Heterologous prion-forming proteins interact to cross-seed aggregation in Saccharomyces cerevisiae

AbstractThe early stages of protein misfolding remain incompletely understood, as most mammalian proteinopathies are only detected after irreversible protein aggregates have formed. Cross-seeding, where one aggregated protein templates the misfolding of a heterologous protein, is one mechanism proposed to stimulate protein aggregation and facilitate disease pathogenesis. Here, we demonstrate the existence of cross-seeding as a crucial step in the formation of the yeast prion [PSI+], formed by the translation termination factor Sup35. We provide evidence for the genetic and physical interaction of the prion protein Rnq1 with Sup35 as a predominant mechanism leading to self-propagating Sup35 aggregation. We identify interacting sites within Rnq1 and Sup35 and determine the effects of breaking and restoring a crucial interaction. Altogether, our results demonstrate that single-residue disruption can drastically reduce the effects of cross-seeding, a finding that has important implications for human protein misfolding disorders.</jats:p

Prion-associated toxicity is rescued by elimination of cotranslational chaperones

The nascent polypeptide-associated complex (NAC) is a highly conserved but poorly characterized triad of proteins that bind near the ribosome exit tunnel. The NAC is the first cotranslational factor to bind to polypeptides and assist with their proper folding. Surprisingly, we found that deletion of NAC subunits in Saccharomyces cerevisiae rescues toxicity associated with the strong [PSI+] prion. This counterintuitive finding can be explained by changes in chaperone balance and distribution whereby the folding of the prion protein is improved and the prion is rendered nontoxic. In particular, the ribosome-associated Hsp70 Ssb is redistributed away from Sup35 prion aggregates to the nascent chains, leading to an array of aggregation phenotypes that can mimic both overexpression and deletion of Ssb. This toxicity rescue demonstrates that chaperone modification can block key steps of the prion life cycle and has exciting implications for potential treatment of many human protein conformational disorders

Rapid generation of hypomorphic mutations

Hypomorphic mutations are a valuable tool for both genetic analysis of gene function and for synthetic biology applications. However, current methods to generate hypomorphic mutations are limited to a specific organism, change gene expression unpredictably, or depend on changes in spatial-temporal expression of the targeted gene. Here we present a simple and predictable method to generate hypomorphic mutations in model organisms by targeting translation elongation. Adding consecutive adenosine nucleotides, so-called polyA tracks, to the gene coding sequence of interest will decrease translation elongation efficiency, and in all tested cell cultures and model organisms, this decreases mRNA stability and protein expression. We show that protein expression is adjustable independent of promoter strength and can be further modulated by changing sequence features of the polyA tracks. These characteristics make this method highly predictable and tractable for generation of programmable allelic series with a range of expression levels

Phase 3 Safety and Efficacy of AZD1222 (ChAdOx1 nCoV-19) Covid-19 Vaccine

BACKGROUND: The safety and efficacy of the AZD1222 (ChAdOx1 nCoV-19) vaccine in a large, diverse population at increased risk for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the United States, Chile, and Peru has not been known. METHODS: In this ongoing, double-blind, randomized, placebo-controlled, phase 3 clinical trial, we investigated the safety, vaccine efficacy, and immunogenicity of two doses of AZD1222 as compared with placebo in preventing the onset of symptomatic and severe coronavirus disease 2019 (Covid-19) 15 days or more after the second dose in adults, including older adults, in the United States, Chile, and Peru. RESULTS: A total of 32,451 participants underwent randomization, in a 2:1 ratio, to receive AZD1222 (21,635 participants) or placebo (10,816 participants). AZD1222 was safe, with low incidences of serious and medically attended adverse events and adverse events of special interest; the incidences were similar to those observed in the placebo group. Solicited local and systemic reactions were generally mild or moderate in both groups. Overall estimated vaccine efficacy was 74.0% (95% confidence interval [CI], 65.3 to 80.5; P CONCLUSIONS: AZD1222 was safe and efficacious in preventing symptomatic and severe Covid-19 across diverse populations that included older adults. (Funded by AstraZeneca and others; ClinicalTrials.gov number, NCT04516746.)

A many-analysts approach to the relation between religiosity and well-being

The relation between religiosity and well-being is one of the most researched topics in the psychology of religion, yet the directionality and robustness of the effect remains debated. Here, we adopted a many-analysts approach to assess the robustness of this relation based on a new cross-cultural dataset (N=10,535 participants from 24 countries). We recruited 120 analysis teams to investigate (1) whether religious people self-report higher well-being, and (2) whether the relation between religiosity and self-reported well-being depends on perceived cultural norms of religion (i.e., whether it is considered normal and desirable to be religious in a given country). In a two-stage procedure, the teams first created an analysis plan and then executed their planned analysis on the data. For the first research question, all but 3 teams reported positive effect sizes with credible/confidence intervals excluding zero (median reported β=0.120). For the second research question, this was the case for 65% of the teams (median reported β=0.039). While most teams applied (multilevel) linear regression models, there was considerable variability in the choice of items used to construct the independent variables, the dependent variable, and the included covariates

