Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 170
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
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.
J Cell Biol ; 218(10): 3355-3371, 2019 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-31519727

RESUMO

Metastasis is a major cause of cancer-related death. Membrane type 1-matrix metalloproteinase (MT1-MMP) is a critical protease for local invasion and metastasis. MT1-MMP is synthesized in the endoplasmic reticulum (ER) and transported in vesicles to invadopodia, specialized subdomains of the plasma membrane, through secretory and endocytic recycling pathways. The molecular mechanism underlying intracellular transport of MT1-MMP has been extensively studied, but is not fully understood. We show that MT1-MMP diverts the SNARE Bet1 from its function in ER-Golgi transport, to promote MT1-MMP trafficking to the cell surface, likely to invadopodia. In invasive cells, Bet1 is localized in MT1-MMP-positive endosomes in addition to the Golgi apparatus, and forms a novel SNARE complex with syntaxin 4 and endosomal SNAREs. MT1-MMP may also use Bet1 for its export from raft-like structures in the ER. Our results suggest the recruitment of Bet1 at an early stage after MT1-MMP expression promotes the exit of MT1-MMP from the ER and its efficient transport to invadopodia.


Assuntos
Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteínas SNARE/metabolismo , Humanos , Transporte Proteico , Células Tumorais Cultivadas
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.
Proteomics ; 19(13): e1900094, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31115157

RESUMO

The olfactory conditioning of the bee proboscis extension reflex (PER) is extensively used as a paradigm in associative learning of invertebrates but with limited molecular investigations. To investigate which protein changes are linked to olfactory conditioning, a non-sophisticated conditioning model is applied using the PER in the honeybee (Apis mellifera). Foraging honeybees are assigned into three groups based on the reflex behavior and training: conditioned using 2-octanone (PER-conditioned), and sucrose and water controls. Thereafter, the brain synaptosomal proteins are isolated and analyzed by quantitative proteomics using stable isotope labeling (TMT). Additionally, the complex proteome dataset of the bee brain is generated with a total number of 5411 protein groups, including key players in neurotransmitter signaling. The most significant categories affected during olfactory conditioning are associated with "SNARE interactions in vesicular transport" (BET1 and VAMP7), ABC transporters, and fatty acid degradation pathways.


Assuntos
Abelhas/fisiologia , Encéfalo/fisiologia , Condicionamento Clássico/fisiologia , Olfato/fisiologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Marcação por Isótopo , Proteoma/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteínas R-SNARE/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.
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
10.
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
11.
Methods Mol Biol ; 1860: 175-189, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30317504

RESUMO

Intracellular membrane fusion is mediated by the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins that are highly conserved and tightly regulated by a variety of factors. The exocyst complex is one of the multi-subunit tethering complexes and functions in the tethering of the secretory vesicles to the plasma membrane. We have found that the yeast Sec3, a subunit of the exocyst, binds to the t-SNARE protein Sso2 and promotes its interaction with another t-SNARE protein, Sec9. Here, we describe the structural analysis and in vitro membrane fusion assays, by which we found that Sec3 binding leads to a conformational change within Sso2, and facilitates SNARE assembly and the membrane fusion.


Assuntos
Fusão de Membrana , Modelos Moleculares , Proteínas Qa-SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Motivos de Aminoácidos/genética , Lipossomos/química , Lipossomos/metabolismo , Mutagênese , Ligação Proteica , Proteínas Qa-SNARE/química , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/isolamento & purificação , Proteínas Qc-SNARE/química , Proteínas Qc-SNARE/isolamento & purificação , Proteínas Qc-SNARE/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação
12.
IUBMB Life ; 71(3): 364-375, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30550624

