Biology subjects

Wagner, A.

Publications and source records attributed to Wagner, A..

At least 91 records · Page 5Linked to original sources

bioRxiv2021

Yeast proteins reversibly aggregate like amphiphilic molecules

More than a hundred proteins in yeast reversibly aggregate and phase-separate in response to various stressors, such as nutrient depletion and heat shock. We know little about the sequence and structural features behind this ability, which has not been characterized on a proteome-wide level. To identify the distinctive features of aggregation-prone regions, we apply machine learning algorithms to genome-scale limited proteolysis-mass spectrometry data from 96 yeast proteins that phase-separate upon heat shock. We find that the aggregation-prone regions (APRs) of our study proteins are significantly enriched in aliphatic residues and depleted in positively charged amino acids. Aggregator proteins with longer APRs show a greater propensity to aggregate, a relationship that can be explained by equilibrium statistical thermodynamics. Altogether, our observations suggest that proteome-wide reversible protein aggregation is mediated by sequence-encoded properties. Aggregating proteins resemble supra-molecular amphiphiles, where APRs are the hydrophobic parts, and non-APRs are the hydrophilic parts.

molecular biology↗

bioRxiv2021

GroEL/S helps purge deleterious mutations and reduce genetic diversity during adaptive protein evolution

Chaperones are proteins that help other proteins fold. They also affect the adaptive evolution of their client proteins by buffering deleterious mutations and increasing the genetic diversity of evolving proteins. We study how the bacterial chaperone GroE (GroEL + GroES) affects the evolution of green fluorescent protein (GFP). To this end we subjected GFP to multiple rounds of mutation and selection for its color phenotype in four replicate E. coli populations, and studied its evolutionary dynamics through high-throughput sequencing and mutant engineering. We evolved GFP both under stabilizing selection for its ancestral (green) phenotype, and to directional selection for a new (cyan) phenotype. We did so both under low and high expression of the chaperone GroE. In contrast to prevailing wisdom, we observe that GroE does not just buffer but also helps purge deleterious mutations from evolving populations. In doing so, GroE helps reduce the genetic diversity of evolving populations. In addition, it causes phenotypic heterogeneity in mutants with the same genotype, potentiating their effect in some cells, and buffering it in others. Our observations show that chaperones can affect adaptive evolution through more than one mechanism. HighlightsO_LIGroE reduces genetic diversity C_LIO_LIGroE potentiates the effect of deleterious mutations C_LIO_LIGroE intensifies purifying selection and leads to higher activity of client proteins C_LI

evolutionary biology↗

bioRxiv2021

Selection on growth rate and local adaptation drive genomic adaptation during experimental range expansions in the protist Tetrahymena thermophila

O_LIPopulations that expand their range can undergo rapid evolutionary adaptation of life-history traits, dispersal behaviour, and adaptation to the local environment. Such adaptation may be aided or hindered by sexual reproduction, depending on the context. C_LIO_LIHowever, few studies have investigated the genomic causes and consequences or genetic architecture of such adaptation during range expansions. C_LIO_LIWe here studied genomic adaptation during experimental range expansions of the protist Tetrahymena thermophila in landscapes with a uniform environment or a pH-gradient. Specifically, we investigated two aspects of genomic adaptation during range expansion. Firstly, we investigated the genetic architecture of adaptation in terms of the underlying numbers of allele frequency changes from standing genetic variation and de novo variants. We focused on how sexual reproduction may alter this genetic architecture. Secondly, identified genes subject to selection caused by the expanding range itself, and directional selection due to the presence or absence of the pH-gradient. We focused this analysis on alleles with large frequency changes that occurred in parallel in more than one population to identify the most likely candidate targets of selection. C_LIO_LIWe found that sexual reproduction altered genetic architecture both in terms of de novo variants and standing genetic variation. However, sexual reproduction affected allele frequency changes in standing genetic variation only in the absence of long-distance gene flow. Adaptation to the range expansion affected genes involved in cell divisions and DNA repair, whereas adaptation to the pH-gradient additionally affected genes involved in ion balance, and oxidoreductase reactions. These genetic changes may result from selection on growth and adaptation to low pH. C_LIO_LIOur results suggest that the evolution of life-history and the adaptation to the local environment has a genetic basis during our range expansion experiment. In the absence of gene flow, sexual reproduction may have aided genetic adaptation. Gene flow may have swamped expanding populations with maladapted alleles, thus reducing the extent of evolutionary adaptation during range expansion. Sexual reproduction also altered the genetic architecture of our evolving populations via de novo variants, possibly by purging deleterious mutations or by revealing fitness benefits of rare genetic variants. C_LI

evolutionary biology↗

bioRxiv2021

Ecology drives the evolution of diverse siderophore-production strategies in the opportunistic human pathogen Pseudomonas aeruginosa

