Intramembrane Proteolysis: Theme and Variations

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Science  20 Aug 2004:
Vol. 305, Issue 5687, pp. 1119-1123
DOI: 10.1126/science.1096187


Proteases that reside in cellular membranes apparently wield water to hydrolyze the peptide bonds of substrates despite their water-excluding environment. Although these intramembrane proteases bear little or no sequence resemblance to classical water-soluble proteases, they have ostensibly converged on similar hydrolytic mechanisms. Identification of essential amino acid residues of these proteases suggests that they use residue combinations for catalysis in the same way as their soluble cousins. In contrast to classical proteases, however, the catalytic residues of intramembrane proteases lie within predicted hydrophobic transmembrane domains. Elucidating the biological functions of intramembrane proteases, identifying their substrates, and understanding how they hydrolyze peptide bonds within membranes will shed light on the ways these proteases regulate crucial biological processes and contribute to disease.

Nature is an endless combination and repetition of very few laws. She hums the old well-known air through innumerable variations. –Ralph Waldo Emerson

In musical composition, a simple harmonized melody can be repeated many times with varied treatment so that at least some semblance of the general melodic or harmonic theme is evident. Analogous themes and variations occur in biochemistry, in which general mechanisms and conserved catalytic amino acid residues can be found among many different types of enzymes. For example, the study of proteases reveals how nature exploits a relatively simple process in myriad ways. Proteases catalyze the hydrolysis of the amide bonds that link amino acids together into peptides and proteins, and this process requires the concerted effort of key residues within the enzyme's active site. The context in which these conserved catalytic residues are found determines the substrate to be cleaved and can affect the rate, location, and timing of the substrate's hydrolysis.

Proteases are classified into four general types based on their catalytic residues and mechanism of action: (i) serine/threonine proteases, (ii) cysteine proteases, (iii) aspartyl proteases, and (iv) metalloproteases. Each of these four protease categories contains hundreds of known examples with representatives in all forms of life (1). Until recently, all proteases identified were water-soluble enzymes: Either the entire enzyme is normally found in an aqueous environment, or a membrane anchor holds down an otherwise aqueous protease. However, a new cadre of proteases has been discovered that seem to be embedded within the hydrophobic environment of the lipid bilayer. Despite the water-excluding environment of the lipid bilayer, these intramembrane proteases somehow are able to hydrolyze their transmembrane substrates (Fig. 1). The substrates themselves also are unusual: They are typically folded into an α-helix, a conformation that makes the backbone amide bonds inaccessible to nucleophilic attack because of steric hindrance by the amino acid side chains. The intramembrane-cleaving proteases (I-CLiPs) (2) therefore must create a microenvironment for water and the hydrophilic residues needed for catalysis, then bend or unwind their substrates to make the amide bonds susceptible to hydrolysis. Nevertheless, despite the novelty of being membrane-embedded and cleaving transmembrane domains, the I-CLiPs apparently are variations on old, familiar themes found in protease biochemistry: The essential catalytic residues of these I-CHiPs are virtually the same as those found in aqueous proteases.

Fig. 1.

Membrane topology of I-CLiPs and their substrates. (A) S2P contains conserved HEXXH and LDG (74) motifs found in metalloproteases. SREBP is first cleaved by S1P in the luminal loop. The regulatory domain (Reg) interacts with the cholesterol-sensing SCAP protein to ensure that S1P proteolysis occurs only when cholesterol levels are low. Subsequent intramembrane proteolysis releases this transcription factor, which then switches on expression of genes required for cholesterol and fatty acid synthesis. (B) Presenilin is processed into two pieces, an N-terminal fragment (NTF, dark orange) and a C-terminal fragment (CTF, light orange) that remain associated. Each fragment donates one aspartate essential for γ-secretase activity. APP and other substrates are first cleaved in the extracellular domain (APP by β-secretase, Notch by a metalloprotease), and the remnant is cleaved twice within the membrane by γ-secretase to produce the Aβ peptide of Alzheimer's disease (secreted) and the intracellular domain (freed into the cytosol). Inset: Presenilin interacts with three other membrane proteins (nicastrin, Aph-1, and Pen-2) to form active γ-secretase. [The model is redrawn from Fraering et al. (45).] Although the stoichiometry of these components is unclear, evidence suggests that two presenilin molecules lie within a single γ-secretase complex (53). NCT, nicastrin. (C) SPP, like presenilin, contains two aspartates that are essential for protease activity. Signal peptides are removed from membrane proteins by signal peptidase (SP) and these peptides are released from the membrane by SPP-mediated intramembrane proteolysis. (D) Rhomboids contain a conserved serine, histidine, and asparagines that compose a putative catalytic triad of a serine protease. Rhomboid-1 cleaves within the transmembrane region of the Drosophila EGF-like growth factor Spitz.

