蛋白質(zhì)降解與修飾理論與實(shí)驗(yàn)1

1、會(huì)計(jì)學(xué)1蛋白質(zhì)降解與修飾理論與實(shí)驗(yàn)蛋白質(zhì)降解與修飾理論與實(shí)驗(yàn)1Proteins constitute essential components of cellshttp:/ 溶酶體 (Lysosomes)Non-selective Hydrolyze proteins from autophagy Digest proteins from endocytosis/Autophagy Membrane proteins Extracellular proteins 2. 泛素-蛋白酶體通路Selective and (Ubiquitin-Proteasome Pathway) ATP-Depen

2、dent Regulate biochemical reactions Discard unnecessary proteins Degrade damaged or misfolded proteins Present internalized antigens in immunological responses3. 非泛素-蛋白酶體通路Selective and ImmunoproteasomeATP-independent REGgamma dependent protein degradationThe 26S proteasome is a multisubunit complex

3、, consisting of the 20S catalytic particle and the 19S regulatory particle, and selectively degrades ubiquitinated proteins in the presence of ATP. Upon immune responses, cells generate special types of proteasomes, immunoproteasomes, which possess distinct regulatory particle (11S ) and catalytic p

4、article (with different catalytic subunits), for more efficient antigen presentation. Novel protein degradation short-cut pathway was recently disclosed. A representative is the REGgamma-mediated protein degradation.Proteasomes are responsible for degradation of most cellular proteinsa1a2a3a4a7a6a5b

5、1b2b3b4b1b2b3b4a4a3a2a1Proteasome core19 S Proteasome capRpn3Rpn6Rpn11Rpn12Rpn7Rpn5Rpn9Rpn8Rpn10Rpn1Rpn2Rpt1Rpt2 Rpt6 Rpt4 Rpt5Rpt3LidBaseb5 b6 b711 S alternativeProteasome capREGa, REGb, REGgA barrel-shaped cylinder composed of four stacked rings. Each ring contains seven distinct polypeptide subun

6、itsThe -rings modulate the entrance of protein substrates, whereas the b-rings execute the action of proteolysis20S ProteasomeThree of the seven b-subunits have functional threonine protease sites, each generating different proteolytic activitiesb1b2b3b4b5b6b7Chymotypsin-likeCaspase-like Trypsin-lik

7、e20S catalytic core a1a2a3a4a6a7a5b1b2b3b4b7b6b5a7a6a5a4abE1-E1E2-E2底物E3ATPAMP+PPiUbUbUb蛋白酶體泛素蛋白酶體通路Ub: 泛素 (Ubiquitin)，一高度保守的，由76個(gè)氨基酸組成的多肽。E1: 泛素激活酶 (Ubiquitin-activating enzyme)，人類僅有2種E1。E2: 泛素載體蛋白 (Ubiquitin-carrier protein)， 人類約有 30 種E2s。E3: 泛素蛋白連接酶 (Ubiquitin-protein ligase)， 人類有1000多種E3s 。 Aaro

8、n Ciechanover Avram Hershko Irwin Rose 阿龍.西查諾瓦 阿夫拉姆.赫?？?歐文.羅斯 篇數(shù)年份1975年，Goldstein誤將泛素（Ubiquitin)當(dāng)作胸腺激素而發(fā)現(xiàn)。 1977年，Goldknopf和Busch認(rèn)定組蛋白2A與泛素以異肽鍵（Isopeptide Bond）結(jié)合。 1977年，Goldberg證明人類細(xì)胞中存在一可溶的，直接依賴于能量的非溶 酶體類蛋白酶 。1978年，Ciechanover 和 Hershko發(fā)現(xiàn)了APF-1是Goldberg系統(tǒng)中蛋白酶 系的必要成分，結(jié)果發(fā)表在BBRC。1980年，Ciechanover，Hers

