A FBXO7/EYA2-SCFFBXW7 axis promotes AXL-mediated maintenance of mesenchymal and immune evasion phenotypes of cancer cells.

A mesenchymal tumor phenotype associates with immunotherapy resistance, although the mechanism is unclear. Here, we identified FBXO7 as a maintenance regulator of mesenchymal and immune evasion phenotypes of cancer cells. FBXO7 bound and stabilized SIX1 co-transcriptional regulator EYA2, stimulating mesenchymal gene expression and suppressing IFNα/ß, chemokines CXCL9/10, and antigen presentation machinery, driven by AXL extracellular ligand GAS6. Ubiquitin ligase SCFFBXW7 antagonized this pathway by promoting EYA2 degradation. Targeting EYA2 Tyr phosphatase activity decreased mesenchymal phenotypes and enhanced cancer cell immunogenicity, resulting in attenuated tumor growth and metastasis, increased infiltration of cytotoxic T and NK cells, and enhanced anti-PD-1 therapy response in mouse tumor models. FBXO7 expression correlated with mesenchymal and immune-suppressive signatures in patients with cancer. An FBXO7-immune gene signature predicted immunotherapy responses. Collectively, the FBXO7/EYA2-SCFFBXW7 axis maintains mesenchymal and immune evasion phenotypes of cancer cells, providing rationale to evaluate FBXO7/EYA2 inhibitors in combination with immune-based therapies to enhance onco-immunotherapy responses.

Analysis of the FBXO7 promoter reveals overlapping Pax5 and c-Myb binding sites functioning in B cells.

Fbxo7 is a key player in the differentiation and function of numerous blood cell types, and in neurons, oligodendrocytes and spermatocytes. In an effort to gain insight into the physiological and pathological settings where Fbxo7 is likely to play a key role, we sought to define the transcription factors which direct FBXO7 expression. Using sequence alignments across 28 species, we defined the human FBXO7 promoter and found that it contains two conserved regions enriched for multiple transcription factor binding sites. Many of these have roles in either neuronal or haematopoietic development. Using various FBXO7 promoter reporters, we found ELF4, Pax5 and c-Myb have functional binding sites that activate transcription. We find endogenous Pax5 is bound to the FBXO7 promoter in pre-B cells, and that the exogenous expression of Pax5 represses Fbxo7 transcription in early pro-B cells.

Nedd8 hydrolysis by UCH proteases in Plasmodium parasites.

Plasmodium parasites are the causative agents of malaria, a disease with wide public health repercussions. Increasing drug resistance and the absence of a vaccine make finding new chemotherapeutic strategies imperative. Components of the ubiquitin and ubiquitin-like pathways have garnered increased attention as novel targets given their necessity to parasite survival. Understanding how these pathways are regulated in Plasmodium and identifying differences to the host is paramount to selectively interfering with parasites. Here, we focus on Nedd8 modification in Plasmodium falciparum, given its central role to cell division and DNA repair, processes critical to Plasmodium parasites given their unusual cell cycle and requirement for refined repair mechanisms. By applying a functional chemical approach, we show that deNeddylation is controlled by a different set of enzymes in the parasite versus the human host. We elucidate the molecular determinants of the unusual dual ubiquitin/Nedd8 recognition by the essential PfUCH37 enzyme and, through parasite transgenics and drug assays, determine that only its ubiquitin activity is critical to parasite survival. Our experiments reveal interesting evolutionary differences in how neddylation is controlled in higher versus lower eukaryotes, and highlight the Nedd8 pathway as worthy of further exploration for therapeutic targeting in antimalarial drug design.

Fbxl17 is rearranged in breast cancer and loss of its activity leads to increased global O-GlcNAcylation.

