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Identification of a novel lipin homologue from the parasitic protozoan Trypanosoma brucei.

ABSTRACT: Arginine methylation is a post-translational modification that expands the functional diversity of proteins. Kinetoplastid parasites contain a relatively large group of protein arginine methyltransferases (PRMTs) compared to other single celled eukaryotes. Several T. brucei proteins have been shown to serve as TbPRMT substrates in vitro, and a great number of proteins likely to undergo methylation are predicted by the T. brucei genome. This indicates that a large number of proteins whose functions are modulated by arginine methylation await discovery in trypanosomes. Here, we employed a yeast two-hybrid screen using as bait the major T. brucei type I PRMT, TbPRMT1, to identify potential substrates of this enzyme.We identified a protein containing N-LIP and C-LIP domains that we term TbLpn. These domains are usually present in a family of proteins known as lipins, and involved in phospholipid biosynthesis and gene regulation. Far western and co-immunoprecipitation assays confirmed the TbPRMT1-TbLpn interaction. We also demonstrated that TbLpn is localized mainly to the cytosol, and is methylated in vivo. In addition, we showed that, similar to mammalian and yeast proteins with N-LIP and C-LIP domains, recombinant TbLpn exhibits phosphatidic acid phosphatase activity, and that two conserved aspartic acid residues present in the C-LIP domain are critical for its enzymatic activity.This study reports the characterization of a novel trypanosome protein and provides insight into its enzymatic activity and function in phospholipid biosynthesis. It also indicates that TbLpn functions may be modulated by arginine methylation.

SUBMITTER: Pelletier M

PROVIDER: S-EPMC3654991 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Identification of a novel lipin homologue from the parasitic protozoan Trypanosoma brucei.

Pelletier Michel M Frainier Alyssa S AS Munini Dominic N DN Wiemer Jenna M JM Karpie Amber R AR Sattora Jeff J JJ

BMC microbiology 20130508

<h4>Background</h4>Arginine methylation is a post-translational modification that expands the functional diversity of proteins. Kinetoplastid parasites contain a relatively large group of protein arginine methyltransferases (PRMTs) compared to other single celled eukaryotes. Several T. brucei proteins have been shown to serve as TbPRMT substrates in vitro, and a great number of proteins likely to undergo methylation are predicted by the T. brucei genome. This indicates that a large number of pro ...[more]

PMID: 23656927

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Project description:Genetic exchange has been described in several kinetoplastid parasites, but the most well-studied mating system is that of Trypanosoma brucei, the causative organism of African sleeping sickness. Sexual reproduction takes place in the salivary glands (SG) of the tsetse vector and involves meiosis and production of haploid gametes. Few genetic crosses have been carried out to date and consequently there is little information about the mating compatibility of different trypanosomes. In other single-celled eukaryotes, mating compatibility is typically determined by a system of two or more mating types (MT). Here we investigated the MT system in T. brucei.We analysed a large series of F1, F2 and back crosses by pairwise co-transmission of red and green fluorescent cloned cell lines through experimental tsetse flies. To analyse each cross, trypanosomes were cloned from fly SG containing a mixture of both parents, and genotyped by microsatellites and molecular karyotype. To investigate mating compatibility at the level of individual cells, we directly observed the behaviour of SG-derived gametes in intra- or interclonal mixtures of red and green fluorescent trypanosomes ex vivo.Hybrid progeny were found in all F1 and F2 crosses and most of the back crosses. The success of individual crosses was highly variable as judged by the number of hybrid clones produced, suggesting a range of mating compatibilities among F1 progeny. As well as hybrids, large numbers of recombinant genotypes resulting from intraclonal mating (selfers) were found in some crosses. In ex vivo mixtures, red and green fluorescent trypanosome gametes were observed to pair up and interact via their flagella in both inter- and intraclonal combinations. While yellow hybrid trypanosomes were frequently observed in interclonal mixtures, such evidence of cytoplasmic exchange was rare in the intraclonal mixtures.The outcomes of individual crosses, particularly back crosses, were variable in numbers of both hybrid and selfer clones produced, and do not readily fit a simple two MT model. From comparison of the behaviour of trypanosome gametes in inter- and intraclonal mixtures, we infer that mating compatibility is controlled at the level of gamete fusion.

Project description:Drug resistance in pathogenic protozoa is very often caused by changes to the 'transportome' of the parasites. In Trypanosoma brucei, several transporters have been implicated in uptake of the main classes of drugs, diamidines and melaminophenyl arsenicals. The resistance mechanism had been thought to be due to loss of a transporter known to carry both types of agents: the aminopurine transporter P2, encoded by the gene TbAT1. However, although loss of P2 activity is well-documented as the cause of resistance to the veterinary diamidine diminazene aceturate (DA; Berenil(®)), cross-resistance between the human-use arsenical melarsoprol and the diamidine pentamidine (melarsoprol/pentamidine cross resistance, MPXR) is the result of loss of a separate high affinity pentamidine transporter (HAPT1). A genome-wide RNAi library screen for resistance to pentamidine, published in 2012, gave the key to the genetic identity of HAPT1 by linking the phenomenon to a locus that contains the closely related T. brucei aquaglyceroporin genes TbAQP2 and TbAQP3. Further analysis determined that knockdown of only one pore, TbAQP2, produced the MPXR phenotype. TbAQP2 is an unconventional aquaglyceroporin with unique residues in the "selectivity region" of the pore, and it was found that in several MPXR lab strains the WT gene was either absent or replaced by a chimeric protein, recombined with parts of TbAQP3. Importantly, wild-type AQP2 was also absent in field isolates of T. b. gambiense, correlating with the outcome of melarsoprol treatment. Expression of a wild-type copy of TbAQP2 in even the most resistant strain completely reversed MPXR and re-introduced HAPT1 function and transport kinetics. Expression of TbAQP2 in Leishmania mexicana introduced a pentamidine transport activity indistinguishable from HAPT1. Although TbAQP2 has been shown to function as a classical aquaglyceroporin it is now clear that it is also a high affinity drug transporter, HAPT1. We discuss here a possible structural rationale for this remarkable ability.

Dataset Information

Identification of a novel lipin homologue from the parasitic protozoan Trypanosoma brucei.

Publications

Identification of a novel lipin homologue from the parasitic protozoan Trypanosoma brucei.

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