背景:研究证实,含血小板反应蛋白模体的金属蛋白酶与整合素前体8(A disintegrin and metalloproteinase with thrombospondin motifs 8,ADAMTS8)对纤维化具有一定的调控作用,因此,深入探讨ADAMTS8对增生性瘢痕的作用机制具有重要临床意...背景:研究证实,含血小板反应蛋白模体的金属蛋白酶与整合素前体8(A disintegrin and metalloproteinase with thrombospondin motifs 8,ADAMTS8)对纤维化具有一定的调控作用,因此,深入探讨ADAMTS8对增生性瘢痕的作用机制具有重要临床意义。目的:探讨ADAMTS8对增生性瘢痕的调控作用。方法:①免疫组化染色检测人体正常皮肤组织与增生性瘢痕组织中Ⅰ型胶原、Ⅲ型胶原、α-平滑肌肌动蛋白及ADAMTS8的表达。Western blot检测人体正常皮肤组织与增生性瘢痕组织中ADAMTS8蛋白表达,以增生性瘢痕为阳性样本,正常皮肤为阴性样本,绘制受试者工作特征曲线,分析ADAMTS8预测区分正常皮肤与增生性瘢痕的能力。②通过STRING 12.0平台对ADAMTS8构建蛋白质-蛋白质相互作用网络,对获取的靶点进行GO功能富集和KEGG通路富集分析。③分离培养人增生性瘢痕组织成纤维细胞,将第3-6代成纤维细胞分3组处理:对照组常规培养,Ad-NC组转染空载体腺病毒,Ad-ADAMTS8组转染含ADAMTS8过表达的腺病毒,CCK-8法、EdU染色检测细胞增殖活力,流式细胞术、TUNEL染色检测细胞凋亡情况。结果与结论:①免疫组化染色显示,增生性瘢痕中Ⅰ型胶原、Ⅲ型胶原、α-平滑肌肌动蛋白表达高于正常皮肤(P<0.001),ADAMTS8表达低于正常皮肤(P<0.001)。Western blot检测显示,增生性瘢痕中ADAMTS8蛋白表达低于正常皮肤(P<0.001)。受试者工作特征曲线显示,ADAMTS8预测发生增生性瘢痕的曲线下面积为0.86,说明ADAMTS8具有良好区分增生性瘢痕和正常皮肤的能力。②通过STRING数据库筛选出排名前41的基因,KEGG富集显示ADAMTS8主要参与细胞外基质受体相互作用、磷脂酰肌醇-3-激酶-蛋白激酶B信号通路及胞葬作用等生物学过程和关键机制;GO富集显示ADAMTS8参与凋亡相关的通路富集,包括成纤维细胞生长因子受体信号通路的负向调控、成纤维细胞生长因子的结合、细胞凋亡的负向调控和细胞凋亡过程。③CCK-8法、EdU染色显示,过表达ADAMTS8可抑制增生性瘢痕成纤维细胞增殖;流式细胞术、TUNEL染色显示,过表达ADAMTS8可促进增生性瘢痕成纤维细胞凋亡。④结果表明,ADAMTS8在人增生性瘢中表达降低,ADAMTS8过表达可抑制增生性瘢痕成纤维细胞增殖、促进增生性瘢痕成纤维细胞凋亡。展开更多
目的探究同型半胱氨酸(homocysteine,Hcy)调控血小板反应蛋白解整合素金属肽酶8(a disintegrin and metalloproteinase with thrombospondin type 1 motif 8,ADAMTS8)介导铁死亡在动脉粥样硬化(atherosclerosis,AS)中的作用及其机制。...目的探究同型半胱氨酸(homocysteine,Hcy)调控血小板反应蛋白解整合素金属肽酶8(a disintegrin and metalloproteinase with thrombospondin type 1 motif 8,ADAMTS8)介导铁死亡在动脉粥样硬化(atherosclerosis,AS)中的作用及其机制。方法将ApoE^(-/-)小鼠(n=12)随机分为2组,设立正常饮食组(ND组,n=6)和高蛋氨酸饮食组(HMD组,n=6);体外培养RAW264.7巨噬细胞,设立对照组(Control组,0μmol·L^(-1) Hcy)和Hcy干预组(Hcy组,100μmol·L^(-1) Hcy),Hcy干预的巨噬细胞转染腺病毒后设置ADAMTS8过表达阴性对照组(Hcy+Ad-NC)和ADAMTS8过表达组(Hcy+Ad-ADAMTS8),Hcy干预的巨噬细胞转染干扰片段后设置干扰片段对照组(Hcy+si-NC)、ADAMTS8干扰片段组(Hcy+si-ADAMTS8),Hcy干预的巨噬细胞ADAMTS8过表达组(Hcy+Ad-ADAMTS8)和Hcy干预的巨噬细胞ADAMTS8过表达+铁死亡抑制剂组(Fer-1+Hcy+Ad-ADAMTS8);免疫荧光染色观察ADAMTS8、xCT、ACSL4表达差异;Pearson相关系数分析ADAMTS8与铁死亡相关指标xCT、ACSL4的相关性;Western blot检测铁死亡指标xCT、ACSL4表达变化;丙二醛(malondialdehyde,MDA)和超氧化物歧化酶(superoxide dismutase,SOD)活性评估细胞氧化水平及抗氧化能力。结果与ND相比,HMD组ADAMTS8表达明显增高,铁死亡相关蛋白xCT的表达水平下降,而ACSL4表达水平明显上升;与Control组相比,Hcy组中ADAMTS8蛋白表达增高,xCT蛋白表达降低,而ACSL4蛋白表达升高,MDA水平升高而SOD活性明显降低;相关性分析显示ADAMTS8表达水平与xCT表达水平呈负相关,与ACSL4表达水平呈正相关;与Hcy+si-NC组相比,Hcy+si-ADAMTS8组中xCT蛋白表达水平明显升高,ACSL4表达量降低,MDA水平下降,SOD活性增强;相反,与Hcy+Ad-NC组相比,Hcy+Ad-ADAMTS8组中xCT蛋白表达水平降低,ACSL4表达量增加,MDA水平增强,SOD活性下降;与Hcy+Ad-ADAMTS8组相比,Fer-1+Hcy+Ad-ADAMTS8组中xCT蛋白表达水平升高,ACSL4表达量降低,MDA水平下降,SOD活性增强。结论ADAMTS8调控铁死亡影响巨噬细胞内氧化与抗氧化平衡,进而促进Hcy致AS的发生发展。展开更多
