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IF102-1B – RGGT Inhibition

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IF102-1B – RGGT Inhibition View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Access to Research and Communications Annals Phosphonocarboxylates inhibit the second geranylgeranyl addition by Rab Geranylgeranyl Transferase Rudi A. Baron1*, Richard Tavaré1§, Ana C. Figueiredo1, Katarzyna Błażewska2, Boris A. Kashemirov2, Charles E. McKenna2, Frank H. Ebetino3, Adam Taylor4, Michael J. Rogers4, Fraser P. Coxon4, and Miguel C. Seabra1,5,6¶ 1 Molecular Medicine, National Heart and Lung Institute, Imperial College London, London SW7 2AZ, UK, 2 Department of Chemistry, University of Southern California, Los Angeles, California 90089-0744, 3 Procter & Gamble Pharmaceuticals, Cincinnati, Ohio, USA, 4 Bone & Musculoskeletal Programme, Institute of Medical Sciences, University of Aberdeen, Aberdeen, AB25 2ZD, UK, 5Instituto Gulbenkian de Ciência, 2780-156 Oeiras, Portugal, 6Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169-056 Lisboa, Portugal * Present address: Cerenis Therapeutics, 31682 Labège, France § Present address: Kings College London, Division of Imaging sciences, The Rayne Institute, 4th floor, Lambeth Wing, St. Thomas Hospital, London SE1 7EH ¶Address correspondence to Miguel C. Seabra: email: [email protected] Running title: Mechanism of RGGT inhibition by bisphosphonates Rab geranylgeranyl transferase demonstrate directly that in the presence of (RGGT) catalyzes the post-translational (+)-3-IPEHPC, Rab-CC and Rab-CXC geranylgeranyl (GG) modification of (usually) proteins are modified by only a single GG two C-terminal cysteines in Rab GTPases. addition. The presence of (+)-3-IPEHPC Here we studied the mechanism of the Rab resulted in a preference for the Rab N- geranylgeranylation reaction by terminal cysteine to be modified first, bisphosphonate analogs in which one suggesting an order of cysteine phosphonate group is replaced by a geranylgeranylation in RGGT catalysis. Our carboxylate (phosphonocarboxylate, PC). The results further suggest that the inhibitor binds phosphonocarboxylates used were 3-PEHPC, to a site distinct from the GGPP-binding site which was previously reported, and (+)-3- on RGGT. We suggest that IPEHPC, a >25-fold more potent related phosphonocarboxylate inhibitors bind to a compound as measured by both IC50 and Ki. GG-cysteine binding site adjacent to the (+)-3-IPEHPC behaves as a mixed-type active site, which is necessary to align the inhibitor with respect to GG pyrophosphate monoGG-Rab for the second GG addition. (GGPP) and an uncompetitive inhibitor with These inhibitors may represent a novel respect to Rab substrates. We propose that therapeutic approach in Rab-mediated phosphonocarboxylates prevent only the diseases. second GG transfer onto Rabs based on the following evidence. First, geranylgeranylation Most proteins of the Ras-like GTPase of Rab proteins ending with a single cysteine superfamily need to be post-translationally motif such as CaaX, is not affected by the modified by prenyl groups in order to associate inhibitors, either in vitro or in vivo. Second, with cellular membranes and to activate the addition of an -aaX sequence onto Rab- downstream effectors (1). Protein prenylation CC proteins protects the substrate from involves the formation of a thioether link inhibition by the inhibitors. Third, we between conserved C-terminal cysteines in protein substrates and farnesyl pyrophosphate report the surprising finding that (FPP) or geranylgeranyl pyrophosphate (GGPP) phosphonocarboxylate inhibitors of RGGT act (2,3). These prenyl pyrophosphates, which by inhibiting only the second Rab originate from the mevalonate pathway are geranylgeranylation event, therefore providing utilized by three different prenyltransferase specificity towards Rab proteins with a double enzymes (2,3). Farnesyl transferase (FT) and cysteine C-terminal motif. Geranylgeranyl transferase type I (GGT-I) transfer FPP or GGPP, respectively, to a cysteine Experimental procedures residue in the context of a C-terminal CaaX motif, where C is a cysteine, a is an aliphatic Plasmid constructs – Human REP1 residue and X is any amino acid. X contributes cDNA was cloned into pFastBacHTb and significantly to substrate specificity in FT and produced as described previously (13). Rat GGT-I (2). Rab geranylgeranyl transferase RGGT was prepared as described previously (RGGT) is distinct from FT and GGT-I in that it (13). Canine Rab1a, human Rab27a, human specifically recognizes Rab proteins, which vary Rab5a, human Rab18, human Rab6a were in their C-terminus containing CCXX, XCXC, amplified by PCR and cloned into pET14b and XXCC, CCXXX, CaaX and other motifs (3). human Rab13 and mouse Rab23 were cloned RGGT exhibits exquisite specificity for Rabs into