Presenilin/gamma-secretase-mediated cleavage of the voltage-gated sodium channel beta2-subunit regulates cell adhesion and migration

The voltage-gated sodium channel beta2-subunit (beta2) is a member of the IgCAM superfamily and serves as both an adhesion molecule and an auxiliary subunit of the voltage-gated sodium channel. Here we found that beta2 undergoes ectodomain shedding followed by presenilin (PS)-dependent gamma-secretase-mediated cleavage. 12-O-Tetradecanoylphorbol-13-acetate treatment or expression of an alpha-secretase enzyme, ADAM10, resulted in ectodomain cleavage of beta2 in Chinese hamster ovary cells. Subsequent cleavage of the remaining 15-kDa C-terminal fragment (beta2-CTF) was independently inhibited by three specific gamma-secretase inhibitors, expression of the dominant negative form of PS1, and in PS1/PS2 knock-out cells. gamma-Secretase inhibitor treatment also increased endogenous beta2-CTF levels in neuroblastoma cells and mouse primary neuronal cultures. In a cell-free gamma-secretase assay, we detected gamma-secretase activity-dependent generation of a 12 kDa beta2 intracellular domain (ICD), which was loosely associated with the membrane fraction. To assess the functional role of beta2 processing by gamma-secretase, we tested whether N-[N-(3,5-difluorophenylacetyl-l-alanyl)]-S-phenylglycine t-butylester (DAPT), a specific gamma-secretase inhibitor, would alter beta2-mediated cell adhesion and migration. We found that DAPT inhibited cell-cell aggregation and migration in a wound healing assay carried out with Chinese hamster ovary cells expressing beta2. DAPT also reduced migration of neuroblastoma cells in a modified Boyden chamber assay. Since DAPT treatment resulted in increased beta2-CTF levels, we also tested whether beta2-CTFs or beta2-ICDs would directly affect cell migration by overexpressing recombinant proteins. Interestingly, elevated levels of beta2-CTFs, but not ICDs, also blocked cell migration by 81 to 93%. Together, our findings show for the first time that beta2 is a PS/gamma-secretase substrate and gamma-secretase mediated cleavage of beta2-CTF is required for cell-cell adhesion and migration of beta2-expressing cells.     

TrkA receptor activation by nerve growth factor induces shedding of the p75 neurotrophin receptor followed by endosomal gamma-secretase-mediated release of the p75 intracellular domain

Neurotrophins are trophic factors that regulate important neuronal functions. They bind two unrelated receptors, the Trk family of receptor-tyrosine kinases and the p75 neurotrophin receptor (p75). p75 was recently identified as a new substrate for gamma-secretase-mediated intramembrane proteolysis, generating a p75-derived intracellular domain (p75-ICD) with signaling capabilities. Using PC12 cells as a model, we studied how neurotrophins activate p75 processing and where these events occur in the cell. We demonstrate that activation of the TrkA receptor upon binding of nerve growth factor (NGF) regulates the metalloprotease-mediated shedding of p75 leaving a membrane-bound p75 C-terminal fragment (p75-CTF). Using subcellular fractionation to isolate a highly purified endosomal fraction, we demonstrate that p75-CTF ends up in endosomes where gamma-secretase-mediated p75-CTF cleavage occurs, resulting in the release of a p75-ICD. Moreover, we show similar structural requirements for gamma-secretase processing of p75 and amyloid precursor protein-derived CTFs. Thus, NGF-induced endocytosis regulates both signaling and proteolytic processing of p75.     

Pigment epithelium-derived factor inhibits angiogenesis via regulated intracellular proteolysis of vascular endothelial growth factor receptor 1

