RESUMEN
OTU deubiquitinase with linear linkage specificity (OTULIN) regulates inflammation and cell death by deubiquitinating linear ubiquitin chains generated by the linear ubiquitin chain assembly complex (LUBAC). Biallelic loss-of-function mutations causes OTULIN-related autoinflammatory syndrome (ORAS), while OTULIN haploinsuffiency has not been associated with spontaneous inflammation. However, herein, we identify two patients with the heterozygous mutation p.Cys129Ser in OTULIN. Consistent with ORAS, we observed accumulation of linear ubiquitin chains, increased sensitivity to TNF-induced death, and dysregulation of inflammatory signaling in patient cells. While the C129S mutation did not affect OTULIN protein stability or binding capacity to LUBAC and linear ubiquitin chains, it did ablate OTULIN deubiquitinase activity. Loss of activity facilitated the accumulation of autoubiquitin chains on LUBAC. Altered ubiquitination of LUBAC inhibits its recruitment to the TNF receptor signaling complex, promoting TNF-induced cell death and disease pathology. By reporting the first dominant negative mutation driving ORAS, this study expands our clinical understanding of OTULIN-associated pathology.
Asunto(s)
Inflamación , Ubiquitina , Humanos , Muerte Celular , Membrana Celular , Enzimas Desubicuitinizantes , Inflamación/genética , Síndrome , Complejos de Ubiquitina-Proteína LigasaRESUMEN
Phosphatidylinositol 3-kinase α, a heterodimer of catalytic p110α and one of five regulatory subunits, mediates insulin- and insulin like growth factor-signaling and, frequently, oncogenesis. Cellular levels of the regulatory p85α subunit are tightly controlled by regulated proteasomal degradation. In adipose tissue and growth plates, failure of K48-linked p85α ubiquitination causes diabetes, lipodystrophy and dwarfism in mice, as in humans with SHORT syndrome. Here we elucidated the structures of the key ubiquitin ligase complexes regulating p85α availability. Specificity is provided by the substrate receptor KBTBD2, which recruits p85α to the cullin3-RING E3 ubiquitin ligase (CRL3). CRL3KBTBD2 forms multimers, which disassemble into dimers upon substrate binding (CRL3KBTBD2-p85α) and/or neddylation by the activator NEDD8 (CRL3KBTBD2~N8), leading to p85α ubiquitination and degradation. Deactivation involves dissociation of NEDD8 mediated by the COP9 signalosome and displacement of KBTBD2 by the inhibitor CAND1. The hereby identified structural basis of p85α regulation opens the way to better understanding disturbances of glucose regulation, growth and cancer.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ia , Complejos de Ubiquitina-Proteína Ligasa , Ubiquitina-Proteína Ligasas , Animales , Humanos , Ratones , Proteínas Cullin/metabolismo , Insulina/metabolismo , Unión Proteica , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Fosfatidilinositol 3-Quinasa Clase Ia/química , Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismoRESUMEN
Woodhouse-Sakati syndrome consists of hypogonadism, diabetes mellitus, alopecia, ECG abnormalities, and dystonia. This condition is caused by the loss of function of the DCAF17 gene. Most of the patients have been reported from Greater Middle Eastern countries. We report a 38 male from southern India who presented with syncope and massive hemoptysis due to ruptured bronchopulmonary collaterals. He also had alopecia, cataracts, recently diagnosed diabetes and hypogonadism. Whole exome sequencing showed a novel homozygous truncating variant in the DCAF17 gene. Despite embolization of the aortopulmonary collaterals, the patient died of recurrent hemoptysis.
