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Kinases that synthesize inositol phosphates (IPs) and pyrophosphates (PP-IPs) control numerous biological processes in eukaryotic cells. Herein, we extend this cellular signaling repertoire to viruses. We have biochemically and structurally characterized a minimalist inositol phosphate kinase (i.e., TvIPK) encoded by Terrestrivirus, a nucleocytoplasmic large ("giant") DNA virus (NCLDV). We show that TvIPK can synthesize inositol pyrophosphates from a range of scyllo- and myo-IPs, both in vitro and when expressed in yeast cells. We present multiple crystal structures of enzyme/substrate/nucleotide complexes with individual resolutions from 1.95 to 2.6 Å. We find a heart-shaped ligand binding pocket comprising an array of positively charged and flexible side chains, underlying the observed substrate diversity. A crucial arginine residue in a conserved "G-loop" orients the γ-phosphate of ATP to allow substrate pyrophosphorylation. We highlight additional conserved catalytic and architectural features in TvIPK, and support their importance through site-directed mutagenesis. We propose that NCLDV inositol phosphate kinases may have assisted evolution of inositol pyrophosphate signaling, and we discuss the potential biogeochemical significance of TvIPK in soil niches. Synopsis: Inositol phosphate kinases regulate many biological processes in eukaryotes, but recent discoveries of archaeal homologs suggest an ancient evolutionary origin. Here, characterization of a primordial ATP-dependent inositol pyrophosphate kinase encoded by the giant virus genome of Terrestrivirus provides insights into the evolution of inositol phosphate kinases. Terrestrivirus TvIPK specifically pyrophosphorylates a range of myo- and scyllo-based inositol polyphosphates both in vitro and when ectopically expressed in yeast. X-ray crystallographic analysis of TvIPK in complex with its substrates or products reveals conserved catalytic and architectural features. Positively charged and flexible side chains allow recognition of the diverse range of substrates. Pyrophosphorylation depends on ATP γ-phosphate orientation via an Arg residues in a conserved "G-loop." TvIPK, an ATP-dependent inositol pyrophosphate kinase from Terrestrivirus, a putative member of the Mimiviridae family of giant viruses, offers insights into the evolution of inositol phosphate signaling. [ABSTRACT FROM AUTHOR]

Inositol pyrophosphates (PP−IPs) are densely phosphorylated messenger molecules involved in numerous biological processes. PP−IPs contain one or two pyrophosphate group(s) attached to a phosphorylated myo‐inositol ring. 5PP−IP5 is the most abundant PP−IP in human cells. To investigate the function and regulation by PP−IPs in biological contexts, metabolically stable analogs have been developed. Here, we report the synthesis of a new fluorinated phosphoramidite reagent and its application for the synthesis of a difluoromethylene bisphosphonate analog of 5PP−IP5. Subsequently, the properties of all currently reported analogs were benchmarked using a number of biophysical and biochemical methods, including co‐crystallization, ITC, kinase activity assays and chromatography. Together, the results showcase how small structural alterations of the analogs can have notable effects on their properties in a biochemical setting and will guide in the choice of the most suitable analog(s) for future investigations. [ABSTRACT FROM AUTHOR]

Inositol phosphate signaling in plants is of substantial agricultural interest, with a considerable focus on the inositol tris/tetrakisphosphate kinase (ITPK) family of inositol phosphate kinases. Historically, the 4–6 isoforms of ITPKs that higher plants each express have been studied for their multiplexing a metabolic pathway to synthesize inositol hexakisphosphate (ie InsP6 or phytate), through the phosphorylation and dephosphorylation of multiple inositol phosphates, including Ins(1,3,4,5,6)P5 (inositol‐1,3,4,5,6‐pentakisphosphate). A more recent discovery is ITPK‐catalyzed phosphorylation of InsP6 to inositol pyrophosphates, which regulate plant immunity and phosphate homeostasis. However, a molecular‐based explanation for these alternate catalytic activities has been missing, because no plant ITPK structure has previously been solved. Herein, we provide biochemical and structural analyses of ITPKs from Zea mays and Glycine max. For this work we introduce a simple, enzyme‐coupled microplate‐based assay of InsP6 kinase activity that should promote more general access to this important field. Furthermore, a ZmITPK1/InsP6 crystal complex is described at a resolution of 2.6 Å, which identifies a number of catalytically important residues; their functionality is confirmed by mutagenesis. We further demonstrate that ZmITPK1 adds a β‐phosphate to the 3‐position of Ins(1,2,3,4,5)P5, yielding a candidate signal for regulating phosphate homeostasis. An impactful discovery is our description of a 29‐residue catalytic specificity element; by interchanging this element between GmITPK1 and GmITPK2, we demonstrate how its isoform‐specific sequence specifically determines whether the host protein phosphorylates InsP6, without substantially affecting Ins(1,3,4,5,6)P5 metabolism. Our structural rationalization of key catalytic differences between alternate ITPK isoforms will complement future research into their functional diversity. [ABSTRACT FROM AUTHOR]

