The Saccharomyces cerevisiae mitochondrial DNA polymerase and its contribution to the knowledge about human POLG‐related disorders

Most eukaryotes possess a mitochondrial genome, called mtDNA. In animals and fungi, the replication of mtDNA is entrusted by the DNA polymerase γ, or Pol γ. The yeast Pol γ is composed only of a catalytic subunit encoded by MIP1. In humans, Pol γ is a heterotrimer composed of a catalytic subunit homolog to Mip1, encoded by POLG, and two accessory subunits. In the last 25 years, more than 300 pathological mutations in POLG have been identified as the cause of several mitochondrial diseases, called POLG-related disorders, which are characterized by multiple mtDNA deletions and/or depletion in affected tissues. In this review, at first, we summarize the biochemical properties of yeast Mip1, and how mutations, especially those introduced recently in the N-terminal and C-terminal regions of the enzyme, affect the in vitro activity of the enzyme and the in vivo phenotype connected to the mtDNA stability and to the mtDNA extended and point mutability. Then, we focus on the use of yeast harboring Mip1 mutations equivalent to the human ones to confirm their pathogenicity, identify the phenotypic defects caused by these mutations, and find both mechanisms and molecular compounds able to rescue the detrimental phenotype. A closing chapter will be dedicated to other polymerases found in yeast mitochondria, namely Pol ζ, Rev1 and Pol η, and to their genetic interactions with Mip1 necessary to maintain mtDNA stability and to avoid the accumulation of spontaneous or induced point mutations

Modopathies Caused by Mutations in Genes Encoding for Mitochondrial RNA Modifying Enzymes: Molecular Mechanisms and Yeast Disease Models

first_pagesettingsOrder Article Reprints This is an early access version, the complete PDF, HTML, and XML versions will be available soon. Open AccessReview Modopathies Caused by Mutations in Genes Encoding for Mitochondrial RNA Modifying Enzymes: Molecular Mechanisms and Yeast Disease Models by Martina Magistrati 1,†ORCID,Alexandru Ionut Gilea 1,†,Camilla Ceccatelli Berti 1,2ORCID,Enrico Baruffini 1,*ORCID andCristina Dallabona 1ORCID 1 Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parco Area delle Scienze 11/A, 43124 Parma, Italy 2 Department of Medicine and Surgery, University of Parma, Via Gramsci 14, 43126 Parma, Italy * Author to whom correspondence should be addressed. † These authors contributed equally to this work. Int. J. Mol. Sci. 2023, 24(3), 2178; https://doi.org/10.3390/ijms24032178 Received: 28 December 2022 / Revised: 17 January 2023 / Accepted: 20 January 2023 / Published: 22 January 2023 (This article belongs to the Special Issue RNA Regulatory Networks 2.0) Download Review Reports Versions Notes Abstract In eukaryotes, mitochondrial RNAs (mt-tRNAs and mt-rRNAs) are subject to specific nucleotide modifications, which are critical for distinct functions linked to the synthesis of mitochondrial proteins encoded by mitochondrial genes, and thus for oxidative phosphorylation. In recent years, mutations in genes encoding for mt-RNAs modifying enzymes have been identified as being causative of primary mitochondrial diseases, which have been called modopathies. These latter pathologies can be caused by mutations in genes involved in the modification either of tRNAs or of rRNAs, resulting in the absence of/decrease in a specific nucleotide modification and thus on the impairment of the efficiency or the accuracy of the mitochondrial protein synthesis. Most of these mutations are sporadic or private, thus it is fundamental that their pathogenicity is confirmed through the use of a model system. This review will focus on the activity of genes that, when mutated, are associated with modopathies, on the molecular mechanisms through which the enzymes introduce the nucleotide modifications, on the pathological phenotypes associated with mutations in these genes and on the contribution of the yeast Saccharomyces cerevisiae to confirming the pathogenicity of novel mutations and, in some cases, for defining the molecular defects

