• Journal of Microbiology and Biotechnology
• /
• 제20권2호
• /
• pp.301-310
• /
• 2010

Bacillus subtilis strains produce a broad spectrum of bioactive peptides. The lipopeptide surfactin belongs to one well-known class, which includes amphiphilic membrane-active biosurfactants and peptide antibiotics. Both the srfA promoter and the ComP-ComA signal transduction system are an important part of the factor that results in the production of surfactin. Bs-M49, obtained by means of low-energy ion implantation in wild-type Bs-916, produced significantly lower levels of surfactin, and had no obvious effects against R. solani. Occasionally, we found strain Bs-M49 decreased spore formation and the development of competence. Blast comparison of the sequences from Bs-916 and M49 indicate that there is no difference in the srfA operon promoter PsrfA, but there are differences in the coding sequence of the comA gene. These differences result in three missense mutations within the M49 ComA protein. RT-PCR analyses results showed that the expression levels of selected genes involved in competence and sporulation in both the wild-type Bs-916 and mutant M49 strains were significantly different. When we integrated the comA ORF into the chromosome of M49 at the amyE locus, M49 restored hemolytic activity and antifungal activity. Then, HPLC analyses results also showed the comA-complemented strain had a similar ability to produce surf actin with wild-type strain Bs-916. These data suggested that the mutation of three key amino acids in ComA greatly affected the biological activity of Bacillus subtilis. ComA protein 3D structure prediction and motif search prediction indicated that ComA has two obvious motifs common to response regulator proteins, which are the N-terminal response regulator receiver motif and the C-terminal helix-turn-helix motif. The three residues in the ComA N-terminal portion may be involved in phosphorylation activation mechanism. These structural prediction results implicate that three mutated residues in the ComA protein may play an important role in the formation of a salt-bridge to the phosphoryl group keeping active conformation to subsequent regulation of the expression of downstream genes.

• International Journal of Molecular Medicine
• /
• 제43권5호
• /
• pp.2177-2186
• /
• 2019

The epidemiological, animal and cell effects of plant metabolites suggest versatile health benefits of flavonoids. However, whether flavonoids affect the deleterious biological activity of oxygenated cholesterol molecules remains to be elucidated. The present study investigated the effects of 4'-O-methylalpinumisoflavone (mAI) isolated from Maclura tricuspidata (Cudrania tricuspidata) on the 27-hydroxycholesterol (27OHChol)-induced activation of monocytes/macrophages using human THP-1 cells. mAI dose-dependently impaired the expression of C-C motif chemokine ligand (CCL)2 chemokine and the migration of monocytic cells enhanced by 27OHChol. mAI downregulated the surface and cellular levels of CD14 and inhibited the release of soluble CD14. This isoflavone significantly weakened the lipopolysaccharide responses that were enhanced in the presence of 27OHChol, and inhibited the transcription and secretion of the active gene product of matrix metalloproteinase-9. mAI also suppressed the expression of C-C motif chemokine receptor 5 ligands, including CL3 and CCL4, and M1-phenotype markers induced by 27OHChol. Furthermore, mAI impaired phosphorylation of the nuclear factor-κB p65 subunit without affecting the phosphorylation of Akt. These results indicate that mAI inhibits the activation of monocytes/macrophages to the immunostimulatory phenotype in a milieu rich in 27OHChol, suggesting potential benefits of the flavonoid for the treatment of diseases in which the pathogenesis is linked to 27OHChol-induced inflammatory responses.

• Molecules and Cells
• /
• 제39권11호
• /
• pp.827-833
• /
• 2016

Regulator of calcineurin 1 (RCAN1) binds to calcineurin through the PxIxIT motif, which is evolutionarily conserved. SP repeat phosphorylation in RCAN1 is required for its complete function. The specific interaction between RCAN1 and calcineurin is critical for calcium/calmodulin-dependent regulation of calcineurin serine/threonine phosphatase activity. In this study, we investigated two available deletion rcan-1 mutants in Caenorhabditis elegans, which proceed differently for transcription and translation. We found that rcan-1 may be required for calcineurin activity and possess calcineurin-independent function in body growth and egg-laying behavior. In the genetic background of enhanced calcineurin activity, the rcan-1 mutant expressing a truncated RCAN-1 which retains the calcineurin-binding PxIxIT motif but misses SP repeats stimulated growth, while rcan-1 lack mutant resulted in hyperactive egg-laying suppression. These data suggest rcan-1 has unknown functions independent of calcineurin, and may be a stimulatory calcineurin regulator under certain circumstances.

