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Introduction 1.1 Post-Translational Modifications

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Introduction 1.1 Post-Translational Modifications Chapter I: Introduction 1.1 Post-translational modifications Post-translational modifications (PTMs) are responsible for dynamic regulation of protein structure and function (Jensen et al., 2006). One example of the function of post-translational modifications in the dynamic regulation of protein structure and function is signal transduction; post-translational modification of key regulatory proteins in signal transduction pathways controls cellular proliferation, metabolism, gene expression, cytoskeletal organization and apoptosis (Jensen et al., 2006). Post-translational modifications not only change the mass of the protein, they are also often responsible for changes in charge, structure and conformation. This leads to changes in the biological activity of proteins like enzymes, the binding affinity of proteins to receptors and other protein-protein interactions (Hoffmann et al., 2008) (Murray et al., 1998). For example, the interaction of the cytokine TGF-beta with its receptor complex results in the phosphorylation of SMAD family proteins which activates these proteins to regulate target gene expression (Massague et al., 2000). Post-translational modified amino acid residues can act as binding sites on proteins for a specific recognition domain on other proteins. For example, phosphorylated tyrosine residues can bind to SH2 or PTB domains (Pawson et al., 1997). Depending on the type of PTM they can be very abundant and have a large number of target proteins, whereas other PTM`s have only a few target proteins. Moreover, some PTM`s are connected with several different residues and not only with one residue. For example multi-site phosphorylation that primarily targets Serine, Threonine and Tyrosine residues is a crucial mechanism for the regulation of protein localization and functional activity (Cohen et al., 2000). In the following table 1 most commonly studied post-translational modifications and their biological functions are highlighted. 1 Post-translational modification Function Acetylation Regulates protein-protein and protein-ligand interactions as well as protein function Leads to protein stability through the protection of the N-terminus from proteolytic degradation (Han et al., 1992) (Seo et al., 2004) Acylation Mediator of protein-protein and protein- membrane interaction Directs cytosolic proteins to membranes and activates them (Beck et al., 2006) Amidation Regulates intracellular signal transduction, DNA repair and replication, transcription and apoptosis Responsible for protein stability through the protection of the C-terminus and for ligand- receptor interactions (Han et al., 1992) Simple glycosylation Histone remodelling, cell growth and division, gene transcription, apoptosis and proteasomal degradation Regulates cellular responses to hormones and initiates a protective response to stress (Slawson et al., 2006) (Ohtsubo et al., 2006) Complex glycosylation Promotes or inhibits intra- or intermolecular protein interactions Involved in protein folding and intracellular trafficking Involved in cell-cell communication, molecular and cellular homeostasis, receptor activation, signal transduction and endocytosis (Walsh et al., 2006) Hydroxylation Mediates structural stability, hypoxic sensing and blood coagulation 2 (Zagorska et al., 2004) Methylation Methylation on lysine regulates chromatin structure, activation and repression of gene transcription Arginine methylation is involved in transcriptional regulation and signal transduction (Lee et al., 2005) Nitrosylation Mediates redox based signalling Modulates energy metabolism Can inhibit or promote apoptosis (Foster et al., 2003) (Spickett et al., 2006) Oxidation Associated with protein damage Table 1: Common post-translational modifications and their functions. Table adapted from Hoffman et al., 2008 Phosphorylation is the most commonly occurring form of post-translational modification with up to 30% of all proteins phosphorylated (Cohen et al., 2001). It is the objective of this work to study how phosphorylation of cellular proteins impact on the generation, loading and presentation of peptide antigens to cytotoxic T lymphocytes. Thus phosphorylation will be discussed in more detail in the next sections. 