Drug Drop Test: How to Quickly Identify Potential Therapeutic Compounds for Mitochondrial Diseases Using Yeast Saccharomyces cerevisiae

: Mitochondrial diseases (MDs) refer to a group of clinically and genetically heterogeneous pathologies characterized by defective mitochondrial function and energy production. Unfortunately, there is no effective treatment for most MDs, and current therapeutic management is limited to relieving symptoms. The yeast Saccharomyces cerevisiae has been efficiently used as a model organism to study mitochondria-related disorders thanks to its easy manipulation and well-known mitochondrial biogenesis and metabolism. It has been successfully exploited both to validate alleged pathogenic variants identified in patients and to discover potential beneficial molecules for their treatment. The so-called "drug drop test", a phenotype-based high-throughput screening, especially if coupled with a drug repurposing approach, allows the identification of molecules with high translational potential in a cost-effective and time-saving manner. In addition to drug identification, S. cerevisiae can be used to point out the drug's target or pathway. To date, drug drop tests have been successfully carried out for a variety of disease models, leading to very promising results. The most relevant aspect is that studies on more complex model organisms confirmed the effectiveness of the drugs, strengthening the results obtained in yeast and demonstrating the usefulness of this screening as a novel approach to revealing new therapeutic molecules for MDs

<i>Saccharomyces cerevisiae</i> as a Tool for Studying Mutations in Nuclear Genes Involved in Diseases Caused by Mitochondrial DNA Instability

Mitochondrial DNA (mtDNA) maintenance is critical for oxidative phosphorylation (OXPHOS) since some subunits of the respiratory chain complexes are mitochondrially encoded. Pathological mutations in nuclear genes involved in the mtDNA metabolism may result in a quantitative decrease in mtDNA levels, referred to as mtDNA depletion, or in qualitative defects in mtDNA, especially in multiple deletions. Since, in the last decade, most of the novel mutations have been identified through whole-exome sequencing, it is crucial to confirm the pathogenicity by functional analysis in the appropriate model systems. Among these, the yeast Saccharomyces cerevisiae has proved to be a good model for studying mutations associated with mtDNA instability. This review focuses on the use of yeast for evaluating the pathogenicity of mutations in six genes, MPV17/SYM1, MRM2/MRM2, OPA1/MGM1, POLG/MIP1, RRM2B/RNR2, and SLC25A4/AAC2, all associated with mtDNA depletion or multiple deletions. We highlight the techniques used to construct a specific model and to measure the mtDNA instability as well as the main results obtained. We then report the contribution that yeast has given in understanding the pathogenic mechanisms of the mutant variants, in finding the genetic suppressors of the mitochondrial defects and in the discovery of molecules able to improve the mtDNA stability

A yeast-based repurposing approach revealed modulation of dNTP pool as a therapeutic target to treat mitochondrial DNA depletion syndromes

Mitochondrial DNA Depletion Syndromes (MDS) are a group of clinically heterogenous and often severe diseases characterized by a reduction of the mitochondrial DNA (mtDNA) copy number in affected tissues. There are still no satisfactory therapies and, since mitochondrial diseases, taken individually, are rare, therapeutic strategies with potential general applicability to several mitochondrial diseases would be desirable. Yeast has proved to be an excellent model for the study of the mechanisms underlying mitochondrial pathologies and also for the discovery of new therapies thanks to the development of a high–throughput yeast-based assay. We identified ten drugs active against MPV17 disorder modelled in yeast, whose homologous gene is SYM1. MPV17/SYM1 encodes a non-selective channel in the inner mitochondrial membrane whose physiological role and nature of the cargo remains elusive. Recessive mutations in this gene cause a hepatocerebral form of MDS and Navajo neurohepatopathy. All the ten molecules identified determine a concomitant increase of both mitochondrial dNTP pool and mtDNA stability strongly suggesting that the reduced availability of DNA synthesis precursors is the cause of the mtDNA deletion/depletion in Sym1 deficiency. We also assessed the effect of these molecules on mtDNA stability of two additional MDS yeast models characterized by mutations in MIP1 and RNR2, orthologs of the human genes POLG and RRM2B, respectively, extending the potential use of these drugs to other MDS patients. As a drug repurposing approach of FDA-approved drugs was used, this could speed up a possible clinical use of the drugs

