Microscale Thermophoresis Research Articles

Characterizing the interactions between the LPS transport protein LptC and the ABC transporter LptB2FG.

Synthesis of the essential glycolipid lipopolysaccharide (LPS) in Gram-negative bacteria is completed at the outer leaflet of the inner membrane (IM). Following this process, seven LPS transport (Lpt) proteins facilitate the movement of LPS to the outer membrane (OM). LPS transport to the OM is an essential process and disruption at any stage has detrimental effects on bacterial viability. LptB2FG, the first component of the Lpt system, is a type IV ABC transporter that provides the driving force for LPS extraction from the IM and subsequent transport across a stable protein bridge to the outer leaflet of the OM. LptC is a periplasmic protein anchored to the IM by a single transmembrane helix, which intercalates between the LptF and LptG transmembrane domains, and has been shown to regulate the ATPase activity of LptB2FG. Despite a high level of structural homology between the periplasmic domains of LptF and LptG, the crystal and cryo-EM structures of LptB2FG in complex with LptC show preferential binding of the periplasmic domain of LptC to the periplasmic domain of LptF. Therefore, the role of LptG in this transport process remains unknown. Additionally, we identified six partial loss of function LptC mutations during an in vivo exploratory mutagenesis study. To further investigate the LptB2FGC complex, site-directed spin labeling electron paramagnetic resonance (EPR) spectroscopy, microscale thermophoresis (MST), and an in vitro ATPase assay were employed to characterize differences between the binding partners of periplasmic LptF and LptG along with the mechanistic effects of the LptC loss of function mutants on partner binding and ATPase function or regulation. This work provides evidence suggesting a previously undefined interaction between LptC and LptG while also exploring the role of partial loss of function LptC mutants.

Biophysical characterization of pathogenic missense mutation in the ARID domain of ARID1A.

The ARID1A, AT- rich interactive domain containing protein 1A, is frequently mutated in cancer (e.g., 6% of every cancer and 45% of all ovarian cancer). ARID1A is a member of the Switch/Sucrose non-Fermentable (SWI/SNF) complex responsible for chromatin remodeling and gene expression. Within ARID1A lies an ancient and highly conserved DNA-binding domain (ARID domain) of ∼100 amino acids with a helix-turn-helix motif. The Cancer Genome Atlas (TCGA) reports on mutations in the ARID domain for which 37% of the mutations are frameshift, another 37% are missense and remaining are stop gain. Here we use experimental and computational approaches to identify how missense mutation impaired the ARID-to-DNA interactions by impacting protein stability, binding affinity, and structural dynamics potentially explaining their pathogenicity. Specifically, we identified the most mutated variants in the TCGA (i.e., R1020S, M1022K, K1047Q, G1063V, Q1098H). Next, we used web server bioinformatic pathogenic predictors tools (SAMPDI, CUPSAT, SAMBA-3D, SAAMBE-SEQ, SAAFEC, SAAFEC-SEQ, nMCS) to compute biophysical properties such as the protein stability and DNA binding energy and their likelihood for pathogenicity. We used denaturation assays to experimentally determine the stability of selected variants and compare it with the wildtype. To determine the binding affinity, we run electrophoresis mobility shift assays, intrinsic fluorescence, fluorescence anisotropy, and microscale thermophoresis approaches. We compare experimental and bioinformatic tools to decipher the subtle impacts of missense mutation in the ARID domain. Our findings will help us understand the impact of missense mutations in the structure-function relationship.

Revealing the arrangement of the intracellular termini of ASIC1a using transition metal ion FRET.

