Meltome Analysis of Geobacillus thermoleoverans ARTRW1
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ABSTRACT: Thermal Proteome Profiling (TPP) analysis has been applied to a thermophilic bacterial proteome, recently isolated strain of Geobacillus thermoloverans, ARTW1 and thermal stability of more than 1000 proteins were presented. The thermal proteome was investigated in terms of thermostable enzymes that are relevant to industrial applications.
Project description:Phenotype-based screening can identify unique small molecules that elicit a desired cellular response, but additional approaches are required to characterize their targets and mechanisms of action. Here we show that a compound termed LCS3, which selectively impairs the growth of human lung adenocarcinoma (LUAD) cells, induces oxidative stress. To identify the target that mediates this effect, we used thermal proteome profiling (TPP) and uncovered the disulfide reductases GSR and TXNRD1 as targets. We confirmed through enzymatic assays that LCS3 inhibits disulfide reductase activity through a reversible, uncompetitive mechanism. Further, we demonstrate that LCS3-sensitive LUAD cells are sensitive to the synergistic inhibition of glutathione and thioredoxin pathways. Lastly, a genome-wide CRISPR knockout screen identified NQO1 loss as a mechanism of LCS3 resistance. This work highlights the ability of TPP to uncover targets of small molecules identified by high-throughput screens and demonstrates the potential therapeutic utility of inhibiting disulfide reductases in LUAD.
Project description:Heat shock proteins are responsible for protein folding in cells. HSP90 is one of the most important chaperones in human cells, and inhibiting HSP90 is a potential strategy for cancer therapy. Multiple HSP90 inhibitors have been in clinical trials. However, none of them has been approved for disease treatment due to unexpected cellular toxicity. Hence, a more comprehensive investigation of cellular responses to HSP90 inhibitors can aid in a better understanding of the molecular mechanisms of the cytotoxicity and side effects of these inhibitors. Protein thermal stability shift, which represents protein structure and interaction alterations, can provide another level of information complementary to commonly used abundance-based proteomics analysis. In this work, we systematically investigated cell responses to different HSP90 inhibitors through global quantification of protein thermal stability changes using thermal proteome profiling, together with quantification of protein abundance changes. Besides the targets and potential off-targets of the drugs, proteins with significant thermal stability changes under the HSP90 inhibition are found to be related to cell stress responses and the translation process. Moreover, proteins with thermal stability shifts under the inhibition are upstream of those with altered expressions. Importantly, the current results indicate that the inhibition of HSP90 results in a strong perturbance of cell transcription and translation processes. This study provides a different perspective to achieve a better understanding of cellular response to chaperone inhibition.
Project description:A azopodophyllotoxin small molecule, SU056, was identified as a novel inhibitor of Y Box Binding Protein 1 and may inhibit progression of ovarian cancer.
Project description:Determining the mechanisms of action of drug molecules that modulate circadian rhythms is critical to develop novel compounds to treat clock disorders. Here we employed Phenotypic Proteomic Profiling (PPP) integrating multipronged proteomics approaches including global proteome, phosphoproteome, kinome mapping, and proteome-wide profiling of thermal stability (TPP) to systematically determine convergent molecular targets of four circadian period lengthening compounds (Longdaysin, Roscovitine, Purvalanol A, and SP600125) in human cells. We demonstrate convergent changes in phosphorylation level and activity of several proteins and kinases involved in vital signaling pathways including MAPK, NGF, BCR, AMPK, and mTOR signaling by the compounds. Kinome profiling using desthiobiotin-ATP enrichment quantitative proteomics and radiometric assays further indicated inhibition of CKId, ERK1/2, CDK2/7, TNIK and STK26 activity as a common mechanism of action for the compounds. Pharmacological or genetic inhibition of several convergent kinases resulted in circadian period lengthening, establishing them as novel bone fide circadian targets. TPP analysis using live cells revealed binding of these drugs to clock regulatory kinases, signaling molecules, and ubiquitination mediator (F-box) proteins. Phenotypic proteomic profiling thus establishes a set of novel circadian clock effectors.
Project description:Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related deaths, characterized by highly invasion and metastasis. Aldo-keto reductase family 1 member C1 (AKR1C1) plays an important role in cancer cell proliferation and metastasis and has gained attention as an anticancer drug target. Here we report that the natural sesquiterpene lactone alantolactone (ALA) was shown to bind directly to AKR1C1 through the Proteome Integral Solubility Alteration (PISA) analysis, a label-free target identification approach based on thermal proteome profiling.
Project description:We have developed a mass spectrometry-based approach that allowed us to quantitatively monitor protein stability across a broad range of temperatures at the proteome scale (meltome). We profiled the meltomes of several microorganisms and eukaryotic species including human, allowing to investigate the determinants of protein thermostability and survival in various environmental niches. Moreover, we will make Meltome Atlas a valuable, publicly available resource for the biological community to investigate their proteins of interest in the context of protein thermostbility.
Project description:Sponges (Porifera) are early-branching Metazoa who do not possess muscles or neurons, however are able to undergo a whole-body movement that involves the closure of their canal system and collapse of an epithelial tent. In this study we profile proteomic responses of the freshwater sponge Spongilla lacustris during nitric oxide (NO) and agitation induced movements to elucidate the early evolution of coordination. Specifically, we measure condition-dependent changes in protein thermal stability and abundance using Thermal proteome profiling (TPP). These changes are the result of proteins undergoing stabilizing or destabilizing conformational changes broadly caused by e.g. the binding or dissociation of small molecules to the proteins, the formation or loss of protein-protein interactions or a change in post-translational modifications.
Project description:In this study, we developed a model system for the assessment of small molecule-protein interactions using intact cells treated with a commercially available highly specific mitogen-activated protein kinase (MEK) 1/2 inhibitor to prepare a set of unfractionated test samples labeled with tandem mass tags. Our objective was to improve qualitative and quantitative aspects of MS-TSA as well as its efficiency and accuracy. We evaluated individually and for the first time in combination ΦSDM, FAIMS, and SIILCC as an improved MS-based acquisition approach for thermal stability assays (iMAATSA). PSM-level filtering was preliminarily investigated as an approach to reduce melting curve variation and improve the accuracy of Tm measurements.
Project description:Temperature is a universal environmental constraint and organisms have evolved diverse mechanisms of thermotolerance. A central feature of thermophiles relative to mesophiles is a universal shift in protein stability, implying that it is a major constituent of thermotolerance. However, organisms have also evolved extensive buffering systems, such as those that disaggregate and refold denatured proteins and enable survival of heat shock. Here, we show that both cellular and protein structural changes contribute to divergence in protein thermostability between two closely related Saccharomyces species that differ by 8°C in their thermotolerance. Using thermal proteomic profiling we find that 85% of S. cerevisiae proteins are more stable than their S. uvarum homologs and there is an average shift of 1.6°C in temperature induced protein aggregation. In an interspecific hybrid of the two species, S. cerevisiae proteins retain their thermostability, while the thermostability of their S. uvarum homologs is enhanced, indicating that cellular context contributes to protein stability differences. By purifying orthologous proteins we show that amino acid substitutions underlie melting temperature differences for two proteins, Guk1 and Aha1. Amino acid substitutions are also computationally predicted to contribute to stability differences for most of the proteome. Our results imply that coordinated changes in protein thermostability impose a significant constraint on the time scales over which thermotolerance can evolve.