F-box only protein 9 and its role in cancer.

The F-box proteins (FBP), substrate recognition subunit of the SCF (Skp1-Cullin1-F-box protein complex) E3 ligase, play important roles in the ubiquitylation and subsequent degradation of the target proteins from several cellular processes. Disorders of F-box protein-mediated proteolysis lead to human malignancies. FBP plays an important role in many cellular processes, including cell proliferation, cell cycle, apoptosis, migration, invasion, and metastasis, suggesting that it can be associated with tumorigenesis, cancer development and progression. However, the expression and function of FBXO9 (F-box only protein 9) differ in various types of human cancer. Due to the ability to regulate the stability and activity of oncogenes and tumor-suppressor genes, and the physiological functions of many of the F-box proteins remain subtle, further genetic and mechanistic studies will elaborate and help define FBXO9's role. Targeting F-box protein or F-box protein signaling pathways could be an effective strategy for preventing or treating human cancer. This review is presented to summarize the part of FBXO9 in different types of human cancer and its regulation mechanism, and to pave the way to design FBXO9-targeting anticancer therapies.

FBXL19-AS1 aggravates the progression of hepatocellular cancer by downregulating KLF2.

PURPOSE: To uncover the role of FBXL19-AS1 in aggravating the progression of hepatocellular cancer (HCC) by downregulating kruppel-likefactor2 (KLF2). METHODS: FBXL19-AS1 level in HCC tissues and adjacent normal tissues were firstly determined. Its level in HCC with different tumor sizes (≤ 5 cm or > 5 cm) and different tumor stages (stage I-II or III-IV) was examined as well. Subcellular distribution of FBXL19-AS1 was detected. The regulatory effect of FBXL19-AS1 on viability, apoptosis and cell cycle progression of HCC cells was assessed. RNA immunoprecipitation (RIP) assay was conducted to explore the interaction between FBXL19-AS1 with EZH2 and SUZ12. Moreover, chromatin immunoprecipitation (ChIP) assay was carried out to identify the recruitment ability of FBXL19-AS1 on EZH2 and H3K27me3. Finally, the potential role of KLF2 in FBXL19-AS1-mediated HCC proliferation was investigated. RESULTS: FBXL19-AS1 was highly expressed in HCC tissues, especially in those larger than 5 cm in tumor size and worse tumor stage. FBXL19-AS1 was mainly distributed in nucleus and interacted with EZH2 and SUZ12. Knockdown of FBXL19-AS1 suppressed proliferation, cell cycle progression and induced apoptosis of HCC cells. Moreover, silence of FBXL19-AS1 attenuated the recruitment ability of EZH2 on KLF2. Knockdown of KLF2 reversed the regulatory effect of FBXL19-AS1 on proliferative ability of HCC cells. CONCLUSIONS: Long non-coding RNA (lncRNA) FBXL19-AS1 is upregulated in HCC. It accelerates proliferative ability, cell cycle progression and suppresses apoptosis of tumor cells through interacting with KLF2, thus aggravating the progression of HCC.

TP53/p53-FBXO22-TFEB controls basal autophagy to govern hormesis.

