Abstract
IntroductionTransporter proteins are essential for the movement of small molecules across lipid membranes, enabling the proper function of metabolic processes, relied upon by many obligate intracellular pathogens, including Microsporidia a human infecting fungus. Microsporidia utilise nucleotide transporter proteins (NTT) to scavenge nucleotides including ATP, having lost the pathways to synthesise them de novo. Functional studies of transporter proteins rely on uptake assays using radioisotopes to label putative substrates, posing a risk to a researcher’s health. Raman spectroscopy is an alternative method that could be used for the study of transporter proteins. This study reports the development of a method using Raman microscopy and alkyne labelled substrates for the study of transporter proteins taking advantage of the alkynes peak that occurs in the biologically silent region of the Raman spectra.
Methods
The expression levels of NTT transporters from the Microsporidia species Trachipleistophora hominis NTTs were monitored by Western blot analysis. This identified ThNTT4 as a reliable transporter with consistently high expression. ThNTT4 expression was optimised for use in uptake assays, whereby alkyne labelled substrates were detected by Raman spectroscopy as an alternative to radioisotopes. The alkyne labelled ATP analogue, N6pATP, was used to test whether Raman spectroscopy could detect transport of ATP by ThNTT4 in a time dependent manner. Further uptakes with alkyne labelled ATP analogue 2-EATP were performed, and competition assays completed using N6pATP and thymidine analogue 5-EdU were used alongside varying ratios of unlabelled substrates to test for inhibition and specificity of the ThNTT4 for its substrates.
Results
Initial comparisons of ThNTT1-4 showed ThNTT4 to be a consistently highly expressed transporter suitable for use as a model transporter compared to ThNTT1-3. Modifications of the commonly used uptake assays were made incorporating Raman spectroscopy as a detection method into novel method we have named ‘Transporter assay utilising Raman spectroscopy’(TAURS). The results showed that all three alkyne labelled substrates produced Raman peaks within the biological silent region of a Raman spectrum allowing for their use as substrate labels. ThNTT4 was able to transport N6pATP above that of an empty pET-16b vector control. Comparable results were found for the transport of 2-EATP. Competition assays showed an excess of unlabelled ATP inhibited the uptake of N6pATP by ThNTT4 yet replacing non labelled ATP with thymidine restored transport of N6pATP. Further studies showed no uptake above the empty vector control of 5-EdU a pyrimidine of which ThNTT4 has been shown unable to transport.
Discussion
Raman spectroscopy when used with alkyne labelled substrates provides an alternative to radioisotope labelling for detecting transport. This study has shown using an uptake assay utilising Raman spectroscopy(TAURS) that ThNTT4 has a specificity for ATP and has also confirmed that ThNTT4 is unable to transport thymidine a pyrimidine for which thNTT4 has been shown unable to transport. The results in this study show similarity to those produced in the functional study of ThNTT4 which used radioisotopes, in their ability to show transport of a particular substrate, however further work needed to show rates of transport and quantifiable comparisons to radioisotopes. Such a transporter uptake assay using Raman spectroscopy has here been shown as an alternative to the labelling of substrates with radioisotopes for detecting transport. The Raman microscopy method to measure substrate uptake by a transporter, offers researchers a new and alternative approach that is safe, accessible, which can potentially revolutionise the field of transporter protein study.
| Date of Award | 25 May 2023 |
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| Original language | English |
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| Supervisor | Paul Dean (Supervisor), Safwan Akram (Supervisor) & Meez Islam (Supervisor) |