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A novel bacterial display vector based on Escherichia coli has been engineered for recombinant protein production and purification. Accordingly, a construct harboring the enhanced green fluorescent protein (EGFP) and the ice nucleation protein (INP) was designed to produce EGFP via the surface display in E. coli cells. The fusion EGFP-expressed cells were then investigated using fluorescence measurement, SDS- and native-PAGE before and after TEV protease digestion. The displayed EGFP was obtained with a recovery of 57.7 % as a single band on SDS-PAGE. Next, the efficiency of the cell surface display for mutant EGFP (EGFP S202H/Q204H) was examined in sensing copper ions. Under optimal conditions, a satisfactorily linear range for copper ions concentrations up to 10 nM with a detection limit of 0.073 nM was obtained for cell-displayed mutant EGFP (mEGFP). In the presence of bacterial cell lysates and purified mEGFP, response to copper was linear in the 2-10 nM and 0.1-2 μM concentration range, respectively, with a 1.3 nM and 0.14 μM limit of detection. The sensitivity of bacterial cell lysates and surface-displayed mEGFP in the detection of copper ions is higher than the purified mEGFP., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

We present an advanced electrochemical immunosensor designed to detect the vascular endothelial growth factor (VEGF) precisely. The sensor is constructed on a modified porous gold electrode through a fabrication process involving the deposition of silver and gold on an FTO substrate. Employing thermal annealing and a de-alloying process, the silver is eliminated from the electrode, producing a reproducible porous gold substrate. Utilizing a well-defined protocol, we immobilize the heavy-chain (VHH) antibody against VEGF on the gold substrate, facilitating VEGF detection through various electrochemical methods. Remarkably, this immunosensor performs well, featuring an impressive detection limit of 0.05 pg/mL and an extensive linear range from 0.1 pg/mL to 0.1 µg/mL. This emphasizes it's to measure biomarkers across a wide concentration spectrum precisely. The robust fabrication methodology in this research underscores its potential for widespread application, offering enhanced precision, reproducibility, and remarkable detection capabilities for the developed immunosensor., (© 2024. The Author(s).)

