Regulation of ferroptosis by lipid metabolism.

Ferroptosis is an iron-dependent lethal mechanism that can be activated in disease and is a proposed target for cancer therapy. Ferroptosis is defined by the overwhelming accumulation of membrane lipid peroxides. Ferroptotic lipid peroxidation is initiated on internal membranes and then appears at the plasma membrane, triggering lethal ion imbalances and membrane permeabilization. Sensitivity to ferroptosis is governed by the levels of peroxidizable polyunsaturated lipids and associated lipid metabolic enzymes. A different network of enzymes and endogenous metabolites restrains lipid peroxidation by interfering with the initiation or propagation of this process. This emerging understanding is informing new approaches to treat disease by modulating lipid metabolism to enhance or inhibit ferroptosis.

Context-dependent regulation of ferroptosis sensitivity.

Ferroptosis is an important mediator of pathophysiological cell death and an emerging target for cancer therapy. Whether ferroptosis sensitivity is governed by a single regulatory mechanism is unclear. Here, based on the integration of 24 published chemical genetic screens combined with targeted follow-up experimentation, we find that the genetic regulation of ferroptosis sensitivity is highly variable and context-dependent. For example, the lipid metabolic gene acyl-coenzyme A (CoA) synthetase long chain family member 4 (ACSL4) appears far more essential for ferroptosis triggered by direct inhibition of the lipid hydroperoxidase glutathione peroxidase 4 (GPX4) than by cystine deprivation. Despite this, distinct pro-ferroptotic stimuli converge upon a common lethal effector mechanism: accumulation of lipid peroxides at the plasma membrane. These results indicate that distinct genetic mechanisms regulate ferroptosis sensitivity, with implications for the initiation and analysis of this process in vivo.

Ferroptosis regulation by the NGLY1/NFE2L1 pathway.

SignificanceFerroptosis is an oxidative form of cell death whose biochemical regulation remains incompletely understood. Cap'n'collar (CNC) transcription factors including nuclear factor erythroid-2-related factor 1 (NFE2L1/NRF1) and NFE2L2/NRF2 can both regulate oxidative stress pathways but are each regulated in a distinct manner, and whether these two transcription factors can regulate ferroptosis independent of one another is unclear. We find that NFE2L1 can promote ferroptosis resistance, independent of NFE2L2, by maintaining the expression of glutathione peroxidase 4 (GPX4), a key protein that prevents lethal lipid peroxidation. NFE2L2 can also promote ferroptosis resistance but does so through a distinct mechanism that appears independent of GPX4 protein expression. These results suggest that NFE2L1 and NFE2L2 independently regulate ferroptosis.

Reactivity-Based Probe of the Iron(II)-Dependent Interactome Identifies New Cellular Modulators of Ferroptosis.

Ferroptosis is an iron-dependent form of cell death resulting from loss or inhibition of cellular machinery that protects from the accumulation of lipid hydroperoxides. Ferroptosis likely serves a tumor suppressing function in normal cellular homeostasis, but certain cancers exploit and become highly dependent on specific nodes of the pathway, presumably to survive under conditions of increased oxidative stress and elevated labile ferrous iron levels. Here we introduce Ferroptosis Inducing Peroxide for Chemoproteomics-1 (FIPC-1), a reactivity-based probe that couples Fenton-type reaction with ferrous iron to subsequent protein labeling via concomitant carbon-centered radical generation. We show that FIPC-1 induces ferroptosis in susceptible cell types and labels cellular proteins in an iron-dependent fashion. Use of FIPC-1 in a quantitative chemoproteomics workflow reproducibly enriched protein targets in the thioredoxin, oxidoreductase, and protein disulfide isomerase (PDI) families, among others. In further interrogating the saturable targets of FIPC-1, we identified the PDI family member P4HB and the functionally uncharacterized protein NT5DC2, a member of the haloacid dehalogenase (HAD) superfamily, as previously unrecognized modulators of ferroptosis. Knockdown of these target genes sensitized cells to known ferroptosis inducers, while PACMA31, a previously reported inhibitor of P4HB, directly induced ferroptosis and was highly synergistic with erastin. Overall, this study introduces a new reactivity-based probe of the ferrous iron-dependent interactome and uncovers new targets for the therapeutic modulation of ferroptosis.

