[Show abstract][Hide abstract] ABSTRACT: γ-Secretase is composed of four proteins that are obligatory for protease activity: presenilin, nicastrin, Aph1, and Pen-2. Despite the progress toward understanding the function of these individual subunits, there is no information available pertaining to the modulation of γ-secretase in response to environmental changes in cells. Here, we show that hypoxia upregulates γ-secretase activity through a direct interaction with Hif-1α, revealing an unconventional function for Hif-1α as an enzyme subunit, which is distinct from its canonical role as a transcription factor. Moreover, hypoxia-induced cell invasion and metastasis are alleviated by either γ-secretase inhibitors or a dominant-negative Notch coactivator, indicating that γ-secretase/Notch signaling plays an essential role in controlling these cellular processes. The present study reveals a mechanism in which γ-secretase can achieve temporal control through conditional interactions with regulatory proteins, such as Hif-1α, under select physiological and pathological conditions.
[Show abstract][Hide abstract] ABSTRACT: γ-Secretase undergoes endoproteolysis of its catalytic subunit, presenilin (PS), to form PS N-terminal and C-terminal fragments (PS1-NTF/CTF), which generate the active site. PS endoproteolysis, catalyzed by presenilinase (PSase), remains poorly understood and requires novel chemical approaches for its mechanistic study. CBAP is a dual inhibitor that suppresses both γ-secretase and PSase activities. To probe γ-secretase and PSase activity in cells, we have synthesized the clickable photoaffinity probe CBAP-BPyne. We found that CBAP-BPyne specifically labels PS1-NTF and signal peptide peptidase (SPP). CBAP-BPyne is a valuable tool to directly study the mechanism of endoproteolysis.
Medicinal Chemistry Communication 03/2014; 5(3):338-341. · 2.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Notch pathway plays a crucial role in cell fate decisions through controlling various cellular processes. Overactive Notch signal contributes to cancer development from leukemias to solid tumor. γ-Secretase is an intramembrane protease responsible for the final proteolytic step of Notch that releases the membrane-tethered Notch fragment for signaling. Therefore, γ-Secretase is an attractive drug target in treating Notch-mediated cancers. However, the absence of high-throughput γ-Secretase assay using Notch substrate has limited the identification and development of γ-Secretase inhibitors that specifically target the Notch signaling pathway. Here, we report on the development of a 1536-well γ-Secretase assay using a biotinylated recombinant Notch1 substrate. We effectively assimilated and miniaturized this newly developed Notch1 substrate with the AlphaLISA detection technology and demonstrated its robustness with a calculated Z score of 0.66. We further validated this optimized assay by performing a pilot screening against a chemical library consisting of ~5,600 chemicals and identified known γ-secretase inhibitors e.g. DAPT, and Calpeptin; as well as a novel γ-secretase inhibitor referred to as KD-I-085. This assay is the first reported 1536-well AlphaLISA format and represents a novel high-throughput Notch1-γ-secretase assay, which provides an unprecedented opportunity to discover Notch-selective γ-secretase inhibitors that can be potentially used for the treatment of cancer and other human disorders.
[Show abstract][Hide abstract] ABSTRACT: γ-Secretase is an intra-membrane aspartyl protease that cleaves the amyloid precursor protein (APP) to produce neurotoxic β-amyloid peptides (i.e., Aβ42) that have been implicated in the pathogenesis of Alzheimers disease (AD). Small molecule γ-secretase modulators (GSMs) have emerged as potential disease modifying treatments for AD because they reduce the formation of Aβ42 while not blocking the processing of γ-secretase substrates. We developed clickable GSM photoaffinity probes with the goal of identifying the target of various classes of GSMs and to better understand their mechanism of action. Here, we demonstrate that the photoaffinity probe E2012-BPyne specifically labels the N-terminal fragment of presenilin-1 (PS1-NTF) in cell membranes as well as in live cells and primary neuronal cultures. The labeling is competed in the presence of the parent imidazole GSM E2012, but not with acid GSM-1, allosteric GSI BMS-708163 or substrate docking site peptide inhibitor pep11 providing evidence that these compounds have distinct binding sites. Surprisingly, we found that the cross-linking of E2012-BPyne to PS1-NTF is significantly enhanced in the presence of the active-site directed GSI L458. In contrast, L458 does not affect the labeling of the acid GSM photoprobe GSM-5. We also observed that E2012-BPyne specifically labels PS1-NTF (active γ-secretase) but not full-length PS1 (inactive γ-secretase) in ANP.24 cells. Taken together our results support the hypothesis that multiple binding sites within the γ-secretase complex exist, each of which may contribute to different modes of modulatory action.
