内质网分子伴侣GRP94对伪狂犬病毒增殖的调节作用

为探究葡萄糖调节蛋白94(GRP94)对伪狂犬病毒(Pseudorabies virus,PRV)增殖的影响,以及GRP94与PRV结构蛋白之间的相互作用。利用慢病毒转染构建GRP94基因过表达的BHK-21细胞,以及CRISPR/Cas9技术构建GRP94基因敲除的BHK-21细胞,检测PRV感染不同细胞的增殖水平,比较内质网应激相关蛋白表达水平,探索GRP94与PRV结构蛋白之间的互作关系。结果表明,GRP94基因过表达细胞株显著有利于PRV增殖,而GRP94基因缺失细胞株对PRV增殖无显著影响。蛋白免疫印迹结果显示,在GRP94基因缺失细胞株中,GRP78表达水平显著上调。免疫共沉淀结果表明,GRP94与gB、gD、gL具有相互作用。GRP94基因过表达有利于PRV增殖,检测未折叠蛋白反应(unfolded protein response, UPR)相关蛋白,研究GRP94在病毒增殖过程中的具体作用,可为PRV与内质网应激提供分子方面的研究基础,于对抗PRV病毒的感染具有重要意义。     

内质网应激在肺泡上皮间质转分化发生中的作用

目的 探讨衣霉素诱导的内质网应激在肺泡上皮细胞-间质转分化(EMT)中的影响及作用机制.方法 以质量浓度为1.25、2.5、5.0 mg/L的衣霉素(TM)分别处理人肺腺癌上皮细胞A549 24 h,MTT法检测细胞活力;RT-PCR和免疫印迹法检测GRP78、CHOP、E-钙粘蛋白(E-cadherin)、Smad2/3、转录因子Snail的表达;以5μg/L TGF-β1分别处理A549细胞24 h或48h后,免疫印迹法检测GRP78和p-elF2α的表达.结果 1.25 mg/L的TM处理A549细胞24 h后,GRP78 mRNA和蛋白表达升高,CHOPmRNA表达上调;E-cadherin蛋白表达下降,Smad2/3活化,Snail表达上调.而TGF-β1处理A549 24 h,GRP78和p-elF2α表达均上调.结论 衣霉素诱导的内质网应激可介导A549细胞发生EMT,同时EMT发生过程中伴随着内质网应激的发生.     

糖络宁对糖尿病周围神经病变大鼠坐骨神经功能及内质网应激IRE1α-XBP-1-CHOP通路的影响

目的 探讨糖络宁对糖尿病周围神经病变（diabetic peripheral neuropathy,DPN）大鼠内质网应激（endoplasmic reticulum stress,ER stress）IRE1α-XBP-1-CHOP通路的影响。方法 建立DPN大鼠模型,设置空白对照组（Control）、模型对照组（Model）、氧化三甲胺组（TMAO）、糖络宁低剂量组（LTLN）和糖络宁高剂量组（HTLN）,分别灌胃给药12周。采用放射免疫试剂盒测定稳态胰岛素抵抗指数;采用透射电镜和劳克坚劳蓝染色观察坐骨神经结构;测定鼠尾热痛阈值、神经传导速度和蛋白基因产物9.5（protein gene product 9.5,PGP 9.5）蛋白的表达并检测周围神经功能;采用Western blot法检测葡萄糖调节蛋白78（glucose-regulated protein 78,GRP 78）、X盒结合蛋白1（X-box binding protein1,XBP-1）、凋亡蛋白C/EBP同源蛋白（CCAAT/enhancer binding protein Homologous protein,CHOP）、B细胞淋巴瘤-2（B cell lymphoma-2,Bcl-2）、Bcl-2相关X蛋白（Bcl-2 associated X protein,Bax）和磷酸化真核翻译起始因子2α（phospho-ukaryotic translation initiation factor 2α,P-eIF 2α）的蛋白表达;通过免疫荧光组织化学法检测肌醇激酶1（inositol requiring enzyme1α,IRE 1α）、生长停滞及DNA损伤基因34（growth-arrest and DNA damage-inducible gene 34,GADD 34）和内质网氧化蛋白（endoplasmic oxidoreductin-1-like,Ero1α）指标。结果 与模型组比,糖络宁高、低剂量组能够有效改善坐骨神经的结构和功能,并能够影响内质网应激IRE1α-XBP-1-CHOP凋亡通路提高GRP 78、Bcl-2和P-eIF 2α的表达（P<0.05）,降低P-IRE1α、XBP-1、CHOPGADD 34、Bax和Ero1α的表达（P<0.05）。结论 糖络宁能够通过抑制ER stress中IRE1α-XBP-1-CHOP凋亡途径相关蛋白的表达,从而减轻ER stress诱导的细胞凋亡,改善坐骨神经的结构和功能从而防治DPN。     

