Jing Wang

BACKGROUND: Given the persistently high morbidity and mortality of heart failure (HF), targeting myocardial remodeling, particularly pathological hypertrophy and fibrosis, has become a major therapeutic priority. RhoA (Ras homolog gene family member A), a small GTPase governing cytoskeletal reorganization and cell migration, plays a pivotal role in this process. However, RhoA has long been considered undruggable because of its high-affinity binding to GDP/GTP and the absence of well-defined druggable pockets. METHODS: Structural analyses comparing RhoA-GTP and RhoA-GDP conformations, combined with surface plasmon resonance-based screening, were used to identify a RhoA inhibitor. The underlying mechanism was validated in cultured cells and 3-dimensional myocardial tissue models. Therapeutic efficacy was assessed across multiple species of HF models and supported by multiomics analyses linking RhoA activation to human HF. Key findings were further confirmed by multiplex immunohistochemistry and pulldown assays in human heart specimens. RESULTS: We identified an unrecognized cryptic pocket adjacent to GDP in RhoA. A natural product, AH001, selectively occupied this pocket and interacted with GDP, thereby stabilizing the interaction between RhoA and its endogenous inhibitor, RhoGDIα (Rho GDP-dissociation inhibitor 1). AH001 suppressed downstream signaling by reducing MRTFA (myocardin-related transcription factor A) nuclear translocation and downregulating fibrosis- and hypertrophy-related proteins. Moreover, AH001 disrupted pathological crosstalk between Mrtfa + cardiomyocytes and fibroblasts. Consequently, AH001 markedly attenuated myocardial remodeling in multiple HF animal models, as well as in 3-dimensional myocardial tissue models. CONCLUSIONS: These findings establish pharmacological inhibition of RhoA activation as a viable strategy to mitigate myocardial remodeling in HF and provide a conceptual framework for developing reversible inhibitors against previously undruggable small GTPases.

Copper (Cu) performs essential structural and functional roles in all aerobic organisms, but identifying natural cuproproteins through traditional affinity methods is hindered by the variety of copper ligand combinations and binding locations in proteins. Here, using ATOX1 as a bait, fused with the APEX2 or TurboID proximity labeling enzymes, with quantitative proteomics in cancer and normal cells (H1299 and HEK293T), we predicted 682 highly enriched candidate Cu‐binding proteins. Among them, we functionally validated the novel cuproprotein, thioredoxin reductase 1 (TrxR1). Through cryo‐electron microscopy (cryo‐EM), we resolved three copper‐binding sites across its N‐ and C‐terminal redox centers. Functional assays demonstrated that copper inhibits TrxR1 reductase activity and impairs the denitrosylation function of its substrate, thioredoxin 1 (Trx1), consequently inhibiting caspase‐3 cleavage and apoptosis. Beyond expanding the scope of annotated cuproproteins, this study provides a versatile strategy for cuproprotein discovery and defines the copper functions in modulating apoptosis through the TrxR1/Trx1 regulatory module.

Copper (Cu) performs essential structural and functional roles in all aerobic organisms, but identifying natural cuproproteins through traditional affinity methods is hindered by the variety of copper ligand combinations and binding locations in proteins. Here, using ATOX1 as a bait, fused with the APEX2 or TurboID proximity labeling enzymes, with quantitative proteomics in cancer and normal cells (H1299 and HEK293T), we predicted 682 highly enriched candidate Cu‐binding proteins. Among them, we functionally validated the novel cuproprotein, thioredoxin reductase 1 (TrxR1). Through cryo‐electron microscopy (cryo‐EM), we resolved three copper‐binding sites across its N‐ and C‐terminal redox centers. Functional assays demonstrated that copper inhibits TrxR1 reductase activity and impairs the denitrosylation function of its substrate, thioredoxin 1 (Trx1), consequently inhibiting caspase‐3 cleavage and apoptosis. Beyond expanding the scope of annotated cuproproteins, this study provides a versatile strategy for cuproprotein discovery and defines the copper functions in modulating apoptosis through the TrxR1/Trx1 regulatory module.

