Functional and Extracellular Production and Antitumor Activity of Mouse Alpha Klotho in Model Microalga Chlamydomonas reinhardtii.

Klotho is a human protein with versatile functions associated with longevity and well-being. α-Klotho (α-KL) deficiency in the circulatory system is associated with reduced life expectancy with numerous disorders such as chronic kidney disease, atherosclerosis, infertility, skin atrophy, emphysema, and osteoporosis. The antagonistic effects of Klotho protein against intractable cancers have also been well documented over the past two decades. In addition, recent findings have also illuminated the importance of soluble Klotho during cognitive development, oxidative stress, cellular apoptosis, and neurodegenerative disorders. The low-cost and sustainable production of alpha Klotho protein is extremely important for its widespread use against different diseases. Here, we report heterologous, functional, and extracellular production of mouse α-KL (mα-KL) protein in model microalga Chlamydomonas reinhardtii. The secretion of mα-KL into the extracellular environment facilitated downstream processes and warranted low-cost purification in high-titer. Furthermore, the anticarcinogenic efficiency of recombinant mα-KL was examined and validated on Rattus norvegicus AR42J pancreas tumors. Microalgae-based photosynthetic, low-cost, and scalable production of mα-KL could be used to develop a variety of cosmetics, pharmaceuticals, and wellness products, all aimed at serving health and well-being.

Exploring epigenetic drugs as potential inhibitors of SARS-CoV-2 main protease: a docking and MD simulation study.

The COVID-19 pandemic has caused havoc around the globe since 2019 and is considered the largest global epidemic of the twentieth century. Although the first antiviral drug, Remdesivir, was initially introduced against COVID­19, virtually no tangible therapeutic drugs exist to treat SARS-CoV-2 infection. FDA-approved Paxlovid (Nirmatrelvir supplemented by Ritonavir) was recently announced as a promising drug against the SARS-CoV-2 major protease (Mpro). Here we report for the first time the remarkable inhibitory potentials of lead epigenetic-targeting drugs (epi-drugs) against SARS-CoV-2 Mpro. Epi-drugs are promising compounds to be used in combination with cancer chemotherapeutics to regulate gene expression. The search for all known epi-drugs for the specific inhibition of SARS-CoV-2 Mpro was performed for the first time by consensus (three high-order program) molecular docking studies and end-state free energy calculations. Several epi-drugs were identified with highly comparable binding affinity to SARS-CoV-2 Mpro compared to Nirmatrelvir. In particular, potent histone methyltransferase inhibitor EPZ005687 and DNA methyltransferase inhibitor Guadecitabine were prominent as the most promising epi-drug inhibitors for SARS-CoV-2 Mpro. Long Molecular dynamics (MD) simulations (200 ns each) and corresponding MM-GBSA calculations confirmed the stability of the EPZ005687-Mpro complex with MM-GBSA binding free energy (ΔGbind) -48.2 kcal/mol (EPZ005687) compared to Nirmatrelvir (-44.7 kcal/mol). Taken together, the antiviral activities of the highlighted epi-drugs are reported beyond widespread use in combination with anti-cancer agents. The current findings therefore highlight as yet unexplored antiviral potential of epi-drugs suitable for use in patients struggling with chronic immunosuppressive disorders.Communicated by Ramaswamy H. Sarma.

Artificial intelligence-based HDX (AI-HDX) prediction reveals fundamental characteristics to protein dynamics: Mechanisms on SARS-CoV-2 immune escape.

Three-dimensional structure and dynamics are essential for protein function. Advancements in hydrogen-deuterium exchange (HDX) techniques enable probing protein dynamic information in physiologically relevant conditions. HDX-coupled mass spectrometry (HDX-MS) has been broadly applied in pharmaceutical industries. However, it is challenging to obtain dynamics information at the single amino acid resolution and time consuming to perform the experiments and process the data. Here, we demonstrate the first deep learning model, artificial intelligence-based HDX (AI-HDX), that predicts intrinsic protein dynamics based on the protein sequence. It uncovers the protein structural dynamics by combining deep learning, experimental HDX, sequence alignment, and protein structure prediction. AI-HDX can be broadly applied to drug discovery, protein engineering, and biomedical studies. As a demonstration, we elucidated receptor-binding domain structural dynamics as a potential mechanism of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody efficacy and immune escape. AI-HDX fundamentally differs from the current AI tools for protein analysis and may transform protein design for various applications.

