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1.
Proc Natl Acad Sci U S A ; 121(16): e2321447121, 2024 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-38593076

RESUMO

The SNAP receptor (SNARE) proteins syntaxin-1, SNAP-25, and synaptobrevin mediate neurotransmitter release by forming tight SNARE complexes that fuse synaptic vesicles with the plasma membranes in microseconds. Membrane fusion is generally explained by the action of proteins on macroscopic membrane properties such as curvature, elastic modulus, and tension, and a widespread model envisions that the SNARE motifs, juxtamembrane linkers, and C-terminal transmembrane regions of synaptobrevin and syntaxin-1 form continuous helices that act mechanically as semirigid rods, squeezing the membranes together as they assemble ("zipper") from the N to the C termini. However, the mechanism underlying fast SNARE-induced membrane fusion remains unknown. We have used all-atom molecular dynamics simulations to investigate this mechanism. Our results need to be interpreted with caution because of the limited number and length of the simulations, but they suggest a model of membrane fusion that has a natural physicochemical basis, emphasizes local molecular events over general membrane properties, and explains extensive experimental data. In this model, the central event that initiates fast (microsecond scale) membrane fusion occurs when the SNARE helices zipper into the juxtamembrane linkers which, together with the adjacent transmembrane regions, promote encounters of acyl chains from both bilayers at the polar interface. The resulting hydrophobic nucleus rapidly expands into stalk-like structures that gradually progress to form a fusion pore, aided by the SNARE transmembrane regions and without clearly discernible intermediates. The propensity of polyunsaturated lipids to participate in encounters that initiate fusion suggests that these lipids may be important for the high speed of neurotransmitter release.


Assuntos
Fusão de Membrana , Proteínas SNARE , Proteínas SNARE/metabolismo , Simulação de Dinâmica Molecular , Proteínas R-SNARE , Sintaxina 1 , Neurotransmissores , Lipídeos
2.
Proc Natl Acad Sci U S A ; 121(42): e2409636121, 2024 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-39374398

RESUMO

The Ca2+ sensor synaptotagmin-1 (Syt1) triggers neurotransmitter release together with the neuronal sensitive factor attachment protein receptor (SNARE) complex formed by syntaxin-1, SNAP25, and synaptobrevin. Moreover, Syt1 increases synaptic vesicle (SV) priming and impairs spontaneous vesicle release. The Syt1 C2B domain binds to the SNARE complex through a primary interface via two regions (I and II), but how exactly this interface mediates distinct functions of Syt1 and the mechanism underlying Ca2+ triggering of release are unknown. Using mutagenesis and electrophysiological experiments, we show that region II is functionally and spatially subdivided: Binding of C2B domain arginines to SNAP-25 acidic residues at one face of region II is crucial for Ca2+-evoked release but not for vesicle priming or clamping of spontaneous release, whereas other SNAP-25 and syntaxin-1 acidic residues at the other face mediate priming and clamping of spontaneous release but not evoked release. Mutations that disrupt region I impair the priming and clamping functions of Syt1 while, strikingly, mutations that enhance binding through this region increase vesicle priming and clamping of spontaneous release, but strongly inhibit evoked release and vesicle fusogenicity. These results support previous findings that the primary interface mediates the functions of Syt1 in vesicle priming and clamping of spontaneous release and, importantly, show that Ca2+ triggering of release requires a rearrangement of the primary interface involving dissociation of region I, while region II remains bound. Together with biophysical studies presented in [K. Jaczynska et al., bioRxiv [Preprint] (2024). https://doi.org/10.1101/2024.06.17.599417 (Accessed 18 June 2024)], our data suggest a model whereby this rearrangement pulls the SNARE complex to facilitate fast SV fusion.


