Dynamic imaging of protease activity with fluorescently quenched activity-based probes

Protease activity is tightly regulated in both normal and disease conditions. However, it is often difficult to monitor the dynamic nature of this regulation in the context of a live cell or whole organism. To address this limitation, we developed a series of quenched activity-based probes (qABPs) that become fluorescent upon activity-dependent covalent modification of a protease target. These reagents freely penetrate cells and allow direct imaging of protease activity in living cells. Targeted proteases are directly identified and monitored biochemically by virtue of the resulting covalent tag, thereby allowing unambiguous assignment of protease activities observed in imaging studies. We report here the design and synthesis of a selective, cell-permeable qABP for the study of papain-family cysteine proteases. This probe is used to monitor real-time protease activity in live human cells with fluorescence microscopy techniques as well as standard biochemical methods.     

Noninvasive optical imaging of cysteine protease activity using fluorescently quenched activity-based probes

We have generated a series of quenched near-infrared fluorescent activity-based probes (qNIRF-ABPs) that covalently target the papain-family cysteine proteases shown previously to be important in multiple stages of tumorigenesis. These 'smart' probes emit a fluorescent signal only after covalently modifying a specific protease target. After intravenous injection of NIRF-ABPs into mice bearing grafted tumors, noninvasive, whole-body imaging allowed direct monitoring of cathepsin activity. Importantly, the permanent nature of the probes also allowed secondary, ex vivo biochemical profiling to identify specific proteases and to correlate their activity with whole-body images. Finally, we demonstrate that these probes can be used to monitor small-molecule inhibition of protease targets both biochemically and by direct imaging methods. Thus, NIRF-ABPs are (i) potentially valuable new imaging agents for disease diagnosis and (ii) powerful tools for preclinical and clinical testing of small-molecule therapeutic agents in vivo.     

Non-invasive imaging of cysteine cathepsin activity in solid tumors using a 64Cu-labeled activity-based probe

The papain family of cysteine cathepsins are actively involved in multiple stages of tumorigenesis. Because elevated cathepsin activity can be found in many types of human cancers, they are promising biomarkers that can be used to target radiological contrast agents for tumor detection. However, currently there are no radiological imaging agents available for these important molecular targets. We report here the development of positron emission tomography (PET) radionuclide-labeled probes that target the cysteine cathepsins by formation of an enzyme activity-dependent bond with the active site cysteine. These probes contain an acyloxymethyl ketone (AOMK) functional group that irreversibly labels the active site cysteine of papain family proteases attached to a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) tag for labeling with (64)Cu for PET imaging studies. We performed biodistribution and microPET imaging studies in nude mice bearing subcutaneous tumors expressing various levels of cysteine cathepsin activity and found that the extent of probe uptake by tumors correlated with overall protease activity as measured by biochemical methods. Furthermore, probe signals could be reduced by pre-treatment with a general cathepsin inhibitor. We also found that inclusion of a Cy5 tag on the probe increased tumor uptake relative to probes lacking this fluorogenic dye. Overall, these results demonstrate that small molecule activity-based probes carrying radio-tracers can be used to image protease activity in living subjects.     

Evaluation of alpha,beta-unsaturated ketone-based probes for papain-family cysteine proteases

The field of activity-based proteomics makes use of small molecule active site probes to monitor distinct subsets of enzymatic proteins. While a number of reactive functional groups have been applied to activity-based probes (ABPs) that target diverse families of proteases, there remains a continual need for further evaluation of new probe scaffolds and reactive functional groups for use in ABPs. In this study we evaluate the utility of the, alpha,beta-unsaturated ketone reactive group for use in ABPs targeting the papain-family of cysteine proteases. We find that this reactive group shows highly selective labeling of cysteine cathepsins in both intact cells and total cell extracts. We observed a variable degree of background labeling that depended on the type of tag and linker used in the probe synthesis. The relative ease of synthesis of this class of compounds provides the potential for further derivatization to generate new families of cysteine protease ABPs with unique specificity and labeling properties.     

