We have identified short N,C-capped peptides that selectively inhibit the proteasome of the malaria-causing pathogen Plasmodium falciparum. These compounds are highly potent in culture with no toxicity in host cells. One cyclic biphenyl ether compound inhibited intraerythrocytic growth of P. falciparum with an IC50 of 35 nM, and we show that even a pulse treatment with this cyclic peptide induced parasite death due to proteasome inhibition. These compounds represent promising new antimalarial agents that target the essential proteasomal machinery of the parasite without toxicity toward the host.
The great complexity of many human pathologies, such as cancer, diabetes, and neurodegenerative diseases, requires new tools for studies of biological processes on the whole organism level. The discovery of novel biocompatible reactions has tremendously advanced our understanding of basic biology; however, no efficient tools exist for real-time non-invasive imaging of many human proteases that play very important roles in multiple human disorders. We recently reported that the "split luciferin" biocompatible reaction represents a valuable tool for evaluation of protease activity directly in living animals using bioluminescence imaging (BLI). Since BLI is the most sensitive in vivo imaging modality known to date, this method can be widely applied for the evaluation of the activity of multiple proteases, as well as identification of their new peptide-specific substrates. In this unit, we describe several applications of this "split luciferin" reaction for quantification of protease activities in test tube assays and living animals.
The ubiquitin-proteasome system (UPS) is a potential pathway for therapeutic intervention for pathogens such as Plasmodium, the causative agent of malaria. However, due to the essential nature of this proteolytic pathway, proteasome inhibitors must avoid inhibition of the host enzyme complex to prevent toxic side effects. The Plasmodium proteasome is poorly characterized, making rational design of inhibitors that induce selective parasite killing difficult. In this study, we developed a chemical probe that labels all catalytic sites of the Plasmodium proteasome. Using this probe, we identified several subunit selective small molecule inhibitors of the parasite enzyme complex. Treatment with an inhibitor that is specific for the ?5 subunit during blood stage schizogony led to a dramatic decrease in parasite replication while short-term inhibition of the ?2 subunit did not affect viability. Interestingly, coinhibition of both the ?2 and ?5 catalytic subunits resulted in enhanced parasite killing at all stages of the blood stage life cycle and reduced parasite levels in vivo to barely detectable levels. Parasite killing was achieved with overall low host toxicity, something that has not been possible with existing proteasome inhibitors. Our results highlight differences in the subunit dependency of the parasite and human proteasome, thus providing a strategy for development of potent antimalarial drugs with overall low host toxicity.
Proteolytic enzymes are key signaling molecules in both normal physiological processes and various diseases. After synthesis, protease activity is tightly controlled. Consequently, levels of protease messenger RNA and protein often are not good indicators of total protease activity. To more accurately assign function to new proteases, investigators require methods that can be used to detect and quantify proteolysis. In this review, we describe basic principles, recent advances, and applications of biochemical methods to track protease activity, with an emphasis on the use of activity-based probes (ABPs) to detect protease activity. We describe ABP design principles and use case studies to illustrate the application of ABPs to protease enzymology, discovery and development of protease-targeted drugs, and detection and validation of proteases as biomarkers.
The family of cathepsin proteases plays an important physiological role in both normal physiology and in the physiology of many human diseases. This activity, which is upregulated in many cancers, can be exploited for tumor imaging both in vivo and ex vivo. To characterize the behavior of a topically applied quenched fluorescent activity-based probe, GB119, ex vivo, we developed a basic immunohistochemistry technique to identify unquenched GB119 within tissue.
Activity-based probes (ABPs) are small molecules that exclusively form covalent bonds with catalytically active enzymes. In the last decade, they have especially been used in functional proteomics studies of proteases. Here, we present phosphoramidate peptides as a novel type of ABP for serine proteases. These molecules can be made in a straightforward manner by standard Fmoc-based solid-phase peptide synthesis, allowing rapid diversification. The resulting ABPs covalently bind different serine proteases, depending on the amino acid recognition element adjacent to the reactive group. A reporter tag enables downstream gel-based analysis or LC-MS/MS-mediated identification of the targeted proteases. Overall, we believe that these readily accessible probes will provide new avenues for the functional study of serine proteases in complex proteomes.
The induction of hypoxia inducible factors (HIFs) is essential for the adaptation of tumor cells to a low oxygen environment. We found that the expression of the apoptosis inhibitor ARC was induced by hypoxia in a variety of cancer cell types and its induction is primarily HIF1 dependent. Chromatin immunoprecipitation (ChIP) and reporter assays also indicate that the ARC gene is regulated by direct binding of HIF1 to a hypoxia response element (HRE) located at -190 bp upstream of the transcription start site. HIFs play an essential role in the pathogenesis of renal cell carcinoma (RCC) under normoxic conditions, through the loss of the Von Hippel Lindau (VHL) gene. Accordingly, our results show that ARC is not expressed in normal renal tissue, but is highly expressed in 65% of RCC tumors, which also express high levels of Carbonic Anhydrase IX (CAIX), a HIF1-dependent gene. Compared to controls, ARC-deficient RCCs exhibited decreased colony formation and increased apoptosis in vitro. In addition, loss of ARC resulted in a dramatic reduction of RCC tumor growth in SCID mice in vivo. Thus, HIF-mediated increased expression of ARC in RCC can explain how loss of VHL can promote survival early in tumor formation.
The precise targeting of cytotoxic agents to specific cell types or cellular compartments is of significant interest in medicine, with particular relevance for infectious diseases and cancer. Here, we describe a method to exploit aberrant levels of mobile ferrous iron (Fe(II)) for selective drug delivery in vivo. This approach makes use of a 1,2,4-trioxolane moiety, which serves as an Fe(II)-sensitive "trigger," making drug release contingent on Fe(II)-promoted trioxolane fragmentation. We demonstrate in vivo validation of this approach with the Plasmodium berghei model of murine malaria. Malaria parasites produce high concentrations of mobile ferrous iron as a consequence of their catabolism of host hemoglobin in the infected erythrocyte. Using activity-based probes, we successfully demonstrate the Fe(II)-dependent and parasite-selective delivery of a potent dipeptidyl aminopeptidase inhibitor. We find that delivery of the compound in its Fe(II)-targeted form leads to more sustained target inhibition with greatly reduced off-target inhibition of mammalian cathepsins. This selective drug delivery translates into improved efficacy and tolerability. These findings demonstrate the utility of a purely chemical means to achieve selective drug targeting in vivo. This approach may find useful application in parasitic infections and more broadly in any disease state characterized by aberrant production of reactive ferrous iron.
The cysteine cathepsins are a family of proteases that play important roles in both normal cellular physiology and many human diseases. In cancer, the activity of many of the cysteine cathepsins is upregulated and can be exploited for tumor imaging. Here we present the design and synthesis of a new class of quenched fluorescent activity-based probes (qABPs) containing a phenoxymethyl ketone (PMK) electrophile. These reagents show enhanced in vivo properties and broad reactivity resulting in dramatically improved labeling and tumor imaging properties compared to those of previously reported ABPs.
Elucidating the molecular and biochemical details of bacterial infections can be challenging because of the many complex interactions that exist between a pathogen and its host. Consequently, many tools have been developed to aid the study of bacterial pathogenesis. Small molecules are a valuable complement to traditional genetic techniques because they can be used to rapidly perturb genetically intractable systems and to monitor post-translationally regulated processes. Activity-based probes are a subset of small molecules that covalently label an enzyme of interest based on its catalytic mechanism. These tools allow monitoring of enzyme activation within the context of a native biological system and can be used to dissect the biochemical details of enzyme function. This review describes the development and application of activity-based probes for examining aspects of bacterial infection on both sides of the host-pathogen interface.
