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1.
Annu Rev Biophys ; 51: 377-408, 2022 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-35167762

RESUMO

Major recent advances and previous data have led to a plausible model of how key proteins mediate neurotransmitter release. In this model, the soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor (SNARE) proteins syntaxin-1, SNAP-25, and synaptobrevin form tight complexes that bring the membranes together and are crucial for membrane fusion. NSF and SNAPs disassemble SNARE complexes and ensure that fusion occurs through an exquisitely regulated pathway that starts with Munc18-1 bound to a closed conformation of syntaxin-1. Munc18-1 also binds to synaptobrevin, forming a template to assemble the SNARE complex when Munc13-1 opens syntaxin-1 while bridging the vesicle and plasma membranes. Synaptotagmin-1 and complexin bind to partially assembled SNARE complexes, likely stabilizing them and preventing fusion until Ca2+ binding to synaptotagmin-1 causes dissociation from the SNARE complex and induces interactions with phospholipids that help trigger release. Although fundamental questions remain about the mechanism of membrane fusion, these advances provide a framework to investigate the mechanisms underlying presynaptic plasticity.


Assuntos
Proteínas do Tecido Nervoso , Proteínas SNARE , Fusão de Membrana , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Neurotransmissores , Proteínas R-SNARE/química , Proteínas R-SNARE/metabolismo , Proteínas SNARE/química , Proteínas SNARE/metabolismo , Transmissão Sináptica , Sintaxina 1/química , Sintaxina 1/metabolismo
2.
PLoS One ; 17(1): e0258876, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34986150

RESUMO

The androgen receptor (AR) plays a central role in prostate cancer. Development of castration resistant prostate cancer (CRPC) requires androgen-independent activation of AR, which involves its large N-terminal domain (NTD) and entails extensive epigenetic changes depending in part on histone lysine demethylases (KDMs) that interact with AR. The AR-NTD is rich in low-complexity sequences, including a polyQ repeat. Longer polyQ sequences were reported to decrease transcriptional activity and to protect against prostate cancer, although they can lead to muscular atrophy. However, the molecular mechanisms underlying these observations are unclear. Using NMR spectroscopy, here we identify weak interactions between the AR-NTD and the KDM4A catalytic domain, and between the AR ligand-binding domain and a central KDM4A region that also contains low-complexity sequences. We also show that the AR-NTD can undergo liquid-liquid phase separation in vitro, with longer polyQ sequences phase separating more readily. Moreover, longer polyQ sequences hinder nuclear localization in the absence of hormone and increase the propensity for formation of AR-containing puncta in the nucleus of cells treated with dihydrotestosterone. These results lead us to hypothesize that polyQ-dependent liquid-liquid phase separation may provide a mechanism to decrease the transcriptional activity of AR, potentially opening new opportunities to design effective therapies against CRPC and muscular atrophy.


Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Peptídeos/metabolismo , Receptores Androgênicos/genética , Androgênios , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Expressão Gênica/genética , Glutamina/metabolismo , Humanos , Masculino , Peptídeos/genética , Neoplasias da Próstata/genética , Neoplasias de Próstata Resistentes à Castração/genética , Domínios Proteicos/genética , Receptores Androgênicos/metabolismo , Transdução de Sinais/genética , Ativação Transcricional/genética
3.
Science ; 374(6573): eabm4805, 2021 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-34762488

RESUMO

Protein-protein interactions play critical roles in biology, but the structures of many eukaryotic protein complexes are unknown, and there are likely many interactions not yet identified. We take advantage of advances in proteome-wide amino acid coevolution analysis and deep-learning­based structure modeling to systematically identify and build accurate models of core eukaryotic protein complexes within the Saccharomyces cerevisiae proteome. We use a combination of RoseTTAFold and AlphaFold to screen through paired multiple sequence alignments for 8.3 million pairs of yeast proteins, identify 1505 likely to interact, and build structure models for 106 previously unidentified assemblies and 806 that have not been structurally characterized. These complexes, which have as many as five subunits, play roles in almost all key processes in eukaryotic cells and provide broad insights into biological function.


