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1.
Bioorg Med Chem Lett ; 27(14): 3101-3106, 2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-28539220

RESUMO

A series of potent dual JAK1/3 inhibitors have been developed from a moderately selective JAK3 inhibitor. Substitution at the C6 position of the pyrrolopyridazine core with aryl groups provided exceptional biochemical potency against JAK1 and JAK3 while maintaining good selectivity against JAK2 and Tyk2. Translation to in vivo efficacy was observed in a murine model of chronic inflammation. X-ray co-crystal structure determination confirmed the presumed inhibitor binding orientation in JAK3. Efforts to reduce hERG channel inhibition will be described.


Assuntos
Janus Quinase 1/antagonistas & inibidores , Janus Quinase 3/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Piridazinas/química , Pirróis/química , Animais , Sítios de Ligação , Domínio Catalítico , Linhagem Celular , Cristalografia por Raios X , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Humanos , Inflamação/prevenção & controle , Concentração Inibidora 50 , Janus Quinase 1/metabolismo , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/metabolismo , Janus Quinase 3/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Conformação Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Piridazinas/síntese química , Piridazinas/farmacocinética , Pirróis/síntese química , Pirróis/farmacocinética , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , TYK2 Quinase/antagonistas & inibidores , TYK2 Quinase/metabolismo
2.
Medchemcomm ; 8(11): 2093-2099, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30108726

RESUMO

Myeloperoxidase, a mammalian peroxidase involved in the immune system as an anti-microbial first responder, can produce hypochlorous acid in response to invading pathogens. Myeloperoxidase has been implicated in several chronic pathological diseases due to the chronic production of hypochlorous acid, as well as other reactive radical species. A high throughput screen and triaging protocol was developed to identify a reversible inhibitor of myeloperoxidase toward the potential treatment of chronic diseases such as atherosclerosis. The identification and characterization of a reversible myeloperoxidase inhibitor, 7-(benzyloxy)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine is described.

3.
ACS Med Chem Lett ; 6(8): 845-9, 2015 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-26288682

RESUMO

Early hit to lead work on a pyrrolopyridine chemotype provided access to compounds with biochemical and cellular potency against Janus kinase 2 (JAK2). Structure-based drug design along the extended hinge region of JAK2 led to the identification of an important H-bond interaction with the side chain of Tyr 931, which improved JAK family selectivity. The 4,5-dimethyl thiazole analogue 18 demonstrated high levels of JAK family selectivity and was identified as a promising lead for the program.

4.
ACS Med Chem Lett ; 6(8): 850-5, 2015 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-26288683

RESUMO

JAK2 kinase inhibitors are a promising new class of agents for the treatment of myeloproliferative neoplasms and have potential for the treatment of other diseases possessing a deregulated JAK2-STAT pathway. X-ray structure and ADME guided refinement of C-4 heterocycles to address metabolic liability present in dialkylthiazole 1 led to the discovery of a clinical candidate, BMS-911543 (11), with excellent kinome selectivity, in vivo PD activity, and safety profile.

5.
J Mol Biol ; 427(4): 924-942, 2015 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-25579995

RESUMO

The human pregnane X receptor (PXR) is a promiscuous nuclear receptor that functions as a sensor to a wide variety of xenobiotics and regulates expression of several drug metabolizing enzymes and transporters. We have generated "Adnectins", derived from 10th fibronectin type III domain ((10)Fn3), that target the PXR ligand binding domain (LBD) interactions with the steroid receptor co-activator-1 (SRC-1) peptide, displacing SRC-1 binding. Adnectins are structurally homologous to the immunoglobulin superfamily. Three different co-crystal structures of PXR LBD with Adnectin-1 and CCR1 (CC chemokine receptor-1) antagonist Compound-1 were determined. This structural information was used to modulate PXR affinity for a related CCR1 antagonist compound that entered into clinical trials for rheumatoid arthritis. The structures of PXR with Adnectin-1 reveal specificity of Adnectin-1 in not only targeting the interface of the SRC-1 interactions but also engaging the same set of residues that are involved in binding of SRC-1 to PXR. Substituting SRC-1 with Adnectin-1 does not alter the binding conformation of Compound-1 in the ligand binding pocket. The structure also reveals the possibility of using Adnectins as crystallization chaperones to generate structures of PXR with compounds of interest.


