Chemoproteomics Identifies State-Dependent and Proteoform-Selective Caspase-2 Inhibitors.

Caspases are a highly conserved family of cysteine-aspartyl proteases known for their essential roles in regulating apoptosis, inflammation, cell differentiation, and proliferation. Complementary to genetic approaches, small-molecule probes have emerged as useful tools for modulating caspase activity. However, due to the high sequence and structure homology of all 12 human caspases, achieving selectivity remains a central challenge for caspase-directed small-molecule inhibitor development efforts. Here, using mass spectrometry-based chemoproteomics, we first identify a highly reactive noncatalytic cysteine that is unique to caspase-2. By combining both gel-based activity-based protein profiling (ABPP) and a tobacco etch virus (TEV) protease activation assay, we then identify covalent lead compounds that react preferentially with this cysteine and afford a complete blockade of caspase-2 activity. Inhibitory activity is restricted to the zymogen or precursor form of monomeric caspase-2. Focused analogue synthesis combined with chemoproteomic target engagement analysis in cellular lysates and in cells yielded both pan-caspase-reactive molecules and caspase-2 selective lead compounds together with a structurally matched inactive control. Application of this focused set of tool compounds to stratify the functions of the zymogen and partially processed (p32) forms of caspase-2 provide evidence to support that caspase-2-mediated response to DNA damage is largely driven by the partially processed p32 form of the enzyme. More broadly, our study highlights future opportunities for the development of proteoform-selective caspase inhibitors that target nonconserved and noncatalytic cysteine residues.

L-Arginine sulfate reduces irreversible protein binding in immobilized metal affinity chromatography.

Immobilized metal affinity chromatography (IMAC) is a powerful technique for capture and purification of relevant biopharmaceuticals in complex biological matrices. However, protein recovery can be drastically compromised due to surface induced spreading and unfolding of the analyte, leading to fouling of the stationary phase. Here, we report on the kinetics of irreversible adsorption of a protease on an IMAC resin in a time span ranging from minutes to several hours. This trend correlated with the thermal data measured by nano differential scanning calorimetry, and showed a time-dependent change in protein unfolding temperature. Our results highlight that 'soft' proteins show a strong time dependent increase in irreversible adsorption. Furthermore, commonly used co-solvents for preservation of the native protein conformation are tested for their ability to reduce fouling. Thermal data suggests that the amino acid l-arginine is beneficial in preventing unfolding, which was confirmed in batch adsorption experiments. The choice of counter-ions has to be considered when using this amino acid. These results show that l-arginine sulfate decelerates the irreversible adsorption kinetics of proteins on the IMAC stationary phase to a greater extent than l-arginine chloride.

PROFICS: A bacterial selection system for directed evolution of proteases.

Proteases serve as important tools in biotechnology and as valuable drugs or drug targets. Efficient protein engineering methods to study and modulate protease properties are thus of great interest for a plethora of applications. We established PROFICS (PRotease Optimization via Fusion-Inhibited Carbamoyltransferase-based Selection), a bacterial selection system, which enables the optimization of proteases for biotechnology, therapeutics or diagnosis in a simple overnight process. During the PROFICS process, proteases are selected for their ability to specifically cut a tag from a reporter enzyme and leave a native N-terminus. Precise and efficient cleavage after the recognition sequence reverses the phenotype of an Escherichia coli knockout strain deficient in an essential enzyme of pyrimidine synthesis. A toolbox was generated to select for proteases with different preferences for P1' residues (the residue immediately following the cleavage site). The functionality of PROFICS is demonstrated with viral proteases and human caspase-2. PROFICS improved caspase-2 activity up to 25-fold after only one round of mutation and selection. Additionally, we found a significantly improved tolerance for all P1' residues caused by a mutation in a substrate interaction site. We showed that this improved activity enables cells containing the new variant to outgrow cells containing all other mutants, facilitating its straightforward selection. Apart from optimizing enzymatic activity and P1' tolerance, PROFICS can be used to reprogram specificities, erase off-target activity, optimize expression via tags/codon usage, or even to screen for potential drug-resistance-conferring mutations in therapeutic targets such as viral proteases in an unbiased manner.