Chemical-proteomic strategies to investigate cysteine posttranslational modifications

The unique combination of nucleophilicity and redox-sensitivity that is characteristic of cysteine residues results in a variety of posttranslational modifications (PTMs), including oxidation, nitrosation, glutathionylation, prenylation, palmitoylation and Michael adducts with lipid-derived electrophiles (LDEs). These PTMs regulate the activity of diverse protein families by modulating the reactivity of cysteine nucleophiles within active sites of enzymes, and governing protein localization between soluble and membrane-bound forms. Many of these modifications are highly labile, sensitive to small changes in the environment, and dynamic, rendering it difficult to detect these modified species within a complex proteome. Several chemical-proteomic platforms have evolved to study these modifications and enable a better understanding of the diversity of proteins that are regulated by cysteine PTMs. These platforms include: (1) chemical probes to selectively tag PTM-modified cysteines; (2) differential labeling platforms that selectively reveal and tag PTM-modified cysteines; (3) lipid, isoprene and LDE derivatives containing bioorthogonal handles; and (4) cysteine-reactivity profiling to identify PTM-induced decreases in cysteine nucleophilicity. Here, we will provide an overview of these existing chemical-proteomic strategies and their effectiveness at identifying PTM-modified cysteine residues within native biological systems.     

Cystatins, thyropins and inhibitors homologous to propeptides of cysteine proteases

Protein inhibitors of proteolytic enzymes play an important role in regulating the activity of endogenous proteases and in host defense mechanisms against pathogens preventing the deleterious effects of exogenous proteases. In recent years a great interest in protein inhibitors of cysteine proteases has increased due to the extensive growth of knowledge about the contribution of cysteine proteases to pathological processes associated with many human diseases, as well as due to prospects for treatment of these disorders which may arise from the thorough understanding of their inhibitory mechanisms. This paper reviews the most important aspects of three families of cysteine protease inhibitors: cystatins, thyropins and inhibitors homologous to propeptides of cysteine proteases. Special attention is given to structural bases of the interactions between the inhibitors and their target enzymes. The paper presents a general characterization of the families according to the MEROPS classification of protease inhibitors, pointing out new members.     

Specificity of aza-peptide electrophile activity-based probes of caspases

Activity-Based Probes (ABPs) are small molecules that form stable covalent bonds with active enzymes thereby allowing detection and quantification of their activities in complex proteomes. A number of ABPs that target proteolytic enzymes have been designed based on well-characterized mechanism-based inhibitors. We describe here the evaluation of a novel series of ABPs based on the aza-aspartate inhibitory scaffold. Previous in vitro kinetic studies showed that this scaffold has a high degree of selectivity for the caspases, clan CD cysteine proteases activated during apoptotic cell death. Aza-aspartate ABPs containing either an epoxide or Michael acceptor reactive group were potent labels of executioner caspases in apoptotic cell extracts. However they were also effective labels of the clan CD protease legumain and showed unexpected crossreactivity with the clan CA protease cathepsin B. Interestingly, related aza peptides containing an acyloxymethyl ketone reactive group were relatively weak but highly selective labels of caspases. Thus azapeptide electrophiles are valuable new ABPs for both detection of a broad range of cysteine protease activities and for selective targeting of caspases. This study also highlights the importance of confirming the specificity of covalent protease inhibitors in crude proteomes using reagents such as the ABPs described here.     

Chemistry-based functional proteomics: mechanism-based activity-profiling tools for ubiquitin and ubiquitin-like specific proteases

Determining the biological function of newly discovered gene products requires the development of novel functional approaches. To facilitate this task, recent developments in proteomics include small molecular probes that target proteolytic enzyme families including serine, threonine, and cysteine proteases. For the families of ubiquitin (Ub) and ubiquitin-like (UBL)-specific proteases, such tools were lacking until recently. Here, we review the advances made in the development of protein-based active site-directed probes that target proteases specific for ubiquitin and ubiquitin-like proteins. Such probes were applied successfully to discover and characterize novel Ub/UBL-specific proteases. Ub/UBL processing and deconjugation are performed by a diverse set of proteases belonging to several different enzyme families, including members of the ovarian tumor domain (OTU) protease family. A further definition of this family of enzymes will benefit from a directed chemical proteomics approach. Some of the Ub/UBL-specific proteases react with multiple Ub/UBLs and members of the same protease family can recognize multiple Ub/UBLs, underscoring the need for tools that appropriately address enzyme specificity.     

Degradation of T-cell receptor chains in the endoplasmic reticulum is inhibited by inhibitors of cysteine proteases.

