RESUMO
Improper signaling of the IL-36 receptor (IL-36R), a member of the IL-1 receptor family, has been associated with various inflammation-associated diseases. However, the requirements for IL-36R signal transduction remain poorly characterized. This work seeks to define the requirements for IL-36R signaling and intracellular trafficking. In the absence of cognate agonists, IL-36R was endocytosed and recycled to the plasma membrane. In the presence of IL-36, IL-36R increased accumulation in LAMP1+ lysosomes. Endocytosis predominantly used a clathrin-mediated pathway, and the accumulation of the IL-36R in lysosomes did not result in increased receptor turnover. The ubiquitin-binding Tollip protein contributed to IL-36R signaling and increased the accumulation of both subunits of the IL-36R.
Assuntos
Endocitose/fisiologia , Interleucina-1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/metabolismo , Receptores de Interleucina/metabolismo , Transdução de Sinais/fisiologia , Linhagem Celular , Humanos , Interleucina-1/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisossomos/genética , Transporte Proteico/fisiologia , Receptores de Interleucina/genéticaRESUMO
LL-37 is a peptide secreted by human epithelial cells that can lyse bacteria, suppress signaling by Toll-like receptor 4 (TLR4), and enhance signaling to double-stranded RNA (dsRNA) by TLR3. How LL-37 interacts with dsRNA to affect signal transduction by TLR3 is not completely understood. We determined that LL-37 binds dsRNA and traffics to endosomes and releases the dsRNA in a pH-dependent manner. Using dynamic light scattering spectroscopy and cell-based FRET experiments, LL-37 was found to form higher order complexes independent of dsRNA binding. Upon acidification LL-37 will dissociate from a larger complex. In cells, LL-37 has a half-live of â¼ 1 h. LL-37 half-life was increased by inhibiting endosome acidification or inhibiting cathepsins, which include proteases whose activity are activated by endosome acidification. Residues in LL-37 that contact poly(I:C) and facilitate oligomerization in vitro were mapped. Peptide LL-29, which contains the oligomerization region of LL-37, inhibited LL-37 enhancement of TLR3 signal transduction. LL-29 prevented LL-37 · poly(I:C) co-localization to endosomes containing TLR3. These results shed light on the requirements for LL-37 enhancement of TLR3 signaling.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos/química , Transdução de Sinais , Receptor 3 Toll-Like/metabolismo , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/antagonistas & inibidores , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/genética , Sítios de Ligação , Endossomos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Peptídeos/metabolismo , RNA de Cadeia Dupla/metabolismo , Receptor 3 Toll-Like/química , Receptor 3 Toll-Like/genética , Regulação para Cima , CatelicidinasRESUMO
LL-37 is an antimicrobial peptide produced by human cells that can down-regulate the lipopolysaccharide-induced innate immune responses and up-regulate double-stranded (ds) RNA-induced innate responses through Toll-like receptor 3 (TLR3). The murine LL-37 ortholog, mCRAMP, also inhibited lipopolysaccharide-induced responses, but unlike LL-37, it inhibited viral-induced responses in mouse cells. A fluorescence polarization assay showed that LL-37 was able to bind dsRNA better than mCRAMP. In the human lung epithelial cell line BEAS-2B, LL-37, but not mCRAMP, colocalized with TLR3, and the colocalization was increased in the presence of dsRNA. The presence of poly(I:C) increased the accumulation of LL-37 in Rab5 endosomes. Signaling by cells induced with both LL-37 and poly(I:C) was sensitive to inhibitors that affect clathrin-independent trafficking, whereas signaling by poly(I:C) alone was not, suggesting that the LL-37-poly(I:C) complex trafficked to signaling endosomes by a different mechanism than poly(I:C) alone. siRNA knockdown of known LL-37 receptors identified that FPRL1 was responsible for TLR3 signaling induced by LL-37-poly(I:C). These results show that LL-37 and mCRAMP have different activities in TLR3 signaling and that LL-37 can redirect trafficking of poly(I:C) to effect signaling by TLR3 in early endosomes in a mechanism that involves FPRL1.