Journal of Biological Chemistry 02/2013; · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The "Notch-sparing" γ-secretase inhibitor (GSI) BMS-708,163 (Avagacestat) is currently in phase II clinical trials for Alzheimer's disease. Unlike previously failed GSIs, BMS-708,163 is considered to be a promising drug candidate because of its reported Notch-sparing activity for the inhibition of Aβ production over Notch cleavage. We now report that BMS-708,163 binds directly to the presenilin-1 N-terminal fragment and that binding can be challenged by other pan-GSIs, but not by γ-secretase modulators. Furthermore, BMS-708,163 blocks the binding of four different active site-directed GSI photoaffinity probes. We therefore report that this compound acts as a nonselective γ-secretase inhibitor.
[Show abstract][Hide abstract] ABSTRACT: The γ-secretase complex, composed of presenilin, anterior-pharynx-defective 1, nicastrin, and presenilin enhancer 2, catalyzes the intramembranous processing of a wide variety of type I membrane proteins, including amyloid precursor protein (APP) and Notch. Earlier studies have revealed that nicastrin, a type I membrane-anchored glycoprotein, plays a role in γ-secretase assembly and trafficking and has been proposed to bind substrates. To gain more insights regarding nicastrin structure and function, we generated a conformation-specific synthetic antibody and used it as a molecular probe to map functional domains within nicastrin ectodomain. The antibody bound to a conformational epitope within a nicastrin segment encompassing residues 245-630 and inhibited the processing of APP and Notch substrates in in vitro γ-secretase activity assays, suggesting that a functional domain pertinent to γ-secretase activity resides within this region. Epitope mapping and database searches revealed the presence of a structured segment, located downstream of the previously identified DAP domain (DYIGS and peptidase; residues 261-502), that is homologous to a tetratricopeptide repeat (TPR) domain commonly involved in peptide recognition. Mutagenesis analyses within the predicted TPR-like domain showed that disruption of the signature helical structure resulted in the loss of γ-secretase activity but not the assembly of the γ-secretase and that Leu571 within the TPR-like domain plays an important role in mediating substrate binding. Taken together, these studies offer provocative insights pertaining to the structural basis for nicastrin function as a "substrate receptor" within the γ-secretase complex.
Proceedings of the National Academy of Sciences 05/2012; 109(22):8534-9. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Presenilin-1 (PS1) is the catalytic subunit of γ-secretase, and mutations in this protein cause familial Alzheimer Disease (FAD). However, little is known about how these mutations affect the active site of γ-secretase. Here, we show that PS1 mutations alter the S2 subsite within the active site of γ-secretase using a multiple photoaffinity probe approach called "photophore walking." Moreover, we developed a unique in vitro assay with a biotinylated recombinant Notch1 substrate and demonstrated that PS1 FAD mutations directly and significantly reduced γ-secretase activity for Notch1 cleavage. Substitution of the Notch Cys-1752 residue, which interacts with the S2 subsite, with Val, Met, or Ile has little effect on wild-type PS1 but leads to more efficient substrates for mutant PS1s. This study indicates that alteration of this S2 subsite plays an important role in determining the activity and specificity of γ-secretase for APP and Notch1 processing, which provides structural basis and insights on how certain PS1 FAD mutations lead to AD pathogenesis.
