Divergent Synthesis of Multifunctional Molecular Probes To Elucidate the Enzyme Specificity of Dipeptidic γ-Secretase Inhibitors

Fuwa, Haruhiko; Takahashi, Yasuko; Konno, Yu; Watanabe, Naoto; Miyashita, Hiroyuki; Sasaki, Makoto; Natsugari, Hideaki; Kan, Toshiyuki; Fukuyama, Tohru; Tomita, Taisuke; Iwatsubo, Takeshi

doi:10.1021/cb700073y

Cited by 87 publications

(77 citation statements)

References 52 publications

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“…In fact, in agreement with previous results, photoaffinity probes based on transition state analog (Fig. 2C) as well as dipeptidic (DBZ-BpB3) (24) type inhibitors, all of which specifically targeted the endogenous SPP in mammalian cells (5,24,25), specifically labeled 3FSPP observed as a monomer band on SDS-PAGE. These data suggest that 3FSPP polypeptides overexpressed in Sf9 cells reconstituted the intramembrane-cleaving activity in a natural conformation.…”

Section: Proteolytically Active Spp Polypeptides Form a Multimericsupporting

confidence: 92%

“…L-852,505, L-852,646, Dibenzazepine (DBZ), DBZ-BpB3, 31C, and 31C-Bpa were kindly provided by Drs. Yueming Li (Memorial SloanKettering Cancer Center, NY), Haruhiko Fuwa (Tohoku University, Miyagi, Japan) Naoki Umezawa and Tsunehiko Higuchi (Nagoya City University, Aichi, Japan) (23)(24)(25).…”

Section: Methodsmentioning

confidence: 99%

“…For immunoprecipitation, DDM-solubilized fractions were coincubated with primary antibody and protein G-agarose (Invitrogen). Photoaffinity labeling experiments were performed as described previously, with some modifications (24,25,29). Briefly, microsomal fractions or 0.25% DDM-solubilized fractions were incubated with 50 nM photoprobes (i.e.…”

Section: Methodsmentioning

confidence: 99%

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Three-dimensional Structure of the Signal Peptide Peptidase

Miyashita

Maruyama

Isshiki

et al. 2011

Journal of Biological Chemistry

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Signal peptide peptidase (SPP) is an atypical aspartic protease that hydrolyzes peptide bonds within the transmembrane domain of substrates and is implicated in several biological and pathological functions. Here, we analyzed the structure of human SPP by electron microscopy and reconstructed the three-dimensional structure at a resolution of 22 Å . Enzymatically active SPP forms a slender, bullet-shaped homotetramer with dimensions of 85 ؋ 85 ؋ 130 Å . The SPP complex has four concaves on the rhombus-like sides, connected to a large chamber inside the molecule. Intriguingly, the N-terminal region of SPP is sufficient for the tetrameric assembly. Moreover, overexpression of the N-terminal region inhibited the formation of the endogenous SPP tetramer and the proteolytic activity within cells. These data suggest that the homotetramer is the functional unit of SPP and that its N-terminal region, which works as the structural scaffold, has a novel modulatory function for the intramembrane-cleaving activity of SPP.The intramembrane-cleaving proteases (I-CLiPs) 5 that sever the transmembrane domains of their substrates have been identified in a range of organisms and play a variety of roles in

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Section: Proteolytically Active Spp Polypeptides Form a Multimericsupporting

confidence: 92%

Section: Methodsmentioning

confidence: 99%

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Three-dimensional Structure of the Signal Peptide Peptidase

Miyashita

Maruyama

Isshiki

et al. 2011

Journal of Biological Chemistry

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“…By contrast, a carbene precursor -trifluoromethylphenyldiazirine (TPD) -did not cause cell death in the generation of active species (Hashimoto et al, 2001). Never-the-less, benzophenones (such as those attached to -secretase) are sometimes preferred for photoaffinity labeling experiments in vitro (Fuwa et al, 2006(Fuwa et al, & 2007.…”

Section: Selections Of Photophoresmentioning

confidence: 99%

Selective Hydrogenation and Transfer Hydrogenation for Post-Functional Synthesis of Trifluoromethylphenyl Diazirine Derivatives for Photoaffinity Labeling

Hashimoto¹,

Murai²,

Holman³

et al. 2012

Hydrogenation

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Elucidation of protein functions on the basis of structure–activity relationships can reveal the mechanisms of homeostasis functions in life and is one of the greatest interests of scientists. In the human body, many proteins are activated and/or inactivated by ligands to maintain homeostasis. Understanding the mechanism of molecular interactions between small bioactive ligands and proteins is an important step in rational drug design and discovery. ! Photoaffinity labeling, which is one of the most familiar approaches for chemical biology analysis, was initiated using diazocarbonyl derivatives in 1962 (Singh et al., 1962). Many researchers have subsequently tried to establish alternative approaches for the direct identification of target proteins for the bioactive small ligands. These approaches are based on the affinity between the ligand and the target protein (Figure 1). Several reviews are published for the recent applications of photoaffinity labeling (Tomohiro et al., 2005; Hashimoto & Hatanaka, 2008). To archive photoaffinity labeling, researchers have to prepare photoaffinity labeling ligands. The native ligands must be modified by photoreactive compounds (photophores) by organic synthesi

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“…Recently, a novel c-SC inhibitor-based chemical probes which carries photoaffinity and biotin moieties for cross linking and purification respectively (Morohashi et al, 2006;Fuwa et al, 2007), was developed to directly identify the molecular target of the small compounds. Using this technique, dipeptidic inhibitors (DAPT, compound E and DBZ) were developed.…”

Section: C-secretase Inhibitorsmentioning

confidence: 99%

The Recent Updates of Therapeutic Approaches Against Aβ for the Treatment of Alzheimer's Disease

Shao

Zhou

Rudd

et al. 2011

The Anatomical Record

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One of the main neuropathological lesions observed in brain autopsy of Alzheimer's disease (AD) patients is the extracellular senile plaques mainly composed of amyloid-beta (Ab) peptide. Recently, treatment strategies have focused on modifying the formation, clearance, and accumulation of this potentially neurotoxic peptide. b-and c-secretase are responsible for the cleavage of amyloid precursor protein (APP) and the generation of Ab peptide. Treatments targeting these two critical secretases may therefore reduce Ab peptide levels and positive impact on AD. Vaccination is also an advanced approach against Ab. This review focuses on recent advances of our understanding of this key peptide, with emphasis on Ab peptide synthesis, accumulation and neurotoxicity, and current therapies including vaccination and two critical secretase inhibitors. MicroRNAs (miRNAs) are a class of conserved endogenous small noncoding RNAs, known to regulate the expression of complementary messenger RNAs, involved in AD development. We therefore address the relationship of miRNAs in the brain and Ab generation, as a novel therapeutic approach to the treatment of AD while also providing new insights on the etiology of this neurological disorder. Anat Rec, 294:1307Rec, 294: -1318Rec, 294: , 2011. V V C 2011 Wiley-Liss, Inc.

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Divergent Synthesis of Multifunctional Molecular Probes To Elucidate the Enzyme Specificity of Dipeptidic γ-Secretase Inhibitors

Cited by 87 publications

References 52 publications

Three-dimensional Structure of the Signal Peptide Peptidase

Three-dimensional Structure of the Signal Peptide Peptidase

Selective Hydrogenation and Transfer Hydrogenation for Post-Functional Synthesis of Trifluoromethylphenyl Diazirine Derivatives for Photoaffinity Labeling

The Recent Updates of Therapeutic Approaches Against Aβ for the Treatment of Alzheimer's Disease

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