Decreased Proteasome Activity Research Articles

L-Carnitine: An adequate supplement for a multi-targeted anti-wasting therapy in cancer

Tumour growth is associated with weight loss resulting from both adipose and muscle wasting. Administration of L-carnitine (1 g/kg body weight) to rats bearing the AH-130 Yoshida ascites hepatoma, a highly cachectic rat tumour. The treatment results in a significant improvement of food intake and in muscle weight (gastrocnemius, EDL and soleus). These beneficial effects are directly related to improved physical performance (total physical activity, mean movement velocity and total travelled distance). Administration of L-carnitine decreases proteasome activity and the expression of genes related with this activity, such as ubiquitin, C8 proteasome subunit and MuRF-1. Interestingly, L-carnitine treatment also decreases caspase-3 mRNA content therefore suggesting a modulation of apoptosis. Moreover, addition of 50 μM of L-carnitine to isolated EDL muscles results in a significant decrease in the proteolytic rate suggesting a direct effect. It can be concluded that L-carnitine supplementation may be a good approach for a multi-targeted therapy for the treatment of cancer-related cachexia.

Lipopolysaccharide-induced neuroinflammation leads to the accumulation of ubiquitinated proteins and increases susceptibility to neurodegeneration induced by proteasome inhibition in rat hippocampus

BackgroundNeuroinflammation and protein accumulation are characteristic hallmarks of both normal aging and age-related neurodegenerative diseases. However, the relationship between these factors in neurodegenerative processes is poorly understood. We have previously shown that proteasome inhibition produced higher neurodegeneration in aged than in young rats, suggesting that other additional age-related events could be involved in neurodegeneration. We evaluated the role of lipopolysaccharide (LPS)-induced neuroinflammation as a potential synergic risk factor for hippocampal neurodegeneration induced by proteasome inhibition.MethodsYoung male Wistar rats were injected with 1 μL of saline or LPS (5 mg/mL) into the hippocampus to evaluate the effect of LPS-induced neuroinflammation on protein homeostasis. The synergic effect of LPS and proteasome inhibition was analyzed in young rats that first received 1 μL of LPS and 24 h later 1 μL (5 mg/mL) of the proteasome inhibitor lactacystin. Animals were sacrificed at different times post-injection and hippocampi isolated and processed for gene expression analysis by real-time polymerase chain reaction; protein expression analysis by western blots; proteasome activity by fluorescence spectroscopy; immunofluorescence analysis by confocal microscopy; and degeneration assay by Fluoro-Jade B staining.ResultsLPS injection produced the accumulation of ubiquitinated proteins in hippocampal neurons, increased expression of the E2 ubiquitin-conjugating enzyme UB2L6, decreased proteasome activity and increased immunoproteasome content. However, LPS injection was not sufficient to produce neurodegeneration. The combination of neuroinflammation and proteasome inhibition leads to higher neuronal accumulation of ubiquitinated proteins, predominant expression of pro-apoptotic markers and increased neurodegeneration, when compared with LPS or lactacystin (LT) injection alone.ConclusionsOur results identify neuroinflammation as a risk factor that increases susceptibility to neurodegeneration induced by proteasome inhibition. These results highlight the modulation of neuroinflammation as a mechanism for neuronal protection that could be relevant in situations where both factors are present, such as aging and neurodegenerative diseases.

Ethanol and Hepatitis C Virus Suppress Peptide–MHC Class I Presentation in Hepatocytes by Altering Proteasome Function

Previously, we reported that exposure of hepatitis C virus (HCV) core-expressing ethanol (EtOH)-metabolizing cells to EtOH significantly suppresses proteasome activity which exists as 26S (20S and 19S) and as an unassociated 20S particle. The replacement of the constitutive proteasomal subunits with immunoproteasome (IPR) favors antigen processing. Here, we examined the effects of EtOH consumption by HCV core transgenic mice on proteasome activity in hepatocytic lysates and in partially purified 26S proteasome and the impact of these changes on antigen presentation. HCV (-) and HCV (+) core transgenic mice were fed chow diet with or without 20% (v/v) EtOH in water for 4weeks. Following the feeding regimen, hepatocytes were isolated and examined for chymotrypsin-like proteasome activity, oxidative stress, and the presentation of SIINFEKL-H2Kb complex. Additionally, the constitutive proteasome and IPR were purified for further analysis and identification of proteasome-interacting proteins (PIPs). EtOH significantly decreased proteasome activity in hepatocytes of HCV (+) mice, and this finding correlated with oxidative stress and dysregulated methylation reactions. In isolated 26S proteasome, EtOH suppressed proteasome activity equally in HCV (+) and HCV (-) mice. EtOH feeding caused proteasome instability and lowered the content of both constitutive and IPR subunits in the 20S proteasome. In addition, the level of other PIPs, PA28 and UCHL5, were also suppressed after EtOH exposure. Furthermore, in EtOH-fed mice and, especially, in HCV (+) mice, the presentation of SIINFEKL-H2Kb complex in hepatocytes was also decreased. Proteasomal dysfunction induced by EtOH feeding and exacerbated by the presence of HCV structural proteins led to suppression of SIINFEKL-H2Kb presentation in hepatocytes.

