34 results on '"Shang, Jun"'
Search Results
2. Characterization and activity-folding relationship of serine protease from Antarctic krill (Euphausia superba)
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Shang-Jun Yin, Ho-Yeon Lee, Wei Wang, Jinhyuk Lee, and Yong-Doo Park
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Structural Biology ,General Medicine ,Molecular Biology - Published
- 2022
3. Detection of Melanogenesis and Anti-Apoptosis-Associated Melanoma Factors: Array CGH and PPI Mapping Integrating Study
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Shang-Jun Yin, Guo-Ying Qian, Jun-Mo Yang, Yong-Doo Park, and Jinhyuk Lee
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Candidate gene ,Melanoma ,Locus (genetics) ,General Medicine ,Computational biology ,Biology ,medicine.disease ,Biochemistry ,Neoplasm Proteins ,Gene Expression Regulation, Neoplastic ,chemistry.chemical_compound ,Human interactome ,chemistry ,Structural Biology ,Cell Line, Tumor ,YWHAZ ,medicine ,Humans ,Apoptosis Regulatory Proteins ,Gene ,YWHAB ,DNA ,Oligonucleotide Array Sequence Analysis - Abstract
Background: We investigated melanogenesis- and anti-apoptosis-related melanoma factors in melanoma cells (TXM1, TXM18, A375P, and A375SM). Objective: To find melanoma associated hub factor, high-throughput screening-based techniques integrating with bioinformatics were investigated. Methods: Array CGH analysis was conducted with a commercial system. Total genomic DNAs prepared individually from each cell line with control DNA were properly labeled with Cy3-dCTP and Cy5-dCTP and hybridizations and subsequently performed data treatment by the log2 green (G; test) to red (R; reference) fluorescence ratios (G/R). Gain or loss of copy number was judged by spots with log2-transformed ratios. PPI mapping analysis of detected candidate genes based on the array CGH results was conducted using the human interactome in the STRING database. Energy minimization and a short Molecular Dynamics (MD) simulation using the implicit solvation model in CHARMM were performed to analyze the interacting residues between YWHAZ and YWHAB. Results: Three genes (BMP-4, BFGF, LEF-1) known to be involved in melanogenesis were found to lose chromosomal copy numbers, and Chr. 6q23.3 was lost in all tested cell lines. Ten hub genes (CTNNB1, PEX13, PEX14, PEX5, IFNG, EXOSC3, EXOSC1, EXOSC8, UBC, and PEX10) were predicted to be functional interaction factors in the network of the 6q23.3 locus. The apoptosis-associated genes E2F1, p50, BCL2L1, and BIRC7 gained, and FGF2 lost chromosomal copy numbers in the tested melanoma cell lines. YWHAB, which gained chromosomal copy numbers, was predicted to be the most important hub protein in melanoma cells. Molecular dynamics simulations for binding YWHAB and YWHAZ were conducted, and the complex was predicted to be energetically and structurally stable through its 3 hydrogen-bond patterns. The number of interacting residues is 27. Conclusion: Our study compares genome-wide screening interactomics predictions for melanoma factors and offers new information for understanding melanogenesis- and anti-apoptosis-associated mechanisms in melanoma. Especially, YWHAB was newly detected as a core factor in melanoma cells.
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- 2021
4. A study of Pb2+ induced unfolding and aggregation of arginine kinase from Euphausia superba: kinetics and computational simulation integrating study
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Yong-Doo Park, Jinhyuk Lee, Yue-Xiu Si, Chen Zhongfa, Shang-Jun Yin, GyuTae Lim, and Guo-Ying Qian
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biology ,Chemistry ,Kinase ,Euphausia ,Kinetics ,General Medicine ,Metabolism ,Arginine kinase ,biology.organism_classification ,Cell biology ,Computational simulation ,Phosphagen ,Structural Biology ,Environmental stress response ,biology.protein ,Molecular Biology - Abstract
Arginine kinase is a crucial phosphagen kinase in invertebrates, which is associated to the environmental stress response, plays a key role in cellular energy metabolism. In this study, we investig...
- Published
- 2021
5. Inhibitory effect of α-ketoglutaric acid on α-glucosidase: integrating molecular dynamics simulation and inhibition kinetics
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Shang-Jun Yin, Yong-Doo Park, Guo-Ying Qian, Shang-Ling Xiong, Myong-Joon Hahn, Jae-Rin Lee, Gyu Tae Lim, Jun-Mo Yang, and Jinhyuk Lee
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0303 health sciences ,Chemistry ,α glucosidase ,030303 biophysics ,Kinetics ,Type 2 Diabetes Mellitus ,alpha-Glucosidases ,General Medicine ,Inhibition kinetics ,Molecular Dynamics Simulation ,Molecular Docking Simulation ,03 medical and health sciences ,Molecular dynamics ,Ketoglutaric Acid ,Diabetes Mellitus, Type 2 ,Biochemistry ,Structural Biology ,Humans ,Ketoglutaric Acids ,Glycoside Hydrolase Inhibitors ,Molecular Biology ,Inhibitory effect - Abstract
The inhibition of α-glucosidase is used as a key clinical approach to treat type 2 diabetes mellitus and thus, we assessed the inhibitory effect of α-ketoglutaric acid (AKG) on α-glucosidase with both an enzyme kinetic assay and computational simulations. AKG bound to the active site and interacted with several key residues, including ASP68, PHE157, PHE177, PHE311, ARG312, TYR313, ASN412, ILE434 and ARG439, as detected by protein-ligand docking and molecular dynamics simulations. Subsequently, we confirmed the action of AKG on α-glucosidase as mixed-type inhibition with reversible and rapid binding. The relevant kinetic parameter IC
- Published
- 2019
6. The inhibitory effect of pyrogallol on tyrosinase activity and structure: Integration study of inhibition kinetics with molecular dynamics simulation
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Shang-Ling Xiong, Jun-Mo Yang, Jinhyuk Lee, Yue-Xiu Si, Guo-Ying Qian, Shang-Jun Yin, Yong-Doo Park, and Gyu Tae Lim
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Tyrosinase ,02 engineering and technology ,Molecular Dynamics Simulation ,Pyrogallol ,Biochemistry ,03 medical and health sciences ,Molecular dynamics ,chemistry.chemical_compound ,Structural Biology ,Catalytic Domain ,Tyrosinase activity ,Enzyme kinetics ,Molecular Biology ,Inhibitory effect ,030304 developmental biology ,0303 health sciences ,biology ,Monophenol Monooxygenase ,Active site ,Substrate (chemistry) ,General Medicine ,021001 nanoscience & nanotechnology ,Molecular Docking Simulation ,Kinetics ,chemistry ,biology.protein ,Biophysics ,0210 nano-technology - Abstract
Pyrogallol is naturally found in aquatic plant and has been proposed as a substrate of tyrosinase. In this study, we evaluated the dual effect of pyrogallol on tyrosinase as an inhibitor in the presence of L‑DOPA simultaneously via integrating methods of enzyme kinetics and computational molecular dynamics (MD) simulations. Pyrogallol was found to be a reversible inhibitor of tyrosinase in the presence of L‑DOPA and its induced mechanism was the parabolic non-competitive inhibition type (IC50 = 0.772 ± 0.003 mM and Ki = 0.529 ± 0.022 mM). Kinetic measurements by real-time interval assay showed that pyrogallol induced rapid inactivation process composing with slight activations at the low dose. Spectrofluorimetry studies showed that pyrogallol mainly induced regional changes in the active site of tyrosinase accompanying with hydrophobic disruption at high dose. The computational MD simulations further revealed that pyrogallol could interact with several residues near the tyrosinase active site pocket such as HIS61, HIS85, HIS259, ASN260, HIS263, VAL283, and ALA296. Our study provides insight into the mechanism by which hydroxyl group composing pyrogallol inhibit tyrosinase and pyrogallol is a potential natural anti-pigmentation agent.
