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.     

Studies with recombinant Saccharomyces cerevisiae CaaX prenyl protease Rce1p

Eukaryotic proteins with carboxyl-terminal CaaX motifs undergo three post-translational processing reactions-protein prenylation, endoproteolysis, and carboxymethylation. Two genes in yeast encoding CaaX endoproteases, AFC1 and RCE1, have been identified. Rce1p is solely responsible for proteolysis of yeast Ras proteins. When proteolysis is blocked, plasma membrane localization of Ras2p is impaired. The mislocalization of undermodified Ras in the cell suggests that Rce1p is an attractive target for cancer therapeutics. Homologous expression of plasmid-encoded Saccharomyces cerevisiae RCE1 under the control of the GAL1 promoter gave a 370-fold increase in endoprotease activity over an uninduced control. Yeast Rce1p was detected by Western blotting with a yRce1p antibody or with an anti-myc antibody to Rce1p bearing a C-terminal myc-epitope. Membrane preparations were examined for their sensitivity to a variety of protease inhibitors, metal ion chelators, and heavy metals. The enzyme was sensitive to cysteine protease inhibitors, Zn(2+), and Ni(2+). The substrate selectivity of yRce1p was determined for a variety of prenylated CaaX peptides including farnesylated and geranylgeranylated forms of human Ha-Ras, Ki-Ras, N-Ras, and yeast Ras2p, a-mating factor, and Rho2p. Six site-directed mutants of conserved polar and ionic amino acids in yRce1p were prepared. Four of the mutants, H194A, E156A, C251A, and H248A, were inactive. Results from the protease inhibition studies and the site-directed mutagenesis suggest that Rce1p is a cysteine protease.     

Postprenylation CAAX processing is required for proper localization of Ras but not Rho GTPases

The CAAX motif at the C terminus of most monomeric GTPases is required for membrane targeting because it signals for a series of three posttranslational modifications that include isoprenylation, endoproteolytic release of the C-terminal- AAX amino acids, and carboxyl methylation of the newly exposed isoprenylcysteine. The individual contributions of these modifications to protein trafficking and function are unknown. To address this issue, we performed a series of experiments with mouse embryonic fibroblasts (MEFs) lacking Rce1 (responsible for removal of the -AAX sequence) or Icmt (responsible for carboxyl methylation of the isoprenylcysteine). In MEFs lacking Rce1 or Icmt, farnesylated Ras proteins were mislocalized. In contrast, the intracellular localizations of geranylgeranylated Rho GTPases were not perturbed. Consistent with the latter finding, RhoGDI binding and actin remodeling were normal in Rce1- and Icmt-deficient cells. Swapping geranylgeranylation for farnesylation on Ras proteins or vice versa on Rho proteins reversed the differential sensitivities to Rce1 and Icmt deficiency. These results suggest that postprenylation CAAX processing is required for proper localization of farnesylated Ras but not geranygeranylated Rho proteins.     

The CaaX proteases, Afc1p and Rce1p, have overlapping but distinct substrate specificities

Many proteins that contain a carboxyl-terminal CaaX sequence motif, including Ras and yeast a-factor, undergo a series of sequential posttranslational processing steps. Following the initial prenylation of the cysteine, the three C-terminal amino acids are proteolytically removed, and the newly formed prenylcysteine is carboxymethylated. The specific amino acids that comprise the CaaX sequence influence whether the protein can be prenylated and proteolyzed. In this study, we evaluated processing of a-factor variants with all possible single amino acid substitutions at either the a(1), the a(2), or the X position of the a-factor Ca(1)a(2)X sequence, CVIA. The substrate specificity of the two known yeast CaaX proteases, Afc1p and Rce1p, was investigated in vivo. Both Afc1p and Rce1p were able to proteolyze a-factor with A, V, L, I, C, or M at the a(1) position, V, L, I, C, or M at the a(2) position, or any amino acid at the X position that was acceptable for prenylation of the cysteine. Eight additional a-factor variants with a(1) substitutions were proteolyzed by Rce1p but not by Afc1p. In contrast, Afc1p was able to proteolyze additional a-factor variants that Rce1p may not be able to proteolyze. In vitro assays indicated that farnesylation was compromised or undetectable for 11 a-factor variants that produced no detectable halo in the wild-type AFC1 RCE1 strain. The isolation of mutations in RCE1 that improved proteolysis of a-factor-CAMQ, indicated that amino acid substitutions E139K, F189L, and Q201R in Rce1p affected its substrate specificity.     

