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1.
Proc Natl Acad Sci U S A ; 117(35): 21391-21402, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817423

RESUMO

Syntaxin17, a key autophagosomal N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, can associate with ATG8 family proteins SNAP29 and VAMP8 to facilitate the membrane fusion process between the double-membraned autophagosome and single-membraned lysosome in mammalian macroautophagy. However, the inherent properties of Syntaxin17 and the mechanistic basis underlying the interactions of Syntaxin17 with its binding proteins remain largely unknown. Here, using biochemical, NMR, and structural approaches, we systemically characterized Syntaxin17 as well as its interactions with ATG8 family proteins, SNAP29 and VAMP8. We discovered that Syntaxin17 alone adopts an autoinhibited conformation mediated by a direct interaction between its Habc domain and the Qa-SNARE motif. In addition, we revealed that the Qa-SNARE region of Syntaxin17 contains one LC3-interacting region (LIR) motif, which preferentially binds to GABARAP subfamily members. Importantly, the GABARAP binding of Syntaxin17 can release its autoinhibited state. The determined crystal structure of the Syntaxin17 LIR-GABARAP complex not only provides mechanistic insights into the interaction between Syntaxin17 and GABARAP but also reveals an unconventional LIR motif with a C-terminally extended 310 helix for selectively binding to ATG8 family proteins. Finally, we also elucidated structural arrangements of the autophagic Syntaxin17-SNAP29-VAMP8 SNARE core complex, and uncovered its conserved biochemical and structural characteristics common to all other SNAREs. In all, our findings reveal three distinct states of Syntaxin17, and provide mechanistic insights into the Syntaxin17-mediated autophagosome-lysosome fusion process.


Assuntos
Autofagossomos/fisiologia , Lisossomos/fisiologia , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Motivos de Aminoácidos , Proteínas Reguladoras de Apoptose/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Escherichia coli , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo
2.
Mol Cell Endocrinol ; 502: 110674, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31811899

RESUMO

Autophagy plays an essential role in gestational diabetes mellitus (GDM). Death-associated protein kinase-3 (DAPK3) regulates a variety of cellular functions; however, the relationship between DAPK3 and autophagy is unknown. In this study, we aim to investigate whether DAPK3 is associated with autophagy in GDM, and we found that DAPK3 was upregulated in the placenta of GDM patients and extravillous trophoblast cells under high-glucose conditions. Silencing DAPK3 decreased the assembly of the STX17-SNAP29-VAMP8 complex, leading to the blockade of autophagosome-lysosome fusion by mediating synaptosomal-associated protein 29 (SNAP29). Moreover, knockdown of DAPK3 ameliorates cell invasion and mediates autophagy in high glucose, and does not alter the expression of autophagy-related genes in normal glucose. Our study demonstrates the significance of DAPK3 in autophagy and GDM, which may provide new insights into the molecular mechanisms regulating trophoblast invasion.


Assuntos
Autofagossomos/metabolismo , Proteínas Quinases Associadas com Morte Celular/genética , Diabetes Gestacional/genética , Lisossomos/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Linhagem Celular , Movimento Celular , Proteínas Quinases Associadas com Morte Celular/metabolismo , Diabetes Gestacional/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Glucose/efeitos adversos , Humanos , Gravidez , Trofoblastos/citologia , Trofoblastos/efeitos dos fármacos , Trofoblastos/metabolismo , Regulação para Cima
3.
Neuroscience ; 423: 1-11, 2019 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-31682953

RESUMO

Progressive myoclonic epilepsies (PMEs) comprise a group of rare disorders of different genetic aetiologies, leading to childhood-onset myoclonus, myoclonic seizures and subsequent neurological decline. One of the genetic causes for PME, a mutation in the gene coding for Golgi SNAP receptor 2 (GOSR2), gives rise to a PME-subtype prevalent in Northern Europe and hence referred to as North Sea Progressive Myoclonic Epilepsy (NS-PME). Treatment for NS-PME, as for all PME subtypes, is symptomatic; the pathophysiology of NS-PME is currently unknown, precluding targeted therapy. Here, we investigated the pathophysiology of NS-PME. By means of chart review in combination with interviews with patients (n = 14), we found heat to be an exacerbating factor for a majority of NS-PME patients (86%). To substantiate these findings, we designed a NS-PME Drosophila melanogaster model. Downregulation of the Drosophila GOSR2-orthologue Membrin leads to heat-induced seizure-like behaviour. Specific downregulation of GOSR2/Membrin in glia but not in neuronal cells resulted in a similar phenotype, which was progressive as the flies aged and was partially responsive to treatment with sodium barbital. Our data suggest a role for GOSR2 in glia in the pathophysiology of NS-PME.