The endoplasmic reticulum, or an organelle closely associated with it, contains proteases that can be used to remove partially assembled or improperly folded proteins. Very little is known at present about the types of protease that degrade these proteins. The beta chain and cluster of differentiation (CD)3 delta subunit of the human T-cell antigen receptor (TCR) are degraded shortly after synthesis. In this study Chinese hamster ovary (CHO) cells transfected with either beta or delta were incubated with a panel of protease inhibitors, and the rates of degradation of the transfected proteins were followed using chain-specific enzyme-linked immunosorbent assays (ELISAs). Of the protease inhibitors tested, degradation of both chains was highly sensitive to sulfhydryl reagents and peptidyl inhibitors of cysteine proteases. Concentrations of inhibitors that produced near complete inhibition of degradation in the endoplasmic reticulum did not cause gross changes in cellular ATP levels nor did they significantly slow constitutive secretion from CHO cells. The inhibitors did not affect the ability of CHO cells to synthesize and assemble disulphide-linked TCR zeta dimers. We conclude that the protease inhibitors were not toxic to cells and did not affect the biosynthetic activity of the endoplasmic reticulum. Furthermore, they did not alter the ability of the endoplasmic reticulum to deliver its content to the Golgi apparatus. Taken together, these results suggest that the cysteine protease inhibitors slow degradation in the endoplasmic reticulum through an action on cysteine proteases. The results imply that the endoplasmic reticulum contains cysteine proteases that can be used to remove retained proteins.     

A cysteine protease from maize isolated in a complex with cystatin

We recently purified a latent but SDS-activated protease complex (40, 15- or 13-kDa proteins) from maize [Yamada et al. (1998) Plant Cell Physiol. 39: 106]. Here, we revealed that the complex was composed of a cysteine protease (40 kDa) and a cystatin, cysteine protease inhibitor (15- or 13-kDa). This is the first report on the isolation of a complex consisting of a cystatin and a target cysteine protease from plants. Cloning of the cysteine protease revealed that it had low homology (25-30%) to other maize cysteine proteases cloned to date but was highly homologous to other plant cysteine proteases such as rice oryzain alpha (84%) and the homologs (50-80%). The cysteine protease expressed in Escherichia coli showed the same substrate and inhibitor specificities as the protease of the complex, demonstrating that the isolated cDNA clone exactly encodes the protease of the complex. The protease expressed in E. coli itself was active but not latent, probably because it was not bound to cystatin. It is most likely that in vitro activation of the protease complex by SDS is caused by the release of bound cystatin. The mRNA of protease was expressed in various tissues except for seeds.     

The involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants

Programmed cell death (PCD) is a process by which cells in many organisms die. The basic morphological and biochemical features of PCD are conserved between the animal and plant kingdoms. Cysteine proteases have emerged as key enzymes in the regulation of animal PCD. Here, we show that in soybean cells, PCD-activating oxidative stress induced a set of cysteine proteases. The activation of one or more of the cysteine proteases was instrumental in the PCD of soybean cells. Inhibition of the cysteine proteases by ectopic expression of cystatin, an endogenous cysteine protease inhibitor gene, inhibited induced cysteine protease activity and blocked PCD triggered either by an avirulent strain of Pseudomonas syringae pv glycinea or directly by oxidative stress. Similar expression of serine protease inhibitors was ineffective. A glutathione S-transferase-cystatin fusion protein was used to purify and characterize the induced proteases. Taken together, our results suggest that plant PCD can be regulated by activity poised between the cysteine proteases and the cysteine protease inhibitors. We also propose a new role for proteinase inhibitor genes as modulators of PCD in plants.     

Design of cell-permeable, fluorescent activity-based probes for the lysosomal cysteine protease asparaginyl endopeptidase (AEP)/legumain

Asparaginyl endopeptidase (AEP), also known as legumain, is a cysteine protease that has been ascribed roles in antigen presentation yet its exact role in human biology remains poorly understood. We report here, the use of a positional scanning combinatorial library of peptide AOMKs containing a P1 aspartic acid to probe the P2, P3, and P4 subsite specificity of endogenous legumain. Using inhibitor specificity profiles of cathepsin B and legumain, we designed fluorescent ABPs that are highly selective, cell-permeable reagents for monitoring legumain activity in complex proteomes.     

