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Showing papers on "MG132 published in 2019"


Journal ArticleDOI
TL;DR: Evidence is provided that considerable levels of p62-mediated selective autophagy are spontaneously induced, and correlate with ROS-Keap1-NRF2 pathway activity, in virus-transformed cells, and a novel insight into virus-mediated oncogenesis is provided.
Abstract: DNA damage response (DDR) and selective autophagy both can be activated by reactive oxygen/nitrogen species (ROS/RNS), and both are of paramount importance in cancer development. The selective autophagy receptor and ubiquitin (Ub) sensor p62 plays a key role in their crosstalk. ROS production has been well documented in latent infection of oncogenic viruses including Epstein-Barr Virus (EBV). However, p62-mediated selective autophagy and its interplay with DDR have not been investigated in these settings. In this study, we provide evidence that considerable levels of p62-mediated selective autophagy are spontaneously induced, and correlate with ROS-Keap1-NRF2 pathway activity, in virus-transformed cells. Inhibition of autophagy results in p62 accumulation in the nucleus, and promotes ROS-induced DNA damage and cell death, as well as downregulates the DNA repair proteins CHK1 and RAD51. In contrast, MG132-mediated proteasome inhibition, which induces rigorous autophagy, promotes p62 degradation but accumulation of the DNA repair proteins CHK1 and RAD51. However, pretreatment with an autophagy inhibitor offsets the effects of MG132 on CHK1 and RAD51 levels. These findings imply that p62 accumulation in the nucleus in response to autophagy inhibition promotes proteasome-mediated CHK1 and RAD51 protein instability. This claim is further supported by the findings that transient expression of a p62 mutant, which is constitutively localized in the nucleus, in B cell lines with low endogenous p62 levels recaptures the effects of autophagy inhibition on CHK1 and RAD51 protein stability. These results indicate that proteasomal degradation of RAD51 and CHK1 is dependent on p62 accumulation in the nucleus. However, small hairpin RNA (shRNA)-mediated p62 depletion in EBV-transformed lymphoblastic cell lines (LCLs) had no apparent effects on the protein levels of CHK1 and RAD51, likely due to the constitutive localization of p62 in the cytoplasm and incomplete knockdown is insufficient to manifest its nuclear effects on these proteins. Rather, shRNA-mediated p62 depletion in EBV-transformed LCLs results in significant increases of endogenous RNF168-γH2AX damage foci and chromatin ubiquitination, indicative of activation of RNF168-mediated DNA repair mechanisms. Our results have unveiled a pivotal role for p62-mediated selective autophagy that governs DDR in the setting of oncogenic virus latent infection, and provide a novel insight into virus-mediated oncogenesis.

50 citations


Journal ArticleDOI
15 Jan 2019-Oncogene
TL;DR: Downregulation of Agr2 by proteasome inhibition significantly enhanced antitumor activity of bevacizumab, highlighting the importance of AGR2 as a predictive marker for selection of subgroup patients in chemotherapy.
Abstract: Anterior gradient 2 (AGR2), a protein belonging to the protein disulfide isomerase (PDI) family, is overexpressed in multiple cancers and promotes angiogenesis to drive cancer progression. The mechanisms controlling AGR2 abundance in cancer remain largely unknown. Here, we observed that AGR2 expression is significantly suppressed by proteasome inhibitor MG132/bortezomib at mRNA and protein levels in lung cancer cells. MG132-mediated repression of AGR2 transcription was independent of ROS generation and ER stress induction, but partially resulted from the downregulated E2F1. Further investigation revealed that MG132 facilitated polyubiquitinated AGR2 degradation through activation of autophagy, as evidenced by predominant restoration of AGR2 level in cells genetic depletion of Atg5 and Atg7, or by autophagy inhibitors. Activation of autophagy by rapamycin noticeably reduced the AGR2 protein in cells and in the mouse tissue samples administrated with bortezomib. We also provided evidence identifying the K48-linked polyubiquitin chains conjugating onto K89 of AGR2 by an E3 ligase UBR5. In addition, an autophagy receptor NBR1 was demonstrated to be important in polyubiquitinated AGR2 clearance in response to MG132 or bortezomib. Importantly, downregulation of AGR2 by proteasome inhibition significantly enhanced antitumor activity of bevacizumab, highlighting the importance of AGR2 as a predictive marker for selection of subgroup patients in chemotherapy.

47 citations


Journal ArticleDOI
TL;DR: An oncogenic function of NQO1 in sustaining HCC cell proliferation through SIRT6/AKT/XIAP signaling pathway is revealed through inhibiting ubiquitin-mediated 26S proteasome degradation.
Abstract: Our 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. Expression 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. We 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. Collectively, these results reveal an oncogenic function of NQO1 in sustaining HCC cell proliferation through SIRT6/AKT/XIAP signaling pathway.

34 citations


Journal ArticleDOI
TL;DR: Findings indicate that MCPIP1 plays a protective role in sepsis-induced ALI by modulating macrophage polarization through inhibition of the JNK/c-Myc signaling pathway.

