RESUMO
Cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) is a late-onset, autosomal recessive neurodegenerative disorder caused by biallelic AAGGG/ACAGG repeat expansion (AAGGG-exp/ACAGG-exp) in RFC1. The recent identification of patients with CANVAS exhibiting compound heterozygosity for AAGGG-exp and truncating variants supports the loss-of-function of RFC1 in CANVAS patients. We investigated the pathological changes in 2 autopsied patients with CANVAS harboring biallelic ACAGG-exp and AAGGG-exp. RNA fluorescence in situ hybridization of the 2 patients revealed CCTGT- and CCCTT-containing RNA foci, respectively, in neuronal nuclei of tissues with neuronal loss. Our findings suggest that RNA toxicity may be involved in the pathogenesis of CANVAS. ANN NEUROL 2024;95:607-613.
Assuntos
Vestibulopatia Bilateral , Ataxia Cerebelar , Doenças do Sistema Nervoso Periférico , Humanos , Ataxia Cerebelar/genética , Hibridização in Situ Fluorescente , RNA , SíndromeRESUMO
The prevalent human pathogen, mumps virus (MuV; orthorubulavirus parotitidis) causes various complications and serious sequelae, such as meningitis, encephalitis, deafness, and impaired fertility. Direct-acting antivirals (DAAs) targeting MuV which can prevent mumps and mumps-associated complications and sequelae are yet to be developed. Paramyxoviridae family members, such as MuV, possess viral surface hemagglutinin-neuraminidase (HN) protein with sialidase activity which facilitates efficient viral replication. Therefore, to develop DAAs targeting MuV we synthesized MuV sialidase inhibitors. It is proposed that the viral HN has a single functional site for N-acetylneuraminic acid (Neu5Ac) binding and sialidase activity. Further, the known MuV sialidase inhibitor is an analog of Neu5Ac-2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA)-which lacks potency. DANA derivatives with higher MuV sialidase inhibitory potency are lacking. The MuV-HN-Neu5Ac binding site has a hydrophobic cavity adjacent to the C4 position of Neu5Ac. Exploiting this, here, we synthesized DANA derivatives with increasing hydrophobicity at its C4 position and created 3 novel sialidase inhibitors (Compounds 1, 2 and 3) with higher specificity for MuV-HN than DANA; they inhibited MuV replication step to greater extent than DANA. Furthermore, they also inhibited hemagglutination and the MuV infection step. The insight-that these 3 novel DANA derivatives possess linear hydrocarbon groups at the C4-hydroxyl group of DANA-could help develop highly potent sialidase inhibitors with high specificity for MuV sialidase, which may function as direct-acting MuV-specific antivirals.
RESUMO
O-GlcNAc is a unique post-translational modification found in cytoplasmic, nuclear, and mitochondrial proteins. In a limited number of extracellular proteins, O-GlcNAc modifications occur through the action of EOGT, which specifically modifies subsets of epidermal growth factor-like (EGF) domain-containing proteins such as Notch receptors. The abnormalities due to EOGT mutations in mice and humans and the increased EOGT expression in several cancers signify the importance of EOGT pathophysiology and extracellular O-GlcNAc. Unlike intracellular O-GlcNAc monosaccharides, extracellular O-GlcNAc extends to form elongated glycan structures. However, the enzymes involved in the O-GlcNAc glycan extension have not yet been reported. In our study, we comprehensively screened potential galactosyltransferase and sialyltransferase genes related to the canonical O-GlcNAc glycan pathway and revealed the essential roles of B4GALT1 and ST3GAL4 in O-GlcNAc glycan elongation in human HEK293 cells. These findings were confirmed by sequential glycosylation of Drosophila EGF20 in vitro by EOGT, ß4GalT-1, and ST3Gal-IV. Thus, the findings from our study throw light on the specific glycosyltransferases that mediate O-GlcNAc glycan elongation in human HEK293 cells.