Protein quality control disruption by PKCbetaII in heart failure

Some genetic forms of human cardiomyopathy result from accumulation of damaged proteins. However, it is not known whether damaged proteins contribute to other forms of cardiomyopathies and heart failure (HF). We found that failing human hearts from two different etiologies displayed three fold increases in misfolded proteins, an ~50% decrease in proteasomal activity (the machinery that disposes of them) and an ~5 fold increase in levels and ~2.5 fold increased activity in cardiac protein kinase C betaII (PKCbetaII) relative to control hearts. Myocardial infarction‐induced and hypertension‐induced heart failure models in rats also exhibited a 50% reduction in cardiac proteasomal activity, an ~3 fold increase in damaged and misfolded proteins, as well as an ~3 fold increased PKCbetaII activity. PKCbetaII directly phosphorylated the proteasome and inhibited its activity, in vitro. Finally, we showed that sustained and selective PKCbetaII inhibition with betaIIV5‐3 (but not the selective inhibition of PKCbetaI, delta or epsilon) re‐established cardiac protein quality control and reversed HF in both models. Therefore, accumulation of abnormal proteins, disruption of protein quality control and increased activity of PKCbetaII appear to contribute to the pathophysiology of heart failure, suggesting that PKCbetaII inhibition may benefit patients with heart failure.

Amyloid-β Secretion, Generation, and Lysosomal Sequestration in Response to Proteasome Inhibition: Involvement of Autophagy

The proteasome is important for degradation of worn out and misfolded proteins. Decreased proteasome activity has been implicated in Alzheimer's disease (AD). Proteasome inhibition induces autophagy, but it is still unknown whether autophagy is beneficial or deleterious to AD neurons, as the autophagosome has been suggested as a site of amyloid-β (Aβ) generation. In this study, we investigated the effect of proteasome inhibition on Aβ accumulation and secretion, as well as the processing of amyloid-β protein precursor (AβPP) in AβPP(Swe) transfected SH-SY5Y neuroblastoma cells. We show that proteasome inhibition resulted in autophagy-dependent accumulation of Aβ in lysosomes, and increased levels of intracellular and secreted Aβ. The enhanced levels of Aβ could not be explained by increased amounts of AβPP. Instead, reduced degradation of the C-terminal fragment of AβPP (C99) by the proteasome makes C99 available for γ-secretase cleavage, leading to Aβ generation. Inhibition of autophagy after proteasome inhibition led to reduced levels of intracellular, but not secreted Aβ, and tended to further increase the C99 to AβPP ratio, supporting involvement of the autophagosome in Aβ generation. Furthermore, proteasome inhibition caused a reduction in cellular viability, which was reverted by inhibition of autophagy. Dysfunction of the proteasome could cause lysosomal accumulation of Aβ, as well as increased generation and secretion of Aβ, which is partly facilitated by autophagy. As a decrease in cellular viability was also detected, it is possible that upregulation of autophagy is an unsuccessful rescue mechanism, which instead of being protective, contributes to AD pathogenesis.

