The powerful potential of amino acid menthyl esters for anti-inflammatory and anti-obesity therapies.

Our newly developed menthyl esters of valine and isoleucine exhibit anti-inflammatory properties beyond those of the well-known menthol in macrophages stimulated by lipopolysaccharide (LPS) and in a mouse model of colitis induced by sodium dextran sulfate. Unlike menthol, which acts primarily through the cold-sensitive TRPM8 channel, these menthyl esters displayed unique mechanisms that operate independently of this receptor. They readily penetrated target cells and efficiently suppressed LPS-stimulated tumour necrosis factor-alpha (Tnf) expression mediated by liver X receptor (LXR), a key nuclear receptor that regulates intracellular cholesterol and lipid balance. The menthyl esters showed affinity for LXR and enhanced the transcriptional activity through their non-competitive and potentially synergistic agonistic effect. This effect can be attributed to the crucial involvement of SCD1, an enzyme regulated by LXR, which is central to lipid metabolism and plays a key role in the anti-inflammatory response. In addition, we discovered that the menthyl esters showed remarkable efficacy in suppressing adipogenesis in 3T3-L1 adipocytes at the mitotic clonal expansion stage in an LXR-independent manner as well as in mice subjected to diet-induced obesity. These multiple capabilities of our compounds establish them as formidable allies in the fight against inflammation and obesity, paving the way for a range of potential therapeutic applications.

Production and characterization of an Fv-clasp of rheumatoid factor, a low-affinity human autoantibody.

Rheumatoid factor (RF) is an autoantibody against IgG that affects autoimmune diseases and inhibits the effectiveness of pharmaceuticals and diagnostic agents. Although RFs derived from various germline genes have been identified, little is known about their molecular recognition mechanisms. In this study, the Fv-clasp format was used to prepare YES8c, an RF. We developed an Escherichia coli secretion expression system capable of producing milligram-scale of YES8c Fv-clasp per 1 L of culture. Although YES8c is an autoantibody with very low affinity, the produced Fv-clasp maintained specific binding to IgG. Interestingly, the molecules prepared by E. coli secretion had a higher affinity than those prepared by refolding. In the structure of the YES8c-Fc complex, the N-terminus of the light chain is close to Fc; therefore, it is suggested that the addition of the N-terminal methionine may cause collisions with Fc, resulting in reduced affinity. Our findings suggest that the Fv-clasp, which provides sufficient stability and a high bacterial yield, is a useful format for studying RFs with very low affinity. Furthermore, the Fv-clasp produced from a secretion expression system, which can properly process the N-terminus, would be suitable for analysis of RFs in which the N-terminus may be involved in interactions.

Recovery of the histamine H3 receptor activity lost in yeast cells through error-prone PCR and in vivo selection.

G protein-coupled receptors (GPCRs) are the largest protein family in humans and are important drug targets. Yeast, especially Saccharomyces cerevisiae, is a useful host for modifying the function and stability of GPCRs through protein engineering, which is advantageous for mammalian cells. When GPCRs are expressed in yeast, their function is often impaired. In this study, we performed random mutagenesis using error-prone PCR and then an in vivo screening to obtain mutants that recovered the activity of the human histamine H3 receptor (H3R), which loses its signaling function when expressed in yeast. Four mutations with recovered activity were identified after screening. Three of the mutations were identified near the DRY and NPxxY motifs of H3R, which are important for activation and are commonly found in class A GPCRs. The mutants responded exclusively to the yeast YB1 strain harboring Gi-chimera proteins, showing retention of G protein specificity. Analysis of one of the mutants with recovered activity, C415R, revealed that it maintained its ligand-binding characteristics. The strategy used in this study may enable the recovery of the activity of other GPCRs that do not function in S. cerevisiae and may be useful in creating GPCRs mutants stabilized in their active conformations.

Anti-nanodisc antibodies specifically capture nanodiscs and facilitate molecular interaction kinetics studies for membrane protein.