Bacteria often cooperate by secreting molecules that can be shared as public goods between cells. Because the production of public goods is subject to cheating by mutants that exploit the good without contributing to it, there has been great interest in elucidating the evolutionary forces that maintain cooperation. However, little is known on how bacterial cooperation evolves under conditions where cheating is unlikely of importance. Here we use experimental evolution to follow changes in the production of a model public good, the iron-scavenging siderophore pyoverdine, of the bacterium Pseudomonas aeruginosa. After 1200 generations of evolution in nine different environments, we observed that cheaters only reached high frequency in liquid medium with low iron availability. Conversely, when adding iron to reduce the cost of producing pyoverdine, we observed selection for pyoverdine hyper-producers. Similarly, hyper-producers also spread in populations evolved in highly viscous media, where relatedness between interacting individuals is higher. Whole-genome sequencing of evolved clones revealed that hyper-production is associated with mutations/deletions in genes encoding quorum-sensing communication systems, while cheater clones had mutations in the iron-starvation sigma factor or in pyoverdine biosynthesis genes. Our findings demonstrate that bacterial social traits can evolve rapidly in divergent directions, with particularly strong selection for increased levels of cooperation occurring in environments where individual dispersal is reduced, as predicted by social evolution theory. Moreover, we establish a regulatory link between pyoverdine production and quorum-sensing, showing that increased cooperation at one trait (pyoverdine) can be associated with the loss (quorum-sensing) of another social trait.

evolutionary biology↗

bioRxiv2021

FOXO1 and FOXO3 cooperatively regulate innate lymphoid cell development

The natural killer (NK) and non-cytotoxic innate lymphoid cells (ILC) lineages play vital role in the regulation of the immune system. Yet understanding of mechanisms controlling NK/ILC development remains incomplete. The evolutionary conserved FOXO family of forkhead transcription factors are critical regulators of cellular processes. We found that the loss of FOXO1 and FOXO3 together caused impaired activation of the NK gene expression program and reduced ETS binding already at the common lymphoid progenitor (CLP) level and a block at the ILC progenitor (ILCP) to NK progenitor transition. FOXO controlled NK cell maturation in organ specific manner and their ability to respond to IL-15. At the ILCP level, disruption of the ILC lineage specific gene programs was associated with broad perturbation of the generation of the non-cytotoxic ILC subsets. We concluded that FOXO1 and FOXO3 cooperatively regulate ILC lineage specification at the progenitor level as well as the generation of mature ILCs.

cell biology↗

bioRxiv2020

Novel quaternary structures of the human prion protein globular domain

Prion disease is caused by the misfolding of the cellular prion protein, PrPC, into a self-templating conformer, PrPSc. Nuclear magnetic resonance (NMR) and X-ray crystallography revealed the 3D structure of the globular domain of PrPC and the possibility of its dimerization via an interchain disulfide bridge that forms due to domain swap or by non-covalent association of two monomers. On the contrary, PrPSc is composed by a complex and heterogeneous ensemble of poorly defined conformations and quaternary arrangements that are related to different patterns of neurotoxicity. Targeting PrPC with molecules that stabilize the native conformation of its globular domain emerged as a promising approach to develop anti-prion therapies. One of the advantages of this approach is employing structure-based drug discovery methods to PrPC. Thus, it is essential to expand our structural knowledge about PrPC as much as possible to aid such drug discovery efforts. In this work, we report a crystallographic structure of the globular domain of human PrPC that shows a novel dimeric form and a novel oligomeric arrangement. We use molecular dynamics simulations to explore its structural dynamics and stability and discuss potential implications of these new quaternary structures to the conversion process.

