A subset of 26S proteasomes is activated at critically low ATP concentrations and contributes to myocardial injury during cold ischemia

Molecular mechanisms leading to myocardial injury during warm or cold ischemia are insufficiently understood. Although proteasomes are thought to contribute to myocardial ischemia-reperfusion injury, their roles during the ischemic period remain elusive. Because donor hearts are commonly exposed to prolonged global cold ischemia prior to cardiac transplantation, we evaluated the role and regulation of the proteasome during cold ischemic storage of rat hearts in context of the myocardial ATP content. When measured at the actual tissue ATP concentration, cardiac proteasome peptidase activity increased by 225% as ATP declined during cold ischemic storage of hearts in University of Wisconsin (UW) solution for up to 48 h. Addition of the specific proteasome inhibitor epoxomicin to the UW solution inhibited proteasome activity in the cardiac extracts, significantly reduced edema formation and preserved the ultrastructural integrity of the cardiomyocyte. Utilizing purified 20S/26S proteasome enzyme preparations, we demonstrate that this activation can be attributed to a subset of 26S proteasomes which are stable at ATP concentrations far below physiological levels, that ATP negatively regulates its activity and that maximal activation occurs at ATP concentrations in the low μmol/L range. These data suggest that proteasome activation is a pathophysiologically relevant mechanism of cold ischemic myocardial injury. A subset of 26S proteasomes appears to be a cell-destructive protease that is activated as ATP levels decline. Proteasome inhibition during cold ischemia preserves the ultrastructural integrity of the cardiomyocyte.     

Dynamics of tissue ubiquitin pools and ubiquitin-proteasome pathway component activities during the systemic response to traumatic shock

Based on the biological significance of the ubiquitin-proteasome pathway (UPP) and its potential role during sepsis, burns and ischemia-reperfusion injury, we hypothesized that the systemic response to traumatic shock (TS) is accompanied by tissue-specific UPP alterations. Therefore, we studied tissue ubiquitin pools, chymotryptic- and tryptic-like proteasome peptidase activities and ubiquitin-protein ligation (UbPL) rates in skeletal muscle, heart, lung, liver, spleen and kidney using a clinically relevant porcine model (bilateral femur fracture/hemorrhage followed by fluid resuscitation). TS induced a systemic reduction of tissue-specific high molecular mass ubiquitin-protein conjugates (>50 kDa). Free ubiquitin was unaffected. The dynamic organ patterns of ubiquitin pools paralleled the typical physiological response to TS and resuscitation. Reduction of ubiquitin-protein conjugates was most pronounced in heart and lung (p<0.05 vs. control) and accompanied by significant increases in proteasome peptidase and UbPL activities in these organs. Unlike all other tissues, spleen proteasome peptidase and UbPL activities were significantly reduced 10 h after TS. These findings support the concept that the UPP could play an important role in regulation of cell functions during the early whole-body response to TS. The UPP might be a therapeutic target to improve the metabolic care after TS, particularly in the heart, lung, and spleen.     

High affinity Zn2+ inhibitory site(s) for the trypsin-like peptidase of the 20S proteasome

The effect of Zn²⁺ on three major peptidase activities of the 20S proteasome purified from Xenopus oocytes was kinetically investigated. An extremely low concentration of Zn²⁺ (μM range) strongly inhibited the trypsin-like activity of the 20S proteasome which was fully recoverable by the addition of EDTA. The concentration of Zn²⁺ for half-maximum inhibition (K_0.5) was 0.60 μM which was at least 10 times lower than that of any other divalent cation tested and essentially the same as for proteasomes purified from various other organisms indicating that the inhibition is highly Zn²⁺-specific, reversible, and common to the proteasome regardless of its source. Zn²⁺ at concentrations below 100 μM instantaneously activated the chymotrypsin-like and PGPH activities, and the Zn²⁺ concentration for half-maximum activation was found to be 42-48 μM. These activities were time-dependently inactivated by submillimolar concentrations of Zn²⁺. The inactivation rates were dependent on the concentration of Zn²⁺ and reached a maximum of 1.60-2.40 min⁻¹ for the three peptidase activities under the conditions used. The Zn²⁺ concentration for half-maximum inactivation was found to be 0.70-1.23 mM. This time-dependent inactivation was not reversed by the addition of EDTA or DTT and might not be accompanied by the dissociation of subunits of the 20S proteasome indicating that all activities are inactivated by an identical phenomenon. These results reveal the three types of effects of Zn²⁺ on the 20S proteasome.     