AIM:To investigate the genetic basis of Weill-Marchesani syndrome(WMS)in a Chinese family and clarify the pathogenic mechanism of novel ADAMTS17 mutations.METHODS:Comprehensive clinical assessments and genetic analyse...AIM:To investigate the genetic basis of Weill-Marchesani syndrome(WMS)in a Chinese family and clarify the pathogenic mechanism of novel ADAMTS17 mutations.METHODS:Comprehensive clinical assessments and genetic analyses were performed on a Chinese family with two affected siblings.Whole-exome sequencing(WES)was conducted for the proband and other family members.Bioinformatics tools were used to evaluate the conservation,predicted pathogenicity,and structural effects of the identified ADAMTS17 variants.In addition,protein structure modeling was applied to assess the functional impacts of the mutations.RESULTS:The proband(a 32-year-old male)and his elder sister(42y)presented typical clinical features of WMS,including short stature,brachydactyly,high myopia,ectopia lentis,and secondary glaucoma.WES identified a novel compound heterozygous mutation in ADAMTS17:a splicing mutation(c.451-2A>G)inherited from the father and a missense mutation(c.1043G>A;p.C348Y)inherited from the mother.The splicing mutation disrupted normal mRNA splicing and processing,leading to premature translation termination.The missense mutation,which is located in the metalloprotease catalytic domain,was predicted to abolish a critical disulfide bond,thereby impairing protein stability.Both mutations exhibited high evolutionary conservation and were predicted to be pathogenic by multiple bioinformatics algorithms.CONCLUSION:A novel compound heterozygous mutation in ADAMTS17 is identified in this WMS-affected Chinese family,and its pathogenicity is verified via bioinformatics analysis and protein structural modeling.These findings are expected to facilitate the genetic diagnosis of WMS and deepen the understanding of its molecular pathogenesis.展开更多
