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Post-Translational Modification David Shiuan Department of Life Science and Institute of Biotechnology National Dong Hwa University.

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Presentation on theme: "Post-Translational Modification David Shiuan Department of Life Science and Institute of Biotechnology National Dong Hwa University."— Presentation transcript:

1 Post-Translational Modification David Shiuan Department of Life Science and Institute of Biotechnology National Dong Hwa University

2 Disparity in mRNA and Protein profiles Electrophoresis 18(1997)533-537 Splicing variants In eukaryotic cells, likely 6-8 proteins/gene Post-translational modification 22 different forms of antitrypsin observed in human plasma

3 Posttranslational Modification What is it ? Addition of groups or deletion of parts to make a finished protein What groups ? How much ? Where ? - methyl - acetyl - glyco - phospho

4 Posttranslational Modification What purpose ? - targeting (eg. some lipoproteins) - stability (eg. secreted glycoproteins ) - function (eg. surface glycoproteins) - control of activity (eg. clotting factors, caspases) How can we study it ?

5 (Human Proteome Initiative)

6 Human proteome Initiative These are mainly generated by alternative splicing and post-translational modifications (PTMs)

7 Human Proteome Initiative

8 Human proteome Initiative 2000- Annotation of all known human proteins Annotation of mammalian orthologs of human proteins Annotation of all known human polymorphisms at the protein sequence level Annotation of all known post-translational modifications in human proteins Tight links to structural information

9 HPI Sep 2007


11 Formation of the nascent protein sequence

12 Protein Sorting and Sequence Modifications

13 Posttranslational Modifications

14 Post-Translational Modifications


16 Posttranslational Modification Modification Charge-dependent change Acylation loss of a-amino positive charge Alkylation alteration of a- or e-amino positive group Carboxylmethylation esterification of specific carboxyl group Phoshorylation mainly modify Ser, Thr and Tyr Sulfation mainly modify Tyr Carboxylation bring negative charge Sialyation mainly on Asn, Thr and Ser Proteolytic processing truncation leads to change of pI

17 Posttranslational Modification Location Modification Nucleus acetylation, phosphorylation Lysosome mannose-6-phosphate labelled N-linked sugar Mitochondria N-formyl acylation Golgi N- and O-linked ologosaccharide, sulfation, palimitoylation ER N-linked oligosaccharide, GPI-anchor Cytosol acetylation, methylation, phosphorylation, Ribosome myristoylation Plasma membrane N- and O-glycosylation, GPI-anchor Extraceullar fluid N- and O-glycosylation, acetylation, phosphorylation Extrallular matrix N- and O-glycosylation, phosphorylation, hydroxylation

18 Protein with Max PTM : 303 modifications FUNCTIONFUNCTION: provide a protective, lubricating barrier against particles and infectious agents at mucosal surfaces

19 Pfam graphical view of domain structurePfam graphical view of domain structure of Mucin-16.

20 Posttranslational Modification Examples: Chromatin Structure/function - acetylation Regulation of mitochondrial processes – phosphorylation Evade immune system – glycosylation Gene regulation – glycosylation Recognition - glycosylation

21 Histone and Nucleosome Function The nucleosome not only serves to compact the genetic material but also provides information that affects nuclear functions including DNA replication, repair and transcription. This information is conveyed through numerous combinations of histone post-translational modifications (PTMs) and histone variants. How and when these combinations of PTMs are imposed and to what extent they are determined by the choice of a specific histone variant.

22 In the nucleosome, DNA is wrapped around a histone octamer, comprising a central core made of a tetramer of histones H3–H4 flanked by two dimers of histones H2A–H2B. Histone H3 variants and their interaction with H4

23 Dynamic Change of Chromatin Structure TIBS 26(2001)431 Structural changes in chromatin are facilitated by a variety of nuclear activities that reversibly modify nucleosomes and nucleosome-remodeling complexes - such as histone kinases, methylases, acetylases, histone deacetylases, DNA methylases The nucleus also contains numerous proteins, such as the high mobility group N (HMGN) proteins, which bind to DNA and to nucleosomes and induce structural changes that affect transcription, replication and other DNA-dependent activities

24 Chromatin Remodeling The regulated alteration of chromatin structure, can be accomplished by : (1) covalent modification of histones (2) action of ATP-dependent remodeling complexes. A variety of mechanisms can be used to remodel chromatin; some act locally on a single nucleosome and others act more broadly.

