Fibronectin Type III Domain

纤连蛋白 III 型结构域
  • 文章类型: Journal Article
    角膜伤口愈合需要上皮重组和基质细胞外基质(ECM)重塑,与ECM蛋白如肌腱蛋白C(TnC)调节和维持角膜稳态。TnC的N端球形结构域和C端纤维蛋白原相关结构域被表皮生长因子(EGF)样重复序列分开,和多达15个纤连蛋白III型结构域(Tnfn)。Tnfn1-5及其剪接变体的过表达发生在各种病理中。我们以前已经使用Tn64(与Tnfn1-5同源的单链可变片段抗体)来建立Tnfn1-5在纤维化病理如类风湿性关节炎和后囊混浊中的作用。这里,我们显示Tn64结合Tnfn重复3-5(其构成TnC内可溶性纤连蛋白结合的主要位点)。与其他Tnfn域不同,Tnfn3-5显示对纤连蛋白基质组装没有抑制。相反,Tnfn3-5构建体是促纤维化的并引起纤连蛋白表达增加。我们使用人角膜上皮细胞(HCEC)线检查了通过Tn64与Tnfn3-5结合的角膜上皮和基质伤口愈合,人角膜成纤维细胞(HCFs)的原代培养,和离体角膜器官培养模型。Tn64增强角膜上皮细胞的增殖和粘附,同时抑制角膜成纤维细胞和肌成纤维细胞的迁移。Tn64似乎通过下调TNF-α来减轻炎症,通过限制纤连蛋白聚合来预防角膜纤维化,促进角膜上皮和基质的再生,这表明它可以作为有效的抗纤维化角膜伤口愈合的治疗剂。
    Corneal wound healing requires epithelial reorganization and stromal extracellular matrix (ECM) remodeling, with ECM proteins such as Tenascin C (TnC) regulating and maintaining corneal homeostasis. The N-terminal globular domain and C-terminal fibrinogen-related domains of TnC are separated by epidermal growth factor (EGF)-like repeats, and upto fifteen fibronectin type III domains (Tn fn). Overexpression of Tn fn 1-5 and its splice variants occurs in varied pathologies. We have previously used Tn64 (a single chain variable fragment antibody cognate to Tn fn 1-5) to establish roles of Tn fn 1-5 in fibrotic pathologies such as rheumatoid arthritis and posterior capsular opacification. Here, we show that Tn64 binds to Tn fn repeats 3-5 (which constitute the major site for binding of soluble fibronectin within TnC). Unlike other Tn fn domains, Tn fn 3-5 displays no inhibition of fibronectin matrix assembly. Rather, the Tn fn 3-5 construct is pro-fibrotic and elicits increased expression of fibronectin. We examined corneal epithelial as well as stromal wound healing through Tn64 binding to Tn fn 3-5, using a human corneal epithelial cell (HCEC) line, primary cultures of human corneal fibroblasts (HCFs), and an ex-vivo corneal organ culture model. Tn64 enhanced proliferation and adhesion of corneal epithelial cells, while inhibiting the migration of corneal fibroblasts and myofibroblasts. Tn64 appears to attenuate inflammation through downregulation of TNF-α, prevent corneal fibrosis by limiting fibronectin polymerization, and promote regeneration of corneal epithelia and stroma, suggesting that it could be developed as a therapeutic agent for effective anti-fibrotic corneal wound healing.