The Plasmodium falciparum and P. berghei genomes each contain three dipeptidyl aminopeptidase (dpap) homologs. dpap1 and -3 are critical for asexual growth, but the role of dpap2, the gametocyte-specific homolog, has not been tested. If DPAPs are essential for transmission as well as asexual growth, then a DPAP inhibitor could be used for treatment and to block transmission. To directly analyze the role of DPAP2, a dpap2-minus P. berghei (Pbdpap2?) line was generated. The Pbdpap2? parasites grew normally, differentiated into gametocytes, and generated sporozoites that were infectious to mice when fed to a mosquito. However, Pbdpap1 transcription was >2-fold upregulated in the Pbdpap2? clonal lines, possibly compensating for the loss of Pbdpap2. The role of DPAP1 and -3 in the dpap2? parasites was then evaluated using a DPAP inhibitor, ML4118S. When ML4118S was added to the Pbdpap2? parasites just before a mosquito membrane feed, mosquito infectivity was not affected. To assess longer exposures to ML4118S and further evaluate the role of DPAPs during gametocyte development in a parasite that causes human malaria, the dpap2 deletion was repeated in P. falciparum. Viable P. falciparum dpap2 (Pfdpap2)-minus parasites were obtained that produced morphologically normal gametocytes. Both wild-type and Pfdpap2-negative parasites were sensitive to ML4118S, indicating that, unlike many antimalarials, ML4118S has activity against parasites at both the asexual and sexual stages and that DPAP1 and -3 may be targets for a dual-stage drug that can treat patients and block malaria transmission.
Activity-based probes (ABPs) are reactive small molecules that covalently bind to active enzymes. When tagged with a fluorophore, ABPs serve as powerful tools to investigate enzymatic activity across a wide variety of applications. In this article, detailed protocols are provided for using fluorescent ABPs to biochemically characterize the activity of proteases in vitro. Furthermore, descriptions are provided of how these probes can be applied to image protease activity in live animals and tissues along with subsequent analysis by histology, flow cytometry, and SDS-PAGE.
The mechanistic details of the pathogenesis of Chlamydia, an obligate intracellular pathogen of global importance, have eluded scientists due to the scarcity of traditional molecular genetic tools to investigate this organism. Here we report a chemical biology strategy that has uncovered the first essential protease for this organism. Identification and application of a unique CtHtrA inhibitor (JO146) to cultures of Chlamydia resulted in a complete loss of viable elementary body formation. JO146 treatment during the replicative phase of development resulted in a loss of Chlamydia cell morphology, diminishing inclusion size, and ultimate loss of inclusions from the host cells. This completely prevented the formation of viable Chlamydia elementary bodies. In addition to its effect on the human Chlamydia trachomatis strain, JO146 inhibited the viability of the mouse strain, Chlamydia muridarum, both in vitro and in vivo. Thus, we report a chemical biology approach to establish an essential role for Chlamydia?CtHtrA. The function of CtHtrA for Chlamydia appears to be essential for maintenance of cell morphology during replicative the phase and these findings provide proof of concept that proteases can be targeted for antimicrobial therapy for intracellular pathogens.
Caspases are cysteine proteases that play essential roles in apoptosis and inflammation. Unfortunately, their highly conserved active sites and overlapping substrate specificities make it difficult to use inhibitors or activity-based probes to study the function, activation, localization, and regulation of individual members of this family. Here we describe a strategy to engineer a caspase to contain a latent nucleophile that can be targeted by a probe containing a suitably placed electrophile, thereby allowing specific, irreversible inhibition and labeling of only the engineered protease. To accomplish this, we have identified a non-conserved residue on the small subunit of all caspases that is near the substrate-binding pocket and that can be mutated to a non-catalytic cysteine residue. We demonstrate that an active-site probe containing an irreversible binding acrylamide electrophile can specifically target this cysteine residue. Here we validate the approach using the apoptotic mediator, caspase-8, and the inflammasome effector, caspase-1. We show that the engineered enzymes are functionally identical to the wild-type enzymes and that the approach allows specific inhibition and direct imaging of the engineered targets in cells. Therefore, this method can be used to image localization and activation as well as the functional contributions of individual caspase proteases to the process of cell death or inflammation.
Matrix metalloproteinases (MMPs) are zinc endopeptidases that play roles in numerous pathophysiological processes and therefore are promising drug targets. However, the large size of this family and a lack of highly selective compounds that can be used for imaging or inhibition of specific MMPs members has limited efforts to better define their biological function. Here we describe a protein engineering strategy coupled with small-molecule probe design to selectively target individual members of the MMP family. Specifically, we introduce a cysteine residue near the active-site of a selected protease that does not alter its overall activity or function but allows direct covalent modification by a small-molecule probe containing a reactive electrophile. This specific engineered interaction between the probe and the target protease provides a means to both image and inhibit the modified protease with absolute specificity. Here we demonstrate the feasibility of the approach for two distinct MMP proteases, MMP-12 and MT1-MMP (or MMP-14).
One of the noncellular microenvironmental factors that contribute to malignancy of solid tumors is acidic peritumoral pH. We have previously demonstrated that extracellular acidosis leads to localization of the cysteine pro-tease cathepsin B on the tumor cell membrane and its secretion. The objective of the present study was to determine if an acidic extracellular pH such as that observed in vivo (i.e., pHe 6.8) affects the activity of proteases, e.g., cathepsin B, that contribute to degradation of collagen IV by tumor cells when grown in biologically relevant three-dimensional (3D) cultures. For these studies, we used 1) 3D reconstituted basement membrane overlay cultures of human carcinomas, 2) live cell imaging assays to assess proteolysis, and 3) in vivo imaging of active tumor proteases. At pHe 6.8, there were increases in pericellular active cysteine cathepsins and in degradation of dye-quenched collagen IV, which was partially blocked by a cathepsin B inhibitor. Imaging probes for active cysteine cathepsins localized to tumors in vivo. The amount of bound probe decreased in tumors in bicarbonate-treated mice, a treatment previously shown to increase peritumoral pHe and reduce local invasion of the tumors. Our results are consistent with the acid-mediated invasion hypothesis and with a role for cathepsin B in promoting degradation of a basement membrane protein substrate, i.e., type IV collagen, in an acidic peritumoral environment.
The discovery of biocompatible reactions had a tremendous impact on chemical biology, allowing the study of numerous biological processes directly in complex systems. However, despite the fact that multiple biocompatible reactions have been developed in the past decade, very few work well in living mice. Here we report that D-cysteine and 2-cyanobenzothiazoles can selectively react with each other in vivo to generate a luciferin substrate for firefly luciferase. The success of this "split luciferin" ligation reaction has important implications for both in vivo imaging and biocompatible labeling strategies. First, the production of a luciferin substrate can be visualized in a live mouse by bioluminescence imaging (BLI) and furthermore allows interrogation of targeted tissues using a "caged" luciferin approach. We therefore applied this reaction to the real-time noninvasive imaging of apoptosis associated with caspase 3/7. Caspase-dependent release of free D-cysteine from the caspase 3/7 peptide substrate Asp-Glu-Val-Asp-D-Cys (DEVD-(D-Cys)) allowed selective reaction with 6-amino-2-cyanobenzothiazole (NH(2)-CBT) in vivo to form 6-amino-D-luciferin with subsequent light emission from luciferase. Importantly, this strategy was found to be superior to the commercially available DEVD-aminoluciferin substrate for imaging of caspase 3/7 activity. Moreover, the split luciferin approach enables the modular construction of bioluminogenic sensors, where either or both reaction partners could be caged to report on multiple biological events. Lastly, the luciferin ligation reaction is 3 orders of magnitude faster than Staudinger ligation, suggesting further applications for both bioluminescence and specific molecular targeting in vivo.
