泛素连接酶E3

网络出版时间：２０２１－５－２８１０：１１　网络出版地址：ｈｔｔｐｓ：／／ｋｎｓ．ｃｎｋｉ．ｎｅｔ／ｋｃｍｓ／ｄｅｔａｉｌ／３４．１０８６．Ｒ．２０２１０５２７．１４５７．００６．ｈｔｍｌ靶向Ｅ３泛素连接酶的药物研究进展商廿颍１，赵春阳２，彭　英１（１．中国医学科学院北京协和医学院药物研究所，北京　１０００５０；２．国家药品监督管理局药品审评中心，北京　１０００２２）收稿日期：２０２１－０１－２７，修回日期：２０２１－０３－２３基金项目：国家自然科学基金资助项目（Ｎｏ８２０７３８３５，８１８７２８５５）作者简介：商廿颍（１９９９－），女，博士生，研究方向：神经药理学，Ｅｍａｉｌ：ｓｈａｎｇｎｉａｎｙｉｎｇ＠ｉｍｍ．ａｃ．ｃｎ；赵春阳（１９８８－），女，助理研究员，研究方向：神经药理学，Ｔｅｌ：０１０－８５２４３１６３，Ｅ ｍａｉｌ：ｚｈａｏｃｙｐｕｍｃ＠１６３．ｃｏｍ；彭　英（１９７０－），女，博士，研究员，研究方向：神经药理学，通讯作者，Ｅ ｍａｉｌ：ｙｐｅｎｇ＠ｉｍｍ．ａｃ．ｃｎｄｏｉ：１０．３９６９／ｊ．ｉｓｓｎ．１００１－１９７８．２０２１．０６．００３文献标志码：Ａ文章编号：１００１－１９７８（２０２１）０６－０７４９－０７中国图书分类号：Ｒ ０５；Ｒ３４１ ３１；Ｒ５７４；Ｒ７４２ ５；Ｒ７４５ ７；Ｒ９７７ ３；Ｒ９７７ ６摘要：泛素－蛋白酶体系统是真核细胞蛋白质降解的重要途径之一，在细胞的增殖、分化、凋亡以及ＤＮＡ的修复等生理活动的调节中起着重要作用。

Ｅ３泛素连接酶是泛素－蛋白酶体系统的重要组成部分，决定底物识别的特异性。

Ｅ３泛素连接酶的失调会引起癌症、阿尔茨海默症和帕金森病等多种疾病。

本文对靶向Ｅ３泛素连接酶的药物在癌症、阿尔茨海默病、帕金森病、糖尿病并发症、动脉粥样硬化以及炎症性肠炎等领域的研究进展进行了总结。

目前能够开发成为药物的靶向Ｅ３泛素连接酶的小分子抑制剂或激动剂数量不多，我国在天然产物的研究与开发方面具有突出的优势，很多天然产物都具有靶向Ｅ３泛素连接酶的活性，值得进一步开发应用。

E3泛素连接酶HECTD3真核表达质粒的鉴定目的鉴定E3泛素连接酶（homologous to the E6-associated protein carboxyl terminus domain containing 3，HECTD3）基因的真核表达质粒。

方法提取无内毒素真核表达质粒pcDNA-HECTD3，利用lipofectamine 2000将其转染进卵巢癌细胞SKOV-3细胞，qRT-PCR法和蛋白质免疫印迹技术检测该基因的表达情况。

结果真核表达质粒pcDNA-HECTD3可以显著地提高HECTD3基因在mRNA和蛋白水平的表达量。

结论HECTD3基因的真核表達质粒构建成功，可以用于后续卵巢癌发生发展的分子生物学研究。

标签：E3泛素连接酶；卵巢癌；真核表达质粒卵巢癌是致死率最高的妇科恶性肿瘤，仅2011年1年就导致全世界140000人死亡[1]。

HECTD3 （homologous to the E6-associated protein carboxyl terminus domain containing 3）是一个功能目前尚未阐述清楚的新的E3泛素连接酶[2]。

有报道指出该基因可能在乳腺癌和宫颈癌中介导顺铂的化疗耐受[3]，但尚未有人报道HECTD3在卵巢癌中的作用。

实验室前期构建该基因真核表达质粒pcDNA-HECTD3，本研究将鉴定该真核表达质粒在胞内对HECTD3基因表达情况的影响，为进一步研究HECTD3基因在卵巢癌发生发展中的作用提供有力的工具和手段。

1实验材料卵巢癌细胞SKOV-3和真核表达质粒pcDNA-HECTD3由本室保存。

Trizol、Lipofectamine 2000购自美国invitrogen公司，RPMI-1640、胎牛血清、胰酶购自美国Gibco公司。

逆转录试剂盒cDNA Synthesis Kit （M-MLV Version）、SYBR○RPremix Ex Taq II（Ti RNaseH Plus）购自TaKaRa公司。

