Conformational similarity in the activation of caspase-3 and -7 revealed by the unliganded and inhibited structures of caspase-7

Caspase-mediated apoptosis has important roles in normal cell differentiation and aging and in many diseases including cancer, neuromuscular disorders and neurodegenerative diseases. Therefore, modulation of caspase activity and conformational states is of therapeutic importance. We report crystal structures of a new unliganded conformation of caspase-7 and the inhibited caspase-7 with the tetrapeptide Ac-YVAD-Cho. Different conformational states and mechanisms for substrate recognition have been proposed based on unliganded structures of the redundant apoptotic executioner caspase-3 and -7. The current study shows that the executioner caspase-3 and -7 have similar conformations for the unliganded active site as well as the inhibitor-bound active site. The new unliganded caspase-7 structure exhibits the tyrosine flipping mechanism in which the Tyr230 has rotated to block entry to the S2 binding site similar to the active site conformation of unliganded caspase-3. The inhibited structure of caspase-7/YVAD shows that the P4 Tyr binds the S4 region specific to polar residues at the expense of a main chain hydrogen bond between the P4 amide and carbonyl oxygen of caspase-7 Gln 276, which is similar to the caspase-3 complex. This new knowledge of the structures and conformational states of unliganded and inhibited caspases will be important for the design of drugs to modulate caspase activity and apoptosis.     

昆虫中分离与鉴定的caspase及其作用

天冬氨酸特异性的半胱氨酸蛋白酶(caspase)家族是执行细胞凋亡的主要酶类,对caspase结构及生物学功能的研究有助于更深入的研究细胞凋亡的分子机制。Caspase具有高度保守性,它们具有相似的氨基酸序列、结构和底物特异性。且具有QACRG的五肽活性位点,该活性位点是caspase家族的典型结构。昆虫caspase在caspase依赖型的细胞凋亡中起关键作用,文章介绍和评述昆虫中已经分离、鉴定的caspase及其功能。     

Plasticity of S2-S4 specificity pockets of executioner caspase-7 revealed by structural and kinetic analysis

Many protein substrates of caspases are cleaved at noncanonical sites in comparison to the recognition motifs reported for the three caspase subgroups. To provide insight into the specificity and aid in the design of drugs to control cell death, crystal structures of caspase-7 were determined in complexes with six peptide analogs (Ac-DMQD-Cho, Ac-DQMD-Cho, Ac-DNLD-Cho, Ac-IEPD-Cho, Ac-ESMD-Cho, Ac-WEHD-Cho) that span the major recognition motifs of the three subgroups. The crystal structures show that the S2 pocket of caspase-7 can accommodate diverse residues. Glu is not required at the P3 position because Ac-DMQD-Cho, Ac-DQMD-Cho and Ac-DNLD-Cho with varied P3 residues are almost as potent as the canonical Ac-DEVD-Cho. P4 Asp was present in the better inhibitors of caspase-7. However, the S4 pocket of executioner caspase-7 has alternate regions for binding of small branched aliphatic or polar residues similar to those of initiator caspase-8. The observed plasticity of the caspase subsites agrees very well with the reported cleavage of many proteins at noncanonical sites. The results imply that factors other than the P4-P1 sequence, such as exosites, contribute to the in vivo substrate specificity of caspases. The novel peptide binding site identified on the molecular surface of the current structures is suggested to be an exosite of caspase-7. These results should be considered in the design of selective small molecule inhibitors of this pharmacologically important protease.     

Endoplasmic reticulum stress-induced death of mouse embryonic fibroblasts requires the intrinsic pathway of apoptosis