Nanodisc technology has dramatically advanced the analysis of molecular interactions for membrane proteins. A nanodisc is designed as a vehicle for membrane proteins that provide a native-like phospholipid environment and better thermostability in a detergent-free buffer. This enables the determination of the thermodynamic and kinetic parameters of small molecule binding by surface plasmon resonance. In this study, we generated a nanodisc specific anti-MSP (membrane scaffold protein) monoclonal antibody biND5 for molecular interaction analysis of nanodiscs. The antibody, biND5 bound to various types of nanodiscs with sub-nanomolar to nanomolar affinity. Epitope mapping analysis revealed specific recognition of 8 amino acid residues in the exposed helix-4 structure of MSP. Further, we performed kinetics binding analysis between adenosine A2a receptor reconstituted nanodiscs and small molecule antagonist ZM241385 using biND5 immobilized sensor chips. These results show that biND5 facilitates the molecular interaction kinetics analysis of membrane proteins substituted in nanodiscs.

Genome destabilization-associated phenotypes arising as a consequence of therapeutic treatment are suppressed by Olaparib.

Malignancy is often associated with therapeutic resistance and metastasis, usually arising after therapeutic treatment. These include radio- and chemo-therapies, which cause cancer cell death by inducing DNA double strand breaks (DSBs). However, it is still unclear how resistance to these DSBs is induced and whether it can be suppressed. Here, we show that DSBs induced by camptothecin (CPT) and radiation jeopardize genome stability in surviving cancer cells, ultimately leading to the development of resistance. Further, we show that cytosolic DNA, accumulating as a consequence of genomic destabilization, leads to increased cGAS/STING-pathway activation and, ultimately, increased cell migration, a precursor of metastasis. Interestingly, these genomic destabilization-associated phenotypes were suppressed by the PARP inhibitor Olaparib. Recognition of DSBs by Rad51 and genomic destabilization were largely reduced by Olaparib, while the DNA damage response and cancer cell death were effectively increased. Thus, Olaparib decreases the risk of therapeutic resistance and cell migration of cells that survive radio- and CPT-treatments.

A methodology for creating mutants of G-protein coupled receptors stabilized in active state by combining statistical thermodynamics and evolutionary molecular engineering.

We challenged the stabilization of a G-protein coupled receptor (GPCR) in the active state solely by multiple amino-acid mutations without the agonist binding. For many GPCRs, the free energy of the active state is higher than that of the inactive state. When the inactive state is stabilized through the lowering of its free energy, the apparent midpoint temperature of thermal denaturation Tm exhibits a significant increase. However, this is not always the case for the stabilization of the active state. We constructed a modified version of our methodology combining statistical thermodynamics and evolutionary molecular engineering, which was recently developed for the inactive state. First, several residues to be mutated are determined using our statistical-thermodynamics theory. Second, a gene (mutant) library is constructed using Escherichia coli cells to efficiently explore most of the mutational space. Third, for the mutant screening, the mutants prepared in accordance with the library are expressed in engineered Saccharomyces cerevisiae YB14 cells which can grow only when a GPCR mutant stabilized in the active state has signaling function. For the adenosine A2A receptor tested, the methodology enabled us to sort out two triple mutants and a double mutant. It was experimentally corroborated that all the mutants exhibit much higher binding affinity for G protein than the wild type. Analyses indicated that the mutations make the structural characteristics shift toward those of the active state. However, only slight increases in Tm resulted from the mutations, suggesting the unsuitability of Tm to the stability measure for the active state.

Improvement of the affinity of an anti-rat P2X4 receptor antibody by introducing electrostatic interactions.

