Molecular & Biochemical Parasitology 153 (2007) 115–124

C-terminal of prenylated in trypanosomatids and RNA interference of enzymes required for the post-translational processing pathway of farnesylated proteins John R. Gillespie a, Kohei Yokoyama b,∗, Karen Lu a, Richard T. Eastman c, James G. Bollinger b,d, Wesley C. Van Voorhis a,c, Michael H. Gelb b,d, Frederick S. Buckner a,∗∗ a Department of Medicine, University of Washington, 1959 N.E. Pacific St., Seattle, WA 98195, USA b Department of Chemistry, University of Washington, Seattle, WA 98195, USA c Department of Pathobiology, University of Washington, Seattle, Washington 98195, USA d Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA Received 22 December 2006; received in revised form 17 February 2007; accepted 26 February 2007 Available online 1 March 2007

Abstract The C-terminal “CaaX”-motif-containing proteins usually undergo three sequential post-translational processing steps: (1) attachment of a prenyl group to the residue; (2) proteolytic removal of the last three amino acids “aaX”; (3) methyl esterification of the exposed ␣-carboxyl group of the prenyl-cysteine residue. The Trypanosoma brucei and Leishmania major Ras converting enzyme 1 (RCE1) orthologs of 302 and 285 amino acids-proteins, respectively, have only 13–20% sequence identity to those from other species but contain the critical residues for the activity found in other orthologs. The Trypanosoma brucei a-factor converting enzyme 1 (AFC1) ortholog consists of 427 amino acids with 29–33% sequence identity to those of other species and contains the consensus HExxH zinc-binding motif. The trypanosomatid RCE1 and AFC1 orthologs contain predicted transmembrane regions like other species. Membranes from Sf9 cells expressing the RCE1 ortholog of T. brucei or L. major showed proteolytic activity against farnesylated RAS-CVIM, whereas membranes containing T. brucei AFC1 ortholog were inactive. The results suggest that RCE1 is responsible for proteolytic removal of the C-terminal aaX from prenyl-CaaX proteins in these parasites. All the three enzymatic post-translational processes are thought to be required for proper cellular functioning of CaaX-proteins in eukaryotic cells. We carried out RNA interference experiments in Trypanosoma brucei of the enzymes involved in farnesyl post-translational modification to evaluate their importance in cell proliferation. Knockdown of T. brucei PFT ␤ subunit and RCE1 mRNAs resulted in >20-fold suppression of cell growth and dramatic morphologic changes. Knockdown of PPMT mRNA caused less dramatic effects on growth but induced noticeable changes in cell morphology. © 2007 Published by Elsevier B.V.

Keywords: Trypanosoma brucei; Leishmania major; Protein prenylation; Protein ; Ras converting enzyme; A-factor converting enzyme; RNA interference

1. Introduction

Abbreviations: RCE1, ras converting enzyme 1 ortholog; AFC1, a- Prenylation (farnesylation and geranylgeranylation) and factor converting enzyme 1 ortholog; PFT, protein farnesyltransferase; PPMT, subsequent modifications are required for proper membrane prenyl protein carboxyl methyltransferase; RNAi, RNAinterference; T. brucei, Trypanosoma brucei; L. major, Leishmania major; AdoMet, S-adenosyl-l- targeting and cellular functioning of a number of proteins in eukaryotic cells such as Ras superfamily GTPases [1,2]. ∗ Corresponding author at: Department of Chemistry, University of Washing- For proteins with the C-terminal CaaX motif (where C is ton, Campus Box 351700, Seattle, WA 98195-7185, USA. cysteine, a is usually an aliphatic , and X is a vari- Tel.: +1 206 616 9214; fax: +1 206 685 8681. ety of residues), farnesylation or geranylgeranylation at the ∗∗ Corresponding author. Tel.: +1 206 616 4264; fax: +1 206 685 8665. E-mail addresses: [email protected] (K. Yokoyama), Cys residue by protein farnesyltransferase (PFT) or protein [email protected] (F.S. Buckner). geranylgeranyltransferase-I is a prerequisite for further enzy-

