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1.
Mol Pharmacol ; 97(1): 2-8, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31704717

RESUMO

The thyrotropin (TSH) receptor (TSHR) signals via G proteins of all four classes and ß-arrestin 1. Stimulation of TSHR leads to increasing cAMP production that has been reported as a monotonic dose-response curve that plateaus at high TSH doses. In HEK 293 cells overexpressing TSHRs (HEK-TSHR cells), we found that TSHR activation exhibits an "inverted U-shaped dose-response curve" with increasing cAMP production at low doses of TSH and decreased cAMP production at high doses (>1 mU/ml). Since protein kinase A inhibition by H-89 and knockdown of ß-arrestin 1 or ß-arrestin 2 did not affect the decreased cAMP production at high TSH doses, we studied the roles of TSHR downregulation and of Gi/Go proteins. A high TSH dose (100 mU/ml) caused a 33% decrease in cell-surface TSHR. However, because inhibiting TSHR downregulation with combined expression of a dominant negative dynamin 1 and ß-arrestin 2 knockdown had no effect, we concluded that downregulation is not involved in the biphasic cAMP response. Pertussis toxin, which inhibits activation of Gi/Go, abolished the biphasic response with no statistically significant difference in cAMP levels at 1 and 100 mU/ml TSH. Concordantly, co-knockdown of Gi/Go proteins increased cAMP levels stimulated by 100 mU/ml TSH from 55% to 73% of the peak level. These data show that biphasic regulation of cAMP production is mediated by Gs and Gi/Go at low and high TSH doses, respectively, which may represent a mechanism to prevent overstimulation in TSHR-expressing cells. SIGNIFICANCE STATEMENT: We demonstrate biphasic regulation of TSH-mediated cAMP production involving coupling of the TSH receptor (TSHR) to Gs at low TSH doses and to Gi/o at high TSH doses. We suggest that this biphasic cAMP response allows the TSHR to mediate responses at lower levels of TSH and that decreased cAMP production at high doses may represent a mechanism to prevent overstimulation of TSHR-expressing cells. This mechanism could prevent chronic stimulation of thyroid gland function.


Assuntos
AMP Cíclico/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Receptores da Tireotropina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tireotropina/administração & dosagem , Relação Dose-Resposta a Droga , Regulação para Baixo , Dinamina I/genética , Dinamina I/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Toxina Pertussis/administração & dosagem , Receptores da Tireotropina/genética , Transdução de Sinais/genética , beta-Arrestina 2/genética , beta-Arrestina 2/metabolismo
2.
J Cell Biol ; 218(6): 1928-1942, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31043431

RESUMO

Multiple mechanisms contribute to cancer cell progression and metastatic activity, including changes in endocytic trafficking and signaling of cell surface receptors downstream of gain-of-function (GOF) mutant p53. We report that dynamin-1 (Dyn1) is up-regulated at both the mRNA and protein levels in a manner dependent on expression of GOF mutant p53. Dyn1 is required for the recruitment and accumulation of the signaling scaffold, APPL1, to a spatially localized subpopulation of endosomes at the cell perimeter. We developed new tools to quantify peripherally localized early endosomes and measure the rapid recycling of integrins. We report that these perimeter APPL1 endosomes modulate Akt signaling and activate Dyn1 to create a positive feedback loop required for rapid recycling of EGFR and ß1 integrins, increased focal adhesion turnover, and cell migration. Thus, Dyn1- and Akt-dependent perimeter APPL1 endosomes function as a nexus that integrates signaling and receptor trafficking, which can be co-opted and amplified in mutant p53-driven cancer cells to increase migration and invasion.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Movimento Celular , Dinamina I/metabolismo , Endossomos/metabolismo , Mutação , Proteína Supressora de Tumor p53/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Adesão Celular , Membrana Celular , Dinamina I/genética , Endocitose , Receptores ErbB/genética , Receptores ErbB/metabolismo , Retroalimentação Fisiológica , Humanos , Integrina beta1/genética , Integrina beta1/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Transporte Proteico , Transdução de Sinais , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética
3.
Elife ; 82019 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-31099751

RESUMO

Regulated exocytosis establishes a narrow fusion pore as initial aqueous connection to the extracellular space, through which small transmitter molecules such as ATP can exit. Co-release of polypeptides and hormones like insulin requires further expansion of the pore. There is evidence that pore expansion is regulated and can fail in diabetes and neurodegenerative disease. Here, we report that the cAMP-sensor Epac2 (Rap-GEF4) controls fusion pore behavior by acutely recruiting two pore-restricting proteins, amisyn and dynamin-1, to the exocytosis site in insulin-secreting beta-cells. cAMP elevation restricts and slows fusion pore expansion and peptide release, but not when Epac2 is inactivated pharmacologically or in Epac2-/- (Rapgef4-/-) mice. Consistently, overexpression of Epac2 impedes pore expansion. Widely used antidiabetic drugs (GLP-1 receptor agonists and sulfonylureas) activate this pathway and thereby paradoxically restrict hormone release. We conclude that Epac2/cAMP controls fusion pore expansion and thus the balance of hormone and transmitter release during insulin granule exocytosis.


