Characterization of the α-γ and α-β Complex: Evidence for an In Vivo Functional Role of α-Crystallin as a Molecular Chaperone

Boyle, Daniel; Takemoto, L.

doi:10.1006/exer.1994.1190

Cited by 73 publications

(40 citation statements)

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“…[39][40][41][42][43][44][45][46][62][63][64][65][66][67][68][69][70][71][72][73][74] One of the most important functions is the ability to protect cell from induced apoptosis. 39-46 a-Crystallin and Hsp27 are closely related family members and protect cells from apoptosis induced by a large numbers of stress factors.…”

Section: Antiapoptotic Mechanisms Of A-crystallinsmentioning

confidence: 99%

“…[19][20]61 aA/B-crystallins are initially known as major lens structural proteins that play an essential role in maintaining the transparency of the ocular lens. 62 Later, a-crystallins are found to act as molecular chaperones [63][64][65][66][67] and also display autokinase activity. 68,69 As antiapoptotic regulators, acrystallins are initially shown to protect cells from thermal, 70 osmotic 71 and oxidative insult.…”

Section: Introductionmentioning

confidence: 99%

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Human αA- and αB-crystallins bind to Bax and Bcl-XS to sequester their translocation during staurosporine-induced apoptosis

et al. 2004

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aA-and aB-crystallins are distinct antiapoptotic regulators. Regarding the antiapoptotic mechanisms, we have recently demonstrated that aB-crystallin interacts with the procaspase-3 and partially processed procaspase-3 to repress caspase-3 activation. Here, we demonstrate that human aA-and aBcrystallins prevent staurosporine-induced apoptosis through interactions with members of the Bcl-2 family. Using GST pulldown assays and coimmunoprecipitations, we demonstrated that a-crystallins bind to Bax and Bcl-X S both in vitro and in vivo. Human aA-and aB-crystallins display similar affinity to both proapoptotic regulators, and so are true with their antiapoptotic ability tested in human lens epithelial cells, human retina pigment epithelial cells (ARPE-19) and rat embryonic myocardium cells (H9c2) under treatment of staurosporine, etoposide or sorbitol. Two prominent mutants, R116C in aA-crystallin and R120G, in aB-crystallin display much weaker affinity to Bax and Bcl-X S . Through the interaction, a-crystallins prevent the translocation of Bax and Bcl-X S from cytosol into mitochondria during staurosporine-induced apoptosis. As a result, a-crystallins preserve the integrity of mitochondria, restrict release of cytochrome c, repress activation of caspase-3 and block degradation of PARP. Thus, our results demonstrate a novel antiapoptotic mechanism for a-crystallins. Keywords: aA-crystallin; R116C; aB-crystallin; R120G; Bax; Bcl-X S Abbreviations: a-crystallin, alpha-crystallin; GFP, green fluorescence protein; HaA, human aA-crystallin; HaB, human aB-crystallin; GFP-HaA, green fluorescence and human aAcrystallin fusion protein; GFP-HaB, green fluorescence and human aB-crystallin fusion protein; MEM, Eagle's minimal essential medium; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PMSF, phenylmethylsufonyl fluoride; HLE, human lens epithelial cells; SDS, sodium dodecylsulfate; TBS, tris-buffered saline; TBS-T, tris-buffered saline with tween-20.

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Section: Antiapoptotic Mechanisms Of A-crystallinsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Human αA- and αB-crystallins bind to Bax and Bcl-XS to sequester their translocation during staurosporine-induced apoptosis

et al. 2004

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“…Lyophilization of ␥-crystallin in the presence of ␣-crystallin did not alter the structure of the ␥-crystallin molecule (24). Evidence for an in vivo chaperone-like sequestering function for ␣-crystallin in the lens has recently been obtained by electron microscopy of immunolabeled ␣/␥-and ␣/␤-crystallin complexes in bovine lenses (25). ␣-Crystallin is therefore now of considerable interest both as a lens protein involved in cataractogenesis and as a general mammalian shock protein with a possible role in other disorders.…”

mentioning

confidence: 98%

Effects of Site-directed Mutations on the Chaperone-like Activity of αB-Crystallin