Assuntos
Peptídeos Catiônicos Antimicrobianos/fisiologia , Catelicidinas/fisiologia , Poli I-C/farmacologia , Receptores de Formil Peptídeo/metabolismo , Receptores de Lipoxinas/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Endocitose/efeitos dos fármacos , Endossomos/metabolismo , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Transporte Proteico , Receptores de Formil Peptídeo/fisiologia , Receptores de Lipoxinas/fisiologia , Transdução de Sinais , Receptor 3 Toll-Like/metabolismo , beta-Ciclodextrinas/farmacologiaRESUMO
Toll-like receptors (TLRs) 3, 7, and 9 are innate immune receptors that recognize nucleic acids from pathogens in endosomes and initiate signaling transductions that lead to cytokine production. Activation of TLR9 for signaling requires proteolytic processing within the ectodomain by endosome-associated proteases. Whether TLR3 requires similar proteolytic processing to become competent for signaling remains unclear. Herein we report that human TLR3 is proteolytically processed to form two fragments in endosomes. Unc93b1 is required for processing by transporting TLR3 through the Golgi complex and to the endosomes. Proteolytic cleavage requires the eight-amino acid Loop1 within leucine-rich repeat 12 of the TLR3 ectodomain. Proteolytic cleavage is not required for TLR3 signaling in response to poly(I:C), although processing could modulate the degree of response toward viral double-stranded RNAs, especially in mouse cells. Both the full-length and cleaved fragments of TLR3 can bind poly(I:C) and are present in endosomes. However, although the full-length TLR3 has a half-life in HEK293T cells of 3 h, the cleaved fragments have half-lives in excess of 7 h. Inhibition of TLR3 cleavage by either treatment with cathepsin inhibitor or by a mutation in Loop1 decreased the abundance of TLR3 in endosomes targeted for lysosomal degradation.
Assuntos
Endossomos/metabolismo , Proteólise , Transdução de Sinais/fisiologia , Receptor 3 Toll-Like/metabolismo , Animais , Linhagem Celular , Endossomos/genética , Endossomos/imunologia , Células HEK293 , Meia-Vida , Humanos , Indutores de Interferon/farmacologia , Lisossomos/genética , Lisossomos/imunologia , Lisossomos/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/imunologia , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Mutação , Poli I-C/farmacologia , Ligação Proteica , Estabilidade Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transdução de Sinais/efeitos dos fármacos , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/imunologiaRESUMO
O(2) regulates multicellular development of the social amoeba Dictyostelium, suggesting it may serve as an important cue in its native soil environment. Dictyostelium expresses an HIFα-type prolyl 4-hydroxylase (P4H1) whose levels affect the O(2)-threshold for culmination implicating it as a direct O(2)-sensor, as in animals. But Dictyostelium lacks HIFα, a mediator of animal prolyl 4-hydroxylase signaling, and P4H1 can hydroxylate Pro143 of Skp1, a subunit of E3(SCF)ubiquitin-ligases. Skp1 hydroxyproline then becomes the target of five sequential glycosyltransferase reactions that modulate the O(2)-signal. Here we show that genetically induced changes in Skp1 levels also affect the O(2)-threshold, in opposite direction to that of the modification enzymes suggesting that the latter reduce Skp1 activity. Consistent with this, overexpressed Skp1 is poorly hydroxylated and Skp1 is the only P4H1 substrate detectable in extracts. Effects of Pro143 mutations, and of combinations of Skp1 and enzyme level perturbations, are consistent with pathway modulation of Skp1 activity. However, some effects were not mirrored by changes in modification of the bulk Skp1 pool, implicating a Skp1 subpopulation and possibly additional unknown factors. Altered Skp1 levels also affected other developmental transitions in a modification-dependent fashion. Whereas hydroxylation of animal HIFα results in its polyubiquitination and proteasomal degradation, Dictyostelium Skp1 levels were little affected by its modification status. These data indicate that Skp1 and possibly E3(SCF)ubiquitin-ligase activity modulate O(2)-dependent culmination and other developmental processes, and at least partially mediate the action of the hydroxylation/glycosylation pathway in O(2)-sensing.
