Tissue-specific landscape of protein aggregation and quality control in an aging vertebrate.

Protein aggregation is a hallmark of age-related neurodegeneration. Yet, aggregation during normal aging and in tissues other than the brain is poorly understood. Here, we leverage the African turquoise killifish to systematically profile protein aggregates in seven tissues of an aging vertebrate. Age-dependent aggregation is strikingly tissue specific and not simply driven by protein expression differences. Experimental interrogation in killifish and yeast, combined with machine learning, indicates that this specificity is linked to protein-autonomous biophysical features and tissue-selective alterations in protein quality control. Co-aggregation of protein quality control machinery during aging may further reduce proteostasis capacity, exacerbating aggregate burden. A segmental progeria model with accelerated aging in specific tissues exhibits selectively increased aggregation in these same tissues. Intriguingly, many age-related protein aggregates arise in wild-type proteins that, when mutated, drive human diseases. Our data chart a comprehensive landscape of protein aggregation during vertebrate aging and identify strong, tissue-specific associations with dysfunction and disease.

Identification of protein aggregates in the aging vertebrate brain with prion-like and phase-separation properties.

Protein aggregation, which can sometimes spread in a prion-like manner, is a hallmark of neurodegenerative diseases. However, whether prion-like aggregates form during normal brain aging remains unknown. Here, we use quantitative proteomics in the African turquoise killifish to identify protein aggregates that accumulate in old vertebrate brains. These aggregates are enriched for prion-like RNA-binding proteins, notably the ATP-dependent RNA helicase DDX5. We validate that DDX5 forms aggregate-like puncta in the brains of old killifish and mice. Interestingly, DDX5's prion-like domain allows these aggregates to propagate across many generations in yeast. In vitro, DDX5 phase separates into condensates. Mutations that abolish DDX5 prion propagation also impair the protein's ability to phase separate. DDX5 condensates exhibit enhanced enzymatic activity, but they can mature into inactive, solid aggregates. Our findings suggest that protein aggregates with prion-like properties form during normal brain aging, which could have implications for the age-dependency of cognitive decline.

In vivo protein turnover rates in varying oxygen tensions nominate MYBBP1A as a mediator of the hyperoxia response.

Oxygen deprivation and excess are both toxic. Thus, the body's ability to adapt to varying oxygen tensions is critical for survival. While the hypoxia transcriptional response has been well studied, the post-translational effects of oxygen have been underexplored. In this study, we systematically investigate protein turnover rates in mouse heart, lung, and brain under different inhaled oxygen tensions. We find that the lung proteome is the most responsive to varying oxygen tensions. In particular, several extracellular matrix (ECM) proteins are stabilized in the lung under both hypoxia and hyperoxia. Furthermore, we show that complex 1 of the electron transport chain is destabilized in hyperoxia, in accordance with the exacerbation of associated disease models by hyperoxia and rescue by hypoxia. Moreover, we nominate MYBBP1A as a hyperoxia transcriptional regulator, particularly in the context of rRNA homeostasis. Overall, our study highlights the importance of varying oxygen tensions on protein turnover rates and identifies tissue-specific mediators of oxygen-dependent responses.

Hsp90 shapes adaptation by controlling the fitness consequences of regulatory variation.

The essential stress-responsive chaperone Hsp90 impacts development and adaptation from microbes to humans. Yet despite evidence of its role in evolution, pathogenesis, and oncogenic transformation, the molecular mechanisms by which Hsp90 alters the consequences of mutations remain vigorously debated. Here we exploit the power of nucleotide-resolution genetic mapping in Saccharomyces cerevisiae to uncover more than 1,000 natural variant-to-phenotype associations governed by this molecular chaperone. Strikingly, Hsp90 more frequently modified the phenotypic effects of cis-regulatory variation than variants that altered protein sequence. Moreover, these interactions made the largest contribution to Hsp90-dependent heredity. Nearly all interacting variants-both regulatory and protein-coding-fell within clients of Hsp90 or targets of its direct binding partners. Hsp90 activity affected mutations in evolutionarily young genes, segmental deletions, and heterozygotes, highlighting its influence on variation central to evolutionary novelty. Reconciling the diverse epistatic effects of this chaperone, synthetic transcriptional regulation and reconstructions of natural alleles by genome editing revealed a central role for Hsp90 in regulating the fundamental relationship between activity and phenotype. Our findings establish that non-coding variation is a core driver of Hsp90's influence on heredity, offering a mechanistic explanation for the chaperone's strong effects on evolution and development across species.

