RESUMO
Microbiota from herbivore rumen is of great interest for mining glycoside hydrolases for lignocellulosic biomass biorefinement. We previously isolated a highly active but poorly thermostable xylanase (LXY) from a rumen fluid fosmid library of Hu sheep, a local high-reproductive species in China. In this study, we used a universal enzyme-engineering strategy called SpyTag/SpyCatcher molecular cyclization to improve LXY stability via isopeptide-bond-mediated ligation. Both linear and cyclized LXY (L- and C-LXY, respectively) shared similar patterns of optimal pH and temperature, pH stability, and kinetic constants (km and Vmax). However, the C-LXY showed enhanced thermostability, ion stability, and resilience to aggregation and freeze-thaw treatment than L-LXY, without compromise of its catalytic efficiency. Circular dichroism and intrinsic and 8-anilino-1-naphthalenesulfonic acid-binding fluorescence analysis indicated that the cyclized enzyme was more capable of maintaining its secondary and tertiary structures than the linear enzyme. Taken together, these results promote the cyclized enzyme for potential applications in the feed, food, paper pulp, and bioenergy industries.
Assuntos
Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/genética , Engenharia de Proteínas/métodos , Rúmen/enzimologia , Animais , Catálise , Dicroísmo Circular , Ciclização , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Ovinos , TermodinâmicaRESUMO
Cholesterol biosynthesis is tightly regulated in the cell. For example, high sterol concentrations can stimulate degradation of the rate-limiting cholesterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, HMGCR). HMGCR is broken down by the endoplasmic reticulum membrane-associated protein complexes consisting of insulin-induced genes (Insigs) and the E3 ubiquitin ligase gp78. Here we found that HMGCR degradation is partially blunted in Chinese hamster ovary (CHO) cells lacking gp78 (gp78-KO). To identify other ubiquitin ligase(s) that may function together with gp78 in triggering HMGCR degradation, we performed a small-scale short hairpin RNA-based screening targeting endoplasmic reticulum-localized E3s. We found that knockdown of both ring finger protein 145 (Rnf145) and gp78 genes abrogates sterol-induced degradation of HMGCR in CHO cells. We also observed that RNF145 interacts with Insig-1 and -2 proteins and ubiquitinates HMGCR. Moreover, the tetrapeptide sequence YLYF in the sterol-sensing domain and the Cys-537 residue in the RING finger domain were essential for RNF145 binding to Insigs and RNF145 E3 activity, respectively. Of note, amino acid substitutions in the YLYF or of Cys-537 completely abolished RNF145-mediated HMGCR degradation. In summary, our study reveals that RNF145, along with gp78, promotes HMGCR degradation in response to elevated sterol levels and identifies residues essential for RNF145 function.
Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hidroximetilglutaril-CoA Redutases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteólise , Receptores do Fator Autócrino de Motilidade/metabolismo , Esteróis/farmacologia , Animais , Células CHO , Cricetinae , Cricetulus , Retículo Endoplasmático/efeitos dos fármacos , Humanos , Hidroximetilglutaril-CoA Redutases/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Receptores do Fator Autócrino de Motilidade/genética , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
One new prenylated benzenoid, (±)-chevalieric acid (1), and four new anthraquinone derivatives, (10S,12S)-, (10S,12R)-, (10R,12S)-, and (10R,12R)-chevalierone (2-5), together with ten previously described compounds (6-15), were isolated from the fungus Aspergillus chevalieri (L. Mangin) Thom and Church. The structures of new compounds were elucidated by extensive 1D and 2D nuclear magnetic resonance (NMR), and HRESIMS spectroscopic analysis. The absolute configurations of 2-5 were determined by experimental and calculated electronic circular dichroism (ECD) and DP4+ analysis. Compound 10 showed weak cytotoxicity against human lung cancer cell line A549 with IC50 39.68 µM. Compounds 2-5 exhibited antibacterial activities against the methicillin-resistant Staphylococcus aureus (MRSA) and opportunistic pathogenic bacterium Pseudomonas aeruginosa. The MIC value for compound 6 against MRSA is 44.02 µM. Additionally, Compounds 8, 10, 11 showed weak to moderate inhibitory activities against the ß-secretase (BACE1), with IC50 values of 36.1, 40.9, 34.9 µM, respectively.
RESUMO
The nonselective membrane disruption of antimicrobial peptides (AMPs) helps in combating the antibacterial resistance. But their overall positive charges lead to undesirable hemolysis and toxicity toward normal living cells, as well as the rapid clearance from blood circulation. In consequence, developing smart AMPs to optimize the antimicrobial outcomes is highly urgent. Relying on the local acidity of microbial infection sites, in this work, we designed an acidity-triggered charge reversal nanotherapeutics with adaptable geometrical morphology for bacterial targeting and optimized therapy. C16-A3K4-CONH2 was proposed and the ε-amino groups in lysine residues were acylated by dimethylmaleic amide (DMA), enabling the generated C16-A3K4(DMA)-CONH2 to self-assemble into negatively charged spherical nanostructure, which relieved the protein adsorption and prolonged blood circulation in vivo. After the access of C16-A3K4(DMA)-CONH2 into the microbial infection sites, acid-sensitive ß-carboxylic amide would hydrolyze to regenerate the positive C16-A3K4-CONH2 to destabilize the negatively charged bacterial membrane. In the meanwhile, attractively, the self-assembled spherical nanoparticle transformed to rod-like nanostructure, which was in favor of the efficient binding with bacterial membranes due to the larger contact area. Our results showed that the acid-activated AMP nanotherapeutics exhibited strong and broad-spectrum antimicrobial activities against Yeast, Gram-positive Staphylococcus aureus, Gram-negative Escherichia coli, and methicillin-resistant Staphylococcus aureus (MRSA). Moreover, the biocompatible lipopeptide nanotherapeutics dramatically improved the dermapostasis caused by bacterial infection. The strategy of merging pathology-activated therapeutic function and morphological adaptation to augment therapeutic outcomes shows the great potential for bacterial inhibition. STATEMENT OF SIGNIFICANCE: The overall positive charges of antimicrobial peptides (AMPs) lead to undesirable hemolysis and nonselective toxicity, as well as the rapid clearance from blood circulation. Infection-activated lipopeptide nanotherapeutics with adaptable geometrical morphology were developed to address these issues. The self-assembled lipopeptide was pre-decorated to reverse the positive charge to reduce the hemolysis and nonselective cytotoxicity. After accessing the acidic infection sites, the nanotherapeutics recovered the positive charge to destabilize negatively charged bacterial membranes. Meanwhile, the morphology of self-assembled nanotherapeutics transformed from spherical nanoparticles to rod-like nanostructures in the lesion site, facilitating the improved association with bacterial membranes to boost the therapeutic efficiency. These results provide new design rationale for AMPs developed for bacterial inhibition.
