γ-Secretase-regulated signaling pathways, such as notch signaling, mediate the differentiation of hematopoietic stem cells, development of the immune system, and peripheral immune responses

Notch signaling mediates the fates of numerous cells in both invertebrates and vertebrates. In the immune system, Notch signalling contributes to the generation of hematopoietic stem cells (HSCs), the promotion of HSC self-renewal, T lineage commitment, intrathymic T cell development, and peripheral lymphocyte differentiation/activation. The intracellular domain (ICD) of Notch is released from the cell membrane by γ-secretase and translocates to the nucleus to modulate gene expression. Hence, γ-secretase plays a central role in the regulation of Notch signaling. More than five dozen type 1 transmembrane proteins, including amyloid precursor protein, Notch, and Delta, are substrates for γ-secretase and their ICDs are released from the cell membrane. Therefore, it is highly possible that mechanisms similar to Notch signaling may widely contribute to γ-secretase-regulated signaling. Besides Notch, some transmembrane proteins such as CD44 and CSF-1R, which are important for immune responses, have been reported as substrates for γ-secretase. Since the ICDs of these proteins are also released by γ-secretase from the cell membrane and localize to the nucleus, it is thought that these ICDs modulate gene expression. Thus, γ-secretase-regulated signaling, including Notch signaling, may play a wide range of roles in the immune system.     

γ-Secretase-mediated growth hormone receptor proteolysis: mapping of the intramembranous cleavage site

GH receptor (GHR) undergoes regulated proteolysis by both metalloprotease (α-secretase) and γ-secretase activities. α-Secretase activity regulates GHR availability and sensitivity and generates circulating GH binding protein. The function of γ-secretase cleavage is yet uncertain. We investigated GHR determinants that affect inducible sequential α- and γ-secretase cleavage and thus remnant and stub generation, respectively. Purification and N-terminal sequencing of the stub revealed that γ-secretase cleavage occurs at an ε-site in GHR’s transmembrane domain four residues from the intracellular domain. Mutagenesis revealed that deletion of the proximal two transmembrane residues prevented both α- and γ-secretase-mediated proteolysis and deletion of four residues around the ε-site precluded surface GHR expression and proteolysis. However, point mutations in and around the ε-site affected neither α- or γ-secretase cleavage. We conclude that both cleavages likely occur at the cell surface and sequentially (α-secretase followed by γ-secretase) and that ε-site cleavage by γ-secretase does not require a consensus sequence.     

γ-Secretase-dependent cleavage of E-cadherin by staurosporine in breast cancer cells

E-cadherin is a transmembrane protein that serves as a cell adhesion molecule component of the adherens junction. We previously showed that cadmium induced γ-secretase-dependent E-cadherin cleavage via oxidative stress. In this study, we report that staurosporine (STS)-induced apoptosis induces caspase-2 and/or -8-dependent E-cadherin cleavage. STS increased γ-secretase-dependent cleavage of E-cadherin in breast cancer cells through caspase activation. The ability of the γ-secretase inhibitor DAPT and the caspase inhibitor zVAD-FMK to block E-cadherin cleavage provided support for these results. The cleavage of E-cadherin was blocked by caspase-2 and -8 inhibitors. Immunofluorescence analysis confirmed that, along with the disappearance of E-cadherin staining at the cell surface, the E-cadherin cytoplasmic domain accumulated in the cytosol. In the presence of an inhibitor of γ-secretase or caspase, the cleavage of E-cadherin was partially blocked. Our findings suggest that activation of caspase-2/-8 stimulated the disruption of cadherin-mediated cell-cell contacts in apoptotic cells via γ-secretase activation.     