Assuntos
Dictyostelium/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Oxigênio/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Prolina/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Western Blotting , Glicosilação , Hidroxilação , Proteínas Luminescentes , Espectrometria de Massas , Mutação/genética , Prolina/genética , Proteínas Quinases Associadas a Fase S/isolamento & purificaçãoRESUMO
Pattern recognition receptors represent the first line of defense against invading pathogens. Herpes simplex virus (HSV) encodes multiple ligands detected by these receptors, yet persists in the majority of infected individuals indicating a breakdown in host defense against the virus. Here we identify a novel mechanism through which HSV immediate-early protein ICP0 inhibits TLR-dependent inflammatory response by blocking NF-kappaB and JNK activation downstream of TLR signal activation. This process depends on ICP0-mediated translocation of USP7 (HAUSP) from the nucleus to cytoplasm. We show that nuclear USP7 migrates to the cytoplasm in response to TLR engagement, a process that contributes to termination of TLR response. Cytoplasmic USP7 binds to and deubiquitinates TRAF6 and IKKgamma, thus terminating TLR-mediated NF-kappaB and JNK activation. These findings suggest that USP7 is part of a negative feedback loop regulating TLR signaling and that ICP0 exploits this physiologic process to attenuate innate response to HSV. ICP0 inhibition of the TLR response serves to uncouple the innate and adaptive immune response, thereby playing a key role in HSV pathogenesis and persistence.
Assuntos
Proteínas Imediatamente Precoces/fisiologia , Imunidade Inata/fisiologia , Receptores Toll-Like/fisiologia , Ubiquitina Tiolesterase/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Células Cultivadas , Herpes Simples/genética , Herpes Simples/imunologia , Humanos , Quinase I-kappa B/metabolismo , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Imunidade Inata/genética , NF-kappa B/metabolismo , NF-kappa B/fisiologia , Ligação Proteica/genética , Ligação Proteica/fisiologia , Processamento de Proteína Pós-Traducional/genética , Transporte Proteico , Fator 6 Associado a Receptor de TNF/metabolismo , Ubiquitina/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Peptidase 7 Específica de UbiquitinaRESUMO
Trypanosoma cruzi, the causative agent of Chagas disease, is surrounded by a mucin coat that plays important functions in parasite survival/invasion and is extensively O-glycosylated by Golgi and cell surface glycosyltransferases. The addition of the first sugar, alpha-N-acetylglucosamine (GlcNAc) linked to Threonine (Thr), is catalyzed by a polypeptide alpha-GlcNAc-transferase (pp-alphaGlcNAcT) which is unstable to purification. Here, a comparison of the genomes of T. cruzi and Dictyostelium discoideum, an amoebazoan which also forms this linkage, identified two T. cruzi genes (TcOGNT1 and TcOGNT2) that might encode this activity. Though neither was able to complement the Dictyostelium gene, expression in the trypanosomatid Leishmania tarentolae resulted in elevated levels of UDP-[(3)H]GlcNAc:Thr-peptide GlcNAc-transferase activity and UDP-[(3)H]GlcNAc breakdown activity. The ectodomain of TcOGNT2 was expressed and the secreted protein was found to retain both activities after extensive purification away from other proteins and the endogenous activity. Product analysis showed that (3)H was transferred as GlcNAc to a hydroxyamino acid, and breakdown was due to hydrolysis. Both activities were specific for UDP-GlcNAc relative to UDP-GalNAc and were abolished by active site point mutations that inactivate a related Dictyostelium enzyme and distantly related animal pp-alphaGalNAcTs. The peptide preference and the alkaline pH optimum were indistinguishable from those of the native activity in T. cruzi microsomes. The results suggest that mucin-type O-glycosylation in T. cruzi is initiated by conserved members of CAZy family GT60, which is homologous to the GT27 family of animal pp-alphaGalNAcTs that initiate mucin-type O-glycosylation in animals.
