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β-Site amyloid precursor protein cleavage enzyme (BACE)-1 and BACE-2 are members of a novel family of membrane-bound aspartyl proteases. While BACE-1 is known to cleave β-amyloid precursor protein (βAPP) at the β-secretase site and to be required for the generation of amyloid β-peptide (Aβ), the role of its homologue BACE-2 in amyloidogenesis is less clear. We now demonstrate that BACE-1 and BACE-2 have distinct specificities in cleavage of βAPP in cultured cells. Radiosequencing of the membrane-bound C-terminal cleavage product revealed that BACE-2 cleaves βAPP in the middle of the Aβ domain between phenylalanines 19 and 20, resulting in increased secretion of APPs-α- and p3-like products and reduced production of Aβ species. This cleavage can occur in the Golgi and later secretory compartments. We also demonstrate that BACE-1-mediated cleavage of βAPP at Asp1 of the Aβ domain can occur as early as in the endoplasmic reticulum, while cleavage at Glu11 occurs in later compartments. These data indicate that the distinct specificities of BACE-1 and BACE-2 in their cleavage of βAPP differentially affect the generation of Aβ. [ABSTRACT FROM AUTHOR]

Members of the Signal Peptide-Peptidase (SPP) and Signal Peptide-Peptidase-like (SPPL) family are intramembrane aspartyl-proteases like their well-studied homologs, the presenilins, which comprise the catalytically active subunit within the γ-secretase complex. The lack of in vitro cleavage assays for SPPL proteases limited their biochemical characterization as well as substrate identification and validation. So far, SPPL proteases have been analyzed exclusively in intact cells or membranes, restricting mechanistic analysis to co-expression of enzyme and substrate variants colocalizing in the same subcellular compartments. We describe the details of developing an in vitro cleavage assay for SPPL2b and its model substrate TNFα and analyzed the influence of phospholipids, detergent supplements, and cholesterol on the SPPL2b in vitro activity. SPPL2b in vitro activity resembles mechanistic principles that have been observed in a cellular context, such as cleavage sites and consecutive turnover of the TNFα transmembrane domain. The novel in vitro cleavage assay is functional with separately isolated protease and substrate and amenable to a high throughput plate-based readout overcoming previous limitations and providing the basis for studying enzyme kinetics, catalytic activity, substrate recognition, and the characteristics of small molecule inhibitors. As a proof of concept, we present the first biochemical in vitro characterization of the SPPL2a and SPPL2b specific small molecule inhibitor SPL-707., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Alzheimer's disease (AD) is a multifactorial disorder driven by abnormal amyloid β-peptide (Aβ) levels. In this study, we investigated the role of presenilin-like signal peptide peptidase-like 2b (SPPL2b) in AD pathophysiology and its potential as a druggable target within the Aβ cascade. Exogenous Aβ42 influenced SPPL2b expression in human cell lines and acute mouse brain slices. SPPL2b and its AD-related substrate BRI2 were evaluated in the brains of App NL-G-F knock-in AD mice and human postmortem AD brains. An early high cortical expression of SPPL2b was observed, followed by a downregulation in late AD pathology in App NL-G-F mice, correlating with synaptic loss. To understand the consequences of pathophysiological SPPL2b dysregulation, we found that SPPL2b overexpression significantly increased APP cleavage, while genetic deletion reduced APP cleavage and Aβ production. Notably, postmortem AD brains showed higher levels of SPPL2b's BRI2 substrate compared to healthy control samples. These results strongly support the involvement of SPPL2b in AD pathology. The early Aβ-induced upregulation of SPPL2b may enhance Aβ production in a vicious cycle, further aggravating Aβ pathology. Therefore, SPPL2b emerges as a potential anti-Aβ drug target., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Signal peptide peptidase (SPP) and the four SPP-like proteases SPPL2a, SPPL2b, SPPL2c and SPPL3 constitute a family of aspartyl intramembrane proteases with homology to presenilins. The different members reside in distinct cellular localisations within the secretory pathway and the endo-lysosomal system. Despite individual cleavage characteristics, they all cleave single-span transmembrane proteins with a type II orientation exhibiting a cytosolic N-terminus. Though the identification of substrates is not complete, SPP/SPPL-mediated proteolysis appears to be rather selective. Therefore, according to our current understanding cleavage by SPP/SPPL proteases rather seems to serve a regulatory function than being a bulk proteolytic pathway. In the present review, we will summarise our state of knowledge on SPP/SPPL proteases and in particular highlight recently identified substrates and the functional and/or (patho)-physiological implications of these cleavage events. Based on this, we aim to provide an overview of the current open questions in the field. These are connected to the regulation of these proteases at the cellular level but also in context of disease and patho-physiological processes. Furthermore, the interplay with other proteostatic systems capable of degrading membrane proteins is beginning to emerge., (© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

