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1.
Int J Mol Sci ; 22(23)2021 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-34884763

RESUMO

The adipokine Neuregulin 4 (Nrg4) protects against obesity-induced insulin resistance. Here, we analyze how the downregulation of Nrg4 influences insulin action and the underlying mechanisms in adipocytes. Validated shRNA lentiviral vectors were used to generate scramble (Scr) and Nrg4 knockdown (KD) 3T3-L1 adipocytes. Adipogenesis was unaffected in Nrg4 KD adipocytes, but there was a complete impairment of the insulin-induced 2-deoxyglucose uptake, which was likely the result of reduced insulin receptor and Glut4 protein. Downregulation of Nrg4 enhanced the expression of proinflammatory cytokines. Anti-inflammatory agents recovered the insulin receptor, but not Glut4, content. Proteins enriched in Glut4 storage vesicles such as the insulin-responsive aminopeptidase (IRAP) and Syntaxin-6 as well as TBC1D4, a protein involved in the intracellular retention of Glut4 vesicles, also decreased by Nrg4 KD. Insulin failed to reduce autophagy in Nrg4 KD adipocytes, observed by a minor effect on mTOR phosphorylation, at the time that proteins involved in autophagy such as LC3-II, Rab11, and Clathrin were markedly upregulated. The lysosomal activity inhibitor bafilomycin A1 restored Glut4, IRAP, Syntaxin-6, and TBC1D4 content to those found in control adipocytes. Our study reveals that Nrg4 preserves the insulin responsiveness by preventing inflammation and, in turn, benefits the insulin regulation of autophagy.


Assuntos
Autofagia/fisiologia , Transportador de Glucose Tipo 4/metabolismo , Resistência à Insulina/fisiologia , Neurregulinas/metabolismo , Receptor de Insulina/biossíntese , Células 3T3 , Adipócitos/metabolismo , Animais , Linhagem Celular , Cistinil Aminopeptidase/biossíntese , Citocinas/biossíntese , Desoxiglucose/metabolismo , Regulação para Baixo , Proteínas Ativadoras de GTPase/biossíntese , Inflamação/patologia , Insulina/metabolismo , Camundongos , Neurregulinas/biossíntese , Neurregulinas/genética , Proteínas Qa-SNARE/biossíntese , Interferência de RNA , RNA Interferente Pequeno/genética
2.
Cell ; 184(24): 5950-5969.e22, 2021 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-34741801

RESUMO

The biogenesis of mammalian autophagosomes remains to be fully defined. Here, we used cellular and in vitro membrane fusion analyses to show that autophagosomes are formed from a hitherto unappreciated hybrid membrane compartment. The autophagic precursors emerge through fusion of FIP200 vesicles, derived from the cis-Golgi, with endosomally derived ATG16L1 membranes to generate a hybrid pre-autophagosomal structure, HyPAS. A previously unrecognized apparatus defined here controls HyPAS biogenesis and mammalian autophagosomal precursor membranes. HyPAS can be modulated by pharmacological agents whereas its formation is inhibited upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or by expression of SARS-CoV-2 nsp6. These findings reveal the origin of mammalian autophagosomal membranes, which emerge via convergence of secretory and endosomal pathways, and show that this process is targeted by microbial factors such as coronaviral membrane-modulating proteins.


Assuntos
Autofagossomos/virologia , COVID-19/virologia , Autofagia , COVID-19/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Endossomos/fisiologia , Endossomos/virologia , Complexo de Golgi/fisiologia , Células HEK293 , Células HeLa , Humanos , Fusão de Membrana , Microscopia Confocal , Fagossomos/metabolismo , Fagossomos/virologia , Proteínas Qa-SNARE/biossíntese , Receptores sigma/biossíntese , SARS-CoV-2 , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/biossíntese , Sinaptotagminas/biossíntese , Receptor Sigma-1
3.
Biochim Biophys Acta Gen Subj ; 1863(11): 129396, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31302181

RESUMO

BACKGROUND: Botulinum neurotoxins (BoNTs) prevent synaptic transmission because they hydrolyze synaptic N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). BoNT serotype C (BoNT/C) targets syntaxin-1A and SNAP-25, and is expected to be applied to cosmetic and therapeutic uses. SNAREs are evolutionally conserved proteins and in yeast a syntaxin-1A orthologue Sso1 is involved in exocytosis. The substrate specificity of BoNT/C is strict and it cannot cleave Sso1. METHODS: Domain swapping and mutational screenings were performed to generate functional chimeras SNAREs of syntaxin-1A and Sso1. Such chimeras are expressed in yeast cells and assessed whether they are susceptible to BoNT/C digestion. RESULTS: The Sso1 and syntaxin-1A chimera (Sso1/STX1A), in which the SNARE domain in Sso1 was replaced with that of syntaxin-1A, was not functional in yeast. The functional incompatibility of Sso1/STX1A was attributable to its accumulation in the ER. We found several mutations that could release Sso1/STX1A from the ER to make the chimera functional in yeast. Yeast cells harboring the mutant chimeras grew similarly to wild-type cells. However, unlike wild-type, yeast harboring the mutant chimeras exhibited a severe growth defect upon expression of BoNT/C. Results of further domain swapping analyses suggest that Sso1 is not digested by BoNT/C because it lacks a binding region to BoNT/C (α-exosite-binding region). CONCLUSIONS: We obtained functional Sso1/STX1A chimeras, which can be applied to a yeast cell-based BoNT/C assay. BoNT/C can recognize these chimeras in a similar manner to syntaxin-1A. GENERAL SIGNIFICANCE: The yeast cell-based BoNT/C assay would be useful to characterize and engineer BoNT/C.


