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Vaccinia virus relies on a series of proteolytic cleavage events involving two viral proteins, I7 and G1, to complete its life cycle. Furthermore, G1 itself is cleaved during vaccinia virus infection. However, convincing evidence is lacking to show whether G1 participates in autoproteolysis or is a substrate of another protease. We employed both biochemical and cell-based approaches to investigate G1 cleavage. G1, when expressed in bacteria, rabbit reticulocyte lysates or HeLa cells, was not processed. Moreover, G1 was cleaved in infected cells, but only in the presence of virus late gene expression; cleavage was strongly inhibited by proteasome inhibitors. Thus, these results imply a more complex G1 cleavage reaction than previously envisaged.

Lewy bodies, the histopathological hallmarks of Parkinson's disease (PD), contain insoluble and aggregated alpha-synuclein (aSyn) and many other proteins, proposing a role for failure in protein degradation system in the PD pathogenesis. Proteasomal dysfunction has indeed been linked to PD and aSyn oligomers have been shown to inhibit proteasomes and autophagy. Our recent studies have shown that inhibitors of prolyl oligopeptidase (PREP) can prevent the aggregation and enhance the clearance of accumulated aSyn, and therefore, we wanted to study if PREP inhibition can overcome the aSyn aggregation and toxicity induced by lactacystin, a proteasomal inhibitor. The cells overexpressing human A30P or A53T mutated aSyn were incubated with lactacystin and a PREP inhibitor, KYP-2047, for 48 h. Theafter, the cells were fractioned, and the effects of lactacystin with/without 1 mu M KYP-2047 on aSyn aggregation and ubiquitin accumulation, cell viability and on autophagic markers (p62, Beclin1 and LC3BII) were studied. We found that KYP-2047 attenuated lactacystin-induced cell death in mutant aSyn overexpressing cells but not in non-overexpressing control cells. KYP-2047 reduced significantly SDS-insoluble high-molecular-weight aSyn oligomers that were in line with the cell viability results. In addition, significant reduction in protein accumulation marker, p62, was seen in SDS fraction while LC3BII, a marker for autophagosome formation, was increased, indicating to enhanced autophagy. Our results further streghten the possibilities for PREP inhibitors as a potential drug therapy against synucleinopathies and other protein aggregating diseases. (C) 2016 Elsevier Ireland Ltd. All rights reserved.

Reduced retrograde memory performance at the cognitive level and aggregation/deposition of amyloid beta (Aβ) in the brain at the cellular level are some of the hallmarks of Alzheimer's Disease (AD). A molecular system that participates in the removal of proteins with an altered conformation is the Ubiquitin-Proteasome System (UPS). Impairments of the UPS in wild-type (WT) mice lead to defective clearance of Aβ and prevent long-term plasticity of synaptic transmission. Here we show data whereby in contrast to WT mice, the inhibition of proteasome-mediated protein degradation in an animal model of AD by MG132 or lactacystin restores impaired activity-dependent synaptic plasticity and its associative interaction, synaptic tagging and capture (STC) in vitro, as well as associative long-term memory in vivo. This augmentation of synaptic plasticity and memory is mediated by the mTOR pathway and protein synthesis. Our data offer novel insights into the rebalancing of proteins relevant for synaptic plasticity which are regulated by UPS in AD-like animal models. In addition, the data provide evidence that proteasome inhibitors might be effective in reinstating synaptic plasticity and memory performance in AD, and therefore offer a new potential therapeutic option for AD treatment.

Pseudomonas aeruginosa is an opportunistic pathogen that induces severe lung infections such as ventilator-associated pneumonia and acute lung injury. Under these conditions, the bacterium diminishes epithelial integrity and inhibits tissue repair mechanisms, leading to persistent infections. Understanding the involved bacterial virulence factors and their mode of action is essential for the development of new therapeutic approaches. In our study we discovered a so far unknown effect of the P. aeruginosa lectin LecB on host cell physiology. LecB alone was sufficient to attenuate migration and proliferation of human lung epithelial cells and to induce transcriptional activity of NF-κB. These effects are characteristic of impaired tissue repair. Moreover, we found a strong degradation of β-catenin, which was partially recovered by the proteasome inhibitor lactacystin. In addition, LecB induced loss of cell–cell contacts and reduced expression of the β-catenin targets c-myc and cyclin D1. Blocking of LecB binding to host cell plasma membrane receptors by soluble l-fucose prevented these changes in host cell behavior and signaling, and thereby provides a powerful strategy to suppress LecB function. Our findings suggest that P. aeruginosa employs LecB as a virulence factor to induce β-catenin degradation, which then represses processes that are directly linked to tissue recovery., Highlights • The Pseudomonas aeruginosa lectin LecB reduces lung cell migration and proliferation • LecB also induces NF-kB-mediated inflammation in lung epithelial cells • ß-catenin degradation and reduction of c-myc and cyclin D1 protein levels inhibit cell proliferation and migration • Blocking of LecB binding to host cell receptors with L-fucose suppresses all effects caused by LecB

A Trypanosoma brucei cell line is described that produces a visual readout of proteasome activity. The cell line contains an integrated transgene encoding an ubiquitin-green fluorescent protein (GFP) fusion polypeptide responsive to the addition of proteasome inhibitors. A modified version of T. brucei ubiquitin unable to be recognized by deubiquitinases (UbG76V) was fused to eGFP and constitutively expressed. The fusion protein is unstable but addition of the proteasome inhibitor lactacystin stabilizes it and leads to visually detectable GFP. This cell line can be widely used to monitor the efficiency of inhibitor treatment through detection of GFP accumulation in studies involving proteasome-mediated proteolysis, screening of proteasome inhibitors or other events related to the ubiquitin-proteasome pathway.

In resting cells, the nuclear factor kappa B (NF-κB) family of transcription factors is stabilized by complexation with the cytoplasmic inhibitor of kappa B alpha (IκBα). Extracellular stimuli, such as tumor necrosis factor alpha (TNFα) or bacterial lipopolysaccharide activate NF-κB through IκBα phosphorylation and ubiquitin-proteasomal degradation. Herein, we developed a novel biosensor, by fusing the monomeric fluorescent protein Kusabira-Orange 2 to IκBα (mKO2-IκBα), to study the dynamics and structure-activity relationship of IκBα degradation. Site-specific deletion studies on the IκBα sequence revealed that the C-terminal PEST domain is required in signal-induced proteasomal degradation of IκBα and functions independently from ankyrin repeats. Using deletion mutants, we show that IκBα ankyrin repeats do not affect IκBα degradability but affect its degradation rate. We demonstrate, by both real-time confocal microscopy and western blot analysis, that the half-life of mKO2-IκBα in response to TNFα is approximately 35 min, which is similar to the half-life of endogenous IκBα. Using this biosensor we also show that selective proteasome inhibitors, such as lactacystin and MG132, inhibit degradation and affect the kinetics of IκBα in a dose-dependent manner. The techniques described here can have a range of possible applications, such as facilitating studies associated with IκBα dynamics and biochemical characteristics, as well as the screening of potential proteasome inhibitors.

Human islet amyloid polypeptide (hIAPP) is the principal constituent of amyloid deposits and toxic oligomers in the pancreatic islets. Together with hyperglycemia, hIAPP-derived oligomers and aggregates are important culprits in type 2 diabetes mellitus (T2DM). Here, we explored the role of the cell's main proteolytic complex, the proteasome, in hIAPP turnover in normal and stressed β-cells evoked by chronic hyperglycemia. Moderate inhibition (10–35%) of proteasome activity/function in cultured human islets by the proteasome inhibitor lactacystin enhanced intracellular accumulation of hIAPP. Unexpectedly, prolonged (>1 h) and marked (>50%) impairment of proteasome activity/function had a strong inhibitory effect on hIAPP transcription and secretion from normal and stressed β-cells. This negative compensatory feedback mechanism for controlling IAPP turnover was also observed in the lactacystin-treated rat insulinoma β-cell line (INS 832/13), demonstrating the presence of an evolutionarily conserved mechanism for IAPP production. In line with these in situ studies, our current ex vivo data showed that proteasome activity and hIAPP expression are also down-regulated in islets isolated from T2DM subjects. Gene expression and promoter activity studies demonstrated that the functional proteasome complex is required for efficient activation of the hIAPP promoter and for full expression of IAPP's essential transcription factor, FOXA2. ChIP studies revealed that promoter occupancy of FoxA2 at the rat IAPP promoter region is an important and limiting factor for amylin expression in proteasome-impaired murine cells. This study suggests a novel regulatory pathway in β-cells involving proteasome, FOXA2, and IAPP, which can be possibly targeted to regulate hIAPP levels and islet amyloidosis in T2DM.

