Notoginsenoside Ft1 inhibits colorectal cancer growth by increasing CD8+ T cell proportion in tumor-bearing mice through the USP9X signaling pathway.

The management of colorectal cancer (CRC) poses a significant challenge, necessitating the development of innovative and effective therapeutics. Our research has shown that notoginsenoside Ft1 (Ng-Ft1), a small molecule, markedly inhibits subcutaneous tumor formation in CRC and enhances the proportion of CD8+ T cells in tumor-bearing mice, thus restraining tumor growth. Investigation into the mechanism revealed that Ng-Ft1 selectively targets the deubiquitination enzyme USP9X, undermining its role in shielding ß-catenin. This leads to a reduction in the expression of downstream effectors in the Wnt signaling pathway. These findings indicate that Ng-Ft1 could be a promising small-molecule treatment for CRC, working by blocking tumor progression via the Wnt signaling pathway and augmenting CD8+ T cell prevalence within the tumor environment.

Effects of exercise, metformin, and combination treatments on type 2 diabetic mellitus-induced muscle atrophy in db/db mice: Crosstalk between autophagy and the proteasome

Both exercise and metformin are common effective clinical treatments of type 2 diabetic mellitus. This study investigated the functional role of exercise, metformin, and combination treatment on type 2 diabetic mellitus–induced muscle atrophy. In this experiment, a total of 10 BKS mice were set as the control group. A total of 40 BKS-db/db mice were randomly divided into the control group (db/db); the exercise intervention group (db/db + Ex), which ran on a treadmill at 7–12 m/min, 30–40 min/day, 5 days/week; the metformin administration group (db/db + Met), which was administered 300 mg/kg of metformin solution by gavage daily; and the exercise combined with metformin administration group (db/db + Ex + Met). After 8 weeks of intervention, their tibialis anterior muscles were removed. The levels of insulin signaling pathway proteins, ubiquitin proteasome, and autophagic lysosome–associated proteins were detected using western blot, the expression of MuRF1 and Atrogin-1 was detected using immunohistochemical staining, and the degradation of autophagosomes was detected using double-labeled immunofluorescence. The db/db mice exhibited reduced insulin sensitivity and inhibition of the autophagic–lysosome system, the ubiquitin–proteasome system was activated, and protein degradation was exacerbated, leading to skeletal muscle atrophy. Exercise and metformin and their combined interventions can increase insulin sensitivity, whereas exercise alone showed more effective in inhibiting the ubiquitin–proteasome system, improving autophagy levels, and alleviating skeletal muscle atrophy. Compared with metformin, exercise demonstrated superior improvement of muscle atrophy by promoting the synthesis and degradation of autophagy through the AMPK/ULK1 pathway. However, the combination treatment exhibits no synergistic effect on muscle atrophy. (AU)

E3 ubiquitin ligase rififylin has yin and yang effects on rabbit cardiac transient outward potassium currents (Ito) and corresponding channel proteins.

Genome-wide association studies have reported a correlation between a SNP of the RING finger E3 ubiquitin protein ligase rififylin (RFFL) and QT interval variability in humans (Newton-Cheh et al., 2009). Previously, we have shown that RFFL downregulates expression and function of the human-like ether-a-go-go-related gene potassium channel and corresponding rapidly activating delayed rectifier potassium current (IKr) in adult rabbit ventricular cardiomyocytes. Here, we report that RFFL also affects the transient outward current (Ito), but in a peculiar way. RFFL overexpression in adult rabbit ventricular cardiomyocytes significantly decreases the contribution of its fast component (Ito,f) from 35% to 21% and increases the contribution of its slow component (Ito,s) from 65% to 79%. Since Ito,f in rabbits is mainly conducted by Kv4.3, we investigated the effect of RFFL on Kv4.3 expressed in HEK293A cells. We found that RFFL overexpression reduced Kv4.3 expression and corresponding Ito,f in a RING domain-dependent manner in the presence or absence of its accessory subunit Kv channel-interacting protein 2. On the other hand, RFFL overexpression in Kv1.4-expressing HEK cells leads to an increase in both Kv1.4 expression level and Ito,s, similarly in a RING domain-dependent manner. Our physiologically detailed rabbit ventricular myocyte computational model shows that these yin and yang effects of RFFL overexpression on Ito,f, and Ito,s affect phase 1 of the action potential waveform and slightly decrease its duration in addition to suppressing IKr. Thus, RFFL modifies cardiac repolarization reserve via ubiquitination of multiple proteins that differently affect various potassium channels and cardiac action potential duration.

