RESUMO
Poly(ADP-ribose) polymerase 1 (PARP1) synthesizes poly(ADP-ribose) (PAR), an essential post-translational modification whose function is important in many cellular processes including DNA damage signaling, cell death, and inflammation. All known PAR biology is intracellular, but we suspected it might also play a role in cell-to-cell communication during inflammation. We found that PAR activated cytokine release in human and mouse macrophages, a hallmark of innate immune activation, and determined structure-activity relationships. PAR was rapidly internalized by murine macrophages, while the monomer, ADP-ribose, was not. Inhibitors of Toll-like receptor 2 (TLR2) and TLR4 signaling blocked macrophage responses to PAR, and PAR induced TLR2 and TLR4 signaling in reporter cell lines suggesting it was recognized by these TLRs, much like bacterial pathogens. We propose that PAR acts as an extracellular damage associated molecular pattern that drives inflammatory signaling.
Assuntos
Poli Adenosina Difosfato Ribose/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular , Citocinas/metabolismo , Dimerização , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Microscopia Confocal , Poli Adenosina Difosfato Ribose/química , Relação Estrutura-Atividade , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Poly-ADP-ribosylation is a post-translational modification that regulates processes involved in genome stability. Breakdown of the poly(ADP-ribose) (PAR) polymer is catalysed by poly(ADP-ribose) glycohydrolase (PARG), whose endo-glycohydrolase activity generates PAR fragments. Here we present the crystal structure of PARG incorporating the PAR substrate. The two terminal ADP-ribose units of the polymeric substrate are bound in exo-mode. Biochemical and modelling studies reveal that PARG acts predominantly as an exo-glycohydrolase. This preference is linked to Phe902 (human numbering), which is responsible for low-affinity binding of the substrate in endo-mode. Our data reveal the mechanism of poly-ADP-ribosylation reversal, with ADP-ribose as the dominant product, and suggest that the release of apoptotic PAR fragments occurs at unusual PAR/PARG ratios.
Assuntos
Glicosídeo Hidrolases/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Tetrahymena thermophila/enzimologia , Biocatálise , Sequência Conservada , Cristalografia por Raios X , Ácido Glutâmico/metabolismo , Glicosídeo Hidrolases/química , Humanos , Hidrólise , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese , Poli Adenosina Difosfato Ribose/química , Especificidade por SubstratoRESUMO
Poly(ADP-ribose) (pADPr) is a large, structurally complex polymer of repeating ADP-ribose units. It is biosynthesized from NAD⺠by poly(ADP-ribose) polymerases (PARPs) and degraded to ADP-ribose by poly(ADP-ribose) glycohydrolase. pADPr is involved in many cellular processes and exerts biological function through covalent modification and noncovalent binding to specific proteins. Very little is known about molecular recognition and structure-activity relationships for noncovalent interaction between pADPr and its binding proteins, in part because of lack of access to the polymer on a large scale and to units of defined lengths. We prepared polydisperse pADPr from PARP1 and tankyrase 1 at the hundreds of milligram scale by optimizing enzymatic synthesis and scaling up chromatographic purification methods. We developed and calibrated an anion exchange chromatography method to assign pADPr size and scaled it up to purify defined length polymers on the milligram scale. Furthermore, we present a pADPr profiling method to characterize the polydispersity of pADPr produced by PARPs under different reaction conditions and find that substrate proteins affect the pADPr size distribution. These methods will facilitate structural and biochemical studies of pADPr and its binding proteins.
Assuntos
Bioquímica/métodos , Biopolímeros/biossíntese , Poli Adenosina Difosfato Ribose/biossíntese , Animais , Biocatálise , Biopolímeros/química , Calibragem , Bovinos , Fracionamento Químico , Cromatografia Líquida de Alta Pressão , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/isolamento & purificação , Glicosídeo Hidrolases/metabolismo , Humanos , Poli Adenosina Difosfato Ribose/química , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/metabolismo , Polimerização , Estrutura Terciária de Proteína , Tanquirases/química , Tanquirases/isolamento & purificação , Tanquirases/metabolismoRESUMO
Current models for cleavage plane determination propose that metaphase spindles are positioned and oriented by interactions of their astral microtubules with the cellular cortex, followed by cleavage in the plane of the metaphase plate [1, 2]. We show that in early frog and fish embryos, where cells are unusually large, astral microtubules in metaphase are too short to position and orient the spindle. Rather, the preceding interphase aster centers and orients a pair of centrosomes prior to nuclear envelope breakdown, and the spindle assembles between these prepositioned centrosomes. Interphase asters center and orient centrosomes with dynein-mediated pulling forces. These forces act before astral microtubules contact the cortex; thus, dynein must pull from sites in the cytoplasm, not the cell cortex as is usually proposed for smaller cells. Aster shape is determined by interactions of the expanding periphery with the cell cortex or with an interaction zone that forms between sister-asters in telophase. We propose a model to explain cleavage plane geometry in which the length of astral microtubules is limited by interaction with these boundaries, causing length asymmetries. Dynein anchored in the cytoplasm then generates length-dependent pulling forces, which move and orient centrosomes.
