RESUMO
Ebola virus causes devastating hemorrhagic fever outbreaks for which no approved therapeutic exists. The viral nucleocapsid, which is minimally composed of the proteins NP, VP35, and VP24, represents an attractive target for drug development; however, the molecular determinants that govern the interactions and functions of these three proteins are still unknown. Through a series of mutational analyses, in combination with biochemical and bioinformatics approaches, we identified a region on VP24 that was critical for its interaction with NP. Importantly, we demonstrated that the interaction between VP24 and NP was required for both nucleocapsid assembly and genome packaging. Not only does this study underscore the critical role that these proteins play in the viral replication cycle, but it also identifies a key interaction interface on VP24 that may serve as a novel target for antiviral therapeutic intervention.
Assuntos
Ebolavirus/fisiologia , Nucleocapsídeo/metabolismo , Proteínas Virais/metabolismo , Linhagem Celular , Genoma Viral , Doença pelo Vírus Ebola/virologia , Humanos , Mutação , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Proteínas Virais/química , Proteínas Virais/genética , Montagem de Vírus , Replicação ViralRESUMO
Tyrosine sulfation is a post-translational modification that facilitates protein-protein interaction. Two sulfated tyrosines (Tys173 and Tys177) were recently identified within the second variable (V2) loop of the major HIV-1 envelope glycoprotein, gp120, and shown to contribute to stabilizing the intramolecular interaction between V2 and the third variable (V3) loop. Here, we report that tyrosine-sulfated peptides derived from V2 act as structural and functional mimics of the CCR5 N-terminus and potently block HIV-1 infection. Nuclear magnetic and surface plasmon resonance analyses indicate that a tyrosine-sulfated V2 peptide (pV2α-Tys) adopts a CCR5-like helical conformation and directly interacts with gp120 in a CD4-dependent fashion, competing with a CCR5 N-terminal peptide. Sulfated V2 mimics, but not their non-sulfated counterparts, inhibit HIV-1 entry and fusion by preventing coreceptor utilization, with the highly conserved C-terminal sulfotyrosine, Tys177, playing a dominant role. Unlike CCR5 N-terminal peptides, V2 mimics inhibit a broad range of HIV-1 strains irrespective of their coreceptor tropism, highlighting the overall structural conservation of the coreceptor-binding site in gp120. These results document the use of receptor mimicry by a retrovirus to occlude a key neutralization target site and provide leads for the design of therapeutic strategies against HIV-1.
Assuntos
Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Mimetismo Molecular , Fragmentos de Peptídeos/metabolismo , Receptores CCR5/metabolismo , Sequência de Aminoácidos , Fármacos Anti-HIV/química , Fármacos Anti-HIV/metabolismo , Fármacos Anti-HIV/farmacologia , Sítios de Ligação , Antígenos CD4/química , Antígenos CD4/metabolismo , Proteína gp120 do Envelope de HIV/química , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , Conformação Proteica , Receptores CCR5/química , Tirosina/análogos & derivados , Tirosina/químicaRESUMO
Development of a Plasmodium falciparum (Pf) transmission blocking vaccine (TBV) has the potential to significantly impact malaria control. Antibodies elicited against sexual stage proteins in the human bloodstream are taken up with the blood meal of the mosquitoes and inactivate parasite development in the mosquito. In a phase 1 trial, a leading TBV identified as Pfs25-EPA/Alhydrogel® appeared safe and immunogenic, however, the level of Pfs25-specific antibodies were likely too low for an effective vaccine. Pfs230, a 230-kDa sexual stage protein expressed in gametocytes is an alternative vaccine candidate. A unique 6-cysteine-rich domain structure within Pfs230 have thwarted its recombinant expression and characterization for clinical evaluation for nearly a quarter of a century. Here, we report on the identification, biochemical, biophysical, and immunological characterization of recombinant Pfs230 domains. Rabbit antibodies generated against recombinant Pfs230 domains blocked mosquito transmission of a laboratory strain and two field isolates using an ex vivo assay. A planned clinical trial of the Pfs230 vaccine is a significant step toward the potential development of a transmission blocking vaccine to eliminate malaria.
