RESUMEN
Deep-sea organisms are subjected to extreme conditions; therefore, understanding their adaptive strategies is crucial. We utilize Saccharomyces cerevisiae as a model to investigate pressure-dependent protein regulation and piezo-adaptation. Using yeast deletion library analysis, we identified 6 poorly characterized genes that are crucial for high-pressure growth, forming novel functional modules associated with cell growth. In this study, we aimed to unravel the molecular mechanisms of high-pressure adaptation in S. cerevisiae, focusing on the role of MTC6. MTC6, the gene encoding the novel glycoprotein Mtc6/Ehg2, was found to stabilize tryptophan permease Tat2, ensuring efficient tryptophan uptake and growth under high pressure at 25 MPa. The loss of MTC6 led to promoted vacuolar degradation of Tat2, depending on the Rsp5-Bul1 ubiquitin ligase complex. These findings enhance our understanding of deep-sea adaptations and stress biology, with broad implications for biotechnology, environmental microbiology, and evolutionary insights across species.
Asunto(s)
Sistemas de Transporte de Aminoácidos , Retículo Endoplásmico , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Triptófano , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Triptófano/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Retículo Endoplásmico/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Estabilidad Proteica , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/genética , Vacuolas/metabolismo , Presión Hidrostática , ProteolisisRESUMEN
Accumulating evidence suggests an association between iron metabolism and lung cancer progression. In biological systems, iron is present in either reduced (Fe2+ ; ferrous) or oxidized (Fe3+ ; ferric) states. However, ferrous and ferric iron exhibit distinct chemical and biological properties, the role of ferrous and ferric iron in lung cancer cell growth has not been clearly distinguished. In this study, we manipulated the balance between cellular ferrous and ferric iron status by inducing gene mutations involving the FBXL5-IRP2 axis, a ubiquitin-dependent regulatory system for cellular iron homeostasis, and determined its effects on lung cancer cell growth. FBXL5 depletion (ferrous iron accumulation) was found to suppress lung cancer cell growth, whereas IRP2 depletion (ferric iron accumulation) did not suppress such growth, suggesting that ferrous iron but not ferric iron plays a suppressive role in cell growth. Mechanistically, the depletion of FBXL5 impaired the degradation of the cyclin-dependent kinase inhibitor, p27, resulting in a delay in the cell cycle at the G1/S phase. FBXL5 depletion in lung cancer cells also improved the survival of tumor-bearing mice. Overall, this study highlights the important function of ferrous iron in cell cycle progression and lung cancer cell growth.
Asunto(s)
Proteínas F-Box , Neoplasias Pulmonares , Animales , Ratones , Complejos de Ubiquitina-Proteína Ligasa/química , Complejos de Ubiquitina-Proteína Ligasa/genética , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Neoplasias Pulmonares/genética , Hierro/metabolismo , Ubiquitina/metabolismo , Compuestos Férricos , Proteínas F-Box/genética , Proteínas F-Box/metabolismoRESUMEN
BACKGROUND/AIM: Alteration of F-box and leucine-rich repeat protein 5 (FBXL5), an iron-sensing ubiquitin ligase, might be related with carcinogenesis of hepatocellular carcinoma (HCC), by disturbing cellular iron homeostasis. However, the clinical implications of FBXL5 expression using patient samples need to be elucidated. PATIENTS AND METHODS: We collected HCC tissue samples from two institutes: Samsung Medical Center (n=259) and Hallym University Sacred Heart Hospital (n=115) and evaluated FBXL5 expression using immunohistochemistry. Using cut-off values determined by X-tile software, association between FBXL5 expression and several clinicopathological parameters was investigated. For external validation, the Cancer Genome Atlas (TCGA) cohort was used. RESULTS: The best cutoff value for FBXL5 IHC expression associated with recurrence-free survival (RFS) was 5%. Low FBXL5 expression was found in 18.7% of the total 374 HCCs and was associated with non-viral etiology (p=0.019). Low FBXL5 expression was related with inferior disease-specific survival (DSS, p=0.002) and RFS (p=0.001) and also was an independent prognostic factor for DSS and RFS. In addition, cases with low FBLX5 mRNA levels showed inferior DSS and RFS (p<0.001 and p=0.002, respectively) compared to high FBLX5 mRNA levels in the TCGA cohort. CONCLUSION: Down-regulation of FBXL5 expression in HCCs might be associated with poor prognosis. FBXL5 might be a prognostic biomarker of HCCs and a potential therapeutic target in conjunction with iron homeostasis.