Infertility is such an important issue in society today. In some cases of male infertility, the main cause is oxidative stress and the presence of reactive oxygen species in the environment or in sperm cells. All current techniques that measure oxidative stress, including the nitroblue tetrazolium Test, DNA Fragmentation Index, Malondialdehyde, and Endz Test are qualitative and semi-quantitative. These methods do not have good sensitivity and specificity. Semen samples from 50 infertile patients and 10 normal individuals were collected. The samples were examined for laboratory routine tests according to the WHO 2010 protocol. Oxidative stress tests, including DFI, NBT, and MDA, were performed for these two groups. Bioluminescence inhibition assay was performed for detection of O 2 .- in semen samples by aequorin. The normal individuals showed significantly better semen parameters than the patient's group. Significantly lower O 2 .- levels were seen in the patient's group compared to normal individuals. The cut-off value of O 2 .- levels in normal individuals was determined to be 8 × 10 5 RLU/s with a sensitivity of 100% and a specificity of 100%. Infertile patients, despite having reduced quality of semen parameters, have high O 2 .- levels, and this causes the intensity of bioluminescence to be quenched in these people., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Herein, we describe the potential use of food-grade hexosomes (HEXs) for delivering fish-purified antioxidant peptide (PF10). Using a binary lipid mixture of Dimodan U/citrem, the nanocarriers were produced with a size range of 202.7-569.8 nm and peptide encapsulation efficiency of 64.6-89.3%. These HEXs were also characterized by SAXS and cryo-TEM, and were able to sustain the release of PF10, where only 32.2% released in PBS after 24 h. SAXS findings verified that PF10 modulate the internal structure of HEXs in a pH-dependent manner. Antioxidant assays proved the efficacy of such nano-self-assemblies in maintaining the bioactivity of the loaded peptide. Moreover, the in vitro gastrointestinal stability test indicated that the antioxidant capacity of the free- and PF10-loaded HEXs decreased under SGF/SIF conditions with the reduction in activity being greater for the free PF10. The present findings may provide a useful basis for development of pH-responsive nano-self-assemblies for delivery of antioxidant peptides., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Vascular endothelial growth factor A 165 (VEGF-A 165 ) and VEGF receptor 2 (KDR) are important mediators of angiogenesis. We aimed to express the soluble KDR ligand-binding domain (sKDR1-3) and evaluate its interaction with the VEGF-A 165 receptor-binding domain (VEGFA 165 -RBD). sKDR1-3 DNA was designed and subcloned into pPinkα-HC plasmid. The cassette was transfected into the Pichia pink™ 4 genome by homologous recombination. We optimized the expression of sKDR1-3 under the induction of different methanol concentrations. VEGFA 165 -RBD was expressed in E. coli BL21 harboring pET28a( +)─VEGFA 165 -RBD vector under induction with IPTG with/without lactose. Interaction and biological activity of sKDR1-3 and VEGFA 165 -RBD were investigated by ELISA and anti-proliferation tests. sKDR1-3 migrated on SDS-PAGE gel as a 35-180 kDa protein due to glycosylation. The relative expression level of sKDR1-3 under 1% methanol was higher than 0.5% and 4% methanol induction. IPTG and cysteine were suitable for induction and refolding of VEGFA 165 -RBD. 25 ng sKDR1-3 and 20 ng VEGFA 165 -RBD showed strong binding. sKDR1-3 bound to VEGFA 165 -RBD and VEGF-A 165 with dissociation constants of 0.148 and 0.2 nM, respectively. 4-10 nM concentrations of sKDR1-3 inhibited the proliferation of HUVE cells induced by 5 nM VEGFA 165 -RBD. In consideration, sKDR1-3 in the nanomolar concentration range, is a promising anticancer drug to inhibit angiogenesis., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Curcumin (bis-α,β-unsaturated β-diketone) plays an important role in the prevention of numerous diseases, including diabetes. Curcumin, as an enzyme inhibitor, has ideal structural properties including hydrophobic nature, flexible backbone, and several available hydrogen bond (H-bond) donors and acceptors. In this study, curcumin-fused aldohexose derivatives 3(a-c) were synthesized and used as influential agents in the treatment of diabetes with inhibitory properties against two carbohydrate-hydrolyzing enzymes α-glucosidase (α-Gls) and α-amylase (α-Amy) which are known to be significant therapeutic targets for the reduction of postprandial hyperglycemia. These compounds were isolated, purified, and then spectrally characterized via FT-IR, Mass, 1 H, and 13 C NMR, which strongly confirmed the targeted product's formation. Also, their inhibitory properties against α-Gls and α-Amy were evaluated spectroscopically. The Results indicated that all compounds strongly inhibited α-Amy and α-Gls by mixed and competitive mechanisms, respectively. The intrinsic fluorescence of α-Amy was quenched by the interaction with compounds 1 and 3b through a dynamic quenching mechanism, and the 1 and 3b/α-Amy complexes were spontaneously formed, mainly driven by the hydrophobic interaction and hydrogen bonding. Fourier transform infrared spectra (FT-IR) comprehensively verified that the binding of compounds 1 and 3b to α-Amy would change the conformation and microenvironment of α-Amy, thereby inhibiting the enzyme activity. Docking and molecular dynamics (MD) simulations showed that all compounds interacted with amino acid residues located in the active pocket site of the proteins. In vivo studies confirmed the plasma glucose diminution after the administration of compound 3b to Wistar rats. Accordingly, the results of the current work may prompt the scientific communities to investigate the possibility of compound 3b application in the clinic., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Key Message: Our manuscript is the first to find a link between activity of SAL1/OLD101 against IP 3 and plant leaf senescence regulation and ROS levels assigning a potential biological role for IP 3 . Leaf senescence is a genetically programmed process that limits the longevity of a leaf. We identified and analyzed the recessive Arabidopsis stay-green mutation onset of leaf death 101 (old101). Developmental leaf longevity is extended in old101 plants, which coincided with higher peroxidase activity and decreased H 2 O 2 levels in young 10-day-old, but not 25-day-old plants. The old101 phenotype is caused by a point mutation in SAL1, which encodes a bifunctional enzyme with inositol polyphosphate-1-phosphatase and 3' (2'), 5'-bisphosphate nucleotidase activity. SAL1 activity is highly specific for its substrates 3-polyadenosine 5-phosphate (PAP) and inositol 1, 4, 5-trisphosphate (IP 3 ), where it removes the 1-phosphate group from the IP 3 second messenger. The in vitro activity of recombinant old101 protein against its substrate IP 3 was 2.5-fold lower than that of wild type SAL1 protein. However, the in vitro activity of recombinant old101 mutant protein against PAP remained the same as that of the wild type SAL1 protein. The results open the possibility that the activity of SAL1 against IP 3 may affect the redox balance of young seedlings and that this delays the onset of leaf senescence., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Over the past twenty years, the prevalence of diabetes as one of the most common metabolic diseases has become a public health problem worldwide. Blood glucose control is important in delaying the onset and progression of diabetes-related complications. α-Glycosidase (α- Glu) and α-amylase (α-Amy) are important enzymes in glucose metabolism. Diabetic control through the inhibition of carbohydrate hydrolyzing enzymes is established as an effective strategy., Methods: In this study, curcumin-based benzaldehyde derivatives with high stability, bioavailability, and favorable efficiency were synthesized., Results: The results showed that L13, L8, and L11 derivatives have the highest inhibitory effect on α-Glu with IC 50 values of 18.65, 20.6, and 31.7 μM and, also L11, L13, and L8 derivatives have the highest inhibitory effect on α-Amy with IC 50 value of 14.8, 21.8, and 44.9 μM respectively. Furthermore, enzyme inhibitory kinetic characterization was also performed to understand the mechanism of enzyme inhibition., Conclusion: L13, compared to the other compounds, exhibited acceptable inhibitory activity against both enzymes. The L13 derivative could be an appropriate candidate for further study through the rational drug design to the exploration of a new class of powerful anti-diabetic drugs considering the antioxidant properties of the synthesized compounds. The derivative helps reduce the glycemic index and limits the activity of the major reactive oxygen species (ROS) producing pathways., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Human epidermal growth factor receptor 2 (HER2)-positive breast cancer represents approximately 15-30% of all invasive breast cancers. Despite the recent advances in therapeutic practices of HER2 subtype, drug resistance and tumor recurrence still have remained as major problems. Drug discovery is a long and difficult process, so the aim of this study is to find potential new application for existing therapeutic agents. Gene expression data for breast invasive carcinoma were retrieved from The Cancer Genome Atlas (TCGA) database. The normal and tumor samples were analyzed using Linear Models for Microarray Data (LIMMA) R package in order to find the differentially expressed genes (DEGs). These genes were used as entry for the library of integrated network-based cellular signatures (LINCS) L1000CDS 2 software and suggested 24 repurposed drugs. According to the obtained results, some of these drugs including vorinostat, mocetinostat, alvocidib, CGP-60474, BMS-387032, AT-7519, and curcumin have significant functional similarity and structural correlation with FDA-approved breast cancer drugs. Based on the drug-target network, which consisted of the repurposed drugs and their target genes, the aforementioned drugs had the highest degrees. Moreover, the experimental approach verified curcumin as an effective therapeutic agent for HER2 positive breast cancer. Hence, our work suggested that some repurposed drugs based on gene expression data can be noticed as potential drugs for the treatment of HER2-positive breast cancer., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Horseradish peroxidase (HRP) is usually used as a label enzyme in immunoassay so the method used for HRP detection in enzyme immunoassay (EIA) plays a key role in sensitivity and precision. The catalytic activity of HRP does not strictly follow classic Michaelis-Menten kinetics, probably due to the inactivation of the enzyme at high concentrations of H 2 O 2 . In this paper, a highly sensitive alternative procedure for the HRP assay using H 2 O 2 -sensitive CdTe quantum dots as a chemiluminescence (CL) system is reported. This method can measure a much more accurate and reliable value of K m (187 mM H 2 O 2 ) in comparison with the standard detection method. This system also was applied to thyroid hormone (T4) detection using HRP-based immunoassay. The QD/H 2 O 2 system exhibits a higher linear range of 0.2-16 μg/dL with the improved LOD value of 0.06 μg/dL and selective response to T4, which was better than the commercial colorimetric immunoassay. Meanwhile, the proposed method has been successfully applied to the clinical determination of T4 in the serum samples, and the results confirmed an excellent correlation with the conventional ELISA method (R 2 = 0.9832), indicating the potential applications of the method for clinical diagnosis as well., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Superoxide dismutases (SODs) (EC 1.15.1.1) are well known antioxidant enzymes that play critical roles in cellular defenses of living organisms against harmful superoxide radicals during oxidative stress. This study details on cloning, biochemical and functional characterization of an iron containing type superoxide dismutase (SOD) from a novel thermophilic bacteria Cohnella sp. A01 (CaSOD). The secondary and three dimensional structure of the protein were predicted. CaSOD gene was subsequently cloned into pET-26b(+) expression vector and expression of the recombinant protein (rCaSOD) was optimized in E. coli BL21 (DE3) and the purified recombinant SOD showed a single band with an apparent molecular weight of 26 kDa by SDS-PAGE. The half-life and thermodynamic parameters including ΔH ⁎ , ΔS ⁎ , and ΔG ⁎ were 187 min at 60 °C, 7.3 kJ.mol -1 , -76.8 kJ.mol -1 .°K -1 , and 84.1 kJ.mol -1 , respectively. The rCaSOD exhibited catalytic activity in a very broad range of pH (6.0-10.0) and temperatures (35-75 °C), as well as stability in a broad pH range, from 3.0 to 11.0, and wide range of temperature, different concentrations of detergent agents, metal ions, organic solvents and other chemicals. The results suggest that this novel enzyme could be used for various industrial applications in cosmetic, food, and pharmaceutical industries., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Moonlighting proteins (MPs) are a subclass of multifunctional proteins in which more than one independent or usually distinct function occurs in a single polypeptide chain. Identification of unknown cellular processes, understanding novel protein mechanisms, improving the prediction of protein functions, and gaining information about protein evolution are the main reasons to study MPs. They also play an important role in disease pathways and drug-target discovery. Since detecting MPs experimentally is quite a challenge, most of them are detected randomly. Therefore, introducing an appropriate computational approach to predict MPs seems reasonable., Results: In this study, we introduced a competent model for detecting moonlighting and non-MPs through extracted features from protein sequences. We attempted to set up a well-judged scheme for detecting outlier proteins. Consequently, 37 distinct feature vectors were utilized to study each protein's impact on detecting MPs. Furthermore, 8 different classification methods were assessed to find the best performance. To detect outliers, each one of the classifications was executed 100 times by tenfold cross-validation on feature vectors; proteins which misclassified 90 times or more were grouped. This process was applied to every single feature vector and eventually the intersection of these groups was determined as the outlier proteins. The results of tenfold cross-validation on a dataset of 351 samples (containing 215 moonlighting and 136 non-moonlighting proteins) reveal that the SVM method on all feature vectors has the highest performance among all methods in this study and other available methods. Besides, the study of outliers showed that 57 of 351 proteins in the dataset could be an appropriate candidate for the outlier. Among the outlier proteins, there were non-MPs (such as P69797) that have been misclassified in 8 different classification methods with 16 different feature vectors. Because these proteins have been obtained by computational methods, the results of this study could reduce the likelihood of hypothesizing whether these proteins are non-moonlighting at all., Conclusions: MPs are difficult to be identified through experimentation. Using distinct feature vectors, our method enabled identification of novel moonlighting proteins. The study also pinpointed that a number of non-MPs are likely to be moonlighting.

