RESUMO
Neural circuit function requires mechanisms for controlling neurotransmitter release and the activity of neuronal networks, including modulation by synaptic contacts, synaptic plasticity, and homeostatic scaling. However, how neurons intrinsically monitor and feedback control presynaptic neurotransmitter release and synaptic vesicle (SV) recycling to restrict neuronal network activity remains poorly understood at the molecular level. Here, we investigated the reciprocal interplay between neuronal endosomes, organelles of central importance for the function of synapses, and synaptic activity. We show that elevated neuronal activity represses the synthesis of endosomal lipid phosphatidylinositol 3-phosphate [PI(3)P] by the lipid kinase VPS34. Neuronal activity in turn is regulated by endosomal PI(3)P, the depletion of which reduces neurotransmission as a consequence of perturbed SV endocytosis. We find that this mechanism involves Calpain 2-mediated hyperactivation of Cdk5 downstream of receptor- and activity-dependent calcium influx. Our results unravel an unexpected function for PI(3)P-containing neuronal endosomes in the control of presynaptic vesicle cycling and neurotransmission, which may explain the involvement of the PI(3)P-producing VPS34 kinase in neurological disease and neurodegeneration.
Assuntos
Transmissão Sináptica , Vesículas Sinápticas , Endocitose/fisiologia , Endossomos , Neurotransmissores , Fosfatos de Fosfatidilinositol , Sinapses/fisiologia , Transmissão Sináptica/fisiologiaRESUMO
PIN-FORMEDs (PINs) are auxin efflux carriers that asymmetrically target the plasma membrane (PM) and are critical for forming local auxin gradients and auxin responses. While the cytoplasmic hydrophilic loop domain of PIN (PIN-HL) is known to include some molecular cues (e.g., phosphorylation) for the modulation of PIN's intracellular trafficking and activity, the complexity of auxin responses suggests that additional regulatory modules may operate in the PIN-HL domain. Here, we have identified and characterized a PIN-HL-interacting protein (PIP) called FORMATION OF APLOID AND BINUCLEATE CELL 1C (FAB1C), a phosphatidylinositol-3-phosphate 5-kinase, which modulates PIN's lytic trafficking. FAB1C directly interacts with PIN-HL and is required for the polarity establishment and vacuolar trafficking of PINs. Unphosphorylated forms of PIN2 interact more readily with FAB1C and are more susceptible to vacuolar lytic trafficking compared to phosphorylated forms. FAB1C also affected lateral root formation by modulating the abundance of periclinally localized PIN1 and auxin maximum in the growing lateral root primordium. These findings suggest that a membrane-lipid modifier can target the cargo-including vesicle by directly interacting with the cargo and modulate its trafficking depending on the cargo's phosphorylation status.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Ácidos Indolacéticos/metabolismo , Raízes de Plantas/metabolismo , Transporte ProteicoRESUMO
The erlin1/erlin2 (E1/E2) complex is an endoplasmic reticulum membrane-located assemblage of the proteins erlin1 and erlin2. Here, we demonstrate direct and selective binding of phosphatidylinositol 3-phosphate (PI(3)P) to recombinant erlins and that disruption or deletion of the E1/E2 complex reduces HeLa cell PI(3)P levels by â¼50 %. This reduction correlated with a decrease in autophagic flux, with no effect on the endocytic pathway, and was not due to reduced VPS34 kinase activity, which is critical for maintaining steady-state PI(3)P levels. Pharmacological inhibition of VPS34 and suppression of PI(3)P levels caused a similar reduction in autophagic flux. Overall, these data indicate that by binding to PI(3)P, the E1/E2 complex plays an important role in maintaining the steady-state levels of PI(3)P and, thus, sustains some key PI(3)P-dependent processes, e.g., autophagy.
