Insight into the Conformational Transitions of Serine Acetyl Transferase Isoforms in E. histolytica: Implications for Structural and Functional Balance.

Serine acetyl transferase (SAT) is one of the crucial enzymes in the cysteine biosynthetic pathway and an essential enzyme for the survival of Entamoeba histolytica, the causative agent of amoebiasis. E. histolytica expresses three isoforms of SAT, where SAT1 and SAT2 are inhibited by the final product cysteine, while SAT3 is not inhibited. SAT3 has a slightly elongated C-terminus compared to SAT1. To understand the stability and conformational transition between two secondary structures of proteins, we measured the effect of urea, a chemical denaturant, on two isoforms of SAT (SAT1 and SAT3) of E. histolytica. The effect of urea on the structure and stability of SAT1 and SAT3 was determined by measuring changes in their far-UV circular dichroism (CD), Trp fluorescence, and near-UV absorption spectra. The urea-induced normal transition curves suggested that the structural transition is reversible and follows a two-state process. Analysis of the urea-induced transition of all optical properties for the stability parameters ΔG D° (Gibbs free energy change (ΔG D) in the absence of urea), m (dependence of ΔG D on urea concentration), and C m (midpoint of urea transition) suggested that SAT1 is more stable than SAT3. Characterization of the end product of the urea-induced transition of both proteins by the far-UV CD and Trp-fluorescence and near-UV absorbance suggested that urea causes α-helix to ß-sheet transition and burial of Trp residues, respectively. To support the in vitro findings, 100 ns molecular dynamics simulations (in silico study) were performed. Both the spectroscopic and molecular dynamics approaches clearly indicated that SAT1 is more stable than SAT3. SAT3 has evolved to escape the feedback inhibition to keep producing cysteine, but in the process, it compromises its structural stability relative to SAT1.

SARS-CoV-2 spike protein interactions with amyloidogenic proteins: Potential clues to neurodegeneration.

The post-infection of COVID-19 includes a myriad of neurologic symptoms including neurodegeneration. Protein aggregation in brain can be considered as one of the important reasons behind the neurodegeneration. SARS-CoV-2 Spike S1 protein receptor binding domain (SARS-CoV-2 S1 RBD) binds to heparin and heparin binding proteins. Moreover, heparin binding accelerates the aggregation of the pathological amyloid proteins present in the brain. In this paper, we have shown that the SARS-CoV-2 S1 RBD binds to a number of aggregation-prone, heparin binding proteins including Aß, α-synuclein, tau, prion, and TDP-43 RRM. These interactions suggests that the heparin-binding site on the S1 protein might assist the binding of amyloid proteins to the viral surface and thus could initiate aggregation of these proteins and finally leads to neurodegeneration in brain. The results will help us to prevent future outcomes of neurodegeneration by targeting this binding and aggregation process.

Biochemical and biophysical characterization of the smallest pyruvate kinase from Entamoeba histolytica.

Entamoeba histolytica infection is highly prevalent in developing countries across the globe. The ATP synthesis in this pathogen is solely dependent on the glycolysis pathway where pyruvate kinase (Pyk) catalyzes the final reaction. Here, we have cloned, overexpressed and purified the pyruvate kinase (EhPyk) from E. histolytica. EhPyk is the shortest currently known Pyk till date as it contains only two of the three characterized domains when compared to the other homologues and our phylogenetic analysis places it on a distinct branch from the known type I/II Pyks. Our purification results suggested that it exists as a homodimer in solution. The kinetic characterization showed that EhPyk has maximum activity at pH 7.5 where it exhibited Michaelis-Menten's kinetics for phosphoenolpyruvate with a Km of 0.23 mM, and it lost its activity at both the acidic pH 4.0 and basic pH 10.0. We also determined the key secondary structural elements of EhPyk at different pH values. MD simulation of EhPyk structure at different pH values suggested that it is most stable at pH 7.0, while least stable at pH 10.0 followed by pH 4.0. Together, our computational simulations correlate well with the experimental studies. In summary, this study expands the current understanding of the EhPyk identified earlier in the amoebic genome and provides the first characterization of this bacterially expressed protein.

