Distinct mRNA and protein interactomes highlight functional differentiation of major eIF4F-like complexes from Trypanosoma brucei

Gene expression in pathogenic protozoans of the family Trypanosomatidae has several novel features, including multiple eIF4F-like complexes involved in protein synthesis. The eukaryotic eIF4F complex, formed mainly by eIF4E and eIF4G subunits, is responsible for the canonical selection of mRNAs required for the initiation of mRNA translation. The best-known complexes implicated in translation in trypanosomatids are based on two related pairs of eIF4E and eIF4G subunits (EIF4E3/EIF4G4 and EIF4E4/EIF4G3), whose functional distinctions remain to be fully described. Here, to define interactomes associated with both complexes in Trypanosoma brucei procyclic forms, we performed parallel immunoprecipitation experiments followed by identification of proteins co-precipitated with the four tagged eIF4E and eIF4G subunits. A number of different protein partners, including RNA binding proteins and helicases, specifically co-precipitate with each complex. Highlights with the EIF4E4/EIF4G3 pair include RBP23, PABP1, EIF4AI and the CRK1 kinase. Co-precipitated partners with the EIF4E3/EIF4G4 pair are more diverse and include DRBD2, PABP2 and different zinc-finger proteins and RNA helicases. EIF4E3/EIF4G4 are essential for viability and to better define their role, we further investigated their phenotypes after knockdown. Depletion of either EIF4E3/EIF4G4 mRNAs lead to aberrant morphology with a more direct impact on events associated with cytokinesis. We also sought to identify those mRNAs differentially associated with each complex through CLIP-seq with the two eIF4E subunits. Predominant among EIF4E4-bound transcripts are those encoding ribosomal proteins, absent from those found with EIF4E3, which are generally more diverse. RNAi mediated depletion of EIF4E4, which does not affect proliferation, does not lead to changes in mRNAs or proteins associated with EIF4E3, confirming a lack of redundancy and distinct roles for the two complexes

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to <90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], >300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Distinct mitochondrial HSP70 homologues conserved in various Leishmania species suggest novel biological functions

We report the identification of two distinct homologues of the 70-kDa mitochondrial heat shock protein (mtHSP70) from Leishmania chagasi/Leishmania infantum (Lc2.1 and Lc2.2). in Leishmania species, multiple genes encoding Lc2.2 are present whilst single genes encode Lc2.1. Strikingly, genes encoding Lc2.1-like proteins are absent from Trypanosoma species. Lc2.2 is characterized by a poly-glutamine rich C-terminus, absent from Lc2.1 or mtHSP70 homologues outside the trypanosomatids. Lc2.1 displays unique substitutions within its peptide-binding domain which modify amino acids strictly conserved in cytoplasmic and mitochondrial HSP70 proteins alike. Affinity purified antibodies recognize mainly a single protein in extracts from promastigotes/epimastigotes of various Leishmania/Trypanosoma species. Upon differentiation of Leishmania amazonensis into amastigotes a second protein (presumably Lc2.1) is induced and becomes the predominant mtHSP70 homologue expressed. Subcellular localization of these proteins was investigated and ratified a distribution throughout the mitochondrial matrix. Our results imply novel mtHSP70 functions which evolved within the genus Leishmania. (C) 2008 Elsevier B.V. All rights reserved

Análise do tempo de consolidação do sistema de plantio direto Analysis of consolidation time of notillage system

