Fas-associated Factor Research Articles

Evolution of the Cdk4/6-Cdkn2 system in invertebrates.

The cell cycle is driven by cyclin-dependent kinases (Cdks). The decision whether the cell cycle proceeds is made during G1 phase, when Cdk4/6 functions. Cyclin-dependent kinase inhibitor 2 (Cdkn2) is a specific inhibitor of Cdk4/6, and their interaction depends on D84 in Cdkn2 and R24/31 in Cdk4/6. This knowledge is based mainly on studies in mammalian cells. Here, we comprehensively analyzed Cdk4/6 and Cdkn2 in invertebrates and found that Cdk4/6 was present in most of the investigated phyla, but the distribution of Cdkn2 was rather uneven among and within the phyla. The positive charge of R24/R31 in Cdk4/6 was conserved in all analyzed species in phyla with Cdkn2. The presence of Cdkn2 and the conservation of the positive charge were statistically correlated. We also found that Cdkn2 has been tightly linked to Fas associated factor 1 (Faf1) during evolution. We discuss potential interactions between Cdkn2 and Cdk4/6 in evolution and the possible cause of the strong conservation of the microsynteny.

The balance between antiviral and antibacterial responses during M. tuberculosis infection is regulated by the ubiquitin ligase CBL.

As a first line of host defense, macrophages must be able to effectively sense and respond to diverse types of pathogens, and while a particular type of immune response may be beneficial in some circumstances, it can be detrimental in others. Upon infecting a macrophage, M. tuberculosis (Mtb) induces proinflammatory cytokines that activate antibacterial responses. Surprisingly, Mtb also triggers antiviral responses that actually hinder the ability of macrophages to control Mtb infection. The ubiquitin ligase CBL suppresses these antiviral responses and shifts macrophages toward a more antibacterial state during Mtb infection, however, the mechanisms by which CBL regulates immune signaling are unknown. We found that CBL controls responses to multiple stimuli and broadly suppresses the expression of antiviral effector genes. We then used mass-spectrometry to investigate potential CBL substrates and identified over 46,000 ubiquitylated peptides in Mtb-infected macrophages, as well as roughly 400 peptides with CBL-dependent ubiquitylation. We then performed genetic interaction analysis of CBL and its putative substrates, and identified the Fas associated factor 2 (FAF2) adapter protein as a key signaling molecule protein downstream of CBL. Together, these analyses identify thousands of new ubiquitin-mediated signaling events during the innate immune response and reveal an important new regulatory hub in this response.

Rat Pharmacokinetics and In Vitro Metabolite Identification of KM-819, a Parkinson's Disease Candidate, Using LC-MS/MS and LC-HRMS.

FAF1 (FAS-associated factor 1) is involved in the activation of Fas cell surface death receptors and plays a role in apoptosis and necrosis. In patients with Parkinson's disease, FAF1 is overexpressed in dopaminergic neurons in the substantia nigra. KM-819, an FAF1 inhibitor, has shown potential for preventing dopaminergic neuronal cell death, promoting the degradation of α-synuclein and preventing its accumulation. This study aimed to develop and validate a quantitative analytical method for determining KM-819 levels in rat plasma using liquid chromatography-tandem mass spectrometry. This method was then applied to pharmacokinetic (PK) studies in rats. The metabolic stability of KM-819 was assessed in rat, dog, and human hepatocytes. In vitro metabolite identification and metabolic pathways were investigated in rat, dog, and human hepatocytes. The structural analog of KM-819, namely N-[1-(4-bromobenzyl)-3,5-dimethyl-1H-pyrazol-4-yl]-2-(phenylsulfanyl) acetamide, served as the internal standard (IS). Proteins were precipitated from plasma samples using acetonitrile. Analysis was carried out using a reverse-phase C18 column with a mobile phase consisting of 0.1% formic acid in distilled water and 0.1% formic acid in acetonitrile. The analytical method developed for KM-819 exhibited linearity within the concentration range of 0.002-10 μg/mL in rat plasma. The precision and accuracy of the intra- and inter-day measurements were <15% for the lower limit of quantification (LLOQ) and all quality control samples. KM-819 demonstrated stability under various sample storage conditions (6 h at room temperature (25 °C), four weeks at -20 °C, three freeze-thaw cycles, and pretreated samples in the autosampler). The matrix effect and dilution integrity met the criteria set by the Food and Drug Administration and the European Medicines Agency. This sensitive, rapid, and reliable analytical method was successfully applied in pharmacokinetic studies in rats. Pharmacokinetic analysis revealed the dose-independent kinetics of KM-819 at 0.5-5 mg/kg, with a moderate oral bioavailability of ~20% in rats. The metabolic stability of KM-819 was also found to be moderate in rat, dog, and human hepatocytes. Metabolite identification in rat, dog, and human hepatocytes resulted in the discovery of six, six, and eight metabolites, respectively. Glucuronidation and mono-oxidation have been proposed as the major metabolic pathways. Overall, these findings contribute to a better understanding of the pharmacokinetic characteristics of KM-819, thereby aiding future clinical studies.

