RESUMEN
The pattern recognition receptor RIG-I is critical for Type-I interferon production. However, the global regulation of RIG-I signaling is only partially understood. Using a human genome-wide RNAi-screen, we identified 226 novel regulatory proteins of RIG-I mediated interferon-ß production. Furthermore, the screen identified a metabolic pathway that synthesizes the inositol pyrophosphate 1-IP7 as a previously unrecognized positive regulator of interferon production. Detailed genetic and biochemical experiments demonstrated that the kinase activities of IPPK, PPIP5K1 and PPIP5K2 (which convert IP5 to1-IP7) were critical for both interferon induction, and the control of cellular infection by Sendai and influenza A viruses. Conversely, ectopically expressed inositol pyrophosphate-hydrolases DIPPs attenuated interferon transcription. Mechanistic experiments in intact cells revealed that the expression of IPPK, PPIP5K1 and PPIP5K2 was needed for the phosphorylation and activation of IRF3, a transcription factor for interferon. The addition of purified individual inositol pyrophosphates to a cell free reconstituted RIG-I signaling assay further identified 1-IP7 as an essential component required for IRF3 activation. The inositol pyrophosphate may act by ß-phosphoryl transfer, since its action was not recapitulated by a synthetic phosphonoacetate analogue of 1-IP7. This study thus identified several novel regulators of RIG-I, and a new role for inositol pyrophosphates in augmenting innate immune responses to viral infection that may have therapeutic applications.
Asunto(s)
Regulación de la Expresión Génica/inmunología , Interferón Tipo I/inmunología , Monoéster Fosfórico Hidrolasas/inmunología , Receptores de Ácido Retinoico/inmunología , Transducción de Señal/inmunología , Humanos , Inmunidad Innata/inmunología , Factor 3 Regulador del Interferón/inmunología , ARN Interferente PequeñoRESUMEN
We describe new signalling consequences for PPIP5K1 (diphosphoinositol pentakisphosphate kinase type 1)-mediated phosphorylation of InsP6 and 5-InsP7 to 1-InsP7 and InsP8. In NIH 3T3 cells, either hyperosmotic stress or receptor activation by PDGF (platelet-derived growth factor) promoted translocation of PPIP5K1 from the cytoplasm to the plasma membrane. The PBD1 (polyphosphoinositide-binding domain) in PPIP5K1 recapitulated that translocation. Mutagenesis of PBD1 to reduce affinity for PtdIns(3,4,5)P3 prevented translocation. Using surface plasmon resonance, we found that PBD1 association with vesicular PtdIns(3,4,5)P3 was inhibited by InsP6 and diphosphoinositol polyphosphates. However, the inhibition by PPIP5K1 substrates (IC50: 5-InsP7=5 µM and InsP6=7 µM) was substantially more potent than that of the PPIP5K1 products (IC50: InsP8=32 µM and 1-InsP7=43 µM). This rank order of ligand competition with PtdIns(3,4,5)P3 was also exhibited by the PH (pleckstrin homology) domains of Akt (also known as protein kinase B), GRP1 (general receptor for phosphoinositides 1) and SIN1 (stress-activated protein kinase-interaction protein 1). We propose that, in vivo, PH domain binding of InsP6 and 5-InsP7 suppresses inappropriate signalling ('noise') from stochastic increases in PtdIns(3,4,5)P3. That restraint may be relieved by localized depletion of InsP6 and 5-InsP7 at the plasma membrane following PPIP5K1 recruitment. We tested this hypothesis in insulin-stimulated L6 myoblasts, using mTOR (mechanistic/mammalian target of rapamycin)-mediated phosphorylation of Akt on Ser473 as a readout for SIN1-mediated translocation of mTORC (mTOR complex) 2 to the plasma membrane [Zoncu, Efeyan and Sabatini (2011) Nat. Rev. Mol. Cell Biol. 12, 21-35]. Knockdown of PPIP5K1 expression was associated with a 40% reduction in Ser473 phosphorylation. A common feature of PtdIns(3,4,5)P3-based signalling cascades may be their regulation by PPIP5K1.
