Transcriptome analysis of Control and FBXO38 knockdown in HCT116 and Hela cell lines
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE220879 | GEO | 2024/02/07
REPOSITORIES: GEO
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Project description:F-box only protein 38 (FBXO38), which belongs to SKP1-CUL1-F-box protein (SCF) family, exhibits the ability to mediate Lys48-linked poly-ubiquitination and subsequent proteasome degradations of PD-1.To investigate the effect of FBXO38 knockdown on transcription in tumor, we performed RNA-seq in control and FBXO38 knockdown B16-F10 tumor issue.
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Project description:SKP1, an essential subunit of the SKP1-CUL1-F-box protein (SCF) complex, also known as CUL1-RING ubiquitin ligase (CRL1), a family of E3s present in all eukaryotes, which includes 69 members in humans that mediates the proteasomal degradation of hundreds of cellular regulatory proteins. Here we found that SKP1 is regulated by SUMOylation and phosphorylation. We performed immunoprecipitation followed with LC-MS to identify the binding proteins of SKP1 regulated by its SUMOylation and phosphorylation.
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Project description:Modular SCF (SKP1-CUL1-Fbox) ubiquitin E3 ligases orchestrate multiple cellular pathways in eukaryotes. Their variable SKP1-Fbox substrate receptor (SR) modules enable regulated substrate recruitment and subsequent proteasomal degradation. CAND proteins are essential for the efficient and timely exchange of SRs. To gain structural understanding of the underlying molecular mechanism, we reconstituted a CAND1-driven exchange reaction of substrate-bound SCF alongside its co-E3 ligase DCNL1 and visualised it by cryo-EM. We describe high-resolution structural intermediates including a ternary CAND1-SCF complex, as well as conformational and compositional intermediates representing SR- or CAND1-dissociation. We describe in molecular detail how CAND1-induced conformational changes in CUL1/RBX1 provide an optimised DCNL1 binding site and reveal an unexpected dual role for DCNL1 in CAND1-SCF dynamics. Moreover, a partially dissociated CAND1-SCF conformation accommodates cullin neddylation, leading to CAND1 displacement. Our structural findings, together with functional biochemical assays help formulate a detailed model for CAND-SCF regulation.
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Project description:Cells adapt to ever-changing environmental cues by remodeling their inventories of multiprotein complexes. The cellular repertoire of modular multiprotein SCF (SKP1-CUL1-Fbox protein) E3 ligase complexes - which mediate much protein degradation - requires CAND1 to distribute the limiting CUL1 subunit as needed across the family of ~70 different Fbox proteins. Yet how a single assembly factor coordinately promotes dissociation of idling complexes and formation of numerous distinct multiprotein complexes as needed remains unknown. Here, we address this by obtaining cryo-EM structures of CAND1-bound SCF complexes in multiple states, and correlating effects of mutations on the conformational ensembles and in biochemical and cellular assays. The data suggest CAND1 initially clasps catalytic domains of an inactive SCF, rolls around, and allosterically rocks and destabilizes the SCF interface. New SCF production proceeds in reverse, through SKP1 and the Fbox allosterically reducing interactions with CAND1. The CAND1-SCF conformational ensemble fuels mixing-and-matching of SCF parts in response to substrate availability. Thus, our data reveal the biogenesis of a predominant family of E3 ubiquitin ligases, and the molecular basis for systemwide multiprotein complex assembly.
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Project description:The SCF (Skp1/cullin-1/F-box protein) class of E3 polyubiquitin ligases generates a signal for proteasomal degradation of thousands of proteins in animals. The evolutionary diversification of the F-box protein (FBP) family of substrate receptor subunits has challenged elucidation of these complexes in protists. Using bioinformatics and orthogonal Skp1 interactome methods, we expand the list of putative FBPs in the social amoeba Dictyostelium and show that Skp1 interactions are highly remodeled between growth and development. In addition, a subset of FBPs was less represented when the posttranslational hydroxylation and glycosylation of the Skp1 subunit was abrogated by deletion of the O2-dependent Skp1 prolyl hydroxylase PhyA. The significance of Skp1 modification for SCF activity was indicated by partial rescue of phyA-KO cell development with proteasomal inhibitors.
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Project description:Aberrant centrosome organization with ensuing alterations of microtubule nucleation enables tumor cells to proliferate and invade despite increased genomic instability. CEP192 is a key factors in the initiation process of centrosome duplication and in the control of centrosome microtubule nucleation. However, regulatory means of CEP192 have remained unknown. Here we report that FBXL13, a binding determinant of SCF (SKP1-CUL1-F-Box)-family E3 ubiquitin ligases, is enriched at centrosomes and interacts with the centrosomal proteins Centrin-2, Centrin-3, CEP152, and CEP192. Among these, CEP192 is specifically targeted for proteasomal degredation by FBXL13. Accordingly, induced FBXL13 expression downregulates centrosomal γ-tubulin, and disrupts centrosomal microtubule arrays. In addition, depletion of FBXL13 induces high levels of CEP192 and γ-tubulin at the centrosomes corresponding to defects in cell motility. Together, we characterize FBXL13 as a novel regulator of microtubule nucleation activity and highlight a role in promoting cell motility with potential tumor-promoting implications.
