• BMB Reports
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• 제47권9호
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• pp.488-493
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• 2014

Adaptor protein FADD forms the death inducing signaling complex (DISC) by recruiting the initiating caspases-8 and -10 through homotypic death effector domain (DED) interactions. Cellular FLICE-inhibitory protein (c-FLIP) is an inhibitor of death ligand-induced apoptosis downstream of death receptors, and FADD competes with procaspase-8/10 for recruitment for DISC. However, the mechanism of action of FADD and c-FLIP proteins remain poorly understood at the molecular level. In this study, we provide evidence indicating that the death effector domain (DED) of FADD interacts directly with the death effector domain of human c-FLIP. In addition, we use homology modeling to develop a molecular docking model of FADD and c-FLIP proteins. We also find that four structure-based mutants (E80A, L84A, K169A and Y171A) of c-FLIP DEDs disturb the interaction with FADD DED, and that these mutations lower the stability of the c-FLIP DED.

• BMB Reports
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• 제49권3호
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• pp.159-166
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• 2016

Several members of tumor necrosis factor receptor (TNFR) superfamily that these members activate caspase-8 from death-inducing signaling complex (DISC) in TNF ligand-receptor signal transduction have been identified. In the extrinsic pathway, apoptotic signal transduction is induced in death domain (DD) superfamily; it consists of a hexahelical bundle that contains 80 amino acids. The DD superfamily includes about 100 members that belong to four subfamilies: death domain (DD), caspase recruitment domain (CARD), pyrin domain (PYD), and death effector domain (DED). This superfamily contains key building blocks: with these blocks, multimeric complexes are formed through homotypic interactions. Furthermore, each DD-binding event occurs exclusively. The DD superfamily regulates the balance between death and survival of cells. In this study, the structures, functions, and unique features of DD superfamily members are compared with their complexes. By elucidating structural insights of DD superfamily members, we investigate the interaction mechanisms of DD domains; these domains are involved in TNF ligand-receptor signaling. These DD superfamily members play a pivotal role in the development of more specific treatments of cancer.

• 한국생물정보학회:학술대회논문집
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• 한국생물정보시스템생물학회 2005년도 BIOINFO 2005
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• pp.305-310
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• 2005

Internal motion of the protein has been described in many papers with C$_{\alpha}$ correlation coefficients to find motional correlation and functional characteristics. To describe the secondary structural motion and stability in protein, we have studied molecular dynamics (MD) simulations on FADD Death Domain and FADD Death Effector Domain which have a similar structure but have different functional characteristics. After 10ns MD simulations, the inter-helical motional correlations and the hydrogen bond ratios were compared between the two domains. From these data we could distinctly compare the internal motions of them and could explain the differences in experimental thermodynamic melting behaviors at molecular level.

• Bulletin of the Korean Chemical Society
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• 제27권1호
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• pp.87-92
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• 2006

Fas-associated death domain protein (FADD) recruits and activates procaspase-8 through interactions between the death effector domains of these two proteins. Cellular FLICE-inhibitory protein (c-FLIP) was identified as a molecule with sequence homology to caspase-8. It has been postulated that c-FLIP prevents formation of the competent death-inducing signaling complex in a ligand-dependent manner, through its interaction with FADD and/or caspase-8. However, the interaction of FADD and $c-FLIP_s$ (short form) in apoptosis signaling has been controversially discussed. We show the purification and the characterization of human full-length FADD and $c-FLIP_s$ expressed in Escherichia coli. The purified FADD and $c-FLIP_s$ are shown as homogeneity, respectively, in SDS-PAGE analysis and light-scattering measurements. The folding properties of the $\alpha$-helical structure of FADD and the super-secondary structure of $c-FLIP_s$ proteins were characterized by circular dichroism spectroscopy. Furthermore, we report here a series of biochemical and biophysical data for FADD-$c-FLIP_s$ binding in vitro. The binding of both FADD and $c-FLIP_s$ proteins was detected by BIAcore biosensor, fluorescence measurement, and size-exclusion column (SEC).