Investigation of the role of central metal ion of Cyathus bulleri laccase 1 using guanidinium chloride-induced denaturation.

The structure and folding/unfolding kinetics of Cyathus bulleri laccase 1 (Lcc1), expressed in Pichia pastoris, were analyzed by spectroscopic methods to understand the role of central metal ion. Far UV CD structure analysis revealed major ß-sheet and minor α helical segments present in the Lcc1. A significant change in the spectrum of Lcc1, indicative of unfolding of secondary structures, was observed with increasing concentrations of guanidinium chloride (GdnHCl) during Trp fluorescence, absorption and CD measurements. A similar trend was also noticed for enzyme activity with respect to GdnHCl concentrations. To establish the role of copper ion in the catalytic activity of laccase, a complete removal of copper was carried out and addition of copper was found to restore the structure and activity during the refolding process. The apo form was also reconstituted by addition of zinc ion which restored nearly 84% of enzyme activity, indicating non-essential role of copper in maintaining conformation and activity. Structural studies and inductively coupled plasma mass spectrometry data supported these observations.

Delineating the effect of mutations on the conformational dynamics of N-terminal domain of TDP-43.

The structural integrity of N-terminal domain (NTD) of TAR DNA-binding protein-43 (TDP-43) is essential for the biological functions of TDP-43 involved in neurodegenerative diseases. Here, we used all-atom molecular dynamics (MD) simulations to understand the folding, dynamics and conformational stability of four variants of NTD, the wild type (WT) and three mutants (L27A, L28A and V31R). The deleterious and destabilizing nature of NTD mutants were predicted on DynaMut and SAAFEC server. Results show that predicted mutations modulate the conformational stability and flexibility of NTD. The effect of mutations on the conformational dynamics of NTD was studied through the free-energy landscape (FEL), essential dynamics (ED), hydrogen-bonds and intermolecular contact map. We observed that the mutations disrupt the intermolecular interactions between the dimeric NTD besides affecting the long-range intramolecular contacts, resulting in a less compact structure. ED essentially provides the collective motion of protein and we observed that the mutation increased the overall motions of the protein which might result in protein dysfunction. FEL shows the different conformational transitions of WT and mutants which revealed the structural basis of protein destabilization and unfolding due to mutation. We find that L28A has most of the deleterious effect compared to other two mutations, L27A and V31R. These results obtained here are well correlated with reported experimental studies which show the disruption of protein folding, stability and function with these mutations. Hence this computational study describes the structural details to unravel the mutant effects at the atomistic resolution and has implications for understanding the TDP-43's physiological and pathological role.

Implication of sulfonylurea derivatives as prospective inhibitors of human carbonic anhydrase II.

Selective carbonic anhydrase (CA) inhibitors have gained a lot of importance owing to the implication of specific isoforms of CA in certain diseases like glaucoma, leukemia, cystic fibrosis, and epilepsy. A novel class of sulfonylurea derivatives was synthesized from corresponding sulfonyl chlorides and amines. Compounds with different pendant moieties in the sulfonylurea derivatives show significant interactions with human carbonic anhydrase II (CAII). In vitro evaluation of the sulfonylurea derivatives revealed that three compounds possess admirable inhibitory activity against CAII. Compounds containing methyl (G2), isopropyl (G4) and o-tosyl (G5) groups displayed IC50 (109-137 µm) for CAII. Fluorescence binding and cytotoxicity studies revealed that these compounds are showing good binding affinity (18-34 µM) to CAII and non- toxic to human cells. Further, molecular docking studies of G2, G4 and G5 with CAII showed that these compounds fit nicely in the active site of CAII. Molecular dynamics simulation studies of these compounds complexed with CAII showed that essential interactions were maintained up to 50 ns of simulation. These results indicate the promising nature of the sulfonylurea scaffold towards CAII inhibition and opens scope of hit to-lead optimization for discovery of effective drugs against CAII-associated disorders.

Design, synthesis and biological evaluation of novel pyridine-thiazolidinone derivatives as anticancer agents: Targeting human carbonic anhydrase IX.

