• Journal of Microbiology and Biotechnology
• /
• v.27 no.2
• /
• pp.357-364
• /
• 2017

In this study, we aimed to generate a series of versatile tagging plasmids that can be used in diverse molecular biological studies of the fungal pathogen Cryptococcus neoformans. We constructed 12 plasmids that can be used to tag a protein of interest with a GFP, mCherry, $4{\times}FLAG$, or $6{\times}HA$, along with nourseothricin-, neomycin-, or hygromycin-resistant selection markers. Using this tagging plasmid set, we explored the adenylyl cyclase complex (ACC), consisting of adenylyl cyclase (Cac1) and its associated protein Aca1, in the cAMP-signaling pathway, which is critical for the pathogenicity of C. neoformans. We found that Cac1-mCherry and Aca1-GFP were mainly colocalized as punctate forms in the cell membrane and non-nuclear cellular organelles. We also demonstrated that Cac1 and Aca1 interacted in vivo by co-immunoprecipitation, using $Cac1-6{\times}HA$ and $Aca1-4{\times}FLAG$ tagging strains. Bimolecular fluorescence complementation further confirmed the in vivo interaction of Cac1 and Aca1 in live cells. Finally, protein pull-down experiments using $aca1{\Delta}$::ACA1-GFP and $aca1{\Delta}$::ACA1-GFP $cac1{\Delta}$ strains and comparative mass spectrometry analysis identified Cac1 and a number of other novel ACC-interacting proteins. Thus, this versatile tagging plasmid system will facilitate diverse mechanistic studies in C. neoformans and further our understanding of its biology.

• Proceedings of the Korea Information Processing Society Conference
• /
• 2022.11a
• /
• pp.690-692
• /
• 2022

기존의 질병 관련 연구들은 대부분 유의미하게 변화되는 유전자들을 찾아내고(Differentially Expressed Genes, DEGs), 이들이 연관된 생물학적 패스웨이(biological pathway)를 찾아내는 방향으로 이루어졌다. 더불어 miRNA(microRNA)가 많은 mRNA 의 발현을 조절하며, 실제 면역, 대사 및 세포 사멸을 포함한 여러 필수 생리학적 및 질병에 매우 중요한 역할을 한다고 밝혀지며, 바이오 마커로써의 miRNA 를 찾아내고자 하는 연구가 활발히 진행되기 시작하였다. 하지만 mRNA 나 miRNA 의 독립적인 연구만으로는 명확한 질병과의 연관성이나 기능을 이해하기에는 어려움이 있다. 따라서 본 연구에서는 질병 상태에서 유의미하게 변화되는 miRNA 와 이러한 miRNA 에 의해 조절되는 mRNA 를 함께 고려하여 분석함으로써, 실제 질병의 발병 원인이 되는 생물학적 패스웨이나 메커니즘을 밝히고자 하였다. 또한, miRNA 와 mRNA 의 연관성을 찾기 위해, PPI(protein-protein interaction) 네트워크에 기반을 둔 RWR(Random Walk with Restart Algorithm)를 적용하여, 직접적 연관성뿐 아니라, 유전자 간의 숨겨진 간접적인 패스웨이를 고려하여 분석하기 위한 웹 기반 시스템을 개발하였다. 이 시스템은 mRNA-miRNA 를 함께 고려한 통합 분석을 통해 숨겨진 질병의 메커니즘을 이해하고 치료 방법을 찾아내는 데 크게 공헌할 것이다.

• BMB Reports
• /
• v.29 no.3
• /
• pp.183-191
• /
• 1996

DNA fragments, homologous to the dTDP-D-glucose 4,6-dehydratase gene, obtained from the genomic DNA of Streptomyces antibioticus $T\ddot{u}99$, a producer of the unusual macrolide antibiotic chlorothricin, were cloned and sequenced. This dehydratase gene was designated as oxil. The coding region of the oxil gene is composed of 987 bp, and analysis of the DNA sequence data reveals sequences for the gene products of 329 amino acids (molecular weight of 36,037). The deduced amino acids are 59% identical to the StrE, dTDP-D-glucose 4,6-dehydratase from the streptomycin pathway. The oxil's function was examined by expressing it in E. coli using the T7 RNA polymerase/promoter system (pRSET) to produce an active fusion protein including a his tag. This enzyme shows specificity of substrate, specific only to dTDP-D-glucose.

• BMB Reports
• /
• v.45 no.3
• /
• pp.183-188
• /
• 2012

Participates in actin remodeling through Rac and receptor endocytosis via Rab5. Here, we used yeast two-hybrid system with Eps8 as bait to screen a human brain cDNA library. ITSN2 was identified as the novel binding factor of Eps8. The interaction between ITSN2 and Eps8 was demonstrated by the in vivo co-immunoprecipitation and colocalization assays and the in vitro GST pull-down assays. Furthermore, we mapped the interaction domains to the region between amino acids 260-306 of Eps8 and the coiled-coil domain of ITSN2. In addition, protein stability assays and immunofluorescence analysis showed ITSN2 overexpression induced the degradation of Eps8 proteins, which was markedly alleviated with the lysosome inhibitor NH4Cl treatment. Taken together, our results suggested ITSN2 interacts with Eps8 and stimulates the degradation of Eps8 proteins.