Asunto(s)
Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Animales , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Humanos , Immunoblotting , Ratones , Células 3T3 NIH , Factor de Crecimiento Derivado de Plaquetas/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Resonancia por Plasmón de SuperficieRESUMEN
Ins(1,4,5)P(3) is a classical intracellular messenger: stimulus-dependent changes in its levels elicits biological effects through its release of intracellular Ca(2+) stores. The Ins(1,4,5)P(3) response is "switched off" by its metabolism to a range of additional inositol phosphates. These metabolites have themselves come to be collectively described as a signaling "family". The validity of that latter definition is critically examined in this review. That is, we assess the strength of the hypothesis that Ins(1,4,5)P(3) metabolites are themselves "classical" signals. Put another way, what is the evidence that the biological function of a particular inositol phosphate depends upon stimulus dependent changes in its levels? In this assessment, examples of an inositol phosphate acting as a cofactor (i.e. its function is not stimulus-dependent) do not satisfy our signaling criteria. We conclude that Ins(3,4,5,6)P(4) is, to date, the only Ins(1,4,5)P(3) metabolite that has been validated to act as a second messenger.
Asunto(s)
Células Eucariotas/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Fosfatos de Inositol/metabolismo , Sistemas de Mensajero Secundario , Animales , Calcio/metabolismo , Células Eucariotas/citología , Humanos , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismoRESUMEN
Diphosphoinositol pentakisphosphate kinase 2 (PPIP5K2) is one of the mammalian PPIP5K isoforms responsible for synthesis of diphosphoinositol polyphosphates (inositol pyrophosphates; PP-InsPs), regulatory molecules that function at the interface of cell signaling and organismic homeostasis. The development of drugs that inhibit PPIP5K2 could have both experimental and therapeutic applications. Here, we describe a synthetic strategy for producing naturally occurring 5-PP-InsP4, as well as several inositol polyphosphate analogs, and we study their interactions with PPIP5K2 using biochemical and structural approaches. These experiments uncover an additional ligand-binding site on the surface of PPIP5K2, adjacent to the catalytic pocket. This site facilitates substrate capture from the bulk phase, prior to transfer into the catalytic pocket. In addition to demonstrating a "catch-and-pass" reaction mechanism in a small molecule kinase, we demonstrate that binding of our analogs to the substrate capture site inhibits PPIP5K2. This work suggests that the substrate-binding site offers new opportunities for targeted drug design.
Asunto(s)
Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Fosfatos de Inositol/síntesis química , Fosfatos de Inositol/farmacología , Fosfotransferasas (Aceptor del Grupo Fosfato)/antagonistas & inhibidores , Sitios de Unión/efectos de los fármacos , Biocatálisis , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Fosfatos de Inositol/química , Ligandos , Modelos Moleculares , Conformación Molecular , Fosfotransferasas (Aceptor del Grupo Fosfato)/química , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato , Propiedades de SuperficieRESUMEN
We obtained detailed kinetic characteristics--stoichiometry, reaction rates, substrate affinities and equilibrium conditions--of human PPIP5K2 (diphosphoinositol pentakisphosphate kinase 2). This enzyme synthesizes 'high-energy' PP-InsPs (diphosphoinositol polyphosphates) by metabolizing InsP6 (inositol hexakisphosphate) and 5-InsP7 (5-diphosphoinositol 1,2,3,4,6-pentakisphosphate) to 1-InsP7 (1-diphosphoinositol 2,3,4,5,6-pentakisphosphate) and InsP8 (1,5-bis-diphosphoinositol 2,3,4,6-tetrakisphosphate), respectively. These data increase our insight into the PPIP5K2 reaction mechanism and clarify the interface between PPIP5K catalytic activities and cellular bioenergetic status. For example, stochiometric analysis uncovered non-productive, substrate-stimulated ATPase activity (thus, approximately 2 and 1.2 ATP molecules are utilized to synthesize each molecule of 1-InsP7 and InsP8, respectively). Impaired ATPase activity of a PPIP5K2-K248A mutant increased atomic-level insight into the enzyme's reaction mechanism. We found PPIP5K2 to be fully reversible as an ATP-synthase in vitro, but our new data contradict previous perceptions that significant 'reversibility' occurs in vivo. PPIP5K2 was insensitive to physiological changes in either [AMP] or [ATP]/[ADP] ratios. Those data, together with adenine nucleotide kinetics (ATP Km=20-40 µM), reveal how insulated PPIP5K2 is from cellular bioenergetic challenges. Finally, the specificity constants for PPIP5K2 revise upwards by one-to-two orders of magnitude the inherent catalytic activities of this enzyme, and we show its equilibrium point favours 80-90% depletion of InsP6/5-InsP7.
