Title: Proteolytic ectodomain shedding of membrane proteins in mammals—hardware, concepts, and recent developments
Abstract: Review5 July 2018free access Proteolytic ectodomain shedding of membrane proteins in mammals—hardware, concepts, and recent developments Stefan F Lichtenthaler Corresponding Author [email protected] orcid.org/0000-0003-2211-2575 German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Neuroproteomics, Klinikum rechts der Isar, School of Medicine, and Institute for Advanced Study, Technical University Munich, Munich, Germany Munich Center for Systems Neurology (SyNergy), Munich, Germany Search for more papers by this author Marius K Lemberg Corresponding Author [email protected] orcid.org/0000-0002-0996-1268 Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany Search for more papers by this author Regina Fluhrer Corresponding Author [email protected] orcid.org/0000-0002-9778-4643 German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Biomedizinisches Centrum (BMC), Ludwig-Maximilians University of Munich, Munich, Germany Search for more papers by this author Stefan F Lichtenthaler Corresponding Author stefan.lichtenthal[email protected] orcid.org/0000-0003-2211-2575 German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Neuroproteomics, Klinikum rechts der Isar, School of Medicine, and Institute for Advanced Study, Technical University Munich, Munich, Germany Munich Center for Systems Neurology (SyNergy), Munich, Germany Search for more papers by this author Marius K Lemberg Corresponding Author [email protected] orcid.org/0000-0002-0996-1268 Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany Search for more papers by this author Regina Fluhrer Corresponding Author [email protected] orcid.org/0000-0002-9778-4643 German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Biomedizinisches Centrum (BMC), Ludwig-Maximilians University of Munich, Munich, Germany Search for more papers by this author Author Information Stefan F Lichtenthaler *,1,2,3, Marius K Lemberg *,4 and Regina Fluhrer *,1,5 1German Center for Neurodegenerative Diseases (DZNE), Munich, Germany 2Neuroproteomics, Klinikum rechts der Isar, School of Medicine, and Institute for Advanced Study, Technical University Munich, Munich, Germany 3Munich Center for Systems Neurology (SyNergy), Munich, Germany 4Center for Molecular Biology of Heidelberg University (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany 5Biomedizinisches Centrum (BMC), Ludwig-Maximilians University of Munich, Munich, Germany *Corresponding author. Tel: +49 89 440046426; E-mail: [email protected] *Corresponding author. Tel: +49 6221 545889; E-mail: [email protected] *Corresponding author. Tel: +49 89 440046505; E-mail: [email protected] EMBO J (2018)37:e99456https://doi.org/10.15252/embj.201899456 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract 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. Introduction Membrane proteins are essential for health and disease and have a large variety of fundamental physiological functions. Levels of individual membrane proteins and their functions are tightly controlled through different mechanisms, including post-translational modifications such as proteolytic ectodomain shedding (or briefly shedding). Shedding is a form of limited proteolysis and thus an irreversible post-translational modification (Fig 1). During the shedding process, a protease (referred to as sheddase) cleaves a membrane protein substrate close to or within its transmembrane (TM) domain, resulting in release of the soluble extracellular domain (ectodomain) from the membrane and a fragment that remains bound to the membrane (Fig 1) (Kapeller et al, 1973; Black, 1980a; Ehlers & Riordan, 1991). Some sheddases are also referred to as secretases (Selkoe, 1990), as the cleaved substrate ectodomain may be secreted. Figure 1. Sheddases trigger the release of a wide range of proteins from the membrane(A) Canonical sheddases cleave single-pass TM membrane proteins in their luminal juxtamembrane region, thereby releasing ectodomains from their membrane-integral domains. Ectodomain refers to that part of the protein that is found on the extracellular side of the membrane—in case that the protein localizes to the plasma membrane—or within the lumen of organelles of the secretory and endocytic pathway, which is topologically equivalent to the extracellular space. (B) GPI-anchored proteins are separated from their lipid modification by cleavage within the C-terminus of the protein. (C) Dual-pass and polytopic membrane proteins (not shown) can be cleaved in loops and ectodomains (not shown). Neuregulin-1 