RESUMO
PURPOSE: DNA methylation prominently inactivates tumor suppressor genes and facilitates oncogenesis. Previously, we delineated a chromosome 18 deletion encompassing the erythrocyte membrane protein band 4.1-like 3 (EPB41L3) gene, a progenitor for the tumor suppressor that is differentially expressed in adenocarcinoma of the lung-1 (DAL-1) in gastric cancer (GC). METHODS: Our current investigation aimed to elucidate EPB41L3 expression and methylation in GC, identify regulatory transcription factors, and identify affected downstream pathways. Immunohistochemistry demonstrated that DAL-1 expression is markedly reduced in GC tissues, with its downregulation serving as an independent prognostic marker. RESULTS: High-throughput bisulfite sequencing of 70 GC patient tissue pairs revealed that higher methylation of non-CpGs in the EPB41L3 promoter was correlated with more malignant tumor progression and higher-grade tissue classification. Such hypermethylation was shown to diminish DAL-1 expression, thus contributing to the malignancy of GC phenotypes. The DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) was found to partially restore DAL-1 expression. Moreover, direct binding of the transcription factor CDC5L to the upstream region of the EPB41L3 promoter was identified via chromosome immunoprecipitation (ChIP)-qPCR and luciferase reporter assays. Immunohistochemistry confirmed the positive correlation between CDC5L and DAL-1 protein levels. Subsequent RNA-seq analysis revealed that DAL-1 significantly influences the extracellular matrix and space-related pathways. GC cell RNA-seq post-5-Aza-CdR treatment and single-cell RNA-seq data of GC tissues confirmed the upregulation of AREG and COL17A1, pivotal tumor suppressors, in response to EPB41L3 demethylation or overexpression in GC epithelial cells. CONCLUSION: In conclusion, this study elucidates the association between non-CpG methylation of EPB41L3 and GC progression and identifies the key transcription factors and downstream molecules involved. These findings enhance our understanding of the role of EPB41L3 in gastric cancer and provide a solid theoretical foundation for future research and potential clinical applications.
The EPB41L3 gene, frequently exhibiting haplotype deletions and reduced expression in gastric cancer tissues, points to its potential role as a tumor suppressor. However, tumor suppressor genes are not only influenced by genomic deletions but also by their methylation status. Our study highlights the significantly lower expression of EPB41L3 in gastric cancer compared to adjacent non-cancerous tissues across 262 patients. We also discovered that elevated non-CpG island methylation of EPB41L3 correlates strongly with tumor malignancy progression, based on the analysis of 70 paired gastric cancer samples. Moreover, we identified CDC5L as a crucial transcription factor interacting with the EPB41L3 promoter. Integrative analyses of transcriptomic and single-cell sequencing data further revealed that AREG and COL17A1 are key downstream molecules regulated by DAL-1, with their expression tightly controlled by EPB41L3 methylation and expression levels. These insights enhance our understanding of EPB41L3's role in gastric cancer and could open new avenues for targeted therapies.
Assuntos
Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Regiões Promotoras Genéticas , Neoplasias Gástricas , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Neoplasias Gástricas/metabolismo , Humanos , Metilação de DNA/genética , Feminino , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/genética , Linhagem Celular Tumoral , Idoso , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas dos MicrofilamentosRESUMO
The members of the evolutionary conserved actin-binding Ezrin, Radixin and Moesin (ERM) protein family are involved in numerous key cellular processes in the cytoplasm. In the last decades, ERM proteins, like actin and other cytoskeletal components, have also been shown to be functional components of the nucleus; however, the molecular mechanism behind their nuclear activities remained unclear. Therefore, our primary aim was to identify the nuclear protein interactome of the single Drosophila ERM protein, Moesin. We demonstrate that Moesin directly interacts with the Mediator complex through direct binding to its Med15 subunit, and the presence of Moesin at the regulatory regions of the Hsp70Ab heat shock gene was found to be Med15-dependent. Both Moesin and Med15 bind to heat shock factor (Hsf), and they are required for proper Hsp gene expression under physiological conditions. Moreover, we confirmed that Moesin, Med15 and Hsf are able to bind the monomeric form of actin and together they form a complex in the nucleus. These results elucidate a mechanism by which ERMs function within the nucleus. Finally, we present the direct interaction of the human orthologues of Drosophila Moesin and Med15, which highlights the evolutionary significance of our finding.