Asunto(s)
Catálisis , Metabolismo Energético , Fosfotransferasas (Aceptor del Grupo Fosfato)/química , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Humanos , Inositol/química , Inositol/metabolismo , Fosfatos de Inositol , Cinética , Redes y Vías Metabólicas , Fosfatos/química , Fosfatos/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Especificidad por SustratoRESUMEN
The diphosphoinositol polyphosphates ("inositol pyrophosphates"; PP-InsPs) regulate many cellular processes in eukaryotes, including stress responses, apoptosis, vesicle trafficking, cytoskeletal dynamics, exocytosis, telomere maintenance, insulin signaling and neutrophil activation. Thus, the enzymes that control the metabolism of the PP-InsPs serve important cell signaling roles. In order to fully characterize how these enzymes are regulated, we need to determine the atomic-level architecture of their active sites. Only then can we fully appreciate reaction mechanisms and their modes of regulation. In this review, we summarize published information obtained from the structural analysis of a human diphosphoinositol polyphosphate phosphohydrolase (DIPP), and a human diphosphoinositol polyphosphate kinase (PPIP5K). This work includes the analysis of crystal complexes with substrates, products, transition state analogs, and a novel phosphonoacetate substrate analog.
Asunto(s)
Ácido Anhídrido Hidrolasas/química , Difosfatos/química , Fosfatos de Inositol/química , Fosfotransferasas (Aceptor del Grupo Fosfato)/química , Ácido Anhídrido Hidrolasas/metabolismo , Dominio Catalítico , Difosfatos/metabolismo , Humanos , Fosfatos de Inositol/metabolismo , Simulación del Acoplamiento Molecular , Ácido Fosfonoacético/análogos & derivados , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Transducción de Señal , Especificidad por SustratoRESUMEN
We illuminate the metabolism and the cell-signaling activities of inositol pyrophosphates, by showing that regulation of yeast cyclin-kinase by 1-InsP7 is not conserved for mammalian CDK5, and by kinetically characterizing Ddp1p/DIPP-mediated dephosphorylation of 1-InsP7, 5-InsP7 and InsP8. Each phosphatase exhibited similar Km values for every substrate (range: 35-148 nM). The rank order of kcat values (1-InsP7>5-InsP7=InsP8) was identical for each enzyme, although DIPP1 was 10- to 60-fold more active than DIPP2α/ß and DIPP3α/ß. We demonstrate InsP8 dephosphorylation preferentially progresses through 1-InsP7. Conversely, we conclude that the more metabolically and functionally significant steady-state route of InsP8 synthesis proceeds via 5-InsP7.
