MG132 Treatment Research Articles

SPOP Promotes GLI3‐dependent SHH Signaling in Breast Cancer

Personalized treatment, which is designed to only block oncogenic pathways activated by specific cancer mutations, is rapidly rising as a superior treatment for cancer. One gene that has recently been identified as highly mutated in a variety of cancers is Speckle Type POZ protein (SPOP), a substrate binding subunit of an E3 ubiquitin ligase that has been shown to target Sonic Hedgehog (SHH) downstream effectors for degradation. Interestingly, recent studies have found that SPOP is downregulated in up to 70% of breast cancer tumors and, furthermore, a correlation has been found between high levels of SHH signaling and poor prognostic pathological breast cancer features. Previous studies from our laboratory have shown that SPOP binds directly to the SHH downstream effector GLI3 to target it for degradation in a manner that is disrupted by prostate cancer-associated SPOP mutations. Since SHH signaling has been shown to be a key player in breast cancer oncogenesis, it is likely that SPOP plays a similar role in breast cancer progression. We therefore hypothesize that downregulation of SPOP induces hyper-activated SHH signaling to promote breast cancer progression through stabilization of GLI3. In order to study this, we employed a lentivirus-mediated approach to generate SPOP knockdown breast cancer cells. Next, we carried out western blot analysis to examine GLI3 protein levels when SPOP is downregulated followed by co-immunoprecipitation analysis to examine a GLI3/SPOP interaction. Finally, we used MG132 treatment to block the proteasome pathway in order to determine whether SPOP-mediated degradation of GLI3 functions through the proteasome. Our results indicate that SPOP knockdown promotes upregulation of the SHH downstream effector GLI3 through a direct physical interaction. Furthermore, we show that SPOP likely targets GLI3 for degradation through the proteasome pathway as MG132 treatment promotes GLI3 stabilization even in the presence of SPOP. Our collective results therefore indicate that SPOP targets GLI3 for degradation through the proteasome pathway which, in turn, would cause hyper-activated SHH signaling and subsequent progression of breast cancer oncogenesis.

RNF8 stabilizes MYC via K63‐linked ubiquitination to promote metabolic reprogramming in triple‐negative breast cancer

Triple-negative breast cancer (TNBC) is the most lethal subtype of breast cancer owing to its aggressive nature and the lack of targeted therapy for most patients. MYC, an oncogenic transcription factor, is elevated in TNBC and causes accelerated tumor growth through metabolic reprogramming. However, there is currently no drug available in clinics to inhibit MYC. Ring Finger Protein 8 (RNF8), an E3 ubiquitin ligase, is highly expressed in TNBC and essential for tumor progression. To comprehensively understand the role of RNF8 in TNBC, we conducted RNA-sequencing and found that the downregulation of RNF8 attenuates the activity of the MYC network in human TNBC cell lines. Moreover, we observed the reduced glucose uptake and utilization in TNBC cells with RNF8 knockdown as a result of the decreased expression of MYC and its target genes such as glucose transporter GLUT1 and key glycolytic enzyme HK2. Knockdown of RNF8 also decreased the metabolic influx in the glycolysis pathway as revealed in the isotope tracer studies coupled with mass spectrometry analysis. Importantly, the treatment of proteasome inhibitor MG132 was able to restore MYC protein expression, while cycloheximide chase assay showed that MYC protein has a shorter half-life in TNBC cells with RNF8 deficiency. In vivo ubiquitination assay further demonstrated that RNF8 mediates Lysine (K)63-linked ubiquitination over MYC, suggesting that RNF8 mediates K63-linked ubiquitination to stabilize MYC against proteasomal degradation. Taken together, these results suggest that RNF8 enhances MYC signaling, thereby promoting metabolic reprogramming and tumor progression in TNBC. Targeting RNF8 may be a reasonable alternative to inhibit MYC-driven cancers such as TNBC.

Dynamics of D-amino acid oxidase ain kidney epithelial cells under amino acid starvation.