Assuntos
Cálcio , Neurotransmissores , Proteínas SNARE , Vesículas Sinápticas , Sinaptotagmina I , Sinaptotagmina I/metabolismo , Sinaptotagmina I/genética , Cálcio/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Proteínas SNARE/metabolismo , Proteínas SNARE/genética , Neurotransmissores/metabolismo , Sintaxina 1/metabolismo , Sintaxina 1/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Proteína 25 Associada a Sinaptossoma/genética , Ratos , Ligação Proteica , Transmissão Sináptica
3.
Cell ; 144(2): 282-95, 2011 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-21241895

RESUMO

At a synapse, fast synchronous neurotransmitter release requires localization of Ca(2+) channels to presynaptic active zones. How Ca(2+) channels are recruited to active zones, however, remains unknown. Using unbiased yeast two-hybrid screens, we here identify a direct interaction of the central PDZ domain of the active-zone protein RIM with the C termini of presynaptic N- and P/Q-type Ca(2+) channels but not L-type Ca(2+) channels. To test the physiological significance of this interaction, we generated conditional knockout mice lacking all multidomain RIM isoforms. Deletion of RIM proteins ablated most neurotransmitter release by simultaneously impairing the priming of synaptic vesicles and by decreasing the presynaptic localization of Ca(2+) channels. Strikingly, rescue of the decreased Ca(2+)-channel localization required the RIM PDZ domain, whereas rescue of vesicle priming required the RIM N terminus. We propose that RIMs tether N- and P/Q-type Ca(2+) channels to presynaptic active zones via a direct PDZ-domain-mediated interaction, thereby enabling fast, synchronous triggering of neurotransmitter release at a synapse.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Canais de Cálcio/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Animais , Cálcio/metabolismo , Canais de Cálcio/química , Proteínas de Ligação ao GTP/química , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/química , Neurotransmissores/metabolismo , Terminações Pré-Sinápticas/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Técnicas do Sistema de Duplo-Híbrido
4.
Annu Rev Cell Dev Biol ; 28: 279-308, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23057743

RESUMO

Neurotransmitter release is governed by proteins that have homo-logs in most types of intracellular membrane fusion, including the Sec1/Munc18 protein Munc18-1 and the SNARE proteins syntaxin-1, synaptobrevin/VAMP, and SNAP-25. The SNAREs initiate fusion by forming tight SNARE complexes that bring the vesicle and plasma membranes together. SNARE maintenance in a functional state depends on two chaperone systems (Hsc70/αCSP/SGT and synuclein); defects in these systems lead to neurodegeneration. Munc18-1 binds to an autoinhibitory closed conformation of syntaxin-1, gating formation of SNARE complexes, and also binds to SNARE complexes, which likely underlies the crucial function of Munc18-1 in membrane fusion by an as-yet unclear mechanism. Syntaxin-1 opening is mediated by Munc13s through their MUN domain, which is homologous to diverse tethering factors and may also have a general role in fusion. MUN domain activity is likely modulated in diverse presynaptic plasticity processes that depend on Ca(2+) and RIM proteins, among others.


Assuntos
Fusão de Membrana , Proteínas Munc18/fisiologia , Proteínas SNARE/fisiologia , Animais , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Humanos , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/fisiologia , Proteínas Munc18/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas SNARE/metabolismo
5.
Proc Natl Acad Sci U S A ; 120(41): e2311416120, 2023 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-37782781

RESUMO

An evolutionarily conserved region of the TDP-43 low-complexity domain (LCD) twenty residues in length can adopt either an α-helical or ß-strand conformation. When in the latter conformation, TDP-43 self-associates via the formation of a labile, cross-ß structure. Self-association can be monitored via the formation of phase-separated protein droplets. Exposure of droplets to hydrogen peroxide leads to oxidation of conserved methionine residues distributed throughout the LCD. Oxidation disassembles the cross-ß structure, thus eliminating both self-association and phase separation. Here, we demonstrate that this process reciprocally enables formation of α-helical structure in precisely the same region formerly functioning to facilitate ß-strand-mediated self-association. We further observe that the α-helical conformation allows interaction with a lipid-like detergent and that exposure to lipids enhances the ß-to-α conformational switch. We hypothesize that regulation of this oxidative switch will prove to be important to the control of localized translation within vertebrate cells. The experimental observations reported herein were heavily reliant on studies of 1,6-hexanediol, a chemical agent that selectively dissolves labile structures formed via the self-association of protein domains of low sequence complexity. This aliphatic alcohol is shown to exert its dissociative activity primarily via hydrogen-bonding interactions with carbonyl oxygen atoms of the polypeptide backbone. Such observations underscore the central importance of backbone-mediated protein:protein interactions that facilitate the self-association and phase separation of LCDs.


