Emily R. Hildebrandt

Small-Molecule Inhibitors of the Rce1p CaaX Protease

The Rce1p protease is required for the maturation of the Ras GTPase and certain other isoprenylated proteins and is considered a chemotherapeutic target. To identify new small-molecule inhibitors of Rce1p, the authors screened the National Cancer Institute Diversity Set compound library using in vitro assays to monitor the proteolytic processing of peptides derived from Ras and the yeast a-factor mating pheromone. Of 46 inhibitors initially identified with a Ras-based assay, only 9 were effective in the pheromone-based assay. The IC50 values of these 9 compounds were in the low micromolar range for both yeast (6-35 µM) and human Rce1p (0.4-46 µM). Four compounds were somewhat Rce1p selective in that they partially inhibited the Ste24p protease and did not inhibit Ste14p isoprenylcysteine carboxyl methyltransferase, 2 enzymes also involved in the maturation of isoprenylated proteins. The remaining 5 compounds inhibited all 3 enzymes. The 2 most Rce1p-selective agents were ineffective trypsin inhibitors, further supporting the specificity of these agents for Rce1p. The 5 least specific compounds formed colloidal aggregates, a proposed common feature of promiscuous inhibitors. Interestingly, the most specific Rce1p inhibitor also formed a colloidal aggregate. In vivo studies revealed that treatment of wild-type yeast with 1 compound induced a Ras2p delocalization phenotype that mimics observed effects in rce1 ste24 null yeast. The 9 compounds identified in this study represent new tools for understanding the enzymology of postisoprenylation-modifying enzymes and provide new insight for the future development of Rce1p inhibitors. (Journal of Biomolecular Screening 2007:983-993)

Mutational Analysis of the Ras Converting Enzyme Reveals a Requirement for Glutamate and Histidine Residues

The Ras converting enzyme (RCE) promotes a proteolytic activity that is required for the maturation of Ras, the yeast a-factor mating pheromone, and certain other proteins whose precursors bear a C-terminal CAAX tetrapeptide motif. Despite the physiological importance of RCE, the enzymatic mechanism of this protease remains undefined. In this study, we have evaluated the substrate specificity of RCE orthologs from yeast (Rce1p), worm, plant, and human and have determined the importance of conserved residues toward enzymatic activity. Our findings indicate that RCE orthologs have conserved substrate specificity, cleaving CVIA, CTLM, and certain other CAAX motifs, but not the CASQ motif, when these motifs are placed in the context of the yeast a-factor precursor. Our mutational studies of residues conserved between the orthologs indicate that an alanine substitution at His194 completely inactivates yeast Rce1p enzymatic activity, whereas a substitution at Glu156 or His248 results in marginal activity. We have also determined that residues Glu157, Tyr160, Phe190, and Asn252 impact the substrate selectivity of Rce1p. Computational methods predict that residues influencing Rce1p function are all near or within hydrophobic segments. Combined, our data indicate that yeast Rce1p function requires residues that are invariably conserved among an extended family of prokaryotic and eukaryotic enzymes and that these residues are likely to lie within or immediately adjacent to the transmembrane segments of this membrane-localized enzyme.

Chemical Inhibition of CaaX Protease Activity Disrupts Yeast Ras localization

Proteins possessing a C‐terminal CaaX motif, such as the Ras GTPases, undergo extensive post‐translational modification that includes attachment of an isoprenoid lipid, proteolytic processing and carboxylmethylation. Inhibition of the enzymes involved in these processes is considered a cancer‐therapeutic strategy. We previously identified nine in vitro inhibitors of the yeast CaaX protease Rce1p in a chemical library screen (Manandhar et al., 2007 ). Here, we demonstrate that these agents disrupt the normal plasma membrane distribution of yeast GFP–Ras reporters in a manner that pharmacologically phenocopies effects observed upon genetic loss of CaaX protease function. Consistent with Rce1p being the in vivo target of the inhibitors, we observe that compound‐induced delocalization is suppressed by increasing the gene dosage of RCE1. Moreover, we observe that Rce1p biochemical activity associated with inhibitor‐treated cells is inversely correlated with compound dose. Genetic loss of CaaX proteolysis results in mistargeting of GFP–Ras2p to subcellular foci that are positive for the endoplasmic reticulum marker Sec63p. Pharmacological inhibition of CaaX protease activity also delocalizes GFP–Ras2p to foci, but these foci are not as strongly positive for Sec63p. Lastly, we demonstrate that heterologously expressed human Rce1p can mediate proper targeting of yeast Ras and that its activity can also be perturbed by some of the above inhibitors. Together, these results indicate that disrupting the proteolytic modification of Ras GTPases impacts their in vivo trafficking. Copyright

