Manford, A. G. | Search bioRxiv

bioRxiv2025

Poxvirus attack of anti-viral defense pathways unleashes an effector-triggered NF-κB response

Effector-triggered immunity (ETI) is a form of pathogen sensing that involves detection of pathogen-encoded virulence factors or "effectors". To discover novel ETI pathways in mammals, we developed a screening approach in which individual virulence factors are expressed in human monocytes and transcriptional responses are assessed by RNA-seq. Using this approach, we identify a poxvirus effector, myxoma virus M3.1, which elicits an anti-viral NF-{kappa}B response. We find that NF-{kappa}B is unleashed by an ETI pathway that senses M3.1 attack of two anti-viral complexes: ZAP and TBK1. NF-{kappa}B activation occurs because the proteins inhibited by M3.1-- N4BP1, ZC3H12A, and TBK1--are negative regulators of NF-{kappa}B. Our results illustrate how negative regulators can function as pathogen sensors and establish a systematic approach for the discovery of ETI pathways.

immunology↗

bioRxiv2021

Discovery of a Covalent FEM1B Recruiter for Targeted Protein Degradation Applications

Proteolysis Targeting Chimeras (PROTACs), heterobifunctional compounds that consist of protein-targeting ligands linked to an E3 ligase recruiter, have arisen as a powerful therapeutic modality for targeted protein degradation (TPD). Despite the popularity of TPD approaches in drug discovery, only a small number of E3 ligase recruiters are available for the >600 E3 ligases that exist in human cells. Here, we have discovered a cysteine-reactive covalent ligand, EN106, that targets FEM1B, an E3 ligase recently discovered as the critical component of the cellular response to reductive stress. By targeting Cys186 in FEM1B, EN106 disrupts recognition of the key reductive stress substrate of FEM1B, FNIP1. We further establish that EN106 can be used as a covalent recruiter for FEM1B in TPD applications, in which we demonstrate that a PROTAC linking EN106 to the BET Bromodomain inhibitor JQ1 leads to specific FEM1B- and proteasome-dependent degradation of BRD4 in cells. Our study showcases a covalent ligand that targets a natural E3 ligase-substrate binding site and highlights the utility of covalent ligand screening in expanding the arsenal of E3 ligase recruiters that can be deployed for TPD applications.

biochemistry↗