The 20S proteasome α5 subunit of Arabidopsis thaliana carries an RNase activity and interacts in planta with the lettuce mosaic potyvirus HcPro protein

In plants, the ubiquitin/26S proteasome system (UPS) plays a central role in protein degradation and is involved in many steps of defence mechanisms, regardless of the types of pathogen targeted. In addition to its proteolytic activities, the UPS ribonuclease (RNase) activity, previously detected in 20S proteasome preparations from cauliflower and sunflower (Helianthus annuus), has been shown to specifically target plant viral RNAs in vitro. In this study, we show that recombinant Arabidopsis thaliana proteasomal α(5) subunit expressed in Escherichia coli harbours an RNase activity that degrades Tobacco mosaic virus (TMV, Tobamovirus)- and Lettuce mosaic virus (LMV, Potyvirus)-derived RNAs in vitro. The analysis of mutated forms of the α(5) subunit demonstrated that mutation of a glutamic acid at position 110 affects RNase activity. Furthermore, it was demonstrated, using a bimolecular fluorescence complement assay, that the multifunctional helper component proteinase (HcPro) of LMV, already known to interfere with the 20S proteasome RNase activity in vitro, can interact in vivo with the recombinant α(5) subunit. Further experiments demonstrated that, in LMV-infected lettuce cells, α(5) is partially relocalized to HcPro-containing infection-specific inclusions. Susceptibility analyses of Arabidopsis mutants, knocked out for each At-PAE gene encoding α(5) , showed that one (KO-pae1) of the two mutants exhibited a significantly increased susceptibility to LMV infection. Taken together, these results extend to A. thaliana α(5) the range of HcPro-interacting proteasomal subunits, and suggest that HcPro may modulate its associated RNase activity which may contribute to an antiviral response.     

Inhibition versus induction of apoptosis by proteasome inhibitors depends on concentration

We previously established that NF-kappaB DNA binding activity is required for Sindbis Virus (SV)-induced apoptosis. To investigate whether SV induces nuclear translocation of NF-kappaB via the proteasomal degradation pathway, we utilized MG132, a peptide aldehyde inhibitor of the catalytic subunit of the proteasome. 20 microM MG132 completely abrogated SV-induced NF-kappaB nuclear activity at early time points after infection. Parallel measures of cell viability 48 h after SV infection revealed that 20 microM MG132 induced apoptosis in uninfected cells. In contrast, a lower concentration of MG132 (200 nM) resulted in partial inhibition of SV-induced nuclear NF-kappaB activity and inhibition of SV-induced apoptosis without inducing toxicity in uninfected cells. The specific proteasomal inhibitor, lactacystin, also inhibited SV-induced death. Taken together, these results suggest that the pro-apoptotic and anti-apoptotic functions of peptide aldehyde proteasome inhibitors such as MG-132 depend on the concentration of inhibitor utilized and expand the list of stimuli requiring proteasomal activation to induce apoptosis to include viruses.     

Antiviral Activity of the Proteasome on Incoming Human Immunodeficiency Virus Type 1

Following cell surface receptor binding and membrane fusion, human immunodeficiency virus (HIV) virion cores are released in the cytoplasm. Incoming viral proteins represent potential targets for cytosolic proteases. We show that treatment of target cells with the proteasome inhibitors MG132 and lactacystin increased the efficiency of HIV infection. Proteasome inhibitors were active at the early steps of the viral cycle. Incoming p24^Gag proteins accumulated in the cytosol, and larger amounts of proviral DNA were synthesized. In vitro, purified 20S proteasome degraded HIV virion components. Thus, degradation of incoming viral proteins by the proteasome represents an early intracellular defense against infection.     

Proteasome Inhibitors Cause Induction of Heat Shock Proteins and Trehalose, Which Together Confer Thermotolerance in Saccharomyces cerevisiae