Preconditioning with a mild stressor such as fasting is a promising way to reduce severe side effects from subsequent chemo- or radiotherapy. However, the underlying mechanisms have been largely unexplored. Here, we demonstrate that the TP53/p53-FBXO22-TFEB (transcription factor EB) axis plays an essential role in this process through upregulating basal macroautophagy/autophagy. Mild stress-activated TP53 transcriptionally induced FBXO22, which in turn ubiquitinated KDM4B (lysine-specific demethylase 4B) complexed with MYC-NCOR1 suppressors for degradation, leading to transcriptional induction of TFEB. Upregulation of autophagy-related genes by increased TFEB dramatically enhanced autophagic activity and cell survival upon following a severe stressor. Mitogen-induced AKT1 activation counteracted this process through the phosphorylation of KDM4B, which inhibited FBXO22-mediated ubiquitination. Additionally, fbxo22-/- mice died within 10 h of birth, and their mouse embryonic fibroblasts (MEFs) showed a lowered basal autophagy, whereas FBXO22-overexpressing mice were resistant to chemotherapy. Taken together, these results suggest that TP53 upregulates basal autophagy through the FBXO22-TFEB axis, which governs the hormetic effect in chemotherapy.Abbreviations: BBC3/PUMA: BCL2 binding component 3; CDKN1A/p21: cyclin dependent kinase inhibitor 1A; ChIP-seq: chromatin immunoprecipitation followed by sequencing; DDB2: damage specific DNA binding protein 2; DRAM: DNA damage regulated autophagy modulator; ESR/ER: estrogen receptor 1; FMD: fasting mimicking diet; HCQ: hydroxychloroquine; KDM4B: lysine-specific demethylase 4B; MAP1LC3/LC3: microtubule associated protein 1 light chain 3 alpha; MEFs: mouse embryonic fibroblasts; MTOR: mechanistic target of rapamycin kinase; NCOR1: nuclear receptor corepressor 1; SCF: SKP1-CUL-F-box protein; SQSTM1: sequestosome 1; TFEB: transcription factor EB.

The histone demethylase KDM2B regulates human primordial germ cell-like cells specification.

Germline specification is a fundamental step for human reproduction and this biological phenomenon possesses technical challenges to study in vivo as it occurs immediately after blastocyst implantation. The establishment of in vitro human primordial germ cell-like cells (hPGCLCs) induction system allows sophisticated characterization of human primordial germ cells (hPGCs) development. However, the underlying molecular mechanisms of hPGCLC specification are not fully elucidated. Here, we observed particularly high expression of the histone demethylase KDM2B in male fetal germ cells (FGCs) but not in male somatic cells. Besides, KDM2B shared similar expression pattern with hPGC marker genes in hPGCLCs, suggesting an important role of KDM2B in germ cell development. Although deletion of KDM2B had no significant effects on human embryonic stem cell (hESC)'s pluripotency, loss of KDM2B dramatically impaired hPGCLCs differentiation whereas ectopically expressed KDM2B could efficiently rescue such defect, indicating this defect was due to KDM2B's loss in hPGCLC specification. Mechanistically, as revealed by the transcriptional profiling, KDM2B suppressed the expression of somatic genes thus inhibited somatic differentiation during hPGCLC specification. These data collectively indicate that KDM2B is an indispensable epigenetic regulator for hPGCLC specification, shedding lights on how epigenetic regulations orchestrate transcriptional events in hPGC development for future investigation.

Degradation of STOP1 mediated by the F-box proteins RAH1 and RAE1 balances aluminum resistance and plant growth in Arabidopsis thaliana.

The C2H2-type zinc finger transcription factor sensitive to proton rhizotoxicity 1 (STOP1) is crucial for aluminum (Al) resistance in Arabidopsis. The F-box protein Regulation of AtALMT1 Expression 1 (RAE1) was recently reported to regulate the stability of STOP1. There is a unique homolog of RAE1, RAH1 (RAE1 homolog 1), in Arabidopsis, but the biological function of RAH1 is still not known. In this study, we characterize the role of RAH1 and/or RAE1 in the regulation of Al resistance and plant growth. We demonstrate that RAH1 can directly interact with STOP1 and promote its ubiquitination and degradation. RAH1 is preferentially expressed in root caps and various vascular tissues, and its expression is induced by Al and controlled by STOP1. Mutation of RAH1 in rae1 but not the wild-type (WT) background increases the level of STOP1 protein, leading to increased expression of STOP1-regulated genes and enhanced Al resistance. Interestingly, the rah1rae1 double mutant shows reduced plant growth compared with the WT and single mutants under normal conditions, and introduction of stop1 mutation into the double mutant background can rescue its reduced plant growth phenotype. Our results thus reveal that RAH1 plays an unequally redundant role with RAE1 in the modulation of STOP1 stability and plant growth, and dynamic regulation of the STOP1 level is critical for the balance of Al resistance and normal plant growth.

Downregulation of autophagy by Met30-mediated Atg9 ubiquitination.