Missorting of MAPT/Tau represents one of the early signs of neurodegeneration in Alzheimer disease. The triggers for this are still a matter of debate. Here we investigated the sorting mechanisms of endogenous MAPT in mature primary neurons using microfluidic chambers (MFCs) where cell compartments can be observed separately. Blocking protein degradation pathways with proteasomal or autophagy inhibitors dramatically increased the missorting of MAPT in dendrites on the neuritic side, suggesting that degradation of MAPT in dendrites is a major determinant for the physiological axonal distribution of MAPT. Such missorted dendritic MAPT differed in its phosphorylation pattern from axonal MAPT. By contrast, enhancing autophagy or proteasomal pathways strongly reduced MAPT missorting, thereby confirming the role of protein degradation pathways in the polar distribution of MAPT. Dendritic missorting of MAPT by blocking protein degradation resulted in the loss of spines but not in overall cell toxicity. Inhibition of local protein synthesis in dendrites eliminated the missorting of MAPT, indicating that the accumulation of dendritic MAPT is locally generated. In support of this, a substantial fraction of Mapt/Tau mRNA was detected in dendrites. Taken together, our results indicate that the autophagy and proteasomal pathways play important roles in fine-tuning dendritic MAPT levels and thereby prevent synaptic toxicity caused by MAPT accumulation. Abbreviations Ani: anisomycin; Baf: bafilomycin A(1); BSA: bovine serum albumin; cAMP: cyclic adenosine monophosphate; CHX: cycloheximide; DMSO: dimethyl sulfoxide; DIV: days in vitro; Epo: epoxomicin; E18: embryonic day 18; FISH: fluorescence in situ hybridization; IgG: immunoglobulin; kDa: kilodalton; Lac: lactacystin; LDH: lactate dehydrogenase; MFC: microfluidic chambers; MAPs: microtubule-associated proteins; MAPT/Tau: microtubule-associated protein tau; PVDF: polyvinylidene difluoride; PBS: phosphate-buffered saline; PRKA: protein kinase AMP-activated; RD150: round device 150; RT: room temperature; SDS: sodium dodecyl sulfate; SEM: standard error of the mean; Wor: wortmannin.

ObjectiveTo explore the effects of histone deacetylase 6(HDAC-6) on the PD cell model induced by proteasome inhibitor lactacystin.MethodsHuman neuroblastoma SK-N-SH cells were cultured. The wild type pcDNA3.1-alpha-synuclein eukaryotic expression plasmid was transferred into the cells which then were divided into control group, group L, group T and group T+L. The cells of group L were added with 5 μmol/L lactacystin dissolved indimethylsulfoxide (DMSO) to induce PD cell model with abnormal protein aggregation, the cells of control group were treated with 5 μmol/L DMSO, the cells of group T were treated with 5 μmol/L selective HDAC-6 inhibitor tubacin dissolved in DMSO, and the cells of group T+L were treated with 5 μmol/L lactacystin and 10 μmol/L tubacin dissolved in DMSO. The expression levels of alpha-synuclein oligomers, HSP-27 and HSP-70 were detected by Western blot and the cell survival rate of all the groups was detected by MTT colorimetric assay, and compared 24 h after the cells were treated.ResultsThe expression levels of alpha-synuclein oligomers, HSP-27 and HSP-70 of the cells of group L were significantly higher than the control group, and the cell survival rate was significantly lower (P 0.05).ConclusionsThe expression level of alpha-synuclein oligomers can be improved and the cell survival rate can be reduced by the PD cell model induced by lactacystin and treated with selective HDAC-6 inhibitor tubacin, which means that alpha-synuclein oligomers of the PD cell model induced by lactacystin can be inhibited and the cell survival rate can be improved by HDAC-6, and the mechanism may be related to the increased of HSP-27 and HSP-70.

Abuse of methamphetamine (METH), a powerful addictive amphetamine-type stimulants (ATS), is becoming a global public health problem. The gamma-aminobutyric acid (GABA)ergic system plays a critical role in METH use disorders. By using rat METH conditioned place preference (CPP) model, we previously demonstrated that METH-associated rewarding memory formation was associated with the reduction of GABAAα1 expression in the dorsal straitum (Dstr), however, the underlying mechanism was unclear. In the present study, we found that METH-induced CPP formation was accompanied by a significant increase in the expression of Synovial apoptosis inhibitor 1 (SYVN1), an endoplasmic reticulum (ER)-associated degradation (ERAD) E3 ubiquitin ligase, in the Dstr. The siRNA knockdown of SYVN1 significantly increased GABAAα1 protein levels in both primary cultured neurons and rodent Dstr. Inhibition of proteasomal activity by MG132 and Lactacystin significantly increased GABAAα1 protein levels. We further found that SYVN1 knockdown increased GABAAα1 in the intra-ER, but not in the extra-ER. Accordingly, endoplasmic reticulum stress (ERS)-associated Glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) increased. Thus, this study revealed that SYVN1, as the ERAD E3 ubiquitin ligase, was associated with Dstr GABAAα1 degradation induced by METH conditioned pairing.

Dysferlin is a transmembrane protein implicated in surface membrane repair of muscle cells. Mutations in dysferlin cause the progressive muscular dystrophies Miyoshi myopathy, limb girdle muscular dystrophy 2B, and distal anterior compartment myopathy. Dysferlinopathies are inherited in an autosomal recessive manner, and many patients with this disease harbor mis-sense mutations in at least one of their two pathogenic DYSF alleles. These patients have significantly reduced or absent dysferlin levels in skeletal muscle, suggesting that dysferlin encoded by mis-sense alleles is rapidly degraded by the cellular quality control system. We reasoned that mis-sense mutated dysferlin, if salvaged from degradation, might be biologically functional. We used a dysferlin-deficient human myoblast culture harboring the common R555W mis-sense allele and a DYSF-null allele, as well as control human myoblast cultures harboring either two wild-type or two null alleles. We measured dysferlin protein and mRNA levels, resealing kinetics of laser-induced plasmalemmal wounds, myotube formation, and cellular viability after treatment of the human myoblast cultures with the proteasome inhibitors lactacystin or bortezomib (Velcade). We show that endogenous R555W mis-sense mutated dysferlin is degraded by the proteasomal system. Inhibition of the proteasome by lactacystin or Velcade increases the levels of R555W mis-sense mutated dysferlin. This salvaged protein is functional as it restores plasma membrane resealing in patient-derived myoblasts and reverses their deficit in myotube formation. Bortezomib and lactacystin did not cause cellular toxicity at the regimen used. Our results raise the possibility that inhibition of the degradation pathway of mis-sense mutated dysferlin could be used as a therapeutic strategy for patients harboring certain dysferlin mis-sense mutations.

Lactacystin is a proteasome inhibitor that interferes with several factors involved in heart remodelling. The aim of this study was to investigate whether the chronic administration of lactacystin induces hypertension and heart remodelling and whether these changes can be modified by captopril or melatonin. In addition, the lactacystin-model was compared with NG-nitro-l-arginine-methyl ester (L-NAME)- and continuous light-induced hypertension. Six groups of three-month-old male Wistar rats (11 per group) were treated for six weeks as follows: control (vehicle), L-NAME (40 mg/kg/day), continuous light (24 h/day), lactacystin (5 mg/kg/day) alone, and lactacystin with captopril (100 mg/kg/day), or melatonin (10 mg/kg/day). Lactacystin treatment increased systolic blood pressure (SBP) and induced fibrosis of the left ventricle (LV), as observed in L-NAME-hypertension and continuous light-hypertension. LV weight and the cross-sectional area of the aorta were increased only in L-NAME-induced hypertension. The level of oxidative load was preserved or reduced in all three models of hypertension. Nitric oxide synthase (NOS) activity in the LV and kidney was unchanged in the lactacystin group. Nuclear factor-kappa B (NF-κB) protein expression in the LV was increased in all treated groups in the cytoplasm, however, in neither group in the nucleus. Although melatonin had no effect on SBP, only this indolamine (but not captopril) reduced the concentration of insoluble and total collagen in the LV and stimulated the NO-pathway in the lactacystin group. We conclude that chronic administration of lactacystin represents a novel model of hypertension with collagenous rebuilding of the LV, convenient for testing antihypertensive drugs or agents exerting a cardiovascular benefit beyond blood pressure reduction.