Members of the caspase family are essential for many apoptotic programs. We studied mouse embryonic fibroblasts (MEFs) deficient in caspases 3 and 7 and in caspase 9 to determine the role of these proteases in endoplasmic reticulum (ER) stress-induced apoptosis. Both caspase 3(-/-)/caspase 7(-/-) and caspase 9(-/-) MEFs were resistant to cytotoxicity induced via ER stress and failed to exhibit apoptotic morphology. Specifically, apoptosis induced by increased intracellular calcium was shown to depend only on caspases 3 and 9, whereas apoptosis induced by disruption of ER function depended additionally on caspase 7. Caspase 3(-/-)/caspase 7(-/-) and caspase 9(-/-) MEFs also exhibited decreased loss of mitochondrial membrane potential, which correlated with altered caspase 9 processing, increased induction of procaspase 11, and decreased processing of caspase 12 in caspase 3(-/-)/caspase 7(-/-) cells. Furthermore, disruption of ER function was sufficient to induce accumulation of cleaved caspase 3 and 7 in a heavy membrane compartment, suggesting a potential mechanism for caspase 12 processing and its role as an amplifier in the death pathway. Caspase 8(-/-) MEFs were not resistant to ER stress-induced cytotoxicity, and processing of caspase 8 was not observed upon induction of ER stress. This study thus demonstrates a requirement for caspases 3 and 9 and a key role for the intrinsic pathway in ER stress-induced apoptosis.     

Caspase cleavage of vimentin disrupts intermediate filaments and promotes apoptosis.

Caspases are key mediators of apoptosis. Using a novel expression cloning strategy we recently developed to identify cDNAs encoding caspase substrates, we isolated the intermediate filament protein vimentin as a caspase substrate. Vimentin is preferentially cleaved by multiple caspases at distinct sites in vitro, including Asp85 by caspases-3 and -7 and Asp259 by caspase-6, to yield multiple proteolytic fragments. Vimentin is rapidly proteolyzed by multiple caspases into similar sized fragments during apoptosis induced by many stimuli. Caspase cleavage of vimentin disrupts its cytoplasmic network of intermediate filaments and coincides temporally with nuclear fragmentation. Moreover, caspase proteolysis of vimentin at Asp85 generates a pro-apoptotic amino-terminal fragment whose ability to induce apoptosis is dependent on caspases. Taken together, our findings suggest that caspase proteolysis of vimentin promotes apoptosis by dismantling intermediate filaments and by amplifying the cell death signal via a pro-apoptotic cleavage product.     

Proteases for cell suicide: functions and regulation of caspases.

Caspases are a large family of evolutionarily conserved proteases found from Caenorhabditis elegans to humans. Although the first caspase was identified as a processing enzyme for interleukin-1beta, genetic and biochemical data have converged to reveal that many caspases are key mediators of apoptosis, the intrinsic cell suicide program essential for development and tissue homeostasis. Each caspase is a cysteine aspartase; it employs a nucleophilic cysteine in its active site to cleave aspartic acid peptide bonds within proteins. Caspases are synthesized as inactive precursors termed procaspases; proteolytic processing of procaspase generates the tetrameric active caspase enzyme, composed of two repeating heterotypic subunits. Based on kinetic data, substrate specificity, and procaspase structure, caspases have been conceptually divided into initiators and effectors. Initiator caspases activate effector caspases in response to specific cell death signals, and effector caspases cleave various cellular proteins to trigger apoptosis. Adapter protein-mediated oligomerization of procaspases is now recognized as a universal mechanism of initiator caspase activation and underlies the control of both cell surface death receptor and mitochondrial cytochrome c-Apaf-1 apoptosis pathways. Caspase substrates have bene identified that induce each of the classic features of apoptosis, including membrane blebbing, cell body shrinkage, and DNA fragmentation. Mice deficient for caspase genes have highlighted tissue- and signal-specific pathways for apoptosis and demonstrated an independent function for caspase-1 and -11 in cytokine processing. Dysregulation of caspases features prominently in many human diseases, including cancer, autoimmunity, and neurodegenerative disorders, and increasing evidence shows that altering caspase activity can confer therapeutic benefits.     

Structural definition and substrate specificity of the S28 protease family: the crystal structure of human prolylcarboxypeptidase

Background

The unique S28 family of proteases is comprised of the carboxypeptidase PRCP and the aminopeptidase DPP7. The structural basis of the different substrate specificities of the two enzymes is not understood nor has the structure of the S28 fold been described.

