Eukaryotic initiation factor 5A dephosphorylation is required for translational arrest in stationary phase cells

The protein known as eIF5A (eukaryotic initiation factor 5A) has an elusive role in translation. It has a unique and essential hypusine modification at a conserved lysine residue in most eukaryotes. in addition, this protein is modified by phosphorylation with unknown functions. in the present study we show that a phosphorylated state of eIF5A predominates in exponentially growing Trypanosoma cruzi cells, and extensive dephosphorylation occurs in cells in stationary phase. Phosphorylation occurs mainly at See, as shown in yeast eIF5A. in addition, a novel phosphorylation site was identified at Tyr(21). in exponential cells, T. cruzi eIF5A is partially associated with polysomes, compatible with a proposed function as an elongation factor, and becomes relatively enriched in polysomal fractions in stationary phase. Overexpression of the wild-type eIF5A, or eIF5A with See replaced by an aspartate residue, but not by alanine, increases the rate of cell proliferation and protein synthesis. However, the presence of an aspartate residue instead of See is toxic for cells reaching the stationary phase, which show a less-pronounced protein synthesis arrest and a decreased amount of eIF5A in dense fractions of sucrose gradients. We conclude that eIF5A phosphorylation and dephosphorylation cycles regulate translation according to the growth conditions.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Financiadora de Estudos e Projetos (FINEP)Universidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, BR-04039032 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, BR-04039032 São Paulo, BrazilWeb of Scienc

Identification of an atypical peptidyl-prolyl cis/trans isomerase from trypanosomatids

The parvulin family of peptidyl-prolyl cis/trans isomerases (PPIases) catalyzes the cis/trans isomerization of the peptide bonds preceding Pro residues. Eukaryotic parvulin-type PPIases have been shown to be involved in cell proliferation and cell cycle progression. Here we present the biochemical and molecular characterization of a novel multi-domain parvulin-type PPIase from the human pathogenic Trypanosoma cruzi, annotated as TcPar45. Like most other parvulins, Par45 has an N-terminal extension, but, in contrast to human Pin1, it contains a forkhead-associated domain (FHA) instead of a WW domain at the N-terminal end. Par45 shows a strong preference for a substrate with the basic Arg residue preceding Pro (Suc-Ala-Arg-Pro-Phe-NH-Np: k(cat)/K(M) = 97.1 /M/s), like that found for human Part14. in contrast to human Pin1, but similarly to Par14, Par45 does not accelerate the cis/trans interconversion of acidic substrates containing Glu-Pro bonds. It is preferentially located in the parasite nucleus. Single RNA interference (RNAi)-mediated knock-down showed that there was a growth inhibition in procyclic Trypanosoma brucei cells. These results identify Par45 as a phosphorylation-independent parvulin required for normal cell proliferation in a unicellular eukaryotic cell. (C) 2010 Elsevier B.V. All rights reserved.DAADConsejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET, Argentina)Agencia Nacional de Promocion Cientifica y Tecnologica (ANPCyT, Argentina)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)INGEBI CONICET, Buenos Aires, DF, ArgentinaUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilMax Planck Res Unit Enzymol Prot Folding, Halle, GermanyUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilWeb of Scienc

Differential Proteomic Analysis of Noncardia Gastric Cancer from Individuals of Northern Brazil

Gastric cancer is the second leading cause of cancer-related death worldwide. The identification of new cancer biomarkers is necessary to reduce the mortality rates through the development of new screening assays and early diagnosis, as well as new target therapies. In this study, we performed a proteomic analysis of noncardia gastric neoplasias of individuals from Northern Brazil. The proteins were analyzed by two-dimensional electrophoresis and mass spectrometry. For the identification of differentially expressed proteins, we used statistical tests with bootstrapping resampling to control the type I error in the multiple comparison analyses. We identified 111 proteins involved in gastric carcinogenesis. The computational analysis revealed several proteins involved in the energy production processes and reinforced the Warburg effect in gastric cancer. ENO1 and HSPB1 expression were further evaluated. ENO1 was selected due to its role in aerobic glycolysis that may contribute to the Warburg effect. Although we observed two up-regulated spots of ENO1 in the proteomic analysis, the mean expression of ENO1 was reduced in gastric tumors by western blot. However, mean ENO1 expression seems to increase in more invasive tumors. This lack of correlation between proteomic and western blot analyses may be due to the presence of other ENO1 spots that present a slightly reduced expression, but with a high impact in the mean protein expression. In neoplasias, HSPB1 is induced by cellular stress to protect cells against apoptosis. In the present study, HSPB1 presented an elevated protein and mRNA expression in a subset of gastric cancer samples. However, no association was observed between HSPB1 expression and clinicopathological characteristics. Here, we identified several possible biomarkers of gastric cancer in individuals from Northern Brazil. These biomarkers may be useful for the assessment of prognosis and stratification for therapy if validated in larger clinical study sets.Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP

Structural and functional characterization of TceIF5A, translation factor, in Trypanosoma cruzi

Pouco se sabe sobre os mecanismos envolvidos na adaptacao de protozoarios do genero Trypanosoma em seus diferentes hospedeiros e as vias de sinalizacao celular implicadas. Nestes organismos o controle de expressao genica ocorre majoritariamente ao nivel pos-transcricional desde o processamento do RNA ate a traducao dos mRNAs em proteinas. Nesta tese estudamos o fator de traducao denominado eukaryotic initiation factor 5A (eIF5A). Uma caracteristica unica desta proteina consiste na presenca de um residuo de hipusina, originado de uma espermidina, para um residuo especifico de lisina na proteina. A eIF5A de Trypanosoma cruzi (TceIF5A) foi purificada e caracterizada por espectrometria de massa. Ela apresenta a hipusinacao na Lys 53 e a fosforilacao na Ser 2 conservados. Foram identificados outros sitios de fosforilacao (Y21 e S47) e metilacoes ainda nao conhecidas em outros organismos. TceIF5A apresenta uma distribuicao citossolica, e sua expressao esta aumentada nas formas proliferativas do T. cruzi. Parte da proteina esta associada com polissomos em parasitas em fase de proliferacao. Em fase estacionaria de crescimento TceIF5A e defosforilada e permanece significativamente enriquecida nos polissomos restantes. A superexpressao de TceIF5A aumenta a taxa de proliferacao celular e a sua quantidade relativa ligada aos polissomos A superexpressao da proteina com substituicao da serina 2 por alanina, que previne a fosforilacao nesta posicao, tambem promove um aumento relativo de TceIF5A associados aos polissomos, mas nao favorece o crescimento dos parasitas. Ja a superexpressao de eIF5A com acido aspartico na posicao 2 tem o crescimento afetado antes de chegar na fase estacionaria e apresenta menos polissomos em relacao a monossomos. Concluimos que eIF5A de T. cruzi possui conservadas suas principais caracteristicas, atuando na elongacao da traducao. Sugerimos que a fosforilacao na serina 2 tem um papel na associacao de TceIF5A aos polissomos na fase estacionaria de crescimentoBV UNIFESP: Teses e dissertaçõe

Proteômica e sepse: novas perspectivas para o diagnóstico Proteomics and sepsis: new perspectives for diagnosis

