Hypoxia blocks ferroptosis of hepatocellular carcinoma via suppression of METTL14 triggered YTHDF2-dependent silencing of SLC7A11

Residue hepatocellular carcinoma (HCC) cells enduring hypoxic environment triggered by interventional embolization obtain more malignant potential with little clarified mechanism. The N⁶-methyladenosine (m⁶A) biological activity plays essential roles in diverse physiological processes. However, its role under hypoxic condition remains largely unexplored. RT-qPCR and Western blot were used to evaluate METTL14 expression in hypoxic HCC cells. MDA assay and electronic microscopy photography were used to evaluate ferroptosis. The correlation between SLC7A11 and METTL14 was conducted by bioinformatical analysis. Flow cytometry was used to verify the effect of SLC7A11 on ROS production. Cell counting kit-8 assay was performed to detect cells proliferation ability. Hypoxia triggered suppression of METTL14 in a HIF-1α–dependent manner potently abrogated ferroptosis of HCC cells. Mechanistic investigation identified SLC7A11 was a direct target of METTL14. Both in vitro and in vivo assay demonstrated that METTL14 induced m⁶A modification at 5’UTR of SLC7A11 mRNA, which in turn underwent degradation relied on the YTHDF2-dependent pathway. Importantly, ectopic expression of SLC7A11 strongly blocked METTL14-induced tumour-suppressive effect in hypoxic HCC. Our investigations lay the emphasis on the hypoxia-regulated ferroptosis in HCC cells and identify the HIF-1α /METTL14/YTHDF2/SLC7A11 axis as a potential therapeutic target for the HCC interventional embolization treatment.     

泛素化在调控铁死亡中的作用

铁死亡是一种由脂质过氧化驱动的铁依赖性的新的细胞死亡方式,越来越多的证据表明,铁死亡与各种病理状态有关,如神经退行性疾病、糖尿病肾病、癌症等,脂质过氧化驱动的铁死亡可能促进或抑制这些疾病的发生发展,细胞中抗氧化系统通过抑制脂质过氧化在抵抗铁死亡过程中发挥着重要作用。铁死亡的关键通路有以SLC7A11-GPX4为关键分子的氨基酸代谢通路、以铁蛋白或转铁蛋白为主的铁代谢通路,以及脂质代谢通路。铁死亡的发生受到细胞内蛋白质的调节,这些蛋白质会发生各种翻译后修饰,包括泛素化修饰。泛素-蛋白酶体系统（ubiquitin-proteasome system,UPS）是细胞内主要降解系统之一,通过酶促级联反应催化泛素分子标记待降解蛋白,随后由蛋白酶体识别并降解目标蛋白质。UPS根据其降解底物的不同在调节铁死亡的反应中发挥双重作用。UPS通过促进铁死亡关键分子（如SLC7A11、GPX4、GSH）以及抗氧化系统成分（如NRF2）的泛素化降解从而促进铁死亡,也可以通过促进脂质代谢通路中相关分子（如ACSL4、ALOX15）的泛素化降解从而抑制铁死亡。本综述介绍泛素化修饰在调控铁死亡进程中作用的最新研究进展,总结了已发表的关于E3泛素连接酶和去泛素酶调控铁死亡的研究,归纳了泛素连接酶、去泛素酶调控铁死亡的作用靶点,有助于确定人类疾病中新的预后指标,为这些疾病提供潜在的治疗策略。     

PHGDH Inhibits Ferroptosis and Promotes Malignant Progression by Upregulating SLC7A11 in Bladder Cancer