• The Plant Pathology Journal
• /
• v.21 no.2
• /
• pp.149-157
• /
• 2005

A novel rice (Oryza sativa L.) gene, homologous to Arabidopsis pathogenesis-related NDR1 gene, was cloned from cDNA library prepared from 30 min Magnaporthe grisea -treated rice seedling leaves, and named as OsNDR1. OsNDR1 encoded a 220-aminoacid polypeptide and was highly similar to the Arabidopsis AtNDR1 protein. OsNDR1 is a plasma membrane (PM)-localized protein, and presumes through sequence analysis and protein localization experiment. Overexpression of OsNDR1 promotes the expression of PBZ1 that is essential for the activation of defense/stressrelated gene. The OsNDR1 promoter did not respond significantly to treatments with either SA, PBZ, or ETP. Exogenously applied BTH induces the same set of SAR genes as biological induction, providing further evidence for BTH as a signal. Presumably, BTH is bound by a receptor and the binding triggers a signal transduction cascade that has an ultimate effect on transcription factors that regulate SAR gene expression. Thus OsNDR1 may act as a transducer of pathogen signals and/or interact with the pathogen and is indeed another important step in clarifying the component participating in the defense response pathways in rice.

• Proceedings of the Korean Society for Applied Microbiology Conference
• /
• 2005.06a
• /
• pp.230-232
• /
• 2005

The formation of insoluble aggregation of the recombinant kringle fragment of human apolipoprotein(a), rhLK8, in endoplasmic reticulum was identified as the rate-limiting step in the rhLK8 secretion in Saccharomyces cerevisiae. To analyze the protein secretion pathway, some of yeast genes closely related to protein secretion was rationally selected and their oligomer DNA were arrayed on the chip. The expression profiling of these genes during the induction of rhLK8 in fermentor fed-batch cultures revealed that several foldases including pdi1 gene were up-regulated in the early induction phase, whereas protein transport-related genes were up-regulated in the late induction phase. The coexpression of pdi1 gene increased rhLK8-folding capacity. Hence, the secretion efficiency of rhLK8 in the strain overexpressing pdi1 gene increased by 2-fold comparing in its parental strain. The oligomer DNA chip arrayed with minimum number of the genes selected in this study could be generally applicable to the monitoring system for the heterologous protein secretion and expression in Saccharomyces cerevisiae. With the optimization of fed-batch culture conditions and the alteration of genetic background of host, we obtained extracellular rhLK8 at higher yields than with Pichia pastoris systems, which was a 25-fold increased secretion level of rhLK8 compared to the secretion level at the initiation of this study.

• Asian Pacific Journal of Cancer Prevention
• /
• v.15 no.18
• /
• pp.7547-7552
• /
• 2014

The mir-155 family is not only involved in a diversity of cancers, but also as a regulator of the immune system. However, the evolutionary history of this family is still unclear. The present study indicates that mir-155 evolved independently with lineage-specific gain of miRNAs. In addition, arm switching has occurred in the mir-155 family, and alternative splicing could produce two different lengths of ancestral sequences, implying the alternative splicing can also drive evolution for intragenic miRNAs. Here we screened validated target genes and immunity-related proteins, followed by analyzation of the mir-155 family function by high-throughput methods like the gene ontology (GO) and Kyoto Eneyclopedin of Genes and Genemes (KEGG) pathway enrichment analysis. The high-throughput analysis showed that the CCND1 and EGFR genes were outstanding in being significantly enriched, and the target genes cebpb and VCAM1 and the protein SMAD2 were also vital in mir-155-related immune reponse activities. Therefore, we conclude that the mir-155 family is highly conserved in evolution, and CCND1 and EGFR genes might be potential targets of mir-155 with regard to progress of cancers, while the cebpb and VCAM1 genes and the protein SMAD2 might be key factors in the mir-155 regulated immune activities.

• Genomics & Informatics
• /
• v.21 no.1
• /
• pp.8.1-8.19
• /
• 2023

Cancer of the stomach is the second most frequent cancer-related death worldwide. The survival rate of patients with gastric cancer (GC) remains fragile. There is a requirement to discover biomarkers for prognosis approaches. Helicobacter pylori in the stomach is closely associated with the progression of GC. We identified the genes associated with poor/favorable prognosis in H. pylori-induced GC. Multivariate statistical analysis was applied on the Gene Expression Omnibus (GEO) dataset GSE54397 to identify differentially expressed miRNAs (DEMs) in gastric tissues with H. pylori-induced cancer compared with the H. pylori-positive with non-cancerous tissue. A protein interaction map (PIM) was built and subjected to DEMs targets. The enriched pathways and biological processes within the PIM were identified based on substantial clusters. Thereafter, the most critical genes in the PIM were illustrated, and their prognostic impact in GC was investigated. Considering p-value less than 0.01 and |Log2 fold change| as >1, five microRNAs demonstrated significant changes among the two groups. Gene functional analysis revealed that the ubiquitination system, neddylation pathway, and ciliary process are primarily involved in H. pylori-induced GC. Survival analysis illustrated that the overexpression of DOCK4, GNAS, CTGF, TGF-b1, ESR1, SELE, TIMP3, SMARCE1, and TXNIP was associated with poor prognosis, while increased MRPS5 expression was related to a favorable prognosis in GC patients. DOCK4, GNAS, CTGF, TGF-b1, ESR1, SELE, TIMP3, SMARCE1, TXNIP, and MRPS5 may be considered prognostic biomarkers for H. pylori-induced GC. However, experimental validation is necessary in the future.

