The Synergistic Effect of Reduced Graphene Oxide and Proteasome Inhibitor in the Induction of Apoptosis through Oxidative Stress in Breast Cancer Cell Lines.

Reduced graphene oxide (rGO) and a proteasome inhibitor (MG-132) are some of the most commonly used compounds in various biomedical applications. However, the mechanisms of rGO- and MG-132-induced cytotoxicity remain unclear. The aim of this study was to investigate the anticancer effect of rGO and MG-132 against ZR-75-1 and MDA-MB-231 breast cancer cell lines. The results demonstrated that rGO, MG-132 or a mix (rGO + MG-132) induced time- and dose-dependent cytotoxicity in ZR-75-1 and MDA-MB-231 cells. Apart from that, we found that treatment with rGO and MG-132 or the mix increased apoptosis, necrosis and induction of caspase-8 and caspase-9 activity in both breast cancer cell lines. Apoptosis and caspase activation were accompanied by changes in the ultrastructure of mitochondria in ZR-75-1 and MDA-MB-231 cells incubated with rGO. Additionally, in the analyzed cells, we observed the induction of oxidative stress, accompanied by increased apoptosis and cell necrosis. In conclusion, oxidative stress induces apoptosis in the tested cells. At the same time, both mitochondrial and receptor apoptosis pathways are activated. These studies provided new information on the molecular mechanisms of apoptosis in the ZR-75-1 and MDA-MB-231 breast cancer cell lines.

FBXL19 Targeted STK11 Degradation Enhances Cigarette Smoke-Induced Airway Epithelial Cell Cytotoxicity.

Objective: To investigate the effects of cigarette smoke (CS) on Serine/Threonine Kinase 11 (STK11) and to determine STK11's role in CS-induced airway epithelial cell cytotoxicity.Methods: STK11 expression levels in the lung tissues of smokers with or without COPD and mice exposed to CS or room air (RA) were determined by immunoblotting and RT-PCR. BEAS-2Bs-human bronchial airway epithelial cells were exposed to CS extract (CSE), and the changes in STK11 expression levels were determined by immunoblotting and RT-PCR. BEAS-2B cells were transfected with STK11-specific siRNA or STK11 expression plasmid, and the effects of CSE on airway epithelial cell cytotoxicity were measured. To determine the specific STK11 degradation-proteolytic pathway, BEAS-2Bs were treated with cycloheximide alone or combined with MG132 or leupeptin. Finally, to identify the F-box protein mediating the STK11 degradation, a screening assay was performed using transfection with a panel of FBXL E3 ligase subunits.Results: STK11 protein levels were significantly decreased in the lung tissues of smokers with COPD relative to smokers without COPD. STK11 protein levels were also significantly decreased in mouse lung tissues exposed to CS compared to RA. Exposure to CSE shortened the STK11 mRNA and protein half-life to 4 h in BEAS-2B cells. STK11 protein overexpression attenuated the CSE-induced cytotoxicity; in contrast, its knockdown augmented CSE-induced cytotoxicity. FBXL19 mediates CSE-induced STK11 protein degradation via the ubiquitin-proteasome pathway in cultured BEAS-2B cells. FBXL19 overexpression led to accelerated STK11 ubiquitination and degradation in a dose-dependent manner.Conclusions: Our results suggest that CSE enhances the degradation of STK11 protein in airway epithelial cells via the FBXL19-mediated ubiquitin-proteasomal pathway, leading to augmented cell death.HIGHLIGHTSLung tissues of COPD-smokers exhibited a decreased STK11 RNA and protein expression.STK11 overexpression attenuates CS-induced airway epithelial cell cytotoxicity.STK11 depletion augments CS-induced airway epithelial cell cytotoxicity.CS diminishes STK11 via FBXL19-mediated ubiquitin-proteasome degradation.

Proteasome inhibitors induce apoptosis by superoxide anion generation via NADPH oxidase 5 in human neuroblastoma SH-SY5Y cells.

