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1.
Autophagy ; 20(2): 329-348, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-37776538

RESUMO

Chemotherapeutic resistance is one of the most common reasons for poor prognosis of patients with nasopharyngeal carcinoma (NPC). We found that CENPN can promote the growth, proliferation and apoptosis resistance of NPC cells, but its relationship with chemotherapeutic resistance in NPC is unclear. Here we verified that the CENPN expression level in NPC patients was positively correlated with the degree of paclitaxel (PTX) resistance and a poor prognosis through analysis of clinical cases. VAMP8 expression was significantly increased after knockdown of CENPN by transcriptome sequencing. We found in cell experiments that CENPN inhibited macroautophagy/autophagy and VAMP8 expression and significantly increased PTX resistance. Overexpression of CENPN reduced the inhibitory effects of PTX on survival, cell proliferation, cell cycle progression and apoptosis resistance in NPC cells by inhibiting autophagy. In turn, knockdown of CENPN can affect the phenotype of NPC cells by increasing autophagy to achieve PTX sensitization. Sequential knockdown of CENPN and VAMP8 reversed the PTX-sensitizing effect of CENPN knockdown alone. Experiments in nude mice confirmed that knockdown of CENPN can increase VAMP8 expression, enhance autophagy and increase the sensitivity of NPC cells to PTX. Mechanistic studies showed that CENPN inhibited the translocation of p-CREB into the nucleus of NPC cells, resulting in the decreased binding of p-CREB to the VAMP8 promoter, thereby inhibiting the transcription of VAMP8. These results demonstrate that CENPN may be a marker for predicting chemotherapeutic efficacy and a potential target for inducing chemosensitization to agents such as PTX.Abbreviations: 3-MA: 3-methyladenine; ATG5: autophagy related 5; CENPN: centromere protein N; CQ: chloroquine; CREB: cAMP responsive element binding protein; ChIP: chromatin immunoprecipitation assay; IC50: half-maximal inhibitory concentration; LAMP2A: lysosomal associated membrane protein 2A; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NPC: nasopharyngeal carcinoma; NPG: nasopharyngitis; oeCENPN: overexpressed CENPN; PTX: paclitaxel; RAPA: rapamycin; RNA-seq: transcriptome sequencing; shCENPN: small hairpin RNA expression vector targeting the human CENPN gene; shCENPN-shVAMP8: sequential knockdown targeting the human CENPN gene and VAMP8 gene; shVAMP8: small hairpin RNA expression vector targeting the human VAMP8 gene; TEM: transmission electron microscopy; TIR: tumor inhibitory rate; VAMP8: vesicle associated membrane protein 8.


Assuntos
Neoplasias Nasofaríngeas , Paclitaxel , Animais , Camundongos , Humanos , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/metabolismo , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Neoplasias Nasofaríngeas/tratamento farmacológico , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/metabolismo , Camundongos Nus , Autofagia/genética , Linhagem Celular Tumoral , RNA Interferente Pequeno/farmacologia , Proteínas R-SNARE/metabolismo , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/farmacologia
2.
Int J Mol Sci ; 24(24)2023 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-38139155

RESUMO

The vesicle-associated membrane protein 7 (VAMP7) is a SNARE protein of the longin family involved in a wide range of subcellular trafficking events, including neurite sprouting and elongation. The expression of the human gene SYBL1, encoding VAMP7, is finely regulated by alternative splicing. Among the minor isoforms identified so far, VAMP7j is the one most expressed and modulated in the human brain. Therefore, we focused on gaining functional evidence on VAMP7j, which lacks a functional SNARE motif but retains both the longin and transmembrane domains. In human SH-SY5Y cells, we found VAMP7j to modulate neuritogenesis by mediating transport of L1CAM toward the plasma membrane, in a fashion regulated by phosphorylation of the longin domain. VAMP7-mediated regulation of L1CAM trafficking seems at least to differentiate humans from rats, with VAMP7j CNS expression being restricted to primates, including humans. Since L1CAM is a central player in neuritogenesis and axon guidance, these findings suggest the species-specific splicing of SYBL1 is among the fine tuners of human neurodevelopmental complexity.