γ-Secretase-Dependent Cleavage of E-Cadherin by Staurosporine in Breast Cancer Cells

E-cadherin is a transmembrane protein that serves as a cell adhesion molecule component of the adherens junction. We previously showed that cadmium induced γ-secretase-dependent E-cadherin cleavage via oxidative stress. In this study, we report that staurosporine (STS)-induced apoptosis induces caspase-2 and/or -8-dependent E-cadherin cleavage. STS increased γ-secretase-dependent cleavage of E-cadherin in breast cancer cells through caspase activation. The ability of the γ-secretase inhibitor DAPT and the caspase inhibitor zVAD-FMK to block E-cadherin cleavage provided support for these results. The cleavage of E-cadherin was blocked by caspase-2 and -8 inhibitors. Immunofluorescence analysis confirmed that, along with the disappearance of E-cadherin staining at the cell surface, the E-cadherin cytoplasmic domain accumulated in the cytosol. In the presence of an inhibitor of γ-secretase or caspase, the cleavage of E-cadherin was partially blocked. Our findings suggest that activation of caspase-2/-8 stimulated the disruption of cadherin-mediated cell–cell contacts in apoptotic cells via γ-secretase activation.     

Presenilin-1 processing of ErbB4 in fetal type II cells is necessary for control of fetal lung maturation

Maturation of pulmonary fetal type II cells to initiate adequate surfactant production is crucial for postnatal respiratory function. Little is known about specific mechanisms of signal transduction controlling type II cell maturation. The ErbB4 receptor and its ligand neuregulin (NRG) are critical for lung development. ErbB4 is cleaved at the cell membrane by the γ-secretase enzyme complex whose active component is either presenilin-1 (PSEN-1) or presenilin-2. ErbB4 cleavage releases the 80 kDa intracellular domain (4ICD), which associates with chaperone proteins such as YAP (Yes-associated protein) and translocates to the nucleus to regulate gene expression. We hypothesized that PSEN-1 and YAP have a development-specific expression in fetal type II cells and are important for ErbB4 signaling in surfactant production. In primary fetal mouse E16, E17, and E18 type II cells, PSEN-1 and YAP expression increased at E17 and E18 over E16. Subcellular fractionation showed a strong cytosolic and a weaker membrane location of both PSEN-1 and YAP. This was enhanced by NRG stimulation. Co-immunoprecipitations showed ErbB4 associated separately with PSEN-1 and with YAP. Their association, phosphorylation, and co-localization were induced by NRG. Confocal immunofluorescence and nuclear fractionation confirmed these associations in a time-dependent manner after NRG stimulation. Primary ErbB4-deleted E17 type II cells were transfected with a mutant ErbB4 lacking the γ-secretase binding site. When compared to transfection with wild-type ErbB4, the stimulatory effect of NRG on surfactant protein mRNA expression was lost. We conclude that PSEN-1 and YAP have crucial roles in ErbB4 signal transduction during type II cell maturation.     

Lysine 624 of the amyloid precursor protein (APP) is a critical determinant of amyloid β peptide length: support for a sequential model of γ-secretase intramembrane proteolysis and regulation by the amyloid β precursor protein (APP) juxtamembrane region

γ-Secretase is a multiprotein intramembrane cleaving aspartyl protease (I-CLiP) that catalyzes the final cleavage of the amyloid β precursor protein (APP) to release the amyloid β peptide (Aβ). Aβ is the primary component of senile plaques in Alzheimer's disease (AD), and its mechanism of production has been studied intensely. γ-Secretase executes multiple cleavages within the transmembrane domain of APP, with cleavages producing Aβ and the APP intracellular domain (AICD), referred to as γ and ε, respectively. The heterogeneous nature of the γ cleavage that produces various Aβ peptides is highly relevant to AD, as increased production of Aβ 1-42 is genetically and biochemically linked to the development of AD. We have identified an amino acid in the juxtamembrane region of APP, lysine 624, on the basis of APP695 numbering (position 28 relative to Aβ) that plays a critical role in determining the final length of Aβ peptides released by γ-secretase. Mutation of this lysine to alanine (K28A) shifts the primary site of γ-secretase cleavage from 1-40 to 1-33 without significant changes to ε cleavage. These results further support a model where ε cleavage occurs first, followed by sequential proteolysis of the remaining transmembrane fragment, but extend these observations by demonstrating that charged residues at the luminal boundary of the APP transmembrane domain limit processivity of γ-secretase.     

