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• W2884607067 abstract "•SRCP1 is necessary for Dictyostelium’s resistance to polyglutamine aggregation•SRCP1 is sufficient to convey resistance to polyglutamine aggregation in other organisms•SRCP1 reduces the level of SDS-insoluble but not soluble polyQ-expanded protein•Residues in SRCP1’s C terminus are necessary for its function The polyglutamine (polyQ) diseases are a group of nine neurodegenerative diseases caused by the expansion of a polyQ tract that results in protein aggregation. Unlike other model organisms, Dictyostelium discoideum is a proteostatic outlier, naturally encoding long polyQ tracts yet resistant to polyQ aggregation. Here we identify serine-rich chaperone protein 1 (SRCP1) as a molecular chaperone that is necessary and sufficient to suppress polyQ aggregation. SRCP1 inhibits aggregation of polyQ-expanded proteins, allowing for their degradation via the proteasome, where SRCP1 is also degraded. SRCP1’s C-terminal domain is essential for its activity in cells, and peptides that mimic this domain suppress polyQ aggregation in vitro. Together our results identify a novel type of molecular chaperone and reveal how nature has dealt with the problem of polyQ aggregation. The polyglutamine (polyQ) diseases are a group of nine neurodegenerative diseases caused by the expansion of a polyQ tract that results in protein aggregation. Unlike other model organisms, Dictyostelium discoideum is a proteostatic outlier, naturally encoding long polyQ tracts yet resistant to polyQ aggregation. Here we identify serine-rich chaperone protein 1 (SRCP1) as a molecular chaperone that is necessary and sufficient to suppress polyQ aggregation. SRCP1 inhibits aggregation of polyQ-expanded proteins, allowing for their degradation via the proteasome, where SRCP1 is also degraded. SRCP1’s C-terminal domain is essential for its activity in cells, and peptides that mimic this domain suppress polyQ aggregation in vitro. Together our results identify a novel type of molecular chaperone and reveal how nature has dealt with the problem of polyQ aggregation. The polyglutamine (polyQ) diseases are a group of nine inherited neurodegenerative diseases caused by the expansion of a polyQ repeat in the coding region of specific proteins (Williams and Paulson, 2008Williams A.J. Paulson H.L. Polyglutamine neurodegeneration: protein misfolding revisited.Trends Neurosci. 2008; 31: 521-528Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). PolyQ-expanded proteins misfold and lead to the formation of protein aggregates, ultimately resulting in the loss of specific types of neurons (Paulson et al., 2000Paulson H.L. Bonini N.M. Roth K.A. Polyglutamine disease and neuronal cell death.Proc. Natl. Acad. Sci. USA. 2000; 97: 12957-12958Crossref PubMed Scopus (100) Google Scholar). PolyQ aggregation is thought to be a key early event in polyQ toxicity, and suppression of polyQ aggregation is one potential way to treat these diseases. PolyQ aggregation has been studied in a wide variety of organisms, ranging from yeast to primates (Bates and Davies, 1997Bates G.P. Davies S.W. Transgenic mouse models of neurodegenerative disease caused by CAG/polyglutamine expansions.Mol. Med. Today. 1997; 3: 508-515Abstract Full Text PDF PubMed Scopus (23) Google Scholar, Kazemi-Esfarjani and Benzer, 2000Kazemi-Esfarjani P. Benzer S. Genetic suppression of polyglutamine toxicity in Drosophila.Science. 