RESUMO
Autophagy-linked FYVE protein (ALFY) is a large, multidomain protein involved in the degradation of protein aggregates by selective autophagy. The C-terminal FYVE domain of ALFY has been shown to bind phosphatidylinositol 3-phosphate (PI(3)P); however, ALFY only partially colocalizes with other FYVE domains in cells. Thus, we asked if the FYVE domain of ALFY has distinct membrane binding properties compared to other FYVE domains and whether these properties might affect its function in vivo. We found that the FYVE domain of ALFY binds weakly to PI(3)P containing membranes in vitro. This weak binding is the result of a highly conserved glutamic acid within the membrane insertion loop in the FYVE domain of ALFY that is not present in any other human FYVE domain. In addition, not only does this glutamic acid reduce binding to membranes in vitro and inhibits its targeting to membranes in vivo, but it is also important for the ability of ALFY to clear protein aggregates.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Ácido Glutâmico , Proteínas Relacionadas à Autofagia , Humanos , Fosfatos de FosfatidilinositolRESUMO
Type I inositol polyphosphate-4-phosphatase (INPP4A) belongs to the group of phosphoinositide phosphatases controlling proliferation, apoptosis, and endosome function by hydrolyzing phosphatidylinositol 3,4-bisphosphate. INPP4A produces multiple transcripts encoding shorter and longer INPP4A isoforms with hydrophilic or hydrophobic C-terminus. Biallelic INPP4A truncating variants cause a spectrum of neurodevelopmental disorders ranging from moderate intellectual disability to postnatal microcephaly with developmental and epileptic encephalopathy and (ponto)cerebellar hypoplasia. We report a girl with the novel homozygous INPP4A variant NM_001134224.2:c.2840del/p.(Gly947Glufs*12) (isoform d). She presented with postnatal microcephaly, global developmental delay, visual impairment, myoclonic seizures, and pontocerebellar hypoplasia and died at the age of 27 months. The level of mutant INPP4A mRNAs in proband-derived leukocytes was comparable to controls suggesting production of C-terminally altered INPP4A isoforms. We transiently expressed eGFP-tagged INPP4A isoform a (NM_004027.3) wildtype and p.(Gly908Glufs*12) mutant [p.(Gly947Glufs*12) according to NM_001134224.2] as well as INPP4A isoform b (NM_001566.2) wildtype and p.(Asp915Alafs*2) mutant, previously reported in family members with moderate intellectual disability, in HeLa cells and determined their subcellular distributions. While INPP4A isoform a was preferentially found in perinuclear clusters co-localizing with the GTPase Rab5, isoform b showed a net-like distribution, possibly localizing near and/or on microtubules. Quantification of intracellular localization patterns of the two INPP4A mutants revealed significant differences compared with the respective wildtype and similarity with each other. Our data suggests an important non-redundant function of INPP4A isoforms with hydrophobic or hydrophilic C-terminus in the brain.
Assuntos
Deficiência Intelectual , Microcefalia , Pré-Escolar , Feminino , Humanos , Cerebelo , Células HeLa , Deficiência Intelectual/genética , Microcefalia/genética , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismoRESUMO
Breast cancer (BC) is considered the leading cause of death among females worldwide. Various risk factors contribute to BC development, such as age, genetics, reproductive factors, obesity, alcohol intake, and lifestyle. Obesity is considered to be a pandemic health problem globally, affecting millions of people worldwide. Obesity has been associated with a high risk of BC development. Determining the impact of obesity on BC development risk in women by demonstrating the molecular and genetic association in pre- and post-menopause females and risk to BC initiation is crucial in order to improve the diagnosis and prognosis of BC disease. In epidemiological studies, BC in premenopausal women was shown to be protective in a certain pattern. These altered effects between the two phases could be due to various physiological changes, such as estrogen/progesterone fluctuating levels. In addition, the relationship between BC risk and obesity is indicated by different molecular alterations as metabolic pathways and genetic mutation or epigenetic DNA changes supporting a strong connection between obesity and BC risk. However, these molecular and genetic alteration remain incompletely understood. The aim of this review is to highlight and elucidate the different molecular mechanisms and genetic changes occurring in obese women and their association with BC risk and development.
