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.

Crystallins as Important Pathogenic Targets for Accumulation of Structural Damages Resulting in Protein Aggregation and Cataract Development: Introduction to This Special Issue of Biochemistry (Moscow).

This issue of Biochemistry (Moscow) is dedicated to the role of protein misfolding and aggregation in cataract development. In fact, many genetic mutations or chemical and physical deleterious factors can initiate alterations in the macrostructural order and proper folding of eye lens proteins, which in some cases result in the formation of large light-scattering aggregates, affecting the quality of vision and making lens more prone to cataract development. Diabetes mellitus, which is associated with oxidative stress and mass production of highly reactive compounds, can accelerate unfolding and aggregation of eye lens proteins. This journal issue contains reviews and research articles that describe the destructive effects of mutations and highly reactive metabolites on the structure and function of lens crystallin proteins, as well important molecules in the lens's natural defense system involved in protection against deleterious effects of the physical and chemical factors.

Relationship between the Structure and Chaperone Activity of Human αA-Crystallin after Its Modification with Diabetes-Associated Oxidative Agents and Protective Role of Antioxidant Compounds.

The study was aimed to evaluate the impact of peroxynitrite (PON, oxidative stress agent in diabetes), methylglyoxal (MGO, diabetes-associated reactive carbonyl compound), and their simultaneous application on the structural and functional features of human αA-crystallin (αA-Cry) using various spectroscopy techniques. Additionally, the surface tension and oligomer size distribution of the treated and untreated protein were tested using tensiometric analysis and dynamic light scattering, respectively. Our results indicated that the reaction of PON and MGO with human αA-Cry leads to the formation of new chromophores, alterations in the secondary to quaternary protein structure, reduction in the size of protein oligomers, and significant enhancement in the chaperone activity of αA-Cry. To reverse the effects of the tested compounds, ascorbic acid and glutathione (main components of lens antioxidant defense system) were applied. As expected, the two antioxidant compounds significantly prevented formation of high molecular weight aggregates of αA-Cry (according to SDS-PAGE). Our results suggest that the lens antioxidant defense system, in particular, glutathione, may provide a strong protection against rapid incidence and progression of diabetic cataract by preventing the destructive reactions of highly reactive DM-associated metabolites.

Evaluation of morphology and angiogenesis of breast cancer in BALB/c mice using trypsin inhibitor from Cucumis melo seeds. In vitro and in vivo study.

Introduction: Melon seeds, as an excellent source of protease inhibitors, may have a protective role against tumor progression and angiogenesis. However, their effects on angiogenesis and the mechanism of their action against cancer progression remain elusive. This study aimed to investigate the effect of bioactive compounds of melon seed on the expression of angiogenesis genes in BALB/c mice with breast cancer. Material and methods: Trypsin inhibitor (TI) was purified from the seed powder of Cucumis melo. Half- maximal inhibitory concentration was determined for TI, extract of melon seed powder (EXT), and tamoxifen (TAM) by the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. Also, breast tumor was induced by subcutaneous injection of MC4-L2 cells in BALB/c inbred mice breast tissue. After tumor growth, mice were treated with TI, EXT, and TAM to examine their effects on the tumor characteristics and expression of angiogenesis-related genes including MMP-2, MMP-9, and vascular endothelial growth factor (VEGF) using the reverse transcription polymerase chain reaction method. Results: Trypsin inhibitor, EXT, TAM, and adjuvant treatment of TI + TAM resulted a reduction in expression of MMP-2, MMP-9, and VEGF. All treatments improved the breast tumor characteristics and the necrosis. The real-time polymerase chain reaction method verified the positive effects of the treatments on the breast cancer cell line and tumors. Conclusions: The results indicated that treatments with TI purified from Cucumis melo seeds and also combination therapy of TI and TAM can be considered as an alternative therapy in breast cancer patients. Further studies are warranted.

The congenital cataract-causing mutations P20R and A171T are associated with important changes in the amyloidogenic feature, structure and chaperone-like activity of human αB-crystallin.

