Respuesta humoral en Leishmaniasis del Perú : Proteínas HSP70 y ribosomales P

En la presente tesis, se abordó el clonado de la hsp70 de L.(V.) peruviana y la caracterización de la respuesta humoral en pacientes con Leishmaniasis Tegumentaria Americana, la Enfermedad de Chagas y la enfermedad mixta Chagas/Leishmaniasis. Se determinó el tamaño del inserto de U4 en 1,6 Kpb. Se mostró que U4 presentaba un marco de lectura abierto de 525 pb. que codifican para los 174 aminoácidos C-terminales de la hsp70 de L.(V.) peruviana. U4 presenta una homología de 98% con Lbb1 (97% de identidad y 1% de cambios conservativos) y con RA1 un 87% (74% de identidad y 13% de cambios conservativos). Se determinó la organización genómica de los genes hsp70 por Southern-blot de ADN genómico de clones de L.(V.) peruviana y L.(V.) braziliensis. Se encontró 2 locus genómicos, uno de los cuales contenía un tandem de genes hsp70 para ambas especies de Leishmania sp. Los ADNc de U4 y Lbb1 se subclonaron pGEX1l T para caracterizar la respuesta humoral en Leishmaniasis Tegumentaria Americana, Enf. de Chagas y la Inf. mixta Chagas/Espundia por Ensayo de Placa de Lisis, Western-blot y ELISA. Los resultados obtenidos por las 3 técnicas indican que U4/Lbb1 se pueden usar en el diagnóstico de L. Mucocutánea (Espundia) y junto a JL7, permitiría un diagnóstico diferencial de la enfermedad mixta Chagas/Espundia. El clonado y la caracterización de la respuesta humoral de las proteínas ribosomales P2 de L.(V.) peruviana y L.(V.) braziliensis. El clonado de las proteínas ribosomales ácidas de tipo P2 de L.(V.) peruviana y L.(V.) braziliensis se realizó mediante rastreo de bibliotecas de expresión en l gt11 con sondas de las proteínas ribosomales P2a y P2b de L.(L.) infantum. En la biblioteca de expresión de L.(V.) peruviana se aisló un recombinante con cada sonda. El recombinante LbpP2a tenía un inserto de aprox. 900 pb., presentando un marco de lectura abierto de 327 pb. que codifican 108 aminoácidos, faltándole sólo los 3 primeros residuos N-terminales para estar completa. El recombinante LbpP2a presenta una homología de aminoácidos de 95% (89% de identidad y 6% de cambios conservativos) con la proteína homóloga de L.(L.) infantum, un 87% (74% de identidad y 13% de cambios conservativos) con la de T. cruzi y un 65% (44% de identidad y 21% de cambios conservativos) con la proteína P2 humana. El otro recombinante (LbpP2b ) aislado de la misma biblioteca continua un inserto de 271 pb., presentando un marco de lectura abierto de 144 pb. que codifican los 44 últimos aminoácidos C-terminales. Cuando se compara el recombinante LbpP2b con sus homólogas de otras especies de Trypanosomátidos, la más alta homología la presenta con la proteína ribosomal P2b de L.(L.) infantum con un 90% (81% de identidad y 9% de cambios conservativos), con la P2b de T. cruzi alcanza un 86% (78% de identidad y 8% de cambios conservativos), con la P2b de T. brucei un 78% (66% de identidad y 12% de cambios conservativos) y con la P2 humana un 73% (59% de identidad y 14% de cambios conservativos). En la biblioteca de expresión de L.(V.) braziliensis se aisló un recombinante (LbbP2b -like) con la sonda la P2a de L.(L.) infantum. Este recombinante contiene un inserto de aprox. 750 pb., presentando un marco de lectura abierto de 264 pb. que codifican los 87 aminoácidos C-terminales de una proteína ribosomal P2b atípica de L.(V.) braziliensis. La comparación de aminoácidos es mayor con el recombinante LbpP2b con un 72% (52% de identidad y 20% de cambios conservativos), un 69% con la de L.(L.) infantum (49% de identidad y 20% de cambios conservativos), con la P2 humana un 64% (35% de identidad y 29% de cambios conservativos), con la T. brucei 58% (38% de identidad y 20% de cambios conservativos) y finalmente con la de T. cruzi un 53% (31% de identidad y 22% de cambios conservativos). Al analizar la respuesta humoral de pacientes con L. Mucocutánea contra los recombinantes LbpP2a y LbbP2b -like, se vió que el epítope inmunodominante no se localiza en el extremo C-terminal, mientras que en las proteínas ribosomales P de T. cruzi, se había determinado el extremo C-terminal como el epítope inmunodominante (péptido R-13). De otro lado, pacientes con la Enfermedad de Chagas no reaccionaban con las proteínas ribosomales de tipo P2 de Leishmania sp., indicando que inducen anticuerpos Leishmania-específicos y que no cros-reaccionan con las de T. Cruzi.I have performed the cloning of the hsp70 of Leishmania (Viannia) peruviana (recombinant U4) and characterization of the humoral inmune response in patients with American Tegumentary Leishmaniasis (ATL), Chagas’ disease (CH) and the mixed infection Chagas/Leishmaniasis (CH/L) against this protein. The recombinant U4 has an insert of 1,6 Kbp., presenting an open reading frame (ORF) of 525 bp, coding for the last 174 aminoacids. The recombinant U4 presents a high degree of homology with the recombinant Lbb1 (C-terminal domain of the hsp70 of L.(V.) braziliensis) of 98% (97% identity and 1% non conservative substitutions) and with the recombinant RA1 (C-terminal domain of the hsp70 of T. cruzi) of 87% (74% identity and 13% non conservative substitutions). I found two hsp70 genomic loci in the genome of the related species L.(V.) peruviana and L.(V.) braziliensis as determined by Southern blot. One locus comprised the tandem repeat of the hsp70 genes, which present a repetitive unit of 3,7 Kbp. I have cloned the U4, Lbb1 and RA1 cDNAs in the expression vector pGEX1l T to characterize the humoral inmune response in ATL, CH and CH/L as measured by ELISA, Phage dot array immunoassay and Western blot. I found a high reactivity of Mococutaneous Leishmaniasis sera with the U4/Lbb1 recombinant proteins, whereas most of the Cutaneous Leishmaniasis, Chagasic and non-related sera did not. I have also cloned the ribosomal P2-type proteins of L.(V.) peruviana (LbpP2a and LbpP2b ) and L.(V.) braziliensis (LbbP2b -like) by screening of the expression libraries with P2-type ribosomal proteins from L.(L.) infantum as probes. The recombinant LbpP2a has an insert of 900 bp. An ORF of 327 bp, codifying 108 aminoacids, lacking the three N-terminal residues to be a full length protein. This recombinant presents an homology of 95% (89% identity and 6% conservative substitution) with the homologous of L.(L.) infantum, 87% of homology (74% identity and 13% conservative substitutions) with the corresponding of T. cruzi and 65% of homology (44% identity and 25% conservative substitutions) with the human P2 protein. The recombinant LbpP2b has an insert of 271 bp, presenting on ORF of 144 bp, coding for the last 44 C-terminal aminoacids. It has an homology of 90% (81% identity and 9% conservative substitutions) with the corresponding of L.(L.) infantum, 86% of homology (78% identity and 8% conservative substitutions) with that of T. cruzi, 78% of homology (66% identity and 12% conservative substitutions) with the P2b protein of T. brucei and 73% of homology (59% identity and 14% conservative substitutions) with the human P2 protein. The LbbP2b -like protein has an insert of 750 bp, presenting an ORF of 264 bp. coding for the 87 C-terminal aminoacids. It has 72% of homology (52% identity and 20% conservative substitutions) with that of L.(V.) braziliensis, 69% (49% identity and 20% conservative substitutions) with that of L.(L.) infantum, 64% (35% identity and 29% conservative substitutions) with the human P2 protein, 58% (38% identity and 20% conservative substitutions) with the P2b of T. brucei and a 53% (31% identity and 22% conservative substitutions) with that of T. cruzi. In both Leishmania P2 proteins (LbpP2a and LbbP2b -like) evaluated, the most antigenic epitope was not located at the C-terminus, whereas in the T. cruzi P2 proteins the inmunodominant epitope was the last 13 C-terminal residues (R-13 peptide). Chagasic sera did not react with the ribosomal P2 proteins from Leishmania sp.Fil:Panebra Alvarado, Alfredo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Common ADRB2 Haplotypes Derived from 26 Polymorphic Sites Direct β2-Adrenergic Receptor Expression and Regulation Phenotypes