biochemistry↗

bioRxiv2020

MetamORF: A repository of unique short Open Reading Frames identified by both experimental and computational approaches for gene-level and meta-analysis

The development of high-throughput technologies revealed the existence of non-canonical short open reading frames (sORFs) on most eukaryotic RNAs. They are ubiquitous genetic elements highly conserved across species and suspected to be involved in numerous cellular processes. MetamORF (http://metamorf.hb.univ-amu.fr/) aims to provide a repository of unique sORFs identified in the human and mouse genomes with both experimental and computational approaches. By gathering publicly available sORF data, normalizing it and summarizing redundant information, we were able to identify a total of 1,162,675 unique sORFs. Despite the usual characterization of ORFs as short, upstream or downstream, there is currently no clear consensus regarding the definition of these categories. Thus, the data has been reprocessed using a normalized nomenclature. MetamORF enables new analyses at loci, gene, transcript and ORF levels, that should offer the possibility to address new questions regarding sORF functions in the future. The repository is available through an user-friendly web interface, allowing easy browsing, visualization, filtering over multiple criteria and export possibilities. sORFs could be searched starting from a gene, a transcript, an ORF ID, or looking in a genome area. The database content has also been made available through track hubs at UCSC Genome Browser.

bioinformatics↗

bioRxiv2020

Alterations of multiple alveolar macrophage states in chronic obstructive pulmonary disease

Despite the epidemics of chronic obstructive pulmonary disease (COPD), the cellular and molecular mechanisms of this disease are far from being understood. Here, we characterize and classify the cellular composition within the alveolar space and peripheral blood of COPD patients and control donors using a clinically applicable single-cell RNA-seq technology corroborated by advanced computational approaches for: machine learning-based cell-type classification, identification of differentially expressed genes, prediction of metabolic changes, and modeling of cellular trajectories within a patient cohort. These high-resolution approaches revealed: massive transcriptional plasticity of macrophages in the alveolar space with increased levels of invading and proliferating cells, loss of MHC expression, reduced cellular motility, altered lipid metabolism, and a metabolic shift reminiscent of mitochondrial dysfunction in COPD patients. Collectively, single-cell omics of multi-tissue samples was used to build the first cellular and molecular framework for COPD pathophysiology as a prerequisite to develop molecular biomarkers and causal therapies against this deadly disease.

systems biology↗

bioRxiv2020

The architecture of EMC reveals a path for membrane protein insertion

Approximately 25% of eukaryotic genes code for integral membrane proteins that are assembled at the endoplasmic reticulum. An abundant and widely conserved multi-protein complex termed EMC has been implicated in membrane protein biogenesis, but its mechanism of action is poorly understood. Here, we define the composition and architecture of human EMC using biochemical assays, crystallography of individual subunits, site-specific photocrosslinking, and cryo-EM reconstruction. Our results show that EMCs cytosolic domain contains a large, moderately hydrophobic vestibule that binds a substrates transmembrane domain (TMD). The cytosolic vestibule leads into a lumenally-sealed, lipid-exposed intramembrane groove large enough to accommodate a single substrate TMD. A gap between the cytosolic vestibule and intramembrane groove provides a path for substrate egress from EMC. These findings suggest how EMC facilitates energy-independent membrane insertion of TMDs, explain why only short lumenal domains are translocated by EMC, and constrain models of EMCs proposed chaperone function.

biochemistry↗

bioRxiv2020

Oleic acid Induces Tissue Resident FoxP3 Regulatory T cell Lineage Stability and Suppressive Functions

