Proteome-wide solubility and thermal stability profiling reveals distinct regulatory roles for ATP

Sridharan, S.; Kurzawa, Nils; Werner, Thilo; Günthner, Ina; Helm, Dominic; Huber, Wolfgang; Bantscheff, Marcus; Savitski, Mikhail M.

doi:10.1038/s41467-019-09107-y

Cited by 230 publications

(318 citation statements)

References 63 publications

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“…Under physiological conditions, proteins involved in phase separated membrane-less nuclear organelles-such as the nucleolus-have been shown to contain an insoluble sub-population [42,43]. We found that aggregators included many such proteins in addition to aggregators that were completely soluble in unstressed conditions ( Fig EV2C).…”

Section: Characterization Of Aggregation-prone Proteinsmentioning

confidence: 73%

“…With the 26S proteasome the thermal destabilization was stronger for 19S regulatory particle than for the 20S core particle ( Fig 5F). Interestingly, proteins from the 19S regulatory particle were thermally stabilized when ATP was added to cell lysates [43]. However, the ATP levels were not altered during the heat shock ( Fig EV1: viability measurements based on ATP quantification).…”

Section: Heat Shock-induced Changes In Thermal Stabilitymentioning

confidence: 98%

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Aggregation and Disaggregation Features of the Human Proteome

Määttä

Rettel

Helm

et al. 2020

Preprint

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Protein aggregates have negative implications in disease. While reductionist experiments have increased our understanding of aggregation processes, the systemic view in biological context is still limited. To extend this understanding, we used mass spectrometry-based proteomics to characterize aggregation and disaggregation in human cells after non-lethal heat shock. Aggregation-prone proteins were enriched in nuclear proteins, high proportion of intrinsically disordered regions, high molecular mass, high isoelectric point and hydrophilic amino acids. During recovery, most aggregating proteins disaggregated with a rate proportional to the aggregation propensity: larger loss in solubility was counteracted by faster disaggregation. High amount of intrinsically disordered regions also resulted in faster disaggregation. However, other characteristics enriched in aggregating proteins did not correlate with the disaggregation rates. In addition, we analyzed changes in protein thermal stability after heat shock. Soluble remnants of aggregated proteins were more thermally stable compared to control condition. Our results provide a rich resource of heat stress-related protein solubility data, propose novel roles for intrinsically disordered regions in protein quality control and reveal a protection mechanism to repress protein aggregation in heat stress.

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Section: Characterization Of Aggregation-prone Proteinsmentioning

confidence: 73%

Section: Heat Shock-induced Changes In Thermal Stabilitymentioning

confidence: 98%

Aggregation and Disaggregation Features of the Human Proteome

Määttä

Rettel

Helm

et al. 2020

Preprint

Self Cite

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“…for a series of ligands, which ones interact strongly with protein of interest), rather than rank order comparisons of absolute ligandprotein binding affinity across the proteome. Such biases are an intrinsic feature of chemoproteomic methods and extend even to label-free approaches such as LiP-MS and CETSA, [43][44] whose detection of protein-ligand interactions require ligand binding to alter proteolytic or thermal stability, respectively. Future studies of acyl-CoA/protein interactions will likely benefit from the integration of multiple approaches.…”

