In the RIP process, shedding occurs at a short, but relatively fixed distance from the membrane. Thus, it is not surprising that the ADAM and BACE proteases are themselves transmembrane proteins having their active site in their luminal domains. Over 30 different ADAMs have been identified in metazoans (reviewed in Edwards et al. 2008; Reiss and Saftig 2009). Many of them are proteolytically active and contain the typical amino acid consensus motif (HExGHxxGxxHD) required for zinc binding in the active site. Other ADAM members lack this motif and are assumed to be involved predominantly in cell adhesion. The human genome contains 21 ADAM family members, of which 12 (ADAM8, 9, 10, 12, 15, 17, 19, 20, 21, 28, 30, 33) are proteolytically active. The other ones lack critical amino acids in their active site, which would be required for proteolytic activity. It remains to be tested whether the inactive ADAMs additionally are able to inhibit the active ADAMs and thereby act as inhibitory or inactive ADAMs, similar to what has been suggested for the inactive rhomboid intramembrane proteases (Lemberg and Freeman 2007).
ADAM proteases are type I membrane proteins of about 750–900 amino acids in length. They consist of an N-terminal signal peptide, followed by a prodomain, the zinc-binding metalloprotease domain, a cysteine-rich domain, an epidermal growth factor (EGF) domain, the transmembrane domain and a cytoplasmic domain, which in many cases is proline-rich. An EGF domain is not found in ADAM10. A more detailed description of the ADAM protease domains, their structure and specific functions can be found in recent reviews (Edwards et al. 2008; Reiss and Saftig 2009). During transport through the secretory pathway the ADAMs are complex N-glycosylated. The prodomain is removed by proprotein convertases, such as furin, resulting in the active protease, which mediates proteolysis in the late compartments of the secretory pathway and at the plasma membrane. Importantly, ADAM protease-mediated shedding can occur for substrates on the same cell surface, but can also happen in trans, meaning that an ADAM protease on one cell surface can cleave a substrate residing on the neighboring cell surface. This has been shown for ADAM10 in ephrin-Eph receptor signaling (Janes et al. 2005) and may be a more generally occurring mechanism controlling shedding and the RIP process. Interestingly, ADAM10 itself is also subject to RIP. It is shed by ADAM9 and 15, followed by γ -secretase intramembrane proteolysis and translocation of the ADAM10 ICD into the nucleus, where it is found in nuclear speckles, thought to be involved in gene regulation (Parkin and Harris 2009; Tousseyn et al. 2009).
Among the known RIP substrates, the sheddase is often ADAM10, but in several cases also ADAM17. However, a generalization of this statement is difficult, given that for many RIP substrates the sheddase is not yet known and that many more RIP substrates are likely to be identified. Thus, it is well possible that other ADAMs or metalloproteases turn out to be additional major contributors to the RIP process.
The major substrates for ADAM10 and 17 during development have been identified in the corresponding protease-deficient mice. ADAM17-deficient mice show perinatal lethality and phenotypic changes, such as open eyelids, skin abnormalities and epithelial maturation defects in multiple organs (Peschon et al. 1998). These changes resemble the phenotype of mice lacking the EGF receptor or some of its ligands, such as transforming growth factor α (TGFα), which demonstrated that EGF receptor ligands are major ADAM17 substrates during development. ADAM10-deficient mice show a loss-of-Notch signaling phenotype and die during embryonic development at day 9.5 (Hartmann et al. 2002). Notch is a cell surface receptor that requires RIP for its signal transduction to control cellular differentiation. Ligand-binding induces Notch shedding, followed by γ-secretase mediated intramembrane proteolysis. This releases the Notch intracellular domain into the nucleus, where it acts as a transcriptional activator of Notch downstream target genes, such as hairy and enhancer of split (for review see Fortini and Bilder 2009; Kopan and Ilagan 2009). Recent work established ADAM10 as the relevant protease for the physiological ligand-induced Notch1 shedding and signaling, in agreement with the lack of Notch signaling in ADAM10 knock-out mice. However, under ligand-independent conditions, as it occurs for certain leukemia-linked Notch mutations, Notch cleavage is mediated by ADAM17 (Bozkulak and Weinmaster 2009; van Tetering et al. 2009). Because of the early lethality of ADAM10 and 17-deficient mice, conditional knock-out mice are currently being used to decipher further substrates as well as physiological and pathophysiological functions of both proteases (Horiuchi et al. 2007; Jorissen et al. 2010).
