RESUMEN
Alphaviruses are enveloped, single-stranded, positive-sense RNA viruses that often require transmission between arthropod and vertebrate hosts for their sustained propagation. Most alphaviruses encode an opal (UGA) termination codon in nonstructural protein 3 (nsP3) upstream of the viral polymerase, nsP4. The selective constraints underlying the conservation of the opal codon are poorly understood. Using primate and mosquito cells, we explored the role and selective pressure on the nsP3 opal codon through extensive mutational analysis in the prototype alphavirus, Sindbis virus (SINV). We found that the opal codon is highly favored over all other codons in primate cells under native 37°C growth conditions. However, this preference is diminished in mosquito and primate cells grown at a lower temperature. Thus, the primary determinant driving the selection of the opal stop codon is not host genetics but the passaging temperature. We show that the opal codon is preferred over amber and ochre termination codons because it results in the highest translational readthrough and polymerase production. However, substituting the opal codon with sense codons leads to excessive full-length polyprotein (P1234) production, which disrupts optimal nsP polyprotein processing, delays the switch from minus-strand to positive-strand RNA production, and significantly reduces SINV fitness at 37°C; this fitness defect is relieved at lower temperatures. A naturally occurring suppressor mutation unexpectedly compensates for a delayed transition from minus to genomic RNA production by also delaying the subsequent transition between genomic and sub-genomic RNA production. Our study reveals that the opal stop codon is the best solution for alphavirus replication at 37°C, producing enough nsP4 protein to maximize replication without disrupting nsP processing and RNA replication transitions needed for optimal fitness. Our study uncovers the intricate strategy dual-host alphaviruses use at a single codon to optimize fitness.
RESUMEN
Bovine adenovirus (BAdV)-3 genome encodes a 26 kDa core protein designated as protein VII, which localizes to the nucleus/nucleolus. The requirement of a protein VII-complementing cell line for the replication of VII-deleted BAdV-3 suggests that protein VII is required for the production of infectious progeny virions. An analysis of the BAV.VIId+ virus (only phenotypically positive for protein VII) detected no noticeable differences in the expression and incorporation of viral proteins in the virions. Moreover, protein VII does not appear to be essential for the formation of mature BAV.VIId+. However, protein VII appeared to be required for the efficient assembly of mature BAV.VIId- virions. An analysis of the BAV.VIId- virus (genotypically and phenotypically negative for protein VII) in non-complementing cells detected the inefficient release of virions from endosomes, which affected the expression of viral proteins or DNA replication. Moreover, the absence of protein VII altered the proteolytic cleavage of protein VI of BAV.VIId-. Our results suggest that BAdV-3 protein VII appears to be required for efficient production of mature virions. Moreover, the absence of protein VII produces non-infectious BAdV-3 by altering the release of BAdV-3 from endosomes/vesicles.
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Mastadenovirus , Virión , Replicación Viral , Animales , Virión/metabolismo , Virión/genética , Bovinos , Mastadenovirus/genética , Mastadenovirus/fisiología , Mastadenovirus/metabolismo , Línea Celular , Proteínas Virales/metabolismo , Proteínas Virales/genética , Ensamble de Virus , Proteínas del Núcleo Viral/metabolismo , Proteínas del Núcleo Viral/genética , Replicación del ADNRESUMEN
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant of concern, first identified in November 2021, rapidly spread worldwide and diversified into several subvariants. The Omicron spike (S) protein accumulated an unprecedented number of sequence changes relative to previous variants. In this review, we discuss how Omicron S protein structural features modulate host cell receptor binding, virus entry, and immune evasion and highlight how these structural features differentiate Omicron from previous variants. We also examine how key structural properties track across the still-evolving Omicron subvariants and the importance of continuing surveillance of the S protein sequence evolution over time.
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COVID-19 , Humanos , SARS-CoV-2 , Evasión InmuneRESUMEN
On the basis of known published data, six peptide sequences were selected that are potentially capable of being rapidly cleaved by the endosomal protease cathepsin B. For comparison, the cleavage of common linker sequences, polyglycine and polyglycine-serine, by cathepsin B was also studied. Different ends of these peptides were labeled with sulfoCyanine3 and sulfoCyanine5 fluorescent dyes, between which Förster resonant energy transfer (FRET) is possible. The kinetics of cleavage of peptides by cathepsin B was studied on a multimodal plate reader by FRET signal reduction. FKFL and FRRG cleavage sites have been shown to be the most suitable for potential use in various drug delivery systems. These sites are much more efficiently cleaved under slightly acidic conditions of endosomes than at neutral extracellular pH.
