Cell-cell metabolite exchange creates a pro-survival metabolic environment that extends lifespan.

Metabolism is deeply intertwined with aging. Effects of metabolic interventions on aging have been explained with intracellular metabolism, growth control, and signaling. Studying chronological aging in yeast, we reveal a so far overlooked metabolic property that influences aging via the exchange of metabolites. We observed that metabolites exported by young cells are re-imported by chronologically aging cells, resulting in cross-generational metabolic interactions. Then, we used self-establishing metabolically cooperating communities (SeMeCo) as a tool to increase metabolite exchange and observed significant lifespan extensions. The longevity of the SeMeCo was attributable to metabolic reconfigurations in methionine consumer cells. These obtained a more glycolytic metabolism and increased the export of protective metabolites that in turn extended the lifespan of cells that supplied them with methionine. Our results establish metabolite exchange interactions as a determinant of cellular aging and show that metabolically cooperating cells can shape the metabolic environment to extend their lifespan.

The proteomic landscape of genome-wide genetic perturbations.

Functional genomic strategies have become fundamental for annotating gene function and regulatory networks. Here, we combined functional genomics with proteomics by quantifying protein abundances in a genome-scale knockout library in Saccharomyces cerevisiae, using data-independent acquisition mass spectrometry. We find that global protein expression is driven by a complex interplay of (1) general biological properties, including translation rate, protein turnover, the formation of protein complexes, growth rate, and genome architecture, followed by (2) functional properties, such as the connectivity of a protein in genetic, metabolic, and physical interaction networks. Moreover, we show that functional proteomics complements current gene annotation strategies through the assessment of proteome profile similarity, protein covariation, and reverse proteome profiling. Thus, our study reveals principles that govern protein expression and provides a genome-spanning resource for functional annotation.

Freeing Yeast from Alcohol Addiction (Just) to Make (It) Fat Instead.

Synthetically re-designing eukaryotic metabolism has proven immensely challenging, raising the question of whether evolution has metabolically hardwired eukaryotic cells. Yu et al. now report that, through orchestrating multiple genetic changes and laboratory evolution, Saccharomyces metabolism can be reprogrammed from its evolutionary objective of producing ethanol to produce large amounts of free fatty acids.

Changes of Cell Biochemical States Are Revealed in Protein Homomeric Complex Dynamics.

We report here a simple and global strategy to map out gene functions and target pathways of drugs, toxins, or other small molecules based on "homomer dynamics" protein-fragment complementation assays (hdPCA). hdPCA measures changes in self-association (homomerization) of over 3,500 yeast proteins in yeast grown under different conditions. hdPCA complements genetic interaction measurements while eliminating the confounding effects of gene ablation. We demonstrate that hdPCA accurately predicts the effects of two longevity and health span-affecting drugs, the immunosuppressant rapamycin and the type 2 diabetes drug metformin, on cellular pathways. We also discovered an unsuspected global cellular response to metformin that resembles iron deficiency and includes a change in protein-bound iron levels. This discovery opens a new avenue to investigate molecular mechanisms for the prevention or treatment of diabetes, cancers, and other chronic diseases of aging.

Functional proteomic profiling links deficient DNA clearance with increased mortality in individuals with severe COVID-19 pneumonia.

The factors that influence survival during severe infection are unclear. Extracellular chromatin drives pathology, but the mechanisms enabling its accumulation remain elusive. Here, we show that in murine sepsis models, splenocyte death interferes with chromatin clearance through the release of the DNase I inhibitor actin. Actin-mediated inhibition was compensated by upregulation of DNase I or the actin scavenger gelsolin. Splenocyte death and neutrophil extracellular trap (NET) clearance deficiencies were prevalent in individuals with severe COVID-19 pneumonia or microbial sepsis. Activity tracing by plasma proteomic profiling uncovered an association between low NET clearance and increased COVID-19 pathology and mortality. Low NET clearance activity with comparable proteome associations was prevalent in healthy donors with low-grade inflammation, implicating defective chromatin clearance in the development of cardiovascular disease and linking COVID-19 susceptibility to pre-existing conditions. Hence, the combination of aberrant chromatin release with defects in protective clearance mechanisms lead to poor survival outcomes.

Functional Metabolomics Describes the Yeast Biosynthetic Regulome.