8-Hydroxyquinoline-based inhibitors of the Rce1 protease disrupt Ras membrane localization in human cells.

Ras converting enzyme 1 (Rce1) is an endoprotease that catalyzes processing of the C-terminus of Ras protein by removing -aaX from the CaaX motif. The activity of Rce1 is crucial for proper localization of Ras to the plasma membrane where it functions. Ras is responsible for transmitting signals related to cell proliferation, cell cycle progression, and apoptosis. The disregulation of these pathways due to constitutively active oncogenic Ras can ultimately lead to cancer. Ras, its effectors and regulators, and the enzymes that are involved in its maturation process are all targets for anti-cancer therapeutics. Key enzymes required for Ras maturation and localization are the farnesyltransferase (FTase), Rce1, and isoprenylcysteine carboxyl methyltransferase (ICMT). Among these proteins, the physiological role of Rce1 in regulating Ras and other CaaX proteins has not been fully explored. Small-molecule inhibitors of Rce1 could be useful as chemical biology tools to understand further the downstream impact of Rce1 on Ras function and serve as potential leads for cancer therapeutics. Structure-activity relationship (SAR) analysis of a previously reported Rce1 inhibitor, NSC1011, has been performed to generate a new library of Rce1 inhibitors. The new inhibitors caused a reduction in Rce1 in vitro activity, exhibited low cell toxicity, and induced mislocalization of EGFP-Ras from the plasma membrane in human colon carcinoma cells giving rise to a phenotype similar to that observed with siRNA knockdowns of Rce1 expression. Several of the new inhibitors were more effective at mislocalizing K-Ras compared to a potent farnesyltransferase inhibitor (FTI), which is significant because of the preponderance of K-Ras mutations in cancer.

Ste24p Mediates Proteolysis of Both Isoprenylated and Non-prenylated Oligopeptides.

Rce1p and Ste24p are integral membrane proteins involved in the proteolytic maturation of isoprenylated proteins. Extensive published evidence indicates that Rce1p requires the isoprenyl moiety as an important substrate determinant. By contrast, we report that Ste24p can cleave both isoprenylated and non-prenylated substrates in vitro, indicating that the isoprenyl moiety is not required for substrate recognition. Steady-state enzyme kinetics are significantly different for prenylated versus non-prenylated substrates, strongly suggestive of a role for substrate-membrane interaction in protease function. Mass spectroscopy analyses identify a cleavage preference at bonds where P1′ is aliphatic in both isoprenylated and non-prenylated substrates, although this is not necessarily predictive. The identified cleavage sites are not at a fixed distance position relative to the C terminus. In this study, the substrates cleaved by Ste24p are based on known isoprenylated proteins (i.e. K-Ras4b and the yeast a-factor mating pheromone) and non-prenylated biological peptides (Aβ and insulin chains) that are known substrates of the M16A family of soluble zinc-dependent metalloproteases. These results establish that the substrate profile of Ste24p is broader than anticipated, being more similar to that of the M16A protease family than that of the Rce1p CAAX protease with which it has been functionally associated.

Topology of the yeast Ras converting enzyme as inferred from cysteine accessibility studies.