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Aminoácidos , Catepsina B , Catepsina B/química , Catepsina B/metabolismo , Aminoácidos/metabolismo , Cinética , Péptidos/química , Endosomas/metabolismo , Sistemas de Liberación de MedicamentosRESUMEN
The prevalence of SARS-CoV-2 as a global health threat has called for population-wide vaccination to curb COVID-19. Hence, the World Health Organization (WHO) has approved several platforms of SARS-CoV-2 vaccines for emergency use. Therefore, a more comprehensive study on the immune response induced by vaccines in diverse individuals is still required. Here, we expressed a local variant of SARS-CoV-2 receptor-binding domain (RBD) and protease cleavage site (PCS), playing a vital role in binding and fusion in Rosetta (DE3). We then characterized it through SDS-PAGE analysis and western blotting. Moreover, we compared and monitored ChAdOx1 nCoV-19 vaccination-induced antibody response in convalescent and healthy vaccinated individuals after the first and second vaccine doses through serologic assay against RBD and PCS, which have not yet been compared. We investigated a cohort of 100 sera samples; based on our parameters, 25 serum samples were selected as convalescent samples and 25 serum samples as healthy samples for comparison. These findings demonstrate that most of the convalescent sera show more reactivity with PCS (80%) than with RBD (56%). Interestingly, IgG antibody response against PCS was more significant in both pre- and post-vaccination in convalescent individuals than in healthy individuals. Indeed, anti-RBD antibody titers were most significant in pre-vaccination and post-first vaccination in convalescent individuals than in healthy individuals and not in pre-vaccination and post-second vaccination. Besides monitoring IgG antibody response against COVID-19, these findings could shed light on the progress, assessment, and efficacy evaluation of SARS-CoV-2 vaccines.Communicated by Ramaswamy H. Sarma.
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COVID-19 , Péptido Hidrolasas , Humanos , Glicoproteína de la Espiga del Coronavirus , Vacunas contra la COVID-19 , Formación de Anticuerpos , ChAdOx1 nCoV-19 , COVID-19/prevención & control , Sueroterapia para COVID-19 , SARS-CoV-2 , Endopeptidasas , Inmunoglobulina G , Anticuerpos NeutralizantesRESUMEN
BACKGROUND: Acid and thermal stabilities are important properties for the preparation of acidic protein beverage. It is an important method for enzymatic modification to improve the functional properties of protein. Irpex lacteus protease showed a selective hydrolysis to soy proteins. The purpose of this study was to investigate the mechanism of enzymatic hydrolysis and its effects on acid and thermal stabilities of soy proteins. RESULTS: The I. lacteus protease selectively hydrolyzed the α and α' subunits of the native soybean ß-conglycinin (7S globulin) to produce products that presented as the 55 kDa band upon sodium dodecyl sulfate polyacrylamide gel electrophoresis. The amino acid sequences of 55 kDa polypeptides were analyzed in gel multi-enzyme digestion followed by liquid chromatography-mass spectrometry. By matching the multi-enzyme digestion peptides with the published polypeptide chain sequences of the α and α' subunits, it was confirmed that the 55 kDa polypeptides were formed by eliminating amino acid residues on both sides of the N- and C-terminals. From the published protein structure database (https://www.uniprot.org/), it is known that the cleaved peptide bonds were in extension regions. Non-selective enzyme hydrolysis of both ß-conglycinin (7S globulin) and glycinin (11S globulin), with corresponding drastic increases in the degree of hydrolysis, was observed when the substrates were preheated to the denaturation degree of 40% and above. However, 55 kDa hydrolyzed products and B polypeptides showed some extent of resistance to the proteolysis by I. lacteus protease even if denaturation degree was 100%. Both selective and non-selective hydrolysis of soy proteins by I. lacteus protease improved the acid and heat stabilities under the same hydrolysis conditions (enzyme/substrate ratio, time, and temperature). CONCLUSION: Enzymatic hydrolysis of soybean proteins by the I. lacteus protease can effectively improve the acid and thermal stabilities of proteins. This discovery is significant to avoid aggregation during processing in the beverage industry. In the near future, the protease has potential application value for modification of other proteins. © 2022 Society of Chemical Industry.