High Expression of Fas-Associated Factor 1 Indicates a Poor Prognosis in Non-Small-Cell Lung Cancer.

Fas-associated factor 1 (FAF1) is a death-promoting protein identified as an interaction partner of the death receptor Fas. The downregulation and mutation of FAF1 have been reported in a variety of human tumors, but there have been few studies on lung cancer. Here, we investigated the prognostic significance of FAF1 expression in non-small-cell lung cancer (NSCLC), and whether aberrant FAF1 expression may be involved in the pathogenesis and prognosis of NSCLC. FAF1 expression was examined in NSCLC specimens as well as human lung cancer cell lines. In addition, changes in cell viability and apoptosis upon regulating FAF1 expression were investigated in lung cancer cell lines. As a result, high FAF1 expression was significantly associated with a poor prognosis in NSCLC. In lung cancer cell lines, FAF1 downregulation hindered cell viability and tended to promote early apoptosis. In conclusion, this is the first study of the clinical significance of FAF1 in NSCLC, showing that FAF1 overexpression is associated with a poor prognosis in NSCLC and that FAF1 acts as a dangerous factor rather than an apoptosis promoter in NSCLC.

Shrimp MANF maintains hemocyte viability via interaction with a tyrosine kinase Abl.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a highly conserved cell protective protein. In this study, we explored its functions in shrimp hemocytes. Our results indicated that LvMANF knockdown could cause a decrease in total hemocyte count (THC) and an increase in caspase3/7 activity. To further explore its working mechanism, transcriptomic analyses were performed with wild-type and LvMANF-knockdown hemocytes. Three upregulated genes from transcriptomic data, including FAS-associated factor 2, rho-associated protein kinase 1, and serine/threonine-protein kinase WNK4 were validated with qPCR. Further experiments showed that LvMANF knockdown and tyrosine kinase LvAbl knockdown could decrease tyrosine phosphorylation in shrimp hemocytes. In addition, the interaction between LvMANF and LvAbl was validated with immunoprecipitation. The knockdown of LvMANF would decrease ERK phosphorylation and increase LvAbl expression. Our results suggest intracellular LvMANF may maintain shrimp hemocyte viability by interacting with LvAbl.

Abstract 1728: Exosomal delivery of Fas-associated factor 1 (FAF1), a multifunctional tumor suppressor and genuine exosome cargo, effectively antagonizes tumor growth

Abstract Introduction: Exosomes are extracellular vesicles of 30-150 nm and can stably deliver their cargos to the target with low immunogenicity and efficient transmission. FAF1 is a genuine exosome cargo and, therefore, loaded into exosomes without additional engineering. FAF1 is a tumor suppressive protein with multifunctional roles including death-promotion (both apoptosis and necrosis), cell cycle arrest, anti-inflammation, and anti-angiogenesis. FAF1 also inhibits metastasis via inhibition of TGF-β pathway. Moreover, FAF1 elicits low expression profiles in several types of cancer. Therefore, this study examined whether exosomal delivery of FAF1 is a plausible strategy for anti-tumor therapeutics. Methods: Stable suspension cells originated from HEK293 cells inducibly overexpressing FAF1 were screened using Tet on-off system to avoid cell death due to continuous FAF1 expression. FAF1 was overexpressed by Doxycycline induction and the amounts of FAF1 loaded on exosomes were normalized by exosome markers. The efficacy of exosomal FAF1 was evaluated in mouse xenograft models implanted with various human carcinoma cells. Results: Cell lines showing FAF1 expression with more than 3 fold of control cells were picked. Among FAF1-overexpressing cells, F5-3 cell line showing highest expression of exosomal FAF1 (showing 23 fold of FAF1 loading efficiency compared to parental cells) was used for exosomal source for in vivo experiments. Furthermore, F5-3 produced about 70 times of exosomal FAF1 when compared to that of HEK293 cell in the same volume of culture medium. The exosomal FAF1 produced in F5-3 cells inhibited tumor growth in various cancer types including lung, breast, liver, and pancreas in mouse xenograft models. In addition, exosomal FAF1 consistently demonstrated superior tumor-suppressive potential to soluble FAF1 in all tumors investigated in this study. Also, there were no serious side effects based on weight changes. Conclusion: This study demonstrated the tumor-suppressive potential of exosomal FAF1, demonstrating the feasibility of exosomal delivery of FAF1 as an efficient therapeutic measure. Furthermore, the selective and massive production properties of F5-3 cell realizes efficient and scalable production of exosomal FAF1. Citation Format: Jeong-Hoon Han, DaeHo Park, Bumjun Jung, Hyeong-Gwon Yoo, Kyung-Ok Cho, Eunhee Kim. Exosomal delivery of Fas-associated factor 1 (FAF1), a multifunctional tumor suppressor and genuine exosome cargo, effectively antagonizes tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1728.