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- 2019
7. Characterization and tissue expression analysis of mitochondrial creatine kinases (types I and II) from Pelodiscus sinensis
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Li-Fang Zeng, Jinhyuk Lee, Gyutae Lim, Yu-Fei Yang, Run-Lan Lin, Shang-Jun Yin, Wei Wang, and Yong-Doo Park
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Structural Biology ,General Medicine ,Molecular Biology - Abstract
The aim of this study was to characterize the functions of the mitochondrial creatine kinases in the Chinese soft-shelled turtle Pelodiscus sinensis (PSCK-MT1 and PSCK-MT2) to characterize function in relation to hibernation. Computational prediction via molecular dynamics simulations showed that PSCK-MT1 had stronger kinase- and creatine-binding affinity than PSCK-MT2. We measured PSCK-MT1 and PSCK-MT2 levels in the myocardium, liver, spleen, lung, kidney, and ovary of P. sinensis before and after hibernation and found that the expression of these enzymes was the most significantly upregulated in the ovary. We enumerated the ovarian follicles and evaluated the physiological indices of P. sinensis and discovered that fat was the main form of energy storage in P. sinensis. Moreover, both PSCK-MTs promoted follicular development during hibernation. Immunohistochemistry was used to study follicular development and revealed that both PSCK-MTs were expressed primarily in the follicular fluid and granulosa layer before and after hibernation. We found that PSCK-MT1 and PSCK-MT2 could play important roles in ovarian follicular development under hibernation. Hence, both PSCK-MTs probably function effectively under the conditions of low temperature and oxygen during hibernation. Communicated by Ramaswamy H. Sarma
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- 2021
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8. Tyrosinase-mediated melanogenesis in melanoma cells: Array comparative genome hybridization integrating proteomics and bioinformatics studies
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Myong-Joon Hahn, Yong-Doo Park, Jun-Mo Yang, Guo-Ying Qian, Jae-Rin Lee, and Shang-Jun Yin
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Proteomics ,Spectrometry, Mass, Electrospray Ionization ,Tyrosinase ,Gene Dosage ,02 engineering and technology ,Biology ,Biochemistry ,03 medical and health sciences ,Structural Biology ,Tandem Mass Spectrometry ,Cell Line, Tumor ,Protein Interaction Mapping ,medicine ,Humans ,Molecular Biology ,Gene ,Endoplasmic Reticulum Chaperone BiP ,Melanoma ,030304 developmental biology ,HSPA9 ,Melanins ,0303 health sciences ,Comparative Genomic Hybridization ,Proteomic Profiling ,Monophenol Monooxygenase ,Pigmentation ,Chromosomes, Human, Pair 11 ,Computational Biology ,General Medicine ,021001 nanoscience & nanotechnology ,medicine.disease ,Cell biology ,Clone Cells ,Neoplasm Proteins ,Gene Ontology ,Proteome ,YWHAZ ,0210 nano-technology ,Chromatography, Liquid - Abstract
We investigated the tyrosinase-associated melanogenesis in melanoma cells by using OMICS techniques. We characterized the chromosome copy numbers, including Chr 11q21 where the tyrosinase gene is located, from several melanoma cell lines (TXM13, G361, and SK-MEL-28) by using array CGH. We revealed that 11q21 is stable in TXM13 cells, which is directly related to a spontaneous high melanin pigment production. Meanwhile, significant loss of copy number of 11q21 was found in G361 and SK-MEL-28. We further profiled the proteome of TXM13 cells by LC-ESI-MSMS and detected more than 900 proteins, then predicted 11 hub proteins (YWHAZ; HSP90AA1; HSPA5; HSPA1L; HSPA9; HSP90B1; HSPA1A; HSPA8; FKSG30; ACTB; DKFZp686DQ972) by using an interactomic algorithm. YWHAZ (25% interaction in the network) is thought to be a most important protein as a linking factor between tyrosinase-triggered melanogenesis and melanoma growth. Bioinformatic tools were further applied for revealing various physiologic mechanisms and functional classification. The results revealed clues for the spontaneous pigmentation capability of TXM13 cells, contrary to G361 and SK-MEL-28 cells, which commonly have depigmentation properties during subculture. Our study comparatively conducted the genome-wide screening and proteomic profiling integrated interactomics prediction for TXM13 cells and suggests new insights for studying both melanogenesis and melanoma.
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- 2020
9. A novel bifunctional glucanase exhibiting high production of glucose and cellobiose from rumen bacterium
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De-Ying Gao, Shang-Jun Yin, Qian Wang, Bo He, Jiakun Wang, Li-Chun Qian, Jiawen Cao, and Qian Deng
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Endo-1,3(4)-beta-Glucanase ,Cellobiose ,Rumen ,beta-Glucans ,Bioconversion ,Genetic Vectors ,Lignocellulosic biomass ,Gene Expression ,02 engineering and technology ,Biochemistry ,Substrate Specificity ,03 medical and health sciences ,Hydrolysis ,chemistry.chemical_compound ,Bacterial Proteins ,Structural Biology ,Enzymatic hydrolysis ,Trioses ,Cellulose 1,4-beta-Cellobiosidase ,Escherichia coli ,Animals ,Amino Acid Sequence ,Cloning, Molecular ,Cellulose ,Molecular Biology ,Glucans ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Sheep ,Sequence Homology, Amino Acid ,Chemistry ,General Medicine ,Glucanase ,021001 nanoscience & nanotechnology ,Recombinant Proteins ,Gastrointestinal Microbiome ,Kinetics ,Enzyme ,Glucose ,0210 nano-technology ,Sequence Alignment - Abstract
Herbivores gastrointestinal microbiota is of tremendous interest for mining novel lignocellulosic enzymes for bioprocessing. We previously reported a set of potential carbohydrate-active enzymes from the metatranscriptome of the Hu sheep rumen microbiome. In this study, we isolated and heterologously expressed two novel glucanase genes, Cel5A-h38 and Cel5A-h49, finding that both recombinant enzymes showed the optimum temperatures of 50 °C. Substrate-specificity determination revealed that Cel5A-h38 was exclusively active in the presence of mixed-linked glucans, such as barley β-glucan and Icelandic moss lichenan, whereas Cel5A-h49 (EC 3.2.1.4) exhibited a wider substrate spectrum. Surprisingly, Cel5A-h38 initially released only cellotriose from lichenan and further converted it into an equivalent amount of glucose and cellobiose, suggesting a dual-function as both endo-β-1,3-1,4-glucanase (EC 3.2.1.73) and exo-cellobiohydrolase (EC 3.2.1.91). Additionally, we performed enzymatic hydrolysis of sheepgrass (Leymus chinensis) and rice (Orysa sativa) straw using Cel5A-h38, revealing liberation of 1.91 ± 0.30 mmol/mL and 2.03 ± 0.09 mmol/mL reducing sugars, respectively, including high concentrations of glucose and cellobiose. These results provided new insights into glucanase activity and lay a foundation for bioconversion of lignocellulosic biomass.
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- 2020
10. Comparative studies of the expression of creatine kinase isoforms under immune stress in Pelodiscus sinensis
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Guo-Ying Qian, Jinhyuk Lee, Shang-Jun Yin, Wei Wang, Yong-Doo Park, Yufei Yang, Caiyan Li, and Li-Fang Zeng
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02 engineering and technology ,Biochemistry ,PSCK-M, muscle type of PSCK ,chemistry.chemical_compound ,Adenosine Triphosphate ,Structural Biology ,Sequence Analysis, Protein ,Malondialdehyde ,Protein Isoforms ,Creatine Kinase ,PSCK-B, brain type of PSCK ,Phylogeny ,0303 health sciences ,Immune stress ,biology ,LDH, lactate dehydrogenase ,RACE, rapid-amplification of cDNA ends ,General Medicine ,Bacterial Infections ,021001 nanoscience & nanotechnology ,Catalase ,MD, molecular dynamics ,T-SOD, total superoxide dismutase ,Immunohistochemistry ,Aeromonas hydrophila ,Turtles ,Molecular Docking Simulation ,medicine.anatomical_structure ,Liver ,0210 nano-technology ,IHC, immunohistochemistry ,Gene isoform ,ATP, adenosine triphosphate ,Molecular Dynamics Simulation ,ns, nanosecond ,Isozyme ,Article ,Superoxide dismutase ,03 medical and health sciences ,RT-PCR, reverse transcription polymerase chain reaction ,In vivo ,Stress, Physiological ,Lactate dehydrogenase ,medicine ,Animals ,Molecular Biology ,030304 developmental biology ,MDA, malondialdehyde ,L-Lactate Dehydrogenase ,Superoxide Dismutase ,Myocardium ,Skeletal muscle ,PSCK, CK from Pelodiscus sinensis ,Molecular biology ,ADP, adenosine diphosphate ,PSCK-S, mitochondrial sarcomeric type of PSCK (type II) ,ps, picosecond ,chemistry ,Gene Expression Regulation ,biology.protein ,Pelodiscus sinensis ,Creatine kinase ,CAT, catalase ,CK, creatine kinase ,Spleen - Abstract
The expression and localization of different isoforms of creatine kinase in Pelodiscus sinensis (PSCK) were studied to reveal the role of PSCK isozymes (PSCK-B, PSCK-M, PSCK-S) under bacterial infection-induced immunologic stress. The computational molecular dynamics simulations predicted that PSCK-S would mostly possess a kinase function in a structural aspect when compared to PSCK-B and PSCK-M. The assay of biochemical parameters such as total superoxide dismutase (T-SOD), lactate dehydrogenase (LDH), malondialdehyde (MDA), catalase (CAT), and the content of ATP were measured along with total PSCK activity in different tissue samples under bacterial infection. The expression detections of PSCK isozymes in vitro and in vivo were overall well-matched where PSCK isozymes were expressed differently in P. sinensis tissues. The results showed that PSCK-B mostly contributes to the spleen, followed by the liver and myocardium; PSCK-M mostly contributes to the liver, followed by the myocardium and skeletal muscle, while PSCK-S contributes to the spleen and is uniquely expressed in skeletal muscle. Our study suggests that the various alterations of PSCK isozymes in tissues of P. sinensis are prone to defense the bacterial infection and blocking energetic imbalance before severe pathogenesis turned on in P. sinensis.
