No abstract

Mammalian mitochondria contain a circular genome (mtDNA) which encodes subunits of the oxidative phosphorylation machinery. The replication and maintenance of mtDNA is carried out by a set of nuclear-encoded factors-of which, helicases form an important group. The TWINKLE helicase is the main helicase in mitochondria and is the only helicase required for mtDNA replication. Mutations in TWINKLE cause a number of human disorders associated with mitochondrial dysfunction, neurodegeneration and premature ageing. In addition, a number of other helicases with a putative role in mitochondria have been identified. In this review, we discuss our current knowledge of TWINKLE structure and function and its role in diseases of mtDNA maintenance. We also briefly discuss other potential mitochondrial helicases and postulate on their role(s) in mitochondria.

TWINKLE is the helicase involved in replication and maintenance of mitochondrial DNA (mtDNA) in mammalian cells. Structurally, TWINKLE is closely related to the bacteriophage T7 gp4 protein and comprises a helicase and primase domain joined by a flexible linker region. Mutations in and around this linker region are responsible for autosomal dominant progressive external ophthalmoplegia (adPEO), a neuromuscular disorder associated with deletions in mtDNA. The underlying molecular basis of adPEO-causing mutations remains unclear, but defects in TWINKLE oligomerization are thought to play a major role. In this study, we have characterized these disease variants by single-particle electron microscopy and can link the diminished activities of the TWINKLE variants to altered oligomeric properties. Our results suggest that the mutations can be divided into those that (i) destroy the flexibility of the linker region, (ii) inhibit ring closure and (iii) change the number of subunits within a helicase ring. Furthermore, we demonstrate that wild-type TWINKLE undergoes large-scale conformational changes upon nucleoside triphosphate binding and that this ability is lost in the disease-causing variants. This represents a substantial advancement in the understanding of the molecular basis of adPEO and related pathologies and may aid in the development of future therapeutic strategies.

LonP1 is a mitochondrial matrix protease whose selective substrate specificity is essential for maintaining mitochondrial homeostasis. Recessively inherited, pathogenic defects in LonP1 have been previously reported to underlie cerebral, ocular, dental, auricular and skeletal anomalies (CODAS) syndrome, a complex multisystemic and developmental disorder. Intriguingly, although classical mitochondrial disease presentations are well-known to exhibit marked clinical heterogeneity, the skeletal and dental features associated with CODAS syndrome are pathognomonic. We have applied whole exome sequencing to a patient with congenital lactic acidosis, muscle weakness, profound deficiencies in mitochondrial oxidative phosphorylation associated with loss of mtDNA copy number and MRI abnormalities consistent with Leigh syndrome, identifying biallelic variants in the LONP1 (NM_004793.3) gene; c.1693T > C predicting p.(Tyr565His) and c.2197G > A predicting p.(Glu733Lys); no evidence of the classical skeletal or dental defects observed in CODAS syndrome patients were noted in our patient. In vitro experiments confirmed the p.(Tyr565His) LonP1 mutant alone could not bind or degrade a substrate, consistent with the predicted function of Tyr565, whilst a second missense [p.(Glu733Lys)] variant had minimal effect. Mixtures of p.(Tyr565His) mutant and wild-type LonP1 retained partial protease activity but this was severely depleted when the p.(Tyr565His) mutant was mixed with the p.(Glu733Lys) mutant, data consistent with the compound heterozygosity detected in our patient. In summary, we conclude that pathogenic LONP1 variants can lead to a classical mitochondrial disease presentations associated with severe biochemical defects in oxidative phosphorylation in clinically relevant tissues.

Chloride intracellular channel protein 1 (CLIC1) is a dual-state protein that can exist either as a soluble monomer or in an integral membrane form. The transmembrane domain (TMD), implicated in membrane penetration and pore formation, comprises helix α1 and strand β2 of the N-domain of soluble CLIC1. The mechanism by which the TMD binds, inserts, and oligomerizes in membranes to form a functional chloride channel is unknown. Here we report the secondary, tertiary, and quaternary structural changes of the CLIC1 TMD as it partitions between an aqueous and membrane-mimicking environment. A synthetic 30-mer peptide comprising the TMD was examined in 2,2,2-trifluoroethanol, sodium dodecyl sulfate (SDS) micelles, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes using far-ultraviolet circular dichroism and fluorescence spectroscopy. Data obtained in the presence of SDS micelles and POPC liposomes show that Trp35 and Cys24 have reduced solvent accessibility, indicating that the peptide adopts an inserted orientation. The peptide assumes a helical structure in the presence of these mimetics, consistent with its predicted membrane conformation. This acquisition of secondary structure is concentration-dependent, suggesting an oligomerization event. Stable dimeric and trimeric species were subsequently identified using SDS-polyacrylamide gel electrophoresis. We propose that, in the vicinity of membranes, the mixed α/β TMD in CLIC1 rearranges to form a helix that then likely dimerizes via noncovalent helix-helix interactions to form a membrane-competent protopore complex. Such oligomerization would be essential for forming a functional ion channel, given that each CLIC1 monomer possesses only a single TMD. This work highlights the central role of the TMD in CLIC1 function: It is capable of promoting membrane insertion and dimerization in the absence of the C-domain and large portions of the N-domain.