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.

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.

Myopathy-associated G154S mutation causes important changes in the conformational stability, amyloidogenic properties, and chaperone-like activity of human αB-crystallin.

Glycine to serine substitution at position 154 of human αB-crystallin (αB-Cry) is behind the development of cardiomyopathy and late-onset distal myopathy. The current study was conducted with the aim to investigate the structural and functional features of the G154S mutant αB-Cry using various spectroscopic techniques and microscopic analyses. The secondary and tertiary structures of human αB-Cry were preserved mainly in the presence of G154S mutation, but the mutant protein indicated a reduced chaperone-like activity when γ-Cry as its natural partner in eye lenses was the substrate protein. Moreover, a significant reduction in the enzyme refolding ability and in vivo chaperone activity of the mutant protein were observed. Also, the mutant protein displayed reduced conformational stability upon urea-induced denaturation. Both fluorescence and electron microscopic analyses suggested that G154S mutant protein has an increased susceptibility for amyloid fibril formation. Therefore, the pathomechanism of G154S mutation can be explained by its attenuated chaperone function, decreased conformational stability, and increased amyloidogenic propensity. Some of these important changes may also alter the correct interaction of the mutated αB-Cry with its target proteins in myopathy.

Analysis of the data on titration of native and peroxynitrite modified αA- and αB-crystallins by Cu2+-ions.

The interaction of αA- and αB-crystallins with Cu2+ ion modulates their structure and chaperone-like activity which is important for lens transparency. Theoretical analysis of the dependences of fluorescence intensity of native αA- and αB-crystallins and αA- and αB-crystallins modified by peroxynitrite on concentration of Cu2+ ions has been carried out. It has been shown that one subunit of native αA-crystallin contains two equivalent Cu2+-binding sites. The microscopic dissociation constant for Cu2+-αA-crystallin complex (K diss) was found to be equal to 9.7 µM. For peroxynitrite modified αA-crystallin the K diss value is equal to 17 µM. One subunit of native αB-crystallin contains two non-equivalent Cu2+-binding sites. The corresponding microscopic dissociation constants for Cu2+-αB-crystallin complexes (K 1 and K 2) were found to be equal to 0.94 and 36 µM. For peroxynitrite modified αB-crystallin the K 1 and K 2 values are equal to 4.3 and 70 µM, respectively.

Importance of the positively charged residue at position 54 to the chaperoning function, conformational stability and amyloidogenic nature of human αA-crystallin.

Arginine 54 (R54) in αA-Crystallin (αA-Cry) is highly conserved within different species. Recently, three missense mutations at this hot spot position have been reported to cause congenital cataract disorders. To investigate the impact of charge on structural and functional aspects of αA-Cry, R54 was individually substituted with lysine and aspartate. Replacement of R54 with the positively and negatively charged residues led to structural alteration and reduction in the protein conformational and proteolytic stability. Also, these mutations resulted in important increase in the amyloidogenic propensity of αA-Cry. Additionally, all these changes were more pronounced upon R54D mutation. Keeping the positive charge by R54K mutation, the structural integrity and stability of αA-Cry were partially preserved. Our results suggest that arginine 54 may also participate in salt bridge formation and conformational stabilization of αA-Cry. Also, it seems that unique physicochemical properties of arginine 54 may have a prominent role in the structural integrity, conformational stability and functional aspects of human αA-Cry.

The structural alteration and aggregation propensity of glycated lens crystallins in the presence of calcium: Importance of lens calcium homeostasis in development of diabetic cataracts.

The imbalance of the calcium homeostasis in the lenticular tissues of diabetic patients is an important risk factor for development of cataract diseases. In the current study, the impact of elevated levels of calcium ions were investigated on structure and aggregation propensity of glycated lens crystallins using gel electrophoresis and spectroscopic assessments. The glycated proteins indicated significant resistance against calcium-induced structural insults and aggregation. While, glycated crystallins revealed an increased conformational stability; a slight instability was observed for these proteins upon interaction with calcium ions. Also, in the presence of calcium, the proteolytic pattern of native crystallins was altered and that of glycated protein counterparts remained almost unchanged. According to results of this study it is suggested that the structural alteration of lens crystallins upon glycation may significantly reduce their calcium buffering capacity in eye lenses. Therefore, under chronic hyperglycemia accumulation of this cataractogenic metal ion in the lenticular tissues may subsequently culminate in activation of different pathogenic pathways, leading to development of lens opacity and cataract diseases.

