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1.
J Bioinform Comput Biol ; 17(2): 1950006, 2019 04.
Article in English | MEDLINE | ID: mdl-31057073

ABSTRACT

In contrast to ab-initio protein modeling methodologies, comparative modeling is considered as the most popular and reliable algorithm to model protein structure. However, the selection of the best set of templates is still a major challenge. An effective template-ranking algorithm is developed to efficiently select only the reliable hits for predicting the protein structures. The algorithm employs the pairwise as well as multiple sequence alignments of template hits to rank and select the best possible set of templates. It captures several key sequences and structural information of template hits and converts into scores to effectively rank them. This selected set of templates is used to model a target. Modeling accuracy of the algorithm is tested and evaluated on TBM-HA domain containing CASP8, CASP9 and CASP10 targets. On an average, this template ranking and selection algorithm improves GDT-TS, GDT-HA and TM_Score by 3.531, 4.814 and 0.022, respectively. Further, it has been shown that the inclusion of structurally similar templates with ample conformational diversity is crucial for the modeling algorithm to maximally as well as reliably span the target sequence and construct its near-native model. The optimal model sampling also holds the key to predict the best possible target structure.


Subject(s)
Algorithms , Models, Molecular , Protein Domains , Caspase 10/chemistry , Caspase 8/chemistry , Caspase 9/chemistry , Computational Biology/methods , Protein Conformation
2.
Cell Death Differ ; 26(2): 229-244, 2019 02.
Article in English | MEDLINE | ID: mdl-29748600

ABSTRACT

Apical caspases initiate and effector caspases execute apoptosis. Reagents that can distinguish between caspases, particularly apical caspases-8, 9, and 10 are scarce and generally nonspecific. Based upon a previously described large-scale screen of peptide-based caspase substrates termed HyCoSuL, we sought to develop reagents to distinguish between apical caspases in order to reveal their function in apoptotic cell death paradigms. To this end, we selected tetrapeptide-based sequences that deliver optimal substrate selectivity and converted them to inhibitors equipped with a detectable tag (activity-based probes-ABPs). We demonstrate a strong relationship between substrate kinetics and ABP kinetics. To evaluate the utility of selective substrates and ABPs, we examined distinct apoptosis pathways in Jurkat T lymphocyte and MDA-MB-231 breast cancer lines triggered to undergo cell death via extrinsic or intrinsic apoptosis. We report the first highly selective substrate appropriate for quantitation of caspase-8 activity during apoptosis. Converting substrates to ABPs promoted loss-of-activity and selectivity, thus we could not define a single ABP capable of detecting individual apical caspases in complex mixtures. To overcome this, we developed a panel strategy utilizing several caspase-selective ABPs to interrogate apoptosis, revealing the first chemistry-based approach to uncover the participation of caspase-8, but not caspase-9 or -10 in TRAIL-induced extrinsic apoptosis. We propose that using select panels of ABPs can provide information regarding caspase-8 apoptotic signaling more faithfully than can single, generally nonspecific reagents.


Subject(s)
Caspase 10/isolation & purification , Caspase 8/isolation & purification , Caspase 9/isolation & purification , Peptides/chemistry , Apoptosis/genetics , Caspase 10/chemistry , Caspase 10/genetics , Caspase 3/chemistry , Caspase 3/genetics , Caspase 3/isolation & purification , Caspase 8/chemistry , Caspase 8/genetics , Caspase 9/chemistry , Caspase 9/genetics , Caspase Inhibitors/chemistry , Caspase Inhibitors/pharmacology , Humans , Jurkat Cells , Kinetics , Signal Transduction , Substrate Specificity
3.
J Chem Phys ; 149(7): 072319, 2018 Aug 21.
Article in English | MEDLINE | ID: mdl-30134714

ABSTRACT

In this paper, we report results of using enhanced sampling and blind selection techniques for high-accuracy protein structural refinement. By combining a parallel continuous simulated tempering (PCST) method, previously developed by Zang et al. [J. Chem. Phys. 141, 044113 (2014)], and the structure based model (SBM) as restraints, we refined 23 targets (18 from the refinement category of the CASP10 and 5 from that of CASP12). We also designed a novel model selection method to blindly select high-quality models from very long simulation trajectories. The combined use of PCST-SBM with the blind selection method yielded final models that are better than initial models. For Top-1 group, 7 out of 23 targets had better models (greater global distance test total scores) than the critical assessment of structure prediction participants. For Top-5 group, 10 out of 23 were better. Our results justify the crucial position of enhanced sampling in protein structure prediction and refinement and demonstrate that a considerable improvement of low-accuracy structures is achievable with current force fields.


