Death Effector Domain Research Articles

Molecular characterization of caspase-8-like and its expression induced by microcystin-LR in grass carp (Ctenopharygodon idella)

Caspase-8, an initiator caspase, plays a vital role in apoptosis. In this study, caspase-8-like (named as Cicaspase-8-like), a homologue of caspase-8, was identified in grass carp (Ctenopharygodon idella). The full-length cDNA sequence of CiCaspase-8-like was 1409 bp and contained a 162 bp 5′-UTR, a 239 bp 3′-UTR and a 1008 bp coding sequence. The putative amino acids sequence was 335 residues long, including a large subunit (P20) and a small subunit (P10), but lacking conserved death effector domains. A histidine active site DHSQMDAFVCCVLSHG and a cysteine active-site motif KPKLFFIQACQG were found in P20. Phylogenetic analysis showed that Cicaspase-8-like clustered with the caspase-8 and caspase-8-like of other fish and grouped closely with Carassius auratus caspase-8-like. Quantitative real-time PCR revealed that the Cicaspase-8-like mRNA were expressed constitutively in all tested tissues from healthy grass carp, with high expression level in the blood, spleen, liver and gill, indicating its role in immune reaction. The expression of Cicaspase-8-like mRNA was decreased significantly in the liver because of the stress caused by microcystin-LR (MC-LR) (75 and 100 μg MC-LR/kg BW) at 24 h and 96 h post injection (P < 0.05), but it was increased significantly in grass carp treated with 25 μg MC-LR/kg BW at 24 h (P < 0.05) post injection. Cleaved fragments of Cicaspase-8-like were observed using western blot analysis, and the expression of Cicaspase-8-like protein was increased after MC-LR treatments. Moreover, the expression of both caspase-9 and caspase-3 mRNA increased significantly after treatment with the three doses of MC-LR. TUNEL assay results showed remarkable changes in apoptosis after the MC-LR treatment. These results suggest that Cicaspase-8-like is an important caspase and plays an essential role in MC-LR-induced apoptosis.

Caspase recruitment domains for proteininteractions in cellular signaling (Review).

The caspase recruitment domain (CARD), a well-known protein interaction module, belongs to the death domain (DD) superfamily, which includes DDs, death effector domains, and pyrin domains. The DD superfamily mediates the protein interactions necessary for apoptosis and immune cell signaling pathways. Among these domains, the CARD has been studied extensively as it mediates important cellular signaling events that are associated with various human diseases including cancer, neuro-degenerative diseases and immune disorders. Homo-type and hetero-type CARD-CARD interactions mediate the formation of large signaling complexes, including caspase-activating complexes and downstream signaling complexes. The present review summarizes and discusses the results of structural studies of various CARDs and their complexes. These studies shed light on the mechanisms that control the assembly and disassembly of signaling complexes and provide an improved understanding of cellular signaling processes.

Mechanisms of Procaspase-8 Activation in the Extrinsic Programmed Cell Death Pathway

Caspase-8 performs initiatory functions during the induction of apoptosis through the extrinsic pathway. Apoptosis is a type of programmed cell death that plays an important role in regulating embryogenesis and maintaining homeostasis in the tissue of an adult organism, as well as differentiating and removing damaged cells. Dysregulation of the apoptosis mechanisms is associated with the pathogenesis and progression of a number of oncological and neurodegenerative diseases. Caspase-8 (also called СAP4, FLICE, MACH, MCH5) is one of two members of the death effector domain (DED)-containing caspases. Despite the fact that the role of caspase-8 in apoptosis has been well known since the mid 1990s, we are only now beginning to understand the subtle mechanisms of its activation and regulation in response to the activation of death receptors (DRs). In particular, it was demonstrated that the activation of caspase-8 requires the formation of specific oligomeric structures, which are named DED filaments. In this review, the recent data on the mechanisms of activating initiator caspase-8 in DED filaments are considered that allow us to better understand the subtle mechanisms of the initiation of the programmed cell death.