Background: Bladder cancer (BCa) is a prevalent urologic malignancy that shows a poor prognosis. Abnormal metabolism and its key genes play a critical role in BCa progression. In this study, the role played by PhosphoGlycerol Dehydrogenase (PHGDH), an important molecule of serine metabolism, was investigated with regard to the regulation of ferroptosis in BCa.Methods: The BCa tissues of 90 patients were analyzed by RNA-sequencing for differential pathways and genes. Western blot, qPCR, and IHC were used to determine PHGDH expression in the cell lines (in vitro) and patient tissues (in vivo). R software was used to analyze PHGDH expression, prognosis, and PHGDH+SLC7A11 score. The biological functions of PHGDH were examined through organoids, and in vitro and in vivo experiments. C11 probes, electron microscopy, and ferroptosis inhibitors/ inducers were used to detect cellular ferroptosis levels. Protein profiling, co-IP, and RIP assays were used to screen proteins that might bind to PHGDH. PHGDH-targeted inhibitor NCT-502 was used to evaluate its effect on BCa cells.Results: PHGDH was highly expressed in patients with BCa. Knock-down of PHGDH promoted ferroptosis, while the decreased proliferation of BCa cells. Additionally, PHGDH knock-down downregulated the expression of SLC7A11. Co-IP and mass spectrometry experiments indicate that PHGDH binds to PCBP2, an RNA-binding protein, and inhibits its ubiquitination degradation. PCBP2 in turn stabilizes SLC7A11 mRNA and increases its expression. NCT-502, a PHGDH inhibitor, promotes ferroptosis and inhibits tumor progression in BCa. The PHGDH+ SLC7A11 score was significantly correlated with patient prognosis.Conclusions: To conclude, the PHGDH, via interaction with PCBP2, upregulates SLC7A11 expression. This inhibits ferroptosis and promotes the malignant progression of BCA. The results of this study indicated that NCT-502 could serve as a therapeutic strategy for BCa.     

Tyrosine phosphorylation disrupts elongin interaction and accelerates SOCS3 degradation

The suppressors of cytokine signaling (SOCS) are negative feedback inhibitors of cytokine and growth factor-induced signal transduction. The C-terminal SOCS box region is thought to regulate SOCS protein stability most likely via an elongin C interaction. In the present study, we have found that phosphorylation of SOCS3 at two tyrosine residues in the conserved SOCS box, Tyr204 and Tyr221, can inhibit the SOCS3-elongin C interaction and activate proteasome-mediated SOCS3 degradation. Jak-mediated phosphorylation of SOCS3 decreased SOCS3 protein half-life, and phosphorylation of both Tyr204 and Tyr221 was required to fully destabilize SOCS3. In contrast, a phosphorylation-deficient mutant of SOCS3, Y204F,Y221F, remained stable in the presence of activated Jak2 and receptor tyrosine kinases. SOCS3 stability correlated with the relative amount that bound elongin C, because in vitro phosphorylation of a SOCS3-glutathione S-transferase fusion protein abolished its ability to interact with elongin C. In addition, a SOCS3/SOCS1 chimera that co-precipitates with markedly increased elongin C, was significantly more stable than wild-type SOCS3. The data suggest that interaction with elongin C stabilizes SOCS3 protein expression and that phosphorylation of SOCS box tyrosine residues disrupts the complex and enhances proteasome-mediated degradation of SOCS3.     

Ferroptosis in calcium oxalate kidney stone formation and the possible regulatory mechanism of ANKRD1

The objective of this study was to explore the role of ferroptosis in the formation of calcium oxalate (CaOx) kidney stones and the regulatory mechanism of the ankyrin repeat domain 1 (ANKRD1) gene. The study found that the Nrf2/HO-1 and p53/SLC7A11 signaling pathways were activated in the kidney stone model group, and the expression of the ferroptosis marker proteins SLC7A11 and GPX4 was significantly reduced, while the expression of ACSL4 was significantly increased. The expression of the iron transport-related proteins CP and TF increased significantly, and Fe²⁺ accumulated in the cell. The expression of HMGB1 increased significantly. In addition, the level of intracellular oxidative stress was increased. The gene with the most significant difference caused by CaOx crystals in HK-2 cells was ANKRD1. Silencing or overexpression of ANKRD1 by lentiviral infection technology regulated the expression of the p53/SLC7A11 signaling pathway, which regulated the ferroptosis induced by CaOx crystals. In conclusion, CaOx crystals can mediate ferroptosis through the Nrf2/HO-1 and p53/SLC7A11 pathways, thereby weakening the resistance of HK-2 cells to oxidative stress and other unfavorable factors, enhancing cell damage, and increasing crystal adhesion and CaOx crystal deposition in the kidney. ANKRD1 participates in the formation and development of CaOx kidney stones by activating ferroptosis mediated by the p53/SLC7A11 pathway.     

