Structural insights into assembly and function of GluN1-2C, GluN1-2A-2C, and GluN1-2D NMDARs.

Neurotransmission mediated by diverse subtypes of N-methyl-D-aspartate receptors (NMDARs) is fundamental for basic brain functions and development as well as neuropsychiatric diseases and disorders. NMDARs are glycine- and glutamate-gated ion channels that exist as heterotetramers composed of obligatory GluN1 and GluN2(A-D) and/or GluN3(A-B). The GluN2C and GluN2D subunits form ion channels with distinct properties and spatio-temporal expression patterns. Here, we provide the structures of the agonist-bound human GluN1-2C NMDAR in the presence and absence of the GluN2C-selective positive allosteric potentiator (PAM), PYD-106, the agonist-bound GluN1-2A-2C tri-heteromeric NMDAR, and agonist-bound GluN1-2D NMDARs by single-particle electron cryomicroscopy. Our analysis shows unique inter-subunit and domain arrangements of the GluN2C NMDARs, which contribute to functional regulation and formation of the PAM binding pocket and is distinct from GluN2D NMDARs. Our findings here provide the fundamental blueprint to study GluN2C- and GluN2D-containing NMDARs, which are uniquely involved in neuropsychiatric disorders.

Evolutionary balance between foldability and functionality of a glucose transporter.

Despite advances in resolving the structures of multi-pass membrane proteins, little is known about the native folding pathways of these complex structures. Using single-molecule magnetic tweezers, we here report a folding pathway of purified human glucose transporter 3 (GLUT3) reconstituted within synthetic lipid bilayers. The N-terminal major facilitator superfamily (MFS) fold strictly forms first, serving as a structural template for its C-terminal counterpart. We found polar residues comprising the conduit for glucose molecules present major folding challenges. The endoplasmic reticulum membrane protein complex facilitates insertion of these hydrophilic transmembrane helices, thrusting GLUT3's microstate sampling toward folded structures. Final assembly between the N- and C-terminal MFS folds depends on specific lipids that ease desolvation of the lipid shells surrounding the domain interfaces. Sequence analysis suggests that this asymmetric folding propensity across the N- and C-terminal MFS folds prevails for metazoan sugar porters, revealing evolutionary conflicts between foldability and functionality faced by many multi-pass membrane proteins.

Design, synthesis, and biological activities of 3-((4,6-diphenylpyrimidin-2-ylamino)methylene)-2,3-dihydrochromen-4-ones.

Novel (Z)-3-((4,6-diphenylpyrimidin-2-ylamino)methylene)-2,3-dihydrochromen-4-one derivatives were designed and synthesized to find chemotherapeutic agents. Derivative 9 was selected based on its clonogenicity against cancer cells and synthetic yield for further biological experiments. It showed decreases in aurora kinase A, B, and C phosphorylation from western blot analysis. Derivative 9 upregulated the expression of G1 cell cycle inhibitory proteins including p21 and p27, and G1 progressive cyclin D1, and downregulated G1-to-S progressive cyclins, resulting in cell cycle arrest at the G1/S boundary. It stimulated the cleavage of caspase-9, -3, -7, and poly (ADP-ribose) polymerase, resulting in triggering apoptosis through a caspase-dependent pathway. In addition, derivative 9 inhibited in vivo tumor growth in a syngeneic tumor implantation mouse model. The findings of this study suggest that derivative 9 can be considered as a lead compound for chemotherapeutic agents.

Mass spectrometric investigation of concentration-dependent effect of curcumin and oxidative stress on intracellular glutathione levels.