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Globulinas , Proteínas de Soja , Proteínas de Soja/química , Péptido Hidrolasas/metabolismo , Harina , Glycine max/química , Antígenos de Plantas/metabolismo , Proteínas de Almacenamiento de Semillas/metabolismo , Péptidos/química , Endopeptidasas/metabolismo , Globulinas/químicaRESUMEN
Coronaviruses (CoVs) initiate replication by translation of the positive-sense RNA genome into the replicase polyproteins connecting 16 nonstructural protein domains (nsp1-16), which are subsequently processed by viral proteases to yield mature nsp. For the betacoronavirus murine hepatitis virus (MHV), total inhibition of translation or proteolytic processing of replicase polyproteins results in rapid cessation of RNA synthesis. The nsp5-3CLpro (Mpro) processes nsps7-16, which assemble into functional replication-transcription complexes (RTCs), including the enzymatic nsp12-RdRp and nsp14-exoribonuclease (ExoN)/N7-methyltransferase. The nsp14-ExoN activity mediates RNA-dependent RNA proofreading, high-fidelity RNA synthesis, and replication. To date, the solved partial RTC structures, biochemistry, and models use or assume completely processed, mature nsp. Here, we demonstrate that in MHV, engineered deletion of the cleavage sites between nsp13-14 and nsp14-15 allowed recovery of replication-competent virus. Compared to wild-type (WT) MHV, the nsp13-14 and nsp14-15 cleavage deletion mutants demonstrated delayed replication kinetics, impaired genome production, altered abundance and patterns of recombination, and impaired competitive fitness. Further, the nsp13-14 and nsp14-15 mutant viruses demonstrated mutation frequencies that were significantly higher than with the WT. The results demonstrate that cleavage of nsp13-14 or nsp14-15 is not required for MHV viability and that functions of the RTC/nsp14-ExoN are impaired when assembled with noncleaved intermediates. These data will inform future genetic, structural, biochemical, and modeling studies of coronavirus RTCs and nsp 13, 14, and 15 and may reveal new approaches for inhibition or attenuation of CoV infection. IMPORTANCE Coronavirus replication requires proteolytic maturation of the nonstructural replicase proteins to form the replication-transcription complex. Coronavirus replication-transcription complex models assume mature subunits; however, mechanisms of coronavirus maturation and replicase complex formation have yet to be defined. Here, we show that for the coronavirus murine hepatitis virus, cleavage between the nonstructural replicase proteins nsp13-14 and nsp14-15 is not required for replication but does alter RNA synthesis and recombination. These results shed new light on the requirements for coronavirus maturation and replication-transcription complex assembly, and they may reveal novel therapeutic targets and strategies for attenuation.
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Exorribonucleasas , Aptitud Genética , Virus de la Hepatitis Murina , Proteolisis , ARN Viral , Proteínas no Estructurales Virales , Proteinas del Complejo de Replicasa Viral , Animales , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Ratones , Virus de la Hepatitis Murina/enzimología , Virus de la Hepatitis Murina/genética , Virus de la Hepatitis Murina/crecimiento & desarrollo , Virus de la Hepatitis Murina/fisiología , Mutación , Poliproteínas/química , Poliproteínas/genética , Poliproteínas/metabolismo , ARN Viral/biosíntesis , ARN Viral/genética , Recombinación Genética , Transcripción Genética , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Proteinas del Complejo de Replicasa Viral/química , Proteinas del Complejo de Replicasa Viral/genética , Proteinas del Complejo de Replicasa Viral/metabolismo , Replicación ViralRESUMEN
Substrate sequence specificity is a fundamental characteristic of proteolytic enzymes. Hundreds of proteases are encoded in plant genomes, but the vast majority of them have not been characterized and their distinct specificity remains largely unknown. Here we present our current protocol for profiling sequence specificity of plant proteases using Proteomic Identification of Cleavage Sites (PICS). This simple, cost-effective protocol is suited for detailed, time-resolved specificity profiling of purified or enriched proteases. The isolated active protease or fraction with enriched protease activity together with a suitable control are incubated with split aliquots of proteome-derived peptide libraries, followed by identification of specifically cleaved peptides using quantitative mass spectrometry. Detailed specificity profiles are obtained by alignment of many individual cleavage sites. The chapter covers preparation of complementary peptide libraries from heterologous sources, the cleavage assay itself, as well as mass spectrometry data analysis.