Rho Family GTPase modification and dependence on CAAX motif-signaled posttranslational modification

Rho GTPases (20 human members) comprise a major branch of the Ras superfamily of small GTPases, and aberrant Rho GTPase function has been implicated in oncogenesis and other human diseases. Although many of our current concepts of Rho GTPases are based on the three classical members (RhoA, Rac1, and Cdc42), recent studies have revealed the diversity of biological functions mediated by other family members. A key basis for the functional diversity of Rho GTPases is their association with distinct subcellular compartments, which is dictated in part by three posttranslational modifications signaled by their carboxyl-terminal CAAX (where C represents cysteine, A is an aliphatic amino acid, and X is a terminal amino acid) tetrapeptide motifs. CAAX motifs are substrates for the prenyltransferase-catalyzed addition of either farnesyl or geranylgeranyl isoprenoid lipids, Rce1-catalyzed endoproteolytic cleavage of the AAX amino acids, and Icmt-catalyzed carboxyl methylation of the isoprenylcysteine. We utilized pharmacologic, biochemical, and genetic approaches to determine the sequence requirements and roles of CAAX signal modifications in dictating the subcellular locations and functions of the Rho GTPase family. Although the classical Rho GTPases are modified by geranylgeranylation, we found that a majority of the other Rho GTPases are substrates for farnesyltransferase. We found that the membrane association and/or function of Rho GTPases are differentially dependent on Rce1- and Icmt-mediated modifications. Our results further delineate the sequence requirements for prenyltransferase specificity and functional roles for protein prenylation in Rho GTPase function. We conclude that a majority of Rho GTPases are targets for pharmacologic inhibitors of farnesyltransferase, Rce1, and Icmt.     

Heterologous Expression Studies of Saccharomyces cerevisiae Reveal Two Distinct Trypanosomatid CaaX Protease Activities and Identify Their Potential Targets

