Activity-Based Probes As a Tool for Functional Proteomic Analysis of Proteases
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Review Activity-based probes as a tool for functional proteomic analysis of proteases Expert Rev. Proteomics 5(5), 721–730 (2008) Marko Fonović† and Traditional proteomics methodology allows global ana lysis of protein abundance but does not Matthew Bogyo provide information on the regulation of protein activity. Proteases, in particular, are known for †Author for correspondence their multilayered post-translational activity regulation that can lead to a significant difference Department of Biochemistry, between protease abundance levels and their enzyme activity. To address these issues, the field Molecular and Structural of activity-based proteomics has been established in order to characterize protein activity and Biology, Jožef Stefan Institute, monitor the functional regulation of enzymes in complex proteomes. In this review, we present Jamova Cesta 39, SI-1000 structural features of activity-based probes for proteases and discuss their applications in Ljubljana, Slovenia proteomic profiling of various catalytic classes of proteases. Tel.: +386 014 773 474 [email protected] Keywords: activity-based probe • protease • proteolysis • proteomics All organisms express a large variety of struct- than activity do not always provide sufficient urally and catalytically diverse proteases. information to allow functional assignments of Genome-sequencing projects have revealed the specific proteases. presence of at least 569 human proteases that Traditional proteomics methodology, based on can be divided into several distinct catalytical 2D gel chromatography coupled with mass spec- classes: metallo proteinases, serine, cysteine, trometry (MS), has recently been complemented threonine and aspartic proteases [1] . Interestingly, with non-gel-based methods, such as MudPIT the mouse and rat genomes show even higher and SELDI [3,4]. Chromatography separations protease variety, with 644 and 629 members, employed in these methods significantly increase respectively [2]. Proteases have the unique abil- resolving power and proteome coverage, but they ity to hydrolyze peptide bonds and, therefore, still lack the ability to resolve active and inactive irreversibly modify the function of target pro- populations of enzymes. Activity-based proteo- teins. They play crucial roles in diverse physio- mics is a relatively new subdivision of proteomics logical processes, such as protein turnover, blood that has been developed to characterize protein coagulation, apoptosis, hormone processing and activity and directly monitor the functional regu- bone remodeling. Since proteolytic processing lation of enzymes in complex proteomes. This is an irreversible event, it must be tightly regu- method utilizes small molecule activity-based lated to avoid catastrophic consequences. The probes (ABPs) that covalently modify the enzyme most common way that proteases are regulated active site and enable detection and affinity puri- is by expression as an inactive proenzyme which fication of a target enzyme population (FIGURE 1). requires activation by proteolytic cleavage. The ABPs are highly selective and can be used for ana- activation process can be regulated by cellular lysis of complex samples, such as cell lysates, intact signaling as well as by changes in the micro- cells and even whole organisms. In this review, we environment. If this regulation fails, endo- will outline advances in the development of ABPs genous extra cellular and intracellular protease and highlight the numerous applications of these inhibitors (e.g., serpins and cystatins) prevent reagents for the study of various protease classes any unwanted proteolysis. This multilayered and their roles in human disease. post-translational regulation can lead to signifi- cant differences between overall abundance lev- Structure of activity-based probes els and activity of proteases. Thus, methods that All ABPs share a similar basic design that rely on detection of protease abundance rather incorporates elements required for targeting, www.expert-reviews.com 10.1586/14789450.5.5.721 © 2008 Expert Reviews Ltd ISSN 1478-9450 721 Review Fonović & Bogyo y Labeling with ABP Affinity capture Elution MS/MS Intensit m/z Whole proteome Analysis Identification Expert Rev. Proteomics © Future Science Group (2008) Figure 1. Workflow of activity-based proteomics. Target proteins in the complex proteome are labeled with activity-based probes. Labeled proteins are enriched by affinity purification and identified by mass spectrometry. ABP: Activity-based probes; MS/MS: Tandem mass spectrometry. modification and detection of labeled proteins. Structurally, the linker can be as simple as an extended alkyl or polyethylene those elements can