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.

Complement activation induces excessive T cell cytotoxicity in severe COVID-19.

Severe COVID-19 is linked to both dysfunctional immune response and unrestrained immunopathology, and it remains unclear whether T cells contribute to disease pathology. Here, we combined single-cell transcriptomics and single-cell proteomics with mechanistic studies to assess pathogenic T cell functions and inducing signals. We identified highly activated CD16+ T cells with increased cytotoxic functions in severe COVID-19. CD16 expression enabled immune-complex-mediated, T cell receptor-independent degranulation and cytotoxicity not found in other diseases. CD16+ T cells from COVID-19 patients promoted microvascular endothelial cell injury and release of neutrophil and monocyte chemoattractants. CD16+ T cell clones persisted beyond acute disease maintaining their cytotoxic phenotype. Increased generation of C3a in severe COVID-19 induced activated CD16+ cytotoxic T cells. Proportions of activated CD16+ T cells and plasma levels of complement proteins upstream of C3a were associated with fatal outcome of COVID-19, supporting a pathological role of exacerbated cytotoxicity and complement activation in COVID-19.

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.

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.

TF-FVIIa PAR2-ß-Arrestin Signaling Sustains Organ Dysfunction in Coxsackievirus B3 Infection of Mice.

BACKGROUND: Accumulating evidence implicates the activation of G-protein-coupled PARs (protease-activated receptors) by coagulation proteases in the regulation of innate immune responses. METHODS: Using mouse models with genetic alterations of the PAR2 signaling platform, we have explored contributions of PAR2 signaling to infection with coxsackievirus B3, a single-stranded RNA virus provoking multiorgan tissue damage, including the heart. RESULTS: We show that PAR2 activation sustains correlates of severe morbidity-hemodynamic compromise, aggravated hypothermia, and hypoglycemia-despite intact control of the virus. Following acute viral liver injury, canonical PAR2 signaling impairs the restoration process associated with exaggerated type I IFN (interferon) signatures in response to viral RNA recognition. Metabolic profiling in combination with proteomics of liver tissue shows PAR2-dependent reprogramming of liver metabolism, increased lipid droplet storage, and gluconeogenesis. PAR2-sustained hypodynamic compromise, reprograming of liver metabolism, as well as imbalanced IFN responses are prevented in ß-arrestin coupling-deficient PAR2 C-terminal phosphorylation mutant mice. Thus, wiring between upstream proteases and immune-metabolic responses results from biased PAR2 signaling mediated by intracellular recruitment of ß-arrestin. Importantly, blockade of the TF (tissue factor)-FVIIa (coagulation factor VIIa) complex capable of PAR2 proteolysis with the NAPc2 (nematode anticoagulant protein c2) mitigated virus-triggered pathology, recapitulating effects seen in protease cleavage-resistant PAR2 mice. CONCLUSIONS: These data provide insights into a TF-FVIIa signaling axis through PAR2-ß-arrestin coupling that is a regulator of inflammation-triggered tissue repair and hemodynamic compromise in coxsackievirus B3 infection and can potentially be targeted with selective coagulation inhibitors.

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.

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.

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.

Spermidine reduces neuroinflammation and soluble amyloid beta in an Alzheimer's disease mouse model.

BACKGROUND: Deposition of amyloid beta (Aß) and hyperphosphorylated tau along with glial cell-mediated neuroinflammation are prominent pathogenic hallmarks of Alzheimer's disease (AD). In recent years, impairment of autophagy has been identified as another important feature contributing to AD progression. Therefore, the potential of the autophagy activator spermidine, a small body-endogenous polyamine often used as dietary supplement, was assessed on Aß pathology and glial cell-mediated neuroinflammation. RESULTS: Oral treatment of the amyloid prone AD-like APPPS1 mice with spermidine reduced neurotoxic soluble Aß and decreased AD-associated neuroinflammation. Mechanistically, single nuclei sequencing revealed AD-associated microglia to be the main target of spermidine. This microglia population was characterized by increased AXL levels and expression of genes implicated in cell migration and phagocytosis. A subsequent proteome analysis of isolated microglia confirmed the anti-inflammatory and cytoskeletal effects of spermidine in APPPS1 mice. In primary microglia and astrocytes, spermidine-induced autophagy subsequently affected TLR3- and TLR4-mediated inflammatory processes, phagocytosis of Aß and motility. Interestingly, spermidine regulated the neuroinflammatory response of microglia beyond transcriptional control by interfering with the assembly of the inflammasome. CONCLUSIONS: Our data highlight that the autophagy activator spermidine holds the potential to enhance Aß degradation and to counteract glia-mediated neuroinflammation in AD pathology.

Pyruvate kinase variant of fission yeast tunes carbon metabolism, cell regulation, growth and stress resistance.