Genome-metabolism interactions enable cell growth. To probe the extent of these interactions and delineate their functional contributions, we quantified the Saccharomyces amino acid metabolome and its response to systematic gene deletion. Over one-third of coding genes, in particular those important for chromatin dynamics, translation, and transport, contribute to biosynthetic metabolism. Specific amino acid signatures characterize genes of similar function. This enabled us to exploit functional metabolomics to connect metabolic regulators to their effectors, as exemplified by TORC1, whose inhibition in exponentially growing cells is shown to match an interruption in endomembrane transport. Providing orthogonal information compared to physical and genetic interaction networks, metabolomic signatures cluster more than half of the so far uncharacterized yeast genes and provide functional annotation for them. A major part of coding genes is therefore participating in gene-metabolism interactions that expose the metabolism regulatory network and enable access to an underexplored space in gene function.

Natural proteome diversity links aneuploidy tolerance to protein turnover.

Accessing the natural genetic diversity of species unveils hidden genetic traits, clarifies gene functions and allows the generalizability of laboratory findings to be assessed. One notable discovery made in natural isolates of Saccharomyces cerevisiae is that aneuploidy-an imbalance in chromosome copy numbers-is frequent1,2 (around 20%), which seems to contradict the substantial fitness costs and transient nature of aneuploidy when it is engineered in the laboratory3-5. Here we generate a proteomic resource and merge it with genomic1 and transcriptomic6 data for 796 euploid and aneuploid natural isolates. We find that natural and lab-generated aneuploids differ specifically at the proteome. In lab-generated aneuploids, some proteins-especially subunits of protein complexes-show reduced expression, but the overall protein levels correspond to the aneuploid gene dosage. By contrast, in natural isolates, more than 70% of proteins encoded on aneuploid chromosomes are dosage compensated, and average protein levels are shifted towards the euploid state chromosome-wide. At the molecular level, we detect an induction of structural components of the proteasome, increased levels of ubiquitination, and reveal an interdependency of protein turnover rates and attenuation. Our study thus highlights the role of protein turnover in mediating aneuploidy tolerance, and shows the utility of exploiting the natural diversity of species to attain generalizable molecular insights into complex biological processes.

Virus-induced senescence is a driver and therapeutic target in COVID-19.

Derailed cytokine and immune cell networks account for the organ damage and the clinical severity of COVID-19 (refs. 1-4). Here we show that SARS-CoV-2, like other viruses, evokes cellular senescence as a primary stress response in infected cells. Virus-induced senescence (VIS) is indistinguishable from other forms of cellular senescence and is accompanied by a senescence-associated secretory phenotype (SASP), which comprises pro-inflammatory cytokines, extracellular-matrix-active factors and pro-coagulatory mediators5-7. Patients with COVID-19 displayed markers of senescence in their airway mucosa in situ and increased serum levels of SASP factors. In vitro assays demonstrated macrophage activation with SASP-reminiscent secretion, complement lysis and SASP-amplifying secondary senescence of endothelial cells, which mirrored hallmark features of COVID-19 such as macrophage and neutrophil infiltration, endothelial damage and widespread thrombosis in affected lung tissue1,8,9. Moreover, supernatant from VIS cells, including SARS-CoV-2-induced senescence, induced neutrophil extracellular trap formation and activation of platelets and the clotting cascade. Senolytics such as navitoclax and a combination of dasatinib plus quercetin selectively eliminated VIS cells, mitigated COVID-19-reminiscent lung disease and reduced inflammation in SARS-CoV-2-infected hamsters and mice. Our findings mark VIS as a pathogenic trigger of COVID-19-related cytokine escalation and organ damage, and suggest that senolytic targeting of virus-infected cells is a treatment option against SARS-CoV-2 and perhaps other viral infections.

The metabolic domestication syndrome of budding yeast.

Cellular metabolism evolves through changes in the structure and quantitative states of metabolic networks. Here, we explore the evolutionary dynamics of metabolic states by focusing on the collection of metabolite levels, the metabolome, which captures key aspects of cellular physiology. Using a phylogenetic framework, we profiled metabolites in 27 populations of nine budding yeast species, providing a graduated view of metabolic variation across multiple evolutionary time scales. Metabolite levels evolve more rapidly and independently of changes in the metabolic network's structure, providing complementary information to enzyme repertoire. Although metabolome variation accumulates mainly gradually over time, it is profoundly affected by domestication. We found pervasive signatures of convergent evolution in the metabolomes of independently domesticated clades of Saccharomyces cerevisiae. Such recurring metabolite differences between wild and domesticated populations affect a substantial part of the metabolome, including rewiring of the TCA cycle and several amino acids that influence aroma production, likely reflecting adaptation to human niches. Overall, our work reveals previously unrecognized diversity in central metabolism and the pervasive influence of human-driven selection on metabolite levels in yeasts.

