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The baculovirus-insect cell expression system allows addition of O-fucose to EGF-like domains of glycoproteins, following the action of the protein O-fucosyltransferase 1 named POFUT1. In this study, recombinant Spodoptera frugiperda POFUT1 from baculovirus-infected Sf9 cells was compared to recombinant Mus musculus POFUT1 produced by CHO cells. Contrary to recombinant murine POFUT1 carrying two hybrid and/or complex type N-glycans, Spodoptera frugiperda POFUT1 exhibited paucimannose N-glycans, at least on its highly evolutionary conserved across Metazoa NRT site. The abilities of both recombinant enzymes to add in vitro O -fucose to EGF-like domains of three different recombinant mammalian glycoproteins were then explored. In vitro POFUT1-mediated O-fucosylation experiments, followed by click chemistry and blot analyses, showed that Spodoptera frugiperda POFUT1 was able to add O-fucose to mouse NOTCH1 EGF-like 26 and WIF1 EGF-like 3 domains, similarly to the murine counterpart. As proved by mass spectrometry, full-length human WNT Inhibitor Factor 1 expressed by Sf9 cells was also modified with O-fucose. However, Spodoptera frugiperda POFUT1 was unable to modify the single EGF-like domain of mouse PAMR1 with O-fucose, contrary to murine POFUT1. Absence of orthologous proteins such as PAMR1 in insects may explain the enzyme's difficulty in adding O-fucose to a domain that it never encounters naturally.
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Fucosiltransferases , Proteínas Recombinantes , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Spodoptera/enzimologia , Spodoptera/genética , Spodoptera/metabolismo , Fucosiltransferases/química , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Humanos , Animais , Camundongos , Células CHO , Cricetulus , Células Sf9 , Glicosilação , Sequência Consenso , Fucose/metabolismo , Domínios ProteicosRESUMO
Background: Retinitis pigmentosa (RP) is a complex inherited and progressive degenerative retinal disease. The eyes shut homolog (EYS) is frequently associated with RP is surprisingly high. Exploring the function of EYS is quite difficult due to the unique gene size and species specificity. Gene therapy may provide a breakthrough to treat this disease. Therefore, exploring and clarifying pathogenic mutations of EYS-associated RP has important guiding significance for clinical treatment. Methods: Clinical and molecular genetic data for EYS-associated RP were retrospectively analyzed. Sanger sequencing was applied to identify novel mutations in these patients. Candidate pathogenic variants were subsequently evaluated using bioinformatic tools. Results: A novel pair of compound heterozygous mutations was identified: a novel stop-gain mutation c.2439C>A (p.C813fsX) and a frameshift deletion mutation c.6714delT (p. P2238fsX) of the EYS gene in the RP family. Both of these mutations were rare or absent in the 1000 Genomes Project, dbSNP, and Genome Aggregation Database (gnomAD). These two mutations would result in a lack of multiple functionally important epidermal growth factor-like and Laminin G-like coding regions in EYS. Conclusions: A novel compound heterozygote of the EYS gene in a Chinese family with an autosomal inheritance pattern of RP was identified. Identifying more pathogenic mutations and expanding the mutation spectrum of the EYS gene will contribute to a more comprehensive understanding of the molecular pathogenesis of RP disease that could be gained in the future. It also could provide an important basis for the diagnosis, clinical management, and genetic counseling of the disease.
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População do Leste Asiático , Retinose Pigmentar , Humanos , Estudos Retrospectivos , Mutação/genética , Retinose Pigmentar/genética , Mutação da Fase de Leitura , Proteínas do Olho/genéticaRESUMO
Steroid-induced cataracts (SIC) are defined as cataracts associated with the administration of corticosteroids. Long-term glucocorticoid treatment for inflammatory diseases reportedly increases the risk of SIC, and steroids can induce cataracts by disrupting ocular growth factor balance or homeostasis. In this study, we verified the effect of chondroitin sulfate proteoglycan 5 (CSPG5) using dexamethasone (dexa)-treated human lens epithelial (HLE-B3) cells and the lens epithelium from the anterior capsule of SIC patients obtained during cataract surgery. CSPG5 expression increased in the lens epithelium of SIC patients. The downregulation of CSPG5 suppressed the dexa-induced epithelial-mesenchymal transition (EMT)-related protein expression and motility in HLE-B3 cells. The disruption of the transcription factors EZH2 and B-Myb downregulated CSPG5, dexa-induced fibronectin expression, and cell migration in HLE-B3 cells, reaffirming that CSPG5 expression regulates EMT in lens epithelial cells. Taken together, these results indicate that the steroid-induced effects on lens epithelial cells are mediated via alterations in CSPG5 expression. Therefore, our study emphasizes the potential of CSPG5 as a therapeutic target for the prevention and treatment of SIC.
