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1.
J Virol ; 88(4): 1942-52, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24284328

RESUMO

Genotype II.3 (GII.3) noroviruses are a major cause of sporadic gastroenteritis, particularly in children. The greater incidence of GII.3 noroviruses in the pediatric population compared to the adult demographic suggests development of herd immunity to this genotype, possibly as a consequence of limited evolution of immune epitopes. This study aimed to identify and characterize immune epitopes on the GII.3 capsid protein and to determine the level of immune cross-reactivity within the genotype. A panel of seven GII.3 virus-like particles (VLPs), representing norovirus strains isolated during 1975 to 2008, was tested by enzyme-linked immunosorbent assay (ELISA) for reactivity with human sera and a rabbit anti-GII.3 strain-specific polyclonal serum generated against the 2008 GII.3 VLP. Immunoprecipitation of protease-digested GII.3 VLPs and sequencing of bound peptides via mass spectrometry were used to locate epitopes on the capsid. Two epitopes were investigated further using Mimotopes technology. Serum binding studies demonstrated complete intragenotype GII.3 cross-reactivity using both human and rabbit serum. Six immunoreactive regions containing epitopes were located on the GII.3 capsid protein, two within each capsid domain. Epitopes in the S and P1 domains were highly conserved within GII.3 noroviruses. P2 domain epitopes were variable and contained evolutionarily important residues and histo-blood group antigen (HBGA) binding residues. In conclusion, anti-GII.3 antibody-binding epitopes are highly cross-reactive and mostly conserved within GII.3 strains. This may account for the limited GII.3 prevalence in adults and suggests that a GII.3 strain may be a valuable inclusion in a multivalent pediatric targeted VLP vaccine. Exploration of norovirus immune epitopes is vital for effective vaccine design. IMPORTANCE This study represents an important contribution to the understanding of norovirus immunology in a pediatric genotype. The high cross-reactivity and conservation of GII.3 epitopes suggest development of herd immunity against GII.3 and indicate that a GII.3 strain would be a valuable inclusion in a pediatric targeted multivalent vaccine. Immunological understanding of pediatric norovirus strains is important since norovirus vaccines will likely target high-risk groups such as the pediatric population.


Assuntos
Anticorpos Antivirais/metabolismo , Proteínas do Capsídeo/genética , Gastroenterite/imunologia , Gastroenterite/virologia , Imunidade Coletiva/imunologia , Modelos Moleculares , Norovirus/genética , Sequência de Aminoácidos , Análise de Variância , Animais , Sequência de Bases , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/metabolismo , Cromatografia Líquida , Reações Cruzadas/imunologia , Ensaio de Imunoadsorção Enzimática , Epitopos/genética , Epitopos/imunologia , Epitopos/metabolismo , Humanos , Imunoprecipitação , Dados de Sequência Molecular , Norovirus/imunologia , Coelhos , Alinhamento de Sequência , Análise de Sequência de DNA , Espectrometria de Massas em Tandem
2.
EMBO J ; 28(12): 1732-44, 2009 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-19440203

RESUMO

The N-end rule pathway is conserved from bacteria to man and determines the half-life of a protein based on its N-terminal amino acid. In Escherichia coli, model substrates bearing an N-degron are recognised by ClpS and degraded by ClpAP in an ATP-dependent manner. Here, we report the isolation of 23 ClpS-interacting proteins from E. coli. Our data show that at least one of these interacting proteins--putrescine aminotransferase (PATase)--is post-translationally modified to generate a primary N-degron. Remarkably, the N-terminal modification of PATase is generated by a new specificity of leucyl/phenylalanyl-tRNA-protein transferase (LFTR), in which various combinations of primary destabilising residues (Leu and Phe) are attached to the N-terminal Met. This modification (of PATase), by LFTR, is essential not only for its recognition by ClpS, but also determines the stability of the protein in vivo. Thus, the N-end rule pathway, through the ClpAPS-mediated turnover of PATase may have an important function in putrescine homeostasis. In addition, we have identified a new element within the N-degron, which is required for substrate delivery to ClpA.


Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Redes e Vias Metabólicas , Processamento de Proteína Pós-Traducional , Transaminases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/química , Cromatografia de Afinidade , Dipeptídeos/metabolismo , Endopeptidase Clp/química , Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/química , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Ligação Proteica , Especificidade por Substrato
3.
IUBMB Life ; 65(6): 550-63, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23576524

RESUMO

Histidine-rich glycoprotein (HRG) is a relatively abundant plasma protein that has been implicated in multiple biological processes including immunity, tumor progression, and vascular biology. However, current protocols for purifying HRG from plasma result in the copurification of contaminating proteins and raise questions over the validity of biological activities ascribed to HRG. In this study, we describe a two-step protocol for the large-scale purification of HRG from human plasma using a combination of metal affinity and ion exchange chromatography. The protocol employs a rapid and simple strategy to isolate highly purified HRG that minimizes proteolytic cleavage of the protein. The purification of HRG was assessed at each stage by measuring the amount of HRG immunoreactive protein using a specific monoclonal antibody against total protein, and demonstrated ~1,000-fold purification with an overall yield of ~32%. Mass spectrometry analysis demonstrated that plasma-derived HRG was free of contaminating proteins and gel electrophoresis showed it to have minimal proteolytic degradation. Characterization of protein by physical method showed that the protein exists as a single, monodisperse species. In contrast to the previous studies of HRG purified by different methods, HRG purified using the new procedure demonstrated a reduced profile of functions. Although the HRG retained binding to heparin and phosphatidic acid, it did not interact with necrotic cells or other cellular lipids. These data demonstrate that HRG does not exhibit the broad interactive properties that have been reported previously, suggesting that copurification of HRG-binding partners or other impurities are responsible for some of the reported functional properties. The findings in this study demonstrate that the new purification procedure can provide a ready source of pure HRG to assess ligand specificity and biological function of this important plasma protein.


Assuntos
Proteínas/isolamento & purificação , Proteínas/fisiologia , Sequência de Aminoácidos , Artefatos , Celulose/análogos & derivados , Celulose/química , Cromatografia por Troca Iônica , Dicroísmo Circular , Humanos , Lipídeos de Membrana/química , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
4.
J Colloid Interface Sci ; 606(Pt 2): 1737-1744, 2022 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-34507167

RESUMO

A protein corona forms around nanoparticles when they are intravenously injected into the bloodstream. The composition of the protein corona dictates the interactions between nanoparticles and the biological systems thus their immune evasion, blood circulation, and biodistribution. Here, we report for the first time the impact of nanoparticle stiffness on protein corona formation using a unique emulsion core silica shell nanocapsules library with a wide range of mechanical properties over four magnitudes (700 kPa to 10 GPa). The nanocapsules with different stiffness showed distinct proteomic fingerprints. The protein corona of the stiffest nanocapsules contained the highest amount of complement protein (Complement C3) and immunoglobulin proteins, which contributed to their high macrophage uptake, confirming the important role of nanocapsules stiffness in controlling the protein corona formation thus their in vitro and in vivo behaviors.


Assuntos
Nanopartículas , Coroa de Proteína , Adsorção , Proteômica , Distribuição Tecidual
5.
Mol Omics ; 18(3): 226-236, 2022 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-34989730

RESUMO

The emergence of multidrug-resistant pathogenic bacteria creates a demand for novel antibiotics with distinct mechanisms of action. Advances in next-generation genome sequencing promised a paradigm shift in the quest to find new bioactive secondary metabolites. Genome mining has proven successful for predicting putative biosynthetic elements in secondary metabolite superproducers such as Streptomycetes. However, genome mining approaches do not inform whether biosynthetic gene clusters are dormant or active under given culture conditions. Here we show that using a multi-omics approach in combination with antiSMASH, it is possible to assess the secondary metabolic potential of a Streptomyces strain capable of producing mannopeptimycin, an important cyclic peptide effective against Gram-positive infections. The genome of Streptomyces hygroscopicus NRRL 30439 was first sequenced using PacBio RSII to obtain a closed genome. A chemically defined medium was then used to elicit a nutrient stress response in S. hygroscopicus NRRL 30439. Detailed extracellular metabolomics and intracellular proteomics were used to profile and segregate primary and secondary metabolism. Our results demonstrate that the combination of genomics, proteomics and metabolomics enables rapid evaluation of a strain's performance in bioreactors for industrial production of secondary metabolites.


Assuntos
Streptomyces , Genômica , Família Multigênica , Metabolismo Secundário/genética , Streptomyces/genética , Streptomyces/metabolismo
6.
FEBS J ; 280(1): 233-43, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23167757

