Complete suppression of Htt fibrilization and disaggregation of Htt fibrils by a trimeric chaperone complex
2017; Springer Nature; Volume: 37; Issue: 2 Linguagem: Inglês
10.15252/embj.201797212
ISSN1460-2075
AutoresAnnika Scior, Alexander Buntru, Kristin Arnsburg, Anne Ast, Manuel Iburg, Katrin Juenemann, Maria Lucia Pigazzini, Barbara Mlody, Dmytro Puchkov, Josef Priller, Erich E. Wanker, Alessandro Prigione, Janine Kirstein,
Tópico(s)Signaling Pathways in Disease
ResumoArticle6 December 2017free access Transparent process Complete suppression of Htt fibrilization and disaggregation of Htt fibrils by a trimeric chaperone complex Annika Scior Leibniz-Institute for Molecular Pharmacology (FMP) im Forschungsverbund Berlin, Berlin, Germany Search for more papers by this author Alexander Buntru Max Delbrueck Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Kristin Arnsburg Leibniz-Institute for Molecular Pharmacology (FMP) im Forschungsverbund Berlin, Berlin, Germany Search for more papers by this author Anne Ast Max Delbrueck Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Manuel Iburg Leibniz-Institute for Molecular Pharmacology (FMP) im Forschungsverbund Berlin, Berlin, Germany Search for more papers by this author Katrin Juenemann Leibniz-Institute for Molecular Pharmacology (FMP) im Forschungsverbund Berlin, Berlin, Germany Search for more papers by this author Maria Lucia Pigazzini Leibniz-Institute for Molecular Pharmacology (FMP) im Forschungsverbund Berlin, Berlin, Germany Charité – Universitätsmedizin and NeuroCure Cluster of Excellence, Berlin, Germany Search for more papers by this author Barbara Mlody Max Delbrueck Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Dmytro Puchkov Leibniz-Institute for Molecular Pharmacology (FMP) im Forschungsverbund Berlin, Berlin, Germany Search for more papers by this author Josef Priller Department of Neuropsychiatry and Laboratory of Molecular Psychiatry, Charite Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Erich E Wanker Corresponding Author [email protected] Max Delbrueck Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Alessandro Prigione orcid.org/0000-0001-9457-1952 Max Delbrueck Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Janine Kirstein Corresponding Author [email protected] orcid.org/0000-0003-4990-2497 Leibniz-Institute for Molecular Pharmacology (FMP) im Forschungsverbund Berlin, Berlin, Germany Search for more papers by this author Annika Scior Leibniz-Institute for Molecular Pharmacology (FMP) im Forschungsverbund Berlin, Berlin, Germany Search for more papers by this author Alexander Buntru Max Delbrueck Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Kristin Arnsburg Leibniz-Institute for Molecular Pharmacology (FMP) im Forschungsverbund Berlin, Berlin, Germany Search for more papers by this author Anne Ast Max Delbrueck Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Manuel Iburg Leibniz-Institute for Molecular Pharmacology (FMP) im Forschungsverbund Berlin, Berlin, Germany Search for more papers by this author Katrin Juenemann Leibniz-Institute for Molecular Pharmacology (FMP) im Forschungsverbund Berlin, Berlin, Germany Search for more papers by this author Maria Lucia Pigazzini Leibniz-Institute for Molecular Pharmacology (FMP) im Forschungsverbund Berlin, Berlin, Germany Charité – Universitätsmedizin and NeuroCure Cluster of Excellence, Berlin, Germany Search for more papers by this author Barbara Mlody Max Delbrueck Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Dmytro Puchkov Leibniz-Institute for Molecular Pharmacology (FMP) im Forschungsverbund Berlin, Berlin, Germany Search for more papers by this author Josef Priller Department of Neuropsychiatry and Laboratory of Molecular Psychiatry, Charite Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Erich E Wanker Corresponding Author [email protected] Max Delbrueck Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Alessandro Prigione orcid.org/0000-0001-9457-1952 Max Delbrueck Center for Molecular Medicine, Berlin, Germany Search for more papers by this author Janine Kirstein Corresponding Author [email protected] orcid.org/0000-0003-4990-2497 Leibniz-Institute for Molecular Pharmacology (FMP) im Forschungsverbund