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Biblioteca de Péptidos , Proteoma , Endopeptidasas , Péptido Hidrolasas/metabolismo , Proteolisis , Proteoma/análisis , Proteómica/métodos , Especificidad por Sustrato , Espectrometría de Masas en Tándem/métodosRESUMEN
Complications related to atherosclerosis account for approximately 1 in 4 deaths in the United States and treatment has focused on lowering serum LDL-cholesterol levels with statins. However, approximately 50% of those diagnosed with atherosclerosis have blood cholesterol levels within normal parameters. Human fortilin is an anti-apoptotic protein and a factor in macrophage-mediated atherosclerosis and is hypothesized to protect inflammatory macrophages from apoptosis, leading to subsequent cardiac pathogenesis. Fortilin is unique because it provides a novel drug target for atherosclerosis that goes beyond lowering cholesterol and utilization of a solution nuclear magnetic resonance (NMR) spectroscopy, structure-based drug discovery approach requires milligram quantities of pure, bioactive, recombinant fortilin. Here, we designed expression constructs with different affinity tags and protease cleavage sites to find optimal conditions to obtain the quantity and purity of protein necessary for structure activity relationship studies. Plasmids encoding fortilin with maltose binding protein (MBP), 6-histidine (6His) and glutathione-S-transferase (GST), N- terminal affinity tags were expressed and purified from Escherichia coli (E. coli). Cleavage sites with tobacco etch virus (TEV) protease and human rhinovirus (HRV) 3C protease were assessed. Despite high levels of expression of soluble protein, the fusion constructs were resistant to proteinases without the inclusion of amino acids between the cleavage site and N-terminus. We surveyed constructs with increasing lengths of glycine/serine (GGS) linkers between the cleavage site and fortilin and found that inclusion of at least one GGS insert led to successful protease cleavage and pure fortilin with conserved binding to calcium as measured by NMR.
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Calcio/química , Proteínas Recombinantes de Fusión/genética , Proteína Tumoral Controlada Traslacionalmente 1/genética , Proteasas Virales 3C/química , Sitios de Unión , Calcio/metabolismo , Clonación Molecular , Endopeptidasas/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Histidina/genética , Histidina/metabolismo , Humanos , Proteínas de Unión a Maltosa/genética , Proteínas de Unión a Maltosa/metabolismo , Modelos Moleculares , Oligopéptidos/genética , Oligopéptidos/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteolisis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Solubilidad , Proteína Tumoral Controlada Traslacionalmente 1/química , Proteína Tumoral Controlada Traslacionalmente 1/metabolismoRESUMEN
Development of an effective HIV-1 vaccine has been a great challenge faced by the research community. Recently a novel strategy targeting the viral protease cleavage sites (PCSs) has been tested and shown promising results. This T cell-based vaccine strategy depends on individuals expressing certain HLA class I molecules and since each population has unique distributions of HLA class I alleles, population coverage analysis is required to assess feasibility. Utilizing the validated CD8 T cell epitope data from previous studies we conducted coverage analysis of an HIV-1 vaccine targeting the sequences surrounding all 12-PCSs, Gag-PCSs, and Pol-PCSs. The population coverage, average epitope hit, and minimum number of epitopes recognized by 90% of the population (PC90) was compiled for 66 countries and 16 geographical regions using the web tool provided by "Immune Epitope Database and Analysis Resource". Our analysis shows that the coverage for an HIV-1 vaccine targeting sequences surrounding all 12 PCSs, 5 PCSs in Gag or 6 PCSs in Pol can cover ~ 70% to ~ 100% of the populations analyzed. There was no statistical difference in population coverages for the majority of populations examined when comparing the CD8 T cell epitope sets (12-PCSs, Gag-PCSs, and Pol-PCSs). As expected, vaccines targeting more sequences will have more CD8 T cell epitopes, as the mean average epitope hit for the 12-PCSs, Gag-PCSs, and Pol-PCSs across all countries studied was 9.45, 4.76, and 4.74, respectively, and across all geographical regions was 9.76, 4.99, and 4.92, respectively. The average PC90 for the 12-PCSs, Gag-PCSs, and Pol-PCSs across all countries studied was 2.53, 1.31, and 1.41, respectively, and across all geographical regions was 2.24, 1.23, and 1.29, respectively. Thus, vaccines targeting sequences surrounding the HIV-1 PCSs can cover broad populations; however, whether targeting a subset of the PCSs is sufficient to prevent acquisition requires further preclinical investigation.