The S2P Family: Variation on a Metalloprotease Theme

The first discovery of an I-CLiP arose from studies on the regulation of sterol and fatty acid metabolism. Sterol regulatory element binding proteins (SREBPs) are transcription factors that promote the expression of genes involved in the synthesis of cholesterol and fatty acids (3). Coordinated gene expression is controlled through negative feedback inhibition by cholesterol to ensure that lipids and sterols are produced only when needed. SREBPs are synthesized as precursor proteins that contain four distinct domains: a domain exposed to the cytosol that binds DNA and activates transcription, two transmembrane regions, and a regulatory domain involved in feedback control by cholesterol (Fig. 1A). When cholesterol levels are high, the SREBP precursor is kept in the endoplasmic reticulum (ER) by a multipass membrane protein called SCAP (SREBP cleavage-activating protein) (4) that is bound to a small membrane protein called Insig (5). Reduced cholesterol levels result in dissociation of Insig from SCAP, allowing SCAP to shepherd SREBP to the Golgi apparatus. Proteolysis of SREBP in the Golgi results in release of the transcription factor and its translocation to the nucleus. Control of subcellular localization also regulates the intramembrane serine protease Rhomboid (Fig. 1D): The substrate Spitz is ushered from the ER to the Golgi by a membrane protein called Star. In this case, however, the released Spitz is not a transcription factor, but is rather a secreted growth factor (see below).

Proteolytic release of SREBPs occurs in two steps (Fig. 1A). First, the luminal loop between the two transmembrane regions is cleaved by the membrane-tethered Site-1 protease (S1P) (6). Release of the transcription factor requires subsequent cleavage by the Site-2 protease (S2P), which hydrolyzes an amide bond predicted to lie three residues within the transmembrane domain (7). The requirement for a prior proteolytic event is a common theme among the I-CLiPs. For example, γ-secretase processing of the amyloid β-protein precursor (APP) or the receptor Notch cannot take place without initial shedding of the extracellular domain from the membrane by other enzymes (Fig. 1B). Similarly, proteolysis of remnant signal peptides by signal peptide peptidase (SPP) requires prior processing of nascent membrane proteins by signal peptidase (Fig. 1C). Rhomboid, however, appears to be an exception: It cleaves full-length Spitz without the need for preceding proteolysis.

Complementation cloning has identified S2P as a multipass membrane protein containing a conserved HEXXH sequence characteristic of zinc metalloproteases (8). The two histidines and the glutamate are required for S2P activity, consistent with known metalloprotease biochemistry in which the two histidines coordinate with zinc and the zinc in turn activates the glutamate for interaction with the catalytic water. Further analysis led to the discovery of a conserved aspartate located ∼300 residues from the HEXXH sequence that is likewise critical for S2P activity and thought to be a third residue involved in zinc coordination (9). The involvement of zinc in S2P activity has not been demonstrated, and a cell-free assay for S2P activity has not yet been reported; therefore, S2P has not been directly shown to act as a protease. Nevertheless, extensive genetic analysis has not uncovered any other proteins required for S2P cleavage of SREBP.

Further support for the proteolytic function of S2P comes from the discovery of a family of related proteins in bacteria (10). These prokaryotic proteins are essential for the proteolysis of an otherwise membrane-bound transcription factor needed for sporulation. This factor, σk, controls gene expression in the mother cell after engulfment of the forespore. Cleavage of pro-σk and release of the transcription factor requires the multipass membrane protein SpoIVFB, and this protein likewise contains the HEXXH motif and a second conserved region with an aspartate, both of which are essential for proteolysis. Another bacterial S2P family member, YaeL in Escherichia coli, similarly requires HEXXH and a conserved aspartate to coordinate cell growth and cell division through intramembrane proteolysis of RseA, a factor critical for responding to extracytoplasmic stress (11). The membrane orientations of the substrates SREBP and σk are opposite to each other, correlating with that of their respective enzymes, S2P and SpoIVB, which apparently have opposite orientations (10). This implies that the catalytic region must align with the peptide substrate with proper relative directionality.

