FBXL4: safeguarding against mitochondrial depletion through suppression of mitophagy.

Mitophagy is a critical mitochondrial quality control process that selectively removes dysfunctional or excess mitochondria through the autophagy-lysosome system. The process is tightly controlled to ensure cellular and physiological homeostasis. Insufficient mitophagy can result in failure to remove damaged mitochondria and consequent cellular degeneration, but it is equally important to appropriately restrain mitophagy to prevent excessive mitochondrial depletion. Here, we discuss our recent discovery that the SKP1-CUL1-F-box (SCF)-FBXL4 (F-box and leucine-rich repeat protein 4) E3 ubiquitin ligase localizes to the mitochondrial outer membrane, where it constitutively mediates the ubiquitination and degradation of BNIP3L/NIX and BNIP3 mitophagy receptors to suppress mitophagy. The post-translational regulation of BNIP3L and BNIP3 is disrupted in mitochondrial DNA depletion syndrome 13 (MTDPS13), a multi-systemic disorder caused by mutations in the FBXL4 gene and characterized by elevated mitophagy and mitochondrial DNA/mtDNA depletion in patient fibroblasts. Our results demonstrate that mitophagy is not solely stimulated in response to specific conditions but is instead also actively suppressed through the continuous degradation of BNIP3L and BNIP3 mediated by the SCF-FBXL4 ubiquitin ligase. Thus, cellular conditions or signaling events that prevent the FBXL4-mediated turnover of BNIP3L and BNIP3 on specific mitochondria are expected to facilitate their selective removal.

PPTC7 maintains mitochondrial protein content by suppressing receptor-mediated mitophagy.

PPTC7 is a resident mitochondrial phosphatase essential for maintaining proper mitochondrial content and function. Newborn mice lacking Pptc7 exhibit aberrant mitochondrial protein phosphorylation, suffer from a range of metabolic defects, and fail to survive beyond one day after birth. Using an inducible knockout model, we reveal that loss of Pptc7 in adult mice causes marked reduction in mitochondrial mass and metabolic capacity with elevated hepatic triglyceride accumulation. Pptc7 knockout animals exhibit increased expression of the mitophagy receptors BNIP3 and NIX, and Pptc7-/- mouse embryonic fibroblasts (MEFs) display a major increase in mitophagy that is reversed upon deletion of these receptors. Our phosphoproteomics analyses reveal a common set of elevated phosphosites between perinatal tissues, adult liver, and MEFs, including multiple sites on BNIP3 and NIX, and our molecular studies demonstrate that PPTC7 can directly interact with and dephosphorylate these proteins. These data suggest that Pptc7 deletion causes mitochondrial dysfunction via dysregulation of several metabolic pathways and that PPTC7 may directly regulate mitophagy receptor function or stability. Overall, our work reveals a significant role for PPTC7 in the mitophagic response and furthers the growing notion that management of mitochondrial protein phosphorylation is essential for ensuring proper organelle content and function.

FBXL4 suppresses mitophagy by restricting the accumulation of NIX and BNIP3 mitophagy receptors.

To maintain both mitochondrial quality and quantity, cells selectively remove damaged or excessive mitochondria through mitophagy, which is a specialised form of autophagy. Mitophagy is induced in response to diverse conditions, including hypoxia, cellular differentiation and mitochondrial damage. However, the mechanisms that govern the removal of specific dysfunctional mitochondria under steady-state conditions to fine-tune mitochondrial content are not well understood. Here, we report that SCFFBXL4 , an SKP1/CUL1/F-box protein ubiquitin ligase complex, localises to the mitochondrial outer membrane in unstressed cells and mediates the constitutive ubiquitylation and degradation of the mitophagy receptors NIX and BNIP3 to suppress basal levels of mitophagy. We demonstrate that the pathogenic variants of FBXL4 that cause encephalopathic mtDNA depletion syndrome (MTDPS13) do not efficiently interact with the core SCF ubiquitin ligase machinery or mediate the degradation of NIX and BNIP3. Thus, we reveal a molecular mechanism whereby FBXL4 actively suppresses mitophagy by preventing NIX and BNIP3 accumulation. We propose that the dysregulation of NIX and BNIP3 turnover causes excessive basal mitophagy in FBXL4-associated mtDNA depletion syndrome.

Centrosome Reduction Promotes Terminal Differentiation of Human Cardiomyocytes.