Assuntos
Acetilglucosamina , Galactosiltransferases , Sialiltransferases , Animais , Humanos , Camundongos , Acetilglucosamina/metabolismo , Drosophila/metabolismo , Galactosiltransferases/genética , Glicosiltransferases , Células HEK293 , Polissacarídeos , Receptores Notch/metabolismo , Sialiltransferases/genéticaRESUMO
BACKGROUND: While laparoscopic fundoplication is a standard surgical procedure for patients with esophageal hiatal hernias, the postoperative recurrence of esophageal hiatal hernias is a problem for patients with giant hernias, elderly patients, or obese patients. Although there are some reports indicating that reinforcement with mesh is effective, there are differing opinions regarding the use thereof. The aim of this study is to investigate whether mesh reinforcement is effective for laparoscopic fundoplication in patients with esophageal hiatus hernias. METHODS: The subjects included 280 patients who underwent laparoscopic fundoplication as the initial surgery for giant esophageal hiatal hernias, elderly patients aged 75 years or older, and obese patients with a BMI of 28 or higher, who were considered at risk of recurrent hiatal hernias based on the previous reports. Of the subject patients, 91 cases without mesh and 86 cases following the stabilization of mesh use were extracted to compare the postoperative course including the pathology, symptom scores, surgical outcome, and recurrence of esophageal hiatus hernias. RESULTS: The preoperative conditions indicated that the degree of esophageal hiatal hernias was high in the mesh group (p = 0.0001), while the preoperative symptoms indicated that the score of heartburn was high in the non-mesh group (p = 0.0287). Although the surgical results indicated that the mesh group underwent a longer operation time (p < 0.0001) and a higher frequency of intraoperative complications (p = 0.037), the rate of recurrence of esophageal hiatal hernia was significantly low (p = 0.049), with the rate of postoperative reflux esophagitis also tending to be low (p = 0.083). CONCLUSIONS: Mesh reinforcement in laparoscopic fundoplication for esophageal hiatal hernias contributes to preventing the recurrence of esophageal hiatal hernias when it comes to patient options based on these criteria.
Assuntos
Esofagite Péptica , Hérnia Hiatal , Laparoscopia , Idoso , Humanos , Hérnia Hiatal/complicações , Fundoplicatura/métodos , Telas Cirúrgicas , Laparoscopia/métodos , Esofagite Péptica/complicações , Obesidade/complicaçõesRESUMO
BACKGROUND: Systemic inflammatory response is significant prognostic indicator in patients with various diseases. The relationship between prognostic scoring systems based on the modified Glasgow Prognostic Score (mGPS) and achalasia in patients treated with laparoscopic Hellermyotomy with Dorfundoplication (LHD) remains uninvestigated. This study aimed to examine the role of mGPS in patients with achalasia. METHODS: 457 patients with achalasia who underwent LHD as the primary surgery between September 2005 and December 2020 were included. We divided patients into the mGPS 0 and mGPS 1 or 2 groups and compared the patients' background, pathophysiology, symptoms, surgical outcomes, and postoperative course. RESULTS: mGPS was 0 in 379 patients and 1 or 2 in 78 patients. Preoperative vomiting and pneumonia were more common in patients with mGPS of 1 or 2. There were no differences in surgical outcomes. Postoperative upper gastrointestinal endoscopy revealed that severe esophagitis was more frequently observed in patients with mGPS of 1 or 2 (P < 0.01). The clinical success was 91% and 99% in the mGPS 0 and mGPS 1 or 2 groups, respectively (P < 0.01). CONCLUSIONS: Although severe reflux esophagitis was more common in patients with achalasia with a high mGPS, good clinical success was obtained regardless of the preoperative mGPS.
Assuntos
Acalasia Esofágica , Fundoplicatura , Miotomia de Heller , Laparoscopia , Complicações Pós-Operatórias , Humanos , Acalasia Esofágica/cirurgia , Acalasia Esofágica/diagnóstico , Masculino , Feminino , Pessoa de Meia-Idade , Prognóstico , Laparoscopia/métodos , Miotomia de Heller/métodos , Miotomia de Heller/efeitos adversos , Adulto , Resultado do Tratamento , Fundoplicatura/métodos , Fundoplicatura/efeitos adversos , Complicações Pós-Operatórias/epidemiologia , Estudos Retrospectivos , Idoso , Índice de Gravidade de DoençaRESUMO
Notch signaling is broadly used to regulate cell-fate decisions. We have identified a gene, rumi, with a temperature-sensitive Notch phenotype. At 28 degrees C-30 degrees C, rumi clones exhibit a full-blown loss of Notch signaling in all tissues tested. However, at 18 degrees C only a mild Notch phenotype is evident. In vivo analyses reveal that the target of Rumi is the extracellular domain of Notch. Notch accumulates intracellularly and at the cell membrane of rumi cells but fails to be properly cleaved, despite normal binding to Delta. Rumi is an endoplasmic reticulum-retained protein with a highly conserved CAP10 domain. Our studies show that Rumi is a protein O-glucosyltransferase, capable of adding glucose to serine residues in Notch EGF repeats with the consensus C1-X-S-X-P-C2 sequence. These data indicate that by O-glucosylating Notch in the ER, Rumi regulates its folding and/or trafficking and allows signaling at the cell membrane.