2000; 287: 1837-1840Crossref PubMed Scopus (496) Google Scholar, Meriin et al., 2002Meriin A.B. Zhang X. He X. Newnam G.P. Chernoff Y.O. Sherman M.Y. Huntington toxicity in yeast model depends on polyglutamine aggregation mediated by a prion-like protein Rnq1.J. Cell Biol. 2002; 157: 997-1004Crossref PubMed Scopus (300) Google Scholar, Santarriaga et al., 2015Santarriaga S. Petersen A. Ndukwe K. Brandt A. Gerges N. Bruns Scaglione J. Scaglione K.M. The Social Amoeba Dictyostelium discoideum Is Highly Resistant to Polyglutamine Aggregation.J. Biol. Chem. 2015; 290: 25571-25578Crossref PubMed Scopus (22) Google Scholar, Satyal et al., 2000Satyal S.H. Schmidt E. Kitagawa K. Sondheimer N. Lindquist S. Kramer J.M. Morimoto R.I. Polyglutamine aggregates alter protein folding homeostasis in Caenorhabditis elegans.Proc. Natl. Acad. Sci. USA. 2000; 97: 5750-5755Crossref PubMed Scopus (321) Google Scholar, Scherzinger et al., 1997Scherzinger E. Lurz R. Turmaine M. Mangiarini L. Hollenbach B. Hasenbank R. Bates G.P. Davies S.W. Lehrach H. Wanker E.E. Huntingtin-encoded polyglutamine expansions form amyloid-like protein aggregates in vitro and in vivo.Cell. 1997; 90: 549-558Abstract Full Text Full Text PDF PubMed Scopus (1084) Google Scholar, Tomioka et al., 2017Tomioka I. Ishibashi H. Minakawa E.N. Motohashi H.H. Takayama O. Saito Y. Popiel H.A. Puentes S. Owari K. Nakatani T. et al.Transgenic Monkey Model of the Polyglutamine Diseases Recapitulating Progressive Neurological Symptoms.eNeuro. 2017; 4 (ENEURO.0250-16.2017)Crossref PubMed Scopus (51) Google Scholar). In each case, expression of a polyQ-expanded protein results in the formation of protein aggregates, with the exception of one organism, Dictyostelium discoideum (Malinovska et al., 2015Malinovska L. Palm S. Gibson K. Verbavatz J.M. Alberti S. Dictyostelium discoideum has a highly Q/N-rich proteome and shows an unusual resilience to protein aggregation.Proc. Natl. Acad. Sci. USA. 2015; 112: E2620-E2629Crossref PubMed Scopus (57) Google Scholar, Santarriaga et al., 2015Santarriaga S. Petersen A. Ndukwe K. Brandt A. Gerges N. Bruns Scaglione J. Scaglione K.M. The Social Amoeba Dictyostelium discoideum Is Highly Resistant to Polyglutamine Aggregation.J. Biol. Chem. 2015; 290: 25571-25578Crossref PubMed Scopus (22) Google Scholar). Dictyostelium discoideum has a unique genome among sequenced organisms in that it encodes large numbers of homopolymeric amino acid tracts (Eichinger et al., 2005Eichinger L. Pachebat J.A. Glöckner G. Rajandream M.A. Sucgang R. Berriman M. Song J. Olsen R. Szafranski K. Xu Q. et al.The genome of the social amoeba Dictyostelium discoideum.Nature. 2005; 435: 43-57Crossref PubMed Scopus (1004) Google Scholar). Among the most common homopolymeric amino acid repeats are polyQ repeats, with 1,498 proteins containing 2,528 polyQ tracts of ten or more glutamines (Eichinger et al., 2005Eichinger L. Pachebat J.A. Glöckner G. Rajandream M.A. Sucgang R. Berriman M. Song J. Olsen R. Szafranski K. Xu Q. et al.The genome of the social amoeba Dictyostelium discoideum.Nature. 2005; 435: 43-57Crossref PubMed Scopus (1004) Google Scholar). Endogenous polyQ tracts in Dictyostelium reach well beyond the disease threshold (∼37Q), reaching repeat lengths of 80 glutamines, yet these proteins remain soluble (Santarriaga et al., 2015Santarriaga S. Petersen A. Ndukwe K. Brandt A. Gerges N. Bruns Scaglione J. Scaglione K.M. The Social Amoeba Dictyostelium discoideum Is Highly Resistant to Polyglutamine Aggregation.J. Biol. Chem. 2015; 290: 25571-25578Crossref PubMed Scopus (22) Google Scholar). Moreover, unlike other model organisms, overexpression of a polyQ-expanded huntingtin exon-1 construct (GFPHttex1Q103) does not result in protein aggregation (Malinovska et al., 2015Malinovska L. Palm S. Gibson K. Verbavatz J.M. Alberti S. Dictyostelium discoideum has a highly Q/N-rich proteome and shows an unusual resilience to protein aggregation.Proc. Natl. Acad. Sci. USA. 2015; 112: E2620-E2629Crossref