Photosensitized reactions mediated by the major chromophore arising from glucose decomposition, result in oxidation and cross-linking of lens proteins and activation of the proteasome

Glucose solutions incubated at low oxygen concentration gave rise to the appearance of an absorption band in the UVA-visible region after 10days. Further characterization evidenced that this band was composed by a single chomophore with maximum absorption bands at 335 and 365nm. HPLC/MS and UV spectroscopy assays indicated that this product is composed by five unities of furan. Importantly, the presence of a compound with identical spectral and chromatographic properties was observed in the water-soluble fraction of cataractous human eye lenses. The photo-biological effects of this glucose-derived chromophore (GDC) have been addressed using targets of biological relevance, such as water-soluble proteins from eye lens and the proteasome present in this protein mixture. Increased protein oxidation and protein crosslinking was observed when lens proteins were exposed to UVA-visible light in the presence of GDC under a 5% and 20% oxygen atmosphere. In addition, an increased proteasome peptidase activity was also observed. However, the use of D2O resulted in decreased proteasome activity, suggesting that singlet oxygen promotes the impairment of proteasome activity. Our results suggest that the species generated by Type I and Type II mechanisms have opposite effects on proteasome activity, being Type I a positive activator while Type II lead to impairment of proteasome function.

Association of Plasminogen Activator Inhibitor Type 2 (PAI-2) with Proteasome within Endothelial Cells Activated with Inflammatory Stimuli

Quiescent endothelial cells contain low concentrations of plasminogen activator inhibitor type 2 (PAI-2). However, its synthesis can be rapidly stimulated by a variety of inflammatory mediators. In this study, we provide evidence that PAI-2 interacts with proteasome and affects its activity in endothelial cells. To ensure that the PAI-2·proteasome complex is formed in vivo, both proteins were coimmunoprecipitated from endothelial cells and identified with specific antibodies. The specificity of this interaction was evidenced after (a) transfection of HeLa cells with pCMV-PAI-2 and coimmunoprecipitation of both proteins with anti-PAI-2 antibodies and (b) silencing of the PAI-2 gene using specific small interfering RNA (siRNA). Subsequently, cellular distribution of the PAI-2·proteasome complexes was established by immunogold staining and electron microscopy analyses. As judged by confocal microscopy, both proteins appeared in a diffuse cytosolic pattern, but they also could be found in a dense perinuclear and nuclear location. PAI-2 was not polyubiquitinated, suggesting that it bound to proteasome not as the substrate but rather as its inhibitor. Consistently, increased PAI-2 expression (a) abrogated degradation of degron analyzed after cotransfection of HeLa cells with pCMV-PAI-2 and pd2EGFP-N1, (b) prevented degradation of p53, as evidenced both by confocal microscopy and Western immunoblotting, and (c) inhibited proteasome cleavage of specific fluorogenic substrate. This suggests that PAI-2, in endothelial cells induced with inflammatory stimuli, can inhibit proteasome and thus tilt the balance favoring proapoptotic signaling.

Proteasome activity and autophagosome content in liver are reciprocally regulated by ethanol treatment

The proteasome and autophagy are two major intracellular protein degradation pathways and the regulation of each by ethanol metabolism affects cellular integrity. Using acute and chronic ethanol feeding to mice in vivo, and precision-cut rat liver slices (PCLS) ex vivo, we examined whether ethanol treatment altered these proteolytic pathways. In acute studies, we gave C57Bl/6 mice either ethanol or phosphate-buffered saline (PBS) by gastric intubation and sacrificed them 12h later. PCLS were exposed to 0 or 50mM ethanol for 12 and 24h with or without 4-methylpyrazole (4MP). In chronic studies we pair-fed control and ethanol liquid diets for 4–6weeks to transgenic mice, expressing the green fluorescent protein (GFP) fused to the autophagic marker, microtubule associated protein-1 light chain 3 (LC3). Acute ethanol administration elevated autophagosomes (AVs), as judged by a 1.5-fold increase in LC3II content over PBS-gavaged control mice. Hepatic proteasome activity was unaffected by this treatment. Compared with controls, ethanol exposure for 12 and 24h to PCLS inhibited proteasome activity by 1.5- to 3-fold and simultaneously enhanced AVs by 2- to 5-fold. The decrease in proteasome activity and the rise in AVs both depended on ethanol oxidation as its inhibition by 4-methylpyrazole (4MP) blocked both proteasome inhibition and AV induction. Hepatocytes from mice chronically consuming ethanol exhibited a 1.6-fold decline in proteasome activity, and a 4-fold rise in GFP–LC3 puncta compared with pair-fed control mice. When we exposed hepatocytes from these animals to MG262, a proteasome inhibitor, LC3II puncta per cell further increased 2- to 5-fold over untreated cells. Conclusion: Our findings demonstrate that ethanol metabolism generates oxidants, the levels of which differentially influence the activities of the proteasome and autophagy.