0166-6851/$ – see front matter © 2007 Published by Elsevier B.V. doi:10.1016/j.molbiopara.2007.02.009 116 J.R. Gillespie et al. / Molecular & Biochemical Parasitology 153 (2007) 115–124 matic processing, i.e. the proteolytic removal of the last three of several farnesylated proteins such as K-Ras [34], residues “aaX” followed by methylation of exposed ␣-carboxyl transducin ␥ subunit [35,36], and B [24] has been shown to group of the prenylcysteine [3–5]. be critical for activating downstream effectors. It is also notable Two different enzymes have been discovered to be involved that disruption of PPMT causes a decrease in the half-life of in removal of the tripeptide “aaX” in Saccharomyces cere- RhoA but stabilizes Ras in mammalian cells [37,38]. visiae [6], namely, RCE1 (Ras converting enzyme 1) and AFC1 We report here the cloning and characterization of RCE1 (a-factor converting enzyme 1, also called Zmpste24). These orthologs from the protozoa Trypanosoma brucei (T. brucei) and two enzymes show overlapping but distinct specificity toward Leishmania major (L. major), the caustative agents of African the CaaX sequence [7,8]. RCE1 seems to process most of the sleeping sickness and certain forms of leishmaniasis, respec- naturally occurring CaaX sequences of both farnesyl and ger- tively. We also report the cloning and characterization of the anylgeranyl proteins [7,9–11]. Disruption of the RCE1 gene AFC1 ortholog from T. brucei. Heterologous expression of T. in mice resulted in death at the embryonic stage, and fibrob- brucei and L. major RCE1 and T. brucei AFC1 and in vitro lasts from the mouse embryo failed to proteolyze prenylated enzymatic studies suggest that RCE1 is the enzyme responsible Ras, Rap, and transducin ␥, suggesting that RCE1 is indis- for removal of “aaX” from prenylated proteins in these parasites. pensable for maturation of these GTPases [11]. On the other We have previously reported the cloning of T. brucei PFT ␣ and ␤ hand, AFC1 cleaves the a-factor precursor (at a site that is 26 subunits [39] and PPMT [40]. Thus, the full set of enzymes in the residues upstream of farnesylcysteine) after removal of “aaX” post-translational processing of farnesylated proteins is present and carboxyl methylation [11,12]. Human and S. cerevisiae in this protozoan. In our previous studies, T. brucei and AFC1 orthologs have also been shown to have ability to remove parasites showed high sensitivity to PFT inhibitors compared “aaX” from the farnesylated a-factor precursor in the yeast cells to mammalian cells, suggesting that PFT as well as subsequent lacking the RCE1 gene [7,8], but RCE1 is thought to be the processing enzymes may be excellent drug targets against these major enzyme responsible for removal of “aaX” from proteins parasites [39,41–44]. To evaluate the potential of these enzymes in yeast and mammalian cells. RCE1 and AFC1 orthologs from as targets for the development of anti-parasite drugs, we have several species have been reported [4,9,13–15]. A substrate of carried out RNA interference experiments with T. brucei PFT human AFC1 has been identified to be farnesylated prelamin ␤-subunit, RCE1 and PPMT. A, which undergoes two cleavages like yeast a-factor; removal of “aaX” and then cleavage