Backbone resonance assignments of CPEB3 [1–120], CPEB3 [186–315], and CPEB3 [400–459]

Backbone resonance assignments of CPEB3 [1–120], CPEB3 [186–315], and CPEB3 [400–459] | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Backbone resonance assignments of CPEB3 [1–120], CPEB3 [186–315], and CPEB3 [400–459] Yujin Lee, Harunobu Saito, Masatomo So, Ayako Furukawa, Kenji Sugase This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9240109/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Cytoplasmic polyadenylation element-binding protein 3 (CPEB3) is an RNA-binding protein that is essential for long-term memory formation. Its N-terminal intrinsically disordered region (residues 1–459) exhibits high aggregation propensity and regulates the translation of specific mRNAs, including those encoding AMPA receptor subunits, through processes such as liquid–liquid phase separation and the formation of fibrillar structures. However, the molecular basis of these regulatory mechanisms remains poorly understood. In this study, we present the backbone resonance assignments of three segments within the intrinsically disordered region of CPEB3 (residues 1–120, 186–315, and 400–459). In agreement with sequence-based secondary structure predictions, the 1–120 and 400–459 segments were disordered, whereas the 186–315 segment formed a partial α-helix. CPEB3 Long-term memory IDR Protein aggregation Figures Figure 1 Figure 2 Biological context Cytoplasmic polyadenylation element-binding protein 3 (CPEB3) is an RNA-binding protein that plays a central role in long-term memory maintenance through regulation of local translation at synapses (Fioriti et al. 2015). CPEB3 belongs to the CPEB family of translational regulators that control poly(A) tail length and translation efficiency of target mRNAs in response to cellular signals (Huang et al. 2006). In neurons, CPEB3 has been implicated in the activity-dependent regulation of localized protein synthesis that is required for synaptic plasticity (Pavlopoulos et al. 2011). Under basal neuronal conditions, CPEB3 functions predominantly as a translational repressor, whereas neuronal stimulation converts it into a translational activator (Stephan et al. 2015). This functional transition is accompanied by changes in the self-association state of the protein, linking translational regulation to protein assembly (Drisaldi et al. 2015). CPEB3 consists of 716 amino acids and contains a long N-terminal intrinsically disordered region (IDR; residues 1–459), followed by a structured C-terminal RNA-binding domain comprising two RNA recognition motifs and a zinc-finger domain (Huang et al. 2006). The IDR lacks stable tertiary structure and is enriched in low-complexity sequence, features that are recognized as key determinants of phase separation and aggregation in many RNA-binding proteins (Banani et al. 2017). Within the IDR, two prion-like regions spanning residues 1–169 and 284–449 are critical for self-assembly and aggregation, indicating that multiple segments of the IDR contribute to its assembly properties (Stephan et al. 2015). Recent studies have demonstrated that the CPEB3 IDR can populate multiple assembly states, including liquid-like condensates and more rigid aggregates, depending on cellular context and regulatory inputs (Ramírez de Mingo et al. 2023). Transitions between these assembly states correlate with changes in translational activity, suggesting that structural reorganization of the IDR underlies functional switching between translational repression and activation (Drisaldi et al. 2015). Elucidating the structural basis of these transitions is therefore essential for understanding how CPEB3 couples protein assembly to translational control. However, the intrinsic disorder, conformational heterogeneity, and aggregation propensity of the IDR preclude structural characterization by X-ray crystallography or cryo-electron microscopy. In this context, solution NMR spectroscopy provides a powerful approach for residue-specific characterization of such dynamic systems (Jensen et al. 2013). We previously reported backbone resonance assignments for two segments of the CPEB3 IDR, residues 101–200 and 294–410 (Saito et al. 2025). In this study, we extend these assignments to the remaining three segments, residues 1–120, 186–315, and 400–459, thereby completing the assignments for the full-length N-terminal IDR (residues 1–459). Methods and experiments Protein expression and purification The DNA fragments encoding mouse CPEB3 [1–120], CPEB3 [186–315], and CPEB3 [400–459] were subcloned into a pET-15b vector using In-Fusion cloning. These constructs were designed to overlap with the previously analyzed segments (residues 101–200 and 294–410) by approximately 10 residues to minimize chemical shift perturbations arising from chain termini. The expression vectors encoded an N-terminal His 12 tag followed by a Tobacco Etch Virus (TEV) protease cleavage site and the target proteins. Escherichia coli strain BL21(DE3) cells were transformed with the constructed expression vectors and cultured at 37°C in M9 minimal medium containing 4 g/L D-glucose (Wako), 1 g/L NH 4 Cl (Nacalai Tesque), and 50 mg/L ampicillin (Wako). When the OD 600 reached approximately 0.8, the cells were centrifuged and resuspended in M9 minimal medium containing 2 g/L [ U - 13 C]-D-glucose (Cambridge Isotope Laboratories), 1 g/L 15 NH 4 Cl (Cambridge Isotope Laboratories), and 50 mg/L ampicillin (Wako). Protein expression was induced by the addition of IPTG (Nacalai Tesque) to a final concentration of 1 mM, followed by incubation at 20°C overnight. The harvested cells were resuspended in lysis buffer (50 mM Tris-HCl pH 8.0 at 25°C, 50 mM NaCl, 5 mM β-mercaptoethanol, 0.1 mM PMSF, and 0. % Triton X-100) and sonicated using Q-Sonica 500 (2 s ON/5 s OFF, amplitude 30, for a total of 6 minutes on ice). The cell lysate was centrifuged at 22,140 × g for 20 minutes. For CPEB3 [1–120], the supernatant was loaded onto a HisTrap HP column (Cytiva) after filtration through a 0.45 µm filter. The column was washed with 5 column volumes of equilibration buffer (50 mM Tris-HCl pH 8.0 at 25°C, 50 mM NaCl) and eluted with a 5–300 mM imidazole gradient. For CPEB3 [186–315] and CPEB3 [400–459], which were expressed as inclusion bodies, the cell pellets were washed with wash buffer (50 mM Tris-HCl pH 8.0 at 25°C, 50 mM NaCl, 2 M urea, and 0. % Triton X-100). To solubilize the inclusion bodies, the washed pellets were resuspended in denaturing buffer (50 mM Tris-HCl pH 8.0 at 25°C, 8 M urea, and 5 mM imidazole) and homogenized. The homogenate was centrifuged at 22,140 × g for 20 minutes, and the supernatant was loaded onto a Ni-NTA agarose column (Wako) after filtration through a 0.45 µm filter. The column was washed with 5 column volumes of denaturing buffer and eluted with 300 mM imidazole. The purified proteins were diluted into 50 mM Tris-HCl (pH 8.0) containing 2 M urea. For His-tag cleavage, 2 mg of His 6 -TEV protease was added to the protein samples, and the mixtures were incubated for 4 hours at 30°C. The cleaved His-tag and protease were removed by C18 reversed-phase chromatography, and the target proteins were lyophilized. Protein purity was verified by SDS-PAGE and/or MALDI-TOF MS, and the protein concentration was determined by measuring the absorbance at 280 nm using a DS-11 spectrophotometer (DeNovix). The extinction coefficients ( ε 280 ) were 5,500 M –1 cm –1 for CPEB3 [1–120], 19,480 M –1 cm –1 for CPEB3 [186–315], and 2,980 M –1 cm –1 for CPEB3 [400–459]. The final purified proteins contained an additional N-terminal Gly residue derived from the TEV cleavage site. NMR measurements and data analysis For CPEB3 [1–120], CPEB3 [186–315], and CPEB3 [400–459], the stock samples were buffer-exchanged into 50 mM MES (pH 5.5) containing % D 2 O (Sigma-Aldrich) using Zeba spin columns (Thermo Fisher). The final concentration of the NMR samples was adjusted to 0.5 mM. NMR spectra for CPEB3 [1–120] [186–315], and CPEB3 [400–459] were measured on a 600 MHz AVANCE III HD spectrometer (Bruker) at 298 K. The spectrometer was equipped with a z-gradient TCI cryogenic probe (Bruker). For sequential assignment of the protein backbone, 1 H- 15 N HSQC, HNCO, HN(CA)CO, CBCA(CO)NH, and HNCACB spectra were acquired for each construct (Ikura et al. 1990 ). 1 H chemical shifts were referenced to that of 2,2-dimethyl-2-silapentane-5-sulfonate (DSS, Wako), and 15 N and 13 C chemical shifts were referenced indirectly (Markley et al. 1998 ). All NMR data were processed using NMRPipe (Delaglio et al. 1995 ), and resonance assignments were performed using FLYA (Schmidt and Güntert 2012 ) and CcpNmr Analysis (Skinner et al. 2016 ). Secondary structure propensity calculation The secondary structure propensities of CPEB3 [1–120], CPEB3 [186–315], and CPEB3 [400–459] were calculated using the SSP program (Marsh et al. 2006 ) via NMRbox (Maciejewski et al. 2017 ) based on the measured HN, N, C’, Cα, and Cβ chemical shifts. Additionally, secondary structures of all three CPEB3 constructs were predicted using PSIPRED (Jones 1999 ) based solely on the amino acid sequences. Assignments and data deposition Figure 1 1 H- 15 N HSQC spectra of a CPEB3 [1–120], b CPEB3 [186–315], and c CPEB3 [400–459]. All spectra were acquired in 50 mM MES (pH 5.5) containing 5% D 2 O. Each peak is labeled with its amino acid type (one-letter code) and residue number. We assigned 91.7% of the backbone HN, N, C', and Cα, and Cβ resonances for CPEB3 [1–120] (Fig. 1a) and 100% for both CPEB3 [186–315] and CPEB3 [400–459] (Fig. 1b, c), excluding the HN and N resonances of 24, 17, and 2 Pro residues, respectively, and the N-terminal Gly residue derived from the TEV cleavage site. The 1 H- 15 N HSQC signals of all three CPEB3 constructs were clustered in a narrow 1 H region, which is characteristic of IDR segments. Using the assigned chemical shifts, we calculated the secondary structure propensities (SSP) for CPEB3 [1–120], CPEB3 [186–315], and CPEB3 [400–459]. The calculated SSP propensity for CPEB3 [400–459] indicated that this region is predominantly disordered. In contrast, CPEB3 [1–120] exhibited partial α-helix formation in the region Asp3–Met7. Similarly, CPEB3 [186–315] showed partial α-helix formation