Assuntos
Temperatura Alta , Epilepsias Mioclônicas Progressivas/genética , Epilepsias Mioclônicas Progressivas/fisiopatologia , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Drosophila , Europa (Continente) , Feminino , Humanos , Entrevistas como Assunto , Masculino , Modelos Animais , Mutação , Epilepsias Mioclônicas Progressivas/induzido quimicamente , Neuroglia , Proteínas Qb-SNARE/genética , Proteínas Qb-SNARE/metabolismo , Estudos Retrospectivos
4.
Cell Chem Biol ; 26(9): 1283-1294.e5, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31327703

RESUMO

The proteolytic arm of the protein homeostasis network is maintained by both the ubiquitin-proteasome system (UPS) and autophagy. A well-balanced crosstalk between the two catabolic pathways ensures energy-efficient maintenance of cellular function. Our current understanding of the crosstalk between the UPS and autophagy is centered around substrate ubiquitination. Herein we report an additional method of crosstalk involving ubiquitin-independent 20S proteasome regulation of autophagosome-lysosome fusion. We found that enhancement of 20S proteasome activity increased the degradation of the disordered soluble N-ethylmaleimide-sensitive factor activating protein receptor proteins, synaptosomal-associated protein 29 (SNAP29), and syntaxin 17 (STX17), but not vesicle-associated membrane protein 8. This resulted in a reduction of autophagosome-lysosome fusion, which was ameliorated upon overexpression of both SNAP29 and STX17. In all, we herein present a mechanism of crosstalk between the proteasome and autophagy pathway that is regulated by ubiquitin-independent 20S proteasome-mediated degradation of SNAP29 and STX17.


Assuntos
Autofagia/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Autofagia/genética , Linhagem Celular , Células HEK293 , Células HeLa , Humanos , Lisossomos/metabolismo , Fusão de Membrana , Complexo de Endopeptidases do Proteassoma/fisiologia , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Ubiquitina/metabolismo , Ubiquitinação/fisiologia
5.
Traffic ; 20(9): 661-673, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31297933

RESUMO

Macrophage migration into injured or infected tissue is a key aspect in the pathophysiology of many diseases where inflammation is a driving factor. Membrane-type-1 matrix metalloproteinase (MT1-MMP) cleaves extracellular matrix components to facilitate invasion. Here we show that, unlike the constitutive MT1-MMP surface recycling seen in cancer cells, unactivated macrophages express low levels of MT1-MMP. Upon lipopolysaccharide (LPS) activation, MT1-MMP synthesis dramatically increases 10-fold at the surface by 15 hours. MT1-MMP is trafficked from the Golgi complex to the surface via late endosomes/lysosomes in a pathway regulated by the late endosome/lysosome R-SNAREs VAMP7 and VAMP8. These form two separate complexes with the surface Q-SNARE complex Stx4/SNAP23 to regulate MT1-MMP delivery to the plasma membrane. Loss of either one of these SNAREs leads to a reduction in surface MT1-MMP, gelatinase activity and reduced invasion. Thus, inhibiting MT1-MMP transport through this pathway could reduce macrophage migration and the resulting inflammation.


Assuntos
Membrana Celular/metabolismo , Endossomos/metabolismo , Lisossomos/metabolismo , Ativação de Macrófagos , Metaloproteinase 14 da Matriz/metabolismo , Animais , Movimento Celular , Complexo de Golgi/metabolismo , Camundongos , Transporte Proteico , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Células RAW 264.7
6.
Biol Reprod ; 101(2): 338-346, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31201423

RESUMO

Mammalian oocytes are stored in the ovary for prolonged periods, and arrested in meiotic prophase. During this period, their plasma membranes are constantly being recycled by endocytosis and exocytosis. However, the function of this membrane turnover is unknown. Here, we investigated the requirement for exocytosis in the maintenance of meiotic arrest. Using Trim-away, a newly developed method for rapidly and specifically depleting proteins in oocytes, we have identified the SNARE protein, SNAP23, to be required for meiotic arrest. Degradation of SNAP23 causes premature meiotic resumption in follicle-enclosed oocytes. The reduction in SNAP23 is associated with loss of gap junction communication between the oocyte and surrounding follicle cells. Reduction of SNAP23 protein also inhibits regulated exocytosis in response to a Ca2+ stimulus (cortical granule exocytosis), as measured by lectin staining and cleavage of ZP2. Our results show an essential role for SNAP23 in two key processes that occur in mouse oocytes and eggs.


