RESUMEN
The excessive inflammation caused by the prolonged activation of Toll-like receptor 4 (TLR4) and its downstream signaling pathways leads to sepsis. CD14-mediated endocytosis of TLR4 is the key step to control the amount of TLR4 on cell membrane and the activity of downstream pathways. The actin cytoskeleton is necessary for receptor-mediated endocytosis, but its role in TLR4 endocytosis remains elusive. Here we show that Tropomodulin 1 (Tmod1), an actin capping protein, inhibited lipopolysaccharide (LPS)-induced TLR4 endocytosis and intracellular trafficking in macrophages. Thus it resulted in increased surface TLR4 and the upregulation of myeloid differentiation factor 88 (MyD88)-dependent pathway and the downregulation of TIR domain-containing adaptor-inducing interferon-ß (TRIF)-dependent pathway, leading to the enhanced secretion of inflammatory cytokines, such as TNF-α and IL-6, and the reduced secretion of cytokines, such as IFN-ß. Macrophages deficient with Tmod1 relieved the inflammatory response in LPS-induced acute lung injury mouse model. Mechanistically, Tmod1 negatively regulated LPS-induced TLR4 endocytosis and inflammatory response through modulating the activity of CD14/Syk/PLCγ2/IP3/Ca2+ signaling pathway, the reorganization of actin cytoskeleton, and the membrane tension. Therefore, Tmod1 is a key regulator of inflammatory response and immune functions in macrophages and may be a potential target for the treatment of excessive inflammation and sepsis.
Asunto(s)
Endocitosis , Inflamación , Lipopolisacáridos , Macrófagos , Ratones Endogámicos C57BL , Transducción de Señal , Receptor Toll-Like 4 , Tropomodulina , Animales , Receptor Toll-Like 4/metabolismo , Lipopolisacáridos/farmacología , Ratones , Macrófagos/metabolismo , Macrófagos/inmunología , Inflamación/metabolismo , Inflamación/patología , Tropomodulina/metabolismo , Tropomodulina/genética , Citocinas/metabolismo , Células RAW 264.7 , Factor 88 de Diferenciación Mieloide/metabolismo , Factor 88 de Diferenciación Mieloide/genética , Receptores de Lipopolisacáridos/metabolismo , Masculino , Ratones Noqueados , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/patologíaRESUMEN
BACKGROUND: Bordetella pertussis is the causative agent of whooping cough or pertussis. Although both acellular (aP) and whole-cell pertussis (wP) vaccines protect against disease, the wP vaccine, which is highly reactogenic, is better at preventing colonization and transmission. Reactogenicity is mainly attributed to the lipid A moiety of B. pertussis lipooligosaccharide (LOS). Within LOS, lipid A acts as a hydrophobic anchor, engaging with TLR4-MD2 on host immune cells to initiate both MyD88-dependent and TRIF-dependent pathways, thereby influencing adaptive immune responses. Lipid A variants, such as monophosphoryl lipid A (MPLA) can also act as adjuvants. Adjuvants may overcome the shortcomings of aP vaccines. RESULTS: This work used lipid A modifying enzymes from other bacteria to produce an MPLA-like adjuvant strain in B. pertussis. We created B. pertussis strains with distinct lipid A modifications, which were validated using MALDI-TOF. We engineered a hexa-acylated monophosphorylated lipid A that markedly decreased human TLR4 activation and activated the TRIF pathway. The modified lipooligosaccharide (LOS) promoted IRF3 phosphorylation and type I interferon production, similar to MPLA responses. We generated three other variants with increased adjuvanticity properties and reduced endotoxicity. Pyrogenicity studies using the Monocyte Activation Test (MAT) revealed that these four lipid A variants significantly decreased the IL-6, a marker for fever, response in peripheral blood mononuclear cells (PBMCs). CONCLUSION: These findings pave the way for developing wP vaccines that are possibly less reactogenic and designing adaptable adjuvants for current vaccine formulations, advancing more effective immunization strategies against pertussis.
