Periodic Reporting for period 1 - PHEMDD (Phosphate homeostasis and energy metabolism in Dictyostelium discoideum)
Reporting period: 2017-07-01 to 2019-06-30
In all living systems, homeostasis maintains constancy despite fluctuating environmental conditions. Inorganic phosphate (Pi) is an essential component of life for which the cellular concentration must be tightly controlled. Pi is incorporated in nucleic acids, proteins, lipids, and sugars, serving structural and signaling functions. It is also involved in energy metabolism since the high-energy bonds present in ATP, the main energy currency in the cell, are phosphoanhydride (Pi-Pi) bonds. Alterations in Pi homeostasis or in the levels of Pi storage form (polyphosphate) is linked to many pathological states including myopathy, cardiac dysfunction, platelet dysfunction, hyperparathyroidism, obesity, tumour formation, and cancer. There must then be a system that controls Pi homeostasis in concert with energy metabolism. One excellent candidate to act in such a system is the inositol pyrophosphates (PP-IPs). These are ubiquitously distributed highly phosphorylated molecules that have been described as metabolic messengers, being involved in many processes including cell signalling, gene transcription, growth, proliferation, and regulation of metabolic homeostasis. The purpose of this study is to understand Pi homeostasis by elucidating the relationships between Pi homeostasis, energy metabolism, and PP-IPs, using the social amoeba Dictyostelium discoideum as a model system.
We have uncovered the enzymology behind an alternative “soluble” route to synthesis of inositol phosphates. ITPK1, initially described as an IP3/4 kinase, phosphorylates glucose-6-phosphate derived IP1 to produces higher inositol phosphates. This pathway is totally independent from the synthesis of IP3 by phospholipase C. The elucidation of this pathway was made possible using the polyacrylamide gel electrophoresis analysis of inositol polyphosphates. This technique also allowed us to demonstrate that the levels of inositol polyphosphates is tightly regulated by the metabolic state of the cells. For instance, ATP depletion induced by Pi starvation induces a dramatic ITPK1-dependent increase in inositol polyphosphates. ITPK1 is present not only in animals, plants, and social amoebae, but also in archaeal clades thought to define eukaryogenesis (Asgard), indicating that inositol phosphates had functional roles before the appearance of the eukaryote.
We also have investigated the regulation of inorganic polyphosphate synthesis and the mechanism of its secretion in D. discoideum. We have developed an assay to induce synthesis and secretion on a short time-scale, which allowed us to decipher these biological processes using genetic and pharmacological tools. Our data show that polyphosphate synthesis is induced by high cell density. Moreover, the secretion is a vesicle-mediated process suggesting that the polymer is first translocated across a biological membrane to the interior of an unidentified organelle that by fusing with the plasma membrane release polyphosphate into the environment. In addition, our data demonstrate that, in D. discoideum, inositol polyphosphates do not control directly polyphosphate synthesis.
We also have investigated the regulation of inorganic polyphosphate synthesis and the mechanism of its secretion in D. discoideum. We have developed an assay to induce synthesis and secretion on a short time-scale, which allowed us to decipher these biological processes using genetic and pharmacological tools. Our data show that polyphosphate synthesis is induced by high cell density. Moreover, the secretion is a vesicle-mediated process suggesting that the polymer is first translocated across a biological membrane to the interior of an unidentified organelle that by fusing with the plasma membrane release polyphosphate into the environment. In addition, our data demonstrate that, in D. discoideum, inositol polyphosphates do not control directly polyphosphate synthesis.
This work has redefined the biosynthetic pathway of inositol polyphosphates synthesis in eukaryotes established 30 years ago. It has also strengthened the link between basic metabolism and inositol polyphosphates showing that the glycolytic intermediate glucose-6-phosphate represent, when metabolism slowdown, the main resource to produce inositol phosphates. It is now important to identify how this pathway is regulated under normal conditions and how inositol phosphates synthesis is affected in metabolic disorders.
Although inorganic polyphosphate is present in and secreted by mammalian cells, the enzyme responsible for its synthesis is unknown. We believe that understanding how the polymer is exported to the extracellular medium and how its synthesis is regulated using D. discoideum as a model organism will provide the information needed to identify the mammalian enzyme.
Although inorganic polyphosphate is present in and secreted by mammalian cells, the enzyme responsible for its synthesis is unknown. We believe that understanding how the polymer is exported to the extracellular medium and how its synthesis is regulated using D. discoideum as a model organism will provide the information needed to identify the mammalian enzyme.