Species-wide quantitative transcriptomes and proteomes reveal distinct genetic control of gene expression variation in yeast.

Gene expression varies between individuals and corresponds to a key step linking genotypes to phenotypes. However, our knowledge regarding the species-wide genetic control of protein abundance, including its dependency on transcript levels, is very limited. Here, we have determined quantitative proteomes of a large population of 942 diverse natural Saccharomyces cerevisiae yeast isolates. We found that mRNA and protein abundances are weakly correlated at the population gene level. While the protein coexpression network recapitulates major biological functions, differential expression patterns reveal proteomic signatures related to specific populations. Comprehensive genetic association analyses highlight that genetic variants associated with variation in protein (pQTL) and transcript (eQTL) levels poorly overlap (3%). Our results demonstrate that transcriptome and proteome are governed by distinct genetic bases, likely explained by protein turnover. It also highlights the importance of integrating these different levels of gene expression to better understand the genotype-phenotype relationship.

Spontaneously established syntrophic yeast communities improve bioproduction.

Nutritional codependence (syntrophy) has underexplored potential to improve biotechnological processes by using cooperating cell types. So far, design of yeast syntrophic communities has required extensive genetic manipulation, as the co-inoculation of most eukaryotic microbial auxotrophs does not result in cooperative growth. Here we employ high-throughput phenotypic screening to systematically test pairwise combinations of auxotrophic Saccharomyces cerevisiae deletion mutants. Although most coculture pairs do not enter syntrophic growth, we identify 49 pairs that spontaneously form syntrophic, synergistic communities. We characterized the stability and growth dynamics of nine cocultures and demonstrated that a pair of tryptophan auxotrophs grow by exchanging a pathway intermediate rather than end products. We then introduced a malonic semialdehyde biosynthesis pathway split between different pairs of auxotrophs, which resulted in increased production. Our results report the spontaneous formation of stable syntrophy in S. cerevisiae auxotrophs and illustrate the biotechnological potential of dividing labor in a cooperating intraspecies community.

Inorganic sulfur fixation via a new homocysteine synthase allows yeast cells to cooperatively compensate for methionine auxotrophy.

The assimilation, incorporation, and metabolism of sulfur is a fundamental process across all domains of life, yet how cells deal with varying sulfur availability is not well understood. We studied an unresolved conundrum of sulfur fixation in yeast, in which organosulfur auxotrophy caused by deletion of the homocysteine synthase Met17p is overcome when cells are inoculated at high cell density. In combining the use of self-establishing metabolically cooperating (SeMeCo) communities with proteomic, genetic, and biochemical approaches, we discovered an uncharacterized gene product YLL058Wp, herein named Hydrogen Sulfide Utilizing-1 (HSU1). Hsu1p acts as a homocysteine synthase and allows the cells to substitute for Met17p by reassimilating hydrosulfide ions leaked from met17Δ cells into O-acetyl-homoserine and forming homocysteine. Our results show that cells can cooperate to achieve sulfur fixation, indicating that the collective properties of microbial communities facilitate their basic metabolic capacity to overcome sulfur limitation.

Lysine harvesting is an antioxidant strategy and triggers underground polyamine metabolism.

Both single and multicellular organisms depend on anti-stress mechanisms that enable them to deal with sudden changes in the environment, including exposure to heat and oxidants. Central to the stress response are dynamic changes in metabolism, such as the transition from the glycolysis to the pentose phosphate pathway-a conserved first-line response to oxidative insults1,2. Here we report a second metabolic adaptation that protects microbial cells in stress situations. The role of the yeast polyamine transporter Tpo1p3-5 in maintaining oxidant resistance is unknown6. However, a proteomic time-course experiment suggests a link to lysine metabolism. We reveal a connection between polyamine and lysine metabolism during stress situations, in the form of a promiscuous enzymatic reaction in which the first enzyme of the polyamine pathway, Spe1p, decarboxylates lysine and forms an alternative polyamine, cadaverine. The reaction proceeds in the presence of extracellular lysine, which is taken up by cells to reach concentrations up to one hundred times higher than those required for growth. Such extensive harvest is not observed for the other amino acids, is dependent on the polyamine pathway and triggers a reprogramming of redox metabolism. As a result, NADPH-which would otherwise be required for lysine biosynthesis-is channelled into glutathione metabolism, leading to a large increase in glutathione concentrations, lower levels of reactive oxygen species and increased oxidant tolerance. Our results show that nutrient uptake occurs not only to enable cell growth, but when the nutrient availability is favourable it also enables cells to reconfigure their metabolism to preventatively mount stress protection.