Asunto(s)
Carcinoma Hepatocelular , Proteínas F-Box , Neoplasias Hepáticas , Humanos , Complejos de Ubiquitina-Proteína Ligasa/genética , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Proteínas Repetidas Ricas en Leucina , Hierro/metabolismo , ARN Mensajero , Pronóstico , Proteínas F-Box/genética , Proteínas F-Box/metabolismoRESUMEN
Woodhouse-Sakati syndrome (WSS) is an extremely rare multisystemic disorder with neuroendocrine dysfunctions. It is characterized by hypogonadism, alopecia, diabetes mellitus, intellectual disability and progressive extrapyramidal syndrome along with radiological features of small pituitary gland, progressive frontoparietal white matter changes and abnormal accumulation of iron on globus pallidus. WSS is caused by mutations in DCAF17 gene that encodes for DDB1 and CUL4 associated factor 17. In this study, we report a 17-year-old boy with clinical and radiological features of WSS including mild global developmental delay, mild intellectual disability, sensorineural hearing loss, progressive extrapyramidal syndrome, alopecia, hypogonadotropic hypogonadism and dysmorphic features. Whole exome sequencing analysis revealed a novel potentially pathogenic splice donor site variant (c.458+1G>T) on the intron 4 of DCAF17 gene. Transcript analysis revealed splicing ablation resulting in aberrant splicing of exons 3 and 5 and skipping of exon 4 (c.322_458del). This results in a frameshift and is predicted to cause premature termination of protein synthesis resulting in a protein product of length 120 amino acids (p.[Gly108Ilefs*14]). Our study identified a novel pathogenic variant causing WSS in a patient and expands the spectrum of clinical and genetic characteristics of patients with WSS.
Asunto(s)
Diabetes Mellitus , Hipogonadismo , Discapacidad Intelectual , Masculino , Humanos , Adolescente , Discapacidad Intelectual/genética , Discapacidad Intelectual/patología , Secuenciación del Exoma , Complejos de Ubiquitina-Proteína Ligasa/genética , Proteínas Nucleares/genética , Hipogonadismo/genética , Hipogonadismo/patología , Alopecia/genética , Alopecia/patología , Diabetes Mellitus/genética , Diabetes Mellitus/patología , MutaciónRESUMEN
Cells rapidly remodel their proteomes to align their cellular metabolism to environmental conditions. Ubiquitin E3 ligases enable this response, by facilitating rapid and reversible changes to protein stability, localization, or interaction partners. In Saccharomyces cerevisiae, the GID E3 ligase regulates the switch from gluconeogenic to glycolytic conditions through induction and incorporation of the substrate receptor subunit Gid4, which promotes the degradation of gluconeogenic enzymes. Here, we show an alternative substrate receptor, Gid10, which is induced in response to changes in temperature, osmolarity, and nutrient availability, regulates the ART-Rsp5 ubiquitin ligase pathway, a component of plasma membrane quality control. Proteomic studies reveal that the levels of the adaptor protein Art2 are elevated upon GID10 deletion. A crystal structure shows the basis for Gid10-Art2 interactions, and we demonstrate that Gid10 directs a GID E3 ligase complex to ubiquitinate Art2. Our data suggest that the GID E3 ligase affects Art2-dependent amino acid transport. This study reveals GID as a system of E3 ligases with metabolic regulatory functions outside of glycolysis and gluconeogenesis, controlled by distinct stress-specific substrate receptors.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Complejos de Ubiquitina-Proteína Ligasa , Membrana Celular/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Proteómica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/genética , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Human cytomegalovirus (HCMV) is an important human pathogen and a paradigm of viral immune evasion, targeting intrinsic, innate, and adaptive immunity. We have employed two orthogonal multiplexed tandem mass tag-based proteomic screens to identify host proteins down-regulated by viral factors expressed during the latest phases of viral infection. This approach revealed that the HIV-1 restriction factor Schlafen-11 (SLFN11) was degraded by the poorly characterized, late-expressed HCMV protein RL1, via recruitment of the Cullin4-RING E3 Ubiquitin Ligase (CRL4) complex. SLFN11 potently restricted HCMV infection, inhibiting the formation and spread of viral plaques. Overall, we show that a restriction factor previously thought only to inhibit RNA viruses additionally restricts HCMV. We define the mechanism of viral antagonism and also describe an important resource for revealing additional molecules of importance in antiviral innate immunity and viral immune evasion.
