RESUMO
α-Crystallin maintains the transparency of the lens by preventing the aggregation of damaged proteins. The aim of our work was to study the chaperone-like activity of native α-crystallin in near physiological conditions (temperature, ionic power, pH) using UV-damaged ßL-crystallin as the target protein. α-Crystallin in concentration depended manner inhibits the aggregation of UV-damaged ßL-crystallin. DSC investigation has shown that refolding of denatured UV-damaged ßL-crystallin was not observed under incubation with α-crystallin. α-Crystallin and UV-damaged ßL-crystallin form dynamic complexes with masses from 75 to several thousand kDa. The content of UV-damaged ßL-crystallin in such complexes increases with the mass of the complex. Complexes containing >10% of UV-damaged ßL-crystallin are prone to precipitation whereas those containing <10% of the target protein are relatively stable. Formation of a stable 75â¯kDa complex is indicative of α-crystallin dissociation. We suppose that α-crystallin dissociation is the result of an interaction of comparable amounts of the chaperone-like protein and the target protein. In the lens simultaneous damage of such amounts of protein, mainly ß and gamma-crystallins, is impossible. The authors suggest that in the lens rare molecules of the damaged protein interact with undissociated oligomers of α-crystallin, and thus preventing aggregation.
Assuntos
Cristalino/metabolismo , alfa-Cristalinas/metabolismo , beta-Cristalinas/metabolismo , Chaperonas Moleculares/metabolismo , Agregados Proteicos/fisiologia , Temperatura , Raios UltravioletaRESUMO
The effect of a mixture of N-acetylcarnosine and D-pantethine (1 : 1, m/m) on UV-A induced cataract in rats was studied. It is shown that instillation of a 5% mixture into the eyes or intraperitoneal injections (25 or 150 mg/kg) inhibit the formation of cataracts, starting from 82nd day of the experiment (p < 0.03), after which the protective effect of the mixture significantly increases (p = 0.0003). UV-A irradiation significantly (p < 0.01) increased the content of water-insoluble proteins in the lens. The use of the mixture of N-Acetylcarnosine and D-pantethine prevented (p < 0.001) an increase in the content of water-insoluble proteins caused by UV-A irradiation. Gel permeation chromatography data showed that, in the control group, water insoluble proteins consist of 3 fractions (40 kDa, 100 - 200 kDa, and1000 kDa). UV-A irradiation reduced the amount of protein in fraction 1 and increases the amount of protein in the fractions 2 and 3. The use of the mixture of N-acetylcarnosine and D-pantethine reduced the effects of UV-A light. The authors attribute the effect of the N-acetylcarnosine and D-pantethine mixture to their chaperone-like properties.
Assuntos
Carnosina/análogos & derivados , Catarata/tratamento farmacológico , Cristalino/efeitos dos fármacos , Panteteína/análogos & derivados , Substâncias Protetoras/farmacologia , Agregados Proteicos/efeitos dos fármacos , Lesões Experimentais por Radiação/tratamento farmacológico , Animais , Carnosina/farmacologia , Catarata/etiologia , Catarata/patologia , Cromatografia em Gel , Combinação de Medicamentos , Proteínas do Olho/química , Injeções Intraperitoneais , Cristalino/química , Cristalino/patologia , Cristalino/efeitos da radiação , Masculino , Soluções Oftálmicas , Panteteína/farmacologia , Agregados Proteicos/efeitos da radiação , Lesões Experimentais por Radiação/etiologia , Lesões Experimentais por Radiação/patologia , Ratos , Ratos Wistar , Solubilidade , Raios Ultravioleta/efeitos adversosRESUMO
A comparative experimental study of biomicroscopic appearance of lenses in cataracts of different genesis (age-related, ultraviolet and other radiation-induced or combined) has been performed on animals (mice). It is shown that identical lens opacification can be provoked by aging (endogenous factor), as well as ultraviolet and other radiation exposure (exogenous physical factors). The only differential sign is the severity of the damage. These factors can be arranged in the following ascending order by their damaging ability: aging --> ultraviolet --> gamma rays --> gamma rays + ultraviolet. Anti-cataract effect of a chaperone-like combined drug (N-acetylcarnosin and D-pantetin) has been studied in vivo on a "prolonged" model of induced cataract in rats. The use of the combined drug (1:1 mixture of the two peptides) in the form of ocular instillations and intraperitoneal injections helped slowing the progression of the ultraviolet-induced cataract in vivo.
