RESUMO
Psoriasis is a chronic inflammatory skin disease characterized by the infiltration of T cell and other immune cells to the skin in response to injury or autoantigens. Conventional, as well as unconventional, γδ T cells are recruited to the dermis and epidermis by CCL20 and other chemokines. Together with its receptor CCR6, CCL20 plays a critical role in the development of psoriasiform dermatitis in mouse models. We screened a panel of CCL20 variants designed to form dimers stabilized by intermolecular disulfide bonds. A single-atom substitution yielded a CCL20 variant (CCL20 S64C) that acted as a partial agonist for the chemokine receptor CCR6. CCL20 S64C bound CCR6 and induced intracellular calcium release, consistent with G-protein activation, but exhibited minimal chemotactic activity. Instead, CCL20 S64C inhibited CCR6-mediated T cell migration with nominal impact on other chemokine receptor signaling. When given in an IL-23-dependent mouse model for psoriasis, CCL20 S64C prevented psoriatic inflammation and the up-regulation of IL-17A and IL-22. Our results validate CCR6 as a tractable therapeutic target for psoriasis and demonstrate the value of CCL20 S64C as a lead compound.
Assuntos
Quimiocina CCL20/genética , Dermatite/terapia , Mutagênese Sítio-Dirigida/métodos , Psoríase/terapia , Receptores CCR6/metabolismo , Animais , Terapia Biológica/métodos , Células COS , Quimiocina CCL20/imunologia , Quimiocina CCL20/metabolismo , Chlorocebus aethiops , Cristalografia por Raios X , Dermatite/imunologia , Modelos Animais de Doenças , Epiderme/imunologia , Epiderme/metabolismo , Humanos , Interleucina-23/imunologia , Camundongos , Psoríase/imunologia , Receptores CCR6/imunologia , Linfócitos T/imunologiaRESUMO
Human growth hormone (hGH) and prolactin (hPRL) have a low sequence homology, but both bind and activate hPRL receptors. hGH also binds hGH receptors. hGH has 22 and 20 kDa forms; residues 32-46 have been deleted by alternative RNA splicing to create the smaller form. hGH requires F44 for activity through the hPRL receptor, but not for activity through the hGH receptor. The deletion of F44 from hGH has the same effect as removal of residues 32-46 (approximately 200-fold loss in activity), indicating the importance of F44 in hGH when activating the hPRL receptor. In contrast, when the homologous F50 is deleted from hPRL little or no activity is lost, indicating that this highly conserved phenylalanine is not required for the action of hPRL. Deletion of residues 41-52 (a non-conserved sequence homologous to residues 32-46 of hGH) reduced the activity of hPRL by >14 000-fold. This region is essential for the biological activity of hPRL. As these two proteins have evolved from a common ancestor, they have retained the requirement for this region but need different structural elements to activate hPRL receptors. Such diversity represents an opportunity to fine-tune hormone activity.
Assuntos
Hormônio do Crescimento Humano/metabolismo , Prolactina/metabolismo , Receptores da Prolactina/metabolismo , Sequência de Aminoácidos , Bioensaio , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Hormônio do Crescimento Humano/química , Hormônio do Crescimento Humano/genética , Humanos , Dados de Sequência Molecular , Prolactina/química , Prolactina/genética , Prolactina/farmacologia , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência , Relação Estrutura-AtividadeRESUMO
We describe here the high-level expression of bovine trypsinogen in E. coli, its refolding and activation to beta-trypsin, and the selective incorporation of (15)N-labeled alanine through supplementation of the growth medium. Using this procedure, we expressed (15)N-labeled S195A trypsinogens, both on a wild-type and on a D189S background, in amounts suitable for NMR spectroscopy. 2D [(1)H-(15)N]-HSQC NMR was used to follow conformational changes upon activation of trypsinogen and formation of noncovalent complexes between S195A or S195A/D189S trypsin and protein proteinase inhibitors of different structural families and different sizes, as well as to examine the effects of introduction of the D189S mutation. Spectra of good quality were obtained for both trypsins alone and in complexes of increasing size with the proteinase inhibitors BPTI (total molecular mass 31 kDa), SBTI (total molecular mass 44 kDa), and the serpin alpha(1)-proteinase inhibitor Pittsburgh (alpha(1)PI Pittsburgh) (total molecular mass 69 kDa). Assignments of alanines 55 and 56, close to the active site histidine, and of alanine 195, present in the S195A variant used for most of the studies, were made by mutagenesis. These three alanines, together with two others, probably close to the S1 specificity pocket, were very sensitive to complex formation. In contrast, the remaining 10 alanines were invariant in chemical shift in all 3 of the noncovalent complexes formed, reflecting the conservation of structure in complexes with BPTI and SBTI known from X-ray crystal structures, but also indicating that there is no change in backbone conformation for the noncovalent complex with alpha(1)PI, for which there is no crystal structure. This was true both for S195A and for S195A/D189S trypsins. This high-level expression and labeling approach will be of great use for solution NMR studies on trypsin-serpin complexes, as well as for structural and mechanistic studies on trypsin variants.
Assuntos
Alanina/metabolismo , Escherichia coli/genética , Inibidores da Tripsina/metabolismo , Tripsina/genética , Tripsina/metabolismo , Tripsinogênio/genética , Tripsinogênio/metabolismo , Alanina/genética , Animais , Aprotinina/metabolismo , Ácido Aspártico/genética , Bovinos , Ativação Enzimática/genética , Escherichia coli/enzimologia , Substâncias Macromoleculares , Mutagênese Sítio-Dirigida , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular/métodos , Dobramento de Proteína , Prótons , Serina/genética , Tripsina/biossíntese , Inibidor da Tripsina de Soja de Kunitz/metabolismo , Tripsinogênio/biossínteseRESUMO
We have used [(1)H-(15)N]-HSQC NMR to investigate the structural changes that occur in both serpin and proteinase in forming the kinetically trapped covalent protein-protein complex that is the basis for serpin inhibition of serine proteinases. By alternately using (15)N-alanine specifically-labeled alpha(1)-proteinase inhibitor (alpha(1)PI) Pittsburgh (serpin) and bovine trypsin (proteinase), we were able to selectively monitor structural changes in each component of the 69 kDa complex. Residue-specific assignments of four alanines in the reactive center loop and seven other alanines aided interpretation of the spectral changes in the serpin. We found that the majority of the alanine resonances, including those from reactive center loop residues P12, P11, and P9, were at identical positions in covalent complex and in cleaved alpha(1)PI. Five alanines that are close to the contact region with proteinase showed some chemical shift perturbation compared with cleaved alpha(1)PI, indicating some degree of structural deformation. With (15)N label in the proteinase, an HSQC spectrum was obtained that more closely resembled that of a molten globule, suggesting that the structure of the proteinase had been significantly altered as a result of complex formation. Large increases in line width for all alpha(1)PI resonances in the covalent complex, with the sole exception of two residues in the flexible N-terminal tail, indicate that, unlike the noncovalent alpha(1)PI-anhydroproteinase complex, the covalent complex is a rigid body of effectively increased molecular weight. We conclude that the mutual perturbations of serpin and proteinase result from steric compression and distortion, rather than simple contact effects. This distortion provides a structural basis for the greatly reduced catalytic efficiency of the proteinase in the complex and hence kinetic trapping of the covalent reaction intermediate.
Assuntos
Serina Endopeptidases/química , Inibidores de Serina Proteinase/química , Serpinas/química , Tripsina/química , alfa 1-Antitripsina/química , Alanina/química , Alanina/metabolismo , Animais , Bovinos , Simulação por Computador , Hidrólise , Substâncias Macromoleculares , Modelos Moleculares , Peso Molecular , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular/métodos , Prótons , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/metabolismo , Serpinas/metabolismo , Tripsina/metabolismo , alfa 1-Antitripsina/metabolismoRESUMO
A structural understanding of the nature and scope of serpin inhibition mechanisms has been limited by the inability so far to crystallize any serpin-proteinase complex. We describe here the application of [(1)H-(15)N]-HSQC NMR on uniformly and residue-selectively (15)N-labeled serpin alpha(1)-proteinase inhibitor (Pittsburgh variant with stabilizing mutations) to provide a nonperturbing and exquisitely sensitive means of probing the conformation of the serpin alone and in a noncovalent complex with inactive, serine 195-modified, bovine trypsin. The latter should be a good model both for the few examples of reversible serpin-proteinase complexes and for the initial Michaelis-like complex formed en route to irreversible covalent inhibition. Cleavage of the reactive center loop, with subsequent insertion into beta-sheet A, caused dramatic perturbation of most of the NMR cross-peaks. This was true for both the uniformly labeled and alanine-specifically labeled samples. The spectra of uniformly or leucine- or alanine-specifically labeled alpha(1)-proteinase inhibitor in noncovalent complex with unlabeled inactive trypsin gave almost no detectable chemical shift changes of cross-peaks, but some general increase in line width. Residue-specific assignments of the four alanines in the reactive center loop, at P12, P11, P9, and P4, allowed specific examination of the behavior of the reactive center loop. All four alanines showed higher mobility than the body of the serpin, consistent with a flexible reactive center loop, which remained flexible even in the noncovalent complex with proteinase. The three alanines near the hinge point for insertion showed almost no chemical shift perturbation upon noncovalent complex formation, while the alanine at P4 was perturbed, presumably by interaction with the active site of bound trypsin. Reporters from both the body of the serpin and the reactive center loop therefore indicate that noncovalent complex formation involves no conformational change in the body of the serpin and only minor perturbation of the reactive center loop in the region which contacts proteinase. Thus, despite the large size of serpin and serpin-proteinase complex, 45 and 69 kDa respectively, NMR provides a very sensitive means of probing serpin conformation and mobility, which should be applicable both to noncovalent and to covalent complexes with a range of different proteinases, and probably to other serpins.
Assuntos
Ressonância Magnética Nuclear Biomolecular , Tripsina/química , alfa 1-Antitripsina/química , Alanina/química , Substituição de Aminoácidos/genética , Animais , Sítios de Ligação/genética , Bovinos , Humanos , Hidrólise , Leucina/química , Substâncias Macromoleculares , Mutagênese Sítio-Dirigida , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação Proteica , PrótonsRESUMO
Primate growth hormones (GH) activate both primate and non-primate somatotrophic receptors (GH receptors), but non-primate GHs do not activate primate GH receptors. Previous studies argued the interaction of Asp(171) of human GH and Arg(43) of the receptor produced an attractive ionic interaction. In non-primate GHs, His(170) replaces the homologous Asp(171), producing a repulsive interaction with Arg(43) of the primate receptor which was believed to reduce the attraction of non-primate GH for the human GH receptor, thus providing species specificity. In this report, H170D bovine GH had activity and affinity for human GH receptors approaching those of human GH. In contrast, replacing Asp(171) of human GH with His did not significantly reduce somatotrophic activity, indicating that species specificity is not wholly explained by this residue's interaction with Arg(43) of the receptor. Deletion of either Phe(44) (a residue present only in primate GHs) or residues 32-46 (20-kDa form of human GH) each only marginally reduced somatotrophic activities. But the combination of the D171H mutation with either DeltaPhe(44) or Delta32-46 in human GH reduced binding and activity in a greater than additive fashion, indicated a functional interaction between these distant structural features. In bovine GH addition of phenylalanine at position 44 increased the somatotrophic activity and receptor affinity in cells containing the human GH receptor. The combination of the H170D mutation and the addition of phenylalanine at position 44 created a bovine GH with activity indistinguishable from wild-type human GH. Based on evidence from both bovine and human GHs, the cooperative interaction of these two distant motifs determined the species specificity and indicated that structural plasticity was a critical feature necessary for the species specificity of somatotrophic activity.
Assuntos
Hormônio do Crescimento/metabolismo , Hormônio do Crescimento Humano/metabolismo , Receptores da Somatotropina/metabolismo , Animais , Sítios de Ligação , Bovinos , Eletroforese em Gel de Poliacrilamida/métodos , Hormônio do Crescimento/genética , Hormônio do Crescimento Humano/genética , Humanos , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade da Espécie , Análise Espectral/métodosRESUMO
The expression, folding, and characterization of a series of small proteins with increasingly complex disulfide bond patterns were characterized. A phagemid was prepared from the pT7-7 plasmid to facilitate mutagenic studies with these proteins. cDNAs coding for bovine, rat, and human prolactin; human growth hormone; and bovine alpha-lactalbumin were amplified by PCR using primers that inserted restriction sites at the 5' and 3' ends and reduced the coding sequence to the mature methionyl protein with bacterially preferred codons in the 5' region. The expressed proteins were folded and oxidized by methods that allowed disulfide bond formation to occur either during or following folding. The effectiveness of the folding procedures was determined for each protein by electrophoresis, absorption spectroscopy, and functional studies. The redox conditions required for folding functional proteins varied as the number of disulfide bonds per unit molecular weight increased. Human growth hormone, 22 kDa; human prolactin, 23 kDa; and bovine prolactin, 23 kDa, contain two, three, and three disulfides, respectively, and are folded correctly by air oxidation performed during renaturation under alkaline conditions. Proper disulfide bond formation of rat prolactin, 23 kDa, containing three disulfide bonds required the addition of a reducing agent at the initiation of renaturation. Bovine alpha-lactalbumin, 14 kDa with four disulfide bonds, required complete renaturation prior to the removal of a reducing agent. SDS-gel electrophoresis under nonreducing conditions provided information regarding the proper folding of these proteins. The absorption of 250-nm light by disulfide bonds also provided information regarding the proper folding of rat prolactin and bovine alpha-lactalbumin.
