Search Results (42)

Aims: Treatment of higher-risk myelodysplastic syndromes (HR-MDS) with azacytidine (AZA) exerts significant effects on the epigenome, primarily through DNA demethylation and reactivation of epigenetically silenced genes. Beyond this established mechanism, molecular AZA-linked effects are increasingly being recognized. Materials and methods: Liquid chromatography combined with mass spectrometry (LC-MS/MS) was employed for the accurate assessment of various RNA and DNA modifications pre- and post-AZA treatment of an HR-MDS cohort (N = 8). Mapping of the AZA treatment-responsive regulatory pathways was performed by miRNA-next generation sequencing (NGS), followed by a multi-layered bioinformatic pipeline, integrating miRNA differential expression, gene set enrichment, and network analyses. The precise number of mitochondrial (mt)DNA copies pre- and post-AZA was evaluated by a digital PCR assay. Results: Cell pathways affected by miRNA differential expression patterns pre- and post-AZA treatment discriminated the clinical phenotypes of Responders against Non-Responders to therapy. Intracellular RNA modifications: N6-methyladenosine (m6A), 5-methylcytidine (m5C), N1-methyladenosine (m1A), 2′-O-methylguanosine (Gm) and adenosine-to-inosine (A → I) editing were evaluated for their potential impact in treatment response. Nuclear DNA/mtDNA methylation profiles and mtDNA copy number reduction manifested the mitochondrial features affected by AZA. Our results suggest that neoplastic HSPCs in HR-MDS Responders to AZA adapt by normalizing glycolytic metabolism and enhancing ribosomal activity. The observed reduction of mtDNA content can be associated with improved survival and suppression of malignant progression. Non-Responders, despite experiencing mtDNA depletion, seem unable to coordinate such metabolic reprogramming and remain disadvantaged to AZA therapy. Full article

N⁶-methyladenine and N⁶,N⁶-dimethyladenine are the heterocyclic bases present in the RNA of eukaryotic and bacterial cells and play important regulatory roles. How the degradation of such modified nucleic acids, and the subsequent demethylation of modified heterocyclic bases, occurs in the bacterium Escherichia coli is not established. Here, we investigated the growth of adenine auxotroph strains in a minimal M9 medium supplemented with either N⁶-methyladenine or N⁶,N⁶-dimethyladenine. We found that N⁶-methyladenine supported the growth of ∆purH::Km but not that of the ∆purA::Km strain, whereas N⁶,N⁶-dimethyladenine did not support the growth of either adenine auxotroph. Similar experiments performed using structurally related 2-amino-N⁶-methylpurine and 2-amino-N⁶,N⁶-dimethylpurine bases—using ∆guaA::Km, ∆guaB::Km, and ∆purH::Km guanine auxotrophs—demonstrated that growth of only the ∆guaB::Km mutant was supported by 2-amino-N⁶-methylpurine but not by its dimethylated counterpart. We expressed and purified C-teminus 6xHis tagged E. coli adenine/adenosine deaminases AdeC and Add and tested their substrate specificity. We demonstrated that AdeC protein does not catalyse deamination of either N⁶-methyl- or N⁶,N⁶-dimethyladenine, whereas Add catalyses deamination of N⁶-methyl- but not that of N⁶,N⁶-dimethyladenosine. Based on our findings, biochemical pathways leading to the demodification and return into metabolism of N⁶-methyladenine and 2-amino-N⁶-methylpurine in E. coli are proposed. Full article