The α-helical conformation of the transmembrane substrate renders the amide bonds inaccessible to attack by a catalytic residue or water, requiring some bending or unwinding of the helix before proteolysis takes place. The SREBP substrate contains a conserved asparagine-proline (NP) sequence within its transmembrane region that is critical for proteolytic processing by S2P (12). These two residues have the lowest propensity to form α-helices, suggesting that the NP-containing SREBP transmembrane region may be metastable. After S1P cleavage and dissociation of the other transmembrane region, the NP sequence may facilitate unwinding of the residues immediately upstream, including the leucine-cysteine bond that gets cleaved. Unwinding may result in protrusion of this bond to the membrane surface and access by the active-site residues of S2P. Substrates for SPP and Rhomboid also require helix-disrupting residues, suggesting a general strategy for intramembrane proteolysis and a means of substrate specificity. In contrast, γ-secretase substrates do not appear to contain such residues; thus, this enzyme may be able to carry out helix bending or unwinding.

Presenilin Aspartyl Proteases: Composition for Quartets

A key step in the pathogenesis of Alzheimer's disease is APP proteolysis resulting in the formation of the amyloid-β peptide (Aβ), the principle protein component of the characteristic cerebral plaques of the disease (13). The N terminus of Aβ is produced from APP by the action of β-secretase, which leads to membrane shedding of the large luminal or extracellular APP domain (Fig. 1B). The 99-residue remnant (C99) is then cleaved in the middle of its transmembrane region by γ-secretase, releasing Aβ, and again near the inner leaflet at the S3 or ϵ site to release the APP intracellular domain (AICD). The requirement for prior cleavage is a common theme among the intramembrane proteases, with Rhomboid so far the only exception.

Two contemporaneous observations provided critical clues for the identification of the elusive γ-secretase, a subject of intense interest as a potential therapeutic target. First, knockout of presenilin genes eliminated γ-secretase cleavage of APP (14). Second, the types of compounds that could inhibit γ-secretase contained moieties typically found in aspartyl protease inhibitors (15). These findings led to the identification of two conserved transmembrane aspartates in the multipass presenilin that are critical for γ-secretase cleavage of APP (Fig. 1B), suggesting that presenilins might be the responsible aspartyl proteases (16). Presenilin is cut into two pieces, an N-terminal fragment (NTF) and a C-terminal fragment (CTF), the formation of which is gated by limiting cellular factor(s) (17). The NTF and CTF remain physically associated in a high–molecular weight complex and are metabolically stable (1820). These and other results suggest that the NTF-CTF heterodimer is the biologically active form (21). The NTF and CTF each contribute one of the critical and conserved aspartates, suggesting that the γ-secretase active site might be at the interface between these two presenilin fragments. In strong support of this hypothesis, transition-state analog inhibitors of γ-secretase (compounds designed to interact with the active site of the protease) were found to bind directly to presenilin NTF and CTF (22, 23). However, presenilins are apparently part of a larger multiprotein complex that constitutes γ-secretase (see below).

At the same time presenilins were discovered as susceptibility loci for Alzheimer's disease, they were also shown to be required for Notch signaling (24), a pathway essential for cell differentiation during development and beyond (25). After Notch is synthesized in the ER, the receptor is cleaved in its extracellular domain during its passage through the secretory pathway, and the two pieces so generated remain associated (26). Upon interaction with a cognate ligand, Notch becomes susceptible to a second extracellular proteolysis near the membrane (S2/β in Fig. 1B) (27, 28). The membrane-associated remnant is then cleaved at the S4/γ and S3/ϵ sites by γ-secretase (29), releasing the Notch intracellular domain (NICD). NICD translocates to the nucleus and activates transcription after associating with the nuclear partner CSL (30). Knock-in of a Notch-1 transmembrane mutation that greatly reduces presenilin-mediated proteolysis at S3 leads to a lethal phenotype in mice that is similar to that seen in Notch-1 knockout mice, indicating that efficient γ-secretase cleavage is essential for Notch signaling during development (31). By analogy, it has been suggested that the intracellular domain of APP is likewise implicated in cell signaling, but whether it acts in the nucleus (32) or at the cell membrane before its release by γ-secretase (33) is not yet resolved.

