1、主要内容主要内容 概述概述1.泛素和泛素化泛素和泛素化2.26S蛋白酶体蛋白酶体3.泛素化的生物学意义泛素化的生物学意义4.类泛素蛋白类泛素蛋白5.非泛素化的蛋白降解途径非泛素化的蛋白降解途径AdvancedMGlecture第七章蛋白质降解蛋白降解速率蛋白降解速率肌球蛋白肌球蛋白 血红蛋白血红蛋白晶状体晶体蛋白晶状体晶体蛋白 己糖激酶己糖激酶 AdvancedMGlecture第七章蛋白质降解影响蛋白寿命的因素 内在稳定性(“遗传编码序列)固有的生物物理特征 外界环境 温度 pH 降解活性 特异机制 定位 相互作用蛋白(partners)AdvancedMGlecture第七章蛋白质降解蛋白
2、质的半衰期与N-端氨基酸残基的关系被称为被称为N末端规则，既存在于原核生物，也存在于真核生物中。末端规则，既存在于原核生物，也存在于真核生物中。AdvancedMGlecture第七章蛋白质降解 成熟蛋白N-端的第一个氨基酸第一个氨基酸（除已被切除的N端甲硫氨酸之外，但包括翻译后修饰产物）在蛋白的降解降解中有着举足轻重举足轻重的影响。当某个蛋白质的N端是甲硫氨酸、苷氨酸、甲硫氨酸、苷氨酸、丙氨酸、丝氨酸、苏氨酸和缬氨酸丙氨酸、丝氨酸、苏氨酸和缬氨酸时，表现稳定。其N端为赖氨酸、精氨酸赖氨酸、精氨酸时，表现最不稳定最不稳定，平均2-3分钟就被降解了。AdvancedMGlecture第七章蛋白质
3、降解真核细胞中的两种蛋白降解途径真核细胞中的两种蛋白降解途径 Lysosomes(溶酶体参与的蛋白降解途径溶酶体参与的蛋白降解途径)Receptor mediated endocytosis&phagocytosis Proteasomes:for endogenous proteins(蛋白酶复蛋白酶复合体参与的蛋白质降解途径合体参与的蛋白质降解途径)transcription factors cell cycle cyclins virus coded proteins improperly folded proteins damaged proteins耗能与不耗能的差别耗能与不耗能的差
4、别AdvancedMGlecture第七章蛋白质降解Major pathways for ubiquitin-dependent protein degradation in eukaryotic cells.Mol Cell Neurosci 49(2012)387393AdvancedMGlecture第七章蛋白质降解Death by ProteasesAdvancedMGlecture第七章蛋白质降解 在大肠杆菌中，许多蛋白质的降解是通过一个依赖于ATP的蛋白酶(称为Lon)来实现的。当细胞中存在有错误或半衰期很短的蛋白质时，该蛋白酶就被激活。每切除一个肽键要消耗两个分子ATP。Adva
5、ncedMGlecture第七章蛋白质降解 半衰期(Half-life)蛋白质被降解或变性一半时所需要的平均时间(依赖于你所采用的方法)Turnover 蛋白从其合成到降解的时间间隔(Lifespan)稳定性(Stability)-Subjective property 一个成熟蛋白在某一条件下的天生具有易于变性的趋势，为蛋白的固有属性 变性(Denaturation)一个多肽整个或部分去折叠 降解(Degradation)肽段的蛋白水解 泛素化(Ubiquitination)=Ubiquitylation 蛋白酶(Protease)=peptidase术语术语(Terminology)Adv
6、ancedMGlecture第七章蛋白质降解 有些蛋白的半衰期蛋白的半衰期长达数年，如结晶蛋白；有些蛋白的半衰期只有几分钟，如一些信号传递蛋白。蛋白在稳定性方面的差异差异不是随机的，而是一个程序化是一个程序化过程过程。在发现泛素-蛋白酶体系统之前，细胞中的蛋白质降解被认为主要依赖于溶酶体。AdvancedMGlecture第七章蛋白质降解 2004年诺贝尔化学奖年诺贝尔化学奖：阿龙切哈诺夫(以色列)阿夫拉姆赫尔什科(以色列)欧文罗斯(美)找到了人体细胞控制和调节某种人体蛋白质数量多少的方法。他们发现人体细胞通过给无用蛋白质给无用蛋白质“贴标贴标签签”的方法，帮助人体将那些被贴上标记的蛋白质进行
7、“废物处理废物处理”，使它们自行破裂、自动消亡。泛素调控泛素调控的蛋白质降解的蛋白质降解具有重要的生理意义，它不仅能够清除错误蛋白质，对细胞生长周期、DNA复制以及染色体结构都有重要的调控作用，而且对于理解细胞的许多生理过程和新药的开发具有重要的意义。蛋白质降解是一个有序的过程。蛋白质降解是一个有序的过程。AdvancedMGlecture第七章蛋白质降解 就目前的进展而言，真核细胞控制蛋白质稳定性的主要途径就是通过泛素泛素(ubiquitin)修饰目标蛋白(target protein，也称为底物蛋白substrate protein)，然后这个被泛素标记的蛋白质通过一个叫“26S蛋白酶蛋白
8、酶体体”的蛋白质复合体降解掉。这种通过泛素化途径降解的蛋白质包括细胞周期调节子、转录因子和其他重要的蛋白质。AdvancedMGlecture第七章蛋白质降解1.泛素泛素(Ubiquitin)和泛素化(Ubiquitination)真核蛋白的降解依赖于泛素泛素(Ubiquitin)76 氨基酸残基 高度保守 3 amino acid changes yeast to human 热稳定(Thermostable)1975 年从小牛的胰脏中分离出来AdvancedMGlecture第七章蛋白质降解Ubiquitin AA SequenceMQIFVKTLTGKTITLEVEPSDTIENVKAK