More than 20 years ago, signal peptide peptidase (SPP) and its homologues, the signal peptide peptidase-like (SPPL) proteases have been identified based on their sequence similarity to presenilins, a related family of intramembrane aspartyl proteases. Other than those for the presenilins, no high-resolution structures for the SPP/SPPL proteases are available. Despite this limitation, over the years bioinformatical and biochemical data have accumulated, which altogether have provided a picture of the overall structure and topology of these proteases, their localization in the cell, the process of substrate recognition, their cleavage mechanism, and their function. Recently, the artificial intelligence-based structure prediction tool AlphaFold has added high-confidence models of the expected fold of SPP/SPPL proteases. In this review, we summarize known structural aspects of the SPP/SPPL family as well as their substrates. Of particular interest are the emerging substrate recognition and catalytic mechanisms that might lead to the prediction and identification of more potential substrates and deeper insight into physiological and pathophysiological roles of proteolysis., (© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Signal-Peptide Peptidase Like-3 (SPPL3) is an intramembrane cleaving aspartyl protease that causes secretion of extracellular domains from type-II transmembrane proteins. Numerous Golgi-localized glycosidases and glucosyltransferases have been identified as physiological SPPL3 substrates. By SPPL3 dependent processing, glycan-transferring enzymes are deactivated inside the cell, as their active site-containing domain is cleaved and secreted. Thus, SPPL3 impacts on glycan patterns of many cellular and secreted proteins and can regulate protein glycosylation. However, the characteristics that make a substrate a favourable candidate for SPPL3-dependent cleavage remain unknown. To gain insights into substrate requirements, we investigated the function of a GxxxG motif located in the transmembrane domain of N-acetylglucosaminyltransferase V (GnTV), a well-known SPPL3 substrate. SPPL3-dependent secretion of the substrate's ectodomain was affected by mutations disrupting the GxxxG motif. Using deuterium/hydrogen exchange and NMR spectroscopy, we studied the effect of these mutations on the helix flexibility of the GnTV transmembrane domain and observed that increased flexibility facilitates SPPL3-dependent shedding and vice versa. This study provides first insights into the characteristics of SPPL3 substrates, combining molecular biology, biochemistry, and biophysical techniques and its results will provide the basis for better understanding the characteristics of SPPL3 substrates with implications for the substrates of other intramembrane proteases., (© 2022. The Author(s).)

While chimeric antigen receptor (CAR) T cells targeting CD19 can cure a subset of patients with B cell malignancies, most patients treated will not achieve durable remission. Identification of the mechanisms leading to failure is essential to broadening the efficacy of this promising platform. Several studies have demonstrated that disruption of CD19 genes and transcripts can lead to disease relapse after initial response; however, few other tumor-intrinsic drivers of CAR T cell failure have been reported. Here we identify expression of the Golgi-resident intramembrane protease Signal peptide peptidase-like 3 (SPPL3) in malignant B cells as a potent regulator of resistance to CAR therapy. Loss of SPPL3 results in hyperglycosylation of CD19, an alteration that directly inhibits CAR T cell effector function and suppresses anti-tumor cytotoxicity. Alternatively, over-expression of SPPL3 drives loss of CD19 protein, also enabling resistance. In this pre-clinical model these findings identify post-translational modification of CD19 as a mechanism of antigen escape from CAR T cell therapy., (© 2022. The Author(s).)

Sensing of pathogens by pattern recognition receptors (PRR) is critical to initiate protective host defence reactions. However, activation of the immune system has to be carefully titrated to avoid tissue damage necessitating mechanisms to control and terminate PRR signalling. Dectin-1 is a PRR for fungal β-glucans on immune cells that is rapidly internalised after ligand-binding. Here, we demonstrate that pathogen recognition by the Dectin-1a isoform results in the formation of a stable receptor fragment devoid of the ligand binding domain. This fragment persists in phagosomal membranes and contributes to signal transduction which is terminated by the intramembrane proteases Signal Peptide Peptidase-like (SPPL) 2a and 2b. Consequently, immune cells lacking SPPL2b demonstrate increased anti-fungal ROS production, killing capacity and cytokine responses. The identified mechanism allows to uncouple the PRR signalling response from delivery of the pathogen to degradative compartments and identifies intramembrane proteases as part of a regulatory circuit to control anti-fungal immune responses., (© 2022. The Author(s).)

We report on in vitro wound-healing and cell-growth studies under the influence of radio-frequency (rf) cell stimuli. These stimuli are supplied either by piezoactive surface acoustic waves (SAWs) or by microelectrode-generated electric fields, both at frequencies around 100 MHz. Employing live-cell imaging, we studied the time- and power-dependent healing of artificial wounds on a piezoelectric chip for different cell lines. If the cell stimulation is mediated by piezomechanical SAWs, we observe a pronounced, significant maximum of the cell-growth rate at a specific SAW amplitude, resulting in an increase of the wound-healing speed of up to 135 ± 85% as compared to an internal reference. In contrast, cells being stimulated only by electrical fields of the same magnitude as the ones exposed to SAWs exhibit no significant effect. In this study, we investigate this effect for different wavelengths, amplitude modulation of the applied electrical rf signal, and different wave modes. Furthermore, to obtain insight into the biological response to the stimulus, we also determined both the cell-proliferation rate and the cellular stress levels. While the proliferation rate is significantly increased for a wide power range, cell stress remains low and within the normal range. Our findings demonstrate that SAW-based vibrational cell stimulation bears the potential for an alternative method to conventional ultrasound treatment, overcoming some of its limitations., Competing Interests: The authors declare no competing interest.

