U. of Lleida - Yeast

U. of Lleida - Yeast
Nitric oxide prevents Aft1 activation and metabolic remodeling in Frataxin-deficient yeast
  • Organism: Saccharomyces cerevisiae
  • Instrument: 6420 Triple Quadrupole LC/MS
  • SpikeIn: Yes
Abstract
Yeast frataxin homolog (Yfh1) is the orthologue of human frataxin, a mitochondrial protein whose deficiency causes Friedreich Ataxia. Yfh1 deficiency activates Aft1, a transcription factor governing iron homeostasis in yeast cells. Although the mechanisms causing this activation are not completely understood, it is assumed that it may be caused by iron-sulfur deficiency. However, several evidences indicate that activation of Aft1 occurs in the absence of iron-sulfur deficiency. Besides, Yfh1 deficiency also leads to metabolic remodeling (mainly consisting in a shift from respiratory to fermentative metabolism) and to induction of Yhb1, a nitric oxide (NO) detoxifying enzyme. In this work, we have used conditional Yfh1 mutant yeast strains to investigate the relationship between NO, Aft1 activation and metabolic remodeling. We have observed that NO prevents Aft1 activation caused by Yfh1 deficiency. This phenomenon is not observed when Aft1 is activated by iron scarcity or impaired iron-sulfur biogenesis. In addition, analyzing key metabolic proteins by a targeted proteomics approach, we have observed that NO prevents the metabolic remodeling caused by Yfh1 deficiency. We conclude that Aft1 activation in Yfh1-deficient yeasts is not caused by iron-sulfur deficiency or iron scarcity. Our hypothesis is that Yfh1 deficiency leads to the presence of anomalous iron species that can compromise iron bioavailability and activate a signaling cascade that results in Aft1 activation and metabolic remodeling.
Experiment Description
Yeast cells were resuspended in 50 mM Tris-HCl buffer pH 7,5 plus 1 mM EDTA and a mixture of protease inhibitors and were disrupted using glass beads. After, SDS was added to a final concentration of 2% and protein extracts were vortexed, boiled and centrifuged (12000 rpm 10 min). Proteins were quantified using the Lowry assay and 30 ug were precipitated with cold acetone (9 volumes) and resuspended in 8M urea, 0.1M ammonium bicarbonate. Then, proteins were subjected to reduction by 12 mM DTT and alquilation by 40mM iodoacetamide. Samples were diluted with 0.1 M ammonium bicarbonate to a final concentration of 1.5 M urea and sequencing grade porcine trypsin (Promega) was added to a final enzyme:substrate ratio of 1:100. After digestion, 0,8 ul from a heavy peptide standards mixture was added to the sample. The approximate concentration of each heavy peptide in this mixture was 3,5 uM. Heavy peptides were obtained from JPT (SpikeTidesTM_L). The resulting peptide mix was purified and enriched using C18 columns (Pierce C-18 Spin Columns, Thermo Scientific). Eluted fractions from the C18 column were evaporated using a Concentrator Plus (Eppendorf) and peptides were resuspended in 5% acetonitrile plus 0,1% formic acid. All peptide samples were analyzed on a triple quadrupole spectrometer (Agilent 6420) equipped with an electrospray ion source. Chromatographic separations of peptides were performed on an Agilent 1200 LC system using a Supelco Bioshell A160 Peptide C18 column (1 mm x 15 cm). Peptides (up to 15 micrograms of protein digest) were separated with a linear gradient of acetonitrile/water, containing 0.1% formic acid, at a flow rate of 75 ul/min. A gradient from 5 to 60% acetonitrile in 60 minutes was used. The mass spectrometer was operated in multiple reaction monitoring mode. Transitions were obtained from SRM atlas and imported into Skyline software (MacLean et al, 2010). Each MRM acquisition was performed with Q1 and Q3 operated at unit resolution (0.7 m/z half maximum peak width). Once validated and optimized, the SRM assays were used to quantify all the analyzed peptides using scheduled SRM mode in a single run (retention time window, 120 s; target scan time, 2 sec).
Sample Description
Two yeast strains were used. - BQS201 (also named tetO7-YFH1), in which the YFH1 promoter is replaced by a tet- promoter. - BSS255 (also named tetO7-YFH1 deltaYHB1), which is a nul YHB1 mutant derived from BQS201. Four biological conditions were analyzed: - BQS201 (control condition) - BQS201 plus doxycycline (this drug represses YFH1 expresion). - BQS255 - BQS255 plus doxycycline Yeasts were grown in YPG and doxycycline, where indicated, added for 24 hours.
