Chemical Tools for the Study of Intramembrane Proteases - American

Reviews pubs.acs.org/acschemicalbiology

Chemical Tools for the Study of Intramembrane Proteases Minh T. N. Nguyen,†,§ Tim Van Kersavond,†,§ and Steven H. L. Verhelst*,†,‡ †

Leibniz Institute for Analytical Sciences ISAS, e.V., Otto-Hahn-Str. 6b, 44227 Dortmund, Germany KU Leuven − University of Leuven, Department of Cellular and Molecular Medicine, Laboratory of Chemical Biology, Herestr. 49 Box 802, 3000 Leuven, Belgium

‡

ABSTRACT: Intramembrane proteases (IMPs) reside inside lipid bilayers and perform peptide hydrolysis in transmembrane or juxtamembrane regions of their substrates. Many IMPs are involved in crucial regulatory pathways and human diseases, including Alzheimer’s disease, Parkinson’s disease, and diabetes. In the past, chemical tools have been instrumental in the study of soluble proteases, enabling biochemical and biomedical research in complex environments such as tissue lysates or living cells. However, IMPs place special challenges on probe design and applications, and progress has been much slower than for soluble proteases. In this review, we will give an overview of the available chemical tools for IMPs, including activity-based probes, affinity-based probes, and synthetic substrates. We will discuss how these have been used to increase our structural and functional understanding of this fascinating group of enzymes, and how they might be applied to address future questions and challenges. n 1997, a staggering 161 years after the first description of a protease (pepsin),1 site-2 protease (S2P) was discovered as a first example of an intramembrane protease (IMP, sometimes also referred to as intramembrane cleaving protease or ICLiP).2 IMPs are membrane-embedded enzymes whose active sites are located below the surface of the lipid bilayer. Since the discovery of S2P, several other families of IMPs have been described. Their active site machineries and their mechanisms of peptide bond hydrolysis show surprising similarities to those of soluble protease families, although they are evolutionarily unrelatedan example of convergent evolution.3 Currently, four distinct mechanistic families of IMPs are known: (1) metallo IMPs, such as S2P2 (Figure 1A); (2) the recently discovered glutamyl IMPs, exemplified by Rce14 (Figure 1B); (3) serine IMPs, also known as rhomboid proteases5 (Figure 1C), part of a superfamily of rhomboid proteins,6 which contains a large number of pseudoproteases without proteolytic activity; (4) aspartyl IMPs (also known as GXGD proteases) with presenilin (PS; the active subunit of the γ-secretase complex)7−9 and signal peptide peptidase (SPP)10 as prime examples (Figure 1D). Both soluble proteases and IMPs catalyze the irreversible cleavage of protein substrates. A number of proteases in the human body play a role in food digestion and protein turnover, but many are involved in highly regulatory processes, such as apoptosis, blood coagulation, and antigen presentation. Controlled cleavage of specific substratesa result of the substrate specificity of a protease, regulation of protease activity, and colocalization of protease and substrates eventually leads to a downstream biological effect. Misregulation of these events can cause a wide range of diseases, and additionally, various infectious agents depend on protease

I

© 2015 American Chemical Society

activity for their invasion or survival in the host. Hence, proteases are suitable as drug targets, and nowadays several protease-targeting drugs are clinically applied.11,12 Typical substrates of IMPs consist of membrane proteins with at least one transmembrane helix (TM). They are proteolytically processed at a site within the TM or in a juxtamembrane region. An exception is formed by the substrates of Rce1. Rce1 is an exoprotease that removes the C-terminal tripeptide from its substrates, which are membraneanchored, farnesylated proteins with a CAAX motif at their Cterminus. Although the roles of IMPs are still being uncovered, it is clear that their functions are very diverse. They include biologically important processes such as signaling between eukaryotic cells,5,13 mitochondrial membrane remodelling,14 transcription factor release,2 bacterial quorum sensing,15 and extracytoplasmic stress response in bacteria.16,17 IMPs also play a role or have been implicated in a variety of human diseases, including Alzheimer’s disease, Parkinson’s disease, diabetes, and infection by apicomplexan parasites.18−21 Despite considerable progress in IMP research during the past 15 years, many open questions remain. One of the major questions is how IMPs recognize and subsequently cleave their substrates. On the one hand, the conformational flexibility of substrate TMs appears to play a role for multiple IMP families.22 For example, in rhomboid substrates, residues that decrease helix stability such as glycine and proline seem to be important for cleavage.23−25 On the other hand, a sequence Received: August 31, 2015 Accepted: October 16, 2015 Published: October 16, 2015 2423

DOI: 10.1021/acschembio.5b00693 ACS Chem. Biol. 2015, 10, 2423−2434

Reviews

ACS Chemical Biology

Figure 1. Schematic structures and topologies of the known families of IMPs. The membrane is indicated in dark gray. The cytosol is below the membrane, and the extracellular space/lumen is located above. (A) Metallo IMPs, exemplified by site-2-protease (S2P), contain a zinc ion complexed by histidine and aspartate residues in different TMs. The blue helices indicate the four TMs that are present in all known metallo IMPs; the white TMs are additionally present in the human S2P. (B) The glutamyl IMP Rce1 represents a new type of proteases, for which the mechanism is not yet fully understood, but depends on a histidine and a glutamic acid residue in two TMs. (C) Rhomboids employ a serine−histidine catalytic dyad. Their core structure contains six TMs, with a possible seventh TM located at either the C-terminus as drawn here or the N-terminus. (D) The aspartyl or GXGD IMPs use two aspartate residues for catalysis. Whereas SPP and SPP-like proteases (SPP(L)) are functional by themselves, γsecretase is a complex composed of multiple proteins (PEN2, presenilin (PS), nicastrin (Nct), and Aph-1). PS forms the catalytic core of γ-secretase. Note that the complex can either contain presenilin 1 or presenilin 2. PS is cut into an N-terminal and C-terminal fragment consisting of TM1−6 (PS-NTF; lighter blue) and TM7−9 (PS-CTF; darker blue), respectively. The topology of presenilin is flipped compared with SPP(L).

activity-based probes (ABPs) or affinity-based probes (AfBPs). Furthermore, as several IMPs are involved in medically relevant processes, IMP inhibitors represent potential drugs or drug leads. Here, we will briefly describe the available inhibitors for the different families of IMPs. For more comprehensive overviews of IMP inhibitors, several recent reviews are available.28−30 By far the most effort has been dedicated to γ-secretase inhibitors (GSIs), because of the involvement of γ-secretase in the pathogenesis of Alzheimer’s disease. Although the exact pathogenesis is still unclear, it is believed that neurodegeneration is caused by amyloid beta (Aβ) peptides, generated through cleavage of the amyloid precursor protein. In the typical amyloid plaques of patients, both Aβ40 and Aβ42 are foundwith Aβ42 being the more toxic one and most prone to aggregation. For further information, the reader is referred to a number of reviews.31−33 Among the reported GSIs are (1) transition-state analogs such as L-685,458 (1; Figure 2);34 (2) peptidomimetics, e.g., DAPT (2), and its analogs such as CE and LY-411,575 (3, 4),8,35,36 in which the phenylglycine residue is replaced by a benzodiazepine or dibenzoazepine, respectively; (3) helical peptides mimicking part of a TM;37 and (4) a wide variety of other compounds.28,29 Some GSIs advanced to phase III clinical trials but failed because of side effects, which occurred through the inhibition of cleavage of other γ-secretase substrates including Notch.38 Research has therefore shifted toward “Notch-sparing” GSIs and γ-secretase modulators (GSMs).28,29 The latter modify γ-secretase activity, leading to the formation of shorter Aβ peptides and a lower production of the pathogenic Aβ42. Nonsteroid anti-inflammatory drugs, such as ibuprofen and sulindac sulfide, were the first reported GSMs,39 and later many more followed.40 For rhomboid proteases, a variety of covalent inhibitors based on reactive electrophiles are known: 4-chloroisocoumarins,5,41 fluorophoshonates,42,43 chloromethyl ketones,5,27 βlactams,44,45 and β-lactones46 (5−9, Figure 2). They all form