The AvrPphB effector of Pseudomonas syringae is a papain-like protease that is injected into the host plant cell and cleaves specific kinases to disrupt immune signaling. Here, we used the unique substrate specificity of AvrPphB to generate a specific activity-based probe. This probe displays various AvrPphB isoforms in bacterial extracts, upon secretion and inside the host plant. We show that AvrPphB is secreted as a proprotease and that secretion requires the prodomain, but probably does not involve a pH-dependent unfolding mechanism. The prodomain removal is required for the ability of AvrPphB to trigger a hypersensitive cell death in resistant host plants, presumably since processing exposes a hidden acylation site required for subcellular targeting in the host cell. We detected two active isoforms of AvrPphB in planta, of which the major one localizes exclusively to membranes.
Although there have been numerous advances in our understanding of how apicomplexan parasites such as Toxoplasma gondii enter host cells, many of the signaling pathways and enzymes involved in the organization of invasion mediators remain poorly defined. We recently performed a forward chemical-genetic screen in T. gondii and identified compounds that markedly enhanced infectivity. Although molecular dissection of invasion has benefited from the use of small-molecule inhibitors, the mechanisms underlying induction of invasion by small-molecule enhancers have never been described. Here we identify the Toxoplasma ortholog of human APT1, palmitoyl protein thioesterase-1 (TgPPT1), as the target of one class of small-molecule enhancers. Inhibition of this uncharacterized thioesterase triggered secretion of invasion-associated organelles, increased motility and enhanced the invasive capacity of tachyzoites. We demonstrate that TgPPT1 is a bona fide depalmitoylase, thereby establishing an important role for dynamic and reversible palmitoylation in host-cell invasion by T. gondii.
The past decade has seen rapid growth in the use of diverse compound libraries in classical phenotypic screens to identify modulators of a given process. The subsequent process of identifying the molecular targets of active hits, also called target deconvolution, is an essential step for understanding compound mechanism of action and for using the identified hits as tools for further dissection of a given biological process. Recent advances in omics technologies, coupled with in silico approaches and the reduced cost of whole genome sequencing, have greatly improved the workflow of target deconvolution and have contributed to a renaissance of modern phenotypic profiling. In this review, we will outline how both new and old techniques are being used in the difficult process of target identification and validation as well as discuss some of the ongoing challenges remaining for phenotypic screening.
Legumain or asparaginly endopeptidase (AEP) is a lysosomal cysteine protease with a high level of specificity for cleavage of protein substrates after an asparagine residue. It is also capable of cleaving after aspartic acids sites when in the acidic environment of the lysosome. Legumain expression and activity is linked to a number of pathological conditions including cancer, atherosclerosis and inflammation, yet its biological role in these pathologies is not well-understood. Highly potent and selective inhibitors of legumain would not only be valuable for studying the functional roles of legumain in these conditions, but may have therapeutic potential as well. We describe here the design, synthesis and in vitro evaluation of selective legumain inhibitors based on the aza-asparaginyl scaffold. We synthesized a library of aza-peptidyl inhibitors with various non-natural amino acids and different electrophilic warheads, and characterized the kinetic properties of inactivation of legumain. We also synthesized fluorescently labeled inhibitors to investigate cell permeability and selectivity of the compounds. The inhibitors have second order rate constants of up to 5 × 10(4)M(-1)s(-1) and IC(50) values as low as 4 nM against recombinant mouse legumain. In addition, the inhibitors are highly selective toward legumain and have little or no cross-reactivity with cathepsins. Overall, we have identified several valuable new inhibitors of legumain that can be used to study legumain function in multiple disease models.
To determine the therapeutic efficacy and immunomodulatory effect of an anti-human death receptor 5 (DR5) antibody, TRA-8, in eliminating macrophage subsets in a mouse model of type II collagen-induced arthritis (CIA).
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.
The cysteine peptidase cathepsin B is important in thyroid physiology by being involved in prohormone processing initiated in the follicle lumen and completed in endo-lysosomal compartments. However, cathepsin B has also been localized to the extrafollicular space in thyroid cancer tissue, and is therefore suggested to promote invasiveness and metastasis in thyroid carcinomas through e.g. extracellular matrix degradation.
Toxoplasma gondii is a member of the phylum Apicomplexa that includes several important human pathogens, such as Cryptosporidium and Plasmodium falciparum, the causative agent of human malaria. It is an obligate intracellular parasite that can cause severe disease in congenitally infected neonates and immunocompromised individuals. Despite the importance of attachment and invasion to the success of the parasite, little is known about the underlying mechanisms that drive these processes. Here we describe a screen to identify small molecules that block the process of host cell invasion by the T. gondii parasite. We identified a small molecule that specifically and irreversibly blocks parasite attachment and subsequent invasion of host cells. Using tandem orthogonal proteolysis-activity-based protein profiling, we determined that this compound covalently modifies a single cysteine residue in a poorly characterized protein homologous to the human protein DJ-1. Mutation of this key cysteine residue in the native gene sequence resulted in parasites that were resistant to inhibition of host cell attachment and invasion by the compound. Further analysis of the invasion phenotype confirmed that modification of Cys127 on TgDJ-1 resulted in a block of microneme secretion and motility, even in the presence of direct stimulators of calcium release. Together, our results suggest that TgDJ-1 plays an important role that is likely downstream of the calcium flux required for microneme secretion, parasite motility, and subsequent invasion of host cells.
Although proteases control inflammation and pain, the identity, cellular origin, mechanism of action, and causative role of proteases that are activated during disease are not defined. We investigated the activation and function of cysteine cathepsins (Cat) in colitis.
Proteases are enzymes that cleave peptide bonds in protein substrates. This process can be important for regulated turnover of a target protein but it can also produce protein fragments that then perform other functions. Because the last few decades of protease research have confirmed that proteolysis is an essential regulatory process in both normal physiology and in multiple disease-associated conditions, there has been an increasing interest in developing methods to image protease activity. Proteases are also considered to be one of the few druggable classes of proteins and therefore a large number of small molecule based inhibitors of proteases have been reported. These compounds serve as a starting point for the design of probes that can be used to target active proteases for imaging applications. Currently, several classes of fluorescent probes have been developed to visualize protease activity in live cells and even whole organisms. The two primary classes of protease probes make use of either peptide/protein substrates or covalent inhibitors that produce a fluorescent signal when bound to an active protease target. This review outlines some of the most recent advances in the design of imaging probes for proteases. In particular, it highlights the strengths and weaknesses of both substrate-based and activity-based probes and their applications for imaging cysteine proteases that are important biomarkers for multiple human diseases.
Sentrin specific proteases (SENPs) are responsible for activating and deconjugating SUMO (Small Ubiquitin like MOdifier) from target proteins. It remains difficult to study this posttranslational modification due to the lack of reagents that can be used to block the removal of SUMO from substrates. Here, we describe the identification of small molecule SENP inhibitors and active site probes containing aza-epoxide and acyloxymethyl ketone (AOMK) reactive groups. Both classes of compounds are effective inhibitors of hSENPs 1, 2, 5, and 7 while only the AOMKs efficiently inhibit hSENP6. Unlike previous reported peptide vinyl sulfones, these compounds covalently labeled the active site cysteine of multiple recombinantly expressed SENP proteases and the AOMK probe showed selective labeling of these SENPs when added to complex protein mixtures. The AOMK compound therefore represents promising new reagents to study the process of SUMO deconjugation.
The diverse functional roles that proteases play in basic biological processes make them essential for virtually all organisms. Not surprisingly, proteolysis is also a critical process required for many aspects of pathogenesis. In particular, obligate intracellular parasites must precisely coordinate proteolytic events during their highly regulated life cycle inside multiple host cell environments. Advances in chemical, proteomic and genetic tools that can be applied to parasite biology have led to an increased understanding of the complex events centrally regulated by proteases. In this review, we outline recent advances in our knowledge of specific proteolytic enzymes in two medically relevant apicomplexan parasites: Plasmodium falciparum and Toxoplasma gondii. Efforts over the last decade have begun to provide a map of key proteotolyic events that are essential for both parasite survival and propagation inside host cells. These advances in our molecular understanding of proteolytic events involved in parasite pathogenesis provide a foundation for the validation of new networks and enzyme targets that could be exploited for therapeutic purposes. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.