Assuntos
Exocitose/fisiologia , Oócitos/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Animais , Cálcio/metabolismo , Técnicas de Cultura de Células , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Meiose/fisiologia , Camundongos , Oócitos/efeitos dos fármacos , Folículo Ovariano , Conservantes Farmacêuticos , Proteínas Qb-SNARE/genética , Proteínas Qc-SNARE/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Timerosal/farmacologia
7.
J Biochem Mol Toxicol ; 33(8): e22348, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31066958

RESUMO

Golgi S-nitro-N-acetylpenicillamine receptor complex 1 (GS28) has been implicated in Golgi vesicle transport. We examined the role of GS28 and its molecular mechanisms in sodium nitroprusside (SNP)-induced cell death using GS28 siRNA (siGS28)-transfected HeLa cells. Significant inhibition of cytotoxicity was observed in the cells treated with SNP, and photodegraded SNP showed equal cytotoxicity to SNP. Pretreatment with an ERK inhibitor or siErk1 cotransfection blocked the inhibition in cytotoxicity. Additionally, increased phosphorylation of ERK was maintained in the cells treated with SNP, and Nrf2 level was dependent on ERK phosphorylation. However, pretreatment with a pan-caspase inhibitor had no effect on cytotoxicity or procaspase-3 level. Pretreatment with an autophagy inhibitor or siATG5 cotransfection blocked the inhibition of cytotoxicity. The changes of LC3 corresponded to that in siErk1-cotransfected cells. These data suggest that GS28 has an inductive role in SNP-induced cell death via inhibition of ERK, leading to inhibition of autophagic processes in HeLa cells.


Assuntos
Morte Celular/efeitos dos fármacos , Nitroprussiato/farmacologia , Proteínas Qb-SNARE/metabolismo , Neoplasias do Colo do Útero/patologia , Autofagia/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Células HeLa , Humanos , Fator 2 Relacionado a NF-E2/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Neoplasias do Colo do Útero/metabolismo
8.
Mol Cancer ; 18(1): 78, 2019 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-30943982

RESUMO

BACKGROUND: Emerging evidence indicates that tumor cells release a large amount of exosomes loaded with cargos during tumorigenesis. Exosome secretion is a multi-step process regulated by certain related molecules. Long non-coding RNAs (lncRNAs) play an important role in hepatocellular carcinoma (HCC) progression. However, the role of lncRNA HOTAIR in regulating exosome secretion in HCC cells remains unclear. METHODS: We analyzed the relationship between HOTAIR expression and exosome secretion-related genes using gene set enrichment analysis (GSEA). Nanoparticle tracking analysis was performed to validate the effect of HOTAIR on exosome secretion. The transport of multivesicular bodies (MVBs) after overexpression of HOTAIR was detected by transmission electron microscopy and confocal microscopy analysis of cluster determinant 63 (CD63) with synaptosome associated protein 23 (SNAP23). The mechanism of HOTAIR's regulation of Ras-related protein Rab-35 (RAB35), vesicle associated membrane protein 3 (VAMP3), and SNAP23 was assessed using confocal co-localization analysis, phosphorylation assays, and rescue experiments. RESULTS: We found an enrichment of exosome secretion-related genes in the HOTAIR high expression group. HOTAIR promoted the release of exosomes by inducing MVB transport to the plasma membrane. HOTAIR regulated RAB35 expression and localization, which controlled the docking process. Moreover, HOTAIR facilitated the final step of fusion by influencing VAMP3 and SNAP23 colocalization. In addition, we validated that HOTAIR induced the phosphorylation of SNAP23 via mammalian target of rapamycin (mTOR) signaling. CONCLUSION: Our study demonstrated a novel function of lncRNA HOTAIR in promoting exosome secretion from HCC cells and provided a new understanding of lncRNAs in tumor cell biology.


Assuntos
Carcinoma Hepatocelular/genética , Exossomos/metabolismo , Neoplasias Hepáticas/genética , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , RNA Longo não Codificante/genética , Proteínas rab de Ligação ao GTP/metabolismo , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Progressão da Doença , Exossomos/genética , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Fosforilação , Transporte Proteico , Proteínas Qb-SNARE/genética , Proteínas Qc-SNARE/genética , Transdução de Sinais , Proteínas rab de Ligação ao GTP/genética
9.
Neuroscience ; 420: 41-49, 2019 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-30954670

RESUMO

Successive fusion events between transport vesicles and their target membranes mediate trafficking of secreted, membrane- and organelle-localised proteins. During the initial steps of this process, termed the secretory pathway, COPII vesicles bud from the endoplasmic reticulum (ER) and fuse with the cis-Golgi membrane, thus depositing their cargo. This fusion step is driven by a quartet of SNARE proteins that includes the cis-Golgi t-SNARE Membrin, encoded by the GOSR2 gene. Mis-sense mutations in GOSR2 result in Progressive Myoclonus Epilepsy (PME), a severe neurological disorder characterised by ataxia, myoclonus and seizures in the absence of significant cognitive impairment. However, given the ubiquitous and essential function of ER-to-Golgi transport, why GOSR2 mutations cause neurological dysfunction and not lethality or a broader range of developmental defects has remained an enigma. Here we highlight new work that has shed light on this issue and incorporate insights into canonical and non-canonical secretory trafficking pathways in neurons to speculate as to the cellular and molecular mechanisms underlying GOSR2 PME. This article is part of a Special Issue entitled: SNARE proteins: a long journey of science in brain physiology and pathology: from molecular.


