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Disruption of the TCA cycle reveals an ATF4-mediated integration of redox and amino acid metabolism

Citation

Ryan, Dylan (2022), Disruption of the TCA cycle reveals an ATF4-mediated integration of redox and amino acid metabolism, Dryad, Dataset, https://doi.org/10.5061/dryad.9ghx3ffjz

Abstract

The Tricarboxylic Acid (TCA) cycle is arguably the most critical metabolic cycle in physiology and exists as an essential interface coordinating cellular metabolism, bioenergetics, and redox homeostasis. Despite decades of research, a comprehensive investigation into the consequences of TCA cycle dysfunction remains elusive. Here, we targeted two TCA cycle enzymes, fumarate hydratase (FH), using an FH inhibitor (FHIN-1), and succinate dehydrogenase (SDH), using two inhibitors Atpenin A5 (AA5) and thenoyltrifluoroacetone (TTFA), and combined transcriptomics, and proteomics analyses to fully appraise the consequences of TCA cycle inhibition (TCAi) in murine kidney epithelial cell line.

Data from this study that is included in this Dryad submission is as follows:

1. TruSeq mRNA stranded analysis of murine Fh1fl/fl kidney epithelial cells were treated with vehicle control (DMSO) or 20 micromolar fumarate hydratase inhibitor (FHIN-1) for 24 h. Three biological replicates per condition. 

2. TruSeq mRNA stranded analysis of murine Fh1fl/fl kidney epithelial cells treated with vehicle control (DMSO) or Atpenin A5 (AA5) for 24 h. Three biological replicates per condition. 

3. Label-free proteomic analysis of a murine Fh1fl/fl kidney epithelial cells treated with vehicle control (DMSO) or 20 micromolar FHIN-1 for 24 h. Five biological replicates per condition. 

4. Label-free proteomic analysis of a murine Fh1fl/fl kidney epithelial cells treated with vehicle control (DMSO) and 500 micromolar thenoyltrifluoroacetone (TTFA). Five biological replicates per condition. 

5. Western blot uncropped blots

Methods

TruSeq mRNA stranded

5x104 cells were plated onto 3 replicate 6-cm dishes before the extraction and treated as indicated. RNA isolation was carried using RNeasy kit (Qiagen) following manufacturer’s suggestions and eluted RNA was purified using RNA Clean & Concentrator Kits (Zymo Research). RNA-seq samples libraries were prepared using TruSeq Stranded mRNA (Illumina) following the manufacturer’s description. For the sequencing, the NextSeq 75 cycle high output kit (Illumina) was used and samples spiked in with 1% PhiX. The samples were run using NextSeq 500 sequencer (Illumina). Differential Gene Expression Analysis was done using the counted reads and the R package edgeR version 3.26.5 (R version 3.6.1) for the pairwise comparisons.

Label-free proteomics

Sample preparation

5x104 cells were plated onto 5 replicate 10-cm dishes, grown to confluence and treated as indicated. At the experimental endpoint, cells were washed with PBS on ice and centrifuged at 1500 rpm for 5 mins at 4°C and frozen at -80°C. Cell pellets were lysed, reduced and alkylated in 100 µl of 6M Gu-HCl, 200 mM Tris-HCl pH 8.5, 1 mM TCEP, 1.5 mM Chloroacetamide by probe sonication and heating to 95ºC for 5 min. Protein concentration was measured by a Bradford assay and initially digested with LysC (Wako) with an enzyme to substrate ratio of 1/200 for 4 h at 37 ºC. Subsequently, the samples were diluted 10fold with water and digested with porcine trypsin (Promega) at 37ºC overnight. Samples were acidified to 1% TFA, cleared by centrifugation (16,000 g at RT) and approximately 20 µg of the sample was desalted using a Stage-tip. Eluted peptides were lyophilized, resuspended in 0.1% TFA/water and the peptide concentration was measured by A280 on a nanodrop instrument (Thermo). The sample was diluted to 1 µg/ 5 µl for subsequent analysis.