An accumulation in cells of unfolded proteins is believed to be the common signal triggering the induction of heat shock proteins (hsps). Accordingly, in Saccharomyces cerevisiae, inhibition of protein breakdown at 30°C with the proteasome inhibitor MG132 caused a coordinate induction of many heat shock proteins within 1 to 2 h. Concomitantly, MG132, at concentrations that had little or no effect on growth rate, caused a dramatic increase in the cells’ resistance to very high temperature. The magnitude of this effect depended on the extent and duration of the inhibition of proteolysis. A similar induction of hsps and thermotolerance was seen with another proteasome inhibitor, clasto-lactacystin β-lactone, but not with an inhibitor of vacuolar proteases. Surprisingly, when the reversible inhibitor MG132 was removed, thermotolerance decreased rapidly, while synthesis of hsps continued to increase. In addition, exposure to MG132 and 37°C together had synergistic effects in promoting thermotolerance but did not increase hsp expression beyond that seen with either stimulus alone. Although thermotolerance did not correlate with hsp content, another thermoprotectant trehalose accumulated upon exposure of cells to MG132, and the cellular content of this disaccharide, unlike that of hsps, quickly decreased upon removal of MG132. Also, MG132 and 37°C had additive effects in causing trehalose accumulation. Thus, the resistance to heat induced by proteasome inhibitors is not just due to induction of hsps but also requires a short-lived metabolite, probably trehalose, which accumulates when proteolysis is reduced.     

Proteasome inhibitor MG132 blocks viral DNA replication and assembly of human cytomegalovirus

This study provides evidence that proteasomal activity is required at multiple steps in human cytomegalovirus replication. Electron microscopy revealed that no viral particles were assembled in the presence of proteasome inhibitor MG132. Immunofluorescence and Western blot analyses using MG132 demonstrated that immediate early gene expression was suppressed at low but not high MOI. In contrast, expression of late proteins was completely blocked independent of MOI. Additionally, pulsed-field gel electrophoresis demonstrated that MG132 interferes with cleavage of HCMV DNA. Bromodeoxyuridine incorporation studies showed that de novo viral DNA synthesis is reduced in the presence of MG132. Furthermore, in contrast to previous hypotheses we demonstrated that neither the ND10 components PML and hDaxx nor NFkappaB activation represent the target for MG132.     

Capillary electrophoresis for screening of 20S proteasome inhibitors

A method for studying 20S proteasome inhibitors by capillary electrophoresis (CE) has been developed. Proteasome plays a fundamental role in degrading key regulatory proteins. The 20S proteasome can degrade intrinsically disordered proteins in an ATP-independent manner without additional “helper” molecules. The discovery of new proteasome inhibitors with little or no toxicity is highly desirable in anticancer therapy. In this study, the inhibitory effects of MG132 and MG115 on the 20S proteasome were evaluated by CE for the first time. The optimized CE conditions were as follows: fused-silica capillary of 30 cm effective length and 75 μm internal diameter, pressure injection of 0.5 psi for 5 s, 50 mM Hepes buffer (pH 7.6) with 2% dimethyl sulfoxide, constant voltage of 20 kV, and detection wavelength at 340 nm. Also, the new method was used to study the inhibitory effects of 30 novel peptidyl vinyl ester derivatives of MG132. The 50% inhibition concentrations (IC₅₀ values) of MG132 and MG115 were 40.0 and 84.7 nM, respectively. Two new compounds, XP32 and XP35, showed considerable inhibitory effects on the 20S proteasome. When the concentrations of them were fixed at 172 nM, their inhibition rates were 36.2% and 29.1%, respectively. The results showed that the CE method was powerful, sensitive, and fast and required little sample. It could be employed as one of the reliable drug screening methods for 20S proteasome inhibitors.     

Tau protein degradation is catalyzed by the ATP/ubiquitin-independent 20S proteasome under normal cell conditions

Tau is the major protein exhibiting intracellular accumulation in Alzheimer disease. The mechanisms leading to its accumulation are not fully understood. It has been proposed that the proteasome is responsible for degrading tau but, since proteasomal inhibitors block both the ubiquitin-dependent 26S proteasome and the ubiqutin-independent 20S proteasome pathways, it is not clear which of these pathways is involved in tau degradation. Some involvement of the ubiquitin ligase, CHIP in tau degradation has also been postulated during stress. In the current studies, we utilized HT22 cells and tau-transfected E36 cells in order to test the relative importance or possible requirement of the ubiquitin-dependent 26S proteasomal system versus the ubiquitin-independent 20S proteasome, in tau degradation. By means of ATP-depletion, ubiquitinylation-deficient E36ts20 cells, a 19S proteasomal regulator subunit MSS1-siRNA approaches, and in vitro ubiquitinylation studies, we were able to demonstrate that ubiquitinylation is not required for normal tau degradation.     