Assuntos
Toxinas Botulínicas , Proteínas Qa-SNARE , Proteínas Recombinantes de Fusão , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Sintaxina 1 , Toxinas Botulínicas/biossíntese , Toxinas Botulínicas/genética , Humanos , Proteínas Qa-SNARE/biossíntese , Proteínas Qa-SNARE/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , Sintaxina 1/biossíntese , Sintaxina 1/genética
4.
Biochim Biophys Acta Mol Basis Dis ; 1864(5 Pt A): 1684-1692, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29408595

RESUMO

Syntaxin 3, also known as STX3, is a protein encoded by the STX3 gene in humans. This protein is one of the fundamental components of the exocytotic machinery required for the docking and fusion of secretory granules with the plasma membrane. The roles of STX3 in human breast cancer remains elusive. Here we report that STX3 acts as an oncogenic protein in human breast cancer. We analyzed the expression of STX3 in 148 patients with breast cancer. The mRNA and protein levels of STX3 are significantly up-regulated in human breast cancer compared with matched adjacent non-cancer tissues. The up-regulation of STX3 is correlated with high disease stage and predicts overall and disease-free survival in patients with breast cancer. Lentivirus-mediated knockdown of STX3 represses in vitro proliferation and colony formation and in vivo growth of breast cancer cells, whereas STX3 overexpression promotes the growth of breast cancer cells in vitro and in vivo. We find that STX3 promotes the proliferation of breast cancer cells by increasing the activation of the Akt-mTOR signaling, and Akt inhibitor Ipatasertib or MK-2206 represses STX3 effects on the growth of breast cancer cells. Further mechanism study shows that STX3 binds to PTEN and increases PTEN ubiquitination and degradation, thus leading to activation of the PI3K-Akt-mTOR signaling. Therefore, STX3 promotes the growth of breast cancer cells by regulating the PTEN-PI3K-Akt-mTOR signaling.


Assuntos
Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Qa-SNARE/biossíntese , Transdução de Sinais , Regulação para Cima , Adulto , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Humanos , Células MCF-7 , Pessoa de Meia-Idade , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Qa-SNARE/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
5.
J Cell Biochem ; 119(5): 3853-3863, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29073720

RESUMO

The retinoblastoma is the most common intraocular malignant tumor in infants and children; it is one of the deadliest forms of cancer due to its limited sensitivity to chemotherapy and radiotherapy. In several cancers, chemoresistance is associated with autophagy induction. Non-coding RNAs, including long non-coding RNAs (lncRNA) and microRNAs (miRNAs) have been reported to regulate physiological activities of the cells, including proliferation, apoptosis, migration, as well as autophagy. MALAT1, a well-established lncRNA acts as an oncogene, promotes cancer proliferation, and metastasis via the stimulation of autophagy. In addition to MALAT1, miR-124, a known tumor suppressor, has also been reported to regulate cell apoptosis and autophagy in dopaminergic neurons. In the present study, we investigated the roles of MALAT1 and miR-124 in the regulation of retinoblastoma cell autophagy through evaluating the changes of autophagy-related proteins. Through direct targeting miR-124, MALAT1 promotes retinoblastoma cell autophagy. Further, we investigated whether Syntaxin 17 (STX17), a Soluble NSF Attachment Protein receptor (SNARE) of the autophagosome, is involved in MALAT1/miR-124 regulation of retinoblastoma cell autophagy, and the underlying mechanism. Taken together, we provided novel experimental and theoretical basis for regulation of retinoblastoma cell autophagy, and potential direction of dealing with autophagy-induced chemoresistance of retinoblastoma, which need further in-depth study.