Proteasome assembly defect due to a proteasome subunit beta type 8 (PSMB8) mutation causes the autoinflammatory disorder, Nakajo-Nishimura syndrome

Nakajo-Nishimura syndrome (NNS) is a disorder that segregates in an autosomal recessive fashion. Symptoms include periodic fever, skin rash, partial lipomuscular atrophy, and joint contracture. Here, we report a mutation in the human proteasome subunit beta type 8 gene (PSMB8) that encodes the immunoproteasome subunit β5i in patients with NNS. This G201V mutation disrupts the β-sheet structure, protrudes from the loop that interfaces with the β4 subunit, and is in close proximity to the catalytic threonine residue. The β5i mutant is not efficiently incorporated during immunoproteasome biogenesis, resulting in reduced proteasome activity and accumulation of ubiquitinated and oxidized proteins within cells expressing immunoproteasomes. As a result, the level of interleukin (IL)-6 and IFN-γ inducible protein (IP)-10 in patient sera is markedly increased. Nuclear phosphorylated p38 and the secretion of IL-6 are increased in patient cells both in vitro and in vivo, which may account for the inflammatory response and periodic fever observed in these patients. These results show that a mutation within a proteasome subunit is the direct cause of a human disease and suggest that decreased proteasome activity can cause inflammation.

Cardiac proteasome activity in muscle ring finger-1 null mice at rest and following synthetic glucocorticoid treatment

Muscle ring finger-1 (MuRF1) is a muscle-specific E3 ubiquitin ligase that has been implicated in the regulation of cardiac mass through its control of the ubiquitin proteasome system. While it has been suggested that MuRF1 is required for cardiac atrophy, a resting cardiac phenotype has not been reported in mice with a null deletion [knockout (KO)] of MuRF1. Here, we report that MuRF1 KO mice have significantly larger hearts than age-matched wild-type (WT) littermates at ≥ 6 mo of age and that loss of cardiac mass can occur in the absence of MuRF1. The objective of this study was to determine whether changes in proteasome activity were responsible for the cardiac phenotypes observed in MuRF1 KO mice. Cardiac function, architecture, and proteasome activity were analyzed at rest and following 28 days of dexamethasone (Dex) treatment in 6-mo-old WT and MuRF1 KO mice. Echocardiography demonstrated normal cardiac function in the enlarged hearts in MURF1 KO mice. At rest, heart mass and cardiomyocyte diameter were significantly greater in MuRF1 KO than in WT mice. The increase in cardiac size in MuRF1 KO mice was related to a decrease in proteasome activity and an increase in Akt signaling relative to WT mice. Dex treatment induced a significant loss of cardiac mass in MuRF1 KO, but not WT, mice. Furthermore, Dex treatment resulted in an increase in proteasome activity in KO, but a decrease in WT, mice. In contrast, Akt/mammalian target of rapamycin signaling decreased in MuRF1 KO mice and increased in WT mice in response to Dex treatment. These findings demonstrate that MuRF1 plays an important role in regulating cardiac size through alterations in protein turnover and that MuRF1 is not required to induce cardiac atrophy.

ChemInform Abstract: Regulation of Dopaminergic Neuronal Death by Endogenous Dopamine and Proteasome Activity

AbstractReview: 16 refs.

Aging and calorie restriction modulate yeast redox state, oxidized protein removal, and the ubiquitin–proteasome system

The ubiquitin–proteasome system governs the half-life of most cellular proteins. Calorie restriction (CR) extends the maximum life span of a variety of species and prevents oxidized protein accumulation. We studied the effects of CR on the ubiquitin–proteasome system and protein turnover in aging Saccharomyces cerevisiae. CR increased chronological life span as well as proteasome activity compared to control cells. The levels of protein carbonyls, a marker of protein oxidation, and those of polyubiquitinated proteins were modulated by CR. Controls, but not CR cells, exhibited a significant increase in oxidized proteins. In keeping with decreased proteasome activity, polyubiquitinated proteins were increased in young control cells compared to time-matched CR cells, but were profoundly decreased in aged control cells despite decreased proteasomal activity. This finding is related to a decreased polyubiquitination ability due to the impairment of the ubiquitin-activating enzyme in aged control cells, probably related to a more oxidative microenvironment. CR preserves the ubiquitin–proteasome system activity. Overall, we found that aging and CR modulate many aspects of protein modification and turnover.

