RESUMO
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.
Assuntos
Anti-Inflamatórios , Fármacos Antiobesidade , Receptores X do Fígado , Obesidade , Animais , Camundongos , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Receptores X do Fígado/metabolismo , Receptores X do Fígado/agonistas , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Fármacos Antiobesidade/farmacologia , Fármacos Antiobesidade/uso terapêutico , Adipogenia/efeitos dos fármacos , Ésteres/química , Colite/tratamento farmacológico , Colite/induzido quimicamente , Colite/metabolismo , Humanos , Mentol/farmacologia , Camundongos Endogâmicos C57BL , Lipopolissacarídeos , Fator de Necrose Tumoral alfa/metabolismo , Células 3T3-L1 , Sulfato de Dextrana , Adipócitos/metabolismo , Adipócitos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Canais de Cátion TRPM/metabolismoRESUMO
Doxepin is an antihistamine and tricyclic antidepressant that binds to the histamine H1 receptor (H1R) with high affinity. Doxepin is an 85:15 mixture of the E- and Z-isomers. The Z-isomer is well known to be more effective than the E-isomer, whereas based on the crystal structure of the H1R/doxepin complex, the hydroxyl group of Thr1123.37 is close enough to form a hydrogen bond with the oxygen atom of the E-isomer. The detailed binding characteristics and reasons for the differences remain unclear. In this study, we analyzed doxepin isomers bound to the receptor following extraction from a purified H1R protein complexed with doxepin. The ratio of the E- and Z-isomers bound to wild-type (WT) H1R was 55:45, indicating that the Z-isomer was bound to WT H1R with an approximately 5.2-fold higher affinity than the E-isomer. For the T1123.37V mutant, the E/Z ratio was 89:11, indicating that both isomers have similar affinities. Free energy calculations using molecular dynamics (MD) simulations also reproduced the experimental results of the relative binding free energy differences between the isomers for WT and T1123.37V. Furthermore, MD simulations revealed that the hydroxyl group of T1123.37 did not form hydrogen bonds with the E-isomer, but with the adjacent residues in the binding pocket. Analysis of the receptor-bound doxepin and MD simulations suggested that the hydroxyl group of T1123.37 contributes to the formation of a chemical environment in the binding pocket, which is slightly more favorable for the Z-isomer without hydrogen bonding with doxepin.
Assuntos
Doxepina , Simulação de Dinâmica Molecular , Ligação Proteica , Receptores Histamínicos H1 , Doxepina/química , Doxepina/metabolismo , Receptores Histamínicos H1/química , Receptores Histamínicos H1/metabolismo , Humanos , Ligantes , Ligação de Hidrogênio , Isomerismo , Sítios de Ligação , TermodinâmicaRESUMO
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.
Assuntos
Receptores de Prostaglandina E Subtipo EP4/química , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Regulação Alostérica , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Sítios de Ligação , Caprilatos/química , Caprilatos/metabolismo , Cristalografia por Raios X , Epoprostenol/análogos & derivados , Epoprostenol/química , Epoprostenol/metabolismo , Humanos , Ligantes , Bicamadas Lipídicas , Simulação de Acoplamento Molecular , Naftalenos/química , Naftalenos/metabolismo , Éteres Fenílicos/química , Éteres Fenílicos/metabolismo , Fenilbutiratos/química , Fenilbutiratos/metabolismo , Receptores de Prostaglandina E Subtipo EP4/antagonistas & inibidores , Receptores de Prostaglandina E Subtipo EP4/genética , Spodoptera/genéticaRESUMO
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.
Assuntos
Acetamidas/síntese química , Cisteína/metabolismo , Quinazolinas/síntese química , Acetamidas/química , Acetamidas/farmacologia , Animais , Antineoplásicos , Linhagem Celular , Receptores ErbB , Humanos , Camundongos , Camundongos Nus , Neoplasias , Fosfotransferases/fisiologia , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/antagonistas & inibidores , Quinazolinas/química , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Antígenos HLA-G/química , Modelos Moleculares , Conformação Proteica , Receptores Imunológicos/química , Sítios de Ligação , Antígenos HLA-G/genética , Antígenos HLA-G/imunologia , Humanos , Ligantes , Simulação de Dinâmica Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Ligação Proteica , Isoformas de Proteínas , Receptores Imunológicos/metabolismo , Relação Estrutura-Atividade , Microglobulina beta-2/química , Microglobulina beta-2/metabolismoRESUMO
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.
