RESUMO
α-Synuclein (α-Syn) aggregation is closely associated with Parkinson's disease neuropathology. Physiologically, α-Syn promotes synaptic vesicle (SV) clustering and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly. However, the underlying structural and molecular mechanisms are uncertain and it is not known whether this function affects the pathological aggregation of α-Syn. Here we show that the juxtamembrane region of vesicle-associated membrane protein 2 (VAMP2)-a component of the SNARE complex that resides on SVs-directly interacts with the carboxy-terminal region of α-Syn through charged residues to regulate α-Syn's function in clustering SVs and promoting SNARE complex assembly by inducing a multi-component condensed phase of SVs, α-Syn and other components. Moreover, VAMP2 binding protects α-Syn against forming aggregation-prone oligomers and fibrils in these condensates. Our results suggest a molecular mechanism that maintains α-Syn's function and prevents its pathological amyloid aggregation, the failure of which may lead to Parkinson's disease.
Assuntos
Vesículas Sinápticas , Proteína 2 Associada à Membrana da Vesícula , alfa-Sinucleína , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Proteína 2 Associada à Membrana da Vesícula/genética , Vesículas Sinápticas/metabolismo , Animais , Humanos , Ligação Proteica , Proteínas SNARE/metabolismo , Proteínas SNARE/genética , Camundongos , Ratos , Doença de Parkinson/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/patologia , Neurônios/metabolismo , Neurônios/patologia , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genéticaRESUMO
α-Synuclein (αSYN), a pivotal synaptic protein implicated in synucleinopathies such as Parkinson's disease and Lewy body dementia, undergoes protein phase separation. We reveal that vesicle-associated membrane protein 2 (VAMP2) orchestrates αSYN phase separation both in vitro and in cells. Electrostatic interactions, specifically mediated by VAMP2 via its juxtamembrane domain and the αSYN C-terminal region, drive phase separation. Condensate formation is specific for R-SNARE VAMP2 and dependent on αSYN lipid membrane binding. Our results delineate a regulatory mechanism for αSYN phase separation in cells. Furthermore, we show that αSYN condensates sequester vesicles and attract complexin-1 and -2, thus supporting a role in synaptic physiology and pathophysiology.
Assuntos
Proteína 2 Associada à Membrana da Vesícula , alfa-Sinucleína , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Proteína 2 Associada à Membrana da Vesícula/genética , Humanos , Ligação Proteica , Animais , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Células HEK293 , Eletricidade Estática , Separação de FasesRESUMO
Differentiation of murine epidermal stem/progenitor cells involves the permanent withdrawal from the cell cycle, the synthesis of various protein and lipid components for the cornified envelope, and the controlled dissolution of cellular organelles and nuclei. Deregulated epidermal differentiation contributes to the development of various skin diseases, including skin cancers. With a genome-wide shRNA screen, we identified vesicle-associated membrane protein 2 (VAMP2) as a critical factor involved in skin differentiation. Deletion of VAMP2 leads to aberrant skin stratification and enucleation in vivo. With quantitative proteomics, we further identified an autophagy protein, focal adhesion kinase family interacting protein of 200 kDa (FIP200), as a binding partner of VAMP2. Additionally, we showed that both VAMP2 and FIP200 are critical for murine keratinocyte enucleation and epidermal differentiation. Loss of VAMP2 or FIP200 enhances cutaneous carcinogenesis in vivo. Together, our findings identify important molecular mechanisms underlying epidermal differentiation and skin tumorigenesis.
