PubMed · 12410498
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Jeffrey A Brinker. 2002. The third strike.. https://doi.org/10.1002/ccd.10360
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The alpha and betagamma subunits of heterotrimeric G-proteins contain specific lipid modifications, which are required for their biological function. However, the relevance of these modifications to the interactions within the heterotrimeric G-protein is not fully understood. In order to explore the role of the S-prenyl moiety of the isoprenylated betagamma dimer of retinal transducin, betagamma(t), in the formation of the heterotrimeric complex with the corresponding N-acylated alpha subunit, alpha(t), we employed purified fully processed subunits, which are soluble in aqueous solutions without detergents. Pertussis-toxin-mediated [(32)P]ADP-ribosylation of alpha(t) is strongly stimulated (approximately 10-fold) in the presence of betagamma(t) and can thus serve as a measure for heterotrimer formation. Using this assay, preincubation of alpha(t) with S-prenyl analogues containing farnesyl or geranylgeranyl moieties was found to inhibit heterotrimer formation in a dose-dependent manner. The inhibition was competitive and reversible, as indicated by its reversal upon increase of the betagamma(t) dimer concentration or by removal of the S-prenyl analogue using gel filtration. The competitive nature of the inhibition is supported by the marked attenuation of the inhibition when the S-prenyl analogue was added to alpha(t) together with or after betagamma(t). The inhibition does not involve interaction with the alpha(t) acyl group, since an S-prenyl analogue inhibited the [(32)P]ADP-ribosylation of an unlipidated alpha(t) mutant. These data suggest the existence of a hitherto unrecognized S-prenyl-binding site in alpha(t), which is critical for its interaction with prenylated betagamma(t).
Down Syndrome (DS), one of the major genetic causes of mental retardation, is characterized by disrupted corticogenesis produced, in part, by an abnormal layering of neurons in cortical laminas II and III. Because defects in the normal migration of neurons during corticogenesis can result in delayed cortical radial expansion and abnormalities in cortical layering, we have examined neuronal migration in murine trisomy 16 (Ts16), a mouse model for DS. Using an in vitro assay for chemotaxis, our data demonstrate that the number of acutely dissociated Ts16 cortical neurons migrating in response to glutamate or N-methyl-D-aspartate (NMDA), known chemotactic factors, was decreased compared to normal littermates, suggesting a defect in NMDA receptor- (NMDAR-) mediated events. Ts16 neurons did not lack NMDAR since expression of mRNA and protein for NMDAR subunits was observed in Ts16 cells. However, the number of cells that generated an observable current in response to NMDA was decreased compared to normal littermates. Similar to DS, Ts16 CNS demonstrated an inherent oxidative stress likely caused by the triplication of genes such as SOD1. To determine if the abnormal redox state was a factor in the failure of NMDAR-mediated migration in Ts16, we treated Ts16 neurons with either n-acetyl cysteine (NAC) or dithiothrietol (DTT), known antioxidants. The reduction in NMDAR-mediated migration observed in Ts16 neurons was returned to normal littermate values by NAC or DTT. Our data indicate that oxidative stress may play a key role in the abnormal glutamate-mediated responses during cortical development in the Ts16 mouse and may have an impact on neuronal migration at critical stages.
12-Hydroperoxy-eicosatetraenoic acid (12-HpETE), the main hydroperoxide formed in platelets from arachidonic acid (AA) by 12-lipoxygenase, has been shown to increase the sensitivity of platelets to agonists resulting in increased aggregation. The aim of the present study was to determine the direct effect of low concentrations of 12-HpETE on the signaling pathways leading to AA release from membrane phospholipids and thromboxane A2 (TxA2) formation. Exogenous 12-HpETE activated platelet p38 mitogen-activated protein kinase (p38 MAPK), as assessed by its phosphorylation, at a concentration as low as 100 nM and was much more potent than hydrogen peroxide. Moreover, the incubation of platelets with 100 nM 12-HpETE for 2 min led to the phosphorylation of cytosolic phospholipase A2 (cPLA2). It was associated with a significant decrease in the concentration of AA esterified in phospholipids and an increased concentration of thromboxane B2, the stable catabolite of TxA2. Additionally, decreasing glutathione peroxidase activity pharmacologically favored endogenous 12-HpETE formation and led to an increase in phosphorylated p38 MAPK, while a thiol-reducing agent such as N-acetyl-cysteine fully prevented it. Finally, significant activation of p38 MAPK was also observed in platelets from type 2 diabetic patients with mild hyperglycemia. In conclusion, our data provide a new insight into the mechanism of 12-HpETE-induced platelet priming, suggesting that hydroperoxide-induced p38 MAPK activation could play a relevant role in the exacerbated platelet activation associated with oxidative stress as found in diabetes.