Asunto(s)
Ácido Anhídrido Hidrolasas/química , Ácido Anhídrido Hidrolasas/metabolismo , Fosfatos de Inositol/metabolismo , Humanos , Cinética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismoRESUMEN
Phytases are phosphohydrolytic enzymes that initiate stepwise removal of phosphate from phytate. Simple-stomached species such as swine, poultry, and fish require extrinsic phytase to digest phytate, the major form of phosphorus in plant-based feeds. Consequently, this enzyme is supplemented in these species' diets to decrease their phosphorus excretion, and it has emerged as one of the most effective and lucrative feed additives. This chapter provides a comprehensive review of the evolving course of phytase science and technology. It gives realistic estimates of the versatile roles of phytase in animal feeding, environmental protection, rock phosphorus preservation, human nutrition and health, and industrial applications. It elaborates on new biotechnology and existing issues related to developing novel microbial phytases as well as phytase-transgenic plants and animals. And it targets critical and integrated analyses on the global impact, novel application, and future demand of phytase in promoting animal agriculture, human health, and societal sustainability.
Asunto(s)
6-Fitasa/metabolismo , Ácido Fítico/metabolismo , Animales , Humanos , Modelos Moleculares , Fenómenos Fisiológicos de la Nutrición , Fosfatos/metabolismo , Ácido Fítico/química , Conformación ProteicaRESUMEN
We synthesised analogues of diphosphoinositol polyphosphates (PP-InsPs) in which the diphosphate is replaced by an α-phosphonoacetic acid (PA) ester. Structural analysis revealed that 5-PA-InsP(5) mimics 5-PP-InsP(5) binding to the kinase domain of PPIP5K2; both molecules were phosphorylated by the enzyme. PA-InsPs are promising candidates for further studies into the biology of PP-InsPs.
Asunto(s)
Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Polifosfatos/metabolismo , Sitios de Unión , Biocatálisis , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Fosfatos de Inositol/química , Fosfotransferasas (Aceptor del Grupo Fosfato)/química , Polifosfatos/química , Especificidad por SustratoRESUMEN
Se-dependent glutathione peroxidase-1 (GPX1) and Cu,Zn-superoxide dismutase (SOD1) are two major intracellular antioxidant enzymes. The purpose of this study was to elucidate the biochemical mechanisms for the 40% loss of hepatic GPX1 activity in SOD1(-/-) mice. Compared with the wild type (WT), the SOD1(-/-) mice showed no change in the total amount of GPX1 protein. However, their total enzyme protein exhibited 31 and 38% decreases (P<0.05) in the apparent k(cat) for hydrogen peroxide and tert-butylperoxide (at 2mM GSH), respectively. Most striking, mass spectrometry revealed two chemical forms of the 47th residue of GPX1: the projected native selenocysteine (Sec) and the Se-lacking dehydroalanine (DHA). The hepatic GPX1 protein of the SOD1(-/-) mice contained 38% less Sec and 77% more DHA than that of WT and showed aggravated dissociation of the tetramer structure. In conclusion, knockout of SOD1 elevated the conversion of Sec to DHA in the active site of hepatic GPX1, leading to proportional decreases in the apparent k(cat) and activity of the enzyme protein as a whole. Our data reveal a structural and kinetic mechanism for the in vivo functional dependence of GPX1 on SOD1 in mammals and provide a novel mass spectrometric method for the assay of oxidative modification of the GPX1 protein.
Asunto(s)
Alanina/análogos & derivados , Glutatión Peroxidasa/metabolismo , Selenocisteína/metabolismo , Superóxido Dismutasa/metabolismo , Alanina/biosíntesis , Animales , Electroforesis en Gel de Poliacrilamida , Genotipo , Glutatión Peroxidasa/química , Peróxido de Hidrógeno/metabolismo , Hígado/enzimología , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxidación-Reducción , Estrés Oxidativo , Superóxido Dismutasa/deficiencia , Superóxido Dismutasa-1 , terc-Butilhidroperóxido/metabolismo , Glutatión Peroxidasa GPX1RESUMEN
Aspergillus niger PhyA and Escherichia coli AppA2 are increasingly used in animal feed for phosphorus nutrition and environmental protection. The objective of this study was to determine the impacts of assay conditions on activity estimates of these two phytases and to compare their biochemical characteristics at a pH similar to the stomach environment. The activities of the unpurified AppA2 were more variable than those of PhyA with three commonly used phytase activity assays. The variations associated with AppA2 were accounted for by buffer, pH, and the inclusion of Triton X-100 and BSA by approximately one-third each. At the commonly observed stomach pH of 3.5, the purified AppA2 had a lower affinity to phytate (a higher K(m)), but greater V(max), k(cat), and k(cat)/K(m) than those of PhyA. In summary, differences between AppA2 and PhyA in responses to activity assay conditions and in inherent kinetic properties should be considered in interpreting their feeding efficacy.