Assuntos
Proteínas de Ligação a DNA , Peptídeos , Proteínas de Ligação a DNA/metabolismo , Peptídeos/química , Domínios Proteicos , Metionina/metabolismo , Estresse Oxidativo
6.
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
7.
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
8.
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
9.
Mol Cell ; 54(1): 133-146, 2014 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-24703947

RESUMO

Programmed necrotic cell death induced by the tumor necrosis factor alpha (TNF-α) family of cytokines is dependent on a kinase cascade consisting of receptor-interacting kinases RIP1 and RIP3. How these kinase activities cause cells to die by necrosis is not known. The mixed lineage kinase domain-like protein MLKL is a functional RIP3 substrate that binds to RIP3 through its kinase-like domain but lacks kinase activity of its own. RIP3 phosphorylates MLKL at the T357 and S358 sites. Reported here is the development of a monoclonal antibody that specifically recognizes phosphorylated MLKL in cells dying of this pathway and in human liver biopsy samples from patients suffering from drug-induced liver injury. The phosphorylated MLKL forms an oligomer that binds to phosphatidylinositol lipids and cardiolipin. This property allows MLKL to move from the cytosol to the plasma and intracellular membranes, where it directly disrupts membrane integrity, resulting in necrotic death.


Assuntos
Membrana Celular/enzimologia , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Anticorpos Monoclonais/imunologia , Sítios de Ligação , Cardiolipinas/metabolismo , Membrana Celular/patologia , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Células HT29 , Células HeLa , Humanos , Membranas Intracelulares/enzimologia , Membranas Intracelulares/patologia , Fígado/enzimologia , Fígado/patologia , Lipídeos de Membrana/metabolismo , Necrose , Fosforilação , Conformação Proteica , Proteínas Quinases/genética , Proteínas Quinases/imunologia , Transporte Proteico , Interferência de RNA , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Transdução de Sinais , Especificidade por Substrato , Fatores de Tempo , Transfecção
10.
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
11.
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
12.
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
13.
Nature ; 505(7481): 103-7, 2014 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-24256734

RESUMO

Human body-surface epithelia coexist in close association with complex bacterial communities and are protected by a variety of antibacterial proteins. C-type lectins of the RegIII family are bactericidal proteins that limit direct contact between bacteria and the intestinal epithelium and thus promote tolerance to the intestinal microbiota. RegIII lectins recognize their bacterial targets by binding peptidoglycan carbohydrate, but the mechanism by which they kill bacteria is unknown. Here we elucidate the mechanistic basis for RegIII bactericidal activity. We show that human RegIIIα (also known as HIP/PAP) binds membrane phospholipids and kills bacteria by forming a hexameric membrane-permeabilizing oligomeric pore. We derive a three-dimensional model of the RegIIIα pore by docking the RegIIIα crystal structure into a cryo-electron microscopic map of the pore complex, and show that the model accords with experimentally determined properties of the pore. Lipopolysaccharide inhibits RegIIIα pore-forming activity, explaining why RegIIIα is bactericidal for Gram-positive but not Gram-negative bacteria. Our findings identify C-type lectins as mediators of membrane attack in the mucosal immune system, and provide detailed insight into an antibacterial mechanism that promotes mutualism with the resident microbiota.


Assuntos
Antibacterianos/metabolismo , Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Intestinos/química , Lectinas Tipo C/metabolismo , Porinas/metabolismo , Antibacterianos/química , Antibacterianos/imunologia , Antibacterianos/farmacologia , Antígenos de Neoplasias/química , Antígenos de Neoplasias/imunologia , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/química , Biomarcadores Tumorais/imunologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Microscopia Crioeletrônica , Cristalografia por Raios X , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/imunologia , Bactérias Gram-Negativas/metabolismo , Humanos , Imunidade nas Mucosas/efeitos dos fármacos , Imunidade nas Mucosas/imunologia , Intestinos/imunologia , Intestinos/microbiologia , Lectinas Tipo C/antagonistas & inibidores , Lectinas Tipo C/química , Lectinas Tipo C/imunologia , Lipopolissacarídeos/farmacologia , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/imunologia , Listeria monocytogenes/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Modelos Moleculares , Proteínas Associadas a Pancreatite , Peptidoglicano/metabolismo , Fosfolipídeos/metabolismo , Porinas/antagonistas & inibidores , Porinas/química , Simbiose
14.
Proc Natl Acad Sci U S A ; 114(40): E8518-E8527, 2017 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-28923929

