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.     

Regulated intramembrane proteolysis of Bri2 (Itm2b) by ADAM10 and SPPL2a/SPPL2b

Presenilin, the catalytic component of the gamma-secretase complex, type IV prepilin peptidases, and signal peptide peptidase (SPP) are the founding members of the family of intramembrane-cleaving GXGD aspartyl proteases. SPP-like (SPPL) proteases, such as SPPL2a, SPPL2b, SPPL2c, and SPPL3, also belong to the GXGD family. In contrast to gamma-secretase, for which numerous substrates have been identified, very few in vivo substrates are known for SPP and SPPLs. Here we demonstrate that Bri2 (Itm2b), a type II-oriented transmembrane protein associated with familial British and Danish dementia, undergoes regulated intramembrane proteolysis. In addition to the previously described ectodomain processing by furin and related proteases, we now describe that the Bri2 protein, similar to gamma-secretase substrates, undergoes an additional cleavage by ADAM10 in its ectodomain. This cleavage releases a soluble variant of Bri2, the BRICHOS domain, which is secreted into the extracellular space. Upon this shedding event, a membrane-bound Bri2 N-terminal fragment remains, which undergoes intramembrane proteolysis to produce an intracellular domain as well as a secreted low molecular weight C-terminal peptide. By expressing all known SPP/SPPL family members as well as their loss of function variants, we demonstrate that selectively SPPL2a and SPPL2b mediate the intramembrane cleavage, whereas neither SPP nor SPPL3 is capable of processing the Bri2 N-terminal fragment.     

Nicastrin functions as a gamma-secretase-substrate receptor

gamma-secretase catalyzes the intramembrane cleavage of amyloid precursor protein (APP) and Notch after their extracellular domains are shed by site-specific proteolysis. Nicastrin is an essential glycoprotein component of the gamma-secretase complex but has no known function. We now show that the ectodomain of nicastrin binds the new amino terminus that is generated upon proteolysis of the extracellular APP and Notch domains, thereby recruiting the APP and Notch substrates into the gamma-secretase complex. Chemical- or antibody-mediated blocking of the free amino terminus, addition of purified nicastrin ectodomain, or mutations in the ectodomain markedly reduce the binding and cleavage of substrate by gamma-secretase. These results indicate that nicastrin is a receptor for the amino-terminal stubs that are generated by ectodomain shedding of type I transmembrane proteins. Our data are consistent with a model where nicastrin presents these substrates to gamma-secretase and thereby facilitates their cleavage via intramembrane proteolysis.     

Proteomic profiling of gamma-secretase substrates and mapping of substrate requirements

The presenilin/gamma-secretase complex, an unusual intramembrane aspartyl protease, plays an essential role in cellular signaling and membrane protein turnover. Its ability to liberate numerous intracellular signaling proteins from the membrane and also mediate the secretion of amyloid-beta protein (Abeta) has made modulation of gamma-secretase activity a therapeutic goal for cancer and Alzheimer disease. Although the proteolysis of the prototypical substrates Notch and beta-amyloid precursor protein (APP) has been intensely studied, the full spectrum of substrates and the determinants that make a transmembrane protein a substrate remain unclear. Using an unbiased approach to substrate identification, we surveyed the proteome of a human cell line for targets of gamma-secretase and found a relatively small population of new substrates, all of which are type I transmembrane proteins but have diverse biological roles. By comparing these substrates to type I proteins not regulated by gamma-secretase, we determined that besides a short ectodomain, gamma-secretase requires permissive transmembrane and cytoplasmic domains to bind and cleave its substrates. In addition, we provide evidence for at least two mechanisms that can target a substrate for gamma cleavage: one in which a substrate with a short ectodomain is directly cleaved independent of sheddase association, and a second where a substrate requires ectodomain shedding to instruct subsequent gamma-secretase processing. These findings expand our understanding of the mechanisms of substrate selection as well as the diverse cellular processes to which gamma-secretase contributes.     

