Exploiting cyclodextrins as artificial chaperones to enhance enzyme protection through supramolecular engineering.

We report a method of enzyme stabilisation exploiting the artificial protein chaperone properties of ß-cyclodextrin (ß-CD) covalently embedded in an ultrathin organosilica layer. Putative interaction points of this artificial chaperone system with the surface of the selected enzyme were studied in silico using a protein energy landscape exploration simulation algorithm. We show that this enzyme shielding method allows for drastic enhancement of enzyme stability under thermal and chemical stress conditions, along with broadening the optimal temperature range of the biocatalyst. The presence of the ß-CD macrocycle within the protective layer supports protein refolding after treatment with a surfactant.

A novel TMD-based peroxidase-mimicking nanozyme: From naked eye detection of leukocytosis-related diseases to sensing different bioanalytes.

Being emerged as alternatives to natural enzymes, nanozymes have recently drawn much attention in sensing. Herein, the first multicomponent transition metal dicalchogenide (TMD)-based nanozyme (MCFS/rGO) was synthesized by a facile hydrothermal method and characterized. This peroxidase-mimic nanozyme follows the typical Michaelis-Menten kinetics, showing a higher affinity for H2O2 substrate (Km = 9 µM) compared to that of natural peroxidase (Km = 3700 µM). The remarkable potential of the MCFS/rGO nanozyme to detect H2O2 provided us with a great opportunity to design some simple and fast colorimetric sensing systems. Coupling the efficient peroxidase-mimicking activity of the nanozyme with the H2O2 production capacity of white blood cells (WBCs) leads to the development of a novel, simple, rapid, and efficient colorimetric method to distinguish leukocytosis-related patients from healthy people by the naked eye. This pioneering diagnostic technique can also be utilized to quantitatively measure the WBC count. Moreover, we coupled the mentioned nanozyme-based system with the activity of glucose oxidase enzyme available in different types of honey samples, an innovative mechanism proved to be an effective quality indicator of the samples. Last but not least, the MCFS/rGO nanozyme is also able to determine the quantity of some biologically significant analytes, including glutathione (GSH), ascorbic acid (AA), and mercury ions (Hg2+), of which the limit of detection (LOD) was 9.3 nM, 22.5 nM, and 0.32 µM, respectively. Our results, however, demonstrated the superior performance of the MCFS/rGO nanozyme to determine the first two mentioned bioanalytes compared with other TMDs. Overall, this novel nanozyme-based sensor system can be considered a suitable candidate for developing multipurpose biosensors for medical and biochemical applications.

Hyaluronic acid coated spinel ferrite for combination of chemo and photodynamic therapy: Green synthesis, characterization, and in vitro and in vivo biocompatibility study.

In this project, different photosensitizers were prepared using different ratios of nickel, manganese, and iron. Then, multiple analysis were performed to evaluate their efficiency, and the most suitable one was used to be coated by hyaluronic acid to improve the nano-platform's biocompatibility and target ability. Moreover, another chemical targeting agent (riboflavin) was used to further improve the target ability of the prepared nano-platform. Different spectroscopies and thermal analysis were used to determine the physical and chemical characteristics of the prepared nano-platform. Also, in order to determine the biocompatibility of the nano-platform, in vitro and in vivo tests such as blood hemolysis, blood aggregation and lethal dose were performed. Then, an anti-cancer agent (curcumin) was loaded on the selected nano-platform to makes us able utilizing the synergistic effect of chemotherapy and photodynamic therapy simultaneously. Finally, the cell cytotoxicity results showed that the prepared nano-platform had a great anti-cancer potential which can make it a great candidate as a dual photo and chemo therapy agent for treatment of breast cancers.

Reinforcing our defense or weakening the enemy? A comparative overview of defensive and offensive strategies developed to confront COVID-19.