The CaaX tetrapeptide motif typically directs three sequential posttranslational modifications, namely, isoprenylation, proteolysis, and carboxyl methylation. In all eukaryotic systems evaluated to date, two CaaX proteases (Rce1 and Ste24/Afc1) have been identified. Although the Trypanosoma brucei genome also encodes two putative CaaX proteases, the lack of detectable T. brucei Ste24 activity in trypanosome cell extracts has suggested that CaaX proteolytic activity within this organism is solely attributed to T. brucei Rce1 (J. R. Gillespie et al., Mol. Biochem. Parasitol. 153:115-124. 2007). In this study, we demonstrate that both T. brucei Rce1 and T. brucei Ste24 are enzymatically active when heterologously expressed in yeast. Using a-factor and GTPase reporters, we demonstrate that T. brucei Rce1 and T. brucei Ste24 possess partially overlapping specificities much like, but not identical to, their fungal and human counterparts. Of interest, a CaaX motif found on a trypanosomal Hsp40 protein was not cleaved by either T. brucei CaaX protease when examined in the context of the yeast a-factor reporter but was cleaved by both in the context of the Hsp40 protein itself when evaluated using an in vitro radiolabeling assay. We further demonstrate that T. brucei Rce1 is sensitive to small molecules previously identified as inhibitors of the yeast and human CaaX proteases and that a subset of these compounds disrupt T. brucei Rce1-dependent localization of our GTPase reporter in yeast. Together, our results suggest the conserved presence of two CaaX proteases in trypanosomatids, identify an Hsp40 protein as a substrate of both T. brucei CaaX proteases, support the potential use of small molecule CaaX protease inhibitors as tools for cell biological studies on the trafficking of CaaX proteins, and provide evidence that protein context influences T. brucei CaaX protease specificity.Certain isoprenylated proteins are synthesized as precursors having a highly degenerate C-terminal tetrapeptide CaaX motif (C, cysteine; a, aliphatic amino acid; X, one of several amino acids). This motif typically directs three posttranslational modifications that include covalent attachment of an isoprenoid lipid to the cysteine residue, followed by endoproteolytic removal of the terminal three residues (i.e., aaX), and lastly, carboxyl methyl esterification of the farnesylated cysteine (49, 50). Relevant examples of proteins subject to the above modifications, also referred to as CaaX proteins, include the Ras and Ras-related GTPases, Gγ subunits, prelamin A, members of the Hsp40 family of chaperones, and fungal mating pheromones.Isoprenylation of CaaX proteins is performed by either the farnesyltransferase (FTase) or the geranylgeranyl transferase I (GGTase I). The particular isoprenoid attached, C15 farnesyl or C20 geranylgeranyl, respectively, depends in part on the sequence of the CaaX motif (8, 26, 31). Proteolysis of isoprenylated intermediates is carried out by the otherwise unrelated Rce1p (Ras converting enzyme 1) and Ste24p (sterile mutant 24) enzymes, collectively referred to as CaaX proteases, which are integral membrane proteins residing within the endoplasmic reticulum (3, 40, 45). Studies to elucidate the specificities of the CaaX proteases have often involved reporters designed from biological substrates (e.g., Ras GTPases) (2, 3, 16, 21, 22, 24, 34). Although these studies suggest that isoprenylated CaaX tetrapeptides alone are sufficient for recognition as a substrate, insufficient evidence exists to assert whether this sequence contains all of the necessary information for substrate specificity. Reporters are typically cleaved by either Rce1p or Ste24p. The Saccharomyces cerevisiae a-factor mating pheromone is a rather unusual biological reporter since it is cleaved by both yeast CaaX proteases. Orthologs of the CaaX proteases from humans, worms, and plants can also cleave a-factor when heterologously expressed in yeast, thereby making a-factor a convenient reporter for comparative analyses of CaaX protease activities (3, 5, 6, 36). Where evaluated using the a-factor reporter, Rce1p and Ste24p display partially overlapping target specificity, and this is an expected property of CaaX proteases in all eukaryotic systems (5, 6, 36, 47). Unlike the isoprenylation and proteolysis steps, carboxyl methyl esterification exclusively relies on a single enzyme, the isoprenylcysteine carboxyl methyltransferase (ICMT) (23, 50). A farnesylated cysteine appears to be the sole recognition determinant of the endoplasmic reticulum-localized ICMT (10, 23, 38).Disruption of the posttranslational modifications associated with CaaX proteins is often perceived as an anticancer strategy because of the prominent role of CaaX proteins in cellular transformation (i.e., the Ras GTPases) (49). To date, the most advanced drug discovery efforts have focused on farnesyltransferase inhibitors (FTIs) (9, 53). Inhibitors of the CaaX proteases and ICMT are also being developed (1, 11, 28, 37, 39, 48). Disrupting CaaX protein modifications has therapeutic application to other diseases as well. The relief of prelamin A toxicity by FTIs is a well-documented example (51). Accumulation of the farnesylated but unproteolysed precursor of lamin A results in a progeroid phenotype in individuals lacking ZmpSte24 proteolytic activity. The treatment of parasitic disease is another area under investigation (13). A number of FTIs have been developed that inhibit protozoan FTases, and in vivo testing is a continued effort (15, 32). Although research is less advanced with respect to CaaX protease and ICMT inhibitors, RNA interference experiments on the bloodstream form of Trypanosoma brucei indicate that CaaX processing enzymes are required for viability and proliferation of the parasite (20).In the present study, we evaluated the enzymatic properties of the trypanosomal CaaX proteases. We establish through the use of a variety of in vivo and in vitro assays that T. brucei Rce1 and T. brucei Ste24 are active when heterologously expressed in S. cerevisiae and have partially overlapping substrate specificities. The assays rely on various reporters, specifically the yeast a-factor mating pheromone, a K-Ras4B-based fluorogenic peptide, a green fluorescent protein (GFP)-GTPase fusion, and a T. brucei Hsp40 protein. All but the GTPase reporter could be effectively cleaved by both T. brucei CaaX proteases. We also demonstrate that the trypanosomal CaaX proteases can be targeted for inhibition by small molecules both in vitro and when heterologously expressed in yeast, suggesting that the trypanosomal CaaX proteases may be attractive drug targets for pharmacological inhibition.     

