Disentangling Genetic and Environmental Effects on the Proteotypes of Individuals.

Proteotypes, like genotypes, have been found to vary between individuals in several studies, but consistent molecular functional traits across studies remain to be quantified. In a meta-analysis of 11 proteomics datasets from humans and mice, we use co-variation of proteins in known functional modules across datasets and individuals to obtain a consensus landscape of proteotype variation. We find that individuals differ considerably in both protein complex abundances and stoichiometry. We disentangle genetic and environmental factors impacting these metrics, with genetic sex and specific diets together explaining 13.5% and 11.6% of the observed variation of complex abundance and stoichiometry, respectively. Sex-specific differences, for example, include various proteins and complexes, where the respective genes are not located on sex-specific chromosomes. Diet-specific differences, added to the individual genetic backgrounds, might become a starting point for personalized proteotype modulation toward desired features.

Pervasive Protein Thermal Stability Variation during the Cell Cycle.

Quantitative mass spectrometry has established proteome-wide regulation of protein abundance and post-translational modifications in various biological processes. Here, we used quantitative mass spectrometry to systematically analyze the thermal stability and solubility of proteins on a proteome-wide scale during the eukaryotic cell cycle. We demonstrate pervasive variation of these biophysical parameters with most changes occurring in mitosis and G1. Various cellular pathways and components vary in thermal stability, such as cell-cycle factors, polymerases, and chromatin remodelers. We demonstrate that protein thermal stability serves as a proxy for enzyme activity, DNA binding, and complex formation in situ. Strikingly, a large cohort of intrinsically disordered and mitotically phosphorylated proteins is stabilized and solubilized in mitosis, suggesting a fundamental remodeling of the biophysical environment of the mitotic cell. Our data represent a rich resource for cell, structural, and systems biologists interested in proteome regulation during biological transitions.

Decoding the function of Atg13 phosphorylation reveals a role of Atg11 in bulk autophagy initiation.

Autophagy is initiated by the assembly of multiple autophagy-related proteins that form the phagophore assembly site where autophagosomes are formed. Atg13 is essential early in this process, and a hub of extensive phosphorylation. How these multiple phosphorylations contribute to autophagy initiation, however, is not well understood. Here we comprehensively analyze the role of phosphorylation events on Atg13 during nutrient-rich conditions and nitrogen starvation. We identify and functionally characterize 48 in vivo phosphorylation sites on Atg13. By generating reciprocal mutants, which mimic the dephosphorylated active and phosphorylated inactive state of Atg13, we observe that disrupting the dynamic regulation of Atg13 leads to insufficient or excessive autophagy, which are both detrimental to cell survival. We furthermore demonstrate an involvement of Atg11 in bulk autophagy even during nitrogen starvation, where it contributes together with Atg1 to the multivalency that drives phase separation of the phagophore assembly site. These findings reveal the importance of post-translational regulation on Atg13 early during autophagy initiation, which provides additional layers of regulation to control bulk autophagy activity and integrate cellular signals.

A phosphatase-centric mechanism drives stress signaling response.

Changing environmental cues lead to the adjustment of cellular physiology by phosphorylation signaling networks that typically center around kinases as active effectors and phosphatases as antagonistic elements. Here, we report a signaling mechanism that reverses this principle. Using the hyperosmotic stress response in Saccharomyces cerevisiae as a model system, we find that a phosphatase-driven mechanism causes induction of phosphorylation. The key activating step that triggers this phospho-proteomic response is the Endosulfine-mediated inhibition of protein phosphatase 2A-Cdc55 (PP2ACdc55 ), while we do not observe concurrent kinase activation. In fact, many of the stress-induced phosphorylation sites appear to be direct substrates of the phosphatase, rendering PP2ACdc55 the main downstream effector of a signaling response that operates in parallel and independent of the well-established kinase-centric stress signaling pathways. This response affects multiple cellular processes and is required for stress survival. Our results demonstrate how a phosphatase can assume the role of active downstream effectors during signaling and allow re-evaluating the impact of phosphatases on shaping the phosphorylome.

Reduced proteasome activity in the aging brain results in ribosome stoichiometry loss and aggregation.