Parkinson's disease (PD) is a neurodegenerative disorder, characterized by a loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). Caloric restriction (CR) has been shown to exert ghrelin-dependent neuroprotective effects in the 1-methyl-4-phenyl-1,2,3,6-tetrathydropyridine (MPTP)-based animal model for PD. We here investigated whether CR is neuroprotective in the lactacystin (LAC) mouse model for PD, in which proteasome disruption leads to the destruction of the DA neurons of the SNc, and whether this effect is mediated via the ghrelin receptor. Adult male ghrelin receptor wildtype (WT) and knockout (KO) mice were maintained on an ad libitum (AL) diet or on a 30% CR regimen. After 3 weeks, LAC was injected unilaterally into the SNc, and the degree of DA neuron degeneration was evaluated 1 week later. In AL mice, LAC injection significanty reduced the number of DA neurons and striatal DA concentrations. CR protected against DA neuron degeneration following LAC injection. However, no differences were observed between ghrelin receptor WT and KO mice. These results indicate that CR can protect the nigral DA neurons from toxicity related to proteasome disruption; however, the ghrelin receptor is not involved in this effect.

This article belongs to the Special Issue Ubiquitin System., Spinal muscular atrophy is due to mutations affecting the SMN1 gene coding for the full-length protein (survival motor neuron; SMN) and the SMN2 gene that preferentially generates an exon 7-deleted protein (SMNΔ7) by alternative splicing. To study SMN and SMNΔ7 degradation in the cell, we have used tagged versions at the N- (Flag) or C-terminus (V5) of both proteins. Transfection of those constructs into HeLa cells and treatment with cycloheximide showed that those protein constructs were degraded. Proteasomal degradation usually requires prior lysine ubiquitylation. Surprisingly, lysine-less variants of both proteins tagged either at N- (Flag) or C-terminus (V5) were also degraded. The degradation of the endogenous SMN protein, and the protein constructs mentioned above, was mediated by the proteasome, as it was blocked by lactacystin, a specific and irreversible proteasomal inhibitor. The results obtained allowed us to conclude that SMN and SMNΔ7 proteasomal degradation did not absolutely require internal ubiquitylation nor N-terminal ubiquitylation (prevented by N-terminal tagging). While the above conclusions are firmly supported by the experimental data presented, we discuss and justify the need of deep proteomic techniques for the study of SMN complex components (orphan and bound) turn-over to understand the physiological relevant mechanisms of degradation of SMN and SMNΔ7 in the cell., This work was supported by grants from MINECO SAF-2012-34556 and CIBERNED to José G. Castaño.

Cisplatin is commonly used as a therapeutic agent, despite its known adverse side effects and the occurrence of drug resistance. The development of novel methods for combination therapy with cisplatin is required in order to circumvent these limitations of cisplatin alone. The proteasome inhibitor lactacystin (LAC) has been indicated to produce anti-tumor effects, and has previously been used as an antitumor agent in cancer treatment research; however, its effects in combination with cisplatin treatment are unknown. In the current study, the effects of LAC in combination with cisplatin treatment were investigated in HeLa human cervical cancer (HCC) cells. The results demonstrated that cisplatin treatment inhibited cell growth and induced cell apoptosis. HeLa cell exposure to cisplatin induced endoplasmic reticulum (ER) stress-associated apoptosis, and LAC treatment increased levels of cell apoptosis and the activation of caspase-3. Specifically, LAC treatment increased the cisplatin-induced expression of PDI, GRP78, CHOP, cleaved caspase-4 and cleaved caspase-3. Together, these data indicate that LAC is able to enhance cisplatin cytotoxicity by increasing ER stress-associated apoptosis in HeLa cells.

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide and recent studies implicate a central role for ubiquitin–proteasome system (UPS) impairment in the etiopathogenesis of PD. To explore the possible role of UPS dysfunction in PD and the proteins involved, PC12 cells were treated with 10 μM lactacystin, a 20S proteasome inhibitor, for 24 h. Lactacystin induced cell death and α-synuclein-positive inclusions in cytoplasm. Following two-dimensional difference in-gel electrophoresis (2-D DIGE) which was used to separate the cellular proteins, the proteins that were significantly altered were analyzed and identified. Proteomic study identified 6 differentially expressed proteins between lactacystin-treated and control cells in this study. Four proteins (heat shock 70 kDa protein 8, 78 kDa glucose-regulated protein, serine proteinase inhibitor clade B member 6 and aldehyde reductase) were increased and 2 proteins (peripherin and tyrosine hydroxylase) were decreased following proteasomal inhibition. The results revealed that PC12 cells treated with 10 μM lactacystin for 24 h could be used as a cellular model of PD. The proteins identified in the present indicate not only the damage of proteasomal inhibition to the cells but also the possible responses of the cells. These data show that proteomic study may provide information relevant to biological basis for PD and potential new treatment targets.

Alcohol-induced liver fibrosis and eventually cirrhosis is a leading cause of death. Acetaldehyde, the first metabolite of ethanol, up-regulates expression of the human α2(I) collagen gene ( COL1A2 ). Early acetaldehyde-mediated effects involve phosphorylation and nuclear translocation of SMAD3/4–containing complexes that bind to COL1A2 promoter to induce fibrogenesis. We used human and mouse hepatic stellate cells to elucidate the mechanisms whereby acetaldehyde up-regulates COL1A2 by modulating the role of Ski and the expression of SMADs 3, 4, and 7. Acetaldehyde induced up-regulation of COL1A2 by 3.5-fold, with concomitant increases in the mRNA (threefold) and protein (4.2- and 3.5-fold) levels of SMAD3 and SMAD4, respectively. It also caused a 60% decrease in SMAD7 expression. Ski, a member of the Ski/Sno oncogene family, is colocalized in the nucleus with SMAD4. Acetaldehyde induces translocation of Ski and SMAD4 to the cytoplasm, where Ski undergoes proteasomal degradation, as confirmed by the ability of the proteasomal inhibitor lactacystin to blunt up-regulation of acetaldehyde-dependent COL1A2 , but not of the nonspecific fibronectin gene ( FN1 ). We conclude that acetaldehyde up-regulates COL1A2 by enhancing expression of the transactivators SMAD3 and SMAD4 while inhibiting the repressor SMAD7, along with promoting Ski translocation from the nucleus to cytoplasm. We speculate that drugs that prevent proteasomal degradation of repressors targeting COL1A2 may have antifibrogenic properties.

The ubiquitin–proteasome system is important to maintain pancreatic β-cell function. Inhibition of the proteasome significantly reduced glucose-induced insulin secretion. Key regulators of the stimulus/secretion cascade seem to be affected by protein misfolding if the proteasome is down-regulated as recently reported in humans with Type 2 diabetes. It remains unknown, however, whether the glucose sensor enzyme glucokinase is involved in this process. A direct interaction between glucokinase and ubiquitin could be shown in vivo by FRET, suggesting regulation of glucokinase by the proteasome. After proteasome inhibition glucokinase activity was significantly reduced in MIN6 cells, whereas the protein content was increased, indicating protein misfolding. Enhancing the availability of chaperones by cyclohexamide could induce refolding and restored glucokinase activity. Glucokinase aggregation due to proteasome blocking with MG132, bortezomib, epoxomicin or lactacystin could be detected in MIN6 cells, primary β-cells and hepatocytes using fluorescence-based assays. Glucokinase aggresome formation proceeded microtubule-assisted and was avoided by cyclohexamide. Thus the results of the present study provide support for glucokinase misfolding and aggregation in case of a diminished capacity of the ubiquitin–proteasome system in pancreatic β-cells. In the Type 2 diabetic situation this could contribute to reduced glucose-induced insulin secretion.

