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Doimo, Mara
Publications (4 of 4) Show all publications
Al-Behadili, A., Uhler, J. P., Berglund, A.-K., Peter, B., Doimo, M., Reyes, A., . . . Falkenberg, M. (2018). A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL. Nucleic Acids Research, 46(18), 9471-9483
Open this publication in new window or tab >>A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL
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2018 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, no 18, p. 9471-9483Article in journal (Refereed) Published
Abstract [en]

The role of Ribonuclease H1 (RNase H1) during primer removal and ligation at the mitochondrial origin of light-strand DNA synthesis (OriL) is a key, yet poorly understood, step in mitochondrial DNA maintenance. Here, we reconstitute the replication cycle of L-strand synthesis in vitro using recombinant mitochondrial proteins and model OriL substrates. The process begins with initiation of DNA replication at OriL and ends with primer removal and ligation. We find that RNase H1 partially removes the primer, leaving behind the last one to three ribonucleotides. These 5′-end ribonucleotides disturb ligation, a conclusion which is supported by analysis of RNase H1-deficient patient cells. A second nuclease is therefore required to remove the last ribonucleotides and we demonstrate that Flap endonuclease 1 (FEN1) can execute this function in vitro. Removal of RNA primers at OriL thus depends on a two-nuclease model, which in addition to RNase H1 requires FEN1 or a FEN1-like activity. These findings define the role of RNase H1 at OriL and help to explain the pathogenic consequences of disease causing mutations in RNase H1.

Place, publisher, year, edition, pages
Oxford University Press, 2018
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-154079 (URN)10.1093/nar/gky708 (DOI)000450953200023 ()30102370 (PubMedID)
Funder
Swedish Research Council, VR521-2013-3621Swedish Cancer Society, CAN 2016/816Knut and Alice Wallenberg Foundation
Available from: 2018-12-12 Created: 2018-12-12 Last updated: 2018-12-12Bibliographically approved
Cerqua, C., Morbidoni, V., Desbats, M. A., Doimo, M., Frasson, C., Sacconi, S., . . . Trevisson, E. (2018). COX16 is required for assembly of cytochrome c oxidase in human cells and is involved in copper delivery to COX2. Biochimica et Biophysica Acta - Bioenergetics, 1859(4), 244-252
Open this publication in new window or tab >>COX16 is required for assembly of cytochrome c oxidase in human cells and is involved in copper delivery to COX2
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2018 (English)In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1859, no 4, p. 244-252Article in journal (Refereed) Published
Abstract [en]

Cytochrome c oxidase (COX), complex IV of the mitochondrial respiratory chain, is comprised of 14 structural subunits, several prosthetic groups and metal cofactors, among which copper. Its biosynthesis involves a number of ancillary proteins, encoded by the COX-assembly genes that are required for the stabilization and membrane insertion of the nascent polypeptides, the synthesis of the prosthetic groups, and the delivery of the metal cofactors, in particular of copper. Recently, a modular model for COX assembly has been proposed, based on the sequential incorporation of different assembly modules formed by specific subunits.

We have cloned and characterized the human homologue of yeast COX16. We show that human COX16 encodes a small mitochondrial transmembrane protein that faces the intermembrane space and is highly expressed in skeletal and cardiac muscle. Its knockdown in C. elegans produces COX deficiency, and its ablation in HEK293 cells impairs COX assembly. Interestingly, COX16 knockout cells retain significant COX activity, suggesting that the function of COX16 is partially redundant.

Analysis of steady-state levels of COX subunits and of assembly intermediates by Blue-Native gels shows a pattern similar to that reported in cells lacking COX18, suggesting that COX16 is required for the formation of the COX2 subassembly module. Moreover, COX16 co-immunoprecipitates with COX2. Finally, we found that copper supplementation increases COX activity and restores normal steady state levels of COX subunits in COX16 knockout cells, indicating that, even in the absence of a canonical copper binding motif, COX16 could be involved in copper delivery to COX2.

Place, publisher, year, edition, pages
Elsevier, 2018
Keywords
Cytochrome c oxidase, Mitochondrial respiratory chain, Cytochrome c oxidase assembly, Copper
National Category
Cell Biology
Identifiers
urn:nbn:se:umu:diva-151184 (URN)10.1016/j.bbabio.2018.01.004 (DOI)000430755600003 ()29355485 (PubMedID)2-s2.0-85041568968 (Scopus ID)
Available from: 2018-09-04 Created: 2018-09-04 Last updated: 2018-09-04Bibliographically approved
Montioli, R., Desbats, M. A., Grottelli, S., Doimo, M., Bellezza, I., Voltattorni, C. B., . . . Cellini, B. (2018). Molecular and cellular basis of ornithine δ-aminotransferase deficiency caused by the V332M mutation associated with gyrate atrophy of the choroid and retina. Biochimica et Biophysica Acta - Molecular Basis of Disease, 1864(11), 3629-3638
Open this publication in new window or tab >>Molecular and cellular basis of ornithine δ-aminotransferase deficiency caused by the V332M mutation associated with gyrate atrophy of the choroid and retina
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2018 (English)In: Biochimica et Biophysica Acta - Molecular Basis of Disease, ISSN 0925-4439, E-ISSN 1879-260X, Vol. 1864, no 11, p. 3629-3638Article in journal (Refereed) Published
Abstract [en]

