Fat Mass And Obesity-associated Protein Research Articles

Construction of a label-free fluorescent biosensor for homogeneous detection of m6A eraser FTO in breast cancer tissues

Fat mass and obesity-associated protein (FTO) is a crucial eraser of RNA N6- methyladenosine (m6A) modification, and abnormal FTO expression level is implicated in pathogenesis of numerous cancers. Herein, we demonstrate the construction of a label-free fluorescent biosensor for homogeneous detection of m6A eraser FTO in breast cancer tissues. When FTO is present, it specifically erases the methyl group in m6A, inducing the cleavage of demethylated DNA by endonuclease DpnII and the generation of a single-stranded DNA (ssDNA) with a 3′-hydroxyl group. Subsequently, terminal deoxynucleotidyl transferase (TdT) promotes the incorporation of dTTPs into the ssDNA to obtain a long polythymidine (T) DNA sequence. The resultant long poly (T) DNA sequence can act as a template to trigger hyperbranched strand displacement amplification (HSDA), yielding numerous DNA fragments that may be stained by SYBR Gold to produce an enhanced fluorescence signal. This biosensor processes ultrahigh sensitivity with a detection limit of 1.65 × 10−10 mg/mL (2.6 fM), and it can detect the FTO activity in a single MCF-7 cell. Moreover, this biosensor can screen the FTO inhibitors, evaluate enzyme kinetic parameters, and discriminate the FTO expression levels in the tissues of breast cancer patients and healthy persons.

Structure‐Activity Relationships of 2‐(Arylthio)benzoic Acid FTO Inhibitors

AbstractThe biological role of the fat mass and obesity‐associated protein (FTO) in the initiation and progress of acute myeloid leukemia (AML) has been elucidated, and several representative FTO inhibitors can markedly suppress the proliferation of AML cells. We previously developed FTO inhibitors including FB23. In this study, we adopted bioisosteric replacement of the intramolecular hydrogen bond in FB23 with a sulfur‐oxygen interaction to generate a series of 2‐(arylthio)benzoic acid FTO inhibitors and established their structure‐activity relationships. Compound 8c was the most potent 2‐(arylthio)benzoic acid FTO inhibitor with an IC50 value of 0.3±0.1 μM, which was comparable with that of FB23 in vitro. To enhance the antiproliferative effects in AML cell lines, we applied a prodrug strategy and prepared some esters. 7l, the methyl ester of 8l, exerted a superior inhibitory effect on a panel of AML cancer cell lines. Additionally, 7l treatment notably increased global m6A abundance in AML cells. Collectively, our data suggest that 2‐(arylthio)benzoic acid may be a new lead compound for inhibition of FTO, and the prodrug analog exhibit potential in the treatment of AML.

USP18 Stabilized FTO Protein to Activate Mitophagy in Ischemic Stroke Through Repressing m6A Modification of SIRT6.

Ischemic stroke (IS) is a dangerous cerebrovascular disorder with a significant incidence and death rate. Ubiquitin-specific peptidase 18 (USP18) has been proven to mitigate ischemic brain damage; however, its potential regulatory mechanisms remain unclear. In vivo and in vitro models of IS were established by middle cerebral artery occlusion (MCAO) and oxygen-glucose deprivation/reoxygenation (OGD/R). Neurocyte injury was detected by MTT, LDH, ROS level, mitochondrial membrane potential (Δψm), and flow cytometry. Molecular expression was evaluated by qPCR, Western blotting, and immunofluorescence staining. Molecular mechanisms were determined by Co-IP, RIP, and MeRIP. IS injury was determined by neurological behavior score and TTC staining. Mitophagy was observed by TEM. USP18 and fat mass and obesity-associated protein (FTO) expression declined after OGD/R. Dysfunctional mitochondrial and apoptosis in OGD/R-stimulated neurocytes were eliminated by USP18/FTO overexpression via mitophagy activation. USP18-mediated de-ubiquitination was responsible for increasing FTO protein stability. Up-regulation of FTO protein restrained m6A modification of sirtuin6 (SIRT6) in a YTHDF2-dependent manner to enhance SIRT6 expression and subsequent activation of AMPK/PGC-1α/AKT signaling. FTO induced mitophagy to ameliorate nerve cell damage through SIRT6/AMPK/PGC-1α/AKT pathway. Finally, USP18/FTO overexpression relieved IS in rats via triggering SIRT6/AMPK/PGC-1α/AKT axis-mediated mitophagy. USP18 increased FTO protein stability to trigger SIRT6-induced mitophagy, thus mitigating IS. Our data unravel the novel neuroprotective mechanism of USP18 and suggest its potential as a promising treatment target for IS.