9、I 6 11 29QDKEGIPPDQQRLIFAGKQLEDGRTLSD 31 48 YNIQKESTLHLV LRLRGG 63AdvancedMGlecture第七章蛋白质降解真核细胞依赖于泛素的蛋白降解途径真核细胞依赖于泛素的蛋白降解途径 1.细胞内即将被降解的蛋白首先在细胞内即将被降解的蛋白首先在ATP的的作用下与泛素相连（需要作用下与泛素相连（需要E1,E2,E3三种酶三种酶的参与）的参与）2.将该复合体运送到特定的蛋白降解体系将该复合体运送到特定的蛋白降解体系“26s proteasome”中直到完全降解中直到完全降解 3.ubiquitin recycle在细胞内存在在细胞内存
10、在3 种重要的酶种重要的酶,即泛素活化酶即泛素活化酶(ubiquitin-activating enzyme,简称简称E1)、泛素结合酶、泛素结合酶(ubiquitin-conjugating enzyme,简称简称E2)、泛素蛋白连接酶、泛素蛋白连接酶(ubiquitin-proteinligating enzyme,简称简称E3)。AdvancedMGlecture第七章蛋白质降解AdvancedMGlecture第七章蛋白质降解Ubiquitination(泛素化)Ubiquitinating enzymes E1,E2,E3-thiol ester bond(硫羟酸酯键)Final t
11、arget-isopeptide bond between a lysine(K)residue of the substrate(or the N terminus of the substrate)and ubiquitinAdvancedMGlecture第七章蛋白质降解Ubiquitin Conjugation:A 3 Step MechanismUbiquitin(Ub)activating enzyme E1High energy thiol ester is formed between C-terminal Gly of ubiqutin and a Cys in the E1
12、 active site(ATP/AMP)Ubiquitin conjugating enzymes E2Ub is transferred to a Cys of E2 forming a new thiol esterUbiquitin ligase E3Ub forms isopeptide bond between C-terminal Gly of Ub and-amino group of Lys on a target proteinAdvancedMGlecture第七章蛋白质降解AdvancedMGlecture第七章蛋白质降解Ubiquitin的第一次激活 Ubiquiti
13、n 被乙酰化；E1激活酶Cys上形成-SH化学键；E1转移Ubq 到E2 连接酶上.AdvancedMGlecture第七章蛋白质降解多泛素化(Polyubiquitination)E2 连接酶 is bound by E3 ligase which transfers Ubq to the target proteinAdvancedMGlecture第七章蛋白质降解Why have a 3-step ubiquitination process?Ubiquitin E1(1)E2(12-30)E3(200?)-HECT-type -RING-type -PHD-type -U-box co
14、ntaining-N-末端规则E3连接酶家族AdvancedMGlecture第七章蛋白质降解不同类型的泛素连接酶E3植物学报 Chinese Bulletin of Botany 2011,46(6):606616.AdvancedMGlecture第七章蛋白质降解1）N末端规则E3 家族蛋白质出现的一些使它被降解的特征叫做降解信号；第一类被发现的降解信号是蛋白质N末端出现变性的氨基酸残基，这种现象被称为N 末端规则，识别变性的N 末端残基的E3 被称为N 末端规则E3。此类E3 至少有2 类底物识别位点：一类是特异性识别N 末端精氨酸、赖氨酸和组氨酸残基（型），另一类识别N 末端疏水性残基
15、，包括丙氨酸、色氨酸、亮氨酸、酪氨酸、异柠檬酸（型）。归为四类归为四类AdvancedMGlecture第七章蛋白质降解2）HECT 家族这个家族的第一个成员是E6AP（E6-associated protein），该蛋白为最早发现的E3连接酶。迄今共发现了50种人源的HECT E3均包含C端的350个残基左右的HECT催化结构域和N端的特异性结构域，大体可分为包含WWdomain蛋白(如Nedd4 家族蛋白)和缺少WWdomain蛋白(如E6AP)AdvancedMGlecture第七章蛋白质降解3）环指E3 家族这是最大的E3 家族，包含经典的C3H2C3 或C3HC4 环指结构域。如Sk
16、p/Cullin/F-box(SCF)，目前已获得结构解析的CRL主要有两种，一类是Cul1参与的SCF复合物的晶体结构，另一类是Cul4A参与的DCAF/DDB1/Cul4A/RING复合物的部分晶体结构.4）环指相关E3 家族包括U-box 蛋白、PHD（plant homeo-domain）、FYVE 蛋白。如TIR1-ASK1复合物AdvancedMGlecture第七章蛋白质降解E3连接酶的结构AdvancedMGlecture第七章蛋白质降解Cul1-Rbx1-Skp1-Fboxskp2复合物晶体结构及与E2相互作用的模型DDB1-Cul4A-ROC1泛素连接酶复合物晶体结构RIN
17、G-TYPE E3 ligaseAdvancedMGlecture第七章蛋白质降解AdvancedMGlecture第七章蛋白质降解降解相关信号(degrons)PEST序列(Pro,Glu,Ser,Thr)去泛素化酶类(de-ubiquinating enzyme,DUB)提供额外的调节AdvancedMGlecture第七章蛋白质降解AdvancedMGlecture第七章蛋白质降解Ubiquitin AA SequenceMQIFVKTLTGKTITLEVEPSDTIENVKAKI 6 11 29QDKEGIPPDQQRLIFAGKQLEDGRTLSD 31 48 YNIQKESTLHL
