RESUMO
Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal 5'-phosphate phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5'-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small-molecule screening, protein crystallography, and biolayer interferometry, we discover, visualize, and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain.
Vitamin B6 is an important nutrient for optimal brain function, with deficiencies linked to impaired memory, learning and mood in various mental disorders. In older people, vitamin B6 deficiency is also associated with declining memory and dementia. Although this has been known for years, the precise role of vitamin B6 in these disorders and whether supplements can be used to treat or prevent them remained unclear. This is partly because vitamin B6 is actually an umbrella term for a small number of very similar and interchangeable molecules. Only one of these is 'bioactive', meaning it has a biological role in cells. However, therapeutic strategies aimed at increasing only the bioactive form of vitamin B6 are lacking. Previous work showed that disrupting the gene for an enzyme called pyridoxal phosphatase, which breaks down vitamin B6, improves memory and learning in mice. To investigate whether these effects could be mimicked by drug-like compounds, Brenner, Zink, Witzinger et al. used several biochemical and structural biology approaches to search for molecules that bind to and inhibit pyridoxal phosphatase. The experiments showed that a molecule called 7,8-dihydroxyflavone which was previously found to improve memory and learning in laboratory animals with brain disorders binds to pyridoxal phosphatase and inhibits its activity. This led to increased bioactive vitamin B6 levels in mouse brain cells involved in memory and learning. The findings of Brenner et al. suggest that inhibiting pyridoxal phosphatase to increase vitamin B6 levels in the brain could be used together with supplements. The identification of 7,8-dihydroxyflavone as a promising candidate drug is a first step in the discovery of more efficient pyridoxal phosphatase inhibitors. These will be useful experimental tools to directly study whether increasing the levels of bioactive vitamin B6 in the brain may help those with mental health conditions associated with impaired memory, learning and mood.
Assuntos
Inibidores Enzimáticos , Monoéster Fosfórico Hidrolases , Animais , Camundongos , Humanos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Monoéster Fosfórico Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Hipocampo/metabolismo , Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosfato de Piridoxal/metabolismo , Flavonas/farmacologia , Flavonas/metabolismo , Flavonas/química , Camundongos Endogâmicos C57BLRESUMO
Targeting the intrinsic metabolism of immune or tumor cells is a therapeutic strategy in autoimmunity, chronic inflammation or cancer. Metabolite repair enzymes may represent an alternative target class for selective metabolic inhibition, but pharmacological tools to test this concept are needed. Here, we demonstrate that phosphoglycolate phosphatase (PGP), a prototypical metabolite repair enzyme in glycolysis, is a pharmacologically actionable target. Using a combination of small molecule screening, protein crystallography, molecular dynamics simulations and NMR metabolomics, we discover and analyze a compound (CP1) that inhibits PGP with high selectivity and submicromolar potency. CP1 locks the phosphatase in a catalytically inactive conformation, dampens glycolytic flux, and phenocopies effects of cellular PGP-deficiency. This study provides key insights into effective and precise PGP targeting, at the same time validating an allosteric approach to control glycolysis that could advance discoveries of innovative therapeutic candidates.
Assuntos
Neoplasias , Monoéster Fosfórico Hidrolases , Humanos , Monoéster Fosfórico Hidrolases/metabolismo , GlicóliseRESUMO
Inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase of the family of statins have been suggested as therapeutic options in various tumors. Atorvastatin is a statin with the potential to cross the blood-brain barrier; however, the concentrations necessary for a cytotoxic effect against cancer cells exceed the concentrations achievable via oral administration, which made the development of a novel atorvastatin formulation necessary. We characterized the drug loading and basic physicochemical characteristics of micellar atorvastatin formulations and tested their cytotoxicity against a panel of different glioblastoma cell lines. In addition, activity against tumor spheroids formed from mouse glioma and mouse cancer stem cells, respectively, was evaluated. Our results show good activity of atorvastatin against all tested cell lines. Interestingly, in the three-dimensional (3D) models, growth inhibition was more pronounced for the micellar formulation compared to free atorvastatin. Finally, atorvastatin penetration across a blood-brain barrier model obtained from human induced-pluripotent stem cells was evaluated. Our results suggest that the presented micelles may enable much higher serum concentrations than possible by oral administration; however, if transport across the blood-brain barrier is sufficient to reach the therapeutic atorvastatin concentration for the treatment of glioblastoma via intravenous administration remains unclear.