30 citations


Journal ArticleDOI
22 Jul 2019-Oncogene
TL;DR: It is shown that SOX7 was significantly downregulated in different cancer types, especially in lung and breast cancers, and Panobinostat, an FDA approved pan-HDAC inhibitor, could elevate and restore SoX7 expression in SOX6 silenced lung cancer cells.
Abstract: Apoptosis of cancer cells occurs by a complex gene regulatory network. Here we showed that SOX7 was significantly downregulated in different cancer types, especially in lung and breast cancers. Low expression of SOX7 was associated with advantage stage of cancer with shorter overall survival. Cancer cells with loss of SOX7 promoted cell survival and colony formation, suppressed cellular apoptosis and produced a drug resistant phenotype against a variety of chemo/targeting therapeutic agents. Mechanistically, SOX7 induced cellular apoptosis through upregulation of genes associated with both P38 and apoptotic signaling pathway, as well as preventing the proteasome mediated degradation of pro-apoptotic protein BIM. Treatment of either a proteasome inhibitor MG132 or bortezomib, or with a p-ERK/MEK inhibitor U0126 attenuate the SOX7 promoted BIM degradation. We identified Panobinostat, an FDA approved pan-HDAC inhibitor, could elevate and restore SOX7 expression in SOX7 silenced lung cancer cells. Taken together, these data revealed an unappreciated role of SOX7 in regulation of cellular apoptosis through control of MAPK/ERK-BIM signaling.

29 citations


Journal ArticleDOI
TL;DR: TQ was found to be a novel HIF-1α inhibitor through hypoxia response element (HRE)-luciferase assay-based large screening by using 502 natural compounds containing chemical library, and it was suggested that TQ is a potential anticancer agent targeting Hif-1 α.
Abstract: Several reports have shown that thymoquinone (TQ) effectively attenuates angiogenesis in cancer cells, resulting in suppression of tumor growth. However, it is not yet clear whether TQ reduces hypoxia-inducible factor-1α (HIF-1α) expression in hypoxic cancer cells. Here, we found that TQ was a novel HIF-1α inhibitor through hypoxia response element (HRE)-luciferase assay-based large screening by using 502 natural compounds containing chemical library. TQ reduced HIF-1α protein levels in renal cancer cells; however, it did not affect the HIF-1α protein levels in the presence of proteasome inhibitor, MG132, indicating that the reduction effects of TQ on HIF-1α protein are mediated via the ubiquitination-proteasome dependent pathway. TQ boosted HIF-1α protein degradation, and the mechanism was revealed by inhibiting interaction between HSP90 and HIF-1α. TQ suppressed downstream genes of HIF-1α, indicating negative impact of TQ on HIF-1α transcriptional activities. In addition, TQ altered glucose, lactate, and ATP levels, leading to anaerobic metabolic disturbance. TQ induced apoptosis in hypoxic cancer cells as determined by crystal violet staining and flow cytometry for annexin V-stained cells. Taken together, we suggested that TQ is a potential anticancer agent targeting HIF-1α.

28 citations


Journal ArticleDOI
13 Jun 2019-Cells
TL;DR: Investigation of the functional consequences of inhibiting the UPS and UBA1 on motor neurons highlights the important regulatory role of the UPS in pluripotent stem cell survival and motor neuron differentiation.
Abstract: The ubiquitin proteasome system (UPS) plays an important role in regulating numerous cellular processes, and a dysfunctional UPS is thought to contribute to motor neuron disease. Consequently, we sought to map the changing ubiquitome in human iPSCs during their pluripotent stage and following differentiation to motor neurons. Ubiquitinomics analysis identified that spliceosomal and ribosomal proteins were more ubiquitylated in pluripotent stem cells, whilst proteins involved in fatty acid metabolism and the cytoskeleton were specifically ubiquitylated in the motor neurons. The UPS regulator, ubiquitin-like modifier activating enzyme 1 (UBA1), was increased 36-fold in the ubiquitome of motor neurons compared to pluripotent stem cells. Thus, we further investigated the functional consequences of inhibiting the UPS and UBA1 on motor neurons. The proteasome inhibitor MG132, or the UBA1-specific inhibitor PYR41, significantly decreased the viability of motor neurons. Consistent with a role of the UPS in maintaining the cytoskeleton and regulating motor neuron differentiation, UBA1 inhibition also reduced neurite length. Pluripotent stem cells were extremely sensitive to MG132, showing toxicity at nanomolar concentrations. The motor neurons were more resilient to MG132 than pluripotent stem cells but demonstrated higher sensitivity than fibroblasts. Together, this data highlights the important regulatory role of the UPS in pluripotent stem cell survival and motor neuron differentiation.

28 citations


Journal ArticleDOI
04 Feb 2019-PLOS ONE
TL;DR: Strong evidences are provided that the IAPa Debio 1143, by initially activating the non-canonical NF-kB signaling and subsequently reactivating HIV-1 transcription, represents a new attractive viral latency reversal agent (LRA).
Abstract: Antiretroviral therapy (ART) suppresses HIV replication, but does not cure the infection because replication-competent virus persists within latently infected CD4+ T cells throughout years of therapy. These reservoirs contain integrated HIV-1 genomes and can resupply active virus. Thus, the development of strategies to eliminate the reservoir of latently infected cells is a research priority of global significance. In this study, we tested efficacy of a new inhibitor of apoptosis protein antagonist (IAPa) called Debio 1143 at reversing HIV latency and investigated its mechanisms of action. Debio 1143 activates HIV transcription via NF-kB signaling by degrading the ubiquitin ligase baculoviral IAP repeat-containing 2 (BIRC2), a repressor of the non-canonical NF-kB pathway. Debio 1143-induced BIRC2 degradation results in the accumulation of NF-κB-inducing kinase (NIK) and proteolytic cleavage of p100 into p52, leading to nuclear translocation of p52 and RELB. Debio 1143 greatly enhances the binding of RELB to the HIV-1 LTR. These data indicate that Debio 1143 activates the non-canonical NF-kB signaling pathway by promoting the binding of RELB:p52 complexes to the HIV-1 LTR, resulting in the activation of the LTR-dependent HIV-1 transcription. Importantly, Debio 1143 reverses viral latency in HIV-1 latent T cell lines. Using knockdown (siRNA BIRC2), knockout (CRIPSR NIK) and proteasome machinery neutralization (MG132) approaches, we found that Debio 1143-mediated HIV latency reversal is BIRC2 degradation- and NIK stabilization-dependent. Debio 1143 also reverses HIV-1 latency in resting CD4+ T cells derived from ART-treated patients or HIV-1-infected humanized mice under ART. Interestingly, daily oral administration of Debio 1143 in cancer patients at well-tolerated doses elicited BIRC2 target engagement in PBMCs and induced a moderate increase in cytokines and chemokines mechanistically related to NF-kB signaling. In conclusion, we provide strong evidences that the IAPa Debio 1143, by initially activating the non-canonical NF-kB signaling and subsequently reactivating HIV-1 transcription, represents a new attractive viral latency reversal agent (LRA).

