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.     

Mechanism,specificity, and physiology of signal peptide peptidase (SPP) and SPP-like proteases

Signal peptide peptidase (SPP) and the homologous SPP-like (SPPL) proteases SPPL2a, SPPL2b, SPPL2c and SPPL3 belong to the family of GxGD intramembrane proteases. SPP/SPPLs selectively cleave transmembrane domains in type II orientation and do not require additional co-factors for proteolytic activity. Orthologues of SPP and SPPLs have been identified in other vertebrates, plants, and eukaryotes. In line with their diverse subcellular localisations ranging from the ER (SPP, SPPL2c), the Golgi (SPPL3), the plasma membrane (SPPL2b) to lysosomes/late endosomes (SPPL2a), the different members of the SPP/SPPL family seem to exhibit distinct functions. Here, we review the substrates of these proteases identified to date as well as the current state of knowledge about the physiological implications of these proteolytic events as deduced from in vivo studies. Furthermore, the present knowledge on the structure of intramembrane proteases of the SPP/SPPL family, their cleavage mechanism and their substrate requirements are summarised. This article is part of a Special Issue entitled: Intramembrane Proteases.     

A gamma-secretase-like intramembrane cleavage of TNFalpha by the GxGD aspartyl protease SPPL2b

Gamma-secretase and signal peptide peptidase (SPP) are unusual GxGD aspartyl proteases, which mediate intramembrane proteolysis. In addition to SPP, a family of SPP-like proteins (SPPLs) of unknown function has been identified. We demonstrate that SPPL2b utilizes multiple intramembrane cleavages to liberate the intracellular domain of tumor necrosis factor alpha (TNFalpha) into the cytosol and the carboxy-terminal counterpart into the extracellular space. These findings suggest common principles for regulated intramembrane proteolysis by GxGD aspartyl proteases.     

Consensus analysis of signal peptide peptidase and homologous human aspartic proteases reveals opposite topology of catalytic domains compared with presenilins

The human genome encodes seven intramembrane-cleaving GXGD aspartic proteases. These are the two presenilins that activate signaling molecules and are implicated in Alzheimer's disease, signal peptide peptidase (SPP), required for immune surveillance, and four SPP-like candidate proteases (SPPLs), of unknown function. Here we describe a comparative analysis of the topologies of SPP and its human homologues, SPPL2a, -2b, -2c, and -3. We demonstrate that their N-terminal extensions are located in the extracellular space and, except for SPPL3, are modified with N-glycans. Whereas SPPL2a, -2b, and -2c contain a signal sequence, SPP and SPPL3 contain a type I signal anchor sequence for initiation of protein translocation and membrane insertion. The hydrophilic loops joining the transmembrane regions, which contain the catalytic residues, are facing the exoplasm. The C termini of all these proteins are exposed toward the cytosol. Taken together, our study demonstrates that SPP and its homologues are all of the same principal structure with a catalytic domain embedded in the membrane in opposite orientation to that of presenilins. Other than presenilins, SPPL2a, -2b, -2c, and -3 are therefore predicted to cleave type II-oriented substrate peptides like the prototypic protease SPP.     

Foamy Virus Envelope Protein Is a Substrate for Signal Peptide Peptidase-like 3 (SPPL3)

Signal peptide peptidase (SPP), its homologs, the SPP-like proteases SPPL2a/b/c and SPPL3, as well as presenilin, the catalytic subunit of the γ-secretase complex, are intramembrane-cleaving aspartyl proteases of the GxGD type. In this study, we identified the 18-kDa leader peptide (LP18) of the foamy virus envelope protein (FVenv) as a new substrate for intramembrane proteolysis by human SPPL3 and SPPL2a/b. In contrast to SPPL2a/b and γ-secretase, which require substrates with an ectodomain shorter than 60 amino acids for efficient intramembrane proteolysis, SPPL3 cleaves mutant FVenv lacking the proprotein convertase cleavage site necessary for the prior shedding. Moreover, the cleavage product of FVenv generated by SPPL3 serves as a new substrate for consecutive intramembrane cleavage by SPPL2a/b. Thus, human SPPL3 is the first GxGD-type aspartyl protease shown to be capable of acting like a sheddase, similar to members of the rhomboid family, which belong to the class of intramembrane-cleaving serine proteases.     

