Cloning and Characterization of a Mammalian Prenyl Protein-specific Protease

Abstract

Proteins containing C-terminal "CAAX" sequence motifs undergo three sequential post-translational processing steps: modification of the cysteine with either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenyl lipid, proteolysis of the C-terminal -AAX tripeptide, and methylation of the carboxyl group of the now C-terminal prenylcysteine. A putative prenyl protein protease in yeast, designated Rce1p, was recently identified. In this study, a portion of a putative human homologue of RCE1 (hRCE1) was identified in a human expressed sequence tag data base, and the corresponding cDNA was cloned. Expression of hRCE1 was detected in all tissues examined. Both yeast and human RCE1 proteins were produced in Sf9 insect cells by infection with a recombinant baculovirus; membrane preparations derived from the infected Sf9 cells exhibited a high level of prenyl protease activity. Recombinant hRCE1 so produced recognized both farnesylated and geranylgeranylated proteins as substrates, including farnesyl-Ki-Ras, farnesyl-N-Ras, farnesyl-Ha-Ras, and the farnesylated heterotrimeric G protein G ␥1 subunit, as well as geranylgeranylKi-Ras and geranylgeranyl-Rap1b. The protease activity of hRCE1 activity was specific for prenylated proteins, because unprenylated peptides did not compete for enzyme activity. hRCE1 activity was also exquisitely sensitive to a prenyl peptide analogue that had been previously described as a potent inhibitor of the prenyl protease activity in mammalian tissues. These data indicate that both the yeast and the human RCE1 gene products are bona fide prenyl protein proteases and suggest that they play a major role in the processing of CAAX-type prenylated proteins.A variety of proteins are modified with an isoprenoid lipid at a cysteine that is initially four residues from the C terminus (1-3). Such proteins contain the so-called CAAX motif, in which the "C" is the modified cysteine, the "A" residues are most commonly (but not always) aliphatic amino acids, and the "X" residue can be one of several amino acids. The X residue determines whether the protein is modified by the 15-carbon farnesyl lipid or the 20-carbon geranylgeranyl. If the X residue is a leucine, the protein will be geranylgeranylated; several other residues (e.g. Met, Ser, Ala, and Gln) direct farnesylation. Following prenylation of the protein, two additional processing steps occur (4, 5). First, a specific protease cleaves the -AAX tripeptide from the protein, leaving the prenylated cysteine as the new C terminus. The carboxyl group of the prenylcysteine is then methylated by a specific methyltransferase.It is well established that protein prenylation plays a vital role in the membrane localization and function of most prenylated proteins (6). The role that the proteolysis and methylation of prenyl proteins play in their function, however, is not as well understood. Studies on peptides have demonstrated that each processing step significantly increases the affinity of farnesylated peptides for membranes (7), although the effect of th...

Therapeutic intervention based on protein prenylation and associated modifications

“…Using defined synthetic substrates and membrane-associated or partially purified Rce1p, investigators have shown that Rce1p activity depends on the prenylation status of its substrate, and that Rce1p has a preference for particular CaaX motifs [26][27][28][29][30][31] . Unfortunately, Rce1p has eluded purification, and neither its amino acid sequence nor its biochemical properties reveal straightforward clues about its mechanism of action.…”

On the Physiological Importance of Endoproteolysis of CAAX Proteins

“…In this assay, the level of CAAX endoprotease activity in heart extracts was determined by measuring the ability of the extracts to cleave farnesyl-K-Ras and render it susceptible to methylation by Icmt, thereby producing K-Ras terminating with a farnesylcysteine [ 14 C]methyl ester. Hearts of mice were excised, rinsed with cold phosphate-buffered saline, cut into multiple pieces, and then homogenized and sonicated in a buffer (50 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, 5 mM MgCl 2, pH 7.4) containing protease inhibitors (Roche Applied Science, Nutley, NJ) (6,7). After centrifugation at 1000 ϫ g for 5 min and removal of tissue debris, heart extracts were collected.…”

“…The higher band within the Ras doublet band contains Ras proteins that have not undergone endoproteolytic processing (6). methylation assay (6,7). In this assay, the level of CAAX endoprotease activity in heart extracts was determined by measuring the ability of the extracts to cleave farnesyl-K-Ras and render it susceptible to methylation by Icmt, thereby producing K-Ras terminating with a farnesylcysteine [ 14 C]methyl ester.…”

“…Second, a prenylprotein-specific endoprotease of the endoplasmic reticulum, Ras-converting enzyme 1 (Rce1), clips off the last three amino acids from the protein (i.e. the -AAX) (5)(6)(7). Third, the newly exposed C-terminal isoprenylcysteine is methylesterified by isoprenylcysteine carboxyl methyltransferase (Icmt), a prenylprotein-specific, S-adenosylmethionine-dependent methyltransferase of the endoplasmic reticulum (8 -11).…”

Novel aspects of Ras proteins biology: regulation and implications

“…75 Recently-conducted studies in yeast have led to the identification of two gene products, Ste24p and Rce1p, which partially overlap as C-terminal CAAX proteases. 75,76 The human homologue of Ste24p has been recently cloned, and it appears that this protease can mediate multiple types of proteolytic events. 77 In addition, disruption of the mouse Rce1 gene results in defective Ras processing and mislocalization.…”