Cataract is the major reason for human blindness worldwide. α-Crystallin, as a key chaperone of eye lenses, keeps the lenticular tissues in its transparent state over time. In this study, cataract-causing familial mutations, P20R and A171T, were introduced in CRYАB gene. After successful expression in Escherichia coli and subsequent purification, the recombinant proteins were subjected to extensive structural and functional analyses using various spectroscopic techniques, gel electrophoresis, and electron microscopy. The results of fluorescence and Raman assessments suggest important but discreet conformational changes in human αB-Cry upon these cataractogenic mutations. Furthermore, the mutant proteins exhibited significant secondary structural alteration as revealed by FTIR and Raman spectroscopy. An increase in conformational stability was seen in the human αB-Cry bearing these congenital cataractogenic mutations. The oligomeric size distribution and chaperone-like activity of human αB-Cry were significantly altered by these mutations. The P20R mutant protein was observed to loose most of the chaperone-like activity. Finally, these cataractogenic mutant proteins exhibited an increased propensity to form the amyloid fibrils when incubated under environmental stress. Overall, the structural and functional changes in mutated human αB-Cry proteins can shed light on the pathogenic development of congenital cataracts.

Modulating Insulin Fibrillation Using Engineered B-Chains with Mutated C-Termini.

Stress-induced unfolding and fibrillation of insulin represent serious medical and biotechnological problems. Despite many attempts to elucidate the molecular mechanisms of insulin fibrillation, there is no general agreement on how this process takes place. Several previous studies suggested the importance of the C-terminal region of B-chain in this pathway. Therefore, we generated the T30R and K29R/T30R mutants of insulin B-chain. Recombinantly produced wild-type A-chain and mutant B-chains were combined efficiently in the presence of chaperone αB-crystallin. The mutant B-chains along with the control wild-type insulin were used in a wide range of parallel experiments to compare their fibrillation kinetics, morphology of fibrils, and forces driving the fibril formation. The mutant insulins and their B-chains displayed significant resistance against stress-induced fibrillation, particularly at the nucleation stage, suggesting that the B-chain might be influencing the insulin fibrillation. The fact that the different mature insulins formed larger fibrillar bundles compared to those formed by their B-chains alone suggested the role of A-chain in the lateral association of the insulin fibrils. Overall, in addition to the N-terminal region of the B-chain, which was shown to serve as an important regulator of insulin fibrillation, the C-terminal region of this peptide is also crucial for the control of fibrillation, likely serving as an attachment site engaged in the formation of the nucleus and protofibril. Finally, two mutated insulin variants examined in this study might be of interest to the pharmaceutical sector as, to our knowledge, novel intermediate-acting insulin analogs because of their suitable biological activity and improved stability against stress-induced fibrillation.

The impact of different mutations at Arg54 on structure, chaperone-like activity and oligomerization state of human αA-crystallin: The pathomechanism underlying congenital cataract-causing mutations R54L, R54P and R54C.

A major part of cataractogenic mutations in human αA-Crystallin (αA-Cry) occurs at Arg residues. While Arg54 is highly conserved within different species, the cataractogenic mutations R54L, R54P and R54C have been recently identified in CRYAA gene, encoding human αA-Cry. The detailed structural and functional aspects, stability and amyloidogenic properties of αA-Cry were determined upon the above-mentioned missense mutations, using various spectroscopic techniques, gel electrophoresis, electron microscopy, size exclusion chromatography analyses, and chaperone-like activity assay. The different mutations at Arg54 result in diverse structural alterations among mutant proteins. In addition, the mutant proteins displayed reduced thermal stability, increased amyloidogenic properties and attenuated chaperone-like activity against aggregation of γ-Cry, catalase and lysozyme. The mutant proteins were also capable of forming larger oligomeric complexes with γ-Cry which is the natural partner of α-Cry in the eye lenses. The most significant structural and functional damages were observed upon R54L mutation which was also accompanied with increased oligomeric size distribution of the mutant protein. The cataractogenic nature of R54P mutation can be explained with its detrimental effect on chaperone-like activity, conformational stability and proteolytic digestibility of the mutant protein. Also, R54C αA-Cry displayed an important intrinsic propensity for disulfide protein cross-linking with significantly reduced chaperone-like activity against all client proteins. These mutations revealed a range of detrimental effects on the structure, stability and functional properties of αA-Cry which all together can explain the pathomechanisms underlying development of the associated congenital cataract disorders.

Protective role of antioxidant compounds against peroxynitrite-mediated modification of R54C mutant αA-crystallin.