In this study, an enzyme-triggered system based on β-cyclodextrin (β-CD) has been developed to achieve controlled release of hydrophobic drugs in the presence of maltogenic amylase (MAase). The inclusion complex formation of curcumin (CUR), as a model anticancer compound, with β-CD was characterized by fluorescence and Fourier transform infrared (FTIR) spectroscopy. CUR was loaded into β-CD with an encapsulation efficiency of approximately 30 %. The in vitro profiles of CUR release from β-CD showed that 100 % of the drug was released after one hour incubation in the presence of MAase with cyclodextrin degrading activity. Fluorescence microscopy images indicate a significantly greater cellular uptake of CUR using β-CD-CUR/MAase system compared to β-CD-CUR inclusion complex without MAase. The β-CD-CUR/MAase system exhibited lower IC 50 values and greater anti-proliferative effects in comparison with free CUR and β-CD-CUR in MCF-7 and Huh-7 cancer cells. The results from fluorescence microscopy and flow cytometric assay using the acridine orange/ethidium bromide and Annexin V-PE/7-AAD staining suggest that the β-CD-CUR/MAase system exhibited higher cytotoxic and apoptotic effects on cancer cells compared to other formulations. This triggered release of CUR in the presence of MAase is owing to the β-CD degradation by MAase resulting ring opening and chain scission in β-CD. We demonstrate that this enzyme-mediated controlled release system has a potential application for controlled release of poorly water-soluble drugs or hydrophobic compounds such as CUR., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Future biomedical applications of nanoparticles will encounter these particles with patients' serum which might affect the properties and stability of quantum dots and serum proteins at the desired site of action. Therefore, it is essential to clarify the patient-specific serum components, serve as major interaction partners, the spatial distribution of these, and consequently the time-dependent effects of nanoparticle-protein interaction. Here, a biochemical and structural study was performed on the protein corona formation and the corresponding interaction of different sizes of CdTe QDs with human serum proteins to determine if the mutual effects on optical properties by using electrophoresis, chemiluminescence, and fluorescence spectroscopy. The results revealed that interaction with human serum significantly enhanced the stability and photoluminescence of quantum dots. Structural studies of HSA-coated CdTe QDs also showed that corona formation has no adverse effects on protein structure, and the reduction in fluorescence emissions of HSA is due to the direct quenching of aromatics residues by the quantum dot. Improving nanoparticle properties, as well as the lack of structural changes in HSA, can be very useful in biomedical applications and in vivo studies where stability is important., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Artemin is an abundant thermostable protein in Artemia embryos and it is considered as a highly efficient molecular chaperone against extreme environmental stress conditions. The conformational dynamics of artemin have been suggested to play a critical role in its biological functions. In this study, we have investigated the conformational and functional changes of artemin under heat and oxidative stresses to identify the relationship between its structure and function. The tertiary and quaternary structures of artemin were evaluated by fluorescence measurements, protein cross-linking analysis, and dynamic light scattering. Based on the structural analysis, artemin showed irreversible substantial conformational lability in responses to heat and oxidant, which was mainly mediated through the hydrophobic interactions and dimerization of the chaperone. In addition, the chaperone-like activity of heated and oxidized artemin was examined using lysozyme refolding assay and the results showed that although both factors, i.e. heat and oxidant, at specific levels improved artemin potency, simultaneous incubation with both stressors significantly triggered the chaperone activation. Moreover, the heat-induced dimerization of artemin was found to be the most critical factor for its activation. It was suggested that oxidation presumably acts through stabilizing the dimer structures of artemin through formation of disulfide bridges between the subunits and strengthens its chaperoning efficacy. Accordingly, it is proposed that artemin probably exists in a monomer-oligomer equilibrium in Artemia cysts and environmental stresses and intracellular portion of protein substrates may shift the equilibrium towards the active dimer forms of the chaperone., Competing Interests: MBGG provided some instruments for authors, and contributed in analysis of spectroscopic data. The commercial affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials.

Because of the critical role of vascular endothelial growth factor (VEGF) in angiogenesis and its significantly increased serum levels in early stages of cancer, VEGF is considered an important prognostic biomarker in different cancers. Herein, the amplification power of PCR combined with phage displaying anti-VEGF VHH, a sensitive real-time immunoassay, was precisely designed based on phage display-mediated immuno-PCR (PD-IPCR) for the detection of VEGF. This system benefits from strong and specific binding of antigen and antibody in a sandwich immunosorbent assay platform using avastin (anti-VEGF monoclonal antibody) as the capture antibody. The anti-VEGF phage particles were used as both anti-VEGF agent and DNA template in the PD-IPCR. Anti-VEGF phage ELISA showed a linear range of 3-250 ng/ml and a limit of detection (LOD) of 1.1 ng/ml. Using the PD-IPCR method, the linear range of VEGF detection was found to be 0.06-700 ng/ml, with a detection limit of 3 pg/ml. The recovery rate in serum ranged from 83% to 99%, with a relative standard deviation of 1.2-4.9%. These values indicate that the method has good sensitivity for use in clinical analysis. The proposed method was successfully applied to the clinical determination of VEGF in human serum samples, and the results showed excellent correlation with conventional ELISA (R 2  = 0.995). The novel immunoassay provides a specific and sensitive immunoassay protocol for VEGF detection at very low levels. Graphical abstract.

Background: It often takes more than 10 years and costs more than 1 billion dollars to develop a new drug for a particular disease and bring it to the market. Drug repositioning can significantly reduce costs and time in drug development. Recently, computational drug repositioning attracted a considerable amount of attention among researchers, and a plethora of computational drug repositioning methods have been proposed. This methodology has widely been used in order to address various medical challenges, including cancer treatment. The most common cancers are lung and breast cancers. Thus, suggesting FDA-approved drugs via drug repositioning for breast cancer would help us to circumvent the approval process and subsequently save money as well as time., Methods: In this study, we propose a novel network-based method, named RepCOOL, for drug repositioning. RepCOOL integrates various heterogeneous biological networks to suggest new drug candidates for a given disease., Results: The proposed method showed a promising performance on benchmark datasets via rigorous cross-validation. The final drug repositioning model has been built based on a random forest classifier after examining various machine learning algorithms. Finally, in a case study, four FDA approved drugs were suggested for breast cancer stage II., Conclusion: Results show the potency of the proposed method in detecting true drug-disease relationships. RepCOOL suggested four new drugs for breast cancer stage II namely Doxorubicin, Paclitaxel, Trastuzumab, and Tamoxifen.