Plant Science 180(2011)775–782Contents lists available at ScienceDirectPlantSciencej o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /p l a n t s ciOverexpression of OsRDCP1,a rice RING domain-containing E3ubiquitin ligase,increased tolerance to drought stress in rice (Oryza sativa L.)Hansol Bae a ,1,Sung Keun Kim a ,1,Seok Keun Cho a ,Bin Goo Kang b ,Woo Taek Kim a ,∗a Department of Systems Biology,College of Life Science and Biotechnology,Yonsei University,Seoul 120-749,Republic of Korea bReSEAT Program,Korea Institute of Science and Technology Information,Seoul 130-741,Republic of Koreaa r t i c l e i n f o Article history:Received 5February 2011Received in revised form 21February 2011Accepted 21February 2011Available online 4March 2011Keywords:Drought stress E3ubiquitin ligase Oryza sativaOsRDCP1-overexpressing lines Rice osrdcp1knock-out mutant RING domaina b s t r a c tCaRma1H1was previously identified as a hot pepper drought-induced RING E3Ub ligase.We have identi-fied five putative proteins that display a significant sequence identity with CaRma1H1in the rice genome database (/cgi-bin/RiceGE ).These five rice paralogs possess a single RING motif in their N-terminal regions,consistent with the notion that RING proteins are encoded by a multi-gene family.Therefore,these proteins were named OsRDCPs (Oryza sativa RING domain-containing proteins).Among these paralogs,OsRDCP1was induced by drought stress,whereas the other OsRDCP members were constitutively expressed,with OsRDCP4transcripts expressed at the highest level in rice seedlings.osrdcp1loss-of-function knockout mutant and OsRDCP1-overexpressing transgenic rice plants were developed.Phenotypic analysis showed that wild-type plants and the homozygous osrdcp1G2mutant line displayed similar phenotypes under normal growth conditions and in response to drought stress.This may be due to complementation by other OsRDCP paralogs.In contrast,35S:OsRDCP1T2transgenic rice plants exhib-ited improved tolerance to severe water deficits.Although the physiological function of OsRDCP1remains unclear,there are several possible mechanisms for its involvement in a subset of physiological responses to counteract dehydration stress in rice plants.©2011Elsevier Ireland Ltd.All rights reserved.1.IntroductionTurnover of a wide range of eukaryotic proteins is regulated by the ubiquitin (Ub)-26S proteasome pathway.This pathway plays a critical role in the control of multiple cellular processes as diverse as cell cycle,differentiation,hormone responses,protein trafficking,and responses to environmental stresses [1–3].Multiple Ub chains are attached to the target proteins by E1,E2,and E3enzymes and serve as degradation tags,leading to the proteolysis of targeted proteins by the 26S proteasome complex [4,5].Based on subunit composition,E3s can be classified into two groups.The HECT and RING/U-box E3classes are comprised of single subunits,whereas the SCF and anaphase-promoting-complex (APC)E3ligases consist of multiple polypeptides [1–5].In most cases,E3Ub ligases are responsible for identifying specific target proteins.Abbreviations:ABA,abscisic acid;MeJA,methyl jasmonate;RT-PCR,reverse transcriptase-polymerase chain reaction;SA,salicylic acid;Ub,ubiquitin.∗Corresponding author.Tel.:+82221232661;fax:+8223125657.E-mail address:wtkim@yonsei.ac.kr (W.T.Kim).1These two authors equally contributed to this work and are listed in alphabetical order.A number of RING-containing proteins function as E3Ub ligases in responses to abiotic environmental stresses.Arabidopsis HOS1works as a negative regulator for cold-stress signal transduction [6,7],while XERICO participates positively in salt-and osmotic-stress responses through the overproduction of abscisic acid (ABA)[8].Arabidopsis SDIR1and DRIP RING E3Ub ligases are involved in the regulation of positive and negative drought-stress responses,respectively [9,10].Ryu et al.[11]recently reported that AtAIRP1RING E3is a positive regulator of an ABA-dependent defense mech-anism against drought stress.Compared to the studies of Arabidopsis RING E3ligases,research elucidating the roles of these Ub ligases in crop plants has been scarce.The hot pepper CaRma1H1RING E3ligase confers strong tolerance to dehydration stress when it is ectopically expressed in Arabidopsis [12].More recently,the rice OsDSG1RING protein was reported to regulate high-salt and drought stress responses [13].Collectively,these results are indicative of cellular functions of RING E3Ub ligases in defense against abiotic environmental stresses in higher plants.We aimed to unravel the physiological roles of RING E3Ub lig-ases with respect to abiotic stresses,such as water deficit.In this report,we used rice as a molecular genetic model system and identified five homologous OsRDCP (Oryza sativa RING domain-containing protein)family members,all of which contain a single0168-9452/$–see front matter ©2011Elsevier Ireland Ltd.All rights reserved.doi:10.1016/j.plantsci.2011.02.008776H.Bae et al./Plant Science180(2011)775–782RING motif in their N-terminal regions.Among these rice par-alogs,OsRDCP1was induced by drought stress.Loss-of-function rice mutant plants(osrdcp1)containing a T-DNA copy integrated into the OsRDCP1gene and OsRDCP1-overexpressing transgenic rice plants(35S:OsRDCP1)were developed.Phenotypic analysis showed that wild-type plants and the homozygous osrdcp1G2 mutant line displayed similar phenotypes under normal growth conditions and in response to drought stress.In contrast,OsRDCP1-overexpressing T2transgenic rice plants exhibited markedly improved