Centrosome reduction and redistribution of pericentriolar material (PCM) coincides with cardiomyocyte transitions to a post-mitotic and matured state. However, it is unclear whether centrosome changes are a cause or consequence of terminal differentiation. We validated that centrosomes were intact and functional in proliferative human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), consistent with their immature phenotype. We generated acentrosomal hPSC-CMs, through pharmacological inhibition of centriole duplication, and showed that centrosome loss was sufficient to promote post-mitotic transitions and aspects of cardiomyocyte maturation. As Hippo kinases are activated during post-natal cardiac maturation, we pharmacologically activated the Hippo pathway using C19, which was sufficient to trigger centrosome disassembly and relocalization of PCM components to perinuclear membranes. This was due to specific activation of Hippo kinases, as direct inhibition of YAP-TEAD interactions with verteporfin had no effect on centrosome organization. This suggests that Hippo kinase-centrosome remodeling may play a direct role in cardiac maturation.

PARP1-dependent recruitment of the FBXL10-RNF68-RNF2 ubiquitin ligase to sites of DNA damage controls H2A.Z loading.

The mammalian FBXL10-RNF68-RNF2 ubiquitin ligase complex (FRRUC) mono-ubiquitylates H2A at Lys119 to repress transcription in unstressed cells. We found that the FRRUC is rapidly and transiently recruited to sites of DNA damage in a PARP1- and TIMELESS-dependent manner to promote mono-ubiquitylation of H2A at Lys119, a local decrease of H2A levels, and an increase of H2A.Z incorporation. Both the FRRUC and H2A.Z promote transcriptional repression, double strand break signaling, and homologous recombination repair (HRR). All these events require both the presence and activity of the FRRUC. Moreover, the FRRUC and its activity are required for the proper recruitment of BMI1-RNF2 and MEL18-RNF2, two other ubiquitin ligases that mono-ubiquitylate Lys119 in H2A upon genotoxic stress. Notably, whereas H2A.Z is not required for H2A mono-ubiquitylation, impairment of the latter results in the inhibition of H2A.Z incorporation. We propose that the recruitment of the FRRUC represents an early and critical regulatory step in HRR.

FEM1 proteins are ancient regulators of SLBP degradation.

FEM1A, FEM1B, and FEM1C are evolutionarily-conserved VHL-box proteins, the substrate recognition subunits of CUL2-RING E3 ubiquitin ligase complexes. Here, we report that FEM1 proteins are ancient regulators of Stem-Loop Binding Protein (SLBP), a conserved protein that interacts with the stem loop structure located in the 3' end of canonical histone mRNAs and functions in mRNA cleavage, translation and degradation. SLBP levels are highest during S-phase coinciding with histone synthesis. The ubiquitin ligase complex SCFcyclin F targets SLBP for degradation in G2 phase; however, the regulation of SLBP during other stages of the cell cycle is poorly understood. We provide evidence that FEM1A, FEM1B, and FEM1C interact with and mediate the degradation of SLBP. Cyclin F, FEM1A, FEM1B and FEM1C all interact with a region in SLBP's N-terminus using distinct degrons. An SLBP mutant that is unable to interact with all 4 ligases is expressed at higher levels than wild type SLBP and does not oscillate during the cell cycle. We demonstrate that orthologues of SLBP and FEM1 proteins interact in C. elegans and D. melanogaster, suggesting that the pathway is evolutionarily conserved. Furthermore, we show that FEM1 depletion in C. elegans results in the upregulation of SLBP ortholog CDL-1 in oocytes. Notably, cyclin F is absent in flies and worms, suggesting that FEM1 proteins play an important role in SLBP targeting in lower eukaryotes.

Degradation of Cep68 and PCNT cleavage mediate Cep215 removal from the PCM to allow centriole separation, disengagement and licensing.

An intercentrosomal linker keeps a cell's two centrosomes joined together until it is dissolved at the onset of mitosis. A second connection keeps daughter centrioles engaged to their mothers until they lose their orthogonal arrangement at the end of mitosis. Centriole disengagement is required to license centrioles for duplication. We show that the intercentrosomal linker protein Cep68 is degraded in prometaphase through the SCF(ßTrCP) (Skp1-Cul1-F-box protein) ubiquitin ligase complex. Cep68 degradation is initiated by PLK1 phosphorylation of Cep68 on Ser 332, allowing recognition by ßTrCP. We also found that Cep68 forms a complex with Cep215 (also known as Cdk5Rap2) and PCNT (also known as pericentrin), two PCM (pericentriolar material) proteins involved in centriole engagement. Cep68 and PCNT bind to different pools of Cep215. We propose that Cep68 degradation allows Cep215 removal from the peripheral PCM preventing centriole separation following disengagement, whereas PCNT cleavage mediates Cep215 removal from the core of the PCM to inhibit centriole disengagement and duplication.

SCF ubiquitin ligase-targeted therapies.

