An ambitious EC-funded research initiative on epigenetics advancing towards systems biology

Introduction

Most proteins assemble into multi-subunit complexes to perform their cellular functions. To understand the biological role of a protein of interest, it is therefore important to identify its protein-protein interactions (PPIs). Besides the qualitative identification of PPIs, the protein complex architecture is also of major importance. By distinguishing 2 core-subunits from substoichiometric complex subunits, key subunits and their intrinsic binding domains, enzymatic activities and/or regulatory functions can be identified.

Recent developments in quantitative mass spectrometry (qMS) have made it possible to screen for these protein-protein interactions in a comprehensive and unbiased manner (Vermeulen et al, 2008). Most recent qMS methods are based on label-free quantification, which do not rely on isotope labeling and are therefore ideally suited for PPI identification in any kind of tissue or cell type (Hubner et al, 2010). To determine the protein complex stoichiometry, information on the abundance of interactors needs to be gained. This used to be accomplished by spike-in of isotope labeled reference peptides, a laborious and expensive method. However, recently new computational methods have been developed that are able to approximate the abundance from qMS intensity, one of which is Intensity Based Absolute Quantification (iBAQ) (Schwanhausser et al, 2011). [...]

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Introduction

Protein complexes are central to cellular function. Many important complexes in eukaryotes require recombinant overproduction for a detailed analysis of their structure and function. Baculovirus expression vector systems (BEVS) have become increasingly popular for the production of such specimens, in particular for complexes which depend on a eukaryotic host cell machinery for proper folding, post-translational modification, authentic processing and correct targeting to cell compartments for their activity. The MultiBac system (Fig. 1) is a BEVS specifically designed for producing large eukaryotic complexes with many subunits [Berger et al., Nat. Biotechnol. 2004, Fitzgerald et al., Nat Methods, 2006]. It consists of an array of plasmids which facilitate multigene assembly, a modified baculovirus genome that with optimized protein production properties, and a set of protocols detailing every step from inserting encoding DNAs in the plasmid array to protein production by this technology. The components of the system and the protocols used are continuously being improved, developed and streamlined to simplify handling and improve efficacy [Trowitzsch et al., J. Struct. Biol. 2010, Vijayachandran et al., J. Struct. Biol. 2011]. We believe that our efforts have reduced the previously perceived complexity of the baculovirus/insect cell system to the level of protein expression in E. coli. [...]

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Introduction

Post-translational modifications (PTMs) of histones specify regulatory functions on chromatin through the recruitment of downstream effectors or “readers”, that can specifically recognize different PTMs and translate epigenetic marks into a functionally relevant outcome (reviewed in Taverna et al., 2007). To comprehend the complexity of epigenetic regulation, it is essential to not only catalogue histone PTMs and their patterns, but also to understand roles that histone PTMs and their effectors play in biological processes. An important component of this understanding will come through identification of histone PTM binding proteins. To this end, the peptide pull-down (PPD) assay provides a simple and effective tool to identify and characterize such reader proteins.

The general principle of the PPD is as follows. Biotinylated histone tail peptides containing a specific histone PTM and corresponding control unmodified peptides, are immobilized onto avidin-conjugated beads. The beads are incubated with a sample of interest, such as nuclear extract or purified recombinant protein, and washed to remove unbound proteins. Bound proteins can then be eluted and analyzed by SDS/PAGE and visualized by protein staining. By comparing proteins bound to modified versus unmodified peptides it is possible to identify candidate “reader” proteins for specific histone PTMs. [...]

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Introduction

Green fluorescent proteins (GFP) and derivates thereof are widely used to study protein localization and dynamics in living cells (Heim and Tsien, 1996; Tsien, 1998). The validation and interpretation of these data, however, requires additional information on biochemical properties of the investigated fluorescent fusion proteins e.g. enzymatic activity, DNA binding and interaction with other cellular components. For these biochemical analyses proteins are mostly fused with a small protein tag (e.g. Histidine-tag, c-Myc, FLAG or hemaglutinin). GFP, the most widely used labelling tag in cell biology is rarely used for biochemical analyses although various mono- and polyclonal antibodies are available (Cristea et al., 2005) (Abcam, Cambridge, UK; Sigma, St. Louis, USA.; Roche, Mannheim, Germany) [...]

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Introduction

Here we describe a strategy for isolation of multiprotein complexes from human HeLa S3 cells in a scale and purity optimized for characterization by mass spectrometry. For this purpose, we use stably expressed One-STrEP-tag ® fusion proteins. This approach was successfully used in characterization of histone chaperone Asf1 complexes (Groth et al., 2007), and we have recently optimized it further. Using this protocol we routinely obtain complexes in amounts sufficient for visualising single protein bands by Coomassie Blue staining. [...]

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