at a site 15 residues upstream of 2. Materials and methods farnesylcysteine [12,16]. Failure of the proteolytic processing due to mutations of the prelamin A cleavage site [17] or the 2.1. Materials AFC1 enzyme [18,19], which have been found in the genetic diseases Hutchinson–Gilford and mandibuloacral dys- RAS-CVIM was expressed in E. coli and purified as plasia, results in aberrant accumulation of farnesyl-prelamin A described [44], and T. brucei PFT was produced in the bac- in the nucleus and abnormal nucleus morphology [20–22].A ulovirus/Sf9 insect cell system and purified as described [45]. similar phenotype and defect in prelamin A processing were T. brucei PPMT was expressed in Sf9 cells, and the mem- observed in AFC1 deficient mice, indicating that AFC1 is indis- branes containing the enzyme were prepared as described pensable for the processing of prelamin A [23]. Lamin B1 has [40]. Sf9 cell membranes containing recombinant S. cerevisiae been shown to be processed only by RCE1 based on studies using PPMT (ICMT) were obtained as a generous gift from Dr. C. embryonic fibroblasts from mice with homozygous deficiency Hrycyna (Purdue Univ.). S-Adenosyl-l-[methyl-3H]methionine of either RCE1 or AFC1 [24]. ([3H]AdoMet) was purchased from NEN/Parkin-Elmer. N- Recent studies with mouse embryonic fibroblasts lacking Acetyl-S-farnesyl-l-cysteine (AFC) and RCE1 suggest that removal of “aaX” is required for proper (FPP) were from BIOMOL. Cysmethynil is a generous gift from localization of farnesylated Ras but not geranylgeranylated Dr. P. Casey (Duke Univ.). Rho GTPases [25]. Farnesylated K-Ras is also mislocalized in mammalian cells lacking prenyl-protein specific carboxyl 2.2. Cloning of RCE1 orthologs of T. brucei and L. major, methyltransferase (PPMT, also called as ICMT) [26]. These and an AFC1 ortholog of T. brucei reports and studies with farnesylated and geranylgeranylated short peptides [27,28] suggest the importance of ␣-carboxyl The genes described herein were cloned prior to completion methylation, particularly for farnesylated proteins as an addi- of the genome projects for T. brucei and L. major. All genes were tional hydrophobic element to cause tight and proper membrane cloned by the following methods: first, fragments of the targeted binding. genes were identified in GenBank by TBLASTN search using RCE1 and PPMT localize to the endoplasmic reticulum sequences of RCE1 (GI:6323930) and AFC1 (GI:1352918) of [9,29–31] and act on both farnesylated and geranylgeranylated Saccharomyces cerevisiae; second, the remaining 5- and 3- proteins [32]. Gene disruption of PPMT in mice results in embry- ends of the genes were amplified off cDNA extracted from T. onic lethality [33] as seen with RCE1 disruption. Since the brucei BF427 or L. major Friedlin promastigotes using spliced- carboxyl methylation is thought to be reversible, although a spe- leader RACE and oligo-dT RACE [46]; third, the full length cific esterase has not been found, PPMT has been implicated in genes were PCR amplified from genomic DNA, and 2 clones functional regulation of certain prenylated proteins. Carboxyl were fully sequenced for each target. J.R. Gillespie et al. / Molecular & Biochemical Parasitology 153 (2007) 115–124 117