Herein, we investigated the correlation between curcumin and glutathione (GSH) levels in mammalian cells using gold nanoparticles (AuNPs) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). GSH exists in high concentration in the cytosol and acts as a major antioxidant and reducing agent in organisms. Previous studies showed that curcumin, a well-known antioxidant with anti-inflammatory, anti-proliferative, and anti-carcinogenic activities, affects GSH levels in mammalian cells. However, the correlation between their levels remains controversial and has not yet been completely elucidated. This study used our recent strategy of GSH quantification, where GSH in cell lysate is captured on maleimide groups of AuNPs and analyzed using MALDI-TOF MS with isotopomer GSH (GSH*)-conjugated AuNPs as an internal standard. The comparison between GSH and GSH* relative intensities allows the quantitation of GSH in cells. In this way, GSH levels in mammalian cells were investigated after incubation with curcumin at various concentrations with or without oxidative stress. We observed that intracellular GSH levels were affected by curcumin in a concentration-dependent manner with oxidative stress; GSH levels decrease at a lower curcumin concentration, which can be recovered at increased curcumin concentrations. We also found that the GSH level increased at all curcumin concentrations after a certain incubation time. We believe our strategy can be commonly used to determine GSH levels in cells that are treated differently with various exogenous stimulants like reactive oxygen species, biofunctional natural products, and drug candidates. Graphical abstract.

Cell-cell adhesion in metazoans relies on evolutionarily conserved features of the α-catenin·ß-catenin-binding interface.

Stable tissue integrity during embryonic development relies on the function of the cadherin·catenin complex (CCC). The Caenorhabditis elegans CCC is a useful paradigm for analyzing in vivo requirements for specific interactions among the core components of the CCC, and it provides a unique opportunity to examine evolutionarily conserved mechanisms that govern the interaction between α- and ß-catenin. HMP-1, unlike its mammalian homolog α-catenin, is constitutively monomeric, and its binding affinity for HMP-2/ß-catenin is higher than that of α-catenin for ß-catenin. A crystal structure shows that the HMP-1·HMP-2 complex forms a five-helical bundle structure distinct from the structure of the mammalian α-catenin·ß-catenin complex. Deletion analysis based on the crystal structure shows that the first helix of HMP-1 is necessary for binding HMP-2 avidly in vitro and for efficient recruitment of HMP-1 to adherens junctions in embryos. HMP-2 Ser-47 and Tyr-69 flank its binding interface with HMP-1, and we show that phosphomimetic mutations at these two sites decrease binding affinity of HMP-1 to HMP-2 by 40-100-fold in vitro. In vivo experiments using HMP-2 S47E and Y69E mutants showed that they are unable to rescue hmp-2(zu364) mutants, suggesting that phosphorylation of HMP-2 on Ser-47 and Tyr-69 could be important for regulating CCC formation in C. elegans Our data provide novel insights into how cadherin-dependent cell-cell adhesion is modulated in metazoans by conserved elements as well as features unique to specific organisms.

Structural and functional characterization of Caenorhabditis elegans α-catenin reveals constitutive binding to ß-catenin and F-actin.

Intercellular epithelial junctions formed by classical cadherins, ß-catenin, and the actin-binding protein α-catenin link the actin cytoskeletons of adjacent cells into a structural continuum. These assemblies transmit forces through the tissue and respond to intracellular and extracellular signals. However, the mechanisms of junctional assembly and regulation are poorly understood. Studies of cadherin-catenin assembly in a number of metazoans have revealed both similarities and unexpected differences in the biochemical properties of the cadherin·catenin complex that likely reflect the developmental and environmental requirements of different tissues and organisms. Here, we report the structural and biochemical characterization of HMP-1, the Caenorhabditis elegans α-catenin homolog, and compare it with mammalian α-catenin. HMP-1 shares overall similarity in structure and actin-binding properties, but displayed differences in conformational flexibility and allosteric regulation from mammalian α-catenin. HMP-1 bound filamentous actin with an affinity in the single micromolar range, even when complexed with the ß-catenin homolog HMP-2 or when present in a complex of HMP-2 and the cadherin homolog HMR-1, indicating that HMP-1 binding to F-actin is not allosterically regulated by the HMP-2·HMR-1 complex. The middle (i.e. M) domain of HMP-1 appeared to be less conformationally flexible than mammalian α-catenin, which may underlie the dampened effect of HMP-2 binding on HMP-1 actin-binding activity compared with that of the mammalian homolog. In conclusion, our data indicate that HMP-1 constitutively binds ß-catenin and F-actin, and although the overall structure and function of HMP-1 and related α-catenins are similar, the vertebrate proteins appear to be under more complex conformational regulation.

