Thermoprotection by a cell membrane-localized metacaspase in a green alga.

Caspases are restricted to animals, while other organisms, including plants, possess metacaspases (MCAs), a more ancient and broader class of structurally related yet biochemically distinct proteases. Our current understanding of plant MCAs is derived from studies in streptophytes, and mostly in Arabidopsis (Arabidopsis thaliana) with 9 MCAs with partially redundant activities. In contrast to streptophytes, most chlorophytes contain only 1 or 2 uncharacterized MCAs, providing an excellent platform for MCA research. Here we investigated CrMCA-II, the single type-II MCA from the model chlorophyte Chlamydomonas (Chlamydomonas reinhardtii). Surprisingly, unlike other studied MCAs and similar to caspases, CrMCA-II dimerizes both in vitro and in vivo. Furthermore, activation of CrMCA-II in vivo correlated with its dimerization. Most of CrMCA-II in the cell was present as a proenzyme (zymogen) attached to the plasma membrane (PM). Deletion of CrMCA-II by genome editing compromised thermotolerance, leading to increased cell death under heat stress. Adding back either wild-type or catalytically dead CrMCA-II restored thermoprotection, suggesting that its proteolytic activity is dispensable for this effect. Finally, we connected the non-proteolytic role of CrMCA-II in thermotolerance to the ability to modulate PM fluidity. Our study reveals an ancient, MCA-dependent thermotolerance mechanism retained by Chlamydomonas and probably lost during the evolution of multicellularity.

Resurrection of an ancient inflammatory locus reveals switch to caspase-1 specificity on a caspase-4 scaffold.

Pyroptosis is a mechanism of inflammatory cell death mediated by the activation of the prolytic protein gasdermin D by caspase-1, caspase-4, and caspase-5 in human, and caspase-1 and caspase-11 in mouse. In addition, caspase-1 amplifies inflammation by proteolytic activation of cytokine interleukin-1ß (IL-1ß). Modern mammals of the order Carnivora lack the caspase-1 catalytic domain but express an unusual version of caspase-4 that can activate both gasdermin D and IL-1ß. Seeking to understand the evolutionary origin of this caspase, we utilized the large amount of data available in public databases to perform ancestral sequence reconstruction of an inflammatory caspase of a Carnivora ancestor. We expressed the catalytic domain of this putative ancestor in Escherichia coli, purified it, and compared its substrate specificity on synthetic and protein substrates to extant caspases. We demonstrated that it activates gasdermin D but has reduced ability to activate IL-1ß. Our reconstruction suggests that caspase-1 was lost in a Carnivora ancestor, perhaps upon a selective pressure for which the generation of biologically active IL-1ß by caspase-1 was detrimental. We speculate that later, a Carnivora encountered selective pressures that required the production of IL-1ß, and caspase-4 subsequently gained this activity. This hypothesis would explain why extant Carnivora possess an inflammatory caspase with caspase-1 catalytic function placed on a caspase-4 scaffold.

Extended subsite profiling of the pyroptosis effector protein gasdermin D reveals a region recognized by inflammatory caspase-11.

Pyroptosis is the caspase-dependent inflammatory cell death mechanism that underpins the innate immune response against pathogens and is dysregulated in inflammatory disorders. Pyroptosis occurs via two pathways: the canonical pathway, signaled by caspase-1, and the noncanonical pathway, regulated by mouse caspase-11 and human caspase-4/5. All inflammatory caspases activate the pyroptosis effector protein gasdermin D, but caspase-1 mostly activates the inflammatory cytokine precursors prointerleukin-18 and prointerleukin-1ß (pro-IL18/pro-IL1ß). Here, in vitro cleavage assays with recombinant proteins confirmed that caspase-11 prefers cleaving gasdermin D over the pro-ILs. However, we found that caspase-11 recognizes protein substrates through a mechanism that is different from that of most caspases. Results of kinetics analysis with synthetic fluorogenic peptides indicated that P1'-P4', the C-terminal gasdermin D region adjacent to the cleavage site, influences gasdermin D recognition by caspase-11. Furthermore, introducing the gasdermin D P1'-P4' region into pro-IL18 enhanced catalysis by caspase-11 to levels comparable with that of gasdermin D cleavage. Pro-IL1ß cleavage was only moderately enhanced by similar substitutions. We conclude that caspase-11 specificity is mediated by the P1'-P4' region in its substrate gasdermin D, and similar experiments confirmed that the substrate specificities of the human orthologs of caspase-11, i.e. caspase-4 and caspase-5, are ruled by the same mechanism. We propose that P1'-P4'-based inhibitors could be exploited to specifically target inflammatory caspases.