FTO ameliorates doxorubicin-induced cardiotoxicity by inhibiting ferroptosis via P53–P21/Nrf2 activation in a HuR-dependent m6A manner

Doxorubicin (DOX)-induced cardiotoxicity seriously limits its clinical applicability, and no therapeutic interventions are available. Ferroptosis, an iron-dependent regulated cell death characterised by lipid peroxidation, plays a pivotal role in DOX-induced cardiotoxicity. N6-methyladenosine (m6A) methylation is the most frequent type of RNA modification and involved in DOX-induced ferroptosis, however, its underlying mechanism remains unclear. P21 was recently found to inhibit ferroptosis by interacting with Nrf2 and is regulated in a P53-dependent or independent manner, such as through m6A modification. In the present study, we investigated the mechanism underlying m6A modification in DOX-induced ferroptosis by focusing on P21. Our results show that fat mass and obesity–associated protein (FTO) down-regulation was associated with DOX-induced cardiotoxicity. FTO over-expression significantly improved cardiac function and cell viability in DOX-treated mouse hearts and H9C2 cells. FTO over-expression significantly inhibited DOX-induced ferroptosis, and the Fer-1 inhibition of ferroptosis significantly reduced DOX-induced cardiotoxicity. P21 was significantly upregulated by FTO and activated Nrf2, playing a crucial role in the anti-ferroptotic effect. FTO upregulated P21/Nrf2 in a P53-dependent manner by mediating the demethylation of P53 or in a P53-independent manner by mediating P21/Nrf2 directly. Human antigen R (HuR) is crucial for FTO-mediated regulation of ferroptosis and P53–P21/Nrf2. Notably, we also found that P21 inhibition in turn inhibited HuR and P53 expression, while HuR inhibition further inhibited FTO expression. RNA immunoprecipitation assay showed that HuR binds to the transcripts of FTO and itself. Collectively, FTO inhibited DOX-induced ferroptosis via P21/Nrf2 activation by mediating the m6A demethylation of P53 or P21/Nrf2 in a HuR-dependent manner and constituted a positive feedback loop with HuR and P53–P21. Our findings provide novel insight into key functional mechanisms associated with DOX-induced cardiotoxicity and elucidate a possible therapeutic approach.

Identifying Potent Fat Mass and Obesity-Associated Protein Inhibitors Using Deep Learning-Based Hybrid Procedures

The fat mass and obesity-associated (FTO) protein catalyzes metal-dependent modifications of nucleic acids, namely the demethylation of methyl adenosine inside mRNA molecules. The FTO protein has been identified as a potential target for developing anticancer therapies. Identifying a suitable ligand-targeting FTO protein is crucial to developing chemotherapeutic medicines to combat obesity and cancer. Scientists worldwide have employed many methodologies to discover a potent inhibitor for the FTO protein. This study uses deep learning-based methods and molecular docking techniques to investigate the FTO protein as a target. Our strategy involves systematically screening a database of small chemical compounds. By utilizing the crystal structures of the FTO complexed with ligands, we successfully identified three small-molecule chemical compounds (ZINC000003643476, ZINC000000517415, and ZINC000001562130) as inhibitors of the FTO protein. The identification process was accomplished by employing a combination of screening techniques, specifically deep learning (DeepBindGCN) and Autodock vina, on the ZINC database. These compounds were subjected to comprehensive analysis using 100 nanoseconds of molecular dynamics and binding free energy calculations. The findings of our study indicate the identification of three candidate inhibitors that might effectively target the human fat mass and obesity protein. The results of this study have the potential to facilitate the exploration of other chemicals that can interact with FTO. Conducting biochemical studies to evaluate these compounds’ effectiveness may contribute to improving fat mass and obesity treatment strategies.

