RESUMO
Ebola virus (EBOV) glycoprotein (GP) can be recognized by neutralizing antibodies (NAbs) and is the main target for vaccine design. Here, we first investigate the contribution of the stalk and heptad repeat 1-C (HR1C) regions to GP metastability. Specific stalk and HR1C modifications in a mucin-deleted form (GPΔmuc) increase trimer yield, whereas alterations of HR1C exert a more complex effect on thermostability. Crystal structures are determined to validate two rationally designed GPΔmuc trimers in their unliganded state. We then display a modified GPΔmuc trimer on reengineered protein nanoparticles that encapsulate a layer of locking domains (LD) and a cluster of helper T-cell epitopes. In mice and rabbits, GP trimers and nanoparticles elicit cross-ebolavirus NAbs, as well as non-NAbs that enhance pseudovirus infection. Repertoire sequencing reveals quantitative profiles of vaccine-induced B-cell responses. This study demonstrates a promising vaccine strategy for filoviruses, such as EBOV, based on GP stabilization and nanoparticle display.
Assuntos
Vacinas contra Ebola/administração & dosagem , Glicoproteínas/administração & dosagem , Doença pelo Vírus Ebola/terapia , Proteínas Virais/administração & dosagem , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Antígenos Virais/administração & dosagem , Antígenos Virais/genética , Antígenos Virais/imunologia , Antígenos Virais/ultraestrutura , Linfócitos B/imunologia , Cristalografia por Raios X , Modelos Animais de Doenças , Vacinas contra Ebola/genética , Vacinas contra Ebola/imunologia , Ebolavirus/genética , Ebolavirus/imunologia , Epitopos de Linfócito T/administração & dosagem , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/ultraestrutura , Feminino , Glicoproteínas/genética , Glicoproteínas/imunologia , Glicoproteínas/ultraestrutura , Doença pelo Vírus Ebola/sangue , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/virologia , Humanos , Camundongos , Nanopartículas/química , Domínios Proteicos/genética , Domínios Proteicos/imunologia , Engenharia de Proteínas , Multimerização Proteica/genética , Multimerização Proteica/imunologia , Estabilidade Proteica , Coelhos , Linfócitos T Auxiliares-Indutores/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologia , Proteínas Virais/genética , Proteínas Virais/imunologia , Proteínas Virais/ultraestruturaRESUMO
Secretory signalling glycoprotein (SPX-40) from mammary gland is highly expressed during involution. This protein is involved in a programmed cell death during tissue remodelling which occurs at the end of lactation. SPX-40 was isolated and purified from buffalo (SPB-40) from the samples obtained during involution. One solution of SPB-40 was made by dissolving it in buffer containing 25â¯mM Tris-HCl and 50â¯mM NaCl at pH 8.0. Another solution was made by adding 25% ethanol to the above solution. The biological effects of SPB-40 dissolved in above two solutions were evaluated on MCF-7 breast cancer cell lines. Free SPB-40 indicated significant pro-apoptotic effects while ethanol exposed SPB-40 showed considerably reduced effects on the apoptosis. SPB-40 was crystallized in the native state. The crystals of SPB-40 were soaked in four separate solutions containing 25% acetone, 25% ethanol, 25% butanol and 25% MPD. Four separate data sets were collected and their structures were determined at high resolutions. In all the four structures, the molecules of acetone, ethanol, butanol and MPD respectively were observed in the hydrophobic binding pocket of SPB-40. As a result of which, the conformation of Trp78 was altered thus blocking the binding site in SPB-40 leading to the loss of activity.
Assuntos
Apoptose/efeitos dos fármacos , Glicoproteínas/farmacologia , Glândulas Mamárias Animais/química , Transdução de Sinais/efeitos dos fármacos , Animais , Búfalos , Cristalografia por Raios X , Feminino , Glicoproteínas/química , Glicoproteínas/isolamento & purificação , Glicoproteínas/metabolismo , Humanos , Lactação/metabolismo , Células MCF-7 , Glândulas Mamárias Animais/metabolismo , Relação Estrutura-AtividadeRESUMO
Peptidyl-tRNA hydrolase is an essential enzyme which acts as one of the rescue factors of the stalled ribosomes. It is an esterase that hydrolyzes the ester bond in the peptidyl-tRNA molecules, which are products of ribosome stalling. This enzyme is required for rapid clearing of the peptidyl-tRNAs, the accumulation of which in the cell leads to cell death. Over the recent years, it has been heralded as an attractive drug target for antimicrobial therapeutics. Two distinct classes of peptidyl-tRNA hydrolase, Pth and Pth2, have been identified in nature. This review gives an overview of the structural and functional aspects of Pth, along with its sequence and structural comparison among various species of bacteria. While the mode of binding of the substrate to Pth and the mechanism of hydrolysis are still speculated upon, the structure-based drug design using this protein as the target is still largely unexplored. This review focuses on the structural features of Pth, giving a direction to structure-based drug design on this protein.
