Two DNA fragments encoding PDZ domain （21-110 residues） and BAR domain （ 150-360 residues） from PICK1 （1-416 residues） were amplified by PCR and then introduced into vectors, pET-32M and pMAL-e2X respectively to...Two DNA fragments encoding PDZ domain （21-110 residues） and BAR domain （ 150-360 residues） from PICK1 （1-416 residues） were amplified by PCR and then introduced into vectors, pET-32M and pMAL-e2X respectively to generate recombinant plasmids, pE-pdz and pM-bar. Having been separately transferred into the hosts E. coli BL21 and E. coli JM109, these two strains can express fusion proteins： His-tagged PDZ（PDZ domain） and maltose binding protein-BAR（ MBP-BAR domain） respectively, as confirmed by both SDS-PAGE and Wostem blotting. The interaction between these two domains is dose-dependence, as identified by a pull-down test. Moreover, it has been shown from the ELISA analysis that the actual amount of PDZ bound to MBP-BAR-amylose beads reaches （ 16 ± 0. 5）%, as calculated by the molar ratio of PDZ to MBP-BAR. In addition, the interaction between BAR（bait） and PDZ（prey） in vivo was also examined with a yeast two-hybrid system.展开更多
Pancreatic ductal adenocarcinoma(PDAC) remains a deadly disease with no efficacious treatment options. PDAC incidence is projected to increase, which may be caused at least partially by the obesity epidemic. Significa...Pancreatic ductal adenocarcinoma(PDAC) remains a deadly disease with no efficacious treatment options. PDAC incidence is projected to increase, which may be caused at least partially by the obesity epidemic. Significantly enhanced efforts to prevent or intercept this cancer are clearly warranted. Oncogenic KRAS mutations are recognized initiating events in PDAC development, however, they are not entirely sufficient for the development of fully invasive PDAC.Additional genetic alterations and/or environmental, nutritional, and metabolic signals, as present in obesity, type-2 diabetes mellitus, and inflammation, are required for full PDAC formation. We hypothesize that oncogenic KRAS increases the intensity and duration of the growth-promoting signaling network.Recent exciting studies from different laboratories indicate that the activity of the transcriptional co-activators Yes-associated protein(YAP) and WW-domaincontaining transcriptional co-activator with PDZ-binding motif(TAZ) play a critical role in the promotion and maintenance of PDAC operating as key downstream target of KRAS signaling. While initially thought to be primarily an effector of the tumor-suppressive Hippo pathway, more recent studies revealed that YAP/TAZ subcellular localization and co-transcriptional activity is regulated by multiple upstream signals. Overall, YAP has emerged as a central node of transcriptional convergence in growth-promoting signaling in PDAC cells. Indeed, YAP expression is an independent unfavorable prognostic marker for overall survival of PDAC. In what follows, we will review studies implicating YAP/TAZ in pancreatic cancer development and consider different approaches to target these transcriptional regulators.展开更多