D-amino acid oxidase (DAO) is a flavoenzyme catalyzing the oxidation of D-amino acid (AA)s. In the kidney, its expression is detected in proximal tubules, and DAO is considered to play a role in the conversion of D-form AAs to α-keto acids. LLC-PK1 cells, a pig renal proximal tubule cell line, were used to elucidate the regulation of DAO protein synthesis and degradation. In this study, we showed that trypsinization of LLC-PK1 cells in culture system rapidly reduced the intracellular DAO protein level to ∼33.9% of that before treatment, even within 30 min. Furthermore, we observed that the DAO protein level was decreased when LLC-PK1 cells were subjected to AA starvation. To determine the degradation pathway, we treated the cells with chloroquine and MG132. DAO degradation was found to be inhibited by chloroquine, but not by MG132 treatment. We next examined whether or not DAO was degraded by autophagy. We found that AA starvation led to an increased accumulation of LC3-II, suggesting that DAO protein is degraded by autophagy due to AA starvation conditions. Furthermore, treatment with cycloheximide inhibited DAO protein degradation. Taken together, DAO protein is degraded by autophagy under starvation. The present study revealed the potential dynamics of DAO correlated with renal pathophysiology.

The inhibitive effects of proteasome inhibitor MG-132 on pterygium fibroblasts in vitro and the potential key regulators involved

This study aimed to determine whether MG-132 as a proteasome inhibitor can effectively hinder pterygium progression, and to screen out potential regulators involved in MG-132 mediated process. Human pterygium fibroblasts (HPFs) were derived from pterygium tissues from 5 patients. Cell proliferation was examined by MTT, cell cycle and apoptosis were detected by flow cytometry. The overgrowth pterygium tissues were characterized by H&E staining and IHC compared with normal tissues. Differential mRNA expression with MG-132 treatment was determined by RNA sequencing and analyzed by GO and KEGG pathways. The expression levels of Nrf2, MCPIP1, CDKN1B and XBP1, four genes closely associated with pterygium, were detected by RT-qPCR and western blotting. MG-132 dose-dependently inhibited the growth of HPFs, induced G2/M phase arrest of cell cycle at a certain dose, and also caused cell apoptosis, with the levels of cleaved caspase3, cleaved PARP, Bax and p21 increased. Ki-67 and Bcl-2 were highly expressed while Bax was decreased in pterygium tissues. Total 7199 differentially expressed genes (DEGs) were identified, including HSPA family most significantly increased, and AL590428.1, AL122125.1 and lincRNAs such as FGF14-AS2 decreased. The up-regulated DEGs were mainly enriched in RNA degradation pathway, while down-regulated DEGs were related to the regulation of cell cycle. The expressions of Nrf2 and MCPIP1 were significantly increased, while XBP1 and CDKN1B were decreased. In conclusion, MG-132 inhibited the proliferation and induced apoptosis of HPFs in vitro with 7199 DEGs participated in, which may provide a useful reference for the exploitation of MG-132 in treating pterygium.

Praja2 suppresses the growth of gastric cancer by ubiquitylation of KSR1 and inhibiting MEK-ERK signal pathways.

Gastric cancer (GC) is a common malignant tumor, which has a high incidence and fatality. Therefore, it is important to clarify the molecular mechanism of the occurrence and development for GC and to find more effective treatments and targeted drugs. In this study, we found that the kinase suppressor of Ras1 (KSR1) was increased in GC tissues and cell lines. Silencing of KSR1 inhibited the proliferation, migration and invasion of MKN-45 cells. E3 ligase Praja2 was downregulated in GC tissues and cell lines. In addition, praja2 promoted ubiquitylation of KSR1, but inhibited MEK-ERK signal pathways. Functional analysis indicated overexpression of praja2 inhibited the proliferation, migration and invasion of MKN-45 cells, while MG132 or FGF2 treatment removed the inhibitory effects of praja2 on GC progression. In vivo tumorigenesis experiments indicated praja2 inhibited tumor growth via KSR1-MEK-ERK axis. In conclusion, praja2 promoted the ubiquitylation and degradation of KSR1, which disturbed MEK- ERK signaling and inhibited GC progression. Our study might provide a novel target for GC clinical treatment.