Carpaine alleviates tendinopathy in mice by promoting the ubiquitin-proteasomal degradation of p65 via targeting the E3 ubiquitin ligase LRSAM1.

BACKGROUND: Currently, there are no specific drugs or targets available for the treatment of tendinopathy. However, inflammation has recently been found to play a pivotal role in tendinopathy progression, thereby identifying it as a potential therapeutic target. Carpaine (CA) exhibits potential anti-inflammatory pharmacological properties and may offer a therapeutic option for tendinopathy. PURPOSE: This study aimed to investigate the effectiveness of CA in addressing tendinopathy and uncovering its underlying mechanisms. METHODS: Herein, the efficacy of CA by local administration in vivo in comparison to the first-line drug indomethacin was evaluated in a mouse collagenase-induced tendinopathy (CIT) model. Furthermore, IL-1ß induced a simulated pathological inflammatory microenvironment in tenocytes to investigate its underlying mechanisms in vitro. Further confirmation experiments were performed by overexpressing or knocking down the selective targets of CA in vivo. RESULTS: The findings demonstrated that CA was dose-dependent in treating tendinopathy and that the high-dose group outperformed the first-line drug indomethacin. Mechanistically, CA selectively bound to and enhanced the activity of the E3 ubiquitin ligase LRSAM1 in tendinopathy. This effect mediated the ubiquitination of p65 at lysine 93, subsequently promoting its proteasomal degradation. As a result, the NF-κB pathway was inactivated, leading to a reduction in inflammation of tendinopathy. Consequently, CA effectively mitigated the progression of tendinopathy. Moreover, the LRSAM1 overexpression demonstrated effectiveness in mitigating the tendinopathy progression and its knockdown abolished the therapeutic effects of CA. CONCLUSION: CA attenuates the progression of tendinopathy by promoting the ubiquitin-proteasomal degradation of p65 via increasing the enzyme activity of LRSAM1. The exploration of LRSAM1 has also unveiled a new potential target for treating tendinopathy based on the ubiquitin-proteasomal pathway.

ZmMKK9-ZmMPK20-ZmRIN2 cascade boosts plant heat tolerance by regulating stomatal response.

KEY MESSAGE: Recent research has unveiled that the ZmMKK9-ZmMPK20-ZmRIN2 cascade plays a role in suppressing stomatal opening induced by high temperatures and is a significant contributor to enhancing thermotolerance in plants.

Recent advances in targeted protein degraders as potential therapeutic agents.

Targeted protein degradation (TPD) technology has gradually become widespread in the past 20 years, which greatly boosts the development of disease treatment. Contrary to small inhibitors that act on protein kinases, transcription factors, ion channels, and other targets they can bind to, targeted protein degraders could target "undruggable targets" and overcome drug resistance through ubiquitin-proteasome pathway (UPP) and lysosome pathway. Nowadays, some bivalent degraders such as proteolysis-targeting chimeras (PROTACs) have aroused great interest in drug discovery, and some of them have successfully advanced into clinical trials. In this review, to better understand the mechanism of degraders, we elucidate the targeted protein degraders according to their action process, relying on the ubiquitin-proteasome system or lysosome pathway. Then, we briefly summarize the study of PROTACs employing different E3 ligases. Subsequently, the effect of protein of interest (POI) ligands, linker, and E3 ligands on PROTAC degradation activity is also discussed in detail. Other novel technologies based on UPP and lysosome pathway have been discussed in this paper such as in-cell click-formed proteolysis-targeting chimeras (CLIPTACs), molecular glues, Antibody-PROTACs (Ab-PROTACs), autophagy-targeting chimeras, and lysosome-targeting chimeras. Based on the introduction of these degradation technologies, we can clearly understand the action process and degradation mechanism of these approaches. From this perspective, it will be convenient to obtain the development status of these drugs, choose appropriate degradation methods to achieve better disease treatment and provide basis for future research and simultaneously distinguish the direction of future research efforts.

