Summary of Study ST001986
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001262. The data can be accessed directly via it's Project DOI: 10.21228/M8KM58 This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST001986 |
| Study Title | Fbxo7 promotes Cdk6 activity to inhibit PFKP and glycolysis in T cells |
| Study Summary | Deregulated Fbxo7 expression is associated with many pathologies, including anaemia, male sterility, cancer, and Parkinson’s disease, demonstrating its critical role in a variety of cell types. Although Fbxo7 is an F-box protein that recruits substrates for SCF-type E3 ubiquitin ligases, it also promotes the formation of cyclin D/Cdk6/p27 complexes in an E3-ligase independent fashion. We discovered PFKP, the major gatekeeper of glycolysis, in a screen for Fbxo7 substrates. PFKP has been previously shown to be a critical substrate of Cdk6 for the viability of T-ALL cells experiencing high levels of reactive oxygen species. We investigated the molecular relationships between Fbxo7, Cdk6 and PFKP, and the functional effect Fbxo7 has on T cell metabolism, viability, and activation. Fbxo7 promotes Cdk6-independent ubiquitination and Cdk6-dependent phosphorylation of PFKP. Importantly Fbxo7-deficient cells have reduced Cdk6 activity, and haematopoietic and lymphocytic cell lines show a significant dependency on Fbxo7. CD4+ T cells with reduced Fbxo7 have increased glycolysis, and lower cell viability and activation levels. Metabolomic studies of activated CD4+ T cells confirm increased glycolytic flux in Fbxo7-deficient cells, as well as altered nucleotide biosynthesis and arginine metabolism. We show Fbxo7 expression is glucose-responsive, and we propose Fbxo7 inhibits PFKP and glycolysis via its activation of Cdk6. |
| Institute | University of Cambridge |
| Department | Department of Pathology |
| Laboratory | Laman Lab |
| Last Name | Laman |
| First Name | Heike |
| Address | Tennis Court Road, Cambridge CB2 1QP, UK. |
| hl316@cam.ac.uk | |
| Phone | +44 (0)1223 333722 |
| Submit Date | 2021-10-29 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2022-11-16 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001262 |
| Project DOI: | doi: 10.21228/M8KM58 |
| Project Title: | Fbxo7 promotes Cdk6 activity to inhibit PFKP and glycolysis in T cells |
| Project Type: | T cell biology |
| Project Summary: | Deregulated Fbxo7 expression is associated with many pathologies, including anaemia, male sterility, cancer, and Parkinson’s disease, demonstrating its critical role in a variety of cell types. Although Fbxo7 is an F-box protein that recruits substrates for SCF-type E3 ubiquitin ligases, it also promotes the formation of cyclin D/Cdk6/p27 complexes in an E3-ligase independent fashion. We discovered PFKP, the major gatekeeper of glycolysis, in a screen for Fbxo7 substrates. PFKP has been previously shown to be a critical substrate of Cdk6 for the viability of T-ALL cells experiencing high levels of reactive oxygen species. We investigated the molecular relationships between Fbxo7, Cdk6 and PFKP, and the functional effect Fbxo7 has on T cell metabolism, viability, and activation. Fbxo7 promotes Cdk6-independent ubiquitination and Cdk6-dependent phosphorylation of PFKP. Importantly Fbxo7-deficient cells have reduced Cdk6 activity, and haematopoietic and lymphocytic cell lines show a significant dependency on Fbxo7. CD4+ T cells with reduced Fbxo7 have increased glycolysis, and lower cell viability and activation levels. Metabolomic studies of activated CD4+ T cells confirm increased glycolytic flux in Fbxo7-deficient cells, as well as altered nucleotide biosynthesis and arginine metabolism. We show Fbxo7 expression is glucose-responsive, and we propose Fbxo7 inhibits PFKP and glycolysis via its activation of Cdk6. |
| Institute: | University of Cambridge |
| Department: | Department of Pathology |
| Laboratory: | Laman Lab |
| Last Name: | Heike |
| First Name: | Laman |
| Address: | Tennis Court Road, Cambridge CB2 1QP, UK |
| Email: | hl316@cam.ac.uk |
| Phone: | +44 (0)1223 333722 |
| Contributors: | Rebecca Harris, Ming Yang, Christina Schmidt, Sarbjit Singh, Amarnath Natarajan, Christian Frezza, and Heike Laman |
Subject:
| Subject ID: | SU002067 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | Fbxo7LacZ mice (Fbxo7tm1a(EUCOMM)Hmgu on a C57BL/6J background) were bred as heterozygous crosses |
| Age Or Age Range: | WT, heterozygous and homozygous littermates were harvested between 6-9 weeks. |
| Gender: | Male and female |
| Subject Comments: | All experiments in mice were performed in accordance with the UK Animals (Scientific Procedures) Act 1986 and ARRIVE guidelines. Animal licences were approved by the Home Office and the University of Cambridge's Animal Welfare & Ethical Review Body Standing Committee. Experiments were performed under Home Office licence PPL 70/9001. |
Factors:
Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample groups | Treatment |
|---|---|---|---|
| SA185837 | RH2-09 | Mutant | unlabelled |
| SA185838 | RH2-10 | Mutant | unlabelled |
| SA185839 | RH2-12 | Mutant | unlabelled |
| SA185840 | RH2-08 | Mutant | unlabelled |
| SA185841 | RH2-11 | Mutant | unlabelled |
| SA185842 | RH2-07 | Mutant | unlabelled |
| SA185843 | RH2-03 | Wild type | unlabelled |
| SA185844 | RH2-02 | Wild type | unlabelled |