tolerance to severe water deficits.Although the physi-ological function of OsRDCP1has not yet been determined,there are several possibilities for the involvement of OsRDCP1in a subset of physiological responses to counteract dehydration stress in rice plants.2.Materials and methods2.1.Plant materialsRice(O.sativa var.Japonica cv.Dongjin)seeds were germi-nated on Murashige and Skoog medium containing MS basal salts (Wako Pure Chemical,Osaka,Japan),3%sucrose,0.2%phytogel,and 0.55mM myo-inositol as described previously[14].Seedlings were grown for1–2weeks at27◦C under continuous light,transplanted to soil in a greenhouse,and raised to maturity.The rice mutant line osrdcp1that contained a T-DNA insertion within OsDRCP1was developed from a population of T-DNA-tagging lines produced from O.sativa var.Japonica cv.Dongjin[15,16]using a gene-specific primer M1(5 -TTGACTTTGCACACATAGGACA-3 )and a T-DNA right border primer LBa-1(5 -ACAAGCCGTAAGTGCAAGTG-3 ).Plants homozygous for the T-DNA insertion were identified through mul-tiple PCRs with primers M2(5 -CTGGTGCCATTGTATGGTCGT-3 ) and LBa-1.The35S:OsRDCP1transgenic rice plants overexpress-ing OsRDCP1under the control of the35S CaMV promoter were generated by Agrobacterium-mediated co-cultivation methods as previously earlier[17].Regenerated plants were grown in a green-house at approximately30◦C during the day and20◦C at night.The light/dark cycle in the greenhouse was14/10h[18].2.2.Reverse transcriptase-polymerase chain reaction(RT-PCR)RT-PCR was performed in25␮l reaction buffer(10mM Tris, pH8.0,50mM KCl,1.5mM MgCl2,0.01%gelatin,and200␮M deoxynucleotides)containing1␮l of thefirst-strand cDNA reaction products,1␮M gene-specific primer sets,and2.5units Taq poly-merase(Promega,Madison,WI,USA).Gene-specific primers were designed based on the nucleotide sequences of OsRDCP1,OsRDCP2, OsRDCP3,OsRDCP4,and OsRDCP5(Table1).PCR conditions con-sisted of25cycles of45s at95◦C,1min at60◦C,and90s at72◦C in an automatic thermal cycler(Perkin-Elmer/Cetus,Norwalk,CT, USA).PCR products were separated on1.2%agarose gels and visu-alized under UV light.2.3.Application of abiotic stresses and plant hormonesRice plants were subjected to various abiotic stresses based on the method of Cho et al.[19]with significant modifications as described previously[20].For drought stress treatments,rice seedlings were grown for2weeks on agar plates under light con-ditions,harvested,and dehydrated on Whatman3MMfilter paper at room temperature.The degree of water stress was determined as the decrease in the fresh weight(5–30%)of the seedlings.For high-salinity treatments,2-week-old whole seedlings were soaked in solution containing150mM NaCl for0,2,6,or24h with gentle shaking.For low-temperature treatments,2-week-old plants were incubated at4◦C for0,2,6,or24h.For hormone treatments,leaf Table1Primer sequences for RT-PCR and genotyping PCR.Gene Primer sequenceRT-PCROsRDCP1(Os04g44820)Forward5 -GGTTCTTTGTTCTTGTCAGTGCTG-3Reverse5 -GGAATGGGCACACCATTCAG-3 OsRDCP2(Os02g42690)Forward5 -TGAGACGGCAGCACATGGAG-3Reverse5 -TCACTACTTAACAGCGTCCGCTC-3 OsRDCP3(Os01g56070)Forward5 -CAGCATGTTGATGTGTTCTTGAAG-3Reverse5 -CTACAAACAAGACTTGCAACTGCAC-3 OsRDCP4(Os03g47500)Forward5 -GTTTCACTATGGGTATGGCCATG-3Reverse5 -CATGCAAATATTCTATGAACACTTCATG-3 OsRDCP5(Os12g43930)Forward5 -ATATGGACACGGCCACGGT-3Reverse5 -CTCTAGTTCATTCAGACAGAGCTTCC-3 OsRab16b(Os11g26780)Forward5 -ACAAGGGCAACAACCACCAG-3Reverse5 -GCTTGCAATGGCATCACAAG-3 OsActin(Os03g50885)Forward5 -GTCAGCAACTGGGATGATATGG-3Reverse5 -TCTCCTTGCTCATCCTGTCAG-3 Genotyping PCROsRDCP1M15 -TTGACTTTGCACACATAGGACA-3M25 -CTGGTGCCATTGTATGGTCGT-3 LBa-15 -ACAAGCCGTAAGTGCAAGTG-3tissues of4-week-old plants were sprayed with100␮M salicylic acid(SA),100␮M methyl jasmonate(MeJA),or100␮M ABA and harvested at different time points.For ethylene treatments,light-grown2-week-old intact plants were enclosed in6-l jars containing air or air plus50␮l/l ethylene for various time periods.Proper ethylene concentrations were confirmed by gas chromatography [21].2.4.RNA gel blot analysisTotal RNA was obtained from rice plants as described previ-ously[22].RNA(10␮g per lane)was separated by electrophoresis on1.0%-agarose gels,blotted onto nylon membranefilters(Amer-sham,Arlington Heights,IL,USA),and hybridized with32P-labeled cDNA probes for OsRDCP1,OsEXP,OsLEA3,OsPHGPX,or OsActin under high-stringency hybridization and washing conditions.Blots were visualized by autoradiography at−80◦C using Kodak XAR-5film and an intensifying screen.Hybridization signals were quanti-fied with a PhosphorImager(Fuji,Tokyo,Japan).2.5.In vitro ubiquitination assaysIn vitro ubiquitination assays were conducted as described pre-viously[23]with minor modifications.Briefly,500ng bacterially expressed MBP-OsRDCP1fusion protein was incubated with150␮l ubiquitination reaction buffer(50mM Tris–HCl,pH7.5,2.5mM MgCl2,and0.5mM DTT)containing4mM ATP and20ng each of human E1and human E2UbcH5B.Arabidopsis E1(UBA1)and Arabidopsis E2(UBC8)were previously used for our in vitro ubiqui-tination assays.However,in this study,we obtained better results of rice E3Ub ligase assay with human E1and human E2UbcH5B as described by Park et al.[24].Reaction mixtures were incubated at 30◦C for0–120min,separated by8%SDS–PAGE,and subjected to immuno-blotting using an anti-MBP antibody(New England Bio-Labs,Ipswich,MA,USA)as described previously[25].2.6.Phenotypic analysis of wild-type,osrtbp1mutant,and35S:OsRDCP1transgenic rice plantsWild-type,osrtbp1mutant,and various independent 35S:OsRTBP1transgenic rice plants were subjected to drought stress as described previously[12,23]with modifications.Six-week-old plants were grown in pots under greenhouse conditions. The soil was then allowed to dry by withholding water for15 days until plants displayed wilting.Dehydration sensitivity wasH.Bae et al./Plant Science180(2011)775–782777Fig.1.Identification offive homologous rice RING E3OsRDCP family members.