Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - MitoMethylome (Mitochondrial methylation and its role in health and disease)

Teaser

Mitochondria are the primary site for cellular ATP synthesis and are involved in a variety of metabolic processes. Dysfunction of this organelle has been linked to a broad range of disease states as well as to normal ageing. However, there is currently no treatment for...

Summary

Mitochondria are the primary site for cellular ATP synthesis and are involved in a variety of metabolic processes. Dysfunction of this organelle has been linked to a broad range of disease states as well as to normal ageing. However, there is currently no treatment for mitochondrial dysfunction. Mitochondrial function relies heavily on interactions with the nuclear genome and the cytoplasm, but little is known about how metabolism is coordinated between the different compartments. Increasing evidence indicates that metabolism of one carbon units is important in this context. Results from my laboratory has shown that abnormal intramitochondrial 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 intramitochondrial methylation reactions has not yet been performed. This project will identify and characterise intramitochondrial methylation and associate newly identified sites to their physiological role.
S-adenosylmethionine (SAM) is the predominant intracellular methyl group donor, required for a diverse set of post-translational modifications, nucleotide methylations or the synthesis of co-factors and metabolites. SAM is generated in the one-carbon cycle in the cytosol and needs to be imported into mitochondria via a specific transporter. Disrupting this transporter in model organisms allows me to determine the role of intramitochondrial SAM and its targets.

The main aims of the project are:

1. Develop model systems with intra-mitochondrial SAM deficiency
2. Characterise the molecular and metabolic consequences of a depleted intra-mitochondrial SAM pool
3. Determine the intra-mitochondrial methylated proteome
4. Identify modulators of the mitochondrial methylome

Work performed

To aim 1:
During this first period of the ERC grant my laboratory has generated and characterised genetically modified fruit fly models. This has been achieved by deleting the chromosomal region carrying the mitochondrial SAM transporter (SAMC) and replacing it either with alleles not expressing SAMC at all, or with alleles that will express the same mutations in SAMC as identified in human patients. Additionally, my laboratory has now conditional knockout mouse models that allow for the tissue-specific deletion of the murine SAM transport. We are currently performing the last crosses to generate full body-, heart-, and skeletal muscle-specific SAMC knockout mice.

To aim 2:
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 an untargeted metabolomics analysis. We established measuring intramitochondrial SAM levels, using liquid chromatography, coupled to mass spectrometry (LC/MS), from both fly and human cell line extracts. We will also apply this to different tissues of our SAMC KO mouse models and are currently investigating whether we can use this for diagnostic purposes in our clinic. Our work has shown that severe intramitochondrial SAM deficiency leads to early lethality cause by mainly 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.

To aim 3:
We have developed a novel method to identify posttranslational 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.
My group has now applied this method to identify methylated mitochondrial proteins in flies. We have identified 50 mitochondrial protein targets, many of which are conserved to humans. Only three have previously been reported. One of the novel identified sites has previously been implicated in human disease, it is therefore likely that the lack of methylation at this position is disease-causing. We will further investigate this possibility. Another modification involves a protein involved in mitochondrial RNA turnover, a process I have extensive experience in. My laboratory has several models available for this protein and we will investigate the role of its methylation. All 50 sites will be validated, using targeted proteomics.
The identification of methylation modifications on mitochondrial transcripts is currently being developed.

To aim 4:
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 1 of the fly genome.

Final results

My laboratory has developed several novel genetically modified models with intramitochondrial SAM deficiency, including both SAMC null flies and flies expressing mutant forms of SAMC identified in patients with mitochondrial disease. Additionally, we have established genetically modified moue models, which allow us to generated tissue-specific or complete body KO mouse models for SAMC. None of these models have existed prior to this project.
Further, in order to identify methylated protein targets inside mitochondria, my group has developed an improved method for labelling flies with stable isotope amino acids. This has vastly improved efficiency and sensitivity of current methods and is applicable for the identification of a range of different protein modifications.
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.