Protein click chemistry and its potential for medical applications.

A revolution in chemical biology occurred with the introduction of click chemistry. Click chemistry plays an important role in protein chemistry modifications, providing specific, sensitive, rapid, and easy-to-handle methods. Under physiological conditions, click chemistry often overlaps with bioorthogonal chemistry, defined as reactions that occur rapidly and selectively without interfering with biological processes. Click chemistry is used for the posttranslational modification of proteins based on covalent bond formations. With the contribution of click reactions, selective modification of proteins would be developed, representing an alternative to other technologies in preparing new proteins or enzymes for studying specific protein functions in different biological processes. Click-modified proteins have potential in diverse applications such as imaging, labeling, sensing, drug design, and enzyme technology. Due to the promising role of proteins in disease diagnosis and therapy, this review aims to highlight the growing applications of click strategies in protein chemistry over the last two decades, with a special emphasis on medicinal applications.

Cellulose membrane coated Mo-doped TiO2nanotube sheets for sustained oxidation of biomolecules.

Titanium dioxide nanotubes (TNT) are widely researched materials for the photocatalytic generation of free radicals, which are useful in wastewater treatment. We aimed to prepare Mo-doped TNT sheets, covered with a cellulose membrane to avoid TNT surface inactivation by protein adsorption. We studied the susceptibility of serum albumin (SA) bound to different molar ratios of palmitic acid (PA) to denaturation and fibrillation by this system, which is meant to mimic oxidative stress conditions such as non-alcoholic fatty liver disease. The results demonstrated that cellulose membrane-covered TNT successfully oxidized the SA, identified by structural changes to the protein. Increasing the molar ratio of PA to protein-enhanced thiol group oxidation while protecting the protein against structural changes. Finally, we propose that in this photocatalyzed oxidation system, the protein is oxidized by a non-adsorptive mechanism mediated by H2O2. Therefore, we suggest that this system could be used as a sustained oxidation system to oxidize biomolecules as well as potentially in wastewater treatment.

The Stabilizing Mechanism of Immobilized Metagenomic Xylanases on Bio-Based Hydrogels to Improve Utilization Performance: Computational and Functional Perspectives.

While polysaccharide-based superabsorbent hydrogels (SHs) have attracted increasing interest as proficient carriers in the enzyme immobilization, the nature of the favored interactions between the SHs and enzymes is still unclear. Herein, a combined experimental and computational study was employed to investigate the dominant parameters affecting on the stabilization of two metagenomic xylanases on the SHs. The thermostable enzymes (PersiXyn3 and PersiXyn4) with similar domains were screened, cloned, expressed, and purified from cattle rumen metagenome. Then, the enzymes were immobilized on the carboxymethyl cellulose-g-poly(acrylic acid-co-acrylamide) hydrogel which resulted in increasing their activity and stability. The carboxymethyl cellulose (CMC)-based characteristic of the hydrogel provided high numbers of H-bondings/ionic bridges, causing an improvement in the stability, hydrolysis performance, and reusability of the immobilized enzymes. More specifically, enzyme immobilization resulted in ∼40% increase in the content of the reducing sugars released after treatment of paper pulp. After 16 reuse cycles, the immobilized PersiXyn4 displayed 35.9% activity, but the immobilized PersiXyn3 retained just 8.2% of its initial activity. The comparative investigations illustrated that a higher number of positively charged amino acids in the binding site of the enzyme provided stronger electrostatic attractions between it and negative functionalities of the hydrogel. This was suggested as the main reason for the higher affinity of PersiXyn4 toward hydrogel and explained the better hydrolysis performance and reusability of the immobilized PersiXyn4 on the SH. These findings are essential for designing novel innovative SH carriers and the successful engineering of optimal enzyme assemblies through the prediction of the immobilized enzyme's stabilities.

The impact of water molecules on binding affinity of the anti-diabetic thiazolidinediones for catalase: Kinetic and mechanistic approaches.