Assuntos
Catarata/etiologia , Raios gama/efeitos adversos , Cristalino , Chaperonas Moleculares/toxicidade , Raios Ultravioleta/efeitos adversos , Animais , Catarata/patologia , Modelos Animais de Doenças , Progressão da Doença , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Cristalino/efeitos dos fármacos , Cristalino/patologia , Cristalino/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Acústica , Fatores de RiscoRESUMO
Microscopic lens changes are studied in mice (F1C57B1XCBA) depending on age and after ultraviolet (UV), gamma-irradiation and their combination. In all animals compared to young 3-months animals nonspecific changes due to aging were revealed: microvacuoles in cytoplasm of fiber cells, flattening of epithelial cells and nuclei fragmentation, swelling and confluence of cortical layer cells in anterior and posterior subcapsular zones. In epithelial cells the following changes were noted: nuclei polymorphism, cavities in a cell layer formation of multilayered structures of fibroblast-like cells and cell desquamation. Degree of nuclei vacuolization was significantly lower in groups with gamma- and gamma+UV-irradiation but higher in a group of UV exposure. The number of cells per unit area was significantly decreased in a group of gamma-irradiation after 7 and 10 months of exposure. Aging and UV exposure caused decrease of nuclei area, whereas affect of gamma-irradiation depended on proliferative capacity of cells: size of central cells increased and of peripheral cells reduced. Specific changes were not revealed for any of the factors.
Assuntos
Envelhecimento , Catarata , Raios gama/efeitos adversos , Cristalino , Raios Ultravioleta/efeitos adversos , Envelhecimento/patologia , Animais , Catarata/etiologia , Catarata/patologia , Modelos Animais de Doenças , Relação Dose-Resposta à Radiação , Cristalino/patologia , Cristalino/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Microscopia/métodos , Índice de Gravidade de DoençaRESUMO
Comparative study of lens biomicroscopy in cataract of different etiology (senile, ultraviolet, radioactive and combined ultraviolet- radioactive exposure) is performed in experiment on animals (mice). Lens opacification pattern was showed to be similar in aging as an endogenous factor and ultraviolet (UV) and radioactive exposure as exogenous physical factors. Specificity of these factors is expressed in degree of damage only. Depending on damaging potential the factors may be arranged in a following way (in increasing manner): aging, ultraviolet irradiation, gamma-irradiation, gamma+UV-irradiation.
Assuntos
Envelhecimento/fisiologia , Catarata , Raios gama/efeitos adversos , Cristalino , Raios Ultravioleta/efeitos adversos , Animais , Catarata/diagnóstico , Catarata/etiologia , Catarata/fisiopatologia , Técnicas de Diagnóstico Oftalmológico , Modelos Animais de Doenças , Relação Dose-Resposta à Radiação , Humanos , Cristalino/fisiopatologia , Cristalino/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Microscopia/métodos , Fatores de Risco , Índice de Gravidade de DoençaRESUMO
Using differential electrophoresis protein composition of lens major proteins in hybrid mice F1 (C57B1XCBA) with cataracts of different etiology (senile, ultraviolet, radioactive and combined ultraviolet-radioactive exposure) was studied Changes that may be specific for cataract caused by aging, ultraviolet and/or gamma-irradiation were not revealed in water-soluble and water-insoluble protein fractions.
