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The multibasic furin cleavage site at the S1/S2 boundary of the spike protein is a hallmark of SARS-CoV-2 and plays a crucial role in viral infection. However, the mechanism underlying furin activation and its regulation remain poorly understood. Here, we show that GalNAc-T3 and T7 jointly initiate clustered O-glycosylations in the furin cleavage site of the SARS-CoV-2 spike protein, which inhibit furin processing, suppress the incorporation of the spike protein into virus-like-particles and affect viral infection. Mechanistic analysis reveals that the assembly of the spike protein into virus-like particles relies on interactions between the furin-cleaved spike protein and the membrane protein of SARS-CoV-2, suggesting a possible mechanism for furin activation. Interestingly, mutations in the spike protein of the alpha and delta variants of the virus confer resistance against glycosylation by GalNAc-T3 and T7. In the omicron variant, additional mutations reverse this resistance, making the spike protein susceptible to glycosylation in vitro and sensitive to GalNAc-T3 and T7 expression in human lung cells. Our findings highlight the role of glycosylation as a defense mechanism employed by host cells against SARS-CoV-2 and shed light on the evolutionary interplay between the host and the virus.
Assuntos
COVID-19 , Furina , Mutação , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/química , Humanos , SARS-CoV-2/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Glicosilação , Furina/metabolismo , Furina/genética , COVID-19/virologia , COVID-19/metabolismo , Células HEK293 , N-Acetilgalactosaminiltransferases/metabolismo , N-Acetilgalactosaminiltransferases/genética , Animais , Chlorocebus aethiops , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
OBJECTIVE: To develop and validate a computed tomography (CT)-based scoring system for evaluating the risk of dural defects (DDs) in anterior surgery for cervical ossification of the posterior longitudinal ligament (OPLL). METHODS: We retrospectively analyzed CT imaging features of 114 OPLL patients in our institute who received anterior decompression surgery. Intraoperative DDs were found in 16 patients. A multivariable logistic regression was used to evaluate the predictors. According to the odd ratio of the included risk factors, we developed a CT scoring system for evaluating the risk of DDs in anterior OPLL surgery. The system was further validated in an independent group of 39 OPLL patients. RESULTS: We developed a CT scoring system as follows: hook sign (2 points), K-line (-) (1 point) and broad base (1 point). Thus, the system comprised 4 total points, and patients were at high risks of dural defects when the score ≥3 points. The operating characteristics of a score ≥3 for predicting DDs in the validation group were: sensitivity of 0.83, specificity of 0.94, LR positive of 13.75, LR negative of 0.18 and AUC of 0.886. The discriminatory ability of the proposed score could be demonstrated in the validation cohort. CONCLUSIONS: The relatively simple and easy-to-use scoring system we propose integrates the 3 most reliable spinal CT findings observed in patients with OPLL and a DD. The likelihood to identify the underlying risks of spinal CSF leaks may be useful to triage patients who may benefit from indirect decompression techniques.
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Vértebras Cervicais , Descompressão Cirúrgica , Dura-Máter , Ossificação do Ligamento Longitudinal Posterior , Tomografia Computadorizada por Raios X , Humanos , Ossificação do Ligamento Longitudinal Posterior/cirurgia , Ossificação do Ligamento Longitudinal Posterior/diagnóstico por imagem , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Tomografia Computadorizada por Raios X/métodos , Dura-Máter/cirurgia , Dura-Máter/diagnóstico por imagem , Descompressão Cirúrgica/métodos , Estudos Retrospectivos , Vértebras Cervicais/cirurgia , Vértebras Cervicais/diagnóstico por imagem , Adulto , Fatores de RiscoRESUMO
The frequent mutation of KRAS oncogene in some of the most lethal human cancers has spurred incredible efforts to develop KRAS inhibitors, yet only one covalent inhibitor for the KRASG12C mutant has been approved to date. New venues to interfere with KRAS signaling are desperately needed. Here, we report a "localized oxidation-coupling" strategy to achieve protein-specific glycan editing on living cells for disrupting KRAS signaling. This glycan remodeling method exhibits excellent protein and sugar specificity and is applicable to different donor sugars and cell types. Attachment of mannotriose to the terminal galactose/N-acetyl-D-galactosamine epitopes of integrin αv ß3 , a membrane receptor upstream of KRAS, blocks its binding to galectin-3, suppresses the activation of KRAS and downstream effectors, and mitigates KRAS-driven malignant phenotypes. Our work represents the first successful attempt to interfere with KRAS activity by manipulating membrane receptor glycosylation.