Assuntos
AMP Cíclico/metabolismo , Exocitose , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Insulina/metabolismo , Animais , Proteínas de Transporte/metabolismo , Dinamina I/metabolismo , Humanos , Camundongos Knockout
4.
Virology ; 528: 118-136, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30597347

RESUMO

Although several reports suggest that the entry of infectious bronchitis virus (IBV) depends on lipid rafts and low pH, the endocytic route and intracellular trafficking are unclear. In this study, we aimed to shed greater light on early steps in IBV infection. By using chemical inhibitors, RNA interference, and dominant negative mutants, we observed that lipid rafts and low pH was indeed required for virus entry; IBV mainly utilized the clathrin mediated endocytosis (CME) for entry; GTPase dynamin 1 was involved in virus containing vesicle scission; and the penetration of IBV into cells led to active cytoskeleton rearrangement. By using R18 labeled virus, we found that virus particles moved along with the classical endosome/lysosome track. Functional inactivation of Rab5 and Rab7 significantly inhibited IBV infection. Finally, by using dual R18/DiOC labeled IBV, we observed that membrane fusion was induced after 1 h.p.i. in late endosome/lysosome.


Assuntos
Endocitose , Endossomos/virologia , Vírus da Bronquite Infecciosa/fisiologia , Lisossomos/virologia , Internalização do Vírus , Animais , Linhagem Celular , Chlorocebus aethiops , Clatrina/metabolismo , Citoesqueleto/virologia , Dinamina I/metabolismo , Concentração de Íons de Hidrogênio , Fusão de Membrana , Microdomínios da Membrana/virologia , Interferência de RNA , Células Vero , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo
5.
J Pathol ; 247(2): 177-185, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30350425

RESUMO

Dynamin plays an essential role in maintaining the structure and function of the glomerular filtration barrier. Specifically, dynamin regulates the actin cytoskeleton and the turnover of nephrin in podocytes, and knocking down dynamin expression causes proteinuria. Moreover, promoting dynamin oligomerization with Bis-T-23 restores podocyte function and reduces proteinuria in several animal models of chronic kidney disease. Thus, dynamin is a promising therapeutic target for treating chronic kidney disease. Here, we investigated the pathophysiological role of dynamin under proteinuric circumstances in a rat model and in humans. We found that glomerular Dnm2 and Dnm1 mRNA levels are increased prior to the onset of proteinuria in a rat model of spontaneous proteinuria. Also, in zebrafish embryos, we confirm that knocking down dynamin translation results in proteinuria. Finally, we show that the glomerular expression of dynamin and cathepsin L protein is increased in several human proteinuric kidney diseases. We propose that the increased expression of glomerular dynamin reflects an exhausted attempt to maintain and/or restore integrity of the glomerular filtration barrier. These results confirm that dynamin plays an important role in maintaining the glomerular filtration barrier, and they support the notion that dynamin is a promising therapeutic target in proteinuric kidney disease. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.


Assuntos
Dinamina II/metabolismo , Dinamina I/metabolismo , Nefropatias/metabolismo , Glomérulos Renais/metabolismo , Proteinúria/metabolismo , Adulto , Idoso , Animais , Catepsina L/genética , Catepsina L/metabolismo , Modelos Animais de Doenças , Dinamina I/genética , Dinamina II/genética , Feminino , Taxa de Filtração Glomerular , Humanos , Nefropatias/genética , Nefropatias/fisiopatologia , Glomérulos Renais/fisiopatologia , Masculino , Pessoa de Meia-Idade , Proteinúria/genética , Proteinúria/fisiopatologia , Ratos Endogâmicos Dahl , Ratos Endogâmicos SHR , Fatores de Tempo , Regulação para Cima , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
6.
J Neurosci ; 39(2): 199-211, 2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30381405

RESUMO

Dynamin 1 (dyn1) is required for clathrin-mediated endocytosis in most secretory (neuronal and neuroendocrine) cells. There are two modes of Ca2+-dependent catecholamine release from single dense-core vesicles: full-quantal (quantal) and subquantal in adrenal chromaffin cells, but their relative occurrences and impacts on total secretion remain unclear. To address this fundamental question in neurotransmission area using both sexes of animals, here we report the following: (1) dyn1-KO increased quantal size (QS, but not vesicle size/content) by ≥250% in dyn1-KO mice; (2) the KO-increased QS was rescued by dyn1 (but not its deficient mutant or dyn2); (3) the ratio of quantal versus subquantal events was increased by KO; (4) following a release event, more protein contents were retained in WT versus KO vesicles; and (5) the fusion pore size (d p) was increased from ≤9 to ≥9 nm by KO. Therefore, Ca2+-induced exocytosis is generally a subquantal release in sympathetic adrenal chromaffin cells, implying that neurotransmitter release is generally regulated by dynamin in neuronal cells.SIGNIFICANCE STATEMENT Ca2+-dependent neurotransmitter release from a single vesicle is the primary event in all neurotransmission, including synaptic/neuroendocrine forms. To determine whether Ca2+-dependent vesicular neurotransmitter release is "all-or-none" (quantal), we provide compelling evidence that most Ca2+-induced secretory events occur via the subquantal mode in native adrenal chromaffin cells. This subquantal release mode is promoted by dynamin 1, which is universally required for most secretory cells, including neurons and neuroendocrine cells. The present work with dyn1-KO mice further confirms that Ca2+-dependent transmitter release is mainly via subquantal mode, suggesting that subquantal release could be also important in other types of cells.