SPPL2a and SPPL2b promote intramembrane proteolysis of TNFalpha in activated dendritic cells to trigger IL-12 production

Homologues of signal peptide peptidase (SPPLs) are putative aspartic proteases that may catalyse regulated intramembrane proteolysis of type II membrane-anchored signalling factors. Here, we show that four human SPPLs are each sorted to a different compartment of the secretory pathway. We demonstrate that SPPL2a and SPPL2b, which are sorted to endosomes and the plasma membrane, respectively, are functional proteases that catalyse intramembrane cleavage of tumour necrosis factor alpha (TNFalpha). The two proteases promoted the release of the TNFalpha intracellular domain, which in turn triggers expression of the pro-inflammatory cytokine interleukin-12 by activated human dendritic cells. Our study reveals a critical function for SPPL2a and SPPL2b in the regulation of innate and adaptive immunity.     

Expression and characterization of Drosophila signal peptide peptidase-like (sppL), a gene that encodes an intramembrane protease

Intramembrane proteases of the Signal Peptide Peptidase (SPP) family play important roles in developmental, metabolic and signaling pathways. Although vertebrates have one SPP and four SPP-like (SPPL) genes, we found that insect genomes encode one Spp and one SppL. Characterization of the Drosophila sppL gene revealed that the predicted SppL protein is a highly conserved structural homolog of the vertebrate SPPL3 proteases, with a predicted nine-transmembrane topology, an active site containing aspartyl residues within a transmembrane region, and a carboxy-terminal PAL domain. SppL protein localized to both the Golgi and ER. Whereas spp is an essential gene that is required during early larval stages and whereas spp loss-of-function reduced the unfolded protein response (UPR), sppL loss of function had no apparent phenotype. This was unexpected given that genetic knockdown phenotypes in other organisms suggested significant roles for Spp-related proteases.     

Differential Inhibition of Signal Peptide Peptidase Family Members by Established γ-Secretase Inhibitors

The signal peptide peptidases (SPPs) are biomedically important proteases implicated as therapeutic targets for hepatitis C (human SPP, (hSPP)), plasmodium (Plasmodium SPP (pSPP)), and B-cell immunomodulation and neoplasia (signal peptide peptidase like 2a, (SPPL2a)). To date, no drug-like, selective inhibitors have been reported. We use a recombinant substrate based on the amino-terminus of BRI2 fused to amyloid β 1-25 (Aβ_1-25) (FBA) to develop facile, cost-effective SPP/SPPL protease assays. Co-transfection of expression plasmids expressing the FBA substrate with SPP/SPPLs were conducted to evaluate cleavage, which was monitored by ELISA, Western Blot and immunoprecipitation/MALDI-TOF Mass spectrometry (IP/MS). No cleavage is detected in the absence of SPP/SPPL overexpression. Multiple γ-secretase inhibitors (GSIs) and (Z-LL)₂ ketone differentially inhibited SPP/SPPL activity; for example, IC₅₀ of LY-411,575 varied from 51±79 nM (on SPPL2a) to 5499±122 nM (on SPPL2b), while Compound E showed inhibition only on hSPP with IC₅₀ of 1465±93 nM. Data generated were predictive of effects observed for endogenous SPPL2a cleavage of CD74 in a murine B-Cell line. Thus, it is possible to differentially inhibit SPP family members. These SPP/SPPL cleavage assays will expedite the search for selective inhibitors. The data also reinforce similarities between SPP family member cleavage and cleavage catalyzed by γ-secretase.     