Genome dilution by cell growth drives starvation-like proteome remodeling in mammalian and yeast cells.

Cell size is tightly controlled in healthy tissues and single-celled organisms, but it remains unclear how size influences cell physiology. Increasing cell size was recently shown to remodel the proteomes of cultured human cells, demonstrating that large and small cells of the same type can be biochemically different. Here, we corroborate these results in mouse hepatocytes and extend our analysis using yeast. We find that size-dependent proteome changes are highly conserved and mostly independent of metabolic state. As eukaryotic cells grow larger, the dilution of the genome elicits a starvation-like proteome phenotype, suggesting that growth in large cells is limited by the genome in a manner analogous to a limiting nutrient. We also demonstrate that the proteomes of replicatively-aged yeast are primarily determined by their large size. Overall, our data suggest that genome concentration is a universal determinant of proteome content in growing cells.

Defining the condensate landscape of fusion oncoproteins.

Fusion oncoproteins (FOs) arise from chromosomal translocations in ~17% of cancers and are often oncogenic drivers. Although some FOs can promote oncogenesis by undergoing liquid-liquid phase separation (LLPS) to form aberrant biomolecular condensates, the generality of this phenomenon is unknown. We explored this question by testing 166 FOs in HeLa cells and found that 58% formed condensates. The condensate-forming FOs displayed physicochemical features distinct from those of condensate-negative FOs and segregated into distinct feature-based groups that aligned with their sub-cellular localization and biological function. Using Machine Learning, we developed a predictor of FO condensation behavior, and discovered that 67% of ~3000 additional FOs likely form condensates, with 35% of those predicted to function by altering gene expression. 47% of the predicted condensate-negative FOs were associated with cell signaling functions, suggesting a functional dichotomy between condensate-positive and -negative FOs. Our Datasets and reagents are rich resources to interrogate FO condensation in the future.

Volumetric Flow Assessment in Extracranial Arteries in Patients with 70-99% Internal Carotid Artery Stenosis.

Background: Cerebral blood flow (CBF) can be related to the risk of occurrence of neurological symptoms. Well-developed collateral circulation is a good prognostic factor in patients with cerebrovascular disease. Understanding the mechanisms of collateral circulation may be important in the diagnosis, treatment, and monitoring in this group of patients. The aim of this study covered the assessment of CBF in patients with 70−99% Internal carotid artery (ICA) stenosis, focusing on the circulation pathways and flow volume in extracranial arteries. Materials and methods: 53 patients with 70−99% ICA stenosis (mean age 73.4 ± 7 years old; 17 female, mean age 73.9 ± 7.5 years old; 36 male, mean age 73.2 ± 6.8 years old) were included in the study. In all patients a Doppler ultrasound (DUS) examination, measuring blood flow volume in the internal carotid artery (ICA), external carotid artery (ECA), and vertebral artery (VA), was performed. The cerebral blood flow (CBF) was compared to the previously reported CBF values in the healthy population > 65 years old. Results: Among the study groups three subgroups with flow differences were identified: patients with elevated CBF (significant volumetric flow compensation­26/53, 49%), patients with CBF similar to (mild compensation­17/53, 32%), and patients with CBF lower than (no compensation­10/53, 19%) the healthy, equally aged population. The percentage of patients with significant volumetric flow compensation was the highest in age groups 65−69 years old (62.5%) and >80 years old (60%). In the oldest age group (>80 years old) no patients without flow compensation (0%) were observed. The level of compensation depends on the number of the arteries with compensatory increased flow. In patients with significant volumetric flow compensation, the compensatory increased flow was observed, on average, in 3.31 arteries, in the mild compensation group­in 2.18 arteries, and in the no compensation group only in 1 artery. ICA plays the most important role in the volumetric flow compensation­the increase in the flow volume, in comparison to the reference values, was between 116.7 mL/min and 251.9 mL/min (in the ECA 57.6 mL/min−110.4 mL/min; in the VA 73.9 mL/min−104.9 mL/min). The relative flow increase was highest in the VA: 215−246%, then in the ECA: 163−206%, and finally in the ICA: 148.6−192%. The increased flow was most commonly observed in the VA­57 arteries, in second place in the ECA­42 arteries, and ICA­31 arteries. In patients with unilateral ICA stenosis, the volumetric flow increase was stated more frequently in the ipsilateral ECAs then in the contralateral ones (23 vs. 14). In the VA the opposite tendency was observed (29 contralateral vs. 23 ipsilateral). The ability of volumetric flow compensation decreased significantly with increasing age. Conclusions: Understanding the mechanisms of collateral circulation and their assessment in Doppler ultrasonography may provide a novel and easily accessible tool of identifying and monitoring patients with cerebrovascular disease.

