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1.
Science ; 370(6514): 351-356, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33060361

RESUMO

The mechanistic target of rapamycin complex 1 (mTORC1) couples nutrient sufficiency to cell growth. mTORC1 is activated by exogenously acquired amino acids sensed through the GATOR-Rag guanosine triphosphatase (GTPase) pathway, or by amino acids derived through lysosomal degradation of protein by a poorly defined mechanism. Here, we revealed that amino acids derived from the degradation of protein (acquired through oncogenic Ras-driven macropinocytosis) activate mTORC1 by a Rag GTPase-independent mechanism. mTORC1 stimulation through this pathway required the HOPS complex and was negatively regulated by activation of the GATOR-Rag GTPase pathway. Therefore, distinct but functionally coordinated pathways control mTORC1 activity on late endocytic organelles in response to distinct sources of amino acids.


Assuntos
Aminoácidos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Lisossomos/enzimologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas R-SNARE/metabolismo , Ativação Enzimática , Células HEK293 , Humanos , Pinocitose , Proteólise
2.
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
3.
J Neurosci ; 40(28): 5389-5401, 2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32532887

RESUMO

Spontaneous neurotransmitter release is a fundamental property of synapses in which neurotransmitter filled vesicles release their content independent of presynaptic action potentials (APs). Despite their seemingly random nature, these spontaneous fusion events can be regulated by Ca2+ signaling pathways. Here, we probed the mechanisms that maintain Ca2+ sensitivity of spontaneous release events in synapses formed between hippocampal neurons cultured from rats of both sexes. In this setting, we examined the potential role of vesicle-associated membrane protein 4 (VAMP4), a vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein in spontaneous neurotransmission. Our results show that VAMP4 is required for Ca2+-dependent spontaneous excitatory neurotransmission, with a limited role in spontaneous inhibitory neurotransmission. Key residues in VAMP4 that regulate its retrieval as well as functional clathrin-mediated vesicle trafficking were essential for the maintenance of VAMP4-mediated spontaneous release. Moreover, high-frequency stimulation (HFS) that typically triggers asynchronous release and retrieval of VAMP4 from the plasma membrane also augmentsCa2+-sensitive spontaneous release for up to 30 min in a VAMP4-dependent manner. This VAMP4-mediated link between asynchronous and spontaneous excitatory neurotransmission might serve as a presynaptic substrate for synaptic plasticity coupling distinct forms of release.SIGNIFICANCE STATEMENT Spontaneous neurotransmitter release that occurs independent of presynaptic action potentials (APs) shows significant sensitivity to intracellular Ca2+ levels. In this study, we identify the vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) molecule vesicle-associated membrane protein 4 (VAMP4) as a key component of the machinery that maintains these Ca2+-sensitive fraction of spontaneous release events. Following brief intense activity, VAMP4-dependent synaptic vesicle retrieval supports a pool of vesicles that fuse spontaneously in the long term. We propose that this vesicle trafficking pathway acts to shape spontaneous release and associated signaling based on previous activity history of synapses.


Assuntos
Cálcio/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Proteínas R-SNARE/metabolismo , Vesículas Sinápticas/metabolismo , Potenciais de Ação/fisiologia , Animais , Células Cultivadas , Feminino , Hipocampo/citologia , Masculino , Camundongos , Neurônios/citologia , Técnicas de Patch-Clamp , Proteínas R-SNARE/genética , Ratos , Ratos Sprague-Dawley , Sinapses/metabolismo , Transmissão Sináptica/fisiologia
4.
Nat Med ; 26(3): 408-417, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32161403

RESUMO

The diagnosis of lymphomas and leukemias requires hematopathologists to integrate microscopically visible cellular morphology with antibody-identified cell surface molecule expression. To merge these into one high-throughput, highly multiplexed, single-cell assay, we quantify cell morphological features by their underlying, antibody-measurable molecular components, which empowers mass cytometers to 'see' like pathologists. When applied to 71 diverse clinical samples, single-cell morphometric profiling reveals robust and distinct patterns of 'morphometric' markers for each major cell type. Individually, lamin B1 highlights acute leukemias, lamin A/C helps distinguish normal from neoplastic mature T cells, and VAMP-7 recapitulates light-cytometric side scatter. Combined with machine learning, morphometric markers form intuitive visualizations of normal and neoplastic cellular distribution and differentiation. When recalibrated for myelomonocytic blast enumeration, this approach is superior to flow cytometry and comparable to expert microscopy, bypassing years of specialized training. The contextualization of traditional surface markers on independent morphometric frameworks permits more sensitive and automated diagnosis of complex hematopoietic diseases.