Asunto(s)
Adyuvantes Inmunológicos , Bordetella pertussis , Lípido A , Receptor Toll-Like 4 , Lípido A/análogos & derivados , Lípido A/inmunología , Bordetella pertussis/inmunología , Humanos , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 4/inmunología , Adyuvantes Inmunológicos/farmacología , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/inmunología , Vacuna contra la Tos Ferina/inmunología , Lipopolisacáridos , Factor 3 Regulador del Interferón/metabolismo , Tos Ferina/prevención & control , Tos Ferina/inmunología , Interleucina-6/metabolismo , Interleucina-6/inmunologíaRESUMEN
INTRODUCTION: Neuroinflammation and microglial activation-related dendritic injury contribute to the pathogenesis of Autism Spectrum Disorder (ASD). Previous studies show that Progranulin (PGRN) is a growth factor associated with inflammation and synaptic development, but the role of PGRN in autism and the mechanisms underlying changes in PGRN expression remain unclear. AIMS: To investigate the impact of PGRN in autism, we stereotactically injected recombinant PGRN into the hippocampus of ASD model rats. Additionally, we explored the possibility that sortilin may be the factor behind the alterations in PGRN by utilizing SORT1 knockdown. Ultimately, we aimed to identify potential targets for the treatment of autism. RESULTS: PGRN could alleviate inflammatory responses, protect neuronal dendritic spines, and ameliorate autism-like behaviors. Meanwhile, elevated expression of sortilin and decreased levels of PGRN were observed in both ASD patients and rats. Enhanced sortilin levels facilitated PGRN internalization into lysosomes. Notably, suppressing SORT1 expression amplified PGRN levels, lessened microglial activation, and mitigated inflammation, thereby alleviating autism-like behaviors. CONCLUSION: Collectively, our findings highlight elevated sortilin levels in ASD rat brains, exacerbating dendrite impairment by affecting PGRN expression. PGRN supplementation and SORT1 knockdown hold potential as therapeutic strategies for ASD.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular , Trastorno Autístico , Progranulinas , Ácido Valproico , Animales , Femenino , Humanos , Masculino , Ratas , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Trastorno Autístico/metabolismo , Trastorno Autístico/inducido químicamente , Trastorno Autístico/tratamiento farmacológico , Espinas Dendríticas/efectos de los fármacos , Espinas Dendríticas/patología , Espinas Dendríticas/metabolismo , Hipocampo/metabolismo , Hipocampo/efectos de los fármacos , Microglía/metabolismo , Microglía/efectos de los fármacos , Progranulinas/genética , Ratas Sprague-Dawley , Ácido Valproico/farmacologíaRESUMEN
Beige fat activation involves a fuel switch to fatty acid oxidation following chronic cold adaptation. Mitochondrial acyl-CoA synthetase long-chain family member 1 (ACSL1) localizes in the mitochondria and plays a key role in fatty acid oxidation; however, the regulatory mechanism of the subcellular localization remains poorly understood. Here, we identify an endosomal trafficking component sortilin (encoded by Sort1) in adipose tissues that shows dynamic expression during beige fat activation and facilitates the translocation of ACSL1 from the mitochondria to the endolysosomal pathway for degradation. Depletion of sortilin in adipocytes results in an increase of mitochondrial ACSL1 and the activation of AMPK/PGC1α signaling, thereby activating beige fat and preventing high-fat diet (HFD)-induced obesity and insulin resistance. Collectively, our findings indicate that sortilin controls adipose tissue fatty acid oxidation by substrate fuel selection during beige fat activation and provides a potential targeted approach for the treatment of metabolic diseases.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular , Adipocitos , Coenzima A Ligasas , Dieta Alta en Grasa , Metabolismo Energético , Mitocondrias , Animales , Masculino , Ratones , Células 3T3-L1 , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Adipocitos/metabolismo , Tejido Adiposo Beige/metabolismo , Coenzima A Ligasas/metabolismo , Coenzima A Ligasas/genética , Ácidos Grasos/metabolismo , Resistencia a la Insulina , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Obesidad/metabolismo , Obesidad/genética , Oxidación-Reducción , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Transporte de Proteínas , Transducción de Señal , TermogénesisRESUMEN
Hepatocellular carcinoma (HCC) is known to be lethal disease. However, its prognosis remains poor, primarily because the precise oncogenic mechanisms underlying HCC progression remain elusive, thus hampering effective treatment. Here, we aimed to identify the potential oncogenes in HCC and elucidate the underlying mechanisms of their action. To identify potential candidate genes, an integrative analysis of eight publicly available genomic datasets was performed, and the functional implications of the identified genes were assessed in vitro and in vivo. Sortilin 1 (SORT1) was identified as a potential candidate oncogene in HCC, and its overexpression in HCC cells was confirmed by analyzing spatial transcriptomic and single-cell data. Silencing SORT1 in Huh-7 and Hep3B cells significantly reduced HCC progression in vitro and in vivo. Functional analyses of oncogenic pathways revealed that SORT1 expression regulated the Notch signaling pathway activation and CD133 expression. Furthermore, analysis of epigenetic regulation of the candidate gene and its clinical implications using The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA LIHC) and our HCC cohort (AJOU_HCC cohort) data demonstrated an inverse correlation between the methylation status of the SORT1 promoter region, specifically at the cg16988986 site, and SORT1 mRNA expression, indicating the epigenetic regulation of SORT1 in HCC. In addition, the distinct methylation status of cg16988986 was significantly associated with patient survival. In conclusion, SORT1 plays a pivotal role in HCC by activating the Notch signaling pathway and increasing CD133 expression. These findings suggest SORT1 as a promising therapeutic target for HCC.