The p53-caspase-2 axis in the cell cycle and DNA damage response.

Caspase-2 was discovered almost three decades ago. It was one of the first two mammalian homologs of CED-3, the other being interleukin 1ß-converting enzyme (ICE/caspase-1). Despite high similarity with CED-3 and its fly and mammalian counterparts (DRONC and caspase-9, respectively), the function of caspase-2 in apoptosis has remained enigmatic. A number of recent studies suggest that caspase-2 plays an important role in the regulation of p53 in response to cellular stress and DNA damage to prevent the proliferation and accumulation of damaged or aberrant cells. Here, we review these recent observations and their implications in caspase-2-mediated cellular death, senescence, and tumor suppression.

The Role of Caspase-2 in Regulating Cell Fate.

Caspase-2 is the most evolutionarily conserved member of the mammalian caspase family and has been implicated in both apoptotic and non-apoptotic signaling pathways, including tumor suppression, cell cycle regulation, and DNA repair. A myriad of signaling molecules is associated with the tight regulation of caspase-2 to mediate multiple cellular processes far beyond apoptotic cell death. This review provides a comprehensive overview of the literature pertaining to possible sophisticated molecular mechanisms underlying the multifaceted process of caspase-2 activation and to highlight its interplay between factors that promote or suppress apoptosis in a complicated regulatory network that determines the fate of a cell from its birth and throughout its life.

Enhancing the promiscuity of a member of the Caspase protease family by rational design.

The N-terminal cleavage of fusion tags to restore the native N-terminus of recombinant proteins is a challenging task and up to today, protocols need to be optimized for different proteins individually. Within this work, we present a novel protease that was designed in-silico to yield enhanced promiscuity toward different N-terminal amino acids. Two mutations in the active-site amino acids of human Caspase-2 were determined to increase the recognition of branched amino-acids, which show only poor binding capabilities in the unmutated protease. These mutations were determined by sequential and structural comparisons of Caspase-2 and Caspase-3 and their effect was additionally predicted using free-energy calculations. The two mutants proposed in the in-silico studies were expressed and in-vitro experiments confirmed the simulation results. Both mutants showed not only enhanced activities toward branched amino acids, but also smaller, unbranched amino acids. We believe that the created mutants constitute an important step toward generalized procedures to restore original N-termini of recombinant fusion proteins.

14-3-3 protein binding blocks the dimerization interface of caspase-2.

Among all species, caspase-2 (C2) is the most evolutionarily conserved caspase required for effective initiation of apoptosis following death stimuli. C2 is activated through dimerization and autoproteolytic cleavage and inhibited through phosphorylation at Ser139 and Ser164 , within the linker between the caspase recruitment and p19 domains of the zymogen, followed by association with the adaptor protein 14-3-3, which maintains C2 in its immature form procaspase (proC2). However, the mechanism of 14-3-3-dependent inhibition of C2 activation remains unclear. Here, we report the structural characterization of the complex between proC2 and 14-3-3 by hydrogen/deuterium mass spectrometry and protein crystallography to determine the molecular basis for 14-3-3-mediated inhibition of C2 activation. Our data reveal that the 14-3-3 dimer interacts with proC2 not only through ligand-binding grooves but also through other regions outside the central channel, thus explaining the isoform-dependent specificity of 14-3-3 protein binding to proC2 and the substantially higher binding affinity of 14-3-3 protein to proC2 than to the doubly phosphorylated peptide. The formation of the complex between 14-3-3 protein and proC2 does not induce any large conformational change in proC2. Furthermore, 14-3-3 protein interacts with and masks both the nuclear localization sequence and the C-terminal region of the p12 domain of proC2 through transient interactions in which both the p19 and p12 domains of proC2 are not firmly docked onto the surface of 14-3-3. This masked region of p12 domain is involved in C2 dimerization. Therefore, 14-3-3 protein likely inhibits proC2 activation by blocking its dimerization surface. DATABASES: Structural data are available in the Protein Data Bank under the accession numbers 6SAD and 6S9K.