Journal of Biological Chemistry 03/2012; 287(21):17288-96. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have developed clickable active site-directed photoaffinity probes for γ-secretase which incorporate a photoreactive benzophenone group and an alkyne handle for subsequent click chemistry mediated conjugation with azide-linked reporter tags for visualization (e.g., TAMRA-azide) or enrichment (e.g., biotin-azide) of labeled proteins. Specifically, we synthesized clickable analogs of L646 (2) and L505 (3) and validated specific labeling to presenilin-1N-terminal fragment (PS1-NTF), the active site aspartyl protease component within the γ-secretase complex. Additionally, we were able to identify signal peptide peptidase (SPP) by Western blot analysis. Furthermore, we analyzed the photo-labeled proteins in an unbiased fashion by click chemistry with TAMRA-azide followed by in-gel fluorescence detection. This approach expands the utility of γ-secretase inhibitor (GSI) photoaffinity probes in that labeled proteins can be tagged with any number of azide-linked reporters groups using a single clickable photoaffinity probe for target pull down and/or fluorescent imaging applications.
[Show abstract][Hide abstract] ABSTRACT: Aβ42 is believed to play a causative role in Alzheimer's disease (AD) pathogenesis. γ-Secretase modulators (GSMs) are actively being pursued as potential AD therapeutics because they selectively alter the cleavage site of the amyloid precursor protein (APP) to reduce the formation of Aβ42. However, the binding partner of acid based GSMs was unresolved until now. We have developed clickable photoaffinity probes based on piperidine acetic acid GSM-1 and identified PS1 as the target within the γ-secretase complex. Furthermore, we provide evidence that allosteric interaction of GSMs with PS1 results in a conformational change in the active site of the γ-secretase complex leading to the observed modulation of γ-secretase activity.
ACS Chemical Neuroscience 12/2011; 2(12):705-710. · 3.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The γ-secretase membrane protein complex is responsible for proteolytic maturation of signaling precursors and catalyzes the final step in the production of the amyloid β-peptides implicated in the pathogenesis of Alzheimer disease. The incorporation of PEN-2 (presenilin enhancer 2) into a pre-activation intermediate, composed of the catalytic subunit presenilin and the accessory proteins APH-1 (anterior pharynx-defective 1) and nicastrin, triggers the endoproteolysis of presenilin and results in an active tetrameric γ-secretase. We have determined the three-dimensional reconstruction of a mature and catalytically active γ-secretase using single-particle cryo-electron microscopy. γ-Secretase has a cup-like shape with a lateral belt of ∼40-50 Å in height that encloses a water-accessible internal chamber. Active site labeling with a gold-coupled transition state analog inhibitor suggested that the γ-secretase active site faces this chamber. Comparison with the structure of a trimeric pre-activation intermediate suggested that the incorporation of PEN-2 might contribute to the maturation of the active site architecture.
Journal of Biological Chemistry 03/2011; 286(24):21440-9. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Increase in the generation and deposition of amyloid-β (Aβ) plays a central role in the development of Alzheimer's Disease (AD). Elevation of the activity of γ-secretase, a key enzyme required for the generation for Aβ, can thus be a potential risk factor in AD. However, it is not known whether γ-secretase can be upregulated in vivo. While in vitro studies showed that expression of all four components of γ-secretase (Nicastrin, Presenilin, Pen-2 and Aph-1) are required for upregulation of γ-secretase, it remains to be established as to whether this is true in vivo. To investigate whether overexpressing a single component of the γ-secretase complex is sufficient to elevate its level and activity in the brain, we analyzed transgenic mice expressing either wild type or familial AD (fAD) associated mutant PS1. In contrast to cell culture studies, overexpression of either wild type or mutant PS1 is sufficient to increase levels of Nicastrin and Pen-2, and elevate the level of active γ-secretase complex, enzymatic activity of γ-secretase and the deposition of Aβ in brains of mice. Importantly, γ-secretase comprised of mutant PS1 is less active than that of wild type PS1-containing γ-secretase; however, γ-secretase comprised of mutant PS1 cleaves at the Aβ42 site of APP-CTFs more efficiently than at the Aβ40 site, resulting in greater accumulation of Aβ deposits in the brain. Our data suggest that whereas fAD-linked PS1 mutants cause early onset disease, upregulation of PS1/γ-secretase activity may be a risk factor for late onset sporadic AD.
