YL-109 attenuates sepsis-associated multiple organ injury through inhibiting the ERK/AP-1 axis and pyroptosis by upregulating CHIP.

Sepsis is a severe inflammatory disorder that can lead to life-threatening multiple organ injury. Lipopolysaccharide (LPS)-induced inflammation is the leading cause of multiple organ failure in sepsis. This study aimed to explore the effect of a novel agent, 2-(4-hydroxy-3-methoxyphenyl)-benzothiazole (YL-109), on LPS-induced multiple organ injury and the molecular mechanisms underlying these processes. The results showed that YL-109 protected against LPS-induced high mortality, cardiac dysfunction, pulmonary and intestinal injury through inhibiting the proinflammatory response, NLRP3 expression and pyroptosis-associated indicators in mouse tissues. YL-109 suppressed LPS-initiated cytokine release, pyroptosis and pyroptosis-related protein expression in HL-1, IEC-6 and MLE-12 cells, which was consistent with the results of the in vivo experiments. Mechanistically, YL-109 reduces phosphorylated ERK (extracellular signal-regulated kinase) levels and NF-κB activation, which are achieved through upregulating CHIP (carboxy terminus of Hsc70-interacting protein) expression, thereby inhibiting c-Jun and c-Fos activation as well as NLRP3 expression. As an E3 ligase, CHIP overexpression obviously promoted the degradation of phosphorylated ERK and inhibited the expression of NF-κB-mediated NLRP3 in cells stimulated with LPS. The protective effects of YL-109 against cardiac, pulmonary and intestinal damage, inflammation and pyroptosis caused by LPS were eliminated in CHIP knockout mice. Our results not only reveal the protective effect and molecular mechanism of YL-109 against LPS-mediated organs damage but also provide additional insights into the effect of CHIP on negatively regulating pyroptosis and inflammatory pathways.

Effects of UBE3A on Cell and Liver Metabolism through the Ubiquitination of PDHA1 and ACAT1.

Nonalcoholic fatty liver disease (NAFLD) is substantiated by the reprogramming of liver metabolic pathways that disrupts the homeostasis of lipid and glucose metabolism and thus promotes the progression of the disease. The metabolic pathways associated with NAFLD are regulated at different levels from gene transcription to various post-translational modifications including ubiquitination. Here, we used a novel orthogonal ubiquitin transfer platform to identify pyruvate dehydrogenase A1 (PDHA1) and acetyl-CoA acetyltransferase 1 (ACAT1), two important enzymes that regulate glycolysis and ketogenesis, as substrates of E3 ubiquitin ligase UBE3A/E6AP. We found that overexpression of UBE3A accelerated the degradation of PDHA1 and promoted glycolytic activities in HEK293 cells. Furthermore, a high-fat diet suppressed the expression of UBE3A in the mouse liver, which was associated with increased ACAT1 protein levels, while forced expression of UBE3A in the mouse liver resulted in decreased ACAT1 protein contents. As a result, the mice with forced expression of UBE3A in the liver exhibited enhanced accumulation of triglycerides, cholesterol, and ketone bodies. These results reveal the role of UBE3A in NAFLD development by inducing the degradation of ACAT1 in the liver and promoting lipid storage. Overall, our work uncovers an important mechanism underlying the regulation of glycolysis and lipid metabolism through UBE3A-mediated ubiquitination of PDHA1 and ACAT1 to regulate their stabilities and enzymatic activities in the cell.

Cold-upregulated glycosyltransferase gene 1 (OsCUGT1) plays important roles in rice height and spikelet fertility.

Glycosyltransferases (GTs) regulate many physiological processes and stress responses in plants. However, little is known about the function of GT in rice development. In this study, molecular analyses revealed that the expression of a rice GT gene (Cold-Upregulated Glycosyltransferase Gene 1, CUGT1) is developmentally controlled and stress-induced. OsCUGT1 was knocked out by using the clustered regularly interspaced short palindromic repeats (CRISPR) system to obtain the mutant oscugt1, which showed a severe dwarf and sterility phenotype. Further cytological analyses indicated that the dwarfism seen in the oscugt1 mutant might be caused by fewer and smaller cells. Histological pollen analysis suggests that the spikelet sterility in oscugt1 mutants may be caused by abnormal microsporogenesis. Moreover, multiple transgenic plants with knockdown of OsCUGT1 expression through RNA interference were obtained, which also showed obvious defects in plant height and fertility. RNA sequencing revealed that multiple biological processes associated with phenylpropanoid biosynthesis, cytokinin metabolism and pollen development are affected in the oscugt1 mutant. Overall, these results suggest that rice OsCUGT1 plays an essential role in rice development.

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.

Regulation of O-Linked N-Acetyl Glucosamine Transferase (OGT) through E6 Stimulation of the Ubiquitin Ligase Activity of E6AP.

Glycosyltransferase OGT catalyzes the conjugation of O-linked ß-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.