Inhibitory effect of acarbose on tyrosinase: application of molecular dynamics integrating inhibition kinetics.

Due to its clinical and cosmetic applications, investigators have paid attention to tyrosinase (TYR) inhibitor development. In this study, a TYR inhibition study with acarbose was investigated to gain insights into the regulation of the catalytic function. Biochemical assay results indicated that acarbose was turned to be an inhibitor of TYR in a reversible binding manner and probed as a distinctive mixed-type inhibitor via measurement of double-reciprocal kinetic (Ki = 18.70 ± 4.12 mM). Time-interval kinetic measurement indicated that TYR catalytic function was gradually inactivated by acarbose in a time-dependent behavior displaying with a monophase process that was evaluated by semi-logarithmic plotting. Spectrofluorimetric measurement by integrating with a hydrophobic residue detector (1-anilinonaphthalene-8-sulfonate) showed that the high dose of acarbose derived a conspicuous local structural deformation of the TYR catalytic site pocket. Computational docking simulation showed that acarbose bound to key residues such as HIS61, TYR65, ASN81, HIS244, and HIS259. Our study extends an understanding of the functional application of acarbose and proposes that acarbose is an alternative candidate drug for a whitening agent via direct retardation of TYR catalytic function and it would be applicable for the relevant skin hyperpigmentation disorders concerning the dermatologic clinical purpose.Communicated by Ramaswamy H. Sarma.

Seasonal spermatogenesis, epididymal storage, and creatine kinase expression in Pelodiscus sinensis.

The soft-shelled turtle, Pelodiscus sinensis, is an important economic aquaculture species. Its reproduction exhibits seasonality; however, there is a lack of systematic studies focused on sperm maturation and epididymal storage. The testes and epididymides of P. sinensis were sampled from March to December. The seasonal reproduction and maturation of the spermatozoa were examined by anatomy, hematoxylin and eosin staining, AB-PAS staining, and immunohistochemistry. Spermatogenesis exhibited obvious seasonality in P. sinensis. It was found that the spermatogenic epithelium was most active during June to September, whereas the diameter of the epididymal tubules was smallest during June to October. As key enzymes of ATP metabolism, creatine kinases were highly expressed in the epididymal tubule epithelium during the breeding season, which may be important for the regulation of sperm maturation. In addition, the epididymal tubule epithelium changed with the season in June to September, the epididymal tubule epithelium proliferated to form villous structures, and secreted a large number of glycoproteins, which may be related to the rapid maturation of sperm during the breeding season. In conclusion, this study provided insights into the spermatogenesis of P. sinensis through histological analysis and enriched our understanding of reproduction in reptiles.

Study on the pattern of spermatogenesis during the breeding season of the Chinese soft-shelled turtle, Pelodiscus sinensis (Reptilia: Trionychidae).

The Chinese soft-shelled turtle, Pelodiscus sinensis (Reptilia: Trionychidae) is a typical seasonal breeding species and its spermatogenesis pattern is complex. In this study, the process of sperm cell development was studied using histology. The process of sperm cell development may be divided into six stages based on a combination of different cell types in the seminiferous epithelium. A close examination revealed two patterns of sperm cell development in the seminiferous tubules during the breeding season. The first is a normal sperm cell development pattern, in which the process of sperm cell development and maturation are completed in the seminiferous epithelium without round spermatozoa in the lumen. The second is rapid sperm cell development, in which the first batches of round spermatozoa fall off the seminiferous epithelium before they mature, thus beginning a second batch of sperm cell development. The round sperm cells are shed into the lumen and further mature in the seminiferous tubules and epididymis. This rapid sperm cell development process of the Chinese soft-shelled turtle is rare in other vertebrate species and may be an adaptation to cope with seasonal breeding. The results of this study provide insight into the theory of seasonal reproduction in reptiles.

Characterization and tissue expression analysis of mitochondrial creatine kinases (types I and II) from Pelodiscus sinensis.