Genomics of Helper Component Proteinase Reveals Effective Strategy for <Emphasis Type="Italic">Papaya Ringspot Virus</Emphasis> Resistance

Papaya ringspot virus (PRSV) causes severe economic losses in both cucurbits and papaya throughout the tropics and subtropics. Development of PRSV-resistant transgenic plants faces a major hurdle in achieving resistance against geographically distinct isolates. One of the major reasons of failing to achieve the broad-spectrum PRSV resistance is the involvement of silencing suppressor proteins of viral origin. Here, based on sequence profile of silencing suppressor protein, HcPro, we show that PRSV-HcPro, acts as a suppressor of RNA silencing through micro RNA binding in a dose- dependent manner. In planta expression of PRSV-HcPro affects developmental biology of plants, suggesting the interference of suppressor protein in micro RNA-directed regulatory pathways of plants. Besides facilitating the establishment of PRSV, it showed strong positive synergism with other heterologous viruses as well. This study provides a strategy to develop effective and stable PRSV-resistant transgenic plants.     

dsRNA介导的番木瓜环斑病毒（PRSV）的抗病性研究

以模式植物拟南芥（Arabidopsis thaliana）和烟草（Nicotiana tabacum）及PRSV寄主植物番木瓜（CaricapapayaL.）作为试验材料,开展了番木瓜环斑病毒外壳蛋白基因dsRNA介导的PRSV病原抗性的研究。利用农杆菌介导法将番木瓜环斑病毒外壳蛋白CP基因反向重复表达载体pHellsgate12-CPIR（简称PHG12-CPIR）分别转化到烟草和拟南芥中,获得阳性植株,并利用渗透法和农杆菌介导的瞬时表达体系将pHG12-CPIR载体导入到番木瓜中。对转基因植株进行攻毒试验并分析了其抗病性。在接种3~7d内,在拟南芥和番木瓜上转基因植株的发病情况较轻,而野生型植株叶片与转基因植株相比,均表现出不同程度的黄化、皱缩和枯斑等症状。在接种PRSV后,番木瓜和拟南芥转化植株表现症状的叶片的比例与对照相比,结果显著低于对照,而在烟草植株上症状表现的差异不明显。在3种植物上RT-PCR检测结果显示,在接种番木瓜环斑病毒PRSV后,野生型植株中有高浓度的病毒积累,而转pHG12-CPIR基因植株中几乎没有病毒积累,推测转pHG12-CPIR基因植株中瞬时表达系统已启动RNAi机制抑制了CP基因的表达。     

蛋白酶体活性缺失对拟南芥根发育影响的显微和超微结构观察

以拟南芥根为材料,运用光学和透射电子显微镜分析了蛋白酶体抑制剂MG132对拟南芥根尖伸长区细胞的显微及超微结构的影响。结果发现:(1)微分干涉显微镜观察结果表明,MG132处理将导致拟南芥根部伸长区细胞的细胞质液泡化,并且抑制剂浓度越高细胞质液泡化越明显。(2)半薄切片结合考马斯亮蓝染色结果表明,MG132诱导的液泡中富含蛋白质。(3)免疫荧光标记结合共聚焦显微镜观察结果表明,液泡中的蛋白质主要为泛素缀合蛋白,暗示泛素化蛋白质的积累诱导细胞质自体吞噬的发生。(4)透射电镜观察结果表明,MG132处理的确诱导了自体吞噬作用的发生以及随后发生的自噬起源的细胞质液泡化。该研究结果为泛素/蛋白酶体途径与自体吞噬依赖的蛋白降解系统之间的联系提供了线索。     

A Proteasome Inhibitor-stimulated Nrf1 Protein-dependent Compensatory Increase in Proteasome Subunit Gene Expression Reduces Polycomb Group Protein Level

The polycomb group (PcG) proteins, Bmi-1 and Ezh2, are important epigenetic regulators that enhance skin cancer cell survival. We recently showed that Bmi-1 and Ezh2 protein level is reduced by treatment with the dietary chemopreventive agents, sulforaphane and green tea polyphenol, and that this reduction involves ubiquitination of Bmi-1 and Ezh2, suggesting a key role of the proteasome. In the present study, we observe a surprising outcome that Bmi-1 and Ezh2 levels are reduced by treatment with the proteasome inhibitor, MG132. We show that this is associated with a compensatory increase in the level of mRNA encoding proteasome protein subunits in response to MG132 treatment and an increase in proteasome activity. The increase in proteasome subunit level is associated with increased Nrf1 and Nrf2 level. Moreover, knockdown of Nrf1 attenuates the MG132-dependent increase in proteasome subunit expression and restores Bmi-1 and Ezh2 expression. The MG132-dependent loss of Bmi-1 and Ezh2 is associated with reduced cell proliferation, accumulation of cells in G₂, and increased apoptosis. These effects are attenuated by forced expression of Bmi-1, suggesting that PcG proteins, consistent with a prosurvival action, may antagonize the action of MG132. These studies describe a compensatory Nrf1-dependent, and to a lesser extent Nrf2-dependent, increase in proteasome subunit level in proteasome inhibitor-treated cells and confirm that PcG protein levels are regulated by proteasome activity.     

