• Molecules and Cells
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• 제42권7호
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• pp.546-556
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• 2019

Protein phosphatase 4 (PP4) is a crucial protein complex that plays an important role in DNA damage response (DDR), including DNA repair, cell cycle arrest and apoptosis. Despite the significance of PP4, the mechanism by which PP4 is regulated remains to be elucidated. Here, we identified a novel PP4 inhibitor, protein phosphatase 4 inhibitory protein (PP4IP) and elucidated its cellular functions. PP4IP-knockout cells were generated using the CRISPR/Cas9 system, and the phosphorylation status of PP4 substrates (H2AX, KAP1, and RPA2) was analyzed. Then we investigated that how PP4IP affects the cellular functions of PP4 by immunoprecipitation, immunofluorescence, and DNA double-strand break (DSB) repair assays. PP4IP interacts with PP4 complex, which is affected by DNA damage and cell cycle progression and decreases the dephosphorylational activity of PP4. Both overexpression and depletion of PP4IP impairs DSB repairs and sensitizes cells to genotoxic stress, suggesting timely inhibition of PP4 to be indispensable for cells in responding to DNA damage. Our results identify a novel inhibitor of PP4 that inhibits PP4-mediated cellular functions and establish the physiological importance of this regulation. In addition, PP4IP might be developed as potential therapeutic reagents for targeting tumors particularly with high level of PP4C expression.

• 한국미생물·생명공학회지
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• 제49권4호
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• pp.594-602
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• 2021

The NLRP3 (nucleotide-binding domain, leucine-rich repeat family pyrin domain containing 3) inflammasome plays an important role in the initiation of inflammatory responses, through the recognition of pathogen-associated molecular patterns and tumor progression, including tumor growth and metastasis. In this study, we examined the effects of defective NLRP3 on the growth, migration, and invasiveness of hepatocellular carcinoma (HCC) SK-Hep1 cell. First, HCC SK-Hep1 cells were transfected with human NLRP3 targeting LentiCRISPRv2 vector using the CRISPR-Cas9 system, and NLRP3 deficiency was confirmed by RT-qPCR and western blotting. NLRP3 deficient SK-Hep1 cells showed delayed cell growth and decreased protein expression of PI3K, p-AKT, and pNF-κB when compared to NLRP3 complete SK-Hep1 cells. In addition, NLRP3 deficiency arrested the cell cycle at G1 phase through an increase in p21 and a reduction in CDK6. NLRP3 deficient SK-Hep1 cells also showed significantly delayed cell migration, invasion, and wound healing. The expression of epithelial-mesenchymal transition signaling molecules, such as N-cadherin and MMP-9, was found to be dramatically decreased in NLRP3 deficient SK-Hep1 cells compared to NLRP3 complete SK-Hep1 cells.

• Molecules and Cells
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• 제44권12호
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• pp.911-919
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• 2021

The virus-induced genome editing (VIGE) system aims to induce targeted mutations in seeds without requiring any tissue culture. Here, we show that tobacco rattle virus (TRV) harboring guide RNA (gRNA) edits germ cells in a wild tobacco, Nicotiana attenuata, that expresses Streptococcus pyogenes Cas9 (SpCas9). We first generated N. attenuata transgenic plants expressing SpCas9 under the control of 35S promoter and infected rosette leaves with TRV carrying gRNA. Gene-edited seeds were not found in the progeny of the infected N. attenuata. Next, the N. attenuata ribosomal protein S5 A (RPS5A) promoter fused to SpCas9 was employed to induce the heritable gene editing with TRV. The RPS5A promoter-driven SpCas9 successfully produced monoallelic mutations at three target genes in N. attenuata seeds with TRV-delivered guide RNA. These monoallelic mutations were found in 2%-6% seeds among M₁ progenies. This editing method provides an alternative way to increase the heritable editing efficacy of VIGE.

• The Plant Pathology Journal
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• 제37권4호
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• pp.389-395
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• 2021

Soil is the major source of plant-associated microbes. Several fungal and bacterial species live within plant tissues. Actinomycetes are well known for producing a variety of antibiotics, and they contribute to improving plant health. In our previous report, Streptomyces globisporus SP6C4 colonized plant tissues and was able to move to other tissues from the initially colonized ones. This strain has excellent antifungal and antibacterial activities and provides a suppressive effect upon various plant diseases. Here, we report the genome-wide analysis of antibiotic producing genes in S. globisporus SP6C4. A total of 15 secondary metabolite biosynthetic gene clusters were predicted using antiSMASH. We used the CRISPR/Cas9 mutagenesis system, and each biosynthetic gene was predicted via protein basic local alignment search tool (BLAST) and rapid annotation using subsystems technology (RAST) server. Three gene clusters were shown to exhibit antifungal or antibacterial activity, viz. cluster 16 (lasso peptide), cluster 17 (thiopeptide-lantipeptide), and cluster 20 (lantipeptide). The results of the current study showed that SP6C4 has a variety of antimicrobial activities, and this strain is beneficial in agriculture.

