RESUMO
NOTCH1 is a transmembrane receptor interacting with membrane-tethered ligands on opposing cells that mediate the direct cell-cell interaction necessary for many cell fate decisions. Protein O-fucosyltransferase 1 (POFUT1) adds O-fucose to Epidermal Growth Factor (EGF)-like repeats in the NOTCH1 extracellular domain, which is required for trafficking and signaling activation. We previously showed that POFUT1 S162L caused a 90% loss of POFUT1 activity and global developmental defects in a patient; however, the mechanism by which POFUT1 contributes to these symptoms is still unclear. Compared to controls, POFUT1 S162L patient fibroblast cells had an equivalent amount of NOTCH1 on the cell surface but showed a 60% reduction of DLL1 ligand binding and a 70% reduction in JAG1 ligand binding. To determine if the reduction of O-fucose on NOTCH1 in POFUT1 S162L patient fibroblasts was the cause of these effects, we immunopurified endogenous NOTCH1 from control and patient fibroblasts and analyzed O-fucosylation using mass spectral glycoproteomics methods. NOTCH1 EGF8 to EGF12 comprise the ligand binding domain, and O-fucose on EGF8 and EGF12 physically interact with ligands to enhance affinity. Glycoproteomics of NOTCH1 from POFUT1 S162L patient fibroblasts showed WT fucosylation levels at all sites analyzed except for a large decrease at EGF9 and the complete absence of O-fucose at EGF12. Since the loss of O-fucose on EGF12 is known to have significant effects on NOTCH1 activity, this may explain the symptoms observed in the POFUT1 S162L patient.
Assuntos
Fibroblastos , Fucose , Fucosiltransferases , Receptor Notch1 , Humanos , Fibroblastos/metabolismo , Fucose/metabolismo , Fucosiltransferases/metabolismo , Fucosiltransferases/genética , Receptor Notch1/metabolismo , Receptor Notch1/química , Família de Proteínas EGF/metabolismoRESUMO
Epidermal growth factor (EGF) repeats are present in various proteins and form well-defined structures with three disulfide bonds. One representative protein is the Notch receptor. Each EGF repeat contains unique atypical O-linked glycans, such as O-linked N-acetylglucosamine (O-GlcNAc). To generate a monoclonal antibody against the O-GlcNAc moiety in mouse Notch1, we expressed the recombinant C-terminal His6-tagged Notch1 EGF14-15 protein in HEK293T cells to prepare the immunogen. Most of the proteins were not secreted and showed higher molecular weight ladders in the cell lysate, suggesting protein aggregation. To overcome this issue, we fused Sparcl1 as an extracellular escorting tag to the N-terminus of Notch1 EGF14-15. The fusion protein was efficiently secreted extracellularly without protein aggregates in the lysates. Following PreScission protease treatment, Notch1 EGF14-15 was efficiently released from the escorting tag. Notch1 EGF14-15 prepared using this method was indeed O-GlcNAcylated. The optimal length of the escorting tag was determined by generating deletion mutants to improve the extracellular secretion of EGF14-15. Hence, a large amount of EGF14-15 was successfully prepared from the culture supernatant of HEK293T cells, which were otherwise prone to aggregation.
