Protein Engineering in the Ubiquitin System: Tools for Discovery and Beyond.

Ubiquitin (UB) transfer cascades consisting of E1, E2, and E3 enzymes constitute a complex network that regulates a myriad of biologic processes by modifying protein substrates. Deubiquitinating enzymes (DUBs) reverse UB modifications or trim UB chains of diverse linkages. Additionally, many cellular proteins carry UB-binding domains (UBDs) that translate the signals encoded in UB chains to target proteins for degradation by proteasomes or in autophagosomes, as well as affect nonproteolytic outcomes such as kinase activation, DNA repair, and transcriptional regulation. Dysregulation of the UB transfer pathways and malfunctions of DUBs and UBDs play causative roles in the development of many diseases. A greater understanding of the mechanism of UB chain assembly and the signals encoded in UB chains should aid in our understanding of disease pathogenesis and guide the development of novel therapeutics. The recent flourish of protein-engineering approaches such as unnatural amino acid incorporation, protein semisynthesis by expressed protein ligation, and high throughput selection by phage and yeast cell surface display has generated designer proteins as powerful tools to interrogate cell signaling mediated by protein ubiquitination. In this study, we highlight recent achievements of protein engineering on mapping, probing, and manipulating UB transfer in the cell. SIGNIFICANCE STATEMENT: The post-translational modification of proteins with ubiquitin alters the fate and function of proteins in diverse ways. Protein engineering is fundamentally transforming research in this area, providing new mechanistic insights and allowing for the exploration of concepts that can potentially be applied to therapeutic intervention.

E-commerce development and its contribution to agricultural non-point source pollution control: Evidence from 283 cities in China.

Agricultural non-point source (ANPS) pollution is a pressing environmental issue in developing countries that poses a substantial threat to sustainable development. With the rapid growth of e-commerce and its great penetration and transformation in many socioeconomic sectors, e-commerce plus agriculture is widely regarded as the solution to sustainable agricultural development. However, the environmental impacts of e-commerce on agriculture, as well as the underlying mechanisms have yet to be fully explored and verified. Based on China's practices of e-commerce development and its integration with agriculture, a panel dataset of 283 prefecture-level cities from 2009 to 2019 was collected, and a spatial difference in difference (SDID) model combined with a Durbin model was constructed to examine the local and spatial spillover effects of e-commerce development on ANPS pollution. It is found out that e-commerce development has significant positive environmental impacts achieved by stimulating industrial structure upgrading and promoting green technology innovation, while the mechanism of cultivation scaling up tends to aggravate the ANPS pollution. Spatial analysis demonstrates that e-commerce development also helps alleviate the ANPS pollution of neighboring regions with a decayed effect over a distance. Meanwhile, the impact of e-commerce on mitigating ANPS pollution shows regional heterogeneity. Those developed regions present significant positive effects, while those regions dominated by agriculture economy and without sufficient supporting facilities for e-commerce development, show significant negative effects. Therefore, we argue that the positive environmental contribution of e-commerce is not bound to happen but instead is contingent, while policies should be adapted to local conditions and enhanced to encourage the integration of e-commerce and other socioeconomic sectors, in order to develop beyond the immature pioneering stage.

Differential Degradation of TRA2A and PYCR2 Mediated by Ubiquitin E3 Ligase E4B.

E4B belongs to the U-box E3 ligase family and functions as either an E3 or an E4 enzyme in protein ubiquitination. Transformer2A (TRA2A) and Pyrroline-5-carboxylate reductase 2 (PYCR2) are related to cancer development and are overexpressed in many cancer cells. The degradation of TRA2A and PYCR2 mediated by the ubiquitin-proteasome system (UPS) has not been reported. This study validated that E4B could ubiquitinate TRA2A and PYCR2 as an E3 ligase both in vitro and in the HEK293 cells. E4B mediated the degradation by forming K11- and K48- linked polyubiquitin chains on TRA2A and PYCR2, respectively. E4B regulated the alternative splicing function of TRA2A and affected RSRC2 transcription in the HEK293 cells. Although E4B is highly expressed, it hardly degrades TRA2A and PYCR2 in hepatocellular carcinoma (HCC) cells, suggesting other mechanisms exist for degradation of TRA2A and PYCR2 in the HCC cells. We finally reported that E4B interacted with substrates via its variable region.