in the region Ala226–Ala239. The relatively low SSP scores and broad line widths observed in these regions suggest that these α-helices are transient. These experimental observations are partially consistent with the sequence-based secondary structure predictions using PSIPRED. Direct backbone assignment of the entire 459-residue N-terminal IDR of CPEB3 is highly challenging due to severe signal overlap. However, by integrating the present results with the previously assigned segments (residues 101–200 and 294–410), we have successfully completed the assignment of the full-length IDR. This achievement establishes a solid foundation for investigating the self-aggregation mechanism of CPEB3 using NMR spectroscopy. Declarations Acknowledgements This work was performed in part using the NMR spectrometers with the ultra-high magnetic fields under the Collaborative Research Program of Institute for Protein Research, Osaka University, NMRCR-25-05. This work was supported by JSPS KAKENHI (grant Numbers 22H05088 and 22KK0098), the Japan Keirin-Association grant (JKA; grant number 2023M-363), and the Kyoto University 125th Anniversary Fund “Kusunoki 125”. Author contributions Y.L. and H.S. conducted the experiments, and Y.L. wrote the manuscript. M.S. and A.F. contributed to project supervision, with K.S. serving as the lead supervisor. Data availability The backbone and Cβ chemical shift assignments for CPEB3 [1–120], CPEB3 [186–315], and CPEB3 [400–459] have been deposited in the Biological Magnetic Resonance Data Bank (BMRB, https://bmrb.io) under the accession numbers 53536, 53537, and 53538. Competing Interests The authors declare no competing interests. References Banani SF, Lee HO, Hyman AA, Rosen MK (2017) Biomolecular condensates: organizers of cellular biochemistry. Nat Rev Mol Cell Biol 18:285–298 Delaglio F, Grzesiek S, Vuister GW et al (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293 Drisaldi B, Colnaghi L, Fioriti L et al (2015) MAP2 contributes to memory retention through CPEB3-dependent control of sub-synaptic protein synthesis. Neuron 85:1011–1025 Fioriti L, Myers C, Huang Y-Y et al (2015) The persistence of hippocampal-based memory requires protein synthesis mediated by the prion-like protein CPEB3. Neuron 86:1433–1448 Ford L, Ling E, Kandel ER, Fioriti L (2019) CPEB3 inhibits translation of mRNA targets by localizing them to P bodies. Proc Natl Acad Sci U S A 116:18078–18087 Huang Y-S, Kan M-C, Lin C-L, Richter JD (2006) CPEB3 and CPEB4 in neurons: analysis of RNA-binding specificity and translational control of AMPA receptor GluR2 mRNA. EMBO J 25:4865–4876 Ikura M, Kay LE, Bax A (1990) A novel approach for sequential assignment of 1H, 13C, and 15N spectra of larger proteins: heteronuclear triple-resonance three-dimensional NMR spectroscopy. Application to calmodulin. Biochemistry 29:4659–4667 Jensen MR, Zweckstetter M, Huang J-R, Blackledge M (2013) Exploring free-energy landscapes of intrinsically disordered proteins at atomic resolution using NMR spectroscopy. Chem Rev 114:6632–6660 Jones DT (1999) Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol 292:195–202 Lin P-H, Wu G-W, Lin Y-H et al (2024) TDP-43 amyloid fibril formation via phase separation-related and -unrelated pathways. ACS Chem Neurosci 15:1584–1597 Maciejewski MW, Schuyler AD, Gryk MR et al (2017) NMRbox: A resource for biomolecular NMR computation. Biophys J 112:1529–1534 Markley JL, Bax A, Arata Y et al (1998) Recommendations for the presentation of NMR structures of proteins and nucleic acids. J Mol Biol 280:933–952 Marsh JA, Singh VK, Jia Z, Forman-Kay JD (2006) Sensitivity of secondary structure propensities to sequence differences between alpha- and gamma-synuclein: implications for fibrillation. Protein Sci 15:2795–2804 Molliex A, Temirov J, Lee J et al (2015) Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell 163:123–133 Pavlopoulos E, Trifilieff P, Chevalier A et al (2011) Neuralized1 activates CPEB3: a function for nonproteolytic ubiquitin in synaptic plasticity and memory storage. Cell 147:1369–1383 Ramírez de Mingo A, Lorente-García M et al (2023) Structural insights into the CPEB3 protein: from its intrinsically disordered region to its functional aggregates. Biomolecules 13:1620 Saito H, Lee Y, Ueno M, Sekiyama N, So M, Furukawa A, Sugase K (2025) Backbone resonance assignments of the CPEB3 [101–200] and CPEB3 [294–410]. Biomol NMR Assign 19:109–114 Schmidt E, Güntert P (2012) A new algorithm for reliable and general NMR resonance assignment. J Am Chem Soc 134:12817–12829 Skinner SP, Fogh RH, Boucher W et al (2016) CcpNmr AnalysisAssign: a flexible platform for integrated NMR analysis. J Biomol NMR 66:111–124 Stephan JS, Fioriti L, Lamba N et al (2015) The CPEB3 protein is a functional prion that interacts with the actin cytoskeleton. Cell Rep 11:1772–1785 Sun Y, Zhang S, Hu J et al (2022) Molecular structure of an amyloid fibril formed by FUS low-complexity domain. iScience 25:103701 Wittmer Y, Jami KM, Stowell RK et al (2023) Liquid droplet aging and seeded fibril formation of the Cytotoxic granule associated RNA binding protein TIA1 low complexity domain. J Am