RESUMO
Immunoglobulin superfamily member (IgSF) proteins play a significant role in regulating immune responses with surface expression on all immune cell subsets, making the IgSF an attractive family of proteins for therapeutic targeting in human diseases. We have developed a directed evolution platform capable of engineering IgSF domains to increase affinities for cognate ligands and/or introduce binding to non-cognate ligands. Using this scientific platform, ICOSL domains have been derived with enhanced binding to ICOS and with additional high-affinity binding to the non-cognate receptor, CD28. Fc-fusion proteins containing these engineered ICOSL domains significantly attenuate T cell activation in vitro and in vivo and can inhibit development of inflammatory diseases in mouse models. We also present evidence that engineered ICOSL domains can be formatted to selectively provide costimulatory signals to augment T cell responses. Our scientific platform thus provides a system for developing therapeutic protein candidates with selective biological impact for treatments of a wide array of human disorders including cancer and autoimmune/inflammatory diseases.
Assuntos
Imunoglobulinas/química , Imunoglobulinas/genética , Família Multigênica , Animais , Antígenos CD28/genética , Antígenos CD28/imunologia , Evolução Molecular Direcionada , Feminino , Humanos , Imunoglobulinas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Domínios Proteicos , Linfócitos T/imunologiaRESUMO
Glutamate mediates fast excitatory neurotransmission via ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. The trafficking and gating properties of AMPA receptors (AMPARs) can be amplified by transmembrane AMPAR regulatory proteins (TARPs), which are often expressed in localized brain regions. Herein, we describe the discovery, lead optimization, and preclinical characterization of 5-arylbenzimidazolone and oxindole-based negative modulators of AMPARs associated with TARP γ-8, the primary TARP found in hippocampus. High-throughput screen lead 4 was optimized for potency and brain penetration to provide benzimidazolone 3, JNJ-55511118.1 Replacement of the benzimidazolone core in 3 with an oxindole mitigated reactive metabolite formation and led to the identification of 18 (GluA1/γ-8 pIC50 = 9.7). Following oral dosing in rats, 18 demonstrated robust target engagement in hippocampus as assessed by ex vivo autoradiography (ED50 = 0.6 mg/kg, plasma EC50 = 9 ng/mL).
RESUMO
We describe the computational design of proteins that bind the potent analgesic fentanyl. Our approach employs a fast docking algorithm to find shape complementary ligand placement in protein scaffolds, followed by design of the surrounding residues to optimize binding affinity. Co-crystal structures of the highest affinity binder reveal a highly preorganized binding site, and an overall architecture and ligand placement in close agreement with the design model. We use the designs to generate plant sensors for fentanyl by coupling ligand binding to design stability. The method should be generally useful for detecting toxic hydrophobic compounds in the environment.
Assuntos
Biologia Computacional/métodos , Fentanila/metabolismo , Entorpecentes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Cristalografia por Raios X , Expressão Gênica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Immunohistochemical staining for the lysosome-associated membrane protein 2 (LAMP-2) has been proposed previously as an alternative to electron microscopy to identify hepatic phospholipidosis. This study used LAMP-2 immunohistochemistry (IHC) to diagnose phospholipidosis in rats exhibiting renal tubular injury. Rats were administered toreforant, a histamine H4 receptor antagonist by oral gavage at a dose of 3, 10, or 100 mg/kg/d for 6 months. Hematoxylin and eosin staining revealed renal tubular epithelial cell vacuolation, hypertrophy, degeneration, and luminal dilation in the 100 mg/kg/d group animals. Renal tubular injury was confirmed using kidney injury marker 1 (KIM-1) IHC. The involvement of phosopholipidosis in the renal injury was investigated by LAMP-2. Adipophilin IHC was included to differentiate phospholipidosis from lipidosis. Increased LAMP-2 staining was observed in the 100 mg/kg/d group animals when compared to vehicle group animals. Lysosome-associated membrane protein-2 staining was most prominent in the outer stripe of the outer medulla where KIM-1 staining was also most prominent. By contrast, adipophilin staining was not increased. Phospholipidosis was also confirmed by electron microscopy. These data support the use of LAMP-2 IHC as a diagnostic tool and suggest an association between phospholipidosis and the renal tubular injury caused by toreforant.
