RESUMO
The main protease of severe acute respiratory syndrome coronavirus 2, Mpro, is a key viral protein essential for viral infection and replication. Mpro has been the target of many pharmacological efforts; however, the host-specific regulation of Mpro protein remains unclear. Here, we report the ubiquitin-proteasome-dependent degradation of Mpro protein in human cells, facilitated by the human E3 ubiquitin ligase ZBTB25. We demonstrate that Mpro has a short half-life that is prolonged via proteasomal inhibition, with its Lys-100 residue serving as a potential ubiquitin acceptor. Using in vitro binding assays, we observed ZBTB25 and Mpro bind to each other in vitro, and using progressive deletional mapping, we further uncovered the required domains for this interaction. Finally, we used an orthologous beta-coronavirus infection model and observed that genetic ablation of ZBTB25 resulted in a more highly infective virus, an effect lost upon reconstitution of ZBTB25 to deleted cells. In conclusion, these data suggest a new mechanism of Mpro protein regulation as well as identify ZBTB25 as an anticoronaviral E3 ubiquitin ligase.
Assuntos
Proteases 3C de Coronavírus , Proteínas de Ligação a DNA , SARS-CoV-2 , Humanos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteases Virais/genética , Proteases Virais/metabolismo , Proteínas Virais/metabolismo , SARS-CoV-2/fisiologia , Proteases 3C de Coronavírus/metabolismo , COVID-19/virologiaRESUMO
BACKGROUND AND OBJECTIVE: It is currently recommended that patients avoid large meals prior to their lung function tests. The aim of this study is to determine whether this recommendation is necessary in clinical practice. METHODS: A randomized controlled cross-over trial was conducted. Subjects performed lung function tests (spirometry, measurement of lung volumes and gas transfer) prior to, directly following and 2 h after consuming a large breakfast. On the control arm, subjects performed the same lung function tests while fasting for the duration of the morning. The study subjects comprised 12 healthy subjects, 10 COPD patients and 10 patients with interstitial lung disease. RESULTS: There were no significant differences between measurements on the meal and control days for FEV(1), FVC, TLC or DL(CO). There were no significant changes with time in any of these parameters over the course of either the meal or control morning. CONCLUSIONS: Common measures of lung function are not affected by the prior consumption of a large meal and it is unnecessary to advise patients to avoid a large meal prior to lung function assessment.