Synthesis of avenanthramides using engineered Escherichia coli.

BACKGROUND: Hydroxycinnamoyl anthranilates, also known as avenanthramides (avns), are a group of phenolic alkaloids with anti-inflammatory, antioxidant, anti-itch, anti-irritant, and antiatherogenic activities. Some avenanthramides (avn A-H and avn K) are conjugates of hydroxycinnamic acids (HC), including p-coumaric acid, caffeic acid, and ferulic acid, and anthranilate derivatives, including anthranilate, 4-hydroxyanthranilate, and 5-hydroxyanthranilate. Avns are primarily found in oat grain, in which they were originally designated as phytoalexins. Knowledge of the avns biosynthesis pathway has now made it possible to synthesize avns through a genetic engineering strategy, which would help to further elucidate their properties and exploit their beneficial biological activities. The aim of the present study was to synthesize natural avns in Escherichia coli to serve as a valuable resource. RESULTS: We synthesized nine avns in E. coli. We first synthesized avn D from glucose in E. coli harboring tyrosine ammonia lyase (TAL), 4-coumarate:coenzyme A ligase (4CL), anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT), and anthranilate synthase (trpEG). A trpD deletion mutant was used to increase the amount of anthranilate in E. coli. After optimizing the incubation temperature and cell density, approximately 317.2 mg/L of avn D was synthesized. Avn E and avn F were then synthesized from avn D, using either E. coli harboring HpaBC and SOMT9 or E. coli harboring HapBC alone, respectively. Avn A and avn G were synthesized by feeding 5-hydroxyanthranilate or 4-hydroxyanthranilate to E. coli harboring TAL, 4CL, and HCBT. Avn B, avn C, avn H, and avn K were synthesized from avn A or avn G, using the same approach employed for the synthesis of avn E and avn F from avn D. CONCLUSIONS: Using different HCs, nine avns were synthesized, three of which (avn D, avn E, and avn F) were synthesized from glucose in E. coli. These diverse avns provide a strategy to synthesize both natural and unnatural avns, setting a foundation for exploring the biological activities of diverse avns.

Preparation and evaluation of in situ photocleavable mass tags with facile mass variation for matrix-free laser desorption ionization mass spectrometry.

Mass tags have been used for the precise identification, quantification, and characterization of macrobiomolecules and small organic molecules. Existing research has not yet demonstrated the preparation of a series of trityl-based photocleavable mass tags (PMTs) with similar structures but different molecular weights and mass variability. Herein, we introduce the design and synthesis of trityl-based in situ PMTs that generate heterolytic photocleavable cationic species upon laser irradiation. Mass variation of the PMTs was achieved via a simple conjugation reaction in the final step of synthesis. We prepared a series of PMTs with similar structures but different molecular weights and performed organic matrix-free laser desorption/ionization mass spectrometry (LDI MS) analysis. The practical applicability of the PMTs was evaluated by conjugating PMTs to oligonucleotides and utilizing them for detecting specific oligonucleotide targets combined with a mass signal amplification strategy. Quantitative aspects were also evaluated to verify the capability of the mass tags for multiplexed detection and the quantification of targets. The LDI MS analysis clearly demonstrated in situ heterolytic photocleavage that formed trityl cation peaks with high S/N ratios and high sensitivity. We strongly believe that the developed mass tags and LDI MS are useful alternatives to conventional signal transduction methods used for biosensors, such as surface plasmon resonance, electrochemical redox, and fluorescence.

Ultrasensitive detection of microRNAs using nanoengineered micro gold shells and laser desorption/ionization time-of-flight MS.

We report on a simple, fast, and highly sensitive method that allows femtomolar detection of microRNAs (miRNAs) without the need of polymerase chain reaction using a nanoengineered micro gold shell (µAuS) and laser desorption/ionization time-of-flight (LDI-TOF) mass spectrometry (MS). Target miRNAs are selectively captured by a chemically decorated gold chip and a µAuS, which carries with it a large number of small molecules, termed amplification tags (Am-tags). Subsequent LDI-TOF MS analysis allows a highly amplified mass signal of Am-tags for the presence of target miRNAs in a sample, resulting in ultrasensitive detection.