(A)Schematic representation of predictedfive rice OsRDCP paralogs.The GenBank accession numbers of OsRDCP1,OsRDCP2,OsRDCP3,OsRDCP4,and OsRDCP5are Os04g44820,Os02g42690,Os01g56070,Os03g47500,and Os12g43930,respectively.N-terminal RING domain and C-terminal membrane-anchoring domain are indicated by green bars and red bars,respectively.(B)Sequence alignment of the RING domain of the OsRDCP paralogs.Amino acids identical in allfive proteins are shown in black.Amino-acid residues that are conserved in at least three of thefive sequences are shaded.Putative Zn2+-interacting Cys and His residues are indicated.The numbers on the right indicate the amino-acid residues.(C)Comparison of the predicted amino-acid sequences offive full-length rice OsRDCP paralogs.The green box and red box indicate N-terminal RING domain and C-terminal membrane-anchoring domain,respectively.(For interpretation of the references to color in thisfigure legend,the reader is referred to the web version of the article.)scored as the capacity of plants to resume growth when returned to normal water conditions with re-watering.3.Results and discussion3.1.Identification and expression offive homologous rice OsRDCP gene family membersCaRma1H1was previously identified as a hot pepper drought-induced RING E3Ub ligase[12].Transgenic Arabidopsis plants that ectopically expressed CaRma1H1were highly resistant to severe water deficits.Searches of the rice genome database (/cgi-bin/RiceGE)revealed that there are at leastfive putative proteins that display a significant sequence identity with CaRma1H1and Arabidopsis AtRma1[26]homologs (Fig.1A).Thesefive rice paralogs possess a single RING motif in their N-terminal regions(Fig.1B),consistent with the notion that RING proteins are encoded by a large multi-gene family[2,4,5].In addition,they contain a putative membrane-anchoring domain in their C-terminal regions.Thus,these paralogs were named OsRDCPs (O.sativa RING domain-containing proteins).The predicted OsRDCP paralogs were25–79%identical to each other at the amino acid level (Fig.1C).To examine whether these rice RING E3members were induced by water deficits,2-week-old rice seedlings were subjected to drought stress and expression of each member was monitored by RT-PCR using gene-specific primer sets(Table1).The degree of water stress was monitored as the loss of fresh weight(0–30%)of the seedlings.The results showed that,among thefive paralogs,778H.Bae et al./Plant Science180(2011)775–782Fig.2.RT-PCR analysis offive OsRDCP family members in rice seedlings.Light-grown2-week-old seedlings were subjected to drought stress.Expression of each gene member in shoot and root tissues was analyzed by RT-PCR using gene-specific primer sets(Table1).The degree of water stress was monitored as loss of seedling fresh weight(0or30%).Rice Rab16b was used as a positive control for a drought-induced gene and actin was used as a loading control.OsRDCP1was significantly induced by drought stress in both shoot and root tissues,while the other OsRDCP members were constitu-tively expressed(Fig.2).The level of the OsRDCP4transcript was the highest in rice seedlings.3.2.OsRDCP1is a drought-inducible RING E3Ub ligase in riceThe OsRDCP1gene(GenBank accession number AK070732)is located on chromosome4and contains one intron(2093bp)in the5 -untranslated region(Fig.3A).The coding region of OsRDCP1 is762bp and encodes253amino acids with a molecular mass of 28.1kDa and a calculated p I of7.2.Full-length OsRDCP1is37%, 29%,and31%identical to hot pepper CaRma1H1[12],Arabidopsis AtRma1[26],and human RING protein HsRma1[27],respectively (Fig.3B).In addition,the RING domain of OsRDCP1is48–78%iden-tical to those of hot pepper,Arabidopsis,and human RING proteins. Thesefindings suggest that the domain is critical for Ub ligase activ-ity in rice plants.Like CaRma1H1,AtRma1,and HsRma1,OsRDCP1 has a single putative membrane-anchoring domain in its extreme C-terminus,suggesting that it is a membrane-associated protein.These structural conservations suggest that OsRDCP1is a RING domain-containing E3Ub ligase.Therefore,we characterized the OsRDCP1gene in more detail at the molecular level.Since OsRDCP1 was identified initially as a homolog of water stress-induced hot pepper CaRma1H1,we hypothesized that the expression of OsRDCP1is modulated by abiotic stresses in rice plants.To test this hypothesis,the OsRDCP1mRNA accumulation profile was monitored by RNA gel blot analyses under various abiotic stress conditions.Two-week-old light-grown rice seedlings were sub-jected to dehydration stress.OsRDCP1transcript(approximately 1.2kb)levels were significantly augmented in response to5–30% water loss(Fig.3C).This increase was seen in both shoots and roots with similar degrees of induction.The OsEXP gene,a homolog of the Arabidopsis EXPANSIN gene[28],was included as a positive control for drought stress.OsEXP was induced following20–30%water loss (Fig.3C).Thus,the induction kinetics of OsEXP was distinct from that of OsRDCP1.Significant induction of OsRDCP1transcripts was also detected following cold stress(2,6,and24h at4◦C)(Fig.3D). In contrast,expression of OsRDCP1was unaffected by high salin-ity(150mM NaCl)for at least24h,even though the expression of OsLEA3,a