Macroautophagy/autophagy is a highly conserved eukaryotic molecular process that facilitates the recycling of superfluous cytoplasmic materials, damaged organelles, and invading pathogens, resulting in proper cellular homeostasis and survival during stress conditions. Autophagy is stringently regulated at multiple stages, including control at transcriptional, translational, and posttranslational levels. In this work, we identified a mechanism by which regulation of autophagy is achieved through the posttranslational modification of Atg9. Here, we show that, in order to limit autophagy to a low, basal level during normal conditions, Atg9 is ubiquitinated and subsequently targeted for degradation in a proteasome-dependent manner through the action of the E3 ligase Met30. When cells require increased autophagy flux to respond to nutrient deprivation, the proteolysis of Atg9 is significantly reduced. Overall, this work reveals an additional layer of mechanistic regulation that allows cells to further maintain appropriate levels of autophagy and to rapidly induce this process in response to stress.

Caenorhabditis elegans F-Box Protein Promotes Axon Regeneration by Inducing Degradation of the Mad Transcription Factor.

In Caenorhabditis elegans, axon regeneration is activated by a signaling cascade through the receptor tyrosine kinase (RTK) SVH-2. Axonal injury induces svh-2 gene expression by degradation of the Mad-like transcription factor MDL-1. In this study, we identify the svh-24/sdz-33 gene encoding a protein containing F-box and F-box-associated domains as a regulator of axon regeneration in motor neurons. We find that sdz-33 is required for axon injury-induced svh-2 expression. SDZ-33 targets MDL-1 for poly-ubiquitylation and degradation. Furthermore, we demonstrate that SDZ-33 promotes axotomy-induced nuclear degradation of MDL-1, resulting in the activation of svh-2 expression in animals. These results suggest that the F-box protein is required for RTK signaling in the control of axon regeneration.SIGNIFICANCE STATEMENT In Caenorhabditis elegans, axon regeneration is positively regulated by the growth factor SVH-1 and its receptor tyrosine kinase SVH-2. Expression of the svh-2 gene is induced by axonal injury via the Ets-like transcription factor ETS-4, whose transcriptional activity is inhibited by the Mad-like transcription factor MDL-1. Axon injury leads to the degradation of MDL-1, and this is linked to the activation of ETS-4 transcriptional activity. In this study, we identify the sdz-33 gene encoding a protein containing an F-box domain as a regulator of axon regeneration. We demonstrate that MDL-1 is poly-ubiquitylated and degraded through the SDZ-33-mediated 26S proteasome pathway. These results reveal that an F-box protein promotes axon regeneration by degrading the Mad transcription factor.

Production of ROS by Gallic Acid Activates KDM2A to Reduce rRNA Transcription.

Metformin, which is suggested to have anti-cancer effects, activates KDM2A to reduce rRNA transcription and proliferation of cancer cells. Thus, the specific activation of KDM2A may be applicable to the treatment of cancers. In this study, we screened a food-additive compound library to identify compounds that control cell proliferation. We found that gallic acid activated KDM2A to reduce rRNA transcription and cell proliferation in breast cancer MCF-7 cells. Gallic acid accelerated ROS production and activated AMPK. When ROS production or AMPK activity was inhibited, gallic acid did not activate KDM2A. These results suggest that both ROS production and AMPK activation are required for activation of KDM2A by gallic acid. Gallic acid did not reduce the succinate level, which was required for KDM2A activation by metformin. Metformin did not elevate ROS production. These results suggest that the activation of KDM2A by gallic acid includes mechanisms distinct from those by metformin. Therefore, signals from multiple intracellular conditions converge in KDM2A to control rRNA transcription. Gallic acid did not induce KDM2A-dependent anti-proliferation activity in non-tumorigenic MCF10A cells. These results suggest that the mechanism of KDM2A activation by gallic acid may be applicable to the treatment of breast cancers.

SlTLFP8 reduces water loss to improve water-use efficiency by modulating cell size and stomatal density via endoreduplication.