Shh-Gli2-Runx2 inhibits vascular calcification.

BACKGROUND: In patients with chronic kidney disease (CKD), vascular calcification (VC) is common and is associated with a higher risk of all-cause mortality. Shh, one ligand for Hedgehog (Hh) signaling, participates in osteogenesis and several cardiovascular diseases. However, it remains unclear whether Shh is implicated in the development of VC. METHODS: Inorganic phosphorus 2.6 mM was used to induce vascular smooth muscle cells (VSMCs) calcification. Mice were fed with adenine diet supplement with 1.2% phosphorus to induce VC. RESULTS: Shh was decreased in VSMCs exposed to inorganic phosphorus, calcified arteries in mice fed with an adenine diet, as well as radial arteries from patients with CKD presenting VC. Overexpression of Shh inhibited VSMCs ostosteoblastic differentiation and calcification, whereas its silencing accelerated these processes. Likewise, mice treated with smoothened agonist (SAG; Hh signaling agonist) showed alleviated VC, and mice treated with cyclopamine (CPN; Hh signaling antagonist) exhibited severe VC. Additionally, overexpression of Gli2 significantly reversed the pro-calcification effect of Shh silencing on VSMCs, suggesting that Shh inhibited VC via Gli2. Mechanistically, Gli2 interacted with Runx2 and promoted its ubiquitin proteasomal degradation, therefore protecting against VC. Of interest, the pro-degradation effect of Gli2 on Runx2 was independent of Smurf1 and Cullin4B. CONCLUSIONS: Our study provided deeper insight to the pathogenesis of VC, and Shh might be a novel potential target for VC treatment.

[Hydnocarpin inhibits malignant progression of triple negative breast cancer via CNOT4-mediated ubiquitination and degradation of YAP].

This study aims to investigate the effect and mechanism of hydnocarpin(HC) in treating triple negative breast cancer(TNBC). Cell counting kit-8(CCK-8), xCELLigence real-time cellular analysis(RTCA), and colony formation assay were employed to determine the effects of HC on the proliferation of two TNBC cell lines: MDA-MB-231 and MDA-MB-436. The effects of HC on the migration and invasion of TNBC cells were detected by high-content analysis, wound-healing assay, and Transwell assay. The changes in the epithelial-mesenchymal transition(EMT) and the expression of invasion-and migration-associated proteins [E-cadherin, vimentin, Snail, matrix metalloproteinase-2(MMP-2), and MMP-9] were detected by Western blot. Western blot and RT-qPCR were employed to determine the protein and mRNA levels of Yes-associated protein(YAP) and downstream targets(CTGF and Cyr61). TNBC cells were transfected with Flag-YAP for the overexpression of YAP, and the role of YAP as a key target for HC to inhibit TNBC malignant progression was examined by CCK-8 assay, Transwell assay, and wound-healing assay. The pathway of HC-induced YAP degradation was detected by the co-treatment of proteasome inhibitor with HC and ubiquitination assay. The binding of HC to YAP and the E3 ubiquitin ligase Ccr4-not transcription complex subunit 4(CNOT4) was detected by microscale thermophoresis(MST) assay and drug affinity responsive target stability(DARTS) assay. The results showed that HC significantly inhibited the proliferation, colony formation, invasion, and EMT of TNBC cells. HC down-regulated the protein and mRNA levels of CTGF and Cyr61. HC down-regulated the total protein level of YAP, while it had no effect on the mRNA level of YAP. The overexpression of YAP antagonized the inhibitory effects of HC on the proliferation, migration, and invasion of TNBC cells. HC promoted the degradation of YAP through the proteasome pathway and up-regulated the ubiquitination level of YAP. The results of MST and DARTS demonstrated direct binding between HC, YAP, and CNOT4. The above results indicated that HC inhibited the malignant progression of TNBC via CNOT4-mediated degradation and ubiquitination of YAP.

