAB also restrained NF-kB transcriptional activity and pro-inflammatory cytokine responses (IL-1β, IL-6, and TNF-α). These findings supply a new insight that will aid in clarifying the effect of SAB against cerebral I/R injury and provide the development of SAB as a potential candidate for treating ischemic stroke.

  5. Curcumin Represses NLRP3 Inflammasome Activation via TLR4/MyD88/NF-κB and P2X7R Signaling in PMA-Induced Macrophages

    PubMed Central

    Kong, Fanqi; Ye, Bozhi; Cao, Jiatian; Cai, Xueli; Lin, Lu; Huang, Shanjun; Huang, Weijian; Huang, Zhouqing

    2016-01-01

    Aims: In the NOD-like receptor (NLR) family, the pyrin domain containing 3 (NLRP3) inflammasome is closely related to the progression of atherosclerosis. This study aimed to assess the effects of curcumin on NLRP3 inflammasome in phorbol 12-myristate 13-acetate (PMA)-induced macrophages and explore its underlying mechanism. Methods: Human monocytic THP-1 cells were pretreated with curcumin for 1 h and subsequently induced with PMA for 48 h. Total protein was collected for Western blot analysis. Cytokine interleukin (IL)-1β release and nuclear factor kappa B (NF-κB) p65 translocation were detected by ELISA assay and cellular NF-κB translocation kit, respectively. Results: Curcumin significantly reduced the expression of NLRP3 and cleavage of caspase-1 and IL-1β secretion in PMA-induced macrophages. Moreover, Bay (a NF-κB inhibitor) treatment considerably suppressed the expression of NLRP3 inflammasome in PMA-induced THP-1 cells. Curcumin also markedly inhibited the upregulation of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), phosphorylation level of IκB-α, and activation of NF-κB in PMA-induced macrophages. In addition, purinergic 2X7 receptor (P2X7R) siRNA was administered, and it significantly decreased NLRP3 inflammasome expression in PMA-induced macrophages. Furthermore, curcumin reversed PMA-stimulated P2X7R activation, which further reduced the expression of NLRP3 and cleavage of caspase-1 and IL-1β secretion. Silencing of P2X7R using siRNA also suppressed the activation of NF-κB pathway in PMA-induced macrophages, but P2X7R-silenced cells did not significantly decrease the expression of TLR4 and MyD88. Conclusion: Curcumin inhibited NLRP3 inflammasome through suppressing TLR4/MyD88/NF-κB and P2X7R pathways in PMA-induced macrophages. PMID:27777559

  6. Targeting the genital tract mucosa with a lipopeptide/recombinant adenovirus prime/boost vaccine induces potent and long-lasting CD8+ T cell immunity against herpes: importance of MyD88.

    PubMed

    Zhang, Xiuli; Dervillez, Xavier; Chentoufi, Aziz Alami; Badakhshan, Tina; Bettahi, Ilham; Benmohamed, Lbachir

    2012-11-01

    Targeting of the mucosal immune system of the genital tract with subunit vaccines has failed to induce potent and durable local CD8(+) T cell immunity, which is crucial for protection against many sexually transmitted viral pathogens, including HSV type 2 (HSV-2), which causes genital herpes. In this study, we aimed to investigate the potential of a novel lipopeptide/adenovirus type 5 (Lipo/rAdv5) prime/boost mucosal vaccine for induction of CD8(+) T cell immunity to protect the female genital tract from herpes. The lipopeptide vaccine and the rAdv5 vaccine express the immunodominant HSV-2 CD8(+) T cell epitope (gB(498-505)), and both were delivered intravaginally in the progesterone-induced B6 mouse model of genital herpes. Compared with mice immunized with the homologous lipopeptide/lipopeptide (Lipo/Lipo) vaccine, the Lipo/rAdv5 prime/boost immunized mice 1) developed potent and sustained HSV-specific CD8(+) T cells, detected in both the genital tract draining nodes and in the vaginal mucosa; 2) had significantly lower virus titers; 3) had decreased overt signs of genital herpes disease; and 4) did not succumb to lethal infection (p < 0.005) after intravaginal HSV-2 challenge. Polyfunctional CD8(+) T cells, producing IFN-γ, TNF-α, and IL-2 and exhibiting cytotoxic activity, were associated with protection (p < 0.005). The protective CD8(+) T cell response was significantly compromised in the absence of the adapter MyD88 (p = 0.0001). Taken together, these findings indicate that targeting of the vaginal mucosa with a Lipo/rAdv5 prime/boost vaccine elicits a potent, MyD88-dependent, and long-lasting mucosal CD8(+) T cell protective immunity against sexually transmitted herpes infection and disease.

  7. Alcohol-induced sedation and synergistic interactions between alcohol and morphine: A key mechanistic role for Toll-Like Receptors and MyD88-dependent signalling

    PubMed Central

    Corrigan, Frances; Wu, Yue; Tuke, Jonathan; Coller, Janet K.; Rice, Kenner C.; Diener, Kerrilyn R.; Hayball, John D.; Watkins, Linda R.; Somogyi, Andrew A.; Hutchinson, Mark R.

    2015-01-01

    Increasing evidence demonstrates induction of proinflammatory Toll-like receptor (TLR) 2 and TLR4 signaling by morphine and, TLR4 signaling by alcohol; thus indicating a common site of drug action and a potential novel innate immune-dependent hypothesis for opioid and alcohol drug interactions. Hence, the current study aimed to assess the role of TLR2, TLR4, MyD88 (as a critical TLR-signalling participant), NF-κB, Interleukin-1β (IL-1β; as a downstream proinflammatory effector molecule) and the µ opioid receptor (MOR; as a classical site for morphine action) in acute alcohol-induced sedation (4.5 g/kg) and alcohol (2.5 g/kg) interaction with morphine (5 mg/kg) by assessing the loss of righting reflex (LORR) as a measure of sedation. Wild-type male Balb/c mice and matched genetically-deficient TLR2, TLR4, and MyD88 strains were utilized, together with pharmacological manipulation of MOR, NF-κB, TLR4 and Interleukin-1β. Alcohol induced significant LORR in wild-type mice; this was halved by MyD88 and TLR4 deficiency, and surprisingly nearly completely eliminated by TLR2 deficiency. In contrast, the interaction between morphine and alcohol was found to be MOR-, NF-κB-, TLR2- and MyD88-dependent, but did not involve TLR4 or Interleukin-1β. Morphine-alcohol interactions caused acute elevations in microglial cell counts and NF-κB-p65 positive cells in the motor cortex in concordance with wild-type and TLR2 deficient mouse behavioral data, implicating neuroimmunopharmacological signaling as a pivotal mechanism in this clinically problematic drug-drug interaction. PMID:25542736

  8. Dioscin alleviates alcoholic liver fibrosis by attenuating hepatic stellate cell activation via the TLR4/MyD88/NF-κB signaling pathway

    PubMed Central

    Liu, Min; Xu, Youwei; Han, Xu; Yin, Lianhong; Xu, Lina; Qi, Yan; Zhao, Yanyan; Liu, Kexin; Peng, Jinyong

    2015-01-01

    The present work aimed to investigate the activities and underlying mechanisms of dioscin against alcoholic liver fibrosis (ALF). In vivo liver fibrosis in mice was induced by an alcoholic liquid diet, and in vitro studies were performed on activated HSC-T6 and LX2 cells treated with lipopolysaccharide. Our results showed that dioscin significantly attenuated hepatic stellate cells (HSCs) activation, improved collagen accumulation, and attenuated inflammation through down-regulating the levels of myeloid differentiation factor 88 (MyD88), nuclear factor κB (NF-κB), interleukin (IL)-1, IL-6 and tumour necrosis factor-α by decreasing Toll-like receptor (TLR)4 expression both in vivo and in vitro. TLR4 overexpression was also decreased by dioscin, leading to the markedly down-regulated levels of MyD88, NF-κB, transforming growth factor-β1 (TGF-β1), α-smooth muscle actin (α-SMA) and type I collagen (COL1A1) in cultured HSCs. Suppression of cellular MyD88 by ST2825 or abrogation of NF-κB by pyrrolidine dithiocarbamate eliminated the inhibitory effects of dioscin on the levels of TGF-β1, α-SMA and COL1A1. In a word, dioscin exhibited potent effects against ALF via altering TLR4/MyD88/NF-κB signaling pathway, which provided novel insights into the mechanisms of this compound as an antifibrogenic candidate for the treatment of ALF in the future. PMID:26655640

  9. Blood-Brain Barrier Deterioration and Hippocampal Gene Expression in Polymicrobial Sepsis: An Evaluation of Endothelial MyD88 and the Vagus Nerve.

    PubMed

    Honig, Gerard; Mader, Simone; Chen, Huiyi; Porat, Amit; Ochani, Mahendar; Wang, Ping; Volpe, Bruce T; Diamond, Betty

    2016-01-01

    Systemic infection can initiate or exacerbate central nervous system (CNS) pathology, even in the absence of overt invasion of bacteria into the CNS. Recent epidemiological studies have demonstrated that human survivors of sepsis have an increased risk of long-term neurocognitive decline. There is thus a need for improved understanding of the physiological mechanisms whereby acute sepsis affects the CNS. In particular, MyD88-dependent activation of brain microvascular endothelial cells and a resulting loss of blood-brain barrier integrity have been proposed to play an important role in the effects of systemic inflammation on the CNS. Signaling through the vagus nerve has also been considered to be an important component of CNS responses to systemic infection. Here, we demonstrate that blood-brain barrier permeabilization and hippocampal transcriptional responses during polymicrobial sepsis occur even in the absence of MyD88-dependent signaling in cerebrovascular endothelial cells. We further demonstrate that these transcriptional responses can occur without vagus nerve input. These results suggest that redundant signals mediate CNS responses in sepsis. Either endothelial or vagus nerve activation may be individually sufficient to transmit systemic inflammation to the central nervous system. Transcriptional activation in the forebrain in sepsis may be mediated by MyD88-independent endothelial mechanisms or by non-vagal neuronal pathways.

  10. miR-489 inhibits silica-induced pulmonary fibrosis by targeting MyD88 and Smad3 and is negatively regulated by lncRNA CHRF

    PubMed Central

    Wu, Qiuyun; Han, Lei; Yan, Weiwen; Ji, Xiaoming; Han, Ruhui; Yang, Jingjin; Yuan, Jiali; Ni, Chunhui

    2016-01-01

    Silicosis is an incurable occupational disease associated with inflammation, fibroblast proliferation and the accumulation of extracellular matrix in lung tissues. The dysregulation of lncRNAs and miRNAs has been implicated in many complex diseases; however, the current understanding of their roles in fibrotic lung diseases, especially silicosis, remains limited. Our previous microRNA (miRNA, miR) microarray data have indicated decreased expression levels of miR-489 in lung tissues of silica-induced pulmonary fibrosis. Here, we further explored the role of miR-489 in a mouse model of silicosis. Interestingly, miR-489 levels were reduced in both macrophages that were exposed to silica and fibroblasts that were exposed to TGF-β1. Additionally, the overexpressed miR-489 carried out its anti-fibrotic role by attenuating inflammation and fibrotic progression in vivo. Our molecular study further demonstrated that miR-489 inhibited silica-induced pulmonary fibrosis primarily by repressing its target genes MyD88 and Smad3. Moreover, the up-regulated lncRNA cardiac hypertrophy-related factor (CHRF) reversed the inhibitory effect of miR-489 on MyD88 and Smad3 and then triggered the inflammation and fibrotic signaling pathways. Overall, our data indicate that the CHRF-miR-489-MyD88 Smad3 signaling axis exerts key functions in silica-induced pulmonary fibrosis and may represent a therapeutic target for silicosis. PMID:27506999

  11. LPS-Stimulated Inflammatory Environment Inhibits BMP-2-Induced Osteoblastic Differentiation Through Crosstalk Between TLR4/MyD88/NF-κB and BMP/Smad Signaling

    PubMed Central

    Huang, Ru-Lin; Yuan, Yuwen; Zou, Gang-Ming; Liu, Guangwang; Tu, Jun

    2014-01-01

    Abstract Bone morphogenetic protein-2 (BMP-2) is a novel differentiation factor that is capable of inducing osteoblast differentiation and bone formation, making it an attractive option in treatment of bone defects, fractures, and spine fusions. Inflammation, which was a common situation during bone healing, is recognized to inhibit osteogenic differentiation and bone formation. However, the effect of inflammation on BMP-2-induced osteoblastic differentiation remains ambiguous. In this study, we showed that an inflammatory environment triggered by lipopolysaccharide (LPS) in vitro would suppress BMP-2-induced osteogenic differentiation of bone marrow mesenchymal stem cells, which represented by decreased alkaline phosphatase (ALPase) activity and down-regulated osteogenic genes. In addition, LPS activated nuclear factor-κB (NF-κB) via a TLR4/MyD88-dependent manner and inhibited BMP-2-induced phosphorylation and nuclear translocation of Smad1/5/8. The blocking of NF-κB signaling by pretreatment with specific inhibitors such as BAY-11-7082, TPCK and PDTC, or by transfection with plasmids encoding p65 siRNA or IκBα siRNA could significantly reverse the inhibitory effect of LPS on BMP-2-induced BMP/Smad signaling and osteogenic differentiation. By contrast, even without stimulation of LPS, overexpression of p65 gene showed obvious inhibitory effects on BMP-2-induced BMP/Smad signaling and ALPase activity. These data indicate that the LPS-mediated inflammatory environment inhibits BMP-2-induced osteogenic differentiation, and that the crosstalk between TLR4/MyD88/NF-κB and BMP/Smad signaling negatively modulates the osteoinductive capacity of BMP-2. PMID:24050190

  12. Dexmedetomidine preconditioning may attenuate myocardial ischemia/reperfusion injury by down-regulating the HMGB1-TLR4-MyD88-NF-кB signaling pathway

    PubMed Central

    Zhang, Juan; Meng, Xiao-wen; Ji, Fu-hai

    2017-01-01

    Aims To investigate whether dexmedetomidine (DEX) preconditioning could alleviate the inflammation caused by myocardial ischemia/reperfusion (I/R) injury by reducing HMGB1-TLR4-MyD88-NF-кB signaling. Methods Seventy rats were randomly assigned into five groups: sham group, myocardial I/R group (I/R), DEX+I/R group (DEX), DEX+yohimbine+I/R group (DEX/YOH), and yohimbine+I/R group (YOH). Animals were subjected to 30 min of ischemia induced by occluding the left anterior descending artery followed by 120 min of reperfusion. Myocardial infarct size and histological scores were evaluated. The levels of IL-6 and TNF-α in serum and myocardium were quantified by enzyme-linked immunosorbent assay, and expression of HMGB1, TLR4, MyD88, IκB and NF-κB in the myocardial I/R area were determined with Western blot and immunocytochemistry. Results Myocardial infarct sizes, histological scores, levels of circulating and myocardial IL-6 and TNF-α, the expression of HMGB1, TLR4, MyD88 and NF-κB, and the degradation of IκB were significantly increased in the I/R group compared with the sham group (P<0.01). DEX preconditioning significantly reduced the myocardial infarct size and histological scores (P<0.01 vs. I/R group). Similarly, the serum and myocardial levels of IL-6 and TNF-α, the expression of HMGB1, TLR4, MyD88 and NF-κB, and the degradation of IκB were significantly reduced in the DEX group (P<0.01 vs. I/R group). These effects were partly reversed by yohimbine, a selective α2-adrenergic receptor antagonist, while yohimbine alone had no significant effect on any of the above indicators. Conclusion DEX preconditioning reduces myocardial I/R injury in part by attenuating inflammation, which may be attributed to the downregulation of the HMGB1-TLR4-MyD88-NF-кB signaling pathway mediated by the α2-adrenergic receptor activation. PMID:28222157

  13. The HIV Protease Inhibitor Saquinavir Inhibits HMGB1-Driven Inflammation by Targeting the Interaction of Cathepsin V with TLR4/MyD88

    PubMed Central

    Pribis, John P; Al-Abed, Yousef; Yang, Huan; Gero, Domokos; Xu, Hongbo; Montenegro, Marcelo F; Bauer, Eileen M; Kim, Sodam; Chavan, Sangeeta S; Cai, Changchun; Li, Tunliang; Szoleczky, Petra; Szabo, Csaba; Tracey, Kevin J; Billiar, Timothy R

    2015-01-01

    Extracellular high-mobility group box 1 (HMGB1) (disulfide form), via activation of toll-like receptor 4 (TLR4)-dependent signaling, is a strong driver of pathologic inflammation in both acute and chronic conditions. Identification of selective inhibitors of HMGB1-TLR4 signaling could offer novel therapies that selectively target proximal endogenous activators of inflammation. A cell-based screening strategy led us to identify first generation HIV-protease inhibitors (PI) as potential inhibitors of HMGB1-TLR4 driven cytokine production. Here we report that the first-generation HIV-PI saquinavir (SQV), as well as a newly identified mammalian protease inhibitor STO33438 (334), potently block disulfide HMGB1-induced TLR4 activation, as assayed by the production of TNF-α by human monocyte-derived macrophages (THP-1). We further report on the identification of mammalian cathepsin V, a protease, as a novel target of these inhibitors. Cellular as well as recombinant protein studies show that the mechanism of action involves a direct interaction between cathepsin V with TLR4 and its adaptor protein MyD88. Treatment with SQV, 334 or the known cathepsin inhibitor SID26681509 (SID) significantly improved survival in murine models of sepsis and reduced liver damage following warm liver ischemia/reperfusion (I/R) models, both characterized by strong HMGB1-TLR4 driven pathology. The current study demonstrates a novel role for cathepsin V in TLR4 signaling and implicates cathepsin V as a novel target for first-generation HIV-PI compounds. The identification of cathepsin V as a target to block HMGB1-TLR4-driven inflammation could allow for a rapid transition of the discovery from the bench to the bedside. Disulfide HMGB1 drives pathologic inflammation in many models by activating signaling through TLR4. Cell-based screening identified the mammalian protease cathepsin V as a novel therapeutic target to inhibit TLR4-mediated inflammation induced by extracellular HMGB1 (disulfide

  14. L265P Mutation of the MYD88 Gene Is Frequent in Waldenström’s Macroglobulinemia and Its Absence in Myeloma

    PubMed Central

    Mori, Naoki; Ohwashi, Mari; Yoshinaga, Kentaro; Mitsuhashi, Kenjiro; Tanaka, Norina; Teramura, Masanao; Okada, Michiko; Shiseki, Masayuki; Tanaka, Junji; Motoji, Toshiko

    2013-01-01

    L265P mutation in the MYD88 gene has recently been reported in Waldenström’s macroglobulinemia; however the incidence has been different according to the methods used. To determine the relevance and compare the incidence by different methods, we analyzed the L265P mutation in bone marrow mononuclear cells from lymphoid neoplasms. We first performed cloning and sequencing in 10 patients: 8 Waldenström’s macroglobulinemia; 1 non-IgM-secreting lymphoplasmacytic lymphoma; and 1 low grade B-cell lymphoma with monoclonal IgG protein. The L265P mutation was detected in only 1/8 Waldenström’s macroglobulinemia patients (2 of 9 clones). To confirm these results, direct sequencing was performed in the 10 patients and an additional 17 Waldenström’s macroglobulinemia patients and 1 lymphoplasmacytic lymphoma patient. Nine of 28 patients (7/25 Waldenström’s macroglobulinemia, 1/2 lymphoplasmacytic lymphoma, and B-cell lymphoma) harbored the mutation. We next tested for the mutation with BSiE1 digestion and allele-specific polymerase chain reaction in the 28 patients and 38 patients with myeloma. Aberrant bands corresponding to the mutation were detected by BSiE1 digestion in 19/25 patients with Waldenström’s macroglobulinemia (76%), 1/2 lymphoplasmacytic lymphoma and B-cell lymphoma, but not in the 38 myeloma patients. The L265P mutation was more frequent in patients with Waldenström’s macroglobulinemia than in those with myeloma (p=1.3x10-10). The mutation was detected by allele-specific polymerase chain reaction in 18/25 Waldenström’s macroglobulinemia patients (72%). In the 25 Waldenström’s macroglobulinemia patients, the L265P was more frequently detected by BSiE1 digestion than by direct sequencing (p=5.3x10-4), and in males (15/16, 94%) than in females (4/9, 44%) (p=1.2x10-2). No siginificant difference was observed in the incidence of the L265P mutation between BSiE1 digestion and allele-specific polymerase chain reaction (p=0.32). These

  15. Card9- and MyD88-Mediated Gamma Interferon and Nitric Oxide Production Is Essential for Resistance to Subcutaneous Coccidioides posadasii Infection.

    PubMed

    Hung, Chiung-Yu; Castro-Lopez, Natalia; Cole, Garry T

    2016-04-01

    Coccidioidomycosis is a potentially life-threatening respiratory disease which is endemic to the southwestern United States and arid regions of Central and South America. It is responsible for approximately 150,000 infections annually in the United States alone. Almost every human organ has been reported to harbor parasitic cells of Coccidioides spp. in collective cases of the disseminated form of this mycosis. Current understanding of the mechanisms of protective immunity against lung infection has been largely derived from murine models of pulmonary coccidioidomycosis. However, little is known about the nature of the host response to Coccidioides in extrapulmonary tissue. Primary subcutaneous coccidioidal infection is rare but has been reported to result in disseminated disease. Here, we show that activation of MyD88 and Card9 signal pathways are required for resistance to Coccidioides infection following subcutaneous challenge of C57BL/6 mice, which correlates with earlier findings of the protective response to pulmonary infection. MyD88(-/-) andCard9(-/-) mice recruited reduced numbers of T cells, B cells, and neutrophils to the Coccidioides-infected hypodermis com pared to wild-type mice; however, neutrophils were dispensable for resistance to skin infection. Further studies have shown that gamma interferon (IFN-γ) production and activation of Th1 cells characterize resistance to subcutaneous infection. Furthermore, activation of a phagosomal enzyme, inducible nitric oxide synthase, which is necessary for NO production, is a requisite for fungal clearance in the hypodermis. Collectively, our data demonstrate that MyD88- and Card9-mediated IFN-γ and nitric oxide production is essential for protection against subcutaneous Coccidioides infection.

  16. Salmonid Tollip and MyD88 factors can functionally replace their mammalian orthologues in TLR-mediated trout SAA promoter activation.

    PubMed

    Rebl, Alexander; Rebl, Henrike; Liu, Shuzhen; Goldammer, Tom; Seyfert, Hans-Martin

    2011-01-01

    Many functional details of the piscine Toll-like receptor (TLR) signal-mediated activation of immune defense are still elusive. We used an established reconstitution system of mammalian TLR signaling to examine if this system would allow for pathogen-dependent promoter activation of the serum amyloid A (SAA)-encoding gene from rainbow trout (Oncorhynchus mykiss) and if the key mediators MyD88 and Tollip from trout can functionally substitute for their mammalian orthologues. Cells of the established human embryonic kidney line HEK-293 were transiently co-transfected with vectors expressing bovine TLR2 or TLR4 factors and a reporter gene driven by the promoter of the trout SAA gene. Escherichia coli stimulation increased reporter gene expression more than 3-fold. Deletion series and point mutations identified in the proximal SAA promoter a composite overlapping binding site for NF-κB and CEBP factors as crucial for promoter activation. Overexpression of NF-κB p65, but not of p50 or different members of the CEBP factor family proved this factor as an essential driver for SAA expression. Overexpression of a transdominant-negative mutant of the trout MyD88 factor reduced TLR-mediated SAA promoter activation confirming functional conservation of its TIR domain. Overexpression of the Tollip factor from trout also quenched TLR-mediated NF-κB and TLR4-mediated SAA promoter activation. The MyD88 mutant and Tollip expression studies confirm the functional homology of both piscine factors and their mammalian counterparts. We provide for the first time evidence that also the Tollip-mediated negative loop of TLR signaling may be conserved in non-mammalian organisms.

  17. MYD88 L265P in Waldenström macroglobulinemia, immunoglobulin M monoclonal gammopathy, and other B-cell lymphoproliferative disorders using conventional and quantitative allele-specific polymerase chain reaction

    PubMed Central

    Xu, Lian; Hunter, Zachary R.; Yang, Guang; Zhou, Yangsheng; Cao, Yang; Liu, Xia; Morra, Enrica; Trojani, Alessandra; Greco, Antonino; Arcaini, Luca; Varettoni, Maria; Brown, Jennifer R.; Tai, Yu-Tzu; Anderson, Kenneth C.; Munshi, Nikhil C.; Patterson, Christopher J.; Manning, Robert J.; Tripsas, Christina K.; Lindeman, Neal I.

    2013-01-01

    By whole-genome and/or Sanger sequencing, we recently identified a somatic mutation (MYD88 L265P) that stimulates nuclear factor κB activity and is present in >90% of Waldenström macroglobulinemia (WM) patients. MYD88 L265P was absent in 90% of immunoglobulin M (IgM) monoclonal gammopathy of undetermined significance (MGUS) patients. We therefore developed conventional and real-time allele-specific polymerase chain reaction (AS-PCR) assays for more sensitive detection and quantification of MYD88 L265P. Using either assay, MYD88 L265P was detected in 97 of 104 (93%) WM and 13 of 24 (54%) IgM MGUS patients and was either absent or rarely expressed in samples from splenic marginal zone lymphoma (2/20; 10%), CLL (1/26; 4%), multiple myeloma (including IgM cases, 0/14), and immunoglobulin G MGUS (0/9) patients as well as healthy donors (0/40; P < 1.5 × 10−5 for WM vs other cohorts). Real-time AS-PCR identified IgM MGUS patients progressing to WM and showed a high rate of concordance between MYD88 L265P ΔCT and BM disease involvement (r = 0.89, P = .008) in WM patients undergoing treatment. These studies identify MYD88 L265P as a widely present mutation in WM and IgM MGUS patients using highly sensitive and specific AS-PCR assays with potential use in diagnostic discrimination and/or response assessment. The finding of this mutation in many IgM MGUS patients suggests that MYD88 L265P may be an early oncogenic event in WM pathogenesis. PMID:23321251

  18. Upregulation of chicken TLR4, TLR15 and MyD88 in heterophils and monocyte-derived macrophages stimulated with Eimeria tenella in vitro.

    PubMed

    Zhou, Zuoyong; Wang, Zhiying; Cao, Liting; Hu, Shijun; Zhang, Ze; Qin, Bo; Guo, Zhili; Nie, Kui

    2013-04-01

    Coccidiosis, caused by Eimeria parasites, is a major parasitic disease responsible for great economic losses in the poultry industry. Toll-like receptor (TLR) family is one of the most important innate immune receptors, which involved in pathogen detection by initiating host responses, and it plays important roles in the reduction and clearance of pathogens. Very little information is available about the roles of chicken TLRs (ChTLRs) during Eimeria tenella infection. In the current study, mRNA expression of ChTLRs and associated signal adaptors in heterophils and monocyte-derived macrophages stimulated with E. tenella in vitro were measured by real-time quantitative polymerase chain reaction. The results showed that ChTLR4 and ChTLR15 expression were increased significantly in heterophils and monocyte-derived macrophages following live E. tenella sporozoites stimulation. The heat-killed E. tenella sporozoites stimulated higher expression of ChTLRs and signal adaptors than live sporozoites, the expression of ChTLR4, ChTLR15 and MyD88 in heterophils and monocyte-derived macrophages stimulated with heat-killed E. tenella sporozoites were up-regulated significantly than unstimulated cells. The results suggest that ChTLR4 and ChTLR15 are involved in response to E. tenella infection, and may operate in a MyD88-dependent manner for host defense.

  19. Protective Effects of Celastrol on Diabetic Liver Injury via TLR4/MyD88/NF-κB Signaling Pathway in Type 2 Diabetic Rats

    PubMed Central

    Han, Li-ping; Li, Chun-jun; Sun, Bei; Xie, Yun; Guan, Yue; Ma, Ze-jun; Chen, Li-ming

    2016-01-01

    Immune and inflammatory pathways play a central role in the pathogenesis of diabetic liver injury. Celastrol is a potent immunosuppressive and anti-inflammatory agent. So far, there is no evidence regarding the mechanism of innate immune alterations of celastrol on diabetic liver injury in type 2 diabetic animal models. The present study was aimed at investigating protective effects of celastrol on the liver injury in diabetic rats and at elucidating the possible involved mechanisms. We analyzed the liver histopathological and biochemical changes and the expressions of TLR4 mediated signaling pathway. Compared to the normal control group, diabetic rats were found to have obvious steatohepatitis and proinflammatory cytokine activities were significantly upregulated. Celastrol-treated diabetic rats show reduced hepatic inflammation and macrophages infiltration. The expressions of TLR4, MyD88, NF-κB, and downstream inflammatory factors IL-1β and TNFα in the hepatic tissue of treated rats were downregulated in a dose-dependent manner. We firstly found that celastrol treatment could delay the progression of diabetic liver disease in type 2 diabetic rats via inhibition of TLR4/MyD88/NF-κB signaling cascade pathways and its downstream inflammatory effectors. PMID:27057550

  20. Bovine viral diarrhea virus type 2 in vivo infection modulates TLR4 responsiveness in differentiated myeloid cells which is associated with decreased MyD88 expression.

    PubMed

    Schaut, Robert G; McGill, Jodi L; Neill, John D; Ridpath, Julia F; Sacco, Randy E

    2015-10-02

    Symptoms of bovine viral diarrhea virus (BVDV) infection range from subclinical to severe, depending on strain virulence. Several in vitro studies showed BVDV infection impaired leukocyte function. Fewer studies have examined the effects of in vivo BVDV infection on monocyte/macrophage function, especially with strains of differing virulence. We characterized cytokine production by bovine myeloid cells isolated early or late in high (HV) or low virulence (LV) BVDV2 infection. Given BVDV infection may enhance susceptibility to secondary bacterial infection, LPS responses were examined as well. Monocytes from HV and LV infected calves produced higher levels of cytokines compared to cells from controls. In contrast, monocyte-derived macrophage cytokine levels were generally reduced. Modulated cytokine expression in HV BVDV2 macrophages was associated with decreased MyD88 expression, likely due to its interaction with viral NS5A. These data and those of others, suggest that certain Flaviviridae may have evolved strategies for subverting receptor signaling pathways involving MyD88.

  1. MyD88 mediates the protective effects of probiotics against the arteriolar thrombosis and leukocyte recruitment associated with experimental colitis.

    PubMed

    Souza, Daniele G; Senchenkova, Elena Y; Russell, Janice; Granger, D Neil

    2015-04-01

    Several studies in patients with IBD and in animal models of IBD have revealed a protective effect of probiotics in reducing clinical symptoms of disease and in blunting the gut inflammation that accompanies this condition. However, the mechanism underlying the therapeutic effect of probiotics is currently unknown. Furthermore, the ability of probiotics to influence the enhanced thrombus development that accompanies IBD has not been studied. This study addresses whether the enhanced extraintestinal thrombosis (induced by light/dye injury) associated with experimental colitis is altered by oral treatment with the probiotic preparation VSL#3 or by the absence of microbiota. Colitis was induced by DSS 3% in Swiss Webster mice, germ-free mice, C57BL/6 WT, or Myd88 mice. In some experiments, mice received VSL#3 for 8 days before and during DSS feeding. Swiss Webster mice were also subjected to a chronic model of DSS colitis, and the effect of VSL#3 was evaluated. VSL#3 treatment significantly attenuated the accelerated thrombus formation observed in both acute and chronic models of colitis. VSL#3-treated mice also exhibited attenuated inflammatory response and injury in the colon. The protective effects of VSL#3 on colitis-associated thrombogenesis and inflammation were not evident in MyD88-deficient mice. Our results suggest that improved control of the enteric microflora in IBD may afford protection against the hypercoagulable prothrombotic state that follows this condition.

  2. MyD88 mediates the protective effects of probiotics against the arteriolar thrombosis and leukocyte recruitment associated with experimental colitis

    PubMed Central

    Souza, Daniele G.; Senchenkova, Elena Y.; Russell, Janice; Granger, D. Neil

    2014-01-01

    Several studies in IBD patients and in animal models of IBD have revealed a protective effect of probiotics in reducing clinical symptoms of disease and in blunting the gut inflammation that accompanies this condition. However, the mechanism underlying the therapeutic effect of probiotics is currently unknown. Furthermore, the ability of probiotics to influence the enhanced thrombus development that accompanies IBD has not been studied. This study addresses whether the enhanced extra-intestinal thrombosis (induced by light/dye injury) associated with experimental colitis is altered by oral treatment with the probiotic preparation VSL#3 or by the absence of microbiota. Colitis was induced by DSS 3% in Swiss Webster mice, germ free mice, C57BL/6 WT or Myd88−/− mice. In some experiments, mice received VSL#3 for 8 days before and during DSS feeding. Swiss Webster mice were also subjected to a chronic model of DSS colitis and the effect of VSL#3 was evaluated. VSL#3 treatment significantly attenuated the accelerated thrombus formation observed in both acute and chronic models of colitis. VSL#3-treated mice also exhibited attenuated inflammatory response and injury in the colon. The protective effects of VSL#3 on colitis-associated thrombogenesis and inflammation were not evident in MyD88-deficient mice. Our results suggest that improved control of the enteric microflora in IBD may afford protection against the hypercoagulable, prothrombotic state that follows this condition. PMID:25738377

  3. TLR4-MyD88/Mal-NF-kB axis is involved in infection of HSV-2 in human cervical epithelial cells.

    PubMed

    Liu, Hongya; Chen, Kai; Feng, Wenqiang; Wu, Xinxing; Li, Hui

    2013-01-01

    We have established an in vitro HSV-2 acute infection model with Human cervical epithelial (HCE cells, the primary target and natural host cells for HSV-2) to investigate the role of TLRs-mediated innate immune response to HSV-2. In current study, we found that HSV-2 infection induced activity of NF-kB reporter and expression of cytokines are TLR4-dependent using approaches with shRNA and TLR4 antagonist. Knockdown experiments demonstrated that the adaptor molecules MyD88 and Mal of the TLRs signaling pathway are required in the HSV-2 induced TLR4-dependent NF-kB activation in HCE cells. Western blot assay suggested that knockdown of TLR4 decreased the phosphorylation of IRAK1 and inhibitor of NF-kB (IkB-α) upon HSV-2 infection. Finally, decreased expression of either TLR4 or MyD88/Mal alone or both significantly abolished productions of IL-6 and IFN-β by ELISA analysis. Taken together, our results from the in vitro infection model reveal for the first time that there exists the pathway via TLR4-Mal/MyD88-IRAK1-NF-kB axis in human cervical epithelial cells in response to HSV-2 infection.

  4. Toll-pathway in tiger shrimp (Penaeus monodon) responds to white spot syndrome virus infection: evidence through molecular characterisation and expression profiles of MyD88, TRAF6 and TLR genes.

    PubMed

    Deepika, A; Sreedharan, K; Paria, Anutosh; Makesh, M; Rajendran, K V

    2014-12-01

    The Toll-pathway plays key roles in regulating the innate immune response in invertebrates. Myeloid differentiation factor 88 (MyD88) and Tumour necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) are key molecules in this signalling pathway. To investigate the role of Toll-pathway in innate immune response of shrimp, Penaeus monodon, MyD88 (PmMyD88) and TRAF6 (PmTRAF6) were identified and characterised. PmMyD88 cDNA is 1716 bp long with an open reading frame (ORF) of 1449 bp encoding a putative protein of 482 amino acids, with a death domain, a TIR domain and C-terminal extension domain. PmTRAF6 cDNA is 2563 bp long with an ORF of 1785 bp (594 amino acids) with an N-terminal RING-type zinc finger domain, two TRAF-type zinc finger domains, a coiled region and a MATH domain. In healthy shrimp, PmMyD88, PmTRAF6 and PmToll were detected in 15 tissues with the highest expression in midgut, eyestalk and lymphoid organ, respectively. Responses of these genes to WSSV in experimentally-infected P. monodon as well as in cultured haemocytes and also effect of poly I:C on the gene expression in vitro was investigated at six time-points in seven tissues. PmToll showed significant up-regulation at all time-points of infection in six tissues and until 24 h post-infection in vitro. However, poly I:C-induced haemocytes showed up-regulation of the gene until 48 h post-exposure. WSSV caused significant up-regulation of PmMyD88 in most of the tissues tested. The virus challenge as well as poly I:C induction in vitro also resulted in significant up-regulation of the gene. Up-regulated expression of PmTRAF6 was detected in haemocytes and lymphoid organ at late stage of infection. In vitro virus challenge showed significant up-regulation of PmTRAF6 at almost all time-points whereas no significant change in the expression was observed on poly I:C induction. The responses of these key genes, observed in the present study, suggest that Toll-pathway as a whole may play a crucial

  5. Kaposi's sarcoma-associated herpesvirus microRNAs target IRAK1 and MYD88, two components of the toll-like receptor/interleukin-1R signaling cascade, to reduce inflammatory-cytokine expression.

    PubMed

    Abend, Johanna R; Ramalingam, Dhivya; Kieffer-Kwon, Philippe; Uldrick, Thomas S; Yarchoan, Robert; Ziegelbauer, Joseph M

    2012-11-01

    Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is the causative agent of KS, an important AIDS-associated malignancy. KSHV expresses at least 18 different mature microRNAs (miRNAs). We identified interleukin-1 receptor (IL-1R)-associated kinase 1 (IRAK1) as a potential target of miR-K12-9 (miR-K9) in an array data set examining changes in cellular gene expression levels in the presence of KSHV miRNAs. Using 3'-untranslated region (3'UTR) luciferase reporter assays, we confirmed that miR-K9 and other miRNAs inhibit IRAK1 expression. In addition, IRAK1 expression is downregulated in cells transfected with miR-K9 and during de novo KSHV infection. IRAK1 is an important component of the Toll-like receptor (TLR)/IL-1R signaling cascade. The downregulation of IRAK1 by miR-K9 resulted in the decreased stimulation of NF-κB activity in endothelial cells treated with IL-1α and in B cells treated with a TLR7/8 agonist. Interestingly, miR-K9 had a greater effect on NF-κB activity than did a small interfering RNA (siRNA) targeting IRAK1 despite the more efficient downregulation of IRAK1 expression with the siRNA. We hypothesized that KSHV miRNAs may also be regulating a second component of the TLR/IL-1R signaling cascade, resulting in a stronger phenotype. Reanalysis of the array data set identified myeloid differentiation primary response protein 88 (MYD88) as an additional potential target. 3'UTR luciferase reporter assays and Western blot analysis confirmed the targeting of MYD88 by miR-K5. The presence of miR-K9 and miR-K5 inhibited the production of IL-6 and IL-8 upon the IL-1α stimulation of endothelial cells. These results demonstrate KSHV-encoded miRNAs regulating the TLR/IL-1R signaling cascade at two distinct points and suggest the importance of these pathways during viral infection.

  6. MyD88 dependence of beryllium-induced dendritic cell trafficking and CD4⁺ T-cell priming.

    PubMed

    McKee, A S; Mack, D G; Crawford, F; Fontenot, A P

    2015-11-01

    Beryllium exposure results in beryllium hypersensitivity in a subset of exposed individuals, leading to granulomatous inflammation and fibrosis in the lung. In addition to its antigenic properties, beryllium has potent adjuvant activity that contributes to sensitization via unknown pathways. Here we show that beryllium induces cellular death and release of interleukin (IL)-1α and DNA into the lung. Release of IL-1α was inflammasome independent and required for beryllium-induced neutrophil recruitment into the lung. Beryllium enhanced classical dendritic cell (cDC) migration from the lung to draining lymph nodes (LNs) in an IL-1R-independent manner, and the accumulation of activated cDCs in the LN was associated with increased priming of CD4(+) T cells. DC migration was reduced in Toll-like receptor 9 knockout (TLR9KO) mice; however, cDCs in the LNs of TLR9-deficient mice were highly activated, suggesting a role for more than one innate receptor in the effects on DCs. The adjuvant effects of beryllium on CD4(+) T-cell priming were similar in wild-type, IL-1R-, caspase-1-, TLR2-, TLR4-, TLR7-, and TLR9-deficient mice. In contrast, DC migration, activation, and the adjuvant effects of beryllium were significantly reduced in myeloid differentiation primary response gene 88 knockout (MyD88KO) mice. Collectively, these data suggest that beryllium exposure results in the release of damage-associated molecular patterns that engage MyD88-dependent receptors to enhance pulmonary DC function.

  7. HMGB1 translocation and release mediate cigarette smoke–induced pulmonary inflammation in mice through a TLR4/MyD88-dependent signaling pathway

    PubMed Central

    Cheng, Yao; Wang, Dan; Wang, Bin; Li, Huanan; Xiong, Junjie; Xu, Shuyun; Chen, Quan; Tao, Kun; Yang, Xiaoyan; Zhu, Yu; He, Sirong

    2017-01-01

    We performed studies to determine the role of high-mobility group box 1 (HMGB1) in cigarette smoke (CS)–induced pulmonary inflammation. After mice were exposed to five cigarettes four times a day for 3 d, toll-like receptor 4 (TLR4) expression and TLR4-mediated signaling were significantly up-regulated, and HMGB1 had translocated from the nucleus to the cytoplasm in lung epithelial cells and then been released into the extracellular lung space. On CS exposure, inflammatory cell recruitment and proinflammatory cytokine production were significantly increased in lung tissue and bronchoalveolar lavage, and these effects depended on the TLR4 signaling pathway. HMGB1 inhibition decreased the CS-induced inflammatory response, whereas treatment with exogenous HMGB1 aggravated the damage and increased the phosphorylation of JNK, p38, and IκBα in the lungs of wild-type mice but not in TLR4-knockout mice. Blockade of TLR4 action or TLR4 knockout significantly inhibited HMGB1-induced proinflammatory cytokine production in mouse tracheal epithelial (MTE) cells and lung tissues. In addition, a MyD88 deficiency inhibited JNK, p38, and IκBα phosphorylation, and this effect was associated with the suppressed production of TNF-α and IL-1β in MTE cells and lung tissues in response to CS stimulation. Thus HMGB1 activates the NF-κB and JNK/p38 pathways through TLR4/MyD88-dependent signaling and induces an inflammatory response in lungs exposed to CS. PMID:27807045

  8. Human Pancreatic Cancer Cells Induce a MyD88-Dependent Stromal Response to Promote a Tumor-Tolerant Immune Microenvironment.

    PubMed

    Delitto, Daniel; Delitto, Andrea E; DiVita, Bayli B; Pham, Kien; Han, Song; Hartlage, Emily R; Newby, Brittney N; Gerber, Michael H; Behrns, Kevin E; Moldawer, Lyle L; Thomas, Ryan M; George, Thomas J; Brusko, Todd M; Mathews, Clayton E; Liu, Chen; Trevino, Jose G; Hughes, Steven J; Wallet, Shannon M

    2017-02-01

    Cancer cells exert mastery over the local tumor-associated stroma (TAS) to configure protective immunity within the tumor microenvironment. The immunomodulatory character of pancreatic lysates of patients with cancer differs from those with pancreatitis. In this study, we evaluated the cross-talk between pancreatic cancer and its TAS in primary human cell culture models. Upon exposure of TAS to pancreatic cancer cell-conditioned media, we documented robust secretion of IL6 and IL8. This TAS response was MyD88-dependent and sufficient to directly suppress both CD4(+) and CD8(+) T-cell proliferation, inducing Th17 polarization at the expense of Th1. We found that patients possessed a similar shift in circulating effector memory Th17:Th1 ratios compared with healthy controls. The TAS response also directly suppressed CD8(+) T-cell-mediated cytotoxicity. Overall, our results demonstrate how TAS contributes to the production of an immunosuppressive tumor microenvironment in pancreatic cancer. Cancer Res; 77(3); 672-83. ©2016 AACR.

  9. Low-intensity pulsed ultrasound (LIPUS) inhibits LPS-induced inflammatory responses of osteoblasts through TLR4-MyD88 dissociation.

    PubMed

    Nakao, Juna; Fujii, Yasuyuki; Kusuyama, Joji; Bandow, Kenjiro; Kakimoto, Kyoko; Ohnishi, Tomokazu; Matsuguchi, Tetsuya

    2014-01-01

    Previous reports have shown that osteoblasts are mechano-sensitive. Low-intensity pulsed ultrasound (LIPUS) induces osteoblast differentiation and is an established therapy for bone fracture. Here we have examined how LIPUS affects inflammatory responses of osteoblasts to LPS. LPS rapidly induced mRNA expression of several chemokines including CCL2, CXCL1, and CXCL10 in both mouse osteoblast cell line and calvaria-derived osteoblasts. Simultaneous treatment by LIPUS significantly inhibited mRNA induction of CXCL1 and CXCL10 by LPS. LPS-induced phosphorylation of ERKs, p38 kinases, MEK1/2, MKK3/6, IKKs, TBK1, and Akt was decreased in LIPUS-treated osteoblasts. Furthermore, LIPUS inhibited the transcriptional activation of NF-κB responsive element and Interferon-sensitive response element (ISRE) by LPS. In a transient transfection experiment, LIPUS significantly inhibited TLR4-MyD88 complex formation. Thus LIPUS exerts anti-inflammatory effects on LPS-stimulated osteoblasts by inhibiting TLR4 signal transduction.

  10. Release of IL-12 by dendritic cells activated by TLR ligation is dependent on MyD88 signaling, whereas TRIF signaling is indispensable for TLR synergy.

    PubMed

    Krummen, Mathias; Balkow, Sandra; Shen, Limei; Heinz, Stefanie; Loquai, Carmen; Probst, Hans-Christian; Grabbe, Stephan

    2010-07-01

    Recently, it has been shown that certain combinations of TLR ligands act in synergy to induce the release of IL-12 by DCs. In this study, we sought to define the critical parameters underlying TLR synergy. Our data show that TLR ligands act synergistically if MyD88- and TRIF-dependent ligands are combined. TLR4 uses both of these adaptor molecules, thus activation via TLR4 proved to be a synergistic event on its own. TLR synergy did not affect all aspects of DC activation but enhanced primarily the release of certain cytokines, particularly IL-12, whereas the expression of costimulatory molecules remained unchanged. Consequently, synergistic activation of DC did not affect their ability to induce T cell proliferation but resulted in T(H)1-biased responses in vitro and in vivo. Furthermore, we examined the impact of TLR ligand combinations on primary DC in vitro but observed only modest effects with a combination of CpG + Poly (I:C). However, noticeable synergy in terms of IL-12 production by DCs was detectable in vivo after systemic administration of CpG + Poly (I:C). Finally, we show that synergy is partially dependent on IFNAR signaling but does not require the release of IFNs to the enviroment, suggesting an autocrine action of type I IFNs.

  11. Porcine circovirus type 2 induces type I interferon production via MyD88-IKKα-IRFs signaling rather than NF-κB in porcine alveolar macrophages in vitro.

    PubMed

    Chen, Mengmeng; Han, Junyuan; Zhang, Yaqun; Duan, Dianning; Zhang, Shuxia

    2016-02-01

    Type I interferon (IFN-I) plays important roles in host antiviral responses. The interferon regulatory factor (IRF) and NF-κB transcription factors are thought to be important in the processes of viral secretion and triggering of interferon production. Recently, studies have shown that porcine circovirus type 2 (PCV2) can induce IFN-I production in vivo and in vitro, but the mechanisms underlying the production of PAMs infected with PCV2 remains unknown. Treatment of these cells with BAY11-7082, an inhibitor of NF-κB activation, allowed us to study the secretion of IFN-α and IFN-β in PAMs infected with PCV2. We found that IFN-α expression was induced following virus infection of PAMs. Notably, even after inhibitor treatment of PAMs infected with PCV2, secretion of IFN-α was significantly higher (P<0.05) compared with the PCV2 infection alone group. Our findings suggest that NF-κB plays a minor role in PCV2-induced type I interferon responses. To further characterize the signaling pathway that drives IFN-I expression in PAMs in response to PCV2, we used siRNA to silence the expression of Myeloid differentiation factor 88 (MyD88) and study the role of MyD88-IKKα-IRF signaling in IFN-I production in PAMs induced by PCV2. Our findings show that PCV2 induced IFN-α mRNA transcription, which is associated with the activities of MyD88, IRF7, and IRF3. Thus, PCV2 can induce IFN-I transcription via the MyD88-IKKα-IRF signaling axis.

  12. TLR4-MyD88-TRAF6-TAK1 Complex-Mediated NF-κB Activation Contribute to the Anti-Inflammatory Effect of V8 in LPS-Induced Human Cervical Cancer SiHa Cells.

    PubMed

    He, Aiqin; Ji, Rui; Shao, Jia; He, Chenyun; Jin, Ming; Xu, Yunzhao

    2016-02-01

    The synthetic compound 7-4-[Bis-(2-hydroxyethyl)-amino]-butoxy-5-hydroxy-8-methoxy-2-phenylchromen-4-one (V8) is a novel flavonoid-derived compound. In this study, we investigated the effects of V8 on Toll-like receptor 4 (TLR4)-mediated inflammatory reaction in human cervical cancer SiHa cells and lipopolysaccharide (LPS)-induced TLR4 activity in cervical cancer SiHa (HPV16+) cells, but not in HeLa (HPV18+) and C33A (HPV-) cells. In addition, V8 inhibited LPS-induced expression of TLR4, MyD88, TRAF6 and phosphorylation of TAK1, and their interaction with TLR4 in SiHa cells, resulting in an inhibition of TLR4-MyD88-TRAF6-TAK1 complex. Moreover, V8 blocked LPS-induced phosphorylation of IκB and IKK, resulting in inhibition of the nuclear translocation of P65-NF-κB in SiHa cells. We also found that V8 reduced the expression of NF-κB target genes, such as those for COX-2, iNOS, IL-6, IL-8, CCL-2, and TNF-α in LPS-stimulated SiHa cells. These results suggested that V8 exerted an anti-inflammatory effect on SiHa cells by inhibiting the TLR4-MyD88-TRAF6-TAK1 complex-mediated NF-κB activation.