Assuntos
Coativador 1 de Receptor Nuclear/química , Receptores CCR1/antagonistas & inibidores , Receptores de Esteroides/química , Ureia/análogos & derivados , Valina/análogos & derivados , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Humanos , Lignanas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Receptor de Pregnano X , Ligação Proteica , Estrutura Terciária de Proteína , Receptores CCR1/metabolismo , Alinhamento de Sequência , Ressonância de Plasmônio de Superfície , Ureia/química , Ureia/metabolismo , Ureia/farmacologia , Valina/química , Valina/metabolismo , Valina/farmacologia
6.
J Med Chem ; 57(18): 7550-64, 2014 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-25101488
7.
Eur J Orthop Surg Traumatol ; 24(6): 993-8, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24253959

RESUMO

BACKGROUND: Proximal tibia fractures are difficult to treat especially when soft tissues are compromised by conventional open reduction and internal fixation with high complication rates. Many methods have been tried to manage these fractures. Less invasive stabilization system (LISS) is the latest technology applied for these injuries. This report presents clinical results of the LISS for the treatment of complex proximal tibia fractures. MATERIALS AND METHODS: From June 2007 to May 2010, total of 35 cases of the proximal tibia fractures (19 AO type 41A, 11 type 41B and five AO type 41C) were treated with the LISS technique. Clinical and radiological evaluation was done at 6, 10, 14, 20, 24 weeks and 9, 12, 18 and 24 months, respectively. RESULTS: The mean age of the patients was 50.17 years (range 20-73 years); male patients were 21 and female 14. The mean follow-up time was 31.42 months (range 21-42 months). The patients were evaluated using Knee Society scores, and the mean score was 92.11 (range 84-100); the mean full weight bearing time was 15.8 weeks (range 12-22), and union time was 25.17 weeks (range 20-29). Superficial infections and slight mal-alignment were seen on five patients each. CONCLUSION: The less invasive stabilization internal fixator system can be used successfully to treat complex proximal tibia fractures with minimal complications. It can be an alternative method for the treatment of the proximal tibia fractures.


Assuntos
Fixação Interna de Fraturas/instrumentação , Fixadores Internos , Fraturas da Tíbia/cirurgia , Adulto , Idoso , Articulação do Tornozelo/fisiopatologia , Feminino , Seguimentos , Fixação Interna de Fraturas/efeitos adversos , Consolidação da Fratura , Humanos , Fixadores Internos/efeitos adversos , Articulação do Joelho/fisiopatologia , Tempo de Internação , Masculino , Pessoa de Meia-Idade , Duração da Cirurgia , Estudos Prospectivos , Radiografia , Amplitude de Movimento Articular , Fraturas da Tíbia/diagnóstico por imagem , Fatores de Tempo , Suporte de Carga , Adulto Jovem
8.
J Orthop Surg (Hong Kong) ; 22(3): 299-303, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25550006

RESUMO

PURPOSE: To review the outcomes of 53 patients who underwent minimally invasive plate osteosynthesis (MIPO) for distal tibial fractures. METHODS: Medical records of 31 men and 22 women aged 22 to 78 (mean, 51) years who underwent MIPO using a locking compression plate for distal tibial fractures of the left (n=28) and right (n=25) legs with or without intra-articular extension were reviewed. RESULTS: Patients were followed up for a mean of 26 (range, 24-38) months. The mean time from injury to surgery was 9 (range, 3-12) days. The mean operating time was 105 (range, 75-180) minutes. The mean hospital stay was 16 (range, 8-25) days. Non-weight bearing walking with a crutch was started after a mean of 5.7 (range, 3-9) days. The mean time to callus formation was 12 (range, 8-15) weeks. The mean time to full weight bearing was 15 (range, 8-22) weeks. The mean time to bone union was 25 (range, 20-30) weeks. All except 2 fractures united anatomically. At 10 months, the range of motion of the ankle joint in all patients was similar to the contralateral side. Two patients had malunion but this was not clinically significant. Five patients had superficial infection, and 2 patients had persistent pain. CONCLUSION: MIPO is effective for closed, unstable fractures of the distal tibia. It reduces surgical trauma and preserves fracture haematoma.


