A regulatory module comprising G3BP1-FBXL5-IRP2 axis determines sodium arsenite-induced ferroptosis.

Arsenic contamination is extremely threatening to the global public health. It was reported that sodium arsenite exposure induces serious kidney injury. However, the underlying mechanism is unclear. Ferroptosis is a newly characterized form of iron-dependent programmed cell death, which is implicated in the pathogenesis of various human diseases, including kidney injury. The lethal accumulation of iron-catalyzed lipid peroxidation is the fundamental biochemical characteristic of ferroptosis. Herein we report that sodium arsenite exposure initiates ferroptosis in mammalian HEK293, MEF and HT1080 cells, and induces ferroptosis-associated acute kidney injury in mice. RNA-binding protein G3BP1, the switch component of stress granules, is indispensable for sodium arsenite-induced ferroptosis in a stress granule-independent manner. Mechanistically, G3BP1 stabilizes IRP2, the master regulator of cellular iron homeostasis, through binding to and suppressing the translation of FBXL5 mRNA, which encodes the E3 ligase component to mediate IRP2 ubiquitination and proteasomal degradation. Sodium arsenite intoxication expedites this G3BP1-FBXL5-IRP2 axis and elevates cellular labile free iron, which is responsible for sodium arsenite exposure-induced lipid peroxidation and ferroptotic cell death. In summary, this study highlights a regulatory module comprising G3BP1-FBXL5-IRP2 axis in determining sodium arsenite-induced ferroptosis and ferroptosis-associated acute kidney injury in mice.

Plant systemic acquired resistance compound salicylic acid as a potent inhibitor against SCF (SKP1-CUL1-F-box protein) mediated complex in Fusarium oxysporum by homology modeling and molecular dynamics simulations.

Fusarium oxysporum causes significant economic losses in many crop plants by causing root rot, necrosis, and wilting symptoms. Homology and molecular dynamics studies are promising tools for the detection in F. oxysporum of the systemic resistance compound, salicylic acid, for control of the SKP1-CUL1-F-box protein complex. The structure of SKP1-CUL1-F-box subunit Skp1 from F. oxysporum is produced by Modeler 9v7 for the conduct of docking studies. The Skp1 structure is based on the yeast Cdc4/Skp1 (PDB ID: 3MKS A) crystal structure collected by the Protein data bank. Applying molecular dynamic model simulation methods to the final predicted structure and further evaluated by 3D and PROCHECK test programmers, the final model is verified to be accurate. Applying GOLD 3.0.1, SCF Complex Skp1 is used to prevent stress-tolerant operation. The SKP1-CUL1-F-box model is predicted to be stabilized and tested as a stable docking structure. The predicted model of the SCF structure has been stabilized and confirmed to be a reliable structure for docking studies. The results indicated that GLN8, LYS9, VAL10, TRP11, GLU48, ASN49 in SCF complex are important determinant residues in binding as they have strong hydrogen bonding with salicylic acid, which showed best docking results with SKP1-CUL1-F-box complex subunit Skp1 with docking score 25.25KJ/mol. Insilco studies have been used to determine the mode of action of salicylic acid for Fusarium control. Salicylic acid hinders the SKP1-CUL1-F-box complex, which is important in protein-like interactions through hydrogen bodings. Results from docking studies have shown that the best energy for SKP1-CUL1-F-box was salicylic acid.Communicated by Ramaswamy H. Sarma.

Co-operative binding of SKP1, Cullin1 and Cullin7 to FBXW8 results in Cullin1-SKP1-FBXW8-Cullin7 functional complex formation that monitors cellular function of ß-TrCP1.

