SnapShot: Ferroptosis.

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.

Non-canonical substrate recognition by the human WDR26-CTLH E3 ligase regulates prodrug metabolism.

The yeast glucose-induced degradation-deficient (GID) E3 ubiquitin ligase forms a suite of complexes with interchangeable receptors that selectively recruit N-terminal degron motifs of metabolic enzyme substrates. The orthologous higher eukaryotic C-terminal to LisH (CTLH) E3 complex has been proposed to also recognize substrates through an alternative subunit, WDR26, which promotes the formation of supramolecular CTLH E3 assemblies. Here, we discover that human WDR26 binds the metabolic enzyme nicotinamide/nicotinic-acid-mononucleotide-adenylyltransferase 1 (NMNAT1) and mediates its CTLH E3-dependent ubiquitylation independently of canonical GID/CTLH E3-family substrate receptors. The CTLH subunit YPEL5 inhibits NMNAT1 ubiquitylation and cellular turnover by WDR26-CTLH E3, thereby affecting NMNAT1-mediated metabolic activation and cytotoxicity of the prodrug tiazofurin. Cryoelectron microscopy (cryo-EM) structures of NMNAT1- and YPEL5-bound WDR26-CTLH E3 complexes reveal an internal basic degron motif of NMNAT1 essential for targeting by WDR26-CTLH E3 and degron mimicry by YPEL5's N terminus antagonizing substrate binding. Thus, our data provide a mechanistic understanding of how YPEL5-WDR26-CTLH E3 acts as a modulator of NMNAT1-dependent metabolism.

Inhibition of CPAP-tubulin interaction prevents proliferation of centrosome-amplified cancer cells.

Centrosome amplification is a hallmark of human cancers that can trigger cancer cell invasion. To survive, cancer cells cluster amplified extra centrosomes and achieve pseudobipolar division. Here, we set out to prevent clustering of extra centrosomes. Tubulin, by interacting with the centrosomal protein CPAP, negatively regulates CPAP-dependent peri-centriolar material recruitment, and concurrently microtubule nucleation. Screening for compounds that perturb CPAP-tubulin interaction led to the identification of CCB02, which selectively binds at the CPAP binding site of tubulin. Genetic and chemical perturbation of CPAP-tubulin interaction activates extra centrosomes to nucleate enhanced numbers of microtubules prior to mitosis. This causes cells to undergo centrosome de-clustering, prolonged multipolar mitosis, and cell death. 3D-organotypic invasion assays reveal that CCB02 has broad anti-invasive activity in various cancer models, including tyrosine kinase inhibitor (TKI)-resistant EGFR-mutant non-small-cell lung cancers. Thus, we have identified a vulnerability of cancer cells to activation of extra centrosomes, which may serve as a global approach to target various tumors, including drug-resistant cancers exhibiting high incidence of centrosome amplification.

ABCA3 Deficiency-Variant-Specific Response to Hydroxychloroquine.

Biallelic variants in ABCA3, the gene encoding the lipid transporter ATP-binding cassette subfamily A member 3 (ABCA3) that is predominantly expressed in alveolar type II cells, may cause interstitial lung diseases in children (chILD) and adults. Currently, there is no proven therapy, but, frequently, hydroxychloroquine (HCQ) is used empirically. We hypothesized that the in vitro responsiveness to HCQ might correlate to patients' clinical outcomes from receiving HCQ therapy. The clinical data of the subjects with chILD due to ABCA3 deficiency and treated with HCQ were retrieved from the literature and the Kids Lung Register data base. The in vitro experiments were conducted on wild type (WT) and 16 mutant ABCA3-HA-transfected A549 cells. The responses of the functional read out were assessed as the extent of deviation from the untreated WT. With HCQ treatment, 19 patients had improved or unchanged respiratory conditions, and 20 had respiratory deteriorations, 5 of whom transiently improved then deteriorated. The in vitro ABCA3 functional assays identified two variants with complete response, five with partial response, and nine with no response to HCQ. The variant-specific HCQ effects in vivo closely correlated to the in vitro data. An ABCA3+ vesicle volume above 60% of the WT volume was linked to responsiveness to HCQ; the HCQ treatment response was concentration dependent and differed for variants in vitro. We generated evidence for an ABCA3 variant-dependent impact of the HCQ in vitro. This may also apply for HCQ treatment in vivo, as supported by the retrospective and uncontrolled data from the treatment of chILD due to ABCA3 deficiency.