  13. Impaired Innate Immunity in Tlr4−/− Mice but Preserved CD8+ T Cell Responses against Trypanosoma cruzi in Tlr4-, Tlr2-, Tlr9- or Myd88-Deficient Mice

    PubMed Central

    Tzelepis, Fanny; Klezewsky, Weberton; da Silva, Raquel N.; Neves, Fabieni S.; Cavalcanti, Gisele S.; Boscardin, Silvia; Nunes, Marise P.; Santiago, Marcelo F.; Nóbrega, Alberto; Rodrigues, Maurício M.; Bellio, Maria

    2010-01-01

    The murine model of T. cruzi infection has provided compelling evidence that development of host resistance against intracellular protozoans critically depends on the activation of members of the Toll-like receptor (TLR) family via the MyD88 adaptor molecule. However, the possibility that TLR/MyD88 signaling pathways also control the induction of immunoprotective CD8+ T cell-mediated effector functions has not been investigated to date. We addressed this question by measuring the frequencies of IFN-γ secreting CD8+ T cells specific for H-2Kb-restricted immunodominant peptides as well as the in vivo Ag-specific cytotoxic response in infected animals that are deficient either in TLR2, TLR4, TLR9 or MyD88 signaling pathways. Strikingly, we found that T. cruzi-infected Tlr2−/−, Tlr4−/−, Tlr9−/− or Myd88−/− mice generated both specific cytotoxic responses and IFN-γ secreting CD8+ T cells at levels comparable to WT mice, although the frequency of IFN-γ+CD4+ cells was diminished in infected Myd88−/− mice. We also analyzed the efficiency of TLR4-driven immune responses against T. cruzi using TLR4-deficient mice on the C57BL genetic background (B6 and B10). Our studies demonstrated that TLR4 signaling is required for optimal production of IFN-γ, TNF-α and nitric oxide (NO) in the spleen of infected animals and, as a consequence, Tlr4−/− mice display higher parasitemia levels. Collectively, our results indicate that TLR4, as well as previously shown for TLR2, TLR9 and MyD88, contributes to the innate immune response and, consequently, resistance in the acute phase of infection, although each of these pathways is not individually essential for the generation of class I-restricted responses against T. cruzi. PMID:20442858

  14. Comprehensive Genomic Profiling of Orbital and Ocular Adnexal Lymphomas Identifies Frequent Alterations in MYD88 and Chromatin Modifiers: New Routes to Targeted Therapies

    PubMed Central

    Cani, Andi K.; Soliman, Moaaz; Hovelson, Daniel H.; Liu, Chia-Jen; McDaniel, Andrew S.; Haller, Michaela J.; Bratley, Jarred; Rahrig, Samantha; Li, Qiang; Briceño, César A.; Tomlins, Scott A.; Rao, Rajesh C.

    2016-01-01

    Non-Hodgkin lymphoma of the orbit and ocular adnexa is the most common primary orbital malignancy. Treatments for low- (extra-nodal marginal zone and follicular lymphomas) and high-grade (diffuse large B-cell lymphoma) are associated with local and vision-threatening toxicities. High-grade lymphomas relapse frequently and exhibit poor survival rates. Despite advances in genomic profiling and precision-medicine, orbital and ocular adnexal lymphomas remain poorly characterized molecularly. We performed targeted next-generation sequencing profiling of 38 formalin-fixed, paraffin-embedded, orbital and ocular adnexal lymphomas obtained from a single-center using a panel targeting near-term, clinically-relevant genes. Potentially actionable mutations and copy-number alterations were prioritized based on gain- and loss-of function analyses, catalogued approved and investigational therapies. Of 36 informative samples, including marginal zone lymphomas (n=20), follicular lymphomas (n=9), and diffuse large B-cell lymphomas (n=7), 53% harbored a prioritized alteration (median=1, range 0–5/sample). MYD88 was the most frequently altered gene in our cohort, with potentially clinically-relevant hot-spot gain-of-function mutations identified in 71% of diffuse large B-cell and 25% of marginal zone lymphomas. Prioritized alterations in epigenetic modulators were common and included gain-of-function EZH2 and loss-of-function ARID1A mutations (14% of diffuse large B-cell lymphomas and 22% of follicular lymphomas contained alterations in each of these two genes). Single prioritized alterations were also identified in the histone methyltransferases KMT2B (follicular lymphoma) and KMT3B (diffuse large B-cell lymphoma). Loss-of-function mutations and copy-number alterations in the tumor suppressors TP53 (diffuse large B-cell and follicular lymphoma), CDKN2A (all subtypes), PTEN (diffuse large B-cell lymphoma), ATM (diffuse large B-cell lymphoma) and NF1 (diffuse large B-cell lymphoma

  15. IgM+IgD+CD27+ B cells are markedly reduced in IRAK-4-, MyD88-, and TIRAP- but not UNC-93B-deficient patients.

    PubMed

    Weller, Sandra; Bonnet, Mélanie; Delagreverie, Héloïse; Israel, Laura; Chrabieh, Maya; Maródi, László; Rodriguez-Gallego, Carlos; Garty, Ben-Zion; Roifman, Chaim; Issekutz, Andrew C; Zitnik, Simona Eva; Hoarau, Cyrille; Camcioglu, Yildiz; Vasconcelos, Júlia; Rodrigo, Carlos; Arkwright, Peter D; Cerutti, Andrea; Meffre, Eric; Zhang, Shen-Ying; Alcais, Alexandre; Puel, Anne; Casanova, Jean-Laurent; Picard, Capucine; Weill, Jean-Claude; Reynaud, Claude-Agnès

    2012-12-13

    We studied the distribution of peripheral B-cell subsets in patients deficient for key factors of the TLR-signaling pathways (MyD88, TIRAP/MAL, IL-1 receptor-associated kinase 4 [IRAK-4], TLR3, UNC-93B, TRIF). All TLRs, except TLR3, which signals through the TRIF adaptor, require MyD88 and IRAK-4 to mediate their function. TLR4 and the TLR2 heterodimers (with TLR1, TLR6, and possibly TLR10) require in addition the adaptor TIRAP, whereas UNC-93B is needed for the proper localization of intracellular TLR3, TLR7, TLR8, and TLR9. We found that IgM(+)IgD(+)CD27(+) but not switched B cells were strongly reduced in MyD88-, IRAK-4-, and TIRAP-deficient patients. This defect did not appear to be compensated with age. However, somatic hypermutation of Ig genes and heavy-chain CDR3 size distribution of IgM(+)IgD(+)CD27(+) B cells were not affected in these patients. In contrast, the numbers of IgM(+)IgD(+)CD27(+) B cells were normal in the absence of TLR3, TRIF, and UNC-93B, suggesting that UNC-93B-dependent TLRs, and notably TLR9, are dispensable for the presence of this subset in peripheral blood. Interestingly, TLR10 was found to be expressed at greater levels in IgM(+)IgD(+)CD27(+) compared with switched B cells in healthy patients. Hence, we propose a role for TIRAP-dependent TLRs, possibly TLR10 in particular, in the development and/or maintenance of IgM(+)IgD(+)CD27(+) B cells in humans.

  16. Subversion of innate immune responses by Brucella through the targeted degradation of the TLR signaling adapter, MAL.

    PubMed

    Sengupta, Dola; Koblansky, Alicia; Gaines, Jennifer; Brown, Tim; West, A Phillip; Zhang, Dekai; Nishikawa, Tak; Park, Sung-Gyoo; Roop, R Martin; Ghosh, Sankar

    2010-01-15

    Gram-negative bacteria belonging to the Brucella species cause chronic infections that can result in undulant fever, arthritis, and osteomyelitis in humans. Remarkably, Brucella sp. genomes encode a protein, named TcpB, that bears significant homology with mammalian Toll/IL-1 receptor domains and whose expression causes degradation of the phosphorylated, signal competent form of the adapter MyD88-adapter-like (MAL). This effect of TcpB is mediated through its box 1 region and has no effect on other TLR adapter proteins such as MyD88 or TIR-domain containing adapter protein-inducing IFNbeta. TcpB also does not affect a mutant, signal-incompetent form of MAL that cannot be phosphorylated. Interestingly, the presence of TcpB leads to enhanced polyubiquitination of MAL, which is likely responsible for its accelerated degradation. A Brucella abortus mutant lacking TcpB fails to reduce levels of MAL in infected macrophages. Therefore, TcpB represents a unique pathogen-derived molecule that suppresses host innate-immune responses by specifically targeting an individual adapter molecule in the TLR signaling pathway for degradation.

  17. TLR4/MyD88-Induced CD11b+Gr-1intF4/80+ Non-Migratory Myeloid Cells Suppress Th2 Effector Function in the Lung

    PubMed Central

    Arora, Meenakshi; Poe, Stephanie L.; Oriss, Timothy B.; Krishnamoorthy, Nandini; Yarlagadda, Manohar; Wenzel, Sally E.; Billiar, Timothy R.; Ray, Anuradha; Ray, Prabir

    2010-01-01

    In humans, environmental exposure to a high dose of lipopolysaccharide (LPS) protects from allergic asthma the immunological underpinnings of which are not well understood. In mice, exposure to a high LPS dose blunted house dust mite-induced airway eosinophilia and Th2 cytokine production. While adoptively transferred Th2 cells induced allergic airway inflammation in control mice, they were unable to do so in LPS-exposed mice. LPS promoted the development of a CD11b+Gr1intF4/80+ lung-resident cell resembling myeloid-derived suppressor cells in a TLR4- and MyD88-dependent fashion that suppressed lung dendritic cell (DC)-mediated reactivation of primed Th2 cells. LPS effects switched from suppressive to stimulatory in MyD88-/- mice. Suppression of Th2 effector function was reversed by anti-IL-10 or inhibition of Arginase 1. Lineageneg bone marrow progenitor cells could be induced by LPS to develop into CD11b+Gr1intF4/80+ cells both in vivo and in vitro which when adoptively transferred suppressed allergen-induced airway inflammation in recipient mice. These data suggest that CD11b+Gr1intF4/80+ cells contribute to the protective effects of LPS in allergic asthma by tempering Th2 effector function in the tissue. PMID:20664577

  18. Depurinized milk downregulates rat thymus MyD88/Akt/p38 function, NF-κB-mediated inflammation, caspase-1 activity but not the endonuclease pathway: in vitro/in vivo study

    PubMed Central

    Kocic, Gordana; Veljkovic, Andrej; Kocic, Hristina; Colic, Miodrag; Mihajlovic, Dusan; Tomovic, Katarina; Stojanovic, Svetlana; Smelcerovic, Andrija

    2017-01-01

    The aim of this study was the evaluation of 15 days dietary regimen of depurinized (DP) milk (obtained using our patented technological procedures) or 1.5% fat UHT milk instead of standard chow diet, on rat thymus and bone marrow MyD88/Akt/p38, NF-κB, caspase-1 and endonuclease pathways, in relation to peripheral blood cell composition. To determine whether the reduced mass of the thymus is a consequence of the direct effect of DP/UHT milk on apoptosis of thymocytes, in vitro Annexin-V-FITC/PI assay was performed. Significant decreases in the thymus wet weight, thymocyte MyD88, Akt-1/phospho-Akt-1 kinase, p38/phospho-p38, NF-κB, caspase-1 activity and CD4+/CD8+ antigen expression were obtained, especially in the DP milk group. The activity of thymocyte alkaline and acid DNase increased in the DP but not in the UHT milk group. The level of IL-6 significantly decreased in DP milk treated group, while the level of total TGF-β and IL-6 increased in UHT milk group. Significant differences in hematological parameters were obtained in commercial milk fed group. Observed results about prevention of experimental diabetes in DP pretreated groups may suggest that purine compounds, uric acid and other volatile toxic compounds of commercial milk may suppress oral tolerance, probably via IL-6 and TGF-β cytokine effects. PMID:28176796

  19. Suppression of OVA-alum induced allergy by Heligmosomoides polygyrus products is MyD88-, TRIF-, regulatory T- and B cell-independent, but is associated with reduced innate lymphoid cell activation.

    PubMed

    McSorley, Henry J; Blair, Natalie F; Robertson, Elaine; Maizels, Rick M

    2015-11-01

    The murine intestinal nematode Heligmosomoides polygyrus exerts multiple immunomodulatory effects in the host, including the suppression of allergic inflammation in mice sensitized to allergen presented with alum adjuvant. Similar suppression is attained by co-administration of H. polygyrus excretory/secretory products (HES) with the sensitizing dose of ovalbumin (OVA) in alum. We investigated the mechanism of suppression by HES in this model, and found it was maintained in MyD88xTRIF-deficient mice, implying no role for helminth- or host-derived TLR ligands, or IL-1 family cytokines that signal in a MyD88- or TRIF-dependent manner. We also found suppression was unchanged in µMT mice, which lack B2 B cells, and that suppression was not abrogated when regulatory T cells were depleted in Foxp3.LuciDTR-4 mice. However, reduced IL-5 production was seen in the first 12 h after injection of OVA-alum when HES was co-administered, associated with reduced activation of IL-5(+) and IL-13(+) group 2 innate lymphoid cells. Thus, the suppressive effects of HES on alum-mediated OVA sensitization are reflected in the very earliest innate response to allergen exposure in vivo.

  20. Porcine circovirus type 2 increases IL-1β and IL-10 production via the MyD88-NF-κB signaling pathway in porcine alveolar macrophages in vitro.

    PubMed

    Han, Junyuan; Zhang, Shuxia; Zhang, Yaqun; Chen, Mengmeng; Lv, Yingjun

    2016-07-25

    Porcine alveolar macrophages represent the first line of defense in the porcine lung after infection with porcine circovirus type 2 (PCV2) via the respiratory tract. However, PCV2 infection impairs the microbicidal capability of PAMs and alters cytokine production and/or secretion. Currently, the reason for the imbalance of cytokines has not been fully elucidated and the regulatory mechanisms involved are not clear. Here, we investigated the expression levels and regulation of IL-1β and IL-10 in PAMs following incubation with PCV2 in vitro. Both levels of IL-1β and IL-10 increased in PAM supernatants, and the distribution of NF-κB p65 staining in the nucleus, the expression of MyD88 and p-IκB in the cytoplasm and the DNA-binding activity of NF-κB increased after incubation with PCV2, while the expression of p65 in the cytoplasm of PAMs decreased. However, when PAMs were co-incubated with PCV2 and small interfering RNA targeting MyD88, these effects were reversed. Additionally, mRNA expression levels of Toll-like receptor (TLR)-2, -3, -4, -7, -8 and -9 were increased when PAMs were incubated with PCV2. These findings showed that PCV2 induced increased IL-1β and IL-10 production in PAMs, and these changes in expression were relative to the TLR-MyD88-NF-κB signaling pathway.

  1. LPS Up-Regulates ICAM-1 Expression in Breast Cancer Cells by Stimulating a MyD88-BLT2-ERK-Linked Cascade, Which Promotes Adhesion to Monocytes

    PubMed Central

    Park, Geun-Soo; Kim, Jae-Hong

    2015-01-01

    Monocytes are the major inflammatory cells that infiltrate most solid tumors in humans. The interaction of tumor cells with infiltrating monocytes and their adhesion to these monocytes play a significant role in altering the tumor to become more aggressive. Recently, exposure to lipopolysaccharide (LPS) was suggested to promote cancer cell adhesion to monocytes; however, little is known about the details of the signaling mechanism involved in this process. In this study, we found that LPS up-regulates ICAM-1 expression in MDA-MB-231 breast cancer cells, which facilitates their adhesion to THP-1 monocytes. In addition, we analyzed the signaling mechanism underlying the up-regulation of ICAM-1 and found that the siRNA-mediated depletion of BLT2 markedly suppressed the LPS-induced expression of ICAM-1 in MDA-MB-231 cells and the subsequent adhesion of these cells to THP-1 monocytes. Moreover, we demonstrated that myeloid differentiation primary response gene 88 (MyD88) lies downstream of LPS/TLR4 and upstream of BLT2 and that this ‘MyD88-BLT2’ cascade mediates ERK activation and subsequent ICAM-1 expression, which is critical for the adhesion of MDA-MB-231 cells to THP-1 monocytes. Taken together, our results demonstrate for the first time that LPS up-regulates ICAM-1 expression in breast cancer cells via a MyD88-BLT2-ERK-linked signaling cascade, leading to the increased adhesion of breast cancer cells to monocytes. PMID:26299331

  2. MiR-23a-5p modulates mycobacterial survival and autophagy during mycobacterium tuberculosis infection through TLR2/MyD88/NF-κB pathway by targeting TLR2.

    PubMed

    Gu, Xing; Gao, Yan; Mu, De-Guang; Fu, En-Qing

    2017-03-19

    Autophagy plays a pivotal role in activating the antimicrobial host defense against Mycobacterium tuberculosis (M.tb.). The emerging roles of microRNAs (miRNAs) in regulating immune responses have attracted increasing attention in recent years. Appreciating the potential of host-directed therapies designed to control autophagy during mycobacterial infection, we focused on the influence of miR-23a-5p on the activation of macrophage autophagy during M.tb. infection in bone marrow-derived macrophages (BMDMs) and murine RAW264.7 cells. Here, we demonstrated that M.tb.-infection of macrophages lead to markedly enhanced expression of miR-23a-5p in a time- and dose-dependent manner. Furthermore, forced expression of miR-23a-5p accelerated the survival rate of intracellular mycobacteria, while transfection with miR-23a-5p inhibitors attenuated mycobacterial survival. More importantly, overexpression of miR-23a-5p dramatically prevented M.tb.-induced activation of autophagy in macrophages, whereas inhibitors of miR-23a-5p remarkably accelerated M.tb.-induced autophagy. Mechanistically, miR-23a-5p is able to modulate TLR2/MyD88/NF-κB signaling activity by targeting TLR2 in RAW264.7 cells in response to M.tb.-infection. Collectively, these findings demonstrated that miR-23a-5p modulated the innate host defense by promoting mycobacteria survival and inhibiting the activation of autophagy against M.tb. through TLR2/MyD88/NF-κB pathway by targeting TLR2, which may provide a promising therapeutic target for tuberculosis.

  3. CXC195 suppresses proliferation and inflammatory response in LPS-induced human hepatocellular carcinoma cells via regulating TLR4-MyD88-TAK1-mediated NF-κB and MAPK pathway

    SciTech Connect

    Wang, Yiting; Tu, Qunfei; Yan, Wei; Xiao, Dan; Zeng, Zhimin; Ouyang, Yuming; Huang, Long; Cai, Jing; Zeng, Xiaoli; Chen, Ya-Jie; Liu, Anwen

    2015-01-02

    Highlights: • CXC195 exhibited significant anti-proliferative effect and induced cell cycle arrest in LPS-induced HepG2 cells. • CXC195 suppressed the release of pro-inflammatory mediators in LPS-induced HepG2 cells. • CXC195 regulated TLR4-MyD88-TAK1-mediated NF-κB and MAPK pathway in LPS-induced HepG2 cells. - Abstract: CXC195 showed strong protective effects in neuronal apoptosis by exerting its antioxidant activity. However, the anti-cancer effects of CXC195 is still with limited acquaintance. Here, we investigated the role of CXC195 in lipopolysaccharide (LPS)-induced human hepatocellular carcinoma (HCC) cells lines (HepG2) and the possible signaling pathways. CXC195 exhibited significant anti-proliferative effect and induced cell cycle arrest in LPS-induced HepG2 cells. In addition, CXC195 suppressed the release of pro-inflammatory mediators in LPS-induced HepG2 cells, including TNF-α, iNOS, IL-1β, IL-6, CC chemokine ligand (CCL)-2, CCL-22 and epidermal growth factor receptor (EGFR). Moreover, CXC195 inhibited the expressions and interactions of TLR4, MyD88 and TAK1, NF-κB translocation to nucleus and its DNA binding activity, phosphorylation of ERK1/2, p38 and JNK. Our results suggested that treatment with CXC195 could attenuate the TLR4-mediated proliferation and inflammatory response in LPS-induced HepG2 cells, thus might be beneficial for the treatment of HCC.

  4. DAMP Molecule S100A9 Acts as a Molecular Pattern to Enhance Inflammation during Influenza A Virus Infection: Role of DDX21-TRIF-TLR4-MyD88 Pathway

    PubMed Central

    Tsai, Su-Yu; Segovia, Jesus A.; Chang, Te-Hung; Morris, Ian R.; Berton, Michael T.; Tessier, Philippe A.; Tardif, Mélanie R.; Cesaro, Annabelle; Bose, Santanu

    2014-01-01

    Pathogen-associated molecular patterns (PAMPs) trigger host immune response by activating pattern recognition receptors like toll-like receptors (TLRs). However, the mechanism whereby several pathogens, including viruses, activate TLRs via a non-PAMP mechanism is unclear. Endogenous “inflammatory mediators” called damage-associated molecular patterns (DAMPs) have been implicated in regulating immune response and inflammation. However, the role of DAMPs in inflammation/immunity during virus infection has not been studied. We have identified a DAMP molecule, S100A9 (also known as Calgranulin B or MRP-14), as an endogenous non-PAMP activator of TLR signaling during influenza A virus (IAV) infection. S100A9 was released from undamaged IAV-infected cells and extracellular S100A9 acted as a critical host-derived molecular pattern to regulate inflammatory response outcome and disease during infection by exaggerating pro-inflammatory response, cell-death and virus pathogenesis. Genetic studies showed that the DDX21-TRIF signaling pathway is required for S100A9 gene expression/production during infection. Furthermore, the inflammatory activity of extracellular S100A9 was mediated by activation of the TLR4-MyD88 pathway. Our studies have thus, underscored the role of a DAMP molecule (i.e. extracellular S100A9) in regulating virus-associated inflammation and uncovered a previously unknown function of the DDX21-TRIF-S100A9-TLR4-MyD88 signaling network in regulating inflammation during infection. PMID:24391503

  5. Kaempferol ameliorates H9N2 swine influenza virus-induced acute lung injury by inactivation of TLR4/MyD88-mediated NF-κB and MAPK signaling pathways.

    PubMed

    Zhang, Ruihua; Ai, Xia; Duan, Yongjie; Xue, Man; He, Wenxiao; Wang, Cunlian; Xu, Tong; Xu, Mingju; Liu, Baojian; Li, Chunhong; Wang, Zhijun; Zhang, Ruihong; Wang, Guohua; Tian, Shufei; Liu, Huifeng

    2017-03-02

    Kaempferol, a very common type of dietary flavonoids, has been found to exert antioxidative and anti-inflammatory properties. The purpose of our investigation was designed to reveal the effect of kaempferol on H9N2 influenza virus-induced inflammation in vivo and in vitro. In vivo, BALB/C mice were infected intranasally with H9N2 influenza virus with or without kaempferol treatment to induce acute lung injury (ALI) model. In vitro, MH-S cells were infected with H9N2 influenza virus with or without kaempferol treatment. In vivo, kaempferol treatment attenuated pulmonary edema, the W/D mass ratio, pulmonary capillary permeability, myeloperoxidase (MPO) activity, and the numbers of inflammatory cells. Kaempferol reduced ROS and Malondialdehyde (MDA) production, and increased the superoxide dismutase (SOD) activity. Kaempferol also reduced overproduction of TNF-α, IL-1β and IL-6. In addition, kaempferol decreased the H9N2 viral titre. In vitro, ROS, MDA, TNF-α, IL-1β and IL-6 was also reduced by kaempferol. Moreover, our data showed that kaempferol significantly inhibited the upregulation of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), phosphorylation level of IκBα and nuclear factor-κB (NF-κB) p65, NF-κB p65 DNA binding activity, and phosphorylation level of MAPKs, both in vivo and in vitro. These results suggest that kaempferol exhibits a protective effect on H9N2 virus-induced inflammation via suppression of TLR4/MyD88-mediated NF-κB and MAPKs pathways, and kaempferol may be considered as an effective drug for the potential treatment of influenza virus-induced ALI.

  6. Phylogeny of Toll-Like Receptor Signaling: Adapting the Innate Response

    PubMed Central

    Roach, Jeffrey M.; Racioppi, Luigi; Jones, Corbin D.; Masci, Anna Maria

    2013-01-01

    The Toll-like receptors represent a largely evolutionarily conserved pathogen recognition machinery responsible for recognition of bacterial, fungal, protozoan, and viral pathogen associated microbial patterns and initiation of inflammatory response. Structurally the Toll-like receptors are comprised of an extracellular leucine rich repeat domain and a cytoplasmic Toll/Interleukin 1 receptor domain. Recognition takes place in the extracellular domain where as the cytoplasmic domain triggers a complex signal network required to sustain appropriate immune response. Signal transduction is regulated by the recruitment of different intracellular adaptors. The Toll-like receptors can be grouped depending on the usage of the adaptor, MyD88, into MyD88-dependent and MyD88 independent subsets. Herein, we present a unique phylogenetic analysis of domain regions of these receptors and their cognate signaling adaptor molecules. Although previously unclear from the phylogeny of full length receptors, these analyses indicate a separate evolutionary origin for the MyD88-dependent and MyD88-independent signaling pathway and provide evidence of a common ancestor for the vertebrate and invertebrate orthologs of the adaptor molecule MyD88. Together these observations suggest a very ancient origin of the MyD88-dependent pathway Additionally we show that early duplications gave rise to several adaptor molecule families. In some cases there is also strong pattern of parallel duplication between adaptor molecules and their corresponding TLR. Our results further support the hypothesis that phylogeny of specific domains involved in signaling pathway can shed light on key processes that link innate to adaptive immune response. PMID:23326591

  7. Impaired Cd14 and Cd36 expression, bacterial clearance, and Toll-like receptor 4-Myd88 signaling in caveolin-1-deleted macrophages and mice.

    PubMed

    Tsai, Tsung-Huang; Chen, Shu-Fen; Huang, Tai-Yu; Tzeng, Chun-Fu; Chiang, Ann-Shyn; Kou, Yu Ru; Lee, Tzong-Shyuan; Shyue, Song-Kun

    2011-01-01

    An overwhelming immune response, particularly from macrophages, with gram-negative bacteria-induced sepsis plays a critical role in survival of and organ damage in infected patients. Caveolin-1 (Cav-1), a major structure protein of caveolae, regulates many cellular functions. We examined the vital role of Cav-1 in the response of macrophages and mice to bacteria or LPS exposure. Deletion of Cav-1 decreased the expression of CD14 and CD36 during macrophage differentiation and suppressed their phagocytotic ability. As well, the ability to kill bacteria was inhibited in Cav-1 macrophages and mice peritoneal cavity, tissue, and plasma, which was partly attributed to hindered expression of iNOS induced by bacteria or LPS. Furthermore, deletion of Cav-1 attenuated the expression of Toll-like receptor 4 and myeloid differentiation factor 88 and the activation of nuclear factor κB, all of which impeded the production of inflammatory cytokines in response to bacterial exposure in Cav-1 macrophages and mice. Thus, Cav-1 participates in the regulation of CD14, CD36, Toll-like receptor 4 and myeloid differentiation factor 88 protein expression and is crucial for the immune response of macrophages to bacterial infection. Cav-1 may be a therapeutic target in the treatment of sepsis.

  8. Toll-like receptor 4 activation promotes cardiac arrhythmias by decreasing the transient outward potassium current (Ito) through an IRF3-dependent and MyD88-independent pathway.

    PubMed

    Monnerat-Cahli, Gustavo; Alonso, Hiart; Gallego, Monica; Alarcón, Micaela Lopez; Bassani, Rosana A; Casis, Oscar; Medei, Emiliano

    2014-11-01

    Cardiac arrhythmias are one of the main causes of death worldwide. Several studies have shown that inflammation plays a key role in different cardiac diseases and Toll-like receptors (TLRs) seem to be involved in cardiac complications. In the present study, we investigated whether the activation of TLR4 induces cardiac electrical remodeling and arrhythmias, and the signaling pathway involved in these effects. Membrane potential was recorded in Wistar rat ventricle. Ca(2+) transients, as well as the L-type Ca(2+) current (ICaL) and the transient outward K(+) current (Ito), were recorded in isolated myocytes after 24 h exposure to the TLR4 agonist, lipopolysaccharide (LPS, 1 μg/ml). TLR4 stimulation in vitro promoted a cardiac electrical remodeling that leads to action potential prolongation associated with arrhythmic events, such as delayed afterdepolarization and triggered activity. After 24 h LPS incubation, Ito amplitude, as well as Kv4.3 and KChIP2 mRNA levels were reduced. The Ito decrease by LPS was prevented by inhibition of interferon regulatory factor 3 (IRF3), but not by inhibition of interleukin-1 receptor-associated kinase 4 (IRAK4) or nuclear factor kappa B (NF-κB). Extrasystolic activity was present in 25% of the cells, but apart from that, Ca(2+) transients and ICaL were not affected by LPS; however, Na(+)/Ca(2+) exchanger (NCX) activity was apparently increased. We conclude that TLR4 activation decreased Ito, which increased AP duration via a MyD88-independent, IRF3-dependent pathway. The longer action potential, associated with enhanced Ca(2+) efflux via NCX, could explain the presence of arrhythmias in the LPS group.

  9. Human metapneumovirus M2-2 protein inhibits innate immune response in monocyte-derived dendritic cells.

    PubMed

    Ren, Junping; Liu, Guangliang; Go, Jonathan; Kolli, Deepthi; Zhang, Guanping; Bao, Xiaoyong

    2014-01-01

    Human metapneumovirus (hMPV) is a leading cause of lower respiratory infection in young children, the elderly and immunocompromised patients. Repeated hMPV infections occur throughout life. However, immune evasion mechanisms of hMPV infection are largely unknown. Recently, our group has demonstrated that hMPV M2-2 protein, an important virulence factor, contributes to immune evasion in airway epithelial cells by targeting the mitochondrial antiviral-signaling protein (MAVS). Whether M2-2 regulates the innate immunity in human dendritic cells (DC), an important family of immune cells controlling antigen presenting, is currently unknown. We found that human DC infected with a virus lacking M2-2 protein expression (rhMPV-ΔM2-2) produced higher levels of cytokines, chemokines and IFNs, compared to cells infected with wild-type virus (rhMPV-WT), suggesting that M2-2 protein inhibits innate immunity in human DC. In parallel, we found that myeloid differentiation primary response gene 88 (MyD88), an essential adaptor for Toll-like receptors (TLRs), plays a critical role in inducing immune response of human DC, as downregulation of MyD88 by siRNA blocked the induction of immune regulatory molecules by hMPV. Since M2-2 is a cytoplasmic protein, we investigated whether M2-2 interferes with MyD88-mediated antiviral signaling. We found that indeed M2-2 protein associated with MyD88 and inhibited MyD88-dependent gene transcription. In this study, we also identified the domains of M2-2 responsible for its immune inhibitory function in human DC. In summary, our results demonstrate that M2-2 contributes to hMPV immune evasion by inhibiting MyD88-dependent cellular responses in human DC.

  10. Subversion of Toll-like receptor signaling by a unique family of bacterial Toll/interleukin-1 receptor domain-containing proteins.

    PubMed

    Cirl, Christine; Wieser, Andreas; Yadav, Manisha; Duerr, Susanne; Schubert, Sören; Fischer, Hans; Stappert, Dominik; Wantia, Nina; Rodriguez, Nuria; Wagner, Hermann; Svanborg, Catharina; Miethke, Thomas

    2008-04-01

    Pathogenic microbes have evolved sophisticated molecular strategies to subvert host defenses. Here we show that virulent bacteria interfere directly with Toll-like receptor (TLR) function by secreting inhibitory homologs of the Toll/interleukin-1 receptor (TIR) domain. Genes encoding TIR domain containing-proteins (Tcps) were identified in Escherichia coli CFT073 (TcpC) and Brucella melitensis (TcpB). We found that TcpC is common in the most virulent uropathogenic E. coli strains and promotes bacterial survival and kidney pathology in vivo. In silico analysis predicted significant tertiary structure homology to the TIR domain of human TLR1, and we show that the Tcps impede TLR signaling through the myeloid differentiation factor 88 (MyD88) adaptor protein, owing to direct binding of Tcps to MyD88. Tcps represent a new class of virulence factors that act by inhibiting TLR- and MyD88-specific signaling, thus suppressing innate immunity and increasing virulence.

  11. Human parainfluenza virus type 2 V protein inhibits TRAF6-mediated ubiquitination of IRF7 to prevent TLR7- and TLR9-dependent interferon induction.

    PubMed

    Kitagawa, Yoshinori; Yamaguchi, Mayu; Zhou, Min; Nishio, Machiko; Itoh, Masae; Gotoh, Bin

    2013-07-01

    Paramyxovirus V proteins block Toll-like receptor 7 (TLR7)- and TLR9-dependent signaling leading to alpha interferon production. Our recent study has provided evidence that interaction of the V proteins with IRF7 is important for the blockade. However, the detailed mechanisms still remain unclear. Here we reexamined the interaction of the human parainfluenza virus type 2 (HPIV2) V protein with signaling molecules involved in TLR7/9-dependent signaling. Immunoprecipitation experiments in HEK293T cells transfected with V protein and one of the signaling molecules revealed that the V protein interacted with not only IRF7 but also TRAF6, IKKα, and MyD88. Whereas overexpression of TRAF6 markedly enhanced the level of V protein associating with IRF7, IKKα, and MyD88 in HEK293T cells, the level of V protein associating with TRAF6 was little affected by overexpression of IRF7, IKKα, and MyD88. Moreover, knockdown or knockout of endogenous TRAF6 in HEK293T or mouse embryonic fibroblast cells resulted in dissociation of the V protein from IRF7, IKKα, and MyD88. These results demonstrate that binding of the V protein to IRF7, IKKα, and MyD88 is largely indirect and mediated by endogenous TRAF6. It was found that the V protein inhibited TRAF6-mediated lysine 63 (K63)-linked polyubiquitination of IRF7, which is prerequisite for IRF7 activation. Disruption of the tryptophan-rich motif of the V protein significantly affected its TRAF6-binding efficiency, which correlated well with the magnitude of inhibition of K63-linked polyubiquitination and the resultant activation of IRF7. Taken together, these results suggest that the HPIV2 V protein prevents TLR7/9-dependent interferon induction by inhibiting TRAF6-mediated K63-linked polyubiquitination of IRF7.

  12. Escherichia coli maltose-binding protein (MBP) directly induces mouse Th1 activation through upregulating TLR2 and downregulating TLR4 expressions.

    PubMed

    Wang, Fang; Ni, Weihua; Liu, Guomu; Wang, Juan; Xie, Fei; Yuan, Hongyan; Guo, Yingying; Zhai, RuiPing; Chen, Tanxiu; Li, Qiongshu; Tai, Guixiang

    2015-06-01

    Maltose-binding protein (MBP), a component of the maltose transport system of Escherichia coli, has been commonly thought to have minimal bioactivity. Our previous studies demonstrated that MBP could significantly enhance Bacillus Calmette-Guerin (BCG)-induced T helper 1 (Th1) cell activation in mice. In the present study, we analyzed the effect of MBP on mouse T cells and found that MBP promoted the proliferation and IFN-γ production of CD4(+) T cells, suggesting that MBP directly induces Th1 activation. To explore the mechanism of Th1 activation, the expression of Toll-like receptor 2/4 (TLR2/4) on purified mouse CD4(+) T cells was detected. The results showed that MBP up-regulated TLR2 while down-regulated TLR4 expression, accompanied by a clear increase in MyD88 expression and IκB phosphorylation. Notably, the addition of anti-TLR2 antibody abrogated the MBP-induced CD4(+) T cells proliferation, IFN-γ secretion and MyD88 expression, whereas the addition of anti-TLR4 antibody exhibited a contradictive effect. Besides, the block of either TLR2 or TLR4 both reduced IκB phosphorylation. These results above suggest that TLR2-mediated MyD88-dependent pathway contributes to MBP-induced Th1 activation, while TLR4 appears to counteract this effect via MyD88-independent pathway.

  13. Toll-like receptors are critical for clearance of Brucella and play different roles in development of adaptive immunity following aerosol challenge in mice.

    PubMed

    Pei, Jianwu; Ding, Xicheng; Fan, Yaping; Rice-Ficht, Allison; Ficht, Thomas A

    2012-01-01

    Brucella spp. cause undulant fever in humans and brucellosis in variety of other animals. Both innate and adaptive immunity have been shown to be important in controlling Brucella infection. Toll-like receptors (TLRs) represent a group of pattern recognition receptors (PRRs) that play critical roles in the host innate immune response, as well as development of adaptive immunity. In the current report, we investigated the role of TLR signaling in the clearance of Brucella and development of adaptive immunity in TLR2(-/-), TLR4(-/-), or MyD88(-/-) mice following aerosol exposure to B. melitensis 16 M. Consistent with previous reports, MyD88 is required for efficient clearance of Brucella from all three organs (lung, spleen, and liver). The results reveal Th2-skewed immune responses in TLR2(-/-) mice late in infection and support a TLR2 requirement for efficient clearance of Brucella from the lungs, but not from the spleen or liver. Similarly, TLR4 is required for efficient clearance of Brucella from the lung, but exhibits a minor contribution to clearance from the spleen and no demonstrable contribution to clearance from the liver. Lymphocyte proliferation assays suggest that the TLRs are not involved in the development of cell-mediated memory response to Brucella antigen. Antibody detection reveals that TLR2 and 4 are required to generate early antigen-specific IgG, but not during the late stages of infection. TLR2 and 4 are only transiently required for IgM production and not at all for IgA production. In contrast, MyD88 is essential for antigen specific IgG production late in infection, but is not required for IgM generation over the course of infection. Surprisingly, despite the prominent role for MyD88 in clearance from all tissues, MyD88-knockout mice express significantly higher levels of serum IgA. These results confirm the important role of MyD88 in controlling infection in the spleen while providing evidence of a prominent contribution to protection in

  14. TB, or not TB: that is the question – does TLR signaling hold the answer?

    PubMed Central

    Doherty, Terence M.; Arditi, Moshe

    2004-01-01

    Innate immunity critically depends on signaling by Toll-like receptors (TLRs) that rely heavily on an intracellular adapter protein called myeloid differentiation factor 88 (MyD88). Adaptive immune defenses are generally thought to be orchestrated by innate immune responses and so should require intact TLR-MyD88 signaling pathways. But a surprising new study in MyD88-null mice infected with Mycobacterium tuberculosis challenges this view and instead suggests that MyD88 may not be absolutely required for a normal adaptive immune response. PMID:15599394

  15. TB, or not TB: that is the question -- does TLR signaling hold the answer?

    PubMed

    Doherty, Terence M; Arditi, Moshe

    2004-12-01

    Innate immunity critically depends on signaling by Toll-like receptors (TLRs) that rely heavily on an intracellular adapter protein called myeloid differentiation factor 88 (MyD88). Adaptive immune defenses are generally thought to be orchestrated by innate immune responses and so should require intact TLR-MyD88 signaling pathways. But a surprising new study in MyD88-null mice infected with Mycobacterium tuberculosis challenges this view and instead suggests that MyD88 may not be absolutely required for a normal adaptive immune response.

  16. A Small Molecule that Mimics the BB-Loop in the Toll/IL-1 Receptor Domain of MyD88 Attenuates Staphylococcal Enterotoxin B Induced Pro-Inflammatory Cytokine Production and Toxicity in Mice

    DTIC Science & Technology

    2011-06-01

    to allow the cells to swell. 50 µl of 10% Nonidet P - 40 was added to the cells and briefly centrifuged. The nuclear pellet was resuspended in...protein 10 (IP-10). A conserved sequence, (F/Y)-(V/L/I)-( P /G), called the BB- loop appears in the TIR domain of most members of the TLR/IL-1R family...from 7 structural analogs based on the tripeptide sequence of the BB-loop [(F/Y)-(V/L/I)-( P /G)] of the Toll/IL-1 receptor (TIR) domain binding

  17. The human adaptor SARM negatively regulates adaptor protein TRIF-dependent Toll-like receptor signaling.

    PubMed

    Carty, Michael; Goodbody, Rory; Schröder, Martina; Stack, Julianne; Moynagh, Paul N; Bowie, Andrew G

    2006-10-01

    Toll-like receptors discriminate between different pathogen-associated molecules and activate signaling cascades that lead to immune responses. The specificity of Toll-like receptor signaling occurs by means of adaptor proteins containing Toll-interleukin 1 receptor (TIR) domains. Activating functions have been assigned to four TIR adaptors: MyD88, Mal, TRIF and TRAM. Here we characterize a fifth TIR adaptor, SARM, as a negative regulator of TRIF-dependent Toll-like receptor signaling. Expression of SARM blocked gene induction 'downstream' of TRIF but not of MyD88. SARM associated with TRIF, and 'knockdown' of endogenous SARM expression by interfering RNA led to enhanced TRIF-dependent cytokine and chemokine induction. Thus, the fifth mammalian TIR adaptor SARM is a negative regulator of Toll-like receptor signaling.

  18. A putative TIR domain protein from Helicobacter pylori is dimeric in solution and interacts with human TLR adaptor Myeloid Differentiation Primary Response 88.

    PubMed

    Türköz, Burcu Kaplan

    2017-03-06

    Helicobacter pylori is an important human pathogen capable of causing persistent infection with minimal immune response. The first line of defense during H. pylori infection is through gastric epithelial cells that present Toll like receptors (TLR), a family of bacterial proteins which share homology with the Toll/IL1 receptor (TIR) domain. Bacterial TIR proteins (BTP) mimic human TIR domain proteins and act on MyD88 signaling pathways to suppress TLR signaling. H. pylori might also produce a similar protein. A putative H. pylori BTP was found based on sequence homology and the corresponding gene hp1437 was inserted into an expression vector in fusion with an N-terminal cleavable 6his-tag. The recombinant protein, 6his-HP1437 was purified using nickel affinity chromatography with a yield of 8 mg/ L culture. Oligomerization of HP1437 was investigated by size-exclusion chromatography. Our results show that HP1437 forms dimers in solution similar to other BTPs. Furthermore, GST pull down assays identify an interaction between HP1437 and human TIR domain adaptor MyD88. This study suggests that HP1437 has the characteristic features of BTPs and may play a direct role in reduced immune response against H. pylori by binding to MyD88 and pave the way for an in-depth characterization of this putative novel H. pylori virulence factor.

  19. Protein cold adaptation: Role of physico-chemical parameters in adaptation of proteins to low temperatures.

    PubMed

    Shokrollahzade, Soheila; Sharifi, Fatemeh; Vaseghi, Akbar; Faridounnia, Maryam; Jahandideh, Samad

    2015-10-21

    During years 2007 and 2008, we published three papers (Jahandideh, 2007a, JTB, 246, 159-166; Jahandideh, 2007b, JTB, 248, 721-726; Jahandideh, 2008, JTB, 255, 113-118) investigating sequence and structural parameters in adaptation of proteins to low temperatures. Our studies revealed important features in cold-adaptation of proteins. Here, we calculate values of a new set of physico-chemical parameters and perform a comparative systematic analysis on a more comprehensive database of psychrophilic-mesophilic homologous protein pairs. Our obtained results confirm that psychrophilicity rules are not merely the inverse rules of thermostability; for instance, although contact order is reported as a key feature in thermostability, our results have shown no significant difference between contact orders of psychrophilic proteins compared to mesophilic proteins. We are optimistic that these findings would help future efforts to propose a strategy for designing cold-adapted proteins.

  20. Matricellular Proteins in Cardiac Adaptation and Disease

    PubMed Central

    Frangogiannis, Nikolaos G.

    2015-01-01

    The term “matricellular proteins” describes a family of structurally unrelated extracellular macromolecules that, unlike structural matrix proteins, do not play a primary role in tissue architecture, but are induced following injury and modulate cell:cell and cell:matrix interactions. When released to the matrix, matricellular proteins associate with growth factors, cytokines and other bioactive effectors and bind to cell surface receptors transducing signaling cascades. Matricellular proteins are upregulated in the injured and remodeling heart and play an important role in regulation of inflammatory, reparative, fibrotic and angiogenic pathways. Thrombospondins (TSP)-1, -2 and -4, tenascin-C and –X, secreted protein acidic and rich in cysteine (SPARC), osteopontin, periostin and members of the CCN family (including CCN1 and CCN2/Connective Tissue Growth Factor) are involved in a variety of cardiac pathophysiologic conditions, including myocardial infarction, cardiac hypertrophy and fibrosis, aging-associated myocardial remodeling, myocarditis, diabetic cardiomyopathy and valvular disease. This review manuscript discusses the properties and characteristics of the matricellular proteins and presents our current knowledge on their role in cardiac adaptation and disease. Understanding the role of matricellular proteins in myocardial pathophysiology and identification of the functional domains responsible for their actions may lead to design of peptides with therapeutic potential for patients with heart disease. PMID:22535894

  1. Interleukin-33 and Mast Cells Bridge Innate and Adaptive Immunity: From the Allergologist’s Perspective

    PubMed Central

    Jang, Tae Young; Kim, Young Hyo

    2015-01-01

    Interleukin (IL) 33, a member of the IL-1 superfamily, is an “alarmin” protein and is secreted in its active form from damaged cells undergoing necrotic cell death. Mast cells are one of the main effector cell types in allergic disorders. They secrete a variety of mediators, including T helper 2 cytokines. As mast cells have high-affinity IgE receptors (FcεRI) on their surface, they can capture circulating IgE. IgE-bound mast cells degranulate large amounts of histamine, heparin, and proteases when they encounter antigens. As IL-33 is an important mediator of innate immunity and mast cells play an important role in adaptive immune responses, interactions between the two could link innate and adaptive immunity. IL-33 promotes the adhesion of mast cells to laminin, fibronectin, and vitronectin. IL-33 increases the expression of adhesion molecules, such as intracellular adhesion molecule-1 and vascular cell adhesion molecule-1, in endothelial cells, thus enhancing mast cell adhesion to blood vessel walls. IL-33 stimulates mast cell proliferation by activating the ST2/Myd88 pathway; increases mast cell survival by the activation of survival proteins such as Bcl-XL; and promotes the growth, development, and maturation of mast cell progenitors. IL-33 is also involved in the activation of mature mast cells and production of different proinflammatory cytokines. The interaction of IL-33 and mast cells could have important clinical implications in the field of clinical urology. Epithelial dysfunction and mast cells could play an important role in the pathogenesis of interstitial cystitis. Urinary levels of IL-33 significantly increase in patients with interstitial cystitis. In addition, the number of mast cells significantly increase in the urinary bladders of patients with interstitial cystitis. Therefore, inhibition of mast cell activation and degranulation in response to increase in IL-33 is a potential therapeutic target in the treatment of interstitial cystitis

  2. Viruses are a dominant driver of protein adaptation in mammals

    PubMed Central

    Enard, David; Cai, Le; Gwennap, Carina; Petrov, Dmitri A

    2016-01-01

    Viruses interact with hundreds to thousands of proteins in mammals, yet adaptation against viruses has only been studied in a few proteins specialized in antiviral defense. Whether adaptation to viruses typically involves only specialized antiviral proteins or affects a broad array of virus-interacting proteins is unknown. Here, we analyze adaptation in ~1300 virus-interacting proteins manually curated from a set of 9900 proteins conserved in all sequenced mammalian genomes. We show that viruses (i) use the more evolutionarily constrained proteins within the cellular functions they interact with and that (ii) despite this high constraint, virus-interacting proteins account for a high proportion of all protein adaptation in humans and other mammals. Adaptation is elevated in virus-interacting proteins across all functional categories, including both immune and non-immune functions. We conservatively estimate that viruses have driven close to 30% of all adaptive amino acid changes in the part of the human proteome conserved within mammals. Our results suggest that viruses are one of the most dominant drivers of evolutionary change across mammalian and human proteomes. DOI: http://dx.doi.org/10.7554/eLife.12469.001 PMID:27187613

  3. Mycobacterium tuberculosis heat shock proteins use diverse Toll-like receptor pathways to activate pro-inflammatory signals.

    PubMed

    Bulut, Yonca; Michelsen, Kathrin S; Hayrapetian, Linda; Naiki, Yoshikazu; Spallek, Ralf; Singh, Mahavir; Arditi, Moshe

    2005-06-03

    Although the Toll-like receptors used by Mycobacterium tuberculosis membrane and secreted factors are known, the pathways activated by M. tuberculosis heat shock proteins are not. An efficient immune response against the intracellular pathogen M. tuberculosis is critically dependent on rapid detection of the invading pathogen by the innate immune system and coordinated activation of the adaptive immune response. Macrophage phagocytosis of M. tuberculosis is accompanied by activation of the transcription factor NF-kappaB and secretion of inflammatory mediators that play an important role in granuloma formation and immune protection during M. tuberculosis infection. The interaction between M. tuberculosis and the various Toll-like receptors is complex, and it appears that distinct mycobacterial components may interact with different members of the Toll-like receptor family. Here we show that recombinant, purified, mycobacterial heat shock proteins 65 and 70 induce NF-kappaB activity in a dose-dependent manner in human endothelial cells. Furthermore, we show that whereas mycobacterial heat shock protein 65 signals exclusively through Toll-like receptor 4, heat shock protein 70 also signals through Toll-like receptor 2. Mycobacterial heat shock protein 65-induced NF-kappaB activation was MyD88-, TIRAP-, TRIF-, and TRAM-dependent and required the presence of MD-2. A better understanding of the recognition of mycobacterial heat shock proteins and their role in the host immune response to the pathogen may open the way to a better understanding of the immunological processes induced by this important human pathogen and the host-pathogen interactions and may help in the rational design of more effective vaccines or vaccine adjuvants.

  4. Identification, expression pattern and functional characterization of As-MyD88 in bacteria challenge and during different developmental stages of Artemia sinica.

    PubMed

    Qin, Tong; Zhao, Xinxin; Luan, Hong; Ba, Huazhong; Yang, Lei; Li, Zhenegmin; Hou, Lin; Zou, Xiangyang

    2015-05-01

    Myeloid differentiation factor 88 (MYD88), a key adapter protein in Toll-like receptor signaling, affects the immune response and the formation of the dorsal-ventral axis. Here, the 1555bp full-length cDNA of MyD88 from Artemia sinica (As-MyD88) was obtained. Molecular characterization revealed that the sequence includes an 1182bp open reading frame encoding a predicted protein of 393 amino acids. The predicted protein contains a death domain in the N-terminus, and box1 and 2 motifs of the TIR domain in the C-terminus. Real-time quantitative PCR, Western blotting and immunohistochemistry were used to determine the expression level, protein production and location of As-MYD88 during embryonic development and bacterial challenge. The highest expression level during embryonic development was at the 0h and 5h stages of A. sinica. As-MYD88 was remarkably upregulated after bacterial challenge. Our results suggested that As-MYD88 plays a vital role in response to bacterial challenge, and during post-diapause embryonic development of A. sinica.

  5. The Innate Immune Responses of Colonic Epithelial Cells to Trichuris muris Are Similar in Mouse Strains That Develop a Type 1 or Type 2 Adaptive Immune Response

    PubMed Central

    deSchoolmeester, Matthew L.; Manku, Harinder; Else, Kathryn J.

    2006-01-01

    Trichuris muris resides in intimate contact with its host, burrowing within cecal epithelial cells. However, whether the enterocyte itself responds innately to T. muris is unknown. This study investigated for the first time whether colonic intestinal epithelial cells (IEC) produce cytokines or chemokines following T. muris infection and whether divergence of the innate response could explain differentially polarized adaptive immune responses in resistant and susceptible mice. Increased expression of mRNA for the proinflammatory cytokines gamma interferon (IFN-γ) and tumor necrosis factor and the chemokine CCL2 (MCP-1) were seen after infection of susceptible and resistant strains, with the only difference in expression being a delayed increase in CCL2 in BALB/c IEC. These increases were ablated in MyD88−/− mice, and NF-κB p65 was phosphorylated in response to T. muris excretory/secretory products in the epithelial cell line CMT-93, suggesting involvement of the MyD88-NF-κB signaling pathway in IEC cytokine expression. These data reveal that IEC respond innately to T. muris. However, the minor differences identified between resistant and susceptible mice are unlikely to underlie the subsequent development of a susceptible type 1 (IFN-γ-dominated) or resistant type 2 (interleukin-4 [IL-4]/IL-13-dominated) adaptive immune response. PMID:17057095

  6. The innate immune responses of colonic epithelial cells to Trichuris muris are similar in mouse strains that develop a type 1 or type 2 adaptive immune response.

