MG132 Treatment Research Articles

Altered MARCH1 ubiquination-regulated dendritic cell immune functions during the early stage of zymosan-induced multiple organ dysfunction syndrome (MODS) in mice

Using a zymosan-induced mouse model of multiple organ dysfunction syndrome (MODS), we previously found profound increases in spleen immune cells’ expressions of ubiquitin and MHC-II molecules and increased CD11c+ dendritic cells (DCs) within 24h of zymosan injection. We postulated that the early stage of MODS altered DCs function via an ubiquitination-associated mechanism. We intraperitoneally injected zymosan into 100 male C57BL/6 mice (0.8mg/g) and randomly divided them into 5 groups based on the days after injection (20mice/group): 1d, 3d, 5d, 7d, and 10d. Mice were examined for spleen CD11c+ DC functions at the indicated days. Untreated mice were used for normal spleen tissue and T cell samples. By qPCR, IL-12 and TNF-α mRNA expressions in spleen CD11c+ DCs were significantly increased in MODS 1d mice; on subsequent days post-injection, these mRNA levels gradually returned to control levels. The same patterns were found for MODS mice DCs induction of untreated mouse T cells proliferation and IL-2 and IFN-γ mRNA expressions. When T cell functions were examined using MODS 1d DCs with and without MG132 treatment, an inhibitor of ubiquitinated protein degradation, T cell functional activities were enhanced by DCs treated with MG132. MODS 1d DCs also had significantly reduced MARCH1 mRNA expression, a key ubiquitin ligase that regulates DCs MHC-II expression. Silencing DCs MARCH1 expression with siRNA resulted in enhancing their induction of T cell functional activities. Using co-immunoprecipitation, Western blot, and flow cytometry assays, we deduced that MARCH1 ubiquitinated DC surface MHC-II molecules to regulate DC's immune functions in MODS mice. Our results suggest that aberrant degradation of spleen DCs MARCH1-mediated ubiquitinated proteins is involved during the earliest stage of MODS development.

Hsp60 Chaperonin Acts as Barrier to Pharmacologically Induced Oxidative Stress Mediated Apoptosis in Tumor Cells with Differential stress Response

Mitochondrial functions play a central role in energy metabolism and provide survival fitness to both normal and tumor cells. Mitochondrial chaperonin Hsp60 is involved in both pro- and anti-apoptotic functions, but how Hsp60 senses the mitochondria selective oxidative stress response is unknown. In this study, by using rotenone, an irreversible inhibitor of oxidative phosphorylation against IMR-32 and BC-8 tumor cells containing differential heat shock transcriptional machinery, we studied whether the oxidative stress response is related to Hsp60. The accelerated cytotoxicity in response to rotenone has been correlated with enhanced production of O2•−, H2O2, reactive oxygen species, and Hsp60 translocation from the mitochondria to the cytoplasm. The inability of cells to resist oxidative stress mediated Hsp60 translocation appeared to depend on mitochondrial oxyradical scavenging system and Bax translocation. A delayed oxidative stress response in hsp60 shRNA-treated cells was found to be due to increased mitochondrial translocation of Hsp60 on shRNA pre-sensitization. Overexpression of Hsp60 failed to protect cells from oxidative stress due to a lack of its mitochondrial retention upon post-rotenone treatment. These results also revealed that Hsp60 mitochondrial localization is indispensable for decreasing O2•− levels, but not H2O2 and ROS levels. However, cycloheximide treatment alone induced Hsp60 translocation, while rotenone combination delayed this translocation. In contrast to oxidative stress, MG132 and 17AAG treatments showed mitochondrial retention of Hsp60; however, MG132 combination either with hsp60 shRNA or 17AAG induced its translocation. Additionally, overexpression of Huntingtin gene also resulted in Hsp60 mitochondrial accumulation. We suggest that Hsp60 may act as a barrier to pharmacological targeting of mitochondria.

