Biology subjects

Davis, B. J.

Publications and source records attributed to Davis, B. J..

3 recordsLinked to original sources

bioRxiv2023

Oblique Line Scan Illumination Enables Expansive, Accurate and Sensitive Single Protein Measurements in Solution and in Living Cells

Single-molecule localization microscopy (SMLM) techniques, such as single-molecule tracking (SMT), enable in situ measurements in cells from which data-rich metrics can be extracted. SMT has been successfully applied to a variety of biological questions and model systems, aiming to unravel the spatiotemporal regulation of molecular mechanisms that govern protein function, downstream pathway effects, and cellular function. While powerful, SMLM often suffers from low throughput and illumination inhomogeneity, along with microscope and user-induced technical biases. Due to technical limitations in scaling SMLM techniques, a tradeoff between spatiotemporal resolution and throughput has been made historically, restricting broad application of these technologies. Here we address these limitations using Oblique Line Scan (OLS), a robust single-objective light-sheet based illumination and detection modality that achieves nanoscale spatial resolution and sub-millisecond temporal resolution across a 250 x 190 m field of view. We demonstrate OLS-enabled SMT on Halo-Tagged proteins in living cells capturing protein motion up to 14 m2 /s. By exploiting the adaptability of the acquisition frame rate and the improved rejection of out of focus light, we extend the utility of OLS beyond cellular compartments with in-solution SMT (isSMT) for single-molecule measurement of ligand-protein interactions and disruption of protein-protein interactions (PPI). We illustrate the versatility of OLS by showcasing two-color SMT, STORM, and single molecule fluorescence recovery after photobleaching (FRAP). OLS expands the range of SMLM applications and paves the way for robust, high-throughput single-molecule investigations of protein dynamics required for drug screening and systems biology studies, both in cells and in solution.

biophysics↗

bioRxiv2021

Repurposing Tranexamic Acid as an Anticancer Agent

Tranexamic Acid (TA) is a clinically used antifibrinolytic that acts as a lysine mimetic to block binding of Plasminogen with Plasminogen activators, preventing conversion of Plasminogen to its proteolytically activated form, Plasmin. Previous studies suggested that TA may exhibit anticancer activity by blockade of extracellular Plasmin formation. Plasmin-mediated cleavage of the CDCP1 protein may increase its oncogenic functions through several downstream pathways. Results presented herein demonstrate that TA blocks Plasmin-mediated excision of the extracellular domain of the oncoprotein CDCP1. In vitro studies indicate that TA reduces the viability of a broad array of human and murine cancer cell lines, and breast tumor growth studies demonstrate that TA reduces cancer growth in vivo. Based on the ability of TA to mimic lysine and arginine, we hypothesized that TA may perturb multiple processes that involve Lys/Arg-rich protein sequences, and that TA may alter intracellular signaling pathways in addition to blocking extracellular Plasmin production. Indeed, TA-mediated suppression of tumor cell viability is associated with multiple biochemical actions, including inhibition of protein synthesis, reduced activating phosphorylation of STAT3 and S6K1, decreased expression of the MYC oncoprotein, and suppression of Lys acetylation. These findings suggest that TA or TA analogs may serve as lead compounds and inspire the production of new classes of anticancer agents that function by mimicking Lys and Arg.

cancer biology↗

bioRxiv2021

Identification of the Molecular Targets of Disulfide Bond Disrupting Agents

Breast cancer mortality remains unacceptably high, indicating a need for safer and more effective therapeutic agents. Disulfide bond Disrupting Agents (DDAs) were previously identified as a novel class of anticancer compounds that selectively kill cancers that overexpress the Epidermal Growth Factor Receptor (EGFR) or its family member HER2. DDAs kill EGFR+ and HER2+ cancer cells via the parallel downregulation of EGFR, HER2, and HER3 and activation/oligomerization of Death Receptors 4 and 5 (DR4/5). However, the mechanisms by which DDAs mediate these effects are unknown. Affinity purification analyses employing biotinylated-DDAs reveal that the Protein Disulfide Isomerase (PDI) family members AGR2, PDIA1, and ERp44 are DDA target proteins. Further analyses demonstrate that shRNA-mediated knockdown of AGR2 and ERp44, or expression of ERp44 mutants, enhance basal and DDA-induced DR5 oligomerization. DDA treatment of breast cancer cells disrupts PDIA1 and ERp44 mixed disulfide bonds with their client proteins. Together, the results herein reveal DDAs as the first small molecule, active site inhibitors of AGR2 and ERp44, and demonstrate roles for AGR2 and ERp44 in regulating the activity, stability, and localization of DR4 and DR5, and activation of Caspase 8.

pharmacology and toxicology↗