Assuntos
Fator de Crescimento Epidérmico , Receptores Notch , Humanos , Animais , Camundongos , Fator de Crescimento Epidérmico/química , Células HEK293 , Receptores Notch/metabolismo , Receptor Notch1/química , Acetilglucosamina/metabolismo , Proteínas de Ligação ao Cálcio , Proteínas da Matriz Extracelular/metabolismoRESUMO
BACKGROUND & AIMS: Fatty acid translocase CD36 (CD36/FAT) is a widely expressed membrane protein with multiple immuno-metabolic functions. Genetic CD36 deficiency is associated with increased risk of metabolic dysfunction-associated fatty liver disease (MAFLD) in patients. Liver fibrosis severity mainly affects the prognosis in patients with MAFLD, but the role of hepatocyte CD36 in liver fibrosis of MAFLD remains unclear. METHODS: A high-fat high-cholesterol diet and a high-fat diet with high-fructose drinking water were used to induce nonalcoholic steatohepatitis (NASH) in hepatocyte-specific CD36 knockout (CD36LKO) and CD36flox/flox (LWT) mice. Human hepG2 cell line was used to investigate the role of CD36 in regulating Notch pathway in vitro. RESULTS: Compared to LWT mice, CD36LKO mice were susceptible to NASH diet-induced liver injury and fibrosis. The analysis of RNA-sequencing data revealed that Notch pathway was activated in CD36LKO mice. LY3039478, an inhibitor of γ-secretase, inhibited Notch1 protein S3 cleavage and Notch1 intracellular domain (N1ICD) production, alleviating liver injury and fibrosis in CD36LKO mice livers. Likewise, both LY3039478 and knockdown of Notch1 inhibited the CD36KO-induced increase of N1ICD production, causing the decrease of fibrogenic markers in CD36KO HepG2 cells. Mechanistically, CD36 formed a complex with Notch1 and γ-secretase in lipid rafts, and hence CD36 anchored Notch1 in lipid rafts domains and blocked Notch1/γ-secretase interaction, inhibiting γ-secretase-mediated cleavage of Notch1 and the production of N1ICD. CONCLUSIONS: Hepatocyte CD36 plays a key role in protecting mice from diet-induced liver injury and fibrosis, which may provide a potential therapeutic strategy for preventing liver fibrogenesis in MAFLD.
Assuntos
Antígenos CD36 , Dieta , Hepatócitos , Cirrose Hepática , Hepatopatia Gordurosa não Alcoólica , Fragmentos de Peptídeos , Receptor Notch1 , Animais , Camundongos , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Antígenos CD36/deficiência , Antígenos CD36/genética , Antígenos CD36/metabolismo , Dieta/efeitos adversos , Deleção de Genes , Células Hep G2 , Hepatócitos/metabolismo , Cirrose Hepática/etiologia , Cirrose Hepática/genética , Cirrose Hepática/prevenção & controle , Microdomínios da Membrana , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fenótipo , Receptor Notch1/química , Receptor Notch1/metabolismo , Transdução de Sinais , HumanosRESUMO
NOTCH1 is a transmembrane receptor in metazoans that is linked to a variety of disorders. The receptor contains an extracellular domain (ECD) with 36 tandem epidermal growth factor-like (EGF) repeats. The ECD is responsible for intercellular signaling via protein-ligand interactions with neighboring cells. Each EGF repeat consists of approximately 40 amino acids and 3 conserved disulfide bonds. The Abruptex region (EGF24-29) is critical for NOTCH1 signaling and is known for its missense mutations. Certain EGF repeats are modified with the addition of O-linked glycans and many have calcium binding sites, which give each EGF repeat a unique function. It has been shown that the loss of the O-fucose site of EGF27 alters NOTCH1 activity. To investigate the role of glycosylation in the NOTCH1 signaling pathway, nuclear magnetic resonance spectroscopy has been employed to study the structures of EGF27 and its glycoforms. Here, we report the backbone and sidechain 1H, 15N, and 13C-resonance assignments of the unmodified EGF27 protein and the predicted secondary structure derived from the assigned chemical shifts.
Assuntos
Fator de Crescimento Epidérmico , Receptor Notch1 , Animais , Camundongos , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/metabolismo , Receptor Notch1/química , Receptor Notch1/metabolismo , Ressonância Magnética Nuclear Biomolecular , Glicosilação , Sítios de LigaçãoRESUMO
O-GlcNAc modification of Notch receptors regulates Notch ligand interactions in a manner distinct from other forms of O-glycans on epidermal growth factor (EGF)-like repeats of Notch receptors. Although many proteins, besides Notch receptors, are expected to be O-GlcNAcylated by EGF domain-specific O-GlcNAc transferase (EOGT), only a small number of proteins have been reported to be modified in vivo, and elongated O-GlcNAc glycans have not been extensively explored. To extend our view of the specificity and variety of the glycan modification, we conducted a comprehensive analysis of O-GlcNAc glycans on NOTCH1 in mammals. Mass spectrometric analysis of NOTCH1 fragments expressed in HEK293T cells revealed that several EGF domains with putative O-GlcNAcylation sites were hardly modified with O-GlcNAc. Although amino acid residues before the modification site are preferentially occupied with aromatic residues, Phe and Tyr are preferable to Trp for the apparent modification with O-GlcNAc. Furthermore, a minor form of fucosylated O-GlcNAc glycans was detected in a subset of EGF domains. Fucosylation of O-GlcNAc glycans was enhanced by FUT1, FUT2, or FUT9 expression. The FUT9-dependent Lewis X epitope was confirmed by immunoblotting using an anti-Lewis X antibody. As expected from the similarity in the extended structures between O-Fuc and O-GlcNAc glycans, the Lexis X antigen was detected on NOTCH1 fragments co-expressed with L-Fringe, which mediates elongation of O-Fuc glycans. Our results refined the putative consensus sequence for the EOGT-dependent O-GlcNAc modification in mammals and revealed the structural diversity of functional Notch O-glycans.