Background: The β2-adrenergic receptor (β2AR) is expressed on numerous cell-types including airway smooth muscle cells and cardiomyocytes. Drugs (agonists or antagonists) acting at these receptors for treatment of asthma, chronic obstructive pulmonary disease, and heart failure show substantial interindividual variability in response. The ADRB2 gene is polymorphic in noncoding and coding regions, but virtually all ADRB2 association studies have utilized the two common nonsynonymous coding SNPs, often reaching discrepant conclusions. Methodology/Principal Findings: We constructed the 8 common ADRB2 haplotypes derived from 26 polymorphisms in the promoter, 5′UTR, coding, and 3′UTR of the intronless ADRB2 gene. These were cloned into an expression construct lacking a vector-based promoter, so that β2AR expression was driven by its promoter, and steady state expression could be modified by polymorphisms throughout ADRB2 within a haplotype. “Whole-gene” transfections were performed with COS-7 cells and revealed 4 haplotypes with increased cell surface β2AR protein expression compared to the others. Agonist-promoted downregulation of β2AR protein expression was also haplotype-dependent, and was found to be increased for 2 haplotypes. A phylogenetic tree of the haplotypes was derived and annotated by cellular phenotypes, revealing a pattern potentially driven by expression. Conclusions/Significance: Thus for obstructive lung disease, the initial bronchodilator response from intermittent administration of β-agonist may be influenced by certain β2AR haplotypes (expression phenotypes), while other haplotypes may influence tachyphylaxis during the response to chronic therapy (downregulation phenotypes). An ideal clinical outcome of high expression and less downregulation was found for two haplotypes. Haplotypes may also affect heart failure antagonist therapy, where β2AR increase inotropy and are anti-apoptotic. The haplotype-specific expression and regulation phenotypes found in this transfection-based system suggest that the density of genetic information in the form of these haplotypes, or haplotype-clusters with similar phenotypes can potentially provide greater discrimination of phenotype in human disease and pharmacogenomic association studies

