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Taisuke Tomita

Publications and source records attributed to Taisuke Tomita.

18 recordsLinked to original sources
PubMed2004

Selective reconstitution and recovery of functional gamma-secretase complex on budded baculovirus particles.

In vitro reconstitution of functions of membrane proteins is often hampered by aggregation, misfolding, or lack of post-translational modifications of the proteins attributable to overexpression. To overcome this technical obstacle, we have developed a method to express multimeric integral membrane proteins in extracellular (budded) baculovirus particles that are released from Sf9 cells co-infected with multiple transmembrane proteins. We applied this method to the reconstitution of gamma-secretase, a membrane protease complex that catalyzes the intramembrane cleavage of beta-amyloid precursor protein to release Abeta peptides, the major component of amyloid deposits in Alzheimer brains as well as of Notch. When we co-infected Sf9 cells with human presenilin 1 (PS1), nicastrin, APH-1a, and PEN-2, a high-molecular-weight membrane protein complex that contained PS1 exclusively in its fragment form associated with three other cofactor proteins was reconstituted and recovered in a highly gamma-secretase-active state in budded virus particles, whereas nonfunctional PS1 holoproteins massively contaminated the parental Sf9 cell membranes. The relative gamma-secretase activity (per molar PS1 fragments) was concentrated by approximately 2.5 fold in budded virus particles compared with that in Sf9 membranes. The budded baculovirus system will facilitate structural and functional analyses of gamma-secretase, as well as screening of its binding molecules or inhibitors, and will also provide a versatile methodology for the characterization of a variety of membrane protein complexes.

Amyloid Precursor Protein Secretases↗
PubMed2004

Parallel synthesis of DAPT derivatives and their gamma-secretase-inhibitory activity.

Parallel synthesis of the C-terminal-modified DAPT (1) derivatives was accomplished utilizing our novel resin 7. Condensation reaction of the N-acylamino acid 10 with the amines 11a-o proceeded smoothly to give the corresponding amides 6a-o without any epimerization. Among the analogues, the benzophenonemethyl amide derivative 6o showed 30 times more potent activity than the original DAPT (1).

Amides↗
PubMed2004

Targeted introduction of V642I mutation in amyloid precursor protein gene causes functional abnormality resembling early stage of Alzheimer's disease in aged mice.

While the exact aetiology of Alzheimer's disease (AD) is unknown, distinct genetic mutations have been identified for the rare cases of familial AD (FAD). V642I mutation in amyloid precursor protein (APP) co-segregates with FAD with perfect penetration, and the clinicopathological characteristics of patients with this mutation resemble that of sporadic AD. To examine the pathogenic process of this FAD-linked trait in vivo, we produced a mouse with the corresponding point mutation in the APP gene using homologous recombination and Cre-loxP site-specific recombination ('knock-in' technique). Mice with the heterozygous V642I-APP allele most precisely reflected the genotype of humans bearing this mutation. For the observation period of 2.5 years the mutants stayed apparently indistinguishable from the wild-type littermates. However, behavioural analysis revealed significantly deteriorated long-term memory in mutants when examined for the retention of spatial attention. Interestingly, acquisition of spatial memory was slightly affected but short-term working memory was not deteriorated at all. Histological examination was negative for formation of neuritic plaques or neurofibrillary tangles, whereas the relative amount of longer form of beta-amyloid species A beta 42(43) was significantly increased against that of the shorter form (A beta 40) in the mutant brain homogenates. We conclude that a V642I-APP mutant allele in aged mice confers functional components, but not organic components, of the AD-related phenotype that are observed in the early stage of AD. This V642I-APP knock-in mutant line may serve as a model to study the early pathogenic processes of AD in vivo and to develop therapeutics for this stage.

Age Factors↗
PubMed2004

Biochemical characterization of the Drosophila wingless signaling pathway based on RNA interference.