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Catarata , Cristalino , Humanos , Cristalino/metabolismo , Catarata/induzido quimicamente , Catarata/metabolismo , Epitélio , Células Epiteliais/metabolismo , Proteoglicanas de Sulfatos de CondroitinaRESUMO
BACKGROUND: The tumour microenvironment consists of a complex and dynamic milieu of cancer cells, including tumour-associated stromal cells (leukocytes, fibroblasts, vascular cells, etc.) and their extracellular products. During invasion and metastasis, cancer cells actively remodel the tumour microenvironment and alterations of microenvironment, particularly cancer-associated fibroblasts (CAFs), can promote tumour progression. However, the underlying mechanisms of the CAF formation and their metastasis-promoting functions remain unclear. METHODS: Primary liver fibroblasts and CAFs were isolated and characterized. CAFs in clinical samples were identified by immunohistochemical staining and the clinical significance of CAFs was also analysed in two independent cohorts. A transwell coculture system was used to confirm the role of HCC cells in CAF recruitment and activation. qRT-PCR, western blotting and ELISA were used to screen paracrine cytokines. The role and mechanism of Egfl7 in CAFs were explored via an in vitro coculture system and an in vivo mouse orthotopic transplantation model. RESULTS: We showed that CAFs in hepatocellular carcinoma (HCC) are characterized by the expression of α-SMA and that HCC cells can recruit liver fibroblasts (LFs) and activate them to promote their transformation into CAFs. High α-SMA expression, indicating high CAF infiltration, was correlated with malignant characteristics. It was also an independent risk factor for HCC survival and could predict a poor prognosis in HCC patients. Then, we demonstrated that EGF-like domain multiple 7 (Egfl7) was preferentially secreted by HCC cells, and exhibited high potential to recruit and activate LFs into the CAF phenotype. The ability of Egfl7 to modulate LFs relies upon increased phosphorylation of FAK and AKT via the receptor ανß3 integrin. Strikingly, CAFs activated by paracrine Egfl7 could further remodel the tumour microenvironment by depositing fibrils and collagen and in turn facilitate HCC cell proliferation, invasion and metastasis. CONCLUSION: Our data highlighted a novel role of Egfl7 in remodelling the tumour microenvironment: it recruits LFs and activates them to promote their transformation into CAFs via the ανß3 integrin signaling pathway, which further promotes HCC progression and contributes to poor clinical outcomes in HCC patients. Video Abstract.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Camundongos , Fibroblastos , Integrina beta3 , Peptídeos e Proteínas de Sinalização Intercelular , Microambiente TumoralRESUMO
Scavenger receptors (SRs), as multifunctional pattern recognition receptors, play an important role in innate immunity in mammals, however, their function in fish is limited. Herein, scavenger receptor F2 in Epinephelus coioides (EcSRECII) induced an innate immune response to LPS in GS cells. EcSRECII markedly enhanced LPS-induced NF-κB and IFN-ß signaling pathways, whereas knockdown of EcSRECII significantly inhibited LPS-induced NF-κB and IFN-ß promoter activation. Interestingly, only retain of epidermal growth factor (EGF)/EGF-like domain in EcSRECII resulted in a punctate cytoplasmic distribution, while the C-terminal domain exhibited a distinct cytoskeletal cytoplasmic distribution. Moreover, devoid of this EGF/EGF-like domain fragment more sharply impaired its ability to activate EcSRECII-induced NF-κB activation than deletion of the C-terminal domain region, but both domains significantly induced IFN-ß promoter activation. Full-length EcSRECII and the deletion mutant of C-terminal domain could partly colocalize with lysosomes by LPS derived from V. parahaemolyticus (V.p. LPS) in GS cells, but there was no similar distribution in the deletion mutant of EGF/EGF-like domain. This finding firstly suggested that the N-terminal EGF/EGF-like domain was necessary for the NF-κB signaling pathway to trigger resistance to vibrio infection and its functional exertion may be associated with lysosomes, thus providing insights into the regulation of resistance to vibrio infection in teleosts.