RESUMO

Emerging evidence on efficient tumor growth regulation by endogenous lectins directs interest to determine on a proof-of-principle level the range of information on alterations provided by full-scale analysis using phosphoproteomics. In our pilot study, we tested galectin-4 (gal-4) that is a growth inhibitor for colon cancer cells (CRC), here working with the LS 180 line. In order to cover monitoring of short- and long-term effects stable isotope labeling by amino acids in cell culture-based quantitative phosphoproteomic analyses were conducted on LS 180 cell preparations collected 1 and 72 h after adding gal-4 to the culture medium. After short-term treatment, 981 phosphosites, all of them S/T based, were detected by phosphoproteomics. Changes higher than 1.5-fold were seen for eight sites in seven proteins. Most affected were the BET1 homolog (BET1), whose level of phosphorylation at S50 was about threefold reduced, and centromere protein F (CENPF), extent of phosphorylation at S3119 doubling in gal-4-treated cells. Phosphoproteome analysis after 72 h of treatment revealed marked changes at 33 S/T-based phosphosites from 29 proteins. Prominent increase of phosphorylation was observed for cofilin-1 at position S3. Extent of phosphorylation of the glutamine transporter SLC1A5 at position S503 was decreased by a factor of 3. Altered phosphorylation of BET1, CENPF, and cofilin-1 as well as a significant effect of gal-4 treatment on glutamine uptake by cells were substantiated by independent methods in the Vaco 432, Colo 205, CX 1, and HCT 116 cell lines. With the example of gal-4 which functions as a tumor suppressor in CRC cells, we were able to prove that cell surface binding of the lectin not only markedly influences the cell proteome, but also has a bearing on malignancy-associated intracellular protein phosphorylation. These results underscore the potential of this approach to give further work on elucidating the details of signaling underlying galectin-triggered growth inhibition a clear direction. © 2018 IUBMB Life, 71(3):364-375, 2019.


Assuntos
Antineoplásicos/farmacologia , Galectina 4/farmacologia , Proteínas de Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteoma/metabolismo , Sistema ASC de Transporte de Aminoácidos/genética , Sistema ASC de Transporte de Aminoácidos/metabolismo , Transporte Biológico/efeitos dos fármacos , Isótopos de Carbono , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cofilina 1/genética , Cofilina 1/metabolismo , Glutamina/metabolismo , Células HCT116 , Humanos , Marcação por Isótopo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas de Neoplasias/genética , Isótopos de Nitrogênio , Fosfoproteínas/genética , Fosforilação/efeitos dos fármacos , Proteoma/genética , Proteínas Qc-SNARE/genética , Proteínas Qc-SNARE/metabolismo , Proteínas Recombinantes/farmacologia
13.
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
14.
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
15.
J Biol Chem ; 293(47): 18353-18364, 2018 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-30305396

RESUMO

Multisite phosphorylation of proteins is a common mechanism for signal integration and amplification in eukaryotic signaling networks. Proteins are commonly phosphorylated at multiple sites in an ordered manner, whereby phosphorylation by one kinase primes the substrate by generating a recognition motif for a second kinase. Here we show that substrate priming promotes phosphorylation by Saccharomyces cerevisiae Kin1 and Kin2, kinases that regulate cell polarity, exocytosis, and the endoplasmic reticulum (ER) stress response. Kin1/Kin2 phosphorylated substrates within the context of a sequence motif distinct from those of their most closely related kinases. In particular, the rate of phosphorylation of a peptide substrate by Kin1/Kin2 increased >30-fold with incorporation of a phosphoserine residue two residues downstream of the phosphorylation site. Recognition of phosphorylated substrates by Kin1/Kin2 was mediated by a patch of basic residues located in the region of the kinase αC helix. We identified a set of candidate Kin1/Kin2 substrates reported to be dually phosphorylated at sites conforming to the Kin1/Kin2 consensus sequence. One of these proteins, the t-SNARE protein Sec9, was confirmed to be a Kin1/Kin2 substrate both in vitro and in vivo Sec9 phosphorylation by Kin1 in vitro was enhanced by prior phosphorylation at the +2 position. Recognition of primed substrates was not required for the ability of Kin2 to suppress the growth defect of secretory pathway mutants but was necessary for optimal growth under conditions of ER stress. These results suggest that at least some endogenous protein substrates of Kin1/Kin2 are phosphorylated in a priming-dependent manner.


Assuntos
Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Motivos de Aminoácidos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Peptídeos/química , Peptídeos/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Qc-SNARE/genética , Proteínas Qc-SNARE/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato
16.
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
17.
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
18.
J Clin Invest ; 128(9): 3941-3956, 2018 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-30102258

RESUMO

The t-SNARE protein SNAP23 conventionally functions as a component of the cellular machinery required for intracellular transport vesicle fusion with target membranes and has been implicated in the regulation of fasting glucose levels, BMI, and type 2 diabetes. Surprisingly, we observed that adipocyte-specific KO of SNAP23 in mice resulted in a temporal development of severe generalized lipodystrophy associated with adipose tissue inflammation, insulin resistance, hyperglycemia, liver steatosis, and early death. This resulted from adipocyte cell death associated with an inhibition of macroautophagy and lysosomal degradation of the proapoptotic regulator BAX, with increased BAX activation. BAX colocalized with LC3-positive autophagic vacuoles and was increased upon treatment with lysosome inhibitors. Moreover, BAX deficiency suppressed the lipodystrophic phenotype in the adipocyte-specific SNAP23-KO mice and prevented cell death. In addition, ATG9 deficiency phenocopied SNAP23 deficiency, whereas ATG7 deficiency had no effect on BAX protein levels, BAX activation, or apoptotic cell death. These data demonstrate a role for SNAP23 in the control of macroautophagy and programmed cell death through an ATG9-dependent, but ATG7-independent, pathway regulating BAX protein levels and BAX activation.


