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Hemin is an important iron‐containing porphyrin that has important therapeutic properties to control the symptoms of porphyria attacks that include pain, high blood pressure, rapid heartbeat, mental changes, and so forth. Lysozyme has many applications, which include from therapeutic to preservation of foods. The interaction of hemin with an important model protein, hen egg white lysozyme (HEWL), has been observed in this study through hybrid in silico with biophysical studies. There was a strong 1:1 cooperative binding between hemin and HEWL, which was revealed from the strong quenching of the latter by the former. The binding was favored by the decrease in temperature, which is due to the involvement of static type of quenching mechanism in the interaction. Experimental analyses suggested that the major forces involved in the binding were polar ones, and there was an efficient energy transfer from the fluorophore to the ligand. The combination of far‐UV CD spectroscopy and Rayleigh light scattering experiments established that hemin has the strong tendency to unfold HEWL. An extensive molecular docking investigation involving 2000 docking runs recognized the preferred binding site of hemin within HEWL, and also suggested that apart from the polar forces, hydrophobic interactions have also played an important role in the binding. [ABSTRACT FROM AUTHOR]

Bacillus circulans xylanase (BcX) from the glycoside hydrolase family 11 degrades xylan through a retaining, double‐displacement mechanism. The enzyme is thought to hydrolyze glycosidic bonds in a processive manner and has a large, active site cleft, with six subsites allowing the binding of six xylose units. Such an active site architecture suggests that oligomeric xylose substrates can bind in multiple ways. In the crystal structure of the catalytically inactive variant BcX E78Q, the substrate xylotriose is observed in the active site, as well as bound to the known secondary binding site and a third site on the protein surface. Nuclear magnetic resonance (NMR) titrations with xylose oligomers of different lengths yield nonlinear chemical shift trajectories for active site nuclei resonances, indicative of multiple binding orientations for these substrates for which binding and dissociation are in fast exchange on the NMR timescale, exchanging on the micro‐ to millisecond timescale. Active site binding can be modeled with a 2 : 1 model with dissociation constants in the low and high millimolar range. Extensive mutagenesis of active site residues indicates that tight binding occurs in the glycon binding site and is stabilized by Trp9 and the thumb region. Mutations F125A and W71A lead to large structural rearrangements. Binding at the glycon site is sensed throughout the active site, whereas the weak binding mostly affects the aglycon site. The interactions with the two active site locations are largely independent of each other and of binding at the secondary binding site. [ABSTRACT FROM AUTHOR]

Investigating how transcription factors control complex cellular processes requires tools that enable responses to be visualised at the single-cell level and their cell fate to be followed over time. For example, the tumour suppressor p53 (also called TP53 in humans and TRP53 in mice) can initiate diverse cellular responses by transcriptional activation of its target genes: Puma to induce apoptotic cell death and p21 to induce cell cycle arrest/cell senescence. However, it is not known how these processes are regulated and initiated in different cell types. Also, the context-dependent interaction partners and binding loci of p53 remain largely elusive. To be able to examine these questions, we here developed knock-in mice expressing triple-FLAG-tagged p53 to facilitate p53 pull-down and two p53 response reporter mice, knocking tdTomato and GFP into the Puma/Bbc3 and p21 gene loci, respectively. By crossing these reporter mice into a p53-deficient background, we show that the new reporters reliably inform on p53-dependent and p53-independent initiation of both apoptotic or cell cycle arrest/senescence programs, respectively, in vitro and in vivo. Synopsis: The respective contribution of tumour suppressor p53-activated cellular response pathways to prevention of tumour development remains debated. This resource introduces three novel mouse strains for studying p53's interaction biochemistry and for assessing its contribution to cell cycle arrest and apoptosis in vitro and in vivo. Knock-in mice expressing N-terminally triple-FLAG tagged p53 facilitate identification of p53-interacting proteins and of genomic p53 binding sites. Knock-in mice expressing a p21-IRES-GFP reporter allow in situ imaging of p21-initiated cell cycle arrest and cell senescence in response to p53-dependent and p53-independent stimuli. Knock-in mice expressing a Puma-tdTomato allow in situ imaging of PUMA-initiated apoptotic programs activated in response to p53-dependent and p53-independent stimuli. Novel p53 knock-in and p53 target gene mouse strains enable interaction proteomics, DNA binding studies, as well as the imaging of p53 contributions to cell cycle arrest and apoptosis in situ. [ABSTRACT FROM AUTHOR]

Nucleases of the S1/P1 family have important applications in biotechnology and molecular biology. We have performed structural analyses of SmNuc1 nuclease from Stenotrophomonas maltophilia, including RNA cleavage product binding and mutagenesis in a newly discovered flexible Arg74‐motif, involved in substrate binding and product release and likely contributing to the high catalytic rate. The Arg74Gln mutation shifts substrate preference towards RNA. Purine nucleotide binding differs compared to pyrimidines, confirming the plasticity of the active site. The enzyme–product interactions indicate a gradual, stepwise product release. The activity of SmNuc1 towards c‐di‐GMP in crystal resulted in a distinguished complex with the emerging product 5′‐GMP. This enzyme from an opportunistic pathogen relies on specific architecture enabling high performance under broad conditions, attractive for biotechnologies. [ABSTRACT FROM AUTHOR]

ABSTRACT Botulinum neurotoxins (BoNTs), including serotypes A and E, are potent biotoxins known to cause human poisoning. In addition to the critical protective antigen found in the full BoNT molecule, the receptor binding domain (Hc domain), BoNTs also harbour another essential protective antigen—the light chain‐translocation domain (L‐HN domain). Leveraging these pivotal protective antigens, we genetically engineered a series of inactivated chimeric molecules incorporating L‐HN and Hc domains of BoNT/A and E. The structure of these chimeric molecules, mirror BoNT/A and E, but are devoid of enzyme activity. Experimental findings demonstrated that a lead candidate mEL‐HN‐mAHc harnessing the inactivated protease LCHN/E with the mutated gangliosides binding site Hc/A (mE‐mA) elicited robust immune protection against BoNT/A and E simultaneously in a mouse model, requiring low immune dosages and minimal immunisations. Moreover, mE‐mA exhibited high protective efficacy against BoNT/A and E in guinea pigs and New Zealand white rabbits, resulting in elevated neutralising antibody titres. Furthermore, mE‐mA proved to be a more stable and safer vaccine compared to formaldehyde‐inactivated toxoid. Our data underscore the genetically engineered mE‐mA as a highly effective bivalent vaccine against BoNT/A and E, paving the way for the development of polyvalent vaccines against biotoxins. [ABSTRACT FROM AUTHOR]

TAD boundaries are genomic elements that separate biological processes in neighboring domains by blocking DNA loops that are formed through Cohesin‐mediated loop extrusion. Most TAD boundaries consist of arrays of binding sites for the CTCF protein, whose interaction with the Cohesin complex blocks loop extrusion. TAD boundaries are not fully impermeable though and allow a limited amount of inter‐TAD loop formation. Based on the reanalysis of Nano‐C data, a multicontact Chromosome Conformation Capture assay, we propose a model whereby clustered CTCF binding sites promote the successive stalling of Cohesin and subsequent dissociation from the chromatin. A fraction of Cohesin nonetheless achieves boundary read‐through. Due to a constant rate of Cohesin dissociation elsewhere in the genome, the maximum length of inter‐TAD loops is restricted though. We speculate that the DNA‐encoded organization of stalling sites regulates TAD boundary permeability and discuss implications for enhancer–promoter loop formation and other genomic processes. [ABSTRACT FROM AUTHOR]

