MG132 Treatment Research Articles

Cul5-type Ubiquitin Ligase KLHDC1 Contributes to the Elimination of Truncated SELENOS Produced by Failed UGA/Sec Decoding.

SummaryThe UGA codon signals protein translation termination, but it can also be translated into selenocysteine (Sec, U) to produce selenocysteine-containing proteins (selenoproteins) by dedicated machinery. As Sec incorporation can fail, Sec-containing longer and Sec-lacking shorter proteins co-exist. Cul2-type ubiquitin ligases were recently shown to destabilize such truncated proteins; however, which ubiquitin ligase targets truncated proteins for degradation remained unclear. We report that the Cul5-type ubiquitin ligase KLHDC1 targets truncated SELENOS, a selenoprotein, for proteasomal degradation. SELENOS is involved in endoplasmic reticulum (ER)-associated degradation, which is linked to reactive oxygen species (ROS) production, and the knockdown of KLHDC1 in U2OS cells decreased ER stress-induced cell death. Knockdown of SELENOS increased the cell population with lower ROS levels. Our findings reveal that, in addition to Cul2-type ubiquitin ligases, KLHDC1 is involved in the elimination of truncated oxidoreductase-inactive SELENOS, which would be crucial for maintaining ROS levels and preventing cancer development.

Proteasomal inhibition triggers viral oncoprotein degradation via autophagy-lysosomal pathway.

Epstein-Barr virus (EBV) nuclear oncoprotein EBNA3C is essential for B-cell transformation and development of several B-cell lymphomas particularly those are generated in an immuno-compromised background. EBNA3C recruits ubiquitin-proteasome machinery for deregulating multiple cellular oncoproteins and tumor suppressor proteins. Although EBNA3C is found to be ubiquitinated at its N-terminal region and interacts with 20S proteasome, the viral protein is surprisingly stable in growing B-lymphocytes. EBNA3C can also circumvent autophagy-lysosomal mediated protein degradation and subsequent antigen presentation for T-cell recognition. Recently, we have shown that EBNA3C enhances autophagy, which serve as a prerequisite for B-cell survival particularly under growth deprivation conditions. We now demonstrate that proteasomal inhibition by MG132 induces EBNA3C degradation both in EBV transformed B-lymphocytes and ectopic-expression systems. Interestingly, MG132 treatment promotes degradation of two EBNA3 family oncoproteins–EBNA3A and EBNA3C, but not the viral tumor suppressor protein EBNA3B. EBNA3C degradation induced by proteasomal inhibition is partially blocked when autophagy-lysosomal pathway is inhibited. In response to proteasomal inhibition, EBNA3C is predominantly K63-linked polyubiquitinated, colocalized with the autophagy-lysosomal fraction in the cytoplasm and participated within p62-LC3B complex, which facilitates autophagy-mediated degradation. We further show that the degradation signal is present at the first 50 residues of the N-terminal region of EBNA3C. Proteasomal inhibition reduces the colony formation ability of this important viral oncoprotein, induces apoptotic cell death and increases transcriptional activation of both latent and lytic gene expression which further promotes viral reactivation from EBV transformed B-lymphocytes. Altogether, this study offers rationale to use proteasome inhibitors as potential therapeutic strategy against multiple EBV associated B-cell lymphomas, where EBNA3C is expressed.

IRF3 enhances NF-κB activation by targeting IκBα for degradation in teleost fish

Tightly regulation of NF-κB signaling is essential to innate and adaptive immune responses, but its regulatory mechanism remains unclear in various organisms, especially teleost fish. In this study, we reported that IRF3 attenuates the inhibitory effect of IκBα on NF-κB activation in teleost fish. Overexpression of IRF3 can promote IκBα degradation, whereas its knockdown can relieve degradation of IκBα. IRF3 promoted the degradation of IκBα protein, but this effect could be inhibited by MG132 treatment. IRF3 is crucial for the polyubiquitination and proteasomal degradation of IκBα. Our findings indicate that IRF3 regulates NF-κB pathway by targeting IκBα for ubiquitination and degradation. This study provides novel evidence on the regulation of innate immune signaling pathways in teleost fish and thus provides new insights into the regulatory mechanisms in mammals.

Anaphase-promoting complex/cyclosome-Cdc-20 promotes Zwint-1 degradation.

