Analysis of UVA-Related Alterations upon Aging of Eye Lens Proteins by Mini Two-Dimensional Polyacrylamide Gel Electrophoresis

Weinreb, Orly; Rijk, F. Anke van; Steely, H. Thomas; Dovrat, A.; Bloemendal, H.

doi:10.1159/000055613

Cited by 13 publications

(7 citation statements)

References 37 publications

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“…An interaction of ␣-crystallin with ␤L-crystallin produced filament-like structures, and similar interactions between ␤L-crystallin with ␣A-crystallin (isolated from UV-A-irradiated lenses) showed even more pronounced filament formation (9). A similar study of interaction between ␣-crystallin and ␤L-crystallin at 60°C produced soluble complexes with mean radius of gyration ϳ14 nm, mean molecular mass of ϳ4 ϫ 10 6 Da, and maximum size of 40 nm (10).…”

mentioning

confidence: 78%

Identification of Interaction Sites between Human βA3- and αA/αB-crystallins by Mammalian Two-hybrid and Fluorescence Resonance Energy Transfer Acceptor Photobleaching Methods

Gupta

Srivastava

2009

Journal of Biological Chemistry

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Our recent study has shown that ␤A3-crystallin along with ␤B1-and ␤B2-crystallins were part of high molecular weight complex obtained from young, old, and cataractous lenses suggesting potential interactions between ␣-and ␤-crystallins (Srivastava, O. P., Srivastava, K., and Chaves, J. M. (2008) Mol. Vis. 14, 1872-1885). To investigate this further, this study was carried out to determine the interaction sites of ␤A3-crystallin with ␣A-and ␣B-crystallins. The study employed a mammalian two-hybrid method, an in vivo assay to determine the regions of ␤A3-crystallin that interact with ␣A-and ␣B-crystallins. Five regional truncated mutants of ␤A3-crystallin were generated using specific primers with deletions of N-terminal extension (NT) (named ␤A3-NT), N-terminal extension plus motif I (named ␤A3-NT ؉ I), N-terminal extension plus motifs I and II (named ␤A3-NT ؉ I ؉ II), motif III plus IV (named ␤A3-III ؉ IV), and motif IV (named ␤A3-IV). The mammalian two-hybrid studies were complemented with fluorescence resonance energy transfer acceptor photobleaching studies using the above described mutant proteins, fused with DsRed (Red) and AcGFP fluorescent proteins. The results showed that the motifs III and IV of ␤A3-crystallin were interactive with ␣A-crystallin, and motifs II and III of ␤A3-crystallin primarily interacted with ␣B-crystallin.The structural proteins (crystallins) of the vertebrate lens belong to two families, i.e. ␣-crystallin and ␤-␥ crystallins superfamily. Although ␣-crystallin is made of two primary gene products of ␣A and ␣B-crystallins, the ␤-␥ superfamily is constituted by four acidic (␤A1, ␤A2, ␤A3, and ␤A4) and three basic (␤B1, ␤B2, and ␤B3) ␤-crystallins and six ␥-crystallins (␥A, ␥B, ␥C, ␥D, ␥E, and ␥F) (1, 2). High concentrations of these crystallins and their interactions provide refractive power to the lens for focusing light on to the retina. Both ␣A-and ␣B-crystallins also function as molecular chaperons and prevent aberrant protein interactions and protein unfolding. The ␤-and ␥-crystallins have only structural properties (2-4), except that our results showed that ␤A3 crystallin contains proteinase activity (5, 6). The expressions of the crystallins are both developmentally and spatially regulated (1), and their interactions lead to the transparency of the lens because of short range order of the crystallin matrix (7,8).Previous reports have shown that the ␣-crystallin interacts with other crystallins and intermediate filaments (2). An interaction of ␣-crystallin with ␤L-crystallin produced filament-like structures, and similar interactions between ␤L-crystallin with ␣A-crystallin (isolated from UV-A-irradiated lenses) showed even more pronounced filament formation (9). A similar study of interaction between ␣-crystallin and ␤L-crystallin at 60°C produced soluble complexes with mean radius of gyration ϳ14 nm, mean molecular mass of ϳ4 ϫ 10 6 Da, and maximum size of 40 nm (10). Recently, we dissociated a fraction containing ␤A3-, ␤B1-, and ␤B2-crystallins from the ␣-crystallin fraction o...

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mentioning

confidence: 78%

Identification of Interaction Sites between Human βA3- and αA/αB-crystallins by Mammalian Two-hybrid and Fluorescence Resonance Energy Transfer Acceptor Photobleaching Methods

Gupta

Srivastava

2009

Journal of Biological Chemistry

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“…Mini‐one‐dimensional gel electrophoresis (13% SDS) was carried out under conditions described by Laemmli [19] for normal one‐dimensional electrophoresis. Mini‐two‐dimensional PAGE [20] was performed essentially under conditions as described previously for large gels [21]. Minor modifications were carried out as reported previously [22].…”

Section: Methodsmentioning

confidence: 99%

UV‐A‐related alterations of young and adult lens water‐insoluble α‐crystallin, plasma membranous and cytoskeletal proteins