PLoS ONE 01/2011; 6(11):e28179. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Alzheimer's disease (AD) is characterized pathologically by the abundance of senile plaques and neurofibrillary tangles in the brain. We synthesized over 1200 novel gamma-secretase modulator (GSM) compounds that reduced Abeta(42) levels without inhibiting epsilon-site cleavage of APP and Notch, the generation of the APP and Notch intracellular domains, respectively. These compounds also reduced Abeta(40) levels while concomitantly elevating levels of Abeta(38) and Abeta(37). Immobilization of a potent GSM onto an agarose matrix quantitatively recovered Pen-2 and to a lesser degree PS-1 NTFs from cellular extracts. Moreover, oral administration (once daily) of another potent GSM to Tg 2576 transgenic AD mice displayed dose-responsive lowering of plasma and brain Abeta(42); chronic daily administration led to significant reductions in both diffuse and neuritic plaques. These effects were observed in the absence of Notch-related changes (e.g., intestinal proliferation of goblet cells), which are commonly associated with repeated exposure to functional gamma-secretase inhibitors (GSIs).
[Show abstract][Hide abstract] ABSTRACT: Gamma-secretase is an aspartyl protease that cleaves multiple substrates within their transmembrane domains. Gamma-secretase processes the amyloid precursor protein (APP) to generate gamma-amyloid (Agamma) peptides associated with Alzheimer's disease. Here, we show that APP possesses a substrate inhibitory domain (ASID) that negatively modulates gamma-secretase activity for Agamma production by binding to an allosteric site within the gamma-secretase complex. Alteration of this ASID by deletion or mutation, as is seen with the Flemish mutation (A21G), reduces its inhibitory potency and promotes Agamma production. Notably, peptides derived from ASID show selective inhibition of gamma-secretase activity for Agamma production over Notch1 processing. Therefore, this mode of regulation represents an unprecedented mechanism for modulating gamma-secretase, providing insight into the molecular basis of Alzheimer's disease pathogenesis and a potential strategy for the development of therapeutics.
[Show abstract][Hide abstract] ABSTRACT: Gamma-secretase is a widely expressed multisubunit enzyme complex which is involved in the pathogenesis of Alzheimer disease and hematopoietic malignancies through its aberrant processing of the amyloid precursor protein (APP) and Notch1, respectively. While gamma-secretase has been extensively studied, there is a dearth of information surrounding the activity, composition, and function of gamma-secretase expressed in distinct cellular populations. Here we show that endogenous gamma-secretase complexes of hematopoietic origin are distinct from epithelial derived gamma-secretase complexes. Hematopoietic gamma-secretase has reduced activity for APP and Notch1 processing compared to epithelial gamma-secretase. Characterization of the active complexes with small molecule affinity probes reveals that hematopoietic gamma-secretase has an atypical subunit composition with significantly altered subunit stoichiometry. Furthermore, we demonstrate that these discrete complexes exhibit cell-line specific substrate selectivity suggesting a possible mechanism of substrate regulation. These data underscore the need for studying endogenous gamma-secretase to fully understand of the biology of gamma-secretase and its complexity as a molecular target for the development of disease therapeutics.
[Show abstract][Hide abstract] ABSTRACT: Cancer stem cells (CSCs) are thought to be critical for the engraftment and long-term growth of many tumors, including glioblastoma (GBM). The cells are at least partially spared by traditional chemotherapies and radiation therapies, and finding new treatments that can target CSCs may be critical for improving patient survival. It has been shown that the NOTCH signaling pathway regulates normal stem cells in the brain, and that GBMs contain stem-like cells with higher NOTCH activity. We therefore used low-passage and established GBM-derived neurosphere cultures to examine the overall requirement for NOTCH activity, and also examined the effects on tumor cells expressing stem cell markers. NOTCH blockade by gamma-secretase inhibitors (GSIs) reduced neurosphere growth and clonogenicity in vitro, whereas expression of an active form of NOTCH2 increased tumor growth. The putative CSC markers CD133, NESTIN, BMI1, and OLIG2 were reduced following NOTCH blockade. When equal numbers of viable cells pretreated with either vehicle (dimethyl sulfoxide) or GSI were injected subcutaneously into nude mice, the former always formed tumors, whereas the latter did not. In vivo delivery of GSI by implantation of drug-impregnated polymer beads also effectively blocked tumor growth, and significantly prolonged survival, albeit in a relatively small cohort of animals. We found that NOTCH pathway inhibition appears to deplete stem-like cancer cells through reduced proliferation and increased apoptosis associated with decreased AKT and STAT3 phosphorylation. In summary, we demonstrate that NOTCH pathway blockade depletes stem-like cells in GBMs, suggesting that GSIs may be useful as chemotherapeutic reagents to target CSCs in malignant gliomas.