Intramembrane proteolysis is more than a mechanism to "clean" the membranes from proteins no longer needed. By non-reversibly modifying transmembrane proteins, intramembrane cleaving proteases hold key roles in multiple signaling pathways and often distinguish physiological from pathological conditions. Signal peptide peptidase (SPP) and signal peptide peptidase-like proteases (SPPLs) recently have been associated with multiple functions in the field of signal transduction. SPP/SPPLs together with presenilins (PSs) are the only two families of intramembrane cleaving aspartyl proteases known in mammals. PS1 or PS2 comprise the catalytic center of the γ-secretase complex, which is well-studied in the context of Alzheimer's disease. The mammalian SPP/SPPL family of intramembrane cleaving proteases consists of five members: SPP and its homologous proteins SPPL2a, SPPL2b, SPPL2c, and SPPL3. Although these proteases were discovered due to their homology to PSs, it became evident in the past two decades that no physiological functions are shared between these two families. Based on studies in cell culture models various substrates of SPP/SPPL proteases have been identified in the past years and recently-developed mouse lines lacking individual members of this protease family, will help to further clarify the physiological functions of these proteases. In this review we concentrate on signaling roles of mammalian intramembrane cleaving aspartyl proteases. In particular, we will highlight the signaling roles of PS via its substrates NOTCH, VEGF, and others, mainly focusing on its involvement in vasculature. Delineating also signaling pathways that are affected and/or controlled by SPP/SPPL proteases. From SPP's participation in tumor progression and survival, to SPPL3's regulation of protein glycosylation and SPPL2c's control over cellular calcium stores, various crossovers between proteolytic activity of intramembrane proteases and cell signaling will be described., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Papadopoulou and Fluhrer.)

Ectodomain (EC) shedding defines the proteolytic removal of a membrane protein EC and acts as an important molecular switch in signaling and other cellular processes. Using tumor necrosis factor (TNF)α as a model substrate, we identify a non-canonical shedding activity of SPPL2a, an intramembrane cleaving aspartyl protease of the GxGD type. Proline insertions in the TNFα transmembrane (TM) helix strongly increased SPPL2a non-canonical shedding, while leucine mutations decreased this cleavage. Using biophysical and structural analysis, as well as molecular dynamic simulations, we identified a flexible region in the center of the TNFα wildtype TM domain, which plays an important role in the processing of TNFα by SPPL2a. This study combines molecular biology, biochemistry, and biophysics to provide insights into the dynamic architecture of a substrate's TM helix and its impact on non-canonical shedding. Thus, these data will provide the basis to identify further physiological substrates of non-canonical shedding in the future., Competing Interests: The authors declare no competing interests., (© 2020 The Authors.)

Intramembrane proteolysis describes the cleavage of substrate proteins within their hydrophobic transmembrane segments. Several families of intramembrane proteases have been identified including the aspartyl proteases Signal peptide peptidase (SPP) and its homologues, the SPP-like (SPPL) proteases SPPL2a, SPPL2b, SPPL2c and SPPL3. As presenilin homologues, they employ a similar catalytic mechanism as the well-studied γ-secretase. However, SPP/SPPL proteases cleave transmembrane proteins with a type II topology. The characterisation of SPP/SPPL-deficient mouse models has highlighted a still growing spectrum of biological functions and also promoted the substrate discovery of these proteases. In this review, we will summarise the current hypotheses how phenotypes of these mouse models are linked to the molecular function of the enzymes. At the cellular level, SPP/SPPL-mediated cleavage events rather provide specific regulatory switches than unspecific bulk proteolysis. By this means, a plethora of different cell biological pathways is influenced including signal transduction, membrane trafficking and protein glycosylation.

The lectin-like oxidized LDL receptor 1 (LOX-1) is a key player in the development of atherosclerosis. LOX-1 promotes endothelial activation and dysfunction by mediating uptake of oxidized LDL and inducing pro-atherogenic signaling. However, little is known about modulators of LOX-1-mediated responses. Here, we show that the function of LOX-1 is controlled proteolytically. Ectodomain shedding by the metalloprotease ADAM10 and lysosomal degradation generate membrane-bound N-terminal fragments (NTFs), which we identified as novel substrates of the intramembrane proteases signal peptide peptidase-like 2a and b (SPPL2a/b). SPPL2a/b control cellular LOX-1 NTF levels which, following self-association via their transmembrane domain, can activate MAP kinases in a ligand-independent manner. This leads to an up-regulation of several pro-atherogenic and pro-fibrotic targets including ICAM-1 and the connective tissue growth factor CTGF. Consequently, SPPL2a/b-deficient mice, which accumulate LOX-1 NTFs, develop larger and more advanced atherosclerotic plaques than controls. This identifies intramembrane proteolysis by SPPL2a/b as a novel atheroprotective mechanism via negative regulation of LOX-1 signaling., (© 2019 Mentrup et al.)

Signal peptide peptidase (SPP) and the four homologous SPP-like (SPPL) proteases constitute a family of intramembrane aspartyl proteases with selectivity for type II-oriented transmembrane segments. Here, we analyse the physiological function of the orphan protease SPPL2c, previously considered to represent a non-expressed pseudogene. We demonstrate proteolytic activity of SPPL2c towards selected tail-anchored proteins. Despite shared ER localisation, SPPL2c and SPP exhibit distinct, though partially overlapping substrate spectra and inhibitory profiles, and are organised in different high molecular weight complexes. Interestingly, SPPL2c is specifically expressed in murine and human testis where it is primarily localised in spermatids. In mice, SPPL2c deficiency leads to a partial loss of elongated spermatids and reduced motility of mature spermatozoa, but preserved fertility. However, matings of male and female SPPL2c -/- mice exhibit reduced litter sizes. Using proteomics we identify the sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA2)-regulating protein phospholamban (PLN) as a physiological SPPL2c substrate. Accumulation of PLN correlates with a decrease in intracellular Ca 2+ levels in elongated spermatids that likely contribute to the compromised male germ cell differentiation and function of SPPL2c -/- mice., (© 2019 The Authors.)