Intramembrane proteolysis of GXGD-type aspartyl proteases is slowed by a familial Alzheimer disease-like mutation

More than 150 familial Alzheimer disease (FAD)-associated missense mutations in presenilins (PS1 and PS2), the catalytic subunit of the gamma-secretase complex, cause aberrant amyloid beta-peptide (Abeta) production, by increasing the relative production of the highly amyloidogenic 42-amino acid variant. The molecular mechanism behind this pathological activity is unclear, and different possibilities ranging from a gain of function to a loss of function have been discussed. gamma-Secretase, signal peptide peptidase (SPP) and SPP-like proteases (SPPLs) belong to the same family of GXGD-type intramembrane cleaving aspartyl proteases and share several functional similarities. We have introduced the FAD-associated PS1 G384A mutation, which occurs within the highly conserved GXGD motif of PS1 right next to the catalytically critical aspartate residue, into the corresponding GXGD motif of the signal peptide peptidase-like 2b (SPPL2b). Compared with wild-type SPPL2b, mutant SPPL2b slowed intramembrane proteolysis of tumor necrosis factor alpha and caused a relative increase of longer intracellular cleavage products. Because the N termini of the secreted counterparts remain unchanged, the mutation selectively affects the liberation of the intracellular processing products. In vitro experiments demonstrate that the apparent accumulation of longer intracellular cleavage products is the result of slowed sequential intramembrane cleavage. The longer cleavage products are still converted to shorter peptides, however only after prolonged incubation time. This suggests that FAD-associated PS mutation may also result in reduced intramembrane cleavage of beta-amyloid precursor protein (betaAPP). Indeed, in vitro experiments demonstrate slowed intramembrane proteolysis by gamma-secretase containing PS1 with the G384A mutation. As compared with wild-type PS1, the mutation selectively slowed Abeta40 production, whereas Abeta42 generation remained unaffected. Thus, the PS1 G384A mutation causes a selective loss of function by slowing the processing pathway leading to the benign Abeta40.     

Signal-peptide-peptidase-like 2a (SPPL2a) is targeted to lysosomes/late endosomes by a tyrosine motif in its C-terminal tail

Signal-peptide-peptidase-like 2A (SPPL2a), an aspartyl intramembrane protease, has been implicated in the proteolysis of TNF-alpha, Fas Ligand and Bri2. Here, we show that endogenous SPPL2a - in agreement with overexpression studies - is localised in membranes of lysosomes/late endosomes. Furthermore, we have analysed the molecular determinants for lysosomal sorting of SPPL2a by creating chimaeric constructs between SPPL2a and its plasma membrane localised homologue SPPL2b. Lysosomal transport of SPPL2a critically depends on its cytosolic carboxyterminal tail. A canonical tyrosine-based sorting motif of the YXX? type at position 498 is sufficient to direct SPPL2a to lysosomal/late endosomal compartments. This motif accounts for the differential localisation of the homologous proteases SPPL2a and SPPL2b and thereby influences the access to substrates and biological function of SPPL2a.     

Endogenous tagging reveals a mid-Golgi localization of the glycosyltransferase-cleaving intramembrane protease SPPL3

Numerous Golgi-resident enzymes implicated in glycosylation are regulated by the conserved intramembrane protease SPPL3. SPPL3-catalyzed endoproteolysis separates Golgi enzymes from their membrane anchors, enabling subsequent release from the Golgi and secretion. Experimentally altered SPPL3 expression changes glycosylation patterns, yet the regulation of SPPL3-mediated Golgi enzyme cleavage is not understood and conflicting results regarding the subcellular localization of SPPL3 have been reported. Here, we used precise genome editing to generate isogenic cell lines expressing N- or C-terminally tagged SPPL3 from its endogenous locus. Using these cells, we conducted co-localization analyses of tagged endogenous SPPL3 and Golgi markers under steady-state conditions and upon treatment with drugs disrupting Golgi organization. Our data demonstrate that endogenous SPPL3 is Golgi-resident and found predominantly in the mid-Golgi. We find that endogenous SPPL3 co-localizes with its substrates but similarly with non-substrate type II proteins, demonstrating that in addition to co-localization in the Golgi other substrate-intrinsic properties govern SPPL3-mediated intramembrane proteolysis. Given the prevalence of SPPL3-mediated cleavage among Golgi-resident proteins our results have important implications for the regulation of SPPL3 and its role in the organization of the Golgi glycosylation machinery.     

