Proteasome Peptidase Activities Research Articles

Rapamycin Allosterically Inhibits the Proteasome

Rapamycin is a canonical allosteric inhibitor of the mammalian tarpet of rapamycin (mTOR) kinase with immunosuppressive and proapoptotic activities. We found that in vitro rapamycin also regulates the proteasome, which is an essential intracellular protease of the ubiquitin-proteasome pathway. Rapamycin inhibits proteinase and selected peptidase activities of the catalytic core proteasome at low micromolar concentrations. Moreover, the drug interferes with binding of the 19S cap essential for processing of polyubiquitinylated substrates and with the PA200 proteasome activator to the 20S catalytic core proteasome. These protein complexes are known to bind to specific grooves on the α face region of the 20S core. Treatment with rapamycin affects the conformational dynamics of the proteasomal gate, which is centrally positioned within the α face and allosterically regulated element responsible for the intake of substrates. We showed that rapamycin shares all the proteasome targeting properties not only with other two-domain, closed-ring analogs (rapalogs) but also with its single domain mimics and seco-rapamycin, which is the first in vivo open-ring metabolite of rapamycin that does not affect mTOR. We hypothesize that rapamycin and related compounds bind to the α face and allosterically impact proteasome function. This article discusses the implications of our findings for the mechanism of in vivo actions of rapamycin and for the design of novel allosteric drugs targeting the proteasome.

Regulatory Role of Proteasome in Determination of Platelet Life Span

Limit of platelet life span (8-10 days) is determined by the activity of a putative "internal clock" composed of Bcl-2 family proteins, whereas the role of other molecular players in this process remains obscure. Here, we sought to establish a central role of proteasome in platelet life span regulation. Administration of mice with inhibitors of proteasome peptidase activity induced significant thrombocytopenia. This was associated with enhanced clearance of biotin-labeled platelets from circulation and reduction in average platelet half-life from 66 to 37 h. Cells pretreated in vitro with proteasome inhibitors exhibited augmented annexin V binding and a drop in mitochondrial transmembrane potential indicative of apoptotic cell death and decreased platelet life span. These cells were preferentially phagocytosed by monocyte-derived macrophages, thus linking proteasome activity with platelet survival. The decisive role of proteasome in this process was underscored from enhanced expression of conformationally active Bax in platelets with attenuated proteasome activity, which was consistent with pro-apoptotic phenotype of these cells. The present study establishes a critical role of proteasome in delimiting platelet life span ostensibly through constitutive elimination of the conformationally active Bax. These findings bear potential implications in clinical settings where proteasome peptidase activities are therapeutically targeted.

High glucose and diabetes modulate cellular proteasome function: Implications in the pathogenesis of diabetes complications

The precise link between hyperglycemia and its deleterious effects on retinal and kidney microvasculature, and more specifically loss of retinal perivascular supporting cells including smooth muscle cell/pericytes (SMC/PC), in diabetes are not completely understood. We hypothesized that differential cellular proteasome activity contributes to sensitivity of PC to high glucose-mediated oxidative stress and vascular rarefaction. Here we show that retinal endothelial cells (EC) have significantly higher proteasome peptidase activity compared to PC. High glucose treatment (HGT) increased the level of total ubiquitin-conjugated proteins in cultured retinal PC and EC, but not photoreceptor cells. In addition, in vitro proteasome activity assays showed significant impairment of proteasome chymotrypsin-like peptidase activity in PC, but not EC. The PA28-α/-β and PA28-β/-γ protein levels were also higher in the retina and kidney glomeruli of diabetic mice, respectively. Our results demonstrate, for the first time, that high glucose has direct biological effects on cellular proteasome function, and this modulation might be protective against cellular stress or damage induced by high glucose.