• BMB Reports
• /
• 제44권3호
• /
• pp.199-204
• /
• 2011

Ephrin signaling is involved in various morphogenetic events, such as axon guidance, hindbrain segmentation, and angiogenesis. We conducted a yeast two-hybrid screen using the intracellular domain (ICD) of EphrinB1 to gain biochemical insight into the function of the EphrinB1 ICD. We identified the transcriptional co-repressor xTLE1/Groucho as an EphrinB1 interacting protein. Whole-mount in situ hybridization of Xenopus embryos confirmed the co-localization of EphrinB1 and a Xenopus counterpart to TLE1, xTLE4, during various stages of development. The EphrinB1/xTLE4 interaction was confirmed by co-immunoprecipitation experiments. Further characterization of the interaction revealed that the carboxy-terminal PDZ binding motif of EphrinB1 and the SP domain of xTLE4 are required for binding. Additionally, phosphorylation of EphrinB1 by a constitutively activated fibroblast growth factor receptor resulted in loss of the interaction, suggesting that the interaction is modulated by tyrosine phosphorylation of the EphrinB1 ICD.

• IMMUNE NETWORK
• /
• 제5권3호
• /
• pp.144-149
• /
• 2005

Background: Fc receptor-mediated phagocytosis is a complex process involving the activation of kinases and phosphatases. FcgammaRIIB has been known to transduces inhibitory signals through an immunoreceptor tyrosine-based inhibitory motif (ITIM) in cytoplasmic domains. In this study, we examined the involvement of inositol-phosphatase in the Fc receptor-mediated phagocytosis. Methods: J774 cells were infected using vaccinia viral vector containing SH2 domain-containing inositol-phosphatase (SHIP) cDNA and stimulated with the sensitized sheep red blood cells. Results: Stimulation of J774 cells induced the tyrosine phosphorylation of SHIP which was maximal at 5 minutes. Phosphatidylinositol-3 (PI-3) kinase inhibitor (wortmannin) inhibits J774 cell phagocytosis of sensitized sheep red blood cells in a dose-dependent manner. Heterologious expression of SHIP in J774 cells inhibits phagocytosis of sensitized sheep red blood cells in a dose-dependency manner, but catalytically dead mutants of SHIP has no effect on phagocytosis. Conclusion: These results strongly suggest that the active signals mediated by PI-3 kinase are opposed by inhibitory signals through SHIP in the regulation of Fc receptor-mediated phagocytosis.

• BMB Reports
• /
• 제43권6호
• /
• pp.407-412
• /
• 2010

Homeodomain (HD) is a highly conserved DNA-binding domain composed of helix-turn-helix motif. Drosophila Vnd (Ventral nervous system defective) containing HD acts as a regulator to either enhance or suppress gene expression upon binding to its target sequence. In this study, kinetic analysis of Vnd binding to DNA was performed. The result demonstrates that DNA-binding affinity of the recombinant protein containing HD and NK2-specific domain (NK2-SD) was higher than that of the full-length Vnd. To access whether phosphorylation sites within HD and NK2-SD affect the interaction of the protein with the target sequence, alanine substitutions were introduced. The result shows that S631A mutation within NK2-SD does not contribute significantly to the DNA-binding affinity. However, S571A and T600A mutations within HD showed lower affinity for DNA binding. In addition, DNA-binding analysis using embryonic nuclear protein also demonstrates that Vnd interacts with other nuclear proteins, suggesting the existence of Vnd as a complex.

• Journal of Applied Biological Chemistry
• /
• 제64권2호
• /
• pp.185-192
• /
• 2021

건선(Psoriasis)은 IL-6, CXCL8, TNF-α 및 IFN-γ뿐만 아니라 Th17 세포에서 분비되는 IL-17A 등 다양한 염증성 사이토카인에 의해 표피의 과증식(hyperkeratosis) 및 만성적 염증(inflammation)이 유발되는 난치성 피부 질환이다. 소목(Caesalpinia sappan L.)의 유효성분으로 알려진 브라질린(brazilin)은 항산화, 항염증 및 피부 장벽 개선 등의 효능이 알려졌다. 특히, tumor necrosis factor (TNF)-α 자극 HaCaT 각질형성세포 모델에서 브라질린의 건선 치료 소재 가능성을 보여주었다. 그러나, 직접적인 건선 유발 인자인 C-C motif chemokine ligand (CCL) 20의 조절은 전혀 보고되지 않았다. 따라서, 본 연구에서는 건선 유사 모델을 활용해 CCL20 발현 조절 여부 및 그 기작에 대해 규명하고자 하였다. IL-17A로 자극된 HaCaT 세포에서 브라질린은 CCL20, CXCL8 발현 및 signal transducer and transcription (STAT)3 인산화를 유의하게 억제하였다. 또한, 브라질린은 TNF-α/IL-17A/IFN-γ 3종 사이토카인으로 처리된 조건에서도 STAT3 인산화를 억제하며 염증성 분자(CXCL8, CCL20, IL-1, IL-6, 및 TNF-α)의 발현을 하향 조절하였다. 마지막으로 브라질린은 TNF-α/IL-17A/IFN-γ로 자극된 건선 유사 환경에서 피부 장벽 개선에도 유의적인 영향을 미쳤다. 위 결과들을 통해 우리는 궁극적으로 브라질린이 STAT3 인산화 억제를 통해 CCL20 발현을 하향 조절하며, 건선 유발 사이토카인들의 발현 또한 억제함을 알 수 있었다. 향후, 건선 동물모델 및 임상시험을 통해 브라질린의 건선 개선에 대한 효능이 검증된다면, 건선 환자에게 잠재적인 치료 물질로 사용될 수 있을 것으로 기대된다.