Assuntos
Proteínas de Drosophila , Glicosiltransferases/química , Glicosiltransferases/fisiologia , Receptores Notch/metabolismo , Transdução de Sinais , Alelos , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Cromossomos , Sequência Consenso , Drosophila/química , Drosophila/embriologia , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/deficiência , Embrião não Mamífero , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica , Genes de Insetos , Glucose/metabolismo , Glucosiltransferases/deficiência , Glicosilação , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Homozigoto , Imuno-Histoquímica , Modelos Biológicos , Mutação , Dobramento de Proteína , Estrutura Terciária de Proteína , Interferência de RNA , Receptores Notch/genética , Serina/metabolismo , Solubilidade , Spodoptera/citologia , Spodoptera/genética , Spodoptera/metabolismo , Temperatura , TransgenesRESUMO
Protein glycosylation is a general post-translational modification pathway that controls various biological functions including protein trafficking, cell adhesion, and protein-ligand interaction [...].
Assuntos
Processamento de Proteína Pós-Traducional , Glicosilação , Transporte Proteico , Adesão CelularRESUMO
BACKGROUND: In recent years, the number of patients requiring surgery for intra-thoracic stomach (ITS) has been increasing due to the effects of obesity and gibbus due to aging. The aim of this study is to assess the effects of the degree of hernia on the pathological conditions and surgical outcomes in ITS patients. METHODS: ITS was defined as cases in which over 50% of the stomach had deviated into the mediastinum by esophagogastric fluoroscopy and/or computed tomography, with 65 patients who underwent laparoscopic surgery as the initial surgery included. We compared the pathological conditions and surgical outcomes by dividing the subjects into 3 groups: Group A: 50%- < 75%; Group B: 75%- < 100%; and Group C: 100% (upside-down stomach), depending on the degree of deviation into the mediastinum of the stomach. RESULTS: The breakdown of patients was 33 in Group A, 21 in Group B, and 11 in Group C. Regarding the preoperative pathological conditions, Group C had a high body mass index (BMI) and a low score for factor V according to upper gastrointestinal endoscopy (p = 0.0109, p = 0.0062, respectively). While the surgical results indicated that the operation time was extended depending on the degree of hernia (p = 0.0051), there was no marked difference in other surgical outcomes or the postoperative course among the three groups, with a high degree of satisfaction. CONCLUSIONS: In the case of ITS, although the operation time was extended depending on the degree of the hernia, the surgical outcomes were the same, and overall good results were obtained.
Assuntos
Hérnia Hiatal , Laparoscopia , Humanos , Hérnia Hiatal/complicações , Hérnia Hiatal/cirurgia , Laparoscopia/efeitos adversos , Laparoscopia/métodos , Obesidade/complicações , Estômago/cirurgia , Resultado do TratamentoRESUMO
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
The Notch pathway represents evolutionarily conserved intercellular signaling essential for cell-to-cell communication during development. Dysregulation of Notch signaling has been implicated in various diseases, and its control represents a potential cancer treatment strategy. Notch signaling is initiated by the interaction of NOTCH receptors with their ligands on neighboring cells. Therefore, the truncated NOTCH ectodomain, composed mainly of tandem repeats of epidermal growth factor-like (EGF) domains, serves as a decoy molecule that competes for ligand binding and thus inhibits ligand-dependent Notch signaling. Although full-length NOTCH EGF repeats exhibited potent Notch inhibitory activity, they were poorly produced in the transfected cells. This study evaluated the effect of EGF domain-modifying glycosyltransferases on the secretion of NOTCH EGF repeats. Our results in HEK293T cells revealed that, unlike the effect on endogenous NOTCH receptors, overexpressed EGF domain-specific O-GlcNAc transferase (EOGT) markedly enhanced the secretion of NOTCH1 EGF repeats in an enzyme activity-dependent manner. The co-expression of protein O-glucosyltransferase 1 further manifested the effect of EOGT. The resultant changes in O-glycosylation of NOTCH3 were evaluated by label-free glycopeptide quantification. This study provides an experimental strategy to efficiently generate NOTCH EGF repeats by manipulating the expression of glycosyltransferases that alter the O-glycosylation of EGF domains.