Asunto(s)
Antígeno AC133 , Proteínas Adaptadoras del Transporte Vesicular , Carcinoma Hepatocelular , Neoplasias Hepáticas , Neovascularización Patológica , Receptores Notch , Transducción de Señal , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Línea Celular Tumoral , Receptores Notch/metabolismo , Receptores Notch/genética , Antígeno AC133/metabolismo , Antígeno AC133/genética , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Regulación Neoplásica de la Expresión Génica , Ratones , Masculino , Ratones Desnudos , Metástasis de la Neoplasia , Femenino , Ratones Endogámicos BALB C , Epigénesis Genética , AngiogénesisRESUMEN
The functions of the natural dsRNA sensors TLR3 (TRIF) and RIG-I (MAVS) are crucial during viral challenge and have not been accurately clarified in adaptive immune responses to rotavirus (RV) infection. In this study, we found that RV infection caused severe pathological damage to the small intestine of TLR3-/- and TRIF-/- mice. Our data found that dendritic cells from TLR3-/- and TRIF-/- mice had impaired Ag presentation to the RV and attenuated initiation of T cells upon viral infection. These attenuated functions resulted in impaired CD4+ T and CD8+ T function in mice lacking TLR3-TRIF signaling postinfection. Additionally, attenuated proliferative capacity of T cells from TLR3-/- and TRIF-/- mice was observed. Subsequently, we observed a significant reduction in the absolute number of memory T cells in the spleen and mesenteric lymph node (MLN) of TRIF-/- recipient mice following RV infection in a bone marrow chimeric model. Furthermore, there was reduced migration of type 2 classical dendritic cells from the intestine to MLNs after RV infection in TLR3-/- and TRIF-/- mice. Notably, RV infection resulted in attenuated killing of spleen and MLN tissues in TRIF-/- and MAVS-/- mice. Finally, we demonstrated that RV infection promoted apoptosis of CD8+ T cells in TRIF-/- and TLR3-/-MAVS-/- mice. Taken together, our findings highlight an important mechanism of TLR3 signaling through TRIF in mucosal T cell responses to RV and lay the foundation for the development of a novel vaccine.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Células Dendríticas , Ratones Noqueados , Infecciones por Rotavirus , Rotavirus , Transducción de Señal , Receptor Toll-Like 3 , Animales , Receptor Toll-Like 3/inmunología , Ratones , Infecciones por Rotavirus/inmunología , Transducción de Señal/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Rotavirus/inmunología , Células Dendríticas/inmunología , Ratones Endogámicos C57BL , Mucosa Intestinal/inmunología , Linfocitos T CD8-positivos/inmunología , Inmunidad Mucosa , Presentación de Antígeno/inmunologíaRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Biomarcadores de Tumor , Neoplasias de la Próstata , Sindecano-1 , Neoplasias de la Próstata/patología , Masculino , Humanos , Sindecano-1/análisisRESUMEN
Spinal cord injury (SCI) patients have an increased susceptibility to coronary heart disease (CHD) due to dysregulated lipid deposition. We conducted a comprehensive investigation to gain insights into the specific roles of Apolipoprotein B-100 (APOB-100) in the development of CHD in patients suffering from SCI. First, we established an SCI rat model through semitransection. APOB-100 expression in plasma exosomes obtained from patients were determined. Subsequently, we found APOB-100 affected macrophage polarization when treating co-cultured neurons/macrophages lacking Sortilin with extracellular vesicles derived