JUSTIFICATIVA E OBJETIVOS: O diagnóstico e o tratamento da sepse continuam a desafiar a todos; e desenvolver formas mais precisas de abordagem são absolutamente necessárias. O objetivo deste estudo foi empregar técnicas proteômicas, eletroforese bidimensional e espectrometria de massa, para verificar a expressão diferencial de proteínas, em soro de pacientes com sepse comparado com controles saudáveis. MÉTODO: Amostras de soro de 30 pacientes com sepse, causada por vários tipos de microorganismos e de 30 controles saudáveis foram obtidas para análise. A seguir, foram submetidas a 2D-SDS-PAGE, comparação entre géis, seleção de spots para excisão e digestão com tripsina, sendo os peptídeos analisados por MALDI TOF-TOF. Os espectros obtidos foram processados (Mascot-matrixscience) para identificação de proteínas no NCBInr Data Bank. RESULTADOS: A análise das imagens mostrou vários spots com expressão diferencial nos géis dos pacientes com sepse em relação aos controles. A identificação de proteínas em alguns destes spots encontrou: precursor Orosomucoide 1, Apolipoproteína A-IV, precursor Apolipoproteína A-IV, precursor Haptoglobina, Haptoglobina, proteína Zinc finger, Amilóide sérico A-1, Transtiretina, Nebulin, Complemento C4, Alfa1-Antitripsina, produto protéico não nominado e outros. CONCLUSÕES: Soros de pacientes com diferentes tipos de sepse expressam padrão protéico característico por 2D-SDS-PAGE comparado com controles. A maior expressão foi de proteínas de fase aguda e lipoproteínas. É possível que no futuro, com a proteômica, criar painel diagnóstico de proteínas, encontrar novos biomarcadores e alvos para intervenção terapêutica na sepse. Esta é a primeira descrição, com a proteômica, das alterações na expressão protéica, no soro de pacientes com sepse.<br>BACKGROUND AND OBJECTIVES: The diagnostic and treatment of sepsis continue to challenger all, and, more specific forms to approach are absolutely necessary. The objective of this study was to use proteomics techniques, two-dimensional electrophoresis and mass spectrometry, to verify the differential protein expression between serum of patients with sepsis and health controls. METHODS: Samples of serum the 30 patients with sepsis, caused for different types of microorganisms and serum of 30 health controls were obtained for analysis. Next, were submitted to 2D-SDS-PAGE, gels compared, selection of spots for excision and digestion with trypsin, being the peptides analyzed for MALDI TOF-TOF. The obtained spectrums were processed (Mascot-matrix science) for protein identification in NCBInr Data Bank. RESULTS: Image analyses showed several spots with differential expressions in the gels of the patients with sepsis in relation to the controls. The protein identification of some of these spots founded: Orosomucoid 1 precursor, Apolipoprotein A-IV, Apolipoprotein A-IV precursor, Haptoglobin protein precursor, Haptoglobin, Zinc finger protein, Serum amyloid A-1, Transthyretin, Nebulin, Complement C4, Alpha1-Antitrypsin, Unnamed protein product and others. CONCLUSIONS: Serum of the patients with different types of sepsis express characteristic protein profiles by 2D-SDS-PAGE compared with controls. The most expressed were from acute phase proteins and lipoproteins. It is possible in the future, with proteomics, create diagnostic panel of proteins, finding news biomarkers and targets for therapeutic interventions in sepsis. This is a first description, with proteomics, of the alterations in protein expression, in serum of the patients with sepsis

A Novel Monoclonal Antibody Against the C-terminus of beta-Tubulin Recognizes Endocytic Organelles in Trypanosoma cruzi

Microtubule cytoskeleton is a dynamic structure involved in the maintenance of eukaryote cell shape, motion of cilia and flagellum, and intracellular movement of vesicles and organelles. Many antibodies against tubulins have been described, most of them against the C-terminal portion, which is exposed at the outside of the microtubules. By generating a novel set of monoclonal antibodies against the cytoskeleton of Trypanosoma cruzi, a flagellate protozoan that causes Chagas' disease, we selected a clone (mAb 3G4) that recognizes beta-tubulin. The epitope for mAb 3G4 was mapped by pepscan to a highly conserved sequence motif found between alpha-helices 11 and 12 of the C-terminus of beta-tubulin in eukaryotes. It labels vesicular structures in both T. cruzi and mammalian cells, colocalizing respectively with a major cysteine protease (Cruzipain) and lysosome associated protein (LAMP2) respectively, but it does not label regular microtubules on these cellular models. We propose that the epitope recognized by mAb 3G4 is exposed only in a form of tubulin associated with endosomes.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ Fed Sao Paulo, Dept Microbiol Imunol & Parasitol, BR-04023062 Sao Paulo, BrazilUniv Fed Sao Paulo, Inst Ciencia & Tecnol, BR-12231280 Sao Jose Dos Campos, BrazilForschungszentrum Karlsruhe, KIT, Inst Biol Interfaces, D-76021 Karlsruhe, GermanyUniv Fed Sao Paulo, Dept Microbiol Imunol & Parasitol, BR-04023062 Sao Paulo, BrazilUniv Fed Sao Paulo, Inst Ciencia & Tecnol, BR-12231280 Sao Jose Dos Campos, BrazilWeb of Scienc