β-TrCP1 facilitates cell cycle checkpoint activation, DNA repair, and cell survival through ablation of β-TrCP2 in response to genotoxic stress

F-box proteins β-TrCP1 and β-TrCP2 are paralogs present in the human genome. They control several cellular processes including cell cycle and DNA damage signaling. Moreover, it is reported that they facilitate DNA damage-induced accumulation of p53 by directing proteasomal degradation of MDM2, a protein that promotes p53 degradation. However, the individual roles of β-TrCP1 and β-TrCP2 in the genotoxic stress-induced activation of cell cycle checkpoints and DNA damage repair remain largely unknown. Here, using biochemical, molecular biology, flow cytometric, and immunofluorescence techniques, we show that β-TrCP1 and β-TrCP2 communicate during genotoxic stress. We found that expression levels of β-TrCP1 are significantly increased while levels of β-TrCP2 are markedly decreased upon induction of genotoxic stress. Further, our results revealed that DNA damage-induced activation of ATM kinase plays an important role in maintaining the reciprocal expression levels of β-TrCP1 and β-TrCP2 via the phosphorylation of β-TrCP1 at Ser158. Phosphorylated β-TrCP1 potently promotes the proteasomal degradation of β-TrCP2 and MDM2, resulting in the activation of p53. Additionally, β-TrCP1 impedes MDM2 accumulation via abrogation of its lysine 63-linked polyubiquitination by β-TrCP2. Thus, β-TrCP1 helps to arrest cells at the G2/M phase of the cell cycle and promotes DNA repair upon DNA damage through attenuation of β-TrCP2. Collectively, our findings elucidate an intriguing posttranslational regulatory mechanism of these two paralogs under genotoxic stress and revealed β-TrCP1 as a key player in maintaining the genome integrity through the attenuation of β-TrCP2 levels in response to genotoxic stress.

Histone Deacetylase Inhibitor-Induced CDKN2B and CDKN2D Contribute to G2/M Cell Cycle Arrest Incurred by Oxidative Stress in Hepatocellular Carcinoma Cells via Forkhead Box M1 Suppression.

Forkhead box protein M1 (FOXM1) is a pivotal regulator of G2/M cell cycle progression in many types of cancer. Previously, our study demonstrated that histone deacetylase inhibition (HDACi) sensitizes hepatocellular carcinoma cells (HCC) to oxidative stress through FOXM1 suppression. However, the mechanism underlying its suppression by HDACi still requires elucidation. We hypothesized that HDACi induce genes responsible for destabilizing and inactivating FOXM1. The transcriptome in the HepG2 was revealed by massive analysis of cDNA end (MACE). Expression of mRNA and proteins were analyzed by quantitative real-time PCR (qPCR) and western blot, respectively. Cell cycle was analyzed by fluorescence-activated cell sorting (FACS). Oxidative stress and HDACi suppressed CDK4/6 levels while enhancing CDK inhibitor 2B and 2D (CDKN2B and CDKN2D) expressions in HCC. Palbociclib, a specific inhibitor of CDK4/6, induced G2/M cell cycle arrest in HCC by down-regulating phosphorylation level of FOXM1, and its downstream target genes such as aurora kinase A (AURKA) and polo-like kinase 1 (PLK1). HDACi treatment increased the ubiquitination level of FOXM1 by suppressing ubiquitin-specific peptidase 21 (USP21), which deubiquitinates FOXM1. Inhibiting FOXM1 degradation with MG132 treatment affected neither palbociclib-induced G2/M cell cycle arrest nor expression of its target genes. Double knockdown of CDKN2B and CDKN2D reduced the oxidative stress and HDACi-induced G/2M cell cycle arrest. In conclusion, oxidative stress and HDACi synergistically cause G2/M cell cycle arrest via CDKN2 induction, which sequentially inhibits CDK4/6, FOXM1, and its downstream target genes AURKA, PLK1, and CCNB1 phosphorylation in HCC.

Spironolactone-induced XPB degradation requires TFIIH integrity and ubiquitin-selective segregase VCP/p97

ABSTRACT Mineralocorticoid and androgen receptor antagonist, spironolactone, was recently identified as an inhibitor of nucleotide excision repair (NER), acting via induction of proteolysis of TFIIH component Xeroderma Pigmentosum B protein (XPB). This activity provides a strong rationale for repurposing spironolactone for cancer therapy. Here, we report that the spironolactone-induced XPB proteolysis is mediated through ubiquitin-selective segregase, valosin-containing protein (VCP)/p97. We show that spironolactone induces a dose- and time-dependent degradation of XPB but not XPD, and that the XPB degradation is blocked by VCP/p97 inhibitors DBeQ, NMS-873, and neddylation inhibitor MLN4924. Moreover, the cellular treatment by VCP/p97 inhibitors leads to the accumulation of ubiquitin conjugates of XPB but not XPD. VCP/p97 knockdown by inducible shRNA does not affect XPB level but compromises the spironolactone-induced XPB degradation. Also, VCP/p97 interacts with XPB upon treatment of spironolactone and proteasome inhibitor MG132, while the VCP/p97 adaptor UBXD7 binds XPB and its ubiquitin conjugates. Additionally, ATP analog-mediated inhibition of Cdk7 significantly decelerates spironolactone-induced XPB degradation. Likewise, engaging TFIIH to NER by UV irradiation slows down spironolactone-induced XPB degradation. These results indicate that the spironolactone-induced XPB proteolysis requires VCP/p97 function and that XPB within holo-TFIIH rather than core-TFIIH is more vulnerable to spironolactone-induced proteolysis. AbbreviationsNER: nucleotide excision repair; TFIIH: transcription factor II H; CAK: Cdk-activating kinase (CAK) complex; XPB: Xeroderma Pigmentosum type B; VCP/p97: valosin-containing protein/p97; Cdk7: cyclin-dependent kinase 7; NAE: NEDD8-activating enzyme; IP: immunoprecipitation