Assuntos
Neoplasias Pulmonares , Proteínas Proto-Oncogênicas p21(ras) , Humanos , Neoplasias Pulmonares/patologia , Mutação , Polissacarídeos , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de SinaisRESUMO
The mammalian sterile 20-like (MST) family belongs to the serine/threonine protein kinase (STK) superfamily and participates in a variety of biological processes, such as cell apoptosis, polarity, migration, immune regulation, inflammatory responses, and cancer. In the economically important bighead carp (Hypophthalmichthys nobilis), the STK gene family and immune-related biological functions may be helpful in increasing its economic yield. However, the comprehensive role of STKs in the bighead carp remains unclear. In this study, the five stk sequences from the bighead carp were divided into two classes: stk3/4 and stk24/25/26. Gene structure and motif prediction analyses confirmed that stk is conserved in the bighead carp. Compared to 26 other vertebrate species, teleosts (including bighead carp) possess more stk members because of teleost-specific whole-genome duplication. Synteny analysis revealed that stk3, stk24, stk25, and stk26 have been relatively conserved in bighead carp during evolution. Meanwhile, stk4 was lost in most Cyprinid species, including bighead carp, during evolution. RNA-seq data revealed that STK expression was associated with various pathogens, and the expression of these STKs (Hnstk3, Hnstk24a, Hnstk24b, Hnstk25, and Hnstk26) was different in seven tissues of bighead carp. In addition, we showed that STK expression levels were dramatically altered in the head kidney and that stk24 was involved in defense against Aeromonas hydrophila. This study provides a molecular basis for the analysis of stk function in bighead carp, and can be used as a reference for further phylogenomics.
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Carpas , Cyprinidae , Animais , Carpas/genética , Cyprinidae/genética , Genoma , Sintenia , Genômica , MamíferosRESUMO
Purpose: To explore the relationship between blood-brain barrier (BBB) leakage and brain structure in non-brain metastasis lung cancer (LC) by magnetic resonance imaging (MRI) as well as to indicate the possibility of brain metastasis (BM) occurrence. Patients and methods: MRI were performed in 75 LC patients and 29 counterpart healthy peoples (HCs). We used the Patlak pharmacokinetic model to calculate the average leakage in each brain region according to the automated anatomical labeling (AAL) atlas. The thickness of the cortex and the volumes of subcortical structures were calculated using the FreeSurfer base on Destrieux atlas. We compared the thickness of the cerebral cortex, the volumes of subcortical structures, and the leakage rates of BBB, and evaluated the relationships between these parameters. Results: Compared with HCs, the leakage rates of seven brain regions were higher in patients with advanced LC (aLC). In contrast to patients with early LC (eLC), the cortical thickness of two regions was decreased in aLCs. The volumes of twelve regions were also reduced in aLCs. Brain regions with increased BBB penetration showed negative correlations with thinner cortices and reduced subcortical structure volumes (P<0.05, R=-0.2 to -0.50). BBB penetration was positively correlated with tumor size and with levels of the tumor marker CYFRA21-1 (P<0.05, R=0.2-0.70). Conclusion: We found an increase in BBB permeability in non-BM aLCs that corresponded to a thinner cortical thickness and smaller subcortical structure volumes. With progression in LC staging, BBB shows higher permeability and may be more likely to develop into BM.
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BACKGROUND: Currently, there are increasing surgical treatments for neck pain. However, whether to use cervical brace after operation remains poorly defined. We aim to clear the clinical efficacy of the use of cervical brace after cervical surgery. METHODS: We searched for relevant studies in 8 electronic databases up to March 2021. The mean difference and 95% confidence intervals were used for continuous data. Cochrane Collaboration's tool was used to assess the risk of bias. The data were collected and input into the Review Manager 5.3 software (The Cochrane Collaboration, Copenhagen, Denmark). RESULTS: Four randomized controlled trials were finally included in our study. For pain, the pooled analysis showed that postoperative neck brace compared with no brace can relieve neck pain at all follow-up periods except 6 months. For neck disability index, the result showed that postoperative neck brace compared with no brace can improve neck disability index during the 3 to 12 month follow-up period. However, no significant difference was identified between 2 groups within the follow-up of 6 weeks after surgery. In addition, the result tends to get the opposite at follow-up of 24 months. For 36-Short form health survey Physical Component Summary, there was no significant difference between 2 groups in the early 3 weeks after surgery, but the results were changed after 3 weeks. For 36-short form health survey Mental Component Summary, there appears to be no significant change between 2 groups at all time intervals. CONCLUSION: Wearing a cervical brace after cervical surgery is conducive to improving symptoms after cervical surgery at different stages. However, there is no relevant evidence indicating it can improve the mental health of postoperative patients. Higher quality, large prospective randomized studies are needed to verify the current conclusions.
