The repetitive cytoskeletal protein H49 of Trypanosoma cruzi is a calpain-like protein located at the flagellum attachment zone

Paracoccidioides brasiliensis causes paracoccidioidomycosis, a systemic mycosis in Latin America. Formamidases hydrolyze formamide, putatively plays a role infungal nitrogen metabolism. An abundant 45-kDa protein was identified as the P. brasiliensis formamidase. In this study, recombinant formamidase was over-expressed in bacteria and a polyclonal antibody to this protein was produced. Weidentified a 180-kDa protein species reactive to the antibody produced in miceagainst the P. brasiliensis recombinant purified formamidase of 45 kDa. The180-kDa purified protein yielded a heat-denatured species of 45 kDa. Both protein species of 180 and 45 kDa were identified as formamidase by peptide massfinger printing using MS. The identical mass spectra generated by the 180 and the45-kDa protein species indicated that the fungal formamidase is most likely homotetrameric in its native conformation. Furthermore, the purified formami-dase migrated as a protein of 191 kDa in native polyacrylamide gel electrophoresis, thus revealing that the enzyme forms a homotetrameric structure in its native state. This enzyme is present in the fungus cytoplasm and the cell wall. Use of a yeast two-hybrid system revealed cell wall membrane proteins, in addition to cytosolic proteins interacting with formamidase. These data provide new insights intoformamidase structure as well as potential roles for formamidase and its interaction partners in nitrogen metabolism

Study on the processing of the surface glycoprotein of 82 kDa(GP82) from metacyclic trypomastigotes of Trypanosoma cruzi

Os tripomastigotas metaciclicos de Trypanosoma cruzi sintetizam uma glicoproteina de superficie estagio-especifica de 82 kDa (GP82) que esta envolvida na entrada do parasita nas celulas hospedeiras. A GP82 encontra-se ligada a superficie celular por uma ancora de glicosilfosfatidilinositol (GPI). As proteinas ligadas por GPI sao sintetizadas com uma extremidade C-terminal que e substituida por uma ancora GPI numa reacao de transamidacao que acontece pos-traducionalmente. A sequencia da GP82 que determina a adicao da ancora GPI contem 25 aminoacidos distribuidos no sitio w, seguindo-se uma sequencia espacadora (7 aminoacidos) e uma regiao mais hidrofobica (15 aminoacidos). O sitio w, reponsavel pela clivagem e adicao da ancora GPI, e composto por 3 aminoacidos: o aminoacido w ao qual a ancora GPI e tranferida, e Asp; o aminoacido w+1 e Gly e o aminoacido w+2 e Ser. Visando determinar experimentalmente a sequencia para ancoragem por GPI em T. cruzi, fizemos uma mutacao no putativo sitio de clivagem e adicao (sitio w) da ancora. Por meio de mutacao sitio-dirigida foi gerada uma substituicao do aminoacido no sitio w (posicao 492), onde o codon GAC foi trocado por TCC, assim o motivo de clivagem e adicao Asp-Gly-Ser presente na proteina nativa foi substituido por Ser-Gly-Ser. Para avaliar o processamento de uma proteina com sinal defectiva para adicao de GPI, geramos uma GP82 truncada, eliminando todo o sinal C-terminal incluindo-se o sitio c). As duas construcoes foram clonadas no vetor pTEX e inseridas em epimastigotas (cepa G) por meio de eletroporacao. A incorporacao do plasmidio foi confirmada por analise de Southern blot apos diGestão com Xho I, e a transcricao dos genes transfectados por analise de northem blot. A traducao dos genes exogenos foi analisada por western blot empregando um anticorpo monoclonal especifico para GP82 (MAb-3F6), e a localizacao celular dos produtos traduzidos foi feita por imunofluorescencia e...(au)BV UNIFESP: Teses e dissertaçõe

Lysophosphatidylcholine Triggers TLR2- and TLR4- Mediated Signaling Pathways but Counteracts LPSInduced NO Synthesis in Peritoneal Macrophages by Inhibiting NF-κB Translocation and MAPK/ERK Phosphorylation

