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1、泛素化蛋白修饰 Protein Modification by Ubiquitination北京协和医学院研究生课程：细胞信息与调控2010年10月13日,The Central Dogma（Revised),E1,-E1,E2,-E2,底物,E3,ATP,AMP+PPi,Ub,Ub,Ub,泛素蛋白酶体通路,Ub:泛素(Ubiquitin)，一高度保守的，由76个氨基酸组成的多肽。E1:泛素激活酶(Ubiquitin-activating enzyme)，人类仅有2种E1。E2:泛素载体蛋白(Ubiquitin-carrier protein)，人类约有 30 种E2s。E3:泛素蛋白连接酶(
2、Ubiquitin-protein ligase)，人类有500多种E3s。,9,泛素化(ubiquitination)即蛋白质被泛素(ubiquitin，由76个氨基酸组成的多肽）共价修饰的过程，在几乎所有的真核细胞活动中起着关键作用。泛素 蛋白酶体(proteasome)通路则是真核细胞内最主要的蛋白质降解途径。泛素化调控的细胞活动至少包括:细胞周期（Cell cycle progression）细胞凋亡（Apoptosis）转录调控（Transcriptional regulation）DNA修复（DNA repair）免疫应答（Immune response）蛋白质降解及质量控制（Pr
3、otein degradation and quality control),2,The Nobel Prize in Chemistry 2004 for the discovery of ubiquitin-mediated proteolysis,Aaron Ciechanover Avram Hershko Irwin Rose 阿龙.西查诺瓦 阿夫拉姆.赫希科 欧文.罗斯,3,近年与泛素蛋白酶体相关的PubMed论文数在持续增长,4,篇数,年份,本专题内容,1.泛素蛋白酶体通路的组成及重要性2.泛素化与泛素化酶3.去泛素化酶4.泛素化异常与人类重大疾病的发生5.蛋白酶体构成6.蛋白酶
4、体抑制剂与药物7.调节蛋白酶体活性的新机制8.类泛素蛋白（SUMO,NEDD8,ISG15,APG8,APG12，PUP等）,5,中国协和医科大学出版社，2008年,泛素介导的蛋白质降解Ubiquitin-Mediated Proteolysis主编：邱小波 王琛 王琳芳,参考书,陈竺和诺贝尔奖得主Aaron Ciechanover作序,蛋白质泛素化研究简史,1975年，Goldstein误将泛素（Ubiquitin)当作胸腺激素而发现。1977年，Goldknopf和Busch认定组蛋白2A与泛素以异肽键（Isopeptide Bond）结合。1977年，Goldberg证明人类细胞中存在一
5、可溶的，直接依赖于能量的非溶 酶体类蛋白酶。1978年，Ciechanover 和 Hershko发现了APF-1是Goldberg系统中蛋白酶 系的必要成分，结果发表在BBRC。1980年，Ciechanover，Hershko和Rose等人证明了APF-1与当时已被发 现的泛素是同一物质。1984年，Finley和Varshavsky等发现泛素在细胞周期中的重要性。1987年，Goldberg和Rechsteiner两个小组几乎同时分离出分子量很大的 依赖于ATP并降解泛素化底物的蛋白水解酶。1988年，Goldberg将这种蛋白水解酶命名为Proteasome（蛋白酶体）。1997年，Y
6、eh 等小组发现类泛素蛋白SUMO和NEDD8的功能。2003年，美国FDA批准了用Velcade来治疗多发性骨髓瘤；Velcade 是 在Goldberg发明的蛋白酶体抑制剂MG132的基础上研发而成的。2004年，Ciechanover，Hershko和Rose因发现泛素介导的蛋白质降解共 同获得诺贝尔化学奖。2008年，Darwin等在细菌中发现类泛素化修饰。,7,Figure 8-34 Ubiquitin(泛素）,泛素由76个氨基酸残基组成，其中包括7个赖氨酸残基(K),其C末端可与底物的赖氨酸残基形成异肽键，从而引起底物泛素化。泛素的K11、K29、K48和K63均能参与形成泛素与泛
7、素间的异肽键(Isopeptide bond)。,10,E1与ATP结合后，催化泛素羧基末端腺嘌呤化，同时释放无机焦磷酸（PPi）。E1活化位点的半胱氨酸硫醇激活泛素腺嘌呤核苷酸中间产物上的羧基，形成一个泛素硫酯键。E2激活的重要条件是位于UBC结构域的一个保守半胱氨酸催化位点与泛素羧基端形成一个硫酯键。E3催化被E2活化的泛素C-端甘氨酸与底物或下一个泛素的赖氨酸间形成泛素异肽键(Isopeptide bond)。,11,调节蛋白质内吞，修饰和转录,靶蛋白必须经多聚泛素(多于4个泛素分子)修饰才能被蛋白酶体识别与降解。,泛素化模式（Ubiquitination Modes）,调节蛋白质的活性
8、和定位,K48 polyubiquitylation,K63 polyubiquitylation,Monoubiquitylation,Multiple monoubiquitylation,12,a.RING finger domain（SCF复合体，APC，MDM2，Parkin,和c-Cb1）b.U-box domain（CHIP)c.HECT domain（能与底物形成硫酯键)d.N-End Rule,E3的种类,13,E3泛素连接酶以单体和复合体两种形式存在,14,Amerik and Hochstrasser,2004,去泛素化作用（Deubiquitination）,15,泛素
9、-C-末端水解酶，通常参与蛋白降解后泛素分子的再利用及对多聚泛素链的修饰，也参与由泛素前体产生泛素单体的过程（如UCH37、UCH-L1)。泛素特异性蛋白酶，参与去除蛋白质上的多聚泛素链，也可从短泛素链的末端去掉单个泛素（如USP8)。去泛素化酶属半胱氨酸蛋白酶，目前已发现90多种。,去泛素化酶可分为两类：,去泛素化酶(Deubiquitinating enzyme),16,泛素化途径的异常与人类重大疾病,神经退行性疾病（如帕金森氏症）：Parkin,UCH-L1癌症:BRCA1,CYLD,Mdm2,Nrdp1,pVHL 传染病病原体的入侵、致病机制:E6-AP,17,泛素连接酶Parkin的