Caspar negatively regulates anti-bacterial immunity by controlling the nuclear translocation of Relish in Chinese mitten crab (Eriocheir sinensis)

Caspar, a homolog of the Fas-associated factor 1 (FAF1) family, contains an N-terminal ubiquitin interaction domain, a ubiquitin-like self-association domain, and a C-terminal ubiquitin regulatory domain. Caspar has been reported to be involved in the antibacterial immunity of Drosophila, which is unclear whether it is involved in the antibacterial immune process of crustaceans. In this article, we identified a Caspar gene in Eriocheir sinensis and named it EsCaspar. EsCaspar positively respond to bacterial stimulation and downregulate the expression of certain associated antimicrobial peptides by inhibiting the nuclear translocation of EsRelish. Thus, EsCaspar might be a suppressor of the immune deficiency (IMD) pathway that prevents over-activation of the immune system. Indeed, excess EsCaspar protein in crabs reduced resistance to bacterial infection. In conclusion, EsCaspar is a suppressor of the IMD pathway in crabs that plays a negative regulatory role in antimicrobial immunity.

Expression and Localization of Fas-Associated Factor 1 in Testicular Tissues of Different Ages and Ovaries at Different Reproductive Cycle Phases of Bos grunniens.

Fas-associated factor 1 (FAF1), a member of the Fas family, is involved in biological processes such as apoptosis, inflammation, cell proliferation and proteostasis. This study aimed to explore the biological role of FAF1 in testicular tissue at different ages (juveniles (1 and 2 years old), adults (3, 4, 6, and 7 years old) and old-aged animals (11 years old)) and ovaries during different reproductive cycle phases (follicular, luteal, and pregnancy phases). FAF1 mRNA, relative protein expression and protein expression localization were determined in testes and ovaries using real-time quantification, WB and immunohistochemistry (IHC), respectively. Real-time quantification of testis tissues showed that the relative expression of FAF1 mRNA in testis tissues at 3, 4 and 7 years of age was significantly higher than of those in other ages, and in ovarian tissues was significantly higher in luteal phase ovaries than those in follicular and pregnancy phase ovaries; follicular phase ovaries were the lowest. WB of testis tissues showed that the relative protein expression of FAF1 protein was significantly higher at 11 and 7 years of age; in ovarian tissue, the relative protein expression of FAF1 protein was significantly higher in follicular phase ovaries than in luteal and pregnancy phase ovaries, and lowest in luteal phase ovaries. The relative protein expression of FAF1 at 3, 4 and 7 years of age was the lowest. IHC showed that FAF1 was mainly expressed in spermatozoa, spermatocytes, spermatogonia and supporting cells; in ovarian tissue, FAF1 was expressed in ovarian germ epithelial cells, granulosa cells, cumulus cells and luteal cells. The IHC results showed that FAF1 mRNA and protein were significantly differentially expressed in testes of different ages and ovarian tissues of different reproductive cycle phases, revealing the significance of FAF1 in the regulation of male and female B. grunniens reproductive physiology. Furthermore, our results provide a basis for the further exploration of FAF1 in the reproductive physiology of B. grunniens.