Assuntos
Mucinas/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Animais , Dictyostelium/genética , Dictyostelium/metabolismo , Genoma de Protozoário , Glicosilação , Leishmania/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Protozoários/genética , Trypanosoma cruzi/genética , Difosfato de Uridina/metabolismoRESUMO
T-cell tolerance is a major obstacle to successful cancer immunotherapy; thus, developing strategies to break immune tolerance is a high priority. Here we show that expression of the E3 ubiquitin ligase Grail is upregulated in CD8+ T cells that have infiltrated into transplanted lymphoma tumours, and Grail deficiency confers long-term tumour control. Importantly, therapeutic transfer of Grail-deficient CD8+ T cells is sufficient to repress established tumours. Mechanistically, loss of Grail enhances anti-tumour reactivity and functionality of CD8+ T cells. In addition, Grail-deficient CD8+ T cells have increased IL-21 receptor (IL-21R) expression and hyperresponsiveness to IL-21 signalling as Grail promotes IL-21R ubiquitination and degradation. Moreover, CD8+ T cells isolated from lymphoma patients express higher levels of Grail and lower levels of IL-21R, compared with CD8+ T cells from normal donors. Our data demonstrate that Grail is a crucial factor controlling CD8+ T-cell function and is a potential target to improve cytotoxic T-cell activity.Grail is an E3 ubiquitin ligase that inhibits T-cell receptor signalling in CD4+ T cells. Here the authors show Grail also limits IL-21 receptor expression and function in CD8+ T cells, is overactive in these cells in patients with lymphoma, and promotes tumour development in a lymphoma transplant mouse model.
Assuntos
Linfócitos T CD8-Positivos/enzimologia , Linfoma/imunologia , Ubiquitina-Proteína Ligases/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Humanos , Tolerância Imunológica , Interleucinas/genética , Interleucinas/imunologia , Linfoma/enzimologia , Linfoma/genética , Camundongos , Camundongos Knockout , Infiltração de Neutrófilos , Receptores de Interleucina-21/genética , Receptores de Interleucina-21/imunologia , Ubiquitina-Proteína Ligases/genéticaRESUMO
Signal transducer and activator of transcription 6 (STAT6) transmits signals from cytokines IL-4 and IL-13 and is activated in allergic airway disease. We are developing phosphopeptide mimetics targeting the SH2 domain of STAT6 to block recruitment to phosphotyrosine residues on IL-4 or IL-13 receptors and subsequent Tyr641 phosphorylation to inhibit the expression of genes contributing to asthma. Structure-affinity relationship studies showed that phosphopeptides based on Tyr631 from IL-4Rα bind with weak affinity to STAT6, whereas replacing the pY+3 residue with simple aryl and alkyl amides resulted in affinities in the mid to low nM range. A set of phosphatase-stable, cell-permeable prodrug analogues inhibited cytokine-stimulated STAT6 phosphorylation in both Beas-2B human airway cells and primary mouse T-lymphocytes at concentrations as low as 100 nM. IL-13-stimulated expression of CCL26 (eotaxin-3) was inhibited in a dose-dependent manner, demonstrating that targeting the SH2 domain blocks both phosphorylation and transcriptional activity of STAT6.
Assuntos
Fosfopeptídeos/farmacologia , Fator de Transcrição STAT6/efeitos dos fármacos , Domínios de Homologia de src/efeitos dos fármacos , Animais , Asma/genética , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-13/biossíntese , Interleucina-4/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Pró-Fármacos , Ratos , Receptores de Interleucina-3/efeitos dos fármacos , Receptores de Interleucina-4/efeitos dos fármacos , Relação Estrutura-Atividade , Ativação Transcricional/efeitos dos fármacos , Tirosina/química , Tirosina/metabolismoRESUMO
BACKGROUND & OBJECTIVES: Entamoeba histolytica, the causative agent of amoebiasis and amoebic liver abscess, lyses host cells by direct contact using surface lectins and releases cysteine proteinase (CP). Virulence of E. histolytica is directly related to activity of its CP. The relationship of CP activity and cytotoxicity has not been established. The present study was carried out to explore the events following contact of E. histolytica with target cells. METHODS: Protease activity of E. histolytica was measured by azocaseine and haemoglobin assays, and cysteine proteinase activity was assessed by substrate gel electrophoresis. Target cell lysis was measured by chromium release assay. RESULTS: Protease activity of E. histolytica was increased 2.5-fold following contact with BHK-21 cell line. CP activity of trophozoites alone was visualized at position 56, 35 and 29 kDa in substrate gel electrophoresis. Contact of trophozoites with target cells augmented the cytotoxic activity of amoebic CP. The increase in CP activity seen by substrate gel electrophoresis and cytotoxicity assay was blocked by pretreatment with E 64, a specific CP inhibitor and GalNAc, a contact inhibitor. INTERPRETATION & CONCLUSION: The present data showed the involvement of amoebic CP in cytotoxicity and that the CP activity was enhanced on lectin-mediated contact of E. histolytica to the target cells. Further studies need to be done to understand the mechanism at the molecular level.
