| 1. |
- Purhonen, Janne, et al.
(author)
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A sensitive assay for dNTPs based on long synthetic oligonucleotides, EvaGreen dye and inhibitor-resistant high-fidelity DNA polymerase
- 2020
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In: Nucleic Acids Research. - : Oxford University Press (OUP). - 1362-4962 .- 0305-1048. ; 48:15, s. 87-87
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Journal article (peer-reviewed)abstract
- Deoxyribonucleoside triphosphates (dNTPs) are vital for the biosynthesis and repair of DNA. Their cellular concentration peaks during the S phase of the cell cycle. In non-proliferating cells, dNTP concentrations are low, making their reliable quantification from tissue samples of heterogeneous cellular composition challenging. Partly because of this, the current knowledge related to the regulation of and disturbances in cellular dNTP concentrations derive mostly from cell culture experiments with little corroboration at the tissue or organismal level. Here, we fill the methodological gap by presenting a simple non-radioactive microplate assay for the quantification of dNTPs with a minimum requirement of 4-12 mg of biopsy material. In contrast to published assays, this assay is based on long synthetic single-stranded DNA templates (50-200 nucleotides), an inhibitor-resistant high-fidelity DNA polymerase, and the double-stranded-DNA-binding EvaGreen dye. The assay quantified reliably less than 50 fmol of each of the four dNTPs and discriminated well against ribonucleotides. Additionally, thermostable RNAse HII-mediated nicking of the reaction products and a subsequent shift in their melting temperature allowed near-complete elimination of the interfering ribonucleotide signal, if present. Importantly, the assay allowed measurement of minute dNTP concentrations in mouse liver, heart and skeletal muscle.
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| 2. |
- Purhonen, Janne, et al.
(author)
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Mitochondrial complex III deficiency drives c-MYC overexpression and illicit cell cycle entry leading to senescence and segmental progeria
- 2023
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In: Nature Communications. - 2041-1723. ; 14:1
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Journal article (peer-reviewed)abstract
- Accumulating evidence suggests mitochondria as key modulators of normal and premature aging, yet whether primary oxidative phosphorylation (OXPHOS) deficiency can cause progeroid disease remains unclear. Here, we show that mice with severe isolated respiratory complex III (CIII) deficiency display nuclear DNA damage, cell cycle arrest, aberrant mitoses, and cellular senescence in the affected organs such as liver and kidney, and a systemic phenotype resembling juvenile-onset progeroid syndromes. Mechanistically, CIII deficiency triggers presymptomatic cancer-like c-MYC upregulation followed by excessive anabolic metabolism and illicit cell proliferation against lack of energy and biosynthetic precursors. Transgenic alternative oxidase dampens mitochondrial integrated stress response and the c-MYC induction, suppresses the illicit proliferation, and prevents juvenile lethality despite that canonical OXPHOS-linked functions remain uncorrected. Inhibition of c-MYC with the dominant-negative Omomyc protein relieves the DNA damage in CIII-deficient hepatocytes in vivo. Our results connect primary OXPHOS deficiency to genomic instability and progeroid pathogenesis and suggest that targeting c-MYC and aberrant cell proliferation may be therapeutic in mitochondrial diseases.
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| 3. |
- Purhonen, Janne, et al.
(author)
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NAD+ repletion produces no therapeutic effect in mice with respiratory chain complex III deficiency and chronic energy deprivation
- 2018
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In: FASEB Journal. - 0892-6638. ; 32:11, s. 5913-5926
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Journal article (peer-reviewed)abstract
- Biosynthetic precursors of NAD+ can replenish a decreased cellular NAD+ pool and, supposedly via sirtuin (SIRT) deacetylases, improvemitochondrial function.Wefound decreased hepaticNAD+ concentration and downregulated biosynthesis in Bcs1lp.S78G knock-in mice with respiratory chain complex III deficiency and mitochondrial hepatopathy. Aiming at ameliorating disease progression via NAD+ repletion and improved mitochondrial function, we fed thesemice nicotinamide riboside (NR), aNAD+ precursor. A targetedmetabolomics verified successful administration and suggested enhancedNAD+ biosynthesis in the treated mice, although hepaticNAD+ concentrationwas unchanged at the end point. In contrast to our expectations,NRdid not improve the hepatopathy, hepatic mitochondrial respiration, or survival of Bcs1lp.S78G mice. We linked this lack of therapeutic effect to NAD+-independent activation of SIRT-1 and -3 via AMPK and cAMP signaling related to the starvation-like metabolic state of Bcs1lp.S78G mice. In summary, we describe an unusual metabolic state with NAD+ depletion accompanied by energy deprivation signals, uncompromised SIRT function, and upregulated oxidative metabolism. Our study highlights that the knowledge of the underlying complexmetabolic alterations is criticalwhen designing therapies formitochondrial dysfunction.
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| 4. |
- Purhonen, Janne, et al.