FoxP3 positive regulatory T cells (Tregs) rely on fatty acid {beta}-oxidation (FAO)-driven OXPHOS for differentiation and function. Recent data have demonstrated a role for Tregs in the maintenance of tissue homeostasis with tissue-resident Tregs possessing tissue-specific transcriptomes. However, specific signals that establish these tissue-resident Tregs programs are largely unknown. As Tregs metabolically rely on FAO, and considering the lipid-rich environments of tissues, we hypothesized that environmental lipids drive Treg homeostasis. Using human adipose tissue as a model for tissue residency, we identify oleic acid as the most prevalent free fatty acid in human adipose tissue. Mechanistically, oleic acid amplifies Treg FAO-driven OXPHOS metabolism, creating a positive feedback mechanism that induces the expression of Foxp3 and enhances phosphorylation of STAT5, which acts to stabilize the Treg lineage and increase suppressive function. Comparing the transcriptomic program induced by oleic acid to that of the pro-inflammatory arachidonic acid, we find that Tregs sorted from peripheral blood and adipose of healthy donors transcriptomically resemble the oleic acid in vitro treated Tregs, whereas Tregs obtained from the adipose tissue of relapsing-remitting MS patients more closely resemble an arachidonic acid profile. Finally, we find that oleic acid concentrations are reduced in the fat tissue of MS patients, and exposure of dysfunctional MS Tregs to oleic acid restores defects in their suppressive function. These data demonstrate the importance of fatty acids in regulating tissue inflammatory signals.

immunology↗

bioRxiv2020

K2P channel C-type gating involves asymmetric selectivity filter order-disorder transitions

K2P channels regulate nervous, cardiovascular, and immune system functions1,2 through the action of their selectivity filter (C-type) gate3-6. Although structural studies show K2P conformations that impact activity7-13, no selectivity filter conformational changes have been observed. Here, combining K2P2.1 (TREK-1) X-ray crystallography in different potassium concentrations, potassium anomalous scattering, molecular dynamics, and functional studies, we uncover the unprecedented, asymmetric, potassium-dependent conformational changes underlying K2P C-type gating. Low potassium concentrations evoke conformational changes in selectivity filter strand 1 (SF1), selectivity filter strand 2 (SF2), and the SF2-transmembrane helix 4 loop (SF2-M4 loop) that destroy the S1 and S2 ion binding sites and are suppressed by C-type gate activator ML335. Shortening the uniquely long SF2-M4 loop to match the canonical length found in other potassium channels or disrupting the conserved Glu234 hydrogen bond network supporting this loop blunts C-type gate response to various physical and chemical stimuli. Glu234 network destabilization also compromises ion selectivity, but can be reversed by channel activation, indicating that the ion binding site loss reduces selectivity similar to other channels14. Together, our data establish that C-type gating occurs through potassium-dependent order-disorder transitions in the selectivity filter and adjacent loops that respond to gating cues relayed through the SF2-M4 loop. These findings underscore the potential for targeting the SF2-M4 loop for the development of new, selective K2P channel modulators.

biophysics↗

bioRxiv2020

Natural selection has preserved and enhanced the phase-separation properties of FUS during 160 million years of mammalian evolution

Protein phase separation is essential for the self-assembly of non-membraneous organelles. However, we know little about its ability to change in evolution. Here we studied the evolution of the mammalian RNA binding protein FUS, a protein whose prion-like domain (PLD) is essential for the formation of stress granules through liquid-liquid phase separation. Although the prion-like domain evolves three times as rapidly as the remainder of FUS, it harbors absolutely conserved tyrosine residues that are crucial for phase separation. Ancestral reconstruction shows that the phosphorylation sites within the PLD are subject to stabilizing selection. They toggle among a small number of amino acid states. One exception to this pattern are primates, where the number of such phosphosites has increased through positive selection. In addition, we find frequent glutamine to proline changes that help maintain the unstructured state of FUS that is necessary for phase separation. In summary, natural selection has stabilized the liquid-forming potential of FUS and minimized the propensity of cytotoxic liquid-to-solid phase transitions during 160 million years of mammalian evolution.

evolutionary biology↗

bioRxiv2020

Genotype networks of 80 quantitative Arabidopsis thaliana phenotypes reveal phenotypic evolvability despite pervasive epistasis