γ-Secretase complexes containing caspase-cleaved presenilin-1 increase intracellular Aβ(42) /Aβ(40) ratio

Markers for caspase activation and apoptosis have been shown in brains of Alzheimer's disease (AD) patients and AD-mouse models. In neurons, caspase activation is associated with elevated amyloid β-peptide (Aβ) production. Caspases cleave numerous substrates including presenilin-1 (PS1). The cleavage takes place in the large cytosolic loop of PS1-C-terminal fragment (PS1CTF), generating a truncated PS1CTF lacking half of the loop domain (caspCTF). The loop has been shown to possess important regulatory functions with regard to Aβ(40) and Aβ(42) production. Previously, we have demonstrated that γ-secretase complexes are active during apoptosis regardless of caspase cleavage in the PS1CTF-loop. Here, a PS1/PS2-knockout mouse blastocyst-derived cell line was used to establish stable or transient cell lines expressing either caspCTF or full-length CTF (wtCTF). We show that caspCTF restores γ-secretase activity and forms active γ-secretase complexes together with Nicastrin, Pen-2, Aph-1 and PS1-N-terminal fragment. Further, caspCTF containing γ-secretase complexes have a sustained capacity to cleave amyloid precursor protein (APP) and Notch, generating APP and Notch intracellular domain, respectively. However, when compared to wtCTF cells, caspCTF cells exhibit increased intracellular production of Aβ(42) accompanied by increased intracellular Aβ(42) /Aβ(40) ratio without changing the Aβ secretion pattern. Similarly, induction of apoptosis in wtCTF cells generate a similar shift in intracellular Aβ pattern with increased Aβ(42) /Aβ(40) ratio. In summary, we show that caspase cleavage of PS1 generates a γ-secretase complex that increases the intracellular Aβ(42) /Aβ(40) ratio. This can have implications for AD pathogenesis and suggests caspase inhibitors as potential therapeutic agents.     

Gamma-secretase-regulated proteolysis of the Notch receptor by mitochondrial intermediate peptidase

Notch is a transmembrane receptor that controls a diverse array of cellular processes including cell proliferation, differentiation, survival, and migration. The cellular outcome of Notch signaling is dependent on extracellular and intracellular signals, but the complexities of its regulation are not well understood. Canonical Notch signaling involves ligand association that triggers sequential and regulated proteolysis of Notch at several sites. Ligand-dependent proteolysis at the S2 site removes the bulk of the extracellular domain of Notch. Subsequent γ-secretase-mediated intramembrane proteolysis of the remaining membrane-tethered Notch fragment at the S3 site produces a nuclear-destined Notch intracellular domain (NICD). Here we show that following γ-secretase cleavage, Notch is proteolyzed at a novel S5 site. We have identified this S5 site to be eight amino acids downstream of the S3 site. Biochemical fractionation and purification resulted in the identification of the S5 site protease as the mitochondrial intermediate peptidase (MIPEP). Expression of the MIPEP-cleaved NICD (ΔNICD) results in a decrease in cell viability and mitochondria membrane potential. The sequential and regulated proteolysis by γ-secretase and MIPEP suggests a new means by which Notch function can be modulated.     

The cytosolic domain of protein-tyrosine kinase 7 (PTK7), generated from sequential cleavage by a disintegrin and metalloprotease 17 (ADAM17) and γ-secretase, enhances cell proliferation and migration in colon cancer cells