Assuntos
Antineoplásicos/farmacologia , Atorvastatina/química , Atorvastatina/farmacologia , Glioblastoma/tratamento farmacológico , Antineoplásicos/química , Barreira Hematoencefálica , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Composição de Medicamentos , Difusão Dinâmica da Luz , Glioblastoma/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Micelas , Nanomedicina/métodos , Células-Tronco Neoplásicas/efeitos dos fármacos , Oxazóis/químicaRESUMO
Pyridoxal 5'-phosphate (PLP) is an essential cofactor for neurotransmitter metabolism. Pyridoxal phosphatase (PDXP) deficiency in mice increases PLP and γ-aminobutyric acid levels in the brain, yet how PDXP is regulated is unclear. Here, we identify the Ca2+ - and integrin-binding protein 1 (CIB1) as a PDXP interactor by yeast two-hybrid screening and find a calmodulin (CaM)-binding motif that overlaps with the PDXP-CIB1 interaction site. Pulldown and crosslinking assays with purified proteins demonstrate that PDXP directly binds to CIB1 or CaM. CIB1 or CaM does not alter PDXP phosphatase activity. However, elevated Ca2+ concentrations promote CaM binding and, thereby, diminish CIB1 binding to PDXP, as both interactors bind in a mutually exclusive way. Hence, the PDXP-CIB1 complex may functionally differ from the PDXP-Ca2+ -CaM complex.
RESUMO
Pyridoxal 5'-phosphate (PLP) is an essential cofactor in the catalysis of ~140 different enzymatic reactions. A pharmacological elevation of cellular PLP concentrations is of interest in neuropsychiatric diseases, but whole-body consequences of higher intracellular PLP levels are unknown. To address this question, we have generated mice allowing a conditional ablation of the PLP phosphatase PDXP. Ubiquitous PDXP deletion increased PLP levels in brain, skeletal muscle and red blood cells up to 3-fold compared to control mice, demonstrating that PDXP acts as a major regulator of cellular PLP concentrations in vivo. Neurotransmitter analysis revealed that the concentrations of dopamine, serotonin, epinephrine and glutamate were unchanged in the brains of PDXP knockout mice. However, the levels of γ-aminobutyric acid (GABA) increased by ~20%, demonstrating that elevated PLP levels can drive additional GABA production. Behavioral phenotyping of PDXP knockout mice revealed improved spatial learning and memory, and a mild anxiety-like behavior. Consistent with elevated GABA levels in the brain, PDXP loss in neural cells decreased performance in motor tests, whereas PDXP-deficiency in skeletal muscle increased grip strength. Our findings suggest that PDXP is involved in the fine-tuning of GABA biosynthesis. Pharmacological inhibition of PDXP might correct the excitatory/inhibitory imbalance in some neuropsychiatric diseases.
Assuntos
Ansiedade/metabolismo , Encéfalo/metabolismo , Cognição/fisiologia , Músculo Esquelético/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Fosfato de Piridoxal/metabolismo , Animais , Comportamento Animal , Dopamina/metabolismo , Epinefrina/metabolismo , Eritrócitos/metabolismo , Ácido Glutâmico/metabolismo , Masculino , Memória , Camundongos , Camundongos Knockout , Modelos Animais , Neurotransmissores , Fosfoproteínas Fosfatases , Desempenho Psicomotor , Serotonina/metabolismo , Aprendizagem Espacial , Vitamina B 6/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
Mammalian phosphoglycolate phosphatase (PGP, also known as AUM or glycerol-3-phosphate phosphatase) is a small molecule-directed phosphatase important for metabolite repair and lipid metabolism. Although PGP was first characterized as an enzyme involved in epidermal growth factor (EGF) signaling, PGP protein substrates have remained elusive. Here we show that PGP depletion facilitates fatty acid flux through the intracellular triacylglycerol/fatty acid cycle, and that phosphatidylinositol-4,5-bisphosphate (PIP2), produced in a side branch of this cycle, is critical for the impact of PGP activity on EGF-induced signaling. Loss of endogenous PGP expression amplified both EGF-induced EGF receptor autophosphorylation and Src-dependent tyrosine phosphorylation of phospholipase C-γ1 (PLCγ1). Furthermore, EGF enhanced the formation of circular dorsal ruffles in PGP-depleted cells via Src/PLCγ1/protein kinase C (PKC)-dependent signaling to the cytoskeleton. Inhibition of adipose triglyceride lipase normalized the increased PIP2 content, reduced EGF-dependent PLCγ1 hyperphosphorylation, and decreased the elevated dorsal ruffle formation of PGP-depleted cells. Our data explain how PGP exerts control over EGF-induced cellular protein tyrosine phosphorylation, and reveal an unexpected influence of triacylglycerol turnover on growth factor signaling.