SERBP1 Promotes Stress Granule Clearance by Regulating 26S Proteasome Activity and G3BP1 Ubiquitination and Protects Male Germ Cells from Thermostimuli Damage.

Stress granules (SGs) are membraneless cytoplasmic condensates that dynamically assemble in response to various stressors and reversibly disassemble after stimulus removal; however, the mechanisms underlying SG dynamics and their physiological roles in germ cell development are elusive. Here, we show that SERBP1 (SERPINE1 mRNA binding protein 1) is a universal SG component and conserved regulator of SG clearance in somatic and male germ cells. SERBP1 interacts with the SG core component G3BP1 and 26S proteasome proteins PSMD10 and PSMA3 and recruits them to SGs. In the absence of SERBP1, reduced 20S proteasome activity, mislocalized valosin containing protein (VCP) and Fas associated factor family member 2 (FAF2), and diminished K63-linked polyubiquitination of G3BP1 during the SG recovery period were observed. Interestingly, the depletion of SERBP1 in testicular cells invivo causes increased germ cell apoptosis upon scrotal heat stress. Accordingly, we propose that a SERBP1-mediated mechanism regulates 26S proteasome activity and G3BP1 ubiquitination to facilitate SG clearance in both somatic and germ cell lines.

Featured interactome of homocysteine-inducible endoplasmic reticulum protein uncovers novel binding partners in response to ER stress

Homocysteine-inducible endoplasmic reticulum protein (HERP) is an endoplasmic reticulum (ER)-resident protein and important for the adaptation of cellular protein homeostasis by ER-associated degradation (ERAD) system. HERP interactors are critical for cellular viability and the reaction to ER stress. To explore the exact mechanisms by which HERP performed the biological functions, we conducted an interaction analysis of HERP protein in HeLa cells by co-immunoprecipitation (Co-IP) and liquid chromatography-mass spectrometer (LC-MS)/MS coupled with label-free quantification (LFQ). Among the interactome results, 123 proteins significantly interacted with HERP, which leads to numerous biological processes including protein import into nucleus, ubiquitin-dependent ERAD pathway, negative regulation of apoptotic process, and protein transport from ER, along with multiple pathways including several diseases, protein processing in ER, fatty acid metabolism, and steroid biosynthesis. Furthermore, we selected several prey proteins from the interactome data and confirmed that HERP interacted with ancient ubiquitous protein 1 (AUP1), Fas-associated factor family member 2 (FAF2), tripartite motif containing 47 (TRIM47), acyl-CoA synthetase long-chain family member 3 (ACSL3), sequestosome 1 (SQSTM1), and poly(rC) binding protein 2 (PCBP2) by Co-IP and confocal microscopy experiments, respectively. Moreover, the expression and location of several interacted proteins were obviously altered in response to ER stress induced by Thapsigargin stimulation and Enterovirus 71 infection. In conclusion, our findings revealed that the vital proteins interacted with HERP to mediate signaling transduction, thus providing novel clues for the mechanisms of HERP associated with ERAD and metabolism in response to ER stress under physiological and pathological conditions.

A protein-lipid complex that detoxifies free fatty acids.

Fatty acids (FAs) are well known to serve as substrates for reactions that provide cells with membranes and energy. In contrast to these metabolic reactions, the physiological importance of FAs themselves known as free FAs (FFAs) in cells remains obscure. Since accumulation of FFAs in cells is toxic, cells must develop mechanisms to detoxify FFAs. One such mechanism is to sequester free polyunsaturated FAs (PUFAs) into a droplet-like structure assembled by Fas-Associated Factor 1 (FAF1), a cytosolic protein. This sequestration limits access of PUFAs to Fe2+ , thereby preventing Fe2+ -catalyzed PUFA peroxidation. Consequently, assembly of the FAF1-FFA complex is critical to protect cells from ferroptosis, a cell death pathway triggered by PUFA peroxidation. The observations that free PUFAs in cytosol are not randomly diffused but rather sequestered into a membraneless complex should open new directions to explore signaling pathways by which FFAs regulate cellular physiology.

Targeting lipid metabolism for ferroptotic cancer therapy.

