Ubiquitin-mediated Protein Degradation Research Articles

ATPase Valosin-Containing Protein (VCP) Is Involved During the Replication and Egress of Sialodacryoadenitis Virus (SDAV) in Neurons.

Sialodacryoadenitis virus (SDAV) has been identified as the etiological agent responsible for the respiratory system and salivary gland infections in rats. The existing literature on SDAV infections is insufficient to address the topic adequately, particularly in relation to the central nervous system. In order to ascertain how SDAV gains access to neuronal cells and subsequently exits, our attention was focused on the small molecule valosin-containing protein (VCP), which is an ATPase. VCP is acknowledged for its function in the ubiquitin-mediated proteasomal degradation of proteins, including those of viral origin. To ascertain the potential influence of VCP on SDAV replication and egress, high-content screening was employed to determine the viral titer and protein content. Western blot analysis was employed to ascertain the relative expression of VCP. Real-time imaging of SDAV-infected cells and confocal imaging for qualitative morphological analysis were conducted. The Eeyarestatin I (EerI) inhibitor was employed to disrupt VCP involvement in the endoplasmic reticulum-associated protein degradation pathway (ERAD) in both pre- and post-incubation systems, with concentrations of 5 μM/mL and 25 μM/mL, respectively. We demonstrated for the first time that SDAV productively replicates in cultured primary neurons. VCP expression is markedly elevated during SDAV infection. The application of 5 μM/mL EerI in the post-treatment system yielded a statistically significant inhibition of the SDAV yield. It is likely that this modulates the efficacy of virion assembly by arresting viral proteins in the submembrane area.

Spatheliachromen mitigates methylglyoxal-induced myotube atrophy by activating Nrf2, inhibiting ubiquitin-mediated protein degradation, and restoring mitochondrial function

BackgroundMethylglyoxal (MGO) is a potent precursor of glycative stress that leads to oxidative stress and muscle atrophy in diabetes. Spatheliachromen (FPATM-20), derived from Ficus pumila var. awkeotsang, exhibited potential antioxidant activity. PurposeThis study aimed to evaluate the potential impact and underlying mechanisms of FPATM-20 on MGO-induced myotube atrophy and mitochondrial dysfunction in mouse skeletal C2C12 myotubes. MethodsAtrophic and antioxidant factors were evaluated using immunofluorescence, enzyme-linked immunosorbent assay, and western blotting. Mitochondrial function was assessed using the ATP assay and Seahorse Cell Mito Stress Test. The glycogen content was determined using periodic acid-Schiff staining. Molecular docking was performed to determine the interaction between FPATM-20 and Keap1. ResultsIn myotubes treated with MGO, FPATM-20 activated the Nrf2 pathway, reduced ROS levels, enhanced antioxidant defense, and increased glycogen content. FPATM-20 improved myotube viability and size, upregulated myosin heavy chain (MyHC) expression, modulated ubiquitin-proteasome molecules (nuclear FoxO3a, atrogin-1, MuRF-1, and p62/SQSTM1), and inhibited apoptosis (Bax/Bcl-2 ratio and cleaved caspase 3). Moreover, FPATM-20 restored mitochondrial function, including mitochondrial membrane potential, mitochondrial oxygen consumption rate, and mitochondrial biogenesis pathway (nuclear PGC-1α/TFAM/FNDC5). The inhibition of Nrf2 with ML385 reversed the effects of FPATM-20 on MGO. Furthermore, molecular docking confirmed the binding of FPATM-20 to Keap1, a suppressor of Nrf2, showing the crucial role of Nrf2 in protective effects. ConclusionsFPATM-20 protects myotubes from MGO toxicity by activating the Nrf2 antioxidant defense, reducing protein degradation and apoptosis, and enhancing mitochondrial function. Thus, FPATM-20 may be a novel agent for preventing skeletal muscle atrophy.

