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1 蛋白質的轉譯後修飾作用國立臺灣大學生化科技學系張世宗

2 The Extra Episode of Central Dogma: Post-translational Modification

3 Coupling of transcription and translation in bacteria Lehninger Principles of Biochemistry 5 th edition

4 Directing eukaryotic proteins with the appropriate signals to the ER Lehninger Principles of Biochemistry 5 th edition

5 Some proteins undergo assisted folding Molecular chaperone 分子伴護蛋白, 如 Hsp70, Hsp40 結合疏水性及 unfolded 區域, 或 newly synthesized but not yet folded peptide chains, 以保護其不被降解或防止不當的聚集與沈澱維持某些蛋白質於 unfolded 型態, 以利於穿膜運輸加速蛋白質四級結構的形成 Chaperonin (Cpn) 伴護蛋白, 如 Hsp60, Hsp10, Cpn60, Cpn10 直接促進蛋白質的摺疊 Protein disulfide isomerase (PDI) Catalyzes the interchange, or shuffling, of disulfide bonds until the bonds of native conformation are formed. Catalyzes the elimination of folding intermediates with inappropriate disulfide crosslinks. Peptide prolyl cis-trans isomerase (PPI) Interconversion of cis and trans isomers of Pro residue peptide bonds can be a slow step in the folding of proteins that contain some Pro peptide bonds in the cis conformation.

6 蛋白質轉譯出來之後要靠 chaperone 幫助正確摺疊 Alberts et al (2002) Molecular Biology of the Cell (4e) p.357

7 摺疊不良的蛋白質會被送到蛋白體降解系統 Probing the proteasome pathway (2000) Alfred L. Goldberg, Nature Biotechnology 18,

8 相似的桶狀巨分子卻有相反的功能內部折疊區內部水解區 Proteasome (20S) Chaperonin (GroEL) Weissman et al (1995) Science 268: 524

9 與蛋白質摺疊缺失有關的疾病 A soluble protein is secreted in a misfolded state and converted into an insoluble extracellular amyloid fiber. The diseases are collectively referred to as amyloidoses. Amyloid 類澱粉蛋白 / 澱粉樣蛋白 Amyloidosis 類澱粉變性症 / 澱粉樣變性病 Amyloid plaque 澱粉樣蛋白斑 Type II diabetes Amyloid deposition near the pancreatic islet β cells Alzheimer s disease Extracellular amyloid deposition by neuron amyloid β-peptide derived from amyloid β- peptide precursor protein or APP Intracellular aggregation of misfolded proteins Parkinson s disease Huntington s disease α-synuclein aggregates into Lewy bodies Huntingtin with a long polyglutamine repeat Misfolded leads to degradation and loss of function Cystic fibrosis 囊性纖維化病 Defects in a membrane-bound protein called CFTR CFTR: Cystic fibrosis transmembrane conductance regulator, which act as a channel for chloride ions

10 Formation of disease-causing amyloid fibrils APP Amyloid β-peptide Lehninger Principles of Biochemistry 5 th edition

11 The prion (proteinaceous infectious particle) disease coined in 1982 by Dr. Stanley B. Prusiner Tikvah Alper suggested the hypothesis in 1960s that some transmissible spongiform encephalopathies are caused by an infectious agent consisting solely of proteins.. of prion diseases he pathogenesis o lar biology and th es 21: Prusiner, S. B. (1996). Molecu Trends in Bio chemical Science 左邊為正常的 PrP c 蛋白質結構, 多由 α helices 組成右邊則是呈現 β sheets 構型的致病性 PrP sc 蛋白質結構 PrP, the prion protein, comes in various forms, such as Prp c, the normal cellular prion protein, and PrP sc, the scrapie form

12 Prion Proteinaceous infectious particles Novel proteinaceous infectious particles cause scrapie, Stanley B. Prusiner, Science Apr 9;216(4542):

13 Prp c PrP sc TIBS 21 December 1996 Prp c PrP sc PrP sc PrP sc Annu. Rev. Biochem : Prp c, normal cellular prion protein PrP sc, scrapie form

14 在研究狂牛病這個領域上已經產生兩位諾貝爾醫學暨生理學獎得主 (1976 及 1997) 第一位是 Gajdusek, 他於巴布亞新幾內亞的食人族部落發現庫魯 (kuru) 症, 並推測病源可能是一種慢性作用病毒 (slowacting virus) Prusiner 則是第二位研究該領域獲獎的科學家, 他發現造成羊搔癢症的病原並不是病毒, 它是一種不含 DNA 或 RNA 的物質, 而是一種變異的蛋白質,Prusiner P i 並將此具感染力病原命名為 Prion B 型肝炎表面抗原 Kuru