Pigment epithelium-derived factor (PEDF) has been identified as one of the most potent of endogenous negative regulators of blood vessel growth in the body. Here we report that PEDF is able to inhibit growth factor-induced angiogenesis in microvascular endothelial cells through a novel pathway requiring cleavage and intracellular translocation of the transmembrane domain of the VEGFR-1. Analysis of the subcellular distribution of VEGFR-1 revealed the appearance of an 80-kDa C-terminal domain in the cytosol of cells treated with VEGF and PEDF that correlated with a decrease of the full-length receptor in the nuclear and cytoskeletal fractions. This regulated intramembrane proteolysis is dependent on gamma-secretase because inhibition of gamma-secretase abolished the inhibitory effect of PEDF on VEGF-induced angiogenesis as well as VEGFR-1 cleavage. The addition of PEDF to microvascular endothelial cells significantly increases gamma-secretase activity even in the absence of VEGF, showing that VEGF binding to VEGF-R1 is essential for substrate availability. This increase in activity was associated with translocation of presenilin 1 from the perinuclear region to the cell membrane. PEDF was also able to inhibit VEGF-induced phosphorylation of VEGFR-1. Taken together we have identified two novel pathways by which PEDF inhibits VEGF-induced angiogenesis: regulated intramembrane proteolysis and inhibition of phosphorylation. This confirms the importance of PEDF and VEGFR-1 in the negative regulation of angiogenesis.     

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.     

Identification of proteases involved in the proteolysis of vascular endothelium cadherin during neutrophil transmigration

Transmigration of neutrophils across the endothelium occurs at the cell-cell junctions where the vascular endothelium cadherin (VE cadherin) is expressed. This adhesive receptor was previously demonstrated to be involved in the maintenance of endothelium integrity. We propose that neutrophil transmigration across the vascular endothelium goes in parallel with cleavage of VE cadherin by elastase and cathepsin G present on the surface of neutrophils. This hypothesis is supported by the following lines of evidence. 1) Proteolytic fragments of VE cadherin are released into the culture medium upon adhesion of neutrophils to endothelial cell monolayers; 2) conditioned culture medium, obtained after neutrophil adhesion to endothelial monolayers, cleaves the recombinantly expressed VE cadherin extracellular domain; 3) these cleavages are inhibited by inhibitors of elastase; 4) VE cadherin fragments produced by conditioned culture medium or by exogenously added elastase are identical as shown by N-terminal sequencing and mass spectrometry analysis; 5) both elastase- and cathepsin G-specific VE cadherin cleavage patterns are produced upon incubation with tumor necrosis factor alpha-stimulated and fixed neutrophils; 6) transendothelial permeability increases in vitro upon addition of either elastase or cathepsin G; and 7) neutrophil transmigration is reduced in vitro in the presence of elastase and cathepsin G inhibitors. Our results suggest that cleavage of VE cadherin by neutrophil surface-bound proteases induces formation of gaps through which neutrophils transmigrate.     

Generation of intracellular domain of insulin receptor tyrosine kinase by gamma-secretase

The proteolytic cleavage of a precursor protein into alpha- and beta-subunits by furin is required to form functional insulin receptor (IR). In this study, we examined if IR undergoes the additional presenilin (PS)/gamma-secretase-dependent processing. In cells treated with gamma-secretase inhibitors or expressing the dominant-negative PS1 variant led to the accumulation of an endogenous IR C-terminal fragment. In the presence of proteasome inhibitors, we detected a PS/gamma-secretase cleavage product of the IR, termed the IR intracellular domain (ICD). Cellular fractionation and confocal microscopy analyses showed that the IR-ICD is predominantly detected in the nucleus. These data indicate that IR is a tyrosine kinase receptor, which undergoes PS/gamma-secretase-dependent processing. We also show that the autophosphorylation levels of the IR beta-subunit upon insulin stimulation were decreased by the inactivation of PS/gamma-secretase, raising the possibility that the PS/gamma-secretase proteolysis of IR may play a modulatory role in insulin signaling.     

