Misfolded PrP impairs the UPS by interaction with the 20S proteasome and inhibition of substrate entry
2011; Springer Nature; Volume: 30; Issue: 15 Linguagem: Inglês
10.1038/emboj.2011.224
ISSN1460-2075
AutoresPelagia Deriziotis, Ralph André, David M. Smith, Rob Goold, Kerri J. Kinghorn, Mark Kristiansen, James A. Nathan, Rina Rosenzweig, Dasha Krutauz, Michael H. Glickman, John Collinge, Alfred L. Goldberg, Sarah J. Tabrizi,
Tópico(s)Cellular transport and secretion
ResumoArticle8 July 2011free access Misfolded PrP impairs the UPS by interaction with the 20S proteasome and inhibition of substrate entry Pelagia Deriziotis Pelagia Deriziotis Department of Neurodegenerative Disease, University College London Institute of Neurology, Queen Square, London, UKPresent address: Wellcome Trust Centre for Human Genetics, Roosevelt Drive, University of Oxford, Oxford, UK Search for more papers by this author Ralph André Ralph André Department of Neurodegenerative Disease, University College London Institute of Neurology, Queen Square, London, UK Search for more papers by this author David M Smith David M Smith Department of Cell Biology, Harvard Medical School, Boston, MA, USA Department of Biochemistry, West Virginia University School of Medicine, Medical Center Drive, Morgantown, WV, USA Search for more papers by this author Rob Goold Rob Goold Department of Neurodegenerative Disease, University College London Institute of Neurology, Queen Square, London, UK Search for more papers by this author Kerri J Kinghorn Kerri J Kinghorn Department of Neurodegenerative Disease, University College London Institute of Neurology, Queen Square, London, UK Search for more papers by this author Mark Kristiansen Mark Kristiansen MRC Prion Unit, University College London Institute of Neurology, Queen Square, London, UK Search for more papers by this author James A Nathan James A Nathan Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Rina Rosenzweig Rina Rosenzweig Department of Biology, Technion—Israel Institute of Technology, Haifa, Israel Search for more papers by this author Dasha Krutauz Dasha Krutauz Department of Biology, Technion—Israel Institute of Technology, Haifa, Israel Search for more papers by this author Michael H Glickman Michael H Glickman Department of Biology, Technion—Israel Institute of Technology, Haifa, Israel Search for more papers by this author John Collinge John Collinge Department of Neurodegenerative Disease, University College London Institute of Neurology, Queen Square, London, UK MRC Prion Unit, University College London Institute of Neurology, Queen Square, London, UK Search for more papers by this author Alfred L Goldberg Alfred L Goldberg Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Sarah J Tabrizi Corresponding Author Sarah J Tabrizi [email protected] Department of Neurodegenerative Disease, University College London Institute of Neurology, Queen Square, London, UK Search for more papers by this author Pelagia Deriziotis Pelagia Deriziotis Department of Neurodegenerative Disease, University College London Institute of Neurology, Queen Square, London, UKPresent address: Wellcome Trust Centre for Human Genetics, Roosevelt Drive, University of Oxford, Oxford, UK Search for more papers by this author Ralph André Ralph André Department of Neurodegenerative Disease, University College London Institute of Neurology, Queen Square, London, UK Search for more papers by this author David M Smith David M Smith Department of Cell Biology, Harvard Medical School, Boston, MA, USA Department of Biochemistry, West Virginia University School of Medicine, Medical Center Drive, Morgantown, WV, USA Search for more papers by this author Rob Goold Rob Goold Department of Neurodegenerative Disease, University College London Institute of Neurology, Queen Square, London, UK Search for more papers by this author Kerri J Kinghorn Kerri J Kinghorn Department of Neurodegenerative Disease, University College London Institute of Neurology, Queen Square, London, UK Search for more papers by this author Mark Kristiansen Mark Kristiansen