pGEX-4T-1 as described previously (14). due to the strict requirement of Rab Escort The indicated C-terminal sequence mutants were Protein (REP), which is a general Rab-GDP obtained by using the Quickchange mutagenesis binding protein (4). RGGT is a heterodimeric kit (Stratagene). The sequences of all plasmid enzyme consisting of a 60 kDa α-subunit and a constructs used were confirmed by DNA 38 kDa β-subunit, and shares 30% homology sequencing. with its FT and GGT-I counterparts (5). The α- Recombinant Proteins - The subunit associates with REP in a very small area recombinant proteins RGGT and REP1 were compared to the entire surface occupied by the prepared by infection of Sf9 cells with complex, while the β-subunit binds one molecule recombinant baculoviruses encoding each of GGPP in a large hydrophobic cavity (5,6). subunit of the desired enzyme and purified by Although a large number of specific FT nickel Sepharose-affinity chromatography as and GGT-I inhibitors have been developed as described previously (13,15). Recombinant potential anti-cancer agents (7,8), few inhibitors 6xHis and GST-tagged Rab proteins were of RGGT exist. Recently the purified by nickel sepharose or glutathione phosphonocarboxylate 3-PEHPC, was identified sepharose affinity as described previously as a specific RGGT inhibitor, thereby selectively (16,17). All recombinant proteins were snap preventing the prenylation of Rab proteins in frozen in small aliquots and stored at - 80°C until cells (9,10). This compound was derived from use. the bisphosphonate (BP), risedronate, which is Bisphosphonates - 2-Hydroxy-2- used clinically in the treatment of osteoporosis, phosphono-3-pyridin-3-yl-propionic acid (3- due to its ability to potently inhibit the activity of PEHPC, previously referred to as NE-10790) [1] bone-resorbing osteoclasts (11). Although BPs was a gift from Procter & Gamble such as risedronate prevent Rab prenylation, Pharmaceuticals. 2-Hydroxy-3-imidazo[1,2- they do not act by inhibiting RGGT. Rather, a]pyridin-3-yl-2-phosphonopropionic acid (3- these drugs prevent the synthesis of FPP and IPEHPC) [2] was synthesized and resolved into GGPP by inhibiting FPP synthase, thereby its component enantiomers by methods reported preventing all protein prenylation (12). 3- elsewhere (McKenna et al., manuscript in PEHPC is a weak RGGT inhibitor and preparation).The (+)-enantiomer was used in the consequently a weak inhibitor of bone present study, since this has been shown to be resorption. However, we have recently identified selective towards RGGT, and a more potent a similar phosphonocarboxylate, (+)-3-IPEHPC, inhibitor of this enzyme than the (-)-enantiomer which is at least 25 times more potent than 3- (McKenna et al., manuscript in preparation). The PEHPC as an inhibitor of RGGT. Here, we enantiopurity was determined to be > 98 % by chiral HPLC using a Prontosil AX QN (eluted parameter curve fitting for all inhibitor isocratically with 0.7 M TEAA buffer containing concentrations using the average of duplicate 75 % MeOH at pH 5.8; UV detection at 265 nm, determinations. The error represents the flow rate 3 ml/min). The more active (+)- divergence between fitted curves. enantiomer elutes first. The specific optical Proteolytic digestion of prenylated Rab 25 rotation [α]D +109 ° was measured on a Jasco proteins and peptide analysis - The peptides J100 Polarimeter. were prepared essentially as described N OH previously (19,20). The RGGT mixture (25μl), CO H 2 N 2μM REP1, 50nM RGGT, 5μM Rab proteins, PO3H2 5μM GGPP (specific activity = 1600 PO3H2 N OH dpm/pmole) was precipitated by addition of cold CO2H acetone (1ml) for 1h at 4°C. The pellet was washed with 1ml of cold acetone and 1, 3-PEHPC 2, 3-IPEHPC resuspended in 100μl of 25mM Tris-HCl (pH 8), 4% CHAPS, 1mM EDTA and 3μg of In vitro prenylation assay - RGGT endoproteinase C-Lys (Boehringer Mannheim) activity was measured by determining the and incubated overnight at 37°C. The resulting 3 amount of [ H] GGPP transferred to Rab peptides were isolated on a reverse-phase μRPC proteins (15,18). Unless otherwise indicated, the C2/C18 SC 2.1/10 column (Pharmacia) using a standard reaction mixture contained the SMART System chromatography instrument following concentrations in a final volume of (Pharmacia). The column was equilibrated in 25μl: 50mM sodium HEPES (pH 7.2), 5mM MilliQ water with 0.06% trifluoroacetic acid MgCl2, 1mM Nonidet P-40, 1mM (TFA) at room temperature at a flow rate of 3 dithioerythritol (DTE), 5μM [ H] GGPP 200μl/min. 50μl of sample were injected and the (specific activity = 800 dpm/pmole), 4μM Rab peptides were separated by a 3.5ml linear proteins, 2μM REP1 and 50nM RGGT. After 20 gradient (0-70% acetonitrile) and 2.5ml of 100% min at 37°C, the reaction was stopped by acetonitrile (0.056% TFA).
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