23 citations


Journal ArticleDOI
TL;DR: Direct evidence of the proteasome's importance to schistosome viability is provided and a lead is identified for which future studies will aim to improve the potency, selectivity and safety.
Abstract: Proteases are fundamental to successful parasitism, including that of the schistosome flatworm parasite, which causes the disease schistosomiasis in 200 million people worldwide. The proteasome is receiving attention as a potential drug target for treatment of a variety of infectious parasitic diseases, but it has been understudied in the schistosome. Adult Schistosoma mansoni were incubated with 1 μM concentrations of the proteasome inhibitors bortezomib, carfilzomib, and MG132. After 24 h, bortezomib and carfilzomib decreased worm motility by more than 85% and endogenous proteasome activity by >75%, and after 72 h, they increased caspase activity by >4.5-fold. The association between the engagement of the proteasome target and the phenotypic and biochemical effects recorded encouraged the chromatographic enrichment of the S. mansoni proteasome (Sm20S). Activity assays with fluorogenic proteasome substrates revealed that Sm20S contains caspase-type (β1), trypsin-type (β2), and chymotrypsin-type (β5) activities. Sm20S was screened with 11 peptide epoxyketone inhibitors derived from the marine natural product carmaphycin B. Analogue 17 was 27.4-fold less cytotoxic to HepG2 cells than carmaphycin B and showed equal potency for the β5 subunits of Sm20S, human constitutive proteasome, and human immunoproteasome. However, this analogue was 13.2-fold more potent at targeting Sm20S β2 than it was at targeting the equivalent subunits of the human enzymes. Furthermore, 1 μM 17 decreased both worm motility and endogenous Sm20S activity by more than 90% after 24 h. We provide direct evidence of the proteasome's importance to schistosome viability and identify a lead for which future studies will aim to improve the potency, selectivity, and safety.

23 citations


Journal ArticleDOI
14 May 2019-Mbio
TL;DR: Bortzomib is a novel potential therapeutic for HSV with a defined mechanism of action and the combination of bortezomib with acyclovir demonstrated synergistic inhibitory effects on HSV infection.
Abstract: Viruses commandeer host cell 26S proteasome activity to promote viral entry, gene expression, replication, assembly, and egress. Proteasomal degradation activity is critical for herpes simplex virus (HSV) infection. The proteasome inhibitor bortezomib (also known as Velcade and PS-341) is a clinically effective antineoplastic drug that is FDA approved for treatment of hematologic malignancies such as multiple myeloma and mantle cell lymphoma. Low nanomolar concentrations of bortezomib inhibited infection by HSV-1, HSV-2, and acyclovir-resistant strains. Inhibition coincided with minimal cytotoxicity. Bortezomib did not affect attachment of HSV to cells or inactivate the virus directly. Bortezomib acted early in HSV infection by perturbing two distinct proteasome-dependent steps that occur within the initial hours of infection: the transport of incoming viral nucleocapsids to the nucleus and the virus-induced disruption of host nuclear domain 10 (ND10) structures. The combination of bortezomib with acyclovir demonstrated synergistic inhibitory effects on HSV infection. Thus, bortezomib is a novel potential therapeutic for HSV with a defined mechanism of action. IMPORTANCE Viruses usurp host cell functions to advance their replicative agenda. HSV relies on cellular proteasome activity for successful infection. Proteasome inhibitors, such as MG132, block HSV infection at multiple stages of the infectious cycle. Targeting host cell processes for antiviral intervention is an unconventional approach that might limit antiviral resistance. Here we demonstrated that the proteasome inhibitor bortezomib, which is a clinically effective cancer drug, has the in vitro features of a promising anti-HSV therapeutic. Bortezomib inhibited HSV infection during the first hours of infection at nanomolar concentrations that were minimally cytotoxic. The mechanism of bortezomib’s inhibition of early HSV infection was to halt nucleocapsid transport to the nucleus and to stabilize the ND10 cellular defense complex. Bortezomib and acyclovir acted synergistically to inhibit HSV infection. Overall, we present evidence for the repurposing of bortezomib as a novel antiherpesviral agent and describe specific mechanisms of action.

23 citations


Journal ArticleDOI
TL;DR: It is demonstrated that GADD34 protects hepatocellular carcinoma (HCC) cells from tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–induced apoptosis by stabilizing a BCL-2 family member, myeloid cell leukemia 1 (MCL-1).