Assuntos
Envelhecimento/metabolismo , Catarata , Cristalinas , Raios gama/efeitos adversos , Cristalino , Solubilidade/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Animais , Catarata/etiologia , Catarata/metabolismo , Cristalinas/metabolismo , Cristalinas/efeitos da radiação , Modelos Animais de Doenças , Relação Dose-Resposta à Radiação , Eletroforese/métodos , Cristalino/metabolismo , Cristalino/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Índice de Gravidade de DoençaRESUMO
Four randomized groups of male mice F1 (C57Black/CBA) were investigated: a) UV-irraidated (UV-A, 15 min daily during 9 months, dose 5.6 + 1.2 W/m2), b) gamma-rays irradiated (2 Gy), single, c) influence for combination of UV- and gamma-rays treatment, d) aging. The lens opacities were measured on 7th and 10th month. The expert method based on six grade scale was used for cataract measure. On 7th month the median of lens opacities were Aging group = 0; UV-irradiated group = 2.5; gamma-irradiated group = 4.75; gamma- and UV-irradiated group = 6.0. The difference between all groups was significant (p < 0.004, Kruskall-Wallis ANOVA test). The Conover post hock test has shown the significant difference for all comparison pairs (p < 0.002) with the exception of UV-irradiated group, gamma-irradiated group, gamma- and UV-irradiated group. On 10th month the lens opacities were strongly increased: Aging group = 2.5; UV-irradiated group = 5.0; gamma-irradiated group = 6.5; gamma- and UV-irradiated group = 7.5 (median). The difference between groups was significant (p < 0.0001, Kruskall-Wallis ANOVA test). The Conover post hock test has shown the significant difference for all comparison pairs (p <0.003) with the exception ofthe UV-irradiated group and gamma-irradiated group. The formation of the specific lens opacities for any group was not found. Morphology and protein composition have been investigated on 10th month. The results of morphological changes study show destructive and degenerative impairments of capsule, epithelium cells and lens fibers. However, no specific changes related to some particular actions have been found. In addition, there were no specific changes of protein composition of both water-soluble and water-insoluble fractions estimated with Differential Gel Electrophoresis technique. Obtained data mean that aging, UV-treatment, and gamma-radiation causes the similar changes of lens. It was supposed that UV-treatment or/and gamma-radiation act as an aging factor on the lens.
Assuntos
Envelhecimento , Catarata/etiologia , Raios gama/efeitos adversos , Cristalino/patologia , Cristalino/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Animais , Catarata/patologia , Quimera , Eletroforese , Cristalino/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Proteínas/análise , Irradiação Corporal TotalRESUMO
The structure of the oligomeric protein α-crystallin from bovine eye lens was investigated by small-angle neutron scattering (SANS) with contrast variation. Based on the SANS curves, the match point for α-crystallin (43% D2O) and its average scattering length density at this point (2.4·10(10) cm(-2)) were evaluated. The radius of gyration and the distance distribution functions for α-crystallin were calculated. On the basis of these calculations, it was concluded that α-crystallin is characterized by homogeneous distribution of scattering density in the domains inaccessible for water penetration, and all polypeptide subunits in α-crystallin oligomers undergo equal deuteration. The latter indicates that all α-crystallin subunits are equally accessible for water and presumably for some other low molecular weight substances. These conclusions on the α-crystallin structure (homogeneous distribution of scattering density and equal accessibility of all subunits for low molecular weight substances) should be taken into account when elaborating α-crystallin quaternary structure models.
Assuntos
alfa-Cristalinas/química , Algoritmos , Animais , Bovinos , Medição da Troca de Deutério , Difração de Nêutrons , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Propriedades de SuperfícieRESUMO
The damaging effect of UV radiation (lambda > 260 nm) on bovine alpha-crystallin in solution was studied by small-angle X-ray scattering, gel permeation chromatography, electrophoresis, absorption and fluorescence spectroscopy, and differential scanning calorimetry. The results obtained show that damage to even a large number of subunits within an alpha-crystallin oligomer does not cause significant rearrangement of its quaternary structure, aggregation of oligomers, or the loss of their solubility. Due to the high resistance of its quaternary structure, alpha-crystallin is able to prevent aggregation of destabilized proteins (especially of gamma- and beta-crystallins) and so to maintain lens transparency throughout the life of an animal (the chaperone-like function of alpha-crystallin).
