The SKP1-PPM1B axis regulates the switch between autophagy and unconventional secretion
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Beatrix Ueberheide
PROVIDER: MSV000090691 | MassIVE | Thu Nov 10 12:05:00 GMT 2022
REPOSITORIES: MassIVE
Similar Datasets
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.
2023-09-18 | PXD042178 | Pride
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.
2022-07-22 | PXD033864 | Pride
Project description:Intracellular degradation of proteins and organelles by the autophagy-lysosome system is essential for cellular quality control and energy homeostasis. Besides degradation, endolysosomal organelles can fuse with the plasma membrane and contribute to unconventional secretion. Here, we identify a function for mammalian SKP1 in endolysosomes that is independent of its established role as an essential component of the family of SCF/CRL1 ubiquitin ligases. We found that, under nutrient poor conditions, SKP1 is phosphorylated on Thr131, allowing its interaction with V1 subunits of the vacuolar ATPase (V-ATPase). This event, in turn, promotes V-ATPase assembly to acidify late endosomes and enhance endolysosomal degradation. Under nutrient rich conditions, SUMOylation of phosphorylated SKP1 allows its binding to and dephosphorylation by the PPM1B phosphatase. Dephosphorylated SKP1 interacts with SEC22B, recently shown to participate in unconventional secretion, to promote secretion of the content of less acidified hybrid endosomal/autophagic compartments. The differences in abundance of specific proteins between ECVs of SKP1WT and SKP1T131A cells motivated us to determine possible changes in extracellular vesicles (ECV) content depending on SKP1 phosphorylation. To this effect, we performed MS analysis and comparative proteomics of ECVs from SKP1WT and SKP1T131A cells (Fig. 5I, Table S7). We found a higher number of proteins displaying increased abundance (greater than 0.321 log2 fold change) in SKP1T131A cells (127 proteins) compared to SKP1WT cells (16 proteins). The mass spectrometric raw files for this analysis are included here.
Collectively, our study implicates SKP1 phosphorylation as a switch between autophagy and unconventional secretion in a manner dependent on cellular nutrient status.
Corresponding authors:
ana-maria.cuervo@einsteinmed.edu and michele.pagano@nyulangone.org
2023-07-06 | MSV000092361 | MassIVE
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.
2024-02-07 | GSE246258 | GEO
Project description:Purpose: Ubiquitin-mediated proteolysis by the 26S proteasome is a critical regulatory mechanism controlling many biological processes. The multi-subunit Skp1-Cullin1-F-box (SCF) complexes are designed to select substrates for ubiquitylation in all eukaryotes. Within these complexes, the Skp1 protein is defined as the bridge between the F-box and the Cullin1 proteins. In Arabidopsis thaliana, there are 19 Arabidopsis Skp1-Like (ASK) genes that encode Skp1 proteins. Our previous genomics and evolutionary studies suggest that ASK1 plays a predominant role in the ASK family. To demonstrate this hypothesis, we genetically introduced the same Ds insertion allele of ask1-1 in Ler into the Arabidopsis Col-0 reference genome. The ask1-1 allele was initially obtained from an ask1-1(+/-)ask2-1(+/-)/LerWs double heterozygous mutant (Liu et al., 2004). To identify the breadth and depth of SCF functions, we applied RNA-Seq sequencing to systematically analyze the perturbation of transcriptomes in both floral bud and open flower tissues in this novel ask1 mutant. Method: Two replicates of total RNA were extracted from 10-50 mg of floral buds or open flowers from Col-0, ask1, and ASK1 (WT allele) primary inflorescences five hours after dawn grown under a long-day photoperiod condition. The NucleoSpin® RNA kit (Macherey-Nagel) was used for RNA extraction following the manufacturer’s instructions. 1 μg each from the 12 RNA samples was used for pair end 150-mer deep sequencing using a NovaSeq 6000 platform (Novogene). Adapter and low-quality sequences in the raw FASTQ sequencing data were removed to produce valid sequences using Trimmomatic (phred=33, minimum length=36; (Bolger et al., 2014)), which were further aligned to the A. thaliana Col-0 reference genome (TAIR10; www.arabidopsis.org) via TOPHAT2 (Kim et al., 2013a) to identify accepted hits of each locus. HTSeq was then used to calculate an absolute expression level (counts) of each locus based on the accepted hits (Anders et al., 2015). The resulting counts per locus were normalized to counts per million reads (CPM) among all 12 libraries and used to identify groups of SDEGs in bud and open flower tissues in ask1 and ASK1 through comparing to the WT bud and open flower tissues, respectively, by edgeR (Log2FC ≥ 1; FDR < 0.01%; (Robinson et al., 2010)). Results: In total, 52 and 54 SDEGs were identified to be up-regulated and down-regulated, respectively, in ASK1 BC10F4 bud tissues; and 13 and 3 SDEGs were identified to be up-regulated and down-regulated, respectively, in ASK1 BC10F4 open flower tissues (2-fold change (FC) with a false discovery rate (FDR) < 0.01). However, 306 and 941 SDEGs (in total 1,247) in ask1 bud tissues and 392 and 1,137 SDEGs (in total 1,529) in ask1 open flower tissues were identified to be up-regulated and down-regulated, respectively (2-FC and FDR < 0.01). Conclusions: Compared to Col-0, 527 and 640 SDEGs were identified in Ler and Ws, respectively, which were greatly higher than the number of SDEGs identified in ASK1 floral bud (Fisher’s exact test, p = 6.0e-82 and 7.5e-112, respectively) and open flower tissues (Fisher’s exact test, p = 7.4e-150 and 1.2e-186, respectively). Therefore, we concluded that the genetic background of ASK1 and ask1 are isogenic to Col-0. The large array of SDEGs identified in both floral buds and open flower tissues in ask1 demonstrated a wide range of roles of ASK1-containing SCF complexes.
2020-08-20 | GSE132841 | GEO
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.
2023-04-14 | PXD038661 | Pride
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 constructed control and FBXO38 knockdown by shRNA in Hela and HCT116 cell lines. We found that FBXO38 may interact with the immune pathway in the tumor.
2024-02-07 | GSE220879 | GEO
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: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.
2022-03-02 | PXD006756 | Pride
Project description:The target of rapamycin (TOR) plays a central role in eukaryotic cell growth control. With prevalent hyper-activation of the mTOR pathway in human cancers, novel strategies to enhance TOR pathway inhibition are highly desirable. We used a yeast-based high-throughput chemical genetic screen to identify small-molecule enhancers of rapamycin (SMERs) and used whole genome expression analysis to identify their mechanisms of action. This SuperSeries is composed of the following subset Series: GSE22270: Expression data from Saccharomyces cerevisiae Met30 temperature-sensitive strain GSE22271: Expression data from Saccharomyces cerevisiae temperature-sensitive strains specific for SCF core components (Skp1, Cullin (Cdc53), E2 enzyme (Cdc34)) and the F-box protein Cdc4. Refer to individual Series
2011-06-30 | E-GEOD-22272 | biostudies-arrayexpress