Assuntos
Antagonistas dos Receptores Histamínicos/toxicidade , Nefropatias/diagnóstico , Rim/efeitos dos fármacos , Lipidoses/diagnóstico , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Fosfolipídeos/metabolismo , Receptores Histamínicos H4/antagonistas & inibidores , Injúria Renal Aguda , Animais , Moléculas de Adesão Celular/metabolismo , Feminino , Imuno-Histoquímica , Rim/metabolismo , Rim/patologia , Rim/ultraestrutura , Nefropatias/induzido quimicamente , Nefropatias/metabolismo , Nefropatias/patologia , Lipidoses/induzido quimicamente , Lipidoses/metabolismo , Lipidoses/patologia , Proteína 2 de Membrana Associada ao Lisossomo/análise , Masculino , Microscopia Eletrônica de Transmissão , Perilipina-2/análise , Perilipina-2/metabolismo , Ratos Sprague-DawleyRESUMO
Many viral surface glycoproteins and cell surface receptors are homo-oligomers, and thus can potentially be targeted by geometrically matched homo-oligomers that engage all subunits simultaneously to attain high avidity and/or lock subunits together. The adaptive immune system cannot generally employ this strategy since the individual antibody binding sites are not arranged with appropriate geometry to simultaneously engage multiple sites in a single target homo-oligomer. We describe a general strategy for the computational design of homo-oligomeric protein assemblies with binding functionality precisely matched to homo-oligomeric target sites. In the first step, a small protein is designed that binds a single site on the target. In the second step, the designed protein is assembled into a homo-oligomer such that the designed binding sites are aligned with the target sites. We use this approach to design high-avidity trimeric proteins that bind influenza A hemagglutinin (HA) at its conserved receptor binding site. The designed trimers can both capture and detect HA in a paper-based diagnostic format, neutralizes influenza in cell culture, and completely protects mice when given as a single dose 24 h before or after challenge with influenza.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/ultraestrutura , Modelos Químicos , Simulação de Acoplamento Molecular , Engenharia de Proteínas/métodos , Multimerização Proteica , Sítios de Ligação , Ligação ProteicaRESUMO
The discovery of the histamine H4 receptor (H4R) provided a new avenue for the exploration of the physiological role of histamine, as well as providing a new drug target for the development of novel antihistamines. The first step in this process was the identification of selective antagonists to help unravel the pharmacology of the H4R relative to other histamine receptors. The discovery of the selective H4R antagonist JNJ 7777120 was vital for showing a role for the H4R in inflammation and pruritus. While this compound has been very successful as a tool for understanding the function of the receptor, it has drawbacks, including a short in vivo half-life and hypoadrenocorticism toxicity in rats and dogs, that prevented advancing it into clinical studies. Further research let to the discovery of JNJ 39758979, which, similar to JNJ 7777120, was a potent and selective H4R antagonist and showed anti-inflammatory and anti-pruritic activity preclinically. JNJ 39758979 advanced into human clinical studies and showed efficacy in reducing experimental pruritus and in patients with atopic dermatitis. However, development of this compound was terminated due to the occurrence of drug-induced agranulocytosis. This was overcome by developing another H4R antagonist with a different chemical structure, toreforant, that does not appear to have this side effect. Toreforant has been tested in clinical studies in patients with rheumatoid arthritis, asthma, or psoriasis. In conclusions there have been many H4R antagonists reported in the literature, but only a few have been studied in humans underscoring the difficulty in finding ligands with all of the properties necessary for testing in the clinic. Nevertheless, the clinical data to date suggests that H4R antagonists can be beneficial in treating atopic dermatitis and pruritus.
Assuntos
Antagonistas dos Receptores Histamínicos/farmacologia , Antagonistas dos Receptores Histamínicos/uso terapêutico , Histamina/metabolismo , Receptores Histamínicos/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/metabolismo , HumanosRESUMO
Prediction of protein-protein interaction sites in a protein structure provides important information for elucidating the mechanism of protein function and can also be useful in guiding a modeling or design procedures of protein complex structures. Since prediction methods essentially assess the propensity of amino acids that are likely to be part of a protein docking interface, they can help in designing protein-protein interactions. Here, we introduce BindML and BindML+ protein-protein interaction sites prediction methods. BindML predicts protein-protein interaction sites by identifying mutation patterns found in known protein-protein complexes using phylogenetic substitution models. BindML+ is an extension of BindML for distinguishing permanent and transient types of protein-protein interaction sites. We developed an interactive web-server that provides a convenient interface to assist in structural visualization of protein-protein interactions site predictions. The input data for the web-server are a tertiary structure of interest. BindML and BindML+ are available at http://kiharalab.org/bindml/ and http://kiharalab.org/bindml/plus/ .