Expanding the inhibitor space of the WWP1 and WWP2 HECT E3 ligases

The HECT E3 ubiquitin ligases 1 (WWP1) and 2 (WWP2) are responsible for the ubiquitin-mediated degradation of key tumour suppressor proteins and are dysregulated in various cancers and diseases. Here we expand their limited inhibitor space by identification of NSC-217913 displaying a WWP1 IC50 of 158.3 µM (95% CI = 128.7, 195.1 µM). A structure-activity relationship by synthesis approach aided by molecular docking led to compound 11 which displayed increased potency with an IC50 of 32.7 µM (95% CI = 24.6, 44.3 µM) for WWP1 and 269.2 µM (95% CI = 209.4, 347.9 µM) for WWP2. Molecular docking yielded active site-bound poses suggesting that the heterocyclic imidazo[4,5-b]pyrazine scaffold undertakes a π-stacking interaction with the phenolic group of tyrosine, and the ethyl ester enables strong ion-dipole interactions. Given the therapeutic potential of WWP1 and WWP2, we propose that compound 11 may provide a basis for future lead compound development.

RING finger protein 5 protects against acute myocardial infarction by inhibiting ASK1

BackgroundMyocardial infarction (MI) is a major disease with high morbidity and mortality worldwide. However, existing treatments are far from satisfactory, making the exploration of potent molecular targets more imperative. The E3 ubiquitin ligase RING finger protein 5 (RNF5) has been previously reported to be involved in several diseases by regulating ubiquitination-mediated protein degradation. Nevertheless, few reports have focused on its function in cardiovascular diseases, including MI.MethodsIn this study, we established RNF5 knockout mice through precise CRISPR-mediated genome editing and utilized left anterior descending coronary artery ligation in 9-11-week-old male C57BL/6 mice. Subsequently, serum biochemical analysis and histopathological examination of heart tissues were performed. Furthermore, we engineered adenoviruses for modulating RNF5 expression and subjected neonatal rat cardiomyocytes to oxygen-glucose deprivation (OGD) to mimic ischemic conditions, demonstrating the impact of RNF5 manipulation on cellular viability. Gene and protein expression analysis provided insights into the molecular mechanisms. Statistical methods were rigorously employed to assess the significance of experimental findings.ResultsWe found RNF5 was downregulated in infarcted heart tissue of mice and NRCMs subjected to OGD treatment. RNF5 knockout in mice resulted in exacerbated heart dysfunction, more severe inflammatory responses, and increased apoptosis after MI surgery. In vitro, RNF5 knockdown exacerbated the OGD-induced decline in cell activity, increased apoptosis, while RNF5 overexpression had the opposite effect. Mechanistically, it was proven that the kinase cascade initiated by apoptosis signal-regulating kinase 1 (ASK1) activation was closely regulated by RNF5 and mediated RNF5’s protective function during MI.ConclusionsWe demonstrated the protective effect of RNF5 on myocardial infarction and its function was dependent on inhibiting the activation of ASK1, which adds a new regulatory component to the myocardial infarction associated network and promises to enable new therapeutic strategy.

Arginyltransferase 1 (ATE1)-mediated proteasomal degradation of viral haemagglutinin protein: a unique host defence mechanism.

The extensive protein production in virus-infected cells can disrupt protein homeostasis and activate various proteolytic pathways. These pathways utilize post-translational modifications (PTMs) to drive the ubiquitin-mediated proteasomal degradation of surplus proteins. Protein arginylation is the least explored PTM facilitated by arginyltransferase 1 (ATE1) enzyme. Several studies have provided evidence supporting its importance in multiple physiological processes, including ageing, stress, nerve regeneration, actin formation and embryo development. However, its function in viral pathogenesis is still unexplored. The present work utilizes Newcastle disease virus (NDV) as a model to establish the role of the ATE1 enzyme and its activity in pathogenesis. Our data indicate a rise in levels of N-arginylated cellular proteins in the infected cells. Here, we also explore the haemagglutinin-neuraminidase (HN) protein of NDV as a presumable target for arginylation. The data indicate that the administration of Arg amplifies the arginylation process, resulting in reduced stability of the HN protein. ATE1 enzyme activity inhibition and gene expression knockdown studies were also conducted to analyse modulation in HN protein levels, which further substantiated the findings. Moreover, we also observed Arg addition and probable ubiquitin modification to the HN protein, indicating engagement of the proteasomal degradation machinery. Lastly, we concluded that the enhanced levels of the ATE1 enzyme could transfer the Arg residue to the N-terminus of the HN protein, ultimately driving its proteasomal degradation.