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Braquetes , Cervicalgia , Humanos , Pescoço , Cervicalgia/terapia , Estudos Prospectivos , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
Long interspersed nuclear element 1 (LINE-1) is a dominant autonomous retrotransposon in human genomes which plays a role in affecting the structure and function of somatic genomes, resulting in human disorders including genetic disease and cancer. LINE-1 encoded ORF1p protein which possesses RNA-binding and nucleic acid chaperone activity, and interacts with LINE-1 RNA to form a ribonucleoprotein particle (RNP). ORF1p can be detected in many kinds of tumors and its overexpression has been regarded as a hallmark of histologically aggressive cancers. In this study, we developed an In-Cell Western (ICW) assay in T47D cells to screen the compounds which can decrease the expression of ORF1p. Using this assay, we screened 1,947 compounds from the natural products library of Target Mol and Selleckchem, among which three compounds, Hydroxyprogesterone, 2,2':5',2â³-Terthiophene and Ethynyl estradiol displayed potency in diminishing LINE-1 ORF1p expression level. Further mechanistic studies indicated the compounds act by affecting LINE-1 RNA transcription. Notably, we demonstrated that the compounds have an inhibitory effect on the proliferation of several lung and breast cancer cell lines. Taken together, we established a high throughput screening system for ORF1p expression inhibitors and the identified compounds provide some clues to the development of a novel anti-tumor therapeutic strategy by targeting ORF1p.
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Abnormal expression of mucin-type O-glycosylation has been reported to be associated with a variety of human cancers including gliomas. However, little is known about its contribution to the malignancy of Glioblastoma Multiforme (GBM), the deadliest form of brain tumors. Here, we conducted a detailed analysis of the expression profiles of GALNT gene family, which encode polypeptide-N-acetyl-galactosaminyltransferases (GalNAc-Ts) and are responsible for initiating O-glycans, both in the Cancer Genome Atlas (TCGA) and in the Chinese Glioma Genome Atlas (CGGA) databases. We discovered that GALNT12 is the only member within the GALNT family, whose expression demonstrated significant correlation with a worse prognosis of GBM. Genetic knockdown (KD) and knockout (KO) of GALNT12 in U87 MG, a representative GBM cell line with high GALNT12 expression, confirmed that GALNT12 deficiency leads to decreased cell proliferation, migration and invasion. Mechanism study revealed that GALNT12 KD and KO decreased the level of epidermal growth factor (EGF) and consequently attenuated Akt signaling within the cell. In summary, our results indicated that GALNT12 facilitates the malignant characteristics of GBM by influencing the PI3K/Akt/mTOR axis and may serve as a novel prognosis biomarker and a potential therapeutic target of GBM.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Glioma , N-Acetilgalactosaminiltransferases , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Glioma/patologia , Humanos , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
OBJECTIVES: Endocardial cushions are precursors of the valve septum complex that separates the four heart chambers. Several genes have been implicated in the development of endocardial cushions. Specifically, ERp44 has been found to play a role in the early secretory pathway, but its function in heart development has not been well studied. MATERIALS AND METHODS: In this study, we established conditional and tissue-specific knockout mouse models. The morphology, survival rate, the development of heart and endocardial cushion were under evaluation. The relationship between ERp44 and VEGFA was investigated by transcriptome, qPCR, WB, immunofluorescence and immunohistochemistry. RESULTS: ERp44 knockout (KO) mice were smaller in size, and most mice died during early postnatal life. KO hearts exhibited the typical phenotypes of congenital heart diseases, such as abnormal heart shapes and severe septal and valvular defects. Similar phenotypes were found in cTNT-Cre+/- ; ERp44fl / fl mice, which indicated that myocardial ERp44 principally controls endocardial cushion formation. Further studies demonstrated that the deletion of ERp44 significantly decreased the proliferation of cushion cells and impaired the endocardial-mesenchymal transition (EndMT), which was followed by endocardial cushion dysplasia. Finally, we found that ERp44 was directly bound to VEGFA and controlled its release, further regulating EndMT. CONCLUSION: We demonstrated that ERp44 plays a specific role in heart development. ERp44 contributes to the development of the endocardial cushion by affecting VEGFA-mediated EndMT.