Proteasome inhibition prolongs survival during lethal hemorrhagic shock in rats

Several lines of evidence suggest that proteasomes, the major nonlysosomal proteases in eukaryotes, are involved in the pathophysiology of various disease processes, including ischemia-reperfusion injury and trauma. Recently, we demonstrated that 26S proteasome activity is negatively regulated by adenosine triphosphate (ATP) and that proteasome activation during ischemia contributes to myocardial injury. The regulation of tissue proteasome activity by ATP and the potential of proteasomes as drug targets during hemorrhagic shock, however, are unknown. Thus, we evaluated the regulation of tissue proteasome peptidase activity and the effects of the proteasome inhibitor bortezomib in rat models of hemorrhagic shock. Series 1 includes animals (n = 20) hemorrhaged to a mean arterial blood pressure of 30 mm Hg for up to 45 minutes. Series 2 includes animals hemorrhaged to a mean arterial blood pressure of 30 mm Hg for 30 minutes, followed by bortezomib (0.4 mg/kg) or vehicle administration (n =5 per group) and fluid resuscitation until 75 minutes. Series 3 includes animals that underwent 40% blood volume hemorrhage, followed by 2% blood volume hemorrhage every 15 minutes until death. Bortezomib (0.4 mg/kg) or vehicle were administered 15 minutes after the onset of hemorrhage (n = 6-7 per group). Vital signs were continuously monitored. The heart, lung, and pectoral muscle were analyzed for proteasome peptidase activities and levels of ATP, ubiquitin-protein conjugates, and cytokines (tumor necrosis factor α, interleukin 6, and interleukin 10). In Series 1, proteasome peptidase activities in tissue extracts increased proportional to the decrease in tissue ATP concentrations during hemorrhagic shock. Activation of proteasome peptidase activity with decreases of the ATP assay concentration was also detectable in normal tissue extracts. In Series 2, systemic administration of bortezomib inhibited tissue proteasome activities but did not affect the physiologic response. In Series 3, bortezomib inhibited tissue proteasome activities, increased endogenous ubiquitin-protein conjugates, and prolonged survival time from treatment from 48.5 minutes in the control group to 85 minutes (p = 0.0012). Bortezomib treatment did not affect tissue cytokine levels. Proteasome activation contributes to the pathophysiology of severe hemorrhagic shock. Pharmacologic inhibition of the proteasome may provide a survival advantage during lethal hemorrhagic shock.

Exercise Training Restores Cardiac Protein Quality Control in Heart Failure

Exercise training is a well-known coadjuvant in heart failure treatment; however, the molecular mechanisms underlying its beneficial effects remain elusive. Despite the primary cause, heart failure is often preceded by two distinct phenomena: mitochondria dysfunction and cytosolic protein quality control disruption. The objective of the study was to determine the contribution of exercise training in regulating cardiac mitochondria metabolism and cytosolic protein quality control in a post-myocardial infarction-induced heart failure (MI-HF) animal model. Our data demonstrated that isolated cardiac mitochondria from MI-HF rats displayed decreased oxygen consumption, reduced maximum calcium uptake and elevated H2O2 release. These changes were accompanied by exacerbated cardiac oxidative stress and proteasomal insufficiency. Declined proteasomal activity contributes to cardiac protein quality control disruption in our MI-HF model. Using cultured neonatal cardiomyocytes, we showed that either antimycin A or H2O2 resulted in inactivation of proteasomal peptidase activity, accumulation of oxidized proteins and cell death, recapitulating our in vivo model. Of interest, eight weeks of exercise training improved cardiac function, peak oxygen uptake and exercise tolerance in MI-HF rats. Moreover, exercise training restored mitochondrial oxygen consumption, increased Ca2+-induced permeability transition and reduced H2O2 release in MI-HF rats. These changes were followed by reduced oxidative stress and better cardiac protein quality control. Taken together, our findings uncover the potential contribution of mitochondrial dysfunction and cytosolic protein quality control disruption to heart failure and highlight the positive effects of exercise training in re-establishing cardiac mitochondrial physiology and protein quality control, reinforcing the importance of this intervention as a non-pharmacological tool for heart failure therapy.