Water molecules play a vital role in efficient drug binding to its target. Thiazolidinediones (TZDs), a class of anti-diabetic drugs, are widely used for treatment of type 2 diabetes mellitus. In the present study, the possible contribution of water molecules to the binding of TZDs to catalase, a potential target in the liver, was investigated by different experimental and theoretical methods. These studies indicated that TZDs could significantly improve the catalase catalytic function with a significant contribution from water molecules. As a probe for the differential number of released water molecules during the catalase transition from E to E* states, the activity of TZDs-catalase complexes was demonstrated to be mainly dependent on water activity. However, free catalase decomposed the substrate more independently. In addition, the spectrofluorimetry studies showed that the binding of TZDs to catalase needed the release of water molecules from the enzyme's binding pocket. The thermodynamic studies indicated that the binding enthalpy and entropy of TZDs for catalase were decreased with lower water activity. The favorable process contributes to release of water molecules from the binding pocket through the formation of hydrophobic interactions between catalase and TZDs in an enthalpic manner. Molecular docking simulations confirmed that the depletion of water molecules from the binding cavity is essential for effective interactions between TZDs and catalase.

Antichaperone activity and heme degradation effect of methyl tert-butyl ether (MTBE) on normal and diabetic hemoglobins.

Because of the extensive use of methyl tert-butyl ether (MTBE) as an additive to increase the octane quality of gasoline, the environmental pollution by this compound has increased in recent decades. Environmental release of MTBE may lead to its entry to the blood stream through inhalation or drinking of contaminated water, and its interactions with biological molecules such as proteins. The present study was proposed to comparatively investigate the interactions of MTBE with hemoglobin (Hb) from diabetic and nondiabetic individuals using various spectroscopic methods including UV-visible, fluorescence, chemiluminescence, and circular dichroism. These results demonstrated the effects of MTBE on heme degradation of Hb and the reaction of these degradation products with water generating reactive oxygen species. Interaction of Hb with MTBE enhanced its aggregation rate and decreased lag time, indicating the antichaperone activity of MTBE upon interaction with Hb. Furthermore, the diabetic Hb showed more severe effects of MTBE, including heme degradation, reactive oxygen species production, unfolding, and antichaperone behavior than the nondiabetic Hb. The results from molecular docking suggested that the special interaction site of MTBE in the vicinity of Hb heme group is responsible for heme degradation.

A hidden aggregation-prone structure in the heart of hypoxia inducible factor prolyl hydroxylase.

Prolyl hydroxylase domain-containing protein 2 (PHD2), as one of the most important regulators of angiogenesis and metastasis of cancer cells, is a promising target for cancer therapy drug design. Progressive studies imply that abnormality in PHD2 function may be due to misfolding. Therefore, study of the PHD2 unfolding pathway paves the way for a better understanding of the influence of PHD2 mutations and cancer cell metabolites on the protein folding pathway. We study the unfolding of the PHD2 catalytic domain using differential scanning calorimetry (DSC), fluorescence spectroscopy, and discrete molecular dynamics simulations (DMD). Using computational and experimental techniques, we find that PHD2 undergoes four transitions along the thermal unfolding pathway. To illustrate PHD2 unfolding events in atomic detail, we utilize DMD simulations. Analysis of computational results indicates an intermediate species in the PHD2 unfolding pathway that may enhance aggregation propensity, explaining mutation-independent PHD2 malfunction.

Deferiprone: structural and functional modulating agent of hemoglobin fructation.

Diabetic complication arises from the presence of advanced glycation end products in different sites of the body. Great attention should be paid to recognizing anti-glycation compounds. Here, deferiprone as an oral iron chelator drug administrated in treatment of ß-thalassemic patients was selected to find its effect on the fructation of hemoglobin (Hb). Our results indicated that deferiprone could prevent the AGE and carbonyl formation via inhibition of structural changes in the structure of Hb during the fructation process. Moreover, deferiprone can preserve peroxidase and esterase activities of fructated Hb similar to native Hb. Therefore, deferiprone can be introduced as an anti-glycation drug to prevent the AGE formation.

Effects of 940 Hz EMF on luciferase solution: structure, function, and dielectric studies.