Fast proteomics with dia-PASEF and analytical flow-rate chromatography.

Increased throughput in proteomic experiments can improve accessibility of proteomic platforms, reduce costs, and facilitate new approaches in systems biology and biomedical research. Here we propose combination of analytical flow rate chromatography with ion mobility separation of peptide ions, data-independent acquisition, and data analysis with the DIA-NN software suite, to achieve high-quality proteomic experiments from limited sample amounts, at a throughput of up to 400 samples per day. For instance, when benchmarking our workflow using a 500-µL/min flow rate and 3-min chromatographic gradients, we report the quantification of 5211 proteins from 2 µg of a mammalian cell-line standard at high quantitative accuracy and precision. We further used this platform to analyze blood plasma samples from a cohort of COVID-19 inpatients, using a 3-min chromatographic gradient and alternating column regeneration on a dual pump system. The method delivered a comprehensive view of the COVID-19 plasma proteome, allowing classification of the patients according to disease severity and revealing plasma biomarker candidates.

Cysteine and iron accelerate the formation of ribose-5-phosphate, providing insights into the evolutionary origins of the metabolic network structure.

The structure of the metabolic network is highly conserved, but we know little about its evolutionary origins. Key for explaining the early evolution of metabolism is solving a chicken-egg dilemma, which describes that enzymes are made from the very same molecules they produce. The recent discovery of several nonenzymatic reaction sequences that topologically resemble central metabolism has provided experimental support for a "metabolism first" theory, in which at least part of the extant metabolic network emerged on the basis of nonenzymatic reactions. But how could evolution kick-start on the basis of a metal catalyzed reaction sequence, and how could the structure of nonenzymatic reaction sequences be imprinted on the metabolic network to remain conserved for billions of years? We performed an in vitro screening where we add the simplest components of metabolic enzymes, proteinogenic amino acids, to a nonenzymatic, iron-driven reaction network that resembles glycolysis and the pentose phosphate pathway (PPP). We observe that the presence of the amino acids enhanced several of the nonenzymatic reactions. Particular attention was triggered by a reaction that resembles a rate-limiting step in the oxidative PPP. A prebiotically available, proteinogenic amino acid cysteine accelerated the formation of RNA nucleoside precursor ribose-5-phosphate from 6-phosphogluconate. We report that iron and cysteine interact and have additive effects on the reaction rate so that ribose-5-phosphate forms at high specificity under mild, metabolism typical temperature and environmental conditions. We speculate that accelerating effects of amino acids on rate-limiting nonenzymatic reactions could have facilitated a stepwise enzymatization of nonenzymatic reaction sequences, imprinting their structure on the evolving metabolic network.

Mass spectrometry-based high-throughput proteomics and its role in biomedical studies and systems biology.

There are multiple reasons why the next generation of biological and medical studies require increasing numbers of samples. Biological systems are dynamic, and the effect of a perturbation depends on the genetic background and environment. As a consequence, many conditions need to be considered to reach generalizable conclusions. Moreover, human population and clinical studies only reach sufficient statistical power if conducted at scale and with precise measurement methods. Finally, many proteins remain without sufficient functional annotations, because they have not been systematically studied under a broad range of conditions. In this review, we discuss the latest technical developments in mass spectrometry (MS)-based proteomics that facilitate large-scale studies by fast and efficient chromatography, fast scanning mass spectrometers, data-independent acquisition (DIA), and new software. We further highlight recent studies which demonstrate how high-throughput (HT) proteomics can be applied to capture biological diversity, to annotate gene functions or to generate predictive and prognostic models for human diseases.

The brain reacting to COVID-19: analysis of the cerebrospinal fluid proteome, RNA and inflammation.