Different Degree in Proteasome Malfunction Has Various Effects on Root Growth Possibly through Preventing Cell Division and Promoting Autophagic Vacuolization

The ubiquitin/proteasome pathway plays a vital role in plant development. But the effects of proteasome malfunction on root growth, and the mechanism underlying this involvement remains unclear. In the present study, the effects of proteasome inhibitors on Arabidopsis root growth were studied through the analysis of the root length, and meristem size and cell length in maturation zone using FM4–64, and cell-division potential using GFP fusion cyclin B, and accumulation of ubiquitinated proteins using immunofluorescence labeling, and autophagy activity using LysoTracker and MDC. The results indicated that lower concentration of proteasome inhibitors promoted root growth, whereas higher concentration of inhibitors had the opposite effects. The accumulation of cyclin B was linked to MG132-induced decline in meristem size, indicating that proteasome malfunction prevented cell division. Besides, MG132-induced accumulation of the ubiquitinated proteins was associated with the increasing fluorescence signal of LysoTracker and MDC in the elongation zone, revealing a link between the activation of autophagy and proteasome malfunction. These results suggest that weak proteasome malfunction activates moderate autophagy and promotes cell elongation, which compensates the inhibitor-induced reduction of cell division, resulting in long roots. Whereas strong proteasome malfunction induces severe autophagy and disturbs cell elongation, resulting in short roots.     

Identification of Proteasome Subunit Beta Type 6 (PSMB6) Associated with Deltamethrin Resistance in Mosquitoes by Proteomic and Bioassay Analyses

Deltamethrin (DM) insecticides are currently being promoted worldwide for mosquito control, because of the high efficacy, low mammalian toxicity and less environmental impact. Widespread and improper use of insecticides induced resistance, which has become a major obstacle for the insect-borne disease management. Resistance development is a complex and dynamic process involving many genes. To better understand the possible molecular mechanisms involved in DM resistance, a proteomic approach was employed for screening of differentially expressed proteins in DM-susceptible and -resistant mosquito cells. Twenty-seven differentially expressed proteins were identified by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). Four members of the ubiquitin-proteasome system were significantly elevated in DM-resistant cells, suggesting that the ubiquitin-proteasome pathway may play an important role in DM resistance. Proteasome subunit beta type 6 (PSMB6) is a member of 20S proteasomal subunit family, which forms the proteolytic core of 26S proteasome. We used pharmaceutical inhibitor and molecular approaches to study the contributions of PSMB6 in DM resistance: the proteasome inhibitor MG-132 and bortezomib were used to suppress the proteasomal activity and siRNA was designed to block the function of PSMB6. The results revealed that both MG-132 and bortezomib increased the susceptibility in DM-resistant cells and resistance larvae. Moreover, PSMB6 knockdown decreased cellular viability under DM treatment. Taken together, our study indicated that PSMB6 is associated with DM resistance in mosquitoes and that proteasome inhibitors such as MG-132 or bortezomib are suitable for use as a DM synergist for vector control.     

Lamotrigine Attenuates Proteasome Inhibition-Induced Apoptosis by Suppressing the Activation of the Mitochondrial Pathway and the Caspase-8- and Bid-Dependent Pathways

Proteasome impairment has been shown to be involved in neuronal degeneration. Antiepileptic lamotrigine has been demonstrated to have a neuroprotective effect. However, the effect of lamotrigine on the proteasome inhibition-induced neuronal cell death has not been studied. Therefore, we assessed the effect of lamotrigine on the proteasome inhibition-induced neuronal cell apoptosis in relation to cell death process using differentiated PC12 cells and SH-SY5Y cells. The proteasome inhibitors MG132 and MG115 induced a decrease in the levels of Bid and Bcl-2 proteins, an increase in the levels of Bax and p53, loss of the mitochondrial transmembrane potential, cytochrome c release and activation of caspases (-8, -9 and -3). The addition of lamotrigine reduced the proteasome inhibitor-induced changes in the apoptosis-related protein levels, production of reactive oxygen species, depletion and oxidation of glutathione (GSH), and cell death in both cell lines. Lamotrigine and N-acetylcysteine alone did not affect the levels of 26S proteasome and activity of 20S proteasome. MG132 did not alter the levels of 26S proteasome but decreased activity of 20S proteasome. Lamotrigine and N-acetylcysteine attenuated MG132-induced decrease in the activity of 20S proteasome. The results show that lamotrigine appears to suppress the proteasome inhibitor-induced apoptosis in PC12 cells by suppressing the activation of the mitochondrial pathway and the caspase-8- and Bid-dependent pathways. The suppressive effect of lamotrigine appears to be associated with its inhibitory effect on the production of reactive oxygen species, the depletion and oxidation of GSH and the activity reduction of 20S proteasome.     

