Membrane-embedded protease poses for photoshoot.
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Catalytic Domain↗
Biomedical subjects
Michael S Wolfe
Publications and source records attributed to Michael S Wolfe.
Signal peptide peptidase: biochemical properties and modulation by nonsteroidal antiinflammatory drugs.
Signal peptide peptidase (SPP) is an intramembrane aspartyl protease that cleaves remnant signal peptides after their release by signal peptidase. SPP contains active site motifs also found in presenilin, the catalytic component of the gamma-secretase complex of Alzheimer's disease. However, SPP has a membrane topology opposite that of presenilin, cleaves transmembrane substrates of opposite directionality, and does not require complexation with other proteins. Here we show that, upon isolation of membranes and solubilization with detergent, the biochemical characteristics of SPP are remarkably similar to gamma-secretase. The majority of the SPP-catalyzed cleavages occurred at a single site in a synthetic substrate based on the prolactin (Prl) signal sequence. However, as seen with cleavage of substrates by gamma-secretase, additional cuts at other minor sites are also observed. Like gamma-secretase, SPP is inhibited by helical peptidomimetics and apparently contains a substrate-binding site that is distinct from the active site. Surprisingly, certain nonsteroidal antiinflammatory drugs known to shift the site of proteolysis by gamma-secretase also alter the cleavage site of Prl by SPP. Together, these findings suggest that SPP and presenilin share certain biochemical properties, including a conserved drug-binding site for allosteric modulation of substrate proteolysis.
Amino Acid Sequence↗
c-Myc is an important direct target of Notch1 in T-cell acute lymphoblastic leukemia/lymphoma.
Human acute T-cell lymphoblastic leukemias and lymphomas (T-ALL) are commonly associated with gain-of-function mutations in Notch1 that contribute to T-ALL induction and maintenance. Starting from an expression-profiling screen, we identified c-myc as a direct target of Notch1 in Notch-dependent T-ALL cell lines, in which Notch accounts for the majority of c-myc expression. In functional assays, inhibitors of c-myc interfere with the progrowth effects of activated Notch1, and enforced expression of c-myc rescues multiple Notch1-dependent T-ALL cell lines from Notch withdrawal. The existence of a Notch1-c-myc signaling axis was bolstered further by experiments using c-myc-dependent murine T-ALL cells, which are rescued from withdrawal of c-myc by retroviral transduction of activated Notch1. This Notch1-mediated rescue is associated with the up-regulation of endogenous murine c-myc and its downstream transcriptional targets, and the acquisition of sensitivity to Notch pathway inhibitors. Additionally, we show that primary murine thymocytes at the DN3 stage of development depend on ligand-induced Notch signaling to maintain c-myc expression. Together, these data implicate c-myc as a developmentally regulated direct downstream target of Notch1 that contributes to the growth of T-ALL cells.
Animals↗
The gamma-secretase complex: membrane-embedded proteolytic ensemble.
Gamma-secretase is responsible for the proteolytic processing of a variety of membrane-associated fragments derived from type I integral membrane proteins, including the amyloid beta-protein precursor and the Notch receptor. This enzyme is composed of four different integral membrane proteins: presenilin, nicastrin, Aph-1, and Pen-2. During assembly and maturation of the protease complex, presenilin is endoproteolyzed into two subunits, each of which contributes one aspartate to the active site of an aspartyl protease. Substrate apparently interacts with an initial docking site before passing in whole or in part between the two presenilin subunits to the internal water-containing active site. The ectodomain of nicastrin also interacts with the N-terminus of the substrate as an essential step in substrate recognition and processing. Sites for allosteric regulation on the protease complex allow selective inhibition or modulation of APP processing without interfering with Notch signaling, and such selective agents may represent promising leads for the development of Alzheimer's disease therapeutics. Elucidation of detailed structural features of gamma-secretase and other membrane-embedded proteases is the next frontier in understanding how these enzymes carry out hydrolysis within the lipid bilayer.
Amyloid Precursor Protein Secretases↗
Deducing the transmembrane domain organization of presenilin-1 in gamma-secretase by cysteine disulfide cross-linking.