Journal ArticleDOI
TL;DR: This study suggests that deubiquitinating enzyme BRCC36 inhibition could potentially suppress oxLDL-induced inflammatory process by inhibiting NLRP3 activation and resultant IL-1β secretion.
Abstract: Oxidized Low-Density Lipoprotein (oxLDL) is a well-established pro-inflammatory marker that activates the NLRP3 inflammasome. Ubiquitination plays an important role in modulating the stability and functions of various proteins. BRCC36 is a ubiquitin-modifying enzyme that plays a crucial role in protein stabilization and activation in the cytosol, but its role in OxLDL-induced NLRP3 inflammasome activation is not known. Here, we have investigated the role of deubiquitinating enzyme BRCC36 in regulating NLRP3 inflammasome during oxLDL stimulation. Raw 264.7 murine macrophages were stimulated with oxLDL and effect of BRCC36 deubiquitination activity was assessed by fluorometric assay, and protein expression was assessed by Western blotting. The level of IL-1β measured by ELISA and LDH activity as pyroptotic cell death marker was assessed by fluorometric assay. The results showed that oxLDL increased the level of NLRP3 in macrophages and also the level of active caspase-1 and IL-1β. It also modulated the expression of deubiquitinating enzymes and caused pyroptotic cell death as indicated by LDH release. Inhibiting the proteasomal activity by MG132 and siRNA-mediated silencing of BRCC36 in macrophages potentially suppressed oxLDL-induced NLRP3 inflammasome activation and IL-1β secretion. Furthermore, the inhibition of proteasomal deubiquitinating activity with specific BRCC36 inhibitor G5 also reduced the inflammatory cell death. Taken together, our study suggests that deubiquitinating enzyme BRCC36 inhibition could potentially suppress oxLDL-induced inflammatory process by inhibiting NLRP3 activation and resultant IL-1β secretion.

Journal ArticleDOI
TL;DR: Results indicated that TP induced oxidative damage in NRK-52E cells by facilitating Nrf2 degradation by ubiquitination via the GSK-3β/Fyn pathway.

Journal ArticleDOI
TL;DR: The findings show that HTNV counteracts intrinsic and extrinsic apoptosis induction pathways, providing a defense mechanism utilized by hantaviruses to inhibit cytotoxic cell-mediated eradication of infected cells.

Journal ArticleDOI
TL;DR: Results suggest that degradation of Skp2 by FKB is involved in a functional Cullin1, and provide a rationale for further investigating combination of FKB and Bortezomib for treatment of RB deficient, castration-resistant prostate cancer.
Abstract: Flavokawain B (FKB) has been identified from kava root extracts as a potent apoptosis inducer for inhibiting the growth of various cancer cell lines, including prostate cancer. However, the molecular targets of FKB in prostate cancer cells remain unknown. An in vitro NEDD8 Initiation Conjugation Assay was used to evaluate the neddylation inhibitory activity of FKB. Molecular docking and a cellular thermal shift assay were performed to assess the direct interaction between FKB and the NEDD8 activating enzyme (NAE) complex. Protein neddylation, ubiqutination, stability and expression in cells were assessed with immunoprecipitation and Western blotting methods using specific antibodies. Deletion and site specific mutants and siRNAs were used to evaluate deep mechanisms by which FKB induces Skp2 degradation. Cell growth inhibition and apoptosis induction were measured by MTT, ELISA and Western blotting methods. FKB inhibits NEDD8 conjugations to both Cullin1 and Ubc12 in prostate cancer cell lines and Ubc12 neddylation in an in vitro assay. Molecular docking study and a cellular thermal shift assay reveal that FKB interacts with the regulatory subunit (i.e. APP-BP1) of the NAE. In addition, FKB causes Skp2 degradation in an ubiquitin and proteasome dependent manner. Overexpression of dominant-negative cullin1 (1–452), K720R mutant (the neddylation site) Cullin1 or the F-box deleted Skp2 that losses its binding to the Skp1/Cullin1 complex causes the resistance to FKB-induced Skp2 degradation, whereas siRNA knock-down of Cdh1, a known E3 ligase of Skp2 for targeted degradation, didn’t attenuate the effect of FKB on Skp2 degradation. These results suggest that degradation of Skp2 by FKB is involved in a functional Cullin1. Furthermore, proteasome inhibitors Bortezomib and MG132 transcriptionally down-regulate the expression of Skp2, and their combinations with FKB result in enhanced inhibitory effects on the growth of prostate cancer cell lines via synergistic down-regulation of Skp2 and up-regulation of p27/Kip1 and p21/WAF1 protein expression. FKB also selectively inhibits the growth of RB deficient cells with high expression of Skp2. These findings provide a rationale for further investigating combination of FKB and Bortezomib for treatment of RB deficient, castration-resistant prostate cancer.

Journal ArticleDOI
TL;DR: Insights into the chaperones reported here should enable study of the assembly events that generate the 26S holo-proteasome in Arabidopsis from the collection of 64 or more subunits.

Journal ArticleDOI
TL;DR: It is shown that UBC9, a SUMO-conjugating enzyme, regulates ubiquitination and degradation of Nav1.5, and co-immunoprecipitation showed that U BC9 interacts with Nedd4-2, which acts as an E3 ubiquitin-protein ligase involved in ubiquitine-protein degradation and regulates Nav 1.5 expression levels in asumOylation-independent manner.

Journal ArticleDOI
27 Oct 2019-Cells
TL;DR: ICH induced rapid proteasome over-activation, leading to an exaggeration of the ER stress/proteostasis disruption, and neuroinflammation might be a critical event in acute ICH pathology.
Abstract: Background: Neuroinflammation is a hallmark in intracerebral hemorrhage (ICH) that induces secondary brain injury, leading to neuronal cell death. ER stress-triggered apoptosis and proteostasis disruption caused neuroinflammation to play an important role in various neurological disorders. The consequences of ER stress and proteostasis disruption have rarely been studied during the course of ICH development. Methods: ICH was induced by collagenase VII-S intrastriatal infusion. Animals were sacrificed at 0, 3, 6, 24, and 72 h post-ICH. Rats were determined for body weight changes, hematoma volume, and neurological deficits. Brain tissues were harvested for molecular signaling analysis either for ELISA, immunoblotting, immunoprecipitation, RT-qPCR, protein aggregation, or for histological examination. A non-selective proteasome inhibitor, MG132, was administered into the right striatum three hours prior to ICH induction. Results: ICH-induced acute proteasome over-activation caused the early degradation of the endoplasmic reticulum (ER) chaperone GRP78 and IκB protein. These exacerbations were accompanied by the elevation of pro-apoptotic CCAAT-enhancer-binding protein homologous protein (CHOP) and pro-inflammatory cytokines expression via nuclear factor-kappa B (NF-κB) signal activation. Pre-treatment with proteasome inhibitor MG132 significantly ameliorated the ICH-induced ER stress/proteostasis disruption, pro-inflammatory cytokines, neuronal cells apoptosis, and neurological deficits. Conclusions: ICH induced rapid proteasome over-activation, leading to an exaggeration of the ER stress/proteostasis disruption, and neuroinflammation might be a critical event in acute ICH pathology.