This article explores the role of the NUP1 gene in regulating male fertility in rice. The NUP1 gene encodes a peroxidase enzyme that helps remove reactive oxygen species (ROS) in the anther. When the NUP1 gene is not functioning properly, ROS accumulates, disrupting various metabolic processes and leading to the absence of Ubisch body formation and pollen grain production. This ultimately causes male sterility in rice. The study's findings contribute to our understanding of how ROS homeostasis affects cell metabolism and male fertility in plants. [Extracted from the article]

2D covalent organic frameworks (COFs) are attractive for fluorescence sensing due to their lightweight, robust, and highly ordered porous structures. However, the highly conjugated structures between adjacent layers of covalent organic frameworks can often result in aggregation‐caused quenching (ACQ) properties. Here, the study designs two flexible hydrazone‐linked COFs to suppress ACQ effects, thereby enhancing their luminescent activities. Furthermore, the high density of nitrogen and oxygen atoms on these flexible walls serves as binding sites for hydrogen bonding interactions, indicating sensitivity and selectivity towards 2,4,6‐trinitrophenol detection. [ABSTRACT FROM AUTHOR]

Cross‐species prediction of TF binding remains a major challenge due to the rapid evolutionary turnover of individual TF binding sites, resulting in cross‐species predictive performance being consistently worse than within‐species performance. In this study, a novel Nucleotide‐Level Deep Neural Network (NLDNN) is first proposed to predict TF binding within or across species. NLDNN regards the task of TF binding prediction as a nucleotide‐level regression task, which takes DNA sequences as input and directly predicts experimental coverage values. Beyond predictive performance, it also assesses model performance by locating potential TF binding regions, discriminating TF‐specific single‐nucleotide polymorphisms (SNPs), and identifying causal disease‐associated SNPs. The experimental results show that NLDNN outperforms the competing methods in these tasks. Then, a dual‐path framework is designed for adversarial training of NLDNN to further improve the cross‐species prediction performance by pulling the domain space of human and mouse species closer. Through comparison and analysis, it finds that adversarial training not only can improve the cross‐species prediction performance between humans and mice but also enhance the ability to locate TF binding regions and discriminate TF‐specific SNPs. By visualizing the predictions, it is figured out that the framework corrects some mispredictions by amplifying the coverage values of incorrectly predicted peaks. [ABSTRACT FROM AUTHOR]

Pores containing molecular adapters provide internal selective binding sites, thereby allowing the stochastic sensing of analytes. Herein, we demonstrate that semiaza‐bambusuril (BU) acts as a non‐covalent molecular adapter when lodged within the lumen of the wild‐type α‐hemolysin (WT‐αHL) protein pore. Because the bambusurils are recognized as anion receptors, the anion binding site within the adapter‐nanopore complex allows the detection of chloride anions, thus converting a non‐selective pore into an anion sensor. [ABSTRACT FROM AUTHOR]

Paraoxonase 1 (PON1) is an antioxidant enzyme that prevents lipid oxidation by hydrolysing lipid peroxides in the oxidised low‐density lipoprotein (LDL) structure, bound to high‐density lipoprotein (HDL) in serum, and exhibits esterase and lactonase activity. In this work, the hPON1 enzyme was purified from human serum using a hydrophobic gel with Sepharose 4B‐L‐tyrosine‐naphthylamine, and the affinity of some steroid derivatives previously isolated from fungal steroid biotransformations was examined on the pure hPON1 enzyme. The results indicate that all these derivaties activate the hPON1 enzyme to a different extent. Additionally, the binding potential of the most active five steroid derivatives to the PON1 enzyme to form a stable complex was explored through molecular docking and molecular dynamics (MD) simulation. The compounds with the highest potency in the enzymatic assay, S‐8 and S‐20, had the highest binding potential to the enzyme. The stability of the complexes formed by the two compounds was assessed and compared to the stability of the unbound enzyme structure. The enzyme‐compound complexes generally had similar stability to the unbound enzyme structure. Together with this, the MD simulation revealed that compound S‐8 would remain inside the enzyme's binding site during the simulation period, unlike compound S‐20. This situation varies according to the respective derivatives' functional groups and hydrophobic characteristics. [ABSTRACT FROM AUTHOR]

The interaction between linamarin (LIN), a cyanogenic glycoside, and human serum albumin (HSA) was studied by multiple spectroscopic techniques and molecular docking simulation. All measurements were performed under physiological conditions. The obtained results (including the binding constants, effective quenching constant and a number of binding sites) showed that the complex of HSA‐LIN is formed. The values of Stern‐Volmer constants (6.70×103, 5.53×103 and 1.95×103) indicate that fluorescence quenching of HSA was static. Results of site marker experiments showed that the binding site of LIN is mainly located in site I (subdomain IIA) of HSA. The thermodynamic parameters showed that binding process occurs spontaneously through hydrophobic interactions. Molecular docking results are in good agreement with experimental data. Furthermore, computational results revealed LIN binds in the cavity of TRP 214, that is, subdomain IIA (site I) of HSA. This comprehensive study provides a deeper insight into ligand binding in HSA which may be useful in drug design and pharmacology. [ABSTRACT FROM AUTHOR]

Heart failure (HF) is highly prevalent. Mechanisms underlying HF remain incompletely understood. Splicing factors (SF), which control pre‐mRNA alternative splicing, regulate cardiac structure and function. This study investigated regulation of the splicing factor heterogeneous nuclear ribonucleoprotein‐L (hnRNPL) in the failing heart. hnRNPL protein increased in left ventricular tissue from mice with transaortic constriction‐induced HF and from HF patients. In left ventricular tissue, hnRNPL was detected predominantly in nuclei. Knockdown of the hnRNPL homolog Smooth in Drosophila induced cardiomyopathy. Computational analysis of predicted mouse and human hnRNPL binding sites suggested hnRNPL‐mediated alternative splicing of tropomyosin, which was confirmed in C2C12 myoblasts. These findings identify hnRNPL as a sensor of cardiac dysfunction and suggest that disturbances of hnRNPL affect alternative splicing in HF. [ABSTRACT FROM AUTHOR]

Summary Covalent Bruton's tyrosine kinase‐inhibitors (cBTK‐i) are highly active in MYD88‐mutated (MYD88Mut) Waldenstrom's macroglobulinaemia and suppress nuclear factor kappa‐light‐chain‐enhancer of activated B cells and extracellular signal‐regulated kinases‐1/2 (ERK1/2)‐related signalling. BTKCys481 mutations are associated with cBTK‐i acquired resistance and are accompanied by reactivation of ERK1/2 that promotes inflammatory cytokine secretion and paracrine‐mediated resistance of BTK wild‐type (BTKWT) tumour cells. Pirtobrutinib is a non‐covalent BTK‐inhibitor that binds at non‐BTKCys481 sites. We show that pirtobrutinib blocked p‐ERK1/2, ERK1/2‐driven inflammatory cytokines, and overcame paracrine‐mediated resistance in MYD88Mut lymphoma cells expressing mutated BTKCys481. Our results provide important mechanistic insights for the activity of pirtobrutinib in MYD88Mut lymphomas carrying BTKCys481 mutations. [ABSTRACT FROM AUTHOR]