F-box protein FBXW8 is known to interact with scaffolding protein Cullin1 and Cullin7 to form SCF (SKP1, Cullin and F-box protein) complex. However, detail understanding about the importance of both Cullins for SCF-FBXW8 complex formation as well as its ubiquitin ligase activity remains elusive. Here, we show that, through in vitro and in vivo studies, Cullin1 and Cullin7 increase each other's binding to FBXW8 synergistically. Interestingly, absence of either Cullin results in abrogation of binding of other Cullin to FBXW8. Binding of SKP1 to FBXW8 also increases in the presence of both the Cullins. Thus, SKP1, Cullin1 and Cullin7 are essential to form Cullin1-SKP1-FBXW8-Cullin7 functional ubiquitin ligase complex. Further, using computational, mutational and biochemical analysis, we found that Cullin1 binds to N-terminus of FBXW8 through SKP1 while Cullin7 associates with C-terminus of FBXW8 to form Cullin1-SKP1-FBXW8-Cullin7 functional complex in a cooperative manner. Results showed that Cullin1-SKP1-FBXW8-Cullin7 complex plays a key role in maintaining the basal level expression of ß-TrCP1. Moreover, Cullin1-SKP1-FBXW8-Cullin7 complex promotes cell migration by activating ß-catenin via directing proteasomal degradation of ß-TrCP1. Overall, our study reveals the intriguing molecular mechanism of assembly of SKP1, Cullin1, Cullin7 and FBXW8 to form Cullin1-SKP1-FBXW8-Cullin7 functional complex that control the function of ß-TrCP1.

Cullin-RING Ubiquitin Ligase Regulatory Circuits: A Quarter Century Beyond the F-Box Hypothesis.

Cullin-RING ubiquitin ligases (CRLs) are dynamic modular platforms that regulate myriad biological processes through target-specific ubiquitylation. Our knowledge of this system emerged from the F-box hypothesis, posited a quarter century ago: Numerous interchangeable F-box proteins confer specific substrate recognition for a core CUL1-based RING E3 ubiquitin ligase. This paradigm has been expanded through the evolution of a superfamily of analogous modular CRLs, with five major families and over 200 different substrate-binding receptors in humans. Regulation is achieved by numerous factors organized in circuits that dynamically control CRL activation and substrate ubiquitylation. CRLs also serve as a vast landscape for developing small molecules that reshape interactions and promote targeted ubiquitylation-dependent turnover of proteins of interest. Here, we review molecular principles underlying CRL function, the role of allosteric and conformational mechanisms in controlling substrate timing and ubiquitylation, and how the dynamics of substrate receptor interchange drives the turnover of selected target proteins to promote cellular decision-making.

An expanded auxin-inducible degron toolkit for Caenorhabditis elegans.

The auxin-inducible degron (AID) system has emerged as a powerful tool to conditionally deplete proteins in a range of organisms and cell types. Here, we describe a toolkit to augment the use of the AID system in Caenorhabditis elegans. We have generated a set of single-copy, tissue-specific (germline, intestine, neuron, muscle, pharynx, hypodermis, seam cell, anchor cell) and pan-somatic TIR1-expressing strains carrying a co-expressed blue fluorescent reporter to enable use of both red and green channels in experiments. These transgenes are inserted into commonly used, well-characterized genetic loci. We confirmed that our TIR1-expressing strains produce the expected depletion phenotype for several nuclear and cytoplasmic AID-tagged endogenous substrates. We have also constructed a set of plasmids for constructing repair templates to generate fluorescent protein::AID fusions through CRISPR/Cas9-mediated genome editing. These plasmids are compatible with commonly used genome editing approaches in the C. elegans community (Gibson or SapTrap assembly of plasmid repair templates or PCR-derived linear repair templates). Together these reagents will complement existing TIR1 strains and facilitate rapid and high-throughput fluorescent protein::AID tagging of genes. This battery of new TIR1-expressing strains and modular, efficient cloning vectors serves as a platform for straightforward assembly of CRISPR/Cas9 repair templates for conditional protein depletion.

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.

Structure and Role of BCOR PUFD in Noncanonical PRC1 Assembly and Disease.