Assuntos
Fator de Crescimento Epidérmico/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Transdução de Sinais , Triglicerídeos/metabolismo , Linhagem Celular , Fator de Crescimento Epidérmico/genética , Humanos , Fosfatidilinositol 4,5-Difosfato/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Monoéster Fosfórico Hidrolases/genética , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Triglicerídeos/genéticaRESUMO
Mammalian phosphoglycolate phosphatase (PGP) is thought to target phosphoglycolate, a 2-deoxyribose fragment derived from the repair of oxidative DNA lesions. However, the physiological role of this activity and the biological function of the DNA damage product phosphoglycolate is unknown. We now show that knockin replacement of murine Pgp with its phosphatase-inactive PgpD34N mutant is embryonically lethal due to intrauterine growth arrest and developmental delay in midgestation. PGP inactivation attenuated triosephosphate isomerase activity, increased triglyceride levels at the expense of the cellular phosphatidylcholine content, and inhibited cell proliferation. These effects were prevented under hypoxic conditions or by blocking phosphoglycolate release from damaged DNA. Thus, PGP is essential to sustain cell proliferation in the presence of oxygen. Collectively, our findings reveal a previously unknown mechanism coupling a DNA damage repair product to the control of intermediary metabolism and cell proliferation.
Assuntos
Proliferação de Células/fisiologia , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Dano ao DNA , Reparo do DNA , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Feminino , Técnicas de Introdução de Genes , Glicolatos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oxirredução , Fosfatidilcolinas/metabolismo , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/genética , Gravidez , Triglicerídeos/metabolismo , Triose-Fosfato Isomerase/metabolismoRESUMO
Mammalian phosphatases of the haloacid dehalogenase (HAD) superfamily have emerged as important regulators of physiology and disease. Many of these enzymes are stable homodimers; however, the role of their dimerization is largely unknown. Here, we explore the function of the obligatory homodimerization of chronophin, a mammalian HAD phosphatase known to dephosphorylate pyridoxal 5'-phosphate (PLP) and serine/threonine-phosphorylated proteins. The exchange of two residues in the murine chronophin homodimerization interface (chronophin(A194K,A195K)) yields a constitutive monomer both in vitro and in cells. The catalytic activity of monomeric chronophin toward PLP is strongly impaired. X-ray crystallographic studies of chronophin(A194K,A195K) revealed that dimer formation is essential for an intermolecular arginine-arginine-tryptophan stacking interaction that positions a critical histidine residue in the substrate specificity loop of chronophin for PLP coordination. Analysis of all available crystal structures of HAD hydrolases that are grouped together with chronophin in the C2a-type structural subfamily uncovered a highly conserved mode of dimerization that results in intermolecular contacts involving the substrate specificity loop. Our results explain how the dimerization of HAD hydrolases contributes to their catalytic efficiency and substrate specificity.
Assuntos
Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/metabolismo , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/metabolismo , Fosfato de Piridoxal/metabolismo , Fatores Etários , Regulação Alostérica , Animais , Cristalografia por Raios X , Dimerização , Hidrolases/química , Hidrolases/metabolismo , Camundongos , Fosforilação , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The cellular and molecular mechanisms orchestrating the complex process by which bone marrow megakaryocytes form and release platelets remain poorly understood. Mature megakaryocytes generate long cytoplasmic extensions, proplatelets, which have the capacity to generate platelets. Although microtubules are the main structural component of proplatelets and microtubule sliding is known to drive proplatelet elongation, the role of actin dynamics in the process of platelet formation has remained elusive. Here, we tailored a mouse model lacking all ADF/n-cofilin-mediated actin dynamics in megakaryocytes to specifically elucidate the role of actin filament turnover in platelet formation. We demonstrate, for the first time, that in vivo actin filament turnover plays a critical role in the late stages of platelet formation from megakaryocytes and the proper sizing of platelets in the periphery. Our results provide the genetic proof that platelet production from megakaryocytes strictly requires dynamic changes in the actin cytoskeleton.
