DOI: 10.5061/dryad.9ghx3ffjz
title: Disruption of the TCA cycle reveals an ATF4-mediated integration of redox and amino acid metabolism

description: 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
description: <strong>TruSeq mRNA stranded</strong>
 5x10<sup>4</sup> 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. <strong>Label-free
 proteomics</strong> <strong>Sample
 preparation</strong>
 5x10<sup>4</sup> 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. <strong>Mass spectrometry
 analysis</strong> 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. <strong>Data
 analysis</strong> 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). <strong>Western
 blot</strong> 5x10<sup>4</sup> 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<sup>™</sup> 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<sup>™</sup> gradient gels and run at 160V constant for 1h
 in Bolt<sup>™</sup> 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).
description: 1. TruSeq mRNA stranded analysis of murine
 Fh1<sup>fl/fl</sup> 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
 Fh1<sup>fl/fl</sup> 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 Fh1<sup>fl/fl</sup> 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 Fh1<sup>fl/fl</sup> kidney
 epithelial cells treated with vehicle control (DMSO) and 500 micromolar
 thenoyltrifluoroacetone (TTFA). README file -
 README_4_Label-free_proteomics_TTFA.txt