RESUMO
The carboxyl-terminal segment of G protein-coupled receptors has one or more conserved cysteine residues that are potential sites for palmitoylation. This posttranslational modification contributes to membrane association, internalization, and membrane targeting of proteins. In contrast to other members of the glycoprotein hormone receptor family (the LH and thyroid-stimulating hormone receptors), it is not known whether the follicle-stimulating hormone receptor (FSHR) is palmitoylated and what are the effects of abolishing its potential palmitoylation sites. In the present study, a functional analysis of the FSHR carboxyl-terminal segment cysteine residues was carried out. We constructed a series of mutant FSHRs by substituting cysteine residues with alanine, serine, or threonine individually and together at positions 629 and 655 (conserved cysteines) and 627 (nonconserved). The results showed that all three cysteine residues are palmitoylated but that only modification at Cys629 is functionally relevant. The lack of palmitoylation does not appear to greatly impair coupling to G(s) but, when absent at position 629, does significantly impair cell surface membrane expression of the partially palmitoylated receptor. All FSHR Cys mutants were capable of binding agonist with the same affinity as the wild-type receptor and internalizing on agonist stimulation. Molecular dynamics simulations at a time scale of approximately 100 nsec revealed that replacement of Cys629 resulted in structures that differed significantly from that of the wild-type receptor. Thus, deviations from wild-type conformation may potentially contribute to the severe impairment in plasma membrane expression and the modest effects on signaling exhibited by the receptors modified in this particular position.
Assuntos
Cisteína/análise , Cisteína/fisiologia , Rim/citologia , Rim/embriologia , Receptores do FSH/química , Receptores do FSH/fisiologia , Alanina/análise , Alanina/fisiologia , Sequência de Aminoácidos , Linhagem Celular , Simulação por Computador , AMP Cíclico/metabolismo , Humanos , Rim/metabolismo , Lipoilação/fisiologia , Dados de Sequência Molecular , Mutação/genética , Receptores do FSH/genética , Serina/análise , Serina/fisiologia , Treonina/análise , Treonina/fisiologiaRESUMO
Neurolysin (EP24.16) and thimet oligopeptidase (EP24.15) are closely related metalloendopeptidases. Site-directed mutagenesis of Tyr(613) (EP24.16) or Tyr(612) (EP24.15) to either Phe or Ala promoted a strong reduction of k(cat)/K(M) for both enzymes. These data suggest the importance of both hydroxyl group and aromatic ring at this specific position during substrate hydrolysis by these peptidases. Furthermore, the EP24.15 A607G mutant showed a k(cat)/K(M) of 2x10(5) M(-1) s(-1) for the Abz-GFSIFRQ-EDDnp substrate, similar to that of EP24.16 (k(cat)/K(M)=3x10(5) M(-1) s(-1)) which contains Gly at the corresponding position; the wild type EP24.15 has a k(cat)/K(M) of 2.5x10(4) M(-1) s(-1) for this substrate.
Assuntos
Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Alanina/genética , Alanina/fisiologia , Animais , Sítios de Ligação , Catálise , Cinética , Metaloendopeptidases/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Relação Estrutura-Atividade , Especificidade por Substrato , Tirosina/genética , Tirosina/fisiologiaRESUMO
Several factors that may contribute to the stabilization of the helical structure in proteins, detected in studies made on short synthetic peptides, have been reported. Some of them are: presence of alanine or leucine, ionic-pair bonding, stabilization of the helical dipole moment by appropriate charges at the helix N- and C-caps, and aromatic interactions of amino acids located at positions i, i + 4. An analysis of 54 helical structures from 12 proteins showed that all these stabilizing factors were also present in proteins, but the influence of any of them had a different weight, according to the distribution of the hydrophobic and hydrophilic amino acid residues in the helical sequence. The role of non-sequence depending interactions in helical stability, such as presence of disulfide bridges, or bonding of helical residues to substrate and/or cofactors, was also analysed.
Assuntos
Alanina/fisiologia , Aminoácidos/fisiologia , Estabilidade Enzimática/fisiologia , Leucina/fisiologia , Muramidase/ultraestrutura , Hormônios Pancreáticos/fisiologia , Estrutura Secundária de ProteínaRESUMO
Several factors that may contribute to the stabilization of the helical structure in proteins, detected in studies made on short synthetic peptides, have been reported. Some of them are: presence of alanine or leucine, ionic-pair bonding, stabilization of the helical dipole moment by appropriate charges at the helix N- and C-caps, and aromatic interactions of amino acids located at positions i, i + 4. An analysis of 54 helical structures from 12 proteins showed that all these stabilizing factors were also present in proteins, but the influence of any of them had a different weight, according to the distribution of the hydrophobic and hydrophilic amino acid residues in the helical sequence. The role of non-sequence depending interactions in helical stability, such as presence of disulfide bridges, or bonding of helical residues to substrate and/or cofactors, was also analysed