A targeted and controlled drug delivery system based on β-cyclodextrin (β-CD) for encapsulation and controlled release of hydrophobic drugs in the presence of maltogenic amylase (MAase), as a cyclodextrin-hydrolyzing enzyme, and trastuzumab antibody has been developed. In this study, the inclusion complex of curcumin (CUR), as a model anticancer compound, with β-CD was prepared and we constructed an antibody-enzyme bioconjugate (dextran mediated MAase-Trastuzumab bioconjugate) for controlled and targeted release of CUR at HER2 positive cancer cells (including SKBR3 and BT474). Immunocytochemistry analysis indicated that the MAase-Trastuzumab bioconjugate had significant binding affinities to HER2 positive cancer cells and demonstrated high enzyme activity to degrade β-CD in order to rapid release of CUR on targeted cell surface. Fluorescence microscopy images and cytotoxicity studies represent significantly greater cellular uptake and anti-proliferative effects of CUR by β-CD-CUR/MAase-Trastuzumab bioconjugate compared to free CUR and β-CD-CUR in presence and absence of MAase in HER2 positive cells. The results from flow cytometric assay suggest that the β-CD-CUR/MAase-Trastuzumab conjugate exhibited higher cytotoxic and apoptotic effects on cancer cells compared to other formulation. We demonstrate that this formulation has a potential application for targeted and controlled release of drugs in cancer therapy with increased therapeutic efficiency., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

α-Synuclein fibrillation is now regarded as a major pathogenic process in Parkinson's disease and its proteinaceous deposits are also detected in other neurological disorders including Alzheimer's disease. Therefore anti-amyloidegenic compounds may delay or prevent the progression of synucleinopathies disease. Molecular chaperones are group of proteins which mediate correct folding of proteins by preventing unsuitable interactions which may lead to aggregation. The objective of this study was to investigate the anti-amyloidogenic effect of molecular chaperone artemin on α-synuclein. As the concentration of artemin was increased up to 4 μg/ml, a decrease in fibril formation of α-synuclein was observed using thioflavin T (ThT) fluorescence and congo red (CR) assay. Transmission electron microscopy (TEM) images also demonstrated a reduction in fibrils in the presence of artemin. The secondary structure of α-synuclein was similar to its native form prior to fibrillation when incubated with artemin. A cell-based assay has shown that artemin inhibits α-synuclein aggregation and reduce cytotoxicity, apoptosis and reactive oxygen species (ROS) production. Our results revealed that artemin has efficient chaperon activity for preventing α-synuclein fibril formation and toxicity.

In this study, the role of calcium/calmodulin (Ca 2+ /CaM) and melatonin (Mel) as two signal molecules in inducing systemic salt tolerance of Dracocephalum kotschyi Boiss. was investigated. Salinity stress (100 mM NaCl) reduced plant growth and induced ionic, osmotic, and oxidative damages in D. kotschyi leaves. Detection of cytosolic free Ca 2+ ([Ca 2+ ] cyt ) by the Fura-2 method and the measurement of endogenous Mel by GC-MS demonstrated that salinity induced Ca 2+ burst and increased endogenous Mel content in D. kotschyi leaves. Root pretreatment with 5 mM Ca 2+ or 100 μM Mel recovered plant growth, reduced leaf electrolytic leakage, H 2 O 2, and MDA contents and improved membrane integrity not only at the application site (roots), but also at the untreated distal parts (leaves) under salt stress. Rhizospheric treatment with Mel and Ca 2+ triggered systemic tolerance in D. kotschyi, as judged from improving RWC, increasing proline content, modulating Na + , K + , and Ca 2+ homeostasis, and enhancing the activities of SOD, CAT, APX, and POD in the leaves of salt-stressed plants. Mel augmented [Ca 2+ ] cyt , but the rhizospheric application of Ca 2+ antagonists impaired the latter responses. Furthermore, root pretreatment with Ca 2+ increased Mel content, but the application of p-chlorophenylalanine (as an inhibitor of Mel biosynthesis) decreased the above attributes in the leaves of Ca 2+ -treated plants, leading to an arrest in the Ca 2+ -induced systemic salt tolerance. These novel results suggest that interaction of Ca 2+ /CaM and Mel is involved in overcoming salt-induced ionic, osmotic, and oxidative damages and Ca 2+ and Mel may act as long-distance signals for inducing systemic salt tolerance in D. kotschyi., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Background: Since vascular endothelial growth factor (VEGF) is a significant regulator of cancer angiogenesis, it is essential to develop a technology for its sensitive detection. Herein, we sensitized a chemiluminescence (CL) immunoassay through the combination of H 2 O 2 -sensitive TGA-CdTe quantum dot (QD) as signal transduction, dextran as a cross-linker to prepare enzyme-labeled antigen and the ultrahigh bioactivity of catalase (CAT) as reporter enzyme., Results: Under the optimized experimental conditions, the chemiluminescence enzyme-linked immunosorbent assay (CL-ELISA) method can detect VEGF in the excellent linear range of 2-35,000 pg mL -1 , with a detection limit (S/N = 3) of 0.5 pg mL -1 which was approximately ten times lower than the commercial colorimetric immunoassay. This proposed method has been successfully applied to the clinical determination of VEGF in the human serum samples, and the results illustrated an excellent correlation with the conventional ELISA method (R 2  = 0.997). The suitable recovery rate of the method in the serum ranged from 97 to 107%, with a relative standard deviation of 1.2% to 13.4%., Conclusions: The novel immunoassay proposes a highly sensitive, specific, and stable method for very low levels detection of VEGF that can be used in the primary diagnosis of tumors. With the well-designed sensing platform, this approach has a broad potential to be applied for quantitative analysis of numerous disease-related protein biomarkers for which antibodies are available.

Saccharomyces cerevisiae is being used for long as a rich source of proteins, sugars, nucleotides, vitamins and minerals. Autolyzed and hydrolyzed yeast biomass has found numerous applications in the health food industry as well as livestock feeds. Here, we have compared three lysis methods for production of yeast lysates using autolysis, plasmolysis (ethyl acetate 1.5%), and enzymatic hydrolysis (Alcalase 0.2%). The efficiency of each process was compared according to soluble solid and protein contents, cell lysis monitoring, and release of intracellular materials, cell viability and microscopic analysis. Results showed that plasmolysis by ethyl acetate was found to be more efficient compared to autolysis, with a higher recovery of yeast extract (YE) content. In comparison, the content of released solids and proteins were higher during the enzymatic hydrolysis using Alcalase compared to autolysis and plasmolysis treatments. The highest decrease in optical density of 600 nm was monitored for the hydrolyzed cells. Besides, we defined "Degree of Leakage (DL)" as a new index of the lysis process, referring to the percentage of total released proteins from the cells and it was estimated to about 65.8%, which represents an appropriate indicator of the cell lysis. The biochemical and biophysical properties of the hydrolyzed yeast product as well as its biological activity (free radical scavenging activity and bacterial binding capacity) suggest that Alcalase could be used to accelerate the lysis of yeast cells and release the valuable intracellular components used for foodstuffs, feed and fermentation media applications. Production of baker's yeast lysates using autolysis, plasmolysis, and enzymatic hydrolysis methods.

