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1.
Annegret Ulke-Lemée David Hao Sun Hiroaki Ishida Hans J. Vogel Justin A. MacDonald 《BMC biochemistry》2017,18(1):5
Background
The smoothelin-like 1 protein (SMTNL1) can associate with tropomyosin (Tpm) and calmodulin (CaM), two proteins essential to the smooth muscle contractile process. SMTNL1 is phosphorylated at Ser301 by protein kinase A during calcium desensitization in smooth muscle, yet the effect of SMTNL1 phosphorylation on Tpm- and CaM-binding has yet to be investigated.Results
Using pull down studies with Tpm-Sepharose and CaM-Sepharose, we examined the interplay between Tpm binding, CaM binding, phosphorylation of SMTNL1 and calcium concentration. Phosphorylation greatly enhanced the ability of SMTNL1 to associate with Tpm in vitro; surface plasmon resonance yielded a 10-fold enhancement in K D value with phosphorylation. The effect on CaM binding is more complex and varies with the availability of calcium.Conclusions
Combining both CaM and Tpm with SMTNL1 shows that the binding to both is mutually exclusive.2.
Background
Construction of electrochemical impedance sensors by the self-assembly technique has become a promising strategy for the `label-free' detection of protein-ligand interactions. However, previous impedance sensors are devoid of an inherent electrochemical signal, which limits the standardization of the sensors for protein recognition in a reproducible manner.Results
We designed and synthesized an anthraquinonyl glycoside (AG) where the anthraquinone (AQ) moiety can bind to the surface of a graphene-based working electrode while the glycoside serving as a ligand for lectin. By measuring the inherent voltammetric signal of AQ, the glycosides decorated on the working electrode could be simply quantified to obtain electrodes with a unified signal window. Subsequently, impedance analysis showed that the `standardized' electrodes gave a reproducible electrochemical response to a selective lectin with no signal variation in the presence of unselective proteins.Conclusion
Anthraquinone-modified ligands could be used to facilitate the standardization of electrochemical impedance sensors for the reproducible, selective analysis of ligand-protein interactions.3.
Wenjie Zhang Lei Chen Honggang Xiang Chunhua Hu Weibin Shi Ping Dong Wenjie Lv 《BMC biochemistry》2016,17(1):19
Background
Gamma glutamylcyclotransferase (GGCT) has been proved to be involved in various cancers, but the biological function of GGCT in gastric cancer is still largely unknown.Methods
The expression level of GGCT was evaluated by informatics analyses based on the Oncomine database. GGCT gene was then effectively knocked down via lentivirus mediated short hairpin RNA (shRNA) system. Then a series of functional assays, including MTT, colony formation and flow cytometry analysis were conducted on gastric cancer cells following GGCT knockdown.Results
We found GGCT is commonly up-regulated in gastric cancer tissues. Furthermore, MTT analysis showed that GGCT depletion significantly inhibited cell proliferation in MGC80-3 and AGS cells. Colony formation assay revealed that depletion of GGCT reduced the colony formation ability in gastric cancer cells. What’s more, cell cycle analysis showed that depletion of GGCT induced gastric cancer cell cycle arrested G2/M phase. More importantly, cell apoptosis analysis further revealed that GGCT inhibition induced early and late cell apoptosis in gastric cancer.Conclusion
This study suggests GGCT is essential for gastric cancer proliferation and its downregulation may provide a potential anticancer therapy for gastric cancer.4.