2.3. Expression of the proteins in the Sf9/baculovirus was stopped with 200 ␮l of 10% concentrated aqueous HCl system in ethanol. Incorporation of [3H]methyl group into the pro- tein product was measured by the glass fiber filter method as According to the manufacturer’s instructions for the Bac-to- described for PFT assay [44] using 1 mM HCl in ethanol as Bac Baculovirus expression system (Invitrogen), the genes for washing solvent. Error values of this assay are typically lower RCE1 orthologs of T. brucei (isoform 2) and L. major and an than 10% for the same series of measurement. AFC1 ortholog of T. brucei were transferred to pFastBac, which were then transposed into a bacmid in DH10Bac competent E. 2.5. Prenyl protein carboxyl methyltransferase (PPMT) coli cells. Baculoviruses were plaque-purified and amplified. assay For expression of the proteins, Sf9 cells were infected with the baculovirus carrying either RCE1 or AFC1 gene at mul- PPMT activity was measured as described [40]. The standard tiplicity of infections of 1–3 and were cultured for 2–3 days. reaction mixture (20 ␮l) contains 1 ␮M N-acetyl-farnesyl- The cells were disrupted by a Dounce homogenizer in 50 mM cysteine (AFC), 0.4 ␮Ci (0.3 ␮M) [3H]AdoMet, and Sf9 cell Tris–HCl buffer pH 7.5 containing freshly added 1 mM dithio- membranes containing T. brucei PPMT or yeast PPMT (1 ␮g threitol (DTT) and protease inhibitors (1 mM PMSF, 10 ␮g/ml protein). After incubation at 30 ◦C for 20 min, the reaction mix- each of aprotinin, leupeptin, and pepstatin A). The membrane ture was subjected to hexane-extraction, and amounts of the fraction was obtained by centrifugation at 10,000 × g for 10 min product [3H]methyl ester of AFC were quantified by the method ◦ followed by 120,000 × g for 80 min at 4 C, and suspended in a using 1 N NaOH-treatment/[3H]methanol vapor diffusion small volume of buffer (50 mM Tris–HCl pH 7.5, 1 mM DTT) [47]. using a Dounce homogenizer to give a protein concentration of 10–20 mg/ml. Protein concentration was determined using the 2.6. RNA interference Bradford dye reagent from Bio-Rad. Aliquots of the membrane ◦ suspension were frozen in liquid nitrogen and stored at −80 C. Regions of the T. brucei PFT-␤ subunit gene, RCE1, and PPMT genes for RNAi were selected using the program RNAit 2.4. Prenyl protein endoprotease assay (Table 1) [48]. The amplicons were ligated using TA cloning into the vector p2T7TABlue (gift of D. Horn, London School A substrate farnesyl-RAS-CVIM was prepared by incubating of Hygiene and Tropical Medicine) [49]. T. brucei blood- 10 ␮M (22 ␮g) RAS-CVIM and 20 ␮M farnesyl pyrophosphate stream form parasites expressing the T7 RNA polymerase and with 2 ␮M (28 ␮g) purified T. brucei PFT in buffer (100 ␮l) con- the Tet repressor under a single selection marker were pro- taining 30 mM potassium phosphate pH 7.7, 5 mM DTT, 1 mM vided by G. Cross (Rockefeller University) [50]. The cells ◦ ◦ MgCl2,20␮M ZnCl2. After incubation at 30 C for 2 h, the were grown at 37 C in HMI-9 containing 10% heat-inactivated ◦ reaction mixture was frozen at −80 C and used as a source FCS and 2.5 ␮g/ml G418 under 5% CO2 atmosphere [51]. of the substrate. The prenyl protein endoprotease activity was Mid-log phase T. brucei (2.5 × 107) were suspended in 500 ␮l measured by 2 step-incubation as follows. Standard mixtures for of cytomix (120 mM KCl, 0.15 mM CaCl2, 10 mM potassium prenyl protein endoprotease reaction contain 5 ␮l of the above phosphate pH 7.6, 25 mM Hepes, 2 mM EDTA, 5 mM MgCl2) mixture as a source of farnesyl-RAS-CVIM, Sf9 cell membranes containing 10 ␮gofNotI linearized p2T7TABluewith a target (1 ␮) containing either recombinant RCE1 or AFC1 or gene DNA. The mixture was electroporated in a 4 mm gap none, in 20 ␮l buffer containing 50 mM Tris–HCl pH 7.5, 5 mM cuvette with 1.6 kV and 24 resistance. Cells were resuspended ◦ MgCl2, and 5 mM DTT. After incubation at 30 C for 30 min, the in HMI-9 medium under selection pressure with 2.5 ␮g/ml reaction was stopped by adding 200 mM EDTA (5 ␮l), 60 mM hygromycin and 2.5 ␮g/ml G418. The expression of dsRNA ortho-phenanthroline (1 ␮l), and 6 mM TPCK (1 ␮l). To the was induced by addition of 1 ␮g/ml tetracycline to the cultures reaction mixture was added a solution (3 ␮l) containing 0.4 ␮Ci with 1 × 105 cells/ml. Cultures were passed at a 1:10 dilution (2 ␮M, final concentration) [3H]AdoMet (NEN/Parkin-Elmer) daily, and cell concentrations were monitored using a Perkin- and recombinant yeast PPMT/Sf9 membranes (1 ␮g protein). Elmer ATPLite Luminescence ATP detection Assay System The mixture was incubated at 30 ◦C for 1 h, and the reaction (No. 6016941).

