Published online before print April 29, 2004, doi: 10.1161/01.STR.0000126891.93919.4e
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(Stroke. 2004;35:1506.)
© 2004 American Heart Association, Inc.
Progress Review |
Ubiquitin-Proteasome System and Proteasome Inhibition: New Strategies in Stroke Therapy
From the Department of Physiology (C.W.), University of Texas Southwestern Medical Center, Dallas, TX; and Neurological Section (M.D.N.), SMDN–Center for Cardiovascular Medicine and Cerebrovascular Disease Prevention, Sulmona (L’Aquila), Italy.
Correspondence to Dr Mario Di Napoli, Neurological Section SMDN–Center for Cardiovascular Medicine and Cerebrovascular Disease Prevention, Via Trento, 41 67039, Sulmona (AQ), Italy. E-mail mariodinapoli{at}katamail.com
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Abstract |
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Top
Abstract
IntroductionBackground and Purpose— Proteasomes are large multicatalytic proteinase complexes that are found in the cytosol and in the nucleus of eukaryotic cells with a central role in cellular protein turnover. The ubiquitin-proteasome system (UPS) has a central role in the selective degradation of intracellular proteins. Among the key proteins whose levels are modulated by the proteasome are those involved in the control of inflammatory processes, cell cycle regulation, and gene expression. There are now overwhelming data suggesting that the UPS contributes to cerebral ischemic injury.
Summary of Review— Proteasome inhibition is a potential treatment option for stroke. Thus far, proof of principle has been obtained from studies in several animal models of cerebral ischemia. Treatment with proteasome inhibitors reduces effectively neuronal and astrocytic degeneration, cortical infarct volume, infarct neutrophil infiltration, and NF-
B immunoreactivity with an extension of the neuroprotective effect at least 6 hours after ischemic insult. However, it is clear that the UPS represents a central pathway for the processing and metabolism of multiple proteins with critical roles in cellular function. To avoid eliciting significant side effects associated with complete inhibition of the proteasome and the possible immunosuppressive effects from persistent suppression of NF-B activation, it is critical that we understand how to partially and temporally attenuate proteasome function to elicit the desired therapeutic effect before any large-scale use in humans.
Conclusion— This review highlights the most recent advances in our knowledge on UPS, as well as the early experience of using proteasome inhibition strategies to treat acute stroke.
Key Words: cerebral ischemia • NF-kappa B • inflammation • ubiquitinproteasome
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Introduction |
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The progression and extension of cerebral ischemia are related to several mechanisms, many of which involve an inflammatory response component.1 Recent neuroprotective strategies include targeting inflammatory mediators as a whole, although it has proved difficult because of their redundancy. Alternate inflammatory pathways may circumvent the suppression of a single targeted mediator.2 Targeting inflammatory cascades as a whole is another approach, although it has shown no benefit.3 Novel approaches have now focused on alteration of inflammatory transcriptional factors to simultaneously interfere with the upregulation of multiple inflammatory genes.
One promising approach is to suppress the activation of transcription nuclear factor-kappa B (NF-B) by stabilizing the inhibitory protein IB via inhibition of the ubiquitin (Ub)-proteasome system (UPS).4 UPS is the major nonlysosomal pathway of proteolysis in human cells and accounts for the degradation of most short-lived, misfolded, or damaged proteins, as well as long-lived proteins. This pathway is important in the regulation of a number of key biological regulatory mechanisms.4,5 Inhibitors that target the UPS should suppress the activation of NF-B by stabilizing IB, thereby reducing levels of multiple proinflammatory proteins, providing antiinflammatory effects and, ultimately, successfully attenuating the inflammatory cascade in cerebral ischemia, leading to the reduction of the ischemic damage. As such, proteasome inhibitors are a novel approach to the treatment of cerebral ischemia. They have entered clinical evaluation based on efficacy and action demonstrated in laboratory studies, uncovering insights into the pathophysiology of cerebral ischemia.
