The In Silico Identification of Potential Members of the Ded1/DDX3 Subfamily of DEAD-Box RNA Helicases from the Protozoan Parasite Leishmania infantum and Their Analyses in Yeast

Abstract

DEAD-box RNA helicases are ubiquitous proteins found in all kingdoms of life and that are associated with all processes involving RNA. Their central roles in biology make these proteins potential targets for therapeutic or prophylactic drugs. The Ded1/DDX3 subfamily of DEAD-box proteins is of particular interest because of their important role(s) in translation. In this paper, we identified and aligned the protein sequences of 28 different DEAD-box proteins from the kinetoplast-protozoan parasite Leishmania infantum, which is the cause of the visceral form of leishmaniasis that is often lethal if left untreated, and compared them with the consensus sequence derived from DEAD-box proteins in general, and from the Ded1/DDX3 subfamily in particular, from a wide variety of other organisms. We identified three potential homologs of the Ded1/DDX3 subfamily and the equivalent proteins from the related protozoan parasite Trypanosoma brucei, which is the causative agent of sleeping sickness. We subsequently tested these proteins for their ability to complement a yeast strain deleted for the essential DED1 gene. We found that the DEAD-box proteins from Trypanosomatids are highly divergent from other eukaryotes, and consequently they are suitable targets for protein-specific drugs.

Keywords: DEAD-box; Ded1/DDX3; Leishmania; RNA helicase; Saccharomyces cerevisiae; Trypanosoma brucei; leishmaniasis; trypanosomatid.

Figure 1

Phylogenetic tree of LINF and Ded1/DDX3 proteins. A neighbor-joining tree is shown with the branch lengths and distances as shown. The UniProt identifying numbers are shown for the Ded1 and DDX3 proteins, and the TriTryp numbers for L. infantum. The DDX3 proteins were from humans, mice and Drosophila melanogaster, and the Ded1 proteins from Schizosaccharomyces pombe and S. cerevisiae.

Figure 2

Alignment of the helicase cores of DEAD-box proteins of Leishmania. The protein sequence of the 28 different proteins is shown with representative members of the Ded1/DDX3 subfamily of proteins. Note that LINF_010012800 and LINF_010012900 encode the same protein, and both the long and short forms of LINF_080005700 are shown. LINF_050006300 was found after this work was completed. The DEAD-box consensus was derived from 700 sequences from different organisms as previously described [56]. The capital case letters are conserved amino acids and the lowercase letters indicate functional conservation, where a is an aromatic group, l is an aliphatic residue, h is hydrophobic, o is an alcohol, + is positively charged, p is polar, u is tiny, and x is any residue. The numbers signify the typical distances between motifs. The interactions with the RNA and ATP are as shown, based on the solved crystal structures of the ligand-bound DEAD-box protein Vasa (PDB# 2DB3; [80]). However, these interactions are largely conserved in all the solved crystal structures of DEAD-box proteins. The underlined P shows interactions with the phosphates of ATP, while the adenine is from ATP. The helicase core is defined by 2–3 residues that extend from the amino terminus of the conserved phenylalanine of the Q motif and about 33–35 residues that extend from the carboxyl terminus of motif VI. Similarly, the Ded1/DDX3 subfamily consensus was derived from 96 of the original 229 unique sequences with the closest homology to yeast Ded1. Residues distinctive for this subfamily are shown in gray.

Figure 3

Alignment of the helicase flanking sequences of DEAD-box proteins of Leishmania. The consensus for the Ded1/DDX3 subfamily is as described in Figure 2, where it signifies a negatively charged residue. Aliphatic groups (I, L, V) are largely interchangeable, and we considered them equivalent in this figure. Ø denotes amino acids M, V, I, L, F, or W. The conserved sequences listed as characteristic of this subfamily (top) was based on previous work [77,78]. Distinctive residues for this subfamily are shown in gray. Note that the DDX3 70% consensus was based on a subset of the original sequences as indicated in Figure 2; hence it is not representative of the full diversity of annotated Ded1/DDX3 sequences obtained in Blasts.

Figure 4

Complementation of the genes in a ded1-deletion strain. The listed genes in the HA-p415 centromeric or HA-p424 2µ plasmids were constitutively expressed in the ded1::HIS yeast strain from the strong ADH promoter, and then cultures were serially diluted by factors of 10 and spotted on SD plates containing 5-FOA that were subsequently incubated at the indicated temperatures. Plates were incubated three days at 30 °C and 36 °C, and for 7 days at 18 °C. W303 is a wildtype strain expressing endogenous DED1. TIF1, FAL1, and DBP2 encode yeast DEAD-box proteins that are unrelated to the Ded1/DDX3 subfamily. DDX3X is the human protein and DBP1 is the yeast paralog of DED1 that suppresses the DED1 deletion when overexpressed. ADH_p424 is the empty plasmid. Similar results were obtained with proteins expressed off the p415 plasmid.

Figure 5

Expression of the proteins in yeast. The HA-tagged proteins in the p424 plasmid were expressed off the ADH promoter in the W303 yeast strain. (A) The proteins from the extracted cells were separated on a 10% SDS-PAGE, the separated proteins transferred to nitrocellulose membranes, and then visualized with IgG specific to the HA tag or PGK1. (B) The quantified values of the gels shown in (A). Variations in loading were adjusted relative to the PGK1, and then the values were normalized relative to the expression of HA-Ded1_p415.