Assuntos
Fator de Crescimento Epidérmico , Glicosiltransferases , Animais , Fator de Crescimento Epidérmico/química , Glicosiltransferases/genética , Células HEK293 , Humanos , Mamíferos/metabolismo , Polissacarídeos , Receptor Notch1/química , Receptor Notch1/genética , Receptores Notch/metabolismoRESUMO
Cell-cell interactions regulate intracellular signaling via reciprocal contacts of cell membranes in tissue regeneration and cancer growth, indicating a critical need of membrane-derived tools in studying these processes. Hence, cell-membrane-derived nanoparticles (CMNPs) are produced using tonsil-derived mesenchymal stem cells (TMSCs) from children owing to their short doubling time. As target cell types, laryngeal cancer cells are compared to bone-marrow-derived MSCs (BMSCs) because of their cartilage damaging and chondrogenic characteristics, respectively. Treating spheroids of these cell types with CMNPs exacerbates interspheroid hypoxia with robust maintenance of the cell-cell interaction signature for 7 days. Both cell types prefer a hypoxic environment, as opposed to blood vessel formation that is absent in cartilage but is required for cancer growth. Hence, angiogenesis is inhibited by displaying the Notch-1 aptamer on CMNPs. Consequently, laryngeal cancer growth is suppressed efficiently in contrast to improved chondroprotection observed in a series of cell and animal experiments using a xenograft mouse model of laryngeal cancer. Altogether, CMNPs execute a two-edged sword function of inducing hypoxic cell-cell packing, followed by suppressing angiogenesis to promote laryngeal cancer death and chondrogenesis simultaneously. This study presents a previously unexplored therapeutic strategy for anti-cancer and chondroprotective treatment using CMNPs.
Assuntos
Membrana Celular/química , Nanopartículas/química , Receptor Notch1/química , Animais , Caderinas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/citologia , Portadores de Fármacos/química , Células Endoteliais da Veia Umbilical Humana , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Nanopartículas/uso terapêutico , Nanopartículas/toxicidade , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Tonsila Palatina/citologia , Receptor Notch1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transplante HeterólogoRESUMO
The Notch signaling system links cellular fate to that of its neighbors, driving proliferation, apoptosis, and cell differentiation in metazoans, whereas dysfunction leads to debilitating developmental disorders and cancers. Other than a five-by-five domain complex, it is unclear how the 40 extracellular domains of the Notch1 receptor collectively engage the 19 domains of its canonical ligand, Jagged1, to activate Notch1 signaling. Here, using cross-linking mass spectrometry (XL-MS), biophysical, and structural techniques on the full extracellular complex and targeted sites, we identify five distinct regions, two on Notch1 and three on Jagged1, that form an interaction network. The Notch1 membrane-proximal regulatory region individually binds to the established Notch1 epidermal growth factor (EGF) 8-EGF13 and Jagged1 C2-EGF3 activation sites as well as to two additional Jagged1 regions, EGF8-EGF11 and cysteine-rich domain. XL-MS and quantitative interaction experiments show that the three Notch1-binding sites on Jagged1 also engage intramolecularly. These interactions, together with Notch1 and Jagged1 ectodomain dimensions and flexibility, determined by small-angle X-ray scattering, support the formation of nonlinear architectures. Combined, the data suggest that critical Notch1 and Jagged1 regions are not distal but engage directly to control Notch1 signaling, thereby redefining the Notch1-Jagged1 activation mechanism and indicating routes for therapeutic applications.
