Glucose stress causes mRNA retention in nuclear Nab2 condensates.

Nuclear mRNA export via nuclear pore complexes is an essential step in eukaryotic gene expression. Although factors involved in mRNA transport have been characterized, a comprehensive mechanistic understanding of this process and its regulation is lacking. Here, we use single-RNA imaging in yeast to show that cells use mRNA retention to control mRNA export during stress. We demonstrate that, upon glucose withdrawal, the essential RNA-binding factor Nab2 forms RNA-dependent condensate-like structures in the nucleus. This coincides with a reduced abundance of the DEAD-box ATPase Dbp5 at the nuclear pore. Depleting Dbp5, and consequently blocking mRNA export, is necessary and sufficient to trigger Nab2 condensation. The state of Nab2 condensation influences the extent of nuclear mRNA accumulation and can be recapitulated in vitro, where Nab2 forms RNA-dependent liquid droplets. We hypothesize that cells use condensation to regulate mRNA export and control gene expression during stress.

Proteome-wide structural changes measured with limited proteolysis-mass spectrometry: an advanced protocol for high-throughput applications.

Proteins regulate biological processes by changing their structure or abundance to accomplish a specific function. In response to a perturbation, protein structure may be altered by various molecular events, such as post-translational modifications, protein-protein interactions, aggregation, allostery or binding to other molecules. The ability to probe these structural changes in thousands of proteins simultaneously in cells or tissues can provide valuable information about the functional state of biological processes and pathways. Here, we present an updated protocol for LiP-MS, a proteomics technique combining limited proteolysis with mass spectrometry, to detect protein structural alterations in complex backgrounds and on a proteome-wide scale. In LiP-MS, proteins undergo a brief proteolysis in native conditions followed by complete digestion in denaturing conditions, to generate structurally informative proteolytic fragments that are analyzed by mass spectrometry. We describe advances in the throughput and robustness of the LiP-MS workflow and implementation of data-independent acquisition-based mass spectrometry, which together achieve high reproducibility and sensitivity, even on large sample sizes. We introduce MSstatsLiP, an R package dedicated to the analysis of LiP-MS data for the identification of structurally altered peptides and differentially abundant proteins. The experimental procedures take 3 d, mass spectrometric measurement time and data processing depend on sample number and statistical analysis typically requires ~1 d. These improvements expand the adaptability of LiP-MS and enable wide use in functional proteomics and translational applications.

Calreticulin mutations affect its chaperone function and perturb the glycoproteome.

Calreticulin (CALR) is an endoplasmic reticulum (ER)-retained chaperone that assists glycoproteins in obtaining their structure. CALR mutations occur in patients with myeloproliferative neoplasms (MPNs), and the ER retention of CALR mutants (CALR MUT) is reduced due to a lacking KDEL sequence. Here, we investigate the impact of CALR mutations on protein structure and protein levels in MPNs by subjecting primary patient samples and CALR-mutated cell lines to limited proteolysis-coupled mass spectrometry (LiP-MS). Especially glycoproteins are differentially expressed and undergo profound structural alterations in granulocytes and cell lines with homozygous, but not with heterozygous, CALR mutations. Furthermore, homozygous CALR mutations and loss of CALR equally perturb glycoprotein integrity, suggesting that loss-of-function attributes of mutated CALR chaperones (CALR MUT) lead to glycoprotein maturation defects. Finally, by investigating the misfolding of the CALR glycoprotein client myeloperoxidase (MPO), we provide molecular proof of protein misfolding in the presence of homozygous CALR mutations.

Dynamic 3D proteomes reveal protein functional alterations at high resolution in situ.

Biological processes are regulated by intermolecular interactions and chemical modifications that do not affect protein levels, thus escaping detection in classical proteomic screens. We demonstrate here that a global protein structural readout based on limited proteolysis-mass spectrometry (LiP-MS) detects many such functional alterations, simultaneously and in situ, in bacteria undergoing nutrient adaptation and in yeast responding to acute stress. The structural readout, visualized as structural barcodes, captured enzyme activity changes, phosphorylation, protein aggregation, and complex formation, with the resolution of individual regulated functional sites such as binding and active sites. Comparison with prior knowledge, including other 'omics data, showed that LiP-MS detects many known functional alterations within well-studied pathways. It suggested distinct metabolite-protein interactions and enabled identification of a fructose-1,6-bisphosphate-based regulatory mechanism of glucose uptake in E. coli. The structural readout dramatically increases classical proteomics coverage, generates mechanistic hypotheses, and paves the way for in situ structural systems biology.

