• Molecules and Cells
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• 제26권5호
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• pp.443-453
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• 2008

The Bric-a-brac, Tramtrack, Broad-complex (BTB) domain is a protein-protein interaction domain that is found in many zinc finger transcription factors. BTB containing proteins play important roles in a variety of cellular functions including regulation of transcription, regulation of the cytoskeleton, protein ubiquitination, angiogenesis, and apoptosis. Here, we report the cloning and characterization of a novel human gene, KLHL31, from a human embryonic heart cDNA library. The cDNA of KLHL31 is 5743 bp long, encoding a protein product of 634 amino acids containing a BTB domain. The protein is highly conserved across different species. Western blot analysis indicates that the KLHL31 protein is abundantly expressed in both embryonic skeletal and heart tissue. In COS-7 cells, KLHL31 proteins are localized to both the nucleus and the cytoplasm. In primary cultures of nascent mouse cardiomyocytes, the majority of endogenous KLHL31 proteins are localized to the cytoplasm. KLHL31 acts as a transcription repressor when fused to GAL4 DNA-binding domain and deletion analysis indicates that the BTB domain is the main region responsible for this repression. Overexpression of KLHL31 in COS-7 cells inhibits the transcriptional activities of both the TPA-response element (TRE) and serum response element (SRE). KLHL31 also significantly reduces JNK activation leading to decreased phosphorylation and protein levels of the JNK target c-Jun in both COS-7 and Hela cells. These results suggest that KLHL31 protein may act as a new transcriptional repressor in MAPK/JNK signaling pathway to regulate cellular functions.

• 생명과학회지
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• 제20권6호
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• pp.853-860
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• 2010

DevSR two-component system은 Mycobacterium smegmatis의 redox sensing에 관련된 주요한 regulatory system이다. DevSR system은 DevS histidine kinase와 DevR response regulator로 구성되어 있다. 저산소 조건에서 DevS histidine kinase는 활성화되어 DevR response regulator를 인산화 시키고, 인산화된 DevR response regulator는 DevR regulon의 transcriptional activator로 작용한다. DevS의 kinase activity는 DevS의 N-terminal에 위치한 GAF domain에 존재하는 heme의 ligand-binding state에 의해 결정된다. 본 연구에서는 C-terminal kinase domain의 redox-responsive cysteine (C547)이 DevS kinase activity의 redox-dependent control과 연관이 있음을 밝혔다. 산소가 존재할 때, C547 residue 사이의 disulfide bond의 형성은 DevS kinase activity를 불활성화 시킨다. $\beta$-mercaptoethanol과 dithiothreitol과 같은 환원제를 이용하여 산화된 DevS를 환원시켰을 때, DevS kinase activity가 복원된 것이 관찰되었다. 또한, C547을 alanine으로 치환했을 때, M. smegmatis의 DevS의 sensory 기능을 부분적으로 손상되는 것이 complementation 실험을 통해 in vivo 상에서 증명되었다.

• Interdisciplinary Bio Central
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• 제4권4호
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• pp.12.1-12.6
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• 2012

The advanced glycation endproducts receptor (AGE-R) is a signal transduction receptor for multiligand such as S100b and AGEs. S100b has been demonstrated to activate various cells with important links to atherosclerosis initiation and progression including endothelial cells, and smooth muscle cells via AGE-R, triggering activation of multiple signaling cascades through its cytoplasmic domain. Many studies have suggested AGE-R might even participate in the cardiovascular complications involved in the pathogenesis of type I diabetes. Recently, Small Ubiquitin-Related Modifier 1 (SURM-1 also known as SUMO-1) has been recognized as a protein that plays an important role in cellular post-translational modifications in a variety of cellular processes, such as transport, transcriptional, apoptosis and stability. Computer Database search with SUMOplot Analysis program identified the five potential SURMylation sites in human AGE-R: K43, K44, K123, and K273 reside within the extracellular domain of AGE-R, and lastly K374 resides with the cytosolic domain of AGE-R. The presence of the consensus yKXE motif in the AGE-R strongly suggests that AGE-R may be regulated by SURMylation process. To test this, we decided to determine if AGE-R is SURMylated in living vascular cell system. S100b-stimulated murine aortic vascular smooth muscle cells were used for western blot analysis with relevant antibodies. Taken together, bioinformatics database search and molecular biological approaches suggested AGE-R is SURMylated in living cardiovascular cell system. Whilst SURMylation and AGE-R undoubtedly plays an important role in the cardiovascular biology, it remains unclear as to the exact nature of this contribution under both physiological and pathological conditions.

