AdvancedMGlecture第七章蛋白質(zhì)降解

1、AdvancedMGlecture第七章蛋白質(zhì)降解AdvancedMGlecture第七章蛋白質(zhì)降解蛋白降解速率肌球蛋白 血紅蛋白晶狀體晶體蛋白 己糖激酶 AdvancedMGlecture第七章蛋白質(zhì)降解蛋白降解速率肌球蛋白 血紅蛋白晶狀體晶體蛋白 己糖激酶 Ad影響蛋白壽命的因素內(nèi)在穩(wěn)定性 (“遺傳編碼序列)固有的生物物理特征外界環(huán)境溫度pH降解活性特異機制 定位相互作用蛋白(partners)AdvancedMGlecture第七章蛋白質(zhì)降解影響蛋白壽命的因素內(nèi)在穩(wěn)定性 (“遺傳編碼序列)Advan蛋白質(zhì)的半衰期與N-端氨基酸殘基的關(guān)系被稱為N末端規(guī)則，既存在于原核生物，也存在于真核生物

2、中。 AdvancedMGlecture第七章蛋白質(zhì)降解蛋白質(zhì)的半衰期與N-端氨基酸殘基的關(guān)系被稱為N末端規(guī)則，既存 成熟蛋白N-端的第一個氨基酸（除已被切除的N端甲硫氨酸之外，但包括翻譯后修飾產(chǎn)物）在蛋白的降解中有著舉足輕重的影響。 當(dāng)某個蛋白質(zhì)的N端是甲硫氨酸、苷氨酸、丙氨酸、絲氨酸、蘇氨酸和纈氨酸時，表現(xiàn)穩(wěn)定。 其N端為賴氨酸、精氨酸時，表現(xiàn)最不穩(wěn)定，平均2-3分鐘就被降解了。AdvancedMGlecture第七章蛋白質(zhì)降解 成熟蛋白N-端的第一個氨基酸（除已被切除的N端甲硫氨酸之真核細胞中的兩種蛋白降解途徑Lysosomes (溶酶體參與的蛋白降解途徑)Receptor mediat

3、ed endocytosis & phagocytosis Proteasomes:for endogenous proteins (蛋白酶復(fù)合體參與的蛋白質(zhì)降解途徑)transcription factorscell cycle cyclinsvirus coded proteinsimproperly folded proteins damaged proteins耗能與不耗能的差別AdvancedMGlecture第七章蛋白質(zhì)降解真核細胞中的兩種蛋白降解途徑Lysosomes (溶酶體參與Major pathways for ubiquitin-dependent protein deg

4、radation in eukaryotic cells. Mol Cell Neurosci 49 (2012) 387393AdvancedMGlecture第七章蛋白質(zhì)降解Major pathways for ubiquitin-dDeath by ProteasesAdvancedMGlecture第七章蛋白質(zhì)降解Death by ProteasesAdvancedMGle 在大腸桿菌中，許多蛋白質(zhì)的降解是通過一個依賴于ATP的蛋白酶(稱為Lon)來實現(xiàn)的。 當(dāng)細胞中存在有錯誤或半衰期很短的蛋白質(zhì)時，該蛋白酶就被激活。 每切除一個肽鍵要消耗兩個分子ATP。AdvancedMGlectu

5、re第七章蛋白質(zhì)降解 在大腸桿菌中，許多蛋白質(zhì)的降解是通過一個依賴于ATP的蛋半衰期(Half-life) 蛋白質(zhì)被降解或變性一半時所需要的平均時間 (依賴于你所采用的方法)Turnover 蛋白從其合成到降解的時間間隔(Lifespan )穩(wěn)定性(Stability) - Subjective property 一個成熟蛋白在某一條件下的天生具有易于變性的趨勢，為蛋白的固有屬性變性(Denaturation) 一個多肽整個或部分去折疊降解(Degradation) 肽段的蛋白水解泛素化(Ubiquitination) = Ubiquitylation蛋白酶(Protease) = pepti