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- 2020
11. Biochemical Study of Fibrinolytic Protease from Euphausia superba Possessing Multifunctional Serine Protease Activity
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GyuTae Lim, Jinhyuk Lee, Shang-Jun Yin, Jun-Mo Yang, Guo-Ying Qian, and Yong-Doo Park
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Protein Folding ,medicine.medical_treatment ,Molecular Dynamics Simulation ,Biochemistry ,Arthropod Proteins ,Serine ,03 medical and health sciences ,chemistry.chemical_compound ,Structural Biology ,AEBSF ,medicine ,Animals ,Enzyme kinetics ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Protease ,Molecular mass ,Fibrinolysis ,030302 biochemistry & molecular biology ,General Medicine ,Kinetics ,Enzyme ,chemistry ,Osmolyte ,Glycine ,Serine Proteases ,Euphausiacea - Abstract
Background: Fibrinolytic protease from Euphausia superba (EFP) was isolated. Objective: Biochemical distinctions, regulation of the catalytic function, and the key residues of EFP were investigated. Methods: The serial inhibition kinetic evaluations coupled with measurements of fluorescence spectra in the presence of 4-(2-aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF) was conducted. The computational molecular dynamics (MD) simulations were also applied for a comparative study. Results: The enzyme behaved as a monomeric protein with a molecular mass of about 28.6 kD with Km BApNA = 0.629 ± 0.02 mM and kcat/Km BApNA = 7.08 s-1/mM. The real-time interval measurements revealed that the inactivation was a first-order reaction, with the kinetic processes shifting from a monophase to a biphase. Measurements of fluorescence spectra showed that serine residue modification by AEBSF directly caused conspicuous changes of the tertiary structures and exposed hydrophobic surfaces. Some osmolytes were applied to find protective roles. These results confirmed that the active region of EFP is more flexible than the overall enzyme molecule and serine, as the key residue, is associated with the regional unfolding of EFP in addition to its catalytic role. The MD simulations were supportive to the kinetics data. Conclusion: Our study indicated that EFP has an essential serine residue for its catalyst function and associated folding behaviors. Also, the functional role of osmolytes such as proline and glycine that may play a role in defense mechanisms from environmental adaptation in a krill’s body was suggested.
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- 2020
12. A Knock-Down Cell-Based Study for the Functional Analysis of Chloride Intracellular Channel 1 (CLIC1): Integrated Proteomics and Microarray Study
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Jae-Rin Lee, Yong-Doo Park, Myong-Joon Hahn, Shang-Jun Yin, Bit-Na Lee, Guo-Ying Qian, and Jun-Mo Yang
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Proteomics ,Candidate gene ,Microarray ,Protein Array Analysis ,Computational biology ,Biology ,Biochemistry ,Dermatitis, Atopic ,030207 dermatology & venereal diseases ,03 medical and health sciences ,0302 clinical medicine ,Structural Biology ,Chloride Channels ,FLNA ,Humans ,Gene ,030304 developmental biology ,0303 health sciences ,Nucleophosmin ,MNDA ,General Medicine ,Gene Expression Regulation ,A549 Cells ,Gene Knockdown Techniques ,Function (biology) - Abstract
Background: Previously, we detected that chloride intracellular channel 1 (CLIC1) was involved in the pathogenesis of atopic dermatitis (AD). Objective: In this study, we aimed to use high-throughput screening (HTS) approaches to identify critical factors associated with the function of CLIC1 in knock-down cells. Methods: We down-regulated CLIC1 in human A549 cells via siRNA and then conducted serial HTS studies, including proteomics integrated with a microarray and the implementation of bioinformatics algorithms. Results: Together, these approaches identified several important proteins and genes associated with the function of CLIC1. These proteins and genes included tumor rejection antigen (gp96) 1, nucleophosmin, annexin I, keratin 1 and 10, FLNA protein, enolase 1, and metalloprotease 1, which were found using two-dimensional electrophoresis (2-DE) proteomics. Separately, NTNG1, SEMA5A, CLEC3A, GRPR, GNGT2, GRM5, GRM7, DNMT3B, CXCR5, CCL11, CD86, IL2, MNDA, TLR5, IL23R, DPP6, DLGAP1, CAT, GSTA1, GSTA2, GSTA5, CYP2E1, ADH1A, ESR1, ARRDC3, A1F1, CCL5, CASP8, DNTT, SQSTM1, PCYT1A, and SLCO4C1 were found using a DNA microarray integrated with PPI mapping. Conclusion: CCL11 is thought to be a particularly critical gene among the candidate genes detected in this study. By integrating the datasets and utilizing the strengths of HTS, we obtained new insights into the functional role of CLIC1, including the use of CLIC1-associated applications in the treatment of human diseases such as AD.
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- 2020
13. Functional study of acetaldehyde dehydrogenase 1 (ALDH1) in keratinocytes: microarray integrating bioinformatics approaches
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Yong-Doo Park, Shang-Jun Yin, Min-Woo Park, Bit-Na Lee, Guo-Ying Qian, and Jun-Mo Yang
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Keratinocytes ,biology ,Antibody microarray ,Microarray ,Chemistry ,Aldehyde dehydrogenase ,Computational Biology ,Retinal Dehydrogenase ,macromolecular substances ,General Medicine ,Microarray Analysis ,Aldehyde Dehydrogenase 1 Family ,Dermatitis, Atopic ,Biochemistry ,Structural Biology ,biology.protein ,Humans ,Molecular Biology ,Function (biology) - Abstract
The function of acetaldehyde dehydrogenase 1 (ALDH1) has been gradually elucidated in several diseases, especially in various cancers. However, the role of ALDH1 in skin-related diseases has been mostly unknown. Previously, we found that ALDH1 is involved in the pathogenesis of atopic dermatitis (AD). In this study, we used high-throughput screening (HTS) approaches to identify critical factors associated with ALDH1 in human keratinocytes to reveal its functions in skin. We overexpressed ALDH1 in human HaCaT keratinocytes and then conducted serial HTS studies, a DNA microarray and antibody array integrated with bioinformatics algorithms. Together, those tests identified several novel genes associated with the function of ALDH1 in keratinocytes, as well as AD, including CTSG and CCL11. In particular, GNB3, GHSR, TAS2R9, FFAR1, TAS2R16, CCL21, GPR32, NPFFR1, GPR15, FBXW12, CCL19, EDNRA, FFAR3, and RXFP3 proteins were consistently detected as hub proteins in the PPI maps. By integrating the datasets obtained from these HTS studies and using the strengths of each method, we obtained new insights into the functional role of ALDH1 in skin keratinocytes. The approach used here could contribute to the clinical understanding of ALDH1-associated applications for the treatment of AD.Communicated by Ramaswamy H. Sarma.