Berlin, Berlin, Germany Search for more papers by this author Author Information Annika Scior1, Alexander Buntru2, Kristin Arnsburg1, Anne Ast2, Manuel Iburg1, Katrin Juenemann1, Maria Lucia Pigazzini1,3, Barbara Mlody2, Dmytro Puchkov1, Josef Priller4, Erich E Wanker *,2, Alessandro Prigione2 and Janine Kirstein *,1 1Leibniz-Institute for Molecular Pharmacology (FMP) im Forschungsverbund Berlin, Berlin, Germany 2Max Delbrueck Center for Molecular Medicine, Berlin, Germany 3Charité – Universitätsmedizin and NeuroCure Cluster of Excellence, Berlin, Germany 4Department of Neuropsychiatry and Laboratory of Molecular Psychiatry, Charite Universitätsmedizin Berlin, Berlin, Germany *Corresponding author. Tel: +49 30 9406 2157; E-mail: [email protected] author. Tel: +49 30 94793250; E-mail: [email protected] EMBO J (2018)37:282-299https://doi.org/10.15252/embj.201797212 Correction(s) for this article Complete suppression of Htt fibrillization and disaggregation of Htt fibrils by a trimeric chaperone complex04 October 2021 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 Abstract Huntington's disease (HD) is a neurodegenerative disorder caused by an expanded CAG trinucleotide repeat in the huntingtin gene (HTT). Molecular chaperones have been implicated in suppressing or delaying the aggregation of mutant Htt. Using in vitro and in vivo assays, we have identified a trimeric chaperone complex (Hsc70, Hsp110, and J-protein) that completely suppresses fibrilization of HttExon1Q48. The composition of this chaperone complex is variable as recruitment of different chaperone family members forms distinct functional complexes. The trimeric chaperone complex is also able to resolubilize Htt fibrils. We confirmed the biological significance of these findings in HD patient-derived neural cells and on an organismal level in Caenorhabditis elegans. Among the proteins in this chaperone complex, the J-protein is the concentration-limiting factor. The single overexpression of DNAJB1 in HEK293T cells is sufficient to profoundly reduce HttExon1Q97 aggregation and represents a target of future therapeutic avenues for HD. Synopsis A dynamic chaperone complex of Hsc70, Hsp110 and J-protein dissolves pathological Huntingtin fibrils in vitro and prevents aggregate formation in Huntington's disease patient-derived neurons. Hsc70, a type B J-protein and Hsp110 fully supress fibrilization of huntingtin exon1 for at least 24 h in vitro. The same chaperones can disaggregate preformed Huntingtin (Htt) fibrils. Depletion of these chaperones enhances Htt aggregation in C. elegans and HD patient-derived neural cells. Overexpression of DNAJB1 reduces Htt fibril formation in mammalian cells. Introduction Huntington's disease (HD) is caused by a CAG trinucleotide repeat expansion in the first exon of the Huntingtin gene (HTT), which renders N-terminal fragments of the protein (HttQn) aggregation-prone and ultimately results in β-sheet formation and amyloid fibrilization. The pathogenic threshold of the polyQ expansion in HTT is 35, and the aggregation propensity correlates with the number of glutamine residues (Scherzinger et al, 1999; Gusella & MacDonald, 2000). The aggregation of HttQn is proteotoxic and is associated with cellular dysfunction and neuronal degeneration (Hoffner et al, 2007). Consequently, the search for factors that interfere with the amyloid formation in particular by suppressing the formation of toxic oligomeric and high molecular weight aggregates such as amyloid fibrils represents an attractive therapeutic strategy. Commonly, in response to protein misfolding and aggregation the cell induces the expression of the proteostasis network (PN) that is composed of proteases, molecular chaperones, and many other proteins (Balch et al, 2008). However, the expression of polyQ proteins fails to induce this network (Bersuker et al, 2013). The expression of HttQn triggers a protein aggregation cascade. Oligomers and protofibrils can act as nuclei (seeds) that induce a conformational switch in soluble monomeric HttQn molecules by primary and secondary nucleation events (Scherzinger et al, 1999; Wetzel, 2012; Kakkar et al, 2016). Moreover, HttQn fibrils sequester aggregation-prone endogenous proteins