specificity motif around the scissile bond has been described for two rhomboid proteases.26,27 Overall, the substrate specificity of IMPs is still poorly understood compared with that of soluble proteases. A second question concerns the different biological pathways in which IMPs are involved. Although the roles of some IMPs have been elucidated, there are IMPs in many organisms for which no functions or no substrates are known. An important medically relevant question is, are IMPs druggable? In the case of γ-secretase, the answer seems to be yes (although clinical trials for treatment of Alzheimer’s disease with compounds targeting γ-secretase have failed; see section 2.1). For some other families of IMPs, it is still unclear whether their action can be selectively inhibited by drug-like molecules. The development of chemical tools for IMPs lags behind that of soluble proteases. There are several underlying reasons for this. In general, membrane proteins are more difficult to recombinantly express and purify in comparison with soluble proteins, which is a result of their localization in the membrane and the need for the presence of a detergent in order to perform homogeneous assays. It hampers evaluation of possible tools on purified IMPs and complicates crystallization for structural studies. Thus far, the structures of a few model IMPs have been solved. As mentioned above, it is also poorly understood how the different families of IMPs recognize their substrates, which makes the rational design of probes very difficult. Despite these challenging aspects, chemical tools for IMPs have been developed during the past 15 years. In this review, we will give an overview of the various tools available for the study of IMPs and discuss how these tools can be utilized in specific biochemical and structural applications. We will finish with an outlook and thoughts on how the field might further develop.

2. TOOLS FOR IMPS 2.1. Inhibitors. Inhibitors can be used as chemical tools in basic research or as starting points for the development of 2424


Reviews


Figure 2. Examples of IMP inhibitors. Noncovalently binding γ-secretase inhibitors (1−4), covalent rhomboid protease inhibitors (5−9), S2P inhibitors (10, 11), and an Rce1 inhibitor (12).

The latter spacers are often based on the substrate specificity of the target. ABPs have proven especially useful for studying proteases.58,59 In the case of IMPs, they have thus far only been described for rhomboids. Current rhomboid ABPs are derived from reported inhibitor scaffolds and include rhodamine-tagged fluorosphosphonates (13; Figure 4; based on 6a in Figure 2),46,50,60,61 alkynylated isocoumarins (derived from 5b; Figure 2),49,50 and alkynylated β-lactones (9; Figure 2).46,50 The application of these ABPs will be discussed in more detail in section 3. 2.3. Affinity-based Probes (AfBPs). The main difference between AfBPs and ABPs lies in the nature of the interaction with their targets. In contrast to ABPs, AfBPs do not utilize the enzymatic mechanism for modification of targets. They are derived from potent, reversible inhibitors to which a detection tag is appended. AfBPs can be divided into two classes: covalent and noncovalent. Most covalent AfBPs contain a photoreactive group, which can covalently link the probe to its target enzyme upon irradiation (Figure 3B). The two most commonly used photoreactive moieties are diazirines and benzophenones (Figure 4). Photo-cross-linking AfBPs have been extensively investigated in studies of various soluble enzymes.62 AfBPs without cross-linkers include bead-immobilized and biotinylated inhibitors (Figure 3C). They facilitate the capture of the noncovalent enzyme-probe complex, which is then followed by downstream analysis (such as detection by Western blot). Unlike ABPs, the location of interaction with the target enzyme is not restricted to the active site cleft. AfBPs can therefore be considered as chemical tools complementary to ABPs and are used to address different questions. During the past 15 years, AfBPs have been successfully applied to the study of aspartyl IMPs, both for the discovery of the different

covalent bonds with the active site serine and, in some cases, also with a histidine located in the active site. To date, no rhomboid inhibitors with a noncovalent mechanism have been reported. The reason for this may be the ill-defined recognition motif as well as the low affinity of rhomboids for their substrates (and hence for substrate mimicking inhibitors; reported values of KM of the rhomboid GlpG for TatA-derived substrates are 20−200 μM47,48). Although some inhibitors show selectivity for rhomboids of certain biological species,44,49,50 selective inhibition over soluble proteases is still a challenge that needs to be addressed in future research. Reports on inhibitors of S2P are surprisingly scarce. General metallo protease inhibitors that chelate Zn2+ have been described (such as 10; Figure 2),51 and recent studies reported the HIV-protease inhibitor Nelfinavir (11) and analogs as S2P inhibitors.52 For Rce1, prior to the identification of its catalytic residues and long before it was known to be an IMP, inhibitors have been reported based on a variety of scaffolds including farnesylated, nonhydrolyzable peptide analogs (12).53 2.2. Activity-based Probes (ABPs). ABPs54,55 are small molecule tools that can be used for labeling active enzyme species. Generally, ABPs consist of three parts (Figure 3A): (1) a warhead, which is the reactive part of the ABP and is often derived from known electrophilic inhibitorsit binds covalently to the enzyme by a mechanism-based reaction in which an active site residue attacks the electrophile; (2) a detection or reporter tag, such as a fluorophore or biotin, which enables detection or purification of the covalent enzyme-probe complex;56 (3) a spacer, which separates the two previous elements and can also be used to fine-tune the selectivity of the ABP for certain targets.57 For proteases, spacers may vary from simple alkyl chains (broad spectrum) to peptide fragments (selective) that bind to specificity pockets near the active site. 2425


Reviews


Figure 3. Overview of different molecular tools for IMPs. (A) ABPs use a reactive electrophile that covalently modifies an active site nucleophile in a mechanism-based reaction. Recognition elements on the probe may dock into specificity pockets near the active site. A detection tag, such as a fluorophore or biotin, ensures detection of the covalent probe−protease complex. (B) AfBPs can display a covalent mechanism when conjugated to a photo-cross-linker. (C) Another type of AfBP is a noncovalently binding molecule that is attached to a biotin affinity label (as depicted here) or to an insoluble bead matrix (not depicted). (D) Synthetic substrates are generally designed as fluorescently quenched polypeptides, which contain a cleavage site matching the substrate selectivity of the target IMP.

Figure 4. Examples and mechanisms of chemical tools for IMPs. (A) An example ABP for rhomboids comprises FP-Rh (13), which modifies the active site serine. (B) The most often used photo-cross-linkers are benzophenone and diazirine derivatives. Upon irradiation, benzophenone forms a diradical, which can abstract a hydrogen atom from a nearby CH. The formed radicals then combine to the cross-linked product. Diazirines decompose to a singlet carbene, which inserts into CH or heteroatom-H bonds. (C) Examples of synthetic substrates for different IMPs. The arrows indicate the cleavage sites. Noncapital letters refer to D-amino acids.

subunits occurring in the γ-secretase complex and mapping of the binding sites of substrates and inhibitors. 2.4. Synthetic Substrates. Because of the straightforward and automatable synthesis of polypeptides, synthetic substrates have become a popular tool for activity measurements of soluble proteases. Nowadays, a wide variety of chromogenic, fluorogenic, and luminogenic substrates are commercially available, allowing continuous measurement of protease activity in a sample. Most of these substrates are composed of a peptide consisting of residues N-terminal to the scissile bond (the

nonprimed site, according to the Schechter and Berger nomenclature;63 P1, P2, etc.) connected to a leaving group that generates a signal. However, such peptides are incompatible with proteases that also recognize elements on the primed site (C-terminal to the scissile bond; P1′, P2′, etc.). To circumvent this problem, fluorescently quenched peptides, often referred to as FRET peptides, have been designed. These peptides span across a larger part of the active site cleft of the target protease and incorporate a fluorophore and quencher at either side of the scissile bond (Figure 3D). 2426


Reviews


Figure 5. Structures of AfBPs targeting aspartyl IMPs. The cross-linker parts of the molecules, when present, are colored in red. Compounds 18−20 were used to identify PS as the catalytic subunit of γ-secretase. Note that 18 and 19 are functionalized with benzophenone as a photo-cross-linker, whereas AfBP 20 carries a bromoacetamide group. This electrophile will covalently react with nucleophiles on the protein target if they are in close proximity. Structure 21 represents a noncovalently binding AfBP immobilized on a bead. Compound 22, which has a diazirine photo-cross-linker, was used to identify SPP. AfBP 23 was based on a potent GSM that selectively inhibits formation of Aβ40 and Aβ42 over the shorter Aβ38. AfBPs 24−27 have benzophenone photo-cross-linkers at locations corresponding to the different subsites in a substrate (see structure above the probes; the scissile bond is indicated). Compound 28 represents a molecule that selectively inhibits formation of Aβ42.