Assuntos
Aprendizado Profundo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Mapeamento de Interação de Proteínas , Proteoma/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Aciltransferases/química , Aciltransferases/metabolismo , Segregação de Cromossomos , Biologia Computacional , Simulação por Computador , Reparo do DNA , Evolução Molecular , Recombinação Homóloga , Ligases/química , Ligases/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Biossíntese de Proteínas , Conformação Proteica , Mapas de Interação de Proteínas , Proteoma/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/química , Ubiquitina/química , Ubiquitina/metabolismo
4.
Elife ; 102021 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-34779770

RESUMO

Munc13-1 plays a central role in neurotransmitter release through its conserved C-terminal region, which includes a diacyglycerol (DAG)-binding C1 domain, a Ca2+/PIP2-binding C2B domain, a MUN domain and a C2C domain. Munc13-1 was proposed to bridge synaptic vesicles to the plasma membrane through distinct interactions of the C1C2B region with the plasma membrane: (i) one involving a polybasic face that is expected to yield a perpendicular orientation of Munc13-1 and hinder release; and (ii) another involving the DAG-Ca2+-PIP2-binding face that is predicted to result in a slanted orientation and facilitate release. Here, we have tested this model and investigated the role of the C1C2B region in neurotransmitter release. We find that K603E or R769E point mutations in the polybasic face severely impair Ca2+-independent liposome bridging and fusion in in vitro reconstitution assays, and synaptic vesicle priming in primary murine hippocampal cultures. A K720E mutation in the polybasic face and a K706E mutation in the C2B domain Ca2+-binding loops have milder effects in reconstitution assays and do not affect vesicle priming, but enhance or impair Ca2+-evoked release, respectively. The phenotypes caused by combining these mutations are dominated by the K603E and R769E mutations. Our results show that the C1-C2B region of Munc13-1 plays a central role in vesicle priming and support the notion that two distinct faces of this region control neurotransmitter release and short-term presynaptic plasticity.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurotransmissores/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Transporte Biológico , Fenômenos Biofísicos , Comunicação Celular , Membrana Celular/metabolismo , Células Cultivadas , Hipocampo/citologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos Knockout , Simulação de Dinâmica Molecular , Mutação , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Transmissão Sináptica
5.
J Biomol NMR ; 75(8-9): 347-363, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34505210

RESUMO

The development of methyl transverse relaxation optimized spectroscopy has greatly facilitated the study of macromolecular assemblies by solution NMR spectroscopy. However, limited sample solubility and stability has hindered application of this technique to ongoing studies of complexes formed on membranes by the neuronal SNAREs that mediate neurotransmitter release and synaptotagmin-1, the Ca2+ sensor that triggers release. Since the 1H NMR signal of a tBu group attached to a large protein or complex can be observed with high sensitivity if the group retains high mobility, we have explored the use of this strategy to analyze presynaptic complexes involved in neurotransmitter release. For this purpose, we attached tBu groups at single cysteines of fragments of synaptotagmin-1, complexin-1 and the neuronal SNAREs by reaction with 5-(tert-butyldisulfaneyl)-2-nitrobenzoic acid (BDSNB), tBu iodoacetamide or tBu acrylate. The tBu resonances of the tagged proteins were generally sharp and intense, although tBu groups attached with BDSNB had a tendency to exhibit somewhat broader resonances that likely result because of the shorter linkage between the tBu and the tagged cysteine. Incorporation of the tagged proteins into complexes on nanodiscs led to severe broadening of the tBu resonances in some cases. However, sharp tBu resonances could readily be observed for some complexes of more than 200 kDa at low micromolar concentrations. Our results show that tagging of proteins with tBu groups provides a powerful approach to study large biomolecular assemblies of limited stability and/or solubility that may be applicable even at nanomolar concentrations.