Common ADRB2 Haplotypes Derived from 26 Polymorphic Sites Direct β2-Adrenergic Receptor Expression and Regulation Phenotypes

The beta2-adrenergic receptor (beta2AR) is expressed on numerous cell-types including airway smooth muscle cells and cardiomyocytes. Drugs (agonists or antagonists) acting at these receptors for treatment of asthma, chronic obstructive pulmonary disease, and heart failure show substantial interindividual variability in response. The ADRB2 gene is polymorphic in noncoding and coding regions, but virtually all ADRB2 association studies have utilized the two common nonsynonymous coding SNPs, often reaching discrepant conclusions.We constructed the 8 common ADRB2 haplotypes derived from 26 polymorphisms in the promoter, 5'UTR, coding, and 3'UTR of the intronless ADRB2 gene. These were cloned into an expression construct lacking a vector-based promoter, so that beta2AR expression was driven by its promoter, and steady state expression could be modified by polymorphisms throughout ADRB2 within a haplotype. "Whole-gene" transfections were performed with COS-7 cells and revealed 4 haplotypes with increased cell surface beta2AR protein expression compared to the others. Agonist-promoted downregulation of beta2AR protein expression was also haplotype-dependent, and was found to be increased for 2 haplotypes. A phylogenetic tree of the haplotypes was derived and annotated by cellular phenotypes, revealing a pattern potentially driven by expression.Thus for obstructive lung disease, the initial bronchodilator response from intermittent administration of beta-agonist may be influenced by certain beta2AR haplotypes (expression phenotypes), while other haplotypes may influence tachyphylaxis during the response to chronic therapy (downregulation phenotypes). An ideal clinical outcome of high expression and less downregulation was found for two haplotypes. Haplotypes may also affect heart failure antagonist therapy, where beta2AR increase inotropy and are anti-apoptotic. The haplotype-specific expression and regulation phenotypes found in this transfection-based system suggest that the density of genetic information in the form of these haplotypes, or haplotype-clusters with similar phenotypes can potentially provide greater discrimination of phenotype in human disease and pharmacogenomic association studies

Regulation of actin dynamics by tyrosine phosphorylation: Identification of tyrosine phosphorylation sites within the actin-severing domain of villin

We have previously shown that villin, an epithelial cell actin-binding protein, is tyrosine phosphorylated both in vitro and in vivo and that villin\u27s actin-modifying functions are regulated by phosphorylation. Here as a first step toward understanding the role of villin tyrosine phosphorylation, we sought to identify the major phosphorylation site(s) in human villin and study its role in actin filament assembly. We generated a series of carboxyl-terminal truncation mutants of villin and cloned them in the prokaryotic expression vector pGEX-2T. Full-length villin and the truncation mutants were expressed in TKX1 cells, which carry an inducible tyrosine kinase gene. Using this approach, we identified a region in the amino-terminal actin-severing domain of villin as the site of phosphorylation (amino acids 1-261). Five phosphorylation sites were identified by direct mutation of candidate tyrosines (Y) to phenylalanine (F), namely, Y46, -60, -64, -81, and -256. Changing all of these sites to phenylalanine resulted in a villin mutant that neither was phosphorylated in TKX1 cells nor was a substrate for c-src kinase in an in vitro kinase assay. Using a pyrene actin-based fluorescence assay, we mapped the various phosphorylated tyrosine residues with the actin-nucleating and -depolymerizing functions of villin. Phosphorylation of any one of the identified sites inhibited the actin-nucleating function of villin, whereas phosphorylation at Y46 and/or Y60 increased the actin-severing activity of villin. Since there is significant homology between the amino-terminal end of villin and other actin-severing proteins, the results provide a structural basis for the actin-severing mechanism and help understand the relationship of phosphorylation with this function