Assuntos
Antígenos de Protozoários/química , Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/química , Vacinas Antimaláricas/imunologia , Plasmodium falciparum/química , Plasmodium falciparum/imunologia , Proteínas de Protozoários/química , Proteínas de Protozoários/imunologia , Animais , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/genética , Antígenos de Protozoários/farmacologia , Humanos , Vacinas Antimaláricas/genética , Vacinas Antimaláricas/farmacologia , Malária Falciparum/genética , Malária Falciparum/imunologia , Malária Falciparum/prevenção & controle , Malária Falciparum/transmissão , Plasmodium falciparum/genética , Domínios Proteicos , Proteínas de Protozoários/genética , Proteínas de Protozoários/farmacologia , CoelhosRESUMO
Aminoacyl-tRNA synthetases (aaRS) catalyze both chemical steps that translate the universal genetic code. Rodin and Ohno offered an explanation for the existence of two aaRS classes, observing that codons for the most highly conserved Class I active-site residues are anticodons for corresponding Class II active-site residues. They proposed that the two classes arose simultaneously, by translation of opposite strands from the same gene. We have characterized wild-type 46-residue peptides containing ATP-binding sites of Class I and II synthetases and those coded by a gene designed by Rosetta to encode the corresponding peptides on opposite strands. Catalysis by WT and designed peptides is saturable, and the designed peptides are sensitive to active-site residue mutation. All have comparable apparent second-order rate constants 2.9-7.0E-3 M(-1) s(-1) or â¼750,000-1,300,000 times the uncatalyzed rate. The activities of the two complementary peptides demonstrate that the unique information in a gene can have two functional interpretations, one from each complementary strand. The peptides contain phylogenetic signatures of longer, more sophisticated catalysts we call Urzymes and are short enough to bridge the gap between them and simpler uncoded peptides. Thus, they directly substantiate the sense/antisense coding ancestry of Class I and II aaRS. Furthermore, designed 46-mers achieve similar catalytic proficiency to wild-type 46-mers by significant increases in both kcat and Km values, supporting suggestions that the earliest peptide catalysts activated ATP for biosynthetic purposes.
Assuntos
Trifosfato de Adenosina/química , Aminoacil-tRNA Sintetases/química , Códon/química , Código Genético , Peptídeos/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Aminoacilação , Biocatálise , Domínio Catalítico , Códon/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Evolução Molecular , Expressão Gênica , Cinética , Dados de Sequência Molecular , Mutação , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND: Preeclampsia is a significant cause of maternal and fetal mortality and morbidity worldwide. We previously reported associations between trichothiodystrophy (TTD) nucleotide excision repair (NER) and transcription gene mutations in the fetus and the risk of gestational complications including preeclampsia. TTD NER/transcription genes, XPD, XPB and TTD-A, code for subunits of Transcription Factor (TF)IIH. Interpreting XPD mutations in the context of available biochemical data led us to propose adverse effects on CDK-activating kinase (CAK) subunit of TFIIH and TFIIH-mediated functions as a relevant mechanism in preeclampsia. In order to gain deeper insight into the underlying biologic mechanisms involving TFIIH-mediated functions in placenta, we analyzed NER/transcription and global gene expression profiles of normal and preeclamptic placentas and studied gene regulatory networks. RESULTS: We found high expression of TTD NER/transcription genes in normal human placenta, above the mean of their expression in all organs. XPD and XPB were consistently expressed from 14 to 40 weeks gestation while expression of TTD-A was strongly negatively correlated (r=-0.7, P<0.0001) with gestational age. Analysis of gene expression patterns of placentas from a case-control study of preeclampsia using Algorithm for Reconstruction of Accurate Cellular Networks (ARACNE) revealed GTF2E1, a component of TFIIE which modulates TFIIH, among major regulators of differentially-expressed genes in preeclampsia. The basal transcription pathway was among the largest dysregulated protein-protein interaction networks in this preeclampsia dataset. Within the basal transcription pathway, significantly down-regulated genes besides GTF2E1 included those coding for the CAK complex of TFIIH, namely CDK7, CCNH, and MNAT1. Analysis of other relevant gene expression and gene regulatory network data also underscored the involvement of transcription pathways and identified JUNB and JUND (components of transcription factor AP-1) as transcription regulators of the network involving the TTD genes, GTF2E1, and selected gene regulators implicated in preeclampsia. CONCLUSIONS: Our results indicate that TTD NER/transcription genes are expressed in placenta during gestational periods critical to preeclampsia development. Our overall findings suggest that impairment of TFIIH-mediated function in transcription in placenta is a likely mechanism leading to preeclampsia and provide etiologic clues which may be translated into therapeutic and preventive measures.