Polycomb repression complex 1 (PRC1) is a multiprotein assembly that regulates transcription. The Polycomb group ring finger 1 protein (PCGF1) is central in the assembly of the noncanonical PRC1 variant called PRC1.1 through its direct interaction with BCOR (BCL-6-interacting corepressor) or its paralog, BCOR-like 1 (BCORL1). Previous structural studies revealed that the C-terminal PUFD domain of BCORL1 is necessary and sufficient to heterodimerize with the RAWUL domain of PCGF1 and, together, form a new protein-protein binding interface that associates with the histone demethylase KDM2B. Here, we show that the PUFD of BCOR and BCORL1 differ in their abilities to assemble with KDM2B. Unlike BCORL1, the PUFD of BCOR alone does not stably assemble with KDM2B. Rather, additional residues N-terminal to the BCOR PUFD are necessary for stable association. Nuclear magnetic resonance (NMR) structure determination and 15N T2 relaxation time measurements of the BCOR PUFD alone indicate that the termini of the BCOR PUFD, which are critical for binding PCGF1 and KDM2B, are disordered. This suggests a hierarchical mode of assembly whereby BCOR PUFD termini become structurally ordered upon binding PCGF1, which then allows stable association with KDM2B. Notably, BCOR internal tandem duplications (ITDs) leading to pediatric kidney and brain tumors map to the PUFD termini. Binding studies with the BCOR ITD indicate the ITD would disrupt PRC1.1 assembly, suggesting loss of the ability to assemble PRC1.1 is a critical molecular event driving tumorigenesis.

Identification of Structural Elements of the Lysine Specific Demethylase 2B CxxC Domain Associated with Replicative Senescence Bypass in Primary Mouse Cells.

BACKGROUND: Lysine specific demethylase 2B, KDM2B, regulates genes that participate in cellular development, morphogenesis, differentiation and metabolism as a component of the polycomb repressive complex 1 (PRC1). The CxxC finger of KDM2B is responsible for the DNA binding capacity of this epigenetic regulator, acting as a sampling mechanism across chromatin for gene repression OBJECTIVES: The molecular determinants of the CxxC-DNA interaction remain largely unknown, revealing a significant knowledge gap to be explored. Our goal was to elucidate the key residues of the CxxC domain that contribute to its function as well as to further elaborate on the significance of this domain in the KDM2B role METHODS: By using electrophoresis mobility swift assay, we identified structural elements of CxxC domain that participate in the DNA recognition. We created mouse embryonic fibroblasts overexpressing different truncated and point-mutated mouse KDM2B variants to examine the contribution of the KDM2B domains in replicative senescence bypass RESULTS: In this study, we show that only the CxxC finger is essential for the ability of mKDM2B to bypass replicative senescence in primary cells by ink4A-Arf-ink4B locus repression, and that this is mediated by specific interactions of residues R585, K608 and K616 with non-methylated CpG containing DNA CONCLUSIONS: These results provide new structural insights into the molecular interactions of CxxC and could serve as a stepping-stone for developing domain-specific inhibitors for KDM2B.

Skp1 Dimerization Conceals Its F-Box Protein Binding Site.

Skp1 is an adapter that links F-box proteins to cullin-1 in the Skp1/cullin-1/F-box (SCF) protein family of E3 ubiquitin ligases that targets specific proteins for polyubiquitination and subsequent protein degradation. Skp1 from the amoebozoan Dictyostelium forms a stable homodimer in vitro with a Kd of 2.5 µM as determined by sedimentation velocity studies yet is monomeric in crystal complexes with F-box proteins. To investigate the molecular basis for the difference, we determined the solution NMR structure of a doubly truncated Skp1 homodimer (Skp1ΔΔ). The solution structure of the Skp1ΔΔ dimer reveals a 2-fold symmetry with an interface that buries ∼750 Å2 of predominantly hydrophobic surface. The dimer interface overlaps with subsite 1 of the F-box interaction area, explaining why only the Skp1 monomer binds F-box proteins (FBPs). To confirm the model, Rosetta was used to predict amino acid substitutions that might disrupt the dimer interface, and the F97E substitution was chosen to potentially minimize interference with F-box interactions. A nearly full-length version of Skp1 with this substitution (Skp1ΔF97E) behaved as a stable monomer at concentrations of ≤500 µM and actively bound a model FBP, mammalian Fbs1, which suggests that the dimeric state is not required for Skp1 to carry out a basic biochemical function. Finally, Skp1ΔF97E is expected to serve as a monomer model for high-resolution NMR studies previously hindered by dimerization.