Assuntos
Fator de Crescimento Epidérmico , Receptores Notch , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Glicopeptídeos , Glicosilação , Células HEK293 , Humanos , Ligantes , Receptores Notch/química , Receptores Notch/metabolismo , Sequências de Repetição em TandemRESUMO
PURPOSE: Recently, various magnetic resonance imaging (MRI) modalities have been developed to easily detect carotid and aortic plaques, but these techniques are time-consuming and vulnerable to motion artifacts. We investigated the utility of a gradient echo MRI technique known as liver acquisition with volume acceleration flexible (LAVA-Flex) to detect carotid and aortic atherosclerotic plaques. METHODS: Ten patients who underwent carotid endarterectomy (CEA) were assessed regarding the correspondence between LAVA-Flex findings and the histopathology of excised carotid plaques. In addition, 47 patients with cryptogenic ischemic stroke underwent LAVA-Flex and transesophageal echocardiography (TEE) for detection of embolic sources in the thoracic aorta. We analyzed the relationship between the thickness of the aortic plaque measured by TEE and the presence of high-intensity lesions on LAVA-Flex. RESULTS: Nine of 10 patients (90.0%) who underwent CEA showed a high-intensity carotid lesion on LAVA-Flex, which corresponded pathologically to plaques containing large lipid cores and hemorrhage. Twenty-four (51.1%) of 47 cryptogenic stroke patients showed a high-intensity lesion in the thoracic aorta on LAVA-Flex; of these, 21 (87.5%) also demonstrated a large plaque (thickness ≥4 mm) on TEE. Twenty-two (95.7%) of 23 patients without a high-intensity lesion on LAVA-Flex demonstrated no large plaque on TEE. LAVA-Flex had a sensitivity of 95.5% and a specificity of 88.0% in patients with large plaques. CONCLUSION: This study showed that LAVA-Flex successfully detected carotid and aortic plaques. This imaging technique may be useful to rapidly diagnose and evaluate carotid and aortic plaques, which are critical risk factors for aortogenic stroke.
Assuntos
Placa Aterosclerótica , Acidente Vascular Cerebral , Angiografia/efeitos adversos , Artérias Carótidas/patologia , Humanos , Imageamento por Ressonância Magnética/métodos , Placa Aterosclerótica/complicações , Placa Aterosclerótica/diagnóstico por imagem , Acidente Vascular Cerebral/etiologiaRESUMO
Estrogen deficiency during menopause causes a variety of neurological symptoms, including depression. The edible Lion's Mane mushroom, Hericium erinaceus (Bull.: Fr.) Pers. (HE), is a medicinal mushroom that has the potential for a neuroprotective effect and ameliorating neurological diseases, such as depression, anxiety, and neurodegenerative diseases. HE contains phytoestrogens, including daidzein and genistein. However, the ameliorating effect of HE on menopausal symptoms is not well understood. Here we investigated the impact of methanol extract of the HE fruiting body on depressive-like behavior in postmenopausal model rats. The activation of estrogen receptor alpha (ERα) causes body weight loss and uterine weight gain. Body weight gain and uterine weight loss by estrogen deficiency in ovariectomized (OVX) rats were reversed with 17ß-estradiol (E2) but not with HE. Thus, the phytoestrogens in HE may hardly activate ERα. Estrogen receptor beta (ERß) is expressed in the brain, and activation of ERß ameliorates menopausal depressive symptoms. Notably, depressive-like behavior in OVX rats evaluated in forced swim test was reduced by administration of not only E2 but also HE for 92 d. Long-term activation of ERα increases the risk of breast and uterine cancers. HE, therefore, may be effective in treating menopausal depression without the risk of carcinogenesis caused by ERα activation.
