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1.
Annu Rev Biochem ; 93(1): 317-338, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-39094034

RESUMEN

Discovered in 1993, inositol pyrophosphates are evolutionarily conserved signaling metabolites whose versatile modes of action are being increasingly appreciated. These include their emerging roles as energy regulators, phosphodonors, steric/allosteric regulators, and G protein-coupled receptor messengers. Through studying enzymes that metabolize inositol pyrophosphates, progress has also been made in elucidating the various cellular and physiological functions of these pyrophosphate-containing, energetic molecules. The two main forms of inositol pyrophosphates, 5-IP7 and IP8, synthesized respectively by inositol-hexakisphosphate kinases (IP6Ks) and diphosphoinositol pentakisphosphate kinases (PPIP5Ks), regulate phosphate homeostasis, ATP synthesis, and several other metabolic processes ranging from insulin secretion to cellular energy utilization. Here, we review the current understanding of the catalytic and regulatory mechanisms of IP6Ks and PPIP5Ks, as well as their counteracting phosphatases. We also highlight the genetic and cellular evidence implicating inositol pyrophosphates as essential mediators of mammalian metabolic homeostasis.


Asunto(s)
Fosfatos de Inositol , Fosfotransferasas (Aceptor del Grupo Fosfato) , Transducción de Señal , Humanos , Fosfatos de Inositol/metabolismo , Animales , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Homeostasis , Metabolismo Energético , Adenosina Trifosfato/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Monoéster Fosfórico Hidrolasas/genética
2.
Cell ; 186(4): 748-763.e15, 2023 02 16.
Artículo en Inglés | MEDLINE | ID: mdl-36758548

RESUMEN

Although many prokaryotes have glycolysis alternatives, it's considered as the only energy-generating glucose catabolic pathway in eukaryotes. Here, we managed to create a hybrid-glycolysis yeast. Subsequently, we identified an inositol pyrophosphatase encoded by OCA5 that could regulate glycolysis and respiration by adjusting 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7) levels. 5-InsP7 levels could regulate the expression of genes involved in glycolysis and respiration, representing a global mechanism that could sense ATP levels and regulate central carbon metabolism. The hybrid-glycolysis yeast did not produce ethanol during growth under excess glucose and could produce 2.68 g/L free fatty acids, which is the highest reported production in shake flask of Saccharomyces cerevisiae. This study demonstrated the significance of hybrid-glycolysis yeast and determined Oca5 as an inositol pyrophosphatase controlling the balance between glycolysis and respiration, which may shed light on the role of inositol pyrophosphates in regulating eukaryotic metabolism.


Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Difosfatos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fosfatos de Inositol/genética , Fosfatos de Inositol/metabolismo , Glucólisis/genética , Respiración , Pirofosfatasas/metabolismo , Glucosa/metabolismo
3.
EMBO J ; 43(3): 462-480, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38216735

RESUMEN

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.


Asunto(s)
Difosfatos , Virus Gigantes , Difosfatos/metabolismo , Virus Gigantes/metabolismo , Fosfatos de Inositol/química , Fosfatos de Inositol/metabolismo , Fosfatos/metabolismo , Saccharomyces cerevisiae/metabolismo
4.
Nature ; 609(7929): 1005-1011, 2022 09.
Artículo en Inglés | MEDLINE | ID: mdl-36131016

RESUMEN

Lysosomes have many roles, including degrading macromolecules and signalling to the nucleus1. Lysosomal dysfunction occurs in various human conditions, such as common neurodegenerative diseases and monogenic lysosomal storage disorders (LSDs)2-4. For most LSDs, the causal genes have been identified but, in some, the function of the implicated gene is unknown, in part because lysosomes occupy a small fraction of the cellular volume so that changes in lysosomal contents are difficult to detect. Here we develop the LysoTag mouse for the tissue-specific isolation of intact lysosomes that are compatible with the multimodal profiling of their contents. We used the LysoTag mouse to study CLN3, a lysosomal transmembrane protein with an unknown function. In children, the loss of CLN3 causes juvenile neuronal ceroid lipofuscinosis (Batten disease), a lethal neurodegenerative LSD. Untargeted metabolite profiling of lysosomes from the brains of mice lacking CLN3 revealed a massive accumulation of glycerophosphodiesters (GPDs)-the end products of glycerophospholipid catabolism. GPDs also accumulate in the lysosomes of CLN3-deficient cultured cells and we show that CLN3 is required for their lysosomal egress. Loss of CLN3 also disrupts glycerophospholipid catabolism in the lysosome. Finally, we found elevated levels of glycerophosphoinositol in the cerebrospinal fluid of patients with Batten disease, suggesting the potential use of glycerophosphoinositol as a disease biomarker. Our results show that CLN3 is required for the lysosomal clearance of GPDs and reveal Batten disease as a neurodegenerative LSD with a defect in glycerophospholipid metabolism.


