1. Isoprenylcysteine carboxyl methyltransferase activity modulates endothelial cell apoptosis
Qing Lu, Julie Newton, Kristina Kramer, Robert Bellas, Elizabeth O Harrington, Sharon Rounds, Kerri L Sheahan Mol Biol Cell . 2003 Mar;14(3):848-57. doi: 10.1091/mbc.e02-07-0390.
Extracellular ATP, adenosine (Ado), and adenosine plus homocysteine (Ado/HC) cause apoptosis of cultured pulmonary artery endothelial cells through the enhanced formation of intracellular S-adenosylhomocysteine and disruption of focal adhesion complexes. Because an increased intracellular ratio of S-adenosylhomocysteine/S-adenosylmethionine favors inhibition of methylation, we hypothesized that Ado/HC might act by inhibition of isoprenylcysteine-O-carboxyl methyltransferase (ICMT). We found that N-acetyl-S-geranylgeranyl-L-cysteine (AGGC) and N-acetyl-S-farnesyl-L-cysteine (AFC), which inhibit ICMT by competing with endogenous substrates for methylation, caused apoptosis. Transient overexpression of ICMT inhibited apoptosis caused by Ado/HC, UV light exposure, or tumor necrosis factor-alpha. Because the small GTPase, Ras, is a substrate for ICMT and may modulate apoptosis, we also hypothesized that inhibition of ICMT with Ado/HC or AGGC might cause endothelial apoptosis by altering Ras activation. We found that ICMT inhibition decreased Ras methylation and activity and the activation of the downstream signaling molecules Akt, ERK-1, and ERK-2. Furthermore, overexpression of wild-type or dominant active H-Ras blocked Ado/HC-induced apoptosis. These findings suggest that inhibition of ICMT causes endothelial cell apoptosis by attenuation of Ras GTPase methylation and activation and its downstream antiapoptotic signaling pathway.
2. Identification of prenylcysteine carboxymethyltransferase in bovine adrenal chromaffin cells
H M De Busser, A R Lagrou, G A Van Dessel Int J Biochem Cell Biol . 2000 Sep;32(9):1007-16. doi: 10.1016/s1357-2725(00)00036-4.
Chromaffin cells from bovine adrenal medulla were examined for the presence of a specific prenylcysteine carboxymethyltransferase by using N-acetyl-S-farnesyl-L-cysteine and N-acetyl-S-geranylgeranyl-L-cysteine as artificial substrates and a crude cell homogenate as the enzyme source. From Michaelis-Menten kinetics the following constants were calculated: K(m) 90 microM and V(max) 3 pmol/min per mg proteins for N-acetyl-S-farnesyl-L-cysteine; K(m) 52 microM and V(max) 3 pmol/min per mg proteins for N-acetyl-S-geranylgeranyl-L-cysteine. Both substrates were methylated to an optimal extent at the pH range 7. 4-8.0. Methylation activity increased linearly up to 20 min incubation time and was dose dependent up to at least 160 microg of protein. Sinefungin and S-adenosylhomocysteine both caused pronounced inhibition, as also to a lesser extent did farnesylthioacetic acid, deoxymethylthioadenosine and 3-deaza-adenosine. Effector studies showed that the methyltransferase activity varied depending on the concentration and chemical nature of the cations present. Monovalent cations were slightly stimulatory, while divalent metallic ions displayed diverging inhibitory effects. The inhibition by cations was validated by the stimulatory effect of the chelators EDTA and EGTA. Sulphydryl reagents inhibited methylation but to different degrees: Hg(2+)-ions: 100%, N-ethylmaleimide: 30%, dithiothreitol: 0% and mono-iodoacetate: 20%. Due to the hydrophobicity of the substrates dimethyl sulfoxide had to be included in the incubation mixture (<4%; still moderate inhibition at more elevated concentrations). The detergents tested affected the methyltransferase activity to a varying degree. The membrane bound character of the methyltransferase was confirmed.
3. Topical N-acetyl-S-farnesyl-L-cysteine inhibits mouse skin inflammation, and unlike dexamethasone, its effects are restricted to the application site
Joel S Gordon, Maxwell B Stock, Jeffry B Stock, Peter M Wolanin, Karl Rouzard, Eduardo Perez, David A Fela, Gopal Sarngadharan, Arnold V Gonzalez J Invest Dermatol . 2008 Mar;128(3):643-54. doi: 10.1038/sj.jid.5701061.
N-acetyl-S-farnesyl-L-cysteine (AFC), a modulator of G protein and G-protein coupled receptor signaling, inhibits neutrophil chemotaxis and other inflammatory responses in cell-based assays. Here, we show topical AFC inhibits in vivo acute inflammation induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and arachidonic acid using the mouse ear model of inflammation. AFC inhibits edema, as measured by ear weight, and also inhibits neutrophil infiltration as assayed by direct counting in histological sections and by measuring myeloperoxidase (MPO) activity as a neutrophil marker. In addition, AFC inhibits in vivo allergic contact dermatitis in a mouse model utilizing sensitization followed by a subsequent challenge with 2,4-dinitrofluorobenzene. Unlike the established anti-inflammatories dexamethasone and indomethacin, AFC's action was restricted to the site of application. In this mouse model, both dexamethasone and indomethacin inhibited TPA-induced edema and MPO activity in the vehicle-treated, contralateral ear. AFC showed no contralateral ear inhibition for either of these end points. A marginally significant decrease due to AFC treatment was seen in TPA-induced epidermal hyperplasia at 24 hours. This was much less than the 90% inhibition of neutrophil infiltration, suggesting that AFC does not act by directly inhibiting protein kinase C.
