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Direct proteome labelling in fruit flies with SILAF reveals differential phosphorylation of mitochondrial proteins upon loss of OXPHOS subunits

ABSTRACT: Drosophila melanogaster has been a workhorse of genetics and cell biology for more than a century. However, proteomic-based methods have been limited due to the complexity and dynamic range of the fly proteome and the lack of efficient labelling methods. Here, we advanced a chemically defined food source into direct stable-isotope labelling of amino acids in flies (SILAF). It allows for the rapid and cost-efficient generation of a large number of larvae or flies, with full incorporation of lysine-[13C6] after six labelling days. SILAF followed by fractionation and enrichment gave proteomic insights at a depth of 7,196 proteins and 8,451 phosphorylation sites, which substantiated metabolic regulation on enzymatic level. We applied SILAF to quantify the mitochondrial phosphoproteome of an early-stage leucine-rich PPR motif-containing protein (LRPPRC)-knockdown fly model of mitochondrial disease that almost exclusively affects protein levels of the oxidative phosphorylation (OXPHOS) system. While the mitochondrial compartment was hypo-phosphorylated, two conserved phospho-sites on OXPHOS subunits NDUFB10 and NDUFA4 were significantly upregulated upon impaired OXPHOS function. The ease and versatility of the method actuates the fruit fly as an appealing model in proteomic and post-translational modification studies and it enlarges potential metabolic applications based on heavy amino acid diets.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Drosophila Melanogaster (fruit Fly)

DISEASE(S): Mitochondrial Metabolism Disease

SUBMITTER: Florian Schober

LAB HEAD: Anna Wredenberg

PROVIDER: PXD009990 | Pride | 2021-02-10

REPOSITORIES: Pride

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Project description:G4C2 hexanucleotide repeat expansions in a non-coding region of the C9orf72 gene are the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). G4C2 insertion length is variable, and patients can carry up to several thousand repeats. Dipeptide repeat proteins (DPR) translated from G4C2 transcripts are thought to be a main driver of toxicity. Experiments in model organisms with relatively short DPR have shown that arginine-rich DPR are most toxic, while polyGlycine-Alanine (GA) DPRs cause only mild toxicity. However, GA is the most abundant DPR in patient brains, and experimental work in animals has generally relied on the use of low numbers of repeats, with DPRs often tagged for in vivo tracking. Whether repeat length, or tagging, affect the toxicity of GA has not been systematically assessed. Therefore, we generated Drosophila fly lines expressing GA100, GA200 or GA400 specifically in adult neurons. Consistent with previous studies, expression of GA100 and GA200 caused only mild toxicity. In contrast, neuronal expression of GA400 drastically reduced climbing ability and survival of the flies, indicating that long GA DPRs can be highly toxic in vivo. This toxicity could be abolished by tagging GA400. Proteomics analysis of fly brains showed a repeat-length-dependent modulation of the brain proteome, with GA400 causing earlier and stronger changes than shorter GA proteins. PolyGA expression up-regulated proteins involved in ER to Golgi trafficking, and down-regulated proteins involved in insulin signalling. Experimental down-regulation of Tango1, a highly conserved regulator of ER-to Golgi transport, partially rescued GA400 toxicity, suggesting that misregulation of this process contributes to polyGA toxicity. Experimentally increasing insulin signaling also rescued GA toxicity. In summary, our data show that long polyGA proteins can be highly toxic in vivo, and that they may therefore contribute to ALS/FTD pathogenesis in humans patients.

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Direct proteome labelling in fruit flies with SILAF reveals differential phosphorylation of mitochondrial proteins upon loss of OXPHOS subunits

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