Table_1.PDF

Introduction<p>Impulse control disorders (ICDs) are frequent non-motor symptoms in Parkinson’s disease (PD), with potential negative effects on the quality of life and social functioning. ICDs are closely associated with dopaminergic therapy, and genetic polymorphisms in several neurotransmitter pathways may increase the risk of addictive behaviors in PD. However, clinical differentiation between patients at risk and patients without risk of ICDs is still troublesome. The aim of this study was to investigate if genetic polymorphisms across several neurotransmitter pathways were associated with ICD status in patients with PD.</p>Methods<p>Whole-exome sequencing data were available for 119 eligible PD patients from the Norwegian ParkWest study. All participants underwent comprehensive neurological, neuropsychiatric, and neuropsychological assessments. ICDs were assessed using the self-report short form version of the Questionnaire for Impulsive-Compulsive Disorders in PD. Single-nucleotide polymorphisms (SNPs) from 17 genes were subjected to regression with elastic net penalization to identify candidate variants associated with ICDs. The area under the curve of receiver-operating characteristic curves was used to evaluate the level of ICD prediction.</p>Results<p>Among the 119 patients with PD included in the analysis, 29% met the criteria for ICD and 63% were using dopamine agonists (DAs). Eleven SNPs were associated with ICDs, and the four SNPs with the most robust performance significantly increased ICD predictability (AUC = 0.81, 95% CI 0.73–0.90) compared to clinical data alone (DA use and age; AUC = 0.65, 95% CI 0.59–0.78). The strongest predictive factors were rs5326 in DRD1, which was associated with increased odds of ICDs, and rs702764 in OPRK1, which was associated with decreased odds of ICDs.</p>Conclusion<p>Using an advanced statistical approach, we identified SNPs in nine genes, including a novel polymorphism in DRD1, with potential application for the identification of PD patients at risk for ICDs.</p

Association of glucocerebrosidase polymorphisms and mutations with dementia in incident Parkinson's disease

PINE was supported by Parkinson’s UK (grant numbers G0502, G0914, G1302), Scottish Government Chief Scientist Office, BMA Doris Hillier Award, the BUPA Foundation, NHS Grampian Endowments, and RS MacDonald Trust. The Norwegian ParkWest study has been funded by the Research Council of Norway (grant number 177966), the Western Norway Regional Health Authority (grant number 911218), Stavanger University Hospital Research Funds (grant number 501611), and the Norwegian Parkinson’s Disease Association. Janete Chung and Kristin Aaser Lunde are supported by the Western Norway Regional Health Authority (grant numbers 911859 and 911830). The NYPUM study has been funded by the Swedish Medical Research Council, the Swedish Parkinson’s Disease Association, the Swedish Parkinson Foundation, Erling Persson Foundation, Kempe Foundation and the Västerbotten County Council. The funding sources had no role in the design and conduct of the study: collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript: and decision to submit the manuscript for publication.Peer reviewedPostprin

Dopaminergic and Opioid Pathways Associated with Impulse Control Disorders in Parkinson’s Disease

IntroductionImpulse control disorders (ICDs) are frequent non-motor symptoms in Parkinson’s disease (PD), with potential negative effects on the quality of life and social functioning. ICDs are closely associated with dopaminergic therapy, and genetic polymorphisms in several neurotransmitter pathways may increase the risk of addictive behaviors in PD. However, clinical differentiation between patients at risk and patients without risk of ICDs is still troublesome. The aim of this study was to investigate if genetic polymorphisms across several neurotransmitter pathways were associated with ICD status in patients with PD.MethodsWhole-exome sequencing data were available for 119 eligible PD patients from the Norwegian ParkWest study. All participants underwent comprehensive neurological, neuropsychiatric, and neuropsychological assessments. ICDs were assessed using the self-report short form version of the Questionnaire for Impulsive-Compulsive Disorders in PD. Single-nucleotide polymorphisms (SNPs) from 17 genes were subjected to regression with elastic net penalization to identify candidate variants associated with ICDs. The area under the curve of receiver-operating characteristic curves was used to evaluate the level of ICD prediction.ResultsAmong the 119 patients with PD included in the analysis, 29% met the criteria for ICD and 63% were using dopamine agonists (DAs). Eleven SNPs were associated with ICDs, and the four SNPs with the most robust performance significantly increased ICD predictability (AUC = 0.81, 95% CI 0.73–0.90) compared to clinical data alone (DA use and age; AUC = 0.65, 95% CI 0.59–0.78). The strongest predictive factors were rs5326 in DRD1, which was associated with increased odds of ICDs, and rs702764 in OPRK1, which was associated with decreased odds of ICDs.ConclusionUsing an advanced statistical approach, we identified SNPs in nine genes, including a novel polymorphism in DRD1, with potential application for the identification of PD patients at risk for ICDs