As a highly potent reactive oxygen and nitrogen species, peroxynitrite (PON) has endogenous production in the eye ball and contributes to a variety of ocular disorders. In the current study the structural characteristics, chaperone-like activity and conformational stability of R54C mutant αA-crystallin (αA-Cry) were studied upon modification with PON and in the presence of three antioxidant compounds such as ascorbic acid (ASA), glutathione (GSH) and N-acetylcysteine (NAC) using gel electrophoresis and different spectroscopy methods. The results of both fluorescence analysis and gel electrophoresis suggested that PON modification leads to dityrosine-mediated intermolecular cross-linking of this cataractogenic mutant protein. Also, the propensity of R54C mutant αA-Cry for disulfide cross-linking was increased upon PON modification. In addition, the PON-modified protein indicated structural alteration, reduced chemical stability and different pattern of proteolysis. Upon modification with PON, mutant αA-Cry displayed a significant increase in the chaperone-like activity against aggregation of γ-crystallin and insulin. In addition, different antioxidant compounds indicated a prominent role in neutralizing the PON damaging effects on structural integrity and stability of this protein. The results of this study may highlight the importance of antioxidant-rich foods or potent antioxidant supplements in protection of lens crystallins against PON-mediated structural damages and cataract development.

Interaction of prodigiosin with HSA and ß-Lg: Spectroscopic and molecular docking studies.

Human serum albumin (HSA) and bovine ß-lactoglobulin (ß-Lg) are both introduced as blood and oral carrier scaffolds with high affinity for a wide range of pharmaceutical compounds. Prodigiosin, a natural three pyrrolic compound produced by Serratia marcescens, exhibits many pharmaceutical properties associated with health benefits. In the present study, the interaction of prodigiosin with HSA and ß-Lg was investigated using fluorescence spectroscopy, circular dichroism (CD) and computational docking. Prodigiosin interacts with the Sudlow's site I of HSA and the calyx of ß-Lg with association constant of 4.41 × 10(4) and 1.99 × 10(4) M(-1) to form 1:1 and 2:3 complexes at 300K, respectively. The results indicated that binding of prodigiosin to HSA and ß-Lg caused strong fluorescence quenching of both proteins through static quenching mechanism. Electrostatic and hydrophobic interactions are the major forces in the stability of PG-HSA complex with enthalpy- and entropy-driving mode, although the formation of prodigiosin-ß-Lg complex is entropy-driven hydrophobic associations. CD spectra showed slight conformational changes in both proteins due to the binding of prodigiosin. Moreover, the ligand displacement assay, pH-dependent interaction and protein-ligand docking study confirmed that the prodigiosin binds to residues located in the subdomain IIA and IIIA of HSA and central calyx of ß-Lg.

Charge manipulation of the human insulin B chain C-terminal to shed light on the complex mechanism of insulin fibrillation.

Insulin fibrillation poses a significant challenge in the development and treatment of diabetes. Current efforts to unravel its mechanisms have thus far remained incomplete. To shed light on the intricate processes behind insulin fibrillation, we employed mutagenesis techniques to introduce additional positive charge residues into the C-terminal region of the insulin B chain which plays an important role in insulin dimerization. We employed our investigation with various spectroscopic methods, electron microscopy, and molecular dynamics simulations. These methods allowed us to explore the structure and fibrillation behavior of the engineered B chains following their expression in a bacterial host and successful purification. This manipulation had a pronounced impact on the oligomerization behavior of the insulin B chain. It appears that these mutations delay the formation of the dimeric state in the process of transitioning to larger oligomers, consequently, leading to an alteration in the kinetics of fibrillation. Our findings also indicated that the mutant insulin B chains (Di-R, Di-K, and Di-H) displayed resistance to the initiation of fibrillation. This resistance can be attributed to the repulsive forces generated by the introduced positive charges, which disrupt the attractive interactions favoring nucleation. Notably, the mutant B chains formed shorter and less abundant oligomers and fibrils, which can be ascribed to the alterations induced by repulsion. Our engineered mutant B chains exhibited enhanced stability against stress-induced fibrillation, hinting at their potential utility in the development of new insulin analogs. This study underscores the significance of the C-terminal region in the initial stages of insulin B chain fibrillation, providing valuable insights into the intricate mechanisms involved and their potential pharmaceutical applications.

Efficient Expression in the Prokaryotic Host System, Purification and Structural Analyses of the Recombinant Human ACE2 Catalytic Subunit as a Hybrid Protein with the B Subunit of Cholera Toxin (CTB-ACE2).