Asunto(s)
Ésteres , Glicerofosfolípidos , Fosfatos de Inositol , Lisosomas , Glicoproteínas de Membrana , Chaperonas Moleculares , Animales , Biomarcadores/líquido cefalorraquídeo , Biomarcadores/metabolismo , Niño , Ésteres/metabolismo , Glicerofosfolípidos/líquido cefalorraquídeo , Glicerofosfolípidos/metabolismo , Humanos , Fosfatos de Inositol/líquido cefalorraquídeo , Fosfatos de Inositol/metabolismo , Enfermedades por Almacenamiento Lisosomal/líquido cefalorraquídeo , Enfermedades por Almacenamiento Lisosomal/genética , Enfermedades por Almacenamiento Lisosomal/metabolismo , Lisosomas/metabolismo , Lisosomas/patología , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Lipofuscinosis Ceroideas Neuronales/líquido cefalorraquídeo , Lipofuscinosis Ceroideas Neuronales/genética , Lipofuscinosis Ceroideas Neuronales/metabolismo
5.
Mol Cell ; 76(1): 138-147.e5, 2019 10 03.
Artículo en Inglés | MEDLINE | ID: mdl-31473102

RESUMEN

Proteasomes are essential in all eukaryotic cells. However, their function and regulation remain considerably elusive, particularly those of less abundant variants. We demonstrate the human 20S proteasome recombinant assembly and confirmed the recombinant complex integrity biochemically and with a 2.6 Å resolution cryo-EM map. To assess its competence to form higher-order assemblies, we prepared and analyzed recombinant human 20S-PA200, a poorly characterized nuclear complex. Its 3.0 Å resolution cryo-EM structure reveals the PA200 unique architecture; the details of its intricate interactions with the proteasome, resulting in unparalleled proteasome α ring rearrangements; and the molecular basis for PA200 allosteric modulation of the proteasome active sites. Non-protein cryo-EM densities could be assigned to PA200-bound inositol phosphates, and we speculate regarding their functional role. Here we open extensive opportunities to study the fundamental properties of the diverse and distinct eukaryotic proteasome variants and to improve proteasome targeting under different therapeutic conditions.


Asunto(s)
Proteínas Nucleares/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Regulación Alostérica , Animales , Sitios de Unión , Humanos , Fosfatos de Inositol/metabolismo , Modelos Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/ultraestructura , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/ultraestructura , Unión Proteica , Conformación Proteica , Proteolisis , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Células Sf9 , Spodoptera , Relación Estructura-Actividad
6.
Proc Natl Acad Sci U S A ; 121(34): e2400912121, 2024 Aug 20.
Artículo en Inglés | MEDLINE | ID: mdl-39145930

RESUMEN

Myo-inositol-1-phosphate synthase (MIPS) catalyzes the NAD+-dependent isomerization of glucose-6-phosphate (G6P) into inositol-1-phosphate (IMP), controlling the rate-limiting step of the inositol pathway. Previous structural studies focused on the detailed molecular mechanism, neglecting large-scale conformational changes that drive the function of this 240 kDa homotetrameric complex. In this study, we identified the active, endogenous MIPS in cell extracts from the thermophilic fungus Thermochaetoides thermophila. By resolving the native structure at 2.48 Å (FSC = 0.143), we revealed a fully populated active site. Utilizing 3D variability analysis, we uncovered conformational states of MIPS, enabling us to directly visualize an order-to-disorder transition at its catalytic center. An acyclic intermediate of G6P occupied the active site in two out of the three conformational states, indicating a catalytic mechanism where electrostatic stabilization of high-energy intermediates plays a crucial role. Examination of all isomerases with known structures revealed similar fluctuations in secondary structure within their active sites. Based on these findings, we established a conformational selection model that governs substrate binding and eventually inositol availability. In particular, the ground state of MIPS demonstrates structural configurations regardless of substrate binding, a pattern observed across various isomerases. These findings contribute to the understanding of MIPS structure-based function, serving as a template for future studies targeting regulation and potential therapeutic applications.