Developing effective strategies to confront coronavirus disease 2019 (COVID-19) has become one of the greatest concerns of the scientific community. In addition to the vast number of global mortalities due to COVID-19, since its outbreak, almost every aspect of human lives has changed one way or another. In the present review, various defensive and offensive strategies developed to confront COVID-19 are illustrated. The Administration of immune-boosting micronutrients/agents, as well as the inhibition of the activity of incompetent gatekeepers, including some host cell receptors (e.g. ACE2) and proteases (e.g. TMPRSS2), are some efficient defensive strategies. Antibody/phage therapies and specifically vaccines also play a prominent role in the enhancement of host defense against COVID-19. Nanotechnology, however, can considerably weaken the virulence of SARS-CoV-2, utilizing fake cellular locks (compounds mimicking cell receptors) to block the viral keys (spike proteins). Generally, two strategies are developed to interfere with the binding of spike proteins to the host cell receptors, either utilizing fake cellular locks to block the viral keys or utilizing fake viral keys to block the cellular locks. Due to their evolutionary conserved nature, viral enzymes, including 3CLpro, PLpro, RdRp, and helicase are highly potential targets for drug repurposing strategy. Thus, various steps of viral replication/transcription can effectively be blocked by their inhibition, leading to the elimination of SARS-CoV-2. Moreover, RNA decoy and CRISPR technologies likely offer the best offensive strategies after viral entry into the host cells, inhibiting the viral replication/assembly in the infected cells and substantially reducing the quantity of viral progeny.

Inhibitory Potential of Acroptilon repens against Key Enzymes involved in Alzheimer and Diabetes, Phytochemical Profile, Radical Scavenging, and Antibacterial Activity.

Background: This study was devoted to assessing the inhibitory potential of acetone, methanol, and ethanol extracts of Acroptilon repens against disease-associated enzymes, as well as their antioxidant/antibacterial activity and phytochemical composition. Methods: Comparative assessment using various antioxidant evaluation methods, including ferric reducing antioxidant power, scavenging ability on 2,2-diphenyl-1-picrylhydrazyl radical and hydrogen peroxide, and reducing power, indicated that the acetone extract presented the highest antioxidant activity, due to its highest total antioxidant content. Results: The total phenolic content and total flavonoids content of these extracts were 3.44 ± 0.32 mg GAE/g DW and 2.09 ± 0.2 mg QE/g DW, respectively. The hydrodistillation essential oil from A. repens was analyzed by gas chromatography-mass spectroscopy, and 17 compounds were identified. All extracts showed good inhibitory activities against disease-related enzyme acetylcholinesterase and α-amylase, with the lowest IC50 for acetonic extract. Extracts of A. repens exhibited inhibiting activities against the Gram-positive bacteria, with the most effect of acetone extract. Conclusion: Our findings suggest A. repens as a promising source of natural antioxidant, antimicrobial, anti-cholinesterase and anti-amylase agents for the management of oxidative damage, and pharmaceutical, food, and cosmeceutical purposes.

Characteristics, dynamics and mechanisms of actions of some major stress-induced biomacromolecules; addressing Artemia as an excellent biological model.

Stress tolerance is one of the most prominent and interesting topics in biology since many macro- and micro-adaptations have evolved in resistant organisms that are worth studying. When it comes to confronting various environmental stressors, the extremophile Artemia is unrivaled in the animal kingdom. In the present review, the evolved molecular and cellular basis of stress tolerance in resistant biological systems are described, focusing on Artemia cyst as an excellent biological model. The main purpose of the review is to discuss how the structure and physicochemical characteristics of protective factors such as late embryogenesis abundant proteins (LEAPs), small heat shock proteins (sHSPs) and trehalose are related to their functions and by which mechanisms, they exert their functions. In addition, some metabolic depressors in Artemia encysted embryos are also mentioned, indirectly playing important roles in stress tolerance. Importantly, a great deal of attention is given to the LEAPs, exhibiting distinctive folding behaviors and mechanisms of actions. For instance, molecular shield function, chaperone-like activity, moonlighting property, sponging and snorkeling capabilities of the LEAPs are delineated here. Moreover, the molecular interplay between some of these factors is mentioned, leading to their synergistic effects. Interestingly, Artemia life cycle adapts to environmental conditions. Diapause is the defense mode of this life cycle, safeguarding Artemia encysted embryos against various environmental stressors. Communicated by Ramaswamy H. Sarma.