Prevalence and Prognosis of Fever Symptoms, Hypo-, and Hyperthermia in Unselected Emergency Patients.

Assessments of history and body temperature are cornerstones of the diagnostic workup in all patients presenting to emergency departments (ED). Yet, the objective measurement of temperature and the subjective perception of fever can differ. This is a secondary exploratory analysis of a consecutive all-comer study, performed at an adult ED in Switzerland. Trained medical students interviewed all patients if fever was present. Altered temperature (>38.0 °C/<36.0 °C) measured at triage using an ear thermometer was used as the reference standard for diagnostic performance. In case of a disagreement between fever symptoms and altered temperature, discordance was noted. Outcome measures for case severity (acute morbidity, hospitalization, intensive care, and in-hospital mortality) were extracted from the electronic health records. Odds ratios (OR) for discordance between signs and symptoms and outcomes were calculated. Among 2183 patients, 325 patients reported fever symptoms. The sensitivity of fever symptoms as a test for altered temperature was 36.3%. Specificity was 91.5%. The negative predictive value was 84.1%, positive likelihood ratio was 4.2 and negative likelihood ratio was 0.7. The adjusted OR for discordance between fever symptoms and altered temperature was 1.71 (95% CI: 1.2-2.44) for acute morbidity, 1.56 (95% CI: 1.13-2.15) for hospitalization, and 1.12 (95% CI: 0.64-1.59) for intensive care. Unadjusted OR for mortality was 1.5 (95% CI: 0.69-3.25). Fever symptoms and altered temperature broadly overlap, but presentations can be stratified according to concordance between signs and symptoms. In case of discordance, the odds for acute morbidity and hospitalization are increased. Discordance may therefore be further investigated as a red flag for a serious outcome.

Proteomics-Based Monitoring of Pathway Activity Reveals that Blocking Diacylglycerol Biosynthesis Rescues from Alpha-Synuclein Toxicity.

Proteinaceous inclusions containing alpha-synuclein (α-Syn) have been implicated in neuronal toxicity in Parkinson's disease, but the pathways that modulate toxicity remain enigmatic. Here, we used a targeted proteomic assay to simultaneously measure 269 pathway activation markers and proteins deregulated by α-Syn expression across a panel of 33 Saccharomyces cerevisiae strains that genetically modulate α-Syn toxicity. Applying multidimensional linear regression analysis to these data predicted Pah1, a phosphatase that catalyzes conversion of phosphatidic acid to diacylglycerol at the endoplasmic reticulum membrane, as an effector of rescue. Follow-up studies demonstrated that inhibition of Pah1 activity ameliorates the toxic effects of α-Syn, indicate that the diacylglycerol branch of lipid metabolism could enhance α-Syn neuronal cytotoxicity, and suggest a link between α-Syn toxicity and the biology of lipid droplets.

ATP as a biological hydrotrope.

Hydrotropes are small molecules that solubilize hydrophobic molecules in aqueous solutions. Typically, hydrotropes are amphiphilic molecules and differ from classical surfactants in that they have low cooperativity of aggregation and work at molar concentrations. Here, we show that adenosine triphosphate (ATP) has properties of a biological hydrotrope. It can both prevent the formation of and dissolve previously formed protein aggregates. This chemical property is manifested at physiological concentrations between 5 and 10 millimolar. Therefore, in addition to being an energy source for biological reactions, for which micromolar concentrations are sufficient, we propose that millimolar concentrations of ATP may act to keep proteins soluble. This may in part explain why ATP is maintained in such high concentrations in cells.

A pH-driven transition of the cytoplasm from a fluid- to a solid-like state promotes entry into dormancy.

Cells can enter into a dormant state when faced with unfavorable conditions. However, how cells enter into and recover from this state is still poorly understood. Here, we study dormancy in different eukaryotic organisms and find it to be associated with a significant decrease in the mobility of organelles and foreign tracer particles. We show that this reduced mobility is caused by an influx of protons and a marked acidification of the cytoplasm, which leads to widespread macromolecular assembly of proteins and triggers a transition of the cytoplasm to a solid-like state with increased mechanical stability. We further demonstrate that this transition is required for cellular survival under conditions of starvation. Our findings have broad implications for understanding alternative physiological states, such as quiescence and dormancy, and create a new view of the cytoplasm as an adaptable fluid that can reversibly transition into a protective solid-like state.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging.