6、dase術(shù)語(Terminology)AdvancedMGlecture第七章蛋白質(zhì)降解半衰期(Half-life) 蛋白質(zhì)被降解或變性一半時所 有些蛋白的半衰期長達數(shù)年，如結(jié)晶蛋白；有些蛋白的半衰期只有幾分鐘，如一些信號傳遞蛋白。蛋白在穩(wěn)定性方面的差異不是隨機的，而是一個程序化過程。 在發(fā)現(xiàn)泛素-蛋白酶體系統(tǒng)之前，細胞中的蛋白質(zhì)降解被認為主要依賴于溶酶體。 AdvancedMGlecture第七章蛋白質(zhì)降解 有些蛋白的半衰期長達數(shù)年，如結(jié)晶蛋白；有些蛋白的 2004年諾貝爾化學(xué)獎：阿龍切哈諾夫(以色列)阿夫拉姆赫爾什科(以色列)歐文羅斯(美) 找到了人體細胞控制和調(diào)節(jié)某種人體蛋白質(zhì)數(shù)量多少的

7、方法。他們發(fā)現(xiàn)人體細胞通過給無用蛋白質(zhì)“貼標(biāo)簽”的方法，幫助人體將那些被貼上標(biāo)記的蛋白質(zhì)進行“廢物處理”， 使它們自行破裂、自動消亡。泛素調(diào)控的蛋白質(zhì)降解具有重要的生理意義，它不僅能夠清除錯誤蛋白質(zhì)，對細胞生長周期、DNA復(fù)制以及染色體結(jié)構(gòu)都有重要的調(diào)控作用，而且對于理解細胞的許多生理過程和新藥的開發(fā)具有重要的意義。蛋白質(zhì)降解是一個有序的過程。AdvancedMGlecture第七章蛋白質(zhì)降解 2004年諾貝爾化學(xué)獎：蛋白質(zhì)降解是一個有序的過程。 就目前的進展而言，真核細胞控制蛋白質(zhì)穩(wěn)定性的主要途徑就是通過泛素(ubiquitin)修飾目標(biāo)蛋白(target protein，也稱為底物蛋白su

8、bstrate protein)，然后這個被泛素標(biāo)記的蛋白質(zhì)通過一個叫“26S蛋白酶體”的蛋白質(zhì)復(fù)合體降解掉。這種通過泛素化途徑降解的蛋白質(zhì)包括細胞周期調(diào)節(jié)子、轉(zhuǎn)錄因子和其他重要的蛋白質(zhì)。AdvancedMGlecture第七章蛋白質(zhì)降解 就目前的進展而言，真核細胞控制蛋白質(zhì)穩(wěn)定性的1. 泛素(Ubiquitin)和泛素化(Ubiquitination)真核蛋白的降解依賴于泛素(Ubiquitin) 76 氨基酸殘基高度保守 3 amino acid changes yeast to human熱穩(wěn)定(Thermostable)1975 年從小牛的胰臟中分離出來AdvancedMGlectur

9、e第七章蛋白質(zhì)降解1. 泛素(Ubiquitin)和泛素化(UbiquitinUbiquitin AA SequenceMQIFVKTLTGKTITLEVEPSDTIENVKAKI 6 11 29QDKEGIPPDQQRLIFAGKQLEDGRTLSD 31 48 YNIQKESTLHLV LRLRGG 63AdvancedMGlecture第七章蛋白質(zhì)降解Ubiquitin AA SequenceAdvancedM真核細胞依賴于泛素的蛋白降解途徑 1. 細胞內(nèi)即將被降解的蛋白首先在ATP的作用下與泛素相連（需要E1, E2, E3三種酶的參與）2. 將該復(fù)合體運送到特定的蛋白降解體系“26s

10、 proteasome”中直到完全降解3. ubiquitin recycle在細胞內(nèi)存在3 種重要的酶,即泛素活化酶(ubiquitin - activating enzyme ,簡稱E1) 、泛素結(jié)合酶(ubiquitin - conjugating enzyme ,簡稱E2) 、泛素蛋白連接酶 (ubiquitin - proteinligating enzyme , 簡稱E3) 。AdvancedMGlecture第七章蛋白質(zhì)降解真核細胞依賴于泛素的蛋白降解途徑在細胞內(nèi)存在3 種重要的酶,AdvancedMGlecture第七章蛋白質(zhì)降解AdvancedMGlecture第七章蛋白質(zhì)降