Effects of Expression of Exogenous Cyclin G1 on Proliferation of Human Endometrial Carcinoma Cells

Cyclin G1 is the only cyclin that has either positive or negative effects on cell growth. Our previous study found decreased expression of cyclin G1 in human endometrial carcinoma tissues compared with normal endometrial tissues. The study aimed to evaluate cyclin G1 expression and its effect on proliferation of human endometrial carcinoma cells (ECCs). Cyclin G1-GFP (green fluorescence protein) plasmid was constructed and transfected into various differentiated human ECCs, including Ishikawa, HEC-1-B and KLE cells, and proliferation of the transfected cells was determined by the CCK-8 method. Exogenous cyclin G1 mRNA and protein were measured by RT-PCR and Western blot, respectively, and GFP signal was monitored by fluorescence microscopy. Chinese hamster ovary (CHO) cells were transfected with the same constructs as a cell control. Cyclin G1-GFP-transfected Ishikawa cells were further treated with MG132, an inhibitor of proteasome, to analyze if low expression of cyclin G1 is related to its abnormal degradation in ECCs. Ectopic expression of exogenous cyclin G1 was found to significantly suppress the proliferation of Ishikawa and HEC-1-B cells but not KLE cells. Compared with cyclin G1-transfected CHO cells, exogenous cyclin G1 protein expression was low in Ishikawa and HEC-1-B cells, and was undetectable in KLE cells. However, all ECC lines and CHO cells expressed similar levels of exogenous cyclin G1 and GFP mRNA. MG132 treatment increased cyclin G1 protein expression in cyclin G1-GFP-transfected Ishikawa cells. This is the first study to present evidence to suggest that cyclin G1 exerts negative control on proliferation of ECCs. Exogenous cyclin G1 shows different protein expression levels in ECCs with different malignancies, and cyclin G1 protein is highly unstable and is rapidly degraded in ECCs.

Abstract P5-04-02: DYRK2 regulates breast cancer invasion via Snail/E-cadherin pathway

Abstract Introduction: Epithelial-Mesenchymal Transition (EMT) is characterized by the loss of cellular adhesion molecule E-cadherin, and plays a fundamental role during early steps of breast cancer invasion. An important regulator of E-cadherin expression is Snail, a zinc finger transcriptional repressor. Snail is posphorylated by GSK3β and then degraded by βTrCP mediated ubiquitination. Here we found another kinase, DYRK2, regulates the stability of Snail. Knockdown of DYRK2 promoted the EMT and cancer invasion both in vitro and in vivo experiments. Methods: Cell lines: MCF-7 cells were transfected with pSuper vector (pSuper control) and pSuper shRNA DYRK2 (shRNA-DYRK2) with G418 to isolate stable cell lines. Immunoblotting: Expression of pertinent molecular markers was determined by western blotting. RT-PCR: RNA was extracted using TRIsure and 500 ng of total RNA was amplified using Primer Script One Step RT-PCR Kit Ver.2. Invasion assay: Various cancer cell lines were seeded on the top of the upper chamber with serum free medium while the bottom chambers were filled with medium containing 10% FBS. In vivo metastasis assay: Cells in PBS were injected into the left ventricle of 7-week-old female nude mice. Metastases to distant organs were confirmed by IVIS2000. Immunohistochemistry: We acquired paraffin-embedded tissue sections from primary breast tumor cores and from Jikei University of Medicine. Immunohistochemistry for DYRK2 was performed. Results: In MCF-7 cells, knockdown of DYRK2 increased Snail in the protein level but not in mRNA level. Upon DYRK2 knockdown, MG132 treatment had no effect on additional increase in Snail. DYRK2 knockdown decreased Snail ubiquitination. Stable DYRK2 depletion led to Snail accumulation and E-cadherin abrogation. Fibroblast marker, Vimentin, emerged in DYRK2 depleted cells. For further analysis, we carried out invasion assays. The invasion potential in DYRK2-depleted cells was substantially higher than that in control cells. In xenograft model, we used nude mice received intracardial injections of pSuper control or shRNA-DYRK2 cells. 6 weeks after injections, a significant increase in bone and lung metastasis was observed in shRNA-DYRK2 group. The patients with tumors expressed low DYRK2 showed significantly poor prognosis. Conclusion: In breast cancer cells, Snail is phosphorylated by DYRK2 and then acquires the ability to be ubiquitinated. In the absence of DYRK2, Snail is unable to be degraded by ubiquitin-proteasome machinery. Accumulation of Snail promotes EMT and cancer invasion, so low expression of DYRK2 leads to poor prognosis in breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-04-02.

