In vitro cleavage of tumor necrosis factor α (TNFα) by Signal-Peptide-Peptidase-like 2b (SPPL2b) resembles mechanistic principles observed in the cellular context.

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.

Signal peptide peptidase-like 2b modulates the amyloidogenic pathway and exhibits an Aß-dependent expression in Alzheimer's disease.

Alzheimer's disease (AD) is a multifactorial disorder driven by abnormal amyloid ß-peptide (Aß) 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 Aß cascade. Exogenous Aß42 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 AppNL-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 AppNL-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 Aß 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 Aß-induced upregulation of SPPL2b may enhance Aß production in a vicious cycle, further aggravating Aß pathology. Therefore, SPPL2b emerges as a potential anti-Aß drug target.

The role of SPP/SPPL intramembrane proteases in membrane protein homeostasis.

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.

Structure and function of SPP/SPPL proteases: insights from biochemical evidence and predictive modeling.

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.

Helical stability of the GnTV transmembrane domain impacts on SPPL3 dependent cleavage.

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.

Antigen glycosylation regulates efficacy of CAR T cells targeting CD19.

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.

Phagosomal signalling of the C-type lectin receptor Dectin-1 is terminated by intramembrane proteolysis.

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 ß-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.

Vibration enhanced cell growth induced by surface acoustic waves as in vitro wound-healing model.

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.

Non-canonical Shedding of TNFα by SPPL2a Is Determined by the Conformational Flexibility of Its Transmembrane Helix.

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.

Signaling Functions of Intramembrane Aspartyl-Proteases.

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.

Physiological functions of SPP/SPPL intramembrane proteases.

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.

Atherogenic LOX-1 signaling is controlled by SPPL2-mediated intramembrane proteolysis.

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.

The intramembrane protease SPPL2c promotes male germ cell development by cleaving phospholamban.

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 Ca2+-ATPase (SERCA2)-regulating protein phospholamban (PLN) as a physiological SPPL2c substrate. Accumulation of PLN correlates with a decrease in intracellular Ca2+ levels in elongated spermatids that likely contribute to the compromised male germ cell differentiation and function of SPPL2c-/- mice.

Signal peptide peptidase-like 2c impairs vesicular transport and cleaves SNARE proteins.

Members of the GxGD-type intramembrane aspartyl proteases have emerged as key players not only in fundamental cellular processes such as B-cell development or protein glycosylation, but also in development of pathologies, such as Alzheimer's disease or hepatitis virus infections. However, one member of this protease family, signal peptide peptidase-like 2c (SPPL2c), remains orphan and its capability of proteolysis as well as its physiological function is still enigmatic. Here, we demonstrate that SPPL2c is catalytically active and identify a variety of SPPL2c candidate substrates using proteomics. The majority of the SPPL2c candidate substrates cluster to the biological process of vesicular trafficking. Analysis of selected SNARE proteins reveals proteolytic processing by SPPL2c that impairs vesicular transport and causes retention of cargo proteins in the endoplasmic reticulum. As a consequence, the integrity of subcellular compartments, in particular the Golgi, is disturbed. Together with a strikingly high physiological SPPL2c expression in testis, our data suggest involvement of SPPL2c in acrosome formation during spermatogenesis.

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.

Signal peptide peptidase and SPP-like proteases - Possible therapeutic targets?

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.

Latest emerging functions of SPP/SPPL intramembrane proteases.

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.

CLN5 is cleaved by members of the SPP/SPPL family to produce a mature soluble protein.

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.

Substrate determinants of signal peptide peptidase-like 2a (SPPL2a)-mediated intramembrane proteolysis of the invariant chain CD74.

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.