Regulation of Armadillo (Arm) protein levels through ubiquitin-mediated degradation plays a central role in the Wingless (Wg) signaling. Although zeste-white3 (Zw3)-mediated Arm phosphorylation has been implicated in its degradation, we have recently shown that casein kinase Ialpha (CKIalpha) also phosphorylates Arm and induces its degradation. However, it remains unclear how CKIalpha and Zw3, as well as other components of the Arm degradation complex, regulate Arm phosphorylation in response to Wg. In particular, whether Wg signaling suppresses CKIalpha- or Zw3-mediated Arm phosphorylation in vivo is unknown. To clarify these issues, we performed a series of RNA interference (RNAi)-based analyses in Drosophila S2R+ cells by using antibodies that specifically recognize Arm phosphorylated at different serine residues. These analyses revealed that Arm phosphorylation at serine-56 and at threonine-52, serine-48, and serine-44, is mediated by CKIalpha and Zw3, respectively, and that Zw3-directed Arm phosphorylation requires CKIalpha-mediated priming phosphorylation. Daxin stimulates Zw3- but not CKIalpha-mediated Arm phosphorylation. Wg suppresses Zw3- but not CKIalpha-mediated Arm phosphorylation, indicating that a vital regulatory step in Wg signaling is Zw3-mediated Arm phosphorylation. In addition, further RNAi-based analyses of the other aspects of the Wg pathway clarified that Wg-induced Dishevelled phosphorylation is due to CKIalpha and that presenilin and protein kinase A play little part in the regulation of Arm protein levels in Drosophila tissue culture cells.

Adaptor Proteins, Signal Transducing↗
PubMed2004

The inhibition of gamma-secretase as a therapeutic approach to Alzheimer's disease.

Alzheimer's disease is a dementing neurodegenerative disorder for which there is no effective treatment at present. Genetic and biological studies provide evidence that the production and deposition of amyloid-beta peptides (Abeta contribute to the etiology of Alzheimer's disease. gamma-Secretase is the pivotal enzyme in generating the C terminus of Abeta which determines its aggregability and speed of deposition. Drugs that regulate the production of Abeta by inhibiting gamma-secretase activity could provide an effective therapy for Alzheimer's disease, although recent studies suggest that gamma-secretase plays important roles in cellular signaling. This review focuses on studies of the gamma-secretase biology and provides the direction for developing effective and selective gamma-secretase inhibitors as drugs for the treatment of Alzheimer's disease.

Alzheimer Disease↗
PubMed2003

A presenilin dimer at the core of the gamma-secretase enzyme: insights from parallel analysis of Notch 1 and APP proteolysis.

Notch receptors and the amyloid precursor protein are type I membrane proteins that are proteolytically cleaved within their transmembrane domains by a presenilin (PS)-dependent gamma-secretase activity. In both proteins, two peptide bonds are hydrolyzed: one near the inner leaflet and the other in the middle of the transmembrane domain. Under saturating conditions the substrates compete with each other for proteolysis, but not for binding to PS. At least some Alzheimer's disease-causing PS mutations reside in proteins possessing low catalytic activity. We demonstrate (i) that differentially tagged PS molecules coimmunoprecipitate, and (ii) that PS N-terminal fragment dimers exist by using a photoaffinity probe based on a transition state analog gamma-secretase inhibitor. We propose that gamma-secretase contains a PS dimer in its catalytic core, that binding of substrate is at a site separate from the active site, and that substrate is cleaved at the interface of two PS molecules.

Amyloid Precursor Protein Secretases↗
PubMed2003

Analysis of neurons created from wild-type and Alzheimer's mutation knock-in embryonic stem cells by a highly efficient differentiation protocol.

