Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.777
Filtrar
1.
Nat Chem Biol ; 16(4): 400-407, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32198492

RESUMO

The signal transduction enzyme phospholipase D1 (PLD1) hydrolyzes phosphatidylcholine to generate the lipid second-messenger phosphatidic acid, which plays roles in disease processes such as thrombosis and cancer. PLD1 is directly and synergistically regulated by protein kinase C, Arf and Rho GTPases, and the membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2). Here, we present a 1.8 Å-resolution crystal structure of the human PLD1 catalytic domain, which is characterized by a globular fold with a funnel-shaped hydrophobic cavity leading to the active site. Adjacent is a PIP2-binding polybasic pocket at the membrane interface that is essential for activity. The C terminus folds into and contributes part of the catalytic pocket, which harbors a phosphohistidine that mimics an intermediate stage of the catalytic cycle. Mapping of PLD1 mutations that disrupt RhoA activation identifies the RhoA-PLD1 binding interface. This structure sheds light on PLD1 regulation by lipid and protein effectors, enabling rationale inhibitor design for this well-studied therapeutic target.


Assuntos
Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipase D/metabolismo , Fosfolipase D/ultraestrutura , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Células COS , Catálise , Domínio Catalítico , Chlorocebus aethiops , Humanos , Lipídeos de Membrana , Fosfatidilcolinas , Ligação Proteica , Proteína Quinase C/metabolismo , Sistemas do Segundo Mensageiro , Transdução de Sinais/efeitos dos fármacos
2.
Nat Commun ; 11(1): 231, 2020 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-31932584

RESUMO

The opening of a fusion pore during exocytosis creates the first aqueous connection between the lumen of a vesicle and the extracellular space. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate the formation of these dynamic structures, and their kinetic transitions are tightly regulated by accessory proteins at the synapse. Here, we utilize two single molecule approaches, nanodisc-based planar bilayer electrophysiology and single-molecule FRET, to address the relationship between SNARE complex assembly and rapid (micro-millisecond) fusion pore transitions, and to define the role of accessory proteins. Synaptotagmin (syt) 1, a major Ca2+-sensor for synaptic vesicle exocytosis, drove the formation of an intermediate: committed trans-SNARE complexes that form large, stable pores. Once open, these pores could only be closed by the action of the ATPase, NSF. Time-resolved measurements revealed that NSF-mediated pore closure occurred via a complex 'stuttering' mechanism. This simplified system thus reveals the dynamic formation and dissolution of fusion pores.


Assuntos
Cálcio/metabolismo , Fusão de Membrana , Proteínas Sensíveis a N-Etilmaleimida/metabolismo , Proteínas SNARE/metabolismo , Sinaptotagmina I/metabolismo , Animais , Exocitose , Cinética , Bicamadas Lipídicas/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Multimerização Proteica , Ratos , Proteínas SNARE/química , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Vesículas Sinápticas/metabolismo , Sinaptotagmina I/genética
3.
Mol Pharmacol ; 97(3): 145-158, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31871302

