Prenylation of mammalian Ras protein in Xenopus oocytes

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Biology

Background:

• Ras protein requires posttranslational modification involving cholesterol biosynthesis intermediates for membrane anchorage and biological activity.
• Inhibitors of mevalonate synthesis, such as lovastatin and compactin, block Ras-induced Xenopus oocyte maturation.

Purpose of the Study:

• To investigate the role of cholesterol biosynthesis intermediates in Ras protein posttranslational modification in Xenopus oocytes.
• To identify the specific isoprenoid moiety involved in Ras prenylation and to explore the utility of Xenopus oocytes as a model system for studying prenylation inhibitors.

Main Methods:

• Xenopus laevis oocyte maturation assays were performed using oncogenic Ras protein.
• Inhibition studies utilized lovastatin, compactin, mevalonic acid, farnesyl diphosphate, and squalene.
• Inhibition of Ras prenylation was assessed in vivo and in vitro using Saccharomyces cerevisiae and Xenopus oocyte extracts with a c-H-ras C-terminal octapeptide.

Main Results:

• Lovastatin and compactin inhibited Ras-induced oocyte maturation, which was rescued by mevalonic acid or farnesyl diphosphate, but not squalene.
• These findings suggest Ras protein is modified by a farnesyl moiety or derivative in Xenopus oocytes.
• A C-terminal c-H-ras octapeptide inhibited Ras biological activity in vivo and Ras prenylation in vitro, indicating competition for prenyltransferase enzymes.

Conclusions:

• Xenopus oocytes are a suitable in vivo system for studying inhibitors of Ras protein posttranslational modification.
• The study confirms the requirement of a farnesyl moiety for Ras protein function in Xenopus oocytes.
• Peptide inhibitors targeting Ras prenylation can be effectively studied in Xenopus oocytes, even those with poor cell membrane permeability.