The Synergistic Effects for Apoptosis of Propolis Extracts and MG-132 on HeLa Ovarian Cancer Cell Line and Its Molecular Mechanism

In this study, we investigated the synergistic effect of apoptosis of propolis extract and MG-132 on HeLa ovarian cancer cells. The cytotoxicity of MG-132 and propolis was confirmed on dose-dependent manner. The LD<SUB>50</SUB> values of MG-132 and propolis showed at 10 μM and 100 μg/mL, respectively. Based on this concentration, in order to investigate the synergistic effect, the apoptosis effect of the mixture of low concentration of MG-132 and propolis extract was confirmed. The concentration of the mixture determined 0.5 μM MG-132 and 10 μg/mL and 25 μg/mL of propolis extracts. Cytotoxicity was measured after treating HeLa cells with a mixture of MG-132 and propolis extract. HeLa cells survived at least 90% in 0.5 μM of MG-132 and 25 μg/mL of propolis. However, it survived less than 80% when treated with a MG-132 and propolis extract. We investigated the level of expression of apoptosis-related proteins by Western blotting. When apoptosis stimulation occurred, apoptosis marker proteins PARP and caspase-3 produced 70 kDa and 19 kDa fragment, respectively. Treatment with a mixture of MG-132 and propolis extract produced PARP and caspase-3, but a single treatment did not. In the treatment of MG-132 and propolis extract, ubiquitination as another apoptosis molecule was expressed very strongly. In the mixture of MG-132 and propolis extract, the apoptosis molecule as well as proliferation protein expression level was changed. JAK showed digested fragments in mixture treatment group. Thus, the interaction of the mixture of MG-132 and propolis extract can induce apoptosis stimulation, and protein expression levels related to apoptosis and proliferation in cells are changed. Mixture of the two molecules could show potential as a cancer treatment.

Crizotinib changes the metabolic pattern and inhibits ATP production in A549 non-small cell lung cancer cells.

Crizotinib, an inhibitor of the hepatocyte growth factor receptor oncogene, has been studied extensively regarding its antitumor and clinically beneficial effects in non-small cell lung cancer (NSCLC). However, crizotinib's effects on cancer cell energy metabolism, which is linked with tumor proliferation and migration, in NSCLC are unclear. Therefore, the present study focused on crizotinib's effect on NSCLC glucose metabolism. Crizotinib's effects on glucose metabolism, proliferation, migration and apoptosis in A549 cells were explored. Several other inhibitors, including 2-DG, rotenone and MG132, were used to define the mechanism of action in further detail. Data showed that crizotinib treatment reduced A549 cell viability, increased glucose consumption and lactate production, while decreased mitochondrial transmembrane potential (Δψm) and ATP production. Crizotinib treatment, combined with rotenone and MG132 treatment, further inhibited ATP production and Δψm and increased reactive oxygen species content. However, crizotinib did not suppress cell proliferation, migration, ATP production, Δψm or mitochondrial-related apoptosis signals further following 2-DG-mediated inhibition of glycolysis. These results indicated that crizotinib induced low mitochondrial function and compensatory high anaerobic metabolism, but failed to maintain sufficient ATP levels. The alternation of metabolic pattern and insufficient ATP supply may serve important roles in the metabolic antitumor mechanism of crizotinib in A549 cells.

Proteasome Inhibitor MG132 is Toxic and Inhibits the Proliferation of Rat Neural Stem Cells but Increases BDNF Expression to Protect Neurons.

