Development of protein binders recognizing oligomerization motifs for therapeutics and biosensors

Abstract

Oligomerization of protein to form supramolecular complexes plays important role in cell signal trandsduction and pathogenesis. A representative example are toxic amyloid oligomeric species found in Alzheimer’s disease and the formation of an inflammasome through oligomerization of adaptor proteins. The proteins prone to oligomerize have heterogeneous and transient nature due to the mixture of various oligomers. Therefor-e, a method for modulation and detection of oligomerizing proteins are needed for the understanding of pathological mechanism and the development of therapeutics. Herein, we designed a protein that binds to monomers of oligomerizing protein, enabling detection and modulation of them. Firstly, we developed an algorithm that can design complementary β-sheet sequences. The formation of β-sheet is a primary step for the oligomerization of amyloids, which is representative pathological process. Therefore, understanding the sequence pattern constituting β-sheet and applying it to design proteins can be of great interest in developing therapeutics and sensor probe for amyloid. We built a database by collecting structural information of proteins and investigated the complementary interaction between neighboring β-sheets through statistical processing. Based on this, we developed an algorithm that can design sequence with favorable β-sheet interaction, and successfully performed experimental validation through split green fluorescent protein as well as retrospective test with known amyloid structure. Secondly, we developed a ratiometric fluorescence sensor protein that can bind to and detect amyloid monomers through β-sheet interaction. Based on the algorithm developed earlier, split green fluorescent protein was redesigned to be complementary to amyloid-β, and then the fluorescence intensity and excitation wavelength were modified by random mutagenesis. The fluorescence excitation wavelength of sensor protein changes upon complementary binding to amyloid-β as model target, enabling efficient detection of amyloidogenic protein. The sensor protein effectively detects amyloid-β and can be used for monitoring and detection of it. Lastly, we developed artificial antibody-based binding protein that can modulate oligomerization degree of adaptor protein ASC which induces the formation of inflammasome. By developing a protein that binds to the PYD domain that regulates the oligomerization of the adaptor protein ASC, we achieved to reveal the mechanism by which the degree of inflammatory response is regulated by the oligomeric state of inflammasome. In this study, we were able to develop a facile detection method taking advantage of the nature of amyloid oligomerization, and to elucidate the regulatory mechanism resulted by oligomeric state. The binding protein developed through this study can be used for understanding pathological role of oligomerizing proteins, diagnosis, and development of therapeutics.