Quantum Mechanics/Molecular Mechanics Study on Caspase-2 Recognition by Peptide Inhibitors.

For a variety of biological and medical reasons, the ongoing development of humane caspase-2 inhibitors is of vital importance. Herein, a hybrid (Quantum Mechanics/Molecular Mechanics - QM/MM), two-layered molecular model is derived in order to understand better the affinity and specificity of peptide inhibitor interaction with caspase-2. By taking care of both the unique structural features and the catalytic activity of human caspase-2, the critical enzyme residues (E217, R378, N379, T380, and Y420) with the peptide inhibitor are treated at QM level (the Self-Consistent-Charge Density-Functional Tight-Binding method with the Dispersion correction (SCC-DFTB-D)), while the remaining part of the complex is treated at MM level (AMBER force field). The QM/MM binding free energies (BFEs) are well-correlated with the experimental observations and indicate that caspase-2 uniquely prefers a penta-peptide such as VDVAD. The sequence of VDVAD is varied in a systematic fashion by considering the physicochemical properties of every constitutive amino acid and its substituent, and the corresponding BFE with the inhibition constant (Ki) is evaluated. The values of Ki for several caspase-2:peptide complexes are found to be within the experimental range (between 0.01 nM and 1 ?M). The affinity order is: VELAD (Ki=0.081 nM) > VDVAD (Ki=0.23 nM) > VEIAD (Ki=0.61 nM) > VEVAD (Ki=3.7 nM) > VDIAD (Ki=4.5 nM) etc. An approximate condition needed to be satisfied by the kinetic parameters (the Michaelis constant - KM and the specificity constant - kcat/KM) for competitive inhibition is reported. The estimated values of kcat/KM, being within the experimentally established range (between 10-4 and 10-1 ?M-1 s-1), indicate that VELAD and VDVAD are most specific to caspase-2. These two particular peptides are nearly 1.5, 3 and 4 times more specific to the receptor than VEIAD, VEVAD and VDIAD respectively. Additional kinetic threshold, aimed to discriminate tightly bound inhibitors, is given.

Identification of TRIM25 as a Negative Regulator of Caspase-2 Expression Reveals a Novel Target for Sensitizing Colon Carcinoma Cells to Intrinsic Apoptosis.

Colorectal cancer (CRC) is one of the most common cancers that is characterized by a high mortality due to the strong metastatic potential of the primary tumor and the high rate of therapy resistance. Hereby, evasion of apoptosis is the primary underlying cause of reduced sensitivity of tumor cells to chemo- and radiotherapy. Using RNA affinity chromatography, we identified the tripartite motif-containing protein 25 (TRIM25) as a bona fide caspase-2 mRNA-binding protein in colon carcinoma cells. Loss-of-function and gain-of-function approaches revealed that TRIM25 attenuates the protein levels of caspase-2 without significantly affecting caspase-2 mRNA levels. In addition, experiments with cycloheximide revealed that TRIM25 does not affect the protein stability of caspase-2. Furthermore, silencing of TRIM25 induced a significant redistribution of caspase-2 transcripts from RNP particles to translational active polysomes, indicating that TRIM25 negatively interferes with caspase-2 translation. Functionally, the elevation in caspase-2 upon TRIM25 depletion significantly increased the sensitivity of colorectal cells to drug-induced intrinsic apoptosis as implicated by increased caspase-3 cleavage and cytochrome c release. Importantly, the apoptosis-sensitizing effects by transient TRIM25 knockdown were rescued by concomitant silencing of caspase-2, demonstrating a critical role of caspase-2. Inhibition of caspase-2 by TRIM25 implies a survival mechanism that critically contributes to chemotherapeutic drug resistance in CRC.

Caspase-2 and p75 neurotrophin receptor (p75NTR) are involved in the regulation of SREBP and lipid genes in hepatocyte cells.