Weinreb

Dovrat

Dunia

et al. 2001

European Journal of Biochemistry

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The damaging effects of UV-A irradiation on lens waterinsoluble a-crystallin, plasma membranous and cytoskeletal proteins derived from bovine lenses were studied. Young and adult bovine lenses were kept viable for 2 months in organ culture. After 24 h of incubation they were irradiated, and analyses of the proteins by one-dimensional and two-dimensional gel electrophoresis followed by Western blotting were carried out at several time intervals. RNA isolation, PCR and Northern blotting were also performed. We identified age-related changes in water-insoluble a-crystallin, the major membrane protein MP26 and the cytoskeletal proteins vimentin, phakinin and actin between control and UV-irradiated lenses. It appeared that adult lenses are more susceptible to UV light than young lenses, and protein modification occurred more frequently in adult lenses. UV-A irradiation affects not only the cytoskeletal structure, as deduced by the abnormal arrangement of actin in the fiber cells, but also leads to degradation of actin mRNA. Furthermore, analysis of the expression of hsp25 and hsp70 revealed some alteration in the protein pattern of adult lenses. We suggest that degradation of the cytoskeletal proteins following irradiation is due to, at least in part, the decreased protective ability of heat shock proteins upon aging.Keywords: a-crystallin; cytoskeletal proteins; lens aging; plasma membrane; UV-A irradiation.UV-A light (300±400 nm) penetrates the cornea and is maximally absorbed by the eye lens [1]. The human lens contains low molecular mass compounds which absorb maximally at < 365 nm [2] At this specific wavelength the lens is susceptible to irradiation. Experiments in organ culture of bovine lenses showed a relationship between UV-A radiation at 365 nm and subsequent lens opacity. It has been shown that epithelial enzymes of bovine lenses (ATPase, hexokinase, catalase and glucose-6-phosphate dehydrogenase), which are involved in metabolism and the defense mechanism against oxidation, are affected by UV-light [3,4]. Transglutaminase, which is a calciumdependent acyltransferase, may be involved in the mechanism by which UV-A causes damage to the eye lens [5]. The small heat shock protein aB-crystallin [6,7] and hsp25 [8] are substrates for the latter enzyme. We recently demonstrated that modifications, aggregation and reduced chaperone-like activity of water-soluble a-crystallin obtained from UV-A irradiated adult bovine lenses [9] affect lens transparency. This process may accumulate during aging.In this study we addressed the question of whether plasma membranous and cytoskeletal proteins from the water-insoluble fraction of young and adult bovine eye lenses are affected differently by UV-A radiation. The plasma membrane±cytoskeleton complex participates in the maintenance of lens shape, and both structures associate with lens crystallins [10]. We restricted our study to cytoskeletal proteins such as vimentin, phakinin (CP49), b-actin and the major lens membrane protein (MP26) which may be modified as a consequenc...

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“…The organ-culture system is used to investigate the effects of several insulting factors on the eye lens, such as hydrogen peroxide and drugs (17,18) ultraviolet-B (UV-B) and UV-A radiation (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32), hyperbaric oxygen (33,34), chemicals (35,36) and temperature stress (37,38). The system is also used to measure ocular irritancy of contact lens solutions (39) and oxidative damage (40).…”

Section: Discussionmentioning

confidence: 99%

Long-term Lens Organ Culture System with a Method for Monitoring Lens Optical Quality¶

Dovrat¹,

Sivak²

2005

Photochem Photobiol

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The Scan Tox System is a method for monitoring lens optical quality (focus or lack of focus) in culture conditions, which mimic conditions inside the eye. The ocular lens is an ideal organ for long-term culture experiments because it has no direct blood supply and no connection to the nervous system. The Scan Tox System makes it possible to keep lenses for long-term studies of up to a few weeks. The use of cultured lenses, mainly bovine, replaces the need for testing the effects of potentially damaging agents on live animals. This optical monitoring apparatus uses a computer-operated scanning laser beam, a video-camera system and a video frame analyzer to record the focal length and transmittance of the cultured lens. The scanner is designed to measure the focal length at points across the diameter of the lens. The lens container permits the lens to be exposed to a vertical laser beam from below. The laser source projects its light onto a plain mirror, which is mounted at 45 degrees C on a carriage assembly. The mirror reflects the laser beam directly up through the test lens. The mirror carriage is connected to a positioning motor, which moves the laser beam across the lens. The camera sees the cross section of the beams and, by examining the image at each position of the mirror, Scan Tox software is able to measure the quality of the lens by calculating the back vertex distance for each beam position. The cultured lenses continue to maintain their original refractive function. When foreign substances are introduced to a cultured lens, the Scan Tox System measures the resulting optical response. This provides a very sensitive means to follow early damage to the eye lens. Because the lens is maintained in an intact state in solutions that are similar to those inside the eye, the lens retains its normal recuperative powers. So in addition to measuring early damage, this system allows measurement of recovery from damage.

show abstract

Analysis of UVA-Related Alterations upon Aging of Eye Lens Proteins by Mini Two-Dimensional Polyacrylamide Gel Electrophoresis

Cited by 13 publications

References 37 publications

Identification of Interaction Sites between Human βA3- and αA/αB-crystallins by Mammalian Two-hybrid and Fluorescence Resonance Energy Transfer Acceptor Photobleaching Methods

Identification of Interaction Sites between Human βA3- and αA/αB-crystallins by Mammalian Two-hybrid and Fluorescence Resonance Energy Transfer Acceptor Photobleaching Methods

UV‐A‐related alterations of young and adult lens water‐insoluble α‐crystallin, plasma membranous and cytoskeletal proteins

Long-term Lens Organ Culture System with a Method for Monitoring Lens Optical Quality¶

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