Targeting Lyn tyrosine kinase through protein fusions encompassing motifs of Cbp (Csk-binding protein) and the SOCS box of SOCS1

The tyrosine kinase Lyn is involved in oncogenic signalling in several leukaemias and solid tumours, and we have previously identified a pathway centred on Cbp [Csk (C-terminal Src kinase)-binding protein] that mediates both enzymatic inactivation, as well as proteasomal degradation of Lyn via phosphorylation-dependent recruitment of Csk (responsible for phosphorylating the inhibitory C-terminal tyrosine of Lyn) and SOCS1 (suppressor of cytokine signalling 1; an E3 ubiquitin ligase). In the present study we show that fusing specific functional motifs of Cbp and domains of SOCS1 together generates a novel molecule capable of directing the proteasomal degradation of Lyn. We have characterized the binding of pY (phospho-tyrosine) motifs of Cbp to SFK (Src-family kinase) SH2 (Src homology 2) domains, identifying those with high affinity and specificity for the SH2 domain of Lyn and that are preferred substrates of active Lyn. We then fused them to the SB (SOCS box) of SOCS1 to facilitate interaction with the ubiquitination-promoting elongin B/C complex. As an eGFP (enhanced green fluorescent protein) fusion, these proteins can direct the polyubiquitination and proteasomal degradation of active Lyn. Expressing this fusion protein in DU145 cancer cells (but not LNCaP or MCF-7 cells), that require Lyn signalling for survival, promotes loss of Lyn, loss of caspase 3, appearance of an apoptotic morphology and failure to survive/expand. These findings show how functional domains of Cbp and SOCS1 can be fused together to generate molecules capable of inhibiting the growth of cancer cells that express high levels of active Lyn.     

Targeting Ferroptosis as a Novel Approach to Alleviate Aortic Dissection

A variety of programmed cell death types have been shown to participate in the loss of smooth muscle cells (SMCs) during the development of aortic dissection (AD), but it is still largely unclear whether ferroptosis is involved in the development of AD. In the present study, we found that the expression of key ferroptosis regulatory proteins, solute carrier family 7 member 11 (SLC7A11), ferroptosis suppressor protein 1 (FSP1) and glutathione peroxidase 4 (GPX4) were downregulated in aortas of Stanford type A AD (TAAD) patients, and liproxstatin-1, a specific inhibitor of ferroptosis, obviously abolished the β-aminopropionitrile (BAPN)-induced development and rupture of AD in mice. Furthermore, the expression of methyltransferase-like 3 (METTL3), a major methyltransferase of RNA m⁶A, was remarkably upregulated in the aortas of TAAD patients, and the protein levels of METTL3 were negatively correlated with SLC7A11 and FSP1 levels in human aortas. Overexpression of METTL3 in human aortic SMCs (HASMCs) inhibited, while METTL3 knockdown promoted SLC7A11 and FSP1 expression. More importantly, overexpression of METTL3 facilitated imidazole ketone erastin- and cystine deprivation-induced ferroptosis, while knockdown of METTL3 repressed ferroptosis of HASMCs. Overexpression of either SLC7A11 or FSP1 largely abrogated the effect of METTL3 on HASMC ferroptosis. Therefore, we have revealed that ferroptosis is a critical cause of AD in both humans and mice and that METTL3 promotes ferroptosis of HASMCs by inhibiting the expression of SLC7A11 and FSP1. Thus, targeting ferroptosis or m⁶A RNA methylation is a potential novel strategy for the treatment of AD.     