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Ubiquitin and Ubiquitination |
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Ub is covalently attached to other proteins via either an isopeptide (via
-amino groups of internal Lys) or peptide bond.5 Ub conjugation (ubiquitination) is performed by 1 of multiple Ub ligases (E3s). E3s transfer Ub from 1 of several Ub-conjugating enzymes (UBCs or E2s), which obtain Ub from the Ub-activating enzyme (UBA or E1). Ub activation requires energy provided by ATP hydrolysis (Figure 1). The recognition of a substrate by an E3 involves various mechanisms: the presence of a specific primary sequence, an N-terminal destabilizing amino acid, phosphorylation of a specific residue, or poorly characterized structural motifs.5 Polyubiquitin chains are often trimmed by multiple deubiquitinating enzymes (DUBs), which counteract the E1-E2-E3 cascade rescuing certain substrates from degradation.6
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20S and 26S Proteasomes |
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The 26S proteasomes are composed of the 20S proteasome core and two 19S caps (PA700 activators). The 20S proteasome is a barrel-shaped molecule made up of 4 stacked rings, 2 outer
-rings and 2 inner ß-rings. Each ring is made up of 7 different subunits of the -type and ß-type, respectively. Cleavage of the peptide bonds is performed in the central catalytic chamber of the 20S proteasome by a nucleophile attack of an N-terminal Thr residue of either ß5, ß2, or ß1 subunits,6,7 displaying different substrate specificity. These activities have been named chymotrypsin-like (ChTL), trypsin-like (TL), and post-glutamyl-peptide hydrolyzing (PGPH), because they cleave peptides after hydrophobic, basic, and acidic residues, respectively.8 20S proteasomes degrade short peptides and unfolded proteins. Unfolded polypeptide chains reach the central chamber by 1 of the termini or in the form of a loop.9 PA700 has an affinity for the poly-Ub chains, bringing ubiquitinated substrates to the proteasome. Because of its "unfoldase" or "inverse chaperone" activity, the PA700 allows the degradation of folded proteins.6,5 Although the target protein itself is unfolded and fed within the central chamber of the proteasome, the poly-Ub chain is removed and disassembled.10
PA700 contains a hexameric ring of AAA ATPases binding the proteasomal -rings: it forms the "base" together with 2 other subunits, while the remaining 9 PA700 subunits form its "lid."11,12 The 26S enzyme has a central role in the UPS; however, the 20S core enzyme may play an important role in the degradation of unfolded proteins. Such proteins become more abundant within the cells in situations of oxidative stress. Although ATP depletion associated with ischemia impairs the 26S proteolytic activity, the 20S proteasome remains active.13,14
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Localization of Proteasomes Within the Cells |
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Proteasomes are present within the nucleus and cytoplasm of all cells, making up to 1% of total cell protein.15 Most proteasomes diffuse freely within cytoplasm and nucleoplasm; however, a fraction is stably associated with different structures. Proteasomes are enriched at discrete subnuclear foci called the PML bodies, whereas in the cytoplasm, proteasome subpopulations are associated with the external surface of the endoplasmic reticulum (ER) and the cytoskeleton16 and the centrosome.17 In situations of impaired UPS function, ubiquitinated proteins accumulate at PML bodies and around the centrosome, forming a single aggregate,18,19 or "aggresome." Formation of such aggregates is achieved in vitro by the action of proteasome inhibitors and by overexpression of certain proteins usually degraded by the UPS.
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Physiological Roles of Proteasomes |
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In yeast, the 20S proteasome subunits and several PA700 subunits are essential.20,21 Block in the cell cycle and induction of apoptosis are obvious effects of proteasome inhibition resulting from the inhibition of the degradation of regulatory proteins.22 Cells treated with proteasome inhibitors accumulate numerous polyubiquitinated proteins. Regulatory proteins are only a fraction of them, most of them being made up of aged structural and functional proteins, which are normally turned over by the UPS. Many polyubiquitinated proteins are the defective newly translated proteins.23 Aggregates of heavily polyubiquitinated proteins may be engulfed by autophagosomes and degraded by the lysosomal pathway, therefore rescuing cells from a death by suffocation with unwanted proteins. It is a matter of discussion whether the formation of protein aggregates is toxic or beneficial in serving to sequester ubiquitinated proteins from wreaking havoc within the cell.24
Peptides generated by proteasomes have from few to 20–30 amino acids. They are further degraded by cytosolic peptidases; however, a fraction of them are delivered to ER-associated TAP peptide transporter. Once in the ER lumen, they bind the assembling MHC class I molecules. Mature MHC class I molecules are exported to the cell surface, where they present the antigenic peptides to immunologically competent cells.25,26
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Role of the UPS in the Inflammatory Pathways: IB Signaling
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Inflammation can be defined as a complex set of interactions among soluble factors and cells that arise in any tissue in response to traumatic, infectious, postischemic, toxic, or autoimmune injury.27 The inflammatory process normally leads to recovery and healing; however, it can often lead to persistent tissue damage, caused by the infiltration and activity of inflammatory cells. An inflammatory response is also present in cerebral ischemia28 and after an acute ischemic stroke as sustained and persistent inflammatory response.29–31
Inflammatory pathways are regulated by a limited number of transcription factors, the most important being NF-B. NF-B is a collective name for dimeric transcription factors of the Rel family. Its most abundant form is the cytoplasmic p65/p50 dimer, bound to IB.32 On stimulation of various cell types by several cytokines (IL-1, TNF-), bacterial lipopolysaccharide, UV radiation, ionizing radiation, or oxidative stress, a signal transduction cascade is activated, leading to the phosphorylation of IB on Ser 32 and 36 by the multimeric IKK (IB kinase) complex.