The prion-related diseases Creutzfeldt-Jakob

encephalopathy, BSE (Mad Cow Disease) 牛海綿狀腦病

and elk Stained section of cerebral cortex from

degeneration, the most characteristic ti

15 The prion-related diseases Creutzfeldt-Jakob Disease and kuru in human Bovine spongiform encephalopathy, BSE (Mad Cow Disease) 牛海綿狀腦病 Scrapie in sheep Chronic Wasting Disease in deer and elk Stained section of cerebral cortex from autopsy of a patient with CJD showes spongiform degeneration, the most characteristic ti neurohistological feature Lehninger Principles of Biochemistry 5 th edition

16 Determined the prion structure by NMR

17 Protein folding and Denaturation ti

18 The ubiquitination cascade E1 E2 E3 Degradation

19 Comparison of ubiquitin and SUMO Small Ubiquitin-like Modifier (SUMO) C-terminal GG motif

20 The mechanism of reversible sumoylation SENP: sentrin- specific protease Processing E1 Sumoylation E2 Desumoylation E3

21 The structural comparison of Ub and SUMO ψkxe motif GG GG Ub SUMO

22 Molecular consequences of sumoylation Interfere interaction Provide a binding site Conformational change More active or inactive

23 SUMO participates in diverse cellular processes DNA damage repair Nuclear transport Chromosome segregation Cell division Hypoxia Stress response Inflammatory response Oncogenesis Flowering time in plants

24 Identification and characterization of the SUMO system in Chlamydomonas reinhardtii

25 Cloning and expression of CrSUMOs CrSUMO96 CrSUMO97 CrSUMO96 CrSUMO97 ψkxe motif 78% 46% GG

26 CrSUMO-96 and CrSUMO-97 share a similar structural feature with some minor differences A C H1 S2 180 O 180 O S2 H1 CrSUMO96 CrSUMO96 B D * ** H1 S2 180 O 180 O ** * S2 H1 CrSUMO97 CrSUMO97

27 CrSUMO-GG can be polysumoylated by animal E1-E2 E2 in E. coli S96-Full S96-GG S97-Full S97-GG E1-E PolySUMO MonoSUMO

28 SENP1-SUMO complex GG SENP1 SUMO

29 Digestion of CrSUMOs-GST by human SENP1 SENP1 CrSUMO96-GG-GST NEM CrSUMO97-GG-GST NEM CrSUMO-GST SENP1 CrSUMO WB: Anti-His WB: Anti-His

30 PolyCrSUMO chains can be deconjugated d by human SENP CrSUMO CrSUMO-2 E1/E SENP PolyCrSUMOs SENP1 MonoCrSUMO

31 CrSUMO-conjugated proteins markedly increased upon heat shock kda min CrS96- conjugates min CrS97- conjugates Free CrS1 Free CrS2 WB: Anti-CrS1 WB: Anti-CrS2 CrSUMO96 and CrSUMO97 react differently in y response to heat shock

32 Surprise!! We found another CrSUMO148

It turns our that it s not that surprising!

CrSUMO148-1GG GG GG GG GG GG GG GG GG GG GG GG GG GG GG

SAE1/SAE2 (E1) - + - + - + - + - + - + - + - + Ubc9 (E2) - - +

33 It turns our that it s not that surprising! CrSUMO148-FL CrSUMO148-4GG CrSUMO148-3GG CrSUMO148-2GG CrSUMO148-1GG GG GG GG GG GG GG GG GG GG GG GG GG GG GG CrSUMO148-4GG CrSUMO148-3GG CrSUMO148-2GG CrSUMO148-1GG SAE1/SAE2 (E1) Ubc9 (E2) E1-CrS148-1GG PolyCrS148-1GG Ubc

34 SENP1 specifically digests CrSUMO148 at the first GG motif Cr rs148-fl SE ENP1 CrS148-FL + SENP1 Time (min) GG 48-2GG 48-3GG 48-4GG CrS1 CrS1 CrS1 CrS1 kda SENP1 CrS148-FL 17 CrS148-1GG Non of the predicted CrSUMO148 intermediate was observed.

35 Credit goes to 中研院生物多樣性中心吳俊宗教授臺灣大學生化科技學系張麗冠辛永誠劉邦宇蔡佳芸唐曉菁陳柔含胡蜜妮

36 國立臺灣大學生化科技學系張世宗

a P 700 P 680 a a Pq cytochrome Pc Fd H + ATP 酶 ATP ATP a a ATP H 2 OCO 2 H 2 O CO 2 CO 2 CO 2 CO 2 RuBP carboxylase 2

1000016 h t t p : / / w w w. k n s i. c o m. t w 248 30 (02)2299-9006 (02)2299-9110. 100 KANG SI 第一期 vol.1 P.1 P.4 P.7 P.8 99 99 99 ( ) -- ATP 1 a P 700 P 680 a a Pq cytochrome Pc Fd H + ATP 酶 ATP ATP