Jianpi Decoction Combined with Medroxyprogesterone Acetate Alleviates Cancer Cachexia and Prevents Muscle Atrophy by Directly Inhibiting E3 Ubiquitin Ligase.

OBJECTIVE: To provide comprehensive evidence for the anti-cancer cachexia effect of Jianpi Decoction (JP) and to explore its mechanism of anti-cancer cachexia. METHODS: A mouse model of colon cancer (CT26)-induced cancer cachexia (CC) was used to investigate the anti-CC effect of JP combined with medroxyprogesterone acetate (MPA). Thirty-six mice were equally divided into 6 groups: normal control, CC, MPA (100 mg•kg-1•d-1), MPA + low-dose (20 mg•kg-1•d-1) JP (L-JP), MPA + medium-dose (30 mg•kg-1•d-1) JP (M-JP), and MPA + high-dose (40 mg•kg-1•d-1) JP (H-JP) groups. After successful modeling, the mice were administered by gavage for 11 d. The body weight and tumor volume were measured and recorded every 2 d starting on the 8th day after implantation. The liver, heart, spleen, lung, kidney, tumor and gastrocnemius muscle of mice were collected and weighed. The pathological changes of the tumor was observed, and the cross-sectional area of the gastrocnemius muscle was calculated. The protein expressions of STAT3 and E3 ubiquitinase in the gastrocnemius muscle were measured by Western blot. In addition, an in vitro C2C12 myotube formation model was established to investigate the role of JP in hindering dexamethasone-induced muscle atrophy. In vitro experiments were divided into control, model, and JP serum groups. After 2-d administration, microscopic photographs were taken and myotube diameters were calculated. Western blot was performed to measure the protein expressions of STAT3 and E3 ubiquitinase. RESULTS: JP combined with MPA restored tumor-induced weight loss (P<0.05, vs. CC) and muscle fiber size (P<0.01, vs. CC). Mechanistically, JP reduced the expression of atrophy-related proteins MuRF1 and MAFbx in tumor-induced muscle atrophy in vivo (P<0.05, vs. CC). In addition, JP reduced the expression of atrophy-related proteins MuRF1 and MAFbx and p-STAT3 phosphorylation (P<0.05 or P<0.01 vs. model group) in C2C12 myotubes treated with dexamethasone in vitro. CONCLUSIONS: Administration of JP combined with MPA restores tumor-induced cachexia conditions. In addition, the profound effect of JP combined with MPA on tumor-induced cachexia may be due to its inhibition of muscle proteolysis (E3 ubiquitinase system).

Jian-Pi-Yin decoction attenuates lactose-induced chronic diarrhea in rats by regulating GLP-1 and reducing NHE3 ubiquitination and phosphorylation.

Objectives: Jian-Pi-Yin decoction (JPY), a prescription derived from the traditional Chinese medicine Shen-Ling-Bai-Zhu-San, has shown good clinical efficacy in the treatment of diarrhea caused by lactose intolerance. However, the mechanism of action of JPY in the treatment of diarrhea is not fully understood. Design: In this study, a rat diarrhea model was induced by high lactose feeding combined with standing on a small platform to investigate the ameliorating effect of JPY on hyper lactose-induced diarrhea in rats and its possible mechanism. Methods: The rat model of hyper lactose diarrhea was given high, medium, and low doses of JPY and the positive control drug Smida by gavage for 1 week. At the same time, NA+-H+ exchanger 3 (NHE3) inhibitor Tenapanor was administered orally for 3 weeks. Body weight, food intake, water intake, grip strength, and severity of diarrhea symptoms were measured in rats throughout the study. The serum, colon, and jejunum tissues of the model and drug-treated rats were collected for histopathological examination and analysis of relevant indicators. Results: JPY significantly alleviated the symptoms of fatigue, diet reduction and diarrhea in the model group. Glucagon-like peptide-1 (GLP-1) and cyclic adenosine monophosphate (cAMP) expression were also down-regulated after JPY treatment. JPY can significantly promote NHE3 in intestinal tissues of rats with diarrhea, and the mechanism is related to the decrease of GLP-1, inhibition of cAMP/PKA pathway activation, an increase of ubiquitin-specific protease 7 (USP7) and USP10 expression, and decrease of NHE3 ubiquitination and phosphorylation. Conclusion: JPY can reduce the expression of GLP-1, reduce the ubiquitination and phosphorylation of NHE3, regulate the expression of NHE3, at least partly improve ion transport in the intestinal epithelium, and improve the imbalance of electrolyte absorption, thus significantly reducing the diarrhea symptoms of rats with high lactose combined with small platform standing. Innovation: In this study, we explored the mechanism of intestinal GLP-1 activation of cAMP/PKA signaling pathway from multiple dimensions, and increased its expression by reducing phosphorylation and ubiquitination of NHE3, thereby treating chronic diarrhea associated with lactose intolerance.