and molecular chaperones and also inhibit the ubiquitin proteasome system (Olzscha et al, 2011; Hipp et al, 2012; Kirstein-Miles et al, 2013; Kim et al, 2016). Such an impairment of the PN leads to further accumulation of misfolded and aggregated proteins that ultimately results in degeneration of the affected neuronal cells (Cicchetti et al, 2011). Molecular chaperones represent an attractive target to prevent the accumulation of proteotoxic amyloid proteins such as HttQn as demonstrated by recent findings. The first observations that chaperones can to a certain extent decrease the aggregation propensity of HttQn in in vitro assays were obtained using bacterial or yeast Hsp70 and Hsp40 (J-protein) chaperones, respectively (Muchowski et al, 2000). In vivo, overexpression of Hsp70, J-protein, Hsp110, or TRiC reduces the aggregation toxicity of Htt in cultured cells, flies, and HD mouse models (Chan et al, 2000; Tam et al, 2006; Kuo et al, 2013; Monsellier et al, 2015; Kakkar et al, 2016). It was also observed that expression of two chaperones (Hsp70/J-protein or Hsp110/J-protein) synergistically suppressed Htt aggregation (Kuo et al, 2013). These findings are in agreement with previous reports indicating that these chaperones form functional complexes (Rampelt et al, 2012; Nillegoda et al, 2015) and suggest that they cooperate in vivo to prevent or reverse polyQ aggregation (Chan et al, 2000; Kuo et al, 2013). Despite these efforts, many open questions remain. Are there specific chaperones or chaperone complexes that recognize distinct moieties of misfolded and aggregated Htt? The diversity within the chaperone families increased in the course of evolution (Brehme et al, 2014). A pronounced expansion in the number of distinct chaperones occurred, for example, within the J-protein family, suggesting an increased functional specialization of chaperones. Do chaperones interfere with the nucleation events of beta-sheet formation or influence seeding activities? Can chaperones resolubilize Htt once it is assembled into amyloid fibrils? To address these questions, we set out to gain mechanistic insights into how chaperones maintain and restore the solubility of HttQn in vitro and in vivo. In this study, we demonstrate that a trimeric chaperone complex composed of a member each of the Hsp70, Hsp110, and type B J-protein family can completely suppress the amyloid fibril formation of HttExon1Q48 and almost completely the fibrilization of HttExon1Q75. We also demonstrate for the first time the disaggregation of HttExon1Q48 fibrils by this trimeric chaperone complex. The composition of the chaperone complex is variable. The combination of different Hsp70 and J-protein chaperones together with Hsp110 leads to distinct chaperone complexes that exhibit different suppression and disaggregation activities. Depletion of these chaperones in HD patient-derived neural progenitor cells (NPCs) leads to a pronounced increase in Htt protein aggregates (Q44). We could confirm the importance of Hsc70, HSP-110, and the J-protein to maintain the solubility of HttQn and related polyQ proteins on an organismal level in Caenorhabditis elegans. We can show that the J-protein is the chaperone component whose concentration is most critical in the in vitro assays and exhibited the strongest effect on HttQ44 upon knockdown in the NPCs. Accordingly, overexpression of a specific J-protein (DNAJB1) can ameliorate the aggregation of HttExon1Q97 in human cell culture. Results FRET-based assay to monitor the fibrilization of HttExon1Q48 To gain mechanistic insight into how molecular chaperones maintain Htt protein species in a soluble state and prevent their self-assembly into amyloid fibrils, we employed a FRET-based HttExon1 aggregation assay. The assay is based on GST-HttExon1Q48 that is fused at the C-terminus to either CyPet or YPet (Nguyen & Daugherty, 2005). These fluorescent proteins represent a potential FRET pair with CyPet being the donor and YPet the acceptor molecule. The globular GST tag fused to the N-terminus inhibits the fibril formation of the pathogenic polyQ stretch in the HttExon1 fragment. The cleavage of this tag with the PreScission protease (PreSP) liberates the HttExon1Q48-CyPet/YPet (from now on referred to as HttExon1Q48) protein and initiates its self-assembly into fibrils (Fig 1A). In this fibrilized form, the fluorescent fusion proteins come into close proximity that enables the energy transfer from CyPet to YPet. Thus, the FRET efficiency between CyPet and YPet reports on the aggregation status of HttExon1Q48 (Fig 1A). Figure 1. Trimeric human chaperone complex can suppress the fibrilization of HttExon1Q48 and resolubilize HttExon1Q48 fibrils Scheme of experimental FRET-based assay for the analysis of fibrilization of HttExon1Q48. In all FRET assays, we use the fluorescently tagged HttExon1Q48-YPet/CyPet proteins yet refer to them as HttExon1Q48 for clarity. TEM images of HttExon1Q48 fibrils at time points 0 and 24 h after addition of PreSP. Analysis of the sedimentation by ultracentrifugation of HttExon1Q48 24 h post-PreSP treatment is depicted below. The supernatant represents the soluble species and the pellet the insoluble HttExon1Q48 protein. The total depicts a sample before the centrifugation step. Scale bars: 200 nm. FRET analysis of HttExon1Q48 fibrilization. The black curve represents the HttExon1Q48-YPet/CyPet mixtures alone (no chaperone control) in all figures. The relative concentrations of HttExon1Q48 and the chaperones are indicated as ratios in brackets. The first number always refers to HttExon1Q48. The chaperones were added at time point 0 together with HttExon1Q48 and PreSP. The addition of Hsc70, Apg2, and DNAJB1 completely suppresses the fibrilization of HttExon1Q48 (bright red curve). The effect of individual chaperones and chaperone mixtures on the HttExon1Q48 fibrilization is indicated in the figure. The non-pathogenic HttExon1Q23-YPet/CyPet mixtures display no FRET post-PreSP treatment even upon doubling their concentration (dark blue and turquoise curves). TEM analysis of the suppression of HttExon1Q48 fibrilization by Hsc70, Apg2, and DNAJB1. A scheme of the experimental outline is depicted on the right. The red arrow refers to the time point of sample analysis. Scale bar: 100 nm. Suppression of HttExon1Q48 fibrilization by sedimentation analysis in the absence or presence of Hsc70, Apg2, DNAJB1, and ATP. The values refer to the ratio between the fluorescent signal of HttExon1Q48-CyPet in the supernatant (soluble) and pellet (aggregated moiety) fraction. Depicted is the average of three independent experiments with error bars representing the standard deviation. Sedimentation analysis of the disaggregation of HttExon1Q48 by Hsc70, Apg2, and DNAJB1 in the presence or absence of ATP. Depicted are the ratios of supernatant (soluble) to pellet (aggregated HttExon1Q48). Depicted is the average of three independent experiments with error bars representing the standard deviation. TEM analysis of disaggregation of HttExon1Q48 fibrils by Hsc70, Apg2, and DNAJB1. The top left image depicts fibrils after 24 h post-PreSP treatment and the top right after an additional 20 h without addition of chaperones. The bottom row depicts images of HttExon1Q48 fibrils 24 h post-PreSP + 1 h (left), 4 h (middle), and 20 h (right) in the presence of Hsc70, Apg2, DNAJB1, and ATP. A scheme of the experimental outline is depicted on the right. The red arrow refers to the time point of sample analysis. Scale bar: 100 nm. Filter retardation analysis of HttExon1Q97-HA aggregates. Lysates of HEK293T cells expressing HttExon1Q97-HA were probed on the filter membrane with antibodies against HA (HttExon1Q97), DNAJB1, Apg2 (Hsp110), and Hsc70 to detect their association with the respective chaperones (second to fourth membranes) and to confirm the presence of the HttExon1Q97 aggregates (first membrane). Lysates were spotted on the filter in duplicates, and the bottom row depicts the control (cells that do not express HttExon1Q97-HA). Download figure Download PowerPoint We additionally used transmission electron microscopy (TEM) analysis to monitor the aggregation of the HttExon1Q48 proteins in vitro. Images taken 24 h post-PreSP treatment of tagged and untagged HttExon1Q48 proteins show fibrilization of the Htt proteins and validate amyloid formation. Furthermore, they demonstrate that the fluorescent tags do not interfere with amyloid formation (Figs 1B and