Acid-sensing ion channels (ASICs) are proton gated, sodium selective, trimeric ion channels primarily located in the central and peripheral nervous systems. Recently, it has been shown that the C-terminus of ASIC1a binds to the protein receptor interacting serine/threonine kinase 1 (RIPK1) under prolonged acidic conditions and this association triggers necroptotic cell death. It has been hypothesized that at neutral pH, the N- and C-termini interact which prevents the association of RIPK1 with the C-terminus. To test this, we performed microscale thermophoresis using a combination of peptides and purified proteins. We found that the ASIC1a C-terminus binds with high affinity to the RIPK1 death domain but does not bind to the ASIC1a N-terminus. We further investigated the proposed interaction of the ASIC1a N- and C-termini in intact channels in cell membranes using transition metal ion FRET (tmFRET) where we measure quenching of an incorporated small fluorophore unnatural amino acid 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (L-Anap) by metal ions bound to a cysteine reactive chelator. First, we demonstrate that we are able to successfully incorporate L-ANAP at a number of positions in both the N- and C-termini of ASIC1a. Second, we measure tmFRET between multiple positions in the termini which show that the N- and C-termini are not in particularly close proximity and during acidosis they undergo only a small rearrangement that, in fact, moves them closer together. Given this result, we go on to map the topology and dynamics of these regions relative to metal ions incorporated into the plasma membrane via a lipid with an NTA head group. Our results suggest that there must be an alternative mechanism by which ASIC1a binds to RIPK1 only after prolonged acidosis.

Hydroxysafflor yellow A regulates lymphangiogenesis and inflammation via the inhibition of PI3K on regulating AKT/mTOR and NF-κB pathway in macrophages to reduce atherosclerosis in ApoE-/- mice

BackgroundMacrophage-mediated inflammatory infiltration and pathological lymphangiogenesis around atherosclerotic plaques are newly highlighted treatment targets of atherosclerosis. Although the effect of Hydroxysafflor yellow A(HSYA) on atherosclerosis was clear, few studies focus on the regulation of HSYA on such mechanisms. PurposeThis study aimed to uncover the key site of HSYA on improving atherosclerosis by regulating macrophage-induced inflammation and lymphangiogenesis. Study designThis study was designed to explore the new mechanism of HSYA on alleviating atherosclerosis in vitro and in vivo. MethodsWe determined the expression of vascular endothelial growth factor C(VEGF-C) in Raw264.7 cells and high-fat diet fed ApoE knockout (ApoE−/−) mice. Raw264.7 cells were treated with HSYA under the stimulation of LPS and ox-LDL. HFD induced ApoE−/− mice were given different concentrations of HSYA-saline solution by tail vein injection and ATV-saline suspension by gavage. C57/B6j mice fed with chow diet were used for the control group. H&E, oil red O and immunofluorescence staining analysis were used for visualizing the pathological changes. The biological impact of HSYA was evaluated by body weight, lipid metabolism, inflammation levels, and corresponding function indexes of kidney and liver. RT-qPCR and western blot methods were conducted to determine the expression of the inflammation and lymphangiogenesis factors. Molecular docking and microscale thermophoresis analysis were used to verify the combination of HSYA and PI3K. ResultsIn vivo, HSYA reduced the plaque formation, hepatic steatosis and inflammation-related lymphangiogenesis (IAL). It also changed the serum levels of inflammation (VEGF-C, TNF-α, IL-6, VCAM1, MCP1), lipid indexes (LDL, CHOL, TRIG) and relevant lymphangiogenesis (VEGF-C and LYVE-1) and inflammation (VCAM-1 and IL-6) signals in the aorta. In vitro, HSYA regulated Akt/mTOR and NF-κB activation by the inhibition of PI3K in macrophages. ConclusionHSYA affects inflammation and inflammation-associated lymphangiogenesis via suppressing PI3K to affect AKT/mTOR and NF-B pathway activation in macrophages, showing a comprehensive protective effect on atherosclerosis.

Characterization of aspartokinase double mutants using a combination of experiments and simulations

