Cellular stress modulates severity of the inflammatory response in lungs via cell surface BiP.

Inflammation is a central pathogenic feature of the acute respiratory distress syndrome (ARDS) in COVID-19. Previous pathologies such as diabetes, autoimmune or cardiovascular diseases become risk factors for the severe hyperinflammatory syndrome. A common feature among these risk factors is the subclinical presence of cellular stress, a finding that has gained attention after the discovery that BiP (GRP78), a master regulator of stress, participates in the SARS-CoV-2 recognition. Here, we show that BiP serum levels are higher in COVID-19 patients who present certain risk factors. Moreover, early during the infection, BiP levels predict severe pneumonia, supporting the use of BiP as a prognosis biomarker. Using a mouse model of pulmonary inflammation, we observed increased levels of cell surface BiP (cs-BiP) in leukocytes during inflammation. This corresponds with a higher number of neutrophiles, which show naturally high levels of cs-BiP, whereas alveolar macrophages show a higher than usual exposure of BiP in their cell surface. The modulation of cellular stress with the use of a clinically approved drug, 4-PBA, resulted in the amelioration of the lung hyperinflammatory response, supporting the anti-stress therapy as a valid therapeutic strategy for patients developing ARDS. Finally, we identified stress-modulated proteins that shed light into the mechanism underlying the cellular stress-inflammation network in lungs.

A Fluorescence Polarization-Based High-Throughput Screen to Identify the First Small-Molecule Modulators of the Human Adenylyltransferase HYPE/FICD.

The covalent transfer of the AMP portion of ATP onto a target protein-termed adenylylation or AMPylation-by the human Fic protein HYPE/FICD has recently garnered attention as a key regulatory mechanism in endoplasmic reticulum homeostasis, neurodegeneration, and neurogenesis. As a central player in such critical cellular events, high-throughput screening (HTS) efforts targeting HYPE-mediated AMPylation warrant investigation. Herein, we present a dual HTS assay for the simultaneous identification of small-molecule activators and inhibitors of HYPE AMPylation. Employing the fluorescence polarization of an ATP analog fluorophore-Fl-ATP-we developed and optimized an efficient, robust assay that monitors HYPE autoAMPylation and is amenable to automated, high-throughput processing of diverse chemical libraries. Challenging our pilot screen with compounds from the LOPAC, Spectrum, MEGx, and NATx libraries yielded 0.3% and 1% hit rates for HYPE activators and inhibitors, respectively. Further, these hits were assessed for dose-dependency and validated via orthogonal biochemical AMPylation assays. We thus present a high-quality HTS assay suitable for tracking HYPE's enzymatic activity, and the resultant first small-molecule manipulators of HYPE-promoted autoAMPylation.