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.

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.

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.

Treatment with MG132 prevents spontaneous activation of rat oocyte in culture and promotes embryonic development after intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) is an effective reproductive technique for obtaining rat offspring using preserved sperm with low or no motility. However, rat oocytes undergo spontaneous activation immediately after retrieval from the oviduct and poorly develop after ICSI unless it is performed quickly. Here, we evaluated whether treatment with MG132, the proteasome inhibitor, suppresses the spontaneous activation of oocytes before and during ICSI. After retrieval from the oviducts, the rate of development into morula and blastocyst from the oocytes cultured in vitro for 1 h prior to ICSI significantly decreased compared with that from the control oocytes subject to ICSI without culture (7% versus 36%). However, a higher proportion of oocytes treated with MG132 for 0, 1, and 3 h before and during ICSI developed into morulae and blastocysts (70%, 60%, and 52%, respectively). Offspring were obtained from oocytes treated with MG132 for 0 and 1 h before and during ICSI (percentage: 31%). Altogether, MG132 could suppress the spontaneous activation of rat oocytes and increase embryonic development after ICSI.

MG132 Induces Progerin Clearance and Improves Disease Phenotypes in HGPS-like Patients' Cells.

Progeroid syndromes (PS), including Hutchinson-Gilford Progeria Syndrome (HGPS), are premature and accelerated aging diseases, characterized by clinical features mimicking physiological aging. Most classical HGPS patients carry a de novo point mutation within exon 11 of the LMNA gene encoding A-type lamins. This mutation activates a cryptic splice site, leading to the production of a truncated prelamin A, called prelamin A ∆50 or progerin, that accumulates in HGPS cell nuclei and is a hallmark of the disease. Some patients with PS carry other LMNA mutations and are named "HGPS-like" patients. They produce progerin and/or other truncated prelamin A isoforms (∆35 and ∆90). We previously found that MG132, a proteasome inhibitor, induced progerin clearance in classical HGPS through autophagy activation and splicing regulation. Here, we show that MG132 induces aberrant prelamin A clearance and improves cellular phenotypes in HGPS-like patients' cells other than those previously described in classical HGPS. These results provide preclinical proof of principle for the use of a promising class of molecules toward a potential therapy for children with HGPS-like or classical HGPS.

Activation of the ubiquitin-proteasome system contributes to oculopharyngeal muscular dystrophy through muscle atrophy.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset disorder characterized by progressive weakness and degeneration of specific muscles. OPMD is due to extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). Aggregation of the mutant protein in muscle nuclei is a hallmark of the disease. Previous transcriptomic analyses revealed the consistent deregulation of the ubiquitin-proteasome system (UPS) in OPMD animal models and patients, suggesting a role of this deregulation in OPMD pathogenesis. Subsequent studies proposed that UPS contribution to OPMD involved PABPN1 aggregation. Here, we use a Drosophila model of OPMD to address the functional importance of UPS deregulation in OPMD. Through genome-wide and targeted genetic screens we identify a large number of UPS components that are involved in OPMD. Half dosage of UPS genes reduces OPMD muscle defects suggesting a pathological increase of UPS activity in the disease. Quantification of proteasome activity confirms stronger activity in OPMD muscles, associated with degradation of myofibrillar proteins. Importantly, improvement of muscle structure and function in the presence of UPS mutants does not correlate with the levels of PABPN1 aggregation, but is linked to decreased degradation of muscle proteins. Oral treatment with the proteasome inhibitor MG132 is beneficial to the OPMD Drosophila model, improving muscle function although PABPN1 aggregation is enhanced. This functional study reveals the importance of increased UPS activity that underlies muscle atrophy in OPMD. It also provides a proof-of-concept that inhibitors of proteasome activity might be an attractive pharmacological approach for OPMD.

Differential Ubiquitination as an Effective Strategy Employed by the Blood-Brain Barrier for Prevention of Bacterial Transcytosis.