It is impossible to obtain and amplify live neurons from Alzheimer's disease (AD) patients. To establish the neurons harboring AD abnormality, we constructed mouse embryonic stem (ES) cells, in which the AD-causative V642I mutation was introduced to the endogenous amyloid precursor protein (APP) gene, in combination with a protocol to efficiently differentiate ES cells into postmitotic neurons without using a cell sorter. By this protocol, ES cells differentiated into >90% of the central type of adult postmitotic neurons. Neurons derived from V642I-APP knock-in ES cells were indistinguishable from wild-type ES-derived neurons, as determined by the expression of various markers for neuronal differentiation. Notably, V642I-APP knock-in ES cell-derived neurons exhibited significantly increased secretion of Abeta42 without AD-related hyperphosphorylation of tau, indicating that the direct output of the AD-causative mutation is increased Abeta42 secretion. In this study, we analyze created neurons with wild-type and AD genotypes and propose a new strategy for generating neurons for any dominantly inherited neurodegenerative diseases. The strategy can be applied to create human neurons with AD or any other neurodegenerative disease by using human ES cells.

Alzheimer Disease↗
PubMed2003

APP processing and synaptic function.

A large body of evidence has implicated Abeta peptides and other derivatives of the amyloid precursor protein (APP) as central to the pathogenesis of Alzheimer's disease (AD). However, the functional relationship of APP and its proteolytic derivatives to neuronal electrophysiology is not known. Here, we show that neuronal activity modulates the formation and secretion of Abeta peptides in hippocampal slice neurons that overexpress APP. In turn, Abeta selectively depresses excitatory synaptic transmission onto neurons that overexpress APP, as well as nearby neurons that do not. This depression depends on NMDA-R activity and can be reversed by blockade of neuronal activity. Synaptic depression from excessive Abeta could contribute to cognitive decline during early AD. In addition, we propose that activity-dependent modulation of endogenous Abeta production may normally participate in a negative feedback that could keep neuronal hyperactivity in check. Disruption of this feedback system could contribute to disease progression in AD.

Alzheimer Disease↗
PubMed2003

The role of presenilin cofactors in the gamma-secretase complex.

Mutations in presenilin genes account for the majority of the cases of the familial form of Alzheimer's disease (FAD). Presenilin is essential for gamma-secretase activity, a proteolytic activity involved in intramembrane cleavage of Notch and beta-amyloid precursor protein (betaAPP). Cleavage of betaAPP by FAD mutant presenilin results in the overproduction of highly amyloidogenic amyloid beta42 peptides. gamma-Secretase activity requires the formation of a stable, high-molecular-mass protein complex that, in addition to the endoproteolysed fragmented form of presenilin, contains essential cofactors including nicastrin, APH-1 (refs 15-18) and PEN-2 (refs 16, 19). However, the role of each protein in complex formation and the generation of enzymatic activity is unclear. Here we show that Drosophila APH-1 (Aph-1) increases the stability of Drosophila presenilin (Psn) holoprotein in the complex. Depletion of PEN-2 by RNA interference prevents endoproteolysis of presenilin and promotes stabilization of the holoprotein in both Drosophila and mammalian cells, including primary neurons. Co-expression of Drosophila Pen-2 with Aph-1 and nicastrin increases the formation of Psn fragments as well as gamma-secretase activity. Thus, APH-1 stabilizes the presenilin holoprotein in the complex, whereas PEN-2 is required for endoproteolytic processing of presenilin and conferring gamma-secretase activity to the complex.

Alzheimer Disease↗
PubMed2003

Presenilin-dependent gamma-secretase activity mediates the intramembranous cleavage of CD44.

CD44 is the major adhesion molecule for the extracellular matrix components and is implicated in a wide variety of physiological and pathological processes including the regulation of tumor cell growth and metastasis. Our previous studies have shown that CD44 undergoes sequential proteolytic cleavages in the extracellular and transmembrane domains and the cleavage product derived from CD44 intramembranous cleavage acts as a signal transduction molecule. However, the underlying mechanism of the intramembranous cleavage of CD44 remains to be elucidated. In the present study, we report for the first time that CD44 is a substrate of the presenilin (PS)-dependent gamma-secretase. We demonstrate that the intramembranous cleavage of CD44 induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment or mechanical scraping is blocked by gamma-secretase inhibitors in U251MG cells and that this cleavage is also inhibited in PS-deficient mouse embryonic fibroblasts. Furthermore, we showed that PS1 is redistributed to ruffling areas of the plasma membrane similarly to CD44 after TPA treatment, supporting our biochemical observation that PS1 is involved in the intramembranous cleavage of CD44. Our present findings suggest important implications for understanding CD44-dependent signal transduction and a potential role of PS/gamma-secretase activity in the functional regulation of adhesion molecules.

