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.
Email	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

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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.

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.