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., (© 2018 The Authors.)

Signal peptide peptidase (SPP) and the four homologous SPP-like proteases SPPL2a, SPPL2b, SPPL2c and SPPL3 are GxGD-type intramembrane-cleaving proteases (I-CLIPs). In addition to divergent subcellular localisations, distinct differences in the mechanistic properties and substrate requirements of individual family members have been unravelled. SPP/SPPL proteases employ a catalytic mechanism related to that of the γ-secretase complex. Nevertheless, differential targeting of SPP/SPPL proteases and γ-secretase by inhibitors has been demonstrated. Furthermore, also within the SPP/SPPL family significant differences in the sensitivity to currently available inhibitory compounds have been reported. Though far from complete, our knowledge on pathophysiological functions of SPP/SPPL proteases, in particular based on studies in mice, has been significantly increased over the last years. Based on this, inhibition of distinct SPP/SPPL proteases has been proposed as a novel therapeutic concept e.g. for the treatment of autoimmunity and viral or protozoal infections, as we will discuss in this review. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Signal peptide peptidase (SPP) and the four related SPP-like (SPPL) proteases are homologues of the presenilins, which comprise the catalytic centre of the γ-secretase complex. SPP/SPPL proteases are GxGD-type aspartyl intramembrane proteases selective for substrates with a type II membrane topology. Subcellular localisations of SPP/SPPL proteases range from the early secretory pathway to the plasma membrane and the endocytic system. Similarly diverse are their functional roles at the cellular level covering the turnover of signal peptides and membrane proteins, a contribution to the ERAD pathway as well as the regulation of cellular protein glycosylation and certain signaling pathways. Much less well understood are the physiological functions of SPP/SPPL proteases in complex organisms. Whereas a major role of SPPL2a for homeostasis of B cells and dendritic cells has been documented in mice, in vivo functions of SPP and the other SPPLs remain largely elusive to date. SPP/SPPL proteases contribute to regulated intramembrane proteolysis (RIP), a sequential processing of single-spanning transmembrane proteins by an ectodomain sheddase and an intramembrane-cleaving protease (I-CLIP). However, recent studies reported the cleavage of tail-anchored and multi-pass membrane proteins by SPP as well as the capability of SPPL3 to accept substrates without a preceding ectodomain shedding. This revealed that the mechanistic properties within this family are more diverse than initially thought. With this review, we aim to provide an update on recent achievements in defining the function and (patho-) physiological relevance of SPP/SPPL proteases and to highlight open questions in the field., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

The Neuronal ceroid lipofuscinoses (NCLs) are a group of recessive disorders of childhood with overlapping symptoms including vision loss, ataxia, cognitive regression and premature death. 14 different genes have been linked to NCLs (CLN1-CLN14), but the functions of the proteins encoded by the majority of these genes have not been fully elucidated. Mutations in the CLN5 gene are responsible for the Finnish variant late-infantile form of NCL (Finnish vLINCL). CLN5 is translated as a 407 amino acid transmembrane domain containing protein that is heavily glycosylated, and subsequently cleaved into a mature soluble protein. Functionally, CLN5 is implicated in the recruitment of the retromer complex to endosomes, which is required to sort the lysosomal sorting receptors from endosomes to the trans-Golgi network. The mechanism that processes CLN5 into a mature soluble protein is currently not known. Herein, we demonstrate that CLN5 is initially translated as a type II transmembrane protein and subsequently cleaved by SPPL3, a member of the SPP/SPPL intramembrane protease family, into a mature soluble protein consisting of residues 93-407. The remaining N-terminal fragment is then cleaved by SPPL3 and SPPL2b and degraded in the proteasome. This work further characterizes the biology of CLN5 in the hopes of identifying a novel therapeutic strategy for affected children., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The presenilin homologue signal peptide peptidase-like 2a (SPPL2a) is an intramembrane protease of lysosomes/late endosomes which cleaves type II transmembrane proteins. We recently identified CD74, the invariant chain of the MHCII complex, as the first in vivo validated substrate of this protease. In endosomal compartments, CD74 undergoes sequential proteolysis leading to the generation of a membrane-bound N-terminal fragment (NTF) that requires cleavage by SPPL2a for its turnover. In SPPL2a(-/-) mice, this fragment accumulates in B-cells and significantly disturbs their maturation and functionality. To date, the substrate requirements of the protease SPPL2a have not been investigated. In the present study, we systematically analysed the molecular determinants of CD74 with regard to the intramembrane cleavage by SPPL2a. Using domain-exchange experiments, we demonstrate that the intracellular domain (ICD) of CD74 can be substituted without affecting cleavability by SPPL2a. Based on IP-MS analysis of the cleavage product, we report identification of the primary SPPL2a cleavage site between Y52 and F53 within the CD74 transmembrane segment. Furthermore, systematic alanine-scanning mutagenesis of the transmembrane and membrane-proximal parts of the CD74 NTF has been performed. We show that none of the analysed determinants within the CD74 NTF including the residues flanking the primary cleavage site are absolutely essential for SPPL2a cleavage. Importantly, we found that alanine substitution of helix-destabilizing glycines within the transmembrane segment and distinct residues within the luminal membrane-proximal segment led to a reduced efficiency of SPPL2a-mediated processing. Therefore we propose that elements within the transmembrane segment and the luminal juxtamembrane domain facilitate intramembrane proteolysis of CD74 by SPPL2a., (© 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Numerous membrane-bound proteins undergo regulated intramembrane proteolysis. Regulated intramembrane proteolysis is initiated by shedding, and the remaining stubs are further processed by intramembrane-cleaving proteases (I-CLiPs). Neuregulin 1 type III (NRG1 type III) is a major physiological substrate of β-secretase (β-site amyloid precursor protein-cleaving enzyme 1 (BACE1)). BACE1-mediated cleavage is required to allow signaling of NRG1 type III. Because of the hairpin nature of NRG1 type III, two membrane-bound stubs with a type 1 and a type 2 orientation are generated by proteolytic processing. We demonstrate that these stubs are substrates for three I-CLiPs. The type 1-oriented stub is further cleaved by γ-secretase at an ϵ-like site five amino acids N-terminal to the C-terminal membrane anchor and at a γ-like site in the middle of the transmembrane domain. The ϵ-cleavage site is only one amino acid N-terminal to a Val/Leu substitution associated with schizophrenia. The mutation reduces generation of the NRG1 type III β-peptide as well as reverses signaling. Moreover, it affects the cleavage precision of γ-secretase at the γ-site similar to certain Alzheimer disease-associated mutations within the amyloid precursor protein. The type 2-oriented membrane-retained stub of NRG1 type III is further processed by signal peptide peptidase-like proteases SPPL2a and SPPL2b. Expression of catalytically inactive aspartate mutations as well as treatment with 2,2'-(2-oxo-1,3-propanediyl)bis[(phenylmethoxy)carbonyl]-l-leucyl-l-leucinamide ketone inhibits formation of N-terminal intracellular domains and the corresponding secreted C-peptide. Thus, NRG1 type III is the first protein substrate that is not only cleaved by multiple sheddases but is also processed by three different I-CLiPs., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