• 한국미생물·생명공학회지
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• 제51권4호
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• pp.374-389
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• 2023

Organophosphorus (OP) pesticides, particularly malathion, parathion, diazinon, and chlorpyrifos, are widely used in both agricultural and residential contexts. This refractory quality is shared by certain organ phosphorus insecticides, and it may have unintended consequences for certain non-target soil species. Bioremediation cleans organic and inorganic contaminants using microbes and plants. Organophosphate-hydrolyzing enzymes can transform pesticide residues into non-hazardous byproducts and are increasingly being considered viable solutions to the problem of decontamination. When coupled with system analysis, the multi-omics technique produces important data for functional validation and genetic manipulation, both of which may be used to boost the efficiency of bioremediation systems. RNA-guided nucleases and RNA-guided base editors include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR), which are used to alter genes and edit genomes. The review sheds light on key knowledge gaps and suggests approaches to pesticide cleanup using a variety of microbe-assisted methods. Researches, ecologists, and decision-makers can all benefit from having a better understanding of the usefulness and application of systems biology and gene editing in bioremediation evaluations.

• Biomolecules & Therapeutics
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• 제30권2호
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• pp.203-211
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• 2022

Melanogenesis is the production of melanin from tyrosine by a series of enzyme-catalyzed reactions, in which tyrosinase and DOPA oxidase play key roles. The melanin content in the skin determines skin pigmentation. Abnormalities in skin pigmentation lead to various skin pigmentation disorders. Recent research has shown that the expression of EMP2 is much lower in melanoma than in normal melanocytes, but its role in melanogenesis has not yet been elucidated. Therefore, we investigated the role of EMP2 in the melanogenesis of MNT1 human melanoma cells. We examined TRP-1, TRP-2, and TYR expression levels during melanogenesis in MNT1 melanoma cells by gene silencing of EMP2. Western blot and RT-PCR results confirmed that the expression levels of TYR and TRP-2 were decreased when EMP2 expression was knocked down by EMP2 siRNA in MNT1 cells, and these changes were reversed when EMP2 was overexpressed. We verified the EMP2 gene was knocked out of the cell line (EMP2 CRISPR/Cas9) by using a CRISPR/Cas9 system and found that the expression levels of TRP-2 and TYR were significantly lower in the EMP2 CRISPR/Cas9 cell lines. Loss of EMP2 also reduced migration and invasion of MNT1 melanoma cells. In addition, the melanosome transfer from the melanocytes to keratinocytes in the EMP2 KO cells cocultured with keratinocytes was reduced compared to the cells in the control coculture group. In conclusion, these results suggest that EMP2 is involved in melanogenesis via the regulation of TRP-2 expression.

• Journal of Microbiology and Biotechnology
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• 제33권11호
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• pp.1506-1512
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• 2023

Quantitative analysis of adenosine triphosphate (ATP) has been widely used as a diagnostic tool in the food and medical industries. Particularly, the pathogenesis of a few diseases including inflammatory bowel disease (IBD) is closely related to high ATP concentrations. A bioluminescent D-luciferin/luciferase system, which includes a luciferase (FLuc) from the firefly Photinus pyralis as a key component, is the most commonly used method for the detection and quantification of ATP. Here, instead of isolating FLuc produced in recombinant Escherichia coli, we aimed to develop a whole-cell biocatalyst system that does not require extraction and purification of FLuc. To this end, the gene coding for FLuc was introduced into the genome of probiotic Saccharomyces boulardii using the CRISPR/Cas9-based genome editing system. The linear relationship (r² = 0.9561) between ATP levels and bioluminescence generated from the engineered S. boulardii expressing FLuc was observed in vitro. To explore the feasibility of using the engineered S. boulardii expressing FLuc as a whole-cell biosensor to detect inflammation biomarker (i.e., ATP) in the gut, a colitis mouse model was established using dextran sodium sulfate as a colitogenic compound. Our findings demonstrated that the whole-cell biosensor can detect elevated ATP levels during gut inflammation in mice. Therefore, the simple and powerful method developed herein could be applied for non-invasive IBD diagnosis.