Structural basis for Y2 receptor-mediated neuropeptide Y and peptide YY signaling.

Neuropeptide Y (NPY) and its receptors are expressed in various human tissues including the brain where they regulate appetite and emotion. Upon NPY stimulation, the neuropeptide Y1 and Y2 receptors (Y1R and Y2R, respectively) activate GI signaling, but their physiological responses to food intake are different. In addition, deletion of the two N-terminal amino acids of peptide YY (PYY(3-36)), the endogenous form found in circulation, can stimulate Y2R but not Y1R, suggesting that Y1R and Y2R may have distinct ligand-binding modes. Here, we report the cryo-electron microscopy structures of the PYY(3-36)‒Y2R‒Gi and NPY‒Y2R‒Gi complexes. Using cell-based assays, molecular dynamics simulations, and structural analysis, we revealed the molecular basis of the exclusive binding of PYY(3-36) to Y2R. Furthermore, we demonstrated that Y2R favors G protein signaling over ß-arrestin signaling upon activation, whereas Y1R does not show a preference between these two pathways.

Understanding the molecular mechanisms of odorant binding and activation of the human OR52 family.

Structural and mechanistic studies on human odorant receptors (ORs), key in olfactory signaling, are challenging because of their low surface expression in heterologous cells. The recent structure of OR51E2 bound to propionate provided molecular insight into odorant recognition, but the lack of an inactive OR structure limited understanding of the activation mechanism of ORs upon odorant binding. Here, we determined the cryo-electron microscopy structures of consensus OR52 (OR52cs), a representative of the OR52 family, in the ligand-free (apo) and octanoate-bound states. The apo structure of OR52cs reveals a large opening between transmembrane helices (TMs) 5 and 6. A comparison between the apo and active structures of OR52cs demonstrates the inward and outward movements of the extracellular and intracellular segments of TM6, respectively. These results, combined with molecular dynamics simulations and signaling assays, shed light on the molecular mechanisms of odorant binding and activation of the OR52 family.

Fatal systemic disorder caused by biallelic variants in FARSA.

BACKGROUND: Aminoacyl tRNA transferases play an essential role in protein biosynthesis, and variants of these enzymes result in various human diseases. FARSA, which encodes the α subunit of cytosolic phenylalanyl-tRNA synthetase, was recently reported as a suspected causal gene for multiorgan disorder. This study aimed to validate the pathogenicity of variants in the FARSA gene. RESULTS: Exome sequencing revealed novel compound heterozygous variants in FARSA, P347L and R475Q, from a patient who initially presented neonatal-onset failure to thrive, liver dysfunction, and frequent respiratory infections. His developmental milestones were nearly arrested, and the patient died at 28 months of age as a result of progressive hepatic and respiratory failure. The P347L variant was predicted to disrupt heterodimer interaction and failed to form a functional heterotetramer by structural and biochemical analyses. R475 is located at a highly conserved site and is reported to be involved in phenylalanine activation and transfer to tRNA. The R475Q mutant FARSA were co-purified with FARSB, but the mutant enzyme showed an approximately 36% reduction in activity in our assay relative to the wild-type protein. Additional functional analyses on variants from previous reports (N410K, F256L, R404C, E418D, and F277V) were conducted. The R404C variant from a patient waiting for organ transplantation also failed to form tetramers but the E418D, N410K, F256L, and F277V variants did not affect tetramer formation. In the functional assay, the N410K located at the phenylalanine-binding site exhibited no catalytic activity, whereas other variants (E418D, F256L and F277V) exhibited lower ATPase activity than wild-type FARSA at low phenylalanine concentrations. CONCLUSIONS: Our data demonstrated the pathogenicity of biallelic variants in FARSA and suggested the implication of hypomorphic variants in severe phenotypes.

Structural basis of neuropeptide Y signaling through Y1 receptor.