Subject(s)
Caspase 10/chemistry , Caspase 12/chemistry , Molecular Dynamics Simulation , Protein Conformation , Temperature
4.
Cell Rep ; 19(4): 785-797, 2017 04 25.
Article in English | MEDLINE | ID: mdl-28445729

ABSTRACT

Formation of the death-inducing signaling complex (DISC) initiates extrinsic apoptosis. Caspase-8 and its regulator cFLIP control death signaling by binding to death-receptor-bound FADD. By elucidating the function of the caspase-8 homolog, caspase-10, we discover that caspase-10 negatively regulates caspase-8-mediated cell death. Significantly, we reveal that caspase-10 reduces DISC association and activation of caspase-8. Furthermore, we extend our co-operative/hierarchical binding model of caspase-8/cFLIP and show that caspase-10 does not compete with caspase-8 for binding to FADD. Utilizing caspase-8-knockout cells, we demonstrate that caspase-8 is required upstream of both cFLIP and caspase-10 and that DISC formation critically depends on the scaffold function of caspase-8. We establish that caspase-10 rewires DISC signaling to NF-κB activation/cell survival and demonstrate that the catalytic activity of caspase-10, and caspase-8, is redundant in gene induction. Thus, our data are consistent with a model in which both caspase-10 and cFLIP coordinately regulate CD95L-mediated signaling for death or survival.


Subject(s)
Apoptosis/drug effects , Caspase 10/metabolism , Caspase 8/metabolism , Fas Ligand Protein/pharmacology , NF-kappa B/metabolism , CASP8 and FADD-Like Apoptosis Regulating Protein/antagonists & inhibitors , CASP8 and FADD-Like Apoptosis Regulating Protein/genetics , CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , Caspase 10/chemistry , Caspase 10/genetics , Caspase 8/chemistry , Caspase 8/genetics , Cell Line , Cell Survival/drug effects , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Fas-Associated Death Domain Protein/metabolism , HeLa Cells , Humans , Imidazoles/pharmacology , Indoles/pharmacology , Interleukin-8/genetics , Interleukin-8/metabolism , NF-KappaB Inhibitor alpha/metabolism , Oligopeptides/pharmacology , RNA Interference , RNA, Messenger , RNA, Small Interfering/metabolism , Signal Transduction , fas Receptor/metabolism
5.
Bioinformatics ; 33(16): 2496-2503, 2017 Aug 15.
Article in English | MEDLINE | ID: mdl-28419290

ABSTRACT

MOTIVATION: The accurate ranking of predicted structural models and selecting the best model from a given candidate pool remain as open problems in the field of structural bioinformatics. The quality assessment (QA) methods used to address these problems can be grouped into two categories: consensus methods and single-model methods. Consensus methods in general perform better and attain higher correlation between predicted and true quality measures. However, these methods frequently fail to generate proper quality scores for native-like structures which are distinct from the rest of the pool. Conversely, single-model methods do not suffer from this drawback and are better suited for real-life applications where many models from various sources may not be readily available. RESULTS: In this study, we developed a support-vector-machine-based single-model global quality assessment (SVMQA) method. For a given protein model, the SVMQA method predicts TM-score and GDT_TS score based on a feature vector containing statistical potential energy terms and consistency-based terms between the actual structural features (extracted from the three-dimensional coordinates) and predicted values (from primary sequence). We trained SVMQA using CASP8, CASP9 and CASP10 targets and determined the machine parameters by 10-fold cross-validation. We evaluated the performance of our SVMQA method on various benchmarking datasets. Results show that SVMQA outperformed the existing best single-model QA methods both in ranking provided protein models and in selecting the best model from the pool. According to the CASP12 assessment, SVMQA was the best method in selecting good-quality models from decoys in terms of GDTloss. AVAILABILITY AND IMPLEMENTATION: SVMQA method can be freely downloaded from http://lee.kias.re.kr/SVMQA/SVMQA_eval.tar.gz. CONTACT: jlee@kias.re.kr. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Subject(s)
Computational Biology/methods , Models, Molecular , Quality Control , Support Vector Machine , Caspase 10/chemistry , Caspase 10/metabolism , Caspase 8/chemistry , Caspase 8/metabolism , Caspase 9/chemistry , Caspase 9/metabolism , Computational Biology/standards , Protein Conformation
6.
Cell Death Differ ; 24(3): 481-491, 2017 03.
Article in English | MEDLINE | ID: mdl-28106882