MRC Prion Unit, University College London Institute of Neurology, Queen Square, London, UK Search for more papers by this author James A Nathan James A Nathan Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Rina Rosenzweig Rina Rosenzweig Department of Biology, Technion—Israel Institute of Technology, Haifa, Israel Search for more papers by this author Dasha Krutauz Dasha Krutauz Department of Biology, Technion—Israel Institute of Technology, Haifa, Israel Search for more papers by this author Michael H Glickman Michael H Glickman Department of Biology, Technion—Israel Institute of Technology, Haifa, Israel Search for more papers by this author John Collinge John Collinge Department of Neurodegenerative Disease, University College London Institute of Neurology, Queen Square, London, UK MRC Prion Unit, University College London Institute of Neurology, Queen Square, London, UK Search for more papers by this author Alfred L Goldberg Alfred L Goldberg Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Sarah J Tabrizi Corresponding Author Sarah J Tabrizi [email protected] Department of Neurodegenerative Disease, University College London Institute of Neurology, Queen Square, London, UK Search for more papers by this author Author Information Pelagia Deriziotis1,‡, Ralph André1,‡, David M Smith2,3,‡, Rob Goold1, Kerri J Kinghorn1, Mark Kristiansen4, James A Nathan2, Rina Rosenzweig5, Dasha Krutauz5, Michael H Glickman5, John Collinge1,4, Alfred L Goldberg2 and Sarah J Tabrizi *,1 1Department of Neurodegenerative Disease, University College London Institute of Neurology, Queen Square, London, UK 2Department of Cell Biology, Harvard Medical School, Boston, MA, USA 3Department of Biochemistry, West Virginia University School of Medicine, Medical Center Drive, Morgantown, WV, USA 4MRC Prion Unit, University College London Institute of Neurology, Queen Square, London, UK 5Department of Biology, Technion—Israel Institute of Technology, Haifa, Israel ‡These authors contributed equally to this work *Department of Neurodegenerative Disease, University College London Institute of Neurology, Queen Square, London WC1N 3BG, UK. Tel.: +44 845 155 5000/ext. 724434; Fax: +44 207 676 2180; E-mail: [email protected] The EMBO Journal (2011)30:3065-3077https://doi.org/10.1038/emboj.2011.224 Present address: Wellcome Trust Centre for Human Genetics, Roosevelt Drive, University of Oxford, Oxford, UK PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Prion diseases are associated with the conversion of cellular prion protein (PrPC) to toxic β-sheet isoforms (PrPSc), 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. Introduction Prion diseases are fatal neurodegenerative disorders whose pathogenesis is associated with a conformational rearrangement of the normal cellular prion protein (PrPC) to abnormal infectious isoforms (PrPSc) (Prusiner, 1982). Neuropathological findings include spongiform change, marked neuronal loss and astrogliosis, and the accumulation of PrPSc in the brain. Although they share the same amino-acid sequence, PrPC is mainly α-helical, whereas PrPSc is β-sheet rich. Also, PrPSc is less soluble in detergents and more resistant to proteases (Prusiner, 1998). To date, the cause of prion-mediated neurodegeneration remains unclear. Although PrPC is essential for prion disease development, PrPC knockout mice show no gross pathology (Bueler et al, 1993; Mallucci et al, 2002). Therefore, loss of PrPC function is not the cause of prion-mediated cell death and pathology must result from a toxic gain-of-function associated with conversion of PrPC to β-sheet-rich PrPSc. Indeed, many pathways have been proposed to explain precise mechanisms of neuronal cell death in prion disease (Caughey and Baron, 2006; Collinge and Clarke, 2007; Tatzelt and Schatzl, 2007). Evidence suggests that functional impairment in the ubiquitin-proteasome system (UPS) may be important in prion diseases (Deriziotis and Tabrizi, 2008). This pathway catalyses the rapid elimination of misfolded cell proteins and many proteins critical in regulating gene expression