Assuntos
Fixação Interna de Fraturas/métodos , Fraturas da Tíbia/cirurgia , Adulto , Idoso , Placas Ósseas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Procedimentos Cirúrgicos Minimamente Invasivos , Estudos Retrospectivos , Adulto Jovem
9.
Ann Thorac Surg ; 95(6): e155-6, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23706468

RESUMO

Malperfusion of end organs occurs in 20% to 40% patients with acute type A aortic dissection. Because irreversible ischemia is a time-dependent event, expedient diagnosis and treatment are necessary. We herein report successful surgical management of a patient with acute type A aortic dissection causing transient gut ischemia and a rare gall bladder perforation. We implemented one-stage surgical and laparoscopic management approach for the diagnosis and treatment. Increased awareness of this complication and appropriate use of available diagnostic tools may improve the outcome in similar patients. Patients with aortic dissection complicated by visceral ischemia require a prompt sequential and rational multidisciplinary approach for successful management.


Assuntos
Aneurisma Dissecante/complicações , Aneurisma da Aorta Torácica/complicações , Vesícula Biliar/irrigação sanguínea , Isquemia/etiologia , Síndrome de Marfan/complicações , Adulto , Aneurisma Dissecante/diagnóstico por imagem , Aneurisma Dissecante/cirurgia , Angiografia/métodos , Aneurisma da Aorta Torácica/diagnóstico por imagem , Aneurisma da Aorta Torácica/cirurgia , Implante de Prótese Vascular/métodos , Terapia Combinada , Procedimentos Cirúrgicos do Sistema Digestório/métodos , Seguimentos , Humanos , Isquemia/fisiopatologia , Isquemia/cirurgia , Masculino , Síndrome de Marfan/diagnóstico , Medição de Risco , Ruptura Espontânea/etiologia , Ruptura Espontânea/cirurgia , Tomografia Computadorizada por Raios X/métodos , Resultado do Tratamento
10.
J Cardiothorac Surg ; 5: 84, 2010 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-20950491

RESUMO

The prevalence of primary cardiac tumour ranges from 0.0017-0.28% and papillary fibroelastoma is rare but not uncommon benign cardiac neoplasm. Currently, with the advent of higher-resolution imaging technology especially transoesophageal echocardiography such cases being recognized frequently. The clinical presentation of these tumours varies from asymptomatic to severe ischaemic or embolic complications. We herein, present a 50-year-old female patient with a papillary fibroelastoma of the aortic valve arising from the endocardium of the right coronary cusp very close to the commissure between the right and non-coronary cusps. The patient presented with angina-like chest pain and was investigated using echocardiography and CT angiographic modalities in addition to the usual investigations. The differential diagnosis considered was a thrombus, myxoma, Lambl's excrescence and infective vegetation. The surgical management included a prompt resection of the tumour on cardiopulmonary bypass avoiding injury to the aortic valve. The patient recovered well. A review of the literature suggests that the cardiac papillary fibroelastoma is a rare but potentially treatable cause of embolic stroke and other fatal complications, therefore, a strong suspicion; appropriate use of imaging modality, preoperative anticoagulation and urgent surgical resection is warranted. Also, possibility of this diagnosis should be kept in mind while managing cardiac or valvular tumours.


Assuntos
Valva Aórtica , Fibroma/diagnóstico , Neoplasias Cardíacas/diagnóstico , Doenças das Valvas Cardíacas/diagnóstico , Diagnóstico Diferencial , Feminino , Fibroma/patologia , Fibroma/cirurgia , Neoplasias Cardíacas/patologia , Neoplasias Cardíacas/cirurgia , Doenças das Valvas Cardíacas/patologia , Doenças das Valvas Cardíacas/cirurgia , Humanos , Pessoa de Meia-Idade
11.
Case Rep Med ; 20102010.
Artigo em Inglês | MEDLINE | ID: mdl-20886029

RESUMO

Cardiac inflammatory myofibroblastic tumor (IMT) is a rare entity and is associated with distinct clinical, pathological and molecular features. The clinical behavior, natural history, biological potential, management and prognosis of such tumors are unclear. We present herewith an adolescent girl who presented with similar entity involving the junction of the right atrium and the inferior vena cava (IVC) in association with thrombocytosis and IVC thrombosis leading to obstruction of blood flow. Diagnostic tools included imaging and immuno-histopathology studies. Surgical management included resection of the tumor and thrombo-embolectomy of the IVC under cardiopulmonary bypass. This case is unique due to association of complete obstruction of IVC caused by the strategic location of the tumor, thrombosis of vena cava and association of thrombocytosis. These features have not been reported yet in relation to the cardiac IMT. This report will help in better understanding and management of similar cases in terms of planning cannulation of femoral veins or application of total hypothermic circulatory arrest during cardiopulmonary bypass and prompt us to look for recurrence or metastasis during follow up using echocardiography and laboratory investigations. The possibility of IMT should be kept in the differential diagnosis of cardiac tumors especially in children and adolescents.