Assuntos
Reparo do DNA , Feto/metabolismo , Placenta/metabolismo , Pré-Eclâmpsia/patologia , Fator de Transcrição TFIIH/metabolismo , Estudos de Casos e Controles , Regulação para Baixo , Feminino , Idade Gestacional , Células Endoteliais da Veia Umbilical Humana , Humanos , Pré-Eclâmpsia/genética , Gravidez , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Interferente Pequeno/metabolismo , Fator de Transcrição TFIIH/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Transcriptoma , Síndromes de Tricotiodistrofia/genética , Síndromes de Tricotiodistrofia/patologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteína Grupo D do Xeroderma Pigmentoso/metabolismoRESUMO
BACKGROUND: Because amino acid activation is rate-limiting for uncatalyzed protein synthesis, it is a key puzzle in understanding the origin of the genetic code. Two unrelated classes (I and II) of contemporary aminoacyl-tRNA synthetases (aaRS) now translate the code. Observing that codons for the most highly conserved, Class I catalytic peptides, when read in the reverse direction, are very nearly anticodons for Class II defining catalytic peptides, Rodin and Ohno proposed that the two superfamilies descended from opposite strands of the same ancestral gene. This unusual hypothesis languished for a decade, perhaps because it appeared to be unfalsifiable. RESULTS: The proposed sense/antisense alignment makes important predictions. Fragments that align in antiparallel orientations, and contain the respective active sites, should catalyze the same two reactions catalyzed by contemporary synthetases. Recent experiments confirmed that prediction. Invariant cores from both classes, called Urzymes after Ur = primitive, authentic, plus enzyme and representing ~20% of the contemporary structures, can be expressed and exhibit high, proportionate rate accelerations for both amino-acid activation and tRNA acylation. A major fraction (60%) of the catalytic rate acceleration by contemporary synthetases resides in segments that align sense/antisense. Bioinformatic evidence for sense/antisense ancestry extends to codons specifying the invariant secondary and tertiary structures outside the active sites of the two synthetase classes. Peptides from a designed, 46-residue gene constrained by Rosetta to encode Class I and II ATP binding sites with fully complementary sequences both accelerate amino acid activation by ATP ~400 fold. CONCLUSIONS: Biochemical and bioinformatic results substantially enhance the posterior probability that ancestors of the two synthetase classes arose from opposite strands of the same ancestral gene. The remarkable acceleration by short peptides of the rate-limiting step in uncatalyzed protein synthesis, together with the synergy of synthetase Urzymes and their cognate tRNAs, introduce a new paradigm for the origin of protein catalysts, emphasize the potential relevance of an operational RNA code embedded in the tRNA acceptor stems, and challenge the RNA-World hypothesis.
Assuntos
Aminoacil-tRNA Sintetases/genética , Aminoacilação , Evolução Molecular , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/metabolismo , Anticódon/genética , Anticódon/metabolismo , Catálise , Domínio Catalítico , Códon/genética , Códon/metabolismo , Código GenéticoRESUMO
From the catalytic reactions that sustain the global oxygen, nitrogen, and carbon cycles to the stabilization of DNA processing proteins, transition metal ions and metallocofactors play key roles in biology. Although the exquisite interplay between metal ions and protein scaffolds has been studied extensively, the fact that the biological roles of the metals often stem from their placement in the interfaces between proteins and protein subunits is not always recognized. Interfacial metal ions stabilize permanent or transient protein-protein interactions, enable protein complexes involved in cellular signaling to adopt distinct conformations in response to environmental stimuli, and catalyze challenging chemical reactions that are uniquely performed by multisubunit protein complexes. This review provides a structural survey of transition metal ions and metallocofactors found in protein-protein interfaces, along with a series of selected examples that illustrate their diverse biological utility and significance.