Myeloid-derived suppressor cells (MDSCs) are significantly increased in cancer patients and tumor bearing-animals. MDSCs infiltrate into tumors and promote tumor invasion and metastasis. To identify the mediator responsible for the prometastatic property of MDSCs, we used proteomics. We found neutrophilic granule protein (NGP) was decreased >2-fold in MDSCs from metastatic 4T1 tumor-bearing mice compared to nonmetastatic 67NR controls. NGP mRNA levels were decreased in bone marrow and in tumor-infiltrating MDSCs by 45 and 66%, respectively, in 4T1 tumor-bearing mice compared to 67NR controls. Interestingly, 4T1-conditioned medium reduced myeloid cell NGP expression by ? 40%, suggesting that a secreted factor mediates gene reduction. Sequence analysis shows a putative cystatin domain in NGP, and biochemical analysis confirms NGP a novel cathepsin inhibitor. It inhibited cathepsin B activity by nearly 40% in vitro. NGP expression in 4T1 tumor cells suppressed cell invasion, delayed primary tumor growth, and greatly reduced lung metastasis in vivo. A 2.8-fold reduction of cathepsin activity was found in tumors expressing NGP compared to controls. NGP significantly reduced tumor angiogenesis to 12.6 from 19.6 and lymphangiogenesis to 4.6 from 9.1 vessels/field. Necrosis was detectable only in NGP-expressing tumors, and the number of apoptotic cells increased to 22.4 from 8.3 in controls. Taken together, this study identifies a negative regulator of tumor metastasis in MDSCs, NGP, which is down-regulated in metastatic conditions. The finding suggests that malignant tumors promote invasion/metastasis not only through up-regulation of proteases but also down-regulation of protease inhibitors.
Small ubiquitin-related modifier (SUMO) is implicated in the regulation of numerous biological processes including transcription, protein localization, and cell cycle control. Protein modification by SUMO is found in Plasmodium falciparum; however, its role in the regulation of the parasite life cycle is poorly understood. Here we describe functional studies of a SUMO-specific protease (SENP) of P. falciparum, PfSENP1 (PFL1635w). Expression of the catalytic domain of PfSENP1 and biochemical profiling using a positional scanning substrate library demonstrated that this protease has unique cleavage sequence preference relative to the human SENPs. In addition, we describe a class of small molecule inhibitors of this protease. The most potent lead compound inhibited both recombinant PfSENP1 activity and P. falciparum replication in infected human blood. These studies provide valuable new tools for the study of SUMOylation in P. falciparum.
Toll-like receptor (TLR) 9 requires proteolytic processing in the endolysosome to initiate signaling in response to DNA. However, recent studies conflict as to which proteases are required for receptor cleavage. We show that TLR9 proteolysis is a multistep process. The first step removes the majority of the ectodomain and can be performed by asparagine endopeptidase (AEP) or cathepsin family members. This initial cleavage event is followed by a trimming event that is solely cathepsin mediated and required for optimal receptor signaling. This dual requirement for AEP and cathepsins is observed in all cell types that we have analyzed, including mouse macrophages and dendritic cells. In addition, we show that TLR7 and TLR3 are processed in an analogous manner. These results define the core proteolytic steps required for TLR9 function and suggest that receptor proteolysis may represent a general regulatory strategy for all TLRs involved in nucleic acid recognition.
Human strains of Staphylococcus aureus secrete two papain-like proteases, staphopain A and B. Avian strains produce another homologous enzyme, staphopain C. Animal studies suggest that staphopains B and C contribute to bacterial virulence, in contrast to staphopain A, which seems to have a virulence unrelated function. Here we present a detailed study of substrate preferences of all three proteases. The specificity of staphopain A, B and C substrate-binding subsites was mapped using different synthetic substrate libraries, inhibitor libraries and a protein substrate combinatorial library. The analysis demonstrated that the most efficiently hydrolyzed sites, using Schechter and Berger nomenclature, comprise a P2-Gly?Ala(Ser) sequence motif, where P2 distinguishes the specificity of staphopain A (Leu) from that of both staphopains B and C (Phe/Tyr). However, we show that at the same time the overall specificity of staphopains is relaxed, insofar as multiple substrates that diverge from the sequences described above are also efficiently hydrolyzed.
Cathepsin X is a lysosomal cysteine protease that functions as a carboxypeptidase with broad substrate specificity. Cathepsin X was discovered only recently, and its physiological roles are still not well understood. A number of studies suggest that cathepsin X may be involved in a variety of biological processes, including cancer, aging and degenerative conditions of the brain, inflammation, and cellular communication. Here we present the synthesis and characterization of several activity-based probes (ABPs) that target active cathepsin X. These ABPs were used to label cathepsin X in complex lysates, whole cells, and in vivo. Furthermore, we have developed a method for selectively labeling and visualizing active cathepsin X in vitro and in vivo. Overall, the probes developed in this study are valuable tools for the study of cathepsin X function.
An internal cysteine protease domain (CPD) autoproteolytically regulates Clostridium difficile glucosylating toxins by releasing a cytotoxic effector domain into target cells. CPD activity is itself allosterically regulated by the eukaryote-specific molecule inositol hexakisphosphate (InsP(6)). Although allostery controls the function of most proteins, the molecular details underlying this regulatory mechanism are often difficult to characterize. Here we use chemical probes to show that apo-CPD is in dynamic equilibrium between active and inactive states. InsP(6) markedly shifts this equilibrium toward an active conformer that is further restrained upon binding a suicide substrate. Structural analyses combined with systematic mutational and disulfide bond engineering studies show that residues within a ?-hairpin region functionally couple the InsP(6)-binding site to the active site. Collectively, our results identify an allosteric circuit that allows bacterial virulence factors to sense and respond to the eukaryotic environment.
The tumour microenvironment regulates tumour progression and the spread of cancer in the body. Targeting the stromal cells that surround cancer cells could, therefore, improve the effectiveness of existing cancer treatments. Here, we show that magnetic nanoparticle clusters encapsulated inside a liposome can, under the influence of an external magnet, target both the tumour and its microenvironment. We use the outstanding T2 contrast properties (r2=573-1,286 s(-1) mM(-1)) of these ferri-liposomes, which are ?95 nm in diameter, to non-invasively monitor drug delivery in vivo. We also visualize the targeting of the tumour microenvironment by the drug-loaded ferri-liposomes and the uptake of a model probe by cells. Furthermore, we used the ferri-liposomes to deliver a cathepsin protease inhibitor to a mammary tumour and its microenvironment in a mouse, which substantially reduced the size of the tumour compared with systemic delivery of the same drug.
The obligate intracellular parasite pathogen Plasmodium falciparum is the causative agent of malaria, a disease that results in nearly one million deaths per year. A key step in disease pathology in the human host is the parasite-mediated rupture of red blood cells, a process that requires extensive proteolysis of a number of host and parasite proteins. However, only a relatively small number of specific proteolytic processing events have been characterized. Here we describe the application of the Protein Topography and Migration Analysis Platform (PROTOMAP) (Dix, M. M., Simon, G. M., and Cravatt, B. F. (2008) Global mapping of the topography and magnitude of proteolytic events in apoptosis. Cell 134, 679-691; Simon, G. M., Dix, M. M., and Cravatt, B. F. (2009) Comparative assessment of large-scale proteomic studies of apoptotic proteolysis. ACS Chem. Biol. 4, 401-408) technology to globally profile proteolytic events occurring over the last 6-8 h of the intraerythrocytic cycle of P. falciparum. Using this method, we were able to generate peptographs for a large number of proteins at 6 h prior to rupture as well as at the point of rupture and in purified merozoites after exit from the host cell. These peptographs allowed assessment of proteolytic processing as well as changes in both protein localization and overall stage-specific expression of a large number of parasite proteins. Furthermore, by using a highly selective inhibitor of the cysteine protease dipeptidyl aminopeptidase 3 (DPAP3) that has been shown to be a key regulator of host cell rupture, we were able to identify specific substrates whose processing may be of particular importance to the process of host cell rupture. These results provide the first global map of the proteolytic processing events that take place as the human malarial parasite extracts itself from the host red blood cell. These data also provide insight into the biochemical events that take place during host cell rupture and are likely to be valuable for the study of proteases that could potentially be targeted for therapeutic gain.