Chem Soc 145:1580–1592 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 01 May, 2026 Reviews received at journal 01 May, 2026 Reviewers agreed at journal 14 Apr, 2026 Reviewers invited by journal 14 Apr, 2026 Editor assigned by journal 27 Mar, 2026 Submission checks completed at journal 27 Mar, 2026 First submitted to journal 27 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9240109","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":622968621,"identity":"c8ccd6a2-f01c-480c-8fa6-9ed0bb0b95df","order_by":0,"name":"Yujin Lee","email":"","orcid":"","institution":"Kyoto University","correspondingAuthor":false,"prefix":"","firstName":"Yujin","middleName":"","lastName":"Lee","suffix":""},{"id":622968622,"identity":"646b5dd6-553f-45b1-bbe3-6005e3e3e08d","order_by":1,"name":"Harunobu Saito","email":"","orcid":"","institution":"Kyoto University","correspondingAuthor":false,"prefix":"","firstName":"Harunobu","middleName":"","lastName":"Saito","suffix":""},{"id":622968623,"identity":"f57a60e4-2fe8-49d3-8b80-cbfbd3fe50c7","order_by":2,"name":"Masatomo So","email":"","orcid":"","institution":"Kyoto University","correspondingAuthor":false,"prefix":"","firstName":"Masatomo","middleName":"","lastName":"So","suffix":""},{"id":622968624,"identity":"da5c6dcd-2b0d-4774-b36b-ec7fa58023e7","order_by":3,"name":"Ayako Furukawa","email":"","orcid":"","institution":"Kyoto University","correspondingAuthor":false,"prefix":"","firstName":"Ayako","middleName":"","lastName":"Furukawa","suffix":""},{"id":622968625,"identity":"9cd426a5-f59e-4c41-b38f-79a3a40970ba","order_by":4,"name":"Kenji Sugase","email":"data:image/png;base64,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","orcid":"","institution":"Kyoto University","correspondingAuthor":true,"prefix":"","firstName":"Kenji","middleName":"","lastName":"Sugase","suffix":""}],"badges":[],"createdAt":"2026-03-27 04:53:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9240109/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9240109/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107436016,"identity":"998a9158-ec79-45a6-9a58-1b8e165e046f","added_by":"auto","created_at":"2026-04-21 13:17:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1234667,"visible":true,"origin":"","legend":"\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN HSQC spectra of \u003cstrong\u003ea\u003c/strong\u003e CPEB3 [1–120], \u003cstrong\u003eb\u003c/strong\u003e CPEB3 [186–315], and \u003cstrong\u003ec\u003c/strong\u003e CPEB3 [400–459]. All spectra were acquired in 50 mM MES (pH 5.5) containing 5 % D\u003csub\u003e2\u003c/sub\u003eO. Each peak is labeled with its amino acid type (one-letter code) and residue number.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9240109/v1/be78b64532cb64ee70e320b7.png"},{"id":107436017,"identity":"cf5c6bc0-2d1b-4548-b341-9cc6eab8d1f4","added_by":"auto","created_at":"2026-04-21 13:17:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":432971,"visible":true,"origin":"","legend":"\u003cp\u003eSecondary structure analysis of \u003cstrong\u003ea \u003c/strong\u003eCPEB3 [1–120], \u003cstrong\u003eb\u003c/strong\u003e CPEB3 [186–315], and \u003cstrong\u003ec\u003c/strong\u003e CPEB3 [400–459]. The schematics at the top of each panel show the secondary structure predictions based on the amino acid sequences using PSIPRED (Jones 1999). Boxes represent α-helices. The graphs show the secondary structure propensity (SSP) calculated from the measured HN, N, C’, Cα, and Cβ chemical shifts using SSP (Marsh et al. 2006). Gray areas represent unassigned residues.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9240109/v1/b6fb8912ee5aac52325567ad.png"},{"id":107488679,"identity":"e1382e7d-43ae-4f65-ad34-b2997a93193a","added_by":"auto","created_at":"2026-04-22 02:45:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2713379,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9240109/v1/6ee7e60c-238f-46b5-ab3c-37dd8d759c5a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Backbone resonance assignments of CPEB3 [1–120], CPEB3 [186–315], and CPEB3 [400–459]","fulltext":[{"header":"Biological context","content":"\u003cp\u003eCytoplasmic polyadenylation element-binding protein 3 (CPEB3) is an RNA-binding protein that plays a central role in long-term memory maintenance through regulation of local translation at synapses (Fioriti et al. 2015). CPEB3 belongs to the CPEB family of translational regulators that control poly(A) tail length and translation efficiency of target mRNAs in response to cellular signals (Huang et al. 2006). In neurons, CPEB3 has been implicated in the activity-dependent regulation of localized protein synthesis that is required for synaptic plasticity (Pavlopoulos et al. 2011). Under basal neuronal conditions, CPEB3 functions predominantly as a translational repressor, whereas neuronal stimulation converts it into a translational activator (Stephan et al. 2015). This functional transition is accompanied by changes in the self-association state of the protein, linking translational regulation to protein assembly (Drisaldi et al. 2015).