Assuntos
Divisão Celular , Embrião não Mamífero/citologia , Modelos Biológicos , Animais , Centrossomo/ultraestrutura , Embrião não Mamífero/ultraestrutura , Desenvolvimento Embrionário , Feminino , Fertilização , Masculino , Metáfase , Microtúbulos/ultraestrutura , Espermatozoides/ultraestrutura , Fuso Acromático/metabolismo , Fuso Acromático/ultraestrutura , Xenopus laevis , Peixe-Zebra/embriologiaRESUMO
The trace amine-associated receptor 1 (TAAR(1)) is an aminergic G protein-coupled receptor (GPCR) potently activated by 3-iodothyronamine (1), an endogenous derivative of thyroid hormone. Structure-activity relationship studies on 1 and related agonists showed that the rat and mouse species of TAAR(1) accommodated structural modifications and functional groups on the ethylamine portion and the biaryl ether moiety of the molecule. However, the two receptors clearly exhibited distinct, species-specific ligand preferences despite being remarkably similar with 93% sequence similarity. In this study, we generated single and double mutants of rat and mouse TAAR(1) to probe the molecular recognition of agonists and the underlying basis for the ligand selectivity of rat and mouse TAAR(1). Key, nonconserved specificity determinant residues in transmembranes helices 4 and 7 within the ligand binding site appear to be the primary source of a number of the observed ligand preferences. Residue 7.39 in transmembrane 7 dictated the preference for a beta-phenyl ring, while residue 4.56 in transmembrane 4 was partially responsible for the lower potency of 1 and tyramine for the mouse receptor. Additionally, 1 and tyramine were found to have the same binding mode in rat TAAR(1) despite structure-activity relationship data suggesting the possibility of each molecule having different binding orientations. These findings provide valuable insights into the critical binding site residues involved in the ligand-receptor interaction that can influence compound selectivity and functional activity of aminergic GPCRs.
Assuntos
Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Tironinas/farmacologia , Sequência de Aminoácidos , Animais , Humanos , Ligantes , Camundongos , Dados de Sequência Molecular , Proteínas Mutantes/agonistas , Proteínas Mutantes/química , Proteínas Mutantes/genética , Estrutura Secundária de Proteína , Ratos , Receptores Acoplados a Proteínas G/genética , Alinhamento de Sequência , Relação Estrutura-Atividade , Tironinas/química , Tiramina/metabolismo , Tiramina/farmacologiaRESUMO
Amiodarone (Cordarone, Wyeth-Ayerst Pharmaceuticals) is a clinically available drug used to treat a wide variety of cardiac arrhythmias. We report here the synthesis and characterization of a panel of potential amiodarone metabolites that have significant structural similarity to thyroid hormone and its metabolites the iodothyronamines. Several of these amiodarone derivatives act as specific agonists of the G protein-coupled receptor (GPCR) trace amine-associated receptor 1 (TAAR(1)). This result demonstrates a novel molecular target for amiodarone derivatives with potential clinical significance.
Assuntos
Amiodarona/síntese química , Amiodarona/farmacologia , Antiarrítmicos/farmacologia , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Amiodarona/química , Animais , Arritmias Cardíacas/tratamento farmacológico , Técnicas de Química Combinatória , Humanos , Camundongos , Estrutura Molecular , RatosRESUMO
The trace amine-associated receptor 1 (TAAR(1)) is a biogenic amine G protein-coupled receptor (GPCR) that is potently activated by 3-iodothyronamine (1, T(1)AM) in vitro. Compound 1 is an endogenous derivative of the thyroid hormone thyroxine which rapidly induces hypothermia, anergia, and bradycardia when administered to mice. To explore the role of TAAR(1) in mediating the effects of 1, we rationally designed and synthesized rat TAAR(1) superagonists and lead antagonists using the rotamer toggle switch model of aminergic GPCR activation. The functional activity of a ligand is proposed to be correlated to its probable interactions with the rotamer switch residues; agonists allow the rotamer switch residues to toggle to their active conformation, whereas antagonists interfere with this conformational transition. These agonist and antagonist design principles provide a conceptual model for understanding the relationship between the molecular structure of a drug and its pharmacological properties.
Assuntos
Desenho de Fármacos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Animais , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Ratos , Receptores Acoplados a Proteínas G/metabolismo , Relação Estrutura-Atividade , Tironinas/química , Tironinas/metabolismo , Tironinas/farmacologiaRESUMO
3-iodothyronamine (1, T1AM) is a naturally occurring derivative of thyroid hormone that can potently activate the orphan G protein-coupled receptor (GPCR) known as the trace amine-associated receptor 1 (TAAR1). We have previously found that modifying the outer ring of the phenoxyphenethylamine core scaffold of 1 can improve potency and provide potent agonists. In this study, we explored the tolerance of rat and mouse TAAR1 (rTAAR1 and mTAAR1) for structural modifications in the ethylamine portion of 1. We found that incorporating unsaturated hydrocarbon substituents and polar, hydrogen-bond-accepting groups were beneficial for rTAAR1 and mTAAR1, respectively, providing compounds that were equipotent or more potent than 1. Additionally, we have discovered that a naphthyl group is an excellent isosteric replacement for the iodophenyl ring of 1.