Cells balance glycolysis with respiration to support their metabolic needs in different environmental or physiological contexts. With abundant glucose, many cells prefer to grow by aerobic glycolysis or fermentation. Using 161 natural isolates of fission yeast, we investigated the genetic basis and phenotypic effects of the fermentation-respiration balance. The laboratory and a few other strains depended more on respiration. This trait was associated with a single nucleotide polymorphism in a conserved region of Pyk1, the sole pyruvate kinase in fission yeast. This variant reduced Pyk1 activity and glycolytic flux. Replacing the "low-activity" pyk1 allele in the laboratory strain with the "high-activity" allele was sufficient to increase fermentation and decrease respiration. This metabolic rebalancing triggered systems-level adjustments in the transcriptome and proteome and in cellular traits, including increased growth and chronological lifespan but decreased resistance to oxidative stress. Thus, low Pyk1 activity does not lead to a growth advantage but to stress tolerance. The genetic tuning of glycolytic flux may reflect an adaptive trade-off in a species lacking pyruvate kinase isoforms.

Low catalytic activity is insufficient to induce disease pathology in triosephosphate isomerase deficiency.

Triosephosphate isomerase (TPI) deficiency is a fatal genetic disorder characterized by hemolytic anemia and neurological dysfunction. Although the enzyme defect in TPI was discovered in the 1960s, the exact etiology of the disease is still debated. Some aspects indicate the disease could be caused by insufficient enzyme activity, whereas other observations indicate it could be a protein misfolding disease with tissue-specific differences in TPI activity. We generated a mouse model in which exchange of a conserved catalytic amino acid residue (isoleucine to valine, Ile170Val) reduces TPI specific activity without affecting the stability of the protein dimer. TPIIle170Val/Ile170Val mice exhibit an approximately 85% reduction in TPI activity consistently across all examined tissues, which is a stronger average, but more consistent, activity decline than observed in patients or symptomatic mouse models that carry structural defect mutant alleles. While monitoring protein expression levels revealed no evidence for protein instability, metabolite quantification indicated that glycolysis is affected by the active site mutation. TPIIle170Val/Ile170Val mice develop normally and show none of the disease symptoms associated with TPI deficiency. Therefore, without the stability defect that affects TPI activity in a tissue-specific manner, a strong decline in TPI catalytic activity is not sufficient to explain the pathological onset of TPI deficiency.

Tpo1-mediated spermine and spermidine export controls cell cycle delay and times antioxidant protein expression during the oxidative stress response.

Cells counteract oxidative stress by altering metabolism, cell cycle and gene expression. However, the mechanisms that coordinate these adaptations are only marginally understood. Here we provide evidence that timing of these responses in yeast requires export of the polyamines spermidine and spermine. We show that during hydrogen peroxide (H2O2) exposure, the polyamine transporter Tpo1 controls spermidine and spermine concentrations and mediates induction of antioxidant proteins, including Hsp70, Hsp90, Hsp104 and Sod1. Moreover, Tpo1 determines a cell cycle delay during adaptation to increased oxidant levels, and affects H2O2 tolerance. Thus, central components of the stress response are timed through Tpo1-controlled polyamine export.

Cytosine DNA methylation is found in Drosophila melanogaster but absent in Saccharomyces cerevisiae, Schizosaccharomyces pombe, and other yeast species.

The methylation of cytosine to 5-methylcytosine (5-meC) is an important epigenetic DNA modification in many bacteria, plants, and mammals, but its relevance for important model organisms, including Caenorhabditis elegans and Drosophila melanogaster, is still equivocal. By reporting the presence of 5-meC in a broad variety of wild, laboratory, and industrial yeasts, a recent study also challenged the dogma about the absence of DNA methylation in yeast species. We would like to bring to attention that the protocol used for gas chromatography/mass spectrometry involved hydrolysis of the DNA preparations. As this process separates cytosine and 5-meC from the sugar phosphate backbone, this method is unable to distinguish DNA- from RNA-derived 5-meC. We employed an alternative LC-MS/MS protocol where by targeting 5-methyldeoxycytidine moieties after enzymatic digestion, only 5-meC specifically derived from DNA is quantified. This technique unambiguously identified cytosine DNA methylation in Arabidopsis thaliana (14.0% of cytosines methylated), Mus musculus (7.6%), and Escherichia coli (2.3%). Despite achieving a detection limit at 250 attomoles (corresponding to <0.00002 methylated cytosines per nonmethylated cytosine), we could not confirm any cytosine DNA methylation in laboratory and industrial strains of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Saccharomyces boulardii, Saccharomyces paradoxus, or Pichia pastoris. The protocol however unequivocally confirmed DNA methylation in adult Drosophila melanogaster at a value (0.034%) that is up to 2 orders of magnitude below the detection limit of bisulphite sequencing. Thus, 5-meC is a rare DNA modification in drosophila but absent in yeast.