APP intracellular domain formation and unaltered signaling in the presence of familial Alzheimer's disease mutations

One of the cardinal neuropathological findings in brains from Alzheimer's disease (AD) patients is the occurrence of amyloid beta-peptide (Abeta) deposits. The gamma-secretase-mediated intramembrane proteolysis event generating Abeta also results in the release of the APP intracellular domain (AICD), which may mediate nuclear signaling. It was recently shown that AICD starts at a position distal to the site predicted from gamma-secretase cleavage within the membrane. This novel site, the epsilon site, is located close to the inner leaflet of the membrane bilayer. The relationship between proteolysis at the gamma and epsilon sites has not been fully characterized. Here we studied AICD signaling in intact cells using a chimeric C99 molecule and a luciferase reporter system. We show that the release of AICD from the membrane takes place in a compartment downstream of the endoplasmic reticulum, is dependent on presenilin proteins, and can be inhibited by treatment with established gamma-secretase inhibitors. Moreover, we find that AICD signaling remains unaltered from C99 derivatives containing mutations associated with increased Abeta42 production and familial AD. These findings indicate that there are very similar routes for Abeta and AICD formation but that FAD-linked mutations in APP primarily affect gamma-secretase-mediated Abeta42 formation, and not AICD signaling.     

Functional implications of the presenilin dimerization: reconstitution of gamma-secretase activity by assembly of a catalytic site at the dimer interface of two catalytically inactive presenilins

Presenilins are the catalytic components of gamma-secretase, an intramembrane-cleaving protease whose substrates include beta-amyloid precursor protein (betaAPP) and the Notch receptors. These type I transmembrane proteins undergo two distinct presenilin-dependent cleavages within the transmembrane region, which result in the production of Abeta and APP intracellular domain (from betaAPP) and the Notch intracellular domain signaling peptide. Most cases of familial Alzheimer's disease are caused by presenilin mutations, which are scattered throughout the coding sequence. Although the underlying molecular mechanism is not yet known, the familial Alzheimer's disease mutations produce a shift in the ratio of the long and short forms of the Abeta peptide generated by the gamma-secretase. We and others have previously shown that presenilin homodimerizes and suggested that a presenilin dimer is at the catalytic core of gamma-secretase. Here, we demonstrate that presenilin transmembrane domains contribute to the formation of the dimer. In-frame substitution of the hydrophilic loop 1, located between transmembranes I and II, which modulates the interactions within the N-terminal fragment/N-terminal fragment dimer, abolishes both presenilinase and gamma-secretase activities. In addition, by reconstituting gamma-secretase activity from two catalytically inactive presenilin aspartic mutants, we provide evidence of an active diaspartyl group assembled at the interface between two presenilin monomers. Under our conditions, this catalytic group mediates the generation of APP intracellular domain and Abeta but not Notch intracellular domain, therefore suggesting that specific diaspartyl groups within the presenilin catalytic core of gamma-secretase mediate the cleavage of different substrates.     

Effect of gamma-secretase inhibitors on muscarinic receptor-mediated calcium signaling in human salivary epithelial cells

Altered intracellular Ca(2+) signaling has been observed in cells derived from Alzheimer's disease patients, and a possible link between gamma-secretase activity and the content of intracellular Ca(2+) stores has been suggested. To test this hypothesis we studied the effects of several gamma-secretase inhibitors on muscarinic receptor-mediated intracellular calcium release in the human salivary gland cell line HSG. Although several inhibitors in the peptide aldehyde class partially blocked carbachol-induced Ca(2+) transients, these effects did not appear to be due to gamma-secretase inhibition, and overall we found no evidence that inhibition of gamma-secretase activity had any significant effect on agonist-induced intracellular calcium release in HSG cells. In complementary experiments with presenilin-null cells we found that the reconstitution of gamma-secretase activity by transfection with wild-type presenilin 1 likewise had no significant effect on thapsigargin-induced Ca(2+) release. In a test of the specific hypothesis that the level of APP intracellular domain (AICD), the intracellular fragment of the beta-amyloid precursor protein (APP) resulting from gamma-secretase cleavage, can modulate the Ca(2+) content of the endoplasmic reticulum, we were unable to demonstrate any effect of APP small interfering RNA on the magnitude of carbachol-induced intracellular calcium release in HSG cells. Together our data cast considerable doubt on the hypothesis that there is a direct link between gamma-secretase activity and the content of intracellular Ca(2+) stores.     

