RESUMO
We developed a significantly improved genetically encoded quantitative adenosine triphosphate (ATP) sensor to provide real-time dynamics of ATP levels in subcellular compartments. iATPSnFR2 is a variant of iATPSnFR1, a previously developed sensor that has circularly permuted superfolder green fluorescent protein (GFP) inserted between the ATP-binding helices of the ε-subunit of a bacterial F0-F1 ATPase. Optimizing the linkers joining the two domains resulted in a ~fivefold to sixfold improvement in the dynamic range compared to the previous-generation sensor, with excellent discrimination against other analytes, and affinity variants varying from 4 µM to 500 µM. A chimeric version of this sensor fused to either the HaloTag protein or a suitable spectrally separated fluorescent protein provides an optional ratiometric readout allowing comparisons of ATP across cellular regions. Subcellular targeting the sensor to nerve terminals reveals previously uncharacterized single-synapse metabolic signatures, while targeting to the mitochondrial matrix allowed direct quantitative probing of oxidative phosphorylation dynamics.
Assuntos
Trifosfato de Adenosina , Proteínas de Fluorescência Verde , Animais , Humanos , Trifosfato de Adenosina/análise , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Técnicas Biossensoriais/métodos , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , Fosforilação Oxidativa , ATPases Translocadoras de Prótons/metabolismo , ATPases Translocadoras de Prótons/genéticaRESUMO
The two most abundant molecules on synaptic vesicles (SVs) are synaptophysin and synaptobrevin-II (sybII). SybII is essential for SV fusion, whereas synaptophysin is proposed to control the trafficking of sybII after SV fusion and its retrieval during endocytosis. Despite controlling key aspects of sybII packaging into SVs, the absence of synaptophysin results in negligible effects on neurotransmission. We hypothesised that this apparent absence of effect may be because of the abundance of sybII on SVs, with the impact of inefficient sybII retrieval only revealed during periods of repeated SV turnover. To test this hypothesis, we subjected primary cultures of synaptophysin knockout neurons to repeated trains of neuronal activity, while monitoring SV fusion events and levels of vesicular sybII. We identified a significant decrease in both the number of SV fusion events (monitored using the genetically encoded reporter vesicular glutamate transporter-pHluorin) and vesicular sybII levels (via both immunofluorescence and Western blotting) using this protocol. This revealed that synaptophysin is essential to sustain both parameters during periods of repetitive SV turnover. This was confirmed by the rescue of presynaptic performance by the expression of exogenous synaptophysin. Importantly, the expression of exogenous sybII also fully restored SV fusion events in synaptophysin knockout neurons. The ability of additional copies of sybII to fully rescue presynaptic performance in these knockout neurons suggests that the principal role of synaptophysin is to mediate the efficient retrieval of sybII to sustain neurotransmitter release.
Assuntos
Neurônios/metabolismo , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo , Sinaptofisina/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Animais , Células Cultivadas , Hipocampo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Type 2 diabetes (T2D) is a complex metabolic disease associated with obesity, insulin resistance and hypoinsulinemia due to pancreatic ß-cell dysfunction. Reduced mitochondrial function is thought to be central to ß-cell dysfunction. Mitochondrial dysfunction and reduced insulin secretion are also observed in ß-cells of humans with the most common human genetic disorder, Down syndrome (DS, Trisomy 21). To identify regions of chromosome 21 that may be associated with perturbed glucose homeostasis we profiled the glycaemic status of different DS mouse models. The Ts65Dn and Dp16 DS mouse lines were hyperglycemic, while Tc1 and Ts1Rhr mice were not, providing us with a region of chromosome 21 containing genes that cause hyperglycemia. We then examined whether any of these genes were upregulated in a set of ~5,000 gene expression changes we had identified in a large gene expression analysis of human T2D ß-cells. This approach produced a single gene, RCAN1, as a candidate gene linking hyperglycemia and functional changes in T2D ß-cells. Further investigations demonstrated that RCAN1 methylation is reduced in human T2D islets at multiple sites, correlating with increased expression. RCAN1 protein expression was also increased in db/db mouse islets and in human and mouse islets exposed to high glucose. Mice overexpressing RCAN1 had reduced in vivo glucose-stimulated insulin secretion and their ß-cells displayed mitochondrial dysfunction including hyperpolarised membrane potential, reduced oxidative phosphorylation and low ATP production. This lack of ß-cell ATP had functional consequences by negatively affecting both glucose-stimulated membrane depolarisation and ATP-dependent insulin granule exocytosis. Thus, from amongst the myriad of gene expression changes occurring in T2D ß-cells where we had little knowledge of which changes cause ß-cell dysfunction, we applied a trisomy 21 screening approach which linked RCAN1 to ß-cell mitochondrial dysfunction in T2D.