Therapeutic effect of MG-132 on diabetic cardiomyopathy is associated with its suppression of proteasomal activities: roles of Nrf2 and NF-κB

MG-132, a proteasome inhibitor, can upregulate nuclear factor (NF) erythroid 2-related factor 2 (Nrf2)-mediated antioxidative function and downregulate NF-κB-mediated inflammation. The present study investigated whether through the above two mechanisms MG-132 could provide a therapeutic effect on diabetic cardiomyopathy in the OVE26 type 1 diabetic mouse model. OVE26 mice develop hyperglycemia at 2-3 wk after birth and exhibit albuminuria and cardiac dysfunction at 3 mo of age. Therefore, 3-mo-old OVE26 diabetic and age-matched control mice were intraperitoneally treated with MG-132 at 10 μg/kg daily for 3 mo. Before and after MG-132 treatment, cardiac function was measured by echocardiography, and cardiac tissues were then subjected to pathological and biochemical examination. Diabetic mice showed significant cardiac dysfunction, including increased left ventricular systolic diameter and wall thickness and decreased left ventricular ejection fraction with an increase of the heart weight-to-tibia length ratio. Diabetic hearts exhibited structural derangement and remodeling (fibrosis and hypertrophy). In diabetic mice, there was also increased systemic and cardiac oxidative damage and inflammation. All of these pathogenic changes were reversed by MG-132 treatment. MG-132 treatment significantly increased the cardiac expression of Nrf2 and its downstream antioxidant genes with a significant increase of total antioxidant capacity and also significantly decreased the expression of IκB and the nuclear accumulation and DNA-binding activity of NF-κB in the heart. These results suggest that MG-132 has a therapeutic effect on diabetic cardiomyopathy in OVE26 diabetic mice, possibly through the upregulation of Nrf2-dependent antioxidative function and downregulation of NF-κB-mediated inflammation.

Targeting TCTP as a New Therapeutic Strategy in Castration-resistant Prostate Cancer

Heat shock protein 27 (Hsp27) is highly overexpressed in castration-resistant prostate cancer (CRPC) and an antisense inhibitor (OGX-427) is currently in phase II clinical trials. In order to understand mechanisms of action of Hsp27 and find new therapeutic targets specific of CRPC, we screened for Hsp27 client proteins. Here, we report that translationally controlled tumor protein (TCTP) is a new Hsp27 client protein involved in Hsp27 cytoprotection. We found that TCTP expression is absent or weak in normal prostate cells, moderately expressed in 18.5% of treatment naive PC, and becomes uniformly and strongly expressed in 75% of CRPC. To define TCTP function, we developed and worldwide patented a TCTP antisense oligonucleotide (ASO). Interestingly, we found that CRPC progression correlates with TCTP overexpression and loss of P53. TCTP knockdown restored P53 expression and function, suggesting that castration-sensitivity is directly linked to P53 expression. Collectively, these findings provide a new Hsp27 cytoprotection mechanism in CRPC, and preclinical proof-of-concept that combining ASO-mediated TCTP knockdown with castration and/or docetaxel therapy could serve as a novel strategy to treat CRPC, with no or little toxicity for normal prostate cells.