1.2 Phosphorylation With the discovery of rabbit skeletal muscle glycogen phosphorylase, it is now half a century since phosphorylation has been identified as a post-translational modification in eukaryotes (Cozzone et al., 2004), (Krebs et al., 1956). However, the detection of phosphorylation in prokaryotes took another 20 years (Cozzone et al., 1988). Unlike to prokaryotes, where 3 phosphorylation is mostly at histidine, glutamic acid and aspartic acid residues, in eukaryotes, phosphorylation is based on serine, threonine and tyrosine. It is known to be an essential event of signal transduction, activation or inactivation of enzyme activity. The phosphorylation of serine and threonine takes place more abundantly than the phosphorylation of tyrosine with a ratio of 1800 pSer : 200 pThr : 1 pTyr in eukaryotic cells (Mann et al., 2002). Phosphorylation of peptides / proteins is reversible and catalytic where the phosphorylation / dephosphorylation are done by kinases and phosphatases respectively. There are about 500 kinases and around 100 phosphatases encoded in the human genome (Salih et al., 2005), (Cohen et al., 2001). The role of phosphorylation within the cell is enormous. It is linked with regulating cell growth, survival, apoptosis, receptor activation and the corresponding signalling, regulating metabolic pathways and enzyme activity. Phosphorylation is able to regulate protein activities on several levels: a) as a structural element that is necessary for correct protein folding; b) by inducing conformational changes that can result in an increase or inhibition of enzyme activity; c) by acting as docking sites for the assembly of complexes with other proteins; d) as recognition signal for further modification and e) by changing subcellular localization (Lochhead et al., 2005) (Preisinger et al., 2008). Signalling is one of the most important function where phosphorylation of proteins is involved. Here much attention has been focussed on receptor tyrosine kinases (RTK). Activated RTK autophosphorylates tyrosine residues in their intracellular kinase domain. These phosphorylation sites act as docking sites for adaptor proteins that bind to the phosphotyrosines through interaction domains: for instance Src homology domain 2 (SH2) or phosphotyrosine-binding (PTB) domains. The adaptors interact with further binding partners to build a multiprotein signalling complex at the receptor. This regulates distribution, strength and duration of the incoming signal (Kolch et al., 2000); (Pawson et al., 1994); (Seet et al., 2006). One well characterized example is the epidermal growth factor (EGF) receptor signalling pathway. New tools based around phosphopeptide enrichment and high resolution mass spectrometry have provided tremendous new insights into phosphorylation of proteins following various stimuli and allowed studying phosphorylation events at both the local (a single protein or signalling pathway) and global level. 4 1.3 Enrichment and analysis of phosphopeptides / phosphoproteins In recent years, liquid chromatography coupled with mass spectrometry (LC–MS/MS) has become the first choice of analysis in phosphoproteomics. Although 32P-labeling and Edman sequencing have also been used for the characterization of phosphorylation sites (Mann et al., 2002), these methods are more time consuming than MS. Mass spectrometry has a number of advantages over other methods including (a) high sensitivity, (b) the site of the phosphorylation can be unequivocally identified, (c) novel posttranslational modifications can be found, (d) phosphorylation sites in complex mixtures of proteins can be identified, and (e) relative changes in post-translational modification tenure at distinctive sites can be quantified (Larsen et al., 2006). However, prior to analysis it is necessary to enrich phosphopeptides / phosphoproteins bearing in mind the low stoichiometry of phosphorylated proteins / peptides (only a small portion is phosphorylated in the cell) and the reversibility of the modification (Mann et al., 2002). Outlined in this section are some of the methods of enrichment of phosphopeptides (Figure 1). Figure 1. Enrichment methods for phosphopeptides/phosphoproteins. Figure adapted from Reinders et al. (2005). State-of-the-art in phosphoproteomics. Proteomics 5: 4052-4061 5 1.3.1 Affinity chromatography Affinity chromatography is based on resins that interact with the phosphate group of the peptide. Several resins exist, for example immobilized metal affinity chromatography (IMAC), titanium dioxide (TiO2) and zirconium dioxide (ZrO2) chromatography (Jensen et al., 2007). Of these, IMAC is the method most commonly used to enrich phosphopeptides and proteins (Larsen et al., 2005) and was first set up by Porath et al. (Porath et al., 1975). Although the metal ions Fe3+, Ga3+ and Al3+ strongly bind with phosphopetides, Fe3+ -based techniques are more popular. Phosphopeptides bind to the metal ions that are coupled to the column material by iminodiacetic acid (IDA), nitriloacetic acid (NTA) or Tris- (carboxymethyl) - ethylendiamine (TED) linkers, via electrostatic interactions. They are then eluted by salt- and/or pH- gradients after removing non-phosphorylated peptides with a wash step. To avoid very acidic nonphosphorylated peptides from binding to metal ions, the acidic
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