The highly conserved role of γ-secretase in Notch signaling and its importance in development enabled genetic screens in worms that identified two Notch modifiers, a single-pass membrane protein APH-2 (nicastrin) and a multipass protein APH-1 (3436). Nicastrin was independently isolated biochemically as a presenilin-associated protein and later found to be essential for γ-secretase processing of both APP and Notch (37, 38). A saturation screen in Caenorhabditis elegans for presenilin modifiers netted these proteins and added Pen-2. All four proteins (presenilin, nicastrin, Aph-1, and Pen-2) associate with one another (39, 40) and with an immobilized γ-secretase inhibitor (40, 41). Moreover, their coexpression increased γ-secretase activity in both Drosophila and mammalian cells (39, 40) and reconstituted activity in yeast (42). Because yeast have no such protease activity and contain no apparent orthologs of these metazoan proteins, these findings strongly suggest that this quartet of proteins is necessary and sufficient for γ-secretase activity. γ-Secretase is so far unique among intramembrane proteases in being composed of several different proteins: S2P, SPP, and Rhomboid do not appear to require other proteins for the proteolytic event per se. Coexpression, RNA interference, and the identification of assembly intermediates suggest the order in which these four subunits come together (39, 43, 44), and partial dissociation of the protease complex with detergent offers a model for how these subunits interact (Fig. 1B, inset) (45). Nicastrin and Aph-1 together can stabilize full-length presenilin, and the final addition of Pen-2 apparently triggers presenilin endoproteolysis and γ-secretase activity (39). Pen-2 is also required to stabilize the presenilin subunits (46). However, the specific biochemical functions of these presenilin cofactors are presently enigmatic.

Since the discovery that Notch is cleaved by γ-secretase, a plethora of other substrates have been identified, including Erb-B4, E- and N-cadherins, CD44, the low-density lipoprotein receptor, Nectin-1, and the Notch ligands Delta and Jagged (47). Knowledge of the cellular functions of these proteolytic events vary, but in the case of N-cadherin, the produced intracellular domain associates with the transcriptional activator CREB binding protein (CBP) and promotes its migration to the cytosol and degradation by the proteasome (48). Although cellular function can be ascribed in some cases, the ability of γ-secretase to cleave so many different substrates and its apparently poor sequence specificity raise the question of whether a major role of this enzyme is to serve as a general degrading protease for membrane-bound protein remnants. Indeed, γ-secretase appears to be unique among intramembrane proteases in its ability to process so many different substrates: S2P, SPP, and Rhomboid all display some degree of sequence specificity near their respective sites of proteolysis. The broad substrate recognition by γ-secretase is likely related to the fact that, unlike the other intramembrane proteases, the enzyme does not require helix-breaking residues near the cleavage sites within the substrates.

Exactly how do substrates interact with the enzyme? For proteolysis to occur within the boundaries of the membrane, the active site would be expected to be inside the protease complex, sequestering the catalytic water and hydrophilic aspartates from the lipid hydrocarbon tails. As a corollary, the enzyme should have a substrate docking site on the outer surface of the complex, because the substrates are themselves confined to two-dimensional movement within the bilayer. Evidence for such a docking site includes the fact that an APP substrate coelutes with γ-secretase components upon affinity isolation with an immobilized inhibitor directed to the protease active site (41). Moreover, biophysical methods that allow detection of APP-presenilin interactions in cells demonstrate that the interaction occurs in the presence of an active site– directed inhibitor (49). This interaction, however, is prevented by a helical peptide that mimics the APP transmembrane domain and blocks γ-secretase activity by a unique mechanism (50, 51), perhaps by binding to the docking site. Important unresolved issues include the location of the docking site on the protease complex [co-immunoprecipitation of C99 with truncated presenilins suggests that the first and eighth transmembrane regions may contribute to this site (52)], the substrate path from docking site to active site, and how substrate conformation is altered during the enzymatic process. Other I-CLiPs would be predicted to contain substrate docking sites for similar reasons, but this hypothesis remains untested.

An alternative model for γ-secretase that could potentially address these considerations is that the active site forms not within a single presenilin but at the interface of two presenilin molecules composing a dimer. Recent evidence suggests that two presenilin molecules lie at the core of the γ-secretase complex. This includes the coimmunoprecipitation of differently tagged presenilins and the crosslinking of two NTF fragments by modified γ-secretase inhibitors (53). This idea is corroborated by findings on a presenilin-related protein, signal peptide peptidase (see below). Whether S2Ps or Rhomboids are dimers is unknown: Perhaps this is a particular characteristic of the aspartyl I-CLiPs. How the two presenilins might interact within the complex and what this means for substrate processing are important questions for future studies. The issue of the stoichiometry of the γ-secretase complex members is presently unclear. Estimates of the complex size range from 250 kD (40) to 2 MD (54), although most studies suggest 400 to 600 kD (5557). This could mean that some or all components are found at least twice in a single complex or that unidentified modulator proteins are present. These possibilities are not mutually exclusive.