Adult glioblastoma multiforme (GBM) is the most common primary malignant brain tumour caused by multiple molecular factors. N⁶-methyl-adenosine (m⁶A) is an abundant RNA modification that governs cellular RNA metabolism. We hypothesise that changes in m⁶A-modified RNA and regulatory machinery such as the writer proteins, Methyltransferase 3 (METTL3) and WT1-associating protein (WTAP), the demethyltransferase protein, and Alpha-ketoglutarate dependent dioxygenase (FTO), are driving factors of GBM development and treatment resistance. Here, we investigated m⁶A-RNA spatial and quantitative abundance and expression of m⁶A effector proteins directly in GBM tissue and patient-derived low-passage primary adult GBM and low-grade glioma (LGG) cells, and explored the consequences of m⁶A-RNA disruption on GBM invasive capabilities, self-renewal and responsiveness to temozolomide (TMZ). We observed that METTL3, WTAP and FTO transcript and protein expression were significantly increased in cells derived from invasive regions of GBM tumours, and elevated WTAP and FTO expression significantly correlated with poor GBM patient survival. We further found that the abundance of m⁶A-modified RNA in GBM tumours was significant higher in rim and invasive tissue, as well as significantly higher in patient-derived cells from GBM tumour invasive regions. Functional depletion of these effector proteins significantly altered m⁶A levels on and the expression of the pluripotency stem cell marker SOX2 while also impairing self-renewal and cell invasion behaviour and increasing sensitivity to TMZ. The targeting of RNA modification regulatory mechanisms reveals novel therapeutic strategies aimed at improving clinical outcomes for GBM patients. Full article

PCIF1 is primarily recognized as an RNA methyltransferase that mediates N⁶-methylation of cap-proximal adenosine (m⁶Am) and plays diverse roles in gene expression. In this study, we uncover a novel role for PCIF1 as a crucial negative regulator of type I interferon (IFN) induction, a pathway critical for antiviral immunity whose dysregulation leads to inflammatory and autoimmune diseases. We demonstrate that PCIF1 deficiency robustly enhances the poly(I:C)-induced type I IFN response, accompanied by augmented STAT1 activation and interferon-stimulated gene (ISG) expression. Mechanistically, PCIF1 suppresses IFNB1 transcription by attenuating IRF3 phosphorylation and nuclear translocation, as shown by increased nascent IFNB1 mRNA synthesis and promoter activity in PCIF1-deficient cells, without affecting the mRNA stability. Crucially, this suppressive function was independent of PCIF1’s canonical RNA methyltransferase activity, as both wild-type PCIF1 and a methyltransferase-inactive mutant effectively attenuated type I IFN induction. Collectively, our findings establish PCIF1 as a novel methyltransferase-independent suppressor of type I IFN responses, revealing its previously unrecognized non-catalytic function. This discovery offers critical insights into the multifaceted regulation of innate immunity and highlights PCIF1’s non-catalytic function as a promising therapeutic target for modulating antiviral responses and inflammatory diseases. Full article

Glutamate is an excitatory neurotransmitter in the central nervous system (CNS) that mediates synaptic transmission. However, glutamate homeostasis among neural cells is broken in cerebral ischemia. Excessive glutamate triggers N-methyl-d-aspartate receptors (NMDARs) in postsynaptic neurons, leading to intracellular calcium (Ca²⁺) overload and excitoneurotoxicity. At this moment, L-lactate may affect NMDARs and play a protective role in cerebral ischemia. This work proposes that L-lactate regulates glutamate signaling among neural cells. But, dysregulation of L-lactate in glutamate signaling cascades contributes to glutamate excitotoxicity in cerebral ischemia. In detail, L-lactate regulates the glutamine(Gln)-glutamate cycle between astrocytes and presynaptic neurons, which triggers the astroglial L-lactate-sensitive receptor (LLR)-cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway, coordinating astroglial glutamate uptake and neuronal glutamate transmission. L-lactate mediates glutamate signaling and synaptic transmission among neural cells. In addition, L-lactate promotes the function of mitochondrial calcium uniporter complex (MCUC), which quickly depletes intracellular Ca²⁺ in postsynaptic neurons. In addition, L-lactate can promote the conversion of microglia from the pro-inflammatory (M1) to anti-inflammatory (M2) phenotype. Therefore, regulation of L-lactate in glutamate signaling in the CNS might become a preventive target for cerebral ischemia. Full article