18、V LRLRGG 63AdvancedMGlecture第七章蛋白质降解AdvancedMGlecture第七章蛋白质降解Ubiquitin标签的不同类型及功能泛素化介导的非蛋白质降解功能。生物化学与生物物理进展泛素化介导的非蛋白质降解功能。生物化学与生物物理进展Prog Biochem Biophys 2012,39(7):613-621.AdvancedMGlecture第七章蛋白质降解泛素化的基本过程植物学报植物学报 Chinese Bulletin of Botany 2011,46(6):606616.AdvancedMGlecture第七章蛋白质降解Ubiquitylation c
19、ascade and signalingA.Al-Hakim et al./DNA Repair 9(2010)12291240AdvancedMGlecture第七章蛋白质降解Trends in Cell Biology July 2012,Vol.22,No.7AdvancedMGlecture第七章蛋白质降解AdvancedMGlecture第七章蛋白质降解2.蛋白酶体蛋白酶体(PROTEASOME)蛋白酶体被称为“垃圾处理厂”,通常一个人体细胞内大约含有30000 个蛋白酶体,1979年由Goldberg 等人首先分离出来。AdvancedMGlecture第七章蛋白质降解概念：蛋白酶
20、体概念：蛋白酶体是在真核生物和古菌中普遍存在的，在一些原核生物中也存在的一种巨型蛋白质复合物蛋白质复合物。在真核生物中，蛋白酶体位于细胞核和细胞质中。蛋白酶体的主要作用是降解细胞不需要的或受到损伤的蛋白质，这一作用是通过打断肽键的化学反应来实现。蛋白酶体是细胞用来调控特定蛋白质和除去错误折叠蛋白质的主要机制。AdvancedMGlecture第七章蛋白质降解蛋白酶体激活因子和泛素依赖、非泛素依赖降解生物化学与生物物理进展Prog.Biochem.Biophys2011,38(7):593-603.AdvancedMGlecture第七章蛋白质降解 1980s 由Alfred Goldberg&
21、Martin Rechsteiner发现 其结构与GroEL伴侣伴侣素素相似；去折叠和蛋白水解 具有更强的特异性 Why?蛋白酶体AdvancedMGlecture第七章蛋白质降解真核生物蛋白酶体 26S(200 kD)复合体 20S(73 kD)蛋白酶体或多催化蛋白酶复合物(multicatalytic protease complex,MCP)作为关键的蛋白降解组分 19S复合体含有几个ATPases 和一个泛素链结合位点.19S 颗粒作为“盖子”完全的覆盖在20S蛋白酶体上 使底物蛋白去折叠 控制底物进入20S蛋白酶体 激活蛋白水解活性 酵母中，7个亚单位中仅仅 3个具有蛋白水解活性Ad
22、vancedMGlecture第七章蛋白质降解19S和20S蛋白酶体亚单位的特征AdvancedMGlecture第七章蛋白质降解AdvancedMGlecture第七章蛋白质降解真核生物蛋白酶体AdvancedMGlecture第七章蛋白质降解AdvancedMGlecture第七章蛋白质降解酵母蛋白酶体的结构AdvancedMGlecture第七章蛋白质降解Ubiquitin recyclingAdvancedMGlecture第七章蛋白质降解首先，E1 利用ATP 水解释放的能量活化Ub；然后，通过转酰基作用将活化的Ub 转移到E2 上；最后，在E3 协助下将Ub 以48 位赖氨酸连接的
23、方式连接到靶蛋白上。连有多聚泛素链的蛋白质复合物一旦形成，要么以依赖ATP 的方式被26S 蛋白酶体识别并降解，要么在去泛素酶(DUBs)的作用下拆除复合物，释放Ub 和完整的靶蛋白泛素泛素/26S蛋白酶体系统介导的蛋白酶体系统介导的48位赖氨酸连接的泛素链位赖氨酸连接的泛素链标记蛋白的降解标记蛋白的降解生命科学 2011,23(1)AdvancedMGlecture第七章蛋白质降解 蛋白的泛素化(ubiquitylation)途径不仅对其靶蛋白进行降解，而且也是一种细胞核的调节事件。最近几年,已经发现了泛素化与染色与染色质结构，多种信号途径质结构，多种信号途径 以及转录控以及转录控制等制等众
24、多细胞程序相互渗透。Ub-proteasome system是参与控制转录机器(RNA POl II复合体)各组分的分布，含量以及其活性的重要调控者与转录复合体的降解有关。The life of a protein3.依赖于泛素化降解途径的重要意义依赖于泛素化降解途径的重要意义AdvancedMGlecture第七章蛋白质降解1)泛素和染色质泛素和染色质组蛋白的泛素化组蛋白的泛素化 组蛋白组蛋白H2A和和H2B 泛素化泛素化-one of the first recognized markers of trx active chromatin trx：transcription The fir
25、st ubiquitylated protein to be described was histone H2A ubiquitylated forms of histones H2A and H2B were associated specifically with actively transcribed genes Later also H1 and H3 reported to be ubiquitylatedAdvancedMGlecture第七章蛋白质降解泛素化和组蛋白密码泛素化和组蛋白密码 染色质的泛素化 The ubiquitin(Ub)-conjugating enzyme