FBXL5 Regulates IRP2 Stability in Iron Homeostasis via an Oxygen-Responsive [2Fe2S] Cluster.

Cellular iron homeostasis is dominated by FBXL5-mediated degradation of iron regulatory protein 2 (IRP2), which is dependent on both iron and oxygen. However, how the physical interaction between FBXL5 and IRP2 is regulated remains elusive. Here, we show that the C-terminal substrate-binding domain of FBXL5 harbors a [2Fe2S] cluster in the oxidized state. A cryoelectron microscopy (cryo-EM) structure of the IRP2-FBXL5-SKP1 complex reveals that the cluster organizes the FBXL5 C-terminal loop responsible for recruiting IRP2. Interestingly, IRP2 binding to FBXL5 hinges on the oxidized state of the [2Fe2S] cluster maintained by ambient oxygen, which could explain hypoxia-induced IRP2 stabilization. Steric incompatibility also allows FBXL5 to physically dislodge IRP2 from iron-responsive element RNA to facilitate its turnover. Taken together, our studies have identified an iron-sulfur cluster within FBXL5, which promotes IRP2 polyubiquitination and degradation in response to both iron and oxygen concentrations.

Flexibility of the petunia strigolactone receptor DAD2 promotes its interaction with signaling partners.

Strigolactones (SLs) are terpenoid-derived plant hormones that regulate various developmental processes, particularly shoot branching, root development, and leaf senescence. The SL receptor has an unusual mode of action. Upon binding SL, it hydrolyzes the hormone, and then covalently binds one of the hydrolytic products. These initial events enable the SL receptor DAD2 (in petunia) to interact with the F-box protein PhMAX2A of the Skp-Cullin-F-box (SCF) complex and/or a repressor of SL signaling, PhD53A. However, it remains unclear how binding and hydrolysis structurally alters the SL receptor to enable its engagement with signaling partners. Here, we used mutagenesis to alter DAD2 and affect SL hydrolysis or DAD2's ability to interact with its signaling partners. We identified three DAD2 variants whose hydrolytic activity had been separated from the receptor's interactions with PhMAX2A or PhD53A. Two variants, DAD2N242I and DAD2F135A, having substitutions in the core α/ß hydrolase-fold domain and the hairpin, exhibited hormone-independent interactions with PhMAX2A and PhD53A, respectively. Conversely, the DAD2D166A variant could not interact with PhMAX2A in the presence of SL, but its interaction with PhD53A remained unaffected. Structural analyses of DAD2N242I and DAD2D166A revealed only small differences compared with the structure of the WT receptor. Results of molecular dynamics simulations of the DAD2N242I structure suggested that increased flexibility is a likely cause for its SL-independent interaction with PhMAX2A. Our results suggest that PhMAX2A and PhD53A have distinct binding sites on the SL receptor and that its flexibility is a major determinant of its interactions with these two downstream regulators.

Fbxo9 functions downstream of Sox10 to determine neuron-glial fate choice in the dorsal root ganglia through Neurog2 destabilization.