Assuntos
Proteína Jagged-1/metabolismo , Mutação , Domínios e Motivos de Interação entre Proteínas , Receptor Notch1/metabolismo , Animais , Cristalografia por Raios X , Humanos , Proteína Jagged-1/química , Proteína Jagged-1/genética , Ligantes , Camundongos , Ligação Proteica , Receptor Notch1/química , Receptor Notch1/genéticaRESUMO
Biomarker-driven targeted therapies are lacking for head and neck squamous cell carcinoma (HNSCC), which is common and lethal. Efforts to develop such therapies are hindered by a genomic landscape dominated by the loss of tumor suppressor function, including NOTCH1 that is frequently mutated in HNSCC. Clearer understanding of NOTCH1 signaling in HNSCCs is crucial to clinically targeting this pathway. Structural characterization of NOTCH1 mutations in HNSCC demonstrates that most are predicted to cause loss of function, in agreement with NOTCH1's role as a tumor suppressor in this cancer. Experimental manipulation of NOTCH1 signaling in HNSCC cell lines harboring either mutant or wild-type NOTCH1 further supports a tumor suppressor function. Additionally, the loss of NOTCH1 signaling can drive HNSCC tumorigenesis and clinical aggressiveness. Our recent data suggest that NOTCH1 controls genes involved in early differentiation that could have different phenotypic consequences depending on the cancer's genetic background, including acquisition of pseudo-stem cell-like properties. The presence of NOTCH1 mutations may predict response to treatment with an immune checkpoint or phosphatidylinositol 3-kinase inhibitors. The latter is being tested in a clinical trial, and if validated, it may lead to the development of the first biomarker-driven targeted therapy for HNSCC.
Assuntos
Neoplasias de Cabeça e Pescoço/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Transição Epitelial-Mesenquimal , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Mutação/genética , Receptor Notch1/química , Receptor Notch1/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologiaRESUMO
Human NOTCH1 receptor contains 36â epidermal growth factor (EGF)-like repeating domains, in which O-glycosylation status of EGF12 domain regulates the interaction with Notch ligands. Our interest is focused on the effect of specific O-glycosylation states on the structural behavior of EGF11 and EGF10, because they appeared to affect molecular mechanism in receptor-ligand interactions by inducing some conformational alterations in these domains and/or the regions connecting two domains. To understand the structural impact of various O-glycosylation patterns on the pivotal EGF-like repeatsâ 10, 11, and 12, we performed chemical synthesis and NMR studies of site-specifically O-glycosylated EGF11 and EGF10. Our strategy enabled us to synthesize four EGF11 and five EGF10 modules. The specific O-glycosylation states affected in vitro folding of EGF10 more than EGF11, while calcium ion had a larger effect on EGF11 folding. Comprehensive NMR studies shed light on the new type "sugar bridges" crosslinking Thr-O-GlcNAc in the consensus sequence C5-X-X-G-X-(T/S)-G-X-X-C6 and an amino acid in the hinge region between the domains, 445Thr-O-GlcNAc-IIe451 in domainâ 11 and 405Thr-O-GlcNAc-Gln411 in domainâ 10, respectively.
Assuntos
Fator de Crescimento Epidérmico , Receptor Notch1 , Glicosilação , Humanos , Ligantes , Ligação Proteica , Receptor Notch1/química , Receptor Notch1/metabolismoRESUMO
Notch receptors have large extracellular domains containing up to 36 tandem epidermal growth factor-like (EGF) repeats, which facilitate cell signaling by binding ligands on neighboring cells. Notch receptors play major roles in a variety of developmental processes by controlling cell fate decisions. Each EGF repeat consists of about 40 amino acids with 3 conserved disulfide bonds. Many of the EGF repeats are modified by O-linked fucose glycans, and more than half have calcium-binding sites, but the sequences of the EGF repeats vary giving distinct roles to each repeat. EGF repeat 27 (EGF27) from mouse NOTCH1 is modified with O-fucose and is 1 of 7 repeats that is differentially modified by specific Fringe enzymes, which are known to regulate NOTCH1 activation and ligand binding. To better understand the role of EGF27 in NOTCH1 function and regulation, the 3-dimensional structures of EGF27 and its glycoforms are being pursued. E. coli cells were used to produce EGF27 in sufficient quantities for nuclear magnetic resonance analysis. Previous attempts to express the repeat alone and refold the repeat under a steady redox environment were unsuccessful due to low yields and extensive mixed-disulfide bond cross-linking. A new strategy using a cleavable maltose binding protein fusion tag increased the solubility and yield of EGF27. With the fusion tag, EGF27 was refolded to produce the correct disulfide bond arrangement, which was verified enzymatically with the glycosyltransferases, Protein O-fucosyltransferase 1 (POFUT1) and Lunatic Fringe (LFNG).
