蛋白翻譯后修飾課件

1、蛋白翻譯后修飾各物種基因數(shù)量比較細(xì)菌：4400酵母：5800果蠅：14000小鼠：22000人：人：25000橘生淮南則為橘，生于淮北則為枳，葉徒相似，其實(shí)味不同。所以然者何？水土異也！genomic DNAbiological systemmRNAfunctional proteinprotein productssequencingexpression profilingstructural determinationprotein linkage maps(catalog)quantitative protein profilingprotein linkage maps(dynamic

2、)activity profilingpost-translational modification analysisinferred activitydata integrationdata integrationsubcellular localizationtechnologyemergingprototypematurelncRNAmicroRNA中心法則Molecular Biology of the Cell有序？表觀遺傳學(xué)表觀遺傳學(xué) （epigenetics） DNA methylation, Histone modification, RNA interference復(fù)制（復(fù)制

3、（replication）Protein - Protein, Protein - DNA, DNA - Protein蛋白質(zhì)修飾（蛋白質(zhì)修飾（protein modification）Outlines（3W1H）What is Post-translational modification ?What types of Post-translational modifications are exist?What is the biological meanings of protein post-translational modifications?How to detect the P

4、ost-translational modification?What is Post-translational modification (PTM)?Protein modifications- different types, including co-translational and post-translational- inteinsCo- and Post-Translational Modifications Co-translational modifications-occurs during protein synthesis N-terminal processing

5、 N-glycosylation Partial proteolysisPost-translational modifications-occurs after protein synthesis phosphorylation methylation O-glycosylationPosttranslational modification (PTM) is the chemical modification or processing of a protein after its translation. It is one of the later steps in protein b

6、iosynthesis, and thus gene expression, for many proteins.-Numerous （400 types）-Diverse (organisms; proteins) - Preference (e.g., phosphorylation：Ser Thr Tyr)What types of Post-translational modifications are exist?Posttranslational modifications including:Disulfide bond formationChemical modificatio

7、ns: Phosphorylation （1992 Nobel Prize） Acetylation Ubiquitination （2004 Nobel Prize） Glycosylation SUMOylation Methylation Succinylation Proteolytic processingPosttranslational ModificationAcylation （?；饔茫；饔茫?loss of a-amino positive chargeAlkylation alteration of a- or e-amino positive groupCarb

8、oxylmethylation esterification of specific carboxyl groupPhoshorylation mainly modify Ser, Thr and TyrSulfation mainly modify TyrCarboxylation bring negative charge Proteolytic processing truncation leads to change of pI Modification Charge-dependent change Posttranslational ModificationNucleus acet

9、ylation, phosphorylationLysosome mannose-6-phosphate labelled N-linked sugarMitochondria N-formyl acylationGolgi N- and O-linked ologosaccharide, sulfation, palimitoylationER N-linked oligosaccharide, GPI-anchorCytosol acetylation, methylation, phosphorylation, Ribosome myristoylationPlasma membrane

10、 N- and O-glycosylation, GPI-anchorExtracellular fluid N- and O-glycosylation, acetylation, phosphorylation Extracellular matrix N- and O-glycosylation, phosphorylation, hydroxylation Location ModificationWhy protein post-translational modifications?The Biological Meanings Protein processing and mod

11、ification comprise an important third dimension of information, beyond those of DNA sequence and protein sequence. The thousands of component proteins of a cell and their post-translational modifications may change with the cell cycle, environmental conditions, developmental stage, and metabolic sta

12、te. Protein types may similar in a specific time period, but PTMs change all the time Effects of Post-translational Modification (1)PTMs involving addition of functional groups PTMs involving addition by an enzyme in vivo-PTMs involving addition of hydrophobic groups for membrane localization-PTMs i

13、nvolving addition of cofactors for enhanced enzymatic activity-PTMs involving unique modifications of translation factors-PTMs involving addition of smaller chemical groups PTMs involving non-enzymatic additions in vivoEx. Glycation, the addition of a sugar molecule to a protein without the controll

14、ing action of an enzyme PTMs involving non-enzymatic additions in vitroEx. biotinylation, acetylation of conserved lysine residues with a biotin appendageEffects of Post-translational Modification (2) PTMs involving addition of other proteins or peptides - SUMOylation, the covalet linkage to the SUM

15、O protein - Ubiquitination, the covalent linkage to the protein ubiquitin PTMs involving changing the chemical nature of amino acids - Citrullination（瓜氨酸化瓜氨酸化）, or deimination, the conversion of arginine to citrulline- Deamidation（脫酰氨基作用脫酰氨基作用）, the conversion of glutamine to glutamic acid or aspara

