Phase separation of a yeast prion protein promotes cellular fitness

Science ◽  
2018 ◽  
Vol 359 (6371) ◽  
pp. eaao5654 ◽  
Author(s):  
Titus M. Franzmann ◽  
Marcus Jahnel ◽  
Andrei Pozniakovsky ◽  
Julia Mahamid ◽  
Alex S. Holehouse ◽  
...  
Keyword(s):  
Phase Separation ◽  
Prion Protein ◽  
Physiological Function ◽  
Ph Sensor ◽  
Yeast Prion ◽  
Rapid Adaptation ◽  
Yeast Prions ◽  
Stress Sensors ◽  
Domain Phase

Despite the important role of prion domains in neurodegenerative disease, their physiological function has remained enigmatic. Previous work with yeast prions has defined prion domains as sequences that form self-propagating aggregates. Here, we uncovered an unexpected function of the canonical yeast prion protein Sup35. In stressed conditions, Sup35 formed protective gels via pH-regulated liquid-like phase separation followed by gelation. Phase separation was mediated by the N-terminal prion domain and regulated by the adjacent pH sensor domain. Phase separation promoted yeast cell survival by rescuing the essential Sup35 translation factor from stress-induced damage. Thus, prion-like domains represent conserved environmental stress sensors that facilitate rapid adaptation in unstable environments by modifying protein phase behavior.

scholarly journals Liquid-liquid phase separation and aggregation of the prion protein globular domain modulated by a high-affinity DNA aptamer

2019 ◽  
Author(s):  
Carolina O. Matos ◽  
Yulli M. Passos ◽  
Mariana J. do Amaral ◽  
Bruno Macedo ◽  
Matheus Tempone ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Prion Protein ◽  
Liquid Phase Separation ◽  
Cellular Prion Protein ◽  
Globular Domain ◽  
Structural Conversion ◽  
High Affinity ◽  
Liquid Liquid Phase Separation

ABSTRACTStructural conversion of cellular prion protein (PrPC) into scrapie PrP (PrPSc) and subsequent aggregation are key events for the onset of Transmissible Spongiform Encephalopathies (TSEs). Experimental evidences support the role of nucleic acids (NAs) in assisting the protein conversion process. Here, we used the SELEX methodology to identify two 25-mer DNA aptamers against the globular domain of recombinant murine PrP (rPrP90-231), namely A1 and A2. High-affinity binding of A1 and A2 to rPrP was verified by ITC. Aptamers structure was characterized by theoretical predictions, CD, NMR and SAXS, revealing that A1 adopts a hairpin conformation. Aptamer binding caused dynamic aggregation of rPrP90-231, resulting from the ability of rPrP90-231to undergo liquid-liquid phase separation (LLPS). While free rPrP90-231phase separated into large droplets, aptamer binding increased the amount but reduced the size of the condensates. Strikingly, a modified A1 aptamer that does not adopt a hairpin structure induced transition to an ordered state, suggestive of amyloid formation on the surface of the droplets. Our results describe for the first time PrP:NA interaction leading to LLPS and modulation of this effect depending on NA structure and binding stoichiometry, shedding light on the role of NAs in PrP misfolding and TSEs.

Prion protein 2 (dublet) gene (PRND): role in ovine semen capacitation, cryopreservation and fertility

2017 ◽  
Vol 29 (5) ◽  
pp. 985 ◽  
Author(s):  
L. M. Ferreira ◽  
M. Garcia-Herreros ◽  
A. Domingos ◽  
C. C. Marques ◽  
P. Mesquita ◽  
...  
Keyword(s):  
Prion Protein ◽  
Male Fertility ◽  
Gene Polymorphisms ◽  
Physiological Function ◽  
Fluorescein Isothiocyanate ◽  
Cleavage Rate ◽  
Secondary Antibodies ◽  
Fresh Semen ◽  
Low Expression

The aim of the present study was to examine the role of Doppel protein in the capacitation process and fertilising ability of both fresh and frozen–thawed (FT) spermatozoa from rams carrying different prion protein 2 (dublet) (PRND) gene polymorphisms. The detection efficacy of new anti-Doppel monoclonal antibodies and PRND mRNA quantification were also explored in ovine spermatozoa. Three different genotypes (AA, GA, GG) were identified for codon 26 of ovine PRND-c.78G>A. Using flow cytometry, a higher fluorescence was detected in fresh compared with FT sperm samples incubated with anti-Doppel primary and fluorescein isothiocyanate-conjugated secondary antibodies (P < 0.05). Capacitation was affected by semen treatment (fresh and FT) and male PRND genotype (P < 0.05). After IVF, the use of fresh semen resulted in a higher cleavage rate than the use of FT spermatozoa (P = 0.004). IVF using spermatozoa from individuals classified as carriers of the AA or GA PRND genotypes resulted in higher cleavage rates than seen using spermatozoa from GG carriers (P ≤ 0.0006). Finally, using semen from rams with the AA PRND genotype resulted in the highest Day 6 and Day 8 embryo rates (P ≤ 0.04). In conclusion, the results of the present study confirm that the identification of different PRND genotypes is important for studying the sperm capacitation process and for improving sperm cryoresistance and embryo production. Furthermore, the detection of Doppel in ejaculated ovine spermatozoa, along with its low expression after cryopreservation, strongly suggests an important physiological function of this protein in male fertility.

scholarly journals Deciphering the Role of π-Interactions in Polyelectrolyte Complexes Using Rationally Designed Peptides

Polymers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 2074
Author(s):  
Sara Tabandeh ◽  
Cristina Elisabeth Lemus ◽  
Lorraine Leon
Keyword(s):  
Hydrogen Bonding ◽  
Phase Separation ◽  
Liquid Phase ◽  
Charge Density ◽  
Secondary Structures ◽  
Liquid Phase Separation ◽  
Polyelectrolyte Complexes ◽  
Π Interactions ◽  
Liquid Liquid Phase Separation

Electrostatic interactions, and specifically π-interactions play a significant role in the liquid-liquid phase separation of proteins and formation of membraneless organelles/or biological condensates. Sequence patterning of peptides allows creating protein-like structures and controlling the chemistry and interactions of the mimetic molecules. A library of oppositely charged polypeptides was designed and synthesized to investigate the role of π-interactions on phase separation and secondary structures of polyelectrolyte complexes. Phenylalanine was chosen as the π-containing residue and was used together with lysine or glutamic acid in the design of positively or negatively charged sequences. The effect of charge density and also the substitution of fluorine on the phenylalanine ring, known to disrupt π-interactions, were investigated. Characterization analysis using MALDI-TOF mass spectroscopy, H NMR, and circular dichroism (CD) confirmed the molecular structure and chiral pattern of peptide sequences. Despite an alternating sequence of chirality previously shown to promote liquid-liquid phase separation, complexes appeared as solid precipitates, suggesting strong interactions between the sequence pairs. The secondary structures of sequence pairs showed the formation of hydrogen-bonded structures with a β-sheet signal in FTIR spectroscopy. The presence of fluorine decreased hydrogen bonding due to its inhibitory effect on π-interactions. π-interactions resulted in enhanced stability of complexes against salt, and higher critical salt concentrations for complexes with more π-containing amino acids. Furthermore, UV-vis spectroscopy showed that sequences containing π-interactions and increased charge density encapsulated a small charged molecule with π-bonds with high efficiency. These findings highlight the interplay between ionic, hydrophobic, hydrogen bonding, and π-interactions in polyelectrolyte complex formation and enhance our understanding of phase separation phenomena in protein-like structures.

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