11、解Ubiquitination (泛素化)Ubiquitinating enzymes E1,E2, E3 - thiol ester bond(硫羥酸酯鍵)Final target - isopeptide bond between a lysine(K) residue of the substrate (or the N terminus of the substrate) and ubiquitinAdvancedMGlecture第七章蛋白質(zhì)降解Ubiquitination (泛素化)UbiquitinaUbiquitin Conjugation: A 3 Step Mechanis

12、mUbiquitin (Ub) activating enzyme E1High energy thiol ester is formed between C-terminal Gly of ubiqutin and a Cys in the E1 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 G

13、ly of Ub and -amino group of Lys on a target proteinAdvancedMGlecture第七章蛋白質(zhì)降解Ubiquitin Conjugation: A 3 St26S 蛋白酶體降解依賴于泛素的蛋白酶體降解途徑AdvancedMGlecture第七章蛋白質(zhì)降解26S 蛋白酶體降解依賴于泛素的蛋白酶體降解途徑AdvancUbiquitin的第一次激活Ubiquitin 被乙?；?；E1激活酶Cys上形成-SH化學(xué)鍵； E1轉(zhuǎn)移Ubq 到E2 連接酶上.AdvancedMGlecture第七章蛋白質(zhì)降解Ubiquitin的第一次激活Ubiquitin

14、 被乙?；欢喾核鼗?(Polyubiquitination)E2 連接酶 is bound by E3 ligase which transfers Ubq to the target proteinAdvancedMGlecture第七章蛋白質(zhì)降解多泛素化 (Polyubiquitination)E2 連接Why have a 3-step ubiquitination process?UbiquitinE1 (1)E2 (12-30)E3 (200?)- HECT-type - RING-type - PHD-type - U-box containing- N-末端規(guī)則E3連接酶家族A

15、dvancedMGlecture第七章蛋白質(zhì)降解Why have a 3-step ubiquitinati不同類型的泛素連接酶E3植物學(xué)報 Chinese Bulletin of Botany 2011, 46 (6): 606616.兩類AdvancedMGlecture第七章蛋白質(zhì)降解不同類型的泛素連接酶E3植物學(xué)報 Chinese Bulle1） N末端規(guī)則E3 家族蛋白質(zhì)出現(xiàn)的一些使它被降解的特征叫做降解信號； 第一類被發(fā)現(xiàn)的降解信號是蛋白質(zhì)N末端出現(xiàn)變性的氨基酸殘基， 這種現(xiàn)象被稱為N 末端規(guī)則， 識別變性的N 末端殘基的E3 被稱為N 末端規(guī)則E3。此類E3 至少有2 類底物識別

16、位點： 一類是特異性識別N 末端精氨酸、賴氨酸和組氨酸殘基（型），另一類識別N 末端疏水性殘基，包括丙氨酸、色氨酸、亮氨酸、酪氨酸、異檸檬酸（型）。歸為四類AdvancedMGlecture第七章蛋白質(zhì)降解1） N末端規(guī)則E3 家族蛋白質(zhì)出現(xiàn)的一些使它被降解的特征2）HECT 家族這個家族的第一個成員是E6AP（E6-associated protein），該蛋白為最早發(fā)現(xiàn)的E3連接酶。迄今共發(fā)現(xiàn)了50種人源的HECT E3均包含C端的350個殘基左右的HECT催化結(jié)構(gòu)域和N端的特異性結(jié)構(gòu)域，大體可分為包含WWdomain蛋白(如Nedd4 家族蛋白)和缺少WWdomain蛋白(如E6AP)A