The ubiquitin-proteasome system (UPS) is a major proteolytic system that plays an important role in the regulation of various cell processes, such as cell cycle, stress response, and transcriptional regulation, especially in neurons, and dysfunction of UPS is considered to be a cause of neuronal cell death in neurodegenerative diseases. However, the mechanism of neuronal cell death caused by UPS dysfunction has not yet been fully elucidated. In this study, we investigated the mechanism of neuronal cell death induced by proteasome inhibitors using human neuroblastoma SH-SY5Y cells. Z-Leu-D-Leu-Leu-al (MG132), a proteasome inhibitor, induced apoptosis in SH-SY5Y cells in a concentration- and time-dependent manner. Antioxidants N-acetylcysteine and EUK-8 attenuated MG132-induced apoptosis. Apocynin and diphenyleneiodonium, inhibitors of NADPH oxidase (NOX), an enzyme that produces superoxide anions, also attenuated MG132-induced apoptosis. It was also found that MG132 treatment increased the expression of NOX5, a NOX family member, and that siRNA-mediated silencing of NOX5 and BAPTA-AM, which inhibits NOX5 by chelating calcium, suppressed MG132-induced apoptosis and production of reactive oxygen species in SH-SY5Y cells. These results suggest that MG132 induces apoptosis in SH-SY5Y cells through the production of superoxide anion by NOX5.

Mesenchymal stem cells exosomal let-7a-5p improve autophagic flux and alleviate liver injury in acute-on-chronic liver failure by promoting nuclear expression of TFEB.

Acute-on-chronic liver failure is a distinct clinical syndrome characterized by a dysregulated immune response and extensive hepatocyte death without satisfactory therapies. As a cytoplasmic degradative and quality-control process, autophagy was implicated in maintaining intracellular homeostasis, and decreased hepatic autophagy was found in many liver diseases and contributes to disease pathogenesis. Previously, we identified the therapeutic potential of mesenchymal stem cells (MSCs) in ACLF patients; however, the intrinsic mechanisms are incompletely understood. Herein, we showed that MSCs restored the impaired autophagic flux and alleviated liver injuries in ACLF mice, but these effects were abolished when autophago-lysosomal maturation was inhibited by leupeptin (leu), suggesting that MSCs exerted their hepatoprotective function in a pro-autophagic dependent manner. Moreover, we described a connection between transcription factor EB (TFEB) and autophagic activity in this context, as evidenced by increased nuclei translocation of TFEB elicited by MSCs were capable of promoting liver autophagy. Mechanistically, we confirmed that let-7a-5p enriched in MSCs derived exosomes (MSC-Exo) could activate autophagy by targeting MAP4K3 to reduce TFEB phosphorylation, and MAP4K3 knockdown partially attenuates the effect of anti-let-7a-5p oligonucleotide via decreasing the inflammatory response, in addition, inducing autophagy. Altogether, these findings revealed that the hepatoprotective effect of MSCs may partially profit from its exosomal let-7a-5p mediating autophagy repairment, which may provide new insights for the therapeutic target of ACLF treatment.

Efficacy of leupeptin in treating ischemia in a rat hind limb model.

Prolonged tourniquet use can lead to tissue ischemia and can cause progressive muscle and nerve injuries. Such injuries are accompanied by calpain activation and subsequent Wallerian-like degeneration. Several known inhibitors, including leupeptin, are known to impede the activity of calpain and associated tissue damage. We hypothesize that employment of leupeptin in a rat model of prolonged hind limb ischemia can mitigate muscle and nerve injuries. Sprague-Dawley rats (n = 10) weighing between 300-400 g were employed in this study. Their left hind limbs were subjected to blood flow occlusion for a period of 2-h using a neonatal blood pressure cuff. Five rats were given twice weekly intramuscular leupeptin injections, while the other five received saline. After 2 weeks, the animals were euthanized, their sciatic nerves and gastrocnemius muscles were harvested, fixed, stained, and analyzed using NIH Image J software. The administration of leupeptin resulted in larger gastrocnemius muscle fiber cross-sectional areas for the right (non-tourniquet applied) hindlimb as compared to that treated with the saline (p = 0.0110). However, no statistically significant differences were found between these two groups for the injured left hindlimb (p = 0.1440). With regards to the sciatic nerve cross-sectional areas and sciatic functional index, no differences were detected between the leupeptin and control treated groups for both the healthy and injured hindlimbs. This research provides new insights on how to employ leupeptin to inhibit the degenerative effects of calpain and preserve tissues following ischemia resulting from orthopedic or plastic surgery procedures.

