Proteomic Discovery of RNA-Protein Molecular Clamps Using a Thermal Shift Assay with ATP and RNA (TSAR) DOI Creative Commons
Stanley I. Goldstein, Alice C. Fan,

Zi-Hao Wang

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: April 19, 2024

Uncompetitive inhibition is an effective strategy for suppressing dysregulated enzymes and their substrates, but discovery of suitable ligands depends on often-unavailable structural knowledge serendipity. Hence, despite surging interest in mass spectrometry-based target identification, proteomic studies substrate-dependent engagement remain sparse. Herein, we describe the Thermal Shift Assay with ATP RNA (TSAR) as a template proteome-wide ligand binding. Using thermal shift assays, show that simple biochemical additives can facilitate detection native cell lysates. We apply our approach to rocaglates, family molecules specifically clamp eukaryotic translation initiation factor 4A (eIF4A), DEAD-box helicase 3X (DDX3X), potentially other members (DDX) helicases. To identify unexpected interactions, optimized class-specific denaturation window evaluated analog probe dependencies key rocaglate-DDX interactions. report novel DDX targets rocaglate clamping spectrum, confirm DDX3X common several widely studied analogs, provide insights into divergent affinities between synthetic rocaglates. independently validate high-profile including clinical candidate Zotatifin (

Language: Английский

Calcium Signaling Is a Universal Carbon Source Signal Transducer and Effects an Ionic Memory of Past Carbon Sources DOI Open Access

Kobi J. Simpson-Lavy,

Martin Kupiec

International Journal of Molecular Sciences, Journal Year: 2025, Volume and Issue: 26(5), P. 2198 - 2198

Published: Feb. 28, 2025

Glucose is the preferred carbon source for most cells. However, cells may encounter other sources that can be utilized. How match their metabolic gene expression to source, beyond a general glucose repressive system (catabolite repression), remains little understood. By studying effect of up seven different on Snf1 phosphorylation and downstream regulated genes, we searched mechanism identifies sources. We found glycolysis metabolites glucose-6-phosphate (G6P) glucose-1-phosphate (G1P) play central role in adaptation The ratio G1P G6P activates analogue calcium signaling via proton-exporter Pma1 regulate genes. pathway bifurcates with calcineurin-reducing ADH2 (alcohol dehydrogenase) Cmk1-increasing ZWF1 (glucose-6-phosphate expression. Furthermore, not only by present source; it also past were able manipulate this ionic memory obtain high media containing galactose. Our findings provide universal which respond all

Language: Английский

Citations

0
Recent Advances in Mass Spectrometry-Based Bottom-Up Proteomics DOI
Cameron S. Movassaghi, Jie Sun, Yuming Jiang

et al.

Analytical Chemistry, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 25, 2025

Mass spectrometry-based proteomics is about 35 years old, and recent progress appears to be speeding up across all subfields. In this review, we focus on advances over the last two in select areas within bottom-up proteomics, including approaches high-throughput experiments, data analysis using machine learning, drug discovery, glycoproteomics, extracellular vesicle structural proteomics.

Language: Английский

Citations

0
Proteomic Discovery of RNA-Protein Molecular Clamps Using a Thermal Shift Assay with ATP and RNA (TSAR) DOI Creative Commons
Stanley I. Goldstein, Alice C. Fan,

Zi-Hao Wang

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: April 19, 2024

Uncompetitive inhibition is an effective strategy for suppressing dysregulated enzymes and their substrates, but discovery of suitable ligands depends on often-unavailable structural knowledge serendipity. Hence, despite surging interest in mass spectrometry-based target identification, proteomic studies substrate-dependent engagement remain sparse. Herein, we describe the Thermal Shift Assay with ATP RNA (TSAR) as a template proteome-wide ligand binding. Using thermal shift assays, show that simple biochemical additives can facilitate detection native cell lysates. We apply our approach to rocaglates, family molecules specifically clamp eukaryotic translation initiation factor 4A (eIF4A), DEAD-box helicase 3X (DDX3X), potentially other members (DDX) helicases. To identify unexpected interactions, optimized class-specific denaturation window evaluated analog probe dependencies key rocaglate-DDX interactions. report novel DDX targets rocaglate clamping spectrum, confirm DDX3X common several widely studied analogs, provide insights into divergent affinities between synthetic rocaglates. independently validate high-profile including clinical candidate Zotatifin (

Language: Английский

Citations

2