RESUMO

Smooth muscle cells express Kv7.4 and Kv7.5 voltage-dependent potassium channels, which have each been implicated as regulators of smooth muscle contractility, though they display different sensitivities to signaling via cAMP/protein kinase A (PKA) and protein kinase C (PKC). We expressed chimeric channels composed of different components of the Kv7.4 and Kv7.5 α-subunits in vascular smooth muscle cells to determine which components are essential for enhancement or inhibition of channel activity. Forskolin, an activator of the cAMP/PKA pathway, increased wild-type Kv7.5 but not wild-type Kv7.4 current amplitude. Replacing the amino terminus of Kv7.4 with the amino terminus of Kv7.5 conferred partial responsiveness to forskolin. In contrast, swapping carboxy-terminal phosphatidylinositol 4,5-bisphosphate (PIP2) binding domains, or the entire C terminus, was without effect on the forskolin response, but the latter conferred responsiveness to arginine-vasopressin (an inhibitory PKC-dependent response). Serine-to-alanine mutation at position 53 of the Kv7.5 amino terminus abrogated its ability to confer forskolin sensitivity to Kv7.4. Forskolin treatment reduced the sensitivity of Kv7.5 channels to Ciona intestinalis voltage-sensing phosphatase (Ci-VSP)-induced PIP2 depletion, whereas activation of PKC with phorbol-12-myristate-13-acetate potentiated the Ci-VSP-induced decline in Kv7.5 current amplitude. Our findings suggest that PKA-dependent phosphorylation of serine 53 on the amino terminus of Kv7.5 increases its affinity for PIP2, whereas PKC-dependent phosphorylation of the Kv7.5 carboxy terminus is associated with a reduction in PIP2 affinity; these changes in PIP2 affinity have corresponding effects on channel activity. Resting affinities for PIP2 differ for Kv7.4 and Kv7.5 based on differential responsiveness to Ci-VSP activation and different rates of current rundown in ruptured patch recordings. SIGNIFICANCE STATEMENT: Kv7.4 and Kv7.5 channels are known signal transduction intermediates and drug targets for regulation of smooth muscle tone. The present studies identify distinct functional domains that confer differential sensitivities of Kv7.4 and Kv7.5 to stimulatory and inhibitory signaling and reveal structural features of the channel subunits that determine their biophysical properties. These findings may improve our understanding of the roles of these channels in smooth muscle physiology and disease, particularly in conditions where Kv7.4 and Kv7.5 are differentially expressed.


Assuntos
Canais de Potássio KCNQ/química , Canais de Potássio KCNQ/metabolismo , Miócitos de Músculo Liso/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Animais , Cardiotônicos/farmacologia , Linhagem Celular , Colforsina/farmacologia , Humanos , Canais de Potássio KCNQ/genética , Miócitos de Músculo Liso/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
4.
Int J Mol Sci ; 20(18)2019 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-31500374

RESUMO

Alcohol causes diverse acute and chronic symptoms that often lead to critical health problems. Exposure to ethanol alters the activities of sympathetic neurons that control the muscles, eyes, and blood vessels in the brain. Although recent studies have revealed the cellular targets of ethanol, such as ion channels, the molecular mechanism by which alcohol modulates the excitability of sympathetic neurons has not been determined. Here, we demonstrated that ethanol increased the discharge of membrane potentials in sympathetic neurons by inhibiting the M-type or Kv7 channel consisting of the Kv7.2/7.3 subunits, which were involved in determining the membrane potential and excitability of neurons. Three types of sympathetic neurons, classified by their threshold of activation and firing patterns, displayed distinct sensitivities to ethanol, which were negatively correlated with the size of the Kv7 current that differs depending on the type of neuron. Using a heterologous expression system, we further revealed that the inhibitory effects of ethanol on Kv7.2/7.3 currents were facilitated or diminished by adjusting the amount of plasma membrane phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). These results suggested that ethanol and PI(4,5)P2 modulated gating of the Kv7 channel in superior cervical ganglion neurons in an antagonistic manner, leading to regulation of the membrane potential and neuronal excitability, as well as the physiological functions mediated by sympathetic neurons.


Assuntos
Potenciais de Ação , Etanol/metabolismo , Canais de Potássio KCNQ/metabolismo , Neurônios/fisiologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Gânglio Cervical Superior/citologia , Biomarcadores , Membrana Celular/metabolismo , Células Cultivadas , Etanol/farmacologia , Expressão Gênica , Canais de Potássio KCNQ/antagonistas & inibidores , Canais de Potássio KCNQ/genética
5.
Int J Mol Sci ; 20(15)2019 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-31382643