Assuntos
Neoplasias da Mama , Feminino , Humanos , Neoplasias da Mama/etiologia , Neoplasias da Mama/genética , Obesidade/complicações , Obesidade/genética , Fatores de Risco , Estrogênios , Consumo de Bebidas AlcoólicasRESUMO
Ceramide-1-phosphate transfer proteins (CPTPs) are members of the glycolipid transfer protein (GLTP) superfamily that shuttle ceramide-1-phosphate (C1P) between membranes. CPTPs regulate cellular sphingolipid homeostasis in ways that impact programmed cell death and inflammation. CPTP downregulation specifically alters C1P levels in the plasma and trans-Golgi membranes, stimulating proinflammatory eicosanoid production and autophagy-dependent inflammasome-mediated cytokine release. However, the mechanisms used by CPTP to target the trans-Golgi and plasma membrane are not well understood. Here, we monitored C1P intervesicular transfer using fluorescence energy transfer (FRET) and showed that certain phosphoinositides (phosphatidylinositol 4,5 bisphosphate (PI-(4,5)P2) and phosphatidylinositol 4-phosphate (PI-4P)) increased CPTP transfer activity, whereas others (phosphatidylinositol 3-phosphate (PI-3P) and PI) did not. PIPs that stimulated CPTP did not stimulate GLTP, another superfamily member. Short-chain PI-(4,5)P2, which is soluble and does not remain membrane-embedded, failed to activate CPTP. CPTP stimulation by physiologically relevant PI-(4,5)P2 levels surpassed that of phosphatidylserine (PS), the only known non-PIP stimulator of CPTP, despite PI-(4,5)P2 increasing membrane equilibrium binding affinity less effectively than PS. Functional mapping of mutations that led to altered FRET lipid transfer and assessment of CPTP membrane interaction by surface plasmon resonance indicated that di-arginine motifs located in the α-6 helix and the α3-α4 helix regulatory loop of the membrane-interaction region serve as PI-(4,5)P2 headgroup-specific interaction sites. Haddock modeling revealed specific interactions involving the PI-(4,5)P2 headgroup that left the acyl chains oriented favorably for membrane embedding. We propose that PI-(4,5)P2 interaction sites enhance CPTP activity by serving as preferred membrane targeting/docking sites that favorably orient the protein for function.
Assuntos
Fosfatidilinositóis/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Homeostase , Humanos , Modelos Moleculares , Proteínas de Transferência de Fosfolipídeos/química , Conformação Proteica em alfa-HéliceRESUMO
Phosphatidylinositol 3-phosphate (PI(3)P) serves important functions in endocytosis, phagocytosis, and autophagy. PI(3)P is generated by Vps34 of the class III phosphatidylinositol 3-kinase (PI3K) complex. The Vps34-PI3K complex can be divided into Vps34-PI3K class II (containing Vps38, endosomal) and Vps34-PI3K class I (containing Atg14, autophagosomal). Most PI(3)Ps are associated with endosomal membranes. In yeast, the endosomal localization of Vps34 and PI(3)P is tightly regulated by Vps21-module proteins. At yeast phagophore assembly site (PAS) or mammalian omegasomes, PI(3)P binds to WD-repeat protein interacting with phosphoinositide (WIPI) proteins to further recruit two conjugation systems, Atg5-Atg12·Atg16 and Atg8-PE (LC3-II), to initiate autophagy. However, the spatiotemporal regulation of PI(3)P during autophagy remains obscure. Therefore, in this study, we determined the effect of Vps21 on localization and interactions of Vps8, Vps34, Atg21, Atg8, and Atg16 upon autophagy induction. The results showed that Vps21 was required for successive colocalizations and interactions of Vps8-Vps34 and Vps34-Atg21 on endosomes, and Atg21-Atg8/Atg16 on the PAS. In addition to disrupted localization of the PI3K complex II subunits Vps34 and Vps38 on endosomes, the localization of the PI3K complex I subunits Vps34 and Atg14, as well as Atg21, was partly disrupted from the PAS in vps21∆ cells. The impaired PI3K-PI(3)P-Atg21-Atg16 axis in vps21∆ cells might delay autophagy, which is consistent with the delay of early autophagy when Atg21 was absent. This study provides the first insight into the upstream sequential regulation of the PI3K-PI(3)P-Atg21-Atg16 module by Vps21 in autophagy.