In order to obtain novel Human carbonic anhydrase IX (CAIX) inhibitors, a series of pyridine-thiazolidinone derivatives was synthesized and characterized by various spectroscopic techniques. The binding affinity of the compounds was measured by fluorescence binding studies and enzyme inhibition activity using esterase assay of CAIX. It was observed that compound 8 and 11 significantly inhibit the CAIX activity with the IC50 value, 1.61 µM and 1.84 µM, respectively. The binding-affinity of compound 8 and 11 for CAIX was significantly high with their KD values 11.21 µM and 2.32 µM, respectively. Docking studies revealed that compound 8 and 11 efficiently binds in the active site cavity of CA IX by forming sufficient numbers of H-bonds and van der Waals interactions with active side residues. All the compounds were further screened in vitro for anticancer activity and found that compound 8 and 11 exhibit considerable anticancer activity against MCF-7 and HepG-2 cell lines. All these findings suggest that compound 8 and 11 may be further exploited as a novel pharmacophore model for the development of anticancer agents.

Biological evaluation of p-toluene sulphonylhydrazone as carbonic anhydrase IX inhibitors: An approach to fight hypoxia-induced tumors.

To find potential inhibitors of human carbonic anhydrase IX (CAIX), we have successfully deigned, synthesized and characterized three p-toluene sulphonylhydrazone derivatives (1-3). Molecular docking studies provided the structural basis of CAIX inhibition and a deeper insight into the protein-ligand interactions. p-Toluene sulphonylhydrazone derivatives show a well organized conformational compatibility with the active site of CAIX. The protein-ligand complex was stabilized by several non-covalent interactions offered by residues present in the active site cavity. The actual binding affinity of synthesized compounds with CAIX was experimentally measured by fluorescence and isothermal titration calorimetry (ITC). Results of both fluorescence binding and ITC measurements show the binding affinity of p-Toluene sulphonylhydrazone derivatives to the CAIX in the µM range. CAIX enzyme inhibition assay showed the IC50 values in nM range. Though all the three compounds (1-3) showed a good binding with CAIX, compound 2 showed the best inhibition of CAIX activity. These compounds were non-toxic on normal cell lines (HEK-293) and significantly inhibit the proliferation of hypoxic cancer cells. All compounds induce apoptosis in the hypoxic cancer cells. These compounds may be further exploited as promising therapeutic agents to control the hypoxia-induced tumors.

Synthesis, estrogen receptor binding affinity and molecular docking of pyrimidine-piperazine-chromene and -quinoline conjugates.

Substituted 2-amino-7-((6-(4-(2-hydroxyethyl) piperazin-1-yl)-2-methylpyrimidin-4-yl)oxy)-4-phenyl-4H-chromene-3-carbonitriles and 2-amino-7-((6-(4-(2-hydroxyethyl)piperazin-1-yl)-2-methylpyrimidin-4-yl)oxy)-4-phenyl-1,4-dihydroquinoline-3-carbonitriles were synthesized via an efficient multi-component one pot synthesis under mild conditions. These compounds 1-20 were evaluated against human breast cancer cell lines (MCF-7) and human embryonic kidney cells (HEK293) for cytotoxic activities. Among them, compounds 6, 7, 15, 17 and 19 showed better anti-proliferative activities as (IC50 value 48±1.70, 65±1.13, 92±1.18, 30±1.17 and 16±1.10µM) than curcumin drug (48±1.11µM). Molecular docking was also performed with active compounds 6, 7 and 15 against Bcl-2 protein which gave good binding affinity (ΔG=-9.08, -8.29 and -7.70kcal/mol) respectively. Furthermore, the structure-activity relationship (SAR) analysis revealed that the chromene and quinoline moieties, when attached with pyrimide and piperazine moieties, enhanced anti-proliferative activities.

Estimation of thermodynamic stability of human carbonic anhydrase IX from urea-induced denaturation and MD simulation studies.