RESUMO

Protein disulfide isomerase (PDI) family members are important enzymes for the correct folding and maturation of proteins that transit or reside in the endoplasmic reticulum (ER). The human PDI family comprises at least 19 members that differ in cell type expression, substrate specificity and post-translational modifications. PDI family A member 2 (PDIA2, previously known as PDIp) has a similar domain structure to prototypical PDI (also known as PDIA1), but the function and post-translational modifications of PDIA2 remain poorly understood. Unlike most PDI family members, PDIA2 contains three predicted N-linked glycosylation sites. By site-directed mutagenesis and enzymatic deglycosylation, we show here that all three Asn residues within the potential N-linked glycosylation sites of human PDIA2 (N127, N284 and N516) are glycosylated in human cells. Furthermore, mutation of N284 to glycosylation-null Gln increases formation of a highly stable disulfide-bonded PDIA2 dimer. Nevertheless, in HeLa cells, both wild-type and N127/284/516Q mutant PDIA2 proteins localize to the ER, but not the ER-Golgi intermediate compartment, suggesting that glycosylation is important for PDIA2 protein-protein interactions but not subcellular localization. Finally, we identified human major histocompatibility complex class 1 antigens (HLA-A,B,C) as potential binding partners of PDIA2, suggesting an involvement for PDIA2 in antigen presentation in addition to its previously described roles in autoimmunity and Parkinson's disease. These results further characterize this poorly defined member of the PDI family.


Assuntos
Isomerases de Dissulfetos de Proteínas/metabolismo , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Motivos de Aminoácidos , Asparagina/metabolismo , Retículo Endoplasmático/enzimologia , Estabilidade Enzimática , Glicosilação , Células HEK293 , Células HeLa , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Imunoprecipitação , Lectinas de Ligação a Manose/metabolismo , Proteínas de Membrana/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Ligação Proteica , Transporte Proteico
7.
PLoS One ; 7(6): e39248, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22720082

RESUMO

Many insect viruses express caspase inhibitors of the P35 superfamily, which prevent defensive host apoptosis to enable viral propagation. The prototypical P35 family member, AcP35 from Autographa californica M nucleopolyhedrovirus, has been extensively studied. Bacterially purified AcP35 has been previously shown to inhibit caspases from insect, mammalian and nematode species. This inhibition occurs via a pseudosubstrate mechanism involving caspase-mediated cleavage of a "reactive site loop" within the P35 protein, which ultimately leaves cleaved P35 covalently bound to the caspase's active site. We observed that AcP35 purifed from Saccharomyces cerevisae inhibited caspase activity more efficiently than AcP35 purified from Escherichia coli. This differential potency was more dramatic for another P35 family member, MaviP35, which inhibited human caspase 3 almost 300-fold more potently when purified from yeast than bacteria. Biophysical assays revealed that MaviP35 proteins produced in bacteria and yeast had similar primary and secondary structures. However, bacterially produced MaviP35 possessed greater thermal stability and propensity to form higher order oligomers than its counterpart purified from yeast. Caspase 3 could process yeast-purified MaviP35, but failed to detectably cleave bacterially purified MaviP35. These data suggest that bacterially produced P35 proteins adopt subtly different conformations from their yeast-expressed counterparts, which hinder caspase access to the reactive site loop to reduce the potency of caspase inhibition, and promote aggregation. These data highlight the differential caspase inhibition by recombinant P35 proteins purified from different sources, and caution that analyses of bacterially produced P35 family members (and perhaps other types of proteins) may underestimate their activity.


Assuntos
Inibidores de Caspase/farmacologia , Escherichia coli/metabolismo , Saccharomyces cerevisiae/metabolismo , Inibidores de Caspase/isolamento & purificação
8.
Mol Biochem Parasitol ; 166(2): 159-71, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19450733

RESUMO

Several merozoite surface proteins are being assessed as potential components of a vaccine against Plasmodium falciparum, the cause of the most serious form of human malaria. One of these proteins, merozoite surface protein 2 (MSP2), is unusually hydrophilic and contains tandem sequence repeats, characteristics of intrinsically unstructured proteins. A range of physicochemical studies has confirmed that recombinant forms of MSP2 are largely unstructured. Both dimorphic types of MSP2 (3D7 and FC27) are equivalently extended in solution and form amyloid-like fibrils although with different kinetics and structural characteristics. These fibrils have a regular underlying beta-sheet structure and both fibril types stain with Congo Red, but only the FC27 fibrils stain with Thioflavin T. 3D7 MSP2 fibrils seeded the growth of fibrils from 3D7 or FC27 MSP2 monomer indicating the involvement of a conserved region of MSP2 in fibril formation. Consistent with this, digestion of fibrils with proteinase K generated resistant peptides, which included the N-terminal conserved region of MSP2. A monoclonal antibody that reacted preferentially with monomeric recombinant MSP2 did not react with the antigen in situ on the merozoite surface. Glutaraldehyde cross-linking of infected erythrocytes generated MSP2 oligomers similar to those formed by polymeric recombinant MSP2. We conclude that MSP2 oligomers containing intermolecular beta-strand interactions similar to those in amyloid fibrils may be a component of the fibrillar surface coat on P. falciparum merozoites.