RESUMO

Synaptotagmins (Syts) act as Ca2+ sensors in neurotransmitter release by virtue of Ca2+-binding to their two C2 domains, but their mechanisms of action remain unclear. Puzzlingly, Ca2+-binding to the C2B domain appears to dominate Syt1 function in synchronous release, whereas Ca2+-binding to the C2A domain mediates Syt7 function in asynchronous release. Here we show that crystal structures of the Syt7 C2A domain and C2AB region, and analyses of intrinsic Ca2+-binding to the Syt7 C2 domains using isothermal titration calorimetry, did not reveal major differences that could explain functional differentiation between Syt7 and Syt1. However, using liposome titrations under Ca2+ saturating conditions, we show that the Syt7 C2A domain has a very high membrane affinity and dominates phospholipid binding to Syt7 in the presence or absence of l-α-phosphatidylinositol 4,5-diphosphate (PIP2). For Syt1, the two Ca2+-saturated C2 domains have similar affinities for membranes lacking PIP2, but the C2B domain dominates binding to PIP2-containing membranes. Mutagenesis revealed that the dramatic differences in membrane affinity between the Syt1 and Syt7 C2A domains arise in part from apparently conservative residue substitutions, showing how striking biochemical and functional differences can result from the cumulative effects of subtle residue substitutions. Viewed together, our results suggest that membrane affinity may be a key determinant of the functions of Syt C2 domains in neurotransmitter release.


Assuntos
Cálcio/metabolismo , Membrana Celular/metabolismo , Neurotransmissores/metabolismo , Sinaptotagmina I/metabolismo , Sinaptotagminas/metabolismo , Animais , Cristalografia por Raios X , Lipossomos , Ligação Proteica , Domínios Proteicos , Ratos , Transmissão Sináptica , Sinaptotagmina I/química , Sinaptotagmina I/genética , Sinaptotagminas/química , Sinaptotagminas/genética
15.
J Neurosci ; 37(36): 8797-8815, 2017 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-28821673

RESUMO

Munc18-1/UNC-18 is believed to prime SNARE-mediated membrane fusion, yet the underlying mechanisms remain enigmatic. Here, we examine how potential gain-of-function mutations of Munc18-1/UNC-18 affect locomotory behavior and synaptic transmission, and how Munc18-1-mediated priming is related to Munc13-1/UNC-13 and Tomosyn/TOM-1, positive and negative SNARE regulators, respectively. We show that a Munc18-1(P335A)/UNC-18(P334A) mutation leads to significantly increased locomotory activity and acetylcholine release in Caenorhabditis elegans, as well as enhanced synaptic neurotransmission in cultured mammalian neurons. Importantly, similar to tom-1 null mutants, unc-18(P334A) mutants partially bypass the requirement of UNC-13. Moreover, unc-18(P334A) and tom-1 null mutations confer a strong synergy in suppressing the phenotypes of unc-13 mutants. Through biochemical experiments, we demonstrate that Munc18-1(P335A) exhibits enhanced activity in SNARE complex formation as well as in binding to the preformed SNARE complex, and partially bypasses the Munc13-1 requirement in liposome fusion assays. Our results indicate that Munc18-1/UNC-18 primes vesicle fusion downstream of Munc13-1/UNC-13 by templating SNARE complex assembly and acts antagonistically with Tomosyn/TOM-1.SIGNIFICANCE STATEMENT At presynaptic sites, SNARE-mediated membrane fusion is tightly regulated by several key proteins including Munc18/UNC-18, Munc13/UNC-13, and Tomosyn/TOM-1. However, how these proteins interact with each other to achieve the precise regulation of neurotransmitter release remains largely unclear. Using Caenorhabditis elegans as an in vivo model, we found that a gain-of-function mutant of UNC-18 increases locomotory activity and synaptic acetylcholine release, that it partially bypasses the requirement of UNC-13 for release, and that this bypass is synergistically augmented by the lack of TOM-1. We also elucidated the biochemical basis for the gain-of-function caused by this mutation. Thus, our study provides novel mechanistic insights into how Munc18/UNC-18 primes synaptic vesicle release and how this protein interacts functionally with Munc13/UNC-13 and Tomosyn/TOM-1.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas de Transporte/metabolismo , Locomoção/fisiologia , Fosfoproteínas/metabolismo , Proteínas SNARE/metabolismo , Transmissão Sináptica/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Transporte/genética , Mutação/genética , Neurônios , Fosfoproteínas/genética , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular/genética
16.
J Biol Chem ; 291(16): 8516-27, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26846854