Neuropeptide Y (NPY) is highly abundant in the brain and involved in various physiological processes related to food intake and anxiety, as well as human diseases such as obesity and cancer. However, the molecular details of the interactions between NPY and its receptors are poorly understood. Here, we report a cryo-electron microscopy structure of the NPY-bound neuropeptide Y1 receptor (Y1R) in complex with Gi1 protein. The NPY C-terminal segment forming the extended conformation binds deep into the Y1R transmembrane core, where the amidated C-terminal residue Y36 of NPY is located at the base of the ligand-binding pocket. Furthermore, the helical region and two N-terminal residues of NPY interact with Y1R extracellular loops, contributing to the high affinity of NPY for Y1R. The structural analysis of NPY-bound Y1R and mutagenesis studies provide molecular insights into the activation mechanism of Y1R upon NPY binding.

Conformational Dynamics and Functional Implications of Phosphorylated ß-Arrestins.

Arrestins desensitize and/or internalize G-protein-coupled receptors by interacting with phosphorylated receptors. A few studies have reported that arrestins themselves can be phosphorylated, and the phosphorylation status modulates their cellular functions. However, the effects of phosphorylation on arrestin structure have not been studied. Here, we investigated the conformational changes in ß-arrestin-1 and -2 upon incorporation of phospho-mimetic mutations into the known phosphorylation sites (i.e., S412D for ß-arrestin-1 and S14D, T276D, S14D/T276D, S361D, T383D, and S361D/T383D for ß-arrestin-2) by using hydrogen/deuterium-exchange mass spectrometry (HDX-MS). HDX-MS analysis suggested that ß-arrestin-2 S14D/T276D shows an HDX profile similar to the pre-active states, resulting in increased interaction with receptors. Phospho-mimetic mutation at corresponding residues of ß-arrestin-1 (i.e., S13D/T275D) induced similar conformational and functional consequences, and the detailed structural changes related to ß-arrestin-1 S13D/T275D were investigated further by X-ray crystallography.

Watching helical membrane proteins fold reveals a common N-to-C-terminal folding pathway.

To understand membrane protein biogenesis, we need to explore folding within a bilayer context. Here, we describe a single-molecule force microscopy technique that monitors the folding of helical membrane proteins in vesicle and bicelle environments. After completely unfolding the protein at high force, we lower the force to initiate folding while transmembrane helices are aligned in a zigzag manner within the bilayer, thereby imposing minimal constraints on folding. We used the approach to characterize the folding pathways of the Escherichia coli rhomboid protease GlpG and the human ß2-adrenergic receptor. Despite their evolutionary distance, both proteins fold in a strict N-to-C-terminal fashion, accruing structures in units of helical hairpins. These common features suggest that integral helical membrane proteins have evolved to maximize their fitness with cotranslational folding.

Cell-Based Screen Using Amyloid Mimic ß23 Expression Identifies Peucedanocoumarin III as a Novel Inhibitor of α-Synuclein and Huntingtin Aggregates.

Aggregates of disease-causing proteins dysregulate cellular functions, thereby causing neuronal cell loss in diverse neurodegenerative diseases. Although many in vitro or in vivo studies of protein aggregate inhibitors have been performed, a therapeutic strategy to control aggregate toxicity has not been earnestly pursued, partly due to the limitations of available aggregate models. In this study, we established a tetracycline (Tet)-inducible nuclear aggregate (ß23) expression model to screen potential lead compounds inhibiting ß23-induced toxicity. Highthroughput screening identified several natural compounds as nuclear ß23 inhibitors, including peucedanocoumarin III (PCIII). Interestingly, PCIII accelerates disaggregation and proteasomal clearance of both nuclear and cytosolic ß23 aggregates and protects SH-SY5Y cells from toxicity induced by ß23 expression. Of translational relevance, PCIII disassembled fibrils and enhanced clearance of cytosolic and nuclear protein aggregates in cellular models of huntingtin and α-synuclein aggregation. Moreover, cellular toxicity was diminished with PCIII treatment for polyglutamine (PolyQ)-huntingtin expression and α-synuclein expression in conjunction with 6-hydroxydopamine (6-OHDA) treatment. Importantly, PCIII not only inhibited α-synuclein aggregation but also disaggregated preformed α-synuclein fibrils in vitro . Taken together, our results suggest that a Tet-Off ß23 cell model could serve as a robust platform for screening effective lead compounds inhibiting nuclear or cytosolic protein aggregates. Brain-permeable PCIII or its derivatives could be beneficial for eliminating established protein aggregates.

