RESUMO
Efficient expansion of hematopoietic progenitor cells requires, at least, the simultaneous stimulation of the receptors c-kit and gp130. While c-kit is activated by SCF; gp130, in cells which do not express sufficient amounts of IL-6R, can be activated by the complex of soluble IL-6R (sIL-6R) and IL-6. The therapeutic use of IL-6/sIL-6R, however, has been hampered by the high concentrations of the sIL-6R protein required. We have designed a fusion protein of sIL-6R and IL-6, linked by a flexible peptide chain, that was expressed to high levels. On gp130 expressing cells the fusion protein turned out to be fully active at 100 to 1,000-fold lower concentration than the combination of unlinked IL-6 and IL-6R. The fusion protein was used to effectively expand human hematopoietic progenitor cells ex vivo in a dose dependent fashion.
Assuntos
Antígenos CD/química , Divisão Celular/efeitos dos fármacos , Citocinas/química , Células-Tronco Hematopoéticas/citologia , Interleucina-6/química , Estrutura Secundária de Proteína , Receptores de Interleucina/química , Proteínas Recombinantes de Fusão/química , Sequência de Aminoácidos , Antígenos CD/análise , Antígenos CD/biossíntese , Antígenos CD/fisiologia , Antígenos CD34/análise , Carcinoma Hepatocelular , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Citocinas/biossíntese , Citocinas/farmacologia , Desenho de Fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Interleucina-6/biossíntese , Interleucina-6/farmacologia , Neoplasias Hepáticas , Modelos Estruturais , Receptores de Interleucina/biossíntese , Receptores de Interleucina/fisiologia , Receptores de Interleucina-6 , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/farmacologia , Células Tumorais CultivadasRESUMO
Fluorimetric titrations of parvalbumin II (pI 4.2) of pike (Pike II) with Ca2+ and Tb3+ show the CD and EF binding sites to be non-equivalent. The intrinsic binding constants of the strong and the weak sites obtained for Ca2+ are: KsCa = 1.6.10(8) M-1; KwCa = 6.6.10(5) M-1. Differences of the order of 100% were encountered between the Tb3+ binding constants obtained with four different versions of titration. Their average values are: KsTb = 1.9.10(11) M-1; KwTb = 1.0.10(7) M-1. The distances of the strong and the weak sites from the singular Tyr-48, rs = 9.5 A and r2 = 11.5 A, were derived from Förster-type energy transfer and proved compatible with the X-ray structure of parvalbumin III (pI 4.2) of carp (CarpIII). From the distances, it is suggested that CD is the strong and EF the weak metal-binding site of PikeII. Tb3+ was shown by CD spectroscopy to have the same structural effect on PikeII as Ca2+. Removal of the metal ions from PikeII results in a decrease of helix content as monitored by CD spectroscopy. This decrease is larger than that in CarpIII. A concomitant decrease of the fluorescence quantum yield at nearly constant decay time is indicative of mainly static quenching, probably by the non-coordinating carboxylate groups. The maximum helix content is almost completely reestablished upon binding of the first metal ion. However, small changes of the energy transfer in PikeII with one terbium ion bound to the strong site indicate fine structural rearrangements of the strong binding site when Ca2+ is bound to the weak one.
Assuntos
Cálcio/metabolismo , Proteínas Musculares/metabolismo , Parvalbuminas/metabolismo , Salmonidae/metabolismo , Térbio/metabolismo , Animais , Sítios de Ligação , Cinética , Ligantes , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Espectrometria de FluorescênciaRESUMO
The assembly of the insulin hexamer brings the six B13 glutamate side-chains at the centre into close proximity. Their mutual repulsion is unfavourable and zinc co-ordination to B10 histidine is necessary to stabilize the well known zinc-containing hexamers. Since B13 is always a carboxylic acid in all known sequences of hexamer forming insulins, it is likely to be important in the hormone's biology. The mutation of B13 Glu-->Gln leads to a stable zinc-free hexamer with somewhat reduced potency. The structures of the zinc-free B13 Gln hexamer and the 2Zn B13 insulin hexamer have been determined by X-ray analysis and refined with 2.5 A and 2.0 A diffraction data, respectively. Comparisons show that in 2Zn B13 Gln insulin, the hexamer structure (T6) is very like that of the native hormone. On the other hand, the zinc-free hexamer assumes a quaternary structure (T3/R3) seen in the native 4Zn insulin hexamer, and normally associated only with high chloride ion concentrations in the medium. The crystal structures show the B13 Gln side-chains only contact water in contrast to the B13 glutamate in 2Zn insulin. The solvation of the B13 Gln may be associated with this residue favouring helix at B1 to B8. The low potency of the B13 Gln insulin also suggests the residue influences the hormone's conformation.