Quaternary Structure, Salt Sensitivity, and Allosteric Regulation of ß-AMYLASE2 From Arabidopsis thaliana.

The ß-amylase family in Arabidopsis thaliana has nine members, four of which are both plastid-localized and, based on active-site sequence conservation, potentially capable of hydrolyzing starch to maltose. We recently reported that one of these enzymes, BAM2, is catalytically active in the presence of physiological levels of KCl, exhibits sigmoidal kinetics with a Hill coefficient of over 3, is tetrameric, has a putative secondary binding site (SBS) for starch, and is highly co-expressed with other starch metabolizing enzymes. Here we generated a tetrameric homology model of Arabidopsis BAM2 that is a dimer of dimers in which the putative SBSs of two subunits form a deep groove between the subunits. To validate this model and identify key residues, we generated a series of mutations and characterized the purified proteins. (1) Three point mutations in the putative subunit interfaces disrupted tetramerization; two that interfered with the formation of the starch-binding groove were largely inactive, whereas a third mutation prevented pairs of dimers from forming and was active. (2) The model revealed that a 30-residue N-terminal acidic region, not found in other BAMs, appears to form part of the putative starch-binding groove. A mutant lacking this acidic region was active and did not require KCl for activity. (3) A conserved tryptophan residue in the SBS is necessary for activation and may form π-bonds with sugars in starch. (4) Sequence alignments revealed a conserved serine residue next to one of the catalytic glutamic acid residues, that is a conserved glycine in all other active BAMs. The serine side chain points away from the active site and toward the putative starch-binding groove. Mutating the serine in BAM2 to a glycine resulted in an enzyme with a VMax similar to that of the wild type enzyme but with a 7.5-fold lower KM for soluble starch. Interestingly, the mutant no longer exhibited sigmoidal kinetics, suggesting that allosteric communication between the putative SBS and the active site was disrupted. These results confirm the unusual structure and function of this widespread enzyme, and suggest that our understanding of starch degradation in plants is incomplete.

Arabidopsis ß-Amylase2 Is a K+-Requiring, Catalytic Tetramer with Sigmoidal Kinetics.

The Arabidopsis (Arabidopsis thaliana) genome contains nine ß-amylase (BAM) genes, some of which play important roles in starch hydrolysis. However, little is known about BAM2, a plastid-localized enzyme reported to have extremely low catalytic activity. Using conservation of intron positions, we determined that the nine Arabidopsis BAM genes fall into two distinct subfamilies. A similar pattern was found in each major lineage of land plants, suggesting that these subfamilies diverged prior to the origin of land plants. Moreover, phylogenetic analysis indicated that BAM2 is the ancestral member of one of these subfamilies. This finding, along with the conservation of amino acids in the active site of BAM2, suggested that it might be catalytically active. We then identified KCl as necessary for BAM2 activity. Unlike BAM1, BAM3, and BAM5, three Arabidopsis BAMs that all exhibited hyperbolic kinetics, BAM2 exhibited sigmoidal kinetics with a Hill coefficient of over 3. Using multi-angle light scattering, we determined that BAM2 was a tetramer, whereas BAM5 was a monomer. Conserved residues from a diverse set of BAM2 orthologs were mapped onto a homology model of the protein, revealing a large, conserved surface away from the active site that we hypothesize is a secondary carbohydrate-binding site. Introduction of bulky methionine for glycine at two points on this surface reduced catalytic activity significantly without disrupting the tetrameric structure. Expression analysis indicated that BAM2 is more closely coexpressed with other starch degradation enzymes than any other BAM, suggesting that BAM2 may play an important role in starch degradation in plants.