RESUMO
Exploring drug targets based on disease-associated molecular mechanisms during development is crucial for the generation of novel prevention and treatment strategies for neurodevelopmental psychiatric conditions. We report that prefrontal cortex (PFC)-specific postnatal knockdown of DISC1 via in utero electroporation combined with an inducible knockdown expression system drives deficits in synaptic GABAA function and dendritic development in pyramidal neurons, as well as abnormalities in sensorimotor gating, albeit without profound memory deficits. We show for the first time that DISC1 is specifically involved in regulating cell surface expression of α2 subunit-containing GABAA receptors in immature developing neurons, but not after full maturation. Notably, pharmacological intervention with α2/3 subtype-selective GABAA receptor positive allosteric modulators during the early postnatal period ameliorates dendritic deficits and behavioral abnormalities induced by knockdown of DISC1. These findings highlight a critical role of DISC1-mediated disruption of postnatal GABA signaling in aberrant PFC maturation and function.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Receptores de GABA-A/efeitos dos fármacos , Receptores de GABA-A/metabolismo , Animais , Modelos Animais de Doenças , Eletroporação , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/efeitos dos fármacos , Proteínas do Tecido Nervoso/fisiologia , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Subunidades Proteicas , Células Piramidais/metabolismo , Filtro Sensorial/genética , Filtro Sensorial/fisiologiaRESUMO
Fibroblast growth factors (FGFs) are being investigated in human clinical trials as treatments for angina, claudication, and stroke. We designed a molecule structurally unrelated to all FGFs, which potently mimicked basic FGF activity, by combining domains that (1) bind FGF receptors (2) bind heparin, and (3) mediate dimerization. A 26-residue peptide identified by phage display specifically bound FGF receptor (FGFR) 1c extracellular domain but had no homology with FGFs. When fused with the c-jun leucine zipper domain, which binds heparin and forms homodimers, the polypeptide specifically reproduced the mitogenic and morphogenic activities of basic FGF with similar potency (EC50 = 240 pM). The polypeptide required interaction with heparin for activity, demonstrating the importance of heparin for FGFR activation even with designed ligands structurally unrelated to FGF. Our results demonstrate the feasibility of engineering potent artificial agonists for the receptor tyrosine kinases, and have important implications for the design of nonpeptidic ligands for FGF receptors. Furthermore, artificial FGFR agonists may be useful alternatives to FGF in the treatment of ischemic vascular disease.
Assuntos
Desenho de Fármacos , Proteínas Proto-Oncogênicas c-jun/genética , Receptores de Fatores de Crescimento de Fibroblastos/agonistas , Proteínas Recombinantes de Fusão/genética , Células 3T3 , Animais , Linhagem Celular , Dimerização , Fator 2 de Crescimento de Fibroblastos/metabolismo , Heparina/metabolismo , Humanos , Camundongos , Ligação Proteica , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoAssuntos
Isomerases de Aminoácido/química , Isomerases de Aminoácido/metabolismo , Transferases Intramoleculares , Lisina/análise , Sítios de Ligação , Clostridium/enzimologia , Simulação por Computador , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Radicais Livres/análise , Radicais Livres/metabolismo , Modelos Moleculares , Conformação Proteica , Sensibilidade e Especificidade , Espectroscopia de Infravermelho com Transformada de Fourier/métodosRESUMO
Lysine 2,3-aminomutase from Clostridium SB4 has been studied by electron paramagnetic resonance (EPR) spectroscopy at 77 K. Although the reaction catalyzed by this enzyme is similar to rearrangements catalyzed by enzymes requiring adenosylcobalamin, lysine 2,3-aminomutase does not utilize this cofactor. The enzyme instead contains iron-sulfur clusters, cobalt, and pyridoxal phosphate and is activated by S-adenosylmethionine. Subsequent to a reductive incubation procedure that is required to activate the enzyme, EPR studies reveal the appearance of an organic radical signal (g = 2.001) upon addition of both L-lysine and S-adenosylmethionine. The radical signal is complex, having multiple hyperfine transitions. The total radical concentration is proportional to enzyme activity and decreases in parallel with the approach to chemical equilibrium between alpha-lysine and beta-lysine. The signal changes over the time course of the reaction in a way that suggests the presence of more than one radical species, with different relative proportions of species in the steady state and equilibrium state. Isotopic substitution experiments show that unpaired spin density resides on the molecular framework of lysine and that solvent-exchangeable protons do not participate in strong hyperfine coupling to the radical. The results indicate that lysine radicals participate in the rearrangement mechanism.
