Effects of A6E Mutation on Protein Expression and Supramolecular Assembly of Yeast Asparagine Synthetase.

Thunyarat Surasiang,Chalongrat Noree

doi:10.3390/biology10040294

Abstract

Simple SummaryCertain mutations causing 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, we incorporate a mutation, 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 very low abundance of yeast asparagine synthetase and slower yeast growth rate. This suggests that our 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 in order 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 developing any resistance to the conventional asparaginase treatment.Asparagine synthetase deficiency (ASD) has been found to be caused by certain mutations in the gene encoding human asparagine synthetase (ASNS). Among reported mutations, A6E mutation showed the greatest reduction in ASNS abundance. However, the effect of A6E mutation has not yet been tested with yeast asparagine synthetase (Asn1/2p). Here, we constructed a yeast strain by deleting ASN2 from its genome, introducing the A6E mutation codon to ASN1, along with GFP downstream of ASN1. Our mutant yeast construct showed a noticeable decrease of Asn1p(A6E)-GFP levels as compared to the control yeast expressing Asn1p(WT)-GFP. At the stationary phase, the A6E mutation also markedly lowered the assembly frequency of the enzyme. In contrast to Asn1p(WT)-GFP, Asn1p(A6E)-GFP was insensitive to changes in the intracellular energy levels upon treatment with sodium azide during the log phase or fresh glucose at the stationary phase. Our study has 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 a model of ASD. Furthermore, A6E mutation could be introduced to the ASNS gene of acute lymphoblastic leukemia patients to inhibit the upregulation of ASNS by cancer cells, reducing the risk of developing resistance to the asparaginase treatment.

Highlights

Being one of the fundamental processes required for life and survival, the metabolism of any living organism is kept running through the action and network of metabolic enzymes in a well-coordinated and tightly-regulated manner
A6E mutation was originally reported as one of the mutations associated with asparagine synthetase deficiency (ASD), as this mutation adversely affects the protein expression levels of asparagine synthetase, causing neurological defects and small head and brain size in humans [31]
In our previous study and others [17,28], it was found that the assembly of Asn1p into visible intracellular structures is independent of the availability of Asn2p, but not vice versa

Summary

Introduction

Being one of the fundamental processes required for life and survival, the metabolism of any living organism is kept running through the action and network of metabolic enzymes in a well-coordinated and tightly-regulated manner. The synthetase domain is found at the C-terminus of the enzyme, where L-aspartate and Mg-ATP are catalyzed to form the intermediate, β-aspartyl-AMP. Our mutation study using yeast as a model organism suggested that the assembly– disassembly feature of asparagine synthetase is coupled to the regulation and status of its enzymatic activity [28]. The artificial yeast constructs expressing nuclear-targeted and loss-of-function asparagine synthetase showed that the association between the enzyme and the mitotic spindle is not activity-dependent, implying that asparagine synthetase has an unprecedented function in the cell division process, in addition to its well-known function in cellular metabolism [30]

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Conclusion