Clostridium difficile is a leading cause of nosocomial infections. The major virulence factors of this pathogen are the multi-domain toxins TcdA and TcdB. These toxins contain a cysteine protease domain (CPD) that autoproteolytically releases a cytotoxic effector domain upon binding intracellular inositol hexakisphosphate. Currently, there are no known inhibitors of this protease. Here, we describe the rational design of covalent small molecule inhibitors of TcdB CPD. We identified compounds that inactivate TcdB holotoxin function in cells and solved the structure of inhibitor-bound protease to 2.0 Å. This structure reveals the molecular basis of CPD substrate recognition and informed the synthesis of activity-based probes for this enzyme. The inhibitors presented will guide the development of therapeutics targeting C. difficile, and the probes will serve as tools for studying the unique activation mechanism of bacterial toxin CPDs.
Dipeptidyl aminopeptidase 1 (DPAP1) is an essential food vacuole enzyme with a putative role in hemoglobin catabolism by the erythrocytic malaria parasite. Here, the biochemical properties of DPAP1 have been investigated and compared to those of the human ortholog cathepsin C. To facilitate the characterization of DPAP1, we have developed a method for the production of purified recombinant DPAP1 with properties closely resembling those of the native enzyme. Like cathepsin C, DPAP1 is a chloride-activated enzyme that is most efficient in catalyzing amide bond hydrolysis at acidic pH values. The monomeric quaternary structure of DPAP1 differs from the homotetrameric structure of cathepsin C, which suggests that tetramerization is required for a cathepsin C-specific function. The S1 and S2 subsite preferences of DPAP1 and cathepsin C were profiled with a positional scanning synthetic combinatorial library. The S1 preferences bore close similarity to those of other C1-family cysteine peptidases. The S2 subsites of both DPAP1 and cathepsin C accepted aliphatic hydrophobic residues, proline, and some polar residues, yielding a distinct specificity profile. DPAP1 efficiently catalyzed the hydrolysis of several fluorogenic dipeptide substrates; surprisingly, however, a potential substrate with a P2-phenylalanine residue was instead a competitive inhibitor. Together, our biochemical data suggest that DPAP1 accelerates the production of amino acids from hemoglobin by bridging the gap between the endopeptidase and aminopeptidase activities of the food vacuole. Two reversible cathepsin C inhibitors potently inhibited both recombinant and native DPAP1, thereby validating the use of recombinant DPAP1 for future inhibitor discovery and characterization.
High throughput screening (HTS) is one of the primary tools used to identify novel enzyme inhibitors. However, its applicability is generally restricted to targets that can either be expressed recombinantly or purified in large quantities.
The cysteine peptidase cathepsin B is important in thyroid physiology by being involved in thyroid prohormone processing initiated in the follicular lumen and completed in endo-lysosomal compartments. However, cathepsin B has also been localized to the extrafollicular space and is therefore suggested to promote invasiveness and metastasis in thyroid carcinomas through, e.g., ECM degradation. In this study, immunofluorescence and biochemical data from subcellular fractionation revealed that cathepsin B, in its single- and two-chain forms, is localized to endo-lysosomes in the papillary thyroid carcinoma cell line KTC-1 and in the anaplastic thyroid carcinoma cell lines HTh7 and HTh74. This distribution is not affected by thyroid stimulating hormone (TSH) incubation of HTh74, the only cell line that expresses a functional TSH-receptor. Immunofluorescence data disclosed an additional nuclear localization of cathepsin B immunoreactivity. This was supported by biochemical data showing a proteolytically active variant slightly smaller than the cathepsin B proform in nuclear fractions. We also demonstrate that immunoreactions specific for cathepsin V, but not cathepsin L, are localized to the nucleus in HTh74 in peri-nucleolar patterns. As deduced from co-localization studies and in vitro degradation assays, we suggest that nuclear variants of cathepsins are involved in the development of thyroid malignancies through modification of DNA-associated proteins.
Hematopoietic stem cells are retained within discrete bone marrow niches through the effects of cell adhesion molecules and chemokine gradients. However, a small proportion of hematopoietic stem cells can also be found trafficking in the peripheral blood. During induced stem cell mobilization a proteolytic microenvironment is generated, but whether proteases are also involved in physiological trafficking of hematopoietic stem cells is not known. In the present study we examined the expression, secretion and function of the cysteine protease cathepsin X by cells of the human bone marrow.
The widespread resistance of malaria parasites to all affordable drugs has made the identification of new targets urgent. Dipeptidyl aminopeptidases (DPAPs) represent potentially valuable new targets that are involved in hemoglobin degradation (DPAP1) and parasite egress (DPAP3). Here we use activity-based probes to demonstrate that specific inhibition of DPAP1 by a small molecule results in the formation of an immature trophozoite that leads to parasite death. Using computational methods, we designed stable, nonpeptidic covalent inhibitors that kill Plasmodium falciparum at low nanomolar concentrations. These compounds show signs of slowing parasite growth in a murine model of malaria, which suggests that DPAP1 might be a viable antimalarial target. Interestingly, we found that resynthesis and activation of DPAP1 after inhibition is rapid, suggesting that effective drugs would need to sustain DPAP1 inhibition for a period of 2-3 hr.
Macrophages play a key role in innate immune response to pathogens and in tissue homeostasis, inflammation and repair. A serpin A3G (SpiA3G) is highly induced in classically activated macrophages. We show increased localization of SpiA3G in the nucleolus and co-localization with cathepsin L, upon classical, but not alternative activation of macrophages. Despite the increased expression of cathepsin L in the nuclei of classically activated macrophages, no cathepsin activity was detected. Since only pro-inflammatory, but not anti-inflammatory stimuli induce increased nucleolar localization of SpiA3G, we propose that SpiA3g translocation into the nucleolus is important in host defense against pathogens.
Aminopeptidases process the N-terminal amino acids of target substrates by sequential cleavage of one residue at a time. They are found in all cell compartments of prokaryotes and eukaryotes, being implicated in the major proteolytic events of cell survival, defense, growth, and development. We present a new approach for the fast and reliable evaluation of the substrate specificity of individual aminopeptidases. Using solid phase chemistry with the 7-amino-4-carbamoylmethylcoumarin fluorophore, we have synthesized a library of 61 individual natural and unnatural amino acids substrates, chosen to cover a broad spectrum of the possible interactions in the S1 pocket of this type of protease. As proof of concept, we determined the substrate specificity of human, pig, and rat orthologs of aminopeptidase N (CD13), a highly conserved cell surface protease that inactivates enkephalins and other bioactive peptides. Our data reveal a large and hydrophobic character for the S1 pocket of aminopeptidase N that is conserved with aminopeptidase Ns. Our approach, which can be applied in principle to all aminopeptidases, yields useful information for the design of specific inhibitors, and more importantly, reveals a relationship between the kinetics of substrate hydrolysis and the kinetics of enzyme inhibition.