Structural basis for dimerization of the death effector domain of the F122A mutant of Caspase-8

Caspase-8 is an apoptotic protease that is activated by a proximity-induced dimerization mechanism within the death-inducing signaling complex (DISC). The death effector domain (DED) of caspase-8 is involved in protein-protein interactions and is essential for the activation. Here, we report two crystal structures of the dimeric DEDs of the F122A mutant of caspase-8, both of which illustrate a novel domain-swapped dimerization, while differ in the relative orientation of the two subunits and the solvent exposure of the conserved hydrophobic patch Phe122/Leu123. We demonstrate that mutations disrupting the dimerization of the DEDs abrogate the formation of cellular death effector filaments (DEFs) and the induced apoptosis by overexpressed DEDs. Furthermore, such dimerization-disrupting mutations also impair the activation of the full-length caspase-8 and the downstream apoptosis cascade. The structures provide new insights into understanding the mechanism underlying the activation of procaspase-8 within the DISC and DEFs.

Molecular basis of dimerization of initiator caspase was revealed by crystal structure of caspase-8 pro-domain.

The assembly of death-inducing signaling complex (DISC) for activation of initiator caspase is a key step for the receptor-mediated apoptosis signaling. Many death effector domain (DED)-containing proteins are involved in DISC assembly and controlling. One of the main DISC component, caspase-8, contains DED and DED-mediated dimerization and oligomerization in the DISC is critical for the activation of this initiator caspase. There have been intensive studies to understand DED-mediated dimerization and oligomerization for the DISC assembly but no clear answer has been provided and there are many controversial arguments. Here, we suggested novel dimerization process of tandem DED of caspase-8 with crystallographic study.

Novel tumor necrosis factor-α induced protein eight (TNFAIP8/TIPE) family: Functions and downstream targets involved in cancer progression

The tumor necrosis factor (TNF)-α- induced protein 8 (TNFAIP8/TIPE) family is a death effector domain (DED)-containing protein family with four identified members: TNFAIP8 (TIPE), TNFAIP8L1 (TIPE1), TNFAIP8L2 (TIPE2), and TNFAIP8L3 (TIPE3). These proteins were found to play crucial roles in the regulation of immune homeostasis, inflammation, and cancer development. Intensive research in the past two decades revealed a strong correlation of TIPE proteins with the development of various cancers including cancers of the bladder, blood, bone, breast, cervix, colon, esophagus, endometrium, stomach, liver, lung, ovary, pancreas, prostate, and thyroid gland. Also, deregulation of these proteins was found to promote the essential hallmarks of cancer such as survival, tumor growth, proliferation, inhibition of apoptosis, angiogenesis, invasion, migration, and metastasis. Further, differential expression of these proteins in normal and cancer tissues and their association with tumor progression and prognosis signifies the potential diagnostic and prognostic values of TIPE proteins and their importance in cancer therapy. The current review summarizes the literature available thus far on the expression, function, and role of TIPE proteins in the development and maintenance of various cancers.

Phosphorylation of PEA‐15 allosterically induces conformational change suited for FADD binding and negatively regulates apoptosis

Phosphoprotein Enriched in Astrocytes, 15 kD (PEA‐15) is a small non‐catalytic protein, with an N‐terminal death effector domain (DED) and an unstructured C‐terminal tail. PEA‐15 appeals to many biochemists because it regulates two important pathways. Unphosphorylated PEA‐15 interacts with extracellular regulated protein kinase (ERK) 1 and 2, preventing ERK‐dependent cell proliferation. When it is doubly phosphorylated at the two C‐terminal serine residues, PEA‐15 is recruited to the death inducing signaling complex (DISC) when it interacts with Fasassociated death domain (FADD) protein, blocking the normal recruitment and activation of procaspase‐8, which triggers the signal for apoptosis. In our lab, PEA‐15 and FADD are cloned into pET‐28b(+) vector, and are expressed in BL21(DE3) E. Coli competent cells. To mimic the phosphorylation, S104D/S116D mutant protein (PEA‐15DD) was also expressed. The proteins are purified using metal affinity (Ni2+ or Co2+) chromatography followed by ion‐exchange chromatography. The protein structures and interactions between PEA‐15DD and FADD are determined using NMR spectroscopy. Our results showed that the phosphorylation of PEA‐15 changes the conformation of the folded DED, and FADD binding does not further modulate the overall conformation. We concluded that the binding specificity of PEA‐15 is determined by the DED conformation, which is allosterically induced by the C‐terminal phosphorylation states. We further proposed that the balance of various phosphorylation states of PEA‐15 should be strictly regulated in different tissues and cell types in order to maintain the healthy conditions of the cells.Support or Funding InformationNIH AREA R15CA179410This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanisms of recombinant adenovirus-mediated SD-HA fusion protein proliferation inhibition and induced apoptosis of K562 cells