In cancer, many genes are mutated by genome rearrangement, but our understanding of the functional consequences of this remains rudimentary. Here we report the F-box protein encoded by FBXL17 is disrupted in the region of the gene that encodes its substrate-binding leucine rich repeat (LRR) domain. Truncating Fbxl17 LRRs impaired its association with the other SCF holoenzyme subunits Skp1, Cul1 and Rbx1, and decreased ubiquitination activity. Loss of the LRRs also differentially affected Fbxl17 binding to its targets. Thus, genomic rearrangements in FBXL17 are likely to disrupt SCFFbxl17-regulated networks in cancer cells. To investigate the functional effect of these rearrangements, we performed a yeast two-hybrid screen to identify Fbxl17-interacting proteins. Among the 37 binding partners Uap1, an enzyme involved in O-GlcNAcylation of proteins was identified most frequently. We demonstrate that Fbxl17 binds to UAP1 directly and inhibits its phosphorylation, which we propose regulates UAP1 activity. Knockdown of Fbxl17 expression elevated O-GlcNAcylation in breast cancer cells, arguing for a functional role for Fbxl17 in this metabolic pathway.

Loss of FBXO7 results in a Parkinson's-like dopaminergic degeneration via an RPL23-MDM2-TP53 pathway.

The field of Parkinson's disease research has been impeded by the absence of animal models that clearly phenocopy the features of this neurodegenerative condition. Mutations in FBXO7/PARK15 are associated with both sporadic Parkinson's disease and a severe form of autosomal recessive early-onset Parkinsonism. Here we report that conditional deletion of Fbxo7 in the midbrain dopamine neurons results in an early reduction in striatal dopamine levels, together with a slow, progressive loss of midbrain dopamine neurons and onset of locomotor defects. Unexpectedly, a later compensatory response led to a near-full restoration of dopaminergic fibre innervation in the striatum, but nigral cell loss was irreversible. Mechanistically, there was increased expression in the dopamine neurons of FBXO7-interacting protein, RPL23, which is a sensor of ribosomal stress that inhibits MDM2, the negative regulator of p53. A corresponding activated p53 transcriptional signature biased towards pro-apoptotic genes was also observed. These data suggest that the neuroprotective role of FBXO7 involves its suppression of the RPL23-MDM2-p53 axis that promotes cell death in dopaminergic midbrain neurons. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Opposing effects on the cell cycle of T lymphocytes by Fbxo7 via Cdk6 and p27.

G1 phase cell cycle proteins, such as cyclin-dependent kinase 6 (Cdk6) and its activating partners, the D-type cyclins, are important regulators of T-cell development and function. An F-box protein, called F-box only protein 7 (Fbxo7), acts as a cell cycle regulator by enhancing cyclin D-Cdk6 complex formation and stabilising levels of p27, a cyclin-dependent kinase inhibitor. We generated a murine model of reduced Fbxo7 expression to test its physiological role in multiple tissues and found that these mice displayed a pronounced thymic hypoplasia. Further analysis revealed that Fbxo7 differentially affected proliferation and apoptosis of thymocytes at various stages of differentiation in the thymus and also mature T-cell function and proliferation in the periphery. Paradoxically, Fbxo7-deficient immature thymocytes failed to undergo expansion in the thymus due to a lack of Cdk6 activity, while mature T cells showed enhanced proliferative capacity upon T-cell receptor engagement due to reduced p27 levels. Our studies reveal differential cell cycle regulation by Fbxo7 at different stages in T-cell development.

F-box protein interactions with the hallmark pathways in cancer.

F-box proteins (FBP) are the substrate specifying subunit of Skp1-Cul1-FBP (SCF)-type E3 ubiquitin ligases and are responsible for directing the ubiquitination of numerous proteins essential for cellular function. Due to their ability to regulate the expression and activity of oncogenes and tumour suppressor genes, FBPs themselves play important roles in cancer development and progression. In this review, we provide a comprehensive overview of FBPs and their targets in relation to their interaction with the hallmarks of cancer cell biology, including the regulation of proliferation, epigenetics, migration and invasion, metabolism, angiogenesis, cell death and DNA damage responses. Each cancer hallmark is revealed to have multiple FBPs which converge on common signalling hubs or response pathways. We also highlight the complex regulatory interplay between SCF-type ligases and other ubiquitin ligases. We suggest six highly interconnected FBPs affecting multiple cancer hallmarks, which may prove sensible candidates for therapeutic intervention.