Prefoldin is a molecular chaperone composed of six subunits, PFD1–6, and prevents misfolding of newly synthesized nascent polypeptides. Although it is predicted that prefoldin, like other chaperones, modulates protein aggregation, the precise function of prefoldin against protein aggregation under physiological conditions has never been elucidated. In this study, we first established an anti-prefoldin monoclonal antibody that recognizes the prefoldin complex but not its subunits. Using this antibody, it was found that prefoldin was localized in the cytoplasm with dots in co-localization with polyubiquitinated proteins and that the number and strength of dots were increased in cells that had been treated with lactacystin, a proteasome inhibitor, and thapsigargin, an inducer of endoplasmic reticulum stress. Knockdown of prefoldin increased the level of SDS-insoluble ubiquitinated protein and reduced cell viability in lactacystin and thapsigargin-treated cells. Opposite results were obtained in prefoldin-overexpressed cells. It has been reported that mice harboring a missense mutation L110R of MM-1α/PFD5 exhibit neurodegeneration in the cerebellum. Although the prefoldin complex containing L110R MM-1α was properly formed in vitro and in cells derived from L110R MM-1α mice, the levels of ubiquitinated proteins and cytotoxicity were higher in L110R MM-1α cells than in wild-type cells under normal conditions and were increased by lactacystin and thapsigargin treatment, and growth of L110R MM-1α cells was attenuated. Furthermore, the polyubiquitinated protein aggregation level was increased in the brains of L110R MM-1α mice. These results suggest that prefoldin plays a role in quality control against protein aggregation and that dysfunction of prefoldin is one of the causes of neurodegenerative diseases. Background: Prefoldin consisting of six subunits prevents protein misfolding. Results: Prefoldin prevented proteasome inhibitor- and endoplasmic reticulum stress-induced protein aggregation, and mutation of a prefoldin subunit resulted in loss of its activity. Conclusion: In addition to its protein folding activity, prefoldin protects cells from aggregated protein-induced cell death. Significance: Prefoldin is a quality control protein against protein aggregation.

Alpha4 (α4) is a key regulator of protein phosphatase 2A (PP2A) and mTOR in steps essential for cell-cycle progression. α4 forms a complex with PP2A and MID1, a microtubule-associated ubiquitin E3 ligase that facilitates MID1-dependent regulation of PP2A and the dephosphorylation of MID1 by PP2A. Ectopic overexpression of α4 is associated with hepatocellular carcinomas, breast cancer, and invasive adenocarcinomas. Here, we provide data suggesting that α4 is regulated by ubiquitin-dependent degradation mediated by MID1. In cells stably expressing a dominant-negative form of MID1, significantly elevated levels of α4 were observed. Treatment of cells with the specific proteasome inhibitor, lactacystin, resulted in a 3-fold increase in α4 in control cells and a similar level in mutant cells. Using in vitro assays, individual MID1 E3 domains facilitated monoubiquitination of α4, whereas full-length MID1 as well as RING-Bbox1 and RING-Bbox1-Bbox2 constructs catalyzed its polyubiquitination. In a novel non-biased functional screen, we identified a leucine to glutamine substitution at position 146 within Bbox1 that abolished MID1-α4 interaction and the subsequent polyubiquitination of α4, indicating that direct binding to Bbox1 was necessary for the polyubiquitination of α4. The mutant had little impact on the RING E3 ligase functionality of MID1. Mass spectrometry data confirmed Western blot analysis that ubiquitination of α4 occurs only within the last 105 amino acids. These novel findings identify a new role for MID1 and a mechanism of regulation of α4 that is likely to impact the stability and activity level of PP2Ac.

Growing evidence has shown that proNGF-p75NTR-sortilin signaling might be a crucial factor in neurodegeneration, but it remains unclear if it may function in nigral neurons under aging and disease. The purpose of this study is to examine and quantify proNGF and sortilin expression in the substantia nigra and dynamic changes of aging in lactacystin and 6-hydroxydopamine (6-OHDA) rat models of Parkinson’s disease using immunofluorescence, electronic microscopy, western blot and FLIVO staining methods. The expression of proNGF and sortilin was abundantly and selectively identified in tyrosine hydroxylase (TH)-containing dopamine neurons in the substantia nigra. These proNGF/TH, sortilin/TH-positive neurons were densely distributed in the ventral tier, while they were less distributed in the dorsal tier, where calbindin-D28K-containing neurons were numerously located. A correlated decrease of proNGF, sortilin and TH was also detected during animal aging process. While increase of proNGF, sortilin and cleaved (active) caspase-3 expression was found in the lactacystin model, dynamic proNGF and sortilin changes along with dopamine neuronal loss were demonstrated in the substantia nigra of both the lactacystin and 6-OHDA models. This study has thus revealed the presence of the proNGF-sortilin signaling complex in nigral dopamine neurons and its response to aging, lactacystin and 6-OHDA insults, suggesting that it might contribute to neuronal apoptosis or neurodegeneration during pathogenesis and disease progression of Parkinson’s disease; the underlying mechanism and key signaling pathways involved warrant further investigation.

Sf-caspase-1 is the principal effector caspase in Spodoptera frugiperda cells. Like the caspases in other organisms, Sf-caspase-1 is processed by upstream caspases to form an active heterotetramer composed of the p19 and p12 subunits. The regulation of active caspases is crucial for cellular viability. In mammal cells, the subunits and the active form of caspase-3 were rapidly degraded relative to its proenzyme form. In the present study, the S. frugiperda Sf9 cells were transiently transfected with plasmids encoding different fragments of Sf-caspase-1: the pro-Sf-caspase-1 (p37), a prodomain deleted fragment (p31), a fragment containing the large subunit and the prodomain (p25), the large subunit (p19), and the small subunit (p12). Flow cytometry and Western blot analysis revealed that p12, p19, and p25 were unstable in the transfected cells, in contrast to p37 and p31. Lactacystin, a proteasome inhibitor, increased the accumulation of the p19 and p12 subunits, suggesting that the degradation is performed by the ubiquitin-proteasome system. During the activation, the Sf-caspase-1 produces an intermediate form and then undergoes proteolytic processing to form active Sf-caspase-1. We found that both the active and the intermediate form were unstable, indicating that once activated or during its activation, the Sf-caspase-1 was unstable.

The study aimed to analyze the impact of the proteasome inhibitors MG132 (N-carbobenzyoxyl-L-leucyl-L-leucyl-L-leucinal), lactacystin and celastrol on manganese superoxide dismutase (MnSOD), catalase and glutathione-S-transferase-π (GST-π), and on the heat shock protein 70 (Hsp70) in human peripheral blood mononuclear cells (PBMC), exposed to ionizing radiation.Changes in protein levels were analyzed by Western blot. Cellular viability, proteasome activity, level of oxidative stress and apoptosis were determined by standard colorimetric and fluorescence assays.MG132 and lactacystin induced an increase in the intracellular levels of Hsp70. MnSOD was up-regulated by MG132 and celastrol, and GST-π was up-regulated by MG132 and lactacystin. Notably, the proteasome inhibitors significantly modified the protein levels in the irradiated cells and dramatically reduced the intracellular pool of oxidative species. The combined effect of radiation and proteasome inhibition was a dose-dependent up-regulation of the antioxidant enzymes and Hsp70.All three proteasome inhibitors showed antioxidant effects in PBMC and up-regulated the antioxidant enzymes MnSOD, catalase and GST-π and the stress protein Hsp70, modifying the early radiation response, and conferring protection against the effects of ionizing radiation.