HDAC Inhibitor L-Carnitine and Proteasome Inhibitor Bortezomib Synergistically Exert Anti-Tumor Activity In Vitro and In Vivo

Combinations of proteasome inhibitors and histone deacetylases (HDAC) inhibitors appear to be the most potent to produce synergistic cytotoxicity in preclinical trials. We have recently confirmed that L-carnitine (LC) is an endogenous HDAC inhibitor. In the current study, the anti-tumor effect of LC plus proteasome inhibitor bortezomib (velcade, Vel) was investigated both in cultured hepatoma cancer cells and in Balb/c mice bearing HepG2 tumor. Cell death and cell viability were assayed by flow cytometry and MTS, respectively. Gene, mRNA expression and protein levels were detected by gene microarray, quantitative real-time PCR and Western blot, respectively. The effect of Vel on the acetylation of histone H3 associated with the p21cip1 gene promoter was examined by using ChIP assay and proteasome peptidase activity was detected by cell-based chymotrypsin-like (CT-like) activity assay. Here we report that (i) the combination of LC and Vel synergistically induces cytotoxicity in vitro; (ii) the combination also synergistically inhibits tumor growth in vivo; (iii) two major pathways are involved in the synergistical effects of the combinational treatment: increased p21cip1 expression and histone acetylation in vitro and in vivo and enhanced Vel-induced proteasome inhibition by LC. The synergistic effect of LC and Vel in cancer therapy should have great potential in the future clinical trials.

Effect of HIV infection on the expression and the activity of the proteasome in primary CD4 T cells

Background HIV-specific CD8 T cells responses rely on the recognition of peptide-MHC-I complexes by cognate T cell receptors. HIV-derived MHC-I epitopes result from the degradation of viral proteins by the cellular processing machinery including proteasomes and aminopeptidases. Interferon gamma changes proteasome composition and peptidase activities. We hypothesize that HIV infection might affect the expression or activities of the antigen processing machinery, either through a direct effect of the virus or indirectly through cellular activation or from the release of cytokines by surrounding infected cells.

Reperfusion Rather than Ischemia Drives the Formation of Ubiquitin Aggregates After Middle Cerebral Artery Occlusion

Cerebral ischemia leads to accumulation of ubiquitinated protein aggregates. However, the factors triggering ubiquitination and their impact on the outcome of cerebral ischemia remain poorly understood. Here we investigate the relationship between ubiquitin aggregation and duration of ischemia/reperfusion, infarct volume, and proteasomal activity in a mouse model of focal ischemia. Free ubiquitin and ubiquitin aggregate levels were examined by Western blotting in the mouse neocortex and striatum after different periods of ischemia/reperfusion and permanent ischemia induced by middle cerebral artery occlusion. Infarct volumes were measured in thionin-stained brain sections. Proteasome activity was studied by fluorometric peptidase activity assay. Following transient ischemia, ubiquitin aggregates were detected in the ipsilateral neocortex and, to a lesser extent, striatum only after induction of reperfusion. In permanent ischemia, no ubiquitin aggregates were found. Shorter ischemic periods producing no or minimal tissue damage (10-15 minutes) resulted in ubiquitin aggregate levels similar to those produced by ischemia resulting in substantial infarction (30 minutes). Proteasomal impairment was greatest in ischemia without reperfusion, in which no ubiquitin aggregates were detected. The data demonstrate that reperfusion rather than ischemia leads to the appearance of ubiquitinated aggregates, which form in the absence of major tissue damage and are not correlated with decreased proteasomal peptidase activity. Ubiquitin aggregates may form in potentially viable brain tissue, which may be later recruited into infarction by factors independent of ubiquitination.

Cationic Porphyrins Are Reversible Proteasome Inhibitors

The aim of this study is to verify if water-soluble porphyrins can be used as proteasome inhibitors. We have found that cationic porphyrins inhibit proteasome peptidase activities much more effectively than the corresponding anionic derivatives. The relevance of electrostatics in driving porphyin-proteasome interactions has been confirmed by the observation that the inhibitory efficiency of the cationic macrocycles decreases with the number of positive substituents. We have also investigated various metalloporphyrins, which differ due to the different propension of the central metal ion toward axial coordination. Our experimental results indicate that the naked cationic porphyrins are the most active in reversibly inhibiting the three main protease activities of the proteasome in the micromolar range. A spectroscopic characterization of porphyrin-proteasome interactions by UV-vis spectra parallels the results of inhibition assays: the higher the inhibitory effect the stronger the spectroscopic variations are. To interpret the action of porphyrins at a molecular level, we have performed calculations evidencing that cationic porphyrins may hinder the access to the canonical proteolytic site on the proteasome β5 subunit. In particular, an inspection of the top-scoring docking modes shows that the tetracationic porphyrin blocks the catalytic pocket, close to the N termini of the β5 proteasome subunit, more efficiently than its anionic counterpart. Proteasome inhibition activity of porphyrins unites their known anticancer properties making them suitable as a scaffold for the design of novel multitargeted molecules.

