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Ubiquitin and ubiquitin-like proteins (Ubls), such as SUMO, are covalently conjugated to their targets by related, but distinct enzymatic conjugation reactions that involve the dynamic E1-E2-E3 enzyme cascade. E1s activate Ubls by catalyzing Ubl C-terminal adenylation, with the help of ATP, to form a covalent thioester bond. Subsequently, Ubls are transferred to E2 to generate a thioester-linked product. In previous studies, we showed the dynamic processes and thioester intermediates of SUMO with its E1 and E2 conjugating enzymes. Studies of the enzyme specificity of the Ubl conjugation cascade are normally carried out by tedious biochemical processes, and the reaction intermediates are often difficult to capture because they are unstable and have short half-lives. Here, using our recently developed robust quantitative FRET-based technology, we describe systematic investigations of enzymatic specificity and thioester intermediate determination of ubiquitin with its E1-E2 ligases in conjugation with SUMO and its ligases. Our technology easily determined the strong specificity of enzyme-substrate interactions and thioester intermediates in ubiquitination and SUMOylation cascades. The traditional FRET pair ECFP/EYFP lacked adequate signals for these assays. However, in contrast, the highly sensitive FRET pair CyPet/YPet was easily harnessed to define the reaction specificities and intermediates. In addition, the thioester intermediates can be readily monitored by a newly defined FRET index parameter. These results provide an example of a systems biology approach to determine Ubl conjugation specificity and demonstrate that a robust FRET technology can be used to identify enzymes and substrates in other Ubl pathways.

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Various medical treatments for COVID-19 are attempted. After patients are discharged, SARS-CoV-2 recurring cases are reported and the recurrence could profoundly impact patient healthcare and social economics. To date, no data on the effects of medical treatments on recurrence has been published. We analyzed the treatment data of combinations of ten different drugs for the recurring cases in a single medical center, Shenzhen, China. A total of 417 patients were considered and 414 of them were included in this study (3 deaths) with mild-to-critical COVID-19. Patients were treated by 10 different drug combinations and followed up for recurrence for 28 days quarantine after being discharged from the medical center between February and May, 2020. We applied the Synthetic Minority Oversampling Technique (SMOTE) to overcome the rare recurring events in certain age groups and performed Virtual Twins (VT) analysis facilitated by random forest regression for medical treatment-recurrence classification. Among those drug combinations, Methylprednisolone/Interferon/Lopinavir/Ritonavir/Arbidol led to the lowest recurring rate (0.133) as compared to the average recurring rate (0.203). For the younger group (age 20-27) or the older group (age 60-70), the optimal drug combinations are different, but the above combination is still the second best. For obese patients, the combination of Ribavirin/Interferon/Lopinavir/Ritonavir/Arbidol led to the lowest recurring rate for age group of 20-50, whereas the combination of Interferon/Lopinavir/Ritonavir/Arbidol led to lowest recurring rate for age group of 50-70. The insights into combinatorial therapy we provided here shed lights on the use of a combination of (biological and chemical) anti-virus therapy and/or anti-cytokine storm as a potentially effective therapeutic treatment for COVID-19.