HSP70 is a powerful antiapoptotic protein that can block the extrinsic and intrinsic pathways of apoptosis. The present study describes a rapid, sensitive, and inexpensive system using luciferase as a reporter for the functional analysis of apoptotic compounds. For this approach, the co-transformation of Escherichia coli cells was performed with two expression vectors containing Hsp70 and firefly luciferase. It was found that the luciferase inactivated by heat treatment (40-46 °C for 10 min) was approximately reactivated at room temperature and regained 70% of its initial activity before heat inactivation after 60 min. The results show that the reactivation of thermally inactivated luciferase was inhibited in living cells by treatment with VER-155008 and pifitrin-μ as Hsp70 inhibitors, with half-maximal inhibitory concentration of 124 and 384 μM, respectively. The sensitivity of this method for detecting VER-155008 and pifitrin-μ was about 8 and 25 μM, respectively. Also, this reporter system showed no response to doxorubicin and dactinomycin, which bind to DNA, and we used these anticancer compounds as control compounds. Therefore, for the first time, a rapid and simple real-time system using luciferase as a reporter is introduced for the screening of apoptosis-inducing compounds based on suppression of Hsp70 in E. coli cells.

Although the superoxide anion (O 2 -· ) is generated during normal cellular respiration and has fundamental roles in a wide range of cellular processes, such as cell proliferation, migration, apoptosis, and homeostasis, its dysregulation is associated with a variety of diseases. Regarding these prominent roles in biological systems, the development of accurate methods for quantification of superoxide anion has attracted tremendous research attention. Here, we evaluated aequorin, a calcium-dependent photoprotein, as a potential bioluminescent reporter protein of superoxide anion. The mechanism is based on the measurement of aequorin bioluminescence, where the lower the concentration of coelenterazine under the oxidation of superoxide anion, the lower the amount aequorin regeneration, leading to a decrease in bioluminescence. The bioluminescence intensity of aequorin was proportional to the concentration of superoxide anion in the range from 4 to 40 000 pM with a detection limit (S/N = 3) of 1.2 pM, which was 5000-fold lower than those of the chemiluminescence methods. The proposed method exhibited high sensitivity and has been successfully applied to the determination of superoxide anion in the plant cell samples. The results could suggest a photoprotein-based bioluminescence system as a highly sensitive, specific, and simple bioluminescent probe for in vitro detection of superoxide anion.

Single-domain antibodies also known as nanobodies are recombinant antigen-binding domains that correspond to the heavy-chain variable region of camelid antibodies. Previous experimental studies showed that the nanobodies have stable and active structures at high temperatures. In this study, the thermal stability and dynamics of nanobodies have been studied by employing molecular dynamics simulation at different temperatures. Variations in root mean square deviation, native contacts, and solvent-accessible surface area of the nanobodies during the simulation were calculated to analyze the effect of different temperatures on the overall conformation of the nanobody. Then, the thermostability mechanism of this protein was studied through calculation of dynamic cross-correlation matrix, principal component analyses, native contact analyses, and root mean square fluctuation. Our results manifest that the side chain conformation of some residues in the complementarity-determining region 3 (CDR3) and also the interaction between α-helix region of CDR3 and framework2 play a critical role to stabilize the protein at a high temperature. Communicated by Ramaswamy H. Sarma.

Dopamine is a metabolite that plays a key role in the human body and in biomedical and diagnostic applications. Thus, the concentration of this analyte has been considered in various diseases in therapeutic drug monitoring (TDM). In the present study, for the first time, a photoprotein inhibition assay strategy was developed by utilizing aequorin for the direct detection of dopamine as a receptor and reporter simultaneously. The results showed that bioluminescence emission of aequorin was effectively quenched by increasing concentration of dopamine at the range of 1 nM to 100 μM with a detection limit of 53 nM. The viability of this method for the monitoring of dopamine in spiked biological fluids was also established and it was successfully applied for the direct determination of dopamine in a blood serum and urine without preliminary treatment with satisfactory quantitative recovery 90-95% and 82-93%, respectively. The structural investigation using circular dichroism, fluorescence spectroscopy, and docking simulation indicated that, changes in the microenvironment of aromatic residues were significant, while minor conformational alterations of the protein were observed. It seems dopamine inhibits bioluminescence activity with specific binding to the residues involved in the light production., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Bioluminescence of a variety of marine organisms, mostly cnidarians and ctenophores, is carried out by Ca 2+ -dependent photoproteins. The mechanism of light emission operates via the same reaction in both animal families. Despite numerous studies on the ctenophore photoprotein family, the detailed catalytic mechanism and arrangement of amino acid residues surrounding the chromophore in this family are a mystery. Here, we report the crystal structure of Cd 2+ -loaded apo-mnemiopsin1, a member of the ctenophore family, at 2.15 Å resolution and used quantum mechanics/molecular mechanics (QM/MM) to investigate its reaction mechanism. The simulations suggested that an Asp-156-Arg-39-Tyr-202 triad creates a hydrogen-bonded network to facilitate the transfer of a proton from the 2-hydroperoxy group of the chromophore coelenterazine to bulk solvent. We identified a water molecule in the coelenteramide-binding cavity that forms a hydrogen bond with the amide nitrogen atom of coelenteramide, which, in turn, is hydrogen-bonded via another water molecule to Tyr-131. This observation supports the hypothesis that the function of the coelenteramide-bound water molecule is to catalyze the 2-hydroperoxycoelenterazine decarboxylation reaction by protonation of a dioxetanone anion, thereby triggering the bioluminescence reaction in the ctenophore photoprotein family., (© 2019 Molakarimi et al.)

Over the recent decade, the calcium-based assays have gained much popularity in order to discover new drugs. Since breast cancer is the second cause of death in the female population, rapid and effective methods are needed to screen drug compounds with fewer side effects. Human epidermal growth factor receptor 2 (HER2) increases intracellular free Ca 2+ on its signaling pathways. In the present study, BT474 cell line, which overexpresses HER2 receptor, was selected and using fura-2-AM, intracellular Ca 2+ release was investigated. The changes in the concentration of intracellular Ca 2+ were evaluated by variation in the amount of fluorescence intensity. In the presence of epidermal growth factor (EGF), an increase in fluorescence intensity was observed so that after 20 min it raised to the maximum level. After treatment of BT474 cells by lapatinib, as a tyrosine kinase inhibitor (TKI), the signaling pathway of EGFR/HER2 heterodimer was significantly inhibited, which resulted in a decrease in Ca 2+ entry into the cytoplasm and fluorescence emission decreased. The IC 50 value for the effect of lapatinib on BT474 cells was 113.2 nmol/L. Our results suggest this method is a simple, efficient and specific approach and can potentially be useful for screening new drug candidates against EGFR/HER2 heterodimer signaling pathways. Graphical abstract ᅟ.