Influence of a 5-bp Indel Polymorphism at Promoter of the GAS5 lncRNA and Risk of Breast Cancer.

Long non-coding RNAs (lncRNAs) are RNA molecules (>200 nucleotides in length) with no protein-coding capacity. Recent studies have demonstrated that lncRNAs involve in the regulation of their target genes at transcriptional, post-transcriptional and epigenetic levels. The aim of this case-control study was to explore whether growth arrest-specific 5 (GAS5) lncRNA 5-bp Ins/Del (rs145204276) polymorphism is involved in the breast cancer susceptibility. A total of 170 cases and 220 age matched controls were recruited in this study. GAS5 lncRNA polymorphism was genotyped using tetra primers amplification refractory mutation system polymerase chain reaction (T-ARMS-PCR) method. Statistical analysis was performed using SPSS. The distribution of the genotype ins/ins, ins/del and del/del were %75.29, 21.76% and 2.94% and 52.27%, 39.55% and 8.81% in the cases and controls, respectively. The ins/del or del/del genotype had a significantly decreased risk of breast cancer as compared with the ins/ins genotype under a codominant model (OR=0.38, 95%CI 0.24-0.60, p=0.0001; OR= 0.25, 95%CI 0.09-0.69, p=0.008, respectively). Moreover, the deletion allele of this polymorphic site is associated with a protective effect (OR=0.41, 95%CI 0.28-0.60, p=0.0001). Our study provided the first evidence that the deletion allele of GAS5 rs145204276 may have a protective role in mediating individual susceptibility to breast cancer. However, further comprehensive studies are warranted in a larger sample.
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Stress-dependent conformational changes of artemin: Effects of heat and oxidant.

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.

Development of a phage display-mediated immunoassay for the detection of vascular endothelial growth factor.

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 (R2 = 0.995). The novel immunoassay provides a specific and sensitive immunoassay protocol for VEGF detection at very low levels. Graphical abstract.

Modulation of the competition between renaturation and aggregation of lysozyme by additive mixtures.

The effects of 17 kinds of additive mixtures have been studied on refolding and aggregation of a model protein, lysozyme. Most of the prepared mixtures were efficient in inhibiting aggregation of the protein, and, surprisingly, four novel additive mixtures, i.e., lactic acid: l-arginine, lactic acid: l-glutamine, choline chloride: lactic acid, and imidazolium salt: ß-cyclodextrin as well as choline chloride: urea exhibited a more remarkable efficacy in suppressing aggregation. Among these, lactic acid: l-arginine was identified as the most efficient additive, and lactic acid: l-glutamine and choline chloride: lactic acid were inefficient to recover the enzyme activity. In contrast, choline chloride: ethylene glycol: imidazole, choline chloride: glycerol: imidazole, imidazole: betaine: ethylene glycol were found to be less effective mixtures in preventing enzyme aggregation. Totally, it was demonstrated that the protective effects of the mixtures were improved as their concentrations increased. The improvement was more remarkable for imidazolium salt: ß-cyclodextrin and choline chloride: urea, where the denatured lysozyme was reactivated and recovered up to 85% of its initial activity by enhancing their concentrations from 1 to 5% (V/V). It is suggested that such solution additives may be further employed as artificial chaperones to assist protein folding and stability.

A novel milk-clotting cysteine protease from Ficus johannis: Purification and characterization.