Assuntos
Insulina/química , Conformação Proteica , Animais , Cristalização , Glutamatos , Ácido Glutâmico , Glutamina , Humanos , Insulina/metabolismo , Substâncias Macromoleculares , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Suínos , Difração de Raios X , Zinco/químicaRESUMO
The peptide group between residues B24 and B25 of insulin was replaced by an ester bond. This modification only in the backbone was meant to eliminate a structurally important H-bond between the amide proton of B25 and the carbonyl oxygen of A19, and consequently to enhance detachment of the C-terminal B-chain from the body of the molecule, exposing the underlying A-chain. According to a model derived from the effects of side-chain substitutions, main-chain shortening, and crosslinking, this conformational change is prerequisite for receptor binding. Contrary to the expectation that increased flexibility would increase receptor binding and activity, depsi-insulin ([B24-B25 CO-O]insulin) has turned out be only 3-4% potent. In search of an explanation for this observation, the solution structure of depsi-insulin was determined by two-dimensional 1H-NMR spectroscopy. It was found that the loss of the B25-A19 H-bond does not entail detachment of the C-terminal B-chain. On the contrary, it is overcompensated by a gain in hydrophobic interaction achieved by insertion of the Phe B25 side chain into the molecule's core. This is possible because of increased rotational freedom in the backbone owing to the ester bond. Distortion of the B20-B23 turn and an altered direction of the distal B-chain are consequences that also affect self-association. The exceptional position of the B25 side chain is thus the key feature of the depsi-insulin structure. Being buried in the interior, it is not available for guiding the interaction with the receptor, a crucial role attributed to it by the model. This seems to be the main reason why the structure of depsi-insulin represents an inactive conformation.
Assuntos
Insulina/química , Sequência de Aminoácidos , Animais , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptor de Insulina/química , SuínosRESUMO
Mini insulin-like growth factor 1, an inactive insulin-like growth factor 1 mutant lacking the C region, was studied by 2D NMR spectroscopy. Resonances were assigned for almost all protons of the 57 amino acid residues. The 3D structure of the protein was determined by distance geometry methods. Three helical segments; Ala 8-Cys 18, Gly 42-Phe 49, and Leu 54-Cys 61, were identified, corresponding to those present in wild-type insulin-like growth factor 1 and in single-chain insulin. Their relative orientation, however, was found to be changed. This change is connected with a displacement of the Phe 23-Tyr 24-Phe 25-Asn 26 beta-strand-like segment, i.e., of aromatic side chains known to be important for receptor binding. Thus, deletion of the C region of IGF-1 results in a substantial tertiary structural rearrangement that accounts for the loss of receptor affinity.
Assuntos
Fator de Crescimento Insulin-Like I/química , Sequência de Aminoácidos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , SoluçõesRESUMO
This paper reports on an insulin analogue with 12.5-fold receptor affinity, the highest increase observed for a single replacement, and on its solution structure, determined by NMR spectroscopy. The analogue is [D-AlaB26]des-(B27-B30)-tetrapeptide-insulin-B26-amide. C-terminal truncation of the B-chain by four (or five) residues is known not to affect the functional properties of insulin, provided the new carboxylate charge is neutralized. As opposed to the dramatic increase in receptor affinity caused by the substitution of D-Ala for the wild-type residue TyrB26 in the truncated molecule, this very substitution reduces it to only 18% of that of the wild-type hormone when the B-chain is present in full length. The insulin molecule in solution is visualized as an ensemble of conformers interrelated by a dynamic equilibrium. The question is whether the "active" conformation of the hormone, sought after in innumerable structure/function studies, is or is not included in the accessible conformational space, so that it could be adopted also in the absence of the receptor. If there were any chance for the active conformation, or at least a predisposed state to be populated to a detectable extent, this chance should be best in the case of a superpotent analogue. This was the motivation for the determination of the three-dimensional structure of [D-AlaB26]des-(B27-B30)-tetrapeptide-insulin-B26-amide. However, neither the NMR data nor CD spectroscopic comparison of a number of related analogues provided a clue concerning structural features predisposing insulin to high receptor affinity. After the present study it seems more likely than before that insulin will adopt its active conformation only when exposed to the force field of the receptor surface.
Assuntos
Insulina/análogos & derivados , Dicroísmo Circular , Insulina/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , SoluçõesRESUMO
The transmembrane glycoprotein gp130 is the common signal transducing receptor subunit of the interleukin-6-type cytokines. It is a member of the cytokine-receptor superfamily predicted to consist of six domains in its extracellular part. The second and third domain constitute the cytokine-binding module defined by a set of four conserved cysteines and a WSXWS motif, respectively. The three-dimensional structure of the carboxy-terminal domain of this region was determined by multidimensional NMR. The domain consists of seven beta-strands constituting a fibronectin type III-like topology. The structure reveals that the WSDWS motif of gp130 is part of an extended tryptophan/arginine zipper which modulates the conformation of the CD loop.
Assuntos
Antígenos CD/química , Glicoproteínas de Membrana/química , Receptores de Citocinas/química , Sequência de Aminoácidos , Receptor gp130 de Citocina , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Homologia de Sequência de AminoácidosRESUMO
Circular dichroism, and steady-state and time-resolved fluorescence spectroscopy were used to compare the native recombinant human DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) with AGT bound to ds-DNA. Contrary to fluorescence, analysis of the far-UV CD spectra indicated a conformational change of AGT upon binding to DNA: its alpha-helical content is increased by approximately 12%. Analysis of near-UV CD spectra revealed that DNA was also affected, probably being separated into single strands locally.
Assuntos
DNA/química , Metiltransferases/química , Sequência de Aminoácidos , Dicroísmo Circular , DNA/genética , DNA/metabolismo , Humanos , Técnicas In Vitro , Metiltransferases/genética , Metiltransferases/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , O(6)-Metilguanina-DNA Metiltransferase , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de FluorescênciaRESUMO
Synthetic peptides immobilized on cellulose membranes proved to be a powerful tool for the identification of sites in the cytokine IL-6 involved in receptor binding. Similarly, a region in the extracellular part of the IL-6 receptor which is important for interaction with its ligand was identified.
Assuntos
Antígenos CD/química , Antígenos CD/metabolismo , Interleucina-6/química , Interleucina-6/metabolismo , Estrutura Secundária de Proteína , Receptores de Interleucina/química , Receptores de Interleucina/metabolismo , Sequência de Aminoácidos , Antígenos CD/isolamento & purificação , Sítios de Ligação , Clonagem Molecular , Escherichia coli , Humanos , Interleucina-6/isolamento & purificação , Modelos Estruturais , Dados de Sequência Molecular , Peptídeos/química , Receptores de Interleucina/isolamento & purificação , Receptores de Interleucina-6 , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
C-terminally deleted analogs of human interleukin-6 (IL-6) have been constructed at the cDNA level, and after cell-free transcription and translation their biological activity was analyzed. Removal of only 4 amino acids resulted in complete loss of biological activity as determined by the B9 cell proliferation assay. Secondary structure prediction of human IL-6 resulted in 58% helix, 14% beta-structure, and 28% turn and coil (average of 3 independent methods). The circular dichroism of recombinant human IL-6 was measured in the near and far UV. Evaluation of the latter in terms of secondary structures gave 67% helix, 15% beta-structure, and 18% turn and coil.
Assuntos
Interleucina-6 , Animais , Linhagem Celular , Dicroísmo Circular , DNA/análise , Humanos , Interleucina-6/genética , Mutação , Biossíntese de Proteínas , Conformação Proteica , Ratos , Relação Estrutura-Atividade , Transcrição GênicaRESUMO
The sequences of urokinase (UK) and tissue-type plasminogen activator (TPA) were aligned with those of chymotrypsin, trypsin, and elastase according to their 'structurally conserved regions'. In spite of its trypsin-like specificity UK was model-built on the basis of the chymotrypsin structure because of a corresponding disulfide pattern. The extra disulfide bond falls to cysteines 50 and 111d. Insertions can easily be accommodated at the surface. As they occur similarly in both, UK and TPA, a role in plasminogen recognition may be possible. Of the functional positions known to be involved in substrate or inhibitor binding, Asp 97, Lys 143 and Arg 217 (Leu in TPA) may contribute to plasminogen activating specificity. PTI binding may in part be impaired by structural differences at the edge of the binding pocket.
Assuntos
Endopeptidases/análise , Peptídeo Hidrolases/análise , Ativadores de Plasminogênio/análise , Ativador de Plasminogênio Tipo Uroquinase/análise , Sequência de Aminoácidos , Quimotripsina/análise , Humanos , Elastase Pancreática/análise , Conformação Proteica , Relação Estrutura-Atividade , Tripsina/análiseRESUMO
Three mutants of the anaphylatoxin C5a were prepared with positions 2, 64 and 70, respectively, substituted by tryptophan. The last mutant was additionally labelled at Cys27 for fluorescence energy transfer (FET) measurements. The structural integrity and biological activity of the molecules were not affected. Fluorescence anisotropy decay (FAD) measurements showed that the rotational correlation time for tryptophan decreases in the order: [Trp2]rhC5a > [Trp64]rhC5a > [Trp70]rhC5a, indicating an increasing mobility of the side chain. Measurements of the fluorescence energy transfer from Trp70 to the 1,5-AEDANS group at Cys27 yielded a distance distribution of 2.4 +/- 0.8 nm. This value is compatible with the C-terminal chain being arranged as a slightly stretched helix pointing away from the body of the molecule.
Assuntos
Anafilatoxinas/química , Complemento C5a/química , Triptofano/química , Sequência de Aminoácidos , Anafilatoxinas/genética , Dicroísmo Circular , Complemento C5a/genética , Cisteína , Transferência de Energia , Polarização de Fluorescência , Corantes Fluorescentes , Humanos , Dados de Sequência Molecular , Mutação , Naftalenossulfonatos , Proteínas Recombinantes/química , Espectrofotometria Ultravioleta , Reagentes de Sulfidrila , Fatores de Tempo , Triptofano/genéticaRESUMO
Theoretical calculations of CD spectra have generally assumed a single conformation, or a small number of conformers with Boltzmann averaging. Solvent effects on both the conformation and the CD have been neglected. In this work, we have calculated the CD spectrum of cyclo(L-Tyr-L-Tyr) in aqueous solution, taking dynamics and solvation into account. Starting geometries with chi 1 approximately 300 degrees or 60 degrees for both Tyr side chains were derived from MNDO/MOPAC, followed by energy minimization using GROMOS. After addition of 368 water molecules, the system was simulated for 1000 ps at 300 K using GROMOS. In addition to the starting conformer, two other conformers were observed during each simulation. However, each trajectory gave a distinct set of conformers. Rotational strengths were calculated for the cyclic dipeptide at each ps along the trajectories, using the matrix method. The CD spectra calculated from these rotational strengths were averaged over the trajectories. Agreement is very good for the strong negative band near 200 nm, while for the lower energy bands (near 230 and 280 nm), the signs are correct, but the magnitudes are too low. The spectrum calculated from a Boltzmann-weighted average over the in vacuo MNDO/MOPAC conformers was in poor agreement with experiment. Although the solvent did not significantly affect the rotational strength calculated for a given conformer, it is essential to include the solvent in the MD simulations because it affects the relative energies of the conformers and promotes transitions among them.
Assuntos
Dipeptídeos/química , Peptídeos Cíclicos/química , Fenômenos Químicos , Físico-Química , Dicroísmo Circular , Conformação Proteica , Soluções , TemperaturaRESUMO
The biological activity of oligopeptide analogues of C3a is markedly increased by N-terminal attachment of a hydrophobic group as, for instance, 9-fluorenylmethoxycarbonyl (Fmoc), either direct or via a flexible 6-aminohexanoyl (Ahx) spacer. This study presents evidence from fluorescence anisotropy decay measurements that the hydrophobic appendix mediates non-specific binding of the synthetic peptide analogues to phospholipid vesicles. According to quantitative considerations no alternative or additional rate-enhancing mechanisms other than surface diffusion are required to account for the gain in biopotency.