Multiplexed Probing of Proteolytic Enzymes Using Mass Cytometry-Compatible Activity-Based Probes.

The subset of the proteome that contains enzymes in their catalytically active form can be interrogated by using probes targeted toward individual specific enzymes. A subset of such enzymes are proteases that are frequently studied with activity-based probes, small inhibitors equipped with a detectable tag, commonly a fluorophore. Due to the spectral overlap of these commonly used fluorophores, multiplex analysis becomes limited. To overcome this, we developed a series of protease-selective lanthanide-labeled probes compatible with mass cytometry giving us the ability to monitor the activity of multiple proteases in parallel. Using these probes, we were able to identify the distribution of four proteases with different active site geometries in three cell lines and peripheral blood mononuclear cells. This provides a framework for the use of mass cytometry for multiplexed enzyme activity detection.

Extensive peptide and natural protein substrate screens reveal that mouse caspase-11 has much narrower substrate specificity than caspase-1.

Inflammatory cell death, or pyroptosis, is triggered by pathogenic infections or events. It is executed by caspase-1 (in the canonical pyroptosis pathway) or caspase-11 (noncanonical pathway), each via production of a cell-lytic domain from the pyroptosis effector protein gasdermin D through specific and limited proteolysis. Pyroptosis is accompanied by the release of inflammatory mediators, including the proteolytically processed forms of interleukin-1ß (IL-1ß) and IL-18. Given the similar inflammatory outcomes of the canonical and noncanonical pyroptosis pathways, we hypothesized that caspase-1 and -11 should have very similar activities and substrate specificities. To test this hypothesis, we purified recombinant murine caspases and analyzed their primary specificities by massive hybrid combinatorial substrate library (HyCoSuL) screens. We correlated the substrate preferences of each caspase with their activities on the recombinant natural substrates IL-1ß, IL-18, and gasdermin D. Although we identified highly selective and robust peptidyl substrates for caspase-1, we were unable to do so for caspase-11, because caspase-1 cleaved even the best caspase-11 substrates equally well. Caspase-1 rapidly processed pro-IL-1ß and -18, but caspase-11 processed these two pro-ILs extremely poorly. However, both caspase-1 and -11 efficiently produced the cell-lytic domain from the gasdermin D precursor. We hypothesize that caspase-11 may have evolved a specific exosite to selectively engage pyroptosis without directly activating pro-IL-1ß or -18. In summary, comparing the activities of caspase-1 and -11 in HyCoSuL screens and with three endogenous protein substrates, we conclude that caspase-11 has highly restricted substrate specificity, preferring gasdermin D over all other substrates examined.

Recent advances in the development of legumain-selective chemical probes and peptide prodrugs.

Legumain, which is also known as vacuolar processing enzyme (VPE) or asparaginyl endopeptidase (AEP), is a cysteine protease that was first discovered and characterized in the leguminous seeds of the moth bean in the early 1990s. Later, this enzyme was also detected in higher organisms, including eukaryotes. This pH-dependent protease displays the highest activity in acidic endolysosomal compartments; however, legumain also displays nuclear, cytosolic and extracellular activity when stabilized by other proteins or intramolecular complexes. Based on the results from over 25 years of research, this protease is involved in multiple cellular events, including protein degradation and antigen presentation. Moreover, when dysregulated, this protease contributes to the progression of several diseases, with cancer being the well-studied example. Research on legumain biology was undoubtedly facilitated by the use of small molecule chemical tools. Therefore, in this review, I present the historical perspectives and most current strategies for the development of small molecule substrates, inhibitors and activity-based probes for legumain. These tools are of paramount importance in elucidating the roles of legumain in multiple biological processes. Finally, as this enzyme appears to be a promising molecular target for anticancer therapies, the development of legumain-activated prodrugs is also described.