We study the genotype-phenotype maps of 80 quantitative phenotypes in the model plant Arabidopsis thaliana, by representing the genotypes affecting each phenotype as a genotype network. In such a network, each vertex or node corresponds to an individuals genotype at all those genomic loci that affect a given phenotype. Two vertices are connected by an edge if the associated genotypes differ in exactly one nucleotide. The 80 genotype networks we analyze are based on data from genome-wide association studies of 199 A. thaliana accessions. They form connected graphs whose topography differs substantially among phenotypes. We focus our analysis on the incidence of epistasis (non-additive interactions among mutations) because a high incidence of epistasis can reduce the accessibility of evolutionary paths towards high or low phenotypic values. We find epistatic interactions in 67 phenotypes, and in 51 phenotypes every pairwise mutant interaction is epistatic. Moreover, we find phenotype-specific differences in the fraction of accessible mutational paths to maximum phenotypic values. However, even though epistasis affects the accessibility of maximum phenotypic values, the relationships between genotypic and phenotypic change of our analyzed phenotypes are sufficiently smooth that some evolutionary paths remain accessible for most phenotypes, even where epistasis is pervasive. The genotype network representation we use can complement existing approaches to understand the genetic architecture of polygenic traits in many different organisms. Author summaryHow genotypic change relates to phenotypic change is a fundamental problem in evolutionary biology. A smooth relationship between genotypic and phenotypic change facilitates phenotypic evolution, whereas a rugged one hampers it, because evolutionary change on a rugged landscape does not allow a monotonic increase or decrease in phenotypic value. We use data from multiple genome-wide association studies for 80 different phenotypes in the thale cress Arabidopsis thaliana to construct 80 genotype-phenotype maps for these phenotypes. We use a network representation for each map, where a vertex corresponds to a genotype, and where two vertices are connected if they differ in a single nucleotide. We study the incidence of non-additive interactions between mutations as a proxy for the ease to reach extreme phenotypic values of these maps. This incidence varies greatly among phenotypes, but even where it is highest, some monotonic pathways to high or low phenotypic values exist for the vast majority of phenotypes, such that the underlying phenotypes are accessible to adaptive evolution.

evolutionary biology↗

bioRxiv2020

Gene expression noise can promote the fixation of beneficial mutations in fluctuating environments

Nongenetic phenotypic variation can either speed up or slow down adaptive evolution. We show that it can speed up evolution in environments in which available carbon and energy sources change over time. To this end, we use an experimentally validated model of Escherichia coli growth on two alternative carbon sources, glucose and acetate. On the superior carbon source (glucose), all cells achieve high growth rates, while on the inferior carbon source (acetate) only a small fraction of the population manages to initiate growth. Consequently, populations experience a bottleneck when the environment changes from the superior to the inferior carbon source. Growth on the inferior carbon source depends on a circuit under the control of a transcription factor that is repressed in the presence of the superior carbon source. We show that noise in the expression of this transcription factor can increase the probability that cells start growing on the inferior carbon source. In doing so, it can decrease the severity of the bottleneck and increase mean population fitness whenever this fitness is low. A modest amount of noise can also enhance the fitness effects of a beneficial allele. It can accelerate the spreading of such an allele, increase its likelihood of going to fixation, and reduce its fixation time. Central to the adaptation-enhancing principle we identify is the ability of noise to mitigate population bottlenecks. Because such bottlenecks are frequent in fluctuating environments, and because fluctuating environments themselves are ubiquitous, this principle may apply to a broad range of environments and organisms. Author summaryIndividuals that grow in the same environment and share the same genes may still differ in their behaviour and their traits. These differences between individuals arise from uncertainty inherent in all biological processes, and they are found in all domains of life. Although this random individual variability is itself short-lived, it still has the potential to shape evolution in the long term. For example, if a population encounters a harsh environment, random (nongenetic) differences between individuals can cause some individuals to cope better with the new environment than others. These rare individuals may give a population an advantage compared to populations with fewer such differences between individuals. Furthermore, if some of these rare individuals carry a beneficial gene variant, the beneficial effect of this gene variant may become amplified, and consequently spread faster in a population with more random variation. Using a realistic model of cell growth, we show that this mechanism not only works in unfavourable environments that are stable, but also in environments that switch back and forth between a favourable and an unfavourable state. Because many natural environments undergo such periodic changes, and because random differences between individuals are ubiquitous, the mechanism we have identified may be widespread in nature.

evolutionary biology↗

bioRxiv2020

In Silico Modeling of Metabolic State in Single Th17 Cells Reveals Novel Regulators of Inflammation and Autoimmunity