Assuntos
Molécula L1 de Adesão de Célula Nervosa , Neuroblastoma , Animais , Humanos , Ratos , Membrana Celular/metabolismo , Molécula L1 de Adesão de Célula Nervosa/genética , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Neuroblastoma/metabolismo , Crescimento Neuronal , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Proteínas SNARE/metabolismo
3.
J Immunol ; 211(8): 1203-1215, 2023 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-37638825

RESUMO

The induction of CTL responses by vaccines is important to combat infectious diseases and cancer. Biodegradable poly(lactic-co-glycolic acid) (PLGA) microspheres and synthetic long peptides are efficiently internalized by professional APCs and prime CTL responses after cross-presentation of Ags on MHC class I molecules. Specifically, they mainly use the cytosolic pathway of cross-presentation that requires endosomal escape, proteasomal processing, and subsequent MHC class I loading of Ags in the endoplasmic reticulum (ER) and/or the endosome. The vesicle SNARE protein Sec22b has been described as important for this pathway by mediating vesical trafficking for the delivery of ER-derived proteins to the endosome. As this function has also been challenged, we investigated the role of Sec22b in cross-presentation of the PLGA microsphere-encapsulated model Ag OVA and a related synthetic long peptide. Using CRISPR/Cas9-mediated genome editing, we generated Sec22b knockouts in two murine C57BL/6-derived APC lines and found no evidence for an essential role of Sec22b. Although pending experimental evidence, the target SNARE protein syntaxin 4 (Stx4) has been suggested to promote cross-presentation by interacting with Sec22b for the fusion of ER-derived vesicles with the endosome. In the current study, we show that, similar to Sec22b, Stx4 knockout in murine APCs had very limited effects on cross-presentation under the conditions tested. This study contributes to characterizing cross-presentation of two promising Ag delivery systems and adds to the discussion about the role of Sec22b/Stx4 in related pathways. Our data point toward SNARE protein redundancy in the cytosolic pathway of cross-presentation.


Assuntos
Antígenos , Apresentação Cruzada , Proteínas Qa-SNARE , Proteínas R-SNARE , Animais , Camundongos , Apresentação de Antígeno , Antígenos/metabolismo , Células Dendríticas , Endossomos/metabolismo , Microesferas , Peptídeos/metabolismo , Proteínas Qa-SNARE/metabolismo , Proteínas R-SNARE/metabolismo
4.
Cancer Biol Ther ; 24(1): 2230641, 2023 12 31.
Artigo em Inglês | MEDLINE | ID: mdl-37405957

RESUMO

Osteosarcoma is a highly metastatic malignant bone tumor, necessitating the development of new treatments to target its metastasis. Recent studies have revealed the significance of VAMP8 in regulating various signaling pathways in various types of cancer. However, the specific functional role of VAMP8 in osteosarcoma progression remains unclear. In this study, we observed a significant downregulation of VAMP8 in osteosarcoma cells and tissues. Low levels of VAMP8 in osteosarcoma tissues were associated with patients' poor prognosis. VAMP8 inhibited the migration and invasion capability of osteosarcoma cells. Mechanically, we identified DDX5 as a novel interacting partner of VAMP8, and the conjunction of VAMP8 and DDX5 promoted the degradation of DDX5 via the ubiquitin-proteasome system. Moreover, reduced levels of DDX5 led to the downregulation of ß-catenin, thereby suppressing the epithelial-mesenchymal transition (EMT). Additionally, VAMP8 promoted autophagy flux, which may contribute to the suppression of osteosarcoma metastasis. In conclusion, our study anticipated that VAMP8 inhibits osteosarcoma metastasis by promoting the proteasomal degradation of DDX5, consequently inhibiting WNT/ß-catenin signaling and EMT. Dysregulation of autophagy by VAMP8 is also implicated as a potential mechanism. These findings provide new insights into the biological nature driving osteosarcoma metastasis and highlight the modulation of VAMP8 as a potential therapeutic strategy for targeting osteosarcoma metastasis.


Assuntos
Neoplasias Ósseas , Osteossarcoma , Humanos , beta Catenina/metabolismo , Linhagem Celular Tumoral , Via de Sinalização Wnt , Osteossarcoma/patologia , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Movimento Celular , Proliferação de Células , Proteínas R-SNARE/metabolismo
5.
Mol Plant Pathol ; 24(9): 1154-1167, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37278116

RESUMO

The soilborne bacterial pathogen Ralstonia solanacearum is one of the most destructive plant pathogens worldwide, and its infection process involves the manipulation of numerous plant cellular functions. In this work, we found that the R. solanacearum effector protein RipD partially suppressed different levels of plant immunity triggered by R. solanacearum elicitors, including specific responses triggered by pathogen-associated molecular patterns and secreted effectors. RipD localized in different subcellular compartments in plant cells, including vesicles, and its vesicular localization was enriched in cells undergoing R. solanacearum infection, suggesting that this specific localization may be particularly relevant during infection. Among RipD-interacting proteins, we identified plant vesicle-associated membrane proteins (VAMPs). We also found that overexpression of Arabidopsis thaliana VAMP721 and VAMP722 in Nicotiana benthamiana leaves promoted resistance to R. solanacearum, and this was abolished by the simultaneous expression of RipD, suggesting that RipD targets VAMPs to contribute to R. solanacearum virulence. Among proteins secreted in VAMP721/722-containing vesicles, CCOAOMT1 is an enzyme required for lignin biosynthesis, and mutation of CCOAOMT1 enhanced plant susceptibility to R. solanacearum. Altogether our results reveal the contribution of VAMPs to plant resistance against R. solanacearum and their targeting by a bacterial effector as a pathogen virulence strategy.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Ralstonia solanacearum , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Doenças das Plantas/microbiologia , Plantas/metabolismo , Nicotiana/microbiologia , Imunidade Vegetal/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo
6.
Biochem Biophys Res Commun ; 665: 10-18, 2023 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-37148741

RESUMO

Autophagy has bidirectional functions in cancer by facilitating cell survival and death in a context-dependent manner. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are a large family of proteins essential for numerous biological processes, including autophagy; nevertheless, their potential function in cancer malignancy remains unclear. Here, we explored the gene expression patterns of SNAREs in tissues of patients with colorectal cancer (CRC) and discovered that SEC22B expression, a vesicle SNARE, was higher in tumor tissues than in normal tissues, with a more significant increase in metastatic tissues. Interestingly, SEC22B knockdown dramatically decreased CRC cell survival and growth, especially under stressful conditions, such as hypoxia and serum starvation, and decreased the number of stress-induced autophagic vacuoles. Moreover, SEC22B knockdown successfully attenuated liver metastasis in a CRC cell xenograft mouse model, with histological signs of decreased autophagic flux and proliferation within cancer cells. Together, this study posits that SEC22B plays a crucial role in enhancing the aggressiveness of CRC cells, suggesting that SEC22B might be an attractive therapeutic target for CRC.


Assuntos
Neoplasias Colorretais , Proteínas SNARE , Animais , Humanos , Camundongos , Autofagossomos/metabolismo , Autofagia/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Proteínas R-SNARE/metabolismo , Proteínas SNARE/metabolismo
7.
Medicine (Baltimore) ; 102(15): e33546, 2023 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-37058019

RESUMO

YKT6, as a Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein with vesicle trafficking, plays an essential role in the development and progression of tumor. However, the gene of YKT6 has not been fully assessed in pan-cancer studies. We aim to investigate the gene of YKT6 across 33 different types of tumor by using the Cancer Genome Atlas, Gene Expression Omnibus database, and other several kinds of bioinformatic tools. YKT6 is significantly up-regulated in most tumors, and we found that overexpression of YKT6 is positively associated with poor prognosis of overall survival and poor disease-free survival prognosis in several tumors, such as Adrenocortical carcinoma, Bladder Urothelial Carcinoma, Head and Neck squamous cell carcinoma. We also detected distinct associations exist between YKT6 and tumor mutational burden or microsatellite instability with tumors. YKT6 expression was positively related to cancer-associated fibroblasts for TCGA tumors of colon adenocarcinoma and LGG. Furthermore, we discovered a significantly positively correlation between YKT6 expression and endothelial cell in tumors of colon adenocarcinoma, HNSC-HPV+, OV, READ and THCA. While a negative relationship was obtained between YKT6 expression and endothelial cell in KIRC. Moreover, "Syntaxin binding," "SNARE complex," "vesicle fusion" and "DNA replication" are involved in the influence of YKT6 on tumor pathogenesis. Our pan-cancer analysis offers a deep comprehending the gene of YKT6 in tumoeigenesis from viewpoint of clinical tumor samples.


Assuntos
Adenocarcinoma , Carcinoma de Células de Transição , Neoplasias do Colo , Neoplasias da Bexiga Urinária , Humanos , Proteínas R-SNARE/genética , Proteínas SNARE
8.
Biochem Pharmacol ; 212: 115549, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37060961

RESUMO

Ovarian cancer (OC) is the most common gynecological malignancy. Chemotherapy failure is a major challenge in OC treatment. Targeting autophagy is a promising strategy to enhance the cytotoxicity of chemotherapeutic agents. In this study, we found that costunolide (CTD) inhibits autophagic flux and exhibits high therapeutic efficacy for OC treatment in an in vitro model. Mechanistically, CTD inactivates AMPK/mTOR signaling to inhibit autophagy initiation at the early stage and blocks mTORC1-dependent autophagosome-lysosome fusion at the late stage during autophagy by disrupting SNARE complex (STX17-SNAP29-VAMP8) formation, resulting in lethal autophagy arrest in OC cells. Furthermore, CTD sensitizes OC cells to cisplatin (CDDP) by blocking CDDP-induced autophagy both in vitro and in vivo. Together, our data provide novel mechanistic insights into CTD-induced autophagy arrest and suggest a new autophagy inhibitor for effective treatment of OC.


Assuntos
Cisplatino , Neoplasias Ovarianas , Humanos , Feminino , Cisplatino/farmacologia , Cisplatino/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Lisossomos/metabolismo , Transdução de Sinais , Autofagia , Serina-Treonina Quinases TOR/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qb-SNARE/farmacologia , Proteínas Qc-SNARE/metabolismo , Proteínas Qc-SNARE/farmacologia , Proteínas R-SNARE/metabolismo
9.
Sci Adv ; 9(5): eade9585, 2023 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-36724221

RESUMO

Enhancing the intracellular labile iron pool (LIP) represents a powerful, yet untapped strategy for driving ferroptotic death of cancer cells. Here, we show that NRF2 maintains iron homeostasis by controlling HERC2 (E3 ubiquitin ligase for NCOA4 and FBXL5) and VAMP8 (mediates autophagosome-lysosome fusion). NFE2L2/NRF2 knockout cells have low HERC2 expression, leading to a simultaneous increase in ferritin and NCOA4 and recruitment of apoferritin into the autophagosome. NFE2L2/NRF2 knockout cells also have low VAMP8 expression, which leads to ferritinophagy blockage. Therefore, deletion of NFE2L2/NRF2 results in apoferritin accumulation in the autophagosome, an elevated LIP, and enhanced sensitivity to ferroptosis. Concordantly, NRF2 levels correlate with HERC2 and VAMP8 in human ovarian cancer tissues, as well as ferroptosis resistance in a panel of ovarian cancer cell lines. Last, the feasibility of inhibiting NRF2 to increase the LIP and kill cancer cells via ferroptosis was demonstrated in preclinical models, signifying the impact of NRF2 inhibition in cancer treatment.


Assuntos
Ferroptose , Neoplasias Ovarianas , Humanos , Feminino , Ferroptose/genética , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Apoferritinas , Ferro/metabolismo , Homeostase , Ubiquitina-Proteína Ligases/metabolismo , Proteínas R-SNARE/metabolismo
10.
Biol Pharm Bull ; 45(11): 1609-1615, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36328496

RESUMO

Autophagy is a highly conserved intracellular degrading system and its dysfunction is considered related to the cause of neurodegenerative disorders. A previous study showed that the inhibition of endocytosis transport attenuates soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein transport to lysosomes and block autophagy. The other studies demonstrated oxidative stress, one of the inducers of neurodegenerative diseases inhibits endocytosis transport. Thus, we hypothesized that oxidative stress-induced endocytosis inhibition causes alteration of SNARE protein transport to lysosomes and impairs autophagy. Here, we demonstrated that oxidative stress inhibits endocytosis and decreased the lysosomal localization of VAMP8, one of the autophagy-related SNARE proteins in a human neuroblastoma cell line. Moreover, this oxidative stress induction blocked the autophagosome-lysosome fusion step. Since we also observed decreased lysosomal localization of VAMP8 and inhibition of autophagosome-lysosome fusion in endocytosis inhibitor-treated cells, oxidative stress may inhibit VAMP8 trafficking by suppressing endocytosis and impair autophagy. Our findings suggest that oxidative stress-induced inhibition of VAMP8 trafficking to lysosomes is associated with the development of neurodegenerative diseases due to the blocked autophagosome-lysosome fusion, and may provide a new therapeutic target for restoring the autophagic activity.


Assuntos
Autofagia , Lisossomos , Humanos , Autofagia/fisiologia , Lisossomos/metabolismo , Fusão de Membrana , Estresse Oxidativo , Proteínas R-SNARE/metabolismo , Proteínas SNARE/metabolismo , Transporte Biológico
11.
J Neurosci ; 42(42): 8019-8037, 2022 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-36261266

RESUMO

Mutations in the gene encoding vesicle-associated membrane protein B (VAPB) cause a familial form of amyotrophic lateral sclerosis (ALS). Expression of an ALS-related variant of vapb (vapbP58S ) in Drosophila motor neurons results in morphologic changes at the larval neuromuscular junction (NMJ) characterized by the appearance of fewer, but larger, presynaptic boutons. Although diminished microtubule stability is known to underlie these morphologic changes, a mechanism for the loss of presynaptic microtubules has been lacking. By studying flies of both sexes, we demonstrate the suppression of vapbP58S -induced changes in NMJ morphology by either a loss of endoplasmic reticulum (ER) Ca2+ release channels or the inhibition Ca2+/calmodulin (CaM)-activated kinase II (CaMKII). These data suggest that decreased stability of presynaptic microtubules at vapbP58S NMJs results from hyperactivation of CaMKII because of elevated cytosolic [Ca2+]. We attribute the Ca2+ dyshomeostasis to delayed extrusion of cytosolic Ca2+ Suggesting that this defect in Ca2+ extrusion arose from an insufficient response to the bioenergetic demand of neural activity, depolarization-induced mitochondrial ATP production was diminished in vapbP58S neurons. These findings point to bioenergetic dysfunction as a potential cause for the synaptic defects in vapbP58S -expressing motor neurons.SIGNIFICANCE STATEMENT Whether the synchrony between the rates of ATP production and demand is lost in degenerating neurons remains poorly understood. We report that expression of a gene equivalent to an amyotrophic lateral sclerosis (ALS)-causing variant of vesicle-associated membrane protein B (VAPB) in fly neurons decouples mitochondrial ATP production from neuronal activity. Consequently, levels of ATP in mutant neurons are unable to keep up with the bioenergetic burden of neuronal activity. Reduced rate of Ca2+ extrusion, which could result from insufficient energy to power Ca2+ ATPases, results in the accumulation of residual Ca2+ in mutant neurons and leads to alterations in synaptic vesicle (SV) release and synapse development. These findings suggest that synaptic defects in a model of ALS arise from the loss of activity-induced ATP production.


Assuntos
Esclerose Lateral Amiotrófica , Masculino , Animais , Feminino , Esclerose Lateral Amiotrófica/metabolismo , Drosophila/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Calmodulina/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Neurônios Motores/metabolismo , Proteínas R-SNARE/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo
12.
J Biochem ; 172(6): 337-340, 2022 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-36166826

RESUMO

For proper intracellular vesicle transport, it is essential for transport vesicle membranes to fuse with the appropriate target membranes. Ykt6 is a SNARE protein with functions in diverse vesicle transport pathways, including secretory, endocytotic and autophagic pathways. To exert these functions, the association of Ykt6 with vesicle membranes and the change of its conformation from closed to open play key roles. Recent studies have revealed regulatory mechanisms involved in Ykt6 membrane association and conformation change. When in the cytosol, the vicinal cysteine residues within the C-terminal CCAIM sequence of Ykt6 undergo diprenylation (farnesylation of the distal cysteine residues by farnesyltransferase; this is followed by geranylgeranylation of the proximal cysteine residue by geranylgeranyltransferase-III). Phosphorylation of a serine residue within the SNARE domain triggers the conversion of the Ykt6 conformation from closed to open, allowing Ykt6 membrane association. In this commentary, I briefly summarize and discuss the recently revealed regulatory mechanisms of Ykt6 function by diprenylation and phosphorylation.


Assuntos
Cisteína , Proteínas SNARE , Proteínas SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Fosforilação , Cisteína/metabolismo , Fusão de Membrana
13.
J Cell Biol ; 221(11)2022 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-36125415

RESUMO

Upon internalization, many surface membrane proteins are recycled back to the plasma membrane. Although these endosomal trafficking pathways control surface protein activity, the precise regulatory features and division of labor between interconnected pathways are poorly defined. In yeast, we show recycling back to the surface occurs through distinct pathways. In addition to retrograde recycling pathways via the late Golgi, used by synaptobrevins and driven by cargo ubiquitination, we find nutrient transporter recycling bypasses the Golgi in a pathway driven by cargo deubiquitination. Nutrient transporters rapidly internalize to, and recycle from, endosomes marked by the ESCRT-III associated factor Ist1. This compartment serves as both "early" and "recycling" endosome. We show Ist1 is ubiquitinated and that this is required for proper endosomal recruitment and cargo recycling to the surface. Additionally, the essential ATPase Cdc48 and its adaptor Npl4 are required for recycling, potentially through regulation of ubiquitinated Ist1. This collectively suggests mechanistic features of recycling from endosomes to the plasma membrane are conserved.


Assuntos
Endossomos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Ubiquitina , Proteínas de Transporte Vesicular , Adenosina Trifosfatases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Proteínas R-SNARE/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Proteínas de Transporte Vesicular/metabolismo
14.
Proc Natl Acad Sci U S A ; 119(36): e2202730119, 2022 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-36044553

RESUMO

Protein secretion in cancer cells defines tumor survival and progression by orchestrating the microenvironment. Studies suggest the occurrence of active secretion of cytosolic proteins in liver cancer and their involvement in tumorigenesis. Here, we investigated the identification of extended-synaptotagmin 1 (E-Syt1), an endoplasmic reticulum (ER)-bound protein, as a key mediator for cytosolic protein secretion at the ER-plasma membrane (PM) contact sites. Cytosolic proteins interacted with E-Syt1 on the ER, and then localized spatially inside SEC22B+ vesicles of liver cancer cells. Consequently, SEC22B on the vesicle tethered to the PM via Q-SNAREs (SNAP23, SNX3, and SNX4) for their secretion. Furthermore, inhibiting the interaction of protein kinase Cδ (PKCδ), a liver cancer-specific secretory cytosolic protein, with E-Syt1 by a PKCδ antibody, decreased in both PKCδ secretion and tumorigenicity. Results reveal the role of ER-PM contact sites in cytosolic protein secretion and provide a basis for ER-targeting therapy for liver cancer.


Assuntos
Neoplasias Hepáticas , Proteínas R-SNARE , Sinaptotagmina I , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Transporte Proteico , Proteínas R-SNARE/metabolismo , Sinaptotagmina I/metabolismo , Microambiente Tumoral
15.
Int J Mol Sci ; 23(8)2022 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-35457172

RESUMO

Tetanus and Botulinum type B neurotoxins are bacterial metalloproteases that specifically cleave the vesicle-associated membrane protein VAMP at an identical peptide bond, resulting in inhibition of neuroexocytosis. The minute amounts of these neurotoxins commonly used in experimental animals are not detectable, nor is detection of their VAMP substrate sensitive enough. The immune detection of the cleaved substrate is much more sensitive, as we have previously shown for botulinum neurotoxin type A. Here, we describe the production in rabbit of a polyclonal antibody raised versus a peptide encompassing the 13 residues C-terminal with respect to the neurotoxin cleavage site. The antibody was affinity purified and found to recognize, with high specificity and selectivity, the novel N-terminus of VAMP that becomes exposed after cleavage by tetanus toxin and botulinum toxin type B. This antibody recognizes the neoepitope not only in native and denatured VAMP but also in cultured neurons and in neurons in vivo in neurotoxin-treated mice or rats, suggesting the great potential of this novel tool to elucidate tetanus and botulinum B toxin activity in vivo.


Assuntos
Toxinas Botulínicas Tipo A , Tétano , Animais , Anticorpos/metabolismo , Camundongos , Neurotoxinas/metabolismo , Peptídeos/metabolismo , Proteólise , Proteínas R-SNARE/química , Proteínas R-SNARE/metabolismo , Coelhos , Ratos , Toxina Tetânica/química , Toxina Tetânica/metabolismo
16.
Cell Death Dis ; 13(1): 84, 2022 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-35082283

RESUMO

Deregulated lncRNAs play critical roles in tumorigenesis and tumor progression. NR2F1-AS1 is an antisense lncRNA of NR2F1. However, the biological function of NR2F1-AS1 in gastric cancer (GC) remains largely unclear. In this study, we revealed that NR2F1-AS1 and NR2F1 were both positively correlated with the degree of malignancy and predicted poor prognosis in two independent GC cohorts. Besides, NR2F1-AS1 and NR2F1 can respond to Epithelial-to-mesenchymal transition (EMT) signaling in GC, since their expression was increased by TGF-beta treatment and decreased after stable overexpression of OVOL2 in GC cell lines. NR2F1-AS1 and NR2F1 were highly co-expressed in pan-tissues and pan-cancers. Depletion of NR2F1-AS1 compromised the expression level of NR2F1 in GC cells. Furthermore, NR2F1-AS1 knockdown inhibited the proliferation, migration, invasion and G1/S transition of GC cells. More importantly, transcriptome sequencing revealed a novel ceRNA network composed of NR2F1-AS1, miR-29a-3p, and VAMP7 in GC. The overexpression of VAMP7 predicted poor prognosis in GC. Rescue assay confirmed that NR2F1-AS1 promotes GC progression through miR-29a-3p/VAMP7 axis. Our finding highlights that the aberrant expression of NR2F1-AS1 is probably due to the abnormal EMT signaling in GC. LncRNA NR2F1-AS1 plays crucial roles in GC progression by modulating miR-29a-3p/VAMP7 axis, suggesting that NR2F1-AS1 may serve as a potential therapeutic target in GC.


Assuntos
MicroRNAs , RNA Longo não Codificante , Neoplasias Gástricas , Fator I de Transcrição COUP/genética , Fator I de Transcrição COUP/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas R-SNARE/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Neoplasias Gástricas/patologia , Fatores de Transcrição/metabolismo
17.
Cells Dev ; 169: 203759, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34856414

RESUMO

Ykt6 has emerged as a key protein involved in a wide array of trafficking events, and has also been implicated in a number of human pathologies, including the progression of several cancers. It is a complex protein that simultaneously exhibits a high degree of structural and functional homology, and yet adopts differing roles in different cellular contexts. Because Ykt6 has been implicated in a variety of vesicle fusion events, we characterized the role of Ykt6 in oogenesis by observing the phenotype of Ykt6 germline clones. Immunofluorescence was used to visualize the expression of membrane proteins, organelles, and vesicular trafficking markers in mutant egg chambers. We find that Ykt6 germline clones have morphological and actin defects affecting both the nurse cells and oocyte, consistent with a role in regulating membrane growth during mid-oogenesis. Additionally, these egg chambers exhibit defects in bicoid and oskar RNA localization, and in the trafficking of Gurken during mid-to-late oogenesis. Finally, we show that Ykt6 mutations result in defects in late endosomal pathways, including endo- and exocytosis. These findings suggest a role for Ykt6 in endosome maturation and in the movement of membranes to and from the cell surface.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/genética , Proteínas de Drosophila/genética , Fusão de Membrana/fisiologia , Oogênese/genética , Proteínas R-SNARE/genética
18.
Cancer Biother Radiopharm ; 37(8): 650-661, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32818383

RESUMO

Background: Breast cancer is one of the most prevalent cancers that often occur in females. Long noncoding RNA differentiation antagonizing nonprotein coding RNA (DANCR) has been involved in the pathogenesis of various tumors, including breast cancer. This study aimed to investigate the role and underlying mechanism of DANCR in breast cancer. Materials and Methods: The level of DANCR was detected in breast cancer tissues and cells by quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability was evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. Cell apoptosis was assessed using flow cytometry. Cell migration and invasion were estimated by the Transwell assay. The relationship between DANCR, miR-4319, and vesicle-associated membrane protein-associated protein B (VAPB) was confirmed by bioinformatic analysis and dual-luciferase reporter assay. The level of microRNA-4319 (miR-4319) was tested by qRT-PCR. The expression of VAPB was measured by qRT-PCR or Western blot assay. Results: DANCR and VAPB were upregulated, while miR-4319 was downregulated in breast cancer tissues and cells. Knockdown of DANCR hindered proliferation, migration, and invasion and promoted apoptosis of breast cancer cells. DANCR knockdown inhibited breast cancer development through regulating miR-4319. Inhibition of miR-4319 restrained breast cancer cell progression by targeting VAPB. Moreover, DANCR regulated VAPB expression by sponging miR-4319 in breast cancer cells. Conclusion: DANCR facilitated breast cancer cell progression through regulating the miR-4319/VAPB axis, indicating that DANCR might be a potential biomarker and therapeutic target for breast cancer treatment.