3. APPLICATION OF CHEMICAL TOOLS In this section, we will discuss the different application areas of chemical tools for IMPs, which range from facilitating the discovery of IMPs to inhibitor screening, detection of the binding sites of small molecules, and structural characterization of IMPs. 3.1. Discovery of IMPs. Chemical tools have been used to aid the discovery of IMPs or confirm the nature of their catalytic residues. The first IMPs to benefit from this approach were aspartyl IMPs. Although it had been known that the amyloid precursor protein was cleaved by a “γ-secretase activity,” it remained unclear to which catalytic family this protease belonged. Difluoroketone peptidomimetic inhibitors were used to classify γ-secretase as an aspartyl IMP, which formed the starting point for further investigations.67 In 2000, three independent papers reported PS as the catalytic subunit of γ-secretase by using different covalent AfBPs, all of which consisted of transition state analogs (18−20; Figure 5).7−9 In later studies, noncovalent AfBPs were also used to investigate γsecretase. Although these do not reveal the direct binding partner, they allow coisolation of components that are part of the complex. For example, the bead immobilized transition state inhibitor 21 isolated not only the N-terminal fragment and C-terminal fragment of PS (PS-NTF and PS-CTF) but also nicastrin and an endogenous substrate. Based on this finding, it

Synthetic FRET substrates have been reported for three families of IMPs: glutamyl IMPs,4,64 aspartyl IMPs, and rhomboid proteases.44 For glutamyl IMPs, the substrates comprise farnesylated polypeptides derived from Rce1 substrates such as RhoA GTPase (14, Figure 4) or K-Ras.4,64 For γ-secretase, a now commercially available FRET substrate was reported by Farmery et al., based on the cleavage site of the amyloid precursor protein (15).65 Recently, Naing et al. screened different commercially available FRET peptides against the aspartyl IMP of the archaeon M. marisnigri JR1. Interestingly, a peptide originally designed for the soluble aspartyl protease renin (16) could be cleaved by the IMP.66 For rhomboids, synthetic FRET substrate 17 (Figure 4) was based on part of the TM of the Drosophila rhomboid substrate Gurken.44 It was effectively cleaved by AarA, the rhomboid of the bacterium Providencia stuartii. An unexpected “quenched” fluorescent substrate was reported by Dickey et al. It was found that a full TM, based on the AarA substrate TatA, functionalized with a FITC at its N-terminus does not show fluorescence when incorporated into a liposome.47 After cleavage, the fluorescently labeled product is released from the liposome and regains its fluorescence. 2427


Reviews


Figure 6. Mapping of binding sites. (A) Direct labeling of the γ-secretase complex by photo-cross-linking AfBPs. γ-secretase is schematically drawn as a horseshoe-shaped structure, which was revealed by recent cryo-electron-microscopy studies.98,104 The TMs of PS-NTF and PS-CTF are colored in lighter and darker blue. Incubation with an AfBP and subsequent irradiation leads to covalent cross-linking. Upon enrichment of modified proteins by immobilized streptavidin, Western blot with antibodies against the different subunits reveals the specificity of the AfBP. Alternatively, an immunoprecipitation to isolate the different subunits is followed by a biotin blot. Depicted here is the modification of PS-CTF detected by the first mentioned workflow. (B) Competition of small molecule GSIs or GSMs with AfBP-labeling may reveal the absence of labeling, indicating either binding to the same site or allosteric changes in the AfBP binding site.

the aspartyl IMP of the archaeon M. marisnigri JR1. It was found that the turnover rate is slow and that the kinetic parameters are similar in detergent and bicelles, indicating that the hydrophobic environment in which the protease was reconstituted did not have a substantial effect on the cleavage.66 Studies on rhomboid proteases with the fluorescently labeled TM of TatA, reconstituted together in liposomes, revealed slow cleavage kinetics and low affinity of rhomboids for their substrates.47 Similar results were obtained with detergentsolubilized rhomboids and a recombinant FRET protein substrate.48 Synthetic FRET-substrate 17 (Figure 4) was used in a high-throughput screen against rhomboid AarA. From a library of nearly 60 000 molecules, 33 were found to have a significant inhibitory effect. The study identified monocyclic βlactams as novel covalent rhomboid inhibitors with low micromolar potency, and the evaluation of a set of analogs led to the formulation of a detailed structure−activity relationship.44 Because β-lactams have been used as pharmaceuticals for several decades, this scaffold may be suitable for future development of rhomboid-targeting drugs. Besides substrates, ABPs have been used for activity measurement of rhomboid proteases. In contrast to synthetic TatA or Gurken substrates, which do not work for rhomboids from all species, FP-Rh (13, Figure 4) was recently shown to react with rhomboids from different organisms and can be used as a universal probe to detect active rhomboids.50 The first report on rhomboid ABPs stems from 2012 and comprised the use of FP-Rh to probe the activity state of the E. coli rhomboid GlpG and several GlpG mutants. It was found that residues located on the cytoplasmic side of the membrane and a region preceding the TM core are required for optimal active site alignment.60 The same ABP was used to develop an inhibition assay based on fluorescence polarization, leading to the discovery of β-lactones as a new class of covalent rhomboid inhibitors.46 In order to evaluate whether detergent micelles,

was concluded that the initial substrate interaction site is not located at the active center (see also section 3.3).68 SPP was also discovered by using covalent AfBPs. Compound 22 (Figure 5), containing a biotin and a diazirine photo-cross-linker, was based on an inhibitor69 that blocks processing of signal peptides. Enrichment of the photolabeled protein by chromatography and analysis by mass spectrometry revealed a membrane protein of unknown function. Sequence databases showed the occurrence of orthologs in higher eukaryotes with two conserved aspartates in different TMs, uncovering not only SPP but also the SPP-like proteases.10 The discovery of rhomboid proteases is an interesting story by itself and finds its origin in Drosophila genetics. Genetic analysis had shown that the 7-TM containing Rhomboid-1 together with another membrane protein regulates the release of the EGF homologue Spitz. However, the mechanism was unknown. In 2001, the Freeman laboratory uncovered rhomboid proteases as one of the most widespread families of IMPs. Both mutational analysis and the use of the general serine protease inhibitor DCI (5a, Figure 2) aided in the characterization of rhomboids as serine proteases.5 3.2. Activity Measurement and Inhibitor Screening. The ability to measure the activity of a protease does not only allow the determination of kinetic parameters but is also essential for screening inhibitors. Both synthetic substrates and ABPs can be used as a read-out for these purposes. The main difference is that proteases react stoichiometrically with ABPs, but they can turn over multiple substrate molecules, which leads to signal amplification. Synthetic substrates have been used for activity measurement of different classes of IMPs. In the case of glutamyl IMPs, farnesylated FRET-peptides, such as compound 14 (Figure 4), have been used for confirmation of the activity of Rce1 homologues, for inhibitor screening and for determining the potency of inhibitors.4,70,71 FRET peptide 16 was used to assay 2428


Reviews


Figure 7. Structural information on rhomboid proteases. (A) The crystal structure of the E. coli rhomboid GlpG (PDB code: 2IC8) revealed an arrangement of five TMs around a shorter central one (TM4). The active site serine and histidine (in stick representations) are part of TM4 and TM6, respectively, and are located in a hydrophilic cavity. Note that loop L5 covers the active site. (B) A closeup of the active site with rhomboid inhibitor 5b (PBD code: 2XOW). The inhibitor binds to both catalytic residues: S201 and H254. The carbonyl group of the inhibitor points toward the oxyanion hole formed by the side chains of H150 and N154, and backbone atoms of L200 and S201. (C) The location of the S1 pocket was deduced from inhibitor-bound crystal structures. The surface of the pocket is formed by side chains of A182, S185, Q189, F197, A243, and, deeper into the pocket, H141. In the apoenzyme (in cyan), the side chain of M249 fills the S1 pocket. The lifting of loop L5 and the movement of M249 out of the S1 pocket induced upon inhibitor binding are indicated by arrows. (D) The S2′ pocketnot present in the apoenzymeis formed upon binding of a β-lactam inhibitor (not displayed) by rotation of the side chain of W236 (indicated by the arrow). The apoenzyme is depicted in cyan, the inhibitor-bound enzyme in white (PDB code: 3ZMI). The surface of the pocket is formed by side chains of W157, V204, M208, Y205, A233, and I237.

