SnapShot: Ferroptosis

Abstract

Ferroptosis is a regulated form of cell death that occurs when phospholipids with polyunsaturated fatty acyl tails are oxidized in an iron-dependent manner. Research in recent years has uncovered complex cellular networks that induce and suppress lethal lipid peroxidation. This Snapshot provides an overview of ferroptosis-related pathways, including relevant biomolecules and small molecule modulators regulating them.

Cells are the basic building blocks of living systems; the mechanisms governing their division, differentiation, and death are critical for life. Until the mid-20^th century, death of cells was thought to be largely uncontrolled. Recent decades have revealed that regulated cell death is ubiquitous in the development and homeostasis of virtually all multicellular organisms, and is dysregulated in environmental and genetic diseases.

Ferroptosis is a form of regulated cell death driven by iron-dependent lipid peroxidation: ferroptosis can be induced or suppressed by specific pharmacological and genetic perturbations. Peroxidation of phospholipids, which compose the lipid bilayers that make up cellular membranes, is the key driver of ferroptotic death (Stockwell et al., 2017). Regulation of ferroptosis involves controlling the abundance of key phospholipid substrates, the factors that drive their peroxidation, and the factors that eliminate these lipid peroxides (Figure).

The substrates for peroxidation during ferroptosis are phospholipids with polyunsaturated acyl tails (PL-PUFAs) (Figure, bottom left), due to their intrinsic susceptibility to peroxidation chemistry. These PL-PUFAs are generated by enzymes such as ACSL4 and LPCATs (green, Figure, lower left) that activate and incorporate free PUFAs into phospholipids. PUFAs can be scavenged from the environment and from dietary sources, and can be synthesized from the basic building block acetyl CoA, through the action of Acetyl CoA Carboxylase (ACC) (green, Figure, lower left). Energy stress and AMPK suppress ferroptosis by inhibiting ACC (red, Figure, lower left) (Lee et al., 2020).

Once PL-PUFAs are incorporated into membrane environments, iron-dependent enzymes and labile iron use molecular oxygen (O₂) to do a peroxidation reaction, generating PL-PUFA-OOH (Figure, blue pathway). Iron-dependent enzymes found to drive ferroptosis include lipoxygenases and cytochrome P450 oxidoreductase (POR) (Zou et al., 2020). Labile iron is imported through the transferrin receptor 1 (TfR1), and stored in ferritin. Ferritin can be degraded through an autophagy-like process known as ferritinophagy, which releases labile iron and facilitates the peroxidation reaction driving ferroptosis (blue, Figure, upper left). Radiation can also directly stimulate lipid peroxidation, and radiotherapy likely works in part through triggering ferroptosis (Lei et al., 2020; Ye et al., 2020). In contrast, Prominin2 suppresses ferroptosis by facilitating the formation of multivesicular bodies containing ferritin-bound iron and, as a consequence, exporting iron out of cells (blue, Figure, upper left) (Brown et al., 2019).

There are three pathways for eliminating peroxidized PL-PUFAs (red, Figure, center and lower right): the GPX4-glutathione axis (Stockwell et al., 2017), the FSP1-CoQ₁₀ axis (Bersuker et al., 2019; Doll et al., 2019), and the GCH1-BH₄ axis (Kraft et al., 2020):

• GPX4 uses the cysteine-containing tripeptide glutathione to eliminate phospholipid peroxides (red, Figure, center). Glutathione itself is generated from cysteine, which can either be obtained from methionine through the transsulfuration pathway, or from extracellular cystine through system x_c⁻ (black, Figure, top center), which exchanges intracellular glutamate for extracellular cystine; cystine is the oxidized disulfide of the amino acid cysteine (Stockwell et al., 2017). System x_c⁻ is a central hub for regulation of ferroptosis, as CD8⁺-T-cell-derived IFNγ triggers ferroptosis in cancer cells upon immunotherapy by downregulating SLC7A11, one of the two genes that composes system x_c⁻. In contrast, NRF2 upregulates SLC7A11, thereby protecting from ferroptotic cell death.

• Reduced coenzyme Q₁₀, also known as ubiquinol, suppresses the formation of PL-PUFA-OOHs. FSP1 (formerly known as AIFM2) regenerates ubiquinol from ubiquinone (red, Figure, right), which is generated through the mevalonate pathway (Bersuker et al., 2019; Doll et al., 2019). FSP1 can be activated by PPARα (red, Figure, right), which is under the control of the MDM2/MDMX complex (green, Figure, right), independent of p53 (Venkatesh et al., 2020).