Regulation of protein expression is essential for maintaining normal cell function. Proteasomes play important roles in protein degradation and dysregulation of proteasomes is implicated in neurodegenerative disorders. In this study, using a proteasome inhibitor MG132, we showed that proteasome inhibition reduces neural stem cell (NSC) proliferation and is toxic to NSCs. Interestingly, MG132 treatment increased the percentage of neurons in both proliferation and differentiation culture conditions of NSCs. Proteasome inhibition reduced B-cell lymphoma 2 (Bcl-2)/Bcl-2 associated X protein ratio. In addition, MG132 treatment induced cAMP response element-binding protein phosphorylation and increased the expression of brain-derived neurotrophic factor transcripts and proteins. These data suggest that proteasome function is important for NSC survival and differentiation. Moreover, although MG132 is toxic to NSCs, it may increase neurogenesis. Therefore, by modifying MG132 chemical structure and developing none toxic proteasome inhibitors, neurogenic chemicals can be developed to control NSC cell fate.

The mRNA of TCTP functions as a sponge to maintain homeostasis of TCTP protein levels in hepatocellular carcinoma

Translationally controlled tumor protein (TCTP) is a highly conserved protein that accumulated in the tumorigenesis of various malignancies. Despite the important role of TCTP protein in tumor progression, the precise function and underlying mechanistic regulation of TCTP mRNA in hepatocellular carcinoma (HCC) remain unclear. In this study, we found that TCTP protein was overexpressed in HCC patients but TCTP mRNA expression levels were reversed. TCTP knockout HCC cells exhibited attenuated abilities of proliferation, migration, and invasion. The knockdown of TCTP by siRNA effectively reduced TCTP mRNA levels but not protein levels in HCC cells. Moreover, although the constitutive knockdown of TCTP inhibited almost 80% of TCTP protein expression levels in tumors of wildtype transgenic mice (TCTP KD/WT), partial restoration of TCTP protein expression was observed in the tumors of heterozygous TCTP mice (TCTP KD/TCTP±). The blockage of mRNA synthesis with ActD stimulated TCTP protein expression in HCC cells. In contrast, combined treatment with ActD and CHX or MG132 treatment alone did not lead to the TCTP protein accumulation in cells. Furthermore, following the introduction of exogenous TCTP in cells and orthotopic HCC tumor models, the endogenous TCTP protein did not change with the recombinational TCTP expression and kept a rather stable level. Dual-luciferase assays revealed that the coding sequence of TCTP mRNA functions as a sponge to regulate the TCTP protein expression. Collectively, our results indicated that the TCTP mRNA and protein formed a closed regulatory circuit and works as a buffering system to keep the homeostasis of TCTP protein levels in HCC.

Overexpression of Cancer Upregulated Gene 2 (CUG2) Decreases Spry2 Through c-Cbl, Leading to Activation of EGFR and β-Catenin Signaling.

PurposeThe mechanism by which cancer upregulated gene 2 (CUG2) overexpression induces cancer stem cell-like phenotypes is not fully understood. Because the increased activity and expression of epidermal growth factor receptor (EGFR) kinase have been reported in A549 cancer cells overexpressing CUG2 (A549-CUG2) compared with control cells (A549-Vec), the Sprouty2 (Spry2) protein has gained attention as the downstream molecule of EGFR signaling. Therefore, we aim to identify the role of Spry2 in CUG2-overexpressing lung cancer cells.Materials and MethodsSpry2 expression levels were examined in A549-CUG2 and A549-Vec cells by Western blotting and qRT-PCR. Cell migration, invasion, and sphere formation were examined after Spry2 suppression and overexpression. EGFR-Stat1 and Akt-ERK protein phosphorylation levels were detected via immunoblotting. NEK2 kinase and β-catenin reporter assay were performed for downstream of Spry2 signaling.ResultsAlthough A549-CUG2 cells showed lower levels of the Spry2 protein than A549-Vec cells, no difference in levels of Spry2 transcript was observed between both cells via qRT-PCR. Furthermore, MG132 treatment enhanced the protein levels and ubiquitination of Spry2, suggesting that Spry2 protein expression can be regulated via the ubiquitin-proteasome pathway. The enforced expression of c-Cbl, known as the binding partner of Spry2, decreased the Spry2 protein levels, whereas its knockdown oppositely increased them. Epithelial–mesenchymal transition (EMT) and sphere formation were increased in A549-Vec cells during Spry2 siRNA treatment, confirming the role of Spry2 in CUG2-induced oncogenesis. Furthermore, EMT and sphere formation were determined by the Spry2 protein levels through the regulation of EGFR-Stat1 and β-catenin-NEK2-Yap1 signaling pathways.ConclusionCUG2 reduces Spry2 protein levels, the negative signaling molecule of cell proliferation, via c-Cbl, possibly activating the EGFR and β-catenin signaling pathways and, in turn, contributing to the induction of cancer stem cell-like phenotypes.