Amyloid Precursor Protein Secretases↗
PubMed2003

Sulindac sulfide is a noncompetitive gamma-secretase inhibitor that preferentially reduces Abeta 42 generation.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been known to reduce risk for Alzheimer's disease. In addition to the anti-inflammatory effects of NSAIDs to block cylooxygenase, it has been shown recently that a subset of NSAIDs selectively inhibits the secretion of highly amyloidogenic Abeta42 from cultured cells, although the molecular target(s) of NSAIDs in reducing the activity of gamma-secretase for Abeta42 generation (gamma(42)-secretase) still remain unknown. Here we show that sulindac sulfide (SSide) directly acts on gamma-secretase and preferentially inhibits the gamma(42)-secretase activity derived from the 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate-solubilized membrane fractions of HeLa cells, in an in vitro gamma-secretase assay using recombinant amyloid beta precursor protein C100 as a substrate. SSide also inhibits activities for the generation of Abeta40 as well as for Notch intracellular domain at higher concentrations. Notably, SSide displayed linear noncompetitive inhibition profiles for gamma(42)-secretase in vitro. Our data suggest that SSide is a direct inhibitor of gamma-secretase that preferentially affects the gamma(42)-secretase activity.

Amyloid Precursor Protein Secretases↗
PubMed2002

The mechanism of gamma-secretase activities through high molecular weight complex formation of presenilins is conserved in Drosophila melanogaster and mammals.

Mutations in presenilin 1 (PS1) and PS2 genes contribute to the pathogenesis of early onset familial Alzheimer's disease by increasing secretion of the pathologically relevant Abeta42 polypeptides. PS genes are also implicated in Notch signaling through proteolytic processing of the Notch receptor in Caenorhabditis elegans, Drosophila melanogaster, and mammals. Here we show that Drosophila PS (Psn) protein undergoes endoproteolytic cleavage and forms a stable high molecular weight (HMW) complex in Drosophila S2 or mouse neuro2a (N2a) cells in a similar manner to mammalian PS. The loss-of-function recessive point mutations located in the C-terminal region of Psn, that cause an early pupal-lethal phenotype resembling Notch mutant in vivo, disrupted the HMW complex formation, and abolished gamma-secretase activities in cultured cells. The overexpression of Psn in mouse embryonic fibroblasts lacking PS1 and PS2 genes rescued the Notch processing. Moreover, disruption of the expression of Psn by double-stranded RNA-mediated interference completely abolished the gamma-secretase activity in S2 cells. Surprisingly, gamma-secretase activity dependent on wild-type Psn was associated with a drastic overproduction of Abeta1-42 from human betaAPP in N2a cells, but not in S2 cells. Our data suggest that the mechanism of gamma-secretase activities through formation of HMW PS complex, as well as its abolition by loss-of-function mutations located in the C terminus, are highly conserved features in Drosophila and mammals.

Amyloid Precursor Protein Secretases↗
PubMed2002

Presenilins mediate a dual intramembranous gamma-secretase cleavage of Notch-1.

Following ectodomain shedding, Notch-1 undergoes presenilin (PS)-dependent constitutive intramembranous endoproteolysis at site-3. This cleavage is similar to the PS-dependent gamma-secretase cleavage of the beta-amyloid precursor protein (betaAPP). However, topological differences in cleavage resulting in amyloid beta-peptide (Abeta) or the Notch-1 intracellular domain (NICD) indicated independent mechanisms of proteolytic cleavage. We now demonstrate the secretion of an N-terminal Notch-1 Abeta-like fragment (Nbeta). Analysis of Nbeta by MALDI-TOF MS revealed that Nbeta is cleaved at a novel site (site-4, S4) near the middle of the transmembrane domain. Like the corresponding cleavage of betaAPP at position 40 and 42 of the Abeta domain, S4 cleavage is PS dependent. The precision of this cleavage is affected by familial Alzheimer's disease-associated PS1 mutations similar to the pathological endoproteolysis of betaAPP. Considering these similarities between intramembranous processing of Notch and betaAPP, we conclude that these proteins are cleaved by a common mechanism utilizing the same protease, i.e. PS/gamma-secretase.