Lactylation-driven FTO targets CDK2 to aggravate microvascular anomalies in diabetic retinopathy

Diabetic retinopathy (DR) is a leading cause of irreversible vision loss in working-age populations. Fat mass and obesity-associated protein (FTO) is an N6-methyladenosine (m6A) demethylase that demethylates RNAs involved in energy homeostasis, though its influence on DR is not well studied. Herein, we detected elevated FTO expression in vitreous fibrovascular membranes of patients with proliferative DR. FTO promoted cell cycle progression and tip cell formation of endothelial cells (ECs) to facilitate angiogenesis in vitro, in mice, and in zebrafish. FTO also regulated EC-pericyte crosstalk to trigger diabetic microvascular leakage, and mediated EC–microglia interactions to induce retinal inflammation and neurodegeneration in vivo and in vitro. Mechanistically, FTO affected EC features via modulating CDK2 mRNA stability in an m6A-YTHDF2-dependent manner. FTO up-regulation under diabetic conditions was driven by lactate-mediated histone lactylation. FB23-2, an inhibitor to FTO’s m6A demethylase activity, suppressed angiogenic phenotypes in vitro. To allow for systemic administration, we developed a nanoplatform encapsulating FB23-2 and confirmed its targeting and therapeutic efficiency in mice. Collectively, our study demonstrates that FTO is important for EC function and retinal homeostasis in DR, and warrants further investigation as a therapeutic target for DR patients.

Reaction-mediated dual signaling inhibition gated organic photoelectrochemical transistors biosensor for FTO activity detection based on the in situ oxidization of ZnSe/Bi2MoO6 heterojunction

Organic photoelectrochemical transistor (OPECT) biosensing is a new concept that combines photoelectrochemical and organic electrochemical transistor biosensing. It reduces background noise and combines both signal amplification for low abundance biomolecule detection. In this work, an OPECT biosensor for the detection of fat mass and obesity-associated protein (FTO) was constructed, in which ZnSe/Bi2MoO6 was chosen as the gate material, hm6ARNA (containing N6-methyladenine RNA sequence) as the target molecule, and hydroquinone as the electron donor. Under FTO oxidation induced RNA demethylation, the formation of hemin loaded with G-quadruplex on the electrode surface was triggered. With the catalysis of hemin-based DNAzyme towards the oxidation reaction caused by H2O2 towards hydroquinone and ZnSe, the electron donor was consumed and ZnSe was oxidized in situ to form SeO2 which reduced the photo-anodic response. The variation of the gate effectively gated the state of the channel polymer thus producing different channel current responses, a process that was closely related to the content of FTO. In addition, the effects of rhein and potassium perfluorohexanesulfonate on FTO activity were also investigated in this experiment, with IC50 values of 10.62 μM, 31.28 mg/L respectively.

Enzyme-Free Dynamic DNA Reaction Networks for On-Demand Bioanalysis and Bioimaging.