Assuntos
Neurônios , Proteínas SNARE , Substâncias Macromoleculares , Espectroscopia de Ressonância Magnética , Ressonância Magnética Nuclear Biomolecular
6.
Elife ; 102021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34137372

RESUMO

Two proteins called Sec17 and Sec18 may have a larger role in membrane fusion than is commonly assumed in textbook models.


Assuntos
Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfatases , Fusão de Membrana , Proteínas SNARE , Saccharomyces cerevisiae , Proteínas de Transporte Vesicular
7.
Proc Natl Acad Sci U S A ; 118(4)2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33468652

RESUMO

Neurotransmitter release is governed by eight central proteins among other factors: the neuronal SNAREs syntaxin-1, synaptobrevin, and SNAP-25, which form a tight SNARE complex that brings the synaptic vesicle and plasma membranes together; NSF and SNAPs, which disassemble SNARE complexes; Munc18-1 and Munc13-1, which organize SNARE complex assembly; and the Ca2+ sensor synaptotagmin-1. Reconstitution experiments revealed that Munc18-1, Munc13-1, NSF, and α-SNAP can mediate Ca2+-dependent liposome fusion between synaptobrevin liposomes and syntaxin-1-SNAP-25 liposomes, but high fusion efficiency due to uncontrolled SNARE complex assembly did not allow investigation of the role of synaptotagmin-1 on fusion. Here, we show that decreasing the synaptobrevin-to-lipid ratio in the corresponding liposomes to very low levels leads to inefficient fusion and that synaptotagmin-1 strongly stimulates fusion under these conditions. Such stimulation depends on Ca2+ binding to the two C2 domains of synaptotagmin-1. We also show that anchoring SNAP-25 on the syntaxin-1 liposomes dramatically enhances fusion. Moreover, we uncover a synergy between synaptotagmin-1 and membrane anchoring of SNAP-25, which allows efficient Ca2+-dependent fusion between liposomes bearing very low synaptobrevin densities and liposomes containing very low syntaxin-1 densities. Thus, liposome fusion in our assays is achieved with a few SNARE complexes in a manner that requires Munc18-1 and Munc13-1 and that depends on Ca2+ binding to synaptotagmin-1, all of which are fundamental features of neurotransmitter release in neurons.


Assuntos
Proteínas Munc18/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Vesículas Sinápticas/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Sinaptotagmina I/metabolismo , Animais , Cálcio/metabolismo , Regulação da Expressão Gênica , Lipossomos/química , Lipossomos/metabolismo , Fusão de Membrana , Proteínas Munc18/genética , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Neurotransmissores/genética , Neurotransmissores/metabolismo , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Ratos , Transmissão Sináptica , Vesículas Sinápticas/química , Proteína 25 Associada a Sinaptossoma/genética , Sinaptotagmina I/genética , Sintaxina 1/genética , Sintaxina 1/metabolismo , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismo
8.
Nat Commun ; 11(1): 5516, 2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33139696

RESUMO

Assembly of SNARE complexes that mediate neurotransmitter release requires opening of a 'closed' conformation of UNC-64/syntaxin. Rescue of unc-13/Munc13 mutant phenotypes by overexpressed open UNC-64/syntaxin suggested a specific function of UNC-13/Munc13 in opening UNC-64/ syntaxin. Here, we revisit the effects of open unc-64/syntaxin by generating knockin (KI) worms. The KI animals exhibit enhanced spontaneous and evoked exocytosis compared to WT animals. Unexpectedly, the open syntaxin KI partially suppresses exocytosis defects of various mutants, including snt-1/synaptotagmin, unc-2/P/Q/N-type Ca2+ channel alpha-subunit and unc-31/CAPS, in addition to unc-13/Munc13 and unc-10/RIM, and enhanced exocytosis in tom-1/Tomosyn mutants. However, open syntaxin aggravates the defects of unc-18/Munc18 mutants. Correspondingly, open syntaxin partially bypasses the requirement of Munc13 but not Munc18 for liposome fusion. Our results show that facilitating opening of syntaxin enhances exocytosis in a wide range of genetic backgrounds, and may provide a general means to enhance synaptic transmission in normal and disease states.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Exocitose/genética , Lipossomos/metabolismo , Transmissão Sináptica/genética , Sintaxina 1/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Técnicas de Introdução de Genes , Mutação , Neurotransmissores/metabolismo , Sintaxina 1/genética
9.
Elife ; 92020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32808925