Made available in DSpace on 2015-09-28T13:02:39Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) igor_almeida_etal_IOC_2013.pdf: 680569 bytes, checksum: 68dcc939113bc5eb10c5deee819a7ff8 (MD5) Previous issue date: 2013Universidade Federal do Rio de Janeiro. Centro de Ciências da Saúde. Instituto de Bioquímica Médica. Programa de Biologia Molecular e Biotecnologia. Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular- INCT-EM. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Centro de Ciências da Saúde. Instituto de Bioquímica Médica. Programa de Biologia Molecular e Biotecnologia. Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular- INCT-EM. Rio de Janeiro, RJ, Brasil.University of Texas at El Paso. Department of Biological Sciences. The Border Biomedical Research Center. El Paso, Texas, USA.University of Texas at El Paso. Department of Biological Sciences. The Border Biomedical Research Center. El Paso, Texas, USA / Universidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Departamento de Biologia Celular e Molecular Patogênicos. Ribeirão Preto, SP, Brasil.University of Texas at El Paso. Department of Biological Sciences. The Border Biomedical Research Center. El Paso, Texas, USA.University of Texas at El Paso. Department of Biological Sciences. The Border Biomedical Research Center. El Paso, Texas, USA / Universidade de São Paulo. Faculdade de Medicina de Ribeirão Preto. Departamento de Biologia Celular e Molecular Patogênicos. Ribeirão Preto, SP, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunofarmacologia. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Centro de Ciências da Saúde. Instituto de Biofísica Carlos Chagas Filho. Laboratório de Parasitologia Molecular. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Centro de Ciências da Saúde. Instituto de Bioquímica Médica. Programa de Biologia Molecular e Biotecnologia. Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular- INCT-EM. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunofarmacologia. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Centro de Ciências da Saúde. Instituto de Bioquímica Médica. Programa de Biologia Molecular e Biotecnologia. Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular- INCT-EM. Rio de Janeiro, RJ, Brasil.Background: Lysophosphatidylcholine (LPC) is the main phospholipid component of oxidized low-density lipoprotein (oxLDL) and is usually noted as a marker of several human diseases, such as atherosclerosis, cancer and diabetes. Some studies suggest that oxLDL modulates Toll-like receptor (TLR) signaling. However, effector molecules that are present in oxLDL particles and can trigger TLR signaling are not yet clear. LPC was previously described as an attenuator of sepsis and as an immune suppressor. In the present study, we have evaluated the role of LPC as a dual modulator of the TLR-mediated signaling pathway. Methodology/Principal Findings: HEK 293A cells were transfected with TLR expression constructs and stimulated with LPC molecules with different fatty acid chain lengths and saturation levels. All LPC molecules activated both TLR4 and TLR2-1 signaling, as evaluated by NF-қB activation and IL-8 production. These data were confirmed by Western blot analysis of NF-қB translocation in isolated nuclei of peritoneal murine macrophages. However, LPC counteracted the TLR4 signaling induced by LPS. In this case, NF-қB translocation, nitric oxide (NO) synthesis and the expression of inducible nitric oxide synthase (iNOS) were blocked. Moreover, LPC activated the MAP Kinases p38 and JNK, but not ERK, in murine macrophages. Interestingly, LPC blocked LPS-induced ERK activation in peritoneal macrophages but not in TLR-transfected cells. Conclusions/Significance: The above results indicate that LPC is a dual-activity ligand molecule. It is able to trigger a classical proinflammatory phenotype by activating TLR4- and TLR2-1-mediated signaling. However, in the presence of classical TLR ligands, LPC counteracts some of the TLR-mediated intracellular responses, ultimately inducing an anti-inflammatory phenotype; LPC may thus play a role in the regulation of cell immune responses and disease progression

LPC activates JNK and p38, but not ERK, in macrophages.