Assuntos
Anti-Infecciosos , Staphylococcus aureus Resistente à Meticilina , Humanos , Lipopeptídeos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Testes de Sensibilidade Microbiana , Anti-Infecciosos/farmacologia , Bactérias , Hemólise , Amidas , Antibacterianos/farmacologia , Antibacterianos/químicaRESUMO
PURPOSE: The aim of this study was to evaluate the clinical efficacy and the level of DKK1 and alkaline phosphatase (ALP) activity in gingival crevicular fluid (GCF) while taking Er:YAG laser as an adjunctive to scaling and root planning in the treatment of chronic periodontitis (CP). METHODS: Eleven patients with CP were included and there were nineteen pairs of homonym teeth(thirty-eight teeth) in this split-mouth design, and they were randomly assigned to experimental group or control group. In the experimental group, a combination of ultrasonic subgingigval scaling and root planning with hand instrument (SRP) were performed with Er: YAG laser as an adjunctive; in the control group, only SRP was performed. The main variables were bleeding index (BI), probing depth (PD), clinical attachment loss (CAL) which were assessed at baseline (1 week after ultrasonic subgingival scaling), l month and 3 months after treatment. GCF was collected at baseline, l week, l month and 3 months, and the levels of DKK1 and ALP activity were detected at the same time point. The data were analyzed with SPSS 19.0 software package. RESULTS: Both groups showed significant reduction of PD, CAL, BI values 1 month and 3months after treatment, but no significant difference in clinical parameters were found between the two groups. In the experimental group, the activity of ALP reduced to (386.69±146.42), (341.221±171.62), (249.27±98.72) from (396.191±150.55) U/L and the level of DKK1 dropped to (310.34±184.68), (270.04±55.14), (247.31±56.99) from (307.12±45.63) µg/L at the end of 1 week, 1 month, 3 months, respectively. Meanwhile, in the control group, the activity of ALP reduced to (374.72±131.27), (344.42±127.80), (252.36±90.4 ) from (394.09±120.25) U/L and the level of DKK1 dropped to (310.34±84.68), (270.04±55.14), (247.31±56.99) from (305.33±147.40) µg/L at the end of l week, l month, 3months, respectively. There is no significant difference between the two groups at any period for ALP or DKK1. CONCLUSIONS: Er:YAG laser was a safe no-surgical adjunctive therapy in treating chronic periodontitis, further observation is needed to determine its long-term effectiveness.
Assuntos
Fosfatase Alcalina , Periodontite Crônica , Líquido do Sulco Gengival , Peptídeos e Proteínas de Sinalização Intercelular , Fosfatase Alcalina/metabolismo , Biomarcadores/metabolismo , Periodontite Crônica/metabolismo , Periodontite Crônica/terapia , Índice de Placa Dentária , Raspagem Dentária , Seguimentos , Líquido do Sulco Gengival/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Lasers de Estado Sólido , Perda da Inserção Periodontal , Índice Periodontal , Bolsa Periodontal , Aplainamento RadicularRESUMO
PURPOSE: To detect the change of secreted frizzled-related protein-1ï¼SFRP1ï¼ in gingival crevicular fluid during periodontal initial treatment and explore the relationship between SFRP1 and the activity of chronic periodontitis. METHODS: Twenty-two patients with moderate to severe periodontitis were selected, and 5 healthy volunteers were enrolled into the study as control group. The bleeding index(BI),periodontal probing depth(PD)and clinical attachment loss(CAL) were recorded at 1 week after supragingival scaling, one month after subgingival scaling. Gingival crevicular fluid (GCF) was collected at 1 week after supragingival scaling, one week after subgingival scaling, one month after subgingival scaling. The level of SFRP1 in GCF samples was detected by ELISA. SPSS19.0 software package was used for data processing. RESULTS: The amount of SFRP1 in GCF of moderate to severe periodontitis group was (40.80±4.85) pg, and that of normal control was (33.42±2.24) pg at 1 week after supragingival scaling. The amount of SFRP1 in GCF was significantly higher in moderate to severe periodontitis compared to normal control group (P<0.05). In moderate to severe periodontitis group, the amount of SFRP1 in GCF significantly increased at l week after subgingival scaling (45.99±5.23) pg compared to 1 week after supragingival scaling and 1 month after subgingival scaling (36.92±4.00) pg (P<0.05); There was significant decrease in the amount of SFRP1 in GCF at 1 month after subgingival scaling,compared to 1 week after supragingival scaling and l week after subgingival scaling (P<0.05). CONCLUSIONS: The BI, PD in the periodontitis groups were significantly improved at 1 month after initial therapy,and the amount of SFRP1 in GCF changed with different periodontal inflammation stateï¼