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Co-inhibition of Plasmodium falciparum S-adenosylmethionine decarboxylase/ornithine decarboxylase reveals perturbation-specific compensatory mechanisms by transcriptome, proteome, and metabolome analyses.

ABSTRACT: Polyamines are ubiquitous components of all living cells, and their depletion usually causes cytostasis, a strategy employed for treatment of West African trypanosomiasis. To evaluate polyamine depletion as an anti-malarial strategy, cytostasis caused by the co-inhibition of S-adenosylmethionine decarboxylase/ornithine decarboxylase in Plasmodium falciparum was studied with a comprehensive transcriptome, proteome, and metabolome investigation. Highly synchronized cultures were sampled just before and during cytostasis, and a novel zero time point definition was used to enable interpretation of results in lieu of the developmentally regulated control of gene expression in P. falciparum. Transcriptome analysis revealed the occurrence of a generalized transcriptional arrest just prior to the growth arrest due to polyamine depletion. However, the abundance of 538 transcripts was differentially affected and included three perturbation-specific compensatory transcriptional responses as follows: the increased abundance of the transcripts for lysine decarboxylase and ornithine aminotransferase and the decreased abundance of that for S-adenosylmethionine synthetase. Moreover, the latter two compensatory mechanisms were confirmed on both protein and metabolite levels confirming their biological relevance. In contrast with previous reports, the results provide evidence that P. falciparum responds to alleviate the detrimental effects of polyamine depletion via regulation of its transcriptome and subsequently the proteome and metabolome.

SUBMITTER: van Brummelen AC

PROVIDER: S-EPMC3783055 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Co-inhibition of Plasmodium falciparum S-adenosylmethionine decarboxylase/ornithine decarboxylase reveals perturbation-specific compensatory mechanisms by transcriptome, proteome, and metabolome analyses.

van Brummelen Anna C AC Olszewski Kellen L KL Wilinski Daniel D Llinás Manuel M Louw Abraham I AI Birkholtz Lyn-Marie LM

The Journal of biological chemistry 20081210 7

Polyamines are ubiquitous components of all living cells, and their depletion usually causes cytostasis, a strategy employed for treatment of West African trypanosomiasis. To evaluate polyamine depletion as an anti-malarial strategy, cytostasis caused by the co-inhibition of S-adenosylmethionine decarboxylase/ornithine decarboxylase in Plasmodium falciparum was studied with a comprehensive transcriptome, proteome, and metabolome investigation. Highly synchronized cultures were sampled just befor ...[more]

PMID: 19073607

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Project description:Polyamine biosynthesis of the malaria parasite, Plasmodium falciparum, is regulated by a single, hinge-linked bifunctional PfAdoMetDC/ODC [ P. falciparum AdoMetDC (S-adenosylmethionine decarboxylase)/ODC (ornithine decarboxylase)] with a molecular mass of 330 kDa. The bifunctional nature of AdoMetDC/ODC is unique to Plasmodia and is shared by at least three species. The PfAdoMetDC/ODC contains four parasite-specific regions ranging in size from 39 to 274 residues. The significance of the parasite-specific inserts for activity and protein-protein interactions of the bifunctional protein was investigated by a single- and multiple-deletion strategy. Deletion of these inserts in the bifunctional protein diminished the corresponding enzyme activity and in some instances also decreased the activity of the neighbouring, non-mutated domain. Intermolecular interactions between AdoMetDC and ODC appear to be vital for optimal ODC activity. Similar results have been reported for the bifunctional P. falciparum dihydrofolate reductase-thymidylate synthase [Yuvaniyama, Chitnumsub, Kamchonwongpaisan, Vanichtanankul, Sirawaraporn, Taylor, Walkinshaw and Yuthavong (2003) Nat. Struct. Biol. 10, 357-365]. Co-incubation of the monofunctional, heterotetrameric approximately 150 kDa AdoMetDC domain with the monofunctional, homodimeric ODC domain (approximately 180 kDa) produced an active hybrid complex of 330 kDa. The hinge region is required for bifunctional complex formation and only indirectly for enzyme activities. Deletion of the smallest, most structured and conserved insert in the ODC domain had the biggest impact on the activities of both decarboxylases, homodimeric ODC arrangement and hybrid complex formation. The remaining large inserts are predicted to be non-globular regions located on the surface of these proteins. The large insert in AdoMetDC in contrast is not implicated in hybrid complex formation even though distinct interactions between this insert and the two domains are inferred from the effect of its removal on both catalytic activities. Interference with essential protein-protein interactions mediated by parasite-specific regions therefore appears to be a viable strategy to aid the design of selective inhibitors of polyamine metabolism of P. falciparum.

