Functional characterization of two variants in the mitochondrial topoisomerase gene TOP1MT that impact regulation of the mitochondrial genome

Abstract

TOP1MT encodes a mitochondrial topoisomerase that is important for mtDNA regulation, and is involved in mitochondrial replication, transcription and translation. Two variants predicted to affect TOP1MT function (R199C and V338L) were identified by exome sequencing of a newborn with hypertrophic cardiomyopathy. As no pathogenic TOP1MT variants have been confirmed previously, we characterized these variants for their ability to rescue several TOP1MT functions in knockout cells. Consistent with a role for these TOP1MT variants contributing to the patient phenotype, comprehensive characterization suggests that both variants had impaired topoisomerase activity and demonstrates that neither variant was able to restore steady state levels of mitochondrial encoded proteins, nor reduced oxidative phosphorylation. However, the two variants behaved differently in some respects. While the R199C variant was better at restoring transcript levels, the V338L variant was able to restore mtDNA copy number and replication. These findings suggest that the different TOP1MT variants affect distinct TOP1MT functions. Altogether, these findings begin to provide insight into the many roles that TOP1MT plays in the maintenance and expression of the mitochondrial genome, and how impairments in this important protein may lead to human pathology.

Activation of the cGAS-STING innate immune response in cells with deficient mitochondrial topoisomerase TOP1MT

“…We next examined the expression of mtDNA-encoded transcripts, as the supercoiling extent of mtDNA directly influences the transcription of the mitochondrial genome [36][37][38].…”

“…As TOP1MT can also impact the translation of mitochondrial proteins independently of its topoisomerase activity and changes in transcripts [38], we studied the effect of P193L on the…”

“…Given the mitochondrial dysfunction noted in patient derived blood cells, we wanted to examine the ability of TOP1MT's P193L variant to rescue multiple mitochondrial parameters known to be impaired in HCT KO cells [38]. As described previously [38], starting from HCT WT and HCT KO, we generated four different stable cell lines using retroviral constructs expressing TOP1MT and the mCherry fluorescent protein separated by a self-cleavage peptide (TOP1MT-T2A-mCherry), or an empty vector control. This construct leads to expression of TOP1MT, which will be directed to the mitochondria by the mitochondrial targeting sequence, and mCherry, which can be used to monitor transduction.…”

“…Next, we wanted to determine the ability of the P193L variant to rescue the aberrant functions previously identified in HCT KO cells [38], including: reduced nucleoid size, the hyperfused mitochondrial network, and decreases in mitochondrial transcription, mtDNA replication, mitochondrial translation and oxidative phosphorylation.…”

“…One intriguing candidate is TOP1MT, a topoisomerase that helps resolve crossovers and knots that can originate during maintenance of the circular mtDNA genome. TOP1MT is the only human topoisomerase that is solely present in the mitochondria [32][33][34], where it plays important roles in mtDNA replication, transcription, and translation [35][36][37][38]. Interestingly, a wide range of autoantibodies have been described and detected in the serum of SLE patients [39,40] including anti-double-stranded DNA (anti-dsDNA) [41,42] and autoantibodies recognizing the nuclear topoisomerase enzyme TOP1 [43][44][45][46] as well as the mitochondrial TOP1MT [47].…”