We designed a rectangular waveguide exposure system to study the effects of mobile phone frequency (940 MHz) electromagnetic fields (EMF) on luciferase structure and activity. The luciferase activity of exposed samples was significantly higher than that of unexposed samples. Dynamic light scattering of the exposed samples showed smaller hydrodynamic radii compared to unexposed samples (20 nm vs. 47 nm ± 5%). The exposed samples also showed less tendency to form aggregates, monitored by turbidity measurements at l = 360 nm. A microwave dielectric measurement was performed to study the hydration properties of luciferase solutions with a precision network analyzer over frequency ranges from 0.2 to 20 GHz before and after exposure. The change in the dielectric properties of the exposed luciferase solution was related to the disaggregation potency of the applied field. Together, our results suggested that direct interactions with luciferase molecules and its dipole moment were responsible for the reduced aggregation and enhanced luciferase activity upon exposure to the EMF.

Structural and functional alterations of polydopamine-coated hemoglobin: New insights for the development of successful oxygen carriers.

One of the major factors that is currently hindering the development of hemoglobin (Hb)-based oxygen carriers (HBOCs) is the autoxidation of Hb into nonfunctional methemoglobin. Modification with polydopamine (PDA), which is a biocompatible free radical scavenger has shown the ability to protect Hb against oxidation. Due to its tremendous potential in the development of successful HBOCs, herein, we conduct a thorough evaluation of the effect of PDA on the stability, aggregation, structure and function of the underlying Hb. By UV-vis spectrometry we show that PDA can prevent Hb's aggregation while thermal denaturation studies indicate that, although PDA coating resulted in a lower midpoint transition temperature, it was also able to protect the protein from full denaturation. These results are further corroborated by differential scanning calorimetry. Circular dichroism reveals that PDA can promote changes in Hb's secondary structure while, by UV-vis spectroscopy, we show that PDA also interacts with the porphyrin complex located in Hb's hydrophobic pocket. Last but not least, affinity studies show that PDA-coated Hb has a higher capability for oxygen release. Such an effect is further enhanced at lower pH. Importantly, through molecular docking simulations we provide a plausible explanation for the observed experimental results.

A New Mediterranean Flour Moth-Derived Chitosan: Characterization and Co-electrospun Hybrid Fabrication.

In this study, the chitin of adult Mediterranean flour moth (Ephestia kuheniella) (Cht) was extracted and then converted to chitosan by deacetylation process to achieve the chitosan derived from E. kuheniella (Chsfm). The new chitosan-based scaffold was produced using the polyvinyl alcohol (PVA) co-electrospinning technique. The degree of deacetylation was obtained using the distillation-titration and Fourier transform infrared spectroscopy. The surface morphology and crystallinity index of Chsfm were observed using scanning electron microscopy and X-ray diffraction analysis, respectively, and compared with the commercial chitosan (Chsc). Thermogravimetric analysis was used to estimate two chitosans' water content and thermal stability. The average molecular mass analysis was performed using viscometry. Moreover, the minimum inhibitory concentration and DPPH assay were used to study the antimicrobial activity and antioxidant potential of the Chsfm, respectively. Accordingly, Chsfm was smoother with fewer pores and flakes than Chsc, and its crystallinity index was higher than Chsc. The water content and thermal stability were lower and similar for Chsfm compared to Chsc. The average molecular mass of Chsfm was ~ 5.8 kDa, making it classified as low molecular weight chitosan. The antimicrobial activity of Chsfm against a representative Gram-negative bacteria; E. coli resulted to be the same as Chsc. However, less effective than Chsc against a representative Gram-positive bacteria is S. aureus. The Chsfm/PVA ratio scaffold was optimized at 30:70 to fabricate a uniform nanofiber scaffold.

The structural and functional consequences of melatonin and serotonin on human αB-crystallin and their dual role in the eye lens transparency.

Crystallins are the major soluble lens proteins, and α-crystallin, the most important protective protein of the eye lens, has two subunits (αA and αB) with chaperone activity. αB-crystallin (αB-Cry) with a relatively wide tissue distribution has an innate ability to interact effectively with the misfolded proteins, preventing their aggregation. Melatonin and serotonin have also been identified in relatively high concentrations in the lenticular tissues. This study investigated the effect of these naturally occurring compounds and medications on the structure, oligomerization, aggregation, and chaperone-like activity of human αB-Cry. Various spectroscopic methods, dynamic light scattering (DLS), differential scanning calorimetry (DSC), and molecular docking have been used for this purpose. Based on our results, melatonin indicates an inhibitory effect on the aggregation of human αB-Cry without altering its chaperone-like activity. However, serotonin decreases αB-Cry oligomeric size distribution by creating hydrogen bonds, decreases its chaperone-like activity, and at high concentrations increases protein aggregation.