RESUMO

Plasma membrane (PM) lipid composition and domain organization are modulated by polarized exocytosis. Conversely, targeting of secretory vesicles at specific domains in the PM is carried out by exocyst complexes, which contain EXO70 subunits that play a significant role in the final recognition of the target membrane. As we have shown previously, a mature Arabidopsis trichome contains a basal domain with a thin cell wall and an apical domain with a thick secondary cell wall, which is developed in an EXO70H4-dependent manner. These domains are separated by a cell wall structure named the Ortmannian ring. Using phospholipid markers, we demonstrate that there are two distinct PM domains corresponding to these cell wall domains. The apical domain is enriched in phosphatidic acid (PA) and phosphatidylserine, with an undetectable amount of phosphatidylinositol 4,5-bisphosphate (PIP2), whereas the basal domain is PIP2-rich. While the apical domain recruits EXO70H4, the basal domain recruits EXO70A1, which corresponds to the lipid-binding capacities of these two paralogs. Loss of EXO70H4 results in a loss of the Ortmannian ring border and decreased apical PA accumulation, which causes the PA and PIP2 domains to merge together. Using transmission electron microscopy, we describe these accumulations as a unique anatomical feature of the apical cell wall-radially distributed rod-shaped membranous pockets, where both EXO70H4 and lipid markers are immobilized.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Lipídeos de Membrana/genética , Proteínas de Transporte Vesicular/genética , Arabidopsis/química , Proteínas de Arabidopsis/química , Membrana Celular/química , Membrana Celular/genética , Exocitose/genética , Lipídeos de Membrana/metabolismo , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/genética , Tricomas/química , Tricomas/genética , Proteínas de Transporte Vesicular/química
6.
EMBO J ; 38(17): e101289, 2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31368584

RESUMO

Synapse development requires spatiotemporally regulated recruitment of synaptic proteins. In this study, we describe a novel presynaptic mechanism of cis-regulated oligomerization of adhesion molecules that controls synaptogenesis. We identified synaptic adhesion-like molecule 1 (SALM1) as a constituent of the proposed presynaptic Munc18/CASK/Mint1/Lin7b organizer complex. SALM1 preferentially localized to presynaptic compartments of excitatory hippocampal neurons. SALM1 depletion in excitatory hippocampal primary neurons impaired Neurexin1ß- and Neuroligin1-mediated excitatory synaptogenesis and reduced synaptic vesicle clustering, synaptic transmission, and synaptic vesicle release. SALM1 promoted Neurexin1ß clustering in an F-actin- and PIP2-dependent manner. Two basic residues in SALM1's juxtamembrane polybasic domain are essential for this clustering. Together, these data show that SALM1 is a presynaptic organizer of synapse development by promoting F-actin/PIP2-dependent clustering of Neurexin.


Assuntos
Actinas/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Sinapses/metabolismo , Animais , Moléculas de Adesão Celular Neuronais/metabolismo , Células Cultivadas , Células HEK293 , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Camundongos , Proteínas do Tecido Nervoso/genética , Neurogênese
7.
EMBO J ; 38(8): e100312, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-31368593

RESUMO

The small GTPase Rab7 is a key organizer of receptor sorting and lysosomal degradation by recruiting of a variety of effectors depending on its GDP/GTP-bound state. However, molecular mechanisms that trigger Rab7 inactivation remain elusive. Here we find that, among the endosomal pools, Rab7-positive compartments possess the highest level of PI4P, which is primarily produced by PI4K2A kinase. Acute conversion of this endosomal PI4P to PI(4,5)P2 causes Rab7 dissociation from late endosomes and releases a regulator of autophagosome-lysosome fusion, PLEKHM1, from the membrane. Rab7 effectors Vps35 and RILP are not affected by acute PI(4,5)P2 production. Deletion of PI4K2A greatly reduces PIP5Kγ-mediated PI(4,5)P2 production in Rab7-positive endosomes leading to impaired Rab7 inactivation and increased number of LC3-positive structures with defective autophagosome-lysosome fusion. These results reveal a late endosomal PI4P-PI(4,5)P2 -dependent regulatory loop that impacts autophagosome flux by affecting Rab7 cycling and PLEKHM1 association.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagossomos/metabolismo , Endossomos/metabolismo , Lisossomos/metabolismo , Fusão de Membrana , Glicoproteínas de Membrana/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Endocitose , Células HEK293 , Humanos , Ligação Proteica , Transporte Proteico
8.
Biochim Biophys Acta Mol Cell Res ; 1866(10): 1595-1607, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31301364

