Liquid-Liquid Phase Separation-mediated formation of amyloid fibrils from DcpS scavenger enzymes

Background

The Decapping Scavenger (DcpS) enzyme plays a role in mRNA processing within the cell. It is essential for cell viability and can be found in eukaryotes of varying complexity [1]. DcpS acts in the hydrolysis of CAP structures in oligonucleotides or dinucleotides resulting from mRNA degradation by the exosome. However, it has also been associated with colorectal cancer as a biomarker and proposed as a therapeutic target for myeloid leukemia [1,2]. Furthermore, point mutations in DcpS have been reported in association with certain intellectual disabilities [3].

Al-Raqad Syndrome, for instance, involves specific mutations in the DcpS gene, where patients exhibit progressive demyelination of the central and peripheral nervous systems, as well as craniofacial deformities [4]. The damaging effects on the nervous system resemble those observed in other neurodegenerative diseases in which amyloid aggregation plays a key role [3]. It is not yet clear whether DcpS-related proteinopathy results from dysregulation at the expression level or from proteoform alterations (such as inactivation or aggregate formation).

The process of protein aggregation is complex and involves the disruption of the protein’s native structural state and the formation of aggregates with diverse morphologies, structures, and solubilities [5]. Amyloid aggregation is one such pathway, characterized by the formation of insoluble, β-sheet-rich fibrils. During this process, other proteoforms are generated that possess cytotoxic potential and may catalyze cellular damage [6]. In this study, Ferenc-Mrozek and colleagues demonstrate that DcpS, under specific conditions, undergoes amyloid-type protein aggregation in vitro, and that mutant variants with higher homology to those associated with Al-Raqad syndrome exhibit an accelerated aggregation profile. Furthermore, the authors show that the studied proteins also undergo liquid–liquid phase separation.

Key Results

High-temperature effects on the structural stability of human and C. elegans DcpS regarding β-sheet-rich aggregate formation
The conformational transition of DcpS under increasing temperature was analyzed by monitoring intrinsic tryptophan fluorescence and particle size via Dynamic Light Scattering (DLS). Conformational transition and particle size increase occurred at 47 °C and 57 °C for C. elegans and human DcpS, respectively. For both proteins, these temperatures led to the formation of Thioflavin-T–responsive material. For human DcpS, circular dichroism (CD) analysis of secondary structure indicated a transition toward a β-sheet-rich spectrum, with no return to the native conformation upon cooling. In contrast, C. elegans DcpS showed an apparent increase in minima around 208 nm and 218 nm in the CD spectrum upon heating, while still remaining Thioflavin-T–responsive.

Stability analysis of DcpS at physiological temperature (37 °C)
For human DcpS, Thioflavin-T fluorescence intensity increased proportionally with protein concentration during real-time incubation, indicating that both the aggregation onset and intensity depend on concentration. For C. elegans DcpS, decreasing concentration reduced maximum fluorescence intensity without altering the aggregation onset across all conditions. FT-IR endpoint analysis confirmed structural transitions to a β-sheet pattern, and transmission electron microscopy (TEM) revealed fibrillar morphologies similar to amyloid fibers.

Droplet-like protein condensates of DcpS
The potential involvement of DcpS in phase separation processes was investigated using polyethylene glycol (PEG) as a molecular crowding agent. For both human and C. elegans DcpS, 5% PEG induced the formation of spherical and uniform phase-separated droplets, although C. elegans DcpS occasionally exhibited fused particles. Aggregation kinetics in the presence of PEG showed that 5% PEG reduced the maximum Thioflavin-T fluorescence intensity but triggered earlier aggregation, while 10% PEG led to a marked reduction in Thioflavin-T response.

Main Questions

Q1. The authors mention that both proteins undergo transitions to β-sheet–rich structures when exposed to their respective denaturation temperatures. However, when examining the spectra for C. elegans DcpS, the temperature increase appears to move the 208 nm and 218 nm peaks away from zero, suggesting that the protein is “gaining” structure rather than aggregating. How can this be interpreted, considering that deconvolution software may be losing spectral resolution?