25 H3 Barcode Hypotheses Histones can be modified by post-translational modifications (PTMs), including acetylation, methylation, phosphorylation and ubiquitination (mainly in N-terminal) The histone code hypothesis : specific PTMs regulate gene expression by two mechanisms: (1) changing the chromatin structure into activated or repressed transcriptional state (2) acting as a docking site for transcriptional regulators

26 Chromatin Remodeling – mechanisms for transcription-associated structural changes in chromatin

27 Acetylation in Histone H3 Globular Domain Regulates Gene Expression in Yeast Cell 121(2005)375 Lys 56 in histone H3 : in the globular domain and extends toward the DNA major groove/nucleosome K56 acetylation : enriched at certain active genes, such as histones

28 SPT10, a putative acetyltransferase: required for cell cycle-specific K56 acetylation at histone genes Histone H3 K56 acetylation at the entry- exit gate enables recruitment of the SWI/SNF nucleosome remodeling complex and so regulates gene activity Acetylation in Histone H3 Globular Domain Regulates Gene Expression in Yeast Cell 121(2005)375

29 The High Mobility Group N (HMGN) proteins HMGN proteins - a family of nuclear proteins binds to nucleosomes, changes chromatin architecture, enhances transcription/replication HMGN proteins - function modulated by posttranslational modifications HMGN provide insights into the molecular mechanisms by which structural proteins affect DNA-dependent activities in the context of chromatin

30 Effect of HMGN proteins on transcription and replication from in vitro assembled chromatin templates

31 All HMGN proteins contain three functional domains: a bipartite nuclear localization signal (NLS) a nucleosomal binding domain (NBD) a chromatin-unfolding domain (CHUD) Functional domains of the high mobility group N (HMGN) proteins

32 Increasing number of reported mitochondrial kinases, phosphatases and phosphoproteins suggests that phosphorylation may be important in the regulation of mitochondrial processes Pagliarini and Dixon 2006 Signaling processes to and from mitochondria

33 Posttranslational Modifications at the Amino-Terminus * ~50% eukaryotic protein, the N-terminus is acetylated

34 Posttranslational Modifications Addition of Prosthetic Groups

35 Protein Glycosylation The most important and complex form of PTM Approx. 1% mammalian genes Early view about carbohydrates (non- specific, static structures) has been challenged Ann. Rev. Biochem. 72(2003)643

36 Protein Glycosylation Which proteins are decorated with glycans (polysaccharides) ? What are the structures of these glycans? What is their functional significance?

37 List of All Glycoproteins Sep 2007

38 Protein Glycosylation Common in Eukaryotic Proteins


40 N-Linked Glycans N-linked glycans are covalently attached to Asn residues within a consensus sequence (Asn-Xaa- Ser/Thr), enabling prediction of the modification sites by protein sequence analysis All N-linked glycans share a common pentasaccharide core (GlcNAc2Man3) recognized by lectins and N-glycanase enzymes (PNGase F) These reagents have been used to visualize proteins bearing N-linked glycans from cell or tissue lysates and to enrich them for mass spectrometry analysis

41 O-Linked Glycans Comparable tools are lacking for the study of proteins bearing O-linked glycans. Mucin-type, the most prevalent O-linked glycosylation is characterized by an N-acetylgalactosamine (GalNAc) residue -linked to the hydroxyl group of Ser or Thr. GalNAc residue is installed by a family of 24 N-acetyl- galactosaminyltransferases, then further elaborated by a series of glycosyltransferases to generate higher-order O-linked structures. Because of the complex biosynthetic origin, O-linked glycans are not installed at a defined consensus motif and their presence cannot be accurately predicted based on the protein's primary sequence

42 Mucin-Type Proteins Large, abundant, filamentous glycoproteins that are present at the interface between many epithelia and their extracellular environments Mucin consist of at least 50% O-glycans by weight, in mucin domains or PTS regions (riched in Pro, Thr, Ser) These large regions comprise up to 6000 amino acids in length, with short (8–169 amino acids) tandem repeats

43 PNAS 79(1982)2051

44 Probing mucin-type O-linked glycosylation in living animals PNAS 103(2006)4819-4824 Changes in O-linked protein glycosylation are known to correlate with disease states, but are difficult to monitor because of a lack of experimental tools A technique for rapid profiling of O-linked glycoproteins in living animals by metabolic labeling with N- azidoacetylgalactosamine (GalNAz) followed by Staudinger ligation with phosphine probes

45 PNAS 103(2006)4819-4824 Peracetylated N-azidoacetylgalactosamine (Ac4GalNAz), an azido analog of GalNAc, was shown to be metabolized by cultured cells and incorporated into the core position of O-linked glycans. The azide is distinguished from all cellular functionality by its unique chemical reactivity with phosphine probes, a reaction termed the Staudinger ligation. Thus, proteins modified with GalNAz, a marker of O-linked glycans, can be selectively tagged for visualization or enrichment