| SA185845 | RH2-04 | Wild type | unlabelled |
| SA185846 | RH2-05 | Wild type | unlabelled |
| SA185847 | RH2-06 | Wild type | unlabelled |
| SA185848 | RH2-01 | Wild type | unlabelled |
| Showing results 1 to 12 of 12 |
Collection:
| Collection ID: | CO002060 |
| Collection Summary: | Spleens were harvested from WT, heterozygous or homozygous Fbxo7LacZ mice and processed to a single cell suspension. For n=2 of Fig. 3E, cells were incubated in RBC lysis buffer (eBioscience) for 5 minutes prior to centrifugation. For all other data, viable lymphocytes were separated with mouse Lympholyte® Cell Separation Media (Cedarlane Labs). CD4+ T cells were then isolated by negative selection using the MojoSort Mouse CD4 T Cell Isolation Kit (Biolegend). Isolated CD4+ T cells were seeded at 1x106/mL in RPMI supplemented with 10% FBS, 100 U/mL penicillin and streptomycin and 5 µM β-mercaptoethanol. To activate, cells were added to plates coated with 2 µg/mL α-CD3 (clone 145-2C11) and containing 2 µg/mL soluble α-CD28 (clone 37.51) and incubated for the indicated duration. Cell viability and activation were measured by flow cytometry by staining with antibodies to CD4-PE (clone GK1.5), CD25-PE/Cy7 (clone PC61), CD69-FITC (clone H1.2F3) and an eFluor™ 780 fixable viability dye (eBioscience) for 30 min at 4oC in the dark. Samples were analysed on a CytoFLEX S flow cytometer. |
| Sample Type: | T-cells |
Treatment:
| Treatment ID: | TR002079 |
| Treatment Summary: | To activate, cells were added to plates coated with 2 µg/mL α-CD3 (clone 145-2C11) and containing 2 µg/mL soluble α-CD28 (clone 37.51) and incubated for the indicated duration. Cell viability and activation were measured by flow cytometry by staining with antibodies to CD4-PE (clone GK1.5), CD25-PE/Cy7 (clone PC61), CD69-FITC (clone H1.2F3) and an eFluor™ 780 fixable viability dye (eBioscience) for 30 min at 4oC in the dark. Samples were analysed on a CytoFLEX S flow cytometer. For metabolite profiling (Fig. 4C & Supplementary Fig. 3C), murine CD4+ T cells were isolated and activated in a 12 well plate for 48 hours. For 13C stable isotope tracing (Fig. 4D), murine CD4+ T cells were isolated and activated in a 12 well plate with culture media containing 2g/L D-Glucose-1,2-13C2 (Sigma) for 24 hours. |
Sample Preparation:
| Sampleprep ID: | SP002073 |
| Sampleprep Summary: | To extract metabolites, cells were harvested, washed twice in PBS and resuspended in 200 μL ice cold metabolite extraction solution (50% LC–MS grade methanol, 30% LC-MS grade acetonitrile, 20% ultrapure water, 5 μM valine-d8 as internal standard) per 1x106 cells. Cells were incubated in a dry ice-methanol bath for 20 min, then at 4oC with shaking for 15 min. Samples were centrifuged at 13000 rpm for 20 min and the supernatant was collected into autosampler vials for LC-MS analysis. |
Combined analysis:
| Analysis ID | AN003237 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Dionex Ultimate 3000 |
| Column | SeQuant ZIC-pHILIC (150 x 2.1mm,5um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | UNSPECIFIED |
| Units | arbitrary unit |
Chromatography:
| Chromatography ID: | CH002387 |
| Chromatography Summary: | LC-MS chromatographic separation of metabolites was achieved using a Millipore Sequant ZIC-pHILIC analytical column (5 µm, 2.1 × 150 mm) equipped with a 2.1 × 20 mm guard column (both 5 mm particle size) with a binary solvent system. Solvent A was 20 mM ammonium carbonate, 0.05% ammonium hydroxide; Solvent B was acetonitrile. The column oven and autosampler tray were held at 40°C and 4°C, respectively. The chromatographic gradient was run at a flow rate of 0.200 mL/min as follows: 0–2 min: 80% B; 2-17 min: linear gradient from 80% B to 20% B; 17-17.1 min: linear gradient from 20% B to 80% B; 17.1-22.5 min: hold at 80% B. |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | SeQuant ZIC-pHILIC (150 x 2.1mm,5um) |
| Column Temperature: | 40 |
| Flow Gradient: | 0-2 min: 80% B; 2-17 min: linear gradient from 80% B to 20% B; 17-17.1 min: linear gradient from 20% B to 80% B; 17.1-22.5 min: hold at 80% B. |
| Flow Rate: | 0.200 mL/min |
| Solvent A: | 100% water; 20 mM ammonium carbonate; 0.05% ammonium hydroxide |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS003010 |
| Analysis ID: | AN003237 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | The mass spectrometer was operated in full-scan, polarity-switching mode, with the spray voltage set to +4.5 kV/-3.5 kV, the heated capillary held at 320°C, and the auxiliary gas heater held at 280°C. The sheath gas flow was set to 25 units, the auxiliary gas flow was set to 15 units, and the sweep gas flow was set to 0 units. HRMS data acquisition was performed in a range of m/z = 70–900, with the resolution set at 70,000, the AGC target at 1x106, and the maximum injection time (Max IT) at 120 ms. Metabolite identities were confirmed using two parameters: (1) precursor ion m/z was matched within 5 ppm of theoretical mass predicted by the chemical formula; (2) the retention time of metabolites was within 5% of the retention time of a purified standard run with the same chromatographic method. Chromatogram review and peak area integration were performed using the Thermo Fisher software Tracefinder (v.5.0). Correction for natural abundance was performed using the Accucor Package (v.0.2.3) |
| Ion Mode: | UNSPECIFIED |