17、dvancedMGlecture第七章蛋白質(zhì)降解2）HECT 家族這個家族的第一個成員是E6AP（E6-as3） 環(huán)指E3 家族這是最大的E3 家族， 包含經(jīng)典的C3H2C3 或C3HC4 環(huán)指結(jié)構(gòu)域。如Skp/Cullin/F-box (SCF)，目前已獲得結(jié)構(gòu)解析的CRL主要有兩種，一類是Cul1參與的SCF復(fù)合物的晶體結(jié)構(gòu)，另一類是Cul4A參與的DCAF/DDB1/Cul4A/RING復(fù)合物的部分晶體結(jié)構(gòu).4） 環(huán)指相關(guān)E3 家族包括U-box 蛋白、PHD （plant homeo-domain）、FYVE 蛋白。如TIR1-ASK1復(fù)合物AdvancedMGlecture第七章蛋白

18、質(zhì)降解3） 環(huán)指E3 家族這是最大的E3 家族， 包含經(jīng)典的C3HE3連接酶的結(jié)構(gòu)AdvancedMGlecture第七章蛋白質(zhì)降解E3連接酶的結(jié)構(gòu)AdvancedMGlecture第七章蛋白Cul1-Rbx1-Skp1-Fboxskp2復(fù)合物晶體結(jié)構(gòu)及與E2相互作用的模型DDB1-Cul4A-ROC1泛素連接酶復(fù)合物晶體結(jié)構(gòu)RING-TYPE E3 ligaseAdvancedMGlecture第七章蛋白質(zhì)降解Cul1-Rbx1-Skp1-Fboxskp2復(fù)合物晶體結(jié)構(gòu)AdvancedMGlecture第七章蛋白質(zhì)降解AdvancedMGlecture第七章蛋白質(zhì)降解降解相關(guān)信號 (degr

19、ons)PEST序列 (Pro, Glu, Ser, Thr)去泛素化酶類(de-ubiquinating enzyme, DUB) 提供額外的調(diào)節(jié)AdvancedMGlecture第七章蛋白質(zhì)降解降解相關(guān)信號 (degrons)PEST序列 (Pro, GAdvancedMGlecture第七章蛋白質(zhì)降解AdvancedMGlecture第七章蛋白質(zhì)降解Ubiquitin AA SequenceMQIFVKTLTGKTITLEVEPSDTIENVKAKI 6 11 29QDKEGIPPDQQRLIFAGKQLEDGRTLSD 31 48 YNIQKESTLHLV LRLRGG 63Advan

20、cedMGlecture第七章蛋白質(zhì)降解Ubiquitin AA SequenceAdvancedMDifferent types of ubiquitin chainsJournal of Cell Science 125 (3) 539-548AdvancedMGlecture第七章蛋白質(zhì)降解Different types of ubiquitin cUbiquitin標(biāo)簽的不同類型及功能泛素化介導(dǎo)的非蛋白質(zhì)降解功能。生物化學(xué)與生物物理進展Prog Biochem Biophys 2012, 39(7): 613-621.AdvancedMGlecture第七章蛋白質(zhì)降解Ubiquitin

21、標(biāo)簽的不同類型及功能泛素化介導(dǎo)的非蛋白質(zhì)降泛素化的基本過程植物學(xué)報 Chinese Bulletin of Botany 2011, 46 (6): 606616.AdvancedMGlecture第七章蛋白質(zhì)降解泛素化的基本過程植物學(xué)報 Chinese Bulletin Ubiquitylation cascade and signalingA. Al-Hakim et al. / DNA Repair 9 (2010) 12291240AdvancedMGlecture第七章蛋白質(zhì)降解Ubiquitylation cascade and sigTrends in Cell Biology

22、July 2012, Vol. 22, No. 7Current hypothetical roles of K63, M1, and K11 chains in NF-kB activation.AdvancedMGlecture第七章蛋白質(zhì)降解Trends in Cell Biology July 20AdvancedMGlecture第七章蛋白質(zhì)降解AdvancedMGlecture第七章蛋白質(zhì)降解2.蛋白酶體 (PROTEASOME)蛋白酶體被稱為“垃圾處理廠”,通常一個人體細胞內(nèi)大約含有30000 個蛋白酶體, 1979年由Goldberg 等人首先分離出來。AdvancedMGle