PARK2 suppresses the proliferation of high-grade serous ovarian carcinoma via inducing the proteasomal degradation of ZNF703.

High-grade serous ovarian cancer (HGSC) is an aggressive disease with poor prognosis. The oncoprotein ZNF703 is implicated in driving HGSC pathogenesis, but factors regulating its abundance remain unclear. In this study, we aim to investigate the potential connection between ZNF703 dysregulation and ubiquitin-mediated protein degradation in HGSC. Bioinformatics prediction was performed using BioGRID database. HGSC representative cell lines were utilized for in vitro and in vivo studies. Results showed that ZNF703 protein was stabilized upon proteasome inhibition, suggesting a regulation via ubiquitination. The ubiquitin E3 ligase PARK2 was found to interact with ZNF703 in a dose-dependent manner, promoting its polyubiquitination and subsequent proteasomal degradation. Re-expression of PARK2 in HGSC cells led to reduced ZNF703 levels together with decreased Cyclin D1/E1 abundance and G1 cell cycle arrest. ZNF703 overexpression alone increased S phase cells, Cyclin D1/E1 levels, and xenograft tumor growth, while co-expression with PARK2 mitigated these oncogenic effects. Collectively, our findings identify ZNF703 as a bona fide substrate of PARK2, reveal a tumor suppressive function for PARK2 in attenuating ZNF703-mediated G1/S transition and HGSC growth through instigating its degradation. This study elucidates a pivotal PARK2-ZNF703 axis with therapeutic implications for targeted intervention in HGSC.

The Regulatory Impact of CFLAR Methylation Modification on Liver Lipid Metabolism.

Non-alcoholic fatty liver disease (NAFLD) has emerged as the leading cause of chronic liver disease worldwide. Caspase 8 and FADD-like apoptosis regulator (CFLAR) has been identified as a potent factor in mitigating non-alcoholic steatohepatitis (NASH) by inhibiting the N-terminal dimerization of apoptosis signal-regulating kinase 1 (ASK1). While arginine methyltransferase 1 (PRMT1) was previously reported to be associated with increased hepatic glucose production, its involvement in hepatic lipid metabolism remains largely unexplored. The interaction between PRMT1 and CFLAR and the methylation of CFLAR were verified by Co-IP and immunoblotting assays. Recombinant adenoviruses were generated for overexpression or knockdown of PRMT1 in hepatocytes. The role of PRMT1 in NAFLD was investigated in normal and high-fat diet-induced obese mice. In this study, we found a significant upregulation of PRMT1 and downregulation of CFLAR after 48h of fasting, while the latter significantly rebounded after 12h of refeeding. The expression of PRMT1 increased in the livers of mice fed a methionine choline-deficient (MCD) diet and in hepatocytes challenged with oleic acid (OA)/palmitic acid (PA). Overexpression of PRMT1 not only inhibited the expression of genes involved in fatty acid oxidation (FAO) and promoted the expression of genes involved in fatty acid synthesis (FAS), resulting in increased triglyceride accumulation in primary hepatocytes, but also enhanced the gluconeogenesis of primary hepatocytes. Conversely, knockdown of hepatic PRMT1 significantly alleviated MCD diet-induced hepatic lipid metabolism abnormalities and liver injury in vivo, possibly through the upregulation of CFLAR protein levels. Knockdown of PRMT1 suppressed the expression of genes related to FAS and enhanced the expression of genes involved in FAO, causing decreased triglyceride accumulation in OA/PA-treated primary hepatocytes in vitro. Although short-term overexpression of PRMT1 had no significant effect on hepatic triglyceride levels under physiological conditions, it resulted in increased serum triglyceride and fasting blood glucose levels in normal C57BL/6J mice. More importantly, PRMT1 was observed to interact with and methylate CFLAR, ultimately leading to its ubiquitination-mediated protein degradation. This process subsequently triggered the activation of c-Jun N-terminal kinase 1 (JNK1) and lipid deposition in primary hepatocytes. Together, these results suggested that PRMT1-mediated methylation of CFLAR plays a critical role in hepatic lipid metabolism. Targeting PRMT1 for drug design may represent a promising strategy for the treatment of NAFLD.