ABSTRACT

Peptido-mimetic inhibitor of apoptosis protein (IAP) antagonists (Smac mimetics (SMs)) can kill tumour cells by depleting endogenous IAPs and thereby inducing tumour necrosis factor (TNF) production. We found that interferon-γ (IFNγ) synergises with SMs to kill cancer cells independently of TNF- and other cell death receptor signalling pathways. Surprisingly, CRISPR/Cas9 HT29 cells doubly deficient for caspase-8 and the necroptotic pathway mediators RIPK3 or MLKL were still sensitive to IFNγ/SM-induced killing. Triple CRISPR/Cas9-knockout HT29 cells lacking caspase-10 in addition to caspase-8 and RIPK3 or MLKL were resistant to IFNγ/SM killing. Caspase-8 and RIPK1 deficiency was, however, sufficient to protect cells from IFNγ/SM-induced cell death, implying a role for RIPK1 in the activation of caspase-10. These data show that RIPK1 and caspase-10 mediate cell death in HT29 cells when caspase-8-mediated apoptosis and necroptosis are blocked and help to clarify how SMs operate as chemotherapeutic agents.


Subject(s)
Apoptosis/drug effects , Caspase 10/metabolism , Inhibitor of Apoptosis Proteins/metabolism , Interferon-gamma/pharmacology , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Animals , CRISPR-Cas Systems/genetics , Caspase 10/chemistry , Caspase 10/genetics , Caspase 8/chemistry , Caspase 8/genetics , Caspase 8/metabolism , Caspase Inhibitors/pharmacology , Cell Line , Cytokine TWEAK/pharmacology , Drug Synergism , HT29 Cells , Humans , Inhibitor of Apoptosis Proteins/antagonists & inhibitors , Interferon-gamma/genetics , Interferon-gamma/metabolism , Mice , Mice, Knockout , Pentanoic Acids/pharmacology , Protein Kinases/deficiency , Protein Kinases/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/deficiency , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Receptors, Tumor Necrosis Factor, Type I/deficiency , Receptors, Tumor Necrosis Factor, Type I/genetics , Receptors, Tumor Necrosis Factor, Type I/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology
7.
Nature ; 534(7608): 570-4, 2016 06 23.
Article in English | MEDLINE | ID: mdl-27309814

ABSTRACT

Small molecules are powerful tools for investigating protein function and can serve as leads for new therapeutics. Most human proteins, however, lack small-molecule ligands, and entire protein classes are considered 'undruggable'. Fragment-based ligand discovery can identify small-molecule probes for proteins that have proven difficult to target using high-throughput screening of complex compound libraries. Although reversibly binding ligands are commonly pursued, covalent fragments provide an alternative route to small-molecule probes, including those that can access regions of proteins that are difficult to target through binding affinity alone. Here we report a quantitative analysis of cysteine-reactive small-molecule fragments screened against thousands of proteins in human proteomes and cells. Covalent ligands were identified for >700 cysteines found in both druggable proteins and proteins deficient in chemical probes, including transcription factors, adaptor/scaffolding proteins, and uncharacterized proteins. Among the atypical ligand-protein interactions discovered were compounds that react preferentially with pro- (inactive) caspases. We used these ligands to distinguish extrinsic apoptosis pathways in human cell lines versus primary human T cells, showing that the former is largely mediated by caspase-8 while the latter depends on both caspase-8 and -10. Fragment-based covalent ligand discovery provides a greatly expanded portrait of the ligandable proteome and furnishes compounds that can illuminate protein functions in native biological systems.