Assuntos
Epilepsias Mioclônicas Progressivas/genética , Epilepsias Mioclônicas Progressivas/fisiopatologia , Transporte Proteico/genética , Proteínas Qb-SNARE/genética , Proteínas Qb-SNARE/metabolismo , Animais , Complexo de Golgi/metabolismo , Complexo de Golgi/patologia , Humanos , Mutação , Epilepsias Mioclônicas Progressivas/metabolismo , Neurônios/metabolismo , Neurônios/patologia
10.
Genet Test Mol Biomarkers ; 23(5): 299-303, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30945947

RESUMO

Aims: Autophagic eradication of pathogenic microbes, including Mycobacterium tuberculosis (Mtb), is an effective host immune process that protects hosts from developing diseases associated with intracellular pathogens. This study was designed to investigate the association between the single nucleotide polymorphisms (SNPs) of the autophagy-related genes VAMP8 and VTI1B, and the susceptibility to pulmonary tuberculosis (PTB) in a Chinese Han population. Materials and Methods: Two SNPs, rs1010 from the VAMP8 gene and rs15493 from the VTI1B gene, were examined in 202 PTB patients and 216 healthy controls using high-resolution melt-polymerase chain reaction. Results: The rs1010 SNP genotypes AG (p = 0.028) and GG (p = 0.016) were associated with increased susceptibility to PTB. However, the VTI1B rs15493 SNP had no impact on the susceptibility to PTB (p > 0.05). Conclusions: Our study demonstrated that the rs1010 SNP of VAMP8 gene was significantly associated with the susceptibility to PTB. This result suggests that rs1010 genotyping could be used as prognostic biomarker to predict the risk of Mtb infection and/or PTB disease development after Mtb infection in the Chinese Han population.


Assuntos
Proteínas Qb-SNARE/genética , Proteínas R-SNARE/genética , Tuberculose Pulmonar/genética , Adulto , Grupo com Ancestrais do Continente Asiático , Biomarcadores/sangue , Estudos de Casos e Controles , China , Grupos Étnicos , Feminino , Frequência do Gene , Predisposição Genética para Doença , Testes Genéticos/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Proteínas Qb-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Tuberculose Pulmonar/metabolismo
11.
Mol Biol Cell ; 30(9): 1085-1097, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30811271

RESUMO

Syntaxin 11 (stx11) is a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) that is selectively expressed in immune cells; however, its precise role in macrophages is unclear. We showed that stx11 knockdown reduces the phagocytosis of Escherichia coli in interferon-γ-activated macrophages. stx11 knockdown decreased Toll-like receptor 4 (TLR4) localization on the plasma membrane without affecting total expression. Plasma membrane-localized TLR4 was primarily endocytosed within 1 h by lipopolysaccharide (LPS) stimulation and gradually relocalized 4 h after removal of LPS. This relocalization was significantly impaired by stx11 knockdown. The lack of TLR4 transport to the plasma membrane is presumably related to TLR4 degradation in acidic endosomal organelles. Additionally, an immunoprecipitation experiment suggested that stx11 interacts with SNAP-23, a plasma membrane-localized SNARE protein, whose depletion also inhibits TLR4 replenishment in LPS-stimulated cells. Using an intramolecular Förster resonance energy transfer (FRET) probe for SNAP-23, we showed that the high FRET efficiency caused by LPS stimulation is reduced by stx11 knockdown. These findings suggest that stx11 regulates the stimulus-dependent transport of TLR4 to the plasma membrane by cooperating with SNAP-23 in macrophages. Our results clarify the regulatory mechanisms underlying intracellular transport of TLR4 and have implications for microbial pathogenesis and immune responses.