Early release of macrophage migration inhibitory factor after liver ischemia and reperfusion injury in rats

Macrophage migration inhibitory factor (MIF) is an important mediator of ischemia/reperfusion (I/R) injury in heart, brain and intestine. We previously demonstrated that MIF was released during warm/cold ischemia in vitro. However, the role of MIF in liver I/R injury remains unclear. We aimed to test the hypothesis that MIF acts as an early proinflammatory cytokine and could mediate the inflammatory injury in liver I/R. Rats (n = 6 per group) were subjected to 90 min warm ischemia followed by 0.5 h, 6 h and 24 h reperfusion, respectively to liver transplantation (LTx) after 6 h of cold ischemia followed by 24 h of reperfusion. The expression of MIF, its receptor (cluster of differentiation 74 (CD74)) and the downstream inflammatory cytokines (tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β)) were analyzed. Peritoneal macrophages were cultured for 6 h alone or in the presence of effluent from cold-preserved livers or effluent depleted of MIF. Warm I/R increased hepatic MIF-mRNA and protein expression. MIF-protein was released into peripheral circulation in vivo with a maximum at 0.5 h after reperfusion. Induction of MIF-expression was associated with the expression of proinflammatory cytokines and its receptor in both models. MIF released by isolated cold preserved livers, induced TNF-α and IL-1β production by cultured peritoneal macrophages. Intrahepatic upregulation of MIF, release into systemic circulation and the associated upregulation of the proinflammatory mediators suggest a role of MIF in mediating the inflammatory response to I/R injury. Blocking experiments will help to elucidate its role as potential molecular target for preventing hepatic I/R injury.     

Inherent toxicity of organ preservation solutions to cultured hepatocytes

Organ preservation solutions have been designed to protect grafts against the injury inflicted by cold ischemia. However, toxicity of University of Wisconsin (UW) solution during rewarming has been reported. Therefore, we here assessed the toxicity of UW, histidine-tryptophan-ketoglutarate (HTK), Euro-Collins, histidine-lactobionate (HL), sodium-lactobionate-sucrose and Celsior solutions in cultured hepatocytes under hypothermic (4 °C), intermediate (21 °C) and physiological (37 °C) conditions. Marked toxicity of UW, HTK, HL and Euro-Collins solutions was observed at both 37 and 21 °C. With the exception of UW solution, these solutions also increased cell injury during cold incubation (LDH release after 18 h at 4 °C: HTK 76 ± 2%, Euro-Collins 78 ± 17%, HL 81 ± 15%; control: Krebs-Henseleit buffer 20 ± 6%). Testing of individual components using modified Krebs-Henseleit buffers suggested that histidine and phosphate are responsible for (part of) this toxicity. These potential toxicities should be taken into account in the development of future preservation solutions.     

Tyrosine Hydroxylase Is Short-Term Regulated by the Ubiquitin-Proteasome System in PC12 Cells and Hypothalamic and Brainstem Neurons from Spontaneously Hypertensive Rats: Possible Implications in Hypertension

Aberrations in the ubiquitin-proteasome system (UPS) are implicated in the pathogenesis of various diseases. Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamines biosynthesis, is involved in hypertension development. In this study we investigated whether UPS regulated TH turnover in PC12 cells and hypothalamic and brainstem neurons from spontaneously hypertensive rats (SHR) and whether this system was impaired in hypertension. PC12 cells were exposed to proteasome or lysosome inhibitors and TH protein level evaluated by Western blot. Lactacystin, a proteasome inhibitor, induced an increase of 86±15% in TH levels after 30 min of incubation, then it started to decrease up to 6 h to reach control levels and finally it rose up to 35.2±8.5% after 24 h. Bafilomycin, a lysosome inhibitor, did not alter TH protein levels during short times, but it increased TH by 92±22% above basal after 6 h treatment. Before degradation proteasome substrates are labeled by conjugation with ubiquitin. Efficacy of proteasome inhibition on TH turnover was evidenced by accumulation of ubiquitinylated TH after 30 min. Further, the inhibition of proteasome increased the quantity of TH phosphorylated at Ser40, which is essential for TH activity, by 2.7±0.3 fold above basal. TH protein level was upregulated in neurons from hypothalami and brainstem of SHR when the proteasome was inhibited during 30 min, supporting that neuronal TH is also short-term regulated by the proteasome. Since the increased TH levels reported in hypertension may result from proteasome dysfunction, we evaluate proteasme activity. Proteasome activity was significantly reduced by 67±4% in hypothalamic and brainstem neurons from SHR while its protein levels did not change. Present findings show that TH is regulated by the UPS. The impairment in proteasome activity observed in SHR neurons may be one of the causes of the increased TH protein levels reported in hypertension.     