The adenosine A_2A receptor (A_2AR) has been identified as a therapeutic target for treating neurodegenerative diseases and cancer. In recent years, we have highlighted the 2-aminoquinazoline heterocycle as an promising scaffold for designing new A_2AR antagonists, exemplified by 6-bromo-4-(furan-2-yl)quinazolin-2-amine 1 (K_i (hA_2AR) = 20 nM). Here, we report the synthesis of new 2-aminoquinazoline derivatives with substitutions at the C6- and C7-positions, and the introduction of aminoalkyl chains containing tertiary amines at the C2-position to enhance antagonist activity and solubility properties. Compound 5m showed a high affinity for hA_2AR with a K_i value of 5 nM and demonstrated antagonist activity with an IC₅₀ of 6 µM in a cyclic AMP assay. Introducing aminopentylpiperidine and 4-[(piperidin-1-yl)methyl]aniline substituents maintained the binding affinities (9x, K_i = 21 nM; 10d, K_i = 15 nM) and functional antagonist activities (9x, IC₅₀ = 9 µM; 10d, IC₅₀ = 5 µM) of the synthesized compounds while improving solubility. This study provides insights into the future development of A_2AR antagonists for therapeutic applications. Full article

Tozadenant (4-hydroxy-N-(4-methoxy-7-morpholinobenzo[d]thiazol-2-yl)-4-methylpiperidine-1-carboxamide) is a highly selective adenosine A_2A receptor (A_2AR) antagonist and a promising lead structure for the development of A_2AR-selective positron emission tomography (PET) probes. Although several ¹⁸F-labelled tozadenant derivatives showed favorable in vitro properties, recent in vivo PET studies observed poor brain penetration and lower specific binding than anticipated from the in vitro data. While these findings might be attributable to the structural modification associated with ¹⁸F-labelling, they could also reflect inherent properties of the parent compound. However, PET studies with radioisotopologues of tozadenant to evaluate its cerebral pharmacokinetics and brain distribution are still lacking. In the present work, we applied N-Boc-O-desmethyltozadenant as a suitable precursor for the preparation of [O-methyl-¹¹C]tozadenant ([¹¹C]tozadenant) by O-methylation with [¹¹C]methyl iodide followed by acidic deprotection. This approach afforded [¹¹C]tozadenant in radiochemical yields of 18 ± 2%, with molar activities of 50–60 GBq/µmol (1300–1600 mCi/µmol) and radiochemical purities of 95 ± 3%. In addition, in vitro autoradiography in pig and rat brain slices demonstrated the expected striatal accumulation pattern and confirmed the A_2AR specificity of the radioligand, making it a promising tool for in vivo PET studies on the cerebral pharmacokinetics and brain distribution of tozadenant. Full article

The ongoing arms race between viruses and their hosts is constantly evolving. One of the ways in which cells defend themselves against invading viruses is by using restriction factors (RFs), which are cell-intrinsic antiviral mechanisms that block viral replication and transcription. Recent research has identified a specific group of RFs that belong to the cellular epigenetic machinery and are able to restrict the gene expression of certain viruses. These RFs can be referred to as epigenetic restriction factors or eRFs. In this review, eRFs have been classified into two categories. The first category includes eRFs that target viral chromatin. So far, the identified eRFs in this category include the PML-NBs, the KRAB/KAP1 complex, IFI16, and the HUSH complex. The second category includes eRFs that target viral RNA or, more specifically, the viral epitranscriptome. These epitranscriptomic eRFs have been further classified into two types: those that edit RNA bases—adenosine deaminase acting on RNA (ADAR) and pseudouridine synthases (PUS), and those that covalently modify viral RNA—the N6-methyladenosine (m6A) writers, readers, and erasers. We delve into the molecular machinery of eRFs, their role in limiting various viruses, and the mechanisms by which viruses have evolved to counteract them. We also examine the crosstalk between different eRFs, including the common effectors that connect them. Finally, we explore the potential for new discoveries in the realm of epigenetic networks that restrict viral gene expression, as well as the future research directions in this area. Full article