Assuntos
Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Endocitose/fisiologia , Endossomos/metabolismo , Técnicas de Silenciamento de Genes , Macrófagos/metabolismo , Fusão de Membrana , Camundongos , Camundongos Endogâmicos BALB C , Fagocitose/fisiologia , Ligação Proteica , Transporte Proteico , Proteínas Qa-SNARE/genética , Receptor 4 Toll-Like/genética
12.
Sci Rep ; 9(1): 1211, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30718891

RESUMO

Homozygous mutations in SNAP29, encoding a SNARE protein mainly involved in membrane fusion, cause CEDNIK (Cerebral Dysgenesis, Neuropathy, Ichthyosis and Keratoderma), a rare congenital neurocutaneous syndrome associated with short life expectancy, whose pathogenesis is unclear. Here, we report the analysis of the first genetic model of CEDNIK in zebrafish. Strikingly, homozygous snap29 mutant larvae display CEDNIK-like features, such as microcephaly and skin defects. Consistent with Snap29 role in membrane fusion during autophagy, we observe accumulation of the autophagy markers p62 and LC3, and formation of aberrant multilamellar organelles and mitochondria. Importantly, we find high levels of apoptotic cell death during early development that might play a yet uncharacterized role in CEDNIK pathogenesis. Mutant larvae also display mouth opening problems, feeding impairment and swimming difficulties. These alterations correlate with defective trigeminal nerve formation and excess axonal branching. Since the paralog Snap25 is known to promote axonal branching, Snap29 might act in opposition with, or modulate Snap25 activity during neurodevelopment. Our vertebrate genetic model of CEDNIK extends the description in vivo of the multisystem defects due to loss of Snap29 and could provide the base to test compounds that might ameliorate traits of the disease.


Assuntos
Ceratodermia Palmar e Plantar/metabolismo , Síndromes Neurocutâneas/metabolismo , Proteínas SNARE/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Autofagia , Humanos , Ceratodermia Palmar e Plantar/genética , Ceratodermia Palmar e Plantar/fisiopatologia , Fusão de Membrana , Modelos Genéticos , Mutação , Malformações do Sistema Nervoso/metabolismo , Síndromes Neurocutâneas/genética , Síndromes Neurocutâneas/fisiopatologia , Fenótipo , Ligação Proteica , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteínas SNARE/fisiologia , Proteína 25 Associada a Sinaptossoma/metabolismo , Proteína 25 Associada a Sinaptossoma/fisiologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/fisiologia
13.
Pain ; 160(2): 508-527, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30335684

RESUMO

Sensitization of the transient receptor potential ion channel vanilloid 1 (TRPV1) is critically involved in inflammatory pain. To date, manifold signaling cascades have been shown to converge onto TRPV1 and enhance its sensitization. However, many of them also play a role for nociceptive pain, which limits their utility as targets for therapeutic intervention. Here, we show that the vesicle transport through interaction with t-SNAREs homolog 1B (Vti1b) protein promotes TRPV1 sensitization upon inflammation in cell culture but leaves normal functioning of TRPV1 intact. Importantly, the effect of Vti1b can be recapitulated in vivo: Virus-mediated knockdown of Vti1b in sensory neurons attenuated thermal hypersensitivity during inflammatory pain without affecting mechanical hypersensitivity or capsaicin-induced nociceptive pain. Interestingly, TRPV1 and Vti1b are localized in close vicinity as indicated by proximity ligation assays and are likely to bind to each other, either directly or indirectly, as suggested by coimmunoprecipitations. Moreover, using a mass spectrometry-based quantitative interactomics approach, we show that Vti1b is less abundant in TRPV1 protein complexes during inflammatory conditions compared with controls. Alongside, we identify numerous novel and pain state-dependent binding partners of native TRPV1 in dorsal root ganglia. These data represent a unique resource on the dynamics of the TRPV1 interactome and facilitate mechanistic insights into TRPV1 regulation. We propose that inflammation-related differences in the TRPV1 interactome identified here could be exploited to specifically target inflammatory pain in the future.


Assuntos
Regulação da Expressão Gênica/genética , Hiperalgesia/genética , Dor/metabolismo , Proteínas Qb-SNARE/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Cálcio/metabolismo , Capsaicina/farmacologia , Células Cultivadas , Modelos Animais de Doenças , Adjuvante de Freund/toxicidade , Gânglios Espinais/citologia , Humanos , Hiperalgesia/fisiopatologia , Inflamação/induzido quimicamente , Inflamação/complicações , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dor/etiologia , Proteínas Qb-SNARE/genética , Interferência de RNA/fisiologia , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/fisiologia , Transdução de Sinais , Canais de Cátion TRPV/genética
14.
Neuroscience ; 420: 32-40, 2019 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-30471354

RESUMO

Vti proteins are conserved from yeast to humans and regulate intracellular membrane trafficking by providing one specific SNARE domain, the Qb SNARE, to the four helical SNARE bundle that drives membrane fusion. Two mammalian Vti genes, Vti1a and Vti1b are reported to regulate distinct aspects of endolysosomal trafficking and retrograde transport to the Golgi, but have also been implicated in synaptic vesicle secretion. In this review, we summarize the current evidence for the role of Vti proteins in intracellular trafficking in different cells. We propose that, despite some unique aspects, the two mammalian VTI genes have largely redundant functions in neurosecretory cells and recycle molecules required for the sorting of regulated cargo to the Golgi. Defects in this recycling also lead to defects in synaptic transmission and dense core vesicle secretion.


