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Proteolysis within the membrane is catalyzed by a diverse family of proteases immersed within the hydrophobic environment of cellular membranes. These ubiquitous intramembrane-cleaving proteases (I-CLiPs) hydrolyze the transmembrane domains of a large variety of membrane-embedded proteins to facilitate signaling events essential to normal biological functions found in all forms of life. The importance of this unique class of enzyme is highlighted by its central involvement in a variety of human pathologies, including Alzheimer’s disease, Parkinson’s disease, cancer and the virulence of a number of viral, bacterial and fungal pathogens. I-CLiPs therefore represent promising targets for the therapeutic treatment of numerous diseases. The key to understanding the normal biological function of I-CLiPs and capitalizing on their therapeutic potential is through a thorough understanding of the complex catalytic mechanisms that govern this unusual class of enzyme. This is an intrinsically difficult endeavor, given that these enzymes and their substrates reside within lipid membranes, making any in vitro assay technically challenging to design and execute. Here we describe several in vitro enzymatic assays for the study of the Alzheimer’s disease associated γ-secretase protease, which have aided the development of potent γ-secretase-targeting compounds as candidate therapeutics. These assays have also been applied in various forms for the study of other I-CLiPs, providing valuable mechanistic insights into some of the functional similarities and differences between several members of this fascinating family of proteases.
Intramembrane-cleaving proteases (I-CLiPs) are ubiquitous membrane proteins found in all forms of life (Urban, 2013). They catalyze the cleavage of the transmembrane domain of many membrane-embedded proteins to facilitate important cellular signaling events. The first I-CLiP to be discovered, site-2 protease (S2P), was uncovered while investigating the cellular signaling events that regulate cholesterol homeostasis (Rawson et al., 1997). This zinc metalloprotease is also involved in the ER unfolded protein response (Ye et al., 2000). Shortly thereafter, an aspartyl protease named presenilin was found to be responsible for γ-secretase activity (Wolfe et al., 1999). This unique protease is responsible for the intramembrane cleavage of both notch receptors (De Strooper et al., 1999) and amyloid precursor protein (APP) (Li et al., 2000a; Wolfe et al., 1999), putting the enzyme at the center of both metazoan developmental biology and Alzheimer’s disease (AD). Shortly thereafter, a family of serine intramembrane proteases named rhomboid were found to regulate important signaling events driving Drosophila development (Urban et al., 2001). Conspicuously absent, an intramembrane protease utilizing a catalytic cysteine has yet to be identified. Together, the discovery of these three families of intramembrane proteases founded a new field, termed regulated intramembrane proteolysis (RIP) (Brown et al., 2000).
Of the many I-CLiPs distributed ubiquitously throughout nature, the presenilin/γ-secretase complex is arguably the most well-studied intramembrane protease. It’s involvement in notch signaling and the generation of potentially pathogenic amyloid beta (Aβ) peptides via cleavage of APP in Alzheimer’s disease has thrust this unusual protease into the spotlight. γ-secretase is a multicomponent complex comprised of the catalytic presenilin, Pen-2, Aph-1 and nicastrin (Edbauer et al., 2003; Kimberly et al., 2003; Takasugi et al., 2003). Pen-2 and Aph-1 are thought play a scaffolding role within the complex (LaVoie et al., 2003; Takasugi et al., 2003), while nicastrin functions to sterically occlude non-substrates from interacting with the protease (Bolduc et al., 2016). All four components are required for complex assembly and full activity. Presenilin’s requirement of cofactors for activity is unique among other members of the aspartly intramembrane protease family. Other aspartyl I-CLiPs such as signal peptide peptidase (SPP) and SPP-like proteases are thought to function alone, without the need for other protein cofactors or subunits (Voss et al., 2013).
Initially, the notion that proteolysis could occur within the hydrophobic environment of cellular membranes was met with skepticism. How could the catalytic water required for hydrolysis enter the lipid bilayer where water is normally excluded? The purification of recombinant intramembrane proteases and the subsequent development of in vitro enzymatic assays directly demonstrated that these I-CLiPs were indeed catalyzing hydrolysis within the transmembrane domain of their substrates. Later, high-resolution structures of rhomboid (Wang et al., 2006), an archeal S2P homolog (Feng et al., 2007) and more recently γ-secretase (Bai et al., 2015) revealed that the catalytic amino acids of each of these enzymes reside within their membrane-immersed transmembrane domains.
