INTRODUCTION

2.1.1 PROTEIN-PROTEIN INTERACTIONS

The recent expansion in the cloning o f novel genes has resulted in a working draft sequence for >90% of the entire human genome. The inevitable question of the possible function of the identified novel genes must now be addressed. Although the protein sequences themselves may provide some vital information, the in vivo interaction of the proteins is fundamental to discern the complexity of their functioning. One approach, which has been termed ‘guilt by association,’ is to analyse the interaction of novel gene products with known proteins (Oliver, 2000). The development of techniques dynamic enough to allow for the genome-wide screening o f gene products for potential protein-protein interactions presents an arduous challenge.

Protein-protein interactions form the basis o f a multitude of cellular pathways from signal transduction to gene regulation and expression. Associations between two proteins may be transient, such as those observed between kinases and their substrates or of a more permanent nature, as observed in the structural cellular architecture. The detection of protein-protein interactions requires the use of a method sensitive enough to detect transient associations without forfeiting its ability to exclude background.

Conventional in vitro methods for the detection o f protein-protein interactions include co- immunoprecipitation, protein affinity chromatography (including a variation on this theme termed pull down-assays), affinity blotting and immunohistochemistiy. These methods are discussed later (3.1). In addition to in vitro biochemical methods, there are contemporary methodologies which allow the detection of protein-protein interactions in vivo. Some examples of these methodologies are discussed here.

The two-fusion fluorescence resonance energy transfer (FRET) method exploits the properties of green-fluorescent protein (GFP), which was cloned and sequenced by Prasher

et al. (1992) fi-om the cmàdin^asiÀequorea victoria. The protein was found to be inherently fluorescent without the need for any external substrates. Mutant pairs o f GFP, examples of which include cyan-fluorescent protein (GFP) and yellow-fluorescent protein (YFP), can act as donors and acceptors o f fluorophores respectively. Two proteins of interest can be fused to a GFP mutant donor and acceptor pair and co-expressed in a mammalian cell line. Not

only does this method help determine the subeellular localisation o f the fusion proteins but allows for a measurement of their interaction. If these fusion proteins come within close proximity o f each other (~10 nm) the donor fluorophore, in the example given here from a CFP-coupled fusion protein, will be transferred to the acceptor, here the YFP-coupled fusion protein and it is this process that is termed FRET (reviewed in Pollok and Heim, 1999).

In the presence of FRET, the detected donor emission is reduced and the acceptor emission increased as a result o f the energy transfer which is quantifiable. This approach has been used successfully to detect protein-protein interactions an example of which includes the interaction between Bcl-2 and Bax fusion proteins upon co-expression in a mammalian cell line (Mahajan et <3/., 1998).

Another in \i\o method for the identification o f protein-protein interactions is the yeast two- hybrid screen. The method allows for the extensive screening of expression libraries with a known protein to identify novel protein interactions. The system has become a favoured method to isolate binding partners for proteins due to its cost-effective use o f routine molecular biological techniques. The yeast two-hybrid methodology has been used extensively in this thesis so a detailed overview o f the system follows.

2.1.2 TH E BASIS FO R TH E YEAST TW O-HYBRID SYSTEM

The original yeast two-hybrid system was developed by Fields and Song (1989) as a novel genetic system for determining protein-protein interactions. The theoretical origin o f the yeast two-hybrid system is rooted in prior knowledge o f eukaryotic transcriptional regulation. Previous studies had shown that certain yeast transcription factors, like the GAL4 protein, consist of two functionally and physically separable domains, the DNA- binding domain (DNA-BD) and the transcription activation domain (AD). The DNA-BD targets the transcription factor to specific promoter sequences known in yeast as upstream activation sequences (UAS) whereas the AD interacts with the RNA polymerase complex to initiate transcription (Keegan et a l, 1986; Hope and Struhl, 1986). Separation of the DNA-BD and AD abolishes the function of the GAL4 protein to initiate transcription and the two domains are unable to re-associate of their own accord. However, if the two domains are held within close proximity o f each other, the GAL4 transcription factor is

reconstituted (Brent and Ptashne, 1985). In the two-hybrid system, two separate proteins of interest are fused to the DNA-BD and AD respectively. If the proteins interact they function as a bridge between the DNA-BD and AD which reconstitutes the transcription factor and in turn activates the transcription o f reporter genes. Reporter gene expression allows the interaction to be identified by both enzymatic assays and the growth of yeast host cells on nutrient-deficient medium. A schematic o f the yeast two-hybrid system is shown in Figure 2.1.

In this thesis, a GAL4-based assay using the MATCHMAKER two-hybrid system 3 was used (2.2). This system will be the primary focus here. An alternative Lex-A based system known as the ‘interaction trap’ is described in section 2.1.11.

2.1.3 TH E GAL4-BASED YEAST TW O-HYBRID SYSTEM

The stringent regulation of galactose metabolism by the GAL4 transcription factor has been exploited in the GAL4-based yeast two-hybrid system for the control o f reporter gene activation. The genes which encode the metabolism o f galactose in yeast are controlled by two regulatory proteins, GAL4 and GAL 80. In the presence o f galactose, the GAL4 transcription factor binds to the 20 recognized GAL UASs that initiate the transcription of genes for galactose metabolism. In the absence of galactose, GAL80 binds to GAL4 and prevents transcriptional activation. All yeast strains used in the GAL4-based yeast two- hybrid assay are deleted for these endogenous GAL4 and GAL80 genes. Instead, the separated domains of the GAL4 transcription factor, the GAL4 DNA-BD and GAL4 AD fused to two proteins o f interest, are introduced into yeast. Reporter genes which possess a promoter modified to include different GAL4 responsive UASs have been incorporated into yeast strains and are expressed if the GAL4 DNA-BD and AD hybrid fusions interact and reconstitute the active GAL4 transcription factor.