Journal ArticleDOI
TL;DR: KPC1 prevented basal and apoptotic stress‐induced Bax translocation to mitochondria, suggesting that Bax can be a novel target for the antiapoptotic effects of KPC1 on I/R‐induced cardiomyocyte apoptosis and render mechanistic penetration into at least a subset of the mitochondrial effects of kPC1.
Abstract: Bax triggers cell apoptosis by permeabilizing the outer mitochondrial membrane, leading to membrane potential loss and cytochrome c release. However, it is unclear if proteasomal degradation of Bax is involved in the apoptotic process, especially in heart ischemia-reperfusion (I/R)-induced injury. In the present study, KPC1 expression was heightened in left ventricular cardiomyocytes of patients with coronary heart disease (CHD), in I/R-myocardium in vivo and in hypoxia and reoxygenation (H/R)-induced cardiomyocytes in vitro. Overexpression of KPC1 reduced infarction size and cell apoptosis in I/R rat hearts. Similarly, the forced expression of KPC1 restored mitochondrial membrane potential (MMP) and cytochrome c release driven by H/R in H9c2 cells, whereas reducing cell apoptosis, and knockdown of KPC1 by short-hairpin RNA (shRNA) deteriorated cell apoptosis induced by H/R. Mechanistically, forced expression of KPC1 promoted Bax protein degradation, which was abolished by proteasome inhibitor MG132, suggesting that KPC1 promoted proteasomal degradation of Bax. Furthermore, KPC1 prevented basal and apoptotic stress-induced Bax translocation to mitochondria. Bax can be a novel target for the antiapoptotic effects of KPC1 on I/R-induced cardiomyocyte apoptosis and render mechanistic penetration into at least a subset of the mitochondrial effects of KPC1.

Journal ArticleDOI
TL;DR: IGF-1 inhibited porcine primary granulosa cell apoptosis via degradation of pro-apoptotic BimEL, providing a direction for the treatment of infertility caused by ovarian dysplasia, such as polycystic ovary syndrome and the improvement of assisted reproductive technology.
Abstract: Insulin-like growth factor-1 (IGF-1) is an intra-ovarian growth factor that plays important endocrine or paracrine roles during ovarian development. IGF-1 affects ovarian function and female fertility through reducing apoptosis of granulosa cells, yet the underlying mechanism remains poorly characterized. Here, we aimed to address these knowledge gaps using porcine primary granulosa cells and examining the anti-apoptotic mechanisms of IGF-1. IGF-1 prevented the granulosa cell from apoptosis, as shown by TUNEL and Annexin V/PI detection, and gained the anti-apoptotic index, the ratio of Bcl-2/Bax. This process was partly mediated by reducing the pro-apoptotic BimEL (Bcl-2 Interacting Mediator of Cell Death-Extra Long) protein level. Western blotting showed that IGF-1 promoted BimEL phosphorylation through activating p-ERK1/2, and that the proteasome system was responsible for degradation of phosphorylated BimEL. Meanwhile, IGF-1 enhanced the Beclin1 level and the rate of LC3 II/LC3 I, indicating that autophagy was induced by IGF-1. By blocking the proteolysis processes of both proteasome and autophagy flux with MG132 and chloroquine, respectively, the BimEL did not reduce and the phosphorylated BimEL protein accumulated, thereby indicating that both proteasome and autophagy pathways were involved in the degradation of BimEL stimulated by IGF-1. In conclusion, IGF-1 inhibited porcine primary granulosa cell apoptosis via degradation of pro-apoptotic BimEL. This study is critical for us to further understand the mechanisms of follicular survival and atresia regulated by IGF-1. Moreover, it provides a direction for the treatment of infertility caused by ovarian dysplasia, such as polycystic ovary syndrome and the improvement of assisted reproductive technology.