Assuntos
Núcleo Celular , Proteínas de Drosophila , Resposta ao Choque Térmico , Proteínas dos Microfilamentos , Ligação Proteica , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Núcleo Celular/metabolismo , Humanos , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/genética , Complexo Mediador/metabolismo , Complexo Mediador/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Actinas/metabolismo , Regulação da Expressão Gênica , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de MembranaRESUMO
Tuft cells are a rare epithelial cell type that play important roles in sensing and responding to luminal antigens. A defining morphological feature of this lineage is the actin-rich apical "tuft," which contains large fingerlike protrusions. However, details of the cytoskeletal ultrastructure underpinning the tuft, the molecules involved in building this structure, or how it supports tuft cell biology remain unclear. In the context of the small intestine, we found that tuft cell protrusions are supported by long-core bundles that consist of F-actin crosslinked in a parallel and polarized configuration; they also contain a tuft cell-specific complement of actin-binding proteins that exhibit regionalized localization along the bundle axis. Remarkably, in the sub-apical cytoplasm, the array of core actin bundles interdigitates and co-aligns with a highly ordered network of microtubules. The resulting cytoskeletal superstructure is well positioned to support subcellular transport and, in turn, the dynamic sensing functions of the tuft cell that are critical for intestinal homeostasis.
Assuntos
Actinas , Citoesqueleto , Células Epiteliais , Mucosa Intestinal , Microtúbulos , Animais , Mucosa Intestinal/ultraestrutura , Mucosa Intestinal/metabolismo , Mucosa Intestinal/citologia , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Actinas/metabolismo , Camundongos , Intestino Delgado/metabolismo , Intestino Delgado/ultraestrutura , Intestino Delgado/citologia , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Células em TufoRESUMO
Migration of endothelial and many other cells requires spatiotemporal regulation of protrusive and contractile cytoskeletal rearrangements that drive local cell shape changes. Unexpectedly, the small GTPase Rho, a crucial regulator of cell movement, has been reported to be active in both local cell protrusions and retractions, raising the question of how Rho activity can coordinate cell migration. Here, we show that Rho activity is absent in local protrusions and active during retractions. During retractions, Rho rapidly activated ezrin-radixin-moesin proteins (ERMs) to increase actin-membrane attachment, and, with a delay, nonmuscle myosin 2 (NM2). Rho activity was excitable, with NM2 acting as a slow negative feedback regulator. Strikingly, inhibition of SLK/LOK kinases, through which Rho activates ERMs, caused elongated cell morphologies, impaired Rho-induced cell contractions, and reverted Rho-induced blebbing. Together, our study demonstrates that Rho activity drives retractions by sequentially enhancing ERM-mediated actin-membrane attachment for force transmission and NM2-dependent contractility.
Assuntos
Actomiosina , Movimento Celular , Forma Celular , Proteínas do Citoesqueleto , Actomiosina/metabolismo , Humanos , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas rho de Ligação ao GTP/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Membrana/metabolismo , Actinas/metabolismo , Animais , Miosina Tipo II/metabolismoRESUMO
The KRAS oncogene drives many common and highly fatal malignancies. These include pancreatic, lung, and colorectal cancer, where various activating KRAS mutations have made the development of KRAS inhibitors difficult. Here we identify the scaffold protein SH3 and multiple ankyrin repeat domain 3 (SHANK3) as a RAS interactor that binds active KRAS, including mutant forms, competes with RAF and limits oncogenic KRAS downstream signalling, maintaining mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) activity at an optimal level. SHANK3 depletion breaches this threshold, triggering MAPK/ERK signalling hyperactivation and MAPK/ERK-dependent cell death in KRAS-mutant cancers. Targeting this vulnerability through RNA interference or nanobody-mediated disruption of the SHANK3-KRAS interaction constrains tumour growth in vivo in female mice. Thus, inhibition of SHANK3-KRAS interaction represents an alternative strategy for selective killing of KRAS-mutant cancer cells through excessive signalling.