\u003c/p\u003e\n\u003cp\u003eCPEB3 consists of 716 amino acids and contains a long N-terminal intrinsically disordered region (IDR; residues 1\u0026ndash;459), followed by a structured C-terminal RNA-binding domain comprising two RNA recognition motifs and a zinc-finger domain (Huang et al. 2006). The IDR lacks stable tertiary structure and is enriched in low-complexity sequence, features that are recognized as key determinants of phase separation and aggregation in many RNA-binding proteins (Banani et al. 2017). Within the IDR, two prion-like regions spanning residues 1\u0026ndash;169 and 284\u0026ndash;449 are critical for self-assembly and aggregation, indicating that multiple segments of the IDR contribute to its assembly properties (Stephan et al. 2015).\u003c/p\u003e\n\u003cp\u003eRecent studies have demonstrated that the CPEB3 IDR can populate multiple assembly states, including liquid-like condensates and more rigid aggregates, depending on cellular context and regulatory inputs (Ram\u0026iacute;rez de Mingo et al. 2023). Transitions between these assembly states correlate with changes in translational activity, suggesting that structural reorganization of the IDR underlies functional switching between translational repression and activation (Drisaldi et al. 2015). Elucidating the structural basis of these transitions is therefore essential for understanding how CPEB3 couples protein assembly to translational control. However, the intrinsic disorder, conformational heterogeneity, and aggregation propensity of the IDR preclude structural characterization by X-ray crystallography or cryo-electron microscopy. In this context, solution NMR spectroscopy provides a powerful approach for residue-specific characterization of such dynamic systems (Jensen et al. 2013). We previously reported backbone resonance assignments for two segments of the CPEB3 IDR, residues 101\u0026ndash;200 and 294\u0026ndash;410 (Saito et al. 2025). In this study, we extend these assignments to the remaining three segments, residues 1\u0026ndash;120, 186\u0026ndash;315, and 400\u0026ndash;459, thereby completing the assignments for the full-length N-terminal IDR (residues 1\u0026ndash;459).\u003c/p\u003e"},{"header":"Methods and experiments","content":"\n\u003ch3\u003eProtein expression and purification\u003c/h3\u003e\n\u003cp\u003eThe DNA fragments encoding mouse CPEB3 [1\u0026ndash;120], CPEB3 [186\u0026ndash;315], and CPEB3 [400\u0026ndash;459] were subcloned into a pET-15b vector using In-Fusion cloning. These constructs were designed to overlap with the previously analyzed segments (residues 101\u0026ndash;200 and 294\u0026ndash;410) by approximately 10 residues to minimize chemical shift perturbations arising from chain termini. The expression vectors encoded an N-terminal His\u003csub\u003e12\u003c/sub\u003e tag followed by a Tobacco Etch Virus (TEV) protease cleavage site and the target proteins.\u003c/p\u003e \u003cp\u003e \u003cem\u003eEscherichia coli\u003c/em\u003e strain BL21(DE3) cells were transformed with the constructed expression vectors and cultured at 37\u0026deg;C in M9 minimal medium containing 4 g/L D-glucose (Wako), 1 g/L NH\u003csub\u003e4\u003c/sub\u003eCl (Nacalai Tesque), and 50 mg/L ampicillin (Wako). When the OD\u003csub\u003e600\u003c/sub\u003e reached approximately 0.8, the cells were centrifuged and resuspended in M9 minimal medium containing 2 g/L [\u003cem\u003eU\u003c/em\u003e-\u003csup\u003e13\u003c/sup\u003eC]-D-glucose (Cambridge Isotope Laboratories), 1 g/L \u003csup\u003e15\u003c/sup\u003eNH\u003csub\u003e4\u003c/sub\u003eCl (Cambridge Isotope Laboratories), and 50 mg/L ampicillin (Wako). Protein expression was induced by the addition of IPTG (Nacalai Tesque) to a final concentration of 1 mM, followed by incubation at 20\u0026deg;C overnight. The harvested cells were resuspended in lysis buffer (50 mM Tris-HCl pH 8.0 at 25\u0026deg;C, 50 mM NaCl, 5 mM β-mercaptoethanol, 0.1 mM PMSF, and 0. % Triton X-100) and sonicated using Q-Sonica 500 (2 s ON/5 s OFF, amplitude 30, for a total of 6 minutes on ice). The cell lysate was centrifuged at 22,140 \u0026times; \u003cem\u003eg\u003c/em\u003e for 20 minutes.\u003c/p\u003e \u003cp\u003eFor CPEB3 [1\u0026ndash;120], the supernatant was loaded onto a HisTrap HP column (Cytiva) after filtration through a 0.45 \u0026micro;m filter. The column was washed with 5 column volumes of equilibration buffer (50 mM Tris-HCl pH 8.0 at 25\u0026deg;C, 50 mM NaCl) and eluted with a 5\u0026ndash;300 mM imidazole gradient. For CPEB3 [186\u0026ndash;315] and CPEB3 [400\u0026ndash;459], which were expressed as inclusion bodies, the cell pellets were washed with wash buffer (50 mM Tris-HCl pH 8.0 at 25\u0026deg;C, 50 mM NaCl, 2 M urea, and 0. % Triton X-100). To solubilize the inclusion bodies, the washed pellets were resuspended in denaturing buffer (50 mM Tris-HCl pH 8.0 at 25\u0026deg;C, 8 M urea, and 5 mM imidazole) and homogenized. The homogenate was centrifuged at 22,140 \u0026times; \u003cem\u003eg\u003c/em\u003e for 20 minutes, and the supernatant was loaded onto a Ni-NTA agarose column (Wako) after filtration through a 0.45 \u0026micro;m filter. The column was washed with 5 column volumes of denaturing buffer and eluted with 300 mM imidazole. The purified proteins were diluted into 50 mM Tris-HCl (pH 8.0) containing 2 M urea.