Previously, a lipase purified from a Pseudomonas source showed to form amyloid fibril structure very rapidly in the absence of a detectable lag phase. In this process the urea-unfolded enzyme encounters a medium close to physiological, but is unable to fold and, therefore, the main driving force of aggregation lies in the sequence of the protein and in its aggregation-promoting regions (APRs). Two regions with the highest propensity to aggregate were identified. These were Regions 51-57 and 160-172 as they were found with all four prediction algorithms. Two mutants of lipase, F171E and I52E, were selected and their propensity to aggregate was evaluated using thioflavin T (ThT), Congo red binding, circular dichroism, transmission electron microscopy (TEM) and dynamic light scattering. While I52E lipase formed aggregates that were capable of amyloid dye binding, showed a typical β-sheet structure and amorphous/fibrillar morphology, the aggregates formed by the F171E mutant indicated diminished ThT binding, lower light scattering, a smaller content of β-sheet structure and a lower presence of aggregates by TEM imaging. These data indicate that the region of the Sequence 160-172 is an APR region of this protein and lead to the suggestion of strategies aimed at promoting the solubility of this protein., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Mnemiopsin 2 from Mnemiopsis leidyi is a calcium-regulated photoprotein which has luminescence properties in the presence of calcium and coelenterazine. All calcium-regulated photoproteins contain EF-hand loops consisting of 12 individual residues in which the 6th position is occupied by Gly. However, the 6th residue in mneniopsin 2 is Glu rather than Gly. Here, we investigated the structural and functional consequences of substitution of Glu by Gly (E50G variant) using site-directed mutagenesis and spectroscopic procedures. It was revealed that the luminescence activity of the variant was about 17 times greater than that of wild-type (WT) photoprotein. In comparison with WT protein, our variant showed higher optimum temperature and calcium sensitivity as well as slower rate of luminescence decay. Homology modeling and sequence analysis with other known photoproteins showed that EF-hand I loop can affect the luminescence activity of E50G variant. Structural studies using circular dichroism and fluorescence spectroscopy revealed that mutation leads to the reduction in secondary structural content and local structural alterations. Finally, it can be concluded that the activity of E50G variant increases as a result of more flexibility that brought about by Gly essential for adopting the correct conformation for functional activity., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Various cold-adapted organisms produce antifreeze proteins (AFPs), which prevent the freezing of cell fluids by inhibiting the growth of ice crystals. AFPs are currently being recognized in various organisms, living in extremely low temperatures. AFPs have several important applications in increasing freeze tolerance of plants, maintaining the tissue in frozen conditions and producing cold-hardy plants by applying transgenic technology. Substantial differences in the sequence and structure of the AFPs, pose a challenge for researchers to identify these proteins. In this paper, we proposed a novel method to identify AFPs, using supportive vector machine (SVM) by incorporating 4 types of features. Results of the two used benchmark datasets, revealed the strength of the proposed method in AFP prediction. According to the results of an independent test setup, our method outperformed the current state-of-the-art methods. In addition, the comparison results of the discrimination power of different feature types revealed that physicochemical descriptors are the most contributing features in AFP detection. This method has been implemented as a stand-alone tool, named afpCOOL, for various operating systems to predict AFPs with a user friendly graphical interface.

A method is described for the chemiluminescence based determination of the activity of catalase (CAT) using H 2 O 2 -sensitive CdTe quantum dots (QDs). It is based on the finding that the chemiluminescence (CL) of the CdTe/H 2 O 2 system is reduced due to the consumption of H 2 O 2 by the catalytic action of CAT. The Michaelis constant is calculated to be 519 ± 27 mM, showing the potential of the method to accurately measure the K m compared to the standard method. The method does not require QDs to be conjugated to biological/organic molecules and therefore is considered to be a rapid and convenient method for determination of CAT in real samples. At an incubation time of 2 s, the LOD was calculated to be 4.5 unit/mL, with a linear range from 6 to 400 unit/mL. The assay is sensitive, simple, and suitable for practical applications. Graphical abstract Schematic representation of chemiluminescence-based catalase U(CAT) assay using the CdSe QD/H 2 O 2 system. The reduction of H 2 O 2 is reflected by the chemiluminescence of the QDs. A mechanism is put forward based on the changes in chemiluminescence intensity of the QDs by the consumption of H 2 O 2 due to the catalytic action of CAT.

Encysted embryos of Artemia are among the most stress-resistant eukaryotes partly due to the massive amount of a cysteine-rich protein termed artemin. High number of cysteine residues in artemin and their intramolecular spatial positions motivated us to investigate the role of the cysteine residues in the chaperone-like activity of artemin. According to the result of Ellman's assay, there are nine free thiols (seven buried and two exposed) and one disulfide bond per monomer of artemin. Subsequent theoretical analysis of the predicted 3D structure of artemin confirmed the data obtained by the spectroscopic study. Native and reduced/modified forms of artemin were also compared with respect to their efficiency in chaperoning activity, tertiary structure, and stability. Since the alkylation and reduction of artemin diminished its chaperone activity, it appears that its chaperoning potential depends on the formation of intermolecular disulfide bond and the presence of cysteine residues. Comparative fluorescence studies on the structure and stability of the native and reduced protein revealed some differences between them. Due to the redox-dependent functional switching of artemin from the less to more active form, it can be finally suggested as a redox-dependent chaperone.

Iron is a component of many proteins that have crucial roles in plant growth and development, such as ferritin and catalase. Iron also, as a ferromagnetic element, is assumed to be influenced by a static magnetic field (SMF). In the present study, we examined the relationship between ferrous content and gene expression and activity of ferritin and catalase in soybean plants under the influence of 0, 20, and 30 mT SMF for 5 day, 5 h each. Exposure to 20 mT decreased gene expression of Fe transporter, ferrous and H 2 O 2 contents and gene expression, content and activity of ferritin and catalase. Opposite responses were observed under 30 mT treatments. The results suggest that SMF triggered a signaling pathway that is mediated by iron. The structure and activity of purified ferritin and apoferritin from horse spleen, and catalase from bovine liver proteins under SMF were evaluated as well. Secondary structure of proteins were not influenced by SMF (evidenced by far-UV circular dichroism), whereas their tertiary structure, size, and activity were altered (shown by fluorescence spectroscopy and dynamic light-scattering). From these results, it is likely that the number of iron atoms is involved in the nature of influence of SMF on protein structure., (Copyright © 2018. Published by Elsevier GmbH.)

In this study, we report a detailed experimental, binding free energy calculation and molecular dynamics (MD) simulation investigation of the interactions of carboxylic-functionalized multi-walled carbon nanotubes (COOH-f-MWCNTs) with porcine trypsin (pTry). The enzyme exhibits decreased thermostability at 330K in the presence of COOH-f-MWCNTs. Furthermore, the activity of pTry also decreases in the presence of COOH-f-MWCNTs. The restricted diffusion of the substrate to the active site of the enzyme was observed in the experiment. The MD simulation analysis suggested that this could be because of the blocking of the S1 pocket of pTry, which plays a vital role in the substrate selectivity. The intrinsic fluorescence of pTry is quenched with increase in the COOH-f-MWCNTs concentration. Circular dichroism (CD) and UV-visible absorption spectroscopies indicate the ability of COOH-f-MWCNTs to experience conformational change in the native structure of the enzyme. The binding free energy calculations also show that electrostatics, π-cation, and π-π stacking interactions play important roles in the binding of the carboxylated CNTs with pTry. The MD simulation results demonstrated that the carboxylated CNTs adsorb to the enzyme stronger than the CNT without the-COOH groups. Our observations can provide an example of the nanoscale toxicity of COOH-f-MWCNTs for proteins, which is a critical issue for in vivo application of COOH-f-MWCNTs., Competing Interests: The authors have declared that no competing interests exist.