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  • 文章类型: Journal Article
    蛋白质磷酸化是一种普遍的翻译修饰,它的失调与各种疾病有关,包括癌症.尽管意义重大,缺乏具有高特异性和亲和力的FCP/SCP型Ser/Thr蛋白磷酸酶Scp1的特异性抑制剂。在这项研究中,我们专注于adnectin,一种抗体模拟蛋白,旨在鉴定具有广泛结合表面的Scp1特异性结合分子,该分子靶向Scp1的底物识别位点。使用具有随机化FG环的adnectin呈递噬菌体文库进行Scp1的生物淘选。我们成功地鉴定了FG-1Adn,对Scp1表现出高亲和力和特异性。与FG-1肽相关的Scp1结合序列的Ala扫描分析显示疏水性残基,包括芳香氨基酸,在Scp1识别中发挥重要作用。此外,发现FG-1Adn与Scp1共定位在细胞中,特别是在质膜上。此外,Westernblotting分析表明FG-1Adn提高了细胞中靶蛋白Scp1的磷酸化水平,表明FG-1Adn可以抑制Scp1的功能。这些结果表明FG-1Adn可用作Scp1的特异性抑制剂。
    Protein phosphorylation is a prevalent translational modification, and its dysregulation has been implicated in various diseases, including cancer. Despite its significance, there is a lack of specific inhibitors of the FCP/SCP-type Ser/Thr protein phosphatase Scp1, characterized by high specificity and affinity. In this study, we focused on adnectin, an antibody-mimetic protein, aiming to identify Scp1-specific binding molecules with a broad binding surface that target the substrate-recognition site of Scp1. Biopanning of Scp1 was performed using an adnectin-presenting phage library with a randomized FG loop. We succeeded in identifying FG-1Adn, which showed high affinity and specificity for Scp1. Ala scanning analysis of the Scp1-binding sequence in relation to the FG-1 peptide revealed that hydrophobic residues, including aromatic amino acids, play important roles in Scp1 recognition. Furthermore, FG-1Adn was found to co-localize with Scp1 in cells, especially on the plasma membrane. In addition, Western blotting analysis showed that FG-1Adn increased the phosphorylation level of the target protein of Scp1 in cells, indicating that FG-1Adn can inhibit the function of Scp1. These results suggest that FG-1Adn can be used as a specific inhibitor of Scp1.
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  • 文章类型: Journal Article
    背景:含纤维连接蛋白III型结构域3B(FNDC3B),含纤连蛋白III型结构域的蛋白质家族的成员,已在各种恶性肿瘤中显示。然而,FNDC3B在胰腺癌(PC)进展中的确切作用仍有待阐明.
    方法:在本研究中,我们整合了国家生物技术信息中心的数据,癌症基因组图谱,基因型-组织表达数据库,和基因表达综合数据集分析FNDC3B表达及其与各种临床病理参数的关联。随后,基因本体论和京都基因和基因组百科全书,随着基因集富集分析(GSEA),单样品基因组富集分析(ssGSEA)和估计分析被招募以深入研究基于FNDC3B表达的生物学功能和免疫浸润。此外,采用Cox分析和Kaplan-Meier分析进行预后评估.随后,根据Cox分析结果构建列线图以增强FNDC3B的预后能力。最后,初步探讨了FNDC3B在PC细胞中的生物学功能。
    结果:研究表明,与正常胰腺组织相比,FNDC3B在肿瘤组织中的表达明显更高,这种表达与各种临床病理参数显着相关。GSEA揭示了FNDC3B参与与整合素信号通路和细胞粘附相关的生物过程和信号通路。此外,ssGSEA分析显示FNDC3B表达与Th2细胞和中性粒细胞浸润呈正相关,而与浆细胞样树突状细胞和Th17细胞浸润呈负相关。Kaplan-Meier分析进一步支持PC患者中FNDC3B高表达与较短的总生存期有关。疾病特异性生存,和无进展间隔。然而,尽管单因素分析显示FNDC3B表达与PC患者预后之间存在显著相关性,这种关联在多变量分析中不成立.最后,我们的发现强调了FNDC3B表达在调节增殖中的关键作用,迁移,和PC细胞的侵袭能力。
    结论:尽管存在局限性,这项研究的结果强调了FNDC3B作为预后生物标志物的潜力及其在推动PC进展中的关键作用,特别是在协调免疫反应方面。
    BACKGROUND: Fibronectin type III domain containing 3B (FNDC3B), a member of the fibronectin type III domain-containing protein family, has been indicated in various malignancies. However, the precise role of FNDC3B in the progression of pancreatic cancer (PC) still remains to be elucidated.