Efficient Enrichment and Analysis of Vicinal-Diol-Containing Flavonoid Molecules Using Boronic-Acid-Functionalized Particles and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

Detection and quantitation of flavonoids are relatively difficult compared to those of other small-molecule analytes because flavonoids undergo rapid metabolic processes, resulting in their elimination from the body. Here, we report an efficient enrichment method for facilitating the analysis of vicinal-diol-containing flavonoid molecules using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In our strategy, boronic-acid-functionalized polyacrylamide particles were used, where boronic acids bound to vicinal diols to form boronate monoesters at basic pH. This complex remained intact during the enrichment processes, and the vicinal-diol-containing flavonoids were easily separated by centrifugation and subsequent acidic treatments. The selectivity and limit of detection of our strategy were confirmed by mass spectrometry analysis, and the validity was assessed by performing the detection and quantitation of quercetin in mouse organs.

On-Demand Modulation of Bacterial Cell Fates on Multifunctional Dynamic Substrates.

This paper reports unprecedented dynamic surfaces based on zwitterionic low-density self-assembled monolayers (LDSAMs) of alkanethiolates on gold, which integrate three interconvertible states-bacteria-adherable, bactericidal, and nonfouling states-through electrical modulations. The conformations of alkanethiolates were electrically modulated to generate zwitterionic, anionic, and cationic surfaces, which responded differently to bacteria and determined the fate of bacteria. Furthermore, the reversible switching of multifunctions of the surface was realized for killing bacteria and subsequently releasing dead bacteria from the surface. For practical application of our strategy, we examined the selective antibacterial effect of our surface for eradication of mycoplasma contaminants in contaminated mammalian cell cultures.

Combination of Mass Signal Amplification and Isotope-Labeled Alkanethiols for the Multiplexed Detection of miRNAs.

We report a fast and sensitive method for the multiplexed detection of miRNAs by combining mass signal amplification and isotope-labeled signal reporter molecules. In our strategy, target miRNAs are captured specifically by immobilized DNAs on gold nanoparticles (AuNPs), which carry a large number of small molecules, called amplification tags (Am-tags), as the reporter for the detection of target miRNAs. For multiplexed detection, we designed and synthesized four Am-tags containing 0, 4, 8, 12 isotopes so that they had same molecular properties but different molecular weights. By observing the mass signals of the Am-tags on AuNPs decorated along with different probe DNAs, four types of miRNAs in a sample could be easily discriminated, and the relative amounts of these miRNAs could be quantified. The practicability of our strategy was further verified by measuring the expression levels of two miRNAs in HUVECs in response to different CuSO4 concentrations.

Analysis of small biomolecules and xenobiotic metabolism using converted graphene-like monolayer plates and laser desorption/ionization time-of-flight mass spectrometry.

We report a method of small molecule analysis using a converted graphene-like monolayer (CGM) plate and laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS) without organic matrices. The CGM plate was prepared from self-assembled monolayers of biphenyl-4-thiol on gold using electron beam irradiation followed by an annealing step. The above plate was utilized for the LDI-TOF MS analyses of various small molecules and their mixtures, e.g., amino acids, sugars, fatty acids, oligoethylene glycols, and flavonoids. The CGM plate afforded high signal-to-noise ratios, good limits of detection (1pmol to 10fmol), and reusability for up to 30 cycles. As a practical application, the enzymatic activity of the cytochrome P450 2A6 (CYP2A6) enzyme in human liver microsomes was assessed in the 7-hydroxylation of coumarin using the CGM plate without other purification steps. We believe that the prepared CGM plate can be practically used with the advantages of simplicity, sensitivity, and reusability for the matrix-free analysis of small biomolecules.