DAPT analogs 3 and 4 (Figure 2) bind to PS-NTF and to SPP, in contrast to DAPT (2) itself, which only binds PS-CTF. Once the subunits to which AfBPs bind have been mapped, it becomes more straightforward to test where an unlabeled small molecule interacts. Photolabeling by the AfBP is performed in the presence or absence of the compound of interest (Figure 6B). The absence of labeling indicates that the compound either has a binding site that overlaps with the AfBP or that the probe binding site is allosterically altered. Various GSIs compete with AfBPs derived from transition state analogs. An exception is formed by inhibitors that are comprised of substrate-like helical peptides. These do not compete with transition state AfBPs but still disrupt the interaction between substrate and γ-secretase, providing evidence for the existence of an initial substrate docking site.37,75 An elegant AfBP approach termed “photophore walking” was developed by Li and co-workers. Four different AfBPs were designed (24−27, Figure 5), each containing a single benzophenone photo-cross-linker located at positions that correspond to the P2, P1, P1′, and P3′ of a substrate, respectively. Upon interaction with γ-secretase, these can covalently cross-link to the different putative S2−S3′ subsites near the active site. The authors focused on dicoumarin GSI 28 (Figure 5), which noncompetitively inhibits γ-secretase, with the highest potency against Aβ42 formation. Incubation of γsecretase with compound 28 prevented labeling by probes 24 and 25, but not by 26 and 27.76 As probes 24 and 25 bear their photo-cross-linker at locations that correspond to the P2 and P1 positions of a substrate, it was suggested that dicoumarin 28 allosterically induces changes at the supposed S2 and S1 subsites. The same research group used the photophore walking approach to compare the behavior of γ-secretase and SPP. Based on the reactivity of the above-mentioned probes against these two enzymes, it was concluded that γ-secretase and SPP share similar subsites that interact with the P1 and P1′ residues of the probes but have structurally distinct subsites for the P2 and P3′ residues. Additionally, it was shown that GSIs and GSMs have different effects on the two proteases.77 3.4. Structural Studies. Probe labeling and competition experiments, as described in the previous paragraphs, have provided insights into the binding sites of small molecule

which are used in most inhibitor assays, may form a limitation by influencing the rhomboid behavior, Fp-Rh was used to compare GlpG in a micelle and a liposome (lipid bilayer) environment. When tested against a panel of 50 molecules reactive toward serine proteases, the inhibition profile did not show substantial differences, validating micelle-solubilized IMPs as a model system for inhibitor screening.61 3.3. Binding Sites, Allosteric Interactions, and Mechanistic Details. The site where a small molecule binds in order to inhibit a protease or impose an allosteric influence72 does not only give information about the mechanism of inhibition or modulation but may also reveal how the protease of interest recognizes its substrates. ABPs are designed to react with one or more of the catalytic residues. The usage of an active site mutant of the protease can therefore provide evidence for the specificity of this reaction. Similarly, competition with known active-site directed inhibitors is also indicative of binding to the catalytic residues. For example, reaction of an ABP based on the 4-chloroisocoumarin scaffold 5b (Figure 2) with the catalytic serine of the E. coli GlpG was confirmed to be specific by preincubation with DCI or by using the S201A mutant.49 AfBPs with photo-cross-linkers have been used to identify to which of the subunits in the γ-secretase complex GSIs and GSMs bind. Direct labeling strategies of AfBPs derived from GSIs or GSMs provide the most straightforward approach. After incubation and irradiation, antibodies against the different subunits are utilized in immunoprecipitation or Western blot experiments to identify the AfBP-modified subunit (Figure 6). Experiments along these lines have not only aided in the identification of both PS-NTF and PS-CTF as targets of transition-state analog GSIs7−9 but also resulted in the finding that, e.g., compound 23 (Figure 5), a “non-acidic” GSM that potently inhibits the formation of longer Aβ fragments only binds the PS-NTF.73 It often requires extensive synthetic chemistry efforts in order to obtain AfBPs with a photo-cross-linker and a biotin at a suitable position in the molecule. To rapidly access multiple AfBPs, Fuwa et al. implemented a divergent strategy: alkynelabeled GSIs and azide-labeled biotin-photo-cross-linker conjugates were coupled using click chemistry, resulting in a diverse set of probes.74 It was found that AfBPs based on 2429


Reviews


E. coli GlpG) proved to be inactive.89 Hence, the existence of threonine IMPs may be unlikely. Although cysteines have a low occurrence in helices, they can be found at helix−helix interfaces90,91 and will display higher reactivity if they occur in hydrogen bonding with a basic residue. We imagine that in the future chemical tools may help to identify putative cysteine IMPs. Currently, the limited knowledge of substrates is a bottleneck in IMP research. Over 90 substrates of γ-secretase are known,92 but the number is much smaller for other IMPs. The need for identification methods of IMP substrates may be best illustrated by the striking fact that for the best characterized rhomboid (E. coli GlpG) not a single physiological substrate has been reported to date. Although it was notoriously difficult to link a cleavage event in a cell or cell lysate to a particular protease, several chemical proteomics methods have been developed during the past decade in order to address this problem.93,94 One of these methods, called SPECS, has recently been applied to identify substrates of the Golgi residing aspartyl IMP SPPL3.95 SPECS exploits the fact that the majority of membrane proteins are glycosylated and that the ectodomains are shed upon cleavage. By feeding cells in culture with azidelabeled carbohydrates, the shed ectodomains can be isolated from cell culture medium by click chemistry mediated functionalization with a biotin and subsequent enrichment by streptavidin.96 Most of the newly found SPPL3 substrates are involved in protein glycosylation and confirm the role of SPPL3 as a regulator of glycan-modifying enzymes in the Golgi. We foresee that in the future the discovery of IMP substrates will be increasingly accomplished by chemical proteomics methods. It will not only help to define the functional roles of IMPs but also to understand the substrate specificity. Advances in cryo-electron-microscopy have led to structures with higher and higher resolution.97 Together with the continuous efforts in membrane protein crystallization, more structures of IMPs with atomic resolution can be expected. Very recently, a cryo-electron-microscopy structure with atomic resolution of the γ-secretase complex has been reported.98 For rhomboids, the next step will be a bound substrate oras an alternativea peptide-like inhibitor that spans both primed and nonprimed sites. Structures of other IMPs in complex with small molecule inhibitors or modulators will be especially interesting in order to verify the mechanism of action and to confirm or refute whether the principles found in rhomboid structures are generally applicable to IMPs. Certainly, it will refine the current models of how IMPs recognize their substrates. While it is very plausible that the currently reported transition state inhibitors and photo-cross-linking AfBPs bind similar to the aspartyl IMPs as a substrate does, it is unclear which residues on PS and SPP build the different specificity pockets. Tandem mass spectrometry methods have aided in the discovery of the direct targets of AfBPs, butto our knowledgethe exact residues that are covalently modified have not yet been mapped. This will be a challenging task, since the identification of modified tryptic peptides is demanding, as illustrated by a recent study on protein lipidation.99 Membrane proteins have the additional disadvantage of generating longer and more hydrophobic tryptic peptides than soluble proteins. The usage of digestive proteases other than trypsin, as well as developments in instrumentation and bioinformatics may address these difficulties. Currently, probes for soluble proteases are commonly applied in complex systems such as whole cells or animal

