Documenti di Didattica
Documenti di Professioni
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David Shiuan
Department of Life Science and Institute of Biotechnology National Dong Hwa University
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
- methyl
- acetyl - glyco - phospho
Posttranslational Modification
What purpose ?
- targeting (eg. some lipoproteins) - stability (eg. secreted glycoproteins )
These are mainly generated by alternative splicing and post-translational modifications (PTMs)
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
Posttranslational Modifications
Post-Translational Modifications
Post-Translational Modifications
Posttranslational Modification
Modification
Acylation Alkylation Carboxylmethylation Phoshorylation Sulfation Carboxylation Sialyation
Charge-dependent change
loss of a-amino positive charge alteration of a- or e-amino positive group esterification of specific carboxyl group mainly modify Ser, Thr and Tyr mainly modify Tyr bring negative charge mainly on Asn, Thr and Ser
Posttranslational Modification
Location
Nucleus Lysosome Mitochondria Golgi
Modification
acetylation, phosphorylation mannose-6-phosphate labelled N-linked sugar N-formyl acylation 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
Posttranslational Modification
Examples:
Chromatin Structure/function - acetylation Regulation of mitochondrial processes phosphorylation Evade immune system glycosylation Gene regulation glycosylation Recognition - glycosylation
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 H3H4 flanked by two dimers of histones H2AH2B.
Histone H3 variants and their interaction with H4
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
Cell 121(2005)375
Lys 56 in histone H3 : in the globular domain and extends toward the DNA major
groove/nucleosome
SPT10, a putative acetyltransferase: required for cell cycle-specific K56 acetylation at histone genes Histone H3 K56 acetylation at the entryexit gate enables recruitment of the SWI/SNF nucleosome remodeling complex and so regulates gene activity
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
a bipartite nuclear localization signal (NLS) a nucleosomal binding domain (NBD) a chromatin-unfolding domain (CHUD)
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
Posttranslational Modifications
at the Amino-Terminus
Posttranslational Modifications
Addition of Prosthetic Groups
Protein Glycosylation
Protein Glycosylation
Which proteins are decorated with glycans (polysaccharides) ? What are the structures of these glycans? What is their functional significance?
Sep 2007
Protein Glycosylation
N-Linked Glycans
N-linked glycans are covalently attached to Asn residues within a consensus sequence (Asn-XaaSer/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-acetylgalactosaminyltransferases, 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 (8169 amino acids) tandem repeats
PNAS 79(1982)2051
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 Nazidoacetylgalactosamine (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
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)
Dube, Danielle H. et al. (2006) Proc. Natl. Acad. Sci. USA 103, 4819-4824
Dube, Danielle H. et al. (2006) Proc. Natl. Acad. Sci. USA 103, 4819-4824
Flow cytometry analysis of splenocytes from Ac4GalNAz-treated (magenta) or Ac4ManNAz-treated (green) C57BL/6 mice
Copyright 2006 by the National Academy of Sciences
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 ()
Dube, Danielle H. et al. (2006) Proc. Natl. Acad. Sci. USA 103, 4819-4824
Complex sulfation patterns present in glycosaminoglycans are crucial to growth factor activation
Trends Genet 16(20000)206
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
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
Mass Spectrometry
Proteomic Solutions
Chromatography Purification
Native source
Protein Characterisation
Databases/ Bioinformatics
Structure
Function
ETTAN design
cDNA Libraries
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 Proteomics
Western 2D gel , Ab specific to phospho-tyrosine
2003
The MS/MS ions search accepts data in the form of peak lists
Medium___
NC, PVDF
SDS gel, NC, PVDF NC, PVDF
Sensitivity__ _Specificity________
10 ng
50 ng 0.1 mg
specific epitopes
specific precusors may be specific to one monosaccharide vicinal hydroxyl group of sugars vicinal hydroxyl group of sugars all monosaccharide
0.1 mg 1-10 mg 5 mg
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
Secretory Proteins
Nonsecretory Proteins
NetOGlyc 3.1
NetGlyc 1.0
NetPhos 2.0