Preventive role of lens antioxidant defense mechanism against riboflavin-mediated sunlight damaging of lens crystallins.

The main components of sunlight reaching the eye lens are UVA and visible light exerting their photo-damaging effects indirectly by the aid of endogenous photosensitizer molecules such as riboflavin (RF). In this study, lens proteins solutions were incubated with RF and exposed to the sunlight. Then, gel mobility shift analysis and different spectroscopic assessments were applied to examine the structural damaging effects of solar radiation on these proteins. Exposure of lens proteins to direct sunlight, in the presence of RF, leads to marked structural crosslinking, oligomerization and proteolytic instability. These structural damages were also accompanied with reduction in the emission fluorescence of Trp and Tyr and appearance of a new absorption peak between 300 and 400nm which can be related to formation of new chromophores. Also, photo-oxidation of lens crystallins increases their oligomeric size distribution as examined by dynamic light scattering analysis. The above mentioned structural insults, as potential sources of sunlight-induced senile cataract and blindness, were significantly attenuated in the presence of ascorbic acid and glutathione which are two important components of lens antioxidant defense system. Therefore, the powerful antioxidant defense mechanism of eye lens is an important barrier against molecular photo-damaging effects of solar radiations during the life span.

Evaluation of structure, chaperone-like activity and protective ability of peroxynitrite modified human α-Crystallin subunits against copper-mediated ascorbic acid oxidation.

The copper-catalyzed oxidation of ascorbic acid (ASA) to dehydroascorbate (DHA) and hydrogen peroxide plays a central role in pathology of cataract diseases during ageing and in diabetic patients. In the current study, the structural feature, chaperone-like activity and protective ability of peroxynitrite (PON) modified αA- and αB-Crystallin (Cry) against copper-mediated ASA oxidation were studied using different spectroscopic measurements and gel mobility shift assay. Upon PON modification, additional to protein structural alteration, the contents of nitrotyrosine, nitrotryptophan, dityrosine and carbonyl groups were significantly increased. Moreover, αB-Cry demonstrates significantly larger capacity for PON modification than αA-Cry. Also, based on the extent of PON modification, these proteins may display an improved chaperone-like activity and enhanced protective ability against copper-mediated ASA oxidation. In the presence of copper ions, chaperone-like activity of both native and PON-modified α-Cry subunits were appreciably improved. Additionally, binding of copper ions to native and PON-modified proteins results in the significant reduction of their solvent exposed hydrophobic patches. Overall, the increase in chaperone-like activity/ASA protective ability of PON-modified α-Cry and additional enhancement of its chaperoning action with copper ions appear to be an important defense mechanism offered by this protein.

Protective Effects of Acetylation on the Pathological Reactions of the Lens Crystallins with Homocysteine Thiolactone.

Various post-translational lens crystallins modifications result in structural and functional insults, contributing to the development of lens opacity and cataract disorders. Lens crystallins are potential targets of homocysteinylation, particularly under hyperhomocysteinemia which has been indicated in various eye diseases. Since both homocysteinylation and acetylation primarily occur on protein free amino groups, we applied different spectroscopic methods and gel mobility shift analysis to examine the possible preventive role of acetylation against homocysteinylation. Lens crystallins were extensively acetylated in the presence of acetic anhydride and then subjected to homocysteinylation in the presence of homocysteine thiolactone (HCTL). Extensive acetylation of the lens crystallins results in partial structural alteration and enhancement of their stability, as well as improvement of α-crystallin chaperone-like activity. In addition, acetylation partially prevents HCTL-induced structural alteration and aggregation of lens crystallins. Also, acetylation protects against HCTL-induced loss of α-crystallin chaperone activity. Additionally, subsequent acetylation and homocysteinylation cause significant proteolytic degradation of crystallins. Therefore, further experimentation is required in order to judge effectively the preventative role of acetylation on the structural and functional insults induced by homocysteinylation of lens crystallins.

The impact of calcium ion on structure and aggregation propensity of peroxynitrite-modified lens crystallins: new insights into the pathogenesis of cataract disorders.