It has been 10 years since the concept of ferroptosis was put forward and research focusing on ferroptosis has been increasing continuously. Ferroptosis is driven by iron-dependent lipid peroxidation, which can be antagonized by glutathione peroxidase 4 (GPX4), ferroptosis inhibitory protein 1 (FSP1), dihydroorotate dehydrogenase (DHODH) and Fas-associated factor 1 (FAF1). Various cellular metabolic events, including lipid metabolism, can modulate ferroptosis sensitivity. It is worth noting that the reprogramming of lipid metabolism in cancer cells can promote the occurrence and development of tumors. The metabolic flexibility of cancer cells opens the possibility for the coordinated targeting of multiple lipid metabolic pathways to trigger cancer cells ferroptosis. In addition, cancer cells must obtain immortality, escape from programmed cell death including ferroptosis, to promote cancer progression, which provides new perspectives for improving cancer therapy. Targeting the vulnerability of ferroptosis has received attention as one of the significant possible strategies to treat cancer given its role in regulating tumor cell survival. We review the impact of iron and lipid metabolism on ferroptosis and the potential role of the crosstalk of lipid metabolism reprogramming and ferroptosis in antitumor immunity and sum up agents targeting lipid metabolism and ferroptosis for cancer therapy.

The complex of Fas-associated factor 1 with Hsp70 stabilizes the adherens junction integrity by suppressing RhoA activation.

Fas-associated factor 1 (FAF1) is a scaffolding protein that plays multiple functions, and dysregulation of FAF1 is associated with many types of diseases such as cancers. FAF1 contains multiple ubiquitin-related domains (UBA, UBL1, UBL2, UAS, and UBX), each domain interacting with a specific partner. In particular, the interaction of UBL1 with heat shock protein 70 (Hsp70) is associated with tumor formation, although the molecular understanding remains unknown. In this study, the structural analysis revealed that His160 of FAF1 is important for its interaction with Hsp70. The association of Hsp70 with FAF1 is required for the interaction with IQGAP1. FAF1 negatively regulates RhoA activation by FAF1-Hsp70 complex formation, which then interacts with IQGAP1. These steps play a key role in maintaining the stability of cell-to-cell junction. We conclude that FAF1 plays a critical role in the structure and function of adherens junction during tissue homeostasis and morphogenesis by suppressing RhoA activation, which induces the activation of Rho-associated protein kinase, phosphorylation of myosin light chain, formation of actin stress fiber, and disruption of adherens junction. In addition, depletion of FAF1 increased collective invasion in a 3D spheroid cell culture. These results provide insight into how the FAF1-Hsp70 complex acts as a novel regulator of the adherens junction integrity. The complex can be a potential therapeutic target to inhibit tumorigenesis and metastasis.

FAF1 blocks ferroptosis by inhibiting peroxidation of polyunsaturated fatty acids

Iron-dependent peroxidation of polyunsaturated fatty acids (PUFAs) leads to ferroptosis. While detoxification reactions removing lipid peroxides in phospholipids such as that catalyzed by glutathione peroxidase 4 (GPX4) protect cells from ferroptosis, the mechanism through which cells prevent PUFA peroxidation was not completely understood. We previously identified Fas-associated factor 1 (FAF1) as a protein directly interacting with free PUFAs through its UAS domain. Here we report that this interaction is crucial to protect cells from ferroptosis. In the absence of FAF1, cultured cells became sensitive to ferroptosis upon exposure to physiological levels of PUFAs, and mice developed hepatic injury upon consuming a diet enriched in PUFA. Mechanistically, we demonstrate that FAF1 assembles a globular structure that sequesters free PUFAs into a hydrophobic core, a reaction that prevents PUFA peroxidation by limiting its access to iron. Our study suggests that peroxidation of free PUFAs contributes to ferroptosis, and FAF1 acts upstream of GPX4 to prevents initiation of ferroptosis by limiting peroxidation of free PUFAs.

Caspar, an adapter for VAPB and TER94, modulates the progression of ALS8 by regulating IMD/NFκB-mediated glial inflammation in a Drosophila model of human disease.