Patients with COVID-19 can have a variety of neurological symptoms, but the active involvement of central nervous system (CNS) in COVID-19 remains unclear. While routine cerebrospinal fluid (CSF) analyses in patients with neurological manifestations of COVID-19 generally show no or only mild inflammation, more detailed data on inflammatory mediators in the CSF of patients with COVID-19 are scarce. We studied the inflammatory response in paired CSF and serum samples of patients with COVID-19 (n = 38). Patients with herpes simplex virus encephalitis (HSVE, n = 10) and patients with non-inflammatory, non-neurodegenerative neurological diseases (n = 28) served as controls. We used proteomics, enzyme-linked immunoassays, and semiquantitative cytokine arrays to characterize inflammatory proteins. Autoantibody screening was performed with cell-based assays and native tissue staining. RNA sequencing of long-non-coding RNA and circular RNA was done to study the transcriptome. Proteomics on single protein level and subsequent pathway analysis showed similar yet strongly attenuated inflammatory changes in the CSF of COVID-19 patients compared to HSVE patients with, e.g., downregulation of the apolipoproteins and extracellular matrix proteins. Protein upregulation of the complement system, the serpin proteins pathways, and other proteins including glycoproteins alpha-2 and alpha-1 acid. Importantly, calculation of interleukin-6, interleukin-16, and CXCL10 CSF/serum indices suggest that these inflammatory mediators reach the CSF from the systemic circulation, rather than being produced within the CNS. Antibody screening revealed no pathological levels of known neuronal autoantibodies. When stratifying COVID-19 patients into those with and without bacterial superinfection as indicated by elevated procalcitonin levels, inflammatory markers were significantly (p < 0.01) higher in those with bacterial superinfection. RNA sequencing in the CSF revealed 101 linear RNAs comprising messenger RNAs, and two circRNAs being significantly differentially expressed in COVID-19 than in non-neuroinflammatory controls and neurodegenerative patients. Our findings may explain the absence of signs of intrathecal inflammation upon routine CSF testing despite the presence of SARS-CoV2 infection-associated neurological symptoms. The relevance of blood-derived mediators of inflammation in the CSF for neurological COVID-19 and post-COVID-19 symptoms deserves further investigation.

DIA-NN: neural networks and interference correction enable deep proteome coverage in high throughput.

We present an easy-to-use integrated software suite, DIA-NN, that exploits deep neural networks and new quantification and signal correction strategies for the processing of data-independent acquisition (DIA) proteomics experiments. DIA-NN improves the identification and quantification performance in conventional DIA proteomic applications, and is particularly beneficial for high-throughput applications, as it is fast and enables deep and confident proteome coverage when used in combination with fast chromatographic methods.

Metabolic Remodeling in Times of Stress: Who Shoots Faster than His Shadow?

A sudden increase in pentose phosphate pathway (PPP) activity, the fastest known cellular response to oxidative stress, protects cells through timely generation of NADPH. Originally discovered in budding yeast, Kuehne and colleagues demonstrate the conservation of this mechanism in human cells and reveal its importance for skin cells exposed to UV light.

Ribosome profiling reveals ribosome stalling on tryptophan codons and ribosome queuing upon oxidative stress in fission yeast.

Translational control is essential in response to stress. We investigated the translational programmes launched by the fission yeast Schizosaccharomyces pombe upon five environmental stresses. We also explored the contribution of defence pathways to these programmes: The Integrated Stress Response (ISR), which regulates translation initiation, and the stress-response MAPK pathway. We performed ribosome profiling of cells subjected to each stress, in wild type cells and in cells with the defence pathways inactivated. The transcription factor Fil1, a functional homologue of the yeast Gcn4 and the mammalian Atf4 proteins, was translationally upregulated and required for the response to most stresses. Moreover, many mRNAs encoding proteins required for ribosome biogenesis were translationally downregulated. Thus, several stresses trigger a universal translational response, including reduced ribosome production and a Fil1-mediated transcriptional programme. Surprisingly, ribosomes stalled on tryptophan codons upon oxidative stress, likely due to a decrease in charged tRNA-Tryptophan. Stalling caused ribosome accumulation upstream of tryptophan codons (ribosome queuing/collisions), demonstrating that stalled ribosomes affect translation elongation by other ribosomes. Consistently, tryptophan codon stalling led to reduced translation elongation and contributed to the ISR-mediated inhibition of initiation. We show that different stresses elicit common and specific translational responses, revealing a novel role in Tryptophan-tRNA availability.