16、gine to aspartic acid- Eliminylation, the conversion to an alkene by beta-elimination of phosphothreonine and phosphoserine, or dehydration of threonine and serine, as well as by decarboxylation of cysteine- Carbamylation（氨甲?；奔柞；? the conversion of lysine to homocitrulline PTMs involving structura

17、l changes - disulfide bridges, the covalent linkage of two cysteine amino acids - proteolytic cleavage, cleavage of a protein at a peptide bond - racemization（外消旋作用） of proline by prollisomeraseA. Nonenzymatic Reactiondeamidation：Asn, Gln Asp/Gluracemization：Asp, Serdehydroalanine：Cys, phosphor-Sers

18、low oxidation：Cys, His, Metslow cleavage and permutation of peptide bondsreducing sugar reaction with NH2-group of aas or side chains (Lys)：Maillard reaction (Browing reaction)；Schiffs base reaction.B. Enzymatic ReactionN-linked glycosylationCarboxyl methylationS-isoprenylation-Cys1. Irrversible, Un

19、idirectional Reaction (permanently modified) Phosphorylation (protein kinase) / Dephosphorylation (phosphatase)：Ser, Tyr, Thr. Uridylyl and adenylyl transfer in bacterial glutamine synthetase2. Irrversible, Bi-directional Reaction. (Signal Amplification)Protein Protein-nPinPi Protein Phosphotase H2O

20、 nATP Protein Kinases nADP RS-SR + R-SH R-S-S-R + RSH(disulfide isomerase) Coupled with protein-folding process3. Reversible ReactionWhat is the biological meanings of Post-translational modification (PTM)?Jensen Nature Reviews Molecular Cell Biology 7, 391-403 (June 2006) | doi:10.1038/nrm1939Disul

21、fide bond formationMaturation of InsulinTranslocation of Secretory ProteinGlycosylation is the reaction in which a carbohydrate, is attached to a hydroxyl or other functional group of another molecule. (Most, 1%)Glycosylation is a form of co-translational and post-translational modification. Glycans

22、 serve a variety of structural and functional roles in membrane and secreted proteins (ER, Golgi ).Five classes of glycans are produced:N-linked glycans attached to a nitrogen of asparagine or arginine side-chainsO-linked glycans attached to the hydroxy oxygen of serine, threonine, tyrosine, hydroxy

23、lysine, or hydroxyproline side-chains, or to oxygens on lipids such as ceramide（神經(jīng)酰胺） phospho-glycans linked through the phosphate of a phospho-serine;C-linked glycans, a rare form of glycosylation where a sugar is added to a carbon on a tryptophan side-chain glypiation（糖基磷脂酰肌醇化）, which is the addit

24、ion of a GPI anchor that links proteins to lipids through glycan linkages.Example: ABO Blood GroupThe ABO Blood SystemThe ABO Blood SystemBlood Type(genotype)Type A(AA, AO)Type B(BB, BO)Type AB(AB)Type O(OO)AntigensOn RedBloodCell SurfacePlasmaAntibodiesB onlyA onlyNoneA and BA onlyB onlyA and BNone

25、ABO blood group in humanPET (Positron Emission Tomography) Scan （正電子發(fā)射斷層掃描） 18F-2-deoxy-glucose (more than 95% of PET scan in clinics) OHOHHOHHHF18HOHOHPET Scan for Head and Neck Tumorshttp:/ Scan for Breast Cancershttp:/ Scan for Lung Cancershttp:/ Function of protein glycosylation: Aids in proper

26、protein folding Provides protection against proteases Form ECM (extracellular matrix) Employed for signaling （ex. Modulation of immune response） Anchor protein on membraneMost soluble and membrane-bound proteins made in the ER（內(nèi)質(zhì)網(wǎng)） are glycoproteins, in contrast to cytosolic proteins.Glycoprotein sy

27、nthesis is a 3-part process:1.Assembly of the precursor oligosaccharide2.En-bloc transfer to the protein3.Modification of the oligosaccharide by removal of sugarsRole of oligosaccharides in recognition and adhesionPhosphorylation is the addition of a phosphate (PO43-) group to a protein or other org

28、anic molecule. Phosphorylation turns many protein enzymes on and off, causing or preventing the mechanisms of diseases such as cancer and diabetesKinases: specificitySer/Thrkinases: acceptor is OH of Ser or Thr;TyrKinases: acceptor is OH of Tyr Phosphorylation replaces neutral hydroxyl groups on ser

29、ines, threonines, or tyrosines with negatively-charged phosphates with pKs near 1.2 and 6.5. Thus, below pH 5.5, phosphates add a single negative charge; near pH 6.5, they add 1.5 negative charges; above pH 7.5, they add 2 negative charges. The relative amount of each isoform can also easily and rap