from SCI rats, where APOB-100 co-immunoprecipitated with Sortilin. Moreover, APOB-100 upregulation reduced neuronal cell viability and triggered apoptosis by upregulating Sortilin, leading to a decline in the Basso, Beattie, and Bresnahan (BBB) scale, exacerbation of neuron injury, increased macrophage infiltration, and elevated blood lipid-related indicators in SCI rats, which could be reversed by silencing Sortilin. In conclusion, APOB-100 from post-SCI patients' extracellular vesicles upregulates Sortilin, thereby endangering those patients to CHD.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular , Apolipoproteína B-100 , Enfermedad Coronaria , Vesículas Extracelulares , Traumatismos de la Médula Espinal , Animales , Apolipoproteína B-100/metabolismo , Humanos , Vesículas Extracelulares/metabolismo , Ratas , Enfermedad Coronaria/metabolismo , Enfermedad Coronaria/patología , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/patología , Masculino , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Neuronas/metabolismo , Femenino , Macrófagos/metabolismo , Persona de Mediana Edad , Modelos Animales de Enfermedad , Apoptosis , Ratas Sprague-DawleyRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular , Antineoplásicos , Camptotecina , Proliferación Celular , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Camptotecina/farmacología , Camptotecina/química , Camptotecina/síntesis química , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Proliferación Celular/efectos de los fármacos , Péptidos/química , Péptidos/farmacología , Péptidos/síntesis química , Relación Estructura-Actividad , Estructura Molecular , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Movimiento Celular/efectos de los fármacosRESUMEN
Heterozygous loss-of-function mutations in the GRN gene are a common cause of frontotemporal dementia. Such mutations lead to decreased plasma and cerebrospinal fluid levels of progranulin (PGRN), a neurotrophic factor with lysosomal functions. Sortilin is a negative regulator of extracellular PGRN levels and has shown promise as a therapeutic target for frontotemporal dementia, enabling increased extracellular PGRN levels through inhibition of sortilin-mediated PGRN degradation. Here we report the development of a high-affinity sortilin-binding affibody-peptide fusion construct capable of increasing extracellular PGRN levels in vitro. By genetic fusion of a sortilin-binding affibody generated through phage display and a peptide derived from the progranulin C-terminus, an affinity protein (A3-PGRNC15*) with 185-pM affinity for sortilin was obtained. Treating PGRN-secreting and sortilin-expressing human glioblastoma U-251 cells with the fusion protein increased extracellular PGRN levels up to 2.5-fold, with an EC50 value of 1.3 nM. Our results introduce A3-PGRNC15* as a promising new agent with therapeutic potential for the treatment of frontotemporal dementia. Furthermore, the work highlights means to increase binding affinity through synergistic contribution from two orthogonal polypeptide units.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular , Progranulinas , Proteolisis , Proteínas Recombinantes de Fusión , Progranulinas/metabolismo , Progranulinas/genética , Humanos , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/metabolismo , Línea Celular Tumoral , Unión Proteica , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/genética , Péptidos/farmacología , Péptidos/metabolismoRESUMEN
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.