骨关节炎(OA)是一种普遍的退行性关节疾病,其特征是软骨退化和关节炎症。血小板结合蛋白基序的解聚蛋白样金属蛋白酶(a disintegrin and metalloproteinase with thrombospondin motifs,AD⁃AMTS)家族成员在OA的发病机制中受到了广泛关注...骨关节炎(OA)是一种普遍的退行性关节疾病,其特征是软骨退化和关节炎症。血小板结合蛋白基序的解聚蛋白样金属蛋白酶(a disintegrin and metalloproteinase with thrombospondin motifs,AD⁃AMTS)家族成员在OA的发病机制中受到了广泛关注,尤其是ADAMTS⁃4和ADAMTS⁃5,它们降解软骨蛋白聚糖活性远高于其他成员。蛋白聚糖是ECM的重要组成部分,蛋白聚糖的降解导致软骨的结构和功能破坏,是OA发展的关键因素。本文综述了ADAMTS⁃4和ADAMTS⁃5的结构和功能,重点解析二者底物切割位点的序列选择性、三维构象偏好及翻译后修饰、变构效应,和微环境信号对其蛋白水解活性的多层次调控机制,同时回顾了国内外学者对其靶向药的研究及未来展望。展开更多
解整合素金属蛋白酶家族由膜锚定型和分泌型蛋白构成,分别是解整合素金属蛋白酶(a disintegrin and metalloproteinase, ADAMs)和具有血小板反应蛋基序的解整合素金属蛋白酶(a disintegrin-like and metalloproteinase with thrombospon...解整合素金属蛋白酶家族由膜锚定型和分泌型蛋白构成,分别是解整合素金属蛋白酶(a disintegrin and metalloproteinase, ADAMs)和具有血小板反应蛋基序的解整合素金属蛋白酶(a disintegrin-like and metalloproteinase with thrombospondin motifs, ADAMTS)。ADAMs和ADAMTS在多种肿瘤的发生、发展中发挥重要作用。在胃癌中,ADAMs和ADAMTS家族成员的异常表达与肿瘤的增殖、侵袭及转移密切相关,靶向ADAMs和ADAMTS的药物研发有望成为胃癌的有效的治疗手段。本文通过概述ADAMs和ADAMTS在胃癌中的研究进展,为其作为胃癌潜在治疗靶点提供理论依据。展开更多
文摘目的探究同型半胱氨酸(homocysteine,Hcy)调控血小板反应蛋白解整合素金属肽酶8(a disintegrin and metalloproteinase with thrombospondin type 1 motif 8,ADAMTS8)介导铁死亡在动脉粥样硬化(atherosclerosis,AS)中的作用及其机制。方法将ApoE^(-/-)小鼠(n=12)随机分为2组,设立正常饮食组(ND组,n=6)和高蛋氨酸饮食组(HMD组,n=6);体外培养RAW264.7巨噬细胞,设立对照组(Control组,0μmol·L^(-1) Hcy)和Hcy干预组(Hcy组,100μmol·L^(-1) Hcy),Hcy干预的巨噬细胞转染腺病毒后设置ADAMTS8过表达阴性对照组(Hcy+Ad-NC)和ADAMTS8过表达组(Hcy+Ad-ADAMTS8),Hcy干预的巨噬细胞转染干扰片段后设置干扰片段对照组(Hcy+si-NC)、ADAMTS8干扰片段组(Hcy+si-ADAMTS8),Hcy干预的巨噬细胞ADAMTS8过表达组(Hcy+Ad-ADAMTS8)和Hcy干预的巨噬细胞ADAMTS8过表达+铁死亡抑制剂组(Fer-1+Hcy+Ad-ADAMTS8);免疫荧光染色观察ADAMTS8、xCT、ACSL4表达差异;Pearson相关系数分析ADAMTS8与铁死亡相关指标xCT、ACSL4的相关性;Western blot检测铁死亡指标xCT、ACSL4表达变化;丙二醛(malondialdehyde,MDA)和超氧化物歧化酶(superoxide dismutase,SOD)活性评估细胞氧化水平及抗氧化能力。结果与ND相比,HMD组ADAMTS8表达明显增高,铁死亡相关蛋白xCT的表达水平下降,而ACSL4表达水平明显上升;与Control组相比,Hcy组中ADAMTS8蛋白表达增高,xCT蛋白表达降低,而ACSL4蛋白表达升高,MDA水平升高而SOD活性明显降低;相关性分析显示ADAMTS8表达水平与xCT表达水平呈负相关,与ACSL4表达水平呈正相关;与Hcy+si-NC组相比,Hcy+si-ADAMTS8组中xCT蛋白表达水平明显升高,ACSL4表达量降低,MDA水平下降,SOD活性增强;相反,与Hcy+Ad-NC组相比,Hcy+Ad-ADAMTS8组中xCT蛋白表达水平降低,ACSL4表达量增加,MDA水平增强,SOD活性下降;与Hcy+Ad-ADAMTS8组相比,Fer-1+Hcy+Ad-ADAMTS8组中xCT蛋白表达水平升高,ACSL4表达量降低,MDA水平下降,SOD活性增强。结论ADAMTS8调控铁死亡影响巨噬细胞内氧化与抗氧化平衡,进而促进Hcy致AS的发生发展。