We introduce a new method for purifying recombinant proteins expressed in bacteria using a highly specific, inducible, self-cleaving protease tag. This tag is comprised of the Vibrio cholerae MARTX toxin cysteine protease domain (CPD), an autoprocessing enzyme that cleaves exclusively after a leucine residue within the target protein-CPD junction. Importantly, V. cholerae CPD is specifically activated by inositol hexakisphosphate (InsP(6)), a eukaryotic-specific small molecule that is absent from the bacterial cytosol. As a result, when His(6)-tagged CPD is fused to the C-terminus of target proteins and expressed in Escherichia coli, the full-length fusion protein can be purified from bacterial lysates using metal ion affinity chromatography. Subsequent addition of InsP(6) to the immobilized fusion protein induces CPD-mediated cleavage at the target protein-CPD junction, releasing untagged target protein into the supernatant. This method condenses affinity chromatography and fusion tag cleavage into a single step, obviating the need for exogenous protease addition to remove the fusion tag(s) and increasing the efficiency of tag separation. Furthermore, in addition to being timesaving, versatile, and inexpensive, our results indicate that the CPD purification system can enhance the expression, integrity, and solubility of intractable proteins from diverse organisms.
Actin filaments are key components of the eukaryotic cytoskeleton that provide mechanical structure and generate forces during cell shape changes, growth, and migration. Actin filaments are dynamically assembled into higher-order structures at specified locations to regulate diverse functions. The Rab family of small guanosine triphosphatases is evolutionarily conserved and mediates intracellular vesicle trafficking. We found that Rab35 regulates the assembly of actin filaments during bristle development in Drosophila and filopodia formation in cultured cells. These effects were mediated by the actin-bundling protein fascin, which directly associated with active Rab35. Targeting Rab35 to the outer mitochondrial membrane triggered actin recruitment, demonstrating a role for an intracellular trafficking protein in localized actin assembly.
Understanding the ways in which pathogens invade and neutralize their hosts is of great interest from both an academic and a clinical perspective. However, in many cases genetic tools are unavailable or insufficient to fully characterize the detailed mechanisms of pathogenesis. Small molecule approaches are particularly powerful due to their ability to modulate specific biological functions in a highly controlled manner and their potential to broadly target conserved processes across species. Recently, two approaches that make use of small molecules, activity-based protein profiling and high-throughput phenotypic screening, have begun to find applications in the study of pathways involved in pathogenesis. In this Review we highlight ways in which these techniques have been applied to examine bacterial and parasitic pathogenesis and discuss possible ways in which these efforts can be expanded in the near future.
The protozoan parasite Toxoplasma gondii relies on post-translational modification, including proteolysis, of proteins required for recognition and invasion of host cells. We have characterized the T. gondii cysteine protease cathepsin L (TgCPL), one of five cathepsins found in the T. gondii genome. We show that TgCPL is the primary target of the compound morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS), which was previously shown to inhibit parasite invasion by blocking the release of invasion proteins from microneme secretory organelles. As shown by fluorescently labeled LHVS and TgCPL-specific antibodies, TgCPL is associated with a discrete vesicular structure in the apical region of extracellular parasites but is found in multiple puncta throughout the cytoplasm of intracellular replicating parasites. LHVS fails to label cells lacking TgCPL due to targeted disruption of the TgCPL gene in two different parasite strains. We present a structural model for the inhibition of TgCPL by LHVS based on a 2.0 A resolution crystal structure of TgCPL in complex with its propeptide. We discuss possible roles for TgCPL as a protease involved in the degradation or limited proteolysis of parasite proteins involved in invasion.
Toxoplasma gondii is a eukaryotic parasite of the phylum Apicomplexa that is able to infect a wide variety of host cells. During its active invasion process it secretes proteins from discrete secretory organelles: the micronemes, rhoptries and dense granules. Although a number of rhoptry proteins have been shown to be involved in important interactions with the host cell, very little is known about the mechanism of secretion of any Toxoplasma protein into the host cell. We used a chemical inhibitor of phospholipase A2s, 4-bromophenacyl bromide (4-BPB), to look at the role of such lipases in the secretion of Toxoplasma proteins. We found that 4-BPB was a potent inhibitor of rhoptry secretion in Toxoplasma invasion. This drug specifically blocked rhoptry secretion but not microneme secretion, thus effectively showing that the two processes can be de-coupled. It affected parasite motility and invasion, but not attachment or egress. Using propargyl- or azido-derivatives of the drug (so-called click chemistry derivatives) and a series of 4-BPB-resistant mutants, we found that the drug has a very large number of target proteins in the parasite that are involved in at least two key steps: invasion and intracellular growth. This potent compound, the modified "click-chemistry" forms of it, and the resistant mutants should serve as useful tools to further study the processes of Toxoplasma early invasion, in general, and rhoptry secretion, in particular.
Hemoglobin digestion is an essential process for blood-feeding parasites. Using chemical tools, we deconvoluted the intracellular hemoglobinolytic cascade in the tick Ixodes ricinus, a vector of Lyme disease and tick-borne encephalitis. In tick gut tissue, a network of peptidases was demonstrated through imaging with specific activity-based probes and activity profiling with peptidic substrates and inhibitors. This peptidase network is induced upon blood feeding and degrades hemoglobin at acidic pH. Selective inhibitors were applied to dissect the roles of the individual peptidases and to determine the peptidase-specific cleavage map of the hemoglobin molecule. The degradation pathway is initiated by endopeptidases of aspartic and cysteine class (cathepsin D supported by cathepsin L and legumain) and is continued by cysteine amino- and carboxy-dipeptidases (cathepsins C and B). The identified enzymes are potential targets to developing novel anti-tick vaccines.
Recent advances in the field of non-invasive optical imaging have included the development of contrast agents that report on the activity of enzymatic targets associated with disease pathology. In particular, proteases have proven to be ideal targets for development of optical sensors for cancer. Recently developed contrast agents for protease activity include both small peptides and large polymer-based quenched fluorescent substrates as well as fluorescently labeled activity based probes (ABPs). While substrates produce a fluorescent signal as a result of processing by a protease, ABPs are retained at the site of proteolysis due to formation of a permanent covalent bond with the active site catalytic residue. Both methods have potential advantages and disadvantages yet a careful comparison of substrates and ABPs has not been performed. Here we present the results of a direct comparison of commercially available protease substrates with several recently described fluorescent ABPs in a mouse model of cancer. The results demonstrate that fluorescent ABPs show more rapid and selective uptake into tumors as well as overall brighter signals compared to substrate probes. These data suggest that the lack of signal amplification for an ABP is offset by the increased kinetics of tissue uptake and prolonged retention of the probes once bound to a protease target. Furthermore, fluorescent ABPs can be used as imaging reagents with similar or better results as the commercially available protease substrates.
Tumors initiate angiogenesis primarily by secreting vascular endothelial growth factor (VEGF-A(164)). The first new vessels to form are greatly enlarged, pericyte-poor sinusoids, called mother vessels (MV), that originate from preexisting venules. We postulated that the venular enlargement necessary to form MV would require a selective degradation of their basement membranes, rigid structures that resist vascular expansion. To identify the specific proteases responsible for MV formation, we induced angiogenesis in mouse tissues with an adenoviral vector expressing VEGF-A(164) (Ad-VEGF-A(164)) or with VEGF-A-secreting TA3/St mammary tumors. We found that MV formation resulted from greatly increased activity of cathepsins (B>S>L) in venules transitioning into MV, as well as from a reciprocal decrease in the expression of several cysteine protease inhibitors (CPI), stefin A and cystatins B and C, by these same venules. Using a fluorescence probe that selectively binds cellular sites of cathepsin protease activity in vivo, we showed that increased cathepsin activity was localized exclusively to perivenular cells, not to venule endothelial cells. CPI strikingly inhibited angiogenesis in the Matrigel assay, and Ad-VEGF-A(164)-induced angiogenesis was reduced by approximately 50% in cathepsin B-null mice. Thus, VEGF-A, whether expressed by interstitial cells infected with an adenoviral vector or by tumor cells, upsets the normal cathepsin-CPI balance in nearby venules, leading to degradation of their basement membranes, an important first step in angiogenesis.