Sulfatase 2 (Sulf-2) has been previously shown to be upregulated in breast cancer. Sulf-2 removes sulfate moieties on heparan sulfate proteoglycans which in turn modulate heparin binding growth factor signaling. Here we report that matrix detachment resulted in decreased Sulf-2 expression in breast cancer cells and increased cleavage of poly ADP-ribose polymerase. Silencing of Sulf-2 promotes matrix detachment induced cell death in MCF10DCIS cells. In an attempt to identify Sulf-2 specific inhibitor, we found that proteasomal inhibitors such as MG132, Lactacystin and Bortezomib treatment abolished Sulf-2 expression in multiple breast cancer cell lines. Additionally, we show that Bortezomib treatment of MCF10DCIS cell xenografts in mouse mammary fat pads significantly reduced tumor size, caused massive apoptosis and more importantly reduced Sulf-2 levels in vivo. Finally, our immunohistochemistry analysis of Sulf-2 expression in cohort of patient derived breast tumors indicates that Sulf-2 is significantly upregulated in autologous metastatic lesions compared to primary tumors (p < 0.037, Pearson correlation, Chi-Square analysis). In all, our data suggest that Sulf-2 might play an important role in breast cancer progression from ductal carcinoma in situ into an invasive ductal carcinoma potentially by resisting cell death. © 2013 Springer Science+Business Media Dordrecht.

Baculovirus AcMNPV causes proteotoxicity in Sf9 cells as revealed by accumulation of ubiquitinated proteins and aggresomes in the course of infection. Inhibition of proteasomes by lactacystin increased markedly the stock of ubiquitinated proteins indicating a primary role of proteasomes in detoxication. The proteasomes were present in Sf9 cells as 26S and 20S complexes whose protease activity did not change during infection. Proteasome inhibition caused a delay in the initiation of viral DNA replication suggesting an important role of proteasomes at early stages in infection. However, lactacystin did not affect ongoing replication indicating that active proteasomes are not required for genome amplification. At late stages in infection (24–48hpi), aggresomes containing the ubiquitinated proteins and HSP/HSC70s showed gradual fusion with the vacuole-like structures identified as lysosomes by antibody to cathepsin D. This result suggests that lysosomes may assist in protection against proteotoxicity caused by baculoviruses absorbing the ubiquitinated proteins.

Plasminogen activator inhibitor-1 (PAI-1) is a member of serine protease inhibitor family, which regulates the activity of tissue plasminogen activator (tPA). In CNS, tPA/PAI-1 activity is involved in the regulation of a variety of cellular processes such as neuronal development, synaptic plasticity and cell survival. To gain a more insights into the regulatory mechanism modulating tPA/PAI-1 activity in brain, we investigated the effects of proteasome inhibitors on tPA/PAI-1 expression and activity in rat primary astrocytes, the major cell type expressing both tPA and PAI-1. We found that submicromolar concentration of MG132, a cell permeable peptide-aldehyde inhibitor of ubiquitin proteasome pathway selectively upregulates PAI-1 expression. Upregulation of PAI-1 mRNA as well as increased PAI-1 promoter reporter activity suggested that MG132 transcriptionally increased PAI-1 expression. The induction of PAI-1 downregulated tPA activity in rat primary astrocytes. Another proteasome inhibitor lactacystin similarly increased the expression of PAI-1 in rat primary astrocytes. MG132 activated MAPK pathways as well as PI3K/Akt pathways. Inhibitors of these signaling pathways reduced MG132-mediated upregulation of PAI-1 in varying degrees and most prominent effects were observed with SB203580, a p38 MAPK pathway inhibitor. The regulation of tPA/PAI-1 activity by proteasome inhibitor in rat primary astrocytes may underlie the observed CNS effects of MG132 such as neuroprotection.

Forkhead box O1 (FoxO1) is a key molecule for the development and functions of peripheral T cells. However, the precise mechanisms regulating FoxO1 expression in peripheral T cells remain elusive. We previously reported that Zfat(f/f)-CD4Cre mice showed a marked decline in FoxO1 protein levels in peripheral T cells, partially through proteasomal degradation. Here we have identified the precise mechanisms, apart from proteasome-mediated degradation, of the decreased FoxO1 levels in Zfat-deficient T cells. First, we confirmed that tamoxifen-inducible deletion of Zfat in Zfat(f/f)-CreERT2 mice coincidently decreases FoxO1 protein levels in peripheral T cells, indicating that Zfat is essential for maintaining FoxO1 levels in these cells. Although the proteasome-specific inhibitors lactacystin and epoxomicin only moderately increase FoxO1 protein levels, the inhibitors of lysosomal proteolysis bafilomycin A1 and chloroquine restore the decreased FoxO1 levels in Zfat-deficient T cells to levels comparable with those in control cells. Furthermore, Zfat-deficient T cells show increased numbers of autophagosomes and decreased levels of p62 protein, together indicating that Zfat deficiency promotes lysosomal FoxO1 degradation through autophagy. In addition, Zfat deficiency increases the phosphorylation levels of Thr-308 and Ser-473 of Akt and the relative amounts of cytoplasmic to nuclear FoxO1 protein levels, indicating that Zfat deficiency causes Akt activation, leading to nuclear exclusion of FoxO1. Our findings have demonstrated a novel role of Zfat in maintaining FoxO1 protein levels in peripheral T cells by regulating the activities of autophagy and the Akt signaling pathway.

Background The proteasome degrades ubiquitinated proteins and is the major pathway for intracellular protein degradation. The role of the proteasome in endothelial dysfunction observed in septic shock remains unknown. We stimulated primary cultures of human umbilical vein endothelial cells with lipopolysaccharide (LPS) and investigated effects on the proteasome. We hypothesized that proteasome inhibition would decrease endothelial cell activation, oxidative stress, and alter the proteome. Methods Endothelial cells were exposed to LPS (100 ng/mL) for 6 hours with or without lactacystin (5 mM), a proteasome inhibitor. Proteasome content and ubiquitinated proteins were measured by enzyme-linked immunosorbent assay and immunoblot, respectively. Markers of cellular activation, vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, were measured by immunoblot and immunoassay. Superoxide anion production was determined by dihydroethidium assay, and nitrotyrosine (a marker of peroxynitrite) was visualized by immunofluoresence. The endothelial cell proteome was analyzed by 2D gel electrophoresis. Results LPS stimulation of endothelial cells significantly increased proteasome content, whereas the total levels of ubquitinated proteins decreased. This suggests that LPS activates the proteasome system in endothelial cells. LPS increased total content and cell surface expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, whereas proteasome inhibition ameliorated these increases. LPS increased both superoxide anion production and nitrotyrosine staining. Proteasome inhibition decreased both markers of cellular oxidative stress. Proteomic analysis identified two novel proteins upregulated by LPS and normalized with proteasome inhibition as follows: guanine nucleotide binding protein-1 and heterogeneous ribonucleoprotein K transcript variant. Conclusions These results suggest that inhibition of the proteasome diminishes a number of markers of cellular stress induced by LPS. The proteasome may be a promising therapeutic target in clinical situations of severe pro-inflammatory stress.

Rhabdomyosarcoma is a soft tissue sarcoma mainly seen in children. Despite considerable progress within the last few years, therapeutic approaches for this type of tumor are still limited. The respective tumor cells originate from myogenic precursor cells and are characterized by a blockade in their differentiation program. Interestingly, there is a direct inverse correlation between the differentiation status of a specific rhabdomyosarcoma cell and its metastatic potential. Thus, here, we tested whether the ubiquitous transcription factor NF-κB, which regulates myogenic differentiation and is also a promising therapeutic target in the treatment of other types of tumors, might be an interesting candidate for the development of novel rhabdomyosarcoma treatment strategies. For this purpose, we analyzed NF-κB activity (classical pathway) in myoblasts with different differentiation potential, specifically in three different rhabdomyosarcoma cell lines. In addition, we inhibited NF-κB activity in these cells and analyzed the effects on myogenic differentiation. We show that after the induction of differentiation, NF-κB activity declines rapidly in normal myoblasts, but only slightly in rhabdomyosarcoma cells. However, after treatment of the cells with two different small-molecule NF-κB-inhibiting compounds, the IKK inhibitor curcumin and the proteasome inhibitor lactacystin, we found that neither curcumin nor lactacystin promoted myogenic differentiation in either normal myoblasts or rhabdomyosarcoma cells. Taken together, our data suggest that treatment with curcumin or lactacystin might not be a suitable approach in the treatment of rhabdomyosarcoma.

