Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) influences cytotoxicity, translocating to the nucleus during apoptosis. Here we report a signalling pathway in which nitric oxide (NO) generation that follows apoptotic stimulation elicits S-nitrosylation of GAPDH, which triggers binding to Siah1 (an E3 ubiquitin ligase), nuclear translocation and apoptosis. S-nitrosylation of GAPDH augments its binding to Siah1, whose nuclear localization signal mediates translocation of GAPDH. GAPDH stabilizes Siah1, facilitating its degradation of nuclear proteins. Activation of macrophages by endotoxin and of neurons by glutamate elicits GAPDH-Siah1 binding, nuclear translocation and apoptosis, which are prevented by NO deletion. The NO-S-nitrosylation-GAPDH-Siah1 cascade may represent an important molecular mechanism of cytotoxicity.

Besides its role in glycolysis, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) initiates a cell death cascade 1-9 . Diverse apoptotic stimuli activate inducible nitric oxide synthase (iNOS) or neuronal NOS (nNOS), with the generated nitric oxide (NO) S-nitrosylating GAPDH, abolishing its catalytic activity and conferring on it the ability to bind to Siah1, an E3-ubiquitin-ligase with a nuclear localization signal (NLS). The GAPDH-Siah1 protein complex, in turn, translocates to the nucleus and mediates cell death; these processes are blocked by procedures that interfere with GAPDH-Siah1 binding. Nuclear events induced by GAPDH to kill cells have been obscure. Here we show that nuclear GAPDH is acetylated at Lys 160 by the acetyltransferase p300/CREB binding protein (CBP) through direct protein interaction, which in turn stimulates the acetylation and catalytic activity of p300/CBP. Consequently, downstream targets of p300/CBP, such as p53 (refs 10 -15 ), are activated and cause cell death. A dominant-negative mutant GAPDH with the substitution of Lys 160 to Arg (GAPDH-K160R) prevents activation of p300/CBP, blocks induction of apoptotic genes and decreases cell death. Our findings reveal a pathway in which NO-induced nuclear GAPDH mediates cell death through p300/CBP.10Correspondence and requests for materials should be addressed A.S. or S.H.S (E-mail: asawa1@jhmi.edu; E-mail: ssnyder@jhmi.edu). 8 Current address: Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA. 9 These authors contributed equally to this work. AUTHOR CONTRIBUTIONS Ni.S. and M.R.H. were primarily responsible for experimental design and work, data analysis and preparation of figures, and helped to write the manuscript; M.K., M.C., B.-I.B., Ne.S. and B.T. contributed to data acquisition and analysis; T.D. and V.D. helped with the data analysis, provided technical assistance and material support; S.H.S and A.S. supervised the project and wrote the manuscript.Note: Supplementary Information is available on the Nature Cell Biology website. COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests. -(3-(aminomethyl)benzyl)acetamidine (1400W), a selective iNOS inhibitor (Fig. 1a). To ascertain whether p300 and/or CBP are physiologically responsible for GAPDH acetylation in intact cells, we depleted p300 and CBP by RNA interference (RNAi). Depletion of either protein decreased GAPDH acetylation, which was abolished following depletion of both CBP and p300 (Fig. 1b). We also observed acetylation of GAPDH by p300 in vitro ( Supplementary Information, Fig. S1a). Moreover, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric analysis of nuclear GAPDH in HEK293 cells following apoptotic stress revealed acetylation at Lys 160 ( Supplementary Information, Fig. S1b, c). To confirm the site of acetylation in intact cells, we transfected HEK293 cells with GAPDH or a mutant GAPDH (GAPDH-K160R). We observed acetylation of wild-type but not the K160R mutant GAPDH (Fi...

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) participates in a cell death cascade wherein a variety of stimuli activate nitric oxide (NO) synthases with NO nitrosylating GAPDH, conferring on it the ability to bind to Siah, an E3-ubiquitin-ligase, whose nuclear localization signal enables the GAPDH͞Siah protein complex to translocate to the nucleus where degradation of Siah targets elicits cell death. R-(؊)-Deprenyl (deprenyl) ameliorates the progression of disability in early Parkinson's disease and also has neuroprotective actions. We show that deprenyl and a related agent, TCH346, in subnanomolar concentrations, prevent S-nitrosylation of GAPDH, the binding of GAPDH to Siah, and nuclear translocation of GAPDH. In mice treated with the dopamine neuronal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), low doses of deprenyl prevent binding of GAPDH and Siah1 in the dopamineenriched corpus striatum.fforts to prevent neurodegeneration in disorders such as Alzheimer's disease, Parkinson's disease (PD), Huntington's disease, and stroke have proceeded along two pathways. One involves identifying the fundamental molecular causes of these diseases, although even for Huntington's disease whose molecular causation is established, curative therapy has not followed directly. Alternatively, elucidating mechanisms of cell death may lead to agents that prevent neurodegeneration without knowing the specific disease etiology.The monoamine oxidase-B (MAO-B) inhibitor, R-(Ϫ)-Deprenyl (selegiline, hereafter designated deprenyl) (Fig. 1a) has been used in the therapy of PD with the initial goal of elevating dopamine levels (1-4). Deprenyl can delay the progression of disability in early Parkinson's disease, and recent studies also indicated that deprenyl reduces neuronal death in a variety of in vivo and in vitro experimental models. These include death of neuronal cultures induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), nitric oxide (NO), or peroxynitrite, as well as in vivo hypoxia models and peripheral or optic nerve crush (5-13). Neuroprotection by deprenyl has been suggested to be independent of MAO-B (14-16). Such a concept has been reinforced by the fact that the deprenyl derivative TCH346 (Fig. 1a) with no inhibitory action for MAO-B, also displays neuroprotective effects in culture models at concentrations as low as 0.1 pM and in intact animals at oral doses as low as 0.3 g͞kg (17-20).Recently we described a cell death signaling system whereby cell stressors activate inducible or neuronal nitric oxide synthase (NOS) with a specific S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by NO (21). S-nitrosylation of GAPDH abolishes catalytic activity and confers upon GAPDH the ability to bind to Siah1 (hereafter designated Siah), an E3-ubiquitin-ligase whose nuclear localization signal mediates nuclear translocation of the GAPDH͞Siah complex. In the nucleus, GAPDH extends the rapid turnover of Siah, leading to the degradation of selected nuclear targets of Siah and apoptosis. In ...