Inhibition of proteasome reveals basal mitochondrial ubiquitination

Strict quality control for mitochondrial proteins is necessary to ensure cell homeostasis. Two cellular pathways–Ubiquitin Proteasome System (UPS) and autophagy–contribute to mitochondrial homeostasis under stressful conditions. Here, we investigate changes to the mitochondria proteome and to the ubiquitin landscape at mitochondria in response to proteasome inhibition. Treatment of HeLa cells devoid of Parkin, the primary E3 ligase responsible for mitophagy, with proteasome inhibitor MG132 for a few hours caused mitochondrial oxidative stress and fragmentation, reduced energy output, and increased mitochondrial ubiquitination without inducing mitophagy. Overexpression of Parkin did not show any induction of mitophagy in response to MG132 treatment. Analysis of ubiquitin chains on isolated mitochondria revealed predominance of K48, K29 and K63-linked polyubiquitin. Interestingly, of all ubiquitinated mitochondrial proteins detected in response to MG132 treatment, a majority (≥90%) were intramitochondrial irrespective of Parkin expression. However, overall levels of these ubiquitinated mitochondrial proteins did not change significantly upon proteasome inhibition when evaluated by quantitative proteomics (LFQ and SILAC), suggesting that only a small portion are ubiquitinated under basal conditions. Another aspect of proteasome inhibition is significant enrichment of UPS, lysosomal and phagosomal components, and other heat shock proteins associated with isolated mitochondria. Taken together, our study highlights a critical role of UPS for ubiquitinating and removing imported proteins as part of a basal mitochondrial quality control system independent of Parkin. SignificanceAs centers of cellular bioenergetics, numerous metabolic pathways and signaling cascades, the health of mitochondria is of utmost importance for ensuring cell survival. Due to their unique physiology, mitochondria are constantly subjected to damaging oxidative radicals (ROS) and protein import-related stress due to buildup of unfolded aggregate-prone proteins. Thus, for quality control purposes, mitochondria are constantly under surveillance by Autophagy and the Ubiquitin Proteasome System (UPS), both of which share ubiquitin as a common signal. The ubiquitin landscape of mitochondria has been studied in detail under stressful conditions, however, little is known about basal mitochondrial ubiquitination. Our study reveals that the extent of ubiquitination at mitochondria greatly increases upon proteasome inhibition, pointing to a large number of potential substrates for proteasomal degradation. Interestingly, most of the ubiquitination occurs on intramitochondrial proteins, components of the electron transport chain (ETC) and matrix-resident metabolic enzymes in particular. Moreover, numerous cytosolic UPS components, chaperones and autophagy-lysosomal proteins were recruited to mitochondria upon proteasome inhibition. Taken together, this suggests that the levels and functions of mitochondrial proteins are constantly regulated through ubiquitin-dependent proteasomal degradation even under basal conditions. Unclogging mitochondrial import channels may provide a mechanism to alleviate stress associated with mitochondrial protein import or to adapt cells according to their metabolic needs. Therefore, targeting the mitochondrial ubiquitination/deubiquitination machinery, such as improving the therapeutic potency of proteasome inhibitors, may provide an additional therapeutic arsenal against tumors.

Ubiquitin conjugating enzyme E2 M promotes apoptosis in osteoarthritis chondrocytes via Wnt/β-catenin signaling