Assuntos
Artrite Reumatoide , Fator Reumatoide , Complexo Antígeno-Anticorpo , Autoanticorpos , Humanos , Imunoglobulina GRESUMO
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.
Assuntos
ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/metabolismo , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Modelos Moleculares , Conformação Proteica , Subunidades Proteicas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Rotação , Ressonância de Plasmônio de Superfície , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
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.
Assuntos
Autoanticorpos/química , Autoanticorpos/imunologia , Células Germinativas , Fator Reumatoide/metabolismo , Substituição de Aminoácidos , Afinidade de Anticorpos , Sítios de Ligação , Regiões Determinantes de Complementaridade , Cristalografia por Raios X , Epitopos/imunologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunoglobulina G/química , Imunoglobulina G/imunologia , Mutagênese , Conformação Proteica , Receptores Fc , Fator Reumatoide/química , Fator Reumatoide/imunologia , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Anticorpos Monoclonais , Receptores Purinérgicos P2X4 , Animais , Humanos , Camundongos , Domínios Proteicos , Ratos , Receptores Purinérgicos P2X4/análise , Receptores Purinérgicos P2X4/químicaRESUMO
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.
Assuntos
Vírus da Caxumba/metabolismo , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Receptores Virais/metabolismo , Trissacarídeos/química , Trissacarídeos/metabolismo , Animais , Anticorpos Neutralizantes/química , Fusão Celular , Membrana Celular/metabolismo , Chlorocebus aethiops , Cristalografia por Raios X , Epitopos/química , Células HEK293 , Humanos , Lactose/química , Lactose/metabolismo , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica , Domínios Proteicos , Receptores Virais/química , Termodinâmica , Células Vero , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.
Assuntos
Antagonistas Colinérgicos/química , Antagonistas Colinérgicos/farmacologia , Quinuclidinil Benzilato/análogos & derivados , Quinuclidinil Benzilato/química , Quinuclidinil Benzilato/farmacologia , Receptor Muscarínico M2/antagonistas & inibidores , Receptor Muscarínico M2/química , Acetilcolina/análogos & derivados , Acetilcolina/química , Acetilcolina/metabolismo , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Regulação Alostérica , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Antagonistas Colinérgicos/metabolismo , Cristalografia por Raios X , Evolução Molecular , Humanos , Ligantes , Modelos Moleculares , Conformação Proteica , Quinuclidinil Benzilato/metabolismo , Receptor Muscarínico M2/genética , Receptor Muscarínico M2/metabolismo , Tirosina/química , Tirosina/metabolismoRESUMO
The biogenic amine histamine is an important pharmacological mediator involved in pathophysiological processes such as allergies and inflammations. Histamine H(1) receptor (H(1)R) antagonists are very effective drugs alleviating the symptoms of allergic reactions. Here we show the crystal structure of the H(1)R complex with doxepin, a first-generation H(1)R antagonist. Doxepin sits deep in the ligand-binding pocket and directly interacts with Trp 428(6.48), a highly conserved key residue in G-protein-coupled-receptor activation. This well-conserved pocket with mostly hydrophobic nature contributes to the low selectivity of the first-generation compounds. The pocket is associated with an anion-binding region occupied by a phosphate ion. Docking of various second-generation H(1)R antagonists reveals that the unique carboxyl group present in this class of compounds interacts with Lys 191(5.39) and/or Lys 179(ECL2), both of which form part of the anion-binding region. This region is not conserved in other aminergic receptors, demonstrating how minor differences in receptors lead to pronounced selectivity differences with small molecules. Our study sheds light on the molecular basis of H(1)R antagonist specificity against H(1)R.
Assuntos
Doxepina/metabolismo , Antagonistas dos Receptores Histamínicos/metabolismo , Receptores Histamínicos H1/química , Receptores Histamínicos H1/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Doxepina/química , Antagonistas dos Receptores Histamínicos/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Isomerismo , Ligantes , Modelos Moleculares , Fosfatos/química , Fosfatos/metabolismo , Ligação Proteica , Conformação Proteica , Receptores Adrenérgicos beta 2/química , Receptores de Dopamina D3/química , Especificidade por SubstratoRESUMO
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.
