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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