    METHODS: In this study, we integrated data from the National Center for Biotechnology Information, the Cancer Genome Atlas, Genotype-Tissue Expression database, and Gene Expression Omnibus datasets to analyze FNDC3B expression and its association with various clinicopathological parameters. Subsequently, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, along with Gene Set Enrichment Analysis (GSEA), single sample Gene Set Enrichment Analysis (ssGSEA) and estimate analysis were recruited to delve into the biological function and immune infiltration based on FNDC3B expression. Additionally, the prognostic estimation was conducted using Cox analysis and Kaplan-Meier analysis. Subsequently, a nomogram was constructed according to the result of Cox analysis to enhance the prognostic ability of FNDC3B. Finally, the preliminary biological function of FNDC3B in PC cells was explored.
    RESULTS: The study demonstrated a significantly higher expression of FNDC3B in tumor tissues compared to normal pancreatic tissues, and this expression was significantly associated with various clinicopathological parameters. GSEA revealed the involvement of FNDC3B in biological processes and signaling pathways related to integrin signaling pathway and cell adhesion. Additionally, ssGSEA analysis indicated a positive correlation between FNDC3B expression and infiltration of Th2 cells and neutrophils, while showing a negative correlation with plasmacytoid dendritic cells and Th17 cells infiltration. Kaplan-Meier analysis further supported that high FNDC3B expression in PC patients was linked to shorter overall survival, disease-specific survival, and progression-free interval. However, although univariate analysis demonstrated a significant correlation between FNDC3B expression and prognosis in PC patients, this association did not hold true in multivariate analysis. Finally, our findings highlight the crucial role of FNDC3B expression in regulating proliferation, migration, and invasion abilities of PC cells.
    CONCLUSIONS: Despite limitations, the findings of this study underscored the potential of FNDC3B as a prognostic biomarker and its pivotal role in driving the progression of PC, particularly in orchestrating immune responses.
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  • 文章类型: Journal Article
    目的:能够测量肿瘤中PD-L1状态的当天PET显像剂是优化PD-1和PD-L1治疗的重要工具。在这里,我们描述了小说的发现和评价,用于成像PD-L1的基于氟-18标记的大环肽的PET配体。
    方法:[18F]通过铜介导的点击化学合成BMS-986229,以产生具有皮摩尔亲和力的PD-L1PET配体,并作为评估PD-L1表达的体内工具进行测试。
    结果:放射自显影显示L2987(PD-L1())与HT-29(PD-L1(-))肿瘤组织,具有>90%的特异性结合。在人非小细胞肺癌(NSCLC)和食蟹猴脾组织中观察到特异性放射性配体结合(>90%)。灵长类动物PD-L1(+)组织的图像具有高信噪比,在非表达组织中具有低背景信号。PET成像可以清晰地显示体内小鼠模型中PD-L1的表达,L2987(PD-L1(+))的摄取比对照HT-29(PD-L1(-))肿瘤高5倍。此外,该显像剂用于测量PD-L1抑制剂(肽或mAb)的靶标接合,在PD-L1(+)肿瘤中高达97%。
    结论:开发了一种新型的18F标记的大环肽放射性配体,用于PD-L1表达组织的PET成像,当与基于adnectin或mAb的配体直接比较时,在非人灵长类动物模型中显示出一些优势。临床研究目前正在评估[18F]BMS-986229以测量肿瘤中的PD-L1表达。
    OBJECTIVE: A same-day PET imaging agent capable of measuring PD-L1 status in tumors is an important tool for optimizing PD-1 and PD-L1 treatments. Herein we describe the discovery and evaluation of a novel, fluorine-18 labeled macrocyclic peptide-based PET ligand for imaging PD-L1.
