Post-Translational Modification

David ShiuanDepartment of Life Science and

Institute of BiotechnologyNational Dong Hwa University

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

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

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 ?

(Human Proteome Initiative)

Human proteome Initiative

These are mainly generated by alternative splicing and post-translational modifications (PTMs)

Human Proteome Initiative

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

HPI Sep 2007

Formation of the nascent protein sequence

Protein Sorting and Sequence Modifications

Posttranslational Modifications

Post-Translational Modifications

Post-Translational Modifications

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

Posttranslational Modification

Location Modification

Nucleus acetylation, 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 N- and O-glycosylation, GPI-anchorExtraceullar fluid N- and O-glycosylation, acetylation, phosphorylation Extrallular matrix N- and O-glycosylation, phosphorylation, hydroxylation

Protein with Max PTM : 303 modifications

FUNCTION: provide a protective, lubricating barrier against particles and infectious agents at mucosal surfaces

Pfam graphical view of domain structure of Mucin-16.

Posttranslational Modification

Examples:

Chromatin Structure/function - acetylation Regulation of mitochondrial processes –

phosphorylation Evade immune system – glycosylation Gene regulation – glycosylation Recognition - glycosylation

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.

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

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

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.

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

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

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

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

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

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

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

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

Posttranslational Modifications

at the Amino-Terminus

* ~50% eukaryotic protein, the N-terminus is acetylated

Posttranslational Modifications

Addition of Prosthetic Groups

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

Protein Glycosylation

Which proteins are decorated with glycans

(polysaccharides) ?

What are the structures of these glycans?

What is their functional significance?

List of All Glycoproteins Sep 2007

Protein Glycosylation Common in Eukaryotic Proteins

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

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

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

PNAS 79(1982)2051

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

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

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)

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

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 (–)

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

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

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

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

Role of oligosaccharides in recognition and adhesion

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

Mass Spectrometry

Nativesource

ProteinCharacterisation

Databases/Bioinformatics

cDNALibraries

Expr. analysisgene level

ChromatographyPurification

Express, purifyand detect (tags)

Expr. analysisprotein level

Protein profiles/differential anal.

Structure Function

ETTAN design

Proteomic Solutions

Proteomic analysis of post-translational modificationsNature 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.

Phospho – ProteomicsWestern 2D gel , Ab specific to phospho-

tyrosine

2003

MS/MS Ions SearchThe MS/MS ions search accepts data in the form of

peak lists containing mass and intensity pairs

Methods to detect protein modification

Method____ Medium___ Sensitivity__ _Specificity________

MAb NC, PVDF 10 ng specific epitopes

Metabolic SDS gel, NC, 50 ng specific precusorslabelling 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 monosaccharideanalysis

Selective incorporation of glycosylated amino acids into proteins

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

ExPASy – the proteomic server

Secretory Proteins

Nonsecretory Proteins

NetOGlyc 3.1

NetGlyc 1.0

NetPhos 2.0