In this chapter we describe three in vitro enzymatic assays regularly used in our labs for the study of γ-secretase catalysis. We and others have utilized these assays and variations thereof to dissect the intricate catalytic mechanisms that govern these truly unique enzymes.
Detergent-solubilized assays require the solubilization of both the intramembrane protease and its substrate in a detergent system that allows for catalytic activity. In the case of γ-secretase, a relatively weak zwitterionic detergent CHAPSO was chosen, as this detergent allowed for the γ-secretase complex to remain intact, whereas most other detergents dissociated the complex (Li et al., 2000b). These assays have been described for at least one member of each class of intramembrane-cleaving proteases and were the first assays developed for studying I-CLiPs in vitro (Li et al., 2000b; Urban and Wolfe, 2005; Weihofen et al., 2002). The detergents useful for studying these I-CLiPs in a soluble state have been determined empirically. From a technical standpoint, detergent-solubilized assays are easier to design and implement than more complex proteoliposome assays. However, because of their more artificial nature—removing enzyme and substrate from physiologic lipid membranes—detergent-solubilized assays sometimes fail to fully recapitulate key mechanistic features imparted by the intact lipid bilayer.
Although the detergents are used to solubilize enzyme and substrate, the addition of lipids to the reaction mixture has been shown to be necessary for presenilin/γ-secretase activity. Furthermore, certain lipid compositions modulate not only the total activity of the enzyme but also the relative amounts of Aβ species produced from the cleavage of the APP based substrate C100-FLAG (Holmes et al., 2012; Osenkowski et al., 2008). Below is the description of a CHAPSO-solubilized assay for the study of γ-secretase.
An enzyme-incorporated proteoliposome assay involves incorporating an intramembrane protease into a lipid vesicle to generate a proteoliposome and subsequently adding detergent-solubilized substrate to initiate the reaction. Immersing an intramembrane protease into the lipid bilayer of a proteoliposome has been shown to more accurately recapitulate key enzyme-substrate interactions compared to the detergent-solubilized assay. For example, we find that γ-secretase processing of the APP-based substrate C100-FLAG produces an Aβ42/40 ratio closer to the physiologic ratio of ~0.2 when γ-secretase is incorporated into proteoliposomes (Holmes et al., 2012); γ-secretase cleavage of C100-FLAG in the CHAPSO-solubilized assay (above) produces an artificially elevated ratio. Similarly, rhomboid cleavage site-specificity of Spitz substrate is maintained only in the context of the lipid bilayer (Moin and Urban, 2012).
Although the enzyme-incorporated proteoliposome assay allows for the more accurate study of enzyme-substrate-lipid interactions than the detergent-solubilized assay, it still fails to allow for the precise determination of the kinetic variables kcat and Km due to the fact that the substrate is added to the reaction mixture in the detergent-solubilized state (see below). Nevertheless, as described below for γ-secretase, this assay has proved to be a facile and robust means to study I-CLiPs while recapitulating some of the key regulatory features of lipid membranes on catalytic activity.
Both the detergent-solubilized assay and enzyme-incorporated proteoliposome assay outlined above have provided valuable insights into the function and regulation of γ-secretase and other I-CLiPs. However, both assays are artificial in that normally γ-secretase enzyme and its substrates are both confined to a lipid bilayer under physiologic conditions rather than substrate (and also enzyme in the case of the CHAPSO assay) being solubilized in detergent. Studying γ-secretase (or other I-CLiPs) exclusively in artificial detergent-solubilized conditions runs the risk of measuring artifacts and/or missing out on key features of the enzyme’s catalytic mechanism present only when both enzyme and substrate are oriented in a 2-dimensional lipid bilayer.
When studying the kinetic parameters of any enzyme, an in vitro assay must be designed with defined start and stop times for initiating and terminating enzymatic activity. Accomplishing this with an intramembrane protease having both enzyme and substrate incorporated into a proteoliposome is inherently difficult, given that any procedure for generating proteoliposomes is laborious and time-consuming, during which period the enzyme will have free access to substrate. Thus, an inducible proteoliposome assay was needed to study I-CLiPs with a mechanism in place to initiate cleavage only when desired. To this end, Urban and colleagues recently developed an innovative and elegant, yet conceptually simple, inducible proteoliposome assay to study rhomboid intramembrane kinetics (Dickey et al., 2013). As outlined below (Fig. 1), we have adapted this assay to study γ-secretase cleavage of a notch-based substrate (Bolduc et al., 2016).