Assuntos
Estrutura Quaternária de Proteína/efeitos da radiação , Raios Ultravioleta/efeitos adversos , alfa-Cristalinas/química , Animais , Varredura Diferencial de Calorimetria , Bovinos , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Córtex do Cristalino/química , Desnaturação Proteica , Espalhamento a Baixo Ângulo , Espectrometria de Fluorescência , Espectrofotometria , alfa-Cristalinas/isolamento & purificação , alfa-Cristalinas/efeitos da radiaçãoAssuntos
Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/química , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Multimerização Proteica , Estrutura Quaternária de Proteína , Temperatura , alfa-Cristalinas/química , alfa-Cristalinas/metabolismo , Animais , Bovinos , Músculo Esquelético/enzimologia , Ligação Proteica , CoelhosRESUMO
Aggregation ofcrystallins, the lens proteins, is one of the basic stages of cataract formation. Among the protein aggregation models used to study the molecular mechanisms of the initial stages of lenticular opacity, UV-induced aggregation of betaL-crystallin is most close to the in vivo conditions. The carnosine derivative N-acetyl carnosine has been shown to be effective in inhibiting the UV-induced aggregation of betaL-crystallin. Examination of the accumulation kinetics of carbonyl groups in betaL-crystallin under UV irradiation has indicated that neither carnosine nor N-acetyl carnosine fails to affect this parameter--an indicator of oxidative protein damage. By taking into account also the fact that N-acetyl carnosine is not an antioxidant, it can be believed that the molecular mechanism of action of this compound on UV-induced aggregation of betaL is unassociated with its antioxidative properties. The authors hypothesize that the molecular chaperon-like properties similar to those of alpha-crystallin underlie the mechanism of action of the acetyl derivative carnosine. The prospects for searching anticataract agents of a new chaperon-like class are discussed.
Assuntos
Carnosina/análogos & derivados , Catarata/tratamento farmacológico , Cristalino/metabolismo , Chaperonas Moleculares/uso terapêutico , beta-Cristalinas/metabolismo , Animais , Carnosina/uso terapêutico , Catarata/etiologia , Catarata/metabolismo , Bovinos , Modelos Animais de Doenças , Progressão da Doença , Cristalino/efeitos dos fármacos , Cristalino/efeitos da radiação , Masculino , Raios Ultravioleta/efeitos adversos , beta-Cristalinas/efeitos dos fármacos , beta-Cristalinas/efeitos da radiaçãoRESUMO
There is a potential of therapeutic action on certain stages of caractogenesis, in particular on the aggregation of water-soluble proteins of cytoplasmic lens fiber cells, giving rise to insoluble protein complexes. The effect of a combined preparation (N-acetyl carnosine and D-patethine), acting by the chaperon-like mechanism, was studied in vivo on a prolonged rat model of UV-induced cataract. The use of the combined preparation consisting of a mixture of peptides of N-acetyl carnosine and D-patethine in a ratio of 1:1 as ocular instillations and intraperitoneal injections could slow down the development of UV-induced cataract in vivo. Pathomorphological studies suggest that the combined preparation has a protective effect on lens tissue when the rat model of UV-induced cataract is employed.
Assuntos
Carnosina/análogos & derivados , Catarata/tratamento farmacológico , Catarata/etiologia , Cristalinas/metabolismo , Cristalino/metabolismo , Chaperonas Moleculares/uso terapêutico , Animais , Carnosina/uso terapêutico , Catarata/metabolismo , Cristalinas/efeitos dos fármacos , Cristalinas/efeitos da radiação , Modelos Animais de Doenças , Progressão da Doença , Seguimentos , Cristalino/patologia , Cristalino/efeitos da radiação , Masculino , Oligopeptídeos/uso terapêutico , Prognóstico , Ratos , Ratos Wistar , Índice de Gravidade de Doença , Fatores de Tempo , Raios Ultravioleta/efeitos adversosRESUMO
To study the mechanisms of action of new-generation anticataract drugs, it is necessary to have an accessible and adequate model of age-related cataract. A model of UV-induced cataract is pathogenetically closest to that of age-related cataract. A prolonged rat model of UV-induced cataract developing within 10 months is proposed; the clinical features of UV-induced cataract have been established at different stages of its development. A moderate homogeneous cloud-like lenticular opacity was observed at the end of the experiment; a less pronounced homogeneous opacity was seen in the anterior and posterior cortical layers. Cataract development was assessed by the appraisal method using the developed rat lenticular transparency scale, as well as by microdensitometry of biomicroscopic lenticular optical sections. Within the proposed model, the pathomorphological lenticular changes are largely similar to the histological pattern of age-related cataract.
