Doxorubicin (DOX)-related cardiotoxicity has been recognized as a serious complication of cancer chemotherapy. Effective targeted strategies for myocardial protection in addition to DOX treatment are urgently needed. The purpose of this paper was to determine the therapeutic effect of berberine (Ber) on DOX-triggered cardiomyopathy and explore the underlying mechanism. Our data showed that Ber markedly prevented cardiac diastolic dysfunction and fibrosis, reduced cardiac malondialdehyde (MDA) level and increased antioxidant superoxide dismutase (SOD) activity in DOX-treated rats. Moreover, Ber effectively rescued the DOX-induced production of reactive oxygen species (ROS) and MDA, mitochondrial morphological damage and membrane potential loss in neonatal rat cardiac myocytes and fibroblasts. This effect was mediated by increases in the nuclear accumulation of nuclear erythroid factor 2-related factor 2 (Nrf2) and levels of heme oxygenase-1 (HO-1) and mitochondrial transcription factor A (TFAM). We also found that Ber suppressed the differentiation of cardiac fibroblasts (CFs) into myofibroblasts, as indicated by decreased expression of α-smooth muscle actin (α-SMA), collagen I and collagen III in DOX-treated CFs. Pretreatment with Ber inhibited ROS and MDA production and increased SOD activity and the mitochondrial membrane potential in DOX-challenged CFs. Further investigation indicated that the Nrf2 inhibitor trigonelline reversed the protective effect of Ber on both cardiomyocytes and CFs after DOX stimulation. Taken together, these findings demonstrated that Ber effectively alleviated DOX-induced oxidative stress and mitochondrial damage by activating the Nrf2-mediated pathway, thereby leading to the prevention of myocardial injury and fibrosis. The current study suggests that Ber is a potential therapeutic agent for DOX-induced cardiotoxicity that exerts its effects by activating Nrf2.

E3 ubiquitin (UB) ligases E4B and carboxyl terminus of Hsc70-interacting protein (CHIP) use a common U-box motif to transfer UB from E1 and E2 enzymes to their substrate proteins and regulate diverse cellular processes. To profile their ubiquitination targets in the cell, we used phage display to engineer E2-E4B and E2-CHIP pairs that were free of cross-reactivity with the native UB transfer cascades. We then used the engineered E2-E3 pairs to construct “orthogonal UB transfer (OUT)” cascades so that a mutant UB (xUB) could be exclusively used by the engineered E4B or CHIP to label their substrate proteins. Purification of xUB-conjugated proteins followed by proteomics analysis enabled the identification of hundreds of potential substrates of E4B and CHIP in human embryonic kidney 293 cells. Kinase MAPK3 (mitogen-activated protein kinase 3), methyltransferase PRMT1 (protein arginine N-methyltransferase 1), and phosphatase PPP3CA (protein phosphatase 3 catalytic subunit alpha) were identified as the shared substrates of the two E3s. Phosphatase PGAM5 (phosphoglycerate mutase 5) and deubiquitinase OTUB1 (ovarian tumor domain containing ubiquitin aldehyde binding protein 1) were confirmed as E4B substrates, and β-catenin and CDK4 (cyclin-dependent kinase 4) were confirmed as CHIP substrates. On the basis of the CHIP-CDK4 circuit identified by OUT, we revealed that CHIP signals CDK4 degradation in response to endoplasmic reticulum stress.

E3 ubiquitin (UB) ligases are the ending modules of the E1–E2-E3 cascades that transfer UB to cellular proteins and regulate their biological functions. Identifying the substrates of an E3 holds the key to elucidate its role in cell regulation. Here, we construct an orthogonal UB transfer (OUT) cascade to identify the substrates of E6AP, a HECT E3 also known as Ube3a that is implicated in cancer and neurodevelopmental disorders. We use yeast cell surface display to engineer E6AP to exclusively transfer an affinity-tagged UB variant (xUB) to its substrate proteins. Proteomic identification of xUB-conjugated proteins in HEK293 cells affords 130 potential E6AP targets. Among them, we verify that MAPK1, CDK1, CDK4, PRMT5, β-catenin, and UbxD8 are directly ubiquitinated by E6AP in vitro and in the cell. Our work establishes OUT as an efficient platform to profile E3 substrates and reveal the cellular circuits mediated by the E3 enzymes.

Glycosyltransferase OGT catalyzes the conjugation of O-linked β-D-N-acetylglucosamine (O-GlcNAc) to Ser and Thr residues of the cellular proteins and regulates many key processes in the cell. Here, we report the identification of OGT as a ubiquitination target of HECT-type E3 ubiquitin (UB) ligase E6AP, whose overexpression in HEK293 cells would induce the degradation of OGT. We also found that the expression of E6AP in HeLa cells with the endogenous expression of the E6 protein of the human papillomavirus (HPV) would accelerate OGT degradation by the proteasome and suppress O-GlcNAc modification of OGT substrates in the cell. Overall, our study establishes a new mechanism of OGT regulation by the ubiquitin–proteasome system (UPS) that mediates the crosstalk between protein ubiquitination and O-GlcNAcylation pathways underlying diverse cellular processes.

Aim: To investigate undergraduate nursing students' general study engagement using intra‐individual assessment and to evaluate the impact of core self‐evaluation and career calling on study engagement.Design: A descriptive and cross‐sectional design.Methods: Data were collected using a self‐administered questionnaire survey. Four hundred and twenty nursing students from first‐ to fourth (final)‐academic year in Guangzhou Medical University were invited to participate in the spring of 2021. The Cronbach's alpha, one‐way analysis of variance, Bonferroni post hoc analysis, Pearson correlation analysis and multiple stepwise regression analysis were used to analyse the data.Results: The first‐ and fourth‐year undergraduates showed significantly higher levels of study engagement (first‐year undergraduates, 3.52 ± 0.59; fourth‐year undergraduates, 3.54 ± 0.64), core self‐evaluation (first‐year undergraduates, 3.04 ± 0.48; fourth‐year undergraduates, 3.11 ± 0.45) and career calling (first‐year undergraduates, 3.65 ± 0.47; fourth‐year undergraduates, 3.69 ± 0.50) than those of second‐year undergraduates (study engagement, 3.32 ± 0.61; core self‐evaluation, 2.93 ± 0.52; career calling, 3.41 ± 0.50) and third‐year undergraduates (study engagement, 3.16 ± 0.61; core self‐evaluation, 2.88 ± 0.50; career calling, 3.34 ± 0.38). The Pearson correlation analysis among nursing students revealed a significant positive correlation between core self‐evaluation, career calling and study engagement (p < 0.01). A partial mediation effect of career calling was present in the relationship between core self‐evaluation and study engagement.