SOCS2 can enhance interleukin-2 (IL-2) and IL-3 signaling by accelerating SOCS3 degradation

Cytokine responses can be regulated by a family of proteins termed suppressors of cytokine signaling (SOCS) which can inhibit the JAK/STAT pathway in a classical negative-feedback manner. While the SOCS are thought to target signaling intermediates for degradation, relatively little is known about how their turnover is regulated. Unlike other SOCS family members, we find that SOCS2 can enhance interleukin-2 (IL-2)- and IL-3-induced STAT phosphorylation following and potentiate proliferation in response to cytokine stimulation. As a clear mechanism for these effects, we demonstrate that expression of SOCS2 results in marked proteasome-dependent reduction of SOCS3 and SOCS1 protein expression. Furthermore, we provide evidence that this degradation is dependent on the presence of an intact SOCS box and that the loss of SOCS3 is enhanced by coexpression of elongin B/C. This suggests that SOCS2 can bind to SOCS3 and elongin B/C to form an E3 ligase complex resulting in the degradation of SOCS3. Therefore, SOCS2 can enhance cytokine responses by accelerating proteasome-dependent turnover of SOCS3, suggesting a mechanism for the gigantism observed in SOCS2 transgenic mice.     

Structure of the SOCS4-ElonginB/C complex reveals a distinct SOCS box interface and the molecular basis for SOCS-dependent EGFR degradation

Tyrosine kinase signaling is tightly controlled by negative feedback inhibitors including suppressors of cytokine signaling (SOCS). SOCS assemble as SH2 domain substrate recognition modules in ElonginB/C-cullin ubiquitin ligases. In accordance, SOCS4 reduces STAT3 signaling from EGFR through increased receptor degradation. Variable C-termini in SOCS4-SOCS7 exclude these family members from a SOCS2-type domain arrangement in which a strictly conserved C terminus determines domain packing. The structure of the SOCS4-ElonginC-ElonginB complex reveals a distinct SOCS structural class. The N-terminal ESS helix functionally replaces the CIS/SOCS1-SOCS3 family C terminus in a distinct SH2-SOCS box interface that facilitates further interdomain packing between the extended N- and C-terminal regions characteristic for this subfamily. Using peptide arrays and calorimetry the STAT3 site in EGFR (pY(1092)) was identified as a high affinity SOCS4 substrate (K(D) = 0.5 microM) revealing a mechanism for EGFR degradation. SOCS4 also bound JAK2 and KIT with low micromolar affinity, whereas SOCS2 was specific for GH-receptor.     

Resveratrol inhibits ferroptosis and decelerates heart failure progression via Sirt1/p53 pathway activation

Resveratrol is an organic compound widely studied for its therapeutic uses. We investigated whether resveratrol exerts cardioprotective effects by inhibiting ferroptosis via the Sirt1/p53 pathway. A heart failure model was established by aortic coarctation in Sirt1 knockout mice. The superoxide dismutase (SOD), glutathione (GSH) levels and mitochondrial morphology in murine heart tissues were assessed at different time points to determine the role of ferroptosis in heart failure progression. The cardiac function of mice with heart failure was evaluated by determining the brain natriuretic peptide (BNP) and sST2 concentration and conducting echocardiography. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were transfected with the p53 K382R mutant and Sirt1 interference lentiviral vectors. Immunoprecipitation (IP) experiments were performed to investigate whether Sirt1 influences ferroptosis via p53 K382 acetylation and SLC7A11 expression modulation. Resveratrol improved cardiac function in mice and decelerated ferroptosis and fibrosis progression in heart failure. However, the ability of resveratrol to prevent ferroptosis and treat heart failure was lost after silencing Sirt1. Sirt1 reduced ferroptosis by diminishing the levels of p53 K382 acetylation, reducing the degradation of SLC7A11, and increasing the levels of GSH and glutathione peroxidase 4 (GPX4) in cells. In conclusion, by activating the Sirt1/p53 pathway in heart failure, resveratrol decreased the depletion of SLC7A11, inhibited ferroptosis, and improved cardiac function.     

