MUTANAL · Identification and mutational analysis of the nucleobase and nucleoside transporters of Aspergillus nidulans implicated in the transport of antiviral/anticancer drugs
FP6 — Marie Curie Actions (Human Resources and Mobility)
- Duration
- 2005-07-01 → 2006-06-30
- EU contribution
- €34,500
- Participants
- 1
- Scheme
- IIF
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Results in brief
Final Activity Report Summary - MUTANAL (Identification and mutational analysis of the nucleobase and nucleoside transporters of Aspergillus nidulans implicated in the transport of ... drugs)
The research objectives for the period from the 1st to the 12th months of the project were (i) the determination of the substrate specificity of seven nucleobase transporters (Perm1-Perm7) belonging to a distinct NCS1 (Nucleobase/Cation Symporter 1) transporter family and two nucleoside (ENT, CNT) transporters of Aspergillus nidulans and (ii) isolation of the nucleoside CNT and one of the nucleobase transporter genes in order to subject them to mutational analysis for the purpose to reveal the structure-function relationship of these enzymes and their substrate-binding centre. Substrate specificities of five of the seven nucleobase transporters and one of the nucleoside transporters were determined by growth tests of deletion mutants and reinforced by developmental Northern analysis and uptake experiments. Perm1 was identified as exclusive allantoin transporter on the basis of growth test. perm1 deletion mutant was not able to grow when allantoin was applied as sole N-source in the media, while wild type grew normally. The result was reinforced by Northern analysis, which indicated that perm1 is regulated by its substrate: the presence of allantoin in the growth media affected the expression level of perm1 gene positively. Perm4 proved to be a high-affinity, high-capacity uracil permease of germinating conidia on the basis of growth test of deleted perm4 mutant on the toxic 5-fluoro uracil and kinetic uptake experiments, reinforced by developmental Northern analysis. perm4 is not expressed in resting conidiospores, is transcriptionally activated and reaches a peak during the isotrophic growth phase of conidiospore germination, and stays at a basic low level in mycelium. Transcriptional expression is correlated with uracil transport activity. Perm5 is supposed to be involved in thyamine transport on the basis of Northern analysis. Expression level of perm5 is increased from a low basic level to a very high level in response to the presence of thyamine in the media during the isotrophic growth phase of conidiospore germination and stays at a high level in mycelium. Perm6 is involved in guanine transport on the basis of growth test of perm6 deletion mutant, where guanine was applied as sole N-source. perm3 is expressed similarly to perm4, although at an extremely low level, perm3 deletion mutant shows growth ability in presence of the toxic 5-fluoro uracil and kinetic experiments confirmed that Perm3 has minor role in uracil uptake since it is a low-affinity and low-capacity permease. Function of Perm2 and 7 remained unclear. Amongst the nucleoside type transporters, function of CNT was determined as adenosine and exclusive uridine permease on the basis of growth test of cnt deletion mutant on toxic analogues. According to the proposed project we started to develop the random mutagenesis on the cnt and perm1 genes.
Data: CORDIS, © European Union
Project objective
Nucleobases and nucleosides play a key role in nucleic acid and nucleotide metabolism in all cells, act as signalling molecules, neuromodulators and serve as nitrogen source in plants and many micro-organisms. They are synthesised de novo or, when de novo synthesis pathways are lacking, salvaged from the environment (purine auxotroph parasites; specialised mammalian cell types). Transport of nucleobases and nucleosides across biological membranes is mediated by specific transport proteins. Nucleobase- and nucleoside transporters have been identified and characterised in a variety of pro- and eukaryotes. However, due to the limited amount of molecular data, it remained unknown how the secondary structure determines the substrate-specificity of the transporters.Many antiviral and anticancer nucleoside drugs are substrates of nucleoside and some of the nucleobase transporters, such as 5-fluoro uracil, 5-fluoro uridine and their derivatives. Since the transport of nucleoside drugs is critical to their therapeutic effectiveness, understanding the molecular mechanism of the transport process could enable the design of more effective nucleoside drugs and those with better absorption profile. Currently, the rational design of these drugs is hindered by the absence of high-resolution structural data on the transporters. Revealing the structure-activity relationships of transporters could facilitate the discovery of novel drugs with affinity specific for particular transporters.This project intends to investigate the uracil and uridine substrate specific nucleobase and nucleoside transporters of the model organism, A. nidulans, and subject them to mutational analysis using a complex molecular biological strategy to generate and characterise various functional mutant derivatives of these proteins, which will finally lead to a better understanding of the structure and specificity (and the relationship between these two) of the transporters molecules.
Original text from CORDIS.
Participants
- UNIVERSITY OF SZEGED · SZEGEDCoordinatorCity levelHungary
Links
Data: CORDIS, © European Union