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Figure 5 Morphological and molecular features of ferroptosis with PCSK9 deficiency. (A) The central regulator that inhibits ferroptosis is the selenoprotein GPX4, which together with reduced glutathione (GSH), has an antioxidant capacity against lipid reactive oxygen species (ROS), thus blocking ferroptosis (right side). The pathways that lead to the activation and synthesis of GPX4/GSH and hence ferroptosis inhibition include the System Xc--mediated import of cysteine (Cys), production of cysteine by the transsulfuration (TS) pathway and finally, the production of selenocysteine (Se) by the mevalonate pathway. Another important product of the mevalonate pathway is the CoQ10, which can inhibit ferroptosis independently of GPX4. The oxidoreductase FSP1 reduces CoQ10 to ubiquinol (CoQ10 H2) which can trap lipid ROS and then regenerates CoQ10 using NAD(P)H. (left side) The p62/Keap1/Nrf2 pathway plays an important role in inhibiting ferroptosis by activating the expression of antioxidant defense genes NAD(P)H quinone dehydrogenase 1 (NQO-1), FTH-1 and heme oxygenase 1 (HO-1). Under normal conditions, Keap1 binding to Nrf2 induces its ubiquitination and hence proteasome degradation. Under stress conditions, p62 binds to Keap1, the inhibitor of Nrf2, thus stabilizing Nrf2 which can then translocate to the nucleus and induce the expression of antioxidant defense genes. On the other hand, the pathways that lead to lipid ROS accumulation and induction of ferroptosis include: the accumulation of free intracellular ferrous (Fe2+) that can produce hydroxyl and peroxide radicals by the Fenton reaction, hence oxidizing lipids, and tumor protein p53 (p53) activation that inhibits system Xc? uptake of cystine by decreasing the expression of solute carrier family 7 member 11 (SLC7A11), hence affecting GSH/GPX4 antioxidant capacity. It can also activate SAT1 transcription factor to induce lipid peroxidation by increasing ALOX-15 levels; this leads to the production of PUFAs, which are sensitive to lipid peroxidation, resulting in ferroptosis. Abbreviations: ACAC: Acetyl-CoA carboxylase; ACSL4: long-chain fatty Acyl-CoA synthetase 4; Fe3+: ferric cation; FTL: ferritin light chain; GCL: glutamate-cysteine ligase; GSS: glutathione synthetase; HMGCR: 3-hydroxy-3-methylglutaryl-CoA reductase; IPP: isopentenyl pyrophosphate; LD: lipid droplets, PL: phospholipid. (B) TEM photomicrographs of HepG2 cells transduced with PCSK9 shRNA (top panels) and control shRNA (bottom panels) at different magnifications. The arrows point to the mitochondria. The mitochondrial membranes were measured at the higher magnification of 80,000 (between the yellow head arrows) and the results are indicated within brackets. (C) Western blot analyses of proteins involved in different signaling pathways of ferroptosis. Membrane images of each protein and their relative housekeeping genes were combined/fused for comparison purposes. Wells were loaded alternatively with samples from (a) siCTR- and (b) siPCSK9-transfected cells (from left to right). MW stands for molecular weight markers. GAPDH or vinculin were used for protein normalization. Unpaired t-test, * p < 0,05; ** p < 0,01, *** p < 0.001 (n = 3). (D) Histograms showing the percentage of cell growth inhibition induced by siPCSK9 in HepG2 cells normalized to siCTR, which was followed 6 days after treatment with ferrostatin-1 (left panel) and 8 days after treatment with ?-tocopherol (right panel). Average values were obtained by combining values from both PCSK9 siRNAs. Unpaired t-test, * p < 0,05 (n = 1, with 3 technical replicates.).