Angiotensin-converting enzyme 2 (ACE2) has a specific interaction with the coronavirus spike protein, enabling its entry into human cells. This membrane enzyme converts angiotensin II into angiotensin 1-7, which has an essential role in protecting the heart and improving lung function. Many therapeutic properties have been attributed to the human recombinant ACE2 (hrACE2), especially in combating complications related to diabetes mellitus and hypertension, as well as, preventing the coronavirus from entering the target tissues. In the current study, we designed an appropriate gene construct for the hybrid protein containing the ACE2 catalytic subunit and the B subunit of cholera toxin (CTB-ACE2). This structural feature will probably help the recombinant hybrid protein enter the mucosal tissues, including the lung tissue. Optimization of this hybrid protein expression was investigated in BL21 bacterial host cells. Also, the hybrid protein was identified with an appropriate antibody using the ELISA method. A large amount of the hybrid protein (molecular weight of ~ 100 kDa) was expressed as the inclusion body when the induction was performed in the presence of 0.25 mM IPTG and 1% sucrose for 10 h. Finally, the protein structural features were assessed using several biophysical methods. The fluorescence emission intensity and oligomeric size distribution of the CTB-ACE2 suggested a temperature-dependent alteration. The ß-sheet and α-helix were also dominant in the hybrid protein structure, and this protein also displays acceptable chemical stability. In overall, according to our results, the efficient expression and successful purification of the CTB-ACE2 protein may pave the path for its therapeutic applications against diseases such as covid-19, diabetes mellitus and hypertension.

Unveiling the structural and functional consequences of the p.D109G pathogenic mutation in human αB-Crystallin responsible for restrictive cardiomyopathy and skeletal myopathy.

αB-Crystallin (αB-Cry) is expressed in many tissues, and mutations in this protein are linked to various diseases, including cataracts, Alzheimer's disease, Parkinson's disease, and several types of myopathies and cardiomyopathies. The p.D109G mutation, which substitutes a conserved aspartate residue involved in the interchain salt bridges, with glycine leads to the development of both restrictive cardiomyopathy (RCM) and skeletal myopathy. In this study, we generated this mutation in the α-Cry domain (ACD) which is crucial for forming the active chaperone dimeric state, using site-directed mutagenesis. After inducing expression in the bacterial host, we purified the mutant and wild-type recombinant proteins using anion exchange chromatography. Various spectroscopic evaluations revealed significant changes in the secondary, tertiary, and quaternary structures of human αB-Cry caused by this mutation. Furthermore, this pathogenic mutation led to the formation of protein oligomers with larger sizes than those of the wild-type protein counterpart. The mutant protein also exhibited increased chaperone activity and decreased chemical, thermal, and proteolytic stability. Atomic force microscopy (AFM), transmission electron microscopy (TEM), and fluorescence microscopy (FM) demonstrated that p.D109G mutant protein is more prone to forming amyloid aggregates. The misfolding associated with the p.D109G mutation may result in abnormal interactions of human αB-Cry with its natural partners (e.g., desmin), leading to the formation of protein aggregates. These aggregates can interfere with normal cellular processes and may contribute to muscle cell dysfunction and damage, resulting in the pathogenic involvement of the p.D109G mutant protein in restrictive cardiomyopathy and skeletal myopathy.

Understanding the structural and functional changes and biochemical pathomechanism of the cardiomyopathy-associated p.R123W mutation in human αB-crystallin.

αB-crystallin, a member of the small heat shock protein (sHSP) family, is expressed in diverse tissues, including the eyes, brain, muscles, and heart. This protein plays a crucial role in maintaining eye lens transparency and exhibits holdase chaperone and anti-apoptotic activities. Therefore, structural and functional changes caused by genetic mutations in this protein may contribute to the development of disorders like cataract and cardiomyopathy. Recently, the substitution of arginine 123 with tryptophan (p.R123W mutation) in human αB-crystallin has been reported to trigger cardiomyopathy. In this study, human αB-crystallin was expressed in Escherichia coli (E. coli), and the missense mutation p.R123W was created using site-directed mutagenesis. Following purification via anion exchange chromatography, the structural and functional properties of both proteins were investigated and compared using a wide range of spectroscopic and microscopic methods. The p.R123W mutation induced significant alterations in the secondary, tertiary, and quaternary structures of human αB-crystallin. This pathogenic mutation resulted in an increased ß-sheet structure and formation of protein oligomers with larger sizes compared to the wild-type protein. The mutant protein also exhibited reduced chaperone activity and lower thermal stability. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) demonstrated that the p.R123W mutant protein is more prone to forming amyloid aggregates. The structural and functional changes observed in the p.R123W mutant protein, along with its increased propensity for aggregation, could impact its proper functional interaction with the target proteins in the cardiac muscle, such as calcineurin. Our results provide an explanation for the pathogenic intervention of p.R123W mutant protein in the occurrence of hypertrophic cardiomyopathy (HCM).

