Structural and Functional Analyses of the Flavoprotein Disulfide Reductase FN0820 of Fusobacterium nucleatum.

Escherichia coli RclA and Staphylococcus aureus MerA are part of the Group I flavoprotein disulfide reductase (FDR) family and have been implicated in the contribution to bacterial pathogenesis by defending against the host immune response. Fusobacterium nucleatum is a pathogenic, anaerobic Gram-negative bacterial species commonly found in the human oral cavity and gastrointestinal tract. In this study, we discovered that the F. nucleatum protein FN0820, belonging to the Group I FDR family, exhibited a higher activity of a Cu2+-dependent NADH oxidase than E. coli RclA. Moreover, FN0820 decreased the dissolved oxygen level in the solution with higher NADH oxidase activity. We found that L-tryptophan and its analog 5-hydroxytryptophan inhibit the FN0820 activities of NADH oxidase and the concomitant reduction of oxygen. Our results have implications for developing new treatment strategies against pathogens that defend the host immune response with Group I FDRs.

Effect of superspeed sintering on translucency, opalescence, microstructure, and phase fraction of multilayered 4 mol% yttria-stabilized tetragonal zirconia polycrystal and 6 mol% yttria-stabilized partially stabilized zirconia ceramics.

STATEMENT OF PROBLEM: The optical properties of recently developed multilayer zirconia have mainly been studied for the effects of conventional sintering and speed sintering but not as much for the effect of superspeed sintering. As superspeed sintering protocols typically require a higher sintering temperature and higher heating and cooling rates than speed- and conventional sintering protocols, the optical properties of superspeed sintered zirconia may be affected differently. PURPOSE: The purpose of this in vitro study was to investigate the effect of superspeed sintering on the optical properties, microstructure, and phase fraction of multilayered 4 mol% yttria-stabilized (4Y-) and 6 mol% yttria-stabilized (6Y-) zirconia. MATERIAL AND METHODS: Multilayered 4Y- and 6Y-zirconia were sectioned. After conventional and superspeed sintering, the translucency parameter (TP), and opalescence parameter (OP) were measured with a spectrophotometer (n=10). To obtain the grain sizes from the field emission scanning electron microscopy (FE-SEM) images for each layer (n=2), more than 500 (6Y-zirconia) and 800 grains (4Y-zirconia) were measured by linear intercept methods. The phase fractions were obtained through X-ray diffraction (XRD) analysis by using the Rietveld method (n=1). The results were analyzed by 3-way ANOVA and post hoc Tukey honest significant difference tests (TP and OP) and by 3-way ANOVA and post hoc Scheffé tests (grain size) (α=.05). RESULTS: No layers exhibited a significant difference in TP after superspeed sintering, except the dentin layer (DL) and transition layer 2 (T2) of 4Y- and 6Y-zirconia, respectively. The TP increased (P<.05) in DL for superspeed sintered 4Y-zirconia and decreased (P<.05) in T2 for the superspeed sintered 6Y-zirconia. However, the difference in TP by superspeed sintering was lower than the perceptibility thresholds of 50:50%. The OP decreased (P<.05) in the DL and T2 of 4Y-zirconia after superspeed sintering. For 6Y-zirconia, the OP decreased (P<.05) in all layers except for the transition layer 1 (T1) after superspeed sintering. However, the difference in OP values was minimal, with only a 1.1 difference observed for Zolid Gen-X (4Y) and a range of 1.22 to 1.62 for Katana UTML (6Y) when using superspeed sintering. No significant change was found in the grain size after superspeed sintering of either zirconia. Regardless of the sintering speed, the average grain size of the 6Y-zirconia (conventional: 2.09 to 2.21 µm; superspeed: 2.11 to 2.20 µm) was larger than that of the 4Y-zirconia (conventional: 0.50 to 0.52 µm; superspeed: 0.52 to 0.54 µm). Owing to superspeed sintering, the metastable tetragonal (T') phase content increased while the tetragonal (T) phase decreased in 4Y-zirconia; in 6Y-zirconia, the cubic (C) phase content increased, while the T'-phase content decreased. CONCLUSIONS: Superspeed sintering did not result in any clinically significant changes in the translucency and opalescence of 4Y- or 6Y-zirconia.

Heterologous Synthesis and Characterization of Thiocillin IV.

