1、The Nobel Prize in Chemistry,2004“for the discovery of ubiquitin-mediated protein degradation”Avram HershkoIrwin RoseAaron CiechanoverWolf,D.H.“Proteasomes:The World of Regulatory Proteolysis”细胞周期和细胞分裂、分化发育的调节细胞对外环境的应激反应的调节神经网络的形态发生细胞表面受体和离子通道的下调DNA修复免疫和感染反应的调节细胞器的生物生成Functions of Ubiquitination-med
2、iated protein degradationCell Cycle by UbiquitinationE2FMSG1G2DK4/6EK2p21E2FAK2BCDC2p53securinSLBPRbDifferent modification states of p53 and MDM2Daniela Hoeller et al.Ubiquitin and ubiquitin-like proteins in cancer pathogenesis.Nat.Rev.Cancer,2006,6,776NF-B signalling pathwayscanonical pathwayNon-ca
3、nonical pathwayUbiquitinHuman Ub:MQIFVKTLTGKTITLEVEPNDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLADYNIQKESTLHLVLRLRGGYeast Ub:MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGLys-48Gly-76 Ubiquitin-proteasome system(UPS)Daniela Hoeller et al.Nat.Rev.Cancer,2006,6,776Protein Ubiqui
4、tinationRING E3HECT E3Functions of the Ub system Non-proteolytic(mono-Ub or K63 Ub chain)DNA repair,transcription,kinase activation.Proteolytic(K48 or K29 linked Ub chains)1)concentration control:key regulators(e.g.p53,cyclins,HIF)2)protein quality control:misfolded or damaged proteins(e.g.CFTR,prio
5、n,huntingtin)Different types of Lysine modifications(Ubiquitination and ubiquitin-like modifications)Daniela Hoeller et al.Nat.Rev.Cancer,2006,6,776Ubiquitin and ubiquitin-like proteinsUbiquitin-mediated ProteolysisE1Ub-S-E3 cell cycle signal transduction antigen processing tumor suppressionmisfolde
6、d proteinscyclinsCDK inhibitorsIk kBa atranscriptionc-junc-fosp53b b-cateninoncogenesmyc?virusesbacterialmanyE2Ub-S-substratesUb Ub Ub Ub Ub.Lys-48 C terminusCombinatorial nature of UPSAdaptors(a few)Rad23E3s:next wave of drug targetsProteasome inhibitor Velcade:multiple myeloma UPS:targets in anti-
7、cancer therapyE3s:determine substrate specificity,better targetsCancer,Huntingtons disease,spinocerebellar ataxias,Pagets disease of bone,AIDS-related cancer,muscle wasting,cystic fibrosis,Alzheimers disease,Parkinsons disease,hypertension,etc.Two major scientific issues central to our understanding
8、s of E3 ligase1.Mechanisms controlling the assembly and activity of E3 ligases2.Molecular nature of E3 ligases and their substratesTwo families of E3 ubiquitin ligases-S-E2substrateE3?UbUbUbUbUbUbHECT family E3(40)E6APE2substrateUbUbUbUbUbUbHECTRING family E3(1000s)CullinROCE2adaptorsubstrateUbUbUbU