ZW10 interactor (Zwint-1) is an important component of the centromere and can recruit the dynamic protein kinase and dynein to promote chromosome movement and regulate the spindle assembly checkpoint (SAC). Zwint-1 activity is tightly regulated during the cell cycle. However, how the stability of Zwint-1 is regulated has not been clarified. Here, we show that the relative levels of Zwint-1 expression gradually decreased with the progression of cell cycling and decline sharply during mitotic exit. Treatment with cycloheximide reduced the levels of Zwint-1 while treatment with MG132 to inhibit endogenous ubiquitin-proteasome elevated the levels of Zwint-1 in HEK293T cells or Hela cells. Such data suggest that Zwint-1 may be degraded by endogenous ubiquitin-proteasome. Furthermore, induction of cell-division cycle protein 20 (Cdc20) overexpression decreased the levels of Zwint-1, which was abrogated by MG132 treatment. In contrast, Cdc20 silencing promoted the accumulation of Zwint-1. in vivo ubiquitination assay revealed that Cdc20 promoted the formation of Zwint-1 and ubiquitin-proteasome conjugates. Cotransfection with Cdc20 and wild-type Zwint-1, but not Zwint-1ΔD-box , reduced the levels of Zwint-1. Immunoprecipitation and western blot analyses showed that Cdc20 interacted with wild-type Zwint-1, but not Zwint-1ΔD-box although both Zwint-1 and Zwint-1ΔD-box overexpression did not induce mitotic arrest. Collectively, our data indicated that Zwint-1 was ubiquitinated by anaphase-promoting complex/cyclosome (APC/C)-Cdc20 in a D-box-dependent manner. Therefore, the APC/C-Cdc20 controls the stability of Zwint-1, ensuring accurate regulation of the spindle assembly during the cell cycling in HEK293T cells.

Proteasome inhibition creates a chromatin landscape favorable to RNA Pol II processivity

Proteasome inhibition creates a chromatin landscape favorable to RNA Pol II processivity

Melatonin influences the expression and oligomerization of amylin in rat INS-1E cells.

The present study investigated whether melatonin influences the expression/oligomerization of amylin with endoplasmic reticulum (ER) stress in rat insulinoma INS-1E cells. No change in cell survival after exposure to thapsigargin- and tunicamycin-combined melatonin treatment or melatonin-only treatment was observed when compared with the normal control cells. With thapsigargin-only or combined tunicamycin-melatonin treatments, phosphorylation of extracellular signal-regulated kinase (ERK) was significantly increased compared with control and melatonin-only treatments. A significant increase was observed in the levels of ER stress markers, namely, phosphorylated inositol-requiring protein 1α (p-IRE1α), CCAAT enhancer binding proteins (C/EBP)-homologous protein, p-eukaryotic translation initiation factor 2α and cleaved caspase-12, in the thapsigargin-combined melatonin-treated cells as compared with the tunicamycin-combined or only melatonin treatment. The melatonin-only treatment resulted in increased levels of amylin expression/oligomerization in 15-25 kDa and insulin proteins, compared with the thapsigargin- and tunicamycin-combined melatonin treatments. Treatment with ER stress inhibitor 4-phenylbutyric acid (4-PBA) did not suppress amylin expression/oligomerization or insulin production with thapsigargin or tunicamycin treatment. Levels of cleaved caspase-12 were significantly decreased in the thapsigargin- or tunicamycin-4-PBA combination treatments. Therefore, whether melatonin regulates the amylin expression/oligomerization in thapsigargin- or tunicamycin-combined with Bafilomycin A1 (autophagy inhibitor) or MG132 (proteasome inhibitor) treatments were investigated. Amylin expression/oligomerization with melatonin treatment was significantly decreased in the thapsigargin- or tunicamycin-combined Bafilomycin A1 or MG132 treatments. Since these outcomes are involved in cell viability, they indicate that increased cell death leads to decreased amylin expression/oligomerization, however, the effects of melatonin treatment on amylin expression/oligomerization induce proliferation of pancreatic β cells and improve the cellular functions of pancreatic β cells.

Dynamic Interaction of USP14 with the Chaperone HSC70 Mediates Crosstalk between the Proteasome, ER Signaling, and Autophagy.

