Date of Award
2008
Degree Type
Thesis
Degree Name
Doctor of Philosophy
Program
Biochemistry
Supervisor
Dr. Shawn Li
Abstract
Protein-protein interaction via the recognition of a conserved sequence motif in one protein by a modular interaction domain in another provides an effective means to generate complex protein networks in cellular signal transduction. By taking advantage of the modular nature of many regulatory proteins, we attempted to simplify protein protein interactions to the corresponding domain-ligand recognition. Here we developed the peptide array target screening (PATS) method that combines peptide array screening with bioinformatics analysis to decipher, at a genome-wide level, protein networks mediated by modular interaction domains. When applied to a group of 12 Src homology (SH) 3 domains, PATS uncovered a high confidence network that comprised 8 SH3 domain-containingtargetproteinsand284interactingpartners. Comparedtoprotein interactions listed in the online predicted human interaction protein database (OPHID), PATS shared a highly significant overlap (p = 1.92E-05), and also identified a large number of novel interactions.
Different SH2 domains select for distinct phosphopeptides, and the function of a given SH2 domain is often dictated by the specific motifs that it recognizes. Therefore, deciphering the phosphotyrosyl peptide motifrecognized by an SH2 domain is the key to understand its cellular function, and also a critical step to select its potential binding ligands that can be further identified by peptide array screening. We cloned all 120 human SH2 domains and determined the phosphotyrosyl peptide-binding properties of 76 SH2 domains by screening an oriented peptide array library (OPAL). Based on the OPAL data, we developed SMALI (or scoring matrix-assisted ligand identification), a web based program for predicting phosphopeptides preferred by the SH2 domains. When
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applied to SH2D1A/SAP, a protein whose mutation or deletion underlies the X-linked lymphoproliferative syndrome, SMALI not only recapitulated known interactions but also identified a number of novel interacting proteins for this disease-associated protein. Compared to Scansite, a popular motif-scanning program, SMALI exhibited improved accuracy in identifying binding peptides for SH2 domains.
With the capability of SMALI to define the binding motifs for modular interaction domains, PATS can be feasibly extended to the large number of interaction domains encoded by the human genome, and aid in the mapping of the human interactome.
Recommended Citation
Wu, Chenggang, "Mapping Domain-mediated Protein-protein Interaction Networks Using Peptide Arrays" (2008). Digitized Theses. 4933.
https://ir.lib.uwo.ca/digitizedtheses/4933