Carbonic anhydrase IX (CAIX) is a transmembrane glycoprotein, overexpressed in cancer cells under hypoxia condition. In cancerous cells, CAIX plays an important role to combat the deleterious effects of a high rate of glycolytic metabolism. In order to favor tumor survival, CAIX maintains intracellular pH neutral or slightly alkaline and extracellular acidic pH. The equilibrium unfolding and conformational stability of CAIX were measured in the presence of increasing urea concentrations to understand it's structural features under stressed conditions. Two different spectroscopic techniques were used to follow urea-induced denaturation and observed that urea induces a reversible denaturation of CAIX. Coincidence of the normalized transition curves of both optical properties suggesting that denaturation of CAIX is a two-state process, i.e., native state ↔ denatured state. Each denaturation curve was analyzed to estimate thermodynamic parameters, ΔGD0,value of Gibbs free energy change (ΔGD) associated with the urea-induced denaturation, Cm (midpoint of denaturation) and m (=δΔGD/δ[urea]). We further performed molecular dynamics simulation of CAIX for 50ns to see the dynamics of protein structure in the presence of different urea concentrations. An excellent agreement was observed between in silico and in vitro studies.

Design, synthesis, in silico and biological evaluation of novel 2-(4-(4-substituted piperazin-1-yl)benzylidene)hydrazine carboxamides.

A series of novel 2-(4-(4-substituted piperazin-1-yl)benzylidene)hydrazinecarboxamide derivatives has been successfully designed and synthesized to evaluate their potential as carbonic anhydrase (CA) inhibitors. The inhibitory potential of synthesized compounds against human CAI and CAII was evaluated. Compounds 3a-n exhibited [Formula: see text] values between [Formula: see text] against CAI and [Formula: see text] against CAII. Compound 3g was the most active inhibitor, with an [Formula: see text] value of [Formula: see text] against CAII. Molecular docking studies of compound 3g with CAII showed this compound fits nicely in the active site of CAII and it interacts with the zinc ion ([Formula: see text]) along with three histidine residues in the active site. Molecular dynamics simulation studies of compound 3g complexed with CAII also showed essential interactions which were maintained up to 40 ns of simulation. In vivo sub-acute toxicity study using 3g (300 mg/kg) was found non-toxic in adult Wistar rats.

Effect of pH on structure, function, and stability of mitochondrial carbonic anhydrase VA.

Mitochondrial carbonic anhydrase VA (CAVA) catalyzes the hydration of carbon dioxide to produce proton and bicarbonate which is primarily expressed in the mitochondrial matrix of liver, and involved in numerous physiological processes including lipogenesis, insulin secretion from pancreatic cells, ureagenesis, gluconeogenesis, and neuronal transmission. To understand the effect of pH on the structure, function, and stability of CAVA, we employed spectroscopic techniques such as circular dichroism, fluorescence, and absorbance measurements in wide range of pH (from pH 2.0 to pH 11.5). CAVA showed an aggregation at acidic pH range from pH 2.0 to pH 5.0. However, it remains stable and maintains its secondary structure in the pH range, pH 7.0-pH 11.5. Furthermore, this enzyme has an appreciable activity at more than pH 7.0 (7.0 < pH ≤ 11.5) with maximum activity at pH 9.0. The maximal values of kcat and kcat/Km at pH 9.0 are 3.7 × 106 s-1 and 5.5 × 107 M-1 s-1, respectively. However, this enzyme loses its activity in the acidic pH range. We further performed 20-ns molecular dynamics simulation of CAVA to see the dynamics at different pH values. An excellent agreement was observed between in silico and in vitro studies. This study provides an insight into the activity of CAVA in the pH range of subcellular environment.

GdmCl-induced unfolding studies of human carbonic anhydrase IX: a combined spectroscopic and MD simulation approach.