9、hko和Rose等人證明了APF-1與當(dāng)時(shí)已被發(fā) 現(xiàn)的泛素是同一物質(zhì) 。1984年，F(xiàn)inley和Varshavsky等發(fā)現(xiàn)ubiquitin conjugation，以及泛素在 細(xì)胞周期中的重要性。 1986年，Alexander Varshavsky發(fā)現(xiàn)degradation signals (degrons) 以 及 N-end rule。1987年，Goldberg和Rechsteiner兩個(gè)小組幾乎同時(shí)分離出分子量很大的 依賴于ATP并降解泛素化底物的蛋白水解酶。1988年，Goldberg將這種蛋白水解酶命名為Proteasome（蛋白酶體）。1989年， 1997年，Yeh 等小

10、組發(fā)現(xiàn)類泛素蛋白SUMO和NEDD8的功能。2003年，美國FDA批準(zhǔn)了用Velcade來治療多發(fā)性骨髓瘤。2004年，Ciechanover，Hershko和Rose因發(fā)現(xiàn)泛素介導(dǎo)的蛋白質(zhì)降解共 同獲得諾貝爾化學(xué)獎(jiǎng)。 Ubiquitin (泛素）泛素由76個(gè)氨基酸殘基組成，其中包括7個(gè)賴氨酸殘基(K), 其C末端可與底物的賴氨酸殘基形成異肽鍵，從而引起底物泛素化。泛素的K11、K29、K48和K63均能參與形成泛素與泛素間的異肽鍵 (Isopeptide bond)。The C-terminal glycine 76 (Gly76) was identified to attach to t

11、he lysine site of protein substrate during ubiquitination 。失調(diào)蛋白 底物 修飾 腫瘤類別SKP2 p27 (KIP) Polyubiquitylation Malignant melanomaMDM2 p53 Polyubiquitylation Non-small-cell lung cancer, soft-tissue carcinoma, colorectal cancer HAUSP p53, MDM2 De-ubiquitylation Non-small-cell lung cancer lymphoma APC Cyc

12、lin B, Polyubiquitylation Colorectal cancer securin FANCL FANCD2 Monoubiquitylation Fanconi anaemia-related cancers CYLD IKK De-ubiquitylation Cylindromatosis IAP2 BCL10 Polyubiquitylation MALT lymphomas CBL RTKs Multiple Lymphoma, AML and monoubiquitylation gastric carcinoma pVHL HIFPolyubiquitinat

13、ionvon Hippel-Lindau disease泛素化失調(diào)與腫瘤泛素連接酶Mdm2和E6-AP與p53的降解和癌癥人乳頭瘤病毒（HPV）HeLa宮頸癌11S proteasomeImmunoproteasome (PA28a/b) In response to interferon-g, the b1, b2, and b5 subunits are replaced during proteasome de novo biogenesis by b1i (LMP2), b2i (MECL1), and b5i (LMP7) to form the immunoproteasome.

14、PA28a/b complex-a hetero-oligomer complex; interferon-g inducible; nuclear and cytosol localization; function: antigen processing.20S constitutive proteasome a1a2a3a4a6a7a5b1b2b3b4b7b6b5a7a6a5a4a1a2a3a4a6a7a5b2ib3b4b7b6b5ia7a6a5a4+b1iINF-g20S immunoproteasome a6a7a5aaabbbbPA28g complex-a homo-oligom

15、er complex; non-interferon-g inducible; nuclear localization; cellular targets: SRC-3, p21, HCV-core protein.11S proteasome activator ( consist of REG or PA28 a, b or g)-bindsto the a rings of the 20S proteasome, enhancing proteolytic activityin an ATP- and ubiquitin- independent manner.免疫蛋白酶體(Immun

16、oproteasome)負(fù)責(zé)抗原提呈(Antigen Presentation)免疫蛋白酶體的催化顆粒(20S) 有三個(gè)特殊亞單位LMP2、LMP7 和MECL-1。其調(diào)節(jié)顆粒為PA28（11S）。20S顆粒MHC class I antigen presentation pathwayT cellPeptideMHC class IProtein antigenProteasome+/- UbiquitinPeptides (2-24 a.a.)TAP (8-16 a.a.)Endoplasmic ReticulumCD8+Golgi8-10 a.a.Amino acidsERAP1endo

