The mitochondrial inner membrane protein MPV17 prevents uracil accumulation in mitochondrial DNA

Abstract

Mitochondrial inner membrane protein MPV17 is a protein of unknown function that is associated with mitochondrial DNA (mtDNA)-depletion syndrome (MDS). MPV17 loss-of-function has been reported to result in tissue-specific nucleotide pool imbalances, which can occur in states of perturbed folate-mediated one-carbon metabolism (FOCM), but MPV17 has not been directly linked to FOCM. FOCM is a metabolic network that provides one-carbon units for the de novo synthesis of purine and thymidylate nucleotides (e.g. dTMP) for both nuclear DNA (nuDNA) and mtDNA replication. In this study, we investigated the impact of reduced MPV17 expression on markers of impaired FOCM in HeLa cells. Depressed MPV17 expression reduced mitochondrial folate levels by 43% and increased uracil levels, a marker of impaired dTMP synthesis, in mtDNA by 3-fold. The capacity of mitochondrial de novo and salvage pathway dTMP biosynthesis was unchanged by the reduced MPV17 expression, but the elevated levels of uracil in mtDNA suggested that other sources of mitochondrial dTMP are compromised in MPV17-deficient cells. These results indicate that MPV17 provides a third dTMP source, potentially by serving as a transporter that transfers dTMP from the cytosol to mitochondria to sustain mtDNA synthesis. We propose that MPV17 loss-offunction and related hepatocerebral MDS are linked to impaired FOCM in mitochondria by providing insufficient access to cytosolic dTMP pools and by severely reducing mitochondrial folate pools.

MPV17 does not control cancer cell proliferation

“…This result is in agreement with the work of Dalla Rosa and collaborators who showed that proliferating fibroblasts from MPV17-deficient patients do not display any reduced mtDNA content [9]. It is also in accordance with the recent work of Alonzo and collaborators who did not find any reduction of mtDNA content in MPV17-silenced HeLa cells [12].…”

“…In this work we studied the putative role of MPV17 in cancer cell proliferation. While MPV17 silencing did not lead to depletion of mtDNA content in the tested cancer cell lines, in accordance with literature [9] [12], the resulting proliferation phenotypes associated with MPV17 shRNA-mediated knockdown were unsettling and inconclusive, leading us to perform a rescue experiment that eventually excluded a role of MPV17 in cancer cell proliferation. The intriguing and somewhat misleading results we obtained during this investigation clearly demonstrate the need to systematically perform a rescue experiment in order to ascertain the specific nature of the experimental results.…”

“…This long isoform referenced in RefSeq as NM002437.5 encodes the MPV17 protein that we detected on western blot while the translation of all the other MPV17 isoforms is not experimentally demonstrated. However, among the 22 isoforms, we observe that all the transcripts targeted by sh129921 (1, 2, 3, 12, 13, 14, 15, 16 and 18) are also targeted by either sh128669 (15,16) or sh131201 (1,18) or both (2,3,12,13,14). The same kind of observation stands for the transcripts targeted by sh127649 (1,2,3,5,12,13,14,15,16,18,19,21), providing no clear explanation about the opposite proliferation phenotypes observed for each pair of shRNAs (sh129921/sh127649 versus sh128669/131201) (see S5 Fig).…”

“…Although the current knowledge about MPV17 and its homologs have been recently reviewed [6], the function of MPV17 is still obscure. There are evidence supporting that MPV17 might be a channel with stress-dependent gating properties [7] [8] involved in nucleotides homeostasis [9] [3] [10] [11] [12].…”

Opa1overexpression protects from early onsetMpv17-/--related mouse kidney disease

“…Accordingly, high levels of rGTP were detected in mtDNA from Mpv17 -/livers (Moss et al, 2017). Other data showed that reduced MPV17 expression was associated with impaired dTMP synthesis without affecting de novo or salvage synthesis of dTMP, suggesting that MPV17 may be involved in maintaining dTMP levels in mitochondria through a still uncharacterized pathway, which may be involved in transporting dTMP or one of its precursors from the cytosol to mitochondria (Alonzo et al, 2018). In zebrafish, Mpv17 has been related to pyrimidine nucleotide metabolism via impairment of dihydroorotate dehydrogenase (Martorano et al, 2019).…”