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Heavy-Metals-Mediated Phospholipids Scrambling by Human Phospholipid Scramblase 3: A Probable Role in Mitochondrial Apoptosis
journal contribution
posted on 2019-12-10, 14:04 authored by Santosh
Kumar Palanirajan, Sathyanarayana N. GummadiHuman 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.
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affinitybinding motifmetal ions bindPbhPLSCR 3 mediates PL translocationhPLSCR 3 translocated aminophospholipidsROSHgmitochondrial membrane proteincytochrome c releasereactive oxygen speciesMitochondrial Apoptosis Human phospholipid scramblases258Vt-bid-induced cytochrome c releaseEscherichia coli RosettaapoptosiDEhPLSCR 3
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