Post-Translational Modification of GPX4 is a Promising Target for Treating Ferroptosis-Related Diseases

1In 1982, a new glutathione peroxidase was first isolated from the pig liver. Unlike previously discovered glutathione peroxidase 1-3 (GPX1-3), this new enzyme, phospholipid hydroperoxide glutathione peroxidase (PHGPX), acts directly on peroxidized phospholipids in the membrane. GPX1-3 are tetramers that mainly reduce H2O2 and fatty acid hydroperoxide, whereas PHGPX is a monomeric protein and reduces lipid hydroperoxides specifically. Later, PHGPX was renamed glutathione peroxidase 4 (GPX4). Until 2014, GPX4 was identified as the central regulator of ferroptosis, which is an iron-dependent, non-apoptotic, programmed necroptotic cell death form. How to regulate the protein level of GPX4 and its activity has become a hot topic to discover the potential treatment of ferroptosis-related diseases recently.

2The selenoprotein GPX4 has high antioxidant activity. It directly reduces phospholipid hydroperoxide through a conserved catalytic triad of selenocysteine 46, asparagine 81, and tryptophane 136. The reaction kinetics of GPX4 is described as a Ping-Pong pattern that is embodied in two phases (Figure 1). Firstly, the peroxide is reduced through the active site selenocysteine 46 and then converted to the oxidized form (selenenic acid derivative). The second stage is to recharge the oxidized catalytic site via glutathione (GSH). The content of GSH and nicotinamide adenine dinucleotide phosphate (NADPH) directly affect the GPX4 activity.

3Cystine/glutamate transporter (system χC −) on the cell membrane, composed of a catalytic subunit SLC7A11 and a chaperone subunit SLC3A2, pumps cystine from extracellular into cells for GSH biosynthesis. Therefore, classic ferroptosis inducers (FINs) are divided into two classes (Figure 1): class Ⅰ FIN such as sorafenib, sulfasalazine, erastin and its analog imidazole ketone erastin (IKE), can block the GSH synthesis through inhibiting system χC −, and then reduce the GPX4 enzyme activity by GSH depletion; class Ⅱ FIN such as Ras-selective lethal small molecule 3 (RSL3), ML162, and ML210, can directly inhibit GPX4 activity. They eventually lead to the accumulation of lipid peroxides without the reduction of GSH. Apart from these two types of FINs, other methods or compounds can also induce ferroptosis (Figure 1): L-buthionine sulfoximine (BSO) suppresses glutamate-cycteine ligase (GCL) and induces GSH depletion, driving cells to ferroptosis; Cystine starvation leads to glutamate accumulation, GSH biosynthesis reduction, and GPX4 translation blockage, therefore sensitizing cancer cells to ferroptosis. FINs and other compounds that cause ferroptosis drive tumor cell death, sensitizing cancer cells to radiotherapy and chemotherapy in prostate cancer, colorectal cancer, osteosarcoma, and leukemia. Therefore, traditional chemotherapy/radiotherapy combined with FINs treatment is beneficial to reverse multidrug resistance.

4Besides small molecular compounds like FINs, post-translational modifications (PTMs) also provide an emerging perspective to regulate its protein level/activity (Figure 2). Thus, studying and targeting PTMs of GPX4 has a great potential to treat cancers in the future.

5Succination was discovered in 2006 as a non-enzymatic, irreversible protein modification. This post-translational modification is mediated by fumarate. Fumarate is an intermediate of the Krebs cycle in mitochondria. It binds to the sulfhydryl group of the cysteine residue and forms a thioether bond in the absence of an enzyme. An elegant study showed that intracellular fumarate aggregation led to the succination of GPX4 at cysteine 93 (mono- and di-succination), which significantly reduced the enzymatic activity of GPX4 and sensitized cancer cells to FINs. In addition, fumarate hydratase (FH)-inactivated cells were shown to be synthetic lethal with FINs. This study suggests that targeting PTMs of GPX4 is a promising therapeutic strategy for ferroptosis-related diseases for the first time.

6Ubiquitination is a reversible process in which the ubiquitin is added to lysine residue(s) of the substrate protein by three different types of enzymes, E1, E2, and E3. The process of ubiquitination ultimately renders proteins to degradation by the proteasome thereby affecting intracellular protein levels. On the other hand, deubiquitinases (DUBs) remove ubiquitin chains from ubiquitinated proteins. E3 ligase and DUB are both having substrate specificity.

7Proteomic analyses reveal that GPX4 could be ubiquitinated at lysine 107, 162, and 167. However, those hypothetically ubiquitinated residues have not been verified in bench works. Whereas, a study showed that when treating non-small cell lung cancer cells with the DUB inhibitor, pyridinium sulfur palladium complex (PdPT), GPX4 was ubiquitinated and degraded. Subsequently, cancer cell growth was restrained by inducing GPX4-dependent ferroptosis. Later, another study showed that a Parthenolide (PTL)-derived drug DMOCPTL directly bound to the GPX4 active site (selenocysteine 46), and led to GPX4 ubiquitination in triple-negative breast cancer cells. Unfortunately, the above-mentioned studies did not reveal the ubiquitinated residue(s) of GPX4.

8Discovering the E3 ligase is also very important for understanding the regulatory mechanisms of protein ubiquitination. A very recent study discovered that the E3 motif-containing 46 (TRIM46) might be one of the E3 ligases of GPX4 in human retinal capillary endothelial cells with high glucose treatment. Whether GPX4 has other E3 ligases is needed further investigation. These findings indicate that GPX4 can be ubiquitinated under certain circumstances. Importantly, treating cells with PdPT, DMOCPTL, or other similar compounds could potentially kill cancer cells by inducing ferroptosis.

