RESUMO
Many receptors elicit signal transduction by activating multiple intracellular pathways. This transduction can be triggered by a non-specific ligand, which simultaneously activates all the signaling pathways of the receptors. However, the binding of one biased ligand preferentially trigger one pathway over another, in a process called biased signaling. The identification the functional motions related to each of these distinct pathways has a direct impact on the development of new effective and specific drugs. We show here how to detect specific functional motions by considering the case of the NGF/TrkA-Ig2 complex. NGF-mediated TrkA receptor activation is dependent on specific structural motions that trigger the neuronal growth, development, and survival of neurons in nervous system. The R221W mutation in the ngf gene impairs nociceptive signaling. We discuss how the large-scale structural effects of this mutation lead to the suppression of collective motions necessary to induce TrkA activation of nociceptive signaling. Our results suggest that subtle changes in the NGF interaction network due to the point mutation are sufficient to inhibit the motions of TrkA receptors putatively linked to nociception. The methodological approach presented in this article, based jointly on the normal mode analysis and the experimentally observed functional alterations due to point mutations provides an essential tool to reveal the structural changes and motions linked to the disease, which in turn could be necessary for a drug design study.
Assuntos
Modelos Moleculares , Fator de Crescimento Neural/metabolismo , Mutação Puntual , Receptor trkA/genética , Receptor trkA/metabolismo , Transdução de Sinais , Movimento , Fator de Crescimento Neural/química , Ligação Proteica , Conformação Proteica , Receptor trkA/químicaRESUMO
Human kallikrein 6 (KLK6) is highly expressed in the central nervous system and with elevated level in demyelinating disease. KLK6 has a very restricted specificity for arginine (R) and hydrolyses myelin basic protein, protein activator receptors and human ionotropic glutamate receptor subunits. Here we report a previously unreported activity of KLK6 on peptides containing clusters of basic amino acids, as in synthetic fluorogenic peptidyl-Arg-7-amino-4-carbamoylmethylcoumarin (peptidyl-ACC) peptides and FRET peptides in the format of Abz-peptidyl-Q-EDDnp (where Abz=ortho-aminobenzoic acid and Q-EDDnp=glutaminyl-N-(2,4-dinitrophenyl) ethylenediamine), in which pairs or sequences of basic amino acids (R or K) were introduced. Surprisingly, KLK6 hydrolyzed the fluorogenic peptides Bz-A-R↓R-ACC and Z-R↓R-MCA between the two R groups, resulting in non-fluorescent products. FRET peptides containing furin processing sequences of human MMP-14, nerve growth factor (NGF), Neurotrophin-3 (NT-3) and Neurotrophin-4 (NT-4) were cleaved by KLK6 at the same position expected by furin. Finally, KLK6 cleaved FRET peptides derived from human proenkephalin after the KR, the more frequent basic residues flanking enkephalins in human proenkephalin sequence. This result suggests the ability of KLK6 to release enkephalin from proenkephalin precursors and resembles furin a canonical processing proteolytic enzyme. Molecular models of peptides were built into the KLK6 structure and the marked preference of the cut between the two R of the examined peptides was related to the extended conformation of the substrates.
Assuntos
Calicreínas/metabolismo , Cinética , Peptídeo Hidrolases/metabolismo , Peptídeos/química , Aminoácidos Básicos/química , Aminoácidos Básicos/genética , Encefalinas/química , Encefalinas/metabolismo , Transferência Ressonante de Energia de Fluorescência , Furina/química , Furina/metabolismo , Humanos , Hidrólise , Calicreínas/química , Calicreínas/genética , Metaloproteinase 14 da Matriz/química , Metaloproteinase 14 da Matriz/metabolismo , Modelos Moleculares , Fator de Crescimento Neural/química , Fator de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/metabolismo , Neurotrofina 3 , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Peptídeos/metabolismo , Conformação Proteica , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Proteólise , Especificidade por SubstratoRESUMO
Nerve growth factor (NGF) is a member of the neurotrophins, which are important regulators of embryonic development and adult function in the vertebrate nervous systems. The signaling elicited by NGF regulates diverse activities, including survival, axon growth, and synaptic plasticity. NGF action is mediated by engagement with two structurally unrelated transmembrane receptors, p75(NTR) and TrkA, which are co-expressed in a variety of cells. The functional interactions of these receptors have been widely demonstrated and include complex formation, convergence of signaling pathways, and indirect interaction through adaptor proteins. Each domain of the receptors was shown to be important for the formation of TrkA and p75(NTR) complexes, but only the intramembrane and transmembrane domains seemed to be crucial for the creation of high-affinity binding sites. However, whether these occur through a physical association of the receptors is unclear. In the present work, we demonstrate by Förster resonance energy transfer that p75(NTR) and TrkA are physically associated through their intracellular (IC) domains and that this interaction occurs predominantly at the cell membrane and prior to NGF stimulation. Our data suggest that there is a pool of receptors dimerized before NGF stimulus, which could contribute to the high-affinity binding sites. We modeled the three-dimensional structure of the TrkA IC domain by homology modeling, and with this and the NMR-resolved structure of p75(NTR), we modeled the heterodimerization of TrkA and p75(NTR) by docking methods and molecular dynamics. These models, together with the results obtained by Förster resonance energy transfer, provide structural insights into the receptors' physical association.
Assuntos
Fator de Crescimento Neural/química , Receptor de Fator de Crescimento Neural/química , Receptor trkA/química , Animais , Biologia Computacional , Transferência Ressonante de Energia de Fluorescência , Hipocampo/química , Camundongos , Células PC12 , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Ratos , Homologia Estrutural de ProteínaRESUMO
Nerve growth factor (NGF) overexpression and increased production of peroxynitrite occur in several neurodegenerative diseases. We investigated whether NGF could undergo posttranslational oxidative or nitrative modifications that would modulate its biological activity. Compared to native NGF, peroxynitrite-treated NGF showed an exceptional ability to induce p75(NTR)-dependent motor neuron apoptosis at physiologically relevant concentrations. Whereas native NGF requires an external source of nitric oxide (NO) to induce motor neuron death, peroxynitrite-treated NGF induced motor neuron apoptosis in the absence of exogenous NO. Nevertheless, NO potentiated the apoptotic activity of peroxynitrite-modified NGF. Blocking antibodies to p75(NTR) or downregulation of p75(NTR) expression by antisense treatment prevented motor neuron apoptosis induced by peroxynitrite-treated NGF. We investigated what oxidative modifications were responsible for inducing a toxic gain of function and found that peroxynitrite induced tyrosine nitration in a dose-dependent manner. Moreover, peroxynitrite triggered the formation of stable high-molecular-weight oligomers of NGF. Preventing tyrosine nitration by urate abolished the effect of peroxynitrite on NGF apoptotic activity. These results indicate that the oxidation of NGF by peroxynitrite enhances NGF apoptotic activity through p75(NTR) 10,000-fold. To our knowledge, this is the first known posttranslational modification that transforms a neurotrophin into an apoptotic agent.
Assuntos
Apoptose/fisiologia , Neurônios Motores/metabolismo , Fator de Crescimento Neural/química , Ácido Peroxinitroso/farmacologia , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Células Cultivadas , Ensaio de Desvio de Mobilidade Eletroforética , Espectrometria de Massas , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Fator de Crescimento Neural/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Ratos , Receptor de Fator de Crescimento Neural/antagonistas & inibidores , Receptor de Fator de Crescimento Neural/efeitos dos fármacos , Receptor de Fator de Crescimento Neural/metabolismo , Tirosina/metabolismoRESUMO
Beta-nerve growth factor (beta-NGF) is a trophic factor in the nervous system. We aimed to isolate and characterize this protein in view of its potential therapeutic use in neurodegenerative diseases. For purification a two-step ion-exchange procedure was followed. The characterization was performed using separation and immunological techniques, as well as a biological assay. These studies showed that the obtained protein consisted of a mixture of beta-NGF molecules, intact at their NH2-terminal extreme, and molecules which have lost the NH2-terminal octapeptide and exhibit modifications increasing its hydrophobicity. All these molecular species were recognized immunologically and showed biological activity.