and metabolism (Goldberg, 2003). In the UPS, protein substrates are covalently linked to a polyubiquitin chain, which leads to rapid binding and degradation by the 26S proteasome (Glickman and Ciechanover, 2002). This ATP-dependent proteolytic complex consists of the 20S core particle and one or two 19S regulatory particles. The 20S proteasome is a barrel-shaped complex within which substrates are degraded. It is composed of four stacked rings, containing seven α-subunits in the two outer rings and seven β-subunits in the two inner rings. Three of these β-subunits have peptidase activity (Glickman and Ciechanover, 2002): the β1-subunits have caspase-like activity, β2-subunits have trypsin-like activity and β5-subunits have chymotrypsin-like activity. The N-termini of the α-subunits function as a gate into the proteolytic chamber and block substrate entry (Groll et al, 2000; Smith et al, 2007; Gillette et al, 2008; Rabl et al, 2008). The six ATPase subunits that comprise the base of the 19S (Rpt1-6) regulate gate opening in the 20S proteasome, which leads to increased entry and hydrolysis of peptides and unfolded proteins (Kohler et al, 2001; Smith et al, 2005). Impairment of the UPS has also been suggested to contribute to the pathogenesis of neurodegenerative conditions such as Huntington's disease (HD), Parkinson's disease (PD) and Alzheimer's disease (AD) (Rubinsztein, 2006). In these diseases, misfolded proteins accumulate as aggregated intraneuronal inclusions or as neurofibrillary tangles, which contain ubiquitin and proteasomes. Conditional depletion of 26S proteasomes in neurons of the substantia nigra or forebrain in mice results in neurodegeneration with inclusions resembling Lewy bodies in PD (Bedford et al, 2008). In prion disease, prion-infected mouse brains have increased levels of ubiquitin conjugates, which correlate with decreased proteasome function (Kang et al, 2004); and the Hectd2 gene which encodes a ubiquitin ligase, was identified as a gene influencing incubation time for prion disease in mice (Lloyd et al, 2009). Increasing evidence suggests that soluble micro-aggregates of misfolded proteins, rather than larger protein inclusions, are toxic to neurons in these diseases (Rubinsztein, 2006). It is possible that the build-up of such aggregates eventually overwhelms the UPS, causing a functional impairment. We reported that aggregated β-sheet-rich PrP oligomers inhibit the proteolytic activities of the 26S proteasome, an effect specific to PrP in an aggregated, non-native β-sheet conformation (Kristiansen et al, 2007). By contrast, the recombinant protein in a PrPC-like conformation and other fibrillar amyloidogenic proteins was not inhibitory. Using a transgenic mouse model expressing a short-lived reporter protein (Lindsten et al, 2003), we presented further evidence for impairment of the UPS in prion-infected brains (Kristiansen et al, 2007). Therefore, impairment of the UPS may have an important role in prion and other neurodegenerative diseases characterised by accumulation of misfolded proteins, but the biochemical mechanisms underlying this dysfunction remain unclear. Inactivation of any one of the three active sites of the 20S proteasome slows, but does not block, protein degradation; in fact, in order to impair protein degradation significantly, the chymotrypsin-like sites as well as either the caspase-like or the trypsin-like sites need to be inhibited (Kisselev et al, 2006). We found previously that aggregated β-sheet-rich PrP isoforms inhibit peptide hydrolysis by all these sites, although the trypsin-like activity was inhibited to a lesser extent (Kristiansen et al, 2007). There are two possible explanations for these effects. First, the aggregated PrP species may enter the 20S particle and directly inhibit its three active sites. This mechanism seems unlikely as the pore of the 20S barrel does not exceed 2 nm in diameter (Groll et al, 2000); consequently, aggregated proteins should not be able to enter this particle. Alternatively, PrP isoforms may inhibit the entry of protein and