|Organization or Institution||University of Florida|
|Topic||Biochemistry / Chem Bio.|
High-throughput protease reprogramming powered by a suite of integrative vectors.
Ethan W. Slaton1, Samantha G. Martinusen1, Julia Besu2, Cassidy Simas3, Carl A. Denard1
(1) Chemical Engineering, University of Florida, Gainesville FL, (2) Biology, University of Florida, Gainesville FL, (3) Biomedical Engineering, University of Florida, Gainesville FL
Proteases make up approximately 2% of the human proteome, and they are involved in several biological processes, including apoptosis, intracellular signaling, blood coagulation, digestion, inflammation, and disease. Proteases are tightly regulated; when dysregulated, proteases are often involved in diseases, making them critical therapeutic targets. Current protease therapeutic targeting strategies primarily rely on active site binding small molecule inhibitors. Since related proteases share similar active site topologies, active site-targeting small molecules often exhibit off-target toxicity.
Most existing high-throughput screening technologies identify binding molecules that inhibit a protease. However, catalytic inhibition may lead to on-target side effects because proteases have multiple physiological substrates. In addition to disease-associated pathways, target proteases process substrates necessary for cellular health. Therefore, there is a need to modulate proteases beyond catalytic inhibition. In particular, methods to discover or engineer molecules that selectively alter protease activity through substrate specificity would enable new therapeutic opportunities. Such discoveries would also deepen our knowledge of protease modulatory landscapes.
Here we present a high-throughput platform to isolate, characterize, and discover protease modulatory nanobodies that can reprogram a protease. We have developed a suite of integrative vectors (SIVS) to streamline this technology. The high-throughput activity screen for reprogramming proteases (HARP) is a functional screen that utilizes yeast surface display to detect protease activity. Importantly, this platform enables the discovery of substrate-selective modulators. The motivation for developing SIVS is it allows us to insert a protease and substrate cassette into the yeast chromosome. Chromosomal integration will enable us to limit the noise of the fluorescent signals when using flow cytometry. We want to utilize SIVS to achieve multiplex substrate display on the yeast surface. In this manner, we will be able to reprogram protease activity on multiple substrates, including but not limited to finding substrate selective protein-based reprogrammers.
We have developed HARP as a technology to discover protease modulatory nanobodies. This system contains a protease cassette that is inducible by beta-estradiol (βE) and a substrate cassette that is inducible by galactose (pGal). These inducible promoters are essential as they allow us to control which cassettes we express at certain experimental stages. An Endoplasmic Reticulum Retention Signal (ERS), present in both cassettes, will enable us to tune contact time within the Endoplasmic Reticulum (ER), ensuring that the protease has enough time to cleave the substrate successfully. Within the substrate cassette is an AGA2 fusion marker which allows the substrate cassette to migrate to the cell's surface, enabling us to look for activity through yeast surface display. The substrate is flanked on both sides by FLAG and HA epitope tags. Harnessing the AGA1-AGA2 mediated yeast surface display, the substrate cassettes will be stained with fluorescently labeled antibodies specific to the epitope tags present in the cassette. We expect high fluorescent signals from the FLAG and HA tags for inactive proteases when running the cells through a flow cytometer. If the protease is active, we should see a loss of HA signal, meaning we successfully cleaved the substrate. We have seen numerous proteases active within this system, including BACE1, IDE, and MMP8, signifying that our system can screen for activity in various proteases. To expand the functionality of HARP, we can also add a third modulatory cassette that allows us to modulate the activity of the protease.
In a recent example, we show that our system can isolate MMP8 inhibitory nanobodies. We then introduced an extensive nanobody library to look for modulators of the proteases we have tested. We can then isolate these modulators with a gated sort on the flow cytometer to look for modulators.
In the future, we will biochemically characterize these nanobodies. Next-Gen sequencing results will examine how nanobodies' physicochemical properties relate to their function. We will then utilize statistical machine learning to predict function from sequence properties.