Assuntos
Sítios de Ligação , Biologia Computacional/métodos , Domínios e Motivos de Interação entre Proteínas , Proteínas , Software , Algoritmos , Bases de Dados de Proteínas , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Interface Usuário-ComputadorRESUMO
Identifying HCV drug resistance mutations (DRMs) is increasingly important as new direct acting antiviral therapies (DAA) become available. Tagged pooled pyrosequencing (TPP) was originally developed as cost-effective approach for detecting low abundance HIV DRMs. Using 127 HCV-positive samples from a Canadian injection drug user cohort, we demonstrated the suitability and efficiency of TPP for evaluating DRMs in HCV NS5B gene. At a mutation identification threshold of 1%, no nucleoside inhibitor DRMs were detected among these DAA naïve subjects. Clinical NS5B resistance to non-nucleoside inhibitors and interferon/ribavirin was predicted to be low within this cohort. S282T mutation, the primary mutation selected by sofosbuvir in vitro, was not identified while S282G/C/R variants were detected in 9 subjects. Further characterization on these new S282 variants using in silico molecular modeling implied their potential association with resistance. Combining TPP with in silico analysis detects NS5B polymorphisms that may explain differences in treatment outcomes.
Assuntos
Antivirais/farmacologia , Farmacorresistência Viral , Hepacivirus/genética , Hepatite C Crônica/virologia , Proteínas Mutantes/genética , Mutação de Sentido Incorreto , Proteínas não Estruturais Virais/genética , Adulto , Antivirais/uso terapêutico , Estudos de Coortes , Feminino , Hepacivirus/efeitos dos fármacos , Hepacivirus/isolamento & purificação , Hepatite C Crônica/tratamento farmacológico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Abuso de Substâncias por Via Intravenosa/complicações , Resultado do TratamentoRESUMO
The histamine H4 receptor (H(4)R) has been shown to have preclinical involvement in both inflammatory and pruritic responses. JNJ-39758979 [(R)-4-(3-amino-pyrrolidin-1-yl)-6-isopropyl-pyrimidin-2-ylamine] is a potent and selective H(4)R antagonist with a Ki at the human receptor of 12.5 ± 2.6 nM and greater than 80-fold selectivity over other histamine receptors. The compound also exhibited excellent selectivity versus other targets. JNJ-39758979 showed dose-dependent activity in models of asthma and dermatitis consistent with other H(4)R antagonists. Preclinical toxicity studies of up to 6 months in rats and 9 months in monkeys indicated an excellent safety profile, supporting the clinical testing of the compound. An oral formulation of JNJ-39758979 was studied in a phase 1 human volunteer study to assess safety, pharmacokinetics, and pharmacodynamics. The compound was well tolerated, with the exception of dose-dependent nausea, and no safety issues were noted in the phase 1 study. JNJ-39758979 exhibited good pharmacokinetics upon oral dosing with a plasma half-life of 124-157 hours after a single oral dose. In addition, dose-dependent inhibition of histamine-induced eosinophil shape change was detected, suggesting that the H4R was inhibited in vivo. In conclusion, JNJ-39758979 is a potent and selective H(4)R antagonist that exhibited good preclinical and phase 1 safety in healthy volunteers with evidence of a pharmacodynamics effect in humans.