positive control for salt stress responses[29],was up-regulated after a2-h treatment(Fig.3E).We next examined the changes in OsRDCP1mRNA levels in response to various plant hormones.As shown in Fig.3F,OsRDCP1 transcription was activated by100␮M MeJA after a2–6-h incu-bation,with induction kinetics different than that of OsPHGPX,a positive control for MeJA induction[30].On the other hand,expres-sion of the OsRDCP1gene was not affected by100␮M SA(Fig.3F), 100␮M ABA,or20␮l/l ethylene.Thus,OsRDCP1is inducible by a subset of abiotic stresses,including drought,cold,and MeJA.3.3.In vitro self-ubiquitination assaysTo test whether OsRDCP1contained E3Ub ligase activity,full-length OsRDCP1was expressed in Escherichia coli as a fusion protein with maltose binding protein(MBP).Purified MBP-OsRDCP1was incubated at30◦C in the presence or absence of Ub,ATP,human E1,and the human E2protein UbcH5B for various time periods, and subjected to immuno-blot analysis with an anti-MBP antibody. MBP-OsRDCP1produced high-molecular-mass smear ladders in a time-dependent manner(Fig.4A).In contrast,no ubiquitina-tion was detected when the E1,E2,ATP,or Ub was excluded from the reaction mixture(Fig.4B).The MBP-OsRDCP1C54S and MBP-OsRDCP1C86S mutant proteins,in which the Cys54and Cys86 residues,respectively,were replaced with Ser residues,displayed a negligible level of Ub ligase activity(Fig.4C).The Cys54and Cys86 residues are highly conserved among different RING proteins[4,5]. In contrast,mutation of K23to R23did not exert any inhibitory effects on the Ub ligase activity.Collectively,the results depicted in Figs.3and4strongly suggest that rice OsRDCP1is a drought-inducible RING E3Ub ligase.3.4.Isolation of a T-DNA insertion mutant of OsRDCP1and construction of35S:OsRDCP1transgenic rice plantsTo elucidate the possible functions of OsRDCP1,a single loss-of-function rice mutant line containing a T-DNA insertion in the OsRDCP1gene was isolated and referred to as osrdcp1.The inser-tion was annotated within the intron located in the5 -untranslated region of OsRDCP1on chromosome4(line1A-175-10)(Fig.5A).The homozygous mutant line was subsequently identified by multiplex PCR(Fig.5B).RNA gel blot analysis showed that the G2rice mutant seedlings contained an undetectable amount of OsRDCP1mRNA. The osrdcp1mutant plants did not exhibit any detectable mor-phological differences as compared to the wild-type plants under normal growth conditions.This may be due to complementation by other OsRDCP paralogs.Transgenic rice plants that overexpressed OsRDCP1under the control of the CaMV35S promoter were also constructed. Markedly heightened mRNA levels of OsRDCP1were observed inH.Bae et al./Plant Science180(2011)775–782779Fig.3.Structure and expression OsRDCP1.(A)Restriction enzyme map analysis of the rice OsRDCP1gene.Solid bars indicate coding regions and the solid line indicates the intron.The gene-specific probe used for RNA gel blot analysis is indicated.The green bar and red bar indicate N-terminal RING domain and C-terminal membrane-anchoring domain,respectively.(B)Phylogenetic relationship of RING Ub-ligase homologs from rice(OsRDCP1,OsRDCP2,OsRDCP3,OsRDCP4,and OsRDCP5),Arabidopsis(AtRma1, At4g28270,and At4g27470),hot pepper(CaRma1H1),and humans(HsRma1).(C–F)RNA gel blot analyses of OsRDCP1mRNA.Light-grown2-or4-week-old rice plants were subjected to(C)drought(0–30%loss of fresh weight),(D)cold temperatures(4◦C for0–24h),(E)high salinity(150mM NaCl for0–24h),(F)SA(100␮M for0–6h),or MeJA (100␮M for0–6h).Total RNA was isolated,separated by electrophoresis,and blotted.Filters were hybridized with32P-labeled OsRDCP1,OsEXP,OsLEA3,OsPHGPX,or OsActin under high-stringency conditions.(For interpretation of the references to color in thisfigure legend,the reader is referred to the web version of the article.)different independent35S:OsRDCP1T2transgenic lines (Fig.5C).These osrdcp1loss-of-function mutant and OsRDCP1-overexpressing transgenic rice plants were used for drought tolerance analysis.3.5.Over-expression,but not knock-out,of OsRDCP1alteredplant responses to water stressSix-week-old wild-type,G2osrdcp1mutant,and T2 35S:OsRDCP1(lines#8,#9,and#10)plants were grown in pots where water covered up to approximately40%of each plant. These plants were then subjected to drought stress by withholding water until the soil in the pots was completely dry.To maintain constant experimental conditions,the drought stress experiments were carried out in a growth chamber at26◦C and70%humidity, which resulted in the gradual dehydration of the soil.These growth conditions are similar to those of domesticfields where rice plants are cultivated in May and June in South Korea.After12–15days of dehydration,both wild-type and osrtbp1mutant plants displayed wilting(Fig.6).Thus,no significant phenotypic differences were detected between the wild-type and osrtbp1plants.In contrast,the 35S:OsRDCP1transgenic lines exhibited considerably less visual symptoms of drought stress.After15days of drought stress,these transgenic lines appeared healthy and showed reduced desiccation damage relative to the wild-type and osrtbp1plants(Fig.6).After re-watering for5–15days,the wild-type and osrtbp1plants were unable to recover and died(Fig.6).Under these experimental conditions,however,OsRDCP1-overexpressing lines successively survived and continued to grow.These results indicate that the OsRDCP1-overexpressing plants were more tolerant to water deficit as compared to the wild-type and osrdcp1plants.Overall, these results suggest that OsRDCP1is involved in plant responses to unfavorable