Investigation on the role of inositol pyrophosphates (PP-IPs) in DNA repair pathways and telomere dynamics using trypanosomatids as a model

Abstract

Inositol pyrophosphates (PP-IPs) are a specialized group of soluble molecules that have one or two pyrophosphate groups attached to the inositol ring. In model eukaryotes, the best-characterized PP-IPs are PP-IP4, IP7, and IP8. These molecules are involved in several cellular processes, including pathways related to DNA metabolism, such as homologous recombination (HR) and telomere length control. However, the action mechanism of PP-IPs in these pathways are not fully understood. PP-IP4, IP7, and IP8 are synthesized by complementary pathways involving the participation of IP6K and PP-IP5K kinases. Trypanosomatid parasites (e.g., T. cruzi and Leishmania spp.) have orthologous genes for IP6K, but do not have orthologs for PP-IP5K, which prevent the IP8 synthesis. This peculiarity makes them excellent models for the study of PP-IPs, especially IP7 and PP-IP4. Moreover, trypanosomatids apparently do not possess the DNA repair pathway called non-homologous end joining (NHEJ), and they have a divergent and still poorly understood telomeric dynamics. Thus, this project aims to investigate the action mechanisms of PP-IPs in DNA repair and telomeric maintenance using trypanosomatids as a model. Hence, some crucial issues will be addressed: What are the DNA metabolism pathways of and how critical is the participation of IP7 and PP-IP4 in these pathways? What are the main protein-targets of IP7 and PP-IP4 involved in these processes? The possible participation of IP7 or PP-IP4 in the telomeric dynamics could be a consequence of the involvement of these PP-IPs in HR during the alternative lengthening of telomeres (ALT)? To address the main objective and answer these questions, L. braziliensis and T. cruzi strains that do not synthesize IP7 will be generated by knockout (KO) of IP6K using CRISPR-Cas9 approach. These lineages will then be challenged with specific genotoxic agents and their DNA repair capacity and telomeric dynamics will be analyzed. Concomitantly, using the same approach, telomerase-KO and double-KO lineages (IP6K-KO and telomerase-KO) will be generated to investigate whether the possible involvement of IP7 with telomeres would be via ALT or telomerase since the ALT mechanism is similar to HR. For this purpose, the telomere lengthening recovery (after shortening caused by hydrogen peroxide) will be analyzed by flow-fish and single telomere extension (STEX) assay. Finally, using a bold approach, IP7 and PP-IP4 molecules will be synthesized in vitro with the beta-phosphate moiety labeled. These molecules will be used in pyrophosphorylation reactions with protein extracts, where it is expected that there will be hydrolysis of the pyrophosphate portion, and non-enzymatic transfer of the beta-phosphate group to target proteins. The tracking of the target proteins will be carried out by mass spectrometry after pull-down assays. Assays to investigate the chemical/structural function of pyrophosphates in the target proteins will also be performed (e.g., molecular docking, SAXS and cryo-MS). Thus, in addition to broadening our understanding of the role of PP-IPs in crucial pathways of DNA metabolism, the comparative analysis of this project will provide insights into how the essentiality of these molecules has diversified throughout evolution. Pyrophosphorylation by PP-IPs are not enzymatic and represent a new and poor-understood form of post-translational modification. It is noteworthy that as IP7 is only synthesized by IP6K and kinases are excellent druggable targets, this project may provide a new route for the establishment of effective chemotherapies, not only against trypanosomatid parasites (etiological agents of several neglected diseases), but also against any cells whose short-term DNA repair and telomere elongation are essential for proliferation, such as malignant cells. (AU)