Assuntos
Exocitose/fisiologia , Neurônios/metabolismo , Transporte Proteico/fisiologia , Proteínas Qb-SNARE/metabolismo , Transmissão Sináptica/fisiologia , Animais , Humanos , Lisossomos/metabolismo
15.
BMC Med Genomics ; 11(1): 111, 2018 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-30547786

RESUMO

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a major health burden in need for new medication. To identify potential drug targets a genomic study was performed in lipid-laden primary human hepatocyte (PHH) and human hepatoma cell cultures. METHODS: PHH, HuH7 and HepG2 hepatoma cell cultures were treated with lipids and/or TNFα. Intracellular lipid load was quantified with the ORO assay. The Affymetrix HG-U133+ array system was employed to perform transcriptome analysis. The lipid droplet (LD) growth and fusion was determined by fluorescence microscopy. LD associated proteins were imaged by confocal immunofluorescence microscopy and confirmed by Western immunoblotting. Bioinformatics defined perturbed metabolic pathways. RESULTS: Whole genome expression profiling identified 227, 1031 and 571 significant regulated genes. Likewise, the combined lipid and TNFα treatment of PHH, HuH7 and HepG2 cell cultures revealed 154, 1238 and 278 differentially expressed genes. Although genomic responses differed among in-vitro systems, commonalities were ascertained by filtering the data for LD associated gene regulations. Among others the LD-growth and fusion associated cell death inducing DFFA like effector C (CIDEC), perilipins (PLIN2, PLIN3), the synaptosome-associated-protein 23 and the vesicle associated membrane protein 3 were strongly up-regulated. Likewise, the PPAR targets pyruvate-dehydrogenase-kinase-4 and angiopoietin-like-4 were up-regulated as was hypoxia-inducible lipid droplet-associated (HILPDA), flotilin and FGF21. Their inhibition ameliorates triglyceride and cholesterol accumulation. TNFα treatment elicited strong induction of the chemokine CXCL8, the kinases MAP3K8, MAP4K4 and negative regulators of cytokine signaling, i.e. SOCS2&SOCS3. Live cell imaging of DsRED calreticulin plasmid transfected HuH7 cells permitted an assessment of LD growth and fusion and confocal immunofluorescence microscopy evidenced induced LD-associated PLIN2, CIDEC, HIF1α, HILPDA, JAK1, PDK4 and ROCK2 expression. Notwithstanding, CPT1A protein was repressed to protect mitochondria from lipid overload. Pharmacological inhibition of the GTPase-dynamin and the fatty acid transporter-2 reduced lipid uptake by 28.5 and 35%, respectively. Finally, a comparisons of in-vitro/NAFLD patient biopsy findings confirmed common gene regulations thus demonstrating clinical relevance. CONCLUSION: The genomics of fat-laden hepatocytes revealed LD-associated gene regulations and perturbed metabolic pathways. Immunofluorescence microscopy confirmed expression of coded proteins to provide a rationale for therapeutic intervention strategies. Collectively, the in-vitro system permits testing of drug candidates.


Assuntos
Genômica/métodos , Gotículas Lipídicas/química , Hepatopatia Gordurosa não Alcoólica/genética , Carnitina O-Palmitoiltransferase/metabolismo , Células Cultivadas , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Microscopia de Fluorescência , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/patologia , Ácido Oleico/farmacologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Ácido Palmítico/farmacologia , Perilipina-2/metabolismo , Proteínas Qb-SNARE/química , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/química , Proteínas Qc-SNARE/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Proteína 3 Associada à Membrana da Vesícula/química , Proteína 3 Associada à Membrana da Vesícula/metabolismo
16.
Theranostics ; 8(19): 5200-5212, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30555541