Assuntos
Agaricales , Fármacos Neuroprotetores , Animais , Estradiol/farmacologia , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Feminino , Genisteína , Hericium , Humanos , Metanol , Ovariectomia , Fitoestrógenos , Ratos , Aumento de PesoRESUMO
Notch signaling, which was initially identified in Drosophila wing morphogenesis, plays pivotal roles in cell development and differentiation. Optimal Notch pathway activity is essential for normal development and dysregulation of Notch signaling leads to various human diseases, including many types of cancers. In hematopoietic cancers, such as T-cell acute lymphoblastic leukemia, Notch plays an oncogenic role, while in acute myeloid leukemia, it has a tumor-suppressive role. In solid tumors, such as hepatocellular carcinoma and medulloblastoma, Notch may have either an oncogenic or tumor-suppressive role, depending on the context. Aberrant expression of Notch receptors or ligands can alter the ligand-dependent Notch signaling and changes in trafficking can lead to ligand-independent signaling. Defects in any of the two signaling pathways can lead to tumorigenesis and tumor progression. Strikingly, O-glycosylation is one such process that modulates ligand-receptor binding and trafficking. Three types of O-linked modifications on the extracellular epidermal growth factor-like (EGF) repeats of Notch receptors are observed, namely O-glucosylation, O-fucosylation, and O-N-acetylglucosamine (GlcNAc) modifications. In addition, O-GalNAc mucin-type O-glycosylation outside the EGF repeats also appears to occur in Notch receptors. In this review, we first briefly summarize the basics of Notch signaling, describe the latest information on O-glycosylation of Notch receptors classified on a structural basis, and finally describe the regulation of Notch signaling by O-glycosylation in cancer.
Assuntos
N-Acetilglucosaminiltransferases , Neoplasias , Animais , Fucose/química , Glicosilação , N-Acetilglucosaminiltransferases/metabolismo , Receptores Notch/metabolismoRESUMO
Epidermal growth factor (EGF) domain-specific O-GlcNAc transferase (EOGT) is an endoplasmic reticulum (ER)-resident protein that modifies EGF repeats of Notch receptors and thereby regulates Delta-like ligand-mediated Notch signaling. Several EOGT mutations that may affect putative N-glycosylation consensus sites are recorded in the cancer database, but the presence and function of N-glycans in EOGT have not yet been characterized. Here, we identified N-glycosylation sites in mouse EOGT and elucidated their molecular functions. Three predicted N-glycosylation consensus sequences on EOGT are highly conserved among mammalian species. Within these sites, we found that Asn-263 and Asn-354, but not Asn-493, are modified with N-glycans. Lectin blotting, endoglycosidase H digestion, and MS analysis revealed that both residues are modified with oligomannose N-glycans. Loss of an individual N-glycan on EOGT did not affect its endoplasmic reticulum (ER) localization, enzyme activity, and ability to O-GlcNAcylate Notch1 in HEK293T cells. However, simultaneous substitution of both N-glycosylation sites affected both EOGT maturation and expression levels without an apparent change in enzymatic activity, suggesting that N-glycosylation at a single site is sufficient for EOGT maturation and expression. Accordingly, a decrease in O-GlcNAc stoichiometry was observed in Notch1 co-expressed with an N263Q/N354Q variant compared with WT EOGT. Moreover, the N263Q/N354Q variant exhibited altered subcellular distribution within the ER in HEK293T cells, indicating that N-glycosylation of EOGT is required for its ER localization at the cell periphery. These results suggest critical roles of N-glycans in sustaining O-GlcNAc transferase function both by maintaining EOGT levels and by ensuring its proper subcellular localization in the ER.
Assuntos
Retículo Endoplasmático/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Sequência de Aminoácidos , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Edição de Genes , Glicopeptídeos/análise , Glicosilação , Humanos , Camundongos , Mutagênese Sítio-Dirigida , N-Acetilglucosaminiltransferases/deficiência , N-Acetilglucosaminiltransferases/genética , Receptor Notch1/genética , Receptor Notch1/metabolismo , Alinhamento de Sequência , Espectrometria de Massas em Tandem , Tunicamicina/farmacologiaRESUMO
BACKGROUND: Surgical site infections (SSI) are one of the most frequent complications following stoma reversal (SR-SSI) and lead to multiple problems such as decreased mobility of the patients or increased hospital costs. Several risk factors for SR-SSI have been reported, but there are no risk scoring systems for predicting SR-SSI. The current study aimed to analyze the risk factors for SR-SSI and develop a scoring system. MATERIALS AND METHODS: Multivariate analysis of risk factors for SR-SSI was performed in patients who underwent elective SR and were followed-up during the first month after surgery. A logistic regression model was used to identify risk factors and construct a predictive score. RESULTS: Of the 182 patients, 53 (29.1%) developed SSI. In multivariate analysis, three variables as preoperative risk factors were associated with increased SR-SSI incidence: subcutaneous fat thickness (≥ 20 mm) (odds ratio [OR]: 8.46 [95% confidence interval (CI): 3.45-20.7], P <0.001), period from stoma creation (≤ 20 weeks) (OR: 2.88 [95% CI: 1.14-7.28], P = 0.025), and SSI after the primary operation (OR: 3.06 [95% CI: 1.19-7.90], P = 0.021). Each of these variables contributed 2,1, and 1 points to the risk score, respectively. The SR-SSI rate was 2.9%, 20.3%, 34.2%, 54.5%, and 81.8% for the scores of 0,1,2,3, and 4 points, respectively. The area under the receiver operating characteristic curve was 0.773 (95% CI: 0.703-0.844). CONCLUSIONS: A simple clinical scoring system based on three preoperative variables may be useful in predicting the risk of SR-SSI.