17、plasmic reticulum aminopeptidase 1 Transporter associated with antigen processingElectron micrographs of REGg as a heptamerLi et al, EMBO, 20, 2001Introduction to REGg REGg (PA28g, PSME3, or Ki antigen) belongs to the REG or 11S family of proteasome activators. Originally isolated as Ki antigen, REG

18、g is the target of an autoantibody appearing in the serum of systemic lupus erythematosus patients. REGg forms a homoheptamer with a molecular mass of 200 kDa. Its sub-cellular distribution is primarily nuclear. In vitro, REGg is known to stimulate Trypsin-like activity. REGg deficient mice display

19、reduced body size with cell specific mitotic defects, and Increased apoptosis. Our current study indicate that REGg plays important roles in multiple cellular processes, including carcinogenesis, aging, male reproductive function, neuronal function, and metabolism.類泛素蛋白及其同源性u(píng)biquitin-like proteins U

20、biquitin-fold modifier 1 Alzheimers Disease Protein modification by ubiquitin-like protein, PUP, in prokaryotesUbiquitinISG15SumoNEDD8PIASSUMO修飾的功能Targeting RanGAP1to RanBP2SUMO化修飾的底物BRBOutline for Experimental Assays:1. Proteasome activity assays: Fluorogenic peptide cleavage -In vitro proteasome a

21、ctivity -In vivo proteasome activity2. Protein stability/degradation assays: -Measurement of protein decay in vivo -Protein degradation assays3. Cell free ubiquitilation assays.4. In vivo sumoylation assay.5. AutophagyIn vitro proteasome activityBackground:20S proteasome has at least three different

22、 peptidase activity:Chymotrypsin-like (b1), trypsin-like (b2), peptidylglutamyl-peptide hydrolyzing (PGPH, b5) activity. PGPH is also known as caspase-like activity.These cleave on the carboxyl side of hydrophobic, basic and acidic residues, respectively.Background:All three enzymes hydrolyze the pe

23、ptide bond but they have different target amino acids.Trypsin like Arg, Lys basic aa (Boc-LRR)Chymotrypsin F, Yaromatic aa (Suc-LLVY; Suc-FLF)Caspase-like activity Asp, Gluacidic aa (Acctyl-YVAD, Z-LLE)In vitro proteasome activityApplication:1. To Determine the specific proteasome activity.2. To exa

24、mine the action of a specific proteasome activator.3. To explore the regulation of a substrate protein degradation.In vitro proteasome activityLi. et al. EMBO J. 2001 Jul 2;20(13):3359-69. Outline for Experimental Assays:1. Proteasome activity assays: Fluorogenic peptide cleavage -In vitro proteasom

25、e activity -In vivo proteasome activity2. Protein stability/degradation assays: -Measurement of protein decay in vivo -Protein degradation assays3. Cell free ubiquitilation assays.4. In vivo sumoylation assay.5. AutophagyJ Med Chem. 2010 Oct 28;53(20):7452-60.A new fluorogenic peptide determines pro

26、teasome activity in single cells.Urru SA, . R, Salmona M.SourceVia La Masa 19, Milan 20156, Italy.AbstractThe ubiquitin-proteasome system plays a critical role in many diseases, making it an attractive biomarker and therapeutic target. However, the impact of results obtained in vitro using purified

27、proteasome particles or whole cell extracts is limited by the lack of efficient methods to assess proteasome activity in living cells. We have engineered an internally quenched fluorogenic peptide with a proteasome-specific cleavage motif fused to TAT and linked to the fluorophores DABCYL and EDANS.