Asunto(s)
Diabetes Mellitus , Hipogonadismo , Discapacidad Intelectual , Humanos , Masculino , Hemoptisis , Proteínas Nucleares/genética , Diabetes Mellitus/diagnóstico , Diabetes Mellitus/genética , Diabetes Mellitus/patología , Alopecia/complicaciones , Alopecia/diagnóstico , Alopecia/genética , Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/genética , Discapacidad Intelectual/patología , Hipogonadismo/diagnóstico , Hipogonadismo/genética , Hipogonadismo/patología , Complejos de Ubiquitina-Proteína LigasaRESUMEN
Arsenic contamination is extremely threatening to the global public health. It was reported that sodium arsenite exposure induces serious kidney injury. However, the underlying mechanism is unclear. Ferroptosis is a newly characterized form of iron-dependent programmed cell death, which is implicated in the pathogenesis of various human diseases, including kidney injury. The lethal accumulation of iron-catalyzed lipid peroxidation is the fundamental biochemical characteristic of ferroptosis. Herein we report that sodium arsenite exposure initiates ferroptosis in mammalian HEK293, MEF and HT1080 cells, and induces ferroptosis-associated acute kidney injury in mice. RNA-binding protein G3BP1, the switch component of stress granules, is indispensable for sodium arsenite-induced ferroptosis in a stress granule-independent manner. Mechanistically, G3BP1 stabilizes IRP2, the master regulator of cellular iron homeostasis, through binding to and suppressing the translation of FBXL5 mRNA, which encodes the E3 ligase component to mediate IRP2 ubiquitination and proteasomal degradation. Sodium arsenite intoxication expedites this G3BP1-FBXL5-IRP2 axis and elevates cellular labile free iron, which is responsible for sodium arsenite exposure-induced lipid peroxidation and ferroptotic cell death. In summary, this study highlights a regulatory module comprising G3BP1-FBXL5-IRP2 axis in determining sodium arsenite-induced ferroptosis and ferroptosis-associated acute kidney injury in mice.
Asunto(s)
Lesión Renal Aguda , Arsenitos , Proteínas F-Box , Ferroptosis , Compuestos de Sodio , Humanos , Ratones , Animales , ADN Helicasas , Células HEK293 , Proteína 2 Reguladora de Hierro/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN , Hierro/metabolismo , Mamíferos/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Proteínas F-Box/química , Proteínas F-Box/genética , Proteínas F-Box/metabolismoRESUMEN
IMPORTANCE: Inactivation of EP300/CREBB paralogous cellular lysine acetyltransferases (KATs) during the early phase of infection is a consistent feature of DNA viruses. The cell responds by stabilizing transcription factor IRF3 which activates transcription of scores of interferon-stimulated genes (ISGs), inhibiting viral replication. Human respiratory adenoviruses counter this by assembling a CUL4-based ubiquitin ligase complex that polyubiquitinylates RUVBL1 and 2 inducing their proteasomal degradation. This inhibits accumulation of active IRF3 and the expression of anti-viral ISGs, allowing replication of the respiratory HAdVs in the face of inhibition of EP300/CBEBBP KAT activity by the N-terminal region of E1A.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas , Proteínas E1A de Adenovirus , Proteínas Portadoras , ADN Helicasas , Inmunidad Innata , Complejo de la Endopetidasa Proteasomal , Estrés Fisiológico , Humanos , Proteínas E1A de Adenovirus/metabolismo , Adenovirus Humanos/enzimología , Adenovirus Humanos/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Cullin/metabolismo , ADN Helicasas/metabolismo , Interferones/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Estructura Cuaternaria de Proteína , Complejos de Ubiquitina-Proteína Ligasa/química , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitinación , Replicación ViralRESUMEN
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
BACKGROUND: Woodhouse-Sakati syndrome (WSS) is a rare, autosomal recessive genetic disorder with variable clinical manifestations mainly affecting the endocrine and nervous systems. The aim of this study was to systematically review the genetic basis of WSS and report the genetic variants and clinical phenotypes associated with the disease. METHODS: PubMed, Science Direct, Scopus, and Web of Science databases were searched from the time of inception until June 2022. Broad search terms were used to capture the literature describing all genetic variants associated with WSS. The search keywords used are "Woodhouse Sakati" along with the term "mutation" OR "gene" OR "variant" OR "polymorphism". RESULTS: Twenty-five eligible studies were included in this study. One hundred and eighty-five patients in 97 families from 12 different countries were diagnosed with WSS. In patients from the Greater Middle East (GME) region, consanguineous marriages were common (67%). Thirteen different DCAF17 variants were associated with WSS development (including 8 identified in the GME region). The most frequent variant was a frameshift deletion variant (c.436delC, p.Ala147Hisfs*9) unique to Arabs that was reported in 11 cases from Tunisia, Kuwait, Qatar, Bahrain, and Saudi Arabia. There were no clear genotype-phenotype correlations for the different variants. CONCLUSIONS: This systematic review highlights the molecular basis and clinical manifestations of WSS globally, including the GME region, where the disease is prevalent due to consanguinity. Additional studies are now needed to understand the genotype-phenotype correlation for different DCAF17 variants and their impact on the phenotypic heterogeneity observed in WSS patients.