目的分析双硫死亡(disulfidptosis)相关基因人PDZ和LIM域蛋白1(PDZ and LIM domain protein 1,PDLIM1)m RNA在多种肿瘤中的表达及作用。方法通过仙桃学术网站分析PDLIM1 mRNA的表达情况。利用仙桃学术网站和Sangerbox 3.0数据分析平台探...目的分析双硫死亡(disulfidptosis)相关基因人PDZ和LIM域蛋白1(PDZ and LIM domain protein 1,PDLIM1)m RNA在多种肿瘤中的表达及作用。方法通过仙桃学术网站分析PDLIM1 mRNA的表达情况。利用仙桃学术网站和Sangerbox 3.0数据分析平台探究PDLIM1在33种肿瘤中的诊断和预后能力。利用TISIDB数据库分析PDLIM1与临床分级和分期的相关性。在Sangerbox 3.0数据分析平台和Kaplan-Meier Plotter数据库中分析PDLIM1与肿瘤免疫相关性。通过STRING数据库和Cytoscape构建蛋白质互作网络(protein-protein interaction networks,PPI)。利用Sangerbox 3.0数据分析平台进行富集分析。最后利用GSCA(Gene Set Cancer Analysis)网站分析获得PDLIM1 mRNA表达与药物的敏感性。结果PDLIM1 mRNA在33种肿瘤中表达量存在异质性。PDLIM1在胆管癌(CHOL)、多形性胶质母细胞瘤(GBM)、肾透明细胞癌(KIRC)、肺腺癌(LUAD)、卵巢癌(OV)、胰腺癌(PAAD)、黑色素瘤(SKCM)和睾丸生殖细胞肿瘤(TGCT)中具有良好的诊断能力。PDLIM1在胶质瘤(GBMLGG)、脑低级别胶质瘤(LGG)、混合肾癌(KIPAN)、多形性胶质细胞瘤(GBM)、间皮瘤(MESO)、葡萄膜黑色素瘤(UVM)和肾上腺皮质癌(ACC)中高表达预后差,而在肉瘤中低表达预后差。PDLIM1 mRNA表达与头颈鳞状细胞癌(HIVSC)、肾乳头状细胞癌(KIRP)、子宫内膜癌(UCEC)、子宫癌肉瘤(UCS)和葡萄膜黑色素瘤(UVM)的分级,以及与宫颈癌、头颈鳞状细胞癌、子宫内膜癌和脑低级别胶质瘤肿瘤的分期有关。PDLIM1与以前列腺癌为首的36种肿瘤的免疫浸润显著相关,且发现在PDLIM1 mRNA高表达的患者中经免疫治疗后的预后相对较好。PDLIM1在生物体内主要通过参与肌动蛋白细胞骨架、细胞黏附、肿瘤相关途径的调节来发挥作用,对以Isoliquiritigenin为首的多种药物敏感。结论PDLIM1与多种肿瘤的临床预后和免疫浸润等方面密切相关,有望成为一种肿瘤诊断和预后生物标志物或治疗靶点。展开更多
PDZ连接激酶/T-LAK细胞源蛋白激酶(PDZ-binding-kinase/T-LAKcell-originated protein kinase,PBK/TOPK)是一种新近发现的丝-苏氨酸激酶,具有参与调控恶性肿瘤细胞的增殖和周期变化,促进肿瘤细胞转化,并且通过MAPKK信号通路参与调控细胞...PDZ连接激酶/T-LAK细胞源蛋白激酶(PDZ-binding-kinase/T-LAKcell-originated protein kinase,PBK/TOPK)是一种新近发现的丝-苏氨酸激酶,具有参与调控恶性肿瘤细胞的增殖和周期变化,促进肿瘤细胞转化,并且通过MAPKK信号通路参与调控细胞DNA损伤修复.特别是近期发现其具有强烈的细胞恶性诱导潜能而可能成为新的肿瘤治疗靶点.展开更多
基金the National Natural Science Foundation of China(No 30400065)
文摘Two DNA fragments encoding PDZ domain （21-110 residues） and BAR domain （ 150-360 residues） from PICK1 （1-416 residues） were amplified by PCR and then introduced into vectors, pET-32M and pMAL-e2X respectively to generate recombinant plasmids, pE-pdz and pM-bar. Having been separately transferred into the hosts E. coli BL21 and E. coli JM109, these two strains can express fusion proteins： His-tagged PDZ（PDZ domain） and maltose binding protein-BAR（ MBP-BAR domain） respectively, as confirmed by both SDS-PAGE and Wostem blotting. The interaction between these two domains is dose-dependence, as identified by a pull-down test. Moreover, it has been shown from the ELISA analysis that the actual amount of PDZ bound to MBP-BAR-amylose beads reaches （ 16 ± 0. 5）%, as calculated by the molar ratio of PDZ to MBP-BAR. In addition, the interaction between BAR（bait） and PDZ（prey） in vivo was also examined with a yeast two-hybrid system.