PubMed Scopus (57) Google Scholar, Santarriaga et al., 2015Santarriaga S. Petersen A. Ndukwe K. Brandt A. Gerges N. Bruns Scaglione J. Scaglione K.M. The Social Amoeba Dictyostelium discoideum Is Highly Resistant to Polyglutamine Aggregation.J. Biol. Chem. 2015; 290: 25571-25578Crossref PubMed Scopus (22) Google Scholar). Together, this suggests that Dictyostelium encodes novel proteins or pathways to suppress polyQ aggregation. Here, we have analyzed known protein quality control pathways and show that Hsp70, autophagy, and the ubiquitin-proteasome system are not responsible for suppressing polyQ aggregation in Dictyostelium. Using a forward genetic screen, we identified a single Dictyostelium discoideum-specific gene that is necessary for suppressing polyQ aggregation. This gene encodes serine-rich chaperone protein 1 (SRCP1), a small 9.1 kDa protein that suppresses polyQ aggregation. In the presence of SRCP1, aggregation-prone polyQ proteins are degraded via the proteasome, where SRCP1 is also degraded. Upon conditions where polyQ degradation is impaired, SRCP1 also suppresses polyQ aggregation, consistent with a chaperone function for SRCP1. SRCP1 does not contain any identifiable chaperone domains but rather utilizes a C-terminal domain that resembles amyloid (pseudo-amyloid) to suppress aggregation of polyQ-expanded proteins. Together, our findings provide insight into how Dictyostelium discoideum resists polyQ aggregation and identify a new type of molecular chaperone that conveys resistance to polyQ aggregation. Among known protein quality control pathways, molecular chaperones, autophagy, and the ubiquitin-proteasome pathway assist in combating polyQ aggregation (Koyuncu et al., 2017Koyuncu S. Fatima A. Gutierrez-Garcia R. Vilchez D. Proteostasis of Huntingtin in Health and Disease.Int. J. Mol. Sci. 2017; 18: E1568Crossref PubMed Scopus (28) Google Scholar, Nath and Lieberman, 2017Nath S.R. Lieberman A.P. The Ubiquitination, Disaggregation and Proteasomal Degradation Machineries in Polyglutamine Disease.Front. Mol. Neurosci. 2017; 10: 78Crossref PubMed Scopus (25) Google Scholar). To determine if these pathways suppress polyQ aggregation in Dictyostelium, we stably expressed GFPHttex1Q103 in Dictyostelium and inhibited select protein quality control pathways. Inhibition of known protein quality control components, including Hsp70, autophagy, and the ubiquitin-proteasome system, did not lead to an accumulation of GFPHttex1Q103 puncta (Figures S1A–S1J), suggesting that other protein quality control pathways are responsible for Dictyostelium’s unusual resistance to polyQ aggregation. We next utilized a forward genetic screen to identify genes responsible for suppressing polyQ aggregation in Dictyostelium. We performed a restriction enzyme-mediated integration (REMI) screen in Dictyostelium stably expressing GFPHttex1Q103 and coupled it with high-content imaging to identify clonal isolates where a minimum of 5% of the cells contained GFPHttex1Q103 puncta (versus the ∼1% of cells that normally have aggregates). From this screen we identified a single uncharacterized Dictyostelium discoideum-specific gene responsible for suppressing GFPHttex1Q103 aggregation (Figures 1A–1D). The protein encoded by this gene is a member of a large gene family of undefined function that encodes proteins with a serine-rich domain; therefore, we named it SRCP1. To confirm that SRCP1 is responsible for suppressing GFPHttex1Q103 aggregation, we generated SRCP1 knockout Dictyostelium strains. Knocking out SRCP1 led to no obvious growth phenotypes (data not shown), while expression of GFPHttex1Q103 in SRCP1−/− cells resulted in the formation of numerous GFPHttex1Q103 puncta (Figures 1E–1H) that are