Elimination of [PSI+] Prion and Influence on Yeast Cell Growth


 Background

Prions are proteinaceous infectious particles that act as pathogens and cause the development of lethal neurodegenerative diseases in humans and other animals. Yeast Saccharomyces cerevisiae is a widespread model system in which mechanisms of prion induction and elimination have been identified. New and safe substances and methods are being sought to cure cells of prion proteins. It is particularly important that by treating cells from prions and restoring them from the [PSI+] to the [psi−] form, the primary growth of the cells is restored. One of the main objectives of this study was to determine the growth dynamics of S. cerevisiae cells with different [PSI+] prion variants, cells that have lost [PSI+] prion variants, and cells that never had [PSI+] prion variants.
Results

In this research, we applied GuHCl and combined GuHCl and PEF treatment against [PSI+] prion. We evaluated cells culture growth dynamics – optical density and doubling time and determined that method of [PSI+] prion elimination does not affect cell doubling time. Also, we found that both elimination methods affect the optical density reached by [psi−] cells. However, the cells in which the [PSI+] prion has been eliminated by GuHCl alone are able to reach the same optical density as unaffected [psi−] cells and higher optical density than the affected [psi−] cells by GuHCl alone.
Conclusions

These findings indicate the potential long-term positive effect of [PSI+] prion on cell growth, which persists after [PSI+] removal.


Results
In this research, we applied GuHCl and combined GuHCl and PEF treatment against [PSI + ] prion. We

Background
Prions are proteinaceous infectious particles that act as pathogens and cause the development of lethal neurodegenerative diseases in humans and other animals [1]. Prions, unlike other pathogens such as bacteria, viruses, or fungi, are composed of proteins and do not contain nucleic acids. These proteins tend to aggregate and are particularly resistant to a variety of physical, chemical, and biological agents [2]. Due to these unique properties of prions, they are actively studied. Prion protein studies in humans or other mammals are severely hampered by long disease latencies (7-40 years) [3], reproductive time, ethics, and other issues. Therefore, model systems of lower eukaryotes are often used. Yeast Saccharomyces cerevisiae is a widespread model system in which protein structures, functions, and interactions are studied, and can be compared to higher eukaryotic systems due to the high conservatism of biological mechanisms [4]. Mechanisms of prion induction, propagation, aggregates formation, elimination, and interactions of different prion proteins, have been identi ed in the yeast model system [5].
At least 10 prion proteins have been identi ed in S. cerevisiae cells [6][7][8][9][10][11][12][13][14][15][16], including the [PSI + ] prion, which has been known for more than two decades and is still under intense research [17]. The altered structure of the Sup35 protein leads to protein aggregation and the emergence of the [PSI + ] phenotype [18]. The native Sup35 protein is involved in translation termination. Also, it has GTPase activity and is one of the subunits of the translation termination factor. Sup35 together with the Sup45 protein form a translation termination factor complex that recognizes STOP codons in the ribosome (responsible Sup45 subunit) and catalyzes the isolation of the synthesized polypeptide by GTP hydrolysis (responsible Sup35 subunit) [19]. The Sup35 protein is encoded by the SUP35 gene of 2058 base pairs in length, but a mutation in the SUP35 gene is not required for [PSI + ] to occur, so [PSI + ] acts as an epigenetic element that alters only the cell phenotype [20]. Complete loss of function of Sup35 protein is known to be lethal to yeast cells, but a signi cant decrease in Sup35 protein function is tolerated and S. cerevisiae cells can survive [18].
Different prion states were identi ed in both mammalian and yeast cells and were named prion variants.
These variants may form distinct phenotypes that differ in prion protein stability and frequency of spontaneous occurrence in mammals, as well as prion protein and mitotic stability and treatment e cacy in yeast [21]. It is becoming increasingly important to distinguish such prion variants because they can cause pathologically distinct infectious diseases in animals [22]. In the yeast system, the [PSI + ] prion can form at least two different variants. Different ratios of aggregated and soluble Sup35 protein are found in different [PSI + ] variants, leading to suppression of STOP codons of different potency [23]. This phenomenon is well illustrated by the nonsense ade1-14 mutation, which has a premature termination codon in S. cerevisiae cells. The truncated Ade1 protein is expressed at e cient translation termination, i.e., at a low fraction of aggregated Sup35. In this case, the cells accumulate an intermediate product of metabolism -the red pigment. Alternatively, in the presence of a large proportion of the aggregated Sup35p protein the STOP codons are omitted and the complete Ade1 protein is synthesized. Such cells remain white. Cells with a pink and white phenotype were named weak and strong prion variants, respectively [24]. Knowing that different prion variants can lead to the formation of different phenotypes, it becomes necessary to nd out whether these phenotypic differences persist after curing cells of different prion variants.
Research on prion elimination is being carried out intensively. New and safe substances and methods are being sought to cure cells of prion proteins [25]. The goal of most of these studies is to obtain the highest possible cure e ciency and the lowest probability of recurrence of the prion protein. Guanidine hydrochloride (GuHCl) is a chemical compound commonly used to eliminate prions in yeast cells. At low concentrations of GuHCl, ATPase activity of Hsp104 protein is known to be attenuated in vitro [26;27]. Hsp104 is essential for the fragmentation and proliferation of amyloid brils, while decreased activity of this protein results in the formation of long brils that can no longer enter the daughter cells [5]. In addition to prion elimination, physical factors such as the pulsed electric eld (PEF) can be used [28].
Prion proteins do not affect traditional protein degradation agents such as proteases, heat, acids, and so on [29], and the formation of prion proteins is mainly in uenced by various electrostatic interactions [30], so it is likely that PEF may increase the e cacy of treatment against [PSI + ].
The intracellular soluble/insoluble protein ratio changes in [PSI + ] cells because [PSI + ] cells tend to accumulate longer, insoluble proteins. This results in the activation of heat shock and other stress response proteins. In this case, the molecular response pathways to stress are rearranged in the [PSI + ] cell, which strongly affects cell growth [31]. It is particularly important that by treating cells from prions and restoring them from the [PSI + ] to the [psi − ] form, the primary growth of the cells is restored. This would help to understand whether the formation of a prion leads to the occurrence of irreversible changes in the cell.