Assuntos
Sistema de Sinalização das MAP Quinases , Mutação , Proteínas do Tecido Nervoso , Proteínas Proto-Oncogênicas p21(ras) , Animais , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Humanos , Camundongos , Linhagem Celular Tumoral , Feminino , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Sistema de Sinalização das MAP Quinases/genética , Morte Celular/genética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Camundongos Nus , Proteínas dos MicrofilamentosRESUMO
Abnormalities in gamma-aminobutyric acid (GABA)ergic neurotransmission play a role in the pathogenesis of autism, although the mechanisms responsible for alterations in specific brain regions remain unclear. Deficits in social motivation and interactions are core symptoms of autism, likely due to defects in dopaminergic neural pathways. Therefore, investigating the morphology and functional roles of GABAergic neurons within dopaminergic projection areas could elucidate the underlying etiology of autism. The aim of this study was to (1) compare the morphology and arborization of glutamate decarboxylase (GAD)-positive neurons from the midbrain tegmentum; (2) evaluate synaptic activity in primary neurons from the striatum; and (3) assess GABAergic postsynaptic puncta in the ventral striatum of wild-type (WT) and Shank3-deficient mice. We found a significant decrease in the number of short neurites in GAD positive primary neurons from the midbrain tegmentum in Shank3-deficient mice. The application of a specific blocker of GABAA receptors (GABAAR) revealed significantly increased frequency of spontaneous postsynaptic currents (sPSCs) in Shank3-deficient striatal neurons compared to their WT counterparts. The mean absolute amplitude of the events was significantly higher in striatal neurons from Shank3-deficient compared to WT mice. We also observed a significant reduction in gephyrin/GABAAR γ2 colocalization in the striatum of adult male Shank3-deficient mice. The gene expression of collybistin was significantly lower in the nucleus accumbens while gephyrin and GABAAR γ2 were lower in the ventral tegmental area (VTA) in male Shank3-deficient compared to WT mice. In conclusion, Shank3 deficiency leads to alterations in GABAergic neurons and impaired GABAergic function in dopaminergic brain areas. These changes may underlie autistic symptoms, and potential interventions modulating GABAergic activity in dopaminergic pathways may represent new treatment modality.
Assuntos
Corpo Estriado , Neurônios GABAérgicos , Mesencéfalo , Proteínas do Tecido Nervoso , Sinapses , Animais , Neurônios GABAérgicos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/deficiência , Mesencéfalo/metabolismo , Mesencéfalo/patologia , Sinapses/metabolismo , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Biomarcadores/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/deficiência , Glutamato Descarboxilase/metabolismo , Forma Celular , Ácido gama-Aminobutírico/metabolismo , Camundongos Endogâmicos C57BL , Camundongos , Masculino , Camundongos Knockout , Receptores de GABA-A/metabolismo , Proteínas de MembranaRESUMO
Autism Spectrum Disorder (ASD) is a group of neurodevelopmental disorders with heterogeneous symptomatology. Arguably, the most pervasive shortfall of ASD are the deficits in sociability and the animal models of the disorder are expected to exhibit such impairments. The most widely utilized behavioral task for assessing sociability in rodents is the Three-Chamber Social Interaction Test (SIT). However, SIT has been yielding inconsistent results in social interaction behavior across different rodent models of ASD, which could be pointing to the suboptimal methodology of the task. Here, we compared social behavior assessed in SIT and in another prominent sociability behavioral assay, Reciprocal Interaction Test (RCI), in a SH3 and multiple ankyrin repeated domains 3 (SHANK3) mouse model of ASD. Head-to-head comparison showed no association (p = 0.15, 0.25, 0.43) and a fixed bias (p = 0.01, < 0.001, < 0.001) in sociability assessment between the behavioral assays in both wild-type (WT) controls and Shank3B(-/-) mice. Adult Shank3B(-/-) mice of both sexes displayed normative sociability in SIT when compared to the WT controls (p = 0.74) but exhibited less than half of social interaction (p < 0.001) and almost three times more social disinterest (p < 0.001) when compared to WT mice in RCI. At least in the Shank3B(-/-) mouse model of ASD, we presume RCI could be a preferable way of assessing social interaction compared to SIT. Considering the variability of animal models of ASD and the wide palette of tools available for the assessment of their behavior, a consensus approach would be needed for observational and interventional analyses.
Assuntos
Transtorno do Espectro Autista , Modelos Animais de Doenças , Proteínas do Tecido Nervoso , Comportamento Social , Interação Social , Animais , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/psicologia , Masculino , Camundongos , Feminino , Proteínas do Tecido Nervoso/genética , Comportamento Animal/fisiologia , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/genética , Camundongos KnockoutRESUMO
Stromal cells play a critical role in the tumor microenvironment of breast cancer (BC), as they are recruited by tumor cells and regulate the metastatic spread. Though high expression of α-parvin, a member of the parvin family of actin-binding proteins, is reported to be associated with a poor prognosis and metastasis in several cancers, its role in carcinogenesis has not been thoroughly explored. Therefore, we aimed to examine the expression of α-parvin in BC patients by compartmentalizing and quantifying tissues to determine whether α-parvin can be a potential therapeutic target. We performed immunohistochemical (IHC) staining of α-parvin in BC tissues, and the IHC scores were calculated in the overall tissue, stroma, and epithelium using image analysis software. The expression of α-parvin was significantly higher in BC tissues (p = 0.0002) and BC stroma (p < 0.0001) than in normal tissues. Furthermore, all α-parvin scores were significantly positively correlated with the proliferation marker Ki67. The overall and stroma scores are associated with the tumor, (lymph) node, and metastasis (TNM) classification, stage, and grade. These results suggest that high expression of α-parvin in stroma is associated with BCs and might be a new predictive marker for diagnosing BC.