insoluble via filter-trap assay, consistent with a role for SRCP1 in suppressing polyQ aggregation (Figures 1I and 1J). We next wanted to confirm that our results were due to removal of SRCP1 and not an indirect effect. To accomplish this, we transformed SRCP1−/− cells with GFPHttex1Q103 and either RFP or RFPSRCP1. Consistent with SRCP1 suppressing GFPHttex1Q103 aggregation, expression of RFPSRCP1 but not RFP alone prevented GFPHttex1Q103 aggregation (Figures 1K–1M). Together these data are consistent with SRCP1 being responsible for Dictyostelium discoideum’s unusual resistance to polyQ aggregation. We next wanted to determine SRCP1’s endogenous function in Dictyostelium. Because Dictyostelium contain an abnormally high number of homopolymeric amino acid tracts, we hypothesized that SRCP1 played a role in maintaining proteostasis in Dictyostelium’s repeat-rich proteome. To test this hypothesis, we collected lysates from two independent SRCP1−/− cell lines and performed differential centrifugation to separate soluble and insoluble fractions. Consistent with SRCP1 playing an important role in maintaining proteostasis in Dictyostelium, we observed an accumulation of ubiquitinated species in the insoluble fraction of the SRCP1−/− cells, consistent with the presence of polyubiquitinated protein aggregates (Figure 1N). In addition to polyubiquitinated proteins, the insoluble fraction was also enriched for endogenous polyQ proteins in the absence of SRCP1, suggesting that SRCP1 functions, at least in part, to maintain Dictyostelium’s polyQ-rich proteome (Figure 1O). Together these data suggest that SRCP1 plays an important role in maintaining proteostasis in Dictyostelium. Because reducing polyQ aggregation in patients is one potential therapeutic avenue for the polyQ diseases, we next assessed the ability of SRCP1 to suppress GFPHttex1Q74 aggregation in human cells. To test this, we co-transfected HEK293 cells with GFPHttex1Q74 in the presence or absence of RFPSRCP1 and assessed the ability of RFPSRCP1 to prevent GFPHttex1Q74 aggregation. Expression of GFPHttex1Q74 resulted in the formation of GFPHttex1Q74 puncta, while co-expression of RFPSRCP1 led to a dramatic decrease in the number and size of GFPHttex1Q74 puncta (Figures 2A–2C). Importantly, expression of RFPSRCP1 alone did not display any toxicity (Figure S2). PolyQ aggregates migrate more slowly in SDS-PAGE gels and form high-molecular-weight aggregates (Scherzinger et al., 1999Scherzinger E. Sittler A. Schweiger K. Heiser V. Lurz R. Hasenbank R. Bates G.P. Lehrach H. Wanker E.E. Self-assembly of polyglutamine-containing huntingtin fragments into amyloid-like fibrils: implications for Huntington’s disease pathology.Proc. Natl. Acad. Sci. USA. 1999; 96: 4604-4609Crossref PubMed Scopus (577) Google Scholar). To determine if SRCP1 suppressed the formation of polyQ aggregates, we next analyzed the amount of GFPHttex1Q74 aggregates in the presence or absence of RFPSRCP1. Expression of GFPHttex1Q74 resulted in the presence of both soluble GFPHttex1Q74 and GFPHttex1Q74 aggregates (Figure 2D). Co-expression of RFPSRCP1 with GFPHttex1Q74 greatly reduced the amount of GFPHttex1Q74 aggregates as measured by SDS-PAGE, filter-trap assay, and confocal microscopy, while having no effect on the levels of monomeric, soluble GFPHttex1Q74 (Figures 2D–2I). Consistent with SRCP1 having no effect on soluble polyQ protein, SRCP1 did not alter the levels of GFPHttex1Q23, suggesting that SRCP1 selectively affects polyQ-expanded proteins that have a propensity to form aggregates (aggregation-prone) (Figures 2J and 2K). Because the polyQ diseases are neurodegenerative diseases, we next tested if SRCP1 prevented polyQ aggregation in human neurons. To