Asunto(s)
Infecciones por Citomegalovirus/inmunología , Citomegalovirus/inmunología , Evasión Inmune , Proteínas Nucleares/inmunología , Proteolisis , Proteínas del Envoltorio Viral/inmunología , Citomegalovirus/genética , Infecciones por Citomegalovirus/genética , Humanos , Proteínas Nucleares/genética , Complejos de Ubiquitina-Proteína Ligasa/genética , Complejos de Ubiquitina-Proteína Ligasa/inmunología , Proteínas del Envoltorio Viral/genéticaRESUMEN
Proteolysis-targeting chimeras (PROTACs), heterobifunctional compounds that consist of protein-targeting ligands linked to an E3 ligase recruiter, have arisen as a powerful therapeutic modality for targeted protein degradation (TPD). Despite the popularity of TPD approaches in drug discovery, only a small number of E3 ligase recruiters are available for the >600 E3 ligases that exist in human cells. Here, we have discovered a cysteine-reactive covalent ligand, EN106, that targets FEM1B, an E3 ligase recently discovered as the critical component of the cellular response to reductive stress. By targeting C186 in FEM1B, EN106 disrupts recognition of the key reductive stress substrate of FEM1B, FNIP1. We further establish that EN106 can be used as a covalent recruiter for FEM1B in TPD applications by demonstrating that a PROTAC linking EN106 to the BET bromodomain inhibitor JQ1 or the kinase inhibitor dasatinib leads to the degradation of BRD4 and BCR-ABL, respectively. Our study showcases a covalent ligand that targets a natural E3 ligase-substrate binding site and highlights the utility of covalent ligand screening in expanding the arsenal of E3 ligase recruiters suitable for TPD applications.
Asunto(s)
Acetamidas/química , Proteínas de Ciclo Celular/metabolismo , Proteolisis , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Animales , Azepinas/química , Sitios de Unión , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Línea Celular , Cisteína/química , Dasatinib/química , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Ratones , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Inhibidores de Proteínas Quinasas/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Triazoles/química , Complejos de Ubiquitina-Proteína Ligasa/antagonistas & inhibidores , Complejos de Ubiquitina-Proteína Ligasa/genéticaRESUMEN
BACKGROUND: Woodhouse-Sakati syndrome is a rare autosomal recessive disease with endocrine and neuroectodermal aberrations with heterogeneous phenotypes and disease course. The most common phenotypes of the disease are progressive sensorineural hearing loss and alopecia, mild-to-moderate mental retardation and hypogonadism. The disease results from mutations in the DCAF17 gene. METHOD: Here, we reported a large consanguineous pedigree with multiple affected individuals with Woodhouse-Sakati syndrome phenotypes. Laboratory tests confirmed the endocrine perturbance in affected individuals. To find out the underlying genetic change, whole-exome sequencing was carried out. RESULT: Analysis of the exome data identified a splicing-site deletion NM_025000.3:c.1423-1_1425delGACA in DCAF17 gene. Sanger sequencing confirmed the co-segregation of the variant with the disease phenotypes in the family. CONCLUSION: The variant is predicted to cause aberrant splicing, i.e., exon skipping, resulting in the translation of a truncated functionless protein which results in appearance of typical phenotypic features and clinical laboratory findings of Woodhouse-Sakati syndrome in affected members of the family.