Micrococcin P1 and P2 are thiopeptides with a wide range of biological functions including antibacterial and antimalarial activities. We previously demonstrated optimized enzymatic sequences for the exclusive and scalable biosynthesis of micrococcin P2. Thiocillin IV is predicted to be the congener of O-methylated micrococcin P2, but the exact structure has not been elucidated. In this study, we report the first scalable biosynthesis and full structural characterization of thiocillin IV, a 26-membered thiopeptide. This was achieved by generating a recombinant plasmid by inserting tclO, a gene encoding an O-methyltransferase, and genes responsible for micrococcin P2 production and incorporating them into a Bacillus strain. With the incorporation of precursor peptide genes and optimal culture conditions, production reached 2.4 mg/L of culture. The purified thiocillin IV structure was identified as O-methylated micrococcin P2 at the 8-Thr position, and its promising biological activity toward various Gram-positive pathogens was observed. This study provides tclO-mediated site-selective methylation and opens a biotechnological opportunity to produce selective thiopeptides.

Structural analysis of the overoxidized Cu/Zn-superoxide dismutase in ROS-induced ALS filament formation.

Eukaryotic Cu, Zn-superoxide dismutase (SOD1) is primarily responsible for cytotoxic filament formation in amyotrophic lateral sclerosis (ALS) neurons. Two cysteine residues in SOD1 form an intramolecular disulfide bond. This study aims to explore the molecular mechanism of SOD1 filament formation by cysteine overoxidation in sporadic ALS (sALS). In this study, we determined the crystal structure of the double mutant (C57D/C146D) SOD1 that mimics the overoxidation of the disulfide-forming cysteine residues. The structure revealed the open and relaxed conformation of loop IV containing the mutated Asp57. The double mutant SOD1 produced more contagious filaments than wild-type protein, promoting filament formation of the wild-type SOD1 proteins. Importantly, we further found that HOCl treatment to the wild-type SOD1 proteins facilitated their filament formation. We propose a feasible mechanism for SOD1 filament formation in ALS from the wild-type SOD1, suggesting that overoxidized SOD1 is a triggering factor of sALS. Our findings extend our understanding of other neurodegenerative disorders associated with ROS stresses at the molecular level.

Non-canonical ß-adrenergic activation of ERK at endosomes.

G-protein-coupled receptors (GPCRs), the largest family of signalling receptors, as well as important drug targets, are known to activate extracellular-signal-regulated kinase (ERK)-a master regulator of cell proliferation and survival1. However, the precise mechanisms that underlie GPCR-mediated ERK activation are not clearly understood2-4. Here we investigated how spatially organized ß2-adrenergic receptor (ß2AR) signalling controls ERK. Using subcellularly targeted ERK activity biosensors5, we show that ß2AR signalling induces ERK activity at endosomes, but not at the plasma membrane. This pool of ERK activity depends on active, endosome-localized Gαs and requires ligand-stimulated ß2AR endocytosis. We further identify an endosomally localized non-canonical signalling axis comprising Gαs, RAF and mitogen-activated protein kinase kinase, resulting in endosomal ERK activity that propagates into the nucleus. Selective inhibition of endosomal ß2AR and Gαs signalling blunted nuclear ERK activity, MYC gene expression and cell proliferation. These results reveal a non-canonical mechanism for the spatial regulation of ERK through GPCR signalling and identify a functionally important endosomal signalling axis.

Dimerization of ß2-adrenergic receptor is responsible for the constitutive activity subjected to inverse agonism.

Dimerization of beta 2-adrenergic receptor (ß2-AR) has been observed across various physiologies. However, the function of dimeric ß2-AR is still elusive. Here, we revealed that dimerization of ß2-AR is responsible for the constitutive activity of ß2-AR generating inverse agonism. Using a co-immunoimmobilization assay, we found that transient ß2-AR dimers exist in a resting state, and the dimer was disrupted by the inverse agonists. A Gαs preferentially interacts with dimeric ß2-AR, but not monomeric ß2-AR, in a resting state, resulting in the production of a resting cAMP level. The formation of ß2-AR dimers requires cholesterol on the plasma membrane. The cholesterol did not interfere with the agonist-induced activation of monomeric ß2-AR, unlike the inverse agonists, implying that the cholesterol is a specific factor regulating the dimerization of ß2-AR. Our model not only shows the function of dimeric ß2-AR but also provides a molecular insight into the mechanism of the inverse agonism of ß2-AR.

RKIP Induction Promotes Tumor Differentiation via SOX2 Degradation in NF2-Deficient Conditions.