Assuntos
Coxins Endocárdicos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Membrana/genética , Chaperonas Moleculares/genética , Miocárdio/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Proliferação de Células/genética , Proliferação de Células/fisiologia , Cardiopatias Congênitas/genética , Proteínas de Membrana/metabolismo , Mesoderma/metabolismo , Camundongos Knockout , Chaperonas Moleculares/metabolismo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Placental malaria infection is mediated by the binding of the malarial VAR2CSA protein to the placental glycosaminoglycan, chondroitin sulfate. Recombinant subfragments of VAR2CSA (rVAR2) have also been shown to bind specifically and with high affinity to cancer cells and tissues, suggesting the presence of a shared type of oncofetal chondroitin sulfate (ofCS) in the placenta and in tumors. However, the exact structure of ofCS and what determines the selective tropism of VAR2CSA remains poorly understood. In this study, ofCS was purified by affinity chromatography using rVAR2 and subjected to detailed structural analysis. We found high levels of N-acetylgalactosamine 4-O-sulfation (â¼80-85%) in placenta- and tumor-derived ofCS. This level of 4-O-sulfation was also found in other tissues that do not support parasite sequestration, suggesting that VAR2CSA tropism is not exclusively determined by placenta- and tumor-specific sulfation. Here, we show that both placenta and tumors contain significantly more chondroitin sulfate moieties of higher molecular weight than other tissues. In line with this, CHPF and CHPF2, which encode proteins required for chondroitin polymerization, are significantly upregulated in most cancer types. CRISPR/Cas9 targeting of CHPF and CHPF2 in tumor cells reduced the average molecular weight of cell-surface chondroitin sulfate and resulted in a marked reduction of rVAR2 binding. Finally, utilizing a cell-based glycocalyx model, we showed that rVAR2 binding correlates with the length of the chondroitin sulfate chains in the cellular glycocalyx. These data demonstrate that the total amount and cellular accessibility of chondroitin sulfate chains impact rVAR2 binding and thus malaria infection.
Assuntos
Antígenos de Protozoários/metabolismo , Sulfatos de Condroitina/metabolismo , Glicocálix/metabolismo , Malária Falciparum/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Antígenos de Protozoários/química , Antígenos de Protozoários/genética , Sulfatos de Condroitina/química , Sulfatos de Condroitina/genética , Feminino , Glicocálix/química , Glicocálix/genética , Células HEK293 , Células HeLa , Humanos , Malária Falciparum/genética , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/metabolismo , Placenta/metabolismo , Plasmodium falciparum/genética , Gravidez , Proteínas de Protozoários/química , Proteínas de Protozoários/genéticaRESUMO
Recombinant adenoviruses have broad applications for gene delivery and expression. Furthermore, the adenovirus packaging system facilitates the expression of RNA-guided CRISPR/Cas9 nuclease complexes. In this study, we developed a novel system, named AdBlue, for the construction of recombinant adenoviruses using an enzymatic assembly strategy. This system could significantly reduce the time and labor required to generate adenoviral vectors. When applied to CRISPR/Cas9 design, it simplifies the preparation of recombinant adenoviruses carrying nuclease complexes and can induce high levels of site-specific mutagenesis. Our system has outstanding advantages for adenovirus preparation and could be a useful molecular engineering tool for gene delivery and editing.