Assuntos
Fucose , Biossíntese Peptídica , Peptídeos , Receptor Notch1 , Animais , Fucose/química , Fucose/metabolismo , Glicosilação , Camundongos , Peptídeos/química , Peptídeos/genética , Receptor Notch1/biossíntese , Receptor Notch1/química , Receptor Notch1/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Sequências Repetitivas de AminoácidosRESUMO
BACKGROUND: Spontaneous coronary artery dissection (SCAD) is frequently reported as a disorder that primarily affects women without risk factors for cardiovascular disease. Although it has been recognized as one of the genetically mediated vascular disorders, the genetic pathogenesis of SCAD remains obscure to date. CASE PRESENTATION: In this report, we presented a rare case of pregnancy-associated SCAD in a young woman that occurred in multiple coronary arteries within a short period. The initial conservative management and then intravascular ultrasound-guided primary percutaneous coronary intervention (PCI) were adopted to achieve optimal results of revascularization in affected coronary arteries and avoid potential risks for PCI-associated complications. We further performed the whole-exome sequencing and Sanger sequencing and, for the first time, reported a novel heterozygous missense variant, c.4574 C > T (p.Arg1438Cys), in the NOTCH1 gene. This variant has never been documented in the medical literature and was predicted as being potentially damaging or disease-causing variant. CONCLUSIONS: We described a rare case of recurrent SCAD in a young woman after baby delivery. The initial conservative management and PCI with multiple stent implantations were successfully implemented to achieve optimal results of revascularization in coronary arteries. We, for the first time, identified a novel missense variant in the NOTCH1 gene, which appears to be a potential predisposing factor for artery fragility.
Assuntos
Anomalias dos Vasos Coronários/diagnóstico , Anomalias dos Vasos Coronários/etiologia , Suscetibilidade a Doenças , Mutação de Sentido Incorreto , Complicações Cardiovasculares na Gravidez , Receptor Notch1/genética , Doenças Vasculares/congênito , Adulto , Alelos , Sequência de Aminoácidos , Angiografia Coronária , Anomalias dos Vasos Coronários/terapia , Análise Mutacional de DNA , Feminino , Predisposição Genética para Doença , Humanos , Modelos Moleculares , Gravidez , Conformação Proteica , Receptor Notch1/química , Relação Estrutura-Atividade , Avaliação de Sintomas , Ultrassonografia de Intervenção , Doenças Vasculares/diagnóstico , Doenças Vasculares/etiologia , Doenças Vasculares/terapiaRESUMO
Cerebral cavernous malformations (CCM) or cavernomas are slow-flow capillary vascular malformations with a mulberry-like appearance, which are predominantly located in the central nervous system. CCM can occur in a sporadic or a familial form. The latter is inherited in an autosomal dominant manner, and in the majority of the fragile lesions, mutations in the genes CCM1 (KRIT1), CCM2 (OSM), or CCM3 (PDCD10) can be detected. Loss of these genes leads to numerous alterations in endothelial cell signaling resulting in a disturbed vessel architecture and function. Lower activity of Notch signaling occurs upon loss of CCM1, CCM3, or the CCM1-interacting protein ICAP1 in cell culture and animal models. Notch signaling in endothelial cells is an essential regulator of angiogenesis, arterial-venous differentiation, vascular permeability and stability, mural cell recruitment, and flux of metabolites across the vessel wall. The purpose of this chapter is to briefly summarize the current understanding of Notch signaling in familial CCM and to provide a protocol for detecting cleaved Notch1 receptor proteins on paraformaldehyde-fixed paraffin-embedded mouse tissue.
