Fundamental Biochemical Mechanisms of Ferroptosis

System x_c^-/GSH/GPX4 Axis

Ferroptosis Inducers

Ferroptosis Suppressors

Figure 2. Enzymatic mechanisms of lipid peroxidation and ferroptosis.

Free intracellular arachidonic acid and adrenic acid are conjugated to coenzyme A (CoA) by ACSL4. LPCAT3 then catalyzes the esterification of these PUFAs into membrane phospholipids (PLs). LOs, particularly 15-LO, which normally uses free PUFAs as substrates, are then capable of oxidizing PUFA-PLH and generating PLOOHs, such as (e.g., PE-15-HpETE). If GPX4 inadequately reduces PLOOHs to PLOHs, the accumulation of PLOOHs will serve as a lethal signal to trigger ferroptosis.

Conjugated fatty acids (CFAs) also elicit ferroptosis-promoting effects, whereas the incorporation of saturated and monounsaturated fatty acids (SFAs and MUFAs) into cellular membranes counteract the effects of PUFAs and CFAs.

2. Defective Lipid Peroxide Repair Mechanisms

The system x_c^-/GSH/GPX4 axis is a critical pathway for ferroptosis that regulates intracellular glutathione (GSH) content to balance cellular redox (Figure 3).

Figure 3.The system x_c^-/GSH/GPX4 axis controls GSH synthesis.

Cystine Depletion

System x_c^- transports intracellular glutamate to the extracellular space while transporting extracellular cystine into the cell.

Regulation of System x_c^- Expression

Tumor suppressors p53 and BAP1 sensitize cells to ferroptosis by suppressing the transcription of a subunit of the system x_c^-, which reduces the import of cystine, and consequent synthesis of glutathione, a critical cellular antioxidant.

GSH Homeostasis

Cystine is transformed into cysteine for GSH synthesis. Under conditions of cysteine insufficiency, methionine can be converted to cysteine through the transsulfuration pathway to ultimately supplement the cysteine pool (Figure 4).

Figure 4. Methionine can be used as a precursor in the formation of cysteine, which is essential for GSH synthesis.

GSH is highly critical for protecting cells from damage caused by oxidative stress, and defective GSH synthesis increases the sensitivity of cells to ferroptosis due to disrupted oxidation-reduction balance.

GPX4 Dysregulation

Glutathione peroxidase 4 (GPX4) is a lipid repair enzyme that utilizes GSH as a substrate to limit lipid peroxide accumulation (Figure 5). Thus, when the GSH pool is depleted, GPX4 activity is also reduced.

GPX4 is a selenoprotein that functions on biological membranes as a phospholipid hydroperoxidase. It catalyzes the reduction of lipid peroxides at the expense of GSH. When GPX4 activity is hindered, lipid peroxides accumulate and ultimately cause cell death.

The Mevalonate Pathway & FSP1

Activation of squalene synthase (SQS) directs the MVA pathway towards cholesterol synthesis, suppressing the formation of several important non-sterol products like coenzyme Q₁₀ (CoQ₁₀), an important free-radical scavenging antioxidant (Figure 6).

Figure 6. The MVA and biopterin pathways play important roles in inhibiting ferroptosis.

Additionally, GTP cyclohydrolase 1 (GCH1) increases the de novo synthesis of CoQ₁₀ by initiating the synthesis of dihydrobiopterin (BH₂) and tetrahydrobiopterin (BH₄). The actions of these proteins inhibit ferroptosis by acting as radical trapping antioxidants or contributing to the synthesis of reduced CoQ₁₀.

The MVA pathway also plays a key role in GPX4 maturation, as isopentenyl pyrophosphate (IPP) contributes to the insertion of selenocysteine into the catalytic center of GPX4, which is important for its antioxidant activity. Loss of GPX4 function, either through loss of catalytic activity or by its degradation, promotes ferroptosis due to the loss of antioxidant activity.

FSP1

Ferroptosis suppressor protein 1 (FSP1) is an important regulator of ferroptosis that acts in a GSH-independent manner (Figure 7). FSP1 is an NADPH-dependent oxidoreductase that reduces CoQ₁₀ to ubiquinol (CoQ₁₀H₂) and vitamin K to vitamin K hydroquinone (VKH₂). The reduced forms of these molecules act as important reducing agents that are able to detoxify lipid peroxides.

Regulation of FSP1 Expression

The MDM2/MDMX complex reduces expression of FSP1 by inhibiting PPARα activity.