    PubMed

    deSchoolmeester, Matthew L; Manku, Harinder; Else, Kathryn J

    2006-11-01

    Trichuris muris resides in intimate contact with its host, burrowing within cecal epithelial cells. However, whether the enterocyte itself responds innately to T. muris is unknown. This study investigated for the first time whether colonic intestinal epithelial cells (IEC) produce cytokines or chemokines following T. muris infection and whether divergence of the innate response could explain differentially polarized adaptive immune responses in resistant and susceptible mice. Increased expression of mRNA for the proinflammatory cytokines gamma interferon (IFN-gamma) and tumor necrosis factor and the chemokine CCL2 (MCP-1) were seen after infection of susceptible and resistant strains, with the only difference in expression being a delayed increase in CCL2 in BALB/c IEC. These increases were ablated in MyD88-/- mice, and NF-kappaB p65 was phosphorylated in response to T. muris excretory/secretory products in the epithelial cell line CMT-93, suggesting involvement of the MyD88-NF-kappaB signaling pathway in IEC cytokine expression. These data reveal that IEC respond innately to T. muris. However, the minor differences identified between resistant and susceptible mice are unlikely to underlie the subsequent development of a susceptible type 1 (IFN-gamma-dominated) or resistant type 2 (interleukin-4 [IL-4]/IL-13-dominated) adaptive immune response.

  7. The role of adapter proteins in T cell activation.

    PubMed

    Koretzky, G A; Boerth, N J

    1999-12-01

    Engagement of antigen receptors on lymphocytes leads to a myriad of complex signal transduction cascades. Recently, work from several laboratories has led to the identification and characterization of novel adapter molecules, proteins with no intrinsic enzymatic activity but which integrate signal transduction pathways by mediating protein-protein interactions. Interestingly, it appears that many of these adapter proteins play as critical a role as the effector enzymes themselves in both lymphocyte development and activation. This review describes some of the biochemical and molecular features of several of these newly identified hematopoietic cell-specific adapter molecules highlighting their importance in regulating (both positively and negatively) signal transduction mediated by the T cell antigen receptor.

  8. TLR9 played a more important role than TLR2 in the combination of maltose-binding protein and BCG-induced Th1 activation.

    PubMed

    Ni, Weihua; Wang, Fang; Liu, Guomu; Zhang, Nannan; Yuan, Hongyan; Jie, Jing; Tai, Guixiang

    2016-11-01

    Our previous study demonstrated that maltose-binding protein (MBP) combined with BCG induced synergistic mouse Th1 activation in vivo. Here, to explore the mechanism of MBP combined with BCG on Th1 activation, mouse purified CD4(+) T cells were stimulated with MBP and BCG in vitro. The results showed that MBP combined with BCG synergistically increased IFN-γ production, accompanied with the upregulation of TLR2/9 expressions, suggesting that TLR2/9 were involved in the combination-induced Th1 activation. Next, TLR2 antibodies and TLR9 inhibitor were used to further analyze the effects of TLRs in Th1 activation. Results showed TLR2 antibody partly decreased MBP combined with BCG-induced IFN-γ production, MyD88 expression and IκB phosphorylation, indicating that TLR2-mediated MyD88-dependent pathway was involved in the MBP combined with BCG-induced Th1 activation. Moreover, MBP combined with BCG-induced Th1 activation was completely abrogated by TLR9 inhibitor, suggesting that TLR9-mediated MyD88-dependent pathway played a more important role than TLR2 in the combination-induced Th1 activation. Further study showed that TLR9 inhibitor downregulated TLR2 expression, suggesting that TLR9 signaling regulated TLR2 activation to favor Th1 resonse induced by MBP combined with BCG. Collectively, we demonstrated for the first time that the cross-talk of TLR2 and TLR9 triggered Th1 activation collaboratively and our findings provided valuable information about designing more effective adjuvant for cancer therapy.

  9. MAP1S Protein Regulates the Phagocytosis of Bacteria and Toll-like Receptor (TLR) Signaling.

    PubMed

    Shi, Ming; Zhang, Yifan; Liu, Leyuan; Zhang, Tingting; Han, Fang; Cleveland, Joseph; Wang, Fen; McKeehan, Wallace L; Li, Yu; Zhang, Dekai

    2016-01-15

    Phagocytosis is a critical cellular process for innate immune defense against microbial infection. The regulation of phagocytosis process is complex and has not been well defined. An intracellular molecule might regulate cell surface-initiated phagocytosis, but the underlying molecular mechanism is poorly understood (1). In this study, we found that microtubule-associated protein 1S (MAP1S), a protein identified recently that is involved in autophagy (2), is expressed primarily in macrophages. MAP1S-deficient macrophages are impaired in the phagocytosis of bacteria. Furthermore, we demonstrate that MAP1S interacts directly with MyD88, a key adaptor of Toll-like receptors (TLRs), upon TLR activation and affects the TLR signaling pathway. Intriguingly, we also observe that, upon TLR activation, MyD88 participates in autophagy processing in a MAP1S-dependent manner by co-localizing with MAP1 light chain 3 (MAP1-LC3 or LC3). Therefore, we reveal that an intracellular autophagy-related molecule of MAP1S controls bacterial phagocytosis through TLR signaling.

  10. Dietary protein requirements and adaptive advantages in athletes.

    PubMed

    Phillips, Stuart M

    2012-08-01

    Dietary guidelines from a variety of sources are generally congruent that an adequate dietary protein intake for persons over the age of 19 is between 0·8-0·9 g protein/kg body weight/d. According to the US/Canadian Dietary Reference Intakes, the RDA for protein of 0·8 g protein/kg/d is "...the average daily intake level that is sufficient to meet the nutrient requirement of nearly all [~98 %]… healthy individuals..." The panel also states that "...no additional dietary protein is suggested for healthy adults undertaking resistance or endurance exercise." These recommendations are in contrast to recommendations from the US and Canadian Dietetic Association: "Protein recommendations for endurance and strength trained athletes range from 1·2 to 1·7 g/kg/d." The disparity between those setting dietary protein requirements and those who might be considered to be making practical recommendations for athletes is substantial. This may reflect a situation where an adaptive advantage of protein intakes higher than recommended protein requirements exists. That population protein requirements are still based on nitrogen balance may also be a point of contention since achieving balanced nitrogen intake and excretion likely means little to an athlete who has the primary goal of exercise performance. The goal of the present review is to critically analyse evidence from both acute and chronic dietary protein-based studies in which athletic performance, or correlates thereof, have been measured. An attempt will be made to distinguish between protein requirements set by data from nitrogen balance studies, and a potential adaptive 'advantage' for athletes of dietary protein in excess of the RDA.

  11. An Adaptable Investigative Graduate Laboratory Course for Teaching Protein Purification

    ERIC Educational Resources Information Center

    Carroll, Christopher W.; Keller, Lani C.

    2014-01-01

    This adaptable graduate laboratory course on protein purification offers students the opportunity to explore a wide range of techniques while allowing the instructor the freedom to incorporate their own personal research interests. The course design involves two sequential purification schemes performed in a single semester. The first part…

  12. TLR9 re-expression in cancer cells extends the S-phase and stabilizes p16INK4a protein expression

    PubMed Central

    Parroche, P; Roblot, G; Le Calvez-Kelm, F; Tout, I; Marotel, M; Malfroy, M; Durand, G; McKay, J; Ainouze, M; Carreira, C; Allatif, O; Traverse-Glehen, A; Mendiola, M; Pozo-Kreilinger, J J; Caux, C; Tommasino, M; Goutagny, N; Hasan, U A

    2016-01-01

    Toll-like receptor 9 (TLR9) recognizes bacterial, viral or cell damage-associated DNA, which initiates innate immune responses. We have previously shown that TLR9 expression is downregulated in several viral induced cancers including HPV16-induced cervical neoplasia. Findings supported that downregulation of TLR9 expression is involved in loss of anti-viral innate immunity allowing an efficient viral replication. Here we investigated the role of TLR9 in altering the growth of transformed epithelial cells. Re-introducing TLR9 under the control of an exogenous promoter in cervical or head and neck cancer patient-derived cells reduced cell proliferation, colony formation and prevented independent growth of cells under soft agar. Neither TLR3, 7, nor the TLR adapter protein MyD88 expression had any effect on cell proliferation, indicating that TLR9 has a unique role in controlling cell growth. The reduction of cell growth was not due to apoptosis or necrosis, yet we observed that cells expressing TLR9 were slower in entering the S-phase of the cell cycle. Microarray-based gene expression profiling analysis highlighted a strong interferon (IFN) signature in TLR9-expressing head and neck cancer cells, with an increase in IFN-type I and IL-29 expression (IFN-type III), yet neither IFN-type I nor IL-29 production was responsible for the block in cell growth. We observed that the protein half-life of p16INK4a was increased in TLR9-expressing cells. Taken together, these data show for the first time that TLR9 affects the cell cycle by regulating p16INK4a post-translational modifications and highlights the role of TLR9 in the events that lead to carcinogenesis. PMID:27454079

  13. Adaptation in protein fitness landscapes is facilitated by indirect paths

    PubMed Central

    Wu, Nicholas C; Dai, Lei; Olson, C Anders; Lloyd-Smith, James O; Sun, Ren

    2016-01-01

    The structure of fitness landscapes is critical for understanding adaptive protein evolution. Previous empirical studies on fitness landscapes were confined to either the neighborhood around the wild type sequence, involving mostly single and double mutants, or a combinatorially complete subgraph involving only two amino acids at each site. In reality, the dimensionality of protein sequence space is higher (20L) and there may be higher-order interactions among more than two sites. Here we experimentally characterized the fitness landscape of four sites in protein GB1, containing 204 = 160,000 variants. We found that while reciprocal sign epistasis blocked many direct paths of adaptation, such evolutionary traps could be circumvented by indirect paths through genotype space involving gain and subsequent loss of mutations. These indirect paths alleviate the constraint on adaptive protein evolution, suggesting that the heretofore neglected dimensions of sequence space may change our views on how proteins evolve. DOI: http://dx.doi.org/10.7554/eLife.16965.001 PMID:27391790

  14. Faster-X adaptive protein evolution in house mice.

    PubMed

    Kousathanas, Athanasios; Halligan, Daniel L; Keightley, Peter D

    2014-04-01

    The causes of the large effect of the X chromosome in reproductive isolation and speciation have long been debated. The faster-X hypothesis predicts that X-linked loci are expected to have higher rates of adaptive evolution than autosomal loci if new beneficial mutations are on average recessive. Reproductive isolation should therefore evolve faster when contributing loci are located on the X chromosome. In this study, we have analyzed genome-wide nucleotide polymorphism data from the house mouse subspecies Mus musculus castaneus and nucleotide divergence from Mus famulus and Rattus norvegicus to compare rates of adaptive evolution for autosomal and X-linked protein-coding genes. We found significantly faster adaptive evolution for X-linked loci, particularly for genes with expression in male-specific tissues, but autosomal and X-linked genes with expression in female-specific tissues evolve at similar rates. We also estimated rates of adaptive evolution for genes expressed during spermatogenesis and found that X-linked genes that escape meiotic sex chromosome inactivation (MSCI) show rapid adaptive evolution. Our results suggest that faster-X adaptive evolution is either due to net recessivity of new advantageous mutations or due to a special gene content of the X chromosome, which regulates male function and spermatogenesis. We discuss how our results help to explain the large effect of the X chromosome in speciation.

  15. A role for the adaptor proteins TRAM and TRIF in toll-like receptor 2 signaling.

    PubMed

    Nilsen, Nadra J; Vladimer, Gregory I; Stenvik, Jørgen; Orning, M Pontus A; Zeid-Kilani, Maria V; Bugge, Marit; Bergstroem, Bjarte; Conlon, Joseph; Husebye, Harald; Hise, Amy G; Fitzgerald, Katherine A; Espevik, Terje; Lien, Egil

    2015-02-06

    Toll-like receptors (TLRs) are involved in sensing invading microbes by host innate immunity. TLR2 recognizes bacterial lipoproteins/lipopeptides, and lipopolysaccharide activates TLR4. TLR2 and TLR4 signal via the Toll/interleukin-1 receptor adaptors MyD88 and MAL, leading to NF-κB activation. TLR4 also utilizes the adaptors TRAM and TRIF, resulting in activation of interferon regulatory factor (IRF) 3. Here, we report a new role for TRAM and TRIF in TLR2 regulation and signaling. Interestingly, we observed that TLR2-mediated induction of the chemokine Ccl5 was impaired in TRAM or TRIF deficient macrophages. Inhibition of endocytosis reduced Ccl5 release, and the data also suggested that TRAM and TLR2 co-localize in early endosomes, supporting the hypothesis that signaling may occur from an intracellular compartment. Ccl5 release following lipoprotein challenge additionally involved the kinase Tbk-1 and Irf3, as well as MyD88 and Irf1. Induction of Interferon-β and Ccl4 by lipoproteins was also partially impaired in cells lacking TRIF cells. Our results show a novel function of TRAM and TRIF in TLR2-mediated signal transduction, and the findings broaden our understanding of how Toll/interleukin-1 receptor adaptor proteins may participate in signaling downstream from TLR2.

  16. Activation of MyD88 Signaling upon Staphylococcal Enterotoxin Binding to MHC Class II Molecules

    DTIC Science & Technology

    2011-01-20

    with magnetic beads were obtained from Miltenyi Biotech Inc. (Auburn, CA). The FITC-conjugated mAbs anti- CD14 , anti- CD3, and Ig isotype control...and Maloney murine leukemia virus reverse transcriptase was purchased from Perkin Elmer (Waltham, MA). Mouse anti-human CD14 and CD3 mAbs conjugated...harvested from the interface, washed, and suspended in RPMI 1640 medium. Monocytes ( CD14 +) were purified as previously described [33]. Briefly, total

  17. Adaptive Evolution in Rodent Seminal Vesicle Secretion Proteins

    PubMed Central

    Clark, Nathaniel L.; Nguyen, Eric D.; Swanson, Willie J.

    2008-01-01

    Proteins involved in reproductive fitness have evolved unusually rapidly across diverse groups of organisms. These reproductive proteins show unusually high rates of amino acid substitutions, suggesting that the proteins have been subject to positive selection. We sought to identify seminal fluid proteins experiencing adaptive evolution because such proteins are often involved in sperm competition, host immunity to pathogens, and manipulation of female reproductive physiology and behavior. We performed an evolutionary screen of the mouse prostate transcriptome for genes with elevated evolutionary rates between mouse and rat. We observed that secreted rodent prostate proteins evolve approximately twice as fast as nonsecreted proteins, remarkably similar to findings in the primate prostate and in the Drosophila male accessory gland. Our screen led us to identify and characterize a group of seminal vesicle secretion (Svs) proteins and to show that the gene Svs7 is evolving very rapidly, with many amino acid sites under positive selection. Another gene in this group, Svs5, showed evidence of branch-specific selection in the rat. We also found that Svs7 is under selection in primates and, by using three-dimensional models, demonstrated that the same regions have been under selection in both groups. Svs7 has been identified as mouse caltrin, a protein involved in sperm capacitation, the process responsible for the timing of changes in sperm activity and behavior, following ejaculation. We propose that the most likely explanation of the adaptive evolution of Svs7 that we have observed in rodents and primates stems from an important function in sperm competition. PMID:18718917

  18. The Extracellular IFI16 Protein Propagates Inflammation in Endothelial Cells Via p38 MAPK and NF-κB p65 Activation.

    PubMed

    Bawadekar, Mandar; De Andrea, Marco; Lo Cigno, Irene; Baldanzi, Gianluca; Caneparo, Valeria; Graziani, Andrea; Landolfo, Santo; Gariglio, Marisa

    2015-06-01

    The nuclear interferon-inducible-16 (IFI16) protein acts as DNA sensor in inflammasome signaling and as viral restriction factor. Following Herpesvirus infection or UV-B treatment, IFI16 delocalizes from the nucleus to the cytoplasm and is eventually released into the extracellular milieu. Recently, our group has demonstrated the occurrence of IFI16 in sera of systemic-autoimmune patients that hampers biological activity of endothelia through high-affinity membrane binding. As a continuation, we studied the activity of endotoxin-free recombinant IFI16 (rIFI16) protein on primary endothelial cells. rIFI16 caused dose/time-dependent upregulation of IL-6, IL-8, CCL2, CCL5, CCL20, ICAM1, VCAM1, and TLR4, while secretion of IL-6 and IL-8 was amplified with lipopolysaccharide synergy. Overall, cytokine secretion was completely inhibited in MyD88-silenced cells and partially by TLR4-neutralizing antibodies. By screening downstream signaling pathways, we found that IFI16 activates p38, p44/42 MAP kinases, and NF-kB. In particular, activation of p38 is an early event required for subsequent p44/42 MAP kinases activity and cytokine induction indicating a key role of this kinase in IFI16 signaling. Altogether, our data conclude that extracellular IFI16 protein alone or by synergy with lipopolysaccharide acts like Damage-associated molecular patterns propagating "Danger Signal" through MyD88-dependent TLR-pathway.

  19. Adaptive resolution simulation of an atomistic protein in MARTINI water

    NASA Astrophysics Data System (ADS)

    Zavadlav, Julija; Melo, Manuel Nuno; Marrink, Siewert J.; Praprotnik, Matej

    2014-02-01

    We present an adaptive resolution simulation of protein G in multiscale water. We couple atomistic water around the protein with mesoscopic water, where four water molecules are represented with one coarse-grained bead, farther away. We circumvent the difficulties that arise from coupling to the coarse-grained model via a 4-to-1 molecule coarse-grain mapping by using bundled water models, i.e., we restrict the relative movement of water molecules that are mapped to the same coarse-grained bead employing harmonic springs. The water molecules change their resolution from four molecules to one coarse-grained particle and vice versa adaptively on-the-fly. Having performed 15 ns long molecular dynamics simulations, we observe within our error bars no differences between structural (e.g., root-mean-squared deviation and fluctuations of backbone atoms, radius of gyration, the stability of native contacts and secondary structure, and the solvent accessible surface area) and dynamical properties of the protein in the adaptive resolution approach compared to the fully atomistically solvated model. Our multiscale model is compatible with the widely used MARTINI force field and will therefore significantly enhance the scope of biomolecular simulations.

  20. Inflammatory responses induced by lipopolysaccharide are amplified in primary human monocytes but suppressed in macrophages by complement protein C5a.

    PubMed

    Seow, Vernon; Lim, Junxian; Iyer, Abishek; Suen, Jacky Y; Ariffin, Juliana K; Hohenhaus, Daniel M; Sweet, Matthew J; Fairlie, David P

    2013-10-15

    Monocytes and macrophages are important innate immune cells equipped with danger-sensing receptors, including complement and Toll-like receptors. Complement protein C5a, acting via C5aR, is shown in this study to differentially modulate LPS-induced inflammatory responses in primary human monocytes versus macrophages. Whereas C5a enhanced secretion of LPS-induced IL-6 and TNF from primary human monocytes, C5a inhibited these responses while increasing IL-10 secretion in donor-matched human monocyte-derived macrophages differentiated by GM-CSF or M-CSF. Gαi/c-Raf/MEK/ERK signaling induced by C5a was amplified in macrophages but not in monocytes by LPS. Accordingly, the Gαi inhibitor pertussis toxin and MEK inhibitor U0126 blocked C5a inhibition of LPS-induced IL-6 and TNF production from macrophages. This synergy was independent of IL-10, PI3K, p38, JNK, and the differentiating agent. Furthermore, C5a did not inhibit IL-6 production from macrophages induced by other TLR agonists that are selective for Toll/IL-1R domain-containing adapter inducing IFN-β (polyinosinic-polycytidylic acid) or MyD88 (imiquimod), demonstrating selectivity for C5a regulation of LPS responses. Finally, suppression of proinflammatory cytokines IL-6 and TNF in macrophages did not compromise antimicrobial activity; instead, C5a enhanced clearance of the Gram-negative bacterial pathogen Salmonella enterica serovar Typhimurium from macrophages. C5aR is thus a regulatory switch that modulates TLR4 signaling via the Gαi/c-Raf/MEK/ERK signaling axis in human macrophages but not monocytes. The differential effects of C5a are consistent with amplifying monocyte proinflammatory responses to systemic danger signals, but attenuating macrophage cytokine responses (without compromising microbicidal activity), thereby restraining inflammatory responses to localized infections.

  1. Dietary protein for athletes: from requirements to optimum adaptation.

    PubMed

    Phillips, Stuart M; Van Loon, Luc J C

    2011-01-01

    Opinion on the role of protein in promoting athletic performance is divided along the lines of how much aerobic-based versus resistance-based activity the athlete undertakes. Athletes seeking to gain muscle mass and strength are likely to consume higher amounts of dietary protein than their endurance-trained counterparts. The main belief behind the large quantities of dietary protein consumption in resistance-trained athletes is that it is needed to generate more muscle protein. Athletes may require protein for more than just alleviation of the risk for deficiency, inherent in the dietary guidelines, but also to aid in an elevated level of functioning and possibly adaptation to the exercise stimulus. It does appear, however, that there is a good rationale for recommending to athletes protein intakes that are higher than the RDA. Our consensus opinion is that leucine, and possibly the other branched-chain amino acids, occupy a position of prominence in stimulating muscle protein synthesis; that protein intakes in the range of 1.3-1.8 g · kg(-1) · day(-1) consumed as 3-4 isonitrogenous meals will maximize muscle protein synthesis. These recommendations may also be dependent on training status: experienced athletes would require less, while more protein should be consumed during periods of high frequency/intensity training. Elevated protein consumption, as high as 1.8-2.0 g · kg(-1) · day(-1) depending on the caloric deficit, may be advantageous in preventing lean mass losses during periods of energy restriction to promote fat loss.

  2. Phenotypical Temperature Adaptation of Protein Turnover in Desert Annuals 1

    PubMed Central

    Smrcka, Alan V.; Szarek, Stan R.

    1986-01-01

    Protein synthesis and protein degradation rates were measured in three desert annual species at four different experimental temperatures. The taxa chosen for this study were the C3 winter annuals, Bowlesia incana Ruiz & Pavon and Plantago insularis Eastw., and a C4 summer annual, Atriplex elegans (Moq.) D. Dietr. Peak rates of protein synthesis correlated well with the preferred habitat temperatures of B. incana and A. elegans; optima occurred at 25 and 35°C, respectively. Plants of P. insularis showed an optimum protein synthesis rate at 35°C; however, this optimum rate was considerably lower than for the other two species. Higher activation energies for protein synthesis tended to parallel adaptation to higher temperature habitats. Responses of protein degradation to temperature in A. elegans and B. incana were consistent with their natural thermal regimes, when evaluated for the transition from 25 to 35°C. Again, protein degradation in P. insularis shows an intermediate response to temperature during the 25 to 35°C transition. PMID:16664583

  3. Functional constraints on adaptive evolution of protein ubiquitination sites

    PubMed Central

    Lu, Liang; Li, Yang; Liu, Zhongyang; Liang, Fengji; Guo, Feifei; Yang, Shuai; Wang, Dan; He, Yangzhige; Xiong, Jianghui; Li, Dong; He, Fuchu

    2017-01-01

    It is still unclear whether there exist functional constraints on the evolution of protein ubiquitination sites, because most previous studies regarded all protein ubiquitination sites as a whole or only focused on limited structural properties. We tried to clarify the relation between functional constraints and ubiquitination sites evolution. We investigated the evolutionary conservation of human ubiquitination sites in a broad evolutionary scale from G. gorilla to S. pombe, and we found that in organisms originated after the divergence of vertebrate, ubiquitination sites are more conserved than their flanking regions, while the opposite tendency is observed before this divergence time. By grouping the ubiquitination proteins into different functional categories, we confirm that many functional constraints like certain molecular functions, protein tissue expression specificity and protein connectivity in protein-protein interaction network enhance the evolutionary conservation of ubiquitination sites. Furthermore, by analyzing the gains of ubiquitination sites at different divergence time and their functional characters, we validate that the emergences of ubiquitination sites at different evolutionary time were also affected by the uncovered functional constraints. The above results suggest that functional constraints on the adaptive evolution of ubiquitination sites increase the opportunity for ubiquitination to synthetically regulate various cellular and developmental processes during evolution. PMID:28054638

  4. A Comparison of Rosetta Stones in Adapter Protein Families

    PubMed Central

    Kumar, Hulikal Shivashankara Santosh; Kumar, Vadlapudi

    2016-01-01

    The inventory of proteins used in different kingdoms appears surprisingly similar in all sequenced eukaryotic genome. Protein domains represent the basic evolutionary units that form proteins. Domain duplication and shuffling by recombination are probably the most important forces driving protein evolution and hence the complexity of the proteome. While the duplication of whole genes as well as domain encoding exons increases the abundance of domains in the proteome, domain shuffling increases versatility, i.e. the number of distinct contexts in which a domain can occur. In this study we considered five important adapter domain families namely WD40, KELCH, Ankyrin, PDZ and Pleckstrin Homology (PH domain) family for the comparison of Domain versatility, Abundance and domain sharing between them. We used ecological statistics methods such as Jaccard’s Similarity Index (JSI), Detrended Correspondence Analysis, k-Means clustering for the domain distribution data. We found high propensity of domain sharing between PH and PDZ. We found higher abundance of only few selected domains in PH, PDZ, ANK and KELCH families. We also found WD40 family with high versatility and less redundant domain occurrence, with less domain sharing. Hence, the assignments of functions to more orphan WD40 proteins that will help in the identification of suitable drug targets. PMID:28246462

  5. Determinants of Divergent Adaptive Immune Responses after Airway Sensitization with Ligands of Toll-Like Receptor 5 or Toll-Like Receptor 9

    PubMed Central

    Lee, Linda M.; Ji, Ming; Sinha, Meenal; Dong, Matthew B.; Ren, Xin; Wang, Yanli; Lowell, Clifford A.; Ghosh, Sankar; Locksley, Richard M.; DeFranco, Anthony L.

    2016-01-01

    Excessive type 2 helper T cell responses to environmental antigens can cause immunopathology such as asthma and allergy, but how such immune responses are induced remains unclear. We studied this process in the airways by immunizing mice intranasally with the antigen ovalbumin together with either of two Toll-like receptor (TLR) ligands. We found the TLR5 ligand flagellin promoted a type 2 helper T cell response, whereas, a TLR9 ligand CpG oligodeoxyribonucleotide (ODN) promoted a type 1 helper T cell response. CpG ODN induced mRNA encoding interleukin (IL)-12 p40, whereas, flagellin caused IL-33 secretion and induced mRNAs encoding IL-1 and thymic stromal lymphopoietin (TSLP). By using mice deficient in the TLR and IL-1R signaling molecule, myeloid differentiation primary response 88 (MyD88), in conventional dendritic cells (cDCs) and alveolar macrophages (AMs), and by cell sorting different lung populations after 2 hours of in vivo stimulation, we characterized the cell types that rapidly produced inflammatory cytokines in response to TLR stimulation. CpG ODN was likely recognized by TLR9 on cDCs and AMs, which made mRNA encoding IL-12. IL-12 was necessary for the subsequent innate and adaptive interferon-γ production. In contrast, flagellin stimulated multiple cells of hematopoietic and non-hematopoietic origin, including AMs, DCs, monocytes, and lung epithelial cells. AMs were largely responsible for IL-1α, whereas lung epithelial cells made TSLP. Multiple hematopoietic cells, including AMs, DCs, and monocytes contributed to other cytokines, including IL-1β and TNFα. MyD88-dependent signals, likely through IL-1R and IL-33R, and MyD88-independent signals, likely from TSLP, were necessary in cDCs for promotion of the early IL-4 response by CD4 T cells in the draining lymph node. Thus, the cell types that responded to TLR ligands were a critical determinant of the innate cytokines produced and the character of the resulting adaptive immune response in the

  6. The E3 ubiquitin ligase Nrdp1 'preferentially' promotes TLR-mediated production of type I interferon.

    PubMed

    Wang, Chen; Chen, Taoyong; Zhang, Jia; Yang, Mingjin; Li, Nan; Xu, Xiongfei; Cao, Xuetao

    2009-07-01

    E3 ubiquitin ligases are important in both innate and adaptive immunity. Here we report that Nrdp1, an E3 ubiquitin ligase, inhibited the production of proinflammatory cytokines but increased interferon-beta production in Toll-like receptor-triggered macrophages by suppressing adaptor MyD88-dependent activation of transcription factors NF-kappaB and AP-1 while promoting activation of the kinase TBK1 and transcription factor IRF3. Nrdp1 directly bound and polyubiquitinated MyD88 and TBK1, which led to degradation of MyD88 and activation of TBK1. Knockdown of Nrdp1 inhibited the degradation of MyD88 and the activation of TBK1 and IRF3. Nrdp1-transgenic mice showed resistance to lipopolysaccharide-induced endotoxin shock and to infection with vesicular stomatitis virus. Our data suggest that Nrdp1 functions as both an adaptor protein and an E3 unbiquitin ligase to regulate TLR responses in different ways.

  7. Protein phosphorylation and regulation of adaptive responses in bacteria.

    PubMed Central

    Stock, J B; Ninfa, A J; Stock, A M

    1989-01-01

    Bacteria continuously adapt to changes in their environment. Responses are largely controlled by signal transduction systems that contain two central enzymatic components, a protein kinase that uses adenosine triphosphate to phosphorylate itself at a histidine residue and a response regulator that accepts phosphoryl groups from the kinase. This conserved phosphotransfer chemistry is found in a wide range of bacterial species and operates in diverse systems to provide different regulatory outputs. The histidine kinases are frequently membrane receptor proteins that respond to environmental signals and phosphorylate response regulators that control transcription. Four specific regulatory systems are discussed in detail: chemotaxis in response to attractant and repellent stimuli (Che), regulation of gene expression in response to nitrogen deprivation (Ntr), control of the expression of enzymes and transport systems that assimilate phosphorus (Pho), and regulation of outer membrane porin expression in response to osmolarity and other culture conditions (Omp). Several additional systems are also examined, including systems that control complex developmental processes such as sporulation and fruiting-body formation, systems required for virulent infections of plant or animal host tissues, and systems that regulate transport and metabolism. Finally, an attempt is made to understand how cross-talk between parallel phosphotransfer pathways can provide a global regulatory curcuitry. PMID:2556636

  8. BtpB, a novel Brucella TIR-containing effector protein with immune modulatory functions.

    PubMed

    Salcedo, Suzana P; Marchesini, María I; Degos, Clara; Terwagne, Matthieu; Von Bargen, Kristine; Lepidi, Hubert; Herrmann, Claudia K; Santos Lacerda, Thais L; Imbert, Paul R C; Pierre, Philippe; Alexopoulou, Lena; Letesson, Jean-Jacques; Comerci, Diego J; Gorvel, Jean-Pierre

    2013-01-01

    Several bacterial pathogens have TIR domain-containing proteins that contribute to their pathogenesis. We identified a second TIR-containing protein in Brucella spp. that we have designated BtpB. We show it is a potent inhibitor of TLR signaling, probably via MyD88. BtpB is a novel Brucella effector that is translocated into host cells and interferes with activation of dendritic cells. In vivo mouse studies revealed that BtpB is contributing to virulence and control of local inflammatory responses with relevance in the establishment of chronic brucellosis. Together, our results show that BtpB is a novel Brucella effector that plays a major role in the modulation of host innate immune response during infection.

  9. Effects of protein conformation in docking: improved pose prediction through protein pocket adaptation

    NASA Astrophysics Data System (ADS)

    Jain, Ajay N.

    2009-06-01

    Computational methods for docking ligands have been shown to be remarkably dependent on precise protein conformation, where acceptable results in pose prediction have been generally possible only in the artificial case of re-docking a ligand into a protein binding site whose conformation was determined in the presence of the same ligand (the "cognate" docking problem). In such cases, on well curated protein/ligand complexes, accurate dockings can be returned as top-scoring over 75% of the time using tools such as Surflex-Dock. A critical application of docking in modeling for lead optimization requires accurate pose prediction for novel ligands, ranging from simple synthetic analogs to very different molecular scaffolds. Typical results for widely used programs in the "cross-docking case" (making use of a single fixed protein conformation) have rates closer to 20% success. By making use of protein conformations from multiple complexes, Surflex-Dock yields an average success rate of 61% across eight pharmaceutically relevant targets. Following docking, protein pocket adaptation and rescoring identifies single pose families that are correct an average of 67% of the time. Consideration of the best of two pose families (from alternate scoring regimes) yields a 75% mean success rate.

  10. Effects of protein conformation in docking: improved pose prediction through protein pocket adaptation.

    PubMed

    Jain, Ajay N

    2009-06-01

    Computational methods for docking ligands have been shown to be remarkably dependent on precise protein conformation, where acceptable results in pose prediction have been generally possible only in the artificial case of re-docking a ligand into a protein binding site whose conformation was determined in the presence of the same ligand (the "cognate" docking problem). In such cases, on well curated protein/ligand complexes, accurate dockings can be returned as top-scoring over 75% of the time using tools such as Surflex-Dock. A critical application of docking in modeling for lead optimization requires accurate pose prediction for novel ligands, ranging from simple synthetic analogs to very different molecular scaffolds. Typical results for widely used programs in the "cross-docking case" (making use of a single fixed protein conformation) have rates closer to 20% success. By making use of protein conformations from multiple complexes, Surflex-Dock yields an average success rate of 61% across eight pharmaceutically relevant targets. Following docking, protein pocket adaptation and rescoring identifies single pose families that are correct an average of 67% of the time. Consideration of the best of two pose families (from alternate scoring regimes) yields a 75% mean success rate.

  11. Mastoparan, a G protein agonist peptide, differentially modulates TLR4- and TLR2-mediated signaling in human endothelial cells and murine macrophages.

    PubMed

    Lentschat, Arnd; Karahashi, Hisae; Michelsen, Kathrin S; Thomas, Lisa S; Zhang, Wenxuan; Vogel, Stefanie N; Arditi, Moshe

    2005-04-01

    Previous studies have implicated a role for heterotrimeric G protein-coupled signaling in B cells, monocytes, and macrophages stimulated with LPS and have shown that G proteins coimmunoprecipitate with membrane-bound CD14. In this study, we have extended these observations in human dermal microvessel endothelial cells (HMEC) that lack membrane-bound CD14 and in murine macrophages to define further the role of heterotrimeric G proteins in TLR signaling. Using the wasp venom-derived peptide, mastoparan, to disrupt G protein-coupled signaling, we identified a G protein-dependent signaling pathway in HMEC stimulated with TLR4 agonists that is necessary for the activation of p38 phosphorylation and kinase activity, NF-kappaB and IL-6 transactivation, and IL-6 secretion. In contrast, HMEC activation by TLR2 agonists, TNF-alpha, or IL-1beta was insensitive to mastoparan. In the murine macrophage cell line, RAW 264.7, and in primary murine macrophages, G protein dysregulation by mastoparan resulted in significant inhibition of LPS-induced signaling leading to both MyD88-dependent and MyD88-independent gene expression, while TLR2-mediated gene expression was not significantly inhibited. In addition to inhibition of TLR4-mediated MAPK phosphorylation in macrophages, mastoparan blunted IL-1R-associated kinase-1 kinase activity induced by LPS, but not by TLR2 agonists, yet failed to affect phosphorylation of Akt by phosphoinositol-3-kinase induced by either TLR2- or TLR4-mediated signaling. These data confirm the importance of heterotrimeric G proteins in TLR4-mediated responses in cells that use either soluble or membrane-associated CD14 and reveal a level of TLR and signaling pathway specificity not previously appreciated.

  12. Differential effect of early antibiotic intervention on bacterial fermentation patterns and mucosal gene expression in the colon of pigs under diets with different protein levels.

    PubMed

    Zhang, Chuanjian; Yu, Miao; Yang, Yuxiang; Mu, Chunlong; Su, Yong; Zhu, Weiyun

    2017-03-01

    The study aimed to evaluate the effects of early antibiotic intervention (EAI) on bacterial fermentation patterns and mucosal immune markers in the colon of pigs with different protein level diets. Eighteen litters of piglets at day (d) 7 were fed creep feed without or with growth promoting antibiotics until d 42. At d 42, pigs within each group were further randomly assigned to a normal- or low-crude protein (CP) diet. At d 77 and d 120, five pigs per group were slaughtered for analyzing colonic bacteria, metabolites, and mucosal gene expressions. Results showed that low-CP diet increased propionate and butyrate concentrations at d 77 but reduced ammonia and phenol concentrations (P < 0.05). EAI increased p-cresol and indole concentrations under normal-CP diet at d 77 (P < 0.05). Low-CP diet significantly affected (P < 0.05) some bacteria groups (Firmicutes, Clostridium cluster IV, Clostridium cluster XIVa, Escherichia coli, and Lactobacillus), but EAI showed limited effects. Low-CP diet down-regulated gene expressions of pro-inflammatory cytokines, toll-like receptor (TLR4), myeloid differentiating factor 88 (MyD88), and nuclear factor-κB p65 (NF-κB p65) (P < 0.05). EAI up-regulated mRNA expressions of interleukin-8 (IL-8) and interferon-γ (IFN-γ) under normal-CP diet at d 77 (P < 0.05). Furthermore, reductions of E. coli and ammonia under low-CP diet were positively correlated with down-regulated gene expressions of pro-inflammatory cytokines, which were positively correlated with the down-regulated TLR4-MyD88-NF-κB signaling pathway. In conclusion, EAI had short-term effects under normal-CP diet with increased aromatic amino acid fermentation and gene expressions of pro-inflammatory cytokines. Low-CP diet markedly reduced protein fermentation, modified microbial communities, and down-regulated gene expressions of pro-inflammatory cytokines possibly via down-regulating TLR4-MyD88-NF-κB signaling pathway.

  13. Brucella Induces an Unfolded Protein Response via TcpB That Supports Intracellular Replication in Macrophages

    PubMed Central

    Smith, Judith A.; Khan, Mike; Magnani, Diogo D.; Harms, Jerome S.; Durward, Marina; Radhakrishnan, Girish K.; Liu, Yi-Ping; Splitter, Gary A.

    2013-01-01

    Brucella melitensis is a facultative intracellular bacterium that causes brucellosis, the most prevalent zoonosis worldwide. The Brucella intracellular replicative niche in macrophages and dendritic cells thwarts immune surveillance and complicates both therapy and vaccine development. Currently, host-pathogen interactions supporting Brucella replication are poorly understood. Brucella fuses with the endoplasmic reticulum (ER) to replicate, resulting in dramatic restructuring of the ER. This ER disruption raises the possibility that Brucella provokes an ER stress response called the Unfolded Protein Response (UPR). In this study, B. melitensis infection up regulated expression of the UPR target genes BiP, CHOP, and ERdj4, and induced XBP1 mRNA splicing in murine macrophages. These data implicate activation of all 3 major signaling pathways of the UPR. Consistent with previous reports, XBP1 mRNA splicing was largely MyD88-dependent. However, up regulation of CHOP, and ERdj4 was completely MyD88 independent. Heat killed Brucella stimulated significantly less BiP, CHOP, and ERdj4 expression, but induced XBP1 splicing. Although a Brucella VirB mutant showed relatively intact UPR induction, a TcpB mutant had significantly compromised BiP, CHOP and ERdj4 expression. Purified TcpB, a protein recently identified to modulate microtubules in a manner similar to paclitaxel, also induced UPR target gene expression and resulted in dramatic restructuring of the ER. In contrast, infection with the TcpB mutant resulted in much less ER structural disruption. Finally, tauroursodeoxycholic acid, a pharmacologic chaperone that ameliorates the UPR, significantly impaired Brucella replication in macrophages. Together, these results suggest Brucella induces a UPR, via TcpB and potentially other factors, that enables its intracellular replication. Thus, the UPR may provide a novel therapeutic target for the treatment of brucellosis. These results also have implications for other

  14. Pre-Sleep Protein Ingestion to Improve the Skeletal Muscle Adaptive Response to Exercise Training.

    PubMed

    Trommelen, Jorn; van Loon, Luc J C

    2016-11-28

    Protein ingestion following resistance-type exercise stimulates muscle protein synthesis rates, and enhances the skeletal muscle adaptive response to prolonged resistance-type exercise training. As the adaptive response to a single bout of resistance exercise extends well beyond the first couple of hours of post-exercise recovery, recent studies have begun to investigate the impact of the timing and distribution of protein ingestion during more prolonged recovery periods. Recent work has shown that overnight muscle protein synthesis rates are restricted by the level of amino acid availability. Protein ingested prior to sleep is effectively digested and absorbed, and thereby stimulates muscle protein synthesis rates during overnight recovery. When applied during a prolonged period of resistance-type exercise training, protein supplementation prior to sleep can further augment gains in muscle mass and strength. Recent studies investigating the impact of pre-sleep protein ingestion suggest that at least 40 g of protein is required to display a robust increase in muscle protein synthesis rates throughout overnight sleep. Furthermore, prior exercise allows more of the pre-sleep protein-derived amino acids to be utilized for de novo muscle protein synthesis during sleep. In short, pre-sleep protein ingestion represents an effective dietary strategy to improve overnight muscle protein synthesis, thereby improving the skeletal muscle adaptive response to exercise training.

  15. Pre-Sleep Protein Ingestion to Improve the Skeletal Muscle Adaptive Response to Exercise Training

    PubMed Central

    Trommelen, Jorn; van Loon, Luc J. C.

    2016-01-01

    Protein ingestion following resistance-type exercise stimulates muscle protein synthesis rates, and enhances the skeletal muscle adaptive response to prolonged resistance-type exercise training. As the adaptive response to a single bout of resistance exercise extends well beyond the first couple of hours of post-exercise recovery, recent studies have begun to investigate the impact of the timing and distribution of protein ingestion during more prolonged recovery periods. Recent work has shown that overnight muscle protein synthesis rates are restricted by the level of amino acid availability. Protein ingested prior to sleep is effectively digested and absorbed, and thereby stimulates muscle protein synthesis rates during overnight recovery. When applied during a prolonged period of resistance-type exercise training, protein supplementation prior to sleep can further augment gains in muscle mass and strength. Recent studies investigating the impact of pre-sleep protein ingestion suggest that at least 40 g of protein is required to display a robust increase in muscle protein synthesis rates throughout overnight sleep. Furthermore, prior exercise allows more of the pre-sleep protein-derived amino acids to be utilized for de novo muscle protein synthesis during sleep. In short, pre-sleep protein ingestion represents an effective dietary strategy to improve overnight muscle protein synthesis, thereby improving the skeletal muscle adaptive response to exercise training. PMID:27916799

  16. Interleukin-1-receptor-associated kinase 2 (IRAK2)-mediated interleukin-1-dependent nuclear factor kappaB transactivation in Saos2 cells requires the Akt/protein kinase B kinase.

    PubMed Central

    Cenni, Vittoria; Sirri, Alessandra; De Pol, Anto; Maraldi, Nadir Mario; Marmiroli, Sandra

    2003-01-01

    The post-receptor pathway that leads to nuclear factor kappaB (NF-kappaB) activation begins with the assembly of a membrane-proximal complex among the interleukin 1 (IL-1) receptors and the adaptor molecules, myeloid differentiation protein 88 (MyD88), IL-1-receptor-associated kinases (IRAKs) and tumour-necrosis-factor-receptor-associated factor 6. Eventually, phosphorylation of the inhibitor of NF-kappaB (IkappaB) by the IkappaB kinases releases NF-kappaB, which translocates to the nucleus and modulates gene expression. In this paper, we report that IRAK2 and MyD88, but not IRAK1, interact physically with Akt, as demonstrated by co-immunoprecipitation and pull-down experiments. Interestingly, the association of Akt with recombinant IRAK2 is decreased by stimulation with IL-1, and is favoured by pre-treatment with phosphatase. Likewise, Akt association with IRAK2 is increased considerably by overexpression of PTEN (phosphatase and tensin homologue deleted on chromosome 10), while it is completely abrogated by overexpression of phosphoinositide-dependent protein kinase 1. These data indicate that Akt takes part in the formation of the signalling complex that conveys the signal from the IL-1 receptors to NF-kappaB, a step that is much more membrane-proximal than was reported previously. We also demonstrate that Akt activity is necessary for IL-1-dependent NF-kappaB transactivation, since a kinase-defective mutant of Akt impairs IRAK2- and MyD88-dependent, but not IRAK1-dependent, NF-kappaB activity, as monitored by a gene reporter assay. Accordingly, IRAK2 failed to trigger inducible nitric oxide synthase and IL-1beta production in cells expressing dominant-negative Akt. However, NF-kappaB binding to DNA was not affected by inhibition of Akt, indicating that Akt regulates NF-kappaB at a level distinct from the dissociation of p65 from IkappaBalpha and its translocation to the nucleus, possibly involving phosphorylation of the p65 transactivation domain. PMID:12906710

  17. Staphylococcus aureus surface proteins involved in adaptation to oxacillin identified using a novel cell shaving approach.

    PubMed

    Solis, Nestor; Parker, Benjamin L; Kwong, Stephen M; Robinson, Gareth; Firth, Neville; Cordwell, Stuart J

    2014-06-06

    Staphylococcus aureus is a Gram-positive pathogen responsible for a variety of infections, and some strains are resistant to virtually all classes of antibiotics. Cell shaving proteomics using a novel probability scoring algorithm to compare the surfaceomes of the methicillin-resistant, laboratory-adapted S. aureus COL strain with a COL strain in vitro adapted to high levels of oxacillin (APT). APT displayed altered cell morphology compared with COL and increased aggregation in biofilm assays. Increased resistance to β-lactam antibiotics was observed, but adaptation to oxacillin did not confer multidrug resistance. Analysis of the S. aureus COL and APT surfaceomes identified 150 proteins at a threshold determined by the scoring algorithm. Proteins unique to APT included the LytR-CpsA-Psr (LCP) domain-containing MsrR and SACOL2302. Quantitative RT-PCR showed increased expression of sacol2302 in APT grown with oxacillin (>6-fold compared with COL). Overexpression of sacol2302 in COL to levels consistent with APT (+ oxacillin) did not influence biofilm formation or β-lactam resistance. Proteomics using iTRAQ and LC-MS/MS identified 1323 proteins (∼50% of the theoretical S. aureus proteome), and cluster analysis demonstrated elevated APT abundances of LCP proteins, capsule and peptidoglycan biosynthesis proteins, and proteins involved in wall remodelling. Adaptation to oxacillin also induced urease proteins, which maintained culture pH compared to COL. These results show that S. aureus modifies surface architecture in response to antibiotic adaptation.

  18. The MRL proteins: adapting cell adhesion, migration and growth.

    PubMed

    Coló, Georgina P; Lafuente, Esther M; Teixidó, Joaquin

    2012-01-01

    MIG-10, RIAM and Lamellipodin (Lpd) are the founding members of the MRL family of multi-adaptor molecules. These proteins have common domain structures but display distinct functions in cell migration and adhesion, signaling, and in cell growth. The binding of RIAM with active Rap1 and with talin provides these MRL molecules with important regulatory roles on integrin-mediated cell adhesion and migration. Furthermore, RIAM and Lpd can regulate actin dynamics through their binding to actin regulatory Ena/VASP proteins. Recent data generated with the Drosophila MRL ortholog called Pico and with RIAM in melanoma cells indicate that these proteins can also regulate cell growth. As MRL proteins represent a relatively new family, many questions on their structure-function relationships remain unanswered, including regulation of their expression, post-translational modifications, new interactions, involvement in signaling and their knockout mice phenotype.

  19. Adaptive Resistance and Differential Protein Expression of Salmonella enterica Serovar Enteritidis Biofilms Exposed to Benzalkonium Chloride▿

    PubMed Central

    Mangalappalli-Illathu, Anil K.; Korber, Darren R.

    2006-01-01

    The development of adaptive resistance of Salmonella enterica serovar Enteritidis ATCC 4931 biofilms following exposure to benzalkonium chloride (BC) either continuously (1 μg ml−1) or intermittently (10 μg ml−1 for 10 min daily) was examined. Biofilms adapted to BC over a 144-h period could survive a normally lethal BC challenge (500 μg ml−1 for 10 min) and then regrow, as determined by increases in biofilm thickness, total biomass, and the ratio of the viable biomass to the nonviable biomass. Exposure of untreated control biofilms to the lethal BC challenge resulted in biofilm erosion and cell death. Proteins found to be up-regulated following BC adaptation were those involved in energy metabolism (TpiA and Eno), amino acid and protein biosynthesis (WrbA, TrxA, RplL, Tsf, Tuf, DsbA, and RpoZ), nutrient binding (FruB), adaptation (CspA), detoxification (Tpx, SodB, and a probable peroxidase), and degradation of 1,2-propanediol (PduJ and PduA). A putative universal stress protein (YnaF) was also found to be up-regulated. Proteins involved in proteolysis (DegQ), cell envelope formation (RfbH), adaptation (UspA), heat shock response (DnaK), and broad regulatory functions (Hns) were found to be down-regulated following adaptation. An overall increase in cellular protein biosynthesis was deduced from the significant up-regulation of ribosomal subunit proteins, translation elongation factors, and amino acid biosynthesis protein and down-regulation of serine endoprotease. The cold shock response, stress response, and detoxification are suggested to play roles in the adaptive resistance of Salmonella serovar Enteritidis biofilms to BC. PMID:16940079

  20. Adaptive resistance and differential protein expression of Salmonella enterica serovar Enteritidis biofilms exposed to benzalkonium chloride.

    PubMed

    Mangalappalli-Illathu, Anil K; Korber, Darren R

    2006-11-01

    The development of adaptive resistance of Salmonella enterica serovar Enteritidis ATCC 4931 biofilms following exposure to benzalkonium chloride (BC) either continuously (1 microg ml(-1)) or intermittently (10 microg ml(-1) for 10 min daily) was examined. Biofilms adapted to BC over a 144-h period could survive a normally lethal BC challenge (500 microg ml(-1) for 10 min) and then regrow, as determined by increases in biofilm thickness, total biomass, and the ratio of the viable biomass to the nonviable biomass. Exposure of untreated control biofilms to the lethal BC challenge resulted in biofilm erosion and cell death. Proteins found to be up-regulated following BC adaptation were those involved in energy metabolism (TpiA and Eno), amino acid and protein biosynthesis (WrbA, TrxA, RplL, Tsf, Tuf, DsbA, and RpoZ), nutrient binding (FruB), adaptation (CspA), detoxification (Tpx, SodB, and a probable peroxidase), and degradation of 1,2-propanediol (PduJ and PduA). A putative universal stress protein (YnaF) was also found to be up-regulated. Proteins involved in proteolysis (DegQ), cell envelope formation (RfbH), adaptation (UspA), heat shock response (DnaK), and broad regulatory functions (Hns) were found to be down-regulated following adaptation. An overall increase in cellular protein biosynthesis was deduced from the significant up-regulation of ribosomal subunit proteins, translation elongation factors, and amino acid biosynthesis protein and down-regulation of serine endoprotease. The cold shock response, stress response, and detoxification are suggested to play roles in the adaptive resistance of Salmonella serovar Enteritidis biofilms to BC.

  1. Purification, cloning, and functional characterization of a novel immunomodulatory protein from Antrodia camphorata (bitter mushroom) that exhibits TLR2-dependent NF-κB activation and M1 polarization within murine macrophages.