this end, induced pluripotent stem cell (iPSC)-derived neurons were transfected with either GFPHttex1Q74 alone or GFPHttex1Q74 and RFPSRCP1. Similar to HEK293 cells, co-expression of RFPSRCP1 led to a dramatic decrease in GFPHttex1Q74 puncta (Figures 2L and 2M). In addition to human neurons, we also assessed SRCP1’s ability to suppress polyQ aggregation in an intact animal. We injected zebrafish embryos with RNA encoding either GFPHttex1Q74 or GFPHttex1Q74 and RFPSRCP1 and counted the number of GFPHttex1Q74 puncta in spinal-cord neurons. Similar to human cells, co-expression of RFPSRCP1 in zebrafish led to a significant reduction of GFPHttex1Q74 puncta, consistent with SRCP1 suppressing GFPHttex1Q74 aggregation (Figures 2N and 2O). Together, these data demonstrate that SRCP1 is sufficient to suppress polyQ aggregation in human cells and in zebrafish neurons. In human cells co-expression of RFPSRCP1 and GFPHttex1Q74 led to a dramatic decrease in GFPHttex1Q74 aggregates but did not cause a corresponding increase in soluble GFPHttex1Q74 levels (Figures 2D and 2F). This suggests that SRCP1 was either increasing the clearance of GFPHttex1Q74 aggregates or promoting degradation of GFPHttex1Q74 that misfolds prior to aggregation. One well-established route for the clearance of both soluble and aggregated polyQ is autophagy (Koyuncu et al., 2017Koyuncu S. Fatima A. Gutierrez-Garcia R. Vilchez D. Proteostasis of Huntingtin in Health and Disease.Int. J. Mol. Sci. 2017; 18: E1568Crossref PubMed Scopus (28) Google Scholar). To determine if expression of SRCP1 resulted in autophagic degradation of polyQ protein, we transfected HEK293 cells with GFPHttex1Q74 in the presence or absence of RFPSRCP1 and treated with the autophagy inhibitor 3-MA 24 hr post-transfection. Consistent with previous publications, treatment with 3-MA led to a significant increase in soluble and aggregated GFPHttex1Q74 (Qin et al., 2003Qin Z.H. Wang Y. Kegel K.B. Kazantsev A. Apostol B.L. Thompson L.M. Yoder J. Aronin N. DiFiglia M. Autophagy regulates the processing of amino terminal huntingtin fragments.Hum. Mol. Genet. 2003; 12: 3231-3244Crossref PubMed Scopus (233) Google Scholar, Ravikumar et al., 2002Ravikumar B. Duden R. Rubinsztein D.C. Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy.Hum. Mol. Genet. 2002; 11: 1107-1117Crossref PubMed Scopus (930) Google Scholar). However, under conditions of autophagy inhibition, co-expression of RFPSRCP1 did not lead to a significant increase in soluble GFPHttex1Q74 levels, suggesting that SRCP1 does not promote GFPHttex1Q74 clearance via autophagy (Figures 3A–3C). Because inhibiting autophagy did not increase levels of soluble GFPHttex1Q74 in the presence of SRCP1, we next turned our attention to the proteasome, the other major route for GFPHttex1Q74 degradation (Michalik and Van Broeckhoven, 2004Michalik A. Van Broeckhoven C. Proteasome degrades soluble expanded polyglutamine completely and efficiently.Neurobiol. Dis. 2004; 16: 202-211Crossref PubMed Scopus (48) Google Scholar). To determine if the presence of SRCP1 resulted in proteasomal degradation of GFPHttex1Q74, HEK293 cells were transfected with GFPHttex1Q74 in the presence or absence of RFPSRCP1 and treated with proteasome inhibitor 24 hr post-transfection. Proteasome inhibition led to increased levels of both soluble and aggregated GFPHttex1Q74 (Michalik and Van Broeckhoven, 2004Michalik A. Van Broeckhoven C. Proteasome degrades soluble expanded polyglutamine completely and efficiently.Neurobiol. Dis. 2004; 16: 202-211Crossref PubMed Scopus (48) Google Scholar, Miller et al., 2005Miller V.M. Nelson R.F. Gouvion C.M. Williams A. Rodriguez-Lebron E. Harper S.Q. Davidson B.L. Rebagliati M.R. Paulson H.L. CHIP suppresses polyglutamine aggregation and toxicity in vitro and in vivo.J. Neurosci. 