• 한국균학회소식:학술대회논문집
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• 한국균학회 2015년도 추계학술대회 및 정기총회
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• pp.26-26
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• 2015

Phytophthora infestans is the causal agent of potato and tomato late blight, one of the most devastating plant diseases. P. infestans secretes effector proteins that are both modulators and targets of host plant immunity. Among these are the so-called RXLR effectors that function inside plant cells and are characterized by a conserved motif following the N-terminal signal peptide. In contrast, the effector activity is encoded by the C terminal region that follows the RXLR domain. Recently, I performed in planta functional profiling of different RXLR effector alleles. These genes were amplified from a variety of P. infestans isolates and cloned into a Potato virus X (PVX) vector for transient in planta expression. I assayed for R-gene specific induction of hypersensitive cell death. The findings included the discovery of new effector with avirulence activity towards the Solanum bulbocastanum Rpi-blb2 resistance gene. The Rpi-blb2 encodes a protein with a putative CC-NBS-LRR (a coiled-coil-nucleotide binding site and leucine-rich repeat) motif that confers Phytophthora late blight disease resistance. We examined the components required for Rpi-blb2-mediated resistance to P. infestans in Nicotiana benthamiana. Virus-induced gene silencing was used to repress candidate genes in N. benthamiana and to assay against P. infestans infections. NbSGT1 was required for disease resistance to P. infestans and hypersensitive responses (HRs) triggered by co-expression of AVRblb2 and Rpi-blb2 in N. benthamiana. RAR1 and HSP90 did not affect disease resistance or HRs in Rpi-blb2-transgenic plants. To elucidate the role of salicylic acid (SA) in Rpi-blb2-mediated resistance, we analyzed the response of NahG-transgenic plants following P. infestans infection. The increased susceptibility of Rpi-blb2-transgenic plants in the NahG background correlated with reduced SA and SA glucoside levels. Furthermore, Rpi-blb2-mediated HR cell death was associated with $H_2O_2$, but not SA, accumulation. SA affects basal defense and Rpi-blb2-mediated resistance against P. infestans. These findings provide evidence about the roles of SGT1 and SA signaling in Rpi-blb2-mediated resistance against P. infestans.

• Molecules and Cells
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• 제26권2호
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• pp.165-170
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• 2008

Procaspase-8 is activated by forming a death-inducing signaling complex (DISC) with the Fas-associated death domain (FADD) and the Fas receptor, but the mechanism of its activation is not well understood. Procaspase-8 devoid of the death effector domain at its N-terminus (${\Delta}nprocaspase-8$) was reported to be activated by kosmotropic salts, but it has not been induced to form a DISC in vitro because it cannot interact with FADD. Here, we report the production of full-length procaspase-8 and show that it is activated by adding the Fas death domain (Fas-DD) and the FADD forming the cytoplasmic part of the DISC (cDISC). Furthermore, mutations known to affect DISC formation in vivo were shown to have the same effect on procaspase-8 activation in vitro. An antibody that induces Fas-DD association enhanced procaspase-8 activation, suggesting that the Fas ligand is not required for low-level activation of procaspase-8, but that Fas receptor clustering is needed for high-level activation of procaspase-8 leading to cell death. In vitro activation of procaspase-8 by forming a cDISC will be invaluable for investigating activation of ligand-mediated apoptosis and the numerous interactions affecting procaspase-8 activation.

• 생명과학회지
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• 제31권10호
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• pp.954-959
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• 2021

세포 사멸은 항상성을 유지하기 위해 세포군을 조절하는 중요한 메커니즘이며 시스테인 단백질분해효소 중 하나인 카스파제는 세포 사멸 경로의 중요한 중재자이다. Caspase-8은 세포외 자극에 의해 시작되는 외인성 세포자멸 경로의 개시자 카스파제이다. Caspase-8에는 보존된 도메인인 N-말단의 두개의 죽음 이펙터 도메인(DED)과 C-말단의 2개의 촉매 도메인을 가지며, 이는 이러한 외인성 세포자멸 경로에 중요하게 작용한다. 외인성 세포멸사 경로에서, TNF 슈퍼패밀리인 죽음 수용체는 세포 외부로부터의 죽음 수용체 특이적 리간드의 결합에 의해 활성화된다. 활성화된 죽음 수용체가 어댑터 단백질인 Fas-associated death domain 단백질(FADD)을 모집한 후, 죽음 수용체와 FADD의 죽음 도메인(DD)이 서로 결합하고 죽음 수용체와 결합한 FADD가 caspase-8의 전구체 형태인 procaspase-8을 모집한다. FADD와 procaspase-8의 죽음 이펙터 도메인은 서로 결합하고 FADD에 결합된 procaspase-8은 prodomain의 절단에 의해 활성화된다. 이 죽음 수용체-FADD-caspase-8 복합체는 세포사멸 유도 신호복합체(DISC)라고 한다. 세포 FLICE 억제 단백질(c-FLIPs)은 세포사멸을 억제하는 역할과 촉진하는 역할을 모두 수행하여 caspase-8의 활성화를 조절하고 caspase-8 활성화는 caspase-3와 같은 작동자 카스파제를 활성화를 시킨다. 마지막으로 활성화된 작동자 카스파제는 DNA 분해, 핵 응축, 세포막 수포 및 카스파제 기질의 단백질 분해에 작용하여 세포사멸을 완료한다.