23、cture第七章蛋白質(zhì)降解2.蛋白酶體 (PROTEASOME)蛋白酶體被稱為“垃圾處概念：蛋白酶體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種巨型蛋白質(zhì)復(fù)合物。在真核生物中，蛋白酶體位于細胞核和細胞質(zhì)中。 蛋白酶體的主要作用是降解細胞不需要的或受到損傷的蛋白質(zhì)，這一作用是通過打斷肽鍵的化學(xué)反應(yīng)來實現(xiàn)。蛋白酶體是細胞用來調(diào)控特定蛋白質(zhì)和除去錯誤折疊蛋白質(zhì)的主要機制。 AdvancedMGlecture第七章蛋白質(zhì)降解概念：蛋白酶體是在真核生物和古菌中普遍存在的，在一些原核生物蛋白酶體激活因子和泛素依賴、非泛素依賴降解生物化學(xué)與生物物理進展Prog. Biochem. Bioph

24、ys2011, 38(7): 593-603.AdvancedMGlecture第七章蛋白質(zhì)降解蛋白酶體激活因子和泛素依賴、非泛素依賴降解生物化學(xué)與生物物理1980s 由Alfred Goldberg & Martin Rechsteiner發(fā)現(xiàn)其結(jié)構(gòu)與GroEL伴侶素相似；去折疊和蛋白水解具有更強的特異性Why?蛋白酶體AdvancedMGlecture第七章蛋白質(zhì)降解1980s 由Alfred Goldberg & Mart真核生物蛋白酶體26S (200 kD) 復(fù)合體 20S (73 kD) 蛋白酶體或多催化蛋白酶復(fù)合物(multicatalytic protease complex,

25、 MCP) 作為關(guān)鍵的蛋白降解組分 19S復(fù)合體含有幾個ATPases 和一個泛素鏈結(jié)合位點. 19S 顆粒作為“蓋子” 完全的覆蓋在20S蛋白酶體上使底物蛋白去折疊控制底物進入20S蛋白酶體 激活蛋白水解活性酵母中，7個亞單位中僅僅 3個具有蛋白水解活性AdvancedMGlecture第七章蛋白質(zhì)降解真核生物蛋白酶體26S (200 kD) 復(fù)合體 Advan19S和20S蛋白酶體亞單位的特征AdvancedMGlecture第七章蛋白質(zhì)降解19S和20S蛋白酶體亞單位的特征AdvancedMGlecAdvancedMGlecture第七章蛋白質(zhì)降解AdvancedMGlecture第七章

26、蛋白質(zhì)降解真核生物蛋白酶體AdvancedMGlecture第七章蛋白質(zhì)降解真核生物蛋白酶體AdvancedMGlecture第七章蛋白AdvancedMGlecture第七章蛋白質(zhì)降解AdvancedMGlecture第七章蛋白質(zhì)降解酵母蛋白酶體的結(jié)構(gòu)AdvancedMGlecture第七章蛋白質(zhì)降解酵母蛋白酶體的結(jié)構(gòu)AdvancedMGlecture第七章蛋Ubiquitin recyclingAdvancedMGlecture第七章蛋白質(zhì)降解Ubiquitin recyclingAdvancedMGl首先，E1 利用ATP 水解釋放的能量活化Ub；然后，通過轉(zhuǎn)?；饔脤⒒罨腢b 轉(zhuǎn)移到

27、E2 上；最后，在E3 協(xié)助下將Ub 以48 位賴氨酸連接的方式連接到靶蛋白上。連有多聚泛素鏈的蛋白質(zhì)復(fù)合物一旦形成，要么以依賴ATP 的方式被26S 蛋白酶體識別并降解，要么在去泛素酶(DUBs )的作用下拆除復(fù)合物，釋放Ub 和完整的靶蛋白泛素/26S蛋白酶體系統(tǒng)介導(dǎo)的48位賴氨酸連接的泛素鏈標(biāo)記蛋白的降解生命科學(xué) 2011, 23(1)AdvancedMGlecture第七章蛋白質(zhì)降解首先，E1 利用ATP 水解釋放的能量活化Ub；然后，通過轉(zhuǎn)蛋白的泛素化(ubiquitylation)途徑不僅對其靶蛋白進行降解，而且也是一種細胞核的調(diào)節(jié)事件。 最近幾年, 已經(jīng)發(fā)現(xiàn)了泛素化與染色質(zhì)結(jié)構(gòu)，