目的探讨融合蛋白SD-HA能否通过竞争结合BCR-ABL的第177位酪氨酸磷酸化位点（Y177p），调控与其相关的下游信号分子活性，进而抑制K562细胞增殖并诱导凋亡。方法Western blot结合免疫共沉淀技术分析融合蛋白SD-HA与BCR-ABL的Y177p的相互作用，及其对下游信号途径Ras-MAPK和磷脂酰肌醇3-激酶(PI3K)-Akt的影响；Western blot分析融合蛋白SD-HA对细胞膜受体死亡途径级联反应caspase-8、caspase-3和PRAP的影响。结果双向免疫共沉淀实验可检测到融合蛋白SD-HA能与Grb2竞争性结合BCR-ABL Y177p，形成复合物。融合蛋白SD-HA可降低活化Ras、磷酸化MAPK（p-MAPK）、p-ELK的表达水平，抑制Ras-MAPK信号途径；融合蛋白SD-HA还可降低p-Akt及Akt的底物p-GSK的表达水平，抑制PI3K-Akt信号途径，从而抑制K562细胞增殖；通过死亡效应结构域（DED）与caspases-8前体DED结合而寡聚化，激活caspases-8、caspase-3和PRAP，诱导K562细胞凋亡。结论融合蛋白SD-HA抑制BCR-ABL-Y177与Grb2结合的策略，可作为慢性髓性白血病治疗新的切入点。

Tumor Necrosis Factor-α Induced Protein 8: Pathophysiology, Clinical Significance, and Regulatory Mechanism.

Tumor necrosis factor-α-induced protein-8 (TNFAIP8) is the earliest discovered component of TNFAIP8 family [tumor necrosis factor-α-induced protein-8 like (TIPE) family]. TNFAIP8 contains a putative death effector domain (DED) homologous to DED II in FLIP (Fas-associated death domain-like interleukin-1β-converting enzyme-inhibitory protein), which may affect cell survival/death process. Recently, it has been demonstrated that TNFAIP8 could inhibit apoptosis and autophagy in various types of cells. Moreover, TNFAIP8 level fluctuated evidently in patients with inflammatory, malignant, and autoimmune diseases, indicating that it might be an anti-apoptotic and oncogenetic protein. Herein we will review the discovery, gene/protein structure, pathophysiological functions, and clinical significance of TNFAIP8 together with its potential regulatory mechanism.

DDIAS suppresses TRAIL-mediated apoptosis by inhibiting DISC formation and destabilizing caspase-8 in cancer cells.

DNA damage-induced apoptosis suppressor (DDIAS) has an anti-apoptotic function during DNA damage in lung cancer. However, the anti-apoptotic mechanism of DDIAS in cancer cells under other conditions has not been reported. We report here that DDIAS protects cancer cells from tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by two distinct mechanisms in non-small cell lung cancer (NSCLC) and hepatocellular carcinoma (HCC) cells. DDIAS depletion sensitized NSCLC and HCC cells to TRAIL-mediated apoptosis, an effect that was abrogated by pharmacological or genetic inhibition of caspase-8 and was independent of caspase-9, p53, or mitogen-activated protein kinase signaling. Interestingly, we found that the N terminus of DDIAS interacted with the death effector domain of Fas-associated protein death domain (FADD) and prevented its recruitment to the death-inducing signaling complex (DISC), thereby blocking caspase-8 activation. DDIAS knockdown also suppressed epidermal growth factor-induced phosphorylation of p90 ribosomal S6 kinase (RSK) 2 and stabilized caspase-8 by preventing its ubiquitination and proteasomal degradation. This effect was abolished by RSK2 overexpression. Taken together, DDIAS has dual functions in inhibiting DISC formation as well as in destabilizing caspase-8, thereby suppressing TRAIL-mediated apoptosis of cancer cells. Thus, we suggest that DDIAS can serve as an effective therapeutic target in the treatment of NSCLC and HCC.