Mechanisms involved in suppression of osteoclast supportive activity by transforming growth factor-ß1 via the ubiquitin-proteasome system.

Orthodontic treatment requires the regulation of bone remodeling in both compression and tension sides. Transforming growth factor-ß1 (TGF-ß1) is an important coupling factor for bone remodeling. However, the mechanism underlying the TGF-ß1-mediated regulation of the osteoclast-supporting activity of osteoblasts and stromal cells remain unclear. The current study investigated the effect of TGF-ß1 on receptor activator of nuclear factor kappa-B ligand (RANKL) expression in stromal cells induced by 1α,25(OH)2D3 (D3) and dexamethasone (Dex). TGF-ß1 downregulated the expression of RANKL induced by D3 and Dex in mouse bone marrow stromal lineage, ST2 cells. Co-culture system revealed that TGF-ß1 suppressed osteoclast differentiation from bone marrow cell induced by D3 and Dex-activated ST2 cells. The inhibitory effect of TGF-ß1 on RANKL expression was recovered by inhibiting the interaction between TGF-ß1 and the TGF-ß type I/activin receptor or by downregulating of smad2/3 expression. Interestingly, TGF-ß1 degraded the retinoid X receptor (RXR)-α protein which forms a complex with vitamin D receptor (VDR) and regulates transcriptional activity of RANKL without affecting nuclear translocation of VDR and phosphorylation of signal transducer and activator of transcription3 (STAT3). The degradation of RXR-α protein by TGF-ß1 was recovered by a ubiquitin-proteasome inhibitor. We also observed that poly-ubiquitination of RXR-α protein was induced by TGF-ß1 treatment. These results indicated that TGF-ß1 downregulates RANKL expression and the osteoclast-supporting activity of osteoblasts/stromal cells induced by D3 and Dex through the degradation of the RXR-α protein mediated by ubiquitin-proteasome system.

Calpain inhibitor prevents atherosclerosis in apolipoprotein E knockout mice by regulating mRNA expression of genes related to cholesterol uptake and efflux.

PURPOSE: We previously reported that a calpain inhibitor (CAI) prevents the development of atherosclerosis in rats. This study aimed to investigate the effects of CAI (1 mg/kg) on atherosclerosis in apolipoprotein E knockout (ApoE KO) mice that were fed a high-fat diet (HFD) and explore the underlying mechanism by analyzing the expression of genes related to the uptake and efflux of cholesterol. METHODS: Atherosclerotic plaques were evaluated. The activity of calpain in the aorta and that of superoxide dismutase (SOD) in the serum were assessed. Lipid profiles in the serum and liver were examined. Serum oxidized low-density lipoprotein (oxLDL), malondialdehyde (MDA), tumor necrosis factor (TNF-α), and interleukin-6 (IL-6) levels were measured. The mRNA expressions of CD68, TNF-α, IL-6, CD36, scavenger receptor (SR-A), peroxisome proliferator-activated receptor gamma (PPAR-γ), liver-x-receptor alpha (LXR-α), and ATP-binding cassette transporter class A1 (ABCA1) in the aorta and peritoneal macrophages were also evaluated. RESULTS: CAI reduced calpain activity in the aorta. CAI also impeded atherosclerotic lesion formation and mRNA expression of CD68 in the aorta and peritoneal macrophages of ApoE KO mice compared with those of mice receiving HFD. However, CAI had no effect on body weight and lipid levels in both the serum and liver. CAI significantly decreased MDA, oxLDL, TNF-α, and IL-6 levels and increased SOD activity in the serum. Moreover, CAI significantly inhibited the mRNA expression of TNF-α and IL-6 genes in the aorta and peritoneal macrophages. In addition, CAI significantly downregulated the mRNA expression of scavenger receptors CD36 and SR-A and upregulated the expression of genes involved in the cholesterol efflux pathway, i.e., PPAR-γ, LXR-α, and ABCA1 in the aorta and peritoneal macrophages. CONCLUSIONS: CAI inhibited the development of atherosclerotic lesions in ApoE KO mice, and this effect might be related to the reduction of oxidative stress and inflammation and the improvement of cholesterol intake and efflux pathways.