RESUMO

The Ca2+ sensor synaptotagmin-1 and the SNARE complex cooperate to trigger neurotransmitter release. Structural studies elucidated three distinct synaptotagmin-1-SNARE complex binding modes involving 'polybasic', 'primary' and 'tripartite' interfaces of synaptotagmin-1. We investigated these interactions using NMR and fluorescence spectroscopy. Synaptotagmin-1 binds to the SNARE complex through the polybasic and primary interfaces in solution. Ca2+-free synaptotagmin-1 binds to SNARE complexes anchored on PIP2-containing nanodiscs. R398Q/R399Q and E295A/Y338W mutations at the primary interface, which strongly impair neurotransmitter release, disrupt and enhance synaptotagmin-1-SNARE complex binding, respectively. Ca2+ induces tight binding of synaptotagmin-1 to PIP2-containing nanodiscs, disrupting synaptotagmin-1-SNARE interactions. Specific effects of mutations in the polybasic region on Ca2+-dependent synaptotagmin-1-PIP2-membrane interactions correlate with their effects on release. Our data suggest that synaptotagmin-1 binds to the SNARE complex through the primary interface and that Ca2+ releases this interaction, inducing PIP2/membrane binding and allowing cooperation between synaptotagmin-1 and the SNAREs in membrane fusion to trigger release.


Inside the brain, cells called neurons relay messages from one place to another in the form of electrical signals. When an electrical signal reaches a junction between two neurons (known as a synapse) it triggers small particles called calcium ions to enter one of the cells. This influx of calcium causes vesicles to fuse with the membrane surrounding the neuron and release molecules called neurotransmitters into the small gap between the two neurons. These molecules travel across the gap to activate an electrical signal in the second neuron which then carries the message onwards. A protein known as synaptotagmin-1 senses calcium ions at synapses and works together with a group of proteins known as the SNARE complex to help vesicles fuse with the cell membrane. Previous studies have reported three different structures of synaptotagmin-1 bound to the SNARE complex in a different way. But it was unclear which of these binding states actually result in the release of neurotransmitters. To address this question, Voleti, Jaczynska and Rizo studied how and when synptotagmin-1 and the SNARE complex bind together using two approaches known as NMR spectroscopy and fluorescence spectroscopy. The experiments suggest that before calcium enters the synapse, synaptotagmin-1 is already bound to a surface on the SNARE complex. This binding inhibits the release of neurotransmitters and has been reported in previous studies. Adding calcium ions causes synaptotagmin-1 to be released from the SNARE complex. This allows synaptotagmin-1 to interact with the membrane and cooperate with the SNARE complex to trigger vesicle fusion. Finding out how neurons release neurotransmitters at synapses may help us to understand how the brain works. This could provide new insights into how defects in the synapse lead to neurological disorders, such as schizophrenia, and potentially aid the development of new treatments for such conditions.


Assuntos
Cálcio/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas SNARE/metabolismo , Transmissão Sináptica/fisiologia , Sinaptotagmina I/metabolismo , Animais , Membrana Celular/fisiologia , Ratos
10.
Proc Natl Acad Sci U S A ; 117(29): 16992-17002, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32631994

