Target Discovery Using Thermal Proteome Profiling

Sridharan, S.; Günthner, Ina; Becher, Isabelle; Savitski, Mikhail M.; Bantscheff, Marcus

doi:10.1002/9781118970195.ch11

Cited by 12 publications

(11 citation statements)

References 62 publications

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“…In the future, TPP might be adapted to an even broader range of applications and might inspire assays for new purposes. Examples are the deconvolution of enzyme-substrate specificities (preprint: Saei et al, 2018) and the recent development of solubility proteome profiling (SPP) for the study of small molecule effects on proteome solubility (Sridharan et al, 2019b). The latter was first realized when using the 2D-TPP approach to identify ATP-binding proteins in cell extracts.…”

Section: Discussionmentioning

confidence: 99%

“…), as well as drugs on protein phase transition. Furthermore, it was observed that many proteins that have an insoluble subpopulation under native conditions solubilize upon heating (Sridharan et al, 2019b), revealing a classical phase behavior of weakly interacting polymers (Shin & Brangwynne, 2017). Thus, combination of TPP with SPP will be a useful tool to study and establish system-wide principles of protein solubility transition and to provide an unbiased approach to screen drugs that can prevent aberrant solubility changes.…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, cell extracts are generally used to identify direct targets of perturbations (e.g., the protein(s) to which a drug binds). The extracts can be prepared by mechanical disruption of cells, for example, by douncing (Sridharan et al, 2019b) or freeze-thaw cycles (Savitski et al, 2014), which can be further aided by enzymatic digestion of certain cell structures [e.g., addition of DNAse to reduce the viscosity of the lysate (Becher et al, 2018), or lysozyme or zymolyase to digest the bacterial or yeast cell walls (Mateus et al, 2018;Ochoa et al, 2019)]. Care should be taken when preparing cell extracts to ensure that proteins remain in their native form-for example, the temperature should not be increased dramatically, and degradation by proteases should be prevented.…”

Section: Preparation Of the Cellular Materials And Induction Of Perturmentioning

confidence: 99%

“…By determining the melting profile of all detected proteins, TPP was initially developed to find targets and off-targets of drug-like molecules (Savitski et al, 2014(Savitski et al, , 2018Huber et al, 2015;Reinhard et al, 2015;Becher et al, 2016;Mateus et al, 2016Mateus et al, , 2018Kitagawa et al, 2017;Azimi et al, 2018;Hu et al, 2019;Sridharan et al, 2019a)-generally, binding of a drug to a protein leads to a thermal stabilization of the protein (Fig 1). More recently, TPP has been used to identify metabolite-binding proteins, mapping the proteins which interact with different nucleotides (Huber et al, 2015;preprint: Saei et al, 2018;Dziekan et al, 2019;Sridharan et al, 2019b), and unraveling that such interactions can be both promiscuous [e.g., interactions with adenosine triphosphate (ATP)] and very specific [e.g., interactions with thymidine monophosphate (dTMP)]. Binding to nucleic acids also leads to changes in protein thermal stability (Becher et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

“…; or metabolite (preprint:Saei et al, 2018;Dziekan et al, 2019;Sridharan et al, 2019b)], genetic [e.g., gene knock-outBanzhaf et al, 2020)], or enzymatic (preprint:Saei et al, 2018) perturbations; or different cell states [different phase of the cell cycleDai et al, 2018), or growth phase;Fig 2]…”

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confidence: 99%

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Thermal proteome profiling for interrogating protein interactions

Mateus

Kurzawa

Becher

et al. 2020

Molecular Systems Biology

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Thermal proteome profiling (TPP) is based on the principle that, when subjected to heat, proteins denature and become insoluble. Proteins can change their thermal stability upon interactions with small molecules (such as drugs or metabolites), nucleic acids or other proteins, or upon post‐translational modifications. TPP uses multiplexed quantitative mass spectrometry‐based proteomics to monitor the melting profile of thousands of expressed proteins. Importantly, this approach can be performed in vitro, in situ, or in vivo. It has been successfully applied to identify targets and off‐targets of drugs, or to study protein–metabolite and protein–protein interactions. Therefore, TPP provides a unique insight into protein state and interactions in their native context and at a proteome‐wide level, allowing to study basic biological processes and their underlying mechanisms.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Preparation Of the Cellular Materials And Induction Of Perturmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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Thermal proteome profiling for interrogating protein interactions

Mateus

Kurzawa

Becher

et al. 2020

Molecular Systems Biology

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174

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Thermal Proteome Profiling for Drug Target Identification and Probing of Protein States

Sauer,

Bantscheff

2023

Methods in Molecular Biology

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Experimental study of proteome halophilicity using nanoDSF: a proof of concept

2021

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40Adaption to environmental conditions is reflected by protein adaptation. In particular, 41 proteins of extremophiles display distinctive traits ensuring functional, structural and 42 dynamical properties under permanently extreme physical and chemical conditions. While it 43 has mostly been studied with approaches focusing on specific proteins, biophysical 44 approaches have also confirmed this link between environmental and protein adaptation at 45 the more complex and diverse scale of the proteome. However, studies of this type remain 46 challenging and often require large amounts of biological material. 47We report here the use of nanoDSF as a tool to study proteome stability and solubility 48 in cell lysates of the model halophilic archaeon Haloarcula marismortui. Notably, our results 49 show that, as with single halophilic protein studies, proteome stability was correlated to the 50 concentration of NaCl or KCl under which the cells were lysed and hence the proteome 51 exposed. This work highlights that adaptation to environmental conditions can be 52 experimentally observed at the scale of the proteome. Still, we show that the biochemical 53 properties of single halophilic proteins can only be partially extrapolated to the whole 54 proteome. 55Page 2 of 23 Extremophiles 9 310 observed between H. marismortui proteome and MDH. Nonetheless, this stresses that the 311 behaviour of the proteome can only partially be extrapolated from that of a given enzyme. 312Our experimental setup allowed little to no observation of peaks of first derivative of 313 ratio of 350/330nm fluorescence during the cooling step following the heating. When 314 observed, these renaturation peaks were less distinctive than those associated with 315 denaturation. Moreover, no peak of first derivative of light scattering could be observed at all, 316 showing that aggregation process was also mostly irreversible. Since aggregation peaks 317 happened after denaturation peaks during the heating step, irreversible aggregation may be 318 the reason why renaturation was very limited, even with the pure MDH. In the case of the 319 proteome, the limited renaturation could be associated with a specific subset of proteins 320 whose denaturation was reversible, rather than a partial renaturation of the whole proteome. 321 This was unlike the ß-lactamase from the moderately halophilic bacterium Chromohalobacter 322 sp. 560 which has been shown to refold spontaneously after thermal denaturation in 0.2M 323 NaCl (Tokunaga et al. 2004). However, after being expressed in non-halophilic Escherichia coli 324 cells where it has undergone low salt denaturation, H. marismortui MDH can be refolded in 325 vitro by increasing the salinity (Cendrin et al. 1993;Franzetti et al. 2001). Similarly, a 326 nucleoside diphosphate kinase from the extremely halophilic archaea H. salinarum, which has 327 been expressed in E. coli, has also been shown to require high salt concentration for refolding 328 while being stable and active in low salinity conditions (Ishibashi et...

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Target Discovery Using Thermal Proteome Profiling

Cited by 12 publications

References 62 publications

Thermal proteome profiling for interrogating protein interactions

Thermal proteome profiling for interrogating protein interactions

Thermal Proteome Profiling for Drug Target Identification and Probing of Protein States

Experimental study of proteome halophilicity using nanoDSF: a proof of concept

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