Epitranscriptomic RNA modifications play a crucial role in the posttranscriptional regulation of gene expression. N⁶-methyladenosine (m⁶A) is the most prevalent internal modification of eukaryotic RNA and plays a pivotal role in RNA fate. RNA m⁶A modification is regulated by a group of cellular proteins, methyltransferases (writers) and demethylases (erasers), which add and remove the methyl group from adenosine, respectively. m⁶A modification is recognized by a group of cellular RNA-binding proteins (readers) that specifically bind to m⁶A-modified RNA, mediating effects on RNA stability, splicing, transport, and translation. The functional significance of m⁶A modification of viral and cellular RNA is an active area of virology research. In this review, we summarize and analyze the current literature on m⁶A modification of HIV-1 RNA, the multifaceted functions of m⁶A in regulating HIV-1 replication, and the role of viral RNA m⁶A modification in evading innate immune responses to infection. Furthermore, we briefly discuss the future directions and therapeutic implications of mechanistic studies of HIV-1 epitranscriptomic modifications. Full article

Reversible N⁶-adenosine methylation of mRNA, referred to as m⁶A modification, has emerged as an important regulator of post-transcriptional RNA processing. Numerous studies have highlighted its crucial role in the pathogenesis of diverse diseases, particularly cancer. Post-translational modifications of m⁶A-related proteins play a fundamental role in regulating the m⁶A methylome, thereby influencing the fate of m⁶A-methylated RNA. A comprehensive understanding of the mechanisms that regulate m⁶A-related proteins and the factors contributing to the specificity of m⁶A deposition has the potential to unveil novel therapeutic strategies for cancer treatment. This review provides an in-depth overview of our current knowledge of post-translational modifications of m⁶A-related proteins, associated signaling pathways, and the mechanisms that drive the specificity of m⁶A modifications. Additionally, we explored the role of m⁶A-dependent mechanisms in the progression of various human cancers. Together, this review summarizes the mechanisms underlying the regulation of the m⁶A methylome to provide insight into its potential as a novel therapeutic strategy for the treatment of cancer. Full article

Clozapine is listed as one of the most effective antipsychotics and has been approved for treating treatment-resistant schizophrenia (TRS); however, several type A and B adverse reactions, including weight gain, metabolic complications, cardiotoxicity, convulsions, and discontinuation syndromes, exist. The critical mechanisms of clinical efficacy for schizophrenia, TRS, and adverse reactions of clozapine have not been elucidated. Recently, the GABA isomer L-β-aminoisobutyric acid (L-BAIBA), a protective myokine in the peripheral organs, was identified as a candidate novel transmission modulator in the central nervous system (CNS). L-BAIBA activates adenosine monophosphate-activated protein kinase (AMPK) signalling in both the peripheral organs and CNS. Activated AMPK signalling in peripheral organs is an established major target for treating insulin-resistant diabetes, whereas activated AMPK signalling in the hypothalamus contributes to the pathophysiology of weight gain and metabolic disturbances. Clozapine increases L-BAIBA synthesis in the hypothalamus. In addition, the various functions of L-BAIBA in the CNS have recently been elucidated, including as an activator of GABA-B and group-III metabotropic glutamate (III-mGlu) receptors. Considering the expressions of GABA-B and III-mGlu receptors (localised in the presynaptic regions), the activation of GABA-B and III-mGlu receptors can explain the distinct therapeutic advantages of clozapine in schizophrenia or TRS associated with N-methyl-D-aspartate (NMDA) receptor disturbance compared with other atypical antipsychotics via the inhibition of the persistent tonic hyperactivation of thalamocortical glutamatergic transmission in the prefrontal cortex. L-BAIBA has also been identified as a gliotransmitter, and a detailed exploration of the function of L-BAIBA in tripartite synaptic transmission can further elucidate the pathophysiology of effectiveness for treating TRS and/or specific adverse reactions of clozapine. Full article

