Construction and characterization of multi-component HER2 signaling complexes with single-molecule co-immunoprecipitation Developing companion diagnostic kits for BH3 mimetic drugs by observing single molecule protein-protein interaction

Abstract

Cancer cells maintains its aggressive proliferative signals through addiction to specific cellular signaling network which varies from person to person even among same type of cancer. In order to efficiently find drugs that can cure an individual's cancer, researchers need a reliable platform to reconstitute cellular signaling complexes without the enormous time and clinical trials that require victims of false positive drugs. In this study, we introduced the immunoprecipitated based methods that can reconstitute the various cellular signaling complexes. First, we were able to reconstitute the ligand free HER2 complexes with kinase activity with catalyzing phospho-tyrosine. Even though HER2 cannot activate kinase without dimerization with other HER family proteins. Ligand free HER2 complexes are the reasons for the main types of cancer, although the enzymatic mechanism has not been well characterized. Upon tyrosine phosphorylation, only marginal increases of recruited Grb2 which is the one of major cellular signaling component was observed. By the virtue of the invitro assay that allows controlling the binding and reaction of each component composing protein complexes, we were able to find that substantial increases of Grb2 is recruited when scaffold protein phosphorylated by kinases only in specific order. Then, we also reconstitute the HER2-HER3 heterodimer with intact functional structure. Hetero dimer of HER2 and HER3 generates a stronger proliferative signal than other permutations. By using, single-molecule immunoprecipitation, we observed the individual HER2-HER3 heterodimers catalyze tyrosine phosphorylation at an unusually high rate, while simultaneously interacting with multiple copies of downstream signaling effectors. Our results suggest that the high catalytic rate and multi-tasking capability make a concerted contribution to the strong signaling potency of the HER2-HER3 heterodimer

Finally, we challenged to reconstitute the protein complexes that mediate apoptotic signals. Anti-apoptotic proteins frequently overexpressed in cancer and sequesters pro-apoptotic proteins therefore, protecting it from apoptosis. Venetoclax is FDA approved PPI (protein-protein interaction) drug that can specifically inhibit B-cell lymphoma-2 (BCL-2). It works by blocking BH3 grooves of BCL2 that can sequester the other proapoptotic proteins. Efficacy of venetoclax depends on how much antiapoptotic signals are primed toward to specifically by BCL2. Therefore, not only by measuring signals toward BCL2 but quantitative comparison signals between BCL2 family should be used to predict sensitivity toward venetoclax. By using single co-IP methods that can directly images PPI between BCL2 family, we intensively measured and compare the PPI of three antiapoptotic proteins BCL-2, BCL-XL, and MCL-1 with proapoptotic proteins, which are frequently observed overexpressed proteins in various cancer.