High-Content Screening Identifies Cyclosporin A as a Novel ABCA3-Specific Molecular Corrector.

ABCA3 (ATP-binding cassette subfamily A member 3) is a lipid transporter expressed in alveolar type II cells and localized in the limiting membrane of lamellar bodies. It is crucial for pulmonary surfactant storage and homeostasis. Mutations in the ABCA3 gene are the most common genetic cause of respiratory distress syndrome in mature newborns and of interstitial lung disease in children. Apart from lung transplant, there is no cure available. To address the lack of causal therapeutic options for ABCA3 deficiency, a rapid and reliable approach is needed to investigate variant-specific molecular mechanisms and to identify pharmacologic modulators for monotherapies or combination therapies. To this end, we developed a phenotypic cell-based assay to autonomously identify ABCA3 wild-type-like or mutant-like cells by using machine learning algorithms aimed at identifying morphologic differences in wild-type and mutant cells. The assay was subsequently used to identify new drug candidates for ABCA3-specific molecular correction by using high-content screening of 1,280 Food and Drug Administration-approved small molecules. Cyclosporin A was identified as a potent corrector, specific for some but not all ABCA3 variants. Results were validated by using our previously established functional small-format assays. Hence, cyclosporin A may be selected for orphan drug evaluation in controlled repurposing trials in patients.

Mitochondrial Alkbh1 localizes to mtRNA granules and its knockdown induces the mitochondrial UPR in humans and C. elegans.

The Fe(II) and 2-oxoglutarate-dependent oxygenase Alkb homologue 1 (Alkbh1) has been shown to act on a wide range of substrates, like DNA, tRNA and histones. Thereby different enzymatic activities have been identified including, among others, demethylation of N3-methylcytosine (m3C) in RNA- and single-stranded DNA oligonucleotides, demethylation of N1-methyladenosine (m1A) in tRNA or formation of 5-formyl cytosine (f5C) in tRNA. In accordance with the different substrates, Alkbh1 has also been proposed to reside in distinct cellular compartments in human and mouse cells, including the nucleus, cytoplasm and mitochondria. Here, we describe further evidence for a role of human Alkbh1 in regulation of mitochondrial protein biogenesis, including visualizing localization of Alkbh1 into mitochondrial RNA granules with super-resolution 3D SIM microscopy. Electron microscopy and high-resolution respirometry analyses revealed an impact of Alkbh1 level on mitochondrial respiration, but not on mitochondrial structure. Downregulation of Alkbh1 impacts cell growth in HeLa cells and delays development in Caenorhabditis elegans, where the mitochondrial role of Alkbh1 seems to be conserved. Alkbh1 knockdown, but not Alkbh7 knockdown, triggers the mitochondrial unfolded protein response (UPRmt) in C. elegans.

The E3 ligase parkin maintains mitochondrial integrity by increasing linear ubiquitination of NEMO.

Parkin, a RING-between-RING-type E3 ubiquitin ligase associated with Parkinson's disease, has a wide neuroprotective activity, preventing cell death in various stress paradigms. We identified a stress-protective pathway regulated by parkin that links NF-κB signaling and mitochondrial integrity via linear ubiquitination. Under cellular stress, parkin is recruited to the linear ubiquitin assembly complex and increases linear ubiquitination of NF-κB essential modulator (NEMO), which is essential for canonical NF-κB signaling. As a result, the mitochondrial guanosine triphosphatase OPA1 is transcriptionally upregulated via NF-κB-responsive promoter elements for maintenance of mitochondrial integrity and protection from stress-induced cell death. Parkin-induced stress protection is lost in the absence of either NEMO or OPA1, but not in cells defective for the mitophagy pathway. Notably, in parkin-deficient cells linear ubiquitination of NEMO, activation of NF-κB, and upregulation of OPA1 are significantly reduced in response to TNF-α stimulation, supporting the physiological relevance of parkin in regulating this antiapoptotic pathway.