Improving plant water-use efficiency (WUE) is important to plant survival and crop yield in the context of water limitation. In this study, SlTLFP8 (Tubby-like F-box protein 8) was identified as an osmotic-induced gene in tomato. Transgenic tomato with up-regulated expression of SlTLFP8 showed enhanced water-deficient resistance, whereas knockout mutants generated by CRISPR/Cas9 were more sensitive to water deficit. SlTLFP8 overexpression significantly enhanced WUE by suppressing transpiration under both water-sufficient and water-deficient conditions. Further study showed that overexpressing SlTLFP8 significantly increased leaf epidermal cell size and thereby decreased stomatal density 10-20%, conversely SlTLFP8 knockout resulted in decreased cell size and thereby increased stomatal density 20-50%. SlTLFP8 overexpression and knockout modulated ploidy levels in leaf cells. Changes in expression of cell cycle related genes also indicated that SlTLFP8 affected cell size and stomatal density through endocycle transition. Despite changes in stomata density and transpiration, altering the expression of SlTLFP8 did not change photosynthesis. Additionally, biomass was not altered and there was little difference in fruit yield for transgenic and wild type lines under water-sufficient and water-deficient conditions. Our results demonstrate the effect of SlTLFP8 on endoreduplication and the potential of SlTLFP8 for improvement of WUE. BRIEF SUMMERY: This work found a new mechanism of TLP (Tubby like protein) response to water-deficient stress. SlTLFP8, a member of TLP family, regulates water-deficient resistance by modulating water loss via affecting stomatal density. Expression of SlTLFP8 was induced by osmotic stress. Transgenic tomato lines with SlTLFP8 overexpression or SlTLFP8 knockout showed significantly differences in water-use efficiency (WUE) and water-deficient resistance. The difference of leaf water loss caused by transpiration is the main explanation of the difference in WUE and water-deficient resistance. Additionally, overexpressing SlTLFP8 significantly decreased stomatal density, while SlTLFP8 knockout resulted in increased stomatal density, and SlTLFP8 affected stomatal density through endoreduplication and altered epidermal cell size. Despite changes in stomata density, altering the expression of SlTLFP8 did not result in distinct changes in photosynthesis, biomass and yield of tomato.

Protective effect of FBXL10 in myocardial ischemia reperfusion injury via inhibiting endoplasmic reticulum stress.

OBJECTIVE: The aim of the study was to investigate the mechanism and effect of FBXL10 in myocardial ischemia reperfusion injury in vivo and in vitro. METHODS: The myocardial ischemia reperfusion (I/R) model was established by 30 min of coronary occlusion followed by 2 h of reperfusion in rats. Western blot and TUNEL assay were used to measure the apoptosis during I/R. The expression levels of endoplasmic reticulum related proteins in myocardial tissues and H9c2 cells were detected by immunohistochemistry staining and immunofluorescence staining. Flow cytometry and CCK-8 were used to detect the apoptosis and viability of H9c2 cells. RESULTS: The results revealed that FBXL10 significantly reduced myocardial infarction, improved the pathological morphology of myocardium, markedly reduced inflammatory response in the myocardial ischemia reperfusion rats. Moreover the expressions of endoplasmic reticulum stress key proteins were caused by I/R were suppressed significantly by FBXL10 treatment, including CHOP, GRP78, ATF4 and p-PERK. Additionally FBXL10 inhibited the expression of endoplasmic reticulum stress key proteins in H/R H9c2 cells. Furthermore, FBXL10 reduced the levels of apoptotic cells and inflammatory response compared with I/R and H/R group. CONCLUSION: Taken together, we found that FBXL10 could attenuate I/R injury through inhibiting endoplasmic reticulum stress (ERs).

Genetic analysis of the Arabidopsis TIR1/AFB auxin receptors reveals both overlapping and specialized functions.

The TIR1/AFB auxin co-receptors mediate diverse responses to the plant hormone auxin. The Arabidopsis genome encodes six TIR1/AFB proteins representing three of the four clades that were established prior to angiosperm radiation. To determine the role of these proteins in plant development we performed an extensive genetic analysis involving the generation and characterization of all possible multiply-mutant lines. We find that loss of all six TIR1/AFB proteins results in early embryo defects and eventually seed abortion, and yet a single wild-type allele of TIR1 or AFB2 is sufficient to support growth throughout development. Our analysis reveals extensive functional overlap between even the most distantly related TIR1/AFB genes except for AFB1. Surprisingly, AFB1 has a specialized function in rapid auxin-dependent inhibition of root growth and early phase of root gravitropism. This activity may be related to a difference in subcellular localization compared to the other members of the family.