Assuntos
Proteínas Relacionadas à Autofagia , Autofagia , Carcinogênese , Diferenciação Celular , Epiderme , Queratinócitos , Proteína 2 Associada à Membrana da Vesícula , Animais , Camundongos , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Queratinócitos/metabolismo , Queratinócitos/citologia , Epiderme/metabolismo , Epiderme/patologia , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Proteína 2 Associada à Membrana da Vesícula/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinogênese/genética , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/genética , Células Epidérmicas/metabolismo , Núcleo Celular/metabolismo , Camundongos KnockoutRESUMO
BACKGROUND: Neurotransmitter release depends on the fusion of synaptic vesicles with the presynaptic membrane and is mainly mediated by SNARE complex assembly. During the transition of Munc18-1/Syntaxin-1 to the SNARE complex, the opening of the Syntaxin-1 linker region catalyzed by Munc13-1 leads to the extension of the domain 3a hinge loop, which enables domain 3a to bind SNARE motifs in Synaptobrevin-2 and Syntaxin-1 and template the SNARE complex assembly. However, the exact mechanism of domain 3a extension remains elusive. RESULTS: Here, we characterized residues on the domain 3a hinge loop that are crucial for the extension of domain 3a by using biophysical and biochemical approaches and electrophysiological recordings. We showed that the mutation of residues T323/M324/R325 disrupted Munc13-1-mediated SNARE complex assembly and membrane fusion starting from Munc18-1/Syntaxin-1 in vitro and caused severe defects in the synaptic exocytosis of mouse cortex neurons in vivo. Moreover, the mutation had no effect on the binding of Synaptobrevin-2 to isolated Munc18-1 or the conformational change of the Syntaxin-1 linker region catalyzed by the Munc13-1 MUN domain. However, the extension of the domain 3a hinge loop in Munc18-1/Syntaxin-1 was completely disrupted by the mutation, leading to the failure of Synaptobrevin-2 binding to Munc18-1/Syntaxin-1. CONCLUSIONS: Together with previous results, our data further support the model that the template function of Munc18-1 in SNARE complex assembly requires the extension of domain 3a, and particular residues in the domain 3a hinge loop are crucial for the autoinhibitory release of domain 3a after the MUN domain opens the Syntaxin-1 linker region.
Assuntos
Proteínas do Tecido Nervoso , Proteína 2 Associada à Membrana da Vesícula , Camundongos , Animais , Proteínas do Tecido Nervoso/genética , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Sintaxina 1/genética , Sintaxina 1/química , Sintaxina 1/metabolismo , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas SNARE/metabolismo , Ligação ProteicaRESUMO
Migraine is a common and complex neurological disease potentially caused by a polygenic interaction of multiple gene variants. Many genes associated with migraine are involved in pathways controlling the synaptic function and neurotransmitters release. However, the molecular mechanisms underpinning migraine need to be further explored.Recent studies raised the possibility that migraine may arise from the effect of regulatory non-coding variants. In this study, we explored the effect of candidate non-coding variants potentially associated with migraine and predicted to lie within regulatory elements: VAMP2_rs1150, SNAP25_rs2327264, and STX1A_rs6951030. The involvement of these genes, which are constituents of the SNARE complex involved in membrane fusion and neurotransmitter release, underscores their significance in migraine pathogenesis. Our reporter gene assays confirmed the impact of at least two of these non-coding variants. VAMP2 and SNAP25 risk alleles were associated with a decrease and increase in gene expression, respectively, while STX1A risk allele showed a tendency to reduce luciferase activity in neuronal-like cells. Therefore, the VAMP2_rs1150 and SNAP25_rs2327264 non-coding variants affect gene expression, which may have implications in migraine susceptibility. Based on previous in silico analysis, it is plausible that these variants influence the binding of regulators, such as transcription factors and micro-RNAs. Still, further studies exploring these mechanisms would be important to shed light on the association between SNAREs dysregulation and migraine susceptibility.
