Investigation of the possible role of inositol pyrophosphates in DNA repair pathways in Trypanosoma cruzi, the etiologic agent of Chagas Disease

• Grant number: 23/00253-8
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: September 01, 2023
• End date: August 31, 2027
• Field of knowledge: Biological Sciences - Parasitology - Protozoology of Parasites
• Principal Investigator: Marcelo Santos da Silva
• Grantee: Bryan Etindi Abuchery
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 19/10753-2 - Investigation on the role of inositol pyrophosphates (PP-IPs) in DNA repair pathways and telomere dynamics using trypanosomatids as a model, AP.JP

Abstract

Inositol pyrophosphates (PP-IPs) - mainly IP7, and IP8 - are involved in a wide range of cellular processes in eukaryotes, such as apoptosis, cell cycle dynamics, regulation of telomere length, homologous recombination, among others. However, the precise mechanism of action of PP-IPs in pathways related to DNA metabolism is not fully understood. IP7 and IP8 are synthesized by pathways involving the participation of IP6K and PP-IP5K kinases, respectively. Trypanosomatids have an ortholog gene for IP6K, but apparently do not have orthologs for PP-IP5K, which makes them excellent models for the study of PP-IPs. Trypanosoma cruzi (Trypanosomatidae family) is a single-celled eukaryotic parasite and the etiologic agent of Chagas disease, a neglected tropical disease that affects millions of people worldwide. Thus, the main goal of this project is to investigate the possible role of PP-IPs in the main DNA repair pathways (homologous recombination repair - HR, base excision repair - BER, and nucleotide excision repair - NER) using T. cruzi as a model and aiming to identify key pathways that can be used for species-specific interventions. For this, lineages from T. cruzi (CL Brener) knockout for IP6K (loss of function) and overexpressing IP6K (gain of function) will be generated using the CRISPR-Cas9 approach. After confirming the decrease or increase of IP7 in the corresponding lineages, they will be challenged with different genotoxic agents (ionizing radiation, hydrogen peroxide, and UV light), and the DNA repair capacity will be evaluated using classical (fluorescence microscopy, western blot using antibodies against DNA repair players, comet, TUNEL, flow cytometry, growth curves, etc) and innovative approaches. For instance, we intend to adapt and apply a transfection-based method to evaluate if T. cruzi lineages will be able to repair damage localized to exogenous DNA (plasmid). Furthermore, using a bold approach, IP7 molecules will be synthesized in vitro with the ²-phosphate moiety labeled. These molecules will be used in pyrophosphorylation reactions with the objective of tracking their main targets using pull-down and mass spectrometry. After identifying these targets, in vivo and in silico studies will be carried out to investigate the role played by pyrophosphorylation in these players. In summary, this project will contribute significantly to a better understanding of the pyrophosphorylation during DNA repair, a non-enzymatic post-translational modification still little understood. (AU)