April 2009
Volume 50, Issue 13
ARVO Annual Meeting Abstract  |   April 2009
The Chaperone and Anti-Apoptosis Are Intimately Related Functions in Human A-Crystallin
Author Affiliations & Notes
  • N. Pasupuleti
    Ophthalmology and Visual Sciences,
    Case Western Reserve University, Cleveland, Ohio
  • S. Matsuyama
    Comprehensive Cancer Center,
    Case Western Reserve University, Cleveland, Ohio
  • A. Doseff
    Department of Molecular Genetics, The Ohio State University, Columbus, OH, Columbus, Ohio
  • R. Nagaraj
    Ophthalmology and Visual Sciences,
    Case Western Reserve University, Cleveland, Ohio
  • Footnotes
    Commercial Relationships  N. Pasupuleti, None; S. Matsuyama, None; A. Doseff, None; R. Nagaraj, None.
  • Footnotes
    Support  R01EY-09912 and R01EY-016219, P30EY-11373, RPB and OLERF (RHN) NSF-MCB0542244 and RO1 HL075040 (AID) RO1AG031903 (SM)
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 2121. doi:
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      N. Pasupuleti, S. Matsuyama, A. Doseff, R. Nagaraj; The Chaperone and Anti-Apoptosis Are Intimately Related Functions in Human A-Crystallin. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2121.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Alpha-crystallin is a major lens protein of the small heat shock protein family. It exhibits chaperone and anti-apoptotic functions. A previous study from our laboratory demonstrated that an R21A mutant of human A-crystallin is a better chaperone and an R49A mutant is a weaker chaperone relative to the wild type (Wt) protein (Biswas et al., Biochemistry, 45: 4569-4577, 2006). In this study, we have investigated whether the chaperone function of A-crystallin is related to its anti-apoptotic function.

Methods: : We generated CHO cell lines stably expressing Wt, R21A or R49A mutant proteins. Apoptosis was induced by staurosporine, etoposide or doxorubicin. GFP-human Bax, human Bim and human procaspase-3 were overexpressed in the cell lines. Apoptosis was measured by Hoechst staining. Caspase activities were measured spectrophotometrically using specific peptide substrates.

Results: : R21A cells showed better protection against apoptosis than Wt cells. R49A cells showed reduced protection against the apoptotic agents. Transient transfection experiments showed that GFP-Bax translocated to the mitochondria at a higher rate in R49A cells when compared to Wt cells, but in R21A cells GFP-Bax did not translocate to mitochondria. R21A cells showed better protection against apoptosis from Bim overexpression when compared to Wt or R49A cells. Staurosporine treatment increased Akt phosphorylation in R21A cells compared to Wt cells, but it was absent in R49A cells. When compared to Wt cells, R49A cells showed higher activities of caspase-3 and -9, but those activities were lower in R21A cells. Procaspase-3 overexpression followed by treatment with doxorubicin resulted in induction of apoptosis in Wt cells, which was significantly inhibited by R21A, but not by R49A.

Conclusions: : Our data show that the chaperone and anti-apoptotic functions are intimately related functions of human A-crystallin and suggests that in the human lens these two properties are likely responsible for prevention of protein aggregation and apoptosis of epithelial cells that help maintain lens transparency during aging.

Keywords: apoptosis/cell death • crystallins • aging 

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