Loss of NF2 (merlin) has been suggested as a genetic cause of neurofibromatosis type 2 and malignant peripheral nerve sheath tumor (MPNST). Previously, we demonstrated that NF2 sustained TGFß receptor 2 (TßR2) expression and reduction or loss of NF2 activated non-canonical TGFß signaling, which reduced Raf kinase inhibitor protein (RKIP) expression via TßR1 kinase activity. Here, we show that a selective RKIP inducer (novel chemical, Nf18001) inhibits tumor growth and promotes schwannoma cell differentiation into mature Schwann cells under NF2-deficient conditions. In addition, Nf18001 is not cytotoxic to cells expressing NF2 and is not disturb canonical TGFß signaling. Moreover, the novel chemical induces expression of SOX10, a marker of differentiated Schwann cells, and promotes nuclear export and degradation of SOX2, a stem cell factor. Treatment with Nf18001 inhibited tumor growth in an allograft model with mouse schwannoma cells. These results strongly suggest that selective RKIP inducers could be useful for the treatment of neurofibromatosis type 2 as well as NF2-deficient MPNST. IMPLICATIONS: This study identifies that a selective RKIP inducer inhibits tumor growth and promotes schwannoma cell differentiation under NF2-deficient conditions by reducing SOX2 and increasing SOX10 expression.

Diversity-oriented routes to thiopeptide antibiotics: total synthesis and biological evaluation of micrococcin P2.

We report the first total synthesis of micrococcin P2 (MP2, 1) by a diversity-oriented route that incorporates a number of refinements relative to earlier syntheses. Biological data regarding the activity of 1 against a range of human pathogens are also provided. Furthermore, we disclose a chemical property of MP2 that greatly facilitates medicinal chemistry work in the micrococcin area and describe a method to obtain MP2 by fermentation in B. subtilis.

Novel chemical inhibitor against SOD1 misfolding and aggregation protects neuron-loss and ameliorates disease symptoms in ALS mouse model.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective death of motor neurons. Mutations in Cu, Zn-superoxide dismutase (SOD1) causing the gain of its toxic property are the major culprit of familial ALS (fALS). The abnormal SOD1 aggregation in the motor neurons has been suggested as the major pathological hallmark of ALS patients. However, the development of pharmacological interventions against SOD1 still needs further investigation. In this study, using ELISA-based chemical screening with wild and mutant SOD1 proteins, we screened a new small molecule, PRG-A01, which could block the misfolding/aggregation of SOD1 or TDP-43. The drug rescued the cell death induced by mutant SOD1 in human neuroblastoma cell line. Administration of PRG-A01 into the ALS model mouse resulted in significant improvement of muscle strength, motor neuron viability and mobility with extended lifespan. These results suggest that SOD1 misfolding/aggregation is a potent therapeutic target for SOD1 related ALS.

Blue-conversion of organic dyes produces artifacts in multicolor fluorescence imaging.

Multicolor fluorescence imaging is a powerful tool visualizing the spatiotemporal relationship among biomolecules. Here, we report that commonly employed organic dyes exhibit a blue-conversion phenomenon, which can produce severe multicolor image artifacts leading to false-positive colocalization by invading predefined spectral windows, as demonstrated in the case study using EGFR and Tensin2. These multicolor image artifacts become much critical in localization-based superresolution microscopy as the blue-converted dyes are photoactivatable. We provide a practical guideline for the use of organic dyes for multicolor imaging to prevent artifacts derived by blue-conversion.

FluoSTEPs: Fluorescent biosensors for monitoring compartmentalized signaling within endogenous microdomains.

Growing evidence suggests that many essential intracellular signaling events are compartmentalized within kinetically distinct microdomains in cells. Genetically encoded fluorescent biosensors are powerful tools to dissect compartmentalized signaling, but current approaches to probe these microdomains typically rely on biosensor fusion and overexpression of critical regulatory elements. Here, we present a novel class of biosensors named FluoSTEPs (fluorescent sensors targeted to endogenous proteins) that combine self-complementing split green fluorescent protein, CRISPR-mediated knock-in, and fluorescence resonance energy transfer biosensor technology to probe compartmentalized signaling dynamics in situ. We designed FluoSTEPs for simultaneously highlighting endogenous microdomains and reporting domain-specific, real-time signaling events including kinase activities, guanosine triphosphatase activation, and second messenger dynamics in live cells. A FluoSTEP for 3',5'-cyclic adenosine monophosphate (cAMP) revealed distinct cAMP dynamics within clathrin microdomains in response to stimulation of G protein-coupled receptors, showcasing the utility of FluoSTEPs in probing spatiotemporal regulation within endogenous signaling architectures.

Epigallocatechin Gallate Inhibits the Uridylate-Specific Endoribonuclease Nsp15 and Efficiently Neutralizes the SARS-CoV-2 Strain.