Functional analysis of missense DARS2 variants in siblings with leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation

Biallelic pathogenic variants in the nuclear gene DARS2 (MIM# 610956), encoding the mitochondrial enzyme aspartyl-tRNA synthetase (MT-ASPRS) cause leukoencephalopathy with Brain Stem and Spinal Cord Involvement and Lactate Elevation (LBSL) (MIM# 611105), a neurometabolic disorder characterized by progressive ataxia, spasticity, developmental arrest or regression and characteristic brain MRI findings. Most patients exhibit a slowly progressive disease course with motor deterirartion that begins in childhood or adolescence, but can also occasionaly occur in adulthood. More severe LBSL presentations with atypical brain MRI findings have been recently described. Baker's yeast orthologue of DARS2, MSD1, is required for growth on oxidative carbon sources. A yeast with MSD1 knockout (msd1 Delta) demonstrated a complete lack of oxidative growth which could be rescued by wild-type MSD1 but not MSD1 with pathogenic variants. Here we reported two siblings who exhib-ited developmental regression and ataxia with different age of onset and phenotypic severity. Exome sequencing revealed 2 compound heterozygous missense variants in DARS2: c.473A&gt;T (p.Glu158Val) and c.829G&gt;A (p. Glu277Lys); this variant combination has not been previously reported. The msd1 Delta yeast transformed with plas-mids expressing p.Glu259Lys, equivalent to human p.Glu277Lys, showed complete loss of oxidative growth and oxygen consumption, while the strain carrying p.Gln137Val, equivalent to human p.Glu158Val, showed a signif-icant reduction of oxidative growth, but a residual ability to grow was retained. Structural analysis indicated that p.Glu158Val may interfere with protein binding of tRNAAsp, while p.Glu277Lys may impact both homodimerization and catalysis of MT-ASPRS. Our data illustrate the utility of yeast model and in silico analysis to determine pathogenicity of DARS2 variants, expand the genotypic spectrum and suggest intrafamilial variabil-ity in LBSL.(c) 2022 Elsevier Inc. All rights reserved

Pathogenic variants in GCSH encoding the moonlighting H-protein cause combined Nonketotic Hyperglycinemia and Lipoate Deficiency

Maintaining protein lipoylation is vital for cell metabolism. The H-protein encoded by GCSH has a dual role in protein lipoylation required for bioenergetic enzymes including pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase, and in the one-carbon metabolism through its involvement in glycine cleavage enzyme system, intersecting two vital roles for cell survival. Here we report six patients with biallelic pathogenic variants in GCSH and a broad clinical spectrum ranging from neonatal fatal glycine encephalopathy to an attenuated phenotype of developmental delay, behavioral problems, limited epilepsy, and variable movement problems. The mutational spectrum includes one insertion c.293-2_293-1insT, one deletion c.122_(228 + 1_229-1) del, one duplication of exons 4 and 5, one nonsense variant p.Gln76*and four missense p.His57Arg, p.Pro115Leu, and p.Thr148Pro and the previously described p.Met1?. Via functional studies in patient's fibroblasts, molecular modelling, expression analysis in GCSH knock-down COS7 cells and yeast, and in vitro protein studies, we demonstrate for the first time that most variants identified in our cohort produced a hypomorphic effect on both mitochondrial activities, protein lipoylation and glycine metabolism, causing combined deficiency whereas some missense variants affect primarily one function only. The clinical features of the patients reflect the impact of the GCSH changes on any of the two functions analyzed. Our analysis illustrates the complex interplay of functional and clinical impact when pathogenic variants affect a multifunctional protein involved in two metabolic pathways and emphasizes the value of the functional assays to select the treatment and investigate new personalized options