Assuntos
Autofagia , Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Proteínas de Neoplasias/biossíntese , Proteínas Qa-SNARE/biossíntese , RNA Longo não Codificante/metabolismo , RNA Neoplásico/metabolismo , Retinoblastoma/metabolismo , Linhagem Celular Tumoral , Humanos , MicroRNAs/genética , Proteínas de Neoplasias/genética , Proteínas Qa-SNARE/genética , RNA Longo não Codificante/genética , RNA Neoplásico/genética , Retinoblastoma/genética , Retinoblastoma/patologia
6.
Gene ; 593(1): 110-116, 2016 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-27530212

RESUMO

The purpose of our study was to identify the currently lacking molecular mechanism that accounts for the co-occurrence of two seemingly disparate diseases: psoriasis and type II diabetes. We aimed to investigate a panel of 84 genes related to the diabetic regulatory network in psoriasis (Ps), psoriasis type II diabetes (Ps-T2D), type II diabetes (T2D) and healthy control (HC). We hypothesize that such attempts would provide novel diagnostic markers and/or insights into pathogenesis of the disease. A quantitative Real Time-PCR Human Diabetes RT(2) Profiler PCR Array was chosen to explore the expression profile 84 diabetic genes in study subjects. Statistical analysis was carried out using appropriate software. The analysis revealed three candidate genes GSK3B, PTPN1, STX4 that are differentially expressed in study subjects. GSK3B was highly significant in Ps-T2D (P=0.00018, FR=-26.6), followed by Ps (P=0.0028, FR=-14.5) and T2D groups (P=0.032, FR=-5.9). PTPN1 showed significant association only with PS-T2D (P=0.00027, FR=-8.5). STX4 showed significant association with both Ps (P=0.0002, FR=-20) and Ps-T2D (P=0.0016, FR=-11.2). ACE represents an additional marker that showed suggestive association with Ps (P=0.0079, FR=-9.37). Our study highlights the complex genetics of Ps-T2D and present biomarkers for the development of T2D in Ps cases.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Regulação da Expressão Gênica , Glicogênio Sintase Quinase 3 beta/biossíntese , Proteína Tirosina Fosfatase não Receptora Tipo 1/biossíntese , Psoríase/metabolismo , Proteínas Qa-SNARE/biossíntese , Adulto , Biomarcadores/metabolismo , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/genética , Feminino , Glicogênio Sintase Quinase 3 beta/genética , Humanos , Masculino , Pessoa de Meia-Idade , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Psoríase/classificação , Psoríase/genética , Proteínas Qa-SNARE/genética
7.
J Control Release ; 220(Pt A): 316-328, 2015 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-26546271

RESUMO

Inefficient cytosolic delivery and vector toxicity contribute to the limited use of antisense oligonucleotides (ASOs) and siRNA as therapeutics. As anthrax toxin (Atx) accesses the cytosol, the purpose of this study was to evaluate the potential of disarmed Atx to deliver either ASOs or siRNA. We hypothesized that this delivery strategy would facilitate improved transfection efficiency while eliminating the toxicity seen for many vectors due to membrane destabilization. Atx complex formation with ASOs or siRNA was achieved via the in-frame fusion of either Saccharomyces cerevisiae GAL4 or Homo sapien sapien PKR (respectively) to a truncation of Atx lethal factor (LFn), which were used with Atx protective antigen (PA). Western immunoblotting confirmed the production of: LFN-GAL4, LFn-PKR and PA which were detected at ~45.9 kDa, ~37 kDa, and ~83 kDa respectively and small angle neutron scattering confirmed the ability of PA to form an annular structure with a radius of gyration of 7.0 ± 1.0 nm when placed in serum. In order to form a complex with LFn-GAL4, ASOs were engineered to contain a double-stranded region, and a cell free in vitro translation assay demonstrated that no loss of antisense activity above 30 pmol ASO was evident. The in vitro toxicity of both PA:LFn-GAL4:ASO and PA:LFn-PKR:siRNA complexes was low (IC50>100 µg/mL in HeLa and Vero cells) and subcellular fractionation in conjunction with microscopy confirmed the detection of LFn-GAL4 or LFn-PKR in the cytosol. Syntaxin5 (Synt5) was used as a model target gene to determine pharmacological activity. The PA:LFn-GAL4:ASO complexes had transfection efficiency approximately equivalent to Nucleofection® over a variety of ASO concentrations (24h post-transfection) and during a 72 h time course. In HeLa cells, at 200 pmol ASO (with PA:LFN-GAL4), 5.4 ± 2.0% Synt5 expression was evident relative to an untreated control after 24h. Using 200 pmol ASOs, Nucleofection® reduced Synt5 expression to 8.1 ± 2.1% after 24h. PA:LFn-GAL4:ASO transfection of non- or terminally-differentiated THP-1 cells and Vero cells resulted in 35.2 ± 19.1%, 36.4 ± 1.8% and 22.9 ± 6.9% (respectively) Synt5 expression after treatment with 200 pmol of ASO and demonstrated versatility. Nucleofection® with Stealth RNAi™ siRNA reduced HeLa Synt5 levels to 4.6 ± 6.1% whereas treatment with the PA:LFn-PKR:siRNA resulted in 8.5 ± 3.4% Synt5 expression after 24h (HeLa cells). These studies report for the first time an ASO and RNAi delivery system based upon protein toxin architecture that is devoid of polycations. This system may utilize regulated membrane back-fusion for the cytosolic delivery of ASOs and siRNA, which would account for the lack of toxicity observed. High delivery efficiency suggests further in vivo evaluation is warranted.