Assuntos
Neoplasias da Mama , Humanos , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Feminino , Prognóstico , Pessoa de Meia-Idade , Biomarcadores Tumorais/metabolismo , Adulto , Proteínas dos Microfilamentos/metabolismo , Células Estromais/metabolismo , Células Estromais/patologia , Idoso , Microambiente Tumoral , Antígeno Ki-67/metabolismo , Imuno-HistoquímicaRESUMO
Neuronal development is a highly regulated process that is dependent on the correct coordination of cellular responses to extracellular cues. In response to semaphorin axon guidance proteins, the MICAL1 protein is stimulated to produce reactive oxygen species that oxidize actin on specific methionine residues, leading to filamentous actin depolymerization and consequent changes in neuronal growth cone dynamics. Crossing genetically modified mice homozygous for floxed Mical1 (Mical1fl/fl) alleles with transgenic mice expressing Cre recombinase under the control of a tyrosinase gene enhancer/promoter (Tyr::Cre) enabled conditional Mical1 deletion. Immunohistochemical analysis showed Mical1 expression in the cerebellum, which plays a prominent role in the coordination of motor movements, with reduced Mical1 expression in Mical1fl/fl mice co-expressing Tyr::Cre. Analysis of the gaits of mice running on a treadmill showed that both male and female Mical1fl/fl, Tyr::Cre mutant mice had significant alterations to their striding patterns relative to wild-type mice, although the specific aspects of their altered gaits differed between the sexes. Additional motor tests that involved movement on a rotating rod, descending a vertical pole, or crossing a balance beam did not show significant differences between the genotypes, suggesting that the effect of the Mical1fl/fl, Tyr::Cre genetic modifications was only manifested during specific highly coordinated movements that contribute to running. These findings indicate that there is a behavioral consequence in Mical1fl/fl, Tyr::Cre mutant mice that affects motor control as manifested by alterations in their gait.
Assuntos
Monofenol Mono-Oxigenase , Animais , Camundongos , Feminino , Masculino , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo , Marcha/genética , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Camundongos Transgênicos , Cerebelo/metabolismo , Corrida/fisiologia , Camundongos Endogâmicos C57BLRESUMO
Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder characterized by difficulties in social interaction and communication, repetitive behaviors, and restricted interests. Unfortunately, the underlying molecular mechanism behind ASD remains unknown. It has been reported that oxidative and nitrosative stress are strongly linked to ASD. We have recently found that nitric oxide (NOâ¢) and its products play an important role in this disorder. One of the key proteins associated with NO⢠is thioredoxin (Trx). We hypothesize that the Trx system is altered in the Shank3 KO mouse model of autism, which may lead to a decreased activity of the nuclear factor erythroid 2-related factor 2 (Nrf2), resulting in oxidative stress, and thus, contributing to ASD-related phenotypes. To test this hypothesis, we conducted in vivo behavioral studies and used primary cortical neurons derived from the Shank3 KO mice and human SH-SY5Y cells with SHANK3 mutation. We showed significant changes in the levels and activity of Trx redox proteins in the Shank3 KO mice. A Trx1 inhibitor PX-12 decreased Trx1 and Nrf2 expression in wild-type mice, causing abnormal alterations in the levels of synaptic proteins and neurotransmission markers, and an elevation of nitrosative stress. Trx inhibition resulted in an ASD-like behavioral phenotype, similar to that of Shank3 KO mice. Taken together, our findings confirm the strong link between the Trx system and ASD pathology, including the increased oxidative/nitrosative stress, and synaptic and behavioral deficits. The results of this study may pave the way for identifying novel drug targets for ASD.