ADAM10 and 17 have a similar, but not identical, broad substrate specificity and can cleave peptides in vitro at the same peptide bonds, often after a positively charged amino acid at the P1 position (Caescu et al. 2009). This is in contrast to the more pronounced specificity in vivo. For example, in cells and in animals ADAM10, but not ADAM17 is the major sheddase for APP and Notch, as described above. This remarkable specificity in cells suggests the existence of as yet unknown factors, which control the protease specificity in the cellular environment. Generally, ADAM proteases cleave at a short distance from the membrane. For APP the principal determinant of the ADAM10-mediated α-secretase cleavage appears to be an α-helical conformation and the distance of 12 amino acids from the membrane (Sisodia 1992). Interestingly, for APP the initial α-secretase cleavage seems to be followed by a carboxypeptidase cleavage removing the C-terminal, positively charged lysine (Esch et al. 1990; Kuhn et al. 2010). While the functional relevance of this C-terminal truncation is not yet clear, it is interesting to note that the maturation of several prohormones also requires the removal of a C-terminal Lys or Arg by a carboxypeptidase (Steiner 1998).
Tissue inhibitors of metalloproteases (TIMPs) are soluble, secreted proteins, which physiologically inhibit ADAM proteases and matrix metalloproteases (MMPs) by binding to the metalloprotease domains. They show greater selectivity to ADAMs than to matrix metalloproteases (MMPs). TIMP1 inhibits ADAM10, but not ADAM17, whereas TIMP3 efficiently blocks ADAM17 and with a lower efficiency also ADAM10 (Amour et al. 1998, 2000). Small molecule inhibitors with selectivity to either ADAM10 or 17 have been developed (Ludwig et al. 2005). Therapeutically, such molecules are interesting for different diseases, such as inhibition of tumor necrosis factor α (TNFα) shedding in rheumatoid arthritis or for blocking RIP of Notch in leukemias. Clinical trials with ADAM10 and 17 inhibitors are ongoing, but several previous trials were halted because of mechanism-based liver toxicity.
The activity of ADAM proteases can be increased above its constitutive level by diverse stimuli (reviewed in Hayashida et al. 2010). This includes growth factors, cytokines, protein kinase C (PKC) activators, G-protein coupled receptors, calcium ionophores and other natural or experimental stimuli. In many cases, the exact underlying mechanisms are not yet fully elucidated. Interestingly, a recent study showed that phorbol-12-myristate-13-acetate (PMA) leads to the isomerization of disulfide bonds in ADAM17, thereby inducing a structural change and enhanced catalytic activity (Willems et al. 2010). The concept of a structural change induced by activation is also supported by a recent study demonstrating that a tight-binding hydroxamate inhibitor only binds and blocks ADAM17 in stimulated, but not in quiescent cells (Le Gall et al. 2010). Another recent study demonstrated that activated p38 MAPK phosphorylates the cytoplasmic domain of ADAM17 and that this is required for ADAM17-mediated ectodomain shedding of transforming growth factor α (TGFα) family ligands (Xu and Derynck 2010).