SARS-CoV-2, the coronavirus strain that initiated the COVID-19 pandemic, and its subsequent variants present challenges to vaccine development and treatment. As the coronavirus evades the host innate immune response at the initial stage of infection, the disease can have a long nonsymptomatic period. The uridylate-specific endoribonuclease Nsp15 processes the viral genome for replication and cleaves the polyU sequence in the viral RNA to interfere with the host immune system. This study screened natural compounds in vitro to identify inhibitors against Nsp15 from SARS-CoV-2. Three natural compounds, epigallocatechin gallate (EGCG), baicalin, and quercetin, were identified as potential inhibitors. Potent antiviral activity of EGCG was confirmed in plaque reduction neutralization tests with a SARS-CoV-2 strain (PRNT50 = 0.20 µM). Because the compound has been used as a functional food ingredient due to its beneficial health effects, we theorize that this natural compound may help inhibit viral replication while minimizing safety issues.

Emodin induces collagen type I synthesis in Hs27 human dermal fibroblasts.

Fibrillar collagen and elastic fibers are the main components of the dermal extracellular matrix (ECM), which confers mechanical strength and resilience to the skin. In particular, type I collagen produced by fibroblasts is the most abundant collagen that determines the general strength of the ECM, thereby contributing to the prevesntion of the skin-aging process. Although the natural anthraquinone derivative emodin (1,3,8-trihydroxy-6-methylanthraquinone) exerts numerous beneficial effects, including antiviral, anticancer, anti-inflammatory and wound-healing effects in diverse cells, the effect of emodin on collagen expression or skin aging is not fully understood. The present study demonstrated that exposure to emodin increased type I collagen synthesis in a concentration- and time-dependent manner in Hs27 human dermal fibroblasts. Subsequent experiments showed that emodin strongly increased collagen type I levels without altering cell proliferation or cellular matrix metalloproteinase-1 (MMP-1) expression. Additionally, it was determined that increased phosphorylation of 5' AMP-activated protein kinase, following emodin treatment, was responsible for increased type I collagen synthesis. These findings clearly indicate that emodin plays an important role in collagen type I synthesis in dermal fibroblasts, thereby making it a potential drug candidate for treating skin aging and wrinkles.

Improved resolution in single-molecule localization microscopy using QD-PAINT.

Single-molecule localization microscopy (SMLM) has allowed the observation of various molecular structures in cells beyond the diffraction limit using organic dyes. In principle, the SMLM resolution depends on the precision of photoswitching fluorophore localization, which is inversely correlated with the square root of the number of photons released from the individual fluorophores. Thus, increasing the photon number by using highly bright fluorophores, such as quantum dots (QDs), can theoretically fundamentally overcome the current resolution limit of SMLM. However, the use of QDs in SMLM has been challenging because QDs have no photoswitching property, which is essential for SMLM, and they exhibit nonspecificity and multivalency, which complicate their use in fluorescence imaging. Here, we present a method to utilize QDs in SMLM to surpass the resolution limit of the current SMLM utilizing organic dyes. We confer monovalency, specificity, and photoswitchability on QDs by steric exclusion via passivation and ligand exchange with ptDNA, PEG, and casein as well as by DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) via automatic thermally driven hybridization between target-bound docking and dye-bound complementary imager strands. QDs are made monovalent and photoswitchable to enable SMLM and show substantially better photophysical properties than Cy3, with higher fluorescence intensity and an improved resolution factor. QD-PAINT displays improved spatial resolution with a narrower full width at half maximum (FWHM) than DNA-PAINT with Cy3. In summary, QD-PAINT shows great promise as a next-generation SMLM method for overcoming the limited resolution of the current SMLM.

Analysis of transient membrane protein interactions by single-molecule diffusional mobility shift assay.

Various repertoires of membrane protein interactions determine cellular responses to diverse environments around cells dynamically in space and time. Current assays, however, have limitations in unraveling these interactions in the physiological states in a living cell due to the lack of capability to probe the transient nature of these interactions on the crowded membrane. Here, we present a simple and robust assay that enables the investigation of transient protein interactions in living cells by using the single-molecule diffusional mobility shift assay (smDIMSA). Utilizing smDIMSA, we uncovered the interaction profile of EGFR with various membrane proteins and demonstrated the promiscuity of these interactions depending on the cancer cell line. The transient interaction profile obtained by smDIMSA will provide critical information to comprehend the crosstalk among various receptors on the plasma membrane.

The Formosalides: Structure Determination by Total Synthesis.