文摘Pancreatic ductal adenocarcinoma(PDAC) remains a deadly disease with no efficacious treatment options. PDAC incidence is projected to increase, which may be caused at least partially by the obesity epidemic. Significantly enhanced efforts to prevent or intercept this cancer are clearly warranted. Oncogenic KRAS mutations are recognized initiating events in PDAC development, however, they are not entirely sufficient for the development of fully invasive PDAC.Additional genetic alterations and/or environmental, nutritional, and metabolic signals, as present in obesity, type-2 diabetes mellitus, and inflammation, are required for full PDAC formation. We hypothesize that oncogenic KRAS increases the intensity and duration of the growth-promoting signaling network.Recent exciting studies from different laboratories indicate that the activity of the transcriptional co-activators Yes-associated protein(YAP) and WW-domaincontaining transcriptional co-activator with PDZ-binding motif(TAZ) play a critical role in the promotion and maintenance of PDAC operating as key downstream target of KRAS signaling. While initially thought to be primarily an effector of the tumor-suppressive Hippo pathway, more recent studies revealed that YAP/TAZ subcellular localization and co-transcriptional activity is regulated by multiple upstream signals. Overall, YAP has emerged as a central node of transcriptional convergence in growth-promoting signaling in PDAC cells. Indeed, YAP expression is an independent unfavorable prognostic marker for overall survival of PDAC. In what follows, we will review studies implicating YAP/TAZ in pancreatic cancer development and consider different approaches to target these transcriptional regulators.
文摘PDZ连接激酶/T-LAK细胞源蛋白激酶(PDZ-binding-kinase/T-LAKcell-originated protein kinase,PBK/TOPK)是一种新近发现的丝-苏氨酸激酶,具有参与调控恶性肿瘤细胞的增殖和周期变化,促进肿瘤细胞转化,并且通过MAPKK信号通路参与调控细胞DNA损伤修复.特别是近期发现其具有强烈的细胞恶性诱导潜能而可能成为新的肿瘤治疗靶点.
基金Supported by National Basic Research Program of China(973 Program,No.2014CB910200)National Natural Science Foundation of China(No.81370522 and No.31400647)+1 种基金Guangdong Natural Science Foundation(No.S2012010008170)Specialized Research Fund for the Doctoral Program of Higher Education(No.20120001120096)~~
文摘多重PDZ结构域蛋白1型(MUPP1)是一种存在于上皮细胞和神经细胞内含有13个PDZ结构域的重要支架蛋白.在上皮细胞中,MUPP1蛋白在紧密连接结构的形成和上皮细胞的极化过程中发挥重要作用.而在中枢神经系统中,MUPP1基因的1个提前终止突变导致了其最后12个PDZ结构域的缺失,以及严重的先天性脑积水.此外,MUPP1蛋白的表达水平与酒精依赖性和药物戒断的严重性也具有显著的相关性.因此,对MUPP1蛋白所含的PDZ结构域进行纯化和性质鉴定,将有助于深入研究MUPP1蛋白的功能和分子机制.在本文研究中,利用亲和纯化和分子筛技术,对大鼠来源的MUPP1蛋白的第8个PDZ结构域进行了表达和纯化.多角度激光光散射的数据表明:MUPP1-PDZ8结构域在溶液中为单体,分子量为16.4 k D.圆二色谱结果表明,MUPP1-PDZ8结构域具有较好的二级结构折叠,测得其熔解温度为71.6摄氏度,暗示该PDZ结构域在溶液中非常稳定.最后,MUPP1-PDZ8结构域的晶体结构显示,该结构域属于I型PDZ结构域,包含3个α螺旋和6个β折叠.其中GLGL模块、β折叠B上的1 351位亮氨酸,以及α螺旋B上的1 405位异亮氨酸/1 398位组氨酸形成的PDZ结合口袋,可以特异性地与其目标蛋白质的羧基末端相结合.综上所述,本文的研究提供了MUPP1-PDZ8结构域的生化特性,以及该结构域与其目标蛋白质相互作用的分子机制,这将为MUPP1蛋白的功能研究提供生物化学与结构生物学的理论基础.