be divided into the reactive group, linker glycol spacer. Alternatively, the linker can be used to increase probe and tag (FIGURE 2A). specificity by the addition of structural elements (i.e., peptides) that enhance the specificity of the probe. For most protease probes, Reactive group the linker is a short peptide sequence that mimics a true protein The reactive functional group, also termed ‘the warhead’, provides substrate. This peptide linker can be optimized to target distinct a covalent attachment of the probe to the catalytic residue of the subfamilies of proteases by changing its amino acid sequence [14] . protease active site. The majority of protease families have well- A more recent improvement in linker design has been the develop- established catalytic mechanisms that have been defined using ment of a region that can be enzymatically or chemically cleaved to abundant structural and kinetic data. These data have enabled the release probe-labeled proteins. This simplifies the elution process synthesis of a large variety of specific, irreversible small- molecule and decreases background protein contamination during an affinity inhibitors that can be transformed into ABPs by the addition of purification. Various cleavable linker elements have recently been an appropriate reporter tag [5]. Probes for serine, cysteine and reported. Cravatt and coworkers developed a cleavable linker that threonine proteases utilize electrophile groups that form a cova- has a peptide recognition site for tobacco etch virus protease and lent bond with their active site nucleophile (serine, cysteine or applied it to the identification of proteins that had been labeled with threonine residue). The electrophile has to be strong enough a sulfonate ester probe [15,16] . One of the first chemically cleavable to react with the active site nucleophile but not with other free linkers described makes use of a disulfide bond that is readily cleaved nucleophiles in the proteome (FIGURE 2B). Various reactive groups in mild reductive conditions (FIGURE 3A) [17–19] . Disulfide linkers work have been successfully applied to protease probe designs (Tab LE 1). well in some applications, such as profiling of serine hydrolases, but The phosphonate group is highly reactive towards the hydroxyl they are not compatible with reductive buffers that are needed for nucleophile of serine proteases and has therefore found use in a the profiling of cysteine proteases. Disulfides are also quite labile number of probes for both trypsin and chymotrypsin fold serine and prone to disulfide exchange, which can also lead to a premature proteases [6]. The proteasome, on the other hand, has been tar- release and nonspecific labeling of proteins containing free thiol geted with probes containing a vinyl sulfone reactive group even groups [20]. These problems have been addressed with the develop- though this class of compounds was originally designed to react ment of a novel diazo cleavable linker that enables selective elution with cysteine proteases [7]. Interestingly, a,b-epoxy ketones are also under mild reductive conditions (FIGURE 3A) [21] . The diazo linker was known to form highly selective bonds with the N-terminal threo- successfully incorporated into ABPs that were used for labeling and nine of the proteasome [8]. Cysteine proteases are the family most proteomic identification of cysteine and serine proteases [22]. As an extensively studied using ABPs and probes containing a number alternative to cleavage by chemical reduction, acid cleavable linkers of different reactive functional groups, including epoxides, diazo- have been developed and are also commercially available for isotope methyl ketones, acyloxymethyl ketones and vinyl sulfones, have coded affinity tag (ICAT) applications[23–25] . However, the strong been designed [9–11] . Unfortunately, the same electrophile-based acids required for cleavage, such as trifluoroacetic acid, also release approach does not work well for metalloproteinases and aspartic nonspecifically bound proteins. Finally, the linker region can be used proteinases because they do not form covalent intermediates with for isotopic labeling in ICAT-like applications. Incorporation of light their substrates. In this case, probes are designed using reversible and heavy linkers have been demonstrated on the general papain inhibitor scaffolds coupled to a photoreactive group that provides family protease probe DCG-04, which was used for quantitative covalent attachment upon irradiation with UV light [12,13]. profiling of cysteine proteases by direct MS-based methods [26]. Linker Tag The most basic function of the linker is to separate the reactive Incorporation of a tag group into an activity-based probe functional group from the tag. This improves probe accessi bility enables the visualization