The transcription factor Sox10 is a key regulator in the fate determination of a subpopulation of multipotent trunk neural crest (NC) progenitors toward glial cells instead of sensory neurons in the dorsal root ganglia (DRG). However, the mechanism by which Sox10 regulates glial cell fate commitment during lineage segregation remains poorly understood. In our study, we showed that the neurogenic determinant Neurogenin 2 (Neurog2) exhibited transient overlapping expression with Sox10 in avian trunk NC progenitors, which progressively underwent lineage segregation during migration toward the forming DRG. Gain- and loss-of-function studies revealed that the temporary expression of Neurog2 was due to Sox10 regulation of its protein stability. Transcriptional profiling identified Sox10-regulated F-box only protein (Fbxo9), which is an SCF (Skp1-Cul-F-box)-type ubiquitin ligase for Neurog2. Consistently, overexpression of Fbxo9 in NC progenitors down-regulated Neurog2 protein expression through ubiquitination and promoted the glial lineage at the expense of neuronal differentiation, whereas Fbxo9 knockdown resulted in the opposite phenomenon. Mechanistically, we found that Fbxo9 interacted with Neurog2 to promote its destabilization through the F-box motif. Finally, epistasis analysis further demonstrated that Fbxo9 and probably other F-box members mediated the role of Sox10 in destabilizing Neurog2 protein and directing the lineage of NC progenitors toward glial cells rather than sensory neurons. Altogether, these findings unravel a Sox10-Fbxo9 regulatory axis in promoting the glial fate of NC progenitors through Neurog2 destabilization.

Assembly and Regulation of CRL Ubiquitin Ligases.

Cullin-RING ubiquitin ligases (CRLs) determine the substrate specificity of ubiquitination reactions, and substrates are recruited to the cullin core through binding to their cognate substrate receptor modules. Because a family of substrate receptors compete for the same cullin core, the assembly and activity of CRLs are dynamically regulated to fulfill the needs of the cell to adapt to the changing pool of proteins demanding ubiquitination. Cullins are modified by NEDD8, a ubiquitin-like protein. This process, referred to as neddylation, promotes the E3 activity of CRLs by inducing conformational rearrangement in the Cullin-RING catalytic core. Cand1 is a cullin-associated protein whose binding is excluded by cullin neddylation. Although early biochemical studies suggested that Cand1 inhibits CRL activity, genetic studies revealed its positive role in ubiquitination. Emerging evidence from kinetic and quantitative proteomic studies demonstrated that Cand1 stimulates assembly of new Skp1-Cul1-F-box protein (SCF) complexes by exchanging the Skp1-F-box protein substrate receptor modules. Furthermore, aided by refined experimental design as well as computational simulation, an attractive model has been developed in which substrate, neddylation cycle and Cand1-mediated "adaptive exchange" collaborate to maintain the dynamics of the cellular SCF repertoire. Here, we review and discuss recent advances that have deepened our understanding of CRL regulation.

Isolation and characterization of kelch repeat-containing F-box proteins from colored wheat.

F-box proteins play important roles in the regulation of various developmental processes in plants. Approximately 1796 F-box genes have been identified in the wheat genome, but details of their functions remain unknown. Moreover, not much was known about the roles of kelch repeat domain-containing F-box genes (TaKFBs) in wheat. In the present study, we isolated five TaKFBs to investigate the roles of KFBs at different stages of colored wheat grain development. The cDNAs encoding TaKFB1, TaKFB2, TaKFB3, TaKFB4, and TaKFB5 contained 363, 449, 353, 382, and 456 bp open reading frames, respectively. All deduced TaKFBs contained an F-box domain (IPR001810) and a kelch repeat type 1 domain (IPR006652), except TaKFB2. Expression of TaKFBs was elevated during the pigmentation stages of grain development. To clarify how TaKFB and SKP interact in wheat, we investigated whether five TaKFB proteins showed specificity for six SKP proteins using a yeast two-hybrid (Y2H) assay. An Y2H screen was performed to search for proteins capable of binding the TaKFBs and interaction was identified between TaKFB1 and aquaporin PIP1. To examine the subcellular localization of TaKFBs, we transiently expressed TaKFB-green fluorescent protein (GFP) fusions in tobacco leaves; the TaKFB-GFP fusions were detected in the nucleus and the cytoplasm. Y2H and bimolecular fluorescence complementation (BiFC) assays revealed that TaKFB1 specifically interacts with aquaporin PIP1. These results will provide useful information for further functional studies on wheat F-box proteins and their possible roles.