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Project description:F-box proteins are the variable substrate adaptor subunits of the SCF (Skp1, Cul1, F-box) ubiquitin ligases. The family comprise about 69 members with important roles in cancer pathogenesis and cell proliferation. Among the F-box proteins, Fbxl13 is frequently amplified in several cancer patient cohorts suggesting an important role in promoting cancer pathogenesis. However, the main function of Fbxl13 is unknown and its biochemical mechanism of action remains uncharacterised. Here, we show that Fbxl13 specifically interacts with the centrosomal proteins Centrin-2, Centrin-3, Cep152, and Cep192. Fbxl13 is enriched at the centrosome, where it targets Cep192 for ubiquitin mediated proteolysis. In line with this, Fbxl13 overexpression downregulated centrosomal γ-tubulin, and disrupted centrosomal microtubule arrays. Furthermore, depletion of Fbxl13 in U2OS cells corresponds to defects in centrosome duplication and cell motility. Thus, we propose that Fbxl13 is a novel regulator of centrosome duplication and microtubule nucleation activity, highlighting a potential tumourigenic role of Fbxl13 in promoting cell motility.
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Project description:SOX9 is a master transcription factor that regulates development and stem cell programs. However, its potential oncogenic activity and regulatory mechanisms that control SOX9 protein stability are poorly understood. Here we show that SOX9 is a substrate of FBW7, a tumor suppressor and a SCF (Skp1-Cul1-F-box)-type ubiquitin ligase. FBW7 recognizes a conserved degron surrounding threonine 236 (T236) in SOX9 that is phosphorylated by GSK3 kinase. Specifically, FBW7-alpha targets T236-phosphorylated SOX9 for ubiquitylation and proteasomal degradation. Further, we demonstrate that FBW7 inactivation stabilizes SOX9 protein promoting migration, metastasis and treatment resistance in medulloblastoma, one of the most common childhood brain tumors. Notably, expression of mutationally stabilized SOX9-T236/240A in medulloblastoma cells coincides with activation of pro-metastatic genes. FBW7 is frequently downregulated in all medulloblastoma subgroups and mutated specifically in SHH-driven medulloblastoma. In cases where FBW7 mRNA levels are low, SOX9 protein is significantly elevated and this phenotype is associated with metastasis at diagnosis and poor patient outcome. Finally, pharmacological inhibition of PI3K/Akt/mTOR activity destabilizes SOX9 in a GSK3/FBW7-dependent manner, rendering medulloblastoma cells sensitive to cytostatic treatment.
2016-09-13 | GSE84320 | GEO
Project description:We investigated DNA methylation status by RRBS method after oncogenic Braf activation using MEFs isolated from Braf knockin mice. Window-based analysis made it clear that the regions <500 bp from TSS were hypermethylated after Braf activation, whereas those >5000 bp from TSS were less methylated after Braf activation compared with wild-type MEFs. A hallmark of human cancer genomes is global DNA hypomethylation accompanied by focal DNA hypermethylation. Although factors and pathways that mediate DNA hypermethylation in cancer cells have been described, the basis of DNA hypomethylation remains to be determined. Here we show that expression of an activated BRAF oncoprotein, BRAFV600E, is sufficient to promote global DNA hypomethylation in mouse embryonic fibroblasts (MEFs). Conversion of 5-methylcytosine (5-mC) to its oxidation product 5-hydroxymethylcytosine (5-hmC) is thought to be the first step in a pathway leading to complete DNA demethylation. We find that BRAFV600E-directed global DNA hypomethylation occurs through conversion of 5-mC to 5-hmC by TET3, a member of the ten-eleven translocation (TET) family of proteins. In normal cells, TET3 is maintained at low levels resulting from ubiquitination by a SKP1–CUL1–F-box (SCF)-type ubiquitin ligase containing the F-box protein FBXW7, a known tumor suppressor, and subsequent proteasome-mediated degradation. BRAFV600E increases TET3 levels through inhibition of glycogen synthase kinase-3b (GSK3b), a protein kinase that phosphorylates TET3 and is required for SCFFBXW7-mediated ubiquitination. Consistent with these cell culture results, we find increased levels of TET3 and 5-hmC in experimentally induced mouse lung adenomas and patient-derived human colorectal adenomas that contain BRAFV600E. Moreover, we show that RNAi-mediated knockdown of Tet3 results in decreased BRAFV600E-induced tumorigenesis. Finally, to test the generality of our findings, we repeated several key experiments using a KRASG12D oncoprotein rather than BRAFV600E and obtained similar results. Collectively, our results elucidate a mechanism of global DNA hypomethylation promoted by oncogenic BRAF and KRAS, and establish an essential role for TET3 at an early stage of oncogenesis.
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