9、bUbUbMDM2E2p53UbUbUbUbUbUbRINGRING fingerZinc fingerC1C2X2H3X2X2C4C5C6C7C8Zn+Zn+X17X17X2X5C1C2C5C6C6C7X2C/H4C/H3C7C8X2X2X2X(9-27)X(1-3)X(4-48)Zn+Zn+RING:Really Interesting New GeneRING Finger GenesRING Finger GenesC3H2C3(RING-H2)ROC1 CAICRNHIMD LCIECQA NQASATSE ECTVAWG VCNHAFHFHCISRWLKTRQ-V CPLD cel
10、l cycle controlROC2 CAICRVQVMD ACLRCQA EN-KQE DCVVVWG ECNHSFHNCCMSLWVKQNN-R CPLC unknownAPC11 CGICRMAFNG CCPDCKV P-GD DCPLVWG QCSHCFHMHCILKWLHAQQVQQH CPMC mitotsisPRAJA1 CPICCSEYVK-GEVATELPCHHYFHKPCVSIWLQKSG-T CPVC unknown PARKIN CITCTDVRSP-VLVFQCNSRHVICLDCFHLYCVTRLNDRQ 11P CVAGC parkison diseaseC3H
11、C4(RING-HC)c-Cbl CKICAENDKD-VKIEP CGHLMCTSCLTSWQE SEG-QG CPFC signal transductionSiah CPVCFDYVLP-PILQCQAGHLVCNQCRQKL-SC CPTC developmental signalingBRCA1 CPICLELIKE-PVSTKCDHIFCKFCMLKLLNQKKGPSQ CPLC tumor suppressionBARD1 CSRCANILKE-PVCLGGCEHIFCSGCISDCVGS-G CPVC tumor suppressionMURF CPICLEMFSK-PVVIL
12、PCQHNLCRKCANDVFQ18RFR CPSC signal transductionPML CQQCQAEA KC-PKLLPCLHTLCSGCLEASGM-Q CPIC transcription IAP1 CKVCMDKEVS-IVFIPCGHLVVCKDCAPSLRK-CPIC apoptosisIAP2 CKVCMDKEVS-VVFIPCGHLVVCQECAPSLRK-CPIC apoptosisIAP3 CKICMDRNIA-IVFVPCGHLVTCKQCAEAVDK-CPMC apoptosisC2H2C4HDM2 CVICQGRP KN-GCIVHGKTG HLMACFT
13、CAKKLKKRNK-P CPVC control p53Yeast:39Worm:154Fly:135Human:350Arabidopsis:490How Many E3 Ligases?Proteases:550Kinases:520Phosphatses:150DNA polymerases:16E1:16E2:53E3:Cullin-ROC complexes300-500RING proteins450HECT proteins40U-box proteins10-20Ubl ligases?1000Single-subunit RING type E3:Mdm2p53 bindi
14、ngZn1 19 102305 322438 478 491RINGNESNLSNoLS181-185 191-205 464-471Ubc940-59Smurf175-114 p300102-222 ARF210-244 acidic221 274L5/L11/L26284-374 RB273-321 Gankyrin412-437 Mdm2/MdmX438-491 Mdm2*AktS166/S186ATMS395MDM2 structure and interacting proteinsMDM2 ubiquitinates p53MDM2-1+2+3-4+5+6poly ubiquiti
15、nated p53UbcH5+-+-Honda et al.(1997)FEBS Letter 420:25 p53 can be activated by multiple stressesp53MDM2Oncogenic insultsARFDNA damageKinases(ATM,Chk1/2)Normal cellTumor cellMultiple-subunits RING type E3:Cullin-Roc-based ligase(CRL)complexE3 Ubiquitin LigaseUb-S-E2substrateE3UbUbUbUbE3 Ubiquitin Lig
16、aseUb-S-E2substrateE3?UbUbUbUbE3 Ubiquitin LigaseUb-S-E2substrateE3?UbUbUbUbRING Cullin binds ROCCullin 1ROCHuman Cullins and Cullin-related proteinsKipreos et al.(1996)Cell 85:829CUL5780APC2822Nd8CUL1776CUL2745CUL3768CUL4A759CUL4B717ROCPARC,CUL7APC:anaphase-promoting complexfunction to mediate mito
17、sis progressionresponsible for degradation of various mitotic proteins,including cylins and seurinsAPC11 is a RING finger proteinRING fingerZinc fingerC1C2X2H3X2X2C4C5C6C7C8Zn+Zn+X17X17X2X5C1C2C5C6C6C7X2C/H4C/H3C7C8X2X2X2X(9-27)X(1-3)X(4-48)Zn+Zn+RING:Really Interesting New GeneSkp1 brings F-box pro