Cell-specific inhibition of paramyxovirus maturation by proteasome inhibitors

Effects of proteasome inhibitors on the replication of a paramyxovirus in comparison with the effects on replication of an orthomyxovirus and rhabdovirus were investigated. Treatment of Sendai virus (SeV)-infected LLC-MK2 cells with 50 microM MG132 reduced virus growth to ca. 1/10,000, and treatment with different concentrations of MG132 reduced virus growth in a dose-dependent manner. Released amounts of viral proteins were reduced in correspondence with decrease in infectivity. The inhibition of virus maturation was confirmed by an SeV-like particle formation system. Lactacystin also impaired SeV growth and zLL impaired the growth to a lesser extent, suggesting involvement of proteasomes in the restriction of virus growth. In the presence of MG132, localizations of the M protein and viral F and HN glycoproteins on the cell membrane appeared to be partly dissociated, although the viral glycoproteins were normally transported to the cell surface. These results suggest that an early step of SeV assembly was disturbed by proteasome inhibitors. The relationship of the results with ubiquitin is also discussed. SeV maturation was less susceptible and resistant to MG132 in CV1 cells and A549 cells, respectively, indicating cell specificity of the drug effect. Release of vesicular stomatitis virus also showed high susceptibility to MG132 and release of influenza virus A/WSN/33 was only mildly susceptible to the drug in LLC-MK2 cells. Effects of proteasome inhibitors on virus maturation are thus highly cell-specific and partly virus-specific.     

Sulforaphane attenuates postnatal proteasome inhibition and improves spatial learning in adult mice

Proteasomes are known to degrade proteins involved in various processes like metabolism, signal transduction, cell-cycle regulation, inflammation, and apoptosis. Evidence showed that protein degradation has a strong influence on developing neurons as well as synaptic plasticity. Here, we have shown that sulforaphane (SFN) could prevent the deleterious effects of postnatal proteasomal inhibition on spatial reference and working memory of adult mice. One day old Balb/c mice received intracerebroventricular injections of MG132 and SFN. Sham received an equal volume of aCSF. We observed that SFN pre-administration could attenuate MG132 mediated decrease in proteasome and calpain activities. In vitro findings revealed that SFN could induce proteasomal activity by enhancing the expression of catalytic subunit-β5. SFN pre-administration prevented the hippocampus based spatial memory impairments during adulthood, mediated by postnatal MG132 exposure. Histological examination showed deleterious effects of MG132 on pyramidal neurons and granule cell neurons in DG and CA3 sub-regions respectively. Furthermore, SFN pre-administration has shown to attenuate the effect of MG132 on proteasome subunit-β5 expression and also induce the Nrf2 nuclear translocation. In addition, SFN pre-administered mice have also shown to induce expression of pCaMKII, pCreb, and mature/pro-Bdnf, molecules which play a crucial role in spatial learning and memory consolidation. Our findings have shown that proteasomes play an important role in hippocampal synaptic plasticity during the early postnatal period and SFN pre-administration could enhance the proteasomal activity as well as improve spatial learning and memory consolidation.     

beta-Synuclein reduces proteasomal inhibition by alpha-synuclein but not gamma-synuclein