type III is cleaved at two sites in its loop domain, thereby releasing a bioactive peptide from its membrane anchors. (D) As a variation of canonical shedding, in regulated intramembrane proteolysis (RIP), the sheddase-generated membrane-integral fragment is further processed in the plane of the lipid bilayer, releasing an intracellular domain and a short extracellular peptide fragment. In this case, shedding is the first step of two subsequent proteolytic cleavages. (E) Non-canonical sheddases cleave their substrate in or close to the TM domain without requiring any preceding cleavage. Depending on the site of cleavage, the intracellular fragment is released from the lipid bilayer or stays anchored by a slightly shortened TM domain. Download figure Download PowerPoint Shedding is best understood in mammals, where it has emerged as a key cellular mechanism to control not only abundance, but also activation and inactivation of membrane proteins, for example, through release of membrane-bound growth factors and cytokines or through degradation of surface receptors and cell adhesion proteins (e.g., Black et al, 1997; Moss et al, 1997; Peschon et al, 1998; Colombo et al, 2018). Given the large number of substrates, shedding influences many processes in development, physiology, and disease, such as connectivity in the nervous system (e.g., Hattori et al, 2000), cholesterol homeostasis (Sakai et al, 1996, 1998), Alzheimer's disease (e.g., Vassar et al, 1999), and inflammatory disorders (e.g., Black et al, 1997; Moss et al, 1997). Yet, for other membrane proteins, shedding may simply be a mechanism of protein turnover and may not be coupled to (patho)physiological consequences. In the literature, the term shedding sometimes also refers to the non-proteolytic release of membrane proteins and the release of vesicles from the plasma membrane (Black, 1980a,1980b), which are different molecular processes and are not covered here. This review gives an overview of ectodomain shedding, starting with an operational definition of shedding, then highlighting the involved proteases and substrates and their regulation, and finally describing the functional consequences and medical implications of shedding. The aim of this review article is to use selected examples a) to demonstrate that shedding is a fundamental cell biological process, b) to illustrate general principles of shedding that emerge from the comparison of different sheddase families, and c) to highlight new trends and conceptual advances in the field. Definition of ectodomain shedding Shedding occurs for single-span TM proteins (Fig 1A), GPI-anchored proteins (Fig 1B), and proteins with two or more TM domains (Fig 1C). For several substrates, shedding is the first proteolytic cleavage and may be followed by additional proteolytic cleavage(s) within the TM segment. Both cleavages together are conceptually referred to as “regulated intramembrane proteolysis” (Fig 1D) (Brown et al, 2000; Lichtenthaler et al, 2011). In all cases, shedding refers to the release of a protein's ectodomain from the membrane. Initially, the term ectodomain shedding was used in a narrow manner with regard to cellular localization (plasma membrane) (Kapeller et al, 1973; Black, 1980a; Arribas et al, 1996), the position of the cleavage sites within the substrates (lumenal juxtamembrane domain) and the number of proteases and substrates involved (Ehlers & Riordan, 1991; Massague & Pandiella, 1993). However, several key studies over the past years, which will be discussed in more detail below, demonstrated that shedding occurs in all cellular organelles of the secretory and endocytic pathway, happens both outside and even within the substrates’ TM domain (Fig 1E), and is mediated by many more proteases than previously thought, including membrane-bound, intramembrane, and even soluble proteases. Moreover, it now has become clear that shedding impacts on many, if not all single-span membrane proteins and numerous polytopic TM proteins at some stage during their lifetime. In order to reflect these new findings, we propose a broader definition of shedding. Ectodomain shedding is the proteolytic release of the bulk or even the entire ectodomain of a mature membrane protein into the luminal or extracellular space and often alters the substrate's function. Depending on the cellular compartment where shedding occurs, the ectodomain is released into the extracellular space (at the plasma membrane) or into the lumen of the organelles (e.g., Golgi or endosome), which is topologically equivalent to the extracellular space (Schatz & Dobberstein, 1996), and from where