Systemic Perturbation of the ERK Signaling Pathway by the Proteasome Inhibitor, MG132

Inhibition of the ubiquitin-proteasome protein degradation pathway has been identified as a viable strategy for anti-tumor therapy based on its broad effects on cell proliferation. By the same token, the variety of elicited effects confounds the interpretation of cell-based experiments using proteasome inhibitors such as MG132. It has been proposed that MG132 treatment reduces growth factor-stimulated phosphorylation of extracellular signal-regulated kinases (ERKs), at least in part through upregulation of dual specificity phosphatases (DUSPs). Here, we show that the effects of MG132 treatment on ERK signaling are more widespread, leading to a reduction in activation of the upstream kinase MEK. This suggests that MG132 systemically perturbs the intracellular phosphoproteome, impacting ERK signaling by reducing phosphorylation status at multiple levels of the kinase cascade.

Nicotinamide mononucleotide adenylyltransferase maintains active zone structure by stabilizing Bruchpilot

Active zones are specialized presynaptic structures critical for neurotransmission. We show that a neuronal maintenance factor, nicotinamide mononucleotide adenylyltransferase (NMNAT), is required for maintaining active zone structural integrity in Drosophila by interacting with the active zone protein, Bruchpilot (BRP), and shielding it from activity-induced ubiquitin-proteasome-mediated degradation. NMNAT localizes to the peri-active zone and interacts biochemically with BRP in an activity-dependent manner. Loss of NMNAT results in ubiquitination, mislocalization and aggregation of BRP, and subsequent active zone degeneration. We propose that, as a neuronal maintenance factor, NMNAT specifically maintains active zone structure by direct protein-protein interaction.

Proteasome inhibition attenuates heart failure during the late stages of pressure overload through alterations in collagen expression

Although the role of the ubiquitin–proteasome system (UPS) in cardiac hypertrophy induced by pressure overload has been consistently studied, the fundamental importance of the UPS in cardiac fibrosis has received much less attention. Our previous study found that proteasome inhibitor (MG132) treatment attenuated cardiac fibrosis and heart failure during the early and middle stages of pressure overload. However, the effects of this inhibitor on late-stage pressure overload hearts remain unclear and controversial. The present study was designed to investigate the effects and possible mechanisms of MG132 on cardiac fibrosis and dysfunction during the late stages of pressure overload. Male Sprague Dawley rats with abdominal aortic constriction (AAC) or a sham operation received an intraperitoneal injection of MG132 (0.1mgkg−1day−1) or vehicle for 16 weeks. Left ventricular (LV) function, collagen deposition and Ang II levels were evaluated at study termination. Ang II-stimulated adult rat cardiac fibroblasts were utilized to examine the effects of MG132 on collagen synthesis and the relationship between the renin–angiotensin–aldosterone system (RAAS) and the UPS. MG132 treatment attenuated ventricular dysfunction by suppressing cardiac fibrosis rather than inhibiting cardiac hypertrophy during the late-stages of pressure overload. We also found that Ang II activates UPS in the heart and MG132 attenuates Ang II-induced collagen synthesis via suppression of the NF-κB/TGF-β/Smad2 signaling pathways. Proteasome inhibition therefore could provide a new promising therapeutic strategy to prevent cardiac fibrosis and progression of heart failure even during the late-stages of pressure overload.

Potential role for Nrf2 activation in the therapeutic effect of MG132 on diabetic nephropathy in OVE26 diabetic mice

Oxidative stress is a major cause of diabetic nephropathy. Upregulation of the key antioxidative transcription factor, nuclear factor-erythroid 2-related factor 2 (Nrf2), was found to prevent the development of diabetic nephropathy. The present study was designed to explore the therapeutic effect of Nrf2 induced by proteasomal inhibitor MG132 at a low dose (10 μg/kg) on diabetic nephropathy. Transgenic type 1 diabetic (OVE26) mice displayed renal dysfunction with albuminuria by 3 mo of age, at which time MG132 treatment was started. After 3-mo treatment with MG132, renal function, morphology, and biochemical changes were examined with real-time PCR, Western blotting, and immunohistochemical examination. Compared with age-matched, nontreated diabetic mice, MG132-treated diabetic mice showed significant improvements in terms of renal structural and functional alterations. These therapeutic effects were associated with increased Nrf2 expression and transcriptional upregulation of Nrf2-regulated antioxidants. Mechanistic study using human renal tubular HK11 cells confirmed the role of Nrf2, as silencing the Nrf2 gene with its specific siRNA abolished MG132 prevention of high-glucose-induced profibrotic response. Furthermore, diabetes was found to significantly increase proteasomal activity in the kidney, an effect that was significantly attenuated by 3 mo of treatment with MG132. These results suggest that MG132 upregulates Nrf2 function via inhibition of diabetes-increased proteasomal activity, which can provide the basis for the therapeutic effect of MG132 on the kidney against diabetes-induced oxidative damage, inflammation, fibrosis, and eventual dysfunction.