Prionisation
After prion induction, cell colonies containing weak and strong prion variants were isolated (Additional le: Tabe 1). Cell colonies were sorted by diameter -up to 3 mm and more than 3 mm. In the group of up to 3 mm, the color of the colonies was approximately evenly distributed -46.68 ± 2.14% pink colonies, and 53 ± 2.14% white colonies ( Figure 1).
On the other hand, in a group of more than 3 mm, the white color of the colonies was dominant 77. The p-value determined by the Chi-squared test is p < 0.05, therefore the statistically reliable relationship between colony size and its color phenotype was determined (Additional le: Table 3 Table 2).
The weak [PSI + ] prion variant was eliminated with similar e ciency using both GuHCl alone and the combination of GuHCl and PEF (65.2 ± 11.03%). There is no statistically signi cant difference between the effect of the elimination methods used on the removal of the weak [PSI + ] prion (p > 0.05) (Additional le 2: Table 3). However, by eliminating the strong [PSI + ] prion variant, the opposite results were obtained -the strong [PSI + ] prion variant was successfully eliminated using GuHCl alone (33.63 ± 5.73%), but elimination was not successful with the combination of GuHCl and PEF (0%). In this case, a statistically reliable difference between the e ciency of the methods used was obtained (p < 0.05) (Additional le 2: Table 4). GuHCl alone reach statistically signi cantly (p < 0.05) higher OD (6.94 ± 0.32 at 48 h) than the cells treated with the combined method -GuHCl and PEF -5.87 ± 0.33 (at 48 h) (Additional le 3: Table 9).

Cell Growth Dynamics
For the cells that have never had a [PSI + ] prion the highest reached OD was 7.11 ± 0.01 (at 48 h), this value is similar to the cells in which the weak [PSI + ] prion variant was eliminated using GuHCl alone (p > 0.05) (Additional le 3: Table 10).  An additional study was performed: the cells that never had a [PSI + ] prion were exposed to both elimination methods. The highest OD was reached by the cells that were not exposed to any of the elimination methods -7.11 ± 0.01 (at 48 hours) ( Figure 5) (Additional le 3: Tables 6-8).