Long non-coding RNA LncKdm2b regulates cortical neuronal differentiation by cis-activating Kdm2b.

The mechanisms underlying spatial and temporal control of cortical neurogenesis of the brain are largely elusive. Long non-coding RNAs (lncRNAs) have emerged as essential cell fate regulators. Here we found LncKdm2b (also known as Kancr), a lncRNA divergently transcribed from a bidirectional promoter of Kdm2b, is transiently expressed during early differentiation of cortical projection neurons. Interestingly, Kdm2b's transcription is positively regulated in cis by LncKdm2b, which has intrinsic-activating function and facilitates a permissive chromatin environment at the Kdm2b's promoter by associating with hnRNPAB. Lineage tracing experiments and phenotypic analyses indicated LncKdm2b and Kdm2b are crucial in proper differentiation and migration of cortical projection neurons. These observations unveiled a lncRNA-dependent machinery in regulating cortical neuronal differentiation.

LINC01436, regulating miR-585 and FBXO11, is an oncogenic lncRNA in the progression of gastric cancer.

Accumulating studies have indicated that long non-coding RNAs (lncRNAs) are crucial modulators in cancer biology. In this work, we investigated the function and related mechanisms of LINC01436 in the progression of gastric cancer (GC). We demonstrated that LINC01436 was significantly up-regulated in cancerous tissues of GC samples, and its overexpression was correlated with a worse prognosis for the patients. In the GC cell line BGC823 cells, LINC01436 knockdown repressed the proliferation and metastasis of cancer cells; conversely, in GC cell line AGS cells, overexpression of LINC01436 showed the opposite effects. We then demonstrated that miR-585, a tumor suppressor, could bind to both LINC01436 and the 3'-UTR of F-box protein 11 (FBOX11), and LINC01436 was proved to sponge miR-585 and repress it, and indirectly promoted the expression of FBOX11. Collectively, these results suggested that LINC01436 was an oncogenic lncRNA in GC and promoted proliferation and metastasis of GC cell via regulating miR-585 and FBOX11.

FBXL19-AS1 promotes cell proliferation and inhibits cell apoptosis via miR-876-5p/FOXM1 axis in breast cancer.

As the most common cancer and one of the leading causes of cancer-associated mortality, breast cancer continues to need more key molecules to regulate its progression. F-box and leucine-rich repeat protein 19 antisense RNA 1 (known as FBXL19-AS1) is a long non-coding RNA (lncRNA) which has been reported as an oncogene in several types of human cancers. However, the specific downstream targets of FBXL19-AS1 remain unknown. In this study, we set out to find more reliable downstream molecules of FBXL19-AS1 in breast cancer. FBXL19-AS1 was expressed at a high level in breast cancer cells. Loss-of-function experiments revealed that silencing FBXL19-AS1 could impair cell proliferation and induce cell apoptosis in breast cancer. In addition, the location of FBXL19-AS1 in the cytoplasm was detected by fluorescent in situ hybridization assay, while FBXL19-AS1 regulated the expression of Forkhead box M1 (FOXM1) by directly absorbing miR-876-5p. Through rescue assays, it was observed that FOXM1 overexpression recovered the inhibited tumor growth caused by FBXL19-AS1 downregulation. We affirmed the function of FBXL19-AS1 in breast cancer and described the mechanism of the FBXL19-AS1/miR-876-5p/FOXM1 axis. The current work presents the molecular mechanism which underlies FBXL19-AS1 in breast cancer and suggests a comprehensive, feasible FBXL19-AS1-mediated therapeutic approach for treating breast cancer.

FBXO47 regulates telomere-inner nuclear envelope integration by stabilizing TRF2 during meiosis.