The Ras converting enzyme (Rce1p) is an endoprotease that is involved in the post-translational processing of the Ras GTPases and other isoprenylated proteins. Its role in Ras biosynthesis marks Rce1p as an anticancer target. By assessing the chemical accessibility of cysteine residues substituted throughout the Saccharomyces cerevisiae Rce1p sequence, we have determined that yeast Rce1p has eight segments that are protected from chemical modification. Notably, the three residues that are essential for yeast Rce1p function (E156, H194, and H248) are all chemically inaccessible and associated with separate protected segments. By specifically assessing the chemical reactivity and glycosylation potential of the NH2 and COOH termini of Rce1p, we further demonstrate that Rce1p has an odd number of transmembrane spans. Substantial evidence that the most NH2-terminal segment functions as a transmembrane segment with the extreme NH2 terminus projecting into the endoplasmic reticulum (ER) lumen is presented. Because each of the remaining seven segments is too short to contain two spans and is flanked by chemically reactive positions, we infer that these segments are not transmembrane segments but rather represent compact structural features and/or hydrophobic loops that penetrate but do not fully span the bilayer (i.e., re-entrant helices). We thus propose a topological model in which yeast Rce1p contains a single transmembrane helix localized at its extreme NH2 terminus and one or more re-entrant helices and/or compact structural domains that populate the cytosolic face of the ER membrane. Lastly, we demonstrate that the natural cysteine residues of Rce1p are chemically inaccessible and fully dispensable for in vivo enzyme activity, formally eliminating the possibility of a cysteine-based enzymatic mechanism for this protease.

Efficient farnesylation of an extended C-terminal C(x)3X sequence motif expands the scope of the prenylated proteome

Protein prenylation is a post-translational modification that has been most commonly associated with enabling protein trafficking to and interaction with cellular membranes. In this process, an isoprenoid group is attached to a cysteine near the C terminus of a substrate protein by protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I or II (GGTase-I and GGTase-II). FTase and GGTase-I have long been proposed to specifically recognize a four-amino acid CAAX C-terminal sequence within their substrates. Surprisingly, genetic screening reveals that yeast FTase can modify sequences longer than the canonical CAAX sequence, specifically C(x)3X sequences with four amino acids downstream of the cysteine. Biochemical and cell-based studies using both peptide and protein substrates reveal that mammalian FTase orthologs can also prenylate C(x)3X sequences. As the search to identify physiologically relevant C(x)3X proteins begins, this new prenylation motif nearly doubles the number of proteins within the yeast and human proteomes that can be explored as potential FTase substrates. This work expands our understanding of prenylations impact within the proteome, establishes the biologically relevant reactivity possible with this new motif, and opens new frontiers in determining the impact of non-canonically prenylated proteins on cell function.

Protein Isoprenylation in Yeast Targets COOH-Terminal Sequences Not Adhering to the CaaX Consensus

In vitro and in silico studies of the CaaX-type prenyl transferases suggest a wider array of prenylatable sequences than those determined in vivo. Berger and Kim et al. investigate whether this disconnect is due to use of... Protein isoprenylation targets a subset of COOH-terminal Cxxx tetrapeptide sequences that has been operationally defined as a CaaX motif. The specificity of the farnesyl transferase toward each of the possible 8000 combinations of Cxxx sequences, however, remains largely unresolved. In part, it has been difficult to consolidate results stemming from in vitro and in silico approaches that yield a wider array of prenylatable sequences relative to those known in vivo. We have investigated whether this disconnect results from the multistep complexity of post-translational modification that occurs in vivo to CaaX proteins. For example, the Ras GTPases undergo isoprenylation followed by additional proteolysis and carboxymethylation events at the COOH-terminus. By contrast, Saccharomyces cerevisiae Hsp40 Ydj1p is isoprenylated but not subject to additional modification. In fact, additional modifications are detrimental to Ydj1p activity in vivo. We have taken advantage of the properties of Ydj1p and a Ydj1p-dependent growth assay to identify sequences that permit Ydj1p isoprenylation in vivo while simultaneously selecting against nonprenylatable and more extensively modified sequences. The recovered sequences are largely nonoverlapping with those previously identified using an in vivo Ras-based yeast reporter. Moreover, most of the sequences are not readily predicted as isoprenylation targets by existing prediction algorithms. Our results reveal that the yeast CaaX-type prenyltransferases can utilize a range of sequence combinations that extend beyond the traditional constraints for CaaX proteins, which implies that more proteins may be isoprenylated than previously considered.