Cloning and characterization of the genes encoding the ankyrin repeat and SOCS box-containing proteins Asb-1, Asb-2, Asb-3 and Asb-4

Members of the suppressor of cytokine signalling (SOCS) family of proteins have been shown to inhibit cytokine signalling via direct interactions with JAK kinases or activated cytokine receptors. In addition to their novel amino-terminal regions and SH2 domains that mediate these interactions, the SOCS proteins also contain carboxy-terminal regions of homology called the SOCS box. The SOCS box serves to couple SOCS proteins and their binding partners with the elongin B and C complex, possibly targeting them for degradation. Several other families of proteins also contain SOCS boxes but differ from the SOCS proteins in the type of domain or motif they contain upstream of the SOCS box. We report here the cloning, characterization, mapping and expression analysis of four members of the ankyrin repeat and SOCS box-containing (Asb) protein family.     

SOCS-1 and SOCS-3 block insulin signaling by ubiquitin-mediated degradation of IRS1 and IRS2

Inflammation associates with peripheral insulin resistance, which dysregulates nutrient homeostasis and leads to diabetes. Inflammation induces the expression of SOCS proteins. We show that SOCS1 or SOCS3 targeted IRS1 and IRS2, two critical signaling molecules for insulin action, for ubiquitin-mediated degradation. SOCS1 or SOCS3 bound both recombinant and endogenous IRS1 and IRS2 and promoted their ubiquitination and subsequent degradation in multiple cell types. Mutations in the conserved SOCS box of SOCS1 abrogated its interaction with the elongin BC ubiquitin-ligase complex without affecting its binding to IRS1 or IRS2. The SOCS1 mutants also failed to promote the ubiquitination and degradation of either IRS1 or IRS2. Adenoviral-mediated expression of SOCS1 in mouse liver dramatically reduced hepatic IRS1 and IRS2 protein levels and caused glucose intolerance; by contrast, expression of the SOCS1 mutants had no effect. Thus, SOCS-mediated degradation of IRS proteins, presumably via the elongin BC ubiquitin-ligase, might be a general mechanism of inflammation-induced insulin resistance, providing a target for therapy.     

Mesenchymal stem cells protect against ferroptosis via exosome-mediated stabilization of SLC7A11 in acute liver injury

Mesenchymal stem cells (MSCs) have attracted interest for their potential to alleviate liver injury. Here, the protective effect of MSCs on carbon tetrachloride (CCl₄)-induced acute liver injury (ALI) was investigated. In this study, we illustrated a novel mechanism that ferroptosis, a newly recognized form of regulated cell death, contributed to CCl₄-induced ALI. Subsequently, based on the in vitro and in vivo evidence that MSCs and MSC-derived exosomes (MSC-Exo) treatment achieved pathological remission and inhibited the production of lipid peroxidation, we proposed an MSC-based therapy for CCl₄-induced ALI. More intriguingly, treatment with MSCs and MSC-Exo downregulated the mRNA level of prostaglandin-endoperoxide synthase 2 (Ptgs2) and lipoxygenases (LOXs) while it restored the protein level of SLC7A11 in primary hepatocytes and mouse liver, indicating that the inhibition of ferroptosis partly accounted for the protective effect of MSCs and MSC-Exo on ALI. We further revealed that MSC-Exo-induced expression of SLC7A11 protein was accompanied by increasing of CD44 and OTUB1. The aberrant expression of ubiquitinated SLC7A11 triggered by CCl₄ could be rescued with OTUB1-mediated deubiquitination, thus strengthening SLC7A11 stability and thereby leading to the activation of system X_C⁻ to prevent CCl₄-induced hepatocyte ferroptosis. In conclusion, we showed that MSC-Exo had a protective role against ferroptosis by maintaining SLC7A11 function, thus proposing a novel therapeutic strategy for ferroptosis-induced ALI.Subject terms: Hepatotoxicity, Experimental models of disease

Schematic diagram of the protective effect of MSC-derived exosomes on maintaining SLC7A11 function during ferroptosis involved in CCl₄-induced ALI.     