The accumulation of aggregated alpha-synuclein is thought to contribute to the pathogenesis of Parkinson's disease. Recent studies indicate that aggregated alpha-synuclein binds to S6', a component of the 19 S subunit in the 26 S proteasome and inhibits 26 S proteasomal degradation, both ubiquitin-independent and ubiquitin-dependent. The IC(50) of aggregated alpha-synuclein for inhibition of the 26 S ubiquitin-independent proteasomal activity is approximately 1 nm. alpha-Synuclein has two close homologues, termed beta-synuclein and gamma-synuclein. In the present study we compared the effects of the three synuclein homologues on proteasomal activity. The proteasome exists as a 26 S and a 20 S species, with the 26 S proteasome containing the 20 S core and 19 S cap. Monomeric alpha- and beta-synucleins inhibited the 20 S and 26 S proteasomal activities only weakly, but monomeric gamma-synuclein strongly inhibited ubiquitin-independent proteolysis. The IC(50) of monomeric gamma-synuclein for the 20 S proteolysis was 400 nm. In monomeric form, none of the three synuclein proteins inhibited 26 S ubiquitin-dependent proteasomal activity. Although beta-synuclein had no direct effect on proteasomal activity, co-incubating monomeric beta-synuclein with aggregated alpha-synuclein antagonized the inhibition of the 26 S ubiquitin-independent proteasome by aggregated alpha-synuclein when added before the aggregated alpha-synuclein. Co-incubating beta-synuclein with gamma-synuclein had no effect on the inhibition of the 20 S proteasome by monomeric gamma-synuclein. Immunoprecipitation and pull-down experiments suggested that antagonism by beta-synuclein resulted from binding to alpha-synuclein rather than binding to S6'. Pull-down experiments demonstrated that recombinant monomeric beta-synuclein does not interact with the proteasomal subunit S6', unlike alpha-synuclein, but beta-synuclein does bind alpha-synuclein and competes with S6' for binding to alpha-synuclein. Based on these data, we hypothesize that the alpha- and gamma-synucleins regulate proteasomal function and that beta-synuclein acts as a negative regulator of alpha-synuclein.     

Cloning of the papaya ringspot virus (PRSV) replicase gene and generation of PRSV-resistant papayas through the introduction of the PRSV replicase gene

Papaya ringspot virus (PRSV) can cause a destructive disease in papaya (Carica papaya L.). Based on observations that viral replicase (RP) gene confers resistance to virus in other plants, we designed a pair of primers and cloned the RP gene from PRSV by RT-PCR. The 3'-truncated and 5'-extended RP gene fragment was then oriented under the control of the CaMV35 S promoter and nos termination sequence in the mini Ti plasmid vector pRok to construct a plant expression vector, designated pRPTW. Papaya (C. papaya L.) cv. Tai-nong-2 embryogenic calli were transformed by Agrobacterium tumefaciens LBA4404 harboring the pRPTW vector. After selection on 100 mg/ml kanamycin, 20 putative transgenic papayas were regenerated and confirmed by PCR-Southern blot and Southern blot analyses. PRSV inoculation tests showed that the RP gene conferred resistance to PRSV in transgenic papayas and those offspring carrying the RP gene. The consistency of the presence of the RP gene and PRSV resistance indicates that replicase-mediated resistance against PRSV was attained in papaya. Possible mechanisms include RNA-mediated resistance and protein-mediated resistance, as well as others, although further studies are required.     

Aggregated and monomeric alpha-synuclein bind to the S6' proteasomal protein and inhibit proteasomal function

The accumulation of aggregated alpha-synuclein is thought to contribute to the pathophysiology of Parkinson's disease, but the mechanism of toxicity is poorly understood. Recent studies suggest that aggregated proteins cause toxicity by inhibiting the ubiquitin-dependent proteasomal system. In the present study, we explore how alpha-synuclein interacts with the proteasome. The proteasome exists as a 26 S and a 20 S species. The 26 S proteasome is composed of the 19 S cap and the 20 S core. Aggregated alpha-synuclein strongly inhibited the function of the 26 S proteasome. The IC(50) of aggregated alpha-synuclein for ubiquitin-independent 26 S proteasomal activity was 1 nm. Aggregated alpha-synuclein also inhibited 26 S ubiquitin-dependent proteasomal activity at a dose of 500 nm. In contrast, the IC(50) of aggregated alpha-synuclein for 20 S proteasomal activity was > 1 microm. This suggests that aggregated alpha-synuclein selectively interacts with the 19 S cap. Monomeric alpha-synuclein also inhibited proteasomal activity but with lower affinity and less potency. Recombinant monomeric alpha-synuclein inhibited the activity of the 20 S proteasomal core with an IC(50) > 10 microm, exhibited no inhibition of 26 S ubiquitin-dependent proteasomal activity at doses up to 5 microm, and exhibited only partial inhibition (50%) of the 26 S ubiquitin-independent proteasomal activity at doses up to 10 mm. Binding studies demonstrate that both aggregated and monomeric alpha-synuclein selectively bind to the proteasomal protein S6', a subunit of the 19 S cap. These studies suggest that proteasomal inhibition by aggregated alpha-synuclein could be mediated by interaction with S6'.