RESUMEN
Parkin is a RING-between-RING E3 ligase that functions in the covalent attachment of ubiquitin to specific substrates, and mutations in Parkin are linked to Parkinson's disease, cancer and mycobacterial infection. The RING-between-RING family of E3 ligases are suggested to function with a canonical RING domain and a catalytic cysteine residue usually restricted to HECT E3 ligases, thus termed 'RING/HECT hybrid' enzymes. Here we present the 1.58 Å structure of Parkin-R0RBR, revealing the fold architecture for the four RING domains, and several unpredicted interfaces. Examination of the Parkin active site suggests a catalytic network consisting of C431 and H433. In cells, mutation of C431 eliminates Parkin-catalysed degradation of mitochondria, and capture of an ubiquitin oxyester confirms C431 as Parkin's cellular active site. Our data confirm that Parkin is a RING/HECT hybrid, and provide the first crystal structure of an RING-between-RING E3 ligase at atomic resolution, providing insight into this disease-related protein.
Asunto(s)
Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Biocatálisis , Dominio Catalítico , Humanos , Mitocondrias/metabolismo , Modelos Moleculares , Mutación , Enfermedad de Parkinson/enzimología , Enfermedad de Parkinson/genética , Fenilalanina , Estructura Terciaria de ProteínaRESUMEN
AIM: Peroxisome proliferator activated receptors (PPARs) are nuclear receptors involved in glucose and lipid metabolism. Three isoforms of PPARs have been identified with different tissue distribution and biological functions. Although the pharmacology of each receptor is well studied, the physiological effect of simultaneous activation of PPARalpha, gamma and delta is only starting to emerge. We sought to determine the biological effects of a novel PPAR pan activator and elucidate the physiological mechanisms involved. METHODS: Ob/ob, diet-induced obese (DIO) or PPARalpha knockout mice were administered a novel agonist that activates all PPARs to various degrees to determine the effect on body weight, body composition, food intake and energy expenditure. In addition, serum parameters including glucose, insulin, triglycerides and ketone bodies as well as tissue acylcarnitine were evaluated. The effect of the novel agonist on liver and skeletal muscle histopathology was also studied. RESULTS: We report that simultaneous activation of all PPARs resulted in substantial weight loss in ob/ob and DIO mice. Consistent with known PPAR pharmacology, we observed that agonist treatment increased lipid oxidation, although appetite suppression was mainly responsible for the weight loss. Agonist-induced weight loss was completely absent in PPARalpha knockout mice suggesting that PPARalpha pharmacology was the major contributor to weight regulation in mice. CONCLUSIONS: Our work provides evidence that simultaneous activation of PPARalpha, gamma and delta decreases body weight by regulating appetite. These effects of the pan agonist were completely absent in PPARalpha knockout mice, suggesting that PPARalpha pharmacology was the major contributor to weight loss.
Asunto(s)
Depresores del Apetito/farmacología , Obesidad/tratamiento farmacológico , PPAR alfa/agonistas , PPAR delta/agonistas , PPAR gamma/agonistas , Pérdida de Peso/fisiología , Animales , Regulación del Apetito/fisiología , Metabolismo Energético/fisiología , Resistencia a la Insulina/fisiología , Peroxidación de Lípido/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/fisiopatología , PPAR alfa/genética , PPAR gamma/genética , Rosiglitazona , Tiazoles/farmacología , Tiazolidinedionas/farmacologíaRESUMEN
Six classes of nonpeptide bradykinin antagonists were designed using a template derived from structural studies of peptide antagonists. Several compounds from each class were synthesized and assayed for binding to the human bradykinin B2 receptor. Each family showed compounds active at the level of the smallest template peptide; three classes contained compounds with Kd < 8 microM. These results provide diverse leads for a medicinal chemistry-based optimization program.
Asunto(s)
Antagonistas de los Receptores de Bradiquinina , Bradiquinina/antagonistas & inhibidores , Diseño de Fármacos , Receptores de Bradiquinina/metabolismo , Animales , Células CHO , Cricetinae , Humanos , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad Cuantitativa , Receptor de Bradiquinina B2 , Programas InformáticosRESUMEN
The "ELR" (Glu-Leu-Arg) tripeptide sequence near the N-terminus of interleukin-8 (IL-8) contributes a large part of the receptor binding free energy. Prior X-ray and nuclear magnetic resonance (NMR) structures of IL-8 have shown this region of the molecule to be highly mobile. We reasoned that a hydrophobic interaction between the leucine and the neighboring beta-turn might exist in the receptor binding conformation of the N-terminus. To test this hypothesis, we mutated two residues to cysteine and connected the N-terminus to the beta-turn. The mutant retains receptor binding affinity reasonably close to wild type and allows the characterization of a high-affinity conformation that may be useful in the design of small IL-8 mimics. The L5C/H33C mutant is refined to R-values of R = 20.6% and Rfree = 27.7% at 2.35 A resolution. Other receptor binding determinants reside in the "N-loop" found after "ELR" and preceding the first beta-strand. All available structures of IL-8 have been found with one of two distinct N-loop conformations. One of these is relevant for receptor binding, based on NMR results with receptor peptides. The other conformation obscures the receptor-peptide binding surface and may have an undetermined but necessarily different function.