13.
Structure ; 15(8): 1005-13, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17698003

RESUMO

Nicotinamide riboside kinase (NRK) has an important role in the biosynthesis of NAD(+) as well as the activation of tiazofurin and other NR analogs for anticancer therapy. NRK belongs to the deoxynucleoside kinase and nucleoside monophosphate (NMP) kinase superfamily, although the degree of sequence conservation is very low. We report here the crystal structures of human NRK1 in a binary complex with the reaction product nicotinamide mononucleotide (NMN) at 1.5 A resolution and in a ternary complex with ADP and tiazofurin at 2.7 A resolution. The active site is located in a groove between the central parallel beta sheet core and the LID and NMP-binding domains. The hydroxyl groups on the ribose of NR are recognized by Asp56 and Arg129, and Asp36 is the general base of the enzyme. Mutation of residues in the active site can abolish the catalytic activity of the enzyme, confirming the structural observations.


Assuntos
Cristalografia por Raios X , Fosfotransferases (Aceptor do Grupo Álcool)/química , Difosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Antineoplásicos/metabolismo , Sítios de Ligação , Catálise , Escherichia coli/genética , Humanos , Ligações de Hidrogênio , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Mutagênese Sítio-Dirigida , Mutação , Mononucleotídeo de Nicotinamida/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/análise , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/isolamento & purificação , Ligação Proteica , Estrutura Secundária de Proteína , Ribavirina/análogos & derivados , Ribavirina/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
14.
Expert Opin Ther Targets ; 11(5): 695-705, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17465726

RESUMO

Nicotinamide adenine dinucleotide (NAD(+)) has crucial roles in many cellular processes, both as a coenzyme for redox reactions and as a substrate to donate ADP-ribose units. Enzymes involved in NAD(+) metabolism are attractive targets for drug discovery against a variety of human diseases, including cancer, multiple sclerosis, neurodegeneration and Huntington's disease. A small-molecule inhibitor of nicotinamide phosphoribosyltransferase, an enzyme in the salvage pathway of NAD(+) biosynthesis, is presently in clinical trials against cancer. An analog of a kynurenine pathway intermediate is efficacious against multiple sclerosis in an animal model. Indoleamine 2,3-dioxygenase plays an important role in immune evasion by cancer cells and other disease processes. Inhibitors against kynurenine 3-hydroxylase can reduce the production of neurotoxic metabolites while increasing the production of neuroprotective compounds. This review summarizes the existing knowledge on NAD(+) metabolic enzymes, with emphasis on their relevance for drug discovery.


Assuntos
Desenho de Drogas , NAD/metabolismo , Acrilamidas/farmacologia , Acrilamidas/uso terapêutico , Adenosina Difosfato Ribose/metabolismo , Envelhecimento/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/metabolismo , Ensaios Clínicos Fase II como Assunto , ADP-Ribose Cíclica/fisiologia , Dano ao DNA , Inibidores Enzimáticos/farmacologia , Humanos , Cinurenina/metabolismo , Camundongos , NAD/fisiologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Piperidinas/farmacologia , Piperidinas/uso terapêutico , Poli Adenosina Difosfato Ribose/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Sirtuínas/antagonistas & inibidores , Sirtuínas/metabolismo , Triptofano/metabolismo
15.
J Biol Chem ; 282(28): 20584-92, 2007 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-17475623