• Molecules and Cells
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• 제40권4호
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• pp.299-306
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• 2017

The transcriptional activator AphB has been implicated in acid resistance and pathogenesis in the food borne pathogens Vibrio vulnificus and Vibrio cholerae. To date, the full-length AphB crystal structure of V. cholerae has been determined and characterized by a tetrameric assembly of AphB consisting of a DNA binding domain and a regulatory domain (RD). Although acidic pH and low oxygen tension might be involved in the activation of AphB, it remains unknown which ligand or stimulus activates AphB at the molecular level. In this study, we determine the crystal structure of the AphB RD from V. vulnificus under aerobic conditions without modification at the conserved cysteine residue of the RD, even in the presence of the oxidizing agent cumene hydroperoxide. A cysteine to serine amino acid residue mutant RD protein further confirmed that the cysteine residue is not involved in sensing oxidative stress in vitro. Interestingly, an unidentified small molecule was observed in the inter-subdomain cavity in the RD when the crystal was incubated with cumene hydroperoxide molecules, suggesting a new ligand-binding site. In addition, we confirmed the role of AphB in acid tolerance by observing an aphB-dependent increase in cadC transcript level when V. vulnificus was exposed to acidic pH. Our study contributes to the understanding of the AphB molecular mechanism in the process of recognizing the host environment.

• Tuberculosis and Respiratory Diseases
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• 제50권1호
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• pp.52-66
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• 2001