Assuntos
Cricetinae/virologia , Cisteína Endopeptidases/metabolismo , Entamoeba histolytica/patogenicidade , Fígado/virologia , Acetilgalactosamina/química , Animais , Caseínas/metabolismo , Caseínas/farmacologia , Linhagem Celular , Cromo/farmacologia , Eletroforese , Entamebíase/metabolismo , Hemoglobinas/metabolismo , Lectinas/metabolismoRESUMO
A three-dimensional (3D), luminescent, 5-fold interpenetrating metal-organic framework (MOF), [Zn2(fdc)2(bpee)2(H2O)]n·2H2O (1) exhibiting highly selective sensing of nitrobenzene (NB) via a fluorescence quenching mechanism has been demonstrated.
Assuntos
Nitrobenzenos/análise , Compostos Organometálicos/química , Espectrometria de Fluorescência , Complexos de Coordenação/química , Cristalografia por Raios X , Etano/análogos & derivados , Etano/química , Furanos/química , Conformação Molecular , Piridinas/química , Teoria Quântica , Zinco/químicaRESUMO
Respiratory syncytial virus (RSV) is the major cause of bronchiolitis in infants, and a common feature of RSV infections is increased lung permeability. The accumulation of fluid in the infected lungs is caused by changes in the endothelial and epithelial membrane integrity. However, the exact mechanisms of viral-induced fluid extravasation remain unclear. Here, we report that infection of human epithelial cells with RSV results in significant epithelial membrane barrier disruption as assessed by a decrease in transepithelial electrical resistance (TEpR). This decrease in TEpR, which indicates changes in paracellular permeability, was mediated by marked cellular cytoskeletal rearrangement. Importantly, the decrease in TEpR was attenuated by using p38 MAPK inhibitors (SB-203580) but was partially affected by JNK inhibitor SP-600125. Interestingly, treatment of A549 cells with MEK1/2 inhibitor (U-0126) led to a decrease in TEpR in the absence of RSV infection. The changes in TEpR were concomitant with an increase in heat shock protein 27 (Hsp27) phosphorylation and with actin microfilament rearrangement. Thus our data suggest that p38 MAPK and Hsp27 are required for RSV induction of human epithelial membrane permeability.
Assuntos
Proteínas de Choque Térmico/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas de Neoplasias/metabolismo , Mucosa Respiratória/enzimologia , Mucosa Respiratória/virologia , Infecções por Vírus Respiratório Sincicial/metabolismo , Apoptose , Brônquios/citologia , Permeabilidade Capilar/fisiologia , Células Cultivadas , Citoesqueleto/fisiologia , Água Extravascular Pulmonar/metabolismo , Água Extravascular Pulmonar/virologia , Proteínas de Choque Térmico HSP27 , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Chaperonas Moleculares , Mucosa Respiratória/citologia , Infecções por Vírus Respiratório Sincicial/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND & OBJECTIVES: Entamoeba histolytica, the causative agent of amoebiasis and amoebic liver abscess, lyses host cells by direct contact using surface lectins and releases cysteine proteinase (CP). Virulence of E. histolytica is directly related to activity of its CP. The relationship of CP activity and cytotoxicity has not been established. The present study was carried out to explore the events following contact of E. histolytica with target cells. METHODS: Protease activity of E. histolytica was measured by azocaseine and haemoglobin assays, and cysteine proteinase activity was assessed by substrate gel electrophoresis. Target cell lysis was measured by chromium release assay. RESULTS: Protease activity of E. histolytica was increased 2.5-fold following contact with BHK-21 cell line. CP activity of trophozoites alone was visualized at position 56, 35 and 29 kDa in substrate gel electrophoresis. Contact of trophozoites with target cells augmented the cytotoxic activity of amoebic CP. The increase in CP activity seen by substrate gel electrophoresis and cytotoxicity assay was blocked by pretreatment with E 64, a specific CP inhibitor and GalNAc, a contact inhibitor. INTERPRETATION & CONCLUSION: The present data showed the involvement of amoebic CP in cytotoxicity and that the CP activity was enhanced on lectin-mediated contact of E. histolytica to the target cells. Further studies need to be done to understand the mechanism at the molecular level.