The protective mechanisms of blood-brain barrier (BBB) prohibiting entry of pathogens into central nervous system (CNS) are critical for maintenance of brain homeostasis. These include various intracellular defense mechanisms that are vital to block transcytosis of neurotropic pathogens into the CNS. However, mechanistic details of coordination between these defense pathways remain unexplored. In this study, we established that BBB-driven ubiquitination acts as a major intracellular defense mechanism for clearance of Streptococcus pneumoniae, a critical neurotropic pathogen, during transit through BBB. Our findings suggest that the BBB employs differential ubiquitination with either K48- or K63-ubiquitin (Ub) chain topologies as an effective strategy to target S. pneumoniae toward diverse killing pathways. While K63-Ub decoration triggers autophagic killing, K48-Ub directs S. pneumoniae exclusively toward proteasomes. Time-lapse fluorescence imaging involving proteasomal marker LMP2 revealed that in the BBB, the majority of the ubiquitinated S. pneumoniae was cleared by proteasome. Fittingly, inhibition of proteasome and autophagy pathway led to accumulation of K48-Ub- and K63-Ub-marked S. pneumoniae, respectively, and triggered significant increases in intracellular S. pneumoniae burden. Moreover, genetic impairment of either K48- or K63-Ub chain formation demonstrated that although both chain types are key in disposal of intracellular S. pneumoniae, K48-Ub chains and subsequent proteasomal degradation have more pronounced contributions to intracellular S. pneumoniae killing in the BBB. Collectively, these observations, for the first time, illustrated a pivotal role of differential ubiquitination deployed by BBB in orchestrating a symphony of intracellular defense mechanisms for interception and degradation of S. pneumoniae, blocking its entry into the brain, which could be exploited to prevent bacterial CNS infections. IMPORTANCE The blood-brain barrier (BBB) represents a unique cellular barrier that provides structural integrity and protection to the CNS from pathogen invasion. Recently, ubiquitination, which is key for cellular homeostasis, was shown to be involved in pathogen clearance. In this study, we deciphered that the BBB deploys differential ubiquitination as an effective strategy to prevent S. pneumoniae trafficking into the brain. The different ubiquitin chain topologies formed on S. pneumoniae dictated the selection of downstream degradative pathways, namely, autophagy and proteasomes, among which the contribution of the proteasomal system in S. pneumoniae killing is more pronounced. Overall our study revealed how the BBB deploys differential ubiquitination as a strategy for synchronization of various intracellular defense pathways, which work in tandem to ensure the brain's identity as an immunologically privileged site.

Effects of a proteasome inhibitor on the riboflavin production in Ashbya gossypii.

AIMS: Effects of a proteasome inhibitor, MG-132, on the riboflavin production in Ashbya gossypii were investigated to elucidate the relationship of the riboflavin production with flavoprotein homeostasis. METHODS AND RESULTS: The addition of MG-132 to the liquid medium reduced the specific riboflavin production by 79% in A. gossypii at 25 µM after 24 h. The addition of the inhibitor also caused the accumulation of reactive oxygen species and ubiquitinated proteins. These results indicated that MG-132 works in A. gossypii without any genetic engineering and reduces riboflavin production. In the presence of 25 µM MG-132, specific NADH dehydrogenase activity was increased by 1.4-fold compared to DMSO, but specific succinate dehydrogenase (SDH) activity was decreased to 52% compared to DMSO. Additionally, the amount of AgSdh1p (ACR052Wp) was also reduced. Specific riboflavin production was reduced to 22% when 20 mM malonate, a SDH inhibitor, was added to the culture medium. The riboflavin production in heterozygous AgSDH1 gene-disrupted mutant (AgSDH1-/+ ) was reduced to 63% compared to that in wild type. CONCLUSIONS: MG-132 suppresses the riboflavin production and SDH activity in A. gossypii. SDH is one of the flavoproteins involved in the riboflavin production in A. gossypii. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that MG-132 has a negative influence on the riboflavin production and SDH activity in A. gossypii and leads to the elucidation of the connection of the riboflavin production with flavoproteins.

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.