IKK-mediated phosphorylation triggers the ubiquitination of IB by the E3 ligase SCFßTRCP. Ubiquitinated IB is targeted to the 26S proteasome.33 Once IB is degraded, the nuclear localization signal of NF-B is unmasked, allowing its translocation to the nucleus where it binds to promoter regions of several proinflammatory genes, inducing their expression and thus amplifying the inflammatory response (Figure 2).
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The p50 subunit of NF-B is generated from the p105 precursor by limited proteolytic cleavage mediated by the 26S proteasome.34,35 The intensity of NF-B activation depends on various factors, including the variable E3 activity of the SCFßTRCP complex, which is regulated by a reversible covalent modification with the Ub-like protein NEDD8.36 Finally, the activity of the IKK kinase depends on the formation of unusual poly-Ub chains linked by Lys63.37
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UPS in the Central Nervous System |
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UPS components are among the most abundant proteins of the central nervous system (CNS), but the relative levels of individual components show high variability between different regions and cell types.38,39 Ub immunoreactivity is normally diffuse; however, in various pathological conditions, it concentrates in neuronal inclusion bodies, suggesting that neurodegenerative disorders can involve an impairment of the UPS function.38
Neuronal differentiation in vitro is accompanied by increased levels of Ub conjugates, decreased levels of free Ub, enhanced capacity of ubiquitination, increased proteasome activity, and induction of immunoproteasome subunits.38,40–42 Intense Ub immunoreactivity correlates with neuronal differentiation, involving dendrite outgrowth and arborization in vivo.43,44 Ub is also involved in the sodium-dependent uptake of various neurotransmitters in the cerebral cortex.45 Curiously, while dipeptide inhibitors of the N-end rule prevent neurite outgrowth, proteasome inhibitors induce neurite outgrowth,41,46 indicating that UPS role in the CNS goes far beyond a simple degradative role. At higher doses and longer expositions, proteasome inhibitors induce neuronal cell death.47
The C-terminal Ub hydrolase UCH-L1 (PGP 9.5) constitutes 1% to 2% of total soluble brain protein and is used as a marker of neurons and neuroendocrine cells. UPS activity is required for experience-dependent remodeling of the postsynaptic densities in cultured rat hippocampal neurons.48 UPS at the synapse appears to operate at the presynaptic and at the postsynaptic level. The net outcome of inhibiting the UPS is to enhance synaptic transmission.49
Brain proteasomes display the same basic enzymatic activities.50 They have been detected in the cytoplasm, nuclei, dendrites, axons, and synaptic buttons of various CNS cell types, including pyramidal cells, granular cells of the hippocampus, Purkinje cells, and glial cells.51–53 Activity and expression of brain proteasomes decrease in neurodegenerative disorders and with age, contributing to the elevations in protein oxidation, protein aggregation, and neurodegeneration evident in the aging CNS.54,55 Alterations of ubiquitination of specific substrates caused by mutations of appropriate E3s are often associated with different neurological diseases.56–58
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General Properties and Chemistry of Proteasome Inhibitors |
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Several natural and synthetic compounds that act as proteasome inhibitors have been reported.59 Their chemical classes, chemical structures, and mechanisms of action are summarized in Table 1. One of these compounds, the MLN-519 a synthetic analog of the bacterial metabolite lactacystin,60 is under clinical evaluation for inhibiting reperfusion injury after ischemic CNS injury.61 Other natural proteasome inhibitors include eponemycin,62 epoxomycin,63 aclacinomycin A64 and PR-39, an Arg- and Pro-rich porcine polypeptide.65 Additional synthetic proteasome inhibitors are aldehyde derivatives (CEP-161267,25 or MG13266), dipeptide benzamide derivatives (CVT-63467), and dipeptide boronic acid substitutes (Bortezomib,68 NVP-AFB340, and NVP-AFD31469). Another group of vinyl sulfone tripeptide proteasome inhibitors has been described by Bogyo et al.70 Finally, the HIV-1 protease inhibitor, ritonavir, is also a competitive micromolar inhibitor of the proteasome.71
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Changes in the UPS During Ischemia/Reperfusion Injury in the Brain |
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The UPS plays a complex and unambiguous role in the etiopathology of cerebral ischemia/reperfusion, both directly and indirectly, because of its pivot role in many intracellular pathways.59
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Adjusted Changes of Ubiquitin-Proteasome System to Hypoxia |