peptide substrates into the 20S proteasome perhaps by blocking gate opening by the regulatory ATPases. Such an effect should reduce its ability to digest proteins as well as small peptide substrates, but to varying extents. In fact, Kisselev et al (2002) showed that agents or mutations that promote gate opening enhance most dramatically the hydrolysis of hydrophobic and acidic peptides whose breakdown is limited by entry into the particle, but have lesser effects on hydrolysis of basic peptides which is limited by the slow turnover rate of the trypsin-like site (Kisselev et al, 2002). Therefore, an inhibitory effect of the aggregated PrP on gate opening should result in a more marked reduction in the chymotrypsin-like and caspase-like activities, as was observed (Kristiansen et al, 2007). The aim of the present study was to determine how aggregated β-sheet-rich PrP species interact with the proteasome and inhibit its function. Here, we present evidence that they inhibit by decreasing gate opening in the 20S particle, leading to a reduced capacity of the proteasomes to degrade peptides and proteins. We also demonstrate that PrP isoforms directly interact with the 26S proteasome both in vitro and in vivo, and cause an accumulation of key UPS substrates in prion-infected mouse brains. This ability of aggregated β-sheet-rich PrP isoforms to block substrate entry into the 20S proteasome may be a model for understanding pathogenic mechanisms in other neurodegenerative diseases, where there is also an accumulation of misfolded β-sheet-rich proteins and impairment of protein degradation. Results Key UPS substrates accumulate in prion-infected mouse brain Previously, we presented evidence that the UPS is impaired in prion-infected brains of transgenic mice expressing a short-lived reporter protein (Kristiansen et al, 2007). In order to confirm that the UPS was impaired in vivo, we assayed the levels of three important substrates. During inflammation, IκB is a rapidly degraded proteasomal substrate that accumulates upon treatment of cells with proteasome inhibitors (Palombella et al, 1994). Assays of IκBα levels by western blot in RML prion-infected mouse brains revealed a significant accumulation above those in matched controls (Figure 1A). Levels of two additional endogenous proteasomal substrates, the cyclin-dependent kinase inhibitor, p27 (Figure 1B), and the p53 tumour suppressor protein (Figure 1C), were also significantly greater in RML prion-infected mouse brains than in matched controls. Since there was no significant difference in transcript expression between prion-infected and control brains (Supplementary Figure S1), the increased levels of IκBα, p27 and p53 are most likely due to reduced proteasomal function. Figure 1.Accumulation of substrates of the UPS occurs in prion-infected mouse brains. (A) IκBα, (B) p27 and (C) p53 levels are significantly increased, assessed by immunoblot relative to β-actin expression, in four end-stage prion-infected mouse brain homogenates as compared with four uninfected controls (***P<0.001, **P<0.01, and *P<0.05, respectively). Download figure Download PowerPoint PrPSc and 26S proteasome components associate with each other in vivo To investigate whether PrPSc binds to the 26S proteasome during infection in vivo, we tested if anti-PrP antibody (Ab)-coated beads could co-immunoprecipitate 26S components from prion-infected brain. We observed that PrP co-immunoprecipitated both the 20S particle and the 19S subunit, Rpt1, in RML prion-infected mouse brain (Figure 2A). It could also be seen to co-immunoprecipitate another 19S subunit, Rpn7 (Supplementary Figure S2). In similar experiments, anti-20S Ab-coated beads co-immunoprecipitated PrP from an RML prion-infected mouse brain fraction (Figure 2B). The immunoprecipitate was then analysed by immunoblotting with an anti-PrP antibody either directly (Figure 2B, middle) or after proteinase K (PK) digestion (Figure 2B, bottom), which revealed the triplet of protease-resistant bands characteristic of prion disease-associated PrPSc. Control experiments