Increased FUN14 domain containing 1 (FUNDC1) ubiquitination level inhibits mitophagy and alleviates the injury in hypoxia-induced trophoblast cells.

Preeclampsia (PE) is a pregnancy disorder characterized by excessive trophoblast cell death. This study aims to explore the exact mechanism of the ubiquitination level of FUN14 domain containing 1 (FUNDC1) in mitophagy and injury in hypoxic trophoblast cells. In this study, HTR-8/SVneo trophoblast cells were cultured under normoxic and hypoxic conditions and PE mouse model was established. We found low ubiquitination level of FUNDC1 in hypoxic trophoblast cells and placenta of pregnant women with PE. Proteasome inhibitor MG-132 and protease activator MF-094 were added into HTR-8/SVneo trophoblast cells. Proteasome inhibitor MG-132 decreased FUNDC1 ubiquitination level while protease activator MF-094 increased FUNDC1 ubiquitination level. Inhibition of FUNDC1 ubiquitination promoted mitophagy and mitochondrial membrane potential (Δψm) in normoxic trophoblast cells, increased levels of reactive oxygen species (ROS) and malondialdehyde (MDA) and decreased levels of glutathione (GSH) and superoxide dismutase (SOD). In addition, FUNDC1 ubiquitination alleviated cell injury in PE mice in vivo. In conclusion, increased FUNDC1 ubiquitination level inhibited mitophagy and Δψm changes in hypoxic trophoblast cells, and thus alleviated oxidative injury.

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.

Avian Metapneumovirus Subgroup C Induces Mitochondrial Antiviral Signaling Protein Degradation through the Ubiquitin-Proteasome Pathway.

The mitochondrial antiviral signaling (MAVS) protein, a critical adapter, links the upstream recognition of viral RNA to downstream antiviral signal transduction. However, the interaction mechanism between avian metapneumovirus subgroup C (aMPV/C) infection and MAVS remains unclear. Here, we confirmed that aMPV/C infection induced a reduction in MAVS expression in Vero cells in a dose-dependent manner, and active aMPV/C replication was required for MAVS decrease. We also found that the reduction in MAVS occurred at the post-translational level rather than at the transcriptional level. Different inhibitors were used to examine the effect of proteasome or autophagy on the regulation of MAVS. Treatment with a proteasome inhibitor MG132 effectively blocked MAVS degradation. Moreover, we demonstrated that MAVS mainly underwent K48-linked ubiquitination in the presence of MG132 in aMPV/C-infected cells, with amino acids 363, 462, and 501 of MAVS being pivotal sites in the formation of polyubiquitin chains. Finally, E3 ubiquitin ligases for MAVS degradation were screened and identified and RNF5 targeting MAVS at Lysine 363 and 462 was shown to involve in MAVS degradation in aMPV/C-infected Vero cells. Overall, these results reveal the molecular mechanism underlying aMPV/C infection-induced MAVS degradation by the ubiquitin-proteasome pathway.

Substrate ubiquitination retains misfolded membrane proteins in the endoplasmic reticulum for degradation.

To maintain secretory pathway fidelity, misfolded proteins are commonly retained in the endoplasmic reticulum (ER) and selected for ER-associated degradation (ERAD). Soluble misfolded proteins use ER chaperones for retention, but the machinery that restricts aberrant membrane proteins to the ER is unclear. In fact, some misfolded membrane proteins escape the ER and traffic to the lysosome/vacuole. To this end, we describe a model substrate, SZ∗, that contains an ER export signal but is also targeted for ERAD. We observe decreased ER retention when chaperone-dependent SZ∗ ubiquitination is compromised. In addition, appending a linear tetra-ubiquitin motif onto SZ∗ overrides ER export. By screening known ubiquitin-binding proteins, we then positively correlate SZ∗ retention with Ubx2 binding. Deletion of Ubx2 also inhibits the retention of another misfolded membrane protein. Our results indicate that polyubiquitination is sufficient to retain misfolded membrane proteins in the ER prior to ERAD.