Interaction of C-Terminal Truncated Human αA-Crystallins with Target Proteins

Significant portion of alphaA-crystallin in human lenses exists as C-terminal residues cleaved at residues 172, 168, and 162. Chaperone activity, determined with alcohol dehydrogenase (ADH) and betaL-crystallin as target proteins, was increased in alphaA(1-172) and decreased in alphaA(1-168) and alphaA(1-162). The purpose of this study was to show whether the absence of the C-terminal residues influences protein-protein interactions with target proteins.Our hypothesis is that the chaperone-target protein binding kinetics, otherwise termed subunit exchange rates, are expected to reflect the changes in chaperone activity. To study this, we have relied on fluorescence resonance energy transfer (FRET) utilizing amine specific and cysteine specific fluorescent probes. The subunit exchange rate (k) for ADH and alphaA(1-172) was nearly the same as that of ADH and alphaA-wt, alphaA(1-168) had lower and alphaA(1-162) had the lowest k values. When betaL-crystallin was used as the target protein, alphaA(1-172) had slightly higher k value than alphaA-wt and alphaA(1-168) and alphaA(1-162) had lower k values. As expected from earlier studies, the chaperone activity of alphaA(1-172) was slightly better than that of alphaA-wt, the chaperone activity of alphaA(1-168) was similar to that of alphaA-wt and alphaA(1-162) had substantially decreased chaperone activity.Cleavage of eleven C-terminal residues including Arg-163 and the C-terminal flexible arm significantly affects the interaction with target proteins. The predominantly hydrophilic flexible arm appears to be needed to keep the chaperone-target protein complex soluble

Multiple Aggregates and Aggresomes of C-Terminal Truncated Human αA-Crystallins in Mammalian Cells and Protection by αB-Crystallin

Cleavage of 11 (αA162), 5 (αA168) and 1 (αA172) residues from the C-terminus of αA-crystallin creates structurally and functionally different proteins. The formation of these post-translationally modified αA-crystallins is enhanced in diabetes. In the present study, the fate of the truncated αA-crystallins expressed in living mammalian cells in the presence and absence of native αA- or αB-crystallin has been studied by laser scanning confocal microscopy (LSM).YFP tagged αAwt, αA162, αA168 and αA172, were individually transfected or co-transfected with CFP tagged αAwt or αBwt, expressed in HeLa cells and studied by LSM. Difference in protein aggregation was not caused by different level of α-crystallin expression because Western blotting results showed nearly same level of expression of the various α-crystallins. The FRET-acceptor photo-bleaching protocol was followed to study in situ protein-protein interaction. αA172 interacted with αAwt and αBwt better than αA168 and αA162, interaction of αBwt being two-fold stronger than that of αAwt. Furthermore, aggresomes were detected in cells individually expressing αA162 and αA168 constructs and co-expression with αBwt significantly sequestered the aggresomes. There was no sequestration of aggresomes with αAwt co-expression with the truncated constructs, αA162 and αA168. Double immunocytochemistry technique was used for co-localization of γ-tubulin with αA-crystallin to demonstrate the perinuclear aggregates were aggresomes.αA172 showed the strongest interaction with both αAwt and αBwt. Native αB-crystallin provided protection to partially unfolded truncated αA-crystallins whereas native αA-crystallin did not. Aggresomes were detected in cells expressing αA162 and αA168 and αBwt co-expression with these constructs diminished the aggresome formation. Co-localization of γ-tubulin in perinuclear aggregates validates for aggresomes

Protective Role of C-Phycocyanin Against Secondary Changes During Sodium Selenite Mediated Cataractogenesis

Age related cataract is the leading cause of blindness associated with accumulation of oxidative stress in the eye lens. The present investigation reveals the rational of the beneficial effects of the natural compound C-phycocyanin (C-PC) is beneficial when administered to rat pups to protect against the secondary effects of sodium selenite induced cataractogenesis. A single subcutaneous dose of sodium selenite (19 μmol/kg body weight) on the 10th day of postpartum is adequate to induce cataract in rat pups. Serum biochemical parameters, such as the level of electrolytes, mean activities of anti-oxidant enzymes i.e. superoxide dismutase, catalase and reduced glutathione were observed to be significantly altered during selenite induced cataractogenic process. Histopathological examination revealed signs of degradation of normal cell architecture in the liver, kidney and eye lens. Interestingly, the deleterious effects of sodium selenite toxicity were restored with the simultaneous treatment with C-PC. The results suggest that an administration of 200 mg/kg body weight of C-PC has the ability to prevent/alter the secondary changes reflected in the serum biochemical and histological modifications in rats exposed to sodium selenite. These results complement the beneficial role of C-PC of cyanobacterial origin as a efficacious anti-cataractogenic agent against sodium selenite toxicity

Time –dependent spectral changes in the emission spectra of the SITS-labeled αA- crystallins interacting with LYI- labeled βL- crystallin.

<p>The emission spectra of fluorescence- labeled αA crystallin excited at 336 nm were recorded at every 2 min intervals at 0, 2,4,6,8,10,12,14,16,18,20,22,24,26,28 and 30 minutes after mixing of SITS – labeled αA - wt and C-terminal truncated αA-crystallins with LYI – labeled βL- crystallin in a 1∶1 ratio, with a final protein concentration of 1 mg/ml at 62°C. The decrease in fluorescence intensity at 426 nm of SITS-labeled αA- crystallins and concomitant increase in fluorescence intensity at 515 nm of LYI- labeled ADH is indicative of energy transfer due to exchange of subunits between the two labeled proteins.</p

Subunit exchange rate constant (k) of αA-wt and its C-terminal truncated forms interacting with βL-crystallin when increase and decrease respectively (Mean±SE).

<p>Subunit exchange rate constant (k) of αA-wt and its C-terminal truncated forms interacting with βL-crystallin when increase and decrease respectively (Mean±SE).</p

Time –dependent spectral changes in the emission spectra of SITS-labeled αA- crystallins interacting with LYI- labeled ADH.

<p>The emission spectra of fluorescence- labeled αA crystallin excited at 336 nm were recorded at every 2 min intervals at 0, 2,4,6,8,10,12,14,16,18,20,22,24,26,28 and 30 minutes after mixing of SITS – labeled αA - wt and its C-terminal truncated αA_1–172, αA_1–168, and αA_1–162 with LYI – labeled ADH in 1∶1 ratio, with a final protein concentration of 1 mg/ml at 37°C. The decrease in fluorescence intensity at 426 nm of SITS- labeled αA- crystallins and concomitant increase in fluorescence intensity at 515 nm of LYI- labeled ADH is indicative of energy transfer.</p

Effect of C-terminal truncation of αA-crystallin on its chaperone activity as measured with ADH as the target protein.

<p>The chaperone activity of αA-wt (▪) and C-terminal truncated αA_1–172,(▴) αA_1–168 (•) and αA_1–162 (−) ADH alone (♦) determined with ADH as the target protein at 1∶1 ratio (0.14 mg/ml) denatured with EDTA at 37°C.</p

Subunit exchange rate constant (k) of αA-wt and its C-terminal truncated forms interacting with ADH when increase and decrease respectively (Mean±SE).

<p>Subunit exchange rate constant (k) of αA-wt and its C-terminal truncated forms interacting with ADH when increase and decrease respectively (Mean±SE).</p