Assuntos
Isomerases de Aminoácido/química , Desoxiadenosinas/química , Transferases Intramoleculares , Clostridium/enzimologia , Espectroscopia de Ressonância de Spin Eletrônica , Ativação Enzimática , Isótopos , Lisina/químicaRESUMO
Electron paramagnetic resonance (EPR) spectroscopy has been used to characterize an organic radical that appears in the steady state of the reaction catalyzed by lysine 2,3-aminomutase from Clostridium SB4. Results of a previous electron paramagnetic resonance (EPR) study [Ballinger, M. D., Reed, G. H., & Frey, P. A. (1992) Biochemistry 31, 949-953] demonstrated the presence of EPR signals from an organic radical in reaction mixtures of the enzyme. The materialization of these signals depended upon the presence of the enzyme, all of its cofactors, and the substrate, lysine. Changes in the EPR spectrum in response to deuteration in the substrate implicated the carbon skeleton of lysine as host for the radical center. This radical has been further characterized by EPR measurements on samples with isotopically substituted forms of lysine and by analysis of the hyperfine splittings in resolution-enhanced spectra by computer simulations. Changes in the hyperfine splitting patterns in EPR spectra from samples with [2-2H]lysine and [2-13C]-lysine show that the paramagnetic species is a pi-radical with the unpaired spin localized primarily in a p orbital on C2 of beta-lysine. In the EPR spectrum of this radical, the alpha-proton, the beta-nitrogen, and the beta-proton are responsible for the hyperfine structure. Analysis of spectra for reactions initiated with L-lysine, [3,3,4,4,5,5,6,6-2H8]lysine, [2-2H]lysine, perdeuteriolysine, [alpha-15N]lysine, and [alpha-15N,2-2H]lysine permit a self-consistent assignment of hyperfine splittings.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Isomerases de Aminoácido/química , Transferases Intramoleculares , Isomerases de Aminoácido/metabolismo , Simulação por Computador , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres/química , Lisina/químicaRESUMO
The bacterial serine protease, subtilisin BPN', has been mutated so that it will efficiently and selectively cleave substrates containing two consecutive basic (dibasic) residues. Mutants were designed on the basis of both the structure of subtilisin BPN' and considerations of sequence differences between it and eukaryotic homologs, Kex2, PC2, and furin, which are known to cleave dibasic substrates. These eukaryotic proteases have high sequence homology to one another but differ substantially from subtilisin BPN' in loops that interact with the substrate. When these loops were grafted into subtilisin BPN', the mutated enzyme could not be expressed, presumably due to destabilization of the folded enzyme. We noted that several neutral residues in subtilisin BPN' (Gly 166, Ser 33, and Asn 62) that are positioned to interact with a dibasic substrate are acidic residues at analogous positions in Kex2. Mutating these residues individually to either Glu or Asp in subtilisin BPN' resulted in systematic shifts in substrate specificity (kcat/Km) toward basic residues and away from the natural preference for hydrophobic substrates. A combination mutant, where Asn 62 was changed to Asp and Gly 166 was changed to Asp (N62D/G166D), had a larger than additive shift in specificity toward dibasic substrates. This unexpectedly large change was confirmed by detailed analysis with a variety of synthetic substrates. Additional substrate determinants were revealed by sorting a library of phage particles (substrate phage) containing five contiguous randomized residues. This method identified a particularly good substrate (Asn-Leu-Met-Arg-Lys) that was selectively cleaved in the context of a fusion protein by the N62D/G166D subtilisin.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Oligopeptídeos/química , Conformação Proteica , Subtilisinas/química , Subtilisinas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Escherichia coli , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Oligopeptídeos/metabolismo , Mutação Puntual , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Subtilisinas/isolamento & purificaçãoRESUMO
The serine protease, subtilisin BPN', was engineered to cleave proteins after tribasic sequences in a manner that resembles the substrate specificity of furin, one of the mammalian subtilisin homologs that processes prohormones. As a starting point we used a double mutant of subtilisin BPN' (N62D/ G166D) that showed substantial preference for cleaving after sequences having consecutive dibasic residues (namely, at the P1 and P2 substrate positions) [Ballinger et al. (1995) Biochemistry 34, 13312-13319]. Additional specificity for basic residues was engineered at the P4 position by introducing subtilisin-to-furin substitutions at three hydrophobic residues that composed the S4 subsite (Y104, I107, and L126). Initial attempts to incorporate a Y104D or I107E mutation or the Y104D/I107E double mutation into the dibasic specific enzyme failed to generate the processed enzyme. The problem was traced to the inability of the mutant prosubtilisins to process themselves and fold correctly. Replacing the natural processing site sequence (AHAY) with a good furin substrate sequence (RHKR) resulted in expression of the triple subtilisin mutant (N62D/Y104D/G166D) we call "furilisin". Furilisin hydrolyzes synthetic tribasic substrates (succinyl-RAKR-pNA or succinyl-KAKR-pNA) with high catalytic efficiency (kcat/K(m) > 3 x 10(5) M-1 s-1) and discriminates in favor of Arg versus Ala at the P4 position by a factor of 360. The overall specificity change versus the wild-type enzyme was dramatic. For example, succinyl-RAKR-pNA was cleaved approximately 60000 times faster than succinyl-AAPF-pNA, a good substrate for wild-type subtilisin. Similarly, furilisin was inhibited (K1* = 29 nM) by a variant of the turkey ovomucoid third domain inhibitor that contained an engineered furin substrate site (RCKR decreases) [Lu et al. (1993) J. Biol. Chem. 268, 14583-14585] and not by one having a good wild-type subtilisin substrate sequence (ACTL decreases). Interestingly, the extreme changes in substrate specificity resulted from substantial synergy between the engineered subsites. These studies provide a basic example of how to manipulate substrate specificity in a modular fashion, thereby creating an engineered-enzyme that may be useful as a protein processing tool.
Assuntos
Engenharia de Proteínas , Subtilisinas/química , Subtilisinas/metabolismo , Sequência de Aminoácidos , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Escherichia coli/genética , Furina , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Peptídeos/farmacologia , Alinhamento de Sequência , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Especificidade por Substrato , Subtilisinas/genéticaRESUMO
The anaerobic ribonucleoside triphosphate reductase from Escherichia coli reduces CTP to dCTP in the presence of a second protein, named dA1, and a Chelex-treated boiled extract of the bacteria, named RT. The reaction requires S-adenosylmethionine, NADPH, dithiothreitol, ATP, and Mg2+ and K+ ions. It occurs only under anaerobic conditions. We now show that the overall reaction occurs in two steps. The first is an activation of the reductase by dA1 and RT and requires S-adenosylmethionine, NADPH, dithiothreitol, and possibly K+ ions. In the second step, the activated reductase reduces CTP to dCTP with ATP acting as an allosteric effector. During activation, S-adenosylmethionine is cleaved reductively to methionine + 5'-deoxyadenosine. This step is inhibited strongly by S-adenosylhomocysteine and various chelators. The activation of the anaerobic reductase shows a considerable similarity to that of pyruvate formate-lyase (Knappe, J., Neugebauer, F. A., Blaschkowski, H. P., and Gänzler, M. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1332-1335).
Assuntos
Escherichia coli/enzimologia , Ribonucleotídeo Redutases/metabolismo , S-Adenosilmetionina/metabolismo , S-Adenosilmetionina/farmacologia , Anaerobiose , Citidina Trifosfato/metabolismo , Desoxiadenosinas/metabolismo , Ativação Enzimática , Cinética , Metionina/metabolismo , Modelos Biológicos , Oxirredução , S-Adenosil-Homocisteína/farmacologiaRESUMO
The role of pyridoxal 5'-phosphate (PLP) in the radical-mediated amino group migration catalyzed by lysine 2,3-aminomutase from Clostridia SB4 has been investigated by electron spin echo envelope modulation (ESEEM) spectroscopy. This pulsed electron paramagnetic resonance (EPR) method was used to estimate the distance between the unpaired electron in the alpha-radical of beta-lysine, a steady-state intermediate in the reaction, and deuterium at the C4' position of the cofactor, PLP. [4'-2H]PLP was synthesized and exchanged into the enzyme. The steady-state radical was generated in the labeled samples and in samples with unlabeled PLP by addition of L-lysine.H2SO4 to activated enzyme. ESEEM spectra of the samples prepared with [4'-2H]PLP exhibited distinctive low-frequency modulations that were not present in spectra of matched samples with unlabeled PLP. Fourier transformation of the modulations yielded a prominent doublet signal centered about the Larmor frequency of deuterium. The magnitude of the doublet splitting of the 2H ESEEM signal exhibited angle selection across the CW EPR powder pattern. The observed angle selection, as well as simulation of the time domain spectra, indicated that the doublet splitting was due to the combined effects of the 2H hyperfine and nuclear quadrupole interactions. The influences of the quadrupole interaction and of isotropic and dipolar hyperfine interactions were explored by simulations of the ESEEM spectra. The analysis indicates a distance of < 3.5 A between the 2H at C4' of PLP and the radical center at C alpha lysine. The data are most compatible with an aldimine linkage between PLP and the beta-nitrogen of beta-lysine.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Isomerases de Aminoácido/metabolismo , Clostridium/enzimologia , Transferases Intramoleculares , Lisina/metabolismo , Fosfato de Piridoxal/metabolismo , Ânions/farmacologia , Cloretos/farmacologia , Deutério , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres , Lisina/análogos & derivados , Lisina/biossíntese , Modelos Químicos , Sulfatos/farmacologiaRESUMO
Lysine 2,3-aminomutase from Clostridia catalyzes the interconversion of L-lysine and L-beta-lysine. The enzyme contains iron-sulfur clusters and is activated by pyridoxal 5'-phosphate and S-adenosylmethionine, all of which participate in catalysis. Current spectroscopic evidence implicates two substrate-based organic radicals as intermediates in the mechanism. One of these species, the radical N3-(5'-phosphopyridoxylidene)-beta-lysin-2-yl (3), appears in the steady state of the reaction of lysine and has been definitively characterized by EPR and ESEEM spectroscopy. The 2-deuterio form of this radical, 3-2-d, which is generated in the reaction of L-[2-2H]lysine, can be distinguished by line shape analysis from 3. The rate at which the signal for 3-2-d is transformed into that for 3 has been measured by rapid mix-freeze quench kinetic analysis. The rate constant for this process is 24 +/- 8 s-1 at 21 degrees C. This is the rate constant for the turnover of radical 3 and is indistinguishable from the turnover number of lysine 2,3-aminomutase. Therefore, radical 3 is kinetically competent as an intermediate in the reaction of lysine 2,3-aminomutase.