9Alkylation is a chemical process of introducing one or more alkyl groups into a protein or a compound. An alkyl group is an alkane molecule that is missing a hydrogen atom. For example, methyl groups are the simplest alkyl and are added to various proteins including core histones. Previous studies have shown several class Ⅱ FINs such as RSL3 and ML162 exerted covalent inhibition activity. These class Ⅱ FINs bound to selenocysteine 46 residue of GPX4 via an electrophilic alkyl chloride moiety, mediating alkylation on GPX4. Besides selenocysteine 46, GPX4 contains several cysteine residues which are also nucleophilic sites. A report showed that GPX4 was most likely alkylated at cysteine 107 with the addition of withaferin A. In addition, the alkylation of GPX4 resulted in ferroptosis in high-risk neuroblastoma. However, Hassaniia et al did not further investigate the specifically modified residue(s) of GPX4 or which kind(s) of alkylation occurs on GPX4.

10Phosphorylation is one of the most common PTMs involved in the regulation of protein stability and activity. It occurs on serine, tyrosine, and threonine residues of target proteins and is regulated by protein kinases and phosphatases. Phosphorylation of GPX4 has been found in the pig heart, golden hamster’s sperms, and rat testicles. Notably, the mature testicle is the organ with the highest GPX4 activity in mammals, and sperm express high levels of GPX4. Studies showed that GPX4 lost its enzyme activity during the differentiation and maturation of sperm. Meanwhile, the phosphorylation of GPX4 on tyrosine has been found in capacitated sperm of the golden hamster. Several proteomic analyses also revealed that human GPX4 might be phosphorylated at serine 40. These data indicate that phosphorylation of GPX4 participates in the GPX4 activity regulation. However, the exact phosphorylated residues, the corresponding phosphatases, and the biological effects of phosphorylation on GPX4 need to be further investigated.

11Glycosylation (N-linked and O-linked) is under the reaction of glycosyltransferases. Protein glycosylation affects protein stability and folding and leads to changes in protein function. GPX4 has been identified as a membrane glycoprotein in an N-linked glycoproteome study in human sperm. The modification site was at the asparagine residue of GPX4. In addition, deglycosylation treatment was also found to significantly decrease sperm fertilization rate. This study suggests that N-linked glycosylation plays an important role in modifying membrane GPX4 activity and enables sperm to exert their normal fertilization functions.

12In addition to phosphorylation and glycosylation, SUMOylation is also predicted to occur on GPX4. SUMOylation is a small ubiquitin-like modifier (SUMO) connected to the substrate protein through a tertiary enzymatic reaction. It often modifies lysine residues of substrate proteins. SUMOylation regulates the protein-protein interactions, mediates the function and localization of substrate proteins, and increases protein stability. A study using software resources (SUMOplot) for bioinformatics analysis predicted that the residues of GPX4 most likely to be modified by SUMO were lysine 74, 106, and 125. The cationic region of GPX4, including lysine 125, has the potential to interact with the phospholipid bilayer. Then the redox-active center of GPX4 was directed to the H2O2 group in the phospholipid fatty acid chain, resulting in the inhibition of lipid peroxidation of the cell membrane in silico.

13Since GPX4 is a core regulator of ferroptosis, although it is unknown whether the phosphorylation, glycosylation or SUMOylation of GPX4 affect ferroptosis, it would be of interest to investigate it in future translational studies.

14PTMs are an important regulator of protein level and activity with huge potential in translational and clinical research. For instance, S-farnesylation is one of the post-translational protein lipid modifications. Hutchinson–Gilford progeria syndrome (HGPS) is a genetic rare disease caused by progerin formation due to a deficiency of removing a farnesylcysteine-containing peptide from prelamin A. Consequently, prelamin A turned into farnesylated and non-functional progerin instead of lamin A. However, progein was identified to be the critical target of farnesyltransferase inhibitors (FTIs) and one FTI called lonafarnib have been tested in a cohort study which showed a significantly lower mortality rate (hazard ratio, 0.12) after a median of 2.2 years of follow-up. S-palmitoylation is also an important PTM in many oncoproteins such as RAS-family GTPases and epidermal growth factor receptor (EGFR) and tumor suppressors such as melanocortin one receptor (MRC1), which serves as an important target pathway to inhibit tumor growth and prevent tumorigenesis. Inducing palmitoylation in MCR1variant individuals can lower their risk to develop melanoma. Targeting inhibition of palmitoylation has also shown sensitization to gefitinib in cancer cells.

15The investigation of the GPX4 function was far ahead of the emergence of ferroptosis. A variety of PTMs of GPX4 has been observed in both physiological and pathophysiological conditions. Targeting PTMs of GPX4 could be a promising strategy for treating diseases, including ferroptosis-related diseases such as cancers, neurodegenerative disorders, and ischemic-reperfusion diseases as well as other important players in ferroptosis. However, in contrast to other proteins, such as TP53, studies on PTMs of GPX4 have only just started. At this point, we believe it is important to explore several aspects in future studies, such as to discover whether some of the PTMs summarized above affect GPX4 activity in bench works and GPX4 dysfunction-relative diseases. What are the corresponding enzymes of the above-mentioned PTMs? If other PTMs, such as methylation, lipoylation, or newly identified lactation also modify GPX4 protein? Since ferroptosis is an important cell death form in various conditions, and GPX4 is its core regulator, more in-depth studies are urgently needed to assist GPX4-based clinical therapeutic strategies for tumor killing and neuronal protection.