Assuntos
Leucemia/diagnóstico , Leucemia/patologia , Linfoma/diagnóstico , Linfoma/patologia , Análise de Célula Única/métodos , Células-Tronco Hematopoéticas/patologia , Humanos , Laminas/metabolismo , Antígenos Comuns de Leucócito/metabolismo , Células Mieloides/patologia , Proteínas R-SNARE/metabolismo
5.
Biochem Biophys Res Commun ; 524(4): 977-982, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32059845

RESUMO

We previously found that VAMP721/722 SNARE proteins guide secretory vesicles to pathogen-attacking sites during immune responses in Arabidopsis, which suggests that these vesicles should deliver immune molecules. However, the lethality of vamp721 vamp722 double null mutant makes it difficult to understand the nature of cargo transported via VAMP721/722 vesicles. Since VAMP721/722-depleted (VAMP721+/-VAMP722-/- and VAMP721-/-VAMP722+/-) plants show compromised resistance to extracellular pathogens, we assume that an immune protein secreted through the VAMP721/722-engaged exocytosis would be remained more in VAMP721/722-depleted plants than WT. By comparing intracellular proteins between WT and VAMP721/722-depleted plants, we found caffeoyl-CoA O-methyltransferase 1 (CCOAOMT1) involved in the lignin biosynthesis was more abundantly detected in both VAMP721/722-depleted lines than WT. Plants are well-known to deposit secondary cell walls as physical barriers at pathogen-attempting sites. Therefore, extracellular detection of CCOAOMT1 and impaired resistance to Pseudomonas syringae DC3000 in ccoaomt1 plants suggest that plants secrete cell wall-modifying enzymes at least including CCOAOMT1 to reinforce the secondary cell walls for immunity.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Metiltransferases/metabolismo , Proteínas R-SNARE/metabolismo , Arabidopsis/citologia , Parede Celular/metabolismo , Lignina/metabolismo , Vesículas Secretórias/metabolismo
6.
Sci Rep ; 10(1): 2907, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32076023

RESUMO

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


Assuntos
Bioensaio/métodos , Lipídeos/química , Neurônios/metabolismo , Proteolipídeos/metabolismo , Proteínas SNARE/metabolismo , Animais , Bovinos , Cricetulus , Transferência Ressonante de Energia de Fluorescência , Proteínas Qa-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Ratos
7.
J Cell Biol ; 219(3)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-31940424

RESUMO

Phagocytes use their actomyosin cytoskeleton to migrate as well as to probe their environment by phagocytosis or macropinocytosis. Although migration and extracellular material uptake have been shown to be coupled in some immune cells, the mechanisms involved in such coupling are largely unknown. By combining time-lapse imaging with genetics, we here identify the lysosomal Ca2+ channel Trpml as an essential player in the coupling of cell locomotion and phagocytosis in hemocytes, the Drosophila macrophage-like immune cells. Trpml is needed for both hemocyte migration and phagocytic processing at distinct subcellular localizations: Trpml regulates hemocyte migration by controlling actomyosin contractility at the cell rear, whereas its role in phagocytic processing lies near the phagocytic cup in a myosin-independent fashion. We further highlight that Vamp7 also regulates phagocytic processing and locomotion but uses pathways distinct from those of Trpml. Our results suggest that multiple mechanisms may have emerged during evolution to couple phagocytic processing to cell migration and facilitate space exploration by immune cells.