Assuntos
Autofagossomos , Proteínas de Saccharomyces cerevisiae , Animais , Autofagossomos/metabolismo , Autofagia , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Endopeptidases/metabolismo , Mamíferos/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatos de Fosfatidilinositol , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
A main characteristic of sphingolipids is the presence of a very long chain fatty acid (VLCFA) whose function in cellular processes is not yet fully understood. VLCFAs of sphingolipids are involved in the intracellular traffic to the vacuole and the maturation of early endosomes into late endosomes is one of the major pathways for vacuolar traffic. Additionally, the anionic phospholipid phosphatidylinositol-3-phosphate (PtdIns (3)P or PI3P) is involved in protein sorting and recruitment of small GTPase effectors at late endosomes/multivesicular bodies (MVBs) during vacuolar trafficking. In contrast to animal cells, PI3P mainly localizes to late endosomes in plant cells and to a minor extent to a discrete sub-domain of the plant's early endosome (EE)/trans-Golgi network (TGN) where the endosomal maturation occurs. However, the mechanisms that control the relative levels of PI3P between TGN and MVBs are unknown. Using metazachlor, an inhibitor of VLCFA synthesis, we found that VLCFAs are involved in the TGN/MVB distribution of PI3P. This effect is independent from either synthesis of PI3P by PI3-kinase or degradation of PI(3,5)P2 into PI3P by the SUPPRESSOR OF ACTIN1 (SAC1) phosphatase. Using high-resolution live cell imaging microscopy, we detected transient associations between TGNs and MVBs but VLCFAs are not involved in those interactions. Nonetheless, our results suggest that PI3P might be transferable from TGN to MVBs and that VLCFAs act in this process.
Assuntos
Arabidopsis/metabolismo , Endossomos/metabolismo , Ácidos Graxos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Vias Biossintéticas , Esfingolipídeos/metabolismo , Rede trans-Golgi/metabolismoRESUMO
Macrophage phagocytosis is required for effective clearance of invading bacteria and other microbes. Coordinated phosphoinositide signaling is critical both for phagocytic particle engulfment and subsequent phagosomal maturation to a degradative organelle. Phosphatidylinositol 3-phosphate (PtdIns(3)P) is a phosphoinositide that is rapidly synthesized and degraded on phagosomal membranes, where it recruits FYVE domain- and PX motif-containing proteins that promote phagosomal maturation. However, the molecular mechanisms that regulate PtdIns(3)P removal from the phagosome have remained unclear. We report here that a myotubularin PtdIns(3)P 3-phosphatase, myotubularin-related protein-4 (MTMR4), regulates macrophage phagocytosis. MTMR4 overexpression reduced and siRNA-mediated Mtmr4 silencing increased levels of cell-surface immunoglobulin receptors (i.e. Fcγ receptors (FcγRs)) on RAW 264.7 macrophages, associated with altered pseudopodal F-actin. Furthermore, MTMR4 negatively regulated the phagocytosis of IgG-opsonized particles, indicating that MTMR4 inhibits FcγR-mediated phagocytosis, and was dynamically recruited to phagosomes of macrophages during phagocytosis. MTMR4 overexpression decreased and Mtmr4-specific siRNA expression increased the duration of PtdIns(3)P on phagosomal membranes. Macrophages treated with Mtmr4-specific siRNA were more resistant to Mycobacterium marinum-induced phagosome arrest, associated with increased maturation of mycobacterial phagosomes, indicating that extended PtdIns(3)P signaling on phagosomes in the Mtmr4-knockdown cells permitted trafficking of phagosomes to acidic late endosomal and lysosomal compartments. In conclusion, our findings indicate that MTMR4 regulates PtdIns(3)P degradation in macrophages and thereby controls phagocytosis and phagosomal maturation.