<p>Peritoneal macrophages from BALB/c mice were incubated in the absence or presence of different concentrations of LPC mix (Sigma) for 20 min at 37 °C in a 5% CO₂ atmosphere, and the cytoplasm content was homogenized and assayed as follows. The Phospho-MAPK array was used for analysis of enzymatic activation (<b>A</b>). The reaction was visualized with the enhanced chemiluminescent system and subjected to densitometric analysis (***, p< 0.001, ANOVA). Protein levels of the phosphorylated MAPKs JNK (<b>B</b>), p38 (<b>C</b>) and ERK (<b>D</b>) were determined by Western blot. Data is the mean ± S.E. of two different experiments.</p

LPC triggers NF-қB activation through either TLR4- or TLR2/1-dependent signaling pathways.

<p>HEK 293A cells were transfected in three different groups. Groups A and B received expression constructs for TLR4 (<b>A</b>) or TLR2 and TLR1 (<b>B</b>). Both also received MD-2, CD14, and CD36 constructs and the ELAM-1-firefly luciferase and β-actin-<i>Renilla</i> luciferase reporter plasmids. The third group (<b>C</b>) received only the empty vector pDisplay and the luciferase reporter plasmids. Groups A and B were separately stimulated with 0.1, 1, 10, 100 and 200 µM of different types of LPC (Sigma; C14:0, C16:0, C18:0, and C18:1), 100 ng/mL of LPS and 1 nM of Pam3CSK4 (P3C). Group C was stimulated with LPS, Pam3Cys or 0.1, 1, 10 and 100 µM of LPC (C16:0). The agonists were diluted in DMEM medium with 10% bovine fetal serum. After 4 h of incubation, luciferase activity was measured and expressed as the ratio of NF-қB-dependent firefly luciferase activity to the control <i>Renilla</i> luciferase activity. Data is the mean ± S.E. of two different experiments. ** P < 0.01, *** P < 0.001 (One way ANOVA, Parameter, Bonferroni’s Multiple Comparison Test).</p

LPC inhibits NF-қB translocation, iNOS expression, and NO production in LPS-stimulated macrophages.

<p>Peritoneal macrophages from BALB/cmice were incubated in the absence or presence of 1 µg/mL LPS and different concentrations of LPC (Sigma) at 37 °C in a 5% CO₂ atmosphere. After 1 h of incubation, NF-қB translocation (<b>A</b>) was assayed by Western blot analysis. After 24 hours, NO production (<b>B</b>) was assayed by measuring the amount of nitrite in the culture supernatant using the Griess reagent, and iNOS expression (<b>C</b>) was determined by Western blot analysis followed by densitometry (lower panel). Data is the mean ± S.D. of three different experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 (One way ANOVA, Parameter, Bonferroni’s Multiple Comparison Test).</p

LPC triggers IL-8 production through either TLR4- or TLR2/1-dependent signaling pathways.

<p>HEK 293A cells were transfected and stimulated as described on Figure 1. After 20 hours of incubation, IL-8 production was measured by the ELISA assay. Data is the mean ± S.E. of two different experiments. ** P < 0.01, *** P < 0.001 (One way ANOVA, Parameter, Bonferroni’s Multiple Comparison Test).</p

LPC inhibits LPS-induced ERK activation.

<p>Peritoneal macrophages from BALB/c mice were incubated in the absence or presence of 1 µg/mL LPS or in the presence or absence of the indicated concentrations of LPC (Sigma) at 37 °C in a 5% CO₂ atmosphere (<b>A</b>, <b>D</b>, <b>E</b>). In parallel HEK 293A cells with TLR constructs as indicated (<b>B</b>, <b>C</b>). Each group received expression constructs for TLR4 (<b>B</b>) or both TLR2 and TLR1 (<b>C</b>), as well as MD-2, CD14 and CD36 plasmids. The cells were then incubated in the absence or presence of 100 ng/mL LPS or 1 nM Pam3CSK4 (P3C) and 10 or 100 µM of LPC, for 40 min at 37 °C in a 5% CO₂ atmosphere. After incubation either macrophages or HEK cells were homogenized, the protein levels was determined and samples evaluated by Western blot with the use of antibodies against p-ERK (<b>A</b>, <b>B</b>, <b>C</b>), p-JNK (<b>D</b>) and p-P38 (<b>E</b>). Loading controls were run with the use of antibodies raised towards actin. Experiments were performed at least two times with different animals and samples.</p