Bertolini Mayara S, Cline Sabrina E, Chiurillo Miguel A, Mantilla Brian S, Eidex Aharon, Crowe Logan P, Qiu Danye, Jessen Henning J, Saiardi Adolfo, Docampo Roberto
Department of Cellular Biology, Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia, USA.
Department of Biological Sciences, University of Cincinnati, Cincinnati, Ohio, USA.
mBio. 2025 May 14;16(5):e0331824. doi: 10.1128/mbio.03318-24. Epub 2025 Apr 2.
Inositol phosphates are involved in a myriad of biological roles and activities such as Ca signaling, phosphate homeostasis, energy metabolism, and disease pathogenicity. In synthesis of inositol phosphates occurs through the phosphoinositide phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP) into inositol 1,4,5-trisphosphate (IP) and diacylglycerol and further IP phosphorylation by additional kinases that leads to the formation of highly phosphorylated inositol derivatives, known as inositol pyrophosphates. Inositol-tetrakisphosphate 1-kinase (ITPK1) is an enzyme that mediates a PLC-independent inositol polyphosphate synthesis through phosphorylation of inositol monophosphates and other intermediates in the cytosol. In this work, we identified and characterized a ITPK1 (TcITPK1) homolog. The ability of TcITPK1 to act as the mediator for this alternative pathway was established through Δ and Δ Δ yeast complementation assays and SAX-HPLC analyses of radioactively labeled inositol. TcITPK1 localizes to the cytosol, and knockout attempts of revealed that only one allele was replaced by the DNA donor cassette at the specific locus, suggesting that alleles may have lethal effects in epimastigotes. Ablation of phosphoinositide phospholipase C 1 () affected the synthesis of IP from glucose 6-phosphate but did not affect the synthesis of inositol polyphosphates, while ablation of inositol phosphosphingolipid phospholipase () affected the synthesis of inositol polyphosphates, thus revealing that the PLC-independent pathway using either glucose 6-phosphate or inositol phosphoceramide is involved in the synthesis of inositol polyphosphates, while the PLC-dependent pathway is involved in IP formation needed for Ca signaling.
Millions of people are infected with and the current treatment is not satisfactory. Inositol pyrophosphates have been established as important signaling molecules. Our work demonstrates the presence of a phospholipase C-independent pathway for the synthesis of inositol pyrophosphates in . Furthermore, we demonstrate that this pathway starts with the synthesis of inositol monophosphates from glucose 6-phosphate or from inositol phosphoceramide, linking it to carbohydrate and sphingolipid metabolism. The essentiality of the pathway for the survival of infective stages makes it an ideal drug target for treating American trypanosomiasis.
肌醇磷酸参与众多生物学作用和活动，如钙信号传导、磷酸盐稳态、能量代谢和疾病致病性。肌醇磷酸的合成通过磷脂酰肌醇特异性磷脂酶C（PLC）催化磷脂酰肌醇4,5 - 二磷酸（PIP）水解为肌醇1,4,5 - 三磷酸（IP）和二酰基甘油，以及进一步由其他激酶对IP进行磷酸化，从而导致形成高度磷酸化的肌醇衍生物，即肌醇焦磷酸。肌醇四磷酸1 - 激酶（ITPK1）是一种酶，它通过在细胞质中对肌醇单磷酸和其他中间体进行磷酸化来介导不依赖PLC的肌醇多磷酸合成。在这项工作中，我们鉴定并表征了一种ITPK1（TcITPK1）同源物。通过Δ和ΔΔ酵母互补试验以及放射性标记肌醇的SAX - HPLC分析，确定了TcITPK1作为该替代途径介导者的能力。TcITPK1定位于细胞质，对其进行敲除尝试发现只有一个等位基因在特定位点被DNA供体盒取代，这表明等位基因可能对前鞭毛体具有致死作用。敲除磷脂酰肌醇特异性磷脂酶C 1（）影响了从6 - 磷酸葡萄糖合成IP，但不影响肌醇多磷酸的合成，而敲除肌醇磷酸鞘脂磷脂酶（）影响了肌醇多磷酸的合成，从而揭示了使用6 - 磷酸葡萄糖或肌醇磷酸神经酰胺的不依赖PLC的途径参与肌醇多磷酸的合成，而依赖PLC的途径参与钙信号传导所需的IP形成。
数以百万计的人感染了，目前的治疗并不令人满意。肌醇焦磷酸已被确立为重要的信号分子。我们的工作证明了在中存在一条不依赖磷脂酶C的肌醇焦磷酸合成途径。此外，我们证明该途径始于从6 - 磷酸葡萄糖或肌醇磷酸神经酰胺合成肌醇单磷酸，将其与碳水化合物和鞘脂代谢联系起来。该途径对于感染阶段生存的必要性使其成为治疗美洲锥虫病的理想药物靶点。
