LAUSR

Reactive oxygen species (ROS) are produced by a variety of cellular processes including metabolic activity, mitochondria, cellular signaling, and oncogene activity. Methionine sulfoxide reductase A (MsrA) is an antioxidant enzyme that can reduce methionine sulfoxide (MetO) in proteins back to methionine (Met). This enzyme plays a protective role in cells against oxidative damage. Although many proteins with MetO are in vitro substrates of MsrA, no in vivo substrates have been confidently identified. In vitro, MsrA can also function as an oxidase that converts methionine to methionine sulfoxide, but it is also not known if it does so in vivo. The cytosolic form of MsrA is myristoylated, and overexpression of the myristoylated form protects the heart from ischemia-reperfusion injury while overexpression of the non-myristoylated form does not. Here we report a novel binding partner of MsrA, STARD3, identified by use of a human protein microarray. ProtoArray Human Protein Microarray v5.0 contains 9,483 human proteins from multiple protein classes along with 2,016 controls. STARD3 was scored as an interacting protein with high probability. STARD3 contains the star-related lipid transfer (START) domain, and proteins with that domain are implicated in lipid and sterol metabolism. STARD3 is known to bind cholesterol and transport it from the endoplasmic reticulum to the late endosome. We overexpressed both myristoylated MsrA and STARD3 in HEK293 cells and immunoprecipitated cell homogenates with anti-STARD3 antibody. We demonstrated that myristoylated MsrA was co-immunoprecipitated with STARD3, but non-myristoylated form wasn’t. This indicates that myristoylation is required for the interaction. Met307 of STARD3 is known to be essential for lipid binding, and we found that it is also required for the interaction of STARD3 and myristoylated MsrA. Finally, we showed by confocal microscopy that myristoylated MsrA was colocalized with STARD3 at late endosomes/lysosomes. We conclude that myristoylated MsrA binds to STARD3 on the cytoplasmic surface of late endosomes.