Proteomics of the Chloroplast: Systematic Identification and Targeting Analysis of Lumenal and Peripheral Thylakoid Proteins

The soluble and peripheral proteins in the thylakoids of pea were systematically analyzed by using two-dimensional electrophoresis, mass spectrometry, and N-terminal Edman sequencing, followed by database searching. After correcting to eliminate possible isoforms and post-translational modifications, we estimated that there are at least 200 to 230 different lumenal and peripheral proteins. Sixty-one proteins were identified; for 33 of these proteins, a clear function or functional domain could be identified, whereas for 10 proteins, no function could be assigned. For 18 proteins, no expressed sequence tag or full-length gene could be identified in the databases, despite experimental determination of a significant amount of amino acid sequence. Nine previously unidentified proteins with lumenal transit peptides are presented along with their full-length genes; seven of these proteins possess the twin arginine motif that is characteristic for substrates of the TAT pathway. Logoplots were used to provide a detailed analysis of the lumenal targeting signals, and all nuclear-encoded proteins identified on the two-dimensional gels were used to test predictions for chloroplast localization and transit peptides made by the software programs ChloroP, PSORT, and SignalP. A combination of these three programs was found to provide a useful tool for evaluating chloroplast localization and transit peptides and also could reveal possible alternative processing sites and dual targeting. The potential of proteomics for plant biology and homology-based searching with mass spectrometry data is discussed

Exploring the Potential Role of Moonlighting Function of the Surface-Associated Proteins From Mycobacterium bovis BCG Moreau and Pasteur by Comparative Proteomic

Submitted by Sandra Infurna ([email protected]) on 2019-09-03T12:10:25Z No. of bitstreams: 1 TalitaPagani_LeilaMLima_etal_IOC_2019.pdf: 2490553 bytes, checksum: bec0e4e17098191a9db8299c1d950da1 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2019-09-03T12:32:35Z (GMT) No. of bitstreams: 1 TalitaPagani_LeilaMLima_etal_IOC_2019.pdf: 2490553 bytes, checksum: bec0e4e17098191a9db8299c1d950da1 (MD5)Made available in DSpace on 2019-09-03T12:32:35Z (GMT). No. of bitstreams: 1 TalitaPagani_LeilaMLima_etal_IOC_2019.pdf: 2490553 bytes, checksum: bec0e4e17098191a9db8299c1d950da1 (MD5) Previous issue date: 2019Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genômica Funcional e Bioinformática. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genômica Funcional e Bioinformática. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genômica Funcional e Bioinformática. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genômica Funcional e Bioinformática. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório Interdisciplinar de Pesquisas Médicas. Rio de Janeiro, RJ, Brasil / Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo de Meis. Unidade de Espectometria de Massas e Proteômica. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Microbiologia Celular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genômica Funcional e Bioinformática. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Genômica Funcional e Bioinformática. Rio de Janeiro, RJ, Brasil.Surface-associated proteins from Mycobacterium bovis BCG Moreau RDJ are important components of the live Brazilian vaccine against tuberculosis. They are important targets during initial BCG vaccine stimulation and modulation of the host's immune response, especially in the bacterial-host interaction. These proteins might also be involved in cellular communication, chemical response to the environment, pathogenesis processes through mobility, colonization, and adherence to the host cell, therefore performing multiple functions. In this study, the proteomic profile of the surface-associated proteins from M. bovis BCG Moreau was compared to the BCG Pasteur reference strain. The methodology used was 2DE gel electrophoresis combined with mass spectrometry techniques (MALDI-TOF/TOF), leading to the identification of 115 proteins. Of these, 24 proteins showed differential expression between the two BCG strains. Furthermore, 27 proteins previously described as displaying moonlighting function were identified, 8 of these proteins showed variation in abundance comparing BCG Moreau to Pasteur and 2 of them presented two different domain hits. Moonlighting proteins are multifunctional proteins in which two or more biological functions are fulfilled by a single polypeptide chain. Therefore, the identification of such proteins with moonlighting predicted functions can contribute to a better understanding of the molecular mechanisms unleashed by live BCG Moreau RDJ vaccine components

Proteomic Analysis of HCC-1954 and MCF-7 Cell Lines Highlights Crosstalk between αv and β1 Integrins, E-Cadherin and HER-2

