New Developments in Solid Phase Peptide Chemistry Facilitate the Study of the Unique Metalloprotease, Ste24

Abstract

Protein and peptide prenylation is an essential biological process involved in many signal transduction pathways. In its most prevalent form, prenylation involves three enzymatic steps; transfer of an isoprenoid moiety by FTase or GGTaseI to the cysteine of a C-terminal CaaX sequence (where C is cysteine, a is an aliphatic amino acid, and X is a variable amino acid dictating whether a farnesyl or longer geranylgeranyl chain is added), proteolytic removal of the aaX sequence by ZMPSTE24 or RCE1, and finally carboxymethylation of the newly exposed C-terminal cysteine by ICMT enzyme. ZMPSTE24 also catalyzes a second cleavage step upstream of the CaaX site in prelamin A, and mutations abolishing this step lead to progeroid diseases. Ste24 is the yeast homolog of ZMPSTE24 and is the founding member of a unique class of integral membrane metalloproteases. Its precise mechanism of action has yet to be explored fully at the molecular level, and it is unique in that it performs two separate cleavage reactions sequentially at distinct sites in the same substrate molecule. The system historically used for studying prenylation is the mating pheromone a-Factor, a dodecameric peptide with a methyl ester C-terminal farnesylated cysteine. Producing this and other prenylated peptides presents four key challenges: the C-terminal cysteine is prone to epimerization, the terminal methyl ester is not readily available through traditional SPPS, the terminal cysteine has to be chemoselectively modified with a hydrophobic prenyl chain on the C-terminal cysteine, and often there are several cysteines in the sequence which necessitates additional orthogonal protecting group chemistry. In this work, various synthetic methodologies were developed in order to overcome these challenges, and then utilized for the production of a myriad of peptide probes based on the structure of a-Factor. These probes were used to study both cleavage steps of ZMPSTE24, as well as the other enzymes involved in the prenylation pathway.