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- 2020
14. Metabolic responses and arginine kinase expression of juvenile cuttlefish (Sepia pharaonis) under salinity stress
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Wei Song, Shang-Jun Yin, Linmeng Zhang, Yong-Doo Park, Jia-Xin Wan, Li-Li Zhang, Xia-Min Jiang, and Yue-Xiu Si
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0301 basic medicine ,Cuttlefish ,Salinity ,Sepia ,Biochemistry ,Gene Expression Regulation, Enzymologic ,03 medical and health sciences ,chemistry.chemical_compound ,Stress, Physiological ,Structural Biology ,Animals ,Osmotic pressure ,Molecular Biology ,Sepia pharaonis ,chemistry.chemical_classification ,biology ,Glycogen ,Muscles ,Arginine Kinase ,04 agricultural and veterinary sciences ,General Medicine ,Arginine kinase ,biology.organism_classification ,Adaptation, Physiological ,Lactic acid ,030104 developmental biology ,Enzyme ,Liver ,chemistry ,040102 fisheries ,biology.protein ,0401 agriculture, forestry, and fisheries - Abstract
The pharaoh cuttlefish Sepia pharaonis is particularly sensitive to environmental changes in its breeding environment. The breeding of S. pharaonis larvae was carried out in different salinities for 48h, and the changes in survival rate, histological structure, energy metabolism, and anti-oxidative stress parameters were investigated and correlated with arginine kinase (AK) expression changes in muscle and liver tissues. The suitable salinity for larvae cultivation ranged from 24 to 30‰, and the survival rate showed a significant decline at 21‰ salinity. Histological observations of muscle and liver showed that changes in salinity and osmotic pressure had an adverse effect on tissue structure. Measurements of glycogen and lactic acid levels suggested that S. pharaonis could dynamically adjust energy metabolism to provide additional energy under unsuitable salinity. The protein levels and enzyme activities of AK in muscle significantly increased at 21‰ salinity. The results were consistent with prompt replenishment of phosphoarginine stores during salinity stress to maintain a dynamic ATP balance, suggesting that AK plays an important role in the regulation of energy metabolism. This study provides insight into metabolic changes during salinity stress and sheds light on the functional role of AK in S. pharaonis.
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- 2018
15. Screening and analysis of agouti signaling protein interaction partners in Pelodiscus sinensis suggests a role in lipid metabolism
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Xiaoying Zheng, Yue-Xiu Si, Yong-Doo Park, Shang-Jun Yin, Zhang Lili, Guo-Ying Qian, Qian Wang, and Xuanwei Chen
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Apolipoprotein B ,Protein Conformation ,Two-hybrid screening ,Gene Expression ,02 engineering and technology ,Molecular Dynamics Simulation ,Filamin ,Biochemistry ,03 medical and health sciences ,Structure-Activity Relationship ,Structural Biology ,Protein Interaction Mapping ,Animals ,Humans ,Amino Acid Sequence ,Protein Interaction Maps ,Receptor ,Molecular Biology ,Phylogeny ,030304 developmental biology ,0303 health sciences ,biology ,Chemistry ,Lipid metabolism ,General Medicine ,021001 nanoscience & nanotechnology ,Lipid Metabolism ,Turtles ,Melanocortin 4 receptor ,Molecular Docking Simulation ,Docking (molecular) ,biology.protein ,Agouti Signaling Protein ,lipids (amino acids, peptides, and proteins) ,Vitronectin ,0210 nano-technology ,Carrier Proteins ,Protein Binding - Abstract
Agouti signaling protein (ASP) is a secreted paracrine protein that has been widely reported to function in melanogenesis and obesity and could potentially be a core protein that regulates the color and fatty phenotype of P. sinensis. In this study, we screened out interacting proteins of ASP by combined co-immunoprecipitation mass spectrometry (CoIP-MS), yeast two hybrid (Y2H) analysis, and computational predictions. We performed docking of ASP with its well-known receptor melanocortin receptor 4 (MC4R) to predict the binding capacity and to screen out actual ASP interacting proteins, CoIP-MS was performed where identified 32 proteins that could bind with ASP and Y2H confirmed seven proteins binding with ASP directly. CoIP-MS and Y2H screening results including PPI prediction revealed that vitronectin (VTN), apolipoprotein A1 (APOA1), apolipoprotein B (APOB), and filamin B (FLNB) were the key interacting proteins of ASP. VTN, APOA1, and APOB are functional proteins in lipid metabolism and various skin disorders, suggesting ASP may function in lipid metabolism through these partners. This study provided protein-protein interaction information of ASP, and the results will promote further research into the diverse roles of ASP, as well as its binding partners, and their function in different strains of P. sinensis.
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- 2019
16. Analysis of the peptides detected in atopic dermatitis and various inflammatory diseases patients-derived sera
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Hee Seung Yang, Jun-Mo Yang, Ick-Hyun Cho, Shang-Jun Yin, and Yong-Doo Park
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Proteomics ,0301 basic medicine ,Proteome ,Enzyme-Linked Immunosorbent Assay ,Inflammation ,Biochemistry ,Dermatitis, Atopic ,03 medical and health sciences ,0302 clinical medicine ,Structural Biology ,medicine ,Humans ,Molecular Biology ,Zinc finger ,Kininogen ,biology ,General Medicine ,Atopic dermatitis ,medicine.disease ,Blot ,030104 developmental biology ,Gene Expression Regulation ,030220 oncology & carcinogenesis ,Immunology ,Major basic protein ,biology.protein ,medicine.symptom ,Peptides ,Biomarkers - Abstract
Serum proteomics has been applied for the discovery and analysis of biomarkers related to human disease. Serum is an optimal source to identify proteins derived from diseased-tissue compartments. We recently established an integrative method to analyze highly basic proteins that remain unresolved by the general 2D-PAGE method. In this follow-up study, we successfully detected several disease-associated proteins from sera samples obtained from patients with atopic dermatitis (AD). After proteomic analyses, target proteins were validated from AD patient-derived sera using ELISA or Western blotting methods We detected zinc finger CCHC domain containing 10 (ZCCHC10), peptidoglycan recognition protein L (PGRP-L), kininogen, α-1-antitrypsin, and hornerin proteins that are dysregulated in AD patient sera samples, which suggest effective approaches to methodologically analyze the serum proteome. Thus, the integrated proteomic method approach described here could be applicable for the detection of proteins associated with other human diseases. Our present study provides new insights into optimized serum proteomic techniques to understand systemic events of AD.
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- 2018
17. Functional study of 14-3-3 protein epsilon (YWHAE) in keratinocytes: microarray integrating bioinformatics approaches
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Hyunchang Kwak, Ji-Won Han, Jun-Mo Yang, Yong-Doo Park, Shang-Jun Yin, Jae-Rin Lee, Bit-Na Lee, and Myong-Joon Hahn
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Keratinocytes ,0303 health sciences ,Microarray ,Antibody microarray ,Sequence Analysis, RNA ,030303 biophysics ,RNA ,Computational Biology ,General Medicine ,Biology ,Bioinformatics ,Dermatitis, Atopic ,03 medical and health sciences ,14-3-3 Proteins ,Structural Biology ,HaCaT Cells ,Humans ,MAPK15 ,Extracellular Signal-Regulated MAP Kinases ,Molecular Biology ,YWHAE ,Gene ,14-3-3 Protein Epsilon ,Function (biology) - Abstract
Previously, we detected that 14-3-3 protein epsilon (YWHAE) was involved in the pathogenesis of atopic dermatitis (AD) and tyrosinase-mediated pigmentation. In this study, we aimed to identify critical factors associated with YWHAE in human keratinocytes using high-throughput screening (HTS) approaches to reveal its functions in skin. We overexpressed YWHAE in human HaCaT keratinocytes and then conducted serial HTS studies, including RNA sequencing integrated with antibody arrays and the implementation of bioinformatics algorithms. Cumulatively, these approaches identified several novel genes in keratinocytes associated with the function of YWHAE including KRT9, KRT1, KRT6C, BST2, CIB2, APH1B, ACTC1, IFI27, TUBA1A, CAPN6, UTY, MX2, and MAPK15, based on RNA sequencing data, and MAPK1, MMP2, TYK2, NOS3, and CASP3, based on antibody array data. In particular, CD37 is a unique gene that was detected and validated in all the methods applied in this study. By integrating the datasets obtained from these HTS studies and utilizing the strengths of each method, we obtained new insights into the functional role of YWHAE in skin keratinocytes. The approach used here could contribute to the clinical understanding of YWHAE-associated applications in the treatment of AD disease. AbbreviationsDAVIDthe database for annotation, visualization and integrated discoveryHTSHigh-throughput screeningKEGGKyoto Encyclopedia of Genes and GenomesPPIprotein-protein interactionsCommunicated by Ramaswamy H. Sarma.