For the serine protease GlpG rhomboid, Urban and coworkers found that the nucleophilic serine rotated to an inactive orientation within the enzyme active site when the catalytic histidine was protonated at low pH (Dickey et al., 2013). They therefore formed proteoliposomes containing both rhomboid and TatA substrate at low pH to prevent premature substrate cleavage and subsequently raised the pH to initiate the reaction after the proteoliposomes were formed. We took a similar approach with the aspartyl protease γ-secretase, forming the proteoliposomes at a basic pH before initiating the reaction by returning the reaction to neutral pH (details below). Presumably, basic pH conditions deprotonate both catalytic aspartates of γ-secretase, rendering it inactive (Quintero-Monzon et al., 2011). Although this method has only been applied to rhomboid and γ-secretase to date, a similar approach should work for other intramembrane proteases, assuming any pH-induced conformational changes in enzyme are rapidly reversible upon pH neutralization.
Initially, we attempted to use the same N100-FLAG notch based substrate for this inducible proteoliposome assay as we previously used for the detergent-solubilized assays discussed above. However, we encountered two technical hurdles that forced us to use an alternative substrate: 1) the recombinant notch substrate was inefficiently incorporated into vesicles by detergent dilution; 2) the substrate oriented primarily in one direction when inserted into the membrane (N-terminus inside the vesicle). We hypothesized that this may be due the presence of the large and highly charged intracellular domain of the recombinant N100-FLAG substrate. We therefore designed a synthetic substrate in which the intracellular domain of notch is replaced with fluorescein (N43, see below) to allow us to monitor the cleavage reaction. As discussed below, this substrate allowed for efficient proteoliposome incorporation as well as roughly equal N-terminal orientations upon insertion into the lipid bilayer. Urban and colleagues similarly used a short synthetic TatA peptide tagged with fluorescein in their assay. Together, this may argue that future inducible proteoliposome assays developed to study other I-CLiPs should attempt to utilize short synthetic peptides as substrates as well, as they appear to be better behaved in this assay system than are large recombinant substrates.
In contrast to the detergent-solubilized assays, the inducible proteoliposome assay allows for the precise calculation of physiologically meaningful kcat and Km values. In both the detergent-solubilized assay and the γ-secretase-incorporated proteoliposome assay, the substrate is solubilized in detergent prior to being added to the reaction mixture. Km values for substrate must therefore be calculated in terms of molarity here, which makes no physiologic sense, as substrate and enzyme are normally confined to the 2-dementional lipid bilayer within cells. For the inducible proteoliposome assay, intramembrane Km values are calculated and expressed in terms of mole percent with respect to total lipid concentration. γ-secretase and rhomboid have been found to have vastly different intramembrane Km values using this assay, demonstrating these two I-CLiPs have evolved very different mechanisms for substrate recognition (Bolduc et al., 2016; Dickey et al., 2013).
kcat values calculated in the detergent-solubilized or enzyme-incorporated proteoliposome assay can potentially be misleading. Although intramembrane proteolysis by I-CLiPs appears to be an intrinsically slow process (Dickey et al., 2013; Kamp et al., 2015), the detergent-solubilized substrate assays require substrate to partition between detergent and/or detergent-lipid states, which could potentially be rate-limiting in the enzymatic reaction. The inducible proteoliposome assay eliminates the requirement of detergent, therefore allowing for a more accurate calculation of kcat and intramembrane Km values. The description of an inducible proteoliposome assay below was adapted by us from Urban and colleagues for the study of γ-secretase.
Synthetic notch-based substrates for utilization in the inducible proteoliposome assay:
Intramembrane cleaving proteases represent a unique class of enzyme at the forefront of biology and medicine. A thorough understanding of the intramembrane protease catalytic mechanism will be required to fully understand how these proteases govern important biological processes as well as allow researchers to capitalize on the therapeutic potential of these enzymes. This can only be accomplished with robust and facile methods by which to study the activity of these enzymes in vitro. Although the mysteries of the intramembrane protease cleavage mechanism are only now beginning to be unraveled, the enzymatic assays outlined here have proven highly valuable for interrogating the function of γ-secretase and other members of the I-CLiP family.
This work was supported by NIH grant P01 AG15379.