Assuntos
Bactérias/enzimologia , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Hidrólise , Especificidade por SubstratoRESUMO
Wrightia tinctoria globulin (WTG), one of the major seed storage proteins, was isolated for the first time from seeds of the medicinal plant. WTG was extracted and purified to homogeneity in two steps using anion-exchange and size-exclusion chromatographies. On an SDS-PAGE gel under non-reducing conditions, a major band of ~56 kDa was observed; under reducing conditions, however, two major polypeptides, one with molecular weight ~32-34 kDa and the other with molecular weight ~22-26 kDa were observed. Intact mass determination by MALDI-TOF supported this observation. The N-terminal amino acid sequence of WTG matched in NCBI database with an expressed sequence tag obtained from the c-DNA of developing embryo m-RNA of Wrightia tinctoria. The EST sequence was further substantiated by partial de novo internal sequencing using MALDI-TOF/TOF. The high sequence homology with seed storage protein 11S globulin confirmed that WTG is a type of 11S globulin. Circular dichroism analysis showed that the secondary structure of WTG consists predominantly of ß-sheets (44.2%) and moderate content of α-helices (10.3%). WTG showed hemagglutinating property indicating that the protein may possess lectin-like activity. WTG was crystallized at 20 Å°C by the vapour diffusion method using PEG 400 as precipitant. The crystals belonged to the orthorhombic space group P212121 with cell dimensions of a=109.9Å, b=113.2Å and c=202.2Å with six molecules per asymmetric unit. Diffraction data were collected to a resolution of 2.2Å under cryocondition. Preliminary structure solution of WTG indicated the possibility of a hexameric assembly in its asymmetric unit.
Assuntos
Apocynaceae/química , Globulinas/química , Hemaglutinação/efeitos dos fármacos , Proteínas de Armazenamento de Sementes/química , Sequência de Aminoácidos , Western Blotting , Eletroforese em Gel de Poliacrilamida , Eritrócitos/efeitos dos fármacos , Globulinas/isolamento & purificação , Globulinas/farmacologia , Humanos , Dados de Sequência Molecular , Proteínas de Armazenamento de Sementes/isolamento & purificação , Proteínas de Armazenamento de Sementes/farmacologia , Sementes/química , Alinhamento de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Difração de Raios XRESUMO
A Kunitz type dual inhibitor (TKI) of factor Xa (FXa) and trypsin was found in tamarind. It also shows prolongation of blood coagulation time. The deduced 185 amino acid sequence of TKI by cDNA cloning and sequence analysis revealed that it belongs to the Kunitz type soybean trypsin inhibitor (STI) family; however, it has a distorted Kunitz signature sequence due to insertion of Asn15 in the motif. TKI exhibited a competitive inhibitory activity against both FXa (K(i) = 220 nm) and porcine pancreatic trypsin (K(i) = 3.2 nm). The crystal structure of TKI shows a ß-trefoil fold similar to Kunitz STI inhibitors; however, a distinct mobile reactive site, an inserted residue and loop ß7ß8 make it distinct from classical Kunitz inhibitors. The crystal structure of TKI-trypsin and a 3D model of TKI-FXa complex revealed that the distinct reactive site loop probably plays a role in dual inhibition. The reactive site of TKI interacts with an active site and two exosites (36 loop and autolysis loop) of FXa. Apart from Arg66 (P1), Arg64 (P3) is one of the most important residues responsible for the specificity of TKI towards FXa. Along with the reactive site loop (ß4ß5), loops ß1 and ß7ß8 also interact with FXa and could further confer selectivity for FXa. We also present the role of inserted Asn15 in the stabilization of complexes. To the best of our knowledge, this is the first structure of FXa inhibitor belonging to the Kunitz type inhibitor family and its unique structural and sequence features make TKI a novel potent inhibitor. DATABASE: The complete nucleotide of TKI was deposited in the NCBI gene databank with accession no. HQ385502. The atomic coordinates and structure factor files for the structure of TKI and TKI:PPT complex have been deposited in the Protein Data Bank with accession numbers 4AN6 and 4AN7, respectively STRUCTURED DIGITAL ABSTRACT: TKI and TKI bind by x-ray crystallography (View interaction) TKI and PPT bind by x-ray crystallography (View interaction).
Assuntos
Inibidores do Fator Xa , Inibidores de Proteases/farmacologia , Inibidores da Tripsina/farmacologia , Sequência de Aminoácidos , Modelos Moleculares , Dados de Sequência Molecular , Inibidores de Proteases/química , Conformação Proteica , Homologia de Sequência de Aminoácidos , Inibidores da Tripsina/químicaRESUMO
The complex of Tamarindus indica Kunitz-type trypsin inhibitor and porcine trypsin has been crystallized by the sitting-drop vapour-diffusion method using ammonium acetate as precipitant and sodium acetate as buffer. The homogeneity of complex formation was checked by size-exclusion chromatography and further confirmed by reducing SDS-PAGE. The crystals diffracted to 2.0 angstrom resolution and belonged to the tetragonal space group P4(1), with unit-cell parameters a = b = 57.1, c = 120.1 angstrom. Preliminary X-ray diffraction analysis indicated the presence of one unit of inhibitor-trypsin complex per asymmetric unit, with a solvent content of 45%.
Assuntos
Peptídeos/química , Proteínas de Plantas/química , Tamarindus/química , Tripsina/química , Animais , Cristalização , Cristalografia por Raios X , Dados de Sequência Molecular , Sementes/química , Sus scrofa , Tamarindus/anatomia & histologia , Difração de Raios XRESUMO
A protein with chitinase activity has been isolated and purified from tamarind (Tamarindus indica) seeds. N-terminal amino-acid sequence analysis of this protein confirmed it to be an approximately 34 kDa endochitinase which belongs to the acidic class III chitinase family. The protein was crystallized by the vapour-diffusion method using PEG 4000. The crystals belonged to the tetragonal space group P4(1), with two molecules per asymmetric unit. Diffraction data were collected to a resolution of 2.6 A.