In theory, any protein with two distinct modular components where non-covalent re­ association is assessable can be used; these assays are sometimes referred to as protein- ffagment complementation assays (Pelletier et al, 1999). Apart from the yeast two-hybrid system, one example where this premise has been used is the split-ubiquitin sensor method o f detecting protein-protein interactions which is discussed in 2.1.9.

2.1.4 REPORTER GENE ACTIVATION

Reporter genes commonly used in the yeast two-hybrid screen are the lacZ/MELl, ADE2

and HIS3 which are activated by the reconstituted GAL4 transcription factor. The gene lacZ

encodes p-galactosidase which cleaves the chromogenic substrate 5-bromo-4-chloro-3- indolyl-p-D-galactopyranoside (X-Gal) to produce a blue colouration. p-Galactosidase is an intracellular enzyme and is assayed in the presence of X-Gal upon permeabilisation of yeast cells by ffeeze/thawing (2.3.4.5 ). M ELl encodes the secreted enzyme, a - galactosidase, which allows for the direct assessment of reporter gene activity on solid agar media by cleaving the substrate 5-bromo-4-chloro-3-indolyl-a-D-galactopyranoside (X -a - Gal). The HISS gene encodes imidazole glycerol phosphate-dehydratase, an enzyme involved in histidine biosynthesis and allows for the growth o f the yeast cells on histidine- deficient (-H) media.

A recent improvement to the commercial GAL4 based system has been the addition of the AH 109 yeast strain which has been modified to include an additional nutritional reporter

gene,ADE2, as well as HISS. ADE2 encodes phosphoribosylamino-imidazole-carboxylase, an enzyme involved in adenine biosynthesis. This allows further nutritional selection of interacting proteins on adenine-deficient (-A) media (James et al, 1996).

Another current approach has been the use of an oxygen-sensitive fluorophore embedded in a silicone matrix in a 96 well plate format to screen for protein-protein interactions in the yeast two-hybrid system. Here, in the presence o f an interaction, the co-transformed cells proliferate in liquid nutrient deficient-media applied to the silicone matrix and consume oxygen. The decrease in oxygen concentration results in fluorescence which can be measured using a fluorescence plate reader (CLONTECHniques, 2001). This technique is more convenient for use than the a and p galactosidase assays since it avoids the need for additional reagents. The intensity of the fluorescence can also be used in a quantitative manner to determine the strength o f the interaction.

2.1.5 VECTORS USED IN THE GAL4-BASED YEAST TWO-HYBRID ASSAY A. CLONING VECTORS FOR THE EXPRESSION OF HYBRID PROTEINS The generation o f ‘bait’ and ‘fish’ hybrid constructs in the yeast two-hybrid assay requires the use of cloning vectors which encode the GAL4 DNA-BD and AD respectively (2.1.3).

The example of the GAL4 DNA-BD vector used in this thesis is p AS2-1 and the GAL4 AD vector is pGADlO. The characteristics of both o f the cloning vectors are summarised in Table 2.LA. Plasmid maps of pAS2-l and pGADlO are shown in Figures 2.2 and 2.3, respectively. Both plasmids carry the P-lactamase gene {bid) which confers ampicillin resistance in Escherichia coli {E.coli) and allows the plasmid to be maintained and propagated in bacteria. pAS2-l encodes the GAL4 DNA-BD aa 1-147 and contains a multiple cloning site (MCS) with unique restriction sites at the 3' end of the GAL4 DNA-BD open reading frame (ORE). This allows for the insertion of a nucleotide sequence encoding a protein of interest which can then be expressed in yeast as a fusion to the DNA-BD and used as a ‘bait’ in the yeast two-hybrid screen. In pA S2-l, GAL4 DNA-BD fusion proteins are expressed from the full length yfD//7 promoter leading to high levels o f expression. The pAS2-l vector also contains the tryptophan biosynthesis gene {TRPl gene) which allows for plasmid retention in yeast on tryptophan-deficient (-W) media. The GAL4 DNA-BD fusion protein contains an intrinsic nuclear localisation signal which directs the protein to the yeast nucleus. pAS2-1 also carries the wild type CYH2 gene that confers cycloheximide- sensitivity onto transformed yeast cells which carry the cyhr2 allele such as the CG1945 and AH 109 yeast strain. This DNA-BD cloning vector has now been superceded by pGBKT? which has higher-expression levels in all yeast host strains (CLONTECHniques, 1999). Additionally, it has kanamycin bacterial selection allowing for easier segregation o f the vector from the GAL4-AD cDNA insert vector after library screening.

The pGAD 10 vector encodes GAL4 AD aa 768-881. pGADlO also contains a MCS at the 3’ end o f the GAL4 AD ORE which permits the in-ffame sub-cloning o f a gene or genes of interest as for a cDNA library. pGADlO also has been modified to include the nuclear localisation sequence from the SV40 T-antigen. The expression o f the GAL4 AD fusion fi*om pG AD 10 is comparatively low as it is from a truncated version o f the AD Hl promoter. The pGADlO vector contains the leucine biosynthesis gene {LEU2 gene) which allows for its retention in yeast grown on leucine-deficient (-L) media. The pGADlO cloning vector has also now been superceded by pGADT7 which has higher levels of protein expression (CLONTECHniques, 1999).

their expression. The method o f transformation can be the simultaneous transformation of both constructs, often referred to as co-transformation or alternatively sequential transformation of the individual constructs. Sequential transformation requires less plasmid DNA but is more time-consuming (Gietz et ah, 1992). In this thesis, the co-transformation method has been used.