Unraveling the impact of the p.R107L mutation on the structure and function of human αB-Crystallin: Implications for cataract formation.

To date, several pathogenic mutations have been identified in the primary structure of human α-Crystallin, frequently involving the substitution of arginine with a different amino acid. These mutations can lead to the incidence of cataracts and myopathy. Recently, an important cataract-associated mutation has been reported in the functional α-Crystallin domain (ACD) of human αB-Crystallin protein, where arginine 107 (R107) is replaced by a leucine. In this study, we investigated the structure, chaperone function, stability, oligomerization, and amyloidogenic properties of the p.R107L human αB-Crystallin using a number of different techniques. Our results suggest that the p.R107L mutation can cause significant changes in the secondary, tertiary, and quaternary structures of αB-Crystallin. This cataractogenic mutation led to the formation of protein oligomers with larger sizes than the wild-type protein and reduced the chemical and thermal stability of the mutant chaperone. Both fluorescence and microscopic assessments indicated that this mutation significantly altered the amyloidogenic properties of human αB-Crystallin. Furthermore, the mutant protein indicated an attenuated in vitro chaperone activity. The molecular dynamics (MD) simulation confirmed the experimental results and indicated that p.R107L mutation could alter the proper conformation of human αB-Crystallin dimers. In summary, our results indicated that the p.R107L mutation could promote the formation of larger oligomers, diminish the stability and chaperone activity of human αB-Crystallin, and these changes, in turn, can play a crucial role in the development of cataract disorder.

Insights into the dual nature of αB-crystallin chaperone activity from the p.P39L mutant at the N-terminal region.

The substitution of leucine to proline at position 39 (p.P39L) in human αB-crystallin (αB-Cry) has been associated with conflicting interpretations of pathogenicity in cataracts and cardiomyopathy. This study aimed to investigate the effects of the p.P39L mutation on the structural and functional features of human αB-Cry. The mutant protein was expressed in Escherichia coli (E. coli) and purified using anion exchange chromatography. We employed a wide range of spectroscopic analyses, gel electrophoresis, transmission electron microscopy (TEM), and atomic force microscopy (AFM) techniques to investigate the structure, function, stability, and fibrillation propensity of the mutant protein. The p.P39L mutation caused significant changes in the secondary, tertiary, and quaternary structures of human αB-Cry and increased the thermal stability of the protein. The mutant αB-Cry exhibited an increased chaperone activity and an altered oligomeric size distribution, along with an increased propensity to form amyloid aggregates. It is worth mentioning, increased chaperone activity has important positive and negative effects on damaged cells related to cataracts and cardiomyopathy, particularly by interfering in the process of apoptosis. Despite the apparent positive nature of the increased chaperone activity, it is also linked to adverse consequences. This study provides important insights into the effect of proline substitution by leucine at the N-terminal region on the dual nature of chaperone activity in human αB-Cry, which can act as a double-edged sword.

Exploring the significance of potassium homeostasis in copper ion binding to human αB-Crystallin.

αB-Crystallin (αB-Cry) is a small heat shock protein known for its protective role, with an adaptable structure that responds to environmental changes through oligomeric dynamics. Cu(II) ions are crucial for cellular processes but excessive amounts are linked to diseases like cataracts and neurodegeneration. This study investigated how optimal and detrimental Cu(II) concentrations affect αB-Cry oligomers and their chaperone activity, within the potassium-regulated ionic-strength environment. Techniques including isothermal titration calorimetry, differential scanning calorimetry, fluorescence spectroscopy, inductively coupled plasma atomic emission spectroscopy, cyclic voltammetry, dynamic light scattering, circular dichroism, and MTT assay were employed and complemented by computational methods. Results showed that potassium ions affected αB-Cry's structure, promoting Cu(II) binding at multiple sites and scavenging ability, and inhibiting ion redox reactions. Low concentrations of Cu(II), through modifications of oligomeric interfaces, induce regulation of surface charge and hydrophobicity, resulting in an increase in chaperone activity. Subunit dynamics were regulated, maintaining stable interfaces, thereby inhibiting further aggregation and allowing the functional reversion to oligomers after stress. High Cu(II) disrupted charge/hydrophobicity balance, sewing sizable oligomers together through subunit-subunit interactions, suppressing oligomer dissociation, and reducing chaperone efficiency. This study offers insights into how Cu(II) and potassium ions influence αB-Cry, advancing our understanding of Cu(II)-related diseases.