Assuntos
Transtornos de Enxaqueca , Proteína 2 Associada à Membrana da Vesícula , Humanos , Proteína 2 Associada à Membrana da Vesícula/genética , Fusão de Membrana , Alelos , Transtornos de Enxaqueca/genética , Expressão Gênica , Proteína 25 Associada a Sinaptossoma/genéticaRESUMO
Vesicle fusion at the plasma membrane is critical for releasing hormones and neurotransmitters and for delivering the cognate G protein-coupled receptors (GPCRs) to the cell surface. The SNARE fusion machinery that releases neurotransmitters has been well characterized. In contrast, the fusion machinery that delivers GPCRs is still unknown. Here, using high-speed multichannel imaging to simultaneously visualize receptors and v-SNAREs in real time in individual fusion events, we identify VAMP2 as a selective v-SNARE for GPCR delivery. VAMP2 was preferentially enriched in vesicles that mediate the surface delivery of µ opioid receptor (MOR), but not other cargos, and was required selectively for MOR recycling. Interestingly, VAMP2 did not show preferential localization on MOR-containing endosomes, suggesting that v-SNAREs are copackaged with specific cargo into separate vesicles from the same endosomes. Together, our results identify VAMP2 as a cargo-selective v-SNARE and suggest that surface delivery of specific GPCRs is mediated by distinct fusion events driven by distinct SNARE complexes.
Assuntos
Fusão de Membrana , Receptores Acoplados a Proteínas G , Proteínas SNARE , Proteína 2 Associada à Membrana da Vesícula , Membrana Celular/metabolismo , Neurotransmissores/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas SNARE/metabolismo , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
Synaptic accumulation of α-synuclein (α-Syn) oligomers and their interactions with VAMP2 have been reported to be the basis of synaptic dysfunction in Parkinson's disease (PD). α-Syn mutants associated with familial PD have also been known to be capable of interacting with VAMP2, but the exact mechanisms resulting from those interactions to eventual synaptic dysfunction are still unclear. Here, we investigate the effect of α-Syn mutant oligomers comprising A30P, E46K, and A53T on VAMP2-embedded vesicles. Specifically, A30P and A53T oligomers cluster vesicles in the presence of VAMP2, which is a shared mechanism with wild type α-Syn oligomers induced by dopamine. On the other hand, E46K oligomers reduce the membrane mobility of the planar bilayers, as revealed by single-particle tracking, and permeabilize the membranes in the presence of VAMP2. In the absence of VAMP2 interactions, E46K oligomers enlarge vesicles by fusing with one another. Our results clearly demonstrate that α-Syn mutant oligomers have aberrant effects on VAMP2-embedded vesicles and the disruption types are distinct depending on the mutant types. This work may provide one of the possible clues to explain the α-Syn mutant-type dependent pathological heterogeneity of familial PD.
Assuntos
Doença de Parkinson , alfa-Sinucleína , Humanos , alfa-Sinucleína/genética , Transporte Biológico , Dopamina , Mutação , Doença de Parkinson/genética , Proteína 2 Associada à Membrana da Vesícula/genéticaRESUMO
Myelin is required for rapid nerve signaling and is emerging as a key driver of CNS plasticity and disease. How myelin is built and remodeled remains a fundamental question of neurobiology. Central to myelination is the ability of oligodendrocytes to add vast amounts of new cell membrane, expanding their surface areas by many thousand-fold. However, how oligodendrocytes add new membrane to build or remodel myelin is not fully understood. Here, we show that CNS myelin membrane addition requires exocytosis mediated by the vesicular SNARE proteins VAMP2/3. Genetic inactivation of VAMP2/3 in myelinating oligodendrocytes caused severe hypomyelination and premature death without overt loss of oligodendrocytes. Through live imaging, we discovered that VAMP2/3-mediated exocytosis drives membrane expansion within myelin sheaths to initiate wrapping and power sheath elongation. In conjunction with membrane expansion, mass spectrometry of oligodendrocyte surface proteins revealed that VAMP2/3 incorporates axon-myelin adhesion proteins that are collectively required to form nodes of Ranvier. Together, our results demonstrate that VAMP2/3-mediated membrane expansion in oligodendrocytes is indispensable for myelin formation, uncovering a cellular pathway that could sculpt myelination patterns in response to activity-dependent signals or be therapeutically targeted to promote regeneration in disease.