ConspectusThe pursuit of in-depth studying the nature and law of life activity has been dominating current research fields, ranging from fundamental biological studies to applications that concern synthetic biology, bioanalysis, and clinical diagnosis. Motivated by this intention, the spatiotemporally controlled and in situ analysis of living cells has been a prospective branch by virtue of high-sensitivity imaging of key biomolecules, such as biomarkers. The past decades have attested that deoxyribonucleic acid (DNA), with biocompatibility, programmability, and customizable features, is a competitive biomaterial for constructing high-performance molecular sensing tools. To conquer the complexity of the wide extracellular-intracellular distribution of biomarkers, it is a meaningful breakthrough to explore high-efficiently amplified DNA circuits, which excel at operating complex yet captivating dynamic reaction networks for various bioapplications. In parallel, the multidimensional performance improvements of nucleic acid circuits, including the availability, detection sensitivity, and reliability, are critical parameters for realizing accurate imaging and cell regulation in bioanalysis.In this Account, we summarize our recent work on enzyme-free dynamic DNA reaction networks for bioanalysis from three main aspects: DNA circuitry functional extension of molecular recognition for epigenetic analysis and regulation, DNA circuitry amplification ability improvement for sensitive biomarker detection, and site-specific activation of DNA circuitry systems for reliable and accurate cell imaging. In the first part, we have designed an epigenetically responsive deoxyribozyme (DNAzyme) circuitry system for intracellular imaging and gene regulation, which enriches the possible analyzed species by chemically modifying conventional DNAzyme. For example, an exquisite N6-methyladenine (m6A)-caged DNAzyme was built for achieving the precise FTO (fat mass and obesity-associated protein)-directed gene regulation. In addition, varieties of DNAzyme-based nanoplatforms with self-sufficient cofactor suppliers were assembled, which subdued the speed-limiting hardness of DNAzyme cofactors in live-cell applications. In the second part, we have developed a series of hierarchically assembled DNA circuitry systems to improve the signal transduction ability of traditional DNA circuits. First, the amplification ability of the DNAzyme circuit has been significantly enhanced via several heterogeneously or homogeneously concatenated circuitry models. Furthermore, a feedback reaction pathway was integrated into these concatenated circuits, thus dramatically increasing the amplification efficiency. Second, considering the complex cellular environment, we have simplified the redundancy of multicomponents or reaction procedures of traditional cascaded circuits, relying on the minimal component complexity and merely one modular catalytic reaction, which guaranteed high cell-delivering uniformity while fostering reaction kinetics and analysis reliability. In the third part, we have constructed in-cell-selective endogenous-stimulated DNA circuitry systems via the multiply guaranteed molecular recognitions, which could not only eliminate the signal leakage, but could also retain its on-site and multiplex signal amplification. Based on the site-specific activation strategy, more circuitry availability in cellular scenarios has been acquired for reliable and precise biological sensing and regulation. These enzyme-free dynamic DNA reaction networks demonstrate the purpose-to-concreteness engineering for tailored multimolecule recognition and multiple signal amplification, achieving high-gain signal transduction and high-reliability targeted imaging in bioanalysis. We envision that the enzyme-free dynamic DNA reaction network can contribute to more bioanalytical layouts, which will facilitate the progression of clinical diagnosis and prognosis.

FTO Deficiency Alleviates LPS-induced Acute Lung Injury by TXNIP/NLRP3-mediated Alveolar Epithelial Cell Pyroptosis.

N6-methyladenosine (m6A) plays a role in various diseases, but it has rarely been reported in acute lung injury (ALI). The FTO (fat mass and obesity-associated) protein can regulate mRNA metabolism by removing m6A residues. The aim of this study was to examine the role and mechanism of the m6A demethylase FTO in LPS-induced ALI. Lung epithelial FTO-knockout mice and FTO-knockdown/overexpression human alveolar epithelial (A549) cell lines were constructed to evaluate the effects of FTO on ALI. Bioinformatics analysis and a series of invivo and invitro assays were used to examine the mechanism of FTO regulation. Rescue assays were conducted to examine whether the impact of FTO on ALI depended on the TXNIP/NLRP3 pathway. In LPS-induced ALI, RNA m6A modification amounts were upregulated, and FTO expression was downregulated. In vivo, lung epithelial FTO knockout alleviated alveolar structure disorder, tissue edema, and pulmonary inflammation and improved the survival of ALI mice. In vitro, FTO knockdown reduced A549 cell damage and death induced by LPS, whereas FTO overexpression exacerbated cell damage and death. Mechanistically, bioinformatics analysis revealed that TXNIP was a downstream target of FTO. FTO deficiency mitigated pyroptosis in LPS-induced ALI via the TXNIP/NLRP3 pathway. Rescue assays confirmed that the impact of FTO on the TXNIP/NLRP3 pathway was significantly reversed by the TXNIP inhibitor SRI-37330. Deficiency of FTO alleviates LPS-induced ALI via TXNIP/NLRP3 pathway-mediated alveolar epithelial cell pyroptosis, which might be a novel therapeutic strategy for combating ALI.