Shiga-toxigenic Escherichia coli (STEC) produces subtilase cytotoxin (SubAB), which cleaves the molecular chaperone BiP in the endoplasmic reticulum (ER), leading to an ER stress response and then activation of apoptotic signaling pathways. Here, we show that an early event in SubAB-induced apoptosis in HeLa cells is mediated by RNA-dependent protein kinase (PKR)-like ER kinase (PERK), not activating transcription factor 6 (ATF6) or inositol-requiring enzyme 1(Ire1), two other ER stress sensors. PERK knockdown suppressed SubAB-induced eIF2α phosphorylation, activating transcription factor 4 (ATF4) expression, caspase activation, and cytotoxicity. Knockdown of eIF2α by small interfering RNA (siRNA) or inhibition of eIF2α dephosphorylation by Sal003 enhanced SubAB-induced caspase activation. Treatment with proteasome inhibitors (i.e., MG132 and lactacystin), but not a general caspase inhibitor (Z-VAD) or a lysosome inhibitor (chloroquine), suppressed SubAB-induced caspase activation and poly(ADP-ribose) polymerase (PARP) cleavage, suggesting that the ubiquitin-proteasome system controls events leading to caspase activation, i.e., Bax/Bak conformational changes, followed by cytochrome c release from mitochondria. Levels of ubiquitinated proteins in HeLa cells were significantly decreased by SubAB treatment. Further, in an early event, some antiapoptotic proteins, which normally turn over rapidly, have their synthesis inhibited, and show enhanced degradation via the proteasome, resulting in apoptosis. In PERK knockdown cells, SubAB-induced loss of ubiquitinated proteins was inhibited. Thus, SubAB-induced ER stress is caused by BiP cleavage, leading to PERK activation, not by accumulation of ubiquitinated proteins, which undergo PERK-dependent degradation via the ubiquitin-proteasome system.

Purpose To elucidate the effects of tumor necrosis factor (TNF)-α on the expression of inducible nitric oxide synthase (iNOS) in retinal pigment epithelial cells in vitro. Methods ARPE-19 cell lines were cultured with TNF-α stimulation, and then treated with proteasome inhibitors (MG132 or lactacystin) for 30 minutes. The expression of iNOS was determined by RT-PCR and Western blot. The expression of nitric oxide (NO) was determined by an enzyme-linked immunosorbent assay. The interaction of nuclear factor kappa-B (NF-κB) activation and iNOS induction was assessed by electrophoretic mobility shift assay. Results The expression of iNOS in ARPE-19 was induced by TNF-α in a dose-dependent manner. Upregulation of iNOS resulted in increased production of NO. iNOS induced by TNF-α could be inhibited by MG-132 and lactacystin. Supershift assay revealed that NF-κB activation was responsible for iNOS induction. Conclusion TNF-α could induce iNOS expression and NO production in RPE cells, at least in part, via an NF-κB signal pathway.

During the final stage of oocyte growth, the morphology of the oocyte nucleoli changes into a compact structure. The objective of this study was to determine the involvement of the proteasome, which is a large protein complex responsible for degrading intracellular proteins, in the nucleolar compaction. The mean nucleolar diameter of growing porcine oocytes (about 100 µm in diameter) was larger than that of fully grown (120 µm) oocytes (15.5 ± 0.3 vs. 13.2 ± 0.1 µm, P

Dexamethasone (Dex), a ligand for transcriptional enhancement of tyrosine aminotransferase (TAT) and tryptophan 2,3-dioxygenase (TO) genes, (100 nM) maximally increased these mRNA levels at 12 h and 7 h in primary cultured rat hepatocytes and the nuclear fraction, respectively. Lactacystin (5 μM) and epoxomicin (0.5 μM), 26S proteasome inhibitors, significantly suppressed the Dex-dependent maximum increase of TAT and TO mRNA levels in the cells and the nuclear fraction. Electrophoretic mobility shift assay demonstrated that lactacystin did not affect binding of glucocorticoid receptor to glucocorticoid responsive element. Furthermore, lactacystin did not affect the activation of GRE luciferase reporter by Dex transfected to the cells. The results demonstrate that 26S proteasome is positively involved in the Dex-dependent increase of TAT and TO mRNA levels in the cells and suggest that the mechanism of action of 26S proteasome may be degradation of some RNase(s), which breaks down TAT and TO mRNAs.

Background and Purpose: The proteasome is a multi-subunit complex that proteolytically cleaves proteins. The replacement of the constitutive proteasome subunits β1, β2, and/or β5 with the IFNγ-inducible subunits LMP2, MECL1, and/or LMP7 results in the ‘immunoproteasome’. The inducible subunits change the cleavage specificities of the proteasome, but it is unclear whether they have functions in addition to this. The purpose of the present study was to determine the role of the proteasome in general, as well as LMP7 and MECL1 specifically, with regard to cytokine production by activated primary splenocytes. Methods: A LMP7/MECL1-null mouse was engineered to determine the roles of these subunits in cytokine production. Isolated splenocytes from wild-type and LMP7/MECL1–/– mice were treated with lactacystin and activated with PMA and ionomycin and subsequently cytokine mRNA levels were quantified. Results: The present study demonstrates that LMP7/MECL1 regulates the expression of IFNγ, IL4, IL10, IL2Rβ, GATA3, and t-bet. In contrast, the regulation of IL2, IL13, TNFα, and IL2Rα by the proteasome appears to occur independently of LMP7/MECL1. Conclusions: Collectively, the present study demonstrates that LMP7 and MECL1 regulate cytokine expression, suggesting this system represents a novel mechanism for the regulation of cytokines and cytokine signaling.

Paclitaxel (Taxol) is one of the most effective treatment options for patients suffering from a variety of cancers. A major side effect seen in a high percentage of patients treated with paclitaxel is irreversible peripheral neuropathy. We previously reported that prolonged treatment with paclitaxel activates a calcium-dependent enzyme, calpain, which degrades neuronal calcium sensor 1 (NCS-1) and subsequent loss of intracellular calcium signaling. Because it appears that activation of calpain is an early step in this destructive cascade, we proposed that inhibition of calpain will protect against the unwanted side effects of paclitaxel treatment. First, NCS-1 levels and intracellular calcium signaling were found to be protected by the presence of lactacystin, a protesome inhibitor. To reinforce the role of calpain in this process, we showed that increased concentrations of calpastatin, a naturally occurring calpain inhibitor, were protective. Next, we tested two mutated versions of NCS-1 developed with point mutations at the P2 position of the calpain cleavage site of NCS-1 to decrease the likelihood of NCS-1 degradation. One mutant was cleaved more favorably by calpain compared with NCS-1 WT, whereas the other mutant was less favorably cleaved. Expression of either mutated version of NCS-1 in neuroblastoma cells protected intracellular calcium signals from paclitaxel-induced changes. These results support our hypothesis that it is possible to protect cells from paclitaxel-induced degradation of NCS-1 by inhibiting calpain activity.

Insulin receptor substrate-2 (IRS-2) plays a critical role in the survival and function of pancreatic β-cells. Gene disruption of IRS-2 results in failure of the β-cell compensatory mechanism and diabetes. Nonetheless, the regulation of IRS-2 protein expression in β-cells remains largely unknown. Inducible nitric-oxide synthase (iNOS), a major mediator of inflammation, has been implicated in β-cell damage in type 1 and type 2 diabetes. The effects of iNOS on IRS-2 expression have not yet been investigated in β-cells. Here, we show that iNOS and NO donor decreased IRS-2 protein expression in INS-1/832 insulinoma cells and mouse islets, whereas IRS-2 mRNA levels were not altered. Interleukin-1β (IL-1β), alone or in combination with interferon-γ (IFN-γ), reduced IRS-2 protein expression in an iNOS-dependent manner without altering IRS-2 mRNA levels. Proteasome inhibitors, MG132 and lactacystin, blocked the NO donor-induced reduction in IRS-2 protein expression. Treatment with NO donor led to activation of glycogen synthase kinase-3β (GSK-3β) and c-Jun N-terminal kinase (JNK/SAPK) in β-cells. Inhibition of GSK-3β by pharmacological inhibitors or siRNA-mediated knockdown significantly prevented NO donor-induced reduction in IRS-2 expression in β-cells. In contrast, a JNK inhibitor, SP600125, did not effectively block reduced IRS-2 expression in NO donor-treated β-cells. These data indicate that iNOS-derived NO reduces IRS-2 expression by promoting protein degradation, at least in part, through a GSK-3β-dependent mechanism. Our findings suggest that iNOS-mediated decreased IRS-2 expresssion may contribute to the progression and/or exacerbation of β-cell failure in diabetes.