30、idly be determined from staining intensity on 2D gelsProtein PhosphorylationProtein Protein-nPinPi Protein Phosphotase H2O nATP Protein Kinases nADPFunction of protein phosphorylationBiological thermodynamics of energy-requiring reactions Phosphorylation of Na+/K+-ATPase during the transport of sodi

31、um (Na+) and potassium (K+) ions across the cell membrane in regulation to maintain stasis of the bodys water content.Mediates enzyme inhibition Phosphorylation of src tyrosine kinase (pronounced sarc) by C-terminal Src kinase (Csk) induces a conformational change in the enzyme, resulting in a fold

32、in the structure, which masks its kinase domain, and is thus shut off. Important for protein-protein interaction via recognition domains. Phosphorylation of the cytosolic(胞漿) components of NADPH oxidase, a large membrane-bound, multi-protein enzyme present in phagocytic cells, plays an important rol

33、e in the regulation of protein-protein interactions in the enzyme. Important in protein degradation. In the late 1990s, it was recognized that phosphorylation of some proteins causes them to be degraded by the ATP-dependent ubiquitin/proteasome pathway. These target proteins become substrates for pa

34、rticular E3 ubiquitin ligases only when they are phosphorylated.Chk-dependent phosphorylation of XRCC1 in the DNA damage response promotes base excision repairChou et al. EMBO J. 2008 Dec 3;27(23):3140-50Protein MethylationIn the chemical sciences, methylation denotes the addition of a methyl group

35、to a substrate or the substitution of an atom or group by a methyl group. Methylation is a form of alkylation. In biological systems, methylation is catalyzed by enzymes; such methylation can be involved in modification of heavy metals, regulation of gene expression, regulation of protein function,

36、and RNA metabolism. Methylation of heavy metals can also occur outside of biological systems. Protein MethylationPRMT(18)Type 2Type 1PRMT5,7PRMT1,3,4,6,8Symmetrical Di-methylation(SDMA)Asymmetrical Di-methylation(ADMA)MMALys: MonomethylatedDimethylatedtrimethylatedAcetylation describes a reaction th

37、at introduces an acetyl functional group into a chemical compound.Protein AcetylationProtein AcetylationaspirinSalicylic acidProtein UbiquitinationProtein UbiquitinationAntigen processingApoptosisBiogenesis of organellesCell cycle and divisionDNA transcription and repairDifferentiation and developme

38、ntImmune response and inflammationNeural and muscular degenerationMorphogenesis of neural networksModulation of cell surface receptors, ion channels and the secretory pathwayResponse to stress and extracellular modulatorsRibosome biogenesisViral infectionProcess of ubiquitination and proteosome-medi

39、ated protein degrationProtein SUMOylationSUMO (small ubiquitin-related modifier) proteins are small Protein tags that are conjugated to proteins to modify their function The ubiquitin system tags proteins for degradation by the proteosome but SUMO conjugation has a range of other functions, stabiliz

40、ing some proteins and altering their subcellular localization. Sumoylation may also influence ubiquitination and protein stability indirectly.Three different SUMO proteins are conjugated to proteins, SUMO-1, SUMO-2 and SUMO-3.Cross-talk It is now clear that PTMs work in concert, and the crosstalk be

41、tween different modifications determines the final biological read-out. some modifications can influence others, and it appears that specific combinations of these modifications can form a dynamic “code”. Although there are now many examples of these “functional networks”, it is likely that we have

42、just begun to scratch the surface. Better antibodies and novel technologies will help to complete this crosstalk puzzle, for which the specific fine-tuning appears critical to determine life as we know it.Examples of Cross-talkProgrammedSequentialHow To Identify PTMs? Low copy Specificity Fragile of

43、 peptide bonds Diversity SensitivityChallenge for PTM identificationMethods to detect protein modifications Western Blot(combine mutation to characterize modification sites ) 1D or 2D gel （32P） MS (mass spectrometry) - In-source CID - Precursor/parent ion scanning - Neutral loss scan Other methods:-

44、 IF (immunofluorescence) - ChIP(Chromatin immunoprecipitation)PhosphorylationAnalysis of the entire complement of phosphorylated proteins in cells: “phosphoproteome”Qualitative and quantitative information regarding protein phosphorylation Most common sites of phosphorylation: Ser, Thr, Tyr- Ratio:

45、1000/100/1 for serine/threonine/tyrosine- elimination reaction (80Da or 98Da)MS can be used to detect and map locations for phosphorylationMW increase from addition of phosphate grouptreatment with phosphatase allows determination of number of phosphate groupsdigestion and tandem MS allows for deter

