Surasiang Thunyarat, Noree Chalongrat
Institute of Molecular Biosciences, Mahidol University, Salaya, Nakhon Pathom 73170, Thailand.
Biology (Basel). 2021 Apr 3;10(4):294. doi: 10.3390/biology10040294.
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 from its genome, introducing the A6E mutation codon to , along with downstream of . 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 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.
天冬酰胺合成酶缺乏症（ASD）已被发现是由编码人类天冬酰胺合成酶（ASNS）的基因中的某些突变引起的。在已报道的突变中，A6E突变导致ASNS丰度下降最为显著。然而，A6E突变对酵母天冬酰胺合成酶（Asn1/2p）的影响尚未得到测试。在此，我们通过从其基因组中删除，将A6E突变密码子引入，并在其下游引入，构建了一种酵母菌株。与表达Asn1p(WT)-GFP的对照酵母相比，我们构建的突变酵母显示Asn1p(A6E)-GFP水平显著降低。在稳定期，A6E突变也显著降低了该酶的组装频率。与Asn1p(WT)-GFP不同，Asn1p(A6E)-GFP在对数期用叠氮化钠处理或在稳定期用新鲜葡萄糖处理时，对细胞内能量水平的变化不敏感。我们的研究证实，A6E突变对天冬酰胺合成酶蛋白表达水平的影响在单细胞和多细胞真核生物中都是常见的，这表明酵母可以作为ASD的模型。此外，可以将A6E突变引入急性淋巴细胞白血病患者的基因中，以抑制癌细胞对ASNS的上调，降低对天冬酰胺酶治疗产生耐药性的风险。