The m6A demethylases FTO and ALKBH5 aggravate the malignant progression of nasopharyngeal carcinoma by coregulating ARHGAP35

N6-methyladenosine (m6A) is an RNA modification that can be removed by demethylases [fat mass and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5)], which regulate gene expression and cell function. We show that m6A levels and m6A demethylase levels are altered in nasopharyngeal carcinoma (NPC) tissues vs. normal tissues. High FTO and ALKBH5 predict a poor prognosis in NPC patients. Silencing FTO and ALKBH5 inhibited the malignant behavior of patient-derived NPC cells in a short time. However, as time progressed, the inhibitory effect of FTO or ALKBH5 was weakened, and the cosilencing of FTO and ALKBH5 maintained a better inhibitory effect. Combined transcriptome and m6A-seq analysis revealed a downstream target gene that was jointly regulated by FTO and ALKBH5 in NPC, and ARHGAP35 was chosen to do further study. The synergistic silencing of FTO and ALKBH5 increased the methylation level on the mRNA CDS of a new transcription factor (ARHGAP35) and positively regulate the protein coding capacity and mRNA stability of ARHGAP35, thus leading to increased expression of ARHGAP35 and inhibition of the malignant phenotype of tumor cells. Our study revealed that the growth and metastasis of NPC can be stably inhibited through synergistic silencing of the demethylases FTO and ALKBH5, which play a positive role in the treatment of NPC by regulating the downstream transcript ARHGAP35 and increasing its m6A level.

AVCAPIR: A Novel Procalcific PIWI-Interacting RNA in Calcific Aortic Valve Disease.

Calcification of the aortic valve leads to increased leaflet stiffness and consequently results in the development of calcific aortic valve disease (CAVD). However, the underlying molecular and cellular mechanisms of calcification remain unclear. Here, we identified a novel aortic valve calcification-associated PIWI-interacting RNA (piRNA; AVCAPIR) that increases valvular calcification and promotes CAVD progression. Using piRNA sequencing, we identified piRNAs contributing to the pathogenesis of CAVD that we termed AVCAPIRs. High-cholesterol diet-fed ApoE-/- mice with AVCAPIR knockout were used to examine the role of AVCAPIR in aortic valve calcification (AVC). Gain- and loss-of-function assays were conducted to determine the role of AVCAPIR in the induced osteogenic differentiation of human valvular interstitial cells. To dissect the mechanisms underlying AVCAPIR-elicited procalcific effects, we performed various analyses, including an RNA pulldown assay followed by liquid chromatography-tandem mass spectrometry, methylated RNA immunoprecipitation sequencing, and RNA sequencing. RNA pulldown and RNA immunoprecipitation assays were used to study piRNA interactions with proteins. We found that AVCAPIR was significantly upregulated during AVC and exhibited potential diagnostic value for CAVD. AVCAPIR deletion markedly ameliorated AVC in high-cholesterol diet-fed ApoE-/- mice, as shown by reduced thickness and calcium deposition in the aortic valve leaflets, improved echocardiographic parameters (decreased peak transvalvular jet velocity and mean transvalvular pressure gradient, as well as increased aortic valve area), and diminished levels of osteogenic markers (Runx2 and Osterix) in aortic valves. These results were confirmed in osteogenic medium-induced human valvular interstitial cells. Using unbiased protein-RNA screening and molecular validation, we found that AVCAPIR directly interacts with FTO (fat mass and obesity-associated protein), subsequently blocking its N6-methyladenosine demethylase activity. Further transcriptomic and N6-methyladenosine modification epitranscriptomic screening followed by molecular validation confirmed that AVCAPIR hindered FTO-mediated demethylation of CD36 mRNA transcripts, thus enhancing CD36 mRNA stability through the N6-methyladenosine reader IGF2BP1 (insulin-like growth factor 2 mRNA binding protein 1). In turn, the AVCAPIR-dependent increase in CD36 stabilizes its binding partner PCSK9 (proprotein convertase subtilisin/kexin type 9), a procalcific gene, at the protein level, which accelerates the progression of AVC. We identified a novel piRNA that induced AVC through an RNA epigenetic mechanism and provide novel insights into piRNA-directed theranostics in CAVD.

Fat mass and obesity-associated protein inhibit the pathology of rheumatoid arthritis through the NSUN2/SFRP1/Wnt/β-catenin signal axis.

