RESUMEN

Detection of cytosolic DNA by the cyclic GMP-AMP (cGAMP) synthase (cGAS)-stimulator of interferon genes (STING) pathway provides immune defense against pathogens and cancer but can also cause autoimmunity when overactivated. The exonuclease three prime repair exonuclease 1 (TREX1) degrades DNA in the cytosol and prevents cGAS activation by self-DNA. Loss-of-function mutations of the TREX1 gene are linked to autoimmune diseases such as Aicardi-Goutières syndrome, and mice deficient in TREX1 develop lethal inflammation in a cGAS-dependent manner. In order to determine the type of cells in which cGAS activation drives autoinflammation, we generated conditional cGAS knockout mice on the Trex1-/- background. Here, we show that genetic ablation of the cGAS gene in classical dendritic cells (cDCs), but not in macrophages, was sufficient to rescue Trex1-/- mice from all observed disease phenotypes including lethality, T cell activation, tissue inflammation, and production of antinuclear antibodies and interferon-stimulated genes. These results show that cGAS activation in cDC causes autoinflammation in response to self-DNA accumulated in the absence of TREX1.

Asunto(s)

Autoinmunidad , Células Dendríticas , Exodesoxirribonucleasas , Ratones Noqueados , Nucleotidiltransferasas , Fosfoproteínas , Animales , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/deficiencia , Exodesoxirribonucleasas/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/deficiencia , Células Dendríticas/inmunología , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/inmunología , Ratones , Autoinmunidad/inmunología , Malformaciones del Sistema Nervioso/genética , Malformaciones del Sistema Nervioso/inmunología , Enfermedades Autoinmunes del Sistema Nervioso/inmunología , Enfermedades Autoinmunes del Sistema Nervioso/genética , Enfermedades Autoinmunes del Sistema Nervioso/patología , Inflamación/inmunología , Inflamación/genética , Macrófagos/inmunología , Macrófagos/metabolismo , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/genética

RESUMEN

Early, rapid, and accurate diagnostic tests play critical roles not only in the identification/management of individuals infected by SARS-CoV-2, but also in fast and effective public health surveillance, containment, and response. Our aim has been to develop a fast and robust fluorescence in situ hybridization (FISH) detection method for detecting SARS-CoV-2 RNAs by using an HEK 293 T cell culture model. At various times after being transfected with SARS-CoV-2 E and N plasmids, HEK 293 T cells were fixed and then hybridized with ATTO-labeled short DNA probes (about 20 nt). At 4 h, 12 h, and 24 h after transfection, SARS-CoV-2 E and N mRNAs were clearly revealed as solid granular staining inside HEK 293 T cells at all time points. Hybridization time was also reduced to 1 h for faster detection, and the test was completed within 3 h with excellent results. In addition, we have successfully detected 3 mRNAs (E mRNA, N mRNA, and ORF1a (-) RNA) simultaneously inside the buccal cells of COVID-19 patients. Our high-resolution RNA FISH might significantly increase the accuracy and efficiency of SARS-CoV-2 detection, while significantly reducing test time. The method can be conducted on smears containing cells (e.g., from nasopharyngeal, oropharyngeal, or buccal swabs) or smears without cells (e.g., from sputum, saliva, or drinking water/wastewater) for detecting various types of RNA viruses and even DNA viruses at different timepoints of infection.

Asunto(s)

COVID-19 , Hibridación Fluorescente in Situ , ARN Viral , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , Hibridación Fluorescente in Situ/métodos , ARN Viral/genética , COVID-19/diagnóstico , COVID-19/virología , COVID-19/genética , Células HEK293 , Fosfoproteínas/genética , Proteínas de la Envoltura de Coronavirus/genética , ARN Mensajero/genética , Proteínas de la Nucleocápside de Coronavirus/genética

RESUMEN

Structural plasticity of dendritic spines in the nucleus accumbens (NAc) is crucial for learning from aversive experiences. Activation of NMDA receptors (NMDARs) stimulates Ca2+-dependent signaling that leads to changes in the actin cytoskeleton, mediated by the Rho family of GTPases, resulting in postsynaptic remodeling essential for learning. We investigated how phosphorylation events downstream of NMDAR activation drive the changes in synaptic morphology that underlie aversive learning. Large-scale phosphoproteomic analyses of protein kinase targets in mouse striatal/accumbal slices revealed that NMDAR activation resulted in the phosphorylation of 194 proteins, including RhoA regulators such as ARHGEF2 and ARHGAP21. Phosphorylation of ARHGEF2 by the Ca2+-dependent protein kinase CaMKII enhanced its RhoGEF activity, thereby activating RhoA and its downstream effector Rho-associated kinase (ROCK/Rho-kinase). Further phosphoproteomic analysis identified 221 ROCK targets, including the postsynaptic scaffolding protein SHANK3, which is crucial for its interaction with NMDARs and other postsynaptic scaffolding proteins. ROCK-mediated phosphorylation of SHANK3 in the NAc was essential for spine growth and aversive learning. These findings demonstrate that NMDAR activation initiates a phosphorylation cascade crucial for learning and memory.