Asunto(s)
Diabetes Mellitus , Humanos , Epidemiología Molecular , Medio Oriente , Alopecia , Proteínas Nucleares , Complejos de Ubiquitina-Proteína LigasaRESUMEN
Gastrointestinal tumors have become a worldwide health problem with high morbidity and poor clinical outcomes. Chemotherapy and surgery, the main treatment methods, are still far from meeting the treatment needs of patients, and targeted therapy is in urgent need of development. Recently, emerging evidence suggests that kelch-like (KLHL) proteins play essential roles in maintaining proteostasis and are involved in the progression of various cancers, functioning as adaptors in the E3 ligase complex and promoting the specific degradation of substrates. Therefore, KLHL proteins should be taken into consideration for targeted therapy strategy discovery. This review summarizes the current knowledge of KLHL proteins in gastrointestinal tumors and discusses the potential of KLHL proteins as potential drug targets and prognostic biomarkers.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Neoplasias Gastrointestinales , Secuencia Kelch , Humanos , Neoplasias Gastrointestinales/tratamiento farmacológico , Secuencia Kelch/genética , Secuencia Kelch/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismoRESUMEN
The principal component of the protein homeostasis network is the ubiquitin-proteasome system. Ubiquitination is mediated by an enzymatic cascade involving, i.e. E3 ubiquitin ligases, many of which belong to the cullin-RING ligases family. Genetic defects in the ubiquitin-proteasome system components, including cullin-RING ligases, are known causes of neurodevelopmental disorders. Using exome sequencing to diagnose a pediatric patient with developmental delay, pyramidal signs and limb ataxia, we identified a de novo missense variant c.376G>C; p.(Asp126His) in the FEM1C gene encoding a cullin-RING ligase substrate receptor. This variant alters a conserved amino acid located within a highly constrained coding region and is predicted as pathogenic by most in silico tools. In addition, a de novo FEM1C mutation of the same residue p.(Asp126Val) was associated with an undiagnosed developmental disorder, and the relevant variant (FEM1CAsp126Ala) was found to be functionally compromised in vitro. Our computational analysis showed that FEM1CAsp126His hampers protein substrate binding. To further assess its pathogenicity, we used the nematode Caenorhabditis elegans. We found that the FEM-1Asp133His animals (expressing variant homologous to the FEM1C p.(Asp126Val)) had normal muscle architecture yet impaired mobility. Mutant worms were sensitive to the acetylcholinesterase inhibitor aldicarb but not levamisole (acetylcholine receptor agonist), showing that their disabled locomotion is caused by synaptic abnormalities and not muscle dysfunction. In conclusion, we provide the first evidence from an animal model suggesting that a mutation in the evolutionarily conserved FEM1C Asp126 position causes a neurodevelopmental disorder in humans.