Asunto(s)
Dominio Catalítico , Inositol , Mio-Inositol-1-Fosfato Sintasa , Mio-Inositol-1-Fosfato Sintasa/metabolismo , Mio-Inositol-1-Fosfato Sintasa/genética , Mio-Inositol-1-Fosfato Sintasa/química , Inositol/metabolismo , Inositol/química , Fosfatos de Inositol/metabolismo , Glucosa-6-Fosfato/metabolismo , Glucosa-6-Fosfato/química , Modelos Moleculares , Conformación Proteica , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/química
7.
Mol Cell ; 70(5): 936-948.e7, 2018 06 07.
Artículo en Inglés | MEDLINE | ID: mdl-29883610

RESUMEN

Necroptosis is an important form of lytic cell death triggered by injury and infection, but whether mixed lineage kinase domain-like (MLKL) is sufficient to execute this pathway is unknown. In a genetic selection for human cell mutants defective for MLKL-dependent necroptosis, we identified mutations in IPMK and ITPK1, which encode inositol phosphate (IP) kinases that regulate the IP code of soluble molecules. We show that IP kinases are essential for necroptosis triggered by death receptor activation, herpesvirus infection, or a pro-necrotic MLKL mutant. In IP kinase mutant cells, MLKL failed to oligomerize and localize to membranes despite proper receptor-interacting protein kinase-3 (RIPK3)-dependent phosphorylation. We demonstrate that necroptosis requires IP-specific kinase activity and that a highly phosphorylated product, but not a lowly phosphorylated precursor, potently displaces the MLKL auto-inhibitory brace region. These observations reveal control of MLKL-mediated necroptosis by a metabolite and identify a key molecular mechanism underlying regulated cell death.


Asunto(s)
Neoplasias del Colon/enzimología , Fosfatos de Inositol/metabolismo , Proteínas Quinasas/metabolismo , Sitios de Unión , Muerte Celular/efectos de los fármacos , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Neoplasias del Colon/virología , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células HT29 , Herpesvirus Humano 1/patogenicidad , Humanos , Células Jurkat , Mutación , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Quinasas/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología
8.
J Biol Chem ; 300(4): 107116, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38403246

RESUMEN

Inositol phosphates and their metabolites play a significant role in several biochemical pathways, gene expression regulation, and phosphate homeostasis. Among the different inositol phosphates, inositol hexakisphosphate (IP6) is a substrate of inositol hexakisphosphate kinases (IP6Ks), which phosphorylate one or more of the IP6 phosphate groups. Pyrophosphorylation of IP6 leads to the formation of inositol pyrophosphates, high-energy signaling molecules that mediate physiological processes through their ability to modify target protein activities, either by directly binding to their target protein or by pyrophosphorylating protein serine residues. 5-diphosphoinositol pentakisphosphate, the most abundant inositol pyrophosphate in mammals, has been extensively studied and found to be significantly involved in a wide range of physiological processes. Three IP6K (IP6K1, IP6K2, and IP6K3) isoforms regulate IP7 synthesis in mammals. Here, we summarize our current understanding of IP6K1's roles in cytoskeletal remodeling, trafficking, cellular migration, metabolism, gene expression, DNA repair, and immunity. We also briefly discuss current gaps in knowledge, highlighting the need for further investigation.


Asunto(s)
Fosfotransferasas (Aceptor del Grupo Fosfato) , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Animales , Humanos , Fosfatos de Inositol/metabolismo , Citoesqueleto/metabolismo , Mamíferos/metabolismo
9.
PLoS Pathog ; 19(6): e1011423, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-37267431