Anticancer properties of ester derivatives of betulin in human metastatic melanoma cells (Me-45).

BACKGROUND: Betulinic acid and betulin are triterpenes that have anticancer properties in various types of cancer. Unfortunately, the bioavailability and the bio-distribution of betulinic acid and its metabolic precursor, betulin are very low because of poor solubility in aqueous buffers. METHODS: In this study, we examined the anticancer properties of the ester derivatives of betulin compared to their precursors in a malignant melanoma cell line. We assessed five amino acid esters of betulin. The compounds contained four basic amino acids-natural lysine (l-Lys-OH) and three of its derivatives (l-Dap-OH, l-Dab-OH, and l-Orn-OH)-and alanine (l-Ala-OH) as a negative control (amino acid without an amine group in the side chain). The derivatives were more soluble than their precursors (betulin and betulinic acid) in water. The betulin esters were tested in the malignant melanoma cell line Me-45. To evaluate the cytotoxicity, MTT test was performed after 24, 48 and 72 h of incubation with the test compounds at a concentration range of 0.75-100 µM. For analysis of the apoptotic activity, TUNEL assay was performed. Additionally, expression of caspase-3 and PARP-1 was investigated immunocytochemically. RESULTS: The highest biological activity was observed with the lysine ester. The results showed that the highest cytotoxicity and the highest number of positively stained nuclei in metastatic melanoma Me-45 cells were obtained after 72 h of incubation with betulin derivatives containing lysine and ornithine. CONCLUSIONS: The betulin ester derivatives showed enhanced antitumor activity compared to their non-modified precursors. Esters of betulin can be more potent anticancer agents than their precursor as a consequence of the rapid bioavailability and increased concentration in cancer cells.

Small Molecule Active Site Directed Tools for Studying Human Caspases.

Caspases are proteases of clan CD and were described for the first time more than two decades ago. They play critical roles in the control of regulated cell death pathways including apoptosis and inflammation. Due to their involvement in the development of various diseases like cancer, neurodegenerative diseases, or autoimmune disorders, caspases have been intensively investigated as potential drug targets, both in academic and industrial laboratories. This review presents a thorough, deep, and systematic assessment of all technologies developed over the years for the investigation of caspase activity and specificity using substrates and inhibitors, as well as activity based probes, which in recent years have attracted considerable interest due to their usefulness in the investigation of biological functions of this family of enzymes.

Design of ultrasensitive probes for human neutrophil elastase through hybrid combinatorial substrate library profiling.

The exploration of protease substrate specificity is generally restricted to naturally occurring amino acids, limiting the degree of conformational space that can be surveyed. We substantially enhanced this by incorporating 102 unnatural amino acids to explore the S1-S4 pockets of human neutrophil elastase. This approach provides hybrid natural and unnatural amino acid sequences, and thus we termed it the Hybrid Combinatorial Substrate Library. Library results were validated by the synthesis of individual tetrapeptide substrates, with the optimal substrate demonstrating more than three orders of magnitude higher catalytic efficiency than commonly used substrates of elastase. This optimal substrate was converted to an activity-based probe that demonstrated high selectivity and revealed the specific presence of active elastase during the process of neutrophil extracellular trap formation. We propose that this approach can be successfully used for any type of endopeptidase to deliver high activity and selectivity in substrates and probes.

Biochemical Characterization and Substrate Specificity of Autophagin-2 from the Parasite Trypanosoma cruzi.