We have recently developed a mouse monoclonal antibody (12-10H) binding to the head domain region in rat P2X4 receptor (rP2X4R, which is crucial for the pathogenesis of neuropathic pain) expressed on the cell with the highest binding affinity (KD = 20 nM). However, the 12-10H antibody failed to detect endogenously expressed P2X4Rs in microglia isolated from the spinal cord of rats whose spinal nerves were injured. Then, we prepared R5 mutant, in which five arginine residues were introduced into variable regions except for the "hot spot" in the 12-10H antibody to increase electrostatic interactions with the head domain, an anionic region, in rP2X4R. The mutation resulted in an increase of 50-fold in the affinity of the R5 mutant for the head domain with respect to the intact 12-10H antibody. As a result, detection of P2X4Rs endogenously expressed on primary cultured microglial cells originated from the neonatal rat brain and spinal cord microglia isolated from a rat model of neuropathic pain was achieved. These findings suggest a strategy to improve the affinity of a monoclonal antibody for an anionic antigen by the introduction of several arginine residues into variable regions other than the "hot spot" in the paratope.

Structural Basis of a Conventional Recognition Mode of IGHV1-69 Rheumatoid Factors.

Rheumatoid factors (RFs) are autoantibodies that recognize the fragment crystallizable (Fc) region of immunoglobulin G (IgG). Genetically diverse RFs are produced in rheumatoid arthritis patients; however, in hematologic diseases, such as cryoglobulinemia and B cell lymphoma, RFs from a limited combination of heavy chain V-region genes and J-region genes are produced in large quantities and forms immune complexes with IgG. These genetically limited RFs have historically been used for the immunochemical characterization of RFs. Among them, RFs derived from the heavy-chain germline gene IGHV1-69 are the most common. Recently, the crystal structure of an IGHV1-69-derived RF named YES8c was elucidated in complex with human IgG1-Fc. Based on the structure and mutant analyses, a recognition mechanism for the autoantigen (IgG-Fc) common to IGHV1-69-derived RFs was proposed. This review summarizes the immunochemical character of the IGHV1-69-derived RFs, and then focuses on the recognition mechanism of the IGHV1-69-derived RFs, referring the structural features of the IGHV1-69-derived neutralizing antibodies.

An affinity change model to elucidate the rotation mechanism of V1-ATPase.

V-ATPases are ubiquitous proton-transporting ATPases of eukaryotic and prokaryotic membranes that utilize energy from ATP hydrolysis. The hydrophilic catalytic part called V1-ATPase is composed of a ring-shaped hexametric A3B3 complex and a central DF shaft. We previously proposed a rotation mechanism of the Enterococcus hirae V1-ATPase based on the crystal structures of the V1 and A3B3 complexes. However, the driving force that induces the conformational changes of A3B3 and rotation of the DF shaft remains unclear. In this study, we investigated the binding affinity changes between subunits of V1-ATPase by surface plasmon resonance analysis. The binding of ATP to subunit A was found to considerably increase the affinity between the A and B subunits, and thereby ATP binding contributes to forming the A1B1 tight conformation. Furthermore, the DF shaft bound to the reconstituted A1B1 complex with high affinity, suggesting that the tight A1B1 complex is a major binding unit of the shaft in the A3B3 ring complex. Based on these results, we propose that rotation of the V1-ATPase is driven by affinity changes between each subunit via thermal fluctuations.

Structural and Functional Basis for LILRB Immune Checkpoint Receptor Recognition of HLA-G Isoforms.

Human leukocyte Ig-like receptors (LILR) LILRB1 and LILRB2 are immune checkpoint receptors that regulate a wide range of physiological responses by binding to diverse ligands, including HLA-G. HLA-G is exclusively expressed in the placenta, some immunoregulatory cells, and tumors and has several unique isoforms. However, the recognition of HLA-G isoforms by LILRs is poorly understood. In this study, we characterized LILR binding to the ß2-microglobulin (ß2m)-free HLA-G1 isoform, which is synthesized by placental trophoblast cells and tends to dimerize and multimerize. The multimerized ß2m-free HLA-G1 dimer lacked detectable affinity for LILRB1, but bound strongly to LILRB2. We also determined the crystal structure of the LILRB1 and HLA-G1 complex, which adopted the typical structure of a classical HLA class I complex. LILRB1 exhibits flexible binding modes with the α3 domain, but maintains tight contacts with ß2m, thus accounting for ß2m-dependent binding. Notably, both LILRB1 and B2 are oriented at suitable angles to permit efficient signaling upon complex formation with HLA-G1 dimers. These structural and functional features of ligand recognition by LILRs provide novel insights into their important roles in the biological regulations.