Supramolecular self‐assembly with well‐defined building blocks like lipids, deoxyribonucleic acid, or ligands relies on accessible molecular structures and predictable interactions. However, assembling heterogeneous, undefined blocks, such as disordered proteins, amorphous solids, and catecholic derivatives, remains challenging due to their unpredictable assembly, leading to irreversible aggregation, severe precipitation, and unreliable performance. Here, the first programmable, sustainable, and durable self‐assembly strategy of supramolecules with heterogenous is presented, derived blocks via harmonizing multiple molecular interactions. This approach achieves reversible assembly/disassembly, ≈73.7% reduced precipitation, and salt‐ and alkaline‐durability under freeze‐thaw cycles in model catecholic derivatives, functioning effectively as robust adhesive primers and hydrogel interfacial strengtheners. Moreover, through molecular force measurements and computational simulations, the first general criterion and benchmark for high precision supramolecular self‐assembly is proposed, applicable to complex derivatives and interactions: with blocks bearing multiple binding sites existing, the co‐assembling blocks should bear at least two binding sites with minimum binding strength (≈17 to ≈37 kJ mol−1) to prevent disassembly. This study paves the way and provides benchmarks for precision self‐assembly of diverse supramolecules using heterogeneous derivatives for adhesion technology, nanomaterial synthesis and bio‐inspired applications. [ABSTRACT FROM AUTHOR]

Summary The objective was to evaluate the in vitro inhibition of enzymes related to neurodegeneration (prolyl endopeptidase, PEP; acetylcholinesterase, AChE; butyrylcholinesterase, BChE; lipoxygenase, LOX) and the antioxidant activity from fermented milks (FM) with Lactococcus and Lactobacillus spp. All FM presented antioxidant activity (ABTS and ORAC); however, FM with Lactococcus NRRL B‐50571 (FM‐571) presented higher (P < 0.05) oxidative haemolysis inhibition. Additionally, FM‐571 presented the lowest IC₅₀ for LOX (1.78 mg/mL), PEP (3.16 mg/mL), AChE (3.22 mg/mL) and BChE (2.14 mg/mL). Thus, FM‐571 was selected for in silico analysis for the prediction of binding sites of peptides present in this FM with enzymes active sites. Altogether, thirty peptides showed (P < 0.01) high binding potential to AChE, seventeen to BChE and LOX and sixteen to PEP. Thus, FM‐571 shows potential for the development of dairy products with potential neuroprotective effects. [ABSTRACT FROM AUTHOR]

Respiratory complex I is a central metabolic enzyme coupling NADH oxidation and quinone reduction with proton translocation. Despite the knowledge of the structure of the complex, the coupling of both processes is not entirely understood. Here, we use a combination of site‐directed mutagenesis, biochemical assays, and redox‐induced FTIR spectroscopy to demonstrate that the quinone chemistry includes the protonation and deprotonation of a specific, conserved aspartic acid residue in the quinone binding site (D325 on subunit NuoCD in Escherichia coli). Our experimental data support a proposal derived from theoretical considerations that deprotonation of this residue is involved in triggering proton translocation in respiratory complex I. [ABSTRACT FROM AUTHOR]

Prostate cancer (PCa) is a high‐mortality cancer. Docetaxel (DCT) combined with second‐generation anti‐androgens is considered the golden standard therapy for PCa, whose application is limited for DCT resistance (DR). Therefore, exploring the mechanism of DR is of great importance. In this study, PCa cell lines of PC3 and DU145 were employed, and DR cells were constructed by treatment with graded DCT. CircSLC4A7, miR‐1205, and microtubule‐associated protein tau (MAPT) transfections were established. Cell counting kit‐8 assay was performed to evaluate the cell activity and IC50 of DCT. After being treated with DCT, DR was assessed by colony formation assay, flow cytometry analysis, and terminal transferase‐mediated UTP nick end‐labeling assay. Real‐time quantitative PCR and western blotting analysis evaluated the expression levels of genes. The dual‐luciferase reporter gene assay verified the miR‐1205 binding sites with circSLC4A7 and MAPT. An animal experiment was performed to assess the tumor growth influenced by circSLC4A7. After conducting DR cells and isolated exosomes, we found that not only co‐culture with DR cells but also treatment with DR cells' exosomes would promote the DR of normal cells. Moreover, circSLC4A7 was highly expressed in DR cells and their exosomes. CircSLC4A7 overexpression enhanced DR, represented as raised IC50 of DCT, increased colony formation, and decreased cell apoptosis after DCT treatment, while circSLC4A7 knockdown had the opposite effect. MiR‐1205 was confirmed as a circSLC4A7‐sponged miRNA and miR‐1205 inhibitor reversed the effect of sh‐circSLC4A7. MAPT was further identified as a target of miR‐1205 and had a similar effect with circSLC4A7. The effect of circSLC4A7 on DR was also confirmed by xenograft experiments. Collectively, circSLC4A7 in resistant‐cells‐derived exosomes promotes DCT resistance of PCa via miR‐1205/MAPT axis, which may provide a new treatment strategy for DR of PCa. [ABSTRACT FROM AUTHOR]

Ligand binding hotspots are regions of protein surfaces that form particularly favourable interactions with small molecule pharmacophores. Targeting interactions with these hotspots maximises the efficiency of ligand binding. Existing methods are capable of identifying hotspots but often lack assays to quantify ligand binding and direct elaboration at these sites. Herein, we describe a fragment‐based competitive 19F Ligand Based NMR (LB‐NMR) screening platform that enables routine, quantitative ligand profiling focused at ligand‐binding hotspots. As a proof of concept, the method was applied to 4′‐phosphopantetheine adenylyltransferase (PPAT) from Mycobacterium abscessus (Mabs). X‐ray crystallographic characterisation of the hits from a 960‐member fragment screen identified three ligand‐binding hotspots across the PPAT active site. From the fragment hits a collection of 19F reporter candidates were designed and synthesised. By rigorous prioritisation and use of optimisation workflows, a single 19F reporter molecule was generated for each hotspot. Profiling the binding of a set of structurally characterised ligands by competitive 19F LB‐NMR with this suite of 19F reporters recapitulated the binding affinity and site ID assignments made by ITC and X‐ray crystallography. This quantitative mapping of ligand binding events at hotspot level resolution establishes the utility of the fragment‐based competitive 19F LB‐NMR screening platform for hotspot‐directed ligand profiling [ABSTRACT FROM AUTHOR]

Human phosphoglycerate kinase 1(hPGK1) is a key glycolytic enzyme that regulates the balance between ADP and ATP concentrations inside the cell. Phosphorylation of hPGK1 at S203 and S256 has been associated with enzyme import from the cytosol to the mitochondria and the nucleus respectively. These changes in subcellular locations drive tumorigenesis and are likely associated with site‐specific changes in protein stability. In this work, we investigate the effects of site‐specific phosphorylation on thermal and kinetic stability and protein structural dynamics by hydrogen–deuterium exchange (HDX) and molecular dynamics (MD) simulations. We also investigate the binding of 3‐phosphoglycerate and Mg‐ADP using these approaches. We show that the phosphomimetic mutation S256D reduces hPGK1 kinetic stability by 50‐fold, with no effect of the mutation S203D. Calorimetric studies of ligand binding show a large decrease in affinity for Mg‐ADP in the S256D variant, whereas Mg‐ADP binding to the WT and S203D can be accurately investigated using protein kinetic stability and binding thermodynamic models. HDX and MD simulations confirmed the destabilization caused by the mutation S256D (with some long‐range effects on stability) and its reduced affinity for Mg‐ADP due to the strong destabilization of its binding site (particularly in the apo‐state). Our research provides evidence suggesting that modifications in protein stability could potentially enhance the translocation of hPGK1 to the nucleus in cancer. While the structural and energetic basis of its mitochondrial import remain unknown. [ABSTRACT FROM AUTHOR]

