To function effectively proteins must avoid aberrant aggregation, and hence they are expected to be expressed at concentrations safely below their solubility limits. By analyzing proteome-wide mass ...spectrometry data of Caenorhabditis elegans, however, we show that the levels of about three-quarters of the nearly 4,000 proteins analyzed in adult animals are close to their intrinsic solubility limits, indeed exceeding them by about 10% on average. We next asked how aging and functional self-assembly influence these solubility limits. We found that despite the fact that the total quantity of proteins within the cellular environment remains approximately constant during aging, protein aggregation sharply increases between days 6 and 12 of adulthood, after the worms have reproduced, as individual proteins lose their stoichiometric balances and the cellular machinery that maintains solubility undergoes functional decline. These findings reveal that these proteins are highly prone to undergoing concentration-dependent phase separation, which on aging is rationalized in a decrease of their effective solubilities, in particular for proteins associated with translation, growth, reproduction, and the chaperone system.
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Stress-inducible molecular chaperones have essential roles in maintaining protein homeostasis, but the extent to which they affect overall proteome stability remains unclear. Here, we analyze the ...effects of the DnaK (Hsp70) system on protein stability in Escherichia coli using pulse proteolysis combined with quantitative proteomics. We quantify ∼1,500 soluble proteins and find ∼500 of these to be protease sensitive under normal growth conditions, indicating a high prevalence of conformationally dynamic proteins, forming a metastable subproteome. Acute heat stress results in the unfolding of an additional ∼200 proteins, reflected in the exposure of otherwise buried hydrophobic regions. Overexpression of the DnaK chaperone system markedly stabilizes numerous thermo-sensitive proteins, including multiple ribosomal proteins and large, hetero-oligomeric proteins containing the evolutionarily ancient c.37 fold (P loop nucleoside triphosphate hydrolases). Thus, the Hsp70 system, in addition to its known chaperone functions, has a remarkable capacity to stabilize proteins in their folded states under denaturing stress conditions.
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•Acute heat stress causes unfolding of a thermo-sensitive subproteome in E. coli•DnaK (Hsp70) markedly stabilizes most thermo-sensitive proteins•Numerous essential ribosomal proteins are conformationally stabilized by DnaK•Other DnaK-stabilized proteins tend to be large, multi-domain, and hetero-oligomeric
Molecular chaperones function in maintaining protein homeostasis. Zhao et al. analyze proteome stability in Escherichia coli using pulse proteolysis and mass spectrometry. Numerous ribosomal proteins as well as large multi-domain proteins unfold upon heat stress. Overexpression of the DnaK (Hsp70) chaperone system markedly stabilizes thermo-sensitive proteins in their folded conformations.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
In Alzheimer's disease, aggregates of Aβ and tau in amyloid plaques and neurofibrillary tangles spread progressively across brain tissues following a characteristic pattern, implying a ...tissue-specific vulnerability to the disease. We report a transcriptional analysis of healthy brains and identify an expression signature that predicts-at ages well before the typical onset-the tissue-specific progression of the disease. We obtain this result by finding a quantitative correlation between the histopathological staging of the disease and the expression patterns of the proteins that coaggregate in amyloid plaques and neurofibrillary tangles, together with those of the protein homeostasis components that regulate Aβ and tau. Because this expression signature is evident in healthy brains, our analysis provides an explanatory link between a tissue-specific environmental risk of protein aggregation and a corresponding vulnerability to Alzheimer's disease.