Biomolecular Condensation: A New Phase in Cancer Research.

Multicellularity was a watershed development in evolution. However, it also meant that individual cells could escape regulatory mechanisms that restrict proliferation at a severe cost to the organism: cancer. From the standpoint of cellular organization, evolutionary complexity scales to organize different molecules within the intracellular milieu. The recent realization that many biomolecules can "phase-separate" into membraneless organelles, reorganizing cellular biochemistry in space and time, has led to an explosion of research activity in this area. In this review, we explore mechanistic connections between phase separation and cancer-associated processes and emerging examples of how these become deranged in malignancy. SIGNIFICANCE: One of the fundamental functions of phase separation is to rapidly and dynamically respond to environmental perturbations. Importantly, these changes often lead to alterations in cancer-relevant pathways and processes. This review covers recent advances in the field, including emerging principles and mechanisms of phase separation in cancer.

Massive QTL analysis identifies pleiotropic genetic determinants for stress resistance, aroma formation, and ethanol, glycerol and isobutanol production in Saccharomyces cerevisiae.

BACKGROUND: The brewer's yeast Saccharomyces cerevisiae is exploited in several industrial processes, ranging from food and beverage fermentation to the production of biofuels, pharmaceuticals and complex chemicals. The large genetic and phenotypic diversity within this species offers a formidable natural resource to obtain superior strains, hybrids, and variants. However, most industrially relevant traits in S. cerevisiae strains are controlled by multiple genetic loci. Over the past years, several studies have identified some of these QTLs. However, because these studies only focus on a limited set of traits and often use different techniques and starting strains, a global view of industrially relevant QTLs is still missing. RESULTS: Here, we combined the power of 1125 fully sequenced inbred segregants with high-throughput phenotyping methods to identify as many as 678 QTLs across 18 different traits relevant to industrial fermentation processes, including production of ethanol, glycerol, isobutanol, acetic acid, sulfur dioxide, flavor-active esters, as well as resistance to ethanol, acetic acid, sulfite and high osmolarity. We identified and confirmed several variants that are associated with multiple different traits, indicating that many QTLs are pleiotropic. Moreover, we show that both rare and common variants, as well as variants located in coding and non-coding regions all contribute to the phenotypic variation. CONCLUSIONS: Our findings represent an important step in our understanding of the genetic underpinnings of industrially relevant yeast traits and open new routes to study complex genetics and genetic interactions as well as to engineer novel, superior industrial yeasts. Moreover, the major role of rare variants suggests that there is a plethora of different combinations of mutations that can be explored in genome editing.

A prion accelerates proliferation at the expense of lifespan.

In fluctuating environments, switching between different growth strategies, such as those affecting cell size and proliferation, can be advantageous to an organism. Trade-offs arise, however. Mechanisms that aberrantly increase cell size or proliferation-such as mutations or chemicals that interfere with growth regulatory pathways-can also shorten lifespan. Here we report a natural example of how the interplay between growth and lifespan can be epigenetically controlled. We find that a highly conserved RNA-modifying enzyme, the pseudouridine synthase Pus4/TruB, can act as a prion, endowing yeast with greater proliferation rates at the cost of a shortened lifespan. Cells harboring the prion grow larger and exhibit altered protein synthesis. This epigenetic state, [BIG+] (better in growth), allows cells to heritably yet reversibly alter their translational program, leading to the differential synthesis of dozens of proteins, including many that regulate proliferation and aging. Our data reveal a new role for prion-based control of an RNA-modifying enzyme in driving heritable epigenetic states that transform cell growth and survival.