Modulation of Nrf2/HO-1 by Natural Compounds in Lung Cancer.

Oxidative stresses (OSs) are considered a pivotal factor in creating various pathophysiological conditions. Cells have been able to move forward by modulating numerous signaling pathways to moderate the defects of these stresses during their evolution. The company of Kelch-like ECH-associated protein 1 (Keap1) as a molecular sensing element of the oxidative and electrophilic stress and nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) as a master transcriptional regulator of the antioxidant response makes a master cytoprotective antioxidant pathway known as the Keap1/Nrf2 pathway. This pathway is considered a dual-edged sword with beneficial features for both normal and cancer cells by regulating the gene expression of the array of endogenous antioxidant enzymes. Heme oxygenase-1 (HO-1), a critical enzyme in toxic heme removal, is one of the clear state indicators for the duality of this pathway. Therefore, Nrf2/HO-1 axis targeting is known as a novel strategy for cancer treatment. In this review, the molecular mechanism of action of natural antioxidants on lung cancer cells has been investigated by relying on the Nrf2/HO-1 axis.

Disruption of tubulin polymerization and cell proliferation by 1-naphthylarsonic acid.

Arsenical compounds exhibit a differential toxicity to cancer cells. Microtubules are a primary target of a number of anticancer drugs, such as arsenical compounds. The interaction of 1-NAA (1-naphthylarsonic acid) has been investigated on microtubule polymerization under in vitro and cellular conditions. Microtubules were extracted from sheep brain. Transmission electron microscopy was used to show microtubule structure in the presence of 1-NAA. Computational docking method was applied for the discovery of ligand-binding sites on the microtubular proteins. Proliferation of HeLa cells and HF2 (human foreskin fibroblasts) was measured by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay method following their incubation with 1-NAA. Fluorescence microscopic labelling was done with the help of α-tubulin monoclonal antibody and Tunel kit was used to investigate the apoptotic effects of 1-NAA on the HeLa cells. 1-NAA inhibits the tubulin polymerization by the formation of abnormal polymers having high affinity to the inner cell wall.

Insulin fibrillation: Strategies for inhibition.

Insulin and its homologous are the most utilized protein drugs due to their role in diabetic patients' treatment. Insulin forms amyloid-like fibrils in vivo at the injection site. Therefore, the study of its fibrillation mechanism and designing efficient inhibitors have high importance in the pharmaceutical industry. Insulin fibrils are formed at both acidic and neutral pH in vitro. Overall, this process involves the dissociation of hexameric form to monomeric, partially dissociating the native monomeric form, nuclei formation, and finally converting oligomers to large ordered aggregates. Intermediate and terminal species are different pathologically. This review is focused on the research works dedicated to the inhibition of insulin fibril formation. The inhibitors include various polyphenols, natural compounds, nanoparticles, and synthetic chemicals/peptides, as well as the classification of inhibitors targets concerning protein fibrillation. Although most inhibitors stabilize the native structure of the protein and prevent the formation of partially folded species, there are other inhibitors that hinder other steps in the course of fibrillation. Also, several inhibitors were able to dissociate the pre-existing fibrils. Finding inhibition strategies could be beneficial for developing new inhibitors that are more efficient and can block the amyloid pathway in a specific desired stage.

Enzymatically triggered delignification through a novel stable laccase: A mixed in-silico /in-vitro exploration of a complex environmental microbiota.

