Blessings in disguise: biological benefits of prion-like mechanisms

Prions and amyloids are often associated with disease, but related mechanisms provide beneficial functions in nature. Prion-like mechanisms (PriLiMs) are found from bacteria to humans, where they alter the biological and physical properties of prion-like proteins. We have proposed that prions can serve as heritable bet-hedging devices for diversifying microbial phenotypes. Other, more dynamic proteinaceous complexes may be governed by similar self-templating conformational switches. Additional PriLiMs continue to be identified and many share features of self-templating protein structure (including amyloids) and dependence on chaperone proteins. Here, we discuss several PriLiMs and their functions, intending to spur discussion and collaboration on the subject of beneficial prion-like behaviors.National Science Foundation (U.S.) (NSF Fellowship)Howard Hughes Medical Institute (Investigator

Luminidependens (LD) is an Arabidopsis protein with prion behavior

Prion proteins provide a unique mode of biochemical memory through self-perpetuating changes in protein conformation and function. They have been studied in fungi and mammals, but not yet identified in plants. Using a computational model, we identified candidate prion domains (PrDs) in nearly 500 plant proteins. Plant flowering is of particular interest with respect to biological memory, because its regulation involves remembering and integrating previously experienced environmental conditions. We investigated the prion-forming capacity of three prion candidates involved in flowering using a yeast model, where prion attributes are well defined and readily tested. In yeast, prions heritably change protein functions by templating monomers into higher-order assemblies. For most yeast prions, the capacity to convert into a prion resides in a distinct prion domain. Thus, new prion-forming domains can be identified by functional complementation of a known prion domain. The prion-like domains (PrDs) of all three of the tested proteins formed higher-order oligomers. Uniquely, the Luminidependens PrD (LDPrD) fully replaced the prion-domain functions of a well-characterized yeast prion, Sup35. Our results suggest that prion-like conformational switches are evolutionarily conserved and might function in a wide variety of normal biological processes.Howard Hughes Medical InstituteG. Harold and Leila Y. Mathers FoundationEleanor Schwartz Charitable FoundationNational Science Foundation (U.S.). Graduate Research Fellowship Progra

A genetic platform for the study of protein perturbation and prion-based inheritance

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2017.Cataloged from PDF version of thesis.Includes bibliographical references.Proteins mediate every cellular process. In order for life to exist, each protein must be finely tuned to carry out its function at the proper time and place. Because the environment is dynamic and often unpredictable, the regulation of proteins must be responsive to environmental stimuli. Mutations, age, and severe or repeated insults can decrease the quality of protein regulation, leading to disease. The study of protein regulation and its dysfunction in disease are of vital importance. Regulatory and disease phenomena involving protein assembly or aggregation are common but currently understudied on account of their intractability with existing techniques. In order to equip scientists with better tools to tackle these difficult phenomena, my collaborators Ahmad Khalil and Szilvia Kiriakov (Boston University) and I developed the yTRAP platform (standing for: yeast transcriptional reporters of aggregating proteins). yTRAP couples the activity of a synthetic transcriptional activator to a protein's solubility. It enables sensitive measurement of a protein's state within a eukaryotic cellular context, preserving complex interactions that may be lost using in vitro techniques. yTRAP can be measured in high throughput to enable large studies, screens, and selections on aggregation phenomena. The reporter output is modular and can be customized to desired purposes and measurement modalities. Using a fluorescent output, the signal from yTRAP is readily quantifiable. The combination of these desirable properties enables many kinds of previously-impossible studies. Furthermore, because of its exquisite sensitivity, yTRAP can be used to broadly screen for protein perturbation beyond the context of aggregation. For example, it can report on alterations in protein localization, binding partners, or degradation. I applied yTRAP to track yeast prions, which have previously been difficult to study due to a lack of simple and reliable reporters. Prions are protein-based elements of inheritance that have profound implications for the evolution of single-celled organisms. I first utilized yTRAP to identify factors that faithfully switch prion states on and off, thus proving that prion states can be deterministically regulated. I used these factors to create new cellular tools out of prions: 1) a memory device that records elevated temperatures experienced by a yeast population, and 2) an anti-prion drive that eliminates prions from mating partners and progeny. Separately, I conducted an ecological study into the yeast prion [SW*]. I found that [SWI*] confers a 'pioneering' cellular program that encourages migration and diverse mating partners. Loss of the prion confers a protective 'settled' cellular program with growth and survivability advantages. yTRAP greatly facilitated this study through reliable tracking of the prion state. Prion-like phenomena are now ripe for study with yTRAP.by Gregory A. Newby.Ph. D

Efficient C•G-to-G•C base editors developed using CRISPRi screens, target-library analysis, and machine learning

Programmable C•G-to-G•C base editors (CGBEs) have broad scientific and therapeutic potential, but their editing outcomes have proved difficult to predict and their editing efficiency and product purity are often low. We describe a suite of engineered CGBEs paired with machine learning models to enable efficient, high-purity C•G-to-G•C base editing. We performed a CRISPR interference (CRISPRi) screen targeting DNA repair genes to identify factors that affect C•G-to-G•C editing outcomes and used these insights to develop CGBEs with diverse editing profiles. We characterized ten promising CGBEs on a library of 10,638 genomically integrated target sites in mammalian cells and trained machine learning models that accurately predict the purity and yield of editing outcomes (R = 0.90) using these data. These CGBEs enable correction to the wild-type coding sequence of 546 disease-related transversion single-nucleotide variants (SNVs) with >90% precision (mean 96%) and up to 70% efficiency (mean 14%). Computational prediction of optimal CGBE-single-guide RNA pairs enables high-purity transversion base editing at over fourfold more target sites than achieved using any single CGBE variant.US NIH (Grants F31NS115380, U01AI142756, UG3AI150551, RM1HG009490, R35GM118062, R35GM138167 and P30CA072720