RESUMO

Enhancer of Zeste Homolog 2 (EZH2) is the catalytic subunit of Polycomb Repressive Complex 2 (PRC2), which minimally requires two other subunits, EED and SUZ12, for enzymatic activity. EZH2 has been traditionally known to mediate histone H3K27 trimethylation, a hallmark of silent chromatin. Emerging evidence indicates that EZH2 also activates gene expression in cancer cells in a context distinct from canonical PRC2. The molecular mechanism underlying the functional conversion of EZH2 from a gene repressor to an activator is unclear. Here, we show that EZH2 harbors a hidden, partially disordered transactivation domain (TAD) capable of interacting with components of active transcription machinery, mimicking archetypal acidic activators. The EZH2 TAD comprises the SRM (Stimulation-Responsive Motif) and SANT1 (SWI3, ADA2, N-CoR, and TFIIIB 1) regions that are normally involved in H3K27 methylation. The crystal structure of an EZH2-EED binary complex indicates that the EZH2 TAD mediates protein oligomerization in a noncanonical PRC2 context and is entirely sequestered. The EZH2 TAD can be unlocked by cancer-specific EZH2 phosphorylation events to undergo structural transitions that may enable subsequent transcriptional coactivator binding. The EZH2 TAD directly interacts with the transcriptional coactivator and histone acetyltransferase p300 and activates gene expression in a p300-dependent manner in cells. The corresponding TAD may also account for the gene activation function of EZH1, the paralog of EZH2. Distinct kinase signaling pathways that are known to abnormally convert EZH2 into a gene activator in cancer cells can now be understood in a common structural context of the EZH2 TAD.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/química , Células HEK293 , Células HeLa , Humanos , Proteínas Intrinsicamente Desordenadas/química , Fosforilação , Ligação Proteica , Domínios Proteicos
11.
Proc Natl Acad Sci U S A ; 117(12): 6559-6570, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32156735

RESUMO

Secretagogin (SCGN) is a hexa-EF-hand protein that is highly expressed in the pancreas, brain, and gastrointestinal tract. SCGN is known to modulate regulated exocytosis in multiple cell lines and tissues; however, its exact functions and underlying mechanisms remain unclear. Here, we report that SCGN interacts with the plasma membrane SNARE SNAP-25, but not the assembled SNARE complex, in a Ca2+-dependent manner. The crystal structure of SCGN in complex with a SNAP-25 fragment reveals that SNAP-25 adopts a helical structure and binds to EF-hands 5 and 6 of SCGN. SCGN strongly inhibits SNARE-mediated vesicle fusion in vitro by binding to SNAP-25. SCGN promotes the plasma membrane localization of SNAP-25, but not Syntaxin-1a, in SCGN-expressing cells. Finally, SCGN controls neuronal growth and brain development in zebrafish, likely via interacting with SNAP-25 or its close homolog, SNAP-23. Our results thus provide insights into the regulation of SNAREs and suggest that aberrant synapse functions underlie multiple neurological disorders caused by SCGN deficiency.


Assuntos
Exocitose , Secretagoginas/química , Secretagoginas/metabolismo , Animais , Sítios de Ligação , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Cálcio/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Mutação , Ligação Proteica , Conformação Proteica , Secretagoginas/genética , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Peixe-Zebra
12.
Protein Sci ; 29(6): 1440-1458, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32086964

RESUMO

Munc13-1 is crucial for neurotransmitter release and, together with Munc18-1, orchestrates assembly of the neuronal SNARE complex formed by syntaxin-1, SNAP-25, and synaptobrevin. Assembly starts with syntaxin-1 folded into a self-inhibited closed conformation that binds to Munc18-1. Munc13-1 is believed to catalyze the opening of syntaxin-1 to facilitate SNARE complex formation. However, different types of Munc13-1-syntaxin-1 interactions have been reported to underlie this activity, and the critical nature of Munc13-1 for release may arise because of its key role in bridging the vesicle and plasma membranes. To shed light into the mechanism of action of Munc13-1, we have used NMR spectroscopy, SNARE complex assembly experiments, and liposome fusion assays. We show that point mutations in a linker region of syntaxin-1 that forms intrinsic part of the closed conformation strongly impair stimulation of SNARE complex assembly and liposome fusion mediated by Munc13-1 fragments, even though binding of this linker region to Munc13-1 is barely detectable. Conversely, the syntaxin-1 SNARE motif clearly binds to Munc13-1, but a mutation that disrupts this interaction does not affect SNARE complex assembly or liposome fusion. We also show that Munc13-1 cannot be replaced by an artificial tethering factor to mediate liposome fusion. Overall, these results emphasize how very weak interactions can play fundamental roles in promoting conformational transitions and strongly support a model whereby the critical nature of Munc13-1 for neurotransmitter release arises not only from its ability to bridge two membranes but also from an active role in opening syntaxin-1 via interactions with the linker.


