Parasite-specific inserts in the bifunctional S-adenosylmethionine decarboxylase/ornithine decarboxylase of Plasmodium falciparum modulate catalytic activities and domain interactions

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 ≈150 kDa AdoMetDC domain with the monofunctional, homodimeric ODC domain (≈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.

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The cytoplasmic domain of the Plasmodium falciparum ligand EBA-175 is essential for invasion but not protein trafficking

The Journal of Cell Biology ◽

10.1083/jcb.200301046 ◽

2003 ◽

Vol 162 (2) ◽

pp. 317-327 ◽

Cited By ~ 64

Author(s):

Tim-Wolf Gilberger ◽

Jennifer K. Thompson ◽

Michael B. Reed ◽

Robert T. Good ◽

Alan F. Cowman

Keyword(s):

Plasmodium Falciparum ◽

Host Cell ◽

Protein Interactions ◽

Protein Trafficking ◽

Cytoplasmic Domain ◽

Specific Protein ◽

Host Cells ◽

Protein Protein Interactions ◽

Parasite Plasmodium Falciparum ◽

Erythrocyte Binding

The invasion of host cells by the malaria parasite Plasmodium falciparum requires specific protein–protein interactions between parasite and host receptors and an intracellular translocation machinery to power the process. The transmembrane erythrocyte binding protein-175 (EBA-175) and thrombospondin-related anonymous protein (TRAP) play central roles in this process. EBA-175 binds to glycophorin A on human erythrocytes during the invasion process, linking the parasite to the surface of the host cell. In this report, we show that the cytoplasmic domain of EBA-175 encodes crucial information for its role in merozoite invasion, and that trafficking of this protein is independent of this domain. Further, we show that the cytoplasmic domain of TRAP, a protein that is not expressed in merozoites but is essential for invasion of liver cells by the sporozoite stage, can substitute for the cytoplasmic domain of EBA-175. These results show that the parasite uses the same components of its cellular machinery for invasion regardless of the host cell type and invasive form.

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The influence of nerve section on the metabolism of polyamines in rat diaphragm muscle

Biochemical Journal ◽

10.1042/bj1960603 ◽

1981 ◽

Vol 196 (2) ◽

pp. 603-610 ◽

Cited By ~ 17

Author(s):

D Hopkins ◽

K L Manchester

Keyword(s):

Ornithine Decarboxylase ◽

Normal Values ◽

Methionine Adenosyltransferase ◽

Diaphragm Muscle ◽

Regulatory Mechanisms ◽

Polyamine Metabolism ◽

Decarboxylase Activity ◽

Rat Diaphragm ◽

Nerve Section ◽

Adenosylmethionine Decarboxylase

Concentrations of spermidine, spermine and putrescine have been measured in rat diaphragm muscle after unilateral nerve section. The concentration of putrescine increased approx. 10-fold 2 days after nerve section, that of spermidine about 3-fold by day 3, whereas an increase in the concentration of spermine was only observed after 7-10 days. It was not possible to show enhanced uptake of either exogenous putrescine or spermidine by the isolated tissue during the hypertrophy. Consistent with the accumulation of putrescine, activity of ornithine decarboxylase increased within 1 day of nerve section, was maximally elevated by the second day and then declined. Synthesis of spermidine from [14C]putrescine and either methionine or S-adenosylmethionine bt diaphragm cytosol rose within 1 day of nerve section, but by day 3 had returned to normal or below normal values. Activity of adenosylmethionine decarboxylase similarly increased within 1 day of nerve section, but by day 3 had declined to below normal values. Activity of methionine adenosyltransferase was elevated throughout the period studied. The concentration of S-adenosylmethionine was likewise enhanced during hypertrophy. Administration of methylglyoxal bis(guanylhydrazone) produced a marked increase in adenosylmethionine decarboxylase activity and a large increase in putrescine concentration, but did not prevent the rise in spermidine concentration produced by denervation. Possible regulatory mechanisms of polyamine metabolism consistent with the observations are discussed.

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Plasmodium falciparum: Analysis of Protein-Protein Interactions of the 140/130/110-kDa Rhoptry Protein Complex Using Antibody and Mouse Erythrocyte Binding Assays

Experimental Parasitology ◽

10.1006/expr.1993.1075 ◽

1993 ◽

Vol 77 (2) ◽

pp. 179-194 ◽

Cited By ~ 14

Author(s):

T.Y. Samyellowe

Keyword(s):

Plasmodium Falciparum ◽

Protein Interactions ◽

Protein Complex ◽

Protein Protein Interactions ◽

Binding Assays ◽

Mouse Erythrocyte ◽

Erythrocyte Binding ◽

Rhoptry Protein

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Protein–protein interactions between the bilirubin-conjugating UDPglucuronosyltransferase UGT1A1 and its shorter isoform 2 regulatory partner derived from alternative splicing

Biochemical Journal ◽

10.1042/bj20121594 ◽

2013 ◽

Vol 450 (1) ◽

pp. 107-114 ◽

Cited By ~ 5

Author(s):

Mélanie Rouleau ◽

Pierre Collin ◽

Judith Bellemare ◽

Mario Harvey ◽

Chantal Guillemette

Keyword(s):

Complex Formation ◽

Protein Interactions ◽

Disulfide Bonds ◽

Transmembrane Domain ◽

Cysteine Residues ◽

Uridine Diphosphate ◽

Protein Protein Interactions ◽

Reducing Conditions ◽

Alternatively Spliced ◽

Intermolecular Disulfide Bonds

The oligomerization of UGTs [UDP (uridine diphosphate)-glucuronosyltransferases] modulates their enzyme activities. Recent findings also indicate that glucuronidation is negatively regulated by the formation of inactive oligomeric complexes between UGT1A enzymes [i1 (isoform 1)] and an enzymatically inactive alternatively spliced i2 (isoform 2). In the present paper, we assessed whether deletion of the UGT-interacting domains previously reported to be critical for enzyme function might be involved in i1–i2 interactions. The bilirubin-conjugating UGT1A1 was used as a prototype. We also explored whether intermolecular disulfide bonds are involved in i1–i2 interactions and the potential role of selected cysteine residues. Co-immunoprecipitation assays showed that UGT1A1 lacking the SP (signal peptide) alone or also lacking the transmembrane domain (absent from i2) did not self-interact, but still interacted with i2. The deletion of other N- or C-terminal domains did not compromise i1–i2 complex formation. Under non-reducing conditions, we also observed formation of HMWCs (high-molecular-mass complexes) for cells overexpressing i1 and i2. The presence of UGTs in these complexes was confirmed by MS. Mutation of individual cysteine residues throughout UGT1A1 did not compromise i1–i1 or i1–i2 complex formation. These findings are compatible with the hypothesis that the interaction between i1 and i2 proteins (either transient or stable) involves binding of more than one domain that probably differs from those involved in i1–i1 interactions.

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