2005; 25: 9152-9161Crossref PubMed Scopus (193) Google Scholar, Waelter et al., 2001Waelter S. Boeddrich A. Lurz R. Scherzinger E. Lueder G. Lehrach H. Wanker E.E. Accumulation of mutant huntingtin fragments in aggresome-like inclusion bodies as a result of insufficient protein degradation.Mol. Biol. Cell. 2001; 12: 1393-1407Crossref PubMed Scopus (528) Google Scholar) (Figures 3D–3F). However, co-expression of RFPSRCP1 in the presence of proteasome inhibition led to an even further increase in the amount of soluble GFPHttex1Q74 when compared to proteasome inhibition alone. This indicates that in the presence of SRCP1, aggregation-prone, polyQ-expanded protein was being degraded by the proteasome (Figures 3D–3F). Furthermore, in the presence of proteasome inhibition, SRCP1 prevented polyQ aggregation, consistent with SRCP1 functioning as a molecular chaperone (Figures 3D–3F). Together, these data demonstrate that the expression of SRCP1 results in the degradation of aggregation-prone GFPHttex1Q74 via the proteasome, but not the lysosome, and that SRCP1 acts as a molecular chaperone preventing GFPHttex1Q74 aggregation upon conditions where GFPHttex1Q74 degradation is impaired. In the experiments where RFPSRCP1 and GFPHttex1Q74 were co-transfected, we observed that RFPSRCP1 levels were difficult to detect. However, in experiments where RFPSRCP1 was transfected alone, we could easily detect RFPSRCP1. This led us to hypothesize that GFPHttex1Q74 accelerated the turnover of RFPSRCP1. We next analyzed levels of RFPSRCP1 in human cells expressing GFPHttex1Q74, RFPSRCP1, or GFPHttex1Q74 and RFPSRCP1, and found that the presence of GFPHttex1Q74 led to a dramatic decrease in RFPSRCP1 levels (Figures 4A and 4B ). To determine if RFPSRCP1 is degraded by the proteasome, we transfected human cells with RFPSRCP1 in the presence or absence of proteasome inhibition and assessed levels of RFPSRCP1. Proteasome inhibition led to a marked stabilization of RFPSRCP1, consistent with RFPSRCP1 being degraded by the proteasome (Figures 4C and 4D). Together, these data demonstrate that RFPSRCP1 is degraded by the proteasome in a manner that is stimulated by the presence of GFPHttex1Q74. SRCP1’s ability to suppress polyQ aggregation in the presence of proteasome inhibition is consistent with SRCP1 being a molecular chaperone. SRCP1 does not contain a canonical chaperone domain; however, it does contain a serine-rich N terminus. Because DNAJB6 utilizes a serine-rich domain to suppress polyQ aggregation (Kakkar et al., 2016Kakkar V. Månsson C. de Mattos E.P. Bergink S. van der Zwaag M. van Waarde M.A.W.H. Kloosterhuis N.J. Melki R. van Cruchten R.T.P. Al-Karadaghi S. et al.The S/T-Rich Motif in the DNAJB6 Chaperone Delays Polyglutamine Aggregation and the Onset of Disease in a Mouse Model.Mol. Cell. 2016; 62: 272-283Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), we hypothesized that SRCP1’s serine-rich N terminus may be important for suppressing polyQ aggregation. To test this, we generated constructs where all serine and threonine residues in SRCP1’s N terminus were mutated to alanine (Figure 5A). However, mutation of SRCP1’s serine and threonine residues (RFPSRCP1ST1) did not disrupt SRCP1’s ability to suppress polyQ aggregation (Figures 5B–5D). Together, these data demonstrate that SRCP1’s serine-rich domain is dispensable for its ability to suppress polyQ aggregation. Because mutating ∼40% of the residues in SRCP1’s N-terminal serine-rich domain did not cause any measurable defect in SRCP1 activity, we next turned our attention to SRCP1’s C-terminal region to identify a region that may be important for SRCP1’s