A Many-analysts Approach to the Relation Between Religiosity and Well-being

The relation between religiosity and well-being is one of the most researched topics in the psychology of religion, yet the directionality and robustness of the effect remains debated. Here, we adopted a many-analysts approach to assess the robustness of this relation based on a new cross-cultural dataset (N = 10, 535 participants from 24 countries). We recruited 120 analysis teams to investigate (1) whether religious people self-report higher well-being, and (2) whether the relation between religiosity and self-reported well-being depends on perceived cultural norms of religion (i.e., whether it is considered normal and desirable to be religious in a given country). In a two-stage procedure, the teams first created an analysis plan and then executed their planned analysis on the data. For the first research question, all but 3 teams reported positive effect sizes with credible/confidence intervals excluding zero (median reported β = 0.120). For the second research question, this was the case for 65% of the teams (median reported β = 0.039). While most teams applied (multilevel) linear regression models, there was considerable variability in the choice of items used to construct the independent variables, the dependent variable, and the included covariates

NAC deletion relocalizes Ssb to nascent polypeptides and away from prion aggregates.

<p>(A) Fractions from the monosome and polysome peaks of ribosome profile experiments were TCA precipitated prior to SDS-PAGE and Western blotting. More Ssb comigrated with polysomes in the <i>egd1Δegd2Δ</i> strain relative to the WT or to the whole-NAC deletion. Western blots are of the sucrose gradient fractions that contained the monosome and polysome peaks and are representative images from five independent experiments. Quantifications are from five independent experiments, and data are represented as mean ± SEM. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006431#sec017" target="_blank">Materials and Methods</a> for full computational details. (B) WT and NAC deletion strains were subjected to co-immunoprecipitation with an anti-Sup35 antibody. Equal amounts of Sup35 were immunoprecipitated across all strains. “Total” and “unbound” fractions were collected and no differences in Ssb protein expression levels were apparent (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006431#pgen.1006431.s003" target="_blank">S3 Fig</a>) Western blots are representative images from three independent experiments. All strains utilized contained [<i>RNQ</i>+] and the strong [<i>PSI</i>+] variant.</p

NAC subunits affect the yeast chaperone network by altering chaperone pools.

<p>In the presence of the NAC, RAC-Ssb receives nascent polypeptides from the NAC and assists with their folding. Both Ssa and Ssb bind to aggregated Sup35 and affect its ability to form and propagate the [<i>PSI</i>+] prion. When NAC subunits are deleted, Rac-Ssb becomes the first complete chaperone system to interact with nascent polypeptides. The unfolded state of these proteins requires more extensive interaction with RAC-Ssb, thereby sequestering cytosolic Ssb to the ribosome. Thus, less cytosolic Ssb is available to bind to monomeric or aggregated Sup35. This would lead to a relative increase in Ssa1 binding to Sup35 aggregates, which inhibits efficient monomer joining and reduces the ability of Hsp104 to cure the prion.</p

Nonsense suppression and prion status are not changed as a result of NAC subunit deletion.

<p>(A) [<i>PSI</i>+] NAC deletion strains were tested for growth on YPD and SD-Ade to monitor nonsense suppression of the <i>ade1-14</i> allele. (B) Stop codon readthrough is not significantly altered in NAC deletion strains relative to the WT. Expression of PGK(stop)LacZ fusion proteins was monitored by a β-galactosidase assay [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006431#pgen.1006431.ref050" target="_blank">50</a>]. Data are represented as mean ± SEM. (C) Ribosome profiling revealed no differences in ribosome or polysome formation between the NAC deletion strains. (D) SDD-AGE shows that Sup35 aggregates in the NAC deletion strains are not changed relative to the WT, and all strains retain the “strong” strain of the [<i>PSI</i>+] prion.</p

NAC deletion strains retain [<i>PSI</i>+] despite altered Sup35 solubility.

<p>(A) NAC deletion strains containing pCUP1-<i>SUP35-GFP</i> were grown in selective media in the presence of 50μM CuSO4. Two-dimensional images were taken with an Olympus FV1200 laser scanning microscope with a 100X oil immersion objective. The <i>egd1Δegd2Δ</i> strain showed a greater population of aggregates than the WT or other NAC deletion strains. (B) Solubility of Sup35 in [<i>PSI</i>+] lysates of indicated strains. Total (T), supernatant (S), and pellet (P) fractions were subjected to SDS-PAGE and Western blot. With endogenous levels of protein expression, all NAC deletion strains show insoluble Sup35. The <i>egd1Δegd2Δ</i> and <i>egd1Δbtt1Δ</i> strains exhibit more soluble Sup35 than the other NAC deletion strains. (C) NAC deletion strains were grown overnight in selective media; Sup35 overexpression was induced by the addition of CuSO₄ to a final concentration of 50μM at time = 0. Solubility assays were performed on cells collected at indicated timepoints following Sup35 induction. The <i>egd1Δegd2Δ</i> and <i>egd1Δbtt1Δ</i> strains exhibited a defect in joining of nascent Sup35 to existing aggregates.</p