Inositol pyrophosphates are small, diffusible signaling molecules that possess the most concentrated three-dimensional array of phosphate groups in Nature; up to eight phosphates are crammed around a six-carbon inositol ring. This review discusses the physico-chemical properties of these unique molecules, and their mechanisms of action. Also provided is information on the enzymes that regulate the levels and hence the signaling properties of these molecules. This review pursues the idea that many of the biological effects of inositol pyrophosphates can be rationalized by their actions at the interface of cell signaling and metabolism that is essential to cellular and organismal homeostasis. [ABSTRACT FROM AUTHOR]

The inositol pyrophosphate messengers (PP-InsPs) are emerging as an important class of cellular regulators. These molecules have been linked to numerous biological processes, including insulin secretion and cancer cell migration, but how they trigger such a wide range of cellular responses has remained unanswered in many cases. Here, we show that the PP-InsPs exhibit complex speciation behaviour and propose that a unique conformational switching mechanism could contribute to their multifunctional effects. We synthesised non-hydrolysable bisphosphonate analogues and crystallised the analogues in complex with mammalian PPIP5K2 kinase. Subsequently, the bisphosphonate analogues were used to investigate the protonation sequence, metal-coordination properties, and conformation in solution. Remarkably, the presence of potassium and magnesium ions enabled the analogues to adopt two different conformations near physiological pH. Understanding how the intrinsic chemical properties of the PP-InsPs can contribute to their complex signalling outputs will be essential to elucidate their regulatory functions. [ABSTRACT FROM AUTHOR]

The ubiquitous mammalian signaling molecule bis-diphosphoinositol tetrakisphosphate (1,5-(PP)2-myo-InsP4, or InsP8) displays the most congested three-dimensional array of phosphate groups found in nature. The high charge density, the accumulation of unstable P-anhydrides and P-esters, the lack of UV absorbance, and low levels of optical rotation constitute severe obstacles to its synthesis, characterization, and purification. Herein, we describe the first procedure for the synthesis of enantiopure 1,5-(PP)2-myo-InsP4 and 3,5-(PP)2-myo-InsP4 utilizing a C2-symmetric P-amidite for desymmetrization and concomitant phosphitylation followed by a one-pot bidirectional P-anhydride-forming reaction that combines sixteen chemical transformations with high efficiency. The configuration of these materials is unambiguously shown by subsequent X-ray analyses of both enantiomers after being individually soaked into crystals of the kinase domain of human diphosphoinositol pentakisphosphate kinase 2. [ABSTRACT FROM AUTHOR]

Highlights: [•] Understanding inositol pyrophosphate turnover through kinetic study of Ddp1p/DIPPs. [•] k cat values for 1-InsP7 are 5–20-fold higher than those for 5-InsP7 and InsP8. [•] InsP7 does not regulate CDK5, the human homologue of the yeast Pho85 cyclin kinase. [•] Kinetics imply differential expression of DIPP isoforms sets signaling sensitivity. [•] Metabolically and functionally separate routes for InsP8 synthesis and hydrolysis. [ABSTRACT FROM AUTHOR]

Abstract: We have cloned and characterized four Itpk genes from soybean. All four recombinant Itpk proteins showed canonical Ins(1,3,4)P3 5/6-kinase activity, but a kinetic analysis raised questions about its biological significance. Instead, we provide evidence that one alternative biological role for soybean Itpks is to interconvert the Cl− channel inhibitor, Ins(3,4,5,6)P4, and its metabolic precursor, Ins(1,3,4,5,6)P5, within a substrate cycle. The soybean Itpks also phosphorylated Ins(3,4,6)P3 to Ins(1,3,4,6)P4 which was further phosphorylated to Ins(1,3,4,5,6)P5 by soybean Ipk2. Thus, soybean Itpks may participate in an inositol lipid-independent pathway of InsP6 synthesis. [Copyright &y& Elsevier]