연구배경 : 폐상피세포가 능동적으로 IL-8을 분비한다는 것은 주지의 사실이다. NF-${\kappa}B$는 IL-8 발현 조절에 있어서 가장 중요한 역할을 담당하는 전사인자이다. Triptolide는 최근 밝혀진 NF-${\kappa}B$ 억제제로서 중국한약제인 뇌공등 (Tripterygium Wilfordii)에서 추출된약제이다. 연자들은 새로운 NF-${\kappa}B$ 억제제인 triptolide가 폐상피세포에서 NF-${\kappa}B$ 의존성 IL-8 유전자의triptolide가 염증성 폐질환에서 새로운 치료제로서의 가능성을 확인하기 위하여 본 연구를 시행하였다. 방법 : 폐상피세포로서 A549 사용하였고 triptolide는 미국의 Pharamagenesis(Palo Alto, CA)사로부터 제공받았다. NF-${\kappa}B$ 활성유도물질로는 IL-$1{\beta}$(R&D)와 PMA(Sigma)를 이용하였다. IL-8 유전자의 발현은 RT-PCR과 ELISA를 이용하여 측정 하였다. NF-${\kappa}B$의존성 IL-8 유전자의 전사활성을 평가하기 위하여는 IL-8 NF-${\kappa}B$ luciferase construct를 안정적으로 유전자주입한 A549 IL-8 NF-${\kappa}B$ luciferase 세포주를 제조해 사용하였고 NF-${\kappa}B$ DNA 결합은 electromobility shift assay(EMSA)를 이용하였다. p65 전사활성을 assay하기 위해서는 Gal4-p65 fusion protein expression system을 유전자주입과 luciferase assay를 통하여 시행하였다. Transcriptional coactivator의 역할을 규명 하가 위하여서는 CBP(CREB-binding protein)와 SRC-1(steroid receptor coactivator-1) 발현 벡터를 유전자 주입하고 luciferase assay를 이용하여 확인하였다. 결과 : Luciferase assay로 triptolide가 PMA와 IL-$1{\beta}$자극에 의한 IL-8 NF-${\kappa}B$ 활성을 의미있게 감소시킴을 확인하였다. IL-8 ELISA와 RT-PCR로 triptolide가 PMA와 IL-$1{\beta}$ 자극에 의해 유도되는 IL-8 발현을 각각 단백질과 mRNA 수준에서 억제함을 관찰하였다. Triptolide가 PMA와 IL-$1{\beta}$에 의한 IL-8 NF-${\kappa}B$의 전사활성을 억제시킨 반면 EMSA와 $I{\kappa}B{\alpha}$ Western blot을 이용한 실험에서는 triptolide가 NF-${\kappa}B$ DNA 결합과 $I{\kappa}B{\alpha}$의 분해에 전혀 영향을 미치지 못함을 확인하였다. 이러한 전사활성 억제와 DNA 결합 간의 불일치의 원인으로서는 DNA 결합 이후에 발생하는 핵내 에서의 transactivation에 triptolide가 영향을 미치리라고 생각되어 p65 transactivation study를 Gal4-p65T A(p65의 transactivation domain) fusion protein 발현 시스템과 luciferase assay를 이용하여 시행한 결과 triptolide가 p65 transactivation을 억제함으로써 NF-${\kappa}B$를 억제함을 확인하였다. 그러나 CBP나 SRC-1과 같은 coactivator의 역할을 규명하기 위한 유전자주입 실험에서 triptolide에 의한 p65 transactivation 억제에 대해 CBP나 SRC-1의 과발현이 별다른 영향을 미치지 못하였다. 결론 : Triptolide는 폐상피세포에서 NF-${\kappa}B$ 의존성 IL-8 유전자의 전사활성을 억제하고 그 기전은 $I{\kappa}B{\alpha}$ 경로가 아닌 핵내에서의 p65 transactivation 억제에 의해 발생하며 이에는 CBP나 SRC-1과 같은 coactivator가 관여하지 않음을 확인하였다.

• Journal of Microbiology
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• 제37권3호
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• pp.154-158
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• 1999

The arginine residue at position 243 (Arg 243) of the yeast transcription factor, Gcn4p, is invariably conserved among bZIP transcription factors. Using site-directed oligonucleotide saturation mutagenesis involving two-step polymerase chain reaction (PCR) amplification, random mutations were successfully introduced at the codon of 243 in the basic domain of Gcn4p. This mutant library was transformed ito Gcn4p defective yeast strain and selected for the transcriptionally active colonies. All colonies which were transcriptionally active had arginines in the codon 243. In this study, the strand preference by Taq polymerase during mutagenesis was also tested. Oligonucleotides were specially designed to test whether or not the polymerase was preferred using the strand as a template. A population of randomly mutated products were cloned into an appropriate vector and characterized by DNA sequencing analysis. Saturation mutagenesis which was performed efficiently by this method revealed a strong bias in terms of strand preference of Taq polymerase by an approximate ratio of 3 to 1 in this study.

• BMB Reports
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• 제48권8호
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• pp.431-437
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• 2015

Notch signaling plays a pivotal role in cell fate determination, cellular development, cellular self-renewal, tumor progression, and has been linked to developmental disorders and carcinogenesis. Notch1 is activated through interactions with the ligands of neighboring cells, and acts as a transcriptional activator in the nucleus. The Notch1 intracellular domain (Notch1-IC) regulates the expression of target genes related to tumor development and progression. The Notch1 protein undergoes modification after translation by posttranslational modification enzymes. Phosphorylation modification is critical for enzymatic activation, complex formation, degradation, and subcellular localization. According to the nuclear cycle, Notch1-IC is degraded by E3 ligase, FBW7 in the nucleus via phosphorylation-dependent degradation. Here, we summarize the Notch signaling pathway, and resolve to understand the role of phosphorylation in the regulation of Notch signaling as well as to understand its relation to cancer. [BMB Reports 2015; 48(8): 431-437]

