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Inhibition of type I and type II geranylgeranyl-protein transferases by the monoterpene perillyl alcohol in NIH3T3
1Department of Human Oncology, University of Wisconsin-Madison, 53792, U.S.A.
Abstract:
The monoterpene perillyl alcohol has anticancer activities that include both prevention and treatment of a wide variety of cancers in animal models. In purified enzyme studies, perillyl alcohol inhibited farnesyl-protein transferase and type I geranylgeranyl-protein transferase. However, whether and which of the polyprenyl-protein transferases is inhibited by perillyl alcohol in vivo is not known. The previously reported monoterpene-induced inhibition of the incorporation of [14C]mevalonolactone into proteins in cultured cells could be due to an inhibition of one or several enzymes in the mevalonate pathway or to changes in the levels of protein substrates for isoprenylation. In the current study, we first analyzed the levels of individual phosphorylated isoprenoid intermediates between mevalonate and geranylgeranyl pyrophosphate in NIH3T3 cells labeled for 4 hr with [14C]mevalonolactone and found that perillyl alcohol did not inhibit the synthesis of these intermediates. Next, proteins including Ras, RhoA, and Rab6 were immunoprecipitated from NIH3T3 cells. Perillyl alcohol was found to inhibit the incorporation of [14C]mevalonolactone into RhoA and Rab6 but not Ras protein. The cellular levels of these three proteins were constant over the 4-hr treatment period. Finally, the distribution of Ras, Rap1, and Rab6 proteins between the aqueous and the detergent-enriched phases was measured. Rap1 and Rab6 but not Ras from perillyl alcohol-treated NIH3T3 cells accumulated in the aqueous phase. Thus, we conclude that perillyl alcohol can inhibit the in vivo prenylation of specific proteins by type I and type II geranylgeranyl-protein transferases but not farnesyl-protein transferase in NIH3T3 cells.
Insights
Perillyl alcohol, a natural compound, inhibits protein prenylation in cells, affecting specific proteins like RhoA and Rab6. This anticancer agent targets prenyl-protein transferases in vivo, offering new insights into its therapeutic mechanisms.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Perillyl alcohol exhibits anticancer properties in preclinical models.
- In vitro studies show inhibition of farnesyl-protein transferase and type I geranylgeranyl-protein transferase by perillyl alcohol.
- The in vivo targets of perillyl alcohol's inhibition of polyprenyl-protein transferases remain unclear.
Purpose of the Study:
- To investigate the in vivo effects of perillyl alcohol on protein prenylation in cultured cells.
- To determine which specific prenyl-protein transferases are inhibited by perillyl alcohol in NIH3T3 cells.
- To elucidate the mechanism by which perillyl alcohol impacts isoprenoid metabolism and protein modification.
Main Methods:
- Analysis of isoprenoid intermediates in [14C]mevalonolactone-labeled NIH3T3 cells treated with perillyl alcohol.
- Immunoprecipitation of Ras, RhoA, and Rab6 proteins to assess [14C]mevalonolactone incorporation.
- Fractionation of cell lysates to determine the localization of prenylated proteins (Ras, Rap1, Rab6) after perillyl alcohol treatment.
Main Results:
- Perillyl alcohol did not inhibit the synthesis of isoprenoid intermediates between mevalonate and geranylgeranyl pyrophosphate.
- Perillyl alcohol inhibited the prenylation of RhoA and Rab6 proteins, but not Ras protein.
- Prenylation-dependent localization of Rap1 and Rab6 proteins to the detergent phase was impaired by perillyl alcohol, causing their accumulation in the aqueous phase.
Conclusions:
- Perillyl alcohol inhibits the in vivo prenylation of specific proteins, including RhoA and Rab6, in NIH3T3 cells.
- The study suggests that perillyl alcohol targets type I and type II geranylgeranyl-protein transferases in vivo, but not farnesyl-protein transferase.
- These findings clarify the molecular targets of perillyl alcohol's anticancer activity and its impact on protein modification pathways.