Assuntos
Metais/química , Proteínas/química , Proteínas/metabolismo , Animais , Humanos , Metais/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismoRESUMO
The Lyme disease spirochete Borrelia burgdorferi senses and responds to environmental cues as it transits between the tick vector and vertebrate host. Failure to properly adapt can block transmission of the spirochete and persistence in either vector or host. We previously identified BBD18, a novel plasmid-encoded protein of B. burgdorferi, as a putative repressor of the host-essential factor OspC. In this study, we investigate the in vivo role of BBD18 as a regulatory protein, using an experimental mouse-tick model system that closely resembles the natural infectious cycle of B. burgdorferi. We show that spirochetes that have been engineered to constitutively produce BBD18 can colonize and persist in ticks but do not infect mice when introduced by either tick bite or needle inoculation. Conversely, spirochetes lacking BBD18 can persistently infect mice but are not acquired by feeding ticks. Through site-directed mutagenesis, we have demonstrated that abrogation of spirochete infection in mice by overexpression of BBD18 occurs only with bbd18 alleles that can suppress OspC synthesis. Finally, we demonstrate that BBD18-mediated regulation does not utilize a previously described ospC operator sequence required by B. burgdorferi for persistence in immunocompetent mice. These data lead us to conclude that BBD18 does not represent the putative repressor utilized by B. burgdorferi for the specific downregulation of OspC in the mammalian host. Rather, we suggest that BBD18 exhibits features more consistent with those of a global regulatory protein whose critical role occurs during spirochete acquisition by feeding ticks. IMPORTANCE Lyme disease, caused by Borrelia burgdorferi, is the most common arthropod-borne disease in North America. B. burgdorferi is transmitted to humans and other vertebrate hosts by ticks as they take a blood meal. Transmission between vectors and hosts requires the bacterium to sense changes in the environment and adapt. However, the mechanisms involved in this process are not well understood. By determining how B. burgdorferi cycles between two very different environments, we can potentially establish novel ways to interfere with transmission and limit infection of this vector-borne pathogen. We are studying a regulatory protein called BBD18 that we recently described. We found that too much BBD18 interferes with the spirochete's ability to establish infection in mice, whereas too little BBD18 appears to prevent colonization in ticks. Our study provides new insight into key elements of the infectious cycle of the Lyme disease spirochete.
Assuntos
Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/fisiologia , Interações Hospedeiro-Patógeno , Ixodes/microbiologia , Animais , Proteínas de Bactérias/genética , Borrelia burgdorferi/genética , Análise Mutacional de DNA , Feminino , Deleção de Genes , Expressão Gênica , Camundongos , Mutagênese Sítio-DirigidaRESUMO
Computational protein design efforts aim to create novel proteins and functions in an automated manner and, in the process, these efforts shed light on the factors shaping natural proteins. The focus of these efforts has progressed from the interior of proteins to their surface and the design of functions, such as binding or catalysis. Here we examine progress in the development of robust methods for the computational design of non-natural interactions between proteins and molecular targets such as other proteins or small molecules. This problem is referred to as the de novo computational design of interactions. Recent successful efforts in de novo enzyme design and the de novo design of protein-protein interactions open a path towards solving this problem. We examine the common themes in these efforts, and review recent studies aimed at understanding the nature of successes and failures in the de novo computational design of interactions. While several approaches culminated in success, the use of a well-defined structural model for a specific binding interaction in particular has emerged as a key strategy for a successful design, and is therefore reviewed with special consideration.
Assuntos
Simulação por Computador , Modelos Moleculares , Proteínas/química , Substituição de Aminoácidos , Evolução Molecular Direcionada , Ligação de Hidrogênio , Ligação Proteica , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas/genética , SoftwareRESUMO
We previously suggested links between specific XPD mutations in the fetal genome and the risk of placental maldevelopment and preeclampsia, possibly due to impairment of Transcription Factor (TF)IIH-mediated functions in placenta. To identify the underlying mechanisms, we conducted the current integrative analysis of several relevant transcriptome data sources. Our meta-analysis revealed downregulation of TFIIH subunits in preeclamptic placentas. Our overall integrative analysis suggested that, in the presence of hypoxia and oxidative stress, EGFR signaling deficiency, which can be caused by TFIIH impairment as well as by other mechanisms, results in ATF3 upregulation, inducing mediators of clinical symptoms of preeclampsia such as FLT1 and ENG. EGFR- and ATF3-dependent pathways play prominent roles in cancer development. We propose that dysregulation of these canonical cancer molecular pathways occurs in preeclampsia and delineate the relevance of TFIIH, providing etiologic clues which could eventually translate into a therapeutic approach.