Protein-tyrosine kinase 7 (PTK7) is a member of the defective receptor protein-tyrosine kinases and is known to function as a regulator of planar cell polarity during development. Its expression is up-regulated in some cancers including colon carcinomas. A 100-kDa fragment of PTK7 was detected in the culture media from colon cancer cells and HEK293 cells. The shed fragment was named sPTK7-Ig1-7 because its molecular mass was very similar to that of the entire extracellular domain of PTK7 that contains immunoglobulin-like loops 1 to 7 (Ig1-7). The shedding of sPTK7-Ig1-7 was enhanced by treatment with phorbol 12-myristate 13-acetate. In addition to the sPTK7-Ig1-7 found in the culture medium, two C-terminal fragments of PTK7 were detected in the cell lysates: PTK7-CTF1, which includes a transmembrane segment and a cytoplasmic domain, and PTK7-CTF2, which lacks most of the transmembrane segment from PTK7-CTF1. Analysis of PTK7 processing in the presence of various protease inhibitors or after knockdown of potential proteases suggests that shedding of PTK7 into sPTK7-Ig1-7 and PTK7-CTF1 is catalyzed by ADAM17, and further cleavage of PTK7-CTF1 into PTK7-CTF2 is mediated by the γ-secretase complex. PTK7-CTF2 localizes to the nucleus and enhances proliferation, migration, and anchorage-independent colony formation. Our findings demonstrate a novel role for PTK7 in the tumorigenesis via generation of PTK7-CTF2 by sequential cleavage of ADAM17 and γ-secretase.     

A synthetic substrate assay for the gamma-secretase of the β-A4 amyloid of alzheimer's disease

γ-secretase, the endoprotease which releases the C-terminus of βA4 amyloid peptide, cleaves within the hydrophobic transmembrane domain of the amyloid precursor protein. In order to obtain a substrate for γ-secretase, a dodecapeptide which spans the cleavage site was synthesized, labelled with 125-iodine and conjugated to an agarose gel. A radiometric solid-phase assay was developed using this immobilized substrate. Peptide products were separated by reverse-phase HPLC and TLC to allow characterization of the cleavage site(s).     

Determination of the proteolytic cleavage sites of the amyloid precursor-like protein 2 by the proteases ADAM10, BACE1 and γ-secretase

Regulated intramembrane proteolysis of the amyloid precursor protein (APP) by the protease activities α-, β- and γ-secretase controls the generation of the neurotoxic amyloid β peptide. APLP2, the amyloid precursor-like protein 2, is a homolog of APP, which shows functional overlap with APP, but lacks an amyloid β domain. Compared to APP, less is known about the proteolytic processing of APLP2, in particular in neurons, and the cleavage sites have not yet been determined. APLP2 is cleaved by the β-secretase BACE1 and additionally by an α-secretase activity. The two metalloproteases ADAM10 and ADAM17 have been suggested as candidate APLP2 α-secretases in cell lines. Here, we used RNA interference and found that ADAM10, but not ADAM17, is required for the constitutive α-secretase cleavage of APLP2 in HEK293 and SH-SY5Y cells. Likewise, in primary murine neurons knock-down of ADAM10 suppressed APLP2 α-secretase cleavage. Using mass spectrometry we determined the proteolytic cleavage sites in the APLP2 sequence. ADAM10 was found to cleave APLP2 after arginine 670, whereas BACE1 cleaves after leucine 659. Both cleavage sites are located in close proximity to the membrane. γ-secretase cleavage was found to occur at different peptide bonds between alanine 694 and valine 700, which is close to the N-terminus of the predicted APLP2 transmembrane domain. Determination of the APLP2 cleavage sites enables functional studies of the different APLP2 ectodomain fragments and the production of cleavage-site specific antibodies for APLP2, which may be used for biomarker development.     

