Leishmania express a functional Cdc20 homologue

Abstract

Our knowledge concerning the mechanisms of cell cycle regulation in organisms belonging to the Trypanosometidae family is limited. Leishmania donovani are parasitic protozoa that cause kala azar, a fatal form of visceral leishmaniasis in humans. Here we provide evidence that the L. donovani genome contains a Cdc20 homologue. Cdc20 is a regulator of the Anaphase Promoting Complex/Cyclosome (APC/C) that mediates ubiquitin-dependent proteasomal degradation of key cell cycle regulators in eukaryotes. We show that L. donovani Cdc20 protein (LdCdc20p) can complement a lack of yeast Cdc20 protein in Saccharomyces cerevisiae cells, validating the functionality of LdCdc20p. Furthermore, we demonstrate cyclic expression of LdCdc20p and that it contains an active RXXL destruction motif, a distinctive feature of proteins targeted for proteasomal degradation by APC/C. Finally, in line with the proteasome mediating LdCdc20p degradation, promastigotes exposed to proteasome inhibitor display elevated LdCdc20p levels. Taken together our data indicate that Leishmania regulate their cell cycle by ubiquitin-dependent proteasomal degradation mediated by the APC/C.

Introduction

Ubiquitin-dependent degradation of cell cycle regulators such as cyclins is required for cell cycle progress and is mediated by ubiquitin ligases such as the Anaphase Promoting Complex/Cyclosome (APC/C) [1], [2]. The activity and regulation of APC/C, in particular the nature of its activating proteins, is highly conserved from yeast to human. APC/C is activated in a cell cycle specific manner from metaphase until the G1–S transition. Its activity is regulated by phosphorylation and by sequential association with two regulatory proteins – Cdc20 and Cdh1. Cdc20 is an essential protein that binds and activates APC/C in mitosis and is required for the degradation of mitotic APC/C substrates like cyclin A, cyclin B, securin and others [3]. Cdh1 is required for APC/C activation during G1 [4].

Organisms of the genus Leishmania (Leishmania spp.) are intracellular parasitic protozoa of the family Trypanosometidae. Leishmania donovani is the causative agent of kala azar, a fatal form of visceral leishmaniasis where parasites infect humans and reside in the liver, spleen and bone marrow [5], [6]. Until recently, limited information was available concerning cell cycle regulation in Leishmania even though it is likely to influence development inside both vectors and hosts. However, now that genome sequences are available it is expected that conserved cell cycle genes will be identified, which can be evaluated functionally in subsequent studies.

The capability to perform RNA interference (RNAi) assays in Trypanosoma brucei, a member of the Trypanosometidae family, has facilitated identification and biochemical characterization of cell cycle related proteins in this parasite (reviewed in [7]). Proteins identified thus include: two cyclin-like proteins with short half-lives and a putative APC/C destruction box motif [8], [9]; the regulatory protein kinases aurora [10] and polo [11]; two subunits of APC/C, APC1 and APC3 (CDC27) [12]. All of these proteins influence cell cycle progression from mitosis through cytokinesis. In addition, numerous cyclin dependent protein kinases (CDKs) and their activating cyclins have been identified and shown to play roles in cell cycle checkpoints [13], [14], [15]. Finally, in other studies two additional cell cycle activities have been identified, an E3 ligase that controls cell cycle during S phase and G2 comprising the Skp1–Cdc53/Cullin-F-box (SCF) protein complex [16] and an F-box protein termed CFB2 that was shown to be essential for T. brucei cytokinesis [17]. Taken together these data support the premise that Trypanosometidae, like all other eukaryotes, regulate cell cycle using ubiquitin-dependent proteasomal degradation mediated by APC/C and SCF complexes.

Here, we present evidence that L. donovani promastigotes express a Cdc20 (LdCdc20) homologue containing an active RXXL destruction box motif that exhibits a very short half-life and cyclic behavior. This finding corroborates that cell cycle regulation also in this parasite involves APC/C mediated ubiquitin-dependent proteasomal degradation.