RESUMO

Cisplatin resistance significantly affects the survival rate of patients with ovarian cancer. However, the main mechanism underlying cisplatin resistance in ovarian cancer remains unclear. Methods: Immunohistochemistry was used to determine the expression of OGT, OGA and O-GlcNAc in chemoresistant and chemosensitive ovarian cancer tissues. Functional analyses (in vitro and in vivo) were performed to confirm the role of OGT in cisplatin resistance. Autophagy-related proteins were tested by western blot. Transmission electron microscopy and mRFP-GFP-LC3 adenovirus reporter were used for autophagy flux analysis. Immunoprecipitation assay was utilized to detect protein-protein interactions. Results: We found that O-GlcNAc and O-GlcNAc transferase (OGT) levels were significantly lower in chemoresistant ovarian cancer tissues than in chemosensitive tissues, whereas O-GlcNAcase (OGA) levels did not differ. The down-regulation of OGT increased cisplatin resistance in ovarian cancer cells but had no effect on the efficacy of paclitaxel. The down-regulation of OGT improved tumor resistance to cisplatin in a mouse xenograft tumor model. OGT knockdown enhanced cisplatin-induced autophagy, which reduced apoptotic cell death induced by cisplatin, and promoted autolysosome formation. A reduction in O-GlcNAcylated SNAP-29 levels caused by the down-regulation of OGT promoted the formation of the SNARE complex and autophagic flux. Conclusion: Our findings suggest that down-regulation of OGT enhances cisplatin-induced autophagy via SNAP-29, resulting in cisplatin-resistant ovarian cancer. OGT may represent a novel target for overcoming cisplatin resistance in ovarian cancer.


Assuntos
Antineoplásicos/farmacologia , Autofagia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , N-Acetilglucosaminiltransferases/biossíntese , Neoplasias Ovarianas/patologia , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Adenoviridae/genética , Animais , Antineoplásicos/administração & dosagem , Western Blotting , Cisplatino/administração & dosagem , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Genes Reporter , Vetores Genéticos , Xenoenxertos , Humanos , Imuno-Histoquímica , Imunoprecipitação , Camundongos , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Transplante de Neoplasias , Neoplasias Ovarianas/tratamento farmacológico , Coloração e Rotulagem , Resultado do Tratamento
17.
Int J Mol Med ; 42(6): 3278-3290, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30221662

RESUMO

The O­linked ß­N­acetylglucosamine (O­GlcNAc) modification and autophagy are associated with diabetic myocardial injury, however, the molecular mechanisms between the two processes remain to be fully elucidated. The purpose of the present study was to elucidate the molecular regulation of autophagy by O­GlcNAc­modified synaptosomal­associated protein 29 (SNAP29) in diabetic myocardial injury. A rat model of type I diabetes was established via intraperitoneal injection of streptozotocin (STZ; 55 mg/kg). Significant increases in the O­GlcNAc modification and accumulation of the autophagy markers microtubule­associated protein 1 light chain 3α II/I and P62, which suggest that autophagic flux is inhibited, were observed in rats 8 weeks following STZ induction. Subsequently, the selective O­GlcNAcase inhibitor, thiamet G, increased the level of O­GlcNAc modification, which further disrupted autophagic flux; deteriorated cardiac diastolic function, as indicated by an increased left ventricular filling peak velocity/atrial contraction flow peak velocity ratio shown by echocardiography; and exacerbated myocardial abnormalities, as characterized by cardiomyocyte disorganization and fat and interstitial fibrosis accumulation. By contrast, 6­diazo­5­oxo­L­norleucine, an inhibitor of glucosamine fructose­6­phosphate aminotransferase isomerizing 1, acted as an O­GlcNAc antagonist and reduced the level of O­GlcNAc modification, which maintained autophagic flux and improved cardiac diastolic function. In vitro, high glucose (25 mM) was used to stimulate primary neonatal rat cardiomyocytes (NRCMs). Consistent with the myocardium of diabetic rats, it was also shown in the NRCMs that O­GlcNAc modification of SNAP29 negatively regulated autophagic flux. The application of the short hairpin RNA interference lysosome­associated membrane protein (LAMP2) and the autophagy inhibitor 3­methyladenine demonstrated that high glucose inhibited autophagy­mediated degradation rather than affected the initial stage of autophagy. Finally, co­immunoprecipitation was used to determine the role of the O­GlcNAc­modified substrate protein SNAP29, which acted as an SNAP29­syntaxin­17 (STX17)­vesicle­associated membrane protein 8 (VAMP8) complex during disease progression. The present study is the first, to the best of our knowledge, to demonstrate that SNAP29 is an O­GlcNAc substrate and that an increase in O­GlcNAc­modified SNAP29 inhibits SNAP29­STX17­VAMP8 complex formation, thereby inhibiting the degradation of autophagy and exacerbating myocardial injury in type I diabetic rats.