Results

The experimentally phased 2.8 Å crystal structure is presented for human PRCP. PRCP contains an α/β hydrolase domain harboring the catalytic Asp-His-Ser triad and a novel helical structural domain that caps the active site. Structural comparisons with prolylendopeptidase and DPP4 identify the S1 proline binding site of PRCP. A structure-based alignment with the previously undescribed structure of DPP7 illuminates the mechanism of orthogonal substrate specificity of PRCP and DPP7. PRCP has an extended active-site cleft that can accommodate proline substrates with multiple N-terminal residues. In contrast, the substrate binding groove of DPP7 is occluded by a short amino-acid insertion unique to DPP7 that creates a truncated active site selective for dipeptidyl proteolysis of N-terminal substrates.

Conclusion

The results define the structure of the S28 family of proteases, provide the structural basis of PRCP and DPP7 substrate specificity and enable the rational design of selective PRCP modulators.     

Caspase multiplexing: simultaneous homogeneous time-resolved quenching assay (TruPoint) for caspases 1, 3, and 6

Caspases are a group of cysteine proteases involved in apoptosis and inflammation. A multiparametric homogeneous assay capable of measuring activity of three different caspases in a single well of a microtiter plate is described. Different fluorescent europium, samarium, terbium, and dysprosium chelates were coupled to a caspase substrate peptide, their luminescence properties, were analyzed, and their function in a time-resolved fluorescence quenching-based caspase 3 assay was studied. Substrates for caspases 1, 2, 3, 6, and 8 and granzyme B were also synthesized and their specificities for different caspases were determined. By selecting suitable lanthanide chelates and substrates we developed a multiparametric homogeneous time-resolved fluorescence quenching-based assay for caspases 1, 3, and 6. The assay was capable of measuring the activity of both single caspases and a mixture of three caspases mixed in the same well.     

Cleavage and Inactivation of ATM during Apoptosis

The activation of the cysteine proteases with aspartate specificity, termed caspases, is of fundamental importance for the execution of programmed cell death. These proteases are highly specific in their action and activate or inhibit a variety of key protein molecules in the cell. Here, we study the effect of apoptosis on the integrity of two proteins that have critical roles in DNA damage signalling, cell cycle checkpoint controls, and genome maintenance-the product of the gene defective in ataxia telangiectasia, ATM, and the related protein ATR. We find that ATM but not ATR is specifically cleaved in cells induced to undergo apoptosis by a variety of stimuli. We establish that ATM cleavage in vivo is dependent on caspases, reveal that ATM is an efficient substrate for caspase 3 but not caspase 6 in vitro, and show that the in vitro caspase 3 cleavage pattern mirrors that in cells undergoing apoptosis. Strikingly, apoptotic cleavage of ATM in vivo abrogates its protein kinase activity against p53 but has no apparent effect on the DNA binding properties of ATM. These data suggest that the cleavage of ATM during apoptosis generates a kinase-inactive protein that acts, through its DNA binding ability, in a trans-dominant-negative fashion to prevent DNA repair and DNA damage signalling.     

Multiparameter measurement of caspase 3 activation and apoptotic cell death in NT2 neuronal precursor cells using high-content analysis

Caspase activation is a component of a number of neurodegenerative disorders, including stroke. In this study, the authors describe a multiplexed assay for caspase 3 activation, nuclear condensation, and cell viability in a neuronal precursor cell line Ntera-2, injured with staurosporine and etoposide. Using a high-content screening approach, cells were identified by staining with the nuclear stain Hoechst 33342; cell viability was measured by staining cells with YoPro-1, which is taken up by damaged cells but excluded from healthy cells; and caspase 3/7 activation was detected using the cell-permeable probe PhiPhi-Lux, which becomes fluorescent when cleaved by active caspase 3 or 7. These 3 dyes were detected simultaneously using a 4-band pass filter set on a Cellomics Array scan. The authors used peptide-fmk inhibitors selective for a variety of caspases, demonstrating that the injury is mediated primarily through caspase 3 or 7, although other caspases or related proteases may play a minor role. The general caspase inhibitor zVAD-fmkwas able to block cell death and caspase activation with the highest potency. The caspase 3 selective inhibitor DEVD-fmkwas almost as potent as zVAD-fmk; other peptide caspase inhibitors displayed only modest inhibition of cell death. This assay was also used as a high-content screening tool for the evaluation of novel caspase 3 inhibitors for the potential treatment of degenerative disorders.     