A high-content screen for small-molecule regulators of epithelial cell-adhesion molecule (EpCAM) cleavage yields a robust inhibitor.

Epithelial cell-adhesion molecule (EpCAM) is a transmembrane protein that regulates cell cycle progression and differentiation and is overexpressed in many carcinomas. The EpCAM-induced mitogenic cascade is activated via regulated intramembrane proteolysis (RIP) of EpCAM by ADAM and γ-secretases, generating the signaling-active intracellular domain EpICD. Because of its expression pattern and molecular function, EpCAM is a valuable target in prognostic and therapeutic approaches for various carcinomas. So far, several immunotherapeutic strategies have targeted the extracellular domain of EpCAM. However, targeting the intracellular signaling cascade of EpCAM holds promise for specifically interfering with EpCAM's proliferation-stimulating signaling cascade. Here, using a yellow fluorescence protein-tagged version of the C-terminal fragment of EpCAM, we established a high-content screening (HCS) of a small-molecule compound library (n = 27,280) and characterized validated hits that target EpCAM signaling. In total, 128 potential inhibitors were initially identified, of which one compound with robust inhibitory effects on RIP of EpCAM was analyzed in greater detail. In summary, our study demonstrates that the development of an HCS for small-molecule inhibitors of the EpCAM signaling pathway is feasible. We propose that this approach may also be useful for identifying chemical compounds targeting other disorders involving membrane cleavage-dependent signaling pathways.

Targeting TRAF6 E3 ligase activity with a small-molecule inhibitor combats autoimmunity.

Constitutive NF-κB signaling represents a hallmark of chronic inflammation and autoimmune diseases. The E3 ligase TNF receptor-associated factor 6 (TRAF6) acts as a key regulator bridging innate immunity, pro-inflammatory cytokines, and antigen receptors to the canonical NF-κB pathway. Structural analysis and point mutations have unraveled the essential role of TRAF6 binding to the E2-conjugating enzyme ubiquitin-conjugating enzyme E2 N (Ubc13 or UBE2N) to generate Lys63-linked ubiquitin chains for inflammatory and immune signal propagation. Genetic mutations disrupting TRAF6-Ubc13 binding have been shown to reduce TRAF6 activity and, consequently, NF-κB activation. However, to date, no small-molecule modulator is available to inhibit the TRAF6-Ubc13 interaction and thereby counteract NF-κB signaling and associated diseases. Here, using a high-throughput small-molecule screening approach, we discovered an inhibitor of the TRAF6-Ubc13 interaction that reduces TRAF6-Ubc13 activity both in vitro and in cells. We found that this compound, C25-140, impedes NF-κB activation in various immune and inflammatory signaling pathways also in primary human and murine cells. Importantly, C25-140 ameliorated inflammation and improved disease outcomes of autoimmune psoriasis and rheumatoid arthritis in preclinical in vivo mouse models. Hence, the first-in-class TRAF6-Ubc13 inhibitor C25-140 expands the toolbox for studying the impact of the ubiquitin system on immune signaling and underscores the importance of TRAF6 E3 ligase activity in psoriasis and rheumatoid arthritis. We propose that inhibition of TRAF6 activity by small molecules represents a promising novel strategy for targeting autoimmune and chronic inflammatory diseases.

E1B-55K-Mediated Regulation of RNF4 SUMO-Targeted Ubiquitin Ligase Promotes Human Adenovirus Gene Expression.