    PubMed

    Sheu, Fuu; Chien, Po-Jung; Hsieh, Kuang-Yang; Chin, Kah-Lock; Huang, Wan-Ting; Tsao, Chiao-Yin; Chen, Yin-Fang; Cheng, Hui-Chung; Chang, Hui-Hsin

    2009-05-27

    A new immunomodulatory protein, designated ACA, was purified from the mycelium extract of Antrodia camphorata , a well-known folk medicine bitter mushroom in Taiwan, and N-terminally sequenced. By taking advantage of its N-terminal amino acid sequence, the full-length ACA gene was cloned using rapid amplification of cDNA ends (RACE) approach. This gene encodes a 136 amino acid protein that is homologous to the phytotoxic proteins from fungi. On the basis of the data of N-terminal sequencing and N-glycosidase F treatment, the native ACA was confirmed to be a glycoprotein. The similarity in activation of TLR4-deficient macrophages by both the native ACA and recombinant ACA (rACA) suggested that the glycosyl group(s) of the native ACA was insignificant in macrophage activation. Moreover, the failure of rACA to induce TLR2-deficient macrophages and to activate the RAW 264.7 macrophages transfected with the dominate-negative MyD88 (dnMyD88) indicated that the ACA-mediated macrophage activation was TLR2/MyD88 dependent. Microarray assay of the ACA-activated NFκB-related gene expression showed that rACA demonstrated a LPS-mimetic proinflammatory response toward RAW 264.7 macrophages. Furthermore, rACA enhanced phagocytosis activity and CD86 (B7-2) expression as well as induced TNF-α and IL-1β production within murine peritoneal macrophages. A time-dependent induction of mRNA expression of cytokines TNF-α, IL-1β, IL-6, and IL-12 as well as chemokines CCL3, CCL4, CCL5, and CCL10, but not IL-10, CCL17, CCL22, and CCL24, was observed after the ACA treatment of the macrophages. These results proposed that ACA exhibited M1 polarization and differentiation in macrophages. Thus, ACA is an important immunomodulatory protein of A. camphorata.

  2. Gain-of-Function Mutations in the Toll-Like Receptor Pathway: TPL2-Mediated ERK1/ERK2 MAPK Activation, a Path to Tumorigenesis in Lymphoid Neoplasms?

    PubMed Central

    Rousseau, Simon; Martel, Guy

    2016-01-01

    Lymphoid neoplasms form a family of cancers affecting B-cells, T-cells, and NK cells. The Toll-Like Receptor (TLR) signaling adapter molecule MYD88 is the most frequently mutated gene in these neoplasms. This signaling adaptor relays signals from TLRs to downstream effector pathways such as the Nuclear Factor kappa B (NFκB) and Mitogen Activated Protein Kinase (MAPK) pathways to regulate innate immune responses. Gain-of-function mutations such as MYD88[L265P] activate downstream signaling pathways in absence of cognate ligands for TLRs, resulting in increased cellular proliferation and survival. This article reports an analysis of non-synonymous somatic mutations found in the TLR signaling network in lymphoid neoplasms. In accordance with previous reports, mutations map to MYD88 pro-inflammatory signaling and not TRIF-mediated Type I IFN production. Interestingly, the analysis of somatic mutations found downstream of the core TLR-signaling network uncovered a strong association with the ERK1/2 MAPK cascade. In support of this analysis, heterologous expression of MYD88[L265P] in HEK293 cells led to ERK1/2 MAPK phosphorylation in addition to NFκB activation. Moreover, this activation is dependent on the protein kinase Tumor Promoting Locus 2 (TPL2), activated downstream of the IKK complex. Activation of ERK1/2 would then lead to activation, amongst others, of MYC and hnRNPA1, two proteins previously shown to contribute to tumor formation in lymphoid neoplasms. Taken together, this analysis suggests that TLR-mediated ERK1/2 activation via TPL2 may be a novel path to tumorigenesis. Therefore, the hypothesis proposed is that inhibition of ERK1/2 MAPK activation would prevent tumor growth downstream of MYD88[L265]. It will be interesting to test whether pharmacological inhibitors of this pathway show efficacy in primary tumor cells derived from hematologic malignancies such as Waldenstrom's Macroglobulinemia, where the majority of the cells carry the MYD88[L265P

  3. Catalysis of protein folding by chaperones accelerates evolutionary dynamics in adapting cell populations.

    PubMed

    Cetinbaş, Murat; Shakhnovich, Eugene I

    2013-01-01

    Although molecular chaperones are essential components of protein homeostatic machinery, their mechanism of action and impact on adaptation and evolutionary dynamics remain controversial. Here we developed a physics-based ab initio multi-scale model of a living cell for population dynamics simulations to elucidate the effect of chaperones on adaptive evolution. The 6-loci genomes of model cells encode model proteins, whose folding and interactions in cellular milieu can be evaluated exactly from their genome sequences. A genotype-phenotype relationship that is based on a simple yet non-trivially postulated protein-protein interaction (PPI) network determines the cell division rate. Model proteins can exist in native and molten globule states and participate in functional and all possible promiscuous non-functional PPIs. We find that an active chaperone mechanism, whereby chaperones directly catalyze protein folding, has a significant impact on the cellular fitness and the rate of evolutionary dynamics, while passive chaperones, which just maintain misfolded proteins in soluble complexes have a negligible effect on the fitness. We find that by partially releasing the constraint on protein stability, active chaperones promote a deeper exploration of sequence space to strengthen functional PPIs, and diminish the non-functional PPIs. A key experimentally testable prediction emerging from our analysis is that down-regulation of chaperones that catalyze protein folding significantly slows down the adaptation dynamics.

  4. Thermal adaptability of Kluyveromyces marxianus in recombinant protein production

    PubMed Central

    2013-01-01

    Background Kluyveromyces marxianus combines the ease of genetic manipulation and fermentation with the ability to efficiently secrete high molecular weight proteins, performing eukaryotic post-translational modifications. It is able to grow efficiently in a wide range of temperatures. The secretion performances were analyzed in the host K. marxianus L3 in the range between 5°C and 40°C by means of 3 different reporter proteins, since temperature appears a key parameter for production and secretion of recombinant proteins. Results The recombinant strains were able to grow up to 40°C and, along the tested temperature interval (5-40°C), the specific growth rates (μ) were generally lower as compared to those of the untransformed strain. Biomass yields were slightly affected by temperature, with the highest values reached at 15°C and 30°C. The secretion of the endogenous β-fructofuranosidase, used as an internal control, was efficient in the range of the tested temperature, as evaluated by assaying the enzyme activity in the culture supernatants. The endogenous β-fructofuranosidase production was temperature dependent, with the highest yield at 30°C. The heterologous proteins HSA, GAA and Sod1p were all successfully produced and secreted between 5°C and 40°C, albeit each one presented a different optimal production temperature (15, 40, 5-30°C for HSA, GAA and Sod1p, respectively). Conclusions K. marxianus L3 has been identified as a promising and flexible cell factory. In a sole host, the optimization of growth temperatures for the efficient secretion of each individual protein can be carried out over a wide range of temperatures. PMID:23587421

  5. Prolonged Adaptation to a Low or High Protein Diet Does Not Modulate Basal Muscle Protein Synthesis Rates – A Substudy

    PubMed Central

    Hursel, Rick; Martens, Eveline A. P.; Gonnissen, Hanne K. J.; Hamer, Henrike M.; Senden, Joan M. G.; van Loon, Luc J. C.; Westerterp-Plantenga, Margriet S.

    2015-01-01

    Background Based on controlled 36 h experiments a higher dietary protein intake causes a positive protein balance and a negative fat balance. A positive net protein balance may support fat free mass accrual. However, few data are available on the impact of more prolonged changes in habitual protein intake on whole-body protein metabolism and basal muscle protein synthesis rates. Objective To assess changes in whole-body protein turnover and basal muscle protein synthesis rates following 12 weeks of adaptation to a low versus high dietary protein intake. Methods A randomized parallel study was performed in 40 subjects who followed either a high protein (2.4 g protein/kg/d) or low protein (0.4 g protein/kg/d) energy-balanced diet (30/35/35% or 5/60/35% energy from protein/carbohydrate/fat) for a period of 12 weeks. A subgroup of 7 men and 8 women (body mass index: 22.8±2.3 kg/m2, age: 24.3±4.9 y) were selected to evaluate the impact of prolonged adaptation to either a high or low protein intake on whole body protein metabolism and basal muscle protein synthesis rates. After the diet, subjects received continuous infusions with L-[ring-2H5]phenylalanine and L-[ring-2H2]tyrosine in an overnight fasted state, with blood samples and muscle biopsies being collected to assess post-absorptive whole-body protein turnover and muscle protein synthesis rates in vivo in humans. Results After 12 weeks of intervention, whole-body protein balance in the fasted state was more negative in the high protein treatment when compared with the low protein treatment (-4.1±0.5 vs -2.7±0.6 μmol phenylalanine/kg/h;P<0.001). Whole-body protein breakdown (43.0±4.4 vs 37.8±3.8 μmol phenylalanine/kg/h;P<0.03), synthesis (38.9±4.2 vs 35.1±3.6 μmol phenylalanine/kg/h;P<0.01) and phenylalanine hydroxylation rates (4.1±0.6 vs 2.7±0.6 μmol phenylalanine/kg/h;P<0.001) were significantly higher in the high vs low protein group. Basal muscle protein synthesis rates were maintained on a low

  6. Adaptive Protein Evolution in Animals and the Effective Population Size Hypothesis

    PubMed Central

    Galtier, Nicolas

    2016-01-01

    The rate at which genomes adapt to environmental changes and the prevalence of adaptive processes in molecular evolution are two controversial issues in current evolutionary genetics. Previous attempts to quantify the genome-wide rate of adaptation through amino-acid substitution have revealed a surprising diversity of patterns, with some species (e.g. Drosophila) experiencing a very high adaptive rate, while other (e.g. humans) are dominated by nearly-neutral processes. It has been suggested that this discrepancy reflects between-species differences in effective population size. Published studies, however, were mainly focused on model organisms, and relied on disparate data sets and methodologies, so that an overview of the prevalence of adaptive protein evolution in nature is currently lacking. Here we extend existing estimators of the amino-acid adaptive rate by explicitly modelling the effect of favourable mutations on non-synonymous polymorphism patterns, and we apply these methods to a newly-built, homogeneous data set of 44 non-model animal species pairs. Data analysis uncovers a major contribution of adaptive evolution to the amino-acid substitution process across all major metazoan phyla—with the notable exception of humans and primates. The proportion of adaptive amino-acid substitution is found to be positively correlated to species effective population size. This relationship, however, appears to be primarily driven by a decreased rate of nearly-neutral amino-acid substitution because of more efficient purifying selection in large populations. Our results reveal that adaptive processes dominate the evolution of proteins in most animal species, but do not corroborate the hypothesis that adaptive substitutions accumulate at a faster rate in large populations. Implications regarding the factors influencing the rate of adaptive evolution and positive selection detection in humans vs. other organisms are discussed. PMID:26752180

  7. Protein engineering reveals ancient adaptive replacements in isocitrate dehydrogenase

    PubMed Central

    Dean, Antony M.; Golding, G. Brian

    1997-01-01

    Evolutionary analysis indicates that eubacterial NADP-dependent isocitrate dehydrogenases (EC 1.1.1.42) first evolved from an NAD-dependent precursor about 3.5 billion years ago. Selection in favor of utilizing NADP was probably a result of niche expansion during growth on acetate, where isocitrate dehydrogenase provides 90% of the NADPH necessary for biosynthesis. Amino acids responsible for differing coenzyme specificities were identified from x-ray crystallographic structures of Escherichia coli isocitrate dehydrogenase and the distantly related Thermus thermophilus NAD-dependent isopropylmalate dehydrogenase. Site-directed mutagenesis at sites lining the coenzyme binding pockets has been used to invert the coenzyme specificities of both enzymes. Reconstructed ancestral sequences indicate that these replacements are ancestral. Hence the adaptive history of molecular evolution is amenable to experimental investigation. PMID:9096353

  8. Protein Secondary Structure Prediction Using Local Adaptive Techniques in Training Neural Networks

    NASA Astrophysics Data System (ADS)

    Aik, Lim Eng; Zainuddin, Zarita; Joseph, Annie

    2008-01-01

    One of the most significant problems in computer molecular biology today is how to predict a protein's three-dimensional structure from its one-dimensional amino acid sequence or generally call the protein folding problem and difficult to determine the corresponding protein functions. Thus, this paper involves protein secondary structure prediction using neural network in order to solve the protein folding problem. The neural network used for protein secondary structure prediction is multilayer perceptron (MLP) of the feed-forward variety. The training set are taken from the protein data bank which are 120 proteins while 60 testing set is the proteins which were chosen randomly from the protein data bank. Multiple sequence alignment (MSA) is used to get the protein similar sequence and Position Specific Scoring matrix (PSSM) is used for network input. The training process of the neural network involves local adaptive techniques. Local adaptive techniques used in this paper comprises Learning rate by sign changes, SuperSAB, Quickprop and RPROP. From the simulation, the performance for learning rate by Rprop and Quickprop are superior to all other algorithms with respect to the convergence time. However, the best result was obtained using Rprop algorithm.

  9. Optimizing intramuscular adaptations to aerobic exercise: effects of carbohydrate restriction and protein supplementation on mitochondrial biogenesis.

    PubMed

    Margolis, Lee M; Pasiakos, Stefan M

    2013-11-01

    Mitochondrial biogenesis is a critical metabolic adaptation to aerobic exercise training that results in enhanced mitochondrial size, content, number, and activity. Recent evidence has shown that dietary manipulation can further enhance mitochondrial adaptations to aerobic exercise training, which may delay skeletal muscle fatigue and enhance exercise performance. Specifically, studies have demonstrated that combining carbohydrate restriction (endogenous and exogenous) with a single bout of aerobic exercise potentiates the beneficial effects of exercise on markers of mitochondrial biogenesis. Additionally, studies have demonstrated that high-quality protein supplementation enhances anabolic skeletal muscle intracellular signaling and mitochondrial protein synthesis following a single bout of aerobic exercise. Mitochondrial biogenesis is stimulated by complex intracellular signaling pathways that appear to be primarily regulated by 5'AMP-activated protein kinase and p38 mitogen-activated protein kinase mediated through proliferator-activated γ receptor co-activator 1 α activation, resulting in increased mitochondrial DNA expression and enhanced skeletal muscle oxidative capacity. However, the mechanisms by which concomitant carbohydrate restriction and dietary protein supplementation modulates mitochondrial adaptations to aerobic exercise training remains unclear. This review summarizes intracellular regulation of mitochondrial biogenesis and the effects of carbohydrate restriction and protein supplementation on mitochondrial adaptations to aerobic exercise.

  10. Exercise training-induced gender-specific heat shock protein adaptations in human skeletal muscle.

    PubMed

    Morton, James P; Holloway, Kathryn; Woods, Paul; Cable, Nigel T; Burniston, Jatin; Evans, Louise; Kayani, Anna C; McArdle, Anne

    2009-02-01

    This study investigates the effects of short-term endurance training on heat shock protein (HSP) adaptations of male and female human skeletal muscle. The data demonstrate that females did not respond to continuous or interval training in terms of increasing HSP content of the vastus lateralis muscle. In contrast, males displayed HSP adaptations to both training interventions. These data provide a platform for future human studies to examine a potential gender-specific stress response to exercise.

  11. High-resolution mapping of protein concentration reveals principles of proteome architecture and adaptation.

    PubMed

    Levy, Emmanuel D; Kowarzyk, Jacqueline; Michnick, Stephen W

    2014-05-22

    A single yeast cell contains a hundred million protein molecules. How these proteins are organized to orchestrate living processes is a central question in biology. To probe this organization in vivo, we measured the local concentration of proteins based on the strength of their nonspecific interactions with a neutral reporter protein. We first used a cytosolic reporter and measured local concentrations for ~2,000 proteins in S. cerevisiae, with accuracy comparable to that of mass spectrometry. Localizing the reporter to membranes specifically increased the local concentration measured for membrane proteins. Comparing the concentrations measured by both reporters revealed that encounter frequencies between proteins are primarily dictated by their abundances. However, to change these encounter frequencies and restructure the proteome, as in adaptation, we find that changes in localization have more impact than changes in abundance. These results highlight how protein abundance and localization contribute to proteome organization and reorganization.

  12. Adaptation.

    PubMed

    Broom, Donald M

    2006-01-01

    The term adaptation is used in biology in three different ways. It may refer to changes which occur at the cell and organ level, or at the individual level, or at the level of gene action and evolutionary processes. Adaptation by cells, especially nerve cells helps in: communication within the body, the distinguishing of stimuli, the avoidance of overload and the conservation of energy. The time course and complexity of these mechanisms varies. Adaptive characters of organisms, including adaptive behaviours, increase fitness so this adaptation is evolutionary. The major part of this paper concerns adaptation by individuals and its relationships to welfare. In complex animals, feed forward control is widely used. Individuals predict problems and adapt by acting before the environmental effect is substantial. Much of adaptation involves brain control and animals have a set of needs, located in the brain and acting largely via motivational mechanisms, to regulate life. Needs may be for resources but are also for actions and stimuli which are part of the mechanism which has evolved to obtain the resources. Hence pigs do not just need food but need to be able to carry out actions like rooting in earth or manipulating materials which are part of foraging behaviour. The welfare of an individual is its state as regards its attempts to cope with its environment. This state includes various adaptive mechanisms including feelings and those which cope with disease. The part of welfare which is concerned with coping with pathology is health. Disease, which implies some significant effect of pathology, always results in poor welfare. Welfare varies over a range from very good, when adaptation is effective and there are feelings of pleasure or contentment, to very poor. A key point concerning the concept of individual adaptation in relation to welfare is that welfare may be good or poor while adaptation is occurring. Some adaptation is very easy and energetically cheap and

  13. Measuring and Detecting Molecular Adaptation in Codon Usage Against Nonsense Errors During Protein Translation

    PubMed Central

    Gilchrist, Michael A.; Shah, Premal; Zaretzki, Russell

    2009-01-01

    Codon usage bias (CUB) has been documented across a wide range of taxa and is the subject of numerous studies. While most explanations of CUB invoke some type of natural selection, most measures of CUB adaptation are heuristically defined. In contrast, we present a novel and mechanistic method for defining and contextualizing CUB adaptation to reduce the cost of nonsense errors during protein translation. Using a model of protein translation, we develop a general approach for measuring the protein production cost in the face of nonsense errors of a given allele as well as the mean and variance of these costs across its coding synonyms. We then use these results to define the nonsense error adaptation index (NAI) of the allele or a contiguous subset thereof. Conceptually, the NAI value of an allele is a relative measure of its elevation on a specific and well-defined adaptive landscape. To illustrate its utility, we calculate NAI values for the entire coding sequence and across a set of nonoverlapping windows for each gene in the Saccharomyces cerevisiae S288c genome. Our results provide clear evidence of adaptation to reduce the cost of nonsense errors and increasing adaptation with codon position and expression. The magnitude and nature of this adaptation are also largely consistent with simulation results in which nonsense errors are the only selective force driving CUB evolution. Because NAI is derived from mechanistic models, it is both easier to interpret and more amenable to future refinement than other commonly used measures of codon bias. Further, our approach can also be used as a starting point for developing other mechanistically derived measures of adaptation such as for translational accuracy. PMID:19822731

  14. Metabolic and proteomic study of NS0 myeloma cell line following the adaptation to protein-free medium.

    PubMed

    de la Luz-Hernández, K R; Rojas-del Calvo, L; Rabasa-Legón, Y; Lage-Castellanos, A; Castillo-Vitlloch, A; Díaz, J; Gaskell, S

    2008-07-21

    Proteomics and metabolomics technologies are potentially useful tool for the study of the very complex process of cell adaptation to protein-free medium. In this work, we used the iTRAQ technology to analyze different protein levels in adapted and non-adapted NS0 myeloma cell line. Several proteins with differential expression profile were characterized and quantified. Carbohydrate metabolism, protein synthesis and membrane transport were the principal pathways that change after the adaptation. Changes in lactate production rate with respect to glucose consumption rate were observed according to the changes observed by proteomic.

  15. Influence of Histidine-Containing Tags on the Biodistribution of ADAPT Scaffold Proteins.

    PubMed

    Lindbo, Sarah; Garousi, Javad; Åstrand, Mikael; Honarvar, Hadis; Orlova, Anna; Hober, Sophia; Tolmachev, Vladimir

    2016-03-16

    Engineered scaffold proteins (ESP) are high-affinity binders that can be used as probes for radionuclide imaging. Histidine-containing tags enable both efficient purification of ESP and radiolabeling with (99m)Tc(CO)3. Earlier studies demonstrated that the use of a histidine-glutamate-histidine-glutamate-histidine-glutamate (HE)3-tag instead of the commonly used hexahistidine (H6)-tag reduces hepatic uptake of radiolabeled ESP and short peptides. Here, we investigated the influence of histidine-containing tags on the biodistribution of a novel type of ESP, ADAPTs. A series of anti-HER2 ADAPT probes having H6- or (HE)3-tags in the N-termini were prepared. The constructs, (HE)3-ADAPT6 and H6-ADAPT6, were labeled with two different nuclides, (99m)Tc or (111)In. The labeling with (99m)Tc(CO)3 utilized the histidine-containing tags, while (111)In was attached through a maleimido derivative of DOTA conjugated to the N-terminus. For (111)In-labeled ADAPTs, the use of (HE)3 provided a significantly (p < 0.05) lower hepatic uptake at 1 h after injection, but there was no significant difference in hepatic uptake of (111)In-(HE)3-ADAPT6 and H6-ADAPT6 at later time points. Interestingly, in the case of (99m)Tc, (99m)Tc(CO)3-H6-ADAPT6 provided significantly (p < 0.05) lower uptake in a number of normal tissues and was more suitable as an imaging probe. Thus, the influence of histidine-containing tags on the biodistribution of the novel ADAPT scaffold proteins was different compared to its influence on other ESPs studied so far. Apparently, the effect of a histidine-containing tag on the biodistribution is highly dependent on the scaffold composition of the ESP.

  16. Biomolecular Mechanism, Cloning, Sequencing and Analysis of Adaptive Reflection cDNAs and Proteins from Squid

    DTIC Science & Technology

    2010-05-03

    drivers of these adaptive changes in reflectance that we will investigate include electromotive ion fluxes, pH change and exposures to metal ions and...found in L. pealeii. As is the case for the other reflectin proteins (16), Ref-Lp1 and 2 contain a series of conserved subdomains (SDs). Ref-Lp1

  17. Assessment of the protein quality of 15 new northern adapted cultivars of quality protein maize using amino acid analysis.

    PubMed

    Zarkadas, C G; Hamilton, R I; Yu, Z R; Choi, V K; Khanizadeh, S; Rose, N G; Pattison, P L

    2000-11-01

    Amino acid determinations were carried out on 15 new northern adapted cultivars of quality protein maize (QPM) containing opaque-2 modifier genes to ascertain whether their amino acid scoring patterns could be used to select high-lysine QPM genotypes and to assess their protein quality. Total protein in these cultivars ranged from 8.0 to 10.2% compared to two commercial maize varieties, Dekalb DK435 (7.9%) and Pioneer 3925 (10.3%). Four of these QPM genotypes, QPM-C26, QPM-C21, QPM-C79, and QPM-C59, contained high levels of lysine (4.43-4.58 g of lysine/100 g of protein), whereas the remaining varied from 3.43 to 4.21 g of lysine/100 g of protein, compared to Dekalb DK435 and Pioneer 3925, which contained 2.9 and 3. 1 g of lysine/100 g of protein, respectively. Although lysine is the first limiting amino acid in QPM inbreds, the high-lysine QPM genotypes may supply approximately 70.2-72.6% of human protein requirements, compared to 46.2% for Dekalb DK435 and 50.1% for Pioneer 3925, 55-63% for oats, and 59-60.3% for barley. Northern adapted QPM genotypes may have the potential to increase their lysine content even further, either by an increase in specific high-lysine-containing nonzein proteins, such as the synthesis of factor EF-1a, or by a further reduction in the 19 and 22 kDa alpha-zein in the endosperm or both. This knowledge could assist maize breeders in the selection of new high-performance QPM genotypes with improved protein quality and quantity.

  18. Adaptation of Chinese hamster ovary cells to low culture temperature: cell growth and recombinant protein production.

    PubMed

    Yoon, Sung Kwan; Hong, Jong Kwang; Choo, Seung Ho; Song, Ji Yong; Park, Hong Woo; Lee, Gyun Min

    2006-04-20

    Recombinant Chinese hamster ovary (rCHO) cells producing erythropoietin (EPO) and rCHO cells producing follicle-stimulating hormone (FSH) showed a significant increase in specific productivity (q) when grown at 32 degrees C compared to 37 degrees C. However, low culture temperature suppressed cell growth, and therefore, did not increase volumetric productivity as much as q. In an attempt to increase the volumetric productivity through improvement of hypothermic growth, EPO producing rCHO (CHO-EPO) cells and FSH producing rCHO (CHO-FSH) cells were adapted at 32 degrees C in a repeated batch mode using spinner flasks. Cell growth of both CHO-EPO and CHO-FSH gradually improved during adaptation at 32 degrees C. Specific growth rates of CHO-EPO and CHO-FSH cells at 32 degrees C, through adaptation, were increased by 73% and 20%, respectively. During adaptation at 32 degrees C, mRNA levels of cold-inducible RNA-binding protein (CIRP) of both rCHO cell lines did not change significantly, suggesting that CIRP expression may not be the only cause for growth suppression at low culture temperature. Unlike cell growth, the recombinant protein production of both rCHO cell lines was not increased during adaptation due to decreased specific productivities. The specific EPO productivity and specific FSH productivity were decreased by 49% and 22%, respectively. Southern blot analyses showed that the decreased specific productivities were not due to the loss of foreign gene copies. Taken together, improvement of hypothermic cell growth by adaptation does not appear to be applicable for enhanced recombinant protein production, since specific productivity decreases during adaptation to the low culture temperature.

  19. Dynamics and Adaptive Benefits of Protein Domain Emergence and Arrangements during Plant Genome Evolution

    PubMed Central

    Kersting, Anna R.; Bornberg-Bauer, Erich; Moore, Andrew D.; Grath, Sonja

    2012-01-01

    Plant genomes are generally very large, mostly paleopolyploid, and have numerous gene duplicates and complex genomic features such as repeats and transposable elements. Many of these features have been hypothesized to enable plants, which cannot easily escape environmental challenges, to rapidly adapt. Another mechanism, which has recently been well described as a major facilitator of rapid adaptation in bacteria, animals, and fungi but not yet for plants, is modular rearrangement of protein-coding genes. Due to the high precision of profile-based methods, rearrangements can be well captured at the protein level by characterizing the emergence, loss, and rearrangements of protein domains, their structural, functional, and evolutionary building blocks. Here, we study the dynamics of domain rearrangements and explore their adaptive benefit in 27 plant and 3 algal genomes. We use a phylogenomic approach by which we can explain the formation of 88% of all arrangements by single-step events, such as fusion, fission, and terminal loss of domains. We find many domains are lost along every lineage, but at least 500 domains are novel, that is, they are unique to green plants and emerged more or less recently. These novel domains duplicate and rearrange more readily within their genomes than ancient domains and are overproportionally involved in stress response and developmental innovations. Novel domains more often affect regulatory proteins and show a higher degree of structural disorder than ancient domains. Whereas a relatively large and well-conserved core set of single-domain proteins exists, long multi-domain arrangements tend to be species-specific. We find that duplicated genes are more often involved in rearrangements. Although fission events typically impact metabolic proteins, fusion events often create new signaling proteins essential for environmental sensing. Taken together, the high volatility of single domains and complex arrangements in plant genomes

  20. Evolutionary Dynamics on Protein Bi-stability Landscapes can Potentially Resolve Adaptive Conflicts

    PubMed Central

    Sikosek, Tobias; Bornberg-Bauer, Erich; Chan, Hue Sun

    2012-01-01

    Experimental studies have shown that some proteins exist in two alternative native-state conformations. It has been proposed that such bi-stable proteins can potentially function as evolutionary bridges at the interface between two neutral networks of protein sequences that fold uniquely into the two different native conformations. Under adaptive conflict scenarios, bi-stable proteins may be of particular advantage if they simultaneously provide two beneficial biological functions. However, computational models that simulate protein structure evolution do not yet recognize the importance of bi-stability. Here we use a biophysical model to analyze sequence space to identify bi-stable or multi-stable proteins with two or more equally stable native-state structures. The inclusion of such proteins enhances phenotype connectivity between neutral networks in sequence space. Consideration of the sequence space neighborhood of bridge proteins revealed that bi-stability decreases gradually with each mutation that takes the sequence further away from an exactly bi-stable protein. With relaxed selection pressures, we found that bi-stable proteins in our model are highly successful under simulated adaptive conflict. Inspired by these model predictions, we developed a method to identify real proteins in the PDB with bridge-like properties, and have verified a clear bi-stability gradient for a series of mutants studied by Alexander et al. (Proc Nat Acad Sci USA 2009, 106:21149–21154) that connect two sequences that fold uniquely into two different native structures via a bridge-like intermediate mutant sequence. Based on these findings, new testable predictions for future studies on protein bi-stability and evolution are discussed. PMID:23028272

  1. Adapt

    NASA Astrophysics Data System (ADS)

    Bargatze, L. F.

    2015-12-01

    Active Data Archive Product Tracking (ADAPT) is a collection of software routines that permits one to generate XML metadata files to describe and register data products in support of the NASA Heliophysics Virtual Observatory VxO effort. ADAPT is also a philosophy. The ADAPT concept is to use any and all available metadata associated with scientific data to produce XML metadata descriptions in a consistent, uniform, and organized fashion to provide blanket access to the full complement of data stored on a targeted data server. In this poster, we present an application of ADAPT to describe all of the data products that are stored by using the Common Data File (CDF) format served out by the CDAWEB and SPDF data servers hosted at the NASA Goddard Space Flight Center. These data servers are the primary repositories for NASA Heliophysics data. For this purpose, the ADAPT routines have been used to generate data resource descriptions by using an XML schema named Space Physics Archive, Search, and Extract (SPASE). SPASE is the designated standard for documenting Heliophysics data products, as adopted by the Heliophysics Data and Model Consortium. The set of SPASE XML resource descriptions produced by ADAPT includes high-level descriptions of numerical data products, display data products, or catalogs and also includes low-level "Granule" descriptions. A SPASE Granule is effectively a universal access metadata resource; a Granule associates an individual data file (e.g. a CDF file) with a "parent" high-level data resource description, assigns a resource identifier to the file, and lists the corresponding assess URL(s). The CDAWEB and SPDF file systems were queried to provide the input required by the ADAPT software to create an initial set of SPASE metadata resource descriptions. Then, the CDAWEB and SPDF data repositories were queried subsequently on a nightly basis and the CDF file lists were checked for any changes such as the occurrence of new, modified, or deleted

  2. Fast automated protein NMR data collection and assignment by ADAPT-NMR on Bruker spectrometers

    NASA Astrophysics Data System (ADS)

    Lee, Woonghee; Hu, Kaifeng; Tonelli, Marco; Bahrami, Arash; Neuhardt, Elizabeth; Glass, Karen C.; Markley, John L.

    2013-11-01

    ADAPT-NMR (Assignment-directed Data collection Algorithm utilizing a Probabilistic Toolkit in NMR) supports automated NMR data collection and backbone and side chain assignment for [U-13C, U-15N]-labeled proteins. Given the sequence of the protein and data for the orthogonal 2D 1H-15N and 1H-13C planes, the algorithm automatically directs the collection of tilted plane data from a variety of triple-resonance experiments so as to follow an efficient pathway toward the probabilistic assignment of 1H, 13C, and 15N signals to specific atoms in the covalent structure of the protein. Data collection and assignment calculations continue until the addition of new data no longer improves the assignment score. ADAPT-NMR was first implemented on Varian (Agilent) spectrometers [A. Bahrami, M. Tonelli, S.C. Sahu, K.K. Singarapu, H.R. Eghbalnia, J.L. Markley, PLoS One 7 (2012) e33173]. Because of broader interest in the approach, we present here a version of ADAPT-NMR for Bruker spectrometers. We have developed two AU console programs (ADAPT_ORTHO_run and ADAPT_NMR_run) that run under TOPSPIN Versions 3.0 and higher. To illustrate the performance of the algorithm on a Bruker spectrometer, we tested one protein, chlorella ubiquitin (76 amino acid residues), that had been used with the Varian version: the Bruker and Varian versions achieved the same level of assignment completeness (98% in 20 h). As a more rigorous evaluation of the Bruker version, we tested a larger protein, BRPF1 bromodomain (114 amino acid residues), which yielded an automated assignment completeness of 86% in 55 h. Both experiments were carried out on a 500 MHz Bruker AVANCE III spectrometer equipped with a z-gradient 5 mm TCI probe. ADAPT-NMR is available at http://pine.nmrfam.wisc.edu/ADAPT-NMR in the form of pulse programs, the two AU programs, and instructions for installation and use.

  3. Fast automated protein NMR data collection and assignment by ADAPT-NMR on Bruker spectrometers.

    PubMed

    Lee, Woonghee; Hu, Kaifeng; Tonelli, Marco; Bahrami, Arash; Neuhardt, Elizabeth; Glass, Karen C; Markley, John L

    2013-11-01

    ADAPT-NMR (Assignment-directed Data collection Algorithm utilizing a Probabilistic Toolkit in NMR) supports automated NMR data collection and backbone and side chain assignment for [U-(13)C, U-(15)N]-labeled proteins. Given the sequence of the protein and data for the orthogonal 2D (1)H-(15)N and (1)H-(13)C planes, the algorithm automatically directs the collection of tilted plane data from a variety of triple-resonance experiments so as to follow an efficient pathway toward the probabilistic assignment of (1)H, (13)C, and (15)N signals to specific atoms in the covalent structure of the protein. Data collection and assignment calculations continue until the addition of new data no longer improves the assignment score. ADAPT-NMR was first implemented on Varian (Agilent) spectrometers [A. Bahrami, M. Tonelli, S.C. Sahu, K.K. Singarapu, H.R. Eghbalnia, J.L. Markley, PLoS One 7 (2012) e33173]. Because of broader interest in the approach, we present here a version of ADAPT-NMR for Bruker spectrometers. We have developed two AU console programs (ADAPT_ORTHO_run and ADAPT_NMR_run) that run under TOPSPIN Versions 3.0 and higher. To illustrate the performance of the algorithm on a Bruker spectrometer, we tested one protein, chlorella ubiquitin (76 amino acid residues), that had been used with the Varian version: the Bruker and Varian versions achieved the same level of assignment completeness (98% in 20 h). As a more rigorous evaluation of the Bruker version, we tested a larger protein, BRPF1 bromodomain (114 amino acid residues), which yielded an automated assignment completeness of 86% in 55 h. Both experiments were carried out on a 500 MHz Bruker AVANCE III spectrometer equipped with a z-gradient 5 mm TCI probe. ADAPT-NMR is available at http://pine.nmrfam.wisc.edu/ADAPT-NMR in the form of pulse programs, the two AU programs, and instructions for installation and use.

  4. Intra-plastid protein trafficking: how plant cells adapted prokaryotic mechanisms to the eukaryotic condition.

    PubMed

    Celedon, Jose M; Cline, Kenneth

    2013-02-01

    Protein trafficking and localization in plastids involve a complex interplay between ancient (prokaryotic) and novel (eukaryotic) translocases and targeting machineries. During evolution, ancient systems acquired new functions and novel translocation machineries were developed to facilitate the correct localization of nuclear encoded proteins targeted to the chloroplast. Because of its post-translational nature, targeting and integration of membrane proteins posed the biggest challenge to the organelle to avoid aggregation in the aqueous compartments. Soluble proteins faced a different kind of problem since some had to be transported across three membranes to reach their destination. Early studies suggested that chloroplasts addressed these issues by adapting ancient-prokaryotic machineries and integrating them with novel-eukaryotic systems, a process called 'conservative sorting'. In the last decade, detailed biochemical, genetic, and structural studies have unraveled the mechanisms of protein targeting and localization in chloroplasts, suggesting a highly integrated scheme where ancient and novel systems collaborate at different stages of the process. In this review we focus on the differences and similarities between chloroplast ancestral translocases and their prokaryotic relatives to highlight known modifications that adapted them to the eukaryotic situation. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.

  5. Life at the border: Adaptation of proteins to anisotropic membrane environment

    PubMed Central

    Pogozheva, Irina D; Mosberg, Henry I; Lomize, Andrei L

    2014-01-01

    This review discusses main features of transmembrane (TM) proteins which distinguish them from water-soluble proteins and allow their adaptation to the anisotropic membrane environment. We overview the structural limitations on membrane protein architecture, spatial arrangement of proteins in membranes and their intrinsic hydrophobic thickness, co-translational and post-translational folding and insertion into lipid bilayers, topogenesis, high propensity to form oligomers, and large-scale conformational transitions during membrane insertion and transport function. Special attention is paid to the polarity of TM protein surfaces described by profiles of dipolarity/polarizability and hydrogen-bonding capacity parameters that match polarity of the lipid environment. Analysis of distributions of Trp resides on surfaces of TM proteins from different biological membranes indicates that interfacial membrane regions with preferential accumulation of Trp indole rings correspond to the outer part of the lipid acyl chain region—between double bonds and carbonyl groups of lipids. These “midpolar” regions are not always symmetric in proteins from natural membranes. We also examined the hydrophobic effect that drives insertion of proteins into lipid bilayer and different free energy contributions to TM protein stability, including attractive van der Waals forces and hydrogen bonds, side-chain conformational entropy, the hydrophobic mismatch, membrane deformations, and specific protein–lipid binding. PMID:24947665

  6. Immunoinhibitory adapter protein Src homology domain 3 lymphocyte protein 2 (SLy2) regulates actin dynamics and B cell spreading.

    PubMed

    von Holleben, Max; Gohla, Antje; Janssen, Klaus-Peter; Iritani, Brian M; Beer-Hammer, Sandra

    2011-04-15

    Appropriate B cell activation is essential for adaptive immunity. In contrast to the molecular mechanisms that regulate positive signaling in immune responses, the counterbalancing negative regulatory pathways remain insufficiently understood. The Src homology domain 3 (SH3)-containing adapter protein SH3 lymphocyte protein 2 (SLy2, also known as hematopoietic adapter-containing SH3 and sterile α-motif (SAM) domains 1; HACS1) is strongly up-regulated upon B cell activation and functions as an endogenous immunoinhibitor in vivo, but the underlying molecular mechanisms of SLy2 function have been elusive. We have generated transgenic mice overexpressing SLy2 in B and T cells and have studied the biological effects of elevated SLy2 levels in Jurkat and HeLa cells. Our results demonstrate that SLy2 induces Rac1-dependent membrane ruffle formation and regulates cell spreading and polarization and that the SLy2 SH3 domain is essential for these effects. Using immunoprecipitation and confocal microscopy, we provide evidence that the actin nucleation-promoting factor cortactin is an SH3 domain-directed interaction partner of SLy2. Consistent with an important role of SLy2 for actin cytoskeletal reorganization, we further show that SLy2-transgenic B cells are severely defective in cell spreading. Together, our findings extend our mechanistic understanding of the immunoinhibitory roles of SLy2 in vivo and suggest that the physiological up-regulation of SLy2 observed upon B cell activation functions to counteract excessive B cell spreading.

  7. Live imaging using adaptive optics with fluorescent protein guide-stars

    PubMed Central

    Tao, Xiaodong; Crest, Justin; Kotadia, Shaila; Azucena, Oscar; Chen, Diana C.; Sullivan, William; Kubby, Joel

    2012-01-01

    Spatially and temporally dependent optical aberrations induced by the inhomogeneous refractive index of live samples limit the resolution of live dynamic imaging. We introduce an adaptive optical microscope with a direct wavefront sensing method using a Shack-Hartmann wavefront sensor and fluorescent protein guide-stars for live imaging. The results of imaging Drosophila embryos demonstrate its ability to correct aberrations and achieve near diffraction limited images of medial sections of large Drosophila embryos. GFP-polo labeled centrosomes can be observed clearly after correction but cannot be observed before correction. Four dimensional time lapse images are achieved with the correction of dynamic aberrations. These studies also demonstrate that the GFP-tagged centrosome proteins, Polo and Cnn, serve as excellent biological guide-stars for adaptive optics based microscopy. PMID:22772285

  8. Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli.

    PubMed

    Yosef, Ido; Goren, Moran G; Qimron, Udi

    2012-07-01

    The clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR/Cas) constitute a recently identified prokaryotic defense mechanism against invading nucleic acids. Activity of the CRISPR/Cas system comprises of three steps: (i) insertion of alien DNA sequences into the CRISPR array to prevent future attacks, in a process called 'adaptation', (ii) expression of the relevant proteins, as well as expression and processing of the array, followed by (iii) RNA-mediated interference with the alien nucleic acid. Here we describe a robust assay in Escherichia coli to explore the hitherto least-studied process, adaptation. We identify essential genes and DNA elements in the leader sequence and in the array which are essential for the adaptation step. We also provide mechanistic insights on the insertion of the repeat-spacer unit by showing that the first repeat serves as the template for the newly inserted repeat. Taken together, our results elucidate fundamental steps in the adaptation process of the CRISPR/Cas system.

  9. Adaptability of protein structures to enable functional interactions and evolutionary implications.

    PubMed

    Haliloglu, Turkan; Bahar, Ivet

    2015-12-01

    Several studies in recent years have drawn attention to the ability of proteins to adapt to intermolecular interactions by conformational changes along structure-encoded collective modes of motions. These so-called soft modes, primarily driven by entropic effects, facilitate, if not enable, functional interactions. They represent excursions on the conformational space along principal low-ascent directions/paths away from the original free energy minimum, and they are accessible to the protein even before protein-protein/ligand interactions. An emerging concept from these studies is the evolution of structures or modular domains to favor such modes of motion that will be recruited or integrated for enabling functional interactions. Structural dynamics, including the allosteric switches in conformation that are often stabilized upon formation of complexes and multimeric assemblies, emerge as key properties that are evolutionarily maintained to accomplish biological activities, consistent with the paradigm sequence→structure→dynamics→function where 'dynamics' bridges structure and function.

  10. Late embryogenesis abundant proteins: versatile players in the plant adaptation to water limiting environments.

    PubMed

    Olvera-Carrillo, Yadira; Luis Reyes, José; Covarrubias, Alejandra A

    2011-04-01

    Late Embryogenesis Abundant (LEA) proteins accumulate at the onset of seed desiccation and in response to water deficit in vegetative plant tissues. The typical LEA proteins are highly hydrophilic and intrinsically unstructured. They have been classified in different families; each one showing distinctive conserved motifs. In this manuscript we present and discuss some of the recent findings regarding their role in plant adaptation to water deficit, as well as those concerning to their possible function, and how it can be related to their intrinsic structural flexibility.

  11. Physical and molecular bases of protein thermal stability and cold adaptation.

    PubMed

    Pucci, Fabrizio; Rooman, Marianne

    2017-02-01

    The molecular bases of thermal and cold stability and adaptation, which allow proteins to remain folded and functional in the temperature ranges in which their host organisms live and grow, are still only partially elucidated. Indeed, both experimental and computational studies fail to yield a fully precise and global physical picture, essentially because all effects are context-dependent and thus quite intricate to unravel. We present a snapshot of the current state of knowledge of this highly complex and challenging issue, whose resolution would enable large-scale rational protein design.

  12. Comparative Proteomics of Mouse Tears and Saliva: Evidence from Large Protein Families for Functional Adaptation

    PubMed Central

    Karn, Robert C.; Laukaitis, Christina M.

    2015-01-01

    We produced a tear proteome of the genome mouse, C57BL/6, that contained 139 different protein identifications: 110 from a two-dimensional (2D) gel with subsequent trypsin digestion, 19 from a one-dimensional (1D) gel with subsequent trypsin digestion and ten from a 1D gel with subsequent Asp-N digestion. We compared this tear proteome with a C57BL/6 mouse saliva proteome produced previously. Sixteen of the 139 tear proteins are shared between the two proteomes, including six proteins that combat microbial growth. Among the 123 other tear proteins, were members of four large protein families that have no counterparts in humans: Androgen-binding proteins (ABPs) with different members expressed in the two proteomes, Exocrine secreted peptides (ESPs) expressed exclusively in the tear proteome, major urinary proteins (MUPs) expressed in one or both proteomes and the mouse-specific Kallikreins (subfamily b KLKs) expressed exclusively in the saliva proteome. All four families have members with suggested roles in mouse communication, which may influence some aspect of reproductive behavior. We discuss this in the context of functional adaptation involving tear and saliva proteins in the secretions of mouse lacrimal and salivary glands, respectively.

  13. Shared and Unique Proteins in Human, Mouse and Rat Saliva Proteomes: Footprints of Functional Adaptation

    PubMed Central

    Karn, Robert C.; Chung, Amanda G.; Laukaitis, Christina M.

    2013-01-01

    The overall goal of our study was to compare the proteins found in the saliva proteomes of three mammals: human, mouse and rat. Our first objective was to compare two human proteomes with very different analysis depths. The 89 shared proteins in this comparison apparently represent a core of highly-expressed human salivary proteins. Of the proteins unique to each proteome, one-half to 2/3 lack signal peptides and probably are contaminants instead of less highly-represented salivary proteins. We recently published the first rodent saliva proteomes with saliva collected from the genome mouse (C57BL/6) and the genome rat (BN/SsNHsd/Mcwi). Our second objective was to compare the proteins in the human proteome with those we identified in the genome mouse and rat to determine those common to all three mammals, as well as the specialized rodent subset. We also identified proteins unique to each of the three mammals, because differences in the secreted protein constitutions can provide clues to differences in the evolutionary adaptation of the secretions in the three different mammals. PMID:24926433

  14. Electrophoresis and spectrometric analyses of adaptation-related proteins in thermally stressed Chromobacterium violaceum.

    PubMed

    Cordeiro, I B; Castro, D P; Nogueira, P P O; Angelo, P C S; Nogueira, P A; Gonçalves, J F C; Pereira, A M R F; Garcia, J S; Souza, G H M F; Arruda, M A Z; Eberlin, M N; Astolfi-Filho, S; Andrade, E V; López-Lozano, J L

    2013-10-29

    Chromobacterium violaceum is a Gram-negative proteobacteria found in water and soil; it is widely distributed in tropical and subtropical regions, such as the Amazon rainforest. We examined protein expression changes that occur in C. violaceum at different growth temperatures using electrophoresis and mass spectrometry. The total number of spots detected was 1985; the number ranged from 99 to 380 in each assay. The proteins that were identified spectrometrically were categorized as chaperones, proteins expressed exclusively under heat stress, enzymes involved in the respiratory and fermentation cycles, ribosomal proteins, and proteins related to transport and secretion. Controlling inverted repeat of chaperone expression and inverted repeat DNA binding sequences, as well as regions recognized by sigma factor 32, elements involved in the genetic regulation of the bacterial stress response, were identified in the promoter regions of several of the genes coding proteins, involved in the C. violaceum stress response. We found that 30 °C is the optimal growth temperature for C. violaceum, whereas 25, 35, and 40 °C are stressful temperatures that trigger the expression of chaperones, superoxide dismutase, a probable small heat shock protein, a probable phasing, ferrichrome-iron receptor protein, elongation factor P, and an ornithine carbamoyltransferase catabolite. This information improves our comprehension of the mechanisms involved in stress adaptation by C. violaceum.

  15. The Role of Semidisorder in Temperature Adaptation of Bacterial FlgM Proteins

    PubMed Central

    Wang, Jihua; Yang, Yuedong; Cao, Zanxia; Li, Zhixiu; Zhao, Huiying; Zhou, Yaoqi

    2013-01-01

    Probabilities of disorder for FlgM proteins of 39 species whose optimal growth temperature ranges from 273 K (0°C) to 368 K (95°C) were predicted by a newly developed method called Sequence-based Prediction with Integrated NEural networks for Disorder (SPINE-D). We showed that the temperature-dependent behavior of FlgM proteins could be separated into two subgroups according to their sequence lengths. Only shorter sequences evolved to adapt to high temperatures (>318 K or 45°C). Their ability to adapt to high temperatures was achieved through a transition from a fully disordered state with little secondary structure to a semidisordered state with high predicted helical probability at the N-terminal region. The predicted results are consistent with available experimental data. An analysis of all orthologous protein families in 39 species suggests that such a transition from a fully disordered state to semidisordered and/or ordered states is one of the strategies employed by nature for adaptation to high temperatures. PMID:24314090

  16. [Quasi-adaptive response to alkylating agents in Escherichia coli and Ada-protein functions].

    PubMed

    Vasil'eva, S V; Moshkovskaia, E Iu; Terekhov, A S; Mikoian, V D; Vanin, A F

    2008-01-01

    In 2005 we have described in exponentially growing E. coli cells a new fundamental genetic phenomenon,--quasi-adaptive response to alkylating compounds (quasi-Ada). Phenotypic expression of quasi-Ada is similar to the true Ada response. However, in contrast to the letter, it develops in the course of pretreatment of the cells by a sublethal dose of nonalkylating agent, an NO-containing dinitrosyl iron complex with glutathione (DNICglu). To reveal the mechanisms of quasi-adaptation and its association with the function of the Ada regulatory protein, here we used a unique property of dual gene expression regulation of aidB1 gene, a part of the Ada-regulon, namely its relative independence from Ada protein in anaerobic conditions. Based on the results of aidB1 gene expression analysis an EPR spectra of E. coli MV2176 cells (aidB1::lacZ) in aerobic and anaerobic conditions after the corresponding treatments, we conclude that the function and the spatial structure of meAda and [(Cys-)2Fe+(NO+)2]Ada are identical and thus the nitrosylated protein represents a regulator of the Ada regulon gene expression during quasi-adaptation development.

  17. Kinetic Stability of Proteins in Beans and Peas: Implications for Protein Digestibility, Seed Germination, and Plant Adaptation.

    PubMed

    Xia, Ke; Pittelli, Sandy; Church, Jennifer; Colón, Wilfredo

    2016-10-12

    Kinetically stable proteins (KSPs) are resistant to the denaturing detergent sodium dodecyl sulfate (SDS). Such resilience makes KSPs resistant to proteolytic degradation and may have arisen in nature as a mechanism for organismal adaptation and survival against harsh conditions. Legumes are well-known for possessing degradation-resistant proteins that often diminish their nutritional value. Here we applied diagonal two-dimensional (D2D) SDS-polyacrylamide gel electrophoresis (PAGE), a method that allows for the proteomics-level identification of KSPs, to a group of 12 legumes (mostly beans and peas) of agricultural and nutritional importance. Our proteomics results show beans that are more difficult to digest, such as soybean, lima beans, and various common beans, have high contents of KSPs. In contrast, mung bean, red lentil, and various peas that are highly digestible contain low amounts of KSPs. Identified proteins with high kinetic stability are associated with warm-season beans, which germinate at higher temperatures. In contrast, peas and red lentil, which are cool-season legumes, contain low levels of KSPs. Thus, our results show protein kinetic stability is an important factor in the digestibility of legume proteins and may relate to nutrition efficiency, timing of seed germination, and legume resistance to biotic stressors. Furthermore, we show D2D SDS-PAGE is a powerful method that could be applied for determining the abundance and identity of KSPs in engineered and wild legumes and for advancing basic research and associated applications.

  18. Expression of many immunologically important genes in Mycobacterium tuberculosis-infected macrophages is independent of both TLR2 and TLR4 but dependent on IFN-alphabeta receptor and STAT1.