Biochemical characterization of apoptotic cleavage of KH-type splicing regulatory protein (KSRP)/far upstream element-binding protein 2 (FBP2)

Caspases, Asp-specific cysteine protease, cleave proteins upon apoptosis. To identify and characterize new caspase substrate in the nucleus, the proteome of the rat liver extracts was analyzed after the treatment with caspases. One of the identified proteins was KSRP/FBP2 that is preferentially cleaved by caspase-3 and 7 at two sites after Asp102 and Asp183. The second site was cleaved only in the protein produced in cells, but not in in vitro translated protein. These results indicate that more than the primary sequence may be important for the recognition by caspases.     

The viral nucleocapsid protein of transmissible gastroenteritis coronavirus (TGEV) is cleaved by caspase-6 and -7 during TGEV-induced apoptosis

The transmissible gastroenteritis coronavirus (TGEV), like many other viruses, exerts much of its cytopathic effect through the induction of apoptosis of its host cell. Apoptosis is coordinated by a family of cysteine proteases, called caspases, that are activated during apoptosis and participate in dismantling the cell by cleaving key structural and regulatory proteins. We have explored the caspase activation events that are initiated upon infection of the human rectal tumor cell line HRT18 with TGEV. We show that TGEV infection results in the activation of caspase-3, -6, -7, -8, and -9 and cleavage of the caspase substrates eIF4GI, gelsolin, and alpha-fodrin. Surprisingly, the TGEV nucleoprotein (N) underwent proteolysis in parallel with the activation of caspases within the host cell. Cleavage of the N protein was inhibited by cell-permeative caspase inhibitors, suggesting that this viral structural protein is a target for host cell caspases. We show that the TGEV nucleoprotein is a substrate for both caspase-6 and -7, and using site-directed mutagenesis, we have mapped the cleavage site to VVPD(359) downward arrow. These data demonstrate that viral proteins can be targeted for destruction by the host cell death machinery.     

Conformational restrictions in the active site of unliganded human caspase-3

Caspases are cysteine proteases that play a critical role in the initiation and regulation of apoptosis. These enzymes act in a cascade to promote cell death through proteolytic cleavage of intracellular proteins. Since activation of apoptosis is implicated in human diseases such as cancer and neurodegenerative disorders, caspases are targets for drugs designed to modulate their action. Active caspases are heterodimeric enzymes with two symmetrically arranged active sites at opposite ends of the molecule. A number of crystal structures of caspases with peptides or proteins bound at the active sites have defined the mechanism of action of these enzymes, but molecular information about the active sites before substrate engagement has been lacking. As part of a study of peptidyl inhibitors of caspase-3, we crystallized a complex where the inhibitor did not bind in the active site. Here we present the crystal structure of the unoccupied substrate-binding site of caspase-3. No large conformational differences were apparent when this site was compared with that in enzyme-inhibitor complexes. Instead, the 1.9 A structure reveals critical side chain movements in a hydrophobic pocket in the active site. Notably, the side chain of tyrosine204 is rotated by approximately 90 degrees so that the phenol group occupies the S2 subsite in the active site. Thus, binding of substrate or inhibitors is impeded unless rotation of this side chain opens the area. The positions of these side chains may have important implications for the directed design of inhibitors of caspase-3 or caspase-7.     

Potent and selective nonpeptide inhibitors of caspases 3 and 7 inhibit apoptosis and maintain cell functionality

