Summary of Study ST003959

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 PR002482. The data can be accessed directly via it's Project DOI: 10.21228/M8TC38 This work is supported by NIH grant, U2C- DK119886.

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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.

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Study IDST003959
Study TitleSteady state levels of polar metabolites HEK293T, SDHBKO and UQCRC1KO compared to WT cells.
Study SummaryThis study aimed to investigate changes in relative abundance of polar metabolites in cells with OXPHOS dysfunction using targeted GC-MS analysis. Steady-state analysis of polar metabolites by gas chromatography-mass spectrometry (GC-MS) in HEK293T UQCRC1KO and SDHBKO cell lines enabled the relative quantification of TCA intermediates. The results revealed a significant increase in succinate and a similar behavior in respect to the abundances of other TCA metabolites as was observed in SDHBKO.
Institute
University of Melbourne
Last NameRoopasingam
First NameKugapreethan
Address30 Flemington Rd, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC, Australia.
Emailk.roopasingam@unimelb.edu.au
Phone0434297212
Submit Date2025-05-29
Raw Data AvailableYes
Raw Data File Type(s)gqd
Analysis Type DetailGC-MS
Release Date2025-06-11
Release Version1
Kugapreethan Roopasingam Kugapreethan Roopasingam
https://dx.doi.org/10.21228/M8TC38
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR002482
Project DOI:doi: 10.21228/M8TC38
Project Title:Complex II assembly drives metabolic adaptation to OXPHOS dysfunction.
Project Summary:During acute oxidative phosphorylation (OXPHOS) dysfunction, the reverse activity of succinate dehydrogenase (Complex II) maintains the redox state of Coenzyme Q by utilizing either fumarate or oxygen as terminal electron acceptors. The tendency for one over another has been suggested to be tissue-specific, but the underlying mechanism and consequence of this is unknown. Using quantitative proteomics to screen a panel of HEK293T knockout cell lines, we identified an increase in SDHAF2 protein, a Complex II assembly factor that enhances the flavination of catalytic subunit SDHA, as critical for metabolic adaptation during OXPHOS stress in HEK293T cells. Loss of SDHAF2 during Complex III inhibition resulted in a reduction in Complex II F-site derived reactive oxygen species (ROS), a severe growth impairment, and a net reductive TCA cycle driven by an inability of mitochondria to support additional Complex II assembly. This in turn leads to use of fumarate as terminal electron acceptor at the cost of a ROS-mediated switch to glycolysis. Cell lines adapted to glycolysis did not accumulate SDHAF2 upon OXPHOS stress and exhibited a net reductive TCA cycle and mild growth phenotypes with or without SDHAF2 being present. Thus, our study reveals how Complex II assembly controls a balance between protection of the Q-pool and ROS-meditated signaling during oxidative stress in cells reliant on mitochondrial OXPHOS.
Institute:University of Melbourne
Last Name:Roopasingam
First Name:Kugapreethan
Address:30 Flemington Rd, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC, Australia.
Email:k.roopasingam@unimelb.edu.au
Phone:0434297212

Subject:

Subject ID:SU004096
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Sample source Treatment
SA452749BKO_001_UNK-0022_13032023_22SDHBKO_001 Knock out
SA452750BKO_002_UNK-0032_13032023_32SDHBKO_002 Knock out
SA452751BKO_003_UNK-0026_13032023_26SDHBKO_003 Knock out
SA452752BKO_004_UNK-0017_13032023_17SDHBKO_004 Knock out
SA452753BKO_005_UNK-0015_13032023_15SDHBKO_005 Knock out
SA452754BKO_006_UNK-0007_13032023_7SDHBKO_006 Knock out
SA452755UQKO_001_UNK-0027_13032023_27UQCRC1KO_001 Knock out
SA452756UQKO_002_UNK-0030_13032023_30UQCRC1KO_002 Knock out
SA452757UQKO_003_UNK-0029_13032023_29UQCRC1KO_003 Knock out
SA452758UQKO_004_UNK-0006_13032023_6UQCRC1KO_004 Knock out
SA452759UQKO_005_UNK-0008_13032023_8UQCRC1KO_005 Knock out
SA452760UQKO_006_UNK-0028_13032023_28UQCRC1KO_006 Knock out
SA452761WT_001_UNK-0009_13032023_9Wild type_001 Control
SA452762WT_002_UNK-0033_13032023_33Wild type_002 Control
SA452763WT_003_UNK-0023_13032023_23Wild type_003 Control
SA452764WT_004_UNK-0019_13032023_19Wild type_004 Control
SA452765WT_005_UNK-0020_13032023_20Wild type_005 Control
SA452766WT_006_UNK-0012_13032023_12Wild type_006 Control
Showing results 1 to 18 of 18

Collection:

Collection ID:CO004089
Collection Summary:HEK293T cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific) supplemented with 10 % (v/v) fetal bovine serum (FBS, CellSera), 50 µg/mL uridine (Sigma), and a mixture of 100 µg/mL streptomycin and 100 units/mL penicillin (Thermo Scientific).
Sample Type:Embryonic cells

Treatment:

Treatment ID:TR004105
Treatment Summary:HEK293T knockouts for SDHB and UQCRC1 were made using the CRISPR-Cas9 mediated technology as described in Stroud, D. A. et al. Nature 538, 123–126 (2016).

Sample Preparation:

Sampleprep ID:SP004102
Sampleprep Summary:For steady-state metabolite profiling, polar metabolites were extracted from snap-frozen cells using 600 µL of HPLC grade methanol:chloroform mixture (9:1; [v/v]), along with internal standards (1.66μM 13C5,15N-valine, 1.66μM 13C6-sorbitol), as described above. The clarified supernatants and pooled biological quality controls (PBQC’s) were dried using a CentriVap concentrator (Labconco).

Chromatography:

Chromatography ID:CH004942
Instrument Name:Shimadzu GC-2010
Column Name:Agilent DB-5 GC column (30 m x 0.25 mm, 1 µm)
Column Temperature:100℃
Flow Gradient:N/A
Flow Rate:1 mL/min
Solvent A:N/A
Solvent B:N/A
Chromatography Type:GC

Analysis:

Analysis ID:AN006507
Analysis Type:MS
Chromatography ID:CH004942
Num Factors:18
Num Metabolites:195
Units:Relative abundance
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