Regulated intramembrane proteolysis--lessons from amyloid precursor protein processing

Regulated intramembrane proteolysis (RIP) controls the communication between cells and the extracellular environment. RIP is essential in the nervous system, but also in other tissues. In the RIP process, a membrane protein typically undergoes two consecutive cleavages. The first one results in the shedding of its ectodomain. The second one occurs within its transmembrane domain, resulting in secretion of a small peptide and the release of the intracellular domain into the cytosol. The proteolytic cleavage fragments act as versatile signaling molecules or are further degraded. An increasing number of membrane proteins undergo RIP. These include growth factors, cytokines, cell adhesion proteins, receptors, viral proteins and signal peptides. A dysregulation of RIP is found in diseases, such as leukemia and Alzheimer's disease. One of the first RIP substrates discovered was the amyloid precursor protein (APP). RIP processing of APP controls the generation of the amyloid β-peptide, which is believed to cause Alzheimer's disease. Focusing on APP as the best-studied RIP substrate, this review describes the function and mechanism of the APP RIP proteases with the goal to elucidate cellular mechanisms and common principles of the RIP process in general.     

Regulated proteolytic processing of Tie1 modulates ligand responsiveness of the receptor-tyrosine kinase Tie2

Regulated ectodomain shedding followed by intramembrane proteolysis has recently been recognized as important in cell signaling and for degradation of several type I transmembrane proteins. The receptor-tyrosine kinase Tie1 is known to undergo ectodomain cleavage generating a membrane-tethered endodomain. Here we show Tie1 is a substrate for regulated intramembrane proteolysis. After Tie1 ectodomain cleavage the newly formed 45-kDa endodomain undergoes additional proteolytic processing mediated by gamma-secretase to generate an amino-terminal-truncated 42-kDa fragment that is subsequently degraded by proteasomal activity. This sequential processing occurs constitutively and is stimulated by phorbol ester and vascular endothelial growth factor. To assess the biological significance of regulated Tie1 processing, we analyzed its effects on angiopoietin signaling. Activation of ectodomain cleavage causes loss of phosphorylated Tie1 holoreceptor and generation of phosphorylated receptor fragments in the presence of cartilage oligomeric protein angiopoietin 1. A key function of gamma-secretase is in preventing accumulation of these phosphorylated fragments. We also find that regulated Tie1 processing modulates ligand responsiveness of the Tie-1-associated receptor Tie2. Activation of Tie1 ectodomain cleavage increases cartilage oligomeric protein angiopoietin 1 activation of Tie2. This correlates with increased ability of Tie2 to bind ligand after shedding of the Tie1 extracellular domain. A similar enhancement of ligand activation of Tie2 is seen when Tie1 expression is suppressed by RNA interference. Together these data indicate that Tie1, via its extracellular domain, limits the ability of ligand to bind and activate Tie2. Furthermore the data suggest that regulated processing of Tie1 may be an important mechanism for controlling signaling by Tie2.     

Regulated Intramembrane Proteolysis of the Frontotemporal Lobar Degeneration Risk Factor,TMEM106B,by Signal Peptide Peptidase-like 2a (SPPL2a)

The sequential processing of single pass transmembrane proteins via ectodomain shedding followed by intramembrane proteolysis is involved in a wide variety of signaling processes, as well as maintenance of membrane protein homeostasis. Here we report that the recently identified frontotemporal lobar degeneration risk factor TMEM106B undergoes regulated intramembrane proteolysis. We demonstrate that TMEM106B is readily processed to an N-terminal fragment containing the transmembrane and intracellular domains, and this processing is dependent on the activities of lysosomal proteases. The N-terminal fragment is further processed into a small, rapidly degraded intracellular domain. The GxGD aspartyl proteases SPPL2a and, to a lesser extent, SPPL2b are responsible for this intramembrane cleavage event. Additionally, the TMEM106B paralog TMEM106A is also lysosomally localized; however, it is not a specific substrate of SPPL2a or SPPL2b. Our data add to the growing list of proteins that undergo intramembrane proteolysis and may shed light on the regulation of the frontotemporal lobar degeneration risk factor TMEM106B.     