    METHODS: [18F]BMS-986229 was synthesized via copper mediated click-chemistry to yield a PD-L1 PET ligand with picomolar affinity and was tested as an in-vivo tool for assessing PD-L1 expression.
    RESULTS: Autoradiography showed an 8:1 binding ratio in L2987 (PD-L1 (+)) vs. HT-29 (PD-L1 (-)) tumor tissues, with >90% specific binding. Specific radioligand binding (>90%) was observed in human non-small-cell lung cancer (NSCLC) and cynomolgus monkey spleen tissues. Images of PD-L1 (+) tissues in primates were characterized by high signal-to-noise, with low background signal in non-expressing tissues. PET imaging enabled clear visualization of PD-L1 expression in a murine model in vivo, with 5-fold higher uptake in L2987 (PD-L1 (+)) than in control HT-29 (PD-L1 (-)) tumors. Moreover, this imaging agent was used to measure target engagement of PD-L1 inhibitors (peptide or mAb), in PD-L1 (+) tumors as high as 97%.
    CONCLUSIONS: A novel 18F-labeled macrocyclic peptide radioligand was developed for PET imaging of PD-L1 expressing tissues that demonstrated several advantages within a nonhuman primate model when compared directly to adnectin- or mAb-based ligands. Clinical studies are currently evaluating [18F]BMS-986229 to measure PD-L1 expression in tumors.
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  • 文章类型: Journal Article
    Titin是自然界中发现的最大的蛋白质,跨越脊椎动物横纹肌中的半个肌节。蛋白质有多种功能,包括粗丝的组织,并在肌肉收缩周期中充当分子弹簧。肌动蛋白的错义变体与心脏和骨骼肌病有关。Titin主要由多种重复模式的免疫球蛋白和纤连蛋白III型(Fn3)结构域的串联重复组成;然而,这些域中的绝大多数尚未通过实验确定其高分辨率结构。这里,我们介绍了7个野生型titinFn3结构域和2个在肥厚型心肌病(HCM)患者中报道的罕见错义变异体的晶体结构.所有结构域都呈现典型的Fn3折叠,HCM患者报告的包含变体的结构域保留野生型构象。评估了在HCM患者中发现的五种罕见错义变体对域折叠和稳定性的影响:四种导致7至13°C之间的热失稳,一种阻止了其结构域的折叠。该结构还使我们能够定位其突变与先天性肌病有关的残基的位置,并合理化它们如何传达其有害作用。我们没有发现生理同源二聚体形成的证据,排除了一种假设的titin变体如何发挥病理作用的机制。
    Titin is the largest protein found in nature and spans half a sarcomere in vertebrate striated muscle. The protein has multiple functions, including in the organisation of the thick filament and acting as a molecular spring during the muscle contraction cycle. Missense variants in titin have been linked to both cardiac and skeletal myopathies. Titin is primarily composed of tandem repeats of immunoglobulin and fibronectin type III (Fn3) domains in a variety of repeat patterns; however, the vast majority of these domains have not had their high-resolution structure determined experimentally. Here, we present the crystal structures of seven wild type titin Fn3 domains and two harbouring rare missense variants reported in hypertrophic cardiomyopathy (HCM) patients. All domains present the typical Fn3 fold, with the domains harbouring variants reported in HCM patients retaining the wild-type conformation. The effect on domain folding and stability were assessed for five rare missense variants found in HCM patients: four caused thermal destabilization of between 7 and 13 °C and one prevented the folding of its domain. The structures also allowed us to locate the positions of residues whose mutations have been linked to congenital myopathies and rationalise how they convey their deleterious effects. We find no evidence of physiological homodimer formation, excluding one hypothesised mechanism as to how titin variants could exert pathological effects.
