Systematic analysis of chemical-protein interactions from zebrafish embryo by proteome-wide thermal shift assay, bridging the gap between molecular interactions and toxicity pathways

Lizano-Fallas, Veronica; Amor, Ana Carrasco del; Cristóbal, Susana

doi:10.1016/j.jprot.2021.104382

Cited by 7 publications

(13 citation statements)

References 73 publications

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“…We have previously reported that the identification of targets from hydrophobic chemicals increased in robustness by introducing a step of sedimentation of the microsomal vesicles before the thermal shift assay. This implementation of the method eliminates the risk of the unquantified hydrophobic chemical sequestration inside the microsomes, that is a temperature dependent process, and compromised the maintenance of the stable concentration of chemical and proteome along the thermal shift assay 12,22 . Therefore, our PISA method has been implemented for its application to toxicology to obtain a soluble proteome that would be stable with chemical compounds and without larger pre-fractionation that would increase costs.…”

Section: Resultsmentioning

confidence: 99%

“…One of the advantages of using omics instead of large suites of in vitro bioassays is that with omics only one single analytical assay is performed, and all biological pathways are covered at the same time, gaining in time, resources, and completeness of the acquired knowledge. An example of an omic methodology recently utilized to screen chemicals is the proteome integral solubility alteration (PISA) assay 12 .…”

Section: Introductionmentioning

confidence: 99%

“…This solubility alteration can be probed by applying a stability factor, as temperature in thermal profiling 14 . Upon this principle, in this previous study, PISA was applied for the first time in the field of toxicology and showed its potency by predicting the impact of exposure to individual chemicals, mixtures of chemicals, new chemicals from biodiscovery, and side-effects of newly developed drugs 12 . Even though the PISA methodology offers completeness by assessing the entire proteome, covering all biological pathways, an approach that could extract the uniqueness out of the complete answer is required for transferring and integrate this knowledge into AOPs by the identification of MIEs.…”

Section: Introductionmentioning

confidence: 99%

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Proteome integral solubility alteration assay combined with multi-criteria decision-making analysis for developing adverse outcome pathways

Lizano-Fallas

Amor

Cristóbal

2022

Preprint

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Understanding the biological impact of chemicals is hindered by the high number and diversity of compounds in the market. To simplify the chemical risk assessment, the adverse outcome pathway (AOP) method has arisen as a framework to predict the impact of chemical exposure on human and environmental health. The development of this predictive tool requires knowledge of the molecular interaction between chemicals and protein targets. Those molecular initiating events connect alterations of cellular function with physiological impairment. This strategy aims to focus on the complex biological interaction to predict the impact on health. The high-throughput identification of all chemical targets can be obtained by a proteomics-based thermal shift assay, however, selecting the priority target candidate is a biased process strongly dependent on expert knowledge and literature. Here, we unravel new molecular initiating event from a tested chemical combining the target deconvolution by the proteome integral solubility alteration (PISA) assay, and the target selection by an analytical hierarchy process (AHP) approach. In the proof-of-concept study, we identified by PISA assay 8 protein targets for 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) from the soluble proteome from hepatic cells containing 2824 proteins. The definition of the AHP approach facilitates the selection of heat shock protein beta-1 (Hspb1) as the most suitable protein for developing AOPs. Our results demonstrated that the process of target identification is independent from a chemical characterization, and that the process of data curation and target selection is less sensitive to lack of toxicological information. We anticipate that this innovative integration of methods could decipher the chemical-protein interactions from new chemicals including the new alternative chemicals designed for chemical replacement and that would discover new molecular initiating events to support more sustainable methodologies to gain time and resources in chemicals assessment.SYNOPSISOur combined methodologies can determine the most suitable target to develop adverse outcome pathways from the proteome-wide protein target identification.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Proteome integral solubility alteration assay combined with multi-criteria decision-making analysis for developing adverse outcome pathways

Lizano-Fallas

Amor

Cristóbal

2022

Preprint

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“…The Cellular Thermal Shift Assay relayed on a large collection of antibodies for protein identification and quantitation . However, the introduction of mass spectrometry (MS) to analyze the thermally induced changes of proteome solubility was pivotal to enable the unbiased identification of targets from soluble proteome in methods called Thermal Proteome Profiling (TPP) and later Proteome Integral Solubility Alteration Assay. , The developments of these methodological approaches have recently explored many scientific problems defined by interactions of small chemicals and cellular proteins from biomedicine and analytical chemistry. − At our lab, we have focused on implementations for its application to biodiscovery or toxicology …”