ligands to IMPs, but the information remains relatively imprecise and without atomic resolution. Although membrane protein crystallography is very challenging, several structures of IMPs have been elucidated during the past decade, by either Xray crystallography or cryo-electron-microscopy. This has given the first glimpses into the mechanism of hydrolysis within the membrane. The crystal structure of the E. coli and H. inf luenzae rhomboid GlpG showed that it forms a hydrophilic cavity created by five TMs surrounding a central, slightly shorter TM (Figure 7A).78−81 Crystal structures of other IMPs, including S2P51 and aspartyl IMPs82,83 have followed. These also revealed hydrophilic cavities or channels that ensure a supply of water molecules necessary for substrate hydrolysis. No structures of substrate-enzyme complexes have yet been elucidated. However, crystal structures of the E. coli rhomboid GlpG with bound small molecule inhibitors have significantly improved our knowledge of how substrates might interact. The first rhomboid-inhibitor structure comprised 4-chloro-isocoumarin 5b.41 The structure revealed that this mechanism-based inhibitor forms a covalent bond with the active site serine S201, as expected, and additionally with the other residue of the catalytic dyad, histidine H254 (Figure 7B), confirming the reactivity of the two residues. The structure also revealed the position of the oxyanion hole, later confirmed by several other structures,27,42,43 and a cavity that forms the S1 pocket, which was supported by biochemical data41 and a structure of a more peptide-like inhibitor (Figure 7C).27 Interestingly, in the apoenzyme structure,78 the pocket is blocked by the side chain of M249 located on loop L5, which occupies part of the active site and is displaced upon inhibitor binding (Figure 7C). Other structures have also shown that recognition pockets may only be created upon binding of an inhibitor or substrate, such as the S2′ pocket, which forms by rotation of W236 (Figure 7D).45 Overall, structures of GlpG with and without inhibitors have led to novel insights into the interaction of IMPs with substrates and small molecules. For rhomboids, it has led to different models of how substrates are granted access to the active site.84−86 These include lifting of loop L5, which has been observed in all inhibitor-rhomboid structures, and possible movement of TM579 (not discussed here). The current crystal structures might lead to the future design of more potent inhibitors or chemical probes, possibly aided by computational methods. However, subtle binding-induced conformational changes, as revealed by various inhibitor-rhomboid structures, could complicate this approach.

4. CONCLUSIONS AND OUTLOOK Since 1997, several IMPs have been reported: metallo, aspartyl, serine, and glutamyl IMPs. The first three groups share similar mechanisms of peptide bond hydrolysis with their soluble counterparts. For glutamyl IMPs, further research is needed to uncover its mechanism. According to the current MEROPS database,87 seven distinct catalytic types of soluble proteases exist. This brings up the question whether there might be other, still undiscovered IMPs with mechanisms similar to, for example, soluble threonine or cysteine proteases. Threonine proteases include proteasomal β-subunits as prototypical examples, which utilize an N-terminal threonine as catalytic residue. The catalytic residue of taspase-1, another threonine protease, is also an N-terminal threonine.88 However, localization of an N-terminal threonine within the membrane seems unfavorable, and a “threonine rhomboid” (the S201T mutant of 2430


Reviews

ACS Chemical Biology models.100 Experiments in live cells or in vivo do not only allow the study of the complex regulatory mechanisms in protease biology, but also the evaluation of inhibitors in biologically relevant settings. At an early stage of the drug development process, these approaches are expected to facilitate the selection of hit compounds with better properties and less off-targets than traditional methods.101 To enable such strategies for IMPs, it will be necessary to design ABPs, AfBPs and synthetic substrates with improved selectivity and favorable in vivo properties. Naturally, this will require extensive synthetic chemistry efforts. An increased knowledge of substrate specificity and structural information, possibly combined with molecular modeling, will be invaluable to accelerate this process. Additionally, the currently available chemical tools may enable high throughput screening to find new scaffolds for inhibitor and probe development. We expect that labeling strategies and chemical proteomics methods developed for IMPs will help to characterize other membrane proteins as well. Since membranes form the border between organelles and the rest of the cell or between the cell and the rest of the organism, membrane proteins play a vital role in communication processes between cells or cellular compartments. Examples include ion channels and G-protein coupled receptors, both validated drug targets. The rhomboid pseudoproteases would be the most obvious protein class to start a translation of tools and methods developed for IMPs. The functions of inactive rhomboid homologues are still illdefined, but recent studies suggest that rhomboid pseudoproteases play important regulatory roles. For example, Drosophila iRhom is involved in ER-associated degradation of growth factors.102 Similar to rhomboid proteases, iRhoms also bind type I membrane proteins, and the interaction between iRhoms and clients may resemble that of rhomboid proteases and its substrates.103 Hence, IMP probe strategies that are based on affinity may be readily applied here. Overall, we expect that the near future will bring substantial advances in the application of chemical tools to membrane proteins in general and IMPs in particular.

■

(3) Kinch, L. N., and Grishin, N. V. (2013) Bioinformatics perspective on rhomboid intramembrane protease evolution and function. Biochim. Biophys. Acta, Biomembr. 1828, 2937−2943. (4) Manolaridis, I., Kulkarni, K., Dodd, R. B., Ogasawara, S., Zhang, Z., Bineva, G., O’Reilly, N., Hanrahan, S. J., Thompson, A. J., Cronin, N., Iwata, S., and Barford, D. (2013) Mechanism of farnesylated CAAX protein processing by the intramembrane protease Rce1. Nature 504, 301−305. (5) Urban, S., Lee, J. R., and Freeman, M. (2001) Drosophila Rhomboid-1 defines a family of putative intramembrane serine proteases. Cell 107, 173−182. (6) Freeman, M. (2014) The rhomboid-like superfamily: molecular mechanisms and biological roles. Annu. Rev. Cell Dev. Biol. 30, 235− 254. (7) Li, Y. M., Xu, M., Lai, M. T., Huang, Q., Castro, J. L., DiMuzioMower, J., Harrison, T., Lellis, C., Nadin, A., Neduvelil, J. G., Register, R. B., Sardana, M. K., Shearman, M. S., Smith, A. L., Shi, X. P., Yin, K. C., Shafer, J. A., and Gardell, S. J. (2000) Photoactivated gammasecretase inhibitors directed to the active site covalently label presenilin 1. Nature 405, 689−694. (8) Seiffert, D., Bradley, J. D., Rominger, C. M., Rominger, D. H., Yang, F., Meredith, J. E., Jr., Wang, Q., Roach, A. H., Thompson, L. A., Spitz, S. M., Higaki, J. N., Prakash, S. R., Combs, A. P., Copeland, R. A., Arneric, S. P., Hartig, P. R., Robertson, D. W., Cordell, B., Stern, A. M., Olson, R. E., and Zaczek, R. (2000) Presenilin-1 and −2 are molecular targets for gamma-secretase inhibitors. J. Biol. Chem. 275, 34086−34091. (9) Esler, W. P., Kimberly, W. T., Ostaszewski, B. L., Diehl, T. S., Moore, C. L., Tsai, J. Y., Rahmati, T., Xia, W., Selkoe, D. J., and Wolfe, M. S. (2000) Transition-state analogue inhibitors of gamma-secretase bind directly to presenilin-1. Nat. Cell Biol. 2, 428−434. (10) Weihofen, A., Binns, K., Lemberg, M. K., Ashman, K., and Martoglio, B. (2002) Identification of signal peptide peptidase, a presenilin-type aspartic protease. Science 296, 2215−2218. (11) Turk, B. (2006) Targeting proteases: successes, failures and future prospects. Nat. Rev. Drug Discovery 5, 785−799. (12) Drag, M., and Salvesen, G. S. (2010) Emerging principles in protease-based drug discovery. Nat. Rev. Drug Discovery 9, 690−701. (13) De Strooper, B., Annaert, W., Cupers, P., Saftig, P., Craessaerts, K., Mumm, J. S., Schroeter, E. H., Schrijvers, V., Wolfe, M. S., Ray, W. J., Goate, A., and Kopan, R. (1999) A presenilin-1-dependent gammasecretase-like protease mediates release of Notch intracellular domain. Nature 398, 518−522. (14) McQuibban, G. A., Saurya, S., and Freeman, M. (2003) Mitochondrial membrane remodelling regulated by a conserved rhomboid protease. Nature 423, 537−541. (15) Stevenson, L. G., Strisovsky, K., Clemmer, K. M., Bhatt, S., Freeman, M., and Rather, P. N. (2007) Rhomboid protease AarA mediates quorum-sensing in Providencia stuartii by activating TatA of the twin-arginine translocase. Proc. Natl. Acad. Sci. U. S. A. 104, 1003− 1008. (16) Kanehara, K., Ito, K., and Akiyama, Y. (2002) YaeL (EcfE) activates the sigma(E) pathway of stress response through a site-2 cleavage of anti-sigma(E), RseA. Genes Dev. 16, 2147−2155. (17) Alba, B. M., Leeds, J. A., Onufryk, C., Lu, C. Z., and Gross, C. A. (2002) DegS and YaeL participate sequentially in the cleavage of RseA to activate the sigma(E)-dependent extracytoplasmic stress response. Genes Dev. 16, 2156−2168. (18) Chan, E. Y., and McQuibban, G. A. (2013) The mitochondrial rhomboid protease: its rise from obscurity to the pinnacle of diseaserelevant genes. Biochim. Biophys. Acta, Biomembr. 1828, 2916−2925. (19) Sibley, L. D. (2013) The roles of intramembrane proteases in protozoan parasites. Biochim. Biophys. Acta, Biomembr. 1828, 2908− 2915. (20) Urban, S. (2009) Making the cut: central roles of intramembrane proteolysis in pathogenic microorganisms. Nat. Rev. Microbiol. 7, 411−423.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], [email protected]. Author Contributions §