it may subsequently be secreted into the extracellular space. The proteolytic cut occurs within the extracellular or luminal juxtamembrane (membrane-proximal) region or within the TM anchor of a membrane protein substrate. Cleavage sites within the juxtamembrane region are typically at a short distance of often 10 - 35 amino acids from the TM segment, but more distant cleavage sites are possible and, in fact, the exact cleavage sites have only been determined for few shedding substrates (e.g., summarized for ADAMs and BACE1 in Caescu et al, 2009; Yan, 2017). Several other proteolytic events in cells, such as removal of a signal peptide by signal peptidase (Blobel & Dobberstein, 1975) and proteolytic cleavages by mitochondrial AAA proteases (Levytskyy et al, 2017), formally share similarities to ectodomain shedding, but will not be discussed in this review, as they occur either during protein biosynthesis but not on the mature protein (signal peptidase), or do not occur in the secretory or endocytic pathway (mitochondria). Hardware: canonical sheddases The human genome contains nearly 600 protease-encoding genes (Lopez-Otin & Bond, 2008), and an increasing number of them are recognized to act as sheddases, with some having many shedding substrates and others so far having only a single substrate reported to undergo shedding, as will be discussed below. Proteases are commonly considered as sheddases if they cleave their substrates in the luminal juxtamembrane domain with a short distance to the membrane-anchoring domain (Ehlers & Riordan, 1991). We refer to these proteases as canonical sheddases (Table 1) to distinguish them from the more recently described non-canonical sheddases (described below) that cleave within a substrates’ TM domain or at the membrane boundary (Table 2). Canonical sheddases are typically themselves membrane-bound, but more and more soluble proteases, such as matrix metalloproteases (MMPs), are also reported to mediate shedding, as will be discussed below. In Tables 1 and 2, we additionally distinguish between sheddases whose primary function is ectodomain release and proteases that mostly have non-shedding functions, but can additionally act as secondary or “part-time” sheddases. Some of the best-characterized sheddases, such as “a disintegrin and metalloprotease 10” (ADAM10), ADAM17 (also known as TACE for TNFα-converting enzyme), and “β-site APP cleaving enzyme” (BACE1), have many shedding substrates and act as “full-time” sheddases. In contrast, other proteases, such as matrix metalloproteases (MMPs) or pro-protein convertases, have mostly non-shedding functions, because they cleave soluble proteins (MMPs) or remove pro-peptides (pro-protein convertases) without shedding the whole ectodomain. As will be discussed below, such proteases are increasingly found to additionally act as sheddases on a few selected substrates. This qualifies them as “part-time” sheddases. Table 1. List of canonical, mammalian sheddase families Sheddase type Protease family Protease type Cellular localization References Full-time sheddases ADAM proteases (metalloproteases) ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM19, ADAM20, ADAM21, ADAM28, ADAM30, ADAM33 Membrane-anchored, type I Late secretory pathway and plasma membrane Pruessmeyer and Ludwig (2009), Saftig and Lichtenthaler (2015), Weber and Saftig (2012), Zunke and Rose-John (2017) BACE proteases (aspartyl proteases) BACE1, BACE2 Membrane-anchored, type I Trans-Golgi network and endosomes Barao et al (2016), Dislich and Lichtenthaler (2012), Vassar et al (2014), Yan (2017) Site-1 protease (serine protease), also known as SKI-1 or S1P Membrane-anchored, type I Golgi Seidah et al (2017), Seidah and Prat (2012) Part-time sheddases Meprin β (metalloprotease) Membrane-anchored, type I Broder and Becker-Pauly (2013) MT-MMPs (metalloproteases) MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, MT6-MMP, also named MMP14-MMP17, MMP24, MMP25 Membrane-anchored, type I or GPI-anchored Late secretory pathway and plasma membrane Hayashida et al (2010), Itoh (2015) Pro-protein convertases (serine proteases) PCSK1/3, PCSK2, furin, PCSK4, PCSK5/6, PACE4, PCSK7 and PCSK9 Membrane-anchored, type I or soluble Late secretory pathway and plasma membrane Seidah et al (2017), Seidah and Prat (2012) Transmembrane serine proteases Matriptase, Matriptase-2, Matriptase-3, Polyserase-1, Corin, Hepsin, TMPRSS2, TMPRSS3, TMPRSS4, MSPL, Spinesin, Enteropeptidase, HAT, DESC1, TMPRSS11A, HAT-like 4, HAT-like 5 Membrane-anchored, type II Szabo and Bugge (2011), Tanabe and List (2017) Matrix metalloproteases (MMPs) MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP19, MMP20, MMP21, MMP23, MMP26, MMP27, MMP28 Soluble Extracellular space Freitas-Rodriguez et al (2017), Klein and Bischoff (2011), Peixoto et al (2012) Legumain (δ-secretase) cysteine protease Soluble Zhang et al (2016) Cathepsin S and L (cysteine protease) Soluble Extracellular space Sobotic et al (2015) Family members with known shedding function are indicated in bold and italics. Selected review articles that typically describe the whole protease family are given. Some articles also contain lists of identified substrates. For proteases with few shedding substrates, the original study is cited. Site-1 protease belongs to the family of pro-protein convertases, but is listed separately to highlight that it acts as a full-time sheddase in contrast to the other members of the same family. Table 2. List of non-canonical, mammalian sheddase families Sheddase type Protease family and members Protease type Cellular localization References Full-time sheddases Rhomboid proteases (serine proteases) RHBDL1, RHBDL2, RHBDL3, RHBDL4 Integral multi-pass TM protein Golgi (RHBDL1), plasma membrane (RHBDL2), endosomes (RHBDL3), ER (RHBDL4) Freeman (2014), Lemberg (2013) SPP/SPPL family (aspartyl proteases) SPPL3 SPP, SPPL2a, SPPL2b, SPPL2c, (SPPL3 is a major sheddase; SPP acts as a sheddase only in exceptional cases) Integral multi-pass TM protein ER (SPP), lysosomes (SPPL2a), cell surface (SPPL2b), ER (SPPL2c), Golgi (SPPL3) Kuhn et al (2015), Voss et al (2014) Boname et al (2014), Chen et al (2014) Part-time sheddases Presenilin/γ-secretase (aspartyl protease) Presenilin-1, Presenilin-2 (γ-secretase acts as a sheddase only in exceptional cases) Integral multi-pass TM protein, Plasma membrane, endosomes Laurent et al (2015), Schauenburg et al (2018) Family members with known shedding function are indicated in bold and italics. Selected review articles are given that typically describe the whole protease family. Some articles also contain lists of identified substrates. For proteases with few shedding substrates, the original study is cited. The following paragraphs first describe the canonical sheddases, starting with membrane-bound sheddases, followed by soluble ones. As some sheddases have more than 100 substrates, only selected substrates are listed, in particular those that have been validated under protease-deficient conditions or through in vivo studies. ADAM10 and ADAM17 The best-characterized canonical sheddases, ADAM10 and ADAM17, are most likely active in the trans-Golgi network (TGN), in later secretory pathway compartments, and at the plasma membrane (Fig 2). More than 100 substrates for ADAM10 and similar numbers for ADAM17 have been identified in different tissues and cells using candidate testing and advanced proteomics, although not all of them have been validated under physiological conditions and with in vitro assays (for detailed lists, see e.g., Pruessmeyer & Ludwig, 2009; Weber & Saftig, 2012; Kawahara et al, 2014; Saftig & Lichtenthaler, 2015; Kuhn et al, 2016; Zunke & Rose-John, 2017). Selected substrates are highlighted in Table 3. Substrate cleavage by ADAM10 often happens constitutively under non-stimulated conditions, whereas substrate shedding by ADAM17 is mostly observed, when cells are stimulated, either with physiological activators or phorbol esters such as PMA (phorbol-12-myristat-13-acetat, also known as TPA, 12-O-tetradecanoylphorbol-13-acetat). Figure 2. Cellular localization of sheddasesCatalytically active canonical and non-canonical sheddases not only localize to the cell surface but also to different subcellular compartments. The localization of selected canonical (red) and non-canonical (green) sheddases is indicated. Download figure Download PowerPoint Table 3. Examples of substrates of selected canonical and non-canonical, mammalian sheddases.aa Only such substrates are listed that have been validated, preferentially under sheddase-inactivating conditions or through in vivo experiments. Sheddase Selected substrates References ADAM10 Notch, APP, PrP, EGF, ephrin-A5, N-cadherin, DR6, CD23 Altmeppen et al (2011), Colombo et al (2018), Hartmann et al (2002), Janes et al (2005), Jorissen et al (2010), Kuhn et al (2016, 2010), Pan and Rubin (1997), Postina et al (2004), Reiss et al (2005), Sahin et al (2004), Suh et al (2013), Vincent et al (2001), Weskamp et al (2006) ADAM17 TGFα, TNFα, IL6R, amphiregulin, epiregulin, heparin-binding EGF-like growth factor, L-selectin, TNFR2 Althoff et al (2000), Black et al (1997), Ludwig et al (2005), Moss et al (1997), Peschon et al (1998), Sahin et al (2004) BACE1 APP, NRG1, SEZ6, CHL1 