\u003c/p\u003e \u003cp\u003eFor His-tag cleavage, 2 mg of His\u003csub\u003e6\u003c/sub\u003e-TEV protease was added to the protein samples, and the mixtures were incubated for 4 hours at 30\u0026deg;C. The cleaved His-tag and protease were removed by C18 reversed-phase chromatography, and the target proteins were lyophilized. Protein purity was verified by SDS-PAGE and/or MALDI-TOF MS, and the protein concentration was determined by measuring the absorbance at 280 nm using a DS-11 spectrophotometer (DeNovix). The extinction coefficients (\u003cem\u003eε\u003c/em\u003e\u003csub\u003e280\u003c/sub\u003e) were 5,500 M\u003csup\u003e\u0026ndash;1\u003c/sup\u003e cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e for CPEB3 [1\u0026ndash;120], 19,480 M\u003csup\u003e\u0026ndash;1\u003c/sup\u003e cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e for CPEB3 [186\u0026ndash;315], and 2,980 M\u003csup\u003e\u0026ndash;1\u003c/sup\u003e cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e for CPEB3 [400\u0026ndash;459]. The final purified proteins contained an additional N-terminal Gly residue derived from the TEV cleavage site.\u003c/p\u003e\n\u003ch3\u003eNMR measurements and data analysis\u003c/h3\u003e\n\u003cp\u003eFor CPEB3 [1\u0026ndash;120], CPEB3 [186\u0026ndash;315], and CPEB3 [400\u0026ndash;459], the stock samples were buffer-exchanged into 50 mM MES (pH 5.5) containing % D\u003csub\u003e2\u003c/sub\u003eO (Sigma-Aldrich) using Zeba spin columns (Thermo Fisher). The final concentration of the NMR samples was adjusted to 0.5 mM. NMR spectra for CPEB3 [1\u0026ndash;120] [186\u0026ndash;315], and CPEB3 [400\u0026ndash;459] were measured on a 600 MHz AVANCE III HD spectrometer (Bruker) at 298 K. The spectrometer was equipped with a z-gradient TCI cryogenic probe (Bruker). For sequential assignment of the protein backbone, \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN HSQC, HNCO, HN(CA)CO, CBCA(CO)NH, and HNCACB spectra were acquired for each construct (Ikura et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). \u003csup\u003e1\u003c/sup\u003eH chemical shifts were referenced to that of 2,2-dimethyl-2-silapentane-5-sulfonate (DSS, Wako), and \u003csup\u003e15\u003c/sup\u003eN and \u003csup\u003e13\u003c/sup\u003eC chemical shifts were referenced indirectly (Markley et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). All NMR data were processed using NMRPipe (Delaglio et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1995\u003c/span\u003e), and resonance assignments were performed using FLYA (Schmidt and G\u0026uuml;ntert \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) and CcpNmr Analysis (Skinner et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSecondary structure propensity calculation\u003c/h2\u003e \u003cp\u003eThe secondary structure propensities of CPEB3 [1\u0026ndash;120], CPEB3 [186\u0026ndash;315], and CPEB3 [400\u0026ndash;459] were calculated using the SSP program (Marsh et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) via NMRbox (Maciejewski et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) based on the measured HN, N, C\u0026rsquo;, Cα, and Cβ chemical shifts. Additionally, secondary structures of all three CPEB3 constructs were predicted using PSIPRED (Jones \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) based solely on the amino acid sequences.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAssignments and data deposition\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;1\u003c/b\u003e \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN HSQC spectra of \u003cb\u003ea\u003c/b\u003e CPEB3 [1\u0026ndash;120], \u003cb\u003eb\u003c/b\u003e CPEB3 [186\u0026ndash;315], and \u003cb\u003ec\u003c/b\u003e CPEB3 [400\u0026ndash;459]. All spectra were acquired in 50 mM MES (pH 5.5) containing 5% D\u003csub\u003e2\u003c/sub\u003eO. Each peak is labeled with its amino acid type (one-letter code) and residue number.\u003c/p\u003e \u003cp\u003eWe assigned 91.7% of the backbone HN, N, C', and Cα, and Cβ resonances for CPEB3 [1\u0026ndash;120] (Fig.\u0026nbsp;1a) and 100% for both CPEB3 [186\u0026ndash;315] and CPEB3 [400\u0026ndash;459] (Fig.\u0026nbsp;1b, c), excluding the HN and N resonances of 24, 17, and 2 Pro residues, respectively, and the N-terminal Gly residue derived from the TEV cleavage site. The \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN HSQC signals of all three CPEB3 constructs were clustered in a narrow \u003csup\u003e1\u003c/sup\u003eH region, which is characteristic of IDR segments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUsing the assigned chemical shifts, we calculated the secondary structure propensities (SSP) for CPEB3 [1\u0026ndash;120], CPEB3 [186\u0026ndash;315], and CPEB3 [400\u0026ndash;459]. The calculated SSP propensity for CPEB3 [400\u0026ndash;459] indicated that this region is predominantly disordered. In contrast, CPEB3 [1\u0026ndash;120] exhibited partial α-helix formation in the region Asp3\u0026ndash;Met7. Similarly, CPEB3 [186\u0026ndash;315] showed partial α-helix formation in the region Ala226\u0026ndash;Ala239. The relatively low SSP scores and broad line widths observed in these regions suggest that these α-helices are transient. These experimental observations are partially consistent with the sequence-based secondary structure predictions using PSIPRED.