Assuntos
Antígenos de Bactérias/genética , Toxinas Bacterianas/genética , Técnicas de Silenciamento de Genes , Oligonucleotídeos Antissenso/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Transfecção/métodos , Animais , Antígenos de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Chlorocebus aethiops , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Células HeLa , Humanos , Oligonucleotídeos Antissenso/biossíntese , Proteínas Qa-SNARE/biossíntese , Proteínas Qa-SNARE/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Células Vero , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
8.
Arch Med Res ; 46(1): 47-53, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25523146

RESUMO

BACKGROUND AND AIMS: Obesity is associated with increased adipose tissue inflammation as well as with the development of type 2 diabetes (T2D). Syntaxin 8 (STX8) is a protein required for the transport of endosomes. In this study we analyzed the relationship of STX8 with the presence of T2D in the context of obesity. METHODS: With this purpose, 21 subjects (seven lean [LN], eight obese normoglycemic [OB-NG] and six obese with type 2 diabetes [OB-T2D]) were included in the study. Gene and protein expression levels of STX8 and GLUT4 were analyzed in visceral adipose tissue (VAT). RESULTS: mRNA (p = 0.008) and protein (p <0.001) expression levels of STX8 were significantly increased in VAT of OB-T2D patients. Moreover, gene expression levels of SLC2A4 (GLUT4) were downregulated (p = 0.002) in VAT of obese patients. We found that STX8 was positively correlated (p <0.05) with fasting glucose concentrations, plasma glucose 2 h after an OGTT and C-reactive protein. Interestingly, the expression of STX8 was negatively correlated (p <0.05) with the expression of SLC2A4 in VAT. CONCLUSIONS: Increased STX8 expression in VAT appears to be associated with the presence of T2D in obese patients through a mechanism that may involve GLUT4.


Assuntos
Diabetes Mellitus Tipo 2/patologia , Transportador de Glucose Tipo 4/biossíntese , Gordura Intra-Abdominal/metabolismo , Obesidade/metabolismo , Proteínas Qa-SNARE/biossíntese , Adulto , Biomarcadores/metabolismo , Glicemia/análise , Proteína C-Reativa/metabolismo , Feminino , Expressão Gênica , Humanos , Inflamação/imunologia , Resistência à Insulina/genética , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética
9.
J Cell Sci ; 125(Pt 16): 3827-39, 2012 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-22573826

RESUMO

Integrins are the primary receptors of cells adhering to the extracellular matrix, and play key roles in various cellular processes including migration, proliferation and survival. The expression and distribution of integrins at the cell surface is controlled by endocytosis and recycling. The present study examines the function of syntaxin 6 (STX6), a t-SNARE located in the trans-Golgi network, in integrin trafficking. STX6 is overexpressed in many types of human cancer. We show that depletion of STX6 inhibits chemotactic cell migration and the delivery of the laminin receptor α3ß1 integrin to the cell surface, whereas STX6 overexpression stimulates chemotactic cell migration, integrin delivery, and integrin-initiated activation of focal adhesion kinase. These data indicate that STX6 plays a rate-limiting role in cell migration and integrin trafficking. In STX6-depleted cells, α3ß1 integrin is accumulated in recycling endosomes that contain the v-SNARE VAMP3. Importantly, we show that STX6 and VAMP3 form a v-/t-SNARE complex, VAMP3 is required in α3ß1 integrin delivery to the cell surface, and endocytosed α3ß1 integrin traffics to both VAMP3 and STX6 compartments. Collectively, our data suggest a new integrin trafficking pathway in which endocytosed integrins are transported from VAMP3-containing recycling endosomes to STX6-containing trans-Golgi network before being recycled to the plasma membrane.


Assuntos
Quimiotaxia/fisiologia , Integrinas/metabolismo , Proteínas Qa-SNARE/biossíntese , Proteína 3 Associada à Membrana da Vesícula/metabolismo , Adesão Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Endossomos/metabolismo , Células HeLa , Humanos , Imuno-Histoquímica , Transporte Proteico , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas SNARE/metabolismo , Transfecção , Proteína 3 Associada à Membrana da Vesícula/genética
10.
Reprod Biol Endocrinol ; 10: 17, 2012 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-22390153