Assuntos
Receptores Histamínicos H1/química , Animais , Sítios de Ligação/fisiologia , Doxepina/química , Doxepina/farmacologia , Histamina/química , Histamina/metabolismo , Antagonistas dos Receptores Histamínicos/química , Antagonistas dos Receptores Histamínicos/farmacologia , Antagonistas dos Receptores Histamínicos H1/química , Antagonistas dos Receptores Histamínicos H1/metabolismo , Humanos , Ligação Proteica/efeitos dos fármacos , Receptores Histamínicos H1/metabolismoRESUMO
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.
Assuntos
Autoanticorpos , Fator Reumatoide , Humanos , Fator Reumatoide/genética , Fator Reumatoide/metabolismo , Autoanticorpos/química , Escherichia coli/genética , Escherichia coli/metabolismo , Imunoglobulina G/químicaRESUMO
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.
Assuntos
Receptores Histamínicos H3 , Saccharomyces cerevisiae , Animais , Humanos , Histamina/metabolismo , Mamíferos/metabolismo , Reação em Cadeia da Polimerase , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Receptores Histamínicos/genética , Receptores Histamínicos/metabolismo , Receptores Histamínicos H3/genética , Receptores Histamínicos H3/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
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.
Assuntos
Antineoplásicos , Neoplasias , Humanos , Linhagem Celular Tumoral , DNA , Quebras de DNA de Cadeia Dupla , Neoplasias/tratamento farmacológico , Neoplasias/genética , Fenótipo , Ftalazinas/farmacologia , GenomaRESUMO
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.
Assuntos
Proteínas de Membrana , Nanoestruturas , Proteínas de Membrana/metabolismo , Bicamadas Lipídicas/química , Cinética , Nanoestruturas/química , Fosfolipídeos/metabolismoRESUMO
BACKGROUND: Recent successes in the determination of G-protein coupled receptor (GPCR) structures have relied on the ability of receptor variants to overcome difficulties in expression and purification. Therefore, the quick screening of functionally expressed stable receptor variants is vital. RESULTS: We developed a platform using Saccharomyces cerevisiae for the rapid construction and evaluation of functional GPCR variants for structural studies. This platform enables us to perform a screening cycle from construction to evaluation of variants within 6-7 days. We firstly confirmed the functional expression of 25 full-length class A GPCRs in this platform. Then, in order to improve the expression level and stability, we generated and evaluated the variants of the four GPCRs (hADRB2, hCHRM2, hHRH1 and hNTSR1). These stabilized receptor variants improved both functional activity and monodispersity. Finally, the expression level of the stabilized hHRH1 in Pichia pastoris was improved up to 65 pmol/mg from negligible expression of the functional full-length receptor in S. cerevisiae at first screening. The stabilized hHRH1 was able to be purified for use in crystallization trials. CONCLUSIONS: We demonstrated that the S. cerevisiae system should serve as an easy-to-handle and rapid platform for the construction and evaluation of GPCR variants. This platform can be a powerful prescreening method to identify a suitable GPCR variant for crystallography.
Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Saccharomyces cerevisiae/metabolismo , Cristalização , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Pichia/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores Histamínicos H1/genética , Receptores Histamínicos H1/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
G-protein coupled receptors (GPCRs) play essential roles in regulation of many physiological processes and are one of the major targets of pharmaceutical drugs. The 3D structure can provide important information for the understanding of GPCR function and the design of new drugs. However, the success of structure determination relies largely on the production of recombinant GPCRs, because the expression levels of GPCRs are very low in native tissues except rhodopsin. All non-rhodopsin GPCRs whose structures were determined so far were expressed in insect cells and the availability of other hosts was unknown. Recently, we succeeded to determine the structure of human histamine H(1) receptor (H(1)R) expressed in Pichia pastoris. Here, we report the expression and purification procedures of recombinant H(1)R used in the structural determination. The receptor was designed to possess a N-terminal 19-residue deletion and a replacement of the third cytoplasmic loop with T4-lysozyme. The receptor was verified to show similar binding activities with the receptor expressed in other hosts. The receptor was purified by the immobilized metal ion affinity chromatography and used for the crystallographic study that resulted in the successful structure determination.
Assuntos
Pichia/genética , Receptores Histamínicos H1/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Sequência de Aminoácidos , Cromatografia de Afinidade , Clonagem Molecular , Técnicas de Cultura , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Humanos , Dados de Sequência Molecular , Ligação Proteica , Biossíntese de Proteínas , Proteólise , Pirilamina/química , Receptores Histamínicos H1/química , Receptores Histamínicos H1/isolamento & purificação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Saccharomyces cerevisiaeRESUMO
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.