Influence of leucine on protein metabolism, phosphokinase expression, and cell proliferation in human duodenum

Although leucine increases protein anabolism through the mammalian target of rapamycin (mTOR) pathway in human muscles, its effects on intestinal mucosal proteins remain unknown. We aimed to assess the effects of leucine on duodenal protein metabolism in healthy humans and to elucidate the signaling pathways involved. Eleven healthy volunteers received for 5 h, on 2 occasions and in random order, an enteral supply of maltodextrins (0.25 g . kg(-1) . h(-1)) or maltodextrins and leucine (0.035 g . kg(-1) . h(-1)) simultaneously with a continuous intravenous infusion of [(2)H(5)]phenylalanine (9 μmol . kg(-1) .h(-1)). Endoscopic duodenal biopsy samples were collected and frozen until analyzed. Phenylalanine enrichment was assessed by gas chromatography-mass spectrometry in duodenal protein and in free intracellular amino acid pools used as precursor to calculate the mucosal fractional synthesis rate (FSR). Proteasome proteolytic activities and phosphokinase expression were assessed by using specific fluorogenic substrates or macroarrays, respectively. Leucine supplementation slightly reduced FSR (mean ± SEM: 81.3 ± 6.3%/d) compared with maltodextrins alone (91.7 ± 8.5%/d; P = 0.0537). In addition, total proteasome activity decreased significantly with leucine (236 ± 21 compared with 400 ± 58 relative fluorescence units/μg protein; P < 0.05), with no modification of chymotrypsin-like, trypsin-like, caspase-like, or peptidase activities. Leucine did not affect the mTOR pathway but did increase the phosphorylation states of PI3K, Akt, AMPK, p38 MAPK, JNK, GSK-3α/β, STAT3, and STAT5 and increased cyclin D1 mRNA concentrations, which suggested that leucine may enhance cell proliferation. Enteral leucine supplementation decreased proteasome activity in duodenal mucosa and enhanced cell proliferation through the PI3K/Akt/GSK-3α/β-catenin pathway. This trial was registered at clinicaltrials.gov as NCT01254110.

Cellular stress response pathways and ageing: intricate molecular relationships.

Ageing is driven by the inexorable and stochastic accumulation of damage in biomolecules vital for proper cellular function. Although this process is fundamentally haphazard and uncontrollable, senescent decline and ageing is broadly influenced by genetic and extrinsic factors. Numerous gene mutations and treatments have been shown to extend the lifespan of diverse organisms ranging from the unicellular Saccharomyces cerevisiae to primates. It is becoming increasingly apparent that most such interventions ultimately interface with cellular stress response mechanisms, suggesting that longevity is intimately related to the ability of the organism to effectively cope with both intrinsic and extrinsic stress. Here, we survey the molecular mechanisms that link ageing to main stress response pathways, and mediate age-related changes in the effectiveness of the response to stress. We also discuss how each pathway contributes to modulate the ageing process. A better understanding of the dynamics and reciprocal interplay between stress responses and ageing is critical for the development of novel therapeutic strategies that exploit endogenous stress combat pathways against age-associated pathologies.

The Involvement of SMILE/TMTC3 in Endoplasmic Reticulum Stress Response

BackgroundThestate of operational tolerance has been detected sporadically in some renal transplanted patients that stopped immunosuppressive drugs, demonstrating that allograft tolerance might exist in humans. Several years ago, a study by Brouard et al. identified a molecular signature of several genes that were significantly differentially expressed in the blood of such patients compared with patients with other clinical situations. The aim of the present study is to analyze the role of one of these molecules over-expressed in the blood of operationally tolerant patients, SMILE or TMTC3, a protein whose function is still unknown.Methodology/Principal FindingsWe first confirmed that SMILE mRNA is differentially expressed in the blood of operationally tolerant patients with drug-free long term graft function compared to stable and rejecting patients. Using a yeast two-hybrid approach and a colocalization study by confocal microscopy we furthermore report an interaction of SMILE with PDIA3, a molecule resident in the endoplasmic reticulum (ER). In accordance with this observation, SMILE silencing in HeLa cells correlated with the modulation of several transcripts involved in proteolysis and a decrease in proteasome activity. Finally, SMILE silencing increased HeLa cell sensitivity to the proteasome inhibitor Bortezomib, a drug that induces ER stress via protein overload, and increased transcript expression of a stress response protein, XBP-1, in HeLa cells and keratinocytes.Conclusion/SignificanceIn this study we showed that SMILE is involved in the endoplasmic reticulum stress response, by modulating proteasome activity and XBP-1 transcript expression. This function of SMILE may influence immune cell behavior in the context of transplantation, and the analysis of endoplasmic reticulum stress in transplantation may reveal new pathways of regulation in long-term graft acceptance thereby increasing our understanding of tolerance.