using BSA-coated beads did not precipitate any proteins (Figure 2A and B). These results indicate a specific association between PrPSc and 26S proteasome components in prion-infected brains. Figure 2.PrPSc and 26S components co-immunoprecipitate. (A) 20S proteasomes and 19S components co-precipitate with PrP from prion-infected mouse brain using anti-PrP antibody-coated beads. Immunoprecipitated (IPanti-PrP), control (using beads coated with BSA alone; IPBSA-coated) and unbound (supernatant) fractions, together with starting material (input) and antibody-coated beads alone (Ab-coated beads), were immunoblotted with anti-PrP, anti-20S and anti-Rpt1 antibodies. Boxed areas highlight specific bands as distinct from immunoglobulin chains eluted from the antibody-coated beads. An increased proportion of the IP fraction was loaded for detection of Rpt1. Separate panels showing images as detected by the same antibody are from the same immunoblot. (B) PrPSc co-precipitates with the 20S particle from prion-infected mouse brain using anti-20S antibody-coated beads. IPanti-20S, control (IPBSA-coated) and unbound (supernatant) fractions, and input starting material, were immunoblotted with anti-20S and anti-PrP antibodies. The characteristic triplet of protease-resistant bands revealed by proteinase K (+PK) digestion of the IPanti-20S fraction demonstrates the presence of PrPSc. Aggregated β-PrP binds directly to 20S and 19S components. (C) 20S and 19S components co-precipitate with PrP when human 26S proteasomes are incubated with aggregated β-PrP for 1 h at 37°C and immunoprecipitated using anti-PrP antibody-coated beads. IPanti-PrP, control (IPBSA-coated) and unbound (supernatant) fractions, together with input starting material, were immunoblotted with anti-PrP, anti-20S, anti-Rpt1 and anti-Rpn7 antibodies. (D) 20S proteasome co-precipitates with PrP when human 20S proteasomes are incubated with aggregated β-PrP for 1 h at 37°C, but not when incubated with aggregated α-helical PrP. The IPanti-PrP fraction and input starting material were immunoblotted with an anti-20S antibody. No 20S subunits were detected in the aggregated β-PrP-only control. The anti-20S antibody used to visualise the 20S recognises the following subunits: α5/α7, β1, β5 and β7. Download figure Download PowerPoint Aggregated β-PrP binds directly to human 20S and 26S proteasomes Full-length recombinant mouse PrP (aa23-231) was folded into either an α-helical structure representative of native PrPC (α-PrP), or a predominantly β-sheet species, termed β-PrP, that has similar physico-chemical properties to PrPSc (Jackson et al, 1999b), as described previously (Kristiansen et al, 2007). To determine whether PrP interacts directly with the 26S proteasome, we incubated purified human 26S particles with aggregated β-PrP and then added anti-PrP Ab-coated beads. 20S proteasome and 19S components (Rpt1 and Rpn7) were precipitated by the anti-PrP antibody (Figure 2C). Control experiments using BSA-coated beads did not precipitate significant amounts of any 26S proteasome components (Figure 2C). In similar experiments, human 20S proteasomes were incubated with either aggregated β-PrP or heat-aggregated α-PrP before incubation with the anti-PrP Ab-coated beads. 20S subunits were precipitated by the anti-PrP antibody in the presence of aggregated β-PrP, but not with aggregated α-PrP (Figure 2D). These results demonstrate that aggregated β-PrP has a high affinity for the 20S particle, suggesting a strong interaction between β-sheet-rich PrP species and 20S proteasomes. β-Sheet-rich PrP isoforms inhibit degradation of casein To assess the functional relevance of prion-induced inhibition of the UPS, we measured the effects of different PrP isoforms on the degradation of a model protein substrate, FITC-casein, by purified 20S proteasomes (Kisselev et al, 1999). Pre-incubating yeast 20S proteasomes with aggregated β-PrP markedly reduced the degradation of casein and this inhibitory effect was dependent on the amount of β-PrP added (Figure 3A). When an equal amount of aggregated α-PrP was added, there was little or no inhibition of FITC-casein degradation (Figure 3B), showing that aggregation per se is not sufficient. Figure 3.