Inactivation of the proteasome by 4-hydroxy-2-nonenal is site specific and dependant on 20S proteasome subtypes

The proteasome represents a major intracellular proteolytic system responsible for the degradation of oxidized and ubiquitinated proteins in both the nucleus and cytoplasm. We have previously reported that proteasome undergoes modification by the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) and exhibits declines in peptidase activities during cardiac ischemia/reperfusion. This study was undertaken to characterize the effects of HNE on the structure and function of the 20S proteasome. To assess potential tissue-specific differences in the response to HNE, we utilized purified 20S proteasome from heart and liver, tissues that express different proteasome subtypes. Following incubation of heart and liver 20S proteasome with HNE, changes in the 2D gel electrophoresis patterns and peptidase activities of the proteasome were evaluated. Proteasome subunits were identified by mass spectrometry prior to and following treatment with HNE. Our results demonstrate that specific subunits of the 20S proteasome are targeted for modification by HNE and that modified proteasome exhibits selective alterations in peptidase activities. The results provide evidence for a likely mechanism of proteasome inactivation in response to oxidative stress particularly during cardiac ischemia/reperfusion.     

The moonlighting of RAD23 in DNA repair and protein degradation

A moonlighting protein is one, which carries out multiple, often wholly unrelated, functions. The RAD23 protein is a fascinating example of this, where the same polypeptide and the embedded domains function independently in both nucleotide excision repair (NER) and protein degradation via the ubiquitin-proteasome system (UPS). Hence, through direct binding to the central NER component XPC, RAD23 stabilizes XPC and contributes to DNA damage recognition. Conversely, RAD23 also interacts directly with the 26S proteasome and ubiquitylated substrates to mediate proteasomal substrate recognition. In this function, RAD23 activates the proteolytic activity of the proteasome and engages specifically in well-characterized degradation pathways through direct interactions with E3 ubiquitin-protein ligases and other UPS components. Here, we summarize the past 40 years of research into the roles of RAD23 in NER and the UPS.     

Bone morphogenetic protein-7 (BMP-7) mediates ischemic preconditioning-induced ischemic tolerance via attenuating apoptosis in rat brain

A mild cerebral ischemic insult, also known as ischemic preconditioning (IPC), confers transient tolerance to a subsequent ischemic challenge in the brain. This study was conducted to investigate whether bone morphogenetic protein-7 (BMP-7) is involved in neuroprotection elicited by IPC in a rat model of ischemia. Ischemic tolerance was induced in rats by IPC (15 min middle cerebral artery occlusion, MCAO) at 48 h before lethal ischemia (2 h MCAO). The present data showed that IPC increased BMP-7 mRNA and protein expression after 24 h reperfusion following ischemia in the brain. In rats of ischemia, IPC-induced reduction of cerebral infarct volume and improvement of neuronal morphology were attenuated when BMP-7 was inhibited either by antagonist noggin or short interfering RNA (siRNA) pre-treatment. Besides, cerebral IPC-induced up-regulation of B-cell lymphoma 2 (Bcl-2) and down-regulation of cleaved caspase-3 at 24 h after ischemia/reperfusion (I/R) injury were reversed via inhibition of BMP-7. These findings indicate that BMP-7 mediates IPC-induced tolerance to cerebral I/R, probably through inhibition of apoptosis.     

Expression proteomics of acute promyelocytic leukaemia cells treated with methotrexate

Methotrexate was first introduced as a cytotoxic agent that inhibits nucleotide biosynthesis in various cancer disorders; its molecular mechanism remains elusive. To understand the molecular mechanism by which methotrexate induces apoptosis, we analyzed the resulting intracellular protein changes in methotrexate-treated acute promyelocytic leukaemia (HL-60) cells by cysteine-labeled differential in-gel electrophoresis (CL-DIGE) combined with mass spectrometry. Initial CL-DIGE analysis revealed that 24 proteins were differentially expressed (p < 0.05) in the HL-60 cell proteome after treatment with 2.5 µM methotrexate for 72 h. We found that three structural α4, α5, α7 proteasome subunits, a non-catalytic β3 and two 26S regulatory proteasome subunits were down-regulated in methotrexate-treated HL-60 cells. Western blot analyses further showed that the inhibition of proteasome subunits is accompanied by suppression of NF-κB subunits and promotes the accumulation of ubiquitinated proteins. Furthermore, methotrexate activated unfolded protein response by inducing the expression of endoplasmic reticulum-resident proteins such as calreticulin, protein disulphide isomerase A3 and A4, and 78 kDa glucose regulated protein in a time-dependent manner. Altogether, our findings demonstrated that targeting NF-κB, structural and regulatory proteasome subunits with methotrexate may provide new insight into understanding methotrexate-induced apoptotic activities in HL-60 cells.     