Asunto(s)

Proteínas del Tejido Nervioso , Plasticidad Neuronal , Proteoma , Receptores de N-Metil-D-Aspartato , Animales , Receptores de N-Metil-D-Aspartato/metabolismo , Plasticidad Neuronal/fisiología , Ratones , Fosforilación , Proteoma/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Masculino , Transducción de Señal , Quinasas Asociadas a rho/metabolismo , Quinasas Asociadas a rho/genética , Ratones Endogámicos C57BL , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Aprendizaje/fisiología , Reacción de Prevención/fisiología , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Sinapsis/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Espinas Dendríticas/metabolismo

RESUMEN

BACKGROUND: Plerocercoid larvae of the tapeworm Spirometra mansoni can infect both humans and animals, leading to severe parasitic zoonosis worldwide. Despite ongoing research efforts, our understanding of the developmental process of S. mansoni remains inadequate. To better characterize posttranslational regulation associated with parasite growth, development, and reproduction, a comparative phosphoproteomic study was conducted on the plerocercoid and adult stages of S. mansoni. METHODS: In this study, site-specific phosphoproteomic analysis was conducted via 4D label-free quantitative analysis technology to obtain primary information about the overall phosphorylation status of plerocercoids and adults. RESULTS: A total of 778 differentially abundant proteins (DAPs) were detected between adults and plerocercoids, of which 704 DAPs were upregulated and only 74 were downregulated. DAPs involved in metabolic activity were upregulated in plerocercoid larvae compared with adults, whereas DAPs associated with binding were upregulated in adults. Gene Ontology (GO) and Kyoto Encyclopedia of Genes (KEGG) analyses indicated that most DAPs involved in signal transduction and environmental information processing pathways were highly active in adults. DAPs upregulated in the plerocercoid group were enriched mainly in metabolic activities. The kinases PKACA, GSK3B, and smMLCK closely interact, suggesting potential active roles in the growth and development of S. mansoni. CONCLUSIONS: The dataset presented in this study offers a valuable resource for forthcoming research on signaling pathways as well as new insights into functional studies on the molecular mechanisms of S. mansoni.

Asunto(s)

Fosfoproteínas , Proteoma , Spirometra , Animales , Spirometra/genética , Spirometra/metabolismo , Fosforilación , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Proteínas del Helminto/metabolismo , Proteínas del Helminto/genética , Larva/metabolismo , Larva/genética , Proteómica/métodos

RESUMEN

An imbalance in estrogen signaling is a critical event in breast tumorigenesis. The majority of breast cancers (BCs) are hormone-sensitive; they majorly express the estrogen receptor (ER+) and are activated by 17ß-estradiol (E2). The steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step in steroid biosynthesis. The dysregulation of the epigenetic machinery, modulating E2 levels, is a primary occurrence for promoting breast tumorigenesis. StAR expression, concomitant with E2 synthesis, was reported to be aberrantly high in human and mouse hormone-dependent BC cells compared with their non-cancerous counterparts. However, the mechanism of action of StAR remains poorly understood. We discovered StAR as an acetylated protein and have identified a number of lysine (K) residues that are putatively acetylated in malignant and non-malignant breast cells, using LC-MS/MS (liquid chromatography-tandem mass spectrometry), suggesting they differently influence E2 synthesis in mammary tissue. The treatment of hormone-sensitive MCF7 cells with a variety of histone deacetylase inhibitors (HDACIs), at therapeutically and clinically relevant doses, identified a few additional StAR acetylated lysine residues. Among a total of fourteen StAR acetylomes undergoing acetylation and deacetylation, K111 and K253 were frequently recognized either endogenously or in response to HDACIs. Site-directed mutagenesis studies of these two StAR acetylomes, pertaining to K111Q and K253Q acetylation mimetic states, resulted in increases in E2 levels in ER+ MCF7 and triple negative MB-231 BC cells, compared with their values seen with human StAR. Conversely, these cells carrying K111R and K253R deacetylation mutants diminished E2 biosynthesis. These findings provide novel and mechanistic insights into intra-tumoral E2 regulation by elucidating the functional importance of this uncovered StAR post-translational modification (PTM), involving acetylation and deacetylation events, underscoring the potential of StAR as a therapeutic target for hormone-sensitive BC.