Calcineurin is a serine/threonine protein phosphatase that is highly conserved from yeast to human and plays a critical role in many physiological processes. Regulators of calcineurin (RCANs) are a family of endogenous calcineurin regulators, which are capable of inhibiting the catalytic activity of calcineurin in vivo and in vitro. In this study, we first characterized the biochemical properties of yeast calcineurin and its endogenous regulator Rcn1, a yeast homolog of RCAN1. Our data show that Rcn1 inhibits yeast calcineurin toward pNPP substrate with a noncompetitive mode; and Rcn1 binds cooperatively to yeast calcineurin through multiple low‐affinity interactions at several docking regions. Next, we reinvestigated the mechanism underlying the inhibition of mammalian calcineurin by RCAN1 using a combination of biochemical, biophysical, and computational methods. In contrast to previous observations, RCAN1 noncompetitively inhibits calcineurin phosphatase activity toward both pNPP and phospho‐RII peptide substrates by targeting the enzyme active site in part. Re‐analysis of previously reported kinetic data reveals that the RCAN1 concentrations used were too low to distinguish between the inhibition mechanisms [Chan B et al. (2005) Proc Natl Acad Sci USA 102, 13075]. The results presented in this study provide new insights into the interaction between calcineurin and RCAN1/Rcn1. [ABSTRACT FROM AUTHOR]

Molecular docking is by far the most preferred approach in structure‐based drug design for its effectiveness to predict the scoring and posing of a given bioactive small molecule into the binding site of its pharmacological target. Herein, we present MzDOCK, a new GUI‐based pipeline for Windows operating system, designed with the intent of making molecular docking easier to use and higher reproducible even for inexperienced people. By harmonic integration of python and batch scripts, which employs various open source packages such as Smina (docking engine), OpenBabel (file conversion) and PLIP (analysis), MzDOCK includes many practical options such as: binding site configuration based on co‐crystallized ligands; generation of enantiomers from SMILES input; application of different force fields (MMFF94, MMFF94s, UFF, GAFF, Ghemical) for energy minimization; retention of selectable ions and cofactors; sidechain flexibility of selectable binding site residues; multiple input file format (SMILES, PDB, SDF, Mol2, Mol); generation of reports and of pictures for interactive visualization. Users can download for free MzDOCK at the following link: https://github.com/Muzatheking12/MzDOCK. [ABSTRACT FROM AUTHOR]

The crystal structure of Michaelis complex of the bovine trypsin with N‐α‐benzoyl‐l‐arginine ethyl ester (BAEE) was determined at 2.30 Å resolution. The structure of enzyme‐substrate complex was solved in space group H32. The active site of trypsin was found to be occupied with the N‐α‐benzoyl‐l‐arginine ethyl ester. The hydrolyzed product of substrate molecules was also crystallized with trypsin. The substrate was embedded within the S1 binding site of the enzyme, and showed contacts with Gly‐195, Ser‐216, and Ser‐192. Some water molecules were also found within the vicinity of catalytic residues of enzyme. Interestingly, the hydrolyzed product present within the crystal lattice was found to be with inverted configuration. The crystal structure of trypsin in‐complex with N‐α‐benzoyl‐l‐arginine ethyl ester has never been reported previously. [ABSTRACT FROM AUTHOR]

Clothianidin (CL) is a neonicotinoid insecticide widely used in crop protection against insect pests. However, its effects on photosynthesis remain largely unknown. Here, by investigating the influence of CL at the concentrations of 22 and 110 μg/L on the primary processes of photosynthesis, membrane fluidity and structural changes of pea chloroplasts, we located several primary binding sites of this pesticide. Similar dynamics were observed for both concentrations. However, statistically significant differences were only found at 110 μg/L for all methods used. The light saturated rate of linear electron flow decreased mainly due to the disturbance of electron flow on the acceptor side of photosystem II (PSII) associated with the appearance of QB‐nonreducing centers and empty QB binding sites of PSII. The functioning of the donor side of PSII, the activity of photosystem I (PSI) and the maximum quantum yield of PSII photochemistry (Fv/Fm) were not found to be significantly altered. Increased membrane fluidity and structural alterations of the thylakoid membrane led to a decrease in the development of the proton gradient ΔрН and membrane energization processes. [ABSTRACT FROM AUTHOR]

Klotho is an antiaging protein that has multiple functions. The purpose of this study is to investigate whether soluble klotho plays a role in cellular stress response pathways. We found that klotho deficiency (kl−/−) largely decreased HSF1 levels and impaired heat shock protein expression. Interestingly, recombinant soluble klotho‐induced HSF1 and HSPs such as HSP90, HSP70, and HSP27 in kl−/− mouse embryonic fibroblasts (MEFs). Soluble Klotho treatment also induced cell proliferation and HSF1 promoter activity in MEF kl−/− cells in a concentration‐dependent manner. Furthermore, using point mutagenesis, we identified regulatory/binding sites of transcription factors EGR1 regulated by soluble klotho in the HSF1 promoter. Taken together, our findings unravel the molecular basis of klotho and provide molecular evidence supporting a direct interaction between soluble klotho and HSF1‐mediated stress response pathway. [ABSTRACT FROM AUTHOR]

The regulation of dopamine (DA) removal from the synaptic cleft is a crucial process in neurotransmission and is facilitated by the sodium‐ and chloride‐coupled dopamine transporter DAT. Psychostimulant drugs, cocaine, and amphetamine, both block the uptake of DA, while amphetamine also triggers the release of DA. As a result, they prolong or even amplify neurotransmitter signaling. Atypical inhibitors of DAT lack cocaine‐like rewarding effects and offer a promising strategy for the treatment of drug use disorders. Here, we present the 3.2 Å resolution cryo‐electron microscopy structure of the Drosophila melanogaster dopamine transporter (dDAT) in complex with the atypical non‐competitive inhibitor AC‐4‐248. The inhibitor partially binds at the central binding site, extending into the extracellular vestibule, and locks the transporter in an outward open conformation. Our findings propose mechanisms for the non‐competitive inhibition of DAT and attenuation of cocaine potency by AC‐4‐248 and provide a basis for the rational design of more efficacious atypical inhibitors. [ABSTRACT FROM AUTHOR]

Here, we describe the characterization of a radioligand selective for GluN2B‐containing NMDA receptors, 3‐[3H] 1‐(azetidin‐1‐yl)‐2‐(6‐(4‐fluoro‐3‐methyl‐phenyl)pyrrolo[3,2‐b]pyridin‐1‐yl)ethanone ([3H]‐JNJ‐ GluN2B‐5). In rat cortical membranes, the compound bound to a single site, and the following kinetic parameters were measured; association rate constant Kon = 0.0066 ± 0.0006 min−1 nM−1, dissociation rate constant Koff = 0.0210 ± 0.0001 min−1 indicating calculated KD = Koff/Kon = 3.3 ± 0.4 nM, (mean ± SEM, n = 3). The equilibrium dissociation constant determined from saturation binding experiments in rat cortex was KD of 2.6 ± 0.3 nM (mean ± SEM, n = 3). In contrast to the widely used GluN2B radioligand [3H]‐Ro 25‐6981, whose affinity Ki for sigma 1 and sigma 2 receptors are 2 and 189 nM, respectively, [3H]‐JNJ‐GluN2B‐5 exhibits no measurable affinity for sigma 1 and sigma 2 receptors (Ki > 10 μM for both) providing distinct selectivity advantages. Anatomical distribution of [3H]‐JNJ‐GluN2B‐5 binding sites in rat, mouse, dog, monkey, and human brain tissue was studied using in vitro autoradiography, which showed high specific binding in the hippocampus and cortex and negligible binding in the cerebellum. Enhanced selectivity for GluN2B‐containing receptors translated to a good signal‐to‐noise ratio in both in vitro radioligand binding and in vitro autoradiography assays. In conclusion, [3H]‐JNJ‐GluN2B‐5 is a high‐affinity GluN2B radioligand with excellent signal‐to‐noise ratio and unprecedented selectivity. [ABSTRACT FROM AUTHOR]