Gsk3ß and Tomm20 are substrates of the SCFFbxo7/PARK15 ubiquitin ligase associated with Parkinson's disease.

Fbxo7 is a clinically relevant F-box protein, associated with both cancer and Parkinson's disease (PD). Additionally, SNPs within FBXO7 are correlated with alterations in red blood cell parameters. Point mutations within FBXO7 map within specific functional domains, including near its F-box domain and its substrate recruiting domains, suggesting that deficiencies in SCFFbxo7/PARK15 ubiquitin ligase activity are mechanistically linked to early-onset PD. To date, relatively few substrates of the ligase have been identified. These include HURP (hepatoma up-regulated protein), whose ubiquitination results in proteasome-mediated degradation, and c-IAP1 (inhibitor of apoptosis protein 1), TNF receptor-associated factor 2 (TRAF2), and NRAGE, which are not destabilized as a result of ubiquitination. None of these substrates have been linked directly to PD, nor has it been determined whether they would directly engage neuronal cell death pathways. To discover ubiquitinated substrates of SCFFbxo7 implicated more directly in PD aetiology, we conducted a high-throughput screen using protein arrays to identify new candidates. A total of 338 new targets were identified and from these we validated glycogen synthase kinase 3ß (Gsk3ß), which can phosphorylate α-synuclein, and translocase of outer mitochondrial membrane 20 (Tomm20), a mitochondrial translocase that, when ubiquitinated, promotes mitophagy, as SCFFbxo7 substrates both in vitro and in vivo Ubiquitin chain restriction analyses revealed that Fbxo7 modified Gsk3ß using K63 linkages. Our results indicate that Fbxo7 negatively regulates Gsk3ß activity, rather than its levels or localization. In addition, Fbxo7 ubiquitinated Tomm20, and its levels correlated with Fbxo7 expression, indicating a stabilizing effect. None of the PD-associated mutations in Fbxo7 impaired Tomm20 ubiquitination. Our findings demonstrate that SCFFbxo7 has an impact directly on two proteins implicated in pathological processes leading to PD.

Defective erythropoiesis in a mouse model of reduced Fbxo7 expression due to decreased p27 expression.

During the final stages of erythropoiesis, lineage-restricted progenitors mature over three to five cell divisions, culminating with withdrawal from the cell cycle and the loss of most organelles, including mitochondria and nuclei. Recent genome-wide association studies in human populations have associated several SNPs near or within FBXO7 with erythrocyte phenotypes. Fbxo7 encodes a multi-functional F-box protein known to bind p27 and participate in selective mitophagy. One SNP causes an amino acid substitution (Met115Ile) and is associated with smaller erythrocytes. We find that the less common IIe115 allele of Fbxo7 binds less efficiently to p27, and cells expressing this allele proliferate faster than cells expressing Met115. We show that an erythroleukaemic cell line with reduced Fbxo7 expression fails to stabilize p27 levels, exit the cell cycle, and produce haemoglobin. In addition, mice deficient in Fbxo7 expression are anaemic due to a reduction in erythrocyte numbers, and this is associated with lower p27 levels, increased numbers of late-stage erythroblasts with greater than 2N DNA content, and delayed mitophagy during terminal differentiation. Collectively, these data support an important physiological, cell cycle regulatory role for Fbxo7 during erythropoiesis.

Study of an FBXO7 patient mutation reveals Fbxo7 and PI31 co-regulate proteasomes and mitochondria.

Mutations in FBXO7 have been discovered to be associated with an atypical parkinsonism. We report here a new homozygous missense mutation in a paediatric patient that causes an L250P substitution in the dimerisation domain of Fbxo7. This alteration selectively ablates the Fbxo7-PI31 interaction and causes a significant reduction in Fbxo7 and PI31 levels in patient cells. Consistent with their association with proteasomes, patient fibroblasts have reduced proteasome activity and proteasome subunits. We also show PI31 interacts with the MiD49/51 fission adaptor proteins, and unexpectedly, PI31 acts to facilitate SCFFbxo7-mediated ubiquitination of MiD49. The L250P mutation reduces the SCFFbxo7 ligase-mediated ubiquitination of a subset of its known substrates. Although MiD49/51 expression was reduced in patient cells, there was no effect on the mitochondrial network. However, patient cells show reduced levels of mitochondrial function and mitophagy, higher levels of ROS and are less viable under stress. Our study demonstrates that Fbxo7 and PI31 regulate proteasomes and mitochondria and reveals a new function for PI31 in enhancing the SCFFbxo7 E3 ubiquitin ligase activity.

Knockdown of Fbxo7 reveals its regulatory role in proliferation and differentiation of haematopoietic precursor cells.

Fbxo7 is an unusual F-box protein because most of its interacting proteins are not substrates for ubiquitin-mediated degradation. Fbxo7 directly binds p27 and Cdk6, enhances the level of cyclin D-Cdk6 complexes, and its overexpression causes Cdk6-dependent transformation of immortalised fibroblasts. Here, we test the ability of Fbxo7 to transform haematopoietic pro-B (Ba/F3) cells which, unexpectedly, it was unable to do despite high levels of Cdk6. Instead, reduction of Fbxo7 expression increased proliferation, decreased cell size and shortened G1 phase. Analysis of cell cycle regulators showed that cells had decreased levels of p27, and increased levels of S phase cyclins and Cdk2 activity. Also, Fbxo7 protein levels correlated inversely with those of CD43, suggesting direct regulation of its expression and, therefore, of B cell maturation. Alterations to Cdk6 protein levels did not affect the cell cycle, indicating that Cdk6 is neither rate-limiting nor essential in Ba/F3 cells; however, decreased expression of Cdk6 also enhanced levels of CD43, indicating that expression of CD43 is independent of cell cycle regulation. The physiological effect of reduced levels of Fbxo7 was assessed by creating a transgenic mouse with a LacZ insertion into the Fbxo7 locus. Homozygous Fbxo7(LacZ) mice showed significantly increased pro-B cell and pro-erythroblast populations, consistent with Fbxo7 having an anti-proliferative function and/or a role in promoting maturation of precursor cells.

Identification of F-box only protein 7 as a negative regulator of NF-kappaB signalling.

The nuclear factor κB (NF-κB) signalling pathway controls important cellular events such as cell proliferation, differentiation, apoptosis and immune responses. Pathway activation occurs rapidly upon TNFα stimulation and is highly dependent on ubiquitination events. Using cytoplasmic to nuclear translocation of the NF-κB transcription factor family member p65 as a read-out, we screened a synthetic siRNA library targeting enzymes involved in ubiquitin conjugation and de-conjugation for modifiers of regulatory ubiquitination events in NF-κB signalling. We identified F-box protein only 7 (FBXO7), a component of Skp, Cullin, F-box (SCF)-ubiquitin ligase complexes, as a negative regulator of NF-κB signalling. F-box protein only 7 binds to, and mediates ubiquitin conjugation to cIAP1 and TRAF2, resulting in decreased RIP1 ubiquitination and lowered NF-κB signalling activity.

A Competitive binding mechanism between Skp1 and exportin 1 (CRM1) controls the localization of a subset of F-box proteins.

SCF-type E3 ubiquitin ligases are crucial regulators of cell cycle progression. As the F-box protein is the substrate-specifying subunit of this family of ligases, their availability dictates the timing and the location of the ubiquitination of substrates. We report here our investigation into the regulation of the localization of F-box proteins, in particular Fbxo7, whose mislocalization is associated with human disease. We identified a motif in Fbxo7 that we have characterized as a functional leucine-rich nuclear export sequence (NES), and which allowed binding to the nuclear export protein, exportin 1 (CRM1). Unusually, the NES was embedded within the F-box domain, which is bound by Skp1 and enables the F-box protein to form part of an E3 ubiquitin ligase. The NES of Fbxo7 controlled its localization and was conserved in Fbxo7 homologues in other species. Skp1 binding prevented Fbxo7 from contacting CRM1. We propose that this competitive binding allowed Fbxo7 to accumulate within the nucleus starting at the G1/S transition. More than ten other F-box proteins also contain an NES at the same location in their F-box domains, indicating that this competitive binding mechanism may contribute to the regulation of a sixth of the known F-box proteins.