SummaryUSP14 is a deubiquitinating enzyme associated with the proteasome important for protein degradation. Here we show that upon proteasome inhibition or expression of the mutant W58A-USP14, association of USP14 with the 19S regulatory particle is disrupted. MS-based interactomics revealed an interaction of USP14 with the chaperone, HSC70, in neuroblastoma cells. Proteasome inhibition enhanced binding of USP14 to HSC70, and to XBP1u and IRE1α proteins, demonstrating a role in the unfolded protein response. Striatal neurons expressing mutant huntingtin exhibited reduced USP14 and HSC70 levels, whereas inhibition of HSC70 downregulated USP14. Furthermore, proteasome inhibition or use of the mutant W58A-USP14 facilitated the interaction of USP14 with the autophagy protein, GABARAP. Functionally, overexpression of W58A-USP14 increased GABARAP positive autophagosomes in striatal neurons, and this was abrogated using the HSC70 inhibitor, VER-155008. Modulation of the USP14-HSC70 axis may represent a potential therapeutic target in HD to beneficially influence multiple proteostasis pathways.

Genome-Wide Small Interfering RNA Screening Reveals a Role for Cullin3-Really Interesting New Gene Ligase Signaling in Heterologous Sensitization of Adenylyl Cyclase.

Heterologous sensitization of adenylyl cyclase (AC) is revealed as enhanced or exaggerated AC/cAMP signaling that occurs following persistent activation of Gα i/o-coupled receptors. This paradoxical phenomenon was discovered more than 40 years ago and was proposed as a cellular mechanism to explain the adaptive changes that occur following chronic exposure to drugs of abuse. However, the underlying molecular mechanisms of heterologous sensitization of AC remain largely unknown. In the present study, we performed a genome-wide cell-based RNA interference screen as an unbiased approach to identify genes associated with heterologous sensitization of AC. Following a series of validation and confirmation assays, three genes that form an E3 ligase complex, cullin3 (CUL3), neural precursor-cell-expressed and developmentally downregulated 8 (NEDD8), and really interesting new gene (RING)-box protein 1 (RBX1), were identified as specific modulators of heterologous sensitization of AC. Furthermore, based on the downstream actions of these genes, we evaluated the activity of proteasome inhibitors as well as the specific NEDD8-activating enzyme inhibitor, MLN4924 (Pevonedistat), in AC sensitization. We demonstrate that MG-132 and bortezomib treatments could mimic the inhibitory effects observed with gene knockdown, and MLN4924 was potent and efficacious in blocking the development of heterologous sensitization of endogenous and recombinant AC isoforms, including AC1, AC2, AC5, and AC6. Together, by using genetic and pharmacological approaches, we identified, for the first time, cullin3-RING ligases and the protein degradation pathway as essential modulators for heterologous sensitization of AC. SIGNIFICANCE STATEMENT: Through a genome-wide cell-based RNA interference screening, we identified three genes that form an E3 ligase complex, cullin3, neural precursor-cell-expressed and developmentally downregulated 8 (NEDD8), and really interesting new gene-box protein 1, as specific modulators of heterologous sensitization of AC. The effect of cullin3, NEDD8, or really interesting new gene-box protein 1 small interfering RNAs on heterologous sensitization was recapitulated by proteasome inhibitors, MG132 and bortezomib, and the specific NEDD8-activating enzyme inhibitor, MLN4924. These results suggest a novel hypothesis in which protein degradation is involved in the sensitization of AC signaling that occurs following chronic activation of Gαi/o-coupled receptors.

NQO1 potentiates apoptosis evasion and upregulates XIAP via inhibiting proteasome-mediated degradation SIRT6 in hepatocellular carcinoma

BackgroundOur previous study has demonstrated that NAD(P)H: quinone oxidoreductase 1 (NQO1) is significantly upregulated in human liver cancer where it potentiates the apoptosis evasion of liver cancer cell. However, the underlying mechanisms of the oncogenic function of NQO1 in HCC have not been fully elucidated.MethodsExpression of NQO1, SIRT6, AKT and X-linked inhibitor of apoptosis protein (XIAP) protein were measured by western blotting and immunohistochemistry. Additionally, the interaction between NQO1 and potential proteins were determined by immunoprecipitation assays. Furthermore, the effect of NQO1 and SIRT6 on tumor growth was determined in cell model and orthotopic tumor implantation model.ResultsWe found that NQO1 overexpression in HCC enhanced SIRT6 protein stability via inhibiting ubiquitin-mediated 26S proteasome degradation. High level of SIRT6 reduced acetylation of AKT which resulted in increased phosphorylation and activity of AKT. Activated AKT subsequently phosphorylated anti-apoptotic protein XIAP at Ser87 which determined its protein stability. Reintroduction of SIRT6 or AKT efficiently rescued NQO1 knock-out-mediated inhibition of growth and induction of apoptosis. In orthotopic mouse model, NQO1 knock-out inhibited tumor growth and induced apoptosis while this effect was effectively rescued by SIRT6 overexpression or MG132 treatment partially.ConclusionsCollectively, these results reveal an oncogenic function of NQO1 in sustaining HCC cell proliferation through SIRT6/AKT/XIAP signaling pathway.