Isorhamnetin and anti-PD-L1 antibody dual-functional mesoporous silica nanoparticles improve tumor immune microenvironment and inhibit YY1-mediated tumor progression.

BACKGROUND: The immune checkpoint inhibitor (ICI) anti-PD-L1 monoclonal antibody can inhibit the progress of hepatocellular carcinoma (HCC). Epithelial-mesenchymal transformation (EMT) can promote tumor migration and the formation of immune-suppression microenvironment, which affects the therapeutic effect of ICI. Yin-yang-1 (YY1) is an important transcription factor regulating proliferation, migration and EMT of tumor cells. This work proposed a drug-development strategy that combined the regulation of YY1-mediated tumor progression with ICIs for the treatment of HCC. METHODS: We first studied the proteins that regulated YY1 expression by using pull-down, co-immunoprecipitation, and duo-link assay. The active compound regulating YY1 content was screened by virtual screening and cell-function assay. Isorhamnetin (ISO) and anti-PD-L1 antibody dual-functional mesoporous silica nanoparticles (HMSN-ISO@ProA-PD-L1 Ab) were prepared as an antitumor drug to play a synergistic anti-tumor role. RESULTS: YY1 can specifically bind with the deubiquitination enzyme USP7. USP7 can prevent YY1 from ubiquitin-dependent degradation and stabilize YY1 expression, which can promote the proliferation, migration and EMT of HCC cells. Isorhamnetin (ISO) were screened out, which can target USP7 and promote YY1 ubiquitin-dependent degradation. The cell experiments revealed that the HMSN-ISO@ProA-PD-L1 Ab nanoparticles can specifically target tumor cells and play a role in the controlled release of ISO. HMSN-ISO@ProA-PD-L1 Ab nanoparticles inhibited the growth of Hepa1-6 transplanted tumors and the effect was better than that of PD-L1 Ab treatment group and ISO treatment group. HMSN-ISO@ProA-PD-L1 Ab nanoparticles also exerted a promising effect on reducing MDSC content in the tumor microenvironment and promoting T-cell infiltration in tumors. CONCLUSIONS: The isorhamnetin and anti-PD-L1 antibody dual-functional nanoparticles can improve tumor immune microenvironment and inhibit YY1-mediated tumor progression. This study demonstrated the possibility of HCC treatment strategies based on inhibiting USP7-mediated YY1 deubiquitination combined with anti-PD-L1 monoclonal Ab.

Hyperthermia maintains death receptor expression and promotes TRAIL-induced apoptosis.