Assuntos
Dissulfetos , Hormônio do Crescimento Humano/química , Lactalbumina/química , Prolactina/química , Dobramento de Proteína , Animais , Bioensaio , Bovinos , Cromatografia por Troca Iônica , Feminino , Vetores Genéticos , Hormônio do Crescimento Humano/genética , Hormônio do Crescimento Humano/isolamento & purificação , Humanos , Lactalbumina/genética , Lactalbumina/isolamento & purificação , Glândulas Mamárias Animais/metabolismo , Hipófise/metabolismo , Plasmídeos , Reação em Cadeia da Polimerase/métodos , Prolactina/genética , Prolactina/isolamento & purificação , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espectrofotometria Ultravioleta , Células Tumorais CultivadasRESUMO
Human growth hormone (hGH) stimulates somatogenic and lactogenic actions through the GH and prolactin (PRL) receptors, respectively. In contrast, non-primate GHs stimulate only somatotropic action. Phe44, of the human GH sequence is present in all hormones stimulating lactogenic action and absent in all hormones stimulating only somatotropic action. We speculate that the presence of Phe44 is a feature necessary for specifying lactogenic activity. In this report, the role of Phe44 was investigated by its deletion or substitution with alanine or leucine. Deletion of Phe44 or substitution with leucine did not significantly change the structure of hGH as determined by circular dichroism, absorbance, and fluorescence spectroscopies. In contrast, substitution of alanine perturbed the structure. Deletion of Phe44 reduced binding affinity for the lactogenic receptor, resulting in a reduced activation. Substitution with either alanine or leucine partially restored lactogenic receptor binding affinity, which correlated with the hormones' activity in the Nb2 rat lymphoma cells. All the recombinant hGHs had similar somatotropic activities in FDC-P1 cells transfected with the hGH receptor. These data indicate that the hydrophobic side chain of Phe44 is required for lactogenic receptor binding and activation but is unnecessary for somatotropic action.
Assuntos
Hormônio do Crescimento/química , Receptores da Prolactina/metabolismo , Receptores da Somatotropina/metabolismo , Alanina/química , Animais , Bioensaio , Dicroísmo Circular , Clonagem Molecular , Hormônio do Crescimento/metabolismo , Humanos , Lactação , Leucina/química , Fenilalanina/química , Estrutura Secundária de Proteína , Ratos , Proteínas Recombinantes , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Relação Estrutura-AtividadeRESUMO
Phosphorylated prolactin has been identified and isolated from bovine pituitaries. The biological activity of this phosphoprotein is severely reduced in comparison with nonphosphorylated prolactin. The sites of phosphorylation are serines 26, 34, and 90, and the stoichiometry is 1:1:10, respectively. In this report, the phosphoserine residues have been individually replaced with glutamic acid in recombinant methionyl bovine prolactins in order to mimic phosphorylation at each site. Substitution of glutamic acid for serine at positions 26, 34, and 90 reduced protein helical contents by 10, 6, and 14%, respectively. UV absorbances for S26E and S34E bovine prolactins were blue-shifted, similar to the biological isolates of phosphorylated bovine prolactin, but the biological activities of the S26E and S34E mutants (ED50 values of 16.3 and 18.8 pM, respectively) were similar to that of wild-type prolactin (ED50 value of 18.6 pM) in the Nb2 rat lymphoma assay. S90E bovine prolactin had the greatest reduction in helical content but showed similar UV and fluorescent spectra to the wild-type bovine prolactin. The biological activity of S90E bovine prolactin (ED50 value of 672 pM) was reduced to an activity similar to that of phosphorylated bovine prolactin. The data indicate that the phosphorylation of serine 90 is responsible for the reduction in biological activity.