RESUMEN

The mature HIV-1 capsid is stabilized by host and viral determinants. The capsid protein CA binds to the cellular metabolites inositol hexakisphosphate (IP6) and its precursor inositol (1, 3, 4, 5, 6) pentakisphosphate (IP5) to stabilize the mature capsid. In target cells, capsid destabilization by the antiviral compounds lenacapavir and PF74 reveals a HIV-1 infectivity defect due to IP5/IP6 (IP5/6) depletion. To test whether intrinsic HIV-1 capsid stability and/or host factor binding determines HIV-1 insensitivity to IP5/6 depletion, a panel of CA mutants was assayed for infection of IP5/6-depleted T cells and wildtype cells. Four CA mutants with unstable capsids exhibited dependence on host IP5/6 for infection and reverse transcription (RTN). Adaptation of one such mutant, Q219A, by spread in culture resulted in Vpu truncation and a capsid three-fold interface mutation, T200I. T200I increased intrinsic capsid stability as determined by in vitro uncoating of purified cores and partially reversed the IP5/6-dependence in target cells for each of the four CA mutants. T200I further rescued the changes to lenacapavir sensitivity associated with the parental mutation. The premature dissolution of the capsid caused by the IP5/6-dependent mutations imparted a unique defect in integration targeting that was rescued by T200I. Collectively, these results demonstrate that T200I restored other capsid functions after RTN for the panel of mutants. Thus, the hyperstable T200I mutation stabilized the instability defects imparted by the parental IP5/6-dependent CA mutation. The contribution of Vpu truncation to mutant adaptation was linked to BST-2 antagonization, suggesting that cell-to-cell transfer promoted replication of the mutants. We conclude that interactions at the three-fold interface are adaptable, key mediators of capsid stability in target cells and are able to antagonize even severe capsid instability to promote infection.


Asunto(s)
Fármacos Anti-VIH , Seropositividad para VIH , VIH-1 , Humanos , Cápside/metabolismo , VIH-1/genética , VIH-1/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Fosfatos de Inositol/metabolismo , Fármacos Anti-VIH/farmacología
10.
Cell ; 143(6): 897-910, 2010 Dec 10.
Artículo en Inglés | MEDLINE | ID: mdl-21145457

RESUMEN

The inositol pyrophosphate IP7 (5-diphosphoinositolpentakisphosphate), formed by a family of three inositol hexakisphosphate kinases (IP6Ks), modulates diverse cellular activities. We now report that IP7 is a physiologic inhibitor of Akt, a serine/threonine kinase that regulates glucose homeostasis and protein translation, respectively, via the GSK3ß and mTOR pathways. Thus, Akt and mTOR signaling are dramatically augmented and GSK3ß signaling reduced in skeletal muscle, white adipose tissue, and liver of mice with targeted deletion of IP6K1. IP7 affects this pathway by potently inhibiting the PDK1 phosphorylation of Akt, preventing its activation and thereby affecting insulin signaling. IP6K1 knockout mice manifest insulin sensitivity and are resistant to obesity elicited by high-fat diet or aging. Inhibition of IP6K1 may afford a therapeutic approach to obesity and diabetes.


Asunto(s)
Fosfatos de Inositol/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Aumento de Peso , Adipogénesis , Envejecimiento/metabolismo , Animales , Técnicas de Cultivo de Célula , Dieta , Difosfatos/metabolismo , Inositol/metabolismo , Insulina/metabolismo , Resistencia a la Insulina , Ratones , Obesidad/metabolismo , Fosforilación , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética
11.
Biochemistry ; 63(7): 939-951, 2024 04 02.
Artículo en Inglés | MEDLINE | ID: mdl-38507812

RESUMEN

MshA is a GT-B glycosyltransferase catalyzing the first step in the biosynthesis of mycothiol. While many GT-B enzymes undergo an open-to-closed transition, MshA is unique because its 97° rotation is beyond the usual range of 10-25°. Molecular dynamics (MD) simulations were carried out for MshA in both ligand bound and unbound states to investigate the effect of ligand binding on localized protein dynamics and its conformational free energy landscape. Simulations showed that both the unliganded "opened" and liganded "closed" forms of the enzyme sample a wide degree of dihedral angles and interdomain distances with relatively low overlapping populations. Calculation of the free energy surface using replica exchange MD for the apo "opened" and an artificial generated apo "closed" structure revealed overlaps in the geometries sampled, allowing calculation of a barrier of 2 kcal/mol for the open-to-closed transition in the absence of ligands. MD simulations of fully liganded MshA revealed a smaller sampling of the dihedral angles. The localized protein fluctuation changes suggest that UDP-GlcNAc binding activates the motions of loops in the 1-l-myo-inositol-1-phosphate (I1P)-binding site despite little change in the interactions with UDP-GlcNAc. Circular dichroism, intrinsic fluorescence spectroscopy, and mutagenesis studies were used to confirm the ligand-induced structural changes in MshA. The results support a proposed mechanism where UDP-GlcNAc binds with rigid interactions to the C-terminal domain of MshA and activates flexible loops in the N-terminal domain for binding and positioning of I1P. This model can be used for future structure-based drug development of inhibitors of the mycothiol biosynthetic pathway.