RESUMO

Multiple neurodegenerative diseases are caused by the aggregation of the human α-Synuclein (α-Syn) protein. α-Syn possesses high structural plasticity and the capability of interacting with membranes. Both features are not only essential for its physiological function but also play a role in the aggregation process. Recently it has been proposed that α-Syn is able to form lipid-protein particles reminiscent of high-density lipoproteins. Here, we present a method to obtain a stable and homogeneous population of nanometer-sized particles composed of α-Syn and anionic phospholipids. These particles are called α-Syn lipoprotein (nano)particles to indicate their relationship to high-density lipoproteins formed by human apolipoproteins in vivo and of in vitro self-assembling phospholipid bilayer nanodiscs. Structural investigations of the α-Syn lipoprotein particles by circular dichroism (CD) and magic angle solid-state nuclear magnetic resonance (MAS SS-NMR) spectroscopy establish that α-Syn adopts a helical secondary structure within these particles. Based on cryo-electron microscopy (cryo-EM) and dynamic light scattering (DLS) α-Syn lipoprotein particles have a defined size with a diameter of ∼23 nm. Chemical cross-linking in combination with solution-state NMR and multiangle static light scattering (MALS) of α-Syn particles reveal a high-order protein-lipid entity composed of ∼8-10 α-Syn molecules. The close resemblance in size between cross-linked in vitro-derived α-Syn lipoprotein particles and a cross-linked species of endogenous α-Syn from SH-SY5Y human neuroblastoma cells indicates a potential functional relevance of α-Syn lipoprotein nanoparticles.


Assuntos
Lipoproteínas HDL/química , Nanopartículas/química , Fosfolipídeos/química , alfa-Sinucleína/química , Linhagem Celular Tumoral , Humanos , Ressonância Magnética Nuclear Biomolecular
17.
J Biomol NMR ; 66(4): 281-293, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27988858

RESUMO

Neurotransmitter release depends critically on the neuronal SNARE complex formed by syntaxin-1, SNAP-25 and synaptobrevin, as well as on other proteins such as Munc18-1, Munc13-1 and synaptotagmin-1. Although three-dimensional structures are available for these components, it is still unclear how they are assembled between the synaptic vesicle and plasma membranes to trigger fast, Ca2+-dependent membrane fusion. Methyl TROSY NMR experiments provide a powerful tool to study complexes between these proteins, but assignment of the methyl groups of the SNARE complex is hindered by its limited solubility. Here we report the assignment of the isoleucine, leucine, methionine and valine methyl groups of the four SNARE motifs of syntaxin-1, SNAP-25 and synaptobrevin within the SNARE complex based solely on measurements of lanthanide-induced pseudocontact shifts. Our results illustrate the power of this approach to assign protein resonances without the need of triple resonance experiments and provide an invaluable tool for future structural studies of how the SNARE complex binds to other components of the release machinery.


Assuntos
Elementos da Série dos Lantanídeos/química , Espectroscopia de Ressonância Magnética , Complexos Multiproteicos/química , Neurônios , Ressonância Magnética Nuclear Biomolecular , Proteínas SNARE/química , Animais , Marcação por Isótopo , Espectroscopia de Ressonância Magnética/métodos , Complexos Multiproteicos/metabolismo , Neurônios/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Ratos , Proteínas SNARE/metabolismo
18.
Nature ; 468(7322): 400-5, 2010 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-20927106