    PubMed

    Shi, Shuangping; Blumenthal, Antje; Hickey, Christopher M; Gandotra, Sheetal; Levy, David; Ehrt, Sabine

    2005-09-01

    Macrophages respond to several subcellular products of Mycobacterium tuberculosis (Mtb) through TLR2 or TLR4. However, primary mouse macrophages respond to viable, virulent Mtb by pathways largely independent of MyD88, the common adaptor molecule for TLRs. Using microarrays, quantitative PCR, and ELISA with gene-disrupted macrophages and mice, we now show that viable Mtb elicits the expression of inducible NO synthase, RANTES, IFN-inducible protein 10, immune-responsive gene 1, and many other key genes in macrophages substantially independently of TLR2, TLR4, their combination, or the TLR adaptors Toll-IL-1R domain-containing adapter protein and Toll-IL-1R domain-containing adapter inducing IFN-beta. Mice deficient in both TLR2 and TLR4 handle aerosol infection with viable Mtb as well as congenic controls. Viable Mtb also up-regulates inducible NO synthase, RANTES, IFN-inducible protein 10, and IRG1 in macrophages that lack mannose receptor, complement receptors 3 and 4, type A scavenger receptor, or CD40. These MyD88, TLR2/4-independent transcriptional responses require IFN-alphabetaR and STAT1, but not IFN-gamma. Conversely, those genes whose expression is MyD88 dependent do not depend on IFN-alphabetaR or STAT1. Transcriptional induction of TNF is TLR2/4, MyD88, STAT1, and IFN-alphabetaR independent, but TNF protein release requires the TLR2/4-MyD88 pathway. Thus, macrophages respond transcriptionally to viable Mtb through at least three pathways. TLR2 mediates the responses of a numerically minor set of genes that collectively do not appear to affect the course of infection in mice; regulation of TNF requires TLR2/4 for post-transcriptional control, but not for transcriptional induction; and many responding genes are regulated through an unknown, TLR2/4-independent pathway that may involve IFN-alphabetaR and STAT1.

  19. Emergence of tissue sensitivity to Hox protein levels underlies the evolution of an adaptive morphological trait

    PubMed Central

    Refki, Peter Nagui; Armisén, David; Crumière, Antonin Jean Johan; Viala, Séverine; Khila, Abderrahman

    2014-01-01

    Growth control scales morphological attributes and, therefore, provides a critical contribution to the evolution of adaptive traits. Yet, the genetic mechanisms underlying growth in the context of specific ecological adaptations are poorly understood. In water striders, adaptation to locomotion on the water surface is associated with allometric and functional changes in thoracic appendages, such that T2-legs, used as propelling oars, are longer than T3-legs, used as steering rudders. The Hox gene Ubx establishes this derived morphology by elongating T2-legs but shortening T3-legs. Using gene expression assays, RNAi knockdown, and comparative transcriptomics, we demonstrate that the evolution of water surface rowing as a novel means of locomotion is associated with the evolution of a dose-dependent promoting-repressing effect of Ubx on leg growth. In the water strider Limnoporus dissortis, T3-legs express six to seven times higher levels of Ubx compared to T2-legs. Ubx RNAi shortens T2-legs and the severity of this phenotype increases with increased depletion of Ubx protein. Conversely, Ubx RNAi lengthens T3-legs but this phenotype is partially rescued when Ubx protein is further depleted. This dose-dependent effect of Ubx on leg growth is absent in non-rowing relatives that retain the ancestral relative leg length. We also show that the spatial patterns of expression of dpp, wg, hh, egfr, dll, exd, hth, and dac are unchanged in Ubx RNAi treatments. This indicates that the dose-dependent opposite effect of Ubx on T2- and T3-legs operates without any apparent effect on the spatial expression of major leg patterning genes. Our data suggest that scaling of adaptive allometries can evolve through changes in the levels of expression of Hox proteins early during ontogeny, and in the sensitivity of the tissues that express them, without any major effects on pattern formation. PMID:24886828

  20. Median Modified Wiener Filter for nonlinear adaptive spatial denoising of protein NMR multidimensional spectra.

    PubMed

    Cannistraci, Carlo Vittorio; Abbas, Ahmed; Gao, Xin

    2015-01-26

    Denoising multidimensional NMR-spectra is a fundamental step in NMR protein structure determination. The state-of-the-art method uses wavelet-denoising, which may suffer when applied to non-stationary signals affected by Gaussian-white-noise mixed with strong impulsive artifacts, like those in multi-dimensional NMR-spectra. Regrettably, Wavelet's performance depends on a combinatorial search of wavelet shapes and parameters; and multi-dimensional extension of wavelet-denoising is highly non-trivial, which hampers its application to multidimensional NMR-spectra. Here, we endorse a diverse philosophy of denoising NMR-spectra: less is more! We consider spatial filters that have only one parameter to tune: the window-size. We propose, for the first time, the 3D extension of the median-modified-Wiener-filter (MMWF), an adaptive variant of the median-filter, and also its novel variation named MMWF*. We test the proposed filters and the Wiener-filter, an adaptive variant of the mean-filter, on a benchmark set that contains 16 two-dimensional and three-dimensional NMR-spectra extracted from eight proteins. Our results demonstrate that the adaptive spatial filters significantly outperform their non-adaptive versions. The performance of the new MMWF* on 2D/3D-spectra is even better than wavelet-denoising. Noticeably, MMWF* produces stable high performance almost invariant for diverse window-size settings: this signifies a consistent advantage in the implementation of automatic pipelines for protein NMR-spectra analysis.

  1. Median Modified Wiener Filter for nonlinear adaptive spatial denoising of protein NMR multidimensional spectra

    PubMed Central

    Cannistraci, Carlo Vittorio; Abbas, Ahmed; Gao, Xin

    2015-01-01

    Denoising multidimensional NMR-spectra is a fundamental step in NMR protein structure determination. The state-of-the-art method uses wavelet-denoising, which may suffer when applied to non-stationary signals affected by Gaussian-white-noise mixed with strong impulsive artifacts, like those in multi-dimensional NMR-spectra. Regrettably, Wavelet's performance depends on a combinatorial search of wavelet shapes and parameters; and multi-dimensional extension of wavelet-denoising is highly non-trivial, which hampers its application to multidimensional NMR-spectra. Here, we endorse a diverse philosophy of denoising NMR-spectra: less is more! We consider spatial filters that have only one parameter to tune: the window-size. We propose, for the first time, the 3D extension of the median-modified-Wiener-filter (MMWF), an adaptive variant of the median-filter, and also its novel variation named MMWF*. We test the proposed filters and the Wiener-filter, an adaptive variant of the mean-filter, on a benchmark set that contains 16 two-dimensional and three-dimensional NMR-spectra extracted from eight proteins. Our results demonstrate that the adaptive spatial filters significantly outperform their non-adaptive versions. The performance of the new MMWF* on 2D/3D-spectra is even better than wavelet-denoising. Noticeably, MMWF* produces stable high performance almost invariant for diverse window-size settings: this signifies a consistent advantage in the implementation of automatic pipelines for protein NMR-spectra analysis. PMID:25619991

  2. Adaptive Evolution of Eel Fluorescent Proteins from Fatty Acid Binding Proteins Produces Bright Fluorescence in the Marine Environment

    PubMed Central

    Gruber, David F.; Gaffney, Jean P.; Mehr, Shaadi; DeSalle, Rob; Sparks, John S.; Platisa, Jelena; Pieribone, Vincent A.

    2015-01-01

    We report the identification and characterization of two new members of a family of bilirubin-inducible fluorescent proteins (FPs) from marine chlopsid eels and demonstrate a key region of the sequence that serves as an evolutionary switch from non-fluorescent to fluorescent fatty acid-binding proteins (FABPs). Using transcriptomic analysis of two species of brightly fluorescent Kaupichthys eels (Kaupichthys hyoproroides and Kaupichthys n. sp.), two new FPs were identified, cloned and characterized (Chlopsid FP I and Chlopsid FP II). We then performed phylogenetic analysis on 210 FABPs, spanning 16 vertebrate orders, and including 163 vertebrate taxa. We show that the fluorescent FPs diverged as a protein family and are the sister group to brain FABPs. Our results indicate that the evolution of this family involved at least three gene duplication events. We show that fluorescent FABPs possess a unique, conserved tripeptide Gly-Pro-Pro sequence motif, which is not found in non-fluorescent fatty acid binding proteins. This motif arose from a duplication event of the FABP brain isoforms and was under strong purifying selection, leading to the classification of this new FP family. Residues adjacent to the motif are under strong positive selection, suggesting a further refinement of the eel protein’s fluorescent properties. We present a phylogenetic reconstruction of this emerging FP family and describe additional fluorescent FABP members from groups of distantly related eels. The elucidation of this class of fish FPs with diverse properties provides new templates for the development of protein-based fluorescent tools. The evolutionary adaptation from fatty acid-binding proteins to fluorescent fatty acid-binding proteins raises intrigue as to the functional role of bright green fluorescence in this cryptic genus of reclusive eels that inhabit a blue, nearly monochromatic, marine environment. PMID:26561348

  3. Heat shock proteins and exercise adaptations. Our knowledge thus far and the road still ahead.

    PubMed

    Henstridge, Darren C; Febbraio, Mark A; Hargreaves, Mark

    2016-03-15

    By its very nature, exercise exerts a challenge to the body's cellular homeostatic mechanisms. This homeostatic challenge affects not only the contracting skeletal muscle but also a number of other organs and results over time in exercise-induced adaptations. Thus it is no surprise that heat shock proteins (HSPs), a group of ancient and highly conserved cytoprotective proteins critical in the maintenance of protein and cellular homeostasis, have been implicated in exercise/activity-induced adaptations. It has become evident that HSPs such as HSP72 are induced or activated with acute exercise or after chronic exercise training regimens. These observations have given scientists an insight into the protective mechanisms of these proteins and provided an opportunity to exploit their protective role to improve health and physical performance. Although our knowledge in this area of physiology has improved dramatically, many questions still remain unanswered. Further understanding of the role of HSPs in exercise physiology may prove beneficial for therapeutic targeting in diseased patient cohorts, exercise prescription for disease prevention, and training strategies for elite athletes.

  4. High protein flexibility and reduced hydration water dynamics are key pressure adaptive strategies in prokaryotes

    PubMed Central

    Martinez, N.; Michoud, G.; Cario, A.; Ollivier, J.; Franzetti, B.; Jebbar, M.; Oger, P.; Peters, J.

    2016-01-01

    Water and protein dynamics on a nanometer scale were measured by quasi-elastic neutron scattering in the piezophile archaeon Thermococcus barophilus and the closely related pressure-sensitive Thermococcus kodakarensis, at 0.1 and 40 MPa. We show that cells of the pressure sensitive organism exhibit higher intrinsic stability. Both the hydration water dynamics and the fast protein and lipid dynamics are reduced under pressure. In contrast, the proteome of T. barophilus is more pressure sensitive than that of T. kodakarensis. The diffusion coefficient of hydration water is reduced, while the fast protein and lipid dynamics are slightly enhanced with increasing pressure. These findings show that the coupling between hydration water and cellular constituents might not be simply a master-slave relationship. We propose that the high flexibility of the T. barophilus proteome associated with reduced hydration water may be the keys to the molecular adaptation of the cells to high hydrostatic pressure. PMID:27595789

  5. High protein flexibility and reduced hydration water dynamics are key pressure adaptive strategies in prokaryotes

    NASA Astrophysics Data System (ADS)

    Martinez, N.; Michoud, G.; Cario, A.; Ollivier, J.; Franzetti, B.; Jebbar, M.; Oger, P.; Peters, J.

    2016-09-01

    Water and protein dynamics on a nanometer scale were measured by quasi-elastic neutron scattering in the piezophile archaeon Thermococcus barophilus and the closely related pressure-sensitive Thermococcus kodakarensis, at 0.1 and 40 MPa. We show that cells of the pressure sensitive organism exhibit higher intrinsic stability. Both the hydration water dynamics and the fast protein and lipid dynamics are reduced under pressure. In contrast, the proteome of T. barophilus is more pressure sensitive than that of T. kodakarensis. The diffusion coefficient of hydration water is reduced, while the fast protein and lipid dynamics are slightly enhanced with increasing pressure. These findings show that the coupling between hydration water and cellular constituents might not be simply a master-slave relationship. We propose that the high flexibility of the T. barophilus proteome associated with reduced hydration water may be the keys to the molecular adaptation of the cells to high hydrostatic pressure.

  6. ApoCanD: Database of human apoptotic proteins in the context of cancer.

    PubMed

    Kumar, Rahul; Raghava, Gajendra P S

    2016-02-10

    In the past decade, apoptosis pathway has gained a serious consideration being a critical cellular process in determining the cancer progression. Inverse relationship between cancer progression and apoptosis rate has been well established in the literature. It causes apoptosis proteins under the investigative scanner for developing anticancer therapies, which certainly got a success in the case of few apoptosis proteins as drug targets. In the present study, we have developed a dedicated database of 82 apoptosis proteins called ApoCanD. This database comprises of crucial information of apoptosis proteins in the context of cancer. Genomic status of proteins in the form of mutation, copy number variation and expression in thousands of tumour samples and cancer cell lines are the major bricks of this database. In analysis, we have found that TP53 and MYD88 are the two most frequently mutated proteins in cancer. Availability of other information e.g. gene essentiality data, tertiary structure, sequence alignments, sequences profiles, post-translational modifications makes it even more useful for the researchers. A user-friendly web interface is provided to ameliorate the use of ApoCanD. We anticipate that, this database will facilitate the research community working in the field of apoptosis and cancer. The database can be accessed at: http://crdd.osdd.net/raghava/apocand.

  7. Homeodomain Protein Otp and Activity-Dependent Splicing Modulate Neuronal Adaptation to Stress

    PubMed Central

    Amir-Zilberstein, Liat; Blechman, Janna; Sztainberg, Yehezkel; Norton, William H.J.; Reuveny, Adriana; Borodovsky, Nataliya; Tahor, Maayan; Bonkowsky, Joshua L.; Bally-Cuif, Laure; Chen, Alon; Levkowitz, Gil

    2015-01-01

    SUMMARY Regulation of corticotropin-releasing hormone (CRH) activity is critical for the animal’s adaptation to stressful challenges, and its dysregulation is associated with psychiatric disorders in humans. However, the molecular mechanism underlying this transcriptional response to stress is not well understood. Using various stress paradigms in mouse and zebrafish, we show that the hypothalamic transcription factor Orthopedia modulates the expression of CRH as well as the splicing factor Ataxin 2-Binding Protein-1 (A2BP1/Rbfox-1). We further show that the G protein coupled receptor PAC1, which is a known A2BP1/Rbfox-1 splicing target and an important mediator of CRH activity, is alternatively spliced in response to a stressful challenge. The generation of PAC1-hop messenger RNA isoform by alternative splicing is required for termination of CRH transcription, normal activation of the hypothalamic-pituitary-adrenal axis and adaptive anxiety-like behavior. Our study identifies an evolutionarily conserved biochemical pathway that modulates the neuronal adaptation to stress through transcriptional activation and alternative splicing. PMID:22284183

  8. Temperature adaptation at homologous sites in proteins from nine thermophile-mesophile species pairs.

    PubMed

    McDonald, John H

    2010-07-12

    Whether particular amino acids are favored by selection at high temperatures over others has long been an open question in protein evolution. One way to approach this question is to compare homologous sites in proteins from one thermophile and a closely related mesophile; asymmetrical substitution patterns have been taken as evidence for selection favoring certain amino acids over others. However, most pairs of prokaryotic species that differ in optimum temperature also differ in genome-wide GC content, and amino acid content is known to be associated with GC content. Here, I compare homologous sites in nine thermophilic prokaryotes and their mesophilic relatives, all with complete published genome sequences. After adjusting for the effects of differing GC content with logistic regression, 139 of the 190 pairs of amino acids show significant substitutional asymmetry, evidence of widespread adaptive amino acid substitution. The patterns are fairly consistent across the nine pairs of species (after taking the effects of differing GC content into account), suggesting that much of the asymmetry results from adaptation to temperature. Some amino acids in some species pairs deviate from the overall pattern in ways indicating that adaptation to other environmental or physiological differences between the species may also play a role. The property that is best correlated with the patterns of substitutional asymmetry is transfer free energy, a measure of hydrophobicity, with more hydrophobic amino acids favored at higher temperatures. The correlation of asymmetry and hydrophobicity is fairly weak, suggesting that other properties may also be important.

  9. Substrate adaptabilities of Thermotogae mannan binding proteins as a function of their evolutionary histories.

    PubMed

    Boucher, Nathalie; Noll, Kenneth M

    2016-09-01

    The Thermotogae possess a large number of ATP-binding cassette (ABC) transporters, including two mannan binding proteins, ManD and CelE (previously called ManE). We show that a gene encoding an ancestor of these was acquired by the Thermotogae from the archaea followed by gene duplication. To address the functional evolution of these proteins as a consequence of their evolutionary histories, we measured the binding affinities of ManD and CelE orthologs from representative Thermotogae. Both proteins bind cellobiose, cellotriose, cellotetraose, β-1,4-mannotriose, and β-1,4-mannotetraose. The CelE orthologs additionally bind β-1,4-mannobiose, laminaribiose, laminaritriose and sophorose while the ManD orthologs additionally only weakly bind β-1,4-mannobiose. The CelE orthologs have higher unfolding temperatures than the ManD orthologs. An examination of codon sites under positive selection revealed that many of these encode residues located near or in the binding site, suggesting that the proteins experienced selective pressures in regions that might have changed their functions. The gene arrangement, phylogeny, binding properties, and putative regulatory networks suggest that the ancestral mannan binding protein was a CelE ortholog which gave rise to the ManD orthologs. This study provides a window on how one class of proteins adapted to new functions and temperatures to fit the physiologies of their new hosts.

  10. Massively parallel sampling of lattice proteins reveals foundations of thermal adaptation

    NASA Astrophysics Data System (ADS)

    Venev, Sergey V.; Zeldovich, Konstantin B.

    2015-08-01

    Evolution of proteins in bacteria and archaea living in different conditions leads to significant correlations between amino acid usage and environmental temperature. The origins of these correlations are poorly understood, and an important question of protein theory, physics-based prediction of types of amino acids overrepresented in highly thermostable proteins, remains largely unsolved. Here, we extend the random energy model of protein folding by weighting the interaction energies of amino acids by their frequencies in protein sequences and predict the energy gap of proteins designed to fold well at elevated temperatures. To test the model, we present a novel scalable algorithm for simultaneous energy calculation for many sequences in many structures, targeting massively parallel computing architectures such as graphics processing unit. The energy calculation is performed by multiplying two matrices, one representing the complete set of sequences, and the other describing the contact maps of all structural templates. An implementation of the algorithm for the CUDA platform is available at http://www.github.com/kzeldovich/galeprot and calculates protein folding energies over 250 times faster than a single central processing unit. Analysis of amino acid usage in 64-mer cubic lattice proteins designed to fold well at different temperatures demonstrates an excellent agreement between theoretical and simulated values of energy gap. The theoretical predictions of temperature trends of amino acid frequencies are significantly correlated with bioinformatics data on 191 bacteria and archaea, and highlight protein folding constraints as a fundamental selection pressure during thermal adaptation in biological evolution.

  11. The use of amphipols as universal molecular adapters to immobilize membrane proteins onto solid supports

    PubMed Central

    Charvolin, Delphine; Perez, Jean-Baptiste; Rouvière, Florent; Giusti, Fabrice; Bazzacco, Paola; Abdine, Alaa; Rappaport, Fabrice; Martinez, Karen L.; Popot, Jean-Luc

    2009-01-01

    Because of the importance of their physiological functions, cell membranes represent critical targets in biological research. Membrane proteins, which make up ≈1/3 of the proteome, interact with a wide range of small ligands and macromolecular partners as well as with foreign molecules such as synthetic drugs, antibodies, toxins, or surface recognition proteins of pathogenic organisms. Whether it is for the sake of basic biomedical or pharmacological research, it is of great interest to develop tools facilitating the study of these interactions. Surface-based in vitro assays are appealing because they require minimum quantities of reagents, and they are suitable for multiplexing and high-throughput screening. We introduce here a general method for immobilizing functional, unmodified integral membrane proteins onto solid supports, thanks to amphipathic polymers called “amphipols.” The key point of this approach is that functionalized amphipols can be used as universal adapters to associate any membrane protein to virtually any kind of support while stabilizing its native state. The generality and versatility of this strategy is demonstrated by using 5 different target proteins, 2 types of supports (chips and beads), 2 types of ligands (antibodies and a snake toxin), and 2 detection methods (surface plasmon resonance and fluorescence microscopy). PMID:19116278

  12. Temperature dependent mistranslation in a hyperthermophile adapts proteins to lower temperatures

    PubMed Central

    Schwartz, Michael H.; Pan, Tao

    2016-01-01

    All organisms universally encode, synthesize and utilize proteins that function optimally within a subset of growth conditions. While healthy cells are thought to maintain high translational fidelity within their natural habitats, natural environments can easily fluctuate outside the optimal functional range of genetically encoded proteins. The hyperthermophilic archaeon Aeropyrum pernix (A. pernix) can grow throughout temperature variations ranging from 70 to 100°C, although the specific factors facilitating such adaptability are unknown. Here, we show that A. pernix undergoes constitutive leucine to methionine mistranslation at low growth temperatures. Low-temperature mistranslation is facilitated by the misacylation of tRNALeu with methionine by the methionyl-tRNA synthetase (MetRS). At low growth temperatures, the A. pernix MetRS undergoes a temperature dependent shift in tRNA charging fidelity, allowing the enzyme to conditionally charge tRNALeu with methionine. We demonstrate enhanced low-temperature activity for A. pernix citrate synthase that is synthesized during leucine to methionine mistranslation at low-temperature growth compared to its high-fidelity counterpart synthesized at high-temperature. Our results show that conditional leucine to methionine mistranslation can make protein adjustments capable of improving the low-temperature activity of hyperthermophilic proteins, likely by facilitating the increasing flexibility required for greater protein function at lower physiological temperatures. PMID:26657639

  13. MSG5, a novel protein phosphatase promotes adaptation to pheromone response in S. cerevisiae.

    PubMed Central

    Doi, K; Gartner, A; Ammerer, G; Errede, B; Shinkawa, H; Sugimoto, K; Matsumoto, K

    1994-01-01

    Pheromone-stimulated yeast cells and haploid gpa1 deletion mutants arrest their cell cycle in G1. Overexpression of a novel gene called MSG5 suppresses this inhibition of cell division. Loss of MSG5 function leads to a diminished adaptive response to pheromone. Genetic analysis indicates that MSG5 acts at a stage where the protein kinases STE7 and FUS3 function to transmit the pheromone-induced signal. Since loss of MSG5 function causes an increase in FUS3 enzyme activity but not STE7 activity, we propose that MSG5 impinges on the pathway at FUS3. Sequence analysis suggests that MSG5 encodes a protein tyrosine phosphatase. This is supported by the finding that recombinant MSG5 has phosphatase activity in vitro and is able to inactivate autophosphorylated FUS3. Thus MSG5 might stimulate recovery from pheromone by regulating the phosphorylation state of FUS3. Images PMID:8306972

  14. In vitro strain adaptation of CWD prions by serial protein misfolding cyclic amplification.

    PubMed

    Meyerett, Crystal; Michel, Brady; Pulford, Bruce; Spraker, Terry R; Nichols, Traci A; Johnson, Theodore; Kurt, Timothy; Hoover, Edward A; Telling, Glenn C; Zabel, Mark D

    2008-12-20

    We used serial protein misfolding cyclic amplification (sPMCA) to amplify the D10 strain of CWD prions in a linear relationship over two logs of D10 dilutions. The resultant PMCA-amplified D10 induced terminal TSE disease in CWD-susceptible Tg(cerPrP)1536 mice with a survival time approximately 80 days shorter than the original D10 inoculum, similar to that produced by in vivo sub-passage of D10 in Tg(cerPrP)1536 mice. Both in vitro-amplified and mouse-passaged D10 produced brain lesion profiles, glycoform ratios and conformational stabilities significantly different than those produced by the original D10 inoculum in Tg(cerPrP)1536 mice. These findings demonstrate that sPMCA can amplify and adapt prion strains in vitro as effectively and much more quickly than in vivo strain adaptation by mouse passage. Thus sPMCA may represent a powerful tool to assess prion strain adaptation and species barriers in vitro.

  15. PSB27: A thylakoid protein enabling Arabidopsis to adapt to changing light intensity.

    PubMed

    Hou, Xin; Fu, Aigen; Garcia, Veder J; Buchanan, Bob B; Luan, Sheng

    2015-02-03

    In earlier studies we have identified FKBP20-2 and CYP38 as soluble proteins of the chloroplast thylakoid lumen that are required for the formation of photosystem II supercomplexes (PSII SCs). Subsequent work has identified another potential candidate functional in SC formation (PSB27). We have followed up on this possibility and isolated mutants defective in the PSB27 gene. In addition to lack of PSII SCs, mutant plants were severely stunted when cultivated with light of variable intensity. The stunted growth was associated with lower PSII efficiency and defective starch accumulation. In response to high light exposure, the mutant plants also displayed enhanced ROS production, leading to decreased biosynthesis of anthocyanin. Unexpectedly, we detected a second defect in the mutant, namely in CP26, an antenna protein known to be required for the formation of PSII SCs that has been linked to state transitions. Lack of PSII SCs was found to be independent of PSB27, but was due to a mutation in the previously described cp26 gene that we found had no effect on light adaptation. The present results suggest that PSII SCs, despite being required for state transitions, are not associated with acclimation to changing light intensity. Our results are consistent with the conclusion that PSB27 plays an essential role in enabling plants to adapt to fluctuating light intensity through a mechanism distinct from photosystem II supercomplexes and state transitions.

  16. Protein restriction does not impair adaptations induced in cardiomyocytes by exercise in rats.

    PubMed

    Cabral, C A C; Natali, A J; Natali, A Y; Novaes, R D; Lavorato, V N; Drumond, L R; Carneiro Júnior, M A; Silva, M F; Quintão-Junior, J F; Gontijo, L N; Silva, C H O; Felix, L B; Silva, M E

    2013-11-01

    The effect of a treadmill running program on physical performance and morphofunctional adaptations was investigated in control and malnourished rats. Male 4-week old Wistar rats were randomized in groups of 12 animals: control trained (CT), control sedentary (CS), malnourished trained (MT) and malnourished sedentary (MS). Control and malnourished animals received chow with 12% protein or 6% protein, respectively. Trained groups were subjected to a treadmill running program for 8 weeks. Physical performance, biochemical parameters, cardiomyocytes morphology and biomechanics were determined. Malnourished animals presented reduction in body mass, serum levels of total protein, albumin and hemoglobin compared to the control groups. At 1 and 3 Hz cardiomyocytes from CT and MT showed higher cell shortening, speed of contraction and relaxation compared to the other groups. At 3 Hz cardiomyocytes from MS showed reduction in cell shortening and speed of contraction compared to CS. Protein restriction does not prevent the improvement in physical performance or cardiomyocytes biomechanical efficiency and growth in response to exercise. These findings could represent a modulatory effect of exercise to maintain cardiomyocyte growth at the expense of reducing the rate of body growth in order to ensure proper cellular function in conditions of cardiovascular overload imposed by exercise.

  17. Adaptability of Protein Structures to Enable Functional Interactions and Evolutionary Implications

    PubMed Central

    Haliloglu, Turkan; Bahar, Ivet

    2015-01-01

    Several studies in recent years have drawn attention to the ability of proteins to adapt to intermolecular interactions by conformational changes along structure-encoded collective modes of motions. These so-called soft modes, primarily driven by entropic effects, facilitate, if not enable, functional interactions. They represent excursions on the conformational space along principal low-ascent directions/paths away from the original free energy minimum, and they are accessible to the protein even prior to protein-protein/ligand interactions. An emerging concept from these studies is the evolution of structures or modular domains to favor such modes of motion that will be recruited or integrated for enabling functional interactions. Structural dynamics, including the allosteric switches in conformation that are often stabilized upon formation of complexes and multimeric assemblies, emerge as key properties that are evolutionarily maintained to accomplish biological activities, consistent with the paradigm sequence → structure → dynamics → function where ‘dynamics’ bridges structure and function. PMID:26254902

  18. Reciprocal Influence of Protein Domains in the Cold-Adapted Acyl Aminoacyl Peptidase from Sporosarcina psychrophila

    PubMed Central

    Parravicini, Federica; Natalello, Antonino; Papaleo, Elena; De Gioia, Luca; Doglia, Silvia Maria; Lotti, Marina; Brocca, Stefania

    2013-01-01

    Acyl aminoacyl peptidases are two-domain proteins composed by a C-terminal catalytic α/β-hydrolase domain and by an N-terminal β-propeller domain connected through a structural element that is at the N-terminus in sequence but participates in the 3D structure of the C-domain. We investigated about the structural and functional interplay between the two domains and the bridge structure (in this case a single helix named α1-helix) in the cold-adapted enzyme from Sporosarcina psychrophila (SpAAP) using both protein variants in which entire domains were deleted and proteins carrying substitutions in the α1-helix. We found that in this enzyme the inter-domain connection dramatically affects the stability of both the whole enzyme and the β-propeller. The α1-helix is required for the stability of the intact protein, as in other enzymes of the same family; however in this psychrophilic enzyme only, it destabilizes the isolated β-propeller. A single charged residue (E10) in the α1-helix plays a major role for the stability of the whole structure. Overall, a strict interaction of the SpAAP domains seems to be mandatory for the preservation of their reciprocal structural integrity and may witness their co-evolution. PMID:23457536

  19. Subfamily-specific adaptations in the structures of two penicillin-binding proteins from Mycobacterium tuberculosis

    DOE PAGES

    Prigozhin, Daniil M.; Krieger, Inna V.; Huizar, John P.; ...

    2014-12-31

    Beta-lactam antibiotics target penicillin-binding proteins including several enzyme classes essential for bacterial cell-wall homeostasis. To better understand the functional and inhibitor-binding specificities of penicillin-binding proteins from the pathogen, Mycobacterium tuberculosis, we carried out structural and phylogenetic analysis of two predicted D,D-carboxypeptidases, Rv2911 and Rv3330. Optimization of Rv2911 for crystallization using directed evolution and the GFP folding reporter method yielded a soluble quadruple mutant. Structures of optimized Rv2911 bound to phenylmethylsulfonyl fluoride and Rv3330 bound to meropenem show that, in contrast to the nonspecific inhibitor, meropenem forms an extended interaction with the enzyme along a conserved surface. Phylogenetic analysis shows thatmore » Rv2911 and Rv3330 belong to different clades that emerged in Actinobacteria and are not represented in model organisms such as Escherichia coli and Bacillus subtilis. Clade-specific adaptations allow these enzymes to fulfill distinct physiological roles despite strict conservation of core catalytic residues. The characteristic differences include potential protein-protein interaction surfaces and specificity-determining residues surrounding the catalytic site. Overall, these structural insights lay the groundwork to develop improved beta-lactam therapeutics for tuberculosis.« less

  20. Chronic Phencyclidine Increases Synapsin-1 and Synaptic Adaptation Proteins in the Medial Prefrontal Cortex

    PubMed Central

    Pickering, Chris; Ericson, Mia; Söderpalm, Bo

    2013-01-01

    Phencyclidine (PCP) mimics many aspects of schizophrenia, yet the underlying mechanism of neurochemical adaptation for PCP is unknown. We therefore used proteomics to study changes in the medial prefrontal cortex in animals with PCP-induced behavioural deficits. Male Wistar rats were injected with saline or 5 mg/kg phencyclidine for 5 days followed by two days of washout. Spontaneous alternation behaviour was tested in a Y-maze and then proteins were extracted from the medial prefrontal cortex. 2D-DIGE analysis followed by spot picking and protein identification with mass spectrometry then provided a list of differentially expressed proteins. Treatment with 5 mg/kg phencyclidine decreased the percentage of correct alternations in the Y-maze compared to saline-treated controls. Proteomics analysis of the medial prefrontal cortex found upregulation of 6 proteins (synapsin-1, Dpysl3, Aco2, Fscn1, Tuba1c, and Mapk1) and downregulation of 11 (Bin1, Dpysl2, Sugt1, ApoE, Psme1, ERp29, Pgam1, Uchl1, Ndufv2, Pcmt1, and Vdac1). A trend to upregulation was observed for Gnb4 and Capza2, while downregulation trends were noted for alpha-enolase and Fh. Many of the hits in this study concur with recent postmortem data from schizophrenic patients and this further validates the use of phencyclidine in preclinical translational research. PMID:23738220

  1. ADP ribosylation adapts an ER chaperone response to short-term fluctuations in unfolded protein load

    PubMed Central

    Petrova, Kseniya; Tomba, Giulia; Vendruscolo, Michele

    2012-01-01

    Gene expression programs that regulate the abundance of the chaperone BiP adapt the endoplasmic reticulum (ER) to unfolded protein load. However, such programs are slow compared with physiological fluctuations in secreted protein synthesis. While searching for mechanisms that fill this temporal gap in coping with ER stress, we found elevated levels of adenosine diphosphate (ADP)–ribosylated BiP in the inactive pancreas of fasted mice and a rapid decline in this modification in the active fed state. ADP ribosylation mapped to Arg470 and Arg492 in the substrate-binding domain of hamster BiP. Mutations that mimic the negative charge of ADP-ribose destabilized substrate binding and interfered with interdomain allosteric coupling, marking ADP ribosylation as a rapid posttranslational mechanism for reversible inactivation of BiP. A kinetic model showed that buffering fluctuations in unfolded protein load with a recruitable pool of inactive chaperone is an efficient strategy to minimize both aggregation and costly degradation of unfolded proteins. PMID:22869598

  2. APS, an adapter protein with a PH and SH2 domain, is a substrate for the insulin receptor kinase.

    PubMed Central

    Ahmed, Z; Smith, B J; Kotani, K; Wilden, P; Pillay, T S

    1999-01-01

    APS (adapter protein with a PH and SH2 domain) is the newest member of a family of tyrosine kinase adapter proteins including SH2-B and Lnk. We previously identified SH2-B as an insulin-receptor-binding protein and substrate [Kotani, Wilden and Pillay (1998) Biochem J. 335, 103-109]. Here we show that APS interacts with the insulin receptor kinase activation loop through its SH2 domain and insulin stimulates the tyrosine-phosphorylation of APS. Furthermore, the phosphorylation of activation-loop tyrosine residues 1158 and 1162 are required for this interaction. PMID:10417330

  3. Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

    PubMed Central

    Nilvebrant, Johan; Åstrand, Mikael; Georgieva-Kotseva, Maria; Björnmalm, Mattias; Löfblom, John; Hober, Sophia

    2014-01-01

    The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein. PMID:25089830

  4. Adaptive GDDA-BLAST: fast and efficient algorithm for protein sequence embedding.

    PubMed

    Hong, Yoojin; Kang, Jaewoo; Lee, Dongwon; van Rossum, Damian B

    2010-10-22

    A major computational challenge in the genomic era is annotating structure/function to the vast quantities of sequence information that is now available. This problem is illustrated by the fact that most proteins lack comprehensive annotations, even when experimental evidence exists. We previously theorized that embedded-alignment profiles (simply "alignment profiles" hereafter) provide a quantitative method that is capable of relating the structural and functional properties of proteins, as well as their evolutionary relationships. A key feature of alignment profiles lies in the interoperability of data format (e.g., alignment information, physio-chemical information, genomic information, etc.). Indeed, we have demonstrated that the Position Specific Scoring Matrices (PSSMs) are an informative M-dimension that is scored by quantitatively measuring the embedded or unmodified sequence alignments. Moreover, the information obtained from these alignments is informative, and remains so even in the "twilight zone" of sequence similarity (<25% identity). Although our previous embedding strategy was powerful, it suffered from contaminating alignments (embedded AND unmodified) and high computational costs. Herein, we describe the logic and algorithmic process for a heuristic embedding strategy named "Adaptive GDDA-BLAST." Adaptive GDDA-BLAST is, on average, up to 19 times faster than, but has similar sensitivity to our previous method. Further, data are provided to demonstrate the benefits of embedded-alignment measurements in terms of detecting structural homology in highly divergent protein sequences and isolating secondary structural elements of transmembrane and ankyrin-repeat domains. Together, these advances allow further exploration of the embedded alignment data space within sufficiently large data sets to eventually induce relevant statistical inferences. We show that sequence embedding could serve as one of the vehicles for measurement of low-identity alignments

  5. Different genome stability proteins underpin primed and naïve adaptation in E. coli CRISPR-Cas immunity.

    PubMed

    Ivančić-Baće, Ivana; Cass, Simon D; Wearne, Stephen J; Bolt, Edward L

    2015-12-15

    CRISPR-Cas is a prokaryotic immune system built from capture and integration of invader DNA into CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci, termed 'Adaptation', which is dependent on Cas1 and Cas2 proteins. In Escherichia coli, Cascade-Cas3 degrades invader DNA to effect immunity, termed 'Interference'. Adaptation can interact with interference ('primed'), or is independent of it ('naïve'). We demonstrate that primed adaptation requires the RecG helicase and PriA protein to be present. Genetic analysis of mutant phenotypes suggests that RecG is needed to dissipate R-loops at blocked replication forks. Additionally, we identify that DNA polymerase I is important for both primed and naive adaptation, and that RecB is needed for naïve adaptation. Purified Cas1-Cas2 protein shows specificity for binding to and nicking forked DNA within single strand gaps, and collapsing forks into DNA duplexes. The data suggest that different genome stability systems interact with primed or naïve adaptation when responding to blocked or collapsed invader DNA replication. In this model, RecG and Cas3 proteins respond to invader DNA replication forks that are blocked by Cascade interference, enabling DNA capture. RecBCD targets DNA ends at collapsed forks, enabling DNA capture without interference. DNA polymerase I is proposed to fill DNA gaps during spacer integration.

  6. Bacillus anthracis TIR Domain-Containing Protein Localises to Cellular Microtubule Structures and Induces Autophagy

    PubMed Central

    Carlsson, Emil; Thwaite, Joanne E.; Jenner, Dominic C.; Spear, Abigail M.; Flick-Smith, Helen; Atkins, Helen S.; Ding, Jeak Ling

    2016-01-01

    Toll-like receptors (TLRs) recognise invading pathogens and mediate downstream immune signalling via Toll/IL-1 receptor (TIR) domains. TIR domain proteins (Tdps) have been identified in multiple pathogenic bacteria and have recently been implicated as negative regulators of host innate immune activation. A Tdp has been identified in Bacillus anthracis, the causative agent of anthrax. Here we present the first study of this protein, designated BaTdp. Recombinantly expressed and purified BaTdp TIR domain interacted with several human TIR domains, including that of the key TLR adaptor MyD88, although BaTdp expression in cultured HEK293 cells had no effect on TLR4- or TLR2- mediated immune activation. During expression in mammalian cells, BaTdp localised to microtubular networks and caused an increase in lipidated cytosolic microtubule-associated protein 1A/1B-light chain 3 (LC3), indicative of autophagosome formation. In vivo intra-nasal infection experiments in mice showed that a BaTdp knockout strain colonised host tissue faster with higher bacterial load within 4 days post-infection compared to the wild type B. anthracis. Taken together, these findings indicate that BaTdp does not play an immune suppressive role, but rather, its absence increases virulence. BaTdp present in wild type B. anthracis plausibly interact with the infected host cell, which undergoes autophagy in self-defence. PMID:27391310

  7. Immune response to Candida albicans is preserved despite defect in O-mannosylation of secretory proteins.

    PubMed

    Corbucci, Cristina; Cenci, Elio; Skrzypek, Franck; Gabrielli, Elena; Mosci, Paolo; Ernst, Joachim F; Bistoni, Francesco; Vecchiarelli, Anna

    2007-12-01

    The PMT gene family in Candida albicans encodes five isoforms of the protein mannosyltransferases that initiate O-mannosylation of secretory proteins. Mutations at the Pmt level have been associated with differences in pathogenicity, e.g. in contrast to pmt5/pmt5, pmt2/PMT2 mutants showed poor virulence. Our objective was to determine whether these differences were related to the capacity of pmt2/PMT2 and pmt5/pmt5 to (i) express differences in selected virulence factors, and (ii) stimulate the natural immune system. The results show that pmt mutants (i) form hyphae in serum, (ii) show defective production of proteases but not of phospholipases with respect to the parental strain, (iii) undergo mycelial transition in the kidneys of hematogenously infected animals, (iv) are phagocytosed and killed by macrophages similar to the parental strain, although neutrophils are unable to destroy pmt5/pmt5, (v) engage TLR4 and stimulate MyD88 leading to NF-kappaB activation, and (vi) stimulate cytokine production by macrophages. Collectively our findings suggest that the defect in protein O-mannosylation in C. albicans cause attenuation of the virulence although the antigenic factors that retain the capacity to stimulate an efficient immune response are preserved.

  8. Structural adaptation of tooth enamel protein amelogenin in the presence of SDS micelles

    PubMed Central

    Chandrababu, Karthik Balakrishna; Dutta, Kaushik; Lokappa, Sowmya Bekshe; Ndao, Moise; Evans, John Spencer; Moradian-Oldak, Janet

    2014-01-01

    Amelogenin, the major extracellular matrix protein of developing tooth enamel is intrinsically disordered. Through its interaction with other proteins and mineral, amelogenin assists enamel biomineralization by controlling the formation of highly organized enamel crystal arrays. We used circular dichroism (CD), dynamic light scattering (DLS), fluorescence and NMR spectroscopy to investigate the folding propensity of recombinant porcine amelogenin rP172 following its interaction with SDS, at levels above critical micelle concentration. The rP172-SDS complex formation was confirmed by DLS, while an increase in the structure moiety of rP172 was noted through CD and fluorescence experiments. Fluorescence quenching analyses performed on several rP172 mutants where all but one Trp was replaced by Tyr at different sequence regions confirmed that the interaction of amelogenin with SDS micelles occurs via the N-terminal region close to Trp25 where helical segments can be detected by NMR. NMR spectroscopy and structural refinement calculations using CS-Rosetta modelling confirm that the highly conserved N-terminal domain is prone to form helical structure when bound to SDS micelles. Our findings reported here reveal interactions leading to significant changes in the secondary structure of rP172 upon treatment with SDS. These interactions may reflect the physiological relevance of the flexible nature of amelogenin and its sequence specific helical propensity that might enable it to structurally adapt with charged and potential targets such as cell surface, mineral, and other proteins during enamel biomineralization. PMID:24114119

  9. RNA binding proteins mediate the ability of a fungus to adapt to the cold.

    PubMed

    Fang, Weiguo; St Leger, Raymond J

    2010-03-01

    Little is known about how fungi adapt to chilling. In eubacteria, cold shock proteins (CSPs) facilitate translation by destabilizing RNA secondary structure. Animals and plants have homologous cold shock domains within proteins, and additional glycine-rich RNA binding proteins (GRPs), but their role in stress resistance is poorly understood. In this study, we identified GRP homologues in diverse fungi. However, only Aspergillus clavatus and Metarhizium anisopliae possessed cold shock domains. Both M. anisopliae's small eubacteria-like CSP (CRP1) and its GRP (CRP2) homologue were induced by cold. Disrupting either Crp1 or Crp2 greatly reduced metabolism and conidial germination rates at low temperatures, and decreased tolerance to freezing. However, while both Crp1 and Crp2 reduced freezing-induced production of reactive oxygen species, only Crp1 protected cells against H(2)O(2) and increased M. anisopliae's virulence to caterpillars. Unlike CRP2, CRP1 rescued the cold-sensitive growth defects of an Escherichia coli CSP deletion mutant, and CRP1 also demonstrated transcription anti-termination activity, so CRP1 can regulate transcription and translation at low temperature. Expressing either Crp1 or Crp2 in yeast increased metabolism at cold temperatures and Crp1 improved tolerance to freezing. Thus besides providing a model relevant to many biological systems, Crp1 and Crp2 have potential applications in biotechnology.

  10. Functional Validation of Hydrophobic Adaptation to Physiological Temperature in the Small Heat Shock Protein αA-crystallin

    PubMed Central

    Posner, Mason; Kiss, Andor J.; Skiba, Jackie; Drossman, Amy; Dolinska, Monika B.; Hejtmancik, J. Fielding; Sergeev, Yuri V.

    2012-01-01

    Small heat shock proteins (sHsps) maintain cellular homeostasis by preventing stress and disease-induced protein aggregation. While it is known that hydrophobicity impacts the ability of sHsps to bind aggregation-prone denaturing proteins, the complex quaternary structure of globular sHsps has made understanding the significance of specific changes in hydrophobicity difficult. Here we used recombinant protein of the lenticular sHsp α A-crystallin from six teleost fishes environmentally adapted to temperatures ranging from -2°C to 40°C to identify correlations between physiological temperature, protein stability and chaperone-like activity. Using sequence and structural modeling analysis we identified specific amino acid differences between the warm adapted zebrafish and cold adapted Antarctic toothfish that could contribute to these correlations and validated the functional consequences of three specific hydrophobicity-altering amino acid substitutions in αA-crystallin. Site directed mutagenesis of three residues in the zebrafish (V62T, C143S, T147V) confirmed that each impacts either protein stability or chaperone-like activity or both, with the V62T substitution having the greatest impact. Our results indicate a role for changing hydrophobicity in the thermal adaptation of α A-crystallin and suggest ways to produce sHsp variants with altered chaperone-like activity. These data also demonstrate that a comparative approach can provide new information about sHsp function and evolution. PMID:22479631

  11. Optimizing Intramuscular Adaptations to Aerobic Exercise: Effects of Carbohydrate Restriction and Protein Supplementation on Mitochondrial Biogenesis12

    PubMed Central

    Margolis, Lee M.; Pasiakos, Stefan M.

    2013-01-01

    Mitochondrial biogenesis is a critical metabolic adaptation to aerobic exercise training that results in enhanced mitochondrial size, content, number, and activity. Recent evidence has shown that dietary manipulation can further enhance mitochondrial adaptations to aerobic exercise training, which may delay skeletal muscle fatigue and enhance exercise performance. Specifically, studies have demonstrated that combining carbohydrate restriction (endogenous and exogenous) with a single bout of aerobic exercise potentiates the beneficial effects of exercise on markers of mitochondrial biogenesis. Additionally, studies have demonstrated that high-quality protein supplementation enhances anabolic skeletal muscle intracellular signaling and mitochondrial protein synthesis following a single bout of aerobic exercise. Mitochondrial biogenesis is stimulated by complex intracellular signaling pathways that appear to be primarily regulated by 5′AMP-activated protein kinase and p38 mitogen-activated protein kinase mediated through proliferator-activated γ receptor co-activator 1 α activation, resulting in increased mitochondrial DNA expression and enhanced skeletal muscle oxidative capacity. However, the mechanisms by which concomitant carbohydrate restriction and dietary protein supplementation modulates mitochondrial adaptations to aerobic exercise training remains unclear. This review summarizes intracellular regulation of mitochondrial biogenesis and the effects of carbohydrate restriction and protein supplementation on mitochondrial adaptations to aerobic exercise. PMID:24228194

  12. Effectively explore metastable states of proteins by adaptive nonequilibrium driving simulations

    NASA Astrophysics Data System (ADS)

    Wan, Biao; Xu, Shun; Zhou, Xin

    2017-03-01

    Nonequilibrium drivings applied in molecular dynamics (MD) simulations can efficiently extend the visiting range of protein conformations, but might compel systems to go far away from equilibrium and thus mainly explore irrelevant conformations. Here we propose a general method, called adaptive nonequilibrium simulation (ANES), to automatically adjust the external driving on the fly, based on the feedback of the short-time average response of system. Thus, the ANES approximately keeps the local equilibrium but efficiently accelerates the global motion. We illustrate the capability of the ANES in highly efficiently exploring metastable conformations in the deca-alanine peptide and find that the 0.2 -μ s ANES approximately captures the important states and folding and unfolding pathways in the HP35 solution by comparing with the result of the recent 398 -μ s equilibrium MD simulation on Anton [S. Piana et al., Proc. Natl. Acad. Sci. USA 109, 17845 (2012), 10.1073/pnas.1201811109].

  13. Angiotensin II induces kidney inflammatory injury and fibrosis through binding to myeloid differentiation protein-2 (MD2)

    PubMed Central

    Xu, Zheng; Li, Weixin; Han, Jibo; Zou, Chunpeng; Huang, Weijian; Yu, Weihui; Shan, Xiaoou; Lum, Hazel; Li, Xiaokun; Liang, Guang

    2017-01-01

    Growing evidence indicates that angiotensin II (Ang II), a potent biologically active product of RAS, is a key regulator of renal inflammation and fibrosis. In this study, we tested the hypothesis that Ang II induces renal inflammatory injury and fibrosis through interaction with myeloid differentiation protein-2 (MD2), the accessory protein of toll-like receptor 4 (TLR4) of the immune system. Results indicated that in MD2−/− mice, the Ang II-induced renal fibrosis, inflammation and kidney dysfunction were significantly reduced compared to control Ang II-infused wild-type mice. Similarly, in the presence of small molecule MD2 specific inhibitor L6H21 or siRNA-MD2, the Ang II-induced increases of pro-fibrotic and pro-inflammatory molecules were prevented in tubular NRK-52E cells. MD2 blockade also inhibited activation of NF-κB and ERK. Moreover, MD2 blockade prevented the Ang II-stimulated formation of the MD2/TLR4/MyD88 signaling complex, as well as the increased surface binding of Ang II in NRK-52E cells. In addition, Ang II directly bound recombinant MD2 protein, rather than TLR4 protein. We conclude that MD2 is a significant contributor in the Ang II-induced kidney inflammatory injury in chronic renal diseases. Furthermore, MD2 inhibition could be a new and important therapeutic strategy for preventing progression of chronic renal diseases. PMID:28322341

  14. Different genome stability proteins underpin primed and naïve adaptation in E. coli CRISPR-Cas immunity

    PubMed Central

    Ivančić-Baće, Ivana; Cass, Simon D; Wearne, Stephen J; Bolt, Edward L

    2015-01-01

    CRISPR-Cas is a prokaryotic immune system built from capture and integration of invader DNA into CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci, termed ‘Adaptation’, which is dependent on Cas1 and Cas2 proteins. In Escherichia coli, Cascade-Cas3 degrades invader DNA to effect immunity, termed ‘Interference’. Adaptation can interact with interference (‘primed’), or is independent of it (‘naïve’). We demonstrate that primed adaptation requires the RecG helicase and PriA protein to be present. Genetic analysis of mutant phenotypes suggests that RecG is needed to dissipate R-loops at blocked replication forks. Additionally, we identify that DNA polymerase I is important for both primed and naive adaptation, and that RecB is needed for naïve adaptation. Purified Cas1-Cas2 protein shows specificity for binding to and nicking forked DNA within single strand gaps, and collapsing forks into DNA duplexes. The data suggest that different genome stability systems interact with primed or naïve adaptation when responding to blocked or collapsed invader DNA replication. In this model, RecG and Cas3 proteins respond to invader DNA replication forks that are blocked by Cascade interference, enabling DNA capture. RecBCD targets DNA ends at collapsed forks, enabling DNA capture without interference. DNA polymerase I is proposed to fill DNA gaps during spacer integration. PMID:26578567

  15. Architectural adaptation and protein expression patterns of Salmonella enterica serovar Enteritidis biofilms under laminar flow conditions.