Subject(s)
Cysteine/metabolism , Drug Evaluation, Preclinical/methods , Proteome/chemistry , Proteome/metabolism , Small Molecule Libraries/metabolism , Small Molecule Libraries/pharmacology , T-Lymphocytes/metabolism , Apoptosis , Caspase 10/chemistry , Caspase 10/metabolism , Caspase 8/chemistry , Caspase 8/metabolism , Cells, Cultured , Enzyme Precursors/chemistry , Enzyme Precursors/metabolism , Humans , Ligands , Peptide Fragments/chemistry , Peptide Fragments/metabolism , T-Lymphocytes/chemistry , Transcription Factors/chemistry , Transcription Factors/metabolism
8.
J Mol Model ; 22(4): 72, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26969678

ABSTRACT

The prediction of domain/linker residues in protein sequences is a crucial task in the functional classification of proteins, homology-based protein structure prediction, and high-throughput structural genomics. In this work, a novel consensus-based machine-learning technique was applied for residue-level prediction of the domain/linker annotations in protein sequences using ordered/disordered regions along protein chains and a set of physicochemical properties. Six different classifiers-decision tree, Gaussian naïve Bayes, linear discriminant analysis, support vector machine, random forest, and multilayer perceptron-were exhaustively explored for the residue-level prediction of domain/linker regions. The protein sequences from the curated CATH database were used for training and cross-validation experiments. Test results obtained by applying the developed PDP-CON tool to the mutually exclusive, independent proteins of the CASP-8, CASP-9, and CASP-10 databases are reported. An n-star quality consensus approach was used to combine the results yielded by different classifiers. The average PDP-CON accuracy and F-measure values for the CASP targets were found to be 0.86 and 0.91, respectively. The dataset, source code, and all supplementary materials for this work are available at https://cmaterju.org/cmaterbioinfo/ for noncommercial use.


Subject(s)
Caspase 10/chemistry , Caspase 8/chemistry , Caspase 9/chemistry , Computational Biology/methods , Support Vector Machine , Bayes Theorem , Databases, Protein , Decision Trees , Discriminant Analysis , Humans , Neural Networks, Computer , Protein Domains , Sequence Analysis, Protein , Structural Homology, Protein
9.
Proteins ; 84(8): 1021-8, 2016 Aug.
Article in English | MEDLINE | ID: mdl-25737479

ABSTRACT

We present a Model Quality Assessment Program (MQAP), called MQAPsingle, for ranking and assessing the absolute global quality of single protein models. MQAPsingle is quasi single-model MQAP, a method that combines advantages of both "pure" single-model MQAPs and clustering MQAPs. This approach results in higher accuracy compared to the state-of-the-art single-model MQAPs. Notably, the prediction for a given model is the same regardless if this model is submitted to our server alone or together with other models. Proteins 2016; 84:1021-1028. © 2015 Wiley Periodicals, Inc.


Subject(s)
Caspase 10/chemistry , Computational Biology/methods , Models, Molecular , Software , Benchmarking , Humans , Internet , Protein Conformation
10.
Gene ; 570(2): 288-94, 2015 Oct 10.
Article in English | MEDLINE | ID: mdl-26048002