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- 2019
18. An OMICS-based study of the role of C3dg in keratinocytes: RNA sequencing, antibody-chip array, and bioinformatics approaches
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Ye-Jin Chang, Yong-Doo Park, Myong-Joon Hahn, Jun-Mo Yang, Bit-Na Lee, Li-Li Zhang, Shang-Jun Yin, Hyunchang Kwak, and Jae-Rin Lee
- Subjects
Keratinocytes ,Microarray ,Protein Array Analysis ,02 engineering and technology ,Bioinformatics ,Biochemistry ,Antibodies ,03 medical and health sciences ,Structural Biology ,medicine ,Humans ,Molecular Biology ,Gene ,030304 developmental biology ,0303 health sciences ,biology ,Sequence Analysis, RNA ,Critical factors ,RNA ,Computational Biology ,General Medicine ,Atopic dermatitis ,Hep G2 Cells ,021001 nanoscience & nanotechnology ,Omics ,medicine.disease ,Peptide Fragments ,HaCaT ,Complement C3b ,biology.protein ,Antibody ,0210 nano-technology ,Algorithms - Abstract
Previously, we have identified the C3dg protein as an important player in the pathogenesis of atopic dermatitis (AD). In this study, we aimed to identify critical factors associated with C3dg in human keratinocytes based on high-throughput screening (HTS) approaches. We overexpressed C3dg in HaCaT human keratinocytes and conducted serial HTS studies, including RNA sequencing analysis integrated with antibody-chip arrays and implementation of bioinformatics algorithms (PPI mappings). Cumulatively, these approaches identified several novel C3dg-associated genes and proteins that are thought to be significantly involved in skin diseases including AD. These novel genes and proteins included LPA, PROZ, BLK, CLDN11, and FGF22, which are believed to play important roles in C3dg-associated skin functions in keratinocytes, as well as genes related to the two important pathways of systemic lupus erythematosus and Staphylococcus aureus infection. In particular, FGF22 is a unique gene that was detected and validated in all methods applied in this study. By integrating the datasets obtained from these HTS studies and utilizing the strengths of each method, we obtained new insights into the functional role of C3dg in keratinocytes. The approach used here contributes to clinical understanding of C3dg-associated applications and may also be applicable to treatment of AD.
- Published
- 2019
19. Kinetics for Zinc Ion Induced Sepia Pharaonis Arginine Kinase Inactivation and Aggregation
- Author
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Yue-Xiu Si, Jinhyuk Lee, Guo-Ying Qian, Xia-Min Jiang, Juan-Ge Cheng, Shang-Jun Yin, and Yong-Doo Park
- Subjects
inorganic chemicals ,0301 basic medicine ,Conformational change ,Sepia ,Arginine ,chemistry.chemical_element ,Zinc ,Molecular Dynamics Simulation ,Biochemistry ,Article ,Aggregation ,03 medical and health sciences ,Structural Biology ,Animals ,Amino Acid Sequence ,Proline ,Cloning, Molecular ,Binding site ,chemistry.chemical_classification ,Binding Sites ,biology ,Arginine Kinase ,Zn2+ ,General Medicine ,Arginine kinase ,inhibition ,Kinetics ,030104 developmental biology ,Enzyme ,chemistry ,Glycine ,biology.protein ,Sepia pharaonis ,Energy Metabolism ,Hydrophobic and Hydrophilic Interactions - Abstract
Arginine kinase is an essential enzyme which is closely related to energy metabolism in marine invertebrates. Arginine kinase provides a significant role in quick response to environmental change and stress. In this study, we simulated a tertiary structure of Sepia pharaonis arginine kinase (SPAK) based on the gene sequence and conducted the molecular dynamics simulations between SPAK and Zn(2+). Using these results, the Zn(2+) binding sites were predicted and the initial effect of Zn(2+) on the SPAK structure was elucidated. Subsequently, the experimental kinetic results were compared with the simulation results. Zn(2+) markedly inhibited the activity of SPAK in a manner of non-competitive inhibitions for both arginine and ATP. We also found that Zn(2+) binding to SPAK resulted in tertiary conformational change accompanying with the hydrophobic residues exposure. These changes caused SPAK aggregation directly. We screened two protectants, glycine and proline, which effectively prevented SPAK aggregation and recovered the structure and activity. Overall, our study suggested the inhibitory effect of Zn(2+) on SPAK and Zn(2+) can trigger SPAK aggregation after exposing large extent of hydrophobic surface. The protective effects of glycine and proline against Zn(2+) on SPAK folding were also demonstrated.
- Published
- 2016
20. Effects of cadmium on the cuttlefishSepia pharaonis’arginine kinase: unfolding kinetics integrated with computational simulations
- Author
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Shang-Jun Yin, Yong-Doo Park, Feng Zhao, Xia-Min Jiang, Jinhyuk Lee, Yue-Xiu Si, and Guo-Ying Qian
- Subjects
Models, Molecular ,0301 basic medicine ,Protein Folding ,030103 biophysics ,Arginine ,Molecular Conformation ,Context (language use) ,Molecular Dynamics Simulation ,Protein aggregation ,Protein Aggregates ,03 medical and health sciences ,Enzyme activator ,Structural Biology ,Animals ,Amino Acid Sequence ,Enzyme Inhibitors ,Molecular Biology ,Sepia pharaonis ,Binding Sites ,biology ,Chemistry ,Osmolar Concentration ,Decapodiformes ,Temperature ,Arginine Kinase ,General Medicine ,Hydrogen-Ion Concentration ,Arginine kinase ,biology.organism_classification ,Enzyme Activation ,Kinetics ,Biochemistry ,Osmolyte ,Glycine ,biology.protein ,Cadmium ,Protein Binding - Abstract
Arginine kinase is closely associated with adaptation to environmental stresses such as high salinity and heavy metal ion levels in marine invertebrates. In this study, the effects of Cd(2+) on the cuttlefish Sepia pharaonis' arginine kinase (SPAK) were investigated. SPAK was isolated from the muscles of S. pharaonis and upon further purification, showed a single band on SDS-PAGE. Cd(2+) effectively inactivated SPAK, and the double-reciprocal kinetics indicated that Cd(2+) induced non-competitive inhibition of arginine and ATP. Spectrofluorometry results showed that Cd(2+) induced tertiary structure changes in SPAK with the exposure of hydrophobic surfaces that directly induced SPAK aggregation. The addition of osmolytes, glycine, and proline successfully blocked SPAK aggregation and restored the conformation and activity of SPAK. Molecular dynamics simulations involving SPAK and Cd(2+) showed that Cd(2+) partly blocks the entrance of ATP to the active site, and this result is consistent with the experimental results showing Cd(2+)-induced inactivation of SPAK. These results demonstrate the effect of Cd(2+) on SPAK enzymatic function and unfolding, including aggregation and the protective effects of osmolytes on SPAK folding. This study provides concrete evidence of the toxicity of Cd(2+) in the context of the metabolic enzyme SPAK, and it illustrates the toxic effects of heavy metals and detoxification mechanisms in cuttlefish.
- Published
- 2015
21. Effects of osmolytes on Pelodiscus sinensis creatine kinase: A study on thermal denaturation and aggregation
- Author
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Wei Wang, Yong-Doo Park, Shang-Jun Yin, Yue-Xiu Si, Jinhyuk Lee, Qin-Xin Jin, Nai-Yun Fang, and Guo-Ying Qian
- Subjects
Models, Molecular ,Protein Denaturation ,Protein Conformation ,Molecular Sequence Data ,Biochemistry ,Mass Spectrometry ,chemistry.chemical_compound ,Structural Biology ,Animals ,Amino Acid Sequence ,Proline ,Molecular Biology ,biology ,Dimethyl sulfoxide ,Creatine Kinase, MM Form ,Spectrofluorometer ,General Medicine ,Turtles ,Enzyme Activation ,Kinetics ,Poikilotherm ,chemistry ,Docking (molecular) ,Osmolyte ,biology.protein ,Thermodynamics ,Creatine kinase ,Sequence Alignment ,Adenosine triphosphate - Abstract
The protective effect of osmolytes on the thermal denaturation and aggregation of Pelodiscus sinensis muscle creatine kinase (PSCK) was investigated by a combination of spectroscopic methods and thermodynamic analysis. Our results demonstrated that the addition of osmolytes, such as glycine and proline, could prevent thermal denaturation and aggregation of PSCK in a concentration-dependent manner. When the concentration of glycine and proline increased in the denatured system, the relative activation was significantly enhanced; meanwhile, the aggregation of PSCK during thermal denaturation was decreased. Spectrofluorometer results showed that glycine and proline significantly decreased the tertiary structural changes of PSCK and that thermal denaturation directly induced PSCK aggregation. In addition, we also built the 3D structure of PSCK and osmolytes by homology models. The results of computational docking simulations showed that the docking energy was relatively low and that the clustering groups were spread to the surface of PSCK, indicating that osmolytes directly protect the surface of the protein. P. sinensis are poikilothermic and quite sensitive to the change of ambient temperature; however, there were few studies on the thermal denaturation of reptile CK. Our study provides important insight into the protective effects of osmolytes on thermal denaturation and aggregation of PSCK.