• IMMUNE NETWORK
• /
• 제2권2호
• /
• pp.72-78
• /
• 2002

Background: The precise mechanism by which CTLA-4 regulates T cell immune responses is still not fully understood. Previously we proposed that CTLA-4 could downregulate T cell function by modulating a signaling cascade initiated from the T cell receptor complex. The evidence for this notion comes from our findings that CTLA-4 associated with the T cell receptor zeta (TCR zeta) chain, and hence regulated TCR zeta phosphorylation by co-associated SHP-2 tyrosine phosphatase (1). In this report, we investigated whether any other signaling molecules could be involved in the CTLA-4 signaling pathway. Methods: We have taken biochemical approaches, such as immunoprecipitation followed by autoradiography or immunoblotting, to identify the molecules associated with CTLA-4. To perform these assays, we used activated primary T cells and ectopically transfected 293 cells. Various truncation mutants of CTLA-4 were used to map the interaction site on CTLA-4. Results: We found that in addition to TCR zeta and SHP-2, a recently cloned small adaptor molecule, SAP (SLAM-associated protein), was also able to associate with CTLA-4. We identified the domain of SAP association in CTLA-4 being a motif involving GVYVKM. This motif has been previously found to bind SHP-2 through its phosphorylated tyrosine interaction with SH-2 domain of SHP-2. Indeed, co-expression of SAP and SHP-2 reduced their binding to CTLA-4 significantly, suggesting that SAP and SHP-2 compete for the common binding site, GVYVKM. Thus, by blocking SHP-2 recruitment SAP could function as a negative regulator of CTLA-4. Conclusion: Taken together, our data suggest the existence of complicate signaling cascade in regulating CTLA-4 function, and further provide evidence that SAP can act either as a positive or negative regulator depending on the nature of the associating receptors.

• The Korean Journal of Physiology and Pharmacology
• /
• 제15권1호
• /
• pp.31-36
• /
• 2011

To understand the roles of purinergic receptors and cellular molecules below the receptors in the vascular inflammatory response, we determined if extracellular nucleotides up-regulated chemokine expression in vascular smooth muscle cells (VSMCs). Human aortic smooth muscle cells (AoSMCs) abundantly express $PSY_1$, $PSY_6$, and $PSY_{11}$ receptors, which all respond to extracellular nucleotides. Exposure of human AoSMCs to $NAD^+$, an agonist of the human $PSY_{11}$ receptor, and $NADP^+$ as well as ATP, an agonist for $PSY_1$ and $PSY_{11}$ receptors, caused increase in chemokine (C-C motif) ligand 2 gene (CCL2) transcript and CCL2 release; however, UPT did not affect CCL2 expression. CCL2 release by $NAD^+$ and $NADP^+$ was inhibited by a concentration dependent manner by suramin, an antagonist of P2-purinergic receptors. $NAD^+$ and $NADP^+$ activated protein kinase C and enhanced phosphorylation of mitogen-activated protein kinases and Akt. $NAD^+$- and $NADP^+$-mediated CCL2 release was significantly attenuated by SP6001250, U0126, LY294002, Akt inhibitor IV, RO318220, GF109203X, and diphenyleneiodium chloride. These results indicate that extracellular nucleotides can promote the proinflammatory VSMC phenotype by up-regulating CCL2 expression, and that multiple cellular elements, including phosphatidylinositol 3-kinase, Akt, protein kinase C, and mitogen-activated protein kinases, are involved in that process.

• 대한의생명과학회지
• /
• 제19권3호
• /
• pp.266-269
• /
• 2013

Hox genes encode transcription factors important for anterior-posterior body patterning at early stages of embryonic development. However, the precise mechanisms by which signal pathways are stimulated to regulate Hox gene expression are not clear. In the previous study, protein kinase B alpha (Akt1) has been identified as a putative upstream regulator of Hox genes, and Akt1 has shown to regulate Gcn5, a prototypical histone acetyltransferase (HAT), in a negative way in mouse embryonic fibroblast (MEF) cells. Since the activity of HAT such as the CBP/p300, and PCAF (a Gcn5 homolog), was down-regulated by Akt through a phosphorylation at the Akt consensus substrate motif (RXRXXS/T), the amino acid sequence of Gcn5 protein was analyzed. Mouse Gcn5 contains an Akt consensus substrate motif as RQRSQS sequence while human Gcn5 does not have it. In order to see whether Akt1 directly binds to Gcn5, immunoprecipitation with anti-Akt1 antibody was carried out in wild-type (WT) mouse embryonic fibroblast (MEF) cells, and then western blot analysis was performed with anti-Akt1 and anti-Gcn5 antibodies. Gcn5 protein was detected in the Akt1 immunoprecipitated samples of MEFs. This result demonstrates that Akt1 directly binds to Gcn5, which might have contributed the down regulation of the 5' Hoxc gene expressions in wild type MEF cells.