Proteolytic regulation of the urokinase receptor/CD87 on monocytic cells by neutrophil elastase and cathepsin G

The urokinase receptor (CD87) participates to the pericellular proteolytic potential of migrating cells and to the recruitment of leukocytes during inflammation. It consists of three structurally homologous domains, with the C-terminal domain D3 attached to cell membranes through a GPI anchor. CD87 is susceptible to an endoproteolytic processing removing the N-terminal domain D1 and generating truncated D2D3 membrane species, thus modulating CD87-associated functions. Full-length or truncated CD87 can be also released from cells via juxtamembrane cleavage by phospholipases and/or by yet unidentified proteinases. Using a recombinant CD87 and the CD87-positive monocytic U937 cell line and isolated blood monocytes, we show by protein immunoblotting and flow immunocytometry that the human neutrophil serine-proteinases elastase and cathepsin G cleave CD87 within the D1-D2 linker sequence, while in addition cathepsin G is highly efficient in cleaving the C terminus of D3. The combination of cathepsin G and elastase provided by degranulated neutrophils results in enzymatic cooperation leading to the release from monocytic cells of a truncated D2D3 species resembling that previously described in pathological body fluids. Using mass spectrometry analysis, the proteolytic fragmentation of synthetic peptides mapping the D1-D2 linker and D3 C-terminal domains identifies potential cleavage sites for each enzyme and suggests the existence of a mechanism regulating the CD87(D1-D2)-associated chemotactic activity. Finally, isolated or combined elastase and cathepsin G drastically reduce the capacity of cells to bind urokinase. Secretable leukocyte serine-proteinases are thus endowed with high potential for the regulation of CD87 expression and function on inflammatory cells.     

Proteases produced by activated neutrophils are able to release soluble CD23 fragments endowed with proinflammatory effects.

Polymorphonuclear neutrophils (PMNs) are the major source of proteolytic activities involved mainly in tissue injuries observed in chronic inflammatory disorders. High levels of soluble forms of CD23 (the low-affinity receptor for IgE) were found in biological fluids from these patients, and recent reports focused on a CD23-mediated regulation of inflammatory response. In this context, we show here that co-culture of activated PMN with CD23+ B cells resulted in a drastic release of soluble CD23 fragments from the cell surface. This cleavage was inhibited by serine proteases inhibitors, including a1-antitrypsin. We next demonstrated that purified human leukocyte elastase or cathepsin G efficiently cleaved membrane CD23 on B cells with a high specificity. Soluble fragments released by serine proteases-mediated CD23 proteolysis stimulated resting monocytes to produce oxidative burst and proinflammatory cytokine without any co-stimulatory signal. This work strongly supports the idea that the capacity of PMN-derived proteases to release soluble forms of CD23 participates in the inflammatory process mediated by these cells.     

A ligand-induced extracellular cleavage regulates gamma-secretase-like proteolytic activation of Notch1

Gamma-secretase-like proteolysis at site 3 (S3), within the transmembrane domain, releases the Notch intracellular domain (NICD) and activates CSL-mediated Notch signaling. S3 processing occurs only in response to ligand binding; however, the molecular basis of this regulation is unknown. Here we demonstrate that ligand binding facilitates cleavage at a novel site (S2), within the extracellular juxtamembrane region, which serves to release ectodomain repression of NICD production. Cleavage at S2 generates a transient intermediate peptide termed NEXT (Notch extracellular truncation). NEXT accumulates when NICD production is blocked by point mutations or gamma-secretase inhibitors or by loss of presenilin 1, and inhibition of NEXT eliminates NICD production. Our data demonstrate that S2 cleavage is a ligand-regulated step in the proteolytic cascade leading to Notch activation.     

Presenilin-dependent intramembrane proteolysis of CD44 leads to the liberation of its intracellular domain and the secretion of an Abeta-like peptide