During regulated intramembrane proteolysis (RIP) a membrane-spanning substrate protein is cleaved by an ectodomain sheddase and an intramembrane cleaving protease. A cytoplasmic intracellular domain (ICD) is liberated, which can migrate to the nucleus thereby influencing transcriptional regulation. Signal peptide peptidase-like (SPPL) 2a and 2b have been implicated in RIP of type II transmembrane proteins. Even though SPPL2a might represent a potential pharmacological target for treatment of B-cell-mediated autoimmunity, no specific and potent inhibitors for this enzyme are currently available. We report here on the first quantitative cell-based assay for measurement of SPPL2a/b activity. Demonstrating the failure of standard Gal4/VP16 reporter assays for SPPL2a/b analysis, we have devised a novel system employing β-galactosidase (βGal) complementation. This is based on detecting nuclear translocation of the proteolytically released substrate ICDs, which results in specific restoration of βGal activity. Utilizing this potentially high-throughput compatible new setup, we demonstrate nuclear translocation of the ICDs from integral membrane protein 2B (ITM2B), tumor necrosis factor (TNF) and CD74 and identify secreted frizzled-related protein 2 (SFRP2) as potential transcriptional downstream target of the CD74 ICD. We show that the presented assay is easily adaptable to other intramembrane proteases and therefore represents a valuable tool for the functional analysis and development of new inhibitors of this class of enzymes., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Signal peptide peptidase-like 3 (Sppl3) is a Golgi-resident intramembrane-cleaving protease that is highly conserved among multicellular eukaryotes pointing to pivotal physiological functions in the Golgi network which are only beginning to emerge. Recently, Sppl3 was shown to control protein N-glycosylation, when the key branching enzyme N-acetylglucosaminyltransferase V (GnT-V) and other medial/trans Golgi glycosyltransferases were identified as first physiological Sppl3 substrates. Sppl3-mediated endoproteolysis releases the catalytic ectodomains of these enzymes from their type II membrane anchors. Protein glycosylation is a multistep process involving numerous type II membrane-bound enzymes, but it remains unclear whether only few of them are Sppl3 substrates or whether Sppl3 cleaves many of them and thereby controls protein glycosylation at multiple levels. Therefore, to systematically identify Sppl3 substrates we used Sppl3-deficient and Sppl3-overexpression cell culture models and analyzed them for changes in secreted membrane protein ectodomains using the proteomics "secretome protein enrichment with click sugars (SPECS)" method. SPECS analysis identified numerous additional new Sppl3 candidate glycoprotein substrates, several of which were biochemically validated as Sppl3 substrates. All novel Sppl3 substrates adopt a type II topology. The majority localizes to the Golgi network and is implicated in Golgi functions. Importantly, most of the novel Sppl3 substrates catalyze the modification of N-linked glycans. Others contribute to O-glycan and in particular glycosaminoglycan biosynthesis, suggesting that Sppl3 function is not restricted to N-glycosylation, but also functions in other forms of protein glycosylation. Hence, Sppl3 emerges as a crucial player of Golgi function and the newly identified Sppl3 substrates will be instrumental to investigate the molecular mechanisms underlying the physiological function of Sppl3 in the Golgi network and in vivo. Data are available via ProteomeXchange with identifier PXD001672., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Intramembrane proteolysis has emerged as a key mechanism required for membrane proteostasis and cellular signaling. One of the intramembrane-cleaving proteases (I-CLiPs), γ-secretase, is also intimately implicated in Alzheimer's disease, a major neurodegenerative disease and leading cause of dementia. High-resolution crystal structural analyses have revealed that I-CLiPs harbor their active sites buried deeply in the membrane bilayer. Surprisingly, however, the key kinetic constants of these proteases, turnover number kcat and catalytic efficiency kcat/KM, are largely unknown. By investigating the kinetics of intramembrane cleavage of the Alzheimer's disease-associated β-amyloid precursor protein in vitro and in human embryonic kidney cells, we show that γ-secretase is a very slow protease with a kcat value similar to those determined recently for rhomboid-type I-CLiPs. Our results indicate that low turnover numbers may be a general feature of I-CLiPs., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