Glycine Transporter 2 (GlyT2) inhibitors have shown considerable potential as analgesics for the treatment of neuropathic pain but also display considerable side effects. One potential source of side effects is irreversible inhibition. In this study, we have characterized the mechanism of ORG25543 inhibition of GlyT2 by first considering three potential ligand binding sites on GlyT2—the substrate site, the vestibule allosteric site and the lipid allosteric site. The three sites were tested using a combination of molecular dynamics simulations and analysis of the inhibition of glycine transport of a series point mutated GlyT2 using electrophysiological methods. We demonstrate that the lipid allosteric site on GlyT2 is the most likely binding site for ORG25543. We also demonstrate that cholesterol derived from the cell membrane can form specific interactions with inhibitor‐bound transporters to form an allosteric network of regulatory sites. These observations will guide the future design of GlyT2 inhibitors with the objective of minimising on‐target side effects and improving the therapeutic window for the treatment of patients suffering from neuropathic pain. [ABSTRACT FROM AUTHOR]

Variants in the 5′ UTR of ANKRD26 are a common cause of inherited thrombocytopenia (ANKRD26‐RT), and are associated with sustained ANKRD26 expression, which inhibits megakaryocyte maturation and proplatelet formation. ANKRD26 expression is controlled by the binding of a RUNX1/FLI1 complex to the 5′ UTR. To date, all reported ANKRD26‐RD associated variants have been within the RUNX1 binding site and a 22 base pair flanking region. Here, we report a novel variant in the 5′ UTR of ANKRD26, c.‐107C>T. This variant is in the FLI1 binding site, and is predicted to disrupt FLI1 binding due to loss of a hydrogen bond with FLI1. Differentiated PBMCs from affected family members showed impaired megakaryocyte maturation and proplatelet formation and sustained expression of ANKRD26, and platelets from affected family members had higher ANKRD26 expression than control platelets. The variant increased activity of the ANKRD26 promotor in a reporter assay. We also provide evidence that the previously reported c.‐140C>G ANKRD26 5′ UTR variant is benign and not associated with thrombocytopenia. Identification of the c.‐107C>T variant extends the range of the regulatory region in the 5′ UTR of ANKRD26 that is associated with ANKRD26‐RT. [ABSTRACT FROM AUTHOR]

Long noncoding RNAs (LncRNAs) play essential roles in regulating gene expression in various biological processes. However, the function of lncRNAs in vascular smooth muscle cell (VSMC) transformation remains to be explained. In this work, we discover that a new bone marrow protein (BMP) signaling target, lncRNA RP11‐301G19.1, is significantly induced in BMP7‐treated VSMCs through lncRNA microarray analysis. Addition of BMP signaling inhibitor LDN‐193189 attenuates the expression of ACTA2 and SM‐22α, as well as the mRNA level of RP11‐301G19.1. Furthermore, lncRNA RP11‐301G19.1 is critical to the VSMC differentiation and is directly activated by SMAD1/9. Mechanistically, knocking down of RP11‐301G19.1 leads to the decrease of ATOH8, another BMP target, while the forced expression of RP11‐301G19.1 reactivates ATOH8. In addition, miR‐17‐5p, a miRNA negatively regulated by BMP‐7, contains predicted binding sites for lncRNA RP11‐301G19.1 and ATOH8 3′UTR. Accordingly, overexpression of miR‐17‐5p decreases the levels of them. Together, our results revealed the role of lncRNA RP11‐301G19.1 as a miRNA sponge to upregulate ATOH8 in VSMC phenotype transformation. [ABSTRACT FROM AUTHOR]

The frequent use of anti‐inflammatory drugs and the side effects of existing drugs keep the need for new compounds constant. For this purpose, flurbiprofen and ibuprofen‐like compounds, which are frequently used anti‐inflammatory compounds in this study, were synthesized and their structures were elucidated. Like ibuprofen and flurbiprofen, the compounds contain a residue of phenylacetic acid. On the other hand, it contains a secondary amine residue. Thus, it is planned to reduce the acidity, which is the biggest side effect of NSAI drugs, even a little bit. The estimated ADME parameters of the compounds were evaluated. Apart from internal use, local use of anti‐inflammatory compounds is also very important. For this reason, the skin permeability values of the compounds were also calculated. And it has been found to be compatible with reference drugs. The COX enzyme inhibitory effects of the obtained compounds were tested by in vitro experiments. Compound 2a showed significant activity against COX‐1 enzyme with an IC50 = 0.123 + 0.005 μM. The interaction of the compound with the enzyme active site was clarified by molecular dynamics studies. [ABSTRACT FROM AUTHOR]

This document is a corrigendum for a scientific article titled "A Magnesium Binding Site And The Anomeric Effect Regulate The Abiotic Redox Chemistry Of Nicotinamide Nucleotides." The authors acknowledge an error in the NMR resonance assignment for reduced nicotinamide mononucleotide (NMNH) in their manuscript. They provide the correct peaks and labeling for the C(2)H of 1,4-NMNH. The overall findings of the paper remain unaffected by this correction. The document also includes additional figures with corrected labeling for reference. [Extracted from the article]

Genetic and epigenetic alterations are cancer hallmark characteristics. However, the role of inherited cancer predisposition alleles in co‐opting lineage factor epigenetic reprogramming and tumor progression remains elusive. Here the FinnGen cohort phenome‐wide analysis, along with multiple genome‐wide association studies, has consistently identified the rs339331‐RFX6/6q22 locus associated with prostate cancer (PCa) risk across diverse populations. It is uncovered that rs339331 resides in a reprogrammed androgen receptor (AR) binding site in PCa tumors, with the T risk allele enhancing AR chromatin occupancy. RFX6, an AR‐regulated gene linked to rs339331, exhibits synergistic prognostic value for PCa recurrence and metastasis. This comprehensive in vitro and in vivo studies demonstrate the oncogenic functions of RFX6 in promoting PCa cell proliferation and metastasis. Mechanistically, RFX6 upregulates HOXA10 that profoundly correlates with adverse PCa outcomes and is pivotal in RFX6‐mediated PCa progression, facilitating the epithelial‐mesenchymal transition (EMT) and modulating the TGFβ/SMAD signaling axis. Clinically, HOXA10 elevation is associated with increased EMT scores, tumor advancement and PCa recurrence. Remarkably, reducing RFX6 expression restores enzalutamide sensitivity in resistant PCa cells and tumors. This findings reveal a complex interplay of genetic and epigenetic mechanisms in PCa pathogenesis and drug resistance, centered around disrupted prostate lineage AR signaling and abnormal RFX6 expression. [ABSTRACT FROM AUTHOR]