Aspartokinase (AK) is synergistically suppressed by Thr and Lys in the Corynebacterium metabolic pathway. Site-directed mutations can significantly improve AK inhibition. Our previous studies confirmed that sites 379 and 380 were important sites affecting enzyme activity, so we further screen the double mutants with excellent enzymatic properties from sites 379 and 380, and discuss the difference of enzyme activity between the double mutants and single mutants. Here, a double mutant, T379L/A380 M, with improved enzyme activity (2.74-fold) was obtained. Enzymatic property experiments showed that the optimum temperature of T379L/A380 M increased from 26 °C (recombinant Escherichia coli; WT-AK) to 45 °C and that the optimal pH decreased from 8.0 (WT-AK) to 7.5. Further, the half-life decreased from 4.5 to 3.32 h. These enzymatic properties were better than other mutant strains. Inhibition was diminished with low concentrations of Lys, and Lys + Thr presented an activating role. Subsequently, the reasons for the improved AK enzyme activity were illustrated with microscale thermophoresis (MST) experiments and molecular dynamic (MD) simulation by measuring ligand affinity and AK conformational changes. MST showed that the affinity between T379L/A380 M and Lys decreased, but the affinity between T379L/A380 M and Asp increased, promoting T379L/A380 M enzyme activity. MD experiments showed that T379L/A380 M enhanced the Asp-ATP affinity and catalyzed the transfer of residues S192 and D193 to Asp, promoting T379L/A380 M enzyme activity. However, the mutation did not cause fluctuations in the substrate Asp and ATP pockets. This might be why the enzyme activity was inferior to that of the single mutants (T379L and A380 M).

Characterization of a new CCCTC-binding factor binding site as a dual regulator of Epstein-Barr virus latent infection.

Distinct viral gene expression characterizes Epstein-Barr virus (EBV) infection in EBV-producing marmoset B-cell (B95-8) and EBV-associated gastric carcinoma (SNU719) cell lines. CCCTC-binding factor (CTCF) is a structural chromatin factor that coordinates chromatin interactions in the EBV genome. Chromatin immunoprecipitation followed by sequencing against CTCF revealed 16 CTCF binding sites in the B95-8 and SNU719 EBV genomes. The biological function of one CTCF binding site (S13 locus) located on the BamHI A right transcript (BART) miRNA promoter was elucidated experimentally. Microscale thermophoresis assay showed that CTCF binds more readily to the stable form than the mutant form of the S13 locus. EBV BART miRNA clusters encode 22 miRNAs, whose roles are implicated in EBV-related cancer pathogenesis. The B95-8 EBV genome lacks a 11.8-kb EcoRI C fragment, whereas the SNU719 EBV genome is full-length. ChIP-PCR assay revealed that CTCF, RNA polymerase II, H3K4me3 histone, and H3K9me3 histone were more enriched at S13 and S16 (167-kb) loci in B95-8 than in the SNU719 EBV genome. 4C-Seq and 3C-PCR assays using B95-8 and SNU719 cells showed that the S13 locus was associated with overall EBV genomic loci including 3-kb and 167-kb region in both EBV genomes. We generated mutations in the S13 locus in bacmids with or without the 11.8-kb BART transcript unit (BART(+/-)). The S13 mutation upregulated BART miRNA expression, weakened EBV latency, and reduced EBV infectivity in the presence of EcoRI C fragment. Another 3C-PCR assay using four types of BART(+/-)·S13(wild-type(Wt)/mutant(Mt)) HEK293-EBV cells revealed that the S13 mutation decreased DNA associations between the 167-kb region and 3-kb in the EBV genome. Based on these results, CTCF bound to the S13 locus along with the 11.8-kb EcoRI C fragment is suggested to form an EBV 3-dimensional DNA loop for coordinated EBV BART miRNA expression and infectivity.

ABT1 modifies SMARD1 pathology via interactions with IGHMBP2 and stimulation of ATPase and helicase activity.

