Cytoplasmic and Nuclear Anti-Apoptotic Roles of αB-Crystallin in Retinal Pigment Epithelial Cells

Abstract

In addition to its well-characterized role in the lens, αB-crystallin performs other functions. Methylglyoxal (MGO) can alter the function of the basement membrane of retinal pigment epithelial (RPE) cells. Thus, if MGO is not efficiently detoxified, it can induce adverse reactions in RPE cells. In this study, we examined the mechanisms underlying the anti-apoptotic activity of αB-crystallin in the human retinal pigment epithelial cell line ARPE-19 following MGO treatment using various assays, including nuclear staining, flow cytometry, DNA electrophoresis, pulse field gel electrophoresis, western blot analysis, confocal microscopy and co-immunoprecipitation assays. To directly assess the role of phosphorylation of αB-crystallin, we used site-directed mutagenesis to convert relevant serine residues to alanine residues. Using these techniques, we demonstrated that MGO induces apoptosis in ARPE-19 cells. Silencing αB-crystallin sensitized ARPE-19 cells to MGO-induced apoptosis, indicating that αB-crystallin protects ARPE-19 cells from MGO-induced apoptosis. Furthermore, we found that αB-crystallin interacts with the caspase subtypes, caspase-2L, -2S, -3, -4, -7, -8, -9 and -12 in untreated control ARPE-19 cells and that MGO treatment caused the dissociation of these caspase subtypes from αB-crystallin; transfection of S19A, S45A or S59A mutants caused the depletion of αB-crystallin from the nuclei of untreated control RPE cells leading to the release of caspase subtypes. Additionally, transfection of these mutants enhanced MGO-induced apoptosis in ARPE-19 cells, indicating that phosphorylation of nuclear αB-crystallin on serine residues 19, 45 and 59 plays a pivotal role in preventing apoptosis in ARPE-19 cells. Taken together, these results suggest that αB-crystallin prevents caspase activation by physically interacting with caspase subtypes in the cytoplasm and nucleus, thereby protecting RPE cells from MGO-induced apoptosis.

Protein polymer nanoparticles engineered as chaperones protect against apoptosis in human retinal pigment epithelial cells

“…Once internalized, these protein polymer fusions would have direct access to misfolding client proteins inside the cytosol and the nucleus. Jeong and coworkers have previously reported that αB-crystallin physically interacts with caspase subtypes in the nucleus, which protects RPE from apoptosis [48]. Similarly, Dastoor and coworkers reported that endogenous heat shock cognate protein70 (Hsc70) translocates from the cytoplasm to the nucleus when oxidative stress is applied [49].…”

Small heat shock proteins: Role in cellular functions and pathology

“…αB-crystallin interacts with the caspase subtypes, caspase-2L, -2S, -3, -4, -7, -8, -9 and -12 in control ARPE-19 cells, and MGO treatment leads to dissociation of caspase subtypes from αB-crystallin [257]. Phosphorylation of αB-crystallin on serine residues 19, 45 and 59 plays an important role in preventing apoptosis in ARPE-19 cells [257]. However, it has also been reported that S59-phosphorylated-αB-crystallin preferentially interacts with and sequesters the anti-apoptotic molecule, Bcl-2 and hence S59-phosphorylation down-regulates the anti-apoptotic function of αB-crystallin [258].…”

“…αB-crystallin interacts with the caspase subtypes, caspase-2L, -2S, -3, -4, -7, -8, -9 and -12 in control ARPE-19 cells, and MGO treatment leads to dissociation of caspase subtypes from αB-crystallin [257]. Phosphorylation of αB-crystallin on serine residues 19, 45 and 59 plays an important role in preventing apoptosis in ARPE-19 cells [257].…”

“…apoptosis [257]. αB-crystallin interacts with the caspase subtypes, caspase-2L, -2S, -3, -4, -7, -8, -9 and -12 in control ARPE-19 cells, and MGO treatment leads to dissociation of caspase subtypes from αB-crystallin [257].…”

“…Hsp27 inhibits the release of the second mitochondria-derived activator of caspases, Smac, which binds to the inhibitor of apoptosis proteins (IAPs) and promotes apoptosis via activation of caspases[255]. αB-crystallin and Hsp27 interact with procaspase-3 and prevent its maturation to active caspase-3[253,257,[259][260][261][262]. αB-crystallin, HspB2 and Hsp27 are also found to localize to mitochondria[247,274,275], probably protecting their integrity and viability.…”