Rab GTPases containing a CAAX motif are processed post-geranylgeranylation by proteolysis and methylation

Post-translational modification by protein prenylation is required for membrane targeting and biological function of monomeric GTPases. Ras and Rho proteins possess a C-terminal CAAX motif (C is cysteine, A is usually an aliphatic residue, and X is any amino acid), in which the cysteine is prenylated, followed by proteolytic cleavage of the AAX peptide and carboxyl methylation by the Rce1 CAAX protease and Icmt methyltransferase, respectively. Rab GTPases usually undergo double geranylgeranylation within CC or CXC motifs. However, very little is known about processing and membrane targeting of Rabs that naturally contain a CAAX motif. We show here that a variety of Rab-CAAX proteins undergo carboxyl methylation, both in vitro and in vivo, with one exception. Rab38(CAKS) is not methylated in vivo, presumably because of the inhibitory action of the lysine residue within the AAX motif for cleavage by Rce1. Unlike farnesylated Ras proteins, we observed no targeting defects of overexpressed Rab-CAAX proteins in cells deficient in Rce1 or Icmt, as reported for geranylgeranylated Rho proteins. However, endogenous geranylgeranylated non-methylated Rab-CAAX and Rab-CXC proteins were significantly redistributed to the cytosol at steady-state levels and redistribution correlates with higher affinity of RabGDI for non-methylated Rabs in Icmt-deficient cells. Our data suggest a role for methylation in Rab function by regulating the cycle of Rab membrane recruitment and retrieval. Our findings also imply that those Rabs that undergo post-prenylation processing follow an indirect targeting pathway requiring initial endoplasmic reticulum membrane association prior to specific organelle targeting.     

Engineered variants of the Ras effector protein RASSF5 (NORE1A) promote anticancer activities in lung adenocarcinoma

Within the superfamily of small GTPases, Ras appears to be the master regulator of such processes as cell cycle progression, cell division, and apoptosis. Several oncogenic Ras mutations at amino acid positions 12, 13, and 61 have been identified that lose their ability to hydrolyze GTP, giving rise to constitutive signaling and eventually development of cancer. While disruption of the Ras/effector interface is an attractive strategy for drug design to prevent this constitutive activity, inhibition of this interaction using small molecules is impractical due to the absence of a cavity to which such molecules could bind. However, proteins and especially natural Ras effectors that bind to the Ras/effector interface with high affinity could disrupt Ras/effector interactions and abolish procancer pathways initiated by Ras oncogene. Using a combination of computational design and in vitro evolution, we engineered high-affinity Ras-binding proteins starting from a natural Ras effector, RASSF5 (NORE1A), which is encoded by a tumor suppressor gene. Unlike previously reported Ras oncogene inhibitors, the proteins we designed not only inhibit Ras-regulated procancer pathways, but also stimulate anticancer pathways initiated by RASSF5. We show that upon introduction into A549 lung carcinoma cells, the engineered RASSF5 mutants decreased cell viability and mobility to a significantly greater extent than WT RASSF5. In addition, these mutant proteins induce cellular senescence by increasing acetylation and decreasing phosphorylation of p53. In conclusion, engineered RASSF5 variants provide an attractive therapeutic strategy able to oppose cancer development by means of inhibiting of procancer pathways and stimulating anticancer processes.     