RESUMO

Influenza A viruses cause a highly contagious respiratory disease in humans and are responsible for periodic widespread epidemics with high mortality rates. The influenza A virus NS1 protein (NS1A) plays a key role in countering host antiviral defense and in virulence. The 73-residue N-terminal domain of NS1A (NS1A-(1-73)) forms a symmetric homodimer with a unique six-helical chain fold. It binds canonical A-form double-stranded RNA (dsRNA). Mutational inactivation of this dsRNA binding activity of NS1A highly attenuates virus replication. Here, we have characterized the unique structural features of the dsRNA binding surface of NS1A-(1-73) using NMR methods and describe the 2.1-A x-ray crystal structure of the corresponding dsRNA binding domain from human influenza B virus NS1B-(15-93). These results identify conserved dsRNA binding surfaces on both NS1A-(1-73) and NS1B-(15-93) that are very different from those indicated in earlier "working models" of the complex between dsRNA and NS1A-(1-73). The combined NMR and crystallographic data reveal highly conserved surface tracks of basic and hydrophilic residues that interact with dsRNA. These tracks are structurally complementary to the polyphosphate backbone conformation of A-form dsRNA and run at an approximately 45 degrees angle relative to the axes of helices alpha2/alpha2'. At the center of this dsRNA binding epitope, and common to NS1 proteins from influenza A and B viruses, is a deep pocket that includes both hydrophilic and hydrophobic amino acids. This pocket provides a target on the surface of the NS1 protein that is potentially suitable for the development of antiviral drugs targeting both influenza A and B viruses.


Assuntos
Vírus da Influenza A/química , Vírus da Influenza B/química , Dobramento de Proteína , RNA de Cadeia Dupla/química , RNA Viral/química , Proteínas não Estruturais Virais/química , Cristalografia por Raios X , Dimerização , Humanos , Vírus da Influenza A/metabolismo , Vírus da Influenza A/patogenicidade , Vírus da Influenza B/metabolismo , Vírus da Influenza B/patogenicidade , Influenza Humana/metabolismo , Influenza Humana/mortalidade , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , RNA de Cadeia Dupla/metabolismo , RNA Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo
16.
Nat Struct Mol Biol ; 13(7): 582-8, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16783377

RESUMO

Nicotinamide phosphoribosyltransferase (NMPRTase) has a crucial role in the salvage pathway of NAD+ biosynthesis, and a potent inhibitor of NMPRTase, FK866, can reduce cellular NAD+ levels and induce apoptosis in tumors. We have determined the crystal structures at up to 2.1-A resolution of human and murine NMPRTase, alone and in complex with the reaction product nicotinamide mononucleotide or the inhibitor FK866. The structures suggest that Asp219 is a determinant of substrate specificity of NMPRTase, which is confirmed by our mutagenesis studies. FK866 is bound in a tunnel at the interface of the NMPRTase dimer, and mutations in this binding site can abolish the inhibition by FK866. Contrary to current knowledge, the structures show that FK866 should compete directly with the nicotinamide substrate. Our structural and biochemical studies provide a starting point for the development of new anticancer agents.


Assuntos
Antineoplásicos/farmacologia , Pentosiltransferases/antagonistas & inibidores , Acrilamidas/farmacologia , Sequência de Aminoácidos , Animais , Cristalização , Humanos , Cinética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Nicotinamida Fosforribosiltransferase , Pentosiltransferases/química , Piperidinas/farmacologia , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
17.
Structure ; 13(10): 1443-52, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16216576

RESUMO

Taspase1 catalyzes the proteolytic processing of the mixed lineage leukemia (MLL) nuclear protein, which is required for maintaining Hox gene expression patterns. Chromosomal translocations of the MLL gene are associated with leukemia in infants. Taspase1, a threonine aspartase, is a member of the type 2 asparaginase family, but is the only protease in this family. We report here the crystal structures of human activated Taspase1 and its proenzyme, as well as the characterization of the effects of mutations in the active site region using a newly developed fluorogenic assay. The structure of Taspase1 has significant differences from other asparaginases, especially near the active site. Mutation of the catalytic nucleophile, Thr234, abolishes autocatalytic processing in cis but does not completely block proteolysis in trans. The structure unexpectedly showed the binding of a chloride ion in the active site, and our kinetic studies confirm that chlorides ions are inhibitors of this enzyme at physiologically relevant concentrations.