SPP Aspartyl Proteases: Variation on a Variation

The concept of presenilin as the catalytic component for γ-secretase was considerably strengthened when signal peptide peptidase (SPP) was found to be a similar intramembrane aspartyl protease. SPP clears remnant signal peptides from the membrane after their production by signal peptidase (Fig. 1C). However, this process apparently also plays a role in immune surveillance, during which signal peptides from the major histocompatibility complex (MHC) type I molecules are cleaved by SPP and the peptide products are presented on the cell surface as an indication to natural killer cells that MHC synthesis is proceeding normally (58). In addition, SPP is exploited by the hepatitis C virus for the maturation of its core protein, suggesting that this protease may be a suitable target for antiviral therapy (59). SPP was identified by affinity labeling with a peptidomimetic inhibitor, and the protein sequence displayed parallels with presenilin (Fig. 1C) (60). SPP contains two conserved aspartates, each predicted to lie in the middle of a transmembrane domain, and the aspartate-containing sequences resemble those found in presenilins. The predicted topology of SPP also resembles that of presenilins, placing the key aspartates in the same relative position to each other in the membrane. As with S2P compared with its bacterial relatives, the orientation of the aspartate-containing transmembrane domains of SPP is apparently opposite to that of presenilins, again in correlation with the orientation of SPP substrates, which is opposite that of γ-secretase substrates. Before the identification of SPP, a computational search for presenilin-like proteins netted an entire family of so-called presenilin homologs (PSHs) (61); however, it is not yet clear if all of these proteins have catalytic activity.

SPP appears to be less complicated than γ-secretase. Expression of human SPP in yeast reconstituted the protease activity, suggesting that the protein has activity on its own and does not require other mammalian protein cofactors (60). Moreover, unlike presenilins, SPP is not processed into two pieces. Thus, SPP may be a more tractable enzyme for understanding this type of intramembrane aspartyl protease and may shed light on γ-secretase structure and function. Indeed, the catalytic sites of the two proteases appear remarkably similar: their activities are inhibited by some of the same active site– directed peptidomimetics (51, 62). SPP forms a homodimer very rapidly in cells, and this dimer is stable enough to allow isolation and analysis (63). Moreover, this dimer can be specifically labeled by a transition-state analog inhibitor, suggesting that the dimer is catalytically active. The functional importance of this dimer and how it is assembled remains to be determined. However, another SPP-like protein, PSH4, also forms a stable homodimer (64), suggesting that this may be a general property of the PSH family. In terms of substrate recognition, however, SPP does display an important difference with γ-secretase: the apparent requirement for helix-breaking residues that should facilitate the ability of the enzyme to access the site of hydrolysis (65). In this respect, SPP shows more similarity to S2P and Rhomboid.

Rhomboid Serine Proteases: Playing Solo?

The study of a conserved growth factor signaling pathway also led to intramembrane proteolysis. Epidermal growth factor (EGF) receptor ligands are synthesized as single-pass membrane proteins, but signaling requires proteolytic release and secretion of the ligand for interaction with its cognate receptor. In vertebrates, this is accomplished by membrane-tethered metalloproteases. Genetic analysis in Drosophila, however, identified two essential players, dubbed Star and Rhomboid-1, in the proteolysis of the EGF ortholog Spitz. No other components are apparently required. Full-length Spitz remains in the ER until it is ushered by Star to the Golgi apparatus, where it encounters Rhomboid-1 (66). Rhomboid-mediated proteolysis in the Golgi is then followed by secretion for intercellular communication. But how does Rhomboid allow cleavage of Spitz?

Mutational analysis of conserved nonglycine residues revealed a requirement for a serine, a histidine, and an asparagine, which together might serve as a catalytic triad typically found in serine proteases (Fig. 1D) (67). These three residues are predicted to reside about the same depth within the membrane and thus have the potential to interact with each other. Consistent with this idea, the cleavage site of Spitz was estimated to be at an equivalent depth in the transmembrane region, and Spitz cleavage was sensitive only to serine protease inhibitors. Moreover, a careful analysis of concentration dependence revealed that expression of catalytic amounts of Rhomboid-1 still allowed Spitz proteolysis. Taken together, these findings suggest that Rhomboid-1 is a novel intramembrane serine protease.