Proteasome inhibitor MG132 suppresses pancreatic ductal adenocarcinoma-cell migration by increasing ESE3 expression.

The clinical significance of the proteasome inhibitor MG132 has been examined in numerous human cancer types; however, its influence on the metastasis and progression of pancreatic cancer is yet to be determined. In the present study, the effect of MG132 treatment on pancreatic ductal adenocarcinoma (PDAC) cell lines (SW1990 and PANC-1) was examined. Compared with the control groups, MG132 treatment resulted in higher expression levels of ETS homologous factor (ESE3), a crucial member of the E26 transformation-specific family that is central to various differentiation and development processes in epithelial tissues. MG132 treatment also increased the nuclear translocation of ESE3. Mechanistically, MG132 further inhibited the invasion and migration of PDAC cells by promoting E-cadherin expression, which not only plays an important role in cell-cell adhesion, but is also a direct target of ESE3. Furthermore, subsequent knockdown experiments, using short interfering RNAs, demonstrated that MG132 upregulated E-cadherin via an increase in ESE3 expression. The results of the present study support the hypothesis that MG132 treatment inhibits PDAC metastasis, highlighting the potential of MG132 as a therapeutic agent for the treatment of patients with PDAC.

SUMOylation and deacetylation affect NF-κB p65 activity induced by high glucose in human lens epithelial cells.

To explore the effects of IκBα SUMOylation and NF-κB p65 deacetylation on NF-κB p65 activity induced by high glucose in cultured human lens epithelial cells (HLECs). HLECs (SRA01/04) were cultured with 5.5, 25, and 50 mmol/L glucose media for 24h, and with 50 mmol/L glucose media for 0, 12, and 24h respectively. SUMO1 and SIRT1 expressions were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot (WB). IκBα and NF-κB p65 expressions were detected by WB. With NAC, DTT, MG132 or Resveratrol (RSV) treatment, SUMO1 and SIRT1 expressions were detected by WB. Protein expression localizations were examined by immunofluorescence and co-immunofluorescence. The effects of SUMO1 or SIRT1 overexpression, as well as MG132 and RSV, on the nuclear expression and activity of IκBα and NF-κB p65 were analyzed by immunoblot and dual luciferase reporter gene assay. SUMO1 and SIRT1 expressions were influenced by high glucose in mRNA and protein levels, which could be blocked by NAC or DTT. SUMO1 was down-regulated by using MG132, and SIRT1 was up-regulated under RSV treatment. IκBα nuclear expression was attenuated and NF-κB p65 was opposite under high glucose, while IκBα and NF-κB p65 location was transferred to the nucleus. SUMO1 or SIRT1 overexpression and MG132 or RSV treatment affected the nuclear expression and activity of IκBα and NF-κB p65 under high glucose condition. IκBα SUMOylation and NF-κB p65 deacetylation affect NF-κB p65 activity in cultured HLECs under high glucose, and presumably play a significant role in controlling diabetic cataract.

Expression of heterogeneous nuclear ribonucleo-protein A2B1 in mouse cerebellar development and human medulloblastoma

