Discovery of RNA-Protein Molecular Clamps Using Proteome-Wide Stability Assays

Journal of Proteome Research, Journal Year: 2025, Volume and Issue: unknown

Published: March 12, 2025

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 a the ligand binding. Using proteome integral solubility alteration (PISA) assays, show that simple biochemical additives can enable detection RNA-protein-small molecule complexes native cell lysates. We apply our approach to rocaglates, molecules specifically clamp RNA eukaryotic translation initiation factor 4A (eIF4A), DEAD-box helicase 3X (DDX3X), potentially other members (DDX) family. To identify unexpected interactions, used class-specific thermal window compared ATP analog base dependencies key rocaglate-DDX interactions. report novel DDX targets high-profile rocaglates-including clinical candidate Zotatifin-and validate findings using limited proteolysis-mass spectrometry fluorescence polarization (FP) experiments. also provide insight into divergent DDX3X affinities between synthetic rocaglates. Taken together, study provides model screening uncompetitive inhibitors chemical proteomics uncovers actionable clamping targets, clearing path toward characterization molecular clamps associated helicases.

Language: Английский

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Transducin-like enhancer of split 3 protects against lipopolysaccharide-induced inflammation through DDX5-ATF1-PPP2R5A signaling

Journal of Advanced Research, Journal Year: 2025, Volume and Issue: unknown

Published: March 1, 2025

Sepsis consists of life-threatening multi-organ dysfunction caused by an excessive systemic inflammatory response to infection. Therefore, identifying negative regulators innate inflammation is crucial for treating this condition. In study, we aimed understand how transducin-like enhancer split 3 (TLE3) regulates responses. We detected Tle3 changes in sepsis patients analyzing public databases, which were confirmed septic survivors, mouse models, and macrophages using Western blotting, qRT-PCR, immunohistochemistry staining. investigated the role mechanism TLE3 utilizing bone marrow-transplantation (BMT) adenovirus-infected mice. Furthermore, Protein-Protein Docking, BiFC, LC-MS/MS analysis, CUT & Tag-seq, CHIP experiments utilized disclose underlying involving macrophage inflammation. found that transcript upregulated peripheral blood samples survivors decreased non-survivors, suggesting critical outcomes. also lipopolysaccharide (LPS)-stimulated human monocyte-derived (MDMs), murine marrow-derived (BMDMs), Gain-of- loss-of-function LPS-stimulated BMDMs, MDMs, models showed alleviates LPS-induced cytokine production, as well nuclear factor kappa-light-chain-enhancer activated B cells (NF-κB) mitogen-activated protein kinase (MAPK) activation macrophages, protects against acute inflammation, injury, death sepsis. Mechanistically, interacts with transcriptional coactivator, DEAD-box helicase 5 (DDX5), promoting its retention cytoplasm ultimately decreasing transcription DDX5/ activating 1 (ATF1)-targeted gene Ppp2r5a. TLE3-DDX5-ATF1 axis downregulates PPP2R5A, a regulatory subunit phosphatase 2A (PP2A), thereby increasing PP2A activity dephosphorylation NF-κB MAPK. Our study shows represents novel suppressor signaling macrophages.

Language: Английский

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RNA helicases, DDX5 and DDX17, facilitate lytic reactivation of gammaherpesviruses

PLoS Pathogens, Journal Year: 2025, Volume and Issue: 21(4), P. e1013009 - e1013009

Published: April 21, 2025

Human gammaherpesviruses comprise of Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), are oncogenic viruses that cause life-long infections. The utilize an extensive virus-host interaction network for facilitating viral replication, whereby virus-encoded proteins modulate host processes. Thus, identifying targets aid in gammaherpesviral replication will help develop therapies to combat these viruses. We identified DDX5 DDX17 interact with protein kinases, KSHV-encoded vPK EBV-encoded BGLF4. found required lytic reactivation loss both decreased KSHV EBV reactivation. Depletion lowered the transcription RTA, key gene drives cascade, due reduced occupancy Brg1, a chromatin remodeler, at RTA promoter. Consequently, inhibition Brg1 Here we demonstrate how hijack function two promote their cycle.

Language: Английский

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HNRNPU promotes the progression of triple-negative breast cancer via RNA transcription and alternative splicing mechanisms

Cell Death and Disease, Journal Year: 2022, Volume and Issue: 13(11)

Published: Nov. 8, 2022

Abstract Triple-negative breast cancer (TNBC) is a great detriment to women’s health due the lack of effective therapeutic targets. In this study, we employed an integrated genetic screen identify pivotal oncogenic factor, heterogeneous nuclear ribonucleoprotein U (HNRNPU), which required for progression TNBC. We elucidated pro-oncogenic role HNRNPU, can induce proliferation and migration TNBC cells via its association with DEAD box helicase 5 (DDX5) protein. Elevated levels HNRNPU-DDX5 complex prohibited intron retention minichromosome maintenance protein 10 (MCM10) pre-mRNA, decreased nonsense-mediated mRNA decay, activated Wnt/β-catenin signalling; on other hand, located in transcriptional start sites (TSS) LIM domain only 4 (LMO4) upregulation promoted transcription LMO4, consequently activating PI3K-Akt-mTOR signalling. Our data highlight synergetic effects HNRNPU RNA splicing regulating suggest that may act as potential molecular target treatment