Assuntos
Amiloide/química , Antígenos de Protozoários/química , Malária Falciparum/parasitologia , Plasmodium falciparum/química , Proteínas de Protozoários/química , Sequência de Aminoácidos , Amiloide/genética , Amiloide/imunologia , Animais , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Humanos , Malária Falciparum/imunologia , Dados de Sequência Molecular , Plasmodium falciparum/genética , Plasmodium falciparum/imunologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia
9.
J Biol Chem ; 282(32): 23104-16, 2007 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-17526486

RESUMO

The binding of CD4 and chemokine receptors to the gp120 attachment glycoprotein of human immunodeficiency virus triggers refolding of the associated gp41 fusion glycoprotein into a trimer of hairpins with a 6-helix bundle (6HB) core. These events lead to membrane fusion and viral entry. Here, we examined the functions of the fusion peptide-proximal polar segment and membrane-proximal Trp-rich region (MPR), which are exterior to the 6HB. Alanine substitution of Trp(666), Trp(672), Phe(673), and Ile(675) in the MPR reduced entry by up to 120-fold without affecting gp120-gp41 association or cell-cell fusion. The L537A polar segment mutation led to the loss of gp120 from the gp120-gp41 complex, reduced entry by approximately 10-fold, but did not affect cell-cell fusion. Simultaneous Ala substitution of Leu(537) with Trp(666), Trp(672), Phe(673), or Ile(675) abolished entry with 50-80% reductions in cell-cell fusion. gp120-gp41 complexes of fusion-defective double mutants were resistant to soluble CD4-induced shedding of gp120, suggesting that their ability to undergo receptor-induced conformational changes was compromised. Consistent with this idea, a representative mutation, L537A/W666A, led to an approximately 80% reduction in lipophilic fluorescent dye transfer between gp120-gp41-expressing cells and receptor-expressing targets, indicating a block prior to the lipid-mixing phase. The L537A/W666A double mutation increased the chymotrypsin sensitivity of the polar segment in a trimer of hairpins model, comprising the 6HB core, the polar segment, and MPR linked N-terminally to maltose-binding protein. The data indicate that the polar segment and MPR of gp41 act synergistically in forming a fusion-competent gp120-gp41 complex and in stabilizing the membrane-interactive end of the trimer of hairpins.


Assuntos
Proteína gp41 do Envelope de HIV/química , Alanina/química , Sequência de Aminoácidos , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Dimerização , Vetores Genéticos , Glicoproteínas/química , Proteína gp120 do Envelope de HIV/química , Humanos , Leucina/química , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Homologia de Sequência de Aminoácidos
10.
Biochem J ; 363(Pt 1): 105-15, 2002 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-11903053

RESUMO

Porphyromonas gingivalis is an anaerobic, asaccharolytic Gram-negative rod associated with chronic periodontitis. We have undertaken a proteomic study of the outer membrane of P. gingivalis strain W50 using two-dimensional gel electrophoresis and peptide mass fingerprinting. Proteins were identified by reference to the pre-release genomic sequence of P. gingivalis available from The Institute for Genomic Research. Out of 39 proteins identified, five were TonB-linked outer membrane receptors, ten others were putative integral outer membrane proteins and four were putative lipoproteins. Pyroglutamate was found to be the N-terminal residue of seven of the proteins, and was predicted to be the N-terminal residue of 13 additional proteins. The RgpA, Kgp and HagA polyproteins were identified as fully processed domains in outer membranes prepared in the presence of proteinase inhibitors. Several domains were found to be C-terminally truncated 16-57 residues upstream from the N-terminus of the following domain, at a residue penultimate to a lysine. This pattern of C-terminal processing was not detected in a W50 strain isogenic mutant lacking the lysine-specific proteinase Kgp. Construction of another W50 isogenic mutant lacking the arginine-specific proteinases indicated that RgpB and/or RgpA were also involved in domain processing. The C-terminal adhesin of RgpA, designated RgpA27, together with RgpB and two newly identified proteins designated P27 and P59 were found to migrate on two-dimensional gels as vertical streaks at a molecular mass 13-42 kDa higher than that calculated from their gene sequences. The electrophoretic behaviour of these proteins, together with their immunoreactivity with a monoclonal antibody that recognizes lipopolysaccharide, is consistent with a modification that could anchor the proteins to the outer membrane.


Assuntos
Proteínas de Bactérias , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Hemaglutininas/química , Hemaglutininas/metabolismo , Porphyromonas gingivalis/metabolismo , Adesinas Bacterianas , Sequência de Aminoácidos , Western Blotting , Adesão Celular , Membrana Celular/metabolismo , Eletroforese em Gel Bidimensional , Cisteína Endopeptidases Gingipaínas , Lectinas , Lipopolissacarídeos/metabolismo , Dados de Sequência Molecular , Mutação , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos
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