    PubMed

    Mangalappalli-Illathu, Anil K; Lawrence, John R; Swerhone, George D W; Korber, Darren R

    2008-03-31

    Salmonella enterica serovar Enteritidis is a significant biofilm-forming pathogen. The influence of a 10-fold difference in nutrient laminar flow velocity on the dynamics of Salmonella Enteritidis biofilm formation and protein expression profiles were compared in order to ascertain how flow velocity influenced biofilm structure and function. Low-flow (0.007 cm s(-1)) biofilms consisted of diffusely-arranged microcolonies which grew until merging by approximately 72 h. High-flow (0.07 cm s(-1)) biofilms were significantly thicker (36+/-3 microm (arithmetic mean+/-standard error; n=225) versus 16+/-2 microm for low-flow biofilms at 120 h) and consisted of large bacterial mounds interspersed by water channels. Lectin-binding analysis of biofilm exopolymers revealed a significantly higher (P<0.05) proportion of N-acetylgalactosamine (GalNAc) in low-flow biofilms (55.2%), relative to only 1.2% in high-flow biofilms. Alternatively, the proportions of alpha-L-fucose and N-acetylglucosamine (GlcNAc2)-N-acetylneuraminic acid (NeuNAc) polymer-conjugates were significantly higher (P<0.05) in high-flow biofilms (69.1% and 29.6%, respectively) than low-flow biofilms (33.1% and 11.7%, respectively). Despite an apparent flow rate-based physiologic effect on biofilm structure and exopolymer composition, no major shift in whole-cell protein expression patterns was seen between 168 h-old low-flow and high-flow biofilms, and notably did not include any response involving the stress response proteins, DnaK, SodB, and Tpx. Proteins involved in degradation and energy metabolism (PduA, GapA, GpmA, Pgk, and RpiA), RNA and protein biosynthesis (Tsf, TufA, and RpoZ), cell processes (Crr, MalE, and PtsH), and adaptation (GrcA), and some hypothetical proteins (YcbL and YnaF) became up-regulated in both biofilm systems relative to a 168 h-old planktonic cell control. Our results indicate that Salmonella Enteritidis biofilms altered their structure and extracellular glycoconjugate composition

  16. Flavin-Induced Oligomerization in Escherichia coli Adaptive Response Protein AidB

    SciTech Connect

    Hamill, Michael J.; Jost, Marco; Wong, Cintyu; Elliott, Sean J.; Drennan, Catherine L.

    2011-11-21

    The process known as 'adaptive response' allows Escherichia coli to respond to small doses of DNA-methylating agents by upregulating the expression of four proteins. While the role of three of these proteins in mitigating DNA damage is well understood, the function of AidB is less clear. Although AidB is a flavoprotein, no catalytic role has been established for the bound cofactor. Here we investigate the possibility that flavin plays a structural role in the assembly of the AidB tetramer. We report the generation and biophysical characterization of deflavinated AidB and of an AidB mutant that has greatly reduced affinity for flavin adenine dinucleotide (FAD). Using fluorescence quenching and analytical ultracentrifugation, we find that apo AidB has a high affinity for FAD, as indicated by an apparent dissociation constant of 402.1 {+-} 35.1 nM, and that binding of substoichiometric amounts of FAD triggers a transition in the AidB oligomeric state. In particular, deflavinated AidB is dimeric, whereas the addition of FAD yields a tetramer. We further investigate the dimerization and tetramerization interfaces of AidB by determining a 2.8 {angstrom} resolution crystal structure in space group P3{sub 2} that contains three intact tetramers in the asymmetric unit. Taken together, our findings provide strong evidence that FAD plays a structural role in the formation of tetrameric AidB.

  17. Adaptive evolution of the venom-targeted vWF protein in opossums that eat pitvipers.

    PubMed

    Jansa, Sharon A; Voss, Robert S

    2011-01-01

    The rapid evolution of venom toxin genes is often explained as the result of a biochemical arms race between venomous animals and their prey. However, it is not clear that an arms race analogy is appropriate in this context because there is no published evidence for rapid evolution in genes that might confer toxin resistance among routinely envenomed species. Here we report such evidence from an unusual predator-prey relationship between opossums (Marsupialia: Didelphidae) and pitvipers (Serpentes: Crotalinae). In particular, we found high ratios of replacement to silent substitutions in the gene encoding von Willebrand Factor (vWF), a venom-targeted hemostatic blood protein, in a clade of opossums known to eat pitvipers and to be resistant to their hemorrhagic venom. Observed amino-acid substitutions in venom-resistant opossums include changes in net charge and hydrophobicity that are hypothesized to weaken the bond between vWF and one of its toxic snake-venom ligands, the C-type lectin-like protein botrocetin. Our results provide the first example of rapid adaptive evolution in any venom-targeted molecule, and they support the notion that an evolutionary arms race might be driving the rapid evolution of snake venoms. However, in the arms race implied by our results, venomous snakes are prey, and their venom has a correspondingly defensive function in addition to its usual trophic role.

  18. Object-adapted trapping and shape-tracking to probe a bacterial protein chain motor

    NASA Astrophysics Data System (ADS)

    Roth, Julian; Koch, Matthias; Rohrbach, Alexander

    2015-03-01

    The helical bacterium Spiroplasma is a motile plant and anthropod pathogen which swims by propagating pairs of kinks along its cell body. As a well suited model system for bacterial locomotion, understanding the cell's molecular motor is of vital interest also regarding the combat of bacterial diseases. The extensive deformations related to these kinks are caused by a contractile cytoskeletal protein ribbon representing a linear motor in contrast to common rotary motors as, e.g., flagella. We present new insights into the working of this motor through experiments with object-adapted optical traps and shape-tracking techniques. We use the given laser irradiation from the optical trap to hinder bacterial energy (ATP) production through the production of O2 radicals. The results are compared with experiments performed under the influence of an O2-Scavenger and ATP inhibitors, respectively. Our results show clear dependences of the kinking properties on the ATP concentration inside the bacterium. The experiments are supported by a theoretical model which we developed to describe the switching of the ribbon's protein subunits.

  19. Phospholipids and protein adaptation of Pseudomonas sp. to the xenoestrogen tributyltin chloride (TBT).

    PubMed

    Bernat, Przemysław; Siewiera, Paulina; Soboń, Adrian; Długoński, Jerzy

    2014-09-01

    A tributyltin (TBT)-resistant strain of Pseudomonas sp. isolated from an overworked car filter was tested for its adaptation to TBT. The isolate was checked for organotin degradation ability, as well as membrane lipid and cellular protein composition in the presence of TBT. The phospholipid profiles of bacteria, grown with and without increased amounts of TBT, were characterized using liquid chromatography/electrospray ionization/mass spectrometry. The strain reacted to the biocide by changing the composition of its phospholipids. TBT induced a twofold decline in the amounts of many molecular species of phosphatidylglycerol and an increase in the levels of phosphatidic acid (by 58%) and phosphatidylethanolamine (by 70%). An increase in the degree of saturation of phospholipid fatty acids of TBT exposed Pseudomonas sp. was observed. These changes in the phospholipid composition and concentration reflect the mechanisms which support optimal lipid ordering in the presence of toxic xenobiotic. In the presence of TBT the abundances of 16 proteins, including TonB-dependent receptors, porins and peroxidases were modified, which could indicate a contribution of some enzymes to TBT resistance.

  20. Positive and negative regulation of antigen receptor signaling by the Shc family of protein adapters.

    PubMed

    Finetti, Francesca; Savino, Maria Teresa; Baldari, Cosima T

    2009-11-01

    The Shc adapter family includes four members that are expressed as multiple isoforms and participate in signaling by a variety of cell-surface receptors. The biological relevance of Shc proteins as well as their variegated function, which relies on their highly conserved modular structure, is underscored by the distinct and dramatic phenotypic alterations resulting from deletion of individual Shc isoforms both in the mouse and in two model organisms, Drosophila melanogaster and Caenorhabditis elegans. The p52 isoform of ShcA couples antigen and cytokine receptors to Ras activation in both lymphoid and myeloid cells. However, the recognition of the spectrum of activities of p52ShcA in the immune system has been steadily expanding in recent years to other fundamental processes both at the cell and organism levels. Two other Shc family members, p66ShcA and p52ShcC/Rai, have been identified recently in T and B lymphocytes, where they antagonize survival and attenuate antigen receptor signaling. These developments reveal an unexpected and complex interplay of multiple Shc proteins in lymphocytes.

  1. Adaptive evolution of tight junction protein claudin-14 in echolocating whales.

    PubMed

    Xu, Huihui; Liu, Yang; He, Guimei; Rossiter, Stephen J; Zhang, Shuyi

    2013-11-10

    Toothed whales and bats have independently evolved specialized ultrasonic hearing for echolocation. Recent findings have suggested that several genes including Prestin, Tmc1, Pjvk and KCNQ4 appear to have undergone molecular adaptations associated with the evolution of this ultrasonic hearing in mammals. Here we studied the hearing gene Cldn14, which encodes the claudin-14 protein and is a member of tight junction proteins that functions in the organ of Corti in the inner ear to maintain a cationic gradient between endolymph and perilymph. Particular mutations in human claudin-14 give rise to non-syndromic deafness, suggesting an essential role in hearing. Our results uncovered two bursts of positive selection, one in the ancestral branch of all toothed whales and a second in the branch leading to the delphinid, phocoenid and ziphiid whales. These two branches are the same as those previously reported to show positive selection in the Prestin gene. Furthermore, as with Prestin, the estimated hearing frequencies of whales significantly correlate with numbers of branch-wise non-synonymous substitutions in Cldn14, but not with synonymous changes. However, in contrast to Prestin, we found no evidence of positive selection in bats. Our findings from Cldn14, and comparisons with Prestin, strongly implicate multiple loci in the acquisition of echolocation in cetaceans, but also highlight possible differences in the evolutionary route to echolocation taken by whales and bats.

  2. Comparative proteome profiling of bovine and human Staphylococcus epidermidis strains for screening specifically expressed virulence and adaptation proteins.

    PubMed

    Siljamäki, Pia; Varmanen, Pekka; Kankainen, Matti; Pyörälä, Satu; Karonen, Taru; Iivanainen, Antti; Auvinen, Petri; Paulin, Lars; Laine, Pia K; Taponen, Suvi; Simojoki, Heli; Sukura, Antti; Nyman, Tuula A; Savijoki, Kirsi

    2014-08-01

    The present study reports a comparative proteome cataloging of a bovine mastitis and a human-associated Staphylococcus epidermidis strain with a specific focus on surfome (cell-wall bound and extracellular) proteins. Protein identification by 1DE coupled with LC-MS/MS analyses resulted in 1400 and 1287 proteins from the bovine (PM221) and human (ATCC12228) strains, respectively, covering over 50% of all predicted and more than 30% of all predicted surfome proteins in both strains. Comparison of the identification results suggests elevated levels of proteins involved in adherence, biofilm formation, signal transduction, house-keeping functions, and immune evasion in PM221, whereas ATCC12228 was more effective in expressing host defense evasion proteases, skin adaptation lipases, hemagglutination, and heavy-metal resistance proteins. Phenotypic analyses showed that only PM221 displays protein- and DNA-mediated adherent growth, and that PM221 was more efficient in cleaving tributyrin, a natural compound of milk fat under low CO2 conditions. These findings are in line with the identification data and suggest that distinct expression of lipases and adhesive surfome proteins could lead to the observed phenotypes. This study is the first extensive survey of S. epidermidis proteomes to date, providing several protein candidates to be examined for their roles in adaptation and virulence in vivo. All MS data have been deposited in the ProteomeXchange with identifier PXD000404 (http://proteomecentral.proteomexchange.org/dataset/PXD000404).

  3. A Staphylococcus aureus TIR domain protein virulence factor blocks TLR2-mediated NF-κB signaling.

    PubMed

    Askarian, Fatemeh; van Sorge, Nina M; Sangvik, Maria; Beasley, Federico C; Henriksen, Jørn R; Sollid, Johanna U E; van Strijp, Jos A G; Nizet, Victor; Johannessen, Mona

    2014-01-01

    Signaling through Toll-like receptors (TLRs), crucial molecules in the induction of host defense responses, requires adaptor proteins that contain a Toll/interleukin-1 receptor (TIR) domain. The pathogen Staphylococcus aureus produces several innate immune-evasion molecules that interfere with the host's innate immune response. A database search analysis suggested the presence of a gene encoding a homologue of the human TIR domain in S. aureus MSSA476 which was named staphylococcal TIR domain protein (TirS). Ectopic expression of TirS in human embryonic kidney, macrophage and keratinocyte cell lines interfered with signaling through TLR2, including MyD88 and TIRAP, NF-κB and/or mitogen-activated protein kinase pathways. Moreover, the presence of TirS reduced the levels of cytokines MCP-1 and G-CSF secreted in response to S. aureus. The effects on NF-κB pathway were confirmed using S. aureus MSSA476 wild type, an isogenic mutant MSSA476ΔtirS, and complemented MSSA476ΔtirS +pTirS in a Transwell system where bacteria and host cells were physically separated. Finally, in a systematic mouse infection model, TirS promoted bacterial accumulation in several organs 4 days postinfection. The results of this study reveal a new S. aureus virulence factor that can interfere with PAMP-induced innate immune signaling in vitro and bacterial survival in vivo.

  4. Evolutionary adaptation of an AraC-like regulatory protein in Citrobacter rodentium and Escherichia species.

    PubMed

    Tan, Aimee; Petty, Nicola K; Hocking, Dianna; Bennett-Wood, Vicki; Wakefield, Matthew; Praszkier, Judyta; Tauschek, Marija; Yang, Ji; Robins-Browne, Roy

    2015-04-01

    The evolution of pathogenic bacteria is a multifaceted and complex process, which is strongly influenced by the horizontal acquisition of genetic elements and their subsequent expression in their new hosts. A well-studied example is the RegA regulon of the enteric pathogen Citrobacter rodentium. The RegA regulatory protein is a member of the AraC/XylS superfamily, which coordinates the expression of a gene repertoire that is necessary for full pathogenicity of this murine pathogen. Upon stimulation by an exogenous, gut-associated signal, namely, bicarbonate ions, RegA activates the expression of a series of genes, including virulence factors, such as autotransporters, fimbriae, a dispersin-like protein, and the grlRA operon on the locus of enterocyte effacement pathogenicity island. Interestingly, the genes encoding RegA homologues are distributed across the genus Escherichia, encompassing pathogenic and nonpathogenic subtypes. In this study, we carried out a series of bioinformatic, transcriptional, and functional analyses of the RegA regulons of these bacteria. Our results demonstrated that regA has been horizontally transferred to Escherichia spp. and C. rodentium. Comparative studies of two RegA homologues, namely, those from C. rodentium and E. coli SMS-3-5, a multiresistant environmental strain of E. coli, showed that the two regulators acted similarly in vitro but differed in terms of their abilities to activate the virulence of C. rodentium in vivo, which evidently was due to their differential activation of grlRA. Our data indicate that RegA from C. rodentium has strain-specific adaptations that facilitate infection of its murine host. These findings shed new light on the development of virulence by C. rodentium and on the evolution of virulence-regulatory genes of bacterial pathogens in general.

  5. Evolutionary Adaptation of an AraC-Like Regulatory Protein in Citrobacter rodentium and Escherichia Species

    PubMed Central

    Tan, Aimee; Petty, Nicola K.; Hocking, Dianna; Bennett-Wood, Vicki; Wakefield, Matthew; Praszkier, Judyta; Tauschek, Marija; Yang, Ji

    2015-01-01

    The evolution of pathogenic bacteria is a multifaceted and complex process, which is strongly influenced by the horizontal acquisition of genetic elements and their subsequent expression in their new hosts. A well-studied example is the RegA regulon of the enteric pathogen Citrobacter rodentium. The RegA regulatory protein is a member of the AraC/XylS superfamily, which coordinates the expression of a gene repertoire that is necessary for full pathogenicity of this murine pathogen. Upon stimulation by an exogenous, gut-associated signal, namely, bicarbonate ions, RegA activates the expression of a series of genes, including virulence factors, such as autotransporters, fimbriae, a dispersin-like protein, and the grlRA operon on the locus of enterocyte effacement pathogenicity island. Interestingly, the genes encoding RegA homologues are distributed across the genus Escherichia, encompassing pathogenic and nonpathogenic subtypes. In this study, we carried out a series of bioinformatic, transcriptional, and functional analyses of the RegA regulons of these bacteria. Our results demonstrated that regA has been horizontally transferred to Escherichia spp. and C. rodentium. Comparative studies of two RegA homologues, namely, those from C. rodentium and E. coli SMS-3-5, a multiresistant environmental strain of E. coli, showed that the two regulators acted similarly in vitro but differed in terms of their abilities to activate the virulence of C. rodentium in vivo, which evidently was due to their differential activation of grlRA. Our data indicate that RegA from C. rodentium has strain-specific adaptations that facilitate infection of its murine host. These findings shed new light on the development of virulence by C. rodentium and on the evolution of virulence-regulatory genes of bacterial pathogens in general. PMID:25624355

  6. Specific adaptation of gastric emptying to diets with differing protein content in the rat: is endogenous cholecystokinin implicated?

    PubMed Central

    Shi, G; Leray, V; Scarpignato, C; Bentouimou, N; Varannes, S; Cherbut, C; Galmiche, J

    1997-01-01

    Background—Recent studies indicate that gastric emptying may be influenced by patterns of previous nutrient intake. Endogenous cholecystokinin (CCK), whose synthesis and release can be affected by dietary intake, has a major role in the regulation of gastric emptying. 
Aims—To evaluate the influence of diets with differing protein content on gastric emptying of differing liquid test meals and plasma CCK levels in the rat and to check whether the inhibitory effect of exogenous CCK on gastric emptying is modified after long term intake of diets with differing protein content. 
Methods—Rats were fed for three weeks with high protein, medium protein (regular), or low protein diet. On day 22 gastric emptying of a peptone meal was studied. In addition, basal and postprandial CCK levels after the different dietary regimens were measured by bioassay. The time course of dietary adaptation was studied and its specificity assessed through the use of different (peptone, glucose, and methylcellulose) test meals. The effect of exogenous CCK-8 on gastric emptying was studied at the end of the adaptation period (three weeks).
Results—Feeding the animals with a high protein diet for three weeks resulted in a significant (p<0.05) acceleration (by 21.2(8.2)%) of gastric emptying while feeding with a low protein diet was followed by a significant (p<0.05) delay (by 24.0 (6.2)%) in the emptying rate. When the time course of the effect of dietary adaptation on gastric emptying was studied, it appeared that at least two weeks are required for dietary protein to be effective. The regulatory effect of dietary protein on gastric emptying proved to be dependent on meal composition. Only the emptying rate of a protein containing meal (40% peptone) was significantly modified by previous dietary intake. No significant (p>0.05) changes were observed with glucose and methylcellulose meals whose emptying rates were similar in rats receiving a high protein or low protein diet. A

  7. Immune adjuvant effect of a Toxoplasma gondii profilin-like protein in autologous whole-tumor-cell vaccination in mice

    PubMed Central

    Pyo, Kyoung-Ho; Lee, You-Won; Lim, Sun Min; Shin, Eun-Hee

    2016-01-01

    Profilin-like protein in Toxoplasma gondii (TgPLP) is a Toll-like receptor (TLR) agonist. In this study, we investigated whether TgPLP has an adjuvant effect on immune function in autologous whole-tumor-cell vaccine (AWV) treatment. Mice vaccinated with AWV together with recombinant TgPLP protein had smaller CT26 tumors and increased survival. TgPLP treatment strongly increased the production of IL-12 through MyD88 signaling and several chemokines, including CCL5, CCL12, and XCL1, in bone marrow-derived macrophages (BMMs). In addition, TgPLP increased the phagocytosis of tumor cells by BMMs and promoted immune cell mobility on a tumor-matrigel scaffold. TgPLP triggered immune responses as demonstrated by increased expression of antigen presenting cell markers (MHC class I and II, B7.1, and B7.2) in BMMs and increased IL-12 and IFN-γ expression in mice. Mice vaccinated with AWV and TgPLP had more immune cells (CD4+ and CD8+ T cells, natural killer cells, and macrophages) in the spleen and higher total IgG and IgG2a concentrations in the blood than mice vaccinated with AWV alone. These findings suggest that TgPLP is a TLR-based vaccine adjuvant that enhances antitumor immune responses during vaccination with AWV. PMID:27687589

  8. Inhibition of TLR4 signaling by Brucella TIR-containing protein TcpB-derived decoy peptides.

    PubMed

    Ke, Yuehua; Li, Wenna; Wang, Yufei; Yang, Mingjuan; Guo, Jinpeng; Zhan, Shaoxia; Du, Xinying; Wang, Zhoujia; Yang, Min; Li, Juan; Li, Wenfeng; Chen, Zeliang

    2016-09-01

    Brucella spp. avoid host immune recognition and thus, weaken the immune response to infection. The Toll/interleukin-1 receptor (TIR) domain-containing protein (TcpB/Btp1) of Brucella spp. is thought to be involved in blocking host innate immune responses by binding to adaptors downstream of Toll-like receptors. In this study, based on the observation that TcpB binds to the host target proteins, MAL, through the TIR domain, we examined decoy peptides from TcpB TIR domains and found that TB-8 and TB-9 substantially inhibit lipopolysaccharide (LPS)-induced signaling in vitro and in vivo. Both these peptides share a common loop, the DD loop, indicating a novel structural region mediating TIR interactions. The inhibition of LPS signaling by TB-8 and TB-9 shows no preference to MyD88-dependent cytokines, such as TNF-α and IL-1β or TRIF-dependent cytokines including IFN-β and IL-6. Furthermore, these two peptides rescue the virulence of Brucella ΔtcpB mutants at the cellular level, indicating key roles of the DD loop in Brucella pathogenesis. In conclusion, identification of inhibitors from the bacterial TIR domains is helpful not only for illustrating interacting mechanisms between TIR domains and bacterial pathogenesis, but also for developing novel signaling inhibitors and therapeutics for human inflammatory diseases.

  9. Cth2 Protein Mediates Early Adaptation of Yeast Cells to Oxidative Stress Conditions.

    PubMed

    Castells-Roca, Laia; Pijuan, Jordi; Ferrezuelo, Francisco; Bellí, Gemma; Herrero, Enrique

    2016-01-01

    Cth2 is an mRNA-binding protein that participates in remodeling yeast cell metabolism in iron starvation conditions by promoting decay of the targeted molecules, in order to avoid excess iron consumption. This study shows that in the absence of Cth2 immediate upregulation of expression of several of the iron regulon genes (involved in high affinity iron uptake and intracellular iron redistribution) upon oxidative stress by hydroperoxide is more intense than in wild type conditions where Cth2 is present. The oxidative stress provokes a temporary increase in the levels of Cth2 (itself a member of the iron regulon). In such conditions Cth2 molecules accumulate at P bodies-like structures when the constitutive mRNA decay machinery is compromised. In addition, a null Δcth2 mutant shows defects, in comparison to CTH2 wild type cells, in exit from α factor-induced arrest at the G1 stage of the cell cycle when hydroperoxide treatment is applied. The cell cycle defects are rescued in conditions that compromise uptake of external iron into the cytosol. The observations support a role of Cth2 in modulating expression of diverse iron regulon genes, excluding those specifically involved in the reductive branch of the high-affinity transport. This would result in immediate adaptation of the yeast cells to an oxidative stress, by controlling uptake of oxidant-promoting iron cations.

  10. Cth2 Protein Mediates Early Adaptation of Yeast Cells to Oxidative Stress Conditions

    PubMed Central

    Ferrezuelo, Francisco; Bellí, Gemma; Herrero, Enrique

    2016-01-01

    Cth2 is an mRNA-binding protein that participates in remodeling yeast cell metabolism in iron starvation conditions by promoting decay of the targeted molecules, in order to avoid excess iron consumption. This study shows that in the absence of Cth2 immediate upregulation of expression of several of the iron regulon genes (involved in high affinity iron uptake and intracellular iron redistribution) upon oxidative stress by hydroperoxide is more intense than in wild type conditions where Cth2 is present. The oxidative stress provokes a temporary increase in the levels of Cth2 (itself a member of the iron regulon). In such conditions Cth2 molecules accumulate at P bodies-like structures when the constitutive mRNA decay machinery is compromised. In addition, a null Δcth2 mutant shows defects, in comparison to CTH2 wild type cells, in exit from α factor-induced arrest at the G1 stage of the cell cycle when hydroperoxide treatment is applied. The cell cycle defects are rescued in conditions that compromise uptake of external iron into the cytosol. The observations support a role of Cth2 in modulating expression of diverse iron regulon genes, excluding those specifically involved in the reductive branch of the high-affinity transport. This would result in immediate adaptation of the yeast cells to an oxidative stress, by controlling uptake of oxidant-promoting iron cations. PMID:26824473

  11. Vaccinia Virus Immunomodulator A46: A Lipid and Protein-Binding Scaffold for Sequestering Host TIR-Domain Proteins

    PubMed Central

    Radakovics, Katharina; Smith, Terry K.; Bobik, Nina; Round, Adam; Djinović-Carugo, Kristina; Usón, Isabel

    2016-01-01

    Vaccinia virus interferes with early events of the activation pathway of the transcriptional factor NF-kB by binding to numerous host TIR-domain containing adaptor proteins. We have previously determined the X-ray structure of the A46 C-terminal domain; however, the structure and function of the A46 N-terminal domain and its relationship to the C-terminal domain have remained unclear. Here, we biophysically characterize residues 1–83 of the N-terminal domain of A46 and present the X-ray structure at 1.55 Å. Crystallographic phases were obtained by a recently developed ab initio method entitled ARCIMBOLDO_BORGES that employs tertiary structure libraries extracted from the Protein Data Bank; data analysis revealed an all β-sheet structure. This is the first such structure solved by this method which should be applicable to any protein composed entirely of β-sheets. The A46(1–83) structure itself is a β-sandwich containing a co-purified molecule of myristic acid inside a hydrophobic pocket and represents a previously unknown lipid-binding fold. Mass spectrometry analysis confirmed the presence of long-chain fatty acids in both N-terminal and full-length A46; mutation of the hydrophobic pocket reduced the lipid content. Using a combination of high resolution X-ray structures of the N- and C-terminal domains and SAXS analysis of full-length protein A46(1–240), we present here a structural model of A46 in a tetrameric assembly. Integrating affinity measurements and structural data, we propose how A46 simultaneously interferes with several TIR-domain containing proteins to inhibit NF-κB activation and postulate that A46 employs a bipartite binding arrangement to sequester the host immune adaptors TRAM and MyD88. PMID:27973613

  12. Immunogenicity of recombinant proteins consisting of Plasmodium vivax circumsporozoite protein allelic variant-derived epitopes fused with Salmonella enterica Serovar Typhimurium flagellin.

    PubMed

    Leal, Monica Teixeira Andrade; Camacho, Ariane Guglielmi Ariza; Teixeira, Laís Helena; Bargieri, Daniel Youssef; Soares, Irene Silva; Tararam, Cibele Aparecida; Rodrigues, Mauricio M

    2013-09-01

    A Plasmodium falciparum circumsporozoite protein (CSP)-based recombinant fusion vaccine is the first malaria vaccine to reach phase III clinical trials. Resistance to infection correlated with the production of antibodies to the immunodominant central repeat region of the CSP. In contrast to P. falciparum, vaccine development against the CSP of Plasmodium vivax malaria is far behind. Based on this gap in our knowledge, we generated a recombinant chimeric protein containing the immunodominant central repeat regions of the P. vivax CSP fused to Salmonella enterica serovar Typhimurium-derived flagellin (FliC) to activate the innate immune system. The recombinant proteins that were generated contained repeat regions derived from each of the 3 different allelic variants of the P. vivax CSP or a fusion of regions derived from each of the 3 allelic forms. Mice were subcutaneously immunized with the fusion proteins alone or in combination with the Toll-like receptor 3 (TLR-3) agonist poly(I·C), and the anti-CSP serum IgG response was measured. Immunization with a mixture of the 3 recombinant proteins, each containing immunodominant epitopes derived from a single allelic variant, rather than a single recombinant protein carrying a fusion of regions derived from each of 3 allelic forms elicited a stronger immune response. This response was independent of TLR-4 but required TLR-5/MyD88 activation. Antibody titers significantly increased when poly(I·C) was used as an adjuvant with a mixture of the 3 recombinant proteins. These recombinant fusion proteins are novel candidates for the development of an effective malaria vaccine against P. vivax.

  13. ADAPT, a Novel Scaffold Protein-Based Probe for Radionuclide Imaging of Molecular Targets That Are Expressed in Disseminated Cancers.

    PubMed

    Garousi, Javad; Lindbo, Sarah; Nilvebrant, Johan; Åstrand, Mikael; Buijs, Jos; Sandström, Mattias; Honarvar, Hadis; Orlova, Anna; Tolmachev, Vladimir; Hober, Sophia

    2015-10-15

    Small engineered scaffold proteins have attracted attention as probes for radionuclide-based molecular imaging. One class of these imaging probes, termed ABD-Derived Affinity Proteins (ADAPT), has been created using the albumin-binding domain (ABD) of streptococcal protein G as a stable protein scaffold. In this study, we report the development of a clinical lead probe termed ADAPT6 that binds HER2, an oncoprotein overexpressed in many breast cancers that serves as a theranostic biomarker for several approved targeting therapies. Surface-exposed amino acids of ABD were randomized to create a combinatorial library enabling selection of high-affinity binders to various proteins. Furthermore, ABD was engineered to enable rapid purification, to eradicate its binding to albumin, and to enable rapid blood clearance. Incorporation of a unique cysteine allowed site-specific conjugation to a maleimido derivative of a DOTA chelator, enabling radionuclide labeling, ¹¹¹In for SPECT imaging and ⁶⁸Ga for PET imaging. Pharmacologic studies in mice demonstrated that the fully engineered molecule (111)In/⁶⁸Ga-DOTA-(HE)3-ADAPT6 was specifically bound and taken up by HER2-expressing tumors, with a high tumor-to-normal tissue ratio in xenograft models of human cancer. Unbound tracer underwent rapid renal clearance followed by high renal reabsorption. HER2-expressing xenografts were visualized by gamma-camera or PET at 1 hour after infusion. PET experiments demonstrated feasibility for discrimination of xenografts with high or low HER2 expression. Our results offer a preclinical proof of concept for the use of ADAPT probes for noninvasive in vivo imaging.

  14. Bovine viral diarrhea virus type 2 in vivo infection modulates TLR4 responsiveness in differentiated Myeloid cells which is associated with decreased MyD88 expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bovine viral diarrhea virus (BVDV) causes clinical signs in cattle ranging from mild to severe acute infection which can lead to increased susceptibility to secondary bacteria. In this study we examined the effects of BVDV genotype 2 (BVDV2) infection on the ability of myeloid lineage cells derived...

  15. IL-33-induced alterations in murine intestinal function and cytokine responses are MyD88, STAT6, and IL-13-dependent

    Technology Transfer Automated Retrieval System (TEKTRAN)

    IL-33 is a recently identified cytokine member of the IL-1 family. The biological activities of IL-33 are associated with promotion of Th2 and inhibition of Th1/Th17 immune responses. Exogenous IL-33 induces a typical “type 2” immune response in the gastrointestinal tract, yet the underlying mechani...

  16. Therapeutic Inhibition of Pro-Inflammatory Signaling and Toxicity to Staphylococcal Enterotoxin B by a Synthetic Dimeric BB-Loop Mimetic of MyD88

    DTIC Science & Technology

    2012-07-27

    C57BL/6 mice were obtained from Charles River (NCI-Frederick, Frederick, MD). Cell Isolation and Purification Peripheral blood mononuclear cells (MNC...7]. Consequently, both super- antigenic exotoxins (SEs) and bacterial LPS ( endotoxin ) have been implicated in the pathogenesis of TSS, supported by...enterotoxin B (SEB) and SEA was purchased from Porton Down, Inc. (Salisbury, UK) and stored at 250uC. SEB or SEA was endotoxin free and prepared under GMP

  17. Molecular cloning, characterization and expression analysis of TLR9, MyD88 and TRAF6 genes in common carp (Cyprinus carpio)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Induction of innate immune pathways is critical for early host defense but there is limited understanding of how teleost fish recognize pathogen molecules and activate these pathways. In mammals, cells of the innate immune system detect pathogenic molecular structures using pattern recognition rece...

  18. The negatively charged regions of lactoferrin binding protein B, an adaptation against anti-microbial peptides.

    PubMed

    Morgenthau, Ari; Beddek, Amanda; Schryvers, Anthony B

    2014-01-01

    Lactoferrin binding protein B (LbpB) is a bi-lobed membrane bound lipoprotein that is part of the lactoferrin receptor complex in a variety of Gram-negative pathogens. Despite high sequence diversity among LbpBs from various strains and species, a cluster of negatively charged amino acids is invariably present in the protein's C-terminal lobe in all species except Moraxella bovis. The function of LbpB in iron acquisition has yet to be experimentally demonstrated, whereas in vitro studies have shown that LbpB confers protection against lactoferricin, a short cationic antimicrobial peptide released from the N- terminus of lactoferrin. In this study we demonstrate that the negatively charged regions can be removed from the Neisseria meningitidis LbpB without compromising stability, and this results in the inability of LbpB to protect against the bactericidal effects of lactoferricin. The release of LbpB from the cell surface by the autotransporter NalP reduces the protection against lactoferricin in the in vitro killing assay, attributed to removal of LbpB during washing steps, but is unlikely to have a similar impact in vivo. The protective effect of the negatively charged polysaccharide capsule in the killing assay was less than the protection conferred by LbpB, suggesting that LbpB plays a major role in protection against cationic antimicrobial peptides in vivo. The selective release of LbpB by NalP has been proposed to be a mechanism for evading the adaptive immune response, by reducing the antibody binding to the cell surface, but may also provide insights into the primary function of LbpB in vivo. Although TbpB and LbpB have been shown to be major targets of the human immune response, the selective release of LbpB suggests that unlike TbpB, LbpB may not be essential for iron acquisition, but important for protection against cationic antimicrobial peptides.

  19. Adaptive aneuploidy protects against thiol peroxidase deficiency by increasing respiration via key mitochondrial proteins.

    PubMed

    Kaya, Alaattin; Gerashchenko, Maxim V; Seim, Inge; Labarre, Jean; Toledano, Michel B; Gladyshev, Vadim N

    2015-08-25

    Aerobic respiration is a fundamental energy-generating process; however, there is cost associated with living in an oxygen-rich environment, because partially reduced oxygen species can damage cellular components. Organisms evolved enzymes that alleviate this damage and protect the intracellular milieu, most notably thiol peroxidases, which are abundant and conserved enzymes that mediate hydrogen peroxide signaling and act as the first line of defense against oxidants in nearly all living organisms. Deletion of all eight thiol peroxidase genes in yeast (∆8 strain) is not lethal, but results in slow growth and a high mutation rate. Here we characterized mechanisms that allow yeast cells to survive under conditions of thiol peroxidase deficiency. Two independent ∆8 strains increased mitochondrial content, altered mitochondrial distribution, and became dependent on respiration for growth but they were not hypersensitive to H2O2. In addition, both strains independently acquired a second copy of chromosome XI and increased expression of genes encoded by it. Survival of ∆8 cells was dependent on mitochondrial cytochrome-c peroxidase (CCP1) and UTH1, present on chromosome XI. Coexpression of these genes in ∆8 cells led to the elimination of the extra copy of chromosome XI and improved cell growth, whereas deletion of either gene was lethal. Thus, thiol peroxidase deficiency requires dosage compensation of CCP1 and UTH1 via chromosome XI aneuploidy, wherein these proteins support hydroperoxide removal with the reducing equivalents generated by the electron transport chain. To our knowledge, this is the first evidence of adaptive aneuploidy counteracting oxidative stress.

  20. Plant natriuretic peptides induce proteins diagnostic for an adaptive response to stress

    PubMed Central

    Turek, Ilona; Marondedze, Claudius; Wheeler, Janet I.; Gehring, Chris; Irving, Helen R.

    2014-01-01

    In plants, structural and physiological evidence has suggested the presence of biologically active natriuretic peptides (PNPs). PNPs are secreted into the apoplast, are systemically mobile and elicit a range of responses signaling via cGMP. The PNP-dependent responses include tissue specific modifications of cation transport and changes in stomatal conductance and the photosynthetic rate. PNP also has a critical role in host defense responses. Surprisingly, PNP-homologs are produced by several plant pathogens during host colonization suppressing host defense responses. Here we show that a synthetic peptide representing the biologically active fragment of the Arabidopsis thaliana PNP (AtPNP-A) induces the production of reactive oxygen species in suspension-cultured A. thaliana (Col-0) cells. To identify proteins whose expression changes in an AtPNP-A dependent manner, we undertook a quantitative proteomic approach, employing tandem mass tag (TMT) labeling, to reveal temporal responses of suspension-cultured cells to 1 nM and 10 pM PNP at two different time-points post-treatment. Both concentrations yield a distinct differential proteome signature. Since only the higher (1 nM) concentration induces a ROS response, we conclude that the proteome response at the lower concentration reflects a ROS independent response. Furthermore, treatment with 1 nM PNP results in an over-representation of the gene ontology (GO) terms “oxidation-reduction process,” “translation” and “response to salt stress” and this is consistent with a role of AtPNP-A in the adaptation to environmental stress conditions. PMID:25505478

  1. Changes in the receptorbinding haemagglutinin protein of wild-type morbilliviruses are not required for adaptation to Vero cells.

    PubMed

    Nielsen, Line; Andersen, Mads Klindt; Jensen, Tove Dannemann; Blixenkrone-Møller, Merete; Bolt, Gert

    2003-10-01

    We examined the consequences of isolation and adaptation to Vero cells for the receptorbinding haemagglutinin (H) gene of four syncytia-forming isolates of canine distemper virus (CDV) and of a dolphin morbillivirus isolate. A Vero-adapted CDV isolate exhibited biased hypermutation, since 11 out of 12 nucleotide differences to other isolates from the same epidemic were U-C transitions. Most of these transitions appeared to have taken place during in vitro cultivation. Previously, biased hypermutation in morbilliviruses has almost exclusively been described for subacute sclerosing panencephalitis and measles inclusion body encephalitis, which are rare measles virus brain infections. Amino acid changes in the H proteins were not required for Vero cell adaptation, suggesting that Vero cells express receptors for wild-type morbilliviruses. This strongly indicate the existence of other morbillivirus receptors than CD46 and CDw150.

  2. Adaptive changes in protein expression in Escherichia coli as a consequence of growth in milk

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteria must adapt to the environment in the mammary gland and the pressures exerted by the host immune system in order to survive. Understanding bacterial adaptation to their environment could become a foundation to research into better therapeutics for treatment of bacterial infections. As a fi...

  3. [The possible role of the elements of protein secondary structure in adaptation to the action of ionizing radiation].

    PubMed

    Kharchenko, L I; Pavlovskaia, T E

    1997-01-01

    Changes in the secondary structure of enzymes induced by gamma-rays 60Co at doses not exceeding one ionization per macromolecule were studied to elucidate a possible role of radiation-chemical processes in the evolution of proteins. The data on the comparative radioresistance of various types of secondary protein structures, alpha-helix, parallel and anti-parallel beta-structures, and beta-turn, were obtained by the method of circular dichroism. It was shown that beta-turns were resistant against radiation, alpha-helix was relatively stable, and beta-layer underwent significant changes. The importance of these structural types in the evolution of proteins is discussed. A special role of beta-turn as structural elements fixing the confirmation of macromolecules and therefore responsible for adaptation of the protein structure against a constant radiation background is proposed.

  4. Surface proteins of Setaria cervi induce inflammation in macrophage through Toll-like receptor 4 (TLR4)-mediated signalling pathway.

    PubMed

    Mukherjee, Su; Mukherjee, Sa; Bhattacharya, S; Sinha Babu, S P

    2017-01-01

    Lymphatic filariasis is a vectorborne parasitic disease that results in morbidities, disabilities and socio-economic loss each year globally. Inflammatory consequences associated with any form of filariasis have drawn special attention. However, the molecular insight behind the inflammation of host macrophage (MФ) is considered as one of the shaded areas in filarial research. Herein, major emphasis was given to study the signalling pathway of MФ inflammation induced by surface proteins (SPs) of filarial parasite through in vitro and in vivo approaches. Twenty-four hours of in vitro stimulation of Raw MФs with endotoxin-free SPs of Setaria cervi resulted in the secretion of pro-inflammatory cytokines (TNF-α and IL-1β) that revealed induction of inflammation, which was found to be elicited from classical NF-кB activation. Moreover, this NF-кB activation was found to be signalled from TLR4 and mediated by the downstream signalling intermediates, viz. MyD88, pTAK1 and NEMO. In vivo studies in adult Wistar rats, experimentally injected with SPs, clearly supported the outcomes of in vitro experiments by showing higher degree of inflammation rather classical activation of the peritoneal MФs. Therefore, SPs from S. cervi cuticle could be responsible for the induction of pro-inflammatory response in MФ, which appears to be propagated through TLR4-NF-кB route.

  5. Imiquimod directly inhibits Hedgehog signalling by stimulating adenosine receptor/protein kinase A-mediated GLI phosphorylation

    PubMed Central

    Wolff, F; Loipetzberger, A; Gruber, W; Esterbauer, H; Aberger, F; Frischauf, A M

    2013-01-01

    Imiquimod (IMQ), a nucleoside analogue of the imidazoquinoline family, is used in the topical treatment of basal cell carcinoma (BCC) and other skin diseases. It is reported to be a TLR7 and TLR8 agonist and, as such, initiates a Th1 immune response by activating sentinel cells in the vicinity of the tumour. BCC is a hedgehog (HH)-driven malignancy with oncogenic glioma-associated oncogene (GLI) signalling activated in a ligand-independent manner. Here we show that IMQ can also directly repress HH signalling by negatively modulating GLI activity in BCC and medulloblastoma cells. Further, we provide evidence that the repressive effect of IMQ on HH signalling is not dependent on TLR/MYD88 signalling. Our results suggest a mechanism for IMQ engaging adenosine receptors (ADORAs) to control GLI signalling. Pharmacological activation of ADORA with either an ADORA agonist or IMQ resulted in a protein kinase A (PKA)-mediated GLI phosphorylation and reduction in GLI activator levels. The activation of PKA and HH pathway target gene downregulation in response to IMQ were abrogated by ADORA inhibition. Furthermore, activated Smoothened signalling, which positively signals to GLI transcription factors, could be effectively counteracted by IMQ. These results reveal a previously unknown mode of action of IMQ in the treatment of BCC and also suggest a role for ADORAs in the regulation of oncogenic HH signalling. PMID:23995793

  6. Mutation of a cuticular protein, BmorCPR2, alters larval body shape and adaptability in silkworm, Bombyx mori.

    PubMed

    Qiao, Liang; Xiong, Gao; Wang, Ri-xin; He, Song-zhen; Chen, Jie; Tong, Xiao-ling; Hu, Hai; Li, Chun-lin; Gai, Ting-ting; Xin, Ya-qun; Liu, Xiao-fan; Chen, Bin; Xiang, Zhong-huai; Lu, Cheng; Dai, Fang-yin

    2014-04-01

    Cuticular proteins (CPs) are crucial components of the insect cuticle. Although numerous genes encoding cuticular proteins have been identified in known insect genomes to date, their functions in maintaining insect body shape and adaptability remain largely unknown. In the current study, positional cloning led to the identification of a gene encoding an RR1-type cuticular protein, BmorCPR2, highly expressed in larval chitin-rich tissues and at the mulberry leaf-eating stages, which is responsible for the silkworm stony mutant. In the Dazao-stony strain, the BmorCPR2 allele is a deletion mutation with significantly lower expression, compared to the wild-type Dazao strain. Dysfunctional BmorCPR2 in the stony mutant lost chitin binding ability, leading to reduced chitin content in larval cuticle, limitation of cuticle extension, abatement of cuticle tensile properties, and aberrant ratio between internodes and intersegmental folds. These variations induce a significant decrease in cuticle capacity to hold the growing internal organs in the larval development process, resulting in whole-body stiffness, tightness, and hardness, bulging intersegmental folds, and serious defects in larval adaptability. To our knowledge, this is the first study to report the corresponding phenotype of stony in insects caused by mutation of RR1-type cuticular protein. Our findings collectively shed light on the specific role of cuticular proteins in maintaining normal larval body shape and will aid in the development of pest control strategies for the management of Lepidoptera.

  7. Decoupling of rapid and adaptive evolution among seminal fluid proteins in heliconius butterflies with divergent mating systems.

    PubMed

    Walters, James R; Harrison, Richard G

    2011-10-01

    Reproductive proteins often diverge rapidly between species. This pattern is frequently attributed to postmating sexual selection. Heliconius butterflies offer a good opportunity to examine this hypothesis by contrasting patterns of reproductive protein evolution between clades with divergent mating systems. Pupal-mating Heliconius females typically mate only once, limiting opportunity for postmating sexual selection. In contrast, adult-mating females remate throughout life. Reproductive protein evolution is therefore predicted to be slower and show little evidence of positive selection in the pupal-mating clade. We examined this prediction by sequencing 18 seminal fluid protein genes from a dozen Heliconius species and a related outgroup. Two proteins exhibited dN/dS > 1, implicating positive selection in the rapid evolution of at least a few Heliconius seminal fluid proteins. However, contrary to predictions, the average evolutionary rate of seminal fluid proteins was greater among pupal-mating Heliconius. Based on these results, we suggest that positive selection and relaxed constraint can generate conflicting patterns of reproductive protein evolution between mating systems. As predicted, some loci may show elevated evolutionary rates in promiscuous taxa relative to monandrous taxa resulting from adaptations to postmating sexual selection. However, when monandry is derived (as in Heliconius), the opposite pattern may result from relaxed selective constraints.

  8. Mechanism of bacterial interference with TLR4 signaling by Brucella Toll/interleukin-1 receptor domain-containing protein TcpB.

    PubMed

    Alaidarous, Mohammed; Ve, Thomas; Casey, Lachlan W; Valkov, Eugene; Ericsson, Daniel J; Ullah, M Obayed; Schembri, Mark A; Mansell, Ashley; Sweet, Matthew J; Kobe, Bostjan

    2014-01-10

    Upon activation of Toll-like receptors (TLRs), cytoplasmic Toll/interleukin-1 receptor (TIR) domains of the receptors undergo homo- or heterodimerization. This in turn leads to the recruitment of adaptor proteins, activation of transcription factors, and the secretion of pro-inflammatory cytokines. Recent studies have described the TIR domain-containing protein from Brucella melitensis, TcpB (BtpA/Btp1), to be involved in virulence and suppression of host innate immune responses. TcpB interferes with TLR4 and TLR2 signaling pathways by a mechanism that remains controversial. In this study, we show using co-immunoprecipitation analyses that TcpB interacts with MAL, MyD88, and TLR4 but interferes only with the MAL-TLR4 interaction. We present the crystal structure of the TcpB TIR domain, which reveals significant structural differences in the loop regions compared with other TIR domain structures. We demonstrate that TcpB forms a dimer in solution, and the crystal structure reveals the dimerization interface, which we validate by mutagenesis and biophysical studies. Our study advances the understanding of the molecular mechanisms of host immunosuppression by bacterial pathogens.

  9. Signal Regulatory Protein alpha (SIRPalpha)+ Cells in the Adaptive Response to ESAT-6/CFP-10 Protein of Tuberculous Mycobacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10(CFP-10) are co-secreted proteins of Mycobacterium tuberculosis complex mycobacteria (includes M. bovis, the zoonotic agent of bovine tuberculosis) involved in phagolysosome escape of the bacillus and, potentially, in the eff...

  10. Expression of Iron-Related Proteins Differentiate Non-Cancerous and Cancerous Breast Tumors

    PubMed Central

    Pizzamiglio, Sara; De Bortoli, Maida; Taverna, Elena; Signore, Michele; Veneroni, Silvia; Chi-shing Cho, William; Orlandi, Rosaria; Verderio, Paolo; Bongarzone, Italia

    2017-01-01

    We have previously reported hepcidin and ferritin increases in the plasma of breast cancer patients, but not in patients with benign breast disease. We hypothesized that these differences in systemic iron homeostasis may reflect alterations in different iron-related proteins also play a key biochemical and regulatory role in breast cancer. Thus, here we explored the expression of a bundle of molecules involved in both iron homeostasis and tumorigenesis in tissue samples. Enzyme-linked immunosorbent assay (ELISA) or reverse-phase protein array (RPPA), were used to measure the expression of 20 proteins linked to iron processes in 24 non-cancerous, and 56 cancerous, breast tumors. We found that cancerous tissues had higher level of hepcidin than benign lesions (p = 0.012). The univariate analysis of RPPA data highlighted the following seven proteins differentially expressed between non-cancerous and cancerous breast tissue: signal transducer and transcriptional activator 5 (STAT5), signal transducer and activator of transcription 3 (STAT3), bone morphogenetic protein 6 (BMP6), cluster of differentiation 74 (CD74), transferrin receptor (TFRC), inhibin alpha (INHA), and STAT5_pY694. These findings were confirmed for STAT5, STAT3, BMP6, CD74 and INHA when adjusting for age. The multivariate statistical analysis indicated an iron-related 10-protein panel effective in separating non-cancerous from cancerous lesions including STAT5, STAT5_pY694, myeloid differentiation factor 88 (MYD88), CD74, iron exporter ferroportin (FPN), high mobility group box 1 (HMGB1), STAT3_pS727, TFRC, ferritin heavy chain (FTH), and ferritin light chain (FTL). Our results showed an association between some iron-related proteins and the type of tumor tissue, which may provide insight in strategies for using iron chelators to treat breast cancer. PMID:28216608

  11. PSB27: A thylakoid protein enabling Arabidopsis to adapt to changing light intensity

    SciTech Connect

    Hou, Xin; Garcia, Veder J.; Buchanan, Bob B.; Luan, Sheng

    2016-08-22

    complex that might be PSII supercomplex. Under objective 2, we have found that FKBP16-2 interacted with PSB27 that was further pursuited and published a research article in PNAS (attached). Under Objective 3, we have identified several mutants of other FKBPs in the thyalkoid lumen that should be further studied if future funding is available. Under Objective 4, we have started to build a network of lumenal proteins that play a number of roles in photosynthesis. For example, the CYP37 and CYP28 are linked to chloroplast signaling to nucleus, critical for controlling plant response to high light and adaptation to climate change. Unfortunately these studies have been terminated due to funding shortage.

  12. The Negatively Charged Regions of Lactoferrin Binding Protein B, an Adaptation against Anti-Microbial Peptides

    PubMed Central

    Morgenthau, Ari; Beddek, Amanda; Schryvers, Anthony B.