Serotonin Influences Insulin Secretion in Rat Insulinoma INS-1E Cells.

Serotonin or 5-hydroxytryptamine (5-HT) is a monoamine that plays a critical role in insulin secretion, energy metabolism, and mitochondrial biogenesis. However, the action of serotonin in insulin production and secretion by pancreatic β cells has not yet been elucidated. Here, we investigated how exogenous nanomolar serotonin concentrations regulate insulin synthesis and secretion in rat insulinoma INS-1E cells. Nanomolar serotonin concentrations (10 and 50 nM) significantly increased insulin protein expression above the constant levels in untreated control cells and decreased insulin protein levels in the media. The reductions in insulin protein levels in the media may be associated with ubiquitin-mediated protein degradation. The levels of membrane vesicle trafficking-related proteins including Rab5, Rab3A, syntaxin6, clathrin, and EEA1 proteins were significantly decreased by serotonin treatment compared to the untreated control cells, whereas the expressions of Rab27A, GOPC, and p-caveolin-1 proteins were significantly reduced by serotonin treatment. In this condition, serotonin receptors, Gαq-coupled 5-HT2b receptor (Htr2b), and ligand-gated ion channel receptor Htr3a were significantly decreased by serotonin treatment. To confirm the serotonylation of Rab3A and Rab27A during insulin secretion, we investigated the protein levels of Rab3A and Rab27A, in which transglutaminase 2 (TGase2) serotonylated Rab3A but not Rab27A. The increases in ERK phosphorylation levels were consistent with increases in the expression of p-Akt. Also, the expression level of the Bcl-2 protein was significantly increased by 50 and 100 nM serotonin treatment compared to the untreated control cells, whereas the levels of Cu/Zn-SOD and Mn-SOD proteins decreased. These results indicate that nanomolar serotonin treatment regulates the insulin protein level but decreases this level in media through membrane vesicle trafficking-related proteins (Rab5, Rab3A, syntaxin6, clathrin, and EEA1), the Akt/ERK pathway, and Htr2b/Htr3a in INS-1E cells.

Deoxynivalenol induces m6A-mediated upregulation of p21 and growth arrest of mouse hippocampal neuron cells in vitro

Hippocampal neurons maintain the ability of proliferation throughout life to support neurogenesis. Deoxynivalenol (DON) is a mycotoxin that exhibits brain toxicity, yet whether and how DON affects hippocampal neurogenesis remains unknown. Here, we use mouse hippocampal neuron cells (HT-22) as a model to illustrate the effects of DON on neuron proliferation and to explore underlying mechanisms. DON exposure significantly inhibits the proliferation of HT-22 cells, which is associated with an up-regulation of cell cycle inhibitor p21 at both mRNA and protein levels. Global and site-specific m6A methylation levels on the 3’UTR of p21 mRNA are significantly increased in response to DON treatment, whereas inhibition of m6A hypermethylation significantly alleviates DON-induced cell cycle arrest. Further mechanistic studies indicate that the m6A readers YTHDF1 and IGF2BP1 are responsible for m6A-mediated increase in p21 mRNA stability. Meanwhile, 3’UTR of E3 ubiquitin ligase TRIM21 mRNA is also m6A hypermethylated, and another m6A reader YTHDF2 binds to the m6A sites, leading to decreased TRIM21 mRNA stability. Consequently, TRIM21 suppression impairs ubiquitin-mediated p21 protein degradation. Taken together, m6A-mediated upregulation of p21, at both post-transcriptional and post-translational levels, contributes to DON-induced inhibition of hippocampal neuron proliferation. These results may provide new insights for epigenetic therapy of neurodegenerative diseases.Graphical abstractDON inhibits the proliferation of HT-22 cells.RNA m6A hypermethylation on the transcript of p21 enhances the mRNA stability in a YTHDF1- and IGF2BP1-dependent manner, which leads to the upregulation of p21.RNA m6A hypermethylation on the transcript of TRIM21 decreases the mRNA stability in a YTHDF2-dependent manner, which contributes to prevent p21 ubiquitin-mediated degradation.High expression of p21 contributes to inhibit cell proliferation.