Cystamine prevents ischemia-reperfusion injury by inhibiting polyamination of RhoA

Transglutaminase2 (TGase2) activates Rho-associated kinase (ROCK), an important mediator of ischemia-reperfusion (IR) injury, through polyamination of RhoA. Cystamine, an oxidized dimer of cysteamine inhibits the transamidation activity of TGase2. We examined whether addition of cystamine to an organ preservation solution protects rat cardiomyocyte cells (H9C2) from cell death in IR injury. H9C2 cells were stored under hypoxic conditions at 4 °C in laboratory-made preservation solution (SNU) or SNU solution supplemented with cystamine (SNU-C1), and cell preservation in the two solutions was compared by measuring the release of lactate dehydrogenase. The cells were preserved more effectively in SNU-C1 than in SNU solution. Cystamine inhibited the intracellular activity of TGase2 which increased during cold storage or reoxygenation. The inhibition of TGase2 by cystamine reduced the polyamination of RhoA, the interaction between RhoA and ROCK2, and F-actin formation. Cystamine also prevented the activation of caspases during cold storage. These results suggest that addition of cystamine to the organ preservation solution significantly enhances cardiomyocytes preservation apparently by inhibiting TGase2-mediated RhoA-ROCK pathway and that TGase2 may play an important role in IR injury by regulating ROCK.     

Misfolded PrP impairs the UPS by interaction with the 20S proteasome and inhibition of substrate entry

Prion diseases are associated with the conversion of cellular prion protein (PrP(C)) to toxic β-sheet isoforms (PrP(Sc)), which are reported to inhibit the ubiquitin-proteasome system (UPS). Accordingly, UPS substrates accumulate in prion-infected mouse brains, suggesting impairment of the 26S proteasome. A direct interaction between its 20S core particle and PrP isoforms was demonstrated by immunoprecipitation. β-PrP aggregates associated with the 20S particle, but did not impede binding of the PA26 complex, suggesting that the aggregates do not bind to its ends. Aggregated β-PrP reduced the 20S proteasome's basal peptidase activity, and the enhanced activity induced by C-terminal peptides from the 19S ATPases or by the 19S regulator itself, including when stimulated by polyubiquitin conjugates. However, the 20S proteasome was not inhibited when the gate in the α-ring was open due to a truncation mutation or by association with PA26/PA28. These PrP aggregates inhibit by stabilising the closed conformation of the substrate entry channel. A similar inhibition of substrate entry into the proteasome may occur in other neurodegenerative diseases where misfolded β-sheet-rich proteins accumulate.     

Proteasome function is required to maintain muscle cellular architecture

BACKGROUND INFORMATION: Protein degradation via the UPS (ubiquitin-proteasome system) plays critical roles in muscle metabolism and signalling pathways. The present study investigates temporal requirements of the UPS in muscle using conditional expression of mutant proteasome beta subunits to cause targeted inhibition of proteasome function. RESULTS AND CONCLUSIONS: The Drosophila GeneSwitch system was used, with analyses of the well-characterized larval somatic body wall muscles. This method acutely disrupts proteasome function and causes rapid accumulation of polyubiquitinated proteins, specifically within the muscle. Within 12 h of transgenic proteasome inhibition, there was a gross disorganization of muscle architecture and prominent muscle atrophy, progressing to the arrest of all co-ordinated movement by 24 h. Progressive muscle architecture changes include rapid loss of sarcomere organization, loss of nuclei spacing/patterning, vacuole formation and the accumulation of nuclear and cytoplasmic aggregates at the ultrastructural level. At the neuromuscular junction, the highly specialized muscle membrane folds of the subsynaptic reticulum were rapidly lost. Within 24 h after transgenic proteasome inhibition, muscles contained numerous autophagosomes and displayed highly elevated expression of the endoplasmic reticulum chaperone GRP78 (glucose-regulated protein of 78 kDa), indicating that the loss of muscle maintenance correlates with induction of the unfolded protein response. Taken together, these results demonstrate that the UPS is acutely required for maintenance of muscle and neuromuscular junction architecture, and provides a Drosophila genetic model to mechanistically evaluate this requirement.     