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., (© 2014 The Authors.)

The invariant chain (CD74) mediates targeting of the MHCII complex to endosomal compartments, where CD74 undergoes degradation allowing MHCII to acquire peptides. We demonstrated recently that intramembrane proteolysis of the final membrane-bound N-terminal fragment (NTF) of CD74 is catalyzed by Signal-peptide-peptidase-like 2a (SPPL2a) and that this process is indispensable for development and function of B lymphocytes in mice. In SPPL2a(-/-) mice, homeostasis of these cells is disturbed by the accumulation of the unprocessed CD74 NTF. So far, evidence for this essential role of SPPL2a is restricted to mice. Nevertheless, inhibition of SPPL2a has been suggested as novel approach to target B cells for treating autoimmunity. Here, we characterize human B cell lines with a homozygous microdeletion on chromosome 15. We demonstrate that this deletion disrupts the SPPL2a genomic locus and leads to loss of SPPL2a transcript. Lymphoblastoid cell lines from patients with this deletion exhibit absence of SPPL2a at the protein level and show an accumulation of the CD74 NTF comparable to B cells from SPPL2a(-/-) mice. By this means, we present evidence that the role of SPPL2a in CD74 proteolysis is conserved in human B cells and provide support for modulation of SPPL2a activity as a therapeutic concept., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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 similar 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 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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Intramembrane proteases are critically involved in signal transduction and membrane protein turnover. Signal-peptide-peptidase-like 2a (SPPL2A), a presenilin-homologue residing in lysosomes/late endosomes, cleaves type II-oriented transmembrane proteins. We recently identified SPPL2A as the enzyme controlling turnover and functions of the invariant chain (CD74) of the major histocompatibility complex II (MHCII) and demonstrated critical importance of this process for B cell development. Surprisingly, we found that SPPL2A is critical for formation of dental enamel. In Sppl2a knockout mice, enamel of the erupted incisors was chalky white and rapidly eroded after eruption. SPPL2A was found to be expressed in enamel epithelium during secretory and maturation stage amelogenesis. Mineral content of enamel in Sppl2a⁻/⁻ incisors was inhomogeneous and reduced by ∼20% compared to wild-type mice with the most pronounced reduction at the mesial side. Frequently, disruption of the enamel layer and localized detachment of the most superficial enamel layer was observed in the knockout incisors leading to an uneven enamel surface. In Sppl2a null mice, morphology and function of secretory stage ameloblasts were not noticeably different from that of wild-type mice. However, maturation stage ameloblasts showed reduced height and a characteristic undulation of the ameloblast layer with localized adherence of the cells to the outer enamel. This was reflected in a delayed and incomplete resorption of the proteinaceous enamel matrix. Thus, we conclude that intramembrane proteolysis by SPPL2A is essential for maintaining cellular homeostasis of ameloblasts. Because modulation of SPPL2A activity appears to be an attractive therapeutic target to deplete B cells and treat autoimmunity, interference with tooth enamel formation should be investigated as a possible adverse effect of pharmacological SPPL2A inhibitors in humans., (Copyright © 2013 American Society for Bone and Mineral Research.)

The successive events of shedding and regulated intramembrane proteolysis are known to comprise a fundamental biological process of type I and II membrane proteins (e.g. amyloid precursor protein, Notch receptor and pro-tumor necrosis factor-α). Some of the resulting fragments were shown to be involved in important intra- and extracellular signalling events. Although shedding of the human transferrin receptor-1 (TfR1) has been known for > 30 years and soluble TfR1 is an accepted diagnostic marker, the fate of the remaining N-terminal fragment (NTF) remains unknown. In the present study, we demonstrate for the first time that TfR1-NTF is subject to regulated intramembrane proteolysis and, using MALDI-TOF-TOF-MS, we have identified the cleavage site as being located C-terminal from Gly-84. We showed that the resulting C-terminal peptide is extracellularly released after regulated intramembrane proteolysis and it was detected as a monomer with an internal disulfide bridge. We further identified signal peptide peptidase-like 2a and mainly signal peptide peptidase-like 2b as being responsible for the intramembrane proteolysis of TfR1-NTF., (© 2013 The Authors Journal compilation © 2013 FEBS.)