Manuela Crisan Liliana Halip Paulina Bourosh Sergiu Adrian Chicu Yurii Chumakov 《Chemistry Central journal》2017,11(1):129
Background
Nitroaromatic and chloronitroaromatic compounds have been a subject of great interest in industry and recently in medical-pharmaceutic field. 2-Chloro-4-nitro/2-chloro-5-nitrobenzoic acids and 4-nitrobenzoic acid are promising new agents for the treatment of main infectious killing diseases in the world: immunodeficiency diseases and tuberculosis.Results
New ethanolamine nitro/chloronitrobenzoates were synthesized and characterized by X-ray crystallography, UV–vis, FT-IR and elementary analysis techniques. The toxicity of the compounds prepared and correspondent components was evaluated using Hydractinia echinata as test system. A significant lower toxicity was observed for nitro-derivative compared with chloronitro-derivatives and individual components. Crystallographic studies, together with the chemical reactivity and stability profiles resulted from density functional theory and ab initio molecular orbital calculations, explain the particular behavior of ethanolamine 4-nitrobenzoate in biological test.Conclusions
The experimental and theoretical data reveal the potential of these compounds to contribute to the design of new active pharmaceutical ingredients with lower toxicity.5.
Ulaganathan Sivagnanam Shweta Narayana Murthy Sathyanarayana N. Gummadi 《BMC biochemistry》2016,17(1):10
Background
Human phospholipid scramblase 1 (hPLSCR1) was initially identified as a Ca2+ dependent phospholipid translocator involved in disrupting membrane asymmetry. Recent reports revealed that hPLSCR1 acts as a multifunctional signaling molecule rather than functioning as scramblase. hPLSCR1 is overexpressed in a variety of tumor cells and is known to interact with a number of protein molecules implying diverse functions.Results
In this study, the nuclease activity of recombinant hPLSCR1 and its biochemical properties have been determined. Point mutations were generated to identify the critical region responsible for the nuclease activity. Recombinant hPLSCR1 exhibits Mg2+ dependent nuclease activity with an optimum pH and temperature of 8.5 and 37 °C respectively. Experiments with amino acid modifying reagents revealed that histidine, cysteine and arginine residues were crucial for its function. hPLSCR1 has five histidine residues and point mutations of histidine residues to alanine in hPLSCR1 resulted in 60 % loss in nuclease activity. Thus histidine residues could play a critical role in the nuclease activity of hPLSCR1.Conclusions
This is the first report on the novel nuclease activity of the multi-functional hPLSCR1. hPLSCR1 shows a metal dependent nuclease activity which could play a role in key cellular processes that needs to be further investigated.6.
Background
Luciferases, enzymes that catalyze bioluminescent reactions in different organisms, have been extensively used for bioanalytical purposes. The most well studied bioluminescent system is that of firefly and other beetles, which depends on a luciferase, a benzothiazolic luciferin and ATP, and it is being widely used as a bioanalytical reagent to quantify ATP. Protein kinases are proteins that modify other proteins by transferring phosphate groups from a nucleoside triphosphate, usually ATP.Methods
Here, we used a red-light emitting luciferase from Phrixotrix hirtus railroad worm to determine the activity of kinases in a coupled assay, based on luminescence that is generated when luciferase is in the presence of its substrate, the luciferin, and ATP.Results
In this work we used, after several optimization reactions, creatine kinase isoforms as well as ?NEK7 protein kinase in the absence or presence of ATP analogous inhibitors to validate this new luminescence method.Conclusion
With this new approach we validated a luminescence method to quantify kinase activity, with different substrates and inhibition screening tests, using a novel red-light emitting luciferase as a reporter enzyme.7.
Mohamed Bourass Adil Touimi Benjelloun Mohammed Benzakour Mohammed Mcharfi Mohammed Hamidi Si Mohamed Bouzzine Mohammed Bouachrine 《Chemistry Central journal》2016,10(1):67
Background
Novel six organic donor-π-acceptor molecules (D-π-A) used for Bulk Heterojunction organic solar cells (BHJ), based on thienopyrazine were studied by density functional theory (DFT) and time-dependent DFT (TD-DFT) approaches, to shed light on how the π-conjugation order influence the performance of the solar cells. The electron acceptor group was 2-cyanoacrylic for all compounds, whereas the electron donor unit was varied and the influence was investigated.Methods
The TD-DFT method, combined with a hybrid exchange-correlation functional using the Coulomb-attenuating method (CAM-B3LYP) in conjunction with a polarizable continuum model of salvation (PCM) together with a 6-31G(d,p) basis set, was used to predict the excitation energies, the absorption and the emission spectra of all molecules.Results
The trend of the calculated HOMO–LUMO gaps nicely compares with the spectral data. In addition, the estimated values of the open-circuit photovoltage (Voc) for these compounds were presented in two cases/PC60BM and/PC71BM.Conclusion
The study of structural, electronics and optical properties for these compounds could help to design more efficient functional photovoltaic organic materials.8.
