Effects of A6E mutation on protein expression and structure formation of Asn1p-GFP in Saccharomyces cerevisiae

Name: Thunyarat Surasiang

LCSH: Asparagin

MeSH: Gene expression

Abstract: Asparagine synthetase deficiency (ASD) is caused by certain mutations in the ASNS gene coding for human asparagine synthetase (ASNS). Among 30 identified mutations, A6E mutation strikingly impaired the abundance of human ASNS. However, this mutation, associated with ASD in humans, has not been tested in the yeast system yet. So, this study aims to investigate the effects of A6E mutation on (1) protein abundance, and (2) supramolecular structure formation of yeast asparagine synthetase (Asn1p) tagged with a green fluorescent protein (GFP), allowing both the protein detection with anti-GFP and the direct visualization under a fluorescence microscope. The genome of yeast BY4741 was engineered by first knocking out ASN2 to prevent the complication in the characterization of A6E mutation effects on its paralog, ASN1 (as a major contributor to the asparagine biosynthesis), and then introducing A6E mutation codon to the chromosomal ASN1, along with GFP. Interestingly, A6E mutation showed a negative impact on protein abundance of Asn1p-GFP in all yeast growth stages. At the stationary phase, A6E mutation also markedly lowered the assembly frequency of the enzyme. In contrast to Asn1p(WT)-GFP, Asn1p(A6E)-GFP was insensitive to rapid changes in the intracellular energy levels upon treatment with sodium azide during log-phase or glucose addition at stationary phase. The results confirmed that the effect of A6E mutation on protein expression levels of asparagine synthetase is common in both unicellular and multicellular eukaryotes, suggesting that yeast could be used as an alternative model of ASD. IMPLICATION OF THE THESIS. Certain mutations causing an extremely low abundance of asparagine synthetase (the enzyme responsible for producing asparagine, one of the amino acids required for normal growth and development) have been identified in humans with neurological problems and small head and brain size. Currently, yeast is becoming more popular in modeling many human diseases. In this study, a mutation was incorporated, associated with human asparagine synthetase deficiency, into the yeast asparagine synthetase gene to demonstrate that this mutation can also show similar effects as those observed in humans, leading to a very low abundance of yeast asparagine synthetase and slower yeast growth rate. This suggests that the yeast system can be alternatively used to initially screen for any drugs that can help rescue the protein levels of asparagine synthetase before applying them to further studies in mammals and humans. Furthermore, this mutation might specifically be introduced into the asparagine synthetase gene of the target cancer cells to suppress the overproduction of asparagine synthetase within these abnormal cells, therefore inhibiting the growth of cancer, which might be helpful for patients with blood cancer to prevent them from developing any resistance to the conventional asparaginase treatment.

Mahidol University. Mahidol University Library and Knowledge Center

Address: NAKHONPATHOM

Email: liwww@mahidol.ac.th

Name: Chalongrat Noree

Role: Thesis Advisors

Name: Poochit Nonejuie

Role: Thesis Advisors

Name: Panadda Boonserm

Role: Thesis Advisors

Created: 2021

Modified: 2025-06-04

Issued: 2025-06-04

วิทยานิพนธ์/Thesis

application/pdf

eng

DegreeName: Master of Science

Level: Master's Degree

Descipline: Molecular Genetics and Genetic Engineering

Grantor: Mahidol University

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