(author)
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Quantification of all 12 canonical ribonucleotides by real-time fluorogenic in vitro transcription
- 2024
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In: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 52:1
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Journal article (peer-reviewed)abstract
- Enzymatic methods to quantify deoxyribonucleoside triphosphates have existed for decades. In contrast, no general enzymatic method to quantify ribonucleoside triphosphates (rNTPs), which drive almost all cellular processes and serve as precursors of RNA, exists to date. ATP can be measured with an enzymatic luminometric method employing firefly luciferase, but the quantification of other ribonucleoside mono-, di-, and triphosphates is still a challenge for a non-specialized laboratory and practically impossible without chromatography equipment. To allow feasible quantification of ribonucleoside phosphates in any laboratory with typical molecular biology and biochemistry tools, we developed a robust microplate assay based on real-time detection of the Broccoli RNA aptamer during in vitro transcription. The assay employs the bacteriophage T7 and SP6 RNA polymerases, two oligonucleotide templates encoding the 49-nucleotide Broccoli aptamer, and a high-affinity fluorogenic aptamer-binding dye to quantify each of the four canonical rNTPs. The inclusion of nucleoside mono- and diphosphate kinases in the assay reactions enabled the quantification of the mono- and diphosphate counterparts. The assay is inherently specific and tolerates concentrated tissue and cell extracts. In summary, we describe the first chromatography-free method to quantify ATP, ADP, AMP, GTP, GDP, GMP, UTP, UDP, UMP, CTP, CDP and CMP in biological samples.
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| 5. |
- Rajendran, Jayasimman, et al.
(author)
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Alternative oxidase-mediated respiration prevents lethal mitochondrial cardiomyopathy
- 2018
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In: EMBO Molecular Medicine. - : EMBO. - 1757-4676 .- 1757-4684. ; 2018
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Journal article (peer-reviewed)abstract
- Alternative oxidase (AOX) is a non-mammalian enzyme that can bypass blockade of the complex III-IV segment of the respiratory chain (RC). We crossed a Ciona intestinalis AOX transgene into RC complex III (cIII)-deficient Bcs1lp.S78G knock-in mice, displaying multiple visceral manifestations and premature death. The homozygotes expressing AOX were viable, and their median survival was extended from 210 to 590 days due to permanent prevention of lethal cardiomyopathy. AOX also prevented renal tubular atrophy and cerebral astrogliosis, but not liver disease, growth restriction, or lipodystrophy, suggesting distinct tissue-specific pathogenetic mechanisms. Assessment of reactive oxygen species (ROS) production and damage suggested that ROS were not instrumental in the rescue. Cardiac mitochondrial ultrastructure, mitochondrial respiration, and pathological transcriptome and metabolome alterations were essentially normalized by AOX, showing that the restored electron flow upstream of cIII was sufficient to prevent cardiac energetic crisis and detrimental decompensation. These findings demonstrate the value of AOX, both as a mechanistic tool and a potential therapeutic strategy, for cIII deficiencies.
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| 6. |
- Tegelberg, Saara, et al.
(author)
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Respiratory chain complex III deficiency due to mutated BCS1L : A novel phenotype with encephalomyopathy, partially phenocopied in a Bcs1l mutant mouse model
- 2017
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In: Orphanet Journal of Rare Diseases. - : Springer Science and Business Media LLC. - 1750-1172. ; 12:1
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Journal article (peer-reviewed)abstract
- Background: Mitochondrial diseases due to defective respiratory chain complex III (CIII) are relatively uncommon. The assembly of the eleven-subunit CIII is completed by the insertion of the Rieske iron-sulfur protein, a process for which BCS1L protein is indispensable. Mutations in the BCS1L gene constitute the most common diagnosed cause of CIII deficiency, and the phenotypic spectrum arising from mutations in this gene is wide. Results: A case of CIII deficiency was investigated in depth to assess respiratory chain function and assembly, and brain, skeletal muscle and liver histology. Exome sequencing was performed to search for the causative mutation(s). The patient's platelets and muscle mitochondria showed respiration defects and defective assembly of CIII was detected in fibroblast mitochondria. The patient was compound heterozygous for two novel mutations in BCS1L, c.306A > T and c.399delA. In the cerebral cortex a specific pattern of astrogliosis and widespread loss of microglia was observed. Further analysis showed loss of Kupffer cells in the liver. These changes were not found in infants suffering from GRACILE syndrome, the most severe BCS1L-related disorder causing early postnatal mortality, but were partially corroborated in a knock-in mouse model of BCS1L deficiency. Conclusions: We describe two novel compound heterozygous mutations in BCS1L causing CIII deficiency. The pathogenicity of one of the mutations was unexpected and points to the importance of combining next generation sequencing with a biochemical approach when investigating these patients. We further show novel manifestations in brain, skeletal muscle and liver, including abnormality in specialized resident macrophages (microglia and Kupffer cells). These novel phenotypes forward our understanding of CIII deficiencies caused by BCS1L mutations.
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