• Proceedings of the Microbiological Society of Korea Conference
• /
• 2007.05a
• /
• pp.120-122
• /
• 2007

All living organisms use numerous signal-transduction pathways to sense and respond to their environments and thereby survive and proliferate in a range of biological niches. Molecular dissection of these signalling networks has increased our understanding of these communication processes and provides a platform for therapeutic intervention when these pathways malfunction in disease states, including infection. Owing to the expanding availability of sequenced genomes, a wealth of genetic and molecular tools and the conservation of signalling networks, members of the fungal kingdom serve as excellent model systems for more complex, multicellular organisms. Here, we employed Cryptococcus neoformans as a model system to understand how fungal-signalling circuits operate at the molecular level to sense and respond to a plethora of environmental stresses, including osmoticshock, UV, high temperature, oxidative stress and toxic drugs/metabolites. The stress-activated p38/Hog1 MAPK pathway is structurally conserved in many organisms as diverse as yeast and mammals, but its regulation is uniquely specialized in a majority of clinical Cryptococcus neoformans serotype A and D strains to control differentiation and virulence factor regulation. C. neoformans Hog1 MAPK is controlled by Pbs2 MAPK kinase (MAPKK). The Pbs2-Hog1 MAPK cascade is controlled by the fungal "two-component" system that is composed of a response regulator, Ssk1, and multiple sensor kinases, including two-component.like (Tco) 1 and Tco2. Tco1 and Tco2 play shared and distinct roles in stress responses and drug sensitivity through the Hog1 MAPK system. Furthermore, each sensor kinase mediates unique cellular functions for virulence and morphological differentiation. We also identified and characterized the Ssk2 MAPKKK upstream of the MAPKK Pbs2 and the MAPK Hog1 in C. neoformans. The SSK2 gene was identified as a potential component responsible for differential Hog1 regulation between the serotype D sibling f1 strains B3501 and B3502 through comparative analysis of their meiotic map with the meiotic segregation of Hog1-dependent sensitivity to the fungicide fludioxonil. Ssk2 is the only polymorphic component in the Hog1 MAPK module, including two coding sequence changes between the SSK2 alleles in B3501 and B3502 strains. To further support this finding, the SSK2 allele exchange completely swapped Hog1-related phenotypes between B3501 and B3502 strains. In the serotype A strain H99, disruption of the SSK2 gene dramatically enhanced capsule biosynthesis and mating efficiency, similar to pbs2 and hog1 mutations. Furthermore, ssk2, pbs2, and hog1 mutants are all hypersensitive to a variety of stresses and completely resistant to fludioxonil. Taken together, these findings indicate that Ssk2 is the critical interface protein connecting the two-component system and the Pbs2-Hog1 pathway in C. neoformans.

• Journal of Plant Biotechnology
• /
• v.50
• /
• pp.225-231
• /
• 2023

Cold stress is one of the most vulnerable environmental stresses that affect plant growth and crop yields. With the recent advancements in genetic approaches using Arabidopsis and other model systems, genes involved in cold-stress response have been identified and the key cold signaling factors have been characterized. Exposure to low-temperature stress triggers the activation of a set of genes known as cold regulatory (COR) genes. This activation process plays a crucial role in enhancing the resistance of plants to cold and freezing stress. The inducer of the C-repeatbinding factor (CBF) expression 1-CBF module (ICE1-CBF module) is a key cold signaling pathway regulator that enhances the expression of downstream COR genes; however, this signaling module in Panax ginseng remains elusive. Here, we identified cold-signaling-related genes, PgCBF1, PgCBF3, and PgICE1 and conducted functional genomic analysis with a heterologous system. We confirmed that the overexpression of cold- PgCBF3 in the cbf1/2/3 triple Arabidopsis mutant compensated for the cold stress-induced deficiency of COR15A and salt-stress tolerance. In addition, nuclearlocalized PgICE1 has evolutionarily conserved phosphorylation sites that are modulated by brassinsteroid insensitive 2 (PgBIN2) and sucrose non-fermenting 1 (SNF1)-related protein kinase 3 (PgSnRK3), with which it physically interacted in a yeast two-hybrid assay. Overall, our data reveal that the regulators identified in our study, PgICE1 and PgCBFs, are evolutionarily conserved in the P. ginseng genome and are functionally involved in cold and abiotic stress responses.