10、失活与帕金森氏症（AR-JP),Current Opinion in Neurobiology 2004,14:384389,18,Current Opinion in Neurobiology 2004,14:384389,Parkin Substrates,19,UCH-L1为一去泛素化酶，它在帕金森氏症病人中的突变可能导致其酶活性的丧失，减少了泛素自由单体的供应及底物蛋白的降解，进而导致黑质病变。,帕金森氏症中的去泛素化,20,失调蛋白 底物 修饰 肿瘤类别SKP2 p27(KIP)Polyubiquitylation Malignant melanomaMDM2 p53 Polyubi
11、quitylation Non-small-cell lung cancer,soft-tissue carcinoma,colorectal cancer HAUSP p53,MDM2 De-ubiquitylation Non-small-cell lung cancer lymphoma APC Cyclin B,Polyubiquitylation Colorectal cancer securin FANCL FANCD2 Monoubiquitylation Fanconi anaemia-related cancers CYLD IKK De-ubiquitylation Cyl
12、indromatosis IAP2 BCL10 Polyubiquitylation MALT lymphomas CBL RTKs Multiple Lymphoma,AML and monoubiquitylation gastric carcinoma pVHL HIFPolyubiquitinationvon Hippel-Lindau disease,泛素化失调与肿瘤,21,CYLD具有TRAF2（TNF-receptor-associated factor 2，为 I B 的泛素连接酶)结合位点，可促进 I B 的去泛素化。,去泛素化酶CYLD的突变与圆柱瘤Cylindromato
13、s的发生相关,22,CYLD促进IB的去泛素化，从而抑制NF-B的活性,23,N ENGL J MED 2004,350:187-188,圆柱瘤Cylindromatos也称“头帕肿瘤综合症”(turban tumour syndrome),其患者细胞CYLD的两个等位基因都发生了突变，结果使患者细胞中CYLD丧失功能，导致核转录因子NF-B的过度活化；启动基因的转录，导致细胞的过度增殖,24,泛素连接酶Mdm2和E6-AP与p53的降解和癌症,25,Ubiquitination occurs on histones H1,H2A,H2B and H3.This modification on
14、 different histones plays distinct roles in regulation of chromatin structures,and hence gene expression and genome stability,Ubiquitination and transcriptional regulation,Ubiquitination of histone H2A and H2B has opposite effects on transcription.Ubiquitination of H2B is associated with gene activa
15、tion,while H2A ubiquitination contributes to gene silencing,Wang et al.,NATURE|VOL 431|14 OCTOBER 2004,Lee et al.,Cell 131,10841096,2007,Histone Crosstalk between H2B Mono-ubiquitination and H3 Methylation Mediated by COMPASS,Cps35 Is Required for Translating Histone Crosstalk between H2B Monoubiqui
16、tination and H3 Methylation by COMPASS,1.Nrdp1 是一新的含有RING finger 结构域的泛素连接酶。,Nrdp1 是一促进ErbB3及BRUCE泛素化和降解的泛素连接酶,2.ErbB3 是表皮生长因子受体家族(EGFR family)成员之一，在生长，发育及细胞存活中起着关键的作用。该家族与许多 肿瘤密切相关，已成为癌症治疗的重要标靶之一。3.BRUCE/Apollon 是一含有BIR domain（凋亡抑制蛋白的标志性结构域）和E2 domain 的巨型(530 kDa)膜蛋白,26,Nrdp1促进细胞凋亡并可能与癌症和帕金森氏症相关,1.Q
17、iu*&Goldberg*(2002),PNAS2.Qiu*et al.(2004),EMBO J3.Qiu*&Goldberg*(2005),J.Biol.Chem.4.Liu,Qiu*et al.Manuscript in preparation,27,本专题内容,1.泛素蛋白酶体通路的组成及重要性2.泛素化与泛素化酶3.去泛素化酶4.泛素化异常与人类重大疾病的发生5.蛋白酶体构成6.蛋白酶体抑制剂与药物7.调节蛋白酶体活性的新机制8.类泛素蛋白,28,The Biochemical Roles of Protein Degradation in a Cell,1.溶酶体(Lysosome