water-stress conditions.Previous studies indicated that transgenic rice plants over-expressing stress-related proteins,including Ca2+-dependent protein kinase and trehalose-metabolizing enzymes,became more tolerant to abiotic stresses[31,32].In addition,constitutive expres-sion of stress-inducible transcription factors conferred improved resistance to water stress in rice.For example,enhanced toler-ance to drought and high salinity was observed in transgenic rice plants that over-expressed CBF3/DREB1A and ABF3,Arabidop-sis transcription factors in ABA-independent and ABA-dependent stress-response pathways,respectively[33].Transgenic rice plants were developed that constitutively expressed plant-specific NAC transcription factors,resulting in increased resistance to drought and salt stresses with the concomitant induction of a large num-ber of stress-related genes[34,35].Based on these previous results, along with our current data,we considered the possibility that780H.Bae et al./Plant Science180(2011)775–782Fig.4.In vitro self-ubiquitination analysis of OsRDCP1.(A)Bacterially expressed MBP-OsRDCP1was incubated at30◦C for0–120min in the presence of E1,E2,ATP,and Ub. Reaction mixtures were subjected to immuno-blot analysis with the anti-MBP antibody.(B)MBP-OsRDCP1was incubated for60min in the presence or absence of E1,E2, ATP,and/or Ub.(C)Wild-type MBP-OsRDCP1and its various mutants were analyzed in Ub ligase assays.K23R,C54S,and C86S are mutant proteins in which the Lys23,Cys54, and Val86residues were changed to Arg,Ser,and Ser,respectively.Fig.5.Molecular characterization of the loss-of-function osrdcp1T-DNA mutant line and OsRDCP1-overexpressing transgenic rice plants.(A)Schematic diagram of OsRDCP1 with the T-DNA insertion.Solid bars and the solid line indicate exons and the intron,respectively.Integration of T-DNA in the intron is shown as a triangle.Gene-specific (M1and M2)and T-DNA-specific(LBa-1)primers used in genotyping PCRs are indicated with arrows.(B)Genotyping of the osrdcp1mutant plants.Gene-specific and T-DNA-specific primer sets used for genomic PCR are shown on the left.W,wild-type;T,T-DNA insertion.(C)RNA gel blot analysis of OsRDCP1mRNA in wild-type,the homozygous G1osrdcp1mutant line,and several independent35S:OsRDCP1T1transgenic rice plants.rRNAs are shown as loading controls.H.Bae et al./Plant Science180(2011)775–782781Fig.6.Overexpression of OsRDCP1increased tolerance to drought stress in transgenic rice plants.Six-week-old wild-type,G2osrdcp1mutant,and T235S:OsRDCP1(lines#8, #9,and#10)plants were grown in pots where water covered approximately40%of each plant.These plants were then subjected to drought stress by withholding water for 15days to dry soil completely.Thereafter,plants were re-watered and their growth and morphologies were monitored for another15days.OsRDCP1,a stress-inducible RING E3Ub ligase,may be involved in turn-over or degradation of a negative transcription factor(s)that inhibits the expression of water stress-induced genes.Constitutive expression of OsRDCP1in rice cells may quicken the ubiquitin-dependent degradation of a certain transcriptional repressor(s), thereby enhancing the cellular level of stress-responsive proteins. The elevated levels of stress-responsive proteins would enhance tolerance against water stress.Alternatively,over-expression of OsRDCP1may induce the ubiquitination and subsequent degrada-tion pathway of a protein(s)that is responsible for the inactivation or degradation of water stress-related proteins,which would also result in increased levels of defense proteins in rice cells.These hypotheses are partially supported by recent results that rice RING E3OsDSG1and U-box E3OsPUB15are involved in the regulation of salt,drought,and oxidative stress responses[13,24].Though the actual mechanism remains to be defined,our results suggest that OsRDCP1plays a critical role in rice cellular responses to water deficits.In addition to OsRDCP1,there are four homologous OsRDCP paralogs(Fig.1).We speculate that these paralogs may also play a role in cellular processes in rice.This assumption is consistent with the notion that osrdcp1knock-out mutant plants display nor-mal phenotypes possibly due to complementation by other OsRDCP members.The basal levels of mRNAs for these paralogs are quite low except for OsRDCP4transcript(Fig.2).Thus,we are making an effort to obtain knock-out mutant and overexpressing transgenic rice plants to analyze rice RING-domain containing OsRDCP E3Ub ligase members.4.Concluding remarksHigher plants are continuously faced with diverse environmen-tal stresses during their life cycle.Dehydration stress can seriously impair the growth and development of many soil plants and is often responsible for large reductions in crop yields globally[36–40]. Thus,it is of immense importance to unravel the roles of stress-inducible genes and develop stress tolerant crop plants.Although the physiological function of OsRDCP1remains to be elucidated, we propose that OsRDCP1is involved in a subset of physiological responses that counteract dehydration stress in rice plants.Thus, OsRDCP1may be a useful target protein for the development of drought-tolerant rice plants.Future experiments will focus on pre-cisely defining the cellular and physiological roles of OsRDCP1with respect to drought tolerance.AcknowledgementsThis work was supported by grants from the National Research Foundation(Project No.2009-0078317funded by the Min-istry of Education,Science,and Technology,Republic of Korea), the BioGreen21Program(funded by the Rural Development。

