Thrombopoietin receptor-based protein-protein interaction screening (THROPPIS).

As protein-protein interactions (PPIs) are involved in many cellular events, development of mammalian cytosolic PPI detection systems is important for drug discovery as well as understanding biological phenomena. We have previously reported a c-kit-based PPI screening (KIPPIS) system, in which proteins of interest were fused with a receptor tyrosine kinase c-kit, leading to intracellular PPI-dependent cell growth. However, it has not been investigated whether PPI can be detected using other receptors. In this study, we employed a thrombopoietin receptor, which belongs to the Type I cytokine receptor family, to develop a thrombopoietin receptor-based PPI screening (THROPPIS) system. To improve the sensitivity of THROPPIS, we examined two strategies of (i) localization of the chimeric receptors on the cell membrane, and (ii) addition of a helper module to the chimeric receptors. Intriguingly, the nonlocalized chimeric receptor showed the best performance of THROPPIS. Furthermore, the addition of the helper module dramatically improved the detection sensitivity. In total, 5 peptide-domain interactions were detected successfully, demonstrating the versatility of THROPPIS. In addition, a peptide-domain interaction was detected even when insulin receptor or epidermal growth factor receptor was used as a signaling domain, demonstrating that this PPI detection system can be extended to other receptors.

A Novel Cell-Based Intracellular Protein-Protein Interaction Detection Platform (SOLIS) for Multimodality Screening.

Intervention in protein-protein interactions (PPIs) has tremendous effects in the molecular therapy of many diseases. To fulfill the requirements for targeting intracellular proteins, here we develop SOS-localization-based interaction screening (SOLIS), which elaborately mimics signaling via the Ras-mitogen-activated protein kinase pathway. SOLIS employs two chimeric proteins in which a membrane localization motif (CaaX) is fused at the C-terminus of a protein of interest and the catalytic domain of SOS is fused at the C-terminus of another protein of interest. Interaction between the two proteins of interest induces membrane localization of the SOS chimera and cell proliferation. Thus, the SOLIS system enables enrichment of superior binders based on cell proliferation in an intracellular PPI-dependent manner. This was verified by three major modalities against intracellular PPIs (small molecules, peptide aptamers, and intrabodies). The system worked over a broad range of affinities (KD = 0.32-140 nM). In a screening of a site-directed randomized library, novel intrabody clones were selected on the basis of the potency of cell proliferation. Three other PPI detection methods (NanoBiT, SPR, and pull-down assays) were employed to characterize the SOLIS system, and several intrabody clones were judged as false negatives in these assays. SOLIS signals would be less sensitive to the orientation/conformation of the chimeric proteins, and this feature emerges as the advantage of SOLIS as a mammalian cytosolic PPI detection system with few false negatives.