Assuntos
Proteínas dos Microfilamentos , Fator 2 Relacionado a NF-E2 , Proteínas do Tecido Nervoso , Tiorredoxinas , Animais , Camundongos , Tiorredoxinas/metabolismo , Tiorredoxinas/genética , Humanos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/genética , Linhagem Celular Tumoral , Transtorno do Espectro Autista/metabolismo , Transtorno do Espectro Autista/genética , Neurônios/metabolismo , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Masculino , Células Cultivadas , Estresse OxidativoRESUMO
BACKGROUND: The Coiled-Coil Domain-Containing Protein 88 A (CCDC88A) gene encodes the actin-binding protein Girdin, which plays important roles in maintaining the actin cytoskeleton and in cell migration and was recently associated with a specific form of epileptic encephalopathy. Biallelic protein-truncating variants of CCDC88A have been considered responsible for progressive encephalopathy with edema, hypsarrhythmia, and optic atrophy (PEHO)-like syndrome. To date, only three consanguineous families with loss-of-function homozygous variants in the CCDC88A gene have been reported. The described patients share many clinical features, such as microcephaly, neonatal hypotonia, seizures, profound developmental delay, face and limb edema, and dysmorphic features, with a similar appearance of the eyes, nose, mouth, and fingers. CASE PRESENTATION: We report on a child from a nonconsanguineous family who presented with profound global developmental delay, severe epilepsy, and brain malformations, including subcortical band heterotopia. The patient harbored two heterozygous pathogenic variants in the trans configuration in the CCDC88A gene, which affected the coiled-coil and C-terminal domains. CONCLUSIONS: We detail the clinical and cerebral imaging data of our patient in the context of previously reported patients with disease-causing variants in the CCDC88A gene, emphasizing the common phenotypes, including cortical malformations, that warrant screening for sequence variants in this gene.
Assuntos
Fenótipo , Humanos , Masculino , Proteínas dos Microfilamentos/genética , Atrofia Óptica/genética , Atrofia Óptica/diagnóstico , Proteínas de Transporte Vesicular/genética , Lactente , Feminino , Epilepsia/genética , Edema Encefálico , Espasmos Infantis , Doenças NeurodegenerativasRESUMO
Formin HOmology Domain 2-containing (FHOD) proteins are a subfamily of actin-organizing formins important for striated muscle development in many animals. We showed previously that absence of the sole FHOD protein, FHOD-1, from Caenorhabditis elegans results in thin body wall muscles with misshapen dense bodies that serve as sarcomere Z-lines. We demonstrate here that mutations predicted to specifically disrupt actin polymerization by FHOD-1 similarly disrupt muscle development, and that FHOD-1 cooperates with profilin PFN-3 for dense body morphogenesis, and with profilins PFN-2 and PFN-3 to promote body wall muscle growth. We further demonstrate that dense bodies in worms lacking FHOD-1 or PFN-2/PFN-3 are less stable than in wild-type animals, having a higher proportion of dynamic protein, and becoming distorted by prolonged muscle contraction. We also observe accumulation of actin and actin depolymerization factor/cofilin homologue UNC-60B in body wall muscle of these mutants. Such accumulations may indicate targeted disassembly of thin filaments dislodged from unstable dense bodies, possibly accounting for the abnormally slow growth and reduced body wall muscle strength in fhod-1 mutants. Overall, these results implicate FHOD protein-mediated actin assembly in forming stable sarcomere Z-lines, and identify profilin as a new contributor to FHOD activity in striated muscle development.
Assuntos
Actinas , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Forminas , Contração Muscular , Profilinas , Sarcômeros , Animais , Caenorhabditis elegans/metabolismo , Profilinas/metabolismo , Profilinas/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Sarcômeros/metabolismo , Contração Muscular/fisiologia , Forminas/metabolismo , Actinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/genética , Mutação/genética , Desenvolvimento Muscular/fisiologia , Citoesqueleto de Actina/metabolismo , Músculo Estriado/metabolismo , Músculos/metabolismo , Fatores de Despolimerização de Actina/metabolismoRESUMO
Enveloped viruses enter the host cell by fusing at the cell membrane or entering the cell via endocytosis and fusing at the endosome. Conventional inhibitors target the viral fusion protein to inactivate it for inducing fusion. These target-specific vis-à-vis virus-specific inhibitors fail to display their inhibitory efficacy against emerging and remerging viral infections. This necessitates the need to develop broad-spectrum entry inhibitors that are effective irrespective of the virus. Using a broad range of targeting techniques, the fusion inhibitors can modify the physical characteristics of the viral membrane, making it less prone to fusion. We have previously shown that two tryptophan-aspartic acid (WD)-containing hydrophobic peptides, TG-23 and GG-21, from coronin 1, a phagosomal protein, inhibit membrane fusion by modulating membrane organization and dynamics. In the present work, we designed two WD-containing hydrophilic peptides, QG-22 and AG-22, using coronin 1 as a template and evaluated their fusion inhibitory efficacies in the absence and presence of membrane cholesterol. Our results demonstrate that QG-22 and AG-22 inhibit membrane fusion irrespective of the concentration of membrane cholesterol. Our measurements of depth-dependent membrane organization and dynamics reveal that they impede fusion by enhancing the acyl chain order. Overall, our results validate the hypothesis of designing fusion inhibitors by modulating the membrane's physical properties. In addition, it demonstrates that chain hydrophobicity might not be a critical determinant for the development of peptide-based fusion inhibitors.