TRIM38 negatively regulates TLR3-mediated IFN-β signaling by targeting TRIF for degradation.

Toll-like receptors (TLRs) mediated immune response is crucial for combating pathogens and must be tightly controlled. Tripartite motif (TRIM) proteins are a family of proteins that is involved in a variety of biological and physiological processes. Some members of the TRIM family are important in the regulation of innate immunity. Although it has been shown that TRIM38 negatively regulates innate immunity, the mechanisms by which it does so have not been fully addressed. In this study, we demonstrated that TRIM38 negatively regulates Toll-like receptor 3 (TLR3)-mediated type I interferon signaling by targeting TIR domain-containing adaptor inducing IFN-β (TRIF). We found that overexpression of TRIM38 inhibits TLR3-mediated type I interferon signaling, whereas knockdown of TRIM38 has the reverse effects. We further showed that TRIM38 targets TRIF, a critical adaptor protein downstream of TLR3. TRIF is co-immunoprecipitated with TRIM38, and domain mapping experiments show that PRYSPRY of TRIM38 interacts with the N-terminus of TRIF. Overexpression of TRIM38 decreased expression of overexpressed and endogenous TRIF. This effect could be inhibited by MG132 treatment. Furthermore, the RING/B-box domain of TRIM38 is critical for K48-linked polyubiquitination and proteasomal degradation of TRIF. Collectively, our results suggest that TRIM38 may act as a novel negative regulator for TLR3-mediated type I interferon signaling by targeting TRIF for degradation.

A Proteasome Inhibitor-stimulated Nrf1 Protein-dependent Compensatory Increase in Proteasome Subunit Gene Expression Reduces Polycomb Group Protein Level

The polycomb group (PcG) proteins, Bmi-1 and Ezh2, are important epigenetic regulators that enhance skin cancer cell survival. We recently showed that Bmi-1 and Ezh2 protein level is reduced by treatment with the dietary chemopreventive agents, sulforaphane and green tea polyphenol, and that this reduction involves ubiquitination of Bmi-1 and Ezh2, suggesting a key role of the proteasome. In the present study, we observe a surprising outcome that Bmi-1 and Ezh2 levels are reduced by treatment with the proteasome inhibitor, MG132. We show that this is associated with a compensatory increase in the level of mRNA encoding proteasome protein subunits in response to MG132 treatment and an increase in proteasome activity. The increase in proteasome subunit level is associated with increased Nrf1 and Nrf2 level. Moreover, knockdown of Nrf1 attenuates the MG132-dependent increase in proteasome subunit expression and restores Bmi-1 and Ezh2 expression. The MG132-dependent loss of Bmi-1 and Ezh2 is associated with reduced cell proliferation, accumulation of cells in G(2), and increased apoptosis. These effects are attenuated by forced expression of Bmi-1, suggesting that PcG proteins, consistent with a prosurvival action, may antagonize the action of MG132. These studies describe a compensatory Nrf1-dependent, and to a lesser extent Nrf2-dependent, increase in proteasome subunit level in proteasome inhibitor-treated cells and confirm that PcG protein levels are regulated by proteasome activity.