46 Copyright ©2006 by the National Academy of Sciences Dube, Danielle H. et al. (2006) Proc. Natl. Acad. Sci. USA 103, 4819-4824 Fig. 1. Profiling mucin-type O-linked glycoproteins by metabolic labeling with an azido GalNAc analog (Ac4GalNAz) followed by Staudinger ligation with a phosphine probe (Phos-FLAG)

47 Copyright ©2006 by the National Academy of Sciences Dube, Danielle H. et al. (2006) Proc. Natl. Acad. Sci. USA 103, 4819-4824 Fig. 2. Ac4GalNAz is metabolized in vivo Flow cytometry analysis of splenocytes from Ac4GalNAz-treated (magenta) or Ac4ManNAz-treated (green) C57BL/6 mice Suggesting that GalNAz is metabolically incorporated into cell surface glycans

48 Copyright ©2006 by the National Academy of Sciences Dube, Danielle H. et al. (2006) Proc. Natl. Acad. Sci. USA 103, 4819-4824 Fig. 3. Analysis of GalNAz-labeled glycoproteins on cells and in tissues. (A) Western blot analysis of tissue lysates from B6D2F1 mice administered Ac4GalNAz (+) or vehicle (–)

49 Glycosylation and Protein Functions HIV evades the immune system by evolving a dynamically changing shield of carbohydrates Nature 422(2003)307 Complex sulfation patterns present in glycosaminoglycans are crucial to growth factor activation Trends Genet 16(20000)206 O-GlcNac glycosylation regulate transcription factors such as CREB JACS 125(2003)6612

50 Protein Glycosylation - Biological Significance Oligosaccharides may be a tissue-specific marker Carbohydrates may alter the polarity and solubility Steric interaction between protein and oligosaccharides dictates certain protein 3D structure The bulkiness and negative charge of oligosaccharide chain may protect protein from the attack by proteolytic enzymes

51 The Sugar Code Carbohydrates as Informational Molecule Information: intracellular targeting of proteins, cell-cell interactions, tissue development, extracellular signals Improved methods for structural analysis Sugar code - The unique complex structure of oligosaccharide on glycoprotein read by protein

52 Lectins carbohydrate-binding proteins Lectins read sugar code and mediate many biological processes : [1] Cell-cell recognition [2] Signaling [3] Adhesion [4] Intracellular targeting of newly synthesized proteins

53 Role of oligosaccharides in recognition and adhesion


55 Working with Carbohydrate Oligosaccharides removed from protein or lipid conjugates Stepwise degradations with specific reagents (eg. O- or N- glycosidase) that reveal bond position and stereochemistry Mixture separated by chromatography Overall composition and analysis by GC, Mass and NMR

56 Mass Spectrometry

57 Native source Protein Characterisation Databases/ Bioinformatics cDNA Libraries Expr. analysis gene level Chromatography Purification Express, purify and detect (tags) Expr. analysis protein level Protein profiles/ differential anal. Structure Function ETTAN design Proteomic Solutions

58 Proteomic analysis of post- translational modifications Nature Biotechnology 21, 255 - 261 (2003) The combination of function- or structure-based purification of modified 'subproteomes', such as phosphorylated proteins or modified membrane proteins, with mass spectrometry is proving particularly successful. To map modification sites in molecular detail, novel mass spectrometric peptide sequencing and analysis technologies hold tremendous potential. Finally, stable isotope labeling strategies in combination with mass spectrometry have been applied successfully to study the dynamics of modifications.

59 Phospho – Proteomics Western 2D gel, Ab specific to phospho-tyrosine

60 2003

61 MS/MS Ions Search The MS/MS ions search accepts data in the form of peak lists containing mass and intensity pairs


63 Methods to detect protein modification Method____ Medium___ Sensitivity__ _Specificity________ MAb NC, PVDF 10 ng specific epitopes Metabolic SDS gel, NC, 50 ng specific precusors labelling PVDF Lectins NC, PVDF 0.1 mg may be specific to one monosaccharide Digoxenin NC, PVDF 0.1 mg vicinal hydroxyl group of sugars PAS stain gel, NC, 1-10 mg vicinal hydroxyl group PVDF of sugars Monosaccharide PVDF 5 mg all monosaccharide analysis

64 Selective incorporation of glycosylated amino acids into proteins

65 Conclusion - PTM Despite many important contributions, the diverse roles of glycosylation and other covalent modifications are only beginning to be understood. Detailed studies of their biological effects have been hindered by the dynamic nature and complexicity of PTMs in vivo. Hsieh-Wilson 2004

66 ExPASy – the proteomic server


68 Secretory Proteins

69 Nonsecretory Proteins




73 NetOGlyc 3.1



76 NetGlyc 1.0


78 NetPhos 2.0

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