A progressive loss of protein homeostasis is characteristic of aging and a driver of neurodegeneration. To investigate this process quantitatively, we characterized proteome dynamics during brain aging in the short-lived vertebrate Nothobranchius furzeri combining transcriptomics and proteomics. We detected a progressive reduction in the correlation between protein and mRNA, mainly due to post-transcriptional mechanisms that account for over 40% of the age-regulated proteins. These changes cause a progressive loss of stoichiometry in several protein complexes, including ribosomes, which show impaired assembly/disassembly and are enriched in protein aggregates in old brains. Mechanistically, we show that reduction of proteasome activity is an early event during brain aging and is sufficient to induce proteomic signatures of aging and loss of stoichiometry in vivo. Using longitudinal transcriptomic data, we show that the magnitude of early life decline in proteasome levels is a major risk factor for mortality. Our work defines causative events in the aging process that can be targeted to prevent loss of protein homeostasis and delay the onset of age-related neurodegeneration.

Novel interconnections of HOG signaling revealed by combined use of two proteomic software packages.

Modern quantitative mass spectrometry (MS)-based proteomics enables researchers to unravel signaling networks by monitoring proteome-wide cellular responses to different stimuli. MS-based analysis of signaling systems usually requires an integration of multiple quantitative MS experiments, which remains challenging, given that the overlap between these datasets is not necessarily comprehensive. In a previous study we analyzed the impact of the yeast mitogen-activated protein kinase (MAPK) Hog1 on the hyperosmotic stress-affected phosphorylome. Using a combination of a series of hyperosmotic stress and kinase inhibition experiments, we identified a broad range of direct and indirect substrates of the MAPK. Here we re-evaluate this extensive MS dataset and demonstrate that a combined analysis based on two software packages, MaxQuant and Proteome Discoverer, increases the coverage of Hog1-target proteins by 30%. Using protein-protein proximity assays we show that the majority of new targets gained by this analysis are indeed Hog1-interactors. Additionally, kinetic profiles indicate differential trends of Hog1-dependent versus Hog1-independent phosphorylation sites. Our findings highlight a previously unrecognized interconnection between Hog1 signaling and the RAM signaling network, as well as sphingolipid homeostasis.

Species comparison of liver proteomes reveals links to naked mole-rat longevity and human aging.

BACKGROUND: Mammals display a wide range of variation in their lifespan. Investigating the molecular networks that distinguish long- from short-lived species has proven useful to identify determinants of longevity. Here, we compared the livers of young and old long-lived naked mole-rats (NMRs) and the phylogenetically closely related, shorter-lived, guinea pigs using an integrated omics approach. RESULTS: We found that NMR livers display a unique expression pattern of mitochondrial proteins that results in distinct metabolic features of their mitochondria. For instance, we observed a generally reduced respiration rate associated with lower protein levels of respiratory chain components, particularly complex I, and increased capacity to utilize fatty acids. Interestingly, we show that the same molecular networks are affected during aging in both NMRs and humans, supporting a direct link to the extraordinary longevity of both species. Finally, we identified a novel detoxification pathway linked to longevity and validated it experimentally in the nematode Caenorhabditis elegans. CONCLUSIONS: Our work demonstrates the benefits of integrating proteomic and transcriptomic data to perform cross-species comparisons of longevity-associated networks. Using a multispecies approach, we show at the molecular level that livers of NMRs display progressive age-dependent changes that recapitulate typical signatures of aging despite the negligible senescence and extraordinary longevity of these rodents.

Capturing protein communities by structural proteomics in a thermophilic eukaryote.

The arrangement of proteins into complexes is a key organizational principle for many cellular functions. Although the topology of many complexes has been systematically analyzed in isolation, their molecular sociology in situ remains elusive. Here, we show that crude cellular extracts of a eukaryotic thermophile, Chaetomium thermophilum, retain basic principles of cellular organization. Using a structural proteomics approach, we simultaneously characterized the abundance, interactions, and structure of a third of the C. thermophilum proteome within these extracts. We identified 27 distinct protein communities that include 108 interconnected complexes, which dynamically associate with each other and functionally benefit from being in close proximity in the cell. Furthermore, we investigated the structure of fatty acid synthase within these extracts by cryoEM and this revealed multiple, flexible states of the enzyme in adaptation to its association with other complexes, thus exemplifying the need for in situ studies. As the components of the captured protein communities are known-at both the protein and complex levels-this study constitutes another step forward toward a molecular understanding of subcellular organization.

Bactericidal effect of tetracycline in E. coli strain ED1a may be associated with ribosome dysfunction.