• GCH1 generates the metabolite tetrahydrobiopterin (BH₄), which has a dual function in generating reduced CoQ₁₀ (ubiquinol), and remodeling lipids to disfavor lipid peroxidation (Kraft et al., 2020). Furthermore, monounsaturated fatty acids (MUFAs), when incorporated into phospholipids through the action of ACSL3, act through an unknown mechanism to suppress ferroptosis (red, Figure, lower right).

There are several ferroptosis-inducing compounds, lipids, and proteins (see Ferroptosis Inducers Table), as well as inhibitors of ferroptosis (see Ferroptosis Inhibitors Table). Lipid peroxidation and key ferroptosis regulators can be detected using dyes, assays, molecular markers and antibodies (see Ferroptosis-Related Assays and Tools).

Ferroptosis Inducers
Inducers	Mode of Action
erastin, PE, IKE, erastin analogs, sulfasalazine, glutamate, sorafenib	inhibit system xc− => GSH depletion
INFγ	downregulates expression of system xc−
(1S,3R)-RSL3, ML162, DPI compounds	inhibit GPX4 => lipid peroxidation
cystine/cysteine depriviation buthionine sulfoximine (BSO)	inhibit GCS => GSH depletion
FIN56	depletes CoQ10; decreases GPX4 levels
FINO2	induces lipid peroxidation; indirectly inhibits GPX4
statins	block CoQ10 synthesis by mevalonate pathway inhib.
phospholipids with two PUFA tails	induce lipid peroxidation

Ferroptosis Inhibitors
Inhibitors	Mode of Action
ferrostatins, liproxtatins	block lipid peroxidation
vitamin E, alpha-tocopherol, trolox	block propagation of lipid peroxidation
CoQ10, idebenone	block lipid peroxidation
deferoxamine (DFO), cyclopirox, deferiprone	deplete iron
dihydrobiopterin (BH2), tetrahydrobiopterin (BH4)	antioxidant effect; lipid remodeling
deuterated PUFAs (D-PUFAs), MUFAs	block lipid peroxidation
lipoxygenase inhibitors (e.g. CDC, baicalein and zileuton)	block lipid peroxidation induced by lipoxygenases

Ferroptosis-Related Assays and Tools
Assays and Tools	Reagent method
BODIPY 581/591 C11	sensor for lipid ROS levels
LiperFluo	fluorescent lipid peroxide reagent
TBARS assay	detects MDA products of lipid per.
FENIX (fluorescence-enabled inhibited autoxidation)	microplate-based assay to detect phospholipid peroxidation
metabolomics / lipidomics	detects metabolites and lipids
GSH/GSSG ratio detection kit	quantifies glutathione levels
Fe2+/Fe3+ quantification kit	measures ferrous and ferric iron
anti-TfR1(3B8 2A1 & 3F3-FMA)	detect TfR1, a ferroptosis marker
anti-MDA adduct (1F83)	detects malondialdehyde
anti-4-HNE (ab46545)	detects 4-hydroxynonenal

Our increasing understanding of the mechanisms underlying the connections between metabolism, lipid peroxidation, and ferroptosis, the availability of tools to study this form of cell death, and its emerging physiological functions, promise a wealth of future advances in exploiting ferroptosis for the understanding and treatment of disease.

Abbreviations

ACC

acetyl coenzyme A carboxylase

ACSL3

acyl coenzyme A synthetase long-chain family member 3

ACSL4

acyl coenzyme A synthetase long-chain family member 4

AMPK

AMP-activated protein kinase

BH₂

dihydrobiopterin

BH₄

tetrahydrobiopterin

BSO

buthionine sulfoximine

CoA

coenzyme A

Fe

iron

FSP1

ferroptosis suppressor protein 1

GCH1

GTP cyclohydrolase 1

GCS

glutamylcysteine synthetase

GLS

glutaminase

GPX4

glutathione peroxidase 4

GSH/GSSH

glutathione

GSR

glutathione S-reductase

GSS

glutathione synthetase

HMG CoA

3-hydroxy-3-methylglutaryl CoA

HNE

hydroxynonenal

IKE

imidazole ketone erastin

INFγ

interferon gamma

LPCAT3

lysophosphatidylcholine acyltransferase 3

LOX

lipoxygenase

MDA

malondialdehyde

MDM2

mouse double minute 2

MDMX

mouse double minute 4

MUFA

monounsaturated fatty acid

NRF2

nuclear factor E2-related factor 2

OXPHOS

oxidative phosphorylation

PPARα

peroxisome proliferator activated receptor alpha

PE

piperazine erastin

PL

phospholipid

POR

cytochrome P450 oxidoreductase

PUFA

polyunsaturated fatty acid

TBARS

thiobarbituric acid reactive substances

TCA

tricarboxylic acid

TfR1

transferrin receptor 1

YAP

Yes-associated protein