Lipid-induced toxicity is part of several human diseases, but the mechanisms involved are not fully understood. Fatty liver is characterized by the expression of different growth and tissue factors. The neurotrophin, nerve growth factor (NGF) and its pro-form, pro-NGF, are present in fatty liver together with p75 neurotrophin receptor (p75NTR). Stimulation of human Huh7 hepatocyte cells with NGF and pro-NGF induced Sterol-regulator-element-binding protein-2 (SREBP2) activation and increased Low-Density Lipoprotein Receptor (LDLR) expression. We observed that phosphorylation of caspase-2 by p38 MAPK was essential for this regulation involving a caspase-3-mediated cleavage of SREBP2. RNA sequencing showed that several genes involved in lipid metabolism were altered in p75NTR-deficient mouse liver. The same lipogenic genes were downregulated in p75NTR gene-engineered human Huh7 cells and reciprocally upregulated by stimulation of p75NTRs. In the knock-out mice the serum cholesterol and triglyceride levels were reduced, suggesting a physiological role of p75NTRs in whole-body lipid metabolism. Taken together, this study shows that p75NTR signaling influences a network of genes involved in lipid metabolism in liver and hepatocyte cells. Modulation of p75NTR signaling may be a target to consider in various metabolic disorders accompanied by increased lipid accumulation.

14-3-3 protein masks the nuclear localization sequence of caspase-2.

Caspase-2 is an apical protease responsible for the proteolysis of cellular substrates directly involved in mediating apoptotic signaling cascades. Caspase-2 activation is inhibited by phosphorylation followed by binding to the scaffolding protein 14-3-3, which recognizes two phosphoserines located in the linker between the caspase recruitment domain and the p19 domains of the caspase-2 zymogen. However, the structural details of this interaction and the exact role of 14-3-3 in the regulation of caspase-2 activation remain unclear. Moreover, the caspase-2 region with both 14-3-3-binding motifs also contains the nuclear localization sequence (NLS), thus suggesting that 14-3-3 binding may regulate the subcellular localization of caspase-2. Here, we report a structural analysis of the 14-3-3ζ:caspase-2 complex using a combined approach based on small angle X-ray scattering, NMR, chemical cross-linking, and fluorescence spectroscopy. The structural model proposed in this study suggests that phosphorylated caspase-2 and 14-3-3ζ form a compact and rigid complex in which the p19 and the p12 domains of caspase-2 are positioned within the central channel of the 14-3-3 dimer and stabilized through interactions with the C-terminal helices of both 14-3-3ζ protomers. In this conformation, the surface of the p12 domain, which is involved in caspase-2 activation by dimerization, is sterically occluded by the 14-3-3 dimer, thereby likely preventing caspase-2 activation. In addition, 14-3-3 protein binding to caspase-2 masks its NLS. Therefore, our results suggest that 14-3-3 protein binding to caspase-2 may play a key role in regulating caspase-2 activation. DATABASE: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.ww pdb.org (PDB ID codes 6GKF and 6GKG).

Human procaspase-2 phosphorylation at both S139 and S164 is required for 14-3-3 binding.

Procaspase-2 phosphorylation at several residues prevents its activation and blocks apoptosis. This process involves procaspase-2 phosphorylation at S164 and its binding to the scaffolding protein 14-3-3. However, bioinformatics analysis has suggested that a second phosphoserine-containing motif may also be required for 14-3-3 binding. In this study, we show that human procaspase-2 interaction with 14-3-3 is governed by phosphorylation at both S139 and S164. Using biochemical and biophysical approaches, we show that doubly phosphorylated procaspase-2 and 14-3-3 form an equimolar complex with a dissociation constant in the nanomolar range. Furthermore, our data indicate that other regions of procaspase-2, in addition to phosphorylation motifs, may be involved in the interaction with 14-3-3.

Characterization of three caspases and their pathogen-induced expression pattern in Portunus trituberculatus.