Asunto(s)

Neoplasias de la Mama , Estradiol , Fosfoproteínas , Humanos , Acetilación/efectos de los fármacos , Estradiol/farmacología , Estradiol/metabolismo , Femenino , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Células MCF-7 , Inhibidores de Histona Desacetilasas/farmacología , Procesamiento Proteico-Postraduccional , Lisina/metabolismo , Línea Celular Tumoral

RESUMEN

Somatic embryogenesis (SE) is a biotechnological tool used to generate new individuals and is the preferred method for rapid plant regeneration. However, the molecular basis underlying somatic cell regeneration through SE is not yet fully understood, particularly regarding interactions between the proteome and post-translational modifications. Here, we performed association analysis of high-throughput proteomics and phosphoproteomics in three representative samples (non-embryogenic calli, NEC; primary embryogenic calli, PEC; globular embryos, GE) during the initiation of plant regeneration in cotton, a pioneer crop for genetic biotechnology applications. Our results showed that protein accumulation is positively regulated by phosphorylation during SE, as revealed by correlation analyses. Of the 1418 proteins that were differentially accumulated in the proteome and the 1106 phosphoproteins that were differentially regulated in the phosphoproteome, 115 proteins with 229 phosphorylation sites overlapped (co-differential). Furthermore, seven dynamic trajectory patterns of differentially accumulated proteins (DAPs) and the correlated differentially regulated phosphoproteins (DRPPs) pairs with enrichment features were observed. During the initiation of plant regeneration, functional enrichment analysis revealed that the overlapping proteins (DAPs-DRPPs) were considerably enriched in cellular nitrogen metabolism, spliceosome formation, and reproductive structure development. Moreover, 198 DRPPs (387 phosphorylation sites) were specifically regulated at the phosphorylation level and showed four patterns of stage-enriched phosphorylation susceptibility. Furthermore, enrichment annotation analysis revealed that these phosphoproteins were significantly enriched in endosomal transport and nucleus organization processes. During embryogenic differentiation, we identified five DAPs-DRPPs with significantly enriched characteristic patterns. These proteins may play essential roles in transcriptional regulation and signaling events that initiate plant regeneration through protein accumulation and/or phosphorylation modification. This study enriched the understanding of key proteins and their correlated phosphorylation patterns during plant regeneration, and also provided a reference for improving plant regeneration efficiency.

Asunto(s)

Regulación de la Expresión Génica de las Plantas , Gossypium , Fosfoproteínas , Proteínas de Plantas , Proteómica , Regeneración , Gossypium/metabolismo , Gossypium/genética , Gossypium/crecimiento & desarrollo , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Proteómica/métodos , Regeneración/genética , Regeneración/fisiología , Fosforilación , Proteoma/metabolismo , Técnicas de Embriogénesis Somática de Plantas/métodos , Procesamiento Proteico-Postraduccional

RESUMEN

Acute liver failure (ALF) is characterized by the rapidly progressive deterioration of hepatic function, which, without effective medical intervention, results in high mortality and morbidity. Here, using proteomic and transcriptomic analyses in murine ALF models, we found that the expression of multiple splicing factors was downregulated in ALF. Notably, we found that KH-type splicing regulatory protein (KHSRP) has a protective effect in ALF. Knockdown of KHSRP resulted in dramatic splicing defects, such as intron retention, and led to the exacerbation of liver injury in ALF. Moreover, we demonstrated that KHSRP directly interacts with splicing factor 3b subunit 1 (SF3B1) and enhances the binding of SF3B1 to the intronic branch sites, thereby promoting pre-mRNA splicing. Using splicing inhibitors, we found that Khsrp protects against ALF by regulating pre-mRNA splicing in vivo. Overall, our findings demonstrate that KHSRP is an important splicing activator and promotes the expression of genes associated with ALF progression by interacting with SF3B1; thus, KHSRP could be a possible target for therapeutic intervention in ALF.

Asunto(s)

Fallo Hepático Agudo , Precursores del ARN , Factores de Empalme de ARN , Empalme del ARN , Proteínas de Unión al ARN , Factores de Empalme de ARN/metabolismo , Factores de Empalme de ARN/genética , Animales , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Empalme del ARN/genética , Fallo Hepático Agudo/metabolismo , Fallo Hepático Agudo/genética , Precursores del ARN/metabolismo , Precursores del ARN/genética , Ratones , Humanos , Ratones Endogámicos C57BL , Masculino , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Modelos Animales de Enfermedad , Unión Proteica , Transactivadores

RESUMEN

Metabolic rewiring during the proliferation-to-quiescence transition is poorly understood. Here, using a model of contact inhibition-induced quiescence, we conducted 13C-metabolic flux analysis in proliferating (P) and quiescent (Q) mouse embryonic fibroblasts (MEFs) to investigate this process. Q cells exhibit reduced glycolysis but increased TCA cycle flux and mitochondrial respiration. Reduced glycolytic flux in Q cells correlates with reduced glycolytic enzyme expression mediated by yes-associated protein (YAP) inhibition. The increased TCA cycle activity and respiration in Q cells is mediated by induced mitochondrial pyruvate carrier (MPC) expression, rendering them vulnerable to MPC inhibition. The malate-to-pyruvate flux, which generates NADPH, is markedly reduced by modulating malic enzyme 1 (ME1) dimerization in Q cells. Conversely, the malate dehydrogenase 1 (MDH1)-mediated oxaloacetate-to-malate flux is reversed and elevated in Q cells, driven by high mitochondrial-derived malate levels, reduced cytosolic oxaloacetate, elevated MDH1 levels, and a high cytoplasmic NAD+/NADH ratio. Transcriptomic analysis revealed large number of genes are induced in Q cells, many of which are associated with the extracellular matrix (ECM), while YAP-dependent and cell cycle-related genes are repressed. The results suggest that high TCA cycle flux and respiration in Q cells are required to generate ATP and amino acids to maintain de-novo ECM protein synthesis and secretion.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Ciclo del Ácido Cítrico , Inhibición de Contacto , Fibroblastos , Glucólisis , Malato Deshidrogenasa , Mitocondrias , Transcriptoma , Proteínas Señalizadoras YAP , Animales , Proteínas Señalizadoras YAP/metabolismo , Ratones , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Fibroblastos/metabolismo , Malato Deshidrogenasa/metabolismo , Malato Deshidrogenasa/genética , Mitocondrias/metabolismo , Malatos/metabolismo , Proliferación Celular , Ácido Pirúvico/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/genética

