Matthew J. Young

Biochemical analysis of human POLG2 variants associated with mitochondrial disease

Defects in mitochondrial DNA (mtDNA) maintenance comprise an expanding repertoire of polymorphic diseases caused, in part, by mutations in the genes encoding the p140 mtDNA polymerase (POLG), its p55 accessory subunit (POLG2) or the mtDNA helicase (C10orf2). In an exploration of nuclear genes for mtDNA maintenance linked to mitochondrial disease, eight heterozygous mutations (six novel) in POLG2 were identified in one control and eight patients with POLG-related mitochondrial disease that lacked POLG mutations. Of these eight mutations, we biochemically characterized seven variants [c.307G>A (G103S); c.457C>G (L153V); c.614C>G (P205R); c.1105A>G (R369G); c.1158T>G (D386E); c.1268C>A (S423Y); c.1423_1424delTT (L475DfsX2)] that were previously uncharacterized along with the wild-type protein and the G451E pathogenic variant. These seven mutations encode amino acid substitutions that map throughout the protein, including the p55 dimer interface and the C-terminal domain that interacts with the catalytic subunit. Recombinant proteins harboring these alterations were assessed for stimulation of processive DNA synthesis, binding to the p140 catalytic subunit, binding to dsDNA and self-dimerization. Whereas the G103S, L153V, D386E and S423Y proteins displayed wild-type behavior, the P205R and R369G p55 variants had reduced stimulation of processivity and decreased affinity for the catalytic subunit. Additionally, the L475DfsX2 variant, which possesses a C-terminal truncation, was unable to bind the p140 catalytic subunit, unable to bind dsDNA and formed aberrant oligomeric complexes. Our biochemical analysis helps explain the pathogenesis of POLG2 mutations in mitochondrial disease and emphasizes the need to quantitatively characterize the biochemical consequences of newly discovered mutations before classifying them as pathogenic.

Human mitochondrial DNA replication machinery and disease.

The human mitochondrial genome is replicated by DNA polymerase γ in concert with key components of the mitochondrial DNA (mtDNA) replication machinery. Defects in mtDNA replication or nucleotide metabolism cause deletions, point mutations, or depletion of mtDNA. The resulting loss of cellular respiration ultimately induces mitochondrial genetic diseases, including mtDNA depletion syndromes (MDS) such as Alpers or early infantile hepatocerebral syndromes, and mtDNA deletion disorders such as progressive external ophthalmoplegia, ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy. Here we review the current literature regarding human mtDNA replication and heritable disorders caused by genetic changes of the POLG, POLG2, Twinkle, RNASEH1, DNA2, and MGME1 genes.

Polg2 is essential for mammalian embryogenesis and is required for mtDNA maintenance

Mammalian mitochondrial DNA (mtDNA) is replicated by the heterotrimeric Pol γ comprised of a single catalytic subunit, encoded by Polg, and a homodimeric accessory subunit encoded by the Polg2 gene. While the catalytic subunit has been shown to be essential for embryo development, genetic data regarding the accessory subunit are lacking in mammalian systems. Here, we describe the generation of heterozygous (Polg2(+/-)) and homozygous (Polg2(-/-)) knockout (KO) mice. Polg2(+/-) mice are haplosufficient and develop normally with no discernable difference in mitochondrial function through 2 years of age. In contrast, the Polg2(-/-) is embryonic lethal at day 8.0-8.5 p.c. with concomitant loss of mtDNA and mtDNA gene products. Electron microscopy shows severe ultra-structural defects and loss of organized cristae in mitochondria of the Polg2(-/-) embryos as well as an increase in lipid accumulation compared with both wild-type (WT) and Polg2(+/-) embryos. Our data indicate that Polg2 function is critical to mammalian embryogenesis and mtDNA replication, and that a single copy of Polg2 is sufficient to sustain life.

Phylogenetic and coevolutionary analysis of the β-barrel protein family comprised of mitochondrial porin (VDAC) and Tom40 ☆

Beta-barrel proteins are the main transit points across the mitochondrial outer membrane. Mitochondrial porin, the voltage-dependent, anion-selective channel (VDAC), is responsible for the passage of small molecules between the mitochondrion and the cytosol. Through interactions with other mitochondrial and cellular proteins, it is involved in regulating organellar and cellular metabolism and likely contributes to mitochondrial structure. Tom40 is part of the translocase of the outer membrane, and acts as the channel for passage of preproteins during their import into the organelle. These proteins appear to share a common evolutionary origin and structure. In the current study, the evolutionary relationships between and within both proteins were investigated through phylogenetic analysis. The two groups have a common origin and have followed independent, complex evolutionary pathways, leading to the generation of paralogues in animals and plants. Structures of diverse representatives were modeled, revealing common themes rather than sites of high identity in both groups. Within each group, intramolecular coevolution was assessed, revealing a new set of sites potentially involved in structure-function relationships in these molecules. A weak link between Tom40 and proteins related to the mitochondrial distribution and morphology protein, Mdm10, was identified. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.