Laccases have been broadly applied as a multitasking biocatalyst in various industries, but their applications tend to be limited by easy deactivation, lack of adequate stability, and susceptibility under complex conditions. Identifying stable laccase as a green-biocatalyst is crucial for developing cost-effective biorefining processes. In this direction, we attempted in-silico screening a stable metagenome-derived laccase (PersiLac1) from tannery wastewater in a complex environment. The laccase exhibited high thermostability, retaining 53.19% activity after 180 min at 70 °C, and it was stable in a wide range of pH (4.0-9.0). After 33 days of storage at 50°C, pH 6.0, the enzyme retained 71.65% of its activity. Various metal ions, inhibitors, and organic solvents showed that PersiLac1 has a stable structure. The stable PersiLac1 could successfully remove lignin and phenolic from quinoa husk and rice straw. In the separate hydrolysis and fermentation process (SHF) after 72 h, hydrolysis was obtained 100% and 73.4% for quinoa husk and rice straw, and fermentation by the S. cerevisiae was be produced 41.46 g/L and 27.75g/L ethanol, respectively. Results signified that the novel lignin-degrading enzyme was confirmed to have great potential for industrial application as a green-biocatalyst based on enzymatically triggered to delignification and detoxify lignocellulosic biomass.

The novel homologue of the human α-glucosidase inhibited by the non-germinated and germinated quinoa protein hydrolysates after in vitro gastrointestinal digestion.

Quinoa (Chenopodium quinoa Willd) is a potential source of protein with ideal amino acid profiles which its bioactive compounds can be improved during germination and gastrointestinal digestion. The present investigation studies the impact of germination for 24 hr and simulated gastrointestinal digestion on α-glucosidase inhibitory activity of the quinoa protein and bioactive peptides against the novel homologue of human α-glucosidase, PersiAlpha-GL1. The sprouted quinoa after gastroduodenal digestion was the most effective α-glucosidase inhibitor showing 81.10% α-glucosidase inhibition at concentration 4 mg/ml with the half inhibition rate (IC50 ) of 0.07 mg/ml. Based on the kinetic analysis, both the germinated and non-germinated samples before and after digestion were competitive-type inhibitors of α-glucosidase. Results of this study showed the improved α-glucosidase inhibitory activity of the quinoa bioactive peptides after germination and gastrointestinal digestion and highlighted the potential of metagenome-derived PersiAlpha-GL1 as a novel homologue of the human α-glucosidase for developing the future anti-diabetic drugs. PRACTICAL APPLICATIONS: This study aimed to evaluate the effect of germination and gastrointestinal digestion of the quinoa protein and bioactive peptides on α-glucosidase inhibitory activity against the novel PersiAlpha-GL1. Metagenomic data were used to identify the novel α-glucosidase structurally and functionally homologue of human intestinal. The results showed the highest inhibition on PersiAlpha-GL1 by a germinated quinoa after gastroduodenal digestion and confirmed the potential of PersiAlpha-GL1 to enhance the effectiveness of the anti-diabetic drugs for industrial application.

The oxidative and neurotoxic potentials of the ambient PM2.5 extracts: The efficient multi-solvent extraction method.

The health effects of ambient air particulate matter with a diameter of ≤2.5 µm (PM2.5) on the central nervous system are well known and the induced oxidative stress has been shown as their main neuropathologic outcome. Ambient air PM2.5 sampling methods mostly use air sampler systems that collect PM2.5 on filters, which is followed by a PM2.5 extraction approach. Inefficient extraction may lead to compositional bias and unreal interpretation of the results. This study aimed to compare our proposed multi-solvent extraction (MSE) approach for PM2.5 extraction with a conventional aqueous extraction (AqE) method using the analysis of oxidative effects and cytotoxicity in the human neuroblastoma SH-SY5Y cell line. Ambient PM2.5 samples were collected from an urban traffic location in Tehran city, the capital of Iran, using a high-volume sampler. The developed MSE method was proved to have superior advantages over the AqE method including an increased extraction efficiency (as much as 96 against 48% for PMms and PMaq, respectively), and decreased artifacts and compositional biases. Ambient PM2.5, besides PMms and PMaq were analyzed for water-soluble ions, metals, and major elements. Dithiothreitol, ascorbic acid, lipid peroxidation, and cell viability assays on SH-SY5Y cells represented the significantly higher oxidative potential for PMms compared to PMaq. The increased cytotoxicity may occur because of the increased oxidative potential of PMms and possibly is associated with higher efficiency of the MSE over the AqE method for removal of total redox-active PM components.