Gamma-secretase is a founding member of membrane-embedded aspartyl proteases that cleave substrates within transmembrane domains, and this enzyme is an important target for the development of therapeutics for Alzheimer's disease. The structure of gamma-secretase and its precise catalytic mechanism still remain largely unknown. Gamma-secretase is a complex of four integral membrane proteins, with presenilin (PS) as the catalytic component. To gain structural and functional information about the nine-transmembrane domain (TMD) presenilin, we employed a cysteine mutagenesis/disulfide cross-linking approach. Here we report that native Cys92 is close to both Cys410 and Cys419, strongly implying that TMD1 and TMD8 are adjacent to each other. This structural arrangement also suggests that TMD8 is distorted from an ideal helix. Importantly, binding of an active site directed inhibitor, but not a docking site directed inhibitor, reduces the ability of the native cysteine pairs of PS1 to cross-link upon oxidation. These findings suggest that the conserved cysteines of TMD1 and TMD8 contribute to or allosterically interact with the active site of gamma-secretase.
Amino Acid Sequence↗
Stabilization of the tau exon 10 stem loop alters pre-mRNA splicing.
Neurofibrillary tangles containing filaments of the microtubule-associated protein tau are found in a variety of neurodegenerative diseases. Mutations in the tau gene itself cause frontotemporal dementia with parkinsonism, demonstrating the critical role of tau in pathogenesis. Many of these mutations in tau are silent, are found at the 5'-splice site of exon 10, and lead to increased inclusion of exon 10. These silent mutations are predicted to destabilize a stem loop structure at the exon 10 5'-splice site; however, the existence of this stem loop under physiological conditions and its role in splice regulation are controversial. Here we show that base changes that stabilize this stem loop in vitro substantially decrease exon 10 inclusion in a wild type tau minigene and rescue the increase in exon 10 splicing caused by a dementia-causing point mutation. Moreover, we probed the intracellular structure of the tau stem loop with antisense RNA and demonstrate that the stability of the stem loop dictates antisense effectiveness. Together these results validate the stem loop as a bona fide structure regulating tau exon 10 splicing.
Exons↗
Electron microscopic structure of purified, active gamma-secretase reveals an aqueous intramembrane chamber and two pores.
Gamma-secretase is an intramembrane-cleaving aspartyl protease required for the normal development of metazoans because it processes Notch within cellular membranes to release its signaling domain. More than two dozen additional substrates of diverse functions have been reported, including the Notch ligands Delta and Jagged, N- and E-cadherins, and a sodium channel subunit. The protease is causally implicated in Alzheimer's disease because it releases the neurotoxic amyloid beta-peptide (Abeta) from its precursor, APP. Gamma-secretase occurs as a large complex containing presenilin (bearing the active site aspartates), nicastrin, Aph-1, and Pen-2. Because the complex contains at least 18 transmembrane domains, crystallographic approaches to its structure are difficult and remote. We recently purified the human complex essentially to homogeneity from stably expressing mammalian cells. Here, we use EM and single-particle image analysis on the purified enzyme, which produces physiological ratios of Abeta40 and Abeta42, to obtain structural information on an intramembrane protease. The 3D EM structure revealed a large, cylindrical interior chamber of approximately 20-40 A in length, consistent with a proteinaceous proteolytic site that is occluded from the hydrophobic environment of the lipid bilayer. Lectin tagging of the nicastrin ectodomain enabled proper orientation of the globular, approximately 120-A-long complex within the membrane and revealed approximately 20-A pores at the top and bottom that provide potential exit ports for cleavage products to the extra- and intracellular compartments. Our reconstructed 3D map provides a physical basis for hydrolysis of transmembrane substrates within a lipid bilayer and release of the products into distinct subcellular compartments.
Amyloid Precursor Protein Secretases↗
Alzheimer protease hitches a ride.
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Alzheimer Disease↗
Shutting down Alzheimer's.