Pyrimidine-fused heterocycle derivatives as a novel class of inhibitors for α-glucosidase.

The needs for diverse inhibitors of α-glucosidase (α-Gls) encouraged us to synthesize five different poly-hydroxy functionalized pyrimidine-fused heterocyclic (PHPFH) molecules, having either aliphatic or aromatic side chains (C1-C5) and their inhibitory activities were examined spectroscopically against yeast and mouse intestinal α-Gls. The results revealed that aromatic substitution of the synthetic compounds has significant impact on their inhibitory properties. Moreover C3 with the substituted moiety as 4-(4-aminophenylsulfonyl) phenyl (4-APSP) revealed strong inhibitory activity with non-competitive and competitive inhibition modes against yeast and mouse α-Gls, respectively. Furthermore, in the presence of increasing concentration of C3, both Trp and 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence intensities of yeast α-Gls were gradually decreased, suggesting that C3 binding induced significant structural alteration which was accompanied with the reduction of hydrophobic surfaces. Also, the interaction between yeast α-Gls and C3 was proved to be spontaneous and driven mainly by hydrophobic forces. Overall, this study suggests that aromatic substitution on pyrimidine-fused heterocyclic (PFH) scaffold may represent a novel class of promising inhibitors of α-Gls.

Investigating the role of double mutations R12C/P20R, and R12C/R69C on structure, chaperone-like activity, and amyloidogenic properties of human αB-crystallin.

α-crystallin is a structurally essential small heat shock protein (sHSP) with a chaperone-like activity which maintains transparency of the lenticular tissues during a period of time that is as long as human life. α-crystallin is a multimeric protein consisting of αA and αB subunits, with 57 % homology. The CRYAB gene on chromosome 11 encodes human αB-crystallin (αB-Cry), which contains 175 amino acid residues. In the current study, the cataractogenic mutations R12C, P20R, R69C, and double mutations R12C/P20R and R12C/P20R were embedded into the human CRYAB gene. Following successful expression in the prokaryotic system and purification, a number of spectroscopic techniques, gel electrophoresis, dynamic light scattering (DLS), and transmission electron microscopy (TEM) were applied to assess the role of these mutations on the structure, amyloidogenicity, and biological function of human αB-Cry. The created mutations caused significant changes in the structure, and oligomeric state of human αB-Cry. These mutations, particularly R12C, R12C/P20R, and R12C/R69C, dramatically enhanced the tendency of this protein for the amyloid fibril formation and reduced its chaperone-like activity. Since double mutations R12C/P20R and R12C/P20R were able to intensely change the protein's structure and chaperone function, it can be suggested that they may play a destructive role in a cumulative manner. Our findings indicated that the simultaneous presence of two pathogenic mutations may have a cumulative destructive impacts on the structure and function of human αB-Cry and this observation is likely related to the disease severity of the mutated proteins.

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.

Optimizing expression, purification, structural and functional assessments of a novel dimeric incretin (GLP-1cpGLP-1).

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that reduces postprandial glycemic excursions by enhancing insulin secretion. In this study, a new dimeric GLP-1 analogue (GLP-1cpGLP-1) was designed by inserting human insulin C-peptide (CP) in the middle of a dimer of [Gly8] GLP-1 (7-36). Then, the dimeric incretin (GLP-1cpGLP-1) was ligated to human αB-crystallin (αB-Cry) to create a hybrid protein, abbreviated as αB-GLP-1cpGLP-1. The constructed gene was well expressed in the bacterial host system. After specific chemical release from the hybrid protein, the dimeric incretin was purified by size exclusion chromatography (SEC). Finally, the RP-HPLC analysis indicated a purity of >99 % for the dimeric incretin. The secondary structure assessments by various spectroscopic methods, and in silico analysis suggested that the dimeric incretin has α-helical rich structure. The dynamic light scattering (DLS) analysis indicates that our dimeric incretin forms large oligomeric structures. This incretin analogue significantly reduced blood glucose levels in both healthy and diabetic mice while effectively triggering insulin release. The size exclusion HPLC also indicates the interaction of the new incretin analogue with human serum albumin, the main carrier protein in the bloodstream. Consistent with the results obtained from the biological activity assessments, this significant interaction indicates its potential as a viable therapeutic agent with a long-lasting effect. The results of our research represent a significant breakthrough in the successful design of an active incretin dimer capable of effectively controlling blood sugar levels and inducing insulin secretion in the realm of diabetes treatment.