Dihydroisocoumarins and Phenylglycosides from Scorzonera longiana, Their Antimicrobial Activities and Molecular Docking Studies.

Five new phenyl dihydroisocoumarin glycosides (1-5) and two known compounds (6-7) were identified from the butanol fraction of Scorzonera longiana. The structures of 1-7 were elucidated based on spectroscopic methods. Antimicrobial, antitubercular, and antifungal evaluation of compounds 1-7 were carried out using the microdilution method against nine microorganisms. Compound 1 was active only against Mycobacterium smegmatis (Ms) with a MIC value of 14.84 µg/mL. All tested compounds (1-7) were active against Ms but only compounds 3-7 were active against fungi (C. albicans, S. cerevisiae) with MIC values of 25.0-125 µg/mL. In addition, molecular docking studies were conducted against Ms DprE1 (PDB ID: 4F4Q), Mycobacterium tuberculosis (Mbt) DprE1 (PDB ID: 6HEZ), and arabinosyltransferase C (EmbC, PDB ID: 7BVE) enzymes. Compounds 2, 5, and 7 are the most effective Ms 4F4Q inhibitors. Compound 4 was the most promising inhibitory activity on Mbt DprE with the lowest binding energy of -9,9 kcal/mol.

The effect of friction stir process on the mechanical, tribological, and biocompatibility properties of AZ31B magnesium alloy as a biomaterial: A pilot study.

Recently, AZ31B magnesium alloy has been widely employed in automotive, aerospace, and bio implant industries due to its light-weight and biocompatibility properties. However, the equilibrium of ductility and strength of this material and the negativity brought by its poor wear behavior have limited its versatile use. Friction stir processing (FSP) has been commonly used as severe plastic deformation method for improving mechanical and tribological properties of metal sheets. The effect of this method on the biocompatibility of materials is a matter of curiosity that should be emphasized. So, the present study aims to investigate the effect of friction stir process on the mechanical, tribological, and biocompatibility properties of AZ31B magnesium alloy. It is observed that FSP enhanced the tensile properties of the alloy but decreased its elongation. It was determined that the base material exhibited ductile character on the fracture surface of the specimens, and mixed ductile/brittle fracture was evident with the FSP. In the FSP zone, the hardness value was improved by 17% compared to the base material. Also, the wear performance of the alloy enhanced in ambient air and Simulated Body Fluid (SBF) solution. Wear properties in SBF solution were better due to less adhesive bonds between the friction surfaces. This assessment was supported by SEM images of the wear path and surface of counter bodies. On the other hand, FSPed AZ31B alloy materials with improved strength properties were not cytotoxic for human gingival fibroblasts, and these results may suggest that the materials are safe for clinical uses.

Homology modeling and heterologous expression of highly alkaline subtilisin-like serine protease from Bacillus halodurans C-125.