RESUMO

The rapid and precise clearance of apoptotic cells (efferocytosis) involves a series of phagocytic processes through which apoptotic cells are recognized, engulfed, and degraded within phagocytes. The Rho-family GTPases critically rearrange the cytoskeleton for these phagocytic processes, but we know little about the mechanisms by which regulatory proteins control the spatiotemporal activities of the Rho-family GTPases. Here, we identify ArhGAP12 as a functional GTPase-activating protein (GAP) of Rac1 during Stabilin-2 mediated efferocytosis. ArhGAP12 constitutively forms a complex with the phosphatidylserine receptor, Stabilin-2, via direct interaction with the downstream protein, GULP, but is released from the complex when Stabilin-2 interacts with apoptotic cells. When the phagocytic cup is closed and the apoptotic cell is surrounded by the phagosomal membrane, ArhGAP12 localizes to the phagocytic cup via a specific interaction with phosphatidylinositol-4,5-bisphosphate, which is transiently biosynthesized in the phagocytic cup. Down-regulation of ArhGAP12 results in sustained Rac1 activity, arrangement of F-actin, and delayed phagosome-lysosome fusion. Our results collectively suggest that ArhGAP12 carries dual roles in Stabilin-2 mediated efferocytosis: it binds to GULP/Stabilin-2 and switches off Rac1 basal activity and switches on the Rac1 by releasing itself from the complex. In addition, the spatiotemporal membrane targeting of ArhGAP12 inactivates Rac1 in a time-specific and spatially coordinated manner to orchestrate phagosome maturation. This may shed light on how other RhoGAPs spatiotemporally inactivate Rac or Cdc42 during phagocytosis by various cells, in different circumstances.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fagocitose/fisiologia , Fagossomos/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Moléculas de Adesão Celular Neuronais/farmacologia , Linhagem Celular , Regulação para Baixo , Proteínas Ativadoras de GTPase/genética , Técnicas de Silenciamento de Genes , Humanos , Lisossomos/metabolismo , Camundongos , Fagócitos , Fagocitose/efeitos dos fármacos , Fosfatidilinositol 4,5-Difosfato/metabolismo
9.
Cells ; 8(7)2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31261688

RESUMO

The many functions of phosphoinositides in cytosolic signaling were extensively studied; however, their activities in the cell nucleus are much less clear. In this review, we summarize data about their nuclear localization and metabolism, and review the available literature on their involvements in chromatin remodeling, gene transcription, and RNA processing. We discuss the molecular mechanisms via which nuclear phosphoinositides, in particular phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2), modulate nuclear processes. We focus on PI(4,5)P2's role in the modulation of RNA polymerase I activity, and functions of the nuclear lipid islets-recently described nucleoplasmic PI(4,5)P2-rich compartment involved in RNA polymerase II transcription. In conclusion, the high impact of the phosphoinositide-protein complexes on nuclear organization and genome functions is only now emerging and deserves further thorough studies.


Assuntos
Núcleo Celular/metabolismo , Eucariotos/genética , Genoma , Fosfatidilinositol 4,5-Difosfato/metabolismo , RNA Polimerase II/metabolismo , RNA Polimerase I/metabolismo , Núcleo Celular/genética , Montagem e Desmontagem da Cromatina , Eucariotos/metabolismo , Ligação Proteica/fisiologia , Processamento Pós-Transcricional do RNA , Transcrição Genética
10.
Int J Mol Sci ; 20(12)2019 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-31248120

RESUMO

Polyphosphoinositides (PPIns) are a family of seven lipid messengers that regulate a vast array of signalling pathways to control cell proliferation, migration, survival and differentiation. PPIns are differentially present in various sub-cellular compartments and, through the recruitment and regulation of specific proteins, are key regulators of compartment identity and function. Phosphoinositides and the enzymes that synthesise and degrade them are also present in the nuclear membrane and in nuclear membraneless compartments such as nuclear speckles. Here we discuss how PPIns in the nucleus are modulated in response to external cues and how they function to control downstream signalling. Finally we suggest a role for nuclear PPIns in liquid phase separations that are involved in the formation of membraneless compartments within the nucleus.