During meiosis, telomere attachment to the inner nuclear envelope is required for proper pairing of homologous chromosomes and recombination. Here, we identified F-box protein 47 (FBXO47) as a regulator of the telomeric shelterin complex that is specifically expressed during meiotic prophase I. Knockout of Fbxo47 in mice leads to infertility in males. We found that the Fbxo47 deficient spermatocytes are unable to form a complete synaptonemal complex. FBXO47 interacts with TRF1/2, and the disruption of Fbxo47 destabilizes TRF2, leading to unstable telomere attachment and slow traversing through the bouquet stage. Our findings uncover a novel mechanism of FBXO47 in telomeric shelterin subunit stabilization during meiosis.

The FBXW2-MSX2-SOX2 axis regulates stem cell property and drug resistance of cancer cells.

SOX2 is a key transcription factor that plays critical roles in maintaining stem cell property and conferring drug resistance. However, the underlying mechanisms by which SOX2 level is precisely regulated remain elusive. Here we report that MLN4924, also known as pevonedistat, a small-molecule inhibitor of neddylation currently in phase II clinical trials, down-regulates SOX2 expression via causing accumulation of MSX2, a known transcription repressor of SOX2 expression. Mechanistic characterization revealed that MSX2 is a substrate of FBXW2 E3 ligase. FBXW2 binds to MSX2 and promotes MSX2 ubiquitylation and degradation. Likewise, FBXW2 overexpression shortens the protein half-life of MSX2, whereas FBXW2 knockdown extends it. We further identified hypoxia as a stress condition that induces VRK2 kinase to facilitate MSX2-FBXW2 binding and FBXW2-mediated MSX2 ubiquitylation and degradation, leading to SOX2 induction via derepression. Biologically, expression of FBXW2 or SOX2 promotes tumor sphere formation, which is blocked by MSX2 expression. By down-regulating SOX2 through inactivation of FBXW2 E3 ligase, MLN4924 sensitizes breast cancer cells to tamoxifen in both in vitro and in vivo cancer cell models. Thus, a negative cascade of the FBXW2-MSX2-SOX2 axis was established, which regulates stem cell property and drug resistance. Finally, an inverse correlation of expression was found between FBXW2 and MSX2 in lung and breast cancer tissues. Collectively, our study revealed an anticancer mechanism of MLN4924. By inactivating FBXW2, MLN4924 caused MSX2 accumulation to repress SOX2 expression, leading to suppression of stem cell property and sensitization of breast cancer cells to tamoxifen.

KDM2B in polycomb repressive complex 1.1 functions as a tumor suppressor in the initiation of T-cell leukemogenesis.

KDM2B together with RING1B, PCGF1, and BCOR or BCORL1 comprise polycomb repressive complex 1.1 (PRC1.1), a noncanonical PRC1 that catalyzes H2AK119ub1. It binds to nonmethylated CpG islands through its zinc finger-CxxC DNA binding domain and recruits the complex to target gene loci. Recent studies identified the loss of function mutations in the PRC1.1 gene, BCOR and BCORL1 in human T-cell acute lymphoblastic leukemia (T-ALL). We previously reported that Bcor insufficiency induces T-ALL in mice, supporting a tumor suppressor role for BCOR. However, the function of BCOR responsible for tumor suppression, either its corepressor function for BCL6 or that as a component of PRC1.1, remains unclear. We herein examined mice specifically lacking the zinc finger-CxxC domain of KDM2B in hematopoietic cells. Similar to Bcor-deficient mice, Kdm2b-deficient mice developed lethal T-ALL mostly in a NOTCH1-dependent manner. A chromatin immunoprecipitation sequence analysis of thymocytes revealed the binding of KDM2B at promoter regions, at which BCOR and EZH2 colocalized. KDM2B target genes markedly overlapped with those of NOTCH1 in human T-ALL cells, suggesting that noncanonical PRC1.1 antagonizes NOTCH1-mediated gene activation. KDM2B target genes were expressed at higher levels than the others and were marked with high levels of H2AK119ub1 and H3K4me3, but low levels of H3K27me3, suggesting that KDM2B target genes are transcriptionally active or primed for activation. These results indicate that PRC1.1 plays a key role in restricting excessive transcriptional activation by active NOTCH1, thereby acting as a tumor suppressor in the initiation of T-cell leukemogenesis.