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Bass , Vibrioses , Animais , NF-kappa B/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Lipopolissacarídeos/farmacologia , Cisteína , Transdução de Sinais , Bass/genética , Receptores Depuradores , Vibrioses/veterinária , Lisossomos/metabolismo , Mamíferos/metabolismoRESUMO
Although vaccines are one of the environmentally friendly means to prevent the spread of ticks, there is currently no commercial vaccine effective against Haemaphysalis longicornis ticks. In this study, we identified, characterized, localized, and evaluated the expression patterns, and tested the immunogenic potential of a homologue of Rhipicephalus microplus ATAQ in H. longicornis (HlATAQ). HlATAQ was identified as a 654 amino acid-long protein present throughout the midgut and in Malpighian tubule cells and containing six full and one partial EGF-like domains. HlATAQ was genetically distant (homology < 50%) from previously reported ATAQ proteins and was expressed throughout tick life stages. Its expression steadily increased (p < 0.001) during feeding, reached a peak, and then decreased slightly with engorgement. Silencing of HlATAQ did not result in a phenotype that was significantly different from the control ticks. However, H. longicornis female ticks fed on a rabbit immunized with recombinant HlATAQ showed significantly longer blood-feeding periods, higher body weight at engorgement, higher egg mass, and longer pre-oviposition and egg hatching periods than control ticks. These findings indicate that the ATAQ protein plays a role in the blood-feeding-related physiological processes in the midgut and Malpighian tubules and antibodies directed against it may affect these tissues and disrupt tick engorgement and oviposition.
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Marfan syndrome, an autosomal dominant disorder of connective tissue, is primarily caused by mutations in the fibrillin-1 (FBN1) gene, which encodes the protein fibrillin-1. The protein is composed of epidermal growth factor-like (EGF-like) domains, transforming growth factor beta-binding protein-like (TB) domains, and hybrid (Hyb) domains and is an important component of elastin-related microfibrils in elastic fiber tissue. In this study, we report a cysteine to tyrosine substitution in two different domains of fibrillin-1, both of which cause Marfan syndrome with ocular abnormalities, in two families. Using protease degradation and liquid chromatography-tandem mass spectrometry analyses, we explored the different effects of substitution of cysteine by tyrosine in an EGF-like and a calcium-binding (cb) EGF-like domain on protein stability. The results showed that cysteine mutations in the EGF domain are more likely to result in altered proteolytic sensitivity and thermostability than those in the cbEGF domain. Furthermore, cysteine mutations can lead to new enzymatic sites exposure or hidden canonical cleavage sites. These results indicate the differential clinical phenotypes and molecular pathogenesis of Marfan syndrome caused by cysteine mutations in different fibrillin-1 domains. These results strongly suggest that failure to form disulfide bonds and abnormal proteolysis of fibrillin-1 caused by cysteine mutations may be an important factor underlying the pathogenesis of diseases caused by fibrillin-1 mutations, such as Marfan syndrome.
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Neuregulins (NRGs) are a family of six related physiological ligands all containing a receptor-binding epidermal growth factor (EGF)-like domain that mediate their binding to cellular receptors. Neuregulin-1 (NRG1) is the main physiological ligand to HER3. NRG1 fusion (NRG1+) was first reported in a breast cancer cell line and NRG2 fusions have recently been identified in solid tumors. It is postulated that NRG1 fusions, through mostly transmembrane fusion partners, result in NRG1 being concentrated in proximity to HER3, leading to its constitutive activation and oncogenesis. Recently, a monoclonal antibody that disrupts the binding of NRG1 to HER3 and HER3/HER2 heterodimerization has resulted in NRG1+ tumor shrinkage, suggesting that 'ligand-fusion' may be a novel mechanism of oncogenesis.