Disruption of the mouse Rce1 gene results in defective Ras processing and mislocalization of Ras within cells

Little is known about the enzyme(s) required for the endoproteolytic processing of mammalian Ras proteins. We identified a mouse gene (designated Rce1) that shares sequence homology with a yeast gene (RCE1) implicated in the proteolytic processing of Ras2p. To define the role of Rce1 in mammalian Ras processing, we generated and analyzed Rce1-deficient mice. Rce1 deficiency was lethal late in embryonic development (after embryonic day 15.5). Multiple lines of evidence revealed that Rce1-deficient embryos and cells lacked the ability to endoproteolytically process Ras proteins. First, Ras proteins from Rce1-deficient cells migrated more slowly on SDS-polyacrylamide gels than Ras proteins from wild-type embryos and fibroblasts. Second, metabolic labeling of Rce1-deficient cells revealed that the Ras proteins were not carboxymethylated. Finally, membranes from Rce1-deficient fibroblasts lacked the capacity to proteolytically process farnesylated Ha-Ras, N-Ras, and Ki-Ras or geranylgeranylated Ki-Ras. The processing of two other prenylated proteins, the farnesylated Ggamma1 subunit of transducin and geranylgeranylated Rap1B, was also blocked. The absence of endoproteolytic processing and carboxymethylation caused Ras proteins to be mislocalized within cells. These studies indicate that Rce1 is responsible for the endoproteolytic processing of the Ras proteins in mammals and suggest a broad role for this gene in processing other prenylated CAAX proteins.     

Inhibition of the CaaX proteases Rce1p and Ste24p by peptidyl (acyloxy)methyl ketones

The CaaX proteases Rce1p and Ste24p can independently promote a proteolytic step required for the maturation of certain isoprenylated proteins. Although functionally related, Rce1p and Ste24p are unrelated in primary sequence. They have distinct enzymatic properties, which are reflected in part by their distinct inhibitor profiles. Moreover, Rce1p has an undefined catalytic mechanism, whereas Ste24p is an established zinc-dependent metalloprotease. This study demonstrates that both enzymes are inhibited by peptidyl (acyloxy)methyl ketones (AOMKs), making these compounds the first documented dual specificity inhibitors of the CaaX proteases. Further investigation of AOMK-mediated inhibition reveals that varying the peptidyl moiety can significantly alter the inhibitory properties of AOMKs toward Rce1p and Ste24p and that these enzymes display subtle differences in sensitivity to AOMKs. This observation suggests that this compound class could potentially be engineered to be selective for either of the CaaX proteases. We also demonstrate that the reported sensitivity of Rce1p to TPCK is substrate-dependent, which significantly alters the interpretation of certain reports having used TPCK sensitivity for mechanistic classification of Rce1p. Finally, we show that an AOMK inhibits the isoprenylcysteine carboxyl methyltransferase Ste14p. In sum, our observations raise important considerations regarding the specificity of agents targeting enzymes involved in the maturation of isoprenylated proteins, some of which are being developed as anti-cancer therapeutic agents.     

Restricted Substrate Specificity for the Geranylgeranyltransferase-I Enzyme in Cryptococcus neoformans: Implications for Virulence

