The Active Site Cysteine of the Proapoptotic Protein Glyceraldehyde-3-phosphate Dehydrogenase Is Essential in Oxidative Stress-induced Aggregation and Cell Death

Abstract

Recent studies have revealed that the redox-sensitive glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is involved in neuronal cell death that is triggered by oxidative stress. GAPDH is locally deposited in disulfidebonded aggregates at lesion sites in certain neurodegenerative diseases. In this study, we investigated the molecular mechanism that underlies oxidative stress-induced aggregation of GAPDH and the relationship between structural abnormalities in GAPDH and cell death. Under nonreducing in vitro conditions, oxidants induced oligomerization and insoluble aggregation of GAPDH via the formation of intermolecular disulfide bonds. Because GAPDH has four cysteine residues, including the active site Cys 149 , we prepared the cysteine-substituted mutants C149S, C153S, C244A, C281S, and C149S/C281S to identify which is responsible for disulfide-bonded aggregation. Whereas the aggregation levels of C281S were reduced compared with the wild-type enzyme, neither C149S nor C149S/C281S aggregated, suggesting that the active site cysteine plays an essential role. Oxidants also caused conformational changes in GAPDH concomitant with an increase in ␤-sheet content; these abnormal conformations specifically led to amyloid-like fibril formation via disulfide bonds, including Cys 149 . Additionally, continuous exposure of GAPDH-overexpressing HeLa cells to oxidants produced disulfide bonds in GAPDH leading to both detergent-insoluble and thioflavin-S-positive aggregates, which were associated with oxidative stress-induced cell death. Thus, oxidative stresses induce amyloid-like aggregation of GAPDH via aberrant disulfide bonds of the active site cysteine, and the formation of such abnormal aggregates promotes cell death.In both prokaryotic and eukaryotic cells, glyceraldehyde-3-phosphate dehydrogenase (GAPDH 2 ; EC 1.2.1.12) plays a central role in glycolysis, catalyzing the reversible conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate in a reaction that is accompanied by the reduction of NAD ϩ to NADH. Mammalian GAPDH is a homotetramer composed of four identical subunits. Recent studies show that mammalian GAPDH has diverse activities unrelated to its glycolytic function (1, 2), including roles in membrane fusion, microtubule bundling, nuclear RNA transport (2), regulation of Ca 2ϩ homeostasis (3), and transcription (4). In addition to the various functions of GAPDH described above, particular attention is paid to its role in apoptosis (5-7). Although the proapoptotic role(s) of GAPDH seems to depend upon its accumulation in the particulate fractions, including the nucleus (5, 7), the detailed mechanism is still unclear.Recently, it has been suggested that a wide variety of neurodegenerative diseases are characterized by the accumulation of intracellular and extracellular protein aggregates (8,9). An initializing event in protein aggregation is thought to be the formation of an abnormal oligomer. For instance, ␤-amyloid and ␣-synuclein undergo conformational changes in Alzheimer disea...

Oxidation of 3,4-Dihydroxyphenylacetaldehyde, a Toxic Dopaminergic Metabolite, to a Semiquinone Radical and an ortho-Quinone

“…Also, reductive gel conditions indicate that radical-induced cross-linking is due to oxidative damage above and beyond simple disulfide rearrangements. The protein aggregates formed here may involve stable sulfhydryl cross-links (46) or other types of oxidative damage such as peroxidized proteins or protein carbonyls. A high increase (22%) in protein reactivity was evident with the radical-quinone mixture, indicating that redox cycling and ROS production may contribute to the observed results.…”

Glyceraldehyde-3-phosphate Dehydrogenase Aggregate Formation Participates in Oxidative Stress-induced Cell Death

“…Strain W3CG (20). These recombinant GAPDH proteins were expressed and then purified as previously described (15). Briefly, the transformants were cultured for 2 h at 37°C in M63 minimal medium containing both 50 g/ml ampicillin and 15 g/ml tetracycline, and then 0.2% (w/v) L-(ϩ)arabinose was added to the medium.…”

“…The cloning of human wild-type (WT) GAPDH cDNA was performed as reported previously (15). For bacterial expression, cDNA was cloned into pBAD-HisA (Invitrogen) using the SacIKpnI sites.…”

“…Similar to A␤ and ␣-synuclein, GAPDH is also amyloidogenic (9 -14). We previously reported the molecular mechanism underlying oxidative stressinduced amyloid-like aggregation of GAPDH using the purified rabbit GAPDH and demonstrated the critical role of the active site cysteine in its aggregation (15). The active site cysteine also plays a key role in nuclear translocation of GAPDH during oxidative stress-mediated cell damage (3).…”

Different carbon sources affect lifespan and protein redox state during Saccharomyces cerevisiae chronological ageing

“…The formation of such abnormal aggregates promotes cell death, thus revealing a new role for this classic glycolytic enzyme [47]. One of the spots identified corresponds to Pdc1p, an enzyme strongly expressed in yeast cells growing on fermentative carbon sources and is thought to have a limited expression in cells grown in respiratory carbon sources.…”