28、多種信號途徑 以及轉(zhuǎn)錄控制等眾多細胞程序相互滲透。Ub-proteasome system是參與控制轉(zhuǎn)錄機器(RNA POl II復(fù)合體)各組分的分布，含量以及其活性的重要調(diào)控者與轉(zhuǎn)錄復(fù)合體的降解有關(guān)。The life of a proteinTranscriptionProteolysis3. 依賴于泛素化降解途徑的重要意義AdvancedMGlecture第七章蛋白質(zhì)降解蛋白的泛素化(ubiquitylation)途徑不僅對其靶蛋1) 泛素和染色質(zhì)組蛋白的泛素化組蛋白H2A和H2B 泛素化- one of the first recognized markers of trx active

29、 chromatin trx：transcriptionThe first ubiquitylated protein to be described was histone H2Aubiquitylated forms of histones H2A and H2B were associated specifically with actively transcribed genesLater also H1 and H3 reported to be ubiquitylatedAdvancedMGlecture第七章蛋白質(zhì)降解1) 泛素和染色質(zhì)組蛋白的泛素化組蛋白H2A和H2B 泛素化泛

30、素化和組蛋白密碼染色質(zhì)的泛素化 The ubiquitin (Ub)-conjugating enzyme 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 componen

31、t) can ubiquitylate the linker histone H1; might relate to the role of this TAF in transcriptional activation.泛素化 = 組蛋白密碼的重要組分Mechanism?Direct structural role by loosening chromatin structureOr as ”tag” recognized by proteins such as the proteasome or HDAC6AdvancedMGlecture第七章蛋白質(zhì)降解泛素化和組蛋白密碼染色質(zhì)的泛素化 M

32、echanism?Adv2) 泛素化對RNAPII的調(diào)控DNA damage - use of RNAPII to direct repair to active genesRegulation 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.

33、 Elongating RNAPII, with a unique pattern of CTD phosphorylation, encounters a damaged DNA segment. Here 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

34、polymerase, recruitment of the repair machinery and restoration of DNA integrity.Rsp5 is also a co-activator for the steroid hormone receptors ?AdvancedMGlecture第七章蛋白質(zhì)降解2) 泛素化對RNAPII的調(diào)控DNA damage - u3) 泛素化對轉(zhuǎn)錄因子的調(diào)節(jié)三種策略-Controlling the localization of the TF-Controlling the activity of the TF-Controll

35、ing the abundance of the TFAdvancedMGlecture第七章蛋白質(zhì)降解3) 泛素化對轉(zhuǎn)錄因子的調(diào)節(jié)三種策略AdvancedMGle泛素化對轉(zhuǎn)錄因子的調(diào)控- 3種策略和3種模型A Regulating location. As with NFkB, 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 SUM

36、O (S) can directly conjugate to activators and sequester(扣押，阻礙) them into nuclear bodies. B Regulating activity.Ubiquitylation can regulate the association of activators with co-activator proteins either directly, by blocking the association of an activator with its essential cofactor, or indirectly

37、, by facilitating the exchange of cofactors with an activator.AdvancedMGlecture第七章蛋白質(zhì)降解泛素化對轉(zhuǎn)錄因子的調(diào)控- 3種策略和3種模型A RegulThe canonical nuclear factor-kappa B (NF-kB) activation pathway.Trends in Cell Biology July 2012, Vol. 22, No. 7AdvancedMGlecture第七章蛋白質(zhì)降解The canonical nuclear factor-kC Destroying TFs

