蛋白翻译后修饰课件.pptx

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1、蛋白翻译后修饰,各物种基因数量比较,细菌：4400,酵母：5800,果蝇：14000,小鼠：22000,人：25000,Genotype,Phenotype,橘生淮南则为橘，生于淮北则为枳，叶徒相似，其实味不同。所以然者何？水土异也！,genomic DNA,mRNA,protein products,sequencing,expression profiling,structural determination,protein linkage maps(catalog),quantitative protein profiling,protein linkage maps(dynamic),

2、activity profiling,post-translational modification analysisinferred activity,data integration,data integration,subcellular localization,technology,emerging,prototype,mature,lncRNAmicroRNA,中心法则,Molecular Biology of the Cell,有序？,表观遗传学（epigenetics）DNA methylation,Histone modification,RNA interference,复

3、制（replication）Protein-Protein,Protein-DNA,DNA-Protein,蛋白质修饰（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 Post-translat

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

5、 Partial proteolysis,Protein modification,Post-translational modificationsoccurs after protein synthesis phosphorylation methylation O-glycosylation,Posttranslational modification(PTM)is the chemical modification or processing of a protein after its translation.It is one of the later steps in protei

6、n biosynthesis,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 modifications:Pho

7、sphorylation（1992 Nobel Prize）Acetylation Ubiquitination（2004 Nobel Prize）Glycosylation SUMOylation Methylation Succinylation Proteolytic processing,Posttranslational Modification,Acylation（酰化作用）loss of a-amino positive chargeAlkylation alteration of a-or e-amino positive groupCarboxylmethylation es

8、terification 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 Modification,Nucleus acetylation,phosphoryl

9、ationLysosome 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 N-and O-glycosylation,GPI

10、-anchorExtracellular fluid N-and O-glycosylation,acetylation,phosphorylation Extracellular matrix N-and O-glycosylation,phosphorylation,hydroxylation,Location Modification,Why protein post-translational modifications?The Biological Meanings,Complexity of the Proteome,Protein processing and modificat

11、ion 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 state.Protein

12、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 vivoPTMs involving addition of hydrophobic groups for membrane localizationPTMs involving additio

13、n of cofactors for enhanced enzymatic activityPTMs involving unique modifications of translation factorsPTMs involving addition of smaller chemical groupsPTMs involving non-enzymatic additions in vivoEx.Glycation,the addition of a sugar molecule to a protein without the controlling action of an enzy

14、mePTMs involving non-enzymatic additions in vitroEx.biotinylation,acetylation of conserved lysine residues with a biotin appendage,Effects of Post-translational Modification(2),PTMs involving addition of other proteins or peptides-SUMOylation,the covalet linkage to the SUMO protein-Ubiquitination,th

15、e covalent linkage to the protein ubiquitinPTMs involving changing the chemical nature of amino acids-Citrullination（瓜氨酸化）,or deimination,the conversion of arginine to citrulline-Deamidation（脱酰氨基作用）,the conversion of glutamine to glutamic acid or asparagine to aspartic acid-Eliminylation,the convers

16、ion to an alkene by beta-elimination of phosphothreonine and phosphoserine,or dehydration of threonine and serine,as well as by decarboxylation of cysteine-Carbamylation（氨甲酰化）,the conversion of lysine to homocitrullinePTMs involving structural changes-disulfide bridges,the covalent linkage of two cy

17、steine amino acids-proteolytic cleavage,cleavage of a protein at a peptide bond-racemization（外消旋作用）of proline by prollisomerase,A.Nonenzymatic Reaction,deamidation：Asn,Gln Asp/Gluracemization：Asp,Serdehydroalanine：Cys,phosphor-Serslow oxidation：Cys,His,Metslow cleavage and permutation of peptide bon

18、dsreducing sugar reaction with NH2-group of aas or side chains(Lys)：Maillard reaction(Browing reaction)；Schiffs base reaction.,B.Enzymatic Reaction,N-linked glycosylationCarboxyl methylationS-isoprenylation-Cys,1.Irrversible,Unidirectional Reaction(permanently modified),Phosphorylation(protein kinas

19、e)/Dephosphorylation(phosphatase)：Ser,Tyr,Thr.Uridylyl and adenylyl transfer in bacterial glutamine synthetase,2.Irrversible,Bi-directional Reaction.(Signal Amplification),RS-SR+R-SH R-S-S-R+RSH(disulfide isomerase)Coupled with protein-folding process,3.Reversible Reaction,What is the biological mea

20、nings of Post-translational modification(PTM)?,Jensen Nature Reviews Molecular Cell Biology 7,391-403(June 2006)|doi:10.1038/nrm1939,Disulfide bond formationMaturation of Insulin,Translocation of Secretory Protein,Glycosylation,Glycosylation is the reaction in which a carbohydrate,is attached to a h

21、ydroxyl or other functional group of another molecule.(Most,1%)Glycosylation is a form of co-translational and post-translational modification.Glycans serve a variety of structural and functional roles in membrane and secreted proteins(ER,Golgi).Five classes of glycans are produced:N-linked glycans

22、attached to a nitrogen of asparagine or arginine side-chainsO-linked glycans attached to the hydroxy oxygen of serine,threonine,tyrosine,hydroxylysine,or hydroxyproline side-chains,or to oxygens on lipids such as ceramide（神经酰胺）phospho-glycans linked through the phosphate of a phospho-serine;C-linked