Due to the need for calf rennet alternatives, many attempts have been made to find new proteases. A novel cysteine protease with milk-clotting activity was purified from Ficus johannis by cation exchange chromatography. The protease was stable in various pH (3.0-10.5) with the optimum at 6.5 and showed its maximum activity at 60 °C. The Km and Vmax values of the enzyme were obtained to be 0.604 mg/ml and 0.0273 µmol Tyr/min, respectively. The purified protease exhibited considerable activity towards κ-casein in comparison to α-casein and ß-casein. The enzyme was almost completely active in the presence of high salt concentrations. Besides, it had high stability against autodigestion. The content of free amino acids was determined by HPLC, where leucine, lysine, valine, γ-aminobutyric acid and tyrosine were the most abundant amino acids. The cheese manufactured by using the purified protease showed similar textural properties and physico-chemical compositions to cheese produced using commercial rennet. Considering the special characteristics, including high milk-clotting activity, considerable stability over wide ranges of pH and temperature, resistance towards solvents, salts, and surfactants, the new protease might be the promising candidate for the dairy industry as well as other food and biotechnological industries.

Generation and screening of efficient neutralizing single domain antibodies (VHHs) against the critical functional domain of anthrax protective antigen (PA).

Since anthrax is an acute infectious disease, detection and neutralization of the toxins of pathogenic Bacillus anthracis are of great importance. The critical role of protective antigen (PA) component of tripartite anthrax toxin in toxin entry into the host cell cytosol provided a great deal of effort to generate monoclonal antibodies against this constitute. Regarding the importance of anthrax detection/neutralization and unique physicochemical and pharmacological features of VHHs as single domain antibodies, the present study aimed to generate VHHs against the receptor binding domain of PA, termed PAD4. After camel immunization, a gene repertoire of VHH fragments with a diversity of 4.7×108 clones was produced, followed by constructing a VHH phage display library. A stringent successive biopanning was then carried out to isolate the phages displaying high affinity VHHs against PAD4.Polyclonal and monoclonal Enzyme-linked immunosorbent assay (ELISA) verified binding specificity of phages to the target protein. Modeling of VHHs together with the docking simulation studies, illustrated the binding site of antibodies on antigen. Docking analysis revealed that all selected VHHs potently cover the key functional residues of PAD4. Since the selected VHHs could cover and block the receptor binding loops of PA, they could be proposed as hopeful anti-Anthrax candidates.

A conformation-based phage-display panning to screen neutralizing anti-VEGF VHHs with VEGFR2 mimicry behavior.

The potency of VEGF-based anti-angiogenic strategies in cancer therapy and the brilliant characteristics of VHHs motivated us to directly block VEGF binding to its receptor with neutralizing single domain antibodies, thereby fading away the VEGF signaling pathway. Considering with high resolution crystal structure of VEGF-RBD/VEGFR2 complex, we could adopt a combinatorial screening strategy: stringent panning and competition ELISA, to direct the panning procedure to dominantly screen the favorable binders that bind and block the key functional regions of VEGF. Based on competition assay, the majority of the screened clones (82%) showed the VEGFR2 mimicry behavior for binding to VEGF molecule. The phage pool gets enriched in favor of sequences that bind the receptor binding sites of VEGF. Different immunoassays and molecular docking simulation verified that all selected VHHs could bind and cover the receptor binding sites of VEGF. Consequently, some modifications in panning procedure with considering the structural features and detailed information of functional regions of a protein antigen, led us to successfully trap the high-affinity specific binders against its hot functional regions. Since the selected VHHs could cover the receptor binding site of VEGF and block VEGF binding to the receptor, they might be promising candidates for anti-angiogenic therapies.

Development of a highly-potent anti-angiogenic VEGF8-109 heterodimer by directed blocking of its VEGFR-2 binding site.