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Neoplasias , Fatores de Crescimento Neural/metabolismo , Neuregulina-1 , Carcinogênese/genética , Fusão Gênica , Humanos , Ligantes , Neoplasias/genética , Neoplasias/patologia , Neuregulina-1/genética , Neuregulina-1/metabolismoRESUMO
The fusion genes containing neuregulin-1 (NRG1) are newly described potentially actionable oncogenic drivers. Initial clinical trials have shown a positive response to targeted treatment in some cases of NRG1 rearranged lung adenocarcinoma, cholangiocarcinoma, and pancreatic carcinoma. The cost-effective large scale identification of NRG1 rearranged tumors is an open question. We have tested a data-drilling approach by performing a retrospective assessment of a de-identified molecular profiling database of 3263 tumors submitted for fusion testing. Gene fusion detection was performed by RNA-based targeted next-generation sequencing using the Archer Fusion Plex kits for Illumina (ArcherDX Inc., Boulder, CO). Novel fusion transcripts were confirmed by a custom-designed RT-PCR. Also, the aberrant expression of CK20 was studied immunohistochemically. The frequency of NRG1 rearranged tumors was 0.2% (7/3263). The most common histologic type was lung adenocarcinoma (n = 5). Also, renal carcinoma (n = 1) and prostatic adenocarcinoma (n = 1) were found. Identified fusion partners were of a wide range (CD74, SDC4, TNC, VAMP2, UNC5D), with CD74, SDC4 being found twice. The UNC5D is a novel fusion partner identified in prostate adenocarcinoma. There was no co-occurrence with the other tested fusions nor KRAS, BRAF, and the other gene mutations specified in the applied gene panels. Immunohistochemically, the focal expression of CK20 was present in 2 lung adenocarcinomas. We believe it should be considered as an incidental finding. In conclusion, the overall frequency of tumors with NRG1 fusion was 0.2%. All tumors were carcinomas. We confirm (invasive mucinous) lung adenocarcinoma as being the most frequent tumor presenting NRG1 fusion. Herein novel putative pathogenic gene fusion UNC5D-NRG1 is described. The potential role of immunohistochemistry in tumor identification should be further addressed.
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Adenocarcinoma/genética , Neoplasias Pulmonares/genética , Neuregulina-1/genética , Proteínas de Fusão Oncogênica/genética , Neoplasias da Próstata/genética , Receptores de Superfície Celular/genética , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Diferenciação de Linfócitos B/genética , Feminino , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Neoplasias da Próstata/patologia , Sindecana-4/genética , Tenascina/genética , Proteína 2 Associada à Membrana da Vesícula/genéticaRESUMO
BACKGROUND: Glioma, the most frequent primary tumor of the central nervous system, has poor prognosis. The epidermal growth factor receptor (EGFR) pathway and angiogenesis play important roles in glioma growth, invasion, and recurrence. The present study aimed to use proteomic methods to probe into the role of the EGF-EGFR-angiogenesis axis in the tumorigenesis of glioma and access the therapeutic efficacy of selumetinib on glioma. METHODS: Proteomic profiling was used to characterize 200 paired EGFR-positive and EGFR-negative glioma tissues of all pathological types. The quantitative mass spectrometry data were used for systematic analysis of the proteomic profiles of 10 EGFR-positive and 10 EGFR-negative glioma cases. Consensus-clustering analysis was used to screen target proteins. Immunofluorescence analysis, cell growth assay, and intracranial xenograft experiments were used to verify and test the therapeutic effect of selumetinib on glioma. RESULTS: Advanced proteomic screening demonstrated that the expression of EGF-like domain multiple 7 (EGFL7) was higher in EGFR-positive tumor tissues than in EGFR-negative tumor tissues. In addition, EGFL7 could act as an activator in vitro and in vivo to promote glioma cell proliferation. EGFL7 was associated strongly with EGFR and prognosis. EGFL7 knockdown effectively suppressed glioma cell proliferation. Selumetinib treatment showed tumor reduction effect in EGFR-positive glioblastoma xenograft mouse model. CONCLUSIONS: EGFL7 is a potential diagnostic biomarker and therapeutic target of glioma. Selumetinib could target the EGFR pathway and possibly improve the prognosis of EGFR-positive glioma.