Assuntos
Actomiosina/metabolismo , Movimento Celular , Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Hemócitos/metabolismo , Lisossomos/metabolismo , Macrófagos/metabolismo , Fagocitose , Canais de Receptores Transientes de Potencial/metabolismo , Actomiosina/genética , Animais , Animais Geneticamente Modificados , Cálcio/metabolismo , Sinalização do Cálcio , Citoesqueleto/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/imunologia , Hemócitos/imunologia , Lisossomos/genética , Macrófagos/imunologia , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Fatores de Tempo , Canais de Receptores Transientes de Potencial/genética
8.
Cells ; 8(12)2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31861136

RESUMO

Syntaxin 16, a Qa-SNARE (soluble N-ethylmaleimide-sensitive factor activating protein receptor), is involved in a number of membrane-trafficking activities, particularly transport processes at the trans-Golgi network (TGN). Recent works have now implicated syntaxin 16 in the autophagy process. In fact, syntaxin 16 appears to have dual roles, firstly in facilitating the transport of ATG9a-containing vesicles to growing autophagosomes, and secondly in autolysosome formation. The former involves a putative SNARE complex between syntaxin 16, VAMP7 and SNAP-47. The latter occurs via syntaxin 16's recruitment by Atg8/LC3/GABARAP family proteins to autophagosomes and endo-lysosomes, where syntaxin 16 may act in a manner that bears functional redundancy with the canonical autophagosome Qa-SNARE syntaxin 17. Here, I discuss these recent findings and speculate on the mechanistic aspects of syntaxin 16's newly found role in autophagy.


Assuntos
Autofagia/fisiologia , Sintaxina 16/metabolismo , Animais , Autofagossomos/metabolismo , Autofagossomos/fisiologia , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas de Transporte/metabolismo , Humanos , Lisossomos/metabolismo , Fusão de Membrana/fisiologia , Proteínas de Membrana/metabolismo , Transporte Proteico/fisiologia , Proteínas Qa-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Sintaxina 16/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Rede trans-Golgi/metabolismo , Rede trans-Golgi/fisiologia
9.
PLoS One ; 14(11): e0223254, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31714914

RESUMO

Down syndrome (DS) is a chromosomal disorder caused by trisomy of chromosome 21 (Ts21). Unbalanced karyotypes can lead to dysfunction of the proteostasis network (PN) and disrupted proteostasis is mechanistically associated with multiple DS comorbidities. Autophagy is a critical component of the PN that has not previously been investigated in DS. Based on our previous observations of PN disruption in DS, we investigated possible dysfunction of the autophagic machinery in human DS fibroblasts and other DS cell models. Following induction of autophagy by serum starvation, DS fibroblasts displayed impaired autophagic flux indicated by autophagolysosome accumulation and elevated p62, NBR1, and LC3-II abundance, compared to age- and sex-matched, euploid (CTL) fibroblasts. While lysosomal physiology was unaffected in both groups after serum starvation, we observed decreased basal abundance of the Soluble N-ethylmaleimide-sensitive-factor Attachment protein Receptor (SNARE) family members syntaxin 17 (STX17) and Vesicle Associated Membrane Protein 8 (VAMP8) indicating that decreased autophagic flux in DS is due at least in part to a possible impairment of autophagosome-lysosome fusion. This conclusion was further supported by the observation that over-expression of either STX17 or VAMP8 in DS fibroblasts restored autophagic degradation and reversed p62 accumulation. Collectively, our results indicate that impaired autophagic clearance is a characteristic of DS cells that can be reversed by enhancement of SNARE protein expression and provides further evidence that PN disruption represents a candidate mechanism for multiple aspects of pathogenesis in DS and a possible future target for therapeutic intervention.