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Vacunas contra el SIDA , Infecciones por VIH , VIH-1 , Epítopos de Linfocito T , Infecciones por VIH/prevención & control , VIH-1/genética , Humanos , Péptido Hidrolasas , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Adenoviruses have served as a model for investigating viral-cell interactions and discovering different cellular processes, such as RNA splicing and DNA replication. In addition, the development and evaluation of adenoviruses as the viral vectors for vaccination and gene therapy has led to detailed investigations about adenovirus biology, including the structure and function of the adenovirus encoded proteins. While the determination of the structure and function of the viral capsid proteins in adenovirus biology has been the subject of numerous reports, the last few years have seen increased interest in elucidating the structure and function of the adenovirus core proteins. Here, we provide a review of research about the structure and function of the adenovirus core proteins in adenovirus biology.
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Adenoviridae/genética , Proteínas Virales/genética , Infecciones por Adenoviridae/virología , Animales , Proteínas de la Cápside/genética , Replicación del ADN/genética , ADN Viral/genética , Humanos , Replicación Viral/genéticaRESUMEN
A protocol is described for the isolation of recombinant polyhistidine-tagged membrane proteins from overexpressing Escherichia coli cells. The gene encoding a target membrane protein is cloned into an expression plasmid and then introduced into E. coli cells for overexpression. Membranes from bacterial cells are isolated and the tagged target membrane protein is solubilized in detergent and subsequently bound to an affinity matrix. Tagged proteins are commonly eluted by an excess of a solute that competes for the binding to the matrix. Alternatively, amino acid sequence-specific proteases can be used to cleave off the affinity purification tag directly on the purification column (i.e., on-column cleavage). This selectively releases the target protein and allows subsequent elution. Importantly, this step represents an additional purification step and can significantly increase the purity of the isolated protein.
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Cromatografía de Afinidad/métodos , Proteínas de la Membrana/aislamiento & purificación , Péptido Hidrolasas/metabolismo , Proteínas Recombinantes de Fusión/aislamiento & purificación , Secuencia de Aminoácidos , Membrana Celular/química , Clonación Molecular/métodos , Endopeptidasas/química , Endopeptidasas/metabolismo , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Histidina/química , Histidina/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Organismos Modificados Genéticamente , Péptido Hidrolasas/química , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transformación BacterianaRESUMEN
Cysteine cathepsins are critical components of the adaptive immune system involved in the generation of epitopes for presentation on human leukocyte antigen (HLA) molecules and have been implicated in degradation of autoantigens. Immunoglobulin variable regions with somatic mutations and random complementarity region 3 amino acid composition are inherently immunogenic. T cell reactivity towards immunoglobulin variable regions has been investigated in relation to specific diseases, as well as reactivity to therapeutic monoclonal antibodies. Yet, how the immunoglobulins, or the B cell receptors, are processed in endolysosomal compartments of professional antigen presenting cells has not been described in detail. Here we present in silico and in vitro experimental evidence suggesting that cysteine cathepsins S, L and B may have important roles in generating peptides fitting HLA class II molecules, capable of being presented to T cells, from monoclonal antibodies as well as from central nervous system proteins including a well described autoantigen. By combining neural net models with in vitro proteomics experiments, we further suggest how such degradation can be predicted, how it fits with available cellular models, and that it is immunoglobulin heavy chain variable family dependent. These findings are relevant for biotherapeutic drug design as well as to understand disease development. We also suggest how these tools can be improved, including improved machine learning methodology.