RESUMEN

Anaplastic thyroid carcinoma (ATC) is a highly aggressive human malignancy without effective treatment. Yes-associated protein (YAP) is a critical effector of the Hippo pathway, which is essential in thyroid carcinogenesis. However, the underlying mechanisms of aberrant YAP expression in ATC are not completely understood. Ubiquitylation-related enzyme siRNA screening identified the ubiquitin protein ligase E3 component n-recognin 1 (UBR1) as a stabilizer of YAP in ATC cells. UBR1 deficiency reduced YAP protein levels and its target gene expression. UBR1 directly interacted with YAP and promoted its monoubiquitylation, competitively suppressing its polyubiquitylation and resulting in extended protein half-life. UBR1 depletion reduced ATC cell proliferation and migration in vitro. Xenograft tumor studies also suggested that UBR1 knockdown suppressed ATC cell growth in vivo. Furthermore, exogenous YAP expression partially reversed the inhibitive effects of UBR1 depletion on ATC cell proliferation and migration. Our studies demonstrated that UBR1 directly interacts with YAP and stabilized it in a monoubiquitylation-dependent manner, consequently promoting ATC tumorigenesis, suggesting that UBR1 might be a potentially therapeutic target for ATC treatment.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Movimiento Celular , Proliferación Celular , Carcinoma Anaplásico de Tiroides , Factores de Transcripción , Ubiquitinación , Proteínas Señalizadoras YAP , Humanos , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Animales , Línea Celular Tumoral , Movimiento Celular/genética , Carcinoma Anaplásico de Tiroides/metabolismo , Carcinoma Anaplásico de Tiroides/patología , Carcinoma Anaplásico de Tiroides/genética , Proteínas Señalizadoras YAP/metabolismo , Proteínas Señalizadoras YAP/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Ratones , Estabilidad Proteica , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patología , Neoplasias de la Tiroides/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Progresión de la Enfermedad , Ratones Desnudos , Regulación Neoplásica de la Expresión Génica , Fosfoproteínas/metabolismo , Fosfoproteínas/genética

RESUMEN

Genes of the Sprouty family (Spry1-4) are feedback inhibitors of receptor tyrosine kinases, especially of Ret and the FGF receptors. As such, they play distinct and overlapping roles in embryo morphogenesis and are considered to be tumor suppressors in adult life. Genetic experiments in mice have defined in great detail the role of these genes during embryonic development, however their function in adult mice is less clearly established. Here we generate adult-onset, whole body Spry1/2/4 triple knockout mice. Tumor incidence in triple mutant mice is comparable to that of wild type littermates of up to one year of age, indicating that Sprouty loss per se is not sufficient to initiate tumorigenesis. On the other hand, triple knockout mice do not gain weight as they age, show less visceral fat, and have lower plasma glucose levels than wild type littermates, despite showing similar food intake and slightly reduced motor function. They also show alopecia, eyelid inflammation, and mild hyperthyroidism. Finally, triple knockout mice present phosphaturia and hypophosphatemia, suggesting exacerbated signaling downstream of FGF23. In conclusion, triple knockout mice develop a series of endocrine abnormalities but do not show increased tumor incidence.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Factor-23 de Crecimiento de Fibroblastos , Proteínas de la Membrana , Ratones Noqueados , Fosfoproteínas , Animales , Ratones , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Femenino , Masculino , Enfermedades del Sistema Endocrino/genética , Enfermedades del Sistema Endocrino/metabolismo , Proteínas del Tejido Nervioso , Proteínas Serina-Treonina Quinasas