RESUMO

BACKGROUND: Polycystic Ovary Syndrome (PCOS) is an endocrine-metabolic disorder commonly associated with insulin resistance (IR). Previous studies indicate about the expression of molecules involved in the insulin pathway in endometria of women with PCOS-IR. Therefore, the aim of the present study was to evaluate the effect of insulin and testosterone in the expression of these proteins in the endometria and immortal endometrial stromal cell line (T-HESCs). METHODS: We examined the protein levels of Munc18c, PKC zeta, phospho-PKC Zeta, and Syntaxin-4. Protein levels were assessed by Western Blot and/or immunohistochemistry in proliferative endometria (NPE = 6) and in PCOS endometria with insulin resistance (PCOSE-IR = 6). We also evaluated whether high concentrations of insulin (100 nM) and/or testosterone (100 nM), during a 24 h stimulatory period, affected the expression of these proteins in an immortal endometrial stromal cell line (T-HESCs). Once stimulated, proteins were extracted from cells and were assessed by Western Blot analysis. Immunocytochemistry was performed to detect AR in T-HESC cells. RESULTS: Western Blot data showed decreased expression (p < 0,05) of Munc18c and phospho-PKC Zeta in PCOS-IR endometria (PCOSE-IR) with respect to the control (NPE). In the in vitro study, Western Blot analysis showed decreased levels of Munc18c, PKC Zeta and phospho-PKC Zeta with the different hormonal treatments when compared to the control condition (no hormonal stimulation) (p < 0,05). The AR was present in the endometrial stromal cell line (T-HESC). CONCLUSION: The conditions of hyperinsulinism and hyperandrogenism present in PCOS-IR patients modulate the expression and/or phosphorylation of the proteins involved in the insulin pathway at the endometrial level. These data extend to the T-HESCs cells results, where insulin and testosterone exert an effect on both the expression and phosphorylation of proteins present in the pathway.


Assuntos
Endométrio/metabolismo , Insulina/metabolismo , Proteínas Munc18/biossíntese , Síndrome do Ovário Policístico/metabolismo , Proteína Quinase C/biossíntese , Proteínas Qa-SNARE/biossíntese , Adulto , Linhagem Celular , Feminino , Humanos , Receptores Androgênicos/biossíntese
11.
Planta ; 235(4): 841-50, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22089884

RESUMO

The bacterial merC gene from the Tn21-encoded mer operon is a potential molecular tool for improving the efficiency of metal phytoremediation. Arabidopsis SNARE molecules, including SYP111, SYP121, and AtVAM3 (SYP22), were attached to the C-terminus of MerC to target the protein to various organelles. The subcellular localization of transiently expressed GFP-fused MerC-SYP111, MerC-SYP121, and MerC-AtVAM3 was examined in Arabidopsis suspension-cultured cells. We found that GFP-MerC-SYP111 and GFP-MerC-SYP121 localized to the plasma membrane, whereas GFP-AtVAM3 localized to the vacuolar membranes. These results demonstrate that SYP111/SYP121 and AtVAM3 target foreign molecules to the plasma membrane and vacuolar membrane, respectively. To enhance the efficiency and potential of plants to sequester and accumulate cadmium from contaminated sites, transgenic Arabidopsis plants expressing MerC, MerC-SYP111, MerC-SYP121, or MerC-AtVAM3 were generated. The transgenic plants that expressed MerC, MerC-SYP121, or MerC-AtVAM3 appeared to be normal, whereas the transgenic that expressed MerC-SYP111 exhibited severe growth defects. The transgenic plants expressing merC-SYP121 were more resistant to cadmium than the wild type and accumulated significantly more cadmium. Thus, the expression of MerC-SYP121 in the plant plasma membrane may provide an ecologically compatible approach for the phytoremediation of cadmium pollution.


Assuntos
Proteínas de Arabidopsis/biossíntese , Arabidopsis/metabolismo , Cádmio/metabolismo , Proteínas de Transporte de Cátions/biossíntese , Proteínas Qa-SNARE/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Proteínas SNARE/biossíntese , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Biodegradação Ambiental , Proteínas de Transporte de Cátions/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Engenharia Genética , Variação Genética , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas Qa-SNARE/genética , Proteínas Recombinantes de Fusão/genética , Proteínas SNARE/genética , Transformação Genética
12.
Behav Genet ; 40(5): 680-93, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20563839

RESUMO

Consolidation of aversive operant conditioning into long-term memory (LTM) requires CREB-dependent de novo protein synthesis. The newly synthesized proteins are distributed to the synapses in neurons that are involved in memory formation and storage. Accumulating evidence indicates that the presynaptic release mechanisms also play a role in long-term synaptic plasticity. Our understanding of whether the presynaptic proteins undergo de novo synthesis during long-term memory formation is limited. In this study, we investigated the involvement of syntaxin-1, a presynaptic exocytotic protein, and dynamin-1, an endocytotic protein, in the formation of long-term memory. We took advantage of a well-established aversive operant conditioning model of aerial respiratory behavior in the fresh water pond snail Lymnaea stagnalis, and demonstrated that the LTM formation is associated with increased expression of syntaxin-1 and dynamin-1, coincident with elevated levels of CREB1. Partial knockdown of CREB1 gene by double stranded RNA inhibition (dsRNAi) prior to operant conditioning prevented snails from memory consolidation, and reduced the expression of syntaxin-1 and dynamin-1 at both mRNA and protein levels. These findings suggest that CREB1-mediated gene expression is required for the LTM-induced up-regulation of synaptic proteins, syntaxin-1 and dynamin-1, in L. stagnalis. Our study thus offers new insights into the molecular mechanisms that mediate CREB1-dependent long-term memory formation.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Dinamina I/biossíntese , Memória/fisiologia , Terminações Pré-Sinápticas/fisiologia , Proteínas Qa-SNARE/biossíntese , Animais , Condicionamento Clássico/fisiologia , AMP Cíclico , Lymnaea , Neurônios/fisiologia , Interferência de RNA/fisiologia , RNA Mensageiro/metabolismo , Sinapses/fisiologia
13.
Neuroscience ; 166(3): 832-41, 2010 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-20060037