Assuntos
Diabetes Mellitus Tipo 2/genética , Síndrome de Down/genética , Insulina/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Musculares/genética , Trifosfato de Adenosina/metabolismo , Aneuploidia , Animais , Proteínas de Ligação ao Cálcio , Cromossomos Humanos Par 21/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Síndrome de Down/metabolismo , Síndrome de Down/patologia , Regulação da Expressão Gênica , Glucose/metabolismo , Humanos , Hiperglicemia/genética , Hiperglicemia/metabolismo , Hiperglicemia/patologia , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas Musculares/metabolismo , Biossíntese de Proteínas/genéticaRESUMO
Central nerve terminals contain a small number of synaptic vesicles (SVs) that must sustain the fidelity of neurotransmission across a wide range of stimulation intensities. For this to be achieved, nerve terminals integrate a number of complementary endocytosis modes whose activation spans the breadth of these neuronal stimulation patterns. Two such modes are ultrafast endocytosis and activity-dependent bulk endocytosis, which are triggered by stimuli at either end of the physiological range. Both endocytosis modes generate endosomes directly from the nerve terminal plasma membrane, before the subsequent production of SVs from these structures. This review will discuss the current knowledge relating to the molecular mechanisms involved in the generation of SVs from nerve terminal endosomes, how this relates to other mechanisms of SV production and the functional role of such SVs.
Assuntos
Sistema Nervoso Central/fisiologia , Endossomos/fisiologia , Terminações Nervosas/fisiologia , Vesículas Sinápticas/fisiologia , Animais , Membrana Celular/fisiologia , Endocitose/fisiologiaRESUMO
We investigated the role of all arrestin-like proteins of Aspergillus nidulans in respect to growth, morphology, sensitivity to drugs and specifically for the endocytosis and turnover of the uric acid-xanthine transporter UapA. A single arrestin-like protein, ArtA, is essential for HulA(Rsp) (5) -dependent ubiquitination and endocytosis of UapA in response to ammonium or substrates. Mutational analysis showed that residues 545-563 of the UapA C-terminal region are required for efficient UapA endocytosis, whereas the N-terminal region (residues 2-123) and both PPxY motives are essential for ArtA function. We further show that ArtA undergoes HulA-dependent ubiquitination at residue Lys-343 and that this modification is critical for UapA ubiquitination and endocytosis. Lastly, we show that ArtA is essential for vacuolar turnover of transporters specific for purines (AzgA) or l-proline (PrnB), but not for an aspartate/glutamate transporter (AgtA). Our results are discussed within the frame of recently proposed mechanisms on how arrestin-like proteins are activated and recruited for ubiquitination of transporters in response to broad range signals, but also put the basis for understanding how arrestin-like proteins, such as ArtA, regulate the turnover of a specific transporter in the presence of its substrates.
Assuntos
Arrestina/metabolismo , Endocitose , Proteínas Fúngicas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Ubiquitinação , Arrestina/genética , Aspergillus nidulans/efeitos dos fármacos , Aspergillus nidulans/genética , Aspergillus nidulans/crescimento & desenvolvimento , Aspergillus nidulans/metabolismo , Análise Mutacional de DNA , Endocitose/fisiologia , Proteínas Fúngicas/genética , Proteínas de Membrana Transportadoras/genética , Redes e Vias Metabólicas , Prolina/metabolismo , Transporte Proteico/fisiologia , Purinas/metabolismo , Especificidade por Substrato , Ubiquitinação/fisiologia , Vacúolos/metabolismoRESUMO
Although certain drivers of familial Parkinson's disease (PD) compromise mitochondrial integrity, whether metabolic deficits underly other idiopathic or genetic origins of PD is unclear. Here, we demonstrate that phosphoglycerate kinase 1 (PGK1), a gene in the PARK12 susceptibility locus, is rate limiting in neuronal glycolysis and that modestly increasing PGK1 expression boosts neuronal adenosine 5'-triphosphate production kinetics that is sufficient to suppress PARK20-driven synaptic dysfunction. We found that this activity enhancement depends on the molecular chaperone PARK7/DJ-1, whose loss of function significantly disrupts axonal bioenergetics. In vivo, viral expression of PGK1 confers protection of striatal dopamine axons against metabolic lesions. These data support the notion that bioenergetic deficits may underpin PD-associated pathologies and point to improving neuronal adenosine 5'-triphosphate production kinetics as a promising path forward in PD therapeutics.