• Journal of Pharmaceutical Investigation
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• 제34권2호
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• pp.101-106
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• 2004

This work aims at examining the cellular uptake behavior of poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles derivatized with a protein transduction domain (PTD) using HeLa cells. For this purpose, $Tat_{49-57}$ peptide derived from transcriptional activation (Tat) protein of HIV type-1 was covalently conjugated to the terminal end of PLGA. Nanoparticles were ten prepared with the $Tat_{49-57}-PLGA$ conjugates by a spontaneous phase inversion method. The prepared particles had a mean diameter of ca. 84 nm, as measured by dynamic light scattering. The interaction of the Tat-PLGA nanoparticles with cells was examined by using confocal laser scanning microscopy. It was found tat Tat-PLGA nanoparticles incubated with HeLa cells could efficiently translocate into cytoplasm, while plain PLGA nanoparticles showed negligible cellular uptake. In addition, even at $4^{\circ}C$ or in the presence of sodium azide significant cellular internalization of Tat-PLGA nanoparticles was still observed. These results indicate that a non-endocytotic translocation mechanism might be involved in the cellular uptake of Tat-PLGA nanoparticles.

• 한국발생생물학회지:발생과생식
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• 제14권2호
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• pp.91-97
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• 2010

FOXL2는 winged-helix/forkhead(FH) 도메인 전사인자로서 FOXL2 유전자에 돌연변이가 발생할 경우 blepharophimosis-ptosis-epicanthus inversus syndrome이라 불리는 BPES 질병이 유발되게 된다. BPES는 상염색체 우성인 유전적 질환이다. BPES type I의 환자는 조기난소부전증(POF)과 안검하수 증상이 함께 나타나는 반면, BPES type II의 경우 안검하수 및 소안검 등 안면기형만이 유발된다. FOXL2 단백질이 결여된 난소에서 granulosa 세포의 분화가 멈추는 것으로 보아 FOXL2가 정상적인 난소의 folliculogenesis에 필수적인 역할을 하고 있음을 시사한다. 이전의 연구 결과에서, 본 연구진은 FOXL2와 상호작용하는 단백질에 대한 스크리닝을 통해 스테로이드 합성효소인 CYP19 전사활성에 영향을 미치는 steroidogenic factor-1(SF-1)을 동정하였다. 이번 연구를 통해 FOXL2가 CYP19의 전사를 향상시키고, SF-1에 의한 CYP19의 전사를 더욱 촉진시킨다는 것을 증명하였다. 이와 반대로, BPES 타입 I과 II에서 발견된 FOXL2의 돌연변이형들은 SF-1에 의해 증가된 CYP19의 전사활성을 향상시키는 능력이 감소함을 보여주었다. 본 실험을 통해 FOXL2 돌연변이에 의해 유발되어지는 BPES 질환의 병리생리학적인 이해에 대해 도움을 줄 수 있는 FOXL2의 야생형과 돌연변이형 사이의 서로 다른 기능적인 차이점을 규명하였다.

• 한국균학회소식:학술대회논문집
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• 한국균학회 2014년도 춘계학술대회 및 임시총회
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• pp.27-28
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• 2014