Project description:S-adenosylmethionine decarboxylase (PfAdoMetDC) from Plasmodium falciparum is a prospective antimalarial drug target. The production of recombinant PfAdoMetDC for biochemical validation as a drug target is important. The production of PfAdoMetDC in Escherichia coli has been reported to result in unsatisfactory yields and poor quality product. The co-expression of recombinant proteins with molecular chaperones has been proposed as one way to improve the production of the former in E. coli. E. coli heat shock proteins DnaK, GroEL-GroES and DnaJ have previously been used to enhance production of some recombinant proteins. However, the outcomes were inconsistent. An Hsp70 chimeric protein, KPf, which is made up of the ATPase domain of E. coli DnaK and the substrate binding domain of P. falciparum Hsp70 (PfHsp70) has been previously shown to exhibit chaperone function when it was expressed in E. coli cells whose resident Hsp70 (DnaK) function was impaired. We proposed that because of its domain constitution, KPf would most likely be recognised by E. coli Hsp70 co-chaperones. Furthermore, because it possesses a substrate binding domain of plasmodial origin, KPf would be primed to recognise recombinant PfAdoMetDC expressed in E. coli. First, using site-directed mutagenesis, followed by complementation assays, we established that KPf with a mutation in the hydrophobic residue located in its substrate binding cavity was functionally compromised. We further co-expressed PfAdoMetDC with KPf, PfHsp70 and DnaK in E. coli cells either in the absence or presence of over-expressed GroEL-GroES chaperonin. The folded and functional status of the produced PfAdoMetDC was assessed using limited proteolysis and enzyme assays. PfAdoMetDC co-expressed with KPf and PfHsp70 exhibited improved activity compared to protein co-expressed with over-expressed DnaK. Our findings suggest that chimeric KPf may be an ideal Hsp70 co-expression partner for the production of recombinant plasmodial proteins in E. coli.

Project description:S-adenosylmethionine decarboxylase (AdoMetDC/SpeD) is a key polyamine biosynthetic enzyme required for conversion of putrescine to spermidine. Autocatalytic self-processing of the AdoMetDC/SpeD proenzyme generates a pyruvoyl cofactor from an internal serine. Recently, we discovered that diverse bacteriophages encode AdoMetDC/SpeD homologs that lack AdoMetDC activity and instead decarboxylate L-ornithine or L-arginine. We reasoned that neofunctionalized AdoMetDC/SpeD homologs were unlikely to have emerged in bacteriophages and were probably acquired from ancestral bacterial hosts. To test this hypothesis, we sought to identify candidate AdoMetDC/SpeD homologs encoding L-ornithine and L-arginine decarboxylases in bacteria and archaea. We searched for the anomalous presence of AdoMetDC/SpeD homologs in the absence of its obligatory partner enzyme spermidine synthase, or the presence of two AdoMetDC/SpeD homologs encoded in the same genome. Biochemical characterization of candidate neofunctionalized genes confirmed lack of AdoMetDC activity, and functional presence of L-ornithine or L-arginine decarboxylase activity in proteins from phyla Actinomycetota, Armatimonadota, Planctomycetota, Melainabacteria, Perigrinibacteria, Atribacteria, Chloroflexota, Sumerlaeota, Omnitrophota, Lentisphaerota, and Euryarchaeota, the bacterial candidate phyla radiation and DPANN archaea, and the δ-Proteobacteria class. Phylogenetic analysis indicated that L-arginine decarboxylases emerged at least three times from AdoMetDC/SpeD, whereas L-ornithine decarboxylases arose only once, potentially from the AdoMetDC/SpeD-derived L-arginine decarboxylases, revealing unsuspected polyamine metabolic plasticity. Horizontal transfer of the neofunctionalized genes appears to be the more prevalent mode of dissemination. We identified fusion proteins of bona fide AdoMetDC/SpeD with homologous L-ornithine decarboxylases that possess two, unprecedented internal protein-derived pyruvoyl cofactors. These fusion proteins suggest a plausible model for the evolution of the eukaryotic AdoMetDC.

Dataset Information

Co-inhibition of Plasmodium falciparum S-adenosylmethionine decarboxylase/ornithine decarboxylase reveals perturbation-specific compensatory mechanisms by transcriptome, proteome, and metabolome analyses.

Publications

Co-inhibition of Plasmodium falciparum S-adenosylmethionine decarboxylase/ornithine decarboxylase reveals perturbation-specific compensatory mechanisms by transcriptome, proteome, and metabolome analyses.

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