18、tein to Cul1Bai et al.(1996)Cell 86:263-274Cullin 1Skp1F-boxROC1E2UbCullin 1Skp1F-boxROC1E2UbsubstrateNdF-box proteins:substrate receptorsF-box proteinsWLOCDC4FE2UbCullin 1Roc1Skp1UbUbUbUbUbUbUbiquitination of CDK inhibitor SIC1 by SCFCDC4-ROC ligasePPSIC1SCF-ROC E3 ubiquitin ligaseZheng et al.(2002
19、)Nature 416:703Rbx1/Roc1Cul1 CTDCul1Skp1Skp2 F-boxRepeat 1Repeat 2Repeat 3NNCCCNCSCF-ROC ligasesE2UbCullin 1Roc1UbUbUbUbUbUbUbUbUbUbUbUbUbUbUbUbUbUbSkp1Skp2p27FSkp1 Fbp 4Substrate 4FSkp1 Fbp 5Substrate 5FSkp1 Fbp 6Substrate 6FSkp1 Fbp 7Substrate 7FSkp1 Fbp 63Substrate 50Fb b-TrcPIk kBa aF Fbp 3Subst
20、rate 3FHuman Cullins and Cullin-related proteinsKipreos et al.(1996)Cell 85:829CUL5780APC2822Nd8CUL1SKP1776CUL2745CUL3768CUL4A759CUL4B717?ROCUbUbUbUbUbUbUbUbUbUbUbUbBCR(BTB-CUL3-ROC)E3 ligasesCullin 3Roc1E2UbUbUbUbUbUbUbBTB4Substrate 4BTBBTB5Substrate 5BTBBTB6Substrate 6BTBBTB5Substrate 5BTBBTB6Subs
21、trate 6BTBBTB200Substrate 200BTBMEI-1MEL26BTBNRF2Keap1BTBSubstrate3BTB3BTBCullin4:DDB1-DCAF-ROC ligasesE2UbCullin 4Roc1UbUbUbUbUbUbUbUbUbUbUbUbUbUbUbUbUbUbDDB1DDB2XPCWDDDB1 DCAF 4Substrate 4WDDDB1 DCAF 5Substrate 5WDDDB1 DCAF 6Substrate 6WDDDB1 DCAF 7Substrate 7WDDDB1 DCAF 60Substrate 60WDCSACSBWD D
22、CAF 3Substrate 3WDDDB1:UV-damaged DNA binding protein1DCAFs:DDB1-CUL4 associated factorsHow many cullin ligases?CUL1:63 F-box proteins(Skp1 as adaptor)CUL2/5:40 VHL/SOCS proteins(Elongin B/C as adaptor)CUL3:205 BTB proteins(BTB themselves as adaptors)CUL4A:60 DWD/WD40/DCAF proteins(DDB1 as adaptor)C
23、UL4B:?CUL7:?PARC:?1.Cullin:scaffold proteins2.ROC:RING finger,link cullin with E23.Skp1,Elongin B/C,BTB,DDB1:adaptor,link cullin with substrate receptor4.F-box,VHL,SOCS,BTB,DWD proteins:substrate receptors,link adaptor to substrate5.Substrate:E3 targets(largely unknown,so far,only 20 have been ident
24、ified)Pattern of Cullin-ROC ligases assembly and substrate recruitmentCullin-ROC ligases vs.built-in RING ligases MDM2E2Ubp53RINGUbUbUbUbUbCUL3ROCE2UbBTBsubstrateUbUbUbUbUbCUL1ROCE2UbSKP1FPPsubstrateUbUbUbUbUbHECT domain type E3Family of HECT type E3 ubiquitin ligasesHERC family:6 members,HERC1-6Ned
25、d4 family:9 membersSingle HECT E3s:E6AP,EDD,ARF-BP1/HECTH9HERC:HECT and RCC1-like domainRLD:RCC1-like domainNedd4:neural precursor cells-expressed developmentally downregulated 4Huge MW(80500 kDa),difficult to studyMore than 500 kDa100120 kDaARF-BP1/MuleNedd4 family E3sFunction of the catalytic acti