Selective and reversible modification of kinase cysteines with chlorofluoroacetamides.

Irreversible inhibition of disease-associated proteins with small molecules is a powerful approach for achieving increased and sustained pharmacological potency. Here, we introduce α-chlorofluoroacetamide (CFA) as a novel warhead of targeted covalent inhibitor (TCI). Despite weak intrinsic reactivity, CFA-appended quinazoline showed high reactivity toward Cys797 of epidermal growth factor receptor (EGFR). In cells, CFA-quinazoline showed higher target specificity for EGFR than the corresponding Michael acceptors in a wide concentration range (0.1-10 µM). The cysteine adduct of the CFA derivative was susceptible to hydrolysis and reversibly yielded intact thiol but was stable in solvent-sequestered ATP-binding pocket of EGFR. This environment-dependent hydrolysis can potentially reduce off-target protein modification by CFA-based drugs. Oral administration of CFA quinazoline NS-062 significantly suppressed tumor growth in a mouse xenograft model. Further, CFA-appended pyrazolopyrimidine irreversibly inhibited Bruton's tyrosine kinase with higher target specificity. These results demonstrate the utility of CFA as a new class warheads for TCI.

Evidence for detection of rat P2X4 receptor expressed on cells by generating monoclonal antibodies recognizing the native structure.

P2X purinergic receptors are ATP-driven ionic channels expressed as trimers and showing various functions. A subtype, the P2X4 receptor present on microglial cells is highly involved in neuropathic pain. In this study, in order to prepare antibodies recognizing the native structure of rat P2X4 (rP2X4) receptor, we immunized mice with rP2X4's head domain (rHD, Gln111-Val167), which possesses an intact structure stabilized by S-S bond formation (Igawa and Abe et al. FEBS Lett. 2015), as an antigen. We generated five monoclonal antibodies with the ability to recognize the native structure of its head domain, stabilized by S-S bond formation. Site-directed mutagenesis revealed that Asn127 and Asp131 of the rHD, in which combination of these amino acid residues is only conserved in P2X4 receptor among P2X family, were closely involved in the interaction between rHD and these antibodies. We also demonstrated the antibodies obtained here could detect rP2X4 receptor expressed in 1321N1 human astrocytoma cells.

Insight into the interaction between PriB and DnaT on bacterial DNA replication restart: Significance of the residues on PriB dimer interface and highly acidic region on DnaT.

When the replisome collapses at a DNA damage site, a sequence-independent replication restart system is required. In Escherichia coli, PriA, PriB, and DnaT assemble in an orderly fashion at the stalled replication fork and achieve the reloading of the replisome. PriB-DnaT interaction is considered a significant step in the replication restart. In this study, we examined the contribution of the residues Ser20, His26 and Ser55, which are located on the PriB dimer interface. These residues are proximal to Glu39 and Arg44, which are important for PriB-DnaT interaction. Mutational analyses revealed that His26 and Ser20 of PriB are important for the interaction with DnaT, and that the Ser55 residue of PriB might have a role in negatively regulating the DnaT binding. These residues are involved in not only the interaction between PriB and DnaT but also the dissociation of single-stranded DNA (ssDNA) from the PriB-ssDNA complex due to DnaT binding. Moreover, NMR study indicates that the region Asp66-Glu76 on the linker between DnaT domains is involved in the interaction with wild-type PriB. These findings provide significant information about the molecular mechanism underlying replication restart in bacteria.

Ligand binding to human prostaglandin E receptor EP4 at the lipid-bilayer interface.

