Protection Against Protein Aggregation by Alpha-Crystallin as a Mechanism of Preconditioning

Abstract

Anesthetic preconditioning occurs when cells previously exposed to inhaled anesthetics are protected against subsequent injury. We hypothesize that inhaled anesthetics may cause slight protein misfolding that involves site-specific dehydration, stimulating cytoprotective mechanisms. Human neuroblastoma cells were exposed to ethanol (as the dehydration agent) followed by quantitative analysis of the expression of five heat shock genes: DNAJC5G, CRYAA, HSPB2, HSF4 and HSF2. There was an ethanol-induced upregulation of all genes except HSF4, similar to previous observations using isoflurane. CRYAA (the gene for alphaA-crystallin) exhibited a 23.19 and 17.15-fold increase at 24 and 48 h post ethanol exposure, respectively. Additionally, we exposed glyceraldehyde 3-phosphate dehydrogenase to ethanol, which altered oligomeric subspecies and caused protein aggregation in a concentration-dependent manner. Ethanol-mediated dehydration-induced protein aggregation was prevented by incubation with alpha-crystallin. These data indicate that ethanol mimics the effects of isoflurane presumably through a cellular preconditioning mechanism that involves dehydration-induced protein aggregation.

Oxidation of an Exposed Methionine Instigates the Aggregation of Glyceraldehyde-3-phosphate Dehydrogenase

“…There are numerous reports investigating the mechanism of GAPDH aggregation (11,13,22,23,(43)(44)(45)(46)(47) with several studies stipulating an essential role for intermolecular disulfide bond formation (11,13,22,23). These models of GAPDH aggregation are supported by the observation that insoluble disulfide-cross-linked forms of GAPDH have been found in vivo (11)(12)(13)18).…”

Functional Diversity

“…Upon gentle mixing, the solution became rapidly turbid (data not shown), suggesting the slight compromise to the GAPDH structure had a disasterous effect on the a-cystallin causing it to quickly cooperatively unfold and produce insoluble aggregates. When the experiments were repeated with a newly purchased a-cystallin, the result that was originally expected did occur, namely that a-crystallin was not only resistant to unfolding but actually protected GAPDH against the noxious effects of ethanol and agitation [152]. This accidental discovery using 'old' protein may provide some hidden insight on how GAPDH may play a degenerative role particularly in older tissues or otherwise compromised tissues.…”

Dynamic Oligomeric Properties

“…The protective effects of a-crystallin on ethanol-induced GAPDH aggregation was also demonstrated by an in vitro test [35]. Fresh GAPDH (15 mM) was prepared in a 50 mM sodium phosphate buffer (pH ¼ 7.4) containing 0.3 mM EDTA and gently mixed with and without ethanol (1.5 %) at room temperature for 90 min.…”