Characterization of a Trypanosoma cruzi acetyltransferase: cellular location, activity and structure

Abstract

Trypanosomatids are widespread parasites that cause three major tropical diseases. In trypanosomatids, as in most other organisms, acetylation is a common protein modification that is important in multiple, diverse processes. This paper describes a new member of the Trypanosoma cruzi acetyltransferase family. The gene is single copy and orthologs are also present in the other two sequenced trypanosomatids, Trypanosoma brucei and Leishmania major. This protein (TcAT-1) has the essential motifs present in members of the GCN5-related acetyltransferase (GNAT) family, as well as an additional motif also found in some enzymes from plant and animal species. The protein is evolutionarily more closely related to this group of enzymes than to histone acetyltransferases. The native protein has a cytosolic cellular location and is present in all three life-cycle stages of the parasite. The recombinant protein was shown to have autoacetylation enzymatic activity.

Introduction

Trypanosoma cruzi is a protozoan parasite that causes Chagas disease in humans. The parasite adopts mainly three different forms (stages) through its life-cycle: epimastigote in the insect vector and trypomastigote and amastigote in humans. This severe disease affects several million people in South- and Central America and the available treatment suffer from serious drawbacks. The genome of T. cruzi was recently sequenced, and this has increased the pace of research into parasite biology.

Acetylation is a co- or post-translational modification that affects a majority of eukaryotic proteins, and often alters their properties in different ways [1], [2]. The main consequences of acetylation include effects on protein stability, protein-protein interaction and DNA binding [3]. Acetyltransferases (ATs) are grouped into superfamilies. Members of the PCAF/GCN5 superfamily, also referred to as GCN5-related N-acetyltransferases (GNAT), have characteristic protein sequence motifs that are involved in the binding of acetyl-coenzyme A (AcCoA) [4], [5], [6]. They acetylate substrates such as arylalkylamines, aminoglycosides and histones [7]. Acetylated histone tails do not bind as tightly to DNA, which allows transcription factors to interact with regulatory sequences. Other members of this family participate in N-terminal acetylation of proteins, which involves the transfer of an acetyl group from AcCoA to the α-NH₂ group of a protein or peptide (N^α-acetylation). About 80–90% of cytosolic mammalian proteins are acetylated at the N-terminus [8]. There are three main N-terminal acetyltransferases (NATs) in Saccharomyces cerevisiae: NatA, NatB and NatC, whose catalytic subunits are Ard1p, Nat3p and Mak3p, respectively.

Some members of this superfamily can also acetylate internal residues (N^ɛ-acetylation). In contrast to N^α-acetylation, this modification is typically reversible through the action of deacetylases.

In the malaria parasite, Plasmodium falciparum, the PfGCN5 protein has been shown to be essential for gene regulation during parasite development and could be used as a drug target [9]. The ARD1 orthologue of Trypanosoma brucei has been characterized and has been found to be essential for cellular viability in both the mammalian and insect-stages [10]. A number of acetylated molecules have been detected in T. brucei (lysine 40 of flagellar alpha-tubulin [11], histones [12]) and in T. cruzi (tubulin [13], histones [14], [15], alkylglycerol in glycosylphosphatidylinositol-anchors [16]), and a T. cruzi serine acetyltransferase was characterised previously [17]. Recently, the presence of several acetylated proteins in the three stages of T. cruzi was determined by mass spectrometry [18]. These included: beta tubulin, histone H3, disulfide isomerase, translation elongation factor eEF-1 alpha, a ribosomal protein, actin1, triose phosphate isomerase and cyclophilin A.

We here describe the characterization of a protein from T. cruzi that was identified by sequence homology and predicted structure to be related to acetyltransferases from several species. We show that it is expressed and remains in the cytoplasm and that it is capable of autoacetylation.