EV1A). Using the FRET-based Htt fibrilization assay, we observed an initial decrease in FRET efficiency that accounts for the monomerization of the GST-tagged Htt proteins. After a lag phase of about 3 h, the FRET efficiency sharply increases, reflecting the fibrilization process (elongation phase). The FRET signal reaches a plateau within the next 2 h and remains there for the entire duration of the experiment (Fig 1C; black curve). TEM analysis of samples of the plateau phase shows only fibrilized Htt that can be sedimented with ultracentrifugation (Fig 1B), indicating that HttExon1Q48 is converted into amyloid fibrils. We used a sedimentation approach to further validate the time course of fibrilization. For that, we used the CFP fluorescence of HttExon1Q48-CyPet as readout for the abundance of the protein in the soluble and insoluble fractions and could confirm the aggregation of HttExon1Q48 at the respective time points post-PreSP treatment (Fig EV1B), mirroring the FRET fibrilization curve (Fig 1C; black curve). As a control, we employed HttExon1Q23-CyPet/YPet constructs whose polyQ length is below the pathogenic threshold of 35Q residues required to form Htt fibrils. As expected, the HttExon1Q23-CyPet/YPet proteins do not exhibit any FRET upon PreSP treatment even when we doubled the HttExon1Q23 concentration (Fig 1C; dark blue and turquoise curves). Taken together, we conclude that the FRET assay provides a quantitative and reliable method to monitor the fibrilization of HttExon1Q48. Click here to expand this figure. Figure EV1. Controls for Htt fibrilization and the kinetics, point mutations in HSP-1 and HSP-110, and analysis of ATPase rates of HSP-70s and HSP-110 TEM image of untagged HttExon1Q48 24 h post-PreSP treatment. Scale bar: 200 nm. Sedimentation analysis of the kinetics of HttExon1Q48-CyPet fibrilization. Depicted are the ratios of the supernatant (soluble HttExon1Q48-CyPet) and the pellet (insoluble, aggregated HttExon1Q48-CyPet) at the indicated time points. The graph shows the average of two independent analyses. Analysis of the GST-cleavage reaction of GST-HttExon1Q48 via SDS–PAGE in the presence (bottom) and absence (top) of HSP-1, DNJ-13, and HSP-110. The time points are indicated on top, and the migration of the full-length and cleaved GST-HttExon1Q48 protein is indicated on the right. SDS–PAGE and Coomassie staining of all purified proteins used in this study. CD analysis of HSP-1 (red) and the point mutants HSP-1_D10S (blue) and HSP-1_K71E (green) and the blank control (black). CD analysis of HSP-110 (red) and the point mutants HSP-110_D7S (blue) and HSP-110_N578Y/E581A (green) and the blank control (black). Basal ATPase rates of HSP-1 and HSP-110 and its point mutants. The ATPase rate is indicated on the y-axis in pmol ATP/μM Hsp/min (N = 2). ATPase rates of the Hsp70s: HSP-1 (white), F44E5.4 (light gray), C12C8.1 (dark gray), and F11F1.1 (black) alone and in the presence of the DNJ proteins, DNJ-12, DNJ-13, DNJ-19, and DNJ-24 (N = 2). Sedimentation analysis of disaggregation of HttExon1Q48 fibrils by HSP-1, HSP-110, and DNJ-13 in the presence or absence of ATP after 12 h. Depicted are the ratios of the supernatant (soluble HttExon1Q48) and the pellet (insoluble, aggregated HttExon1Q48). The error bars represent the standard deviation of three independent experiments (N = 3). Download figure Download PowerPoint A distinct trimeric chaperone complex completely suppresses and reverses HttExon1Q48 fibril formation Recently, a metazoan disaggregation complex was identified that has the capacity to disaggregate amorphous aggregates as well as α-synuclein fibrils in vitro (Rampelt et al, 2012; Gao et al, 2015; Nillegoda et al, 2015). Disaggregation in higher eukaryotes requires a member of the Hsp70 chaperone family, a corresponding J-protein and a member of the Hsp110 protein family (Rampelt et al, 2012). Complete suppression of amyloid fibril formation has not been demonstrated yet. Therefore, we set out to first test the ability of human chaperones to suppress the formation of HttExon1Q48 amyloid fibrils by using the aforementioned FRET-based aggregation assay. First, we analyzed human chaperones that exhibited in vitro disaggregation activity