Asunto(s)
Trastornos del Neurodesarrollo , Complejo de la Endopetidasa Proteasomal , Animales , Humanos , Niño , Proteínas Cullin/metabolismo , Acetilcolinesterasa , Habla , Ubiquitina-Proteína Ligasas/genética , Trastornos del Neurodesarrollo/genética , Ubiquitina/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Ataxia/genética , Complejos de Ubiquitina-Proteína LigasaRESUMEN
During mammalian spermatogenesis, the ubiquitin proteasome system maintains protein homoeostasis (proteastasis) and spermatogenic cellular functions. DCAF17 is a substrate receptor in the ubiquitin CRL4 E3 Ligase complex, absence of which causes oligoasthenoteratozoospermia in mice resulting in male infertility. To determine the molecular phenomenon underlying the infertility phenotype caused by disrupting Dcaf17, we performed RNA-sequencing-based gene expression profiling of 3-weeks and 8-weeks old Dcaf17 wild type and Dcaf17 disrupted mutant mice testes. At three weeks, 44% and 56% differentially expressed genes (DEGs) were up- and down-regulated, respectively, with 32% and 68% DEGs were up- and down-regulated, respectively at 8 weeks. DEGs include protein coding genes and lncRNAs distributed across all autosomes and the X chromosome. Gene ontology analysis revealed major biological processes including proteolysis, regulation of transcription and chromatin remodelling are affected due to Dcaf17 disruption. We found that Dcaf17 disruption up-regulated several somatic genes, while germline-associated genes were down-regulated. Up to 10% of upregulated, and 12% of downregulated, genes were implicated in male reproductive phenotypes. Moreover, a large proportion of the up-regulated genes were highly expressed in spermatogonia and spermatocytes, while the majority of downregulated genes were predominantly expressed in round spermatids. Collectively, these data show that the Dcaf17 disruption affects directly or indirectly testicular proteastasis and transcriptional signature in mouse.
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Espermatogénesis , Testículo , Complejos de Ubiquitina-Proteína Ligasa , Animales , Masculino , Ratones , Fertilidad/genética , Espermátides/metabolismo , Espermatogénesis/genética , Testículo/metabolismo , Transcriptoma , Ubiquitina/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismoRESUMEN
The ubiquitin E3 ligase substrate adapter cereblon (CRBN) is a target of thalidomide and lenalidomide1, therapeutic agents used in the treatment of haematopoietic malignancies2-4 and as ligands for targeted protein degradation5-7. These agents are proposed to mimic a naturally occurring degron; however, the structural motif recognized by the thalidomide-binding domain of CRBN remains unknown. Here we report that C-terminal cyclic imides, post-translational modifications that arise from intramolecular cyclization of glutamine or asparagine residues, are physiological degrons on substrates for CRBN. Dipeptides bearing the C-terminal cyclic imide degron substitute for thalidomide when embedded within bifunctional chemical degraders. Addition of the degron to the C terminus of proteins induces CRBN-dependent ubiquitination and degradation in vitro and in cells. C-terminal cyclic imides form adventitiously on physiologically relevant timescales throughout the human proteome to afford a degron that is endogenously recognized and removed by CRBN. The discovery of the C-terminal cyclic imide degron defines a regulatory process that may affect the physiological function and therapeutic engagement of CRBN.