RESUMEN

A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes mild-to-severe respiratory symptoms, including acute respiratory distress. Despite remarkable efforts to investigate the virological and pathological impacts of SARS-CoV-2, many of the characteristics of SARS-CoV-2 infection still remain unknown. The interferon-inducible ubiquitin-like protein ISG15 is covalently conjugated to several viral proteins to suppress their functions. It was reported that SARS-CoV-2 utilizes its papain-like protease (PLpro) to impede ISG15 conjugation, ISGylation. However, the role of ISGylation in SARS-CoV-2 infection remains unclear. We aimed to elucidate the role of ISGylation in SARS-CoV-2 replication. We observed that the SARS-CoV-2 nucleocapsid protein is a target protein for the HERC5 E3 ligase-mediated ISGylation in cultured cells. Site-directed mutagenesis reveals that the residue K374 within the C-terminal spacer B-N3 (SB/N3) domain is required for nucleocapsid-ISGylation, alongside conserved lysine residue in MERS-CoV (K372) and SARS-CoV (K375). We also observed that the nucleocapsid-ISGylation results in the disruption of nucleocapsid oligomerization, thereby inhibiting viral replication. Knockdown of ISG15 mRNA enhanced SARS-CoV-2 replication in the SARS-CoV-2 reporter replicon cells, while exogenous expression of ISGylation components partially hampered SARS-CoV-2 replication. Taken together, these results suggest that SARS-CoV-2 PLpro inhibits ISGylation of the nucleocapsid protein to promote viral replication by evading ISGylation-mediated disruption of the nucleocapsid oligomerization.IMPORTANCEISG15 is an interferon-inducible ubiquitin-like protein that is covalently conjugated to the viral protein via specific Lys residues and suppresses viral functions and viral propagation in many viruses. However, the role of ISGylation in SARS-CoV-2 infection remains largely unclear. Here, we demonstrated that the SARS-CoV-2 nucleocapsid protein is a target protein for the HERC5 E3 ligase-mediated ISGylation. We also found that the residue K374 within the C-terminal spacer B-N3 (SB/N3) domain is required for nucleocapsid-ISGylation. We obtained evidence suggesting that nucleocapsid-ISGylation results in the disruption of nucleocapsid-oligomerization, thereby suppressing SARS-CoV-2 replication. We discovered that SARS-CoV-2 papain-like protease inhibits ISG15 conjugation of nucleocapsid protein via its de-conjugating enzyme activity. The present study may contribute to gaining new insight into the roles of ISGylation-mediated anti-viral function in SARS-CoV-2 infection and may lead to the development of more potent and selective inhibitors targeted to SARS-CoV-2 nucleocapsid protein.

Asunto(s)

COVID-19 , Proteínas de la Nucleocápside de Coronavirus , Proteasas Similares a la Papaína de Coronavirus , Citocinas , SARS-CoV-2 , Ubiquitina-Proteína Ligasas , Ubiquitinas , Replicación Viral , Humanos , Ubiquitinas/metabolismo , Ubiquitinas/genética , SARS-CoV-2/inmunología , SARS-CoV-2/metabolismo , Citocinas/metabolismo , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Proteínas de la Nucleocápside de Coronavirus/genética , COVID-19/virología , COVID-19/inmunología , COVID-19/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteasas Similares a la Papaína de Coronavirus/metabolismo , Células HEK293 , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Evasión Inmune , Proteínas de la Nucleocápside/metabolismo , Proteasas 3C de Coronavirus/metabolismo , Péptidos y Proteínas de Señalización Intracelular

RESUMEN

The availability of data from profiling of cancer patients with multiomics is rapidly increasing. However, integrative analysis of such data for personalized target identification is not trivial. Multiomics2Targets is a platform that enables users to upload transcriptomics, proteomics, and phosphoproteomics data matrices collected from the same cohort of cancer patients. After uploading the data, Multiomics2Targets produces a report that resembles a research publication. The uploaded matrices are processed, analyzed, and visualized using the tools Enrichr, KEA3, ChEA3, Expression2Kinases, and TargetRanger to identify and prioritize proteins, genes, and transcripts as potential targets. Figures and tables, as well as descriptions of the methods and results, are automatically generated. Reports include an abstract, introduction, methods, results, discussion, conclusions, and references and are exportable as citable PDFs and Jupyter Notebooks. Multiomics2Targets is applied to analyze version 3 of the Clinical Proteomic Tumor Analysis Consortium (CPTAC3) pan-cancer cohort, identifying potential targets for each CPTAC3 cancer subtype. Multiomics2Targets is available from https://multiomics2targets.maayanlab.cloud/.

Asunto(s)

Neoplasias , Fosfoproteínas , Proteómica , Transcriptoma , Humanos , Proteómica/métodos , Neoplasias/genética , Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Estudios de Cohortes , Perfilación de la Expresión Génica/métodos , Programas Informáticos , Biología Computacional/métodos

RESUMEN

Cells sense and respond to various mechanical forces from the extracellular matrix primarily by modulating the actin cytoskeleton. Mechanical forces can be translated into biochemical signals in a process called mechanotransduction. Yes-associated protein (YAP) is an effector of Hippo signaling and a mediator of mechanotransduction, but how mechanical forces regulate Hippo signaling is still an open question. We propose that retinoic acid-induced protein 14 (RAI14) responds to mechanical forces and regulates Hippo signaling. RAI14 positively regulates the activity of YAP. RAI14 interacts with NF2, a key component of the Hippo pathway, and the interaction occurs on filamentous actin. When mechanical forces are kept low in cells, NF2 dissociates from RAI14 and filamentous actin, resulting in increased interactions with LATS1 and activation of the Hippo pathway. Clinical data show that tissue stiffness and expression of RAI14 and YAP are upregulated in tumor tissues and that RAI14 is strongly associated with adverse outcome in patients with gastric cancer. Our data suggest that RAI14 links mechanotransduction with Hippo signaling and mediates Hippo-related biological functions such as cancer progression.

