healthy individuals are potential biomarkers that can be used in diagnosis of the disease.
Figure 2.55 Protein microarrays to detect protein-drug interactions. Therapeutic drugs or other small molecules tagged with a fluorescent dye are applied to purified proteins arrayed on a solid support.
Protein−Protein Interactions
Proteins typically function as complexes comprised of different interacting protein subunits. Important cellular processes such as DNA replication, energy metabolism, and signal transduction are carried out by large multiprotein complexes. Thousands of protein-protein interactions occur in a cell. Some of these are short-lived, while others form stable multicomponent complexes that may interact with other complexes. Determining the functional interconnections among the members of a proteome is not an easy task. Several strategies have been developed to examine protein interactions, including protein microarrays, two-hybrid systems, and tandem affinity purification methods.
The two-hybrid method that was originally devised for studying the yeast proteome has been used extensively to determine pairwise protein—protein interactions in both eukaryotes and prokaryotes. The underlying principle of this assay is that the physical connection between two proteins reconstitutes an active transcription factor that initiates the expression of a reporter gene. The transcription factors employed for this purpose have two domains. One domain (DNA-binding domain) binds to a specific DNA site, and the other domain (activation domain) activates transcription (Fig. 2.56A). The two domains are not required to be part of the same protein to function as an effective transcription factor. However, the activation domain alone will not bind to RNA polymerase to activate transcription. Connection with the DNA-binding domain is necessary to place the activation domain in the correct orientation and location to initiate transcription of the reporter gene by RNA polymerase.
Figure 2.56 Two-hybrid assay for detecting pairwise protein interactions. (A) The DNA binding domain of a transcription factor binds to a specific sequence in the regulatory region of a gene which orients and localizes the activation domain that is required for the initiation of transcription of the gene by RNA polymerase. (B) The coding sequences for the DNA binding domain and the activation domain are fused to DNA X and DNA Y, respectively, and both constructs (hybrid genes) are introduced into a cell. After translation, the DNA binding domain-protein X fusion protein binds to the regulatory sequence of a reporter gene. However, protein Y (prey) does not interact with protein X (bait) and the reporter gene is not transcribed because the activation domain does not, on its own, associate with RNA polymerase. (C) The coding sequence for the activation domain is fused to the DNA for protein Z (DNA Z) and transformed into a cell containing the DNA binding domain-DNA X fusion construct. The proteins encoded by the hybrid genes interact and the activation domain is properly oriented to initiate transcription of the reporter gene demonstrating a specific protein-protein interaction.
For a two-hybrid assay, the coding sequences of the DNA-binding and activation domains of a specific transcription factor are cloned into separate vectors (Fig. 2.56). Often, the Gal4 transcriptional factor from Saccharomyces cerevisiae or the bacterial LexA transcription factor is used. A DNA (or cDNA generated from a eukaryotic mRNA) sequence that is cloned in frame with the DNA-binding domain sequence produces a fusion (hybrid) protein and is referred to as the “bait.” This is the target protein for which interacting proteins are to be identified. Another DNA sequence is cloned into another vector in frame with the activation domain coding sequence. A protein attached to the activation domain is called the “prey” and potentially interacts with the bait protein. Host yeast cells are transformed with both bait and prey DNA constructs. After expression of the fusion proteins, if the bait and prey do not interact, then there is no transcription of the reporter gene (Fig. 2.56B). However, if the bait and prey proteins interact, then the DNA-binding and activation domains are also brought together. This enables the activation domain to make contact with RNA polymerase and activate transcription of the reporter gene (Fig. 2.56C). The product of an active reporter gene may produce a colorimetric response or may allow a host cell to proliferate in a specific medium.
For a whole-proteome protein interaction study, two libraries are prepared, each containing thousands of cDNAs generated from total cellular mRNA (or genomic DNA fragments in a study of proteins from a prokaryote). To construct the bait library, cDNAs are cloned into the vector adjacent to the DNA sequence for the DNA-binding domain of the transcription factor Gal4 and then introduced into yeast cells. To construct the prey library, the cDNAs are cloned into the vector containing the sequence for the activation domain, and the constructs are transferred to yeast cells. The libraries are typically screened for bait-prey protein interactions in one of two ways. In one method, a prey library of yeast cells is arrayed on a grid. The prey library is then screened for the production of proteins that interact with a bait protein by introducing individual bait constructs to the arrayed clones by mating (Fig. 2.57A). Alternatively, each yeast clone in a bait library is mated en masse with a mixture of strains in the prey library, and then positive interactions are identified by screening colonies on plates for activation of the reporter gene (Fig. 2.57B). Challenges with using the two-hybrid system for large-scale determination of protein−protein interactions include the inability to clone all possible protein coding genes in frame with the activation and DNA-binding domains, which leads to missed interactions (false negatives), and the detection of interactions that do not normally occur in their natural environments within the original cells and therefore are not biologically relevant (false positives). Nonetheless, this approach has been used to successfully identify interacting proteins in a wide range of organisms from bacteria to humans.
Figure 2.57 Large-scale screens for protein interactions using the yeast two-hybrid system. Two libraries are prepared, one containing genomic DNA fragments fused to the coding sequence for the DNA-binding domain of a transcription factor (bait library) and another containing genomic DNA fragments fused to the activation domain of the transcription factor (prey library). Two methods are commonly used to screen for pairwise protein interactions. (A) Individual yeast strains in the bait library are mated with each yeast strain in an arrayed prey library. Resulting strains in the array that produce bait and prey proteins that interact are detected by assaying for reporter gene activation (activated cells growing in a multiwell plate are indicated in green). (B) Yeast strains in the prey library are mated en masse with individual strains in the bait library. The mixture of strains are screened for reporter gene activity that identifies strains with interacting bait and prey proteins (green).
Instead of studying pairwise protein interactions, the tandem affinity purification tag procedure is designed to capture multiprotein complexes and then identify the components with MS (Fig. 2.58). In this method, a DNA (or cDNA) sequence that encodes the bait protein is fused to a DNA sequence that encodes two small peptides (tags) separated by a protease cleavage site. The peptide tags bind with a high affinity to specific molecules and facilitate purification of the target protein. A “two-tag” system allows two successive rounds of affinity binding to ensure that the target and its associated proteins are free of any nonspecific proteins. Alternatively, a “one-tag” system with a small protein tag that is immunoprecipitated with a specific antibody requires only