ABSTRACT

Caspase 10 is an initiator caspase in death cascades of death receptor mediated apoptotic signaling. We identified and molecularly characterized a novel homolog of caspase 10 from black rockfish (Sebastes schlegelii) and designated as RfCasp10. The complete coding region of RfCasp10 was found to consist of 1659 bps, encoding a 553 amino acid protein with a predicted molecular mass of 61.7 kDa. The characteristic caspase family domain architecture, including death effecter domains (DEDs), was clearly identified in RfCasp10. Moreover, the RfCasp10 gene was found to contain 13 exons. Our pairwise sequence alignment confirmed the prominent sequence similarity of RfCasp10 with its fish homologs, and phylogenetic reconstruction affirmed its homology and substantial evolutionary relationship with known caspases 10 similitudes, in particular with those of teleosts. As detected by qPCR, RfCasp10 was markedly expressed in blood tissues under physiological conditions, whereas its expression was found to be upregulated under pathogenic stress, elicited by Streptococcus iniae and polyinosinic:polycytidylic acid in blood, liver, and spleen tissues. Collectively, our study suggests the plausible elicitation of RfCasp10 mediated apoptosis in immune relevant tissues of black rockfish as a host immune response to a bacterial or viral infection.


Subject(s)
Caspase 10/genetics , Fishes/genetics , Gene Expression Regulation, Enzymologic , Stress, Physiological/genetics , Transcription, Genetic , Amino Acid Sequence , Animals , Caspase 10/chemistry , Molecular Sequence Data , Sequence Homology, Amino Acid
11.
J Mol Biol ; 427(15): 2562-2576, 2015 Jul 31.
Article in English | MEDLINE | ID: mdl-26055539

ABSTRACT

The relative distance and orientation in contacting residue pairs plays a significant role in protein folding and stabilization. We hereby devise a new knowledge-based, coarse-grained contact potential, so-called ICOSA, by correlating inter-residue contact distance and orientation in evaluating pair-wise inter-residue interactions. The rationale of our approach is to establish icosahedral local coordinates to estimate the statistical residue contact distributions in all spherical triangular shells within a sphere. We extend the theory of finite ideal gas reference state to icosahedral local coordinates. ICOSA incorporates long-range contact interactions, which is critical to ICOSA sensitivity and is justified in statistical rigor. With only backbone atoms information, ICOSA is at least comparable to all-atom, fine-grained potentials such as Rosetta, DFIRE, I-TASSER, and OPUS in discriminating near-natives from misfold protein conformations in the Rosetta and I-TASSER protein decoy sets. ICOSA also outperforms a set of widely used coarse-grained potentials and is comparable to all-atom, fine-grained potentials in identifying CASP10 models.


Subject(s)
Algorithms , Computational Biology/methods , Models, Molecular , Protein Conformation , Protein Interaction Mapping/methods , Caspase 10/chemistry , Knowledge Bases , Protein Interaction Maps , Sequence Analysis, Protein/methods , Software
12.
PLoS One ; 8(7): e69648, 2013.
Article in English | MEDLINE | ID: mdl-23894517

ABSTRACT

Protein structure refinement refers to the process of improving the qualities of protein structures during structure modeling processes to bring them closer to their native states. Structure refinement has been drawing increasing attention in the community-wide Critical Assessment of techniques for Protein Structure prediction (CASP) experiments since its addition in 8(th) CASP experiment. During the 9(th) and recently concluded 10(th) CASP experiments, a consistent growth in number of refinement targets and participating groups has been witnessed. Yet, protein structure refinement still remains a largely unsolved problem with majority of participating groups in CASP refinement category failed to consistently improve the quality of structures issued for refinement. In order to alleviate this need, we developed a completely automated and computationally efficient protein 3D structure refinement method, i3Drefine, based on an iterative and highly convergent energy minimization algorithm with a powerful all-atom composite physics and knowledge-based force fields and hydrogen bonding (HB) network optimization technique. In the recent community-wide blind experiment, CASP10, i3Drefine (as 'MULTICOM-CONSTRUCT') was ranked as the best method in the server section as per the official assessment of CASP10 experiment. Here we provide the community with free access to i3Drefine software and systematically analyse the performance of i3Drefine in strict blind mode on the refinement targets issued in CASP10 refinement category and compare with other state-of-the-art refinement methods participating in CASP10. Our analysis demonstrates that i3Drefine is only fully-automated server participating in CASP10 exhibiting consistent improvement over the initial structures in both global and local structural quality metrics. Executable version of i3Drefine is freely available at http://protein.rnet.missouri.edu/i3drefine/.