Background
BTBD10 binds to Akt and protein phosphatase 2A (PP2A) and inhibits the PP2A-mediated dephosphorylation of Akt, thereby keeping Akt activated. Previous studies have suggested that BTBD10 plays an important role in preventing motor neuronal death and accelerating the growth of pancreatic beta cells. Because levels of BTBD10 expression are much lower in many non-nervous tissues than nervous tissues, there may be a relative of BTBD10 that has BTBD10-like function in non-neuronal cells.Results
A 419-amino-acid BTBD10-like protein, named KCTD20 (potassium channel tetramerization protein domain containing 20), was to found to bind to all Akt isoforms and PP2A. Overexpression of KCTD20 increased Akt phosphorylation at Thr308, as BTBD10 did, which suggests that KCTD20 as well as BTBD10 positively regulates the function of Akt. KCTD20 was ubiquitously expressed in non-nervous as well as nervous tissues.Conclusions
KCTD20 is a positive regulator of Akt and may play an important role in regulating the death and growth of some non-nervous and nervous cells.9.
Background
The potential for a compound to cause hepatotoxicity and nephrotoxicity is a matter of extreme interest for human health risk assessment. To assess liver and kidney toxicity, repeated-dose toxicity (RDT) studies are conducted mainly on rodents. However, these tests are expensive, time-consuming and require large numbers of animals. For early toxicity screening, in silico models can be applied, reducing the costs, time and animals used. Among in silico approaches, structure–activity relationship (SAR) methods, based on the identification of chemical substructures (structural alerts, SAs) related to a particular activity (toxicity), are widely employed.Results
We identified and evaluated some SAs related to liver and kidney toxicity, using RDT data on rats taken from the hazard evaluation support system (HESS) database. We considered only SAs that gave the best percentages of true positives (TP).Conclusions
It was not possible to assign an unambiguous mode of action for all the SAs, but a mechanistic explanation is provided for some of them. Such achievements may help in the early identification of liver and renal toxicity of substances.10.
Wesam H. Abdulaal 《BMC biochemistry》2018,19(1):10
Background
Generally, proteases in medicinal plants had different therapeutic effects such as anti-inflammatory effect; modulate the immune response and inhibitory effect toward tumor growth. In this study, protease was purified and characterized from miswak roots, as medicinal plant and natural toothbrush.Results
Physical and chemical characterization of cysteine protease P1 were studied such as pH optimum (6.5), optimum temperature (50?°C), thermal stability (50?°C) and Km (3.3?mg azocasein/ml). The enzyme digested some proteins in the order of caseine > haemoglobin > egg albumin >gelatin > bovine serum albumin. Hg2+ had strong inhibitory effect on enzyme activity compared with other metal ions. Kinetic of inhibition for determination the type of protease was studied. Iodoactamide and p-Hydroximercuribenzaoic acid (p-HMB) caused strong inhibitory effect on enzyme activity indicating the enzyme is cysteine protease.Conclusions
The biochemical characterization of this enzyme will be display the suitable conditions for using of this enzyme in toothpaste in the future and the enzyme may be used in other applications.11.