A novel Hsp70 chaperone from Rutilus frisii kutum was identified, cloned, expressed, purified and its functional characteristics revealed. The 3D structure of Hsp70 from Rutilus kutum was constructed using the crystal structure of E. coli Hsp70 as the template, with 47% sequence identity. The in vitro ATPase activity assay after 60min, ATP hydrolysis of purified recombinant Hsp70 (8μM) was improved by binding to denatured thermally luciferase (3μM) about 2.5-fold compared with that of Hsp70 alone. Based on the results, it was found that the purified Hsp70 chaperone was able to considerably suppress heat-induced aggregation of luciferase by binding to DnaJ co-chaperone (5μM) more than 70% after 10min at 42°C. In addition, Hsp70 DnaJ complex improved the refolding of heat-shocked luciferase nearly 40% after 60min at 25°C. It was concluded that Hsp70 protein from Rutilus frisii kutum has the critical role in preventing heat-induced aggregation of luciferase and refolding of heat-denatured luciferase was strictly dependent on the activity of Hsp70, thus, this protein can potentially be used for improving the functional properties of luciferase in various applications., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Escherichia coli is a common host that is widely used for producing recombinant proteins. However, it is a simple approach for production of heterologous proteins; the major drawbacks in using this organism include incorrect protein folding and formation of disordered aggregated proteins as inclusion bodies. Co-expression of target proteins with certain molecular chaperones is a rational approach for this problem. Aequorin is a calcium-activated photoprotein that is often prone to form insoluble inclusion bodies when overexpressed in E. coli cells resulting in low active yields. Therefore, in the present research, our main aim is to increase the soluble yield of aequorin as a model protein and minimize its inclusion body content in the bacterial cells. We have applied the chaperone-assisted protein folding strategy for enhancing the yield of properly folded protein with the assistance of artemin as an efficient molecular chaperone. The results here indicated that the content of the soluble form of aequorin was increased when it was co-expressed with artemin. Moreover, in the co-expressing cells, the bioluminescence activity was higher than the control sample. We presume that this method might be a potential tool to promote the solubility of other aggregation-prone proteins in bacterial cells.

As a Ca 2+ -regulated photoprotein, aequorin (Aeq) contains four EF-hand motifs, the second one lacks the standard sequence for Ca 2+ coordination and doesn't bind to Ca 2+ . Here, we replaced this loop with a functional loop. According to structural studies, although the global stability of modified aequorin (4EFAeq) is higher than that of Aeq; increasing the local flexibility accompanied by internal structural rearrangements in 4EFAeq result in its penetrability to urea and acrylamide. A fast decay rate was observed for 4EFAeq. Assuming the presence of intermediate states in the luminescent reaction, this observation indicate that the loop replacement leads to the lowering of the half-life of intermediate states which results in increasing the rate of conformational switching of 4EFAeq to light emitting form. However, considerable reduction in initial luminescence intensity of 4EFAeq suggests that the number of functional complexes is reduced. Our findings demonstrate that the conformational effects of the second loop in Aeq elicit a delicate balance between local flexibility and global stability which may be considered as an important functional parameter in photoproteins. It was also concluded that evolutionary conservation of EF-hand ΙΙ in the current form is a consequence of priority of intensity to decay rate in bioluminescent organisms., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Herein, we investigated the effect of the chitosan nanoparticles (CsNP) on the structure, dynamics, and activity of trypsin. The enzyme activity in complex with the nanoparticles slightly increased, which represents the interactions between the nanoparticles and the enzyme. The kinetic parameters of the enzyme, K m and k cat , increased after adding the nanoparticles, resulting in a slight increase in the catalytic efficiency (k cat /K m ). However, the effect of the nanoparticles on the kinetic stability of trypsin has not exhibited significant variations. Fluorescence spectroscopy did not show remarkable changes in the trypsin conformation in the presence of the nanoparticles. The circular dichroism (CD) spectroscopy results also revealed the secondary structure of trypsin attached to the nanoparticles slightly changed. Furthermore, we used molecular dynamics (MD) simulation to find more information about the interaction mechanisms between the nanoparticles and trypsin. The root mean square deviation (RMSD) of Cα atoms results have shown that in the presence of the nanoparticles, trypsin was stable. The simulation and the calculation of the binding free energy demonstrate that the nonpolar interactions are the most important forces for the formation of stable nanoparticle-trypsin complex. This study has explicitly elucidated that the nanoparticles have not considerable effect on the trypsin., (Copyright © 2017. Published by Elsevier B.V.)

Photoinactivation is a common phenomenon in bioluminescence ctenophore photoproteins (e.g mnemiopsin, berovin and BfosPP) with still unknown mechanism. The activity of coelenterate photoproteins (e.g aequorin), which has high structural similarity with ctenophore photoproteins, is not affected by light. Recently, we have characterized the effects of light on ctenophore photoprotein mnemiopsin, in different conformations, which has demonstrated light induced structural changes, uniquely secondary structures, of both apo and holo mnemiopsin. This paper is further expansion of our previous work, by applying molecular dynamics simulations to investigate photoinactivation related dynamics of berovin at atomistic level, in comparison with aequorin, under the influence of electric component of electromagnetic field. The results have indicated that the intense electric filed could influence structure of both berovin and aequorin but in different manner, whereas moderate electric field only effects on berovin's structure remarkably. In this case, increased helicity of residues E180-M193 and decreased helical contents of L38-D46 and L125-D138 segments are considerable in berovin as well as flexibility elevation of calcium binding loops. These changes cause structural expansion of berovin, especially at N-terminal domain, in direction of electric field. In conclusion, the induced structural changes of mentioned helical parts together with elevated fluctuation of their adjacent segments, N26-D46 and M193-Y206, indicate the influence of light on substrate stabilizing residues, Arg41 and Y204. This condition could presumably leads to inactivation of bioluminescence reaction due to separation of substrate from the cavity of the protein., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Bioluminescence in Ca2+-binding photoproteins is an intramolecular reaction triggered by the addition of Ca2+. A comparative study has been done on Ca2+-depleted and Ca2+-loaded apo-mnemiopsin to understand the structural transition of the photoprotein by Ca2+ binding. Ca2+ is removed by TCA (trichloroacetic acid) precipitation to obtain Ca2+-depleted apomnemiopsin., Method: UV-visible, CD and fluorescence spectroscopic studies demonstrate that the addition of Ca2+ is brought about by the overall structure of apo-mnemiopsin becomes more open in a concentration- dependent manner without significantly influencing the secondary structure and indicate that the Ca2+-depleted form of apo-mnemiopsin, in contrast to most other EF-hand calcium binding proteins, adopt a closed conformation when compared to the Ca2+-loaded form. On the other hand, dynamic quenching and limited proteolysis analysis revealed that Ca2+-loaded apo-mnemiopsin became much more flexible than Ca2+ free apo-mnemiopsin., Results: It seems that increased flexibility of the protein, which occurs due to calcium binding, is a critical factor in oxidative decarboxylation reaction on coelenterazine and consequently light emission., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Photoproteins are responsible for light emission in a variety of marine ctenophores and coelenterates. The mechanism of light emission in both families occurs via the same reaction. However, the arrangement of amino acid residues surrounding the chromophore, and the catalytic mechanism of light emission is unknown for the ctenophore photoproteins. In this study, we used quantum mechanics/molecular mechanics (QM/MM) and site-directed mutagenesis studies to investigate the details of the catalytic mechanism in berovin, a member of the ctenophore family. In the absence of a crystal structure of the berovin-substrate complex, molecular docking was used to determine the binding mode of the protonated (2-hydroperoxy) and deprotonated (2-peroxy anion) forms of the substrate to berovin. A total of 13 mutants predicted to surround the binding site were targeted by site-directed mutagenesis which revealed their relative importance in substrate binding and catalysis. Molecular dynamics simulations and MM-PBSA (Molecular Mechanics Poisson-Boltzmann/surface area) calculations showed that electrostatic and polar solvation energy are +115.65 and -100.42 kcal/mol in the deprotonated form, respectively. QM/MM calculations and pKa analysis revealed the deprotonated form of substrate is unstable due to the generation of a dioxetane intermediate caused by nucleophilic attack of the substrate peroxy anion at its C3 position. This work also revealed that a hydrogen bonding network formed by a D158- R41-Y204 triad could be responsible for shuttling the proton from the 2- hydroperoxy group of the substrate to bulk solvent.