Asunto(s)
Aciltransferasas , Autofagosomas , Homólogo de la Proteína 1 Relacionada con la Autofagia , Proteínas Relacionadas con la Autofagia , Autofagia , Péptidos y Proteínas de Señalización Intracelular , Lipoilación , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Autofagia/fisiología , Humanos , Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Fosforilación , Aciltransferasas/metabolismo , Aciltransferasas/genética , Autofagosomas/metabolismo , Células HEK293 , Fosfatos de Fosfatidilinositol/metabolismo , Animales , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Transporte de Proteínas , Proteínas de Transporte VesicularRESUMEN
BACKGROUND: Brain recovery mechanisms after injuries like aneurysmal subarachnoid hemorrhage (aSAH), ischemic stroke (IS), and traumatic brain injury (TBI) involve brain plasticity, synaptic regeneration, and neuroinflammation. We hypothesized that serum levels of the p75 neurotrophic receptor (p75NTR) and associated signaling proteins, as well as differentially expressed (DE) microRNAs, could predict recovery outcomes irrespective of injury type. METHODS: A prospective patient cohort with ischemic stroke (IS, n = 30), aneurysmal subarachnoid hemorrhage (aSAH, n = 31), and traumatic brain injury (TBI, n = 13) were evaluated (total n = 74). Serum samples were collected at two post-injury intervals (early: 1-3 days, late: 4-8 days), and outcomes were assessed after three months using the modified Rankin Scale (mRS), categorizing outcomes as favorable (mRS 0-3) or unfavorable (mRS 4-6). Six proteins were measured using ELISAs: p75NTR, NGF, sortilin, IL1ß, TNFα, and cyclophilin. DE microRNAs were identified using DESeq2, and their target genes were predicted. Serum molecules between patients with differing outcomes were compared using a Kolmogorov-Smirnov test, 2-tailed t-test and multivariate linear discriminant analysis (LDA). RESULTS: Favorable (n = 46) and unfavorable (n = 28) outcome cohorts were balanced with age and sex (p = 0.25 and 0.63). None of the studied proteins correlated with age. Combinatory LDA of the six protein biomarkers indicated strong prognostic value for favorable outcomes (OR 2.09; AUC = 70.3%, p = 0.0058). MicroRNA expression changes over time were identified in the aSAH, TBI, and IS groups (p < 0.05, FDR corrected). Twenty-three microRNAs were commonly DE across all brain injury groups when comparing favorable and unfavorable outcomes (p < 0.05). LDA of four microRNAs targeting the studied proteins showed high prognostic accuracy (OR 11.7; AUC = 94.1%, p = 0.016). CONCLUSIONS: The combined prognostic microRNA and protein biomarker models demonstrated accurate outcome prognostication across diverse injury types, implying the presence of a common recovery mechanism. DE microRNAs were found to target the studied molecules, suggesting a potential mechanistic role in recovery. Further investigation is warranted to study these molecules in prognostication, as well as therapeutic targets for enhancing recovery.
Asunto(s)
Biomarcadores , MicroARN Circulante , Plasticidad Neuronal , Humanos , Masculino , Femenino , Persona de Mediana Edad , Estudios Prospectivos , Biomarcadores/sangre , MicroARN Circulante/sangre , Anciano , Plasticidad Neuronal/fisiología , Adulto , Hemorragia Subaracnoidea/sangre , Lesiones Traumáticas del Encéfalo/sangre , Lesiones Traumáticas del Encéfalo/diagnóstico , Estudios de Cohortes , Enfermedades Neuroinflamatorias/sangre , Accidente Cerebrovascular Isquémico/sangre , Receptores de Factor de Crecimiento Nervioso/sangre , Receptores de Factor de Crecimiento Nervioso/genética , Recuperación de la Función/fisiología , Pronóstico , Proteínas del Tejido Nervioso , Proteínas Adaptadoras del Transporte VesicularRESUMEN
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.