Assuntos
Pirimidinas/farmacologia , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Tiazóis/farmacologia , Animais , Anti-Inflamatórios/efeitos adversos , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/farmacologia , Asma/tratamento farmacológico , Asma/imunologia , Asma/patologia , Forma Celular/efeitos dos fármacos , Dermatite de Contato/tratamento farmacológico , Dermatite de Contato/etiologia , Dermatite de Contato/imunologia , Método Duplo-Cego , Eosinófilos/efeitos dos fármacos , Eosinófilos/patologia , Feminino , Humanos , Isotiocianatos , Macaca fascicularis , Masculino , Ovalbumina , Pirimidinas/efeitos adversos , Pirimidinas/farmacocinética , Ratos , Receptores Histamínicos , Receptores Histamínicos H4 , Tiazóis/efeitos adversos , Tiazóis/farmacocinéticaRESUMO
Cisplatin (CDDP) is known to produce renal proximal tubule injury. Various renal biomarkers have been related to CDDP nephrotoxicity in previous research, but the temporal and spatial relationship of these biomarkers to injury reversal has not been well defined. In this study, the progression and reversal of renal histopathology findings relative to serum and urinary biomarker changes were examined during a 4-week postdose period following single intraperitoneal administration of CDDP (1 mg/kg) or 0.9% saline. Degeneration, vacuolation, inflammation, and regeneration of the S3 segment of proximal tubules were evident 72 hours following CDDP administration. Tubular degeneration and regeneration were also observed at 1 and 1.5 weeks but at lower incidences and/or severity indicating partial reversal. Complete histologic reversal was observed by 2 weeks following CDDP administration. Urinary kidney injury molecule 1 (KIM-1), α-glutathione-S-transferase (α-GST), and albumin levels increased at 72 hours postdosing, concurrently with the earliest histologic evidence of tubule injury. Changes in urinary KIM-1 correlated with KIM-1 immunostaining in the proximal tubular epithelial cells. No significant changes in serum biomarkers occurred except for a minimal increase in urea nitrogen at 1.5 weeks postdosing. Of the novel renal biomarkers examined, urinary KIM-1, α-GST, and albumin showed excellent concordance with CDDP-induced renal injury progression and reversal; and these biomarkers were more sensitive than traditional serum biomarkers in detecting early, acute renal tubular damage confirmed by histopathology. Furthermore, urinary KIM-1, α-GST, and albumin outperformed other biomarkers in correlating with the time of maximum histologic injury.
Assuntos
Biomarcadores/sangue , Biomarcadores/urina , Cisplatino/administração & dosagem , Túbulos Renais Proximais/efeitos dos fármacos , Albuminas/metabolismo , Animais , Moléculas de Adesão Celular/metabolismo , Clusterina/urina , Glutationa Transferase/metabolismo , Imuno-Histoquímica , Isoenzimas/metabolismo , Túbulos Renais Proximais/patologia , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Protein-protein interactions (PPIs) are involved in diverse functions in a cell. To optimize functional roles of interactions, proteins interact with a spectrum of binding affinities. Interactions are conventionally classified into permanent and transient, where the former denotes tight binding between proteins that result in strong complexes, whereas the latter compose of relatively weak interactions that can dissociate after binding to regulate functional activity at specific time point. Knowing the type of interactions has significant implications for understanding the nature and function of PPIs. In this study, we constructed amino acid substitution models that capture mutation patterns at permanent and transient type of protein interfaces, which were found to be different with statistical significance. Using the substitution models, we developed a novel computational method that predicts permanent and transient protein binding interfaces (PBIs) in protein surfaces. Without knowledge of the interacting partner, the method uses a single query protein structure and a multiple sequence alignment of the sequence family. Using a large dataset of permanent and transient proteins, we show that our method, BindML+, performs very well in protein interface classification. A very high area under the curve (AUC) value of 0.957 was observed when predicted protein binding sites were classified. Remarkably, near prefect accuracy was achieved with an AUC of 0.991 when actual binding sites were classified. The developed method will be also useful for protein design of permanent and transient PBIs.
Assuntos
Modelos Biológicos , Mapas de Interação de Proteínas , Proteínas/química , Proteínas/metabolismo , Algoritmos , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Bases de Dados de Proteínas , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas/genéticaRESUMO
The efferent ducts represent an important site of toxicity in the male reproductive tract but are not routinely examined in toxicity studies. This article describes a primary efferent duct toxicity that resulted in secondary testicular changes in rats. Male rats were administered LTI-1, a leukotriene A4 hydrolase inhibitor, at doses up to 250 mg/kg/d for 3 month or 150 mg/kg/d for 6 month. At the highest dose levels, testicular changes were predominantly unilateral and characterized by diffuse dilation or atrophy of the seminiferous tubules. These testicular changes correlated with granulomatous inflammation in the corresponding efferent ducts, suggesting that the mechanism for the testicular changes involves obstruction and impaired fluid reabsorption in the efferent ducts. Subsequent buildup in fluid volume and back-pressure upstream of the blockage cause dilation of the seminiferous tubules, which, in its late stages, progress to tubular atrophy. There are important differences in efferent duct anatomy between rats and larger mammals, including humans, such that the latter are less susceptible to testicular injury by this mechanism. Because of the limited relevance of this rat-specific finding to humans, it is important to distinguish testicular changes secondary to efferent duct toxicity from primary drug-induced testicular toxicity.