The genome of the parasite Trypanosoma cruzi encodes two copies of autophagy-related cysteine proteases, Atg4.1 and Atg4.2. T. cruzi autophagin-2 (TcAtg4.2) carries the majority of proteolytic activity and is responsible for processing Atg8 proteins near the carboxyl terminus, exposing a conserved glycine. This enables progression of autophagy and differentiation of the parasite, which is required for successful colonization of humans. The mechanism of substrate hydrolysis by Atg4 was found to be highly conserved among the species as critical mutations in the TcAtg4.2, including mutation of the conserved Gly-244 residue in the hinge region enabling flexibility of the regulatory loop, and deletion of the regulatory loop, completely abolished processing capacity of the mutants. Using the positional scanning-substrate combinatorial library (PS-SCL) we determined that TcAtg4.2 tolerates a broad spectrum of amino acids in the P4 and P3 positions, similar to the human orthologue autophagin-1 (HsAtg4B). In contrast, both human and trypanosome Atg4 orthologues exhibited exclusive preference for aromatic amino acid residues in the P2 position, and for Gly in the P1 position, which is absolutely conserved in the natural Atg8 substrates. Using an extended P2 substrate library, which also included the unnatural amino acid cyclohexylalanine (Cha) derivative of Phe, we generated highly selective tetrapeptide substrates acetyl-Lys-Lys-Cha-Gly-AFC (Ac-KKChaG-AFC) and acetyl-Lys-Thr-Cha-Gly-AFC (Ac-KTChaG-AFC). Althoughthese substrates were cleaved by cathepsins, making them unsuitable for analysis of complex cellular systems, they were recognized exclusively by TcAtg4.2, but not by HsAtg4B nor by the structurally related human proteases SENP1, SENP2, and UCH-L3.

Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease.

Plants lack aspartate-specific cell death proteases homologous to animal caspases. Instead, a subtilisin-like serine-dependent plant protease named phytaspase shown to be involved in the accomplishment of programmed death of plant cells is able to hydrolyze a number of peptide-based caspase substrates. Here, we determined the substrate specificity of rice (Oryza sativa) phytaspase by using the positional scanning substrate combinatorial library approach. Phytaspase was shown to display an absolute specificity of hydrolysis after an aspartic acid residue. The preceding amino acid residues, however, significantly influence the efficiency of hydrolysis. Efficient phytaspase substrates demonstrated a remarkable preference for an aromatic amino acid residue in the P3 position. The deduced optimum phytaspase recognition motif has the sequence IWLD and is strikingly hydrophobic. The established pattern was confirmed through synthesis and kinetic analysis of cleavage of a set of optimized peptide substrates. An amino acid motif similar to the phytaspase cleavage site is shared by the human gastrointestinal peptide hormones gastrin and cholecystokinin. In agreement with the established enzyme specificity, phytaspase was shown to hydrolyze gastrin-1 and cholecystokinin at the predicted sites in vitro, thus destroying the active moieties of the hormones.

Structural and functional insights into caseinolytic proteases reveal an unprecedented regulation principle of their catalytic triad.

Caseinolytic proteases (ClpPs) are large oligomeric protein complexes that contribute to cell homeostasis as well as virulence regulation in bacteria. Although most organisms possess a single ClpP protein, some organisms encode two or more ClpP isoforms. Here, we elucidated the crystal structures of ClpP1 and ClpP2 from pathogenic Listeria monocytogenes and observe an unprecedented regulation principle by the catalytic triad. Whereas L. monocytogenes (Lm)ClpP2 is both structurally and functionally similar to previously studied tetradecameric ClpP proteins from Escherichia coli and Staphylococcus aureus, heptameric LmClpP1 features an asparagine in its catalytic triad. Mutation of this asparagine to aspartate increased the reactivity of the active site and led to the assembly of a tetradecameric complex. We analyzed the heterooligomeric complex of LmClpP1 and LmClpP2 via coexpression and subsequent labeling studies with natural product-derived probes. Notably, the LmClpP1 peptidase activity is stimulated 75-fold in the complex providing insights into heterooligomerization as a regulatory mechanism. Collectively, our data point toward different preferences for substrates and inhibitors of the two ClpP enzymes and highlight their structural and functional characteristics.

Recent advances and concepts in substrate specificity determination of proteases using tailored libraries of fluorogenic substrates with unnatural amino acids.