28、 This peptide penetrates cell membranes and is rapidly cleaved by the proteasomal chymotrypsin-like activity, generating a quantitative fluorescent reporter of in vivo proteasome activity as assessed by time-lapse or flow cytometry fluorescence analysis. This reporter is an innovative tool for monit

29、oring proteasomal proteolytic activities in physiological and pathological conditions.In vivo proteasome activityOutline for Experimental Assays:1. Proteasome activity assays: Fluorogenic peptide cleavage -In vitro proteasome activity -In vivo proteasome activity2. Protein stability/degradation assa

30、ys: -Measurement of protein decay in vivo -Protein degradation assays3. Cell free ubiquitilation assays.4. In vivo sumoylation assay.5. AutophagyMeasurement of protein decay in vivoBackground:In E. Coli, 2-6% proteins are degraded per hour at steady state.During starvation, 20-40% proteins can be de

31、graded. Two methods are commonly used to determine a proteins half-life, namely radioactive pulse-chase analysis and cycloheximide chase.Other methods: photoactivatable fluorescent proteins (PAFPs).DisadvantageCycloheximide chase strongly affects cellular metabolism that should be considered a serio

32、us problem for this approach.PAFPs needs protein fusion, which may influence some protein turnover. Measurement of protein decay in vivoApplication:1. To Determine the half life of a specific protein.2. To analyze the mechanisms how a specific protein is degraded (in combination with other exp).3. T

33、o define the causal relations between a destructive enzyme and a substrate protein.Radioactive pulse-chase analysisIn biochemistry and molecular biology, a pulse-chase analysis is a method for examining a cellular process occurring over time by successively exposing the cells to a labeled compound (

34、pulse) and then to the same compound in an unlabeled form (chase). - Starve the cells- Pulse chase with 35-S-Met or Lue for 5-30 min.- Chase with “cold” Met or Lue.- By the end of pulse (0 time), collect samples in a time course,lyse the cells, perform immunoprecipitation and autoradiography.Animati

35、onOutline for Experimental Assays:1. Proteasome activity assays: Fluorogenic peptide cleavage -In vitro proteasome activity -In vivo proteasome activity2. Protein stability/degradation assays: -Measurement of protein decay in vivo -Protein degradation assays3. Cell free ubiquitilation assays.4. In v

36、ivo sumoylation assay.5. AutophagyProtein degradation assaysBackground:Traditional in vitro approaches, for ubiquitin-dependent and Ub-independent systems, are to use purified protein components orpartially purified protein complexes to carry out in vitro protein degradation in test tubes.In vivo as

37、says were recently employed by engineering stable celllines containing dual luciferase-fusion protein reporter, including ubiquitin-fusion substrate, ubiquitin-E3 substrates, ubiquitin-independent. Protein degradation assaysApplication:1. Determine proteasome-substrate relations.2. Regulatory mechan

38、isms of protein degradation.3. High-throughput screening of inhibitors in protein degradation.Mol Cell. 2007 Jun 22;26(6):831-42. IRES: internal ribosome entry site Outline for Experimental Assays:1. Proteasome activity assays: Fluorogenic peptide cleavage -In vitro proteasome activity -In vivo prot

39、easome activity2. Protein stability/degradation assays: -Measurement of protein decay in vivo -Protein degradation assays3. Cell free ubiquitilation assays4. In vivo sumoylation assay.5. AutophagyIn vitro ubiquitylationApplication:1.To define a specific protein substrate of a given E3 ligase.2.To de

40、termine the activity of a putative E1/E2/E3 enzyme.3.To examine the action of a regulatory protein at certain condition.4.Detection of auto-, mono-, or poly-ubiquitination of proteins.In vitro ubiquitylation(example)Methods:Substrate protein (p53) was incubated with E3 (GST-MDM2) and HA-REG for two

41、hours at 37 in presence of E1 (UBE1), E2 (Ubch5a) and ubiquitin in the reaction buffer.Reaction buffer:50 mM Tris-cl (pH 7.5), 50 mM NaCl, 4 mM MgCl, 2 mM ATP, 1mM DTTREG enhance p53 ubiquitylationE1: 110ng; E2: 170ng; MDM2:10ng; ub: 5ug; REG:100ngJ Cell Sci. 2010 Dec 1;123(Pt 23):4076-84. 實(shí)驗(yàn)小細(xì)節(jié)：1.