26、Rad6 ubiquitylates K123 in the core of histone H2B.This modification promotes the methylation of another histone,H3,at two positions,K4 and K79.These modifications,in turn,are required for telomeric-gene silencing.TAFII250(TFIID component)can ubiquitylate the linker histone H1;might relate to the ro
27、le of this TAF in transcriptional activation.泛素化=组蛋白密码的重要组分Mechanism?Direct structural role by loosening chromatin structure Or as”tag”recognized by proteins such as the proteasome or HDAC6AdvancedMGlecture第七章蛋白质降解2)泛素化对泛素化对RNAPII的调控的调控DNA damage-use of RNAPII to direct repair to active genesRegulat
28、ion of trx-coupled repair(TCR)by ubiquitylation of RNA polymerase II.Transcription-coupled repair(TCR)is the mechanism through which mutations in actively transcribed genes are preferentially repaired.Elongating RNAPII,with a unique pattern of CTD phosphorylation,encounters a damaged DNA segment.Her
29、e the stalled polymerase recruits the Ub-ligase Rsp5,which in turn ubiquitylates the largest subunit of pol II.Ubiquitylation is followed by the proteasomal destruction of at least one subunit of polymerase,recruitment of the repair machinery and restoration of DNA integrity.Rsp5 is also a co-activa
30、tor for the steroid hormone receptors?AdvancedMGlecture第七章蛋白质降解3)泛素化对转录因子的调节泛素化对转录因子的调节三种策略三种策略-Controlling the localization of the TF-Controlling the activity of the TF-Controlling the abundance of the TFAdvancedMGlecture第七章蛋白质降解泛素化对转录因子的调控泛素化对转录因子的调控-3种策略和种策略和3种模型种模型A Regulating location.As with N
31、FkB,the TF can be maintained outside the nucleus by interactions with an inhibitor(IkB)that is destroyed by the Ubproteasome system.Another Ub-family member SUMO(S)can directly conjugate to activators and sequester(扣押，阻碍)them into nuclear bodies.B Regulating activity.Ubiquitylation can regulate the
32、association of activators with co-activator proteins either directly,by blocking the association of an activator with its essential cofactor,or indirectly,by facilitating the exchange of cofactors with an activator.AdvancedMGlecture第七章蛋白质降解Trends in Cell Biology July 2012,Vol.22,No.7AdvancedMGlectur
33、e第七章蛋白质降解C Destroying TFs when not needed-shutting off proteolysis then gives a rapid response Beta-连环素(catenin)and wnt-signallingPhosphorylationubiquitylationDegradationSignallingInactivation of GSKbStabilization of b-cateninRapid AccumulationTo the nucleusAdvancedMGlecture第七章蛋白质降解Regulating TF abu