Language: Английский

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Post-transcriptional regulation of DEAD-box RNA helicases in hematopoietic malignancies

Genes & Diseases, Journal Year: 2024, Volume and Issue: 11(5), P. 101252 - 101252

Published: Feb. 28, 2024

Hematopoiesis represents a meticulously regulated and dynamic biological process. Genetic aberrations affecting blood cells, induced by various factors, frequently give rise to hematological tumors. These instances are often accompanied multitude of abnormal post-transcriptional regulatory events, including RNA alternative splicing, localization, degradation, storage. Notably, regulation plays pivotal role in preserving hematopoietic homeostasis. The DEAD-Box helicase genes emerge as crucial intricately involved sustaining normal hematopoiesis through diverse mechanisms such modification, ribosome assembly. This review consolidates the existing knowledge on DEAD-box helicases regulating underscores pathogenicity mutant malignant hematopoiesis. Emphasis is placed elucidating both positive negative contributions within system.

Language: Английский

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Proteomic Discovery of RNA-Protein Molecular Clamps Using a Thermal Shift Assay with ATP and RNA (TSAR)

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

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RH20, a phase-separated RNA helicase protein, facilitates plant resistance to viruses

Plant Science, Journal Year: 2024, Volume and Issue: 347, P. 112176 - 112176

Published: July 4, 2024

Language: Английский

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DDX21 functions as a potential novel oncopromoter in pancreatic ductal adenocarcinoma: a comprehensive analysis of the DExD box family

Discover Oncology, Journal Year: 2024, Volume and Issue: 15(1)

Published: Aug. 3, 2024

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal tumor with an ill-defined pathogenesis. DExD box (DDX) family genes are widely distributed and involved in various RNA metabolism cellular biogenesis; their dysregulation associated aberrant processes malignancies. However, the prognostic significance expression patterns of DDX PDAC not fully understood. The present study aimed to explore clinical value PDAC. Differentially expressed were identified. related signatures further investigated using LASSO Cox regression analysis. DDX21 protein was analyzed UALCAN human atlas (HPA) online tools confirmed 40 paired normal tissues through Tissue Microarrays (TMA). independent determined construction nomogram models calibration curves. functional roles gene ontology (GO), Kyoto Encyclopedia Genes Genomes (KEGG), Gene Set Enrichment Analysis (GSEA). Cell proliferation, invasion, migration assessed Counting Kit-8, colony formation, Transwell, wound healing assays. Upregulation (DDX10, DDX21, DDX60, DDX60L) significantly poor prognosis patients based on survival recurrence time. Considering profile values signature-related genes, finally selected for exploration. overexpressed at both mRNA levels compared pancreatic tissues. expression, pathological stage, residual significant indicators Moreover, enrichment analysis revealed that co-expressed predominantly metabolism, helicase activity, ribosome biogenesis, cell cycle, cancer-related pathways, such as PI3K/Akt signaling pathway TGF-β pathway. Furthermore, vitro experiments knockdown reduced MIA PaCa-2 viability, migration, invasion. Four could relatively precisely predict Specifically, upregulation may signal unfavorable by negatively affecting biological properties cells. be considered candidate therapeutic target

Language: Английский

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DEAD-Box Helicase 17 Promotes Amyloidogenesis by Regulating BACE1 Translation

Brain Sciences, Journal Year: 2023, Volume and Issue: 13(5), P. 745 - 745

Published: April 29, 2023

Amyloidogenesis is one of the key pathophysiological changes in Alzheimer's disease (AD). Accumulation toxic Aβ results from catalytic processing β-amyloid precursor protein (APP) associated converting enzyme 1 (BACE1) activity. It reported that dead-box helicase 17 (DDX17) controls RNA metabolism and involved development multiple diseases. However, whether DDX17 might play a role amyloidogenesis has not been documented. In present study, we found level was significantly increased HEK SH-SY5Y cells stably express full-length APP (HEK-APP Y5Y-APP) brain APP/PS1 mice, an animal model AD. knockdown, as opposed to overexpression, markedly reduced levels BACE1 peptide (Aβ) Y5Y-APP cells. We further DDX17-mediated enhancement selectively attenuated by translation inhibitors. Specifically, interacted with 5' untranslated region (5'UTR) mRNA, deletion 5'UTR abolished effect on luciferase activity or BACE1. Here, show enhanced expression AD amyloidogenesis; through 5'UTR-dependent translation, serve important mediator contributing progression

Language: Английский

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The DEAD-box RNA helicase DDX5 (p68) and β-catenin: The crucial regulators of FOXM1 gene expression in arbitrating colorectal cancer

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, Journal Year: 2023, Volume and Issue: 1866(2), P. 194933 - 194933

Published: March 29, 2023

Language: Английский

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