Assuntos
Núcleo Celular/metabolismo , Metabolismo dos Lipídeos , Fosfatidilinositóis/metabolismo , Animais , Fenômenos Químicos , Biologia Computacional , Humanos , Espaço Intranuclear/metabolismo , Redes e Vias Metabólicas , Membrana Nuclear/metabolismo , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositóis/química , Transdução de Sinais
11.
Dev Cell ; 49(6): 894-906.e12, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31105010

RESUMO

RhoA stimulates cell contractility by recruiting downstream effectors to the cortical plasma membrane. We now show that direct binding by anillin is required for effective signaling: this antagonizes the otherwise labile membrane association of GTP-RhoA to promote effector recruitment. However, since its binding to RhoA blocks access by other effectors, we demonstrate that anillin must also concentrate membrane phosphoinositide-4,5-P2 (PIP2) to promote signaling. We propose and test a sequential pathway where GTP-RhoA first binds to anillin and then is retained at the membrane by PIP2 after it disengages from anillin. Importantly, re-binding of membrane GTP-RhoA to anillin, regulated by the cortical density of anillin, creates cycles through this pathway. These cycles repeatedly reset the dissociation kinetics of GTP-RhoA, substantially increasing its dwell time to recruit effectors. Thus, anillin regulates RhoA signaling by a paradigm of kinetic scaffolding that may apply to other signals whose efficacy depends on their cortical dwell times.


Assuntos
Neoplasias da Mama/metabolismo , Movimento Celular/efeitos dos fármacos , Proteínas Contráteis/farmacologia , Citocinese/fisiologia , Guanosina Trifosfato/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Citocinese/efeitos dos fármacos , Feminino , Humanos , Cinética , Células MCF-7 , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/genética
12.
Mol Biol Cell ; 30(12): 1555-1574, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30969890

RESUMO

Eukaryotic cell survival requires maintenance of plasma membrane (PM) homeostasis in response to environmental insults and changes in lipid metabolism. In yeast, a key regulator of PM homeostasis is target of rapamycin (TOR) complex 2 (TORC2), a multiprotein complex containing the evolutionarily conserved TOR protein kinase isoform Tor2. PM localization is essential for TORC2 function. One core TORC2 subunit (Avo1) and two TORC2--associated regulators (Slm1 and Slm2) contain pleckstrin homology (PH) domains that exhibit specificity for binding phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P2). To investigate the roles of PtdIns4,5P2 and constituent subunits of TORC2, we used auxin-inducible degradation to systematically eliminate these factors and then examined localization, association, and function of the remaining TORC2 components. We found that PtdIns4,5P2 depletion significantly reduced TORC2 activity, yet did not prevent PM localization or disassembly of TORC2. Moreover, truncated Avo1 (lacking its C-terminal PH domain) was still recruited to the PM and supported growth. Even when all three PH-containing proteins were absent, the remaining TORC2 subunits were PM-bound. Revealingly, Avo3 localized to the PM independent of both Avo1 and Tor2, whereas both Tor2 and Avo1 required Avo3 for their PM anchoring. Our findings provide new mechanistic information about TORC2 and pinpoint Avo3 as pivotal for TORC2 PM localization and assembly in vivo.


Assuntos
Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas do Domínio Armadillo/química , Proteínas do Domínio Armadillo/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Domínios Proteicos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo
13.
Plant Cell Physiol ; 60(7): 1514-1524, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30989198