Apart from signaling pathways intracellular protein trafficking is increasingly appreciated as a mechanism to control the RIP process. The involvement of protein trafficking comes from the fact that the RIP proteases and the RIP substrates are membrane proteins. In contrast to soluble proteases and their substrates, the membrane proteins cannot freely diffuse, but must be present in close proximity within the same cellular compartment for proteolysis to occur. As a consequence, the amount of shedding and intramembrane proteolysis depends on the intracellular transport of substrate and protease. Details have been reviewed recently for the RIP process in general (Sannerud and Annaert 2009) or at the example of the α-secretase cleavage of APP (Lichtenthaler 2011).
BACE1 is a glycosylated type I membrane protein with 501 amino acids. The N-terminal signal peptide is followed by a prodomain, a catalytic domain comprising the two catalytic aspartic acid residues [amino acids 93–96 (DTGS in the single letter amino acid code) and amino acids 289–292 (DSGT in the single letter amino acid code)] characteristic for aspartyl proteases, a transmembrane domain and a short cytoplasmic tail. The crystal structure of the BACE1 ectodomain shows a conserved general folding of aspartyl proteases (Hong et al. 2000). Upon trafficking through the secretory pathway, the propeptide is cleaved by furin, leading to the active (mature) BACE1 protease (Bennett et al. 2000; Capell et al. 2000; Huse et al. 2000; Creemers et al. 2001). BACE1 forms dimers, which show a higher activity than the monomers (Schmechel et al. 2004; Westmeyer et al. 2004). BACE1 is ubiquitously expressed, with higher expression levels observed in brain and in neurons (Vassar et al. 1999), which explains why neurons are particularly vulnerable in AD because of the increased BACE1-mediated Αβ generation. BACE1 expression in mice is very high in the nervous system right after birth and then decreases to very low levels (Willem et al. 2006). BACE1 localizes to the Golgi, the trans-Golgi network and to the endosomes (Vassar et al. 1999; Capell et al. 2000; Huse et al. 2000). In vivo BACE1 seems to be specifically active within acidic cellular compartments such as the late Golgi and specifically endosomes and lysosomes. BACE1 activity has also been observed in other compartments, however, this seems to occur only upon over-expression of the protease (Huse et al. 2002).
BACE1 is a major drug target for AD, as its inhibition lowers Αβ generation. Potent BACE1 inhibitors have been developed, but mostly do not reach sufficiently high concentrations in the brain. Nevertheless, a first inhibitor is currently being tested in a phase II clinical trial for AD (Vassar et al. 2009). Furthermore, new generation BACE1 inhibitors with significantly improved pharmacokinetics have been developed recently, and may soon be tested in patients. Other preclinical strategies to inhibit BACE1 activity consist of targeting BACE1 with antibodies and with modified drugs, which are specifically delivered to endosomes, where BACE1 cleaves APP (Chang et al. 2007; Rajendran et al. 2008; Mitterreiter et al. 2010). Some of these drugs indirectly inhibit BACE1, by locally raising the membrane-proximal pH in the endosome to levels above the pH-optimum of 4.5 for BACE1.