46、mination of phosphorylation sitesMapping post-translational modifications using mass spectrometrySilver Staining Western Blotting（immunostaining）Two dimensional gel images Proteomics in Practice: A Laboratory Manual of Proteome AnalysisPART II: COURSE MANUAL Step 1: Sample Preparation Step 2: Isoele

47、ctric Focusing Step 3: SDS Polyacrylamide Gel Electrophoresis Step 4: Staining of the Gels Step 5: Scanning of Gels and Image Analysis Step 6: 2D DIGE Step 7: Spot Excision Step 8: Sample Destaining Step 9: In-gel Digestion Step 10: Microscale Purification Step 11: Peptide DigestionStep 12: MS Analy

48、sis Step 13: Calibration of the MALDI-TOF MS Step 14: Preparing for a Database Search Step 15: PMF Database Search Unsuccessful Enrichment strategies to analyze phosphoproteins/peptides Anti-pY quite successful Anti-pS and anti-pT not as successful, but may be used Negatively charged phosphate group

49、s bind to postively charged metal ions (e.g., Fe3+, Ga3+) immobilized to a chromatographic support Limitation: non-specific binding to acidic side chains (D, E)PhosphorylationLiebler, D. C. (2002) Introduction to Proteomics, Humana PressUse of immobilized metal affinity chromatography (IMAC) to isol

50、ate phosphopeptides from a peptide digest.Others 2D-PP (two-dimensional phosphopeptide mapping ) RP-HPLC CE (capillary electrophoresis) Immunoprecipitation Chemically modified CHIPPeppermintStick phosphoprotein molecular weight standards separated on a 13% SDS polyacrylamide gel. The markers contain

51、 (from largest to smallest) beta-galactosidase, bovine serum albumin (BSA), ovalbumin, beta-casein, avidin and lysozyme. Ovalbumin and beta-casein are phosphorylated. The gel was stained with Pro-Q Diamond phosphoprotein gel stain (blue) followed by SYPRO Ruby protein gel stain (red). The digital im

52、ages were pseudocolored.PhosphoVisualization of total protein and phosphoproteins in a 2-D gelProteins from a Jurkat T-cell lymphoma line cell lysate were separated by 2-D gel electrophoresis and stained with Pro-Q Diamond phosphoprotein gel stain () followed by SYPRO Ruby protein gel stain (). Afte

53、r each dye staining, the gel was imaged and the resulting composite image was digitally pseudocolored and overlaid.T.H. Steinberg et al., Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology, Proteomics 2003, 3, 1128-1144Isotope-coded glycosylation-site-specific taggin

54、g(IGOT)Case studyFEN-1 is a structure-specific and multifunctional nuclease (FEN)(EXO)(GEN)Okazaki fragment maturationLong-patch base excision repair pathwayRepeat sequence expansionsApoptotic DNA fragmentationReplication fork resolutionTelomere stabilityFEN1 is involved in multiple DNA metabolism p

55、athwaysZheng, L, et al., Nucleic Acids Res, 2010.FEN1 in Long-patch base excision repairDynamic subcellular localization of FEN1Zheng, L, et al., Nucleic Acids Res, 2010.c-myc-FEN-1mitotrackermergeWTH83K354A Lysine354 is a key residue for FEN1 nuclear localizationProteinprotein interactions mediate

56、FEN1s actions in different DNA metabolic pathwaysBiochemicalactivityBiochemical pathwaysPredicted clinical consequences due to activity deficiencyFEN1. RNA primer removal2. Long patch base excision repairEmbryonic lethalityEXO1. DNA double strand break repair2. Apoptotic DNA fragmentation1. Cancers2

57、. AutoimmunityGEN1. Rescue of stalled replication forks2. Apoptotic DNA fragmentation1. Cancers2. AutoimmunityFEN1 mutations link the mutator gene to human diseaseMethylation of Fen1 suppresses nearby phosphorylation and facilitates PCNA bindingPRMT(18)Type 2Type 1PRMT5,7PRMT1,3,4,6,8Protein arginin

58、e methyltransferases (PRMT) and arginine methylation.A). PRMT family contains 8 sequence-related members which can be divided into two groups. B). Gourp1 catalyzes the formation of type 1 methylation while group 2 catalyzes the formation of type 2 methylation. B.Symmetrical Di-methylation(SDMA)Asymm

59、etrical Di-methylation(ADMA)MMAA.FEN1 is methylated by PRMT5 in vitro and in vivo bed ccIdentification and validation of FEN1 methylation sitesMethylation of FEN1 at Arg192 inhibits its phosphorylation at Ser187Methylation of FEN1 ensures its interaction with PCNA via suppression of its phosphorylationCo-localization of FEN-1 with PCNA or BrdUMethylation defect retards Okazaki fragment maturationMethylation defect retards DNA repli