Assuntos
Epididimo/efeitos dos fármacos , Epóxido Hidrolases/antagonistas & inibidores , Epitélio Seminífero/efeitos dos fármacos , Epitélio Seminífero/patologia , Doenças Testiculares/patologia , Testículo/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Epididimo/metabolismo , Epididimo/patologia , Epóxido Hidrolases/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Epitélio Seminífero/metabolismo , Doenças Testiculares/induzido quimicamente , Testículo/metabolismo , Testículo/patologiaRESUMO
Protein-protein binding events mediate many critical biological functions in the cell. Typically, functionally important sites in proteins can be well identified by considering sequence conservation. However, protein-protein interaction sites exhibit higher sequence variation than other functional regions, such as catalytic sites of enzymes. Consequently, the mutational behavior leading to weak sequence conservation poses significant challenges to the protein-protein interaction site prediction. Here, we present a phylogenetic framework to capture critical sequence variations that favor the selection of residues essential for protein-protein binding. Through the comprehensive analysis of diverse protein families, we show that protein binding interfaces exhibit distinct amino acid substitution as compared with other surface residues. On the basis of this analysis, we have developed a novel method, BindML, which utilizes the substitution models to predict protein-protein binding sites of protein with unknown interacting partners. BindML estimates the likelihood that a phylogenetic tree of a local surface region in a query protein structure follows the substitution patterns of protein binding interface and nonbinding surfaces. BindML is shown to perform well compared to alternative methods for protein binding interface prediction. The methodology developed in this study is very versatile in the sense that it can be generally applied for predicting other types of functional sites, such as DNA, RNA, and membrane binding sites in proteins.
Assuntos
Substituição de Aminoácidos , Modelos Moleculares , Complexos Multiproteicos/química , Domínios e Motivos de Interação entre Proteínas , Software , Algoritmos , Sequência de Aminoácidos , Sítios de Ligação , Simulação por Computador , Sequência Conservada , Filogenia , Ligação Proteica , Curva ROC , Alinhamento de Sequência , Análise de Sequência de ProteínaRESUMO
BACKGROUND: Listeria adhesion protein (LAP) is a housekeeping bifunctional enzyme consisting of N-terminal acetaldehyde dehydrogenase (ALDH) and C-terminal alcohol dehydrogenase (ADH). It aids Listeria monocytogenes in crossing the epithelial barrier through a paracellular route by interacting with its host receptor, heat shock protein 60 (Hsp60). To gain insight into the binding interaction between LAP and Hsp60, LAP subdomain(s) participating in the Hsp60 interaction were investigated. METHODS: Using a ModBase structural model, LAP was divided into 4 putative subdomains: the ALDH region contains N1 (Met(1)-Pro(223)) and N2 (Gly(224)-Gly(411)), and the ADH region contains C1 (Gly(412)-Val(648)) and C2 (Pro(649)-Val(866)). Each subdomain was cloned and overexpressed in Escherichia coli and purified. Purified subdomains were used in ligand overlay, immunofluorescence, and bead-based epithelial cell adhesion assays to analyze each domain's affinity toward Hsp60 protein or human ileocecal epithelial HCT-8 cells. RESULTS: The N2 subdomain exhibited the greatest affinity for Hsp60 with a K(D) of 9.50±2.6 nM. The K(D) of full-length LAP (7.2±0.5 nM) to Hsp60 was comparable to the N2 value. Microspheres (1 µm diameter) coated with N2 subdomain showed significantly (P<0.05) higher binding to HCT-8 cells than beads coated with other subdomains and this binding was inhibited when HCT-8 cells were pretreated with anti-Hsp60 antibody to specifically block epithelial Hsp60. Furthermore, HCT-8 cells pretreated with purified N2 subdomain also reduced L. monocytogenes adhesion by about 4 log confirming its involvement in interaction with epithelial cells. CONCLUSION: These data indicate that the N2 subdomain in the LAP ALDH domain is critical in initiating interaction with mammalian cell receptor Hsp60 providing insight into the molecular mechanism of pathogenesis for the development of potential anti-listerial control strategies.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Chaperonina 60/metabolismo , Listeria/genética , Listeria/metabolismo , Álcool Desidrogenase/química , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Aldeído Oxirredutases/química , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Proteínas de Bactérias/genética , Linhagem Celular , Chaperonina 60/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