Proteolytic ectodomain shedding of membrane proteins in mammals—hardware,concepts, and recent developments

Proteolytic removal of membrane protein ectodomains (ectodomain shedding) is a post‐translational modification that controls levels and function of hundreds of membrane proteins. The contributing proteases, referred to as sheddases, act as important molecular switches in processes ranging from signaling to cell adhesion. When deregulated, ectodomain shedding is linked to pathologies such as inflammation and Alzheimer's disease. While proteases of the “a disintegrin and metalloprotease” (ADAM) and “beta‐site APP cleaving enzyme” (BACE) families are widely considered as sheddases, in recent years a much broader range of proteases, including intramembrane and soluble proteases, were shown to catalyze similar cleavage reactions. This review demonstrates that shedding is a fundamental process in cell biology and discusses the current understanding of sheddases and their substrates, molecular mechanisms and cellular localizations, as well as physiological functions of protein ectodomain shedding. Moreover, we provide an operational definition of shedding and highlight recent conceptual advances in the field. While new developments in proteomics facilitate substrate discovery, we expect that shedding is not a rare exception, but rather the rule for many membrane proteins, and that many more interesting shedding functions await discovery.     

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.     

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.     

gamma-Secretase cleavage and binding to FE65 regulate the nuclear translocation of the intracellular C-terminal domain (ICD) of the APP family of proteins

Regulated intramembrane proteolysis (RIP) of the amyloid precursor protein (APP) produces amyloid beta-protein (Abeta), the probable causative agent of Alzheimer's disease (AD), and is therefore an important target for therapeutic intervention. However, there is a burgeoning consensus that gamma-secretase, one of the proteases that generates Abeta, is also critical for the signal transduction of APP and a growing list of other receptors. APP is a member of a gene family that includes two amyloid precursor-like proteins, APLP1 and APLP2. Although APP and the APLPs undergo similar proteolytic processing, there is little information about the role of their gamma-secretase-generated intracellular domains (ICDs). Here, we show that APLP1 and 2 undergo presenilin-dependent RIP similar to APP, resulting in the release of a approximately 6 kDa ICD for each protein. Each of the ICDs are degraded by an insulin degrading enzyme-like activity, but they can be stabilized by members of the FE65 family and translocate to the nucleus. Given that modulation of APP processing is a therapeutic target and that the APLPs are processed in a manner similar to APP, any strategy aimed at altering APP proteolysis will have to take into account possible effects on signaling by APLP 1 and 2.     

Nectin-1alpha,an immunoglobulin-like receptor involved in the formation of synapses,is a substrate for presenilin/gamma-secretase-like cleavage

Nectin-1 is a member of the immunoglobulin superfamily and a Ca(2+)-independent adherens junction protein involved in synapse formation. Here we show that nectin-1alpha undergoes intramembrane proteolytic processing analogous to that of the Alzheimer's disease amyloid precursor protein, mediated by a presenilin (PS)-dependent gamma-secretase-like activity. 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment of Chinese hamster ovary cells activated a first proteolytic event, resulting in ectodomain shedding of nectin-1alpha. Subsequent cleavage of the remaining 26-kDa membrane-anchored C-terminal fragment (CTF) was inhibited independently by three specific gamma-secretase inhibitors and by expression of the dominant negative form of PS1. The PS/gamma-secretase-like cleavage product was detected in vivo following proteasome inhibitor treatment of cells. An in vitro gamma-secretase assay confirmed the generation of a 24-kDa nectin-1alpha intracellular domain, peripherally associated with the membrane fraction. We also found nectin-1alpha to interact with the N-terminal fragment of PS1. Finally, gamma-secretase inhibition resulted in beta-catenin release from cell junctions, concomitantly with the accumulation of the 26-kDa nectin-1alpha CTF, suggesting that high levels of nectin-1alpha CTF interfere with TPA-induced remodeling of cell-cell junctions. Our results are consistent with a previously reported role for PS/gamma-secretase in adherens junction function involving cleavage of cadherins. Similar to nectin-1, other members of the immunoglobulin superfamily involved in synapse formation may also serve as substrates for PS/gamma-secretase-like intramembrane proteolytic activity.     