Caspases are a family of proteases involved in many important biological processes including apoptosis and inflammation. In this study, we analyzed the expression patterns and effects on immune response in various tissues of the edible crab Portunus trituberculatus. PtCas 2, PtCas 3 and PtCas 4 share overall sequence identities of 55.88%-74.86%, 8.47%-46.54% and 20.11%-50.87%, respectively, with their other crustacean species. PtCas 2, PtCas 3 and PtCas 4 have the same caspase domain and catalytic site found in known caspases. The expression levels of the three caspases differed between tissues. Following bacterial and viral infection, the expression levels of the three caspases reached a maximum level at 24 h post-infection (hpi) in case of bacteria, whereas it was 48 hpi in virus. Moreover, the WSSV, Vibrio alginolyticus or V. parahaemolyticus induced the activities of PtCas 2-4 in a time-dependent manner. These results indicate an involvement of caspases in bacterial and viral induced immune response and demonstrate for the first time that PtCas 2, PtCas 3 and PtCas 4 are essential for optimal response to bacterial and virus infection in crabs.

Apoptosis inhibitor 5 is an endogenous inhibitor of caspase-2.

Caspases are key enzymes responsible for mediating apoptotic cell death. Across species, caspase-2 is the most conserved caspase and stands out due to unique features. Apart from cell death, caspase-2 also regulates autophagy, genomic stability and ageing. Caspase-2 requires dimerization for its activation which is primarily accomplished by recruitment to high molecular weight protein complexes in cells. Here, we demonstrate that apoptosis inhibitor 5 (API5/AAC11) is an endogenous and direct inhibitor of caspase-2. API5 protein directly binds to the caspase recruitment domain (CARD) of caspase-2 and impedes dimerization and activation of caspase-2. Interestingly, recombinant API5 directly inhibits full length but not processed caspase-2. Depletion of endogenous API5 leads to an increase in caspase-2 dimerization and activation. Consistently, loss of API5 sensitizes cells to caspase-2-dependent apoptotic cell death. These results establish API5/AAC-11 as a direct inhibitor of caspase-2 and shed further light onto mechanisms driving the activation of this poorly understood caspase.

Fatty acid synthase inhibition engages a novel caspase-2 regulatory mechanism to induce ovarian cancer cell death.

Blockade of fatty acid synthase (FASN), a key enzyme involved in de novo lipogenesis, results in robust death of ovarian cancer cells. However, known FASN inhibitors have proven to be poor therapeutic agents due to their ability to induce cachexia. Therefore, we sought to identify additional targets in the pathway linking FASN inhibition and cell death whose modulation might kill ovarian cancer cells without the attendant side effects. Here, we show that the initiator caspase-2 is required for robust death of ovarian cancer cells induced by FASN inhibitors. REDD1 (also known as Rtp801 or DDIT4), a known mTOR inhibitor previously implicated in the response to FASN inhibition, is a novel caspase-2 regulator in this pathway. REDD1 induction is compromised in ovarian cancer cells that do not respond to FASN inhibition. Inhibition of FASN induced an ATF4-dependent transcriptional induction of REDD1; downregulation of REDD1 prevented orlistat-induced activation of caspase-2, as monitored by its cleavage, proteolytic activity and dimerization. Abrogation of REDD1-mediated suppression of mTOR by TSC2 RNAi protected FASN inhibitor-sensitive ovarian cancer cells (OVCA420 cells) from orlistat-induced death. Conversely, suppression of mTOR with the chemical inhibitors PP242 or rapamycin-sensitized DOV13, an ovarian cancer cell line incapable of inducing REDD1, to orlistat-induced cell death through caspase-2. These findings indicate that REDD1 positively controls caspase-2-dependent cell death of ovarian cancer cells by inhibiting mTOR, placing mTOR as a novel upstream regulator of caspase-2 and supporting the possibility of manipulating mTOR to enhance caspase-2 activation in ovarian cancer.

Caspase-2 protocols.

Caspase-2 has been shown to function in apoptosis and in some non-apoptotic pathways, including tumor suppression and aging. Caspase-2 has some unique features and is the only caspase that constitutively localizes to the nucleus, although its nuclear function remains unknown. During apoptosis signaling, caspase-2 rapidly homodimerizes, which leads to its activation and proteolytic processing. The activation of caspase-2 can be measured by assessing its dimerization and/or cleavage of the caspase-2 zymogen and its substrates. This chapter outlines commonly used methods to purify recombinant caspase-2 and assess its activity and function in vitro and in cultured cells or tissue extracts.