Section: Discussionmentioning

confidence: 99%

A systems chemoproteomic analysis of acyl-CoA/protein interaction networks

Levy

Montgomery

Sardiu

et al. 2019

Preprint

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CoA metabolite interactions, enabling the identification of biological processes susceptible to altered acetyl-CoA levels. Finally, we utilize systems-level analyses to assess the features of novel protein networks that may interact with acyl-CoAs and demonstrate a strategy for high-confidence annotation of direct acetyl-CoA binding proteins and AT enzymes in human proteomes. Overall our studies illustrate the power of integrating chemoproteomics and systems biology analysis methods and provide a novel resource for understanding the diverse signaling roles of acyl-CoAs in biology and disease. Results Validation of CATNIP for the global study of acyl-CoA/protein interactionsIn order to deeply sample acyl-CoA/protein interactions on a proteome-wide scale, we initially set out to integrate CoA-based protein capture methods with LC-MS/MS ( Fig. 2a). In this workflow, whole cell extracts are first incubated with Lys-CoA Sepharose. This affinity matrix enables active site-dependent enrichment of many different classes of CoA-binding proteins, 8 making it ideal for broad profiling studies. Next, enriched proteins are subjected to tryptic digest and analyzed using MudPIT (multidimensional protein identification technology), a proteomics platform that combines strong cation exchange and C18 reverse phase chromatography to pre-fractionate tryptic peptides, followed by ionization and data-dependent MS/MS. 9 The separation afforded by this approach significantly decreases sample complexity, allowing the identification of rare, low abundance peptides from complex proteomic mixtures. To facilitate the identification of acyl-CoA/protein interactions, competition experiments are performed in which proteomes are pre-incubated with a CoA metabolite prior to capture. 10 Decreased enrichment in competition samples compared to controls (as assessed by quantitative spectral counting) signifies that the CoA metabolite interacts with a protein of interest.These interacting proteins can then be further classified into pharmacological or biological networks using either conventional metrics (fold-change, gene ontology, etc) or systems-based analysis tools.As an initial model, we explored the utility of CATNIP to globally profile acetyl-CoA/protein interactions in unfractionated HeLa cell proteomes. Proteomes were pre-incubated with acetyl-CoA or vehicle (buffer) control, followed by enrichment using Lys-CoA Sepharose. These experiments assessed competition at 3, 30, and 300 µM acetyl-CoA, which spans the physiological concentration range of acetyl-CoA in the cytosol and mitochondria. Protein capture in each condition was quantified using distributed normalized spectral abundance factor (dNSAF), a label-free metric that normalizes spectral counts relative to overall protein length ( Fig. S1a-c , Table S1). 11 Each condition was analyzed in triplicate, constituting 12 experiments, >144 hours of instrument time, and over 1.1 million non-redundant peptide spectra collected. We limited our analysis to highconfidence protein identifications (>4...

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“…We chose the AKT1 (protein kinase B) as a model system due to its importance in metabolism, proliferation, cell survival, growth and angiogenesis. In AKT1 SIESTA experiment (data in Supplementary Data 6), ATP was used at 500 µM, at which concentration it only acts as a cosubstrate 34 . 31% (123/396) of the proteins annotated in Uniprot as ATP binders were also verified in our experiment.…”

Section: Siesta Identified and Ranked Many Novel Putative Substrates mentioning

confidence: 99%

System-wide identification and prioritization of enzyme substrates by thermal analysis (SIESTA)

Saei

Beusch

Sabatier

et al. 2018

Preprint

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Despite the immense importance of enzyme-substrate reactions, there is a lack of generic and unbiased tools for identifying and prioritizing substrate proteins which are modulated in the structural and functional levels through modification. Here we describe a high-throughput unbiased proteomic method called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that enzymatic posttranslational modification of substrate proteins might change their thermal stability. SIESTA successfully identifies several known and novel substrate candidates for selenoprotein thioredoxin reductase 1, protein kinase B (AKT1) and poly-(ADP-ribose) polymerase-10 systems in up to a depth of 7179 proteins. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, open new opportunities in investigating the effect of PTMs on signal transduction, and facilitate drug discovery.

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Proteome-wide solubility and thermal stability profiling reveals distinct regulatory roles for ATP

Cited by 230 publications

References 63 publications

Aggregation and Disaggregation Features of the Human Proteome

Aggregation and Disaggregation Features of the Human Proteome

A systems chemoproteomic analysis of acyl-CoA/protein interaction networks

System-wide identification and prioritization of enzyme substrates by thermal analysis (SIESTA)

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