Assuntos
Isomerases de Aminoácido/metabolismo , Clostridium/enzimologia , Transferases Intramoleculares , Isomerases de Aminoácido/química , Espectroscopia de Ressonância de Spin Eletrônica , Cinética , Lisina/análogos & derivados , Lisina/metabolismo , Estrutura Molecular , Especificidade por SubstratoRESUMO
Individual residues of the heregulinbeta (HRG) egf domain were mutated to alanine and displayed monovalently on phagemid particles as gene III fusion proteins. Wild type HRGbeta egf domain displayed on phage was properly folded as evidenced by its ability to bind ErbB3 and ErbB4 receptor-IgG fusion proteins with affinities close to those measured for bacterially produced HRGbeta egf domain. Binding to ErbB3 and ErbB4 receptors was affected by mutation of residues throughout the egf domain; including the NH2 terminus (His2 and Leu3), the two beta-turns (Val15-Gly18 and Gly42-Gln46), and some discontinuous residues (including Leu3, Val4, Phe13, Val23, and Leu33) that form a patch on the major beta-sheet and the COOH-terminal region (Tyr48 and Met50-Phe53). Binding affinity was least changed by mutations throughout the Omega-loop and the second strand of the major beta-sheet. More mutants had greater affinity loss for ErbB3 compared with ErbB4 implying that it has more stringent binding requirements. Many residues important for HRG binding to its receptors correspond to critical residues for epidermal growth factor (EGF) and transforming growth factor alpha binding to the EGF receptor. Specificity may be determined in part by bulky groups that prevent binding to the unwanted receptor. All of the mutants tested were able to induce phosphorylation and mitogen-activated protein kinase activation through ErbB4 receptors and were able to modulate a transphosphorylation signal from ErbB3 to ErbB2 in MCF7 cells. An understanding of binding similarities and differences among the EGF family of ligands may facilitate the development of egf-like analogs with broad or narrow specificity.
Assuntos
Proteínas de Transporte/metabolismo , Receptores ErbB/metabolismo , Glicoproteínas/metabolismo , Neuregulina-1 , Proteínas Proto-Oncogênicas/metabolismo , Alanina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/ultraestrutura , Ativação Enzimática , Glicoproteínas/química , Glicoproteínas/ultraestrutura , Humanos , Ligantes , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Fosforilação , Ligação Proteica , Estrutura Secundária de Proteína , Receptor ErbB-3 , Receptor ErbB-4 , Transdução de Sinais , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Heregulins (HRGs) are epidermal growth factor (egf) domain containing polypeptide growth factors that bind and activate several members of the ErbB receptor family. Although HRG can bind to ErbB3 and ErbB4 homodimers, the highest affinity and most intracellularly active receptor complexes are hetero-oligomers containing ErbB2. The HRGbeta egf domain was displayed on the surface of M13 phage to facilitate mutagenic analysis and optimize for binding to a homodimeric ErbB3-immunoglobulin (IgG) fusion. Nine libraries were constructed in which virtually the entire sequence was randomized in stretches of four to six amino acids. These were selected separately for binding to immobilized ErbB3-IgG. Analysis of the resulting sequences revealed some areas that diverged radically from the wild-type, whereas others showed strong conservation. The degree of wild-type conservation correlated strongly with the functional importance of the residues as determined by alanine scanning mutagenesis (Jones, J. T., Ballinger, M. D., Pisacane, P. I., Lofgren, J. A., Fitzpatrick, V. D., Fairbrother, W. J., Wells, J. A., and Sliwkowski, M. X. (1998) J. Biol. Chem. 273, 11667-11674). Some variants from several libraries showed significant improvements in binding affinity to the ErbB3-IgG. These optimized segments were combined in various ways in the same molecule to generate variants (containing up to 16 mutations) that had >50-fold higher affinity than wild-type HRGbeta. The optimized variants stimulated ErbB2 phophorylation on MCF7 cells at levels similar to wild-type. This indicates wild-type affinity is optimized for potency and that factors other than affinity for ErbB3 are limiting. These variants showed enhanced affinity toward the ErbB4 homodimer, suggesting these receptors use very similar binding determinants despite them having 65% sequence identity.