Mapping the protein phosphorylation sites in human mitochondrial complex I (NADH: Ubiquinone oxidoreductase): A bioinformatics study with implications for brain aging and neurodegeneration

In eukaryotes, mitochondrial complex I (NADH: ubiquinone oxidoreductase; CI) is central to oxidative phosphorylation (OXPHOS). Mammalian CI is a 45 subunit complex that forms supercomplexes with other OXPHOS complexes. Since CI defects are associated with aging and neurodegeneration, it is pertinent to understand its structure-function relationship. Although genetic mutations could lower CI activity causing mitochondrial dysfunction in several pathologies, post-translational modifications (PTMs) have emerged as a key mechanism contributing to altered CI activity. Among non-oxidative PTMs, protein phosphorylation is the most intricate regulatory mechanism controlling CI structure and function during normal physiology, aging and neurodegeneration. To comprehend this, we carried out a comprehensive bioinformatics analysis of protein phosphorylation of human CI subunits using software-based prediction of phosphorylation (phospho) sites and associated kinases. Phosphorylation was higher among core subunits and active domains of the complex. Among the subunits, NDUFS1 displayed significantly higher number as well as percent phospho sites compared to others. Analysis of the subunits containing iron-sulfur (Fe-S) cluster, NADH and FMN binding sites and quinone binding sites indicated the presence of phospho sites in close proximity to the binding sites of these cofactors with potential functional implications. Phosphoproteomics experiment in rat and human muscle mitochondria identified specific phospho sites in CI subunits, thereby validating the bioinformatic analysis. Molecular modeling of CI subunits indicated structural implications following phosphorylation. We surmise that protein phosphorylation, a transient and regulatory event could influence the structure-function relationship of CI thereby impinging on bioenergetics and ultimately contributing to aging and neurodegeneration.