The anti-apoptotic function of human αA-crystallin is directly related to its chaperone activity

N Pasupuleti,S Matsuyama,O Voss,K Song,D Danielpour,R H Nagaraj,A I Doseff

doi:10.1038/cddis.2010.3

N Pasupuleti, S Matsuyama + Show 5 more

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https://doi.org/10.1038/cddis.2010.3

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Abstract

αA-crystallin is a molecular chaperone and an antiapoptotic protein. This study investigated the mechanism of inhibition of apoptosis by human αA-crystallin and determined if the chaperone activity of αA-crystallin is required for the antiapoptotic function. αA-crystallin inhibited chemical-induced apoptosis in Chinese hamster ovary (CHO) cells and HeLa cells by inhibiting activation of caspase-3 and -9. In CHO cells, it inhibited apoptosis induced by the overexpression of human proapoptotic proteins, Bim and Bax. αA-crystallin inhibited doxorubicin-mediated activation of human procaspase-3 in CHO cells and it activated the PI3K/Akt cell survival pathway by promoting the phosphorylation of PDK1, Akt and phosphatase tensin homologue in HeLa cells. The phosphoinositide 3 kinase (PI3K) activity was increased by αA-crystallin overexpression but the protein content was unaltered. Downregulation of PI3K by the expression of a dominant-negative mutant or inhibition by LY294002 abrogated the ability of αA-crystallin to phosphorylate Akt. These antiapoptotic functions of αA-crystallin were enhanced in a mutant protein (R21A) that shows increased chaperone activity than the wild-type (Wt) protein. Interestingly, a mutant protein (R49A) that shows decreased chaperone activity was far weaker than the Wt protein in its antiapoptotic functions. Together, our study results show that αA-crystallin inhibits apoptosis by enhancing PI3K activity and inactivating phosphatase tensin homologue and that the antiapoptotic function is directly related to its chaperone activity.

Highlights

A-Crystallin is a major structural protein of the lens; it accounts for nearly 40% of the total protein in the lens and is required for maintenance of lens transparency.[1]
Our data suggest that the two cell types expressed similar levels of each aA-crystallin variant
Similar results were obtained in transiently transfected HeLa cells; R21A was better and R49A was worse than Wt in preventing apoptosis induced by either staurosporine (Figure 2c) or etoposide (Figure 2d). These results indicate that R21A has enhanced and R49A has reduced antiapoptotic activity, relative to Wt protein, and that the gain or loss of antiapoptotic function coincides with the chaperone function of aA-crystallin

Summary

Introduction

A-Crystallin is a major structural protein of the lens; it accounts for nearly 40% of the total protein in the lens and is required for maintenance of lens transparency.[1]. It has been reported that cells overexpressing aA- or aB-crystallin are more resistant to thermal,[5] osmotic[6] and oxidative stress.[7] In addition, recent studies have shown that a-crystallin prevents apoptosis induced by a variety of agents, including staurosporine,[8,9] tumor necrosis factor-a,10 UVA light,[8] hydrogen peroxide[11] and etoposide.[9]. The robust effects of a-crystallin against these apoptosis inducers prompted many researchers to investigate molecular mechanisms by which a-crystallin functions as an antiapoptotic protein. Induced apoptosis in lens epithelial cells is inhibited by aA- and aB-crystallins through regulation of PKC-a, RAF/MEK/ERK and Akt signaling pathways,[17] suggesting additional mechanisms of apoptosis inhibition. Whether this property applies for aA-crystallin is not known

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