Table 1 Oligonucleotide primers for RNAi constructs Target RNAi nucleotide position Primers Northern probe position

TbPFT -␤ 338–783 Sense: GCTGAGATGCTCGGAATCACAG 824–1597 Antisense: CCTGGTAATAACCGCGACACAA TbRCE1 51–591 Sense: GGCATCTTTTTATGGCGGAAG 607–970 Antisense: CCACGATATTCCCAAGCCACAG TbPPMT 58–529 Sense: TTTATGTTTTACGCTGGGTGTTTC 547–824 Antisense: TTCGGGATGACGCAATATTGAGTA 118 J.R. Gillespie et al. / Molecular & Biochemical Parasitology 153 (2007) 115–124

Northern blots were performed using parasite total RNA. (GenBank accession numbers DQ831038 and DQ831039). Blots were hybridized with 32P-labeled DNA probes made to The isoforms are of identical length (909 bp), but differ at regions of the gene separate from the regions targeted by the 9 nucleotides, and encode predicted proteins that differ at RNAi constructs (Table 1). The nitrocellulose membranes were 2 residues. The isoforms identified in these studies are the exposed to X-ray films, and amounts of the target RNAs were same length as, but differ slightly in sequence from the cor- quantified by densitometry and normalized against the ␤-tubulin responding gene deposited by the T. brucei Genome Project, band. designated “CAAX prenyl protease 2, putative” (systematic Metabolic labeling of T. brucei (107 bloodstream form cells) name: Tb927.3.1540). A single isoform of the Leishmania RCE1 was done with 100 ␮Ci [3H]mevalonolactone (1.6 ␮М) per sam- ortholog was cloned from Leishmania genomic DNA (858 bp, ple [52]. Proteins extracted from cell pellets were separated on GenBank accession number DQ831041), based on the matching 12.5% SDS-PAGE and imaged by fluorography [53]. sequence (LmjF26.2690) annotated by the Leishmania Genome Project (675 bp), which lacks a part of the 5-end. The addi-  3. Results and discussion tional sequence on the 5 -end of the gene was determined by the spliced-leader RACE method on cDNA. 3.1. Cloning and analyses of amino acid sequences of Sequence similarity between the orthologs from T. brucei RCE1 orthologs from T. brucei and L. major or L. major and other species (human, S. cerevisiae, and D. melanogaster are shown) is only 13–20%, and that between these Two isoforms of the T. brucei RCE1 gene were identified 2 trypanosomatids is 30%. T. brucei and L. major sequences con- by sequencing multiple clones amplified from genomic DNA tain several predicted transmembrane helices (shown as shaded

Fig. 1. Multiple sequence alignments. (A) RCE1 orthologs: Human (GenBank accession number: NP 005124), D. melanogaster (CAB64383), S. cerevisiae (NP 014001), T. brucei (isoform 2), and L. major. The residues highlighted in black boxes are known to be crucial for enzyme activity based on site-directed mutation analyses [8,54], and those indicated by boxes are required for the full activity [8]. (B) AFC1 orthologs: Human (AAH37283), D. melanogaster (AAF57922), S. cerevisiae (NP 012651), and T. brucei. The zinc binding HExxH motif is indicated with the black-boxed residues. W340 next to this motif and N265 (numbers in human ortholog) residues are indicated by boxes, which have been found to be mutated in human patients with mandibuloacral dysplasia or progeria deseases, which are associated with accumulation of farnesylated prelamin A [18,19]. Transmembrane helical regions (shaded residues) were predicted with the TMHMM-2.0 program (http://www.cbs.dtu.dk/services/TMHMM-2.0/). J.R. Gillespie et al. / Molecular & Biochemical Parasitology 153 (2007) 115–124 119

