Analysis of mitochondrial biogenesis regulation by oxidative stress.

Of all the causes of metabolic and neurological disorders, oxidative stress distinguishes itself by its sweeping effect on the dynamic cellular redox homeostasis and, in its wake, exposing the vulnerabilities of the protein machinery of the cell. High levels of Reactive Oxygen Species (ROS) that mitochondria produce during ATP synthesis can damage mtDNA, lipids, and essential mitochondrial proteins. ROS majorly oxidizes cysteine and methionine amino acids in peptides, which can lead to protein unfolding or misfolding of proteins, which ultimately can have a toll on their function. As mitochondrial biogenesis relies on the continuous import of nuclear-encoded proteins into mitochondria mediated by mitochondrial protein import complexes, oxidative stress triggered by mitochondria can rapidly and detrimentally affect mitochondrial biogenesis and homeostasis. Functional Mge1 is a homodimer and acts as a cochaperone and a nucleotide exchange factor of mitochondrial heat shock protein 70 (mHsp70), crucial for mitochondrial protein import. Oxidative stress like ROS, oxidizes Met 155 in Mge1, compromising its ability to dimerize and interact with mHsp70. The cell employs Methionine sulphoxide reductase 2 (Mxr2), a member of the methionine sulphoxide reductase family, to reduce oxidized Met 155 and thereby restore the essential function of Mge1. Oxidation of methionine as a regulated post-translational modification has been gaining traction. Future high throughput studies that can scan the entire mitochondrial proteome to interrogate methionine oxidation and reversal may increase the repertoire of mitochondrial proteins undergoing regulated oxidation and reduction. In this chapter, we describe the methods followed in our laboratory to study the oxidation of Mge1 and its reduction by Mxr2 in vitro.