Protein Qnality Control

概述了蛋白质品质管理中涉及的分子伴侣、激发未折叠蛋白反应(unfolded protein response,UPR)和内质网相关性蛋白质 降解途径(ER-associated degradation,ERAD)等的研究进展,并探讨了该领域存在的问题以及发展前景.指出蛋白质的生命过程经历生成、折叠、组装和降解,每个过程都有严格控制.内质网中,各种蛋白质合成、折叠并经修饰形成具有一定构象的功能性蛋白.其在内质网折叠受阻碍时,未折叠的蛋白聚集,激发UPR,使一系列分子伴侣和蛋白质折叠所需修饰酶类表达上调,帮助其完成折叠和装配.如果这些蛋白仍不能正确折叠,则进入ERAD被降解.     

富硒酵母干预对高脂小鼠GRP78基因表达的影响

目的:探讨富硒酵母干预对高脂大鼠内质网应激基因GRP78表达的影响.方法:选取40只6周龄小鼠作为研究对象,建立高脂小鼠模型,并随机分为4组(a:对照组,b:高脂日粮组,c:高脂日粮+低剂量富硒酵母组,d:高脂日粮+高剂量富硒酵母组),观察肝脏脂肪变性程度,检测肝脏脂质氧化MDA水平和GRP78基因的mRNA表达水平.结果:高脂日粮可以诱发小鼠肝脏指数增大和脂肪变性,饲喂富硒酵母后,小鼠的肥胖和肝脏脂肪变性得到了明显改善;高脂日粮组与其他组相比肝脏中MDA和GRP78基因的表达水平显著升高,高剂量硒酵母对高脂诱发的MDA升高和GRP78基因的表达水平均有拮抗作用.结论:硒能逆转高脂小鼠的肝脏损伤,内质网应激信号通路在其中发挥了作用,该应激可能是通过提高氧化水平而诱导的.     

蛋白质分子的一生——蛋白质的品质管理(英文)

概述了蛋白质品质管理中涉及的分子伴侣、激发未折叠蛋白反应(unfolded protein response, UPR)和内质网相关性蛋白质降解途径(ER-associated degradation, ERAD)等的研究进展,并探讨了该领域存在的问题以及发展前景。指出蛋白质的生命过程经历生成、折叠、组装和降解,每个过程都有严格控制。内质网中,各种蛋白质合成、折叠并经修饰形成具有一定构象的功能性蛋白。其在内质网折叠受阻碍时,未折叠的蛋白聚集,激发 UPR,使一系列分子伴侣和蛋白质折叠所需修饰酶类表达上调,帮助其完成折叠和装配。如果这些蛋白仍不能正确折叠,则进入 ERAD 被降解。     