The purpose of this study is to investigate whether fat mass and obesity-associated protein (FTO) and NOL1/NOP2/Sun domain family member 2 (NSUN2) mediated RNA methylation is associated with RA pathology. We studied the anti-rheumatoid arthritis (RA) mechanism mediated by FTO and NSUN2 in RA samples and collagen-induced arthritis (CIA) rats using real time qPCR (RT-qPCR), western blot, immunofluorescence, and other methods. The expression of NSUN2 was significantly increased in both RA patients and CIA rats compared with normal controls. Knockdown of NSUN2 blocked the Wnt/β-catenin signaling pathway and inhibited RA pathological factors such as MMP3, fibronectin, and interleukins. FTO overexpression inhibited RA by inhibiting the expression of NSUN2, up-regulating the level of SFRP1 protein, and blocking the Wnt/β-catenin signaling pathway. NSUN2 overexpression interfered with the inhibitory effects of FTO on the Wnt/β-catenin signaling pathway and RA pathology, which further verified that FTO inhibited RA through the NSUN2/SFRP1/Wnt/β-catenin signal axis. FTO and NSUN2 are important factors of RA, and this work provides new potential diagnostic biomarkers and therapeutic targets for RA. We also reveal a gene expression regulation pattern of the interaction between m6A and m5C. revealing the pathogenesis of RA from the perspective of RNA methylation.

Fat mass and obesity-associated (FTO) gene is essential for insulin secretion and β-cell function: In vitro studies using INS-1 cells and human pancreatic islets

AimsIn this study, we investigated the role of the FTO gene in pancreatic β-cell biology and its association with type 2 diabetes (T2D). To address this issue, human pancreatic islets and rat INS-1 (832/13) cells were used to perform gene silencing, overexpression, and functional analysis of FTO expression; levels of FTO were also measured in serum samples obtained from diabetic and obese individuals. ResultsThe findings revealed that FTO expression was reduced in islets from hyperglycemic/diabetic donors compared to normal donors. This reduction correlated with decreased INS and GLUT1 expression and increased PDX1, GCK, and SNAP25 expression. Silencing of Fto in INS-1 cells impaired insulin release and mitochondrial ATP production and increased apoptosis in pro-apoptotic cytokine-treated cells. However, glucose uptake and reactive oxygen species production rates remained unaffected. Downregulation of key β-cell genes was observed following Fto-silencing, while Glut2 and Gck were unaffected. RNA-seq analysis identified several dysregulated genes involved in metal ion binding, calcium ion binding, and protein serine/threonine kinase activity. Furthermore, our findings showed that Pdx1 or Mafa-silencing did not influence FTO protein expression. Overexpression of FTO in human islets promoted insulin secretion and upregulated INS, PDX1, MAFA, and GLUT1 expression. Serum FTO levels did not significantly differ between individuals with diabetes or obesity and their healthy counterparts. ConclusionThese findings suggest that FTO plays a crucial role in β-cell survival, metabolism, and function and point to a potential therapeutic utility of FTO in T2D patients.

BMP9-ID1 Pathway Attenuates N6-Methyladenosine Levels of CyclinD1 to Promote Cell Proliferation in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a highly lethal malignant neoplasm, and the involvement of bone morphogenetic protein 9 (BMP9) has been implicated in the pathogenesis of liver diseases and HCC. Our goal was to investigate the role of BMP9 signaling in regulating N6-methyladenosine (m6A) methylation and cell cycle progression, and evaluate the therapeutic potential of BMP receptor inhibitors for HCC treatment. We observed that elevated levels of BMP9 expression in tumor tissues or serum samples from HCC patients were associated with a poorer prognosis. Through in vitro experiments utilizing the m6A dot blotting assay, we ascertained that BMP9 reduced the global RNA m6A methylation level in Huh7 and Hep3B cells, thereby facilitating their cell cycle progression. This effect was mediated by an increase in the expression of the inhibitor of DNA-binding protein 1 (ID1). Additionally, using methylated RNA immunoprecipitation qPCR(MeRIP-qPCR), we showed that the BMP9-ID1 pathway promoted CyclinD1 expression by decreasing the m6A methylation level in the 5' UTR of mRNA. This occurred through the upregulation of the fat mass and obesity-associated protein (FTO) in Huh7 and Hep3B cells. In our in vivo mouse xenograft models, we demonstrated that blocking the BMP receptor with LDN-212854 effectively suppressed HCC growth and induced global RNA m6A methylation. Overall, our findings indicate that the BMP9-ID1 pathway promotes HCC cell proliferation by down-regulating the m6A methylation level in the 5' UTR of CyclinD1 mRNA. Targeting the BMP9-ID1 pathway holds promise as a potential therapeutic strategy for treating HCC.