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Proteínas de Ligação ao Cálcio , Família de Proteínas EGF , Fator de Crescimento Epidérmico , Glioma , Adulto , Animais , Benzimidazóis/farmacologia , Movimento Celular , Fatores de Crescimento Endotelial/metabolismo , Receptores ErbB/metabolismo , Feminino , Humanos , Masculino , Camundongos , Recidiva Local de Neoplasia , Proteômica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The shells of the Pacific oyster Crassostrea gigas contain calcite crystals with three types of microstructures: prismatic, chalky, and foliated layers. Many shell matrix proteins were annotated from the shells of C. gigas; however, it is unclear which SMPs play important roles in their shell mineralization. The matrix proteins have never been reported from the chalky layer. In this study, we identified a chalky layer-derived EGF-like domain-containing protein (CgELC) from the chalky layer of C. gigas shells. The gene sequence of the CgELC was encoded under CGI_ 10,017,544 of the C. gigas genome database. Only peptide fragments in the N-terminal region of CGI_ 10,017,544 were detected by LC-MS/MS analyses, suggesting that CGI_ 10,017,544 was digested at the predicted protease digestion dibasic site by post-translational modification to become a mature CgELC protein. We produced three types of CgELC recombinant proteins, namely, the full length CgELC, as well as the N-terminal and C-terminal parts of CgELC (CgELC-N or -C, respectively), for in vitro crystallization experiments. In the presence of these recombinant proteins, the aggregation of polycrystalline calcite was observed. Some fibrous organic components seemed to be incorporated into the calcite crystals in the presence of the r-CgELC protein. We also noted different features in the crystallization between CgELC-N and CgELC-C; some crystals were inhibited crystal plane formation and contained many columnar prisms inside the crystals (CgELC-N) and formed numerous holes on their surfaces (CgELC-C). These results suggest that CgELC is involved in crystal aggregation and incorporated into calcite crystals.
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Crassostrea/metabolismo , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/metabolismo , Exoesqueleto/metabolismo , Animais , Calcificação Fisiológica/fisiologia , Carbonato de Cálcio/metabolismo , Cristalização/métodos , Domínios Proteicos/fisiologiaRESUMO
BACKGROUND: Protein O-fucosyltransferase 1 (POFUT1) overexpression, which is observed in many cancers such as colorectal cancer (CRC), leads to a NOTCH signaling dysregulation associated with the tumoral process. In rare CRC cases, with no POFUT1 overexpression, seven missense mutations were found in human POFUT1. METHODS: Recombinant secreted forms of human WT POFUT1 and its seven mutated counterparts were produced and purified. Their O-fucosyltransferase activities were assayed in vitro using a chemo-enzymatic approach with azido-labeled GDP-fucose as a donor substrate and NOTCH1 EGF-LD26, produced in E. coli periplasm, as a relevant acceptor substrate. Targeted mass spectrometry (MS) was carried out to quantify the O-fucosyltransferase ability of all POFUT1 proteins. FINDINGS: MS analyses showed a significantly higher O-fucosyltransferase activity of six POFUT1 variants (R43H, Y73C, T115A, I343V, D348N, and R364W) compared to WT POFUT1. INTERPRETATION: This study provides insights on the possible involvement of these seven missense mutations in colorectal tumors. The hyperactive forms could lead to an increased O-fucosylation of POFUT1 protein targets such as NOTCH receptors in CRC patients, thereby leading to a NOTCH signaling dysregulation. It is the first demonstration of gain-of-function mutations for this crucial glycosyltransferase, modulating NOTCH activity, as well as that of other potential glycoproteins.