Assuntos
Neurônios , Doença de Parkinson , Fosfoglicerato Quinase , Fosfoglicerato Quinase/metabolismo , Fosfoglicerato Quinase/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/patologia , Animais , Neurônios/metabolismo , Camundongos , Humanos , Glicólise , Trifosfato de Adenosina/metabolismo , Metabolismo EnergéticoRESUMO
Phosphoglycerate kinase 1 (PGK1), the first ATP producing glycolytic enzyme, has emerged as a therapeutic target for Parkinson's Disease (PD), since a potential enhancer of its activity was reported to significantly lower PD risk. We carried out a suppressor screen of hypometabolic synaptic deficits and demonstrated that PGK1 is a rate limiting enzyme in nerve terminal ATP production. Increasing PGK1 expression in mid-brain dopamine neurons protected against hydroxy-dopamine driven striatal dopamine nerve terminal dysfunction in-vivo and modest changes in PGK1 activity dramatically suppressed hypometabolic synapse dysfunction in vitro. Furthermore, PGK1 is cross-regulated by PARK7 (DJ-1), a PD associated molecular chaperone, and synaptic deficits driven by PARK20 (Synaptojanin-1) can be reversed by increasing local synaptic PGK1 activity. These data indicate that nerve terminal bioenergetic deficits may underly a spectrum of PD susceptibilities and the identification of PGK1 as the limiting enzyme in axonal glycolysis provides a mechanistic underpinning for therapeutic protection.
RESUMO
Inhibitors of histone deacetylases (HDACs) have received special attention as novel anticancer agents. Among various types of synthetic inhibitors, benzamides constitute an important class, and one is an approved drug (chidamide). Here, we present a novel class of HDAC inhibitors containing the N-(2-aminophenyl)-benzamide functionality as the zinc-binding group linked to various cap groups, including the amino acids pyroglutamic acid and proline. We have identified benzamides that inhibit HADC1 and HDAC2 at nanomolar concentrations, with antiproliferative activity at micromolar concentrations against A549 and SF268 cancer cell lines. Docking studies shed light on the mode of binding of benzamide inhibitors to HDAC1, whereas cellular analysis revealed downregulated expression of EGFR mRNA and protein. Two benzamides were investigated in a mouse model of bleomycin-induced pulmonary fibrosis, and both showed efficacy on a preventative dosing schedule. N-(2-Aminophenyl)-benzamide inhibitors of class I HDACs might lead to new approaches for treating fibrotic disorders.
Assuntos
Antineoplásicos , Inibidores de Histona Desacetilases , Camundongos , Animais , Linhagem Celular , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Inibidores de Histona Desacetilases/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antineoplásicos/química , Benzamidas/farmacologia , Benzamidas/uso terapêutico , Benzamidas/química , Linhagem Celular TumoralRESUMO
The field of bioactive lipids is ever expanding with discoveries of novel lipid molecules that promote human health. Adopting a lipidomic-assisted approach, two new families of previously unrecognized saturated hydroxy fatty acids (SHFAs), namely, hydroxystearic and hydroxypalmitic acids, consisting of isomers with the hydroxyl group at different positions, were identified in milk. Among the various regio-isomers synthesized, those carrying the hydroxyl at the 7- and 9-positions presented growth inhibitory activities against various human cancer cell lines, including A549, Caco-2, and SF268 cells. In addition, 7- and 9-hydroxystearic acids were able to suppress ß-cell apoptosis induced by proinflammatory cytokines, increasing the possibility that they can be beneficial in countering autoimmune diseases, such as type 1 diabetes. 7-(R)-Hydroxystearic acid exhibited the highest potency both in cell growth inhibition and in suppressing ß-cell death. We propose that such naturally occurring SHFAs may play a role in the promotion and protection of human health.
Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ácidos Graxos/farmacologia , Animais , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Citocinas/farmacologia , Ácidos Graxos/síntese química , Ácidos Graxos/química , Expressão Gênica/efeitos dos fármacos , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Leite/química , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Ácidos Esteáricos/farmacologia , Estereoisomerismo , Espectrometria de Massas em TandemRESUMO
The accurate formation of synaptic vesicles (SVs) and incorporation of their protein cargo during endocytosis is critical for the maintenance of neurotransmission. During intense neuronal activity, a transient and acute accumulation of SV cargo occurs at the plasma membrane. Activity-dependent bulk endocytosis (ADBE) is the dominant SV endocytosis mode under these conditions; however, it is currently unknown how ADBE mediates cargo retrieval. We examined the retrieval of different SV cargo molecules during intense stimulation using a series of genetically encoded pH-sensitive reporters in neuronal cultures. The retrieval of only one reporter, VAMP4-pHluorin, was perturbed by inhibiting ADBE. This selective recovery was confirmed by the enrichment of endogenous VAMP4 in purified bulk endosomes formed by ADBE. VAMP4 was also essential for ADBE, with a cytoplasmic di-leucine motif being critical for this role. Therefore, VAMP4 is the first identified ADBE cargo and is essential for this endocytosis mode to proceed.