• BMB Reports
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• 제54권11호
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• pp.563-568
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• 2021

Cancer cells predominantly generate energy via glycolysis, even in the presence of oxygen, to support abnormal cell proliferation. Suppression of PDHA1 by PDK1 prevents the conversion of cytoplasmic pyruvate into Acetyl-CoA. Several PDK inhibitors have been identified, but their clinical applications have not been successful for unclear reasons. In this study, endogenous PDHA1 in A549 cells was silenced by the CRISPR/Cas9 system, and PDHA1^WT and PDHA1^3SD were transduced. Since PDHA1^3SD cannot be phosphorylated by PDKs, it was used to evaluate the specific activity of PDK inhibitors. This study highlights that PDHA1^WT and PDHA1^3SD A549 cells can be used as a cell-based PDK inhibitor-distinction system to examine the relationship between PDH activity and cell death by established PDK inhibitors. Leelamine, huzhangoside A and otobaphenol induced PDH activity-dependent apoptosis, whereas AZD7545, VER-246608 and DCA effectively enhanced PDHA1 activity but little toxic to cancer cells. Furthermore, the activity of phosphomimetic PDHA1 revealed the complexity of its regulation, which requires further in-depth investigation.

• The Korean Journal of Physiology and Pharmacology
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• 제22권4호
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• pp.457-465
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• 2018

The expression of BCL-2 interacting cell death suppressor (BIS), an anti-stress or anti-apoptotic protein, has been shown to be regulated at the transcriptional level by heat shock factor 1 (HSF1) upon various stresses. Recently, HSF1 was also shown to bind to BIS, but the significance of these protein-protein interactions on HSF1 activity has not been fully defined. In the present study, we observed that complete depletion of BIS using a CRISPR/Cas9 system in A549 non-small cell lung cancer did not affect the induction of heat shock protein (HSP) 70 and HSP27 mRNAs under various stress conditions such as heat shock, proteotoxic stress, and oxidative stress. The lack of a functional association of BIS with HSF1 activity was also demonstrated by transient downregulation of BIS by siRNA in A549 and U87 glioblastoma cells. Endogenous BIS mRNA levels were significantly suppressed in BIS knockout (KO) A549 cells compared to BIS wild type (WT) A549 cells at the constitutive and inducible levels. The promoter activities of BIS and HSP70 as well as the degradation rate of BIS mRNA were not influenced by depletion of BIS. In addition, the expression levels of the mutant BIS construct, in which 14 bp were deleted as in BIS-KO A549 cells, were not different from those of the WT BIS construct, indicating that mRNA stability was not the mechanism for autoregulation of BIS. Our results suggested that BIS was not required for HSF1 activity, but was required for its own expression, which involved an HSF1-independent pathway.

• Journal of Veterinary Science
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• 제22권3호
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• pp.36.1-36.12
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• 2021

Background: Mouse hepatitis virus (MHV) A59 is a highly infectious pathogen and starts in the respiratory tract and progresses to systemic infection in laboratory mice. The complement system is an important part of the host immune response to viral infection. It is not clear the role of the classical complement pathway in MHV infection. Objectives: The purpose of this study was to determine the importance of the classical pathway in coronavirus pathogenesis by comparing C1qa KO mice and wild-type mice. Methods: We generated a C1qa KO mouse using CRISPR/Cas9 technology and compared the susceptibility to MHV A59 infection between C1qa KO and wild-type mice. Histopathological and immunohistochemical changes, viral loads, and chemokine expressions in both mice were measured. Results: MHV A59-infected C1qa KO mice showed severe histopathological changes, such as hepatocellular necrosis and interstitial pneumonia, compared to MHV A59-infected wild-type mice. Virus copy numbers in the olfactory bulb, liver, and lungs of C1qa KO mice were significantly higher than those of wild-type mice. The increase in viral copy numbers in C1qa KO mice was consistent with the histopathologic changes in organs. These results indicate that C1qa deficiency enhances susceptibility to MHV A59 systemic infection in mice. In addition, this enhanced susceptibility effect is associated with dramatic elevations in spleen IFN-γ, MIP-1 α, and MCP-1 in C1qa KO mice. Conclusions: These data suggest that C1qa deficiency enhances susceptibility to MHV A59 systemic infection, and activation of the classical complement pathway may be important for protecting the host against MHV A59 infection.