Assuntos
Adipócitos/citologia , Adipócitos/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteína X Associada a bcl-2/metabolismo , Células 3T3-L1 , Animais , Apoptose/fisiologia , Autofagia/fisiologia , Proteína 7 Relacionada à Autofagia/deficiência , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/deficiência , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Lipodistrofia/metabolismo , Lipodistrofia/patologia , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteólise , Proteínas Qb-SNARE/deficiência , Proteínas Qb-SNARE/genética , Proteínas Qc-SNARE/deficiência , Proteínas Qc-SNARE/genética , Proteínas de Transporte Vesicular/deficiência , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteína X Associada a bcl-2/deficiência , Proteína X Associada a bcl-2/genética
19.
Nat Commun ; 9(1): 3492, 2018 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-30154410

RESUMO

The Unc-51 like autophagy activating kinase 1 (ULK1) complex plays a central role in the initiation stage of autophagy. However, the function of ULK1 in the late stage of autophagy is unknown. Here, we report that ULK1, a central kinase of the ULK1 complex involved in autophagy initiation, promotes autophagosome-lysosome fusion. PKCα phosphorylates ULK1 and prevents autolysosome formation. PKCα phosphorylation of ULK1 does not change its kinase activity; however, it decreases autophagosome-lysosome fusion by reducing the affinity of ULK1 for syntaxin 17 (STX17). Unphosphorylated ULK1 recruited STX17 and increased STX17's affinity towards synaptosomal-associated protein 29 (SNAP29). Additionally, phosphorylation of ULK1 enhances its interaction with heat shock cognate 70 kDa protein (HSC70) and increases its degradation through chaperone-mediated autophagy (CMA). Our study unearths a key mechanism underlying autolysosome formation, a process in which the kinase activity of PKCα plays an instrumental role, and reveals the significance of the mutual regulation of macroautophagy and CMA in maintaining the balance of autophagy.


Assuntos
Autofagossomos/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Western Blotting , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/genética , Microscopia Eletrônica de Transmissão , Fosforilação , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/genética , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/genética , Proteínas Qc-SNARE/metabolismo , Sinaptossomos/metabolismo
20.
Gene ; 673: 217-224, 2018 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-29908998

RESUMO

OBJECTIVE: Cervical cancer (CC) is one of the most common gynecologic tumors in women worldwide, with poor prognosis and low survival rate. In this study, we identified SNAP23 as a potential tumor suppressor gene in CC. METHODS: The expression of SNAP23 in tissues and cell lines were measured by qRT-PCR, western blot and IHC. Knockdown of SNAP23 by siRNA and ectopic expression of SNAP23 by overexpression plasmid were performed to observe the biological function of SNAP23 in CC. Xenograft nude mice models were established to measure its function in vivo. RESULTS: SNAP23 was downregulated in CC tissues and had a negative correlation with advanced clinical characteristics. Ectopic expression of SNAP23 suppressed malignant phonotype of CC while knockdown of SNAP23 promoted the progression of CC in vitro. The flow cytometry analysis revealed that SNAP23 exerted its tumor suppressor activity via inducing G2/M cell cycle arrest. Moreover, xenograft tumor models showed that SNAP23 suppresses tumor growth in vivo. CONCLUSIONS: Our results revealed that SNAP23 suppressed progression of CC and induced cell cycle G2/M arrest via upregulating p21cip1 and downregulating CyclinB1.


Assuntos
Ciclo Celular , Ciclina B1/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Proteínas Qb-SNARE/genética , Proteínas Qc-SNARE/genética , Neoplasias do Colo do Útero/genética , Animais , Divisão Celular , Linhagem Celular Tumoral , Movimento Celular , Biologia Computacional , Progressão da Doença , Feminino , Fase G2 , Células HeLa , Humanos , Camundongos , Camundongos Nus , Invasividade Neoplásica , Transplante de Neoplasias , Fenótipo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , RNA Interferente Pequeno/metabolismo , Regulação para Cima , Neoplasias do Colo do Útero/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA