RESUMO
Autophagy is a highly conserved process from yeast to mammals in which intracellular materials are engulfed by a double-membrane organelle called autophagosome and degrading materials by fusing with the lysosome. The process of autophagy is regulated by sequential recruitment and function of autophagy-related (Atg) proteins. Genetic hierarchical analyses show that the ULK1 complex comprised of ULK1-FIP200-ATG13-ATG101 translocating from the cytosol to autophagosome formation sites as a most upstream ATG factor; this translocation is critical in autophagy initiation. However, how this translocation occurs remains unclear. Here, we show that ULK1 is palmitoylated by palmitoyltransferase ZDHHC13 and translocated to the autophagosome formation site upon autophagy induction. We find that the ULK1 palmitoylation is required for autophagy initiation. Moreover, the ULK1 palmitoylated enhances the phosphorylation of ATG14L, which is required for activating PI3-Kinase and producing phosphatidylinositol 3-phosphate, one of the autophagosome membrane's lipids. Our results reveal how the most upstream ULK1 complex translocates to the autophagosome formation sites during autophagy.
Assuntos
Aciltransferases , Autofagossomos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Autofagia , Peptídeos e Proteínas de Sinalização Intracelular , Lipoilação , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Autofagia/fisiologia , Humanos , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fosforilação , Aciltransferases/metabolismo , Aciltransferases/genética , Autofagossomos/metabolismo , Células HEK293 , Fosfatos de Fosfatidilinositol/metabolismo , Animais , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Transporte Proteico , Proteínas de Transporte VesicularRESUMO
Recently, the sortilin receptor (SORT1) was found to be preferentially over-expressed on the surface of many cancer cells, which makes SORT1 a novel anticancer target. The SORT1 binding proprietary peptide TH19P01 could achieve the SORT1-mediated cancer cell binding and subsequent internalization. Inspired by the peptide-drug conjugate (PDC) strategy, the TH19P01-camptothecin (CPT) conjugates were designed, efficiently synthesized, and evaluated for their anticancer potential in this study. The water solubility, in vitro anticancer activity, time-kill kinetics, cellular uptake, anti-migration activity, and hemolysis effects were systematically estimated. Besides, in order to monitor the release of CPT from conjugates in real-time, the CPT/Dnp-based "turn on" hybrid peptide was designed, which indicted that CPT could be sustainably released from the hybrid peptide in both human serum and cancer cellular environments. Strikingly, compared with free CPT, the water solubility, cellular uptake, and selectivity towards cancer cells of hybrid peptide LYJ-2 have all been significantly enhanced. Moreover, unlike free CPT or TH19P01, LYJ-2 exhibited selective anti-proliferative and anti-migration effects against SORT1-positive MDA-MB-231 cells. Collectively, this study not only established efficient strategies to improve the solubility and anticancer potential of chemotherapeutic agent CPT, but also provided important references for the future development of TH19P01 based PDCs targeting SORT1.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular , Antineoplásicos , Camptotecina , Proliferação de Células , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camptotecina/farmacologia , Camptotecina/química , Camptotecina/síntese química , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Proliferação de Células/efeitos dos fármacos , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/síntese química , Relação Estrutura-Atividade , Estrutura Molecular , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Movimento Celular/efeitos dos fármacosRESUMO
The diagnosis of prostate cancer using histopathology is reliant on the accurate interpretation of prostate tissue sections. Current standards rely on the assessment of Haematoxylin and Eosin (H&E) staining, which can be difficult to interpret and introduce inter-observer variability. Here, we present a digital pathology atlas and online resource of prostate cancer tissue micrographs for both H&E and the reinterpretation of samples using a novel set of three biomarkers as an interactive tool, where clinicians and scientists can explore high resolution histopathology from various case studies. The digital pathology prostate cancer atlas when used in conjunction with the biomarkers, will assist pathologists to accurately grade prostate cancer tissue samples.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Biomarcadores Tumorais , Neoplasias da Próstata , Sindecana-1 , Neoplasias da Próstata/patologia , Masculino , Humanos , Sindecana-1/análiseRESUMO
Synaptotagmin-1 (Syt1) is a calcium sensor that regulates synaptic vesicle fusion in synchronous neurotransmitter release. Syt1 interacts with negatively charged lipids and the SNARE complex to control the fusion event. However, it remains incompletely understood how Syt1 mediates Ca2+-trigged synaptic vesicle fusion. Here, we discovered that Syt1 undergoes liquid-liquid phase separation (LLPS) to form condensates both in vitro and in living cells. Syt1 condensates play a role in vesicle attachment to the PM and efficiently recruit SNAREs and complexin, which may facilitate the downstream synaptic vesicle fusion. We observed that Syt1 condensates undergo a liquid-to-gel-like phase transition, reflecting the formation of Syt1 oligomers. The phase transition can be blocked or reversed by Ca2+, confirming the essential role of Ca2+ in Syt1 oligomer disassembly. Finally, we showed that the Syt1 mutations causing Syt1-associated neurodevelopmental disorder impair the Ca2+-driven phase transition. These findings reveal that Syt1 undergoes LLPS and a Ca2+-sensitive phase transition, providing new insights into Syt1-mediated vesicle fusion.
Assuntos
Cálcio , Vesículas Sinápticas , Sinaptotagmina I , Sinaptotagmina I/metabolismo , Sinaptotagmina I/genética , Cálcio/metabolismo , Humanos , Animais , Vesículas Sinápticas/metabolismo , Multimerização Proteica , Proteínas SNARE/metabolismo , Proteínas SNARE/genética , Transição de Fase , Mutação/genética , Células HEK293 , Fusão de Membrana , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Separação de FasesRESUMO
Toll-like receptors (TLRs) are pattern recognition receptors that trigger host immune responses against various pathogens by detecting evolutionarily conserved pathogen-associated molecular patterns (PAMPs). TLR21 is a member of the Toll-like receptor family, and emerging data suggest that it recognises unmethylated CpG DNA and is considered a functional homologue of mammalian TLR9. However, little is known regarding the role of TLR21 in the fish immune response. In the present study, we isolated the cDNA sequence of TLR21 from the largemouth bass (Micropterus salmoides) and termed it MsTLR21. The MsTLR21 gene contained an open reading frame (ORF) of 2931 bp and encodes a polypeptide of 976 amino acids. The predicted MsTLR21 protein has two conserved domains, a conserved leucine-rich repeats (LRR) domain and a C-terminal Toll-interleukin (IL) receptor (TIR) domain, similar to those of other fish and mammals. In healthy largemouth bass, the TLR21 transcript was broadly expressed in all the examined tissues, with the highest expression levels in the gills. After challenge with Nocardia seriolae and polyinosinic polycytidylic acid (Poly[I:C]), the expression of TLR21 mRNA was upregulated or downregulated in all tissues tested. Overexpression of TLR21 in 293T cells showed that it has a positive regulatory effect on nuclear factor-kappaB (NF-κB) and interferons-ß (IFN-ß) activity. Subcellular localisation analysis showed that TLR21 was expressed in the cytoplasm. We performed pull-down assays and determined that TLR21 did not interact with myeloid differentiation primary response gene 88 (Myd88); however, it interacted with TIR domain-containing adaptor inducing interferon-ß (TRIF). Taken together, these findings suggest that MsTLR21 plays important roles in TLR/IL-1R signalling pathways and the immune response to pathogen invasion.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular , Sequência de Aminoácidos , Bass , Doenças dos Peixes , Proteínas de Peixes , NF-kappa B , Filogenia , Animais , Bass/imunologia , Bass/genética , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Proteínas de Peixes/química , NF-kappa B/genética , NF-kappa B/metabolismo , NF-kappa B/imunologia , Doenças dos Peixes/imunologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/imunologia , Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Transdução de Sinais/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Alinhamento de Sequência/veterinária , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Fator 88 de Diferenciação Mieloide/imunologia , Fator 88 de Diferenciação Mieloide/química , Perfilação da Expressão Gênica/veterinária , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia , Receptores Toll-Like/química , Receptores Toll-Like/metabolismo , Sequência de BasesRESUMO
Autophagy is a highly conserved catabolic mechanism by which unnecessary or dysfunctional cellular components are removed. The dysregulation of autophagy has been implicated in various neurodegenerative diseases, including Alzheimer's disease (AD). Understanding the molecular mechanism(s)/molecules that influence autophagy may provide important insights into developing therapeutic strategies against AD and other neurodegenerative disorders. Engulfment adaptor phosphotyrosine-binding domain-containing protein 1 (GULP1) is an adaptor that interacts with amyloid precursor protein (APP) to promote amyloid-ß peptide production via an unidentified mechanism. Emerging evidence suggests that GULP1 has a role in autophagy. Here, we show that GULP1 is involved in autophagy through an interaction with autophagy-related 14 (ATG14), which is a regulator of autophagosome formation. GULP1 potentiated the stimulatory effect of ATG14 on autophagy by modulating class III phosphatidylinositol 3-kinase complex 1 (PI3KC3-C1) activity. The effect of GULP1 is attenuated by a GULP1 mutation (GULP1m) that disrupts the GULP1-ATG14 interaction. Conversely, PI3KC3-C1 activity is enhanced in cells expressing APP but not in those expressing an APP mutant that does not bind GULP1, which suggests a role of GULP1-APP in regulating PI3KC3-C1 activity. Notably, GULP1 facilitates the targeting of ATG14 to the endoplasmic reticulum (ER). Moreover, the levels of both ATG14 and APP are elevated in the autophagic vacuoles (AVs) of cells expressing GULP1, but not in those expressing GULP1m. APP processing is markedly enhanced in cells co-expressing GULP1 and ATG14. Hence, GULP1 alters APP processing by promoting the entry of APP into AVs. In summary, we unveil a novel role of GULP1 in enhancing the targeting of ATG14 to the ER to stimulate autophagy and, consequently, APP processing.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Precursor de Proteína beta-Amiloide , Proteínas Relacionadas à Autofagia , Autofagia , Humanos , Precursor de Proteína beta-Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Células HEK293 , Ligação Proteica , Doença de Alzheimer/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMO
The epithelial-mesenchymal transition (EMT) is a genetic reprogramming that tumor cells utilize for metastasis. Epsin-3 (EPN3) is an endocytic adapter protein involved in clathrin-mediated endocytosis and had been previously linked to EMT in breast cancer and glioma metastasis. In this study, identified the role of epsin-3 in lung adenocarcinoma and metastasis and epsin-3 levels identified using an expression profile analysis of patient data indicated the protein was abnormally overexpressed in lung adenocarcinoma patients and this was directly linked to disease severity. Gene knockdowns of EPN3 in human adenocarcinoma cell line A549 and the non-small cell lung carcinoma cell line H1299 decreased the levels of mesenchymal markers, including vimentin (VIM), N-cadherin (NCAD) and embryonic transcription factors like zinc finger E-box binding homeobox 1(ZEB1), snail, and the key molecules of Wnt pathway such as ß-catenin and resulted in increased expression of the epithelial marker E-cadherin (ECAD). Our data links EPN3 to the EMT process in lung cancer and inhibition of its expression reduced the metastatic and invasive ability of lung adenocarcinoma cells by inhibiting the EMT process.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular , Adenocarcinoma de Pulmão , Movimento Celular , Transição Epitelial-Mesenquimal , Neoplasias Pulmonares , Invasividade Neoplásica , Humanos , Transição Epitelial-Mesenquimal/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/genética , Movimento Celular/genética , Linhagem Celular Tumoral , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Regulação Neoplásica da Expressão Gênica , Feminino , Células A549 , Caderinas/metabolismo , Caderinas/genética , Masculino , Via de Sinalização Wnt , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenocarcinoma/genética , Pessoa de Meia-Idade , beta Catenina/metabolismoRESUMO
α-Synuclein (αSYN), a pivotal synaptic protein implicated in synucleinopathies such as Parkinson's disease and Lewy body dementia, undergoes protein phase separation. We reveal that vesicle-associated membrane protein 2 (VAMP2) orchestrates αSYN phase separation both in vitro and in cells. Electrostatic interactions, specifically mediated by VAMP2 via its juxtamembrane domain and the αSYN C-terminal region, drive phase separation. Condensate formation is specific for R-SNARE VAMP2 and dependent on αSYN lipid membrane binding. Our results delineate a regulatory mechanism for αSYN phase separation in cells. Furthermore, we show that αSYN condensates sequester vesicles and attract complexin-1 and -2, thus supporting a role in synaptic physiology and pathophysiology.
Assuntos
Proteína 2 Associada à Membrana da Vesícula , alfa-Sinucleína , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Proteína 2 Associada à Membrana da Vesícula/genética , Humanos , Ligação Proteica , Animais , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Células HEK293 , Eletricidade Estática , Separação de FasesRESUMO
TLR4 and TNFR1 signalling promotes potent proinflammatory signal transduction events, thus, are often hijacked by pathogenic microorganisms. We recently reported that myeloid cells retaliate Yersinia blockade of TAK1/IKK signalling by triggering RIPK1-dependent caspase-8 activation that promotes downstream GSDMD and GSDME-mediated pyroptosis in macrophages and neutrophils respectively. However, the upstream signalling events for RIPK1 activation in these cells are not well defined. Here, we demonstrate that unlike in macrophages, RIPK1-driven pyroptosis and cytokine priming in neutrophils are driven through TNFR1 signalling, while TLR4-TRIF signalling is dispensable. Furthermore, we demonstrate that activation of RIPK1-dependent pyroptosis in neutrophils during Yersinia infection requires IFN-γ priming, which serves to induce surface TNFR1 expression and amplify soluble TNF secretion. In contrast, macrophages utilise both TNFR1 and TLR4-TRIF signalling to trigger cell death, but only require TRIF but not autocrine TNFR1 for cytokine production. Together, these data highlight the emerging theme of cell type-specific regulation in cell death and immune signalling in myeloid cells.
Assuntos
Macrófagos , Neutrófilos , Piroptose , Proteína Serina-Treonina Quinases de Interação com Receptores , Receptores Tipo I de Fatores de Necrose Tumoral , Transdução de Sinais , Receptor 4 Toll-Like , Macrófagos/metabolismo , Neutrófilos/metabolismo , Animais , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Camundongos , Receptor 4 Toll-Like/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Camundongos Endogâmicos C57BL , Interferon gama/metabolismo , Camundongos KnockoutRESUMO
The release of neurotransmitters (NTs) at central synapses is dependent on a cascade of protein interactions, specific to the presynaptic compartment. Among those dedicated molecules, the cytosolic complexins play an incompletely defined role as synaptic transmission regulators. Complexins are multidomain proteins that bind soluble N-ethylmaleimide sensitive factor attachment protein receptor complexes, conferring both inhibitory and stimulatory functions. Using systematic mutagenesis and comparing reconstituted in vitro membrane fusion assays with electrophysiology in cultured neurons from mice of either sex, we deciphered the function of the N-terminus of complexin (Cpx) II. The N-terminus (amino acid 1-27) starts with a region enriched in hydrophobic amino acids (1-12), which binds lipids. Mutants maintaining this hydrophobic character retained the stimulatory function of Cpx, whereas exchanges introducing charged residues perturbed both spontaneous and evoked exocytosis. Mutants in the more distal region of the N-terminal domain (amino acid 11-18) showed a spectrum of effects. On the one hand, mutation of residue A12 increased spontaneous release without affecting evoked release. On the other hand, replacing D15 with amino acids of different shapes or hydrophobic properties (but not charge) not only increased spontaneous release but also impaired evoked release. Most surprising, this substitution reduced the size of the readily releasable pool, a novel function for Cpx at mammalian synapses. Thus, the exact amino acid composition of the Cpx N-terminus fine-tunes the degree of spontaneous and evoked NT release.
Assuntos
Proteínas do Tecido Nervoso , Vesículas Sinápticas , Animais , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/genética , Camundongos , Masculino , Feminino , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/química , Mutação , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/química , Fusão de Membrana/fisiologia , Fusão de Membrana/genética , Células Cultivadas , Fenótipo , Neurônios/metabolismo , Transmissão Sináptica/genética , Transmissão Sináptica/fisiologia , Camundongos Endogâmicos C57BL , Exocitose/fisiologia , Exocitose/genéticaRESUMO
Intestinal ischemia/reperfusion (I/R) injury is a serious condition that causes intestinal dysfunction and can be fatal. Previous research has shown that toll-like receptor 4 (TLR4) inhibitors have a protective effect against this injury. This study aimed to investigate the protective effects of TLR4 inhibitors, specifically cyclobenzaprine, ketotifen, amitriptyline, and naltrexone, in rats with intestinal (I/R) injury. Albino rats were divided into seven groups: vehicle control, sham-operated, I/R injury, I/R-cyclobenzaprine (10 mg/kg body weight), I/R-ketotifen (1 mg/kg body weight), I/R-amitriptyline (10 mg/kg body weight), and I/R-naltrexone (4 mg/kg body weight) groups. Anesthetized rats (urethane 1.8 g/kg) underwent 30 min of intestinal ischemia by occluding the superior mesenteric artery (SMA), followed by 2 h of reperfusion. Intestinal tissue samples were collected to measure various parameters, including malondialdehyde (MDA), nitric oxide synthase (NO), myeloperoxidase (MPO), superoxide dismutase (SOD), TLR4, intercellular adhesion molecule-1 (ICAM-1), nuclear factor kappa bp65 (NF-ĸBP65), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), macrophages CD68, myeloid differentiation factor 88 (MYD88), and toll interleukin receptor-domain-containing adaptor-inducing interferon ß (TRIF). The use of TLR4 inhibitors significantly reduced MDA, MPO, and NO levels, while increasing SOD activity. Furthermore, it significantly decreased TLR4, ICAM-1, TNF-α, MCP-1, MYD88, and TRIF levels. These drugs also showed partial restoration of normal cellular structure with reduced inflammation. Additionally, there was a decrease in NF-ĸBP65 and macrophages CD68 staining compared to rats in the I/R groups. This study focuses on how TLR4 inhibitors enhance intestinal function and protect against intestinal (I/R) injury by influencing macrophages CD86 through (MYD88-TRIF) pathway, as well as their effects on oxidation and inflammation.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular , Fator 88 de Diferenciação Mieloide , Traumatismo por Reperfusão , Transdução de Sinais , Receptor 4 Toll-Like , Animais , Receptor 4 Toll-Like/metabolismo , Receptor 4 Toll-Like/antagonistas & inibidores , Fator 88 de Diferenciação Mieloide/metabolismo , Fator 88 de Diferenciação Mieloide/antagonistas & inibidores , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo , Ratos , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Masculino , Transdução de Sinais/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Intestinos/patologiaRESUMO
OBJECTIVES: This study aims to elucidate the role of circUSP9X (Circular RNA Ubiquitin Specific Peptidase 9 X-Linked) in the development of venous thrombosis in the lower extremities. METHODS: An animal model of Deep Vein Thrombosis (DVT) and a hypoxic model of Human Umbilical Vein Endothelial Cells (HUVECs) treated with Cobalt (II) Chloride (CoCl2) were developed. The expression levels of circUSP9X, microRNA-148b-3p (miR-148b-3p), and SRC Kinase Signaling Inhibitor 1 (SRCIN1) were quantified using quantitative reverse transcription Polymerase Chain Reaction and Western blot analysis. Cell cytotoxicity, viability, apoptosis, and inflammation in HUVECs were assessed via Lactate Dehydrogenase (LDH) assay, MTT assay, flow cytometry, Enzyme-Linked Immunosorbent Assay, and Western blot, respectively. Hematoxylin and Eosin staining were employed for histopathological examination of the venous tissues in the animal model. The interaction between circUSP9X, miR-148b-3p, and SRCIN1 was further explored through dual-luciferase reporter assays and RNA Immunoprecipitation experiments. RESULTS: The present findings reveal a significant upregulation of circUSP9X and SRCIN1 and a concurrent downregulation of miR-148b-3p in DVT cases. Knockdown of circUSP9X or overexpression of miR-148b-3p ameliorated CoCl2-induced apoptosis in HUVECs, reduced LDH release, enhanced cellular viability, and mitigated inflammation. Conversely, overexpression of circUSP9X intensified CoCl2's cytotoxic effects. The effects of manipulating circUSP9X expression were counteracted by the corresponding modulation of miR-148b-3p and SRCIN1 levels. Additionally, circUSP9X knockdown effectively inhibited the formation of DVT in the mouse model. A competitive binding mechanism of circUSP9X for miR-148b-3p, modulating SRCIN1 expression, was identified. CONCLUSION: circUSP9X promotes the formation of DVT through the regulation of the miR-148b-3p/SRCIN1 axis.
Assuntos
Modelos Animais de Doenças , Células Endoteliais da Veia Umbilical Humana , MicroRNAs , Regulação para Cima , Trombose Venosa , Animais , Humanos , Masculino , Camundongos , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Apoptose/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , MicroRNAs/metabolismo , RNA Circular/genética , Regulação para Cima/efeitos dos fármacosRESUMO
Rheumatoid fibroblast-like synoviocytes (RFLS) have an important role in the inflammatory pathogenesis of rheumatoid arthritis (RA). Toll-like receptor 3 (TLR3) is upregulated in RFLS; its activation leads to the production of interferon-ß (IFN-ß), a type I IFN. IFN-stimulated gene 56 (ISG56) is induced by IFN and is involved in innate immune responses; however, its role in RA remains unknown. Therefore, the purpose of this study was to investigate the role of TLR3-induced ISG56 in human RFLS. RFLS were treated with polyinosinic-polycytidylic acid (poly I:C), which served as a TLR3 ligand. ISG56, melanoma differentiation-associated gene 5 (MDA5), and C-X-C motif chemokine ligand 10 (CXCL10) expression were measured using quantitative reverse transcription-polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay. Using immunohistochemistry, we found that ISG56 was expressed in synovial tissues of patients with RA and osteoarthritis. Under poly I:C treatment, ISG56 was upregulated in RFLS. In addition, we found that the type I IFN-neutralizing antibody mixture suppressed ISG56 expression. ISG56 knockdown decreased CXCL10 expression and MDA5 knockdown decreased ISG56 expression. In addition, we found that ISG56 was strongly expressed in the synovial cells of patients with RA. TLR3 signaling induced ISG56 expression in RFLS and type I IFN was involved in ISG56 expression. ISG56 was also found to be associated with CXCL10 expression, suggesting that ISG56 may be involved in TLR3/type I IFN/CXCL10 axis, and play a role in RA synovial inflammation.
Assuntos
Artrite Reumatoide , Quimiocina CXCL10 , Poli I-C , Transdução de Sinais , Sinoviócitos , Receptor 3 Toll-Like , Humanos , Receptor 3 Toll-Like/metabolismo , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Poli I-C/farmacologia , Sinoviócitos/metabolismo , Quimiocina CXCL10/metabolismo , Helicase IFIH1 Induzida por Interferon/metabolismo , Helicase IFIH1 Induzida por Interferon/genética , Células Cultivadas , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas de Ligação a RNA , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Reguladoras de ApoptoseRESUMO
A subset of neuroendocrine tumors (NETs) can cause an excessive secretion of hormones, neuropeptides, and biogenic amines into the bloodstream. These so-called functional NETs evoke a hormone-related disease and lead to several different syndromes, depending on the factors released. One of the most common functional syndromes, carcinoid syndrome, is characterized mainly by over-secretion of serotonin. However, what distinguishes functional from non-functional tumors on a molecular level remains unknown. Here, we demonstrate that the expression of sortilin, a widely expressed transmembrane receptor involved in intracellular protein sorting, is significantly increased in functional compared to non-functional NETs and thus can be used as a biomarker for functional NETs. Furthermore, using a cell line model of functional NETs, as well as organoids, we demonstrate that inhibition of sortilin reduces cellular serotonin concentrations and may therefore serve as a novel therapeutic target to treat patients with carcinoid syndrome.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular , Tumores Neuroendócrinos , Serotonina , Feminino , Humanos , Masculino , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Síndrome do Carcinoide Maligno/metabolismo , Tumores Neuroendócrinos/metabolismo , Tumores Neuroendócrinos/patologia , Serotonina/metabolismo , Pessoa de Meia-Idade , Animais , CamundongosRESUMO
RIPK1 is a multi-functional kinase that regulates cell death and inflammation and has been implicated in the pathogenesis of inflammatory diseases. RIPK1 acts in a kinase-dependent and kinase-independent manner to promote or suppress apoptosis and necroptosis, but the underlying mechanisms remain poorly understood. Here, we show that a mutation (R588E) disrupting the RIPK1 death domain (DD) caused perinatal lethality induced by ZBP1-mediated necroptosis. Additionally, these mice developed postnatal inflammatory pathology, which was mediated by necroptosis-independent TNFR1, TRADD, and TRIF signaling, partially requiring RIPK3. Our biochemical mechanistic studies revealed that ZBP1- and TRIF-mediated activation of RIPK3 required RIPK1 kinase activity in wild-type cells but not in Ripk1R588E/R588E cells, suggesting that DD-dependent oligomerization of RIPK1 and its interaction with FADD determine the mechanisms of RIPK3 activation by ZBP1 and TRIF. Collectively, these findings revealed a critical physiological role of DD-dependent RIPK1 signaling that is important for the regulation of tissue homeostasis and inflammation.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular , Inflamação , Necroptose , Proteínas de Ligação a RNA , Proteína Serina-Treonina Quinases de Interação com Receptores , Transdução de Sinais , Animais , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Camundongos , Inflamação/metabolismo , Inflamação/imunologia , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Proteína de Domínio de Morte Associada a Fas/genética , Morte Celular , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Domínios Proteicos , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Apoptose , Mutação , Proteína de Domínio de Morte Associada a Receptor de TNFRESUMO
Breast cancer is a leading cause of cancer mortality in women worldwide. Using the Infinium MethylationEPIC BeadChip, we analyzed plasma sample methylation to identify the SRCIN1 gene in breast cancer patients. We assessed SRCIN1-related roles and pathways for their biomarker potential. To verify the methylation status, quantitative methylation-specific PCR (qMSP) was performed on genomic DNA and circulating cell-free DNA samples, and mRNA expression analysis was performed using RTâqPCR. The results were validated in a Western population; for this analysis, the samples included plasma samples from breast cancer patients from the USA and from The Cancer Genome Atlas (TCGA) cohort. To study the SRCIN1 pathway, we conducted cell viability assays, gene manipulation and RNA sequencing. SRCIN1 hypermethylation was identified in 61.8% of breast cancer tissues from Taiwanese patients, exhibiting specificity to this malignancy. Furthermore, its presence correlated significantly with unfavorable 5-year overall survival outcomes. The levels of methylated SRCIN1 in the blood of patients from Taiwan and the USA correlated with the stage of breast cancer. The proportion of patients with high methylation levels increased from 0% in healthy individuals to 63.6% in Stage 0, 80% in Stage I and 82.6% in Stage II, with a sensitivity of 78.5%, an accuracy of 90.3% and a specificity of 100%. SRCIN1 hypermethylation was significantly correlated with increased SRCIN1 mRNA expression (p < 0.001). Knockdown of SRCIN1 decreased the viability of breast cancer cells. SRCIN1 silencing resulted in the downregulation of ESR1, BCL2 and various cyclin protein expressions. SRCIN1 hypermethylation in the blood may serve as a noninvasive biomarker, facilitating early detection and prognosis evaluation, and SRCIN1-targeted therapies could be used in combination regimens for breast cancer patients.
Assuntos
Biomarcadores Tumorais , Neoplasias da Mama , Proliferação de Células , Metilação de DNA , Humanos , Neoplasias da Mama/genética , Neoplasias da Mama/sangue , Neoplasias da Mama/patologia , Neoplasias da Mama/diagnóstico , Metilação de DNA/genética , Feminino , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/sangue , Proliferação de Células/genética , Prognóstico , Pessoa de Meia-Idade , Regulação Neoplásica da Expressão Gênica , Detecção Precoce de Câncer , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/sangue , Linhagem Celular Tumoral , AdultoRESUMO
In the present study, we have explored the involvement of Toll-like Receptor 4 (TLR4) in atrial fibrillation (AF), by using a meta-analysis of publicly available human transcriptomic data. The meta-analysis revealed 565 upregulated and 267 downregulated differentially expressed genes associated with AF. Pathway enrichment analysis highlighted a significant overrepresentation in immune-related pathways for the upregulated genes. A significant overlap between AF differentially expressed genes and TLR4-modulated genes was also identified, suggesting the potential role of TLR4 in AF-related transcriptional changes. Additionally, the analysis of other Toll-like receptors (TLRs) revealed a significant association with TLR2 and TLR3 in AF-related gene expression patterns. The examination of MYD88 and TICAM1, genes associated with TLR4 signalling pathways, indicated a significant yet nonspecific enrichment of AF differentially expressed genes. In summary, this study offers novel insights into the molecular aspects of AF, suggesting a pathophysiological role of TLR4 and other TLRs. By targeting these specific receptors, new treatments might be designed to better manage AF, offering hope for improved outcomes in affected patients.