Asunto(s)
Corynebacterium glutamicum , Cisteína , Glicopéptidos , Glicosiltransferasas , Inositol , Glicosiltransferasas/metabolismo , Ligandos , Fosfatos de Inositol/metabolismo , Uridina Difosfato/metabolismo , Conformación Proteica , Simulación de Dinámica Molecular
12.
J Biol Chem ; 299(3): 102928, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36681123

RESUMEN

Inositol pyrophosphates regulate diverse physiological processes; to better understand their functional roles, assessing their tissue-specific distribution is important. Here, we profiled inositol pyrophosphate levels in mammalian organs using an originally designed liquid chromatography-mass spectrometry (LC-MS) protocol and discovered that the gastrointestinal tract (GIT) contained the highest levels of diphosphoinositol pentakisphosphate (IP7) and its precursor inositol hexakisphosphate (IP6). Although their absolute levels in the GIT are diet dependent, elevated IP7 metabolism still exists under dietary regimens devoid of exogenous IP7. Of the major GIT cells, enteric neurons selectively express the IP7-synthesizing enzyme IP6K2. We found that IP6K2-knockout mice exhibited significantly impaired IP7 metabolism in the various organs including the proximal GIT. In addition, our LC-MS analysis displayed that genetic ablation of IP6K2 significantly impaired IP7 metabolism in the gut and duodenal muscularis externa containing myenteric plexus. Whole transcriptome analysis of duodenal muscularis externa further suggested that IP6K2 inhibition significantly altered expression levels of the gene sets associated with mature neurons, neural progenitor/stem cells, and glial cells, as well as of certain genes modulating neuronal differentiation and functioning, implying critical roles of the IP6K2-IP7 axis in developmental and functional regulation of the enteric nervous system. These results collectively reveal an unexpected role of mammalian IP7-a highly active IP6K2-IP7 pathway is conducive to the enteric nervous system.


Asunto(s)
Sistema Nervioso Entérico , Fosfatos de Inositol , Transcriptoma , Animales , Ratones , Difosfatos/análisis , Difosfatos/metabolismo , Sistema Nervioso Entérico/crecimiento & desarrollo , Sistema Nervioso Entérico/metabolismo , Fosfatos de Inositol/análisis , Fosfatos de Inositol/metabolismo , Ratones Noqueados , Neuronas/enzimología , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Ácido Fítico/metabolismo , Tracto Gastrointestinal/metabolismo
13.
Hum Mol Genet ; 31(8): 1183-1196, 2022 04 22.
Artículo en Inglés | MEDLINE | ID: mdl-34673953

RESUMEN

Endocytosis is a fundamentally important process through which material is internalized into cells from the extracellular environment. In the renal proximal tubule, endocytosis of the abundant scavenger receptor megalin and its co-receptor cubilin play a vital role in retrieving low molecular weight proteins from the renal filtrate. Although we know much about megalin and its ligands, the machinery and mechanisms by which the receptor is trafficked through the endosomal system remain poorly defined. In this study, we show that inositol phosphatase interacting protein of 27 kDa (Ipip27A), an interacting partner of the Lowe syndrome protein oculocerebrorenal syndrome of Lowe (OCRL), is required for endocytic traffic of megalin within the proximal renal tubule of zebrafish larvae. Knockout of Ipip27A phenocopies the endocytic phenotype seen upon loss of OCRL, with a deficit in uptake of both fluid-phase and protein cargo, which is accompanied by a reduction in megalin abundance and altered endosome morphology. Rescue and co-depletion experiments indicate that Ipip27A functions together with OCRL to support proximal tubule endocytosis. The results therefore identify Ipip27A as a new player in endocytic traffic in the proximal tubule in vivo and support the view that defective endocytosis underlies the renal tubulopathy in Lowe syndrome and Dent-2 disease.