O sistema de plantio direto já está consagrado como sistema conservacionista de cultivo agrícola, o que motivou este trabalho, através do qual se visa analisar o tempo de consolidação deste sistema, considerando-se cultivos de milho com plantio direto implantado entre 2 e 14 anos. Esta análise foi feita por meio da medição da área das estruturas presentes no perfil cultural do solo com um SIG e, posteriormente avaliada pelo método estatístico de componentes principais. Constatou-se que as estruturas onde o plantio direto foi implantado há menos de 8 anos assemelharam-se às estruturas do sistema de plantio convencional, e quando implantado há mais de 9 anos, assemelharam-se às estruturas do perfil de floresta, concluindo-se que o período de consolidação do sistema de plantio direto situa-se entre o 9º e o 10º ano após a sua implantação, para as condições da área estudada. O excesso de detalhamento na nomenclatura das unidades morfologicamente homogêneas dificultou a análise dos dados.<br>The no-tillage system has already been acclaimed as a conservational system of agricultural cultivation, what has motivated this work, through which it was aimed to analyze the consolidation time of this system, considering maize plantations with no-tillage system implemented between 2 to 14 years. This analysis has been done through the measuring of areas of the present structures in the cropping profile with GIS and later, evaluated by the statistical method of principal components. The structures where no-tillage system has been implemented for less than eight years are similar to conventional cultivation, and where it has been implemented for more than nine years are similar to forest cultivation, leading to the conclusion that the period of consolidation of no-tillage system is between the ninth and tenth year after its implementation, for the conditions of the analyzed area. The excess of details in the nomenclature of morphological homogeneous units makes the data analysis difficult

The eIF4E subunits of two distinct trypanosomatid eIF4F complexes are subjected to differential post-translational modifications associated to distinct growth phases in culture

Submitted by Kamylla Nascimento ([email protected]) on 2017-12-12T13:37:36Z No. of bitstreams: 1 art. The eIF4E subunits - pereira.pdf: 917659 bytes, checksum: a81b3d2e421e124a6f8dde22830114a4 (MD5)Approved for entry into archive by Kamylla Nascimento ([email protected]) on 2017-12-12T13:59:10Z (GMT) No. of bitstreams: 1 art. The eIF4E subunits - pereira.pdf: 917659 bytes, checksum: a81b3d2e421e124a6f8dde22830114a4 (MD5)Made available in DSpace on 2017-12-12T13:59:10Z (GMT). No. of bitstreams: 1 art. The eIF4E subunits - pereira.pdf: 917659 bytes, checksum: a81b3d2e421e124a6f8dde22830114a4 (MD5) Previous issue date: 2013O financiamento pessoal aos membros da equipe foi fornecido pelas agências brasileiras de financiamento CNPq , FACEPE e CAPES . O trabalho de laboratório foi parcialmente financiado com subsídios da FACEPE ( APQ-0557-2.02 / 10 ) e os programas PAPES IV e V da FIOCRUZ. A assistência à cultura de parasitas foi fornecida por B. Lima.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, Brasil.Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, Brasil.The eukaryotic eIF4F complex, the cap binding complex, functions during translation initiation through interactions mediated by its three subunits (eIF4E, eIF4G and eIF4A), other initiation factors and the ribosome. In trypanosomatids, various eIF4E and eIF4G homologues were identified, with two eIF4F-like complexes confirmed (EIF4E4/EIF4G3/EIF4AI and EIF4E3/EIF4G4/EIF4AI). Here, the expression pattern of these complexes was investigated during Leishmania amazonensis and Trypanosoma brucei growth. The two sets of eIF4E and eIF4G homologues were found represented by phosphorylated isoforms with multiple phosphorylation events targeting the two eIF4E homologues. Expression of these multiple isoforms was differentially affected by inhibitors of mRNA synthesis/processing and translation. Phosphorylated EIF4E4 was consistently associated with early/active growth phases in both organisms studied. In T. brucei phosphorylation of both EIF4E3 and 4, overexpressed as HA-tagged fusions, was partially mapped to their N-terminuses. Our results indicate that phosphorylation is associated with a further layer of complexity in translation initiation in trypanosomatids

Distinct mRNA and protein interactomes highlight functional differentiation of major eIF4F-like complexes from Trypanosoma brucei.