Assuntos
Glândulas Suprarrenais/metabolismo , Células Cromafins/metabolismo , Dinamina I/fisiologia , Neurotransmissores/metabolismo , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo , Glândulas Suprarrenais/citologia , Animais , Cálcio/farmacologia , Catecolaminas/metabolismo , Dinamina I/genética , Endocitose/fisiologia , Exocitose/efeitos dos fármacos , Feminino , Técnicas In Vitro , Masculino , Camundongos , Camundongos Knockout , Mutação/genética , Vesículas Secretórias/metabolismo
7.
Int J Oncol ; 54(2): 550-558, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30570111

RESUMO

Dynamin copolymerizes with cortactin to form a ring­like complex that bundles and stabilizes actin filaments. Actin bundle formation is crucial for generation of filopodia and lamellipodia, which guide migration, invasion, and metastasis of cancer cells. However, it is unknown how the dynamin­cortactin complex regulates actin bundle formation. The present study investigated phosphorylation of cortactin by cyclin­dependent kinase 5 (CDK5) and its effect on actin bundle formation by the dynamin­cortactin complex. CDK5 directly phosphorylated cortactin at T145/T219 in vitro. Phosphomimetic mutants in which one or both of these threonine residues was substituted by aspartate were used. The three phosphomimetic mutants (T145D, T219D and T145DT219D) had a decreased affinity for F­actin. Furthermore, electron microscopy demonstrated that these phosphomimetic mutants could not form a ring­like complex with dynamin 1. Consistently, the dynamin 1­phosphomimetic cortactin complexes exhibited decreased actin­bundling activity. Expression of the phosphomimetic mutants resulted in not only aberrant lamellipodia and short filopodia but also cell migration in NG108­15 glioma­derived cells. These results indicate that phosphorylation of cortactin by CDK5 regulates formation of lamellipodia and filopodia by modulating dynamin 1/cortactin­dependent actin bundling. Taken together, these findings suggest that CDK5 is a potential molecular target for anticancer therapy.


Assuntos
Actinas/metabolismo , Cortactina/metabolismo , Quinase 5 Dependente de Ciclina/metabolismo , Dinamina I/metabolismo , Glioma/metabolismo , Pseudópodes/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Fosforilação
8.
Nat Commun ; 9(1): 4634, 2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30401830

RESUMO

Mitochondria and peroxisomes proliferate by division. During division, a part of their membrane is pinched off by constriction of the ring-shaped mitochondrial division (MD) and peroxisome-dividing (POD) machinery. This constriction is mediated by a dynamin-like GTPase Dnm1 that requires a large amount of GTP as an energy source. Here, via proteomics of the isolated division machinery, we show that the 17-kDa nucleoside diphosphate kinase-like protein, dynamin-based ring motive-force organizer 1 (DYNAMO1), locally generates GTP in MD and POD machineries. DYNAMO1 is widely conserved among eukaryotes and colocalizes with Dnm1 on the division machineries. DYNAMO1 converts ATP to GTP, and disruption of its activity impairs mitochondrial and peroxisomal fissions. DYNAMO1 forms a ring-shaped complex with Dnm1 and increases the magnitude of the constricting force. Our results identify DYNAMO1 as an essential component of MD and POD machineries, suggesting that local GTP generation in Dnm1-based machinery regulates motive force for membrane severance.


Assuntos
Dinaminas/metabolismo , Guanosina Trifosfato/metabolismo , Mitocôndrias/metabolismo , Peroxissomos/metabolismo , Trifosfato de Adenosina/metabolismo , Divisão Celular , Dinamina I/metabolismo , Dinaminas/genética , Eucariotos , GTP Fosfo-Hidrolases/metabolismo , Dinâmica Mitocondrial , Núcleosídeo-Difosfato Quinase/metabolismo , Proteômica , Rodófitas , Alinhamento de Sequência
9.
Nature ; 560(7717): 258-262, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30069048