Changes in 20S subunit composition are largely responsible for altered proteasomal activities in experimental autoimmune encephalomyelitis

We recently reported that the proteasomal peptidase activities are altered in the cerebellum of mice with myelin oligodendrocyte glycoprotein (MOG) peptide-induced experimental autoimmune encephalomyelitis (EAE). To determine whether these fluctuations are caused by proteasome activation/inactivation and/or changes in the levels of individual β subunits, we characterized the proteasome subunit composition by western blotting. The results show that the rise in proteasomal peptidase activity in acute EAE correlates with an augmented expression of inducible β subunits whereas the decline in activity in chronic EAE correlates with a reduction in the amount of standard β subunits. Using pure standard (s) and immuno (i) 20S particles for calibration, we determined that the changes in the levels of catalytic subunits account for all of the fluctuations in peptidase activities in EAE. The i-20S and s-20S proteasome were found to degrade carbonylated β-actin with similar efficiency, suggesting that the amount of protein carbonyls in EAE may be controlled by the activity of both core particles. We also found an increase in proteasome activator 11S regulatory particle and a decrease in inhibitor proteasome inhibitor with molecular mass of 31 kDa levels in acute EAE, reflecting a response to inflammation. Elevated levels of 19S regulatory particle and 11S regulatory particle in chronic EAE, however, may occur in response to diminished proteasomal activity in this phase. These findings are central towards understanding the altered proteasomal physiology in inflammatory demyelinating disorders.

Bowman-Birk inhibitors, proteasome peptidase activities and colorectal pre neoplasias induced by 1,2-dimethylhydrazine in Swiss mice

Bowman-Birk inhibitors (BBIs) are protein molecules containing two inhibitory domains for enzymes similar to trypsin and chymotrypsin. Interest in these inhibitors arose from their properties against the cancer chemically induced by 1,2-dimethylhydrazine (DMH). In this study the effect of two BBI preparations (from Glycine max and Macrotyloma axillare) were evaluated for the prevention of colorectal neoplasia induced by intraperitoneal injections of DMH, given at a dose of 30mg/kg, during 12weeks. Mice treated with DMH presented histopathological alterations consistent with tumor development, augmented CD44 expression and increased proteasome peptidase activities. Lysosomal fractions, obtained from the intestines, were chromatographed in a Sepharose-BBI column and increased activity for trypsin and chymotrypsin-like proteases recovered from DMH-treated animals. In parallel, mice treated for eight weeks with BBIs showed a decrease in the chymotrypsin and trypsin-like proteasome activities compared to animals fed on normal diet. For the groups receiving simultaneous treatment with DMH and BBIs, dysplasic lesions were not observed and proteasome peptidase activities were similar to the control group after the 24th week. These results suggest that the mechanism by which BBIs could prevent the appearance of pre neoplastic lesions is associated with inhibition of both the lysosomal and proteasome-dependent proteolytic pathways.

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.

Chronic cerebral hypoperfusion in rats causes proteasome dysfunction and aggregation of ubiquitinated proteins

The deposition of abnormal protein aggregates is a feature of several neurodegenerative diseases. We have employed a rat model to investigate whether chronic cerebral hypoperfusion (CCH) induces proteasome dysfunction and the accumulation of ubiquitinated proteins and aggregates in the CNS. Protein aggregation was analyzed by ethanolic phosphotungstic acid (EPTA) electron microscopy (EM), immunogold EM, laser-scanning confocal microscopy, and Western blotting. Proteasome peptidase activity was studied by peptidase activity assays. EPTA EM and immunogold EM revealed that CCH led to the accumulation of protein aggregates in rat hippocampal CA1 neurons. High-resolution confocal microscopy demonstrated the presence of ubiquitin-positive protein aggregates surrounding nuclei and along dendrites. Western blotting revealed that levels of free ubiquitin were significantly reduced and that levels of ubiquitinated proteins were markedly increased in the hippocampus of CCH rats. Direct activity measurements revealed that proteasome peptidase activity in the hippocampal region of rats was decreased after CCH induction. These data suggest that reduced proteasome activity following CCH could impair the removal of abnormally folded proteins via the ubiquitin–proteasome pathway, leading to the accumulation of potentially toxic protein aggregates that could contribute to neurodegeneration.

