Chemical inhibition of CaaX protease activity disrupts yeast Ras localization

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Medicine

Background:

• Ras GTPases are proteins with a C-terminal CaaX motif.
• These proteins undergo post-translational modifications including isoprenoid lipid attachment, proteolytic processing, and carboxylmethylation.
• Inhibiting enzymes in these pathways is a cancer-therapeutic strategy.

Purpose of the Study:

• To investigate the effects of inhibiting the yeast CaaX protease Rce1p on Ras GTPase distribution and trafficking.
• To validate identified Rce1p inhibitors and explore their therapeutic potential.

Main Methods:

• Chemical library screen to identify Rce1p inhibitors.
• Yeast GFP-Ras reporter system to monitor protein distribution.
• Gene dosage experiments to confirm target specificity.
• Biochemical assays to measure Rce1p activity.
• Confocal microscopy to analyze protein localization.

Main Results:

• Identified nine in vitro inhibitors of yeast Rce1p.
• Inhibitors disrupted normal plasma membrane distribution of GFP-Ras reporters, mimicking genetic loss of CaaX protease function.
• Compound-induced delocalization was suppressed by increased RCE1 gene dosage.
• Rce1p activity correlated inversely with inhibitor dose.
• Loss of CaaX proteolysis or pharmacological inhibition mistargeted GFP-Ras2p to endoplasmic reticulum-associated foci.
• Human Rce1p mediated proper yeast Ras targeting and was inhibited by some compounds.

Conclusions:

• Disrupting proteolytic modification of Ras GTPases impacts their in vivo trafficking.
• Rce1p inhibitors offer a promising avenue for cancer therapy by interfering with Ras protein localization.
• The study validates Rce1p as a target for therapeutic intervention.