Journal ArticleDOI
Ting Yuan1, Feng Zhang1, Xiangxiang Zhou1, Ying Li1, Ya Zhang1, Yangyang Xu1, Xin Wang1 
TL;DR: The present results indicated that the PI3K/AKT pathway is associated with the activation of BAG3 expression in DLBCL cells, and is involved in the protective response against proteasome inhibition.
Abstract: Proteasome inhibitors represent a novel class of drugs that have clinical efficacy against hematological and solid cancer types, including acute myeloid leukaemia, myelodysplastic syndrome an non-small cell lung cancer. It has been demonstrated that the anti-apoptotic protein B-cell lymphoma-2-associated athanogene 3 (BAG3) is induced by proteasome inhibitors in various cancer cells and serves an important role in chemotherapy resistance. The phosphatidylinositol 3-kinase (PI3K)/RAC-α serine/threonine-protein kinase (AKT) pathway is constitutively activated in a number of lymphoid malignancy types, including diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma. In the present study, the aim was to elucidate the role of the PI3K/AKT signaling pathway in the induction of BAG3, following exposure to a proteasome inhibitor in DLBCL cell lines. Bortezomib and MG132 were used as proteasome inhibitors. Western blotting was used to evaluate the roles of proteasome inhibitors and the PI3K/AKT pathway in BAG3 induction in DLBCL cells (LY1 and LY8), and LY294002 was used to block the PI3K/AKT pathway. Cell viability was detected using a Cell Counting Kit-8 assay. Apoptosis of LY1 and LY8 cells was quantified by Annexin V/7-amino-actinomycin D flow cytometry. The BAG3 protein was markedly induced upon exposure to bortezomib and MG132 in a dose-dependent manner. The PI3K/AKT inhibitor LY294002 significantly suppressed the induction of BAG3 by proteasome inhibitors. Inhibition of the PI3K/AKT pathway decreased the proliferation and increased the apoptosis induced by proteasome inhibitors. The present results indicated that the PI3K/AKT pathway is associated with the activation of BAG3 expression in DLBCL cells, and is involved in the protective response against proteasome inhibition.

Journal ArticleDOI
TL;DR: It is found that astrocytes can engulf and degrade α-Syn aggregates via the proteasome and autophagy pathways, and it is shown that ginkgolide B and bilobalide enhanceAstrocytic clearance of α- Syn, which gives an insight into the novel therapy for PD in future.
Abstract: The accumulation of aggregated forms of the α-Synuclein (α-Syn) is associated with the pathogenesis of Parkinson's disease (PD), and the efficient clearance of aggregated α-Syn represents a potential approach in PD therapy. Astrocytes are the most numerous glia cells in the brain and play an essential role in supporting brain functions in PD state. In the present study, we demonstrated that cultured primary astrocytes engulfed and degraded extracellular aggregated recombinant human α-Syn. Meanwhile, we observed that the clearance of α-Syn by astrocytes was abolished by proteasome inhibitor MG132 and autophagy inhibitor 3-methyladenine (3MA). We further showed that intracellular α-Syn was reduced after ginkgolide B (GB) and bilobalide (BB) treatment, and the decrease was reversed by MG132 and 3MA. More importantly, GB and BB reduced indirect neurotoxicity to neurons induced by α-Syn-stimulated astrocytic conditioned medium. Together, we firstly find that astrocytes can engulf and degrade α-Syn aggregates via the proteasome and autophagy pathways, and further show that GB and BB enhance astrocytic clearance of α-Syn, which gives us an insight into the novel therapy for PD in future.

Journal ArticleDOI
TL;DR: Kelch repeat and BTB domain-containing protein 11 negatively modulates osteoclast differentiation by controlling Cullin3-mediated ubiquitination of NFATc1 by the proteasome.
Abstract: Kelch repeat and BTB domain-containing protein 11 (KBTBD11) is a member of the KBTBD subfamily of proteins that possess a BTB domain and Kelch repeats. Despite the presence of the Kbtbd11 gene in mammalian genomes, there are few reports about KBTBD11 at present. In this study, we identified the novel protein KBTBD11 as a negative regulator of osteoclast differentiation. We found that expression of KBTBD11 increased during osteoclastogenesis. Small-interfering-RNA-mediated knockdown of KBTBD11 enhanced osteoclast formation, and markedly increased the expression of several osteoclast marker genes compared with control cells. Conversely, KBTBD11 overexpression impaired osteoclast differentiation, and decreased the expression of osteoclast marker genes. Among six major signaling pathways regulating osteoclast differentiation, KBTBD11 predominantly influenced the nuclear factor of activated T cell cytoplasmic-1 (NFATc1) pathway. Mechanistically, KBTBD11 was found to interact with an E3 ubiquitin ligase, Cullin3. Further experiments involving immunoprecipitation and treatment with MG132, a proteasome inhibitor, showed that the KBTBD11–Cullin3 promotes ubiquitination and degradation of NFATc1 by the proteasome. Considering that NFATc1 is an essential factor for osteoclast differentiation, the KBTBD11 and Cullin3 probably regulate the levels of NFATc1 through the ubiquitin-proteasome degradation system. Thus, KBTBD11 negatively modulates osteoclast differentiation by controlling Cullin3-mediated ubiquitination of NFATc1.

Journal ArticleDOI
TL;DR: Oleandrin sensitizes osteosarcoma cells to cisplatin in synergistic or additive manners, which is consistent with the changes of the intracellular accumulation of platinum and Pt‐DNA adducts.
Abstract: A major problem in osteosarcoma treatment is cisplatin resistance. We have reported the anti-osteosarcoma effect of oleandrin; however, whether oleandrin sensitizes osteosarcoma to cisplatin is unknown. We investigated the chemosensitization of oleandrin and potential mechanisms in osteosarcoma cells U-2OS, SaOS-2, and MG-63. The median-effect analysis demonstrated that cisplatin + oleandrin exerted synergistic (U-2OS and MG-63) or additive effects (SaOS-2), which were consistent with the changes of the intracellular accumulation of platinum (Pt) and Pt-DNA adducts. Immunohistochemistry staining showed that the expression level of the mature form CTR1, the major influx transporter of cisplatin, was low in osteosarcoma tissue. However, oleandrin with or without cisplatin significantly increased the expression and membrane localization of the mature CTR1. Furthermore, CTR1 knockdown reversed the synergistic effect and decreased cisplatin uptake. The mRNA microarray analysis suggested that oleandrin downregulated the expression of proteasome-related genes, which was verified by the proteasome activity assay. Besides, the proteasome inhibitor MG132 upregulated the expression of the mature CTR1 in U-2OS and MG-63 cells. Overall, we conclude that oleandrin sensitizes osteosarcoma cells to cisplatin in synergistic or additive manners. The synergy results from the enhanced cisplatin uptake via oleandrin-mediated inhibition of proteasome activity and subsequent blockage of the mature CTR1 degradation.

