为了观察突触后致密物质-95(postsynaptic density 95,PSD-95)结构域PDZ1过表达对缺氧缺糖诱导的海马神经元凋亡的影响,本研究采用培养21d的Sprague-Dawley大鼠海马神经元,加入PDZ1腺病毒颗粒感染24h,收集细胞进行免疫沉淀、免疫印迹实...为了观察突触后致密物质-95(postsynaptic density 95,PSD-95)结构域PDZ1过表达对缺氧缺糖诱导的海马神经元凋亡的影响,本研究采用培养21d的Sprague-Dawley大鼠海马神经元,加入PDZ1腺病毒颗粒感染24h,收集细胞进行免疫沉淀、免疫印迹实验;缺氧缺糖1.5h后,用DAPI染色后荧光显微镜观察细胞凋亡,并收集细胞进行免疫印迹实验。结果显示:(1)PDZ1在海马神经元中过表达;(2)过表达的PDZ1使缺氧缺糖诱导的海马神经元凋亡数量减少(P<0.05);(3)过表达的PDZ1使PSD-95与GluR6结合减少;(4)PDZ1过表达抑制由缺氧缺糖诱导的大鼠海马神经元MLK3和JNK1/2磷酸化。以上结果提示,PDZ1过表达能拮抗缺氧缺糖诱导的海马神经元凋亡。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
Spermatozoa are not mature until they transit the epididymis where they acquire motility and the ability to fertilize an egg through sequential modifications. The epididymis has three functional regions, caput, corpus...Spermatozoa are not mature until they transit the epididymis where they acquire motility and the ability to fertilize an egg through sequential modifications. The epididymis has three functional regions, caput, corpus, and cauda, and the luminal proteins of the epididymis play important roles in the above modifications. However, the proteins with differential enrichment between the caput and cauda are still largely unknown. To reveal the functions of the caput and cauda during sperm maturation, luminal proteins from caput and cauda of mice were analyzed by isobaric tag for relative and absolute quantitation （iTRAQ）. Overall, 128 differentially enriched proteins were found, of which 46 were caput enriched and 82 were cauda enriched. Bioinformatic analysis showed that lipid metabolism was active in the caput; while anion- and cation-binding activity and phosphorus and organophosphate metabolism were active in the cauda. A new epididymal luminal protein, the caput-enriched PDZ domain containing 1 （Pdzkl）, also named Na^＋/H^＋ exchange regulatory cofactor 3 （NHERF3）, which plays a critical role in cholesterol metabolism and carnitine transport, was found in the lipid metabolism. Western blotting and immunofluorescence analyses showed that Pdzkl was expressed in the epididymis but not in the testis, and localized at the middle piece of the sperm tail. Pdzkl protein level was also reduced in the spermatozoa in case of asthenozoospermic patients compared with that in normozoospermic men, suggesting that Pdzkl may participate in sperm maturation regulation and may be associated with male infertility. These results may provide new insights into the mechanisms of sperm maturation and male infertility.展开更多
基金supported by the National Natural Science Foundation of China(No.30330190)Educational Science Foundation of Jiangsu Province+2 种基金China(No.07KJB310118)Science Foundation of Xuzhou Medical CollegeChina(No.07KJ15)
文摘为了观察突触后致密物质-95(postsynaptic density 95,PSD-95)结构域PDZ1过表达对缺氧缺糖诱导的海马神经元凋亡的影响,本研究采用培养21d的Sprague-Dawley大鼠海马神经元,加入PDZ1腺病毒颗粒感染24h,收集细胞进行免疫沉淀、免疫印迹实验;缺氧缺糖1.5h后,用DAPI染色后荧光显微镜观察细胞凋亡,并收集细胞进行免疫印迹实验。结果显示:(1)PDZ1在海马神经元中过表达;(2)过表达的PDZ1使缺氧缺糖诱导的海马神经元凋亡数量减少(P<0.05);(3)过表达的PDZ1使PSD-95与GluR6结合减少;(4)PDZ1过表达抑制由缺氧缺糖诱导的大鼠海马神经元MLK3和JNK1/2磷酸化。以上结果提示,PDZ1过表达能拮抗缺氧缺糖诱导的海马神经元凋亡。
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.
基金This work was supported by the National Natural Science Foundation of China Grants （81430027 and 81671510 to FS 81501309 to GSW） and the National Basic Research Program of China （2014CB943100 to FS）.
文摘Spermatozoa are not mature until they transit the epididymis where they acquire motility and the ability to fertilize an egg through sequential modifications. The epididymis has three functional regions, caput, corpus, and cauda, and the luminal proteins of the epididymis play important roles in the above modifications. However, the proteins with differential enrichment between the caput and cauda are still largely unknown. To reveal the functions of the caput and cauda during sperm maturation, luminal proteins from caput and cauda of mice were analyzed by isobaric tag for relative and absolute quantitation （iTRAQ）. Overall, 128 differentially enriched proteins were found, of which 46 were caput enriched and 82 were cauda enriched. Bioinformatic analysis showed that lipid metabolism was active in the caput; while anion- and cation-binding activity and phosphorus and organophosphate metabolism were active in the cauda. A new epididymal luminal protein, the caput-enriched PDZ domain containing 1 （Pdzkl）, also named Na^＋/H^＋ exchange regulatory cofactor 3 （NHERF3）, which plays a critical role in cholesterol metabolism and carnitine transport, was found in the lipid metabolism. Western blotting and immunofluorescence analyses showed that Pdzkl was expressed in the epididymis but not in the testis, and localized at the middle piece of the sperm tail. Pdzkl protein level was also reduced in the spermatozoa in case of asthenozoospermic patients compared with that in normozoospermic men, suggesting that Pdzkl may participate in sperm maturation regulation and may be associated with male infertility. These results may provide new insights into the mechanisms of sperm maturation and male infertility.