Assuntos
Autofagia , Síndrome de Down/metabolismo , Síndrome de Down/patologia , Proteínas SNARE/metabolismo , Linhagem Celular , Feminino , Regulação da Expressão Gênica , Humanos , Lactente , Recém-Nascido , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/metabolismo , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Transporte Proteico , Proteínas R-SNARE/metabolismo , Proteínas de Ligação a RNA/metabolismo
10.
Neuron ; 104(5): 869-884.e11, 2019 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-31648898

RESUMO

Age-related neurodegenerative disorders are characterized by a slow, persistent accumulation of aggregated proteins. Although cells can elicit physiological responses to enhance cellular clearance and counteract accumulation, it is unclear how pathogenic proteins evade this process in disease. We find that Parkinson's disease α-synuclein perturbs the physiological response to lysosomal stress by impeding the SNARE protein ykt6. Cytosolic ykt6 is normally autoinhibited by a unique farnesyl-mediated regulatory mechanism; however, during lysosomal stress, it activates and redistributes into membranes to preferentially promote hydrolase trafficking and enhance cellular clearance. α-Synuclein aberrantly binds and deactivates ykt6 in patient-derived neurons, thereby disabling the lysosomal stress response and facilitating protein accumulation. Activating ykt6 by small-molecule farnesyltransferase inhibitors restores lysosomal activity and reduces α-synuclein in patient-derived neurons and mice. Our findings indicate that α-synuclein creates a permissive environment for aggregate persistence by inhibiting regulated cellular clearance and provide a therapeutic strategy to restore protein homeostasis by harnessing SNARE activity.


Assuntos
Lisossomos/metabolismo , Neurônios/metabolismo , Doença de Parkinson/metabolismo , Proteínas R-SNARE/metabolismo , alfa-Sinucleína/metabolismo , Animais , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transporte Proteico/fisiologia , Estresse Fisiológico/fisiologia
11.
Plant Cell ; 31(12): 3015-3032, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31597687

RESUMO

Plant phospholipase Ds (PLDs), essential regulators of phospholipid signaling, function in multiple signal transduction cascades; however, the mechanisms regulating PLDs in response to pathogens remain unclear. Here, we found that Arabidopsis (Arabidopsis thaliana) PLDδ accumulated in cells at the entry sites of the barley powdery mildew fungus, Blumeria graminis f. sp hordei Using fluorescence recovery after photobleaching and single-molecule analysis, we observed higher PLDδ density in the plasma membrane after chitin treatment; PLDδ also underwent rapid exocytosis. Fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy showed that the interaction between PLDδ and the microdomain marker AtREMORIN1.3 (AtREM1.3) increased in response to chitin, indicating that exocytosis facilitates rapid, efficient sorting of PLDδ into microdomains upon pathogen stimulus. We further unveiled a trade-off between brefeldin A (BFA)-resistant and -sensitive pathways in secretion of PLDδ under diverse conditions. Upon pathogen attack, PLDδ secretion involved syntaxin-associated VAMP721/722-mediated exocytosis sensitive to BFA. Analysis of phosphatidic acid (PA), hydrogen peroxide, and jasmonic acid (JA) levels and expression of related genes indicated that the relocalization of PLDδ is crucial for its activation to produce PA and initiate reactive oxygen species and JA signaling pathways. Together, our findings revealed that the translocation of PLDδ to papillae is modulated by exocytosis, thus triggering PA-mediated signaling in plant innate immunity.plantcell;31/12/3015/FX1F1fx1.


Assuntos
Arabidopsis/imunologia , Membrana Celular/metabolismo , Imunidade Inata , Fosfolipase D/metabolismo , Doenças das Plantas/imunologia , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/metabolismo , Ascomicetos/patogenicidade , Brefeldina A/imunologia , Brefeldina A/metabolismo , Quitina/imunologia , Quitina/metabolismo , Ciclopentanos/metabolismo , Exocitose/efeitos dos fármacos , Exocitose/imunologia , Peróxido de Hidrogênio/metabolismo , Imunidade Inata/efeitos dos fármacos , Oxilipinas/metabolismo , Ácidos Fosfatídicos/metabolismo , Fosfolipase D/genética , Doenças das Plantas/microbiologia , Proteínas Qa-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Transdução de Sinais/imunologia , Transdução de Sinais/fisiologia
12.
Nat Commun ; 10(1): 4306, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31541089