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Low-density lipoprotein receptor-related protein 4 (LRP4) is a multi-functional protein implicated in bone, kidney and neurological diseases including Cenani-Lenz syndactyly (CLS), sclerosteosis, osteoporosis, congenital myasthenic syndrome and myasthenia gravis. Why different LRP4 mutation alleles cause distinct and even contrasting disease phenotypes remain unclear. Herein, we utilized the zebrafish model to search for pathways affected by a deficiency of LRP4. The lrp4 knockdown in zebrafish embryos exhibits cyst formations at fin structures and the caudal vein plexus, malformed pectoral fins, defective bone formation and compromised kidney morphogenesis; which partially phenocopied the human LRP4 mutations and were reminiscent of phenotypes resulting form a perturbed Notch signaling pathway. We discovered that the Lrp4-deficient zebrafish manifested increased Notch outputs in addition to enhanced Wnt signaling, with the expression of Notch ligand jagged1b being significantly elevated at the fin structures. To examine conservatism of signaling mechanisms, the effect of LRP4 missense mutations and siRNA knockdowns, including a novel missense mutation c.1117C > T (p.R373W) of LRP4, were tested in mammalian kidney and osteoblast cells. The results showed that LRP4 suppressed both Wnt/ß-Catenin and Notch signaling pathways, and these activities were perturbed either by LRP4 missense mutations or by a knockdown of LRP4. Our finding underscore that LRP4 is required for limiting Jagged-Notch signaling throughout the fin/limb and kidney development, whose perturbation representing a novel mechanism for LRP4-related diseases. Moreover, our study reveals an evolutionarily conserved relationship between LRP4 and Jagged-Notch signaling, which may shed light on how the Notch signaling is fine-tuned during fin/limb development.
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Regulação da Expressão Gênica no Desenvolvimento , Proteínas Relacionadas a Receptor de LDL/genética , Receptores Notch/metabolismo , Proteínas Serrate-Jagged/metabolismo , Transdução de Sinais , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Nadadeiras de Animais/embriologia , Nadadeiras de Animais/metabolismo , Animais , Extremidades/embriologia , Extremidades/fisiologia , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Rim/embriologia , Rim/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Mutação , Mutação de Sentido Incorreto , Organogênese , Via de Sinalização Wnt , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
BACKGROUND: Signal peptide-CUB-EGF-like domain-containing protein 1 (SCUBE1) is expressed in vascular endothelium and human platelets. SCUBE1 levels are increased in acute arterial thrombosis. Multiple myeloma patients are also at increased risk of arterial thrombotic events. This study aimed to measure SCUBE1 levels in newly diagnosed multiple myeloma patients and to define whether SCUBE1 could be a useful marker determining the arterial thrombotic risk. METHODS: SCUBE1 levels of 32 newly diagnosed multiple myeloma patients and 41 healthy control subjects were measured by enzyme-linked immunosorbent assay. RESULTS: Plasma SCUBE1 levels of multiple myeloma patients and control subjects were 6.22 ± 0.9 and 7.95 ± 1.1 ng/ml, respectively. In the patient group, SCUBE1 levels were significantly lower compared to control subjects (p <0.001). CONCLUSIONS: SCUBE1 level measuring does not help to predict the arterial thrombotic risk in multiple myeloma patients. The significantly lower levels of SCUBE1 in multiple myeloma patients are likely to be due to defective platelets and/or increased TNF-α cytokines and is another proof of platelet dysfunction seen in these patients. HIPPOKRATIA 2019, 23(1): 21-24.
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OBJECTIVE: Lung adenocarcinoma is a non-small cell lung cancer, a common cancer in both genders, and has poor clinical outcome. Our aim was to evaluate the role of epidermal growth factor (EGF)-like domain multiple 6 (EGFL6) and its prognostic significance in lung adenocarcinoma. METHODS: EGFL6 expression was studied by immunohistochemical staining of specimens from 150 patients with lung adenocarcinoma. The correlation between clinicopathological features and EGFL6 expression was quantitatively analysed. We used Kaplan-Meier analysis and Cox proportional hazard models to examine the prognostic value of EGFL6 in terms of overall survival. RESULTS: No significant correlation was found between EGFL6 expression and clinical parameters. However, patients with high levels of EGFL6 expression showed a tendency towards poor prognosis, with borderline statistical significance. Grouping the patients according to a medium age value revealed a significant association between high EGFL6 expression and poor clinical outcome in young patients. This finding was further confirmed by grouping the patients into three groups according to age. HR in patients with high EGFL6 expression was higher in younger patients than in older patients. CONCLUSION: High EGFL6 expression may serve as a marker for poor prognosis of lung adenocarcinoma, especially in younger patients.