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Protein misfolding in Dictyostelium: Using a freak of nature to gain insight into a universal problem.

Prion-like proteins can undergo conformational rearrangements from an intrinsically disordered to a highly ordered amyloid state. This ability to change conformation is encoded in distinctive domains, termed prion domains (PrDs). Previous work suggests that PrDs change conformation to affect protein function and create phenotypic diversity. More recent work shows that PrDs can also undergo many weak interactions when disordered, allowing them to organize the intracellular space into dynamic compartments. However, mutations within PrDs and altered aggregation properties have also been linked to age-related diseases in humans. Thus, the physiological role of prion-like proteins, the mechanisms regulating their conformational promiscuity and the links to disease are still unclear. Here, we summarize recent work with prion-like proteins in Dictyostelium discoideum. This work was motivated by the finding that D. discoideum has the highest content of prion-like proteins of all organisms investigated to date. Surprisingly, we find that endogenous and exogenous prion-like proteins remain soluble in D. discoideum and do not misfold and aggregate. We provide evidence that this is due to specific adaptations in the protein quality control machinery, which may allow D. discoideum to tolerate its highly aggregation-prone proteome. We predict that D. discoideum will be an important model to study the function of prion-like proteins and their mechanistic links to disease.

Promiscuous interactions and protein disaggregases determine the material state of stress-inducible RNP granules.

RNA-protein (RNP) granules have been proposed to assemble by forming solid RNA/protein aggregates or through phase separation into a liquid RNA/protein phase. Which model describes RNP granules in living cells is still unclear. In this study, we analyze P bodies in budding yeast and find that they have liquid-like properties. Surprisingly, yeast stress granules adopt a different material state, which is reminiscent of solid protein aggregates and controlled by protein disaggregases. By using an assay to ectopically nucleate RNP granules, we further establish that RNP granule formation does not depend on amyloid-like aggregation but rather involves many promiscuous interactions. Finally, we show that stress granules have different properties in mammalian cells, where they show liquid-like behavior. Thus, we propose that the material state of RNP granules is flexible and that the solid state of yeast stress granules is an adaptation to extreme environments, made possible by the presence of a powerful disaggregation machine.

Dictyostelium discoideum has a highly Q/N-rich proteome and shows an unusual resilience to protein aggregation.

Many protein-misfolding diseases are caused by proteins carrying prion-like domains. These proteins show sequence similarity to yeast prion proteins, which can interconvert between an intrinsically disordered and an aggregated prion state. The natural presence of prions in yeast has provided important insight into disease mechanisms and cellular proteostasis. However, little is known about prions in other organisms, and it is not yet clear whether the findings in yeast can be generalized. Using bioinformatics tools, we show that Dictyostelium discoideum has the highest content of prion-like proteins of all organisms investigated to date, suggesting that its proteome has a high overall aggregation propensity. To study mechanisms regulating these proteins, we analyze the behavior of several well-characterized prion-like proteins, such as an expanded version of human huntingtin exon 1 (Q103) and the prion domain of the yeast prion protein Sup35 (NM), in D. discoideum. We find that these proteins remain soluble and are innocuous to D. discoideum, in contrast to other organisms, where they form cytotoxic cytosolic aggregates. However, when exposed to conditions that compromise molecular chaperones, these proteins aggregate and become cytotoxic. We show that the disaggregase Hsp101, a molecular chaperone of the Hsp100 family, dissolves heat-induced aggregates and promotes thermotolerance. Furthermore, prion-like proteins accumulate in the nucleus, where they are targeted by the ubiquitin-proteasome system. Our data suggest that D. discoideum has undergone specific adaptations that increase the proteostatic capacity of this organism and allow for an efficient regulation of its prion-like proteome.

Filament formation by metabolic enzymes is a specific adaptation to an advanced state of cellular starvation.