Here we report heterologous expression, enzymatic characterization and structure homology modeling of a subtilisin-like alkaline serine protease (ASP) from Bacillus halodurans C-125. Encoding gene was successfully obtained by PCR and cloned into pMA0911 shuttle vector under the control of strong HpaII promoter and expressed extracellularly. ASP enzyme was successfully expressed in B. subtilis WB800 cell line lacking eight extracellular proteases and produced extracellularly in the culture medium. Km, Vmax and specific activity parameters of the recombinantly produced ASP were identified as 0.2899 mg/ml, 76.12 U/ml and 9500 U/mg, respectively. The purified enzyme revealed remarkable proteolytic activity at highly alkaline conditions with a pH optimum 12.0 and notable thermostability with temperature optimum at 60 °C. Furthermore, substrate-free enzyme revealed remarkable pH stability at pH 12.0 and maintained 93% of its initial activity when incubated at 37 °C for 24 h and 60% of its initial activity upon incubation at 60 °C for 1 h. Theoretically calculated molecular mass of ASP protein was confirmed through SDS-PAGE and western blot analysis (Mw: 28.3 kDa). The secondary and tertiary structures of ASP protein were also identified through homology modeling and further examined in detail. ASP harbors a typical S8/S53 peptidase domain comprising 17 ß-sheets and 9 α-helixes within its secondary structure. The structure dynamics analysis of modeled 3D structure further revealed that transient inactivating propeptide chain is the most dynamic region of ASP enzyme with 8.52 Å2 ß-Factor value. Additional residue-dependent fluctuation plot analysis also confirmed the elevated structure dynamics patterning of ASP N-terminus which could be the potential prerequisite for the autonomous propeptide removal of alkaline serine peptidases. Yet the functional domain of ASP becomes quite stable after autonomous exclusion of its propeptide. Although the sequence homology between ASP and commercial detergent additive B. lentus protease (PDB ID:1GCI) was moderate (65.4% sequence similarity), their overlaid 3D structures revealed much higher similarity (98.14%) within 0.80 Å RMSD. In conclusions, with remarkable pH stability, notable thermostability and particularly high specific activity at extreme alkaline conditions, the unveiled ASP protein stands out as a novel protease candidate for various industrial sectors such as textile, detergent, leather, feed, waste, pharmaceutical and others.

The Detailed Comparison of Cell Death Detected by Annexin V-PI Counterstain Using Fluorescence Microscope, Flow Cytometry and Automated Cell Counter in Mammalian and Microalgae Cells.

The evaluation of cell wellness is an important task for molecular biology research. This mainly comprises the assessment for morphology and viability of culturing cells. Annexin V-Propidium iodide counterstaining has been currently one of the common and easy methods to discriminate apoptotic and necrotic cell profiles. The method is operated by fluorescence-based detection of counterstain via laser beam-employed instruments including flow cytometer, fluorescence microscope and automated cell counter. The detection is primarily conducted based on the same principle; however the efficiency of instruments may vary. Here we evaluated the efficiency of those instruments for the clear-cut detection of cell death through various mammalian and microalgae cell lines. To the best of our knowledge, this is the first study revealing comparative analyses of apoptotic and necrotic cells in mammalian and microalgae cells using Annexin V-PI counterstain detected by flow cytometer, fluorescence microscope and automated cell counter. Fluorescence microscope and cell counter instruments were also tested and compared for the traditional trypan blue-based cell viability detection performance. For these, cell death was induced by UV-irradiation and/or bee venom for mammalian (pancreatic cancer, metastatic breast cancer and mouse fibroblasts) and microalgae cells (Chlorella vulgaris), respectfully. Findings postulated that automated cell counter and fluorescence microscopy revealed similar patterns for the detection by both counterstain and trypan blue in mammalian cells. Interestingly, flow cytometry did provide an accurate and significant detection for only one mammalian cell line when UV-treatment was followed by routine Annexin V-Propidium iodide counterstaining. Unlike, only flow cytometry revealed a significant change in the detection of death of microalgae cells by Annexin V-Propidium iodide method, but both Annexin and conventional trypan blue methods were not applicable for the automated cell counter and microscopic detections for microalgae cells. The related outputs propose that the obtaining reliable quantitation strongly depends on cell type and instruments used. These suggest the necessity of optimization and validation endeavors before any cell death detection initiative. The analytical outcomes present insights into detailed assessment of cell death detection of eukaryotic cells and provide a direction to researchers to consider.

Improved pulp bleaching potential of Bacillus subtilis WB800 through overexpression of three lignolytic enzymes from various bacteria.