Myocardial protection by propolis during prolonged hypothermic preservation

Oxidative stress is involved in the pathogenesis of ischemia-reperfusion during myocardial transplantation. Therefore, graft preservation solutions may be improved by supplementation with antioxidants to minimize graft dysfunction caused by cold ischemic injury. Propolis is a polyphenol-rich substance which has an important antioxidant activity. The protective effect of propolis against oxidative stress induced by prolonged cold preservation of heart was investigated. Mice were subjected to a hypothermic model of ischemia in which hearts were preserved for 24 h at 4 °C in Krebs-Hensleit (KH) solution in the absence or presence of propolis concentrations (50, 150 and 250 μg/ml). Levels of released Lactate dehydrogenase (LDH), Creatine phosphokinase (CPK) and Troponine-I (Trop I) were assessed in the preservation solution and histological assessement of heart ischemia injuries was performed. Oxidative stress biomarkers malondialdehyde (MDA) and advanced oxidation protein products (AOPP) and antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) were assessed in cardiac tissue. Mitochondria were isolated from stored hearts and production of reactive oxygen species (ROS) was tested. Propolis supplementation protected efficiently hearts during preservation by reducing significantly levels of lipids and proteins oxidation and restoring activities of antioxidant enzymes. Also, propolis preserved tissue integrity altered by hypothermic ischemia in a concentration-dependent manner. Propolis reduced significantly the rate of H₂O₂ produced by mitochondrial respiration, the best antioxidant effect being obtained at the highest propolis concentration (250 μg/ml). Algerian propolis is a non-temperature sensitive scavenger that protects heart from oxidative damage induced by prolonged hypothermic ischemia.     

Detection and possible role of proteasomes in the bronchoalveolar space of the injured lung

Recent observations suggest the presence of 20S proteasomes (20S) in the lung epithelial lining fluid. However, the physiological relevance of 20S in the alveolar space and possible contribution to disease processes are unknown. Thus, we evaluated whether extracellular proteasomes could have a pathophysiological role in the injured lung using a rat model of lung contusion (LC). Bronchoalveolar lavage fluids (BALF) were obtained at various time points for up to 168 h after LC or sham procedure. Enzyme activities, ELISA and Western blots indicated enzymatically active 20S, the 19S subunit Rpt5 and ubiquitin in BALF. 20S and ubiquitin increased significantly after LC, peaked at 24 h and normalized within 168 h. Mg(2+)/ATP-dependent peptidase activities were detectable 6-24 h after LC. BALF after LC also contained ubiquitin-protein-ligase activity. Addition of Mg(2+)/ATP to BALF after LC led to significant proteolysis and could be prevented with epoxomicin and EDTA. These data suggest for the first time that the Mg(2+)/ATP-dependent 26S proteasome complex exists outside the cell, is released into the lung epithelial lining fluid after LC and contributes to the proteolysis of the bulk of protein in the alveolar space of the injured lung. We infer that proteasome complexes may have a pathophysiological role during lung edema clearance.     

Sulforaphane enhances proteasomal and autophagic activities in mice and is a potential therapeutic reagent for Huntington's disease

The ubiquitin proteasome system (UPS) is impaired in Huntington's disease, a devastating neurodegenerative disorder. Sulforaphane, a naturally occurring compound, has been shown to stimulate UPS activity in cell cultures. To test whether sulforaphane enhances UPS function in vivo, we treated UPS function reporter mice ubiquitously expressing the green fluorescence protein (GFP) fused to a constitutive degradation signal that promotes its rapid degradation in the conditions of a healthy UPS. The modified GFP is termed GFP UPS reporter (GFPu). We found that both GFPu and ubiquitinated protein levels were significantly reduced and the three peptidase activities of the proteasome were increased in the brain and peripheral tissues of the mice. Interestingly, sulforaphane treatment also enhanced autophagy activity in the brain and the liver. To further examine whether sulforaphane promotes mutant huntingtin (mHtt) degradation, we treated Huntington's disease cells with sulforaphane and found that sulforaphane not only enhanced mHtt degradation but also reduced mHtt cytotoxicity. Sulforaphane‐mediated mHtt degradation was mainly through the UPS pathway as the presence of a proteasome inhibitor abolished this effect. Taken together, these data indicate that sulforaphane activates protein degradation machineries in both the brain and peripheral tissues and may be a therapeutic reagent for Huntington's disease and other intractable disorders.