Human adenovirus (HAdV) E1B-55K is a multifunctional regulator of productive viral replication and oncogenic transformation in nonpermissive mammalian cells. These functions depend on E1B-55K's posttranslational modification with the SUMO protein and its binding to HAdV E4orf6. Both early viral proteins recruit specific host factors to form an E3 ubiquitin ligase complex that targets antiviral host substrates for proteasomal degradation. Recently, we reported that the PML-NB-associated factor Daxx represses efficient HAdV productive infection and is proteasomally degraded via a SUMO-E1B-55K-dependent, E4orf6-independent pathway, the details of which remained to be established. RNF4, a cellular SUMO-targeted ubiquitin ligase (STUbL), induces ubiquitinylation of specific SUMOylated proteins and plays an essential role during DNA repair. Here, we show that E1B-55K recruits RNF4 to the insoluble nuclear matrix fraction of the infected cell to support RNF4/Daxx association, promoting Daxx PTM and thus inhibiting this antiviral factor. Removing RNF4 from infected cells using RNA interference resulted in blocking the proper establishment of viral replication centers and significantly diminished viral gene expression. These results provide a model for how HAdV antagonize the antiviral host responses by exploiting the functional capacity of cellular STUbLs. Thus, RNF4 and its STUbL function represent a positive factor during lytic infection and a novel candidate for future therapeutic antiviral intervention strategies.IMPORTANCE Daxx is a PML-NB-associated transcription factor that was recently shown to repress efficient HAdV productive infection. To counteract this antiviral measurement during infection, Daxx is degraded via a novel pathway including viral E1B-55K and host proteasomes. This virus-mediated degradation is independent of the classical HAdV E3 ubiquitin ligase complex, which is essential during viral infection to target other host antiviral substrates. To maintain a productive viral life cycle, HAdV E1B-55K early viral protein inhibits the chromatin-remodeling factor Daxx in a SUMO-dependent manner. In addition, viral E1B-55K protein recruits the STUbL RNF4 and sequesters it into the insoluble fraction of the infected cell. E1B-55K promotes complex formation between RNF4- and E1B-55K-targeted Daxx protein, supporting Daxx posttranslational modification prior to functional inhibition. Hence, RNF4 represents a novel host factor that is beneficial for HAdV gene expression by supporting Daxx counteraction. In this regard, RNF4 and other STUbL proteins might represent novel targets for therapeutic intervention.

Luciferase Advisor: High-Accuracy Model To Flag False Positive Hits in Luciferase HTS Assays.

Firefly luciferase is an enzyme that has found ubiquitous use in biological assays in high-throughput screening (HTS) campaigns. The inhibition of luciferase in such assays could lead to a false positive result. This issue has been known for a long time, and there have been significant efforts to identify luciferase inhibitors in order to enhance recognition of false positives in screening assays. However, although a large amount of publicly accessible luciferase counterscreen data is available, to date little effort has been devoted to building a chemoinformatic model that can identify such molecules in a given data set. In this study we developed models to identify these molecules using various methods, such as molecular docking, SMARTS screening, pharmacophores, and machine learning methods. Among the structure-based methods, the pharmacophore-based method showed promising results, with a balanced accuracy of 74.2%. However, machine-learning approaches using associative neural networks outperformed all of the other methods explored, producing a final model with a balanced accuracy of 89.7%. The high predictive accuracy of this model is expected to be useful for advising which compounds are potential luciferase inhibitors present in luciferase HTS assays. The models developed in this work are freely available at the OCHEM platform at http://ochem.eu .

Modulation of human endogenous retrovirus (HERV) transcription during persistent and de novo HIV-1 infection.

BACKGROUND: The human genome contains multiple LTR elements including human endogenous retroviruses (HERVs) that together account for approximately 8-9% of the genomic DNA. At least 40 different HERV groups have been assigned to three major HERV classes on the basis of their homologies to exogenous retroviruses. Although most HERVs are silenced by a variety of genetic and epigenetic mechanisms, they may be reactivated by environmental stimuli such as exogenous viruses and thus may contribute to pathogenic conditions. The objective of this study was to perform an in-depth analysis of the influence of HIV-1 infection on HERV activity in different cell types. RESULTS: A retrovirus-specific microarray that covers major HERV groups from all three classes was used to analyze HERV transcription patterns in three persistently HIV-1 infected cell lines of different cellular origins and in their uninfected counterparts. All three persistently infected cell lines showed increased transcription of multiple class I and II HERV groups. Up-regulated transcription of five HERV taxa (HERV-E, HERV-T, HERV-K (HML-10) and two ERV9 subgroups) was confirmed by quantitative reverse transcriptase PCR analysis and could be reversed by knock-down of HIV-1 expression with HIV-1-specific siRNAs. Cells infected de novo by HIV-1 showed stronger transcriptional up-regulation of the HERV-K (HML-2) group than persistently infected cells of the same origin. Analysis of transcripts from individual members of this group revealed up-regulation of predominantly two proviral loci (ERVK-7 and ERVK-15) on chromosomes 1q22 and 7q34 in persistently infected KE37.1 cells, as well as in de novo HIV-1 infected LC5 cells, while only one single HML-2 locus (ERV-K6) on chromosome 7p22.1 was activated in persistently infected LC5 cells. CONCLUSIONS: Our results demonstrate that HIV-1 can alter HERV transcription patterns of infected cells and indicate a correlation between activation of HERV elements and the level of HIV-1 production. Moreover, our results suggest that the effects of HIV-1 on HERV activity may be far more extensive and complex than anticipated from initial studies with clinical material.

