Evolution of KIPPIS as a versatile platform for evaluating intracellularly functional peptide aptamers

Abstract

Chimeric proteins have been widely used to evaluate intracellular protein–protein interactions (PPIs) in living cells with various readouts. By combining an interleukin-3-dependent murine cells and chimeric proteins containing a receptor tyrosine kinase c-kit, we previously established a c-kit-based PPI screening (KIPPIS) system to evaluate and select protein binders. In the KIPPIS components, proteins of interest are connected with a chemically inducible helper module and the intracellular domain of the growth-signaling receptor c-kit, which detects PPIs based on cell proliferation as a readout. In this system, proteins of interest can be incorporated into chimeric proteins without any scaffold proteins, which would be advantageous for evaluating interaction between small peptides/domains. To prove this superiority, we apply KIPPIS to 6 peptide aptamer–polypeptide pairs, which are derived from endogenous, synthetic, and viral proteins. Consequently, all of the 6 peptide aptamer–polypeptide interactions are successfully detected by cell proliferation. The detection sensitivity can be modulated in a helper ligand-dependent manner. The assay results of KIPPIS correlate with the activation levels of Src, which is located downstream of c-kit-mediated signal transduction. Control experiments reveal that KIPPIS clearly discriminates interacting aptamers from non-interacting ones. Thus, KIPPIS proves to be a versatile platform for evaluating the binding properties of peptide aptamers.

A growth-based platform for detecting domain–peptide interactions in the cytoplasm of mammalian cells

“…PPI between the target proteins triggers receptor dimerization and subsequent growth signaling, leading to cell growth as a readout. Since we employed c-Kit or thrombopoietin receptor as the intracellular signaling domain, we named c-Kit-based PPI screening (KIPPIS) [12][13][14] or thrombopoietin receptor-based PPI screening (THROPPIS) 15 , respectively. KIPPIS and THROP-PIS constitutively express chimeric receptors through stable retroviral transduction, which prevents off-target signals compared to the conventional PPI detection systems using transient overexpression.…”