Conversion of dietary inositol into propionate and acetate by commensal Anaerostipes associates with host health.

We describe the anaerobic conversion of inositol stereoisomers to propionate and acetate by the abundant intestinal genus Anaerostipes. A inositol pathway was elucidated by nuclear magnetic resonance using [13C]-inositols, mass spectrometry and proteogenomic analyses in A. rhamnosivorans, identifying 3-oxoacid CoA transferase as a key enzyme involved in both 3-oxopropionyl-CoA and propionate formation. This pathway also allowed conversion of phytate-derived inositol into propionate as shown with [13C]-phytate in fecal samples amended with A. rhamnosivorans. Metabolic and (meta)genomic analyses explained the adaptation of Anaerostipes spp. to inositol-containing substrates and identified a propionate-production gene cluster to be inversely associated with metabolic biomarkers in (pre)diabetes cohorts. Co-administration of myo-inositol with live A. rhamnosivorans in western-diet fed mice reduced fasting-glucose levels comparing to heat-killed A. rhamnosivorans after 6-weeks treatment. Altogether, these data suggest a potential beneficial role for intestinal Anaerostipes spp. in promoting host health.

Resolving the Communication GAPs Upstream of TORC1.

In this issue of Developmental Cell, Yang et al. (2020) report that both nutrient- and growth factor-dependent signaling impinge upon the RAG GTPases which in turn control TSC residency time on the lysosome membrane and ultimately mTORC1 activity.

Analysis of metabolically labeled inositol phosphate messengers by NMR.

Inositol phosphates (InsPs) are an important group of eukaryotic messengers and mediate a wide range of processes. To elucidate the biological functions of these molecules, robust techniques to characterize inositol phosphate metabolism at the cellular level are highly sought after. This chapter provides a detailed protocol for the preparation of 13C-labeled myo-inositol, its use for metabolic labeling of mammalian and yeast cells, and the quantitative analysis of intracellular InsP pools from cell extracts using NMR spectroscopy.

Delivery of myo-Inositol Hexakisphosphate to the Cell Nucleus with a Proline-Based Cell-Penetrating Peptide.

Inositol hexakisphosphate (InsP6 ) is a central member of the inositol phosphate messengers in eukaryotic cells. Tools to manipulate the level of InsP6 , particularly with compartment selectivity, are needed to enable functional cellular studies. We present cationic octa-(4S)guanidiniumproline (Z8) for the delivery of InsP6 into the cell nucleus. CD spectroscopy, binding affinity, dynamic light scattering, and computational studies revealed that Z8 binds tightly to InsP6 and upon binding undergoes a conformational change from a PPII-helical structure to a structure that forms aggregates. The unique conformational features of the cationic oligoproline enable complex formation and cellular delivery of InsP6 with considerably greater efficacy than the flexible counterpart octaarginine.

Scalable Chemoenzymatic Synthesis of Inositol Pyrophosphates.

The inositol pyrophosphates (PP-InsPs) are an important group of cellular messengers that influence a broad range of biological processes. To elucidate the functions of these high-energy metabolites at the biochemical level, access to the purified molecules is required. Here, a robust and scalable strategy for the synthesis of various PP-InsPs [5PP-InsP5, 1PP-InsP5, and 1,5(PP)2-InsP4] is reported, relying on the highly active inositol hexakisphosphate kinase A from Entamoeba histolytica and the kinase domain of human diphosphoinositol pentakisphosphate kinase 2. A facile purification procedure using precipitation with Mg2+ ions and an optional strong anion exchange chromatography on an FPLC system afforded PP-InsPs in high purity. Furthermore, the newly developed protocol could be applied to simplify the synthesis of radiolabeled 5PP-InsP5-ß32P, which is a valuable tool for studying protein pyrophosphorylation. The chemoenzymatic method for obtaining PP-InsPs is readily amenable to both chemists and biologists and will thus foster future research on the multiple signaling functions of PP-InsP molecules.

Two bifunctional inositol pyrophosphate kinases/phosphatases control plant phosphate homeostasis.

Many eukaryotic proteins regulating phosphate (Pi) homeostasis contain SPX domains that are receptors for inositol pyrophosphates (PP-InsP), suggesting that PP-InsPs may regulate Pi homeostasis. Here we report that deletion of two diphosphoinositol pentakisphosphate kinases VIH1/2 impairs plant growth and leads to constitutive Pi starvation responses. Deletion of phosphate starvation response transcription factors partially rescues vih1 vih2 mutant phenotypes, placing diphosphoinositol pentakisphosphate kinases in plant Pi signal transduction cascades. VIH1/2 are bifunctional enzymes able to generate and break-down PP-InsPs. Mutations in the kinase active site lead to increased Pi levels and constitutive Pi starvation responses. ATP levels change significantly in different Pi growth conditions. ATP-Mg2+ concentrations shift the relative kinase and phosphatase activities of diphosphoinositol pentakisphosphate kinases in vitro. Pi inhibits the phosphatase activity of the enzyme. Thus, VIH1 and VIH2 relay changes in cellular ATP and Pi concentrations to changes in PP-InsP levels, allowing plants to maintain sufficient Pi levels.

Harnessing 13C-labeled myo-inositol to interrogate inositol phosphate messengers by NMR.

Inositol poly- and pyrophosphates (InsPs and PP-InsPs) are an important group of metabolites and mediate a wide range of processes in eukaryotic cells. To elucidate the functions of these molecules, robust techniques for the characterization of inositol phosphate metabolism are required, both at the biochemical and the cellular level. Here, a new tool-set is reported, which employs uniformly 13C-labeled compounds ([13C6]myo-inositol, [13C6]InsP5, [13C6]InsP6, and [13C6]5PP-InsP5), in combination with commonly accessible NMR technology. This approach permitted the detection and quantification of InsPs and PP-InsPs within complex mixtures and at physiological concentrations. Specifically, the enzymatic activity of IP6K1 could be monitored in vitro in real time. Metabolic labeling of mammalian cells with [13C6]myo-inositol enabled the analysis of cellular pools of InsPs and PP-InsPs, and uncovered high concentrations of 5PP-InsP5 in HCT116 cells, especially in response to genetic and pharmacological perturbation. The reported method greatly facilitates the analysis of this otherwise spectroscopically silent group of molecules, and holds great promise to comprehensively analyze inositol-based signaling molecules under normal and pathological conditions.