Aging has been associated with a progressive decline of proteostasis, but how this process affects proteome composition remains largely unexplored. Here, we profiled more than 5,000 proteins along ...the lifespan of the nematode C. elegans. We find that one-third of proteins change in abundance at least 2-fold during aging, resulting in a severe proteome imbalance. These changes are reduced in the long-lived daf-2 mutant but are enhanced in the short-lived daf-16 mutant. While ribosomal proteins decline and lose normal stoichiometry, proteasome complexes increase. Proteome imbalance is accompanied by widespread protein aggregation, with abundant proteins that exceed solubility contributing most to aggregate load. Notably, the properties by which proteins are selected for aggregation differ in the daf-2 mutant, and an increased formation of aggregates associated with small heat-shock proteins is observed. We suggest that sequestering proteins into chaperone-enriched aggregates is a protective strategy to slow proteostasis decline during nematode aging.
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•Proteome profiling reveals loss of proteome balance during C. elegans aging•Proteome imbalances alter protein stoichiometries and cause proteostasis stress•Changes in protein abundance drive extensive protein aggregation during aging•Sequestration of surplus proteins in chaperone-associated aggregates is protective
Quantitative proteomic analysis in C. elegans reveals widespread proteome imbalance associated with aging. Extended lifespan is correlated with increased formation of chaperone-associated aggregates, suggesting that sequestering aberrant proteins delays proteostasis decline during aging.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Alzheimer’s disease, which is the most common form of dementia, is characterized by the aggregation of the amyloid β peptide (Aβ) and by an impairment of calcium homeostasis caused by excessive ...activation of glutamatergic receptors (excitotoxicity). Here, we studied the effects on calcium homeostasis caused by the formation of Aβ oligomeric assemblies. We found that Aβ oligomers cause a rapid influx of calcium ions (Ca2+) across the cell membrane by rapidly activating extrasynaptic N-methyl-d-aspartate (NMDA) receptors and, to a lower extent, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. We also observed, however, that misfolded oligomers do not interact directly with these receptors. Further experiments with lysophosphatidylcholine and arachidonic acid, which cause membrane compression and stretch, respectively, indicated that these receptors are activated through a change in membrane tension induced by the oligomers and transmitted mechanically to the receptors via the lipid bilayer. Indeed, lysophosphatidylcholine is able to neutralize the oligomer-induced activation of the NMDA receptors, whereas arachidonic acid activates the receptors similarly to the oligomers with no additive effects. An increased rotational freedom observed for a fluorescent probe embedded within the membrane in the presence of the oligomers also indicates a membrane stretch. These results reveal a mechanism of toxicity of Aβ oligomers in Alzheimer’s disease through the perturbation of the mechanical properties of lipid membranes sensed by NMDA and AMPA receptors.
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The accumulation of protein deposits in neurodegenerative diseases has been hypothesized to depend on a metastable subproteome vulnerable to aggregation. To investigate this phenomenon and the ...mechanisms that regulate it, we measured the solubility of the proteome in the mouse Neuro2a cell line under six different protein homeostasis stresses: 1) Huntington’s disease proteotoxicity, 2) Hsp70, 3) Hsp90, 4) proteasome, 5) endoplasmic reticulum (ER)-mediated folding inhibition, and 6) oxidative stress. Overall, we found that about one-fifth of the proteome changed solubility with almost all of the increases in insolubility were counteracted by increases in solubility of other proteins. Each stress directed a highly specific pattern of change, which reflected the remodeling of protein complexes involved in adaptation to perturbation, most notably, stress granule (SG) proteins, which responded differently to different stresses. These results indicate that the protein homeostasis system is organized in a modular manner and aggregation patterns were not correlated with protein folding stability (ΔG). Instead, distinct cellular mechanisms regulate assembly patterns of multiple classes of protein complexes under different stress conditions.
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Unsupervised clustering of biomarkers derived from noninvasive samples such as nasal fluid is less evaluated as a tool for describing asthma endotypes.
We sought to evaluate whether protein ...expression in nasal fluid would identify distinct clusters of patients with asthma with specific lower airway molecular phenotypes.
Unsupervised clustering of 168 nasal inflammatory and immune proteins and Shapley values was used to stratify 43 patients with severe asthma (endotype of noneosinophilic asthma) using a 2 “modeling blocks” machine learning approach. This algorithm was also applied to nasal brushings transcriptomics from U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Diseases Outcomes). Feature reduction and functional gene analysis were used to compare proteomic and transcriptomic clusters. Gene set variation analysis provided enrichment scores of the endotype of noneosinophilic asthma protein signature within U-BIOPRED sputum and blood.
The nasal protein machine learning model identified 2 severe asthma endotypes, which were replicated in U-BIOPRED nasal transcriptomics. Cluster 1 patients had significant airway obstruction, small airways disease, air trapping, decreased diffusing capacity, and increased oxidative stress, although only 4 of 18 were current smokers. Shapley identified 20 cluster-defining proteins. Forty-one proteins were significantly higher in cluster 1. Pathways associated with proteomic and transcriptomic clusters were linked to TH1, TH2, neutrophil, Janus kinase-signal transducer and activator of transcription, TLR, and infection activation. Gene set variation analysis of the nasal protein and gene signatures were enriched in subjects with sputum neutrophilic/mixed granulocytic asthma and in subjects with a molecular phenotype found in sputum neutrophil-high subjects.
Protein or gene analysis may indicate molecular phenotypes within the asthmatic lower airway and provide a simple, noninvasive test for non–type 2 immune response asthma that is currently unavailable.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
•The Wide Field-of-View Nematode Tracking Platform can monitor for more than 5.000 animals in parallel.•The platform extracts features important for characterising the behaviour of C. elegans, a ...widely used model organism in biomedical research.•The platform provides a high power of detection for small changes in behaviour and reduces the risk of false positive results.•The platform capabilities are illustrated by screening potential drugs for neurodegenerative disorders such as Parkinson’s and Alzheimer’s disease.•Full details for assembling the platform, and the provision of open source codes makes it readily accessible to the community.
The nematode worm C. elegans is a model organism widely used for studies of genetics and of human disease. The health and fitness of the worms can be quantified in different ways, such as by measuring their bending frequency, speed or lifespan. Manual assays, however, are time consuming and limited in their scope providing a strong motivation for automation.
We describe the development and application of an advanced machine vision system for characterising the behaviour of C. elegans, the Wide Field-of-View Nematode Tracking Platform (WF-NTP), which enables massively parallel data acquisition and automated multi-parameter behavioural profiling of thousands of worms simultaneously.
We screened more than a million worms from several established models of neurodegenerative disorders and characterised the effects of potential therapeutic molecules for Alzheimer’s and Parkinson’s diseases. By using very large numbers of animals we show that the sensitivity and reproducibility of behavioural assays is very greatly increased. The results reveal the ability of this platform to detect even subtle phenotypes.
The WF-NTP method has substantially greater capacity compared to current automated platforms that typically either focus on characterising single worms at high resolution or tracking the properties of populations of less than 50 animals.
The WF-NTP extends significantly the power of existing automated platforms by combining enhanced optical imaging techniques with an advanced software platform. We anticipate that this approach will further extend the scope and utility of C. elegans as a model organism.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
The formation of misfolded protein oligomers during early stages of amyloid aggregation and the activation of neuroinflammatory responses are two key events associated with neurodegenerative ...diseases. Although it has been established that misfolded oligomers are involved in the neuroinflammatory process, the links between their structural features and their functional effects on the immune response remain unknown. To explore such links, we took advantage of two structurally distinct soluble oligomers (type A and B) of protein HypF-N and compared the elicited microglial inflammatory responses. By using confocal microscopy, protein pull-down, and high-throughput mass spectrometry, we found that, even though both types bound to a common pool of microglial proteins, type B oligomerswith a lower solvent-exposed hydrophobicityshowed enhanced protein binding, correlating with the observed inflammatory response. Furthermore, the interactome associated with inflammatory-mediated neurodegeneration revealed previously unidentified receptors and signaling molecules likely to be involved in the oligomer-elicited innate immune response.
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