Asunto(s)
Interleucina-8/química , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Animales , Antígenos CD/química , Cristalografía por Rayos X , Humanos , Interleucina-8/genética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Unión Proteica , Conformación Proteica , Receptores de Citocinas/química , Receptores de Interleucina/química , Receptores de Interleucina-8ARESUMEN
Results from protein mutagenesis and x-ray crystallographic studies of the multidomain protein Vascular Cell Adhesion Molecule (VCAM) were used to design cyclic octapeptides that retain the critical structural and binding elements of the epitope of VCAM in the interaction with the integrin alpha 4 beta 1 (VLA-4). Changes in the activities of peptide analogues correlated with the relative activities of protein mutants of VCAM, and predicted the properties of two new mutants that bound alpha 4 beta 1 with improved affinity vs wild type protein. The nmr structures of two peptides revealed a high degree of similarity to the structure of the VCAM binding epitope. These results demonstrate that a compact binding epitope identified via protein structure-function studies may be transferred to a synthetically accessible small peptide with the key structure-activity relationships intact.
Asunto(s)
Epítopos/química , Integrinas/metabolismo , Péptidos Cíclicos/química , Unión Proteica , Receptores Mensajeros de Linfocitos/metabolismo , Sitios de Unión , Unión Competitiva , Integrina alfa4beta1 , Espectroscopía de Resonancia Magnética , Metotrexato/química , Modelos Moleculares , Fragmentos de Péptidos/química , Relación Estructura-Actividad , Molécula 1 de Adhesión Celular Vascular/químicaRESUMEN
The migration, adhesion, and subsequent extravasation of leukocytes into inflamed tissues contribute to the pathogenesis of a variety of inflammatory diseases including asthma, rheumatoid arthritis, inflammatory bowel disease, and multiple sclerosis. The integrin adhesion receptor alpha 4 beta 1 expressed on leukocytes binds to the extracellular matrix protein fibronectin and to the cytokine inducible vascular cell adhesion molecule-1 (VCAM-1) at inflamed sites. Binding of alpha 4 beta 1 to VCAM-1 initiates firm adhesion of the leukocyte to the vascular endothelium followed by extravasation into the tissue. Monoclonal antibodies generated against either alpha 4 beta 1 or VCAM-1 can moderate this inflammatory response in a variety of animal models. Recently peptides containing a consensus LDV sequence based on the connecting segment-1 (CS-1) of fibronectin and cyclic peptides containing an RCD motif have shown promise in modulating leukocyte migration and inflammation presumably by blocking the interaction of alpha 4 beta 1 with VCAM-1. Here we describe novel, highly potent, cyclic peptides that competitively inhibit alpha 4 beta 1 binding to VCAM-1 and fibronectin at sub nanomolar concentrations. The structure of a representative analog was determined via NMR spectroscopy and used to facilitate optimization of peptide leads. The peptides discussed here utilize similar functional groups as the binding epitope of VCAM-1, inhibit lymphocyte migration in vivo, and are highly selective for alpha 4 beta 1. Furthermore the structure--activity relationships described here have provided a template for the structure-based design of small molecule antagonists of alpha 4 beta 1-mediated cell adhesion processes.
Asunto(s)
Antiinflamatorios no Esteroideos/síntesis química , Integrinas/antagonistas & inhibidores , Linfocitos/fisiología , Péptidos Cíclicos/síntesis química , Receptores Mensajeros de Linfocitos/antagonistas & inhibidores , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Anticuerpos Monoclonales/inmunología , Unión Competitiva , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Integrina alfa4beta1 , Integrinas/inmunología , Integrinas/metabolismo , Linfocitos/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Ratones , Modelos Moleculares , Estructura Molecular , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Péptidos Cíclicos/farmacología , Receptores Mensajeros de Linfocitos/inmunología , Receptores Mensajeros de Linfocitos/metabolismo , Relación Estructura-Actividad , Molécula 1 de Adhesión Celular Vascular/química , Molécula 1 de Adhesión Celular Vascular/inmunología , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Chemokines play an important role in inflammation. The mechanism via which they bind to more than one receptor and activate them is not well understood. The chemokines are thought to interact with their receptors via two distinct sites, one necessary for binding and the other for activation of signal transduction. In this study we have used alanine scanning mutagenesis to identify residues on RANTES that specifically interact with its receptors CCR1, CCR3, and CCR5 for binding and activation. Residues within a potential receptor binding site known as the N-loop (residues 12-20) and near the N-terminus of RANTES were individually mutated to alanine. The results of this study show that, within the N-loop, the side chain of R17 is necessary for RANTES binding to CCR1, F12 for binding to CCR3, and F12 and I15 for binding to CCR5, thus forming distinct but overlapping binding epitopes. In addition, our finding that P2 is necessary for binding to CCR5 is the first to show that a residue near the N-terminus of a CC-chemokine is involved in binding to a receptor. We have also found that P2, D6, and T7 near the N-terminus are involved in activating signal transduction via CCR1, P2 and Y3 via CCR3, and Y3 and D6 via CCR5. These results indicate that RANTES interacts with each of its receptors in a distinct and specific manner and provide further evidence to support the two-site model of interaction between chemokines and their receptors.
