Biology subjects

Slaton, E. W.

Publications and source records attributed to Slaton, E. W..

4 recordsLinked to original sources

bioRxiv2026

A Yeast Surface Display Platform for Screening Dimeric Mammalian Receptors

Discovering proteins that modulate receptor activity remains a key challenge in the field of protein design and engineering. Traditionally, identifying proteins that interact with receptors often relies on binding as a selection criterion, yielding limited information about the function of discovered binders in a library, including the ability to activate or block signaling cascades associated with the receptor of interest. As a result, extensive downstream characterization is required to assess the biological relevance of discovered binders. To address this issue, we have developed a high-throughput screening system to screen dimeric mammalian receptors using yeast surface display. We demonstrate the programmed dimerization of the extracellular domains of mammalian receptors in yeast via engineered induction pathways, thereby enabling receptor expression and the secretion of associated native cytokines. This surface expression of the involved subunits for the protein receptor and cytokine-induced dimerization activity indicates that the receptor has been activated and is expected to trigger a DNA-driven signaling cascade within a mammalian cell. This system provides a modular platform technology that advances existing yeast-display systems, demonstrating the effectiveness of these high-throughput platforms for screening the function of mammalian receptors. This work is expected to provide a rapid, cost-effective approach to the molecular discovery of novel biologics for targeting dimeric mammalian receptors.

synthetic biology↗

bioRxiv2025

High-throughput Activity Reprogramming of Proteases (HARP)

Developing potent and selective protease inhibitors remains a grueling, iterative, and often unsuccessful endeavor. Although macromolecular inhibitors can achieve single-enzyme specificity, platforms used for macromolecular inhibitor discovery are optimized for high-affinity binders, requiring extensive downstream biochemical characterization to isolate rare inhibitors. Here, we developed the High-throughput Activity Reprogramming of Proteases (HARP) platform, HARP is a yeast-based functional screen that isolates protease-inhibitory macromolecules from large libraries by coupling their inhibition of endoplasmic reticulum-resident proteases to a selectable phenotype on the cell surface. Endowed with high dynamic range and resolution, HARP enabled the isolation of low-nanomolar-range inhibitory nanobodies against tobacco etch virus protease and human kallikrein 6, including a rare 7.6 nM KI TEVp uncompetitive inhibitor. Structural modeling and deep sequencing all provide insights into the molecular determinants of inhibitors and reinforce HARPs foundational findings. Overall, HARP is a premier platform for discovering modulatory macromolecules from various synthetic scaffolds against enzyme targets. Graphical AbstractO_ST_ABSWorkflow of HARPC_ST_ABSA yeast-based reporter of the interaction between a protease, its canonical substrate, and a modulator library within the yeast cell. Quantifying this interaction occurs by fluorescently labeling the displayed substrate cassettes on the surface of the cells, where the desired function (correlating with phenotype) can be selected using fluorescent-activated cell sorting (FACS). Isolated populations are sequenced and purified in preparation for secondary characterization to determine modulator effects and interaction strengths between the modulator and the protease target.

synthetic biology↗

bioRxiv2023

Modular and integrative activity reporters enhance biochemical studies in the yeast ER.

The yeast endoplasmic reticulum sequestration and screening (YESS) system is a generalizable platform that has become highly useful to investigate post-translational modification enzymes (PTM-enzymes). This system enables researchers to profile and engineer the activity and substrate specificity of PTM-enzymes and to discover inhibitor-resistant enzyme mutants. In this study, we expand the capabilities of YESS by transferring its functional components to integrative plasmids. The YESS integrative system yields uniform protein expression and protease activities in various configurations, allows one to integrate activity reporters at two independent loci and to split the system between integrative and centromeric plasmids. We characterize these integrative reporters with two viral proteases, Tobacco etch virus (TEVp) and 3-chymotrypsin like protease (3CLpro), in terms of coefficient of variance, signal-to-noise ratio and fold-activation. Overall, we provide a framework for chromosomal-based studies that is modular, enabling rigorous high-throughput assays of PTM-enzymes in yeast.

synthetic biology↗

bioRxiv2023

A comprehensive survey of coronaviral main protease active site diversity in 3D: Identifying and analyzing drug discovery targets in search of broad specificity inhibitors for the next coronavirus pandemic

Although the rapid development of therapeutic responses to combat SARS-CoV-2 represents a great human achievement, it also demonstrates untapped potential for advanced pandemic preparedness. Cross-species efficacy against multiple human coronaviruses by the main protease (MPro) inhibitor nirmatrelvir raises the question of its breadth of inhibition and our preparedness against future coronaviral threats. Herein, we describe sequence and structural analyses of 346 unique MPro enzymes from all coronaviruses represented in the NCBI Virus database. Cognate substrates of these representative proteases were inferred from their polyprotein sequences. We clustered MPro sequences based on sequence identity and AlphaFold2-predicted structures, showing approximate correspondence with known viral subspecies. Predicted structures of five representative MPros bound to their inferred cognate substrates showed high conservation in protease:substrate interaction modes, with some notable differences. Yeast-based proteolysis assays of the five representatives were able to confirm activity of three on inferred cognate substrates, and demonstrated that of the three, only one was effectively inhibited by nirmatrelvir. Our findings suggest that comprehensive preparedness against future potential coronaviral threats will require continued inhibitor development. Our methods may be applied to candidate coronaviral MPro inhibitors to evaluate in advance the breadth of their inhibition and identify target coronaviruses potentially meriting advanced development of alternative countermeasures.

molecular biology↗