RESUMO

The mucus layer is the first line of innate host defense in the gut that protects the epithelium by spatially separating commensal bacteria. MUC2 mucin is produced and stored by goblet cells that is constitutively exocytosed or hyper secreted upon sensing a threat. How coordinated mucus exocytosis maintains homeostasis in the intestinal epithelium and modulates the immunological landscape remains elusive. Here we describe how the vesicle SNARE protein VAMP8 coordinates mucin exocytosis from goblet cells. Vamp8-/- exhibit a mild pro-inflammatory state basally due to an altered mucus layer and increased encounters with microbial antigens. Microbial diversity shifts to a detrimental microbiota with an increase abundance of pathogenic and mucolytic bacteria. To alleviate the heavy microbial burden and inflammatory state basally, Vamp8-/- skews towards tolerance. Despite this, Vamp8-/- is highly susceptible to both chemical and infectious colitis demonstrating the fragility of the intestinal mucosa without proper mucus exocytosis mechanisms.


Assuntos
Colo/metabolismo , Exocitose/fisiologia , Células Caliciformes/metabolismo , Mucosa Intestinal/metabolismo , Mucina-2/metabolismo , Proteínas R-SNARE/metabolismo , Animais , Biodiversidade , Colo/patologia , Citocinas/metabolismo , Células Caliciformes/patologia , Homeostase , Humanos , Intestinos/patologia , Camundongos Knockout , Microbiota , Mucina-2/genética , Muco/metabolismo , Fenótipo , Proteínas R-SNARE/genética , Proteínas SNARE/metabolismo
13.
Nutrients ; 11(9)2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31491968

RESUMO

Distinct environmental insults might interact with fructose consumption and contribute to the development of metabolic disorders. To address whether in utero glucocorticoid exposure and fructose intake modulate metabolic responses, adult female Wistar rats were exposed to dexamethasone (DEX) during pregnancy, and the offspring were administered fructose at a later time. Briefly, dams received DEX during the third period of pregnancy, while control dams remained untreated. Offspring born to control and DEX-treated mothers were defined as CTL-off and DEX-off, respectively, while untreated animals were designated CTL-off-CTL and DEX-off-CTL. CLT-off and DEX-off treated with 10% fructose in the drinking water for 8 weeks are referred to as CTL-off-FRU and DEX-off-FRU. We found that fructose promoted glucose intolerance and whole-body gluconeogenesis in both CTL-off-FRU and DEX-off-FRU animals. On the other hand, hepatic lipid accumulation was significantly stimulated in DEX-off-FRU rats when compared to the CTL-off-FRU group. The DEX-off-FRU group also displayed impaired very-low-density lipoprotein (VLDL) production and reduced hepatic expression of apoB, mttp, and sec22b. DEX-off-FRU has lower hepatic levels of autophagy markers. Taken together, our results support the unprecedented notion that in utero glucocorticoid exposure exacerbates hepatic steatosis caused by fructose consumption later in life.


Assuntos
Dexametasona/toxicidade , Açúcares da Dieta/toxicidade , Fígado Gorduroso/induzido quimicamente , Frutose/toxicidade , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal , Animais , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Feminino , Idade Gestacional , Gluconeogênese/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Lipoproteínas VLDL/metabolismo , Fígado/metabolismo , Fígado/patologia , Masculino , Gravidez , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Ratos Wistar
14.
J Virol ; 93(22)2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31484747