Mass spectrometry analysis

The tryptic peptides were analyzed on a Fusion Lumos mass spectrometer connected to an Ultimate Ultra3000 chromatography system (both Thermo Scientific, Germany) incorporating an autosampler. 5 μL of the tryptic peptides, for each sample, was loaded on an Aurora column (Ionoptiks, Melbourne Australia) and separated by an increasing acetonitrile gradient, using a 150-min reverse-phase gradient (from 3%–40% Acetonitrile) at a flow rate of 400 nL/min. The mass spectrometer was operated in positive ion mode with a capillary temperature of 220°C, with a potential of 1500 V applied to the column. Data were acquired with the mass spectrometer operating in automatic data-dependent switching mode, with MS resolution of 240k, with a cycle time of 1 s and MS/MS HCD fragmentation/analysis performed in the ion trap. Mass spectra were analyzed using the MaxQuant Software package in biological triplicate. Label-free quantitation was performed using MaxQuant. All the samples were analyzed as biological triplicates.

Data analysis

Data were analyzed using the MaxQuant software package. Raw data files were searched against a human database (Uniprot Homo sapiens), using a mass accuracy of 42.5 ppm and 0.01 false discovery rate (FDR) at both peptide and protein level. Every single file was considered as separate in the experimental design; the replicates of each condition were grouped for the subsequent statistical analysis. Carbamidomethylation was specified as fixed modification while methionine oxidation and acetylation of protein N-termini were specified as variable. Subsequently, missing values were replaced by a normal distribution (1.8 π shifted with a distribution of 0.3 π) in order to allow the following statistical analysis. Results were cleaned for reverse and contaminants and a list of significant changes was determined based on average ratio and t-test. LFQ-analyst was used to perform differential expression analysis after pre-processing with MaxQuant (Shah et al., 2020).

Western blot

5x104 cells were plated onto 6- or 12-well plates, left to adhere overnight and treated for indicated times. At the experimental endpoint, cells were counted on a parallel counting plate using a CASY counter, washed in 1X PBS and then lysed on ice with the appropriate volume (100 mL per 100,000 cells) of 4X Bolt Loading buffer (Thermo Scientific) diluted to 1X with RIPA buffer (150mM NaCl, 1%NP-40, Sodium deoxycholate (DOC) 0.5%, sodium dodecyl phosphate (SDS) 0.1%, 25mM Tris) supplemented with protease and phosphates inhibitors (Protease inhibitor cocktail, Phosphatase inhibitor cocktail 2/3) (Sigma-Aldrich) and containing 4% β-mercaptoethanol. Protein samples were then heated at 70˚C for 10 minutes, briefly centrifuged and stored at -20C for future use. Samples were loaded onto 4-12% Bis-Tris Bolt gradient gels and run at 160V constant for 1h in Bolt MES 1X running buffer (Thermo Scientific). Dry transfer of the proteins onto a nitrocellulose membrane was done using iBLOT2 (Thermo Scientific) for 12 minutes at 20V. Membranes were incubated in blocking buffer for 1h (either 5% BSA or 5% milk in TBS 1X +0.01 % Tween-20, TBST 1X). Primary antibodies were incubated in blocking buffer ON at 4°C under gentle agitation. On the following day, the membranes were washed three times in TBST 1X for 5 mins and then secondary antibodies (conjugated with 680 or 800 nm fluorophores) (Li-Cor) incubated for 1 h at room temperature at 1:2000 dilution in blocking buffer. Images were acquired and quantified using Image Studio lite 5.2 (Li-Cor) on Odyssey CLx instrument (Li-Cor).

Usage Notes

1. TruSeq mRNA stranded analysis of murine Fh1fl/fl kidney epithelial cells were treated with vehicle control (DMSO) or 20 micromolar fumarate hydratase inhibitor (FHIN-1) for 24 h. Three biological replicates per condition. 

README file - README_1_TruSeq_mRNA_stranded_FHIN1

2. TruSeq mRNA stranded analysis of murine Fh1fl/fl kidney epithelial cells treated with vehicle control (DMSO) or Atpenin A5 (AA5) for 24 h. Three biological replicates per condition. 

README file - README_2_TruSeq_mRNA_stranded_AA5

3. Label-free proteomic analysis of a murine Fh1fl/fl kidney epithelial cells treated with vehicle control (DMSO) or 20 micromolar FHIN-1 for 24 h. 

README file - README_3_Label-free_proteomics_FHIN1.txt

4. Label-free proteomic analysis of a murine Fh1fl/fl kidney epithelial cells treated with vehicle control (DMSO) and 500 micromolar thenoyltrifluoroacetone (TTFA).

README file - README_4_Label-free_proteomics_TTFA.txt

Funding

European Research Council, Award: Award: 819920