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.

Characterization and activity-folding relationship of serine protease from Antarctic krill (Euphausia superba).

Euphausia superba (Antarctic krill) serine protease (ESP) was investigated to gain insights into the activity-structural relationship, folding behavior, and regulation of the catalytic function. We purified ESP from the krill muscle and characterized biochemical distinctions via enzyme kinetics. Studies of inhibition kinetics and unfolding in the presence of a serine residue modifier, such as phenylmethanesulfonyl fluoride, were conducted. Structural characterizations were measured by spectrofluorimetry, including 1-anilinonaphthalene-8-sulfonate dye labeling for hydrophobic residues. The computational simulations such as docking and molecular dynamics were finally conducted to detect key residues and folding behaviors in a nano-second range. The kinetic parameters of ESP were measured as KmBANH = 0.97 ± 0.15 mM and kcat/KmBANH = 4.59 s-1/mM. The time-interval kinetics measurements indicated that ESP inactivation was transformed from a monophase to a biphase process to form a thermodynamically stable state. Spectrofluorimetry measurements showed that serine is directly connected to the regional folding of ESP. Several osmolytes such as proline and glycine only partially protected the inactive form of ESP by serine modification. Computational molecular dynamics and docking simulations showed that three serine residues (Ser183, Ser188, and Ser207) and Cys184, Val206, and Gly209 are key residues of catalytic functions. Our study revealed the functional roles of serine residues as key residues of catalytic function at the active site and of the structural conformation as key folding factors, where ESP displays a flexible property of active site pocket compared to the overall structure.Communicated by Ramaswamy H. Sarma.

A study of Pb2+ induced unfolding and aggregation of arginine kinase from Euphausia superba: kinetics and computational simulation integrating study.

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 investigated the Pb2+-induced inhibition and aggregation of Euphausia superba arginine kinase (ESAK) and found that significantly inactivated ESAK in a dose-dependent manner (IC50 = 0.058 ± 0.002 mM). Spectrofluorimetry results showed that Pb2+ induced tertiary structural changes via the internal polarity increased and the non-polarity decreased in ESAK and directly induced ESAK aggregation. The ESAK aggregation process induced by Pb2+ occurred with multi-phase kinetics. The addition of osmolytes did not show protective effect on Pb2+-induced inactivation of ESAK. The computational molecular dynamics (MD) simulation showed that three Pb2+ interrupt the entrance of the active site of ESAK and it could be the reason on the loss of activity of ESAK. Several important residues of ESAK were detected that were importantly contributed the conformation and catalytic function of ESAK. Our study showed that Pb2+-induced misfolding of ESAK and the complete loss of activity irreversibly, which cannot be recovered by osmolytes.Communicated by Ramaswamy H. Sarma.

Seasonal expression of cytoplasmic creatine kinase in the epididymal epithelium of Pelodiscus sinensis.

During hibernation of Pelodiscus sinensis, sperm mature and are stored in the epididymis. We investigated seasonal changes in the morphology of epithelial cells of the epididymis of P. sinensis and changes in expression of cytoplasmic creatine kinase (CK). We found that the epididymal epithelium proliferates rapidly to form multiple layers from June to September, while the epididymal epithelial cells are arranged in a single layer from October to May. From the March before the mating period to the end of the mating period in September, a large amount of neutral glycoprotein is secreted in the epididymal epithelium and in the sperm aggregation area; after October, the glycoprotein in the epididymis decreases. At sperm maturation, cytoplasmic CK is expressed abundantly in the villous epithelium, which is formed by proliferation of epididymal epithelial cells. During hibernation and reproduction, the epididymal epithelium of P. sinensis exhibits different proliferation and secretion patterns as the animal adapts to two types of sperm storage. Cytoplasmic CK may participate in regulating the energy metabolism of the epididymal epithelium; it is an important enzyme for regulating sperm maturation.