These authors contributed equally.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We acknowledge funding by the Deutsche Forschungsgemeinschaft DFG and the Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen. We thank M. Teese for useful discussions.

■

REFERENCES

(1) Schwann, T. (1836) Ueber das Wesen des Verdauungsprocesses. Ann. Pharm. Chem. 20, 28−34. (2) Rawson, R. B., Zelenski, N. G., Nijhawan, D., Ye, J., Sakai, J., Hasan, M. T., Chang, T. Y., Brown, M. S., and Goldstein, J. L. (1997) Complementation cloning of S2P, a gene encoding a putative metalloprotease required for intramembrane cleavage of SREBPs. Mol. Cell 1, 47−57. 2431


Reviews

ACS Chemical Biology (21) De Strooper, B., and Annaert, W. (2010) Novel research horizons for presenilins and gamma-secretases in cell biology and disease. Annu. Rev. Cell Dev. Biol. 26, 235−260. (22) Langosch, D., Scharnagl, C., Steiner, H., and Lemberg, M. K. (2015) Understanding intramembrane proteolysis: from protein dynamics to reaction kinetics. Trends Biochem. Sci. 40, 318−327. (23) Moin, S. M., and Urban, S. (2012) Membrane immersion allows rhomboid proteases to achieve specificity by reading transmembrane segment dynamics. eLife 1, e00173. (24) Urban, S., and Freeman, M. (2003) Substrate specificity of rhomboid intramembrane proteases is governed by helix-breaking residues in the substrate transmembrane domain. Mol. Cell 11, 1425− 1434. (25) Akiyama, Y., and Maegawa, S. (2007) Sequence features of substrates required for cleavage by GlpG, an Escherichia coli rhomboid protease. Mol. Microbiol. 64, 1028−1037. (26) Strisovsky, K., Sharpe, H. J., and Freeman, M. (2009) Sequencespecific intramembrane proteolysis: identification of a recognition motif in rhomboid substrates. Mol. Cell 36, 1048−1059. (27) Zoll, S., Stanchev, S., Began, J., Skerle, J., Lepsik, M., Peclinovska, L., Majer, P., and Strisovsky, K. (2014) Substrate binding and specificity of rhomboid intramembrane protease revealed by substrate-peptide complex structures. EMBO J. 33, 2408−2421. (28) Wolfe, M. S. (2012) gamma-Secretase inhibitors and modulators for Alzheimer’s disease. J. Neurochem. 120 (Suppl 1), 89−98. (29) Golde, T. E., Koo, E. H., Felsenstein, K. M., Osborne, B. A., and Miele, L. (2013) gamma-Secretase inhibitors and modulators. Biochim. Biophys. Acta, Biomembr. 1828, 2898−2907. (30) Wolf, E. V., and Verhelst, S. H. L. (2015) Inhibitors of rhomboid proteases. Biochimie, DOI: 10.1016/j.biochi.2015.07.007. (31) Benilova, I., Karran, E., and De Strooper, B. (2012) The toxic Abeta oligomer and Alzheimer’s disease: an emperor in need of clothes. Nat. Neurosci. 15, 349−357. (32) Roychaudhuri, R., Yang, M., Hoshi, M. M., and Teplow, D. B. (2009) Amyloid beta-protein assembly and Alzheimer disease. J. Biol. Chem. 284, 4749−4753. (33) Masters, C. L., and Selkoe, D. J. (2012) Biochemistry of amyloid beta-protein and amyloid deposits in Alzheimer disease. Cold Spring Harbor Perspect. Med. 2, a006262. (34) Shearman, M. S., Beher, D., Clarke, E. E., Lewis, H. D., Harrison, T., Hunt, P., Nadin, A., Smith, A. L., Stevenson, G., and Castro, J. L. (2000) L-685,458, an aspartyl protease transition state mimic, is a potent inhibitor of amyloid beta-protein precursor gammasecretase activity. Biochemistry 39, 8698−8704. (35) Dovey, H. F., John, V., Anderson, J. P., Chen, L. Z., de Saint Andrieu, P., Fang, L. Y., Freedman, S. B., Folmer, B., Goldbach, E., Holsztynska, E. J., Hu, K. L., Johnson-Wood, K. L., Kennedy, S. L., Kholodenko, D., Knops, J. E., Latimer, L. H., Lee, M., Liao, Z., Lieberburg, I. M., Motter, R. N., Mutter, L. C., Nietz, J., Quinn, K. P., Sacchi, K. L., Seubert, P. A., Shopp, G. M., Thorsett, E. D., Tung, J. S., Wu, J., Yang, S., Yin, C. T., Schenk, D. B., May, P. C., Altstiel, L. D., Bender, M. H., Boggs, L. N., Britton, T. C., Clemens, J. C., Czilli, D. L., Dieckman-McGinty, D. K., Droste, J. J., Fuson, K. S., Gitter, B. D., Hyslop, P. A., Johnstone, E. M., Li, W. Y., Little, S. P., Mabry, T. E., Miller, F. D., and Audia, J. E. (2001) Functional gamma-secretase inhibitors reduce beta-amyloid peptide levels in brain. J. Neurochem. 76, 173−181. (36) Wong, G. T., Manfra, D., Poulet, F. M., Zhang, Q., Josien, H., Bara, T., Engstrom, L., Pinzon-Ortiz, M., Fine, J. S., Lee, H. J., Zhang, L., Higgins, G. A., and Parker, E. M. (2004) Chronic treatment with the gamma-secretase inhibitor LY-411,575 inhibits beta-amyloid peptide production and alters lymphopoiesis and intestinal cell differentiation. J. Biol. Chem. 279, 12876−12882. (37) Das, C., Berezovska, O., Diehl, T. S., Genet, C., Buldyrev, I., Tsai, J. Y., Hyman, B. T., and Wolfe, M. S. (2003) Designed helical peptides inhibit an intramembrane protease. J. Am. Chem. Soc. 125, 11794−11795.