Asunto(s)
Síndrome Oculocerebrorrenal , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Endocitosis/genética , Endosomas/genética , Endosomas/metabolismo , Femenino , Humanos , Fosfatos de Inositol/metabolismo , Túbulos Renales Proximales/metabolismo , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Masculino , Síndrome Oculocerebrorrenal/genética , Síndrome Oculocerebrorrenal/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Proteínas/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo
14.
Biochem Soc Trans ; 52(2): 567-580, 2024 04 24.
Artículo en Inglés | MEDLINE | ID: mdl-38629621

RESUMEN

The maintenance of phosphate homeostasis serves as a foundation for energy metabolism and signal transduction processes in all living organisms. Inositol pyrophosphates (PP-InsPs), composed of an inositol ring decorated with monophosphate and diphosphate moieties, and inorganic polyphosphate (polyP), chains of orthophosphate residues linked by phosphoanhydride bonds, are energy-rich biomolecules that play critical roles in phosphate homeostasis. There is a complex interplay between these two phosphate-rich molecules, and they share an interdependent relationship with cellular adenosine triphosphate (ATP) and inorganic phosphate (Pi). In eukaryotes, the enzymes involved in PP-InsP synthesis show some degree of conservation across species, whereas distinct enzymology exists for polyP synthesis among different organisms. In fact, the mechanism of polyP synthesis in metazoans, including mammals, is still unclear. Early studies on PP-InsP and polyP synthesis were conducted in the slime mould Dictyostelium discoideum, but it is in the budding yeast Saccharomyces cerevisiae that a clear understanding of the interplay between polyP, PP-InsPs, and Pi homeostasis has now been established. Recent research has shed more light on the influence of PP-InsPs on polyP in mammals, and the regulation of both these molecules by cellular ATP and Pi levels. In this review we will discuss the cross-talk between PP-InsPs, polyP, ATP, and Pi in the context of budding yeast, slime mould, and mammals. We will also highlight the similarities and differences in the relationship between these phosphate-rich biomolecules among this group of organisms.


Asunto(s)
Homeostasis , Fosfatos de Inositol , Polifosfatos , Polifosfatos/metabolismo , Animales , Fosfatos de Inositol/metabolismo , Humanos , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfato/metabolismo , Dictyostelium/metabolismo , Transducción de Señal
15.
Nature ; 560(7719): 509-512, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-30069050

RESUMEN

A short, 14-amino-acid segment called SP1, located in the Gag structural protein1, has a critical role during the formation of the HIV-1 virus particle. During virus assembly, the SP1 peptide and seven preceding residues fold into a six-helix bundle, which holds together the Gag hexamer and facilitates the formation of a curved immature hexagonal lattice underneath the viral membrane2,3. Upon completion of assembly and budding, proteolytic cleavage of Gag leads to virus maturation, in which the immature lattice is broken down; the liberated CA domain of Gag then re-assembles into the mature conical capsid that encloses the viral genome and associated enzymes. Folding and proteolysis of the six-helix bundle are crucial rate-limiting steps of both Gag assembly and disassembly, and the six-helix bundle is an established target of HIV-1 inhibitors4,5. Here, using a combination of structural and functional analyses, we show that inositol hexakisphosphate (InsP6, also known as IP6) facilitates the formation of the six-helix bundle and assembly of the immature HIV-1 Gag lattice. IP6 makes ionic contacts with two rings of lysine residues at the centre of the Gag hexamer. Proteolytic cleavage then unmasks an alternative binding site, where IP6 interaction promotes the assembly of the mature capsid lattice. These studies identify IP6 as a naturally occurring small molecule that promotes both assembly and maturation of HIV-1.


Asunto(s)
VIH-1/metabolismo , Fosfatos de Inositol/metabolismo , Virión/metabolismo , Ensamble de Virus , Arginina/metabolismo , Cápside/química , Cápside/metabolismo , Cristalografía por Rayos X , VIH-1/química , VIH-1/genética , Técnicas In Vitro , Lisina/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Virión/química , Virión/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/química , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo
16.
Nature ; 556(7702): 520-524, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29670288

RESUMEN

Neuropeptide Y (NPY) receptors belong to the G-protein-coupled receptor superfamily and have important roles in food intake, anxiety and cancer biology 1,2 . The NPY-Y receptor system has emerged as one of the most complex networks with three peptide ligands (NPY, peptide YY and pancreatic polypeptide) binding to four receptors in most mammals, namely the Y1, Y2, Y4 and Y5 receptors, with different affinity and selectivity 3 . NPY is the most powerful stimulant of food intake and this effect is primarily mediated by the Y1 receptor (Y1R) 4 . A number of peptides and small-molecule compounds have been characterized as Y1R antagonists and have shown clinical potential in the treatment of obesity 4 , tumour 1 and bone loss 5 . However, their clinical usage has been hampered by low potency and selectivity, poor brain penetration ability or lack of oral bioavailability 6 . Here we report crystal structures of the human Y1R bound to the two selective antagonists UR-MK299 and BMS-193885 at 2.7 and 3.0 Å resolution, respectively. The structures combined with mutagenesis studies reveal the binding modes of Y1R to several structurally diverse antagonists and the determinants of ligand selectivity. The Y1R structure and molecular docking of the endogenous agonist NPY, together with nuclear magnetic resonance, photo-crosslinking and functional studies, provide insights into the binding behaviour of the agonist and for the first time, to our knowledge, determine the interaction of its N terminus with the receptor. These insights into Y1R can enable structure-based drug discovery that targets NPY receptors.