Assuntos
Autofagia/fisiologia , Cardiomiopatias/metabolismo , Diabetes Mellitus Experimental/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Animais , Autofagia/genética , Cardiomiopatias/patologia , Células Cultivadas , Diabetes Mellitus Experimental/patologia , Ecocardiografia Doppler , Imunoprecipitação , Masculino , Microscopia Eletrônica de Transmissão , Miócitos Cardíacos/patologia , Proteínas Qa-SNARE/genética , Proteínas Qb-SNARE/genética , Proteínas Qc-SNARE/genética , Proteínas R-SNARE/genética , Ratos , Ratos Sprague-Dawley
18.
Proc Natl Acad Sci U S A ; 115(40): 10004-10009, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30217892

RESUMO

The pathogenic strategy of Escherichia coli and many other gram-negative pathogens relies on the translocation of a specific set of proteins, called effectors, into the eukaryotic host cell during infection. These effectors act in concert to modulate host cell processes in favor of the invading pathogen. Injected by the type III secretion system (T3SS), the effector arsenal of enterohemorrhagic E. coli (EHEC) O157:H7 features at least eight individual NleG effectors, which are also found across diverse attaching and effacing pathogens. NleG effectors share a conserved C-terminal U-box E3 ubiquitin ligase domain that engages with host ubiquitination machinery. However, their specific functions and ubiquitination targets have remained uncharacterized. Here, we identify host proteins targeted for ubiquitination-mediated degradation by two EHEC NleG family members, NleG5-1 and NleG2-3. NleG5-1 localizes to the host cell nucleus and targets the MED15 subunit of the Mediator complex, while NleG2-3 resides in the host cytosol and triggers degradation of Hexokinase-2 and SNAP29. Our structural studies of NleG5-1 reveal a distinct N-terminal α/ß domain that is responsible for interacting with host protein targets. The core of this domain is conserved across the NleG family, suggesting this domain is present in functionally distinct NleG effectors, which evolved diversified surface residues to interact with specific host proteins. This is a demonstration of the functional diversification and the range of host proteins targeted by the most expanded effector family in the pathogenic arsenal of E. coli.


Assuntos
Infecções por Escherichia coli/metabolismo , Escherichia coli O157 , Proteínas de Escherichia coli , Infecções por Escherichia coli/patologia , Escherichia coli O157/química , Escherichia coli O157/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Células HEK293 , Células HeLa , Hexoquinase/metabolismo , Humanos , Complexo Mediador/metabolismo , Domínios Proteicos , Proteólise , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Células U937
19.
Nat Commun ; 9(1): 3421, 2018 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-30143604

RESUMO

The SNAREs Vti1a/1b are implicated in regulated secretion, but their role relative to canonical exocytic SNAREs remains elusive. Here, we show that synaptic vesicle and dense-core vesicle (DCV) secretion is indeed severely impaired in Vti1a/b-deficient neurons. The synaptic levels of proteins that mediate secretion were reduced, down to 50% for the exocytic SNARE SNAP25. The delivery of SNAP25 and DCV-cargo into axons was decreased and these molecules accumulated in the Golgi. These defects were rescued by either Vti1a or Vti1b expression. Distended Golgi cisternae and clear vacuoles were observed in Vti1a/b-deficient neurons. The normal non-homogeneous distribution of DCV-cargo inside the Golgi was lost. Cargo trafficking out of, but not into the Golgi, was impaired. Finally, retrograde Cholera Toxin trafficking, but not Sortilin/Sorcs1 distribution, was compromised. We conclude that Vti1a/b support regulated secretion by sorting secretory cargo and synaptic secretion machinery components at the Golgi.


Assuntos
Proteínas Qb-SNARE/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Axônios/metabolismo , Exocitose/fisiologia , Camundongos , Camundongos Knockout , Neurônios/citologia , Neurônios/metabolismo , Transporte Proteico/fisiologia , Receptores de Superfície Celular/metabolismo
20.
J Cell Biol ; 217(10): 3656-3669, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30097514

RESUMO

Autophagy mediates the bulk degradation of cytoplasmic material, particularly during starvation. Upon the induction of autophagy, autophagosomes form a sealed membrane around cargo, fuse with a lytic compartment, and release the cargo for degradation. The mechanism of autophagosome-vacuole fusion is poorly understood, although factors that mediate other cellular fusion events have been implicated. In this study, we developed an in vitro reconstitution assay that enables systematic discovery and dissection of the players involved in autophagosome-vacuole fusion. We found that this process requires the Atg14-Vps34 complex to generate PI3P and thus recruit the Ypt7 module to autophagosomes. The HOPS-tethering complex, recruited by Ypt7, is required to prepare SNARE proteins for fusion. Furthermore, we discovered that fusion requires the R-SNARE Ykt6 on the autophagosome, together with the Q-SNAREs Vam3, Vam7, and Vti1 on the vacuole. These findings shed new light on the mechanism of autophagosome-vacuole fusion and reveal that the R-SNARE Ykt6 is required for this process.


Assuntos
Autofagossomos/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Fusão de Membrana , Proteínas R-SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/genética , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/genética , Proteínas Qb-SNARE/metabolismo , Proteínas R-SNARE/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Vacúolos/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
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