Prostaglandin E receptor EP4, a G-protein-coupled receptor, is involved in disorders such as cancer and autoimmune disease. Here, we report the crystal structure of human EP4 in complex with its antagonist ONO-AE3-208 and an inhibitory antibody at 3.2 Å resolution. The structure reveals that the extracellular surface is occluded by the extracellular loops and that the antagonist lies at the interface with the lipid bilayer, proximal to the highly conserved Arg316 residue in the seventh transmembrane domain. Functional and docking studies demonstrate that the natural agonist PGE2 binds in a similar manner. This structural information also provides insight into the ligand entry pathway from the membrane bilayer to the EP4 binding pocket. Furthermore, the structure reveals that the antibody allosterically affects the ligand binding of EP4. These results should facilitate the design of new therapeutic drugs targeting both orthosteric and allosteric sites in this receptor family.

Crystal structure of the human angiotensin II type 2 receptor bound to an angiotensin II analog.

Angiotensin II (AngII) plays a central role in regulating human blood pressure, which is mainly mediated by interactions between AngII and the G-protein-coupled receptors (GPCRs) AngII type 1 receptor (AT1R) and AngII type 2 receptor (AT2R). We have solved the crystal structure of human AT2R binding the peptide ligand [Sar1, Ile8]AngII and its specific antibody at 3.2-Å resolution. [Sar1, Ile8]AngII interacts with both the 'core' binding domain, where the small-molecule ligands of AT1R and AT2R bind, and the 'extended' binding domain, which is equivalent to the allosteric modulator binding site of muscarinic acetylcholine receptor. We generated an antibody fragment to stabilize the extended binding domain that functions as a positive allosteric modulator. We also identified a signature positively charged cluster, which is conserved among peptide-binding receptors, to locate C termini at the bottom of the binding pocket. The reported results should help with designing ligands for angiotensin receptors and possibly to other peptide GPCRs.

Structure-function analyses of a stereotypic rheumatoid factor unravel the structural basis for germline-encoded antibody autoreactivity.

Rheumatoid factors (RFs) are autoantibodies against the fragment-crystallizable (Fc) region of IgG. In individuals with hematological diseases such as cryoglobulinemia and certain B cell lymphoma forms, the RFs derived from specific heavy- and light-chain germline pairs, so-called "stereotypic RFs," are frequently produced in copious amounts and form immune complexes with IgG in serum. Of note, many structural details of the antigen recognition mechanisms in RFs are unclear. Here we report the crystal structure of the RF YES8c derived from the IGHV1-69/IGKV3-20 germline pair, the most common of the stereotypic RFs, in complex with human IgG1-Fc at 2.8 Å resolution. We observed that YES8c binds to the CH2-CH3 elbow in the canonical antigen-binding manner involving a large antigen-antibody interface. On the basis of this observation, combined with mutational analyses, we propose a recognition mechanism common to IGHV1-69/IGKV3-20 RFs: (1) the interaction of the Leu432-His435 region of Fc enables the highly variable complementarity-determining region (CDR)-H3 to participate in the binding, (2) the hydrophobic tip in the CDR-H2 typical of IGHV1-69 antibodies recognizes the hydrophobic patch on Fc, and (3) the interaction of the highly conserved RF light chain with Fc is important for RF activity. These features may determine the putative epitope common to the IGHV1-69/IGKV3-20 RFs. We also showed that some mutations in the binding site of RF increase the affinity to Fc, which may aggravate hematological diseases. Our findings unravel the structural basis for germline-encoded antibody autoreactivity.

Structural Analysis of the Histamine H1 Receptor.

The crystal structure of the human histamine H1 receptor (H1R) has been determined in complex with its inverse agonist doxepin, a first-generation antihistamine. The crystal structure showed that doxepin sits deeply inside the ligand-binding pocket and predominantly interacts with residues highly conserved among other aminergic receptors. This binding mode is considered to result in the low selectivity of the first-generation antihistamines for H1R. The crystal structure also revealed the mechanism of receptor inactivation by the inverse agonist doxepin. On the other hand, the crystal structure elucidated the anion-binding site near the extracellular portion of the receptor. This site consists of residues not conserved among other aminergic receptors, which are specific for H1R. Docking simulation and biochemical experimentation demonstrated that a carboxyl group on the second-generation antihistamines interacts with the anion-binding site. These results imply that the anion-binding site is a key site for the development of highly selective antihistamine drugs.

