Limits...
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.

Show MeSH

Related in: MedlinePlus

Measurement of AdoMetDC in cell lysates in the presence of excess exogenous prozyme.AdoMetDC activity was measured in the absence (orange) and presence (green) of added recombinant prozyme (1 µM) for 427 parental cells, AdoMetDC RNAi cells (−Tet control and +Tet induction of RNAi for 4 days), and prozyme cKO cells (expressing prozyme (+Tet), and not expressing prozyme (−Tet) collected day 2 after Tet removal). Errors represent the standard error of the mean (n = 3).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2562514&req=5

ppat-1000183-g004: Measurement of AdoMetDC in cell lysates in the presence of excess exogenous prozyme.AdoMetDC activity was measured in the absence (orange) and presence (green) of added recombinant prozyme (1 µM) for 427 parental cells, AdoMetDC RNAi cells (−Tet control and +Tet induction of RNAi for 4 days), and prozyme cKO cells (expressing prozyme (+Tet), and not expressing prozyme (−Tet) collected day 2 after Tet removal). Errors represent the standard error of the mean (n = 3).

Mentions: In order to determine if AdoMetDC and prozyme are present at similar concentrations, or if one is in excess over the other we measured AdoMetDC activity in cell lysates in the presence and absence of exogenous recombinant prozyme (1 µM). AdoMetDC activity in 427 parental cells was compared to the AdoMetDC RNAi cells (±Tet) and to the prozyme cKO cells (±Tet) (Figure 4). In the control cells (427 cells or AdoMetDC RNAi (−Tet)) the addition of prozyme stimulates AdoMetDC by 5–8-fold, demonstrating that under normal growth conditions prozyme is present in limiting concentration in comparison to AdoMetDC. Knockdown of AdoMetDC (RNAi +Tet) or of prozyme (cKO–Tet), reduced AdoMetDC activity by 60% and 80%, respectively, showing that while neither knockdown approach completely eliminates the protein targets, the prozyme cKO is more efficient at depleting AdoMetDC activity than the induction of AdoMetDC RNAi. The addition of exogenous prozyme has a minimal effect on the activity of the Tet induced AdoMetDC RNAi cells, however activity is increased significantly for the prozyme cKO cells (−Tet), showing that added prozyme can restore activity to the lysate that lacks endogenous prozyme. These data demonstrate that prozyme is present in limiting concentration under typical growth conditions of the wild-type 427 cells, positioning the cell to increase pathway flux by upregulating prozyme levels.


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

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

Measurement of AdoMetDC in cell lysates in the presence of excess exogenous prozyme.AdoMetDC activity was measured in the absence (orange) and presence (green) of added recombinant prozyme (1 µM) for 427 parental cells, AdoMetDC RNAi cells (−Tet control and +Tet induction of RNAi for 4 days), and prozyme cKO cells (expressing prozyme (+Tet), and not expressing prozyme (−Tet) collected day 2 after Tet removal). Errors represent the standard error of the mean (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000183-g004: Measurement of AdoMetDC in cell lysates in the presence of excess exogenous prozyme.AdoMetDC activity was measured in the absence (orange) and presence (green) of added recombinant prozyme (1 µM) for 427 parental cells, AdoMetDC RNAi cells (−Tet control and +Tet induction of RNAi for 4 days), and prozyme cKO cells (expressing prozyme (+Tet), and not expressing prozyme (−Tet) collected day 2 after Tet removal). Errors represent the standard error of the mean (n = 3).
Mentions: In order to determine if AdoMetDC and prozyme are present at similar concentrations, or if one is in excess over the other we measured AdoMetDC activity in cell lysates in the presence and absence of exogenous recombinant prozyme (1 µM). AdoMetDC activity in 427 parental cells was compared to the AdoMetDC RNAi cells (±Tet) and to the prozyme cKO cells (±Tet) (Figure 4). In the control cells (427 cells or AdoMetDC RNAi (−Tet)) the addition of prozyme stimulates AdoMetDC by 5–8-fold, demonstrating that under normal growth conditions prozyme is present in limiting concentration in comparison to AdoMetDC. Knockdown of AdoMetDC (RNAi +Tet) or of prozyme (cKO–Tet), reduced AdoMetDC activity by 60% and 80%, respectively, showing that while neither knockdown approach completely eliminates the protein targets, the prozyme cKO is more efficient at depleting AdoMetDC activity than the induction of AdoMetDC RNAi. The addition of exogenous prozyme has a minimal effect on the activity of the Tet induced AdoMetDC RNAi cells, however activity is increased significantly for the prozyme cKO cells (−Tet), showing that added prozyme can restore activity to the lysate that lacks endogenous prozyme. These data demonstrate that prozyme is present in limiting concentration under typical growth conditions of the wild-type 427 cells, positioning the cell to increase pathway flux by upregulating prozyme levels.

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