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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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The effects of Prozyme knockout on blood form T. brucei.(A) Cell growth curves. Log cell number versus days in culture. Triangle, Flag-tagged prozyme Expressed (prozyme cKO line +1 µg/ml Tet); circle, prozyme Not expressed (prozyme cKO line no Tet); square, prozyme Not expressed plus spermidine (prozyme cKO line no Tet, +0.1 mM Spd). Complete cell death was observed by day 6. Data is displayed in Log units. (B) Western blot analysis of polyamine/trypanothione biosynthetic enzymes (20 µg total protein). Time corresponds to days in the absence of Tet, with (left) or without (right) spermidine (0.1 mM). Flag-antibody detects Flag-tagged prozyme. (C) Analysis of intracellular polyamine levels. (D) Analysis of intracellular thiol levels. The polyamines Put and Spd, and thiols, GSH, GSH-Spd, and T(SH)2 are shown as a percentage of control values (prozyme cKO +Tet). The concentration data (nmoles/108 cells) are provided in Table S2. Errors represent the standard error of the mean (n = 3).
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ppat-1000183-g003: The effects of Prozyme knockout on blood form T. brucei.(A) Cell growth curves. Log cell number versus days in culture. Triangle, Flag-tagged prozyme Expressed (prozyme cKO line +1 µg/ml Tet); circle, prozyme Not expressed (prozyme cKO line no Tet); square, prozyme Not expressed plus spermidine (prozyme cKO line no Tet, +0.1 mM Spd). Complete cell death was observed by day 6. Data is displayed in Log units. (B) Western blot analysis of polyamine/trypanothione biosynthetic enzymes (20 µg total protein). Time corresponds to days in the absence of Tet, with (left) or without (right) spermidine (0.1 mM). Flag-antibody detects Flag-tagged prozyme. (C) Analysis of intracellular polyamine levels. (D) Analysis of intracellular thiol levels. The polyamines Put and Spd, and thiols, GSH, GSH-Spd, and T(SH)2 are shown as a percentage of control values (prozyme cKO +Tet). The concentration data (nmoles/108 cells) are provided in Table S2. Errors represent the standard error of the mean (n = 3).

Mentions: The importance of prozyme to growth of blood form T. brucei was evaluated by the generation of a prozyme conditional knock out (cKO) cell line. T. brucei is a diploid organism, thus to generate the KO line the first prozyme allele was replaced with T7 polymerase and a G418 selectable marker, a Tet responsive FLAG-tagged prozyme gene was integrated into the rRNA locus, and finally the second prozyme allele was replaced by the Tet repressor gene and a hygromycin selectable marker (Figure S1B). Southern blotting confirmed the correct integration of the three vectors (Figure S4). Prozyme cKO cells maintained in the presence of Tet expressed the FLAG-tagged prozyme protein and had similar growth rates to wild-type 427 cells (data not shown). Upon removal of Tet, prozyme expression was reduced to undetectable levels, leading to a rapid arrest of cell growth (day 2) followed by cell death (day 6) (Figure 3A and 3B). Unlike the AdoMetDC RNAi line, the addition of exogenous spermidine did not restore cell growth. These data demonstrate that prozyme is essential for the growth of blood form parasites, and they show that the low activity of homodimeric AdoMetDC (<0.1% of AdoMetDC/prozyme heterodimer activity) is insufficient to promote cell growth. In addition the data confirm that the AdoMetDC/prozyme heterodimer is the functional configuration of AdoMetDC in the cell.


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

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

The effects of Prozyme knockout on blood form T. brucei.(A) Cell growth curves. Log cell number versus days in culture. Triangle, Flag-tagged prozyme Expressed (prozyme cKO line +1 µg/ml Tet); circle, prozyme Not expressed (prozyme cKO line no Tet); square, prozyme Not expressed plus spermidine (prozyme cKO line no Tet, +0.1 mM Spd). Complete cell death was observed by day 6. Data is displayed in Log units. (B) Western blot analysis of polyamine/trypanothione biosynthetic enzymes (20 µg total protein). Time corresponds to days in the absence of Tet, with (left) or without (right) spermidine (0.1 mM). Flag-antibody detects Flag-tagged prozyme. (C) Analysis of intracellular polyamine levels. (D) Analysis of intracellular thiol levels. The polyamines Put and Spd, and thiols, GSH, GSH-Spd, and T(SH)2 are shown as a percentage of control values (prozyme cKO +Tet). The concentration data (nmoles/108 cells) are provided in Table S2. Errors represent the standard error of the mean (n = 3).
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Related In: Results  -  Collection

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ppat-1000183-g003: The effects of Prozyme knockout on blood form T. brucei.(A) Cell growth curves. Log cell number versus days in culture. Triangle, Flag-tagged prozyme Expressed (prozyme cKO line +1 µg/ml Tet); circle, prozyme Not expressed (prozyme cKO line no Tet); square, prozyme Not expressed plus spermidine (prozyme cKO line no Tet, +0.1 mM Spd). Complete cell death was observed by day 6. Data is displayed in Log units. (B) Western blot analysis of polyamine/trypanothione biosynthetic enzymes (20 µg total protein). Time corresponds to days in the absence of Tet, with (left) or without (right) spermidine (0.1 mM). Flag-antibody detects Flag-tagged prozyme. (C) Analysis of intracellular polyamine levels. (D) Analysis of intracellular thiol levels. The polyamines Put and Spd, and thiols, GSH, GSH-Spd, and T(SH)2 are shown as a percentage of control values (prozyme cKO +Tet). The concentration data (nmoles/108 cells) are provided in Table S2. Errors represent the standard error of the mean (n = 3).
Mentions: The importance of prozyme to growth of blood form T. brucei was evaluated by the generation of a prozyme conditional knock out (cKO) cell line. T. brucei is a diploid organism, thus to generate the KO line the first prozyme allele was replaced with T7 polymerase and a G418 selectable marker, a Tet responsive FLAG-tagged prozyme gene was integrated into the rRNA locus, and finally the second prozyme allele was replaced by the Tet repressor gene and a hygromycin selectable marker (Figure S1B). Southern blotting confirmed the correct integration of the three vectors (Figure S4). Prozyme cKO cells maintained in the presence of Tet expressed the FLAG-tagged prozyme protein and had similar growth rates to wild-type 427 cells (data not shown). Upon removal of Tet, prozyme expression was reduced to undetectable levels, leading to a rapid arrest of cell growth (day 2) followed by cell death (day 6) (Figure 3A and 3B). Unlike the AdoMetDC RNAi line, the addition of exogenous spermidine did not restore cell growth. These data demonstrate that prozyme is essential for the growth of blood form parasites, and they show that the low activity of homodimeric AdoMetDC (<0.1% of AdoMetDC/prozyme heterodimer activity) is insufficient to promote cell growth. In addition the data confirm that the AdoMetDC/prozyme heterodimer is the functional configuration of AdoMetDC in the cell.

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