Serine 111 Phosphorylation Regulates OCT4A Protein Subcellular Distribution and Degradation

Embryonic stem cell self-renewal properties are attributed to critical amounts of OCT4A, but little is known about its post-translational regulation. Sequence analysis revealed that OCT4A contains five putative ERK1/2 phosphorylation sites. Consistent with the hypothesis that OCT4A is a putative ERK1/2 substrate, we demonstrate that OCT4A interacts with ERK1/2 by using both in vitro GST pulldown and in vivo co-immunoprecipitation assays. MS analysis identified phosphorylation of OCT4A at Ser-111. To investigate the possibility that ERK1/2 activation can enhance OCT4A degradation, we analyzed endogenous ubiquitination in cells transfected with FLAG-OCT4A alone or with constitutively active MEK1 (MEK1(CA)), and we observed that the extent of OCT4 ubiquitination was clearly increased when MEK1(CA) was coexpressed and that this increase was more evident after MG132 treatment. These results suggest an increase in OCT4A ubiquitination downstream of MEK1 activation, and this could account for the protein loss observed after FGF2 treatment and MEK1(CA) transfection. Understanding and controlling the mechanism by which stem cells balance self-renewal would substantially advance our knowledge of stem cells.

Effects of Combined Treatment of MG132 and Scriptaid on Early and Term Development of Porcine Somatic Cell Nuclear Transfer Embryos

Although improving, the efficiency of producing offspring by somatic cell nuclear transfer (SCNT) is still low (<1.5%). Our laboratory has demonstrated that histone deacetylase inhibitor (Scriptaid) treatment of reconstructed embryos enhances blastocyst formation and cloning efficiency in pigs. It has also been shown that proteasomal inhibitor MG132 treatment for 2 h after activation of oocytes increases blastocyst rate and pregnancy rate. The current experiment was carried out to determine the effects of combined MG132 and Scriptaid treatment on early embryo development in vitro and on term development in vivo. Immediately after electrofusion and activation, SCNT oocytes were treated with 0, 1, or 10 μM MG132 for 2 h in the presence of 500 nM Scriptaid, washed and treated with Scriptaid for an additional 14 to 15 h, then cultured in porcine zygote medium 3 (PZM3) until day 6. There was no difference in percent cleavage (58.1 ± 7.2%, 62.7 ± 7.2%, and 62.5 ± 7.2%) on day 2, or total cell number (23.1 ± 2.2, 24.0 ± 2.0, and 24.5 ± 2.3 for the 0, 1, and 10 μM MG132 groups, respectively) on day 6 among the three groups. Interestingly, there was no difference in percentage of blastocysts between the 0 (18.5±4.7%) and 1 (25.1 ± 4.7%) μM MG132 treatment groups; however, compared with the 10 μM MG132 group (14.0 ± 4.7%), more embryos from the 1 μM MG132 group developed into blastocysts (p<0.05). To determine the effects on term development in vivo, two MG132 groups were included (0 and 1 μM MG132), and embryos were treated as above and transferred into synchronized surrogates after treatment. There was no difference in the oocyte-donor cell fusion rate, number of embryos transferred, pregnancy rate at days 28, 60, and at term, pigs delivered per embryo transfer, litter size, body weight at birth, nor cloning efficiency between the Scriptaid-alone control and MG132+Scriptaid combined groups. In summary, the combined treatment of MG132 and Scriptaid did not improve term development compared to Scriptaid treatment alone.

Quiescent fibroblasts are protected from proteasome inhibition–mediated toxicity

Proteasome inhibition is used as a treatment strategy for multiple types of cancers. Although proteasome inhibition can induce apoptotic cell death in actively proliferating cells, it is less effective in quiescent cells. In this study, we used primary human fibroblasts as a model system to explore the link between the proliferative state of a cell and proteasome inhibition-mediated cell death. We found that proliferating and quiescent fibroblasts have strikingly different responses to MG132, a proteasome inhibitor; proliferating cells rapidly apoptosed, whereas quiescent cells maintained viability. Moreover, MG132 treatment of proliferating fibroblasts led to increased superoxide anion levels, juxtanuclear accumulation of ubiquitin- and p62/SQSTM1-positive protein aggregates, and apoptotic cell death, whereas MG132-treated quiescent cells displayed fewer juxtanuclear protein aggregates, less apoptosis, and higher levels of mitochondrial superoxide dismutase. In both cell states, reducing reactive oxygen species with N-acetylcysteine lessened protein aggregation and decreased apoptosis, suggesting that protein aggregation promotes apoptosis. In contrast, increasing cellular superoxide levels with 2-methoxyestradiol treatment or inhibition of autophagy/lysosomal pathways with bafilomycin A1 sensitized serum-starved quiescent cells to MG132-induced apoptosis. Thus, antioxidant defenses and the autophagy/lysosomal pathway protect serum-starved quiescent fibroblasts from proteasome inhibition-induced cytotoxicity.