Total synthesis allowed the constitution of the cytotoxic marine macrolides of the formosalide family to be confirmed and their previously unknown stereostructure to be assigned with confidence. The underlying blueprint was inherently modular to ensure that each conceivable isomer could be reached. This flexibility derived from the use of strictly catalyst controlled transformations to set the stereocenters, except for the anomeric position, which is under thermodynamic control; as an extra safety measure, all stereogenic centers were set prior to ring closure to preclude any interference of the conformation adopted by the macrolactone rings of the different diastereomers. Late-stage macrocyclization by ring-closing alkyne metathesis was followed by a platinum-catalyzed transannular 6-exo-dig hydroalkoxylation/ketalization to craft the polycyclic frame. The side chain featuring a very labile unsaturation pattern was finally attached to the core by Stille coupling.

Secretomics to Discover Regulators in Diseases.

Secretory proteins play important roles in the cross-talk of individual functional units, including cells. Since secretory proteins are essential for signal transduction, they are closely related with disease development, including metabolic and neural diseases. In metabolic diseases, adipokines, myokines, and hepatokines are secreted from respective organs under specific environmental conditions, and play roles in glucose homeostasis, angiogenesis, and inflammation. In neural diseases, astrocytes and microglia cells secrete cytokines and chemokines that play roles in neurotoxic and neuroprotective responses. Mass spectrometry-based secretome profiling is a powerful strategy to identify and characterize secretory proteins. This strategy involves stepwise processes such as the collection of conditioned medium (CM) containing secretome proteins and concentration of the CM, peptide preparation, mass analysis, database search, and filtering of secretory proteins; each step requires certain conditions to obtain reliable results. Proteomic analysis of extracellular vesicles has become a new research focus for understanding the additional extracellular functions of intracellular proteins. Here, we provide a review of the insights obtained from secretome analyses with regard to disease mechanisms, and highlight the future prospects of this technology. Continued research in this field is expected to provide valuable information on cell-to-cell communication and uncover new pathological mechanisms.

Direct visualization of single-molecule membrane protein interactions in living cells.

Interactions between membrane proteins are poorly understood despite their importance in cell signaling and drug development. Here, we present a co-immunoimmobilization assay (Co-II) enabling the direct observation of membrane protein interactions in single living cells that overcomes the limitations of currently prevalent proximity-based indirect methods. Using Co-II, we investigated the transient homodimerizations of epidermal growth factor receptor (EGFR) and beta-2 adrenergic receptor (ß2-AR) in living cells, revealing the differential regulation of these receptors' dimerizations by molecular conformations and microenvironment in a plasma membrane. Co-II should provide a simple, rapid, and robust platform for visualizing both weak and strong protein interactions in the plasma membrane of living cells.

One-Pot Generation of Bicyclo[3.1.0]hexanols and Cyclohexanones by Double Interrupted Nazarov Reactions.

Organoaluminum-mediated double interrupted Nazarov cyclization to access bicyclo[3.1.0]hexanols via nucleophilic methyl attack followed by Simmons-Smith-type electrophilic cyclopropanation is reported. These alcohols can undergo ring opening to afford cyclohexanones or cyclohexenones, broadening the range of scaffolds available via interrupted Nazarov reaction beyond the usual cyclopentanoid products. Throughout the sequence, a total of four new C-C bonds are formed, along with four new stereogenic centers.

Akkermansia muciniphila-derived extracellular vesicles influence gut permeability through the regulation of tight junctions.

The gut microbiota has an important role in the gut barrier, inflammation and metabolic functions. Studies have identified a close association between the intestinal barrier and metabolic diseases, including obesity and type 2 diabetes (T2D). Recently, Akkermansia muciniphila has been reported as a beneficial bacterium that reduces gut barrier disruption and insulin resistance. Here we evaluated the role of A. muciniphila-derived extracellular vesicles (AmEVs) in the regulation of gut permeability. We found that there are more AmEVs in the fecal samples of healthy controls compared with those of patients with T2D. In addition, AmEV administration enhanced tight junction function, reduced body weight gain and improved glucose tolerance in high-fat diet (HFD)-induced diabetic mice. To test the direct effect of AmEVs on human epithelial cells, cultured Caco-2 cells were treated with these vesicles. AmEVs decreased the gut permeability of lipopolysaccharide-treated Caco-2 cells, whereas Escherichia coli-derived EVs had no significant effect. Interestingly, the expression of occludin was increased by AmEV treatment. Overall, these results imply that AmEVs may act as a functional moiety for controlling gut permeability and that the regulation of intestinal barrier integrity can improve metabolic functions in HFD-fed mice.