Journal ArticleDOI
TL;DR: The inhibition of melanogenesis in B16F10 mouse melanoma cells by TQ treatment resulted from the inhibition of the β‑catenin pathway and confirmed that TQtreatment inhibition in zebrafish was confirmed.
Abstract: Thymoquinone (TQ) is a component found in the seeds of Nigella sativa, an annual plant growing on the Mediterranean coast, and is known for its anticancer and anti‑inflammatory effects. However, to date, at least to the best of our knowledge, limited studies are available examining the molecular mechanisms through which TQ inhibits melanogenesis. Accordingly, this study aimed to treat B16F10 mouse melanoma cells with TQ to investigate its apparent effects and its molecular regulatory mechanisms. Treatment of the B16F10 cells with 10, 15 and 20 µM of TQ for 48 h resulted in a dose‑dependent decrease in the expression of microphthalmia‑associated transcription factor (MITF), tyrosinase expression and tyrosinase activity, and these treatments simultaneously led to a decrease in the protein expression and transcription of β‑catenin, a Wnt signaling pathway protein. Pre‑treatment of the cells with the proteasome inhibitor, MG132, to confirm the inhibition of melanogenesis through the β‑catenin pathway by TQ treatment resulted in an increase in the expression of β‑catenin that was initially reduced by TQ, and the expression and activity of MITF and tyrosinase also increased. Pre‑treatment with LiCl, which is known to inactivate glycogen synthase kinase 3β (GSK3β) by inducing the phosphorylation of the Ser‑9 site, resulted in an increased phospho‑GSK3β expression accompanied by β‑catenin that was initially reduced by TQ, and the recovery of the expression and activity of tyrosinase was also confirmed. The transfection of S37A cDNA into B16F10 cells that overexpress β‑catenin resulted in the recovery of β‑catenin expression that was initially reduced by TQ, and this treatment also recovered the expression and activity of tyrosinase. When zebrafish eggs were treated with 1, 2.5 and 5 µM of TQ at 10 h following fertilization, their melanin content decreased in a dose‑dependent manner. On the whole, these findings demonstrated that the inhibition of melanogenesis in B16F10 mouse melanoma cells by TQ treatment resulted from the inhibition of the β‑catenin pathway and confirmed that TQ treatment inhibited melanogenesis in zebrafish.

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TL;DR: Levels and activity of circulatory proteasome and immunoproteasome (inflammatory subtype) in preeclampsia and hemolysis, elevated liver enzymes, and thrombocytopenia (HELLP) syndrome were examined to identify women with hypertensive pregnancy disorders and sPE and HELLP were characterized by significant upregulation in circulating levels and lytic activity of the prote asome that was partially explained by placental immunoproteinasome upregulation.
Abstract: Excessive accumulation of misfolded proteins was recently demonstrated in preeclampsia. We examined levels and activity of circulatory proteasome and immunoproteasome (inflammatory subtype) in preeclampsia and hemolysis, elevated liver enzymes, and thrombocytopenia (HELLP) syndrome. We analyzed samples from women with hypertensive pregnancy disorders (n=115), including preeclampsia with severe features (sPE) and HELLP syndrome, and normotensive controls (n=45). Plasma proteasome and immunoproteasome immunoreactivity were determined by quantifying the α-subunit of the 20S core and β5i (proteasome subunit beta 8 [PSMB8]), respectively. Plasma proteasome activity was analyzed with fluorogenic substrates. MG132, lactacystin, and ONX0914 were used to inhibit the circulating proteasome and immunoproteasome, respectively. Plasma cytokine profiles were evaluated by multiplex immunoassay. Placental expression of β5 (constitutive proteasome) and β5i (immunoproteasome) was interrogated by immunohistochemistry. Women with sPE had increased plasma 20S levels ( P<0.001) and elevated lytic activities (chymotrypsin-like 7-fold, caspase-like 4.2-fold, trypsin-like 2.2-fold; P <0.001 for all) compared with pregnant controls. Women with features of HELLP displayed the highest plasma proteasome levels and activity, which correlated with decreased IFN-γ (interferon-γ), and increased IL (interleukin)-8 and IL-10. In sPE and HELLP, chymotrypsin-like activity was suppressed by proteasome inhibitors including ONX0914. Compared with gestational age-matched controls, sPE placentas harbored increased β5 and β5i immunostaining in trophoblasts. β5i signal was elevated in HELLP with predominant staining in villous core, extravillous trophoblasts in placental islands, and extracellular vesicles in intervillous spaces. Pregnancy represents a state of increased proteostatic stress. sPE and HELLP were characterized by significant upregulation in circulating levels and lytic activity of the proteasome that was partially explained by placental immunoproteasome upregulation.

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TL;DR: P pulsed stable isotope labeling with amino acids in cell culture (SILAC) is applied to study changes in the synthesis of specific proteins in human lung adenocarcinoma cells in which eEF2K had been depleted by an inducible shRNA to suggest concomitant inhibition of HSP90 and eEF1K could be a strategy to decrease cancer cell survival.