Structural and Biochemical Analysis of the Dual Inhibition of MG-132 against SARS-CoV-2 Main Protease (Mpro/3CLpro) and Human Cathepsin-L.

After almost two years from its first evidence, the COVID-19 pandemic continues to afflict people worldwide, highlighting the need for multiple antiviral strategies. SARS-CoV-2 main protease (Mpro/3CLpro) is a recognized promising target for the development of effective drugs. Because single target inhibition might not be sufficient to block SARS-CoV-2 infection and replication, multi enzymatic-based therapies may provide a better strategy. Here we present a structural and biochemical characterization of the binding mode of MG-132 to both the main protease of SARS-CoV-2, and to the human Cathepsin-L, suggesting thus an interesting scaffold for the development of double-inhibitors. X-ray diffraction data show that MG-132 well fits into the Mpro active site, forming a covalent bond with Cys145 independently from reducing agents and crystallization conditions. Docking of MG-132 into Cathepsin-L well-matches with a covalent binding to the catalytic cysteine. Accordingly, MG-132 inhibits Cathepsin-L with nanomolar potency and reversibly inhibits Mpro with micromolar potency, but with a prolonged residency time. We compared the apo and MG-132-inhibited structures of Mpro solved in different space groups and we identified a new apo structure that features several similarities with the inhibited ones, offering interesting perspectives for future drug design and in silico efforts.

Dynamics of AQP4 upon exposure to seropositive patient serum before and after Rituximab therapy in Neuromyelitis Optica: A cell-based study.

Neuromyelitis Optica (NMO) is an autoimmune inflammatory disease that affects the optic nerves and spinal cord. The autoantibody is generated against the abundant water channel protein of the brain, Aquaporin 4 (AQP4). Of the two isoforms of AQP4, the shorter one (M23) often exists as a supramolecular assembly known as an orthogonal array of particles (OAPs). There have been debates about the fate of these AQP4 clusters upon binding to the antibody, the exact mechanism of its turnover, and the proteins associated with the process. Recently several clinical cases of NMO were reported delineating the effect of Rituximab (RTX) therapy. Extending these reports at the cell signaling level, we developed a glioma based cellular model that mimicked antibody binding and helped us track the subsequent events including a variation of AQP4 levels, alterations in cellular morphology, and the changes in downstream signaling cascades. Our results revealed the extent of perturbations in the signaling pathways related to stress involving ERK, JNK, and AKT1 together with markers for cell death. We could also decipher the possible routes of degradation of AQP4, post-exposure to antibody. We further investigated the effect of autoantibody on AQP4 transcriptional level and involvement of FOXO3a and miRNA-145 in the regulation of transcription. This study highlights the differential outcome at the cellular level when treated with the serum of the same patient pre and post RTX therapy and for the first time mechanistically describes the effect of RTX.

MINDY1 promotes breast cancer cell proliferation by stabilizing estrogen receptor α.