Trisaccharide containing α2,3-linked sialic acid is a receptor for mumps virus.

Mumps virus (MuV) remains an important pathogen worldwide, causing epidemic parotitis, orchitis, meningitis, and encephalitis. Here we show that MuV preferentially uses a trisaccharide containing α2,3-linked sialic acid in unbranched sugar chains as a receptor. Crystal structures of the MuV attachment protein hemagglutinin-neuraminidase (MuV-HN) alone and in complex with the α2,3-sialylated trisaccharide revealed that in addition to the interaction between the MuV-HN active site residues and sialic acid, other residues, including an aromatic residue, stabilize the third sugar of the trisaccharide. The importance of the aromatic residue and the third sugar in the MuV-HN-receptor interaction was confirmed by computational energy calculations, isothermal titration calorimetry studies, and glycan-binding assays. Furthermore, MuV-HN was found to bind more efficiently to unbranched α2,3-sialylated sugar chains compared with branched ones. Importantly, the strategically located aromatic residue is conserved among the HN proteins of sialic acid-using paramyxoviruses, and alanine substitution compromised their ability to support cell-cell fusion. These results suggest that not only the terminal sialic acid but also the adjacent sugar moiety contribute to receptor function for mumps and these paramyxoviruses. The distribution of structurally different sialylated glycans in tissues and organs may explain in part MuV's distinct tropism to glandular tissues and the central nervous system. In the crystal structure, the epitopes for neutralizing antibodies are located around the α-helices of MuV-HN that are not well conserved in amino acid sequences among different genotypes of MuV. This may explain the fact that MuV reinfection sometimes occurs.

Micro-scale and rapid expression screening of highly expressed and/or stable membrane protein variants in Saccharomyces cerevisiae.

Purification of milligram quantities of target proteins is required for structural and biophysical studies. However, mammalian membrane proteins, many of which are important therapeutic targets, are too unstable to be expressed in heterologous hosts and to be solubilized by detergents. One of the most promising ways to overcome these limitations is to stabilize the membrane proteins by generating variants via introduction of truncated flexible regions, fusion partners, and site-directed mutagenesis. Therefore, an effective screening strategy is a key to obtaining successful protein stabilization. Herein, we report the micro-scale and high-throughput screening of stabilized membrane protein variants using Saccharomyces cerevisiae as a host. All steps of the screening, including cultivation and disruption of cells, solubilization of the target protein, and the pretreatment for fluorescence-detected size exclusion chromatography (FSEC), could be performed in a 96-well microplate format. We demonstrated that the dispersion among wells was small, enabling detection of a small but important improvement in the protein stability. We also demonstrated that the thermally stable mutants of a human G protein-coupled receptor could be distinguished based on an increase of the peak height in the FSEC profile, which was well correlated with increased ligand binding activity of the protein. This strategy represents a significant platform for handling numerous mutants, similar to alanine scanning.

Screening of stable G-protein-coupled receptor variants in Saccharomyces cerevisiae.

G-protein-coupled receptors (GPCRs) are not only the largest protein family, but as a whole, they represent the largest group of therapeutic drug targets. Recent successes in the determination of GPCR structures have relied on the stabilization of receptors to overcome the difficulties in expression and purification. Although a large quantity of purified protein is needed for structural determination, the majority of wild-type GPCRs are too unstable to express and purify on a large scale. Therefore, rapid screening of highly expressed stable receptor "variants" is crucial. It has been demonstrated that fusing green fluorescent protein (GFP) to a target membrane protein facilitates the evaluation of the physical properties of the membrane protein in detergent. Furthermore, the budding yeast Saccharomyces cerevisiae enables rapid construction of an expression vector via its own efficient homologous recombination system. Herein, we describe the protocols for rapid construction and screening of stable GPCR variants using GFP and S. cerevisiae.