Assuntos
Fagocitose , Fagossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Actinas/metabolismo , Animais , Endossomos/metabolismo , Humanos , Imunoglobulina G/imunologia , Lisossomos/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Mycobacterium marinum/patogenicidade , Proteínas Tirosina Fosfatases não Receptoras/antagonistas & inibidores , Proteínas Tirosina Fosfatases não Receptoras/genética , Células RAW 264.7 , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores de IgG/metabolismo , Transdução de SinaisRESUMO
Phosphoinositides (PIs) form a minor class of phospholipids with crucial functions in cell physiology, ranging from cell signalling and motility to a role as signposts of compartmental membrane identity. Phosphatidylinositol 3-phosphates are present at the plasma membrane and within the endolysosomal system, where they serve as key regulators of both cell signalling and of intracellular membrane traffic. Here, we provide an overview of the metabolic pathways that regulate cellular synthesis of PI 3-phosphates at distinct intracellular sites and discuss the mechanisms by which these lipids regulate cell signalling and membrane traffic. Finally, we provide a framework for how PI 3-phosphate metabolism is integrated into the cellular network.
Assuntos
Membrana Celular/metabolismo , Fenômenos Fisiológicos Celulares , Membranas Intracelulares/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Transdução de Sinais , Redes Reguladoras de Genes , Redes e Vias MetabólicasRESUMO
MAIN CONCLUSION: Genome-wide identification, together with gene expression patterns and promoter region analysis of FYVE and PHOX proteins in Physcomitrella patens, emphasized their importance in regulating mainly developmental processes in P. patens. Phosphatidylinositol 3-phosphate (PtdIns3P) is a signaling phospholipid, which regulates several aspects of plant growth and development, as well as responses to biotic and abiotic stresses. The mechanistic insights underlying PtdIns3P mode of action, specifically through effector proteins have been partially explored in plants, with main focus on Arabidopsis thaliana. In this study, we searched for genes coding for PtdIns3P-binding proteins such as FYVE and PHOX domain-containing sequences from different photosynthetic organisms to gather evolutionary insights on these phosphoinositide binding domains, followed by an in silico characterization of the FYVE and PHOX gene families in the moss Physcomitrella patens. Phylogenetic analysis showed that PpFYVE proteins can be grouped in 7 subclasses, with an additional subclass whose FYVE domain was lost during evolution to higher plants. On the other hand, PpPHOX proteins are classified into 5 subclasses. Expression analyses based on RNAseq data together with the analysis of cis-acting regulatory elements and transcription factor (TF) binding sites in promoter regions suggest the importance of these proteins in regulating stress responses but mainly developmental processes in P. patens. The results provide valuable information and robust candidate genes for future functional analysis aiming to further explore the role of this signaling pathway mainly during growth and development of tip growing cells and during the transition from 2 to 3D growth. These studies would identify ancestral regulatory players undertaken during plant evolution.
Assuntos
Bryopsida/genética , Evolução Molecular , Proteínas de Plantas/genética , Cromossomos de Plantas/genética , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Genes de Plantas , Anotação de Sequência Molecular , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas/genética , Domínios Proteicos , Estresse Fisiológico/genéticaRESUMO
Dictyostelium cells are professional phagocytes that are capable of handling particles of variable shapes and sizes. Here we offer long bacteria that challenge the uptake mechanism to its limits and report on the responses of the phagocytes if they are unable to engulf the particle by closing the phagocytic cup. Reasons for failure may be a length of the particle much larger than the phagocyte's diameter, or competition with another phagocyte. A cell may simultaneously release a particle and engulf another one. The final phase of release can be fast, causing the phagosome membrane to turn inside-out and to form a bleb. Myosin-II may be involved in the release by generating tension at the plasma membrane, it does however not accumulate on the phagosome to act there directly in expelling the particle. Labeling with GFP-2FYVE indicates that processing of the phagosome with phosphatidylinositol 3-phosphate begins at the base of a long phagosome already before closure of the cup. The decision of releasing the particle can be made even at the stage of the processed phagosome.