Proper cellular localization is required for the function of many proteins. The CaaX prenyltransferases (where CaaX indicates a cysteine followed by two aliphatic amino acids and a variable amino acid) direct the subcellular localization of a large group of proteins by catalyzing the attachment of hydrophobic isoprenoid moieties onto C-terminal CaaX motifs, thus facilitating membrane association. This group of enzymes includes farnesyltransferase (Ftase) and geranylgeranyltransferase-I (Ggtase-1). Classically, the variable (X) amino acid determines whether a protein will be an Ftase or Ggtase-I substrate, with Ggtase-I substrates often containing CaaL motifs. In this study, we identify the gene encoding the β subunit of Ggtase-I (CDC43) and demonstrate that Ggtase-mediated activity is not essential. However, Cryptococcus neoformans CDC43 is important for thermotolerance, morphogenesis, and virulence. We find that Ggtase-I function is required for full membrane localization of Rho10 and the two Cdc42 paralogs (Cdc42 and Cdc420). Interestingly, the related Rac and Ras proteins are not mislocalized in the cdc43Δ mutant even though they contain similar CaaL motifs. Additionally, the membrane localization of each of these GTPases is dependent on the prenylation of the CaaX cysteine. These results indicate that C. neoformans CaaX prenyltransferases may recognize their substrates in a unique manner from existing models of prenyltransferase specificity. It also suggests that the C. neoformans Ftase, which has been shown to be more important for C. neoformans proliferation and viability, may be the primary prenyltransferase for proteins that are typically geranylgeranylated in other species.     

Modulation of the inhibitor properties of dipeptidyl (acyloxy)methyl ketones toward the CaaX proteases

Dipeptidyl (acyloxy)methyl ketones (AOMKs) have been identified as mechanism-based inhibitors of certain cysteine proteases. These compounds are also inhibitors of the integral membrane proteins Rce1p and Ste24p, which are proteases that independently mediate a cleavage step associated with the maturation of certain isoprenylated proteins. The enzymatic mechanism of Rce1p is ill-defined, whereas Ste24p is a zinc metalloprotease. Rce1p is required for the proper processing of the oncoprotein Ras and is viewed as a potential target for cancer therapy. In this study, we synthesized a small library of dipeptidyl AOMKs to investigate the structural elements that contribute to the inhibitor properties of this class of molecules toward Rce1p and Ste24p. The compounds were evaluated using a fluorescence-based in vitro proteolysis assay. The most potent dipeptidyl AOMKs contained an arginine residue and the identity of the benzoate group strongly influenced potency. A ‘warhead’ free AOMK inhibited Rce1p and Ste24p. The data suggest that the dipeptidyl AOMKs are not mechanism-based inhibitors of Rce1p and Ste24p and corroborate the hypothesis that Rce1p is not a cysteine protease.     

Genetic evidence for in vivo cross-specificity of the CaaX-box protein prenyltransferases farnesyltransferase and geranylgeranyltransferase-I in Saccharomyces cerevisiae.

Two protein prenyltransferase enzymes, farnesyltransferase (FTase) and geranylgeranyltransferase-I (GGTase-I), catalyze the covalent attachment of a farnesyl or geranylgeranyl lipid group to the cysteine of a CaaX sequence (cysteine [C], two aliphatic amino acids [aa], and any amino acid [X]. In vitro studies reported here confirm previous reports that CaaX proteins with a C-terminal serine are farnesylated by FTase and those with a C-terminal leucine are geranylgeranylated by GGTase-I. In addition, we found that FTase can farnesylate CaaX proteins with a C-terminal leucine and can transfer a geranylgeranyl group to some CaaX proteins. Genetic data indicate that FTase and GGTase-I have the same substrate preferences in vivo as in vitro and also show that each enzyme can prenylate some of the preferred substrates of the other enzyme in vivo. Specifically, the viability of yeast cells lacking FTase is due to prenylation of Ras proteins by GGTase-I. Although this GGTase-I dependent prenylation of Ras is sufficient for growth, it is not sufficient for mutationally activated Ras proteins to exert deleterious effects on growth. The dependence of the activated Ras phenotype on FTase can be bypassed by replacing the C-terminal serine with leucine. This altered form of Ras appears to be prenylated by both GGTase-I and FTase, since it produces an activated phenotype in a strain lacking either FTase or GGTase-I. Yeast cells can grow in the absence of GGTase-I as long as two essential substrates are overexpressed, but their growth is slow. Such strains are dependent on FTase for viability and are able to grow faster when FTase is overproduced, suggesting that FTase can prenylate the essential substrates of GGTase-I when they are overproduced.     