[Show abstract][Hide abstract] ABSTRACT: gamma-Secretase cleaves multiple substrates within the transmembrane domain that include the amyloid precursor protein as well as the Notch family of receptors. These substrates are associated with Alzheimer disease and cancer. Despite extensive investigation of this protease, little is known regarding the regulation of gamma-secretase specificity. To discover selective inhibitors for drug development and for probing the mechanisms of gamma-secretase specificity, we screened chemical libraries and consequently developed a di-coumarin family of inhibitors that preferentially inhibit gamma-secretase-mediated production of Abeta42 over other cleavage activities. These coumarin dimer-based compounds interact with gamma-secretase by binding to an allosteric site. By developing a multiple photo-affinity probe approach, we demonstrate that this allosteric binding causes a conformational change within the active site of gamma-secretase at the S2 and S1 sub-sites that leads to selective inhibition of Abeta42. In conclusion, by using these di-coumarin compounds, we reveal a mechanism by which gamma-secretase specificity is regulated and provide insights into the molecular basis by which familial presenilin mutations may affect the active site and specificity of gamma-secretase. Furthermore, this class of selective inhibitors provides the basis for development of Alzheimer disease therapeutic agents.
Proceedings of the National Academy of Sciences 11/2009; 106(48):20228-33. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: RHBDL2, a human homolog of the rhomboids, belongs to a unique class of serine intramembrane proteases; little is known about its function and regulation. Here, we show that RHBDL2 is produced as a proenzyme and that the processing of RHBDL2 is required for its cellular protease activity. The processing of RHBDL2 was shown by both Western blot and immunofluorescence analysis. We have demonstrated that a highly conserved Arg residue on loop 1 of RHBDL2 plays a critical role in the activation of the proenzyme. The activation of RHBDL2 is catalyzed by a protease that is sensitive to a class of sulfonamide compounds. Furthermore, endogenous RHBDL2 exists as the processed form and treatment of cells with a sulfonamide inhibitor led to an accumulation of the full length of RHBDL2. Therefore, this study has demonstrated that RHBDL2 activity is regulated by proenzyme activation, revealed a role for the conserved WR residues in loop 1 in RHBDL2 activity, and provided critical insights into the regulation and function of this human rhomboid protease.
Journal of Molecular Biology 10/2009; 394(5):815-25. · 3.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: gamma-Secretase is an aspartyl protease that cleaves multiple substrates including the amyloid precursor protein (APP) and the Notch proteins. Abnormal proteolysis of APP is involved in the pathogenesis of Alzheimer's disease (AD) and overactive Notch signaling plays an oncogenic role in a variety of cancers. gamma-Secretase has emerged as a promising target for drug development in the treatment of AD and cancer. Assays with increased capacity for high-throughput screening would allow for quicker screening of chemical libraries and facilitate inhibitor development. We have developed a homogeneous time-resolved fluorescence (HTRF)-based assay that makes use of a novel biotinylated recombinant APP substrate and solubilized membrane preparation as the source of the gamma-secretase enzyme. The assay was miniaturized to a 1536-well format and validated in a pilot screen against a library of approximately 3,000 compounds. The overall assay performance was robust due to a calculated Z' factor of 0.74 and its demonstrated ability to identify known gamma-secretase inhibitors such as pepstatin A. This validated assay can readily be used for primary screening against large chemical libraries searching for novel inhibitors of gamma-secretase activity that may represent potential therapeutics for AD and a variety of neoplasms.
Assay and Drug Development Technologies 09/2009; 7(5):461-70. · 1.90 Impact Factor