Suppression of p65 phosphorylation coincides with inhibition of IκBα polyubiquitination and degradation

Transcription factor nuclear factor‐κB (NF‐κB) is held in the cytoplasm in an inactive state by IκB inhibitors. Oncogenic activation of NF‐κB is achieved by stimulus‐induced ubiquitination and subsequent proteasome‐mediated degradation of IκBα. Once released from the inhibitor, NF‐κB/p65 enters the nucleus. A pre‐requisite for cytokine‐induced IκBα ubiquitination and degradation is the phosphorylation of IκBα at S32/S36. Phosphorylation of IκBα alone, however, is not sufficient to trigger its degradation, suggesting other events must be required for regulating IκBα degradation. In this study, we tested the hypothesis that phosphorylation of p65 at 536 is required for TNF‐α induced IκBα proteolysis that in turn controls p65 nuclear translocation. We observed that, without affecting IκBα phosphorylation, MEK1 inhibitor U0126 treatment inhibited not only p65‐S536 phosphorylation but also TNF‐α‐induced polyubiquitination of IκBα thereby inhibiting IκBα degradation. With p65 S536 phosphorylation mutants and mimics, we further observed that the structural mutation of p65 serine 536 to alanine inhibited the recruitment of ubiquitin to the p65‐containing complex. As a consequence of suppressing polyubiquitination of the p65‐containing complex, degradation of p65 phosphorylation mutant‐bound IκBα was also inhibited. Accordingly, the nuclear translocation of phosphorylation‐impaired p65 was significantly reduced. These findings suggest that p65 phosphorylation plays a key role in stimulus‐induced IκBα ubiquitination. Published 2005 Wiley‐Liss, Inc.

Effects of the S288c genetic background and common auxotrophic markers on mitochondrial DNA function in Saccharomyces cerevisiae

Saccharomyces cerevisiae is a valuable model organism for the study of eukaryotic processes. Throughout its development as a research tool, several strain backgrounds have been utilized and different combinations of auxotrophic marker genes have been introduced into them, creating a useful but non‐homogeneous set of strains. The ade2 allele was used as an auxotrophic marker, and for ‘red–white’ screening for respiratory competence. his3 alleles that influence the expression of MRM1 have been used as selectable markers, and the MIP1[S] allele, found in the commonly used S228c strain, is associated with mitochondrial DNA defects. The focus of the current work was to examine the effects of these alleles, singly and in combination, on the maintenance of mitochondrial function. The combination of the ade2 and MIP1[S] alleles is associated with a slight increase in point mutations in mitochondrial DNA. The deletion in the his3Δ200 allele, which removes the promoter for MRM1, is associated with loss of respiratory competence at 37 °C in the presence of either MIP1 allele. Thus, multiple factors can contribute to the maintenance of mitochondrial function, reinforcing the concept that strain background is an important consideration in both designing experiments and comparing results obtained by different research groups. Copyright

A p.R369G POLG2 mutation associated with adPEO and multiple mtDNA deletions causes decreased affinity between polymerase γ subunits

Human mitochondrial DNA (mtDNA) polymerase γ (pol γ) is the sole enzyme required to replicate and maintain the integrity of the mitochondrial genome. It comprises two subunits, a catalytic p140 subunit and a smaller p55 accessory subunit encoded by the POLG2 gene. We describe the molecular characterization of a potential dominant POLG2 mutation (p.R369G) in a patient with adPEO and multiple mtDNA deletions. Biochemical studies of the recombinant mutant p55 protein showed a reduced affinity to the pol γ p140 subunit, leading to impaired processivity of the holoenzyme complex but did not show sensitivity to N-ethylmalaimide (NEM) inhibition, inferring a novel disease mechanism.

Purification and functional characterization of human mitochondrial DNA polymerase gamma harboring disease mutations

More than 150 different point mutations in POLG, the gene encoding the human mitochondrial DNA polymerase gamma (pol gamma), cause a broad spectrum of childhood and adult onset diseases like Alpers syndrome, ataxia-neuropathy syndrome and progressive external ophthalmoplegia. These disease mutations can affect the pol gamma enzymes properties in numerous ways, thus potentially influencing the severity of the disease. Hence, a detailed characterization of disease mutants will greatly assist researchers and clinicians to develop a clear understanding of the functional defects caused by these mutant enzymes. Experimental approaches for characterizing the wild-type (WT) and mutant pol gamma enzymes are extensively described in this manuscript. The methods start with construction and purification of the recombinant wild-type and mutant forms of pol gamma protein, followed by assays to determine its structural integrity and thermal stability. Next, the biochemical characterization of these enzymes is described in detail, which includes measuring the purified enzymes catalytic activity, its steady-state kinetic parameters and DNA binding activity, and determining the physical and functional interaction of these pol gamma proteins with the p55 accessory subunit.

POLG2 disease variants: analyses reveal a dominant negative heterodimer, altered mitochondrial localization and impaired respiratory capacity

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 ∼110-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 polγs 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.

The carboxyl‐terminal extension on fungal mitochondrial DNA polymerases: identification of a critical region of the enzyme from Saccharomyces cerevisiae

Fungal mitochondrial DNA (mtDNA) polymerases, in comparison to their metazoan counterparts, harbour unique carboxyl‐terminal extensions (CTEs) of varying lengths and unknown function. To determine the essential regions of the 279 residue CTE of the yeast enzyme (Mip1p), several CTE‐truncation variants were expressed in Saccharomyces cerevisiae. The respiratory competence of mip1Δ175 cells, in which Mip1p lacks the C‐terminal 175 residues, is indistinguishable from that of wild‐type. In contrast, strains harbouring Mip1pΔ351 and Mip1pΔ279 rapidly lose mtDNA. Approximately one in six mip1Δ216 transformants grew on glycerol, albeit poorly. Fluorescence microscopy and Southern blot analysis revealed lower levels of mtDNA in these cells, and the rapid loss of mtDNA during fermentative, but not respiratory, growth. Therefore, only the polymerase‐proximal segment of the Mip1p CTE is necessary for mitochondrial function. Comparison of this essential segment with the sequences of other fungal mtDNA polymerases revealed novel features shared among the mtDNA polymerases of the Saccharomycetales. Copyright