RESUMO

The endoplasmic reticulum (ER)-resident proteins vesicle-associated membrane protein (VAMP)-associated protein A and B (VAPA and VAPB) have been reported to be necessary for efficient hepatitis C virus (HCV) replication, but the specific mechanisms are not well understood. VAPs are known to recruit lipid transfer proteins to the ER, including oxysterol binding protein (OSBP), which has been previously shown to be necessary for cholesterol delivery to the HCV replication organelle in exchange for phosphatidylinositol 4-phosphate [PI(4)P]. Here, we show that VAPA and VAPB are redundant for HCV infection and that dimerization is not required for their function. In addition, we identify the phosphatidylinositol transfer protein Nir2 as an effector of VAPs to support HCV replication. We propose that Nir2 functions to replenish phosphoinositides at the HCV replication organelle to maintain elevated steady-state levels of PI(4)P, which is removed by OSBP. Thus, Nir2, along with VAPs, OSBP, and the phosphatidylinositol 4-kinase, completes a cycle of phosphoinositide flow between the ER and viral replication organelles to drive ongoing viral replication.IMPORTANCE Hepatitis C virus (HCV) is known for its ability to modulate phosphoinositide signaling pathways for its replication. Elevated levels of phosphatidylinositol 4-phosphate [PI(4)P] in HCV replication organelles (ROs) recruits lipid transfer proteins (LTPs), like oxysterol-binding protein (OSBP). OSBP exchanges PI(4)P with cholesterol, thus removing PI(4)P from the HCV RO. Here, we found that the phosphatidylinositol transfer protein Nir2 acts as an LTP and may replenish PI at the HCV RO by interacting with VAMP-associated proteins (VAPs), enabling continuous viral replication during chronic infection. Therefore, the coordination of OSBP, Nir2, and VAPs completes our understanding of the phosphoinositide cycle between the ER and HCV ROs.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas do Olho/metabolismo , Hepacivirus/fisiologia , Hepatite C Crônica/metabolismo , Hepatite C Crônica/virologia , Proteínas de Membrana/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas R-SNARE/metabolismo , 1-Fosfatidilinositol 4-Quinase/metabolismo , Transporte Biológico , Proteínas de Transporte/metabolismo , Colesterol/metabolismo , Retículo Endoplasmático/metabolismo , Células HEK293 , Hepacivirus/metabolismo , Humanos , Organelas/metabolismo , Organelas/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Replicação Viral/fisiologia
15.
Sci Rep ; 9(1): 12686, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31481661

RESUMO

Studies in humans and pre-clinical animal models show milk-derived miRNAs reflect mammary gland function during lactation. The zinc transporter SLC30A2/ZnT2 plays a critical role in mammary gland function; ZnT2-null mice have profound defects in mammary epithelial cell (MEC) polarity and secretion, resulting in sub-optimal lactation. Non-synonymous genetic variation in SLC30A2 is common in humans, and several common ZnT2 variants are associated with changes in milk components that suggest breast dysfunction in women. To identify novel mechanisms through which dysfunction might occur, milk-derived miRNA profiles were characterized in women harboring three common genetic variants in SLC30A2 (D103E, T288S, and Exon 7). Expression of ten miRNAs differed between genotypes, and contributed to distinct spatial separation. Studies in breast milk and cultured MECs confirmed expression of ZnT2 variants alters abundance of protein levels of several predicted mRNA targets critical for breast function (PRLR, VAMP7, and SOX4). Moreover, bioinformatic analysis identified two novel gene networks that may underlie normal MEC function. Thus, we propose that genetic variation in genes critical for normal breast function such as SLC30A2 has important implications for lactation performance in women, and that milk-derived miRNAs can be used to identify novel mechanisms and for diagnostic potential.


Assuntos
Proteínas de Transporte de Cátions/genética , MicroRNAs/metabolismo , Leite Humano/metabolismo , Adolescente , Adulto , Animais , Proteínas de Transporte de Cátions/deficiência , Proteínas de Transporte de Cátions/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Genótipo , Humanos , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Camundongos , Camundongos Knockout , Polimorfismo Genético , Mapas de Interação de Proteínas/genética , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Fatores de Transcrição SOXC/genética , Fatores de Transcrição SOXC/metabolismo , Adulto Jovem
16.
Sci Rep ; 9(1): 11434, 2019 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-31391476

RESUMO

The highly conserved SNARE protein SEC22B mediates diverse and critical functions, including phagocytosis, cell growth, autophagy, and protein secretion. However, these characterizations have thus far been limited to in vitro work. Here, we expand our understanding of the role Sec22b plays in vivo. We utilized Cre-Lox mice to delete Sec22b in three tissue compartments. With a germline deletion of Sec22b, we observed embryonic death at E8.5. Hematopoietic/endothelial cell deletion of Sec22b also resulted in in utero death. Notably, mice with Sec22b deletion in CD11c-expressing cells of the hematopoietic system survive to adulthood. These data demonstrate Sec22b contributes to early embryogenesis through activity both in hematopoietic/endothelial tissues as well as in other tissues yet to be defined.


