Heavy-Metals-Mediated Phospholipids Scrambling by Human Phospholipid Scramblase 3: A Probable Role in Mitochondrial Apoptosis

Human phospholipid scramblases are a family of four homologous transmembrane proteins (hPLSCR1-4) mediating Phospholipids (PLs) translocation in plasma membrane upon CA²⁺ activation. hPLSCR3, the only homologue localized to mitochondria, plays a vital role in mitochondrial structure, function, maintenance, and Apoptosis. Upon CA²⁺ activation, hPLSCR3 mediates PL translocation at the mitochondrial membrane enhancing t-bid-induced cytochrome c release and Apoptosis. Mitochondria are important target organelles for heavy-metals-induced apoptotic signaling cascade and are the central executioner of Apoptosis to trigger. Pb²⁺ and Hg²⁺ toxicity mediates Apoptosis by increased Reactive Oxygen Species (ROS) and cytochrome c release from mitochondria. To discover the role of hPLSCR3 in heavy metal toxicity, hPLSCR3 was overexpressed as a recombinant protein in Escherichia coli Rosetta (DE3) and purified by affinity chromatography. The biochemical assay using synthetic proteoliposomes demonstrated that hPLSCR3 translocated aminophospholipids in the presence of micromolar concentrations of Pb²⁺ and Hg²⁺. A point mutation in the CA²⁺-binding motif (F258V) led to a ∼60% loss in the functional activity and decreased binding affinities for Pb²⁺ and Hg²⁺ implying that the divalent heavy metal ions bind to the CA²⁺-binding motif. This was further affirmed by the characteristic spectra observed with stains-all dye. The conformational changes upon heavy metal binding were monitored by circular dichroism, intrinsic tryptophan fluorescence, and light-scattering studies. Our results revealed that Pb²⁺ and Hg²⁺ bind to hPLSCR3 with higher affinity than CA²⁺ thus mediating scramblase activity. To summarize, this is the first biochemical evidence for heavy metals binding to the mitochondrial membrane protein leading to bidirectional translocation of PLs specifically toward phosphatidylethanolamine.