Section: Introductionmentioning

confidence: 99%

Nonionic Surfactants can Modify the Thermal Stability of Globular and Membrane Proteins Interfering with the Thermal Proteome Profiling Principles to Identify Protein Targets

et al. 2023

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The membrane proteins are essential targets for understanding cellular function. The unbiased identification of membrane protein targets is still the bottleneck for a system-level understanding of cellular response to stimuli or perturbations. It has been suggested to enrich the soluble proteome with membrane proteins by introducing nonionic surfactants in the solubilization solution. This strategy aimed to simultaneously identify the globular and membrane protein targets by thermal proteome profiling principles. However, the thermal shift assay would surpass the cloud point temperature from the nonionic surfactants frequently utilized for membrane protein solubilization. It is expected that around the cloud point temperature, the surfactant micelles would suffer structural modifications altering protein solubility. Here, we show that the presence of nonionic surfactants can alter protein thermal stability from a mixed, globular, and membrane proteome. In the presence of surfactant micelles, the changes in protein solubility analyzed after the thermal shift assay was affected by the thermally dependent modification of the micellar size and its interaction with proteins. We demonstrate that the introduction of nonionic surfactants for the solubilization of membrane proteins is not compatible with the principles of target identification by thermal proteome profiling methodologies. Our results lead to exploring thermally independent strategies for membrane protein solubilization to assure confident membrane protein target identification. The proteome-wide thermal shift methods have already shown their capability to elucidate mechanisms of action from pharma, biomedicine, analytical chemistry, or toxicology, and finding strategies, free from surfactants, to identify membrane protein targets would be the next challenge.

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“…The developments of these methodological approaches have recently explored many scientific problems defined by interactions of small chemicals and cellular proteins from biomedicine, analytical chemistry 7 . At our lab, we have focused on implementation for its application to biodiscovery 8 or toxicology 9 .…”

Section: Introductionmentioning

confidence: 99%

Nonionic surfactants can modify the thermal stability of globular and membrane proteins interfering with the thermal proteome profiling principles to identify protein targets

Berlin

Lizano-Fallas

Amor

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

The membrane proteins are essential targets to understand cellular function. The unbiased identification of membrane protein targets is still the bottleneck for a system-level understanding of cellular response to stimuli or perturbations. It has been suggested to enrich the soluble proteome with membrane proteins by introducing nonionic surfactants in the solubilization solution. This strategy was aiming to simultaneous identify the globular and membrane protein targets by thermal proteome profiling principles. However, the thermal shift assay would surpass the cloud point temperature from the nonionic surfactants frequently utilized for membrane protein solubilization. It is expected that around the cloud point temperature, the surfactant micelles would suffer structural modifications altering protein solubility. Here, we show that the presence of nonionic surfactants can alter protein thermal stability from a mixed, globular and membrane, proteome. In the presence of surfactant micelles, the changes in proteins solubility analyzed after the thermal shift assay were affected by the thermal dependent modification of the micellar size, and its interaction with proteins. We demonstrate that the introduction of nonionic surfactants for the solubilization of membrane proteins is not compatible with the principles of target identification by thermal proteome profiling methodologies. Our results lead to explore thermal-independent strategies for membrane protein solubilization to assure confident membrane protein target identification. The proteome-wide thermal shift methods have already shown their capability to elucidate mechanisms of action from pharma, biomedicine, analytical chemistry, or toxicology and finding strategies, free from surfactants, to identify membrane protein targets would be the next challenge.

show abstract

Systematic analysis of chemical-protein interactions from zebrafish embryo by proteome-wide thermal shift assay, bridging the gap between molecular interactions and toxicity pathways

Cited by 7 publications

References 73 publications

Proteome integral solubility alteration assay combined with multi-criteria decision-making analysis for developing adverse outcome pathways

Proteome integral solubility alteration assay combined with multi-criteria decision-making analysis for developing adverse outcome pathways

Nonionic Surfactants can Modify the Thermal Stability of Globular and Membrane Proteins Interfering with the Thermal Proteome Profiling Principles to Identify Protein Targets

Nonionic surfactants can modify the thermal stability of globular and membrane proteins interfering with the thermal proteome profiling principles to identify protein targets

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