Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 2 de 2
Filter
Add more filters










Database
Language
Publication year range
1.
Methods Mol Biol ; 2116: 139-159, 2020.
Article in English | MEDLINE | ID: mdl-32221920

ABSTRACT

Trypanosoma cruzi is a pathogenic protozoan that still has an impact on public health, despite the decrease in the number of infection cases along the years. T. cruzi possesses an heteroxenic life cycle in which it differentiates in at least four forms. Among the differentiation processes, metacyclogenesis has been exploited in different views by researchers. An intriguing question that rises is how metacyclogenesis is triggered and controlled by cell signaling and which are the differentially expressed proteins and posttranslational modifications involved in this process. An important cell signaling pathway is the protein phosphorylation, and it is reinforced in T. cruzi in which the gene expression control occurs almost exclusively posttranscriptionally. Additionally, the number of protein kinases in T. cruzi is relatively high compared to other organisms. A way to approach these questions is evaluating the cells through phosphoproteomics and proteomics. In this chapter, we will describe the steps from the cell protein extraction, digestion and fractionation, phosphopeptide enrichment, to LC-MS/MS analysis as well as a brief overview on peptide identification. In addition, a published method for in vitro metacyclogenesis will be detailed.


Subject(s)
Phosphoproteins/analysis , Proteomics/methods , Protozoan Proteins/analysis , Trypanosoma cruzi/physiology , Chromatography, Liquid/methods , Gene Expression Regulation, Developmental , Life Cycle Stages/genetics , Parasitology/methods , Phosphoproteins/metabolism , Phosphorylation/physiology , Protozoan Proteins/metabolism , Tandem Mass Spectrometry/methods
2.
Cell Stress Chaperones ; 24(5): 927-936, 2019 09.
Article in English | MEDLINE | ID: mdl-31368045

ABSTRACT

Phosphorylation is an important event in cell signaling that is modulated by kinases and phosphatases. In Trypanosoma cruzi, the etiological agent of Chagas disease, approximately 2% of the protein-coding genes encode for protein kinases. This parasite has a heteroxenic life cycle with four different development stages. In the midgut of invertebrate vector, epimastigotes differentiate into metacyclic trypomastigotes in a process known as metacyclogenesis. This process can be reproduced in vitro by submitting parasites to nutritional stress (NS). Aiming to contribute to the elucidation of mechanisms that trigger metacyclogenesis, we applied super-SILAC (super-stable isotope labeling by amino acids in cell culture) and LC-MS/MS to analyze different points during NS. This analysis resulted in the identification of 4205 protein groups and 3643 phosphopeptides with the location of 4846 phosphorylation sites. Several phosphosites were considered modulated along NS and are present in proteins associated with various functions, such as fatty acid synthesis and the regulation of protein expression, reinforcing the importance of phosphorylation and signaling events to the parasite. These modulated sites may be triggers of metacyclogenesis.


Subject(s)
Chagas Disease/parasitology , Life Cycle Stages/physiology , Proteome/metabolism , Proteomics/methods , Protozoan Proteins/metabolism , Trypanosoma cruzi/metabolism , Animals , Phosphorylation
SELECTION OF CITATIONS
SEARCH DETAIL