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Adenocarcinoma/genética , Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Glicoproteínas de Membrana/genética , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Idoso , Proteínas de Ligação ao Cálcio , Moléculas de Adesão Celular , Feminino , Humanos , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Invasividade Neoplásica , Recidiva Local de Neoplasia , Estadiamento de Neoplasias , Prognóstico , Estudos Retrospectivos , Análise de Sobrevida , Taiwan/epidemiologiaRESUMO
Epidermal growth factor-like domain-containing protein 7 (EGFL7), a member of the epidermal growth factor (EGF)-like protein family, is a potent angiogenic factor expressed in many different cell types. EGFL7 plays a vital role in controlling vascular angiogenesis during embryogenesis, organogenesis, and maintaining skeletal homeostasis. It regulates cellular functions by mediating the main signaling pathways (Notch, integrin) and EGF receptor cascades. Accumulating evidence suggests that Egfl7 plays a crucial role in cancer biology by modulating tumor angiogenesis, metastasis, and invasion. Dysregulation of Egfl7 has been frequently found in several types of cancers, such as malignant glioma, colorectal carcinoma, oral and oesophageal cancers, gastric cancer, hepatocellular carcinoma, pancreatic cancer, breast cancer, lung cancer, osteosarcoma, and acute myeloid leukemia. In addition, altered expression of miR-126, a microRNA associated with Egfl7, was found to play an important role in oncogenesis. More recently, our study has shown that EGFL7 is expressed in both the osteoclast and osteoblast lineages and promotes endothelial cell activities via extracellular signal-regulated kinase (ERK), signal transducer and activator of transcription 3 (STAT3), and integrin signaling cascades, indicative of its angiogenic regulation in the bone microenvironment. Thus, understanding the role of EGFL7 may provide novel insights into the development of improved diagnostics and therapeutic treatment for cancers and skeletal pathological disorders, such as ischemic osteonecrosis and bone fracture healing.
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Fatores de Crescimento Endotelial/genética , MicroRNAs/genética , Neoplasias/genética , Neovascularização Patológica/genética , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Proteínas de Ligação ao Cálcio , Linhagem da Célula/genética , Movimento Celular/genética , Família de Proteínas EGF , Fraturas Ósseas/genética , Fraturas Ósseas/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/classificação , Neoplasias/patologia , Neovascularização Patológica/patologia , Osteonecrose/genética , Osteonecrose/patologia , Transdução de SinaisRESUMO
BACKGROUND: Marfan syndrome (MFS) is a heritable disorder of connective tissue resulting from pathogenic variants of the fibrillin-1 gene (FBN1). Neonatal Marfan syndrome (nMFS) is rare and the most severe form of MFS, involving rapidly progressive cardiovascular dysfunction leading to death during early childhood. The constant enrichment of the nMFS mutation spectrum is helpful to improve our understanding of genotype-phenotype correlations in the disease. Herein, we report a novel dominant mutation in exon 26 of FBN1 (c.3331 T > C) in a sporadic case with nMFS. CASE PRESENTATION: An 8-month-old Han Chinese girl presented with the classic nMFS phenotype, including prominent manifestations of bone overgrowth, aortic root dilatation, and multiple cardiac valve dysfunctions. Genetic analysis revealed that she was heterozygous for a de novo FBN1 missense mutation (c.3331 T > C). The mutation leads to the substitution of a highly conserved FBN1 cysteine residue (p.Cys1111Arg), which is likely to severely perturb the FBN1 structure because of an alteration of the disulfide bond pattern in the calcium-binding epidermal growth factor-like (cbEGF) 12 domain. This variant was absent in 208 ethnically matched controls, providing further evidence that it may be causative of nMFS. An analysis of nMFS-associated mutations from the UMD-FBN1 database indicates that those de novo mutations altering disulfide bonds or Ca(2+) binding sites of the cbEGF domains encoded by exons 25-33, and a lack of phenotypic heterogeneity may be associated with an increased risk for nMFS. CONCLUSION: We diagnosed an infant with rare nMFS showing rapidly progressive cardiovascular dysfunction and widely systemic features. As the only causal FBN1 mutation identified in the patient, the missense mutation c.3331 T > C (p.Cys1111Arg) was associated with the severe phenotype of MFS. However, the pathogenicity of the novel mutation needs further confirmation in other patients with nMFS. Our review of the prominent characteristics of nMFS mutations relative to classic or incomplete MFS-related mutations will be helpful for the recognition of novel nMFS-associated variants.