Breast cancer is the most commonly diagnosed malignant tumor among females. Estrogen receptor α (ERα) is initially expressed in 70% of breast cancers and is a well-known target of endocrine therapy for ERα-positive breast cancer. In the present study, we identified MINDY1, a member belongs to the motif interacting with Ubcontaining novel DUB family (MINDY), as a potential deubiquitylase of ERα in breast cancer. There was a positive correlation between ERα and MINDY1 protein levels in human breast cancer tissues. We found that high expression of MINDY1 was associated with poor prognosis. MINDY1 interacted with ERα, thereby mediating the deubiquitination of ERα and increased its stability in a deubiquitylation activity-dependent manner. MINDY1 depletion significantly decreased the ERα protein level and ERα signaling activity in breast cancer cells. Specifically, MINDY1 associated with the N-terminal of ERα via its catalytic domain, thus inhibiting K48-specific poly-ubiquitination process on ERα protein. In addition, MINDY1 depletion led to growth inhibition and cell cycle arrest of ERα-positive breast cancer cells. Finally, overexpression of ERα could rescue the MINDY1 depletion-induced growth inhibition both in vitro and in vivo, suggesting that MINDY1 promotes breast carcinogenesis through increasing ERα stability. Overall, our study proposed a novel post-translational mechanism of ERα in supporting breast cancer progression. Targeting the MINDY1 may prove to be a promising strategy for patients with ERα-positive breast cancer.

Identification of Phosphorylated Calpain 3 in Rat Brain Mitochondria under mPTP Opening.

The protein phosphorylation of the membrane-bound mitochondrial proteins has become of interest from the point of view of its regulatory role of the function of the respiratory chain, opening of the mitochondrial permeability transition pore (mPTP), and initiation of apoptosis. Earlier, we noticed that upon phosphorylation of proteins in some proteins, the degree of their phosphorylation increases with the opening of mPTP. Two isoforms of myelin basic protein and cyclic nucleotide phosphodiesterase were identified in rat brain non-synaptic mitochondria and it was concluded that they are involved in mPTP regulation. In the present study, using the mass spectrometry method, the phosphorylated protein was identified as Calpain 3 in rat brain non-synaptic mitochondria. In the present study, the phosphoprotein Calpain-3 (p94) (CAPN3) was identified in the rat brain mitochondria as a phosphorylated truncated form of p60-62 kDa by two-dimensional electrophoresis and mass spectrometry. We showed that the calpain inhibitor, calpeptin, was able to suppress the Ca2+ efflux from mitochondria, preventing the opening of mPTP. It was found that phosphorylated truncated CALP3 with a molecular weight of 60-62 contains p-Tyr, which indicates the possible involvement of protein tyrosine phosphatase in this process.

ERK and p38 MAPK inhibition controls NF-E2 degradation and profibrotic signaling in renal proximal tubule cells.

AIMS: Transforming growth factor-ß (TGF-ß) mediates fibrotic manifestations of diabetic nephropathy. We demonstrated proteasomal degradation of anti-fibrotic protein, nuclear factor-erythroid derived 2 (NF-E2), in TGF-ß treated human renal proximal tubule (HK-11) cells and in diabetic mouse kidneys. The current study examined the role of mitogen-activated protein kinase (MAPK) pathways in mediating NF-E2 proteasomal degradation and stimulating profibrotic signaling in HK-11 cells. MAIN METHODS: HK-11 cells were pretreated with vehicle or appropriate proteasome and MAPK inhibitors, MG132 (0.5 µM), SB203580 (1 µM), PD98059 (25 µM) and SP600125 (10 µM), respectively, followed by treatment with/without TGF-ß (10 ng/ml, 24 h). Cell lysates and kidney homogenates from FVB and OVE26 mice treated with/without MG132 were immunoblotted with appropriate antibodies. pUse vector and pUse-NF-E2 cDNA were transfected in HK-11 cells and effects of TGF-ß on JNK MAPK phosphorylation (pJNK) was examined. KEY FINDINGS: We demonstrated activation of p38, ERK, and JNK MAPK pathways in TGF-ß treated HK-11 cells. Dual p38 and ERK MAPK blockade prevented TGF-ß-induced pSer82Hsp27, fibronectin and connective tissue growth factor (CTGF) expression while preserving NF-E2 expression. Blockade of JNK MAPK inhibited TGF-ß-induced CTGF expression without preserving NF-E2 expression. MG132 treatment prevented TGF-ß-induced pJNK in HK-11 cells and in type 1 diabetic OVE26 mouse kidneys, demonstrating that TGF-ß- and diabetes-induced pJNK occurs downstream of proteasome activation. A direct role for NF-E2 in modulating pJNK activation was demonstrated by NF-E2 over-expression. SIGNIFICANCE: ERK and p38 MAPK promotes NF-E2 proteasomal degradation while proteasome activation promotes pJNK and profibrotic signaling in renal proximal tubule cells.

