Heterozygous p.Y955C mutation in DNA polymerase γ leads to alterations in bioenergetics, complex I subunit expression, and mtDNA replication.
Heterozygous p.Y955C mutation in DNA polymerase γ leads to alterations in bioenergetics, complex I subunit expression, and mtDNA replication.
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DOI：
10.1016/j.jbc.2022.102196
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发表时间：
2022-08
影响因子：
4.8
通讯作者：
Young, Matthew J.
中科院分区：
文献类型：
--
作者：
Rahman, Md Mostafijur;Young, Carolyn K. J.;Goffart, Steffi;Pohjoismaki, Jaakko L. O.;Young, Matthew J.
在人体细胞中，ATP 是通过氧化磷酸化机制产生的，如果没有线粒体 DNA (mtDNA) 编码的蛋白质，氧化磷酸化机制就无法运行。 DNA 聚合酶 γ (Polγ) 与其他因素一起修复和复制多拷贝 mtDNA 基因组。 Polγ催化亚基由POLG基因编码，该基因的突变会导致mtDNA基因组不稳定和疾病。研究疾病突变的分子效应的障碍包括缺乏患者样本和缺乏可用的突变模型；因此，我们开发了一种具有最常见的常染色体显性 POLG 突变 c.2864A>G/p.Y955C 的人类 SJCRH30 成肌细胞系模型，因为具有这种突变的个体可能会出现进行性骨骼肌无力。使用靶向测序，我们检测到突变的转换频率为 50%，证实了杂合 Y955C 取代。我们发现突变细胞在含葡萄糖的培养基中生长缓慢，并且与亲本细胞系相比线粒体生物能降低。此外，在含半乳糖的培养基中培养 Y955C 细胞以强制线粒体功能增强了这些生物能缺陷。此外，我们还发现突变细胞系中复合物 I NDUFB8 和 ND3 蛋白水平降低，并且 mtDNA 的维持严重受损（即拷贝数较低、类核较少以及 Y955C 特异性复制中间体的积累）。最后，我们发现突变细胞对线粒体毒物 2'-3'-双脱氧胞苷的敏感性增加。我们预计 POLG Y955C 细胞系将成为一个强大的系统，用于识别新的线粒体毒物和治疗线粒体功能障碍的疗法。
In human cells, ATP is generated using oxidative phosphorylation machinery, which is inoperable without proteins encoded by mitochondrial DNA (mtDNA). The DNA polymerase gamma (Polγ) repairs and replicates the multicopy mtDNA genome in concert with additional factors. The Polγ catalytic subunit is encoded by the POLG gene, and mutations in this gene cause mtDNA genome instability and disease. Barriers to studying the molecular effects of disease mutations include scarcity of patient samples and a lack of available mutant models; therefore, we developed a human SJCRH30 myoblast cell line model with the most common autosomal dominant POLG mutation, c.2864A>G/p.Y955C, as individuals with this mutation can present with progressive skeletal muscle weakness. Using on-target sequencing, we detected a 50% conversion frequency of the mutation, confirming heterozygous Y955C substitution. We found mutated cells grew slowly in a glucose-containing medium and had reduced mitochondrial bioenergetics compared with the parental cell line. Furthermore, growing Y955C cells in a galactose-containing medium to obligate mitochondrial function enhanced these bioenergetic deficits. Also, we show complex I NDUFB8 and ND3 protein levels were decreased in the mutant cell line, and the maintenance of mtDNA was severely impaired (i.e., lower copy number, fewer nucleoids, and an accumulation of Y955C-specific replication intermediates). Finally, we show the mutant cells have increased sensitivity to the mitochondrial toxicant 2′-3′-dideoxycytidine. We expect this POLG Y955C cell line to be a robust system to identify new mitochondrial toxicants and therapeutics to treat mitochondrial dysfunction.
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影响因子：
4.4
作者：
Ciesielska EJ;Kim S;Bisimwa HM;Grier C;Rahman MM;Young CKJ;Young MJ;Oliveira MT;Ciesielski GL
通讯作者：
Ciesielski GL
影响因子：
4.6
作者：
Belmonte FR;Martin JL;Frescura K;Damas J;Pereira F;Tarnopolsky MA;Kaufman BA
通讯作者：
Kaufman BA
影响因子：
4.3
作者：
Farnum, Gregory A.;Nurminen, Anssi;Kaguni, Laurie S.
通讯作者：
Kaguni, Laurie S.
影响因子：
6.1
作者：
Chen, Chun;Vincent, Amy E.;Reeve, Amy K.
通讯作者：
Reeve, Amy K.
影响因子：
9.8
作者：
Elliott, Hannah R.;Samuels, David C.;Eden, James A.;Relton, Caroline L.;Chinnery, Patrick F.
通讯作者：
Chinnery, Patrick F.