Abstract 2074: Hsp27 inhibition activates the unfold protein response and autophagy pathway through inhibition of proteasome activity in prostate cancer

Abstract Introduction and objectives: Hsp27 is a stress-activated, multifunctional chaperone that inhibits treatment-induced apoptosis and is highly expressed in castrate-resistant prostate cancer. Hsp27 is induced by accumulation of unfolded protein aggregates following various stress conditions, and promotes folding of proteins to their native state or degrading misfolded proteins via the ubiquitin-proteasome pathway (UPP). The specific signaling that occurs between the endoplasmic reticulum (ER) and the nucleus in response to ER stress is known as the unfolded protein response (UPR). During the UPR, accumulated unfolded protein is either correctly refolded or degraded by the UPP, but when unfolded protein levels exceed a threshold, damaged cells are committed to cell death. Recently, there has been an increased general interest in understanding the interactions of ER stress, proteasome and autophagy in protein clearance. However, it is still unclear whether ER stress-mediated autophagy is involved in cell survival or cell death. We hypothesize that inhibition of Hsp27 will enhance ER stress and treatment-induced cancer cell death by increasing unfolded protein burden. We set out to characterize the role of Hsp27 in ER stress and UPR in prostate cancer. Methods: Effects of Hsp27 silencing using siRNA or antisense inhibitor (OGX-427) or proteasome inhibition (MG132) on PC3 cell apoptosis and putative targets of UPR were compared. Proteasome activity was measured using fluorescent substrate of proteasome in Hsp27 inhibited or overexpressed PC3 cells. We also evaluated autophagy activity after Hsp27 knockdown and cell viability after combination treatment with Hsp27 knockdown +/- inhibition of autophagy by 3-methyladenine (3-MA). Results: Hsp27 expression and other components of the UPR were activated in PC-3 cells after treatment with MG132 with accumulation of ubiquitinated proteins. Hsp27 knockdown was associated with decreased proteasome activity, increased ubiquitinated protein levels, and activation of UPR, similar to that seen with MG132. In contrast, Hsp27 overexpressing PC3 cells exhibited increased proteasome activity and resistance to MG-132 induced cell death and UPR activation compared to control cells. The inhibition of Hsp27 also led to increased autophagy activity. Furthermore, the combination treatment with Hsp27 silencing and 3MA significantly reduced PC3 cell viability and induced apoptosis. Conclusions: These results suggest that Hsp27 enhances the catalytic activity of the proteasome and increases the degradation rate of ubiquitinated proteins. Moreover, the inhibition of both Hsp27 and autophagy enhances prostate cancer cell death and represents a useful therapeutic strategy to target for anti-cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2074. doi:10.1158/1538-7445.AM2011-2074

Proteasomal non‐catalytic subunit PSMD2 as a potential therapeutic target in association with various clinicopathologic features in lung adenocarcinomas

We previously identified PSMD2, a subunit of the 19S regulatory complex of proteasomes, as a constituent of a signature associated with the acquisition of metastatic phenotype and poor prognosis in lung cancers. In the present study, we found that knockdown of PSMD2 decreased proteasome activity, and induced growth inhibition and apoptosis in lung cancer cell lines. These effects of siRNA-mediated PSMD2 inhibition were associated with changes in the balance between phosphorylated AKT and p38, as well as with induction of p21. In addition, patients with higher PSMD2 expression had poorer prognosis and a small fraction of lung cancer specimens carried increased copies of PSMD2. Notably, our findings clearly illustrate that lung adenocarcinomas can be divided into two groups; those with and without general upregulation of proteasome pathway genes including PSMD2. This general upregulation was significantly more prevalent in the non-terminal respiratory unit (non-TRU)-type, a recently proposed genetically and clinicopathologically relevant expression profile-defined classification of adenocarcinomas (P < 0.001 by Fisher's exact test). Patients with adenocarcinomas with general upregulation had significantly shorter survival after potentially curative resection (P = 0.0001 by log-rank test) independent of disease stage, as shown by multivariate Cox regression analysis. Our results suggest that PSMD2 may be a good molecular target candidate and that other co-regulated proteasome pathway genes and/or their common regulator(s) might also be potential targets, warranting future study including elucidation of the underlying common regulatory mechanism.