Amyotrophic lateral sclerosis (ALS) is a fatal, late-onset, progressive motor neurodegenerative disorder. A key pathological feature of the disease is the presence of heavily ubiquitinated protein inclusions. Both the unfolded protein response and the ubiquitin–proteasome system appear significantly impaired in patients and animal models of ALS. We have studied cellular and molecular mechanisms involved in ALS using a vesicle-associated membrane protein-associated protein B (VAPB/ALS8) Drosophila model [Moustaqim-Barrette, A., Lin, Y.Q., Pradhan, S., Neely, G.G., Bellen, H.J. and Tsuda, H. (2014) The ALS 8 protein, VAP, is required for ER protein quality control. Hum. Mol. Genet., 23, 1975–1989], which mimics many systemic aspects of the human disease. Here, we show that VAPB, located on the cytoplasmic face of the endoplasmic reticulum membrane, interacts with Caspar, an orthologue of human fas associated factor 1 (FAF1). Caspar, in turn, interacts with transitional endoplasmic reticulum ATPase (TER94), a fly orthologue of ALS14 (VCP/p97, valosin-containing protein). Caspar overexpression in the glia extends lifespan and also slows the progression of motor dysfunction in the ALS8 disease model, a phenomenon that we ascribe to its ability to restrain age-dependent inflammation, which is modulated by Relish/NFκB signalling. Caspar binds to VAPB via an FFAT motif, and we find that Caspar’s ability to negatively regulate NFκB signalling is not dependent on the VAPB:Caspar interaction. We hypothesize that Caspar is a key molecule in the pathogenesis of ALS. The VAPB:Caspar:TER94 complex appears to be a candidate for regulating both protein homeostasis and NFκB signalling, with our study highlighting a role for Caspar in glial inflammation. We project human FAF1 as an important protein target to alleviate the progression of motor neuron disease.

Pharmacological Intervention Targeting FAF1 Restores Autophagic Flux for α-Synuclein Degradation in the Brain of a Parkinson's Disease Mouse Model.

α-Synuclein accumulation is implicated in the pathogenesis of neurodegenerative diseases, including Parkinson's disease (PD). Previously, we reported that Fas-associated factor 1 (FAF1), which plays a role in PD pathogenesis, potentiates α-synuclein accumulation through autophagy impairment in dopaminergic neurons. In this study, we show that KM-819, a FAF1-targeting compound, which has completed phase I clinical trials, interferes with α-synuclein accumulation in the mouse brain, as well as in human neuronal cells (SH-SY5Ys). KM-819 suppressed the accumulation of monomeric, oligomeric, and aggregated forms of α-synuclein in neuronal cells. Furthermore, KM-819 restored the turnover rate of α-synuclein in FAF1-overexpressing SH-SY5Y cells, implicating KM-819-mediated reconstitution of the α-synuclein degradative pathway. In addition, KM-819 reconstituted autophagic flux in FAF1-transfected SH-SY5Y cells, also suppressing α-synuclein-induced mitochondrial dysfunction. Moreover, oral administration of KM-819 also interfered with α-synuclein accumulation in the midbrain of mice overexpressing FAF1 via an adeno-associated virus system. Consistently, KM-819 reduced α-synuclein accumulation in both the hippocampus and the midbrain of human A53T α-synuclein transgenic mice. Collectively, these data imply that KM-819 may have therapeutic potential for patients with PD.

USP7 and VCPFAF1 define the SUMO/Ubiquitin landscape at the DNA replication fork.

SummaryThe AAA+ ATPase VCP regulates the extraction of SUMO and ubiquitin-modified DNA replication factors from chromatin. We have previously described that active DNA synthesis is associated with a SUMO-high/ubiquitin-low environment governed by the deubiquitylase USP7. Here, we unveil a functional cooperation between USP7 and VCP in DNA replication, which is conserved from Caenorhabditis elegans to mammals. The role of VCP in chromatin is defined by its cofactor FAF1, which facilitates the extraction of SUMOylated and ubiquitylated proteins that accumulate after the block of DNA replication in the absence of USP7. The inactivation of USP7 and FAF1 is synthetically lethal both in C. elegans and mammalian cells. In addition, USP7 and VCP inhibitors display synergistic toxicity supporting a functional link between deubiquitylation and extraction of chromatin-bound proteins. Our results suggest that USP7 and VCPFAF1 facilitate DNA replication by controlling the balance of SUMO/Ubiquitin-modified DNA replication factors on chromatin.

FAF1 downregulation by Toxoplasma gondii enables host IRF3 mobilization and promotes parasite growth.