Asunto(s)
Alopecia/genética , Arritmias Cardíacas/genética , Enfermedades de los Ganglios Basales/genética , Diabetes Mellitus/genética , Hipogonadismo/genética , Discapacidad Intelectual/genética , Mutación/genética , Proteínas Nucleares/genética , Complejos de Ubiquitina-Proteína Ligasa/genética , Adolescente , Alopecia/patología , Alopecia/fisiopatología , Arritmias Cardíacas/patología , Arritmias Cardíacas/fisiopatología , Enfermedades de los Ganglios Basales/patología , Enfermedades de los Ganglios Basales/fisiopatología , Niño , Consanguinidad , Análisis Mutacional de ADN , Diabetes Mellitus/patología , Diabetes Mellitus/fisiopatología , Facies , Femenino , Humanos , Hipogonadismo/patología , Hipogonadismo/fisiopatología , Discapacidad Intelectual/patología , Discapacidad Intelectual/fisiopatología , Masculino , Linaje , Isoformas de Proteínas/genética , Cuero Cabelludo/patologíaRESUMEN
Woodhouse-Sakati syndrome (WSS) is a rare autosomal recessive neuroendocrine and ectodermal disorder caused by variants in the DCAF17 gene. In Qatar, the c.436delC variant has been reported as a possible founder pathogenic variant with striking phenotypic heterogeneity. In this retrospective study, we report on the clinical and molecular characteristics of additional 58 additional Qatari patients with WSS and compare them to international counterparts' findings. A total of 58 patients with WSS from 32 consanguineous families were identified. Ectodermal and endocrine (primary hypogonadism) manifestations were the most common presentations (100%), followed by diabetes mellitus (46%) and hypothyroidism (36%). Neurological manifestations were overlapping among patients with intellectual disability (ID) being the most common (75%), followed by sensorineural hearing loss (43%) and both ID and aggressive behavior (10%). Distinctive facial features were noted in all patients and extrapyramidal manifestations were uncommon (8.6%). This study is the largest to date on Qatari patients with WSS and highlights the high incidence and clinical heterogeneity of WSS in Qatar due to a founder variant c.436delC in the DCAF17 gene. Early suspicion of WSS among Qatari patients with hypogonadism and ID, even in the absence of other manifestations, would shorten the diagnostic odyssey, guide early and appropriate management, and avoid potential complications.
Asunto(s)
Diabetes Mellitus , Hipogonadismo , Discapacidad Intelectual , Alopecia , Animales , Arritmias Cardíacas , Enfermedades de los Ganglios Basales , Diabetes Mellitus/diagnóstico , Humanos , Hipogonadismo/diagnóstico , Hipogonadismo/genética , Discapacidad Intelectual/diagnóstico , Proteínas Nucleares/genética , Linaje , Qatar/epidemiología , Estudios Retrospectivos , Complejos de Ubiquitina-Proteína Ligasa/genéticaRESUMEN
BACKGROUND: Sepsis is a life-threatening disease caused by the dysregulated host response to the infection and the major cause of death of patients in the intensive care unit (ICU). OBJECTIVE: Early diagnosis of sepsis could significantly reduce in-hospital mortality. Though generated from infection, the development of sepsis follows its own psychological process and disciplines, alters with gender, health status and other factors. Hence, the analysis of mass data by bioinformatics tools and machine learning is a promising method for exploring early diagnosis. METHODS: We collected miRNA and mRNA expression data of sepsis blood samples from Gene Expression Omnibus (GEO) and ArrayExpress databases, screened out differentially expressed genes (DEGs) by R software, predicted miRNA targets on TargetScanHuman and miRTarBase websites, conducted Gene Ontology (GO) term and KEGG pathway enrichment analysis based on overlapping DEGs. The STRING database and Cytoscape were used to build protein-protein interaction (PPI) network and predict hub genes. Then we constructed a Random Forest model by using the hub genes to assess sample type. RESULTS: Bioinformatic analysis of GEO dataset revealed 46 overlapping DEGs in sepsis. The PPI network analysis identified five hub genes, SOCS3, KBTBD6, FBXL5, FEM1C and WSB1. Random Forest model based on these five hub genes was used to assess GSE95233 and GSE95233 datasets, and the area under the curve (AUC) of ROC was 0.900 and 0.7988, respectively, which confirmed the efficacy of this model. CONCLUSION: The integrated analysis of gene expression in sepsis and the effective Random Forest model built in this study may provide promising diagnostic methods for sepsis.