38、when not needed- shutting off proteolysis then gives a rapid responseBeta-連環(huán)素(catenin) and wnt-signallingPhosphorylationubiquitylationDegradationSignallingInactivation of GSKbStabilization of b-cateninRapid AccumulationTo the nucleusAdvancedMGlecture第七章蛋白質(zhì)降解C Destroying TFs when not neeRegulating TF

39、 abundance - model1Regulating 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, whi

40、ch elicits a rapid induction of target genes.Examplesp53 - later lectureWnt-signallingAdvancedMGlecture第七章蛋白質(zhì)降解Regulating TF abundance - modRegulating TF abundance - model2Regulating abundance II - trx-coupled destructionIn this model, activators are destroyed during the act of transcriptional activ

41、ation as a way of limiting uncontrolled activation by any one DNA-bound transcription factor. Nature Reviews Molecular Cell Biology 2003，4:192-201AdvancedMGlecture第七章蛋白質(zhì)降解Regulating TF abundance - modeLink: trx activation degradationTFs often unstableTAD overlaps closely with degronsDegron = domain

42、that signals ubiquitinationMyc and many othersStrong activators = rapidly degradedWeak activators = more stableQ-rich, N-richMutant TADs with activation lost = stabilizedLink: activation - degradationTAD degronStrong TAD = highly unstableTrx Activation Domain (TADs)AdvancedMGlecture第七章蛋白質(zhì)降解Link: trx

43、 activation degradTADs and degrons overlapMarking and destroying active TFs are part of into the activation process itself. A functional relationship between Trx Activation Domain (TADs) and degradation signals (DEGRONS).The transcriptional activation domains (TADs) and degradation signals (degrons)

44、 overlap in 19 unstable transcription factorsNature Reviews Molecular Cell Biology 2003，4:192-201AdvancedMGlecture第七章蛋白質(zhì)降解TADs and degrons overlapMarkinTranscriptional activation- risky business?Evidence indicates that marking and destroying active TFs are part of into the activation process itself.

45、 Kamikaze activatorsAdvancedMGlecture第七章蛋白質(zhì)降解Transcriptional activation- rSrb10 (Cdk8) also targets activator (Gcn4p) sentencing（宣判） it for destructionUbiquitinatedDegradedCTDAdvancedMGlecture第七章蛋白質(zhì)降解Srb10 (Cdk8) also targets actiGcn4 = targeted by Srb10, on the way to destructionGcn4 is phosphoryla

46、ted by Srb10Phospho-Gcn4p is recognized by Ub-ligase complex SCFCdc4WD40 repeats mediates substrate recognitionUbiquitinylation of Gcn4pAdvancedMGlecture第七章蛋白質(zhì)降解Gcn4 = targeted by Srb10, on tAdvancedMGlecture第七章蛋白質(zhì)降解AdvancedMGlecture第七章蛋白質(zhì)降解A ”black widow” model-3Why should Srb10 destroy the activat

47、or?= Activators are destroyed as a direct consequence of recruiting the basal trx machinery to a promoterBasal trx machinery can mark the activators it has encountered, sentencing them to an early deathAdvancedMGlecture第七章蛋白質(zhì)降解A ”black widow” model-3Why sho4) 細胞周期進程中泛素化降解途徑的調(diào)節(jié)AdvancedMGlecture第七章蛋白質(zhì)

48、降解4) 細胞周期進程中泛素化降解途徑的調(diào)節(jié)AdvancedMG研究蛋白是否通過泛素途徑降解？MG132MG132是強效的,可逆的，細胞通透性 的蛋白酶體抑制劑。 AdvancedMGlecture第七章蛋白質(zhì)降解研究蛋白是否通過泛素途徑降解？MG132MG132是強效的,4. Ubiquitin-like ProteinsAdvancedMGlecture第七章蛋白質(zhì)降解4. Ubiquitin-like ProteinsAdva SUMO and Ubiquitin Alignments of ubiquitin and SUMO-1 indicate only 18% identical