Assuntos
Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Domínios e Motivos de Interação entre Proteínas , Receptor Notch1/metabolismo , Transdução de Sinais , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Biomarcadores , Células Endoteliais/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/etiologia , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Humanos , Imuno-Histoquímica , Proteína KRIT1/genética , Proteína KRIT1/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Neovascularização Patológica/metabolismo , Pericitos/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptor Notch1/químicaRESUMO
Biochemical and genetic studies have indicated that O-linked glycosylation such as O-glucose (Glc), fucose (Fuc), and N-acetylglucosamine (GlcNAc) is critical for Notch signaling; however, it is not fully understood how O-glycans regulate the Notch receptor function. Notch receptors are type-I transmembrane proteins with large extracellular domains (ECD), containing 29-36 epidermal growth factor-like (EGF) repeats. Here, we analyzed O-Glc glycans on NOTCH1 and NOTCH2 expressed in HEK293T cells using an Orbitrap Fusion mass spectrometer and successfully revealed the structures and stoichiometries of all 17 EGF repeats of NOTCH1 with the O-Glc consensus sequence (C1-X-S-X-(P/A)-C2), and 16 out of 17 EGF repeats of NOTCH2 with the same consensus sequence. High levels of O-Glc attachment and xylosyl elongation were detected on most NOTCH1 and NOTCH2 EGF repeats. When both glucoside xylosyltransferases, GXYLT1 and GXYLT2, responsible for the xylosyl elongation of O-glucose, were genetically deleted, the expression of endogenous NOTCH1 and NOTCH2 on the surface of HEK293T cells did not change, but the cell surface expression of overexpressed NOTCH1 and NOTCH2 decreased compared with that in the wild type cells. In vitro secretion assays consistently showed a reduced secretion of both the NOTCH1 and NOTCH2 ECDs in GXYLT1 and GXYLT2 double knockout cells compared with the wild type cells, suggesting a significant role of the elongation of O-Glc glycans on the Notch ECDs in the quality control of Notch receptors.
Assuntos
Membrana Celular/metabolismo , Glucose/metabolismo , Polissacarídeos/metabolismo , Receptor Notch1/química , Receptor Notch1/metabolismo , Receptor Notch2/química , Receptor Notch2/metabolismo , Xilose/metabolismo , Sequência de Aminoácidos , Animais , Fator de Crescimento Epidérmico/química , Células HEK293 , Humanos , Camundongos , Domínios Proteicos , Transporte ProteicoRESUMO
Adams-Oliver syndrome (AOS) is a rare hereditary disorder characterized by aplasia cutis congenita (ACC) and terminal transverse limb defects. The etiology of AOS has remained largely unknown, although mutations in the notch receptor 1 (NOTCH1) gene are most common genetic alteration associated with this disease. In this study, we aimed to identify the case of a 6-year-old boy, who presented with large ACC of the scalp and aortic valve stenosis, suggesting the possibility of AOS. Whole-exome sequencing identified a novel, de novo, in-frame deletion in the NOTCH1 gene (NOTCH1 c.1292_1294del, p.Asn431del) in the patient. The p.Asn431del variant was evaluated by several in silico analyses, which predicted that the mutant was likely to be pathogenic. In addition, molecular modeling with the PyMOL Molecular Graphics System suggested that the NOTCH1-N431del destabilizes calcium ion chelation, leading to decreased receptor-ligand binding efficiency. Quantitative reverse transcription PCR showed further significant downregulation of the Notch target genes, hes-related family bHLH transcription factor with YRPW motif 1 (HEY1) and hes family bHLH transcription factor 1 (HES1), suggesting that this mutation causes disease through dysregulation of the Notch signaling pathway. Our study provides evidence that the NOTCH1-N431del mutation is responsible for this case of AOS. To our knowledge, this is the first report of a patient with AOS caused by NOTCH1 mutation in Asia, and this information will be useful for providing the family with genetic counseling that can help to guide their future plans.
Assuntos
Displasia Ectodérmica/genética , Mutação da Fase de Leitura , Deformidades Congênitas dos Membros/genética , Receptor Notch1/genética , Dermatoses do Couro Cabeludo/congênito , Sequência de Aminoácidos , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Criança , China , Humanos , Masculino , Modelos Moleculares , Conformação Proteica , Receptor Notch1/química , Proteínas Repressoras/genética , Dermatoses do Couro Cabeludo/genética , Fatores de Transcrição HES-1/genéticaRESUMO
Aberrant Notch signaling plays a pivotal role in T-cell acute lymphoblastic leukemia (T-ALL) and chronic lymphocytic leukemia (CLL). Amplitude and duration of the Notch response is controlled by ubiquitin-dependent proteasomal degradation of the Notch1 intracellular domain (NICD1), a hallmark of the leukemogenic process. Here, we show that HDAC3 controls NICD1 acetylation levels directly affecting NICD1 protein stability. Either genetic loss-of-function of HDAC3 or nanomolar concentrations of HDAC inhibitor apicidin lead to downregulation of Notch target genes accompanied by a local reduction of histone acetylation. Importantly, an HDAC3-insensitive NICD1 mutant is more stable but biologically less active. Collectively, these data show a new HDAC3- and acetylation-dependent mechanism that may be exploited to treat Notch1-dependent leukemias.