34、ndance-model1 Regulating abundance I-constitutive turnover.By maintaining an activator in a constitutively unstable form,cells are primed for a transcriptional response when appropriate.In this model,a signal from outside the nucleus leads to a transient stabilization of the activator,which elicits
35、a rapid induction of target genes.Examples p53-later lecture Wnt-signallingAdvancedMGlecture第七章蛋白质降解Regulating TF abundance-model2 Regulating abundance II-trx-coupled destruction In this model,activators are destroyed during the act of transcriptional activation as a way of limiting uncontrolled act
36、ivation by any one DNA-bound transcription factor.Nature Reviews Molecular Cell Biology 2003，4:192-201AdvancedMGlecture第七章蛋白质降解Link:trx activation degradation TFs often unstable TAD overlaps closely with degrons Degron=domain that signals ubiquitination Myc and many others Strong activators=rapidly
37、degraded Weak activators=more stable Q-rich,N-rich Mutant TADs with activation lost=stabilizedLink:activation-degradationTAD degronStrong TAD=highly unstableTrx Activation Domain(TADs)AdvancedMGlecture第七章蛋白质降解TADs and degrons overlapMarking and destroying active TFs are part of into the activation p
38、rocess itself.A functional relationship between Trx Activation Domain(TADs)and degradation signals(DEGRONS).The transcriptional activation domains(TADs)and degradation signals(degrons)overlap in 19 unstable transcription factorsNature Reviews Molecular Cell Biology 2003，4:192-201AdvancedMGlecture第七章
39、蛋白质降解Transcriptional activation-risky business?Evidence indicates that marking and destroying active TFs are part of into the activation process itself.Kamikaze activatorsAdvancedMGlecture第七章蛋白质降解Srb10(Cdk8)also targets activator(Gcn4p)sentencing（宣判）it for destructionUbiquitinatedDegradedCTDAdvanced
40、MGlecture第七章蛋白质降解Gcn4=targeted by Srb10,on the way to destruction Gcn4 is phosphorylated by Srb10 Phospho-Gcn4p is recognized by Ub-ligase complex SCFCdc4 WD40 repeats mediates substrate recognition Ubiquitinylation of Gcn4pAdvancedMGlecture第七章蛋白质降解AdvancedMGlecture第七章蛋白质降解A”black widow”model-3 Why
41、should Srb10 destroy the activator?=Activators are destroyed as a direct consequence of recruiting the basal trx machinery to a promoter Basal trx machinery can mark the activators it has encountered,sentencing them to an early deathAdvancedMGlecture第七章蛋白质降解4)细胞周期进程中泛素化降解途径的调节细胞周期进程中泛素化降解途径的调节Advanc
42、edMGlecture第七章蛋白质降解研究蛋白是否通过泛素途径降解？MG132MG132是强效的,可逆的，细胞通透性 的蛋白酶体抑制剂。AdvancedMGlecture第七章蛋白质降解4.Ubiquitin-like ProteinsAdvancedMGlecture第七章蛋白质降解 SUMO and Ubiquitin Alignments of ubiquitin and SUMO-1 indicate only 18%identical in amino acid sequence These two types proteins have remarkably similar sec