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Catepsinas/química , Catepsinas/metabolismo , Cisteína/metabolismo , Inmunoglobulina G/química , Inmunoglobulina G/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Inmunoglobulina G/genética , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/metabolismo , Conformación Molecular , Unión Proteica , Proteolisis , Reproducibilidad de los Resultados , Relación Estructura-ActividadRESUMEN
The formation of disulfide bonds in proteins is an important post-translational modification that is critical for stabilizing the native structures of proteins, particularly proteins exposed to oxidizing environments. For this reason, most cysteines in secreted proteins or protein domains on the surface of the cell are in disulfides, whereas most cysteines in the cytoplasm are in the unmodified -SH form. Disulfide linkages must be experimentally determined, as they cannot be predicted from amino acid sequence. These assignments provide insights into three-dimensional structure and contribute to the understanding of structural-functional relationships. This unit details a series of protocols that have been applied successfully to map disulfide bonds in proteins. The general strategy involves chemical or proteolytic cleavage of the protein followed by chromatographic separation of the resultant peptides. Mass spectrometry is used to identify disulfide-containing peptides and determine sites of disulfide linkage. A partial reduction and alkylation strategy for mapping disulfide linkages in peptides with multiple disulfide bonds is also presented. © 2019 by John Wiley & Sons, Inc.
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Disulfuros/análisis , Péptidos/análisis , Proteínas/química , Alquilación , Cromatografía Líquida de Alta Presión , Cisteína/química , Disulfuros/química , Mapeo Peptídico , Péptidos/química , Desnaturalización Proteica , Dominios Proteicos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The foot-and-mouth disease virus capsid precursor, P1-2A, is cleaved by the 3C protease (3Cpro) to VP0, VP3, VP1 and 2A. The P1-2A precursor (wt or mutant) was expressed alone or with 3Cpro and processing of P1-2A was determined. The VP2 K217R and VP3 I2P substitutions (near the VP0/VP3 junction) strongly reduced the processing at this junction by 3Cpro while the substitution VP2 K217E blocked cleavage. At the VP3/VP1 junction, the substitutions VP3 Q2221P and VP1 T1P each severely inhibited processing at this site. Blocking cleavage at either junction did not prevent processing elsewhere in P1-2A. These modifications were also introduced into full-length FMDV RNA; only wt and the VP2 K217R mutant were viable. Uncleaved VP0-VP3 and the processed products were observed within cells infected with the mutant virus. The VP0-VP3 was not incorporated into empty capsids or virus particles. The three junctions within P1-2A are processed by 3Cpro independently.
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Proteínas de la Cápside/metabolismo , Cisteína Endopeptidasas/metabolismo , Virus de la Fiebre Aftosa/enzimología , Virus de la Fiebre Aftosa/fisiología , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Virales/metabolismo , Proteasas Virales 3C , Animales , ProteolisisRESUMEN
Simian immunodeficiency virus (SIV) infection of Mauritian cynomolgus macaques (MCMs) is an increasingly important nonhuman primate model for HIV vaccine research. We previously reported that in MCMs anti-SIV antibodies can be naturally developed without exogenous infection or vaccination, and that a vaccine targeting SIV protease cleavage sites (PCS) can cross-induce antibodies to non-PCS SIV antigens. We speculate that this is potentially caused by the existence of endogenous SIV-like antigens. External stimuli (such as environmental factors and vaccination) may induce expression of endogenous SIV-like antigens to elicit these antibodies. Database and mass spectrometry analyses were conducted to search for such antigens. We identified endogenous SIV-like DNA sequences in cynomolgus macaque genome and non-PCS peptide homologous to SIV Env protein in PBMCs of a PCS-vaccinated monkey. Our preliminary insights suggest that endogenous SIV-like antigens may be one of the possible reasons for the natural and cross-inducible SIV antibodies in MCMs.
RESUMEN
HIV preferentially infects activated CD4+ T cells and mutates rapidly. The classical vaccine approach aimed to generate broad immune responses to full HIV proteins largely failed to address the potential adverse impact of increased number of activated CD4+ T cells as viral targets. Learning from natural immunity observed in a group of HIV resistant Kenyan female sex workers, we are testing a novel vaccine approach. It focuses immune response to the highly conserved sequences surrounding the HIV protease cleavage sites (PCS) to disrupt viral maturation, while limiting excessive immune activation. Our pilot studies using nonhuman primate SIV infection models suggest that this approach is feasible and promising.