Gyrate atrophy (GA) is a rare recessive disorder characterized by progressive blindness, chorioretinal degeneration and systemic hyperornithinemia. GA is caused by point mutations in the gene encoding ornithine δ-aminotransferase (OAT), a tetrameric pyridoxal 5′-phosphate-dependent enzyme catalysing the transamination of l-ornithine and α-ketoglutarate to glutamic–γ-semialdehyde and l-glutamate in mitochondria. More than 50 OAT variants have been identified, but their molecular and cellular properties are mostly unknown. A subset of patients is responsive to pyridoxine administration, although the mechanisms underlying responsiveness have not been clarified. Herein, we studied the effects of the V332M mutation identified in pyridoxine-responsive patients. The Val332-to-Met substitution does not significantly affect the spectroscopic and kinetic properties of OAT, but during catalysis it makes the protein prone to convert into the apo-form, which undergoes unfolding and aggregation under physiological conditions. By using the CRISPR/Cas9 technology we generated a new cellular model of GA based on HEK293 cells knock-out for the OAT gene (HEK-OAT_KO). When overexpressed in HEK-OAT_KO cells, the V332M variant is present in an inactive apodimeric form, but partly shifts to the catalytically-competent holotetrameric form in the presence of exogenous PLP, thus explaining the responsiveness of these patients to pyridoxine administration. Overall, our data represent the first integrated molecular and cellular analysis of the effects of a pathogenic mutation in OAT. In addition, we validated a novel cellular model for the disease that could prove instrumental to define the molecular defect of other GA-causing variants, as well as their responsiveness to pyridoxine and other putative drugs.

Place, publisher, year, edition, pages
Elsevier, 2018
Keywords
Rare disease, Pyridoxal phosphate, Ornithine aminotransferase, Pathogenic mutation, Pyridoxine
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-153117 (URN)10.1016/j.bbadis.2018.08.032 (DOI)000447477400007 ()30251682 (PubMedID)2-s2.0-85052646089 (Scopus ID)
Available from: 2018-11-12 Created: 2018-11-12 Last updated: 2019-01-09Bibliographically approved
Fonseca, L. V., Doimo, M., Calderan, C., Desbats, M. A., Acosta, M. J., Cerqua, C., . . . Salviati, L. (2018). Mutations in COQ8B (ADCK4) found in patients with steroid-resistant nephrotic syndrome alter COQ8B function. Human Mutation, 39(3), 406-414
Open this publication in new window or tab >>Mutations in COQ8B (ADCK4) found in patients with steroid-resistant nephrotic syndrome alter COQ8B function
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2018 (English)In: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 39, no 3, p. 406-414Article in journal (Refereed) Published
Abstract [en]

Mutations in COQ8B cause steroid-resistant nephrotic syndrome with variable neurological involvement. In yeast, COQ8 encodes a protein required for coenzyme Q (CoQ) biosynthesis, whose precise role is not clear. Humans harbor two paralog genes: COQ8A and COQ8B (previously termed ADCK3 and ADCK4). We have found that COQ8B is a mitochondrial matrix protein peripherally associated with the inner membrane. COQ8B can complement a Delta COQ8 yeast strain when its mitochondrial targeting sequence (MTS) is replaced by a yeast MTS. This model was employed to validate COQ8B mutations, and to establish genotype-phenotype correlations. All mutations affected respiratory growth, but there was no correlation between mutation type and the severity of the phenotype. In fact, contrary to the case of COQ2, where residual CoQ biosynthesis correlates with clinical severity, patients harboring hypomorphic COQ8B alleles did not display a different phenotype compared with those with null mutations. These data also suggest that the system is redundant, and that other proteins (probably COQ8A) may partially compensate for the absence of COQ8B. Finally, a COQ8B polymorphism, present in 50% of the European population (NM_024876.3:c.521A > G, p.His174Arg), affects stability of the protein and could represent a risk factor for secondary CoQ deficiencies or for other complex traits.

Place, publisher, year, edition, pages
John Wiley & Sons, 2018
Keywords
coenzyme Q deficiency, steroid-resistant nephrotic syndrome, yeast, COQ8B, mitochondrial phropathy
National Category
Medical Genetics
Identifiers
urn:nbn:se:umu:diva-145360 (URN)10.1002/humu.23376 (DOI)000424807600010 ()29194833 (PubMedID)
Available from: 2018-03-13 Created: 2018-03-13 Last updated: 2018-06-09Bibliographically approved
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