BACKGROUND: Infection by the pandemic influenza A (H1N1/09) virus resulted in significant pathology among specific ethnic groups worldwide. Natural Killer (NK) cells are important in early innate immune responses to viral infections. Activation of NK cells, in part, depend on killer-cell immunoglobulin-like receptors (KIR) and HLA class I ligand interactions. To study factors involved in NK cell dysfunction in overactive immune responses to H1N1 infection, KIR3DL1/S1 and KIR2DL2/L3 allotypes and cognate HLA ligands of H1N1/09 intensive-care unit (ICU) patients were determined. METHODOLOGY AND FINDINGS: KIR3DL1/S1, KIR2DL2/L3, and HLA -B and -C of 51 H1N1/09 ICU patients and 105 H1N1-negative subjects (St. Theresa Point, Manitoba) were characterized. We detected an increase of 3DL1 ligand-negative pairs (3DL1/S1(+) Bw6(+) Bw4(-)), and a lack of 2DL1 HLA-C2 ligands, among ICU patients. They were also significantly enriched for 2DL2/L3 ligand-positive pairs (P<0.001, Pc<0.001; Odds Ratio:6.3158, CI95%:2.481-16.078). Relative to St. Theresa aboriginals (STh) and Venezuelan Amerindians (VA), allotypes enriched among aboriginal ICU patients (Ab) were: 2DL3 (Ab>VA, P=0.024, Pc=0.047; Odds Ratio:2.563, CI95%:1.109-5.923), 3DL1*00101 (Ab>VA, P<0.001, Pc<0.001), 3DL1*01502 (Ab>STh, P=0.034, Pc=0.268), and 3DL1*029 (Ab>STh, P=0.039, Pc=0.301). Aboriginal patients ligand-positive for 3DL1/S1 and 2DL1 had the lowest probabilities of death (R(d)) (R(d)=28%), compared to patients that were 3DL1/S1 ligand-negative (R(d)=52%) or carried 3DL1*029 (R(d)=52%). Relative to Caucasoids (CA), two allotypes were enriched among non-aboriginal ICU patients (NAb): 3DL1*00401 (NAb>CA, P<0.001, Pc<0.001) and 3DL1*01502 (CAAssuntos
Vírus da Influenza A Subtipo H1N1/isolamento & purificação
, Influenza Humana/metabolismo
, Unidades de Terapia Intensiva
, Receptores KIR2DL2/metabolismo
, Receptores KIR2DL3/metabolismo
, Receptores KIR3DL1/metabolismo
, APACHE
, Sequência de Bases
, Primers do DNA
, Humanos
, Influenza Humana/mortalidade
, Influenza Humana/virologia
, Reação em Cadeia da Polimerase
, Probabilidade
, Receptores KIR2DL2/genética
, Receptores KIR2DL3/genética
, Receptores KIR3DL1/genética
RESUMO
SUMMARY: We present 3D-SURFER, a web-based tool designed to facilitate high-throughput comparison and characterization of proteins based on their surface shape. As each protein is effectively represented by a vector of 3D Zernike descriptors, comparison times for a query protein against the entire PDB take, on an average, only a couple of seconds. The web interface has been designed to be as interactive as possible with displays showing animated protein rotations, CATH codes and structural alignments using the CE program. In addition, geometrically interesting local features of the protein surface, such as pockets that often correspond to ligand binding sites as well as protrusions and flat regions can also be identified and visualized. AVAILABILITY: 3D-SURFER is a web application that can be freely accessed from: http://dragon.bio.purdue.edu/3d-surfer CONTACT: dkihara@purdue.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Biologia Computacional/métodos , Proteínas/química , Software , Sítios de Ligação , Bases de Dados de Proteínas , Conformação Proteica , Proteínas/metabolismo , Propriedades de SuperfícieRESUMO
The mapping of physicochemical characteristics onto the surface of a protein provides crucial insights into its function and evolution. This information can be further used in the characterization and identification of similarities within protein surface regions. We propose a novel method which quantitatively compares global and local properties on the protein surface. We have tested the method on comparison of electrostatic potentials and hydrophobicity. The method is based on 3D Zernike descriptors, which provides a compact representation of a given property defined on a protein surface. Compactness and rotational invariance of this descriptor enable fast comparison suitable for database searches. The usefulness of this method is exemplified by studying several protein families including globins, thermophilic and mesophilic proteins, and active sites of TIM beta/alpha barrel proteins. In all the cases studied, the descriptor is able to cluster proteins into functionally relevant groups. The proposed approach can also be easily extended to other surface properties. This protein surface-based approach will add a new way of viewing and comparing proteins to conventional methods, which compare proteins in terms of their primary sequence or tertiary structure.