49、 in amino acid sequence These two types proteins have remarkably similar secondary and tertiary structuresUnlike the ubiquitin system, which primarily targets substrate proteins to the proteasome, SUMO-1 conjugation has diverse cellular functions AdvancedMGlecture第七章蛋白質(zhì)降解 SUMO and Ubiquitin Alignmen

50、tsSUMO (Small Ubiquitin-like Modifier)Human SUMO1: 101 aa; 11.6 kD kD; PDB1A5RAdvancedMGlecture第七章蛋白質(zhì)降解SUMO (Small Ubiquitin-like MoSUMOylationSUMO = small ubiquitin related modifier (1996) Ubl (ubiquitin-like protein)-specific proteases (Ulp) in yeast and Sentrin-specificproteases (SENP) in mammals

51、AdvancedMGlecture第七章蛋白質(zhì)降解SUMOylationSUMO = small ubiquiSUMO-1 (small ubiquitin-related modifier)peptide of 101 residues / mature polypeptide 98 residuesfunction ubiquitin NOT tagged for degradationRather stabilized or ”targeted” to sub-nuclear structuresWhat is SUMO-1 ?AdvancedMGlecture第七章蛋白質(zhì)降解SUMO-

52、1 (small ubiquitin-relateSmall Ubiquitin MOdifierLink: isopeptide bondbetween the C-terminal Glycine of SUMO and the e-amino group of a lysine residue in the target protein.Structurecharacteristic ubiquitin-fold + unique unstructured N-terminal extension of up to 22 residues - possible protein inter

53、action site?GGXKEThe SUMO-1 proteinAdvancedMGlecture第七章蛋白質(zhì)降解Small Ubiquitin MOdifierGGXKEUnlike ubiquitin modification which targets proteins for degradation, SUMOylation 1. increases a proteins lifetime. 2. change a proteins location in the cell SUMO modification of proteins has many functions. Amo

54、ng 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第七章蛋白質(zhì)降解Unlike ubiquitin modification Largely nuclear target proteins NLS +

55、KxE A short peptide that contains the KxE motif and a NLS suffices(足夠) to produce a SUMO conjugate in vivo. Mutated NLS abolish SumoylationSP100, HDAC4, MDM2Many nuclear targetsAdvancedMGlecture第七章蛋白質(zhì)降解Largely nuclear target proteinSumoylation - functional rolesAntagonizing(拮抗) other modificationsSU

56、MO modification of IkBa stabilizes this NF-kB inhibitor by blocking ubiquitylation at the same acceptor site.Confer new interactionsConformational change New interaction surface enhance or inhibit interactions Altering the subcellular localization of the proteinSumoylation causes the relocalization

57、of the nuclear import factor RanGAP1 from the cytoplasm to the nuclear pore complex (NPC).Many Trx factors associated with PML nuclear bodiesAdvancedMGlecture第七章蛋白質(zhì)降解Sumoylation - functional rolesExamples of SUMO functionProteinSUMO effect RanGAP Causes nuclear translocation IkB Blocks Ub-conjugatio

58、n site,prevents degradation c-Jun Inhibits transcriptional activity p53 and mdm2 Blocks mdm2 self-ubiquitination, prevents degradation SUMO-p53 in DNA binding domain apoptotic activityAdvancedMGlecture第七章蛋白質(zhì)降解Examples of SUMO functionProteSumoylation - consequencesAdvancedMGlecture第七章蛋白質(zhì)降解Sumoylatio

59、n - consequencesAdvaSumoylation co-repressor recruitmentSumoylated TF promotes binding of co-repressor. AdvancedMGlecture第七章蛋白質(zhì)降解Sumoylation co-repressor recNPC passageA substrate that contains a NLS might be sumoylated at the nuclear pore by the E3 ligase activity of RanBP2, after which it might be

60、 de-modified by a Ulp1-type SUMO protease that resides at the nucleoplasmic face of the nuclear pore complex (NPC), or by a Ulp2-type, nucleoplasmic protease.Nuclear dynamic modification Once inside the nucleus, substrates might undergo SUMO modification that is mediated by PIAS or Pc2 E3 ligases. S