Nitrogen‐containing heterocyclic molecules feature metal resource‐independence, flexible conjugation structure and fast charge storage kinetics, making them promising to be used as the electrode material in lithium‐ion batteries. However, an insufficiently high capacity (<400 mAh g−1) seriously threatens their practical application. Herein, a novel molecule, viz. 3,6‐bis(2‐pyrimidinyl)‐1,2,4,5‐tetrazine (DPmT), is developed by inserting an electron‐withdrawing π‐bridge unit of s‐tetrazine between two pyrimidine rings, in which a dense assembly of multiple active sites is realized. The DTmP exhibits a remarkable atom economy of 40 g (mol e)−1, which refers to the molecule mass per unit of charge transferred. The cathode material of DPmT yields a high capacity of 653 mAh g−1 and an energy density of 1188 Wh kg−1 at 50 mA g−1 at 70 °C. And 80% capacity retention is achieved after 500 cycles at 500 mA g−1, confirming its superior cyclability. Spectroscopy studies and theoretical calculations are performed to investigate the charge storage process. First, the C═N and N═N are claimed as plausible binding sites for the lithium ions. Second, the introduction of s‐tetrazine significantly enhances molecular planarity, thereby promoting charge delocalization and molecular stability. This work provides a novel strategy for designing atom‐economic multi‐charge electrode materials with high capacity. [ABSTRACT FROM AUTHOR]

Synthetic structures mimicking the transport function of natural ion channel proteins have a wide range of applications, including therapeutic treatments, separation membranes, sensing, and biotechnologies. However, the development of polymer‐based artificial channels has been hampered due to the limitation on available models. In this study, we demonstrate the great potential of bottlebrush polymers as accessible and versatile molecular scaffolds for developing efficient artificial ion channels. Adopting the bottlebrush configuration enhanced ion transport activity of the channels compared to their linear analogs. Matching the structure of lipid bilayers, the bottlebrush channel with a hydrophilic‐hydrophobic‐hydrophilic triblock architecture exhibited the highest activity among the series. Functionalized with urea groups, these channels displayed high anion selectivity. Additionally, we illustrated that the transport properties could be fine‐tuned by modifying the chemistry of ion binding sites. This work not only highlights the importance of polymer topology control in channel design, but also reveals the great potential for further developing bottlebrush channels with customized features and diverse functionalities. [ABSTRACT FROM AUTHOR]

ABSTRACT Many chemoreceptors contain a C‐terminal pentapeptide at the end of a linker. In Escherichia coli, this pentapeptide forms a high‐affinity binding site for CheR and phosphorylated CheB, and its removal interferes with chemoreceptor adaptation. Analysis of chemoreceptors revealed significant variation in their pentapeptide sequences, and bacteria often possess multiple chemoreceptors with differing pentapeptides. To assess whether this sequence variation alters CheR affinity and chemotaxis, we used Pectobacterium atrosepticum SCRI1043 as a model. SCRI1043 has 36 chemoreceptors, with 19 of them containing a C‐terminal pentapeptide. We show that the affinity of CheR for the different pentapeptides varies up to 11‐fold (KD 90 nM to 1 μM). Pentapeptides with the highest and lowest affinities differ only in a single amino acid. Deletion of the cheR gene abolishes chemotaxis. The replacement of the pentapeptide in the PacC chemoreceptor with those of the highest and lowest affinities significantly reduced chemotaxis to its cognate chemoeffector, L‐Asp. Altering the PacC pentapeptide also reduced chemotaxis to L‐Ser, but not to nitrate, which are responses mediated by the nontethered PacB and PacN chemoreceptors, respectively. Changes in the pentapeptide sequence thus modulate the response of the cognate receptor and that of another chemoreceptor. [ABSTRACT FROM AUTHOR]

Stable homogeneous two‐electron water oxidation electrocatalysts are highly demanded to understand the precise mechanism and reaction intermediates of electrochemical H2O2 production. Here we report a tetranuclear manganese complex with a cubane structure which can electrocatalyze water oxidation to hydrogen peroxide under alkaline and neutral conditions. Such a complex demonstrates an optimal Faradaic efficiency (FE) of 87 %, which is amongst (if not) the highest FE(H2O2) of reported homogeneous and heterogeneous electrocatalysts. In addition, active species were identified and co‐catalysts were excluded through ESI‐MS characterization. Furthermore, we identified water binding sites and isolated one‐electron oxidation intermediate by chemical oxidation of the catalyst in the presence of water substrates. It is evident that efficient proton‐accepting electrolytes avoid rapid proton building‐up at electrode and substantially improve reaction rate and selectivity. Accordingly, we propose a two‐electron catalytic cycle model for water oxidation to hydrogen peroxide with the bioinspired molecular electrocatalyst. The present work is expected to provide an ideal platform to elucidate the two‐electron WOR mechanism at the atomic level. [ABSTRACT FROM AUTHOR]

The thiamine diphosphate (ThDP)‐binding motif, characterized by the canonical GDG(X)24‐27N sequence, is highly conserved among ThDP‐dependent enzymes. We investigated a ThDP‐dependent lyase (JanthE from Janthinobacterium sp. HH01) with an unusual cysteine (C458) replacing the first glycine of this motif. JanthE exhibits a high substrate promiscuity and accepts long aliphatic α‐keto acids as donors. Sterically hindered aromatic aldehydes or non‐activated ketones are acceptor substrates, giving access to a variety of secondary and tertiary alcohols as carboligation products. The crystal structure solved at a resolution of 1.9 Å reveals that C458 is not primarily involved in cofactor binding as previously thought for the canonical glycine. Instead, it coordinates methionine 406, thus ensuring the integrity of the active site and the enzyme activity. In addition, we have determined the long‐sought genuine tetrahedral intermediates formed with pyruvate and 2‐oxobutyrate in the pre‐decarboxylation states and deciphered the atomic details for their stabilization in the active site. Collectively, we unravel an unexpected role for the first residue of the ThDP‐binding motif and unlock a family of lyases that can perform valuable carboligation reactions. [ABSTRACT FROM AUTHOR]

Summary The Fc region of antibodies is vital for most of their physiological functions, many of which are engaged through binding to a range of Fc receptors. However, these same interactions are not always helpful or wanted when therapeutic antibodies are directed against self‐antigens, and can sometimes cause catastrophic adverse reactions. Over the past 40 years, there have been intensive efforts to “silence” unwanted binding to Fc‐gamma receptors, resulting in at least 45 different variants which have entered clinical trials. One of the best known is “LALA” (L234A/L235A). However, neither this, nor most of the other variants in clinical use are completely silenced, and in addition, the biophysical properties of many of them are compromised. I review the development of different variants to see what we can learn from their biological properties and use in the clinic. With the rise of powerful new uses of antibody therapy such as bispecific T‐cell engagers, antibody‐drug conjugates, and checkpoint inhibitors, it is increasingly important to optimize the Fc region as well as the antibody binding site in order to achieve the best combination of safety and efficacy. [ABSTRACT FROM AUTHOR]