Assuntos
Histona Desacetilases/metabolismo , Leucemia/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Linhagem Celular Tumoral , Inibidores de Histona Desacetilases/farmacologia , Humanos , Leucemia/enzimologia , Lisina/metabolismo , Camundongos , Mutação , Peptídeos Cíclicos/farmacologia , Estabilidade Proteica , Receptor Notch1/química , Receptor Notch1/genéticaRESUMO
Triple-negative breast cancer (TNBC) accounts for nearly one-quarter of all breast cancer cases, but effective targeted therapies for this disease remain elusive because TNBC cells lack expression of the three most common receptors seen on other subtypes of breast cancer. Here, we exploit TNBC cells' overexpression of Notch-1 receptors and Bcl-2 anti-apoptotic proteins to provide an effective targeted therapy. Prior studies have shown that the small molecule drug ABT-737, which inhibits Bcl-2 to reinstate apoptotic signaling, is a promising candidate for TNBC therapy. However, ABT-737 is poorly soluble in aqueous conditions, and its orally bioavailable derivative causes severe thrombocytopenia. To enable targeted delivery of ABT-737 to TNBC and enhance its therapeutic efficacy, we encapsulated the drug in poly(lactic-co-glycolic acid) nanoparticles (NPs) that were functionalized with Notch-1 antibodies to produce N1-ABT-NPs. The antibodies in this NP platform enable both TNBC cell-specific binding and suppression of Notch signaling within TNBC cells by locking the Notch-1 receptors in a ligand unresponsive state. This Notch inhibition potentiates the effect of ABT-737 by up-regulating Noxa, resulting in effective killing of TNBC cells. We present the results of in vitro studies that demonstrate N1-ABT-NPs can preferentially bind TNBC cells versus noncancerous breast epithelial cells to effectively regulate Bcl-2 and Notch signaling to induce cell death. Further, we show that N1-ABT-NPs can accumulate in subcutaneous TNBC xenograft tumors in mice following systemic administration to reduce tumor burden and extend animal survival. Together, these findings demonstrate that NP-mediated co-delivery of Notch-1 antibodies and ABT-737 is a potent treatment strategy for TNBC that may improve patient outcomes with further development and implementation.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Compostos de Bifenilo/farmacologia , Nanopartículas/química , Nitrofenóis/farmacologia , Receptor Notch1/química , Sulfonamidas/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Compostos de Bifenilo/química , Compostos de Bifenilo/metabolismo , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Feminino , Humanos , Neoplasias Mamárias Experimentais/diagnóstico por imagem , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Nus , Nanopartículas/metabolismo , Nitrofenóis/química , Nitrofenóis/metabolismo , Imagem Óptica , Piperazinas/química , Piperazinas/metabolismo , Piperazinas/farmacologia , Receptor Notch1/metabolismo , Sulfonamidas/química , Sulfonamidas/metabolismo , Neoplasias de Mama Triplo Negativas/diagnóstico por imagem , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
BACKGROUND: Congenital heart disease (CHD) is the most common birth defect which can arises from different genetic defects. The genetic heterogeneity of this disease leads to restricted success in candidate genes screening method. Emerging approaches such as next-generation sequencing (NGS)-based genetic analysis might provide a better understating of CHD etiology in the patients who are left undiagnosed. To this aim, in this study, we survived the causes of CHD in an Iranian family who was consanguineous and had two affected children. METHODS: Affected individuals of this family were checked previously by PCR-direct sequencing for six candidate genes (NKX2-5, ZIC3, NODAL, FOXH1, GJA1, GATA4) and had not revealed any reported CHD causative mutations. Whole-exome sequencing (WES) was performed on this family probond to determine the underlying cause of CHD, and the identified variants were confirmed and segregated by Sanger sequencing. RESULTS: We identified one heterozygous missense mutation, c.T6797C (p.Phe2266Ser), in the NOTCH1 gene, which seems to be the most probably disease causing of this family patients. This mutation was found to be novel and not reported on 1000 Genomes Project, dbSNP, and ExAC. CONCLUSION: Worldwide, mutations in NOTCH1 gene are considered as one of the most known causes of CHD. The found NOTCH1 variant in this family affected individuals was the first report from Iran. Yet again, this result indicates the importance of NOTCH1 screening in CHD patients.