Assuntos
Desenvolvimento Embrionário , Células Endoteliais/metabolismo , Sistema Hematopoético/embriologia , Proteínas R-SNARE/metabolismo , Animais , Embrião de Mamíferos , Feminino , Masculino , Camundongos , Camundongos Knockout , Proteínas R-SNARE/genética
17.
Thromb Res ; 181: 99-105, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31382081

RESUMO

INTRODUCTION: Platelet hyper-reactivity has been associated with thrombosis and high levels of human vesicle-associated membrane protein 8 (VAMP8) and serotonin transporter (SERT). Two polymorphisms (rs1010 of VAMP8 gene and in SERT gene (SLC6A4)) are associated with arterial thrombosis. AIM: To determine if levels of serotonin, SERT and/or VAMP8 and these polymorphisms are associated with the risk of venous thrombosis. MATERIAL AND METHODS: A total of 324 individuals were included in the RETROVE Study (Riesgo de Enfermedad TROmboembólica VEnosa). VAMP8, SERT and serotonin were determined by ELISA; polymorphisms of SLC6A4 and VAMP8 by polymerase chain reaction (PCR) and real time PCR. The venous thrombotic risk was calculated by a logistic regression method to estimate the crude and adjusted OR (adjusted for sex, age, body mass index and venous thrombosis risk co-factors). RESULTS: Statistically significant high levels of VAMP8 and SERT were found in patients, but not in controls. In contrast, serotonin showed lower levels in patients than in controls. When individuals were studied by gender, only women exhibited a statistically significant difference: the OR for VAMP8 was 3.25 (1.61-6.56 95% CI). The adjusted OR did not change. The OR for SERT was 2.76 (1.36-5.60 95% CI), the adjusted OR was maintained also. For serotonin with OR of 2.62 (1.40-4.92 95% CI), the adjusted OR was not significant. In contrast males did not show significant differences. No statistically differences between patients and controls were found for both polymorphisms. CONCLUSIONS: VAMP8 and SERT levels are associated with venous thrombosis in a female Spanish population.


Assuntos
Proteínas R-SNARE/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Trombose Venosa/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade , Fatores de Risco , Espanha , Trombose Venosa/patologia
18.
Front Immunol ; 10: 1855, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31447853

RESUMO

Cytotoxic T lymphocytes kill infected or malignant cells through the directed release of cytotoxic substances at the site of target cell contact, the immunological synapse. While genetic association studies of genes predisposing to early-onset life-threatening hemophagocytic lymphohistiocytosis has identified components of the plasma membrane fusion machinery, the identity of the vesicular components remain enigmatic. Here, we identify VAMP7 as an essential component of the vesicular fusion machinery of primary, human T cells. VAMP7 co-localizes with granule markers throughout all stages of T cell maturation and simultaneously fuses with granule markers at the IS. Knock-down of VAMP7 expression significantly decreased the killing efficiency of T cells, without diminishing early T cell receptor signaling. VAMP7 exerts its function in a SNARE complex with Syntaxin11 and SNAP-23 on the plasma membrane. The identification of the minimal fusion machinery in T cells provides a starting point for the development of potential drugs in immunotherapy.


Assuntos
Degranulação Celular/imunologia , Grânulos Citoplasmáticos/imunologia , Proteínas R-SNARE/imunologia , Linfócitos T Citotóxicos/imunologia , Células Cultivadas , Grânulos Citoplasmáticos/metabolismo , Humanos , Sinapses Imunológicas/imunologia , Sinapses Imunológicas/metabolismo , Proteínas R-SNARE/metabolismo , Vesículas Secretórias/imunologia , Vesículas Secretórias/metabolismo , Linfócitos T Citotóxicos/metabolismo
19.
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
20.
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
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