Assuntos
Fibrilação Atrial , Receptor 4 Toll-Like , Humanos , Fibrilação Atrial/genética , Fibrilação Atrial/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Transcriptoma , Transdução de Sinais/genética , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Proteínas Adaptadoras de Transporte VesicularRESUMO
Lipophagy, a form of autophagy specific to the degradation of lipid droplets (LDs), plays an important role in the maintenance of cellular homeostasis and metabolic processes. A recent study has identified ATG14 (autophagy related 14) as a molecule that targets LDs and marks them for degradation via lipophagy; a process that is inhibited by the binding of STX18 (syntaxin 18) to ATG14 in mammalian cells. The exact mechanism of regulation of lipophagy, and subsequently of cellular LD levels, is still under investigation; however, dysregulation of this process has been linked to a number of disease phenotypes. An imbalance of lipid levels can result in a wide variety of conditions depending on the cell/tissue type in which they occur. In cells of the retinal pigment epithelium, lipid accumulation can result in dry age-related macular degeneration, in hepatocytes it can result in nonalcoholic fatty liver diseases and in neural cells it can result in the pathogenesis of neurodegenerative conditions such as Alzheimer and Parkinson diseases. Based upon its wide range of implications in diseases, modulation of lipophagy is currently being further investigated for its potential as a treatment for a variety of conditions ranging from viral infection to developmental illnesses.
Assuntos
Gotículas Lipídicas , Animais , Humanos , Proteínas Adaptadoras de Transporte Vesicular , Autofagia/fisiologia , Proteínas Relacionadas à Autofagia/metabolismo , Gotículas Lipídicas/metabolismo , Metabolismo dos LipídeosRESUMO
Relevant studies have indicated the association of HCG18 with tumour occurrence and progression. In this study, we observed that PM2.5 can enhance the growth of lung adenocarcinoma cells by modulating the expression of HCG18. Further investigations, including overexpression and knockout experiments, elucidated that HCG18 suppresses miR-195, which in turn upregulates the expression of ATG14, resulting in the upregulation of autophagy. Consequently, exposure to PM2.5 leads to elevated HCG18 expression in lung tissues, which in turn increases Atg14 expression and activates autophagy pathways through inhibition of miR-195, thereby contributing to oncogenesis.
Assuntos
Adenocarcinoma de Pulmão , Proteínas Relacionadas à Autofagia , Autofagia , Progressão da Doença , Neoplasias Pulmonares , MicroRNAs , Material Particulado , Humanos , Células A549 , Proteínas Adaptadoras de Transporte Vesicular/efeitos dos fármacos , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/metabolismo , Autofagia/genética , Proteínas Relacionadas à Autofagia/efeitos dos fármacos , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Material Particulado/efeitos adversos , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Antígenos HLA/efeitos dos fármacos , Antígenos HLA/metabolismoRESUMO
Systemic exposure to starch-coated iron oxide nanoparticles (IONPs) can stimulate antitumor T cell responses, even when little IONP is retained within the tumor. Here, we demonstrate in mouse models of metastatic breast cancer that IONPs can alter the host immune landscape, leading to systemic immune-mediated disease suppression. We report that a single intravenous injection of IONPs can inhibit primary tumor growth, suppress metastases, and extend survival. Gene expression analysis revealed the activation of Toll-like receptor (TLR) pathways involving signaling via Toll/Interleukin-1 receptor domain-containing adaptor-inducing IFN-ß (TRIF), a TLR pathway adaptor protein. Requisite participation of TRIF in suppressing tumor progression was demonstrated with histopathologic evidence of upregulated IFN-regulatory factor 3 (IRF3), a downstream protein, and confirmed in a TRIF knockout syngeneic mouse model of metastatic breast cancer. Neither starch-coated polystyrene nanoparticles lacking iron, nor iron-containing dextran-coated parenteral iron replacement agent, induced significant antitumor effects, suggesting a dependence on the type of IONP formulation. Analysis of multiple independent clinical databases supports a hypothesis that upregulation of TLR3 and IRF3 correlates with increased overall survival among breast cancer patients. Taken together, these data support a compelling rationale to re-examine IONP formulations as harboring anticancer immune (nano)adjuvant properties to generate a therapeutic benefit without requiring uptake by cancer cells.