Proteasome inhibition induces macrophage apoptosis via mitochondrial dysfunction.

Dysfunction of the ubiquitin-proteasome system has been linked to the pathogenesis of a variety of diseases. Proteasome inhibition not only exerts antitumor effects but also affects inflammatory signaling pathways. MG132, a proteasome inhibitor, has been shown to induce tumor cell apoptosis. However, its role in the induction of macrophage apoptosis remains unknown. In our study, we investigated the mechanism of the proapoptotic effects of MG132 in macrophages. Our data showed that MG132 treatment induced mitochondrial reactive oxygen species (ROS) generation and loss of mitochondrial membrane potential in macrophages. We found that proteasome inhibition induced a significant increase in the apoptosis rate, as evidenced by cleavage of caspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP). Moreover, (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenyl-phosphonium chloride (Mito-TEMPO) attenuated MG132-induced apoptosis. In conclusion, proteasome inhibition by MG132 can induce macrophage apoptosis by promoting the production of ROS and mitochondrial dysfunction.

TFEB phosphorylation on Serine 211 is induced by autophagy in human synovial fibroblasts and by p62/SQSTM1 overexpression in HEK293 cells.

Autophagy receptor p62/SQSTM1 signals a complex network that links autophagy-lysosomal system to proteasome. Phosphorylation of p62 on Serine 349 (P-Ser349 p62) is involved in a cell protective, antioxidant pathway. We have shown previously that P-Ser349 p62 occurs and is rapidly degraded during human synovial fibroblasts autophagy. In this work we observed that fingolimod (FTY720), used as a medication for multiple sclerosis, induced coordinated expression of p62, P-Ser349 p62 and inhibitory TFEB form, phosphorylated on Serine 211 (P-Ser211 TFEB), in human synovial fibroblasts. These effects were mimicked and potentiated by proteasome inhibitor MG132. In addition, FTY720 induced autophagic flux, LC3B-II up-regulation, Akt phosphorylation inhibition on Serine 473 but down-regulated TFEB, suggesting stalled autophagy. FTY720 decreased cytoplasmic fraction contained TFEB but induced TFEB in nuclear fraction. FTY720-induced P-Ser211 TFEB was mainly found in membrane fraction. Autophagy and VPS34 kinase inhibitor, autophinib, further increased FTY720-induced P-Ser349 p62 but inhibited concomitant expression of P-Ser211 TFEB. These results suggested that P-Ser211 TFEB expression depends on autophagy. Overexpression of GFP tagged TFEB in HEK293 cells showed concomitant expression of its phosphorylated form on Serine 211, that was down-regulated by autophinib. These results suggested that autophagy might be autoregulated through P-Ser211 TFEB as a negative feedback loop. Of interest, overexpression of p62, p62 phosphorylation mimetic (S349E) mutant and phosphorylation deficient mutant (S349A) in HEK293 cells markedly induced P-Ser211 TFEB. These results showed that p62 is involved in regulation of TFEB phosphorylation on Serine 211 but that this involvement does not depend on p62 phosphorylation on Serine 349.

Enzalutamide-induced Proteolytic Degradation of the Androgen Receptor in Prostate Cancer Cells Is Mediated Only to a Limited Extent by the Proteasome System.

