Oxidation of HRas cysteine thiols by metabolic stress prevents palmitoylation in vivo and contributes to endothelial cell apoptosis

Abstract

Here we demonstrate a new paradigm in redox signaling, whereby oxidants resulting from metabolic stress directly alter protein palmitoylation by oxidizing reactive cysteine thiolates. In mice fed a high-fat, high-sucrose diet and in cultured endothelial cells (ECs) treated with high palmitate and high glucose (HPHG), there was decreased HRas palmitoylation on Cys181/184 (61±24% decrease for cardiac tissue and 38±7.0% in ECs). This was due to oxidation of Cys181/184, detected using matrix-assisted laser desorption/ionization time of flight (MALDI TOF)-TOF. Decrease in HRas palmitoylation affected its compartmentalization and Ras binding domain binding activity, with a shift from plasma membrane tethering to Golgi localization. Loss of plasma membrane-bound HRas decreased growth factor-stimulated ERK phosphorylation (84±8.6% decrease) and increased apoptotic signaling (24±6.5-fold increase) after HPHG treatment that was prevented by overexpressing wild-type but not C181/184S HRas. The essential role of HRas in metabolic stress was made evident by the similar effects of expressing an inactive dominant negative N17-HRas or a MEK inhibitor. Furthermore, the relevance of thiol oxidation was demonstrated by overexpressing manganese superoxide dismutase, which improved HRas palmitoylation and ERK phosphorylation, while lessening apoptosis in HPHG treated ECs.

Chemical proteomics reveals ADP-ribosylation of small GTPases during oxidative stress

“…Our study provides the first example of endogenous ADP-ribosylation of small GTPases in mammalian cells, which has only been described during bacterial infection by ADP-ribosyltransferase toxins 13 . Interestingly, other studies have suggested that Hras Cys181 and 184 can also be oxidized and modified with glutathione during oxidative stress 47 , which suggests that these key Cys residues in the C-terminal hypervariable region of Hras are dynamically modified by S-fatty-acylation, S-glutathiolation, ADP-ribosylation and are highly sensitive to metabolic changes in cells. Our studies also provide another example of dynamic interplay between S-fatty-acylation and other posttranslational modifications 48 , which have been observed for example with S-palmitoylated and S-nitrosylated PSD-95 49 and suggested for other proteins from PTM-selective proteomic studies 50 .…”

Direct Detection of S-Palmitoylation by Mass Spectrometry

“…They further proposed that direct competition from oxidative PTMs of the C-terminal reactive cysteines prevents H-Ras palmitoylation, leading to endothelial dysfunction. 6 …”

Regulation of the cardiac Na+ pump by palmitoylation of its catalytic and regulatory subunits

“…The presence of PLM facilitates de-glutathionylation of Cys 46 of β1-subunit, with concomitant glutathionylation of PLM at Cys 42 [39], one of the cysteine residues in PLM that may also be palmitoylated. Thus the presence of PLM activates the pump in the sense that it is protected from oxidantinduced inhibition as a result of the reduced glutathionylation of Cys 46 .…”

“…Indeed, although the structural-sequence determinants of protein palmitoylation remain rather poorly characterized, it is well established that cysteine residues with low pK a values display high acylation rates in vitro [43], and low-pK a solvent-exposed cysteine residues are the very same sites that are also likely to be susceptible to glutathionylation. Competition between palmitoylation and oxidation/glutathionylation has been reported for proteins such as caveolin 1 [44], CD81 (cluster of differentiation 81) [45], H-ras and eNOS (endothelial nitric oxide synthase) [46]. Hence there may exist a set of proteins that are rendered insensitive to oxidation/glutathionylation by palmitoylation of key low-pK a cysteine residues, or even for whom palmitoylation and glutathionylation induce opposite effects in terms of changes in activity, as appears to be the case for the Na + pump.…”