Asunto(s)

Vía de Señalización Hippo , Mecanotransducción Celular , Proteínas Serina-Treonina Quinasas , Transducción de Señal , Factores de Transcripción , Humanos , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción/metabolismo , Neurofibromina 2/metabolismo , Neurofibromina 2/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Neoplasias Gástricas/genética , Proteínas Señalizadoras YAP/metabolismo , Actinas/metabolismo , Unión Proteica , Fosfoproteínas/metabolismo , Fosfoproteínas/genética

RESUMEN

Small-cell lung cancer (SCLC) is the most lethal type of lung cancer. Paradoxically, this tumor displays an initial exquisite response to chemotherapy; however, at relapse, the tumor is highly resistant to subsequent available therapies. Here, we report that the expression of three prime repair exonuclease 1 (TREX1) is strongly induced in chemoresistant SCLCs. Assay for transposase-accessible chromatin using sequencing and chromatin immunoprecipitation sequencing revealed a significant increase in chromatin accessibility and transcriptional activity of TREX1 gene locus in chemoresistant SCLCs. Analyses of human SCLC tumors and patient-derived xenografts (PDX) also showed an increase in TREX1 expression in postchemotherapy samples. TREX1 depletion caused the activation of cyclic GMP-AMP synthase stimulator of interferon gene pathway due to cytoplasmic accumulation of damage-associated double-stranded DNA, inducing immunogenicity and enhancing the sensitivity of drug-resistant cells to chemotherapy. These findings suggest TREX1 upregulation may partially contribute to the survival of resistant cells, and its inhibition may represent a promising therapeutic strategy to enhance antitumor immunity and potentiate the efficacy of chemotherapy and/or immunotherapy in chemoresistant SCLCs. Significance: In this study, we show that targeting TREX1 induces an innate immune response and resensitizes SCLC cells to chemotherapy, representing a promising novel target for "immunologically" cold tumors, such as SCLC.

Asunto(s)

Resistencia a Antineoplásicos , Exodesoxirribonucleasas , Inmunidad Innata , Neoplasias Pulmonares , Fosfoproteínas , Carcinoma Pulmonar de Células Pequeñas , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Humanos , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/inmunología , Carcinoma Pulmonar de Células Pequeñas/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/genética , Resistencia a Antineoplásicos/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ratones , Animales , Línea Celular Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos

RESUMEN

SARS-CoV-2 is the causative virus of COVID-19, which has been responsible for millions of deaths worldwide since its discovery. After its emergence, several variants have been identified that challenge the efficacy of the available vaccines. Previously, we generated and evaluated a vaccine based on a recombinant Bacillus Calmette-Guérin (rBCG) expressing the nucleoprotein (N) of SARS-CoV-2 (rBCG-N-SARS-CoV-2). This protein is a highly immunogenic antigen and well conserved among variants. Here, we tested the administration of this vaccine with recombinant N and viral Spike proteins (S), or Receptor Binding Domain (RBD-Omicron variant), plus a booster with the recombinant proteins only, as a novel and effective strategy to protect against SARS-CoV-2 variants. METHODS: BALB/c mice were immunized with rBCG-N-SARS-CoV-2 and recombinant SARS-CoV-2 proteins in Alum adjuvant, followed by a booster with recombinant proteins to assess the safety and virus-specific cellular and humoral immune responses against SARS-CoV-2 antigens. RESULTS: Immunization with rBCG-N-SARS-CoV-2 + recombinant proteins as a vaccine was safe and promoted the activation of CD4+ and CD8+ T cells that recognize SARS-CoV-2 N, S, and RBD antigens. These cells were able to secrete cytokines with an antiviral profile. This immunization strategy also induced robust titers of specific antibodies against N, S, and RBD and neutralizing antibodies of SARS-CoV-2. CONCLUSIONS: Co-administration of the rBCG-N-SARS-CoV-2 vaccine with recombinant SARS-CoV-2 proteins could be an effective alternative to control particular SARS-CoV-2 variants. Due to its safety and capacity to induce virus-specific immune responses, we believe the rBCG-N-SARS-CoV-2 + Proteins vaccine could be an attractive candidate to protect against this virus, especially in newborns.