Ribosomes translate the genetic code into proteins. Recent technical advances have facilitated in situ structural analyses of ribosome functional states inside eukaryotic cells and the minimal bacterium Mycoplasma. However, such analyses of Gram-negative bacteria are lacking, despite their ribosomes being major antimicrobial drug targets. Here we compare two E. coli strains, a lab E. coli K-12 and human gut isolate E. coli ED1a, for which tetracycline exhibits bacteriostatic and bactericidal action, respectively. Using our approach for close-to-native E. coli sample preparation, we assess the two strains by cryo-ET and visualize their ribosomes at high resolution in situ. Upon tetracycline treatment, these exhibit virtually identical drug binding sites, yet the conformation distribution of ribosomal complexes differs. While K-12 retains ribosomes in a translation-competent state, tRNAs are lost in the vast majority of ED1a ribosomes. These structural findings together with the proteome-wide abundance and thermal stability assessments indicate that antibiotic responses are complex in cells and can differ between different strains of a single species, thus arguing that all relevant bacterial strains should be analyzed in situ when addressing antibiotic mode of action.

Co-translational binding of importins to nascent proteins.

Various cellular quality control mechanisms support proteostasis. While, ribosome-associated chaperones prevent the misfolding of nascent chains during translation, importins were shown to prevent the aggregation of specific cargoes in a post-translational mechanism prior the import into the nucleoplasm. Here, we hypothesize that importins may already bind ribosome-associated cargo in a co-translational manner. We systematically measure the nascent chain association of all importins in Saccharomyces cerevisiae by selective ribosome profiling. We identify a subset of importins that bind to a wide range of nascent, often uncharacterized cargoes. This includes ribosomal proteins, chromatin remodelers and RNA binding proteins that are aggregation prone in the cytosol. We show that importins act consecutively with other ribosome-associated chaperones. Thus, the nuclear import system is directly intertwined with nascent chain folding and chaperoning.

Co-translational assembly orchestrates competing biogenesis pathways.

During the co-translational assembly of protein complexes, a fully synthesized subunit engages with the nascent chain of a newly synthesized interaction partner. Such events are thought to contribute to productive assembly, but their exact physiological relevance remains underexplored. Here, we examine structural motifs contained in nucleoporins for their potential to facilitate co-translational assembly. We experimentally test candidate structural motifs and identify several previously unknown co-translational interactions. We demonstrate by selective ribosome profiling that domain invasion motifs of beta-propellers, coiled-coils, and short linear motifs may act as co-translational assembly domains. Such motifs are often contained in proteins that are members of multiple complexes (moonlighters) and engage with closely related paralogs. Surprisingly, moonlighters and paralogs assemble co-translationally in only some but not all of the relevant biogenesis pathways. Our results highlight the regulatory complexity of assembly pathways.

Glycogen accumulation, central carbon metabolism, and aging of hematopoietic stem and progenitor cells.

Inspired by recent proteomic data demonstrating the upregulation of carbon and glycogen metabolism in aging human hematopoietic stem and progenitor cells (HPCs, CD34+ cells), this report addresses whether this is caused by elevated glycolysis of the HPCs on a per cell basis, or by a subpopulation that has become more glycolytic. The average glycogen content in individual CD34+ cells from older subjects (> 50 years) was 3.5 times higher and more heterogeneous compared to younger subjects (< 35 years). Representative glycolytic enzyme activities in HPCs confirmed a significant increase in glycolysis in older subjects. The HPCs from older subjects can be fractionated into three distinct subsets with high, intermediate, and low glucose uptake (GU) capacity, while the subset with a high GU capacity could scarcely be detected in younger subjects. Thus, we conclude that upregulated glycolysis in aging HPCs is caused by the expansion of a more glycolytic HPC subset. Since single-cell RNA analysis has also demonstrated that this subpopulation is linked to myeloid differentiation and increased proliferation, isolation and mechanistic characterization of this subpopulation can be utilized to elucidate specific targets for therapeutic interventions to restore the lineage balance of aging HPCs.

Cell-specific proteome analyses of human bone marrow reveal molecular features of age-dependent functional decline.