Assuntos
Adenoviridae/genética , Sistemas CRISPR-Cas , Edição de Genes/métodos , Vetores Genéticos , Linhagem Celular , Endonucleases/genética , Técnicas de Inativação de Genes , Técnicas de Transferência de Genes , Terapia Genética , Humanos , RNA Guia de CinetoplastídeosRESUMO
Long interspersed nuclear element-1 (LINE-1) is the only active autonomous transposon in the human genome. Its transposition frequently induces host genome instability, leading to a variety of genetic diseases, including cancers. The host factors play important roles in inhibiting LINE-1 retrotransposition. As an important component of the immune system, the host factor SLFN14 has antiviral activity. Our laboratory shows that SLFN14 possesses potent inhibitory activity against LINE-1 retrotransposition. To explore the potential mechanism of SLFN14 inhibition, we analyzed its effects on transcription, translation, reverse transcription and insertion in the LINE-1 replication cycle. We confirmed that SLFN14 could suppress the LINE-1 mRNA level by affecting its transcription and degradation, thereby diminishing the protein and cDNA levels of LINE-1, which eventually block the LINE-1 retrotransposition. Further, by mapping the active domains of SLFN14, we found its inhibitory activity on LINE-1 being closely related to its endoribonuclease and ribosome binding domains. These results demonstrate the mechanism of SLFN14 in regulating LINE-1 replication, which further provide new insights for improving the regulation network of host factors for controlling genomic instability caused by LINE-1 replication.
Assuntos
Endorribonucleases , Genoma Humano , Elementos Nucleotídeos Longos e Dispersos , Endorribonucleases/fisiologia , Instabilidade Genômica , Humanos , Elementos Nucleotídeos Longos e Dispersos/genética , RNA Mensageiro , Transcrição ReversaRESUMO
Increased immature neovessels contribute to plaque growth and instability. Here, we investigated a method to establish functional and stable neovessel networks to increase plaque stability. Rabbits underwent aortic balloon injury and were divided into six groups: sham, vector and lentiviral transfection with vascular endothelial growth factor-A (VEGF)-A, fibroblast growth factor (FGF)-2, platelet-derived growth factor (PDGF)-BB and FGF-2 + PDGF-BB. Lentivirus was percutaneously injected into the media-adventitia of the abdominal aorta by intravascular ultrasound guidance, and plaque-rupture rate, plaque-vulnerability index and plaque neovessel density at the injection site were evaluated. Confocal microscopy, Prussian Blue assay, Evans Blue, immunofluorescence and transmission electron microscopy were used to assess neovessel function and pericyte coverage. To evaluate the effect of FGF-2/PDGF-BB on pericyte migration, we used the mesenchymal progenitor cell line 10T1/2 as an in vitro model. VEGF-A- and FGF-2-overexpression increased the number of immature neovessels, which caused intraplaque haemorrhage and inflammatory cell infiltration, eventually resulting in the plaque vulnerability; however, FGF-2/PDGF-BB induced mature and functional neovessels, through increased neovessel pericyte coverage. Additionally, in vitro analysis of 10T1/2 cells revealed that FGF-2/PDGF-BB induced epsin-2 expression and enhanced the VEGF receptor-2 degradation, which negatively regulated pericyte function consistent with the in vivo data. These results showed that the combination of FGF-2 and PDGF-BB promoted the function and maturation of plaque neovessels, thereby representing a novel potential treatment strategy for vulnerable plaques.
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Becaplermina/administração & dosagem , Fator 2 de Crescimento de Fibroblastos/administração & dosagem , Vetores Genéticos/administração & dosagem , Lentivirus/genética , Neovascularização Patológica/prevenção & controle , Placa Aterosclerótica/terapia , Proteínas Adaptadoras de Transporte Vesicular , Animais , Becaplermina/genética , Becaplermina/metabolismo , Movimento Celular , Fator 2 de Crescimento de Fibroblastos/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Vetores Genéticos/genética , Masculino , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Fosforilação , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , CoelhosRESUMO
Various angiogenic factors have been shown to play important roles in intraplaque angiogenesis, while little is known about the dynamic expression change and interplay between various angiogenic factors and intraplaque angiogenesis under high cholesterol conditions. New Zealand rabbits underwent balloon injury of the abdominal artery and then were assigned to a control group (n = 15, normal chow) or high cholesterol group (n = 25, 1% high cholesterol diet). At weeks 4, 6, 8, 10, and 12 after acclimation, rabbits (high cholesterol group, n = 5; control group, n = 3) were euthanized. No lesions were observed in the control group. From week 4 to week 12, the expression of vascular endothelial growth factor A (VEGF-A), VEGF receptor 2 (VEGFR-2), fibroblast growth factor 2 (FGF-2), FGF receptor 1 (FGFR-1), platelet-derived growth factor-BB (PDGF-BB), and tumor necrosis factor alpha (TNF-α), the vulnerability index (VI) and the microvessel density (MVD) were significantly elevated in the high cholesterol group; however, PDGF receptor ß (PDGFR-ß) expression showed little change. Analysis by double-label immunofluorescence (CD31 and Ng2) and FITC-dextran indicated that the neovessels within the plaque were leaky due to a lack of pericytes. As indicated by Pearson's correlation analysis, there was a highly positive correlation between the VI, MVD, macrophage content, and TNF-α level, and the levels of VEGF-A/VEGFR-2 and FGF-2/FGFR-1. However, no correlations were observed between PDGFR-ß levels and the VI or MVD. High expression of VEGF-A/VEGFR-2 and FGF-2/FGFR-1 but not of PDGF-BB/PDGFR-ß may contribute to immature and inflammatory intraplaque angiogenesis and plaque instability in a rabbit model of atherosclerosis.