The concept of protein folding/unfolding and its impacts on human health.

Proteins have evolved in specific 3D structures and play different functions in cells and determine various reactions and pathways. The newly synthesized amino acid chains once depart ribosome must crumple into three-dimensional structures so can be biologically active. This process of protein that makes a functional molecule is called protein folding. The protein folding is both a biological and a physicochemical process that depends on the sequence of it. In fact, this process occurs more complicated and in some cases and in exposure to some molecules like glucose (glycation), mistaken folding leads to amyloid structures and fatal disorders called conformational diseases. Such conditions are detected by the quality control system of the cell and these abnormal proteins undergo renovation or degradation. This scenario takes place by the chaperones, chaperonins, and Ubiquitin-proteasome complex. Understanding of protein folding mechanisms from different views including experimental and computational approaches has revealed some intermediate ensembles such as molten globule and has been subjected to biophysical and molecular biology attempts to know more about prevalent conformational diseases.

Application of free and immobilized novel bifunctional biocatalyst in biotransformation of recalcitrant lignocellulosic biomass.

Herein, an innovative, green, and practical biocatalyst was developed using conjugation of a novel bifunctional mannanase/xylanase biocatalyst (PersiManXyn1) to the modified cellulose nanocrystals (CNCs). Firstly, PersiManXyn1 was multi-stage in-silico screened from rumen macrobiota, and then cloned, expressed, and purified. Next, CNCs were synthesized from sugar beet pulp using enzymatic and acid hydrolysis processes, and then Fe3O4 NPs were anchored on their surface to produce magnetic CNCs (MCNCs). This hybrid was modified by dopamine providing DA/MCNCs nano-carrier. The bifunctional PersiManXyn1 demonstrated the superior hydrolysis activity on corn cob compared with the monofunctional xylanase enzyme (PersiXyn2). Moreover, the immobilization of PersiManXyn1 on the nano-carrier resulted in an improvement of the thermal stability, kinetic parameters (Kcat), and storage stability of the enzyme. Incorporation of the Fe3O4 NPs on the CNCs made magnetic nano-carrier with high magnetization value (25.8 emu/g) which exhibited rapid response toward the external magnetic fields. Hence, the immobilized biocatalyst could be easily separated from the products by a magnet, and reused up to 8 cycles with maintaining more than 50% of its original activity. The immobilized PersiManXyn1 generated 22.2%, 38.7%, and 35.1% more reducing sugars after 168 h hydrolysis of the sugar beet pulp, coffee waste, and rice straw, respectively, compared to the free enzyme. Based on the results, immobilization of the bifunctional PersiManXyn1 exhibited the superb performance of the enzyme to improve the conversion of the lignocellulosic wastes into high value products and develop the cost-competition biomass operations.

Conformational and thermodynamic characterization of the premolten globule state occurring during unfolding of the molten globule state of cytochrome c.

It has already been shown that the mutant Leu94Gly of horse cytochrome c exists in a molten globule (MG) state. We have carried out studies of reversible folding and unfolding induced by LiCl of this mutant at pH 6.0 and 25 °C by observing changes in the difference molar absorption coefficient at 402 nm, the mean residue ellipticity at 222 nm, and the difference mean residue ellipticity at 409 nm. This process is a three-state process when measured by these probes. The stable folding intermediate state has been characterized by far- and near-UV circular dichroism, tryptophan fluorescence, 8-anilino-1-naphthalenesulfonic acid binding, and dynamic light scattering measurements, which led us to conclude that the intermediate is a premolten globule (PMG). Analysis of the reversible unfolding transition curves for the stability of different states in terms of the Gibbs free energy change at pH 6.0 and 25 °C led us to conclude that the MG state is more stable than the PMG state by 5.4 ± 0.1 kcal mol(-1), whereas the PMG state is more stable than the denatured (D) state by only 1.1 ± 0.1 kcal mol(-1). A comparison of the conformational and thermodynamic properties of the LiCl-induced PMG state at pH 6.0 with those of the PMG state induced by NaCl at pH 2.0 suggests that a similar PMG state is obtained under both denaturing conditions. Differential scanning calorimetry measurements suggest that heat induces a reversible two-state transition between MG and D states.