Effect of ubiquitination on peptidase activities of proteasomes in genotoxic stress

Effect of ubiquitination on peptidase activities of proteasomes in genotoxic stress

Prolongation of myocardial viability by proteasome inhibition during hypothermic organ preservation

Recently, we provided evidence for a possible role of the cardiac proteasome during ischemia, suggesting that a subset of 26S proteasomes is a cell-destructive protease, which is activated as the cellular energy supply declines. Although proteasome inhibition during cold ischemia (CI) reduced injury of ischemic hearts, it remains unknown whether these beneficial effects are maintained throughout reperfusion, and thus, may have pathophysiological relevance. Therefore, we evaluated the effects of epoxomicin (specific proteasome inhibitor) in a rat heterotopic heart transplantation model. Donor hearts were arrested with University of Wisconsin solution (UW) and stored for 12 h/24 h in 4 °C UW ± epoxomicin, followed by transplantation. Efficacy of epoxomicin was confirmed by proteasome peptidase activity measurements and analyses of myocardial ubiquitin pools. After 12hCI, troponin I content of UW was lower with epoxomicin. Although all hearts after 12hCI started beating spontaneously, addition of epoxomicin to UW during CI reduced cardiac edema and preserved the ultrastructural integrity of the post-ischemic cardiomyocyte. After 24hCI in UW ± epoxomicin, hearts did not regain contractility. When hearts were perfused with epoxomicin during cardioplegia, the cardiac proteasome was inhibited immediately, all of these hearts started beating after 24hCI in UW plus epoxomicin and cardiac edema and myocardial ultrastructure were comparable to hearts after 12hCI. Epoxomicin did not affect markers of lipid peroxidation or neutrophil infiltration in post-ischemic hearts. These data further support the concept that proteasome activation during ischemia is of pathophysiological relevance and suggest proteasome inhibition as a promising approach to improve organ preservation strategies.

Physiological levels of ATP negatively regulate proteasome function

Intracellular protein degradation by the ubiquitin-proteasome system is ATP dependent, and the optimal ATP concentration to activate proteasome function in vitro is ∼100 μM. Intracellular ATP levels are generally in the low millimolar range, but ATP at a level within this range was shown to inhibit proteasome peptidase activities in vitro. Here, we report new evidence that supports a hypothesis that intracellular ATP at the physiological levels bidirectionally regulates 26S proteasome proteolytic function in the cell. First, we confirmed that ATP exerted bidirectional regulation on the 26S proteasome in vitro, with the optimal ATP concentration (between 50 and 100 μM) stimulating proteasome chymotrypsin-like activities. Second, we found that manipulating intracellular ATP levels also led to bidirectional changes in the levels of proteasome-specific protein substrates in cultured cells. Finally, measures to increase intracellular ATP enhanced, while decreasing intracellular ATP attenuated the ability of proteasome inhibition to induce cell death. These data strongly suggest that endogenous ATP within the physiological concentration range can exert a negative impact on proteasome activities, allowing the cell to rapidly upregulate proteasome activity on ATP reduction under stress conditions.

Crystal Structure of Yeast Rpn14, a Chaperone of the 19 S Regulatory Particle of the Proteasome

The ubiquitin-proteasome pathway is a major proteolytic system in eukaryotic cells and regulates various cellular processes. The 26 S proteasome, the central enzyme of this pathway, consists of a proteolytic core particle and two 19 S regulatory particles (RPs) composed of ATPase (Rpt) and non-ATPase (Rpn) subunits. Growing evidence indicates that proteasome assembly is assisted by a variety of chaperones. In particular, it has been reported recently that Nas2, Nas6, Rpn14, and Hsm3 bind specific Rpt subunits, thereby contributing to the formation of 19 S RP. Rpn14 transiently binds to the C-terminal domain of the Rpt6 subunit (Rpt6-C) during maturation of the ATPase ring of 19 S RP. In this study, we determined the crystal structure of yeast Rpn14 at 2.0 A resolution, which revealed that this chaperone consists of a unique N-terminal domain with unknown function and a C-terminal domain assuming a canonical seven-bladed beta-propeller fold. The Rpt6-binding site on Rpn14 was predicted based on structural comparison with the complex formed between Nas6 and Rpt3-C. The top face of Rpn14 exhibits a highly acidic surface area, whereas the putative interacting surface of Rpt6-C is basic. By inspection of structural data along with genetic and biochemical data, we determined the specific residues of Rpn14 and Rpt6 for complementary charge interactions that are required for 19 S RP assembly.