The clinical successes of proteasome inhibitors for the treatment of cancer have highlighted the therapeutic potential of targeting this protein degradation system. However, proteasome inhibitors prevent the degradation of numerous proteins, which may cause adverse effects. Increased specificity could be achieved by inhibiting the components of the ubiquitin-proteasome system that target specific subsets of proteins for degradation. F-box proteins are the substrate-targeting subunits of SKP1-CUL1-F-box protein (SCF) ubiquitin ligase complexes. Through the degradation of a plethora of diverse substrates, SCF ubiquitin ligases control a multitude of processes at the cellular and organismal levels, and their dysregulation is implicated in many pathologies. SCF ubiquitin ligases are characterized by their high specificity for substrates, and these ligases therefore represent promising drug targets. However, the potential for therapeutic manipulation of SCF complexes remains an underdeveloped area. This Review explores and discusses potential strategies to target SCF-mediated biological processes to treat human diseases.

Mechanisms and function of substrate recruitment by F-box proteins.

S phase kinase-associated protein 1 (SKP1)-cullin 1 (CUL1)-F-box protein (SCF) ubiquitin ligase complexes use a family of F-box proteins as substrate adaptors to mediate the degradation of a large number of regulatory proteins involved in diverse processes. The dysregulation of SCF complexes and their substrates contributes to multiple pathologies. In the 14 years since the identification and annotation of the F-box protein family, the continued identification and characterization of novel substrates has greatly expanded our knowledge of the regulation of substrate targeting and the roles of F-box proteins in biological processes. Here, we focus on the evolution of our understanding of substrate recruitment by F-box proteins, the dysregulation of substrate recruitment in disease and potential avenues for F-box protein-directed disease therapies.

FBXO11 targets BCL6 for degradation and is inactivated in diffuse large B-cell lymphomas.

BCL6 is the product of a proto-oncogene implicated in the pathogenesis of human B-cell lymphomas. By binding specific DNA sequences, BCL6 controls the transcription of a variety of genes involved in B-cell development, differentiation and activation. BCL6 is overexpressed in the majority of patients with aggressive diffuse large B-cell lymphoma (DLBCL), the most common lymphoma in adulthood, and transgenic mice constitutively expressing BCL6 in B cells develop DLBCLs similar to the human disease. In many DLBCL patients, BCL6 overexpression is achieved through translocation (~40%) or hypermutation of its promoter (~15%). However, many other DLBCLs overexpress BCL6 through an unknown mechanism. Here we show that BCL6 is targeted for ubiquitylation and proteasomal degradation by a SKP1­CUL1­F-box protein (SCF) ubiquitin ligase complex that contains the orphan F-box protein FBXO11 (refs 5, 6). The gene encoding FBXO11 was found to be deleted or mutated in multiple DLBCL cell lines, and this inactivation of FBXO11 correlated with increased levels and stability of BCL6. Similarly, FBXO11 was either deleted or mutated in primary DLBCLs. Notably, tumour-derived FBXO11 mutants displayed an impaired ability to induce BCL6 degradation. Reconstitution of FBXO11 expression in FBXO11-deleted DLBCL cells promoted BCL6 ubiquitylation and degradation, inhibited cell proliferation, and induced cell death. FBXO11-deleted DLBCL cells generated tumours in immunodeficient mice, and the tumorigenicity was suppressed by FBXO11 reconstitution. We reveal a molecular mechanism controlling BCL6 stability and propose that mutations and deletions in FBXO11 contribute to lymphomagenesis through BCL6 stabilization. The deletions/mutations found in DLBCLs are largely monoallelic, indicating that FBXO11 is a haplo-insufficient tumour suppressor gene.

Phosphorylation of Ser72 is dispensable for Skp2 assembly into an active SCF ubiquitin ligase and its subcellular localization.

F-box proteins are the substrate recognition subunits of SCF (Skp1, Cul1, F-box protein) ubiquitin ligase complexes. Skp2 is a nuclear F-box protein that targets the CDK inhibitor p27 for ubiquitin- and proteasome-dependent degradation. In G(0) and during the G(1) phase of the cell cycle, Skp2 is degraded via the APC/C(Cdh1) ubiquitin ligase to allow stabilization of p27 and inhibition of CDKs, facilitating the maintenance of the G(0)/G(1) state. APC/C(Cdh1) binds Skp2 through an N-terminal domain (amino acids 46-94 in human Skp2). It has been shown that phosphorylation of Ser64 and Ser72 in this domain dissociates Skp2 from APC/C. More recently, it has instead been proposed that phosphorylation of Skp2 on Ser72 by Akt/PKB allows Skp2 binding to Skp1, promoting the assembly of an active SCF(Skp2) ubiquitin ligase, and Skp2 relocalization/retention into the cytoplasm, promoting cell migration via an unknown mechanism. According to these reports, a Skp2 mutant in which Ser72 is substituted with Ala is unable to promote cell proliferation and loses its oncogenic potential. Given the contrasting reports, we revisited these results and conclude that phosphorylation of Skp2 on Ser72 does not control Skp2 binding to Skp1 and Cul1, has no influence on SCF(Skp2) ubiquitin ligase activity, and does not affect the subcellular localization of Skp2.

