MPV17 Loss Causes Deoxynucleotide Insufficiency and Slow DNA Replication in Mitochondria.
MPV17 Loss Causes Deoxynucleotide Insufficiency and Slow DNA Replication in Mitochondria.
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DOI：
10.1371/journal.pgen.1005779
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发表时间：
2016-01
期刊：
影响因子：
4.5
通讯作者：
Spinazzola A
中科院分区：
文献类型：
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作者：
Dalla Rosa I;Cámara Y;Durigon R;Moss CF;Vidoni S;Akman G;Hunt L;Johnson MA;Grocott S;Wang L;Thorburn DR;Hirano M;Poulton J;Taylor RW;Elgar G;Martí R;Voshol P;Holt IJ;Spinazzola A
MPV17是一种线粒体内膜蛋白，其功能障碍通过未知机制导致线粒体DNA异常和疾病。脱氧核苷三磷酸（dNTP）池的扰动是线粒体基因组不稳定的公认原因，因此，我们确定了DNA拷贝数和dNTP水平在两个模型的MPV 17缺陷的线粒体。在Mpv17消融小鼠中，肝脏线粒体显示dGTP和dTTP水平大幅下降，线粒体DNA严重耗竭，而在DNA水平接近正常的肾脏和大脑线粒体中，dNTP池未发生显著变化。Mpv17-/-肝脏中线粒体dNTPs的缺乏减缓了细胞器中的DNA复制，如复制中间体水平升高所证明的。MPV17突变患者的静止成纤维细胞重现了Mpv17-/-小鼠的主要受影响组织的关键特征，显示出蛋白质的实际缺失、dNTP水平降低和线粒体DNA耗竭。值得注意的是，脱氧核苷补充剂预防和挽救了患者静止成纤维细胞中的线粒体DNA丢失。因此，我们的研究确定了线粒体中dNTP不足是MPV 17缺乏症中线粒体DNA耗竭的原因，并确定了脱氧核苷补充剂作为MPV 17相关疾病的潜在治疗策略。此外，参与线粒体脱氧核苷酸稳态的因子表达的变化表明MPV 17疾病模型中核苷酸代谢的重塑，这表明缺乏功能性MPV 17的线粒体具有受限的嘌呤线粒体补救途径。线粒体DNA耗竭综合征（MDS）是一种以线粒体DNA（mtDNA）拷贝数减少和呼吸链酶活性降低为特征的遗传异质性疾病。线粒体DNA的缺失与几个基因的突变有关，这些基因编码直接参与线粒体DNA复制的蛋白质或调节线粒体脱氧核苷酸库稳态的因子。然而，对于某些基因，连接突变和mtDNA缺失的机制尚不清楚。其中一个这样的基因是MPV17，其功能丧失导致人类，小鼠和酵母的mtDNA异常。在这里，我们表明，MPV 17功能障碍导致线粒体中DNA合成前体的短缺，减缓了细胞器中的DNA复制。在小鼠组织和人类细胞中，mtDNA拷贝数不仅与dNTP池大小相关，而且生长培养基中补充脱氧核苷可防止静止期MPV 17缺陷细胞中的mtDNA拷贝数耗尽并恢复mtDNA拷贝数。因此，我们的研究将MPV17缺陷、线粒体dNTP不足和线粒体中的缓慢复制与人类疾病中表现的mtDNA耗竭联系起来，并将MPV17相关疾病牢固地置于由脱氧核苷酸扰动引起的mtDNA疾病类别中。MPV17患者来源细胞中mtDNA丢失的预防和逆转为目前无法治疗的疾病确定了潜在的治疗策略。
MPV17 is a mitochondrial inner membrane protein whose dysfunction causes mitochondrial DNA abnormalities and disease by an unknown mechanism. Perturbations of deoxynucleoside triphosphate (dNTP) pools are a recognized cause of mitochondrial genomic instability; therefore, we determined DNA copy number and dNTP levels in mitochondria of two models of MPV17 deficiency. In Mpv17 ablated mice, liver mitochondria showed substantial decreases in the levels of dGTP and dTTP and severe mitochondrial DNA depletion, whereas the dNTP pool was not significantly altered in kidney and brain mitochondria that had near normal levels of DNA. The shortage of mitochondrial dNTPs in Mpv17-/- liver slows the DNA replication in the organelle, as evidenced by the elevated level of replication intermediates. Quiescent fibroblasts of MPV17-mutant patients recapitulate key features of the primary affected tissue of the Mpv17-/- mice, displaying virtual absence of the protein, decreased dNTP levels and mitochondrial DNA depletion. Notably, the mitochondrial DNA loss in the patients’ quiescent fibroblasts was prevented and rescued by deoxynucleoside supplementation. Thus, our study establishes dNTP insufficiency in the mitochondria as the cause of mitochondrial DNA depletion in MPV17 deficiency, and identifies deoxynucleoside supplementation as a potential therapeutic strategy for MPV17-related disease. Moreover, changes in the expression of factors involved in mitochondrial deoxynucleotide homeostasis indicate a remodeling of nucleotide metabolism in MPV17 disease models, which suggests mitochondria lacking functional MPV17 have a restricted purine mitochondrial salvage pathway. Mitochondrial DNA depletion syndrome (MDS) is a genetically heterogeneous condition characterized by a decrease of mitochondrial DNA (mtDNA) copy number and decreased activities of respiratory chain enzymes. Depletion of mtDNA has been associated with mutations in several genes, which encode either proteins directly involved in mtDNA replication or factors regulating the homeostasis of the mitochondrial deoxynucleotide pool. However, for some genes the mechanism linking mutations and mtDNA depletion is not known. One such gene is MPV17, whose loss-of-function causes mtDNA abnormalities in human, mouse and yeast. Here we show that MPV17 dysfunction leads to a shortage of the precursors for DNA synthesis in the mitochondria, slowing DNA replication in the organelle. Not only does mtDNA copy number correlate with dNTP pool size in both mouse tissues and human cells, deoxynucleoside supplementation of the growth medium prevents depletion and restores mtDNA copy number in quiescent MPV17-deficient cells. Hence, our study links MPV17 deficiency, insufficiency of mitochondrial dNTPs, and slow replication in mitochondria to depletion of mtDNA manifesting in the human disease, and places MPV17-related disease firmly in the category of mtDNA disorders caused by deoxynucleotide perturbation. The prevention and reversal of mtDNA loss in MPV17 patient-derived cells identifies potential therapeutic strategy for a currently untreatable disease.