Asunto(s)
MicroARNs , Sepsis , Biología Computacional/métodos , Expresión Génica , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Aprendizaje Automático , Mapas de Interacción de Proteínas/genética , Sepsis/diagnóstico , Sepsis/genética , Complejos de Ubiquitina-Proteína Ligasa/genéticaRESUMEN
PURPOSE: Woodhouse-Sakati syndrome is a rare autosomal recessive syndrome caused by homozygous mutations in the DCAF17 gene, characterized by marked neurologic and endocrine manifestations in the setting of brain iron accumulation and white matter lesions on neuroimaging. Here, we report electrophysiologic profiles in Woodhouse-Sakati syndrome and their possible value in understanding disease pathophysiology and phenotypic variability. METHODS: Thirteen genetically confirmed Woodhouse-Sakati syndrome patients were evaluated via different evoked potential (EP) modalities, including brainstem auditory EPs, pattern reversal visual EPs, and somatosensory EPs to tibial and/or median nerves. RESULTS: All EP modalities showed variable abnormalities. Pattern reversal visual EPs were recorded in all patients, with nine patients exhibiting abnormal results. From those, seven patients showed prolonged P100 latencies after stimulation of right and left eyes for each in turn. Two patients showed P100 latency abnormality after single eye stimulation recorded from midoccipital electrode. Median somatosensory EPs were recorded in 10 patients, with 6 patients having a prolonged cortical N19 response. Tibial somatosensory EP was performed for 11 patients, and 8 patients showed abnormal results where P37 cortical response was absent or prolonged, whereas peripheral potentials at the popliteal fossa were normal. Brainstem auditory EPs were abnormal only in two patients, with prolonged wave III and V latencies. Five patients with hearing impairment presented with normal brainstem auditory EP results. CONCLUSIONS: Multiple EP abnormalities are observed in Woodhouse-Sakati syndrome patients, mainly in pattern reversal visual EPs and somatosensory EPs. These findings indicate potential myelin dysfunction that has a role in the underlying pathophysiology, disease course, and phenotypic variability.
Asunto(s)
Enfermedades de los Ganglios Basales , Hipogonadismo , Discapacidad Intelectual , Alopecia , Arritmias Cardíacas , Enfermedades de los Ganglios Basales/genética , Enfermedades de los Ganglios Basales/patología , Diabetes Mellitus , Potenciales Evocados/fisiología , Potenciales Evocados Auditivos , Potenciales Evocados Somatosensoriales/fisiología , Potenciales Evocados Visuales , Humanos , Hipogonadismo/genética , Hipogonadismo/patología , Discapacidad Intelectual/genética , Discapacidad Intelectual/patología , Proteínas Nucleares/genética , Complejos de Ubiquitina-Proteína Ligasa/genéticaRESUMEN
Proteins encoded by U-box type ubiquitin ligase (PUB) genes in rice are known to play an important role in plant responses to abiotic and biotic stresses. Functional analysis has revealed a detailed molecular mechanism involving PUB proteins in relation to abiotic and biotic stresses. In this study, characteristics of 77 OsPUB genes in rice were identified. Systematic and comprehensive analyses of the OsPUB gene family were then performed, including analysis of conserved domains, phylogenetic relationships, gene structure, chromosome location, cis-acting elements, and expression patterns. Through transcriptome analysis, we confirmed that 16 OsPUB genes show similar expression patterns in drought stress and blast infection response pathways. Numerous cis-acting elements were found in promoter sequences of 16 OsPUB genes, indicating that the OsPUB genes might be involved in complex regulatory networks to control hormones, stress responses, and cellular development. We performed qRT-PCR on 16 OsPUB genes under drought stress and blast infection to further identify the reliability of transcriptome and cis-element analysis data. It was confirmed that the expression pattern was similar to RNA-sequencing analysis results. The transcription of OsPUB under various stress conditions indicates that the PUB gene might have various functions in the responses of rice to abiotic and biotic stresses. Taken together, these results indicate that the genome-wide analysis of OsPUB genes can provide a solid basis for the functional analysis of U-box E3 ubiquitin ligase genes. The molecular information of the U-box E3 ubiquitin ligase gene family in rice, including gene expression patterns and cis-acting regulatory elements, could be useful for future crop breeding programs by genome editing.