(38) Schor, N. F. (2011) What the halted phase III gamma-secretase inhibitor trial may (or may not) be telling us. Ann. Neurol. 69, 237− 239. (39) Weggen, S., Eriksen, J. L., Das, P., Sagi, S. A., Wang, R., Pietrzik, C. U., Findlay, K. A., Smith, T. E., Murphy, M. P., Bulter, T., Kang, D. E., Marquez-Sterling, N., Golde, T. E., and Koo, E. H. (2001) A subset of NSAIDs lower amyloidogenic Abeta42 independently of cyclooxygenase activity. Nature 414, 212−216. (40) Crump, C. J., Johnson, D. S., and Li, Y. M. (2013) Development and mechanism of gamma-secretase modulators for Alzheimer’s disease. Biochemistry 52, 3197−3216. (41) Vinothkumar, K. R., Strisovsky, K., Andreeva, A., Christova, Y., Verhelst, S., and Freeman, M. (2010) The structural basis for catalysis and substrate specificity of a rhomboid protease. EMBO J. 29, 3797− 3809. (42) Xue, Y., and Ha, Y. (2012) Catalytic mechanism of rhomboid protease GlpG probed by 3,4-dichloroisocoumarin and diisopropyl fluorophosphonate. J. Biol. Chem. 287, 3099−3107. (43) Xue, Y., Chowdhury, S., Liu, X., Akiyama, Y., Ellman, J., and Ha, Y. (2012) Conformational change in rhomboid protease GlpG induced by inhibitor binding to its S′ subsites. Biochemistry 51, 3723−3731. (44) Pierrat, O. A., Strisovsky, K., Christova, Y., Large, J., Ansell, K., Bouloc, N., Smiljanic, E., and Freeman, M. (2011) Monocyclic betalactams are selective, mechanism-based inhibitors of rhomboid intramembrane proteases. ACS Chem. Biol. 6, 325−335. (45) Vinothkumar, K. R., Pierrat, O. A., Large, J. M., and Freeman, M. (2013) Structure of rhomboid protease in complex with betalactam inhibitors defines the S2′ cavity. Structure 21, 1051−1058. (46) Wolf, E. V., Zeissler, A., Vosyka, O., Zeiler, E., Sieber, S., and Verhelst, S. H. (2013) A new class of rhomboid protease inhibitors discovered by activity-based fluorescence polarization. PLoS One 8, e72307. (47) Dickey, S. W., Baker, R. P., Cho, S., and Urban, S. (2013) Proteolysis inside the membrane is a rate-governed reaction not driven by substrate affinity. Cell 155, 1270−1281. (48) Arutyunova, E., Panwar, P., Skiba, P. M., Gale, N., Mak, M. W., and Lemieux, M. J. (2014) Allosteric regulation of rhomboid intramembrane proteolysis. EMBO J. 33, 1869−1881. (49) Vosyka, O., Vinothkumar, K. R., Wolf, E. V., Brouwer, A. J., Liskamp, R. M., and Verhelst, S. H. L. (2013) Activity-based probes for rhomboid proteases discovered in a mass spectrometry-based assay. Proc. Natl. Acad. Sci. U. S. A. 110, 2472−2477. (50) Wolf, E. V., Zeissler, A., and Verhelst, S. H. L. (2015) Inhibitor fingerprinting of rhomboid proteases by activity-based protein profiling reveals inhibitor selectivity and rhomboid autoprocessing. ACS Chem. Biol. 10, 2325−2333. (51) Feng, L., Yan, H., Wu, Z., Yan, N., Wang, Z., Jeffrey, P. D., and Shi, Y. (2007) Structure of a site-2 protease family intramembrane metalloprotease. Science 318, 1608−1612. (52) Guan, M., Su, L., Yuan, Y. C., Li, H., and Chow, W. A. (2015) Nelfinavir and nelfinavir analogs block site-2 protease cleavage to inhibit castration-resistant prostate cancer. Sci. Rep. 5, 9698. (53) Gilbert, B. A., Ma, Y. T., and Rando, R. R. (1995) Inhibitors of prenylated protein endoprotease. Methods Enzymol. 250, 206−215. (54) Cravatt, B. F., Wright, A. T., and Kozarich, J. W. (2008) Activity-based protein profiling: from enzyme chemistry to proteomic chemistry. Annu. Rev. Biochem. 77, 383−414. (55) Heal, W. P., Dang, T. H. T., and Tate, E. W. (2011) Activitybased probes: discovering new biology and new drug targets. Chem. Soc. Rev. 40, 246−257. (56) Sadaghiani, A. M., Verhelst, S. H. L., and Bogyo, M. (2007) Tagging and detection strategies for activity-based proteomics. Curr. Opin. Chem. Biol. 11, 20−28. (57) Haedke, U., Kuttler, E. V., Vosyka, O., Yang, Y., and Verhelst, S. H. L. (2013) Tuning probe selectivity for chemical proteomics applications. Curr. Opin. Chem. Biol. 17, 102−109. 2432


Reviews

ACS Chemical Biology (58) Serim, S., Haedke, U., and Verhelst, S. H. L. (2012) Activitybased probes for the study of proteases: recent advances and developments. ChemMedChem 7, 1146−1159. (59) Sanman, L. E., and Bogyo, M. (2014) Activity-based profiling of proteases. Annu. Rev. Biochem. 83, 249−273. (60) Sherratt, A. R., Blais, D. R., Ghasriani, H., Pezacki, J. P., and Goto, N. K. (2012) Activity-Based Protein Profiling of the Escherichia coli GlpG Rhomboid Protein Delineates the Catalytic Core. Biochemistry 51, 7794−7803. (61) Wolf, E. V., Seybold, M., Hadravova, R., Strisovsky, K., and Verhelst, S. H. L. (2015) Activity-based protein profiling of rhomboid proteases in liposomes. ChemBioChem 16, 1616−1621. (62) Geurink, P. P., Prely, L. M., van der Marel, G. A., Bischoff, R., and Overkleeft, H. S. (2011) Photoaffinity Labeling in Activity-Based Protein Profiling. Top. Curr. Chem. 324, 85−113. (63) Schechter, I., and Berger, A. (1967) On the size of the active site in proteases. I. Papain. Biochem. Biophys. Res. Commun. 27, 157−162. (64) Hollander, I., Frommer, E., and Mallon, R. (2000) Human rasconverting enzyme (hRCE1) endoproteolytic activity on K-ras-derived peptides. Anal. Biochem. 286, 129−137. (65) Farmery, M. R., Tjernberg, L. O., Pursglove, S. E., Bergman, A., Winblad, B., and Naslund, J. (2003) Partial purification and characterization of gamma-secretase from post-mortem human brain. J. Biol. Chem. 278, 24277−24284. (66) Naing, S. H., Vukoti, K. M., Drury, J. E., Johnson, J. L., Kalyoncu, S., Hill, S. E., Torres, M. P., and Lieberman, R. L. (2015) Catalytic Properties of Intramembrane Aspartyl Protease Substrate Hydrolysis Evaluated Using a FRET Peptide Cleavage Assay. ACS Chem. Biol. 10, 2166. (67) Wolfe, M. S., Xia, W., Moore, C. L., Leatherwood, D. D., Ostaszewski, B., Rahmati, T., Donkor, I. O., and Selkoe, D. J. (1999) Peptidomimetic probes and molecular modeling suggest that Alzheimer’s gamma-secretase is an intramembrane-cleaving aspartyl protease. Biochemistry 38, 4720−4727. (68) Esler, W. P., Kimberly, W. T., Ostaszewski, B. L., Ye, W., Diehl, T. S., Selkoe, D. J., and Wolfe, M. S. (2002) Activity-dependent isolation of the presenilin- gamma -secretase complex reveals nicastrin and a gamma substrate. Proc. Natl. Acad. Sci. U. S. A. 99, 2720−2725. (69) Weihofen, A., Lemberg, M. K., Ploegh, H. L., Bogyo, M., and Martoglio, B. (2000) Release of signal peptide fragments into the cytosol requires cleavage in the transmembrane region by a protease activity that is specifically blocked by a novel cysteine protease inhibitor. J. Biol. Chem. 275, 30951−30956. (70) Manandhar, S. P., Hildebrandt, E. R., and Schmidt, W. K. (2007) Small-molecule inhibitors of the Rce1p CaaX protease. J. Biomol. Screening 12, 983−993. (71) Porter, S. B., Hildebrandt, E. R., Breevoort, S. R., Mokry, D. Z., Dore, T. M., and Schmidt, W. K. (2007) Inhibition of the CaaX proteases Rce1p and Ste24p by peptidyl (acyloxy)methyl ketones. Biochim. Biophys. Acta, Mol. Cell Res. 1773, 853−862. (72) Merdanovic, M., Monig, T., Ehrmann, M., and Kaiser, M. (2013) Diversity of allosteric regulation in proteases. ACS Chem. Biol. 8, 19−26. (73) Ebke, A., Luebbers, T., Fukumori, A., Shirotani, K., Haass, C., Baumann, K., and Steiner, H. (2011) Novel gamma-secretase enzyme modulators directly target presenilin protein. J. Biol. Chem. 286, 37181−37186. (74) Fuwa, H., Takahashi, Y., Konno, Y., Watanabe, N., Miyashita, H., Sasaki, M., Natsugari, H., Kan, T., Fukuyama, T., Tomita, T., and Iwatsubo, T. (2007) Divergent synthesis of multifunctional molecular probes to elucidate the enzyme specificity of dipeptidic gammasecretase inhibitors. ACS Chem. Biol. 2, 408−418. (75) Kornilova, A. Y., Das, C., and Wolfe, M. S. (2003) Differential effects of inhibitors on the gamma-secretase complex. Mechanistic implications. J. Biol. Chem. 278, 16470−16473. (76) Shelton, C. C., Zhu, L., Chau, D., Yang, L., Wang, R., Djaballah, H., Zheng, H., and Li, Y. M. (2009) Modulation of gamma-secretase specificity using small molecule allosteric inhibitors. Proc. Natl. Acad. Sci. U. S. A. 106, 20228−20233.