In this study, the role of ubiquitin conjugating enzyme E2 M (UBE2M) and molecular mechanisms associated with osteoarthritis (OA) were explored. Cartilage tissues and corresponding healthy tissues from OA patients were isolated. Our data suggested that the expression level of UBE2M in OA patients was significantly higher compared to that in healthy individuals (P < 0.01). The apoptosis of human OA chondrocytes was inhibited when silencing UBE2M and increased when overexpressing UBE2M. XAV939, as a tankyrase 1 inhibitor, could block the signaling pathway of Wnt/β-catenin, which significantly reversed the change introduced by UBE2M. The expression level of cytoplasmic β-catenin in siUBE2M cells dramatically increased, and the expression levels of nuclear β-catenin, cleaved caspase-3 (C-caspase-3), and MMP13 remarkably downregulated. Moreover, the ubiquitination of Axin was enhanced by the overexpression of UBE2M. The expression level of Axin significantly decreased in OA chondrocytes with UBE2M overexpression and increased after MG132 treatment. Moreover, UBE2M enhanced the apoptosis of OA chondrocytes by activating the Axin-dependent Wnt/β-catenin pathway. In this process, UBE2M downregulated Axin in an ubiquitination-dependent degradation pathway and subsequently activated Wnt/β-catenin signaling.

P97 Inhibitor CB-5083 Blocks ERAD in Trypanosoma brucei

Misfolded proteins trapped in the endoplasmic reticulum (ER) are specifically recognized and retrotranslocated to the cytosol by the ER-Associated Degradation (ERAD) system and delivered to the proteasome for destruction. This process was recently described in Trypanosoma brucei (T. brucei) using the misfolded epitope tagged Transferrin Receptor subunits ESAG7:Ty and HA:ESAG6 (HA:E6). Critical to this work was the proteasomal inhibitor MG132. However, MG132 has off-target inhibitory effects on lysosomal Cathepsin L that could cause misinterpretation of turnover results. Here, we evaluate an orally bioavailable p97 inhibitor, CB-5083, for use in T. brucei. p97 is a ubiquitous protein involved in many cellular events including the membrane extraction step of ERAD. CB-5083 strongly inhibits turnover of HA:E6, with comparable protein recovery to MG132 treatment. Interestingly, little deglycosylated cytoplasmic species accumulates, though it normally emerges with MG132 treatment. This suggests that CB-5083 blocks ERAD upstream of the proteasome, as expected for inhibition of the trypanosomal p97 orthologue TbVCP. Under CB-5083 treatment, HA:E6 is also strongly membrane-associated, suggesting ER localization. Finally, we provide an experimental example where CB-5083 treatment offers clarity to the off-target effects of MG132 treatment.

Arabidopsis COPPER TRANSPORTER 1 undergoes degradation in a proteasome-dependent manner.

The essential nutrient copper is toxic in excess. Therefore, plants must tightly control copper uptake and distribution. Arabidopsis thaliana high-affinity copper transporters (COPTs) mediate copper uptake, partitioning, and redistribution. Here we show that COPT1 localizes to the plasma membrane and endoplasmic reticulum in stably transgenic plants expressing a COPT1-green fluorescent protein (GFP) fusion protein, and the fusion protein is rapidly degraded upon plant exposure to excess copper. MG132 treatment largely abolished copper-induced degradation of COPT1, implying a link between the proteasome and COPT1 activity in modulating copper uptake. Co-immunoprecipitation analyses revealed that COPT1 cannot be ubiquitinated in the presence of excess copper and MG132. Through site-directed mutagenesis, we identified Lys159 in the C-terminal cytoplasmic tail of COPT1 as critical for copper acquisition, but not for copper-mediated down-regulation of COPT1, in plants. Furthermore, pharmacological analysis showed that treatment with a vesicle trafficking inhibitor or a V-ATPase inhibitor does not alter the subcellular dynamics of COPT1-GFP, consistent with the absence of a connection between the endosomal recycling/vacuolar system and COPT1 degradation. Together, our data suggest that proteasomal degradation rather than vacuolar proteolysis is important for the regulation of copper transport to maintain copper homeostasis in plants.

LncRNA DRAIC inhibits proliferation and metastasis of gastric cancer cells through interfering with NFRKB deubiquitination mediated by UCHL5