{gamma}-Protocadherins, presenilin-mediated release of C-terminal fragment promotes locus expression

gamma-Protocadherins (gamma-pcdhs) are type I membrane-spanning glycoproteins, widely expressed in the mammal and required for survival. These cell adhesion molecules are expressed from a complex locus comprising 22 functional variable exons arranged in tandem, each encoding extracellular, transmembrane and intracellular sequence, and three exons for an invariant C-terminal domain (gamma-ICD). However, the signaling mechanisms that lie downstream of gamma-pcdhs have not been elucidated. Here we report that gamma-pcdhs are subject to presenilin-dependent intramembrane cleavage (PS-IP), accompanied by shedding of the extracellular domain. The cleaved intracellular domain (gamma-ICD) translocates to the cell nucleus and was detected in subsets of cortical neurons. Notably, gene-targeted mice lacking functional gamma-ICD sequence showed severely reduced gamma-pcdh mRNA levels and neonatal lethality. Most importantly, inhibition of gamma-secretase decreased gamma-pcdh locus expression. Luciferase reporter assays demonstrated that gamma-pcdh promoter activity is increased by gamma-ICD. These results reveal an intracellular signaling mechanism for gamma-pcdhs and identify a novel vital target for the gamma-secretase complex.     

The α-helical content of the transmembrane domain of the British dementia protein-2 (Bri2) determines its processing by signal peptide peptidase-like 2b (SPPL2b)

Regulated intramembrane proteolysis is a widely accepted concept describing the processing of various transmembrane proteins via ectodomain shedding followed by an intramembrane cleavage. The resulting cleavage products can be involved in reverse signaling. Presenilins, which constitute the active center of the γ-secretase complex, signal peptide peptidase (SPP), and its homologues, the SPP-like (SPPL) proteases are members of the family of intramembrane-cleaving aspartyl proteases of the GXGD-type. We recently demonstrated that Bri2 (itm2b) is a substrate for regulated intramembrane proteolysis by SPPL2a and SPPL2b. Intramembrane cleavage of Bri2 is triggered by an initial shedding event catalyzed by A Disintegrin and Metalloprotease 10 (ADAM10). Additionally primary sequence determinants within the intracellular domain, the transmembrane domain and the luminal juxtamembrane domain are required for efficient cleavage of Bri2 by SPPL2b. Using mutagenesis and circular dichroism spectroscopy we now demonstrate that a high α-helical content of the Bri2 transmembrane domain (TMD) reduces cleavage efficiency of Bri2 by SPPL2b, while the presence of a GXXXG dimerization motif influences the intramembrane cleavage only to a minor extent. Surprisingly, only one of the four conserved intramembrane glycine residues significantly affects the secondary structure of the Bri2 TMD and thereby its intramembrane cleavage. Other glycine residues do not influence the α-helical content of the transmembrane domain nor its intramembrane processing.     

A TAG on to the neurogenic functions of APP

The proteolytic processing of amyloid β precursor protein (APP) has long been studied because of its association with the pathology of Alzheimer's disease (AD). The ectodomain of APP is shed by α- or β-secretase cleavage. The remaining membrane bound stub can then undergo regulated intramembrane proteolysis (RIP) by γ-secretase. This cleavage can release amyloid β (Aβ) from the stub left by β-secretase cleavage but also releases the APP intracellular domain (AICD) after α- or β-secretase cleavage. The physiological functions of this proteolytic processing are not well understood. We compare the proteolytic processing of APP to the ligand-dependent RIP of Notch. In this review, we discuss recent evidence suggesting that TAG1 is a functional ligand for APP. The interaction between TAG1 and APP triggers γ-secretase-dependent release of AICD. TAG1, APP and Fe65 colocalise in the neurogenic ventricular zone and in fetal neural progenitor cells in vitro. Experiments in TAG1, APP and Fe65 null mice as well as TAG1 and APP double-null mice demonstrate that TAG1 induces a γ-secretase- and Fe65-dependent suppression of neurogenesis.     