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Imidas , Proteolisis , Complejos de Ubiquitina-Proteína Ligasa , Humanos , Asparagina/química , Dipéptidos/farmacología , Glutamina/química , Imidas/química , Imidas/metabolismo , Lenalidomida/farmacología , Ligandos , Péptido Hidrolasas/metabolismo , Proteolisis/efectos de los fármacos , Proteoma/metabolismo , Talidomida/farmacología , Ubiquitinación/efectos de los fármacos , Secuencias de Aminoácidos , CiclizaciónRESUMEN
Despite the high prevalence of hypospadias and cryptorchidism, the genetic basis for these conditions is only beginning to be understood. Using array-comparative-genomic-hybridization (aCGH), potassium-channel-tetramerization-domain-containing-13 (KCTD13) encoded at 16p11.2 was identified as a candidate gene involved in hypospadias, cryptorchidism and other genitourinary (GU) tract anomalies. Copy number variants (CNVs) at 16p11.2 are among the most common syndromic genomic variants identified to date. Many patients with CNVs at this locus exhibit GU and/or neurodevelopmental phenotypes. KCTD13 encodes a substrate-specific adapter of a BCR (BTB-CUL3-RBX1) E3-ubiquitin-protein-ligase complex (BCR (BTB-CUL3-RBX1) E3-ubiquitin-protein-ligase complex (B-cell receptor (BCR) [BTB (the BTB domain is a conserved motif involved in protein-protein interactions) Cullin3 complex RING protein Rbx1] E3-ubiqutin-protein-ligase complex), which has essential roles in the regulation of cellular cytoskeleton, migration, proliferation, and neurodevelopment; yet its role in GU development is unknown. The prevalence of KCTD13 CNVs in patients with GU anomalies (2.58%) is significantly elevated when compared with patients without GU anomalies or in the general population (0.10%). KCTD13 is robustly expressed in the developing GU tract. Loss of KCTD13 in cell lines results in significantly decreased levels of nuclear androgen receptor (AR), suggesting that loss of KCTD13 affects AR sub-cellular localization. Kctd13 haploinsufficiency and homozygous deletion in mice cause a significant increase in the incidence of cryptorchidism and micropenis. KCTD13-deficient mice exhibit testicular and penile abnormalities together with significantly reduced levels of nuclear AR and SOX9. In conclusion, gene-dosage changes of murine Kctd13 diminish nuclear AR sub-cellular localization, as well as decrease SOX9 expression levels which likely contribute in part to the abnormal GU tract development in Kctd13 mouse models and in patients with CNVs in KCTD13.
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Criptorquidismo , Hipospadias , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Andrógenos , Animales , Criptorquidismo/genética , Dosificación de Gen , Homocigoto , Humanos , Masculino , Ratones , Proteínas Nucleares/metabolismo , Potasio , Receptores Androgénicos/genética , Receptores de Antígenos de Linfocitos B/genética , Eliminación de Secuencia , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinas/genética , Anomalías UrogenitalesRESUMEN
Viruses evolve mechanisms to exploit cellular pathways that increase viral fitness, e.g., enhance viral replication or evade the host cell immune response. The ubiquitin-proteosome system, a fundamental pathway-regulating protein fate in eukaryotes, is hijacked by all seven classes of viruses. Members of the Cullin-RING family of ubiquitin (Ub) ligases are frequently co-opted by divergent viruses because they can target a broad array of substrates by forming multisubunit assemblies comprised of a variety of adapters and substrate receptors. For example, the linker subunit DDB1 in the cullin 4-RING (CRL4)-DDB1 Ub ligase (CRL4DDB1) interacts with an H-box motif found in several unrelated viral proteins, including the V protein of simian virus 5 (SV5-V), the HBx protein of hepatitis B virus (HBV), and the recently identified pUL145 protein of human cytomegalovirus (HCMV). In HCMV-infected cells, pUL145 repurposes CRL4DDB1 to target STAT2, a protein vital to the antiviral immune response. However, the details of how these divergent viral sequences hijack DDB1 is not well understood. Here, we use a combination of binding assays, X-ray crystallography, alanine scanning, cell-based assays, and computational analysis to reveal that viral H-box motifs appear to bind to DDB1 with a higher affinity than the H-box motifs from host proteins DCAF1 and DDB2. This analysis reveals that viruses maintain native hot-spot residues in the H-box motif of host DCAFs and also acquire favorable interactions at neighboring residues within the H-box. Overall, these studies reveal how viruses evolve strategies to produce high-affinity binding and quality interactions with DDB1 to repurpose its Ub ligase machinery. IMPORTANCE Many different viruses modulate the protein machinery required for ubiquitination to enhance viral fitness. Specifically, several viruses hijack the cullin-RING ligase CRL4DDB1 to degrade host resistance factors. Human cytomegalovirus (HCMV) encodes pUL145 that redirects CRL4DDB1 to evade the immune system through the targeted degradation of the antiviral immune response protein STAT2. However, it is unclear why several viruses bind specific surfaces on ubiquitin ligases to repurpose their activity. We demonstrate that viruses have optimized H-box motifs that bind DDB1 with higher affinity than the H-box of native binders. For viral H-boxes, native interactions are maintained, but additional interactions that are absent in host cell H-boxes are formed, indicating that rewiring CRL4DDB1 creates a selective advantage for the virus. The DDB1-pUL145 peptide structure reveals that water-mediated interactions are critical to the higher affinity. Together, our data present an interesting example of how viral evolution can exploit a weakness in the ubiquitination machinery.