Abstract: Studies [Zhou, D., Chen, L.-M., Hernandez, L., Shears, S.B., and Galán, J.E. (2001) A Salmonella inositol polyphosphatase acts in conjunction with other bacterial effectors to promote host-cell actin cytoskeleton rearrangements and bacterial internalization. Mol. Microbiol. 39, 248–259] with engineered Salmonella mutants showed that deletion of SopE attenuated the pathogen’s ability to deplete host-cell InsP5 and remodel the cytoskeleton. We pursued these observations: In SopE-transfected host-cells, membrane ruffling was induced, but SopE did not dephosphorylate InsP5, nor did it recruit PTEN (a cytosolic InsP5 phosphatase) for this task. However, PTEN strengthened SopE-mediated membrane ruffling. We conclude SopE promotes host-cell InsP5 hydrolysis only with the assistance of other Salmonella proteins. Our demonstration that Salmonella-mediated cytoskeletal modifications are independent of inositolphosphates will focus future studies on elucidating alternate pathogenic consequences of InsP5 metabolism, including ion channel conductance and apoptosis. [Copyright &y& Elsevier]

Abstract: Ins(1,4,5,6)P4, a biologically active cell constituent, was recently advocated as a substrate of human Ins(3,4,5,6)P4 1-kinase (hITPK1), because stereochemical factors were believed relatively unimportant to specificity [Miller, G.J., Wilson, M.P., Majerus, P.W. and Hurley, J.H. (2005) Specificity determinants in inositol polyphosphate synthesis: crystal structure of inositol 1,3,4-triphosphate 5/6-kinase. Mol. Cell. 18, 201–212]. Contrarily, we provide three examples of hITPK1 stereospecificity. hITPK1 phosphorylates only the 1-hydroxyl of both Ins(3,5,6)P3 and the meso-compound, Ins(4,5,6)P3. Moreover, hITPK1 has >13,000-fold preference for Ins(3,4,5,6)P4 over its enantiomer, Ins(1,4,5,6)P4. The biological significance of hITPK1 being stereospecific, and not physiologically phosphorylating Ins(1,4,5,6)P4, is reinforced by our demonstrating that Ins(1,4,5,6)P4 is phosphorylated (K m =0.18μM) by inositolphosphate-multikinase. [Copyright &y& Elsevier]

A central feature of Salmonella pathogenicity is the bacterium's ability to enter into non-phagocytic cells. Bacterial internalization is the consequence of cellular responses characterized by Cdc42- and Rac-dependent actin cytoskeleton rearrangements. These responses are triggered by the co-ordinated function of bacterial proteins delivered into the host cell by a specialized protein secretion system termed type III. We report here that SopB, a Salmonella inositol polyphosphatase delivered to the host cell by this secretion system, mediates actin cytoskeleton rearrangements and bacterial entry in a Cdc42-dependent manner. SopB exhibits overlapping functions with two other effectors of bacterial entry, the Rho family GTPase exchange factors SopE and SopE2. Thus, Salmonella strains deficient in any one of these proteins can enter into cells at high efficiency, whereas a strain lacking all three effectors is completely defective for entry. Consistent with an important role for inositol phosphate metabolism in Salmonella-induced cellular responses, a catalytically defective mutant of SopB failed to stimulate actin cytoskeleton rearrangements and bacterial entry. Furthermore, bacterial infection of intestinal cells resulted in a marked increase in Ins(1,4,5,6)P4, a consumption of InsP5 and the activation of phospholipase C. In agreement with the in vivo findings, purified SopB specifically dephosphorylated InsP5 to Ins(1,4,5,6)P4 in vitro. Surprisingly, the inositol phosphate fluxes induced by Salmonella were not caused exclusively by SopB. We show that the SopB-independent inositol phosphate fluxes are the consequence of the SopE-dependent activation of an endogenous inositol phosphatase. The ability of Salmonella to stimulate Rho GTPases signalling and inositol phosphate metabolism through alternative mechanisms is an example of the remarkable ability of this bacterial pathogen to manipulate host cellular functions. [ABSTRACT FROM AUTHOR]