Peer reviewed: TrueAcknowledgements: The authors are grateful to Nancy Standart for critical review of the manuscript. They also acknowledge the substantial support from the FIOCRUZ Technical Platform program through the Platforms for the high throughput SOLID sequencing and the facility for Protein Purification and Characterization and Proteomics at the Carlos Chagas Institute (Fiocruz Parana), as well as the DNA Sequencing and Electron Microscopy Platforms at the Aggeu Magalhaes Institute (Fiocruz Pernambuco).Gene expression in pathogenic protozoans of the family Trypanosomatidae has several novel features, including multiple eIF4F-like complexes involved in protein synthesis. The eukaryotic eIF4F complex, formed mainly by eIF4E and eIF4G subunits, is responsible for the canonical selection of mRNAs required for the initiation of mRNA translation. The best-known complexes implicated in translation in trypanosomatids are based on two related pairs of eIF4E and eIF4G subunits (EIF4E3/EIF4G4 and EIF4E4/EIF4G3), whose functional distinctions remain to be fully described. Here, to define interactomes associated with both complexes in Trypanosoma brucei procyclic forms, we performed parallel immunoprecipitation experiments followed by identification of proteins co-precipitated with the four tagged eIF4E and eIF4G subunits. A number of different protein partners, including RNA binding proteins and helicases, specifically co-precipitate with each complex. Highlights with the EIF4E4/EIF4G3 pair include RBP23, PABP1, EIF4AI and the CRK1 kinase. Co-precipitated partners with the EIF4E3/EIF4G4 pair are more diverse and include DRBD2, PABP2 and different zinc-finger proteins and RNA helicases. EIF4E3/EIF4G4 are essential for viability and to better define their role, we further investigated their phenotypes after knockdown. Depletion of either EIF4E3/EIF4G4 mRNAs lead to aberrant morphology with a more direct impact on events associated with cytokinesis. We also sought to identify those mRNAs differentially associated with each complex through CLIP-seq with the two eIF4E subunits. Predominant among EIF4E4-bound transcripts are those encoding ribosomal proteins, absent from those found with EIF4E3, which are generally more diverse. RNAi mediated depletion of EIF4E4, which does not affect proliferation, does not lead to changes in mRNAs or proteins associated with EIF4E3, confirming a lack of redundancy and distinct roles for the two complexes

Image3_Distinct mRNA and protein interactomes highlight functional differentiation of major eIF4F-like complexes from Trypanosoma brucei.PDF

Gene expression in pathogenic protozoans of the family Trypanosomatidae has several novel features, including multiple eIF4F-like complexes involved in protein synthesis. The eukaryotic eIF4F complex, formed mainly by eIF4E and eIF4G subunits, is responsible for the canonical selection of mRNAs required for the initiation of mRNA translation. The best-known complexes implicated in translation in trypanosomatids are based on two related pairs of eIF4E and eIF4G subunits (EIF4E3/EIF4G4 and EIF4E4/EIF4G3), whose functional distinctions remain to be fully described. Here, to define interactomes associated with both complexes in Trypanosoma brucei procyclic forms, we performed parallel immunoprecipitation experiments followed by identification of proteins co-precipitated with the four tagged eIF4E and eIF4G subunits. A number of different protein partners, including RNA binding proteins and helicases, specifically co-precipitate with each complex. Highlights with the EIF4E4/EIF4G3 pair include RBP23, PABP1, EIF4AI and the CRK1 kinase. Co-precipitated partners with the EIF4E3/EIF4G4 pair are more diverse and include DRBD2, PABP2 and different zinc-finger proteins and RNA helicases. EIF4E3/EIF4G4 are essential for viability and to better define their role, we further investigated their phenotypes after knockdown. Depletion of either EIF4E3/EIF4G4 mRNAs lead to aberrant morphology with a more direct impact on events associated with cytokinesis. We also sought to identify those mRNAs differentially associated with each complex through CLIP-seq with the two eIF4E subunits. Predominant among EIF4E4-bound transcripts are those encoding ribosomal proteins, absent from those found with EIF4E3, which are generally more diverse. RNAi mediated depletion of EIF4E4, which does not affect proliferation, does not lead to changes in mRNAs or proteins associated with EIF4E3, confirming a lack of redundancy and distinct roles for the two complexes.</p