Dislich et al (2015), Esterhazy et al (2011), Hemming et al (2009), Kuhn et al (2012), Stutzer et al (2013), Zhou et al (2012) BACE2 TMEM27, PMEL17 Esterhazy et al (2011), Rochin et al (2013) Meprin β CD99, APP Arolas et al (2012), Bedau et al (2017), Jefferson et al (2011) MT1-MMP CD44, syndecan, RANKL Endo et al (2003), Hikita et al (2006), Kajita et al (2001), Tam et al (2004) MT3-MMP NgR1 Ferraro et al (2011), Sanz et al (2018) MT5-MMP N-cadherin, APP Baranger et al (2016), Folgueras et al (2009), Porlan et al (2014), Willem et al (2015) MMP9, MMP12 N-cadherin, NLG1 Dwivedi et al (2009), Peixoto et al (2012) PC7 Transferrin receptor Guillemot et al (2013), Wang and Pei (2001) Site-1 protease (S1P, SKI-1) SREBP, ATF6, GlcNAc-1-phosphotransferase Marschner et al (2011), Sakai et al (1998), Seidah et al (2017), Ye et al (2000) RHBDL2 Thrombomodulin, EGF, BCAM, Spint-1, CLCP1 Adrain et al (2011), Cheng et al (2011), Johnson et al (2017), Lohi et al (2004) RHBDL4 ERAD substrates, APP Fleig et al (2012), Johnson et al (2017), Paschkowsky et al (2016) SPP XBP1u Chen et al (2014) SPPL3 GnT-V and other glycan-modifying enzymes Kuhn et al (2015), Voss et al (2014) γ-secretase BCMA Laurent et al (2015) Sheddases with many substrates and examples of recent studies are listed. a Only such substrates are listed that have been validated, preferentially under sheddase-inactivating conditions or through in vivo experiments. ADAM10 is essential for ligand-dependent shedding of the Notch1 receptor and its subsequent signaling (Pan & Rubin, 1997; Bozkulak & Weinmaster, 2009; van Tetering et al, 2009), which is required for embryonic development but also in several adult tissues (Sato et al, 2012; reviewed in Alabi et al, 2018). Additionally, it acts as α-secretase for the amyloid precursor protein (APP) thereby preventing the generation of the neurotoxic amyloid-β peptide (Lammich et al, 1999; Postina et al, 2004; Jorissen et al, 2010; Kuhn et al, 2010; Suh et al, 2013), and is, thus, considered a drug target for Alzheimer's disease. Numerous phenotypes have been identified in ADAM10-deficient mice, for example, in the nervous system (Prox et al, 2013), but the many substrates still need to be assigned to the individual phenotypes and functions. It is also possible that some phenotypes are not just caused by the loss of cleavage of a single, but of multiple substrates simultaneously. ADAM17 has a key function in tissue homeostasis through cleavage of several members of the epidermal growth factor (EGF) receptor (EGFR) ligand family, including TGFα (Peschon et al, 1998), and may be a drug target for EGFR-dependent tumors (e.g., Schmidt et al, 2018). Additionally, ADAM17 has a fundamental role in inflammation by being the major sheddase for the cytokine tumor necrosis factor α (TNFα). Thus, ADAM17 is considered a major drug target for inflammatory diseases such as sepsis, rheumatoid arthritis, and inflammatory bowel disease (reviewed in Rose-John, 2013). Besides ADAM10 and ADAM17, the ADAM family has ten more members with proven or assumed proteolytic activity (Weber & Saftig, 2012), but only few (or in some cases no) physiological substrates for them have been identified to date (Table 1). BACE1 and BACE2 Another class of sheddases in the endomembrane system are BACE1 and BACE2 (Fig 2), which were initially identified as APP sheddases (Hussain et al, 1999; Sinha et al, 1999; Vassar et al, 1999; Yan et al, 1999, 2001; Lin et al, 2000; Fluhrer et al, 2002). Candidate approaches and, more recently, proteomic studies identified more than 40 substrates and substrate candidates each for BACE1 and BACE2 (see Table 3 for selected substrates) (Hemming et al, 2009; Esterhazy et al, 2011; Kuhn et al, 2012; Zhou et al, 2012; Stutzer et al, 2013; Dislich et al, 2015), but many of them have not yet been validated under physiological conditions. Since BACE1 acts as the major β-secretase for APP and catalyzes formation of the pathogenic amyloid-β peptide, several inhibitors targeting BACE1 are currently in advanced clinical trials for Alzheimer's disease. However, BACE1 has additional functions in neurobiology, including in myelination, muscle spindle formation and maintenance, synapse formation, and axon targeting (Hu et al, 2006; Willem et al, 2006; Rajapaksha et al, 2011; Cao et al, 2012; Hitt et al, 2012; Cheret et al, 2013; Barao et al, 2015; Zhu et al, 2018). For most substrates, the functional consequences of their cleavage by BACE1 have not yet been investigated, largely for lack of tools such as antibodies targeted to the substrates’ ectodomains or intracellular domains, or because little is known