Adenosine (Ado), a nucleoside bridging intracellular metabolism with intercellular communication, plays an essential role in regulating processes such as sleep and seizure. While the functions of extracellular Ado (“eAdo”) are well documented, our knowledge about the distribution and regulatory functions of intracellular Ado (“iAdo”) is limited by a lack of methods for detecting iAdo in vivo. Here, we develop HypnoS, a genetically encoded fluorescent sensor for iAdo characterized by its high sensitivity, specificity, spatiotemporal resolution, and rapid response (sub-seconds). HypnoS enables real-time visualization of iAdo dynamics in live cultures, acute brain slices, flies, and freely moving mice. Using HypnoS for dual-color mesoscopic imaging in mice, we show that seizure-induced iAdo waves propagated across the cortex, following calcium signals. Additionally, two-photon imaging reveals that iAdo decays more rapidly in astrocytes than in neurons during seizures. Moreover, by recording iAdo dynamics in the basal forebrain during the sleep-wake cycle, we observe that iAdo signals are present during wakefulness and rapid eye movement (REM) sleep, regulated by equilibrative nucleoside transporters (ENT1/2). Thus, HypnoS is a versatile and powerful tool for investigating the biological functions of iAdo across a range of physiological and pathological states.

Glutamine is the most abundant amino acid in human blood and muscle, and is integral to a wide variety of functions in cancer cells. However, the inability to monitor the subcellular distribution of glutamine in real‐time has obscured understanding of glutamine metabolism under physiological and pathological conditions. Here, we report the development of a genetically encoded fluorescent sensor and demonstrate how this GlnBP‐cpYFP fusion “GlutaR sensor” undergoes glutamine‐induced conformational changes reflected in detectable fluorescence responses. Obtained after iterative screening of approximately 1,600 variants, GlutaR exhibits a ratiometric readout, fast response kinetics, and high responsivity (R _488/405 of ~1000 %), and we demonstrate its selectivity for monitoring glutamine fluctuations in multiple cell types. Additionally, using digitonin permeabilization of GlutaR HeLa cells, we generated a calibration curve and performed in situ titration to quantify free glutamine concentrations in subcellular compartments (cytosol, nucleus, mitochondria). Subsequently, we applied GlutaR to investigate how chemical and genetic inhibition of glutamine synthetase (GS) and glutaminase (GLS) differentially alter glutamine levels in subcellular compartments. Finally, we demonstrate GlutaR's ability to monitor dynamic glutamine levels in muscle and liver tissues of diabetic mice in vivo . These findings collectively demonstrate GlutaR as a versatile tool for the spatiotemporal characterization of glutamine metabolism in living cells and tissues.

Glutamine is the most abundant amino acid in human blood and muscle, and is integral to a wide variety of functions in cancer cells. However, the inability to monitor the subcellular distribution of glutamine in real‐time has obscured understanding of glutamine metabolism under physiological and pathological conditions. Here, we report the development of a genetically encoded fluorescent sensor and demonstrate how this GlnBP‐cpYFP fusion “GlutaR sensor” undergoes glutamine‐induced conformational changes reflected in detectable fluorescence responses. Obtained after iterative screening of approximately 1,600 variants, GlutaR exhibits a ratiometric readout, fast response kinetics, and high responsivity (R _488/405 of ~1000 %), and we demonstrate its selectivity for monitoring glutamine fluctuations in multiple cell types. Additionally, using digitonin permeabilization of GlutaR HeLa cells, we generated a calibration curve and performed in situ titration to quantify free glutamine concentrations in subcellular compartments (cytosol, nucleus, mitochondria). Subsequently, we applied GlutaR to investigate how chemical and genetic inhibition of glutamine synthetase (GS) and glutaminase (GLS) differentially alter glutamine levels in subcellular compartments. Finally, we demonstrate GlutaR's ability to monitor dynamic glutamine levels in muscle and liver tissues of diabetic mice in vivo . These findings collectively demonstrate GlutaR as a versatile tool for the spatiotemporal characterization of glutamine metabolism in living cells and tissues.