Fbxo7 promotes Cdk6 activity to inhibit PFKP and glycolysis in T cells.

Fbxo7 is associated with cancer and Parkinson's disease. Although Fbxo7 recruits substrates for SCF-type ubiquitin ligases, it also promotes Cdk6 activation in a ligase-independent fashion. We discovered PFKP, the gatekeeper of glycolysis, in a screen for Fbxo7 substrates. PFKP is an essential Cdk6 substrate in some T-ALL cells. We investigated the molecular relationship between Fbxo7, Cdk6, and PFKP, and the effect of Fbxo7 on T cell metabolism, viability, and activation. Fbxo7 promotes Cdk6-independent ubiquitination and Cdk6-dependent phosphorylation of PFKP. Importantly, Fbxo7-deficient cells have reduced Cdk6 activity, and hematopoietic and lymphocytic cells show high expression and significant dependency on Fbxo7. CD4+ T cells with reduced Fbxo7 show increased glycolysis, despite lower cell viability and activation levels. Metabolomic studies of activated CD4+ T cells confirm increased glycolytic flux in Fbxo7-deficient cells, alongside altered nucleotide biosynthesis and arginine metabolism. We show Fbxo7 expression is glucose-responsive at the mRNA and protein level and propose Fbxo7 inhibits PFKP and glycolysis via its activation of Cdk6.

Distinct roles for cyclins E and A during DNA replication complex assembly and activation.

Initiation of DNA replication is regulated by cyclin-dependent protein kinase 2 (Cdk2) in association with two different regulatory subunits, cyclin A and cyclin E (reviewed in ref. 1). But why two different cyclins are required and why their order of activation is tightly regulated are unknown. Using a cell-free system for initiation of DNA replication that is based on G1 nuclei, G1 cytosol and recombinant proteins, we find that cyclins E and A have specialized roles during the transition from G0 to S phase. Cyclin E stimulates replication complex assembly by cooperating with Cdc6, to make G1 nuclei competent to replicate in vitro. Cyclin A has two separable functions: it activates DNA synthesis by replication complexes that are already assembled, and it inhibits the assembly of new complexes. Thus, cyclin E opens a 'window of opportunity' for replication complex assembly that is closed by cyclin A. The dual functions of cyclin A ensure that the assembly phase (G1) ends before DNA synthesis (S) begins, thereby preventing re-initiation until the next cell cycle.

The E3 ubiquitin ligase SCF(Fbxo7) mediates proteasomal degradation of UXT isoform 2 (UXT-V2) to inhibit the NF-κB signaling pathway.

BACKGROUND: Ubiquitously eXpressed Transcript isoform 2 (UXTV2) is a prefoldin-like protein involved in NF-κB signaling, apoptosis, and the androgen and estrogen response. UXT-V2 is a cofactor in the NF-κB transcriptional enhanceosome, and its knockdown inhibits TNF-α -induced NF-κB activation. Fbxo7 is an F-box protein that interacts with SKP1, Cullin1 and RBX1 proteins to form an SCF(Fbxo7) E3 ubiquitin ligase complex. Fbxo7 negatively regulates NF-κB signaling through TRAF2 and cIAP1 ubiquitination. METHODS: We combine co-immunoprecipitation, ubiquitination in vitro and in vivo, cycloheximide chase assay, ubiquitin chain restriction analysis and microscopy to investigate interaction between Fbxo7 and overexpressed UXT-V2-HA. RESULTS: The Ubl domain of Fbxo7 contributes to interaction with UXTV2. This substrate is polyubiquitinated by SCF(Fbxo7) with K48 and K63 ubiquitin chain linkages in vitro and in vivo. This post-translational modification decreases UXT-V2 stability and promotes its proteasomal degradation. We further show that UXTV1, an alternatively spliced isoform of UXT, containing 12 additional amino acids at the N-terminus as compared to UXTV2, also interacts with and is ubiquitinated by Fbxo7. Moreover, FBXO7 knockdown promotes UXT-V2 accumulation, and the overexpression of Fbxo7-ΔF-box protects UXT-V2 from proteasomal degradation and enhances the responsiveness of NF-κB reporter. We find that UXT-V2 colocalizes with Fbxo7 in the cell nucleus. CONCLUSIONS: Together, our study reveals that SCF(Fbxo7) mediates the proteasomal degradation of UXT-V2 causing the inhibition of the NF-κB signaling pathway. GENERAL SIGNIFICANCE: Discovering new substrates of E3 ubiquitin-ligase SCF(Fbxo7) contributes to understand its function in different diseases such as cancer and Parkinson.