Asunto(s)
Oryza/enzimología , Oryza/genética , Proteínas de Plantas/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Sequías , Oryza/crecimiento & desarrollo , Oryza/metabolismo , Filogenia , Fitomejoramiento , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Estrés Fisiológico , Transcriptoma , Complejos de Ubiquitina-Proteína Ligasa/genética , UbiquitinaciónRESUMEN
Toxoplasma gondii is an obligate intracellular apicomplexan parasite causing lethal diseases in immunocompromised patients. UBL-UBA shuttle proteins (DDI1, RAD23, and DSK2) are important components of the ubiquitin-proteasome system. By degrading ubiquitinated proteins, UBL-UBA shuttle proteins regulate many cellular processes. However, the specific processes regulated by UBL-UBA shuttle proteins remain elusive. Here, we revealed that the deletion of shuttle proteins results in a selective accumulation of ubiquitinated proteins in the nucleus and aberrant DNA replication. ROP18 was mistargeted and accumulated in the shuttle protein mutant strain, resulting in the recruitment of immunity-related GTPases to the parasitophorous vacuole membrane (PVM). Furthermore, the mistargeting of ROP18 and the recruitment of Irgb6 to the PVM were also observed in the DDI1 mutant strain. DDI1 is a nonclassical UBL-UBA shuttle protein homologous to the HIV-1 protease. Molecular docking showed that DDI1 was a potential target of HIV-1 protease inhibitors. However, these inhibitors blocked the growth of T gondii in vitro but not in vivo. In conclusion, the Toxoplasma UBL-UBA shuttle protein regulates several important cellular processes and the mistargeting of ROP18 may be a representative of the abnormal homeostasis caused by shuttle protein mutation.
Asunto(s)
Indinavir/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitina/metabolismo , Animales , Replicación del ADN , Femenino , Inhibidores de la Proteasa del VIH/farmacología , Humanos , Ratones , Ratones Endogámicos BALB C , Proteínas Serina-Treonina Quinasas/genética , Proteínas Protozoarias/genética , Toxoplasma/efectos de los fármacos , Complejos de Ubiquitina-Proteína Ligasa/genética , UbiquitinaciónRESUMEN
CENP-A (centromeric protein A), a histone H3 variant, specifies centromere identity and is essential to centromere maintenance. Little is known about how protein levels of CENP-A are controlled in mammalian cells. Here, we report that the phosphorylation of CENP-A Ser68 primes the ubiquitin-proteasome-mediated proteolysis of CENP-A during mitotic phase in human cultured cells. We identify two major polyubiquitination sites that are responsible for this phosphorylation-dependent degradation. Substituting the two residues, Lys49 and Lys124, with arginines abrogates proper CENP-A degradation and results in CENP-A mislocalization to non-centromeric regions. Furthermore, we find that DCAF11 (DDB1 and CUL4 associated factor 11/WDR23) is the E3 ligase that specifically mediates the observed polyubiquitination. Deletion of DCAF11 hampers CENP-A degradation and causes its mislocalization. We conclude that the Ser68 phosphorylation plays an important role in regulating cellular CENP-A homeostasis via DCAF11-mediated degradation to prevent ectopic localization of CENP-A during the cell cycle.
Asunto(s)
Ciclo Celular , Proteína A Centromérica/metabolismo , Proteínas Cullin/metabolismo , Proteínas de Unión al ADN/metabolismo , Serina/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitinación , Animales , Centrómero , Proteína A Centromérica/química , Proteína A Centromérica/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas Cullin/genética , Proteínas de Unión al ADN/genética , Femenino , Histonas/genética , Histonas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Nucleosomas , Fosforilación , Proteolisis , Serina/química , Serina/genética , Ubiquitina/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/genéticaRESUMEN
Geminin and its binding partner Cdt1 are essential for the regulation of DNA replication. Here we show that the CULLIN3 E3 ubiquitin ligase adaptor protein SPOP binds Geminin at endogenous level and regulates DNA replication. SPOP promotes K27-linked non-degradative poly-ubiquitination of Geminin at lysine residues 100 and 127. This poly-ubiquitination of Geminin prevents DNA replication over-firing by indirectly blocking the association of Cdt1 with the MCM protein complex, an interaction required for DNA unwinding and replication. SPOP is frequently mutated in certain human cancer types and implicated in tumorigenesis. We show that cancer-associated SPOP mutations impair Geminin K27-linked poly-ubiquitination and induce replication origin over-firing and re-replication. The replication stress caused by SPOP mutations triggers replication catastrophe and cell death upon ATR inhibition. Our results reveal a tumor suppressor role of SPOP in preventing DNA replication over-firing and genome instability and suggest that SPOP-mutated tumors may be susceptible to ATR inhibitor therapy.