TRAF6 promotes ubiquitination and regulated intramembrane proteolysis of IL-1R1

It has recently been shown that Interleukin-1 receptor, type 1, an essential regulator of inflammation and inate immunity, undergoes regulated intramembrane proteolysis (RIP). Although IL-1R1-mediated intracellular signalling has been well studied, very little is known about how RIP of IL-1R1 is modulated. In this study, by using wild-type TRAF6 and TRAF6 mutants that are defective in its ubiquitin ligase activity, we show for the first time that TRAF6 induces ubiquitination of IL-1R1. We further demonstrate that of all TRAF family members examined, TRAF6 preferentially ubiquitinates IL-1R1. Moreover, we show that TRAF6 ubiquitin ligase activity and ubiquitination of IL-1R1 are positively correlated with IL-1R1 ectodomain shedding and subsequent gamma-secretase cleavage. Our results indicate that TRAF6-mediated ubiquitination of IL-1R1 has a decisive role in IL-1R1 signalling and propose a molecular mechanism whereby TRAF6 promotes ubiquitination and RIP of IL-1R1 through its ubiquitin ligase activity.     

Failure of the interaction between presenilin 1 and the substrate of gamma-secretase to produce Abeta in insect cells

Aggregates of beta-amyloid peptide (Abeta) are the major component of the amyloid core of the senile plaques observed in Alzheimer's disease (AD). Abeta results from the amyloidogenic processing of its precursor, the amyloid precursor protein (APP), by beta- and gamma-secretase activities. If beta-secretase has recently been identified and termed BACE, the identity of gamma-secretase is still obscure. Studies with knock-out mice showed that presenilin 1 (PS1), of which mutations are known to be the first cause of inherited AD, is mandatory for the gamma-secretase activity. However, the proteolytic activity of PS1 remains a matter of debate. Here we used transfected Sf9 insect cells, a cellular model lacking endogenous beta- and/or gamma-secretase activities, to characterize the role of BACE and PS1 in the amyloidogenic processing of human APP. We show that, in Sf9 cells, BACE performs the expected beta-secretase cleavage of APP, generating C99. We also show that C99, which is a substrate of gamma-secretase, tightly binds to the human PS1. Despite this interaction, Sf9 cells still do not produce Abeta. This strongly argues against a direct proteolytic activity of PS1 in APP processing, and points toward an implication of PS1 in trafficking/presenting its substrate to the gamma-secretase.     

Phorbol 12-myristate 13-acetate-induced release of the colony-stimulating factor 1 receptor cytoplasmic domain into the cytosol involves two separate cleavage events

The colony-stimulating factor 1 (CSF-1) receptor is a protein-tyrosine kinase that regulates cell division, differentiation, and development. In response to phorbol 12-myristate 13-acetate (PMA), the CSF-1 receptor is subject to proteolytic processing. Use of chimeric receptors indicates that the CSF-1 receptor is cleaved at least two times, once in the extracellular domain and once in the transmembrane domain. Cleavage in the extracellular domain results in ectodomain shedding while the cytoplasmic domain remains associated with the membrane. Intramembrane cleavage depends on the sequence of the transmembrane domain and results in the release of the cytoplasmic domain. This process can be blocked by gamma-secretase inhibitors. The cytoplasmic domain localizes partially to the nucleus, displays limited stability, and is degraded by the proteosome. CSF-1 receptors are continuously subject to down-modulation and regulated intramembrane proteolysis (RIP). RIP is stimulated by granulocyte-macrophage-CSF, CSF-1, interleukin-2 (IL-2), IL-4, lipopolysaccharide, and PMA and may provide the CSF-1 receptor with an additional mechanism for signal transduction.     

Bcl-2 family: life-or-death switch

Proteolytic cleavage of the amyloid protein from the amyloid protein precursor (APP) by APP secretases is a key event in Alzheimer's disease (AD) pathogenesis. alpha-Secretases cleave APP within the amyloid sequences, whereas beta- and gamma-secretases cleave on the N- and C-terminal ends respectively. The transmembrane aspartyl protease BACE has been identified as beta-secretase and several proteases (ADAM-10, TACE, PC7) may be alpha-secretases. A number of studies have suggested that presenilins could be gamma-secretases, although this remains to be demonstrated conclusively. Inhibition of beta- and gamma-secretase, or stimulation of alpha-secretase, is a rational strategy for therapeutic intervention in AD.     