Assuntos
Estomas Cirúrgicos , Infecção da Ferida Cirúrgica , Procedimentos Cirúrgicos Eletivos/efeitos adversos , Humanos , Razão de Chances , Estudos Retrospectivos , Fatores de Risco , Estomas Cirúrgicos/efeitos adversos , Infecção da Ferida Cirúrgica/diagnóstico , Infecção da Ferida Cirúrgica/epidemiologia , Infecção da Ferida Cirúrgica/etiologiaRESUMO
O-Linked glycosylation such as O-fucose, O-glucose, and O-N-acetylglucosamine are considered to be unusual. As suggested by the high levels of evolutional conservation, these O-glycans are fundamentally important for life. In the last two decades, our understanding of the importance of these glycans has greatly advanced. In particular, identification of the glycosyltransferases responsible for the biosynthesis of these glycans has accelerated basic research on the functional significance and molecular mechanisms by which these O-glycans regulate protein functions as well as clinical research on human diseases due to changes in these types of O-glycosylation. Notably, Notch receptor signaling is modified with and regulated by these types of O-glycans. Here, we summarize the current view of the structures and the significance of these O-glycans mainly in the context of Notch signaling regulation and human diseases.
Assuntos
Fucose , Receptores Notch , Glucose , Glicosilação , Humanos , Polissacarídeos , Receptores Notch/metabolismo , Transdução de SinaisRESUMO
The Notch-signaling pathway is normally activated by Notch-ligand interactions. A recent structural analysis suggested that a novel O-linked hexose modification on serine 435 of the mammalian NOTCH1 core ligand-binding domain lies at the interface with its ligands. This serine occurs between conserved cysteines 3 and 4 of Epidermal Growth Factor-like (EGF) repeat 11 of NOTCH1, a site distinct from those modified by protein O-glucosyltransferase 1 (POGLUT1), suggesting that a different enzyme is responsible. Here, we identify two novel protein O-glucosyltransferases, POGLUT2 and POGLUT3 (formerly KDELC1 and KDELC2, respectively), which transfer O-glucose (O-Glc) from UDP-Glc to serine 435. Mass spectrometric analysis of NOTCH1 produced in HEK293T cells lacking POGLUT2, POGLUT3, or both genes showed that either POGLUT2 or POGLUT3 can add this novel O-Glc modification. EGF11 of NOTCH2 does not have a serine residue in the same location for this O-glucosylation, but EGF10 of NOTCH3 (homologous to EGF11 in NOTCH1 and -2) is also modified at the same position. Comparison of the sites suggests a consensus sequence for modification. In vitro assays with POGLUT2 and POGLUT3 showed that both enzymes modified only properly folded EGF repeats and displayed distinct acceptor specificities toward NOTCH1 EGF11 and NOTCH3 EGF10. Mutation of the O-Glc modification site on EGF11 (serine 435) in combination with sensitizing O-fucose mutations in EGF8 or EGF12 affected cell-surface presentation of NOTCH1 or reduced activation of NOTCH1 by Delta-like1, respectively. This study identifies a previously undescribed mechanism for fine-tuning the Notch-signaling pathway in mammals.