18、s)Non-selective Hydrolyze proteins from autophagy Digest proteins from endocytosis Membrane proteins Extracellular proteins 2.泛素-蛋白酶体通路Selective and(Ubiquitin-Proteasome Pathway)ATP-Dependent Regulate biochemical reactions Discard unnecessary proteins Degrade damaged or misfolded proteins Present in
19、ternalized antigens in immunological responses,29,20S 催化颗粒,19S调节颗粒,19S,10 nm,电镜下的26S蛋白酶体(Proteasome),Yifan Cheng,Xiao-Bo Qiu et al.,unpublished data,30,26S 蛋白酶体的亚基组成,31,32,免疫蛋白酶体(Immunoproteasome)负责抗原提呈(Antigen Presentation),免疫蛋白酶体的催化颗粒(20S)有三个特殊亚单位LMP2、LMP7 和MECL-1。其调节颗粒为PA28（11S）。,20S颗粒,33,MHC cla
20、ss I antigen presentation pathway,T cell,PeptideMHC class I,Protein antigen,Proteasome+/-Ubiquitin,Peptides(2-24 a.a.),TAP(8-16 a.a.),Endoplasmic Reticulum,CD8+,Golgi,8-10 a.a.,Amino acids,ERAP1,34,35,The proteasome inhibitor,Velcade(Botezomib/PS-341),has been approved by FDA to treat multiple myelo
21、ma and mantle cell lymphoma.,36,中药雷公藤中的抗癌活性成分雷公藤红素Celastrol是一种蛋白酶体抑制剂，它能通过控制癌细胞的蛋白酶体活性进而诱发癌细胞凋亡。Yang,H.et al.,Cancer Res.(2006),37,1、蛋白酶体亚单位hRpn13能激活存在于蛋白酶 体的去泛素化酶UCH37。2、c-Abl通过引起蛋白酶体磷酸化而抑制其活性。,蛋白酶体的调控,38,hRpn13能激活去泛素化酶UCH37,control,+hRpn13,S5a,+hRpn13+Ub-H,Qiu,X.B.*et al.,EMBO J.（2006）25:5742-5753
22、.,39,其它研究表明Rpn13可作为泛素受体而结合于泛素链的近端,Husnjak et al.Nature 453:481,2008Schrener et al.Nature 453:548,2008,41,c-Abl通过引起蛋白酶体磷酸化而抑制其活性,Liu,X.,Huang,W.,Li.,C.,Li,P.,Yuan,J.,Li,X.,Qiu,X.B.,Ma,Q.,and Cao,C.(2006).Molecular Cell.22,317-327.,42,The 26S proteasome is a multisubunit complex,consisting of the 20S
23、catalytic particle and the 19S regulatory particle,and selectively degrades ubiquitinated proteins in the presence of ATP.Proteasomes are responsible for degradation of most cellular proteins,while lysosomes degrade extracellular proteins and most membrane proteins.Upon immune responses,cells genera
24、te special types of proteasomes,immunoproteasomes,which possess distinct regulatory particle(11S)and catalytic particle(with different catalytic subunits),for more efficient antigen presentation.,Proteasomes are responsible for degradation of most cellular proteins,本专题内容,1.泛素蛋白酶体通路的组成及重要性2.泛素化与泛素化酶3
25、.去泛素化酶4.泛素化异常与人类重大疾病的发生5.蛋白酶体构成6.蛋白酶体抑制剂与药物7.调节蛋白酶体活性的新机制8.类泛素蛋白（SUMO,NEDD8,ISG15,ATG8,PUP等）,43,类泛素蛋白及其同源性,44,Ubiquitin-Like Protein Involved in the Proteasome Pathway of Mycobacterium tuberculosis Pearce et al.(2008)Science 322:1104PUP-conjugation is found remarkably in mycobacteria and allied acti
26、nobacterias,and is sporadically present in other lineages,such as verrucomicrobia,nitrospirae,deltaproteobacteria and planctomycetes.Iyer et al.(2008)Biology Direct,3:45,Protein modification by ubiquitin-like protein,PUP,in prokaryotes,PUP is predicted to encode a 64amino acid protein with a molecul