Background
Phosphatase of regenerating liver-3 (PRL-3 or PTP4A3) has been implicated in controlling cancer cell proliferation, motility, metastasis, and angiogenesis. Deregulated expression of PRL-3 is highly correlated with cancer progression and predicts poor survival. Although PRL-3 was categorized as a tyrosine phosphatase, its cellular substrates remain largely unknown.Results
We demonstrated that PRL-3 interacts with integrin β1 in cancer cells. Recombinant PRL-3 associates with the intracellular domain of integrin β1 in vitro. Silencing of integrin α1 enhances PRL-3-integrin β1 interaction. Furthermore, PRL-3 diminishes tyrosine phosphorylation of integrin β1 in vitro and in vivo. With site-specific anti-phosphotyrosine antibodies against residues in the intracellular domain of integrin β1, tyrosine-783, but not tyrosine-795, is shown to be dephosphorylated by PRL-3 in a catalytic activity-dependant manner. Phosphorylation of Y783 is potentiated by ablation of PRL-3 or by treatment with a chemical inhibitor of PRL-3. Conversely, depletion of integrin α1 decreases the phosphorylation of this site.Conclusions
Our results revealed a direct interaction between PRL-3 and integrin β1 and characterized Y783 of integrin β1 as a bona fide substrate of PRL-3, which is negatively regulated by integrin α1.12.
Peter C. Loewen Jacek Switala James P. Wells Fang Huang Anthony T. Zara John S. Allingham Michele C. Loewen 《BMC biochemistry》2018,19(1):8
Background
Stilbene cleaving oxygenases (SCOs), also known as lignostilbene-α,β-dioxygenases (LSDs) mediate the oxidative cleavage of the olefinic double bonds of lignin-derived intermediate phenolic stilbenes, yielding small modified benzaldehyde compounds. SCOs represent one branch of the larger carotenoid cleavage oxygenases family. Here, we describe the structural and functional characterization of an SCO-like enzyme from the soil-born, bio-control agent Pseudomonas brassicacearum.Methods
In vitro and in vivo assays relying on visual inspection, spectrophotometric quantification, as well as liquid-chormatographic and mass spectrometric characterization were applied for functional evaluation of the enzyme. X-ray crystallographic analyses and in silico modeling were applied for structural investigations.Results
In vitro assays demonstrated preferential cleavage of resveratrol, while in vivo analyses detected putative cleavage of the straight chain carotenoid, lycopene. A high-resolution structure containing the seven-bladed β-propeller fold and conserved 4-His-Fe unit at the catalytic site, was obtained. Comparative structural alignments, as well as in silico modelling and docking, highlight potential molecular factors contributing to both the primary in vitro activity against resveratrol, as well as the putative subsidiary activities against carotenoids in vivo, for future validation.Conclusions
The findings reported here provide validation of the SCO structure, and highlight enigmatic points with respect to the potential effect of the enzyme’s molecular environment on substrate specificities for future investigation.13.
Gatta T Campanella L Coluzza C Mambro V Postorino P Tomassetti M Visco G 《Chemistry Central journal》2012,6(Z2):S2
Background and methods
Several standard powdered black pigments were characterized by means of thermogravimetry TG-DTG and allied techniques. These pigments were used to make standard plaster frescoes at this purpose prepared. The latter ones were subjected to Raman and reflectance analysis. The results obtained, together with TG data, were chemometrically processed and used to identify an analogous standard fresco fabricated by an unknown commercial black pigment, obtaining excellent results.Results
The same colorimetric and reflectometric techniques, coupled with suitable chemometric techniques, were then successfully used to identify the type of black pigment present in an ancient roman fresco of the Imperial Age (30 B.C.).Conclusion
TG-DTG resulted useful techniques to autenticate powdered black pigments.Colorimetry and Raman, but also the only colorimetry, were useful to identify an ancient black pigment in situ.14.