The first tropical sea cucumber caspase-8 from Holothuria leucospilota: Molecular characterization, involvement of apoptosis and inducible expression by immune challenge

In this study, the first tropical sea cucumber caspase-8 named HLcaspase-8 was identified from Holothuria leucospilota. The full-length cDNA of HLcaspase-8 is 2293 bp in size, containing a 245 bp 5′-untranslated region (UTR), a 521 bp 3′-UTR and a 1527 bp open reading frame (ORF) encoding a protein of 508 amino acids with a deduced molecular weight of 57.47 kDa. Besides the common signatures of caspase family including conserved cysteine active site pentapeptide motif QACQG, P20 domain and P10 domain, HLcaspase-8 also contains a characteristic DED domain. The over-expression of HLcaspase-8 in HEK293T cells showed that HLcaspase-8 protein could induce apoptosis and the apoptosis could be promoted by TNF-α, indicating that the apoptosis induced by HLcaspase-8 might also be triggered via a receptor-mediated pathway. Moreover, the expression of HLcaspase-8 in in vitro experiments performed in coelomocytes was significantly up-regulated by lipopolysaccharides (LPS) or polyriboinosinic-polyribocytidylic Acid [poly (I:C)] challenge, suggesting that the sea cucumber caspase-8 might play some important roles in the innate immune defense against bacterial and viral infections.

Expression and significance of long non-coding RNA metastasis associated in lung denocarcinoma transcript 1 in the process of hyperoxia-induced lung injury in preterm infants

Objective To explore the protective effect of long non-coding RNA (lncRNA) metastasis associated in lung denocarcinoma transcript 1 (MALAT1) involved in hyperoxia-induced lung injury in preterm infants. Methods This study had downloaded chip data set GSE25286 (Mouse GEO Genome 430 2.0 Array) from gene expression database gene expression omnibus (GEO), according to the state of hyperoxia exposure, the MALAT1 mRNA expression in rats normal lung tissues and hyperoxic lung tissues was compared at day 14th and 29th.In chip data set GSE43830(Human Exon 1.0 ST Arrays) from GEO, the expression of multiple genes[cell division cycle 6(CDC6), death effector domain containing 2(DEDD2), and Cyclin B1(CCNB1)] in WI38 cells (lung fibroblasts) was compared before and after MALAT1 was knockout.At the same time, the peripheral blood samples of premature infants were collected to verify.Totally 40 premature infants were hospitalized in the department of neonatology in our hospital from Jan 2015 to Dec 2016, the peripheral blood samples of 40 premature infants were collected.RNA was extracted and Real time-PCR was performed after reverse transcription, clinical data of these 40 cases were retrospectively analyzed. Results (1) By using Affymetrix Expression console and Affymetrix Transcriptome analysis console software source files of the chip of pretreatment and difference expression gene screening, the expression of lncRNA MALAT1 gene in lung tissues of hyperoxia lung injury mice significantly upregulated[fold change(FC)=2.33, P=0.001]. (2) After MALAT1 in WI38 cell was knockout, MALAT1 expression was significantly reduced(FC=-15.6, P=0.000), the expression of CDC6(FC=-2.37, P=0.001) and CCNB1(FC=-2.16, P=0.002) were down regulated, DEDD2 expression was up regulated(FC=2.46, P=0.000). (3) The results of peripheral blood samples from preterm infants showed that the expression of MALAT1 was significantly increased in preterm infants with hyperoxia-induced lung injury(0.375 5±0.081 9, t=4.634, P=0.015), compared with normal preterm infants(0.273 4±0.067 3). Conclusion Through inhibiting cell apoptosis, lncRNA MALAT1 can protect preterm infants with hyperoxia-induced lung injury, it may provide a new strategy for prevention and treatment of hyperoxia-induced lung injury in premature infants. Key words: Long noncoding RNA; Neonates; Lung injury; Metastasis associated in lung denocarcinoma transcript 1; Hyperoxia