In addition to APP, several other BACE1 substrates have been identified over the past few years, namely neuregulin-1, the P-selectin glycoprotein ligand-1, the amyloid precursor like proteins 1 and 2, the sialyltransferase ST6Gal I, β-subunits of voltage-gated sodium channels, the interleukin-1 receptor 2, and the low density lipoprotein receptor-related protein (Kitazume et al. 2002; Lichtenthaler et al. 2003; Li and Sudhof 2004; von Arnim et al. 2005; Wong et al. 2005; Willem et al. 2006; Kuhn et al. 2007). This demonstrates, that BACE1 has a broader role in the RIP process. However, these substrates should be discussed with some care, since only in a few cases, BACE1 deficiency could be directly related to functional consequences associated with the respective substrate. Moreover, a number of potential substrates were identified only upon their over-expression or over-expression of BACE1. So far clear phenotypic consequences of a BACE1 knockout could only be related to cleavage of neuregulin-1 type III (NRG1). BACE1 knockout mice show a prominent hypomyelination in the peripheral nervous system during postnatal development, which results from reduced processing of NRG1 (Hu et al. 2006; Willem et al. 2006). Interestingly, myelination of the peripheral nervous system occurs postnatally, which coincides with the significant up-regulation of BACE1 expression. NRG1 is believed to be a hairpin protein with two transmembrane domains located in the plasma membrane of axons. Signaling to its receptors (ErbB2/ErbB3) on Schwann cells is known to require endoproteolysis of the luminal/extracellular loop. This cleavage may specifically be required to allow access to the EGF domain. Indeed, unprocessed NRG1 accumulates in BACE1 knockout animals, demonstrating that NRG1 is currently the only in vivo verified BACE1 substrate with a physiological correlate. The remaining myelination observed in the PNS of homozygous BACE1 knockout mice is because of another sheddase probably cleaving in the vicinity of BACE1. The nature of this sheddase is currently unknown, however, it is likely that one of the ADAM proteases may be involved. NRG1 has also been linked to schizophrenia, and, indeed BACE1-deficient mice show schizophrenia-like behavioral changes (Savonenko et al. 2008). If the latter is related to the developmental phenotype remains however speculative. Additional functions of BACE1 in the CNS, such as epileptic seizures, are less well understood (Hitt et al. 2010; Hu et al. 2010).
Although several of the above listed BACE1 substrates are also cleaved by metalloproteases, such as ADAM proteases, and apparently such a cleavage may allow the remaining myelination observed in BACE1 knockout mice, both cleavages may also have fundamentally different functional consequences. In case of APP, the cleavage sites of BACE1 and ADAM10 are 16 amino acids apart. Recent evidence suggests that BACE1 cleavage generates a secreted APP ectodomain, which may act proapoptotically (Nikolaev et al. 2009), whereas the secreted ectodomain generated by ADAM10 appears to have a neurotrophic and neuroprotective function (Furukawa et al. 1996; Meziane et al. 1998). Additionally, BACE1 leads to Αβ generation, whereas ADAM10 cleavage occurs within the Αβ domain and thus prevents Αβ generation. In contrast to the ADAM proteases, which seem to be less dependent on specific amino acid motifs around the cleavage site, BACE1 has a more pronounced substrate specificity and prefers a leucine at the P1 position (Citron et al. 1995; Gruninger-Leitch et al. 2002). For APP the BACE1 cleavage site is further away from the membrane than the ADAM10 cleavage site, but this is not the case for all membrane proteins undergoing shedding by an ADAM protease and by BACE1. For example, for the cell adhesion protein P-selectin glycoprotein ligand-1 the cleavage site by BACE1 is closer to the membrane than the cleavage site by an α-secretase like protease (Lichtenthaler et al. 2003).
Expression of BACE1 is controlled by multiple mechanisms. Several transcription factors regulate BACE1 expression, such as nuclear factor kappa B (NFκB), peroxisome proliferator-activated receptor-γ (PPARγ) and Yin Yang-1 (YY1) (reviewed in Rossner et al. 2006). At the translational level three different mechanisms were identified to control BACE1 expression and affect the RIP-mediated Αβ generation. The 5′ untranslated region of the BACE1 mRNA strongly suppresses BACE1 translation (De Pietri Tonelli et al. 2004; Lammich et al. 2004; Rogers et al. 2004). Energy deprivation, which occurs in AD brains, is able to relieve the translational suppression, resulting in increased BACE1 levels and enhanced Αβ generation in mice (O’Connor et al. 2008). A second mechanism is a naturally occurring antisense RNA, which binds to the open reading frame of the BACE1 mRNA and increases BACE1 translation (Faghihi et al. 2008). Finally, microRNAs inhibit BACE1 mRNA translation (Hebert et al. 2008; Wang et al. 2008). All three mechanisms appear to be altered in AD, providing an explanation for the 2- to 5-fold increase in BACE1 protein levels observed in AD brains.