- Published
- 2013
22. The effect of validamycin A on tyrosinase: Inhibition kinetics and computational simulation
- Author
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Jinhyuk Lee, Jun-Mo Yang, Guo-Ying Qian, Sunyoung Ji, Yue-Xiu Si, Zhi-Jiang Wang, and Shang-Jun Yin
- Subjects
Binding Sites ,Monophenol Monooxygenase ,Protein Conformation ,Chemistry ,Tyrosinase ,Kinetics ,General Medicine ,Inhibition kinetics ,Molecular Dynamics Simulation ,Validamycin ,Biochemistry ,Protein tertiary structure ,Enzyme Activation ,Molecular Docking Simulation ,Molecular dynamics ,chemistry.chemical_compound ,Structural Biology ,Docking (molecular) ,Enzyme Inhibitors ,Molecular Biology ,Inositol ,Protein Binding ,Validamycin A - Abstract
In this study, we investigated validamycin A as a tyrosinase inhibitor based on its structural properties. We found that the reversible inhibition of tyrosinase by validamycin A occurred in a mixed-type manner with Ki = 5.893 ± 0.038 mM, as determined by integrating kinetics studies and computational simulations. Time-interval tyrosinase studies showed that the inhibition followed first-order kinetics with two phases. Fluorescence measurements of ANS binding showed that validamycin A induced changes in the tertiary protein structure of tyrosinase. To obtain further insight, computational docking and molecular dynamics were applied, and the results indicated that HIS85, HIS244, GLU256, HIS259, and ASN260 of tyrosinase interacted with validamycin A. This strategy of predicting tyrosinase inhibition based on hydroxyl group numbers might be useful in the design and screening of potential tyrosinase inhibitors.
- Published
- 2013
23. Kinetics for Cu(2+) induced Sepia pharaonis arginine kinase inactivation and aggregation
- Author
-
Li-Li Zhang, Yong-Doo Park, Feng Wu, Yang-Yong Fu, Xiao-Yu Shi, Shang-Jun Yin, and Yue-Xiu Si
- Subjects
0301 basic medicine ,Sepia ,Arginine ,Proline ,Glycine ,010501 environmental sciences ,Protein aggregation ,01 natural sciences ,Biochemistry ,Protein Structure, Secondary ,03 medical and health sciences ,Enzyme activator ,chemistry.chemical_compound ,Protein Aggregates ,Non-competitive inhibition ,Adenosine Triphosphate ,Structural Biology ,Animals ,Molecular Biology ,Protein Kinase Inhibitors ,0105 earth and related environmental sciences ,biology ,Circular Dichroism ,General Medicine ,Arginine Kinase ,Arginine kinase ,Protein Structure, Tertiary ,Enzyme Activation ,Kinetics ,030104 developmental biology ,Spectrometry, Fluorescence ,chemistry ,Osmolyte ,biology.protein ,Adenosine triphosphate - Abstract
Arginine kinase plays an important role in cellular energy metabolism and is closely related to the environmental stress response in marine invertebrates. We studied the Cu(2+)-mediated inhibition and aggregation of Sepia pharaonis arginine kinase (SPAK) and found that Cu(2+) markedly inhibited the SPAK activity along with mixed-type inhibition against the arginine substrate and noncompetitive inhibition against the ATP cofactor. Spectrofluorimetry results showed that Cu(2+) induced a tertiary structure change in SPAK, resulting in exposure of the hydrophobic surface and increased aggregation. Cu(2+)-mediated SPAK aggregation followed first-order kinetics consistent with monophasic and a biphasic processes. Addition of osmolytes, including glycine and proline, effectively blocked SPAK aggregation and restored SPAK activity. Our results demonstrated the effects of Cu(2+) on SPAK catalytic function, conformation, and aggregation, as well as the protective effects of osmolytes on SPAK folding. This study provided important insights into the role of Cu(2+) as a negative effector of the S. pharaonis metabolic enzyme AK and the possible responses of cephalopods to unfavorable environmental conditions.
- Published
- 2016
24. The effect of fucoidan on tyrosinase: computational molecular dynamics integrating inhibition kinetics
- Author
-
Sangho Oh, Guo-Ying Qian, Yong-Doo Park, Jinhyuk Lee, Jun-Mo Yang, Yue-Xiu Si, Shang-Jun Yin, and Zhi-Jiang Wang
- Subjects
Spectrometry, Mass, Electrospray Ionization ,Molecular model ,Protein Conformation ,Pentamer ,Tyrosinase ,Kinetics ,Molecular Dynamics Simulation ,Random hexamer ,Ligands ,Polysaccharide ,chemistry.chemical_compound ,Polysaccharides ,Structural Biology ,Catalytic Domain ,Carbohydrate Conformation ,Enzyme Inhibitors ,Molecular Biology ,chemistry.chemical_classification ,biology ,Monophenol Monooxygenase ,Fucoidan ,Algal Proteins ,Active site ,General Medicine ,Spectrometry, Fluorescence ,Biochemistry ,chemistry ,Spectrophotometry ,Fucus ,biology.protein ,Thermodynamics ,Hydrophobic and Hydrophilic Interactions ,Copper ,Protein Binding - Abstract
Fucoidan is a complex sulfated polysaccharide extracted from brown seaweed and has a wide variety of biological activities. In this study, we investigated the inhibitory effect of fucoidan on tyrosinase via a combination of inhibition kinetics and computational simulations. Fucoidan reversibly inhibited tyrosinase in a mixed-type manner. Time-interval kinetics showed that the inhibition was processed as first order with biphasic processes. For further insight, we simulated dockings with various sizes of molecular models (monomer to decamer) of fucoidan and showed that the best binding energy change results were obtained from the pentamer (-1.89 kcal/mol) and the hexamer (-1.97 kcal/mol) models of AutoDock Vina. The molecular dynamics simulation confirmed the binding mechanisms between tyrosinase and fucoidan and suggested that fucoidan mostly interacts with several residues including copper ions located in the active site. Our study suggests that fucoidan might be a potential natural antipigment agent.
- Published
- 2012
25. Effect of hesperetin on tyrosinase: Inhibition kinetics integrated computational simulation study
- Author
-
Shang-Jun Yin, Su-Fang Wang, Hae Young Chung, Jun-Mo Yang, Yong-Doo Park, Li Yan, Yue-Xiu Si, Guo-Ying Qian, Daeui Park, and Zhi-Jiang Wang
- Subjects
Models, Molecular ,Tyrosinase ,Flavonoid ,Inhibition kinetics ,Biochemistry ,Anilino Naphthalenesulfonates ,Levodopa ,chemistry.chemical_compound ,Structural Biology ,Catalytic Domain ,Computer Simulation ,Chelation ,Molecular Biology ,Histidine ,Flavonoids ,chemistry.chemical_classification ,Binding Sites ,biology ,Monophenol Monooxygenase ,Chemistry ,Hesperidin ,Hesperetin ,food and beverages ,Active site ,General Medicine ,Flavones ,Kinetics ,Spectrometry, Fluorescence ,Models, Chemical ,Spectrophotometry ,Docking (molecular) ,biology.protein ,Algorithms - Abstract
Tyrosinase inhibitors have potential applications in medicine, cosmetics and agriculture to prevent hyperpigmentation or browning effects. Some of the flavonoids mostly found in herbal plants and fruits are revealed as tyrosinase inhibitors. We studied the inhibitory effects of one such flavonoid, hesperetin, on mushroom tyrosinase using inhibition kinetics and computational simulation. Hesperetin reversibly inhibited tyrosinase in a competitive manner with K i = 4.03 ± 0.26 mM. Measurements of ANS-binding fluorescence showed that hesperetin induced the hydrophobic disruption of tyrosinase. For further insight, we used the docking algorithms to simulate binding between tyrosinase and hesperetin. Simulation was successful (binding energies for Dock6.3: −34.41 kcal/mol and for AutoDock4.2: −5.67 kcal/mol) and showed that a copper ion coordinating with 3 histidine residues (HIS61, HIS85, and HIS259) within the active site pocket was chelated via hesperetin binding. Our study provides insight into the inhibition of tyrosinase in response to flavonoids. A combination of inhibition kinetics and computational prediction may facilitate the identification of potential natural tyrosinase inhibitors such as flavonoids and the prediction of their inhibitory mechanisms.