β-Sheet-rich PrP species inhibit casein degradation by purified 20S proteasomes. (A) Increasing concentrations of aggregated β-PrP (▪=control; •=250 μg/ml; □=500 μg/ml; ○=1 mg/ml) decrease the degradation rate of FITC-labelled casein by wild-type yeast 20S proteasomes. (B) Aggregated α-helical PrP causes much less inhibition than aggregated β-PrP (▪=wild-type 20S alone; □=1 mg/ml α-PrP; ○=1 mg/ml β-PrP). The molar ratio in these reactions of 1 mg/ml PrP to yeast 20S proteasomes is 69:1 based upon the total amount of free monomeric PrP added. Each graph is representative of at least three independent experiments. Download figure Download PowerPoint Low concentrations of aggregated PrP inhibit peptide hydrolysis by the 20S proteasome Our previous data suggested that the inhibitory species are small aggregates of oligomeric β-sheet-rich PrP (Kristiansen et al, 2007). To further define this inhibition, we monitored the chymotrypsin-like activity of wild-type yeast 20S proteasomes after incubation with increasing amounts of aggregated β-PrP. Half-maximal inhibition of the 20S particles was observed at between 90 and 180 nM aggregated β-PrP, where concentrations are calculated based upon the total amount of free monomeric protein added (Figure 4A). However, because most of the β-PrP is aggregated, the actual number of free molecules in solution must be much lower. Since the concentration of 20S proteasomes in the reactions was 9 nM, the affinity of aggregated β-PrP for the proteasome must be quite high. Figure 4.β-Sheet-rich PrP species inhibit wild-type yeast 20S proteasomes, but not an open-gated 20S mutant. (A) Half-maximal inhibition of wild-type yeast 20S proteasomes (9 nM) is observed between 90 and 180 nM aggregated β-PrP (molar concentrations are based upon the free monomeric protein added). (B) Aggregated β-PrP (20 μg/ml) or PrPSc semi-purified from RML prion-infected ScGT-1 cells (20 μg/ml) inhibits all three peptidase activities of the wild-type yeast 20S proteasomes (P<0.001 versus respective controls). (C) Aggregated β-PrP and semi-purified PrPSc have no inhibitory effect on the activities of the open-gated α3ΔN yeast 20S mutant. (D) As expected for an open-gated mutant, α3ΔN 20S particles show much higher basal chymotrypsin-like activity compared with wild-type 20S. (E) Chymotrypsin-like activity is abolished in both wild-type 20S and α3ΔN 20S mutant proteasomes after pre-treatment with 50 μM epoxomicin (EPOX), a specific proteasome inhibitor. (F) The inhibitory effect of aggregated β-PrP on the trypsin-like activity of the 26S proteasome was seen at low substrate concentrations and not at high concentrations. Human 26S proteasomes were incubated with or without 20 μg/ml aggregated β-PrP, before the addition of Boc-LRR-amc substrate (ranging from 20 to 100 μM) to monitor trypsin-like activity. Download figure Download PowerPoint β-Sheet-rich PrP isoforms inhibit wild-type 20S proteasomes, but not an open-gated mutant As protein aggregates cannot enter the narrow 13 Å channel into the 20S proteasome, and therefore should not interact directly with its proteolytic sites, we tested whether aggregated β-sheet-rich PrP isoforms inhibit opening of the gated substrate entry channel in the 20S proteasome α-ring. To examine this possibility, we tested the ability of β-sheet-rich PrP isoforms to inhibit a variant of the 20S proteasome that contains a constitutively open gate (α3ΔN-20S) by means of a nine residue truncation of its α3 N-terminus (Groll et al, 2000). This deletion prevents formation of the closed-gate conformation. Proteasome inhibitors that block the proteolytic sites can readily inhibit the 'open-gated' mutant 20S proteasome, but if agents affect gate opening, they should not be able to influence the peptidase activity of this mutant. When aggregated β-PrP or PrPSc isolated from prion-infected mouse hypothalamic neuronal cells (ScGT-1; Kristiansen et al, 2007) were