for α−synuclein fibrils (Gao et al, 2015). This included the constitutive Hsc70 protein, the Hsp110 protein Apg2, and the class B J-protein DNAJB1. All chaperones were purified without additional tags. The purities of all chaperones and HttExon1Qn variants used in this study were assessed by SDS–PAGE and Coomassie staining (Fig EV1D). We then mixed HttExon1Q48 with the respective chaperones just prior to PreSP treatment. The addition of individual chaperones did not affect the fibrilization kinetics of HttExon1Q48 (Fig 1C). The proteins Hsc70 and DNAJB1 together, however, suppressed the aggregation of HttExon1Q48 for about 15 h. The additional presence of Apg2 (Hsp110) led to a complete suppression of HttExon1Q48 fibrilization for the entire duration of the experiment (20 h; red curve). We refer from now on only to a complete suppression if the chaperones fully inhibit any FRET signal of the HttExon1Q48-CyPet/YPet pair over the complete time period of the experiment that lasts usually between 20 and 30 h. The three chaperones functionally cooperate to suppress the HttExon1Q48 fibrilization and are from now on referred to as chaperone complex. The full suppression of aggregation required ATP and a sevenfold excess of Hsc70 over HttExon1Q48 protein. The ideal ratio between the chaperones Hsc70:DNAJB1:Apg2 for this activity is 2:1:1 (Fig 1C and data not shown). We confirmed the suppression of fibrilization by TEM and sedimentation analyses (Fig 1D and E). We analyzed the GST-cleavage reaction in a time course reaction in the presence and absence of the chaperones to exclude the possibility that the addition of the chaperones might inhibit or delay the PreSP cleavage reaction that liberates the HttExon1Q48 from the GST tag. Importantly, we did not observe an adverse effect of the chaperones on the GST-cleavage reaction (Fig EV1C). Next, we asked if the same chaperone complex could also disaggregate preformed HttExon1Q48 fibrils. We incubated Hsc70, Apg2, and DNAJB1 and ATP with HttExon1Q48 fibrils and analyzed samples after 1, 4, and 20 h by TEM. As can be seen in Fig 1G, the addition of the chaperones results in a decrease in fibrils over time. No fibrillar structures were visible at the 20-h time point. We confirmed the TEM data of the disaggregation using first a sedimentation analysis of HttExon1Q48 in the presence or absence of chaperones and ATP (Fig 1F) and second by using a filter retardation analysis that allows the detection of SDS-resistant amyloid proteins (Fig 4E). Taken together, these studies show that the chaperone complex Hsc70, Apg2, and DNAJB1 can suppress and reverse the aggregation of HttExon1Q48 in vitro. To demonstrate a physical interaction of all three chaperones with the aggregated Htt moiety, HttExon1Q97-HA aggregates from HEK293T cells were isolated via a filter retardation assay and probed with antibodies against Hsc70 (HSPA8), DNAJB1 and Apg2 (HSPA4). All three chaperones were found to be associated with the aggregated HttExon1Q97-HA moiety isolated from HEK293T cells (Fig 1H). Suppression of HttExon1Q48 fibrilization is conserved in metazoan For the subsequent studies, we employed the C. elegans orthologs of the three human chaperones HSP-1 (Hsc70), HSP-110 (Apg2), and DNJ-13 (DNAJB1) as these proteins allow us to complement the in vitro data with in vivo analyses of suppression and disaggregation of HttQn in a living animal. This is of particular importance as Huntington's disease is a late onset neurodegenerative disease and the chaperone capacity to maintain Htt proteins soluble can be studied in an aging animal model such as C. elegans (Morley et al, 2002; Kirstein et al, 2015). The chaperome of C. elegans is of similar complexity as the human chaperone, yet has the advantage of encoding only one cytosolic HSP-110 protein that allows depletion of disaggregase activity by RNAi-mediated knockdown of a single gene (Table EV1; Nikolaidis & Nei, 2004; Rampelt et al, 2012; Brehme et al, 2014). As observed for the human chaperones (Fig 1C), incubation of HttExon1Q48 with the individual nematode orthologous chaperones, HSP-1 (Hsc70), HSP-110 (Apg2), and DNJ-13 (DNAJB1) alone, did not affect the aggregation kinetics (Fig 2A). However, when HSP-1 and