chaperone function. One striking feature of SRCP1’s C-terminal region is a highly hydrophobic, glycine-rich region that resembles amyloid. Peptides that resemble amyloid can form mixed amyloid with amyloid-forming proteins and influence amyloid formation (Cheng et al., 2012Cheng P.N. Liu C. Zhao M. Eisenberg D. Nowick J.S. Amyloid β-sheet mimics that antagonize protein aggregation and reduce amyloid toxicity.Nat. Chem. 2012; 4: 927-933Crossref PubMed Scopus (176) Google Scholar, Sato et al., 2006Sato T. Kienlen-Campard P. Ahmed M. Liu W. Li H. Elliott J.I. Aimoto S. Constantinescu S.N. Octave J.N. Smith S.O. Inhibitors of amyloid toxicity based on beta-sheet packing of Abeta40 and Abeta42.Biochemistry. 2006; 45: 5503-5516Crossref PubMed Scopus (171) Google Scholar). To determine if SRCP1 encodes any predicted amyloidogenic domains, we utilized in silico approaches, including Tango, FISH Amyloid, FoldAmyloid, PASTA 2.0, and AmylPred2, and identified two potential amyloid-forming regions in SRCP1’s C-terminal region, which we termed pseudo-amyloid domains (Figures 6A and 6B ; Figure S3). We next mutated regions within SRCP1’s pseudo-amyloid domains (RFPSRCP161–70A and RFPSRCP171–80A) and determined their ability to suppress GFPHttex1Q74 aggregation. Consistent with SRCP1’s first pseudo-amyloid domain being important for suppressing polyQ aggregation, the RFPSRCP161–70A mutant lost the ability to suppress GFPHttex1Q74 aggregation, while the RFPSRCP171–80A mutant retained full activity (Figures 6C–6E). This is consistent with amino acids 61–70 but not 71–80 of SRCP1 containing critical residues for SRCP1 function (Figures 6C–6E). To gain more detailed insight into residues important for SRCP1 function, we next mutated individual amino acid residues in SRCP1 from 61–70 to alanine and determined their ability to suppress GFPHttex1Q74 aggregation via fluorescence microscopy. Two individual point mutations, RFPSRCP1V65A and RFPSRCP1I69A, resulted in a decrease in SRCP1 function, consistent with an important role for suppressing polyQ aggregation (data not shown; Figure 6F). We next wanted to determine if SRCP1 could directly suppress polyQ aggregation in vitro. Because we were unable to generate soluble recombinant SRCP1 protein, we generated a 20-amino-acid peptide that mimics SRCP1’s C-terminal pseudo-amyloid region. This peptide remained soluble in vitro (data not shown) and was sufficient to suppress aggregation of HttQ46 in vitro as measured by Thioflavin-T fluorescence (Figure 6G). We next wanted to confirm that our SRCP1-derived peptide was inhibiting HttQ46 aggregation and not disrupting the binding of Thioflavin-T to HttQ46 aggregates. To accomplish this, we analyzed HttQ46 aggregate formation at a 5-hr time point by electron microscopy (EM) and found that the addition of the SRCP1 peptide decreased HttQ46 fiber formation and led to the appearance of spherical structures similar to previously described soluble oligomers (Tsigelny et al., 2008Tsigelny I.F. Crews L. Desplats P. Shaked G.M. Sharikov Y. Mizuno H. Spencer B. Rockenstein E. Trejo M. Platoshyn O. et al.Mechanisms of hybrid oligomer formation in the pathogenesis of combined Alzheimer’s and Parkinson’s diseases.PLoS ONE. 2008; 3: e3135Crossref PubMed Scopus (216) Google Scholar) (Figure 6H). The presence of these spherical structures and decrease in fibers also correlated with a decrease in HttQ46 aggregates as measured by filter-trap assay and an increase in smaller species via dynamic light scattering (DLS) (Figures 6I and 6J). This, coupled with the observation that the 1.5:1 and 3:1 molar ratios of peptide to HttQ46 resulted in a delay but not prevention of HttQ46 aggregation (Figure 6G), led us to hypothesize