SMA with respiratory distress type 1 (SMARD1) and Charcot-Marie-Tooth type 2S (CMT2S) are results of mutations in immunoglobulin mu DNA binding protein 2 (IGHMBP2). IGHMBP2 is a UPF1-like helicase with proposed roles in several cellular processes, including translation. This study examines activator of basal transcription 1 (ABT1), a modifier of SMARD1-nmd disease pathology. Microscale thermophoresis and dynamic light scattering demonstrate that IGHMBP2 and ABT1 proteins directly interact with high affinity. The association of ABT1 with IGHMBP2 significantly increases the ATPase and helicase activity as well as the processivity of IGHMBP2. The IGHMBP2/ABT1 complex interacts with the 47S pre-rRNA 5' external transcribed spacer and U3 small nucleolar RNA (snoRNA), suggesting that the IGHMBP2/ABT1 complex is important for pre-rRNA processing. Intracerebroventricular injection of scAAV9-Abt1 decreases FVB-Ighmbp2nmd/nmd disease pathology, significantly increases lifespan, and substantially decreases neuromuscular junction denervation. To our knowledge, ABT1 is the first disease-modifying gene identified for SMARD1. We provide a mechanism proposing that ABT1 decreases disease pathology in FVB-Ighmbp2nmd/nmd mutants by optimizing IGHMBP2 biochemical activity (ATPase and helicase activity). Our studies provide insight into SMARD1 pathogenesis, suggesting that ABT1 modifies IGHMBP2 activity as a means to regulate pre-rRNA processing.

Analyzing the interactome of human CK2β in prostate carcinoma cells reveals HSP70-1 and Rho guanin nucleotide exchange factor 12 as novel interaction partners.

CK2β is the non-catalytic modulating part of the S/T-protein kinase CK2. However, the overall function of CK2β is poorly understood. Here, we report on the identification of 38 new interaction partners of the human CK2β from lysates of DU145 prostate cancer cells using photo-crosslinking and mass spectrometry, whereby HSP70-1 was identified with high abundance. The KD value of its interaction with CK2β was determined as 0.57 μM by microscale thermophoresis, this being the first time, to our knowledge, that a KD value of CK2β with another protein than CK2α or CK2α' was quantified. Phosphorylation studies excluded HSP70-1 as a substrate or activity modulator of CK2, suggesting a CK2 activity independent interaction of HSP70-1 with CK2β. Co-immunoprecipitation experiments in three different cancer cell lines confirmed the interaction of HSP70-1 with CK2β in vivo. A second identified CK2β interaction partner was Rho guanin nucleotide exchange factor 12, indicating an involvement of CK2β in the Rho-GTPase signal pathway, described here for the first time to our knowledge. This points to a role of CK2β in the interaction network affecting the organization of the cytoskeleton.

DNAzyme-Based Microscale Thermophoresis Sensor.

Microscale thermophoresis (MST) technology has emerged as a powerful growing method in a molecular interaction study by measuring fluorescence responses of molecules inside a capillary to infrared (IR) laser heating with the benefits of rapid ratiometric measurement, separation-free, no immobilization, and low sample consumption. Combining the advantages of RNA-cleaving DNAzymes in target recognition and enzymatic catalysis and the strength of MST technology for fluorescence signaling, here, we reported a DNAzyme-based MST method for sensitive target detection. We introduced a fluorescein terminal label at the RNA-cleaving DNAzyme, and the substrate was linked to DNAzyme together with a poly-T sequence in a unimolecular design or not conjugated with DNAzyme in a bimolecular design. The presence of the cofactor activated DNAzyme to catalytically cleave the substrate, causing molecular structure alteration and significant changes in MST signals. This DNAzyme MST sensor enabled sensitively detecting activator targets Pb2+ and l-histidine, with a detection limit of 49 pM Pb2+ and 3.9 μM l-histidine. This biosensing strategy is universal and promising for wide applications.

A Potential Off-Target Effect of the Wnt/β-Catenin Inhibitor KYA1797K: PD-L1 Binding and Checkpoint Inhibition