Assuntos
Proteínas do Tecido Nervoso/metabolismo , Sintaxina 1/metabolismo , Animais , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Mutação Puntual , Conformação Proteica , Ratos , Sintaxina 1/química , Sintaxina 1/genética
13.
Sci Rep ; 10(1): 2907, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32076023

RESUMO

Reconstitution assays with proteoliposomes provide a powerful tool to elucidate the mechanism of neurotransmitter release, but it is important to understand how these assays report on membrane fusion, and recent studies with yeast vacuolar SNAREs uncovered asymmetry in the results of lipid mixing assays. We have investigated whether such asymmetry also occurs in reconstitution assays with the neuronal SNAREs, using syntaxin-1-SNAP-25-containing liposomes and liposomes containing synaptobrevin (T and V liposomes, respectively), and fluorescent probes to monitor lipid and content mixing simultaneously. Switching the fluorescent probes placed on the T and V liposomes, we observed a striking asymmetry in both lipid and content mixing stimulated by a fragment spanning the two C2 domains of synaptotagmin-1, or by a peptide that spans the C-terminal half of the synaptobrevin SNARE motif. However, no such asymmetry was observed in assays performed in the presence of Munc18-1, Munc13-1, NSF and αSNAP, which coordinate the assembly-disassembly cycle of neuronal SNARE complexes. Our results show that switching fluorescent probes between the two types of liposomes provides a useful approach to better understand the reactions that occur between liposomes and detect heterogenous behavior in these reactions.


Assuntos
Bioensaio/métodos , Lipídeos/química , Neurônios/metabolismo , Proteolipídeos/metabolismo , Proteínas SNARE/metabolismo , Animais , Bovinos , Cricetulus , Transferência Ressonante de Energia de Fluorescência , Proteínas Qa-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Ratos
14.
Biophys J ; 118(3): 643-656, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31952804

RESUMO

Synaptotagmin-1 (Syt1) is a calcium sensor protein that is critical for neurotransmission and is therefore extensively studied. Here, we use pairs of optically trapped beads coated with SNARE-free synthetic membranes to investigate Syt1-induced membrane remodeling. This activity is compared with that of Doc2b, which contains a conserved C2AB domain and induces membrane tethering and hemifusion in this cell-free model. We find that the soluble C2AB domain of Syt1 strongly affects the probability and strength of membrane-membrane interactions in a strictly Ca2+- and protein-dependent manner. Single-membrane loading of Syt1 yielded the highest probability and force of membrane interactions, whereas in contrast, Doc2b was more effective after loading both membranes. A lipid-mixing assay with confocal imaging reveals that both Syt1 and Doc2b are able to induce hemifusion; however, significantly higher Syt1 concentrations are required. Consistently, both C2AB fragments cause a reduction in the membrane-bending modulus, as measured by a method based on atomic force microscopy. This lowering of the energy required for membrane deformation may contribute to Ca2+-induced fusion.


Assuntos
Proteínas de Ligação ao Cálcio , Cálcio , Fusão de Membrana , Proteínas do Tecido Nervoso , Sinaptotagmina I , Cálcio/metabolismo , Humanos , Ligação Proteica , Proteínas SNARE/metabolismo , Transmissão Sináptica , Sinaptotagmina I/metabolismo
15.
Nucleic Acids Res ; 47(22): 11623-11636, 2019 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-31647098

RESUMO

Alternative splicing is emerging as an oncogenic mechanism. In prostate cancer, generation of constitutively active forms of androgen receptor (AR) variants including AR-V7 plays an important role in progression of castration-resistant prostate cancer (CRPC). AR-V7 is generated by alternative splicing that results in inclusion of cryptic exon CE3 and translation of truncated AR protein that lacks the ligand binding domain. Whether AR-V7 can be a driver for CRPC remains controversial as the oncogenic mechanism of AR-V7 activation remains elusive. Here, we found that KDM4B promotes AR-V7 and identified a novel regulatory mechanism. KDM4B is phosphorylated by protein kinase A under conditions that promote castration-resistance, eliciting its binding to the splicing factor SF3B3. KDM4B binds RNA specifically near the 5'-CE3, upregulates the chromatin accessibility, and couples the spliceosome to the chromatin. Our data suggest that KDM4B can function as a signal responsive trans-acting splicing factor and scaffold that recruits and stabilizes the spliceosome near the alternative exon, thus promoting its inclusion. Genome-wide profiling of KDM4B-regulated genes also identified additional alternative splicing events implicated in tumorigenesis. Our study defines KDM4B-regulated alternative splicing as a pivotal mechanism for generating AR-V7 and a contributing factor for CRPC, providing insight for mechanistic targeting of CRPC.


Assuntos
Processamento Alternativo/genética , Regulação Neoplásica da Expressão Gênica/genética , Histona Desmetilases com o Domínio Jumonji/genética , Neoplasias de Próstata Resistentes à Castração/genética , Receptores Androgênicos/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Cromatina/metabolismo , Células HEK293 , Humanos , Masculino , Isoformas de Proteínas/genética , Receptores Androgênicos/metabolismo , Spliceossomos/genética
16.
Nat Commun ; 10(1): 4326, 2019 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-31548544

RESUMO

Munc18-1 and Munc13-1 orchestrate assembly of the SNARE complex formed by syntaxin-1, SNAP-25 and synaptobrevin, allowing exquisite regulation of neurotransmitter release. Non-regulated neurotransmitter release might be prevented by αSNAP, which inhibits exocytosis and SNARE-dependent liposome fusion. However, distinct mechanisms of inhibition by αSNAP were suggested, and it is unknown how such inhibition is overcome. Using liposome fusion assays, FRET and NMR spectroscopy, here we provide a comprehensive view of the mechanisms underlying the inhibitory functions of αSNAP, showing that αSNAP potently inhibits liposome fusion by: binding to syntaxin-1, hindering Munc18-1 binding; binding to syntaxin-1-SNAP-25 heterodimers, precluding SNARE complex formation; and binding to trans-SNARE complexes, preventing fusion. Importantly, inhibition by αSNAP is avoided only when Munc18-1 binds first to syntaxin-1, leading to Munc18-1-Munc13-1-dependent liposome fusion. We propose that at least some of the inhibitory activities of αSNAP ensure that neurotransmitter release occurs through the highly-regulated Munc18-1-Munc13-1 pathway at the active zone.


Assuntos
Proteínas Munc18/fisiologia , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/fisiologia , Vesículas Sinápticas/metabolismo , Animais , Bovinos , Cricetulus , Escherichia coli/genética , Fusão de Membrana , Proteínas Munc18/química , Proteínas Munc18/metabolismo , Conformação Proteica , Ratos , Proteínas SNARE/metabolismo , Proteínas SNARE/fisiologia , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/genética , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Sintaxina 1/química , Sintaxina 1/metabolismo
18.
Elife ; 82019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30816091