Fig. 1. (Continued ). sequences) as do other species orthologs (Fig. 1A). Human and 3.2. Cloning and amino acid sequence analysis of an AFC1 yeast enzymes have been shown to localize to the endoplas- ortholog from T. brucei mic reticulum [9,31]. Mutational analyses of the S. cerevisiae enzyme and other orthologs [8,54] revealed several critical The gene encoding the T. brucei AFC1 ortholog is 1281 bp residues for enzyme activity and CaaX sequence specificity, (GenBank accession number DQ831040) and corresponds to the which are indicated by residues highlighted in black boxes in gene Tb09.211.0680 (designated as “CAAX prenyl protease 1, Fig. 1A. These residues, E156, H194, and H248 (residue num- putative”). The T. brucei AFC1 ortholog shows 46% sequence bers in S. cerevisiae), are all conserved in both trypanosomatids. identity to the L. major ortholog and 29–33% identity to three Several additional residues, E157, Y160, F190, H197, and N252, other species and contains five predicted transmembrane helices indicated in boxes in Fig. 1A, have also been shown to be (Fig. 1B). AFC1 is a zinc-metalloprotease containing the HExxH required for the full activity [8]. All of these residues are con- consensus motif (indicated by black boxes in Fig. 1B), which is served, except that Y160 is changed to Phe in several species also found in the T. brucei ortholog. Mutation of the Trp residue including T. brucei (Fig. 1A). immediately adjacent to this motif (W340 of the human protein) 120 J.R. Gillespie et al. / Molecular & Biochemical Parasitology 153 (2007) 115–124

was found in a patient with mandibuloacral dysplasia in which accumulation of unprocessed farnesylated prelamin A occurs. Importance of this residue in the enzyme activity was shown by mutational analysis with the yeast ortholog [18]. This Trp is conserved in the T. brucei ortholog (Fig. 1B). Mutation of N265 was also found in similar progeroid disease [19], and this residue is also conserved in the parasite enzyme. Both AFC1 and RCE1 of S. cerevisiae and human show proteolytic activity to remove the aaX tripeptide from farnesylated a-factor CaaX variants although AFC1 acts on more restricted CaaX sequences [7,8], but there is no apparent homology seen between the RCE1 and AFC1 sequences.