Analysis of the minimal specificity of caspase-2 and identification of Ac-VDTTD-AFC as a caspase-2-selective peptide substrate.

Caspase-2 is an evolutionarily conserved but enigmatic protease whose biological role remains poorly understood. To date, research into the functions of caspase-2 has been hampered by an absence of reagents that can distinguish its activity from that of the downstream apoptotic caspase, caspase-3. Identification of protein substrates of caspase-2 that are efficiently cleaved within cells may also provide clues to the role of this protease. We used a yeast-based transcriptional reporter system to define the minimal substrate specificity of caspase-2. The resulting profile enabled the identification of candidate novel caspase-2 substrates. Caspase-2 cleaved one of these proteins, the cancer-associated transcription factor Runx1, although with relatively low efficiency. A fluorogenic peptide was derived from the sequence most efficiently cleaved in the context of the transcriptional reporter. This peptide, Ac-VDTTD-AFC, was efficiently cleaved by purified caspase-2 and auto-activating caspase-2 in mammalian cells, and exhibited better selectivity for caspase-2 relative to caspase-3 than reagents that are currently available. We suggest that this reagent, used in parallel with the traditional caspase-3 substrate Ac-DEVD-AFC, will enable researchers to monitor caspase-2 activity in cell lysates and may assist in the determination of stimuli that activate caspase-2 in vivo.

[Caspase-2: what do we know today?].

Apoptosis (programmed cell death) is essential machinery for multicellular organisms. Apoptosis plays an important role in cell differentiation, damaged cell elimination and immune system homeostasis. This review is focused on various mechanisms of signal transduction through caspase-2 which believed to be one of the most enigmatical protease involved in apoptosis. Caspase-2 is activated upon stimulation by such agents as genotoxic stress, death receptors ligation, ER stress, metabolic changes, etc. In addition, caspase-2 may act as a tumor suppressor and has been implicated in cell response to oxidative stress and neurodegenerative progression during ischemic brain damage. Thus, variety of signal pathways triggered by caspase-2 place this protease apart from other members of the family and suggests a prominent role in apoptosis. Here, we analyse different functions of this unique caspase and discuss possible applications of accumulated knowledge in advanced oncology and medicine.

Determining the contributions of caspase-2, caspase-8 and effector caspases to intracellular VDVADase activities during apoptosis initiation and execution.

Apoptosis signaling crucially depends on caspase activities. Caspase-2 shares features of both initiator and effector caspases. Opinions are divided on whether caspase-2 activity is established during apoptosis initiation or execution in response to DNA damage, death receptor stimulation, or heat shock. So far, approaches towards measuring caspase-2 activity were restricted to analyses in cell homogenates and extracts, yielded inconsistent results, and were often limited in sensitivity, thereby contributing to controversies surrounding the role of caspase-2 during apoptosis. Furthermore, caspases overlap in substrate specificities, and caspase-8 as well as effector caspases may cleave the optimal VDVAD recognition motif as well. We therefore generated a highly sensitive Förster resonance energy transfer (FRET) substrate to determine the relative contribution of these caspases to VDVADase activity non-invasively inside living cells. We observed limited proteolysis of the substrate during apoptosis initiation in response to death receptor stimulation by FasL, TNFα and TRAIL. However, this activity was attributable to caspase-8 rather than caspase-2. Likewise, no caspase-2-specific activity was detected during apoptosis initiation in response to genotoxic stress (cisplatin, 5-FU), microtubule destabilization (vincristine), or heat shock. The contribution of caspase-2 to proteolytic activities during apoptosis execution was insignificant. Since even residual, ectopically introduced caspase-2 activity could readily be detected inside living cells in our measurements, we conclude, in contrast to several previous studies, that caspase-2 activity does not contribute to apoptosis in the scenarios investigated, and that instead caspase-8 and effector caspases are the most significant VDVADases during canonical apoptosis signaling.