Assuntos
Dictyostelium/citologia , Fagocitose , Bactérias/citologia , Fagócitos/citologia , Fagossomos/metabolismoRESUMO
"Too much of a good thing" perfectly describes the dilemma that living organisms face with metals. The tight control of metal homeostasis in cells depends on the trafficking of metal transporters between membranes of different compartments. However, the mechanisms regulating the location of transport proteins are still largely unknown. Developing Arabidopsis thaliana seedlings require the natural resistance-associated macrophage proteins (NRAMP3 and NRAMP4) transporters to remobilize iron from seed vacuolar stores and thereby acquire photosynthetic competence. Here, we report that mutations in the pleckstrin homology (PH) domain-containing protein AtPH1 rescue the iron-deficient phenotype of nramp3nramp4 Our results indicate that AtPH1 binds phosphatidylinositol 3-phosphate (PI3P) in vivo and acts in the late endosome compartment. We further show that loss of AtPH1 function leads to the mislocalization of the metal uptake transporter NRAMP1 to the vacuole, providing a rationale for the reversion of nramp3nramp4 phenotypes. This work identifies a PH domain protein as a regulator of plant metal transporter localization, providing evidence that PH domain proteins may be effectors of PI3P for protein sorting.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Metais/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Raízes de Plantas/metabolismo , Sequência de Aminoácidos , Arabidopsis/crescimento & desenvolvimento , Sequência de Bases , Transporte de Íons , Mutação , Fenótipo , Raízes de Plantas/crescimento & desenvolvimentoRESUMO
Phosphatidylinositol 3-phosphate (PI(3)P) is the predominant phosphoinositide species in early endosomes and autophagosomes, in which PI(3)P dictates traffic of these organelles. Phosphoinositide levels are tightly regulated by lipid-kinases and -phosphatases; however, a phosphatase that converts PI(3)P back to phosphatidylinositol in the endosomal and autophagosomal compartments is not fully understood. We investigated the subcellular distribution and functions of myotubularin-related protein-4 (MTMR4), which is distinct among other MTMRs in that it possesses a PI(3)P-binding FYVE domain, in lung alveolar epithelium-derived A549 cells. MTMR4 was localized mainly in late endosomes and autophagosomes. MTMR4 knockdown markedly suppressed the motility, fusion, and fission of PI(3)P-enriched structures, resulting in decreases in late endosomes, autophagosomes, and lysosomes, and enlargement of PI(3)P-enriched early and late endosomes. In amino acid- and serum-starved cells, MTMR4 knockdown decreased both autophagosomes and autolysosomes and markedly increased PI(3)P-containing autophagosomes and late endosomes, suggesting that the fusion with lysosomes of autophagosomes and late endosomes might be impaired. Notably, MTMR4 knockdown inhibited the nuclear translocation of starvation stress responsive transcription factor-EB (TFEB) with reduced expression of lysosome-related genes in starved cells. These findings indicate that MTMR4 is essential for the integrity of endocytic and autophagic pathways.
RESUMO
The intermediate conductance calcium-activated potassium channel (KCa3.1) mediates proliferation of many cell types including fibroblasts, and is a molecular target for intervention in various cell proliferative diseases. Our previous study showed that reduction of KCa3.1 channel expression by lyso-globotriaosylceramide (lyso-Gb3) inhibits differentiation into myofibroblasts and collagen synthesis, which might lead to development of ascending thoracic aortic aneurysm secondary to Fabry disease. However, how lyso-Gb3 downregulates KCa3.1 channel expression is unknown. Therefore, we aimed to investigate the underlying mechanisms of lyso-Gb3-mediated KCa3.1 channel downregulation, focusing on the cAMP signaling pathway. We found that lyso-Gb3 increased the intracellular cAMP concentration by upregulation of adenylyl cyclase 6 and inhibited ERK 1/2 phosphorylation through the protein kinase A (PKA) pathway, leading to the inhibition of KCa3.1 channel synthesis, not the exchange protein directly activated by cAMP (Epac) pathway. Moreover, lyso-Gb3 suppressed expression of class II phosphatidylinositol 3-kinase C2ß (PI3KC2ß) by PKA activation, which reduces the production of phosphatidylinositol 3-phosphate [PI(3)P], and the reduced membrane surface expression of KCa3.1 channel was recovered by increasing the intracellular levels of PI(3)P. Consequently, our findings that lyso-Gb3 inhibited both KCa3.1 channel synthesis and surface expression by increasing intracellular cAMP, and controlled surface expression through changes in PI3KC2ß-mediated PI(3)P production, suggest that modulation of PKA and PI3KC2ß activity to control of KCa3.1 channel expression can be an alternative important target to attenuate ascending thoracic aortic aneurysms in Fabry disease.