NEDD4L mediates ITGB4 ubiquitination and degradation to suppress esophageal carcinoma progression

The ubiquitination-mediated protein degradation exerts a vital role in the progression of multiple tumors. NEDD4L, which belongs to the E3 ubiquitin ligase NEDD4 family, is related to tumor genesis, metastasis and drug resistance. However, the anti-tumor role of NEDD4L in esophageal carcinoma, and the potential specific recognition substrate remain unclear. Based on public esophageal carcinoma database and clinical sample data, it was discovered in this study that the expression of NEDD4L in esophageal carcinoma was apparently lower than that in atypical hyperplastic esophageal tissue and esophageal squamous epithelium. Besides, patients with high expression of NEDD4L in esophageal carcinoma tissue had longer progression-free survival than those with low expression. Experiments in vivo and in vitro also verified that NEDD4L suppressed the growth and metastasis of esophageal carcinoma. Based on co-immunoprecipitation and proteome analysis, the NEDD4L ubiquitination-degraded protein ITGB4 was obtained. In terms of the mechanism, the HECT domain of NEDD4L specifically bound to the Galx-β domain of ITGB4, which modified the K915 site of ITGB4 in an ubiquitination manner, and promoted the ubiquitination degradation of ITGB4, thus suppressing the malignant phenotype of esophageal carcinoma.

OsATL32 ubiquitinates the reactive oxygen species-producing OsRac5-OsRbohB module to suppress rice immunity.

Ubiquitination-mediated protein degradation is integral to plant immunity, with E3 ubiquitin ligases acting as key factors in this process. Here, we report the functions of OsATL32, a plasma membrane-localized Arabidopsis Tóxicos En Levadura (ATL)-type E3 ubiquitin ligase, in rice (Oryza sativa) immunity and its associated regulatory network. We found that the expression of OsATL32 is downregulated in both compatible and incompatible interactions between rice and the rice blast fungus Magnaporthe oryzae. The OsATL32 protein level declines in response to infection by a compatible M. oryzae strain or to chitin treatment. OsATL32 negatively regulates rice resistance to blast and bacterial leaf blight diseases, as well as chitin-triggered immunity. Biochemical and genetic studies revealed that OsATL32 suppresses pathogen-induced reactive oxygen species (ROS) accumulation by mediating ubiquitination and degradation of the ROS-producing OsRac5-OsRbohB module, which enhances rice immunity against M. oryzae. The protein phosphatase PHOSPHATASE AND TENSIN HOMOLOG enhances rice blast resistance by dephosphorylating OsATL32 and promoting its degradation, preventing its negative effect on rice immunity. This study provides insights into the molecular mechanism by which the E3 ligase OsATL32 targets a ROS-producing module to undermine rice immunity.

Overexpression of E3 ligase RING finger protein BrRING509 enhances salt stress tolerance in transgenic Arabidopsis plants

Salinity is an important abiotic stress that has an impact on crop yields and quality. Ubiquitin-mediated protein degradation enables plants to effectively withstand environmental stresses. The E3 ligases have been identified as a crucial component of the ubiquitination pathway, which regulates plant responses to abiotic stresses. The RING finger E3 ligase family of proteins has a critical role in plant growth and response to many abiotic stresses. In this study, we identified an important salt-responsive gene, BrRING509 in Brassica rapa. It is localized throughout the plasma membrane and nucleus. The functional results suggest that BrRING509 significantly increases salt stress tolerance in Arabidopsis (Arabidopsis thaliana) transgenic plants. Overexpression of BrRING509 could increase the survival and root length of transgenic seedlings under different salt concentrations. Additionally, ectopic expression of BrRING509 significantly increases the expression level of salt-responsive genes, including AtRD22 and AtRD29A in Arabidopsis transgenic plants subjected to salt stress treatments. The findings of this study indicate that BrRING509 plays a positive role in salt stress tolerance and might be an important candidate gene for the adaptation of Brassica plants in salt-stress environments.

The Protein Response of Salt-Tolerant Zygosaccharomyces rouxii to High-Temperature Stress during the Lag Phase.