Journal ArticleDOI
TL;DR: Six variants with the accessory subunit CDC50A in HEK293T cells expressed with the effect of disease‐associated missense mutations on ATP8A2 fold in a native‐like conformation, but lack key amino acid residues required for ATP‐dependent lipid transport.
Abstract: ATP8A2 is a P4-ATPase (adenosine triphosphate) that actively flips phosphatidylserine and phosphatidylethanolamine from the exoplasmic to the cytoplasmic leaflet of cell membranes to generate and maintain phospholipid asymmetry. Mutations in the ATP8A2 gene have been reported to cause severe autosomal recessive neurological diseases in humans characterized by intellectual disability, hypotonia, chorea, and hyperkinetic movement disorders with or without optic and cerebellar atrophy. To determine the effect of disease-associated missense mutations on ATP8A2, we expressed six variants with the accessory subunit CDC50A in HEK293T cells. The level of expression, cellular localization, and functional activity were analyzed by western blot analysis, immunofluorescence microscopy, and ATPase activity assays. Two variants (p.Ile376Met and p.Lys429Met) expressed at normal ATP8A2 levels and preferentially localized to the Golgi-recycling endosomes, but were devoid of ATPase activity. Four variants (p.Lys429Asn, pAla544Pro, p.Arg625Trp, and p.Trp702Arg) expressed poorly, localized to the endoplasmic reticulum, and lacked ATPase activity. The expression of these variants was increased twofold by the addition of the proteasome inhibitor MG132. We conclude that the p.Ile376Met and p.Lys429Met variants fold in a native-like conformation, but lack key amino acid residues required for ATP-dependent lipid transport. In contrast, the p.Lys429Asn, pAla544Pro, p.Arg625Trp, and p.Trp702Arg variants are highly misfolded and undergo rapid proteosomal degradation.

Journal ArticleDOI
TL;DR: The potential apoptosis induction of RT with a mechanism of action involving the targeting of Mcl-1 for ubiquitin-proteasomal degradation is reported, beneficial for the development of efficient anti-cancer strategies or targeted therapies.
Abstract: Among malignancies, lung cancer is the major cause of cancer death. Despite the advance in lung cancer therapy, the five-year survival rate is extremely restricted due to therapeutic failure and disease relapse. Targeted therapies selectively inhibiting certain molecules in cancer cells have been accepted as promising ways to control cancer. In lung cancer, evidence has suggested that the myeloid cell leukemia 1 (Mcl-1) protein, an anti-apoptotic member of the Bcl-2 family, is a target for drug action. Herein, we report the Mcl-1 targeting activity of renieramycin T (RT), a marine-derived tetrahydroisoquinoline alkaloid that was isolated from the Thai blue sponge Xestospongia sp. RT was shown to be dominantly toxic to lung cancer cells compared to the normal cells in the lung. The cytotoxicity of this compound toward lung cancer cells was mainly exerted through apoptosis induction. For the mechanism of action, we found that RT mediated activation of p53 protein and caspase-9 and -3 activations. While others Bcl-2 family proteins (Bcl-2, Bak, and Bax) were minimally changed in response to RT, Mcl-1 protein was dramatically diminished. We further performed the cycloheximide experiment and found that the half-life of Mcl-1 was significantly shortened by RT treatment. When MG132, a potent selective proteasome inhibitor, was utilized, it could restore the Mcl-1 level. Furthermore, immunoprecipitation analysis revealed that RT significantly increased the formation of Mcl-1-ubiquitin complex compared to the non-treated control. In conclusion, we report the potential apoptosis induction of RT with a mechanism of action involving the targeting of Mcl-1 for ubiquitin-proteasomal degradation. As Mcl-1 is critical for cancer cell survival and chemotherapeutic failure, this novel information regarding the Mcl-1-targeted compound would be beneficial for the development of efficient anti-cancer strategies or targeted therapies.

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TL;DR: It is found that the HSP90α inhibitor NVP-AUY922 (922) effectively downregulated and destabilized the IGF-1Rβ protein, substantially reduced the levels of downstream signaling molecules (p-AKT, AKT and p-ERK1/2), and resulted in growth inhibition and apoptosis in IGF- 1Rβ-overexpressing PC cells.
Abstract: Enhancement of insulin-like growth factor 1 receptor (IGF-IR) degradation by heat shock protein 90 (HSP90) inhibitor is a potential antitumor therapeutic strategy. However, very little is known about how IGF-IR protein levels are degraded by HSP90 inhibitors in pancreatic cancer (PC). We found that the HSP90α inhibitor NVP-AUY922 (922) effectively downregulated and destabilized the IGF-1Rβ protein, substantially reduced the levels of downstream signaling molecules (p-AKT, AKT and p-ERK1/2), and resulted in growth inhibition and apoptosis in IGF-1Rβ-overexpressing PC cells. Preincubation with a proteasome or lysosome inhibitor (MG132, 3 MA or CQ) mainly led to IGF-1Rβ degradation via the lysosome degradation pathway, rather than the proteasome-dependent pathway, after PC cells were treated with 922 for 24 h. These results might be associated with the inhibition of pancreatic cellular chymotrypsin–peptidase activity by 922 for 24 h. Interestingly, 922 induced autophagic flux by increasing LC3II expression and puncta formation. However, knockdown of the crucial autophagy component AGT5 and the chemical inhibitor 3 MA-blocked 922-induced autophagy did not abrogate 922-triggered IGF-1Rβ degradation. Furthermore, 922 could enhance chaperone-mediated autophagy (CMA) activity and promote the association between HSP/HSC70 and IGF-1Rβ or LAMP2A in coimmunoprecipitation and immunofluorescence analyses. Silencing of LAMP2A to inhibit CMA activity reversed 922-induced IGF-1Rβ degradation, suggesting that IGF-1Rβ degradation by 922 was partially dependent on the CMA pathway rather than macroautophagy. This finding is mirrored by the identification of the KFERQ-like motif in IGF-1Rβ. These observations support the potential application of 922 for IGF-1Rβ-overexpressing PC therapy and first identify the role of the CMA pathway in IGF-1Rβ degradation by an HSP90 inhibitor.