Assuntos
Oligodendroglia , Proteína 2 Associada à Membrana da Vesícula , Axônios/fisiologia , Proteínas da Mielina/metabolismo , Bainha de Mielina/metabolismo , Oligodendroglia/metabolismo , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
Fusion of transmitter-containing vesicles with plasma membranes at the synaptic and neuromuscular junctions mediates neurotransmission and muscle contractions, respectively, thereby underlying all thoughts and actions. The fusion process is driven by the coupled folding and assembly of three synaptic SNARE proteins--syntaxin-1 and SNAP-25 on the target plasma membrane (t-SNAREs) and VAMP2 on the vesicular membrane (v-SNARE) into a four-helix bundle. Their assembly is chaperoned by Munc18-1 and many other proteins to achieve the speed and accuracy required for neurotransmission. However, the physiological pathway of SNARE assembly and its coupling to membrane fusion remains unclear. Here, we review recent progress in understanding SNARE assembly and membrane fusion, with a focus on results obtained by single-molecule manipulation approaches and electric recordings of single fusion pores. We describe two pathways of synaptic SNARE assembly, their associated intermediates, energetics, and kinetics. Assembly of the three SNAREs in vitro begins with the formation of a t-SNARE binary complex, on which VAMP2 folds in a stepwise zipper-like fashion. Munc18-1 significantly alters the SNARE assembly pathway: syntaxin-1 and VAMP2 first bind on the surface of Munc18-1 to form a template complex, with which SNAP-25 associates to conclude SNARE assembly and displace Munc18-1. During membrane fusion, multiple trans-SNARE complexes cooperate to open a dynamic fusion pore in a manner dependent upon their copy number and zippering states. Together, these results demonstrate that stepwise and cooperative SNARE assembly drive stagewise membrane fusion.
Assuntos
Fusão de Membrana , Proteínas SNARE , Cinética , Fusão de Membrana/fisiologia , Proteínas Munc18/química , Proteínas Munc18/genética , Proteínas Munc18/metabolismo , Proteínas Qa-SNARE , Proteínas SNARE/química , Proteínas SNARE/metabolismo , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
Invasion in various cancer cells requires coordinated delivery of signaling proteins, adhesion proteins, actin-remodeling proteins and proteases to matrix-degrading structures called invadopodia. Vesicular trafficking involving SNAREs plays a crucial role in the delivery of cargo to the target membrane. Screening of 13 SNAREs from the endocytic and recycling route using a gene silencing approach coupled with functional assays identified syntaxin 7 (STX7) as an important player in MDA-MB-231 cell invasion. Total internal reflection fluorescence microscopy (TIRF-M) studies revealed that STX7 resides near invadopodia and co-traffics with MT1-MMP (also known as MMP14), indicating a possible role for this SNARE in protease trafficking. STX7 depletion reduced the number of invadopodia and their associated degradative activity. Immunoprecipitation studies revealed that STX7 forms distinct SNARE complexes with VAMP2, VAMP3, VAMP7, STX4 and SNAP23. Depletion of VAMP2, VAMP3 or STX4 abrogated invadopodia formation, phenocopying what was seen upon lack of STX7. Whereas depletion of STX4 reduced MT1-MMP level at the cell surfaces, STX7 silencing significantly reduced the invadopodia-associated MT1-MMP pool and increased the non-invadosomal pool. This study highlights STX7 as a major contributor towards the invadopodia formation during cancer cell invasion. This article has an associated First Person interview with the first author of the paper.