Abnormal depolarization of neuronal membranes called paroxysmal depolarization shift (PDS) represents a cellular correlate of interictal spikes. The mechanisms underlying the generation of PDSs or PDS clusters remain obscure. This study aimed to investigate the role of ionotropic glutamate receptors (iGluRs) in the generation of PDS and dependence of the PDS pattern on neuronal membrane potential. We have shown that significant depolarization or hyperpolarization (by more than ±50 mV) of a single neuron does not change the number of individual PDSs in the cluster, indicating the involvement of an external stimulus in PDS induction. Based on this data, we have suggested reliable protocols for stimulating single PDS or PDS clusters. Furthermore, we have found that AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors are necessary for PDS generation since AMPAR antagonist NBQX completely suppresses bicuculline-induced paroxysmal activity. In turn, antagonists of NMDA (N-methyl-D-aspartate) and kainate receptors (D-AP5 and UBP310, respectively) caused a decrease in the amplitude of the first action potential in PDSs and in the amplitude of the oscillations of intracellular Ca²⁺ concentration occurring alongside the PDS cluster generation. The effects of the NMDAR (NMDA receptor) and KAR (kainate receptor) antagonists indicate that these receptors are involved only in the modulation of paroxysmal activity. We have also shown that agonists of some G_i-coupled receptors, such as A₁ adenosine (A₁Rs) or cannabinoid receptors (CBRs) (N₆-cyclohexyladenosine and WIN 55,212-2, respectively), completely suppressed PDS generation, while the A₁R agonist even prevented it. We hypothesized that the dynamics of extracellular glutamate concentration govern paroxysmal activity. Fine-tuning of neuronal activity via action on G_i-coupled receptors or iGluRs paves the way for the development of new approaches for epilepsy pharmacotherapy. Full article

N6-adenosine methylation (m⁶A) is a prevalent form of RNA modification found in the expressed transcripts of many eukaryotic organisms. Moreover, m⁶A methylation is a dynamic and reversible process that requires the functioning of various proteins and their complexes that are evolutionarily conserved between species and include methylases, demethylases, and m⁶A-binding proteins. Over the past decade, the m⁶A methylation process in plants has been extensively studied and the understanding thereof has drastically increased, although the regulatory function of some components relies on information derived from animal systems. Notably, m⁶A has been found to be involved in a variety of factors in RNA processing, such as RNA stability, alternative polyadenylation, and miRNA regulation. The circadian clock in plants is a molecular timekeeping system that regulates the daily and rhythmic activity of many cellular and physiological processes in response to environmental changes such as the day-night cycle. The circadian clock regulates the rhythmic expression of genes through post-transcriptional regulation of mRNA. Recently, m⁶A methylation has emerged as an additional layer of post-transcriptional regulation that is necessary for the proper functioning of the plant circadian clock. In this review, we have compiled and summarized recent insights into the molecular mechanisms behind m⁶A modification and its various roles in the regulation of RNA. We discuss the potential role of m⁶A modification in regulating the plant circadian clock and outline potential future directions for the study of mRNA methylation in plants. A deeper understanding of the mechanism of m⁶A RNA regulation and its role in plant circadian clocks will contribute to a greater understanding of the plant circadian clock. Full article

Based on a screening of a chemical library of A_2A adenosine receptor (AR) antagonists, a series of di- and tri-substituted adenine derivatives were synthesized and tested for their ability to inhibit the activity of the enzyme casein kinase 1 delta (CK1δ) and to bind adenosine receptors (ARs). Some derivatives, here called “dual anta-inhibitors”, demonstrated good CK1δ inhibitory activity combined with a high binding affinity, especially for the A_2AAR. The N⁶-methyl-(2-benzimidazolyl)-2-dimethyamino-9-cyclopentyladenine (17, IC₅₀ = 0.59 μM and KiA_2A = 0.076 μM) showed the best balance of A_2AAR affinity and CK1δ inhibitory activity. Computational studies were performed to simulate, at the molecular level, the protein–ligand interactions involving the compounds of our series. Hence, the dual anta-inhibitor 17 could be considered the lead compound of new therapeutic agents endowed with synergistic effects for the treatment of chronic neurodegenerative and cancer diseases. Full article