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1.
Nat Methods ; 17(5): 495-503, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32284610

RESUMEN

We have used a mass spectrometry-based proteomic approach to compile an atlas of the thermal stability of 48,000 proteins across 13 species ranging from archaea to humans and covering melting temperatures of 30-90 °C. Protein sequence, composition and size affect thermal stability in prokaryotes and eukaryotic proteins show a nonlinear relationship between the degree of disordered protein structure and thermal stability. The data indicate that evolutionary conservation of protein complexes is reflected by similar thermal stability of their proteins, and we show examples in which genomic alterations can affect thermal stability. Proteins of the respiratory chain were found to be very stable in many organisms, and human mitochondria showed close to normal respiration at 46 °C. We also noted cell-type-specific effects that can affect protein stability or the efficacy of drugs. This meltome atlas broadly defines the proteome amenable to thermal profiling in biology and drug discovery and can be explored online at http://meltomeatlas.proteomics.wzw.tum.de:5003/ and http://www.proteomicsdb.org.


Asunto(s)
Regulación de la Expresión Génica , Células Procariotas/metabolismo , Proteínas/química , Proteínas/metabolismo , Proteoma/análisis , Temperatura de Transición , Animales , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Humanos , Mitocondrias/metabolismo , Estabilidad Proteica , Programas Informáticos , Especificidad de la Especie
2.
Cell Chem Biol ; 29(11): 1639-1648.e4, 2022 11 17.
Artículo en Inglés | MEDLINE | ID: mdl-36356585

RESUMEN

DNA-binding proteins are promising therapeutic targets but are notoriously difficult to drug. Here, we evaluate a chemoproteomic DNA interaction platform as a complementary strategy for parallelized compound profiling. To enable this approach, we determined the proteomic binding landscape of 92 immobilized DNA sequences. Perturbation-induced activity changes of captured transcription factors in disease-relevant settings demonstrated functional relevance of the enriched subproteome. Chemoproteomic profiling of >300 cysteine-directed compounds against a coverage optimized bead mixture, which specifically captures >150 DNA binders, revealed competition of several DNA-binding proteins, including the transcription factors ELF1 and ELF2. We also discovered the first compound that displaces the DNA-repair complex MSH2-MSH3 from DNA. Compound binding to cysteine 252 on MSH3 was confirmed using chemoproteomic reactive cysteine profiling. Overall, these results suggested that chemoproteomic DNA bead pull-downs enable the specific readout of transcription factor activity and can identify functional "hotspots" on DNA binders toward expanding the druggable proteome.


Asunto(s)
Cisteína , Proteínas de Unión al ADN , Proteómica , Factores de Transcripción , Proteoma
3.
J Biomol Screen ; 21(4): 414-21, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26637553

RESUMEN

Fragment-based lead discovery has proved to be an effective alternative to high-throughput screenings in identifying chemical matter that can be developed into robust lead compounds. The search for optimal combinations of biophysical techniques that can correctly and efficiently identify and quantify binding can be challenging due to the physicochemical properties of fragments. In order to minimize the time and costs of screening, optimal combinations of biophysical techniques with maximal information content, sensitivity, and robustness are needed. Here we describe an approach utilizing automated microscale thermophoresis (MST) affinity screening to identify fragments active against MEK1 kinase. MST identified multiple hits that were confirmed by X-ray crystallography but not detected by orthogonal methods. Furthermore, MST also provided information about ligand-induced aggregation and protein denaturation. The technique delivered a large number of binders while reducing experimentation time and sample consumption, demonstrating the potential of MST to execute and maximize the efficacy of fragment screening campaigns.


Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , MAP Quinasa Quinasa 1/química , Inhibidores de Proteínas Quinasas/química , Bibliotecas de Moléculas Pequeñas/química , Cristalografía por Rayos X , Difusión , Descubrimiento de Drogas , Expresión Génica , Ensayos Analíticos de Alto Rendimiento/instrumentación , Humanos , Ligandos , MAP Quinasa Quinasa 1/antagonistas & inhibidores , Modelos Moleculares , Unión Proteica , Desnaturalización Proteica , Resonancia por Plasmón de Superficie , Temperatura
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