Assuntos
Glucosiltransferases/metabolismo , Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Receptor Notch3/metabolismo , Transdução de Sinais/fisiologia , Animais , Glucosiltransferases/genética , Glicosilação , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Transporte Proteico/fisiologia , Receptor Notch1/genética , Receptor Notch2/genética , Receptor Notch3/genética , Sequências Repetitivas de AminoácidosRESUMO
Notch signaling receptors, ligands, and their downstream target genes are dysregulated in pancreatic ductal adenocarcinoma (PDAC), suggesting a role of Notch signaling in pancreatic tumor development and progression. However, dysregulation of Notch signaling by post-translational modification of Notch receptors remains poorly understood. Here, we analyzed the Notch-modifying glycosyltransferase involved in the regulation of the ligand-dependent Notch signaling pathway. Bioinformatic analysis revealed that the expression of epidermal growth factor (EGF) domain-specific O-linked N-acetylglucosamine (EOGT) and Lunatic fringe (LFNG) positively correlates with a subset of Notch signaling genes in PDAC. The lack of EOGT or LFNG expression inhibited the proliferation and migration of Panc-1 cells, as observed by the inhibition of Notch activation. EOGT expression is significantly increased in the basal subtype, and low expression of both EOGT and LFNG predicts better overall survival in PDAC patients. These results imply potential roles for EOGT- and LFNG-dependent Notch signaling in PDAC.
Assuntos
Adenocarcinoma/genética , Carcinoma Ductal Pancreático/genética , Glicosiltransferases/genética , N-Acetilglucosaminiltransferases/genética , Adenocarcinoma/patologia , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Biologia Computacional , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Receptores Notch/genética , Transdução de Sinais/genéticaRESUMO
The Notch signaling pathway is highly conserved and essential in animal development and tissue homeostasis. Regulation of Notch signaling is a crucial process for human health. Ligands initiate a signal cascade by binding to Notch receptors expressed on the neighboring cell. Notch receptors interact with ligands through their epidermal growth factor-like repeats (EGF repeats). Most EGF repeats are modified by O-glycosylation with residues, such as O-linked N-acetylglucosamine (O-GlcNAc), O-fucose, and O-glucose. A recent study revealed the distinct roles of these O-glycans in ligand binding, processing, and trafficking of Notch receptors. In particular, O-GlcNAc glycans are essential for Delta-like (DLL) ligand-mediated Notch signaling. In this study, we showed that O-GlcNAc promotes Notch1 trafficking to the cell surfaces under the condition that O-fucose and O-glucose are removed from consecutive EGF repeats of Notch1. Through in vitro experiments, we showed that O-GlcNAc mediates the stability of EGF domains in the same manner as O-fucose and O-glucose. Thus, O-GlcNAc on EGF domains possesses a shared function in the stability of EGF domains and Notch1 trafficking.
Assuntos
Fator de Crescimento Epidérmico/química , Espaço Extracelular/metabolismo , Glucosamina/metabolismo , Dobramento de Proteína , Receptores Notch/química , Receptores Notch/metabolismo , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Drosophila/metabolismo , Fucose/metabolismo , Glucose/metabolismo , Células HEK293 , Humanos , Camundongos , Proteínas Mutantes/metabolismo , Mutação/genética , N-Acetilglucosaminiltransferases/metabolismo , Polissacarídeos/metabolismo , Domínios Proteicos , Estabilidade Proteica , Transporte ProteicoRESUMO
Recently, a recessively inherited intronic repeat expansion in replication factor C1 (RFC1) was identified in cerebellar ataxia with neuropathy and bilateral vestibular areflexia syndrome (CANVAS). Here, we describe a Japanese case of genetically confirmed CANVAS with autonomic failure and auditory hallucination. The case showed impaired uptake of iodine-123-metaiodobenzylguanidine and 123I-ioflupane in the cardiac sympathetic nerve and dopaminergic neurons, respectively, by single-photon emission computed tomography. Long-read sequencing identified biallelic pathogenic (AAGGG)n nucleotide repeat expansion in RFC1 and heterozygous benign (TAAAA)n and (TAGAA)n expansions in brain expressed, associated with NEDD4 (BEAN1). Enrichment of the repeat regions in RFC1 and BEAN1 using a Cas9-mediated system clearly distinguished between pathogenic and benign repeat expansions. The haplotype around RFC1 indicated that the (AAGGG)n expansion in our case was on the same ancestral allele as that of European cases. Thus, long-read sequencing facilitates precise genetic diagnosis of diseases with complex repeat structures and various expansions.