Assuntos
Ácido Aspártico , Peptídeos , Triptofano , Triptofano/química , Triptofano/farmacologia , Ácido Aspártico/química , Ácido Aspártico/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/antagonistas & inibidores , Fusão de Membrana/efeitos dos fármacos , Desenho de Fármacos , Interações Hidrofóbicas e Hidrofílicas , Colesterol/químicaRESUMO
Social thermoregulation is a means of maintaining homeostatic body temperature. While adult mice are a model organism for studying both social behavior and energy regulation, the relationship between huddling and core body temperature (Tb) is poorly understood. Here, we develop a behavioral paradigm and computational tools to identify active-huddling and quiescent-huddling as distinct thermal substates. We find that huddling is an effective thermoregulatory strategy in female but not male groups. At 23 °C (room temperature), but not 30 °C (near thermoneutrality), huddling facilitates large reductions in Tb and Tb-variance. Notably, active-huddling is associated with bidirectional changes in Tb, depending on its proximity to bouts of quiescent-huddling. Further, group-housed animals lacking the synaptic scaffolding gene Shank3b have hyperthermic Tb and spend less time huddling. In contrast, individuals lacking the cold-sensing gene Trpm8 have hypothermic Tb - a deficit that is rescued by increased huddling time. These results reveal how huddling behavior facilitates acute adjustments of Tb in a state-dependent manner.
Assuntos
Regulação da Temperatura Corporal , Proteínas do Tecido Nervoso , Canais de Cátion TRPM , Animais , Camundongos , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Masculino , Feminino , Regulação da Temperatura Corporal/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Mutação , Camundongos Endogâmicos C57BL , Temperatura Corporal , Comportamento Social , Comportamento Animal , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Camundongos KnockoutRESUMO
miR-31 is a highly conserved microRNA that plays crucial roles in cell proliferation, migration and differentiation. We discovered that miR-31 and some of its validated targets are enriched on the mitotic spindle of the dividing sea urchin embryo and mammalian cells. Using the sea urchin embryo, we found that miR-31 inhibition led to developmental delay correlated with increased cytoskeletal and chromosomal defects. We identified miR-31 to directly suppress several actin remodeling transcripts, including ß-actin, Gelsolin, Rab35 and Fascin. De novo translation of Fascin occurs at the mitotic spindle of sea urchin embryos and mammalian cells. Importantly, miR-31 inhibition leads to a significant a increase of newly translated Fascin at the spindle of dividing sea urchin embryos. Forced ectopic localization of Fascin transcripts to the cell membrane and translation led to significant developmental and chromosomal segregation defects, highlighting the importance of the regulation of local translation by miR-31 at the mitotic spindle to ensure proper cell division. Furthermore, miR-31-mediated post-transcriptional regulation at the mitotic spindle may be an evolutionarily conserved regulatory paradigm of mitosis.
Assuntos
MicroRNAs , Biossíntese de Proteínas , Fuso Acromático , Animais , MicroRNAs/metabolismo , MicroRNAs/genética , Fuso Acromático/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/genética , Mitose/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Desenvolvimento Embrionário/genética , Embrião não Mamífero/metabolismo , Segregação de Cromossomos/genética , Actinas/metabolismo , Actinas/genética , Ouriços-do-Mar/embriologia , Ouriços-do-Mar/genética , Ouriços-do-Mar/metabolismoRESUMO
Plastin-3 (PLS3) encodes T-plastin, an actin-bundling protein mediating the formation of actin filaments by which numerous cellular processes are regulated. Loss-of-function genetic defects in PLS3 are reported to cause X-linked osteoporosis and childhood-onset fractures. However, the molecular etiology of PLS3 remains elusive. Functional compensation by actin-bundling proteins ACTN1, ACTN4, and FSCN1 was investigated in zebrafish following morpholino-mediated pls3 knockdown. Primary dermal fibroblasts from six patients with a PLS3 variant were also used to examine expression of these proteins during osteogenic differentiation. In addition, Pls3 knockdown in the murine MLO-Y4 cell line was employed to provide insights in global gene expression. Our results showed that ACTN1 and ACTN4 can rescue the skeletal deformities in zebrafish after pls3 knockdown, but this was inadequate for FSCN1. Patients' fibroblasts showed the same osteogenic transdifferentiation ability as healthy donors. RNA-seq results showed differential expression in Wnt1, Nos1ap, and Myh3 after Pls3 knockdown in MLO-Y4 cells, which were also associated with the Wnt and Th17 cell differentiation pathways. Moreover, WNT2 was significantly increased in patient osteoblast-like cells compared to healthy donors. Altogether, our findings in different bone cell types indicate that the mechanism of PLS3-related pathology extends beyond actin-bundling proteins, implicating broader pathways of bone metabolism.
