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A cascade of three enzymes, E1−E2−E3, is responsible for transferring ubiquitin to target proteins, which controls many different aspects of cellular signaling. The role of the E2 has been largely overlooked, despite influencing substrate identity, chain multiplicity, and topology. Here we report a method—targeted charging of ubiquitin to E2 (tCUbE)—that can track a tagged ubiquitin through its entire enzymatic cascade in living mammalian cells. We use this approach to reveal new targets whose ubiquitination depends on UbcH5a E2 activity. We demonstrate that tCUbE can be broadly applied to multiple E2s and in different human cell lines. tCUbE is uniquely suited to examine E2−E3‐substrate cascades of interest and/or piece together previously unidentified cascades, thereby illuminating entire branches of the UPS and providing critical insight that will be useful for identifying new therapeutic targets in the UPS. [ABSTRACT FROM AUTHOR]

In this paper, we empirically evaluate the effect of climate change on agricultural technical efficiency, considering the moderating effect of mitigation efforts based on emission reduction in China. In particular, we directly measure the intensity of mitigation efforts using the climate change mitigation index. We then apply the two-stage double bootstrap DEA method to build up the empirical model, effectively avoiding biased estimation using the traditional two-stage DEA method. Our results show that the intensity of mitigation measures moderates the impacts of global warming on efficiency and mild mitigation measures are better than drastic ones. [ABSTRACT FROM AUTHOR]

A cascade of three enzymes, E1-E2-E3, is responsible for transferring ubiquitin to target proteins, which controls many different aspects of cellular signaling. The role of the E2 has been largely overlooked, despite influencing substrate identity, chain multiplicity, and topology. Here we report a method-targeted charging of ubiquitin to E2 (tCUbE)-that can track a tagged ubiquitin through its entire enzymatic cascade in living mammalian cells. We use this approach to reveal new targets whose ubiquitination depends on UbcH5a E2 activity. We demonstrate that tCUbE can be broadly applied to multiple E2s and in different human cell lines. tCUbE is uniquely suited to examine E2-E3-substrate cascades of interest and/or piece together previously unidentified cascades, thereby illuminating entire branches of the UPS and providing critical insight that will be useful for identifying new therapeutic targets in the UPS., (© 2024 Wiley-VCH GmbH.)