Dynamin 1 mediates fission of endocytic synaptic vesicles in the brain and has two major splice variants, Dyn1xA and Dyn1xB, which are nearly identical apart from the extended C-terminal region of Dyn1xA. Despite a similar set of binding partners, only Dyn1xA is enriched at endocytic zones and accelerates vesicle fission during ultrafast endocytosis. Here, we report that Dyn1xA achieves this localization by preferentially binding to Endophilin A1 through a newly defined binding site within its long C-terminal tail extension. Endophilin A1 binds this site at higher affinity than the previously reported site, and the affinity is determined by amino acids within the Dyn1xA tail but outside the binding site. This interaction is regulated by the phosphorylation state of two serine residues specific to the Dyn1xA variant. Dyn1xA and Endophilin A1 colocalize in patches near the active zone, and mutations disrupting Endophilin A binding to the long tail cause Dyn1xA mislocalization and stalled endocytic pits on the plasma membrane during ultrafast endocytosis. Together, these data suggest that the specificity for ultrafast endocytosis is defined by the phosphorylation-regulated interaction of Endophilin A1 with the C-terminal extension of Dyn1xA. Synopsis: A splice variant of Dynamin 1, Dyn1xA, is required for ultrafast endocytosis of synaptic vesicles, but how this specificity arises is unclear, given that another variant, Dyn1xB, is nearly identical. Using a combination of proteomics, nuclear magnetic resonance, superresolution microscopy, and time-resolved electron microscopy imaging, this study reveals how Dyn1xA mediates its unique function. A high-affinity Endophilin A1 binding site exists within the extended tail of Dyn1xA that spans a splice boundary and is absent in Dyn1xB. Dyn1xA-specific amino acids near the SH3-binding PxxP motif enhance Endophilin A1 affinity to the nanomolar range. Mutations disrupting the specific interaction between Dyn1xA and Endophilin A cause defective Dyn1xA molecular condensate formation around the endocytic zone and defective ultrafast endocytosis. Ultrafast endocytosis of synaptic vesicles is mediated by the unique tail extension of the splice variant Dynamin 1xA, which contains a high-affinity binding site for endophilin A1. [ABSTRACT FROM AUTHOR]

Peptidoglycan DL‐endopeptidases locally cleave the peptide stem of peptidoglycan in the bacterial cell wall. This process facilitates bacterial growth and division by loosening the rigid peptidoglycan layer. IseA binds to the active site of multiple DL‐endopeptidases and inhibits excessive peptidoglycan degradation that leads to cell lysis. To better understand how IseA inhibits DL‐endopeptidase activity, we determined the crystal structure of the peptidoglycan DL‐endopeptidase CwlO/IseA complex and compared it with that of the peptidoglycan DL‐endopeptidase LytE/IseA complex. Structural analyses showed significant differences between the hydrophobic pocket‐binding residues of the DL‐endopeptidases (F361 of CwlO and W237 of LytE). Additionally, binding assays showed that the F361 mutation of CwlO to the bulkier hydrophobic residue, tryptophan, increased its binding affinity for IseA, whereas mutation to alanine reduced the affinity. These analyses revealed that the hydrophobic pocket‐binding residue of DL‐endopeptidases determines IseA‐binding affinity and is required for substrate‐mimetic inhibition by IseA. [ABSTRACT FROM AUTHOR]

A water‐soluble prism[5]arene host can form endo‐cavity complexes with hydrophilic organic substances in water by displacing frustrated water molecules from its deep cavity. Water molecules structured at both rims of the prismarene host can mediate hydrogen bonding interactions with the guest. Water‐mediated hydrogen bonding interactions were invoked here to elucidate the elevated binding affinities and selectivity of the prismarene host toward hydrophilic organic guests. We show that water at the interface of a host‐guest complex can act as an extension of the host structure, facilitating the accommodation of neutral guests within the binding site. This study highlights the crucial role of water in facilitating supramolecular interactions between a deep‐cavity prismarene host and organic hydrophilic guests in aqueous medium. [ABSTRACT FROM AUTHOR]

The human Atg8 family member GABARAP is involved in numerous autophagy‐related and ‐unrelated processes. We recently observed that specifically the deficiency of GABARAP enhances epidermal growth factor receptor (EGFR) degradation upon ligand stimulation. Here, we report on two putative LC3‐interacting regions (LIRs) within EGFR, the first of which (LIR1) is selected as a GABARAP binding site in silico. Indeed, in vitro interaction studies reveal preferential binding of LIR1 to GABARAP and GABARAPL1. Our X‐ray data demonstrate interaction of core LIR1 residues FLPV with both hydrophobic pockets of GABARAP suggesting canonical binding. Although LIR1 occupies the LIR docking site, GABARAP Y49 and L50 appear dispensable in this case. Our data support the hypothesis that GABARAP affects the fate of EGFR at least in part through direct binding. [ABSTRACT FROM AUTHOR]

Multimeric aptamers have gained more attention than their monomeric counterparts due to providing more binding sites for target analytes, leading to increased affinity. This work attempted to engineer the surface‐based generation of multimeric aptamers by employing the room temperature rolling circle amplification (RCA) technique and chemically modified primers for developing a highly sensitive and selective electrochemical aptasensor. The multimeric aptamers, generated through surface RCA, are hybridized to modified spacer primers, facilitating the positioning of the aptamers in the proximity of sensing surfaces. These multimeric aptamers can be used as bio‐receptors for capturing specific targets. The surface amplification process was fully characterized, and the optimal amplification time for biosensing purposes was determined, using SARS‐CoV‐2 spike protein (SP). Interestingly, multimeric aptasensors produced considerably higher response signals and affinity (more than 10‐fold), as well as higher sensitivity (almost 4‐fold) compared to monomeric aptasensors. Furthermore, the impact of surface structures on the response signals was studied by utilizing both flat working electrodes (WEs) and nano‐/microislands (NMIs) WEs. The NMIs multimeric aptasensors showed significantly higher sensitivity in buffer and saliva media with the limit of detection less than 2 fg/ml. Finally, the developed NMIs multimeric aptasensors were clinically challenged with several saliva patient samples. [ABSTRACT FROM AUTHOR]

Background: circRNA NFIX has been shown to exist as an oncogene in glioma. But its expression and role in NSCLC (non‐small cell lung cancer) are still unclear. This research aimed to discover the expression and function of circRNA NFIX in NSCLC. Methods: In this research, qRT‐PCR was utilized to investigate the expression levels of circRNA NFIX, miRNA‐214‐3p, and TRIAP1 in NSCLC tissues and cell lines. The binding sites between circRNA NFIX/TRIAP1 and miRNA‐214‐3p were predicted using the Starbase. These interactions were further validated using a double luciferase reporter assay. Cell proliferation and apoptosis were assessed through MTT and flow cytometry, respectively. The expression of apoptosis‐related proteins was measured by western blot assay. Results: miRNA‐214‐3p could link with circRNA NFIX. circRNA NFIX was upregulated, while miRNA‐214‐3p was downregulated in NSCLC cell lines and clinical samples. Besides, suppression of circRNA NFIX repressed cell proliferation and induced apoptosis in NSCLC cells by upregulating miRNA‐214‐3p expression. Besides, the data indicated that TRIAP1 was a target of miRNA‐214‐3p, and it was negatively regulated by miRNA‐214‐3p in NSCLC cells. The excessive expression of miRNA‐214‐3p suppressed NSCLC cell proliferation and increased apoptosis. In addition, overexpression of TRIAP1 significantly reversed the effects on NSCLC cells caused by miRNA‐214‐3p mimic. Conclusion: circRNA NFIX silencing repressed the proliferation of NSCLC cells and induced cell apoptosis by regulating the miR‐214‐3p/TRIAP1 axis, which was a potential diagnostic and therapeutic target for NSCLC. [ABSTRACT FROM AUTHOR]