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Although brief and acute hypoxia does not impair proteasome function,72 a clear inhibitory effect of hypoxia on proteasome function is evident after prolonged hypoxic periods73 and in the presence of inflammatory mediators.74,75 Repeated and intermittent episodes of hypoxia decrease markedly proteasomal activity in aged Sprague-Dawley rat brain.76 Hypoxia stabilizes the HIF-1
component of the dimeric hypoxia inducible factor (HIF) transcription factor. Under normoxic conditions, HIF-1 is ubiquitinated by the Von Hippel-Lindau (VHL) E3 and degraded by the proteasome, whereas the other HIF subunit—HIF-1ß/ARNT (aryl hydrocarbon receptor nuclear translocator)—is constitutively expressed.73 HIF-1 coordinates the response to prolonged hypoxia, which pertains to glycolysis, glucose transport, vasodilation, and angiogenesis.73 Proteasome inhibitors prevent HIF-1 degradation,77,78 resulting in accelerated angiogenesis in vitro.79 This mechanism could contribute to the rescue of the penumbra of an ischemic lesion. |
Adjusted Changes of Ubiquitin-Proteasome System to Ischemia |
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In the postischemic hippocampus, conjugated Ub accumulates and free Ub is depleted.72,80,81 The accumulation of conjugated Ub may reflect hypofunction of downstream proteasome activity that normally degrades ubiquitinated proteins. Moreover, direct injection of a proteasome inhibitor into the lateral ventricles of the rat-induced DNA fragmentation in various CNS areas, suggesting that suppression of proteasome is able to induce neuronal apoptosis.82 Therefore, it is reasonable to speculate that proteasome malfunction may in part underlie the molecular events of the ischemia-induced neuronal death. Decreased proteasome activity at the ischemic core and the surrounding tissues allows accumulation of oxidized proteins, resulting in formation of protein aggregates, ER stress, impairment of cell function, and eventually cell death. In an experimental ischemia of rat brains, a 60% elevation of Ub conjugate levels in the ischemic compared with the nonischemic animals was observed within 1 hour of recovery. The conjugate immunoreactivity remained at this level for 6 hours but eventually decreased to control levels by 24 hours of recovery.72,80 Increased formation of poly-Ub conjugates was accompanied with a significant increase in the transcription levels of poly-Ub genes.83
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Adjusted Changes of Ubiquitin-Proteasome System to ATP Depletion |
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Ischemic ATP depletion affects the ubiquitination cascade itself.84–86 One-hour transient focal cerebral ischemia induces marked depletion of the E3 parkin protein levels but does not affect the levels of several E2s. Upregulation of the expression of parkin protects cells from injury induced by ER stress; therefore, parkin depletion may increase the sensitivity of neurons to ER stress and the aggregation of ubiquitinated proteins during the reperfusion period.87 At the ischemic core, the ATP-dependent and Ub-dependent degradation mediated by the 26S proteasome is impaired, whereas the ATP-independent and Ub-independent degradation mediated by 20S proteasome proceeds without obstacles. Many 26S proteasomes dissociate under these conditions into 20S proteasomes and PA700 caps. After ischemia in the gerbil cortex, the 26S proteasome ChTL activity decreases, whereas the 20S proteasome ChTL activity increases.88 Moreover, while in most regions the 26S proteasome activity is recovered after reperfusion, in certain regions (eg, the CA1 region of the hippocampus) PA700 and 20S proteasomes are not fully able to reassociate, indicating the occurrence of irreversible biochemical changes. This probably underlies the delayed neuronal cell death in such regions, which has many features common with neurodegeneration.86
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Adjusted Changes of Ubiquitin-Proteasome System to Intracellular pH Levels |
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The cellular site of action of low pH is not completely resolved. Effects on UPS that appear to exist but have not yet been extensively investigated appear the most likely and are quite reasonable. Proteasome inhibition is correlated with hypoxia-evoked decreases in extracellular and intracellular pH.74 Certainly, less specifically, pH may act by altering proteasome subunits at critical times, by direct transient denaturation or indirectly by enhancement of free radical formation (via iron delocalization and the Fenton reaction),89 or more specifically by altering subunit displacement of the proteosomal complex catalytic activity and altering the action of Ub-protein-ligase complexes.90,91
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Adjusted Changes of UPS to Intracellular Ca2+ Levels |
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The effects of proteasome inhibitors on intracellular Ca2+ levels were tested in murine neocortical cultures and resulted in widespread neuronal death associated with a reduction in intracellular free calcium associated with intracellular calcium starvation.92
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UPS and Mitochondrial Function |
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