Assuntos
Neoplasias da Mama , Podossomos , Proteínas Qa-SNARE , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Feminino , Humanos , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 14 da Matriz/metabolismo , Invasividade Neoplásica , Podossomos/metabolismo , Transporte Proteico , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas SNARE/metabolismo , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Proteína 3 Associada à Membrana da Vesícula/metabolismoRESUMO
BACKGROUND: Early-life Pb exposure can cause behavioral and cognitive problems and induce symptoms of hyperactivity, impulsivity, and inattention in children. Studies showed that blood lead levels were highly correlated with neuropsychiatric disorders, and effects of neurotoxicity might persist and affect the incidence of neurodegenerative diseases, for example Alzheimer's disease (AD). OBJECTIVE: To explore possible mechanisms of developmental Pb-induced neuropsychiatric dysfunctions. METHODS: Children were divided into low blood lead level (BLL) group (0-50.00µg/L) and high BLL group (>â50.00µg/L) and blood samples were collected. miRNA array was used to testify miRNA expression landscape between two groups. Correlation analysis and real-time PCR were applied to find miRNAs that altered in Pb and neuropsychiatric diseases. Animal models and cell experiments were used to confirm the effect of miRNAs in response to Pb, and siRNA and luciferase experiments were conducted to examine their effect on neural functions. RESULTS: miRNA array data and correlation analysis showed that miR-34b was the most relevant miRNA among Pb neurotoxicity and neuropsychiatric disorders, and synapse-associated membrane protein 2 (VAMP2) was the target gene regulating synapse function. In vivo and in vitro studies showed Pb exposure injured rats' cognitive abilities and induced upregulation of miR-34b and downregulation of VAMP2, resulting in decreases of hippocampal synaptic vesicles. Blockage of miR-34b mitigated Pb's effects on VAMP2 in vitro. CONCLUSION: Early-life Pb exposure might exert synapse-toxic effects via inhibiting VAMP2 mediated by upregulation of miR-34b and shed a light on the underlying relationship between Pb neurotoxicity and developmental neuropsychiatric disorders.
Assuntos
Chumbo , MicroRNAs , Animais , Humanos , Chumbo/metabolismo , Chumbo/toxicidade , MicroRNAs/metabolismo , Ratos , Sinapses/metabolismo , Regulação para Cima , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
BACKGROUND: Excessive proliferation and migration of airway smooth muscle cells (ASMCs) directly lead to airway remodeling in asthma. However, the role of circular RNAs (circRNAs) in airway remodeling remains unclear. This study aimed to investigate the regulatory role and mechanism of circ_CSNK1E in ASMCs proliferation and migration. METHODS: In this study, RNA-sequencing was used to analyze cicRNAs expression in asthma samples. ASMCs were treated with 25 ng/ml PDGF-BB to establish a model of asthma in vitro. Then, we used RT-qPCR to assess circRNAs, microRNAs (miRNAs) and messenger RNAs (mRNAs) expression. Besides, CCK-8, colony formation, wound healing and transwell chamber assays were carried out to explore cell proliferation and migration. Subcellular localization assay was used to detect the location of circRNA. Next, bioinformatics, luciferase reporter and RIP assays were performed to evaluate the relationship among circ_CSNK1E, miRNA-34a-5p and VAMP2. RESULTS: circ_CSNK1E expression was found to be significantly up-regulated in asthma samples and PDGF-BB-induced ASMCs. Functional experiments revealed that inhibition of circRNA_CSNK1E suppressed proliferation and migration of ASMCs stimulated by PDGF-BB. Next, we found that circRNA_CSNK1E served as a sponge for miR-34a-5p in ASMCs, and miR-34a-5p mimic suppressed proliferation and migration of ASMCs. Moreover, VAMP2 was confirmed as a direct target of miR-34a-5p. At last, inhibition of circRNA_CSNK1E suppressed proliferation and migration of ASMCs stimulated by PDGF-BB through miR-34a-5p/VAMP2 axis. CONCLUSION: Collectively, these findings clarified the importance of circ_CSNK1E/miRNA-34a-5p/VAMP2 axis for the proliferation and migration of ASMCs. These indicated that inhibition of circ_CSNK1E might be a potential target for the treatment of airway remodeling in asthma.