A novel TNFAIP8 gene mediates l-arginine metabolism in Apostichopus japonicus

Tumor necrosis factor (TNF)-α-induced protein 8 (TNFAIP8) family is a newly identified protein with vital roles in maintaining immune homeostasis. In the current study, we first cloned and characterized a TNFAIP8 gene from the invertebrate sea cucumber Apostichopus japonicus. The gene was designated as AjTNFAIP8. The full-length cDNA of AjTNFAIP8 was 1455 bp long and encoded a matured protein of 201 amino acid residues. Structural analysis indicated that AjTNFAIP8 had a death effector domain (DED)-like domain and composed of six α-helices. Multiple sequence alignment and phylogenetic analysis supported that AjTNFAIP8 is a new member of the TNFAIP8 family. Analysis of basal transcription in five tissues revealed the constitutive expression of AjTNFAIP8 in the detected tissues with highest expression in the respiratory tree and minimum expression in the tentacle. Vibrio splendidus infection and LPS stimulation could significantly downregulate the mRNA expression of AjTNFAIP8. More importantly, the transcription of pro-inflammatory molecule NOS and its production of NO content were significantly increased after AjTNFAIP8 silencing, with the suppression of agmatinase transcript and arginase activity. These results clearly indicated that AjTNFAIP8 is an essential negative regulator in innate immunity. Basic information for further exploration of the functional mechanisms of TNFAIP8 family in other marine invertebrate is provided.

The molluscum contagiosum virus death effector domain containing protein MC160 RxDL motifs are not required for its known viral immune evasion functions

The molluscum contagiosum virus (MCV) uses a variety of immune evasion strategies to antagonize host immune responses. Two MCV proteins, MC159 and MC160, contain tandem death effector domains (DEDs). They are reported to inhibit innate immune signaling events such as NF-κB and IRF3 activation, and apoptosis. The RxDL motif of MC159 is required for inhibition of both apoptosis and NF-κB activation. However, the role of the conserved RxDL motif in the MC160 DEDs remained unknown. To answer this question, we performed alanine mutations to neutralize the arginine and aspartate residues present in the MC160 RxDL in both DED1 and DED2. These mutations were further modeled against the structure of the MC159 protein. Surprisingly, the RxDL motif was not required for MC160's ability to inhibit MAVS-induced IFNβ activation. Further, unlike previous results with the MC159 protein, mutations within the RxDL motif of MC160 had no effect on the ability of MC160 to dampen TNF-α-induced NF-κB activation. Molecular modeling predictions revealed no overall changes to the structure in the MC160 protein when the amino acids of both RxDL motifs were mutated to alanine (DED1=R67A D69A; DED2=R160A D162A). Taken together, our results demonstrate that the RxDL motifs present in the MC160 DEDs are not required for known functions of the viral protein.

C-FLIPL Modulated Wnt/β-Catenin Activation via Association with TIP49 Protein

Cellular FLICE-like inhibitory protein (c-FLIPL) is a key inhibitory protein in the extrinsic apoptotic pathway. Recent studies showed that c-FLIPL could translocate into the nucleus and might be involved in the Wnt signaling pathway. The nuclear function of c-FLIPL was still unclear. Here we found a novel c-FLIPL-associated protein TIP49, which is a nuclear protein identified as a cofactor in the transcriptional regulation of β-catenin. They had co-localization in the nucleus and the DED domain of c-FLIPL was required for the association with TIP49. By performing ChIP experiments, C-FLIPL was detected in the ITF-2 locus and facilitated TIP49 accumulation in the formation of complexes at the T-cell-specific transcription factor site of human ITF-2 promoter. When TIP49 knockdown, c-FLIPL-driven Wnt activation, and cell proliferation were inhibited, suggesting that a role of nuclear c-FLIPL involved in modulation of the Wnt pathway was in a TIP49-dependent manner. Elevated expression of c-FLIPL and TIP49 that coincided in human lung cancers were analyzed in silico using the Oncomine database. Their high expressions were reconfirmed in six lung cancer cell lines and correlated with cell growth. The association of c-FLIPL and TIP49 provided an additional mechanism involved in c-FLIPL-mediated functions, including Wnt activation.