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Aterosclerose/metabolismo , Fator 2 de Crescimento de Fibroblastos/biossíntese , Neovascularização Patológica/metabolismo , Placa Aterosclerótica/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/biossíntese , Fator A de Crescimento do Endotélio Vascular/biossíntese , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Animais , Aterosclerose/patologia , Becaplermina/biossíntese , Modelos Animais de Doenças , Inflamação/metabolismo , Inflamação/patologia , Masculino , Neovascularização Patológica/patologia , Pericitos/metabolismo , Pericitos/patologia , Placa Aterosclerótica/patologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Coelhos , Receptor beta de Fator de Crescimento Derivado de Plaquetas/biossíntese , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The low-density lipoprotein receptor (LDLR) and related receptors are important for the transport of diverse biomolecules across cell membranes and barriers. Their functions are especially relevant for cholesterol homeostasis and diseases, including neurodegenerative and kidney disorders. Members of the LDLR-related protein family share LDLR class A (LA) repeats providing binding properties for lipoproteins and other biomolecules. We previously demonstrated that short linker regions between these LA repeats contain conserved O-glycan sites. Moreover, we found that O-glycan modifications at these sites are selectively controlled by the GalNAc-transferase isoform, GalNAc-T11. However, the effects of GalNAc-T11-mediated O-glycosylation on LDLR and related receptor localization and function are unknown. Here, we characterized O-glycosylation of LDLR-related proteins and identified conserved O-glycosylation sites in the LA linker regions of VLDLR, LRP1, and LRP2 (Megalin) from both cell lines and rat organs. Using a panel of gene-edited isogenic cell line models, we demonstrate that GalNAc-T11-mediated LDLR and VLDLR O-glycosylation is not required for transport and cell-surface expression and stability of these receptors but markedly enhances LDL and VLDL binding and uptake. Direct ELISA-based binding assays with truncated LDLR constructs revealed that O-glycosylation increased affinity for LDL by â¼5-fold. The molecular basis for this observation is currently unknown, but these findings open up new avenues for exploring the roles of LDLR-related proteins in disease.
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Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Receptores de LDL/metabolismo , Acetilgalactosamina/metabolismo , Animais , Células CHO , Membrana Celular/metabolismo , Cricetulus , Drosophila , Glicosilação , Células HEK293 , Células Hep G2 , Humanos , Ligantes , Lipoproteínas/metabolismo , Polissacarídeos/metabolismo , Ligação Proteica , Transporte Proteico , Ratos , Proteínas Recombinantes/metabolismoRESUMO
AIMS: Cardiac pressure and humoral factors induce cardiac hypertrophy and fibrosis, which are characterized by increased stiffness, reduced contractility and altered perfusion. Angiotensin II (AngII) is well known to promote this pathology. Angiotensin-converting enzyme (ACE) 2, which cleaves AngII and forms Ang-(1-7), exerts protective anti-hypertrophy and anti-fibrosis effects. A disintegrin and metalloproteinase 17 (ADAM17), a membrane-bound enzyme reported to cleave ACE2, may participate in the pathological process of AngII perfusion-induced heart damage. However, researchers have not clearly determined whether dickkopf-3 (DKK3) regulates the ADAM17/ACE2 pathway and, if so, whether DKK3-mediated regulation is related to the glycogen synthase kinase-3ß (GSK-3ß)/ß-catenin pathway. In this study, we explored whether DKK3 overexpression ameliorates the development of AngII-induced cardiac fibrosis and hypertrophy through the ADAM17/ACE2 and GSK-3ß/ß-catenin pathways. METHODS: Mice were injected with a DKK3-overexpressing adenovirus or vehicle and then infused with AngII or saline using subcutaneously implanted mini-pumps for four weeks. Hearts were stained with hematoxylin-eosin, Masson's trichrome and immunohistochemical markers for histology. Primary fibroblasts were treated with the adenovirus and AngII and then examined using western blotting, EdU (5-ethynyl-2'-deoxyuridine) assays and immunofluorescence. Additionally, siRNA silencing was performed to study the role of DKK3 and the involved pathways. RESULTS: AngII-induced cardiac hypertrophy and interstitial and perivascular fibrosis were less severe in DKK3-overexpressing mice than in control mice. Moreover, the expression levels of fibrotic genes, such as collagen I and III, and the hypertrophic genes atrial natriuretic peptide (ANP) and beta-myosin heavy chain (ß-MHC) were decreased. DKK3 overexpression also exerted a protective effect by inhibiting ADAM17 phosphorylation, thus increasing ACE2 expression and subsequently promoting AngII degradation. Furthermore, this process was mediated by the inhibition of GSK-3ß and ß-catenin and decreased translocation of ß-catenin to the nucleus. On the other hand, the DKK3 knockdown by siRNA achieved opposite results. CONCLUSION: DKK3 overexpression substantially alleviated AngII infusion-induced cardiac hypertrophy and fibrosis by regulating ADAM17/ACE2 pathway activity and inhibiting the GSK-3ß/ß-catenin pathway.