about the substrates. While BACE1 is highly expressed in the nervous system, its homolog BACE2 is strongly expressed in pancreas (Vassar et al, 1999). In vivo experiments using BACE2-deficient mice revealed that BACE2 regulates pancreatic β-cell function and mass through cleavage of “transmembrane protein 27” (TMEM27) (Esterhazy et al, 2011), making BACE2 a potential drug target for diabetes, which needs to be further evaluated. Another BACE2 substrate is PMEL17, the cleavage of which is required for pigment production in melanocytes and thus, for pigmentation of hair, skin, and mucosa, at least in rodents (Rochin et al, 2013). Meprin β Meprin β is a homodimeric TM metalloprotease. Soluble and TM substrates as well as substrate candidates were identified proteomically from cell lines overexpressing or exposed to soluble meprin β (Bien et al, 2012; Jefferson et al, 2013). Several TM proteins were cleaved within their ectodomain at a large distance from the membrane, which is not seen as a shedding event, as a large part of the ectodomain remains. However, meprin β also acts as a sheddase and cleaves close to the TM domain in CD99 to promote transendothelial cell migration, and in APP, for which it acts as an alternative β-secretase (Jefferson et al, 2011; Arolas et al, 2012; Bedau et al, 2017). To what extent meprin β may contribute to Alzheimer's disease still needs to be explored in more detail. Membrane-type matrix metalloproteases (MT-MMPs) The six MT-MMPs are a subgroup of the larger MMP family. They are assumed to be active at the plasma membrane and are mostly known for their cleavage of soluble substrates (see Table 1), in particular extracellular matrix proteins, such as collagens and fibronectin (Itoh, 2015). Increasingly, they are reported to also act as canonical sheddases for TM proteins (see Table 3 and reviewed in Hayashida et al, 2010; Itoh, 2015), and more shedding substrates are likely to be identified in the future. For example, MT1-MMP sheds RANKL (Receptor activator of NF-kappaB ligand) and negatively regulates osteoclastogenesis (Hikita et al, 2006). MT3-MMP was recently shown to shed the GPI-anchored Nogo receptor 1, which promotes excitatory synapse formation in vitro and in vivo (Sanz et al, 2018). MT5-MMP sheds N-cadherin and controls peripheral thermal nociception, presumably through modulation of cell adhesion between mast cells and sensory fibers (Folgueras et al, 2009). MT5-MMP shedding of N-cadherin also controls adhesion of neuronal stem cells to ependymocytes and thereby stem cell quiescence versus proliferation (Folgueras et al, 2009; Porlan et al, 2014). MT5-MMP was recently furthermore identified as the APP η-secretase, and its inactivation reduced inflammation and amyloid pathology in an Alzheimer's disease mouse model (Willem et al, 2015; Baranger et al, 2016). However, the APP η-secretase cleavage is more distant (~120 amino acids) from the membrane than most other shedding events. It is not yet clear for all examples mentioned above how exactly the MT-MMPs contribute to the indicated (patho)physiological processes, and it is likely that more shedding functions of MT-MMPs will be discovered. Pro-protein convertases, including site-1-protease (S1P) Pro-protein convertases are a family of nine soluble and membrane-bound serine proteases that are commonly found in the TGN and later compartments of the secretory pathway (Fig 2). Several of them, such as furin, remove pro-peptides from soluble or membrane-bound inactive protein precursors (reviewed in Seidah & Prat, 2012), including ADAM and BACE proteases. These pro-peptide cleavages are not considered as shedding event, since they often occur several hundred amino acids distant from the substrates’ TM domains and, therefore, do not remove the majority of the substrates’ ectodomains. Yet, pro-protein convertases are increasingly reported to additionally act as sheddases for selected substrates. For example, PCSK7 sheds the transferrin receptor (Guillemot et al, 2013), whereas furin or another pro-protein convertase sheds MT5-MMP (Wang & Pei, 2001), with both cleavages occurring < 25 amino acids away from the substrates’ TM domains. Thus, while the functional consequences of these shedding events are not yet fully understood, pro-protein convertases can act as “part-time” sheddases. One family member, site-1 protease (S1P), also known as subtilins/kexin-isozyme 1, stands out from the other family members in that it functions primarily as a sheddase (reviewed in Seidah et al, 2017). Known substrates of this Golgi-resident protease include viral proteins as well as the latent transcription factors