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Fibrilina-1/genética , Síndrome de Marfan/genética , Mutação de Sentido Incorreto , Feminino , Marcadores Genéticos , Humanos , Lactente , Recém-Nascido , Síndrome de Marfan/diagnóstico , FenótipoRESUMO
Milk fat globule-epidermal growth factor-factor 8 (MFG-E8), as its name suggests, is a major glycoprotein component of milk fat globules secreted by the mammary epithelium. Although its role in milk fat production is unclear, MFG-E8 has been shown to act as a bridge linking apoptotic cells to phagocytes for removal of these dying cells. MFG-E8 is capable of bridging these two very different cell types via interactions through both its epidermal growth factor (EGF)-like domain(s) and its lectin-type C domains. The EGF-like domain interacts with αVß3 and αVß5 integrins on the surface of phagocytes, whereas the C domains bind phosphatidylserine found on the surface of apoptotic cells. In an attempt to purify full-length, recombinant MFG-E8 expressed in either insect cells or CHO cells, we find that it is highly aggregated. Systematic truncation of the domain architecture of MFG-E8 indicates that the C domains are mainly responsible for the aggregation propensity. Addition of Triton X-100 to the conditioned cell culture media allowed partial recovery of non-aggregated, full-length MFG-E8. A more comprehensive detergent screen identified CHAPS as a stabilizer of MFG-E8 and allowed purification of a significant portion of non-aggregated, full-length protein. The CHAPS-stabilized recombinant MFG-E8 retained its natural ability to bind both αVß3 and αVß5 integrins and phosphatidylserine suggesting that it is properly folded and active. Herein we describe an efficient purification method for production of non-aggregated, full-length MFG-E8.
Assuntos
Antígenos de Superfície , Expressão Gênica , Proteínas do Leite , Animais , Antígenos de Superfície/biossíntese , Antígenos de Superfície/química , Antígenos de Superfície/genética , Antígenos de Superfície/isolamento & purificação , Células CHO , Cricetinae , Cricetulus , Humanos , Camundongos , Proteínas do Leite/biossíntese , Proteínas do Leite/química , Proteínas do Leite/genética , Proteínas do Leite/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Células Sf9 , SpodopteraRESUMO
Tenascin-C is a large, multimodular, extracellular matrix glycoprotein that exhibits a very restricted pattern of expression but an enormously diverse range of functions. Here, we discuss the importance of deciphering the expression pattern of, and effects mediated by, different forms of this molecule in order to fully understand tenascin-C biology. We focus on both post transcriptional and post translational events such as splicing, glycosylation, assembly into a 3D matrix and proteolytic cleavage, highlighting how these modifications are key to defining tenascin-C function.
Assuntos
Encéfalo/metabolismo , Regulação da Expressão Gênica/fisiologia , Redes Reguladoras de Genes/fisiologia , Transdução de Sinais/fisiologia , Tenascina/metabolismo , Animais , Humanos , Processamento de Proteína Pós-Traducional/fisiologiaRESUMO
Complement-mediated cytolysis is the important effect of immune response, which results from the assembly of terminal complement components (C5b-9). Among them, α subunit of C8 (C8α) is the first protein that traverses the lipid bilayer, and then initiates the recruitment of C9 molecules to form pore on target membranes. In this article, a full-length cDNA of C8α (CpC8α) is identified from the whitespotted bamboo shark (Chiloscyllium plagiosum) by RACE. The CpC8α cDNA is 2183 bp in length, encoding a protein of 591 amino acids. The deduced CpC8α exhibits 89%, 49% and 44% identity with nurse shark, frog and human orthologs, respectively. Sequence alignment indicates that the C8α is well conserved during the evolution process from sharks to mammals, with the same modular architecture as well as the identical cysteine composition in the mature protein. Phylogenetic analysis places CpC8α and nurse shark C8α in cartilaginous fish clade, in parallel with the teleost taxa, to form the C8α cluster with higher vertebrates. Hydrophobicity analysis also indicates a similar hydrophobicity of CpC8α to mammals. Finally, expression analysis revealed CpC8α transcripts were constitutively highly expressed in shark liver, with much less expression in other tissues. The well conserved structure and properties suggests an analogous function of CpC8α to mammalian C8α, though it remains to be confirmed by further study.