Assuntos
Proteína ADAM17/metabolismo , Angiotensina II/farmacologia , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Transdução de Sinais , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Angiotensina I , Enzima de Conversão de Angiotensina 2 , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Cardiomegalia/fisiopatologia , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Inflamação/patologia , Metaloproteinases da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos , Peptidil Dipeptidase A/metabolismo , Perfusão , Fosforilação/efeitos dos fármacos , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
AIM: Myocardial infarction (MI) is one of the leading causes of death in elderly people. Expanding the knowledge of the molecular mechanisms underlying MI is of profound importance to developing a cure for MI. The CUGBP- and ETR-3-like factor (CELF) proteins, a family of RNA-binding proteins, play key roles in RNA metabolism. To determine the functions and molecular mechanisms of CELF proteins in MI, an animal model of acute myocardial infarction (AMI) was used in our study. RESULTS: We found that the CUG triplet repeat RNA-binding protein 1 (CUGBP1)/CELF1 expression levels were decreased in AMI-injured hearts, and further studies showed that two highly conserved adenylate-uridylate-rich (AU-rich) elements in the 3'UTR of CUGBP1 were responsible for the decreased CUGBP1 expression. Upon AMI, human antigen R (HuR) was relocated to the cytoplasm from the nucleus and interacted with these AU-rich elements to affect the expression of CUGBP1. Reintroduction of CUGBP1 via gene delivery by recombinant adenovirus improved cardiac function in AMI mice. Our studies also indicated that CUGBP1 protected cardiomyocytes from ischemia-induced injury through the promotion of angiogenesis and inhibition of apoptosis by regulating the vascular endothelial growth factor-A gene. Innovation and Conclusion: Our studies indicate a role for CUGBP1 in cardiac disease and reveal a novel MI post-transcriptional gene regulatory mechanism. The reconstitution of CUGBP1 could be developed as a potential therapeutic option for the management of MI. Antioxid. Redox Signal. 27, 1013-1026.
Assuntos
Proteínas CELF1/genética , Proteínas CELF1/metabolismo , Proteína Semelhante a ELAV 1/metabolismo , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Regiões 3' não Traduzidas , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Humanos , Camundongos , Infarto do Miocárdio/genéticaRESUMO
Bromodomain-containing protein 7 (BRD7) is a tumour suppressor that is known to regulate many pathological processes including cell growth, apoptosis and cell cycle. Endoplasmic reticulum (ER) stress-induced apoptosis plays a key role in diabetic cardiomyopathy (DCM). However, the molecular mechanism of hyperglycaemia-induced myocardial apoptosis is still unclear. We intended to determine the role of BRD7 in high glucose (HG)-induced apoptosis of cardiomyocytes. In vivo, we established a type 1 diabetic rat model by injecting a high-dose streptozotocin (STZ), and lentivirus-mediated short hairpin RNA (shRNA) was used to inhibit BRD7 expression. Rats with DCM exhibited severe myocardial remodelling, fibrosis, left ventricular dysfunction and myocardial apoptosis. The expression of BRD7 was up-regulated in the heart of diabetic rats, and inhibition of BRD7 had beneficial effects against diabetes-induced heart damage. In vitro, H9c2 cardiomyoblasts was used to investigate the mechanism of BRD7 in HG-induced apoptosis. Treating H9c2 cardiomyoblasts with HG elevated the level of BRD7 via activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and increased ER stress-induced apoptosis by detecting spliced/active X-box binding protein 1 (XBP-1s) and C/EBP homologous protein (CHOP). Furthermore, down-regulation of BRD7 attenuated HG-induced expression of CHOP via inhibiting nuclear translocation of XBP-1s without affecting the total expression of XBP-1s. In conclusion, inhibition of BRD7 appeared to protect against hyperglycaemia-induced cardiomyocyte apoptosis by inhibiting ER stress signalling pathway.