Carbonic anhydrase IX (CAIX) is a transmembrane glycoprotein, associated with tumor, acidification which leads to the cancer, and is considered as a potential biomarker for hypoxia-induced cancers. The overexpression of CAIX is linked with hypoxia condition which is mediated by the transcription of hypoxia-induced factor (HIF-1). To understand the biophysical properties of CAIX, we have carried out a reversible isothermal denaturation of CAIX-induced by GdmCl at pH 8.0 and 25°C. Three different spectroscopic probes, the far-UV CD at 222 nm ([θ]222), Trp fluorescence emission at 342 nm (F342) and difference molar absorption coefficient at 287 nm (Δε287) were used to estimate stability parameters, [Formula: see text] (Gibbs free energy change in the absence of GdmCl; Cm (midpoint of the denaturation curve), i.e. molar GdmCl concentration ([GdmCl]) at which ΔGD = 0; and m, the slope (=∂ΔGD/∂[GdmCl])). GdmCl induces a reversible denaturation of CAIX. Coincidence of the normalized transition curves of all optical properties suggests that unfolding/refolding of CAIX is a two-state process. We further performed molecular dynamics simulation of CAIX for 40 ns to see the dynamics of protein structure in different GdmCl concentrations. An excellent agreement was observed between in silico and in vitro studies.

Characterization of folding intermediates during urea-induced denaturation of human carbonic anhydrase II.

Knowledge of folding/unfolding pathway is fundamental basis to study protein structure and stability. Human carbonic anhydrase II (HCAII) is a ∼29kDa, ß-sheet dominated monomeric protein of 259 amino acid residues. In the present study, the urea-induced denaturation of HCAII was carried out which was a tri-phasic process, i.e., N (native) ↔ XI ↔ XII ↔ D (denatured) with stable intermediates XI and XII populated around 2 and 4M urea, respectively. The far-UV CD was used to characterize the intermediate states (XI and XII) for secondary structural content, near-UV CD for tertiary structure, dynamic light scattering for hydrodynamic radius and ANS fluorescence spectroscopy for the presence of exposed hydrophobic patches. Based on these experiments, we concluded that urea-induced XI state has characteristics of molten globule state while XII state bears characteristics features of pre-molten globule state. Characterization of the intermediates on the folding pathway will contribute to a deeper understanding of the structure-function relationship of HCAII. Furthermore, this system may provide an excellent model to study urea stress and the strategies adopted by the organisms to combat such a stress.

GdnHCl-induced unfolding intermediate in the mitochondrial carbonic anhydrase VA.

Carbonic anhydrase VA (CAVA) is a mitochondrial enzyme belonging to the α-family of CAs, which is involved in several physiological processes including ureagenesis, lipogenesis, gluconeogenesis and neuronal transmission. Here, we have tried to understand the folding mechanism of CAVA using guanidine hydrochloride (GdnHCl)-induced denaturation at pH 8.0 and 25°C. The conformational stability was measured from the GdnHCl-induced denaturation study of CAVA monitored by circular dichroism (CD) and fluorescence measurements. On increasing the concentration of GdnHCl up to 5.0, a stable intermediate was observed between the concentrations 3.25M to 3.40M of the denaturant. However, CAVA gets completely denatured at 4.0M GdnHCl. The existence of a stable intermediate state was validated by 1-anilinonaphthalene-8-sulfonic acid (ANS binding) fluorescence and near-UV CD measurements. In silico studies were also performed to analyse the effect of GdnHCl on the structure and stability of CAVA under explicit conditions. Molecular dynamics simulations for 40ns were carried out and a well-defined correlation was established for both in vitro and in silico studies.

Design and synthesis of a novel class of carbonic anhydrase-IX inhibitor 1-(3-(phenyl/4-fluorophenyl)-7-imino-3H-[1,2,3]triazolo[4,5d]pyrimidin 6(7H)yl)urea.

Carbonic anhydrase IX (CAIX) is a promising target in cancer therapy especially in the case of hypoxia-induced tumors. The selective inhibition of CA isozymes is a challenging task in drug design and discovery process. Here, we performed fluorescence-binding studies and inhibition assay combined with molecular docking and molecular dynamics (MD) simulation analyses to determine the binding affinity of two synthesized triazolo-pyrimidine urea derived (TPUI and TPUII) compounds with CAIX and CAII. Fluorescence binding results are showing that molecule TPUI has an excellent binding-affinity for CAIX (kD=0.048µM). The TPUII also exhibits an appreciable binding affinity (kD=7.52µM) for CAIX. TPUI selectively inhibits CAIX as compared to TPUII in the 4-NPA assay. Docking studies show that TPUI is spatially well-fitted in the active site cavity of CAIX, and is involve in H-bond interactions with His94, His96, His119, Thr199 and Thr200. MD simulation studies revealed that TPUI efficiently binds to CAIX and essential active site residual interaction is consistent during the entire simulation of 40ns. These studies suggest that TPUI appeared as novel class of CAIX inhibitor, and may be used as a lead molecule for the development of potent and selective CAIX inhibitor for the hypoxia-induced cancer therapy.