Overexpression of human epidermal growth factor receptor-2 (HER-2) occurs in 20% of all breast cancer subtypes, especially those that present the worst prognostic outcome through a very invasive and aggressive tumour. HCC-1954 (HER-2+) is a highly invasive, metastatic cell line, whereas MCF-7 is mildly aggressive and non-invasive. We investigated membrane proteins from both cell lines that could have a pivotal biological significance in metastasis. Membrane protein enrichment for HCC-1954 and MCF-7 proteomic analysis was performed. The samples were analysed and quantified by mass spectrometry. High abundance membrane proteins were confirmed by Western blot, immunofluorescence, and flow cytometry. Protein interaction prediction and correlations with the Cancer Genome Atlas (TCGA) patient data were conducted by bioinformatic analysis. In addition, β1 integrin expression was analysed by Western blot in cells upon trastuzumab treatment. The comparison between HCC-1954 and MCF-7 membrane-enriched proteins revealed that proteins involved in cytoskeleton organisation, such as HER-2, αv and β1 integrins, E-cadherin, and CD166 were more abundant in HCC-1954. β1 integrin membrane expression was higher in the HCC-1954 cell line resistant after trastuzumab treatment. TCGA data analysis showed a trend toward a positive correlation between HER-2 and β1 integrin in HER-2+ breast cancer patients. Differences in protein profile and abundance reflected distinctive capabilities for aggressiveness and invasiveness between HCC-1954 and MCF-7 cell line phenotypes. The higher membrane β1 integrin expression after trastuzumab treatment in the HCC-1954 cell line emphasised the need for investigating the contribution of β1 integrin modulation and its effect on the mechanism of trastuzumab resistance

Unveiling the Trypanosoma cruzi Nuclear Proteome

Submitted by sandra infurna ([email protected]) on 2015-11-18T15:21:01Z No. of bitstreams: 1 dario_kalume_etal_IOC_2015.pdf: 1125751 bytes, checksum: 42d50fb57df989c4f31cc780e6cb1fc5 (MD5)Approved for entry into archive by sandra infurna ([email protected]) on 2015-11-18T15:30:32Z (GMT) No. of bitstreams: 1 dario_kalume_etal_IOC_2015.pdf: 1125751 bytes, checksum: 42d50fb57df989c4f31cc780e6cb1fc5 (MD5)Made available in DSpace on 2015-11-18T15:30:32Z (GMT). No. of bitstreams: 1 dario_kalume_etal_IOC_2015.pdf: 1125751 bytes, checksum: 42d50fb57df989c4f31cc780e6cb1fc5 (MD5) Previous issue date: 2015University of Brasilia. Institute of Biology. Department of Cell Biology. Campus Darcy Ribeiro, Asa Norte, Brasília, DF, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório Interdisciplinas de Pesquisas Médicas. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Imunofarmacologia. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Centro de Desenvolvimento Tecnológico em Saúde. Rio de Janeiro, RJ, Brasil.University of Brasilia. Institute of Biology. Department of Cell Biology. Campus Darcy Ribeiro, Asa Norte, Brasília, DF, Brasil.University of Brasilia. Institute of Biology. Department of Cell Biology. Campus Darcy Ribeiro, Asa Norte, Brasília, DF, Brasil.University of Brasilia. Institute of Biology. Department of Cell Biology. Campus Darcy Ribeiro, Asa Norte, Brasília, DF, Brasil.University of Brasilia. Institute of Biology. Department of Cell Biology. Campus Darcy Ribeiro, Asa Norte, Brasília, DF, Brasil.University of Brasilia. Institute of Biology. Department of Cell Biology. Campus Darcy Ribeiro, Asa Norte, Brasília, DF, Brasil.University of Brasilia. Institute of Biology. Department of Cell Biology. Campus Darcy Ribeiro, Asa Norte, Brasília, DF, Brasil.University of Brasilia. Institute of Biology. Department of Cell Biology. Campus Darcy Ribeiro, Asa Norte, Brasília, DF, Brasil.Replication of Trypanosoma cruzi, the etiological agent of Chagas disease, displays peculiar features, such as absence of chromosome condensation and closed mitosis. Although previous proteome and subproteome analyses of T. cruzi have been carried out, the nuclear subproteome of this protozoan has not been described. Here, we report, for the first time to the best of our knowledge, the isolation and proteome analysis of T. cruzi nuclear fraction. For that, T. cruzi epimastigote cells were lysed and subjected to cell fractionation using two steps of sucrose density gradient centrifugation. The purity of the nuclear fraction was confirmed by phase contrast and fluorescence microscopy. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) allowed the identification of 864 proteins. Among those, 272 proteins were annotated as putative uncharacterized, and 275 had not been previously reported on global T. cruzi proteome analysis. Additionally, to support our enrichment method, bioinformatics analysis in DAVID was carried out. It grouped the nuclear proteins in 65 gene clusters, wherein the clusters with the highest enrichment scores harbor members with chromatin organization and DNA binding functions

<i>T</i>. <i>cruzi</i> epimastigote subcellular fractionation.

<p>Sucrose gradient steps and isolated fractions are indicated.</p

SDS-PAGE of <i>T</i>. <i>cruzi</i> subcellular fractions.

<p>Epimastigote cells were lysed and subjected to cell fractionation as described in Material and Methods. Cytosolic, kinetoplastic and nuclear samples (30μg/lane) were subjected to 13% T SDS-PAGE. The gel was stained with Coomassie Brilliant Blue.</p

Phase contrast and fluorescence microscopy analysis of <i>T</i>. <i>cruzi</i> nuclear fraction.

<p>A—Phase contrast, B—DAPI staining, C—Merge. The round structures correspond to nuclei.</p

The cluster of most enriched uncharacterized putative proteins by DAVID bioinformatics resources.

<p>The cluster of most enriched uncharacterized putative proteins by DAVID bioinformatics resources.</p