C-Terminal Proteolysis of Prenylated Proteins in Trypanosomatids And

Molecular & Biochemical Parasitology 153 (2007) 115–124 C-terminal proteolysis of prenylated proteins 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 cysteine 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 protein 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 farnesyltransferase; 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 methionine 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 amino acid, 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 methylation of several farnesylated proteins such as K-Ras [34], residues “aaX” followed by methylation of exposed ␣-carboxyl transducin ␥ subunit [35,36], and lamin 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 malaria 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 progeria 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 farnesyl pyrophosphate 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

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