Caspases have been strongly implicated to play an essential role in apoptosis. A critical question regarding the role(s) of these proteases is whether selective inhibition of an effector caspase(s) will prevent cell death. We have identified potent and selective non-peptide inhibitors of the effector caspases 3 and 7. The inhibition of apoptosis and maintenance of cell functionality with a caspase 3/7-selective inhibitor is demonstrated for the first time, and suggests that targeting these two caspases alone is sufficient for blocking apoptosis. Furthermore, an x-ray co-crystal structure of the complex between recombinant human caspase 3 and an isatin sulfonamide inhibitor has been solved to 2.8-A resolution. In contrast to previously reported peptide-based caspase inhibitors, the isatin sulfonamides derive their selectivity for caspases 3 and 7 by interacting primarily with the S(2) subsite, and do not bind in the caspase primary aspartic acid binding pocket (S(1)). These inhibitors blocked apoptosis in murine bone marrow neutrophils and human chondrocytes. Furthermore, in camptothecin-induced chondrocyte apoptosis, cell functionality as measured by type II collagen promoter activity is maintained, an activity considered essential for cartilage homeostasis. These data suggest that inhibiting chondrocyte cell death with a caspase 3/7-selective inhibitor may provide a novel therapeutic approach for the prevention and treatment of osteoarthritis, or other disease states characterized by excessive apoptosis.     

Inflammasome-activated caspase 7 cleaves PARP1 to enhance the expression of a subset of NF-κB target genes

Caspase 1 is part of the inflammasome, which is assembled upon pathogen recognition, while caspases 3 and/or 7 are mediators of apoptotic and nonapoptotic functions. PARP1 cleavage is a hallmark of apoptosis yet not essential, suggesting it has another physiological role. Here we show that after LPS stimulation, caspase 7 is activated by caspase 1, translocates to the nucleus, and cleaves PARP1 at the promoters of a subset of NF-κB target genes negatively regulated by PARP1. Mutating the PARP1 cleavage site D214 renders PARP1 uncleavable and inhibits PARP1 release from chromatin and chromatin decondensation, thereby restraining the expression of cleavage-dependent NF-κB target genes. These findings propose an apoptosis-independent regulatory role for caspase 7-mediated PARP1 cleavage in proinflammatory gene expression and provide insight into inflammasome signaling.     

Homology modeling of nematode Caenorhabditis elegans CED3 protein-inhibitor complex

CED3 protein, the product of a gene necessary for programmed cell death in the nematode Caenorhabditis elegans, is related to a highly specific cysteine protease family i.e., caspases. A tertiary-structural model has been constructed of a complex of the CED3 protein with tetrapeptide-aldehyde inhibitor, Ac-DEVD-CHO. The conformation of CED3 protein active site and the general binding features of inhibitor residues are similar to those observed in other caspases. The loop segment (Phe380-Pro387) binds with the P4 Asp in a different fashion compared to caspase-3. The comparative modeling of active sites from caspase-3 and CED3 protein indicated that although these enzymes require Asp at the position P4, variation could occur in the binding of this residue at the S4 subsite. This model allowed the definition of substrate specificity of CED3 protein from the structural standpoint and provided insight in designing of mutants for structure-function studies of this classical caspase homologue.     

Mutational analyses of the p35-caspase interaction. A bowstring kinetic model of caspase inhibition by p35

Apoptosis is a highly regulated multistep process for programmed cellular destruction. It is centered on the activation of a group of intracellular cysteine proteases known as caspases. The baculoviral p35 protein effectively blocks apoptosis through its broad spectrum caspase inhibition. It harbors a caspase recognition sequence within a highly protruding reactive site loop (RSL), which gets cleaved by a target caspase before the formation of a tight complex. The crystal structure of the post-cleavage complex between p35 and caspase-8 shows that p35 forms a thioester bond with the active site cysteine of the caspase. The covalent bond is prevented from hydrolysis by the N terminus of p35, which repositions into the active site of the caspase to eliminate solvent accessibility of the catalytic residues. Here, we report mutational analyses of the pre-cleavage and post-cleavage p35/caspase interactions using surface plasmon resonance biosensor measurements, pull-down assays and kinetic inhibition experiments. The experiments identify important structural elements for caspase inhibition by p35, including the strict requirement for a Cys at the N terminus of p35 and the rigidity of the RSL. A bowstring kinetic model for p35 function is derived in which the tension generated in the bowstring system during the pre-cleavage interaction is crucial for the fast post-cleavage conformational changes required for inhibition.     