AtFACE-2, a functional prenylated protein protease from Arabidopsis thaliana related to mammalian Ras-converting enzymes

Eukaryotic proteins containing a CAAX (A is aliphatic amino acid) C-terminal tetrapeptide sequence generally undergo a lipid modification, the addition of a prenyl group. Proteins that are modified by prenylation, such as Ras GTPases, can be subsequently modified by a proteolytic event that removes a C-terminal tripeptide (AAX). Two distinct proteases have been identified that are involved in the CAAX proteolytic step, FACE-1/Ste24 and FACE-2/Rce1. These proteases have different enzymatic properties, substrate specificities, and biological functions. However, a proposal has been made that plants lack a FACE-2/Rce1-type protease. Here, we describe the isolation of a cDNA from Arabidopsis thaliana that encodes a 311-aa protein with characteristics that are similar to the FACE-2/Rce1 group of enzymes. Northern blot analysis demonstrates widespread expression of this gene in plant tissues. Heterologous expression of the A. thaliana cDNA in yeast restores CAAX proteolytic activity to yeast lacking native CAAX proteases. The recombinant protein produced in this system displays an in vivo substrate specificity profile distinct from AtSte24 and cleaves a farnesylated CAAX tetrapeptide in vitro. These results provide evidence for the existence of a previously unsuspected plant FACE-2/Rce1 ortholog and support the evolutionary conservation of dual CAAX proteolytic systems in eukaryotes.     

Photoaffinity labeling of Ras converting enzyme 1 (Rce1p) using a benzophenone-containing peptide substrate

Isoprenylation is a post-translational modification that increases protein hydrophobicity and helps target certain proteins to membranes. Ras converting enzyme 1 (Rce1p) is an endoprotease that catalyzes the removal of a three residue fragment from the C-terminus of isoprenylated proteins. To obtain structural information about this membrane protein, photoaffinity labeling agents are being prepared and employed. Here, we describe the synthesis of a benzophenone-containing peptide substrate analogue for Rce1p. Using a continuous spectrofluorometric assay, this peptide was shown to be a substrate for Rce1p. Mass spectrometry was performed to confirm the site of cleavage and structure of the processed probe. Photolysis of the biotinylated compound in the presence of membranes containing Rce1p followed by streptavidin pull-down and Western blot analysis indicated that Rce1p had been labeled by the probe. Photolysis in the presence of both the biotinylated, benzophenone-containing probe and a farnesylated peptide competitor reduced the extent of labeling, suggesting that labeling is occurring in the active site.     

Absence of the CAAX Endoprotease Rce1: Effects on Cell Growth and Transformation

After isoprenylation, the Ras proteins and other CAAX proteins undergo two additional enzymatic modifications-endoproteolytic release of the last three amino acids of the protein by the protease Rce1 and methylation of the carboxyl-terminal isoprenylcysteine by the methyltransferase Icmt. This postisoprenylation processing is thought to be important for the association of Ras proteins with membranes. Blocking postisoprenylation processing, by inhibiting Rce1, has been suggested as a potential approach for retarding cell growth and blocking cellular transformation. The objective of this study was to develop a cell culture system for addressing these issues. We generated mice with a conditional Rce1 allele (Rce1(flox)) and produced Rce1(flox/flox) fibroblasts. Cre-mediated excision of Rce1 (thereby producing Rce1(Delta/Delta) fibroblasts) eliminated Ras endoproteolytic processing and methylation and caused a partial mislocalization of truncated K-Ras and H-Ras fusion proteins within cells. Rce1(Delta/Delta) fibroblasts grew more slowly than Rce1(flox/flox) fibroblasts. The excision of Rce1 also reduced Ras-induced transformation, as judged by the growth of colonies in soft agar. The excision of Rce1 from a Rce1(flox/flox) skin carcinoma cell line also significantly retarded the growth of cells, and this effect was exaggerated by cotreatment of the cells with a farnesyltransferase inhibitor. These studies support the idea that interference with postisoprenylation processing retards cell growth, limits Ras-induced transformation, and sensitizes tumor cells to a farnesyltransferase inhibitor.     