Hypoxia-inducible factor 1alpha (HIF-1alpha)-mediated hypoxia increases BACE1 expression and beta-amyloid generation

The incidence of Alzheimer disease (AD) and vascular dementia is greatly increased following cerebral ischemia and stroke in which hypoxic conditions occur in affected brain areas. beta-Amyloid peptide (Abeta), which is derived from the beta-amyloid precursor protein (APP) by sequential proteolytic cleavages from beta-secretase (BACE1) and presenilin-1 (PS1)/gamma-secretase, is widely believed to trigger a cascade of pathological events culminating in AD and vascular dementia. However, a direct molecular link between hypoxic insults and APP processing has yet to be established. Here, we demonstrate that acute hypoxia increases the expression and the enzymatic activity of BACE1 by up-regulating the level of BACE1 mRNA, resulting in increases in the APP C-terminal fragment-beta (betaCTF) and Abeta. Hypoxia has no effect on the level of PS1, APP, and tumor necrosis factor-alpha-converting enzyme (TACE, an enzyme known to cleave APP at the alpha-secretase cleavage site). Sequence analysis, mutagenesis, and gel shift studies revealed binding of HIF-1 to the BACE1 promoter. Overexpression of HIF-1alpha increases BACE1 mRNA and protein level, whereas down-regulation of HIF-1alpha reduced the level of BACE1. Hypoxic treatment fails to further potentiate the stimulatory effect of HIF-1alpha overexpression on BACE1 expression, suggesting that hypoxic induction of BACE1 expression is primarily mediated by HIF-1alpha. Finally, we observed significant reduction in BACE1 protein levels in the hippocampus and the cortex of HIF-1alpha conditional knock-out mice. Our results demonstrate an important role for hypoxia/HIF-1alpha in modulating the amyloidogenic processing of APP and provide a molecular mechanism for increased incidence of AD following cerebral ischemic and stroke injuries.     

Linking receptor-mediated endocytosis and cell signaling: evidence for regulated intramembrane proteolysis of megalin in proximal tubule

Megalin, a member of the low density lipoprotein receptor gene family, is required for efficient protein absorption in the proximal tubule. Recent studies have shown that the low density lipoprotein receptor-related protein, another member of this gene family, is proteolytically processed by gamma-secretase implying a role for low density lipoprotein receptor-related protein in a Notchlike signaling pathway. This pathway has been shown to involve: 1) metalloprotease-mediated ectodomain shedding and gamma-secretase-mediated intramembrane proteolysis of some receptors. Experiments were performed to determine whether megalin undergoes similar processing. By immunocytochemistry, immunoblotting, and a fluorogenic enzyme assay presenilin-1 (required for gamma-secretase activity) and gamma-secretase activity were found in the brush border of proximal kidney tubules where megalin is localized. Using a fluorogenic peptide containing an amyloid precursor protein gamma-secretase cleavage site and Compound E, a specific gamma-secretase inhibitor, we found high levels of gamma-secretase activity in renal brush border membrane vesicles. Immunoblotting analysis of renal microsomes and opossum kidney proximal tubule (OKP) cells using antibodies directed to the cytosolic domain of megalin showed a 35-40-kDa, membrane-associated, carboxyl-terminal fragment of megalin (MCTF). When cells were incubated with 200 nm phorbol 12-myristate 13-acetate, the appearance of the MCTF increased 2.5-fold and was blocked by metalloprotease inhibitors. When the cells were incubated with gamma-secretase inhibitor Compound E, it caused a 2-fold increase in MCTF. Finally, incubating the cells with 1 microm vitamin D-binding protein resulted in a 25% increase in the appearance of the MCTF. In summary, the MCTF is produced by protein kinase C regulated, metalloprotease-mediated ectodomain shedding and is the substrate for gamma-secretase. We postulate that the enzymatic processing of megalin represents part of a novel ligand-dependent signaling pathway in the proximal tubule that links receptor-mediated endocytosis with cell signaling.