Inhibitor of apoptosis proteins in Drosophila: gatekeepers of death

Regulation of apoptosis is crucial to ensure cellular viability, and failure to do so is linked to several human pathologies. The apoptotic cell death programme culminates in the activation of caspases, a family of highly specific cysteine proteases essential for the destruction of the cell. Although best known for their role in executing apoptosis, caspases also play important signalling roles in non-apoptotic processes, such as regulation of actin dynamics, innate immunity, cell proliferation, differentiation and survival. Under such conditions, caspases are activated without killing the cell. Caspase activation and activity is subject to complex regulation, and various cellular and viral inhibitors have been identified that control the activity of caspases in their apoptotic and non-apoptotic roles. Members of the Inhibitor of APoptosis (IAP) protein family ensure cell viability in Drosophila by directly binding to caspases and regulating their activities in a ubiquitin-dependent manner. The observation that IAPs are essential for cell survival in Drosophila, and are frequently deregulated in human cancer, contributing to tumourigenesis, chemoresistance, disease progression and poor patient survival, highlights the importance of this family of caspase regulators in health and disease. Here we summarise recent advances from Drosophila that start to elucidate how the cellular response to caspase activation is modulated by IAPs and their regulators.     

Possible involvement of caspase-like family in maintenance of cytoskeleton integrity

Caspases, a family of cysteine proteases, are the key effector proteins of apoptosis. These proteases cleave cellular proteins and are responsible for the destruction of the cell body during apoptosis. They are also involved in the activation of other proteins, such as cytokines. In this study, we demonstrate a novel function for these proteases. Z-Asp-CH2-DCB (Z-Asp), a general caspase inhibitor, blocked cell spreading on collagen-coated plates in a dose-dependent manner but did not affect cell viability. Caspase 3-like activity but not caspase 1-like activity was detected in adherent cells on both collagen-coated and poly-L-lysine-coated plates but not in suspended cells. The caspase 3-like activity was significantly inhibited by Z-Asp. However, only Z-Asp, not specific caspase inhibitors (Z-DEVD for caspase 3, Z-YVAD for caspase 1), was effective in the suppression of cell spreading. The inhibitory effect of Z-Asp was blocked by a phosphokinase C activator, PMA, and a Rho activator, lysophosphatidic acid (LPA), while neither a Rac activator, bradykinin, nor a Cdc42 activator, sphingosine-1 -phosphate, was effective. Immunoprecipitation demonstrated that Z-Asp downregulated the expression of focal adhesion kinase (FAK) protein, downstream of Rho signaling, in adherent cells. Our results suggest that not caspase 1 or 3 but another yet unknown caspase(s) plays an important role in the maintenance of cytoskeleton integrity via FAK protein expression, implying a new function for caspases.     

Phage display and structural studies reveal plasticity in substrate specificity of caspase‐3a from zebrafish

The regulation of caspase‐3 enzyme activity is a vital process in cell fate decisions leading to cell differentiation and tissue development or to apoptosis. The zebrafish, Danio rerio, has become an increasingly popular animal model to study several human diseases because of their transparent embryos, short reproductive cycles, and ease of drug administration. While apoptosis is an evolutionarily conserved process in metazoans, little is known about caspases from zebrafish, particularly regarding substrate specificity and allosteric regulation compared to the human caspases. We cloned zebrafish caspase‐3a (casp3a) and examined substrate specificity of the recombinant protein, Casp3a, compared to human caspase‐3 (CASP3) by utilizing M13 bacteriophage substrate libraries that incorporated either random amino acids at P5‐P1′ or aspartate fixed at P1. The results show a preference for the tetrapeptide sequence DNLD for both enzymes, but the P4 position of zebrafish Casp3a also accommodates valine equally well. We determined the structure of zebrafish Casp3a to 2.28Å resolution by X‐ray crystallography, and when combined with molecular dynamics simulations, the results suggest that a limited number of amino acid substitutions near the active site result in plasticity of the S4 sub‐site by increasing flexibility of one active site loop and by affecting hydrogen‐bonding with substrate. The data show that zebrafish Casp3a exhibits a broader substrate portfolio, suggesting overlap with the functions of caspase‐6 in zebrafish development.