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Alzheimer Disease↗
gamma-Secretase substrate selectivity can be modulated directly via interaction with a nucleotide-binding site.
gamma-Secretase is an unusual protease with an intramembrane catalytic site that cleaves many type I membrane proteins, including the amyloid beta-protein (Abeta) precursor (APP) and the Notch receptor. Genetic and biochemical studies have identified four membrane proteins as components of gamma-secretase: heterodimeric presenilin composed of its N- and C-terminal fragments, nicastrin, Aph-1, and Pen-2. Here we demonstrated that certain compounds, including protein kinase inhibitors and their derivatives, act directly on purified gamma-secretase to selectively block cleavage of APP- but not Notch-based substrates. Moreover, ATP activated the generation of the APP intracellular domain and Abeta, but not the generation of the Notch intracellular domain by the purified protease complex, and was a direct competitor of the APP-selective inhibitors, as were other nucleotides. In accord, purified gamma-secretase bound specifically to an ATP-linked resin. Finally, a photoactivable ATP analog specifically labeled presenilin 1-C-terminal fragments in purified gamma-secretase preparations; the labeling was blocked by ATP itself and APP-selective gamma-secretase inhibitors. We concluded that a nucleotide-binding site exists within gamma-secretase, and certain compounds that bind to this site can specifically modulate the generation of Abeta while sparing Notch. Drugs targeting the gamma-secretase nucleotide-binding site represent an attractive strategy for safely treating Alzheimer disease.
Adenosine Triphosphate↗
Growth hormone receptor is a target for presenilin-dependent gamma-secretase cleavage.
Growth hormone receptor (GHR) is a cytokine receptor superfamily member that binds growth hormone (GH) via its extracellular domain and signals via interaction of its cytoplasmic domain with JAK2 and other signaling molecules. GHR is a target for inducible metalloprotease-mediated cleavage in its perimembranous extracellular domain, a process that liberates the extracellular domain as the soluble GH-binding protein and leaves behind a cell-associated GHR remnant protein containing the transmembrane and cytoplasmic domains. GHR metalloproteolysis can be catalyzed by tumor necrosis factor-alpha-converting enzyme (ADAM-17) and is associated with down-modulation of GH signaling. We now study the fate of the GHR remnant protein. By anti-GHR cytoplasmic domain immunoblotting, we observed that the remnant induced in response to phorbol ester or platelet-derived growth factor has a reliable pattern of appearance and disappearance in both mouse preadipocytes endogenously expressing GHR and transfected fibroblasts expressing rabbit GHR. Lactacystin, a specific proteasome inhibitor, did not appreciably change the time course of remnant appearance or clearance but allowed detection of the GHR stub, a receptor fragment slightly smaller than the remnant but containing the C terminus of the remnant (receptor cytoplasmic domain). In contrast, MG132, another (less specific) proteasome inhibitor, strongly inhibited remnant clearance and prevented stub appearance. Inhibitors of gamma-secretase, an aspartyl protease, also prevented the appearance of the stub, even in the presence of lactacystin, and concomitantly inhibited remnant clearance in the same fashion as MG132. In addition, mouse embryonic fibroblasts derived from presenilin 1 and 2 (PS1/2) knockouts recapitulated the gamma-secretase inhibitor studies, as compared with their littermate controls (PS1/2 wild type). Confocal microscopy indicated that the GHR cytoplasmic domain became localized to the nucleus in a fashion dependent on PS1/2 activity. These data indicate that the GHR is subject to sequential proteolysis by metalloprotease and gamma-secretase activities and may suggest GH-independent roles for the GHR.
ADAM Proteins↗
The initial substrate-binding site of gamma-secretase is located on presenilin near the active site.
gamma-Secretase is a structurally enigmatic multiprotein complex that catalyzes intramembrane proteolysis of a variety of substrates, including the amyloid beta-protein precursor of Alzheimer's disease and the Notch receptor essential to cell differentiation. The active site of this transmembrane aspartyl protease apparently lies at the interface between two subunits of presenilin-1 (PS1); however, evidence suggests the existence of an initial substrate-binding site that is distinct from the active site. Here, we report that photoaffinity probes based on potent helical peptide inhibitors and designed to mimic the amyloid beta-protein precursor substrate bind specifically to the PS subunit interface, at a site close to the active site. The location of the helical peptide-binding site suggests that substrate passes between the two PS1 subunits to access the active site. An aggressive Alzheimer-causing mutation in PS1 strongly reduced photolabeling by a transition-state analogue but not by helical peptides, providing biochemical evidence that the pathological effect of this PS mutation is due to alteration of the active-site topography.