Cellular metabolism, a key regulator of immune responses, is difficult to study with current technologies in individual cells Here, we present Compass, an algorithm to characterize the metabolic state of cells based on single-cell RNA-Seq and flux balance analysis. We applied Compass to associate metabolic states with functional variability (pathogenic potential) amongst Th17 cells and recovered a metabolic switch between glycolysis and fatty acid oxidation, akin to known differences between Th17 and Treg cells, as well as novel targets in amino-acid pathways, which we tested through targeted metabolic assays. Compass further predicted a particular glycolytic reaction (phosphoglycerate mutase -- PGAM) that promotes an anti-inflammatory Th17 phenotype, contrary to the common understanding of glycolysis as pro-inflammatory. We demonstrate that PGAM inhibition leads non-pathogenic Th17 cells to adopt a pro-inflammatory transcriptome and induce autoimmunity in vivo. Compass is broadly applicable for characterizing metabolic states of cells and relating metabolic heterogeneity to other cellular phenotypes.

genomics↗

bioRxiv2020

Metabolic and Epigenomic Regulation of Th17/Treg Balance by the Polyamine Pathway

Cellular metabolism can orchestrate immune cell function. We previously demonstrated that lipid biosynthesis represents one such gatekeeper to Th17 cell functional state. Utilizing Compass, a transcriptome-based algorithm for prediction of metabolic flux, we constructed a comprehensive metabolic circuitry for Th17 cell function and identified the polyamine pathway as a candidate metabolic node, the flux of which regulates the inflammatory function of T cells. Testing this prediction, we found that expression and activities of enzymes of the polyamine pathway were enhanced in pathogenic Th17 cells and suppressed in regulatory T cells. Perturbation of the polyamine pathway in Th17 cells suppressed canonical Th17 cell cytokines and promoted the expression of Foxp3, accompanied by dramatic shift in transcriptome and epigenome, transitioning Th17 cells into a Treg-like state. Genetic and chemical perturbation of the polyamine pathway resulted in attenuation of tissue inflammation in an autoimmune disease model of central nervous system, with changes in T cell effector phenotype.

immunology↗

bioRxiv2019

Sex and gene flow modulate evolution during rangeexpansions in the protist Tetrahymena thermophila

At species range edges, individuals often face novel environmental conditions that may limit range expansion until populations adapt. The potential to adapt depends on genetic variation upon which selection can act. However, populations at species range edges are often genetically depauperated. One mechanism to increase genetic variation is to reshuffle existing variation through sex. During range expansions, sex can, however, act as a double-edged sword. The gene swamping hypothesis predicts that for populations expanding along an abiotic gradient, sex can hinder adaptation if asymmetric dispersal leads to numerous maladapted dispersers from the range core swamping the range edge. In this study, we experimentally tested the gene swamping hypothesis by performing replicated range expansions in landscapes with or without an abiotic pH-gradient, using the ciliate Tetrahymena thermophila, while simultaneously manipulating the occurrence of gene flow and sex. We show that sex accelerated evolution of local adaptation in the absence of gene flow, but hindered it in the presence of gene flow. The effect of sex, however, was independent of the pH-gradient, indicating that not only abiotic gradients but also the biotic gradient in population density leads to gene swamping. Overall, our results show that gene swamping can affect adaptation in life-history strategies.

evolutionary biology↗

bioRxiv2019

Short residence times of DNA-bound transcription factors can reduce gene expression noise and increase the transmission of information in a gene regulation system

Gene expression noise is not just ubiquitous but also variable, and we still do not understand some of the most elementary factors that affect it. Among them is the residence time of a transcription factor (TF) on DNA, the mean time that a DNA-bound TF remains bound. Here, we use a stochastic model of transcriptional regulation to study how this residence time affects gene expression. We find that the effect of residence time on gene expression depends on the level of induction of the gene. At high levels of induction, residence time has no effect on gene expression. However, as the level of induction decreases, short residence times reduce gene expression noise. The reason is that fast on-off TF binding dynamics prevent long periods where proteins are predominantly synthesized or degraded, which can cause excessive fluctuations in gene expression. As a consequence, short residence times can help a gene regulation system acquire information about the cellular environment it operates in. Our predictions are consistent with the observation that experimentally measured residence times are usually modest and lie between seconds to minutes.

systems biology↗