23、 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 addition of a GPI anchor that links proteins to lipids through glycan linkages.,Example:ABO Blood Group,ABO blood group in human,PET(Positron Emission Tomography)Scan

24、（正电子发射断层扫描）,18F-2-deoxy-glucose(more than 95%of PET scan in clinics),PET Scan for Head and Neck Tumorshttp:/,PET Scan for Breast Cancershttp:/,PET Scan for Lung Cancershttp:/,Function of protein glycosylation:Aids in proper protein foldingProvides protection against proteasesForm ECM(extracellular m

25、atrix)Employed for signaling（ex.Modulation of immune response）Anchor protein on membraneMost soluble and membrane-bound proteins made in the ER（内质网）are glycoproteins,in contrast to cytosolic proteins.Glycoprotein synthesis is a 3-part process:Assembly of the precursor oligosaccharideEn-bloc transfer

26、 to the proteinModification of the oligosaccharide by removal of sugars,Role of oligosaccharides in recognition and adhesion,Phosphorylation is the addition of a phosphate(PO43-)group to a protein or other organic molecule.Phosphorylation turns many protein enzymes on and off,causing or preventing t

27、he mechanisms of diseases such as cancer and diabetesKinases:specificitySer/Thrkinases:acceptor is OH of Ser or Thr;TyrKinases:acceptor is OH of TyrPhosphorylation replaces neutral hydroxyl groups on serines,threonines,or tyrosines with negatively-charged phosphates with pKs near 1.2 and 6.5.Thus,be

28、low 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 rapidly be determined from staining intensity on 2D gels,Protein Phosphorylation,Function of protein phosphory

29、lation,Biological thermodynamics of energy-requiring reactions Phosphorylation of Na+/K+-ATPase during the transport of sodium(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 kin

30、ase(pronounced sarc)by C-terminal Src kinase(Csk)induces a conformational change in the enzyme,resulting in a fold 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(胞浆)components of N

31、ADPH oxidase,a large membrane-bound,multi-protein enzyme present in phagocytic cells,plays an important role 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 de

32、graded by the ATP-dependent ubiquitin/proteasome pathway.These target proteins become substrates for particular E3 ubiquitin ligases only when they are phosphorylated.,Chk-dependent phosphorylation of XRCC1 in the DNA damage response promotes base excision repair,Chou et al.EMBO J.2008 Dec 3;27(23):

33、3140-50,Protein Methylation,In the chemical sciences,methylation denotes the addition of a methyl group 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 invol

34、ved in modification of heavy metals,regulation of gene expression,regulation of protein function,and RNA metabolism.Methylation of heavy metals can also occur outside of biological systems.,Protein Methylation,Lys:MonomethylatedDimethylatedtrimethylated,Acetylation describes a reaction that introduc

35、es an acetyl functional group into a chemical compound.,Protein Acetylation,Protein Acetylation,Protein Ubiquitination,Protein Ubiquitination,Antigen processingApoptosisBiogenesis of organellesCell cycle and divisionDNA transcription and repairDifferentiation and developmentImmune response and infla

36、mmationNeural and muscular degeneration,Morphogenesis of neural networksModulation of cell surface receptors,ion channels and the secretory pathwayResponse to stress and extracellular modulatorsRibosome biogenesisViral infection,Process of ubiquitination and proteosome-mediated protein degration,Pro

37、tein 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,stabilizing some proteins and alteri

38、ng 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.,Home Work other modifications？,Cross-talk,It is now clear that PTMs work in concert,and the crosstalk be

39、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 just

40、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-talk,Programmed,Sequential,How To Identify PTMs?,Low copySpecificity Fragile of pep

41、tide bondsDiversitySensitivity,Challenge for PTM identification,Methods 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:-IF(immunofluore

42、scence)-ChIP(Chromatin immunoprecipitation),Phosphorylation,Analysis 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:1000/100/1 for serine/t

43、hreonine/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 determination of phosphorylat

44、ion sites,Mapping post-translational modifications using mass spectrometry,Silver Staining Western Blotting（immunostaining）,Two dimensional gel images,Proteomics in Practice:A Laboratory Manual of Proteome Analysis,PART II:COURSE MANUAL Step 1:Sample Preparation Step 2:Isoelectric Focusing Step 3:SD

45、S 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 Analysis Step 13:Calibration of the MAL

46、DI-TOF MS Step 14:Preparing for a Database Search Step 15:PMF Database Search Unsuccessful,Enrichment strategies to analyze phosphoproteins/peptides,Phosphospecific antibodiesAnti-pY quite successfulAnti-pS and anti-pT not as successful,but may be usedImmobilized metal affinity chromatography(IMAC)N

47、egatively charged phosphate groups bind to postively charged metal ions(e.g.,Fe3+,Ga3+)immobilized to a chromatographic supportLimitation:non-specific binding to acidic side chains(D,E),Phosphorylation,Liebler,D.C.(2002)Introduction to Proteomics,Humana Press,Use of immobilized metal affinity chroma

48、tography(IMAC)to isolate phosphopeptides from a peptide digest.,Others,2D-PP(two-dimensional phosphopeptide mapping)RP-HPLC CE(capillary electrophoresis)ImmunoprecipitationChemically modifiedCHIP,Phosphoprotein Stain,PeppermintStick phosphoprotein molecular weight standards separated on a 13%SDS pol

49、yacrylamide gel.The markers contain(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 sta

50、in(red).The digital images were pseudocolored.,Phospho,Phosphoprotein Stain,Visualization 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(blue)fo