Asunto(s)

Anticuerpos Antivirales , Vacuna BCG , Vacunas contra la COVID-19 , COVID-19 , Ratones Endogámicos BALB C , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Animales , Ratones , SARS-CoV-2/inmunología , SARS-CoV-2/genética , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , COVID-19/prevención & control , COVID-19/inmunología , Vacunas contra la COVID-19/inmunología , Vacunas contra la COVID-19/administración & dosificación , Vacuna BCG/inmunología , Vacuna BCG/administración & dosificación , Vacuna BCG/genética , Femenino , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Inmunización Secundaria , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/administración & dosificación , Inmunidad Humoral , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/genética , Proteínas de la Nucleocápside de Coronavirus/inmunología , Proteínas de la Nucleocápside de Coronavirus/genética , Linfocitos T CD8-positivos/inmunología , Fosfoproteínas/inmunología , Fosfoproteínas/genética , Adyuvantes Inmunológicos/administración & dosificación , Inmunidad Celular

RESUMEN

The tandem Tudor-like domain-containing protein Spindlin1 (SPIN1) is a transcriptional coactivator with critical functions in embryonic development and emerging roles in cancer. However, the involvement of SPIN1 in DNA damage repair has remained unclear. Our study shows that SPIN1 is recruited to DNA lesions through its N-terminal disordered region that binds to Poly-ADP-ribose (PAR), and facilitates homologous recombination (HR)-mediated DNA damage repair. SPIN1 promotes H3K9me3 accumulation at DNA damage sites and enhances the interaction between H3K9me3 and Tip60, thereby promoting the activation of ATM and HR repair. We also show that SPIN1 increases chemoresistance. These findings reveal a novel role for SPIN1 in the activation of H3K9me3-dependent DNA repair pathways, and suggest that SPIN1 may contribute to cancer chemoresistance by modulating the efficiency of double-strand break (DSB) repair.

Asunto(s)

Proteínas de Ciclo Celular , Resistencia a Antineoplásicos , Histonas , Lisina Acetiltransferasa 5 , Fosfoproteínas , Unión Proteica , Humanos , Resistencia a Antineoplásicos/genética , Histonas/metabolismo , Lisina Acetiltransferasa 5/metabolismo , Lisina Acetiltransferasa 5/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Roturas del ADN de Doble Cadena , Reparación del ADN por Recombinación , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/genética , Poli Adenosina Difosfato Ribosa/metabolismo , Línea Celular Tumoral , Daño del ADN , Reparación del ADN , Proteínas Asociadas a Microtúbulos

RESUMEN

Steroidogenesis occurs locally in peripheral tissues and via adrenal and gonadal glands' biosynthesis. The C2C12 mouse myoblast cell line and rat skeletal muscles harbor a local steroidogenesis pathway for glucocorticoids, and corticosterone is biosynthesized from skeletal muscle cells. However, Cyp11a1 and StAR protein expressions are not observed in C2C12 cells or rat muscular tissues. In this context, this study investigated the relationship between DNA methylation and key steroidogenic genes. Bioinformatics analysis of methylated DNA immune precipitation showed that C2C12 myoblasts and myotubes did not have remarkable DNA methylated regions in the gene-body of Cyp11a1. However, a highly methylated region in the CpG island was detected in the intronic enhancer of Ad4BP/SF-1, known as the transcriptional factor for steroidogenic genes. After C2C12 myoblasts treatment with 5-aza-2-deoxycytidine, the gene expressions of Ad4BP/SF-1, Cyp11a1, and StAR were significantly time- and concentration-dependent upregulated. To clarify the contribution of Ad4BP/SF-1 on Cyp11a1 and StAR transcripts, we silenced Ad4BP/SF-1 during the 5-aza-2-deoxycytidine treatment in C2C12 myoblasts, resulting in significant suppression of both Cyp11a1 and StAR. Additionally, pregnenolone levels in the supernatants of C2C12 cells were enhanced by 5-aza-2-deoxycytidine treatment, whereas pregnenolone production by C2C12 myoblasts was significantly suppressed by Ad4BP/SF-1 knockdown. These results indicate that DNA methylation of Ad4BP/SF-1 might be involved in the downregulation of steroidogenic genes, such as Cyp11a1 and StAR in C2C12 myoblasts.

Asunto(s)

Enzima de Desdoblamiento de la Cadena Lateral del Colesterol , Islas de CpG , Metilación de ADN , Mioblastos , Fosfoproteínas , Animales , Metilación de ADN/genética , Metilación de ADN/efectos de los fármacos , Ratones , Mioblastos/metabolismo , Mioblastos/efectos de los fármacos , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Línea Celular , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Islas de CpG/genética , Decitabina/farmacología , Ratas , Azacitidina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo

RESUMEN

BACKGROUND: Retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations (RVCL-S) is a rare autosomal dominant systemic microvascular disorder attributed to TREX1 (three-prime repair exonuclease-1) gene mutations, often proned to misdiagnosed. METHODS: We reported a case of RVCL-S coexisting with systemic lupus erythematosus due to a mutation in the TREX1 gene. This study provided a summary and discussion of previously documented cases related to TREX1 mutations or RVCL-S. RESULTS: A 39-year-old female patient visited the clinic due to progressive memory loss and speech difficulties. Magnetic resonance imaging results showed corpus callosum atrophy and multiple subcortical calcifications in both brain hemispheres. Genetic testing revealed a TREX1 gene mutation (c.294dupA). Treatment with immunosuppressive therapy for 2 months led to improvements in communication and mobility. We also summarized previously reported cases providing an overview of TREX1 gene mutation or RCVL-S. CONCLUSION: Our case establishes a compelling foundation for future RVCL-S diagnosis and treatment paradigms. Notably, conducting systemic immunity screening in patients with RVCL-S emerges as a strategic approach to prevent potential diagnostic oversights.