Asunto(s)
Proteínas Cullin , Infecciones por Citomegalovirus , Proteínas de Unión al ADN , Proteínas Virales , Proteínas Cullin/metabolismo , Infecciones por Citomegalovirus/inmunología , Proteínas de Unión al ADN/metabolismo , Humanos , Unión Proteica , Conformación Proteica , Factor de Transcripción STAT2/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Proteínas Virales/metabolismoRESUMEN
Targeting nuclear factor-kappa B (NF-κB) represents a highly viable strategy against chemoresistance in cancers as well as cell death. Ubiquitination, including linear ubiquitination mediated by the linear ubiquitin chain assembly complex (LUBAC), is emerging as a crucial mechanism of overactivated NF-κB signaling. Ovarian tumor family deubiquitinase OTULIN is the only linear linkage-specific deubiquitinase; however, the molecular mechanisms of how it counteracts LUBAC-mediated NF-κB activation have been largely unknown. Here, we identify Lys64/66 of OTULIN for linear ubiquitination facilitated in a LUBAC-dependent manner as a necessary event required for OTULIN-LUBAC interaction under unstressed conditions, which becomes deubiquitinated by OTULIN itself in response to genotoxic stress. Furthermore, this self-deubiquitination of OTULIN occurs intermolecularly, mediated by OTULIN dimerization, resulting in the subsequent dissociation of OTULIN from the LUBAC complex and NF-κB overactivation. Oxidative stress induces OTULIN dimerization via cysteine-mediated covalent disulfide bonds. Our study reveals that the status of the physical interaction between OTULIN and LUBAC is a crucial determining factor for the genotoxic NF-κB signaling, as measured by cell survival and proliferation, while OTULIN loss of function resulting from its dimerization and deubiquitination leads to a dissociation of OTULIN from the LUBAC complex. Of note, similar molecular mechanisms apply to the inflammatory NF-κB signaling in response to tumor necrosis factor α. Hence, a fuller understanding of the detailed molecular mechanisms underlying the disruption of the OTULIN-LUBAC interaction will be instrumental for developing future therapeutic strategies against cancer chemoresistance and necroptotic processes pertinent to numerous human diseases.
Asunto(s)
Daño del ADN , Enzimas Desubicuitinizantes , Endopeptidasas , Inflamación , FN-kappa B , Complejos de Ubiquitina-Proteína Ligasa , Enzimas Desubicuitinizantes/genética , Enzimas Desubicuitinizantes/metabolismo , Resistencia a Antineoplásicos , Endopeptidasas/genética , Endopeptidasas/metabolismo , Humanos , Inflamación/enzimología , Inflamación/genética , FN-kappa B/metabolismo , Necroptosis , Multimerización de Proteína , Transducción de Señal , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , UbiquitinaciónRESUMEN
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.