RESUMO

Pathogenic fungi from the genus Colletotrichum form invasive hyphae; the hyphae are surrounded by an extra-invasive hyphal membrane (EIHM), which is continuous with the plant plasma membrane. Although the EIHM plays a crucial role as the interface between plant and fungal cells, its precise function during Colletotrichum infection remains elusive. Here, we show that enrichment of phosphoinositides (PIs) has a crucial role in Colletotrichum infection. We observed the localization of PIs in Arabidopsis thaliana cells infected by A. thaliana-adapted Colletotrichum higginsianum (Ch), and found that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] was extremely enriched in the EIHM during Ch infection. We also found that phosphatidylinositol 4-phosphate-5 kinase (PIP5K), which catalyzes production of PI(4,5)P2, also accumulated at the EIHM. The overexpression of PIP5K3 in A. thaliana increased hyphal invasion by Ch. An exocytic factor, EXO84b, was targeted to the EIHM during Ch infection, although endocytic factors such as CLATHRIN LIGHT CHAIN 2 and FLOTILLIN 1 did not. Intriguingly, the interfacial membranes between A. thaliana and powdery mildew- or downy mildew-causing pathogens did not accumulate PI(4,5)P2. These results suggest that Ch could modify the PI(4,5)P2 levels in the EIHM to increase the exocytic membrane/protein supply of the EIHM for successful infection. Our results also suggest that PI(4,5)P2 biosynthesis is a promising target for improved defense against Colletotrichum infection.


Assuntos
Arabidopsis/microbiologia , Colletotrichum , Hifas/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Doenças das Plantas/microbiologia , Membrana Celular/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 4,5-Difosfato/fisiologia , Folhas de Planta/microbiologia , Tabaco/microbiologia
14.
Biomolecules ; 9(4)2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30999671

RESUMO

HspA1A is a cytosolic molecular chaperone essential for cellular homeostasis. HspA1A also localizes at the plasma membrane (PM) of tumor and stressed cells. However, it is currently unknown how this cytosolic protein translocates to the PM. Taking into account that HspA1A interacts with lipids, including phosphatidylserine (PS), and that lipids recruit proteins to the PM, we hypothesized that the interaction of HspA1A with PS allows the chaperone to localize at the PM. To test this hypothesis, we subjected cells to mild heat-shock and the PM-localized HspA1A was quantified using confocal microscopy and cell surface biotinylation. These experiments revealed that HspA1A's membrane localization increased during recovery from non-apoptotic heat-shock. Next, we selectively reduced PS targets by overexpressing the C2 domain of lactadherin (Lact-C2), a known PS-biosensor, and determined that HspA1A's membrane localization was greatly reduced. In contrast, the reduction of PI(4,5)P2 availability by overexpression of the PLCδ-PH biosensor had minimal effects on HspA1A's PM-localization. Implementation of a fluorescent PS analog, TopFluor-PS, established that PS co-localizes with HspA1A. Collectively, these results reveal that HspA1A's PM localization and anchorage depend on its selective interaction with intracellular PS. This discovery institutes PS as a new and dynamic partner in the cellular stress response.


Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Fosfatidilserinas/metabolismo , Membrana Celular/metabolismo , Células HEK293 , Células HeLa , Resposta ao Choque Térmico , Humanos , Fosfatidilinositol 4,5-Difosfato/metabolismo , Transporte Proteico
15.
Int J Mol Sci ; 20(8)2019 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-31022885

RESUMO

Transient receptor potential melastatin member 4 (TRPM4) and 5 (TRPM5) channels are Ca2+-activated nonselective cation channels. Intracellular Ca2+ is the most important regulator for them to open, though PI(4,5)P2, a membrane phosphoinositide, has been reported to regulate their Ca2+-sensitivities. We previously reported that negatively-charged amino acid residues near and in the TRP domain are necessary for the normal Ca2+ sensitivity of TRPM4. More recently, a cryo-electron microscopy structure of Ca2+-bound (but closed) TRPM4 was reported, proposing a Ca2+-binding site within an intracellular cavity formed by S2 and S3. Here, we examined the functional effects of mutations of the amino acid residues related to the proposed Ca2+-binding site on TRPM4 and also TRPM5 using mutagenesis and patch clamp techniques. The mutations of the amino acid residues of TRPM4 and TRPM5 reduced their Ca2+-sensitivities in a similar way. On the other hand, intracellular applications of PI(4,5)P2 recovered Ca2+-sensitivity of desensitized TRPM4, but its effect on TRPM5 was negligible. From these results, the Ca2+-binding sites of TRPM4 and TRPM5 were shown to be formed by the same amino acid residues by functional analyses, but the impact of PI(4,5)P2 on the regulation of TRPM5 seemed to be smaller than that on the regulation of TRPM4.


