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Regulated expression of an essential allosteric activator of polyamine biosynthesis in African trypanosomes.

Willert EK, Phillips MA - PLoS Pathog. (2008)

Bottom Line: Further, trypanosomatid AdoMetDC is activated by heterodimer formation with a catalytically dead homolog termed prozyme, found only in these species.Changes in protein stability do not appear to account for the increased steady-state protein levels, as both enzymes are stable in the presence of cycloheximide.In conclusion, we describe the first evidence for regulation of polyamine biosynthesis in T. brucei and we demonstrate that the unique regulatory subunit of AdoMetDC is a key component of this regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA.

ABSTRACT
Trypanosoma brucei is the causative agent of African sleeping sickness. The polyamine biosynthetic pathway has the distinction of being the target of the only clinically proven anti-trypanosomal drug with a known mechanism of action. Polyamines are essential for cell growth, and their metabolism is extensively regulated. However, trypanosomatids appear to lack the regulatory control mechanisms described in other eukaryotic cells. In T. brucei, S-adenosylmethionine decarboxylase (AdoMetDC) and ornithine decarboxylase (ODC) are required for the synthesis of polyamines and also for the unique redox-cofactor trypanothione. Further, trypanosomatid AdoMetDC is activated by heterodimer formation with a catalytically dead homolog termed prozyme, found only in these species. To study polyamine regulation in T. brucei, we generated inducible AdoMetDC RNAi and prozyme conditional knockouts in the mammalian blood form stage. Depletion of either protein led to a reduction in spermidine and trypanothione and to parasite death, demonstrating that prozyme activation of AdoMetDC is essential. Under typical growth conditions, prozyme concentration is limiting in comparison to AdoMetDC. However, both prozyme and ODC protein levels were significantly increased relative to stable transcript levels by knockdown of AdoMetDC or its chemical inhibition. Changes in protein stability do not appear to account for the increased steady-state protein levels, as both enzymes are stable in the presence of cycloheximide. These observations suggest that prozyme and ODC are translationally regulated in response to perturbations in the pathway. In conclusion, we describe the first evidence for regulation of polyamine biosynthesis in T. brucei and we demonstrate that the unique regulatory subunit of AdoMetDC is a key component of this regulation. The data support ODC and AdoMetDC as the key control points in the pathway and the likely rate-limiting steps in polyamine biosynthesis.

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Comparison of prozyme and ODC protein and mRNA levels upon AdoMetDC knockdown.The AdoMetDC RNAi line was induced with Tet, and samples were collected over the time range and conditions described in Figure 2. A representative data set is displayed. Samples were visualized on a Typhoon 8610 scanner and quantitated as described in Materials and Methods. (A) Western analysis. (B) Northern blot analysis. (C) Quantitation of relative ODC and prozyme protein (solid symbols) and mRNA (open symbols) levels. Protein and mRNA levels were normalized to tubulin controls and are displayed as a fold change relative to uninduced control cells (−Tet). AdoMetDC, green triangles; prozyme, purple squares; and ODC, orange circles. The plotted data can be found in Tables S3 and S4. Errors represent the standard error of the mean.
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ppat-1000183-g005: Comparison of prozyme and ODC protein and mRNA levels upon AdoMetDC knockdown.The AdoMetDC RNAi line was induced with Tet, and samples were collected over the time range and conditions described in Figure 2. A representative data set is displayed. Samples were visualized on a Typhoon 8610 scanner and quantitated as described in Materials and Methods. (A) Western analysis. (B) Northern blot analysis. (C) Quantitation of relative ODC and prozyme protein (solid symbols) and mRNA (open symbols) levels. Protein and mRNA levels were normalized to tubulin controls and are displayed as a fold change relative to uninduced control cells (−Tet). AdoMetDC, green triangles; prozyme, purple squares; and ODC, orange circles. The plotted data can be found in Tables S3 and S4. Errors represent the standard error of the mean.

Mentions: We undertook quantitative analysis of the effects on protein and mRNA levels to gain mechanistic insight into the observed induction of prozyme and ODC upon knockdown of AdoMetDC or prozyme. Protein amounts were determined by quantitative Western blotting with fluorescent secondary antibodies, and Northern blots were developed by phosphorimaging; the density of these signals was then quantitated using imaging software and the effects determined relative to tubulin controls (Figure 5A and 5B and Table S3 show a representative data set). AdoMetDC protein and mRNA levels were reduced by 70–80% compared to controls by induction of RNAi, consistent with the activity data. In response, as observed in Figures 2 and 3, both prozyme and ODC protein levels are induced. The induction of prozyme is consistently more robust and occurs earlier in the time course than for ODC. Prozyme protein increased by an average of 25-fold (range 12–40-fold, n = 3), while ODC increased 7-fold (range 5–10-fold, n = 3) (day 4 +Tet; Figure 5C and Table S4). In the presence of spermidine, knockdown of AdoMetDC also led to induction of prozyme and ODC protein, with observed average increases of 10 and 5-fold, respectively (day 4 +Tet, +Spd). Finally, quantitative analysis of the prozyme cKO line showed that the loss of prozyme expression also led to induction of ODC, with ODC protein levels increasing by 4- and 5-fold (day 2 and 3 without Tet, respectively) (Figure S5 and Table S4).