Taspase1 is a threonine protease responsible for cleaving MLL (Mixed-Lineage Leukemia) to achieve proper HOX gene expression. Subsequent studies identified additional Taspase1 substrates including Transcription Factor IIA (TFIIA) and Drosophila HCF. Taspase1 is essential for cell proliferation and is overexpressed in many cancer cell lines. Currently no small molecule inhibitors of this enzyme have been described. Here, we report the synthesis and evaluation of vinyl sulfone, vinyl ketone, epoxy ketone, and boronic acid inhibitors designed based on the preferred Taspase1 cleavage site (Ac-Ile-Ser-Gln-Leu-Asp). Specifically, we evaluated compounds in which the reactive warhead is positioned in place of the P1 aspartic acid side chain as well as at the C-terminus of the peptide. Interestingly, both classes of inhibitors were effective and vinyl ketones and vinyl sulfones showed the greatest potency for the target protease. These results suggest that Taspase1 has unique substrate recognition properties that could potentially be exploited in the design of potent and selective inhibitors of this enzyme.
Caspase-8 is a proapoptotic protease that suppresses neuroblastoma metastasis by inducing programmed cell death. Paradoxically, caspase-8 can also promote cell migration among nonapoptotic cells; here, we show that caspase-8 can promote metastasis when apoptosis is compromised. Migration is enhanced by caspase-8 recruitment to the cellular migration machinery following integrin ligation. Caspase-8 catalytic activity is not required for caspase-8-enhanced cell migration; rather, caspase-8 interacts with a multiprotein complex that can include focal adhesion kinase and calpain 2 (CPN2), enhancing cleavage of focal adhesion substrates and cell migration. Caspase-8 association with CPN2/calpastatin disrupts calpastatin-mediated inhibition of CPN2. In vivo, knockdown of either caspase-8 or CPN2 disrupts metastasis among apoptosis-resistant tumors. This unexpected molecular collaboration provides an explanation for the continued or elevated expression of caspase-8 observed in many tumors.
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.
MARTX toxins modulate the virulence of a number of Gram-negative Vibrio species. This family of toxins is defined by the presence of a cysteine protease domain (CPD), which proteolytically activates the Vibrio cholerae MARTX toxin. Although recent structural studies of the CPD have uncovered a new allosteric activation mechanism, the mechanism of CPD substrate recognition or toxin processing is unknown. Here we show that interdomain cleavage of MARTXVc enhances effector domain function. We also identify the first small-molecule inhibitors of this protease domain and present the 2.35-A structure of the CPD bound to one of these inhibitors. This structure, coupled with biochemical and mutational studies of the toxin, reveals the molecular basis of CPD substrate specificity and underscores the evolutionary relationship between the CPD and the clan CD caspase proteases. These studies are likely to prove valuable for devising new antitoxin strategies for a number of bacterial pathogens.
Small molecules offer unprecedented opportunities for plant research since plants respond to, metabolize, and react with a diverse range of endogenous and exogenous small molecules. Many of these small molecules become covalently attached to proteins. To display these small molecule targets in plants, we introduce a two-step labelling method for minitagged small molecules. Minitags are small chemical moieties (azide or alkyne) that are inert under biological conditions and have little influence on the membrane permeability and specificity of the small molecule. After labelling, proteomes are extracted under denaturing conditions and minitagged proteins are coupled to reporter tags through a click chemistry reaction. We introduce this two-step labelling procedure in plants by studying the well-characterized targets of E-64, a small molecule cysteine protease inhibitor. In contrast to biotinylated E-64, minitagged E-64 efficiently labels vacuolar proteases in vivo. We displayed, purified and identified targets of a minitagged inhibitor that targets the proteasome and cysteine proteases in living plant cells. Chemical interference assays with inhibitors showed that MG132, a frequently used proteasome inhibitor, preferentially inhibits cysteine proteases in vivo. The two-step labelling procedure can be applied on detached leaves, cell cultures, seedlings and other living plant tissues and, when combined with photoreactive groups, can be used to identify targets of herbicides, phytohormones and reactive small molecules selected from chemical genetic screens.
The newly excysted juvenile (NEJ) stage of the Fasciola hepatica lifecycle occurs just prior to invasion into the wall of the gut of the host, rendering it an important target for drug development. The cathepsin B enzymes from NEJ flukes have recently been demonstrated to be crucial to invasion and migration by the parasite. Here we characterize one of the cathepsin B enzymes (recombinant FhcatB1) from NEJ flukes. FhcatB1 has biochemical properties distinct from mammalian cathepsin B enzymes, with an atypical preference for Ile over Leu or Arg residues at the P(2) substrate position and an inability to act as an exopeptidase. FhcatB1 was active across a broad pH range (optimal activity at pH 5.5-7.0) and resistant to inhibition by cystatin family inhibitors from sheep and humans, suggesting that this enzyme would be able to function in extracellular environments in its mammalian hosts. It appears, however, that the FhcatB1 protease functions largely as a digestive enzyme in the gut of the parasite, due to the localization of a specific, fluorescently labeled inhibitor with an Ile at the P(2) position. Molecular modelling and dynamics were used to predict the basis for the unusual substrate specificity: a P(2) Ile residue positions the substrate optimally for interaction with catalytic residues of the enzyme, and the enzyme lacks an occluding loop His residue crucial for exopeptidase activity. The unique features of the enzyme, particularly with regard to its specificity and likely importance to a vital stage of the parasites life cycle, make it an excellent target for therapeutic inhibitors or vaccination.
Imaging agents that enable direct visualization and quantification of apoptosis in vivo have great potential value for monitoring chemotherapeutic response as well as for early diagnosis and disease monitoring. We describe here the development of fluorescently labeled activity-based probes (ABPs) that covalently label active caspases in vivo. We used these probes to monitor apoptosis in the thymi of mice treated with dexamethasone as well as in tumor-bearing mice treated with the apoptosis-inducing monoclonal antibody Apomab (Genentech). Caspase ABPs provided direct readouts of the kinetics of apoptosis in live mice, whole organs and tissue extracts. The probes produced a maximum fluorescent signal that could be monitored noninvasively and that coincided with the peak in caspase activity, as measured by gel analysis. Overall, these studies demonstrate that caspase-specific ABPs have the potential to be used for noninvasive imaging of apoptosis in both preclinical and clinical settings.
Cathepsin B (EC 126.96.36.199) and other cysteine proteases are synthesized as zymogens, which are processed to their mature forms autocatalytically or by other proteases. Autocatalytic processing was suggested to be a bimolecular process, whereas initiation of the processing has not yet been clarified. Procathepsin B was shown by zymography to hydrolyze the synthetic substrate 7-N-benzyloxycarbonyl-L-arginyl-L-arginylamide-4-methylcoumarin (Z-Arg-Arg-NH-MEC), suggesting that procathepsin B is catalytically active. The activity-based probe DCG-04, which is an E-64-type inhibitor, was found to label both mature cathepsin B and its zymogen, confirming the zymography data. Mutation analyses in the linker region between the propeptide and the mature part revealed that autocatalytic processing of procathepsin B is largely unaffected by mutations in this region, including mutations to prolines. On the basis of these results, a model for autocatalytic activation of cysteine cathepsins is proposed, involving propeptide dissociation from the active-site cleft as the first step during zymogen activation. This unimolecular conformational change is followed by a bimolecular proteolytic removal of the propeptide, which can be accomplished in one or more steps. Such activation, which can be also facilitated by glycosaminoglycans or by binding to negatively charged surfaces, may have important physiological consequences because cathepsin zymogens were often found secreted in various pathological states.