Background
Enzymes display high reactivity and selectivity under natural conditions, but may suffer from decreased efficiency in industrial applications. A strategy to address this limitation is to immobilize the enzyme. Mesoporous silica materials offer unique properties as an immobilization support, such as high surface area and tunable pore size.Results
The performance of a commercially available feruloyl esterase, E-FAERU, immobilized on mesoporous silica by physical adsorption was evaluated for its transesterification ability. We optimized the immobilization conditions by varying the support pore size, the immobilization buffer and its pH. Maximum loading and maximum activity were achieved at different pHs (4.0 and 6.0 respectively). Selectivity, shown by the transesterification/hydrolysis products molar ratio, varied more than 3-fold depending on the reaction buffer used and its pH. Under all conditions studied, hydrolysis was the dominant activity of the enzyme. pH and water content had the greatest influence on the enzyme selectivity and activity. Determined kinetic parameters of the enzyme were obtained and showed that Km was not affected by the immobilization but kcat was reduced 10-fold when comparing the free and immobilized enzymes. Thermal and pH stabilities as well as the reusability were investigated. The immobilized biocatalyst retained more than 20% of its activity after ten cycles of transesterification reaction.Conclusions
These results indicate that this enzyme is more suited for hydrolysis reactions than transesterification despite good reusability. Furthermore, it was found that the immobilization conditions are crucial for optimal enzyme activity as they can alter the enzyme performance.15.
Background
The marine invertebrate starfish was found to contain a novel α-N-acetylgalactosaminidase, α-GalNAcase II, which catalyzes removal of terminal α-N-acetylgalactosamine (α-GalNAc), in addition to a typical α-N-acetylgalactosaminidase, α-GalNAcase I, which catalyzes removal of terminal α-N-acetylgalactosamine (α-GalNAc) and, to a lesser extent, galactose. The interrelationship between α-GalNAcase I and α-GalNAcase II and the molecular basis of their differences in substrate specificity remain unknown.Results
Chemical and structural comparisons between α-GalNAcase I and II using immunostaining, N-terminal amino acid sequencing and peptide analysis showed high homology to each other and also to other glycoside hydrolase family (GHF) 27 members. The amino acid sequence of peptides showed conserved residues at the active site as seen in typical α-GalNAcase. Some substitutions of conserved amino acid residues were found in α-GalNAcase II that were located near catalytic site. Among them G171 and A173, in place of C171 and W173, respectively in α-GalNAcase were identified to be responsible for lacking intrinsic α-galactosidase activity of α-GalNAcase II. Chemical modifications supported the presence of serine, aspartate and tryptophan as active site residues. Two tryptophan residues (W16 and W173) were involved in α-galactosidase activity, and one (W16) of them was involved in α-GalNAcase activity.Conclusions
The results suggested that α-GalNAcase I and II are closely related with respect to primary and higher order structure and that their structural differences are responsible for difference in substrate specificities.16.
Background
The production of β-lactamases by bacteria is the most common mechanism of resistance to the widely prescribed β-lactam antibiotics. β-lactamase inhibitory protein (BLIP) competitively inhibits class A β-lactamases via two binding loops that occlude the active site. It has been shown that BLIP Tyr50 is a specificity determinant in that substitutions at this position result in large differential changes in the relative affinity of BLIP for class A β-lactamases.Results
In this study, the effect of systematic substitutions at BLIP position 50 on binding to class A β-lactamases was examined to further explore the role of BLIP Tyr50 in modulating specificity. The results indicate the sequence requirements at position 50 are widely different depending on the target β-lactamase. Stringent sequence requirements were observed at Tyr50 for binding Bacillus anthracis Bla1 while moderate requirements for binding TEM-1 and relaxed requirements for binding KPC-2 β-lactamase were seen. These findings cannot be easily rationalized based on the β-lactamase residues in direct contact with BLIP Tyr50 since they are identical for Bla1 and KPC-2 suggesting that differences in the BLIP-β-lactamase interface outside the local environment of Tyr50 influence the effect of substitutions.Conclusions
Results from this study and previous studies suggest that substitutions at BLIP Tyr50 may induce changes at the interface outside its local environment and point to the complexity of predicting the impact of substitutions at a protein-protein interaction interface.17.