Q2. The FT-IR analyses are excellent, but why were they not performed for the heat-induced aggregation experiments?

Q3. According to Supplementary Figure 6, the presence of 5% and 10% PEG seems to reduce aggregation as measured by Thioflavin-T fluorescence when compared to the control. How should this be interpreted given that Thioflavin-T primarily detects mature fibrils?

Minor

Q1. Circular dichroism spectra could be presented more interpretively if the authors plotted intensity at ~208 nm (y-axis) against temperature (x-axis). Since ~208 nm is particularly sensitive to β-sheet transitions, this visualization would provide a clearer and more didactic interpretation of the structural changes.
Q2 – Wouldn’t it be interesting to evaluate whether PEG alone interferes with Thioflavin T fluorescence?

Reference
1 – Grassi F, Singh M, Moussaud S, Rodriguez GV, Ali Z, Janssen K, Cheray M, Leonard E, Andersson M, de Milito A, Qian H, Walfridsson J, Höglund A. DCPS is a synthetic lethal therapeutic target in acute myeloid leukemia expressing low levels of FHIT. Leukemia. 2025 Aug;39(8):2021-2025. doi: 10.1038/s41375-025-02661-z. Epub 2025 Jun 26. PMID: 40571744; PMCID: PMC12310507.
2 – Jan Dimberg, Shamoun L, Johansson G, Landerholm K, Wågsäter D. Emerging role and clinical implication of mRNA scavenger decapping enzyme in colorectal cancer. Pathol Res Pract. 2024 Jan;253:155009. doi: 10.1016/j.prp.2023.155009. Epub 2023 Dec 5. PMID: 38064867.
3 – Iltaf Ahmed, Rebecca Buchert, Mi Zhou, Xinfu Jiao, Kirti Mittal, Taimoor I. Sheikh, Ute Scheller, Nasim Vasli, Muhammad Arshad Rafiq, M. Qasim Brohi, Anna Mikhailov, Muhammad Ayaz, Attya Bhatti, Heinrich Sticht, Tanveer Nasr, Melissa T. Carter, Steffen Uebe, André Reis, Muhammad Ayub, Peter John, Megerditch Kiledjian, John B. Vincent, Rami Abou Jamra, Mutations in DCPS and EDC3 in autosomal recessive intellectual disability indicate a crucial role for mRNA decapping in neurodevelopment, Human Molecular Genetics, Volume 24, Issue 11, 1 June 2015, Pages 3172–3180, https://doi.org/10.1093/hmg/ddv069
4 – Calista K.L. Ng, Mohammad Shboul, Valerio Taverniti, Carine Bonnard, Hane Lee, Ascia Eskin, Stanley F. Nelson, Mohammed Al-Raqad, Samah Altawalbeh, Bertrand Séraphin, Bruno Reversade, Loss of the scavenger mRNA decapping enzyme DCPS causes syndromic intellectual disability with neuromuscular defects, Human Molecular Genetics, Volume 24, Issue 11, 1 June 2015, Pages 3163–3171, https://doi.org/10.1093/hmg/ddv067
5 – Meisl G, Yang X, Hellstrand E, Frohm B, Kirkegaard JB, Cohen SI, Dobson CM, Linse S, Knowles TP. Differences in nucleation behavior underlie the contrasting aggregation kinetics of the Aβ40 and Aβ42 peptides. Proc Natl Acad Sci U S A. 2014 Jul 1;111(26):9384-9. doi: 10.1073/pnas.1401564111. Epub 2014 Jun 17. PMID: 24938782; PMCID: PMC4084462.
6 – Rinauro, D.J., Chiti, F., Vendruscolo, M. et al. Misfolded protein oligomers: mechanisms of formation, cytotoxic effects, and pharmacological approaches against protein misfolding diseases. Mol Neurodegeneration 19, 20 (2024). https://doi.org/10.1186/s13024-023-00651-2

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