Research on programmed cell death has always been a hot field in the life sciences, both continuously and hotly"Ferroptosis"(Tweet: What is ferroptosis and how is it detected?)Here comes the "one-on-one" you wanted). Or is it the "Copper Death" that is being generously colored in the field of bioinformatics (tweet: Airborne"Hot search"Copper Death丨Unlock a new way of cell death), are involved"Ion transport"。The Solute Carriers (SLC) transporter family is an important family of membrane transporters that play an important role in the transport of glucose, amino acids, and metal ions during transport (Figure 1) [1].
Figure 1The SLC family of proteins transports different ions as well as amino acids[1].
SLC7A11 --- a double-edged sword in the regulatory pathway of death
SLC family memberThe SLC7A11 transporter plays an important role in maintaining intracellular glutathione levels and protecting cells from oxidative stress-induced cell death, which has a recognized pro-survival effect[3]. However, it has been shown that cystine uptake by glioblastoma cells via System XC- (where SLC7A11 is the catalytic subunit) under glucose-deprivation conditions rapidly induces NADPH depletion, reactive oxygen species accumulation, and cell death.
In 2020, the Gambo Yi team published an article in Nature Cell Biology that high SLC7A11 expression promoted cell death under glucose starvation conditionsSLC7A11 acts as a double-edged sword in regulating cellular redox homeostasis and cell death survival
Figure 2High expression of SLC7A11 promotes cell death under glucose-starved conditions[5].
Cystine, consisting of SLC7A11 and SLC3A2, is a glutamate reverse transporter System XC -, which transfers intracellular glutamate out in a 1:1 ratio in exchange for extracellular cystine (CYS2).
Why does the high expression of SLC7A11 "promote" death?
This is because cystine is an insoluble amino acid, and in order to prevent the toxic build-up of highly insoluble cystine within the cell, SLC7A11High cells need to rapidly reduce cystine to cysteine, a process that requires a large amount of NADPH from the glucose-pentose phosphate pathway (PPP), which has a negative effect on the cell.
The NadpH pool is highly depleted and causes such cells to produce glucose and pentose phosphate pathway (PPP)-dependent (Figure 2) [4,5]. Therefore,When glucose** is limited and redox power is insufficient, the abnormal accumulation of cystine or other disulfide molecules in SLC7A11High cells induces disulfide stress to trigger cell death
In February of this year, the article Actin Cytoskeleton Vulnerability to Disulfide Stress Mediates Disulfidptosis published by Gamboyi's team shed more detail on this mechanism of death, which the study showedThe sensitivity of the actin cytoskeleton to disulfide stress mediates disulfideptosisand proposed a strategy to target disulfide in cancer.
Unique cell death patterns of SLC7A11-High cells
Disulfidetis is a novel mode of death that is different from ferroptosis, apoptosis, etc., in SLC7A11High cells,Neither ferroptosis, apoptosis, cell necrosis, nor autophagy inhibitors can rescue glucose starvation-induced cell death(Fig. 3A-b), but reducing agents of disulfur stress, such as dithiothreitol (DTT), mercaptoethanol (2ME), and TCEP, can completely inhibit glucose starvation-induced cell death in SLC7A11High cells (Fig. 3D). In addition, thiol oxidants (diamine and diethyl maleate) promote cell death in SLC7A11High cells under glucose starvation and lead to a sharp accumulation of intracellular disulfide molecules (such as cystine and glutamylcystine, which are further increased after diamine treatment) (Figure 3C). Transmission electron microscopy (TEM) analysis showed that glucose starvation led to cystine accumulation in the cytoplasm of SLC7A11HIGH cells (Figure 3E). The above results show that the cell death caused by disulfide stress is different from ferroptosis, apoptosis, etc., so what are its characteristics?