3.3. Expression of RCE1 orthologs of T. brucei and L. Fig. 3. Endoprotease activity on farnesyl-RAS-CVIM by T. brucei RCE1 and major, and the AFC1 ortholog of T. brucei in the L. major RCE1 orthologs but not by the T. brucei AFC1 ortholog. The indicated amount of Sf9 cell membranes containing recombinant L. major RCE1 (᭹), T. Sf9/baculovirus system brucei RCE1 (), T. brucei AFC1 (), or no recombinant protein () was incu- bated at 30 ◦C for 30 min with farnesyl-RAS-CVIM. The proteolyzed product The recombinant proteins were produced using the Sf9 was measured after conversion to [3H]methyl-esterified protein by incubation cell/baculovirus expression system, and the membrane fractions with yeast PPMT as described in “Materials and Methods”. The scale of enzyme were subjected to SDS-PAGE analysis (Fig. 2). The observed activity shown on the right Y-axis is only for L. major RCE1, and that of the left Y-axis is for others. molecular masses of the expressed proteins were ∼38 kDa for the T. brucei and L. major RCE1 orthologs and ∼40 kDa for the T. brucei AFC1 ortholog, while calculated molecular masses of farnesyl-RAS-CVIM was used as a substrate to measure the pro- the amino acid sequences deduced from the cDNA were 33.9, teolytic activity removing the last three amino acids by a coupled 31.6, and 48.9 kDa for T. brucei RCE1, L. major RCE1, and T. assay utilizing PPMT since removal of “aaX” is required for the brucei AFC1 orthologs, respectively. carboxyl methylation of the C-terminal prenylcysteine. The pro- teolyzed product was radiolabeled by a second incubation with RAS-CVIM protein was efficiently farnesylated by puri- 3 fied recombinant T. brucei PFT as described [45]. The product [ H]AdoMet and yeast PPMT. As shown in Fig. 3, membranes from Sf9 cells overexpressing the recombinant RCE1 ortholog of both T. brucei and L. major proteolyzed farnesylated RAS- CVIM. The enzyme activity of T. brucei and L. major RCE1 associated with the membranes and was not detectable in the cytosolic fraction from Sf9 cells. Both T. brucei and L. major RCE1were unable to utilize non-prenylated RAS-CVIM as a substrate, thus showing specificity for prenylated “CaaX” pro- teins. Incubation of membranes from Sf9 cells co-expressing T. brucei RCE1 and PPMT with farnesyl-RAS-CVIM and [3H]AdoMet resulted in incorporation of the radioactivity into the protein (Fig. 4), showing that these two enzymes catalyze the sequential modifications on prenylated “CaaX-proteins” in the membranes. Membranes containing the T. brucei AFC1 ortholog did not have significant activity on farnesylated RAS-CVIM compared to control Sf9 cell membranes. Since orthologs to yeast a-factor or mammalian prelamin A (the natural substrates for AFC1 in these organisms) have not been found in trypanosomatids, we do not have putative natural substrate(s) with which to test T. brucei AFC1. Therefore, the specific role of AFC1 in T. brucei has yet to be elucidated, but this protein appears to have undetectable endoprotease activity for a farnesylated CaaX protein or peptide.

3.4. RNA interference of T. brucei protein prenylation genes Fig. 2. SDS-PAGE analysis of recombinant T. brucei RCE1, L. major RCE1, ␮ and T. brucei AFC1. Membranes (7.5 g protein) from Sf9 cells expressing L. The T. brucei PFT-␤ subunit gene and the two prenyl pro- major RCE1 (lane 1), none (lane 2), T. brucei RCE1 (lane 3), and T. brucei AFC1 (lane 4) were subjected to SDS-PAGE analysis on a 12.5% gel. Proteins tein processing genes (RCE1 and PPMT) were subjected to were stained by Coomassie blue. Arrow heads indicate putative bands of the gene silencing by overexpression of dsRNA from tetracycline- recombinant proteins. regulated constructs. mRNA levels at 72 h post-induction were J.R. Gillespie et al. / Molecular & Biochemical Parasitology 153 (2007) 115–124 121