Assuntos
Neoplasias da Mama , MicroRNAs , RNA Longo não Codificante , Humanos , Feminino , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias da Mama/genética , Brometos/metabolismo , Regulação Neoplásica da Expressão Gênica , Proliferação de Células/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Apoptose/genética , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
19.
Cancer Lett ; 525: 179-197, 2022 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-34752845

RESUMO

The transient receptor potential melastatin-subfamily member 7 (TRPM7) is a ubiquitous cation channel possessing kinase activity. TRPM7 mediates a variety of physiological responses by conducting flow of cations such as Ca2+, Mg2+, and Zn2+. Here, we show that the activation of TRPM7 channel stimulated by chemical agonists of TRPM7, Clozapine or Naltriben, inhibited autophagy via mediating Zn2+ release to the cytosol, presumably from the intracellular Zn2+-accumulating vesicles where TRPM7 localizes. Zn2+ release following the activation of TRPM7 disrupted the fusion between autophagosomes and lysosomes by disturbing the interaction between Sxt17 and VAMP8 which determines fusion status of autophagosomes and lysosomes. Ultimately, the disrupted fusion resulting from stimulation of TRPM7 channels arrested autophagy. Functionally, we demonstrate that the autophagy inhibition mediated by TRPM7 triggered cell death and suppressed metastasis of cancer cells in vitro, more importantly, restricted tumor growth and metastasis in vivo, by evoking apoptosis, cell cycle arrest, and reactive oxygen species (ROS) elevation. These findings represent a strategy for stimulating TRPM7 to combat cancer.


Assuntos
Neoplasias/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteínas R-SNARE/genética , Canais de Cátion TRPM/genética , Apoptose/efeitos dos fármacos , Autofagossomos/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Clozapina/farmacologia , Humanos , Lisossomos/efeitos dos fármacos , Naltrexona/análogos & derivados , Naltrexona/farmacologia , Metástase Neoplásica , Neoplasias/genética , Transdução de Sinais/efeitos dos fármacos , Canais de Cátion TRPM/agonistas , Zinco/farmacologia
20.
J Cell Sci ; 135(5)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-34821359

RESUMO

Macroautophagy, the degradation and recycling of cytosolic components in the lysosome, is an important cellular mechanism. It is a membrane-mediated process that is linked to vesicular trafficking events. The sorting nexin (SNX) protein family controls the sorting of a large array of cargoes, and various SNXs impact autophagy. To improve our understanding of their functions in vivo, we screened all Drosophila SNXs using inducible RNA interference in the fat body. Significantly, depletion of Snazarus (Snz) led to decreased autophagic flux. Interestingly, we observed altered distribution of Vamp7-positive vesicles with Snz depletion, and the roles of Snz were conserved in human cells. SNX25, the closest human ortholog to Snz, regulates both VAMP8 endocytosis and lipid metabolism. Through knockout-rescue experiments, we demonstrate that these activities are dependent on specific SNX25 domains and that the autophagic defects seen upon SNX25 loss can be rescued by ethanolamine addition. We also demonstrate the presence of differentially spliced forms of SNX14 and SNX25 in cancer cells. This work identifies a conserved role for Snz/SNX25 as a regulator of autophagic flux and reveals differential isoform expression between paralogs.


Assuntos
Proteínas de Drosophila , Nexinas de Classificação , Animais , Autofagia/genética , Drosophila/metabolismo , Endocitose , Humanos , Transporte Proteico , Proteínas R-SNARE , Nexinas de Classificação/genética , Nexinas de Classificação/metabolismo
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