Assuntos
Membrana Celular/metabolismo , Classe II de Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Glicolipídeos/administração & dosagem , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/biossíntese , Fosfatos de Fosfatidilinositol/metabolismo , Esfingolipídeos/administração & dosagem , Animais , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/efeitos dos fármacos , Camundongos , Células NIH 3T3RESUMO
The internalization of some oomycete and fungal pathogen effectors into host plant cells has been reported to be blocked by proteins that bind to the effectors' cell entry receptor, phosphatidylinositol-3-phosphate (PI3P). This finding suggested a novel strategy for disease control by engineering plants to secrete PI3P-binding proteins. In this study, we tested this strategy using the chocolate tree Theobroma cacao. Transient expression and secretion of four different PI3P-binding proteins in detached leaves of T. cacao greatly reduced infection by two oomycete pathogens, Phytophthora tropicalis and Phytophthora palmivora, which cause black pod disease. Lesion size and pathogen growth were reduced by up to 85%. Resistance was not conferred by proteins lacking a secretory leader, by proteins with mutations in their PI3P-binding site, or by a secreted PI4P-binding protein. Stably transformed, transgenic T. cacao plants expressing two different PI3P-binding proteins showed substantially enhanced resistance to both P. tropicalis and P. palmivora, as well as to the fungal pathogen Colletotrichum theobromicola. These results demonstrate that secretion of PI3P-binding proteins is an effective way to increase disease resistance in T. cacao, and potentially in other plants, against a broad spectrum of pathogens.
Assuntos
Cacau/microbiologia , Colletotrichum/fisiologia , Resistência à Doença , Proteínas de Ligação a Fosfato/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Phytophthora/fisiologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Cacau/genética , Regulação da Expressão Gênica de Plantas , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteínas de Ligação a Fosfato/química , Phytophthora/patogenicidade , Folhas de Planta/microbiologia , Proteínas de Plantas/química , Plantas Geneticamente Modificadas , Domínios Proteicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Transformação GenéticaRESUMO
We report a new step in the fertilization in Xenopus laevis which has been found to involve activation of Src tyrosine kinase to stimulate phospholipase C-γ (PLC-γ) which increases inositol 1,4,5-trisphosphate (IP3) to release intracellular calcium ([Ca](i)). Molecular species analysis and mass measurements suggested that sperm activate phospholipase D (PLD) to elevate phosphatidic acid (PA). We now report that PA mass increased 2.7 fold by 1 min after insemination and inhibition of PA production by two methods inhibited activation of Src and PLCγ, increased [Ca](i) and other fertilization events. As compared to 14 other lipids, PA specifically bound Xenopus Src but not PLCγ. Addition of synthetic PA activated egg Src (an action requiring intact lipid rafts) and PLCγ as well as doubling the amount of PLCγ in rafts. In the absence of elevated [Ca](i), PA addition elevated IP3 mass to levels equivalent to that induced by sperm (but twice that achieved by calcium ionophore). Finally, PA induced [Ca](i) release that was blocked by an IP3 receptor inhibitor. As only PLD1b message was detected, and Western blotting did not detect PLD2, we suggest that sperm activate PLD1b to elevate PA which then binds to and activates Src leading to PLCγ stimulation, IP3 elevation and [Ca](i) release. Due to these and other studies, PA may also play a role in membrane fusion events such as sperm-egg fusion, cortical granule exocytosis, the elevation of phosphatidylinositol 4,5-bisphosphate and the large, late increase in sn 1,2-diacylglycerol in fertilization.
