Molecular mechanisms of mitochondrial DNA deletion formation

描述（由申请人提供）：在pif 1基因敲除小鼠中观察到的线粒体功能障碍，加上人类线粒体疾病人群中罕见pif 1变体的富集，表明PIF 1活性对于维持线粒体呼吸功能和预防疾病至关重要。我们的目标是确定关键的线粒体PIF 1功能，通常防止青少年复合物I缺乏症和年龄相关的线粒体DNA（mtDNA）缺失的形成，以扩大我们对PIF 1在人类健康中的作用的理解。PIF 1是定位于细胞核和线粒体的G-四链体（GQ）解旋酶。G-四链体是在核酸中形成的富含鸟嘌呤的二级结构，并且在细胞核中引起DNA不稳定并干扰翻译。我们最近通过免疫细胞化学在培养细胞的线粒体中检测到GQ，提出了令人兴奋的可能性， 这些结构可能在线粒体功能障碍的发展中发挥作用。支持我们的想法，GQ在人类线粒体疾病中发挥作用，我们最近发现，人类mtDNA缺失断点的位置与G-四链体显着相关。我们推测线粒体核酸中GQ分辨率的丧失导致了pif 1基因敲除小鼠中线粒体呼吸的缺陷和mtDNA的不稳定性。我们将使用pif 1酵母、培养的pif 1敲除小鼠细胞和KO骨骼肌中的互补试验来检验这一假设。我们建议将明确定义的DNA和RNA导向的GQ和非GQ解旋酶靶向pif 1缺陷的线粒体，以确定复合物I活性和mtDNA稳定性所需的活性。然后使用腺相关病毒（AAV）递送在小鼠pif 1 KO骨骼肌中体内测试强候选物。我们还将通过检查pif 1空和解旋酶补充细胞的复合物I转录丰度、翻译和呼吸复合物组装来确定线粒体蛋白质生产中的任何缺陷步骤。通过将GQ的免疫检测与下一代测序相结合，将确定WT、空和解旋酶互补肌肉中GQ丰度与mtDNA缺失负荷之间的关系。该策略还将鉴定pif 1无效和挽救样品中GQ位置与缺失断点位置之间的关联。这些机制的研究将支持我们的追求PIF 1作为一个人类疾病基因的患者记录的mtDNA缺失或复合物I缺乏症。重要的是，我们在线粒体疾病患者中发现了罕见的PIF 1纯合和复合杂合突变，这突出了PIF 1作为一种潜在的人类疾病基因。线粒体GQ结构形成和PIF 1活性之间的平衡为人类疾病提供了新的但非常有前途的见解，这里提出的实验有可能开辟线粒体医学研究的新领域。

DESCRIPTION (provided by applicant): The mitochondrial dysfunction observed in the pif1 knockout mouse, coupled with the enrichment of rare pif1 variants in the human mitochondrial disease population, suggest that PIF1 activity is essential for maintaining mitochondrial respiratory function and the prevention of disease. Our objective is to identify the crucial mitochondrial PIF1 functions that normally prevent juvenile complex I deficiency and age-related formation of mitochondrial DNA (mtDNA) deletions to expand our understanding of the role of PIF1 in human health. PIF1 is a G-quadruplex (GQ) helicase that localizes to the nucleus and mitochondria. G-quadruplexes are guanine-rich secondary structures that form in nucleic acids, and in the nucleus cause DNA instability and interfere with translation. We recently detected GQ in the mitochondria of cultured cells by immunocytochemistry, raising the exciting possibility that these structures could play a role in the development of mitochondrial dysfunction. Supporting our idea that GQ play a role in human mitochondrial disease, we recently showed that the location of human mtDNA deletion breakpoints associates significantly with G-quadruplexes. We hypothesize that the loss of the GQ resolution in mitochondrial nucleic acids contributes to the deficits in mitochondrial respiration and the mtDNA instability seen in the pif1 null mouse. We will test this hypothesis using complementation assays in pif1 yeast, cultured pif1 null mouse cells, and KO skeletal muscle. We propose to target well defined DNA- and RNA-directed GQ and non-GQ helicases to pif1-deficient mitochondria to identify the activities required for complex I activity and mtDNA stability. Strong candidates will then be tested in mouse pif1 KO skeletal muscle in vivo using adeno-associated virus (AAV) delivery. We will also identify any defective steps in mitochondrial protein production by examining pif1 null and helicase-complemented cells for complex I transcript abundance, translation, and respiratory complex assembly. The relationship between GQ abundance and mtDNA deletion load in WT, null, and helicase-complimented muscle will be determined by coupling immunodetection of GQ with next generation sequencing. This strategy will also identify associations between GQ position and deletion breakpoint location in pif1 null and rescued samples. These mechanistic studies will support our pursuit of PIF1 as a human disease gene in patients with documented mtDNA deletions or complex I deficiency. Importantly, we have identified rare PIF1 homozygous and compound heterozygous mutations in human patients with mitochondrial disease, which highlights PIF1 as a potential human disease gene. The balance between mitochondrial GQ structure formation and PIF1 activity offers novel yet highly promising insight into human disease, and the experiments proposed here have the potential to open a new field of research in mitochondrial medicine.