A novel corepressor, BCoR-L1, represses transcription through an interaction with CtBP.

Corepressors play a crucial role in negative gene regulation and are defective in several diseases. BCoR is a corepressor for the BCL6 repressor protein. Here we describe and functionally characterize BCoR-L1, a homolog of BCoR. When tethered to a heterologous promoter, BCoR-L1 is capable of strong repression. Like other corepressors, BCoR-L1 associates with histone deacetylase (HDAC) activity. Specifically, BCoR-L1 coprecipitates with the Class II HDACs, HDAC4, HDAC5, and HDAC7, suggesting that they are involved in its role as a transcriptional repressor. BCoR-L1 also interacts with the CtBP corepressor through a CtBP-interacting motif in its amino terminus. Abrogation of the CtBP binding site within BCoR-L1 partially relieves BCoR-L1-mediated transcriptional repression. Furthermore, BCoR-L1 is located on the E-cadherin promoter, a known CtBP-regulated promoter, and represses the E-cadherin promoter activity in a reporter assay. The inhibition of BCoR-L1 expression by RNA-mediated interference results in derepression of E-cadherin in cells that do not normally express E-cadherin, indicating that BCoR-L1 contributes to the repression of an authentic endogenous CtBP target.

Cytokine secretion via cholesterol-rich lipid raft-associated SNAREs at the phagocytic cup.

Lipopolysaccharide-activated macrophages rapidly synthesize and secrete tumor necrosis factor alpha (TNFalpha) to prime the immune system. Surface delivery of membrane carrying newly synthesized TNFalpha is controlled and limited by the level of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin 4 and SNAP-23. Many functions in immune cells are coordinated from lipid rafts in the plasma membrane, and we investigated a possible role for lipid rafts in TNFalpha trafficking and secretion. TNFalpha surface delivery and secretion were found to be cholesterol-dependent. Upon macrophage activation, syntaxin 4 was recruited to cholesterol-dependent lipid rafts, whereas its regulatory protein, Munc18c, was excluded from the rafts. Syntaxin 4 in activated macrophages localized to discrete cholesterol-dependent puncta on the plasma membrane, particularly on filopodia. Imaging the early stages of TNFalpha surface distribution revealed these puncta to be the initial points of TNFalpha delivery. During the early stages of phagocytosis, syntaxin 4 was recruited to the phagocytic cup in a cholesterol-dependent manner. Insertion of VAMP3-positive recycling endosome membrane is required for efficient ingestion of a pathogen. Without this recruitment of syntaxin 4, it is not incorporated into the plasma membrane, and phagocytosis is greatly reduced. Thus, relocation of syntaxin 4 into lipid rafts in macrophages is a critical and rate-limiting step in initiating an effective immune response.

The t-SNARE syntaxin 4 is regulated during macrophage activation to function in membrane traffic and cytokine secretion.

Activation of macrophages with lipopolysaccharide (LPS) induces the rapid synthesis and secretion of proinflammatory cytokines, such as tumor necrosis factor (TNFalpha), for priming the immune response. TNFalpha plays a key role in inflammatory disease; yet, little is known of the intracellular trafficking events leading to its secretion. In order to identify molecules involved in this secretory pathway, we asked whether any of the known trafficking proteins are regulated by LPS. We found that the levels of SNARE proteins were rapidly and significantly up- or downregulated during macrophage activation. A subset of t-SNAREs (Syntaxin 4/SNAP23/Munc18c) known to control regulated exocytosis in other cell types was substantially increased by LPS in a temporal pattern coinciding with peak TNFalpha secretion. Syntaxin 4 formed a complex with Munc18c at the cell surface of macrophages. Functional studies involving the introduction of Syntaxin 4 cDNA or peptides into macrophages implicate this t-SNARE in a rate-limiting step of TNFalpha secretion and in membrane ruffling during macrophage activation. We conclude that, in macrophages, SNAREs are regulated in order to accommodate the rapid onset of cytokine secretion and for membrane traffic associated with the phenotypic changes of immune activation. This represents a novel regulatory role for SNAREs in regulated secretion and in macrophage-mediated host defense.