Diminishing potential to replace damaged tissues is a hallmark for ageing of somatic stem cells, but the mechanisms remain elusive. Here, we present proteome-wide atlases of age-associated alterations in human haematopoietic stem and progenitor cells (HPCs) and five other cell populations that constitute the bone marrow niche. For each, the abundance of a large fraction of the ~12,000 proteins identified is assessed in 59 human subjects from different ages. As the HPCs become older, pathways in central carbon metabolism exhibit features reminiscent of the Warburg effect, where glycolytic intermediates are rerouted towards anabolism. Simultaneously, altered abundance of early regulators of HPC differentiation reveals a reduced functionality and a bias towards myeloid differentiation. Ageing causes alterations in the bone marrow niche too, and diminishes the functionality of the pathways involved in HPC homing. The data represent a valuable resource for further analyses, and for validation of knowledge gained from animal models.

Identifying protein kinase-specific effectors of the osmostress response in yeast.

The budding yeast Saccharomyces cerevisiae reacts to increased external osmolarity by modifying many cellular processes. Adaptive signaling relies primarily on the high-osmolarity glycerol (HOG) pathway, which is closely related to the mammalian p38 mitogen-activated protein kinase (MAPK) pathway in core architecture. To identify target proteins of the MAPK Hog1, we designed a mass spectrometry-based high-throughput experiment to measure the impact of Hog1 activation or inhibition on the Scerevisiae phosphoproteome. In addition, we analyzed how deletion of RCK2, which encodes a known effector protein kinase target of Hog1, modulated osmotic stress-induced phosphorylation. Our results not only provide an overview of the diversity of cellular functions that are directly and indirectly affected by the activity of the HOG pathway but also enabled an assessment of the Hog1-independent events that occur under osmotic stress conditions. We extended the number of putative Hog1 direct targets by analyzing the modulation of motifs consisting of serine or threonine followed by a proline (S/T-P motif) and subsequently validated these with an in vivo interaction assay. Rck2 appears to act as a central hub for many Hog1-mediated secondary phosphorylation events. This study clarifies many of the direct and indirect effects of HOG signaling and its stress-adaptive functions.

Light-sensing via hydrogen peroxide and a peroxiredoxin.

Yeast lacks dedicated photoreceptors; however, blue light still causes pronounced oscillations of the transcription factor Msn2 into and out of the nucleus. Here we show that this poorly understood phenomenon is initiated by a peroxisomal oxidase, which converts light into a hydrogen peroxide (H2O2) signal that is sensed by the peroxiredoxin Tsa1 and transduced to thioredoxin, to counteract PKA-dependent Msn2 phosphorylation. Upon H2O2, the nuclear retention of PKA catalytic subunits, which contributes to delayed Msn2 nuclear concentration, is antagonized in a Tsa1-dependent manner. Conversely, peroxiredoxin hyperoxidation interrupts the H2O2 signal and drives Msn2 oscillations by superimposing on PKA feedback regulation. Our data identify a mechanism by which light could be sensed in all cells lacking dedicated photoreceptors. In particular, the use of H2O2 as a second messenger in signalling is common to Msn2 oscillations and to light-induced entrainment of circadian rhythms and suggests conserved roles for peroxiredoxins in endogenous rhythms.

A Staphylococcus aureus Proteome Overview: Shared and Specific Proteins and Protein Complexes from Representative Strains of All Three Clades.

Staphylococcus aureus is an important model organism and pathogen. This S. aureus proteome overview details shared and specific proteins and selected virulence-relevant protein complexes from representative strains of all three major clades. To determine the strain distribution and major clades we used a refined strain comparison combining ribosomal RNA, MLST markers, and looking at highly-conserved regions shared between strains. This analysis shows three sub-clades (A-C) for S. aureus. As calculations are complex and strain annotation is quite time consuming we compare here key representatives of each clade with each other: model strains COL, USA300, Newman, and HG001 (clade A), model strain N315 and Mu50 (clade B) and ED133 and MRSA252 (clade C). We look at these individual proteomes and compare them to a background of 64 S. aureus strains. There are overall 13,284 S. aureus proteins not part of the core proteome which are involved in different strain-specific or more general complexes requiring detailed annotation and new experimental data to be accurately delineated. By comparison of the eight representative strains, we identify strain-specific proteins (e.g., 18 in COL, 105 in N315 and 44 in Newman) that characterize each strain and analyze pathogenicity islands if they contain such strain-specific proteins. We identify strain-specific protein repertoires involved in virulence, in cell wall metabolism, and phosphorylation. Finally we compare and analyze protein complexes conserved and well-characterized among S. aureus (a total of 103 complexes), as well as predict and analyze several individual protein complexes, including structure modeling in the three clades.