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Vacunas contra el SIDA , Infecciones por VIH/prevención & control , Proteasa del VIH/inmunología , Proteasa del VIH/metabolismo , VIH-1/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Secuencia Conservada/genética , Secuencia Conservada/inmunología , Femenino , Infecciones por VIH/epidemiología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Proteasa del VIH/genética , VIH-1/enzimología , VIH-1/genética , Humanos , Inmunidad Innata , Kenia/epidemiología , Macaca mulatta , Proyectos Piloto , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Virus de la Inmunodeficiencia de los Simios/inmunologíaRESUMEN
Fibroblast growth factor (FGF) 9 has oncogenic activity and plays an important role in the development of ovarian, lung, prostate, and gastric cancers. In the present study, with the aim of reducing the cost of utilizing growth factors in cancer research, a simple and efficient method for the preparation of recombinant human (rh)FGF9 in Escherichia coli was established. The rhFGF9 fusion protein (6 × His-TEV-rhFGF9) and the native protein released by tobacco etch virus (TEV) protease were obtained using a Ni-NTA system, with > 95% purity. Both purified forms of rhFGF9, with and without fusion tags, significantly stimulated the proliferation of NIH3T3 cells. The FGF9 subfamily, including FGF9, FGF16, and FGF20, in addition to rhFGF16, rhFGF9, and rhFGF20, were shown to stimulate the proliferation and migration of HuH7 human hepatocellular carcinoma (HCC) cells. Mechanistic studies revealed that the stimulation of HuH7 cell proliferation and migration with rhFGF9 and rhFGF20 were associated with the activation of the extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB) pathways and matrix metalloproteinase-26 (MMP26). Inhibition of the ERK and NF-κB pathways blocked cell migration, and NF-κB was demonstrated to be regulated by ERK. Therefore, the present study demonstrates a simple method for the preparation of biologically active rhFGF9 protein. Furthermore, the results indicate that exogenous rhFGF9- and rhFGF20-activated ERK/NF-κB signal transduction pathways play important roles in the regulation of HCC cell proliferation and migration, and this discovery helps to find the potential for new solutions of the treatment of liver cancer.
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Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Factor 9 de Crecimiento de Fibroblastos/metabolismo , Hepatocitos/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Animales , Línea Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Factor 9 de Crecimiento de Fibroblastos/genética , Factor 9 de Crecimiento de Fibroblastos/aislamiento & purificación , Expresión Génica , Humanos , Ratones , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
Recombinant expression of disulfide-reticulated peptides and proteins is often challenging. We describe a method that exploits the periplasmic disulfide-bond forming machinery of Escherichia coli and combines this with a cleavable, solubility-enhancing fusion tag to obtain higher yields of correctly folded target protein than is achievable via cytoplasmic expression. The protocols provided herein cover all aspects of this approach, from vector construction and transformation to purification of the cleaved target protein and subsequent quality control.
Asunto(s)
Disulfuros/química , Escherichia coli/genética , Péptidos/química , Péptidos/genética , Periplasma/genética , Cromatografía de Afinidad/métodos , Cromatografía Líquida de Alta Presión/métodos , Disulfuros/aislamiento & purificación , Disulfuros/metabolismo , Electroforesis en Gel de Poliacrilamida/métodos , Péptidos/aislamiento & purificación , Plásmidos/genética , Pliegue de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Solubilidad , Transformación GenéticaRESUMEN
Scorpion venom peptide blockers (KTx) of potassium channels are a valuable tool for structure-functional studies and prospective candidates for medical applications. Low yields of recombinant KTx hamper their wide application. We developed convenient and efficient bioengineering approach to a large-scale KTx production that meets increasing demands for such peptides. Maltose-binding protein was used as a carrier for cytoplasmic expression of folded disulfide-rich KTx in E. coli. TEV protease was applied for in vitro cleavage of the target peptide from the carrier. To produce KTx with retained native N-terminal sequence, the last residue of TEV protease cleavage site (CSTEV) was occupied by the native N-terminal residue of a target peptide. It was shown that decreased efficiency of hydrolysis of fusion proteins with non-canonical CSTEV can be overcome without by-product formation. Disulfide formation and folding of a target peptide occurred in cytoplasm eliminating the need for renaturation procedure in vitro. Advantages of this approach were demonstrated by producing six peptides with three disulfide bonds related to four KTx sub-families and achieving peptide yields of 12-22mg per liter of culture. The developed approach can be of general use for low-cost production of various KTx, as well as other disulfide-rich peptides and proteins.