A chemical bleaching process of paper pulps gives off excessive amount of chlorinated organic wastes mostly released to environment without exposing complete bioremediaton. Recent alternative and eco-friendly approaches toward pulp bleaching appear more responsive to environmental awareness. Here we report, direct use of a recombinant Bacillus subtilis bacterium for pulp bleaching, endowed with three ligninolytic enzymes from various bacteria. In addition, efficient bleaching performance from glutathione-S-transferase (GST) biocatalyst tested for the first time in pulp bleaching applications was also achieved. Simultaneous and extracellular overproduction of highly active GST, laccase, and lignin peroxidase catalysts were also performed by Bacillus cells. Both enhanced bleaching success and improved delignification rates were identified when enzyme combinations tested on both pine kraft and waste paper pulps, ranging from 69.75% to 79.18% and 60.89% to 74.65%, respectively. Furthermore, when triple enzyme combination applied onto the papers from pine kraft and waste pulps, the best ISO brightness values were identified as 66.45% and 64.67%, respectively. The delignification rates of pulp fibers exposed to various enzymatic bleaching sequences were comparatively examined under SEM. In conclusion, the current study points out that in near future, a more fined-tuned engineering of pulp-colonizing bacteria may become a cost-effective and environmentally friendly alternative to chemical bleaching.

Redesigning pH optimum of Geobacillus sp. TF16 endoxylanase through in silico designed DNA swapping strategy.

Thermoalkaliphilic xylanases are highly desired and of great importance due to their vast potential in paper pulp and bleaching processes. Here, we report rapid, cost-effective, and result-oriented combinatorial potential of in silico DNA swapping strategy to engineer the pH optimum of industrially crucial enzymes, particularly engineering of Geobacillus sp. TF16 endoxylanase for alkaline environments. The 3D structures of Geobacillus sp. TF16 and donor Bacillus halodurans C-125 endoxylanases were firstly predicted, analyzed, and compared for their similarities before any in silico design of mutants. Reasonably, to improve its alkaline pH tolerance, the corresponding regions in Geobacillus sp.TF16 endoxylanase were further engineered by swapping with negatively-charged amino acid-rich regions from B. halodurans C-125 endoxylanase. Through only two of four in silico-designed mutants, the optimum pH of GeoTF16 endoxylanase was improved from 8.5 to 10.0. Moreover, as compared to GeoTF16 parental enzyme, both GeoInt3 and GeoInt4 mutants revealed (i) enhanced biobleaching performance, (ii) improved adaptability to alkaline conditions, and (iii) better activity for broader pH range. Unlike GeoTF16 losing activity at pH 11.0 completely, GeoInt4 retained 60% and 40% of its activity at pH 11.0 and 12.0, respectively. Thus, GeoInt4 stands out as a more competent biocatalyst that is suitable for alkaline environments of diverse industrial applications. The current study represents an efficient protein engineering strategy to adapt industrial catalysts to diverse processing conditions. Further comprehensive and fine-tuned research efforts may result in biotechnologically more promising outcomes.

Phytoestrogens and Mycoestrogens Induce Signature Structure Dynamics Changes on Estrogen Receptor α.

Endocrine disrupters include a broad spectrum of chemicals such as industrial chemicals, natural estrogens and androgens, synthetic estrogens and androgens. Phytoestrogens are widely present in diet and food supplements; mycoestrogens are frequently found in grains. As human beings and animals are commonly exposed to phytoestrogens and mycoestrogens in diet and environment, it is important to understand the potential beneficial or hazardous effects of estrogenic compounds. Many bioassays have been established to study the binding of estrogenic compounds with estrogen receptor (ER) and provided rich data in the literature. However, limited assays can offer structure information with regard to the ligand/ER complex. Our current study surveys the global structure dynamics changes for ERα ligand binding domain (LBD) when phytoestrogens and mycoestrogens bind. The assay is based on the structure dynamics information probed by hydrogen deuterium exchange mass spectrometry and offers a unique viewpoint to elucidate the mechanism how phytoestrogens and mycoestrogens interact with estrogen receptor. The cluster analysis based on the hydrogen deuterium exchange (HDX) assay data reveals a unique pattern when phytoestrogens and mycoestrogens bind with ERα LBD compared to that of estradiol and synthetic estrogen modulators. Our study highlights that structure dynamics could play an important role in the structure function relationship when endocrine disrupters interact with estrogen receptors.

HDX-analyzer: a novel package for statistical analysis of protein structure dynamics.

