POLG2 disease variants: analyses reveal a dominant negative heterodimer, altered mitochondrial localization and impaired respiratory capacity.
Hum Mol Genet
; 24(18): 5184-97, 2015 Sep 15.
Article
em En
| MEDLINE
| ID: mdl-26123486
ABSTRACT
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) POLG codes for the 140-kilodalton (kDa) catalytic subunit, p140 and (2) POLG2 encodes the â¼110-kDa homodimeric accessory subunit, p55. Specific mutations are associated with POLG- or POLG2-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 with POLG2-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 function in vitro, demonstrating that the POLG2 c.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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
DNA Polimerase Dirigida por DNA
Limite:
Humans
Idioma:
En
Revista:
Hum Mol Genet
Assunto da revista:
BIOLOGIA MOLECULAR
/
GENETICA MEDICA
Ano de publicação:
2015
Tipo de documento:
Article
País de afiliação:
Estados Unidos