The FBXL family of F-box proteins: variations on a theme.

The ubiquitin-proteasome system (UPS) is responsible for the rapid targeting of proteins for degradation at 26S proteasomes and requires the orchestrated action of E1, E2 and E3 enzymes in a well-defined cascade. F-box proteins (FBPs) are substrate-recruiting subunits of Skp1-cullin1-FBP (SCF)-type E3 ubiquitin ligases that determine which proteins are ubiquitinated. To date, around 70 FBPs have been identified in humans and can be subdivided into distinct families, based on the protein-recruiting domains they possess. The FBXL subfamily is defined by the presence of multiple leucine-rich repeat (LRR) protein-binding domains. But how the 22 FBPs of the FBXL family achieve their individual specificities, despite having highly similar structural domains to recruit their substrates, is not clear. Here, we review and explore the FBXL family members in detail highlighting their structural and functional similarities and differences and how they engage their substrates through their LRRs to adopt unique interactomes.

A Conserved Requirement for Fbxo7 During Male Germ Cell Cytoplasmic Remodeling.

Fbxo7 is the substrate-recognition subunit of an SCF-type ubiquitin E3 ligase complex. It has physiologically important functions in regulating mitophagy, proteasome activity and the cell cycle in multiple cell types, like neurons, lymphocytes and erythrocytes. Here, we show that in addition to the previously known Parkinsonian and hematopoietic phenotypes, male mice with reduced Fbxo7 expression are sterile. In these males, despite successful meiosis, nuclear elongation and eviction of histones from chromatin, the developing spermatids are phagocytosed by Sertoli cells during late spermiogenesis, as the spermatids undergo cytoplasmic remodeling. Surprisingly, despite the loss of all germ cells, there was no evidence of the symplast formation and cell sloughing that is typically associated with spermatid death in other mouse sterility models, suggesting that novel cell death and/or cell disposal mechanisms may be engaged in Fbxo7 mutant males. Mutation of the Drosophila Fbxo7 ortholog, nutcracker (ntc) also leads to sterility with germ cell death during cytoplasmic remodeling, indicating that the requirement for Fbxo7 at this stage is conserved. The ntc phenotype was attributed to decreased levels of the proteasome regulator, DmPI31 and reduced proteasome activity. Consistent with the fly model, we observe a reduction in PI31 levels in mutant mice; however, there is no alteration in proteasome activity in whole mouse testes. Our results are consistent with findings that Fbxo7 regulates PI31 protein levels, and indicates that a defect at the late stages of spermiogenesis, possibly due to faulty spatial dynamics of proteasomes during cytoplasmic remodeling, may underlie the fertility phenotype in mice.

Structure and Function of Fbxo7/PARK15 in Parkinson's Disease.

Fbxo7/PARK15 has well-defined roles, acting as part of a Skp1-Cul1-F box protein (SCF)- type E3 ubiquitin ligase and also having SCF-independent activities. Mutations within FBXO7 have been found to cause an early-onset Parkinson's disease, and these are found within or near to its functional domains, including its F-box domain (FBD), its proline rich region (PRR), and its ubiquitinlike domain (Ubl). We highlight recent advances in our understanding of Fbxo7 function in Parkinson's disease, with respect to these mutations and where they occur in the Fbxo7 protein. We hypothesize that many of Fbxo7 functions contribute to its role in PD pathogenesis.