Myocardin is a transcriptional co-activator of serum response factor (SRF) and can be degraded through ubiquitin-proteasome system. Our preliminary studies unexpectedly revealed that accumulation of myocardin in response to proteasome inhibition by MG132 or lactacystin resulted in decrease of transcriptional activity of myocardin as indicated by reduced expression of SMC contractile marker genes (SM α-actin, SM22, and calponin) and muscle-enriched microRNAs (miR-143/145 and miR-1/133a), and reduced contractility of human vascular smooth muscle cells (SMCs) embedded in collagen gel lattices, suggesting that myocardin degradation is required for its transcriptional activity. Further studies using chromatin immunoprecipitation assay revealed that proteasome inhibition, although increased the occupancy of myocardin and SRF on the promoter of SM α-actin gene, abolished myocardin-dependent recruitment of RNA polymerase II. We further examined the degradation of myocardin in epithelioid and spindle-shaped SMCs and revealed that myocardin in more differentiated spindle-shaped SMCs was more quickly degraded and had shorter half-life than in epithelioid SMCs. In neointimal lesions, we found that stabilization of myocardin protein was companied by downregulation of transcripts of ubiquitin and proteasome subunits, further illustrating the mechanism underlying reduction of myocardin transcriptional activity. In summary, our results have suggested that proteasomal degradation of myocardin is required for its transcriptional activity.

Regulator of G protein signaling protein 4 (RGS4) acts as a GTPase accelerating protein to modulate μ- and δ- opioid receptor (MOR and DOR, respectively) signaling. In turn, exposure to MOR agonists leads to changes in RGS4 at the mRNA and/or protein level. Here we have used human neuroblastoma SH-SY5Y cells that endogenously express MOR, DOR, and RGS4 to study opioid-mediated down-regulation of RGS4. Overnight treatment of SH-SY5Y cells with the MOR agonist DAMGO or the DOR agonist DPDPE decreased RGS4 protein by ∼60% accompanied by a profound loss of opioid receptors but with no change in RGS4 mRNA. The decrease in RGS4 protein was prevented by the pretreatment with pertussis toxin or the opioid antagonist naloxone. The agonist-induced down-regulation of RGS4 proteins was completely blocked by treatment with the proteasome inhibitors MG132 or lactacystin or high concentrations of leupeptin, indicating involvement of ubiquitin-proteasome and lysosomal degradation. Polyubiquitinated RGS4 protein was observed in the presence of MG132 or the specific proteasome inhibitor lactacystin and promoted by opioid agonist. The loss of opioid receptors was not prevented by MG132, demonstrating a different degradation pathway. RGS4 is a GTPase accelerating protein for both Gαi/o and Gαq proteins. After overnight treatment with DAMGO to reduce RGS4 protein, signaling at the Gαi/o-coupled DOR and the Gαq-coupled M3 muscarinic receptor (M3R) was increased but not signaling of the α2 adrenergic receptor or bradykinin BK2 receptor, suggesting the development of cross-talk between the DOR and M3R involving RGS4.

It is recognized that Wnt pathways regulate bone metabolism. We have previously shown that tumor necrosis factor-α (TNF-α) stimulates synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, via p44/p42 mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase (PI3-kinase)/Akt in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of Wnt3a on TNF-α-stimulated IL-6 synthesis in these cells. Wnt3a, which alone did not affect the IL-6 levels, significantly suppressed the TNF-α-stimulated IL-6 release. Lithium Chloride (LiCl), which is an inhibitor of GSK3β, markedly reduced the TNF-α-stimulated IL-6 release, similar to the results with Wnt3a. The suppression by Wnt3a or LiCl was also observed in the intracellular protein levels of IL-6 elicited by TNF-α. Wnt3a failed to affect the TNF-α-induced phosphorylation of p44/p42 MAP kinase, Akt, IκB or NFκB. Either Wnt3a or LiCl failed to reduce, rather increased the IL-6 mRNA expression stimulated by TNF-α. Lactacystin, a proteasome inhibitor, and bafilomycin A1, a lysosomal protease inhibitor, significantly restored the suppressive effect of Wnt3a on TNF-α-stimulated IL-6 release. Taken together, our results strongly suggest that Wnt3a regulates IL-6 release stimulated by TNF-α at post-transcriptional level in osteoblasts.

In mammals, expression of the three alternatively spliced exons of the tyrosine phosphatase CD45 is regulated by the developmental and activation state of the cell. In comparison, the channel catfish, Ictalurus punctatus, CD45 homolog contains 18 functional alternatively spliced exons. Since very little is known about CD45 regulation in ectothermic vertebrates, this study examines the regulation of catfish CD45 mRNA isoform expression in clonal T and B cells in response to stimulation. Results show that mitogenic stimulation using catfish serum or concanavalin A induced expression of mRNAs for small CD45 isoforms, and isoform message expression was growth curve dependent, i.e. cells in logarithmic phase express message for smaller CD45 isoforms, whereas stationary phase cells express message for longer CD45 isoforms. In addition, cells treated with the protein synthesis inhibitor cycloheximide expressed message for longer CD45 isoforms, and treatment with lactacystin, which blocks protein degradation, rescued smaller isoform message expression. Collectively these data suggested that expression of CD45 isoforms, in catfish, at least at the mRNA level, is "constitutively dynamic" and highly dependent on extracellular stimuli.

Cachectic muscle wasting is a frequent complication of many inflammatory conditions, due primarily to excessive muscle catabolism. However, the pathogenesis and intervention strategies against it remain to be established. Here, we tested the hypothesis that Toll-like receptor 4 (TLR4) is a master regulator of inflammatory muscle catabolism. We demonstrate that TLR4 activation by lipopolysaccharide (LPS) induces C2C12 myotube atrophy via up-regulating autophagosome formation and the expression of ubiquitin ligase atrogin-1/MAFbx and MuRF1. TLR4-mediated activation of p38 MAPK is necessary and sufficient for the up-regulation of atrogin1/MAFbx and autophagosomes, resulting in myotube atrophy. Similarly, LPS up-regulates muscle autophagosome formation and ubiquitin ligase expression in mice. Importantly, autophagy inhibitor 3-methyladenine completely abolishes LPS-induced muscle proteolysis, while proteasome inhibitor lactacystin partially blocks it. Furthermore, TLR4 knockout or p38 MAPK inhibition abolishes LPS-induced muscle proteolysis. Thus, TLR4 mediates LPS-induced muscle catabolism via coordinate activation of the ubiquitin-proteasome and the autophagy-lysosomal pathways.—Doyle, A., Zhang, G., Abdel Fattah, E. A., Eissa, N. T., Li, Y.-P. Toll-like receptor 4 mediates lipopolysaccharide-induced muscle catabolism via coordinate activation of ubiquitin-proteasome and autophagy-lysosome pathways.