Diphosphoinositol pentakisphosphate (PP-InsP5 or ‘InsP7’) and bisdiphosphoinositol tetrakisphosphate ([PP]2-InsP4 or ‘InsP8’) are the most highly phosphorylated members of the inositol-based cell signaling family. We have purified a rat hepatic diphosphoinositol polyphosphate phosphohydrolase (DIPP) that cleaves a β-phosphate from the diphosphate groups in PP-InsP5 (Km=340 nM) and [PP]2-InsP4 (Km=34 nM). Inositol hexakisphophate (InsP6) was not a substrate, but it inhibited metabolism of both [PP]2-InsP4 and PP-InsP5 (IC50=0.2 and 3 μM, respectively). Microsequencing of DIPP revealed a ‘MutT’ domain, which in other contexts guards cellular integrity by dephosphorylating 8-oxo-dGTP, which causes AT to CG transversion mutations. The MutT domain also metabolizes some nucleoside phosphates that may play roles in signal transduction. The rat DIPP MutT domain is conserved in a novel recombinant human uterine DIPP. The nucleotide sequence of the human DIPP cDNA was aligned to chromosome 6; the candidate gene contains at least four exons. The dependence of DIPP's catalytic activity upon its MutT domain was confirmed by mutagenesis of a conserved glutamate residue. DIPP's low molecular size, Mg2+ dependency and catalytic preference for phosphoanhydride bonds are also features of other MutT-type proteins. Because overlapping substrate specificity is a feature of this class of proteins, our data provide new directions for future studies of higher inositol phosphates. [ABSTRACT FROM AUTHOR]

Bis­diphosphoinositol tetrakisphosphate ([PP]2-InsP4 or ‘InsP8’) is a ‘high-energy’ inositol phosphate; we report that its metabolism is receptor-regulated in DDT1 MF-2 smooth muscle cells. This conclusion arose by pursuing the mechanism by which F­ decreased cellular levels of [PP]2-InsP4 up to 70%. A similar effect was induced by elevating cyclic nucleotide levels, either with IBMX or by application of either Bt2cAMP (EC50 = 14.7 μM), Bt2cGMP (EC50 = 7.9 μM) or isoproterenol (EC50 = 0.4 nM). Isoproterenol (1 μM) decreased [PP]2-InsP4 levels 25% by 5 min, and 71% by 60 min. This novel, agonist-mediated regulation of [PP]2-InsP4 turnover was very specific; isoproterenol did not decrease the cellular levels of either inositol pentakisphosphate, inositol hexakisphosphate or other diphosphorylated inositol polyphosphates. Bradykinin, which activated phospholipase C, did not affect [PP]2-InsP4 levels. Regulation of [PP]2-InsP4 turnover by both isoproterenol and cell-permeant cyclic nucleotides was unaffected by inhibitors of protein kinases A and G. The effectiveness of the kinase inhibitors was confirmed by their ability to block phosphorylation of the cAMP response element-binding protein. Our results indicate a new signaling action of cAMP, and furnish an important focus for future research into the roles of diphosphorylated inositol phosphates in signal transduction. [ABSTRACT FROM AUTHOR]

Pregnenolone efflux from bovine adrenal cortex mitochondria was measured with the aid of rapid separation of the organelles from the incubation by centrifugation through silicon oil. In the absence of bovine serum albumin or cytosolic supernatant (100 000 x g), an equilibrium distribution of newly synthesized pregnenolone was attained after 2 min at 30 C; 20% of total pregnenolone was located outside the mitochondria. The presence of 13 mg albumin/ml or 13 mg supernatant protein ml increased the exogenous proportion of pregnenolone to over 90% and 75% respectively without affecting the rate of cholesterol side-chain cleavage. The binding of pregnenolone by both albumin and supernatant was non-specific and non-saturable. It is suggested that in vivo the mitochondrial membranes do not present a significant barrier to pregnenolone efflux from the organelle. [ABSTRACT FROM AUTHOR]

Examines the effect of azastene, cyanoketone and trilostane upon respiration and cleavage of the cholesterol side chain in mitochondria from bovine adrenal cortex. Prevention of pregnenolone metabolism; Assay of cholesterol side-chain cleavage; Function of cyanoketone and trilostane as inhibitors of pregnenolone metabolism; Incubation with adenosine diphosphate.

A letter to the editor is presented in response to the article "Alterations of oxidative-phosphorylation reactions in mitochondria isolated from hypothyroid-rat Iiver" by I. Ezawa, published in a previous issue.