Assuntos
Asma , MicroRNAs , RNA Circular , Proteína 2 Associada à Membrana da Vesícula , Remodelação das Vias Aéreas , Asma/genética , Asma/metabolismo , Becaplermina/farmacologia , Movimento Celular/genética , Proliferação de Células/genética , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Miócitos de Músculo Liso/metabolismo , RNA Circular/genética , RNA Circular/metabolismo , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
To investigate the effect of mifepristone on PD-L1 through miR-127-3p/VAMP2 axis to inhibit the malignant biological behavior of ovarian cancer cells. Western blotting was used to detect the protein expression of VAMP2, PD-L1, CyclinD1, Cl-caspase-3 and Bax; qRT-PCR was used to detect the expression of miR-127-3p; double luciferase reporter gene was used to verify the targeted binding of miR-127-3p to VAMP2. The results showed that mifepristone up-regulated the expression of miR-127-3p and mifepristone could significantly inhibit the proliferation of ovarian cancer SKOV3 cells and A2780 cells, promote apoptosis, inhibit the expression of PD-L1, down regulate the expression of CyclinD1 and up regulate the expression of cl-caspase-3 and Bax; silencing miR-127-3p could restore the effects of mifepristone on the proliferation and apoptosis of SKOV3 cells and A2780 cells, as well as the expression of PD-L1, CyclinD1, Cl-caspase-3 and Bax protein; our study confirmed that mifepristone can regulate the expression of VAMP2 and PD-L1 through miR-127-3p and VAMP2 can positively regulate the expression of PD-L1; finally, we found that mifepristone can down regulate PD-L1 through miR-127-3p/VAMP2 axis, inhibit proliferation and promote apoptosis of ovarian cancer cells. Mifepristone can down regulate PD-L1 through miR-127-3p/VAMP2 axis and inhibit the progression of ovarian cancer cells.
Assuntos
Antígeno B7-H1/metabolismo , MicroRNAs/metabolismo , Mifepristona/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Apoptose/efeitos dos fármacos , Antígeno B7-H1/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Antagonistas de Hormônios/uso terapêutico , Humanos , MicroRNAs/genética , Neoplasias Ovarianas/metabolismo , Proteína 2 Associada à Membrana da Vesícula/genéticaRESUMO
Synaptobrevin-2 is one of the key players of neuronal exocytosis. Together with Syntaxin-1A and SNAP25, it forms the core membrane fusion machinery that is responsible for neurotransmitter release and, therefore, signal transmission between neurons. However, in the absence of interaction partners, Synaptobrevin-2 is largely unstructured and exhibits an inherent flexibility. In this graphical review, we provide an overview on the structural states of Synaptobrevin-2 in the absence and in the presence of interaction partners. For this, we first depict its natural habitat, namely the presynaptic nerve terminal, and gather biophysical properties that are likely responsible for its structural diversity. We then provide an overview on key findings describing the disorder-to-order transition of Synaptobrevin-2 from a mostly unstructured protein to a highly structured protein complex component.
Assuntos
Exocitose , Proteína 2 Associada à Membrana da Vesícula , Exocitose/fisiologia , Neurônios/metabolismo , Proteína 2 Associada à Membrana da Vesícula/química , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
Angelman syndrome (AS) is a neurogenetic disorder characterized by severe developmental delay with absence of speech, happy disposition, frequent laughter, hyperactivity, stereotypies, ataxia and seizures with specific EEG abnormalities. There is a 10-15% of patients with an AS phenotype whose genetic cause remains unknown (Angelman-like syndrome, AS-like). Whole-exome sequencing (WES) was performed on a cohort of 14 patients with clinical features of AS and no molecular diagnosis. As a result, we identified 10 de novo and 1 X-linked pathogenic/likely pathogenic variants in 10 neurodevelopmental genes (SYNGAP1, VAMP2, TBL1XR1, ASXL3, SATB2, SMARCE1, SPTAN1, KCNQ3, SLC6A1 and LAS1L) and one deleterious de novo variant in a candidate gene (HSF2). Our results highlight the wide genetic heterogeneity in AS-like patients and expands the differential diagnosis.