The human aryl hydrocarbon receptor (AhR), a ligand‐dependent transcription factor, plays a pivotal role in a diverse array of pathways in biological and pathophysiological events. This position AhR as a promising target for both carcinogenesis and antitumor strategies. In this study we utilized computational modeling to screen and identify FDA‐approved drugs binding to the allosteric site between α2 of bHLH and PAS‐A domains of AhR, with the aim of inhibiting its canonical pathway activity. Our findings indicated that nilotinib effectively fits into the allosteric pocket and forms interactions with crucial residues F82, Y76, and Y137. Binding free energy value of nilotinib is the lowest among top hits and maintains stable within its pocket throughout entire (MD) simulations time. Nilotinib has also substantial interactions with F295 and Q383 when it binds to orthosteric site and activate AhR. Surprisingly, it does not influence AhR nuclear translocation in the presence of AhR agonists; instead, it hinders the formation of the functional AhR‐ARNT‐DNA heterodimer assembly, preventing the upregulation of regulated enzymes like CYP1A1. Importantly, nilotinib exhibits a dual impact on AhR, modulating AhR activity via the PAS‐B domain and working as a noncompetitive allosteric antagonist capable of blocking the canonical AhR signaling pathway in the presence of potent AhR agonists. These findings open a new avenue for the repositioning of nilotinib beyond its current application in diverse diseases mediated via AhR. [ABSTRACT FROM AUTHOR]

SUMMARY: Structural maintenance of chromosome (SMC) complexes play roles in cohesion, condensation, replication, transcription, and DNA repair. Their cores are composed of SMC proteins with a unique structure consisting of an ATPase head, long arm, and hinge. SMC complexes form long rod‐like structures, which can change to ring‐like and elbow‐bent conformations upon binding ATP, DNA, and other regulatory factors. These SMC dynamic conformational changes are involved in their loading, translocation, and DNA loop extrusion. Here, we examined the binding and role of the PpNSE5 regulatory factor of Physcomitrium patens PpSMC5/6 complex. We found that the PpNSE5 C‐terminal half (aa230–505) is required for binding to its PpNSE6 partner, while the N‐terminal half (aa1–230) binds PpSMC subunits. Specifically, the first 71 amino acids of PpNSE5 were required for binding to PpSMC6. Interestingly, the PpNSE5 binding required the PpSMC6 head‐proximal joint region and PpSMC5 hinge‐proximal arm, suggesting a long distance between binding sites on PpSMC5 and PpSMC6 arms. Therefore, we hypothesize that PpNSE5 either links two antiparallel SMC5/6 complexes or binds one SMC5/6 in elbow‐bent conformation, the later model being consistent with the role of NSE5/NSE6 dimer as SMC5/6 loading factor to DNA lesions. In addition, we generated the P. patens Ppnse5KO1 mutant line with an N‐terminally truncated version of PpNSE5, which exhibited DNA repair defects while keeping a normal number of rDNA repeats. As the first 71 amino acids of PpNSE5 are required for PpSMC6 binding, our results suggest the role of PpNSE5–PpSMC6 interaction in SMC5/6 loading to DNA lesions. [ABSTRACT FROM AUTHOR]

SUMMARY Being a bona fide actin bundler, Arabidopsis villin5 (VLN5) plays a crucial role in regulating actin stability and organization within pollen tubes. Despite its significance, the precise mechanism through which VLN5 bundles actin filaments has remained elusive. Through meticulous deletion analysis, we have unveiled that the link between gelsolin repeat 6 (G6) and the headpiece domain (VHP), rather than VHP itself, is indispensable for VLN5‐mediated actin bundling. Further refinement of this region has pinpointed a critical sequence spanning from Val763 to Ser823, essential for VLN5's actin‐bundling activity. Notably, the absence of Val763‐Ser823 in VLN5 results in decreased filamentous decoration within pollen tubes and a diminished ability to rescue actin bundling defects in vln2vln5 mutant pollen tubes compared to intact VLN5. Moreover, our findings highlight that the Val763‐Ser823 sequence harbors a binding site for F‐actin, suggesting that this linker‐based F‐actin binding site, in conjunction with the F‐actin binding site localized in G1–G6, enables a single VLN5 to concurrently bind to two adjacent actin filaments. Therefore, our study unveils a novel mechanism by which VLN5 bundles actin filaments. [ABSTRACT FROM AUTHOR]

The Mycobacterium tuberculosis trifunctional enzyme (MtTFE) is an α2β2 tetrameric enzyme in which the α‐chain harbors the 2E‐enoyl‐CoA hydratase (ECH) and 3S‐hydroxyacyl‐CoA dehydrogenase (HAD) active sites, and the β‐chain provides the 3‐ketoacyl‐CoA thiolase (KAT) active site. Linear, medium‐chain and long‐chain 2E‐enoyl‐CoA molecules are the preferred substrates of MtTFE. Previous crystallographic binding and modeling studies identified binding sites for the acyl‐CoA substrates at the three active sites, as well as the NAD binding pocket at the HAD active site. These studies also identified three additional CoA binding sites on the surface of MtTFE that are different from the active sites. It has been proposed that one of these additional sites could be of functional relevance for the substrate channeling (by surface crawling) of reaction intermediates between the three active sites. Here, 226 fragments were screened in a crystallographic fragment‐binding study of MtTFE crystals, resulting in the structures of 16 MtTFE–fragment complexes. Analysis of the 121 fragment‐binding events shows that the ECH active site is the 'binding hotspot' for the tested fragments, with 41 binding events. The mode of binding of the fragments bound at the active sites provides additional insight into how the long‐chain acyl moiety of the substrates can be accommodated at their proposed binding pockets. In addition, the 20 fragment‐binding events between the active sites identify potential transient binding sites of reaction intermediates relevant to the possible channeling of substrates between these active sites. These results provide a basis for further studies to understand the functional relevance of the latter binding sites and to identify substrates for which channeling is crucial. [ABSTRACT FROM AUTHOR]

Background: Psoriasis is a chronic inflammatory skin disease that can cause systemic inflammation in various organs. Rutin has been suggested to fight psoriasis, but the signaling pathways by which it works need to be explored. Materials and methods: HaCaT cells co‐stimulated with interleukin (IL)‐17, IL‐22, tumor necrosis factor‐alpha (TNF‐α), IL‐1α, and oncostatin M (M5) were used as an in vitro cell model of psoriasis. The proliferation and viability of HaCaT cells were determined by 5‐ethynyl‐2′‐deoxyuridine and cell counting assays. Relative mRNA levels of IL‐6, TNF‐α, chemokines (CXCL1 and CXCL2), and anti‐microbial peptides (S100A7 and S100A8) were detected by reverse transcriptase‐quantitative PCR. Release of IL‐6 and TNF‐α from HaCaT cells was measured by enzyme‐linked immunosorbent assay. Keratin1, Keratin5, p‐JAK2, and p‐STAT3 protein levels were estimated with western blotting. Molecular docking predicted binding sites for Rutin and STAT3. Results: Rutin treatment undercut M5‐urged viability increase and proliferation boost in HaCaT cells. Moreover, M5 stimulation mediated upregulation of IL‐6, TNF‐α, CXCL1, CXCL2, S100A7, and S100A8 was partially reversed after Rutin treatment. In addition, M5 stimulation induced downregulation of Keratin1 and Keratin5 proteins as well as upregulation of p‐JAK2 and p‐STAT3 proteins were attenuated in response to Rutin treatment, manifesting that Rutin treatment inhibited M5‐promoted aberrant differentiation and impaired M5‐mediated activation of the JAK2/STAT3 signaling in HaCaT cells. Molecular docking discovered that residues GLN326 and ASP334 in STAT3 might bind to Rutin. Conclusion: Rutin treatment blocked the JAK2/STAT3 signaling, thus attenuating psoriasis‐related inflammation and anomalous differentiation in keratinocytes. [ABSTRACT FROM AUTHOR]