Background
YedY, a molybdoenzyme belonging to the sulfite oxidase family, is found in most Gram-negative bacteria. It contains a twin-arginine signal sequence that is cleaved after its translocation into the periplasm. Despite a weak reductase activity with substrates such as dimethyl sulfoxide or trimethylamine N-oxide, its natural substrate and its role in the cell remain unknown. Although sequence conservation of the YedY family displays a strictly conserved hydrophobic C-terminal residue, all known studies on Escherichia coli YedY have been performed with an enzyme containing a 6 histidine-tag at the C-terminus which could hamper enzyme activity.Results
In this study, we demonstrate that the tag fused to the C-terminus of Rhodobacter sphaeroides YedY is detrimental to the enzyme’s reductase activity and results in an eight-fold decrease in catalytic efficiency. Nonetheless this C-terminal tag does not influence the properties of the molybdenum active site, as assayed by EPR spectroscopy. When a cleavable His-tag was fused to the N-terminus of the mature enzyme in the absence of the signal sequence, YedY was expressed and folded with its cofactor. However, when the signal sequence was added upstream of the N-ter tag, the amount of enzyme produced was approximately ten-fold higher.Conclusion
Our study thus underscores the risk of using a C-terminus tagged enzyme while studying YedY, and presents an alternative strategy to express signal sequence-containing enzymes with an N-terminal tag. It brings new insights into molybdoenzyme maturation in R. sphaeroides showing that for some enzymes, maturation can occur in the absence of the signal sequence but that its presence is required for high expression of active enzyme.18.
Background
Mycobacterium tuberculosis topoisomerase I (MtTOP1) and Escherichia coli topoisomerase I have highly homologous transesterification domains, but the two enzymes have distinctly different C-terminal domains. To investigate the structure-function of MtTOP1 and to target its activity for development of new TB therapy, it is desirable to have a rapid genetic assay for its catalytic activity, and potential bactericidal consequence from accumulation of its covalent complex.Results
We show that plasmid-encoded recombinant MtTOP1 can complement the temperature sensitive topA function of E. coli strain AS17. Moreover, expression of MtTOP1-G116 S enzyme with the TOPRIM mutation that inhibits DNA religation results in SOS induction and loss of viability in E. coli. The absence of cysteine residues in the MtTOP1 enzyme makes it an attractive system for introduction of potentially informative chemical or spectroscopic probes at specific positions via cysteine mutagenesis. Such probes could be useful for development of high throughput screening (HTS) assays. We employed the AS17 complementation system to screen for sites in MtTOP1 that can tolerate cysteine substitution without loss of complementation function. These cysteine substitution mutants were confirmed to have retained the relaxation activity. One such mutant of MtTOP1 was utilized for fluorescence probe incorporation and fluorescence resonance energy transfer measurement with fluorophore-labeled oligonucleotide substrate.Conclusions
The DNA relaxation and cleavage complex accumulation of M. tuberculosis topoisomerase I can be measured with genetic assays in E. coli, facilitating rapid analysis of its activities, and discovery of new TB therapy targeting this essential enzyme.19.
Antonio Cassano Alberto Manganaro Todd Martin Douglas Young Nadège Piclin Marco Pintore Davide Bigoni Emilio Benfenati 《Chemistry Central journal》2010,4(Z1):S4
Background
The new REACH legislation requires assessment of a large number of chemicals in the European market for several endpoints. Developmental toxicity is one of the most difficult endpoints to assess, on account of the complexity, length and costs of experiments. Following the encouragement of QSAR (in silico) methods provided in the REACH itself, the CAESAR project has developed several models.Results
Two QSAR models for developmental toxicity have been developed, using different statistical/mathematical methods. Both models performed well. The first makes a classification based on a random forest algorithm, while the second is based on an adaptive fuzzy partition algorithm. The first model has been implemented and inserted into the CAESAR on-line application, which is java-based software that allows everyone to freely use the models.Conclusions
The CAESAR QSAR models have been developed with the aim to minimize false negatives in order to make them more usable for REACH. The CAESAR on-line application ensures that both industry and regulators can easily access and use the developmental toxicity model (as well as the models for the other four endpoints).20.