RESUMO

Ribbon synapses of the vertebrate retina are specialized synapses that release neurotransmitter by synaptic vesicle exocytosis in a manner that is proportional to the level of depolarization of the cell. This release property is different from conventional neurons, in which the release of neurotransmitter occurs as a short-lived burst triggered by an action potential. Synaptic vesicle exocytosis is a calcium regulated process that is dependent on a set of interacting synaptic proteins that form the so-called SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex. Syntaxin 3B has been identified as a specialized SNARE molecule in ribbon synapses of the rodent retina. However, the best physiologically-characterized neuron that forms ribbon-style synapses is the rod-dominant or Mb1 bipolar cell of the goldfish retina. We report here the molecular characterization of syntaxin 3B from the goldfish retina. Using a combination of reverse transcription (RT) polymerase chain reaction (PCR) and immunostaining with a specific antibody, we show that syntaxin 3B is highly enriched in the plasma membrane of bipolar cell synaptic terminals of the goldfish retina. Using membrane capacitance measurements we demonstrate that a peptide derived from goldfish syntaxin 3B inhibits synaptic vesicle exocytosis. These experiments demonstrate that syntaxin 3B is an important factor for synaptic vesicle exocytosis in ribbon synapses of the vertebrate retina.


Assuntos
Exocitose , Proteínas Qa-SNARE/fisiologia , Retina/fisiologia , Vesículas Sinápticas/fisiologia , Animais , Membrana Celular/metabolismo , Carpa Dourada , Técnicas de Patch-Clamp , Proteínas Qa-SNARE/biossíntese , Células Bipolares da Retina/metabolismo
14.
Behav Genet ; 39(2): 183-91, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19107586

RESUMO

C57BL/6J and DBA/2J inbred mouse strains have been extensively studied for the genetic dissection of alcohol-related phenotypes. We have previously found Syntaxin 12 to be associated with alcohol preference in C57BL/6J and DBA/2J due to its strain-specific and ethanol responsive expression in the male brain. In the current study, we combined genetic and expression analyses to assess the segregation of Syntaxin 12 c.*1370G>A polymorphism with its strain-specific expression and alcohol preference in an F (2) population (N = 427) derived from C57BL/6J and DBA/2J strains. Syntaxin 12 c.*1370G>A polymorphism was found to segregate with alcohol preference in the B6D2F2 population and a correlation was identified between Syntaxin 12 expression and alcohol preference in the selected B6D2F2 males (r = -0.473, r (2) = 0.22). We followed up our analysis in the BXD RI lines using resources from WebQTL and the Mouse Phenome Database. Our study detected significant associations of Syntaxin 12 molecular variants with its level of expression and alcohol preference in B6D2F2 males. Overall, our findings support a role for Syntaxin 12 as a potential contributor to alcohol preference in mice.


Assuntos
Consumo de Bebidas Alcoólicas/genética , Alcoolismo/genética , Polimorfismo Genético , Proteínas Qa-SNARE/biossíntese , Proteínas Qa-SNARE/fisiologia , Animais , Encéfalo/metabolismo , Feminino , Regulação da Expressão Gênica , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Fenótipo , Síndrome de Abstinência a Substâncias/genética
15.
Traffic ; 9(10): 1629-52, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18764818

RESUMO

Overexpression of the Golgi and endoplasmic reticulum (ER) syntaxins SYP31 and SYP81 strongly inhibits constitutive secretion. By comparing the secreted reporter alpha-amylase with the ER-retained reporter alpha-amylase-HDEL, it was concluded that SYP81 overexpression inhibits both retrograde and anterograde transport, while SYP31 overexpression mainly affected anterograde transport. Of the other interacting SNAREs investigated, only the overexpression of MEMB11 led to an inhibition of protein secretion. Although the position of a fluorescent tag does not influence the correct localization of the fusion protein, only N-terminal-tagged SYP31 retained the ability of the untagged SNARE to inhibit transport. C-terminal-tagged SYP31 failed to exhibit this effect. Overexpression of both wild-type and N-terminal-tagged syntaxins caused standard Golgi marker proteins to redistribute into the ER. Nevertheless, green fluorescent protein (GFP)-SYP31 was still visible as fluorescent punctae, which, unlike SYP31-GFP, were resistant to brefeldin A treatment. Immunogold electron microscopy showed that endogenous SYP81 is not only present at the ER but also in the cis Golgi, indicating that this syntaxin cycles between these two organelles. However, when expressed at non-inhibitory levels, YFP-SYP81 was seen to locate principally to subdomains of the ER. These punctate structures were physically separated from the Golgi, suggesting that they might possibly reflect the position of ER import sites.


Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Nicotiana/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Qa-SNARE/metabolismo , Via Secretória/fisiologia , Clonagem Molecular , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/genética , Retículo Endoplasmático/fisiologia , Complexo de Golgi/enzimologia , Complexo de Golgi/genética , Complexo de Golgi/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Microscopia Imunoeletrônica , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/fisiologia , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Plasmídeos , Transporte Proteico/fisiologia , Protoplastos/enzimologia , Protoplastos/metabolismo , Proteínas Qa-SNARE/biossíntese , Proteínas Qa-SNARE/genética , Nicotiana/enzimologia , Nicotiana/genética , Nicotiana/fisiologia , alfa-Amilases/metabolismo
16.
Biochem Biophys Res Commun ; 371(3): 366-70, 2008 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-18439908

RESUMO

Synip and Stx4 complex plays a key role in GLUT4 vesicle trafficking and fusion with plasma membrane. The interaction of Synip with Stx4 prevents interaction of VAMP2 located in GLUT4 vesicle with Stx4 in basal state. Insulin induces the dissociation of the Synip and Stx4 complex, and then triggers VAMP2 to interact with Stx4 to form the SNARE complex, thus promoting the vesicle fusion. In this report, we adopt a novel system for co-expression of the Synip and Stx4 by using two common vectors pGEX6p-1 and pET28a(+) to investigate their expression, purification, and interaction. Through this co-expression system, we successfully co-expressed the Synip and Stx4 complex with high yield, and co-purified at an approximate 1:1 molar ratio with high purity (95%). We also demonstrate that the 1-28 residues of Stx4 are dispensable for interaction with Synip using this co-expression system.


Assuntos
Proteínas Qa-SNARE/biossíntese , Proteínas Qa-SNARE/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas de Transporte Vesicular/biossíntese , Proteínas de Transporte Vesicular/isolamento & purificação , Animais , Linhagem Celular , Cromatografia em Gel , Vetores Genéticos/genética , Métodos , Camundongos , Estrutura Terciária de Proteína , Proteínas Qa-SNARE/genética , Proteínas Recombinantes/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteínas de Transporte Vesicular/genética
17.
J Immunol ; 179(6): 3397-401, 2007 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-17785771

RESUMO

Little is known about the regulatory roles of specific soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in cytotoxic lymphocytes. Recent information suggests that mutations in the SNARE protein syntaxin 11 result in a form of familial hemophagocytic lymphohistiocytosis (FHL). Because genetic abnormalities in key granule components (e.g., perforin) or in regulators of secretion (e.g., Munc13-4) underlie the other identified forms of FHL, we assessed whether syntaxin 11 might also serve a related regulatory role. We determined that syntaxin 11 is expressed in NK cells and activated CTLs and is located in discrete membrane-associated structures in the cytoplasm. Enhanced expression of syntaxin 11 augments the secretion and killing of tumor targets, and suppression of syntaxin 11 expression inhibits these functions. Our data identify and characterize a role for syntaxin 11 in granule exocytosis and in the generation of cell-mediated killing. These results also provide new insights on the mechanisms of hemopoietic dysregulation in FHL.


Assuntos
Testes Imunológicos de Citotoxicidade , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Proteínas Qa-SNARE/fisiologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Células Clonais , Citoplasma/metabolismo , Grânulos Citoplasmáticos/metabolismo , Exocitose/imunologia , Humanos , Células Jurkat , Células K562 , Proteínas Qa-SNARE/biossíntese , Linfócitos T Citotóxicos/citologia
18.
Acta Histochem ; 109(6): 454-60, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17485106

RESUMO

Mast cells (MCs) play a crucial role in inflammatory reactions. Their presence and number in the peritoneal cavity is important to overcome and enhance resistance to peritoneal infection. When MCs are activated they release a variety of biological mediators from their granules, such as histamine, that contribute to the appropriate and rapid local immune response. Granular content is released using a process of compound exocytosis, also termed degranulation. SNAP-23 and syntaxin 4 are plasma membrane proteins involved in degranulation of rat MCs. Their presence, however, has not been studied in MCs of other rodent species. The aim of the present study was to investigate using immunocytochemistry whether SNAP-23 and syntaxin 4 are present in peritoneal MCs of the mouse and hamster. In addition, the diameter, percentage and histamine content of these cells were also analyzed. Our results demonstrate that SNAP-23 and syntaxin 4 are present in the mouse and hamster peritoneal MCs, suggesting that proteins involved in the secretory process in MCs are conserved among species. Likewise, we conclude that peritoneal MCs of mouse and hamster are heterogeneous in size, percentage and histamine content.