Chemical probes for protein arginine methyltransferases.

Protein arginine methyltransferases (PRMTs) catalyze the transfer of methyl groups to specific arginine residues of their substrates using S-adenosylmethionine as a methyl donor, contributing to regulation of many biological processes including transcription, and DNA damage repair. Dysregulation of PRMT expression is often associated with various diseases including cancers. Different methods have been used to characterize the activities of PRMTs and determine their kinetic parameters including mass spectrometry, radiometric, and antibody-based assays. Here, we present kinetic characterization of PRMTs using a radioactivity-based assay for better comparison along with previously reported values. We also report on full characterization of PRMT9 activity with SAP145 peptide as substrate. We further review the potent, selective and cell-active PRMT inhibitors discovered in recent years to provide a better understanding of available tools to investigate the roles these proteins play in health and disease.

Systematic analysis of F-box proteins reveals a new branch of the yeast mating pathway.

The mating pathway in yeast Saccharomyces cerevisiae has long been used to reveal new mechanisms of signal transduction. The pathway comprises a pheromone receptor, a heterotrimeric G protein, and intracellular effectors of morphogenesis and transcription. Polarized cell growth, in the direction of a potential mating partner, is accomplished by the G-protein ßγ subunits and the small G-protein Cdc42. Transcription induction, needed for cell-cell fusion, is mediated by Gßγ and the mitogen-activated protein kinase (MAPK) scaffold protein Ste5. A potential third pathway is initiated by the G-protein α subunit Gpa1. Gpa1 signaling was shown previously to involve the F-box adaptor protein Dia2 and an endosomal effector protein, the phosphatidylinositol 3-kinase Vps34. Vps34 is also required for proper vacuolar sorting and autophagy. Here, using a panel of reporter assays, we demonstrate that mating pheromone stimulates vacuolar targeting of a cytoplasmic reporter protein and that this process depends on Vps34. Through a systematic analysis of F-box deletion mutants, we show that Dia2 is required to sustain pheromone-induced vacuolar targeting. We also found that other F-box proteins selectively regulate morphogenesis (Ydr306, renamed Pfu1) and transcription (Ucc1). These findings point to the existence of a new and distinct branch of the pheromone-signaling pathway, one that likely leads to vacuolar engulfment of cytoplasmic proteins and recycling of cellular contents in preparation for mating.

p300 Acetylates JHDM1A to inhibit osteosarcoma carcinogenesis.

JHDM1A participates in cancer development via demethylate dimethyl histone H3 lysine 36 (H3K36me2). p300 is an intrinsic acetyltransferase. This study explored the acetyltransferase activity of p300 on JHDM1A and analyzed the JHDM1A acetylation on H3K36me2 demethylation in osteosarcoma. Co-immunoprecipitation (CoIP) and immunoblotting assay found that p300 directly acetylated JHDM1A at K409 residue in osteosarcoma MG-63 and HOS cells. Nucleosomes and mononucleosomes were prepared and found that acetylation of JHDMIA disrupted its association with nucleosomes and thereby impaired its capability to induce H3K36me2 demethylation. Moreover, chromatin immunoprecipitation (ChIP) assay discovered that the input levels of H3K36me2 in the promoter regions of p21 and puma were increased after acetylation of JHDM1A, which raised the p21 and puma mRNA levels in the cells. Finally, the analysis of JHDM1A acetylation on osteosarcoma cell proliferation and invasion, along with tumor growth pointed out that acetylation of JHDMIA inhibited the proliferation and invasion of osteosarcoma HOS cells, as well as suppressed the tumor growth of osteosarcoma. In conclusion, the outcomes of our research verified that p300 could directly acetylate JHDM1A at K409 site, which reduces the demethylation of H3K36me2, enhanced the transcription of p21 and puma, and thereby inhibited the growth and metastasis of osteosarcoma.

GGTase3 is a newly identified geranylgeranyltransferase targeting a ubiquitin ligase.

Protein prenylation is believed to be catalyzed by three heterodimeric enzymes: FTase, GGTase1 and GGTase2. Here we report the identification of a previously unknown human prenyltransferase complex consisting of an orphan prenyltransferase α-subunit, PTAR1, and the catalytic ß-subunit of GGTase2, RabGGTB. This enzyme, which we named GGTase3, geranylgeranylates FBXL2 to allow its localization at cell membranes, where this ubiquitin ligase mediates the polyubiquitylation of membrane-anchored proteins. In cells, FBXL2 is specifically recognized by GGTase3 despite having a typical carboxy-terminal CaaX prenylation motif that is predicted to be recognized by GGTase1. Our crystal structure analysis of the full-length GGTase3-FBXL2-SKP1 complex reveals an extensive multivalent interface specifically formed between the leucine-rich repeat domain of FBXL2 and PTAR1, which unmasks the structural basis of the substrate-enzyme specificity. By uncovering a missing prenyltransferase and its unique mode of substrate recognition, our findings call for a revision of the 'prenylation code'.

An Oxygen-Dependent Interaction between FBXL5 and the CIA-Targeting Complex Regulates Iron Homeostasis.

The iron-sensing protein FBXL5 is the substrate adaptor for a SKP1-CUL1-RBX1 E3 ubiquitin ligase complex that regulates the degradation of iron regulatory proteins (IRPs). Here, we describe a mechanism of FBXL5 regulation involving its interaction with the cytosolic Fe-S cluster assembly (CIA) targeting complex composed of MMS19, FAM96B, and CIAO1. We demonstrate that the CIA-targeting complex promotes the ability of FBXL5 to degrade IRPs. In addition, the FBXL5-CIA-targeting complex interaction is regulated by oxygen (O2) tension displaying a robust association in 21% O2 that is severely diminished in 1% O2 and contributes to O2-dependent regulation of IRP degradation. Together, these data identify a novel oxygen-dependent signaling axis that links IRP-dependent iron homeostasis with the Fe-S cluster assembly machinery.

Lysine-specific demethylase 2A enhances binding of various nuclear factors to CpG-rich genomic DNAs by action of its CXXC-PHD domain.

The lysine-specific demethylase 2A gene (KDM2A) is ubiquitously expressed and its transcripts consist of several alternatively spliced forms, including KDM2A and the shorter form N782 that lacks the 3' end encoding F-box and LRR. KDM2A binds to numerous CpG-rich genomic loci and regulates various cellular activities; however, the mechanism of the pleiotropic function is unknown. Here, we identify the mechanism of KDM2A played by its CXXC-PHD domain. KDM2A is necessary for a rapid proliferation of post-natal keratinocytes while its 3' end eclipses the stimulatory effect. EGFP-N782 binds to chromatin together with the XRCC5/6 complex, and the CXXC-PHD domain regulates the CpG-rich IGFBPL1 promoter. In vitro, CXXC-PHD enhances binding of nuclear extract ORC3 to the CpG-rich promoter, but not to the AT-rich DIP2B promoter to which ORC3 binds constitutively. Furthermore, CXXC-PHD recruits 94 nuclear factors involved in replication, ribosome synthesis, and mitosis, including POLR1A to the IGFBPL1 promoter. This recruitment is unprecedented; however, the result suggests that these nuclear factors bind to their cognate loci, as substantiated by the result that CXXC-PHD recruits POLR1A to the rDNA promoter. We propose that CXXC-PHD promotes permissiveness for nuclear factors to interact, but involvement of the XRCC5/6 complex in the recruitment is undetermined.