Asunto(s)

Exodesoxirribonucleasas , Leucoencefalopatías , Lupus Eritematoso Sistémico , Mutación , Humanos , Femenino , Adulto , Exodesoxirribonucleasas/genética , Lupus Eritematoso Sistémico/diagnóstico , Lupus Eritematoso Sistémico/complicaciones , Lupus Eritematoso Sistémico/genética , Leucoencefalopatías/diagnóstico , Leucoencefalopatías/genética , Leucoencefalopatías/etiología , Fosfoproteínas/genética , Errores Diagnósticos/prevención & control , Imagen por Resonancia Magnética , Vasculitis Retiniana/diagnóstico , Vasculitis Retiniana/etiología , Enfermedades de la Retina , Enfermedades Vasculares , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias

RESUMEN

Calorie restriction (CR) and treatment with rapamycin (RM), an inhibitor of the mTORC1 growth-promoting signaling pathway, are known to slow aging and promote health from worms to humans. At the transcriptome and proteome levels, long-term CR and RM treatments have partially overlapping effects, while their impact on protein phosphorylation within cellular signaling pathways have not been compared. Here we measured the phosphoproteomes of soleus, tibialis anterior, triceps brachii and gastrocnemius muscles from adult (10 months) and 30-month-old (aged) mice receiving either a control, a calorie restricted or an RM containing diet from 15 months of age. We reproducibly detected and extensively analyzed a total of 6960 phosphosites, 1415 of which are not represented in standard repositories. We reveal the effect of these interventions on known mTORC1 pathway substrates, with CR displaying greater between-muscle variation than RM. Overall, CR and RM have largely consistent, but quantitatively distinct long-term effects on the phosphoproteome, mitigating age-related changes to different degrees. Our data expands the catalog of protein phosphorylation sites in the mouse, providing important information regarding their tissue-specificity, and revealing the impact of long-term nutrient-sensing pathway inhibition on mouse skeletal muscle.

Asunto(s)

Envejecimiento , Restricción Calórica , Músculo Esquelético , Sirolimus , Animales , Fosforilación , Envejecimiento/metabolismo , Sirolimus/farmacología , Ratones , Músculo Esquelético/metabolismo , Músculo Esquelético/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Transducción de Señal/efectos de los fármacos , Proteoma/metabolismo , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Proteínas Musculares/metabolismo

RESUMEN

Introduction: Human Cytomegalovirus (HCMV) is a betaherpesvirus that causes severe disease in immunocompromised transplant recipients. Immunotherapy with CD8 T cells specific for HCMV antigens presented on HLA class-I molecules is explored as strategy for long-term relief to such patients, but the antiviral effectiveness of T cell preparations cannot be efficiently predicted by available methods. Methods: We developed an Assay for Rapid Measurement of Antiviral T-cell Activity (ARMATA) by real-time automated fluorescent microscopy and used it to study the ability of CD8 T cells to neutralize HCMV and control its spread. As a proof of principle, we used TCR-transgenic T cells specific for the immunodominant HLA-A02-restricted tegumental phosphoprotein pp65. pp65 expression follows an early/late kinetic, but it is not clear at which stage of the virus cycle it acts as an antigen. We measured control of HCMV infection by T cells as early as 6 hours post infection (hpi). Results: The timing of the antigen recognition indicated that it occurred before the late phase of the virus cycle, but also that virion-associated pp65 was not recognized during virus entry into cells. Monitoring of pp65 gene expression dynamics by reporter fluorescent genes revealed that pp65 was detectable as early as 6 hpi, and that a second and much larger bout of expression occurs in the late phase of the virus cycle by 48 hpi. Since transgenic (Tg)-pp65 specific CD8 T cells were activated even when DNA replication was blocked, our data argue that pp65 acts as an early virus gene for immunological purposes. Discussion: ARMATA does not only allow same day identification of antiviral T-cell activity, but also provides a method to define the timing of antigen recognition in the context of HCMV infection.

Asunto(s)

Linfocitos T CD8-positivos , Infecciones por Citomegalovirus , Citomegalovirus , Fosfoproteínas , Proteínas de la Matriz Viral , Linfocitos T CD8-positivos/inmunología , Citomegalovirus/inmunología , Citomegalovirus/genética , Fosfoproteínas/inmunología , Fosfoproteínas/genética , Humanos , Proteínas de la Matriz Viral/inmunología , Proteínas de la Matriz Viral/genética , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/virología , Regulación Viral de la Expresión Génica , Antígenos Virales/inmunología , Antígeno HLA-A2/inmunología , Antígeno HLA-A2/genética