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand activates apoptotic pathways and could potentially be used in anticancer treatments. However, oral squamous cell carcinoma cells are known to be resistant to tumor necrosis factor-related apoptosis-inducing ligand-induced cell death. It has been previously reported that hyperthermia upregulates tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in other cancers. As such, we evaluated whether hyperthermia upregulates tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in a tumor necrosis factor-related apoptosis-inducing ligand-resistant oral squamous cell carcinoma cell line. METHODS: The oral squamous cell carcinoma cell line HSC3 was cultured and divided into hyperthermia and control groups. We investigated the antitumor effects of recombinant human tumor necrosis factor-related apoptosis-inducing ligand using cell proliferation and apoptosis assays. Additionally, we measured death receptor 4 and 5 levels, and determined death receptor ubiquitination status, as well as E3 ubiquitin ligase targeting of death receptor in both hyperthermia and control groups before recombinant human tumor necrosis factor-related apoptosis-inducing ligand administration. RESULTS: Treatment with recombinant human tumor necrosis factor-related apoptosis-inducing ligand produced greater inhibitory effects in the hyperthermia group than in the control group. Moreover, death receptor protein expression in the hyperthermia group was upregulated on the cell surface (and overall), although death receptor mRNA was downregulated. The half-life of death receptor was several hours longer in the hyperthermia group; concomitantly, E3 ubiquitin ligase expression and death receptor ubiquitination were downregulated in this group. CONCLUSION: Our findings suggested that hyperthermia enhances apoptotic signaling by tumor necrosis factor-related apoptosis-inducing ligand via the suppression of death receptor ubiquitination, which upregulates death receptor expression. These data suggest that the combination of hyperthermia and tumor necrosis factor-related apoptosis-inducing ligand has implications in developing a novel treatment strategy for oral squamous cell carcinoma.

Attenuation of skeletal muscle atrophy via acupuncture, electro-acupuncture, and electrical stimulation.

Background: Accelerated skeletal muscle wasting is a shared trait among many pathologies and aging. Acupuncture has been used as a therapeutic intervention to control pain; however, little is known about its effects on skeletal muscle atrophy and function. The study's purpose was to compare the effects of acupuncture, electro-acupuncture, and electrical stimulation on cast-induced skeletal muscle atrophy. Methods: Forty female Sprague Dawley rats were randomly divided into groups: Control, casted (CAST), CAST+Acupuncture (CAST-A), 4) CAST+Electro-acupuncture (CAST-EA), and CAST+Electrical stimulation (CAST-ES) (n = 8). Plaster casting material was wrapped around the left hind limb. Acupuncture and electro-acupuncture (10 Hz, 6.4 mA) treatments were applied by needling acupoints (stomach-36 and gallbladder-34). Electrical stimulation (10 Hz, 6.4 mA) was conducted by needling the lateral and medial gastrocnemius muscles. Treatments were conducted for 15 min, three times/week for 14 days. Muscle atrophy F-box (MAFbx), muscle RING finger 1 (MuRF1), and contractile properties were assessed. Results: Fourteen days of cast-immobilization decreased muscle fiber CSA by 56% in the CAST group (p = 0.00); whereas, all treatment groups demonstrated greater muscle fiber CSA than the CAST group (p = 0.00). Cast-immobilization increased MAFbx and MuRF1 protein expression in the CAST group (p<0.01) while the CAST-A, CAST-EA, and CAST-ES groups demonstrated lower levels of MAFbx and MuRF1 protein expression (p<0.02) compared to the CAST group. Following fourteen days of cast-immobilization, peak twitch tension did not differ between the CAST-A and CON groups (p = 0.12). Conclusion: Skeletal muscle atrophy, induced by 14 days of cast-immobilization, was significantly attenuated by acupuncture, electro-acupuncture, or electrical stimulation.

Morusinol extracted from Morus alba induces cell cycle arrest and apoptosis via inhibition of DNA damage response in melanoma by CHK1 degradation through the ubiquitin-proteasome pathway.

