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- Nasizadeh, Sima
(författare)
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A Study on Trypanosomal Polyamine Biosynthetic Enzymes
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The polyamines, putrescine, spermidine, and spermine, are essential for all forms of life. The polyamine biosynthetic pathway has been shown to be a potential target for drugs against various trypanosomal parasitic diseases. The two main enzymes in the biosynthesis of polyamines are ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC). ODC catalyzes the first step of polyamine biosynthesis. Mammalian ODC is tightly regulated by polyamines and has a very fast turnover rate. The degradation of this enzyme is 26S proteasome dependent, but ubiquitin independent. The polyamines induce the degradation of mammalian ODC by stimulating the synthesis of a protein termed antizyme, which binds to and targets the enzyme for degradation by the 26S proteasome. Most trypanosomal ODCs, e.g. Leishmania donovani ODC and Trypanosoma brucei ODC are metabolically stable, but, interestingly, Crithidia fasciculata ODC has a fast turnover. In this thesis, the mechanisms involved in the rapid turnover of C. fasciculata ODC are examined. The degradation of C. fasciculata ODC was shown to be rapid also in mammalian systems, whereas that of L. donovani ODC was not. The rapid turnover of the enzyme was markedly reduced by inhibition of the 26S proteasome, indicating the involvement of this proteolytic system in the degradation of C. fasciculata ODC. However, unlike the mammalian ODC, C. fasciculata ODC was not down-regulated by polyamines, suggesting an antizyme independent degradation mechanism. As a result of analyzing the turnover of a series of chimeric ODCs between C. fasciculata ODC and L. donovani ODC, it was found that the central part of the former enzyme is important for its rapid degradation. The characterization of C. fasciculata AdoMetDC, the other main polyamine biosynthetic enzyme, revealed that also this enzyme has an extremely fast turnover (ca 3 min). Furthermore, no polyamine-mediated feedback regulation of AdoMetDC was observed in the parasite. As an alternative to using polyamine biosynthesis inhibitors, CHO cells expressing the trypanosomal ODCs were used to analyze the effects of different polyamine pools on cell cycle progression. It was shown that CHO cells expressing L. donovani ODC had a reduced polyamine synthesis, resulting in a decrease in cellular putrescine and spermidine levels (compared to wild-type CHO cells). On the other hand, the spermine content was increased. The changes in polyamine content were reflected in changes in cell growth kinetics.
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| 3. |
- Nasizadeh, Sima, et al.
(författare)
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Importance of polyamines in cell cycle kinetics as studied in a transgenic system.
- 2005
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 1090-2422 .- 0014-4827. ; 308:May 26, s. 254-264
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Tidskriftsartikel (refereegranskat)abstract
- 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 G(1), S, and G(2) + 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.
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| 4. |
- Nasizadeh, Sima, et al.
(författare)
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Proteasomal degradation of a trypanosomal ornithine decarboxylase
- 2003
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Ingår i: Cellular Physiology and Biochemistry. - : S. Karger AG. - 1015-8987 .- 1421-9778. ; 13:5, s. 321-328
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Tidskriftsartikel (refereegranskat)abstract
- 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 downregulated 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. Copyright (C) 2003 S. Karger AG, Basel.
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