Assuntos
Proteínas de Neoplasias/metabolismo , Placenta/metabolismo , Pré-Eclâmpsia/metabolismo , Lesões Pré-Cancerosas/metabolismo , Fatores de Transcrição/metabolismo , Transcriptoma , Adulto , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Gravidez , Adulto JovemRESUMO
BACKGROUND: Influenza A viruses possess RNA genomes that mutate frequently in response to immune pressures. The mutations in the hemagglutinin genes are particularly significant, as the hemagglutinin proteins mediate attachment and fusion to host cells, thereby influencing viral pathogenicity and species specificity. Large-scale influenza A genome sequencing efforts have been ongoing to understand past epidemics and pandemics and anticipate future outbreaks. Sequencing efforts thus far have generated nearly 9,000 distinct hemagglutinin amino acid sequences. DESCRIPTION: Comparative models for all publicly available influenza A hemagglutinin protein sequences (8,769 to date) were generated using the Rosetta modeling suite. The C-alpha root mean square deviations between a randomly chosen test set of models and their crystallographic templates were less than 2 Å, suggesting that the modeling protocols yielded high-quality results. The models were compiled into an online resource, the Hemagglutinin Structure Prediction (HASP) server. The HASP server was designed as a scientific tool for researchers to visualize hemagglutinin protein sequences of interest in a three-dimensional context. With a built-in molecular viewer, hemagglutinin models can be compared side-by-side and navigated by a corresponding sequence alignment. The models and alignments can be downloaded for offline use and further analysis. CONCLUSIONS: The modeling protocols used in the HASP server scale well for large amounts of sequences and will keep pace with expanded sequencing efforts. The conservative approach to modeling and the intuitive search and visualization interfaces allow researchers to quickly analyze hemagglutinin sequences of interest in the context of the most highly related experimental structures, and allow them to directly compare hemagglutinin sequences to each other simultaneously in their two- and three-dimensional contexts. The models and methodology have shown utility in current research efforts and the ongoing aim of the HASP server is to continue to accelerate influenza A research and have a positive impact on global public health.
Assuntos
Bases de Dados de Proteínas , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Conformação Proteica , Alinhamento de Sequência , Análise de Sequência de Proteína , SoftwareRESUMO
The malaria parasite, Plasmodium falciparum, and related parasites use a variety of proteins with Duffy-Binding Like (DBL) domains to bind glycoproteins on the surface of host cells. Among these proteins, the 175 kDa erythrocyte binding antigen, EBA-175, specifically binds to glycophorin A on the surface of human erythrocytes during the process of merozoite invasion. The domain responsible for glycophorin A binding was identified as region II (RII) which contains two DBL domains, F1 and F2. The crystal structure of this region revealed a dimer that is presumed to represent the glycophorin A binding conformation as sialic acid binding sites and large cavities are observed at the dimer interface. The dimer interface is largely composed of two loops from within each monomer, identified as the F1 and F2 ß-fingers that contact depressions in the opposing monomers in a similar manner. Previous studies have identified a panel of five monoclonal antibodies (mAbs) termed R215 to R218 and R256 that bind to RII and inhibit invasion of erythrocytes to varying extents. In this study, we predict the F2 ß-finger region as the conformational epitope for mAbs, R215, R217, and R256, and confirm binding for the most effective blocking mAb R217 and R215 to a synthetic peptide mimic of the F2 ß-finger. Localization of the epitope to the dimerization and glycan binding sites of EBA-175 RII and site-directed mutagenesis within the predicted epitope are consistent with R215 and R217 blocking erythrocyte invasion by Plasmodium falciparum by preventing formation of the EBA-175- glycophorin A complex.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Protozoários/imunologia , Mapeamento de Epitopos , Eritrócitos/microbiologia , Glicoforinas/metabolismo , Plasmodium falciparum/fisiologia , Multimerização Proteica , Proteínas de Protozoários/imunologia , Especificidade de Anticorpos , Antígenos de Protozoários/química , Antígenos de Protozoários/metabolismo , Sítios de Ligação , Biologia Computacional , Glicoforinas/química , Humanos , Modelos Moleculares , Fragmentos de Peptídeos/imunologia , Biblioteca de Peptídeos , Plasmodium falciparum/imunologia , Plasmodium falciparum/metabolismo , Polissacarídeos/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismoRESUMO
Infectious bursal disease virus (IBDV) causes an economically significant disease of chickens worldwide. Very virulent IBDV (vvIBDV) strains have emerged and induce as much as 60% mortality. The molecular basis for vvIBDV pathogenicity is not understood, and the relative contributions of the two genome segments, A and B, to this phenomenon are not known. Isolate 94432 has been shown previously to be genetically related to vvIBDVs but exhibits atypical antigenicity and does not cause mortality. Here the full-length genome of 94432 was determined, and a reverse genetics system was established. The molecular clone was rescued and exhibited the same antigenicity and reduced pathogenicity as isolate 94432. Genetically modified viruses derived from 94432, whose vvIBDV consensus nucleotide sequence was restored in segment A and/or B, were produced, and their pathogenicity was assessed in specific-pathogen-free chickens. We found that a valine (position 321) that modifies the most exposed part of the capsid protein VP2 critically modified the antigenicity and partially reduced the pathogenicity of 94432. However, a threonine (position 276) located in the finger domain of the virus polymerase (VP1) contributed even more significantly to attenuation. This threonine is partially exposed in a hydrophobic groove on the VP1 surface, suggesting possible interactions between VP1 and another, as yet unidentified molecule at this amino acid position. The restored vvIBDV-like pathogenicity was associated with increased replication and lesions in the thymus and spleen. These results demonstrate that both genome segments influence vvIBDV pathogenicity and may provide new targets for the attenuation of vvIBDVs.
Assuntos
Infecções por Birnaviridae/veterinária , Vírus da Doença Infecciosa da Bursa/genética , Vírus da Doença Infecciosa da Bursa/patogenicidade , Doenças das Aves Domésticas/virologia , Proteínas Estruturais Virais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Infecções por Birnaviridae/mortalidade , Infecções por Birnaviridae/patologia , Infecções por Birnaviridae/virologia , Embrião de Galinha , Galinhas/virologia , Simulação de Dinâmica Molecular , Doenças das Aves Domésticas/patologia , Análise de Sequência de RNA , Baço/virologia , Timo/virologia , Proteínas Estruturais Virais/químicaRESUMO
The nonstructural protein p7 of classical swine fever virus (CSFV) is a small hydrophobic polypeptide with an apparent molecular mass of 6 to 7 kDa. The protein contains two hydrophobic stretches of amino acids interrupted by a short charged segment that are predicted to form transmembrane helices and a cytosolic loop, respectively. Using reverse genetics, partial in-frame deletions of p7 were deleterious for virus growth, demonstrating that CSFV p7 function is critical for virus production in cell cultures. A panel of recombinant mutant CSFVs was created using alanine scanning mutagenesis of the p7 gene harboring sequential three- to six-amino-acid residue substitutions spanning the entire protein. These recombinant viruses allowed the identification of the regions within p7 that are critical for virus production in vitro. In vivo, some of these viruses were partially or completely attenuated in swine relative to the highly virulent parental CSFV Brescia strain, indicating a significant role of p7 in CSFV virulence. Structure-function analyses in model membranes emulating the endoplasmic reticulum lipid composition confirmed that CSFV p7 is a pore-forming protein, and that pore-forming activity resides in the C-terminal transmembrane helix. Therefore, p7 is a viroporin which is clearly involved in the process of CSFV virulence in swine.
