Browsing by Subject "protein-protein interactions"
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Item Discovery of small molecule modulators of protein-protein interactions by FRET-based high-throughput screening and structure-based drug design(2019-08) Lo, Chih HungProtein–protein interactions (PPIs) are of pivotal importance in the regulation of biological systems and are consequently implicated in the development of disease states. Here, we investigated two classes of protein, including a transmembrane protein (tumor necrosis factor receptor 1 (TNFR1)) and intrinsically disordered proteins (tau and huntingtin (HTT)), which are implicated in autoimmune diseases and neurodegenerative diseases respectively. Receptor-specific inhibition of TNFR1 signaling is a highly sought after strategy for treatment of inflammatory diseases such as rheumatoid arthritis. In this study, we investigated the structure-function relationship of TNFR1 by engineering a TNFR1 fluorescence resonance energy transfer (FRET) biosensor to monitor the structural and conformational changes of the receptor. We have also shown using small-molecule tool compounds, that the disruption of receptor-receptor interactions (competitive inhibition) and perturbation of the receptor conformational dynamics (allosteric inhibition) are both feasible approaches to inhibit TNFR1 signaling. We have also made a major discovery showing that long-range structural couplings, between TNFR1 membrane distal and proximal domains, mediated through the ligand-binding loop, determine the conformational states of the receptor that act as a molecular switch in receptor function. In addition to deepening the understanding of a novel mechanism in TNF receptor activation, we have optimized a lead compound through medicinal chemistry by improving its potency by more than sixty-fold to the nanomolar range, thereby advancing therapeutic developments in these clinically important targets. The heterogeneity of tau and HTT pathology is one of the major challenges that plagues current clinical trials, hence impeding the discovery of a cure for Alzheimer’s disease (AD) and Huntington’s disease (HD). We have engineered novel FRET biosensors of these proteins to target the ensemble of heterogeneous protein oligomers or aggregates in cells. The biosensors are not only capable of monitoring oligomer conformations, but can also be used as a high-throughput screening platform. Using these technologies, we have discovered small-molecule inhibitors of tau oligomerization or HTT aggregation that rescue cell cytotoxicity with nanomolar potency.Item Quantitative Fluorescence Studies in Living Cells: Extending Fluorescence Fluctuation Spectroscopy to Peripheral Membrane Proteins(2015-05) Smith, ElizabethThe interactions of peripheral membrane proteins with both membrane lipids and proteins are vital for many cellular processes including membrane trafficking, cellular signaling, and cell growth/regulation. Building accurate biophysical models of these processes requires quantitative characterization of the behavior of peripheral membrane proteins, yet methods to quantify their interactions inside living cells are very limited. Because peripheral membrane proteins usually exist both in membrane-bound and cytoplasmic forms, the separation of these two populations is a key challenge. This thesis aims at addressing this challenge by extending fluorescence fluctuation spectroscopy (FFS) to simultaneously measure the oligomeric state of peripheral membrane proteins in the cytoplasm and at the plasma membrane. We developed a new method based on z-scan FFS that accounts for the fluorescence contributions from cytoplasmic and membrane layers by incorporating a fluorescence intensity z-scan through the cell. H-Ras-EGFP served as a model system to demonstrate the feasibility of the technique. The resolvability and stability of z-scanning was determined as well as the oligomeric state of H-Ras-EGFP at the plasma membrane and in the cytoplasm. Further, we successfully characterized the binding affinity of a variety of proteins to the plasma membrane by quantitative analysis of the z-scan fluorescence intensity profile. This analysis method, which we refer to as z-scan fluorescence profile deconvoution, was further used in combination with dual-color competition studies to determine the lipid specificity of protein binding. Finally, we applied z-scan FFS to provide insight into the early assembly steps of the HTLV-1 retrovirus.