One of the key questions in biology is how the metabolism of a cell responds to changes in the environment. In budding yeast, starvation causes a drop in intracellular pH, but the functional role of this pH change is not well understood. Here, we show that the enzyme glutamine synthetase (Gln1) forms filaments at low pH and that filament formation leads to enzymatic inactivation. Filament formation by Gln1 is a highly cooperative process, strongly dependent on macromolecular crowding, and involves back-to-back stacking of cylindrical homo-decamers into filaments that associate laterally to form higher order fibrils. Other metabolic enzymes also assemble into filaments at low pH. Hence, we propose that filament formation is a general mechanism to inactivate and store key metabolic enzymes during a state of advanced cellular starvation. These findings have broad implications for understanding the interplay between nutritional stress, the metabolism and the physical organization of a cell.

Fission yeast does not age under favorable conditions, but does so after stress.

BACKGROUND: Many unicellular organisms age: as time passes, they divide more slowly and ultimately die. In budding yeast, asymmetric segregation of cellular damage results in aging mother cells and rejuvenated daughters. We hypothesize that the organisms in which this asymmetry is lacking, or can be modulated, may not undergo aging. RESULTS: We performed a complete pedigree analysis of microcolonies of the fission yeast Schizosaccharomyces pombe growing from a single cell. When cells were grown under favorable conditions, none of the lineages exhibited aging, which is defined as a consecutive increase in division time and increased death probability. Under favorable conditions, few cells died, and their death was random and sudden rather than following a gradual increase in division time. Cell death correlated with the inheritance of Hsp104-associated protein aggregates. After stress, the cells that inherited large aggregates aged, showing a consecutive increase in division time and an increased death probability. Their sisters, who inherited little or no aggregates, did not age. CONCLUSIONS: We conclude that S. pombe does not age under favorable growth conditions, but does so under stress. This transition appears to be passive rather than active and results from the formation of a single large aggregate, which segregates asymmetrically at the subsequent cell division. We argue that this damage-induced asymmetric segregation has evolved to sacrifice some cells so that others may survive unscathed after severe environmental stresses.

Protein disorder, prion propensities, and self-organizing macromolecular collectives.

Eukaryotic cells are partitioned into functionally distinct self-organizing compartments. But while the biogenesis of membrane-surrounded compartments is beginning to be understood, the organizing principles behind large membrane-less structures, such as RNA-containing granules, remain a mystery. Here, we argue that protein disorder is an essential ingredient for the formation of such macromolecular collectives. Intrinsically disordered regions (IDRs) do not fold into a well-defined structure but rather sample a range of conformational states, depending on the local conditions. In addition to being structurally versatile, IDRs promote multivalent and transient interactions. This unique combination of features turns intrinsically disordered proteins into ideal agents to orchestrate the formation of large macromolecular assemblies. The presence of conformationally flexible regions, however, comes at a cost, for many intrinsically disordered proteins are aggregation-prone and cause protein misfolding diseases. This association with disease is particularly strong for IDRs with prion-like amino acid composition. Here, we examine how disease-causing and normal conformations are linked, and discuss the possibility that the dynamic order of the cytoplasm emerges, at least in part, from the collective properties of intrinsically disordered prion-like domains. This article is part of a Special Issue entitled: The emerging dynamic view of proteins: Protein plasticity in allostery, evolution and self-assembly.

Molecular chaperones and stress-inducible protein-sorting factors coordinate the spatiotemporal distribution of protein aggregates.

Acute stress causes a rapid redistribution of protein quality control components and aggregation-prone proteins to diverse subcellular compartments. How these remarkable changes come about is not well understood. Using a phenotypic reporter for a synthetic yeast prion, we identified two protein-sorting factors of the Hook family, termed Btn2 and Cur1, as key regulators of spatial protein quality control in Saccharomyces cerevisiae. Btn2 and Cur1 are undetectable under normal growth conditions but accumulate in stressed cells due to increased gene expression and reduced proteasomal turnover. Newly synthesized Btn2 can associate with the small heat shock protein Hsp42 to promote the sorting of misfolded proteins to a peripheral protein deposition site. Alternatively, Btn2 can bind to the chaperone Sis1 to facilitate the targeting of misfolded proteins to a juxtanuclear compartment. Protein redistribution by Btn2 is accompanied by a gradual depletion of Sis1 from the cytosol, which is mediated by the sorting factor Cur1. On the basis of these findings, we propose a dynamic model that explains the subcellular distribution of misfolded proteins as a function of the cytosolic concentrations of molecular chaperones and protein-sorting factors. Our model suggests that protein aggregation is not a haphazard process but rather an orchestrated cellular response that adjusts the flux of misfolded proteins to the capacities of the protein quality control system.