Figure 3Patterns of cell death under glucose-starved conditions[5].
a.Cell death in SLC7A11-High cells treated with DFO, Fer-1, Z-VAD, NEC-1, NEC-2 and CQ. b.Cells overexpressing SLC7A11 were cultured in glucose-free medium and treated with Z-VAD, FER-1 and TCEP for indicated times. c.UMRC6 cells were cultured in medium with or without DTT, 2ME, or TCEP. d.Accumulation of cystine and glutamylcystine in UMRC6 cells. e.Typical TEM image of UMRC6 cells.
Disulfide --- is associated with the actin cytoskeleton
The authors' team hypothesized that under glucose-starved conditions, the increase in NADPH depletion and disulfide stress in SLC7A11-high cells induces the formation of disulfide bonds in redox-sensitive proteins (under normal conditions, the reducing environment of the cytoplasm prevents the formation of disulfide bonds in cytosolic proteins), which may disrupt the activity or function of the corresponding oxidized proteins, thereby impairing cell viability.
To test this hypothesis, the authors' team quantified disulfide proteomic alterations in SLC7A11High cells induced by glucose starvation by stable isotope labeling of amino acids (Figure 4A). Forward and reverse labeling analyses identified 90 cysteine sites. In addition, gene ontology analysis showed that in the proteins of disulfide bonds induced by glucose starvation,Actin cytoskeleton and cell adhesion-related biological processes or pathways are significantly enriched(Fig. 4c), the authors' team also found at least 17 actin cytoskeletal proteins in top proteins with increased disulfide bonds after glucose starvation (Fig. 4b) and that most of these proteins contain cysteine sites for disulfide bonds (Fig. 4D-E). This suggests that glucose starvation in SLC7A11High cells may induce disulfide bonds in actin cytoskeletal proteins (Figure 4C).
Figure 4Disulfide bond formation in actin cytoskeletal proteins under glucose starvation conditions[5].
a.Methods used to identify disulfide-containing scatterplots of disulfide-containing peptides in forward and reverse experiments. c.Gene Ontology (GO) enrichment analysis. d.Increase in the number of different dithiocysteine-containing sites for proteins containing disulfide bonds.
Actin cytoskeletal dynamics of SLC7A11-HIGH cells
As the formation of disulfide bonds affects the electrophoretic mobility of proteins under non-reducing conditions. The authors' team examined the mobility of actin cytoskeletal proteins and found that multiple actin cytoskeletal proteins exhibited slower migration in UMRC6 cells after glucose starvation, suggesting that these actin cytoskeletal proteins formed multiple intermolecular disulfide bonds under glucose-starved conditions (Fig. 5A).
The authors' team also further investigated the actin cytoskeletal dynamics of SLC7A11High cells after glucose starvation. In sugar-containing medium SLC7A11HIGH cells, actin filaments (F-actin) are predominantly in the cellular cortex and stress fibers;However, glucose starvation induces significant changes in cell morphology: cell contraction and F-actin contraction。Co-staining of F-Actin with the membrane dye Cellmask revealed that glucose starvation resulted in the separation of F-actin from the plasma membrane in SLC7A11High cells (Figure 5B-D), and that glucose starvation-induced changes in the cytoskeletal morphology of actin in these cells were SLC7A11-dependent (Figure 5C), which could be eliminated by cystine-starved DG or 2ME (Figure 5F) treatment. Glucose starvation-induced aberrant disulfide bonds of the actin backbone protein in high cells may result in subsequent F-actin contraction and detachment from the plasma membrane.
Figure 5Actin dynamics due to aberrant disulfide bond formation under glucose-starved conditions[5].
a-b.Schematic diagram of glutathione-ated SLC7A11-HIGH-mediated cystine uptake. c.Fluorescently stained for F-actin in sugar-free medium of WT and SLC7A11-KO UMRC6 cells. d.Cells cultured in sugar-free medium were fluorescently stained with F-actin and membranes. e.Dextrose-contained, dextrose-free, dextrose-and-cystine-free (GLC) or cystine-free (GLC) medium were cultured and fluorescently stained for F-actin. f.Fluorescent staining of F-actin in cells cultured with 2ME addition in sugar-containing or sugar-free medium.