Cells make different proteins to perform different tasks. Each protein is a long chain of building blocks called amino acids that must fold into a particular shape before it can be useful. Some proteins can fold in more than one way, a normal form and a 'prion' form. Prions are unusual in that they can force normally folded proteins with the same amino acid sequence as them to refold into new prions. This means that a single prion can make many more that are inherited when cells divide. Some prions can cause disease, but others may be beneficial. Pus4 is a yeast protein that is typically involved in modifying ribonucleic acids, molecules that help translate genetic information into new proteins. Sometimes Pus4 can adopt a beneficial prion conformation called [BIG⁺]. When yeast cells have access to plenty of nutrients, [BIG⁺] helps them grow faster and larger, but this comes at the cost of a shorter lifespan. Garcia, Campbell et al. combined computational modeling and experiments in baker's yeast (Saccharomyces cerevisiae) to investigate the role of [BIG⁺]. They found that the prion accelerated protein production, leading to both faster growth and a shorter lifespan in these cells, even without any changes in gene sequence. Garcia, Campbell et al.'s findings explain the beneficial activity of prion proteins in baker's yeast cells. The results also describe how cells balance a tradeoff between growth and lifespan without any changes in the genome. This helps to highlight that genetics do not always explain the behaviors of cells, and further methods are needed to better understand cell biology.

Protein aggregation and the evolution of stress resistance in clinical yeast.

Protein aggregation, particularly in its prion-like form, has long been thought to be detrimental. However, recent studies have identified multiple instances where protein aggregation is important for normal physiological functions. Combining mass spectrometry and cell biological approaches, we developed a strategy for the identification of protein aggregates in cell lysates. We used this approach to characterize prion-based traits in pathogenic strains of the yeast Saccharomyces cerevisiae isolated from immunocompromised human patients. The proteins that we found, including the metabolic enzyme Cdc19, the translation elongation factor Yef3 and the fibrillarin homologue Nop1, are known to assemble under certain physiological conditions. Yet, such assemblies have not been reported to be stable or heritable. Our data suggest that some proteins which aggregate in response to stress have the capacity to acquire diverse assembled states, certain ones of which can be propagated across generations in a form of protein-based epigenetics. This article is part of the theme issue 'How does epigenetics influence the course of evolution?'

The Hunt for Ancient Prions: Archaeal Prion-Like Domains Form Amyloid-Based Epigenetic Elements.

Prions, proteins that can convert between structurally and functionally distinct states and serve as non-Mendelian mechanisms of inheritance, were initially discovered and only known in eukaryotes, and consequently considered to likely be a relatively late evolutionary acquisition. However, the recent discovery of prions in bacteria and viruses has intimated a potentially more ancient evolutionary origin. Here, we provide evidence that prion-forming domains exist in the domain archaea, the last domain of life left unexplored with regard to prions. We searched for archaeal candidate prion-forming protein sequences computationally, described their taxonomic distribution and phylogeny, and analyzed their associated functional annotations. Using biophysical in vitro assays, cell-based and microscopic approaches, and dye-binding analyses, we tested select candidate prion-forming domains for prionogenic characteristics. Out of the 16 tested, eight formed amyloids, and six acted as protein-based elements of information transfer driving non-Mendelian patterns of inheritance. We also identified short peptides from our archaeal prion candidates that can form amyloid fibrils independently. Lastly, candidates that tested positively in our assays had significantly higher tyrosine and phenylalanine content than candidates that tested negatively, an observation that may help future archaeal prion predictions. Taken together, our discovery of functional prion-forming domains in archaea provides evidence that multiple archaeal proteins are capable of acting as prions-thus expanding our knowledge of this epigenetic phenomenon to the third and final domain of life and bolstering the possibility that they were present at the time of the last universal common ancestor.

Protein self-assembly: A new frontier in cell signaling.