RESUMO

Membrane fission is a fundamental process in the regulation and remodelling of cell membranes. Dynamin, a large GTPase, mediates membrane fission by assembling around, constricting and cleaving the necks of budding vesicles1. Here we report a 3.75 Å resolution cryo-electron microscopy structure of the membrane-associated helical polymer of human dynamin-1 in the GMPPCP-bound state. The structure defines the helical symmetry of the dynamin polymer and the positions of its oligomeric interfaces, which were validated by cell-based endocytosis assays. Compared to the lipid-free tetramer form2, membrane-associated dynamin binds to the lipid bilayer with its pleckstrin homology domain (PHD) and self-assembles across the helical rungs via its guanine nucleotide-binding (GTPase) domain3. Notably, interaction with the membrane and helical assembly are accommodated by a severely bent bundle signalling element (BSE), which connects the GTPase domain to the rest of the protein. The BSE conformation is asymmetric across the inter-rung GTPase interface, and is unique compared to all known nucleotide-bound states of dynamin. The structure suggests that the BSE bends as a result of forces generated from the GTPase dimer interaction that are transferred across the stalk to the PHD and lipid membrane. Mutations that disrupted the BSE kink impaired endocytosis. We also report a 10.1 Å resolution cryo-electron microscopy map of a super-constricted dynamin polymer showing localized conformational changes at the BSE and GTPase domains, induced by GTP hydrolysis, that drive membrane constriction. Together, our results provide a structural basis for the mechanism of action of dynamin on the lipid membrane.


Assuntos
Biopolímeros/química , Biopolímeros/metabolismo , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Microscopia Crioeletrônica , Dinamina I/metabolismo , Dinamina I/ultraestrutura , Biopolímeros/genética , Membrana Celular/química , Dinamina I/química , Dinamina I/genética , Endocitose/genética , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Humanos , Hidrólise , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Mutantes/ultraestrutura , Mutação , Domínios Proteicos , Multimerização Proteica
10.
Brain ; 141(8): 2343-2361, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29961886

RESUMO

Autosomal recessive spinal muscular atrophy (SMA), the leading genetic cause of infant lethality, is caused by homozygous loss of the survival motor neuron 1 (SMN1) gene. SMA disease severity inversely correlates with the number of SMN2 copies, which in contrast to SMN1, mainly produce aberrantly spliced transcripts. Recently, the first SMA therapy based on antisense oligonucleotides correcting SMN2 splicing, namely SPINRAZATM, has been approved. Nevertheless, in type I SMA-affected individuals-representing 60% of SMA patients-the elevated SMN level may still be insufficient to restore motor neuron function lifelong. Plastin 3 (PLS3) and neurocalcin delta (NCALD) are two SMN-independent protective modifiers identified in humans and proved to be effective across various SMA animal models. Both PLS3 overexpression and NCALD downregulation protect against SMA by restoring impaired endocytosis; however, the exact mechanism of this protection is largely unknown. Here, we identified calcineurin-like EF-hand protein 1 (CHP1) as a novel PLS3 interacting protein using a yeast-two-hybrid screen. Co-immunoprecipitation and pull-down assays confirmed a direct interaction between CHP1 and PLS3. Although CHP1 is ubiquitously present, it is particularly abundant in the central nervous system and at SMA-relevant sites including motor neuron growth cones and neuromuscular junctions. Strikingly, we found elevated CHP1 levels in SMA mice. Congruently, CHP1 downregulation restored impaired axonal growth in Smn-depleted NSC34 motor neuron-like cells, SMA zebrafish and primary murine SMA motor neurons. Most importantly, subcutaneous injection of low-dose SMN antisense oligonucleotide in pre-symptomatic mice doubled the survival rate of severely-affected SMA mice, while additional CHP1 reduction by genetic modification prolonged survival further by 1.6-fold. Moreover, CHP1 reduction further ameliorated SMA disease hallmarks including electrophysiological defects, smaller neuromuscular junction size, impaired maturity of neuromuscular junctions and smaller muscle fibre size compared to low-dose SMN antisense oligonucleotide alone. In NSC34 cells, Chp1 knockdown tripled macropinocytosis whereas clathrin-mediated endocytosis remained unaffected. Importantly, Chp1 knockdown restored macropinocytosis in Smn-depleted cells by elevating calcineurin phosphatase activity. CHP1 is an inhibitor of calcineurin, which collectively dephosphorylates proteins involved in endocytosis, and is therefore crucial in synaptic vesicle endocytosis. Indeed, we found marked hyperphosphorylation of dynamin 1 in SMA motor neurons, which was restored to control level by the heterozygous Chp1 mutant allele. Taken together, we show that CHP1 is a novel SMA modifier that directly interacts with PLS3, and that CHP1 reduction ameliorates SMA pathology by counteracting impaired endocytosis. Most importantly, we demonstrate that CHP1 reduction is a promising SMN-independent therapeutic target for a combinatorial SMA therapy.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Glicoproteínas de Membrana/fisiologia , Proteínas dos Microfilamentos/fisiologia , Atrofia Muscular Espinal/fisiopatologia , Animais , Atrofia/fisiopatologia , Calcineurina/metabolismo , Proteínas de Ligação ao Cálcio/fisiologia , Linhagem Celular , Modelos Animais de Doenças , Dinamina I/metabolismo , Endocitose/fisiologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Neurônios Motores/metabolismo , Junção Neuromuscular/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Monoéster Fosfórico Hidrolases/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Peixe-Zebra
11.
Sci Rep ; 8(1): 7233, 2018 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-29740148