Assuntos
Vírus da Febre Suína Clássica/metabolismo , Vírus da Febre Suína Clássica/patogenicidade , Peste Suína Clássica/virologia , Proteínas não Estruturais Virais/metabolismo , Motivos de Aminoácidos , Animais , Linhagem Celular , Vírus da Febre Suína Clássica/química , Vírus da Febre Suína Clássica/genética , Suínos , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , VirulênciaRESUMO
Thrombospondin repeat (TSR)-like domains are structures involved with cell adhesion. Plasmodium falciparum proteins containing TSR domains play crucial roles in parasite development. In particular, the preerythrocytic P. falciparum circumsporozoite protein is involved in hepatocyte invasion. The importance of these domains in two other malaria proteins, the merozoite-specific thrombospondin-related anonymous protein (MTRAP) and the thrombospondin-related apical membrane protein (PTRAMP), were assessed using near-full-length recombinant proteins composed of the extracellular domains produced in Escherichia coli. MTRAP is thought to be released from invasive organelles identified as micronemes during merozoite invasion to mediate motility and host cell invasion through an interaction with aldolase, an actin binding protein involved in the moving junction. PTRAMP function remains unknown. In this study, the conformation of recombinant MTRAP (rMTRAP) appeared to be a highly extended protein (2 nm by 33 nm, width by length, respectively), whereas rPTRAMP had a less extended structure. Using an erythrocyte binding assay, rMTRAP but not rPTRAMP bound human erythrocytes; rMTRAP binding was mediated through the TSR domain. MTRAP- and in general PTRAMP-specific antibodies failed to inhibit P. falciparum development in vitro. Altogether, MTRAP is a highly extended bifunctional protein that binds to an erythrocyte receptor and the merozoite motor.
Assuntos
Genes de Protozoários , Proteínas de Membrana/química , Plasmodium falciparum/química , Proteínas de Protozoários/química , Adolescente , Adulto , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/imunologia , Fenômenos Biofísicos , Cromatografia Líquida de Alta Pressão/métodos , Biologia Computacional , Eritrócitos/imunologia , Eritrócitos/parasitologia , Escherichia coli/química , Frutose-Bifosfato Aldolase/química , Humanos , Glicoproteínas de Membrana/química , Proteínas de Membrana/imunologia , Microscopia de Força Atômica , Pessoa de Meia-Idade , Dados de Sequência Molecular , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/imunologia , Ligação Proteica , Redobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Protozoários/imunologia , Ratos , Receptores Imunológicos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico , Ultracentrifugação , Adulto JovemRESUMO
Mutations in XPD (ERCC2), XPB (ERCC3), and TTD-A (GTF2H5), genes involved in nucleotide excision repair and transcription, can cause several disorders including trichothiodystrophy (TTD) and xeroderma pigmentosum (XP). In this study, we tested the hypothesis that mutations in the XPD gene affect placental development in a phenotype-specific manner. To test our hypothesis and decipher potential biologic mechanisms, we compared all XPD-associated TTD (n=43) and XP (n=37) cases reported in the literature with respect to frequencies of gestational complications. Our genetic epidemiologic investigations of TTD and XP revealed that the exact genetic abnormality was relevant to the mechanism leading to gestational complications such as preeclampsia. Through structural mapping, we localized the preeclampsia-associated mutations to a C-terminal motif and the helicase surfaces of XPD, most likely affecting XPD's binding to cdk-activating kinase (CAK) and p44 subunits of transcription factor (TF) IIH. Our results suggested a link between TTD- but not XP-associated XPD mutations, placental maldevelopment and risk of pregnancy complications, possibly due to impairment of TFIIH-mediated functions in placenta. Our findings highlight the importance of the fetal genotype in development of gestational complications, such as preeclampsia. Therefore, future studies of genetic associations of preeclampsia and other placental vascular complications may benefit from focusing on genetic variants within the fetal DNA.
Assuntos
Fenótipo , Placenta/metabolismo , Fator de Transcrição TFIIH/genética , Proteína Grupo D do Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/genética , Feminino , Genótipo , Humanos , Pré-Eclâmpsia/metabolismo , Gravidez , Fator de Transcrição TFIIH/metabolismo , Síndromes de Tricotiodistrofia/genética , Xeroderma Pigmentoso/metabolismo , Proteína Grupo D do Xeroderma Pigmentoso/metabolismoRESUMO
The 1918 to 1919 "Spanish" influenza pandemic virus killed up to 50 million people. We report here clinical, pathological, bacteriological, and virological findings in 68 fatal American influenza/pneumonia military patients dying between May and October of 1918, a period that includes ~4 mo before the 1918 pandemic was recognized, and 2 mo (September-October 1918) during which it appeared and peaked. The lung tissues of 37 of these cases were positive for influenza viral antigens or viral RNA, including four from the prepandemic period (May-August). The prepandemic and pandemic peak cases were indistinguishable clinically and pathologically. All 68 cases had histological evidence of bacterial pneumonia, and 94% showed abundant bacteria on Gram stain. Sequence analysis of the viral hemagglutinin receptor-binding domain performed on RNA from 13 cases suggested a trend from a more "avian-like" viral receptor specificity with G222 in prepandemic cases to a more "human-like" specificity associated with D222 in pandemic peak cases. Viral antigen distribution in the respiratory tree, however, was not apparently different between prepandemic and pandemic peak cases, or between infections with viruses bearing different receptor-binding polymorphisms. The 1918 pandemic virus was circulating for at least 4 mo in the United States before it was recognized epidemiologically in September 1918. The causes of the unusually high mortality in the 1918 pandemic were not explained by the pathological and virological parameters examined. These findings have important implications for understanding the origins and evolution of pandemic influenza viruses.