Cflarb Complemented the Function of Cflara to Allow Cflara Knock out Zebrafish To Normal Development

Cellular FLICE-inhibitory protein (cFLIP, cflara) is a regulator of death receptor (DR)-induced apoptosis and NF-κB activation. cFLIP is known to prevent activation of the caspase cascade by binding to FADD/caspase-8. Up-regulated cFLIP has been identified in many tumor types, and therefore restoring apoptosis by silencing cFLIP may be one of the more potent strategies in cancer therapeutics. The zebrafish cFLIP gene, cflara, has 2 death effector domains (DEDs) and a single caspase-like domain. Expression of cflara was detected in the zebrafish embryo by RT-PCR and whole-mount in situ hybridization. To study the in vivo function of cflara, we generated a cflara knockout mutant zebrafish using transcription activator-like effector nucleases (TALENs). Frame shift mutation is caused by a 10-bp deletion in the first DED domain. By inbreeding the F1 generation, a homozygous mutant fish was produced and confirmed by PCR. Knockout of cflara leads to abnormal heart development and embryonic lethality in mice. However, mutant zebrafish did not show any differences from wild type in heartbeat rate, survival rate or development. Zebrafish have analogues of both cflara and cflarb. Quantitative PCR showed that cflarb mRNA levels of mutant zebrafish were higher than those in the wild type. In a chemical exposure experiment, mutant zebrafish larvae showed a longer survival rate compared with wild type after CoCl2 treatment. However, no significant difference was observed from cisplatin treatment. This data suggests that cflarb may contribute to normal development and causes a difference in chemical resistance.

Design and experimental validation of small-molecule inhibitors of the FADD protein

CD95 is one of the best-studied members of the death receptor family. Activation of CD95 leads to the induction of the cell death program, apoptosis, via formation of the death-inducing signaling complex (DISC). The Fas-Associated Death Domain (FADD) is a key adaptor protein in the formation of the CD95 DISC and activation of procaspase 8 in the receptor complex. FADD comprises the death domain and the death effector domain (DED). The death domain is needed for the interactions of FADD with CD95, while DED is necessary for the recruitment of procaspases 8 and 10 and the cFLIP protein into the DISC. The search for specific inhibitors that would prevent the interactions of FADD with other core proteins of the DISC is important for the studies of the structure and function of this complex, investigation of apoptosis mechanisms, and development of new treatments for neurodegenerative diseases. In the course of this work, an in silico screening for low-molecular-weight inhibitors that would selectively interact with FADD DED was performed. This work required the molecular modeling of the protein complexes and virtual screening of potential FADD inhibitors. In addition, a new technology to test the activity of these inhibitors was developed. The computational and experimental analysis allowed us to characterize the optimum conformation of FADD for the design of small molecules that would bind to the region of the amino acid residue F25. We presume that further optimization of the structures of the chemical compounds that can bind within the hydrophobic pocket next to F25 of FADD will allow the development of new candidate inhibitors of programmed cell death.