Assuntos
Diferenciação Celular , Glicoproteínas de Membrana , Proteínas dos Microfilamentos , Osteogênese , Peixe-Zebra , Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Animais , Osteogênese/genética , Humanos , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/genética , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Fibroblastos/metabolismo , Osteoblastos/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Técnicas de Silenciamento de GenesRESUMO
Osteoarthritis (OA) is the most common form of arthritic disease, and phenotypic modification of chondrocytes is an important mechanism that contributes to the loss of cartilage homeostasis. This study identified that Fascin actin-bundling protein 1 (FSCN1) plays a pivotal role in regulating chondrocytes phenotype and maintaining cartilage homeostasis. Proteome-wide screening revealed markedly upregulated FSCN1 protein expression in human OA cartilage. FSCN1 accumulation was confirmed in the superficial layer of OA cartilage from humans and mice, primarily in dedifferentiated-like chondrocytes, associated with enhanced actin stress fiber formation and upregulated type I and III collagens. FSCN1-inducible knockout mice exhibited delayed cartilage degeneration following experimental OA surgery. Mechanistically, FSCN1 promoted actin polymerization and disrupted the inhibition of Decorin on TGF-ß1, leading to excessive TGF-ß1 production and ALK1/Smad1/5 signaling activation, thus, accelerated chondrocyte dedifferentiation. Intra-articular injection of FSCN1-overexpressing adeno-associated virus exacerbated OA progression in mice, which was mitigated by an ALK1 inhibitor. Moreover, FSCN1 inhibitor NP-G2-044 effectively reduced extracellular matrix degradation in OA mice, cultured human OA chondrocytes, and cartilage explants by suppressing ALK1/Smad1/5 signaling. These findings suggest that targeting FSCN1 represents a promising therapeutic approach for OA.
Assuntos
Proteínas de Transporte , Condrócitos , Proteínas dos Microfilamentos , Osteoartrite , Animais , Humanos , Masculino , Camundongos , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Condrócitos/metabolismo , Condrócitos/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Osteoartrite/patologia , Osteoartrite/metabolismo , Osteoartrite/genética , Fenótipo , Receptores Odorantes , Transdução de SinaisRESUMO
BACKGROUND: The actin-binding protein anillin (ANLN) functions as an oncogene in various cancers but has not been fully studied in oral squamous cell carcinoma (OSCC). This study aimed to investigate the expression of ANLN in OSCC tissues and cell lines, to better understand its role in mediating proliferative, angiogenic, invasive, and metastatic capabilities in this type of cancer. METHODS: ANLN mRNA and protein levels were assessed using qPCR and western immunoblotting. The expression intensity of ANLN was evaluated using immunohistochemical (IHC) staining. Biological functional assays were employed to characterize the behavior of OSCC cells influenced by ANLN. Additionally, comprehensive bioinformatics analysis, including GO analysis and KEGG enrichment analysis, was performed on differentially expressed genes in ANLN-mediated pathways. RESULTS: OSCC tumors and cell lines exhibited higher ANLN expression. Silencing of ANLN significantly suppressed OSCC cell proliferation, as evidenced by a significant reduction in the Ki-67 index both in vitro and in vivo. The migration and invasive ability of OSCC cells were markedly diminished, coinciding with a decrease in epithelial-mesenchymal transition activity. ANLN was also found to promote angiogenic activity in OSCC cells, partly through synergistic effects mediated by vascular endothelial growth factor A (VEGFA). Downregulation of ANLN expression led to decreased VEGFA levels, resulting in reduced angiogenesis characterized by fewer vascular branches. CONCLUSIONS: Our findings highlight the promising role of ANLN as a biomarker for both diagnostic and prognostic in OSCC. Targeting ANLN with inhibitory strategies could impede the oncogenesis processes at the core of OSCC development, presenting significant opportunities for advancing therapeutic interventions.