Fig. 4. [3H]Methylation of farnesyl-RAS-CVIM by membranes from Sf9 cells co-expressing T. brucei RCE1 and T. brucei PPMT. Membranes (2 ␮g pro- tein) containing recombinant T. brucei RCE1 (᭹), T. brucei PPMT (), or both () were incubated at 30 ◦C for the indicated time with farnesyl-RAS- CVIM and [3H]S-AdoMet. The amount of product was measured by counting 3H-incorporation into protein. decreased by 74, 63, and 63% for the PFT-␤, RCE1, and PPMT genes, respectively (Fig. 5, insets). Knockdown of the PFT-␤ and RCE1 mRNA transcripts led, respectively, to ∼35-fold and 23- fold inhibition of growth at day 5 compared to controls (Fig. 5). Knockdown of the PPMT mRNA transcripts led to 2–3-fold inhibition of growth at day 5. Similar results were observed for multiple clones of each construct (data not shown). The morphology of cells was similar for all three targets sub- jected to knockdown with enlargement and formation of multiple flagellae (Fig. 6). The morphological changes were very similar to those observed with treatment of bloodstream-form T. bru- cei with PFT inhibitors [52]. The number of cells with altered morphology from RNAi was most pronounced against PFT- ␤ (∼85%) followed by RCE1 (∼70%), and least with PPMT (∼50%). This correlates to the degree of growth inhibition. To examine effects on in vivo prenylation events, cells subjected to RNAi were metabolically labeleled with 3H- mevalonolactone which is incorporated into isoprenoids and subsequently into prenylated proteins [52]. RNAi against PFT-␤, led to reduced intensity of at least five protein bands as observed by fluorography (see arrows in Fig. 7). This demonstrates that the mRNA knockdown led to impaired protein prenylation for each of these gene products. The affected bands correspond closely to those bands inhibited by PFT inhibitors shown in previous studies [52], affirming that the changes are specific to inhibition of protein farnesylation. There appears to be little effect of the RNAi on the intense low MW proteins that are believed to be substrates of geranylgeranyltransferase type II [52]. For proteins from the cells treated with RNAi against RCE1 or PPMT (Fig. 7), Fig. 5. Effects of gene silencing of protein prenylation enzymes on T. brucei SDS-PAGE migration shifts of incompletely processed prenyl cell growth. Bloodstream forms of T. brucei were induced to express dsRNA proteins were not detectable presumably because the structural against the target genes: PFT-␤ subunit, RCE1, or PPMT. dsRNA expression changes of these proteins compared to fully processed prenyl was induced by the addition (+) of tetracycline (TCN) on day 1, and growth was compared to control cultures without (−) TCN. Growth was also compared to proteins are small. However, significant reduction of several the background “single marker” (SM) strain (absence of the RNAi expression radiolabeled proteins with apparent molecular weights of 63, 48, construct). Cumulative cell densities are shown on a log-scale as the product 40, 31, and a doublet at 26 kDa was observed (Fig. 7). Most of of the cell number and the total dilution. Insets show Northern blots of RNA these appear to correspond to protein bands reduced by the PFT- taken from parasites 72 h after initiation of the experiment. The Northern blots ␤ ␤ ␤ RNAi, which may represent reduction of protein expression were probed for the target genes and -tubulin for normalization. PFT- RNA levels were reduced to 23.7% of controls; RCE1 RNA was 37.5% of controls; or altered turnover of incompletely processed prenyl proteins as and PPMT RNA was 36.9% of controls. 122 J.R. Gillespie et al. / Molecular & Biochemical Parasitology 153 (2007) 115–124

Fig. 6. Effects of gene silencing of protein prenylation enzymes on T. brucei cell morphology. Photomicrographs are shown of the cultured cells at 72 h. Examples of abnormal morphologies in the cultures with gene silencing are shown in high-magnification on the far right next to the respective low magnification photos. Normal morphology of bloodstream form cultures is demonstrated in the photos of the “single marker” (SM) parasites (bottom). The latter parasites contain the heterologous T7 RNA polymerase and Tet repressor, but no RNAi construct. has been described for some mammalian prenyl proteins [37,38]. very important for growth of bloodstream form T. brucei.Inthe There may be two bands (∼55 and ∼75 kDa) with increased case of the PPMT RNAi experiment where cell growth was not radioactivity associated with RNAi (Fig. 7). This could be due dramatically affected with 63% knockdown of the target RNA, to increased expression of the proteins as reported in human can- it seems likely that this gene is more dispensable for normal cell cer cells in which PFT and protein geranylgeranyltransferase-I growth than are the PFT or RCE1 genes. Combining the finding inhibitors upregulate some GTPases [55,56]. of the PFT RNAi experiments with our observations that small The failure to completely arrest T. brucei cell growth with molecular weight inhibitors of PFT cause T. brucei cell death knockdown of PFT-␤ or RCE1 may be due to the incomplete within ∼48 h [52] supports the contention that PFT is an essen- silencing of the gene targets. Nonetheless, the observed magni- tial T. brucei enzyme. It seems likely that RCE1 is essential in tude of growth inhibition supports the notion that these genes are T. brucei, although specific T. brucei RCE1 inhibitors are not J.R. Gillespie et al. / Molecular & Biochemical Parasitology 153 (2007) 115–124 123

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