42、酶、蛋白的保存2. 微量蛋白添加，減小樣品間差異建議分裝保存，避免反復(fù)凍融；自己純化的蛋白要加甘油保護(hù)液。共同的成分制作Mix，均分；稀釋，減小成分誤差。Outline for Experimental Assays:1. Proteasome activity assays: Fluorogenic peptide cleavage -In vitro proteasome activity -In vivo proteasome activity2. Protein stability/degradation assays: -Measurement of protein decay in

43、 vivo -Protein degradation assays3. Cell free ubiquitilation assays.4. In vivo sumoylation assay.5. AutophagyBackground：SUMO: Small ubiquitin-like modifiers. Three functional isoforms of SUMO family members have been recognized, SUMO1, SUMO2 and SUMO3. In vivo sumoylation assay is an essential evide

44、nce to prove that a protein is SUMO modified. Over-expression of 6His-tag SUMO is commonly used for in vivo sumoylation assay. 富集。Over-expression of E2 or E3 with his-SUMO can make it much easier to see the SUMO conjugation bands in sumoylation assay.His-tagNi-NTA AgaroseproteinInteraction between N

45、i-NTA and a 6xHis-tagged proteinBackground：Ni-NTA Agarose（QIAGEN lot#130187170): nickel-nitrilotriacetic acid. NTA occupies four of the six ligand binding sites in the coordination sphere of the nickel ion, leaving two sites free to interact with the 6xHis tag .Preparation:1. Generate expression vec

46、tor of target protein.2. Generate His-tagged SUMO construct.3. SUMO E2/E3 expression vector(s).4. Check expression of each components in designated cells.Protocol （denature）Cell lysis preparation1.Grow cells in 10cm or 6cm dishes and harvest cells into 1.5ml tubes after transfection 24h.2.Take out 1

47、/5 cell suspension for input . 4/5 cells are suspended in 5ml lysis buffer (buffer A).Nickel affinaty chromatography to purify SUMO conjugates3. Add 30ul Ni-NTA agarose beads and rotate for 4h at RT or 10-12h at 4.Spin down beads and transfer to 1.5ml tubes. Wash with buffer A, B, C+0.2%triton-100 a

48、nd C for 5min each.Incubate with 50ul elution buffer for 20min at RT with shaking at 500 rpm. Load with loading buffer.Western blottingbuffer A/ lysis buffer (6M guanidinium-HCl, 0.1M Na2HPO4/NaH2PO4 ph 8.0, 0.01M Tris-HCl ph 8.0, 5mM imidazole, 10mM -mecaptoethanol ) buffer B (8M Urea, 0.1M Na2HPO4

49、/NaH2PO4 ph 8.0, 0.01M Tris-HCl ph 8.0, 10mM -mecaptoethanol )buffer C (8M Urea, 0.1M Na2HPO4/NaH2PO4 ph 6.3, 0.01M Tris-HCl ph 6.3, 10mM -mecaptoethanol ) elution buffer( 200mM imidazole, 0.15M Tris-HCl ph 6.7, 30% glycerol, 5%SDS, 0.72M -mecaptoethanol Cell Res. 2011 May;21(5):807-16. Epub 2011 Ma

50、r 29. Outline for Experimental Assays:1. Proteasome activity assays: Fluorogenic peptide cleavage -In vitro proteasome activity -In vivo proteasome activity2. Protein stability/degradation assays: -Measurement of protein decay in vivo -Protein degradation assays3. Cell free ubiquitilation assays.4. In vivo sumoylation assay.5. AutophagyAtg proteins that have thu