Assuntos
Cálcio/metabolismo , Fertilização , Ácidos Fosfatídicos/metabolismo , Fosfolipase C gama/metabolismo , Xenopus laevis/metabolismo , Quinases da Família src/metabolismo , 1-Butanol/química , Sequência de Aminoácidos , Animais , Diglicerídeos/química , Ativação Enzimática , Exocitose , Feminino , Concentração Inibidora 50 , Lipídeos/química , Masculino , Microdomínios da Membrana , Dados de Sequência Molecular , Ligação Proteica , Isoformas de Proteínas/metabolismo , Espermatozoides/metabolismo , Fatores de Tempo , XenopusRESUMO
Induction of autophagy requires the ULK1 protein kinase complex and the Vps34 lipid kinase complex. PtdIns3P synthesised by Vps34 accumulates in omegasomes, membrane extensions of the ER within which some autophagosomes form. The ULK1 complex is thought to target autophagosomes independently of PtdIns3P, and its functional relationship to omegasomes is unclear. Here we show that the ULK1 complex colocalises with omegasomes in a PtdIns3P-dependent way. Live-cell imaging of Atg13 (a ULK1 complex component), omegasomes and LC3 establishes and annotates for the first time a complete sequence of steps leading to autophagosome formation, as follows. Upon starvation, the ULK1 complex forms puncta associated with the ER and sporadically with mitochondria. If PtdIns3P is available, these puncta become omegasomes. Subsequently, the ULK1 complex exits omegasomes and autophagosomes bud off. If PtdIns3P is unavailable, ULK1 puncta are greatly reduced in number and duration. Atg13 contains a region with affinity for acidic phospholipids, required for translocation to punctate structures and autophagy progression.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Autofagia/genética , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Classe III de Fosfatidilinositol 3-Quinases/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Fagossomos/metabolismo , Fosfatos de Fosfatidilinositol/química , Fosfatos de Fosfatidilinositol/genética , Fosfatos de Fosfatidilinositol/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Vacúolos/metabolismoRESUMO
Mutations in the gene encoding comparative gene identification 58 (CGI-58)/α/ß hydrolase domain 5 (ABHD5) cause Chanarin-Dorfman syndrome, characterized by excessive triacylglycerol storage in cells and tissues. CGI-58 has been identified as a coactivator of adipose TG lipase (ATGL) and a lysophosphatidic acid acyltransferase (LPAAT). We developed a molecular model of CGI-58 structure and then mutated predicted active site residues and performed LPAAT activity assays of recombinant WT and mutated CGI-58. When mutations of predicted catalytic residues failed to reduce LPAAT activity, we determined that LPAAT activity was due to a bacterial contaminant of affinity purification procedures, plsC, the sole LPAAT in Escherichia coli Purification protocols were optimized to reduce plsC contamination, in turn reducing LPAAT activity. When CGI-58 was expressed in SM2-1(DE3) cells that lack plsC, lysates lacked LPAAT activity. Additionally, mouse CGI-58 expressed in bacteria as a glutathione-S-transferase fusion protein and human CGI-58 expressed in yeast lacked LPAAT activity. Previously reported lipid binding activity of CGI-58 was revisited using protein-lipid overlays. Recombinant CGI-58 failed to bind lysophosphatidic acid, but interestingly, bound phosphatidylinositol 3-phosphate [PI(3)P] and phosphatidylinositol 5-phosphate [PI(5)P]. Prebinding CGI-58 with PI(3)P or PI(5)P did not alter its coactivation of ATGL in vitro. In summary, purified recombinant CGI-58 that is functional as an ATGL coactivator lacks LPAAT activity.