26、vities of HECT E3sMost come from Nedd4 familyCancer Metastasis Rev 2007,26(3-4):587-604.Known functions and substrates of Nedd4 family调控蛋白质运输、跨膜受体的信号转导、病毒芽生、骨发育及骨重塑、胚胎发育等过程膜受体：如TGF-RI、Notch、EGFR、VEGFR2、IGF-1R、CXCR4等内吞机器组分：如Cbl、Endophilin、Eps15、Hrs等转录因子：如Smads、KLF5、Runx2、AP-1、p53/p63/p73等其他底物：如PTEN、M
27、EKK2、RhoA、RNA pol II、Dvl-1等Regulation of the catalytic activities of HECT E3sBBRC,2007,354,329Mol.Cell,2005,19,297PNAS,2006,103,1717Cell,2007,130,651C2HECTE2 CCUbWWsMH2NTDSmad7PYC2HECTWWsDE2 recruitment by Smad7Phosphorylation-mediatedrelief of auto-inhibitionC2-HECT auto-inhibitionTargeting of WW d
28、omains linker of HECT type E3 ligase Smurf1 for activation by CKIP-1Human Nedd4 family of E3 ubiquitin-protein ligasesBioEssays 2006,28,617Ca2+&p-lipid bindingSubcellular localizationSubstrate binding&specificityE2 binding(N-lobe)Ubiquitin binding(C-lobe)C2HECTWWsSmurf1Smurf1 specifically suppresses
29、 osteoblast activity and adult bone formationSmurf1:Smad ubiquitination regulatory factor 1NATURE,1999,400,687JBC,2004,279,12854Cell,2005,121,8799.Physiological(blue)and pharmacological(orange)stimulators and inhibitors of bone formation and resorption are listed.The relative impact,where known,is r
30、epresented by the thickness of the arrows.Solid lines are current therapies and dotted lines putative ones.Determinants of skeletal homeostasis and bone massOsteoblast成骨细胞成骨细胞Osteoclast破骨细胞破骨细胞Regulation of the catalytic activities of HECT E3sBBRC,2007,354,329Mol.Cell,2005,19,297PNAS,2006,103,1717Ce
31、ll,2007,130,651C2HECTE2 CCUbWWsMH2NTDSmad7PYC2HECTWWsDE2 recruitment by Smad7Phosphorylation-mediatedrelief of auto-inhibitionC2-HECT auto-inhibitionQ:Whether and how Smurf1 ligase activity is regulated remains unclear.CKIP-1 is a novel Smurf1 interacting partnerScience 2005,307,1621Physiological fu
32、nction of CKIP-1 is unknown CKIP-1:casein kinase 2 interacting protein-1 Implicated in of PI3K-regulated muscle cell differentiation Regulation of AP-1 activity and apoptosis Regulation of cytoskeleton reorganization Recruitment of nuclear ATM and CK2 kinases to plasma membraneJBC,2000JBC,2004MCB,20
33、04EMBO J.,2005MCB,2005Cell.Signal.,2006Cell.Signal.,2007Cancer Res.,2007 PH LZCKIP-1PH:pleckstrin homology domainLZ:leucine zipperCKIP-1 is not a substrate of Smurf1CKIP-1 promotes Smurf1 self-degradationCKIP-1 promotes Smurf1 auto-ubiquitinationCKIP-1 enhances the trans-E3 activity of Smurf1towards