Assuntos
Catarata/tratamento farmacológico , Catarata/etiologia , Cristalinas/metabolismo , Cristalino/metabolismo , Chaperonas Moleculares/uso terapêutico , Animais , Catarata/metabolismo , Cristalinas/efeitos dos fármacos , Cristalinas/efeitos da radiação , Modelos Animais de Doenças , Progressão da Doença , Seguimentos , Cristalino/patologia , Cristalino/efeitos da radiação , Masculino , Prognóstico , Ratos , Ratos Wistar , Índice de Gravidade de Doença , Fatores de Tempo , Raios Ultravioleta/efeitos adversosRESUMO
Thermal aggregation of rabbit skeletal muscle glycogen phosphorylase b (Phb) has been investigated using dynamic light scattering under conditions of a constant rate of temperature increase (1 K/min). The linear behavior of the dependence of the hydrodynamic radius on temperature for Phb aggregation is consistent with the idea that thermal aggregation of proteins proceeds in the kinetic regime wherein the rate of aggregation is limited by diffusion of the interacting particles (the regime of "diffusion-limited cluster-cluster aggregation"). In the presence of alpha-crystallin, a protein exhibiting chaperone-like activity, the dependence of the hydrodynamic radius on temperature follows the exponential law; this suggests that the aggregation process proceeds in the kinetic regime where the sticking probability for colliding particles becomes lower than unity (the regime of "reaction-limited cluster-cluster aggregation"). Based on analysis of the ratio between the light scattering intensity and the hydrodynamic radius of Phb aggregates, it has been concluded that the addition of alpha-crystallin results in formation of smaller size starting aggregates. The data on differential scanning calorimetry indicate that alpha-crystallin interacts with the intermediates of the unfolding process of the Phb molecule. The proposed scheme of thermal denaturation and aggregation of Phb includes the stage of reversible dissociation of dimers of Phb into monomers, the stage of the formation of the starting aggregates from the denatured monomers of Phb, and the stage of the sticking of the starting aggregates and higher order aggregates. Dissociation of Phb dimer into monomers at elevated temperatures has been confirmed by analytical ultracentrifugation.
Assuntos
Glicogênio Fosforilase/química , Músculo Esquelético/enzimologia , Fosforilase b/química , alfa-Cristalinas/farmacologia , Algoritmos , Animais , Varredura Diferencial de Calorimetria , Bovinos , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Modelos Químicos , Conformação Proteica/efeitos dos fármacos , Desnaturação Proteica/efeitos dos fármacos , Coelhos , alfa-Cristalinas/químicaRESUMO
Studies of molecular mechanisms of chaperone-like activity of alpha-crystallin became an active field of research over last years. However, fine interactions between alpha-crystallin and the damaged protein and their complex organization remain largely uncovered. Complexation between alpha- and betaL-crystallins was studied with thermal denaturation of betaL-crystallin at 60 degrees C using small-angle X-ray scattering (SAXS), light scattering, gel-permeation chromatography and electrophoresis. A mixed solution of alpha- and betaL-crystallins in concentrations about 10 mg/ml incubated at 60 degrees C was found to contain their soluble complexes with mean radius of gyration approximately 14 nm, mean molecular weight approximately 4000 kDA and maximal size approximately 40 nm. In pure betaL-crystallin solution, complexes were not observed at 60 degrees C. In SAXS studies, transitions in the alpha-crystallin quaternary structure at 60 degrees C were shown to occur and result in a double increase of the molecular weight. It suggests that during the temperature-induced denaturation of betaL-crystallin it binds with modified alpha-crystallin or, alternatively, alpha-betaL-crystallin complexation and alpha-crystallin modifications are concurrent. Estimates of the alpha-betaL-crystallin dimensions and relative contents of alpha- and betaL-crystallins in the complex suggest that several alpha-crystallin molecules are involved in complex formation.
Assuntos
Cristalinas/química , Temperatura Alta , Espalhamento de Radiação , SoluçõesRESUMO
The supramolecular structure of crystallins in intact ocular lenses of carp, frog and rat as well as in the interior (nuclear) and outer (cortical) parts of these lenses was studied by the small-angle X-ray scattering method. The results show that the supramolecular structure of crystallins substantially varies both in lenses of different vertebrate species and in various parts of the same lens. In carp lens and in the cortical part of rat lens, crystallins have an ordered supramolecular structure, as indicated by a small-angle X-ray diffraction maximum in the region of Bragg distances 15-20 nm, whereas in frog lens and in the nuclear part of rat lens, the supramolecular structure of these proteins is disordered. The power-law X-ray scattering by rat lens nucleus may be evidence of fractal structures in the lens. A comparison of these results with literary data indicates that there is no obvious correlation between the type of supramolecular structure of crystallins and their polypeptide composition in lenses of different vertebrate species. The results suggest that the supramolecular ordering (short-range order) of crystallins is not a necessary condition for lens transparency.