- Published
- 2012
26. Trifluoroethanol-induced Activity and Structural Changes in Bos taurus Copper- and Zinc-containing Superoxide Dismutase
- Author
-
Long Shi, Zhi-Rong Lü, Hai-Meng Zhou, Fei Zou, Daeui Park, Hae Young Chung, Yue-Xiu Si, Shang-Jun Yin, Yong Xia, Yong-Doo Park, Ming Zhang, and Guo-Ying Qian
- Subjects
chemistry.chemical_classification ,biology ,Superoxide Dismutase ,Stereochemistry ,Kinetics ,chemistry.chemical_element ,Active site ,Trifluoroethanol ,General Medicine ,Biochemistry ,Copper ,Enzyme assay ,Enzyme Activation ,Chemical kinetics ,Superoxide dismutase ,Crystallography ,Enzyme ,chemistry ,Structural Biology ,Catalytic Domain ,biology.protein ,Animals ,Cattle ,Binding site - Abstract
Superoxide dismutase (SOD, EC 1.15.1.1) plays an important antioxidant defense role in organisms exposed to oxygen. Copper- and zinc-containing SOD (Cu/Zn-SOD) catalysis and the change in folding behavior of this enzyme in response to inactivators are therefore of interest. We studied the inhibitory effects of trifluoroethanol (TFE) on the activity and conformation of a Cu/Zn-SOD from Bos taurus. We found that TFE inactivated the enzyme and disrupted the tertiary and secondary structures of Cu/Zn-SOD. Kinetic studies showed that TFE-induced inactivation of Cu/Zn-SOD follows first-order reaction kinetics and that TFE binding sites are distinct from the copper- and zinc-containing active site. These structural changes occurred prior to enzyme activity loss. A computational docking simulation of Cu/Zn-SOD and TFE (binding energy of Dock 6.3: -11.52 kcal/mol) suggested that THR37, ASP40, and GLU119, which are located near the active site, interact with TFE. Evaluation of the ligand binding kinetics of Cu/Zn-SOD during unfolding in the presence of TFE combined with computational prediction allowed us to gain insight into the inactivation of Cu/Zn-SOD.
- Published
- 2011
27. A folding study of Antarctic krill (Euphausia superba) alkaline phosphatase using denaturants
- Author
-
Yong-Doo Park, Shang-Jun Yin, Yue-Xiu Si, Wei Wang, Jinhyuk Lee, Jun-Mo Yang, Guo-Ying Qian, and Zhi-Jiang Wang
- Subjects
Protein Denaturation ,Protein Folding ,Krill ,Euphausia ,Biochemistry ,chemistry.chemical_compound ,Structural Biology ,Animals ,Urea ,Sodium dodecyl sulfate ,Molecular Biology ,Guanidine ,Chromatography ,biology ,Active site ,Sodium Dodecyl Sulfate ,General Medicine ,biology.organism_classification ,Alkaline Phosphatase ,Enzyme Activation ,chemistry ,Antarctic krill ,Sephadex ,biology.protein ,Alkaline phosphatase ,Thermodynamics ,Euphausiacea - Abstract
To gain insight into the structural and folding mechanisms of Antarctic krill alkaline phosphatase (ALP), the enzyme was properly purified by (NH4)2SO4 fractionation and by both Sephadex G-75 and DEAE anion exchange chromatography. The purified enzyme (62.6 kDa; 2.62 unit/mg) was unstable at temperatures exceeding 30°C. Denaturants, such as sodium dodecyl sulfate (SDS), guanidine HCl, and urea, were applied to evaluate the folding mechanism, including kinetics and thermodynamics, of krill ALP. Sodium dodecyl sulfate elicited no significant effect on ALP activity even at excessively high concentrations (300 mM), whereas guanidine HCl and urea effectively inactivated the enzyme at concentrations of 2 and 3.5 M, respectively. Kinetic studies showed that the enzymatic inhibition by guanidine HCl and urea represented a first-order reaction that was a monophasic unfolding process. This process was found to be associated with conformational changes without significant transient free-energy changes. Additionally, the overall structural changes occurred proximally to the active site pocket. Our study provides new insight into ALP of the Antarctic krill, which lives in extreme environmental conditions.
- Published
- 2014
28. Purification, characterization, and unfolding studies of arginine kinase from Antarctic krill
- Author
-
Jun-Mo Yang, Guo-Ying Qian, Yue-Xiu Si, Wei Wang, Jin-Jie Song, Yong-Doo Park, Shang-Jun Yin, Nai-Yun Fang, and Zhi-Jiang Wang
- Subjects
Protein Folding ,Kinetics ,Biochemistry ,chemistry.chemical_compound ,Structural Biology ,Enzyme Stability ,Animals ,Sodium dodecyl sulfate ,Molecular Biology ,chemistry.chemical_classification ,biology ,Molecular mass ,Temperature ,General Medicine ,Arginine Kinase ,Arginine kinase ,biology.organism_classification ,Fluorescence ,Monomer ,Enzyme ,chemistry ,Antarctic krill ,biology.protein ,Euphausiacea - Abstract
The regulation of enzymatic activity and unfolding studies of arginine kinase (AK) from various invertebrates have been the focus of investigation. To gain insight into the structural and folding mechanisms of AK from Euphausia superba (ESAK), we purified ESAK from muscle properly. The enzyme behaved as a monomeric protein with a molecular mass of about 40 kDa and had pH and temperature optima of 8.0 and 30 °C, respectively. The K m Arg and K m ATP for the synthesis of phosphoarginine were 0.30 and 0.47 mM, respectively, and k cat / K m A r g was 282.7 s−1/mM. A study of the inhibition kinetics of structural unfolding in the denaturant sodium dodecyl sulfate (SDS) was conducted. The results showed that ESAK was almost completely inactivated by 1.0 mM SDS. The kinetics analyzed via time-interval measurements revealed that the inactivation was a first-order reaction, with the kinetic processes shifting from a monophase to biphase as SDS concentrations increased. Measurements of intrinsic and 1-anilinonaphthalene-8-sulfonate-binding fluorescence showed that SDS concentrations lower than 5 mM did not induce conspicuous changes in tertiary structures, while higher concentrations of SDS exposed hydrophobic surfaces and induced conformational changes. These results confirmed that the active region of AK is more flexible than the overall enzyme molecule.
- Published
- 2013
29. The effect of Zn(2+) on Pelodiscus sinensis creatine kinase: unfolding and aggregation studies
- Author
-
Jinhyuk Lee, Yue-Xiu Si, Caiyan Li, Wei Wang, Jun-Mo Yang, Su-Fang Wang, Guo-Ying Qian, Shang-Jun Yin, and Tae-Rae Kim
- Subjects
inorganic chemicals ,Protein Folding ,DNA, Complementary ,Kinetics ,Molecular Sequence Data ,Glycine ,Creatine ,chemistry.chemical_compound ,Structural Biology ,Complementary DNA ,Catalytic Domain ,Animals ,Proline ,Amino Acid Sequence ,Molecular Biology ,Creatine Kinase ,Phylogeny ,biology ,Active site ,General Medicine ,Turtles ,Zinc ,chemistry ,Biochemistry ,Osmolyte ,biological sciences ,health occupations ,biology.protein ,bacteria ,Creatine kinase ,Hydrophobic and Hydrophilic Interactions ,Sequence Alignment - Abstract
We studied the effects of Zn(2+) on creatine kinase from the Chinese soft-shelled turtle, Pelodiscus sinensis (PSCK). Zn(2+) inactivated the activity of PSCK (IC(50) = .079 ± .004 mM) following first-order kinetics consistent with multiple phases. The spectrofluorimetry results showed that Zn(2+) induced significant tertiary structural changes of PSCK with exposure to hydrophobic surfaces and that Zn(2+) directly induced PSCK aggregation. The addition of osmolytes such as glycine, proline, and liquaemin successfully blocked PSCK aggregation, recovering the conformation and activity of PSCK. We measured the ORF gene sequence of PSCK by rapid amplification of cDNA end and simulated the 3D structure of PSCK. The results of molecular dynamics simulations showed that eight Zn(2+) bind to PSCK and one Zn(2+) is predicted to bind in a plausible active site of creatine and ATP. The interaction of Zn(2+) with the active site could mostly block the activity of PSCK. Our study provides important insight into the action of Zn(2+) on PSCK as well as more insights into the PSCK folding and ligand-binding mechanisms, which could provide important insight into the metabolic enzymes of P. sinensis.