Shedding of glycan‐modifying enzymes by signal peptide peptidase‐like 3 (SPPL3) regulates cellular N‐glycosylation

Protein N‐glycosylation is involved in a variety of physiological and pathophysiological processes such as autoimmunity, tumour progression and metastasis. Signal peptide peptidase‐like 3 (SPPL3) is an intramembrane‐cleaving aspartyl protease of the GxGD type. Its physiological function, however, has remained enigmatic, since presently no physiological substrates have been identified. We demonstrate that SPPL3 alters the pattern of cellular N‐glycosylation by triggering the proteolytic release of active site‐containing ectodomains of glycosidases and glycosyltransferases such as N‐acetylglucosaminyltransferase V, β‐1,3 N‐acetylglucosaminyltransferase 1 and β‐1,4 galactosyltransferase 1. Cleavage of these enzymes leads to a reduction in their cellular activity. In line with that, reduced expression of SPPL3 results in a hyperglycosylation phenotype, whereas elevated SPPL3 expression causes hypoglycosylation. Thus, SPPL3 plays a central role in an evolutionary highly conserved post‐translational process in eukaryotes.     

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.     

Intramembrane Proteolysis by Signal Peptide Peptidases: A Comparative Discussion of GXGD-type Aspartyl Proteases

Intramembrane-cleaving proteases are required for reverse signaling and membrane protein degradation. A major class of these proteases is represented by the GXGD-type aspartyl proteases. GXGD describes a novel signature sequence that distinguishes these proteases from conventional aspartyl proteases. Members of the family of the GXGD-type aspartyl proteases are the Alzheimer disease-related γ-secretase, the signal peptide peptidases and their homologs, and the bacterial type IV prepilin peptidases. We will describe the major biochemical and functional properties of the signal peptide peptidases and their relatives. We then compare these properties with those of γ-secretase and discuss common mechanisms but also point out a number of substantial differences.During the last years, a number of intramembrane-cleaving proteases termed I-CLiPs³ have been identified (1). I-CLiPs are generally involved in regulated intramembrane proteolysis (2). Upon shedding of a large part of the ectodomain of membrane proteins, the remaining membrane-retained stub is cleaved by specialized proteases within the hydrophobic lipid membrane. Generally, this cleavage can have two predominant biological functions: first, signaling via the liberated ICD within the substrate-expressing cell (reverse signaling) (2); and second, degradation of membrane-retained stubs, which are not required for any further biological function (3). I-CLiPs of three protease classes, metalloproteases, serine proteases, and aspartyl proteases, have been discovered so far (see accompanying minireview by Wolfe (44)).Intramembrane-cleaving aspartyl proteases are represented by the class of the GXGD-type proteases (4). These are unconventional aspartyl proteases that, like the conventional aspartyl proteases, utilize two critical aspartyl residues for peptide bond cleavage. However, in contrast to the conventional proteases, the critical aspartyl residues are located within two TMDs (Fig. 1A). Moreover, these aspartyl residues are embedded in active-site motifs that are completely different from those of conventional aspartyl proteases. The class of GXGD-type aspartyl proteases is currently represented by three different protease families, the most prominent of which is the PS family, providing the catalytically active subunit of γ-secretase (Fig. 1A) (4). PS/γ-secretase is the I-CLiP that liberates amyloid β-peptide, the major component of senile plaques in Alzheimer disease patients (5). In addition, the bacterial type IV prepilin peptidases also belong to the class of the GXGD-type proteases (6). Besides these two protease families, two additional subfamilies of related proteases that also belong to the GXGD-type aspartyl protease family have been identified. These include SPP as well as the SPP homologs, the SPP-like (SPPL) proteases (Fig. 1A) (7, 8).Open in a separate windowFIGURE 1.A, schematic representation of SPPL2a/b, a member of the SPP/SPPL family, and PS, the catalytic core of theγ-secretase. Note the opposite topology of the active sites (indicated by arrows) of the two proteases and their substrates, APP for PS and TNFα for SPPL2a/b. B, proteolytic processing of APP and TNFα. Shedding releases the extracellular part of APP (APPs) and TNFα (TNFα soluble). In the case of APP, a C-terminal fragment (APP CTF), and in case of TNFα, an N-terminal fragment (TNFα NTF) are produced. These membrane-bound fragments are substrate to intramembrane cleavage by PS or SPPL2a/b, respectively, releasing small peptides to the extracellular space (Aβ and TNFα C-domain, respectively) and to the cytosol (APP intracellular domain (AICD) and TNFα ICD), respectively). TNFα FL, full-length TNFα.We will first describe the biochemical, functional, and structural properties of SPP family members. By comparison of these properties, we will then identify common mechanisms of intramembrane proteolysis by GXGD-type proteases but also point out some fundamental differences.     

