RESUMEN
The membrane proximal external region (MPER) of HIV-1 envelope glycoprotein (gp) 41 is an attractive vaccine target for elicitation of broadly neutralizing antibodies (bNAbs) by vaccination. However, current details regarding the quaternary structural organization of the MPER within the native prefusion trimer [(gp120/41)3] are elusive and even contradictory, hindering rational MPER immunogen design. To better understand the structural topology of the MPER on the lipid bilayer, the adjacent transmembrane domain (TMD) was appended (MPER-TMD) and studied. Membrane insertion of the MPER-TMD was sensitive both to the TMD sequence and cytoplasmic residues. Antigen binding of MPER-specific bNAbs, in particular 10E8 and DH511.2_K3, was significantly impacted by the presence of the TMD. Furthermore, MPER-TMD assembly into 10-nm diameter nanodiscs revealed a heterogeneous membrane array comprised largely of monomers and dimers, as enumerated by bNAb Fab binding using single-particle electron microscopy analysis, arguing against preferential trimeric association of native MPER and TMD protein segments. Moreover, introduction of isoleucine mutations in the C-terminal heptad repeat to induce an extended MPER α-helical bundle structure yielded an antigenicity profile of cell surface-arrayed Env variants inconsistent with that found in the native prefusion state. In line with these observations, electron paramagnetic resonance analysis suggested that 10E8 inhibits viral membrane fusion by lifting the MPER N-terminal region out of the viral membrane, mandating the exposure of residues that would be occluded by MPER trimerization. Collectively, our data suggest that the MPER is not a stable trimer, but rather a dynamic segment adapted for structural changes accompanying fusion.
Asunto(s)
Membrana Celular/virología , Proteína gp41 de Envoltorio del VIH/química , VIH-1/inmunología , Anticuerpos Neutralizantes/inmunología , Membrana Celular/inmunología , Anticuerpos Anti-VIH/inmunología , Proteína gp41 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/química , VIH-1/genética , Humanos , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/inmunología , Dominios ProteicosRESUMEN
Insulin and related proteins, which have been found not only in mammals, birds, reptiles, amphibians, fish, and cephalochordate, but also in mollusca, insects, and Caenorhabditis elegans, form a large protein family, the insulin superfamily. In comparing their amino acid sequences, a common sequence characteristic, the insulin structural motif, is found in all members of the superfamily. The structural motif is deduced to be the sequence basis of the identical disulfide linkages and similar three-dimensional structures of the superfamily. The insulin superfamily provides a series of disulfide-containing proteins for the studies of in vitro oxidative folding. The in vitro folding pathways of insulin-like growth factor-1 (IGF-1), porcine insulin precursor (PIP), human proinsulin, and Amphioxus insulin-like peptide (AILP) have been established by capture and analysis of the folding intermediates during their in vitro oxidative folding process. The family also provides an excellent system for study of the sequence structure relation: insulin and IGF-1 share high amino acid sequence homology, but they have evolved different folding behaviors. The sequence determinants of their different folding behaviors have been revealed by analyzing the folding behaviors of those global and local insulin/IGF-1 hybrids.
Asunto(s)
Insulina/metabolismo , Pliegue de Proteína , Secuencia de Aminoácidos , Animales , Humanos , Insulina/química , Factor I del Crecimiento Similar a la Insulina/metabolismo , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Oxidación-Reducción , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
We use the procedure established for 'disulfide stability analysis in redox system' to investigate the unfolding process of porcine insulin precursor (PIP). Six major unfolding intermediates have been captured, in which four contain two disulfides, two contain one disulfide. Based on the characterization and analysis of the intermediates an unfolding pathway has been proposed, by which the native PIP unfolded through in turn 2SS and 1SS intermediates into fully reduced form. Besides, the comparison of the intermediates captured in PIP unfolding process with those intermediates captured in its refolding process revealed that some intermediates captured during both unfolding/refolding processes of PIP have identical disulfide pairing pattern, from which we suggest that the unfolding/refolding processes of PIP share some common intermediates but flow in the opposite direction.