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  • 文章类型: Journal Article
    合成结合蛋白是使用非抗体蛋白作为起始支架的人制造的结合蛋白。分子显示技术,如噬菌体展示,能够构建大型组合库及其高效排序,因此,对合成结合蛋白的发展至关重要。单体是一组基于III型纤连蛋白(FN3)结构域的合成结合蛋白的基础系统。自1998年的原始报告以来,单体和相关的基于FN3的系统已经稳步完善,和目前的方法能够快速产生有效和选择性的结合分子,甚至具有挑战性的目标。FN3结构域是小的(~90个氨基酸)和自主的,在结构上与常规免疫球蛋白(Ig)结构域相似。与Ig域不同,然而,FN3缺乏二硫键但高度稳定。FN3的这些属性在噬菌体和其他展示系统的设计中提出了独特的机遇和挑战。组合库,和图书馆排序策略。本文回顾了我们建立单体开发管道的关键技术创新,重点是噬菌体展示方法。这些可以深入了解分子展示技术和蛋白质-蛋白质相互作用的分子机制,其应当广泛地适用于旨在产生高性能结合蛋白的多种系统。
    Synthetic binding proteins are human-made binding proteins that use non-antibody proteins as the starting scaffold. Molecular display technologies, such as phage display, enable the construction of large combinatorial libraries and their efficient sorting and, thus, are crucial for the development of synthetic binding proteins. Monobodies are the founding system of a set of synthetic binding proteins based on the fibronectin type III (FN3) domain. Since the original report in 1998, the monobody and related FN3-based systems have steadily been refined, and current methods are capable of rapidly generating potent and selective binding molecules to even challenging targets. The FN3 domain is small (∼90 amino acids) and autonomous and is structurally similar to the conventional immunoglobulin (Ig) domain. Unlike the Ig domain, however, the FN3 lacks a disulfide bond but is highly stable. These attributes of FN3 present unique opportunities and challenges in the design of phage and other display systems, combinatorial libraries, and library sorting strategies. This article reviews key technological innovations in the establishment of our monobody development pipeline, with an emphasis on phage display methodology. These give insights into the molecular mechanisms underlying molecular display technologies and protein-protein interactions, which should be broadly applicable to diverse systems intended for generating high-performance binding proteins.
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  • 文章类型: Journal Article
    本研究旨在探讨含纤维连接蛋白III型结构域1(FNDC1)在非小细胞肺癌(NSCLC)中的作用。以及控制其表达的机制。通过qRT-PCR检测组织和细胞样品中FNDC1及相关基因的表达水平。采用Kaplan-Meier分析FNDC1水平与NSCLC患者总生存期的相关性。功能实验,如CCK-8增殖,菌落形成,EDU染色,进行迁移和侵袭实验以研究FNDC1在调节NSCLC细胞恶性程度中的功能作用。使用生物信息学工具和双荧光素酶报告基因测定来鉴定NSCLC细胞中FNDC1的miRNA调节因子。我们的数据揭示了FNDC1在NSCLC肿瘤组织癌细胞系中mRNA和蛋白质水平的上调,与正常同行相比。FNDC1表达较高的NSCLC患者总生存期较差。FNDC1敲低显著抑制增殖,NSCLC细胞的迁移和侵袭,对管的形成有抑制作用。我们进一步证明miR-143-3p是FNDC1的上游调节因子,并且miR-143-3p表达在NSCLC样品中被抑制。类似于FNDC1击倒,miR-143-3p过表达抑制生长,NSCLC细胞的迁移和侵袭。FNDC1过表达可以部分挽救miR-143-3p过表达的作用。FNDC1沉默也抑制了小鼠模型中NSCLC细胞的肿瘤发生。总之,FNDC1促进NSCLC细胞的恶性原型。miR-143-3p是NSCLC细胞中FNDC1的负调节因子,这可能是NSCLC的一个有希望的治疗靶点。
    The study aimed to explore the functional role of fibronectin type III domain containing 1 (FNDC1) in nonsmall cell lung cancer (NSCLC), as well as the mechanism governing its expression. The expression levels of FNDC1 and related genes in tissue and cell samples were detected by qRT-PCR. Kaplan-Meier analysis was employed to analyze the association between FNDC1 level and the overall survival of NSCLC patients. Functional experiments such as CCK-8 proliferation, colony formation, EDU staining, migration and invasion assays were conducted to investigate the functional role of FNDC1 in regulating the malignancy of NSCLC cells. Bioinformatic tools and dual-luciferase