RESUMO

Restrictive anorexia nervosa is associated with reduced eating and severe body weight loss leading to a cachectic state. Hypothalamus plays a major role in the regulation of food intake and energy homeostasis. In the present study, alterations of hypothalamic proteome and particularly of proteins involved in energy and mitochondrial metabolism have been observed in female activity-based anorexia (ABA) mice that exhibited a reduced food intake and a severe weight loss. In the hypothalamus, mitochondrial dynamic was also modified during ABA with an increase of fission without modification of fusion. In addition, increased dynamin-1, and LC3II/LC3I ratio signed an activation of autophagy while protein synthesis was increased. In conclusion, proteomic analysis revealed an adaptive hypothalamic protein response in ABA female mice with both altered mitochondrial response and activated autophagy.


Assuntos
Anorexia Nervosa/genética , Dinamina I/genética , Hipotálamo/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Dinâmica Mitocondrial/genética , Proteoma/genética , Aconitato Hidratase/genética , Aconitato Hidratase/metabolismo , Animais , Anorexia , Anorexia Nervosa/metabolismo , Anorexia Nervosa/fisiopatologia , Autofagia/genética , Modelos Animais de Doenças , Dinamina I/metabolismo , Ingestão de Alimentos/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hipotálamo/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/metabolismo , Condicionamento Físico Animal , Biossíntese de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteoma/metabolismo , Receptores de Enterotoxina/genética , Receptores de Enterotoxina/metabolismo , Transdução de Sinais , Perda de Peso/genética
12.
Neurosci Lett ; 675: 59-63, 2018 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-29604406

RESUMO

Neurons have well-developed membrane microdomains called "rafts" that are recovered as a detergent-resistant low-density membrane microdomain fraction (DRM). NAP-22 is one of the major protein components of neuronal DRM and localizes in the presynaptic region. In order to know the role of NAP-22 in the synaptic transmission, NAP-22 binding proteins in the cytosol were searched with an affinity screening with NAP-22 as a bait and several protein bands were detected. Using mass-analysis and western blotting, one of the main band of ∼90 kDa was identified as dynamin I. The GTPase activity of dynamin I was partly inhibited by NAP-22 expressed in bacteria and this inhibition was recovered by the addition of calmodulin, a NAP-22 binding protein. The GTPase activity of dynamin was known to be activated with acidic membrane lipids such as phosphatidylserine and the addition of NAP-22, a phosphatidylserine binding protein, inhibited the activation of the GTPase by this lipid. Since NAP-22 localizes on the presynaptic plasma membrane and on synaptic vesicles, these results suggest the participation of NAP-22 in the membrane cycling through binding to dynamin and acidic membrane lipids at the presynaptic region.


Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Dinamina I/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Terminações Pré-Sinápticas/metabolismo , Animais , Proteínas de Transporte/metabolismo , Células Cultivadas , Ratos Wistar
13.
PLoS Biol ; 16(4): e2005377, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29668686

RESUMO

Dynamin Guanosine Triphosphate hydrolases (GTPases) are best studied for their role in the terminal membrane fission process of clathrin-mediated endocytosis (CME), but they have also been proposed to regulate earlier stages of CME. Although highly enriched in neurons, dynamin-1 (Dyn1) is, in fact, widely expressed along with Dyn2 but inactivated in non-neuronal cells via phosphorylation by glycogen synthase kinase-3 beta (GSK3ß) kinase. Here, we study the differential, isoform-specific functions of Dyn1 and Dyn2 as regulators of CME. Endogenously expressed Dyn1 and Dyn2 were fluorescently tagged either separately or together in two cell lines with contrasting Dyn1 expression levels. By quantitative live cell dual- and triple-channel total internal reflection fluorescence microscopy, we find that Dyn2 is more efficiently recruited to clathrin-coated pits (CCPs) than Dyn1, and that Dyn2 but not Dyn1 exhibits a pronounced burst of assembly, presumably into supramolecular collar-like structures that drive membrane scission and clathrin-coated vesicle (CCV) formation. Activation of Dyn1 by acute inhibition of GSK3ß results in more rapid endocytosis of transferrin receptors, increased rates of CCP initiation, and decreased CCP lifetimes but did not significantly affect the extent of Dyn1 recruitment to CCPs. Thus, activated Dyn1 can regulate early stages of CME that occur well upstream of fission, even when present at low, substoichiometric levels relative to Dyn2. Under physiological conditions, Dyn1 is activated downstream of epidermal growth factor receptor (EGFR) signaling to alter CCP dynamics. We identify sorting nexin 9 (SNX9) as a preferred binding partner to activated Dyn1 that is partially required for Dyn1-dependent effects on early stages of CCP maturation. Together, we decouple regulatory and scission functions of dynamins and report a scission-independent, isoform-specific regulatory role for Dyn1 in CME.