Introduction: The quest for small molecule inhibitors of the PD-1/PD-L1 checkpoint continues in parallel to the extensive development of monoclonal antibodies directed against this immune checkpoint. Drug screening strategies are being set up to identify novel PD-L1 inhibitors. Methods: A virtual screening based on molecular docking with the PD-L1 protein dimer has been performed to identify a new binder. Binding of the identified ligand to PD-L1 has been validated experimentally using a microscale thermophoresis (MST) assay. The cellular effect of the compound was evidenced using a fluorescence resonance energy transfer (FRET) assay based on activation of tyrosine phosphatase SHP-2. Results: We have identified the potent Wnt/β-catenin inhibitor KYA1797K as a weak PD-L1 binder. Molecular docking suggested that the compound can bind to the interface of a PD-L1 dimer, with a geometry superimposable to that of the reference PD-L1 inhibitor BMS-202. The atypical 2-thioxo-4-thiazolidinone motif of KYA1797K, derived from the natural product rhodanine, plays a major role in the interaction with PD-L1. Binding of KYA1797K to recombinant hPD-L1 was validated experimentally, using MST. The drug was found to bind modestly but effectively to hPD-L1. The FRET assay confirmed the weak capacity of KYA1797K to interfere with the activation of SHP-2 upon its interaction with human PD-1. Discussion: Collectively, the data show that KYA1797K could function as a weak modulator of the PD-1/PD-L1 checkpoint. This effect may contribute, at least partially, to the reported capacity of the β-catenin inhibitor to downregulate PD-L1 in cancer cells. The work also underlines the interest to further consider the rhodanine moiety as a chemical motif for the design of new PD-L1 binders.

Design, synthesis, bioactivity and mechanism of action of novel myricetin derivatives containing amide and hydrazide

A series of myricetin derivatives containing amide and hydrazide were designed and synthesized. All the compounds were characterized by NMR and HRMS. Bioactivity test showed that some of the target compounds had excellent anti-tobacco mosaic virus (TMV) activity. In particular, the median effective concentration (EC50) values of the anti-TMV curative and protective activities of N-(2-(2-(2-((5,7-dimethoxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-4H-chromen-3-yl)oxy)acetyl)hydrazineyl)-2-oxoethyl)-4-(trifluoromethyl)benzamide (G9) were 202.3 and 164.0 μg/mL respectively, superior to ningnanmycin (329.1, 230.3 μg/mL). Microscale thermophoresis (MST) and molecular docking showed that G9 had an excellent binding affinity with tobacco mosaic virus coat protein (TMV-CP) (Kd = 0.158 ± 0.024 μM), which was better than that of ningnanmycin (Kd = 2.074 ± 0.818 μM). Moreover, there were many interaction forces between G9 and the key amino acid residues of TMV-CP. The chlorophyll content and peroxidase (POD) activity of tobacco leaves treated with G9 increased significantly, indicating that G9 could improve the photosynthesis of tobacco leaves and stimulate the resistance of tobacco leaves to TMV. The insecticidal activity of G9 against Mythimna separata (M. separate) was found to be 95.2% at 200 μg/mL, which was close to bufenozide (100%). The insecticidal activity of myricetin was significantly improved after the introduction of active groups of amide and hydrazide, which could be further explored.

Coronavirus Inhibitors Targeting nsp16.

During the past three decades, humans have been confronted with different new coronavirus outbreaks. Since the end of the year 2019, COVID-19 threatens the world as a rapidly spreading infectious disease. For this work, we targeted the non-structural protein 16 (nsp16) as a key protein of SARS-CoV-2, SARS-CoV-1 and MERS-CoV to develop broad-spectrum inhibitors of nsp16. Computational methods were used to filter candidates from a natural product-based library of 224,205 compounds obtained from the ZINC database. The binding of the candidates to nsp16 was assessed using virtual screening with VINA LC, and molecular docking with AutoDock 4.2.6. The top 9 compounds were bound to the nsp16 protein of SARS-CoV-2, SARS-CoV-1, and MERS-CoV with the lowest binding energies (LBEs) in the range of -9.0 to -13.0 kcal with VINA LC. The AutoDock-based LBEs for nsp16 of SARS-CoV-2 ranged from -11.42 to -16.11 kcal/mol with predicted inhibition constants (pKi) from 0.002 to 4.51 nM, the natural substrate S-adenosyl methionine (SAM) was used as control. In silico results were verified by microscale thermophoresis as in vitro assay. The candidates were investigated further for their cytotoxicity in normal MRC-5 lung fibroblasts to determine their therapeutic indices. Here, the IC50 values of all three compounds were >10 µM. In summary, we identified three novel SARS-CoV-2 inhibitors, two of which showed broad-spectrum activity to nsp16 in SARS-CoV-2, SARS-CoV-1, and MERS-CoV. All three compounds are coumarin derivatives that contain chromen-2-one in their scaffolds.