A Label‐Free Deoxyribozyme Sensor for m6A Demethylase Activity Detection†

Comprehensive SummaryA label‐free deoxyribozyme (DNAzyme) biosensor for m6A demethylase activity detection is developed. When demethylase FTO (Fat mass and obesity‐associated protein), an important m6A demethylase of ALKBH demethylase family is present, the m6A group is specifically demethylated, and the cleavage activity of DNAzyme is restored. This allows the cleavage product that contains G‐quadruplex sequence to bind to thioflavin T (ThT) and generate fluorescence signals. The biosensor shows high specificity and sensitivity, and fast reaction speed. Our study demonstrates a new design of allosteric DNAzyme for sensing. This method represents the first label‐free nucleic acid biosensor for FTO assay, providing a feasible route towards inhibitors screening.

RNA m6A dynamic modification mediated by nucleus-localized FTO is involved in follicular reserve.

In eukaryotic organisms, the most common internal modification of messenger RNA (mRNA) is N6-methyladenosine (m6A). This modification can be dynamically and reversibly controlled by specific enzymes known as m6A writers and erasers. The fat-mass and obesity-associated protein (FTO) catalyzes RNA demethylation and plays a critical role in various physiological and pathological processes. Our research identified dynamic alterations in both m6A and FTO during the assembly of primordial follicles, with an inverse relationship observed for m6A levels and nuclear-localized FTO expression. Application of Fto small interfering RNA (siRNA) altered the expression of genes related to cell proliferation, hormone regulation, and cell chemotaxis, and affected RNA alternative splicing. Overexpression of the full-length Fto gene led to changes in m6A levels, alternative splicing of Cdk5, cell proliferation, cell cycle progression, and proportion of primordial follicles. Conversely, overexpression of Fto lacking a nuclear localization signal (NLS) did not significantly alter m6A levels or primordial follicle assembly. These findings suggest that FTO, localized in the nucleus but not in the cytoplasm, regulates RNA m6A demethylation and plays a role in cell proliferation, cell cycle progression, and primordial follicle assembly. These results highlight the potential of m6A and its eraser FTO as possible biomarkers and therapeutic targets.

The FTO-CMPK2 Pathway in Fibroblast-like Synoviocytes Modulates Rheumatoid Arthritis Synovial Inflammation and Cartilage Homeostasis via mtDNA Regulation.

In rheumatoid arthritis (RA), a debilitating autoimmune disorder marked by chronic synovial inflammation and progressive cartilage degradation, fibroblast-like synoviocytes (FLS) are key pathogenic players. Current treatments targeting these cells are limited. Our study focused on the Fat Mass and Obesity-associated protein (FTO), known for its roles in cell proliferation and inflammatory response modulation, and its involvement in RA. We specifically examined the inflammatory regulatory roles of FTO and CMPK2, a mitochondrial DNA synthesis protein, in FLS. Utilizing a combination of in vitro and in vivo methods, including FTO inhibition and gene knockdown, we aimed to understand FTO's influence on RA progression and chondrocyte functionality. Our findings showed that increased FTO expression in RA synovial cells enhanced their proliferation and migration and decreased senescence and apoptosis. Inhibiting FTO significantly slowed the disease progression in our models. Our research also highlighted that the FTO-CMPK2 pathway plays a crucial role in regulating synovial inflammation through the mtDNA-mediated cGAS/STING pathway, affecting chondrocyte homeostasis. This study indicates that targeting the FTO-CMPK2 axis could be a promising new therapeutic strategy for managing RA.

Protective effect of vitexin against high fat-induced vascular endothelial inflammation through inhibiting trimethylamine N-oxide-mediated RNA m6A modification.