The conversion of proteins from their soluble states into well-organized amyloid fibrils has received abundant attention. This process typically consists of three stages: lag, growth and plateau phases. In this study, the process of amyloid fibril formation by lipase from Pseudomonas sp. after diluting out urea was examined by Thioflavin T (ThT) fluorescence, Congo red (CR) binding, 8-anilinonaphthalene-1-sulfonic acid (ANS) binding, dynamic light scattering (DLS), circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopies, X-ray diffraction (XRD) and transmission electron microscopy (TEM). To exclude the presence of preformed aggregates in the pure lipase sample, aforementioned assays were also performed for the protein unfolded in urea before dilution. The aggregates formed immediately after dilution were found to bind to ThT and CR and contain a significant amount of β-sheet structure, as determined by far-UV CD and FTIR spectroscopies, as well as XRD analysis. Moreover, these aggregates present, at least in part, a fibrillar morphology, as deduced with TEM. This examination showed that lipase fibril formation proceeds quickly after dilution, within a few seconds, without a detectable lag phase. We also investigated bacterial inclusion bodies formed after expression of lipase in E. coli, providing evidence for the existence of rapidly formed amyloid-like structural and tinctorial properties in the lipase-containing inclusion bodies., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Withania coagulans fruit has traditionally been used as milk coagulant. The present study reports the purification and characterization of an aspartic protease from W. coagulans fruit. The enzyme was purified via fractional ammonium sulfate precipitation and cation exchange chromatography. SDS-PAGE analysis revealed the presence of a monomeric protein with molecular weight of 31kDa. Proteolytic activity (PA) of the protease was evaluated using casein, and the milk-clotting activity (MCA) was analyzed by skim milk. The K m and V max values of the enzyme for casein were obtained to be 1.29mg/ml and 0.035μmol Tyr/min, respectively. Optimal temperature and pH were 65°C and 5.5, respectively. After incubation of enzyme at 65°C for 1h, 73% of PA was remained which demonstrated high thermal stability of the enzyme. Mass spectrometry analysis of the purified protease and enzyme assays in the presence of protease inhibitors indicated that aspartic protease was the only responsible enzyme in milk coagulation. Furthermore, by investigating the effect of salts on enzyme activity, it was observed that both NaCl and CaCl 2 reduced enzyme activity. These characteristics of the protease suggest that the enzyme may be suitable for producing low salt content cheeses., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Insect acetylcholinesterase (AChE) is the principal target for organophosphate (OP) and carbamate (CB) insecticides. In this research, an AChE from third instar larvae of elm left beetle, Xanthogaleruca luteola was purified by affinity chromatography. The enzyme was purified 75.29-fold with a total yield of 8.51%. As shown on denaturing SDS-PAGE, the molecular mass of purified AChE was 70kDa. The enzyme demonstrated maximum activity at pH7 and 35°C. Furthermore, a series of temephos (Tem) derivatives with the general structure of P(O)XP(O) (1-44) were prepared, synthesized and characterized by 31 P, 13 C, 1 H NMR and FT-IR spectral techniques. The toxicity of 36 new Tem derivatives was screened on the third instar larvae and the compound compound 1,2 cyclohexane-N,N'-bis(N,N'-piperidine phosphoramidate) exhibited the highest insecticidal potential. The method of kinetic analysis is applied in order to obtain the maximum velocity (V max ), the Michaelis constant (K m ) and the parameters characterizing the inhibition type for inhibitors with >75% mortality in preliminary bioassay. The inhibition mechanism was mixed and inhibitory constant (K i ) was calculated as 4.70μM -1 min -1 for this compound. Quantitative structure-activity relationship (QSAR) equations of these compounds indicated that the electron orbital energy has major effect on insecticidal properties., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Artemin is an abundant thermostable protein in Artemia encysted embryos under environmental stresses. It is confirmed that high regulatory expression of artemin is relevant to stress resistance in this crustacean. Here, the protective role of artemin from Artemia urmiana has been investigated on survival of bacterial cells under salt and oxidative shocks. Also, for continuous monitoring of the effect of artemin in prevention of proteins aggregation/inactivation, co-expression of artemin and luciferase (as an intracellular reporter) in bacterial cells was performed. According to the results, residual activity of luciferase in artemin expressing E. coli cells exposing to different concentrations of H 2 O 2 and NaCl was significantly higher than non-expressing cells. The luciferase activity was rapidly lost in control cells under salt treatments while in co-transformed cells, the activity was considerably retained at higher salt concentrations. Also, analysis from cell viability assays showed that artemin-expressing cells exhibited more resistance to both stress conditions. In the present study, we document for the first time that artemin can protect proteins and bacterial cells against oxidative and salt stress conditions. These results can declare the resistance property of this crustacean against harsh environmental conditions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Maltogenic amylases (MAases, EC 3.2.1.133) have been gotten much attention due to their various applications in industry and commercial processes. MAases belong to subfamily 20 of glycoside hydrolase family 13 (NPase or CDases subfamily) and they have important differences with other members of the family. This enzyme consists of two subunits which form two active sites in the dimer form by binding of the central domain of each subunit to the N domain of the next one (domain-swapping dimeric structure). Allosterism is a possible way of regulating enzymatic activity and no evidence has been found regarding to the cooperativity and correlation between MAases subunits, therefore in this study the allosteric behavior of MAases from a native strain (Geobacillus sp. Gh6) was investigated. Unlike other members of α-amylase family, MAases showed positive cooperativity between their subunits and the enzyme exhibited sigmoidal nature towards all three cyclodextrin (CD) substrates with a Hill constant (nH) value equal to 2, 1.6 and 1.1 for α-CD, β-CD and γ-CD, respectively. On further analysis, the effect of glucose and maltose as MAases allosteric effectors in the presence of β-CD substrate showed that these two effectors had a biphasic effect; while they stimulated the enzyme activity at low concentrations (with a decrease in Hill constant), these metabolites acted as allosteric inhibitors at higher concentrations. Due to the key role of MAases in carbohydrate metabolization, an efficient regulating system for this enzyme is required. In this experiment, for the first time the allosteric properties of MAases were observed and investigated., (Copyright © 2016 Elsevier Inc. All rights reserved.)