Assuntos
Autopsia , Influenza Humana/mortalidade , Antígenos Virais/análise , História do Século XX , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/imunologia , Influenza Humana/epidemiologia , Influenza Humana/história , Dados de Sequência Molecular , RNA Viral/análiseRESUMO
The commitment of Plasmodium merozoites to invade red blood cells (RBCs) is marked by the formation of a junction between the merozoite and the RBC and the coordinated induction of the parasitophorous vacuole. Despite its importance, the molecular events underlying the parasite's commitment to invasion are not well understood. Here we show that the interaction of two parasite proteins, RON2 and AMA1, known to be critical for invasion, is essential to trigger junction formation. Using antibodies (Abs) that bind near the hydrophobic pocket of AMA1 and AMA1 mutated in the pocket, we identified RON2's binding site on AMA1. Abs specific for the AMA1 pocket blocked junction formation and the induction of the parasitophorous vacuole. We also identified the critical residues in the RON2 peptide (previously shown to bind AMA1) that are required for binding to the AMA1 pocket, namely, two conserved, disulfide-linked cysteines. The RON2 peptide blocked junction formation but, unlike the AMA1-specific Ab, did not block formation of the parasitophorous vacuole, indicating that formation of the junction and parasitophorous vacuole are molecularly distinct steps in the invasion process. Collectively, these results identify the binding of RON2 to the hydrophobic pocket of AMA1 as the step that commits Plasmodium merozoites to RBC invasion and point to RON2 as a potential vaccine candidate.
Assuntos
Merozoítos/metabolismo , Plasmodium falciparum/metabolismo , Plasmodium falciparum/patogenicidade , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/imunologia , Sítios de Ligação , Sequência Conservada/genética , Cisteína/metabolismo , Citocalasina D/farmacologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Frutose-Bifosfato Aldolase/química , Frutose-Bifosfato Aldolase/metabolismo , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Merozoítos/efeitos dos fármacos , Merozoítos/ultraestrutura , Modelos Biológicos , Dados de Sequência Molecular , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/ultraestrutura , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas de Protozoários/química , Relação Estrutura-AtividadeRESUMO
Selective binding by metalloproteins to their cognate metal ions is essential to cellular survival. How proteins originally acquired the ability to selectively bind metals and evolved a diverse array of metal-centered functions despite the availability of only a few metal-coordinating functionalities remains an open question. Using a rational design approach (Metal-Templated Interface Redesign), we describe the transformation of a monomeric electron transfer protein, cytochrome cb(562), into a tetrameric assembly ((C96)RIDC-1(4)) that stably and selectively binds Zn(2+) and displays a metal-dependent conformational change reminiscent of a signaling protein. A thorough analysis of the metal binding properties of (C96)RIDC-1(4) reveals that it can also stably harbor other divalent metals with affinities that rival (Ni(2+)) or even exceed (Cu(2+)) those of Zn(2+) on a per site basis. Nevertheless, this analysis suggests that our templating strategy simultaneously introduces an increased bias toward binding a higher number of Zn(2+) ions (four high affinity sites) versus Cu(2+) or Ni(2+) (two high affinity sites), ultimately leading to the exclusive selectivity of (C96)RIDC-1(4) for Zn(2+) over those ions. More generally, our results indicate that an initial metal-driven nucleation event followed by the formation of a stable protein architecture around the metal provides a straightforward path for generating structural and functional diversity.