上笼应激对绍兴鸭十二指肠组织结构、抗氧化能力及基因m RNA表达量的影响

将80只200日龄的绍兴蛋鸭随机分为2组,即地面平养组和上笼应激组,在上笼后第1、2、4、7和10天对十二指肠的组织损伤程度以及抗氧化等指标进行测定,并通过实时荧光聚合酶链式反应(real-time fluorescence quantitative polymerase chain reaction, RT-PCR)检测内质网应激、炎症和凋亡相关基因mRNA的相对表达量,为蛋鸭的规模化养殖和上笼应激诱发症的科学防预提供理论依据。结果发现:1)随着上笼应激时间的增加,试验组十二指肠的损伤程度逐渐增加,主要表现为炎症细胞浸润和肠腺上皮细胞脱落。2)上笼应激过程中,试验组十二指肠丙二醛(malondialdehyde, MDA)含量,总抗氧能力(total antioxidant capacity, T-AOC)、超氧化物歧化酶(superoxide dismutase, SOD)和谷胱甘肽过氧化物酶(glutathione peroxidase, GSH-Px)活性呈现波动性变化,而过氧化氢酶(catalase, CAT)活性在上笼应激后第7天显著增加(P0.05)。3)RT-PCR检测结果显示:与对照组相比,内质网应激相关基因葡萄糖调节蛋白78(glucose regulated protein 78, GRP78)和CCAAT/增强子结合蛋白同源蛋白(CCAAT/enhancer-binding protein-homologous protein, CHOP)mRNA表达水平呈现波动性变化,且在上笼后第2和10天显著增加(P0.05);凋亡相关基因Bcl-2相关X蛋白(Bcl-2 associated X protein, Bax)mRNA表达水平在上笼后第7和10天显著增加(P0.05),而半胱氨酸天冬氨酸特异性蛋白酶3(cysteinecontaining aspartatespecific proteases-3, Caspase3)m RNA表达量较对照组差异不显著(P0.05);炎症相关基因环氧合酶-2(cyclooxygenase-2, COX-2)m RNA水平显著上调,且在上笼后第10天显著增加(P0.05),而诱导型一氧化氮合酶(inducible nitric oxide, iNOS)表达水平则呈现先下降后上升的趋势。综上表明,上笼应激过程引起绍兴鸭十二指肠不同程度的组织损伤,同时内质网应激强度上调,最终导致细胞凋亡和炎症反应的产生。     

蛋白质的品质管理

概述了蛋白质品质管理中涉及的分子伴侣、激发未折叠蛋白反应(unfolded protein response,UPR)和内质网相关性蛋白质降解途径(ER-associated degradation,ERAD)等的研究进展,并探讨了该领域存在的问题以及发展前景。指出蛋白质的生命过程经历生成、折叠、组装和降解,每个过程都有严格控制。内质网中,各种蛋白质合成、折叠并经修饰形成具有一定构象的功能性蛋白。其在内质网折叠受阻碍时,未折叠的蛋白聚集,激发UPR,使一系列分子伴侣和蛋白质折叠所需修饰酶类表达上调,帮助其完成折叠和装配。如果这些蛋白仍不能正确折叠,则进入ERAD被降解。     

Nrf2对小鼠非酒精性脂肪性肝炎保护作用机制的研究

通过饲喂小鼠蛋氨酸-胆碱缺乏饲料(methionine choline-deficient diet,MCD)获得小鼠非酒精性脂肪肝炎(nonalcoholic steatohepatities,NASH)模型。随机分为对照组,模型组及姜黄素诱导Nrf2(nuclear factor-erythroid 2 related factor 2,Nrf2)组。给予姜黄素诱导Nrf2组0.9ml/d姜黄素剂量进行灌胃,对照组、模型组给予相应体积的生理盐水灌胃。模型组和姜黄素诱导Nrf2组均饲喂MCD饲料。饲喂4周后,通过HE染色和油红O染色检测小鼠非酒精性脂肪性肝炎情况。检测血清和肝组织中甘油三酯(TG)和胆固醇(Chol)含量;检测肝组织中葡萄糖调节蛋白-78(glucose-regulated protein-78,GRP-78),固醇调节元件结合蛋白-1c(sterol regulatory element binding protein-1c,SREBP-1c)、磷酸化双链RNA依赖的蛋白激酶样内质网激酶(phospho-PRK-1ike ER kinase,p-PERK)的含量。结果表明:与模型组相比,姜黄素诱导Nrf2组血清中TG和Chol水平升高;肝组织中TG和Chol含量降低;同时肝组织中GRP78表达上调,SREBP1的前体和成熟体均有所下降;PERK磷酸化水平降低。因此,Nrf2对蛋氨酸-胆碱缺乏所引起的NASH具有一定的保护作用,而这种保护作用可能与减轻内质网应激相关。     