African swine fever virus protease, a new viral member of the SUMO-1-specific protease family

African swine fever virus (ASFV) is a complex DNA virus that employs polyprotein processing at Gly-Gly-Xaa sites as a strategy to produce several major core components of the viral particle. The virus gene S273R encodes a 31-kDa protein that contains a "core domain" with the conserved catalytic residues characteristic of SUMO-1-specific proteases and the adenovirus protease. Using a COS cell expression system, it was found that protein pS273R is capable of cleaving the viral polyproteins pp62 and pp220 in a specific way giving rise to the same intermediates and mature products as those produced in ASFV-infected cells. Furthermore, protein pS273R, like adenovirus protease and SUMO-1-specific enzymes, is a cysteine protease, because its activity is abolished by mutation of the predicted catalytic histidine and cysteine residues and is inhibited by sulfhydryl-blocking reagents. Protein pS273R is expressed late after infection and is localized in the cytoplasmic viral factories, where it is found associated with virus precursors and mature virions. In the virions, the protein is present in the core shell, a domain where the products of the viral polyproteins are also located. The identification of the ASFV protease will allow a better understanding of the role of polyprotein processing in virus assembly and may contribute to our knowledge of the emerging family of SUMO-1-specific proteases.     

SP3-Enabled Rapid and High Coverage Chemoproteomic Identification of Cell-State–Dependent Redox-Sensitive Cysteines

Proteinaceous cysteine residues act as privileged sensors of oxidative stress. As reactive oxygen and nitrogen species have been implicated in numerous pathophysiological processes, deciphering which cysteines are sensitive to oxidative modification and the specific nature of these modifications is essential to understanding protein and cellular function in health and disease. While established mass spectrometry-based proteomic platforms have improved our understanding of the redox proteome, the widespread adoption of these methods is often hindered by complex sample preparation workflows, prohibitive cost of isotopic labeling reagents, and requirements for custom data analysis workflows. Here, we present the SP3-Rox redox proteomics method that combines tailored low cost isotopically labeled capture reagents with SP3 sample cleanup to achieve high throughput and high coverage proteome-wide identification of redox-sensitive cysteines. By implementing a customized workflow in the free FragPipe computational pipeline, we achieve accurate MS1-based quantitation, including for peptides containing multiple cysteine residues. Application of the SP3-Rox method to cellular proteomes identified cysteines sensitive to the oxidative stressor GSNO and cysteine oxidation state changes that occur during T cell activation.     

SP3-Enabled Rapid and High Coverage Chemoproteomic Identification of Cell-State–Dependent Redox-Sensitive Cysteines

Proteinaceous cysteine residues act as privileged sensors of oxidative stress. As reactive oxygen and nitrogen species have been implicated in numerous pathophysiological processes, deciphering which cysteines are sensitive to oxidative modification and the specific nature of these modifications is essential to understanding protein and cellular function in health and disease. While established mass spectrometry-based proteomic platforms have improved our understanding of the redox proteome, the widespread adoption of these methods is often hindered by complex sample preparation workflows, prohibitive cost of isotopic labeling reagents, and requirements for custom data analysis workflows. Here, we present the SP3-Rox redox proteomics method that combines tailored low cost isotopically labeled capture reagents with SP3 sample cleanup to achieve high throughput and high coverage proteome-wide identification of redox-sensitive cysteines. By implementing a customized workflow in the free FragPipe computational pipeline, we achieve accurate MS1-based quantitation, including for peptides containing multiple cysteine residues. Application of the SP3-Rox method to cellular proteomes identified cysteines sensitive to the oxidative stressor GSNO and cysteine oxidation state changes that occur during T cell activation.     