Molecular and functional characterization of caspase-8 from the big-belly seahorse (Hippocampus abdominalis)

Apoptosis is a physiological process that can also participate in host immune defense mechanisms, including tumor growth suppression along with homeostasis and maturation of immune cells. Caspases are known to be involved in cellular apoptotic signaling; among them, caspase-8 plays an important role in the initiation phase of the apoptotic death cascade. In the current study, we molecularly characterized a caspase-8 homolog (designated as HaCasp-8) from Hippocampus abdominalis. The HaCasp-8 gene harbors a 1476 bp open reading frame (ORF) that codes for a protein of 492 amino acids (aa) with a predicted molecular mass of 55 kDa. HaCasp-8 houses the typical domain architecture of known initiator caspases, including the death effector domain and the carboxyl-terminal catalytic domain. As expected, phylogenetic analysis reflected a closer evolutionary relationship of HaCasp-8 with its teleostean similitudes. The results of our qPCR assays confirmed the ubiquitous expression of HaCasp-8 in physiologically important tissues examined, with pronounced expression levels in ovary tissues, followed by blood cells. HaCasp-8 expression at the mRNA level was found to be significantly modulated by lipopolysaccharide, polyinosinic:polycytidylic acid, Streptococcus iniae, and Edwardsiella tarda injection. Overexpression of HaCasp-8 could trigger a significant level of cell death in HEK293T cells, suggesting its putative role in cell death. Taken together, our findings suggest that HaCasp-8 is an important component in the caspase cascade, and its expression can be significantly modulated under pathogen stress conditions in the big-belly seahorse.

TIPE2 (Tumor Necrosis Factor α-induced Protein 8-like 2) Is a Novel Negative Regulator of TAK1 Signal

TIPE2 (TNF-α-induced protein 8-like 2) is a novel death effector domain protein and is a negative regulator of the innate and adaptive immune response. Although it has been demonstrated that caspase-8 contributes to the negative regulation of TIPE2, the negative regulatory mechanism is not entirely understood. Here, we demonstrate that TIPE2 interacts with TGF-β-activated kinase 1 (TAK1), a crucial regulatory molecule of inflammatory and immune signals, and consequently acts as a powerful negative regulator of TAK1. The interaction between endogenous TIPE2 and TAK1 was observed in RAW264.7 macrophage-like cells and mouse primary cells derived from spleen and thymus. The TIPE2 amino acid 101–140 region interacted with TAK1 by binding to the amino acid 200–291 region of the internal kinase domain of TAK1. TIPE2 interfered with the formation of the TAK1-TAB1-TAB2 complex and subsequently inhibited activation of TAK1 and its downstream molecules. Importantly, silencing TIPE2 through RNA interference attenuated the inhibitory action of TIPE2 on LPS- and TNF-α-stimulated TAK1 activity. Exogenous TIPE2 101–140, the region that interacts with TAK1, also inhibited LPS- and TNF-α-stimulated NF-κB reporter activity. Interestingly, cell-permeable TIPE2 protein maintained its binding ability with TAK1 and exhibited the same inhibitory action of native TIPE2 on TLR4 signaling in vitro. Thus, cell-permeable TIPE2 protein is a potential candidate for intracellular protein therapy for TAK1-related diseases. The present study demonstrates that TIPE2 acts as a novel negative regulator of inflammatory and immune responses through TAK1 signaling.

Cryo-EM Structure of Caspase-8 Tandem DED Filament Reveals Assembly and Regulation Mechanisms of the Death-Inducing Signaling Complex

Caspase-8 activation can be triggered by death receptor-mediated formation of the death-inducing signaling complex (DISC) and by the inflammasome adaptor ASC. Caspase-8 assembles with FADD at the DISC and with ASC at the inflammasome through its tandem death effector domain (tDED), which is regulated by the tDED-containing cellular inhibitor cFLIP and the viral inhibitor MC159. Here we present the caspase-8 tDED filament structure determined by cryoelectron microscopy. Extensive assembly interfaces not predicted by the previously proposed linear DED chain model were uncovered, and were further confirmed by structure-based mutagenesis in filament formation invitro and Fas-induced apoptosis and ASC-mediated caspase-8 recruitment in cells. Structurally, the two DEDs in caspase-8 use quasi-equivalent contacts to enable assembly. Using the tDED filament structure as a template, structural analyses reveal the interaction surfaces between FADD and caspase-8 and the distinct mechanisms of regulation by cFLIP and MC159 through comingling and capping, respectively.