Assuntos
Cristalografia por Raios X , Endopeptidases/química , Endopeptidases/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Cloretos/metabolismo , Cloretos/farmacologia , Dimerização , Endopeptidases/genética , Ativação Enzimática , Precursores Enzimáticos/química , Escherichia coli/genética , Corantes Fluorescentes , Histona-Lisina N-Metiltransferase , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Peptídeo Hidrolases/genética , Conformação Proteica , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Homologia de Sequência de Aminoácidos , Análise Espectral Raman , Água/química
18.
J Biol Chem ; 279(30): 31664-70, 2004 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-15123616

RESUMO

The Toll/interleukin-1 receptor (TIR) domain is conserved in the intracellular regions of Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs) as well as in several cytoplasmic adapter molecules. This domain has crucial roles in signal transduction by these receptors for host immune response. Here we report the crystal structure at 2.3-A resolution of the TIR domain of human IL-1RAPL, the first structure of a TIR domain of the IL-1R superfamily. There are large structural differences between this TIR domain and that of TLR1 and TLR2. Helix alphaD in IL-1RAPL is almost perpendicular to its equivalent in TLR1 or TLR2. The BB loop contains a hydrogen bond unique to IL-1RAPL between Thr residues at the 8th and 10th positions. The structural and sequence diversity among these domains may be important for specificity in the signal transduction by these receptors. A dimer of the TIR domain of IL-1RAPL is observed in the crystal, although this domain is monomeric in solution. Residues in the dimer interface are mostly unique to IL-1RAPL, which is consistent with the distinct functional roles of this receptor. Our functional studies show IL-1RAPL can activate JNK but not the ERK or the p38 MAP kinases, whereas its close homolog, TIGIRR, cannot activate JNK. Deletion mutagenesis studies show that the activation of JNK by IL-1RAPL does not depend on the integrity of its TIR domain, suggesting a distinct mechanism of signaling through this receptor.


Assuntos
Glicoproteínas de Membrana/química , Receptores de Superfície Celular/química , Receptores de Interleucina-1/química , Sequência de Aminoácidos , Linhagem Celular , Cristalografia por Raios X , Dimerização , Humanos , Técnicas In Vitro , Proteína Acessória do Receptor de Interleucina-1 , Proteínas Quinases JNK Ativadas por Mitógeno , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Receptor 1 Toll-Like , Receptor 2 Toll-Like , Receptores Toll-Like
19.
Acta Crystallogr D Biol Crystallogr ; 58(Pt 2): 225-32, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11807246

RESUMO

Lactoferrin is an iron-binding protein. In the iron-bound state, the two domains of each lobe are invariably closed over an Fe(3+) ion. On the other hand, the structures of iron-free forms of lactoferrins from various species show different domain orientations. In order to determine the effects of external conditions such as pH, temperature and the presence of other additive agents on the crystal packing and consequently the influence of crystal packing forces on the final conformations of the two lobes of apolactoferrin, the structure of equine apolactoferrin has been determined at 303 K. The equine apolactoferrin was crystallized at 303 K using a microdialysis setup in which the concentration of protein was kept at 70 mg ml(-1) in 0.025 M Tris-HCl pH 8.0 with a reservoir containing 19% ethanol in the same buffer. The structure has been determined by molecular replacement using equine diferric lactoferrin as a model and was refined to an R factor of 0.23. The value of the overall B factor in the present structure is 81.3 A(2). The overall structure of the protein is similar to its earlier structure based on crystals grown at 277 K as well as that of diferric equine lactoferrin. The N and C lobes have been found to be slightly differently oriented (4.9 and 7.1 degrees, respectively) compared with the structures of equine diferric lactoferrin and apolactoferrin analyzed at 277 K, but the domain orientations in the two structures are identical as they remain closed over the empty iron-binding cleft. Overall, the structures of equine diferric lactoferrin, equine apolactoferrin at 277 K and the present structure of equine apolactoferrin at 303 K display identical domain orientations, suggesting that variation in the temperature of crystal growth, data collection and the processes of iron binding and iron release do not influence the arrangements of domains in equine lactoferrin.


Assuntos
Apoproteínas/química , Lactoferrina/química , Animais , Apoproteínas/metabolismo , Cristalografia por Raios X , Cavalos , Concentração de Íons de Hidrogênio , Ferro/química , Ferro/metabolismo , Lactoferrina/metabolismo , Modelos Moleculares , Conformação Proteica , Homologia de Sequência de Aminoácidos
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