Huynh, L. T. | Search bioRxiv

bioRxiv2026

High-Throughput Screening Identifies Proteasome Inhibitors as Potentiators of Type III Interferon JAK-STAT Signaling and Antiviral Immunity

Type III interferon (IFN3, IFN{lambda}) drives antiviral defense at epithelial barriers with a more favorable safety profile than Type I interferon (IFN1), but weaker signaling limits its therapeutic potential. To identify small molecules that potentiate IFN-JAK-STAT signaling, we developed a high-throughput live-cell assay measuring STAT1/STAT2 phosphorylation in A549 cells and screened ~8,640 compounds. This identified proteasome inhibitors (MG132, delanzomib, ixazomib, oprozomib, bortezomib) as selective potentiators of IFN3-induced STAT activation, with minimal effect on IFN1 signaling. Proteasome inhibition enhanced IFN3 antiviral activity to levels approaching IFN1. Mechanistically, proteasome inhibition increased cell-surface IL10R{beta}, the limiting IFN3 receptor subunit, thereby increasing receptor JAK-STAT signaling. It did not alter IFN1 receptor levels but protected IFNAR2 from late ligand-induced downregulation. These findings establish receptor subunit availability as a rate-limiting, druggable node governing IFN3 signaling strength and demonstrate a scalable platform for identifying cytokine-selective signaling potentiators, with implications for mucosal antiviral therapeutics.

immunology↗

bioRxiv2026

N-terminal Trimming Supports Proteostasis Under Secretory Protein Mislocalization Stress

Secretory proteins that fail to enter the endoplasmic reticulum (ER) are mislocalized to the cytosol, where they threaten cellular proteostasis. How cells manage these mislocalized proteins remains poorly understood. Here, we show that inefficient Sec61 translocon activity induces a transcriptional stress response, which is enhanced by blocking NRF-1-dependent adaptation of proteasomal capacity. To maintain proteasomal capacity, thermodynamically stable mislocalized proteins persist in the cytosol of HepG2 and HEK293T cells, while mislocalized proteins that are inherently more aggregation-prone are preferentially ubiquitinated and degraded. This triaging process is accomplished through N-terminal trimming of the signal sequence of thermodynamically stable proteins, which simultaneously reduces their aggregation and prevents proteasomal degradation. These findings reveal a stability-dependent cytosolic triaging mechanism that matches substrate burden to proteasome capacity when ER import is challenged.

cell biology↗