3. Iron Oxidation of PUFAs Initiates Ferroptosis

Regulation of Iron Levels & Metabolism

Excessive iron metabolism contributes to ferroptosis by producing oxidative stress. Circulating iron, in the form of ferric iron (Fe³⁺) bound to transferrin, is transported into cells through the membrane-bound transferrin receptor 1 (TfR1) (Figure 8). Within endosome compartments, Fe³⁺ is reduced to ferrous iron (Fe²⁺) by the metalloreductase STEAP3 and released into a labile iron pool in the cytoplasm via the divalent metal transporter 1 (DMT1) (Figure 4). Extra iron is stored in ferritin, an iron storage protein complex. Ferritinophagy and mitophagy also increase intracellular iron availability, promoting ferroptosis.

Any increase in iron uptake or reduced capacity for iron storage contributes to iron overload and the potential to generate highly reactive hydroxyl radicals through the Fenton reaction. These radicals can oxidize PUFAs in lipid membranes, creating lipid hydroperoxides.

Metabolic Disturbances in Ferroptosis

E-Cadherin Signaling

Active proliferation is a marker of metabolically active cells. E-cadherin binding between cells mediates contact inhibition of proliferation when cells reach confluence (Figure 9). Contact inhibition of cell growth is essential for normal development, and uncontrolled cell growth is a characteristic of cancer. E-cadherin promotes contact inhibition through the Hippo signaling pathway. Binding of E-cadherin activates Hippo via NF2/Merlin, resulting in phosphorylation of YAP and TAZ, targeting it for proteasomal degradation and preventing their nuclear translation.

Figure 9. E-cadherin promotes contact inhibition of cell proliferation.

In the absence of Hippo signaling, YAP and TAZ are translocated to the nucleus, where they promote the activity of several genes involved in ferroptosis, including ACSL4 (e.g., lipogenesis) and the transferrin receptor (e.g., iron uptake).

Energy Balance in Ferroptosis

mTORC1 and AMPK are master regulators of cellular energy status and metabolism that coordinate opposing functions. In energy-abundant conditions, mTORC1 is activated, whereas in low energy conditions, such as glucose scarcity, AMPK is activated. Both mTORC1 and AMPK can have pro- or anti-ferroptotic effects, depending on cellular context.  

mTORC1

mTORC1 activation promotes the synthesis of proteins such as GPX4 and transsulfuration pathway enzymes (Figure 10). PI3K-Akt-mediated mTORC1 activation also encourages resistance to ferroptosis by promoting SREBP1-mediated increases in stearoyl-CoA desaturase 1 (SCD1), which catalyzes the conversion of SFAs to MUFAs.

Figure 10. mTOR activation promotes MUFA synthesis, inhibiting ferroptosis.

Accordingly, inhibition of mTORC1 is an intriguing strategy for sensitizing cancer cells to ferroptosis induction. However, mTORC1 inhibition has also been shown to inhibit ferroptosis by increasing levels of GSH.

AMPK

AMPK signaling prevents ferroptosis by suppressing lipogenesis through inhibition of acetyl-CoA carboxylase (ACC), an enzyme responsible for the conversion of acetyl-CoA to malonyl-CoA, a key step in fatty acid synthesis (Figure 11).

Figure 11. AMPK signaling inhibits ferroptosis by downregulating the synthesis of new lipids.

This leads to decreased production of PUFAs, a major initiator of ferroptosis. On the other hand, AMPK activation downregulates MUFA production via SREBP, increasing susceptibility to ferroptosis.

Mitochondrial Energy Production & Ferroptosis

Glutaminolysis

Glutaminolysis converts glutamine to glutamate, which fuels the TCA cycle. This increases the rate of oxidative metabolism in mitochondria, which causes the accumulation of ROS and promotes ferroptosis (Figure 12).

Figure 12. Mitochondrial dynamics influence ferroptosis.

Mitochondrial ROS can hyperoxidize peroxiredoxin 3 (PRDX3), a thiol-based peroxidase with important roles in protection against oxidative stress. Hyperoxidized PRDX3 has been shown to block cystine uptake, limiting the synthesis of GSH and promoting ferroptosis. 

The Malate-Aspartate Shuttle

The malate-aspartate shuttle, which is important for providing energy for biosynthesis and maintaining redox balance, transfers reducing equivalents in the form of NADH from the cytosol into the mitochondria to facilitate oxidative phosphorylation (Figure X).

Disruption of glutamic-oxaloacetic transaminase 1 (GOT1), also known as aspartate aminotransferase (AST), within this shuttle has been implicated in creating metabolic vulnerabilities for cystine, glutathione, and lipid antioxidant function and has been exploited to trigger ferroptosis in cancer cells.

Next Steps Towards Therapeutics

While important for controlling damage brought on by the oxidative stress response, ferroptosis has also been implicated in several pathological conditions in the brain, kidney, liver, and heart. This indicates a necessity for the development of therapeutics to inhibit the process. Indeed, much more work must be done to fully understand the underworking of ferroptosis. With expertise in bioactive lipid synthesis and protein, antibody, and assay development, Cayman is here to help in this endeavor by providing much-needed research tools.

Suggested Reading

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