电针内关、百会穴对脑缺血/再灌注大鼠GRP78和Caspase-12基因表达的影响

目的 观察电针内关、百会穴对脑缺血/再灌注（Ischemia/Reperfusion,I/R）大鼠脑GRP78和Caspase-12基因表达的影响,探讨针刺发挥脑保护作用是否与内质网应激凋亡通路有关。方法 100只大鼠随机分为5组,每组20只,即正常组、假手术组、手术造模组、依达拉奉组和针刺干预组。采用线栓法建立大鼠大脑中动脉阻塞（middle cerebral artery occlusion,MCAO）脑I/R模型。采用TUNEL染色法,检测大鼠神经细胞凋亡指数;采用实时定量多聚酶链式反应（Real-Time polymerase chain reaction,RT-PCR）,检测GRP78、Caspase-12 mRNA表达。结果 与正常组和假手术组相比,手术造模组、依达拉奉组和针刺干预组大鼠细胞凋亡指数升高,GRP78和Caspase-12 mRNA表达增多,差异具有统计学意义（P<0.05或P<0.01）;与手术造模组相比,依达拉奉组和针刺干预组大鼠细胞凋亡指数和Caspase-12 mRNA表达均明显降低（P<0.01）,GRP78 mRNA表达明显升高（P<0.01）;与依达拉奉组相比,针刺干预组大鼠各项指标差异无统计学意义（P>0.05）。结论 针刺内关、百会穴可以有效抑制脑缺血神经元细胞凋亡,其保护机制可能上调内质网应激保护性GRP78表达,同时抑制促凋亡Caspase-12表达有关。     

玉米种胚内质网胁迫相关基因对人工老化处理的响应

【目的】在植物中,内质网胁迫（endoplasmic reticulum stress,ERS）和未折叠蛋白应答（unfolded protein response,UPR）参与环境胁迫响应过程,然而,玉米种子老化过程中内质网胁迫相关基因表达情况尚未见报道。文章利用基因数字表达谱技术探究玉米种子老化过程中内质网胁迫相关基因表达规律,以期为揭示种子衰老的分子机制提供理论依据。【方法】以玉米杂交种郑单958种子为材料,采用高温（45℃）高湿（相对湿度100%）的方法进行人工老化处理。分别提取未老化处理（对照）和老化处理3 d的玉米种胚总RNA,利用Illumina HiSeqTM 2000平台进行高通量测序。去除原始数据中的接头序列、包含模糊碱基的序列以及低质量序列,获得Clean reads,利用短序列比对软件SOAPaligner/ SOAP2将Clean Reads分别比对到玉米参考基因组和参考基因序列,采用RPKM（reads per kb per million reads）方法计算基因的表达量,根据FDR（false discovery rate）＜0.001和|log2 ratio(T/CK)|≥1的标准筛选差异表达的基因,对获得的差异表达基因（differentially expressed genes,DEGs）进行KEGG（kyoto encyclopedia of genes and genomes）数据库功能注释分析,筛选出响应人工老化的内质网胁迫相关差异表达基因。利用qRT-PCR技术定量分析内质网胁迫相关基因在不同人工老化时间内的表达特性。【结果】基因数字表达谱鉴定结果表明,有104个差异表达基因在人工老化过程中参与内质网蛋白质加工（protein processing in endoplasmic reticulum）通路,其中内质网胁迫相关基因有97个（81个上调表达,16个下调表达）。对差异表达基因功能注释分析表明,内质网胁迫的标志性蛋白基因BiP以及分子伴侣蛋白基因CRT、CNT和GRP94等显著上调表达。参与内质网相关性降解（endoplasmic reticulum-associated degradation,ERAD）途径的有83个差异表达基因（70个上调,13个下调）,其中启动ERAD途径的关键酶基因EDEM （ER degradation enhancing mannosidase I-like protein）下调,参与蛋白泛素化的E2泛素结合酶基因UbcH5、E3泛素连接酶基因Hrd1和Doa10等也发生显著的表达变化。qRT-PCR结果表明,内质网胁迫相关基因在不同人工老化时间内表现表达多样性和复杂性。【结论】人工老化处理能造成玉米种胚细胞发生内质网胁迫。细胞通过上调分子伴侣基因表达和诱导ERAD途径响应内质网胁迫,但ERAD途径受阻可能引起错误折叠蛋白聚集,从而进一步加剧细胞损伤,最终导致种子活力降低甚至丧失。     

Proapoptotic BAX and BAK modulate the unfolded protein response by a direct interaction with IRE1alpha

Accumulation of misfolded protein in the endoplasmic reticulum (ER) triggers an adaptive stress response-termed the unfolded protein response (UPR)-mediated by the ER transmembrane protein kinase and endoribonuclease inositol-requiring enzyme-1alpha (IRE1alpha). We investigated UPR signaling events in mice in the absence of the proapoptotic BCL-2 family members BAX and BAK [double knockout (DKO)]. DKO mice responded abnormally to tunicamycin-induced ER stress in the liver, with extensive tissue damage and decreased expression of the IRE1 substrate X-box-binding protein 1 and its target genes. ER-stressed DKO cells showed deficient IRE1alpha signaling. BAX and BAK formed a protein complex with the cytosolic domain of IRE1alpha that was essential for IRE1alpha activation. Thus, BAX and BAK function at the ER membrane to activate IRE1alpha signaling and to provide a physical link between members of the core apoptotic pathway and the UPR.     