reporter assay were used to identify the miRNA regulator of FNDC1 in NSCLC cells. Our data revealed the upregulation of FNDC1 at mRNA and protein levels in NSCLC tumor tissues cancer cell lines, compared with normal counterparts. NSCLC patients with higher FNDC1 expression suffered from a poorer overall survival. FNDC1 knockdown significantly suppressed the proliferation, migration and invasion of NSCLC cells, and had an inhibitory effect on tube formation. We further demonstrated that miR-143-3p was an upstream regulator of FNDC1 and miR-143-3p expression was repressed in NSCLC samples. Similar to FNDC1 knockdown, miR-143-3p overexpression inhibited the growth, migration and invasion of NSCLC cells. FNDC1 overexpression could partially rescue the effect of miR-143-3p overexpression.  FNDC1 silencing also suppressed the tumorigenesis of NSCLC cells in mouse model. In conclusion, FNDC1 promotes the malignant prototypes of NSCLC cells. miR-143-3p is a negative regulator of FNDC1 in NSCLC cells, which may serve as a promising therapeutic target in NSCLC.
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  • 文章类型: Journal Article
    哺乳动物分泌的铁蛋白已经有很好的记录,具有能够定位到细胞膜并促进通过内吞作用将铁输送到细胞的蛋白质。然而,软体动物循环液中铁蛋白的存在及其功能在很大程度上仍然未知。在这项研究中,我们的目的是通过使用下拉测定法来研究阿克蛤中铁蛋白(SbFn)的潜在相互作用蛋白。我们的发现揭示了一种胰岛素样生长因子1型受体(IGF-1R)的存在,其能够与SbFn结合并被命名为SbIGF-1R。发现SbIGF-1R由两个富含亮氨酸的重复结构域(L结构域)组成,富含半胱氨酸的结构域,三个纤连蛋白III型结构域,跨膜结构域,和酪氨酸激酶结构域。观察到SbIGF-1R的胞外域以字母\'A\'的形状形成对称的反平行同源二聚体,纤连蛋白III型结构域作为其“腿”。SbIGF-1R基因的mRNA表达在龙葵的各种组织中普遍存在,在血细胞中的表达水平最高,如通过qRT-PCR确定的。使用共聚焦显微镜和酵母双杂交测定法,进一步验证了SbIGF-1R与SbFn的相互作用。结果表明,SbFn与SbIGF-1R共定位在细胞膜上,并且它们的相互作用预计发生在SbIGF-1R的FNIII结构域上。总之,我们的发现强调了推定受体的鉴定,SbIGF-1R,对于SbFn,证明IGF-1R在龙雀中的多功能性。
    The ferritin secreted by mammals has been well documented, with the protein capable of localizing to cell membranes and facilitating the delivery of iron to cells through endocytosis. However, the presence of ferritin in the circulatory fluid of mollusks and its functions remain largely unknown. In this study, we aimed to investigate the potential interacting proteins of ferritin in the ark clam (SbFn) through the use of a pull-down assay. Our findings revealed the presence of an insulin-like growth factor type 1 receptor (IGF-1R) in ark clams, which was capable of binding to SbFn and was named SbIGF-1R. SbIGF-1R was found to be composed of two leucine-rich repeat domains (L domain), a cysteine-rich domain, three fibronectin type III domains, a transmembrane domain, and a tyrosine kinase domain. The ectodomain of SbIGF-1R was observed to form a symmetrical antiparallel homodimer in the shape of the letter \'A\', with the fibronectin type III domains serving as its \'legs\'. The mRNA expression of SbIGF-1R gene was detected ubiquitously in various tissues of the ark clam, with the highest expression levels found in hemocytes, as determined by qRT-PCR. Using a confocal microscopic and yeast two-hybrid assays, the interaction between SbIGF-1R and SbFn was further verified. The results showed that SbFn co-localized with SbIGF-1R on the cell membrane, and their interaction was expected to occur on the FNIII domains of the SbIGF-1R. In conclusion, our findings highlight the identification of a putative receptor, SbIGF-1R, for SbFn, demonstrating the versatility of IGF-1R in ark clams.