Detection of Melanogenesis and Anti-Apoptosis-Associated Melanoma Factors: Array CGH and PPI Mapping Integrating Study.

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.

A novel bifunctional glucanase exhibiting high production of glucose and cellobiose from rumen bacterium.

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 ß-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-ß-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.

Functional study of acetaldehyde dehydrogenase 1 (ALDH1) in keratinocytes: microarray integrating bioinformatics approaches.

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.

A Knock-Down Cell-Based Study for the Functional Analysis of Chloride Intracellular Channel 1 (CLIC1): Integrated Proteomics and Microarray Study.

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.

Tyrosinase-mediated melanogenesis in melanoma cells: Array comparative genome hybridization integrating proteomics and bioinformatics studies.

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.

Biochemical Study of Fibrinolytic Protease from Euphausia superba Possessing Multifunctional Serine Protease Activity.

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.

Comparative studies of the expression of creatine kinase isoforms under immune stress in Pelodiscus sinensis.

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.

Inhibitory effect of α-ketoglutaric acid on α-glucosidase: integrating molecular dynamics simulation and inhibition kinetics.

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 IC50 was measured (IC50 = 1.738 ± 0.041 mM), and the dissociation constant was determined (Ki Slope = 0.46 ± 0.04 mM). Regarding the relationship between structure and activity, a high AKG concentration induced the slight modulation of the shape of the active site, as monitored by hydrophobic exposure. This tertiary conformational change was linked to AKG inhibition and mostly involved regional changes in the active site. Our study provides insight into the functional role of AKG due to its structural property of a hydroxyphenyl ring that interacts with the active site. We suggest that similar hydroxyphenyl ring-containing compounds targeting key residues in the active site might be potential α-glucosidase inhibitors. AbbreviationsAKGalpha-ketoglutaric acidpNPG4-nitrophenyl-α-d-glucopyranosideANS1-anilinonaphthalene-8-sulfonateMDmolecular dynamics.Communicated by Ramaswamy H. Sarma.

Functional study of 14-3-3 protein epsilon (YWHAE) in keratinocytes: microarray integrating bioinformatics approaches.

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.

Screening and analysis of agouti signaling protein interaction partners in Pelodiscus sinensis suggests a role in lipid metabolism.

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.

Characterization of Thermostable and Chimeric Enzymes via Isopeptide Bond-Mediated Molecular Cyclization.

Mannooligosaccharides are released by mannan-degrading endo-ß-1,4-mannanase and are known as functional additives in human and animal diets. To satisfy demands for biocatalysis and bioprocessing in crowed environments, in this study, we employed a recently developed enzyme-engineering system, isopeptide bond-mediated molecular cyclization, to modify a mesophilic mannanase from Bacillus subtilis. The results revealed that the cyclized enzymes showed enhanced thermostability and ion stability and resilience to aggregation and freeze-thaw treatment by maintaining their conformational structures. Additionally, by using the SpyTag/SpyCatcher system, we generated a mannanase-xylanase bifunctional enzyme that exhibited a synergistic activity in substrate deconstruction without compromising substrate affinity. Interestingly, the dual-enzyme ring conformation was observed to be more robust than the linear enzyme but inferior to the single-enzyme ring conformation. Taken together, these findings provided new insights into the mechanisms of molecular cyclization on stability improvement and will be useful in the production of new functional oligosaccharides and feed additives.

An OMICS-based study of the role of C3dg in keratinocytes: RNA sequencing, antibody-chip array, and bioinformatics approaches.

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.

The inhibitory effect of pyrogallol on tyrosinase activity and structure: Integration study of inhibition kinetics with molecular dynamics simulation.

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 L­DOPA 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 L­DOPA and its induced mechanism was the parabolic non-competitive inhibition type (IC50 = 0.772 ±â€¯0.003 mM and Ki = 0.529 ±â€¯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.