Assuntos
Complemento C3a/análogos & derivados , Complemento C3a/metabolismo , Sequência de Aminoácidos , Polarização de Fluorescência/métodos , Cinética , Matemática , Membranas/metabolismo , Modelos Teóricos , Dados de Sequência Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/metabolismo , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Crosslinked insulins have proved to be valuable for structure-function studies and as proinsulin models. In the first part of the paper, a short review of the literature on analytical investigations, the preparation of A1-B1- and A1-B29-crosslinked derivatives, their biological activities in vivo and in vitro, and CD-spectral properties is given. The results of reduction/reoxidation studies with insulin derivatives containing irreversible and cleavable crosslinks are summarized. In the second part, new A1-B29-crosslinked monomers and 3 symmetrical dimers, linked between A1-A'1, B1-B'1 and B29-B'29, are described, as well as some results of tritium-labelling and of enzymatic degradation experiments with A1-B29-linked insulins.
Assuntos
Insulina , Sequência de Aminoácidos , Fenômenos Químicos , Química , Dissulfetos , Marcação por Isótopo/métodos , Substâncias Macromoleculares , Métodos , Modelos Moleculares , Oxirredução , Proinsulina , Relação Estrutura-Atividade , TrítioRESUMO
The structure of an acid proteinase from Endothia parasitica has been solved by x-ray diffraction using multiple isomorphous replacement. A 3 A resolution map was interpreted in terms of a bilobal structure with a long 25 A cleft. The secondary structure is mostly distorted beta-sheet. The circular dichroism was measured and model curves for different secondary structures were fitted by least squares indicating a large component of beta-structure. The structure was seen to be homologous with that of the acid proteinase from R. Chinensis and hence with pepsin and chymosin. A rotation function against diffraction data from chymosin crystals confirm confirm this and suggested an approach to the solution of this structure.
Assuntos
Ascomicetos/enzimologia , Quimosina , Peptídeo Hidrolases , Xylariales/enzimologia , Dicroísmo Circular , Análise de Fourier , Modelos Moleculares , Conformação Proteica , Difração de Raios XAssuntos
Cistina , Polipeptídeo Pancreático , Triptofano , Tirosina , Dicroísmo Circular , Conformação ProteicaAssuntos
Antígenos CD , Interleucina-6/química , Receptores de Interleucina/metabolismo , Animais , Sítios de Ligação , Receptor gp130 de Citocina , Humanos , Interleucina-6/metabolismo , Ativação Linfocitária , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Camundongos , Receptores de Interleucina-6 , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Inorganic anions and phenolic compounds make the subunits of insulin hexamers undergo the T----R transition whereby the extended N-terminal B chain becomes helical and the octahedral metal coordination tetrahedral. The role of the metal ions is permissive. With cresol the transition is also undergone by metal-free hexamers. For coordinative reasons only zinc insulin can be transformed by moderate concentrations of inorganic anions. At higher concentrations and particularly with cresol transformation is also possible if Zn2+ is replaced by other metal ions. Owing to its d--d transitions in the visible cobalt lends itself as a spectroscopic probe for studying the interdependence of transformation and coordination. The transformation-related change in coordination is reflected in both the isotropic absorption and the CD spectrum. Cresol achieves T6----R6 transformation whereas that induced by SCN- ions is T6----T'3R3 with only the axial metal-binding site being realized in the R3 trimer. The spectral effects of the transformation of the two trimers are not additive; an extra contribution seems to be indicative of trimer/trimer interaction. Oxidation of 2 Co2+ insulin to a certain extent affects the structure of insulin; a characteristic positive band appears at 251 nm. Because of its extremely stable and exclusively octahedral complexes the Co3+ ion most strongly withstands transformation. The oxidation of tetrahedrally liganded Co2+ ions in R3 trimers proceeds with reduced velocity. Independent transformation of the Zn2+ trimers is possible in Zn2+/Co3+ metal hybrids of insulin.