Human phospholipid scramblases are single-pass transmembrane proteins (hPLSCR1-5) mediating phospholipids (PLs) translocation in plasma membrane upon Ca2+ activation. hPLSCR3, the only homolog localized to mitochondria, plays a vital role in fat metabolism, mitochondrial structure, function, maintenance, and apoptosis. Upon Ca2+ activation, hPLSCR3 mediates cardiolipin (CL) translocation at the mitochondrial membrane, enhancing truncated Bid (t-Bid) induced cytochrome c release and apoptosis. The role of scramblases in PL translocation is heavily debated, and direct biochemical evidence for PL translocation by hPLSCR3 is yet to be reported. We proposed that metal ion-induced conformational change and the aggregation of the protein are essential for the PL translocation by hPLSCR3. Purifying functionally active form of hPLSCRs in sufficient quantity is a major bottleneck in establishing its structure and understanding the functional mechanism.
Recombinant hPLSCRs form inclusion bodies (IBs) when overexpressed in E. coli. Different additives were added to recover hPLSCRs from IBs, of which 0.3% N-lauroyl sarcosine (NLS) recovered ~50% of bioactive hPLSCRs from IBs. E. coli C43 (DE3) gave higher yields of purified protein (7.76 mg/g cell) followed by E. coli BL21 (DE3) pLysS (5.87 mg/g cell). NLS removal before affinity-based purification is essential as the detergent interferes with the matrix binding. Detergent removal by adsorption onto hydrophobic polystyrene beads was ten times faster than the conventional dialysis method. It was found to be an efficient and alternate tool to dialysis in detergent removal without significantly altering the structure and function of the membrane protein.
Functional assay using synthetic proteoliposomes revealed that recombinant hPLSCR3 translocates amino phospholipids such as NBD-PE & NBD-PS but not neutral phospholipids in the presence of Ca2+ and Mg2+. A point mutation in the calcium-binding motif of hPLSCR3 (F258V) resulted in decreased Ca2+ binding affinity and a ~ 50% loss in functional activity. Mitochondria are important target organelles for heavy metals induced apoptotic signaling cascade. Pb2+ and Hg2+ toxicity mediate apoptosis by increased reactive oxygen species (ROS) and cytochrome c release from mitochondria. The functional assay using synthetic proteoliposomes demonstrated that hPLSCR3 translocated amino phospholipids 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. Metal ion-induced conformational changes were monitored by intrinsic tryptophan fluorescence, circular dichroism, surface hydrophobicity changes, and aggregation studies. Based on our results, we suggest that the metal ion-induced conformational change and the protein aggregation are essential for the phospholipid translocation by hPLSCR3. Pb2+ and Hg2+ (µM) bind to hPLSCR3 with higher affinity than Ca2+ (mM), thus mediating scramblase activity.
A plausible role of phospholipid scramblase 3 (PLSCR3) in Pb2+ and Hg2+ induced apoptosis was investigated with human embryonic kidney (HEK 293) cells. After 12 h of exposure, ~30-40% of the cells was in the early stage of apoptosis with increased reactive oxygen species (ROS), decreased mitochondrial membrane potential, and increased intracellular calcium levels. Also, ~20% of the cardiolipin localized within the inner mitochondrial membrane was translocated to the outer mitochondrial membrane enhancing t-Bid mediated cytochrome c release from the mitochondria. The endogenous expression levels of PLSCR3, caspase 8, and caspase 3 were upregulated in Pb2+ and Hg2+ induced apoptosis. The activation and upregulation of PLSCR3 mediate cardiolipin (CL) translocation playing a potential role in initiating the heavy metal-induced apoptosis. Therefore, PLSCR3 could be the linker between mitochondria and heavy metal apoptosis. To summarize, this is the first biochemical evidence for heavy metals-induced PL translocation by the mitochondrial membrane protein.
Publications:
1. Sivagnanam, U., Palanirajan, S. K., and Gummadi, S. N. (2017). “The role of human phospholipid scramblases in apoptosis: An overview,” Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1864(12), 2261–2271.
2. Palanirajan, S. K., Sivagnanam, U., Murugan, S., and Gummadi, S. N. (2018). “In vitro reconstitution and biochemical characterization of human phospholipid scramblase 3: phospholipid specificity and metal ion binding studies,” Biological Chemistry, 399(4), 361–374.
3. Palanirajan, S. K., and Gummadi, S. N. (2018). “Rapid method for an enhanced recovery of biologically active human phospholipid scramblase1 from inclusion bodies,” Analytical Biochemistry, 556, 104–111.
4. Palanirajan, S. K., and Gummadi, S. N. (2020). “Heavy-Metals-Mediated Phospholipids Scrambling by Human Phospholipid Scramblase 3: A Probable Role in Mitochondrial Apoptosis,” Chemical Research in Toxicology, 33(2), 553–564.
5. Palanirajan, S. K., Govindasamy, P., and Gummadi, S. N. (2020). “Polystyrene adsorbents: rapid and efficient surrogate for dialysis in membrane protein purification,” Scientific Reports, 10(1), 16334.