Assuntos
Cálcio/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Canais de Cátion TRPM/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células HEK293 , Humanos , Masculino , Modelos Moleculares , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Ratos , Canais de Cátion TRPM/química , Canais de Cátion TRPM/genética
16.
Nat Commun ; 10(1): 1193, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30867420

RESUMO

Amyloid ß (Aß) oligomer-induced aberrant neurotransmitter release is proposed to be a crucial early event leading to synapse dysfunction in Alzheimer's disease (AD). In the present study, we report that the release probability (Pr) at the synapse between the Schaffer collateral (SC) and CA1 pyramidal neurons is significantly reduced at an early stage in mouse models of AD with elevated Aß production. High nanomolar synthetic oligomeric Aß42 also suppresses Pr at the SC-CA1 synapse in wild-type mice. This Aß-induced suppression of Pr is mainly due to an mGluR5-mediated depletion of phosphatidylinositol-4,5-bisphosphate (PIP2) in axons. Selectively inhibiting Aß-induced PIP2 hydrolysis in the CA3 region of the hippocampus strongly prevents oligomeric Aß-induced suppression of Pr at the SC-CA1 synapse and rescues synaptic and spatial learning and memory deficits in APP/PS1 mice. These results first reveal the presynaptic mGluR5-PIP2 pathway whereby oligomeric Aß induces early synaptic deficits in AD.


Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Região CA1 Hipocampal/fisiopatologia , Fragmentos de Peptídeos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Sinapses/metabolismo , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/genética , Animais , Região CA1 Hipocampal/metabolismo , Região CA1 Hipocampal/patologia , Cognição/fisiologia , Modelos Animais de Doenças , Embrião de Mamíferos , Humanos , Masculino , Aprendizagem em Labirinto/fisiologia , Memória/fisiologia , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/genética , Presenilina-1/genética , Presenilina-1/metabolismo , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/patologia , Cultura Primária de Células , Multimerização Proteica , Células Piramidais/metabolismo , Células Piramidais/patologia , Receptor de Glutamato Metabotrópico 5/metabolismo , Sinapses/patologia
17.
J Physiol Sci ; 69(3): 513-521, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30900190

RESUMO

Voltage-gated potassium channels are expressed in a wide variety of excitable and non-excitable cells and regulate numerous cellular functions. The activity of ion channels can be modulated by direct interaction or/and functional coupling with other proteins including auxiliary subunits, scaffold proteins and the cytoskeleton. Here, we evaluated the influence of the actin-based cytoskeleton on the Kv2.1 channel using pharmacological and electrophysiological methods. We found that disruption of the actin-based cytoskeleton by latrunculin B resulted in the regulation of the Kv2.1 inactivation mechanism; it shifted the voltage of half-maximal inactivation toward negative potentials by approximately 15 mV, accelerated the rate of closed-state inactivation, and delayed the recovery rate from inactivation. The actin cytoskeleton stabilizing agent phalloidin prevented the hyperpolarizing shift in the half-maximal inactivation potential when co-applied with latrunculin B. Additionally, PIP2 depletion (a strategy that regulates Kv2.1 inactivation) after cytoskeleton disruption does not regulate further the inactivation of Kv2.1, which suggests that both factors could be regulating the Kv2.1 channel by a common mechanism. In summary, our results suggest a role for the actin-based cytoskeleton in regulating Kv2.1 channels.