RESUMO

Munc13-1 plays a crucial role in neurotransmitter release. We recently proposed that the C-terminal region encompassing the C1, C2B, MUN and C2C domains of Munc13-1 (C1C2BMUNC2C) bridges the synaptic vesicle and plasma membranes through interactions involving the C2C domain and the C1-C2B region. However, the physiological relevance of this model has not been demonstrated. Here we show that C1C2BMUNC2C bridges membranes through opposite ends of its elongated structure. Mutations in putative membrane-binding sites of the C2C domain disrupt the ability of C1C2BMUNC2C to bridge liposomes and to mediate liposome fusion in vitro. These mutations lead to corresponding disruptive effects on synaptic vesicle docking, priming, and Ca2+-triggered neurotransmitter release in mouse neurons. Remarkably, these effects include an almost complete abrogation of release by a single residue substitution in this 200 kDa protein. These results show that bridging the synaptic vesicle and plasma membranes is a central function of Munc13-1.


Assuntos
Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Neurotransmissores/metabolismo , Animais , Membrana Celular/metabolismo , Células Cultivadas , Vesículas Citoplasmáticas/metabolismo , Membranas Intracelulares/metabolismo , Camundongos , Proteínas do Tecido Nervoso/genética , Ratos
19.
Elife ; 82019 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-30657450

RESUMO

Neurotransmitter release requires formation of trans-SNARE complexes between the synaptic vesicle and plasma membranes, which likely underlies synaptic vesicle priming to a release-ready state. It is unknown whether Munc18-1, Munc13-1, complexin-1 and synaptotagmin-1 are important for priming because they mediate trans-SNARE complex assembly and/or because they prevent trans-SNARE complex disassembly by NSF-αSNAP, which can lead to de-priming. Here we show that trans-SNARE complex formation in the presence of NSF-αSNAP requires both Munc18-1 and Munc13-1, as proposed previously, and is facilitated by synaptotagmin-1. Our data also show that Munc18-1, Munc13-1, complexin-1 and likely synaptotagmin-1 contribute to maintaining assembled trans-SNARE complexes in the presence of NSF-αSNAP. We propose a model whereby Munc18-1 and Munc13-1 are critical not only for mediating vesicle priming but also for precluding de-priming by preventing trans-SNARE complex disassembly; in this model, complexin-1 also impairs de-priming, while synaptotagmin-1 may assist in priming and hinder de-priming.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Munc18/química , Proteínas Sensíveis a N-Etilmaleimida/química , Proteínas do Tecido Nervoso/química , Proteína 25 Associada a Sinaptossoma/química , Sinaptotagminas/química , Animais , Células CHO , Cálcio/química , Cricetinae , Cricetulus , Microscopia Crioeletrônica , Citoplasma/química , Transferência Ressonante de Energia de Fluorescência , Cinética , Mutação , Proteínas R-SNARE/química , Ratos , Sintaxina 1/química
20.
Mol Biol Cell ; 29(19): 2259-2279, 2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-30044717

RESUMO

Cytokinesis is a complicated yet conserved step of the cell-division cycle that requires the coordination of multiple proteins and cellular processes. Here we describe a previously uncharacterized protein, Ync13, and its roles during fission yeast cytokinesis. Ync13 is a member of the UNC-13/Munc13 protein family, whose animal homologues are essential priming factors for soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex assembly during exocytosis in various cell types, but no roles in cytokinesis have been reported. We find that Ync13 binds to lipids in vitro and dynamically localizes to the plasma membrane at cell tips during interphase and at the division site during cytokinesis. Deletion of Ync13 leads to defective septation and exocytosis, uneven distribution of cell-wall enzymes and components of cell-wall integrity pathway along the division site and massive cell lysis during cell separation. Interestingly, loss of Ync13 compromises endocytic site selection at the division plane. Collectively, we find that Ync13 has a novel function as an UNC-13/Munc13 protein in coordinating exocytosis, endocytosis, and cell-wall integrity during fission yeast cytokinesis.


Assuntos
Citocinese , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Membrana Celular/metabolismo , Parede Celular/metabolismo , Endocitose , Epistasia Genética , Exocitose , Glucosiltransferases/metabolismo , Lipídeos de Membrana/metabolismo , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Mutação/genética , Transporte Proteico
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