Alzheimer's disease (AD)-associated gamma-secretase is a presenilin (PS)- dependent proteolytic activity involved in the intramembraneous cleavage of the beta-amyloid precursor protein, Notch, LDL receptor-related protein, E-cadherin, and ErbB-4. This cut produces the corresponding intracellular domains (ICD), which are required for nuclear signaling of Notch and probably ErbB-4, the beta-amyloid precursor protein, E-cadherin, and the LDL receptor-related protein as well. We have now investigated CD44, a cell surface adhesion molecule, which also undergoes an intramembraneous cleavage to liberate its ICD. We demonstrate that this cleavage requires a PS-dependent gamma-secretase activity. A loss-of-function PS1 mutation, a PS1/PS2 knockout, as well as two independent and highly specific gamma-secretase inhibitors, abolish this cleavage. Surprisingly, small peptides similar to the amyloid beta-peptide (Abeta) are generated by an additional cut in the middle of the transmembrane region of CD44. Like Abeta, these CD44 beta-peptides are generated in a PS-dependent manner. These findings therefore suggest a dual intramembraneous cleavage mechanism mediated by PS proteins. The dual cleavage mechanism is required for nuclear signaling as well as removal of remaining transmembrane domains, a general function of PS in membrane protein metabolism.     

Functional domains in presenilin 1: the Tyr-288 residue controls gamma-secretase activity and endoproteolysis

Processing of the Alzheimer amyloid precursor protein (APP) into the amyloid beta-protein and the APP intracellular domain is a proteolysis event mediated by the gamma-secretase complex where presenilin (PS) proteins are key constituents. PS is subjected to an endoproteolytic cleavage, generating a stable heterodimer composed of an N-terminal and a C-terminal fragment. Here we aimed at further understanding the role of PS in endoproteolysis, in proteolytic processing of APP and Notch, and in assembly of the gamma-secretase complex. By using a truncation protocol and alanine scanning, we identified Tyr-288 in the PS1 N-terminal fragment as critical for PS-dependent intramembrane proteolysis. Further mutagenesis of the 288 site identified mutants differentially affecting endoproteolysis and gamma-secretase activity. The Y288F mutant was endoproteolyzed to the same extent as wild type PS but increased the amyloid beta-protein 42/40 ratio by approximately 75%. In contrast, the Y288N mutant was also endoproteolytically processed but was inactive in reconstituting gamma-secretase in PS null cells. The Y288D mutant was deficient in both endoproteolysis and gamma-secretase activity. All three mutant PS1 molecules were incorporated into gamma-secretase complexes and stabilized Pen-2 in PS null cells. Thus, mutations at Tyr-288 do not affect gamma-secretase complex assembly but can differentially control endoproteolysis and gamma-secretase activity.     

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.     

Interleukin-1 receptor type 1 is a substrate for gamma-secretase-dependent regulated intramembrane proteolysis

Biochemical and genetic studies have revealed that the presenilins interact with several proteins and are involved in the regulated intramembrane proteolysis of numerous type 1 membrane proteins, thereby linking presenilins to a range of cellular processes. In this study, we report the characterization of a highly conserved tumor necrosis factor receptor-associated factor-6 (TRAF6) consensus-binding site within the hydrophilic loop domain of presenilin-1 (PS-1). In coimmunoprecipitation studies we indicate that presenilin-1 interacts with TRAF6 and interleukin-1 receptor-associated kinase 2. Substitution of presenilin-1 residues Pro-374 and Glu-376 by site-directed mutagenesis greatly reduces the ability of PS1 to associate with TRAF6. By studying these interactions, we also demonstrate that the interleukin-1 receptor type 1 (IL-1R1) undergoes intramembrane proteolytic processing, mediated by presenilin-dependent gamma-secretase activity. A metalloprotease-dependent proteolytic event liberates soluble IL-1R1 ectodomain and produces an approximately 32-kDa C-terminal domain. This IL-1R1 C-terminal domain is a substrate for subsequent gamma-secretase cleavage, which generates an approximately 26-kDa intracellular domain. Specific pharmacological gamma-secretase inhibitors, expression of dominant negative presenilin-1, or presenilin deficiency independently inhibit generation of the IL-1R1 intracellular domain. Attenuation of gamma-secretase activity also impairs responsiveness to IL-1beta-stimulated activation of the MAPKs and cytokine secretion. Thus, TRAF6 and interleukin receptor-associated kinase 2 are novel binding partners for PS1, and IL-1R1 is a new substrate for presenilin-dependent gamma-secretase cleavage. These findings also suggest that regulated intramembrane proteolysis may be a control mechanism for IL-1R1-mediated signaling.