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  • 文章类型: Journal Article
    化疗耐药是肝癌治疗中的一个巨大挑战,因为对nab-紫杉醇的耐药在很大程度上影响化疗的疗效。含纤维连接蛋白III型结构域蛋白5(FNDC5)在肝细胞癌细胞中的表达增加可以预测肝细胞癌患者肝切除术后的并发症,也可以刺激肝细胞癌细胞的增殖和侵袭;然而,它在肝癌细胞化疗中的作用从未被评估过。因此,本研究旨在探讨FNDC5是否调控肝细胞癌的化疗耐药。我们通过免疫组织化学鉴定肝细胞癌组织比与癌细胞相邻的正常组织具有更高的FNDC5表达。随后,肝癌细胞中FNDC5的敲减导致其在nab-紫杉醇化疗后对细胞死亡的抵抗力降低.相比之下,FNDC5在肝癌细胞中的过表达增加了肝癌细胞对治疗的抗性。此外,FNDC5通过AMPK/mTOR信号通路促进自噬,从而减少nab-紫杉醇诱导的细胞死亡。最后,我们通过动物实验验证了我们的假设。总之,FNDC5可作为预测肝癌化疗疗效的生物标志物nab-紫杉醇化疗,并以此作为治疗靶点克服肝癌化疗中对nab-紫杉醇的耐药性。
    Chemotherapy resistance is a huge challenge in the treatment of hepatocellular carcinoma because resistance to nab-paclitaxel largely affects the efficacy of chemotherapy. An increased expression of fibronectin type III domain-containing protein 5 (FNDC5) in hepatocellular carcinoma cells can predict post-hepatectomy complications in patients with hepatocellular carcinoma and also stimulate proliferation and invasion of hepatocellular carcinoma cells; however, its role in the chemotherapy of hepatocellular carcinoma cells has never been evaluated. Thus, this study aimed to explore whether FNDC5 regulates chemoresistance in hepatocellular carcinoma. We identified by immunohistochemistry that hepatocellular carcinoma tissues had a higher FNDC5 expression than normal tissues adjacent to the cancer cells. Subsequently, knockdown of FNDC5 in hepatocellular carcinoma cells resulted in their diminished resistance to cell death after chemotherapy with nab-paclitaxel. By contrast, overexpression of FNDC5 in hepatocellular carcinoma cells increased the resistance of hepatocellular carcinoma cells to treatment. Moreover, FNDC5 mechanistically promoted autophagy via the AMPK/mTOR signaling pathway, thereby reducing cell death induced by nab-paclitaxel. Finally, we tested our hypothesis by conducting animal experiments. In conclusion, FNDC5 could be used as a biomarker for predicting chemotherapeutic efficacy in hepatocellular carcinoma treated with nab-paclitaxel chemotherapy, and as a therapeutic target to overcome resistance to nab-paclitaxel in hepatocellular carcinoma chemotherapy.