RESUMO

Jasmonates are a family of plant hormones that regulate plant growth, development and responses to stress. The F-box protein CORONATINE INSENSITIVE 1 (COI1) mediates jasmonate signalling by promoting hormone-dependent ubiquitylation and degradation of transcriptional repressor JAZ proteins. Despite its importance, the mechanism of jasmonate perception remains unclear. Here we present structural and pharmacological data to show that the true Arabidopsis jasmonate receptor is a complex of both COI1 and JAZ. COI1 contains an open pocket that recognizes the bioactive hormone (3R,7S)-jasmonoyl-l-isoleucine (JA-Ile) with high specificity. High-affinity hormone binding requires a bipartite JAZ degron sequence consisting of a conserved α-helix for COI1 docking and a loop region to trap the hormone in its binding pocket. In addition, we identify a third critical component of the jasmonate co-receptor complex, inositol pentakisphosphate, which interacts with both COI1 and JAZ adjacent to the ligand. Our results unravel the mechanism of jasmonate perception and highlight the ability of F-box proteins to evolve as multi-component signalling hubs.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Ciclopentanos/metabolismo , Fosfatos de Inositol/metabolismo , Oxilipinas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Aminoácidos/química , Aminoácidos/metabolismo , Arabidopsis/química , Arabidopsis/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Ciclopentanos/química , Proteínas F-Box/química , Proteínas F-Box/metabolismo , Indenos/química , Indenos/metabolismo , Isoleucina/análogos & derivados , Isoleucina/química , Isoleucina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Oxilipinas/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Reguladores de Crescimento de Plantas/química , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de Sinais
19.
Proc Natl Acad Sci U S A ; 110(34): E3243-52, 2013 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-23918375

RESUMO

Synaptotagmin-1 functions as a Ca(2+) sensor in neurotransmitter release through its two C2 domains (the C2A and C2B domain). The ability of synaptotagmin-1 to bridge two membranes is likely crucial for its function, enabling cooperation with the soluble N-ethylmaleimide sensitive factor adaptor protein receptors (SNAREs) in membrane fusion, but two bridging mechanisms have been proposed. A highly soluble synaptotagmin-1 fragment containing both domains (C2AB) was shown to bind simultaneously to two membranes via the Ca(2+)-binding loops at the top of both domains and basic residues at the bottom of the C2B domain (direct bridging mechanism). In contrast, a longer fragment including a linker sequence (lnC2AB) was found to aggregate in solution and was proposed to bridge membranes through trans interactions between lnC2AB oligomers bound to each membrane via the Ca(2+)-binding loops, with no contact of the bottom of the C2B domain with the membranes. We now show that lnC2AB containing impurities indeed aggregates in solution, but properly purified lnC2AB is highly soluble. Moreover, cryo-EM images reveal that a majority of lnC2AB molecules bridge membranes directly. Fluorescence spectroscopy indicates that the bottom of the C2B domain contacts the membrane in a sizeable population of molecules of both membrane-bound C2AB and membrane-bound lnC2AB. NMR data on nanodiscs show that a fraction of C2AB molecules bind to membranes with antiparallel orientations of the C2 domains. Together with previous studies, these results show that direct bridging constitutes the prevalent mechanism of membrane bridging by both C2AB and lnC2AB, suggesting that this mechanism underlies the function of synaptotagmin-1 in neurotransmitter release.


Assuntos
Modelos Moleculares , Sinaptotagmina I/química , Radioisótopos de Carbono , Cromatografia em Gel , Cromatografia por Troca Iônica , Microscopia Crioeletrônica , Escherichia coli , Fluorescência , Espectroscopia de Ressonância Magnética , Mutagênese Sítio-Dirigida , Espectrofotometria , Marcadores de Spin , Trítio
20.
Proc Natl Acad Sci U S A ; 109(10): 3802-7, 2012 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-22355143

RESUMO

Mint adaptor proteins bind to the amyloid precursor protein (APP) and regulate APP processing associated with Alzheimer's disease; however, the molecular mechanisms underlying Mint regulation in APP binding and processing remain unclear. Biochemical, biophysical, and cellular experiments now show that the Mint1 phosphotyrosine binding (PTB) domain that binds to APP is intramolecularly inhibited by the adjacent C-terminal linker region. The crystal structure of a C-terminally extended Mint1 PTB fragment reveals that the linker region forms a short α-helix that folds back onto the PTB domain and sterically hinders APP binding. This intramolecular interaction is disrupted by mutation of Tyr633 within the Mint1 autoinhibitory helix leading to enhanced APP binding and ß-amyloid production. Our findings suggest that an autoinhibitory mechanism in Mint1 is important for regulating APP processing and may provide novel therapies for Alzheimer's disease.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Precursor de Proteína beta-Amiloide/química , Proteínas do Tecido Nervoso/química , Doença de Alzheimer/metabolismo , Bioquímica/métodos , Biofísica/métodos , Cristalografia por Raios X/métodos , Análise Mutacional de DNA , Células HEK293 , Humanos , Cinética , Espectroscopia de Ressonância Magnética/métodos , Conformação Molecular , Neurônios/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Tirosina/química
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