Assuntos
Vesículas Revestidas por Clatrina/metabolismo , Clatrina/metabolismo , Dinamina II/metabolismo , Dinamina I/metabolismo , Endocitose/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Células A549 , Linhagem Celular Tumoral , Clatrina/genética , Vesículas Revestidas por Clatrina/ultraestrutura , Dinamina I/genética , Dinamina II/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulação da Expressão Gênica , Glicogênio Sintase Quinase 3 beta/genética , Humanos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Ligação Proteica , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Transdução de Sinais , Nexinas de Classificação/genética , Nexinas de Classificação/metabolismo , Coloração e Rotulagem/métodos
14.
Mol Genet Genomic Med ; 6(2): 294-300, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29397573

RESUMO

BACKGROUND: Dynamin 1 is a protein involved in the synaptic vesicle cycle, which facilitates the exocytosis of neurotransmitters necessary for normal signaling and development in the central nervous system. Pathogenic variants in DNM1 have been implicated in global developmental delay (DD), severe intellectual disability (ID), and notably, epileptic encephalopathy. All previously reported DNM1 pathogenic variants causing this severe phenotype occur in the GTPase and Middle domains of the dynamin 1 protein. METHODS: We used whole-exome sequencing to characterize the molecular basis of DD and autistic symptoms in two identical siblings. RESULTS: The twin siblings exhibit mild to moderate ID and autistic symptoms but no epileptic encephalopathy. Exome sequencing revealed a genetic variant, c.1603A>G (p.Lys535Glu), in the PH domain of dynamin 1. Previous in vitro studies showed that mutations at Lys535 inhibit endocytosis and impair PH loop binding to PIP2. CONCLUSIONS: Our data suggest a previously undescribed milder phenotype associated with a missense genetic variant in the PH domain of dynamin 1.


Assuntos
Deficiências do Desenvolvimento/genética , Dinamina I/genética , Criança , Dinamina I/fisiologia , Exoma/genética , Feminino , Variação Genética/genética , Humanos , Deficiência Intelectual/genética , Mutação de Sentido Incorreto , Fenótipo , Domínios Proteicos/genética , Gêmeos Monozigóticos/genética , Sequenciamento Completo do Exoma/métodos
16.
Elife ; 72018 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-29357276

RESUMO

Dynamin is a mechanochemical GTPase essential for membrane fission during clathrin-mediated endocytosis. Dynamin forms helical complexes at the neck of clathrin-coated pits and their structural changes coupled with GTP hydrolysis drive membrane fission. Dynamin and its binding protein amphiphysin cooperatively regulate membrane remodeling during the fission, but its precise mechanism remains elusive. In this study, we analyzed structural changes of dynamin-amphiphysin complexes during the membrane fission using electron microscopy (EM) and high-speed atomic force microscopy (HS-AFM). Interestingly, HS-AFM analyses show that the dynamin-amphiphysin helices are rearranged to form clusters upon GTP hydrolysis and membrane constriction occurs at protein-uncoated regions flanking the clusters. We also show a novel function of amphiphysin in size control of the clusters to enhance biogenesis of endocytic vesicles. Our approaches using combination of EM and HS-AFM clearly demonstrate new mechanistic insights into the dynamics of dynamin-amphiphysin complexes during membrane fission.


Assuntos
Dinamina I/metabolismo , Endocitose , Guanosina Trifosfato/metabolismo , Membranas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Humanos , Hidrólise , Microscopia de Força Atômica , Microscopia Eletrônica , Células Sf9 , Spodoptera
17.
J Gerontol A Biol Sci Med Sci ; 73(3): 289-298, 2018 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-28655199

RESUMO

The greatest risk factor for Alzheimer's disease (AD) is advanced age, but the reason for this association remains unclear. Amyloid-ß (Aß) is produced from amyloid precursor protein (APP) primarily after APP is internalized by clathrin-mediated or clathrin-independent endocytosis. Changes in endocytosis in AD have been identified. We hypothesized that endocytic protein expression is altered during ageing, thus influencing the likelihood of developing AD by increasing Aß production. We explored how levels of endocytic proteins, APP, its metabolites, secretase enzymes, and tau varied with age in cortical brain samples from men of three age ranges (young [20-30], middle aged [45-55], and old [70-90]) with no symptoms of dementia. Aß40 and Aß42 were significantly increased in old brains, while APP and secretase expression was unaffected by age. Phosphorylated GSK3ß increased significantly with age, a possible precursor for neurofibrillary tangle production, although phosphorylated tau was undetectable. Significant increases in clathrin, dynamin-1, AP180, Rab-5, caveolin-2, and flotillin-2 were seen in old brains. Rab-5 also increased in middle-aged brains prior to changes in Aß levels. This age-related increase in endocytic protein expression, not described previously, suggests an age-related upregulation of endocytosis which could predispose older individuals to develop AD by increasing APP internalization and Aß generation.