BACKGROUD: Flavonoids have a variety of biological activities, such as anti-inflammation, anti-tumor, anti-thrombosis and so on. Morusinol, as a novel isoprene flavonoid extracted from Morus alba root barks, has the effects of anti-arterial thrombosis and anti-inflammatory in previous studies. However, the anti-cancer mechanism of morusinol remains unclear. PURPOSE: In present study, we mainly studied the anti-tumor effect of morusinol and its mode of action in melanoma. METHODS: The anti-cancer effect of morusinol on melanoma were evaluated by using the MTT, EdU, plate clone formation and soft agar assay. Flow cytometry was used for detecting cell cycle and apoptosis. The É£-H2AX immunofluorescence and the alkaline comet assay were used to detect DNA damage and the Western blotting analysis was used to investigate the expressions of DNA-damage related proteins. Ubiquitination and turnover of CHK1 were also detected by using the immunoprecipitation assay. The cell line-derived xenograft (CDX) mouse models were used in vivo to evaluate the effect of morusinol on tumorigenicity. RESULTS: We demonstrated that morusinol not only had the ability to inhibit cell proliferation, but also induced cell cycle arrest at G0/G1 phase, caspase-dependent apoptosis and DNA damage in human melanoma cells. In addition, morusinol effectively inhibited the growth of melanoma xenografts in vivo. More strikingly, CHK1, which played an important role in maintaining the integrity of cell cycle, genomic stability and cell viability, was down-regulated in a dose- and time-dependent manner after morusinol treatment. Further research showed that CHK1 was degraded by the ubiquitin-proteasome pathway. Whereafter, morusinol-induced cell cycle arrest, apoptosis and DNA damage were partially salvaged by overexpressing CHK1 in melanoma cell lines. Herein, further experiments demonstrated that morusinol increased the sensitivity of dacarbazine (DTIC) to chemotherapy for melanoma in vitro and in vivo. CONCLUSION: Morusinol induces CHK1 degradation through the ubiquitin-proteasome pathway, thereby inducing cell cycle arrest, apoptosis and DNA damage response in melanoma. Our study firstly provided a theoretical basis for morusinol to be a candidate drug for clinical treatment of cancer, such as melanoma, alone or combinated with dacarbazine.

BAG6-A from Fragaria viridis pollen modulates gametophyte development in diploid strawberry.

Male and female gametophyte development processes are essential steps in the life cycles of all land plants. Here, we characterized a gene, FviBAG6-A, screened from the Fragaria viridis (2 n = 2x=14) pollen cDNA library and physically interacted with S-RNase. Ubiquitinated of Sa-RNase might be determined by the interaction of FviBAG6-A in the ubiquitin-proteasome system during fertilization. We found that overexpression of FviBAG6-A in Arabidopsis caused shorter silique length, and decreased silique number. Moreover, overexpression of FviBAG6-A in Fragaria vesca (2 n = 2x=14) led to a greatly reduced seed number, with nearly 80% of the seeds aborted. Analyses of paraffin sections and reactive oxygen species (ROS) content revealed that the majority of severe pollen defects were likely due to the early degradation of the tapetum and middle layer as a result of ROS accumulation and abnormal development of the uninucleate megaspore mother. Moreover, the FviBAG6-A interact with the E3 ligase SIZ1 and contribute to the SUMOylation of FviBAG6-A , which may be induced by the high level of ROS content, further promoting gametophyte abortion in strawberry transgenic lines. This study characterized the FviBAG6-A and reveals its novel function in gametophyte development.

Short-term exposure to an obesogenic diet causes dynamic dysregulation of proteasome-mediated protein degradation in the hypothalamus of female rats.

OBJECTIVES: Previous work has shown that exposure to a high fat diet dysregulates the protein degradation process in the hypothalamus of male rodents. However, whether this occurs in a sex-independent manner is unknown. The objective of this study was to determine the effects of a short-term obesogenic diet on the ubiquitin-proteasome mediated protein degradation process in the hypothalamus of female rats. METHODS: We fed young adult female rats a high fat diet or standard rat chow for 7 weeks. At the end of the 7th week, animals were euthanized and hypothalamus nuclear and cytoplasmic fractions were collected. Proteasome activity and degradation-specific (K48) ubiquitin signaling were assessed. Additionally, we transfected female rats with CRISPR-dCas9-VP64 plasmids in the hypothalamus prior to exposure to the high fat diet in order to increase proteasome activity and determine the role of reduced proteasome function on weight gain from the obesogenic diet. RESULTS: We found that across the diet period, females gained weight significantly faster on the high fat diet than controls and showed dynamic downregulation of proteasome activity, decreases in proteasome subunit expression and an accumulation of degradation-specific K48 polyubiquitinated proteins in the hypothalamus. Notably, while our CRISPR-dCas9 manipulation was able to selectively increase some forms of proteasome activity, it was unable to prevent diet-induced proteasome downregulation or abnormal weight gain. CONCLUSIONS: Collectively, these results reveal that acute exposure to an obesogenic diet causes reductions in the protein degradation process in the hypothalamus of females.