Zygosaccharomyces rouxii used in soy sauce brewing is an osmotolerant and halotolerant yeast, but it is not tolerant to high temperatures and the underlying mechanisms remain poorly understood. Using a synthetic medium containing only Pro as a nitrogen source, the response of Z. rouxii in protein level to high-temperature stress (40 °C, HTS) during the lag phase was investigated. Within the first two h, the total intracellular protein concentration was significantly decreased from 220.99 ± 6.58 μg/mg DCW to 152.63 ± 10.49 μg/mg DCW. The analysis of the amino acid composition of the total protein through vacuum proteolysis technology and HPLC showed that new amino acids (Thr, Tyr, Ser, and His) were added to newborn protein over time during the lag phase under HTS. The nutritional conditions used in this study determined that the main source of amino acid supply for protein synthesis was through amino acid biosynthesis and ubiquitination-mediated protein degradation. Differential expression analysis of the amino acid biosynthesis-related genes in the transcriptome showed that most genes were upregulated under HTS, excluding ARO8, which was consistently repressed during the lag phase. RT-qPCR results showed that high-temperature stress significantly increased the upregulation of proteolysis genes, especially PSH1 (E3 ubiquitin ligase) by 13.23 ± 1.44 fold (p < 0.0001) within 4 h. Overall, these results indicated that Z. rouxii adapt to prolonged high temperatures stress by altering its basal protein composition. This protein renewal was related to the regulation of proteolysis and the biosynthesis of amino acids.

Identification and Expression Analysis of the SKP1-Like Gene Family under Phytohormone and Abiotic Stresses in Apple (Malus domestica).

Ubiquitination participates in plant hormone signaling and stress response to adversity. SKP1-Like, a core component of the SCF (Skp1-Cullin-F-box) complex, is the final step in catalyzing the ubiquitin-mediated protein degradation pathway. However, the SKP1-Like gene family has not been well characterized in response to apple abiotic stresses and hormonal treatments. This study revealed that 17 MdSKP1-Like gene family members with the conserved domain of SKP1 were identified in apples and were unevenly distributed on eight chromosomes. The MdSKP1-Like genes located on chromosomes 1, 10, and 15 were highly homologous. The MdSKP1-like genes were divided into three subfamilies according to the evolutionary affinities of monocotyledons and dicotyledons. MdSKP1-like members of the same group or subfamily show some similarity in gene structure and conserved motifs. The predicted results of protein interactions showed that members of the MdSKP1-like family have strong interactions with members of the F-Box family of proteins. A selection pressure analysis showed that MdSKP1-Like genes were in purifying selection. A chip data analysis showed that MdSKP1-like14 and MdSKP1-like15 were higher in flowers, whereas MdSKP1-like3 was higher in fruits. The upstream cis-elements of MdSKP1-Like genes contained a variety of elements related to light regulation, drought, low temperature, and many hormone response elements, etc. Meanwhile, qRT-PCR also confirmed that the MdSKP1-Like gene is indeed involved in the response of the apple to hormonal and abiotic stress treatments. This research provides evidence for regulating MdSKP1-Like gene expression in response to hormonal and abiotic stresses to improve apple stress resistance.

Mechanism of selective recognition of Lys48-linked polyubiquitin by macrocyclic peptide inhibitors of proteasomal degradation

Post-translational modification of proteins with polyubiquitin chains is a critical cellular signaling mechanism in eukaryotes with implications in various cellular states and processes. Unregulated ubiquitin-mediated protein degradation can be detrimental to cellular homeostasis, causing numerous diseases including cancers. Recently, macrocyclic peptides were developed that selectively target long Lysine-48-linked polyubiquitin chains (tetra-ubiquitin) to inhibit ubiquitin-proteasome system, leading to attenuation of tumor growth in vivo. However, structural determinants of the chain length and linkage selectivity by these cyclic peptides remained unclear. Here, we uncover the mechanism underlying cyclic peptide’s affinity and binding selectivity by combining X-ray crystallography, solution NMR, and biochemical studies. We found that the peptide engages three consecutive ubiquitins that form a ring around the peptide and determined requirements for preferential selection of a specific trimer moiety in longer polyubiquitin chains. The structural insights gained from this work will guide the development of next-generation cyclic peptides with enhanced anti-cancer activity.