Besides functioning as the plasma transporter of retinol and thyroxine, TTR (transthyretin) is a protease, cleaving apoA-I (apolipoprotein A-I) after a phenylalanine residue. In the present study, we further investigated TTR substrate specificity. By using both P-diverse libraries and a library of phosphonate inhibitors, a TTR preference for a lysine residue in P1 was determined, suggesting that TTR might have a dual specificity and that, in addition to apoA-I, other TTR substrates might exist. Previous studies revealed that TTR is involved in the homoeostasis of the nervous system, as it participates in neuropeptide maturation and enhances nerve regeneration. We investigated whether TTR proteolytic activity is involved in these functions. Both wild-type TTR and TTR(prot-) (proteolytically inactive TTR) had a similar effect in the expression of peptidylglycine alpha-amidating mono-oxygenase, the rate-limiting enzyme in neuropeptide amidation, excluding the involvement of TTR proteolytic activity in neuropeptide maturation. However, TTR was able to cleave amidated NPY (neuropeptide Y), probably contributing to the increased NPY levels reported in TTR-knockout mice. To assess the involvement of TTR proteolytic activity in axonal regeneration, neurite outgrowth of cells cultivated with wild-type TTR or TTR(prot-), was measured. Cells grown with TTR(prot-) displayed decreased neurite length, thereby suggesting that TTR proteolytic activity is important for its function as a regeneration enhancer. By showing that TTR is able to cleave NPY and that its proteolytic activity affects axonal growth, the present study shows that TTR has natural substrates in the nervous system, establishing further its relevance in neurobiology.
Localization of proteases to the surface of endothelial cells and remodeling of the extracellular matrix (ECM) are essential to endothelial cell tube formation and angiogenesis. Here, we partially localized active cathepsin B and its cell surface binding partners, S100A/p11 (p11) of the annexin II heterotetramer (AIIt), to caveolae of human umbilical vein endothelial cells (HUVEC). Via a live-cell proteolysis assay, we observed that degradation products of quenched-fluorescent (DQ)-proteins (i.e. gelatin and collagen IV) colocalized intracellularly with caveolin-1 (cav-1) of HUVEC grown in either monolayer cultures or in vitro tube formation assays. Activity-based probes that bind covalently to active cysteine cathepsins and degradation products of DQ-collagen IV partially localized to intracellular vesicles that contained cav-1 and active cysteine cathepsins. Biochemical analyses revealed that the distribution of active cathepsin B in caveolar fractions increased during in vitro tube formation. Pro-uPA, uPAR, MMP-2 and MMP-14, which have been linked with cathepsin B to ECM degradation pathways, were also found to increase in caveolar fractions during in vitro tube formation. Our findings are the first to demonstrate through live-cell imaging ECM degradation in association with active cathepsin B in caveolae of endothelial cells during tube formation.
The expression of the cysteine protease cathepsin B is increased in early stages of human breast cancer.To assess the potential role of cathepsin B in premalignant progression of breast epithelial cells, we employed a 3D reconstituted basement membrane overlay culture model of MCF10A human breast epithelial cells and isogenic variants that replicate the in vivo phenotypes of hyper plasia(MCF10AneoT) and atypical hyperplasia (MCF10AT1). MCF10A cells developed into polarized acinar structures with central lumens. In contrast, MCF10AneoT and MCF10AT1 cells form larger structures in which the lumens are filled with cells. CA074Me, a cell-permeable inhibitor selective for the cysteine cathepsins B and L,reduced proliferation and increased apoptosis of MCF10A, MCF10AneoT and MCF10AT1 cells in 3D culture. We detected active cysteine cathepsins in the isogenic MCF10 variants in 3D culture with GB111, a cell-permeable activity based probe, and established differential inhibition of cathepsin B in our 3D cultures. We conclude that cathepsin B promotes proliferation and premalignant progression of breast epithelial cells. These findings are consistent with studies by others showing that deletion of cathepsin B in the transgenic MMTV-PyMT mice, a murine model that is predisposed to development of mammary cancer, reduces malignant progression.
The marine natural product symplostatin 4 (Sym4) has been recognized as a potent antimalarial agent. However, its mode of action and, in particular, direct targets have to date remained elusive. We report a chemical synthesis of Sym4 and show that Sym4-treatment of P. falciparum-infected red blood cells (RBCs) results in the generation of a swollen food vacuole phenotype and a reduction of parasitemia at nanomolar concentrations. We furthermore demonstrate that Sym4 is a nanomolar inhibitor of the P. falciparum falcipains in infected RBCs, suggesting inhibition of the hemoglobin degradation pathway as Sym4s mode of action. Finally, we reveal a critical influence of the unusual methyl-methoxypyrrolinone (mmp) group of Sym4 for potent inhibition, indicating that Sym4 derivatives with such a mmp moiety might represent viable lead structures for the development of antimalarial falcipain inhibitors.
Legumain is a lysosomal cysteine protease whose biological function remains poorly defined. Legumain activity is up-regulated in most human cancers and inflammatory diseases most likely as the result of high expression in populations of activated macrophages. Within the tumor microenvironment, legumain activity is thought to promote tumorigenesis. To obtain a greater understanding of the role of legumain activity during cancer progression and inflammation, we developed an activity-based probe that becomes fluorescent only upon binding active legumain. This probe is highly selective for legumain, even in the context of whole cells and tissues, and is also a more effective label of legumain than previously reported probes. Here we present the synthesis and application of our probe to the analysis of legumain activity in primary macrophages and in two mouse models of cancer. We find that legumain activity is highly correlated with macrophage activation and furthermore that it is an ideal marker for primary tumor inflammation and early stage metastatic lesions.
The Plasmodium proteasome has been suggested to be a potential antimalarial drug target; however, toxicity of inhibitors has prevented validation of this enzyme in vivo. We report a screen of a library of 670 analogs of the recent US Food and Drug Administration-approved inhibitor, carfilzomib, to identify compounds that selectively kill parasites. We identified one compound, PR3, that has significant parasite killing activity in vitro but dramatically reduced toxicity in host cells. We found that this parasite-specific toxicity is not due to selective targeting of the Plasmodium proteasome over the host proteasome, but instead is due to a lack of activity against one of the human proteasome subunits. Subsequently, we used PR3 to significantly reduce parasite load in Plasmodium berghei infected mice without host toxicity, thus validating the proteasome as a viable antimalarial drug target.
Vacuolar processing enzymes (VPEs) are important cysteine proteases that are implicated in the maturation of seed storage proteins, and programmed cell death during plant-microbe interactions and development. Here, we introduce a specific, cell-permeable, activity-based probe for VPEs. This probe is highly specific for all four Arabidopsis VPEs, and labeling is activity-dependent, as illustrated by sensitivity for inhibitors, pH and reducing agents. We show that the probe can be used for in vivo imaging and displays multiple active isoforms of VPEs in various tissues and in both monocot and dicot plant species. Thus, VPE activity profiling is a robust, simple and powerful tool for plant research for a wide range of applications. Using VPE activity profiling, we discovered that VPE activity is increased during infection with the oomycete pathogen Hyaloperonospora arabidopsidis (Hpa). The enhanced VPE activity is host-derived and EDS1-independent. Sporulation of Hpa is reduced on vpe mutant plants, demonstrating a role for VPE during compatible interactions that is presumably independent of programmed cell death. Our data indicate that, as an obligate biotroph, Hpa takes advantage of increased VPE activity in the host, e.g. to mediate protein turnover and nutrient release.
RgpA and Kgp gingipains are non-covalent complexes of endoprotease catalytic and hemagglutinin-adhesin domains on the surface of Porphyromonas gingivalis. A motif conserved in each domain has been suggested to function as an oligomerization motif. We tested this hypothesis by mutating motif residues to hexahistidine or insertion of hexahistidine tag to disrupt the motif within the Kgp catalytic domain. All modifications led to the secretion of entire Kgp activity into the growth media, predominantly in a form without functional His-tag. This confirmed the role of the conserved motif in correct posttranslational proteolytic processing and assembly of the multidomain complexes.
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