BACKGROUND/AIM: Androgen receptor (AR) degradation is the primary regulator of androgen receptor activity. This study was designed to investigate the influence of the proteasome on AR protein stability after enzalutamide (Enz) treatment. MATERIALS AND METHODS: Cell counting after treatment was utilized to assess the effect of Enz on cell proliferation. Changes in mRNA levels were evaluated using reverse transcription-polymerase chain reaction (RT-PCR). Proteasome activity was assessed by measurement of the chymotrypsin-like activity of the beta-5 subunit of the proteasome. Changes in protein levels after treatment with Enz, MG132 (MG), bortezomib (Bor), or their combination were assessed using western blot analysis. RESULTS: Treatment with Enz led to a significant reduction of cell proliferation and AR protein levels. However, AR mRNA levels were unchanged. Inhibition of proteasome activity by MG counteracts the Enz-mediated AR degradation transiently, whereas Bor showed no inhibition of the Enz-mediated AR degradation. CONCLUSION: Enz-mediated change in AR stability as an early and essential event after treatment was shown. However, investigations of the ubiquitin/proteasome system indicate involvement of several proteases in the Enz-mediated AR degradation process.

Hsf1 activation by proteotoxic stress requires concurrent protein synthesis.

Heat shock factor 1 (Hsf1) activation is responsible for increasing the abundance of protein-folding chaperones and degradation machinery in response to proteotoxic conditions that give rise to misfolded or aggregated proteins. Here we systematically explored the link between concurrent protein synthesis and proteotoxic stress in the budding yeast, Saccharomyces cerevisiae. Consistent with prior work, inhibiting protein synthesis before inducing proteotoxic stress prevents Hsf1 activation, which we demonstrated across a broad array of stresses and validate using orthogonal means of blocking protein synthesis. However, other stress-dependent transcription pathways remained activatable under conditions of translation inhibition. Titrating the protein denaturant ethanol to a higher concentration results in Hsf1 activation in the absence of translation, suggesting extreme protein-folding stress can induce proteotoxicity independent of protein synthesis. Furthermore, we demonstrate this connection under physiological conditions where protein synthesis occurs naturally at reduced rates. We find that disrupting the assembly or subcellular localization of newly synthesized proteins is sufficient to activate Hsf1. Thus, new proteins appear to be especially sensitive to proteotoxic conditions, and we propose that their aggregation may represent the bulk of the signal that activates Hsf1 in the wake of these insults.

MG-132 interferes with iron cellular homeostasis and alters virulence of bovine herpesvirus 1.

Bovine herpesvirus 1 (BoHV-1) requires an iron-replete cell host to replicate efficiently. BoHV-1 infection provokes an increase in ferritin levels and a decrease of transferrin receptor 1 (TfR-1) expression, ultimately lowering iron pool extent. Thus, cells try to limit iron availability for virus spread. It has been demonstrated that MG-132, a proteasome inhibitor, reduces BoHV-1 release. Since ferritin, the major iron storage protein in mammalian cells, undergoes proteasome-mediated degradation, herein, the influence of MG-132 on iron metabolism during BoHV-1 infection was examined. Following infection in bovine cells (MDBK), MG-132 reduced cell death and viral yield. Western blot analysis showed a significant ferritin accumulation, likely due to the inhibition of its proteasome-mediated degradation pathway. In addition, the concomitant down-regulation of TfR-1 expression, observed during infection, was counteracted by proteasome inhibitor. This trend may be explained by enhanced acidic vesicular organelles, detected by acridine orange staining, determining a reduction of intracellular pH, that promotes new synthesis of TfR-1 degraded in a recycling pathway. In addition, MG-132 influences cellular iron distribution during BoHV-1 infection, as revealed by Perls' Prussian blue staining. However, cellular iron content, evaluated by Atomic Absorption Spectrophotometry, resulted essentially unaltered. These findings reveal that MG-132 may contribute to limit cellular iron availability for virus replication thereby enhancing cell survival.

Asparagine sustains cellular proliferation and c­Myc expression in glutamine­starved cancer cells.