34、 its substrates Smad1/5 CKIP-1 enhances the trans-E3 activity of Smurf1towards its substrates Smad1/5 CKIP-1 specifically regulates E3 activity of Smurf1 and degradation of BMP pathway SmadsCKIP-1 interacts with Smurf1 both in vivo&in vitroMapping the interacting regionsNeither Smurf2 nor other memb
35、ers of Nedd4 family interacts with CKIP-1Paradox:binding WW domains v.s.E3-promoting effectPhospho-lipid bindingSubstrate bindingE2 binding(N-lobe)Ubiquitin binding(C-lobe)C2HECTWWsTwo possibilities:1,CKIP-1 and substrate bind to each of two WW domains.2,CKIP-1 binds to the linker region between the
36、 two WW domains.Smurf1CKIP-1 binds to the linker region between WW domains of Smurf1The linker region is highly homologous but different between Smurf1 and Smurf2.Mapping the key amino acids in the linkerAThe linker is essential for binding细节决定成败！The linker is essential for CKIP-1 promoting Smurf1 u
37、biquitinationCKIP-1 enhances the affinity of Smurf1 to its substrate CKIP-1 enhances Smurf1 E3 ligase activity CKIP-1 targets the WW linker of Smurf1 CKIP-1 augments Smurf1-Smad affinityQ:How about in vivo?Fig.S2cGene targeting in mouse embryonic stem cells has become the gold standard for determini
38、ng gene function in mammals.Gene targeting now lies at the centre of functional genomic analysis.Mario R.Capecchi,Nature Review Genetics,2005,6,507-512discovery of principles for introducing specific gene modifications in mice by the use of embryonic stem cells2007 Nobel Prize-Physiology/MedicineEst
39、ablishment of CKIP-1 deficient miceCKIP-1-/-mice display age-dependent increased bone mass(similar to Smurf1-/-mice)CKIP-1-/-Bigger bones Smurf1-/-CKIP-1-/-mice display enhanced osteoblast but not osteoclast activityCKIP-1-/-mice display reduced Smurf1 activityCKIP-1 is required for efficient Smurf1
40、 functionC2HECTMH1MH2Smad1/5E2CCUbSmurf1PYWWWWin the absence of CKIP-1:Smurf1 low activityC2HECTMH1MH2Smad1/5E2CCUbSmurf1PYCKIP-1WWWW in the presence of CKIP-1:Smurf1 high activityAB泛素连接酶Smurf1的新的调控模式Regulator-linker pattern第一次揭示HECT类E3的WW连接区的重要调控功能对于理解E3活性调控机制及应用研究提供了新思路发现了发现了HECT类泛素连接酶类泛素连接酶Smurf1
41、的的WW结构域结构域连接区介导的酶活调控新机制连接区介导的酶活调控新机制Lu K,Yin X et al.,Nat.Cell Biol.,2008,10(4),994-1002.通过通过RNAiRNAi敲低敲低CKIP-1CKIP-1表达、促进骨形成表达、促进骨形成进行骨质疏松治疗的前期研究进行骨质疏松治疗的前期研究Nature,2003,423:316.骨重塑是骨生物学的重要科学问题之一骨重塑是骨生物学的重要科学问题之一1,骨生长如何终止的？骨生长如何终止的？2,矿化为什么限制于骨骼系统？矿化为什么限制于骨骼系统？3,骨重塑如何维持整体骨量的稳态平衡的？骨重塑如何维持整体骨量的稳态平衡的？骨
42、重塑骨重塑骨质疏松骨质疏松骨形成和骨吸收是骨重塑的两个基本过程骨形成和骨吸收是骨重塑的两个基本过程Nature,2003,423:349.成骨细胞破骨细胞骨质疏松药物骨质疏松药物 需要深入开展成骨细胞调控研究需要深入开展成骨细胞调控研究破骨细胞分化与活化：破骨细胞分化与活化：60多个基因多个基因成骨细胞分化与活化：约成骨细胞分化与活化：约15个基因个基因对骨形成的机制了解远没有骨吸收清楚对骨形成的机制了解远没有骨吸收清楚骨形成是当前骨重塑研究中的重点问题骨形成是当前骨重塑研究中的重点问题我国的老年人群及骨质疏松治疗用药现状我国的老年人群及骨质疏松治疗用药现状 中国：中国：6060岁，岁，1.6