Objective: To investigate the expression and potential role of heterogeneous nuclear ribonucleo-protein A2B1 (HNRNPA2B1) in mouse cerebellar development and the significance of HNRNPA2B1 in human medulloblastoma. Methods: The data of HNRNPA2B1 RNA expression in mouse and human cerebella were obtained from databases. Western blot and immunohistochemical staining were performed to detect the protein level of HNRNPA2B1 in mouse cerebella at different ages. The expression level of HNRNPA2B1 in control human cerebellum and medulloblastoma was detected by immunohistochemical staining. m6A-IP-qPCR method was applied to confirm whether HNRNPA2B1 RNA in Daoy cells was modified with m6A.Western blot was used to detect the effect of MG132 treatment on the HNRNPA2B1 protein level in Daoy cells. Results: The level of HNRNPA2B1 protein in postnatal mouse cerebella was higher than that in adult mouse cerebella, with weak HNRNPA2B1 staining in external granular cells while strong staining in mature Purkinje cells and molecular layer. Compared with control normal human cerebella, the RNA expression level of HNRNPA2B1 increased in medulloblastoma, while immunohistochemical staining showed that the mean intensity of HNRNPA2B1 decreased in medulloblastoma. HNRNPA2B1 RNA in medulloblastoma and Daoy cells was modified by m6A. The HNRNPA2B1 protein level in Daoy cells increased upon MG132 treatment. Conclusions: HNRNPA2B1 is dynamically expressed during mouse cerebellar development. Compared with normal human cerebella, HNRNPA2B1 is significantly up-regulated at transcriptional level but obviously down-regulated at translational level in medulloblastoma. These results indicate that HNRNPA2B1 may be involved in cerebellar development process and medulloblastoma tumorigenesis. The m6A methylation in HNRNPA2B1 transcript and protein ubiquitin-proteasome pathway may account for the down-regulation of HNRNPA2B1 at protein level.

Proteasome inhibition suppress microgravity elevated RANK signaling during osteoclast differentiation

Microgravity (µXg) induces bone loss in astronauts during space missions. Therefore, it is necessary to delineate the underlying mechanisms which leads to bone loss for developing countermeasures. Osteoclasts (OCLs) are multinucleated cells, which resorb bone. Previously, we have demonstrated that simulated µXg enhances OCL formation. However, control of osteoclast bone resorption activity under μXg remains unclear. The OCL formation has been shown to be regulated by ubiquitin–proteasome pathway. Hence, we hypothesized that proteasome inhibition could regulate osteoclast differentiation under μXg. In this study, we identified that RAW264.7 preosteoclast cells treated with proteasome inhibitor (MG-132) suppress RANK receptor expression essential for OCL differentiation, but no effect on TRAF-6. We identified that MG-132 treatment abolished K48-linked poly-ubiquitination under μXg. Immunostaining confirms inhibition of protein ubiquitination and RANK expression in preosteoclast cells. Furthermore, proteasome inhibition suppresses the expression of SQSTM1/p62 under both the ground based Xg and μXg conditions. Also, confocal microscopy using Lyso-Tracker demonstrated that proteasomal inhibition suppress the co-localization of p62 and lysosomes. MG-132 inhibited RANKL induced proteasome activity. RAW264.7 cells treated with the proteasome inhibitor showed an increased level of p-c-Jun activity in control cultures, however decreased under µXg. In contrast, c-Fos and NFATc1 expression was decreased. In-addition, mouse bone marrow cultures treated with MG-132 suppress OCL formation and bone resorption activity. Thus, our findings suggest that proteasome inhibition represents a novel therapeutic approach for bone loss under µXg in space environment.

Proteasome serves as pivotal regulator in Angiostrongylus cantonensis-induced eosinophilic meningoencephalitis.

Proteasome primarily degrades the unneeded or damaged proteins by proteolysis. Disruption of the brain barrier and its resulting meningoencephalitis caused by Angiostrongylus cantonensis are important pathological events in non-permissive hosts. In this study, the results showed upregulated proteasome during A. cantonensis infection. Occludin degradation and matrix metalloproteinase-9 (MMP-9) activity were significantly increased in infected mice than in uninfected mice. Moreover, confocal immunoflourescence microscopy showed that occludin was co-localized with MMP-9. The infected-mice were treated with proteasomal activity inhibitor MG132 by 1.5 and 3.0 mg/kg/day, which resulted in significantly reduced protein levels of phosphorylated IκBα (P<0.05) compared with the untreated control. The phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) showed similar result. In addition, MMP-9 activity and occludin degradation were reduced because of MG132 treatment. These results suggested that the proteasome in A. cantonensis infection degraded phosphorylated IκBα, modulated phosphorylated NF-κB, and then regulated the activation of MMP-9 and occludin degradation. Proteasome alterations were presented in eosinophilic meningitis of BALB/c mice and may contribute to the pathophysiology of eosinophilic meningitis by increasing occludin degradation. This molecule would serve as pivotal regulator in A. cantonensis-induced eosinophilic meningoencephalitis.

