Introduction

Phage display provides a high-throughput platform for functional profiling of peptide or protein libraries based on their binding or catalytic activities. To construct a phage-display library, randomized DNA encoding short peptides (for pep-tide library assembly) and cDNA or genomic DNA libraries from an organism (for protein library assembly) are cloned into the phage genome or a phagemid vector as fusions to the phage capsid gene (1). Upon expression of the fusion gene, the foreign peptide or protein is covalently anchored on the phage surface as part of the phage capsid. Thus, a phage-display comprises a library comprises a collection of phage clones, each containing a different fusion and displaying a unique protein on its surface. The library is then subjected to iterative rounds of selection to substantially enrich for phage particles displaying targets with the desired binding or catalytic functions. Finally, the identities of the selected peptides or proteins are determined by sequencing the fusion genes in the enriched population. Thus far, phage display has been used to select peptides with high-binding affinities for target receptors (2), for the identification of peptides as the substrates of enzyme catalysis (3,4), and for the evolution of catalytic antibodies or enzymes with novel or more efficient catalytic activities (5,6,7,8,9,10). Recently, we and others have applied phage display to profile the catalytic activities of posttranslational modification (PTM) enzymes (11,12). Here, we discuss phage display as an approach for high-throughput identification of the down-stream proteins modified by PTM enzymes.

Protein Posttranslational Modification

Protein PTM plays an important role in controlling the structure, function, subcellular localization, and degradation of the modified proteins in the living cell (13). For example, phosphorylation and glycosylation induce conformational changes in target proteins, modulating their enzymatic activities and interactions with other molecules (14,15). Protein biotinylation, lipoylation, and phospho-pantetheinylation install crucial functional groups that directly participate in substrate turnover at the active site of biosynthetic enzymes (16,17,18). Protein lipidation specifies the subcellular localization of the modified target and its trafficking pathway within the cell (19). At the end of the protein lifespan, ubiquitination marks proteins for proteolytic degradation (20).

Since PTM regulates the structure, function, and interactions of the target proteins with other cellular entities, virtually all signaling events rely on tandem PTMs to transmit messages through dynamic signal transduction networks and to synchronize the processes of cell biology. This is evident in the regulation of p53 tumor-suppressor activity by phosphorylation of specific serine and threonine residues and by the acetylation and ubiquitination of lysing residues (21). It is also well-established that histone modification by phosphorylation, methylation, and acetylation affect chromatin structure and consequently transcription (22). Furthermore, PTM is responsible for the activation of many biosynthetic pathways, such as phosphopantetheinylation of fatty acid synthase, nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS), biotinylation of carboxylases, and lipoylation of α-keto acid dehydrogenase (23). These enzymes play important roles in the biosynthesis of metabolites crucial for the normal function of the cell. Thus, profiling the catalytic activity of PTM enzymes and mapping the hierarchy of modification events would begin to elucidate regulation mechanisms essential for normal cell function, and set the stage for developing therapies to target the dysregulated cellular state.

Profiling PTM Enzymes by Phage Display

An immediate challenge for the systematic profiling of PTM enzymes is the complexity of the protein modification cascade and the crosstalk between multiple signal transduction pathways—one PTM enzyme may have multiple downstream targets, and one specific PTM on the target protein may result from the combined action of multiple PTM enzymes from different pathways, further increasing the workload for genome-wide PTM enzyme profiling. To tackle this inherent complexity, high-throughput systems have been designed to identify the downstream targets of specific PTM enzymes. For example, various chemical genetics and proteomics methods have been developed for the screening of PTM activities (24,25). Some methods take advantage of mechanism-based inhibitors or photo crosslinkers that label specific classes of enzymes with unique chemical functionalities (fluorescence or affinity probes) (26,27). Others have exploited the substrate promiscuity of PTM enzymes to modify their downstream target proteins with biotinylated substrates or substrates that can be subsequently conjugated to biotin. In these systems, biotin serves as an affinity handle with which the downstream targets of PTM enzymes can be identified (28,29). In addition, protein or peptide microarrays have been developed as a high-throughput platform for profiling the activities of specific PTM enzymes (30).