BACKGROUND: HDX mass spectrometry is a powerful platform to probe protein structure dynamics during ligand binding, protein folding, enzyme catalysis, and such. HDX mass spectrometry analysis derives the protein structure dynamics based on the mass increase of a protein of which the backbone protons exchanged with solvent deuterium. Coupled with enzyme digestion and MS/MS analysis, HDX mass spectrometry can be used to study the regional dynamics of protein based on the m/z value or percentage of deuterium incorporation for the digested peptides in the HDX experiments. Various software packages have been developed to analyze HDX mass spectrometry data. Despite the progresses, proper and explicit statistical treatment is still lacking in most of the current HDX mass spectrometry software. In order to address this issue, we have developed the HDXanalyzer for the statistical analysis of HDX mass spectrometry data using R, Python, and RPY2. IMPLEMENTATION AND RESULTS: HDXanalyzer package contains three major modules, the data processing module, the statistical analysis module, and the user interface. RPY2 is employed to enable the connection of these three components, where the data processing module is implemented using Python and the statistical analysis module is implemented with R. RPY2 creates a low-level interface for R and allows the effective integration of statistical module for data processing. The data processing module generates the centroid for the peptides in form of m/z value, and the differences of centroids between the peptides derived from apo and ligand-bound protein allow us to evaluate whether the regions have significant changes in structure dynamics or not. Another option of the software is to calculate the deuterium incorporation rate for the comparison. The two types of statistical analyses are Paired Student's t-test and the linear combination of the intercept for multiple regression and ANCOVA model. The user interface is implemented with wxpython to facilitate the data visualization in graphs and the statistical analysis output presentation. In order to evaluate the software, a previously published xylanase HDX mass spectrometry analysis dataset is processed and presented. The results from the different statistical analysis methods are compared and shown to be similar. The statistical analysis results are overlaid with the three dimensional structure of the protein to highlight the regional structure dynamics changes in the xylanase enzyme. CONCLUSION: Statistical analysis provides crucial evaluation of whether a protein region is significantly protected or unprotected during the HDX mass spectrometry studies. Although there are several other available software programs to process HDX experimental data, HDXanalyzer is the first software program to offer multiple statistical methods to evaluate the changes in protein structure dynamics based on HDX mass spectrometry analysis. Moreover, the statistical analysis can be carried out for both m/z value and deuterium incorporation rate. In addition, the software package can be used for the data generated from a wide range of mass spectrometry instruments.

Enzyme structure dynamics of xylanase I from Trichoderma longibrachiatum.

BACKGROUND: Enzyme dynamics has recently been shown to be crucial for structure-function relationship. Among various structure dynamics analysis platforms, HDX (hydrogen deuterium exchange) mass spectrometry stands out as an efficient and high-throughput way to analyze protein dynamics upon ligand binding. Despite the potential, limited research has employed the HDX mass spec platform to probe regional structure dynamics of enzymes. In particular, the technique has never been used for analyzing cell wall degrading enzymes. We hereby used xylanase as a model to explore the potential of HDX mass spectrometry for studying cell wall degrading enzymes. RESULTS: HDX mass spectrometry revealed significant intrinsic dynamics for the xylanase enzyme. Different regions of the enzymes are differentially stabilized in the apo enzyme. The comparison of substrate-binding enzymes revealed that xylohexaose can significantly stabilize the enzyme. Several regions including those near the reaction centres were significantly stabilized during the xylohexaose binding. As compared to xylohexaose, xylan induced relatively less protection in the enzyme, which may be due to the insolubility of the substrate. The structure relevance of the enzyme dynamics was discussed with reference to the three dimensional structure of the enzyme. HDX mass spectrometry revealed strong dynamics-function relevance and such relevance can be explored for the future enzyme improvement. CONCLUSION: Ligand-binding can lead to the significant stabilization at both regional and global level for enzymes like xylanase. HDX mass spectrometry is a powerful high-throughput platform to identify the key regions protected during the ligand binding and to explore the molecular mechanisms of the enzyme function. The HDX mass spectrometry analysis of cell wall degrading enzymes has provided a novel platform to guide the rational design of enzymes.