Down-regulation effects of IFN-α on p11, 5-htr1b and 5-HTR4 protein levels were affected by NH4CL or MG132 treatment in SH-sy5y cells

In previous studies, we found interferon-α (IFN-α) could reduce protein levels of p11, 5-hydroxytryptamine receptor 1b (5-HT1b) and 5-hydroxytryptamine receptor 4 (5-HT4), but does not influence their messenger RNA levels in SH-sy5y cells. Thus, we investigated the post-transcriptional modulation of these molecules by IFN-α. SH-sy5y cells were treated with IFN-α, NH4Cl or MG132 alone or in combination, and then the protein levels of p11, 5-HT1b and 5-HT4 were analyzed by western blots. The regulatory effects of p11 on 5-HT1b and 5-HT4 were also determined in p11 knock-down cells. NH4Cl but not MG132 could reverse the protein level of p11 in IFN-α-treated SH-sy5y cells. MG132 could recover the protein levels of 5-HT1b and 5-HT4 in p11 knock-down cells. The down-regulation effects of IFN-α on p11, 5-HT1b and 5-HT4 were associated with the lysosome and ubiquitin-proteasome-mediated pathways. p11 was identified as a potent regulator to modulate the ubiquitination of 5-HT1b and 5-HT4. Therefore, it could be potential target therapies in IFN-ainduced depression.

Abstract 800: HECT Domain Containing-3 (HECTD3) is a Novel Regulator of Cardiac Hypertrophy and Inflammation

Objective: In this study, using a Yeast two-hybrid screen, we identified the HECT domain-containing E3 ubiquitin ligase 3 (HECTD3) as one of the novel cardiac binding partners of SUMO2. Present study thus focusses on deciphering the cardiac function of HECTD3. Methods and Results: HECTD3-SUMO2 interaction was further validated by co-immunoprecipitation analysis. Interesting, adenovirus-mediated overexpression of HECTD3 in neonatal rat ventricular cardiomyocytes (NRVCMs) resulted in downregulation of endogenous as well as overexpressed SUMO2. Proteasome inhibitor MG132 treatment however inhibited HECTD3-mediated SUMO2 downregulation, suggesting SUMO2 as a likely substrate of HECTD3 in ubiquitin-proteasome system dependent protein degradation. To get further insights into cardiac substrates of HECTD3, we performed mass-spectrometry analysis of NRVCM protein lysate. Interestingly, several proteins from interferon signaling, including signal transducer and activator of transcription-1 (STAT1), were significantly downregulated by HECTD3 overexpression. Functional dissection of HECTD3-SUMO2-STAT1 interaction revealed attenuation of cellular hypertrophy and LPS mediated activation of inflammatory cascade in NRVCMs. Importantly, in vitro findings were consistent in vivo , where, AAV-9 mediated overexpression of HECTD3 substantially reduced cardiac hypertrophy and fibrosis, inhibited activation of STAT1-mediated downstream signaling, and attenuated infiltration of inflammatory cells to the heart after transverse-aortic constriction or Angiotensin II treatment. Conclusions: In conclusion, we describe here a novel cardioprotective mechanism involving the ubiquitin ligase HECTD3, which exerts anti-hypertrophic and anti-inflammatory effects via dual regulation of SUMO2 and its sumoylation target STAT1. This “dual pathway” inhibition may be exploited for the prevention and/or therapy of cardiac hypertrophy and heart failure.

Increased clusterin levels after myocardial infarction is due to a defect in protein degradation systems activity

Clusterin (CLU) is induced in many organs after tissue injury or remodeling. Recently, we show that CLU levels are increased in plasma and left ventricle (LV) after MI, however, the mechanisms involved are not yet elucidated. On the other hand, it has been shown that the activity of the protein degradation systems (PDS) is affected after MI with a decrease in ubiquitin proteasome system (UPS) and an increase in macroautophagy. The aim of this study was to decipher if the increased CLU levels after MI are in part due to the alteration of PDS activity. Rat neonate cardiomyocytes (NCM) were treated with different modulators of UPS and macroautophagy in order to decipher their role in CLU expression, secretion, and degradation. We observed that inhibition of UPS activity in NCM increased CLU mRNA levels, its intracellular protein levels (p-CLU and m-CLU) and its secreted form (s-CLU). Macroautophagy was also induced after MG132 treatment but is not active. The inhibition of macroautophagy induction in MG132-treated NCM increased CLU mRNA and m-CLU levels, but not s-CLU compared to NCM only treated by MG132. We also demonstrate that CLU can be degraded in NCM through proteasome and lysosome by a macroautophagy independent pathway. In another hand, CLU silencing in NCM has no effect either on macroautophagy or apoptosis induced by MG132. However, the overexpression of CLU secreted isoform in H9c2 cells, but not in NCM decreased apoptosis after MG132 treatment. Finally, we observed that increased CLU levels in hypertrophied NCM and in failing human hearts are associated with proteasome inhibition and macroautophagy alteration. All these data suggest that increased CLU expression and secretion after MI is, in part, due to a defect of UPS and macroautophagy activities in the heart and may have a protective effect by decreasing apoptosis induced by proteasome inhibition.