that the SRCP1 peptide delayed but did not prevent HttQ46 aggregation. To test this hypothesis, we analyzed HttQ46 aggregation in the presence or absence of the SRCP1 peptide after a 72-hr incubation. Consistent with the SRCP1 peptide delaying but not preventing aggregation, HttQ46 aggregates were detected by EM and filter-trap analysis (Figures 6K and 6L), and the smaller species observed via DLS at 5 hr were no longer detectable at 72 hr (Figure 6M). To further analyze SRCP1’s pseudo-amyloid domain in vitro, we tested peptides of the two predicted individual domains (amino acids 61–70 or 71–80) within the SRCP1 peptide. Consistent with our cell data, a peptide that encodes amino acids 61–70 was sufficient to suppress HttQ46 aggregation in vitro, whereas a peptide that consists of amino acids 71–80 did not (Figure 6N). Similarly, a peptide consisting of amino acids 61–70 with V65 and I69 mutated to alanine resulted in a loss in activity in accordance with the fluorescent microscopy data in cells (Figures 6F and 6O). Together, these data support a role for SRCP1’s C-terminal pseudo-amyloid domain in contributing to SRCP1’s ability to suppress polyQ aggregation. Because SRCP1 suppressed polyQ aggregation, we next wanted to determine if SRCP1 reversed disease phenotypes associated with huntingtin aggregation. One such phenotype is the degeneration of neurons, which is believed to contribute to the early pathology of Huntington’s disease (HD) (DiFiglia et al., 1997DiFiglia M. Sapp E. Chase K.O. Davies S.W. Bates G.P. Vonsattel J.P. Aronin N. Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain.Science. 1997; 277: 1990-1993Crossref PubMed Scopus (2313) Google Scholar, Li et al., 2001Li H. Li S.-H. Yu Z.-X. Shelbourne P. Li X.-J. Huntingtin aggregate-associated axonal degeneration is an early pathological event in Huntington’s disease mice.J. Neurosci. 2001; 21: 8473-8481Crossref PubMed Google Scholar). To this end, we utilized two independent HD iPSC-derived neurons with either 60 or 180 glutamines (HD iPSC Consortium, 2012HD iPSC ConsortiumInduced pluripotent stem cells from patients with Huntington’s disease show CAG-repeat-expansion-associated phenotypes.Cell Stem Cell. 2012; 11: 264-278Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar). Neurons derived from HD iPSCs exhibit shortened neurites (Chae et al., 2012Chae J.-I. Kim D.-W. Lee N. Jeon Y.-J. Jeon I. Kwon J. Kim J. Soh Y. Lee D.-S. Seo K.S. et al.Quantitative proteomic analysis of induced pluripotent stem cells derived from a human Huntington’s disease patient.Biochem. J. 2012; 446: 359-371Crossref PubMed Scopus (77) Google Scholar, Kaye and Finkbeiner, 2013Kaye J.A. Finkbeiner S. Modeling Huntington’s disease with induced pluripotent stem cells.Mol. Cell. Neurosci. 2013; 56: 50-64Crossref PubMed Scopus (58) Google Scholar, HD iPSC Consortium, 2012HD iPSC ConsortiumInduced pluripotent stem cells from patients with Huntington’s disease show CAG-repeat-expansion-associated phenotypes.Cell Stem Cell. 2012; 11: 264-278Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar), so we tested if SRCP1 could reverse this phenotype. We expressed RFPSRCP1 in HD iPSC-derived neurons and quantified the length of the neurites in the presence or absence of RFPSRCP1. Overexpression of SRCP1 significantly increased neurite length and reversed the phenotype seen in the untreated HD iPSC-derived neurons (Figures 7A and 7B ). This is consistent with SRCP1 preventing aggregation-related phenotypes and suggests that SRCP1 may prevent toxic events associated with human HD. The model organism Dictyostelium discoideum is a proteostatic outlier that naturally encodes for a la" @default.
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