The growth of the cells with a strong prion variant and the cells eliminated from this variant is shown in
Neither the cells affected by GuHCl alone nor the cells affected by combined treatment of GuHCl and PEF reached the same high optical density as the unaffected cells. These differences are statistically signi cant (p < 0.05) (Additional le 3: Table 12 Table 12).
The doubling time of the cells that never had [PSI + ] prion but were affected by elimination factors were 1.85 ± 0.09 hours (affected by GuHCl) and 1.99 ± 0.05 hours (affected by GuHCl and PEF) ( Figure 6C).
Although, the difference between the doubling time of the unaffected cells that never had [PSI + ] prion and the affected cells that never had [PSI + ] prion increased by 0.02 (GuHCl) and 0.16 (GuHCl and PEF) hours, however no statistically signi cant differences (p > 0.05) were found (Additional le 4: Table 13,14). Thus, the method of treatment does not affect the cell doubling time.

Discussion
S. cerevisiae has become a widely used tool for the discovery of new drugs and their mechanisms of action. S. cerevisiae is an excellent tool for modeling protein aggregation that facilitates the study of amyloid or prion diseases in humans or other animals. One of the longest known and studied yeast prions -[PSI + ] -has become a tool for the identi cation of antiprionic compounds [25]. Developed red/white colorimetric assay allows easy separation of the cells without [PSI + ] prion from the cells with [PSI + ] prion. In addition, this assay makes it easy to distinguish the different prion variants by the cell phenotype [23].
In this study, we showed that [PSI + ] prion variants can be distinguished not only by colorimetric analysis but also by another phenotypic trait -the size of the cell colony. We found a correlation (p < 0.

Materials And Methods
The aim of this study is to determine the growth dynamics of S. . Thus, this strain can not de novo produce adenine, tryptophan, histidine, uracil, and leucine-these amino acids must be present in the culture medium. ADE1 mutant phenotypes allow the discrimination of the cells without prions or containing strong or weak prions by the color of the colonies ( Figure 7).
Phenotypes that were used in this research are listed in Table 1. Spontaneous prion induction and selection of different prion variants S. cerevisiae [psi − ] cells were plated on an agarized YPD medium (containing 2% glucose, 2% peptone, 1% yeast extract, 2% agar) and grown for 72 h at 30°C. One colony of the culture was then transferred to 5 mL of fresh YPD medium and grown overnight (~12 h) at 30°C with aeration, at 130 rpm. Overnight culture was washed with distilled water and 10 7 cells were plated on an agarized SC medium (containing 2% glucose, 0.67% yeast nitrogen base without amino acids, 2% agar, and supplemented with the appropriate amino and nucleic acids according to the genotype of the strains, except adenine) and grown for 21 days at 30°C. Grown colonies (> 400 CFU) were analyzed by diameter, then transferred to plates with agarized ¼YPD medium (the same as YPD, but with a reduced amount of yeast extract -0.25%) and were analyzed by colony color -according to these two criteria weak and strong prion variants -[PSI + ]-w, Cell growth dynamics S. cerevisiae cells variants listed in Table 1 were plated on agarized YPD medium and grown for 48 h at 30°C. One colony of the respective culture was then transferred to 5 mL of fresh ¼YPD medium and grown overnight (~12 h) at 30°C with aeration, at 130 rpm. The overnight culture was transferred to 50 mL of fresh YPD medium to the nal optic density (OD) of 0.06 AU at a 600 nm wavelength. Culture samples were taken every 3 h and cells growth was measured with a biophotometer (Eppendorf, Germany). Growth dynamics were observed until the culture reached the stationary growth phase. After growth, cells were plated on an agarized YPD medium to verify that their phenotype ([PSI] status) had not changed. All the experiments were repeated independently 3 times.
Growth rate (gr) and doubling time (DT): The growth rate (gr) was calculated using the following formula: The doubling time (DT) was calculated using the following formula:

Statistical Analysis
At least 3 independent experimental sessions were performed for each unique set of parameters. Statistical analysis was performed for all acquired data. The Chi-squared test and one-tailed t-test were used to estimate the statistical signi cance. The results were considered statistically signi cant at p < Availability of data and materials All data generated or analysed during this study are included in this published article and its supplementary information les.

Competing interests
The authors declare no con ict of interest.