Intracellular fragment of NLRR3 (NLRR3-ICD) stimulates ATRA-dependent neuroblastoma differentiation

We have previously identified neuronal leucine-rich repeat protein-3 (NLRR3) gene which is preferentially expressed in favorable human neuroblastomas as compared with unfavorable ones. In this study, we have found for the first time that NLRR3 is proteolytically processed by secretases and its intracellular domain (NLRR3-ICD) is then released to translocate into cell nucleus during ATRA-mediated neuroblastoma differentiation. According to our present observations, NLRR3-ICD was induced to accumulate in cell nucleus of neuroblastoma SH-SY5Y cells following ATRA treatment. Since the proteolytic cleavage of NLRR3 was blocked by α- or γ-secretase inhibitor, it is likely that NLRR3-ICD is produced through the secretase-mediated processing of NLRR3. Intriguingly, forced expression of NLRR3-ICD in neuroblastoma SK-N-BE cells significantly suppressed their proliferation as examined by a live-cell imaging system and colony formation assay. Similar results were also obtained in neuroblastoma TGW cells. Furthermore, overexpression of NLRR3-ICD stimulated ATRA-dependent neurite elongation in SK-N-BE cells. Together, our present results strongly suggest that NLRR3-ICD produced by the secretase-mediated proteolytic processing of NLRR3 plays a crucial role in ATRA-mediated neuronal differentiation, and provide a clue to develop a novel therapeutic strategy against aggressive neuroblastomas.     

Protocadherin-12 cleavage is a regulated process mediated by ADAM10 protein: evidence of shedding up-regulation in pre-eclampsia

Protocadherins are a group of transmembrane proteins with homophilic binding activity, members of the cadherin superfamily. Apart from their role in adhesion, the cellular functions of protocadherins are essentially unknown. Protocadherin (PCDH)12 was previously identified in invasive trophoblasts and endothelial and mesangial cells in the mouse. Invalidation studies revealed that the protein was required for optimal placental development. In this article, we show that its human homolog is abundantly expressed in various trophoblast subtypes of the human placenta and at lower levels in endothelial cells. We demonstrate that PCDH12 is shed at high rates in vitro. The shedding mechanism depends on ADAM10 and results in reduced cellular adhesion in a cell migration assay. PCDH12 is subsequently cleaved by the γ-secretase complex, and its cytoplasmic domain is rapidly degraded by the proteasome. PCDH12 shedding is regulated by interlinked intracellular pathways, including those involving protein kinase C, PI3K, and cAMP, that either increase or inhibit cleavage. In endothelial cells, VEGF, prostaglandin E(2), or histamine regulates PCDH12 shedding. The extracellular domain of PCDH12 was also detected in human serum and urine, thus providing evidence of PCDH12 shedding in vivo. Importantly, we observed an increase in circulating PCDH12 in pregnant women who later developed a pre-eclampsia, a frequent pregnancy syndrome and a major cause of maternal and fetal morbidity and mortality. In conclusion, we speculate that, like in mice, PCDH12 may play an important role in human placental development and that proteolytic cleavage in response to external factors, such as cytokines and pathological settings, regulates its activity.     

Shedding-Generated Met Receptor Fragments can be Routed to Either the Proteasomal or the Lysosomal Degradation Pathway

The receptor tyrosine kinase Met and its ligand, the hepatocyte growth factor/scatter factor, are essential for embryonic development, whereas deregulation of Met signaling pathways is associated with tumorigenesis and metastasis. The presenilin-regulated intramembrane proteolysis (PS-RIP) is involved in ligand-independent downregulation of Met. This proteolytic process involves shedding of the Met extracellular domain followed by γ-secretase cleavage, generating labile intracellular fragments degraded by the proteasome. We demonstrate here that upon shedding both generated Met N- and C-terminal fragments are degraded directly in the lysosome, with C-terminal fragments escaping γ-secretase cleavage. PS-RIP and lysosomal degradation are complementary, because their simultaneous inhibition induces synergistic accumulation of fragments. Met N-terminal fragments associate with the high-affinity domain of HGF/SF, confirming its decoy activity which could be reduced through their routing to the lysosome at the expense of extracellular release. Finally, the DN30 monoclonal antibody inducing Met shedding promotes receptor degradation through induction of both PS-RIP and the lysosomal pathway. Thus, we demonstrate that Met shedding initiates a novel lysosomal degradation which participates to ligand-independent downregulation of the receptor.