Long viewed as paradigm-shifting, but rare, prions have recently been discovered in all domains of life. Protein sequences that can drive this form of self-assembly are strikingly common in eukaryotic proteomes, where they are enriched in proteins involved in information flow and signal transduction. Although prions were thought to be a consequence of random errors in protein folding, recent studies suggest that prion formation can be a controlled process initiated by defined cellular signals. Many are present in normal biological contexts, yet are invisible to most technologies used to interrogate the proteome. Here, we review mechanisms by which protein self-assembly can create a stable record of past stimuli, altering adaptive responses, and how prion behavior is controlled by signaling processes. We touch on the diverse implications that this has for normal biological function and regulation, ranging from drug resistance in fungi to the innate immune response in humans. Finally, we discuss the potential for prion domains in transcription factors and RNA-binding proteins to orchestrate heritable gene expression changes in response to transient signals, such as during development.

What Has a Century of Quantitative Genetics Taught Us About Nature's Genetic Tool Kit?

The complexity of heredity has been appreciated for decades: Many traits are controlled not by a single genetic locus but instead by polymorphisms throughout the genome. The importance of complex traits in biology and medicine has motivated diverse approaches to understanding their detailed genetic bases. Here, we focus on recent systematic studies, many in budding yeast, which have revealed that large numbers of all kinds of molecular variation, from noncoding to synonymous variants, can make significant contributions to phenotype. Variants can affect different traits in opposing directions, and their contributions can be modified by both the environment and the epigenetic state of the cell. The integration of prospective (synthesizing and analyzing variants) and retrospective (examining standing variation) approaches promises to reveal how natural selection shapes quantitative traits. Only by comprehensively understanding nature's genetic tool kit can we predict how phenotypes arise from the complex ensembles of genetic variants in living organisms.

Both ROSy and Grim: The Landscape of Protein Redox during Aging.

Covalent cysteine modification by reactive oxygen species (ROS) has been implicated in regulating diverse biological processes, yet global understanding of this modification has remained fragmentary. Developing new approaches for detecting cysteine modification, Xiao et al. (2020) recently charted a comprehensive map of cysteine oxidation across tissues and life stages.

A Prion Epigenetic Switch Establishes an Active Chromatin State.

Covalent modifications to histones are essential for development, establishing distinct and functional chromatin domains from a common genetic sequence. Whereas repressed chromatin is robustly inherited, no mechanism that facilitates inheritance of an activated domain has been described. Here, we report that the Set3C histone deacetylase scaffold Snt1 can act as a prion that drives the emergence and transgenerational inheritance of an activated chromatin state. This prion, which we term [ESI+] for expressed sub-telomeric information, is triggered by transient Snt1 phosphorylation upon cell cycle arrest. Once engaged, the prion reshapes the activity of Snt1 and the Set3C complex, recruiting RNA pol II and interfering with Rap1 binding to activate genes in otherwise repressed sub-telomeric domains. This transcriptional state confers broad resistance to environmental stress, including antifungal drugs. Altogether, our results establish a robust means by which a prion can facilitate inheritance of an activated chromatin state to provide adaptive benefit.

Widespread Prion-Based Control of Growth and Differentiation Strategies in Saccharomyces cerevisiae.

Theory and experiments suggest that organisms would benefit from pre-adaptation to future stressors based on reproducible environmental fluctuations experienced by their ancestors, but the mechanisms driving pre-adaptation remain enigmatic. We report that the [SMAUG+] prion allows yeast to anticipate nutrient repletion after periods of starvation, providing a strong selective advantage. By transforming the landscape of post-transcriptional gene expression, [SMAUG+] regulates the decision between two broad growth and survival strategies: mitotic proliferation or meiotic differentiation into a stress-resistant state. [SMAUG+] is common in laboratory yeast strains, where standard propagation practice produces regular cycles of nutrient scarcity followed by repletion. Distinct [SMAUG+] variants are also widespread in wild yeast isolates from multiple niches, establishing that prion polymorphs can be utilized in natural populations. Our data provide a striking example of how protein-based epigenetic switches, hidden in plain sight, can establish a transgenerational memory that integrates adaptive prediction into developmental decisions.