MicroRNAs (miRNAs) play indispensable roles in post-transcriptional gene regulation. The identification of target mRNAs is essential for dissecting the recognition basis, dynamics, and regulatory mechanism of miRNA-mRNA interactions. However, the lack of an unbiased method for detecting weak miRNA-mRNA interactions remains a long-standing obstacle for miRNA research. Here, we develop and provide proof-of-concept evidence demonstrating a chemical G-clamp-enhanced photo-cross-linking strategy for covalent capture of intracellular miRNA targets in different cell lines. This approach relies on an a ryl-diazirine-G-clamp-modified-nucleoside (ARAGON) miRNA probe containing an alkynyl group that improves the thermal stability of miRNA-target mRNA duplex molecules and can rapidly cross-link with the complementary strand upon UV 365 nm activation, enhancing the transient capture of mRNA targets. After validating the accuracy and binding properties of ARAGON-based miRNA probes through the successful enrichment for the known targets of miR-106a, miR-21, and miR-101, we then extend ARAGON's application to screen for previously unknown targets of different miRNAs in various cell lines. Ultimately, results in this study uncover GAB1 as a target of miR-101 in H1299 lung cancer cells and show that miR-101 silencing of GAB1 can promote apoptosis in H1299 cells, suggesting an oncogenic mechanism of GAB1. This study thus provides a powerful and versatile tool for enhanced screening of global miRNA targets in cells to facilitate investigations of miRNA functions in fundamental cellular processes and disease pathogenesis.

The addition of various chemical modifications to RNA introduces an additional layer of complexity to the regulation of gene expression. Among all RNA modifications, N⁶‐methyladenosine (m⁶A) has earned its status as the most abundant and well‐studied post‐transcriptional modification in mammalian mRNA. Nevertheless, understanding the role of m⁶A in shaping the fate of RNA molecules and its influence on gene expression heavily depends on the development and application of detection technologies. Among all m⁶A detection methods, chemical‐based sequencing methods show unique advantages. Our group recently developed an absolute quantification method named GLORI, which employs nitrite and glyoxal to convert adenosine to inosine efficiently. With its potential to emerge as the gold standard for m⁶A detection, GLORI showcases the promise of nitrite‐based approaches. This review provides a comprehensive overview of m⁶A detection techniques based on deamination or nitrosation, evaluating their strengths and limitations. Furthermore, we offer insights into the future directions of innovative approaches in m⁶A profiling.

N⁶-methyladenosine (m⁶A) is the most abundant posttranscriptional chemical modification in mRNA, involved in regulating various physiological and pathological processes throughout mRNA metabolism. Recently, we developed GLORI, a sequencing method that enables the production of a globally absolute-quantitative m⁶A map at single-base resolution. Our technique utilizes the glyoxal- and nitrite-based chemical reaction, which selectively deaminates unmethylated adenosines while leaving m⁶A intact. The RNA library can then be prepared using a modified library construction protocol from enhanced UV crosslinking and immunoprecipitation (eCLIP) or commercial kits. Here we provide a detailed protocol for proper RNA sample handling and provide further guidelines for the use of a tailored bioinformatics pipeline (GLORI-tools) for subsequent data analysis. Compared with current methods, this new method is both exceptionally sensitive and robust, capable of identifying ~80,000 m⁶A sites with 50 Gb sequencing data in mammalian cells. It also provides a quantitative readout for m⁶A sites at single-base resolution. We hope the technique will provide a precise and unbiased tool for investigating m⁶A biology across various fields. Basic expertise in molecular biology and knowledge of bioinformatics are required for the protocol. The entire procedure can be completed within a week, with the library construction process taking ~4 d.