27、ar size of 6.9 kD,类泛素蛋白的结构高度相似,Ubiquitin,ISG15,Sumo,NEDD8,45,Activating enzyme complex has two subunitsAOS1 is similar to the N-terminal half of Ub E1UBA2 is homologous to the C-terminal half of Ub E1 and contains the cysteine necessary for linkage with SUMOConjugating enzyme:UBC9,SUMO(Small ubiquit
28、in-like modifier)101 amino acids,46,SUMO修饰的功能,47,SUMO化修饰的底物,48,Desumoylating enzymes,Ulp1Has both isopeptidase and C-terminal hydrolase activityRequired for G2/M transitionSumo C-terminal hydrolases,e.g.SENP1,49,Nedd8(neural precursor cell-expressed developmentally downregulated)-81 residues,Activat
29、ing enzyme complex(2 subunits)-APP-BP1 and UBA3The APP-BP1 N-terminal half is homologous to the N-terminal half of ubiquitin E1UBA3 is homologous to the C-terminal half of ubiquitin E1 and contains the cysteine required for thiol ester linkage with Nedd8,Conjugating enzyme:UBC12,50,Function of Neddy
30、lation,Substrates(SCF components)Cdc53 in yeastCullin family members in humans,51,Crystal structure,52,Deneddylation:COP9 Signalosome(CSN),500 kDa protein complex8 distinct subunitsCSN5/Jab1 is catalytic component of isopeptidaseHomology to subunits of the proteasome(CSN5=RPN11),53,Other NEDD8-speci
31、fic hydrolases and isopeptidases,UCH-L3 has NEDD8 C-terminal hydrolase activityUSP21 deconjugates ubiquitin and deneddylates from NEDD8 conjugates,54,ISG15(Interferon-stimulated gene 15),Induced by interferon,lipopolysaccharides,or upon viral infectionsE1(ISG15 activating enzyme)UBP43,ISG15-specific
32、 isopeptidase Modified proteins:JAK-STAT proteins,55,Aut7/Apg8/ATG8 in Autophagy,Aut7/Apg8 is necessary for formation of membrane structures in autophagosomesSubstrate:Phosphatidylethanolamine(a phospholipid),56,Aut7/Apg8/ATG8 and Apg12/ATG12 in Autophagy,Aut7=Apg8,57,Deregulated Substrate Modificat
33、ion Tumour type/disease SENP1 De-sumoylation Infantile teratoma,thyroid oncocytic adenoma SUMO RB Sumoylation Retinoblastoma tumour p14ARF Sumoylation Melanoma SUMO1 Sumoylation Anaplastic large-cell lymphoma SUMO2,UBA2 Sumoylation Hepatocellular carcinoma UBC9 BCL2 Sumoylation Ovarian tumour NEDD8
34、Neddylation Prostate malignancy FAT10 Fatylation Hepatocellular carcinoma,stomach,intestinal,colorectal,uterine,cervical and ovarian cancer UBE1L ISGylation Acute promyelocytic leukemia,lung tumour ISG15 ISGylation Melanoma ISG15 ISGylation Adult T-cell leukemia,bladder,breast,colorectal,ovarian and prostate cancer UBP43 De-ISGylation AML _,Aberrant signaling of ubiquitin-like proteins and cancer,Hoeller et al.,6,776-788(2006),58,小结,Wechman et al.,Nat.Rev.Mol.Cell Biol.,6,599-609(2005),59,课外作业题,简述泛素化的主要生物化学特点，并说明它在细胞活动中的作用。简述蛋白酶体的组成及其与其它蛋白酶的主要差别。简单介绍类泛素蛋白的种类及其主要功能。,谢谢大家,60,