    2014-01-01

    Lactoferrin binding protein B (LbpB) is a bi-lobed membrane bound lipoprotein that is part of the lactoferrin receptor complex in a variety of Gram-negative pathogens. Despite high sequence diversity among LbpBs from various strains and species, a cluster of negatively charged amino acids is invariably present in the protein’s C-terminal lobe in all species except Moraxella bovis. The function of LbpB in iron acquisition has yet to be experimentally demonstrated, whereas in vitro studies have shown that LbpB confers protection against lactoferricin, a short cationic antimicrobial peptide released from the N- terminus of lactoferrin. In this study we demonstrate that the negatively charged regions can be removed from the Neisseria meningitidis LbpB without compromising stability, and this results in the inability of LbpB to protect against the bactericidal effects of lactoferricin. The release of LbpB from the cell surface by the autotransporter NalP reduces the protection against lactoferricin in the in vitro killing assay, attributed to removal of LbpB during washing steps, but is unlikely to have a similar impact in vivo. The protective effect of the negatively charged polysaccharide capsule in the killing assay was less than the protection conferred by LbpB, suggesting that LbpB plays a major role in protection against cationic antimicrobial peptides in vivo. The selective release of LbpB by NalP has been proposed to be a mechanism for evading the adaptive immune response, by reducing the antibody binding to the cell surface, but may also provide insights into the primary function of LbpB in vivo. Although TbpB and LbpB have been shown to be major targets of the human immune response, the selective release of LbpB suggests that unlike TbpB, LbpB may not be essential for iron acquisition, but important for protection against cationic antimicrobial peptides. PMID:24465982

  13. Adaptive Control of Two-Photon Excitation of Green Fluorescent Protein with Shaped Femtosecond Pulses

    NASA Astrophysics Data System (ADS)

    Kawano, Hiroyuki; Nabekawa, Yasuo; Suda, Akira; Oishi, Yu; Mizuno, Hideaki; Miyawaki, Atsushi; Midorikawa, Katsumi

    For many years, it has been believed that a Fourier-transform-limited (FTL) laser pulse is the most effective light source for the generation of nonlinear phenomena, since the FTL pulse has the shortest pulse duration, that is, the highest intensity, that can be limited by the spectral width due to the principle of uncertainty. Recently, many reports have been published on the adaptive control of nonlinear phenomena with shaped femtosecond excitation laser pulses [1, 2]. Their reports have shown that the modification of the spectral and temporal phases of excitation pulses can increase or decrease the probabilities and efficiencies of such nonlinear phenomena. This method has been widely applied to studies on the active control of molecular motions or chemical reactions [3,4]. Considering further novel biological applications, we focus on the two-photon excited fluorescence (TPEF) of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria. GFP is spontaneously fluorescent and is relatively nontoxic compared with other organic dyes used as optical markers. Therefore, it has been widely used as a "tag" material for the fluorescence observation of living cells [5]. Two-photon excitation microscopy (TPEM) is a powerful tool for biological real-time observation due to its various advantages, such as a clear contrast, good S/N ratio, and high spatial resolution [7]. From a practical point of view, however, there is a serious problem with TPEM, which is the photobleaching of a dye. The intensity of a fluorescence signal decreases significantly during observation. One of the reasons for this is that the chromophore structure is degraded by intense excitation laser pulses that are required for efficient two-photon excitation. In this study, therefore, we attempted to determine the optimal phase for maximizing the fluorescence efficiency of a GFP variant with excitation laser pulses of minimal intensity. We considered that GFP can be an ideal dye for the

  14. SH3P7 is a cytoskeleton adapter protein and is coupled to signal transduction from lymphocyte antigen receptors.

    PubMed

    Larbolette, O; Wollscheid, B; Schweikert, J; Nielsen, P J; Wienands, J

    1999-02-01

    Lymphocytes respond to antigen receptor engagement with tyrosine phosphorylation of many cellular proteins, some of which have been identified and functionally characterized. Here we describe SH3P7, a novel substrate protein for Src and Syk family kinases. SH3P7 migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 55-kDa protein that is preferentially expressed in brain, thymus, and spleen. It contains multiple amino acid sequence motifs, including two consensus tyrosine phosphorylation sites of the YXXP type and one SH3 domain. A region of sequence similarity, which we named SCAD, was found in SH3P7 and three actin-binding proteins. The SCAD region may represent a new type of protein-protein interaction domain that mediates binding to actin. Consistent with this possibility, SH3P7 colocalizes with actin filaments of the cytoskeleton. Altogether, our data implicate SH3P7 as an adapter protein which links antigen receptor signaling to components of the cytoskeleton.

  15. Structural insights into the cold adaptation of the photosynthetic pigment-protein C-phycocyanin from an Arctic cyanobacterium.

    PubMed

    Su, Hai-Nan; Wang, Qian-Min; Li, Chun-Yang; Li, Kang; Luo, Wei; Chen, Bo; Zhang, Xi-Ying; Qin, Qi-Long; Zhou, Bai-Cheng; Chen, Xiu-Lan; Zhang, Yu-Zhong; Xie, Bin-Bin

    2017-04-01

    The cold adaptation mechanism of phycobiliproteins, the major photosynthetic pigment-proteins in cyanobacteria and red algae, has rarely been studied. Here we reported the biochemical, structural, and molecular dynamics simulation study of the C-phycocyanin from Arctic cyanobacterial strain Pseudanabaena sp. LW0831. We characterized the phycobilisome components of LW0831 and obtained their gene sequences. Compared to the mesophilic counterpart from Arthrospira platensis (Ar-C-PC), LW0831 C-phycocyanin (Ps-C-PC) has a decreased thermostability (∆Tm of -16°C), one of the typical features of cold-adapted enzymes. To uncover its structural basis, we resolved the crystal structure of Ps-C-PC 1 at 2.04Å. Consistent with the decrease in thermostability, comparative structural analyses revealed decreased intra-trimer and inter-trimer interactions in Ps-C-PC 1, compared to Ar-C-PC. However, comparative molecular dynamics simulations indicated that Ps-C-PC 1 shows similar flexibilities to Ar-C-PC for both the (αβ)3 trimer and (αβ)6 hexamer. Therefore, the optimization mode is clearly different from cold-adapted enzymes, which usually have increased flexibilities. Detailed analyses demonstrated different optimization modes for the α and β subunits and it was revealed that hydrophobic interactions are key to this difference, though salt bridges, hydrogen bonds, and surface hydrophobicity are also involved. This study is the first report of the structure of cold-adapted phycobiliproteins and provides insights into the cold-adaptation strategies of non-enzyme proteins.

  16. Self-adaptive differential evolution algorithm incorporating local search for protein-ligand docking

    NASA Astrophysics Data System (ADS)

    Chung, Hwan Won; Cho, Seung Joo; Lee, Kwang-Ryeol; Lee, Kyu-Hwan

    2013-02-01

    Differential Evolution (DE) algorithm is powerful in optimization problems over several real parameters. DE depends on strategies to generate new trial solutions and the associated parameter values for searching performance. In self-adaptive DE, the automatic learning about previous evolution was used to determine the best mutation strategy and its parameter settings. By combining the self-adaptive DE and Hooke Jeeves local search, we developed a new docking method named SADock (Strategy Adaptation Dock) with the help of AutoDock4 scoring function. As the accuracy and performance of SADock was evaluated in self-docking using the Astex diverse set, the introduced SADock showed better success ratio (89%) than the success ratio (60%) of the Lamarckian genetic algorithm (LGA) of AutoDock4. The self-adapting scheme enabled our new docking method to converge fast and to be robust through the various docking problems.

  17. Selective interleukin-1 receptor–associated kinase 4 inhibitors for the treatment of autoimmune disorders and lymphoid malignancy

    PubMed Central

    Kelly, Priscilla N.; Romero, Donna L.; Yang, Yibin; Shaffer, Arthur L.; Chaudhary, Divya; Robinson, Shaughnessy; Miao, Wenyan; Rui, Lixin; Westlin, William F.; Kapeller, Rosana

    2015-01-01

    Pathological activation of the Toll-like receptor signaling adaptor protein MYD88 underlies many autoimmune and inflammatory disease states. In the activated B cell–like (ABC) subtype of diffuse large B cell lymphoma (DLBCL), the oncogenic MYD88 L265P mutation occurs in 29% of cases, making it the most prevalent activating mutation in this malignancy. IRAK4 kinase accounts for almost all of the biological functions of MYD88, highlighting IRAK4 as a therapeutic target for diseases driven by aberrant MYD88 signaling. Using innovative structure-based drug design methodologies, we report the development of highly selective and bioavailable small molecule IRAK4 inhibitors, ND-2158 and ND-2110. These small molecules suppressed LPS-induced TNF production, alleviated collagen-induced arthritis, and blocked gout formation in mouse models. IRAK4 inhibition promoted killing of ABC DLBCL lines harboring MYD88 L265P, by down-modulating survival signals, including NF-κB and autocrine IL-6/IL-10 engagement of the JAK–STAT3 pathway. In ABC DLBCL xenograft models, IRAK4 inhibition suppressed tumor growth as a single agent, and in combination with the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib or the Bcl-2 inhibitor ABT-199. Our findings support pharmacological inhibition of IRAK4 as a therapeutic strategy in autoimmune disorders, in a genetically defined population of ABC DLBCL, and possibly other malignancies dependent on aberrant MYD88 signaling. PMID:26621451

  18. Cardioprotective effect of metformin in lipopolysaccharide-induced sepsis via suppression of toll-like receptor 4 (TLR4) in heart.

    PubMed

    Vaez, Haleh; Rameshrad, Maryam; Najafi, Moslem; Barar, Jaleh; Barzegari, Abolfazl; Garjani, Alireza

    2016-02-05

    Sepsis-induced myocardial dysfunction is a serious organ complication. In the present study, we investigated the effect of metformin on myocardial dysfunction and TLR4 activity in LPS-induced sepsis. Male Wistar rats were randomly divided into 3 groups (n=6): control received normal saline (0.5ml), LPS group received lipopolysaccharide (0.5mg/kg; i.p), and metformin treated group received LPS (0.5mg/kg)+metformin (100mg/kg; i.p). 9h later the hemodynamic parameters were recorded, blood samples were collected, and the hearts were removed and weighted. The concentration of TNF-α, content of MYD88, the phosphorylation of AMPK, and the rate of TLR4 expression in the heart were assessed. In the animals treated with metformin, the preservation of left ventricular function was associated with the reduction of myeloperoxidase activity (31%, P<0.01) in the heart and decrease of TNF-α level both in the serum and heart tissue (20%, P<0.01 and 42%, P<0.05, respectively). It was found that the level of phosphorylated AMPK in heart was significantly upregulated by 43% (P<0.001) in the metformin group while the content of TLRs adapter protein of MyD88 was reduced by 45% (P<0.05). This was associated with a remarkable decrease in the expression of myocardial TLR4. Furthermore, in a mice model of sepsis, coadministration of compound C (20mg/kg) as an AMPK inhibitor reversed the suppressive effects of metformin on TLR4 expression and MYD88 protein level. These results suggest that metformin exhibits cardioprotective effects in sepsis by suppression of TLR4 activity, at least in part through pathways involving AMPK activation.

  19. Caspase-9/-3 activation and apoptosis are induced in mouse macrophages upon ingestion and digestion of Escherichia coli bacteria.

    PubMed

    Häcker, Hans; Fürmann, Christine; Wagner, Hermann; Häcker, Georg

    2002-09-15

    A number of highly virulent, intracellular bacteria are known to induce cell death by apoptosis in infected host cells. In this work we demonstrate that phagocytosis of bacteria from the Escherichia coli laboratory strain K12 DH5alpha is a potent cell death stimulus for mouse macrophages. RAW264.7 mouse macrophages took up bacteria and digested them within 2-4 h as investigated with green fluorescent protein-expressing bacteria. No evidence of apoptosis was seen at 8 h postexposure, but at 24 h approximately 70% of macrophages displayed an apoptotic phenotype by a series of parameters. Apoptosis was blocked by inhibition of caspases or by forced expression of the apoptosis-inhibiting protein Bcl-2. Processing of caspase-3 and caspase-9 but not caspase-8 was seen suggesting that the mitochondrial branch of the apoptotic pathway was activated. Active effector caspases could be detected in two different assays. Because the adapter molecule myeloid differentiation factor 88 (MyD88) has been implicated in apoptosis, involvement of the Toll-like receptor pathway was investigated. In RAW264.7 cells, heat-treated bacteria were taken up poorly and failed to induce significant apoptosis. However, cell activation was almost identical between live and heat-inactivated bacteria as measured by extracellular signal-regulated kinase activation, generation of free radicals, and TNF secretion. Furthermore, primary bone marrow-derived macrophages from wild-type as well as from MyD88-deficient mice underwent apoptosis upon phagocytosis of bacteria. These results show that uptake and digestion of bacteria leads to MyD88-independent apoptosis in mouse macrophages. This form of cell death might have implications for the generation of the immune response.

  20. Proteins Associated with Adaptation of Cultured Tobacco Cells to NaCl 1

    PubMed Central

    Singh, Narendra K.; Handa, Avtar K.; Hasegawa, Paul M.; Bressan, Ray A.

    1985-01-01

    Cultured tobacco cells (Nicotiana tabacum L. cv Wisconsin 38) adapted to grow in medium containing high levels of NaCl or polyethylene glycol (PEG) produce several new or enhanced polypeptide bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The intensities of some of the polypeptide bands (molecular weights of 58, 37, 35.5, 34, 26, 21, 19.5, and 18 kilodaltons) increase with increasing levels of NaCl adaptation, while the intensities of other polypeptide bands (54, 52, 17.5, and 16.5 kilodaltons) are reduced. Enhanced levels of 43- and 26-kilodalton polypeptides are present in both NaCl and PEG-induced water stress adapted cells but are not detectable in unadapted cells. In addition, PEG adapted cells have enhanced levels of 29-, 17.5-, 16.5-, and 11-kilodalton polypeptides and reduced levels of 58-, 54-, 52-, 37-, 35.5-, 34-, 21-, 19.5-, and 18-kilodalton polypeptide bands. Synthesis of 26-kilodalton polypeptide(s) occurs at two different periods during culture growth of NaCl adapted cells. Unadapted cells also incorporate 35S into a 26-kilodalton polypeptide during the later stage of culture growth beginning at midlog phase. The 26-kilodalton polypeptides from adapted and unadapted cells have similar partial proteolysis peptide maps and are immunologically cross-reactive. During adaptation to NaCl, unadapted cells synthesize and accumulate a major 26-kilodalton polypeptide, and the beginning of synthesis corresponds to the period of osmotic adjustment and culture growth. From our results, we suggest an involvement of the 26-kilodalton polypeptide in the adaptation of cultured tobacco cells to NaCl and water stress. Images Fig. 1 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 Fig. 11 Fig. 12 PMID:16664357

  1. Differential compartmentalization of Streptococcus pyogenes virulence factors and host protein binding properties as a mechanism for host adaptation.

    PubMed

    Kilsgård, Ola; Karlsson, Christofer; Malmström, Erik; Malmström, Johan

    2016-11-01

    Streptococcus pyogenes is an important human pathogen responsible for substantial morbidity and mortality worldwide. Although S. pyogenes is a strictly human pathogen with no other known animal reservoir, several murine infection models exist to explore different aspects of the bacterial pathogenesis. Inoculating mice with wild-type S. pyogenes strains can result in the generation of new bacterial phenotypes that are hypervirulent compared to the original inoculum. In this study, we used a serial mass spectrometry based proteomics strategy to investigate if these hypervirulent strains have an altered distribution of virulence proteins across the intracellular, surface associated and secreted bacterial compartments and if any change in compartmentalization can alter the protein-protein interaction network between bacteria and host proteins. Quantitative analysis of the S. pyogenes surface and secreted proteomes revealed that animal passaged strains are associated with significantly higher amount of virulence factors on the bacterial surface and in the media. This altered virulence factor compartmentalization results in increased binding of several mouse plasma proteins to the bacterial surface, a trend that was consistent for mouse plasma from several different mouse strains. In general, both the wild-type strain and animal passaged strain were capable of binding high amounts of human plasma proteins. However, compared to the non-passaged strains, the animal passaged strains displayed an increased ability to bind mouse plasma proteins, in particular for M protein binders, indicating that the increased affinity for mouse blood plasma proteins is a consequence of host adaptation of this pathogen to a new host. In conclusion, plotting the total amount of virulence factors against the total amount of plasma proteins associated to the bacterial surface could clearly separate out animal passaged strains from wild type strains indicating a virulence model that could

  2. Proteomic Analysis of Rhizoctonia solani Identifies Infection-specific, Redox Associated Proteins and Insight into Adaptation to Different Plant Hosts*

    PubMed Central

    Anderson, Jonathan P.; Hane, James K.; Stoll, Thomas; Pain, Nicholas; Hastie, Marcus L.; Kaur, Parwinder; Hoogland, Christine; Gorman, Jeffrey J.; Singh, Karam B.

    2016-01-01

    Rhizoctonia solani is an important root infecting pathogen of a range of food staples worldwide including wheat, rice, maize, soybean, potato and others. Conventional resistance breeding strategies are hindered by the absence of tractable genetic resistance in any crop host. Understanding the biology and pathogenicity mechanisms of this fungus is important for addressing these disease issues, however, little is known about how R. solani causes disease. This study capitalizes on recent genomic studies by applying mass spectrometry based proteomics to identify soluble, membrane-bound and culture filtrate proteins produced under wheat infection and vegetative growth conditions. Many of the proteins found in the culture filtrate had predicted functions relating to modification of the plant cell wall, a major activity required for pathogenesis on the plant host, including a number found only under infection conditions. Other infection related proteins included a high proportion of proteins with redox associated functions and many novel proteins without functional classification. The majority of infection only proteins tested were confirmed to show transcript up-regulation during infection including a thaumatin which increased susceptibility to R. solani when expressed in Nicotiana benthamiana. In addition, analysis of expression during infection of different plant hosts highlighted how the infection strategy of this broad host range pathogen can be adapted to the particular host being encountered. Data are available via ProteomeXchange with identifier PXD002806. PMID:26811357

  3. Proteomic Analysis of Rhizoctonia solani Identifies Infection-specific, Redox Associated Proteins and Insight into Adaptation to Different Plant Hosts.

    PubMed

    Anderson, Jonathan P; Hane, James K; Stoll, Thomas; Pain, Nicholas; Hastie, Marcus L; Kaur, Parwinder; Hoogland, Christine; Gorman, Jeffrey J; Singh, Karam B

    2016-04-01

    Rhizoctonia solaniis an important root infecting pathogen of a range of food staples worldwide including wheat, rice, maize, soybean, potato and others. Conventional resistance breeding strategies are hindered by the absence of tractable genetic resistance in any crop host. Understanding the biology and pathogenicity mechanisms of this fungus is important for addressing these disease issues, however, little is known about howR. solanicauses disease. This study capitalizes on recent genomic studies by applying mass spectrometry based proteomics to identify soluble, membrane-bound and culture filtrate proteins produced under wheat infection and vegetative growth conditions. Many of the proteins found in the culture filtrate had predicted functions relating to modification of the plant cell wall, a major activity required for pathogenesis on the plant host, including a number found only under infection conditions. Other infection related proteins included a high proportion of proteins with redox associated functions and many novel proteins without functional classification. The majority of infection only proteins tested were confirmed to show transcript up-regulation during infection including a thaumatin which increased susceptibility toR. solaniwhen expressed inNicotiana benthamiana In addition, analysis of expression during infection of different plant hosts highlighted how the infection strategy of this broad host range pathogen can be adapted to the particular host being encountered. Data are available via ProteomeXchange with identifier PXD002806.

  4. Degradation of Human PDZ-Proteins by Human Alphapapillomaviruses Represents an Evolutionary Adaptation to a Novel Cellular Niche.

    PubMed

    Van Doorslaer, Koenraad; DeSalle, Rob; Einstein, Mark H; Burk, Robert D

    2015-06-01

    In order to complete their life cycle, papillomaviruses have evolved to manipulate a plethora of cellular pathways. The products of the human Alphapapillomavirus E6 proteins specifically interact with and target PDZ containing proteins for degradation. This viral phenotype has been suggested to play a role in viral oncogenesis. To analyze the association of HPV E6 mediated PDZ-protein degradation with cervical oncogenesis, a high-throughput cell culture assay was developed. Degradation of an epitope tagged human MAGI1 isoform was visualized by immunoblot. The correlation between HPV E6-induced degradation of hMAGI1 and epidemiologically determined HPV oncogenicity was evaluated using a Bayesian approach within a phylogenetic context. All tested oncogenic types degraded the PDZ-containing protein hMAGI1d; however, E6 proteins isolated from several related albeit non-oncogenic viral types were equally efficient at degrading hMAGI1. The relationship between both traits (oncogenicity and PDZ degradation potential) is best explained by a model in which the potential to degrade PDZ proteins was acquired prior to the oncogenic phenotype. This analysis provides evidence that the ancestor of both oncogenic and non-oncogenic HPVs acquired the potential to degrade human PDZ-containing proteins. This suggests that HPV E6 directed degradation of PDZ-proteins represents an ancient ecological niche adaptation. Phylogenetic modeling indicates that this phenotype is not specifically correlated with oncogenic risk, but may act as an enabling phenotype. The role of PDZ protein degradation in HPV fitness and oncogenesis needs to be interpreted in the context of Alphapapillomavirus evolution.

  5. Proteomic Profiling of Cereal Aphid Saliva Reveals Both Ubiquitous and Adaptive Secreted Proteins

    PubMed Central

    Wilkinson, Tom L.

    2013-01-01

    The secreted salivary proteins from two cereal aphid species, Sitobion avenae and Metopolophium dirhodum, were collected from artificial diets and analysed by tandem mass spectrometry. Protein identification was performed by searching MS data against the official protein set from the current pea aphid (Acyrthosiphon pisum) genome assembly and revealed 12 and 7 proteins in the saliva of S. avenae and M. dirhodum, respectively. When combined with a comparable dataset from A. pisum, only three individual proteins were common to all the aphid species; two paralogues of the GMC oxidoreductase family (glucose dehydrogenase; GLD) and ACYPI009881, an aphid specific protein previously identified as a putative component of the salivary sheath. Antibodies were designed from translated protein sequences obtained from partial cDNA sequences for ACYPI009881 and both saliva associated GLDs. The antibodies detected all parent proteins in secreted saliva from the three aphid species, but could only detect ACYPI009881, and not saliva associated GLDs, in protein extractions from the salivary glands. This result was confirmed by immunohistochemistry using whole and sectioned salivary glands, and in addition, localised ACYPI009881 to specific cell types within the principal salivary gland. The implications of these findings for the origin of salivary components and the putative role of the proteins identified are discussed in the context of our limited understanding of the functional relationship between aphid saliva and the plants they feed on. The mass spectrometry data have been deposited to the ProteomeXchange and can be accessed under the identifier PXD000113. PMID:23460852

  6. Proteomic profiling of cereal aphid saliva reveals both ubiquitous and adaptive secreted proteins.

    PubMed

    Rao, Sohail A K; Carolan, James C; Wilkinson, Tom L

    2013-01-01

    The secreted salivary proteins from two cereal aphid species, Sitobion avenae and Metopolophium dirhodum, were collected from artificial diets and analysed by tandem mass spectrometry. Protein identification was performed by searching MS data against the official protein set from the current pea aphid (Acyrthosiphon pisum) genome assembly and revealed 12 and 7 proteins in the saliva of S. avenae and M. dirhodum, respectively. When combined with a comparable dataset from A. pisum, only three individual proteins were common to all the aphid species; two paralogues of the GMC oxidoreductase family (glucose dehydrogenase; GLD) and ACYPI009881, an aphid specific protein previously identified as a putative component of the salivary sheath. Antibodies were designed from translated protein sequences obtained from partial cDNA sequences for ACYPI009881 and both saliva associated GLDs. The antibodies detected all parent proteins in secreted saliva from the three aphid species, but could only detect ACYPI009881, and not saliva associated GLDs, in protein extractions from the salivary glands. This result was confirmed by immunohistochemistry using whole and sectioned salivary glands, and in addition, localised ACYPI009881 to specific cell types within the principal salivary gland. The implications of these findings for the origin of salivary components and the putative role of the proteins identified are discussed in the context of our limited understanding of the functional relationship between aphid saliva and the plants they feed on. The mass spectrometry data have been deposited to the ProteomeXchange and can be accessed under the identifier PXD000113.

  7. Lectin-like ox-LDL receptor-1 (LOX-1)-Toll-like receptor 4 (TLR4) interaction and autophagy in CATH.a differentiated cells exposed to angiotensin II.

    PubMed

    Ding, Zufeng; Liu, Shijie; Wang, Xianwei; Khaidakov, Magomed; Dai, Yao; Deng, Xiaoyan; Fan, Yubo; Xiang, David; Mehta, Jawahar L

    2015-04-01

    Toll-like receptors (TLRs) play an essential role in innate immune response. Expression of TLRs has also been linked to autophagy. As the main receptor for oxidized low-density lipoprotein (ox-LDL) on the cell surface, lectin-like ox-LDL receptor-1 (LOX-1) is upregulated by proinflammatory cytokines and has been linked to the development of autophagy. However, the relationship between LOX-1, autophagy, and TLR4 in neurons has not been defined. Here, we show that Angiotensin II (Ang II) treatment of CATH.a differentiated neuronal cells resulted in the expression of TLR4 (and associated signals MyD88 and Toll/interleukin-1 receptor domain-containing adapter-inducing interferon (TRIF)), LOX-1 autophagy. LOX-1 knockdown (transfection with specific small interfering RNA (siRNA)) resulted in reduced expression of TLR4 (and associated signals MyD88 and TRIF) and P-P38 mitogen-activated protein kinase (MAPK) and autophagy. TLR4 knockdown with siRNA resulted in reduced LOX-1 expression and autophagy, indicating a positive feedback between LOX-1 and TLR4. Knockdown of TRIF as well as MyD88 or inhibition of P38 MAPK also inhibited the expression of LOX-1 and TLR4 and autophagy. Importantly, pretreatment with 3-methyladenine (autophagy inhibitor) enhanced while rapamycin (autophagy inducer) decreased the expression of LOX-1, TLR4, and P-P38 MAPK. These studies suggest the presence of a bidirectional link between LOX-1and TLR4 in cultured CATH.a differentiated cells exposed to Ang II with an important role for autophagy in this link.

  8. Duplication and Adaptive Evolution of a Key Centromeric Protein in Mimulus, a Genus with Female Meiotic Drive.

    PubMed

    Finseth, Findley R; Dong, Yuzhu; Saunders, Arpiar; Fishman, Lila

    2015-10-01

    The fundamental asymmetry of female meiosis creates an arena for genetic elements to compete for inclusion in the egg, promoting the selfish evolution of centromere variants that maximize their transmission to the future egg. Such "female meiotic drive" has been hypothesized to explain the paradoxically complex and rapidly evolving nature of centromeric DNA and proteins. Although theoretically widespread, few cases of active drive have been observed, thereby limiting the opportunities to directly assess the impact of centromeric drive on molecular variation at centromeres and binding proteins. Here, we characterize the molecular evolutionary patterns of CENH3, the centromere-defining histone variant, in Mimulus monkeyflowers, a genus with one of the few known cases of active centromere-associated female meiotic drive. First, we identify a novel duplication of CENH3 in diploid Mimulus, including in lineages with actively driving centromeres. Second, we demonstrate long-term adaptive evolution at several sites in the N-terminus of CENH3, a region with some meiosis-specific functions that putatively interacts with centromeric DNA. Finally, we infer that the paralogs evolve under different selective regimes; some sites in the N-terminus evolve under positive selection in the pro-orthologs or only one paralog (CENH3_B) and the paralogs exhibit significantly different patterns of polymorphism within populations. Our finding of long-term, adaptive evolution at CENH3 in the context of centromere-associated meiotic drive supports an antagonistic, coevolutionary battle for evolutionary dominance between centromeric DNA and binding proteins.

  9. Seasonal proteomic changes reveal molecular adaptations to preserve and replenish liver proteins during ground squirrel hibernation.

    PubMed

    Epperson, L Elaine; Rose, James C; Carey, Hannah V; Martin, Sandra L

    2010-02-01

    Hibernators are unique among mammals in their ability to survive extended periods of time with core body temperatures near freezing and with dramatically reduced heart, respiratory, and metabolic rates in a state known as torpor. To gain insight into the molecular events underlying this remarkable physiological phenotype, we applied a proteomic screening approach to identify liver proteins that differ between the summer active (SA) and the entrance (Ent) phase of winter hibernation in 13-lined ground squirrels. The relative abundance of 1,600 protein spots separated on two-dimensional gels was quantitatively determined using fluorescence difference gel electrophoresis, and 74 unique proteins exhibiting significant differences between the two states were identified using liquid chromatography followed by tandem mass spectrometry (LC-MS/MS). Proteins elevated in Ent hibernators included liver fatty acid-binding protein, fatty acid transporter, and 3-hydroxy-3-methylglutaryl-CoA synthase, which support the known metabolic fuel switch to lipid and ketone body utilization in winter. Several proteins involved in protein stability and protein folding were also elevated in the Ent phase, consistent with previous findings. In contrast to transcript screening results, there was a surprising increase in the abundance of proteins involved in protein synthesis during Ent hibernation, including several initiation and elongation factors. This finding, coupled with decreased abundance of numerous proteins involved in amino acid and nitrogen metabolism, supports the intriguing hypothesis that the mechanism of protein preservation and resynthesis is used by hibernating ground squirrels to help avoid nitrogen toxicity and ensure preservation of essential amino acids throughout the long winter fast.

  10. Multi-omic profiling -of EPO-producing Chinese hamster ovary cell panel reveals metabolic adaptation to heterologous protein production.

    PubMed

    Ley, Daniel; Seresht, Ali Kazemi; Engmark, Mikael; Magdenoska, Olivera; Nielsen, Kristian Fog; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

    2015-11-01

    Chinese hamster ovary (CHO) cells are the preferred production host for many therapeutic proteins. The production of heterologous proteins in CHO cells imposes a burden on the host cell metabolism and impact cellular physiology on a global scale. In this work, a multi-omics approach was applied to study the production of erythropoietin (EPO) in a panel of CHO-K1 cells under growth-limited and unlimited conditions in batch and chemostat cultures. Physiological characterization of the EPO-producing cells included global transcriptome analysis, targeted metabolome analysis, including intracellular pools of glycolytic intermediates, NAD(P)H/NAD(P)(+) , adenine nucleotide phosphates (ANP), and extracellular concentrations of sugars, organic acids, and amino acids. Potential impact of EPO expression on the protein secretory pathway was assessed at multiple stages using quantitative PCR (qPCR), reverse transcription PCR (qRT-PCR), Western blots (WB), and global gene expression analysis to assess EPO gene copy numbers, EPO gene expression, intracellular EPO retention, and differentially expressed genes functionally related to secretory protein processing, respectively. We found no evidence supporting the existence of production bottlenecks in energy metabolism (i.e., glycolytic metabolites, NAD(P)H/NAD(P)(+) and ANPs) in batch culture or in the secretory protein production pathway (i.e., gene dosage, transcription and post-translational processing of EPO) in chemostat culture at specific productivities up to 5 pg/cell/day. Time-course analysis of high- and low-producing clones in chemostat culture revealed rapid adaptation of transcription levels of amino acid catabolic genes in favor of EPO production within nine generations. Interestingly, the adaptation was followed by an increase in specific EPO productivity.

  11. Early origin and adaptive evolution of the GW182 protein family, the key component of RNA silencing in animals.

    PubMed

    Zielezinski, Andrzej; Karlowski, Wojciech M

    2015-01-01

    The GW182 proteins are a key component of the miRNA-dependent post-transcriptional silencing pathway in animals. They function as scaffold proteins to mediate the interaction of Argonaute (AGO)-containing complexes with cytoplasmic poly(A)-binding proteins (PABP) and PAN2-PAN3 and CCR4-NOT deadenylases. The AGO-GW182 complexes mediate silencing of the target mRNA through induction of translational repression and/or mRNA degradation. Although the GW182 proteins are a subject of extensive experimental research in the recent years, very little is known about their origin and evolution. Here, based on complex functional annotation and phylogenetic analyses, we reveal 448 members of the GW182 protein family from the earliest animals to humans. Our results indicate that a single-copy GW182/TNRC6C progenitor gene arose with the emergence of multicellularity and it multiplied in the last common ancestor of vertebrates in 2 rounds of whole genome duplication (WGD) resulting in 3 genes. Before the divergence of vertebrates, both the AGO- and CCR4-NOT-binding regions of GW182s showed significant acceleration in the accumulation of amino acid changes, suggesting functional adaptation toward higher specificity to the molecules of the silencing complex. We conclude that the silencing ability of the GW182 proteins improves with higher position in the taxonomic classification and increasing complexity of the organism. The first reconstruction of the molecular journey of GW182 proteins from the ancestral metazoan protein to the current mammalian configuration provides new insight into development of the miRNA-dependent post-transcriptional silencing pathway in animals.

  12. Amino Acid Substitutions in Cold-Adapted Proteins from Halorubrum lacusprofundi, an Extremely Halophilic Microbe from Antarctica

    PubMed Central

    DasSarma, Shiladitya; Capes, Melinda D.; Karan, Ram; DasSarma, Priya

    2013-01-01

    The halophilic Archaeon Halorubrum lacusprofundi, isolated from the perennially cold and hypersaline Deep Lake in Antarctica, was recently sequenced and compared to 12 Haloarchaea from temperate climates by comparative genomics. Amino acid substitutions for 604 H. lacusprofundi proteins belonging to conserved haloarchaeal orthologous groups (cHOGs) were determined and found to occur at 7.85% of positions invariant in proteins from mesophilic Haloarchaea. The following substitutions were observed most frequently: (a) glutamic acid with aspartic acid or alanine; (b) small polar residues with other small polar or non-polar amino acids; (c) small non-polar residues with other small non-polar residues; (d) aromatic residues, especially tryptophan, with other aromatic residues; and (e) some larger polar residues with other similar residues. Amino acid substitutions for a cold-active H. lacusprofundi β-galactosidase were then examined in the context of a homology modeled structure at residues invariant in homologous enzymes from mesophilic Haloarchaea. Similar substitutions were observed as in the genome-wide approach, with the surface accessible regions of β-galactosidase displaying reduced acidity and increased hydrophobicity, and internal regions displaying mainly subtle changes among smaller non-polar and polar residues. These findings are consistent with H. lacusprofundi proteins displaying amino acid substitutions that increase structural flexibility and protein function at low temperature. We discuss the likely mechanisms of protein adaptation to a cold, hypersaline environment on Earth, with possible relevance to life elsewhere. PMID:23536799

  13. Protein evaluation of four oat (Avena sativa L.) cultivars adapted for cultivation in the south of Brazil.

    PubMed

    Pedó, I; Sgarbieri, V C; Gutkoski, L C

    1999-01-01

    Four oat cultivars adapted for soil and climate conditions in the southern region of Brazil were evaluated for protein nutritive value. Evaluations were done both in vitro and in vivo. In vitro evaluation was done by essential amino acid profile, available lysine, amino acid scoring, and protein digestibility corrected amino acid-scoring (PDCAAS). Nitrogen balance indices and PER were determined in vivo with rats. In all four cultivars (UFP-15, UFP-16, CTC-03, UFRGS-14), lysine was the most limiting amino acid. Available lysine, amino acid score and PDCAAS were highest for cultivar UFRGS-14 and lowest for CTC-03. When compared to casein, only nitrogen retention for UFRGS-14 did not differ statistically (p>0.05); all other indices of protein quality were inferior to casein for the oat cultivars. The oat cultivars tended to be identical among themselves, except for apparent protein digestibility which was significantly higher in the UFRGS-14 and CTC-03 cultivars. On average, the PER values of the oat cultivars were 82% of casein; the net protein utilization was 88% of casein as determined in vivo and 49% by the estimation in vitro (PDCAAS).

  14. Comparative expression study to increase the solubility of cold adapted Vibrio proteins in Escherichia coli.

    PubMed

    Niiranen, Laila; Espelid, Sigrun; Karlsen, Christian R; Mustonen, Milla; Paulsen, Steinar M; Heikinheimo, Pirkko; Willassen, Nils P

    2007-03-01

    Functional and structural studies require gene overexpression and purification of soluble proteins. We wanted to express proteins from the psychrophilic bacterium Vibrio salmonicida in Escherichia coli, but encountered solubility problems. To improve the solubility of the proteins, we compared the effects of six N-terminal fusion proteins (Gb1, Z, thioredoxin, GST, MBP and NusA) and an N-terminal His6-tag. The selected test set included five proteins from the fish pathogen V. salmonicida and two related products from the mesophilic human pathogen Vibrio cholerae. We tested the expression in two different expression strains and at three different temperatures (16, 23 and 37 degrees C). His6-tag was the least effective tag, and these vector constructs were also difficult to transform. MBP and NusA performed best, expressing soluble proteins with all fusion partners in at least one of the cell types. In some cases MBP, GST and thioredoxin fusions resulted in products of incorrect size. The effect of temperature is complex: in most cases level of expression increased with temperature, whereas the effect on solubility was opposite. We found no clear connection between the preferred expression temperature of the protein and the temperature of the original host organism's natural habitat.

  15. Adaptation of aphid stylectomy for analyses of proteins and mRNAs in barley phloem sap.

    PubMed

    Gaupels, Frank; Buhtz, Anja; Knauer, Torsten; Deshmukh, Sachin; Waller, Frank; van Bel, Aart J E; Kogel, Karl-Heinz; Kehr, Julia

    2008-01-01

    Sieve tubes are transport conduits not only for photoassimilates but also for macromolecules and other compounds that are involved in sieve tube maintenance and systemic signalling. In order to gain sufficient amounts of pure phloem exudates from barley plants for analyses of the protein and mRNA composition, a previously described stylectomy set-up was optimized. Aphids were placed in sealed cages, which, immediately after microcauterization of the stylets, were flooded with water-saturated silicon oil. The exuding phloem sap was collected with a capillary connected to a pump. Using up to 30 plants and 600 aphids (Rhopalosiphum padi) in parallel, an average of 10 mul of phloem sap could be obtained within 6 h of sampling. In first analyses of the macromolecular content, eight so far unknown phloem mRNAs were identified by cDNA-amplified fragment length polymorphism. Transcripts in barley phloem exudates are related to metabolism, signalling, and pathogen defence, for example coding for a protein kinase and a pathogen- and insect-responsive WIR1A (wheat-induced resistance 1A)-like protein. Further, one-dimensional gel electrophoresis and subsequent partial sequencing by mass spectrometry led to the identification of seven major proteins with putative functions in stress responses and transport of mRNAs, proteins, and sugars. Two of the discovered proteins probably represent isoforms of a new phloem-mobile sucrose transporter. Notably, two-dimensional electrophoresis confirmed that there are >250 phloem proteins awaiting identification in future studies.

  16. Differential cold-adaptation among protein components of the thioredoxin system in the psychrophilic eubacterium Pseudoalteromonas haloplanktis TAC 125.

    PubMed

    Cotugno, Roberta; Rosaria Ruocco, Maria; Marco, Salvatore; Falasca, Patrizia; Evangelista, Giovanna; Raimo, Gennaro; Chambery, Angela; Di Maro, Antimo; Masullo, Mariorosario; De Vendittis, Emmanuele

    2009-05-01

    Thioredoxin and thioredoxin reductase from the psychrophilic eubacterium Pseudoalteromonas haloplanktis were obtained as recombinant His-tagged proteins (rPhTrx and rPhTrxR, respectively). rPhTrxR is organised as a homodimeric flavoenzyme, whereas rPhTrx is a small monomeric protein, both containing a functional disulfide bridge. However, three additional cysteines are present as free thiols in purified rPhTrxR. When individually tested in specific assays, rPhTrxR and rPhTrx display a full activity at low temperatures, an indispensable requirement for cold-adapted proteins. In particular, rPhTrxR catalyses the NADPH dependent reduction of DTNB and rPhTrx provokes the insulin precipitation in the presence of DTT. The analysis of the effect of temperature on these reactions indicates that rPhTrxR is more cold-adapted than rPhTrx, having a higher psychrophilicity. The combined activity of rPhTrxR and rPhTrx, tested in a reconstituted assay containing NADPH as electrons donor and human insulin as the thioredoxin substrate, demonstrates a direct functional interaction between the purified recombinant components of the thioredoxin system of P. haloplanktis. Furthermore, the NADPH-dependent reduction of rPhTrx catalysed by rPhTrxR is fully reversible and allows the determination of its redox potential, whose value is in the range of other bacterial and archaeal thioredoxins. The analysis of the thermostability of rPhTrxR points to its discrete heat resistance. However, rPhTrx is much more heat resistant, with a half inactivation time of about 4 h at 95 degrees C. This exceptional heat resistance for a psychrophilic protein is significantly decreased by the reduction of the disulfide bridge of rPhTrx. Functionality, thermodependence and thermostability of the P. haloplanktis thioredoxin system point to the relevance of this key mechanism for the preservation of the reduced state of cytoplasmic proteins even in a cold-adapted source.

  17. Computation of the protein molecular mechanism using adaptive dihedral angle increments

    NASA Astrophysics Data System (ADS)

    Diez, Mikel; Petuya, Victor; Urizar, Mónica; Macho, Erik; Altuzarrra, Oscar

    2013-03-01

    Protein motion simulation is still a troublesome problem yet to be solved, especially due to its high computational requirements. The procedure presented in this paper makes use of the proteins' real degrees of freedom (DOFs). The procedure makes no use of any intermediate energy minimization processes that may alter the motion path or result in very high computational cost requirements. In order to reduce the computational cost, presented algorithms make use of the balls and rods approach for protein structure modelization. Also, structures are normalized in order to minimize inaccuracies introduced by experimental methods, providing a more efficient but still accurate structure for motion simulation.

  18. A proteomic adaptation of small intestinal mucosa in response to dietary protein limitation

    PubMed Central

    Qin, Chunfu; Qiu, Kai; Sun, Wenjuan; Jiao, Ning; Zhang, Xin; Che, Lianqiang; Zhao, Haiyi; Shen, Hexiao; Yin, Jingdong

    2016-01-01

    Dietary protein limitation (PL) is not only beneficial to human health but also applied to minimize nitrogen excretion in livestock production. However, the impact of PL on intestinal physiology is largely unknown. In this study, we identified 5275 quantitative proteins using a porcine model in which pigs suffered PL. A total of 202 proteins |log2 fold-change| > 1 were taken as differentially expressed proteins and subjected to functional and pathway enrichment analysis to reveal proteomic alterations of the jejunal mucosa. Combining with the results of western blotting analysis, we found that protein/carbohydrate digestion, intestinal mucosal tight junction and cell adhesion molecules, and the immune response to foreign antigens were increased in the jejunal mucosa of the pigs upon PL. In contrast, amino acid transport, innate and auto immunity, as well as cell proliferation and apoptosis were reduced. In addition, the expression of functional proteins that involved in DNA replication, transcription and mRNA splicing as well as translation were altered in the jejunal mucosa in response to PL. Furthermore, PL may reduce amino acid transport and cell proliferation through the depression of mTOR pathway. This study provides new insights into the molecular mechanisms underlying the small intestinal response to PL. PMID:27841298

  19. Involvement of the 90 kDa heat shock protein during adaptation of Paracoccidioides brasiliensis to different environmental conditions.

    PubMed

    Tamayo, Diana; Muñoz, Jose F; Torres, Isaura; Almeida, Agostinho J; Restrepo, Angela; McEwen, Juan G; Hernández, Orville

    2013-02-01

    HSP90 is a molecular chaperone that participates in folding, stabilization, activation, and assembly of several proteins, all of which are key regulators in cell signaling. In dimorphic pathogenic fungi such as Paracoccidioides brasiliensis, the adaptation to a higher temperature, acid pH and oxidative stress, is an essential event for fungal survival and also for the establishing of the infectious process. To further understand the role of this protein, we used antisense RNA technology to generate a P. brasiliensis isolate with reduced PbHSP90 gene expression (PbHSP90-aRNA). Reduced expression of HSP90 decreased yeast cell viability during batch culture growth and increased susceptibility to acid pH environments and imposed oxidative stress. Also, PbHSP90-aRNA yeast cells presented reduced viability upon interaction with macrophages. The findings presented here suggest a protective role for HSP90 during adaptation to hostile environments, one that promotes survival of the fungus during host-pathogen interactions.

  20. Redox stress proteins are involved in adaptation response of the hyperthermoacidophilic archaeon Sulfolobus solfataricus to nickel challenge

    PubMed Central

    Salzano, Anna M; Febbraio, Ferdinando; Farias, Tiziana; Cetrangolo, Giovanni P; Nucci, Roberto; Scaloni, Andrea; Manco, Giuseppe

    2007-01-01

    Background Exposure to nickel (Ni) and its chemical derivatives has been associated with severe health effects in human. On the contrary, poor knowledge has been acquired on target physiological processes or molecular mechanisms of this metal in model organisms, including Bacteria and Archaea. In this study, we describe an analysis focused at identifying proteins involved in the recovery of the archaeon Sulfolobus solfataricus strain MT4 from Ni-induced stress. Results To this purpose, Sulfolobus solfataricus was grown in the presence of the highest nickel sulphate concentration still allowing cells to survive; crude extracts from treated and untreated cells were compared at the proteome level by using a bi-dimensional chromatography approach. We identified several proteins specifically repressed or induced as result of Ni treatment. Observed up-regulated proteins were largely endowed with the ability to trigger recovery from oxidative and osmotic stress in other biological systems. It is noteworthy that most of the proteins induced following Ni treatment perform similar functions and a few have eukaryal homologue counterparts. Conclusion These findings suggest a series of preferential gene expression pathways activated in adaptation response to metal challenge. PMID:17692131

  1. Subfamily-specific adaptations in the structures of two penicillin-binding proteins from Mycobacterium tuberculosis

    SciTech Connect

    Prigozhin, Daniil M.; Krieger, Inna V.; Huizar, John P.; Mavrici, Daniela; Waldo, Geoffrey S.; Hung, Li -Wei; Sacchettini, James C.; Terwilliger, Thomas C.; Alber, Tom; Mayer, Claudine

    2014-12-31

    Beta-lactam antibiotics target penicillin-binding proteins including several enzyme classes essential for bacterial cell-wall homeostasis. To better understand the functional and inhibitor-binding specificities of penicillin-binding proteins from the pathogen, Mycobacterium tuberculosis, we carried out structural and phylogenetic analysis of two predicted D,D-carboxypeptidases, Rv2911 and Rv3330. Optimization of Rv2911 for crystallization using directed evolution and the GFP folding reporter method yielded a soluble quadruple mutant. Structures of optimized Rv2911 bound to phenylmethylsulfonyl fluoride and Rv3330 bound to meropenem show that, in contrast to the nonspecific inhibitor, meropenem forms an extended interaction with the enzyme along a conserved surface. Phylogenetic analysis shows that Rv2911 and Rv3330 belong to different clades that emerged in Actinobacteria and are not represented in model organisms such as Escherichia coli and Bacillus subtilis. Clade-specific adaptations allow these enzymes to fulfill distinct physiological roles despite strict conservation of core catalytic residues. The characteristic differences include potential protein-protein interaction surfaces and specificity-determining residues surrounding the catalytic site. Overall, these structural insights lay the groundwork to develop improved beta-lactam therapeutics for tuberculosis.

  2. Different BCR/Abl protein suppression patterns as a converging trait of chronic myeloid leukemia cell adaptation to energy restriction.

    PubMed

    Bono, Silvia; Lulli, Matteo; D'Agostino, Vito Giuseppe; Di Gesualdo, Federico; Loffredo, Rosa; Cipolleschi, Maria Grazia; Provenzani, Alessandro; Rovida, Elisabetta; Dello Sbarba, Persio

    2016-12-20

    BCR/Abl protein drives the onset and progression of Chronic Myeloid Leukemia (CML). We previously showed that BCR/Abl protein is suppressed in low oxygen, where viable cells retain stem cell potential. This study addressed the regulation of BCR/Abl protein expression under oxygen or glucose shortage, characteristic of the in vivo environment where cells resistant to tyrosine kinase inhibitors (TKi) persist. We investigated, at transcriptional, translational and post-translational level, the mechanisms involved in BCR/Abl suppression in K562 and KCL22 CML cells. BCR/abl mRNA steady-state analysis and ChIP-qPCR on BCR promoter revealed that BCR/abl transcriptional activity is reduced in K562 cells under oxygen shortage. The SUnSET assay showed an overall reduction of protein synthesis under oxygen/glucose shortage in both cell lines. However, only low oxygen decreased polysome-associated BCR/abl mRNA significantly in KCL22 cells, suggesting a decreased BCR/Abl translation. The proteasome inhibitor MG132 or the pan-caspase inhibitor z-VAD-fmk extended BCR/Abl expression under oxygen/glucose shortage in K562 cells. Glucose shortage induced autophagy-dependent BCR/Abl protein degradation in KCL22 cells. Overall, our results showed that energy restriction induces different cell-specific BCR/Abl protein suppression patterns, which represent a converging route to TKi-resistance of CML cells. Thus, the interference with BCR/Abl expression in environment-adapted CML cells may become a useful implement to current therapy.

  3. Adaptation of aphid stylectomy for analyses of proteins and mRNAs in barley phloem sap

    PubMed Central

    Gaupels, Frank; Buhtz, Anja; Knauer, Torsten; Deshmukh, Sachin; Waller, Frank; van Bel, Aart J. E.; Kogel, Karl-Heinz; Kehr, Julia

    2008-01-01

    Sieve tubes are transport conduits not only for photoassimilates but also for macromolecules and other compounds that are involved in sieve tube maintenance and systemic signalling. In order to gain sufficient amounts of pure phloem exudates from barley plants for analyses of the protein and mRNA composition, a previously described stylectomy set-up was optimized. Aphids were placed in sealed cages, which, immediately after microcauterization of the stylets, were flooded with water-saturated silicon oil. The exuding phloem sap was collected with a capillary connected to a pump. Using up to 30 plants and 600 aphids (Rhopalosiphum padi) in parallel, an average of 10 μl of phloem sap could be obtained within 6 h of sampling. In first analyses of the macromolecular content, eight so far unknown phloem mRNAs were identified by cDNA-amplified fragment length polymorphism. Transcripts in barley phloem exudates are related to metabolism, signalling, and pathogen defence, for example coding for a protein kinase and a pathogen- and insect-responsive WIR1A (wheat-induced resistance 1A)-like protein. Further, one-dimensional gel electrophoresis and subsequent partial sequencing by mass spectrometry led to the identification of seven major proteins with putative functions in stress responses and transport of mRNAs, proteins, and sugars. Two of the discovered proteins probably represent isoforms of a new phloem-mobile sucrose transporter. Notably, two-dimensional electrophoresis confirmed that there are >250 phloem proteins awaiting identification in future studies. PMID:18632729

  4. The matricellular protein cysteine-rich protein 61 (CCN1/Cyr61) enhances physiological adaptation of retinal vessels and reduces pathological neovascularization associated with ischemic retinopathy.

