Extremely rapid turnover of S-adenosylmethionine decarboxylase in Crithidia fasciculata.

The activity of S-adenosylmethionine decarboxylase (AdoMetDC) in Crithidia fasciculata was shown to be correlated to the growth of the parasite. An increase in activity was observed during exponential growth. Inhibition of protein synthesis induced an extremely rapid decay of AdoMetDC activity. The half-life of the enzyme was estimated to be about 3 min, which is the shortest half-life ever recorded for an eukaryotic AdoMetDC. The reduction in AdoMetDC activity was correlated with a decrease in AdoMetDC protein content, demonstrating a rapid turnover of the enzyme. No polyamine-mediated feedback regulation of AdoMetDC was observed in the parasite.

Downstream effects of haemoglobinase inhibition in Plasmodium falciparum-infected erythrocytes.

Blood-stage malarial parasites (Plasmodium falciparum) digest large quantities of host haemoglobin during their asexual development in erythrocytes. The haemoglobin digestion pathway, involving a succession of cleavages by various peptidases, appears to be essential for parasite development and has received much attention as an antimalarial drug target. A variety of peptidase inhibitors that have potent antimalarial activity are believed to inhibit and/or kill parasites by blocking haemoglobin digestion. It has not however been established how such a blockage might lead to parasite death. The answer to this question should lie in identifying the affected physiological function, but the purpose of excess haemoglobin digestion by P. falciparum has for many years been the subject of debate. The process was traditionally believed to be nutritional until Lew et al. [Blood 2003;101:4189-94] suggested that it is linked to volume control of the infected erythrocyte and is necessary to prevent premature osmotic lysis of the host cell. Their model predicts that sufficient inhibition of haemoglobin degradation should result in premature haemolysis. In this study we examined the downstream effects of reduced haemoglobin digestion on osmoprotection and nutrition. We found that inhibitors of haemoglobinases (plasmepsins, falcipains and aminopeptidases) did not cause premature haemolysis. The inhibitors did however block parasite development and this effect corresponded to a strong inhibition of protein synthesis. The effect on protein synthesis (i) occurred at inhibitor concentrations and times of exposure that were relevant to parasite growth inhibition, (ii) was observed with different chemical classes of inhibitor, and (iii) was synergistic when a plasmepsin and a falcipain inhibitor were combined, reflecting the well-established antimalarial synergism of the combination. Taken together, the results suggest that the likely primary downstream effect of inhibition of haemoglobin degradation is amino acid depletion, leading to blockade of protein synthesis, and that the parasite probably degrades globin for nutritional purposes.

Importance of polyamines in cell cycle kinetics as studied in a transgenic system.

Polyamines are organic cations, which are considered essential for normal cell cycle progression. This view is based on results from numerous studies using a variety of enzyme inhibitors or polyamine analogues interfering with either the metabolism or the physiological functions of the polyamines. However, the presence of non-specific effects may be hard to rule out in such studies. In the present study, we have for the first time used a transgenic cell system to analyze the importance of polyamines in cell growth. We have earlier shown that expression of trypanosomal ODC in an ODC-deficient variant of CHO cells (C55.7) supported growth of these otherwise polyamine auxotrophic cells. However, one of the transgenic cell lines grew much slower than the others. As shown in the present study, the level of ODC activity was much lower in these cells, and that was reflected in a reduction of cellular polyamine levels. Analysis of cell cycle kinetics revealed that reduction of growth was correlated to prolongation of the G1, S, and G2+M phases in the cells. Providing exogenous putrescine to the cells resulted in a normalization of polyamine levels as well as cell cycle kinetics indicating a causal relationship.

Proteasomal degradation of a trypanosomal ornithine decarboxylase.

Mammalian ornithine decarboxylase (ODC), which catalyses the first step in polyamine biosynthesis, has a very fast turnover. It is degraded by the 26S proteasome in an ubiquitin-independent process and the degradation is stimulated by polyamines in a feedback control of the enzyme. Interestingly, there is a major difference in the metabolic stability between ODCs from various trypanosomatids. Trypanosoma brucei and Leishmania donovani both contain stable ODCs, whereas Crithidia fasciculata has an ODC with a rapid turnover. In spite of the difference in stability there is a high degree of sequence homology between C. fasciculata ODC and L. donovani ODC. In the present study we demonstrate that C. fasciculata ODC is rapidly degraded also in mammalian systems like CHO cells and rabbit reticulocyte lysate, suggesting that the degradation signals of the enzyme are recognised by the mammalian systems. L. donovani ODC, on the other hand, is degraded very slowly in the same systems. The degradation of C. fasciculata ODC in the mammalian systems is markedly reduced by inhibition of the 26S proteasome. However, unlike mammalian ODC, C. fasciculata ODC is not down-regulated by polyamines. Thus, the turnover of C. fasciculata ODC and L. donovani ODC in the mammalian systems reflects the degradation of the enzyme in the parasites, making such systems potentially useful as complements to parasitic knockout models for further analysis of the mechanisms involved in the rapid degradation of C. fasciculata ODC.