Product feedback regulation implicated in translational control of the Trypanosoma brucei S-adenosylmethionine decarboxylase regulatory subunit prozyme

Abstract

Human African sleeping sickness (HAT) is caused by the parasitic protozoan Trypanosoma brucei. Polyamine biosynthesis is an important drug target in the treatment of HAT. Previously we showed that trypanosomatid S-adenosylmethionine decarboxylase (AdoMetDC), a key enzyme for biosynthesis of the polyamine spermidine, is activated by heterodimer formation with an inactive paralogue termed prozyme. Furthermore, prozyme protein levels were regulated in response to reduced AdoMetDC activity. Herein we show that T. brucei encodes three prozyme transcripts. The 3'UTRs of these transcripts were mapped and chloramphenicol acetyltransferase (CAT) reporter constructs were used to identify a 1.2 kb region that contained a 3'UTR prozyme regulatory element sufficient to upregulate CAT protein levels (but not RNA) upon AdoMetDC inhibition, supporting the hypothesis that prozyme expression is regulated translationally. To gain insight into trans-acting factors, genetic rescue of AdoMetDC RNAi knock-down lines with human AdoMetDC was performed leading to rescue of the cell growth block, and restoration of prozyme protein to wild-type levels. Metabolite analysis showed that prozyme protein levels were inversely proportional to intracellular levels of decarboxylated AdoMet (dcAdoMet). These data suggest that prozyme translation may be regulated by dcAdoMet, a metabolite not previously identified to play a regulatory role.

Figure 1. Structure of the prozyme 3’UTR and the effects of AdoMetDC inhibitors on prozyme mRNAs

(A) The effects of chemical inhibition of AdoMetDC on expression of polyamine biosynthetic enzymes. BSF and PF form T. brucei 427 cells were treated with MDL 73811 (75 nM). Cells were harvested after 48 h incubation and analyzed by western blot. DHODH is included as a loading control. (B) Northern blot analysis of BSF mRNA from T. brucei 427 cells in the presence or in the absence of MDL 73811 with two different probes (probe primers see Table 3S). (C) PCR amplification of prozyme 3’UTR using genomic BSF DNA (gDNA) or cDNA generated with either SuperScript III (SSIII) or MMLV RT. Primer set 2 (Table S3) was used for PCR amplification. (D) Schematic of the prozyme 3’UTRs as mapped by RACE (rapid amplification of cDNA ends). The location of the probes used in northern analysis (part B above) are shown.

Figure 2. The effect of AdoMetDC inhibition on CAT expression levels from prozyme 3’UTR reporter constructs

(A) Schematic of the prozyme 3’UTR fragments that were cloned into pHD1437 reporter construct vector. Number (from 1 to 10) indicates the fragment number and subsequent construct number. Nucleotide positions are displayed. The start and end points of each fragment are shown in Table 4S. The gene is numbered with position 1 representing the first gene specific base after the splice leader in the 5’UTR (Figures S1 and S2) (B) Structure of the reporter gene vector pHD1437: the black line of the structure of pHD1437 shows the plasmid backbone. Other components include a region from the tubulin locus (Tublin), a T7 polymerase promoter (P_T7), a 5’-splice site and UTR from EP1 gene (EP5’), a CAT reporter gene (CAT), 5’UTR from the actin locus (ACT5’), a puromycin selectable marker gene (Pur), and the actin 3’UTR (ACT 3’). (C) CAT reporter plasmids containing different prozyme 3’UTR fragments were transiently transfected into BSF 90–13 cells. The empty pHD1437 vector was transfected as the control (Ctrl). Cells were grown for 3 h before the addition of MDL 73811 (75 nM), and then incubated for additional 16 h before harvesting. The CAT ELISA assay was used to determine the level of CAT enzyme activity (CAT), and mRNA levels were quantitated by qPCR with primers from table 3S (mRNA). The fold change was calculated as M(+)/M(−). Errors represent the standard error of the mean for n=3–4 independent biological replicates.

Figure 3. The effects of chemical inhibitors of AdoMetDC and/or ODC on the expression of polyamine biosynthetic enzymes and intracelluar polyamine and dcAdoMetDC pools

(A) Western blot analysis of polyamine biosynthetic enzymes (40 µg total protein/lane). BSF 427 cells were grown in the absence (−) or presence (+) of MDL 73811 (M) (75 nM) and/or DFMO (D) (12.5 µM) for three days before harvesting. DHODH is shown as a loading control. (B) HPLC analysis of polyamine biosynthesis pathway metabolites for n=4 replicates. Cells were treated as in A. (C) LC-MS/MS analysis of dcAdoMet pools followed by two separate ion peaks (represented by the blue and red bars). Cells were treated as in A except that incubation with drugs was for either 1 (d1) or 3 (d3) days. Data are displayed as the relative peak area. The lower limit of quantitation (LLQ) for dcAdoMet was a relative peak area of 10⁻⁶.

Figure 4. Expression of hAdoMetDC rescues the effects of AdoMetDC knockdown by RNAi

(A) Representative growth curve analysis comparing AdoMetDC RNAi cells (RNAi) with the same cell line transfected with the human AdoMetDC expression construct (RNAi-C). Tet (1 µg/ml) was added on day 0 resulting in the co-induction of AdoMetDC RNAi and human AdoMet expression. Cell numbers were below detection levels for the RNAi induced cells after day 14. Total cell numbers were calculated as cell density × the total dilution factor and are plotted versus time in days after Tet induction. (B) Representative western analysis. Cells were harvested 3 d after Tet induction and analyzed by western using antibodies to prozyme, T. brucei AdoMetDC and human AdoMetDC. DHODH is shown as the loading control (40 µg total protein/lane). (C) LC-MS/MS analysis of intracellular dcAdoMet levels. Cells were harvested as in B. Data are displayed as the relative peak area. Data represent the mean of n=3 independent biological replicates. Two separate ion fragments were quantitated for each condition (red and blue bars). The LLQ for dcAdoMet was a relative peak area of 10⁻⁶(D) LC-MS/MS analysis of intracellular AdoMet, MTA, spermidine and putrescine levels. Cells were harvested as in B. Data represent the ratio to the uninduced AdoMetDC RNAi line and are shown for the highest abundance ion peak of the two measured. Raw data are displayed in Figure S4. Errors represent the standard error of the mean for n=3 independent biological replicates.

Scheme 1. Polyamine metabolic pathway in T. brucei