POLG2 disease variants: analyses reveal a dominant negative heterodimer, altered mitochondrial localization and impaired respiratory capacity.
POLG2 disease variants: analyses reveal a dominant negative heterodimer, altered mitochondrial localization and impaired respiratory capacity.
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POLG2 疾病变异：分析显示显性负异二聚体、线粒体定位改变和呼吸能力受损。
DOI：
10.1093/hmg/ddv240
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发表时间：
2015
影响因子：
3.5
通讯作者：
Copeland,WilliamC
中科院分区：
文献类型：
--
作者：
Young,MatthewJ;Humble,MargaretM;DeBalsi,KarenL;Sun,KathieY;Copeland,WilliamC
人类线粒体DNA（mtDNA）由mtDNA聚合酶γ（polγ）复制和修复。Polγ由两个核基因编码的三个亚基组成：（1）POLG编码140-kDa催化亚基p140;（2）POLG 2编码110-kDa同源二聚体辅助亚基p55。特定的突变与POLG或POLG 2相关疾病有关。在DNA复制过程中，p55辅助亚基与p140结合，并通过阻止polγ从模板上解离来增加持续合成能力。迄今为止，研究表明，同二聚体p55疾病的变异体缺乏刺激p140的能力;然而，目前所有POLG 2相关疾病的患者都是杂合子。在这些患者中，我们预期p55以25%野生型（WT）同源二聚体、25%变异同源二聚体和50%异源二聚体的形式出现。我们报告的串联亲和策略分离p55异源二聚体的发展。WT/G451 E p55异源二聚体在体外损害polγ功能，表明POLG 2c.1352G>A/p.G451E突变编码显性负性蛋白。为了分析HEK 293细胞中疾病突变的亚细胞后果，我们设计了编码用绿色荧光蛋白（GFP）标记的p55疾病变体的质粒。P205 R和L475 DfsX 2 p55变体表现出不规则的弥漫性线粒体荧光，与WT p55不同，它们不能形成与mtDNA类核相关的明显斑点。此外，P205 R和L475 DfsX 2 p55的均质制剂形成异常的可还原多聚体。我们预测，异常蛋白质折叠或聚集或两者都有助于这些疾病的病理生理。在过表达GFP标记的p55变体的稳定细胞系中检查线粒体生物能量学，发现线粒体储备能力受损。
Human mitochondrial DNA (mtDNA) is replicated and repaired by the mtDNA polymerase gamma, polγ. Polγ is composed of three subunits encoded by two nuclear genes: (1)POLGcodes for the 140-kilodalton (kDa) catalytic subunit, p140 and (2)POLG2encodes the ∼110-kDa homodimeric accessory subunit, p55. Specific mutations are associated withPOLG- orPOLG2-related disorders. During DNA replication the p55 accessory subunit binds to p140 and increases processivity by preventing polγ's dissociation from the template. To date, studies have demonstrated that homodimeric p55 disease variants are deficient in the ability to stimulate p140; however, all patients currently identified withPOLG2-related disorders are heterozygotes. In these patients, we expect p55 to occur as 25% wild-type (WT) homodimers, 25% variant homodimers and 50% heterodimers. We report the development of a tandem affinity strategy to isolate p55 heterodimers. The WT/G451E p55 heterodimer impairs polγ functionin vitro, demonstrating that thePOLG2c.1352G>A/p.G451E mutation encodes a dominant negative protein. To analyze the subcellular consequence of disease mutations in HEK293 cells, we designed plasmids encoding p55 disease variants tagged with green fluorescent protein (GFP). P205R and L475DfsX2 p55 variants exhibit irregular diffuse mitochondrial fluorescence and unlike WT p55, they fail to form distinct puncta associated with mtDNA nucleoids. Furthermore, homogenous preparations of P205R and L475DfsX2 p55 form aberrant reducible multimers. We predict that abnormal protein folding or aggregation or both contribute to the pathophysiology of these disorders. Examination of mitochondrial bioenergetics in stable cell lines overexpressing GFP-tagged p55 variants revealed impaired mitochondrial reserve capacity.