During tumorigenesis, oncogene activation and metabolism rewiring are interconnected. Activated c­Myc upregulates several genes involved in glutamine metabolism, making cancer cells dependent on high levels of this amino acid to survive and proliferate. After studying the response to glutamine deprivation in cancer cells, it was found that glutamine starvation not only blocked cellular proliferation, but also altered c­Myc protein expression, leading to a reduction in the levels of the canonical c­Myc isoform and an increase in the expression of c­Myc 1, a c­Myc isoform translated from an in­frame 5' CUG codon. In an attempt to identify nutrients able to counteract glutamine deprivation effects, it was shown that, in the absence of glutamine, asparagine permitted cell survival and proliferation, and maintained c­Myc expression as in glutamine­fed cells, with high levels of canonical c­Myc and c­Myc 1 almost undetectable. In asparagine­fed cells, global protein translation was higher than in glutamine­starved cells, and there was an increase in the levels of glutamine synthetase (GS), whose activity was essential for cellular viability and proliferation. In glutamine­starved asparagine­fed cells, the inhibition of c­Myc activity led to a decrease in global protein translation and GS synthesis, suggesting an association between c­Myc expression, GS levels and cellular proliferation, mediated by asparagine when exogenous glutamine is absent.

Therapeutic Potential for Regulation of the Nuclear Factor Kappa-B Transcription Factor p65 to Prevent Cellular Senescence and Activation of Pro-Inflammatory in Mesenchymal Stem Cells.

Mesenchymal stem cells have an important potential in the treatment of age-related diseases. In the last years, small extracellular vesicles derived from these stem cells have been proposed as cell-free therapies. Cellular senescence and proinflammatory activation are involved in the loss of therapeutic capacity and in the phenomenon called inflamm-aging. The regulators of these two biological processes in mesenchymal stem cells are not well-known. In this study, we found that p65 is activated during cellular senescence and inflammatory activation in human umbilical cord-derived mesenchymal stem cell. To demonstrate the central role of p65 in these two processes, we used small-molecular inhibitors of p65, such as JSH-23, MG-132 and curcumin. We found that the inhibition of p65 prevents the cellular senescence phenotype in human umbilical cord-derived mesenchymal stem cells. Besides, p65 inhibition produced the inactivation of proinflammatory molecules as components of a senescence-associated secretory phenotype (SASP) (interleukin-6 and interleukin-8 (IL-6 and IL-8)). Additionally, we found that the inhibition of p65 prevents the transmission of paracrine senescence between mesenchymal stem cells and the proinflammatory message through small extracellular vesicles. Our work highlights the important role of p65 and its inhibition to restore the loss of functionality of small extracellular vesicles from senescent mesenchymal stem cells and their inflamm-aging signature.

FBX4 mediates rapid cyclin D1 proteolysis upon DNA damage in immortalized esophageal epithelial cells.

It has been implied that deregulation of cyclin D1 turnover under stresses can facilitate genomic instability and trigger tumorigenesis. Much focus has been placed on identifying the E3 ligases responsible for mediating cyclin D1 degradation. However, the findings were quite controversial and cell type-dependent. Little is known about how cyclin D1 is regulated in precancerous cells upon DNA damage and which E3 ligases mediate the effects. Here we found cyclin D1 reduction is an early response to DNA damage in immortalized esophageal epithelial cells, with expression dropping to a low level within 1 h after γ-irradiation. Comparison of temporal expression of cyclin D1 upon DNA damage between immortalized NE083-hTERT and NE083-E6E7, the latter being p53/p21-defective, showed that DNA damage-induced rapid cyclin D1 reduction was p53-independent and occurred before p21 accumulation. Overexpression of cyclin D1 in NE083-E6E7 cells could attenuate G0/G1 cell cycle arrest at 1 h after irradiation. Furthermore, rapid reduction of cyclin D1 upon DNA damage was attributed to proteasomal degradation, as evidenced by data showing that proteasomal inhibition by MG132 blocked cyclin D1 reduction while cycloheximide facilitated it. Inhibition of ATM activation and knockdown of E3 ligase adaptor FBX4 reversed cyclin D1 turnover in immortalized NE083-hTERT cells. Further study showed that knockdown of FBX4 facilitated DNA breaks, as indicated by an increase in γ-H2AX foci in esophageal cancer cells. Taken together, the results substantiated a pivotal role of ATM and FBX4 in cyclin D1 proteolysis upon DNA damage in precancerous esophageal epithelial cells, implying that deregulation of the process may contribute to carcinogenesis of esophageal squamous cell carcinoma.