Alzheimer Disease↗
PubMed2002

Complex N-glycosylated form of nicastrin is stabilized and selectively bound to presenilin fragments.

The transmembrane glycoprotein nicastrin is a component of presenilin (PS) protein complex that is involved in gamma-cleavage of beta APP and site-3 cleavage of Notch. PS undergoes endoproteolysis, and the proteolytic fragments are incorporated into the high molecular weight protein complexes that are highly stabilized. Here we show that Endo H-resistant, N-glycosylated form of nicastrin (p150-NCT) is highly stabilized and selectively bound to PS fragments. Moreover, loss-of-function mutations of nicastrin inhibited formation of fully glycosylated p150-NCT as well as stabilization of nicastrin, suggesting that glycosylation and stabilization of nicastrin polypeptides are tightly correlated with its function.

Amyloid Precursor Protein Secretases↗
PubMed2002

Molecular cloning and characterization of CALP/KChIP4, a novel EF-hand protein interacting with presenilin 2 and voltage-gated potassium channel subunit Kv4.

Presenilin (PS) genes linked to early-onset familial Alzheimer's disease encode polytopic membrane proteins that are presumed to constitute the catalytic subunit of gamma-secretase, forming a high molecular weight complex with other proteins. During our attempts to identify binding partners of PS2, we cloned CALP (calsenilin-like protein)/KChIP4, a novel member of calsenilin/KChIP protein family that interacts with the C-terminal region of PS. Upon co-expression in cultured cells, CALP was directly bound to and co-localized with PS2 in endoplasmic reticulum. Overexpression of CALP did not affect the metabolism or stability of PS complex, and gamma-cleavage of betaAPP or Notch site 3 cleavage was not altered. However, co-expression of CALP and a voltage-gated potassium channel subunit Kv4.2 reconstituted the features of A-type K(+) currents and CALP directly bound Kv4.2, indicating that CALP functions as KChIPs that are known as components of native Kv4 channel complex. Taken together, CALP/KChIP4 is a novel EF-hand protein interacting with PS as well as with Kv4 that may modulate functions of a subset of membrane proteins in brain.

Amino Acid Sequence↗
PubMed2002

A role for presenilin 1 in regulating the delivery of amyloid precursor protein to the cell surface.

Presenilin 1 (PS1) and presenilin 2 play a critical role in the gamma-secretase processing of amyloid precursor protein (APP) and Notch1. Here, we investigate maturation and intracellular trafficking of APP and other membrane proteins in cells expressing an experimental PS1 deletion mutant (deltaM1,2). Stable expression of deltaM1,2 impairs gamma-secretase processing of Notch1 and delays Abeta secretion. Kinetic studies show enhanced O-glycosylation and sialylation of holo-APP and marked accumulation of APP COOH-terminal fragments (CTFs). Surface biotinylation, live staining, and trafficking studies show increased surface accumulation of holo-APP and CTFs in deltaM1,2 cells resulting from enhanced surface delivery of newly synthesized APP. Expression of a loss-of-function PS1 mutant (D385A) or incubation of cells with gamma-secretase inhibitors also increases surface levels of holo-APP and CTFs. In contrast to APP, glycosylation and surface accumulation of another type I membrane protein, nicastrin, are markedly reduced in deltaM1,2 cells. Finally, expression of deltaM1,2 results in the increased assembly and surface expression of nicotinic acetylcholine receptors, illustrating that PS1's influence on protein trafficking extends beyond APP and other type I membrane protein substrates of gamma-secretase. Collectively, our findings provide evidence that PS1 regulates the glycosylation and intracellular trafficking of APP and select membrane proteins.

Amyloid Precursor Protein Secretases↗