Tetrandrine synergizes with MAPK inhibitors in treating KRAS-mutant pancreatic ductal adenocarcinoma via collaboratively modulating the TRAIL-death receptor axis

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies lacking effective therapies. KRAS mutations that occur in over 90% of PDAC are major oncogenic drivers of PDAC. The MAPK signaling pathway plays a central role in KRAS-driven oncogenic signaling. However, pharmacological inhibitors of the MAPK pathway are poorly responded in KRAS-mutant PDAC, raising a compelling need to understand the mechanism behind and to seek new therapeutic solutions. Herein, we perform a screen utilizing a library composed of 800 naturally-derived bioactive compounds to identify natural products that are able to sensitize KRAS-mutant PDAC cells to the MAPK inhibition. We discover that tetrandrine, a natural bisbenzylisoquinoline alkaloid, shows a synergistic effect with MAPK inhibitors in PDAC cells and xenograft models. Mechanistically, pharmacological inhibition of the MAPK pathway exhibits a double-edged impact on the TRAIL-death receptor axis, transcriptionally upregulating TRAIL yet downregulating its agonistic receptors DR4 and DR5, which may explain the limited therapeutic outcomes of MAPK inhibitors in KRAS-mutant PDAC. Of great interest, tetrandrine stabilizes DR4/DR5 protein via impairing ubiquitination-mediated protein degradation, thereby allowing a synergy with MAPK inhibition in inducing apoptosis in KRAS-mutant PDAC. Our findings identify a new combinatorial approach for treating KRAS-mutant PDAC and highlight the role of TRAIL-DR4/DR5 axis in dictating the therapeutic outcome in KRAS-mutant PDAC.

Pancreatic Cancer-Derived Exosomal miR-Let-7b-5p Stimulates Insulin Resistance in Skeletal Muscle Cells Through RNF20/STAT3/FOXO1 Axis Regulation.

Cancers trigger systemic metabolic disorders usually associated with glucose intolerance, which is an initially apparent phenomenon. One of the features of pancreatic cancer (PC) metabolic reprogramming is the crosstalk between PC and peripheral tissues (skeletal muscle and adipose tissues), emphasized by insulin resistance (IR). Our previous study reported that mice pancreatic cancer-derived exosomes could induce skeletal muscle cells (C2C12) IR, and exosomal microRNAs (miRNAs) may exert an important effect. However, the underlying mechanism remains to be further elucidated. qPCR was used to determine the expression of let-7b-5p in normal pancreatic islet cells and PC cells. Exosomes were purified from PC cell culture medium by ultracentrifugation. The role let-7b-5p on IR-mediated by PC cells-derived exosomes was asses by Oil Red O staining using miRNA inhibitor. Western blot assay was performed to examine the expression of IR-related genes and the activation of signaling pathways. A Luciferase experiment was applied to confirm how let-7b-5p regulated the expression of RNF20. IP/WB analysis further determined whether RNF20 promoted STAT3 ubiquitination. Rescue experiment using RNF20 overexpression plasmid was performed to confirm the role of RNF20 on IR-mediated using PC cell-derived exosomes in C2C12 myotube cells. miRNA-let-7b-5p was identified as the key exosomal miRNA, which could promote the IR in C2C12 myotube cells supported the lipid accumulation, the activation of STAT3/FOXO1 axis, and the decreased expression of IRS-1 and GLUT4. RNF20, an E3 ubiquitin ligase, was confirmed as the target gene of let-7b-5p and was found to improve IR by downregulating STAT3 protein expression via ubiquitination-mediated protein degradation. The ectopic expression of RNF20 could effectively attenuate the IR mediated by the pancreatic cancer-derived exosomes in C2C12 myotube cells. Our data suggest that exosomal miRNA-let-7b-5p may promote IR in C2C12 myotube cells by targeting RNF20 to activate the STAT3/FOXO1 axis.