Assuntos
Actinas/metabolismo , Plaquetas/metabolismo , Cofilina 1/metabolismo , Destrina/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Animais , Plaquetas/citologia , Plaquetas/ultraestrutura , Western Blotting , Forma Celular , Tamanho Celular , Sobrevivência Celular , Cofilina 1/genética , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Destrina/genética , Fibrinogênio/metabolismo , Megacariócitos/citologia , Megacariócitos/metabolismo , Megacariócitos/ultraestrutura , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Contagem de Plaquetas , Esplenomegalia/genética , Esplenomegalia/metabolismo , Esplenomegalia/patologia , Trombina/farmacologia , Fatores de TempoRESUMO
Class I(B) phosphoinositide 3-kinase gamma (PI3Kgamma) elicits various immunologic and cardiovascular responses; however, the molecular basis for this signal heterogeneity is unclear. PI3Kgamma consists of a catalytic p110gamma and a regulatory p87(PIKAP) (p87, also p84) or p101 subunit. Hitherto p87 and p101 are generally assumed to exhibit redundant functions in receptor-induced and G protein betagamma (Gbetagamma)-mediated PI3Kgamma regulation. Here we investigated the molecular mechanism for receptor-dependent p87/p110gamma activation. By analyzing GFP-tagged proteins expressed in HEK293 cells, PI3Kgamma-complemented bone marrow-derived mast cells (BMMCs) from p110gamma(-/-) mice, and purified recombinant proteins reconstituted to lipid vesicles, we elucidated a novel pathway of p87-dependent, G protein-coupled receptor (GPCR)-induced PI3Kgamma activation. Although p101 strongly interacted with Gbetagamma, thereby mediating PI3Kgamma membrane recruitment and stimulation, p87 exhibited only a weak interaction, resulting in modest kinase activation and lack of membrane recruitment. Surprisingly, Ras-GTP substituted the missing Gbetagamma-dependent membrane recruitment of p87/p110gamma by direct interaction with p110gamma, suggesting the indispensability of Ras for activation of p87/p110gamma. Consequently, interference with Ras signaling indeed selectively blocked p87/p110gamma, but not p101/p110gamma, kinase activity in HEK293 and BMMC cells, revealing an important crosstalk between monomeric and trimeric G proteins for p87/p110gamma activation. Our data display distinct signaling requirements of p87 and p101, conferring signaling specificity to PI3Kgamma that could open up new possibilities for therapeutic intervention.
Assuntos
Ativação Enzimática/fisiologia , Modelos Moleculares , Fosfatidilinositol 3-Quinases/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , Proteínas ras/metabolismo , Animais , Linhagem Celular , Imunofluorescência , Proteínas de Fluorescência Verde , Humanos , Mastócitos/metabolismo , Camundongos , Microscopia ConfocalRESUMO
1. Selective protein-protein interactions between neurotransmitter transporters and their synaptic targets play important roles in regulating chemical neurotransmission. We screened a yeast two-hybrid library with bait containing the C-terminal amino acids of VGLUT1 and obtained clones that encode endophilin 1 and endophilin 3, proteins considered to play an integral role in glutamatergic vesicle formation. 2. Using a modified yeast plasmid vector to enable more cost-effective screens, we analyzed the selectivity and specificity of this interaction. Endophilins 1 and 3 selectively recognize only VGLUT1 as the C-terminus of VGLUT2 and VGLUT3 do not interact with either endophilin isoform. We mutagenized four conserved stretches of primary sequence in VGLUT1 that includes two polyproline motifs (Pro1, PPAPPP, and Pro2, PPRPPPP), found only in VGLUT1, and two conserved stretches (SEEK, SYGAT), found also in VGLUT2 and VGLUT3. The absence of the VGLUT conserved regions does not affect VGLUT1-endophilin association. Of the two polyproline stretches, only one (Pro2) is required for binding specificity to both endophilin 1 and endophilin 3. 3. We also show that endophilin 1 and endophilin 3 co-localize with VGLUT1 in synaptic terminals of differentiated rat neocortical neurons in primary culture. These results indicate that VGLUT1 and both endophilins are enriched in a class of excitatory synaptic terminals in cortical neurons and there, may interact to play an important role affecting the vesicular sequestration and synaptic release of glutamate.
Assuntos
Aciltransferases/metabolismo , Neocórtex/enzimologia , Proteínas do Tecido Nervoso/metabolismo , Vesículas Sinápticas/enzimologia , Proteína Vesicular 1 de Transporte de Glutamato/metabolismo , Animais , Células Cultivadas , Embrião de Mamíferos , Ácido Glutâmico/metabolismo , Modelos Biológicos , Neocórtex/metabolismo , Ligação Proteica , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Vesículas Sinápticas/metabolismo , Distribuição Tecidual , Técnicas do Sistema de Duplo-HíbridoRESUMO
The calcium sensor protein visinin-like protein-1 (VILIP-1) was isolated from a brain cDNA yeast two-hybrid library using the large cytoplasmic domain of the alpha4 subunit as a bait. VILIP-1 is a myristoylated calcium sensor protein that contains three functional calcium binding EF-hand motifs. The alpha4 subunit residues 302-339 were found to be essential for the interaction with VILIP-1. VILIP-1 coimmunopurified with detergent-solubilized recombinant alpha4beta2 acetylcholine receptors (AChRs) expressed in tsA201 cells and with native alpha4 AChRs isolated from brain. Coexpression of VILIP-1 with recombinant alpha4beta2 AChRs up-regulated their surface expression levels approximately 2-fold and increased their agonist sensitivity to acetylcholine approximately 3-fold. The modulation of the recombinant alpha4beta2 AChRs by VILIP-1 was attenuated in VILIP-1 mutants that lacked the ability to be myristoylated or to bind calcium. Collectively, these results suggest that VILIP-1 represents a novel modulator of alpha4beta2 AChRs that increases their surface expression levels and agonist sensitivity in response to changes in the intracellular levels of calcium.