Amyloid Precursor Protein Secretases↗
Reconstitution of intramembrane proteolysis in vitro reveals that pure rhomboid is sufficient for catalysis and specificity.
Intramembrane proteolysis is a new paradigm in biology that controls signaling events throughout evolution. Hydrolysis of peptide bonds is thought to occur within the normally hydrophobic membrane environment, but insights into this unusual activity have been lacking because of difficulty in recapitulating activity in vitro. We have reconstituted intramembrane proteolysis with a pure recombinant substrate and rhomboid proteins in both detergent micelles and artificial membrane environments. Rhomboid proteins from diverse organisms including two model bacteria, a pathogen, an extremophile, and an animal were robustly active in pure form, proving that rhomboids are a new class of enzymes and do not require cofactors to catalyze intramembrane proteolysis. Rhomboid proteins directly recognized their substrates in vitro by the top of the substrate transmembrane domain, displaying specificity apparently reciprocal to that of gamma-secretase, the only other activity known to cleave type-I transmembrane domains. Rhomboid proteases represent a different evolutionary path to a serine protease mechanism and exhibited an inhibitor profile unlike other serine proteases. Intriguingly, activity was dramatically modulated by different membrane phospholipid environments, suggesting a mechanism for regulating these proteases. This analysis promises to help reveal the biochemical mechanisms and biological roles of this most widely conserved membrane protein family.
Animals↗
Substituted thiazolamide coupled to a redox delivery system: a new gamma-secretase inhibitor with enhanced pharmacokinetic profile.
Inhibition of gamma-secretase, one of the enzymes responsible for the cleavage of the amyloid precursor protein (APP) to produce pathogenic A beta peptides, is an attractive approach for the treatment of Alzheimer's disease. We have designed a new gamma-secretase thiazolamide inhibitor bearing a dihydronicotinoyl moiety as Redox Delivery System which allows specific delivery of the drug to the brain. Through, on the one hand, A beta peptide production measurements by specific in vitro assays (gamma-secretase Cell Free assay and Cell Based assay on HEK 293 APP transfected cells) and, on the other hand, pharmacokinetic studies on animal models, the new inhibitor shows a good pharmacokinetic profile as well as a potent gamma-secretase inhibitory activity in vitro. From the obtained results, it is expected that drug will be mainly delivered to the CNS with low diffusion in the peripheral tissues. Consequently the side effects of this gamma-secretase inhibitor on the immune cells could be reduced.
Alzheimer Disease↗
Pc 4 photodynamic therapy of U87-derived human glioma in the nude rat.
BACKGROUND AND OBJECTIVES: As a potential therapy for malignant glioma, we tested the phthalocyanine photosensitizer Pc 4 for: (1) rapid clearance from the vasculature, (2) specificity for glioma, and (3) tumoricidal photosensitizing capability. STUDY DESIGN/MATERIALS AND METHODS: Parenchymal injection of U87 cells into athymic rat brains (N = 100) was followed after 12 days by tail vein injection of 0.5 mg/kg Pc 4. After 1 day, the tumor was illuminated with either 5 (N = 11) or 30 (N = 16) J/cm(2) red light at 672 nm. Sacrifice was 1 day later. The brains from these 27 animals underwent H&E (necrosis) and TUNEL assay (apoptosis) histology. Pc 4 concentration of explanted brains and tumors (N = 16), and all blood samples (N = 52) were determined by HPLC-MS 1 day post Pc 4 administration. RESULTS: Tumor-specific apoptosis was almost uniformly seen; however, necrosis was found mostly in the high-light-dose group. Pc 4 concentration in bulk tumor averaged 3.8 times greater than in normal brain. CONCLUSIONS: These results warrant expanding this pre-clinical study to seek effective baseline Pc 4 drug- and light-doses and infusion-to-photoirradiation timing that would be necessary for a Pc 4-mediated PDT clinical trial for glioma patients.
