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Proteomic analysis of detergent-solubilized membrane proteins from insect-developmental forms of Trypanosoma cruzi.

ABSTRACT: The cell surface of Trypanosoma cruzi, the etiologic agent of Chagas disease, is covered by a dense layer of glycosylphosphatidylinositol (GPI)-anchored molecules. These molecules are involved in a variety of interactions between this parasite and its mammalian and insect hosts. Here, using the neutral detergent Triton X-114, we obtained fractions rich in GPI-anchored and other membrane proteins from insect developmental stages of T. cruzi. These fractions were analyzed by two-dimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC-MS/MS), resulting in the identification of 98 proteins of metacyclic trypomastigotes and 280 of epimastigotes. Of those, approximately 65% (n=245) had predicted lipid post-translational modification sites (i.e., GPI-anchor, myristoylation, or prenylation), signal-anchor sequence, or transmembrane domains that could explain their solubility in detergent solution. The identification of some of these modified proteins was also validated by immunoblotting. We also present evidence that, in contrast to the noninfective proliferative epimastigote forms, the infective nonproliferative metacyclic trypomastigote forms express a large repertoire of surface glycoproteins, such as GP90 and GP82, which are involved in adhesion and invasion of host cells. Taken together, our results unequivocally show stage-specific protein profiles that appear to be related to the biology of each T. cruzi insect-derived developmental form.

SUBMITTER: Cordero EM

PROVIDER: S-EPMC2752740 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Proteomic analysis of detergent-solubilized membrane proteins from insect-developmental forms of Trypanosoma cruzi.

Cordero Esteban M EM Nakayasu Ernesto S ES Gentil Luciana G LG Yoshida Nobuko N Almeida Igor C IC da Silveira José Franco JF

Journal of proteome research 20090701 7

The cell surface of Trypanosoma cruzi, the etiologic agent of Chagas disease, is covered by a dense layer of glycosylphosphatidylinositol (GPI)-anchored molecules. These molecules are involved in a variety of interactions between this parasite and its mammalian and insect hosts. Here, using the neutral detergent Triton X-114, we obtained fractions rich in GPI-anchored and other membrane proteins from insect developmental stages of T. cruzi. These fractions were analyzed by two-dimensional liquid ...[more]

PMID: 19374451

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Project description:Chromatin associated proteins are key regulators of many important processes in the cell. Trypanosoma cruzi, a protozoa flagellate that causes Chagas disease, alternates between replicative and nonreplicative forms accompanied by a shift on global transcription levels and by changes in its chromatin architecture. Here, we investigated the T. cruzi chromatin proteome using three different protocols and compared it between replicative (epimastigote) and nonreplicative (trypomastigote) forms by high-resolution mass spectrometry. More than 2000 proteins were identified and quantified both in chromatin and nonchromatin extracts. Besides histones and other known nuclear proteins, trypanosomes chromatin also contains metabolic (mainly from carbohydrate pathway), cytoskeleton and many other proteins with unknown functions. Strikingly, the two parasite forms differ greatly regarding their chromatin-associated factors composition and amount. Although the nucleosome content is the same for both life forms (as seen by MNase digestion), the remaining proteins were much less detected in nonreplicative forms, suggesting that they have a naked chromatin. Proteins associated to DNA proliferation, such as PCNA, RPA, and DNA topoisomerases were exclusively found in the chromatin of replicative stages. On the other hand, the nonreplicative stages have an enrichment of a histone H2B variant. Furthermore, almost 20% of replicative stages chromatin-associated proteins are expressed in nonreplicative forms, but located at nonchromatin space. We identified different classes of proteins including phosphatases and a Ran-binding protein, that may shuttle between chromatin and nonchromatin space during differentiation. Seven proteins, including those with unknown functions, were selected for further validation. We confirmed their location in chromatin and their differential expression, using Western blotting assays and chromatin immunoprecipitation (ChIP). Our results indicate that the replicative state in trypanosomes involves an increase of chromatin associated proteins content. We discuss in details, the qualitative and quantitative implication of this chromatin set in trypanosome chromatin biology. Because trypanosomes are early-branching organisms, this data can boost our understanding of chromatin-associated processes in other cell types.

Dataset Information

Proteomic analysis of detergent-solubilized membrane proteins from insect-developmental forms of Trypanosoma cruzi.

Publications

Proteomic analysis of detergent-solubilized membrane proteins from insect-developmental forms of Trypanosoma cruzi.

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