Asunto(s)
6-Fitasa/química , Fosfatasa Ácida/química , Aspergillus niger/enzimología , Bioquímica/métodos , Proteínas de Escherichia coli/química , Proteínas Fúngicas/química , Complejos Multienzimáticos/química , Aspergillus niger/química , Cinética , Ácido Fítico/químicaRESUMEN
We previously identified a number of mutations in Escherichia coli AppA2 phytase for enhancing its thermostability. The objective of the present study was to determine if these mutations (K46E, K65E, G103S, D112N, D144N, S209G, V227A, and G344D) could be sequentially added to further improve the thermostability of AppA2. Compared with the wild-type enzyme, two variants (D144N/V227A and D144N/V227A/G344D) out of the eight resulting mutants showed 15% enhancement in thermostability (as measured by residual activity after being heated at 80 degrees C for 10 min) and 4 to 5 degrees C increases in the melting temperatures (T (m)). Based on the structural predictions with a highly homologous AppA phytase, the substitution D144N introduces a side-chain-side-chain hydrogen bond, thereby stabilizing the loop region (Gln137-Asn144), and the V227A substitution might eliminate structural hindrance between Val222 and Val227 that face each other in the beta-hairpin structure. In addition, overall catalytic efficiency (k (cat)/K (m)) of the two mutants was also improved (P < 0.05) compared to the wild type. However, no further improvement in thermostability was observed by adding other mutations to D144N/V227A/G344D, which might result from unfavorable electrostatic interactions or structural perturbation. In conclusion, our results underscore the potential as well as difficulty of predicting synergistic effects of multiple mutations on thermostability within phytase.
Asunto(s)
6-Fitasa/química , Fosfatasa Ácida/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Complejos Multienzimáticos/química , Mutación , Ingeniería de Proteínas , 6-Fitasa/genética , 6-Fitasa/metabolismo , Fosfatasa Ácida/genética , Fosfatasa Ácida/metabolismo , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Calor , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Modelos Moleculares , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Mutagénesis Sitio-Dirigida , Conformación ProteicaRESUMEN
Phytases are of biotechnological importance as animal feed additives for their ability to catalyze the hydrolysis of phosphate from phytate for absorption by simple-stomached animals, and to reduce their fecal phosphorus excretion. Aspergillus niger PhyB has high catalytic activity at low pHs around 2.5, but has little activity at the commonly observed gastric pH of young animals (3.0-3.5). Our objective was to determine if the pH optima of PhyB could be broadened to a more characteristic pH range in the stomach of young animals through site-directed mutagenesis. We created two mutants, E272K and E272Q, each with a single amino acid substitution of the same residue in the substrate specificity site. Mutants were designed to replace an acidic amino acid, with either a neutral amino acid (E272Q) or basic amino acid (E272K), and were overexpressed in the yeast Pichia pastoris. While the wild-type (WT) pH optimum was 2.5, mutant E272K shifted to a new optimum of pH 3.2. E272K had a concomitant reduction in K (m) of 36-fold at pH 2.5 and 6-fold at pH 3.2 compared to the WT. Our results indicate that the pH optimum of PhyB can be altered to match the stomach pH, along with an improved substrate affinity.