Subject(s)
Caspase 10/chemistry , Models, Molecular , Protein Conformation , Proteins/chemistry , Software , Algorithms , Humans , Protein Binding , Structure-Activity Relationship
13.
Mol Biosyst ; 9(7): 1774-88, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23694935

ABSTRACT

Protein structure space is believed to consist of a finite set of discrete folds, unlike the protein sequence space which is astronomically large, indicating that proteins from the available sequence space are likely to adopt one of the many folds already observed. In spite of extensive sequence-structure correlation data, protein structure prediction still remains an open question with researchers having tried different approaches (experimental as well as computational). One of the challenges of protein structure prediction is to identify the native protein structures from a milieu of decoys/models. In this work, a rigorous investigation of Protein Structure Networks (PSNs) has been performed to detect native structures from decoys/models. Ninety four parameters obtained from network studies have been optimally combined with Support Vector Machines (SVM) to derive a general metric to distinguish decoys/models from the native protein structures with an accuracy of 94.11%. Recently, for the first time in the literature we had shown that PSN has the capability to distinguish native proteins from decoys. A major difference between the present work and the previous study is to explore the transition profiles at different strengths of non-covalent interactions and SVM has indeed identified this as an important parameter. Additionally, the SVM trained algorithm is also applied to the recent CASP10 predicted models. The novelty of the network approach is that it is based on general network properties of native protein structures and that a given model can be assessed independent of any reference structure. Thus, the approach presented in this paper can be valuable in validating the predicted structures. A web-server has been developed for this purpose and is freely available at .


Subject(s)
Caspases/chemistry , Models, Biological , Protein Interaction Maps , Proteins/chemistry , Area Under Curve , Caspase 10/chemistry , Caspase 10/metabolism , Caspases/metabolism , Databases, Protein , Models, Molecular , Protein Conformation , Proteins/metabolism , Reproducibility of Results , Support Vector Machine
14.
J Biol Chem ; 287(25): 21142-51, 2012 Jun 15.
Article in English | MEDLINE | ID: mdl-22528489

ABSTRACT

During the resolution of inflammatory responses, neutrophils rapidly undergo apoptosis. A direct and fast activation of caspase-8 by cathepsin D was shown to be crucial in the initial steps of neutrophil apoptosis. Nevertheless, the activation mechanism of caspase-8 remains unclear. Here, by using site-specific mutants of caspase-8, we show that both cathepsin D-mediated proteolysis and homodimerization of caspase-8 are necessary to generate an active caspase-8. At acidic pH, cathepsin D specifically cleaved caspase-8 but not the initiator caspase-9 or -10 and significantly increased caspase-8 activity in dimerizing conditions. These events were completely abolished by pepstatin A, a pharmacological inhibitor of cathepsin D. The cathepsin D intra-chain proteolysis greatly stabilized the active site of caspase-8. Moreover, the main caspase-8 fragment generated by cathepsin D cleavage could be affinity-labeled with the active site probe biotin-VAD-fluoromethyl ketone, suggesting that this fragment is enzymatically active. Importantly, in an in vitro cell-free assay, the addition of recombinant human caspase-8 protein, pre-cleaved by cathepsin D, was followed by caspase-3 activation. Our data therefore indicate that cathepsin D is able to initiate the caspase cascade by direct activation of caspase-8. As cathepsin D is ubiquitously expressed, this may represent a general mechanism to induce apoptosis in a variety of immune and nonimmune cells.


Subject(s)
Apoptosis/physiology , Caspase 8/metabolism , Cathepsin D/metabolism , Neutrophils/enzymology , Protein Multimerization/physiology , Proteolysis , Caspase 10/chemistry , Caspase 10/genetics , Caspase 10/metabolism , Caspase 8/chemistry , Caspase 8/genetics , Caspase 9/chemistry , Caspase 9/genetics , Caspase 9/metabolism , Cathepsin D/chemistry , Cathepsin D/genetics , Enzyme Activation/physiology , Female , Humans , Male , Neutrophils/cytology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
15.
J Biol Chem ; 286(19): 16910-3, 2011 May 13.
Article in English | MEDLINE | ID: mdl-21454616

ABSTRACT

The generation of peptides presented by MHC class I molecules requires the proteolytic activity of the proteasome and/or other peptidases. The processing of a short vaccinia virus-encoded antigen can take place by a proteasome-independent pathway involving initiator caspase-5 and -10, which generate antigenic peptides recognized by CD8(+) T lymphocytes. In the present study, comparing single versus double enzyme digestions by mass spectrometry analysis, both qualitative and quantitative differences in the products obtained were identified. These in vitro data suggest that each enzyme can use the degradation products of the other as substrate for new cleavages, indicating concerted endoproteolytic activity of caspase-5 and -10.