43、ondary and tertiary structures Unlike the ubiquitin system,which primarily targets substrate proteins to the proteasome,SUMO-1 conjugation has diverse cellular functions AdvancedMGlecture第七章蛋白质降解SUMO(Small Ubiquitin-like Modifier)Human SUMO1:101 aa;11.6 kD kD;PDB1A5RAdvancedMGlecture第七章蛋白质降解SUMOylat
44、ion SUMO=small ubiquitin related modifier(1996)Ubl(ubiquitin-like protein)-specific proteases(Ulp)in yeast and Sentrin-specificproteases(SENP)in mammalsAdvancedMGlecture第七章蛋白质降解 SUMO-1(small ubiquitin-related modifier)peptide of 101 residues/mature polypeptide 98 residues function ubiquitin NOT tagg
45、ed for degradation Rather stabilized or”targeted”to sub-nuclear structuresWhat is SUMO-1?AdvancedMGlecture第七章蛋白质降解 Small Ubiquitin MOdifier Link:isopeptide bond between the C-terminal Glycine of SUMO and the e-amino group of a lysine residue in the target protein.Structure characteristic ubiquitin-f
46、old+unique unstructured N-terminal extension of up to 22 residues-possible protein interaction site?GGXKEThe SUMO-1 proteinAdvancedMGlecture第七章蛋白质降解 Unlike ubiquitin modification which targets proteins for degradation,SUMOylation 1.increases a proteins lifetime.2.change a proteins location in the ce
47、ll SUMO modification of proteins has many functions.Among the most frequent and best studied are protein stability,nuclear-cytosolic transport,transcriptional regulation(mostly transcriptional repression).SUMO-1&SUMO-2/3SUMO(Small Ubiquitin-like Modifier)AdvancedMGlecture第七章蛋白质降解 Largely nuclear tar
48、get proteins NLS+KxE A short peptide that contains the KxE motif and a NLS suffices(足够)to produce a SUMO conjugate in vivo.Mutated NLS abolish Sumoylation SP100,HDAC4,MDM2Many nuclear targetsAdvancedMGlecture第七章蛋白质降解Sumoylation-functional roles Antagonizing(拮抗)other modifications SUMO modification o
49、f IkBa stabilizes this NF-kB inhibitor by blocking ubiquitylation at the same acceptor site.Confer new interactions Conformational change New interaction surface enhance or inhibit interactions Altering the subcellular localization of the protein Sumoylation causes the relocalization of the nuclear
50、import factor RanGAP1 from the cytoplasm to the nuclear pore complex(NPC).Many Trx factors associated with PML nuclear bodiesAdvancedMGlecture第七章蛋白质降解Examples of SUMO functionProteinSUMO effect RanGAP Causes nuclear translocation IkB Blocks Ub-conjugation site,prevents degradation c-Jun Inhibits tra