Assuntos
Proteínas Cromossômicas não Histona/genética , Cardiomiopatias Diabéticas/genética , Hiperglicemia/genética , Fator de Transcrição CHOP/genética , Proteína 1 de Ligação a X-Box/genética , Animais , Apoptose/genética , Proliferação de Células/genética , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Cardiomiopatias Diabéticas/patologia , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/genética , Humanos , Hiperglicemia/patologia , Sistema de Sinalização das MAP Quinases/genética , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , RNA Interferente Pequeno/genética , RatosRESUMO
During placental malaria, Plasmodium falciparum infected erythrocytes sequester in the placenta, causing health problems for both the mother and fetus. The specific adherence is mediated by the VAR2CSA protein, which binds to placental chondroitin sulfate (CS) on chondroitin sulfate proteoglycans (CSPGs) in the placental syncytium. However, the identity of the CSPG core protein and the cellular impact of the interaction have remain elusive. In this study we identified the specific CSPG core protein to which the CS is attached, and characterized its exact placental location. VAR2CSA pull-down experiments using placental extracts from whole placenta or syncytiotrophoblast microvillous cell membranes showed three distinct CSPGs available for VAR2CSA adherence. Further examination of these three CSPGs by immunofluorescence and proximity ligation assays showed that syndecan-1 is the main receptor for VAR2CSA mediated placental adherence. We further show that the commonly used placental choriocarcinoma cell line, BeWo, express a different set of proteoglycans than those present on placental syncytiotrophoblast and may not be the most biologically relevant model to study placental malaria. Syncytial fusion of the BeWo cells, triggered by forskolin treatment, caused an increased expression of placental CS-modified syndecan-1. In line with this, we show that rVAR2 binding to placental CS impairs syndecan-1-related Src signaling in forskolin treated BeWo cells, but not in untreated cells.
Assuntos
Antígenos de Protozoários/metabolismo , Sulfatos de Condroitina/metabolismo , Malária Falciparum/parasitologia , Placenta/parasitologia , Complicações Infecciosas na Gravidez/parasitologia , Sindecana-1/metabolismo , Feminino , Imunofluorescência , Humanos , Imuno-Histoquímica , Malária Falciparum/metabolismo , Espectrometria de Massas , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Placenta/metabolismo , Plasmodium falciparum , Gravidez , Complicações Infecciosas na Gravidez/metabolismoRESUMO
Superparamagnetic iron oxide nanoparticles (SPIONs) as a contrast agent have been widely used in magnetic resonance imaging for tumor diagnosis and theranostics. However, there has been safety concern of SPIONs with cirrhosis related to excess iron-induced oxidative stress. In this study, the impact of iron overload by SPIONs was assessed on a mouse cirrhosis model. A single dose of SPION injection at 0.5 or 5 mg Fe/kg in the cirrhosis group induced a septic shock response at 24 h with elevated serum levels of liver and kidney function markers and extended impacts over 14 days including high levels of serum cholesterols and persistent low serum iron level. In contrast, full restoration of liver functions was found in the normal group with the same dosages over time. Analysis with PCR array of the toxicity pathways revealed the high dose of SPIONs induced significant expression changes of a distinct subset of genes in the cirrhosis liver. All these results suggested that excess iron of the high dose of SPIONs might be a risk factor for cirrhosis because of the marked impacts of elevated lipid metabolism, disruption of iron homeostasis and possibly, aggravated loss of liver functions.