BackgroundLong non-coding RNA (lncRNA) as a widespread and pivotal epigenetic molecule participates in the occurrence and progression of malignant tumors. DRAIC, a kind of lncRNA whose coding gene location is on 15q23 chromatin, has been found to be weakly expressed in a variety of malignant tumors and acts as a suppressor, but its characteristics and role in gastric cancer (GC) remain to be elucidated.MethodsSixty-seven primary GC tissues and paired paracancerous normal tissues were collected. Bioinformatics is used to predict the interaction molecules of DRAIC. DRAIC and NFRKB were overexpressed or interfered exogenously in GC cells by lentivirus or transient transfection. Quantitative real-time PCR (qPCR) and western blotting were used to evaluate the expression of DRAIC, UCHL5 and NFRKB. The combinations of DRAIC and NFRKB or UCHL5 and NFRKB were verified by RNA-IP and Co-IP assays. Ubiquitination-IP and the treatment of MG132 and CHX were used to detect the ubiquitylation level of NFRKB. The CCK-8 and transwell invasion and migration assays measured the proliferation, migration and invasion of GC cells.ResultsDRAIC is down-regulated in GC tissues and cell lines while its potential interacting molecules UCHL5 and NFRKB are up-regulated, and DRAIC is positively correlated with NFRKB protein instead of mRNA. Lower DRAIC and higher UCHL5 and NFRKB indicated advanced progression of GC patients. DRAIC could increase NFRKB protein significantly instead of NFRKB mRNA and UCHL5, and bind to UCHL5. DRAIC combined with UCHL5 and attenuated binding of UCHL5 and NFRKB, meanwhile promoting the degradation of NFRKB via ubiquitination, and then inhibited the proliferation and metastasis of GC cells, which can be rescued by oeNFRKB.ConclusionDRAIC suppresses GC proliferation and metastasis via interfering with the combination of UCHL5 and NFRKB and mediating ubiquitination degradation.

WWP2 ameliorates acute kidney injury by mediating p53 ubiquitylation and degradation.

E3 ubiquitin ligase gene, WWP2, is associated with acute kidney injury (AKI). This research was conducted to explore the role of WWP2 in AKI. AKI cell model was produced in human renal proximal tubular epithelial cell line (HK-2) by ischemia-reperfusion (IR) injury. CCK8 and flow cytometry assay were performed to explore the influence of WWP2 overexpression on cell proliferation and apoptosis of IR-induced HK-2 cells. Quantitative real-time PCR and immunoblotting (IB) were performed to assess the gene and protein expression. Then, the influence of WWP2 on p53 ubiquitylation and degradation was estimated by immunoprecipitation assay. Our data indicated that WWP2 was down-regulated and p53 was up-regulated in IR-induced HK-2 cells. WWP2 overexpression promoted proliferation and inhibited apoptosis of IR-induced HK-2 cells. And WWP2 interacted with p53 and regulated p53 ubiquitylation and degradation. Furthermore, the influence of WWP2 on cell proliferation and apoptosis was rescued by MG132 (proteasome inhibitor) treatment. In conclusion, our work described for the first time the role of WWP2 in AKI, showing that WWP2 ameliorated AKI by mediating p53 ubiquitylation and degradation. Moreover, the study offers some important insights into the occurrence of AKI and WWP2 may be a novel target of AKI treatment. SIGNIFICANCE OF THE STUDY: Our data elaborates that WWP2 has protective effect against AKI by mediating p53 ubiquitylation and degradation. Thus, WWP2 might be a therapeutic target for AKI.

P23 Co‐chaperone Protects Aryl Hydrocarbon Receptor (AHR) from the Autophagy‐Mediated Degradation

The aryl hydrocarbon receptor (AHR) is first discovered as a transcription factor in response to toxic compounds such as dioxins. It also plays important roles in organ development, immune responses, metabolisms and carcinogenesis. Without ligand activation, AHR resides in the cytosol and is accompanied by Hsp90, p23 and XAP2. AHR is degraded via the 26S proteasome after ligand activation. However, it is still unclear how basal AHR protein levels are regulated. We previously discovered that AHR protein levels are lower in p23 stable knockdown (p23KD) cell lines when there is no ligand treatment. Here we report that treatment of proteasome inhibitor MG132 could not restore AHR in p23KD HeLa cells while the autophagy inhibitor chloroquine increased AHR proteins levels to 2.5 to 3‐fold in p23KD HeLa cells and 2‐fold in WT HeLa cells. Similar results were observed when cells were treated with other autophagy inhibitors (bafilomycin A1 and 3MA). There was higher autophagic flux in p23KD HeLa cells than in WT HeLa cells. Results from the co‐immunoprecipitation experiment and proximity ligation assay showed interaction between AHR and LC3B (LC3B‐II), which is an important protein in macroautophagy. These results support that the AHR protein levels are regulated by autophagy and p23 can protect AHR from the autophagy‐mediated degradation. Additional data will be presented to address the mechanisms of basal AHR protein degradation via autophagy.Support or Funding InformationNIH R15ES023104