The pleiotropic pro-inflammatory cytokine tumour necrosis factor alpha (TNF) is synthesised as a transmembrane protein that is subject to palmitoylation. In this study, the roles of this acylation on TNF-mediated biological effects were investigated. We found that the lipid raft partitioning of TNF is regulated by its palmitoylation. Furthermore, we demonstrated that this palmitoylation process interferes with the cleavage/degradation of TNF intracellular fragments but is not involved in the regulation of its ectodomain shedding. Moreover, we found that the palmitoylation of TNF hinders the binding of soluble TNF to TNFR1 and regulates the integration/retention of TNFR1 into lipid rafts. Finally, we demonstrate that the transmembrane forms of wild-type and palmitoylation-defective TNF interact differently with TNFR1 and regulate NFκB activity, Erk1/2 phosphorylation and interleukin-6 synthesis differently, strongly suggesting that palmitoylation of TNF is involved in the regulation of TNFR1 signalling. An evidence for the physiological intervention of this regulation is provided by the fact that, in macrophages, the binding of endogenous soluble TNF to TNFR1 is enhanced by inhibition of palmitoylation. Therefore, our data introduce the new concept that palmitoylation of TNF is one of the means by which TNF-producing cells regulate their sensitivity to soluble TNF., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Regulated intramembrane proteolysis is a central cellular process involved in signal transduction and membrane protein turnover. The presenilin homologue signal-peptide-peptidase-like 2a (SPPL2a) has been implicated in the cleavage of type 2 transmembrane proteins. We show that the invariant chain (li, CD74) of the major histocompatability class II complex (MHCII) undergoes intramembrane proteolysis mediated by SPPL2a. B lymphocytes of SPPL2a(-/-) mice accumulate an N-terminal fragment (NTF) of CD74, which severely impairs membrane traffic within the endocytic system and leads to an altered response to B cell receptor stimulation, reduced BAFF-R surface expression, and accumulation of MHCII in transitional developmental stage T1 B cells. This results in significant loss of B cell subsets beyond the T1 stage and disrupted humoral immune responses, which can be recovered by additional ablation of CD74. Hence, we provide evidence that regulation of CD74-NTF levels by SPPL2a is indispensable for B cell development and function by maintaining trafficking and integrity of MHCII-containing endosomes, highlighting SPPL2a as a promising pharmacological target for depleting and/or modulating B cells.

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.

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.

Nicastrin is a type I transmembrane glycoprotein, which is part of the high molecular weight γ-secretase complex. γ-Secretase is one of the key players associated with the generation of Alzheimer's disease pathology, since it liberates the neurotoxic amyloid β-peptide. Four proteins Nicastrin, anterior pharynx-defective-1 (Aph-1), presenilin enhancer-2 (Pen-2) and Presenilin are essential to form the active γ-secretase complex. Recently it has been shown, that Nicastrin has a key function in stabilizing the mature γ-secretase complex and may also be involved in substrate recognition. So far no structural data for the Nicastrin ectodomain or any other γ-secretase component are available. We therefore used Circular Dichroism (CD) spectroscopy to demonstrate that Nicastrin, similar to its homologues, the Streptomyces griseus aminopeptidase (SGAP) and the transferrin receptor (TfR), adopts a thermostable secondary structure. Furthermore, the Nicastrin ectodomain has an exceptionally high propensity to refold after thermal denaturation. These findings provide evidence to further support the hypothesis that Nicastrin may share evolutionary conserved properties with the aminopeptidase and the transferrin receptor family.

Presenilin (PS1 or PS2) is the catalytic component of the gamma-secretase complex, which mediates the final proteolytic processing step leading to the Alzheimer's disease (AD)-characterizing amyloid beta-peptide. PS is cleaved during complex assembly into its characteristic N- and C-terminal fragments. Both fragments are integral components of physiologically active gamma-secretase and harbor the two critical aspartyl residues of the active site domain. While the minimal subunit composition of gamma-secretase has been defined and numerous substrates were identified, the cellular mechanism of the endoproteolytic cleavage of PS is still unclear. We addressed this pivotal question by investigating whether familial AD (FAD)-associated PS1 mutations affect the precision of PS endoproteolysis in a manner similar to the way that such mutations shift the intramembrane cleavage of gamma-secretase substrates. We demonstrate that all FAD mutations investigated still allow endoproteolysis to occur. However, the precision of PS1 endoproteolysis is affected by PS1 mutations. Comparing the cleavage products generated by a variety of PS1 mutants revealed that specifically cleavages at positions 293 and 296 of PS1 are selectively affected. Systematic mutagenesis around the cleavage sites revealed a stepwise three amino acid spaced cleavage mechanism of PS endoproteolysis reminiscent to the epsilon-, zeta-, and gamma-cleavages described for typical gamma-secretase substrates, such as the beta-amyloid precursor protein. Our findings therefore suggest that intramembranous cleavage by gamma-secretase and related intramembrane-cleaving proteases may generally occur via stepwise endoproteolysis.

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 gamma-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 gamma-secretase and discuss common mechanisms but also point out a number of substantial differences.