Angiogenesis is a hallmark of various pathological conditions and is controlled by a variety of angiogenic factors. Blockade of vascular endothelial growth factor (VEGF) as the most pivotal stimulator of angiogenesis offers a promising therapeutic approach for some diseases, typically cancer. In the present study, a heterodimeric antagonistic VEGF was precisely designed based on structural information of recently-crystallized VEGF/VEGF receptor-2 (VEGFR-2/fetal liver kinase 1/kinase domain region) complex. Directed blocking of kinase domain region occurs via substitution of a VEGF receptor binding site by two peptide segments in one pole, whereas the binding domain of the other pole of VEGF was intact. Candidate peptides for substitution were selected considering to some sequence and structural criteria. A reliable model of modified VEGF was built, refined using molecular dynamics simulation and docked with VEGFR-2. Docking analysis revealed that binding affinity of mutant VEGF was notably diminished, corroborating our design. Heterodimeric VEGF was expressed, refolded and highly purified by two-step affinity chromatography. Dimerization of this antagonist was confirmed using some analytical techniques. Spectroscopic studies assured us to obtain the heterodimeric form of VEGF. Some angiogenic in vitro assays such endothelial cell proliferation and tube formation indicated that this antagonist is not only strongly capable of inhibiting angiogenesis (half maximal inhibitory concentration of 33 and 24 ng · mL(-1) , respectively), but also showed the highest inhibitory effect compared to all other heterodimeric VEGF variants. The high anti-angiogenic potency of this VEGF antagonist may allow its future use as an anti-tumor agent.

Artemin as an efficient molecular chaperone.

Artemin is an abundant thermostable protein in Artemia encysted embryos under stress. It is considered as a stress protein, as its highly regulated expression is associated with stress resistance in this crustacea. In the present study, artemin has been shown to be a potent molecular chaperone with high efficacy. Artemin is capable of inhibiting the chemical aggregation of proteins such as carbonic anhydrase (CA) and horseradish peroxidase (HRP) at unique molar ratios of chaperone to substrates (1:40 and 1:26 for CA and HRP, respectively). Furthermore, it can also enhance refolding yield of these substrates by nearly 50%. The refolding promotion of CA is checked and verified through a sensitive fluorimetric technique. Based on these experiments, artemin showed higher chaperone activity than other chaperones. The evaluation of artemin surface using ANS showed it to be highly hydrophobic, probably resulting in its high efficacy. These results suggest that artemin can be considered a novel low molecular weight chaperone.

Deletion of extra C-terminal segment and its effect on the function and structure of artemin.

Artemin acts as a molecular chaperone by protecting Artemia embryos undergoing encystment from damage, caused by heat or other forms of stress. According to the amino acid sequence alignment, although artemin shows a fair amount of homology with ferritin, it also contains an extra C-terminal. Analysis of the C-terminal extension of artemin model in previous studies has shown that there are some favorable interactions between this region and its surrounding cleft. In the current study we tried to investigate the role of this C-terminal in chaperone activity of artemin. This extra C-terminal (39 residues) was deleted and the truncated gene was cloned and expressed in Escherichia coli. According to in vivo chaperone-like activity studies, both full-length and C-terminal truncated artemin conferred thermotolerance on transfected E. coli cells. However, bacteria expressing truncated derivative of artemin was less resistant than those producing native artemin against heat. Moreover, the activity recovery on carbonic anhydrase (CA), as protein substrate, was less in the presence of truncated artemin than that of full-length artemin. The results demonstrated that C-terminal deletion decreases the ability of artemin for chaperone-like activity. Theoretical investigations showed that deletion of artemin C-terminal extension makes substantial structural alterations in a way that structural stability and overall integrity of artemin decrease.

Sequence and structural analysis of artemin based on ferritin: a comparative study.

Artemia cysts can tolerate extreme environments, partly due to a heat-stable protein called artemin. According to previous studies, artemin shares structural similarity with ferritins. Actually, there is still no strong structural information about artemin three-dimensional (3-D) structure. In this research, the artemin encoding gene from Artemia urmiana was cloned and sequenced. A reliable 3-D model of artemin was initially built using ferritin as template and refined using Molecular Dynamic (MD) Simulation. It is interesting that the proposed model, confirmed by circular dichroism (CD), shows significant differences in secondary structure contents with ferritin. Three conserved regions (ferroxidase center, iron nucleation center and 3-fold channel) in ferritins, cooperating in iron-interaction, have been substantially changed in artemin. Analysis of C-terminal region of the model revealed its major role in preventing artemin from iron-binding due to some suitable interactions. Finally, it is concluded that significant differences between artemin and ferritin, both in conserved regions related to iron-interaction and three-dimensional structure, can justify their functional differences.