ER tubules mark sites of mitochondrial division

Mitochondrial structure and distribution are regulated by division and fusion events. Mitochondrial division is regulated by Dnm1/Drp1, a dynamin-related protein that forms helices around mitochondria to mediate fission. Little is known about what determines sites of mitochondrial fission within the mitochondrial network. The endoplasmic reticulum (ER) and mitochondria exhibit tightly coupled dynamics and have extensive contacts. We tested whether ER plays a role in mitochondrial division. We found that mitochondrial division occurred at positions where ER tubules contacted mitochondria and mediated constriction before Drp1 recruitment. Thus, ER tubules may play an active role in defining the position of mitochondrial division sites.     

ERdj5 is required as a disulfide reductase for degradation of misfolded proteins in the ER

Membrane and secretory proteins cotranslationally enter and are folded in the endoplasmic reticulum (ER). Misfolded or unassembled proteins are discarded by a process known as ER-associated degradation (ERAD), which involves their retrotranslocation into the cytosol. ERAD substrates frequently contain disulfide bonds that must be cleaved before their retrotranslocation. Here, we found that an ER-resident protein ERdj5 had a reductase activity, cleaved the disulfide bonds of misfolded proteins, and accelerated ERAD through its physical and functional associations with EDEM (ER degradation-enhancing alpha-mannosidase-like protein) and an ER-resident chaperone BiP. Thus, ERdj5 is a member of a supramolecular ERAD complex that recognizes and unfolds misfolded proteins for their efficient retrotranslocation.     

EDEM as an acceptor of terminally misfolded glycoproteins released from calnexin

Terminally misfolded proteins in the endoplasmic reticulum (ER) are retrotranslocated to the cytoplasm and degraded by proteasomes through a mechanism known as ER-associated degradation (ERAD). EDEM, a postulated Man8B-binding protein, accelerates the degradation of misfolded proteins in the ER. Here, EDEM was shown to interact with calnexin, but not with calreticulin, through its transmembrane region. Both binding of substrates to calnexin and their release from calnexin were required for ERAD to occur. Overexpression of EDEM accelerated ERAD by promoting the release of terminally misfolded proteins from calnexin. Thus, EDEM appeared to function in the ERAD pathway by accepting substrates from calnexin.     

The unfolded protein response: from stress pathway to homeostatic regulation

The vast majority of proteins that a cell secretes or displays on its surface first enter the endoplasmic reticulum (ER), where they fold and assemble. Only properly assembled proteins advance from the ER to the cell surface. To ascertain fidelity in protein folding, cells regulate the protein-folding capacity in the ER according to need. The ER responds to the burden of unfolded proteins in its lumen (ER stress) by activating intracellular signal transduction pathways, collectively termed the unfolded protein response (UPR). Together, at least three mechanistically distinct branches of the UPR regulate the expression of numerous genes that maintain homeostasis in the ER or induce apoptosis if ER stress remains unmitigated. Recent advances shed light on mechanistic complexities and on the role of the UPR in numerous diseases.     

Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1

Malfolded proteins in the endoplasmic reticulum (ER) induce cellular stress and activate c-Jun amino-terminal kinases (JNKs or SAPKs). Mammalian homologs of yeast IRE1, which activate chaperone genes in response to ER stress, also activated JNK, and IRE1alpha-/- fibroblasts were impaired in JNK activation by ER stress. The cytoplasmic part of IRE1 bound TRAF2, an adaptor protein that couples plasma membrane receptors to JNK activation. Dominant-negative TRAF2 inhibited activation of JNK by IRE1. Activation of JNK by endogenous signals initiated in the ER proceeds by a pathway similar to that initiated by cell surface receptors in response to extracellular signals.