Periodic Reporting for period 2 - MitoMethylome (Mitochondrial methylation and its role in health and disease)
Reporting period: 2018-10-01 to 2020-03-31
To ensure correct cellular function, mitochondria need to communicate with its cytoplasmic and nuclear surrounding, but little is known about how metabolism is coordinated between these different compartments. Increasing evidence indicates that metabolism of one carbon units is important in this context. Results from my laboratory has shown that abnormal intra-mitochondrial methylation can cause complex biochemical and clinical phenotypes in humans, but little is known on the underlying mechanisms. Currently, intramitochondrial methylation reactions have been implicated in a range of processes required for mitochondrial and cellular function, but a detailed survey and investigation of these methylation reactions has not yet been performed. This project will identify and characterise intramitochondrial methylation and associate newly identified sites to their physiological role.
Methylation is the covalent attachment of a methyl group (CH3) by a specific methyl transferase to biomolecules, such as nucleic acids, proteins, lipids, co-factors or metabolites. The predominant intracellular methyl group donor is S-adenosylmethionine (SAM), which is synthesised in the one-carbon cycle in the cytosol. For methylations inside mitochondria, SAM needs to be imported into mitochondria via a specific transporter, and the genetic manipulation of this transporter in model organisms allows me to determine the role of intramitochondrial SAM, its targets, and its physiological consequences.
My laboratory has generated and characterised genetically modified fruit fly models with varying degrees of intra-mitochondrial SAM deficiency. For this we deleted the locus of the mitochondrial SAM transporter (SAMC) and replaced it either with alleles not expressing SAMC at all, or with alleles expressing patient-specific SAMC mutations that have been shown to lead to reduced SAMC activity (Schober et al. 2020a, in preparation). Additionally, we now have conditional knockout mouse models that allow for the tissue-specific deletion of the murine SAM transporter. We are currently performing crosses to generate full body-, heart-, and skeletal muscle-specific SAMC knockout mice. Full-body SAMC KO mice are not born due to embryonic lethality prior to embryonic day 8.5. Heart-specific SAMC KO leads to lethality at 14 days after birth. These are currently being investigated for their molecular and metabolic phenotype. Skeletal-muscle specific KO crosses are ongoing, but no phenotypic data has yet been obtained.
To aim 2: Characterise the molecular and metabolic consequences of a depleted intra-mitochondrial SAM pool.
Fly models either deficient for SAMC or expressing mutant SAMC have been characterised for their mitochondrial function and mitochondrial gene expression. Additionally, global cellular gene expression analysis have been performed in form of transcriptomic gene expression analysis, proteomic analysis, as well as targeted and untargeted metabolomics analysis. We established measuring intramitochondrial SAM levels, using liquid chromatography, coupled to mass spectrometry (LC/MS), from fly, mouse, and human extracts. Our work has shown that severe intramitochondrial SAM deficiency leads to early lethality mainly caused by a lack of essential metabolites (small molecules) such as ubiquinone, also known as Coenzyme Q10 (CoQ10) and lipoic acid. In contrast, milder intramitochondrial SAM deficiencies lead to a more subtle phenotype, affecting other processes, such as reduced stability of specific OXPHOS subunits. We also see a negative impact on iron-sulphur cluster assembly, which has wide reaching consequences on cellular function (Schober et al. 2020a, in preparation).
To aim 3: Determine the intra-mitochondrial methylated proteome.
We have developed a novel method to identify post-translational modifications, including methylations, in the fly. As a proof-of-principle we described the total phosphoproteome (i.e. phosphorylated protein sites) in flies, and characterised its adaptation to two different food conditions and in a mitochondrial disease model with deficient mitochondrial gene expression (Schober et al. 2020b, in preparation).
My group applied this method to identify methylated mitochondrial proteins in flies. We have identified 74 methylated peptide sites on 54 mitochondrial protein targets, 45 of the methylated sites are conserved to human. Only three have previously been reported. My laboratory performed targeted MS validation, confirming methylation of the majority of identified modifications (Schober et al. 2020a, in preparation).
One of the novel identified sites has previously been implicated in human disease, and we are currently investigating whether methylation is important for the disease mechanism. A second interesting modification involves a protein required for mitochondrial RNA turnover. We are currently generating fly models mutating the modified site in question to study its physiological implications. Finally, we are investigating the molecular role of a methylation on a factor involved in iron-sulphur cluster assembly
In total, we are currently generating genetically modified models both in flies and human cell lines for five different methylated sites to study the physiological and molecular relevance of these methylation modifications.
To aim 4: Identify modulators of the mitochondrial methylome.
My laboratory will investigate modulators of intramitochondrial SAM, by crossing fly SAMC mutant flies to commercially available deletion strains. This library of flies carries chromosomes with overlapping sections of the fly genome, allowing for the identification of genes involved in the regulation of SAMC. We have purchased and started crosses with a fly library spanning chromosome 3 of the fly genome.
My group developed and improved a stable-isotope labelling technique to be used in fruit fly models, which is applicable for any researcher interested in in vivo labelling or tracing. The technique vastly improves efficiency and sensitivity of currently available methods and is applicable for the identification of a range of different protein modifications or other labels.
We have identified a number of protein modifications on mitochondrial proteins previously not reported. I expect that we will identify the biological role of these modifications, providing novel insights into mitochondrial biology.