    PubMed

    Hasan, Adeel; Pokeza, Nataliya; Shaw, Lynn; Lee, Hyun-Seung; Lazzaro, Douglas; Chintala, Hemabindu; Rosenbaum, Daniel; Grant, Maria B; Chaqour, Brahim

    2011-03-18

    Retinal vascular damages are the cardinal hallmarks of retinopathy of prematurity (ROP), a leading cause of vision impairment and blindness in childhood. Both angiogenesis and vasculogenesis are disrupted in the hyperoxia-induced vaso-obliteration phase, and recapitulated, although aberrantly, in the subsequent ischemia-induced neovessel formation phase of ROP. Yet, whereas the histopathological features of ROP are well characterized, many key modulators with a therapeutic potential remain unknown. The CCN1 protein also known as cysteine-rich protein 61 (Cyr61) is a dynamically expressed, matricellular protein required for proper angiogenesis and vasculogenesis during development. The expression of CCN1 becomes abnormally reduced during the hyperoxic and ischemic phases of ROP modeled in the mouse eye with oxygen-induced retinopathy (OIR). Lentivirus-mediated re-expression of CCN1 enhanced physiological adaptation of the retinal vasculature to hyperoxia and reduced pathological angiogenesis following ischemia. Remarkably, injection into the vitreous of OIR mice of hematopoietic stem cells (HSCs) engineered to express CCN1 harnessed ischemia-induced neovessel outgrowth without adversely affecting the physiological adaptation of retinal vessels to hyperoxia. In vitro exposure of HSCs to recombinant CCN1 induced integrin-dependent cell adhesion, migration, and expression of specific endothelial cell markers as well as many components of the Wnt signaling pathway including Wnt ligands, their receptors, inhibitors, and downstream targets. CCN1-induced Wnt signaling mediated, at least in part, adhesion and endothelial differentiation of cultured HSCs, and inhibition of Wnt signaling interfered with normalization of the retinal vasculature induced by CCN1-primed HSCs in OIR mice. These newly identified functions of CCN1 suggest its possible therapeutic utility in ischemic retinopathy.

  5. Wiskott-Aldrich syndrome protein is associated with the adapter protein Grb2 and the epidermal growth factor receptor in living cells.

    PubMed Central

    She, H Y; Rockow, S; Tang, J; Nishimura, R; Skolnik, E Y; Chen, M; Margolis, B; Li, W

    1997-01-01

    Src homology domains [i.e., Src homology domain 2 (SH2) and Src homology domain 3 (SH3)] play a critical role in linking receptor tyrosine kinases to downstream signaling networks. A well-defined function of the SH3-SH2-SH3 adapter Grb2 is to link receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR), to the p21ras-signaling pathway. Grb2 has also been implicated to play a role in growth factor-regulated actin assembly and receptor endocytosis, although the underlying mechanisms remain unclear. In this study, we show that Grb2 interacts through its SH3 domains with the human Wiskott-Aldrich syndrome protein (WASp), which plays a role in regulation of the actin cytoskeleton. We find that WASp is expressed in a variety of cell types and is exclusively cytoplasmic. Although the N-terminal SH3 domain of Grb2 binds significantly stronger than the C-terminal SH3 domain to WASp, full-length Grb2 shows the strongest binding. Both phosphorylation of WASp and its interaction with Grb2, as well as with another adapter protein Nck, remain constitutive in serum-starved or epidermal growth factor-stimulated cells. WASp coimmunoprecipitates with the activated EGFR after epidermal growth factor stimulation. Purified glutathione S-transferase-full-length-Grb2 fusion protein, but not the individual domains of Grb2, enhances the association of WASp with the EGFR, suggesting that Grb2 mediates the association of WASp with EGFR. This study suggests that Grb2 translocates WASp from the cytoplasm to the plasma membrane and the Grb2-WASp complex may play a role in linking receptor tyrosine kinases to the actin cytoskeleton. Images PMID:9307968

  6. Thermal adaptation analyzed by comparison of protein sequences from mesophilic and extremely thermophilic Methanococcus species

    NASA Technical Reports Server (NTRS)

    Haney, P. J.; Badger, J. H.; Buldak, G. L.; Reich, C. I.; Woese, C. R.; Olsen, G. J.

    1999-01-01

    The genome sequence of the extremely thermophilic archaeon Methanococcus jannaschii provides a wealth of data on proteins from a thermophile. In this paper, sequences of 115 proteins from M. jannaschii are compared with their homologs from mesophilic Methanococcus species. Although the growth temperatures of the mesophiles are about 50 degrees C below that of M. jannaschii, their genomic G+C contents are nearly identical. The properties most correlated with the proteins of the thermophile include higher residue volume, higher residue hydrophobicity, more charged amino acids (especially Glu, Arg, and Lys), and fewer uncharged polar residues (Ser, Thr, Asn, and Gln). These are recurring themes, with all trends applying to 83-92% of the proteins for which complete sequences were available. Nearly all of the amino acid replacements most significantly correlated with the temperature change are the same relatively conservative changes observed in all proteins, but in the case of the mesophile/thermophile comparison there is a directional bias. We identify 26 specific pairs of amino acids with a statistically significant (P < 0.01) preferred direction of replacement.

  7. A convenient and adaptable microcomputer environment for DNA and protein sequence manipulation and analysis.

    PubMed Central

    Pustell, J; Kafatos, F C

    1986-01-01

    We describe the further development of a widely used package of DNA and protein sequence analysis programs for microcomputers (1,2,3). The package now provides a screen oriented user interface, and an enhanced working environment with powerful formatting, disk access, and memory management tools. The new GenBank floppy disk database is supported transparently to the user and a similar version of the NBRF protein database is provided. The programs can use sequence file annotation to automatically annotate printouts and translate or extract specified regions from sequences by name. The sequence comparison programs can now perform a 5000 X 5000 bp analysis in 12 minutes on an IBM PC. A program to locate potential protein coding regions in nucleic acids, a digitizer interface, and other additions are also described. PMID:3753784

  8. Kisspeptin inhibits a slow afterhyperpolarization current via protein kinase C and reduces spike frequency adaptation in GnRH neurons

    PubMed Central

    Zhang, Chunguang

    2013-01-01

    Kisspeptin signaling via its cognate receptor G protein-coupled receptor 54 (GPR54) in gonadotropin-releasing hormone (GnRH) neurons plays a critical role in regulating pituitary secretion of luteinizing hormone and thus reproductive function. GPR54 is Gq-coupled to activation of phospholipase C and multiple second messenger signaling pathways. Previous studies have shown that kisspeptin potently depolarizes GnRH neurons through the activation of canonical transient receptor potential channels and inhibition of inwardly rectifying K+ channels to generate sustained firing. Since the initial studies showing that kisspeptin has prolonged effects, the question has been why is there very little spike frequency adaption during sustained firing? Presently, we have discovered that kisspeptin reduces spike frequency adaptation and prolongs firing via the inhibition of a calcium-activated slow afterhyperpolarization current (IsAHP). GnRH neurons expressed two distinct IsAHP, a kisspeptin-sensitive and an apamin-sensitive IsAHP. Essentially, kisspeptin inhibited 50% of the IsAHP and apamin inhibited the other 50% of the current. Furthermore, the kisspeptin-mediated inhibition of IsAHP was abrogated by the protein kinase C (PKC) inhibitor calphostin C, and the PKC activator phorbol 12,13-dibutyrate mimicked and occluded any further effects of kisspeptin on IsAHP. The protein kinase A (PKA) inhibitors H-89 and the Rp diastereomer of adenosine 3′,5′-cyclic monophosphorothioate had no effect on the kisspeptin-mediated inhibition but were able to abrogate the inhibitory effects of forskolin on the IsAHP, suggesting that PKA is not involved. Therefore, in addition to increasing the firing rate through an overt depolarization, kisspeptin can also facilitate sustained firing through inhibiting an apamin-insensitive IsAHP in GnRH neurons via a PKC. PMID:23548613

  9. Differential Regulation of Protein- and Polysaccharide-Specific Ig Isotype Production In Vivo in Response to Intact Streptococcus pneumoniae

    DTIC Science & Technology

    2006-01-01

    response [3]. More recently, a normal pathogen-specific IgG isotype response to Borrelia burgdorferi was observed in TLR2-/- mice, although this...was associated with a higher burden of pathogen [113]. However, MyD88-/- mice infected with B. burgdorferi demonstrated elevated B. burgdorferi

  10. Protein-DNA complex is the exclusive malaria parasite component that activates dendritic cells and triggers innate immune responses.

    PubMed

    Wu, Xianzhu; Gowda, Nagaraj M; Kumar, Sanjeev; Gowda, D Channe

    2010-04-15

    Dendritic cells (DCs) play a crucial role in the development of protective immunity to malaria. However, it remains unclear how malaria parasites trigger immune responses in DCs. In this study, we purified merozoites, food vacuoles, and parasite membrane fragments released during the Plasmodium falciparum schizont burst to homogeneity and tested for the activation of bone marrow-derived DCs from wild-type and TLR2(-/-), TLR4(-/-), TLR9(-/-), and MyD88(-/-) C57BL/6J mice. The results demonstrate that a protein-DNA complex is the exclusive parasite component that activates DCs by a TLR9-dependent pathway to produce inflammatory cytokines. Complex formation with proteins is essential for the entry of parasite DNA into DCs for TLR9 recognition and, thus, proteins convert inactive DNA into a potent immunostimulatory molecule. Exogenous cationic polymers, polylysine and chitosan, can impart stimulatory activity to parasite DNA, indicating that complex formation involves ionic interactions. Merozoites and DNA-protein complex could also induce inflammatory cytokine responses in human blood DCs. Hemozoin is neither a TLR9 ligand for DCs nor functions as a carrier of DNA into cells. Additionally, although TLR9 is critical for DCs to induce the production of IFN-gamma by NK cells, this receptor is not required for NK cells to secret IFN-gamma, and cell-cell contact among myeloid DCs, plasmacytoid DCs, and NK cells is required for IFN-gamma production. Together, these results contribute substantially toward the understanding of malaria parasite-recognition mechanisms. More importantly, our finding that proteins and carbohydrate polymers are able to confer stimulatory activity to an otherwise inactive parasite DNA have important implications for the development of a vaccine against malaria.

  11. Elucidation of phenotypic adaptations: Molecular analyses of dim-light vision proteins in vertebrates

    PubMed Central

    Yokoyama, Shozo; Tada, Takashi; Zhang, Huan; Britt, Lyle

    2008-01-01

    Vertebrate ancestors appeared in a uniform, shallow water environment, but modern species flourish in highly variable niches. A striking array of phenotypes exhibited by contemporary animals is assumed to have evolved by accumulating a series of selectively advantageous mutations. However, the experimental test of such adaptive events at the molecular level is remarkably difficult. One testable phenotype, dim-light vision, is mediated by rhodopsins. Here, we engineered 11 ancestral rhodopsins and show that those in early ancestors absorbed light maximally (λmax) at 500 nm, from which contemporary rhodopsins with variable λmaxs of 480–525 nm evolved on at least 18 separate occasions. These highly environment-specific adaptations seem to have occurred largely by amino acid replacements at 12 sites, and most of those at the remaining 191 (≈94%) sites have undergone neutral evolution. The comparison between these results and those inferred by commonly-used parsimony and Bayesian methods demonstrates that statistical tests of positive selection can be misleading without experimental support and that the molecular basis of spectral tuning in rhodopsins should be elucidated by mutagenesis analyses using ancestral pigments. PMID:18768804

  12. Discs large 1 (Dlg1) scaffolding protein participates with clathrin and adaptator protein complex 1 (AP-1) in forming Weibel-Palade bodies of endothelial cells.

    PubMed

    Philippe, Monique; Léger, Thibaut; Desvaux, Raphaëlle; Walch, Laurence

    2013-05-03

    Weibel-Palade bodies (WPBs) are specific cigar-shaped granules that store von Willebrand factor (VWF) for its regulated secretion by endothelial cells. The first steps of the formation of these granules at the trans-Golgi network specifically require VWF aggregation and an external scaffolding complex that contains the adaptator protein complex 1 (AP-1) and clathrin. Discs large 1 (Dlg1) is generally considered to be a modular scaffolding protein implicated in the control of cell polarity in a large variety of cells by specific recruiting of receptors, channels, or signaling proteins to specialized zones of the plasma membrane. We propose here that in endothelial cells, Dlg1, in a complex with AP-1 and clathrin, participates in the biogenesis of WPBs. Supporting data show that Dlg1 colocalizes with microtubules, intermediate filaments, and Golgi markers. Tandem mass spectrometry experiments led to the identification of clathrin as an Dlg1-interacting partner. Interaction was confirmed by in situ proximity ligation assays. Furthermore, AP-1 and VWF immunoprecipitate and colocalize with Dlg1 in the juxtanuclear zone. Finally, Dlg1 depletion by siRNA duplexes disrupts trans-Golgi network morphology and WPB formation. Our results provide the first evidence for an unexpected role of Dlg1 in controlling the formation of specific secretory granules involved in VWF exocytosis in endothelial cells.

  13. Cooperation of two mRNA-binding proteins drives metabolic adaptation to iron deficiency

    PubMed Central

    Puig, Sergi; Vergara, Sandra V.; Thiele, Dennis J.

    2008-01-01

    Summary Iron (Fe) is an essential co-factor for a wide range of cellular processes. We have previously demonstrated that during Fe-deficiency yeast Cth2 is expressed and promotes degradation of a battery of mRNAs leading to reprogramming of Fe-dependent metabolism and Fe-storage. We report that the Cth2-homologous protein, Cth1, is transiently expressed during Fe-deprivation and participates in the response to Fe-deficiency through the degradation of mRNAs primarily involved in mitochondrially-localized activities including respiration and amino acid biosynthesis. In parallel, wild type but not cth1Δ cth2Δ cells accumulate mRNAs encoding proteins that function in glucose import and storage and store high levels of glycogen. In addition, Fe-deficiency leads to Snf1 phosphorylation, a member of the AMP-activated protein kinase family required for the cellular response to glucose starvation. These studies demonstrate a metabolic reprogramming as a consequence of Fe-starvation that is dependent on the coordinated activities of two mRNA-binding proteins. PMID:18522836

  14. Identifying the adaptive mechanism in globular proteins: Fluctuations in densely packed regions manipulate flexible parts

    NASA Astrophysics Data System (ADS)

    Yilmaz, Lutfu Safak; Atilgan, Ali Rana

    2000-09-01

    A low-resolution structural model based on the packing geometry of α-carbons is utilized to establish a connection between the flexible and rigid parts of a folded protein. The former commonly recognizes a complementing molecule for making a complex, while the latter manipulates the necessary conformational change for binding. We attempt analytically to distinguish this control architecture that intrinsically exists in globular proteins. First with two-dimensional simple models, then for a native protein, bovine pancreatic trypsin inhibitor, we explicitly demonstrate that inserting fluctuations in tertiary contacts supported by the stable core, one can regulate the displacement of residues on loop regions. The positional fluctuations of the flexible regions are annihilated by the rest of the protein in conformity with the Le Chatelier-Braun principle. The results indicate that the distortion of the principal nonbonded contacts between highly packed residues is accompanied by that of the slavery fluctuations that are widely distributed over the native structure. These positional arrangements do not appear in a reciprocal relation between a perturbation and the associated response; the effect of a movement of residue i on residue j is not equal to that of the same movement of residue j on residue i.

  15. Pre- versus post-exercise protein intake has similar effects on muscular adaptations

    PubMed Central

    Aragon, Alan; Wilborn, Colin; Urbina, Stacie L.; Hayward, Sara E.; Krieger, James

    2017-01-01

    The purpose of this study was to test the anabolic window theory by investigating muscle strength, hypertrophy, and body composition changes in response to an equal dose of protein consumed either immediately pre- versus post-resistance training (RT) in trained men. Subjects were 21 resistance-trained men (>1 year RT experience) recruited from a university population. After baseline testing, participants were randomly assigned to 1 of 2 experimental groups: a group that consumed a supplement containing 25 g protein and 1 g carbohydrate immediately prior to exercise (PRE-SUPP) (n = 9) or a group that consumed the same supplement immediately post-exercise (POST-SUPP) (n = 12). The RT protocol consisted of three weekly sessions performed on non-consecutive days for 10 weeks. A total-body routine was employed with three sets of 8–12 repetitions for each exercise. Results showed that pre- and post-workout protein consumption had similar effects on all measures studied (p > 0.05). These findings refute the contention of a narrow post-exercise anabolic window to maximize the muscular response and instead lends support to the theory that the interval for protein intake may be as wide as several hours or perhaps more after a training bout depending on when the pre-workout meal was consumed. PMID:28070459

  16. Phosphoproteome Analysis Links Protein Phosphorylation to Cellular Remodeling and Metabolic Adaptation during Magnaporthe oryzae Appressorium Development.

    PubMed

    Franck, William L; Gokce, Emine; Randall, Shan M; Oh, Yeonyee; Eyre, Alex; Muddiman, David C; Dean, Ralph A

    2015-06-05

    The rice pathogen, Magnaporthe oryzae, undergoes a complex developmental process leading to formation of an appressorium prior to plant infection. In an effort to better understand phosphoregulation during appressorium development, a mass spectrometry based phosphoproteomics study was undertaken. A total of 2924 class I phosphosites were identified from 1514 phosphoproteins from mycelia, conidia, germlings, and appressoria of the wild type and a protein kinase A (PKA) mutant. Phosphoregulation during appressorium development was observed for 448 phosphosites on 320 phosphoproteins. In addition, a set of candidate PKA targets was identified encompassing 253 phosphosites on 227 phosphoproteins. Network analysis incorporating regulation from transcriptomic, proteomic, and phosphoproteomic data revealed new insights into the regulation of the metabolism of conidial storage reserves and phospholipids, autophagy, actin dynamics, and cell wall metabolism during appressorium formation. In particular, protein phosphorylation appears to play a central role in the regulation of autophagic recycling and actin dynamics during appressorium formation. Changes in phosphorylation were observed in multiple components of the cell wall integrity pathway providing evidence that this pathway is highly active during appressorium development. Several transcription factors were phosphoregulated during appressorium formation including the bHLH domain transcription factor MGG_05709. Functional analysis of MGG_05709 provided further evidence for the role of protein phosphorylation in regulation of glycerol metabolism and the metabolic reprogramming characteristic of appressorium formation. The data presented here represent a comprehensive investigation of the M. oryzae phosphoproteome and provide key insights on the role of protein phosphorylation during infection-related development.

  17. Pre- versus post-exercise protein intake has similar effects on muscular adaptations.

    PubMed

    Schoenfeld, Brad Jon; Aragon, Alan; Wilborn, Colin; Urbina, Stacie L; Hayward, Sara E; Krieger, James

    2017-01-01

    The purpose of this study was to test the anabolic window theory by investigating muscle strength, hypertrophy, and body composition changes in response to an equal dose of protein consumed either immediately pre- versus post-resistance training (RT) in trained men. Subjects were 21 resistance-trained men (>1 year RT experience) recruited from a university population. After baseline testing, participants were randomly assigned to 1 of 2 experimental groups: a group that consumed a supplement containing 25 g protein and 1 g carbohydrate immediately prior to exercise (PRE-SUPP) (n = 9) or a group that consumed the same supplement immediately post-exercise (POST-SUPP) (n = 12). The RT protocol consisted of three weekly sessions performed on non-consecutive days for 10 weeks. A total-body routine was employed with three sets of 8-12 repetitions for each exercise. Results showed that pre- and post-workout protein consumption had similar effects on all measures studied (p > 0.05). These findings refute the contention of a narrow post-exercise anabolic window to maximize the muscular response and instead lends support to the theory that the interval for protein intake may be as wide as several hours or perhaps more after a training bout depending on when the pre-workout meal was consumed.

  18. Activation of protein kinase D1 in mast cells in response to innate, adaptive, and growth factor signals.

    PubMed

    Murphy, Thomas R; Legere, Henry J; Katz, Howard R

    2007-12-01

    Little is known about the serine/threonine kinase protein kinase D (PKD)1 in mast cells. We sought to define ligands that activate PKD1 in mast cells and to begin to address the contributions of this enzyme to mast cell activation induced by diverse agonists. Mouse bone marrow-derived mast cells (BMMC) contained both PKD1 mRNA and immunoreactive PKD1 protein. Activation of BMMC through TLR2, Kit, or FcepsilonRI with Pam(3)CSK(4) (palmitoyl-3-cysteine-serine-lysine-4), stem cell factor (SCF), and cross-linked IgE, respectively, induced activation of PKD1, as determined by immunochemical detection of autophosphorylation. Activation of PKD1 was inhibited by the combined PKD1 and protein kinase C (PKC) inhibitor Gö 6976 but not by broad-spectrum PKC inhibitors, including bisindolylmaleimide (Bim) I. Pam(3)CSK(4) and SCF also induced phosphorylation of heat shock protein 27, a known substrate of PKD1, which was also inhibited by Gö 6976 but not Bim I in BMMC. This pattern also extended to activation-induced increases in mRNA encoding the chemokine CCL2 (MCP-1) and release of the protein. In contrast, both pharmacologic agents inhibited exocytosis of beta-hexosaminidase induced by SCF or cross-linked IgE. Our findings establish that stimuli representing innate, adaptive, and growth factor pathways activate PKD1 in mast cells. In contrast with certain other cell types, activation of PKD1 in BMMC is largely independent of PKC activation. Furthermore, our findings also indicate that PKD1 preferentially influences transcription-dependent production of CCL2, whereas PKC predominantly regulates the rapid exocytosis of preformed secretory granule mediators.

  19. Differential metabolic and endocrine adaptations in llamas, sheep, and goats fed high- and low-protein grass-based diets.

    PubMed

    Kiani, A; Alstrup, L; Nielsen, M O

    2015-10-01

    This study aimed to elucidate whether distinct endocrine and metabolic adaptations provide llamas superior ability to adapt to low protein content grass-based diets as compared with the true ruminants. Eighteen adult, nonpregnant females (6 llamas, 6 goats, and 6 sheep) were fed either green grass hay with (HP) or grass seed straw (LP) in a cross-over design experiment over 2 periods of 21 d. Blood samples were taken on day 21 in each period at -30, 60, 150, and 240 min after feeding the morning meal and analyzed for plasma contents of glucose, triglyceride, nonesterified fatty acids, β-hydroxy butyrate (BOHB), urea, creatinine, insulin, and leptin. Results showed that llamas vs sheep and goats had higher plasma concentrations of glucose (7.1 vs 3.5 and 3.6 ± 0.18 mmol/L), creatinine (209 vs 110 and 103 ± 10 μmol/L), and urea (6.7 vs 5.6 and 4.9 ± 0.5 mmol/L) but lower leptin (0.33 vs 1.49 and 1.05 ± 0.1 ng/mL) and BOHB (0.05 vs 0.26 and 0.12 ± 0.02 mmol/L), respectively. BOHB in llamas was extremely low for a ruminating animal. Llamas showed that hyperglycemia coexisted with hyperinsulinemia (in general on the HP diet; postprandially on the LP diet). Llamas were clearly hypercreatinemic compared with the true ruminants, which became further exacerbated on the LP diet, where they also sustained plasma urea at markedly higher concentrations. However, llamas had markedly lower leptin concentrations than the true ruminants. In conclusion, llamas appear to have an intrinsic insulin resistant phenotype. Augmentation of creatinine and sustenance of elevated plasma urea concentrations in llamas when fed the LP diet must reflect distinct metabolic adaptations of intermediary protein and/or nitrogen metabolism, not observed in the true ruminants. These features can contribute to explain lower metabolic rates in llamas compared with the true ruminants, which must improve the chances of survival on low protein content diets.

  20. Binding and inhibition of the ternary complex factor Elk-4/Sap1 by the adapter protein Dok-4.

    PubMed

    Hooker, Erika; Baldwin, Cindy; Roodman, Victoria; Batra, Anupam; Takano, Tomoko; Lemay, Serge

    2017-03-08

    The adapter protein Dok-4 has been reported as both activator and inhibitor of Erk and Elk-1, but lack of knowledge about the identity of its partner molecules has precluded any mechanistic insight into these seemingly conflicting properties. We report that Dok-4 interacts with the transactivation domain of Elk-4  through an atypical PTB domain-mediated interaction. Dok-4 possesses a nuclear export signal and can relocalize Elk-4 from nucleus to cytosol, whereas Elk-4 possesses two nuclear localization signals that restrict interaction with Dok-4. Elk-4 protein, unlike Elk-1, is highly unstable in the presence of Dok-4, through both a an interaction-dependent mechanism and a PH domain-dependent but interaction-independent mechanism. This is reversed by proteasome inhibition, depletion of endogenous Dok-4 or lysine-to-arginine mutation of putative Elk-4 ubiquitination sites. Finally, Elk-4 transactivation is potently inhibited by Dok-4 overexpression but enhanced by Dok-4 knockdown in MDCK renal tubular cells, which correlates with  increased basal and EGF-induced expression of Egr-1, Fos and cylcinD1 mRNA and cell proliferation despite reduced Erk activation. Thus, Dok-4 can target Elk-4 activity through multiple mechanisms including binding of the transactivation domain, nuclear exclusion and protein destabilization, without a requirement for inhibition of Erk.

  1. Adaptive mutation in nuclear export protein allows stable transgene expression in a chimaeric influenza A virus vector.

    PubMed

    Kuznetsova, Irina; Shurygina, Anna-Polina; Wolf, Brigitte; Wolschek, Markus; Enzmann, Florian; Sansyzbay, Abylay; Khairullin, Berik; Sandybayev, Nurlan; Stukova, Marina; Kiselev, Oleg; Egorov, Andrej; Bergmann, Michael

    2014-02-01

    The development of influenza virus vectors with long insertions of foreign sequences remains difficult due to the small size and instable nature of the virus. Here, we used the influenza virus inherent property of self-optimization to generate a vector stably expressing long transgenes from the NS1 protein ORF. This was achieved by continuous selection of bright fluorescent plaques of a GFP-expressing vector during multiple passages in mouse B16f1 cells. The newly generated vector acquired stability in IFN-competent cell lines and in vivo in murine lungs. Although improved vector fitness was associated with the appearance of four coding mutations in the polymerase (PB2), haemagglutinin and non-structural (NS) segments, the stability of the transgene expression was dependent primarily on the single mutation Q20R in the nuclear export protein (NEP). Importantly, a longer insert, such as a cassette of 1299 nt encoding two Mycobacterium tuberculosis Esat6 and Ag85A proteins, could substitute for the GFP transgene. Thus, the inherent property of the influenza virus to adapt can also be used to adjust a vector backbone to give stable expression of long transgenes.

  2. Crystal structures of the Toll/Interleukin-1 receptor (TIR) domains from the Brucella protein TcpB and host adaptor TIRAP reveal mechanisms of molecular mimicry.

    PubMed

    Snyder, Greg A; Deredge, Daniel; Waldhuber, Anna; Fresquez, Theresa; Wilkins, David Z; Smith, Patrick T; Durr, Susi; Cirl, Christine; Jiang, Jiansheng; Jennings, William; Luchetti, Timothy; Snyder, Nathaniel; Sundberg, Eric J; Wintrode, Patrick; Miethke, Thomas; Xiao, T Sam

    2014-01-10

    The Toll/IL-1 receptor (TIR) domains are crucial innate immune signaling modules. Microbial TIR domain-containing proteins inhibit Toll-like receptor (TLR) signaling through molecular mimicry. The TIR domain-containing protein TcpB from Brucella inhibits TLR signaling through interaction with host adaptor proteins TIRAP/Mal and MyD88. To characterize the microbial mimicry of host proteins, we have determined the X-ray crystal structures of the TIR domains from the Brucella protein TcpB and the host adaptor protein TIRAP. We have further characterized homotypic interactions of TcpB using hydrogen/deuterium exchange mass spectrometry and heterotypic TcpB and TIRAP interaction by co-immunoprecipitation and NF-κB reporter assays. The crystal structure of the TcpB TIR domain reveals the microtubule-binding site encompassing the BB loop as well as a symmetrical dimer mediated by the DD and EE loops. This dimerization interface is validated by peptide mapping through hydrogen/deuterium exchange mass spectrometry. The human TIRAP TIR domain crystal structure reveals a unique N-terminal TIR domain fold containing a disulfide bond formed by Cys(89) and Cys(134). A comparison between the TcpB and TIRAP crystal structures reveals substantial conformational differences in the region that encompasses the BB loop. These findings underscore the similarities and differences in the molecular features found in the microbial and host TIR domains, which suggests mechanisms of bacterial mimicry of host signaling adaptor proteins, such as TIRAP.

  3. Proteomic Analysis of ABCA1-Null Macrophages Reveals a Role for Stomatin-Like Protein-2 in Raft Composition and Toll-Like Receptor Signaling*

    PubMed Central

    Chowdhury, Saiful M.; Zhu, Xuewei; Aloor, Jim J.; Azzam, Kathleen M.; Gabor, Kristin A.; Ge, William; Addo, Kezia A.; Tomer, Kenneth B.; Parks, John S.; Fessler, Michael B.

    2015-01-01

    Lipid raft membrane microdomains organize signaling by many prototypical receptors, including the Toll-like receptors (TLRs) of the innate immune system. Raft-localization of proteins is widely thought to be regulated by raft cholesterol levels, but this is largely on the basis of studies that have manipulated cell cholesterol using crude and poorly specific chemical tools, such as β-cyclodextrins. To date, there has been no proteome-scale investigation of whether endogenous regulators of intracellular cholesterol trafficking, such as the ATP binding cassette (ABC)A1 lipid efflux transporter, regulate targeting of proteins to rafts. Abca1−/− macrophages have cholesterol-laden rafts that have been reported to contain increased levels of select proteins, including TLR4, the lipopolysaccharide receptor. Here, using quantitative proteomic profiling, we identified 383 proteins in raft isolates from Abca1+/+ and Abca1−/− macrophages. ABCA1 deletion induced wide-ranging changes to the raft proteome. Remarkably, many of these changes were similar to those seen in Abca1+/+ macrophages after lipopolysaccharide exposure. Stomatin-like protein (SLP)-2, a member of the stomatin-prohibitin-flotillin-HflK/C family of membrane scaffolding proteins, was robustly and specifically increased in Abca1−/− rafts. Pursuing SLP-2 function, we found that rafts of SLP-2-silenced macrophages had markedly abnormal composition. SLP-2 silencing did not compromise ABCA1-dependent cholesterol efflux but reduced macrophage responsiveness to multiple TLR ligands. This was associated with reduced raft levels of the TLR co-receptor, CD14, and defective lipopolysaccharide-induced recruitment of the common TLR adaptor, MyD88, to rafts. Taken together, we show that the lipid transporter ABCA1 regulates the protein repertoire of rafts and identify SLP-2 as an ABCA1-dependent regulator of raft composition and of the innate immune response. PMID:25910759

  4. Differential involvement of IFN-beta in Toll-like receptor-stimulated dendritic cell activation.

    PubMed

    Hoshino, Katsuaki; Kaisho, Tsuneyasu; Iwabe, Tomio; Takeuchi, Osamu; Akira, Shizuo

    2002-10-01

    Toll-like receptor (TLR) can activate dendritic cells (DC) through common signaling pathways requiring a cytoplasmic adapter, MyD88. However, the signaling is differentially regulated among TLR family members. TLR4 can activate MyD88-deficient bone marrow-derived DC (BMDC), and lead to induction of IFN-inducible genes and up-regulation of co-stimulatory molecules such as CD40, implying that the MyD88-independent signaling pathway functions downstream of TLR4. Because these effects can also be induced by type I IFN, we have analyzed whether type I IFN is involved in TLR4-induced responses. In response to lipopolysaccharide (LPS), IFN-beta gene expression was augmented in both wild-type and MyD88-deficient BMDC. Expression of all IFN-inducible genes except immune-responsive gene 1 (IRG1) was abolished and CD40 up-regulation was decreased in LPS-stimulated BMDC lacking either IFN-alpha/beta receptor (IFN-alpha/betaR) or signal transducer and activator of transcription 1 (STAT-1). Similar to the LPS response, TLR9 signaling can also induce expression of IFN-beta and IFN-inducible genes, and up-regulation of CD40. However, all these effects were MyD88 dependent. Thus, in TLR4 signaling, IFN-beta expression can be induced either by the MyD88-dependent or -independent pathway, whereas, in TLR9 signaling, it is dependent on MyD88. In CpG DNA-stimulated DC, expression of IFN-inducible genes except IRG1 was dependent on type I IFN signaling as in LPS-stimulated DC. However, in contrast to TLR4 signaling, TLR9 signaling requires type I IFN signaling for CD40 up-regulation. Taken together, this study demonstrates differential involvement of type I IFN in TLR4- and TLR9-induced effects on DC.

  5. Sexual selection and the adaptive evolution of PKDREJ protein in primates and rodents.

    PubMed

    Vicens, Alberto; Gómez Montoto, Laura; Couso-Ferrer, Francisco; Sutton, Keith A; Roldan, Eduardo R S

    2015-02-01

    PKDREJ is a testis-specific protein thought to be located on the sperm surface. Functional studies in the mouse revealed that loss of PKDREJ has effects on sperm transport and the ability to undergo an induced acrosome reaction. Thus, PKDREJ has been considered a potential target of post-copulatory sexual selection in the form of sperm competition. Proteins involved in reproductive processes often show accelerated evolution. In many cases, this rapid divergence is promoted by positive selection which may be driven, at least in part, by post-copulatory sexual selection. We analysed the evolution of the PKDREJ protein in primates and rodents and assessed whether PKDREJ divergence is associated with testes mass relative to body mass, which is a reliable proxy of sperm competition levels. Evidence of an association between the evolutionary rate of the PKDREJ gene and testes mass relative to body mass was not found in primates. Among rodents, evidence of positive selection was detected in the Pkdrej gene in the family Cricetidae but not in Muridae. We then assessed whether Pkdrej divergence is associated with episodes of sperm competition in these families. We detected a positive significant correlation between the evolutionary rates of Pkdrej and testes mass relative to body mass in cricetids. These findings constitute the first evidence of post-copulatory sexual selection influencing the evolution of a protein that participates in the mechanisms regulating sperm transport and the acrosome reaction, strongly suggesting that positive selection may act on these fertilization steps, leading to advantages in situations of sperm competition.

  6. Sexual selection and the adaptive evolution of PKDREJ protein in primates and rodents

    PubMed Central

    Vicens, Alberto; Gómez Montoto, Laura; Couso-Ferrer, Francisco; Sutton, Keith A.; Roldan, Eduardo R.S.

    2015-01-01

    PKDREJ is a testis-specific protein thought to be located on the sperm surface. Functional studies in the mouse revealed that loss of PKDREJ has effects on sperm transport and the ability to undergo an induced acrosome reaction. Thus, PKDREJ has been considered a potential target of post-copulatory sexual selection in the form of sperm competition. Proteins involved in reproductive processes often show accelerated evolution. In many cases, this rapid divergence is promoted by positive selection which may be driven, at least in part, by post-copulatory sexual selection. We analysed the evolution of the PKDREJ protein in primates and rodents and assessed whether PKDREJ divergence is associated with testes mass relative to body mass, which is a reliable proxy of sperm competition levels. Evidence of an association between the evolutionary rate of the PKDREJ gene and testes mass relative to body mass was not found in primates. Among rodents, evidence of positive selection was detected in the Pkdrej gene in the family Cricetidae but not in Muridae. We then assessed whether Pkdrej divergence is associated with episodes of sperm competition in these families. We detected a positive significant correlation between the evolutionary rates of Pkdrej and testes mass relative to body mass in cricetids. These findings constitute the first evidence of post-copulatory sexual selection influencing the evolution of a protein that participates in the mechanisms regulating sperm transport and the acrosome reaction, strongly suggesting that positive selection may act on these fertilization steps, leading to advantages in situations of sperm competition. PMID:25304980

  7. Enhanced Priming of Adaptive Immunity by Mycobacterium smegmatis Mutants with High-Level Protein Secretion

    PubMed Central

    Taylor, Natalie; Bahunde, Faith; Thompson, Afton; Yu, Jae-Sung; Jacobs, William R.; Letvin, Norm L.; Haynes, Barton F.

    2012-01-01

    Mycobacteria have features that make them attractive as potential vaccine vectors. The nonpathogenic and rapidly growing Mycobacterium smegmatis can express both Mycobacterium tuberculosis antigens and heterologous antigens from other pathogens, and it has been used as a viable vector for the development of live vaccines. In order to further improve antigen-specific immunogenicity of M. smegmatis, we screened a random transposon mutant library for mutants displaying enhanced efficiency of protein secretion (“high secretors”) and isolated 61 mutants showing enhanced endogenic and transgenic protein secretion. Sequence analysis identified a total of 54 genes involved in optimal secretion of insert proteins, as well as multiple independent transposon insertions localized within the same genomic loci and operons. The majority of transposon insertions occurred in genes that have no known protein secretion function. These transposon mutants were shown to prime antigen-specific CD8+ T cell responses better than the parental strain. Specifically, upon introducing the simian immunodeficiency virus (SIV) gag gene into these transposon mutant strains, we observed that they primed SIV Gag-specific CD8+ T cell responses significantly better than the control prime immunization in a heterologous prime/boost regimen. Our results reveal a dependence on bacterial secretion of mycobacterial and foreign antigens for the induction of antigen-specific CD8+ T cells in vivo. The data also suggest that these M. smegmatis transposon mutants could be used as novel live attenuated vaccine strains to express foreign antigens, such as those of human immunodeficiency virus type 1 (HIV-1), and induce strong antigen-specific T cell responses. PMID:22787192

  8. The Troll in Toll: Mal and Tram as bridges for TLR2 and TLR4 signaling.

    PubMed

    Sheedy, Frederick J; O'Neill, Luke A J

    2007-08-01

    Signaling by two of the most important bacteria-sensing TLRs, TLR2 and TLR4, involves two adaptor proteins, MyD88 adaptor-like (Mal) and Toll/IL-1 receptor (TIR) domain-containing adaptor-inducing IFN-beta (Trif)-related adaptor molecule (TRAM). Recently, new insights into the functioning of these two adapters have emerged. Mal is required by both TLRs to act as a bridge to recruit the adaptor MyD88, leading ultimately to NF-kappaB activation. Similarly, TRAM acts as a bridge to recruit TRIF to the TLR4 complex, leading to activation of the transcription factor IFN regulatory factor 3. Consistent with Mal and TRAM being key points of control, recent evidence suggests that they are subject to regulation by phosphorylation. Further, a variant in Mal in humans has been found to protect against multiple infectious diseases. Finally, another TIR domain-containing adaptor, sterile alpha and HEAT/armadillo motif protein (SARM), has been shown to act as an inhibitor of TRIF-dependent signaling. These recent discoveries add to the complexity of TLR signaling and highlight specific control mechanisms for TLR2 and TLR4 signaling.

  9. Morphological adaptation and protein modulation of myotendinous junction following moderate aerobic training.

    PubMed

    Curzi, Davide; Baldassarri, Valentina; De Matteis, Rita; Salamanna, Francesca; Bolotta, Alessandra; Frizziero, Antonio; Fini, Milena; Marini, Marina; Falcieri, Elisabetta

    2015-04-01

    Myotendinous junction is the muscle-tendon interface through which the contractile force can be transferred from myofibrils to the tendon extracellular matrix. At the ultrastructural level, aerobic training can modify the distal myotendinous junction of rat gastrocnemius, increasing the contact area between tissues. The aim of this work is to investigate the correlation between morphological changes and protein modulation of the myotendinous junction following moderate training. For this reason, talin, vinculin and type IV collagen amount and spatial distribution were investigated by immunohistochemistry and confocal microscopy. The images were then digitally analyzed by evaluating fluorescence intensity. Morphometric analysis revealed a significant increased thickening of muscle basal lamina in the trained group (53.1 ± 0.4 nm) with respect to the control group (43.9 ± 0.3 nm), and morphological observation showed the presence of an electron-dense area in the exercised muscles, close to the myotendinous junction. Protein concentrations appeared significantly increased in the trained group (talin +22.2%; vinculin +22.8% and type IV collagen +11.8%) with respect to the control group. Therefore, our findings suggest that moderate aerobic training induces/causes morphological changes at the myotendinous junction, correlated to the synthesis of structural proteins of the muscular basal lamina and of the cytoskeleton.

  10. Adaptive expression pattern of different proteins involved in cellular calcium homeostasis in denervated rat vas deferens.

    PubMed

    Quintas, Luis Eduardo M; Cunha, Valéria M N; Scaramello, Christianne B V; da Silva, Cláudia L M; Caricati-Neto, Afonso; Lafayette, Simone S L; Jurkiewicz, Aron; Noël, François

    2005-11-21

    The activity and protein expression of plasma membrane and sarco(endo)plasmic reticulum (Ca2+-Mg2+)ATPases and ryanodine receptors were investigated in surgically denervated rat vas deferens. The function of thapsigargin-sensitive but not thapsigargin-resistant (Ca2+-Mg2+)ATPase (from sarco(endo)plasmic reticulum and plasma membrane, respectively), evidenced by enzyme activity and Ca2+ uptake experiments, was significantly depressed by 30-50% when compared to innervated vas. Western blots showed that such reduction in sarco(endo)plasmic reticulum (Ca2+-Mg2+)ATPase performance was accompanied by a decrement of similar magnitude in sarco(endo)plasmic reticulum (Ca2+-Mg2+)ATPase type 2 protein expression, without any significant change in plasma membrane (Ca2+-Mg2+)ATPase expression. Finally, [3H]ryanodine binding revealed that the density of ryanodine binding sites was reduced by 45% after denervation without modification in affinity. The present findings demonstrate that sarco(endo)plasmic reticulum proteins involved in intracellular calcium homeostasis are clearly down-regulated and brings further evidence of a modified calcium translocation in denervated rat vas deferens.

  11. Heat Shock Partially Dissociates the Overlapping Modules of the Yeast Protein-Protein Interaction Network: A Systems Level Model of Adaptation

    PubMed Central

    Mihalik, Ágoston; Csermely, Peter

    2011-01-01

    Network analysis became a powerful tool giving new insights to the understanding of cellular behavior. Heat shock, the archetype of stress responses, is a well-characterized and simple model of cellular dynamics. S. cerevisiae is an appropriate model organism, since both its protein-protein interaction network (interactome) and stress response at the gene expression level have been well characterized. However, the analysis of the reorganization of the yeast interactome during stress has not been investigated yet. We calculated the changes of the interaction-weights of the yeast interactome from the changes of mRNA expression levels upon heat shock. The major finding of our study is that heat shock induced a significant decrease in both the overlaps and connections of yeast interactome modules. In agreement with this the weighted diameter of the yeast interactome had a 4.9-fold increase in heat shock. Several key proteins of the heat shock response became centers of heat shock-induced local communities, as well as bridges providing a residual connection of modules after heat shock. The observed changes resemble to a ‘stratus-cumulus’ type transition of the interactome structure, since the unstressed yeast interactome had a globally connected organization, similar to that of stratus clouds, whereas the heat shocked interactome had a multifocal organization, similar to that of cumulus clouds. Our results showed that heat shock induces a partial disintegration of the global organization of the yeast interactome. This change may be rather general occurring in many types of stresses. Moreover, other complex systems, such as single proteins, social networks and ecosystems may also decrease their inter-modular links, thus develop more compact modules, and display a partial disintegration of their global structure in the initial phase of crisis. Thus, our work may provide a model of a general, system-level adaptation mechanism to environmental changes. PMID:22022244

  12. "Adapted Linear Interaction Energy": A Structure-Based LIE Parametrization for Fast Prediction of Protein-Ligand Affinities.

    PubMed

    Linder, Mats; Ranganathan, Anirudh; Brinck, Tore

    2013-02-12

    We present a structure-based parametrization of the Linear Interaction Energy (LIE) method and show that it allows for the prediction of absolute protein-ligand binding energies. We call the new model "Adapted" LIE (ALIE) because the α and β coefficients are defined by system-dependent descriptors and do therefore not require any empirical γ term. The best formulation attains a mean average deviation of 1.8 kcal/mol for a diverse test set and depends on only one fitted parameter. It is robust with respect to additional fitting and cross-validation. We compare this new approach with standard LIE by Åqvist and co-workers and the LIE + γSASA model (initially suggested by Jorgensen and co-workers) against in-house and external data sets and discuss their applicabilities.

  13. Mitogen-Activated Protein Kinase Hog1 Mediates Adaptation to G1 Checkpoint Arrest during Arsenite and Hyperosmotic Stress▿

    PubMed Central

    Migdal, Iwona; Ilina, Yulia; Tamás, Markus J.; Wysocki, Robert

    2008-01-01

    Cells slow down cell cycle progression in order to adapt to unfavorable stress conditions. Yeast (Saccharomyces cerevisiae) responds to osmotic stress by triggering G1 and G2 checkpoint delays that are dependent on the mitogen-activated protein kinase (MAPK) Hog1. The high-osmolarity glycerol (HOG) pathway is also activated by arsenite, and the hog1Δ mutant is highly sensitive to arsenite, partly due to increased arsenite influx into hog1Δ cells. Yeast cell cycle regulation in response to arsenite and the role of Hog1 in this process have not yet been analyzed. Here, we found that long-term exposure to arsenite led to transient G1 and G2 delays in wild-type cells, whereas cells that lack the HOG1 gene or are defective in Hog1 kinase activity displayed persistent G1 cell cycle arrest. Elevated levels of intracellular arsenite and “cross talk” between the HOG and pheromone response pathways, observed in arsenite-treated hog1Δ cells, prolonged the G1 delay but did not cause a persistent G1 arrest. In contrast, deletion of the SIC1 gene encoding a cyclin-dependent kinase inhibitor fully suppressed the observed block of G1 exit in hog1Δ cells. Moreover, the Sic1 protein was stabilized in arsenite-treated hog1Δ cells. Interestingly, Sic1-dependent persistent G1 arrest was also observed in hog1Δ cells during hyperosmotic stress. Taken together, our data point to an important role of the Hog1 kinase in adaptation to stress-induced G1 cell cycle arrest. PMID:18552285

  14. Adaptive diffusion kernel learning from biological networks for protein function prediction

    PubMed Central

    Sun, Liang; Ji, Shuiwang; Ye, Jieping

    2008-01-01

    Background Machine-learning tools have gained considerable attention during the last few years for analyzing biological networks for protein function prediction. Kernel methods are suitable for learning from graph-based data such as biological networks, as they only require the abstraction of the similarities between objects into the kernel matrix. One key issue in kernel methods is the selection of a good kernel function. Diffusion kernels, the discretization of the familiar Gaussian kernel of Euclidean space, are commonly used for graph-based data. Results In this paper, we address the issue of learning an optimal diffusion kernel, in the form of a convex combination of a set of pre-specified kernels constructed from biological networks, for protein function prediction. Most prior work on this kernel learning task focus on variants of the loss function based on Support Vector Machines (SVM). Their extensions to other loss functions such as the one based on Kullback-Leibler (KL) divergence, which is more suitable for mining biological networks, lead to expensive optimization problems. By exploiting the special structure of the diffusion kernel, we show that this KL divergence based kernel learning problem can be formulated as a simple optimization problem, which can then be solved efficiently. It is further extended to the multi-task case where we predict multiple functions of a protein simultaneously. We evaluate the efficiency and effectiveness of the proposed algorithms using two benchmark data sets. Conclusion Results show that the performance of linearly combined diffusion kernel is better than every single candidate diffusion kernel. When the number of tasks is large, the algorithms based on multiple tasks are favored due to their competitive recognition performance and small computational costs. PMID:18366736

  15. Functions of heat shock proteins in pathways of the innate and adaptive immune system.

    PubMed

    Binder, Robert Julian

    2014-12-15

    For more than 50 years, heat shock proteins (HSPs) have been studied for their role in protecting cells from elevated temperature and other forms of stress. More recently, several roles have been ascribed to HSPs in the immune system. These include intracellular roles in Ag presentation and expression of innate receptors, as well as extracellular roles in tumor immunosurveillance and autoimmunity. Exogenously administered HSPs can elicit a variety of immune responses that have been used in immunotherapy of cancer, infectious diseases, and autoimmune disease.

  16. HTLV-1 Tax-mediated TAK1 activation involves TAB2 adapter protein

    SciTech Connect

    Yu Qingsheng; Minoda, Yasumasa; Yoshida, Ryoko; Yoshida, Hideyuki; Iha, Hidekatsu; Kobayashi, Takashi; Yoshimura, Akihiko; Takaesu, Giichi

    2008-01-04

    Human T cell leukemia virus type 1 (HTLV-1) Tax is an oncoprotein that plays a crucial role in the proliferation and transformation of HTLV-1-infected T lymphocytes. It has recently been reported that Tax activates a MAPKKK family, TAK1. However, the molecular mechanism of Tax-mediated TAK1 activation is not well understood. In this report, we investigated the role of TAK1-binding protein 2 (TAB2) in Tax-mediated TAK1 activation. We found that TAB2 physically interacts with Tax and augments Tax-induced NF-{kappa}B activity. Tax and TAB2 cooperatively activate TAK1 when they are coexpressed. Furthermore, TAK1 activation by Tax requires TAB2 binding as well as ubiquitination of Tax. We also found that the overexpression of TRAF2, 5, or 6 strongly induces Tax ubiquitination. These results suggest that TAB2 may be critically involved in Tax-mediated activation of TAK1 and that NF-{kappa}B-activating TRAF family proteins are potential cellular E3 ubiquitin ligases toward Tax.

  17. Transgenic zebrafish reporter lines reveal conserved Toll-like receptor signaling potential in embryonic myeloid leukocytes and adult immune cell lineages.

    PubMed

    Hall, Chris; Flores, Maria Vega; Chien, Annie; Davidson, Alan; Crosier, Kathryn; Crosier, Phil

    2009-05-01

    The immune response of a host to an invading pathogen is dependent on the capacity of its immune cell compartment to recognize highly conserved pathogen components using an ancient class of pattern recognition receptors known as Toll-like receptors (TLRs). Initiation of TLR-mediated signaling results in the induction of proinflammatory cytokines that help govern the scale and duration of any ensuing response. Specificity for TLR signaling is, in part, a result of the differential recruitment of intracellular adaptor molecules. Of these, MyD88 is required for the majority of TLR signaling. Zebrafish have been shown to possess TLRs and adaptor molecules throughout early development, including MyD88, strongly suggesting conservation of this ancient defense mechanism. However, information about which embryonic cells/tissues possess this conserved signaling potential is lacking. To help define which embryonic cells, in particular, those of the innate immune system, have the potential for MyD88-dependent, TLR-mediated signaling, we generated transgenic reporter lines using regulatory elements of the myd88 gene to drive the fluorescent reporters enhanced GFP and Discosoma red fluorescent protein 2 within live zebrafish. These lines possess fluorescently marked cells/tissues consistent with endogenous myd88 expression, including a subset of myeloid leukocytes. These innate immune cells were confirmed to express other TLR adaptors including Mal, trif, and Sarm. Live wound-healing and infection assays validated the potential of these myd88-expressing leukocytes to participate in immune responses. These lines will provide a valuable resource for further resolving the contribution of MyD88 to early vertebrate immunity.

  18. Distribution of cold adaptation proteins in microbial mats in Lake Joyce, Antarctica: Analysis of metagenomic data by using two bioinformatics tools.

    PubMed

    Koo, Hyunmin; Hakim, Joseph A; Fisher, Phillip R E; Grueneberg, Alexander; Andersen, Dale T; Bej, Asim K

    2016-01-01

    In this study, we report the distribution and abundance of cold-adaptation proteins in microbial mat communities in the perennially ice