(77) Gertsik, N., Chau, D. M., and Li, Y. M. (2015) gamma-Secretase Inhibitors and Modulators Induce Distinct Conformational Changes in the Active Sites of gamma-Secretase and Signal Peptide Peptidase. ACS Chem. Biol. 10, 1925. (78) Wang, Y., Zhang, Y., and Ha, Y. (2006) Crystal structure of a rhomboid family intramembrane protease. Nature 444, 179−180. (79) Wu, Z., Yan, N., Feng, L., Oberstein, A., Yan, H., Baker, R. P., Gu, L., Jeffrey, P. D., Urban, S., and Shi, Y. (2006) Structural analysis of a rhomboid family intramembrane protease reveals a gating mechanism for substrate entry. Nat. Struct. Mol. Biol. 13, 1084−1091. (80) Lemieux, M. J., Fischer, S. J., Cherney, M. M., Bateman, K. S., and James, M. N. (2007) The crystal structure of the rhomboid peptidase from Haemophilus influenzae provides insight into intramembrane proteolysis. Proc. Natl. Acad. Sci. U. S. A. 104, 750−754. (81) Ben-Shem, A., Fass, D., and Bibi, E. (2007) Structural basis for intramembrane proteolysis by rhomboid serine proteases. Proc. Natl. Acad. Sci. U. S. A. 104, 462−466. (82) Hu, J., Xue, Y., Lee, S., and Ha, Y. (2011) The crystal structure of GXGD membrane protease FlaK. Nature 475, 528−531. (83) Li, X., Dang, S., Yan, C., Gong, X., Wang, J., and Shi, Y. (2013) Structure of a presenilin family intramembrane aspartate protease. Nature 493, 56−61. (84) Strisovsky, K. (2013) Structural and mechanistic principles of intramembrane proteolysis–lessons from rhomboids. FEBS J. 280, 1579−1603. (85) Brooks, C. L., and Lemieux, M. J. (2013) Untangling structurefunction relationships in the rhomboid family of intramembrane proteases. Biochim. Biophys. Acta, Biomembr. 1828, 2862−2872. (86) Urban, S. (2010) Taking the plunge: integrating structural, enzymatic and computational insights into a unified model for membrane-immersed rhomboid proteolysis. Biochem. J. 425, 501−512. (87) Rawlings, N. D., Waller, M., Barrett, A. J., and Bateman, A. (2014) MEROPS: the database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res. 42, D503−509. (88) Hsieh, J. J., Cheng, E. H., and Korsmeyer, S. J. (2003) Taspase1: a threonine aspartase required for cleavage of MLL and proper HOX gene expression. Cell 115, 293−303. (89) Vinothkumar, K. R. (2011) Structure of rhomboid protease in a lipid environment. J. Mol. Biol. 407, 232−247. (90) Eilers, M., Patel, A. B., Liu, W., and Smith, S. O. (2002) Comparison of helix interactions in membrane and soluble alphabundle proteins. Biophys. J. 82, 2720−2736. (91) Walters, R. F., and DeGrado, W. F. (2006) Helix-packing motifs in membrane proteins. Proc. Natl. Acad. Sci. U. S. A. 103, 13658− 13663. (92) Hemming, M. L., Elias, J. E., Gygi, S. P., and Selkoe, D. J. (2008) Proteomic profiling of gamma-secretase substrates and mapping of substrate requirements. PLoS Biol. 6, e257. (93) Agard, N. J., and Wells, J. A. (2009) Methods for the proteomic identification of protease substrates. Curr. Opin. Chem. Biol. 13, 503− 509. (94) auf dem Keller, U., and Schilling, O. (2010) Proteomic techniques and activity-based probes for the system-wide study of proteolysis. Biochimie 92, 1705−1714. (95) Kuhn, P. H., Voss, M., Haug-Kroper, M., Schroder, B., Schepers, U., Brase, S., Haass, C., Lichtenthaler, S. F., and Fluhrer, R. (2015) Secretome analysis identifies novel signal Peptide peptidase-like 3 (Sppl3) substrates and reveals a role of Sppl3 in multiple Golgi glycosylation pathways. Mol. Cell. Proteomics 14, 1584−1598. (96) Kuhn, P. H., Koroniak, K., Hogl, S., Colombo, A., Zeitschel, U., Willem, M., Volbracht, C., Schepers, U., Imhof, A., Hoffmeister, A., Haass, C., Rossner, S., Brase, S., and Lichtenthaler, S. F. (2012) Secretome protein enrichment identifies physiological BACE1 protease substrates in neurons. EMBO J. 31, 3157−3168. (97) Binshtein, E., and Ohi, M. D. (2015) Cryo-electron microscopy and the amazing race to atomic resolution. Biochemistry 54, 3133− 3141. 2433


Reviews

ACS Chemical Biology (98) Bai, X. C., Yan, C., Yang, G., Lu, P., Ma, D., Sun, L., Zhou, R., Scheres, S. H., and Shi, Y. (2015) An atomic structure of human gamma-secretase. Nature 525, 212−217. (99) Broncel, M., Serwa, R. A., Ciepla, P., Krause, E., Dallman, M. J., Magee, A. I., and Tate, E. W. (2015) Multifunctional reagents for quantitative proteome-wide analysis of protein modification in human cells and dynamic profiling of protein lipidation during vertebrate development. Angew. Chem., Int. Ed. 54, 5948−5951. (100) Verdoes, M., Verhelst, S. H. L. Detection of protease activity in cells and animals, Biochim. Biophys. Acta, Proteins Proteomics 2015, DOI: 10.1016/j.bbapap.2015.04.029. (101) Deu, E., Verdoes, M., and Bogyo, M. (2012) New approaches for dissecting protease functions to improve probe development and drug discovery. Nat. Struct. Mol. Biol. 19, 9−16. (102) Zettl, M., Adrain, C., Strisovsky, K., Lastun, V., and Freeman, M. (2011) Rhomboid family pseudoproteases use the ER quality control machinery to regulate intercellular signaling. Cell 145, 79−91. (103) Adrain, C., and Freeman, M. (2012) New lives for old: evolution of pseudoenzyme function illustrated by iRhoms. Nat. Rev. Mol. Cell Biol. 13, 489−498. (104) Sun, L., Zhao, L., Yang, G., Yan, C., Zhou, R., Zhou, X., Xie, T., Zhao, Y., Wu, S., Li, X., and Shi, Y. (2015) Structural basis of human gamma-secretase assembly. Proc. Natl. Acad. Sci. U. S. A. 112, 6003− 6008.

2434


Chemical Tools for the Study of Intramembrane Proteases - American

Recommend Documents