Circulating Proteasomes After Burn Injury

The objective of the study is to test whether circulating proteasomes are increased in burn patients and to assess whether possible alterations are associated with severity of injury, organ failure, and/or clinically relevant outcomes. In this study, plasma was obtained from burn patients on days 0 (admission, n = 50), 1 (n = 36), 3 (n = 35), 5 (n = 28), 7 (n=34), and 30 (n = 10) (controls: 40 volunteers). The 20S/26S proteasome levels were measured by enzyme-linked immunosorbent assay. Proteasome peptidase activity was assessed using a chymotryptic-like peptide substrate in combination with epoxomicin (specific proteasome inhibitor). Percentage of TBSA burned, presence of inhalation injury, development of sepsis/multiple organ failure, and sequential organ failure assessment scores were documented. On admission, plasma proteasome activity was higher in patients than in controls (P = .011). 26S proteasomes were not detectable. The 20S proteasome concentrations (median [25th/75th percentile]) peaked on day 0 (673 [399/1566] ng/mL; control: 195 [149/249] ng/mL, P < .001), gradually declined within 7 days, and fully returned to baseline at day 30 (116.5 [78/196] ng/mL). Elevated 20S proteasomes were associated with the presence of inhalation injury and correlated linearly with %TBSA in patients without inhalation injury. Initial 20S proteasome concentrations discriminated the presence of inhalation injury in patients with (sensitivity 0.88 and specificity 0.71) and without (sensitivity 0.83 and specificity 0.97) cutaneous burns but did not discriminate sepsis/multiple organ failure development or survival. Circulating 20S proteasome is a biomarker of tissue damage. The 20S proteasome plasma concentrations in patients with burns and/or inhalation injury are unlikely to predict outcomes but may be useful for the diagnosis of inhalation injury.

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.

Inhibitory Effect of Flavonoids on 26S Proteasome Activity

Inhibiting proteasomal degradation has been shown to induce apoptosis in tumor cells. Utilization of proteasome inhibition is therefore one approach to anticancer therapy. Some of the flavonoids can induce cell apoptosis via inhibiting proteasome 26S activity. In this study, the inhibition of 26S proteasome from pig red blood cells was analyzed on 12 flavones, 5 flavanones, and 9 isoflavones by using a proteolysis assay. Several flavonoids such as apigenin-6-hydroxy-7-O-beta-D-glucoside, quercetin, rutin, 6-hydroxyapigenin, 5,6,4'-trihydroxy-7,3'-dimethoxyflavone, 5,6,3',4'-tetrahydroxy-7-methoxyflavone, glycitecin, and 6,7,4'-trihydroxyisoflavone inhibited the chymotrypsin-like, caspase-like, or trypsin-like activity of 26S proteasome when Suc-LLVY-AMC, Z-LLE-AMC, and Ac-RLR-AMC were used as substrates. Three peptidase activities of flavonoids were found to be significantly correlated with one another. Flavones had significantly stronger inhibitory effects on chymotrypsin-like and caspase-like activities than flavanones and isoflavones. 5,6,3',4'-Tetrahydroxy-7-methoxyflavone, 5,6,4'-trihydroxy-7,3'-dimethoxyflavone, and quercetin displayed a mixed type inhibition of 26S by Lineweaver-Burk plots analysis. Furthermore, 5,6,3',4'-tetrahydroxy-7-methoxyflavone is found to have a higher inhibitory effect on 26S proteasome activities and is the only flavonoid to inhibit all three peptidase activities, whereas the inhibition of flavonoids was not affected by ubiquitin-induced stimulation of the three peptidase activities of 26S proteasome; 5,6,3',4'-tetrahydroxy-7-methoxyflavone inhibited 75% casein degradation. These results suggest that both the 6-hydroxy and 7-methoxy positions of the flavone may play an important role in targeting 26S activity.