Regulated expression of an essential allosteric activator of polyamine biosynthesis in African trypanosomes.

Willert EK, Phillips MA - PLoS Pathog. (2008)

Comparison of prozyme and ODC protein and mRNA levels upon AdoMetDC knockdown.The AdoMetDC RNAi line was induced with Tet, and samples were collected over the time range and conditions described in Figure 2. A representative data set is displayed. Samples were visualized on a Typhoon 8610 scanner and quantitated as described in Materials and Methods. (A) Western analysis. (B) Northern blot analysis. (C) Quantitation of relative ODC and prozyme protein (solid symbols) and mRNA (open symbols) levels. Protein and mRNA levels were normalized to tubulin controls and are displayed as a fold change relative to uninduced control cells (−Tet). AdoMetDC, green triangles; prozyme, purple squares; and ODC, orange circles. The plotted data can be found in Tables S3 and S4. Errors represent the standard error of the mean.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2562514&req=5

ppat-1000183-g005: Comparison of prozyme and ODC protein and mRNA levels upon AdoMetDC knockdown.The AdoMetDC RNAi line was induced with Tet, and samples were collected over the time range and conditions described in Figure 2. A representative data set is displayed. Samples were visualized on a Typhoon 8610 scanner and quantitated as described in Materials and Methods. (A) Western analysis. (B) Northern blot analysis. (C) Quantitation of relative ODC and prozyme protein (solid symbols) and mRNA (open symbols) levels. Protein and mRNA levels were normalized to tubulin controls and are displayed as a fold change relative to uninduced control cells (−Tet). AdoMetDC, green triangles; prozyme, purple squares; and ODC, orange circles. The plotted data can be found in Tables S3 and S4. Errors represent the standard error of the mean.
Mentions: We undertook quantitative analysis of the effects on protein and mRNA levels to gain mechanistic insight into the observed induction of prozyme and ODC upon knockdown of AdoMetDC or prozyme. Protein amounts were determined by quantitative Western blotting with fluorescent secondary antibodies, and Northern blots were developed by phosphorimaging; the density of these signals was then quantitated using imaging software and the effects determined relative to tubulin controls (Figure 5A and 5B and Table S3 show a representative data set). AdoMetDC protein and mRNA levels were reduced by 70–80% compared to controls by induction of RNAi, consistent with the activity data. In response, as observed in Figures 2 and 3, both prozyme and ODC protein levels are induced. The induction of prozyme is consistently more robust and occurs earlier in the time course than for ODC. Prozyme protein increased by an average of 25-fold (range 12–40-fold, n = 3), while ODC increased 7-fold (range 5–10-fold, n = 3) (day 4 +Tet; Figure 5C and Table S4). In the presence of spermidine, knockdown of AdoMetDC also led to induction of prozyme and ODC protein, with observed average increases of 10 and 5-fold, respectively (day 4 +Tet, +Spd). Finally, quantitative analysis of the prozyme cKO line showed that the loss of prozyme expression also led to induction of ODC, with ODC protein levels increasing by 4- and 5-fold (day 2 and 3 without Tet, respectively) (Figure S5 and Table S4).

Bottom Line: Further, trypanosomatid AdoMetDC is activated by heterodimer formation with a catalytically dead homolog termed prozyme, found only in these species.Changes in protein stability do not appear to account for the increased steady-state protein levels, as both enzymes are stable in the presence of cycloheximide.In conclusion, we describe the first evidence for regulation of polyamine biosynthesis in T. brucei and we demonstrate that the unique regulatory subunit of AdoMetDC is a key component of this regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA.

ABSTRACT
Trypanosoma brucei is the causative agent of African sleeping sickness. The polyamine biosynthetic pathway has the distinction of being the target of the only clinically proven anti-trypanosomal drug with a known mechanism of action. Polyamines are essential for cell growth, and their metabolism is extensively regulated. However, trypanosomatids appear to lack the regulatory control mechanisms described in other eukaryotic cells. In T. brucei, S-adenosylmethionine decarboxylase (AdoMetDC) and ornithine decarboxylase (ODC) are required for the synthesis of polyamines and also for the unique redox-cofactor trypanothione. Further, trypanosomatid AdoMetDC is activated by heterodimer formation with a catalytically dead homolog termed prozyme, found only in these species. To study polyamine regulation in T. brucei, we generated inducible AdoMetDC RNAi and prozyme conditional knockouts in the mammalian blood form stage. Depletion of either protein led to a reduction in spermidine and trypanothione and to parasite death, demonstrating that prozyme activation of AdoMetDC is essential. Under typical growth conditions, prozyme concentration is limiting in comparison to AdoMetDC. However, both prozyme and ODC protein levels were significantly increased relative to stable transcript levels by knockdown of AdoMetDC or its chemical inhibition. Changes in protein stability do not appear to account for the increased steady-state protein levels, as both enzymes are stable in the presence of cycloheximide. These observations suggest that prozyme and ODC are translationally regulated in response to perturbations in the pathway. In conclusion, we describe the first evidence for regulation of polyamine biosynthesis in T. brucei and we demonstrate that the unique regulatory subunit of AdoMetDC is a key component of this regulation. The data support ODC and AdoMetDC as the key control points in the pathway and the likely rate-limiting steps in polyamine biosynthesis.

Show MeSH
Related in: MedlinePlus