Prevention of Chronic Inflammation by Targeting Macrophage Integrin aDb2

• Main Author: Forgey, Cady
• Format: Others
• Language: English
• Published: Digital Commons @ East Tennessee State University 2020
• Subjects: inflammation; macrophage; protein interaction; integrin; adhesion receptor; Molecular Biology
• Online Access: https://dc.etsu.edu/etd/3849; https://dc.etsu.edu/cgi/viewcontent.cgi?article=5324&context=etd

Macrophage integrin aDb2 promotes macrophage retention and accumulation within inflamed tissue, a key event in development of chronic inflammation. Recently, the P5 peptide was identified as a specific inhibitor for integrin aDb2 interaction with 2-(ω-carboxyethyl) pyrole (CEP), a ligand at inflammatory sites. This thesis aims to identify integrin aD I-domain amino acids involved in binding P5 peptide and likewise to CEP. We propose that non-conserved, basic amino acids of the integrin aDb2 I-domain are responsible for binding to P5 peptide and likewise to CEP. Eight amino acids were analyzed by generating six mutant aD I-domains: K180[A], R189[Q], K205[L], HHK223-225[NIT], K233[A], and K246[A]. Mutagenic constructs were created using PCR site-directed mutagenesis, then transformed into E.coli BL21 cells for IPTG-induced protein expression. Of the 6 mutant I-domains analyzed, amino acid K246 was critical in binding to P5 peptide and CEP through ForteBio Protein-Protein Assay, as well as to CEP by cell adhesion assay.