Assuntos
Movimento Celular , Proliferação de Células , Proteínas dos Microfilamentos , Neoplasias Bucais , Invasividade Neoplásica , Neovascularização Patológica , Humanos , Neoplasias Bucais/patologia , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , Proliferação de Células/genética , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Neovascularização Patológica/metabolismo , Movimento Celular/genética , Invasividade Neoplásica/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/genética , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Animais , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Inativação Gênica , Camundongos , Transição Epitelial-Mesenquimal/genética , Feminino , Masculino , AngiogêneseRESUMO
Introduction: Autism spectrum disorder (ASD) is a heterogeneous group of neurodevelopmental Q8 conditions characterized by deficits in social interaction/communication and restrictive/repetitive behaviors. Recent studies highlight the role of immune system dysfunction and inflammation in ASD pathophysiology. Indeed, elevated levels of pro-inflammatory cytokines were described in the brain and peripheral blood of ASD individuals. Despite this, how this pro-inflammatory profile evolves with aging and whether it may be associated with behavioral deficits is unknown. In this work, we explored the impact of aging on motor behavior and inflammation using Shank3b mutant mice, a model for syndromic ASD. Methods: Using RT-qPCR and flow cytometry, we examined the expression of key pro-inflammatory molecules in the cerebellum, bone marrow, spleen, and peripheral blood, comparing adult and old Shank3b +/+, Shank3b +/-, and Shank3b -/- mice. Results and discussion: Our findings revealed genotype- and age-related differences in inflammation and motor behavior, with Shank3b-/- mice exhibiting accelerated aging and motor impairments. Correlations between pro-inflammatory molecules and behavioral deficits suggest that a link may be present between systemic inflammation and ASD-related behaviors, underscoring the potential role of age-related inflammation ("inflammaging") in exacerbating ASD symptoms.
Assuntos
Envelhecimento , Transtorno do Espectro Autista , Modelos Animais de Doenças , Inflamação , Proteínas dos Microfilamentos , Proteínas do Tecido Nervoso , Animais , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/imunologia , Proteínas do Tecido Nervoso/genética , Camundongos , Envelhecimento/imunologia , Envelhecimento/genética , Inflamação/imunologia , Inflamação/genética , Proteínas dos Microfilamentos/genética , Camundongos Knockout , Masculino , Camundongos Endogâmicos C57BL , Sistema Imunitário/imunologia , Sistema Imunitário/metabolismo , Citocinas/metabolismo , Comportamento AnimalRESUMO
BACKGROUND: LARC patients commonly receive adjuvant therapy, however, hidden micrometastases still limit the improvement of OS. This study aims to investigate the impact of VASN in rectal cancer with pulmonary metastasis and understand the underlying molecular mechanisms to guide adjuvant chemotherapy selection. METHODS: Sequencing data from rectal cancer patients with pulmonary metastasis from Sun Yat-sen University Cancer Center (SYSUCC) and publicly available data were meticulously analyzed. The functional role of VASN in pulmonary metastasis was validated in vivo and in vitro. Coimmunoprecipitation (co-IP), immunofluorescence, and rescue experiments were conducted to unravel potential molecular mechanisms of VASN. Moreover, VASN expression levels in tumor samples were examined and analyzed for their correlations with pulmonary metastasis status, tumor stage, adjuvant chemotherapy benefit, and survival outcome. RESULTS: Our study revealed a significant association between high VASN expression and pulmonary metastasis in LARC patients. Experiments in vitro and in vivo demonstrated that VASN could promote the cell proliferation, metastasis, and drug resistance of colorectal cancer. Mechanistically, VASN interacts with the NOTCH1 protein, leading to concurrent activation of the NOTCH and MAPK pathways. Clinically, pulmonary metastasis and advanced tumor stage were observed in 90% of VASN-positive patients and 53.5% of VASN-high patients, respectively, and VASN-high patients had a lower five-year survival rate than VASN-low patients (26.7% vs. 83.7%). Moreover, the Cox analysis and OS analysis indicated that VASN was an independent prognostic factor for OS (HR = 7.4, P value < 0.001) and a predictor of adjuvant therapy efficacy in rectal cancer. CONCLUSIONS: Our study highlights the role of VASN in decreasing drug sensitivity and activating the NOTCH and MAPK pathways, which leads to tumorigenesis and pulmonary metastasis. Both experimental and clinical data support that rectal cancer patients with VASN overexpression detected in biopsies have a higher risk of pulmonary metastasis and adjuvant chemotherapy resistance.