Substrate specificity of proteases can be determined using several methods among which the most frequently used are positional scanning library, proteomics and phage display. Classic approaches can deliver information about preferences for natural amino acids in binding pockets of virtually all proteases. However, recent studies demonstrate the ability to obtain much more information by application of unnatural amino acids to positional scanning library approaches. This knowledge can be used for the design of more active and specific substrates, inhibitors and activity based probes. In this minireview we describe recent strategies and concepts for the design and application of fluorogenic substrates library tailored for exopeptidases and endopeptidases.

Unnatural amino acids increase activity and specificity of synthetic substrates for human and malarial cathepsin C.

Mammalian cathepsin C is primarily responsible for the removal of N-terminal dipeptides and activation of several serine proteases in inflammatory or immune cells, while its malarial parasite ortholog dipeptidyl aminopeptidase 1 plays a crucial role in catabolizing the hemoglobin of its host erythrocyte. In this report, we describe the systematic substrate specificity analysis of three cathepsin C orthologs from Homo sapiens (human), Bos taurus (bovine) and Plasmodium falciparum (malaria parasite). Here, we present a new approach with a tailored fluorogenic substrate library designed and synthesized to probe the S1 and S2 pocket preferences of these enzymes with both natural and a broad range of unnatural amino acids. Our approach identified very efficiently hydrolyzed substrates containing unnatural amino acids, which resulted in the design of significantly better substrates than those previously known. Additionally, in this study significant differences in terms of the structures of optimal substrates for human and malarial orthologs are important from the therapeutic point of view. These data can be also used for the design of specific inhibitors or activity-based probes.

CD20 expression regulates CD37 levels in B-cell lymphoma - implications for immunotherapies.

Rituximab (RTX) plus chemotherapy (R-CHOP) applied as a first-line therapy for lymphoma leads to a relapse in approximately 40% of the patients. Therefore, novel approaches to treat aggressive lymphomas are being intensively investigated. Several RTX-resistant (RR) cell lines have been established as surrogate models to study resistance to R-CHOP. Our study reveals that RR cells are characterized by a major downregulation of CD37, a molecule currently explored as a target for immunotherapy. Using CD20 knockout (KO) cell lines, we demonstrate that CD20 and CD37 form a complex, and hypothesize that the presence of CD20 stabilizes CD37 in the cell membrane. Consequently, we observe a diminished cytotoxicity of anti-CD37 monoclonal antibody (mAb) in complement-dependent cytotoxicity in both RR and CD20 KO cells that can be partially restored upon lysosome inhibition. On the other hand, the internalization rate of anti-CD37 mAb in CD20 KO cells is increased when compared to controls, suggesting unhampered efficacy of antibody drug conjugates (ADCs). Importantly, even a major downregulation in CD37 levels does not hamper the efficacy of CD37-directed chimeric antigen receptor (CAR) T cells. In summary, we present here a novel mechanism of CD37 regulation with further implications for the use of anti-CD37 immunotherapies.

The Staphylococcus aureus leucine aminopeptidase is localized to the bacterial cytosol and demonstrates a broad substrate range that extends beyond leucine.

Staphylococcus aureus is a potent pathogen of humans exhibiting a broad disease range, in part due to an extensive repertoire of secreted virulence factors, including proteases. Recently, we identified the first example of an intracellular protease (leucine aminopeptidase, LAP) that is required for virulence in S. aureus. Disruption of pepZ, the gene encoding LAP, had no affect on the growth rate of bacteria; however, in systemic and localized infection models the pepZ mutant had significantly attenuated virulence. Recently, a contradictory report was published suggesting that LAP is an extracellular enzyme and it is required for growth in S. aureus. Here, we investigate these results and confirm our previous findings that LAP is localized to the bacterial cytosol and is not required for growth. In addition, we conduct a biochemical investigation of purified recombinant LAP, identifying optimal conditions for enzymatic activity and substrate preference for hydrolysis. Our results show that LAP has a broad substrate range, including activity against the dipeptide cysteine-glycine, and that leucine is not the primary target of LAP.

Mechanism and specificity of the human paracaspase MALT1.