• Journal of Microbiology and Biotechnology
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• 제32권8호
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• pp.1034-1040
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• 2022

Fas-associated death domain (FADD) is an adapter molecule that bridges the interaction between receptor-interacting protein 1 (RIP1) and aspartate-specific cysteine protease-8 (caspase-8). As the primary mediator of apoptotic cell death, caspase-8 has two N-terminal death-effector domains (DEDs) and it interacts with other proteins in the DED subfamily through several conserved residues. In the tumor necrosis receptor-1 (TNFR-1)-dependent signaling pathway, apoptosis is triggered by the caspase-8/FADD complex by stimulating receptor internalization. However, the molecular mechanism of complex formation by the DED proteins remains poorly understood. Here, we found that direct DED-DED interaction between FADD and caspase-8 and the structure-based mutations (Y8D/I128A, E12A/I128A, E12R/I128A, K39A/I128A, K39D/I128A, F122A/I128A, and L123A/I128A) of caspase-8 disrupted formation of the stable DED complex with FADD. Moreover, the monomeric crystal structure of the caspase-8 DEDs (F122A/I128A) was solved at 1.7 Å. This study will provide new insight into the interaction mechanism and structural characteristics between FADD and caspase-8 DED subfamily proteins.

• The Plant Pathology Journal
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• 제33권2호
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• pp.163-169
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• 2017

$SIMAPKKK{\alpha}$, a tomato (Solanum lycopersicum) mitogen-activated protein kinase kinase kinase, is a positive regulator of Pto-mediated effector-triggered immunity, which elicits programmed cell death (PCD) in plants. In this study, we examined whether putative phosphorylation sites in the conserved activation segment of the $SIMAPKKK{\alpha}$ kinase domain are critical for eliciting PCD. Three amino acids, $threonine^{353}$, $serine^{360}$ ($Ser^{360}$), or $serine^{364}$ ($Ser^{364}$), in the conserved activation segment of $SIMAPKKK{\alpha}$ kinase domain were substituted to alanine (T353A, S360A, or S364A), and these variants were transiently expressed in tomato and Nicotiana benthamiana plants. Two alanine substitutions, S360A and S364A, completely abolished $SIMAPKKK{\alpha}$ PCD-eliciting activity in both plants, while T353A substitution did not affect its PCD-eliciting activity. $SIMAPKKK{\alpha}$ wild type and variant proteins accumulated to similar levels in plant leaves. However, $SIMAPKKK{\alpha}$ protein with the largest size was missed when either S360A or S364A substitutions were expressed, whereas proteins with the smaller masses were more accumulated than those of full-length of $SIMAPKKK{\alpha}$ and T353A. These results suggest that phosphorylation of $SIMAPKKK{\alpha}$ at $Ser^{360}$ and $Ser^{364}$ is critical for PCD elicitation in plants.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2004년도 The 2004 KSPP Annual Meeting & International Symposium
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• pp.65-67
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• 2004

During initial interactions of bacteria with their host plants, most plants recognize the bacterial infections and repel the pathogen by plant defense mechanism. The most active plant defense mechanism is the hypersensitive response (HR) which is the localized induced cell death in the plant at the site of infection by a pathogen. A primary locus induced in gram-negative phytopathogenic bacteria during this initial interaction is the Hrp locus. The Hrp locus is composed of a cluster of genes that encodes the bacteral Type 111 machinery that is involved in the secretion and translocation of effector proteins to the plant cell. DNA sequence analysis of hrp gene in phytopathogenic bacteria has revealed a Hrp pathogenicity is]and (PAI) with a tripartite mosaic structure. For many gram-negative pathogenic bacteria, colonization of the host's tissue depends on the type III protein secretion system (TTSS) which secrets and translocates effector proteins into the host cell. Effectors can be divided into several groups including broad host range effectors, host specific effectors, disease specific effectors, and effectors inhibit host defenses. The role of effectors carrying LRR domain in plant resistance is very elusive since most known plant resistance gene carry LRR domain. Host specific effectors such as several avr gene products are involved in the determination of the host specificity. Almost all the phytopathogenic Xanthomonas spp. carry avrBs1, avrBs2, and avrBs3 homologs. Some strains of X. oryzae pv. oryzae carry more than 10 copies of avrBs3 homologs. However, the functions of all those avr genes in host specificity are not characterized well.;