DNJ-13 were added together to HttExon1Q48 monomers, we detected a strong delay and overall decrease in fibrilization. The additional presence of HSP-110 led to a complete suppression of HttExon1Q48 fibrilization (Fig 2A) similar to the human proteins (Fig 1C). The suppression requires an excess of chaperones over HttExon1Q48 proteins and complete suppression could be observed at a HSP-1:HttExon1Q48 (monomer) 3.5:1 ratio or higher and is thus more efficient compared to the human chaperones that require an excess of Hsc70:HttExon1Q48 of 7:1 (Figs 1C, and 2A and B). The optimal ratio between the chaperones HSP-1:DNJ-13:HSP-110 is 2:1:1 (Fig 2B). Interestingly, sub-stoichiometric chaperone concentrations of HSP-1 and HSP-110 can be tolerated if DNJ-13 is present in excess (Fig 2B; compare orange with pink curve). TEM analysis revealed that in the presence of HSP-1, HSP-110, DNJ-13, and ATP, no fibrils or any other larger assemblies can be detected, which confirms the data obtained with the FRET assay (Fig 2A and C). Next, we wanted to test if the three chaperones could also suppress the aggregation of HttExon1 harboring a longer polyQ stretch. For that, we analyzed the aggregation of HttExon1Q75 in the presence and absence of the three chaperones over a time course of 0, 5, and 24 h using a filter retardation assay as readout. Indeed, HSP-1, DNJ-13, and HSP-110 could in addition to HttExon1Q48 also almost completely suppress the aggregation of HttExon1Q75 (Fig 2D). Figure 2. Mechanistic insights into the chaperone-mediated suppression of HttExon1Q48 fibrilization by nematode chaperones FRET analysis of the suppression activity of individual nematode chaperones and chaperone complexes of the fibrilization of HttExon1Q48. HSP-1, HSP-110, and DNJ-13 completely suppress the fibrilization analogous to the human orthologs (bright red curve; compare with Fig 1C). Analysis of various chaperone:HttExon1Q48 ratios and effect on suppression efficiency. TEM analysis of suppression of HttExon1Q48 fibrilization by HSP-1, HSP-110, and DNJ-13 taken 24 h post-PreSP treatment (right image; same as image depicted in Fig 1D). The control in the absence of chaperones is shown on the left. A scheme of the experimental outline is depicted above, and the red arrow refers to the time point of sample analysis. Scale bars: 100 nm. Analysis of chaperone-mediated suppression of HttExon1Q48 (left) and HttExon1Q75 (right) by a filter retardation analysis using an Htt antibody. Time points of analysis are indicated on the left and the absence or presence of chaperones on top of the filters. Analysis of the effect of varying concentrations of ATP and the non-hydrolyzable analog, AMP-PNP on the chaperone-mediated suppression of the fibril formation of HttExon1Q48. Addition of apyrase inhibits the suppression activity of the chaperones in a time-dependent manner. Addition of apyrase before PreSP treatment (light blue), 3 h (dark blue), or 5.5 h (purple) after PreSP treatment affects the chaperone-mediated suppression to a different extent. Single point mutations in HSP-1 that diminish the ATPase activity, D10S (green curve), and K71E (purple), yet not in HSP-110_D7S (light blue) negatively affect the suppression activity. The NEF mutant: HSP-110_N578/E581A (turquoise) negatively affects the suppression activity of the chaperones. Analysis of chaperone-mediated suppression of HttExon1Q48 fibrilization in the presence of citrate synthase (CS) aggregates. The addition of CS aggregates to the HttExon1Q48 proteins alone depicted in green does not affect the fibrilization kinetics of HttExon1Q48. Yet, the addition of CS to a sample containing HttExon1Q48 and HSP-1, HSP-110+ DNJ-13 diminishes the suppression activity of the chaperones (purple curve). The sample containing the chaperone mixture with HttExon1Q48 is depicted in red. Download figure Download PowerPoint Suppression of HttExon1Q48 requires ATP hydrolysis by HSP-1 and the NEF activity of HSP-110 The observation that HSP-110 is required for a complete suppression argues for an ATPase cycle-dependent chaperone activity to suppress the fibril formation of HttExon1Q48. Thus, we analyzed the suppression of Ht
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