Functionalized Fullerene for Inhibition of SARS-CoV-2 Variants.

As virus outbreaks continue to pose a challenge, a nonspecific viral inhibitor can provide significant benefits, especially against respiratory viruses. Polyglycerol sulfates recently emerge as promising agents that mediate interactions between cells and viruses through electrostatics, leading to virus inhibition. Similarly, hydrophobic C60 fullerene can prevent virus infection via interactions with hydrophobic cavities of surface proteins. Here, two strategies are combined to inhibit infection of SARS-CoV-2 variants in vitro. Effective inhibitory concentrations in the millimolar range highlight the significance of bare fullerene's hydrophobic moiety and electrostatic interactions of polysulfates with surface proteins of SARS-CoV-2. Furthermore, microscale thermophoresis measurements support that fullerene linear polyglycerol sulfates interact with the SARS-CoV-2 virus via its spike protein, and highlight importance of electrostatic interactions within it. All-atom molecular dynamics simulations reveal that the fullerene binding site is situated close to the receptor binding domain, within 4nm of polyglycerol sulfate binding sites, feasibly allowing both portions of the material to interact simultaneously.

Combining nano-differential scanning fluorimetry and microscale thermophoresis to investigate VDAC1 interaction with small molecules

The mitochondrial voltage-dependent anion channel 1 (VDAC1) plays a central role in metabolism and apoptosis, which makes it a promising therapeutic target. Nevertheless, molecular mechanisms governing VDAC1 functioning remain unclear. Small-molecule ligands specifically interacting with the channel provide an attractive way of exploring its structure-function relationships and can possibly be used as founding stones for future drug-candidates. While around 30 VDAC1 ligands have been identified over the years, various techniques have been used by research teams, making a fair and direct comparison between compounds impossible. To tackle this issue, we performed ligand-binding assays on a representative set of seventeen known VDAC1 ligands using nano-differential scanning fluorimetry and microscale thermophoresis. While all the compounds have been confirmed as VDAC1 ligands by at least one method, combining both technologies lead to the selection of four molecules (cannabidiol, curcumin, DIDS and VBIT4) as chemical starting points for future design of VDAC1 selective ligands.

Coronatine promotes maize water uptake by directly binding to the aquaporin ZmPIP2;5 and enhancing its activity.

Water uptake is crucial for crop growth and development and drought stress tolerance. The water channel aquaporins (AQP) play important roles in plant water uptake. Here, we discovered that a jasmonic acid analog, coronatine (COR), enhanced maize (Zea mays) root water uptake capacity under artificial water deficiency conditions. COR treatment induced the expression of the AQP gene Plasma membrane intrinsic protein 2;5 (ZmPIP2;5). In vivo and in vitro experiments indicated that COR also directly acts on ZmPIP2;5 to improve water uptake in maize and Xenopus oocytes. The leaf water potential and hydraulic conductivity of roots growing under hyperosmotic conditions were higher in ZmPIP2;5-overexpression lines and lower in the zmpip2;5 knockout mutant, compared to wild-type plants. Based on a comparison between ZmPIP2;5 and other PIP2s, we predicted that COR may bind to the functional site in loop E of ZmPIP2;5. We confirmed this prediction by surface plasmon resonance technology and a microscale thermophoresis assay, and showed that deleting the binding motif greatly reduced COR binding. We identified the N241 residue as the COR-specific binding site, which may activate the channel of the AQP tetramer and increase water transport activity, which may facilitate water uptake under hyperosmotic stress.