Biosynthesis of lysosomal endopeptidases

Despite the clear differences between the amino acid sequence and enzymatic specificity of aspartic and cysteine endopeptidases, the biosynthetic processing of lysosomal members of these two families is very similar. With in vitro translation and pulse-chase analysis in tissue culture cells, the biosynthesis of cathepsin D, a aspartic protease, and cathepsins B, H and L, cysteine proteases, are compared. Both aspartic and cysteine endopeptidases undergo cotranslational cleavage of an amino-terminal signal peptide that mediates transport across the endoplasmic reticulum (ER) membrane. Addition of high-mannose carbohydrate also occurs cotranslationally in the lumen of the ER. Proteases of both enzyme classes are initially synthesized as inactive proenzymes possessing amino-terminal activation peptides. Removal of the propeptide generates an active single-chain enzyme. Whether the single-chain enzyme undergoes asymmetric cleavage into a light and a heavy chain appears to be cell type specific. Finally, late during their biosynthesis both classes of enzymes undergo amino acid trimming, losing a few amino acid residues at the cleavage site between the light and heavy chains and/or at their carboxyltermini. During biosynthesis these enzymes are also secreted to some extent. In most cells the secreted enzyme is the proenzyme bearing some complex carbohydrate. Under certain physiological conditions the inactive secreted enzymes may become activated as a result of a conformational change that may or may not result in autolysis. Analysis of the biochemical nature of the various processing steps helps define the cellular pathway followed by newly synthesized proteases targeted to the lysosome.     

Ritonavir does not inhibit calpain in vitro

Ritonavir, an inhibitor of HIV-1 protease, has been reported to also inhibit the Ca2+-dependent cysteine protease, calpain. We have investigated these claims with an in vitro study of the effect of ritonavir on the m-calpain and mu-calpain isoforms. Ritonavir failed to block either autolytic or hydrolytic calpain activity, but remained fully capable of inhibiting the HIV-1 protease. Any calpain-related effects of ritonavir in cells must, therefore, arise by a mechanism other than direct inhibition of calpains.     

Subunit interaction sites between the regulatory and catalytic subunits of cAMP-dependent protein kinase. Heterobifunctional cross-linking reagents lead to photodependent and photoindependent cross-linking

Heterobifunctional cross-linking reagents have been introduced into the catalytic subunit of cAMP-dependent protein kinase as potential probes for identifying specific points of contact between the catalytic (C)-subunit and the type II regulatory (RII) subunit in the holoenzyme complex. Since at least one of the 2 cysteine residues in the C-subunit is known to be in close proximity to the interaction site between the C-subunit and the RII-subunit, these cysteines were chosen initially as targets for covalent modification by two heterobifunctional cross-linking reagents, p-azidophenacyl bromide and N-4-(azidophenylthio)phthalimide. Treatment of the C-subunit with each reagent led to the stoichiometric modification of Cys-199 and Cys-343. In each case, the modified C-subunit was still capable of forming a stable complex with the RII-subunit. Both modified C-subunits also could be covalently cross-linked to the RII-subunit; however, the mechanisms for cross-linking differed. Catalytic subunit modified by p-azidophenacyl bromide was cross-linked to the RII-subunit in a photodependent manner by a mechanism that was maximal when holoenzyme was formed and cAMP was absent. In contrast, the C-subunit modified by N-4-(azidophenylthio)phthalimide was cross-linked to the RII-subunit by a mechanism that was independent of photolysis. In this case, cross-linking was enhanced by the presence of cAMP. This cross-linking was the result of a disulfide interchange between a modified cysteine in the C-subunit and an unmodified cysteine in the RII-subunit.     

Design and synthesis of visible isotope-coded affinity tags for the absolute quantification of specific proteins in complex mixtures

Identification of proteins in complex mixtures by mass spectrometry is most useful when quantitative data is also obtained. We recently introduced isotope-coded affinity tags (ICAT reagents) for the relative quantification of proteins present in two or more biological samples. In this report, we describe a new generation of ICAT reagents that contain the following additional features: (1) a visible tag that allows the electrophoretic position of tagged peptides during separation to be easily monitored; (2) a photocleavable linker that allows most of the tag to be removed prior to mass spectrometric analysis; (3) an isotope tag that contains carbon-13 and nitrogen-15 atoms instead of deuterium to ensure precise comigration of light and heavy tagged peptides by reverse-phase HPLC. These reagents contain an iodoacetyl group that selectively reacts with peptide cysteine residues. Peptide modification chemistry is also reported that allows tagging of peptides that are devoid of cysteine. The synthesis of these visible isotope-coded affinity tags (VICAT reagents), and their reaction with peptides are described in this report. VICAT reagents containing a carbon-14 visible probe or an NBD fluorophore are described. These reagents are most useful for the determination of the absolute quantity of specific target proteins in complex protein mixtures such as serum or cell lysates.