The Intramembrane Proteases Signal Peptide Peptidase-Like 2a and 2b Have Distinct Functions In Vivo

We reported recently that the presenilin homologue signal peptide peptidase-like 2a (SPPL2a) is essential for B cell development by cleaving the N-terminal fragment (NTF) of the invariant chain (li, CD74). Based on this, we suggested that pharmacological modulation of SPPL2a may represent a novel approach to deplete B cells in autoimmune disorders. With regard to reported overlapping substrate spectra of SPPL2a and its close homologue, SPPL2b, we investigated the role of SPPL2b in CD74 NTF proteolysis and its impact on B and dendritic cell homeostasis. In heterologous expression experiments, SPPL2b was found to cleave CD74 NTF with an efficiency simliar to that of SPPL2a. For in vivo analysis, SPPL2b single-deficient and SPPL2a/SPPL2b double-deficient mice were generated and examined for CD74 NTF turnover/accumulation, B cell maturation and functionality, and dendritic cell homeostasis. We demonstrate that in vivo SPPL2b does not exhibit a physiologically relevant contribution to CD74 proteolysis in B and dendritic cells. Furthermore, we reveal that both proteases exhibit divergent subcellular localizations in B cells and different expression profiles in murine tissues. These findings suggest distinct functions of SPPL2a and SPPL2b and, based on a high abundance of SPPL2b in brain, a physiological role of this protease in the central nervous system.     

Signal peptide peptidases: A family of intramembrane-cleaving proteases that cleave type 2 transmembrane proteins

Five genes encode the five human signal peptide peptidases (SPPs), which are intramembrane-cleaving aspartyl proteases (aspartyl I-CLiPs). SPPs have been conserved through evolution with family members found in higher eukaryotes, fungi, protozoa, arachea, and plants. SPPs are related to the presenilin family of aspartyl I-CLiPs but differ in several key aspects. Presenilins (PSENs) and SPPs both cleave the transmembrane region of membrane proteins; however, PSENs cleave type 1 membrane proteins whereas SPPs cleave type 2 membrane proteins. Though the overall homology between SPPs and PSENs is minimal, they are multipass membrane proteins that contain two conserved active site motifs YD and GxGD in adjacent membrane-spanning domains and a conserved PAL motif of unknown function near their COOH-termini. They differ in that the active site YD and GxGD containing transmembrane domains of SPPs are inverted relative to PSENs, thus, orienting the active site in a consistent topology relative to the substrate. At least two of the human SPPs (SPP and SPPL3) appear to function without additional cofactors, but PSENs function as a protease, called γ-secretase, only when complexed with Nicastrin, APH-1 and Pen-2. The biological roles of SPP are largely unknown, and only a few endogenous substrates for SPPs have been identified. Nevertheless there is emerging evidence that SPP family members are highly druggable and may regulate both essential physiologic and pathophysiologic processes. Further study of the SPP family is needed in order to understand their biological roles and their potential as therapeutic targets.     

A C-terminal region of signal peptide peptidase defines a functional domain for intramembrane aspartic protease catalysis

Intramembrane proteolysis is now firmly established as a prominent biological process, and structure elucidation is emerging as the new frontier in the understanding of these novel membrane-embedded enzymes. Reproducing this unusual hydrolysis within otherwise water-excluding transmembrane regions with purified proteins is a challenging prerequisite for such structural studies. Here we show the bacterial expression, purification, and reconstitution of proteolytically active signal peptide peptidase (SPP), a membrane-embedded enzyme in the presenilin family of aspartyl proteases. This finding formally proves that, unlike presenilin, SPP does not require any additional proteins for proteolysis. Surprisingly, the conserved C-terminal half of SPP is sufficient for proteolytic activity; purification and reconstitution of this engineered fragment of several SPP orthologues revealed that this region defines a functional domain for an intramembrane aspartyl protease. The discovery of minimal requirements for intramembrane proteolysis should facilitate mechanistic and structural analysis and help define general biochemical principles of hydrolysis in a hydrophobic environment.     

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.