Dual regulation of hepatocyte apoptosis by reactive oxygen species: Increases in transcriptional expression and decreases in proteasomal degradation of BimEL

Reactive oxygen species (ROS) have a fundamental role in intracellular signaling transduction. We show here that time-dependent extracellular signal-regulated kinase (ERK) activation due to inactivation of protein tyrosine phosphatases was closely linked to hepatocyte apoptosis under sustained exposure to ROS, which is produced through inhibition of ROS-scavenging enzymes. We found, for the first time, that active ERK transcriptionally increased BimEL expression among seven proteins of the Bcl-2 family. Transfection of Bim siRNA inhibited BimEL expression and hepatocyte apoptosis. Although ERK activation also elicited BimEL phosphorylation and subsequent ubiquitination, exposure to ROS for 9 h decreased proteasome activity. Collectively, the amount of BimEL was elevated by its increased expression and decreased degradation, leading to apoptosis. Exposure to ROS for 6 h caused neither reduction of proteasome activity nor hepatocyte apoptosis. These results indicate that the duration of exposure to ROS determines the fate of cells, that is, survival or death, in addition to the species, amounts, and generation sites of ROS.

The case for therapeutic proteostasis modulators

A functional ubiquitin proteasome pathway (UPP) is vital for all eukaryotic cellular systems and therefore any alteration in this critical component of proteostasis machinery has rpotential pathological consequences. A proteostasis imbalance can be induced by environmental pollutants, age or genetic factors. Though the exact underlying mechanisms are unclear, a decrease in proteasome activity weakens the homeostatic cellular capacity to remove proteins that are either misfolded or need to be replenished, which favors the development of neurodegenerative, cardiac and other conformational diseases. In contrast, induction of proteasome activity is an attribute of many diseases including muscle wasting, sepsis, cachexia and uraemia. In the case of misfolded protein disorders, higher degradation of a single protein leads to the pathophysiological consequences due to the absence of functional protein. Therefore, selective proteostasis inhibition is a potential treatment strategy for misfolded protein disorders, while broad-spectrum proteasome inhibitor drugs are designed to target tumor metastasis. In contrast, for muscle wasting and neurodegeneration, the use of proteostasis-activating or modulating compounds could be more effective.

Regulation of Dopaminergic Neuronal Death by Endogenous Dopamine and Proteasome Activity

Parkinson disease is one of the most common neurodegenerative disorders and is characterized by the selective loss of dopaminergic neurons in the substantia nigra. Although endogenous dopamine itself could serve as a vulnerability factor for dopaminergic neurons, the mechanism by which dopamine contributes to dopaminergic neuronal death remains unknown. In addition, although a decrease in proteasome activity was found in patients with sporadic Parkinson disease, the relationship between the ubiquitin-proteasome system and dopaminergic neuronal death remains to be elucidated. Here we provide an overview of the roles of endogenous dopamine and proteasome activity in dopaminergic cell death. Treatment of catecholaminergic PC12 cells with the herbicide paraquat, a potential risk factor for the development of Parkinson disease, induced an increase in dopamine content, and depletion of intracellular dopamine suppressed paraquat-induced cytotoxicity. Although glutathione, which scavenged dopamine oxidation intermediate, provided almost complete protection against dopamine-mediated toxicity, catalase provided only partial protection against cell death caused by dopamine. These data suggest that the generation of dopamine oxidation intermediate, rather than that of reactive oxygen species, plays a pivotal role in dopamine-induced toxicity. Moreover, treatment with paraquat induced a decrease in proteasome activity, and proteasome inhibition suppressed dopamine-mediated cytotoxicity. Suppression of proteasome activity stimulated the NF-E2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway, and elevated γ-glutamylcysteine synthetase mRNA and glutathione content. Furthermore, suppression of the paraquat-induced increase in gluthathione content exacerbated paraquat toxicity. These results suggest that the reduction of proteasome activity may be involved in cellular defense mechanisms against dopamine-mediated paraquat toxicity.