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Analysis of yeast two-hybrid system interactions of Dictyostelium discoideum type-1 protein phosphatase catalytic subunit (PP1c)

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Author(s):
Renato Astolfi Raposo
Total Authors: 1
Document type: Master's Dissertation
Press: São Paulo.
Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
Defense date:
Examining board members:
Aline Maria da Silva; Maria Celia Bertolini; Carla Columbano de Oliveira
Advisor: Aline Maria da Silva
Abstract

Protein phosphatase type-1 (PP1) is a major protein serine/threonine phosphatase (PSTP) which plays as diverse as important physiological roles, such as regulation of carbohydrate metabolism and of cell cycle. The PP1 holoenzyme comprises a conserved catalytic subunit (PP1c) associated with non-catalytic subunits that modulate its subcellular localization, substrate specificity and enzymatic activity. More than 100 proteins that interact with PP1c have been identified in different eukaryotic organisms. Therefore proteins that interact with PP1c are key to the understanding of PP1 different biological roles. The catalytic subunit of PP1 the social amoeba Dictyostelium discoideum (DdPP1c) is encoded by a single copy gene which is expressed throughout the life cycle of this organism. Some proteins that interact with and possibly modulate the activity of D. discoideum PP1 have been identified, using both similarity searches in the genome sequence of this microorganism as yeast two-hybrid screenings using PP1c as bait. With the latter approach, we have selected more than 25 distinct cDNA clones encoding proteins that potentially interact with DdPP1c after screening D. discoideum cDNA libraries from different developmental stages. In this study, we confirmed that the protein product from 11 of these clones interact with the bait DdPP1c based on two-hybrid assays. The other clones encode proteins that either does not interact or promote self-activation of the reporter gene. The clone related to DDB_G0269300 gene that encodes a predicted protein of 423 amino acids with unknown function was selected for further studies. DDB_G0269300 full-length coding sequence was cloned and new yeast two-hybrid assays were performed confirming the specificity of the interaction with DdPP1c. The recombinant protein rDDB_G0269300 was successfully obtained in bacteria and further used for polyclonal antibodies production in mice. The antiserum anti-rDDB_G0269300 is apparently specific for recognition of the corresponding protein in D. discoideum cell extracts collected after 12h and 16h of development. These results agree with RT-qPCR data showing that the levels of DDB_G0269300 transcripts are increased between 8 h and 12 h during the development, which is indicative of its importance during this phase in Dictyostelium life cycle as a DdPP1c potential molecular partner. (AU)