Assuntos
Envelhecimento/fisiologia , Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Endocitose/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Caveolina 2/metabolismo , Clatrina/metabolismo , Dinamina I/metabolismo , Humanos , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Proteínas Monoméricas de Montagem de Clatrina/metabolismo , Fatores de Risco , Regulação para Cima , Proteínas de Transporte Vesicular/metabolismo , Proteínas tau/metabolismo
18.
Elife ; 62017 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-29022874

RESUMO

Dynamin is a large GTPase that forms a helical collar at the neck of endocytic pits, and catalyzes membrane fission (Schmid and Frolov, 2011; Ferguson and De Camilli, 2012). Dynamin fission reaction is strictly dependent on GTP hydrolysis, but how fission is mediated is still debated (Antonny et al., 2016): GTP energy could be spent in membrane constriction required for fission, or in disassembly of the dynamin polymer to trigger fission. To follow dynamin GTP hydrolysis at endocytic pits, we generated a conformation-specific nanobody called dynab, that binds preferentially to the GTP hydrolytic state of dynamin-1. Dynab allowed us to follow the GTPase activity of dynamin-1 in real-time. We show that in fibroblasts, dynamin GTP hydrolysis occurs as stochastic bursts, which are randomly distributed relatively to the peak of dynamin assembly. Thus, dynamin disassembly is not coupled to GTPase activity, supporting that the GTP energy is primarily spent in constriction.


Assuntos
Dinamina I/metabolismo , Fibroblastos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Polimerização , Guanosina Trifosfato/metabolismo , Humanos , Hidrólise , Anticorpos de Domínio Único/metabolismo
19.
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue ; 29(10): 902-906, 2017 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-29017650

RESUMO

OBJECTIVE: To investigate the protective effect of dynamin-related protein 1 (Drp1) in rats with myocardial ischemia/reperfusion injury (IRI). METHODS: Twenty-four healthy male Wistar rats were randomly divided into three groups (n = 8 each): sham group, IRI model group, and Drp1 inhibitor group. The left anterior descending branch of coronary artery was ligated to produce myocardial ischemia for 30 minutes and reperfusion injury model. Sham group was received only threading without ligation. The Drp1 inhibitor group was injected with 1.2 mg/kg mitochondrial division inhibitor 1 (mdivi-1) at 15 minutes before operation. At 3 hours after reperfusion, hemodynamics, serum myocardial enzymes, mitochondrial membrane potential (MMP), hydrogen peroxide (H2O2), reactive oxygen species (ROS) and ATP production were measured in rats. The myocardial tissues were harvested for the determination of the area at risk (AAR) and the infarct area (AI), and the ratio of AI/AAR was calculated. The expression of Drp1 and cytochrome C (Cyt C) was determined by Western Blot. RESULTS: Compared with the sham group, the left ventricular end diastolic pressure (LVEDP), cardiac troponin I (cTnI), MB isoenzyme of creatine kinase (CK-MB), lactate dehydrogenase (LDH), AI/AAR, H2O2, ROS, protein expression of Drp1 and Cyt C were significantly increased, left ventricular end systolic pressure (LVESP), ejection fraction (EF), fractional shortening (FS), MMP, ATP generation, expression of mitochondrial Cyt C were significantly decreased in IRI model group. Compared with IRI model group, LVEDP was significantly decreased in Drp1 inhibitor group [mmHg (1 mmHg = 0.133 kPa): 8.83±1.20 vs. 16.48±1.80], LVESP, EF, FS were significantly increased [LVESP (mmHg): 116.80±9.78 vs. 87.80±8.82, EF: 0.78±0.11 vs. 0.58±0.07, FS: (48.6±4.1)% vs. (32.4±3.2)%]; myocardial enzymes, H2O2 and ROS were significantly decreased in Drp1 inhibitor group [cTnI (ng/L): 31.9±8.8 vs. 49.2±13.7, CK-MB (U/L): 4.83±1.30 vs. 7.48±2.20, LDH (U/L): 1 327.80±280.20 vs. 1 858.80±324.80, H2O2: 6.40±1.40 vs. 8.90±1.50, ROS: 41 916.3±6 295.3 vs. 65 182.6±3 777.8], AI/AAR was significantly decreased (0.38±0.01 vs. 0.62±0.01), MMP and ATP were significantly increased [MMP: 0.78±0.13 vs. 0.38±0.07, ATP (µmol/g): 150.8±12.3 vs. 103.7±8.4], the expression of Drp1 was significantly decreased (0.50±0.02 vs. 0.79±0.05), expression of mitochondria Cyt C was significantly increased (0.64±0.04 vs. 0.21±0.01), and expression of cytoplasmic Cyt C was significantly decreased (0.48±0.03 vs. 0.78±0.04), and the differences were statistically significant (all P < 0.05). CONCLUSIONS: Mitochondrial fission was excessively high during IRI, and its function was significantly decreased. Drp1 inhibitor could inhibit the division of mitochondria, and improve its function and cardiac function.