A high-fat diet (HFD) is a major risk factor for cardiovascular disease. However, the specific effects of a HFD on vascular inflammation and the protective role of vitexin, a bioactive compound derived from food, require further research. This study investigated the protective effects of vitexin intervention against HFD-induced vascular inflammation and its underlying mechanism. The results demonstrated that vitexin intervention significantly reduced body weight, serum total cholesterol, and low-density lipoprotein cholesterol levels in HFD-fed mice. Vitexin also improved vascular pathological changes and the inflammatory status in the mice. Furthermore, vitexin intervention reduced serum TMAO levels in HFD-fed mice by altering the gut microbiota composition. The HFD significantly increased N6-methyladenosine (m6A) levels in aorta tissues, while vitexin intervention reversed this abnormal m6A level. Through metabolite affinity responsive target fluorescence quenching and molecular docking assays, it was found that vitexin could directly bind to fat mass and obesity-associated protein (FTO), potentially promoting m6A demethylation. The dose-response relationship between TMAO and inflammation/m6A was further validated in HUVEC cells and in vivo mouse experiments. Specifically, TMAO increased m6A levels and inflammation, while vitexin inhibited TMAO-mediated m6A modification, exhibiting anti-inflammatory effects. In conclusion, this study demonstrates the protective role of vitexin against HFD-induced vascular inflammation by inhibiting TMAO-mediated RNA m6A modification, laying the foundation for the development of functional foods.

Demethylase FTO inhibits the occurrence and development of triple-negative breast cancer by blocking m 6A-dependent miR-17-5p maturation-induced ZBTB4 depletion.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer, and its mechanisms of occurrence and development remain unclear. In this study, we aim to investigate the role and molecular mechanisms of the demethylase FTO (fat mass and obesity-associated protein) in TNBC. Through analysis of public databases, we identify that FTO may regulate the maturation of miR-17-5p and subsequently influence the expression of zinc finger and BTB domain-containing protein 4 (ZBTB4), thereby affecting the occurrence and progression of TNBC. We screen for relevant miRNAs and mRNAs from the GEO and TCGA databases and find that the FTO gene may play a crucial role in TNBC. In vitro cell experiments demonstrate that overexpression of FTO can suppress the proliferation, migration, and invasion ability of TNBC cells and can regulate the maturation of miR-17-5p through an m 6A-dependent mechanism. Furthermore, we establish a xenograft nude mouse model and collect clinical samples to further confirm the role and impact of the FTO/miR-17-5p/ZBTB4 regulatory axis in TNBC. Our findings unveil the potential role of FTO and its underlying molecular mechanisms in TNBC, providing new perspectives and strategies for the research and treatment of TNBC.

Dephosphorylation Switch DNAzyme-RCA Circuit: A Robust Strategy for the Homogeneous and Reliable Detection of FTO Demethylase.

Fat mass and obesity-associated protein (FTO) plays a crucial role in regulating the dynamic modification of N6-methyladenosine (m6A) in eukaryotic mRNA. Sensitive detection of the FTO level and efficient evaluation of the FTO demethylase activity are of great importance to early cancer diagnosis and anticancer drug discovery, which are currently challenged by limited sensitivity/precision and low throughput. Herein, a robust strategy based on the dephosphorylation switch DNAzyme-rolling circle amplification (RCA) circuit, termed DSD-RCA, is developed for highly sensitive detection of FTO and inhibitor screening. Initially, the catalytic activity of DNAzyme is silenced by engineering with an m6A modification in its catalytic core. Only in the presence of target FTO can the methyl group on DNAzyme be eliminated, resulting in the activation of the catalytic activity of DNAzyme and thus cleaving the hairpin substrate to release numerous primers. Different from the conventional methods that use the downstream cleavage primer with the original 3'-hydroxyl end directly as the RCA primer with the problem of high background signal, which should be compensated by additional separation and wash steps in heterogeneous format, our DSD-RCA assay uses the upstream cleavage primer with a 2',3'-cyclic phosphate terminus at the 3'-end serving as an intrinsically blocked 3' end. Only after a dephosphorylation reaction mediated by T4 polynucleotide kinase can the upstream cleavage primers with a resultant 3'-hydroxyl end be extended by RCA. With the high signal-to-noise ratio and homogeneous property, the proposed platform can sensitively detect FTO with a limit of detection of 31.4 pM, and the relative standard deviations (RSDs %) ranging from 0.8 to 2.0% were much lower than the heterogeneous methods. The DSD-RCA method was applied for analyzing FTO in cytoplasmic lysates from different cell lines and tissues of breast cancer patients and further used for screening FTO inhibitors without the need for separation or cleaning, providing an opportunity for achieving high throughput and demonstrating the potential applications of this strategy in disease diagnostics, drug discovery, and biological applications.