Subject(s)
Antigens, Viral/chemistry , Antigens/metabolism , Caspase 10/chemistry , Caspases/chemistry , Amino Acid Sequence , CD8-Positive T-Lymphocytes/metabolism , Enzyme Inhibitors/pharmacology , Epitopes/chemistry , Humans , Mass Spectrometry/methods , Molecular Sequence Data , Peptides/chemistry
16.
Cell Death Dis ; 2: e125, 2011 Mar 03.
Article in English | MEDLINE | ID: mdl-21368896

ABSTRACT

The cysteine protease caspase-8 is an essential executioner of the death receptor (DR) apoptotic pathway. The physiological function of its homologue caspase-10 remains poorly understood, and the ability of caspase-10 to substitute for caspase-8 in the DR apoptotic pathway is still controversial. Here, we analysed the particular contribution of caspase-10 isoforms to DR-mediated apoptosis in neuroblastoma (NB) cells characterised by their resistance to DR signalling. Silencing of caspase-8 in tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-sensitive NB cells resulted in complete resistance to TRAIL, which could be reverted by overexpression of caspase-10A or -10D. Overexpression experiments in various caspase-8-expressing tumour cells also demonstrated that caspase-10A and -10D isoforms strongly increased TRAIL and FasL sensitivity, whereas caspase-10B or -10G had no effect or were weakly anti-apoptotic. Further investigations revealed that the unique C-terminal end of caspase-10B was responsible for its degradation by the ubiquitin-proteasome pathway and for its lack of pro-apoptotic activity compared with caspase-10A and -10D. These data highlight in several tumour cell types, a differential pro- or anti-apoptotic role for the distinct caspase-10 isoforms in DR signalling, which may be relevant for fine tuning of apoptosis initiation.


Subject(s)
Apoptosis , Caspase 10/metabolism , Isoenzymes/metabolism , Neuroblastoma/enzymology , Neuroblastoma/physiopathology , Receptors, Death Domain/metabolism , Amino Acid Motifs , Caspase 10/chemistry , Caspase 10/genetics , Caspase 8/genetics , Caspase 8/metabolism , Cell Line, Tumor , Humans , Isoenzymes/genetics , Neuroblastoma/genetics , Receptors, Death Domain/genetics , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , TNF-Related Apoptosis-Inducing Ligand/metabolism , Tumor Necrosis Factor-alpha/metabolism
17.
Cell Death Differ ; 18(3): 405-14, 2011 Mar.
Article in English | MEDLINE | ID: mdl-20829884

ABSTRACT

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent with cancer-selective apoptogenic activity. It evokes the canonical caspase-mediated cell death pathway through death-inducing signaling complex (DISC) formation. We identified that Peroxiredoxin 6 (Prx6) interacts with caspase-10 and caspase-8 via the death effector domain (DED). Prx6 suppresses TRAIL-mediated cell death in human cancer cells, but not that induced by intrinsic apoptosis inducers such as etoposide, staurosporine, or A23187. Among Prx1-6 members, only Prx6 binds to DED caspases and is most effective in suppressing TRAIL or DED caspase-induced cell death. The antiapoptotic activity of Prx6 against TRAIL is not likely associated with its peroxidase activity but is associated with its ability to bind to DED caspases. Increased expression of Prx6 enhances the binding of Prx6 to caspase-10 but reduces TRAIL-induced DISC formation and subsequently caspase activation. Interestingly, Prx6 is highly upregulated in metastatic gastric cancer cells, which are relatively resistant to TRAIL as compared with primary cancer cells. Downregulation of Prx6 sensitizes the metastatic cancer cells to TRAIL-induced cell death. Taken together, these results suggest that Prx6 modulates TRAIL signaling as a negative regulator of caspase-8 and caspase-10 in DISC formation of TRAIL-resistant metastatic cancer cells.