Assuntos
Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Canais de Potássio Shab/metabolismo , Actinas/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Potássio/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Tiazolidinas/farmacologia
18.
Nat Cell Biol ; 21(4): 462-475, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30886346

RESUMO

The tumour suppressor p53 (encoded by TP53) protects the genome against cellular stress and is frequently mutated in cancer. Mutant p53 acquires gain-of-function oncogenic activities that are dependent on its enhanced stability. However, the mechanisms by which nuclear p53 is stabilized are poorly understood. Here, we demonstrate that the stability of stress-induced wild-type and mutant p53 is regulated by the type I phosphatidylinositol phosphate kinase (PIPKI-α (also known as PIP5K1A)) and its product phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). Nuclear PIPKI-α binds to p53 upon stress, resulting in the production and association of PtdIns(4,5)P2 with p53. PtdIns(4,5)P2 binding promotes the interaction between p53 and the small heat shock proteins HSP27 (also known as HSPB1) and αB-crystallin (also known as HSPB5), which stabilize nuclear p53. Moreover, inhibition of PIPKI-α or PtdIns(4,5)P2 association results in p53 destabilization. Our results point to a previously unrecognized role of nuclear phosphoinositide signalling in regulating p53 stability and implicate this pathway as a promising therapeutic target in cancer.


Assuntos
Núcleo Celular/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Proteínas de Choque Térmico Pequenas/metabolismo , Humanos , Fosfatidilinositol 4,5-Difosfato/metabolismo , Estabilidade Proteica
19.
Nat Commun ; 10(1): 969, 2019 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-30814505

RESUMO

Cells govern their homeostasis through autophagy by sequestering substrates, ranging from proteins to aggregates and organelles, into autophagosomes for lysosomal degradation. In these processes cells need to coordinate between substrate remodeling and autophagosome formation for efficient engulfment. We found that in Parkin-mediated mitophagy, mitochondria to be turned over first become grape-like mitoaggregates, followed by their disassembly into smaller pieces via the actinomyosin system. At the disassembly step, we observed spatially-associated, synchronous formation of circular F-actin and BATS-labeled autophagy initiation sites near mitochondria, suggesting coordination between substrate downsizing and autophagosome formation during mitophagy. Interestingly, PtdIns(4,5)P2, instead of PtdIns(3)P, regulates this mitophagy-associated formation of circular F-actin and BATS-sites. Selective depletion of PtdIns(4,5)P2 near omegasomes, the endoplasmic reticulum (ER) subdomains involved in autophagosome formation, impaired mitoaggregate disassembly. Our findings demonstrate the presence of a pool of PtdIns(4,5)P2 adjacent to omegasomes, and that they coordinate mitoaggregate disassembly with autophagy initiation during Parkin-mediated mitophagy.


Assuntos
Actinas/metabolismo , Autofagossomos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Autofagossomos/ultraestrutura , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Proteínas Luminescentes/metabolismo , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Ubiquitina-Proteína Ligases/metabolismo
20.
Cell ; 176(6): 1432-1446.e11, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30827685

RESUMO

The presence of DNA in the cytosol of mammalian cells is an unusual event that is often associated with genotoxic stress or viral infection. The enzyme cGAS is a sensor of cytosolic DNA that induces interferon and inflammatory responses that can be protective or pathologic, depending on the context. Along with other cytosolic innate immune receptors, cGAS is thought to diffuse throughout the cytosol in search of its DNA ligand. Herein, we report that cGAS is not a cytosolic protein but rather localizes to the plasma membrane via the actions of an N-terminal phosphoinositide-binding domain. This domain interacts selectively with PI(4,5)P2, and cGAS mutants defective for lipid binding are mislocalized to the cytosolic and nuclear compartments. Mislocalized cGAS induces potent interferon responses to genotoxic stress, but weaker responses to viral infection. These data establish the subcellular positioning of a cytosolic innate immune receptor as a mechanism that governs self-nonself discrimination.


Assuntos
Membrana Celular/fisiologia , Nucleotidiltransferases/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Citosol/fisiologia , DNA Viral/genética , Feminino , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata/fisiologia , Interferons/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Nucleotidiltransferases/fisiologia , Fosfatidilinositol 4,5-Difosfato/fisiologia , Fosfatidilinositóis , Ligação Proteica , Transdução de Sinais/imunologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA