RESUMO
The γ-secretase complexes are intramembrane cleaving proteases involved in the generation of the Aß peptides in Alzheimer's disease. The complex consists of four subunits, with Presenilin harboring the catalytic site. Here, we study the role of the smallest subunit, PSENEN or Presenilin enhancer 2, encoded by the gene Psenen, in vivo and in vitro. We find a profound Notch deficiency phenotype in Psenen-/- embryos confirming the essential role of PSENEN in the γ-secretase complex. We used Psenen-/- fibroblasts to explore the structure-function of PSENEN by the scanning cysteine accessibility method. Glycine 22 and proline 27, which border the membrane domains 1 and 2 of PSENEN, are involved in complex formation and stabilization of γ-secretase. The hairpin structured hydrophobic membrane domains 1 and 2 are exposed to a water-containing cavity in the complex, while transmembrane domain 3 is not water exposed. We finally demonstrate the essential role of PSENEN for the cleavage activity of the complex. PSENEN is more than a structural component of the γ-secretase complex and might contribute to the catalytic mechanism of the enzyme.
Assuntos
Secretases da Proteína Precursora do Amiloide , Animais , Feminino , Masculino , Camundongos , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Proteínas de Membrana/química , Camundongos Endogâmicos C57BL , Presenilina-1/genética , Estrutura Terciária de ProteínaRESUMO
Target identification is a core challenge in chemical genetics. Here we use chemical similarity to computationally predict the targets of 586 compounds that were active in a zebrafish behavioral assay. Among 20 predictions tested, 11 compounds had activities ranging from 1 nM to 10,000 nM on the predicted targets. The roles of two of these targets were tested in the original zebrafish phenotype. Prediction of targets from chemotype is rapid and may be generally applicable.
Assuntos
Simulação por Computador , Avaliação Pré-Clínica de Medicamentos/métodos , Animais , Comportamento Animal/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fenótipo , Relação Estrutura-Atividade , Peixe-ZebraRESUMO
The γ-secretase complex is a member of the family of intramembrane cleaving proteases, involved in the generation of the Aß peptides in Alzheimer disease. One of the four subunits of the complex, presenilin, harbors the catalytic site, although the role of the other three subunits is less well understood. Here, we studied the role of the smallest subunit, Pen-2, in vivo and in vitro. We found a profound Notch-deficiency phenotype in Pen-2-/- embryos confirming the essential role of Pen-2 in the γ-secretase complex. We used Pen-2-/- fibroblasts to investigate the structure-function relation of Pen-2 by the scanning cysteine accessibility method. We showed that glycine 22 and proline 27 in hydrophobic domain 1 of Pen-2 are essential for complex formation and stability of γ-secretase. We also demonstrated that hydrophobic domain 1 and the loop domain of Pen-2 are located in a water-containing cavity and are in short proximity to the presenilin C-terminal fragment. We finally demonstrated the essential role of Pen-2 for the proteolytic activity of the complex. Our study supports the hypothesis that Pen-2 is more than a structural component of the γ-secretase complex and may contribute to the catalytic mechanism of the enzyme.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Presenilina-2/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Animais , Células Cultivadas , Cisteína/genética , Cisteína/metabolismo , Embrião de Mamíferos , Feminino , Glicina/genética , Glicina/metabolismo , Hibridização In Situ , Masculino , Camundongos , Camundongos Knockout , Presenilina-2/genética , Prolina/genética , Prolina/metabolismoRESUMO
Missense mutations in presenilin 1 (PS1) and presenilin 2 (PS2) proteins are a major cause of familial Alzheimer disease. Presenilins are proteins with nine transmembrane (TM) domains that function as catalytic subunits of the γ-secretase complex responsible for the cleavage of the amyloid precursor protein and other type I transmembrane proteins. The water-filled cavity within presenilin is necessary to mediate the intramembrane proteolysis reaction. Consistent with this idea, cysteine-scanning mutagenesis and NMR studies revealed a number of water-accessible residues within TM7 and TM9 of mouse PS1. In addition to γ-secretase function, presenilins also demonstrate a low conductance endoplasmic reticulum Ca(2+) leak function, and many familial Alzheimer disease presenilin mutations impair this function. To map the potential Ca(2+) conductance pore in PS1, we systematically evaluated endoplasmic reticulum Ca(2+) leak activity supported by a series of cysteine point mutants in TM6, TM7, and TM9 of mouse PS1. The results indicate that TM7 and TM9, but not TM6, could play an important role in forming the conductance pore of PS1. These results are consistent with previous cysteine-scanning mutagenesis and NMR analyses of PS1 and provide further support for our hypothesis that the hydrophilic catalytic cavity of presenilins may also constitute a Ca(2+) conductance pore.
Assuntos
Cálcio/metabolismo , Condutividade Elétrica , Mutagênese , Presenilina-1/genética , Presenilina-1/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Cisteína , Retículo Endoplasmático/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Mutação , Porosidade , Presenilina-1/química , Estrutura Terciária de ProteínaRESUMO
Calmodulin (CaM) is an important regulator of Kv7.x (KCNQx) voltage-gated potassium channels. Channels from this family produce neuronal M currents and cardiac and auditory I(KS) currents and harbor mutations that cause arrhythmias, epilepsy, and deafness. Despite extensive functional characterization, biochemical and structural details of the interaction between CaM and the channel have remained elusive. Here, we show that both apo-CaM and Ca(2+)/CaM bind to the C-terminal tail of the neuronal channel Kv7.4 (KCNQ4), which is involved in both hearing and mechanosensation. Interactions between apo-CaM and the Kv7.4 tail involve two C-terminal tail segments, known as the A and B segments, whereas the interaction between Ca(2+)/CaM and the Kv7.4 C-terminal tail requires only the B segment. Biochemical studies show that the calcium dependence of the CaM:B segment interaction is conserved in all Kv7 subtypes. X-ray crystallographic determination of the structure of the Ca(2+)/CaM:Kv7.4 B segment complex shows that Ca(2+)/CaM wraps around the Kv7.4 B segment, which forms an α-helix, in an antiparallel orientation that embodies a variation of the classic 1-14 Ca(2+)/CaM interaction motif. Taken together with the context of prior studies, our data suggest a model for modulation of neuronal Kv7 channels involving a calcium-dependent conformational switch from an apo-CaM form that bridges the A and B segments to a Ca(2+)/CaM form bound to the B-helix. The structure presented here also provides a context for a number of disease-causing mutations and for further dissection of the mechanisms by which CaM controls Kv7 function.
Assuntos
Potenciais de Ação , Cálcio/metabolismo , Calmodulina/química , Canais de Potássio KCNQ/química , Sequência de Aminoácidos , Cálcio/química , Calmodulina/genética , Calmodulina/metabolismo , Cristalografia por Raios X , Humanos , Canais de Potássio KCNQ/genética , Canais de Potássio KCNQ/metabolismo , Dados de Sequência Molecular , Mutação/genética , Ligação Proteica , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
Interactions between calmodulin (CaM) and voltage-gated calcium channels (Ca(v)s) are crucial for Ca(v) activity-dependent feedback modulation. We recently reported an X-ray structure that shows two Ca(2+)/CaM molecules bound to the Ca(v)1.2 C terminal tail, one at the PreIQ region and one at the IQ domain. Surprisingly, the asymmetric unit of the crystal showed a dimer in which Ca(2+)/CaM bridged two PreIQ helixes to form a 4:2 Ca(2+)/CaM:Ca(v) C-terminal tail assembly. Contrary to previous proposals based on a similar crystallographic dimer, extensive biochemical analysis together with subunit counting experiments of full-length channels in live cell membranes failed to find evidence for multimers that would be compatible with the 4:2 crossbridged complex. Here, we examine this possibility further. We find that CaM over-expression has no functional effect on Ca(v)1.2 inactivation or on the stoichiometry of full-length Ca(v)1.2. These data provide further support for the monomeric Ca(v)1.2 stoichiometry. Analysis of the electrostatic surfaces of the 2:1 Ca(2+)/CaM:Ca(V) C-terminal tail assembly reveals notable patches of electronegativity. These could influence various forms of channel modulation by interacting with positively charged elements from other intracellular channel domains.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Calmodulina/biossíntese , Membrana Celular/metabolismo , Expressão Gênica , Multimerização Proteica , Animais , Canais de Cálcio Tipo L/genética , Calmodulina/genética , Membrana Celular/genética , Humanos , Estrutura Terciária de Proteína , Xenopus laevisRESUMO
The gamma-secretase complex is a prime target for pharmacological intervention in Alzheimer's disease and so far drug discovery efforts have yielded a large variety of potent and rather specific inhibitors of this enzymatic activity. However, as gamma-secretase is able to cleave a wide variety of physiological important substrates, the real challenge is to develop substrate-specific compounds. Therefore, obtaining structural information about gamma-secretase is indispensable. As crystal structures of the complex will be difficult to achieve, applied biochemical approaches need to be integrated with structural information obtained from other intramembrane-cleaving proteases. Here we review current knowledge about the structure and function of gamma-secretase and discuss the value of these findings for the mechanistic understanding of this unusual protease.
Assuntos
Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/fisiologia , Secretases da Proteína Precursora do Amiloide/genética , Sítios de Ligação , Humanos , Modelos Biológicos , Mutação , Transdução de SinaisRESUMO
One of the most prominent drug targets for the treatment of Alzheimer disease is gamma-secretase, a multi-protein complex responsible for the generation of the amyloid-beta peptide. The catalytic core of the complex lies on presenilin, a multi-spanning membrane protease, the activity of which depends on two aspartate residues located in transmembrane domains 6 and 7. We have recently shown by cysteine-scanning mutagenesis that these aspartates are facing a water-filled cavity in the lipid bilayer, demonstrating how proteolytic cleavage of the substrates can be taking place within the membrane. Here, we demonstrate that transmembrane domain 9 and hydrophobic domain VII in the large cytoplasmic loop of presenilin are dynamic structural parts of this cavity. Hydrophobic domain VII is associated with transmembrane domain 7 in the membrane, probably facilitating the entrance of water molecules in the catalytic site. Transmembrane domain 9, on the other hand, exhibits a highly flexible structure, potentially involved in the transport of substrates to the catalytic site, as well as in the binding of gamma-secretase inhibitors. The conserved proline-alanine-leucine motif at the cytoplasmic part of this domain is extremely close to the catalytic Asp257 and is crucial for conformational changes leading to the activation of the catalytic site. We, also, identify a unique mutant in this domain (I437C) that specifically blocks amyloid-beta peptide production without affecting the processing of the physiologically indispensable Notch substrate. Our data are finally combined to propose a model for the architectural organization and activation of the catalytic site of presenilin.
Assuntos
Secretases da Proteína Precursora do Amiloide/química , Peptídeos beta-Amiloides/química , Bicamadas Lipídicas/química , Modelos Moleculares , Peptídeos/química , Presenilinas/química , Doença de Alzheimer/enzimologia , Doença de Alzheimer/genética , Substituição de Aminoácidos , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Domínio Catalítico/fisiologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/metabolismo , Mutagênese , Peptídeos/genética , Peptídeos/metabolismo , Presenilinas/genética , Presenilinas/metabolismo , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Estrutura Terciária de Proteína/fisiologiaRESUMO
The gamma-secretase complex is responsible for the proteolysis of integral membrane proteins. Nicastrin has been proposed to operate as the substrate receptor of the complex with the glutamate 332 (Glu(333) in human) serving as the anionic binding site for the alpha-amino-terminal group of substrates. The putative binding site is located within the aminopeptidase-like domain of Nicastrin. The Glu(332) is proposed to function as the counterpart of the exopeptidase Glu located in the active site of these peptidases. Although Glu(332) could bind the alpha-amino-terminal group of substrates, we hypothesized, in analogy with M28-aminopeptidases, that other residues in the putative binding site of Nicastrin should participate in the interaction as well. Surprisingly, mutagenesis of these residues affected the in vivo processing of APP and Notch substrates only weakly. In addition, the E332Q mutation, which completely abolishes the anionic alpha-amino-terminal binding function, remained fully active. When we introduced the previously characterized E332A mutation, we found strongly decreased gamma-secretase complex levels, but the remaining complex appeared as active as the wild-type complex. We confirmed in two independent in vitro assays that the specific enzymatic activity of the E332A mutant was comparable with that of the wild-type complex. Thus, Glu(332) crucially affects complex maturation rather than substrate recognition. Moreover other Nicastrin mutants, designed to either impede or alter substantially the putative binding pocket, affected only marginally gamma-secretase activity. Consequently, these studies indicate that the main role of the Glu(332) is in the maturation and assembly of gamma-secretase rather than in the recognition of the substrates.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/deficiência , Secretases da Proteína Precursora do Amiloide/genética , Animais , Sítios de Ligação , Linhagem Celular , Sequência Conservada , Expressão Gênica , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Humanos , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Gamma-secretase is a multiprotein complex responsible for the intramembranous cleavage of the amyloid precursor protein and other type I transmembrane proteins. Mutations in Presenilin, the catalytic core of this complex, cause Alzheimer disease. Little is known about the structure of the protein and even less about the catalytic mechanism, which involves proteolytic cleavage in the hydrophobic environment of the cell membrane. It is basically unclear how water, needed to perform hydrolysis, is provided to this reaction. Presenilin transmembrane domains 6 and 7 seem critical in this regard, as each bears a critical aspartate contributing to catalytic activity. Current models imply that both aspartyl groups should closely oppose each other and have access to water. This is, however, still to be experimentally verified. Here, we have performed cysteine-scanning mutagenesis of both domains and have demonstrated that several of the introduced residues are exposed to water, providing experimental evidence for the existence of a water-filled cavity in the catalytic core of Presenilin. In addition, we have demonstrated that the two aspartates reside within this cavity and are opposed to each other in the native complex. We have also identified the conserved tyrosine 389 as a critical partner in the catalytic mechanism. Several additional amino acid substitutions affect differentially the processing of gamma-secretase substrates, implying that they contribute to enzyme specificity. Our data suggest the possibility that more selective gamma-secretase inhibitors could be designed.