TNFAIP1 interacts with KCTD10 to promote the degradation of KCTD10 proteins and inhibit the transcriptional activities of NF-κB and AP-1

The broad-complex, tramtrack, and bric-a-brac/poxvirus and zinc finger domain-containing protein tumor necrosis factor, alpha-induced protein 1 (TNFAIP1) was first identified as a gene whose expression can be induced by the tumor necrosis factor alpha. Some studies showed that TNFAIP1 may function in DNA replication, apoptosis and human diseases. However, the definite functions and the mechanisms of TNFAIP1 are poorly known. In this study, we performed a yeast two-hybrid assay and used TNFAIP1 as the bait to screen human brain cDNA library. Potassium channel tetramerisation domain containing 10 (KCTD10) was identified as TNFAIP1-interacting partner. The KCTD10-TNFAIP1 interaction was then confirmed by the in vitro GST pull-down assays and the in vivo co-immunoprecipitation and colocalization assays. In addition, protein degradation and ubiquitin assays revealed TNFAIP1 overexpression resulted in ubiquitin-mediated degradation of KCTD10 proteins, which was significantly alleviated with the proteasome inhibitor MG132 treatment. Furthermore, transient transfection assays with two reporters showed that TNFAIP1 and KCTD10 inhibited the transcriptional activities of nuclear factor kappa B (NF-κB) and activating protein-1 reporters. Taken together, our results indicated the novel interaction and function between KCTD10 and TNFAIP1 in human PDIP1 family.

Severe Acute Respiratory Syndrome Coronavirus Replication Is Severely Impaired by MG132 due to Proteasome-Independent Inhibition of M-Calpain

The ubiquitin-proteasome system (UPS) is involved in the replication of a broad range of viruses. Since replication of the murine hepatitis virus (MHV) is impaired upon proteasomal inhibition, the relevance of the UPS for the replication of the severe acute respiratory syndrome coronavirus (SARS-CoV) was investigated in this study. We demonstrate that the proteasomal inhibitor MG132 strongly inhibits SARS-CoV replication by interfering with early steps of the viral life cycle. Surprisingly, other proteasomal inhibitors (e.g., lactacystin and bortezomib) only marginally affected viral replication, indicating that the effect of MG132 is independent of proteasomal impairment. Induction of autophagy by MG132 treatment was excluded from playing a role, and no changes in SARS-CoV titers were observed during infection of wild-type or autophagy-deficient ATG5(-/-) mouse embryonic fibroblasts overexpressing the human SARS-CoV receptor, angiotensin-converting enzyme 2 (ACE2). Since MG132 also inhibits the cysteine protease m-calpain, we addressed the role of calpains in the early SARS-CoV life cycle using calpain inhibitors III (MDL28170) and VI (SJA6017). In fact, m-calpain inhibition with MDL28170 resulted in an even more pronounced inhibition of SARS-CoV replication (>7 orders of magnitude) than did MG132. Additional m-calpain knockdown experiments confirmed the dependence of SARS-CoV replication on the activity of the cysteine protease m-calpain. Taken together, we provide strong experimental evidence that SARS-CoV has unique replication requirements which are independent of functional UPS or autophagy pathways compared to other coronaviruses. Additionally, this work highlights an important role for m-calpain during early steps of the SARS-CoV life cycle.