Ginsenoside Rg5's Enhancement of Radiosensitivity of Lung Adenocarcinoma via Reducing HSP90 CDC37 Interaction and Promoting Client Protein Degradation

Ginsenoside Rg5 is a rare ginsenoside showing promising tumor-suppressive effects. This study aimed to explore its radio-sensitizing effects and the underlying mechanisms. Human lung adenocarcinoma cell lines A549 and H460 were used for in vitro and in vivo analysis. Bioinformatic molecular docking prediction and following validation by surface plasmon resonance (SPR) technology and cellular thermal shift assay (CETSA) were conducted to explore the binding between ginsenoside Rg5 and HSP90α. The effects of ginsenoside Rg5 on HSP90-CDC37 interaction, the client protein stability and the downstream regulations were further explored. ginsenoside Rg5 can induce cell cycle arrest at the G1 phase and enhance radiation-induced cell apoptosis. Ginsenoside Rg5 could bind to HSP90α with a high affinity. ginsenoside Rg5 might interfere in the HSP90-CDC37-client interaction. Co-IP assays confirmed that ginsenoside Rg5 weakened the HSP90-CDC37 interaction in a dose-dependent manner. Ginsenoside Rg5 reduced irradiation-induced upregulation of the HSP90-CDC37 client proteins, including SRC, CDK4, RAF1 and ULK1 in A549 cell derived xenograft (CDX) tumors. Ginsenoside Rg5 or MRT67307 pre-treatment suppressed irradiation-induced elevation of the LC3-II/β ratio and restored irradiation-induced downregulation of p62 expression. In A549 CDX tumors, ginsenoside Rg5 treatment suppressed LC3 expression and enhanced irradiation-induced DNA damage. ginsenoside Rg5 may be a potential radiosensitizer for lung adenocarcinoma. It interacts with HSP90 and reduces the binding between HSP90 and CDC37, thereby increasing the ubiquitin-mediated proteasomal degradation of the HSP90-CDC37 client proteins.

Pharmacophore-Based Machine Learning Model To Predict Ligand Selectivity for E3 Ligase Binders.

E3 ligases are enzymes that play a critical role in ubiquitin-mediated protein degradation and are involved in various cellular processes. Pharmacophore analysis is a useful approach for predicting E3 ligase binding selectivity, which involves identifying key chemical features necessary for a ligand to interact with a specific protein target cavity. While pharmacophore analysis is not always sufficient to accurately predict ligand binding affinity, it can be a valuable tool for filtering and/or designing focused libraries for screening campaigns. In this study, we present a fast and an inexpensive approach using a pharmacophore fingerprinting scheme known as ErG, which is used in a multi-class machine learning classification model. This model can assign the correct E3 ligase binder to its known E3 ligase and predict the probability of each molecule to bind to different E3 ligases. Practical applications of this approach are demonstrated on commercial libraries such as Asinex for the rational design of E3 ligase binders. The scripts and data associated with this study can be found on GitHub at https://github.com/Fraunhofer-ITMP/E3_binder_Model.

RING finger ubiquitin E3 ligase CsCHYR1 targets CsATAF1 for degradation to modulate the drought stress response of cucumber through the ABA-dependent pathway

CsCHYR1 (CHY ZINC-FINGER AND RING PROTEIN1) encodes a RING (Really Interesting New Gene) finger E3 ubiquitin ligase involved in ubiquitin-mediated protein degradation and plays an important role for cucumber to resist drought stress. Here, we obtain one of the candidate proteins CsCHYR1 that probably interacts with CsATAF1 by yeast-two hybrid screening. Subsequently, it is verified that CsCHYR1 interacts with CsATAF1 and has self-ubiquitination activity. When the cysteine residue at 180 in the RING domain of CsCHYR1 is replaced by serine or alanine, ubiquitin could not be transported from E2 to the substrate. CsCHYR1 ubiquitinates CsATAF1 and affects the stability of CsATAF1 when plants are subjected to drought stress. The expression level of CsCHYR1 is increased by 4-fold after ABA treatment at 9 h. The Atchyr1 mutants perform an ABA-hyposensitive phenotype and have a lower survival rate than Col-0 and CsCHYR1 Atchyr1 lines. In addition, CsCHYR1 interacts with CsSnRK2.6. Therefore, our study reveals a CsSnRK2.6-CsCHYR1-CsATAF1 complex to promote the drought stress response by decreasing CsATAF1 protein accumulation and inducing stomatal closure. Those findings provide new ideas for cucumber germplasm innovation from the perspective of biochemistry and molecular biology.