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 IC(50) values of these 9 compounds were in the low micromolar range for both yeast (6-35 microM) and human Rce1p (0.4-46 microM). 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.     

Characterization of a Peptide that Specifically Blocks the Ras Binding Domain of p75

The neurotrophin receptor p75 interacts with the GTPase Ras. Unstimulated it inactivates Ras while ligand binding induces Ras activation. We developed an inhibitory peptide (ip75RBD) which interferes with the binding domain of Ras of the intracellular domain of p75. ip75RBD inhibits the binding of Ras to the receptor in vitro. It is membrane-permeable and inhibits ligand-induced Ras activation via p75 in vivo but does not influence Ras activation by the stimulated receptor tyrosine kinases Trk and the epidermal growth factor receptor EGFR. The activation of the neutral sphingomyelinase by stimulated p75 is slightly delayed but not inhibited by the peptide. p75-mediated neuronal death induced by NGF or aggregated beta-amyloid_1–42 is reduced. We conclude that ip75RBD specifically blocks the Ras binding site of p75 and can be used to analyze p75-induced Ras signaling.     

Rce1 suppresses invasion and metastasis of hepatocellular carcinoma via epithelial-mesenchymal transition induced by the TGF-β1/H-Ras signaling pathway

Ras converting enzyme 1 (Rce1) plays an important role in invasion and metastasis of malignancy. However, the mechanism has not yet been fully explored in hepatocellular carcinoma (HCC). Primarily, we investigated the expression of Rce1 and H-Ras influence on patient prognosis through the clinical data. Further, we analyzed the regulatory effects of Rce1/H-Ras signal pathway on the epithelial–mesenchymal transition (EMT) in vitro and in vivo. Finally, we screened out the protein which bonds with Rce1 by CO-IP experiment to discuss the mechanism of Rce1 in EMT of HCC. This research revealed a significantly decreased expression of Rce1 in HCC compared with noncancerous tissues (p < .05). In contrast, H-Ras expression was increased in the tumor. The expression of them was a close association with the differentiation and tumor-node-metastasis (TNM) stage of the tumor (p < .001; p = .035, respectively) and Rce1 was an independent prognostic indicator (95%Cl: 0.193–0.821; p = .013). Through targeted regulation of Rce1 by cDNA or small interfering RNA, results show that the lower expression of Rce1 facilitated EMT and promoted the invasion and metastasis of HCC (p < .05). Furthermore, the CO-IP experiment unfolded that Rce1 could bond with farnesyltransferase-β (FNTB) which mediated the expression of H-Ras. Conclusions: Rce1 inhibits EMT via target regulation H-Ras and suppress the early invasion and metastasis of HCC. It may be a potential therapeutic target and prognostic indicator for HCC.     

Chaperone-mediated specificity in Ras and Rap signaling

Abstract

Ras and Rap proteins are closely related small guanosine triphosphatase (GTPases) that share similar effector-binding domains but operate in a very different signaling networks; Ras has a dominant role in cell proliferation, while Rap mediates cell adhesion. Ras and Rap proteins are regulated by several shared processes such as post-translational modification, phosphorylation, activation by guanine exchange factors and inhibition by GTPase-activating proteins. Sub-cellular localization and trafficking of these proteins to and from the plasma membrane are additional important regulatory features that impact small GTPases function. Despite its importance, the trafficking mechanisms of Ras and Rap proteins are not completely understood. Chaperone proteins play a critical role in trafficking of GTPases and will be the focus of the discussion in this work. We will review several aspects of chaperone biology focusing on specificity toward particular members of the small GTPase family. Understanding this specificity should provide key insights into drug development targeting individual small GTPases.