摘要目的：环指蛋白128 (RNF128)是E3泛素连接酶家族成员之一。

其功能的研究主要集中在其对免疫效应T细胞功能的影响，目前已经证实其在平衡外周免疫耐受性和自身免疫疾病方面发挥着关键作用。

最新的研究提示其异常表达可能参与肿瘤发生发展。

但到目前为止，其对非小细胞肺癌临床进程的影响及其潜在的调节机制仍不清楚。

本研究的主要目的是探讨RNF128在NSCLC中表达及与患者预后，并初步探讨其促进NSCLC进展的机制。

材料和方法：通过免疫组化、荧光定量PCR和Western blot测定NSCLC组织和相应癌旁组织中RNF128的RNA和蛋白的表达水平。

使用208例非小细胞肺癌组织微阵列( TMAs )的方法来评估RNF128表达，结合临床病理与患者随访资料分析RNF128表达与患者临床病理特征及预后的关系；调节RNF128表达后研究其表达与NSCLC细胞侵袭、移动、克隆形成和增殖的关系。

Transwell实验与划痕实验被用来评估细胞的侵袭能力。

结果：NSCLC组织中RNF128 mRNA的表达高于癌旁组织（2.60±1.25 vs 0.50±0.02），NSCLC组织中RNF128蛋白表达也高于相应癌旁组织（6.25±0.99 vs 3.77±0.55）。

免疫组织化学检测组织芯片中RNF128表达后分析显示，RNF128在NSCLC组织中的表达异质性明显，大部分癌组织RNF128表达高于相应癌旁组织；根据RNF128免疫染色强度和阳性细胞所占比，NSCLC组织分为RNF128低表达组（52%，108/208）与RNF128高表达组（48%，100/208），结合患者临床病理特征分析显示，RNF128的表达与NSCLC恶性临床病理特征显著相关，如淋巴结转移( P =1.16×10-4)和进展期肿瘤( P=0.012 )，而结合预后分析证实RNF128高表达组的OS明显低于RNF128低表达组( 34.1 % vs 65.9 %，P =1.5×10-5 )。

chip蛋白e3酶类型全文共四篇示例，供读者参考第一篇示例：Chip蛋白和E3酶相互作用在细胞的生物代谢和信号传导中发挥着重要的作用。

Chip蛋白是一种通过HSP90结合并诱导其泛素化降解的E3泛素连接酶。

E3酶是介导泛素蛋白降解的关键酶类。

Chip蛋白通过诱导特定蛋白的泛素化降解，调控了多种信号通路的活性。

在细胞内，Chip蛋白和E3酶的相互作用对于维持细胞内蛋白的稳态具有至关重要的作用。

目前，已经发现Chip蛋白可以与多种E3酶相互作用，比如Cbl、Parkin、PEX4等。

这些E3酶与Chip蛋白的结合形成了复杂的网络，调控了多种生物进程的进行。

Chip蛋白通过结合不同的E3酶，参与了包括蛋白质泛素化降解、细胞信号传导、细胞凋亡等多种过程。

Chip蛋白是一种特殊的E3泛素连接酶，它能够与多个底物结合并调控其泛素化降解。

Chip蛋白通过与HSP90的结合，促使底物的泛素化降解。

HSP90是一种分子伴侣蛋白，参与了多种蛋白的折叠和稳定。

Chip蛋白的结合促使底物的泛素连接酶结合，诱导了泛素化降解。

Chip蛋白和E3酶相互作用在多种疾病的发生发展中发挥着重要作用。

Chip蛋白和Parkin E3酶的相互作用参与了帕金森病的病理过程。

Chip蛋白通过促进Parkin E3酶对α-突触核蛋白的泛素化降解，破坏了突触的结构和功能，导致神经元的损害。

Chip蛋白和Cbl E3酶的相互作用也与多种癌症的发生有关。

Chip蛋白通过促进Cbl对肿瘤抑制基因蛋白的泛素化降解，抑制了细胞凋亡，导致肿瘤细胞的增殖和转移。

第二篇示例：Chip蛋白E3酶是一类与细胞周期、细胞生长、凋亡和基因表达等生物学过程密切相关的重要蛋白酶。

Chip蛋白E3酶可以通过将泛素与底部蛋白共价结合，将其目标蛋白转运到蛋白酶体进行降解，从而调控细胞内蛋白质的稳态水平，维持细胞的正常功能。

一、Chip蛋白E3酶的种类Chip蛋白E3酶主要可以分为以下几类：1. MDM2：MDM2是一种重要的Chip蛋白E3酶，它主要参与调控p53蛋白的降解。

E3泛素连接酶ITCH调节经典Wnt信号转导途径的
分子机制的开题报告
一、研究背景
Wnt信号转导通路是细胞极为重要的信号转导途径之一，它参与了胚胎发育、细胞增殖、分化、凋亡以及肿瘤形成等多种生物学过程。

经典的Wnt信号转导途径分为Wnt/β-catenin依赖性通路和Wnt/β-catenin 非依赖性通路，在正常的生理状态中，Wnt/β-catenin依赖性通路处于关闭状态。