Cross-Kingdom Chemical Communication Drives a Heritable, Mutually Beneficial Prion-Based Transformation of Metabolism

SummaryIn experimental science, organisms are usually studied in isolation, but in the wild, they compete and cooperate in complex communities. We report a system for cross-kingdom communication by which bacteria heritably transform yeast metabolism. An ancient biological circuit blocks yeast from using other carbon sources in the presence of glucose. [GAR+], a protein-based epigenetic element, allows yeast to circumvent this “glucose repression” and use multiple carbon sources in the presence of glucose. Some bacteria secrete a chemical factor that induces [GAR+]. [GAR+] is advantageous to bacteria because yeast cells make less ethanol and is advantageous to yeast because their growth and long-term viability is improved in complex carbon sources. This cross-kingdom communication is broadly conserved, providing a compelling argument for its adaptive value. By heritably transforming growth and survival strategies in response to the selective pressures of life in a biological community, [GAR+] presents a unique example of Lamarckian inheritance

Health-status outcomes with invasive or conservative care in coronary disease

BACKGROUND In the ISCHEMIA trial, an invasive strategy with angiographic assessment and revascularization did not reduce clinical events among patients with stable ischemic heart disease and moderate or severe ischemia. A secondary objective of the trial was to assess angina-related health status among these patients. METHODS We assessed angina-related symptoms, function, and quality of life with the Seattle Angina Questionnaire (SAQ) at randomization, at months 1.5, 3, and 6, and every 6 months thereafter in participants who had been randomly assigned to an invasive treatment strategy (2295 participants) or a conservative strategy (2322). Mixed-effects cumulative probability models within a Bayesian framework were used to estimate differences between the treatment groups. The primary outcome of this health-status analysis was the SAQ summary score (scores range from 0 to 100, with higher scores indicating better health status). All analyses were performed in the overall population and according to baseline angina frequency. RESULTS At baseline, 35% of patients reported having no angina in the previous month. SAQ summary scores increased in both treatment groups, with increases at 3, 12, and 36 months that were 4.1 points (95% credible interval, 3.2 to 5.0), 4.2 points (95% credible interval, 3.3 to 5.1), and 2.9 points (95% credible interval, 2.2 to 3.7) higher with the invasive strategy than with the conservative strategy. Differences were larger among participants who had more frequent angina at baseline (8.5 vs. 0.1 points at 3 months and 5.3 vs. 1.2 points at 36 months among participants with daily or weekly angina as compared with no angina). CONCLUSIONS In the overall trial population with moderate or severe ischemia, which included 35% of participants without angina at baseline, patients randomly assigned to the invasive strategy had greater improvement in angina-related health status than those assigned to the conservative strategy. The modest mean differences favoring the invasive strategy in the overall group reflected minimal differences among asymptomatic patients and larger differences among patients who had had angina at baseline

Initial invasive or conservative strategy for stable coronary disease

BACKGROUND Among patients with stable coronary disease and moderate or severe ischemia, whether clinical outcomes are better in those who receive an invasive intervention plus medical therapy than in those who receive medical therapy alone is uncertain. METHODS We randomly assigned 5179 patients with moderate or severe ischemia to an initial invasive strategy (angiography and revascularization when feasible) and medical therapy or to an initial conservative strategy of medical therapy alone and angiography if medical therapy failed. The primary outcome was a composite of death from cardiovascular causes, myocardial infarction, or hospitalization for unstable angina, heart failure, or resuscitated cardiac arrest. A key secondary outcome was death from cardiovascular causes or myocardial infarction. RESULTS Over a median of 3.2 years, 318 primary outcome events occurred in the invasive-strategy group and 352 occurred in the conservative-strategy group. At 6 months, the cumulative event rate was 5.3% in the invasive-strategy group and 3.4% in the conservative-strategy group (difference, 1.9 percentage points; 95% confidence interval [CI], 0.8 to 3.0); at 5 years, the cumulative event rate was 16.4% and 18.2%, respectively (difference, 121.8 percentage points; 95% CI, 124.7 to 1.0). Results were similar with respect to the key secondary outcome. The incidence of the primary outcome was sensitive to the definition of myocardial infarction; a secondary analysis yielded more procedural myocardial infarctions of uncertain clinical importance. There were 145 deaths in the invasive-strategy group and 144 deaths in the conservative-strategy group (hazard ratio, 1.05; 95% CI, 0.83 to 1.32). CONCLUSIONS Among patients with stable coronary disease and moderate or severe ischemia, we did not find evidence that an initial invasive strategy, as compared with an initial conservative strategy, reduced the risk of ischemic cardiovascular events or death from any cause over a median of 3.2 years. The trial findings were sensitive to the definition of myocardial infarction that was used