Spectroscopic and MD simulation studies on unfolding processes of mitochondrial carbonic anhydrase VA induced by urea.

Carbonic anhydrase VA (CAVA) is primarily expressed in the mitochondria and involved in numerous physiological processes including lipogenesis, insulin secretion from pancreatic cells, ureagenesis, gluconeogenesis and neuronal transmission. To understand the biophysical properties of CAVA, we carried out a reversible urea-induced isothermal denaturation at pH 7.0 and 25°C. Spectroscopic probes, [θ]222 (mean residue ellipticity at 222 nm), F344 (Trp-fluorescence emission intensity at 344 nm) and Δε280 (difference absorption at 280 nm) were used to monitor the effect of urea on the structure and stability of CAVA. The urea-induced reversible denaturation curves were used to estimate [Formula: see text], Gibbs free energy in the absence of urea; Cm, the mid-point of the denaturation curve, i.e. molar urea concentration ([urea]) at which ΔGD = 0; and m, the slope (=∂ΔGD/∂[urea]). Coincidence of normalized transition curves of all optical properties suggests that unfolding/refolding of CAVA is a two-state process. We further performed 40 ns molecular dynamics simulation of CAVA to see the dynamics at different urea concentrations. An excellent agreement was observed between in silico and in vitro studies.

Cloning, expression, purification and characterization of human mitochondrial carbonic anhydrase VA.

Carbonic anhydrase VA (CAVA) is a mitochondrial enzyme that catalyzes the reversible hydration of CO2 to produce HCO3- and proton. CAV is primarily involved in several biosynthetic processes such as ureagenesis, gluconeogenesis and lipogenesis by providing bicarbonate ion. Here, we report a new strategy for cloning, expression and purification for CAVA in the bacterial system followed by its biophysical characterization. The cDNA of CAVA, a 801 nucleotide long that encodes a 267-amino acid polypeptide of molecular mass of 30-kDa (excluding signal peptide), was sub-cloned in the expression vector pET21c and transformed into Escherichia coli strain BL21 (DE3) for expression. The recombinant protein was purified in two steps by Ni-NTA and DEAE weak anion-exchange chromatography under native condition from the supernatant, while inclusion bodies (IBs) were used to get protein under the denatured condition with a relatively high yield. CAVA was purified under denatured conditions in a single step using Ni-NTA chromatography. SDS-PAGE showed a band of 30-kDa, which was further confirmed as CAVA by Western blot and MALDI-TOF/MS. We further performed enzyme activity to ensure that both forms of purified proteins are enzymatically active. Measurements of secondary structure of the native, denatured and renatured proteins were carried out using circular dichroism. The purified protein can be further used for structural and biochemical studies.

Luminol-based chemiluminescent signals: clinical and non-clinical application and future uses.

Chemiluminescence (CL) is an important method for quantification and analysis of various macromolecules. A wide range of CL agents such as luminol, hydrogen peroxide, fluorescein, dioxetanes and derivatives of oxalate, and acridinium dyes are used according to their biological specificity and utility. This review describes the application of luminol chemiluminescence (LCL) in forensic, biomedical, and clinical sciences. LCL is a very useful detection method due to its selectivity, simplicity, low cost, and high sensitivity. LCL has a dynamic range of applications, including quantification and detection of macro and micromolecules such as proteins, carbohydrates, DNA, and RNA. Luminol-based methods are used in environmental monitoring as biosensors, in the pharmaceutical industry for cellular localization and as biological tracers, and in reporter gene-based assays and several other immunoassays. Here, we also provide information about different compounds that may enhance or inhibit the LCL along with the effect of pH and concentration on LCL. This review covers most of the significant information related to the applications of luminol in different fields.