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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
Peroxisomes are ubiquitous organelles that house various metabolic reactions and are essential for human health1-4. Luminal peroxisomal proteins are imported from the cytosol by mobile receptors, which then recycle back to the cytosol by a poorly understood process1-4. Recycling requires receptor modification by a membrane-embedded ubiquitin ligase complex comprising three RING finger domain-containing proteins (Pex2, Pex10 and Pex12)5,6. Here we report a cryo-electron microscopy structure of the ligase complex, which together with biochemical and in vivo experiments reveals its function as a retrotranslocation channel for peroxisomal import receptors. Each subunit of the complex contributes five transmembrane segments that co-assemble into an open channel. The three ring finger domains form a cytosolic tower, with ring finger 2 (RF2) positioned above the channel pore. We propose that the N terminus of a recycling receptor is inserted from the peroxisomal lumen into the pore and monoubiquitylated by RF2 to enable extraction into the cytosol. If recycling is compromised, receptors are polyubiquitylated by the concerted action of RF10 and RF12 and degraded. This polyubiquitylation pathway also maintains the homeostasis of other peroxisomal import factors. Our results clarify a crucial step during peroxisomal protein import and reveal why mutations in the ligase complex cause human disease.
Asunto(s)
Microscopía por Crioelectrón , Peroxisomas , Complejos de Ubiquitina-Proteína Ligasa , Citosol/metabolismo , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/ultraestructura , Peroxinas/química , Peroxinas/metabolismo , Peroxinas/ultraestructura , Factor 2 de la Biogénesis del Peroxisoma/química , Factor 2 de la Biogénesis del Peroxisoma/metabolismo , Factor 2 de la Biogénesis del Peroxisoma/ultraestructura , Peroxisomas/enzimología , Peroxisomas/ultraestructura , Poliubiquitina , Transporte de Proteínas , Dominios RING Finger , Receptores Citoplasmáticos y Nucleares/química , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores Citoplasmáticos y Nucleares/ultraestructura , Complejos de Ubiquitina-Proteína Ligasa/química , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/ultraestructuraRESUMEN
Nedd4/Rsp5 family E3 ligases mediate numerous cellular processes, many of which require the E3 ligase to interact with PY motif containing adaptor proteins. Several arrestin-related trafficking adaptors (ARTs) of Rsp5 were self-ubiquitinated for activation, but the regulation mechanism remains elusive. Remarkably, we demonstrate that Art1, Art4, and Art5 undergo K63-linked di-ubiquitination by Rsp5. This modification enhances the plasma membrane recruitment of Rsp5 by Art1 or Art5 upon substrate induction, required for cargo protein ubiquitination. In agreement with these observations, we find that di-ubiquitin strengthens the interaction between the pombe orthologs of Rsp5 and Art1, Pub1, and Any1. Furthermore, we discover that the homologous to E6AP C-terminus (HECT) domain exosite protects the K63-linked di-ubiquitin on the adaptors from cleavage by the deubiquitination enzyme Ubp2. Together, our study uncovers a novel ubiquitination modification implemented by Rsp5 adaptor proteins, underscoring the regulatory mechanism of how adaptor proteins control the recruitment, and activity of Rsp5 for the turnover of membrane proteins.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Ubiquitina , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Proteínas de Unión a Poli(A)/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Protein ubiquitylation is an important posttranslational modification affecting a wide range of cellular processes. Due to the low abundance of ubiquitylated species in biological samples, considerable effort has been spent on methods to purify and detect ubiquitylated proteins. We have developed and characterized a novel tool for ubiquitin detection and purification based on OtUBD, a high-affinity ubiquitin-binding domain (UBD) derived from an Orientia tsutsugamushi deubiquitylase (DUB). We demonstrate that OtUBD can be used to purify both monoubiquitylated and polyubiquitylated substrates from yeast and human tissue culture samples and compare their performance with existing methods. Importantly, we found conditions for either selective purification of covalently ubiquitylated proteins or co-isolation of both ubiquitylated proteins and their interacting proteins. As proof of principle for these newly developed methods, we profiled the ubiquitylome and ubiquitin-associated proteome of the budding yeast Saccharomyces cerevisiae. Combining OtUBD affinity purification with quantitative proteomics, we identified potential substrates for the E3 ligases Bre1 and Pib1. OtUBD provides a versatile, efficient, and economical tool for ubiquitin research with specific advantages over certain other methods, such as in efficiently detecting monoubiquitylation or ubiquitin linkages to noncanonical sites.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Ubiquitina , Humanos , Proteoma/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
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.