Animals↗
A high-throughput screen to identify inhibitors of amyloid beta-protein precursor processing.
Cerebral accumulation of the amyloid beta-peptide (Abeta) is believed to play a key role in the pathogenesis of Alzheimer's disease (AD). Because Abeta is produced from the proteolysis of amyloid beta-protein precursor (APP) by beta-and gamma-secretases, these enzymes are considered important drug targets for AD. The authors have developed a luciferase-based reporter system that can identify new molecules that inhibit APP processing in a high-throughput manner. Such molecules can help in understanding the biology of APP and APP processing and in developing new drug prototypes for AD. In this system, APP is fused on its C-terminus with Gal4-VP16, a chimeric yeast-viral transcription activator, and luciferase is under control of the yeast Gal4 promoter. Compounds that modulate the luciferase signal may affect the secretases directly, interact with modifiers of these proteases, or interact with APP directly. The authors successfully interfaced this assay with a high-throughput screen, testing approximately 60,000 compounds with diverse chemical structures. In principle, this sensitive, specific, and quantitative assay may be useful for identifying both inhibitors and stimulators of APP processing.
Amyloid Precursor Protein Secretases↗
Stereochemical analysis of (hydroxyethyl)urea peptidomimetic inhibitors of gamma-secretase.
(Hydroxyethyl)urea peptidomimetics systematically altered at positions P2-P3' with hydrophobic D-amino acids were synthesized. An all D-amino acid containing analogue was identified that effectively blocked gamma-secretase activity in a cell-free system (IC50 = 30 nM). Systematic alteration of the stereocenters of a potent compound revealed interdependence between the various positions. Although typically less potent than their L-peptidomimetic counterparts, selected all D-amino acid containing analogues were equipotent to their counterparts in a cell-based assay when incubated for extended times.
Amino Acids↗
Tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma stimulate gamma-secretase-mediated cleavage of amyloid precursor protein through a JNK-dependent MAPK pathway.
The deposition of the amyloid beta (Abeta) peptide in neuritic plaques plays a critical role in the pathogenesis of Alzheimer's disease (AD). Abeta is generated through the proteolysis of amyloid precursor protein (APP) by the sequential actions of beta- and gamma-secretases. Although recent evidence has unveiled much about the biochemical identity and characteristics of gamma-secretase, the mechanism regulating endogenous gamma-secretase activity remains elusive. To identify possible extracellular signals and associated signaling cascades that could regulate APP proteolysis by gamma-secretase activity, we have developed a cell-based reporter gene assay by stably cotransfecting HEK293 cells with the Gal4-driven luciferase reporter gene and the Gal4/VP16-tagged C-terminal fragment of APP (C99-GV), the immediate substrate of gamma-secretase. The cleavage of C99-GV by gamma-secretase releases the transcription factor that activates luciferase expression, providing a quantitative measurement of gamma-secretase activity. Using this reporter assay, we have demonstrated that interferon-gamma, interleukin-1beta, and tumor necrosis factor-alpha can specifically stimulate gamma-secretase activity, concomitant with increased production of Abeta and the intracellular domain of APP (AICD). The gamma-secretase-dependent cleavage of Notch is also enhanced upon the stimulation of these cytokines. The cytokine-enhanced gamma-secretase activity can be suppressed by a potent inhibitor of c-Jun N-terminal kinase (JNK). Furthermore, cells transfected with dominant-positive MEKK1, one of the most potent activators of the JNK cascade, exhibit increased gamma-secretase activity, suggesting that the JNK-dependent mitogen-activated protein kinase pathway could mediate the cytokine-elicited regulation of gamma-secretase. Our studies provide direct evidence that cytokine-elicited signaling cascades control Abeta production by modulating gamma-secretase activity.
Amyloid Precursor Protein Secretases↗