As a highly potent reactive oxygen and nitrogen species, peroxynitrite (PON) has been indicated in the pathogenesis of various ocular disorders. The PON induces mobilization of intra cellular calcium which plays an important function in structure and activity of lens proteins. Moreover, the amount of calcium increases to the pathogenic level in the cataractous lenses. The aim of this study was to assess the impact of calcium ion on structure and aggregation of PON-modified lens crystallins, using spectroscopic techniques and gel mobility shift assay. The PON modification of lens proteins was confirmed with detection of the significantly increased quantity of carbonyl group, dityrosine, nitrotyrosine and nitrotryptophan. Moreover, the modified proteins indicated high levels of solvent exposed hydrophobic surfaces and markedly elevated proteolytic instability which can be explained with their structural alteration upon this type of modification. The results of UV-vis absorption studies suggest that PON-modified lens crystallins are highly sensitive to aggregation in the presence of both physiological and pathological ranges of calcium ion. Also, the results of thioflavin T fluorescence study indicated absence of any ordered aggregate entity in the calcium-induced aggregate samples. The results of gel mobility shift assay demonstrated the importance of calcium ion in the induction of disulfide and dityrosine covalent cross-linking and formation of the oligomeric structure with relatively larger sizes in the PON-modified crystallins compared to the non-modified protein counterparts. Overall, this study may suggest that a simultaneous raise of calcium ion and PON in the eye ball is an important risk factor for development of cataract diseases.

The impact of Hydrogen peroxide on structure, stability and functional properties of Human R12C mutant αA-crystallin: The imperative insights into pathomechanism of the associated congenital cataract incidence.

The oxidative stress in eye lens which occurs during inflammation and under chronic hyperglycemia has been already indicated in the pathogenesis of cataract disorders. The aim of this study was to examine structural and functional properties of R12C mutant αA-Crystallin (αA-Cry) in the presence of hydrogen peroxide. The study was done using different spectroscopic techniques and gel mobility shift assay. According to results of our study, H2O2 oxidation strongly compromises the chaperone function of the R12C mutant but not of wild-type αA-Cry. Also, it affects the structural properties of both wild-type and mutant proteins, albeit to different degree. The H2O2 exposure promotes extensive disulfide mediated oligomerization of the R12C mutant but not of the wild-type as revealed by gel mobility shift assay and dynamic light scattering. Moreover, in the presence of hydrogen peroxide, the mutant protein demonstrates severe conformational and protease instability and increased amyloidogenic propensity. The obtained results suggest that incubation of R12C mutant recombinant αA-Cry with hydrogen peroxide accelerates the molecular events which have been already implicated in the pathomechanism of cataract development. Taken together these results suggest that individuals carrying the R12C mutation are at an increased risk to develop early-onset cataract under condition of oxidative stress.

The binding assessment with human serum albumin of novel six-coordinate Pt(IV) complexes, containing bidentate nitrogen donor/methyl ligands.

The interactions between platinum complexes and human serum albumin (HSA) play crucial roles in the distribution, metabolism, and activity of platinum-based anticancer drugs. Octahedral platinum (IV) complexes represent a significant class of anticancer agents that display molecular pharmacological properties different from cisplatin. In this study, the interaction between two Pt(IV) complexes with the general formula [Pt(X)2Me2 (tbu2bpy)], where tbu2bpy = 4,4'-ditert-butyl-2,2'-bipyridine, with two leaving groups of X = Cl (Com1) or Br (Com2), and HSA were investigated, using Ultraviolet-Visible (UV-Vis) spectroscopy, fluorescence spectroscopy, circular dichroism (CD) and molecular docking simulation. The spectroscopic and thermodynamic data revealed that the HSA/Pt(IV) complexes interactions were spontaneous process and Com2 demonstrated stronger interaction and binding constant in comparison with Com1. Also, the results suggest approximately similar structural alteration of HSA in the presence of these Pt complexes. Molecular docking revealed that both Pt(IV) complexes bind with HSA in subdomain IB, literally the same as each other. This study suggests that variation in the leaving group, displaying differing departure rate, has no significant contribution in denaturing prosperities of the Pt(IV) complexes against HSA.