Assuntos
Mastócitos/metabolismo , Peritônio/metabolismo , Proteínas Qa-SNARE/biossíntese , Proteínas de Transporte Vesicular/biossíntese , Animais , Tamanho Celular , Cricetinae , Imuno-Histoquímica , Masculino , Mesocricetus , Camundongos , Camundongos Endogâmicos BALB C , Peritônio/citologia , Ratos , Ratos Endogâmicos Lew , Especificidade da Espécie
19.
Mol Cancer Ther ; 5(10): 2468-73, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17041090

RESUMO

Prostate-specific membrane antigen (PSMA) is a protein up-regulated in the vast majority of prostate cancers. Antibodies to PSMA have proved highly specific for prostate cancer cells, and the therapeutic potential of such antibodies is currently being assessed in clinical trials. We have previously shown that PSMA at the cell surface of polarized epithelial cells is predominantly expressed at the apical plasma membrane and that microtubule depolymerization abolishes apical PSMA targeting. In the current report, we implicate a functional role for a target membrane soluble N-ethylmaleimide-sensitive factor adaptor protein receptor, syntaxin 3, in the microtubule-dependent apical targeting of PSMA. PSMA and syntaxin 3 are similarly localized to the apical plasma membrane of the prostatic epithelium and Madin-Darby canine kidney cells. Introduction of a point mutation into syntaxin 3 abolishes its polarized distribution and causes PSMA to be targeted in a nonpolarized fashion. Additionally, treatment of polarized Madin-Darby canine kidney cells with vinblastine, a microtubule depolymerizing chemotherapeutic agent, causes both syntaxin 3 and PSMA to redistribute in a nonpolarized fashion. However, following treatment with the microtubule stabilizing chemotherapeutic agent Taxotere, both syntaxin 3 and PSMA continue to localize in a polarized manner at the apical plasma membrane. Thus, microtubule depolymerizing and stabilizing chemotherapeutic drugs might exact similar cytotoxic effects but have disparate effects on protein targeting. This phenomenon might have important clinical implication, especially related to antibody-mediated immunotherapy, and could potentially be exploited for therapeutic benefit.


Assuntos
Antígeno Prostático Específico/biossíntese , Proteínas Qa-SNARE/biossíntese , Moduladores de Tubulina/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Docetaxel , Cães , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Humanos , Rim/citologia , Masculino , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Nocodazol/farmacologia , Mutação Puntual , Próstata/efeitos dos fármacos , Próstata/metabolismo , Antígeno Prostático Específico/imunologia , Proteínas Qa-SNARE/genética , Taxoides/farmacologia , Vimblastina/farmacologia
20.
Neurochem Int ; 48(6-7): 596-603, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16516346

RESUMO

A family of high-affinity transporters controls the extracellular concentration of glutamate in the brain, ensuring appropriate excitatory signaling and preventing excitotoxicity. There is evidence that one of the neuronal glutamate transporters, EAAC1, is rapidly recycled on and off the plasma membrane with a half-life of no more than 5-7 min in both C6 glioma cells and cortical neurons. Syntaxin 1A has been implicated in the trafficking of several neurotransmitter transporters and in the regulation of EAAC1, but it has not been determined if this SNARE protein is required for EAAC1 trafficking. Expression of two different sets of SNARE proteins was examined in C6 glioma with Western blotting. These cells did not express syntaxin 1A, vesicle-associated membrane protein-1 (VAMP1), or synaptosomal-associated protein of 25 kDa (SNAP-25), but did express a family of SNARE proteins that has been implicated in glucose transporter trafficking, including syntaxin 4, vesicle-associated membrane protein-2 (VAMP2), and synaptosomal-associated protein of 23 kDa (SNAP-23). cDNAs encoding variants of SNAP-23 were co-transfected with Myc-tagged EAAC1 to determine if SNAP-23 function was required for maintenance of EAAC1 surface expression. Expression of a dominant-negative variant of SNAP-23 that lacks a domain required for SNARE complex assembly decreased the fraction of EAAC1 found on the cell surface and decreased total EAAC1 expression, while two control constructs had no effect. The dominant-negative variant of SNAP-23 also slowed the rate of EAAC1 delivery to the plasma membrane. These data strongly suggest that syntaxin 1A is not required for EAAC1 trafficking and provide evidence that SNAP-23 is required for constitutive recycling of EAAC1.


Assuntos
Transportador 3 de Aminoácido Excitatório/biossíntese , Neurônios/metabolismo , Proteínas SNARE/biossíntese , Proteínas de Transporte Vesicular/fisiologia , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Células Cultivadas , Transportador 3 de Aminoácido Excitatório/metabolismo , Transporte Proteico , Proteínas Qa-SNARE/biossíntese , Ratos , Proteínas SNARE/genética , Proteína 25 Associada a Sinaptossoma/biossíntese , Sintaxina 1/biossíntese , Proteína 1 Associada à Membrana da Vesícula/biossíntese , Proteína 2 Associada à Membrana da Vesícula/biossíntese , Proteínas de Transporte Vesicular/biossíntese , Proteínas de Transporte Vesicular/genética
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