Intramembrane proteolysis is now widely recognized as an important physiological pathway required for reverse signaling and membrane protein degradation. Aspartyl intramembrane cleaving proteases of the GXGD-type play an important regulatory role in health and disease. Besides gamma-secretase/presenilin, signal peptide peptidase (SPP) and SPP-like (SPPL) peptidases also belong to the family of GXGD-type aspartyl proteases. Although recently the first SPPL2a/b substrates have been identified, very little is known about substrate requirements, which allow them to be efficiently processed within the membrane. We demonstrate that similar to gamma-secretase substrates, intramembrane proteolysis of Bri2 (Itm2b) is greatly facilitated by an initial shedding event mediated by ADAM-10. Serial deletions revealed that the length of the ectodomain negatively correlates with efficient intramembrane proteolysis. Bri3 (Itm2c), which is highly homologous to Bri2, fails to be shed. Failure of shedding of Bri3 is accompanied by a lack of intramembrane proteolysis by SPPL2b. Surprisingly, a low molecular weight membrane-retained stub of Bri3 also fails to be processed by SPPL2b, indicating that shedding per se is not sufficient for subsequent intramembrane proteolysis. Extensive domain swapping analysis reveals that primary sequence determinants within the intracellular domain and the transmembrane domain together with short luminal juxtamembrane sequences are required for efficient intramembrane proteolysis.

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.

Gamma-secretase mediates the final proteolytic cleavage, which liberates amyloid beta-peptide (Abeta), the major component of senile plaques in the brains of Alzheimer disease patients. Therefore, gamma-secretase is a prime target for Abeta-lowering therapeutic strategies. gamma-Secretase is a protein complex composed of four different subunits, presenilin (PS), APH-1, nicastrin, and PEN-2, which are most likely present in a 1:1:1:1 stoichiometry. PS harbors the catalytically active site, which is critically required for the aspartyl protease activity of gamma-secretase. Moreover, numerous familial Alzheimer disease-associated mutations within the PSs increase the production of the aggregation-prone and neurotoxic 42-amino acid Abeta. Nicastrin may serve as a substrate receptor, although this has recently been challenged. PEN-2 is required to stabilize PS within the gamma-secretase complex. No particular function has so far been assigned to APH-1. The four components are sufficient and required for gamma-secretase activity. At least six different gamma-secretase complexes exist that are composed of different variants of PS and APH-1. All gamma-secretase complexes can exert pathological Abeta production. Assembly of the gamma-secretase complex occurs within the endoplasmic reticulum, and only fully assembled and functional gamma-secretase complexes are transported to the plasma membrane. Structural analysis by electron microscopy and chemical cross-linking reveals a water-containing cavity, which allows intramembrane proteolysis. Specific and highly sensitive gamma-secretase inhibitors have been developed; however, they interfere with the physiological function of gamma-secretase in Notch signaling and thus cause rather significant side effects in human trials. Modulators of gamma-secretase, which selectively affect the production of the pathological 42-amino acid Abeta, do not inhibit Notch signaling.

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.

Presenilins (PS) are critical components of the gamma-secretase complex that mediates cleavage of type I membrane proteins including the beta-amyloid precursor protein to generate the amyloid beta-peptide. In addition, PS1 interacts with beta-catenin and facilitates its metabolism. We demonstrate that phosphorylation of serines 353 and 357 by glycogen synthase kinase-3beta (GSK3beta) induces a structural change of the hydrophilic loop of PS1 that can also be mimicked by substitution of the phosphorylation sites by negatively charged amino acids in vitro and in cultured cells. The structural change of PS1 reduces the interaction with beta-catenin leading to decreased phosphorylation and ubiquitination of beta-catenin. The decreased interaction of PS1 with beta-catenin leads to stabilization of beta-catenin thereby increasing its nuclear signaling and the transcription of target genes, including c-MYC. Consistent with increased expression of c-myc, a PS1 mutant that mimics phosphorylated PS1 increased cell proliferation as compared with wild-type PS1. These results indicate a regulatory mechanism in which GSK3beta-mediated phosphorylation induces a structural change of the hydrophilic loop of PS1 thereby negatively modulating the formation of a ternary complex between beta-catenin, PS1, and GSK3beta, which leads to stabilization of beta-catenin.

Signal peptide peptidase (SPP) is an unusual aspartyl protease, which mediates clearance of signal peptides by proteolysis within the endoplasmic reticulum (ER). Like presenilins, which provide the proteolytically active subunit of the gamma-secretase complex, SPP contains a conserved GxGD motif in its C-terminal domain which is critical for its activity. While SPP is known to be an aspartyl protease of the GxGD type, several presenilin homologues/SPP-like proteins (PSHs/SPPL) of unknown function have been identified by database searches. In contrast to SPP and SPPL3, which are both restricted to the endoplasmic reticulum, SPPL2b is targeted through the secretory pathway to endosomes/lysosomes. As suggested by the differential subcellular localization of SPPL2b and SPPL3 distinct phenotypes were found upon antisense gripNA-mediated knockdown in zebrafish. spp and sppl3 knockdowns in zebrafish result in cell death within the central nervous system, whereas reduction of sppl2b expression causes erythrocyte accumulation in an enlarged caudal vein. Moreover, expression of D/A mutants of the putative C-terminal active sites of spp, sppl2,and sppl3 produced phenocopies of the respective knockdown phenotypes. These data suggest that all investigated PSHs/SPPLs are members of the novel family of GxGD aspartyl proteases. More recently, it was shown that SPPL2b utilizes multiple intramembrane cleavages to liberate the TNFalpha intracellular domain into the cytosol and to release the C-terminal counterpart into the lumen. These findings suggest common principles of intramembrane proteolysis by GxGD type aspartyl proteases. In this article, we will review the similarities of SPPs and gamma-secretase based on recent findings by us and others., (2007 S. Karger AG, Basel)