Ellis, J. M. | bioRxiv Science

bioRxiv2025

Enhancing enzymatic bioconjugation efficiency via installation of a substrate recruitment domain

Enzyme mediated bioconjugation provides a method for easy and rapid formation of protein-protein and protein-small molecule conjugates under mild conditions. Promiscuous enzymes are of particular interest because they can catalyze conjugation reactions on a broad set of substrates. However, this promiscuity carries the risk of undesirable off-target modifications. To mitigate this effect, we used computational design to install a substrate recruitment domain (SRD) onto the promiscuous enzyme, tyrosinase. The redesigned tyrosinase, called D42, preferentially modifies tyrosine residues within the peptide core (core) linked to a 6-amino acid recognition motif/sequence (RS) specific for the SRD. Incorporation of the recognition sequence along with a neighboring tyrosine in peptides or proteins allows for rapid D42-mediated conversion of the tyrosine to an orthoquinone, which can be selectively modified with a variety of nucleophiles. We demonstrate the utility of our design system by rapidly installing cytotoxic molecules on a monoclonal antibody. For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/684804v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1a3b274org.highwire.dtl.DTLVardef@18eeedorg.highwire.dtl.DTLVardef@ee0aaeorg.highwire.dtl.DTLVardef@1ff3fa1_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

The Inositol Trisphosphate Receptor (IP3R) is Dispensable for Rotavirus-induced Ca2+ Signaling and Replication but Critical for Paracrine Ca2+ Signals that Prime Uninfected Cells for Rapid Virus Spread

Rotavirus is a leading cause of viral gastroenteritis. A hallmark of rotavirus infection is an increase in cytosolic Ca2+ caused by the nonstructural protein 4 (NSP4). NSP4 is a viral ion channel that releases Ca2+ from the endoplasmic reticulum (ER) and the increase in Ca2+ signaling is critical for rotavirus replication. In addition to NSP4 itself, host inositol 1,4,5- trisphosphate receptor (IP3R) ER Ca2+ channels may contribute to rotavirus-induced Ca2+ signaling and by extension, virus replication. Thus, we set out to determine the role of IP3R Ca2+ signaling during rotavirus infection using IP3R-knockout MA104-GCaMP6s cells (MA104- GCaMP6s-IP3R-KO), generated by CRISPR/Cas9 genome editing. Live Ca2+ imaging showed that IP3R-KO did not reduce Ca2+ signaling in infected cells but eliminated rotavirus-induced intercellular Ca2+ waves (ICWs) and therefore the increased Ca2+ signaling in surrounding, uninfected cells. Further, MA104-GCaMP6s-IP3R-TKO cells showed similar rotavirus susceptibility, single-cycle replication, and viral protein expression as parental MA104- GCaMP6s cells. However, MA104-GCaMP6s-IP3R-TKO cells exhibited significantly smaller rotavirus plaques, decreased multi-round replication kinetics, and delayed virus spread, suggesting that rotavirus-induced ICW Ca2+ signaling stimulates virus replication and spread. Inhibition of ICWs by blocking the P2Y1 receptor also resulted in decreased rotavirus plaque size. Conversely, exogenous expression of P2Y1 in LLC-MK2-GCaMP6s cells, which natively lack P2Y1 and rotavirus ICWs, rescued the generation of rotavirus-induced ICWs and enabled plaque formation. In conclusion, this study shows that NSP4 Ca2+ signals fully support rotavirus replication in individual cells; however, IP3R is critical for rotavirus-induced ICWs and virus spread by priming Ca2+-dependent pathways in surrounding cells. ImportanceMany viruses exploit host Ca2+ signaling to facilitate their replication; however, little is known about how distinct types of Ca2+ signals contribute to the overall dysregulation of Ca2+ signaling or promote virus replication. Using cells lacking IP3R, a host ER Ca2+ channel, we could differentiate between intracellular Ca2+ signals within virus-infected cells and intercellular Ca2+ waves (ICWs), which increase Ca2+ signaling in neighboring, uninfected cells. In infected cells, IP3R was dispensable for rotavirus-induced Ca2+ signaling and replication, suggesting the rotavirus NSP4 viroporin supplies these signals. However, IP3R-mediated ICWs increase rotavirus replication kinetics and spread, indicating that the Ca2+ signals from the ICWs may prime nearby uninfected cells to better support virus replication upon eventual infection. This "pre-emptive priming" of uninfected cells by exploiting host intercellular pathways in the vicinity of virus-infected cells represents a novel mechanism for viral reprogramming of the host to gain a replication advantage.

microbiology↗

bioRxiv2021

Small molecule allosteric inhibitors of RORγt block Th17-dependent inflammation and associated gene expression in vivo.

Retinoic acid receptor-related orphan nuclear receptor (ROR) {gamma}t is a member of the RORC nuclear hormone receptor family of transcription factors. ROR{gamma}t functions as a critical regulator of thymopoiesis and immune responses. ROR{gamma}t is expressed in multiple immune cell populations including Th17 cells, where its primary function is regulation of immune responses to bacteria and fungi through IL-17A production. However, excessive IL-17A production has been linked to numerous autoimmune diseases. Moreover, Th17 cells have been shown to elicit both pro- and anti-tumor effects. Thus, modulation of the ROR{gamma}t/IL-17A axis may represent an attractive therapeutic target for the treatment of autoimmune disorders and some cancers. Herein we report the design, synthesis and characterization of three selective allosteric ROR{gamma}t inhibitors in preclinical models of inflammation and tumor growth. We demonstrate that these compounds can inhibit Th17 differentiation and maintenance in vitro and Th17-dependent inflammation and associated gene expression in vivo, in a dose-dependent manner. Finally, ROR{gamma}t inhibitors were shown to inhibit tumor formation in pancreatic ductal adenocarcinoma (PDAC) organoids.

immunology↗