Excess fatty acids are closely associated with metabolic dysfunction. The deleterious effects of fatty acids relate, in part, to their ability to up-regulate proinflammatory cytokines and propagate a state of systemic inflammation. CREBh is a recently identified transcription factor that appears to be required for hepatic synthesis of C-reactive protein (CRP). Recent data suggest that fatty acids can up-regulate CREBh, thus establishing a potential molecular link between fatty acids and inflammation. The aim of the current study was to examine the nature and mechanisms of fatty acid-mediated regulation of CREBh. H4IIE liver cells were incubated in the absence or presence of varying concentrations (50–500 μM) of albumin-bound, long-chain saturated (palmitate, stearate) or unsaturated (oleate, linoleate) fatty acids (1–16 hours). All fatty acids significantly increased CREBh gene expression via transcriptional mechanisms, at concentrations as low as 50 μM. Palmitate- or oleate-mediated upregulation of CREBh was not inhibited by triacsin C, an inhibitor of long-chain fatty acyl CoA synthetase, or by the PPARα antagonist, MK886. Inhibition of proteasome activity with MG132 or lactacystin, or inclusion of insulin reduced palmitate- and oleate-mediated increases in CREBh mRNA. Finally, we examined fatty acid regulation of CREBh in vivo. Male Wistar rats were exposed to a 4-hour pancreatic clamp combined with infusion of saline or a mixed lipid emulsion. CREBh mRNA and protein were significantly increased in rats exposed to the lipid infusion compared to the saline group. Collectively, these results may have important implications for metabolic diseases characterized by excess fatty acids, insulin resistance and inflammation.

Upregulation of transforming growth factor-beta (TGF-beta) signaling is interrelated with the development of airway remodeling. In this study, we examined the role of two E3 ubiquitin ligases, Arkadia and Smurf2, which are critically required for TGF-beta signaling in airway remodeling. Rats were immunized with ovalbumin (OVA) and then challenged with an OVA aerosol. In in vitro experiments, normal human bronchial epithelial cells were stimulated with TGF-beta(1) with or without the preincubation of Arkadia/Smurf2 small interfering RNA (siRNA) or lactacystin (an inhibitor of proteasomal degradation). In the lungs of OVA-treated rats, a large number of inflammatory cells were present near the airways. An increased subepithelial collagen deposition was associated with high expression levels of Smad7, SnoN and Ski mRNAs, Arkadia, Smurf2, and TGF-beta type I receptor (TbetaRI), but low expression levels of Smad7, SnoN and Ski proteins. Smad7, SnoN and Ski interacted with both Arkadia and Smurf2 while TbetaRI only interacted with Smurf2 but not with Arkadia. In in vitro experiments, the inhibitory effect of TGF-beta(1) on the expression of Smad7, SnoN and Ski was reversed by Arkadia siRNA and lactacystin, whereas the stimulatory effect of TGF-beta(1) on the expression of TbetaRI protein and Smad7/SnoN/Ski mRNAs was not affected. In contrast, Smurf2 siRNA did not influence the effects of TGF-beta(1) on the expression of the above proteins. Our results suggest that Arkadia may contribute to the pathogenesis of airway remodeling through enhancing TGF-beta signaling by inducing the reduction of Smad7, SnoN and Ski proteins in OVA-sensitized and -challenged rats.

Cbl-b is a member of Cbl family of E3 ubiquitin (Ub) ligase. Besides the important role in ubiquitination process, other members of Cbl family have been suggested to show non-ubiquitination-related function in regulation of osteoblastic differentiation. However, the role of Cbl-b in regulation of osteoblastic function has not been known yet. To elucidate the role of Cbl-b in regulation of osteoblastic function, we examined its effects on Runx2, a master gene of osteoblastic differentiation. We co-expressed Cbl-b and Runx2 in osteoblastic cell lines and tested their effects on osteocalcin promoter activity together with the expression of Runx2 and its downstream genes. Luciferase assay demonstrated that Cbl-b synergistically enhances osteocalcin promoter activity in conjunction with the effect on Runx2. Co-transfection of Cbl-b and Runx2 further upregulated Runx2 protein levels without any alteration in Runx2 mRNA expression. The upregulation of Runx2 protein by Cbl-b was inhibited by the treatment with lactacystin, a specific inhibitor of the 26S proteasome. These results indicated that Cbl-b would control Runx2 protein levels at the post-translational event. Moreover, the upregulation of downstream genes of Runx2 such as osteocalcin and alkaline phosphatase mRNA was also observed. These data propose the involvement of Cbl-b in the regulation of osteoblast-related genes expression.

We investigated the regulatory pathways responsible for agonist-induced internalization and down-regulation of G(q) protein-coupled histamine H(1)-receptors in Chinese hamster ovary cells. Histamine-induced internalization and down-regulation of H(1)-receptors were detected as the loss of [(3)H]mepyramine binding sites on intact cells accessible to hydrophilic and hydrophobic H(1)-receptor antagonists, pirdonium and mepyramine, respectively. Pretreatment of cells with 0.1 mM histamine for 30 min at 37 degrees C induced internalization as well as down-regulation of H(1)-receptors, both of which were inhibited either in the presence of an inhibitor against G protein-coupled receptor kinases (ZnCl(2)) or under hypertonic conditions where clathrin-dependent endocytosis is known to be inhibited, but were not affected by inhibitors against caveolae/raft-dependent endocytosis (filipin and nystatin). Down-regulation of H(1)-receptors, but not their internalization, was inhibited by protein kinase C inhibitors (chelerythrin or GF109203X), a ubiquitin E1 inhibitor (UBEI-41) and proteasome inhibitors (lactacystin and MG-132). Neither a Ca(2+)/calmodulin-dependent protein kinase II inhibitor (KN-62) nor lysosomal protease inhibitors (E-64, leupeptin, chloroquine and NH(4)Cl) affected the internalization and down-regulation of H(1)-receptors. These results suggest that H(1)-receptors internalize upon agonist stimulation via G protein-coupled receptor kinase/clathrin-dependent but caveolae/raft-independent mechanisms and are delivered to proteasomes, preferentially to lysosomes, for their prompt down-regulation.

Diabetic retinopathy has been considered a low-grade chronic inflammatory disease. The production of interleukin-1beta (IL-1beta) in the retina is increased, and this finding has been correlated with an increase in blood-retinal barrier permeability, suggesting that IL-1beta might have an important role in the pathogenesis of diabetic retinopathy. However, in this context, no attention has been given to interleukin-1 type I receptor (IL-1RI), which is the receptor responsible for IL-1beta triggered effects. Therefore, we investigated the effect of high glucose and IL-1beta on the IL-1RI regulation in retinal endothelial cells. A time-dependent downregulation of IL-1RI protein levels was detected in retinal endothelial cells exposed (1-24h) to high glucose, mannitol or IL-1beta. Long-term exposure (7days) to high glucose or mannitol also decreased IL-1RI protein content. IL-1RI downregulation was due to its activation by IL-1beta, since it was inhibited by the presence of anti-IL-1RI or anti-IL-1beta antibodies. Moreover, IL-1RI downregulation was prevented by lysosome inhibitors, chloroquine and ammonium chloride, but not by proteasome inhibitors, MG132 and lactacystin. We also found that IL-1RI translocates to the nucleus after high glucose or IL-1beta treatment. In conclusion, our results indicate that high glucose, probably due to osmotic stress, and IL-1beta downregulate IL-1RI in retinal endothelial cells. The downregulation of IL-1RI is triggered by its activation and is due, at least partially, to lysosomal degradation. High glucose and IL-1beta also enhance the translocation of IL-1RI to the nucleus.

Loss of function mutations in the hERG (human ether-a-go-go related gene or KCNH2) potassium channel underlie the proarrhythmic cardiac long QT syndrome type 2. Most often this is a consequence of defective trafficking of hERG mutants to the cell surface, with channel retention and degradation at the endoplasmic reticulum. Here, we identify the Hsp40 type 1 chaperones DJA1 (DNAJA1/Hdj2) and DJA2 (DNAJA2) as key modulators of hERG degradation. Overexpression of the DJAs reduces hERG trafficking efficiency, an effect eliminated by the proteasomal inhibitor lactacystin or with DJA mutants lacking their J domains essential for Hsc70/Hsp70 activation. Both DJA1 and DJA2 cause a decrease in the amount of hERG complexed with Hsc70, indicating a preferential degradation of the complex. Similar effects were observed with the E3 ubiquitin ligase CHIP. Both the DJAs and CHIP reduce hERG stability and act differentially on folding intermediates of hERG and the disease-related trafficking mutant G601S. We propose a novel role for the DJA proteins in regulating degradation and suggest that they act at a critical point in secretory pathway quality control.