Assuntos
Síndrome de Angelman/genética , Sequenciamento do Exoma/métodos , Redes Reguladoras de Genes , Adolescente , Adulto , Criança , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Proteínas de Choque Térmico , Humanos , Lactente , Masculino , Proteínas de Ligação à Região de Interação com a Matriz/genética , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Proteína 2 Associada à Membrana da Vesícula/genética , Adulto JovemRESUMO
Synaptobrevin-2 (Syb2) is a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) that is essential for neurotransmitter release. It is the most numerous protein on a synaptic vesicle (SV) and drives SV fusion via interactions with its cognate SNARE partners on the presynaptic plasma membrane. Synaptophysin (Syp) is the second most abundant protein on SVs; however, in contrast to Syb2, it has no obligatory role in neurotransmission. Syp interacts with Syb2 on SVs, and the molecular nature of its interaction with Syb2 and its physiological role has been debated for decades. However, recent studies have revealed that the sole physiological role of Syp at the presynapse is to ensure the efficient retrieval of Syb2 during SV endocytosis. In this review, current theories surrounding the role of Syp in Syb2 trafficking will be discussed, in addition to the debate regarding the molecular nature of their interaction. A unifying model is presented that describes how Syp controls Syb2 function as part of an integrated mechanism involving key molecular players such as intersectin-1 and AP180/CALM. Finally, key future questions surrounding the role of Syp-dependent Syb2 trafficking will be posed, with respect to brain function in health and disease.
Assuntos
Terminações Pré-Sinápticas/metabolismo , Transporte Proteico/fisiologia , Proteínas SNARE/metabolismo , Sinaptofisina/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Animais , Endocitose/fisiologia , Humanos , Proteínas SNARE/genética , Sinapses/genética , Sinapses/metabolismo , Sinaptofisina/genética , Proteína 2 Associada à Membrana da Vesícula/genéticaRESUMO
We aimed to explore the function and underlying mechanism of highly upregulated in liver cancer (HULC; an long noncoding RNAs) in hepatocellular carcinoma (HCC) and chemosensitivity of oxaliplatin (Oxa). The expression of HULC, miR-383-5p, and vesicle-associated membrane protein-2 (VAMP2) was detected by quantitative real-time polymerase chain reaction. Western blot assay was applied for measuring the protein expression of cyclinD1, cleaved-caspase-3, light Chain 3 I/II, p62, and VAMP2. Cell viability and Oxa IC50 value were determined by Cell Counting Kit-8 assay. A colony formation assay was conducted to evaluate colony formation ability. Cell apoptosis was assessed by flow cytometry. The interaction between miR-383-5p and HULC or VAMP2 was predicted by bioinformatics analysis and verified by dual-luciferase reporter assay and RNA immunoprecipitation assay. The mice xenograft model was established to investigate the roles of HULC in vivo. HULC and VAMP2 were overexpressed whereas miR-383-5p was lowly expressed in HCC tissues. HULC overexpression promoted the progression of HCC cells and inhibited chemosensitivity of Oxa by increasing cell proliferation and protective autophagy and inhibiting apoptosis, whereas HULC silence presented opposite effects. Moreover, miR-383-5p was a direct target of HULC and miR-383-5p reversed the effects of HULC on the progression of HCC cells and chemosensitivity of Oxa. Besides, HULC acted as a molecular sponge of miR-383-5p to regulate VAMP2 expression. HULC promoted the progression of HCC and inhibited Oxa sensitivity by regulating miR-383-5p/VAMP2 axis, elucidating a novel regulatory mechanism for chemosensitivity of Oxa and providing a potential lncRNA-targeted therapy for HCC.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroRNAs/genética , Oxaliplatina/uso terapêutico , RNA Longo não Codificante/genética , Proteína 2 Associada à Membrana da Vesícula/genética , Animais , Antineoplásicos/farmacologia , Apoptose , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Oxaliplatina/farmacologia , Regulação para Cima , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
All membrane fusion reactions proceed through an initial fusion pore, including calcium-triggered release of neurotransmitters and hormones. Expansion of this small pore to release cargo is energetically costly and regulated by cells, but the mechanisms are poorly understood. Here, we show that the neuronal/exocytic calcium sensor Synaptotagmin-1 (Syt1) promotes expansion of fusion pores induced by SNARE proteins. Pore dilation relied on calcium-induced insertion of the tandem C2 domain hydrophobic loops of Syt1 into the membrane, previously shown to reorient the C2 domain. Mathematical modelling suggests that C2B reorientation rotates a bound SNARE complex so that it exerts force on the membranes in a mechanical lever action that increases the height of the fusion pore, provoking pore dilation to offset the bending energy penalty. We conclude that Syt1 exerts novel non-local calcium-dependent mechanical forces on fusion pores that dilate pores and assist neurotransmitter and hormone release.