The paracaspase domain of MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a component of a gene translocation fused to the N-terminal domains of the cellular inhibitor of apoptosis protein 2. The paracaspase itself, commonly known as MALT1, participates in the NF-κB (nuclear factor κB) pathway, probably by driving survival signals downstream of the B-cell antigen receptor through MALT1 proteolytic activity. We have developed methods for the expression and purification of recombinant full-length MALT1 and its constituent catalytic domain alone. Both are activated by dimerization without cleavage, with a similar dimerization barrier to the distantly related cousins, the apical caspases. By using positional-scanning peptidyl substrate libraries we demonstrate that the activity and specificity of full-length MALT1 is recapitulated by the catalytic domain alone, showing a stringent requirement for cleaving after arginine, and with striking peptide length constraints for efficient hydrolysis. Rates of cleavage (kcat/Km values) of optimal peptidyl substrates are in the same order (10(3)-10(4) M(-1)·s(-1)) as for a putative target protein CYLD. Thus MALT1 has many similarities to caspase 8, even cleaving the putative target protein CYLD with comparable efficiencies, but with diametrically opposite primary substrate specificity.

Imaging of proteases using activity-based probes.

Proteases (proteolytic enzymes) are proteins that catalyze one of the most important biochemical reactions, namely the hydrolysis of the peptide bond in peptide and protein substrates. Therefore these molecular biocatalysts participate in virtually all living processes. The proper balance between intact and processed protease substrates enables to maintenance of homeostasis from a single-cell level to the whole living system. However, when the proteolytic activity is altered, this delicate balance is disturbed, which might lead to the development of a plethora of diseases. Given this, monitoring proteolytic activity is indispensable to understanding how proteases operate in disease lesions and how their altered catalytic activity might be harnessed for a better diagnosis and treatment. In this manuscript, we provide a critical review of the recent development of protease chemical probes which are small molecules that detect proteolytic activity by interacting with protease active site, individual proteases as well as complex proteolytic networks.

Selective chemical reagents to investigate the role of caspase 6 in apoptosis in acute leukemia T cells.

Activated effector caspases 3, 6 and 7 are responsible for cleaving a number of target substrates, leading to the ultimate destruction of cells via apoptosis. The functions of caspases 3 and 7 in apoptosis execution have been widely studied over the years with multiple chemical probes for both of these enzymes. In contrast, caspase 6 seems to be largely neglected when compared to the heavily studied caspases 3 and 7. Therefore, the development of new small-molecule reagents for the selective detection and visualization of caspase 6 activity can improve our understanding of molecular circuits of apoptosis and shed new light on how they intertwine with other types of programmed cell death. In this study, we profiled caspase 6 substrate specificity at the P5 position and discovered that, similar to caspase 2, caspase 6 prefers pentapeptide substrates over tetrapeptides. Based on these data, we developed a set of chemical reagents for caspase 6 investigation, including coumarin-based fluorescent substrates, irreversible inhibitors and selective aggregation-induced emission luminogens (AIEgens). We showed that AIEgens are able to distinguish between caspase 3 and caspase 6 in vitro. Finally, we validated the efficiency and selectivity of the synthesized reagents by monitoring lamin A and PARP cleavage via mass cytometry and western blot analysis. We propose that our reagents may provide new research prospects for single-cell monitoring of caspase 6 activity to reveal its function in programmed cell death pathways.

Investigation of osteoclast cathepsin K activity in osteoclastogenesis and bone loss using a set of chemical reagents.

Cathepsin K (CatK) is a lysosomal cysteine protease whose highest expression is found in osteoclasts, which are the cells responsible for bone resorption. Investigations of the functions and physiological relevance of CatK have often relied on antibody-related techniques, which makes studying its activity patterns a challenging task. Hence, we developed a set of chemical tools for the investigation of CatK activity. We show that our probe is a valuable tool for monitoring the proteolytic activation of CatK during osteoclast formation. Moreover, we demonstrate that our inhibitor of CatK impedes osteoclastogenesis and bone resorption and that CatK is stored in its active form in osteoclasts within their lysosomal compartment and mainly in the ruffled borders of osteoclasts. Given that our probe recognizes active CatK within living cells without exhibiting any observed cytotoxicity in the several models tested, we expect that it would be well suited to theranostic applications in CatK-related diseases.