Assuntos
Endopeptidases/química , Proteínas de Membrana/química , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases , Caderinas/química , Membrana Celular/metabolismo , Dissulfetos/química , Inibidores Enzimáticos/farmacologia , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Presenilina-1 , Estrutura Terciária de Proteína , Receptores Notch/metabolismo , Água/químicaRESUMO
Presenilin-1 is a polytopic membrane protein that assembles with nicastrin, PEN-2, and APH-1 into an active gamma-secretase complex required for intramembrane proteolysis of type I transmembrane proteins. Although essential for a correct understanding of structure-function relationships, its exact topology remains an issue of strong controversy. We revisited presenilin-1 topology by inserting glycosylation consensus sequences in human PS1 and expressing the obtained mutants in a presenilin-1 and 2 knock-out background. Based on the glycosylation status of these variants we provide evidence that presenilin-1 traffics through the Golgi after a conformational change induced by complex assembly. Based on our glycosylation variants of presenilin-1 we hypothesize that complex assembly occurs during transport between the endoplasmic reticulum and the Golgi apparatus. Furthermore, our data indicate that presenilin-1 has a nine-transmembrane domain topology with the COOH terminus exposed to the lumen/extracellular surface. This topology is independently underscored by lysine mutagenesis, cell surface biotinylation, and cysteine derivation strategies and is compatible with the different physiological functions assigned to presenilin-1.
Assuntos
Presenilina-1/química , Presenilina-1/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Glicosilação , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Modelos Moleculares , Presenilina-1/genéticaRESUMO
gamma-Secretase-dependent regulated intramembrane proteolysis of amyloid precursor protein (APP) releases the APP intracellular domain (AICD). The question of whether this domain, like the Notch intracellular domain, is involved in nuclear signalling is highly controversial. Although some reports suggest that AICD regulates the expression of KAI1, glycogen synthase kinase-3beta, Neprilysin and APP, we found no consistent effects of gamma-secretase inhibitors or of genetic deficiencies in the gamma-secretase complex or the APP family on the expression levels of these genes in cells and tissues. Finally, we demonstrate that Fe65, an important AICD-binding protein, transactivates a wide variety of different promoters, including the viral simian virus 40 promoter, independent of AICD coexpression. Overall, the four currently proposed target genes are at best indirectly and weakly influenced by APP processing. Therefore, inhibition of APP processing to decrease Abeta generation in Alzheimer's disease will not interfere significantly with the function of these genes.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/deficiência , Precursor de Proteína beta-Amiloide/genética , Animais , Ácido Aspártico Endopeptidases , Células COS , Linhagem Celular , Chlorocebus aethiops , Endopeptidases/metabolismo , Regulação da Expressão Gênica , Células HeLa , Humanos , Membranas/metabolismo , Camundongos , Peptídeo Hidrolases/metabolismo , Processamento de Proteína Pós-Traducional , Estrutura Terciária de ProteínaRESUMO
Mutations in human presenilin (PS) genes cause aggressive forms of familial Alzheimer's disease. Presenilins are polytopic proteins that harbour the catalytic site of the gamma-secretase complex and cleave many type I transmembrane proteins including beta-amyloid precursor protein (APP), Notch and syndecan 3. Contradictory results have been published concerning whether PS mutations cause 'abnormal' gain or (partial) loss of function of gamma-secretase. To avoid the possibility that wild-type PS confounds the interpretation of the results, we used presenilin-deficient cells to analyse the effects of different clinical mutations on APP, Notch, syndecan 3 and N-cadherin substrate processing, and on gamma-secretase complex formation. A loss in APP and Notch substrate processing at epsilon and S3 cleavage sites was observed with all presenilin mutants, whereas APP processing at the gamma site was affected in variable ways. PS1-Delta9 and PS1-L166P mutations caused a reduction in beta-amyloid peptide Abeta40 production whereas PS1-G384A mutant significantly increased Abeta42. Interestingly PS2, a close homologue of PS1, appeared to be a less efficient producer of Abeta than PS1. Finally, subtle differences in gamma-secretase complex assembly were observed. Overall, our results indicate that the different mutations in PS affect gamma-secretase structure or function in multiple ways.