43、71.67亿人，亿人，12%12%，2020亿美元亿美元/年年 国家卫生部：居民慢性疾病患病率，高血压国家卫生部：居民慢性疾病患病率，高血压26.226.2，胃肠炎，胃肠炎10.310.3，骨质疏松，骨质疏松8.88.8，位列第三，位列第三 1994-19981994-1998，中成药，仙灵骨葆胶囊，活性维生素，中成药，仙灵骨葆胶囊，活性维生素D D 1998-1998-，阿仑膦酸盐，雌激素受体调节剂，雌激素替代治疗，阿仑膦酸盐，雌激素受体调节剂，雌激素替代治疗，降钙素，骨化三醇等降钙素，骨化三醇等 2009-2009-，第一个静脉用药双膦酸盐（艾本）在中国上市，第一个静脉用药双膦酸盐（艾本）
44、在中国上市 绝大多数靶向骨吸收，国际上目前只有甲状旁腺激素基于靶绝大多数靶向骨吸收，国际上目前只有甲状旁腺激素基于靶向骨形成，但向骨形成，但PTHPTH用药用药1818个月后出现骨吸收增加个月后出现骨吸收增加 研发新的促进骨合成代谢的新药已成为新的重点和热点方向研发新的促进骨合成代谢的新药已成为新的重点和热点方向（抑制破坏的同时积极推进重建）（抑制破坏的同时积极推进重建）nucleuscytoplasmPMBMP-Smad1/5通路介导骨形成通路介导骨形成BMP:骨形态发生蛋白骨形态发生蛋白Bone formationSmad4Smad1/5PPSmad1/5PPSmad4Smad1/5PPS
45、mad1/5PPCofactorPPPPBMPBMPR IIBMPR ISmad1/5PPSmurf1促促Smad1/5降解、抑制骨形成降解、抑制骨形成Smurf1:Smad ubiquitination regulatory factor 1，HECT类泛素连接酶类泛素连接酶 骨量上升骨量上升骨密度增加骨密度增加骨形成能力增强骨形成能力增强Smurf1-/-nucleuscytoplasmPMBone formationSmad4Smad1/5PPSmad1/5PPSmad4Smad1/5PPSmad1/5PPCofactorPPPPBMPBMPR IIBMPR ISmad1/5PPUbUb
46、UbSmad1/5Smurf1UbZhu H et al.,Nature,1999,400:687-93Zhao M et al.,JBC,2004,279:12854-9Yamashita M et al.,Cell,2005,121:101-13Smurf1活性过高将导致骨质疏松活性过高将导致骨质疏松骨形成骨形成骨吸收骨吸收 特征：骨量减少、骨组织微结构退行性改变、脆性增加特征：骨量减少、骨组织微结构退行性改变、脆性增加 危害：疼痛，骨折，致残，并发症危及生命危害：疼痛，骨折，致残，并发症危及生命 人群：绝经后妇女和老年人群人群：绝经后妇女和老年人群 趋势：社会老龄化加剧，患病率上升，患者
47、年轻化趋势：社会老龄化加剧，患病率上升，患者年轻化骨平衡骨平衡骨质疏松骨质疏松骨失衡骨失衡科学问题科学问题1.Smurf1的连接酶活性是如何受到调控的？的连接酶活性是如何受到调控的？军事医学科学院军事医学科学院 张令强张令强2.Smurf1的调控与骨发育、骨重塑的关系？的调控与骨发育、骨重塑的关系？3.可否基于可否基于Smurf1调控进行骨质疏松治疗研究？调控进行骨质疏松治疗研究？C2HECTMH1MH2Smad1/5E2CCUbSmurf1PYWWWWin the absence of CKIP-1:Smurf1 low activityC2HECTMH1MH2Smad1/5E2CCUbSm
48、urf1PYCKIP-1WWWW in the presence of CKIP-1:Smurf1 high activityCKIP-1靶向靶向Smurf1的的WW连接区连接区增强其泛素连接酶活性增强其泛素连接酶活性ABLu K et al.,Nat.Cell Biol.,2008,10(8),994-1002CKIP-1-/-小鼠：年龄依赖的骨密度和骨量增加小鼠：年龄依赖的骨密度和骨量增加 特异调控特异调控成体成体骨发育与骨重塑骨发育与骨重塑 CKIP-1-/-Bigger bones Smurf1-/-Lu K et al.,Nat.Cell Biol.,2008,10(8),994-1
49、002CKIP-1:casein kinase 2 interacting protein-1CKIP-1-/-小鼠小鼠成骨成骨细胞而非细胞而非破骨破骨细胞活性升高细胞活性升高骨提取物血清、尿成骨细胞分化矿化能力增加CKIP-1在骨组织的表达随年龄增长而上升在骨组织的表达随年龄增长而上升CKIP-1是一个新的骨形成负调控分子是一个新的骨形成负调控分子CKIP-1 siRNA的筛选的筛选建立成骨细胞靶向性建立成骨细胞靶向性siRNA递送系统递送系统动物体内功能评价动物体内功能评价可否下调可否下调CKIP-1促进骨形成、治疗骨质疏松？促进骨形成、治疗骨质疏松？设计合成设计合成 8条条CKIP-1
50、siRNAsi-3可在人、恒河猴、大鼠、小鼠细胞中可在人、恒河猴、大鼠、小鼠细胞中最为有效的干扰最为有效的干扰CKIP-1基因的表达基因的表达 修饰后的修饰后的CKIP-1 siRNA未引起未引起IFN-和和TNF-水平增加水平增加 All of the designed siRNAs were non-immunostimulatory modified by adding two nucleotides(dT*dT)at 3 overhangs and 2-Ome at U and C sites on the siRNA sense-strand.建立了建立了DSS6寡肽与寡肽与Lipo