Cimifugin suppresses type 2 airway inflammation by binding to SPR and regulating its protein expression in a non-enzymatic manner

BackgroundCimifugin is one of the main bioactive components of Yu-Ping-Feng-San, a well-known traditional Chinese medicine, which can effectively relieve Allergic asthma (AA) and atopic dermatitis and reduce recurrence in clinic. However, the underlying mechanism of cimifugin on AA is still unknown. PurposeIn the present study, we aimed to investigate the effect and mechanism of cimifugin on AA. Study designIn vivo and in vitro experimental studies were performed. MethodsThe effect of cimifugin on AA was demonstrated in vivo and in vitro. Sepiapterin reductase (SPR) was predicted as the most potent target of cimifugin in treating AA by reverse docking. Molecular docking and microscale thermophoresis (MST) were used to analyze the direct binding between cimifugin and SPR. Overexpression and interference of SPR were performed to verify whether targeting SPR is a key step of cimifugin in the treatment of AA. QM385, an inhibitor of SPR, was administrated in vivo and in vitro to evaluate the role of SPR in AA. Further, HPLC and cell-free direct hSPR enzyme activity assay were performed to research whether cimifugin regulated SPR by influencing the enzyme activity. Simultaneously, the inhibitors of protein degradation were used in vitro to explore the mechanism of cimifugin on SPR. ResultsWe found cimifugin effectively alleviated AA by reducing airway hyperresponsiveness, inhibiting type 2 cytokines-mediated airway inflammation, and restoring the expression of epithelial barrier proteins. Molecular docking predicted the direct binding ability of cimifugin to SPR, which was further verified by MST. Notably, the therapeutic effect of cimifugin on AA was dampened with SPR interfering, in contrast, the phenotypic features of AA were significantly alleviated with QM385 application both in vivo and in vitro. Interestingly, cimifugin showed no effect on the enzyme activity of SPR, as the level of its substrate sepiapterin was not affected with cimifugin treatment by cell-free enzyme activity assay. Furthermore, we found cimifugin could reduce SPR protein expression without affecting its mRNA expression probably through autophagosome pathway. ConclusionsTo our knowledge, we're reporting for the first time that cimifugin can suppresses type 2 airway inflammation to alleviate AA by directly binding to SPR and regulating its protein expression in a non-enzymatic manner.

The acyl-CoA-binding protein 2 exhibited the highest affinity for palmitoyl-CoA and promoted Monascus pigment production

PurposeThe present study aimed to explore the binding ability of acyl-CoA binding protein 2 to fatty acid acyl-CoA esters and its effect on Monascus pigment production in M. ruber CICC41233.MethodsThe Mracbp2 gene from M. ruber CICC41233 was cloned with a total DNA and cDNA as the templates through the polymerase chain reaction. The cDNA of the Mracbp2 gene fragment was ligated to expression vector pGEX-6P-1 to construct pGEX-MrACBP2, which was expressed in Escherichia coli BL21 to obtain the fusion protein GST-MrACBP2 and then measure the binding ability of fatty acid acyl-CoA esters. Additionally, the DNA of the Mracbp2 gene fragment was ligated to expression vector pNeo0380 to construct pNeo0380-MrACBP2, which was homologously over-expressed in M. ruber CICC41233 to evaluate Monascus pigment production and fatty acid.ResultsThe cloned Mracbp2 gene of the DNA and cDNA sequence was 1525 bp and 1329 bp in length, respectively. The microscale thermophoresis binding assay revealed that the purified GST-MrACBP2 had the highest affinity for palmitoyl-CoA (Kd =70.57 nM). Further, the Mracbp2 gene was homologously overexpressed in M. ruber CICC41233, and a positive transformant M. ruber ACBP-E was isolated. In the Monascus pigments fermentation, the expression level of the Mracbp2 gene was increased by 1.74-fold after 2 days and 2.38-fold after 6 days. The palmitic acid content and biomass in M. ruber ACBP2-E were significantly lower than that in M. ruber CICC41233 on 2 days and 6 days. However, compared with M. ruber CICC41233, the yields of total pigment, ethanol-soluble pigment, and water-soluble pigment in M. ruber ACBP2-E increased by 63.61%, 71.61%, and 29.70%, respectively.ConclusionsThe purified fusion protein GST-MrACBP2 exhibited the highest affinity for palmitoyl-CoA. The Mracbp2 gene was overexpressed in M. ruber CICC41233, which resulted in a decrease in palmitic acid and an increase in Monascus pigments. Overall, the effect of MrACBP2 on the synthesis of fatty acid and Monascus pigment was explored. This paper explored the effect of MrACBP2 on the fatty acid synthesis and the synthesis of Monascus pigment. The results indicated the regulation of fatty acid synthesis could affect Monascus pigment synthesis, providing a novel strategy for improving the yield of Monascus pigment.