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Proteínas Adaptadoras del Transporte Vesicular , Adenocarcinoma del Pulmón , Movimiento Celular , Transición Epitelial-Mesenquimal , Neoplasias Pulmonares , Invasividad Neoplásica , Femenino , Humanos , Masculino , Persona de Mediana Edad , Células A549 , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adenocarcinoma/genética , Adenocarcinoma del Pulmón/patología , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/genética , beta Catenina/metabolismo , Cadherinas/metabolismo , Cadherinas/genética , Línea Celular Tumoral , Movimiento Celular/genética , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Vía de Señalización WntRESUMEN
Mutations in the endosomal Na+/H+ exchanger 6 (NHE6) cause Christianson syndrome, an X-linked neurological disorder. NHE6 functions in regulation of endosome acidification and maturation in neurons. Using yeast two-hybrid screening with the NHE6 carboxyl terminus as bait, we identify Golgi-associated, gamma adaptin ear-containing, ADP-ribosylation factor (ARF) binding protein 1 (GGA1) as an interacting partner for NHE6. We corroborated the NHE6-GGA1 interaction using: coimmunoprecipitation; overexpressed constructs in mammalian cells; and coimmunoprecipitation of endogenously expressed GGA1 and NHE6 from neuroblastoma cells, as well as from the mouse brain. We demonstrate that GGA1 interacts with organellar NHEs (NHE6, NHE7, and NHE9) and that there is significantly less interaction with cell-surface localized NHEs (NHE1 and NHE5). By constructing hybrid NHE1/NHE6 exchangers, we demonstrate the cytoplasmic tail of NHE6 interacts most strongly with GGA1. We demonstrate the colocalization of NHE6 and GGA1 in cultured, primary hippocampal neurons, using super-resolution microscopy. We test the hypothesis that the interaction of NHE6 and GGA1 functions in the localization of NHE6 to the endosome compartment. Using subcellular fractionation experiments, we show that NHE6 is mislocalized in GGA1 KO cells, wherein we find less NHE6 in endosomes, but more NHE6 transport to lysosomes, and more Golgi retention of NHE6, with increased exocytosis to the surface plasma membrane. Consistent with NHE6 mislocalization, and Golgi retention, we find the intraluminal pH in Golgi to be alkalinized in GGA1-null cells. Our study demonstrates a new interaction between NHE6 and GGA1 which functions in the localization of this intracellular NHE to the endosome compartment.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular , Endosomas , Neuronas , Intercambiadores de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/metabolismo , Intercambiadores de Sodio-Hidrógeno/genética , Animales , Endosomas/metabolismo , Humanos , Ratones , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Neuronas/metabolismo , Hipocampo/metabolismo , Transporte de Proteínas , Unión ProteicaRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Precursor de Proteína beta-Amiloide , Proteínas Relacionadas con la Autofagia , Autofagia , Humanos , Precursor de Proteína beta-Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Células HEK293 , Unión Proteica , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular , Secuencia de Aminoácidos , Lubina , Enfermedades de los Peces , Proteínas de Peces , FN-kappa B , Filogenia , Animales , Lubina/inmunología , Lubina/genética , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Proteínas de Peces/química , FN-kappa B/genética , FN-kappa B/metabolismo , FN-kappa B/inmunología , Enfermedades de los Peces/inmunología , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/inmunología , Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Transducción de Señal/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Alineación de Secuencia/veterinaria , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Factor 88 de Diferenciación Mieloide/inmunología , Factor 88 de Diferenciación Mieloide/química , Perfilación de la Expresión Génica/veterinaria , Receptores Toll-Like/genética , Receptores Toll-Like/inmunología , Receptores Toll-Like/química , Receptores Toll-Like/metabolismo , Secuencia de BasesRESUMEN
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.
Asunto(s)
Calcio , Vesículas Sinápticas , Sinaptotagmina I , Sinaptotagmina I/metabolismo , Sinaptotagmina I/genética , Calcio/metabolismo , Humanos , Animales , Vesículas Sinápticas/metabolismo , Multimerización de Proteína , Proteínas SNARE/metabolismo , Proteínas SNARE/genética , Transición de Fase , Mutación/genética , Células HEK293 , Fusión de Membrana , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Separación de FasesRESUMEN
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.
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Macrófagos , Neutrófilos , Piroptosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Receptores Tipo I de Factores de Necrosis Tumoral , Transducción de Señal , Receptor Toll-Like 4 , Macrófagos/metabolismo , Neutrófilos/metabolismo , Animales , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Ratones , Receptor Toll-Like 4/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Ratones Endogámicos C57BL , Interferón gamma/metabolismo , Ratones NoqueadosRESUMEN
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.
Asunto(s)
Proteínas del Tejido Nervioso , Vesículas Sinápticas , Animales , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/genética , Ratones , Masculino , Femenino , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/química , Mutación , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/química , Fusión de Membrana/fisiología , Fusión de Membrana/genética , Células Cultivadas , Fenotipo , Neuronas/metabolismo , Transmisión Sináptica/genética , Transmisión Sináptica/fisiología , Ratones Endogámicos C57BL , Exocitosis/fisiología , Exocitosis/genéticaRESUMEN
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.