Dephosphorylation of Carma1 by PP2A negatively regulates T-cell activation.

The Carma1-Bcl10-Malt1 (CBM) complex bridges T-cell receptor (TCR) signalling to the canonical IκB kinase (IKK)/NF-κB pathway. NF-κB activation is triggered by PKCθ-dependent phosphorylation of Carma1 after TCR/CD28 co-stimulation. PKCθ-phosphorylated Carma1 was suggested to function as a molecular scaffold that recruits preassembled Bcl10-Malt1 complexes to the membrane. We have identified the serine-threonine protein phosphatase PP2A regulatory subunit Aα (PPP2R1A) as a novel interaction partner of Carma1. PPP2R1A is associated with Carma1 in resting as well as activated T cells in the context of the active CBM complex. By siRNA-mediated knockdown and in vitro dephosphorylation, we demonstrate that PP2A removes PKCθ-dependent phosphorylation of Ser645 in Carma1, and show that maintenance of this phosphorylation is correlated with increased T-cell activation. As a result of PP2A inactivation, we find that enhanced Carma1 S645 phosphorylation augments CBM complex formation, NF-κB activation and IL-2 or IFN-γ production after stimulation of Jurkat T cells or murine Th1 cells. Thus, our data define PP2A-mediated dephosphorylation of Carma1 as a critical step to limit T-cell activation and effector cytokine production.

A 3D-microtissue-based phenotypic screening of radiation resistant tumor cells with synchronized chemotherapeutic treatment.

BACKGROUND: Radiation resistance presents a challenge to the effective treatment of cancer. If therapeutic compounds were capable of resensitizing resistant tumours then a concurrent chemo-radiation treatment could be used to overcome radiation resistance. METHODS: We have developed a phenotypic assay to investigate the response of radiation resistant breast cancer cells grown in 3D-microtissue spheroids to combinations of radiation and established chemotherapeutic drugs. The effects were quantified by real time high content imaging of GFP detection area over 14 days. Ten established chemotherapeutic drugs were tested for their ability to enhance the effects of radiation. RESULTS: Of ten analysed chemotherapeutics, vinblastine was the most effective compound, with docetaxel and doxorubicine being less effective in combination with radiation. To investigate the response in a model closer to the in vivo situation we investigated the response of heterotypic 3D microtissues containing both fibroblasts and breast cancer cells. Drug treatment of these heterotypic 3D cultures confirmed treatment with radiation plus vinblastine to be additive in causing breast cancer growth inhibition. We have validated the screen by comparing radiation sensitizing effects of known chemotherapeutic agents. In both monotypic and heterotypic models the concurrent treatment of vinblastine and radiation proved more effective inhibitors of mammary cancer cell growth. The effective concentration range of both vinblastine and radiation are within the range used in treatment, suggesting the 3D model will offer a highly relevant screen for novel compounds. CONCLUSIONS: For the first time comfortable 3D cell-based phenotypic assay is available, that allows high throughput screening of compounds with radiation therapy modulating capacity, opening the field to drug discovery.

Heterogenous nuclear ribonucleoprotein Q increases protein expression from HIV-1 Rev-dependent transcripts.