Asunto(s)
Arginina/análogos & derivados , Dihidropiridinas/química , Dihidropiridinas/metabolismo , Ácidos Difenilacéticos/química , Ácidos Difenilacéticos/metabolismo , Neuropéptido Y/metabolismo , Compuestos de Fenilurea/química , Compuestos de Fenilurea/metabolismo , Receptores de Neuropéptido Y/antagonistas & inhibidores , Receptores de Neuropéptido Y/química , Arginina/química , Arginina/metabolismo , Arginina/farmacología , Sitios de Unión , Cristalografía por Rayos X , Dihidropiridinas/farmacología , Ácidos Difenilacéticos/farmacología , Humanos , Fosfatos de Inositol/metabolismo , Ligandos , Simulación del Acoplamiento Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación , Neuropéptido Y/química , Neuropéptido Y/farmacología , Resonancia Magnética Nuclear Biomolecular , Compuestos de Fenilurea/farmacología , Unión Proteica , Receptores de Neuropéptido Y/agonistas , Receptores de Neuropéptido Y/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato
17.
Mol Cell ; 64(4): 746-759, 2016 11 17.
Artículo en Inglés | MEDLINE | ID: mdl-27863227

RESUMEN

Excitation-transcription coupling, linking stimulation at the cell surface to changes in nuclear gene expression, is conserved throughout eukaryotes. How closely related coexpressed transcription factors are differentially activated remains unclear. Here, we show that two Ca2+-dependent transcription factor isoforms, NFAT1 and NFAT4, require distinct sub-cellular InsP3 and Ca2+ signals for physiologically sustained activation. NFAT1 is stimulated by sub-plasmalemmal Ca2+ microdomains, whereas NFAT4 additionally requires Ca2+ mobilization from the inner nuclear envelope by nuclear InsP3 receptors. NFAT1 is rephosphorylated (deactivated) more slowly than NFAT4 in both cytoplasm and nucleus, enabling a more prolonged activation phase. Oscillations in cytoplasmic Ca2+, long considered the physiological form of Ca2+ signaling, play no role in activating either NFAT protein. Instead, effective sustained physiological activation of NFAT4 is tightly linked to oscillations in nuclear Ca2+. Our results show how gene expression can be controlled by coincident yet geographically distinct Ca2+ signals, generated by a freely diffusible InsP3 message.


Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Fosfatos de Inositol/metabolismo , Factores de Transcripción NFATC/genética , Proteínas Recombinantes de Fusión/genética , Animales , Basófilos/citología , Basófilos/efectos de los fármacos , Basófilos/metabolismo , Bronquios/citología , Bronquios/efectos de los fármacos , Bronquios/metabolismo , Línea Celular , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Leucotrieno C4/farmacología , Factores de Transcripción NFATC/metabolismo , Transporte de Proteínas , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Tapsigargina/farmacología , Transcripción Genética
18.
Proc Natl Acad Sci U S A ; 118(1)2021 01 05.
Artículo en Inglés | MEDLINE | ID: mdl-33443153

RESUMEN

The differentiation of cells depends on a precise control of their internal organization, which is the result of a complex dynamic interplay between the cytoskeleton, molecular motors, signaling molecules, and membranes. For example, in the developing neuron, the protein ADAP1 (ADP-ribosylation factor GTPase-activating protein [ArfGAP] with dual pleckstrin homology [PH] domains 1) has been suggested to control dendrite branching by regulating the small GTPase ARF6. Together with the motor protein KIF13B, ADAP1 is also thought to mediate delivery of the second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP3) to the axon tip, thus contributing to PIP3 polarity. However, what defines the function of ADAP1 and how its different roles are coordinated are still not clear. Here, we studied ADAP1's functions using in vitro reconstitutions. We found that KIF13B transports ADAP1 along microtubules, but that PIP3 as well as PI(3,4)P2 act as stop signals for this transport instead of being transported. We also demonstrate that these phosphoinositides activate ADAP1's enzymatic activity to catalyze GTP hydrolysis by ARF6. Together, our results support a model for the cellular function of ADAP1, where KIF13B transports ADAP1 until it encounters high PIP3/PI(3,4)P2 concentrations in the plasma membrane. Here, ADAP1 disassociates from the motor to inactivate ARF6, promoting dendrite branching.


Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Fosfatidilinositoles/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/fisiología , Proteínas Adaptadoras Transductoras de Señales/fisiología , Animales , Axones/metabolismo , Transporte Biológico/fisiología , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Humanos , Fosfatos de Inositol/metabolismo , Cinesinas/metabolismo , Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/fisiología , Fosfatos de Fosfatidilinositol/metabolismo , Transducción de Señal
19.
Int J Mol Sci ; 25(3)2024 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-38338641

RESUMEN

The natural cyclic AMP antagonist, prostaglandylinositol cyclic phosphate (cyclic PIP), is biosynthesized from prostaglandin E (PGE) and activated inositol phosphate (n-Ins-P), which is synthesized by a particulate rat-liver-enzyme from GTP and a precursor named inositol phosphate (pr-Ins-P), whose 5-ring phosphodiester structure is essential for n-Ins-P synthesis. Aortic myocytes, preincubated with [3H] myo-inositol, synthesize after angiotensin II stimulation (30 s) [3H] pr-Ins-P (65% yield), which is converted to [3H] n-Ins-P and [3H] cyclic PIP. Acid-treated (1 min) [3H] pr-Ins-P co-elutes with inositol (1,4)-bisphosphate in high performance ion chromatography, indicating that pr-Ins-P is inositol (1:2-cyclic,4)-bisphosphate. Incubation of [3H]-GTP with unlabeled pr-Ins-P gave [3H]-guanosine-labeled n-Ins-P. Cyclic PIP synthase binds the inositol (1:2-cyclic)-phosphate part of n-Ins-P to PGE and releases the [3H]-labeled guanosine as [3H]-GDP. Thus, n-Ins-P is most likely guanosine diphospho-4-inositol (1:2-cyclic)-phosphate. Inositol feeding helps patients with metabolic conditions related to insulin resistance, but explanations for this finding are missing. Cyclic PIP appears to be the key for explaining the curative effect of inositol supplementation: (1) inositol is a molecular constituent of cyclic PIP; (2) cyclic PIP triggers many of insulin's actions intracellularly; and (3) the synthesis of cyclic PIP is decreased in diabetes as shown in rodents.


Asunto(s)
Fosfatos de Inositol , Inositol , Prostaglandinas E , Humanos , Ratas , Animales , Inositol/farmacología , Inositol/metabolismo , Fosfatos de Inositol/metabolismo , Guanosina Trifosfato , Guanosina , Fosfatos
20.
J Biol Chem ; 298(8): 102134, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35709985

RESUMEN

Extra-large stimulatory Gα (XLαs) is a large variant of G protein αs subunit (Gαs) that uses an alternative promoter and thus differs from Gαs at the first exon. XLαs activation by G protein-coupled receptors mediates cAMP generation, similarly to Gαs; however, Gαs and XLαs have been shown to have distinct cellular and physiological functions. For example, previous work suggests that XLαs can stimulate inositol phosphate production in renal proximal tubules and thereby regulate serum phosphate levels. In this study, we show that XLαs directly and specifically stimulates a specific isoform of phospholipase Cß (PLCß), PLCß4, both in transfected cells and with purified protein components. We demonstrate that neither the ability of XLαs to activate cAMP generation nor the canonical G protein switch II regions are required for PLCß stimulation. Furthermore, this activation is nucleotide independent but is inhibited by Gßγ, suggesting a mechanism of activation that relies on Gßγ subunit dissociation. Surprisingly, our results indicate that enhanced membrane targeting of XLαs relative to Gαs confers the ability to activate PLCß4. We also show that PLCß4 is required for isoproterenol-induced inositol phosphate accumulation in osteocyte-like Ocy454 cells. Taken together, we demonstrate a novel mechanism for activation of phosphoinositide turnover downstream of Gs-coupled receptors that may have a critical role in endocrine physiology.


Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gs , Fosfatos de Inositol , Fosfolipasa C beta , Membrana Celular/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Fosfatos de Inositol/metabolismo , Isoenzimas/metabolismo , Isoproterenol/farmacología , Fosfolipasa C beta/metabolismo
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