\u003c/p\u003e \u003cp\u003eDirect backbone assignment of the entire 459-residue N-terminal IDR of CPEB3 is highly challenging due to severe signal overlap. However, by integrating the present results with the previously assigned segments (residues 101\u0026ndash;200 and 294\u0026ndash;410), we have successfully completed the assignment of the full-length IDR. This achievement establishes a solid foundation for investigating the self-aggregation mechanism of CPEB3 using NMR spectroscopy.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e This work was performed in part using the NMR spectrometers with the ultra-high magnetic fields under the Collaborative Research Program of Institute for Protein Research, Osaka University, NMRCR-25-05. This work was supported by JSPS KAKENHI (grant Numbers 22H05088 and 22KK0098), the Japan Keirin-Association grant (JKA; grant number 2023M-363), and the Kyoto University 125th Anniversary Fund \u0026ldquo;Kusunoki 125\u0026rdquo;.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor contributions\u003c/strong\u003e Y.L. and H.S. conducted the experiments, and Y.L. wrote the manuscript. M.S. and A.F. contributed to project supervision, with K.S. serving as the lead supervisor.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData availability\u003c/strong\u003e The backbone and C\u0026beta; chemical shift assignments for CPEB3 [1\u0026ndash;120], CPEB3 [186\u0026ndash;315], and CPEB3 [400\u0026ndash;459] have been deposited in the Biological Magnetic Resonance Data Bank (BMRB, https://bmrb.io) under the accession numbers 53536, 53537, and 53538.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting Interests\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBanani SF, Lee HO, Hyman AA, Rosen MK (2017) Biomolecular condensates: organizers of cellular biochemistry. Nat Rev Mol Cell Biol 18:285\u0026ndash;298\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDelaglio F, Grzesiek S, Vuister GW et al (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277\u0026ndash;293\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDrisaldi B, Colnaghi L, Fioriti L et al (2015) MAP2 contributes to memory retention through CPEB3-dependent control of sub-synaptic protein synthesis. Neuron 85:1011\u0026ndash;1025\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFioriti L, Myers C, Huang Y-Y et al (2015) The persistence of hippocampal-based memory requires protein synthesis mediated by the prion-like protein CPEB3. Neuron 86:1433\u0026ndash;1448\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFord L, Ling E, Kandel ER, Fioriti L (2019) CPEB3 inhibits translation of mRNA targets by localizing them to P bodies. Proc Natl Acad Sci U S A 116:18078\u0026ndash;18087\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang Y-S, Kan M-C, Lin C-L, Richter JD (2006) CPEB3 and CPEB4 in neurons: analysis of RNA-binding specificity and translational control of AMPA receptor GluR2 mRNA. EMBO J 25:4865\u0026ndash;4876\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIkura M, Kay LE, Bax A (1990) A novel approach for sequential assignment of 1H, 13C, and 15N spectra of larger proteins: heteronuclear triple-resonance three-dimensional NMR spectroscopy. Application to calmodulin. Biochemistry 29:4659\u0026ndash;4667\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJensen MR, Zweckstetter M, Huang J-R, Blackledge M (2013) Exploring free-energy landscapes of intrinsically disordered proteins at atomic resolution using NMR spectroscopy. Chem Rev 114:6632\u0026ndash;6660\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJones DT (1999) Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol 292:195\u0026ndash;202\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin P-H, Wu G-W, Lin Y-H et al (2024) TDP-43 amyloid fibril formation via phase separation-related and -unrelated pathways. ACS Chem Neurosci 15:1584\u0026ndash;1597\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaciejewski MW, Schuyler AD, Gryk MR et al (2017) NMRbox: A resource for biomolecular NMR computation. Biophys J 112:1529\u0026ndash;1534\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarkley JL, Bax A, Arata Y et al (1998) Recommendations for the presentation of NMR structures of proteins and nucleic acids. J Mol Biol 280:933\u0026ndash;952\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarsh JA, Singh VK, Jia Z, Forman-Kay JD (2006) Sensitivity of secondary structure propensities to sequence differences between alpha- and gamma-synuclein: implications for fibrillation. Protein Sci 15:2795\u0026ndash;2804\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMolliex A, Temirov J, Lee J et al (2015) Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell 163:123\u0026ndash;133\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePavlopoulos E, Trifilieff P, Chevalier A et al (2011) Neuralized1 activates CPEB3: a function for nonproteolytic ubiquitin in synaptic plasticity and memory storage. Cell 147:1369\u0026ndash;1383\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRam\u0026iacute;rez de Mingo A, Lorente-Garc\u0026iacute;a M et al (2023) Structural insights into the CPEB3 protein: from its intrinsically disordered region to its functional aggregates. Biomolecules 13:1620\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaito H, Lee Y, Ueno M, Sekiyama N, So M, Furukawa A, Sugase K (2025) Backbone resonance assignments of the CPEB3 [101\u0026ndash;200] and CPEB3 [294\u0026ndash;410]. Biomol NMR Assign 19:109\u0026ndash;114\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchmidt E, G\u0026uuml;ntert P (2012) A new algorithm for reliable and general NMR resonance assignment. J Am Chem Soc 134:12817\u0026ndash;12829\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSkinner SP, Fogh RH, Boucher W et al (2016) CcpNmr AnalysisAssign: a flexible platform for integrated NMR analysis. J Biomol NMR 66:111\u0026ndash;124\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStephan JS, Fioriti L, Lamba N et al (2015) The CPEB3 protein is a functional prion that interacts with the actin cytoskeleton. Cell Rep 11:1772\u0026ndash;1785\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun Y, Zhang S, Hu J et al (2022) Molecular structure of an amyloid fibril formed by FUS low-complexity domain. iScience 25:103701\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWittmer Y, Jami KM, Stowell RK et al (2023) Liquid droplet aging and seeded fibril formation of the Cytotoxic granule associated RNA binding protein TIA1 low complexity domain. J Am Chem Soc 145:1580\u0026ndash;1592\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biomolecular-nmr-assignments","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnmr","sideBox":"Learn more about [Biomolecular NMR Assignments](http://link.springer.com/journal/12104)","snPcode":"12104","submissionUrl":"https://submission.nature.com/new-submission/12104/3","title":"Biomolecular NMR Assignments","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"CPEB3, Long-term memory, IDR, Protein aggregation","lastPublishedDoi":"10.21203/rs.3.rs-9240109/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9240109/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCytoplasmic polyadenylation element-binding protein 3 (CPEB3) is an RNA-binding protein that is essential for long-term memory formation. Its N-terminal intrinsically disordered region (residues 1\u0026ndash;459) exhibits high aggregation propensity and regulates the translation of specific mRNAs, including those encoding AMPA receptor subunits, through processes such as liquid\u0026ndash;liquid phase separation and the formation of fibrillar structures. However, the molecular basis of these regulatory mechanisms remains poorly understood. In this study, we present the backbone resonance assignments of three segments within the intrinsically disordered region of CPEB3 (residues 1\u0026ndash;120, 186\u0026ndash;315, and 400\u0026ndash;459). In agreement with sequence-based secondary structure predictions, the 1\u0026ndash;120 and 400\u0026ndash;459 segments were disordered, whereas the 186\u0026ndash;315 segment formed a partial α-helix.\u003c/p\u003e","manuscriptTitle":"Backbone resonance assignments of CPEB3 [1–120], CPEB3 [186–315], and CPEB3 [400–459]","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-21 13:17:34","doi":"10.21203/rs.3.rs-9240109/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-01T14:28:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-01T14:27:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"262798092712559215044698309958781239135","date":"2026-04-14T12:16:30+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-14T11:25:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-27T05:12:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-27T05:12:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biomolecular NMR Assignments","date":"2026-03-27T04:49:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biomolecular-nmr-assignments","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnmr","sideBox":"Learn more about [Biomolecular NMR Assignments](http://link.springer.com/journal/12104)","snPcode":"12104","submissionUrl":"https://submission.nature.com/new-submission/12104/3","title":"Biomolecular NMR Assignments","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a2345d2a-7d93-4436-8dec-c9bf1a12e1f0","owner":[],"postedDate":"April 21st, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-01T14:28:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-01T14:27:52+00:00","index":6,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-15T10:40:51+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-21 13:17:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9240109","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9240109","identity":"rs-9240109","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}