BACKGROUND: Heterogenous nuclear ribonucleoproteins (hnRNPs) control many processes of the gene expression machinery including mRNA transcription, splicing, export, stability and translation. Recent data show interaction of the HIV-1 Rev regulatory protein with a subset of hnRNP proteins, that includes hnRNP Q, suggesting that hnRNPs can contribute to regulation of HIV-1 gene expression by Rev. FINDINGS: In this work we address the effect of hnRNP Q on Rev-dependent gene expression. We show that hnRNP Q overexpression increased levels of proteins produced from a Rev-dependent reporter gene in the presence of Rev. Increased protein levels did not correlate with changes in either the levels or the nucleocytoplasmic distribution of Rev-dependent reporter mRNAs. Similar observations were made in persistently HIV-1 infected HeLa cells. In these cells, hnRNP Q overexpression increased levels of the HIV-1 Gag-p24 protein, while levels of viral Rev-dependent mRNAs were not affected. CONCLUSION: Our data indicate that hnRNP Q can stimulate the protein production of Rev-dependent mRNAs without changing mRNA levels and mRNA export, respectively. This suggests that hnRNP Q can boost HIV gene expression at the level of protein production.

The therapeutic potential of targeting regulated non-apoptotic cell death.

Cell death is critical for the development and homeostasis of almost all multicellular organisms. Moreover, its dysregulation leads to diverse disease states. Historically, apoptosis was thought to be the major regulated cell death pathway, whereas necrosis was considered to be an unregulated form of cell death. However, research in recent decades has uncovered several forms of regulated necrosis that are implicated in degenerative diseases, inflammatory conditions and cancer. The growing insight into these regulated, non-apoptotic cell death pathways has opened new avenues for therapeutic targeting. Here, we describe the regulatory pathways of necroptosis, pyroptosis, parthanatos, ferroptosis, cuproptosis, lysozincrosis and disulfidptosis. We discuss small-molecule inhibitors of the pathways and prospects for future drug discovery. Together, the complex mechanisms governing these pathways offer strategies to develop therapeutics that control non-apoptotic cell death.

CellDeathPred: a deep learning framework for ferroptosis and apoptosis prediction based on cell painting.

Cell death, such as apoptosis and ferroptosis, play essential roles in the process of development, homeostasis, and pathogenesis of acute and chronic diseases. The increasing number of studies investigating cell death types in various diseases, particularly cancer and degenerative diseases, has raised hopes for their modulation in disease therapies. However, identifying the presence of a particular cell death type is not an obvious task, as it requires computationally intensive work and costly experimental assays. To address this challenge, we present CellDeathPred, a novel deep-learning framework that uses high-content imaging based on cell painting to distinguish cells undergoing ferroptosis or apoptosis from healthy cells. In particular, we incorporate a deep neural network that effectively embeds microscopic images into a representative and discriminative latent space, classifies the learned embedding into cell death modalities, and optimizes the whole learning using the supervised contrastive loss function. We assessed the efficacy of the proposed framework using cell painting microscopy data sets from human HT-1080 cells, where multiple inducers of ferroptosis and apoptosis were used to trigger cell death. Our model confidently separates ferroptotic and apoptotic cells from healthy controls, with an average accuracy of 95% on non-confocal data sets, supporting the capacity of the CellDeathPred framework for cell death discovery.

Farnesoid X receptor activation by bile acids suppresses lipid peroxidation and ferroptosis.

Ferroptosis is a regulated cell death modality that occurs upon iron-dependent lipid peroxidation. Recent research has identified many regulators that induce or inhibit ferroptosis; yet, many regulatory processes and networks remain to be elucidated. In this study, we performed a chemical genetics screen using small molecules with known mode of action and identified two agonists of the nuclear receptor Farnesoid X Receptor (FXR) that suppress ferroptosis, but not apoptosis or necroptosis. We demonstrate that in liver cells with high FXR levels, knockout or inhibition of FXR sensitized cells to ferroptotic cell death, whereas activation of FXR by bile acids inhibited ferroptosis. Furthermore, FXR inhibited ferroptosis in ex vivo mouse hepatocytes and human hepatocytes differentiated from induced pluripotent stem cells. Activation of FXR significantly reduced lipid peroxidation by upregulating the ferroptosis gatekeepers GPX4, FSP1, PPARα, SCD1, and ACSL3. Together, we report that FXR coordinates the expression of ferroptosis-inhibitory regulators to reduce lipid peroxidation, thereby acting as a guardian of ferroptosis.