- Published
- 2012
30. An integrated study of tyrosinase inhibition by rutin: progress using a computational simulation
- Author
-
Jinhyuk Lee, Li Yan, Jun-Mo Yang, Sangho Oh, Sen Ye, Shang-Jun Yin, Yong-Doo Park, Zhi-Jiang Wang, Yue-Xiu Si, and Guo-Ying Qian
- Subjects
Models, Molecular ,Stereochemistry ,Tyrosinase ,Rutin ,Kinetics ,Molecular Dynamics Simulation ,Fluorescence ,chemistry.chemical_compound ,Structural Biology ,Catalytic Domain ,Humans ,Binding site ,Enzyme Inhibitors ,Molecular Biology ,Histidine ,Cells, Cultured ,Skin ,biology ,Chemistry ,Monophenol Monooxygenase ,Active site ,General Medicine ,Protein Structure, Tertiary ,HaCaT ,Docking (molecular) ,biology.protein ,Hydrophobic and Hydrophilic Interactions ,Copper - Abstract
Tyrosinase inhibition studies have recently gained the attention of researchers due to their potential application values. We simulated docking (binding energies for AutoDock Vina: -9.1 kcal/mol) and performed a molecular dynamics simulation to verify docking results between tyrosinase and rutin. The docking results suggest that rutin mostly interacts with histidine residues located in the active site. A 10 ns molecular dynamics simulation showed that one copper ion at the tyrosinase active site was responsible for the interaction with rutin. Kinetic analyses showed that rutin-mediated inactivation followed a first-order reaction and mono- and biphasic rate constants occurred with rutin. The inhibition was a typical competitive type with K(i) = 1.10±0.25 mM. Measurements of intrinsic and ANS-binding fluorescences showed that rutin showed a relatively strong binding affinity for tyrosinase and one possible binding site that could be a copper was detected accompanying with a hydrophobic exposure of tyrosinase. Cell viability testing with rutin in HaCaT keratinocytes showed that no toxic effects were produced. Taken together, rutin has the potential to be a potent anti-pigment agent. The strategy of predicting tyrosinase inhibition based on hydroxyl group number and computational simulation may prove useful for the screening of potential tyrosinase inhibitors.
- Published
- 2012
31. Folding studies on muscle type of creatine kinase from Pelodiscus sinensis
- Author
-
Guo-Ying Qian, Shang-Jun Yin, Su-Fang Wang, Jun-Mo Yang, Yue-Xiu Si, and Zhi-Jiang Wang
- Subjects
Protein Folding ,Kinetics ,Guanidine hcl ,Muscle type ,Biochemistry ,chemistry.chemical_compound ,Structural Biology ,Animals ,Urea ,Molecular Biology ,Guanidine ,chemistry.chemical_classification ,biology ,Pelodiscus ,Creatine Kinase, MM Form ,Sodium Dodecyl Sulfate ,General Medicine ,biology.organism_classification ,Protein Structure, Tertiary ,Turtles ,Folding (chemistry) ,Enzyme Activation ,Enzyme ,chemistry ,biology.protein ,Creatine kinase - Abstract
A folding study of creatine kinase from Pelodiscus sinensis has not yet been reported. To gain more insight into structural and folding mechanisms of P. sinensis CK (PSCK), denaturants such as SDS, guanidine HCl, and urea were applied in this study. We purified PSCK from the muscle of P. sinensis and conducted inhibition kinetics with structural unfolding studies under various conditions. The results revealed that PSCK was completely inactivated at 1.8 mM SDS, 1.05 M guanidine HCl, and 7.5 M urea. The kinetics via time-interval measurements showed that the inactivation by SDS, guanidine HCl, and urea were all first-order reactions with kinetic processes shifting from monophase to biphase at increasing concentrations. With respect to tertiary structural changes, PSCK was unfolded in different ways; SDS increased the hydrophobicity but retained the most tertiary structural conformation, while guanidine HCl and urea induced conspicuous changes in tertiary structures and initiated kinetic unfolding mechanisms. Our study provides information regarding PSCK and enhances our knowledge of the reptile-derived enzyme folding.
- Published
- 2012
32. The effect of thiobarbituric acid on tyrosinase: inhibition kinetics and computational simulation
- Author
-
Jinhyuk Lee, Zhi Jiang Wang, Sangho Oh, Guo Ying Qian, Sanghyuk Lee, Yong-Doo Park, Yue Xiu Si, Jun-Mo Yang, Seon Mi Sim, Shang Jun Yin, and Su Fang Wang
- Subjects
Models, Molecular ,Stereochemistry ,Thiobarbituric acid ,Protein Conformation ,Tyrosinase ,Inhibition kinetics ,Computational simulation ,chemistry.chemical_compound ,Structure-Activity Relationship ,Structural Biology ,Catalytic Domain ,Mole ,Humans ,Computer Simulation ,Molecular Biology ,IC50 ,Cells, Cultured ,biology ,Dose-Response Relationship, Drug ,Chemistry ,Monophenol Monooxygenase ,Active site ,General Medicine ,Thiobarbiturates ,Kinetics ,Docking (molecular) ,biology.protein ,Copper - Abstract
Tyrosinase plays various roles in organisms and much research has focused on the regulation of tyrosinase activity. We studied the inhibitory effect of thiobarbituric acid (TBA) on tyrosinase. Our kinetic study showed that TBA inhibited tyrosinase in a reversible noncompetitive manner (K(i) 5 14.0 ± 8.5 mM and IC₅₀ 5 8.0 ± 1.0 mM). Intrinsic and ANS-binding fluorescences studies were also performed to gain more information regarding the binding mechanism. The results showed that no tertiary structural changes were obviously observed. For further insight, we predicted the 3D structure of tyrosinase and simulated the docking between tyrosinase and TBA. The docking simulation was successful with significant scores (binding energy for AutoDock4: -5.52 kcal/mol) and suggested that TBA was located in the active site. The 11 ns molecular dynamics simulation convinced that the four HIS residues (residue numbers: 57, 90, 250, and 282) were commonly responsible for the interaction with TBA. Our results provide a new inhibition strategy that works using an antioxidant rather than targeting the copper ions within the tyrosinase active site.
- Published
- 2011
33. Tyrosinase inhibition by isophthalic acid: kinetics and computational simulation
- Author
-
Hai-Meng Zhou, Zhi-Rong Lü, Hae Young Chung, Yong-Doo Park, Jun-Mo Yang, Li Yan, Yue-Xiu Si, Daeui Park, Guo-Ying Qian, and Shang-Jun Yin
- Subjects
Models, Molecular ,Stereochemistry ,Monophenol Monooxygenase ,Tyrosinase ,Kinetics ,Phthalic Acids ,General Medicine ,Inhibition kinetics ,bacterial infections and mycoses ,Biochemistry ,Fluorescence ,Protein tertiary structure ,respiratory tract diseases ,Isophthalic acid ,Computational simulation ,chemistry.chemical_compound ,chemistry ,Structural Biology ,Docking (molecular) ,Computer Simulation ,Enzyme Inhibitors ,skin and connective tissue diseases ,Molecular Biology - Abstract
Using inhibition kinetics and computational simulation, we studied the reversible inhibition of tyrosinase by isophthalic acid (IPA). IPA inhibited tyrosinase in a complex manner with Ki = 17.8 ± 1.8 mM. Measurements of intrinsic and ANS-binding fluorescence showed that IPA induced no changes in tertiary protein structure. For further insight, we predicted the 3D structure of tyrosinase and used a docking algorithm to simulate binding between tyrosinase and IPA. Simulation was successful (binding energies for Dock6.3: −25.19 kcal/mol and for AutoDock4.2: −4.28 kcal/mol), suggesting that IPA interacts with PRO175 or VAL190. This strategy of predicting tyrosinase inhibition based on hydroxyl group number and orientation may prove useful for the screening of potential tyrosinase inhibitors.
- Published
- 2011
34. High-throughput integrated analyses for the tyrosinase-induced melanogenesis: microarray, proteomics and interactomics studies
- Author
-
Zhi-Rong Lü, Eun Young Seo, Yue-Xiu Si, Shang-Jun Yin, Yong-Doo Park, Guo-Ying Qian, Jun-Mo Yang, and Li Yan
- Subjects
Chemical compound microarray ,Genetics ,Melanins ,Proteomics ,Candidate gene ,Microarray ,RNA-BINDING MOTIF PROTEIN 9 ,Monophenol Monooxygenase ,Tyrosinase ,HEK 293 cells ,General Medicine ,Computational biology ,Biology ,Cell Line ,Up-Regulation ,Structural Biology ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Animals ,Humans ,Cloning, Molecular ,Molecular Biology ,Gene ,Oligonucleotide Array Sequence Analysis - Abstract
The tyrosinase gene was overexpressed in HEK293 cells, and then a DNA microarray and proteomic tools were applied to detect the dysregulated genes in highly pigmented cells. The candidate genes from the microarray were compared to the yeast two-hybridization results. Computational prediction via protein-protein interaction mapping suggested the existence of 66 hub genes in melanogenesis. Most importantly, RNA binding motif protein 9 is newly detected as a putative critical melanogenesis-associated gene in this study. The approach of combining the expression data analysis and predicted protein interaction partners performed in large scales can bring more reliable gene targets for understanding pigmentation.
- Published
- 2010
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