Alleviation of Hepatic Steatosis by 4-azidophlorizin via the Degradation of Geranylgeranyl Diphosphate Synthase by the Ubiquitin-Proteasome Pathway in Vivo and in Vitro.

There are no known approved pharmacotherapies for non-alcoholic fatty liver disease (NAFLD) in the clinical setting. Although studies have provided substantial evidence that geranylgeranyl diphosphate synthase (GGPPS) is a potential therapeutic target for the treatment of NAFLD corresponding drug screening is rare. A GGPPS-targeted inhibitor is identified using a structure-based virtual small molecule screening method. The interaction of 4-AZ and GGPPS is detected by microscale thermophoresis. 4-AZ degradation of GGPPS by the ubiquitin-proteasome pathway is detected by western blotting. The anti-steatotic effect of 4-AZ in vivo is detected by CT. Lipid-related gene detection is detected by real-time PCR both in primary hepatocytes and mice. The compound inhibits the accumulation of lipids in primary hepatocytes and decreases lipogenic gene expression through GGPPS. Pharmacological studies show that 4-AZ can attenuate hepatic steatosis and improve liver injury in high-fat diet-induced mice. This data provides a novel application of 4-AZ NAFLD therapy, proving that the inhibition of GGPPS is a novel strategy for the treatment of NAFLD.

The Inhibitory Properties of a Novel, Selective LMTK3 Kinase Inhibitor

Recently, the oncogenic role of lemur tyrosine kinase 3 (LMTK3) has been well established in different tumor types, highlighting it as a viable therapeutic target. In the present study, using in vitro and cell-based assays coupled with biophysical analyses, we identify a highly selective small molecule LMTK3 inhibitor, namely C36. Biochemical/biophysical and cellular studies revealed that C36 displays a high in vitro selectivity profile and provides notable therapeutic effect when tested in the National Cancer Institute (NCI)-60 cancer cell line panel. We also report the binding affinity between LMTK3 and C36 as demonstrated via microscale thermophoresis (MST). In addition, C36 exhibits a mixed-type inhibition against LMTK3, consistent with the inhibitor overlapping with both the adenosine 5'-triphosphate (ATP)- and substrate-binding sites. Treatment of different breast cancer cell lines with C36 led to decreased proliferation and increased apoptosis, further reinforcing the prospective value of LMTK3 inhibitors for cancer therapy.

Sensitive microscale thermophoresis assay for rapid ochratoxin A detection with fluorescently labeled engineered aptamer.

Ochratoxin A (OTA) is a widespread mycotoxin that causes contamination in a variety of foodstuffs and environments, inducing great health risks to humans and animals. Rapid and sensitive detection of OTA is necessary for food safety, environmental health, and risk assessment. Herein, we report an aptamer microscale thermophoresis (MST) assay for OTA, with the unique merits of ratiometric analysis, rapid measurement, simple operation, high sensitivity, low sample consumption, and high throughput. A fluorescein (FAM)-labeled high-affinity DNA aptamer with a G-quadruplex and duplex structure was used as the recognition element for OTA, and MST, which measures the fluorescence responses of the sample solution inside capillaries to a mild temperature increase generated by infrared laser heating, was employed for signal generation. Upon OTA binding, the FAM-labeled aptamer probe underwent changes in conformation and stability, and the bound and unbound aptamer probes showed significant differences in their MST signals. To achieve sensitive detection of OTA with a large signal change, we systematically characterized aptamers with different stem lengths, which had large effects on the MST responses of the aptamer probes to OTA. We found that a 32-mer aptamer with FAM label at the 3' end gave a sensitive MST response to OTA, allowing OTA detection within seconds with a detection limit of 0.98 nM under optimal experimental conditions. This aptamer MST assay shows potential in real sample analysis and broad applications.