KDM2 proteins constrain transcription from CpG island gene promoters independently of their histone demethylase activity.

CpG islands (CGIs) are associated with the majority of mammalian gene promoters and function to recruit chromatin modifying enzymes. It has therefore been proposed that CGIs regulate gene expression through chromatin-based mechanisms, however in most cases this has not been directly tested. Here, we reveal that the histone H3 lysine 36 (H3K36) demethylase activity of the CGI-binding KDM2 proteins contributes only modestly to the H3K36me2-depleted state at CGI-associated gene promoters and is dispensable for normal gene expression. Instead, we discover that KDM2 proteins play a widespread and demethylase-independent role in constraining gene expression from CGI-associated gene promoters. We further show that KDM2 proteins shape RNA Polymerase II occupancy but not chromatin accessibility at CGI-associated promoters. Together this reveals a demethylase-independent role for KDM2 proteins in transcriptional repression and uncovers a new function for CGIs in constraining gene expression.

Toxoplasma F-box protein 1 is required for daughter cell scaffold function during parasite replication.

By binding to the adaptor protein SKP1 and serving as substrate receptors for the SKP1 Cullin, F-box E3 ubiquitin ligase complex, F-box proteins regulate critical cellular processes including cell cycle progression and membrane trafficking. While F-box proteins are conserved throughout eukaryotes and are well studied in yeast, plants, and animals, studies in parasitic protozoa are lagging. We have identified eighteen putative F-box proteins in the Toxoplasma genome of which four have predicted homologs in Plasmodium. Two of the conserved F-box proteins were demonstrated to be important for Toxoplasma fitness and here we focus on an F-box protein, named TgFBXO1, because it is the most highly expressed by replicative tachyzoites and was also identified in an interactome screen as a Toxoplasma SKP1 binding protein. TgFBXO1 interacts with Toxoplasma SKP1 confirming it as a bona fide F-box protein. In interphase parasites, TgFBXO1 is a component of the Inner Membrane Complex (IMC), which is an organelle that underlies the plasma membrane. Early during replication, TgFBXO1 localizes to the developing daughter cell scaffold, which is the site where the daughter cell IMC and microtubules form and extend from. TgFBXO1 localization to the daughter cell scaffold required centrosome duplication but before kinetochore separation was completed. Daughter cell scaffold localization required TgFBXO1 N-myristoylation and was dependent on the small molecular weight GTPase, TgRab11b. Finally, we demonstrate that TgFBXO1 is required for parasite growth due to its function as a daughter cell scaffold effector. TgFBXO1 is the first F-box protein to be studied in apicomplexan parasites and represents the first protein demonstrated to be important for daughter cell scaffold function.

PIP degron-stabilized Dacapo/p21Cip1 and mutations in ago act in an anti- versus pro-proliferative manner, yet both trigger an increase in Cyclin E levels.

During cell cycle progression, the activity of the CycE-Cdk2 complex gates S-phase entry. CycE-Cdk2 is inhibited by CDK inhibitors (CKIs) of the Cip/Kip family, which include the human p21Cip1 and Drosophila Dacapo (Dap) proteins. Both the CycE and Cip/Kip family proteins are under elaborate control via protein degradation, mediated by the Cullin-RING ligase (CRL) family of ubiquitin ligase complexes. The CRL complex SCFFbxw7/Ago targets phosphorylated CycE, whereas p21Cip1 and Dap are targeted by the CRL4Cdt2 complex, binding to the PIP degron. The role of CRL-mediated degradation of CycE and Cip/Kip proteins during CNS development is not well understood. Here, we analyse the role of ago (Fbxw7)-mediated CycE degradation, and of Dap and p21Cip1 degradation during Drosophila CNS development. We find that ago mutants display over-proliferation, accompanied by elevated CycE expression levels. By contrast, expression of PIP degron mutant Dap and p21Cip1 transgenes inhibit proliferation. However, surprisingly, this is also accompanied by elevated CycE levels. Hence, ago mutation and PIP degron Cip/Kip transgenic expression trigger opposite effects on proliferation, but similar effects on CycE levels.