The SOCS-Box of HIV-1 Vif Interacts with ElonginBC by Induced-Folding to Recruit Its Cul5-Containing Ubiquitin Ligase Complex

The HIV-1 viral infectivity factor (Vif) protein recruits an E3 ubiquitin ligase complex, comprising the cellular proteins elongin B and C (EloBC), cullin 5 (Cul5) and RING-box 2 (Rbx2), to the anti-viral proteins APOBEC3G (A3G) and APOBEC3F (A3F) and induces their polyubiquitination and proteasomal degradation. In this study, we used purified proteins and direct in vitro binding assays, isothermal titration calorimetry and NMR spectroscopy to describe the molecular mechanism for assembly of the Vif-EloBC ternary complex. We demonstrate that Vif binds to EloBC in two locations, and that both interactions induce structural changes in the SOCS box of Vif as well as EloBC. In particular, in addition to the previously established binding of Vif''s BC box to EloC, we report a novel interaction between the conserved Pro-Pro-Leu-Pro motif of Vif and the C-terminal domain of EloB. Using cell-based assays, we further show that this interaction is necessary for the formation of a functional ligase complex, thus establishing a role of this motif. We conclude that HIV-1 Vif engages EloBC via an induced-folding mechanism that does not require additional co-factors, and speculate that these features distinguish Vif from other EloBC specificity factors such as cellular SOCS proteins, and may enhance the prospects of obtaining therapeutic inhibitors of Vif function.     

Crystal Structure of <Emphasis Type="Italic">Human</Emphasis> ASB9-2 and Substrate-Recognition of CKB

Human ankyrin repeat and suppressor of cytokine signaling box protein 9 (hASB9) is a specific substrate-recognition subunit of an elongin C-cullin-SOCS box E3 ubiquitin ligase complex. It recognizes its substrate, brain type creatine kinase (CKB), using the ankyrin repeat domain; and facilitates the polyubiquitination of CKB to mediate proteasomal degradation through the SOCS box domain. HASB9-2 is an isoform of hASB9 that contains one ankyrin repeat domain. In this study, the crystal structure of hASB9-2 is shown at 2.2-Å resolution using molecular replacement. Overall, hASB9-2 forms a slightly curved arch with a characteristic L-shaped cross-section. Amino acid substitution analysis based on docking experiments revealed that His103 and Phe107 in hASB9-2 are essential for binding to CKB. Analysis of truncation mutants demonstrated that the first six ankyrin repeats along with the N-terminal region of hASB9-2 contribute to the interaction with CKB.     

The E3 ubiquitin ligase HOIL-1 induces the polyubiquitination and degradation of SOCS6 associated proteins

The suppressor of cytokine signaling (SOCS) proteins are thought to exert their function through the recruitment of interacting-proteins to the ubiquitin/proteasome degradation pathway. All SOCS proteins bind an Elongin BC E3 ubiquitin ligase complex through the common Socs-box. Here, we show that haem-oxidized IRP2 ubiquitin ligase-1 (HOIL-1), another E3 ubiquitin ligase, interacts with SOCS6. The Ubl domain of HOIL-1 and the SH2 and Socs-box domains of SOCS6 are required for the interaction. HOIL-1 expression stabilizes SOCS6 and induces the ubiquitination and degradation of proteins associated with SOCS6. These data suggest that SOCS proteins may interact with different E3 ubiquitin ligases in addition to a common Elongin BC E3 complex.