An intrinsically disordered pathological prion variant Y145Stop converts into self-seeding amyloids via liquid–liquid phase separation
Edited by Michael K. Rosen, The University of Texas Southwestern Medical Center, Dallas, TX, and approved October 5, 2021 (received for review January 20, 2021)
Significance
Biology has evolved to achieve precise spatiotemporal control of crucial cellular functions through liquid–liquid phase separation of highly flexible proteins and nucleic acids into membraneless organelles. However, these liquid-like intracellular condensates can undergo irreversible phase transitions into solid-like aggregates associated with deadly human diseases. Here, we show that a pathological truncation variant of the prion protein comprising the N-terminal unstructured domain spontaneously phase-separates into liquid droplets under physiological conditions. These liquid-like condensates gradually mature into solid-like self-replicable amyloids reminiscent of toxic misfolded proteinaceous aggregates involved in transmissible prion diseases. This aberrant phase-transition propensity is suppressed in the full-length protein, which may indicate an evolutionarily conserved role of an intriguing relationship between different domains in modulating the protein phase behavior.
Abstract
Biomolecular condensation via liquid–liquid phase separation of intrinsically disordered proteins/regions (IDPs/IDRs) along with other biomolecules is proposed to control critical cellular functions, whereas aberrant phase transitions are associated with a range of neurodegenerative diseases. Here, we show that a disease-associated stop codon mutation of the prion protein (PrP) at tyrosine 145 (Y145Stop), resulting in a truncated, highly disordered, N-terminal IDR, spontaneously phase-separates into dynamic liquid-like droplets. Phase separation of this highly positively charged N-terminal segment is promoted by the electrostatic screening and a multitude of weak, transient, multivalent, intermolecular interactions. Single-droplet Raman measurements, in conjunction with an array of bioinformatic, spectroscopic, microscopic, and mutagenesis studies, revealed a highly mobile internal organization within the liquid-like condensates. The phase behavior of Y145Stop is modulated by RNA. Lower RNA:protein ratios promote condensation at a low micromolar protein concentration under physiological conditions. At higher concentrations of RNA, phase separation is abolished. Upon aging, these highly dynamic liquid-like droplets gradually transform into ordered, β-rich, amyloid-like aggregates. These aggregates formed via phase transitions display an autocatalytic self-templating characteristic involving the recruitment and binding-induced conformational conversion of monomeric Y145Stop into amyloid fibrils. In contrast to this intrinsically disordered truncated variant, the wild-type full-length PrP exhibits a much lower propensity for both condensation and maturation into amyloids, hinting at a possible protective role of the C-terminal domain. Such an interplay of molecular factors in modulating the protein phase behavior might have much broader implications in cell physiology and disease.
Data Availability
All study data are included in the article and supporting information.
Acknowledgments
We thank Prof. Witold Surewicz (Case Western Reserve University) for the kind gift of the DNA plasmid of full-length prion protein; Dr. Mahak Sharma (Indian Institute of Science Education and Research [IISER] Mohali) for confocal microscopy; Ms. Pallavi Joshi for preliminary studies; Dr. Shravan Mishra (IISER Mohali) for providing us yeast total RNA; Dr. Mily Bhattacharya (Thapar Institute), Dr. Priyanka Dogra (St. Jude Children’s Research Hospital), Dr. Anupa Majumdar (Max Planck Institute of Molecular Cell Biology and Genetics), and the members of the S.M. laboratory for critically reading this manuscript; and the anonymous reviewers for their insightful comments and valuable suggestions. This study was funded in part by the IISER Mohali, Department of Science and Technology (Nano-Mission grant to S.M. and FIST grant); Department of Biotechnology (fellowship to A. Agarwal); Council of Scientific and Industrial Research (fellowship to S.K.R.); and the Ministry of Education, Government of India (Centre of Excellence grant to S.M.).
Supporting Information
Appendix 01 (PDF)
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Movie S1.
A video of fluorescently-labeled Y145Stop droplets showing fusion events.
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© 2021. Published under the PNAS license.
Data Availability
All study data are included in the article and supporting information.
Submission history
Accepted: September 23, 2021
Published online: November 5, 2021
Published in issue: November 9, 2021
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Acknowledgments
We thank Prof. Witold Surewicz (Case Western Reserve University) for the kind gift of the DNA plasmid of full-length prion protein; Dr. Mahak Sharma (Indian Institute of Science Education and Research [IISER] Mohali) for confocal microscopy; Ms. Pallavi Joshi for preliminary studies; Dr. Shravan Mishra (IISER Mohali) for providing us yeast total RNA; Dr. Mily Bhattacharya (Thapar Institute), Dr. Priyanka Dogra (St. Jude Children’s Research Hospital), Dr. Anupa Majumdar (Max Planck Institute of Molecular Cell Biology and Genetics), and the members of the S.M. laboratory for critically reading this manuscript; and the anonymous reviewers for their insightful comments and valuable suggestions. This study was funded in part by the IISER Mohali, Department of Science and Technology (Nano-Mission grant to S.M. and FIST grant); Department of Biotechnology (fellowship to A. Agarwal); Council of Scientific and Industrial Research (fellowship to S.K.R.); and the Ministry of Education, Government of India (Centre of Excellence grant to S.M.).
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This article is a PNAS Direct Submission.
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The authors declare no competing interest.
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An intrinsically disordered pathological prion variant Y145Stop converts into self-seeding amyloids via liquid–liquid phase separation, Proc. Natl. Acad. Sci. U.S.A.
118 (45) e2100968118,
https://doi.org/10.1073/pnas.2100968118
(2021).
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