incubated with wild-type 20S proteasomes, both caused a significant reduction in all three peptidase activities (Figure 4B). By contrast, in the α3ΔN mutant 20S particles, these PrP species had no effect on peptide hydrolysis by the three different active sites (Figure 4C). To confirm that the mutant α3ΔN 20S proteasomes had an open gate, we compared its basal activity with that of the wild-type 20S particle. As expected, these open-gated mutants showed much higher basal activity (Figure 4D). In addition, the peptidase activities we monitored were specific to the proteasome, since addition of the specific proteasome inhibitor, epoxomicin, completely abolished the chymotrypsin-like activity of these preparations (Figure 4E). These data imply that aggregated β-sheet-rich PrP species inhibit gate opening in the 20S proteasome, which can account for the previously described inhibition of 26S proteasomal function (Kristiansen et al, 2007). Aggregated β-PrP binds open-gated mutant 26S proteasomes As was found using the 20S particles (Figure 4), aggregated β-PrP significantly reduced the chymotrypsin-like activity of wild-type 26S proteasomes, but did not inhibit the activity of open-gated α3ΔN 26S particles (Figure 5A). Possibly this failure to inhibit the open-gated α3ΔN proteasomes could be due to an inability to bind to the mutant particles. To assess whether aggregated β-PrP associates directly with open-gated α3ΔN 26S proteasomes, we used anti-PrP Ab-coated beads to test if the α3ΔN mutant 26S could be co-immunoprecipitated from these reactions. When wild-type and α3ΔN 26S proteasomes were incubated with aggregated β-PrP, and then incubated with anti-PrP Ab-coated beads, the 20S core components were precipitated (Figure 5B). Control experiments using BSA-coated beads did not precipitate the 20S components (Figure 5B). Thus, although aggregated β-PrP binds to the constitutively open-gated α3ΔN mutant proteasomes, it fails to inhibit substrate entry and degradation. Therefore, β-PrP must inhibit proteasome function by acting on the gating mechanism. Figure 5.Aggregated β-PrP binds directly to both wild-type yeast 26S proteasomes and an open-gated 26S mutant. (A) Aggregated β-PrP (50 μg/ml) inhibits chymotrypsin-like activity of wild-type yeast 26S proteasome (P<0.001 versus relative control), but not the open-gated α3ΔN 26S mutant. (B) 20S components co-precipitate with PrP when either wild-type or α3ΔN yeast 26S proteasomes are incubated with aggregated β-PrP for 1 h at 37°C and immunoprecipitated using anti-PrP antibody-coated beads. Immunoprecipitated (IPanti-PrP) and control (IPBSA-coated) fractions, together with the starting material (input), were immunoblotted with anti-PrP and anti-20S antibodies. Download figure Download PowerPoint Effects on trypsin-like activity also suggest reduced substrate entry These findings, and ones below, indicate that misfolded PrP inhibits the 20S proteasome gating mechanism, and therefore should reduce the hydrolysis of all substrates. However, in most experiments, peptide substrates of the chymotrypsin and caspase sites are inhibited more by aggregated β-PrP than those of the trypsin sites (see Figures 4B and 6). In fact, the rate of substrate cleavage by the chymotrypsin-like and caspase-like sites is more sensitive to gate opening than by the trypsin-like site (Kisselev et al, 2002). This is because gate opening will only enhance substrate hydrolysis if its rate of diffusion into the 20S particle is slower than its rate of cleavage by the active sites, and the trypsin-like site has a much lower turnover rate than the other active sites. Consequently, the entry of its substrates is typically not rate limiting for degradation, but can be made rate limiting by lowering the concentration of its substrates, for example, Boc-LRR-amc (Kisselev et al, 2002). If the β-sheet-rich PrP isoforms inhibit the cleavage of this substrate of the trypsin-like sites by blocking gate opening, the aggregated β-PrP should reduce its hydrolysis at low, but n
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