What determines Rhomboid substrate specificity, and how is this proteolytic event regulated? Most of the Spitz transmembrane region could be swapped with that of a non-substrate protein without affecting cleavage by Rhomboid; however, the N-terminal quarter of the transmembrane region is critical for substrate recognition (68). Indeed, incorporation of this substrate motif into Delta allowed this Notch ligand to be processed by Rhomboid. Further examination of the substrate motif led to the tentative identification of a critical glycine-alanine, suggesting that, as with S2P and SPP, Rhomboid seems to require helix-destabilizing residues within the transmembrane domain of its substrates. Rhomboid activity is distinguished from that of the other I-CLiPs because Rhomboid does not require prior substrate cleavage by another protease. Rhomboid regulation apparently occurs mainly by translocation of the substrate from the Golgi to the ER (mediated by Star) and control of Rhomboid transcription.

Like S2P, Rhomboid genes have been conserved throughout evolution. In spite of overall low homology with eukaryotic Rhomboids, a number of bacterial Rhomboids are capable of cleaving Drosophila Rhomboid substrates, and mutation of the putative catalytic triad residues abolished protease activity, illustrating the evolutionary conservation of the serine protease function of Rhomboid (69). The natural substrates for the bacterial Rhomboids are unknown. As for substrates of eukaryotic Rhomboid-1 homologs, two mitochondrial membrane proteins have been identified as substrates for yeast Rhomboid RBD1 (7072). RBD1-mediated release of one of these substrates is essential for remodeling the mitochondrial membrane, and the human ortholog of RBD1, PARL, could restore substrate proteolysis, proper growth rates, and mitochondrial morphology in a yeast RBD1 mutant (71), suggesting that the role of these Rhomboids in mitochondrial function has been evolutionarily conserved. This finding expands the role of intramembrane proteolysis beyond cell signaling, implicating such proteases in a broad range of biological functions.

Conclusions and Perspective

I-CLiPs are multipass membrane enzymes that apparently hydrolyze transmembrane substrates, and these proteases possess essential residues that reside within the boundaries of the lipid bilayer. But apart from this, what do they have in common? All appear to recapitulate the mechanisms of soluble proteases, although this has not been formally demonstrated. Such proof demands a high-resolution crystal structure, a primary challenge for this field of investigation. All I-CLiPs are predicted to contain an initial substrate docking site, but to date evidence for such a docking site has only been provided for γ-secretase. The I-CLiPs discovered so far are important biologically and are closely regulated, but the means of control vary. They are all involved in cell signaling, but some release specialized transcription factors (S2P), whereas others generate transcriptional modulators and/or contribute to degradation of their substrates (γ-secretase and SPP). Other I-CLiPs generate fragments that can act as immune system surveillance molecules (SPP) or secreted growth factors (Rhomboid). Membrane topology seems to dictate the types of substrates that can be cleaved, but this concept remains speculative. Most I-CLiPs appear to require helix-breaking residues near the cleavage sites of their substrates, although γ-secretase may be an exception.

The next step is to identify substrates for I-CLiP family members whose functions are unknown. For instance, although an entire family of PSHs and Rhomboids have been discovered, natural substrates are known only for a handful of these proteins. The conservation of putative catalytic residues implies conservation of proteolytic function, but the search for substrates is far from trivial. A computational approach for sequence motifs that are apparently required for substrate proteolysis by Rhomboids led to identification of adhesion proteins in the protozoan pathogen Toxoplasma as potential substrates (68). A similar approach led to the identification of the anticoagulant cell-surface protein thrombospondin as a substrate for a human rhomboid (73), but again, the physiological significance of this finding is unknown. Another key issue is understanding the specific mechanisms of these proteases (for example, elucidating conformational changes that take place in both enzyme and substrate during proteolysis, determining if these changes require the input of energy, and identifying enzyme residues that directly interact with substrate). Such understanding should facilitate the identification of new members of this protease class and the design of specific inhibitors. New members will likely display interesting variations on themes already evident from the study of I-CLiPs, enzymes that have transposed the well-known tune of proteolysis into the context of cellular membranes.

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