Asunto(s)
Geminina/metabolismo , Proteínas Nucleares/genética , Proteínas Represoras/genética , Complejos de Ubiquitina-Proteína Ligasa/genética , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Replicación del ADN/genética , Replicación del ADN/fisiología , Geminina/genética , Humanos , Masculino , Ratones , Ratones SCID , Proteínas de Mantenimiento de Minicromosoma/genética , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Mutación/genética , Ubiquitinación/genética , Ubiquitinación/fisiologíaRESUMEN
Although general anesthesia is indispensable during modern surgical procedures, the mechanism by which inhalation anesthetics act on the synaptic membrane at the molecular and cellular level is largely unknown. In this study, we used yeast cells to examine the effect of isoflurane, an inhalation anesthetic, on membrane proteins. Bap2, an amino acid transporter localized on the plasma membrane, was endocytosed when yeast cells were treated with isoflurane. Depletion of RSP5, an E3 ligase, prevented this endocytosis and Bap2 was ubiquitinated in response to isoflurane, indicating an ubiquitin-dependent process. Screening all the Rsp5 binding adaptors showed that Art2 plays a central role in this process. These results suggest that isoflurane affects Bap2 via an Art2-Rsp5-dependent ubiquitination system.
Asunto(s)
Endocitosis/fisiología , Isoflurano/farmacología , Sistemas de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos/fisiología , Membrana Celular/metabolismo , Endocitosis/efectos de los fármacos , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Isoflurano/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Ubiquitina/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/genética , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Autoimmune liver diseases (AILD) often lead to transformation of the liver tissues into hepatocellular carcinoma (HCC). Considering the drawbacks of surgical procedures in such cases, need of successful non-invasive therapeutic strategies and treatment modalities for AILD-associated-HCC still exists. Due to the lack of clear, sufficient knowledge about factors mediating AILD-to-HCC transition, an in silico approach was adopted to delineate the underlying molecular deterministic factors. Parallel enrichment analyses on two different public microarray datasets (GSE159676 and GSE62232) pinpointed the core transcriptional regulators as key players. Correlation between the expression kinetics of these transcriptional modules in AILD and HCC was found to be positive primarily with the advancement of hepatic fibrosis. Most of the regulatory interactions were operative during early (F0-F1) and intermediate fibrotic stages (F2-F3), while the extent of activity in the regulatory network considerably diminished at late stage of fibrosis/cirrhosis (F4). Additionally, most of the transcriptional targets with higher degrees of connectivity in the regulatory network (namely DCAF11, PKM2, DGAT2 and BCAT1) may be considered as potential candidates for biomarkers or clinical targets compared to their low-connectivity counterparts. In summary, this study uncovers new possibilities in the designing of novel prognostic and therapeutic regimen for autoimmunity-associated malignancy of liver in a disease progression-dependent fashion.