然而，失控的Wnt/β-catenin依赖性通路活化已被证明与多种肿瘤的发生、发展、转移有关。

ITCH是一种泛素连接酶，在Wnt信号转导途径中扮演重要的调节作用。

ITCH能够泛素化并降解Wnt信号通路中的调节蛋白，如Dishevelled和LEF-1等，从而抑制Wnt/β-catenin信号通路的活性。

一些研究还表明ITCH能够影响Wnt/β-catenin非依赖性通路的正常运作。

因此，ITCH在细胞中的功能和分子机制已经成为研究的关键点。

二、研究内容
本研究旨在探究ITCH在Wnt信号转导途径中的分子机制，具体研究内容包括以下方面：
（1）ITCH是否能够调节Wnt/β-catenin依赖性通路的活性。

（2）ITCH对Wnt/β-catenin非依赖性通路的调节作用及其分子机制。

（3）ITCH在Wnt信号转导途径中的上游调节机制。

三、研究意义
通过深入研究ITCH在Wnt信号转导途径中的分子机制，可以更好地了解ITCH的功能以及其在肿瘤等相关疾病中的作用。

另一方面，该研
究可以为寻找与Wnt信号转导途径相关的新的靶向治疗方法和抗癌药物提供新的思路和理论基础。

网络出版时间：２０２３－０９－２７１５：３０：１３　网络出版地址：ｈｔｔｐｓ：／／ｌｉｎｋ．ｃｎｋｉ．ｎｅｔ／ｕｒｌｉｄ／３４．１０８６．Ｒ．２０２３０９２６．１４２６．００６Ｅ３泛素连接酶在结直肠癌中的作用机制研究进展李　芳１，王　珏１，晏睿阳１，李凯杨１，沈　慧１，王　丽１，张　静１，张云清２（延安大学１．医学院，２．附属医院病理科，陕西延安　７１６０００）收稿日期：２０２３－０７－１０，修回日期：２０２３－０８－１０基金项目：国家自然科学基金资助项目（Ｎｏ８２２６０５３０）；陕西省自然科学基础研究计划项目（Ｎｏ２０２２ＪＱ ９０７）；陕西省高校科协青年人才托举计划项目（Ｎｏ２０２１０３０９）；２０２２年省级大学生创新创业训练计划项目（ＮｏＳ２０２２１０７１９０８９）作者简介：李　芳（１９９０－），女，博士，讲师，研究方向：结直肠癌分子调控机制，Ｅ ｍａｉｌ：１８７９２８７３１９８＠１６３．ｃｏｍ；张　静（１９８２－），男，博士，教授，研究方向：肿瘤药理学，共同通信作者，Ｅ ｍａｉｌ：ｙａｄｘｚｊ＠ｙａｕ．ｅｄｕ．ｃｎ张云清（１９７８－），男，硕士，副主任医师，研究方向：肿瘤致病机制，通信作者，Ｅ ｍａｉｌ：ｚｈａｎｇｙｑ２８８１１２３＠１６３．ｃｏｍｄｏｉ：１０．１２３６０／ＣＰＢ２０２２１２０２２文献标志码：Ａ文章编号：１００１－１９７８（２０２３）１０－１８１１－０４中国图书分类号：Ｒ３２９ ２５；Ｒ３２９ ２８；Ｒ３４１ ３１；Ｒ７３５ ３５；Ｒ９７７ ６摘要：结直肠癌（ｃｏｌｏｒｅｃｔａｌｃａｎｃｅｒ，ＣＲＣ）作为全球发病率和致死率最高的恶性肿瘤之一，其致病机制十分复杂，至今尚未完全阐明。

泛素化在ＣＲＣ的发生发展过程中扮演重要角色，其调控作用主要依赖于Ｅ３泛素连接酶泛素化修饰底物蛋白使之活性改变或发生泛素—蛋白酶体降解。

该文就ＲＩＮＧ（ｒｅａｌｌｙｉｎｔｅｒｅｓｔｉｎｇｎｅｗｇｅｎｅ）型和ＨＥＣＴ（ｈｏｍｏｌｏｇｏｕｓｔｏＥ６ＡＰＣ ｔｅｒｍｉｎｕｓ）型Ｅ３泛素连接酶在ＣＲＣ细胞增殖、凋亡、迁移、侵袭及化疗敏感性中的作用机制及这两类Ｅ３泛素连接酶的靶向抑制剂相关研究进展作一综述，为ＣＲＣ致病机制研究及其靶向治疗提供新的思路。

E3泛素连接酶CHIP与上皮性癌关系研究陈万涛【摘要】Ubiquitin modification is termed as an important way for thepost-translational regulation of cellular protein, which is found involved in a serial of biological process, such as cell cycle, DNA repair signaling transduction, transcriptional regulation, and so on. Recently, ubiquitin modifications occurred in the malignant tumors have been observed and studied broadly. The specificity of ubiquitin modification rely on the E3 ubiquitin ligases for its specific action with certain substrates, dysfunctionof which is shown to be related to the pathological processes of many diseases, including malignant tumors. The further analysis for the function of E3 ubiquitin ligases will strengthen our knowledge on the molecular mechanisms of carcinogenesis and development of malignant tumors, which might also provide biological targets for molecular classification and clinical treatment. Now a review was conducted for the function of one E3 ubiquitin ligase, carboxy terminus of Hsc70 interacting protein (CHIP), especially for its relationship with epithelial cancers.%泛素化修饰是机体一种非常重要的蛋白质翻译后修饰方式，其广泛参与细胞周期、DNA修复、信号转导、转录调控等生物学过程。