The above results suggest that glucose starvation induces aberrant disulfide bonds of actin cytoskeletal proteins, and F-actin collapses in an SLC7A11-dependent manner in SLC7A11High cells. The identification of new death mechanisms has led to a fundamental understanding of cellular homeostasis, so what is the greatest significance of "disulfide death"?
Disulfide death, what is the significance of the study?
Glucose is the starting substance for glycolysis, which is transported by the glucose transporter (GLUT) family across the cell membrane, targeting glucose transporters (GLUT) is an interesting target for potential cancer** interventions.
In a 2020 article, Gan Boyi's team and others found that cancer cells with high SLC7A11 expression are particularly sensitive to glucose transporter glut inhibitors. The GLUT inhibitors KL-11743 or BAY-876 effectively inhibit glucose uptake, similar to glucose starvation. In SLC7A11-overexpressing cells, Glut1 inhibitor treatment increased the NADP+ NADPH ratio (Figure 6A,B) and elicited intense cell death in UMRC6 cells (Figure 6C). In addition, GLUT inhibition induces the collapse of the disulfide-bonded actin backbone protein and F-actin networks.
In animal models, BAY876** reduced the growth of SLC7A11HIGH NCI-H226 xenograft tumors (Fig. 6D), BAY-876-treated tumors exhibited frequent cell death (Fig. 6E), and BAY-876-treated tumors exhibited more disulfide bonding in actin cytoskeletal proteins. These results suggest that:: Glut inhibitors induce disulfide status and cell death in SLC7A11High cancer cells, and disulfide death in cancer cells may be a key factor in mediating the efficacy of Glut inhibitors** SLC7A11High tumors.
Figure 6Glut inhibitor induction
SLC7A11 is highly expressed, cell death[5]A, glucose uptake levels. b.NADP+ NADPH ratio CProportion of dead cells. BAY-876 was treated with DFO, FER-1, Z-VAD, NEC-1, NEC-2 and CQ at indicated concentrations for 7h. d.Change in tumor volume over time in the NCI-H226 xenograft model. e.The weight of the NCI-H226 xenograft and HE staining and immunohistochemical staining of the NCI-H226 tumor region.
Summary
The authors' team found that in the case of high expression of SLC7A11, glucose starvation restricting PPP production of NADPH leads to the accumulation of small molecule disulfides (including cystine), causing a series of redox defects and cell death.
Figure 7Diagram of the mechanism of disulfide death.
And based on the understanding of the mechanism of disulfidetosis, it was also found that Glut inhibition-induced disulfidetis may be an effective strategy for tumors with high expression of SLC7A11, which often occur in human cancers[6]. The elucidation of a unique cell death mechanism called disulfideptosis provides a key framework for targeting ** cancers.
References
1. wenxin song, et al. solute carrier transporters: the metabolic gatekeepers of immune cells. acta pharm sin b. 2020 jan;10(1):61-78.
2. xiaoguang liu, yilei zhang , li zhuang, et al. nadph debt drives redox bankruptcy: slc7a11/xct-mediated cystine uptake as a double-edged sword in cellular redox regulation . genes dis. 2020 nov 25;8(6):731-745.
3. takeo goji, et al. cystine uptake through the cystine/glutamate antiporter xct triggers glioblastoma cell death under glucose deprivation. j biol chem. 2017 dec 1;292(48):19721-19732.
4. xiaoguang liu, kellen olszewski, yilei zhang, et al. cystine transporter regulation of pentose phosphate pathway dependency and disulfide stress exposes a targetable metabolic vulnerability in cancer. nat cell biol. 2020 apr;22(4):476-486.
5. xiaoguang liu, litong nie, et al. actin cytoskeleton vulnerability to disulfide stress mediates disulfidptosis. nat cell biol. 2023 feb 6.
6. pran**i koppula, li zhuang, boyi gan. cystine transporter slc7a11/xct in cancer: ferroptosis, nutrient dependency, and cancer therapy. protein cell. 2021 aug;12(8):599-620.