Assuntos
Predisposição Genética para Doença , Cardiopatias Congênitas/genética , Mutação/genética , Receptor Notch1/genética , Adulto , Sequência de Aminoácidos , Sequência de Bases , Criança , Família , Feminino , Humanos , Lactente , Irã (Geográfico) , Masculino , Linhagem , Receptor Notch1/química , SíndromeRESUMO
NOTCH proteins constitute a receptor family with a widely conserved role in cell cycle, growing and development regulation. NOTCH1, the best characterised member of this family, regulates the expression of key genes in cell growth and angiogenesis, playing an essential role in cancer development. These observations provide a relevant rationale to propose the inhibition of the intracellular domain of NOTCH1 (Notch1-IC) as a strategy for treating various types of cancer. Notch1-IC stability is mainly controlled by post-translational modifications. FBXW7 ubiquitin E3 ligase-mediated degradation is considered one of the most relevant, being the previous phosphorylation at Thr-2512 residue required. In the present study, we describe for the first time a new regulation mechanism of the NOTCH1 signalling pathway mediated by DYRK2. We demonstrate that DYRK2 phosphorylates Notch1-IC in response to chemotherapeutic agents and facilitates its proteasomal degradation by FBXW7 ubiquitin ligase through a Thr-2512 phosphorylation-dependent mechanism. We show that DYRK2 regulation by chemotherapeutic agents has a relevant effect on the viability, motility and invasion capacity of cancer cells expressing NOTCH1. In summary, we reveal a novel mechanism of regulation for NOTCH1 which might help us to better understand its role in cancer biology.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptor Notch1/metabolismo , Linhagem Celular , Dano ao DNA , Humanos , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Domínios Proteicos , Receptor Notch1/química , TYK2 Quinase , Quinases DyrkRESUMO
Notch, an essential factor in tissue development and homoeostasis, has been reported to play an oncogenic function in a variety of cancers. Here, we report ubiquitin-specific protease 8 (USP8) as a novel deubiquitylase of Notch1 intracellular domain (NICD). USP8 specifically stabilizes and deubiquitylates NICD through a direct interaction. The inhibition of USP8 downregulated the Notch signalling pathway via NICD destabilization, resulting in the retardation of cellular growth, wound closure, and colony forming ability of breast cancer cell lines. These phenomena were restored by the reconstitution of NICD or USP8, supporting the direct interaction between these two proteins. The expression levels of NICD and USP8 proteins were positively correlated in patients with advanced breast cancer. Taken together, our results suggest that USP8 functions as a positive regulator of Notch signalling, offering a therapeutic target for breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Endopeptidases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Receptor Notch1/química , Receptor Notch1/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinogênese , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Feminino , Deleção de Genes , Humanos , Pessoa de Meia-Idade , Ligação Proteica , Domínios Proteicos , Estabilidade Proteica , Transdução de Sinais , Ensaio Tumoral de Célula-Tronco , Regulação para Cima , CicatrizaçãoRESUMO
The release and nuclear translocation of the intracellular domain of Notch receptor (NICD) is the prerequisite for Notch signaling-mediated transcriptional activation. NICD is subjected to various posttranslational modifications including ubiquitination. Here, we surprisingly found that NUMB proteins stabilize the intracellular domain of NOTCH1 receptor (N1ICD) by regulating the ubiquitin-proteasome machinery, which is independent of NUMB's role in modulating endocytosis. BAP1, a deubiquitinating enzyme (DUB), was further identified as a positive N1ICD regulator, and NUMB facilitates the association between N1ICD and BAP1 to stabilize N1ICD. Intriguingly, BAP1 stabilizes N1ICD independent of its DUB activity but relying on the BRCA1-inhibiting function. BAP1 strengthens Notch signaling and maintains stem-like properties of cortical neural progenitor cells. Thus, NUMB enhances Notch signaling by regulating the ubiquitinating activity of the BAP1-BRCA1 complex.