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  • 文章类型: Journal Article
    微生物Vpr样蛋白酶是具有多种功能的细胞外多结构域枯草杆菌酶,可以形成寡聚体,但它们的成熟和寡聚化机制仍有待阐明。这里,我们报告了一种来自嗜热细菌短芽孢杆菌的新型Vpr样蛋白酶(BTV)。WF146.BTV前体包含信号肽,N端前肽,具有插入的蛋白酶相关(PA)结构域的枯草杆菌蛋白酶样催化结构域,两个串联的纤连蛋白III型结构域(Fn1和Fn2),和C端前肽。BTV原形(pro-BTV)可以通过两个缺少N-或C-末端前肽的中间体自动加工成成熟形式(mBTV),分别,C端前肽延迟了酶的自催化成熟。相比之下,pro-BTV通过来自菌株WF146的蛋白酶TSS更有效地加工成mBTV。纯化的mBTV是一种依赖Ca2+的热稳定蛋白酶,在60°C下显示最佳活性,并在60°C下孵育8小时后保留60%以上的活性。PA结构域对于酶的稳定性很重要,并通过限制蛋白质底物进入活性位点而有助于BTV的底物特异性。BTV的原形和成熟形式作为单体和同源二聚体存在,分别,二聚化由Fn1和Fn2结构域介导。BTV的N端前肽不仅充当分子内伴侣和酶抑制剂,而且还抑制酶的同二聚化。N-末端前肽的去除导致酶的结构调整,从而促进酶的二聚化。重要性Vpr样蛋白酶广泛分布于细菌和真菌中,并参与处理单抗生素,降解胶原蛋白,角蛋白,和纤维蛋白,和微生物的发病机理。解剖单个结构域在酶成熟和寡聚化中的作用对于理解这些多结构域蛋白酶的作用机制至关重要。我们的结果表明,短杆菌的细胞外Vpr样蛋白酶BTV的异催化成熟。WF146比酶的自催化成熟更有效。此外,我们发现,C端串联纤连蛋白III型结构域而不是PA结构域介导成熟BTV的二聚化,而N端前肽抑制BTV原聚体的二聚化。这项研究为Vpr样蛋白酶的激活和寡聚化机制提供了新的见解。
    Microbial Vpr-like proteases are extracellular multidomain subtilases with diverse functions and can form oligomers, but their maturation and oligomerization mechanisms remain to be elucidated. Here, we report a novel Vpr-like protease (BTV) from thermophilic bacterium Brevibacillus sp. WF146. The BTV precursor comprises a signal peptide, an N-terminal propeptide, a subtilisin-like catalytic domain with an inserted protease-associated (PA) domain, two tandem fibronectin type III domains (Fn1 and Fn2), and a C-terminal propeptide. The BTV proform (pro-BTV) could be autoprocessed into the mature form (mBTV) via two intermediates lacking the N- or C-terminal propeptide, respectively, and the C-terminal propeptide delays the autocatalytic maturation of the enzyme. By comparison, pro-BTV is more efficiently processed into mBTV by protease TSS from strain WF146. Purified mBTV is a Ca2+-dependent thermostable protease, showing optimal activity at 60°C and retaining more than 60% of activity after incubation at 60°C for 8 h. The PA domain is important for enzyme stability and contributes to the substrate specificity of BTV by restricting the access of protein substrates to the active site. The proform and mature form of BTV exist as a monomer and a homodimer, respectively, and the dimerization is mediated by the Fn1 and Fn2 domains. The N-terminal propeptide of BTV not only acts as intramolecular chaperone and enzymatic inhibitor but also inhibits the homodimerization of the enzyme. The removal of the N-terminal propeptide leads to a structural adjustment of the enzyme and thus promotes enzyme dimerization. IMPORTANCE Vpr-like proteases are widely distributed in bacteria and fungi and are involved in processing lantibiotics, degrading collagen, keratin, and fibrin, and pathogenesis of microbes. The dissection of the roles of individual domains in enzyme maturation and oligomerization is crucial for understanding the action mechanisms of these multidomain proteases. Our results demonstrate that hetero-catalytic maturation of the extracellular Vpr-like protease BTV of Brevibacillus sp. WF146 is more efficient than autocatalytic maturation of the enzyme. Moreover, we found that the C-terminal tandem fibronectin type III domains rather than the PA domain mediate the dimerization of mature BTV, while the N-terminal propeptide inhibits the dimerization of the BTV proform. This study provides new insight into the activation and oligomerization mechanisms of Vpr-like proteases.
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