Asunto(s)
Proinsulina/química , Pliegue de Proteína , Animales , Tampones (Química) , Cromatografía Líquida de Alta Presión , Glutatión , Disulfuro de Glutatión , Espectrometría de Masas , Oxidación-Reducción , Proinsulina/aislamiento & purificación , Conformación Proteica , Desnaturalización Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , PorcinosRESUMEN
Contributions of the evolutionarily conserved A16Leu and B17Leu to insulin foldability were characterized by evaluating folding properties of single-chain insulin analogs. The results showed A16Leu had much more significant effects on the foldability of insulin than B17Leu.
Asunto(s)
Insulina/química , Leucina/análisis , Pliegue de Proteína , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Disulfuros/química , Insulina/genética , Insulina/aislamiento & purificación , Insulina/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proinsulina/química , Proinsulina/genética , Proinsulina/aislamiento & purificación , Proinsulina/metabolismo , Conformación Proteica , Transporte de Proteínas , PorcinosRESUMEN
Insulin is a double-chain (designated A and B chain respectively) protein hormone containing three disulfides, while insulin is synthesized in vivo as a single-chain precursor and folded well before being released from B-cells. Although the structure and function of insulin have been well characterized, the progress in oxidative folding pathway studies of insulin has been very slow, mainly due to the difficulties brought about by its disulfide-linked double-chain structure. To overcome these difficulties, we recently studied the in vitro oxidative folding process of two single-chain insulins: porcine insulin precursor (PIP) and human proinsulin (HPI). Based on the analysis of the intermediates captured during folding process, the folding pathways have been proposed for PIP and HPI separately. Similarities between the two folding pathways disclose some common principles that govern the insulin folding process. The following unfolding studies of PIP and HPI further indicate that C-peptide might also function during the folding of proinsulin. Here, we gave a brief review on in vitro folding/unfolding process of insulin and single-chain insulin. The implication of these studies on protein folding has also been discussed.
Asunto(s)
Insulina/química , Insulina/metabolismo , Pliegue de Proteína , Secuencia de Aminoácidos , Animales , Disulfuros/química , Humanos , Insulina/genética , Datos de Secuencia Molecular , Oxidación-Reducción , Conformación Proteica , Desnaturalización Proteica , Precursores de Proteínas/química , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , PorcinosRESUMEN
Designing an immunogen for effective neutralizing antibody induction against diverse primary isolates of human immunodeficiency virus type 1 (HIV-1) is a high priority for HIV-1 vaccine development. Soluble gp120 envelope (Env) glycoprotein subunit vaccines elicit high titers of antibodies that neutralize T cell line-adapted (TCLA) strains but the antibodies possess poor neutralizing activity against many primary isolates. Previously, we generated soluble trimeric recombinant gp140 from the HIV-1 primary isolate ADA. Here we compared monomeric ADAgp120 and trimeric ADAgp140 as immunogens for neutralizing antibody responses in guinea pigs. Both immunogens generated a neutralizing antibody response that was detectable against the vaccine strain and several heterologous strains. The magnitude of this response was significantly greater in ADAgp140-immunized animals when measured against the TCLA strain, MN, and the R5 primary isolate, Bal. Two additional isolates (SS1196 and Bx08) were neutralized equally by sera from both groups of animals whereas other isolates were neutralized weakly or not at all. Despite equal titers of V3 loop specific binding antibodies in sera from both groups of animals, neutralization of ADA by sera from gp140-immunized animals was insensitive to the presence of ADA-V3 peptide, whereas addition of this peptide to sera from gp120- immunized animals blocked all detectable neutralizing activity against ADA. These results support the idea that trimeric gp140 is an improved immunogen compared to monomeric gp120 but that additional improvements are required to afford broad protection against a spectrum of heterologous primary HIV-1 isolates. This ADAgp140 immunogen may be considered a starting point from which to engineer additional improvements for cross-reactive neutralizing antibody induction.
Asunto(s)
Vacunas contra el SIDA/inmunología , Productos del Gen env/inmunología , Anticuerpos Anti-VIH/sangre , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Vacunas contra el SIDA/administración & dosificación , Secuencia de Aminoácidos , Animales , Dimerización , Productos del Gen env/administración & dosificación , Productos del Gen env/química , Cobayas , Proteína gp120 de Envoltorio del VIH/administración & dosificación , Proteína gp120 de Envoltorio del VIH/química , Infecciones por VIH/inmunología , VIH-1/metabolismo , Humanos , Inmunización , Datos de Secuencia Molecular , Pruebas de Neutralización , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia HumanaRESUMEN
Insulin receptor is a transmembrane protein consisting of four subunits, that form a heterotetramer(alpha(2)beta(2))with molecular weight of 350 kD. Because the extracellular subunit(alpha)consists of 731 residues and a cysteine-rich domain, it is difficult to express and crystallize such a large ligand-binding subunit, thus hampering further study on "insulin-receptor" complex. Based on the fact that the domains L1 and L2 of the alpha subunit, consisted of 119 and 118 residues, contained the high and low affinity insulin binding sites, respectively, the cDNAs of L1 and L2 were obtained from a human placental cDNA library by PCR. The cDNAs of L1, L2 and L1-(Ala)(10)-L2(designed ten-alanine-connected L1 and L2)were cloned, respectively, into an expression plasmid pET-3a, and E.coli BL21(DE3)transformants with such plasmids were successfully induced to express the goal proteins. The expression products were isolated and purified by the washing and solubilization of inclusion body, gel filtration chromatography and ion exchange chromatography. Each final product displayed a single band, corresponding the purity above 99%, in SDS-PAGE. These products have also been confirmed respectively as the L1, L2 and L1-(Ala)(10)-L2 by DNA sequencing, amino acid composition analysis and N-terminal amino acid sequencing.
RESUMEN
Recombinant single-chain insulin (PIP) contains three disulfide bonds. In the presence of denaturants and thiol reagents, the native structure of PIP was disturbed and its native disulfides were shuffled to form a mixture of scrambled isomers which have different degrees of unfolding. In this paper the unfolding degrees of PIP in urea or guanidine hydrochloride containing 0.2 mmol/L 2-mercaptoethanol was analyzed by reverse-phase HPLC and far-UV circular dichroism(CD). The peptide mapping of PIP scrambles demonstrated that PIP had shuffled its native disulfides under the condition we used. Among others, a major non-natural PIP disulfide isomer was purified and its refolding in vitro was investigated. These results show that PIP has only one thermodynamically stable disulfide linkage, and the non-natural disulfide isomers could refold in vitro efficiently to from native PIP. On basis of these, the differences between PIP, IGF-I and insulin on unfolding and refolding were discussed.
RESUMEN
The human immunodeficiency virus-1 (HIV-1) envelope glycoprotein (Env) is comprised of non-covalently associated gp120/gp41 subunits that form trimeric spikes on the virion surface. Upon binding to host cells, Env undergoes a series of structural transitions, leading to gp41 rearrangement necessary for fusion of viral and host membranes. Until now, the prefusion state of gp41 ectodomain (e-gp41) has eluded molecular and structural analysis, and thus assessment of the potential of such an e-gp41 conformer to elicit neutralizing antibodies has not been possible. Considering the importance of gp120 amino (C1) and carboxyl (C5) segments in the association with e-gp41, we hypothesize that these regions are sufficient to maintain e-gp41 in a prefusion state. Based on the available gp120 atomic structure, we designed several truncated gp140 variants by including the C1 and C5 regions of gp120 in a gp41 ectodomain fragment. After iterative cycles of protein design, expression and characterization, we obtained a variant truncated at Lys(665) that stably folds as an elongated trimer under physiologic conditions. Several independent biochemical/biophysical analyses strongly suggest that this mini-Env adopts a prefusion e-gp41 configuration that is strikingly distinct from the postfusion trimer-of-hairpin structure. Interestingly, this prefusion mini-Env, lacking the fragment containing the 2F5/4E10 neutralizing monoclonal antibody binding sites, displays no detectable HIV-neutralizing epitopes when employed as an immunogen in rabbits. The result of this immunogenicity study has important implications for HIV-1 vaccine design efforts. Moreover, this engineered mini-Env protein should facilitate three-dimensional structural studies of the prefusion e-gp41 and serve to guide future attempts at pharmacologic and immunologic intervention of HIV-1.
Asunto(s)
Productos del Gen env/química , Proteína gp41 de Envoltorio del VIH/química , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Baculoviridae/metabolismo , Sitios de Unión , Línea Celular , Reactivos de Enlaces Cruzados/farmacología , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Productos del Gen env/metabolismo , Genes Reporteros , Vectores Genéticos , Glicosilación , Proteínas Fluorescentes Verdes/metabolismo , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteína gp41 de Envoltorio del VIH/metabolismo , VIH-1/metabolismo , Concentración de Iones de Hidrógeno , Inmunoprecipitación , Insectos , Luz , Luciferasas/metabolismo , Lisina/química , Microscopía Electrónica , Modelos Biológicos , Modelos Genéticos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Péptido Hidrolasas/metabolismo , Polisacáridos/química , Unión Proteica , Conformación Proteica , Ingeniería de Proteínas/métodos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Conejos , Dispersión de Radiación , Resonancia por Plasmón de Superficie , Factores de Tiempo , Tripsina/farmacología , Productos del Gen env del Virus de la Inmunodeficiencia HumanaRESUMEN
We have investigated the in vitro refolding process of human proinsulin (HPI) and an artificial mini-C derivative of HPI (porcine insulin precursor, PIP), and found that they have significantly different disulfide-formation pathways. HPI and PIP differ in their amino acid sequences due to the presence of the C-peptide linker found in HPI, therefore suggesting that the C-peptide linker may be responsible for the observed difference in folding behaviour. However, the manner in which the C-peptide contributes to this difference is still unknown. We have used both the disulfide scrambling method and a redox-equilibrium assay to assess the stability of the disulfide bridges. The results show that disulfide reshuffling is easier to induce in HPI than in PIP by the addition of thiol reagent. Thus, the C-peptide may affect the unique folding pathway of HPI by allowing the disulfide bonds of HPI to be easily accessible. The detailed processes of HPI unfolding by reduction of its disulfide bonds and by disulfide scrambling methods were also investigated. In the reductive unfolding process no accumulation of intermediates was detected. In the process of unfolding by disulfide scrambling, HPI gradually rearranged its disulfide bonds to form three major isomers G1, G2 and G3. The most abundant isomer, G1, contains the B7-B19 disulfide bridge. Based on far-UV CD spectra, native gel analysis and cleavage by endoproteinase V8, the G1 isomer has been shown to resemble the intermediate P4 found in the refolding process of HPI. Finally, the major isomer G1 is allowed to refold to native protein HPI by disulfide rearrangement, which indicates that a similar molecular mechanism may exist for the unfolding and refolding process of HPI.
Asunto(s)
Fragmentos de Péptidos/química , Proinsulina/química , Pliegue de Proteína , Secuencia de Aminoácidos , Disulfuros/química , Humanos , Datos de Secuencia MolecularRESUMEN
Human insulin is a double-chain peptide that is synthesized in vivo as a single-chain human proinsulin (HPI). We have investigated the disulfide-forming pathway of a single-chain porcine insulin precursor (PIP). Here we further studied the folding pathway of HPI in vitro. While the oxidized refolding process of HPI was quenched, four obvious intermediates (namely P1, P2, P3, and P4, respectively) with three disulfide bridges were isolated and characterized. Contrary to the folding pathway of PIP, no intermediates with one- or two-disulfide bonds could be captured under different refolding conditions. CD analysis showed that P1, P2, and P3 retained partially structural conformations, whereas P4 contained little secondary structure. Based on the time-dependent distribution, disulfide pair analysis, and disulfide-reshuffling process of the intermediates, we have proposed that the folding pathway of HPI is significantly different from that of PIP. These differences reveal that the C-peptide not only facilitates the folding of HPI but also governs its kinetic folding pathway of HPI. Detailed analysis of the molecular folding process reveals that there are some similar folding mechanisms between PIP and HPI. These similarities imply that the initiation site for the folding of PIP/HPI may reside in the central alpha-helix of the B-chain. The formation of disulfide A20-B19 may guide the transfer of the folding information from the B-chain template to the unstructured A-chain. Furthermore, the implications of this in vitro refolding study on the in vivo folding process of HPI have been discussed.