Abstract:

Genetically-encoded biosensors based on fluorescence proteins (FPs) and fluorescence resonance energy transfer (FRET) have enabled the specific targeting and visualization of signaling events in live cells with high spatiotemporal resolutions. Single-molecule FRET biosensors have been successfully developed to monitor the activity of a variety of signaling molecules, including tyrosine/serine/threonine kinases. We have a developed a general high-throughput screening (HTS) method based on directed evolution to develop sensitive and specific FRET biosensors. We have first applied a yeast library and screened for a mutated binding domain for phosphorylated peptide sequence. When this mutated binding domain and the peptide sequence are connected by a linker and then concatenated in between a pair of FRET FPs, a drastic increase in sensitivity can be achieved. It has also been increasingly clear that controlling protein functions using lights and chemical compounds to trigger allosteric conformational changes can be applied to manipulate protein functions and control cellular behaviors. In this work, we first engineered a novel class of machinery molecules which can provide a surveillance of the intracellular space, visualizing the spatiotemporal patterns of molecular events and automatically triggering corresponding molecular actions to guide cellular functions. We have adopted a modular assembly approach to develop these machinery molecules. As a proof-of-concept, we engineered such a molecule for the sensing of intracellular tyrosine phosphorylation based on fluorescence resonance energy transfer (FRET) and the consequent activation of a tyrosine phosphatase (PTP) Shp2, which plays a critical and positive role in various pathophysiological processes. We have further integrated this machinery molecule to the “don’t eat me” CD47 receptor SIRPa on macrophages such that the engagement of SIRPa and its activation of naturally negative signals will be rewired to turn on the positive Shp2 action to facilitate phagocytosis of red blood cells and target tumor cells, initiated by the specific antigen-targeting antibodies and their interaction with Fcg receptors. Because of the modular design of our engineered molecule, our approach can be extended to perform a broad range of cell-based imaging and immunotherapies, and hence highlight the translational power in bridging the fundamental molecular engineering to clinical medicine. We have also integrated with lights and other means to manipulate the molecular activation of genes and enzymes, which allowed us to control the cellular functions of immunocells with high precision in space and time. As such, we can integrate fundamental science and engineering principles for biomedical and clinical applications.

Biography: 

Dr. Peter Yingxiao Wang, Professor, Dept. of Bioengineering, Institute of Engineering in Medicine, USA; Email: yiw015@eng.ucsd.edu; Phone: 217-898-2810

Dr. Wang obtained his bachelor’s and master’s degrees in Mechanics and Fluid Mechanics from Peking University, Beijing, P.R. China, in 1992 and 1996, respectively. He received his Ph.D. degree in Bioengineering from the University of California, San Diego Jacobs School of Engineering in 2002 and continued his postdoctoral work at UC San Diego working under Bioengineering Professor Shu Chien and Professor Roger Y. Tsien in the Department of Pharmacology. He is current a professor at the department of Bioengineering at UCSD and a fellow of American Institute of Medical and Biological Engineering (AIMBE). Before joining the UC San Diego faculty in 2012, he was an associate professor at the University of Illinois, Urbana-Champaign (UIUC), Department of Bioengineering and a full-time faculty member in the Beckman Institute for Advanced Science and Technology at the University of Illinois. He was also affiliated with the Department of Molecular and Integrative Physiology, Neuroscience Program, the Center for Biophysics and Computational Biology, and Institute of Genomic Biology at UIUC. Dr. Wang is the recipient of the Wallace H. Coulter Early Career Award (both Phase I and Phase II), the National Science Foundation CAREER Award, and National Institutes of Health Independent Scientist Award. His research is supported by the National Institutes of Health, National Science Foundation, and private foundations.