Subject(s)
Caspase 10/chemistry , Caspase 10/metabolism , Caspase 8/chemistry , Caspase 8/metabolism , Death Domain Receptor Signaling Adaptor Proteins/metabolism , Peroxiredoxin VI/metabolism , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Cell Death/drug effects , Drug Resistance, Neoplasm/drug effects , Enzyme Activation/drug effects , HeLa Cells , Humans , Neoplasm Metastasis , Protein Binding/drug effects , Protein Structure, Tertiary , Signal Transduction/drug effects , Stomach Neoplasms/pathology , Tumor Necrosis Factor-alpha/pharmacology , Up-Regulation
18.
Biochim Biophys Acta ; 1770(11): 1528-37, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17822854

ABSTRACT

Caspase-10 (also known as Mch4 and FLICE2) is an initiator caspase in the death receptor (DR)-dependent apoptotic pathway. So far six splice variants (caspase-10a-f) have been identified. Here we describe a novel isoform of the caspase-10 family named caspase-10g that is widely expressed in normal human tissues and various cell lines. Caspase-10g consists of 247 amino acids and does not contain the large or small subunit. A caspase-10g-specific exon is present between exon 5 and exon 6, which results in a protein product truncated shortly after the death-effector domain (DED)-containing prodomain. We further show that overexpression of caspase-10g dramatically enhances NF-kappaB activity in a dose- and time-dependent manner. Moreover, caspase-10g, unlike the protease-active caspase-10a, only promotes slight apoptosis when overexpressed in mammalian cells and it has no effect on caspase-10a-mediated apoptosis. Taken together, these results suggest that caspase-10g, as a novel prodomain-only isoform of caspase-10, may play a regulatory role preferentially in the NF-kappaB pathways.


Subject(s)
Caspase 10/chemistry , Caspase 10/genetics , Cloning, Molecular , NF-kappa B/metabolism , Alternative Splicing/genetics , Amino Acid Sequence , Base Sequence , Caspase 10/biosynthesis , Caspase 10/physiology , Gene Expression Regulation, Enzymologic , HeLa Cells , Humans , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/physiology , Jurkat Cells , Molecular Sequence Data , NF-kappa B/physiology , Protein Structure, Tertiary/genetics , Signal Transduction/genetics
19.
Fish Shellfish Immunol ; 23(6): 1266-74, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17768069

ABSTRACT

We isolated and sequenced caspase-10 cDNA and gene from Japanese flounder, Paralichthys olivaceus. The Japanese flounder (JF)-caspase-10 cDNA consisted of 2282 bp and encoded 495 amino acid residues. The characteristic death effector domains (DEDs) of caspases were observed in JF-caspase-10 as well as the three aspartic acid residues (D-186, -382 and -392), which are potential cleavage sites for the large and small subunit structures. The amino acid residue (His-325) and pentapeptide (QACQG), which are involved in catalytic activity, were absolutely conserved in Japanese flounder-caspase-10. JF-caspase-10 gene has a length of 6.6 kb and consists of 11 exons and 10 introns similar to that of human. The strong expression of JF-caspase-10 mRNA was detected in the gills, peripheral blood leukocytes, spleen and posterior kidney, while the weak expression was observed in the head kidney, heart, intestine, skin and stomach. The over-expression analysis of JF-caspase-10 in Japanese flounder cell line HINAE was shown to induce apoptosis 24h post-transfection using TUNEL assay.


Subject(s)
Caspase 10/genetics , Flounder/genetics , Flounder/immunology , Gene Expression Regulation, Enzymologic/immunology , Amino Acid Sequence , Animals , Caspase 10/chemistry , Fas Ligand Protein/metabolism , Gene Expression Profiling/veterinary , Gene Order , Molecular Sequence Data , Phylogeny , Sequence Alignment , Sequence Homology, Amino Acid
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