文摘AIM:To investigate the genetic basis of Weill-Marchesani syndrome(WMS)in a Chinese family and clarify the pathogenic mechanism of novel ADAMTS17 mutations.METHODS:Comprehensive clinical assessments and genetic analyses were performed on a Chinese family with two affected siblings.Whole-exome sequencing(WES)was conducted for the proband and other family members.Bioinformatics tools were used to evaluate the conservation,predicted pathogenicity,and structural effects of the identified ADAMTS17 variants.In addition,protein structure modeling was applied to assess the functional impacts of the mutations.RESULTS:The proband(a 32-year-old male)and his elder sister(42y)presented typical clinical features of WMS,including short stature,brachydactyly,high myopia,ectopia lentis,and secondary glaucoma.WES identified a novel compound heterozygous mutation in ADAMTS17:a splicing mutation(c.451-2A>G)inherited from the father and a missense mutation(c.1043G>A;p.C348Y)inherited from the mother.The splicing mutation disrupted normal mRNA splicing and processing,leading to premature translation termination.The missense mutation,which is located in the metalloprotease catalytic domain,was predicted to abolish a critical disulfide bond,thereby impairing protein stability.Both mutations exhibited high evolutionary conservation and were predicted to be pathogenic by multiple bioinformatics algorithms.CONCLUSION:A novel compound heterozygous mutation in ADAMTS17 is identified in this WMS-affected Chinese family,and its pathogenicity is verified via bioinformatics analysis and protein structural modeling.These findings are expected to facilitate the genetic diagnosis of WMS and deepen the understanding of its molecular pathogenesis.
文摘骨关节炎(OA)是一种普遍的退行性关节疾病,其特征是软骨退化和关节炎症。血小板结合蛋白基序的解聚蛋白样金属蛋白酶(a disintegrin and metalloproteinase with thrombospondin motifs,AD⁃AMTS)家族成员在OA的发病机制中受到了广泛关注,尤其是ADAMTS⁃4和ADAMTS⁃5,它们降解软骨蛋白聚糖活性远高于其他成员。蛋白聚糖是ECM的重要组成部分,蛋白聚糖的降解导致软骨的结构和功能破坏,是OA发展的关键因素。本文综述了ADAMTS⁃4和ADAMTS⁃5的结构和功能,重点解析二者底物切割位点的序列选择性、三维构象偏好及翻译后修饰、变构效应,和微环境信号对其蛋白水解活性的多层次调控机制,同时回顾了国内外学者对其靶向药的研究及未来展望。
文摘解整合素金属蛋白酶家族由膜锚定型和分泌型蛋白构成,分别是解整合素金属蛋白酶(a disintegrin and metalloproteinase, ADAMs)和具有血小板反应蛋基序的解整合素金属蛋白酶(a disintegrin-like and metalloproteinase with thrombospondin motifs, ADAMTS)。ADAMs和ADAMTS在多种肿瘤的发生、发展中发挥重要作用。在胃癌中,ADAMs和ADAMTS家族成员的异常表达与肿瘤的增殖、侵袭及转移密切相关,靶向ADAMs和ADAMTS的药物研发有望成为胃癌的有效的治疗手段。本文通过概述ADAMs和ADAMTS在胃癌中的研究进展,为其作为胃癌潜在治疗靶点提供理论依据。