Beauveria bassiana (Cordycipitaceae, Hypocreales, Ascomycota) is an anamorphic fungus having a potential to be used as a biological control agent because it parasitizes a wide range of arthropod hosts including termites, aphids, beetles and many other insects. A number of bioactive secondary metabolites (SMs) have been isolated from B. bassiana and functionally verified. Among them, beauvericin and bassianolide are cyclic depsipeptides with antibiotic and insecticidal effects belonging to the enniatin family. Non-ribosomal peptide synthetases (NRPSs) play a crucial role in the synthesis of these secondary metabolites. NRPSs are modularly organized multienzyme complexes in which each module is responsible for the elongation of proteinogenic and non-protein amino acids, as well as carboxyl and hydroxyacids. A minimum of three domains are necessary for one NRPS elongation module: an adenylation (A) domain for substrate recognition and activation; a tholation (T) domain that tethers the growing peptide chain and the incoming aminoacyl unit; and a condensation (C) domain to catalyze peptide bond formation. Some of the optional domains include epimerization (E), heterocyclization (Cy) and oxidation (Ox) domains, which may modify the enzyme-bound precursors or intermediates. In the present study, we analyzed genomes of B. bassiana and its allied species in Hypocreales to verify the distribution of NRPS-encoding genes involving biosynthesis of beauvericin and bassianolide, and to unveil the evolutionary processes of the gene clusters. Initially, we retrieved completely or partially assembled genomic sequences of fungal species belonging to Hypocreales from public databases. SM biosynthesizing genes were predicted from the selected genomes using antiSMASH program. Adenylation (A) domains were extracted from the predicted NRPS, NRPS-like and NRPS-PKS hybrid genes, and used them to construct a phylogenetic tree. Based on the preliminary results of SM biosynthetic gene prediction in B. bassiana, we analyzed the conserved gene orders of beauvericin and bassianolide biosynthetic gene clusters among the hypocrealean fungi. Reciprocal best blast hit (RBH) approach was performed to identify the regions orthologous to the biosynthetic gene cluster in the selected fungal genomes. A clear recombination pattern was recognized in the inferred A-domain tree in which A-domains in the 1st and 2nd modules of beauvericin and bassianolide synthetases were grouped in CYCLO and EAS clades, respectively, suggesting that two modules of each synthetase have evolved independently. In addition, inferred topologies were congruent with the species phylogeny of Cordycipitaceae, indicating that the gene fusion event have occurred before the species divergence. Beauvericin and bassianolide synthetases turned out to possess identical domain organization as C-A-T-C-A-NM-T-T-C. We also predicted precursors of beauvericin and bassianolide synthetases based on the extracted signature residues in A-domain core motifs. The result showed that the A-domains in the 1st module of both synthetases select D-2-hydroxyisovalerate (D-Hiv), while A-domains in the 2nd modules specifically activate L-phenylalanine (Phe) in beauvericin synthetase and leucine (Leu) in bassianolide synthetase. antiSMASH ver. 2.0 predicted 15 genes in the beauvericin biosynthetic gene cluster of the B. bassiana genome dispersed across a total length of approximately 50kb. The beauvericin biosynthetic gene cluster contains beauvericin synthetase as well as kivr gene encoding NADPH-dependent ketoisovalerate reductase which is necessary to convert 2-ketoisovalarate to D-Hiv and a gene encoding a putative Gal4-like transcriptional regulator. Our syntenic comparison showed that species in Cordycipitaceae have almost conserved beauvericin biosynthetic gene cluster although the gene order and direction were sometimes variable. It is intriguing that there is no region orthologous to beauvericin synthetase gene in Cordyceps militaris genome. It is likely that beauvericin synthetase was present in common ancestor of Cordycipitaceae but selective gene loss has occurred in several species including C. militaris. Putative bassianolide biosynthetic gene cluster consisted of 16 genes including bassianolide synthetase, cytochrome P450 monooxygenase, and putative Gal4-like transcriptional regulator genes. Our synteny analysis found that only B. bassiana possessed a bassianolide synthetase gene among the studied fungi. This result is consistent with the groupings in A-domain tree in which bassianolide synthetase gene found in B. bassiana was not grouped with NRPS genes predicted in other species. We hypothesized that bassianolide biosynthesizing cluster genes in B. bassiana are possibly acquired by horizontal gene transfer (HGT) from distantly related fungi. The present study showed that B. bassiana is the only species capable of producing both beauvericin and bassianolide. This property led to B. bassiana infect multiple hosts and to be a potential biological control agent against agricultural pests.