Assuntos
Proteínas SNARE/metabolismo , Sinaptotagmina I/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Cálcio/metabolismo , Fusão Celular , Membrana Celular , Regulação da Expressão Gênica/fisiologia , Células HeLa , Humanos , Lipoproteínas , Modelos Biológicos , Modelos Moleculares , Nanoestruturas , Conformação Proteica , Proteínas SNARE/genética , Sinaptotagmina I/genética , Proteína 2 Associada à Membrana da Vesícula/genéticaRESUMO
Evidence from multiple systems indicates that vesicle SNARE (soluble NSF attachment receptor) proteins are involved in synaptic vesicle endocytosis, although their exact action at the level of single vesicles is unknown. Here we interrogate the role of the main synaptic vesicle SNARE mediating fusion, synaptobrevin-2 (also called VAMP2), in modulation of single synaptic vesicle retrieval. We report that in the absence of synaptobrevin-2, fast and slow modes of single synaptic vesicle retrieval are impaired, indicating a role of the SNARE machinery in coupling exocytosis to endocytosis of single synaptic vesicles. Ultrafast endocytosis was impervious to changes in the levels of synaptobrevin-2, pointing to a separate molecular mechanism underlying this type of recycling. Taken together with earlier studies suggesting a role of synaptobrevin-2 in endocytosis, these results indicate that the machinery for fast synchronous release couples fusion to retrieval and regulates the kinetics of endocytosis in a Ca2+-dependent manner.
Assuntos
Cálcio/metabolismo , Endocitose/genética , Proteínas SNARE/genética , Vesículas Sinápticas/metabolismo , Proteína 2 Associada à Membrana da Vesícula/genética , Animais , Exocitose/genética , Hipocampo/citologia , Hipocampo/metabolismo , Camundongos Knockout , Microscopia de Fluorescência/métodos , Neurônios/citologia , Neurônios/metabolismo , Proteínas SNARE/metabolismo , Sinapses/genética , Sinapses/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
The accurate retrieval of synaptic vesicle (SV) proteins during endocytosis is essential for the maintenance of neurotransmission. Synaptophysin (Syp) and synaptobrevin-II (SybII) are the most abundant proteins on SVs. Neurons lacking Syp display defects in the activity-dependent retrieval of SybII and a general slowing of SV endocytosis. To determine the role of the cytoplasmic C terminus of Syp in the control of these two events, we performed molecular replacement studies in primary cultures of Syp knockout neurons using genetically encoded reporters of SV cargo trafficking at physiological temperatures. Under these conditions, we discovered, 1) no slowing in SV endocytosis in Syp knockout neurons, and 2) a continued defect in SybII retrieval in knockout neurons expressing a form of Syp lacking its C terminus. Sequential truncations of the Syp C-terminus revealed a cryptic interaction site for the SNARE motif of SybII that was concealed in the full-length form. This suggests that a conformational change within the Syp C terminus is key to permitting SybII binding and thus its accurate retrieval. Furthermore, this study reveals that the sole presynaptic role of Syp is the control of SybII retrieval, since no defect in SV endocytosis kinetics was observed at physiological temperatures.