Plater¹,

Goode

Crabbe

1996

Journal of Biological Chemistry

140

115

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Recombinant ␣B-crystallin has been shown to exhibit chaperone-like activity, suppressing the thermal aggregation of ␥-crystallin and aggregation of the reduced insulin B chain conferring thermotolerance to Escherichia coli BL21(DE3) cells. Mutations were made in three specific areas of the ␣B-crystallin, the N terminus D2G, the conserved phenylalanine-rich region, F24R, F27R, F27A, and the two C-terminal lysines K174L/ K175L, K174G/K175G. Biophysical characterization of the mutant ␣B-crystallins using far-UV CD revealed no change in secondary structural elements. Tryptophan fluorescence demonstrated global structural changes. Heat stability of the mutant ␣B-crystallins was not significantly affected as indicated by tryptophan fluorescence of heat-treated proteins.Mutations within the phenylalanine-rich region abolish the chaperone-like activity as measured by both in vivo and in vitro assays. Proteins with mutations at the C terminus demonstrated no significant chaperone-like activity, failing to confer thermotolerance on E. coli and demonstrating no significant inhibition of protein aggregation in either ␥-crystallin or reduced insulin B chain assays. The N-terminal mutation D2G demonstrated a significant reduction in efficiency of the chaperone-like activity although some thermotolerance was conferred in the E. coli assay. In vitro assays showed that complete inhibition of aggregation was only achieved at 10-fold higher concentrations of D2G than that required by the native ␣B-crystallin.Consistent changes in the chaperone-like activity of the site-directed mutants were demonstrated by the three assays. The results suggested that both chargecharge and hydrophobic interactions are important in protein binding by ␣B-crystallin and that the conserved RLFDQFF region is vital for chaperone-like activity.

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“…␣-Crystallin, like other sHSPs, acts like a molecular chaperone in preventing the aggregation of other proteins (8 -10). An intense research activity on the structure and function of ␣-crystallin has grown since then (11)(12)(13)(14)(15)(16)(17)(18)(19)(20), and the subject matter has been extensively reviewed in recent years (21)(22)(23).…”

mentioning

confidence: 99%

Role of ATP on the Interaction of α-Crystallin with Its Substrates and Its Implications for the Molecular Chaperone Function

Biswas

Das

2004

Journal of Biological Chemistry

110

160

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ATP plays a significant role in the function of molecular chaperones of the large heat shock protein families. However, its role in the functions of chaperones of the small heat shock protein families is not understood very well. We report here a study on the role of ATP on the structure and function of the major eye lens chaperone ␣-crystallin. Our in vitro study shows that at physiological temperature, ATP induces the association of ␣-crystallin with substrate proteins. The association process is reversible and low affinity in nature with unit binding stoichiometry. 4,4 -Dianilino-1,1 -binaphthyl-5,5-disulfonic acid, dipotassium salt, binding studies show that ATP induces the exposure of additional hydrophobic sites on ␣-crystallin, but no appreciable enhancement of the same was observed for the substrate protein ␥-crystallin or carbonic anhydrase. An equilibrium unfolding study reveals that ATP at 3 mM concentration stabilizes the ␣-crystallin structure by 4.5 kJ/mol. The compactness induced by ATP makes it more resistant to tryptic cleavage. ATP-induced association of chaperone ␣-crystallin with substrate enhanced its aggregation prevention ability and also enhanced the refolding yield of lactate dehydrogenase from the unfolded state. Our results suggest that the binding of ATP to ␣-crystallin and not its hydrolysis is required for all these effects, as replacement of ATP by its nonhydrolyzable analogue adenosine-5 -O-(3-thiotriphosphate), tetralithium salt, reproduced all the results faithfully. The implication of the ATP-induced reversible protein-protein association at physiological temperatures on the functional role of ␣-crystallin in vivo is discussed.␣-Crystallin is the major protein component of the vertebrate eye lens and is composed of two subunits, ␣A and ␣B, 20 kDa each having 57% sequence homology between them. Both subunits associate to form a large oligomer with an average molecular mass of 800 kDa. It is a key member of the small heat shock protein (sHSP) 1 superfamily having a conserved core

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Characterization of the α-γ and α-β Complex: Evidence for an In Vivo Functional Role of α-Crystallin as a Molecular Chaperone

Cited by 73 publications

References 0 publications

Human αA- and αB-crystallins bind to Bax and Bcl-XS to sequester their translocation during staurosporine-induced apoptosis

Human αA- and αB-crystallins bind to Bax and Bcl-XS to sequester their translocation during staurosporine-induced apoptosis

Effects of Site-directed Mutations on the Chaperone-like Activity of αB-Crystallin

Role of ATP on the Interaction of α-Crystallin with Its Substrates and Its Implications for the Molecular Chaperone Function

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