Identification of the target-proteins of the inositol pyrophosphates PP-IP4 and IP7 in Leishmania braziliensis

Abstract

In model eukaryotes, inositol pyrophosphates (PP-IPs) - mainly PP-IP4, IP7 and IP8 - are involved in a wide range of processes, such as regulation of telomere length, and homologous recombination (HR). However, the PP-IPs target-proteins, as well as their action mechanisms remains unknown. PP-IP4, IP7 and IP8 are synthesized by pathways involving the participation of IP6K and PP-IP5K kinases. Trypanosomatids has an ortholog gene for IP6K, but apparently does not have orthologs for PP-IP5K, i.e., they do not synthesize IP8, which make them excellent models for the study of PP-IP4 e IP7. Thus, this MSc research plan consists of amplifying, cloning, expressing and purifying recombinant IP6K from L. braziliensis in order to use it to synthesize in vitro PP-IP4- and IP7-labeled and then use it to track target-proteins. Briefly, the recombinant IP6Kwill be used to generates PP-IP4- and IP7-labeled from, respectively, I(3,4,5,6)P5 and IP6 isolated from L. braziliensis and a commercial kit containing ATP labeled (³-[(Propargyl)-imido]-ATP). As a control, using the same approach, IP5K recombinant will also be generated and then used to synthesize IP6-labeled (which is a non-pyrophosphate) from I(3,4,5,6)P5 isolated, using the same commercial kit containing ³-[(Propargyl)-imido]-ATP. After obtaining these molecules, L. braziliensis extracts will be used to perform in vitro pyrophosphorylation reactions, where it is expected that only there will be the hydrolysis of the pyrophosphate portion from PP-IP4- and IP7-labeled, and non-enzymatic transfer of the ²-phosphate groups to the target-proteins. Next, the target proteins pyrophosphorilated will be able to react with azide couped to biotin, allowing their capture by pull-down using streptavidin beads. The proteins captured will be identified by mass spectrometry (LC-MS/MS). It is worth emphasizing that this approach will be of fundamental importance to answer the essential questions raised by the main project. (AU)