Assuntos
Dinamina I/metabolismo , Isquemia Miocárdica , Traumatismo por Reperfusão Miocárdica , Animais , Masculino , Ratos , Ratos Wistar
20.
Mol Hum Reprod ; 23(10): 657-673, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-29044420

RESUMO

STUDY QUESTION: Does dynamin regulate human sperm acrosomal exocytosis? SUMMARY ANSWER: Our studies of dynamin localization and function have implicated this family of mechanoenzymes in the regulation of progesterone-induced acrosomal exocytosis in human spermatozoa. WHAT IS KNOWN ALREADY: Completion of an acrosome reaction is a prerequisite for successful fertilization in all studied mammalian species. It follows that failure to complete this unique exocytotic event represents a common aetiology in the defective spermatozoa of male infertility patients that have failed IVF in a clinical setting. Recent studies have implicated the dynamin family of mechanoenzymes as important regulators of the acrosome reaction in murine spermatozoa. The biological basis of this activity appears to rest with the ability of dynamin to polymerize around newly formed membrane vesicles and subsequently regulate the rate of fusion pore expansion. To date, however, the dynamin family of GTPases have not been studied in the spermatozoa of non-rodent species. Here, we have sought to examine the presence and functional significance of dynamin in human spermatozoa. STUDY DESIGN, SIZE, DURATION: Dynamin expression was characterized in the testis and spermatozoa of several healthy normozoospermic individuals. In addition, we assessed the influence of selective dynamin inhibition on the competence of human spermatozoa to undergo a progesterone-induced acrosome reaction. A minimum of five biological and technical replicates were performed to investigate both inter- and intra-donor variability in dynamin expression and establish statistical significance in terms of the impact of dynamin inhibition. PARTICIPANTS/MATERIALS, SETTING, METHODS: The expression and the localization of dynamin in the human testis, epididymis and mature spermatozoa were determined through the application of immunofluorescence, immunoblotting and/or electron microscopy. Human semen samples were fractionated via density gradient centrifugation and the resultant populations of good and poor quality spermatozoa were induced to capacitate and acrosome react in the presence or absence of selective dynamin inhibitors. The acrosome integrity of live spermatozoa was subsequently assessed via the use of fluorescently conjugated Arachis hypogea lectin (PNA). The influence of dynamin phosphorylation and the regulatory kinase(s) responsible for this modification in human spermatozoa were also assessed via the use of in situ proximity ligation assays and pharmacological inhibition. In all experiments, ≥100 spermatozoa were assessed/treatment group and all graphical data are presented as the mean values ± SEM, with statistical significance being determined by ANOVA. MAIN RESULTS AND THE ROLE OF CHANCE: Dynamin 1 (DNM1) and DNM2, but not DNM3, were specifically localized to the acrosomal region of the head of human spermatozoa, an ideal position from which to regulate acrosomal exocytosis. In keeping with this notion, pharmacological inhibition of DNM1 and DNM2 was able to significantly suppress the rates of acrosomal exocytosis stimulated by progesterone. Furthermore, our comparison of dynamin expression in good and poor quality spermatozoa recovered from the same ejaculate, revealed a significant reduction in the amount of DNM2 in the latter subpopulation of cells. In contrast, DNM1 was detected at equivalent levels in both subpopulations of spermatozoa. Such findings are of potential significance given that the poor quality spermatozoa proved refractory to the induction of a progesterone stimulated acrosome reaction. In seeking to identify the regulatory influence of progesterone on DNM2 function, we were able to establish that the protein is a substrate for CDK1-dependent phosphorylation. The functional significance of DNM2 phosphorylation was illustrated by the fact that pharmacological inhibition of CDK1 elicited a concomitant suppression of both DNM2-Ser764 phosphorylation and the overall rates of progesterone-induced acrosomal exocytosis. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: This was an in vitro study performed mainly on ejaculated human spermatozoa. This experimental paradigm necessarily eliminates the physiological contributions of the female reproductive tract that would normally support capacitation and acrosomal responsiveness. WIDER IMPLICATIONS OF THE FINDINGS: This study identifies a novel causative link between dynamin activity and the ability of human spermatozoa to complete a progesterone-induced acrosome reaction. Such findings encourage a more detailed analysis of the contribution of dynamin dysregulation as an underlying aetiology in infertile males whose spermatozoa are unable to penetrate the zona pellucida. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by a National Health and Medical Research Council of Australia Project Grant (APP1103176) awarded to B.N. and E.A.M. The authors report no conflict of interest.


Assuntos
Reação Acrossômica/efeitos dos fármacos , Proteína Quinase CDC2/genética , Dinamina I/genética , Dinaminas/genética , Progesterona/farmacologia , Espermatozoides/efeitos dos fármacos , Reação Acrossômica/fisiologia , Animais , Encéfalo/metabolismo , Proteína Quinase CDC2/metabolismo , Dinamina I/antagonistas & inibidores , Dinamina I/metabolismo , Dinaminas/antagonistas & inibidores , Dinaminas/metabolismo , Epididimo/citologia , Epididimo/metabolismo , Exocitose/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Hidrazonas/farmacologia , Masculino , Mecanotransdução Celular , Camundongos , Naftóis/farmacologia , Fosforilação/efeitos dos fármacos , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Espermatozoides/metabolismo , Espermatozoides/ultraestrutura , Testículo/citologia , Testículo/metabolismo
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