Assuntos
Domínio Catalítico , Biologia Computacional/métodos , Proteínas/química , Modelos Moleculares , Conformação Proteica , Estrutura Terciária de Proteína , Software , Relação Estrutura-Atividade , Propriedades de SuperfícieRESUMO
Characterization and identification of similar tertiary structure of proteins provides rich information for investigating function and evolution. The importance of structure similarity searches is increasing as structure databases continue to expand, partly due to the structural genomics projects. A crucial drawback of conventional protein structure comparison methods, which compare structures by their main-chain orientation or the spatial arrangement of secondary structure, is that a database search is too slow to be done in real-time. Here we introduce a global surface shape representation by three-dimensional (3D) Zernike descriptors, which represent a protein structure compactly as a series expansion of 3D functions. With this simplified representation, the search speed against a few thousand structures takes less than a minute. To investigate the agreement between surface representation defined by 3D Zernike descriptor and conventional main-chain based representation, a benchmark was performed against a protein classification generated by the combinatorial extension algorithm. Despite the different representation, 3D Zernike descriptor retrieved proteins of the same conformation defined by combinatorial extension in 89.6% of the cases within the top five closest structures. The real-time protein structure search by 3D Zernike descriptor will open up new possibility of large-scale global and local protein surface shape comparison.
Assuntos
Algoritmos , Bases de Dados de Proteínas , Estrutura Terciária de Proteína , Alinhamento de Sequência/métodos , SoftwareRESUMO
Experimentally determined protein tertiary structures are rapidly accumulating in a database, partly due to the structural genomics projects. Included are proteins of unknown function, whose function has not been investigated by experiments and was not able to be predicted by conventional sequence-based search. Those uncharacterized protein structures highlight the urgent need of computational methods for annotating proteins from tertiary structures, which include function annotation methods through characterizing protein local surfaces. Toward structure-based protein annotation, we have developed VisGrid algorithm that uses the visibility criterion to characterize local geometric features of protein surfaces. Unlike existing methods, which only concerns identifying pockets that could be potential ligand-binding sites in proteins, VisGrid is also aimed to identify large protrusions, hollows, and flat regions, which can characterize geometric features of a protein structure. The visibility used in VisGrid is defined as the fraction of visible directions from a target position on a protein surface. A pocket or a hollow is recognized as a cluster of positions with a small visibility. A large protrusion in a protein structure is recognized as a pocket in the negative image of the structure. VisGrid correctly identified 95.0% of ligand-binding sites as one of the three largest pockets in 5616 benchmark proteins. To examine how natural flexibility of proteins affects pocket identification, VisGrid was tested on distorted structures by molecular dynamics simulation. Sensitivity decreased approximately 20% for structures of a root mean square deviation of 2.0 A to the original crystal structure, but specificity was not much affected. Because of its intuitiveness and simplicity, the visibility criterion will lay the foundation for characterization and function annotation of local shape of proteins.
Assuntos
Estrutura Terciária de Proteína , 3-alfa-Hidroxiesteroide Desidrogenase (B-Específica)/química , Acetilcolinesterase/química , Algoritmos , Argininossuccinato Sintase/química , Sítios de Ligação , Simulação por Computador , Venenos Elapídicos/química , Proteínas de Choque Térmico HSP90/química , Ligantes , Modelos Moleculares , Oxirredutases/química , Proteínas de Ligação a Fosfato/química , Proteínas de Transferência de Fosfolipídeos/química , Sensibilidade e Especificidade , Tetra-Hidrofolato Desidrogenase/químicaRESUMO
MINER is web-based software for phylogenetic motif (PM) identification. PMs are sequence regions (fragments) that conserve the overall familial phylogeny. PMs have been shown to correspond to a wide variety of catalytic regions, substrate-binding sites and protein interfaces, making them ideal functional site predictions. The MINER output provides an intuitive interface for interactive PM sequence analysis and structural visualization. The web implementation of MINER is freely available at http://www.pmap.csupomona.edu/MINER/. Source code is available to the academic community on request.