The Arabidopsis F-box protein AtFBS1 interacts with 14-3-3 proteins

AtFBS1 is an F-box protein whose transcript accumulates in response to biotic and abiotic stresses. Previous evidence suggests that a postranscriptional event regulates AtFBS1 expression [1]. We now found that AtFBS1 interacts with 14-3-3 proteins through its amino-terminus and the F-box motif. Deletion of any of these regions abolishes the interaction between AtFBS1 and 14-3-3 proteins. On the other hand, the treatment with the proteasome inhibitor MG132 or the deletion of the F-box from AtFBS1 increases β-glucuronidase (GUS) activity in plants containing a translational fusion of AtFBS1 with the GUS reporter gene, indicating that AtFBS1 is degraded by the 26S proteasome. MG132 treatment of seedlings containing a gene fusion between AtFBS1 and the TAP (Tandem Affinity Purification) cassette causes an increase in the half-life of the protein. In an attempt to understand the role of 14-3-3 interactions, we treated Arabidopsis seedlings with 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranosyl 5′-monophosphate (AICAR), an inhibitor of 14-3-3 client interactions. We observed an increase in AtFBS1-TAP stability as a consequence of AICAR treatment. Based on these data we propose that 14-3-3 proteins promote the dimerization of SCFAtFBS1. This also may enhance the AtFBS1 autoubiquitination activity and its degradation by the 26S proteasome. AICAR also affects Cullin1 (CUL1) modification by RUB1, which would provide an additional element to the effect of this compound on AtFBS1 stability.

Translational Homeostasis via the mRNA Cap-Binding Protein, eIF4E

Translational control of gene expression plays a key role in many biological processes. Consequently, the activity of the translation apparatus is under tight homeostatic control. eIF4E, the mRNA 5' cap-binding protein, facilitates cap-dependent translation and is a major target for translational control. eIF4E activity is controlled by a family of repressor proteins, termed 4E-binding proteins (4E-BPs). Here, we describe the surprising finding that despite the importance of eIF4E for translation, a drastic knockdown of eIF4E caused only minor reduction in translation. This conundrum can be explained by the finding that 4E-BP1 is degraded in eIF4E-knockdown cells. Hypophosphorylated 4E-BP1, which binds to eIF4E, is degraded, whereas hyperphosphorylated 4E-BP1 is refractory to degradation. We identified the KLHL25-CUL3 complex as the E3 ubiquitin ligase, which targets hypophosphorylated 4E-BP1. Thus, the activity of eIF4E is under homeostatic control via the regulation of the levels of its repressor protein 4E-BP1 through ubiquitination.

Androgen deprivation causes truncation of the C‐terminal region of androgen receptor in human prostate cancer LNCaP cells

The androgen receptor (AR) acts as a ligand-dependent transcription factor, whereas mutant AR lacking the C-terminal ligand-binding domain functions in a ligand-independent manner. In the present study we report that the C-terminal truncated AR, which we named AR-NH1 (the N-terminal fragment of AR cleaved in the neighborhood of helix 1 of the ligand-binding domain), is produced in LNCaP prostatic carcinoma cells. The AR-NH1 of ~90 kDa was observed in an androgen-independent LNCaP subline and was further accumulated by the proteasome inhibitor MG132. MG132 treatment caused the accumulation of AR-NH1 even in parent LNCaP cells. AR-NH1 was produced in the absence of ligand or in the presence of the AR antagonist bicalutamide, whereas AR agonists suppressed its production. AR-NH1 was detected with different AR antibodies recognizing amino acid residues 1-20 and 300-316 and was also generated from exogenous AR. Both siRNA-mediated AR knockdown and treatment with a serine protease inhibitor (4-(2-aminoethyl)-benzenesulfonyl fluoride) reduced AR-NH1 levels. According to the predicted cleavage site (between amino acid residues 660-685) and its nuclear localization, it is assumed that AR-NH1 functions as a constitutively active transcription factor. These data suggest that AR-NH1 is produced under hormone therapy and contributes to the development of castration-resistant prostate cancer due to its ligand-independent transcriptional activity.