Fas‐associated factor 1 (FAF1) has gained a reputation as a member of the FAS death‐inducing signalling complex. However, the role of FAF1 in the immunity response is not fully understood. Here, we report that, in the human retinal pigment epithelial (RPE) cell line ARPE‐19 cells, FAF1 expression level was downregulated by Toxoplasma gondii infection, and PI3K/AKT inhibitors reversed T. gondii‐induced FAF1 downregulation. In silico analysis for the FAF1 promoter sequence showed the presence of a FOXO response element (FRE), which is a conserved binding site for FOXO1 transcription factor. In accordance with the finding, FOXO1 overexpression potentiated, whereas FOXO1 depletion inhibited intracellular FAF1 expression level. We also found that FAF1 downregulation by T. gondii is correlated with enhanced IRF3 transcription activity. Inhibition of PI3K/AKT pathway with specific inhibitors had no effect on the level of T. gondii‐induced IRF3 phosphorylation but blocked IRF3 nuclear import and ISGs transcription. These results suggest that T. gondii can downregulate host FAF1 in PI3K/AKT/FOXO1‐dependent manner, and the event is essential for IRF3 nuclear translocation to active the transcription of ISGs and thereby T. gondii proliferation.

Up-regulation of pro-angiogenic molecules and events does not relate with an angiogenic switch in metastatic osteosarcoma cells but to cell survival features.

Osteosarcoma (OS) is the most frequent malignant bone tumor, affecting predominantly children. Metastases represent a major clinical challenge and an estimated 80% would present undetectable micrometastases at diagnosis. The identification of metastatic traits and molecules would impact in micrometastasis management. We demonstrated that OS LM7 metastatic cells secretome was able to induce microvascular endothelium cell rearrangements, an angiogenic-related trait. A proteomic analysis indicated a gain in angiogenic-related pathways in these cells, as compared to their parental-non-metastatic OS SAOS2 cells counterpart. Further, factors with proangiogenic functions like VEGF and PDGF were upregulated in LM7 cells. However, no differential angiogenic response was induced by LM7 cells in vivo. Regulation of the Fas-FasL axis is key for OS cells to colonize the lungs in this model. Analysis of the proteomic data with emphasis in apoptosis pathways and related processes revealed that the percentage of genes associated with those, presented similar levels in SAOS2 and LM7 cells. Further, the balance of expression levels of proteins with pro- and antiapoptotic functions in both cell types was subtle. Interestingly and of relevance to the model, Fas associated Factor 1 (FAF1), which participates in Fas signaling, was present in LM7 cells and was not detected in SAOS2 cells. The subtle differences in apoptosis-related events and molecules, together with the reported cell-survival functions of the identified angiogenic factors and the increased survival features that we observed in LM7 cells, suggest that the gain in angiogenesis-related pathways in metastatic OS cells would relate to a prosurvival switch rather to an angiogenic switch as an advantage feature to colonize the lungs. OS metastatic cells also displayed higher adhesion towards microvascular endothelium cells suggesting an advantage for tissue colonization. A gain in angiogenesis pathways and molecules does not result in major angiogenic potential. Together, our results suggest that metastatic OS cells would elicit signaling associated to a prosurvival phenotype, allowing homing into the hostile site for metastasis. During the gain of metastatic traits process, cell populations displaying higher adhesive ability to microvascular endothelium, negative regulation of the Fas-FasL axis in the lung parenchyma and a prosurvival switch, would be selected. This opens a new scenario where antiangiogenic treatments would affect cell survival rather than angiogenesis, and provides a molecular panel of expression that may help in distinguishing OS cells with different metastatic potential.

FAF1 Blocks Ferroptosis by Inhibiting Peroxidation of Polyunsaturated Fatty Acids

Iron-catalyzed peroxidation of polyunsaturated fatty acids (PUFAs) leads to ferroptosis. While scavenging reactions removing lipid peroxides such as that catalyzed by Gpx4 protect cells from ferroptosis, the mechanism through which cells prevent the initial physiological event in ferroptosis – the peroxidation of PUFAs, is not known. We previously identified Fas-associated factor 1 (FAF1) as a protein directly interacting with PUFAs through its UAS domain. Here we report that the UAS domain of FAF1 and PUFAs form a globular structure that sequesters PUFAs into a hydrophobic core. This sequestration prevents PUFA peroxidation by limiting their access to iron. In the absence of FAF1-mediated protection, cultured cells became sensitive to ferroptosis upon exposure to PUFAs, and mice developed hepatic injury upon consuming a diet enriched in arachidonic acid (AA), a PUFA. Our study suggests that the FAF1-mediated sequestration of PUFAs from iron-catalyzed peroxidation acts upstream of Gpx4 to protect cells from ferroptosis.