Asunto(s)
Quimiocinas/metabolismo , Epítopos/metabolismo , Receptores de Quimiocina , Receptores de Citocinas/metabolismo , Células 3T3 , Animales , Quimiocina CCL5/genética , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Unión Proteica/genética , Receptores CCR1 , Receptores CCR3 , Receptores CCR5 , Receptores del VIH/metabolismoRESUMEN
The characteristic CXC chemokine disulfide core of interleukin-8 (IL-8) has been rearranged in a variant replacing the 9-50 disulfide with a 9-38 disulfide. The new variant has been characterized by its binding affinity to IL-8 receptors A and B and the erythrocyte receptor DARC. This variant binds the three receptors with affinities between 500- and 2,500-fold lower than wild-type IL-8. Binding affinity results are also reported for the variant with alanine substituted for both cysteines 9 and 50. The Glu38-->Cys/Cys50-->Ala IL-8 crystallizes in space group P2(1)2(1)2(1) with cell parameters a = 46.4, b = 49.2, and c = 69.5 A, and has been refined to an R-value of 19.4% for data from 10 to 2 A resolution. Analysis of the structure confirms the new disulfide arrangement and suggests that changes at Ile10 may be the principal cause of the lowered affinities.
Asunto(s)
Antígenos CD/metabolismo , Cisteína/genética , Disulfuros/química , Interleucina-8/química , Interleucina-8/metabolismo , Receptores de Interleucina/metabolismo , Sitios de Unión/genética , Unión Competitiva , Simulación por Computador , Cristalografía por Rayos X , Eritrocitos/metabolismo , Interleucina-8/genética , Modelos Moleculares , Mutación , Neutrófilos/metabolismo , Conformación Proteica , Receptores de Interleucina-8A , Agua/químicaRESUMEN
The effect of sodium halide salts on the hydrolysis of three of the dansyl (Dns) peptide substrates described in the previous paper (Yang & Van Wart, 1994) by thermolysin have been studied. Increasing concentrations of sodium chloride decrease the KM value for the hydrolysis of the tripeptides Dns-Gly-Phe-Ala and Dns-Ala-Phe-Ala but leave kcat unaltered. This kinetic behavior is described by a nonessential activation mechanism in which chloride binds preferentially to the enzyme-substrate complex. Similar trends are found for the sodium bromide and fluoride salts. In contrast, sodium chloride decreases both KM and kcat almost equally for the hydrolysis of Dns-Ala-Ala-Phe-Ala, leaving kcat/KM unchanged. Thus, chloride is an uncompetitive inhibitor of this substrate. Molecular modeling studies have been carried out in order to explain the effect of chloride on the binding of these dansyl peptides. The decrease in KM for the hydrolysis of all three substrates is attributed to an interaction of chloride with Arg-203 located in the active site to stabilize the enzyme-substrate complexes. The differential effect of chloride on the kcat values for the hydrolysis of the dansyl tripeptides vs dansyl tetrapeptide is related to differences in binding on the Pn side of the substrates. The tripeptides are predicted to bind to the active site of thermolysin in a single low-energy conformation. However, there are two populations of low-energy binding modes for the tetrapeptide, one of which is believed to be a more productive binding mode.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Aniones/metabolismo , Compuestos de Dansilo/metabolismo , Péptidos/metabolismo , Termolisina/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Compuestos de Dansilo/química , Hidrólisis , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Compuestos de Sodio/química , Compuestos de Sodio/metabolismo , Especificidad por SustratoRESUMEN
RS-93427, a novel analog of prostacyclin, increased adenylate cyclase activity in human platelet membranes (EC50 = 42 nM) to approximately the same maximum level as that produced by prostacyclin (EC50 = 87 nM). The concentration-response curve for RS-93427 appeared to be monophasic. However, a selective prostaglandin D2 antagonist (BW A868C) significantly reduced the stimulation of adenylate cyclase produced by low concentrations of RS-93427 (3.2 to 32 nM). RS-93520, a stereoisomer of RS-93427, also stimulated adenylate cyclase activity but in a biphasic pattern. BW A868C reduced the activation produced by low concentrations of RS-93520 with a 100-fold shift in the response curve. Maximum stimulation by RS-93520 (4.5-fold) was less than that obtained with prostaglandin D2 (7.3-fold). Thus, the stimulation of adenylate cyclase activity by low concentrations of RS-93520 is due to an interaction with prostaglandin D2 receptors while the activation by RS-93427 is mediated by both prostacyclin and prostaglandin D2 receptors. Additional data in support of these conclusions was obtained when these prostaglandins were tested as inhibitors of ADP-induced platelet aggregation in the presence or absence of BW A868C. The potent stimulation of prostaglandin receptors with chimeric molecules provides some insight into the structural features required for receptor activation.