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Enfermedades Autoinmunes/complicaciones , Carcinoma Hepatocelular/etiología , Simulación por Computador , Cirrosis Hepática/complicaciones , Neoplasias Hepáticas/etiología , Enfermedades Autoinmunes/genética , Biomarcadores , Carcinoma Hepatocelular/genética , Proteínas Portadoras/análisis , Proteínas Portadoras/genética , Colangitis Esclerosante/complicaciones , Colangitis Esclerosante/genética , Biología Computacional , Diacilglicerol O-Acetiltransferasa/análisis , Diacilglicerol O-Acetiltransferasa/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Cirrosis Hepática/genética , Cirrosis Hepática Biliar/complicaciones , Cirrosis Hepática Biliar/genética , Hepatopatías/complicaciones , Hepatopatías/genética , Neoplasias Hepáticas/genética , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Hormonas Tiroideas/análisis , Hormonas Tiroideas/genética , Transaminasas/análisis , Transaminasas/genética , Complejos de Ubiquitina-Proteína Ligasa/análisis , Complejos de Ubiquitina-Proteína Ligasa/genética , Proteínas de Unión a Hormona TiroideRESUMEN
Ribonucleoside monophosphate (rNMP) incorporation in genomic DNA poses a significant threat to genomic integrity. In addition to repair, DNA damage tolerance mechanisms ensure replication progression upon encountering unrepaired lesions. One player in the tolerance mechanism is Rad5, which is an E3 ubiquitin ligase and helicase. Here, we report a new role for yeast Rad5 in tolerating rNMP incorporation, in the absence of the bona fide ribonucleotide excision repair pathway via RNase H2. This role of Rad5 is further highlighted after replication stress induced by hydroxyurea or by increasing rNMP genomic burden using a mutant DNA polymerase (Pol ε - Pol2-M644G). We further demonstrate the importance of the ATPase and ubiquitin ligase domains of Rad5 in rNMP tolerance. These findings suggest a similar role for the human Rad5 homologues helicase-like transcription factor (HLTF) and SNF2 Histone Linker PHD RING Helicase (SHPRH) in rNMP tolerance, which may impact the response of cancer cells to replication stress-inducing therapeutics.
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ADN Helicasas/metabolismo , Ribonucleótidos/metabolismo , Adenosina Trifosfatasas/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Daño del ADN , ADN Helicasas/química , ADN Helicasas/genética , Genómica/métodos , Mutación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estrés Fisiológico , Complejos de Ubiquitina-Proteína Ligasa/genética , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Levaduras/fisiologíaAsunto(s)
Alopecia/diagnóstico por imagen , Alopecia/genética , Arritmias Cardíacas/diagnóstico por imagen , Arritmias Cardíacas/genética , Enfermedades de los Ganglios Basales/diagnóstico por imagen , Enfermedades de los Ganglios Basales/genética , Mapeo Cromosómico/métodos , Diabetes Mellitus/diagnóstico por imagen , Diabetes Mellitus/genética , Predisposición Genética a la Enfermedad/genética , Hipogonadismo/diagnóstico por imagen , Hipogonadismo/genética , Discapacidad Intelectual/diagnóstico por imagen , Discapacidad Intelectual/genética , Adolescente , Humanos , Masculino , Proteínas Nucleares/genética , Complejos de Ubiquitina-Proteína Ligasa/genéticaRESUMEN
Nuclear dot protein 52 kDa (NDP52, also known as CALCOCO2) functions as a selective autophagy receptor. The linear ubiquitin chain assembly complex (LUBAC) specifically generates the N-terminal Met1-linked linear ubiquitin chain, and regulates innate immune responses, such as nuclear factor-κB (NF-κB), interferon (IFN) antiviral, and apoptotic pathways. Although NDP52 and LUBAC cooperatively regulate bacterial invasion-induced xenophagy, their functional crosstalk remains enigmatic. Here we show that NDP52 suppresses canonical NF-κB signaling through the broad specificity of ubiquitin-binding at the C-terminal UBZ domain. Upon TNF-α-stimulation, NDP52 associates with LUBAC through the HOIP subunit, but does not disturb its ubiquitin ligase activity, and has a modest suppressive effect on NF-κB activation by functioning as a component of TNF-α receptor signaling complex I. NDP52 also regulates the TNF-α-induced apoptotic pathway, but not doxorubicin-induced intrinsic apoptosis. A chemical inhibitor of LUBAC (HOIPIN-8) cancelled the increased activation of the NF-κB and IFN antiviral pathways, and enhanced apoptosis in NDP52-knockout and -knockdown HeLa cells. Upon Salmonella-infection, colocalization of Salmonella, LC3, and linear ubiquitin was detected in parental HeLa cells to induce xenophagy. Treatment with HOIPIN-8 disturbed the colocalization and facilitated Salmonella expansion. In contrast, HOIPIN-8 showed little effect on the colocalization of LC3 and Salmonella in NDP52-knockout cells, suggesting that NDP52 is a weak regulator in LUBAC-mediated xenophagy. These results indicate that the crosstalk between NDP52 and LUBAC regulates innate immune responses, apoptosis, and xenophagy.