Thioester and Oxyester Linkages in the Ubiquitin System.

The traditional textbook describes ubiquitylation as the conjugation of ubiquitin to a target by forming a covalent bond connecting ubiquitin's carboxy-terminal glycine residue with an acceptor amino acid like lysine or amino-terminal methionine in the substrate protein. While this adequately depicts a significant fraction of cellular ubiquitylation processes, a growing number of ubiquitin modifications do not follow this rule. Recent data demonstrate that ubiquitin can also be efficiently attached to other amino acids, such as cysteine, serine, and threonine, via ester bonding. Initially observed for a virus-encoded ubiquitin ligase, which targets a cysteine residue in a host protein to initiate its degradation, ester-linked ubiquitylation is now shown to also drive regular cellular processes. These ubiquitylation events expand the complexity and diversity of ubiquitin signaling and broaden the capability of cellular messages in the so-called ubiquitin code. Still, questions on the prevalence, relevance, and involvement in physiological and cellular functions await clearing. In this review, we aim to summarize our knowledge on ester-linked ubiquitylation and introduce experimental strategies to circumvent technical issues that complicate analysis of this uncommon posttranslational modification.

Saikosaponin D alleviates cancer cachexia by directly inhibiting STAT3.

Cancer cachexia is a metabolic syndrome that is characterized by progressive loss of skeletal muscle mass, and effective therapeutics have yet to be developed. Saikosaponin D (SSD), a major bioactive component of Radix Bupleuri, exhibits antiinflammatory, anti-tumor, anti-oxidant, anti-viral, and hepatoprotective effects. In this study, we demonstrated that SSD is a promising agent for the treatment of cancer cachexia. SSD could alleviate TCM-induced myotube atrophy and inhibit the expression of E3 ubiquitin ligases muscle RING-finger containing protein-1 (MuRF1) and muscle atrophy Fbox protein (Atrogin-1/MAFbx) in vitro. Moreover, SSD suppressed the progression of cancer cachexia, with significant improvements in the loss of body weight, gastrocnemius muscle, and tibialis anterior muscle mass in vivo. Mechanism investigations demonstrated that SSD could directly bind to STAT3 and specifically inhibit its phosphorylation as well as its transcriptional activity. Overexpression of STAT3 partially abolished the inhibitory effect of SSD on myotube atrophy, indicating that the therapeutic effect of SSD was attributed to STAT3 inhibition. These findings provide novel strategies for treatment of cancer cachexia by targeting STAT3, and SSD may be a promising drug candidate for cancer cachexia.

Dioscin Inhibits Ubiquitin-Proteasome System in Cancer Cells / 肿瘤防治研究

Objective To explore the antitumor small molecules targeting the ubiquitin–proteasome system (UPS) on the basis of active molecules from traditional Chinese medicine. Methods UbG76V-GFP stably expressing cell line was constructed to screen novel small molecule inhibitors targeting UPS. The fluorogenic substrates of Suc-LLVY-AMC, Z-LLE-AMC, and Boc-LRR-AMC were used to assess the effect of dioscin on the 20S proteasome hydrolase activity. The Ub-AMC substrate was used to evaluate the effect of dioscin on the intracellular deubiquitinating enzyme activity. Western blot was used to detect the effect of dioscin on intracellular ubiquitination levels. CCK-8 and colony formation assays were used to detect the inhibitory effect of dioscin on the tumor cell proliferation. Results Dioscin is a UPS inhibitor discovered through the UbG76V-GFP reporter system. It enhances intracellular ubiquitination and inhibits tumor cell proliferation and colony formation by targeting deubiquitinating enzymes. Conclusion Dioscin could significantly inhibit tumor cell proliferation by targeting ubiquitin–proteasome.