Cullin-4B E3 ubiquitin ligase mediates Apaf-1 ubiquitination to regulate caspase-9 activity.

Apoptotic protease-activating factor 1 (Apaf-1) is a component of apoptosome, which regulates caspase-9 activity. In addition to apoptosis, Apaf-1 plays critical roles in the intra-S-phase checkpoint; therefore, impaired expression of Apaf-1 has been demonstrated in chemotherapy-resistant malignant melanoma and nuclear translocation of Apaf-1 has represented a favorable prognosis of patients with non-small cell lung cancer. In contrast, increased levels of Apaf-1 protein are observed in the brain in Huntington’s disease. The regulation of Apaf-1 protein is not yet fully understood. In this study, we show that etoposide triggers the interaction of Apaf-1 with Cullin-4B, resulting in enhanced Apaf-1 ubiquitination. Ubiquitinated Apaf-1, which was degraded in healthy cells, binds p62 and forms aggregates in the cytosol. This complex of ubiquitinated Apaf-1 and p62 induces caspase-9 activation following MG132 treatment of HEK293T cells that stably express bcl-xl. These results show that ubiquitinated Apaf-1 may activate caspase-9 under conditions of proteasome impairment.

Ubiquitination status does not affect Vps34 degradation

Vps34 (vacuolar protein-sorting 34) plays important role in autophagy and endosomal trafficking. These processes are closely associated protein ubiquitination and degradation. We have hypothesized that Vps34 ubiquitination status would also control its degradation. Here, we report that our results did not support this assumption. In cells transiently transfected with ubiquitin (UB) constructs contained different lysine residues (Ks), Vps34 ubiquitination could occur regardless of the presence of any Ks in UB. However, Vps34 protein levels were not significantly altered in cells transiently transfected with these UB mutants. We further found that Vps34 protein was altered by pharmacological manipulation of E2/E3 activity; yet this effect was not significantly affected by UB overexpression. In vivo experiments revealed that in APP/PS1 mice, an animal model of Alzheimer's disease (AD), although ubiquitination of Vps34 was significantly reduced, Vps34 protein levels remained unchanged. Vps34 indeed was subjected to proteasomal or lysosomal degradation, as prolonged treatment of proteasomal inhibitor MG132 or lysosomal inhibitor chloroquine elevated Vps34 protein levels. We conclude that unlike most of other proteins, Vps34 ubiquitination is not closely associated with its degradation.

Oxymatrine ameliorates agomelatine-induced hepatocyte injury through promoting proteasome-mediated CHOP degradation

BackgroundThe novel antidepressant drug agomelatine has been observed to cause adverse effect of hepatotoxicity in clinical applications. This study was designed to explore protective agents and investigated the underlying mechanism on L02 cells. MethodL02 cells were treated with agomelatine and oxymatrine (OMT) and cell apoptosis were analyzed through flow cytometric analysis, CCK-8 assay and TUNEL assay. In a separate experiment, the expressions of ER stress-related proteins were determined by western blot. In addition, MG132, chloroquine (CQ) and bafilomycinA1(BafA1) were used to investigate the potential pathway participating in CHOP degradation. ResultsOMT significantly rescued agomelatine-induced hepatocyte apoptosis. Agomelatine treatment resulted in accumulation of CHOP protein in L02 cells, and this phenomenon could be significantly reduced by OMT, whereas abolished by MG132 treatment. ConclusionWe have demonstrated for the first time that OMT ameliorates the hepatocyte toxicity induced by agomelatine through decreasing CHOP on protein level. The underlying mechanism was proved to involve the molecular events that OMT promotes CHOP degradation via proteasome pathway. Overall, these results suggest that using OMT in combination with agomelatine may provide a safety strategy for clinical depression treatment.