Created on 11/2/17, 1:12 PM
Clustergrammer Heatmap
 
Download
7memberCommunity_PlateB.sky.zip2025-07-02 21:47:56501212247
7memberCommunity_PlateA.sky.zip2025-07-02 21:47:56501212247
7memberCommunity_PlateD.sky.zip2025-07-02 21:47:56501212247
7memberCommunity_PlateC.sky.zip2025-07-02 21:47:56501212247
7memberCommunity_PlateB.sky.zip2025-07-02 21:47:56501212247
210723-ATI-barley-Pt14_2025-02-24_01-08-00.sky.zip2025-07-02 20:45:211010198733
210423-ATI-barley-Pt13_2025-02-24_01-07-11.sky.zip2025-07-02 20:45:201010194027
210416-ATI-barley-Pt12_2025-02-24_01-06-33.sky.zip2025-07-02 20:45:181010193843
210415-ATI-barley-Pt11_2025-02-24_01-05-41.sky.zip2025-07-02 20:45:161010194029
210302-ATI-barley-Pt10_2025-02-24_01-05-05.sky.zip2025-07-02 20:45:151010194234
210224-ATI-barley-Pt9_2025-02-24_01-04-22.sky.zip2025-07-02 20:45:131010194031
210217-ATI-barley-Pt8_2025-02-24_01-03-46.sky.zip2025-07-02 20:45:121010194029
210210-ATI-barley-Pt7_2025-02-24_01-03-02.sky.zip2025-07-02 20:45:101010194631
210203-ATI-barley-Pt6_2025-02-24_01-02-20.sky.zip2025-07-02 20:45:091010195031
210129-ATI-barley-Pt5_2025-02-24_01-01-22.sky.zip2025-07-02 20:45:071010195831
200731-ATI-barley-Pt4_2025-02-24_01-00-26.sky.zip2025-07-02 20:45:061010198719
200717-ATI-barley-Pt3_2025-02-24_00-58-01.sky.zip2025-07-02 20:45:041010198755
200709-ATI-barley-Pt2-Response_2025-02-24_00-56-48.sky.zip2025-07-02 20:45:01101019957
200709-ATI-barley-Pt2_2025-02-24_00-55-11.sky.zip2025-07-02 20:45:011010199136
200602-ATI-barley-Pt1_2025-02-24_00-25-35.sky.zip2025-07-02 20:44:591010199331
TPAD_CSF1A_Batch1-multiNF-DIANN.sky.zip2025-07-01 17:49:472,85327,12832,575259,53243
TPAD_CSF1A_Batch1-multiNF-DIANN-grouped.sky.zip2025-07-01 17:41:312,67226,43931,769253,11943
TPAD_CSF1B_Batch2-multiNF-DIANN.sky.zip2025-07-01 15:26:132,85327,12832,575259,53241
TPAD_CSF1B_Batch2-multiNF-DIANN-grouped.sky.zip2025-07-01 15:17:422,67226,43931,769253,11941
TPAD_CSF2A_Batch3-multiNF-DIANN.sky.zip2025-07-01 14:13:302,85327,12832,575259,53243
TPAD_CSF2A_Batch3-multiNF-DIANN-grouped.sky.zip2025-07-01 14:04:582,67226,43931,769253,11943
TPAD_CSF2B_Batch4-multiNF-DIANN.sky.zip2025-07-01 11:21:372,85327,12832,575259,53243
TPAD_CSF2B_Batch4-multiNF-DIANN-grouped.sky.zip2025-07-01 11:09:482,67226,43931,769253,11943
TPAD_CSF3A_Batch5-multiNF-DIANN.sky.zip2025-06-30 15:31:332,85327,12832,575259,53243
TPAD_CSF3A_Batch5-multiNF-DIANN-grouped.sky.zip2025-06-30 15:23:252,67226,43931,769253,11943
TPAD_CSF3B_Batch6-multiNF-DIANN.sky.zip2025-06-30 14:43:542,85327,12832,575259,53241
TPAD_CSF3B_Batch6-multiNF-DIANN-grouped.sky.zip2025-06-30 14:36:142,67226,43931,769253,11941
TPAD_CSF4_Batch7-multiNF-DIANN.sky.zip2025-06-30 13:55:172,85327,12832,575259,53242
TPAD_CSF4_Batch7-multiNF-DIANN-grouped.sky.zip2025-06-30 13:46:482,67226,43931,769253,11942
TPAD_CSF5_Batch8-multiNF-DIANN.sky.zip2025-06-30 13:02:062,85327,12832,575259,53254
TPAD_CSF5_Batch8-multiNF-DIANN-grouped.sky.zip2025-06-30 11:52:172,67226,43931,769253,11954
METRIC_Eclipse_PSB_All_QCs_2025-06-27_01-27-04.sky.zip2025-06-26 22:27:151666233
Exp_CSF_GPF_A_2024-05-06_21-16-47.sky.zip2025-06-25 18:12:201,8297,3787,37855,0401
2024-05 CSF LIT GPF-MSFragger-3.sky.zip2025-06-25 18:12:201,0825,0956,01940,4016
240207_gpf_2024-06-04_10-24-18.sky.zip2025-06-25 18:12:201,2886,98410,593126,0861
230305_ev2_prm_final_inj1_2024-06-04_07-32-10.sky.zip2025-06-25 18:12:207222,0892,08914,44942
230124_p2_neo_30min_3500targets_opt_trans_pepleveldilution_2024-06-04_06-58-16.sky.zip2025-06-25 18:12:201,0273,5013,50111,42041
CSF_neurod105_assay_individuals_manual_2024-06-03_15-26-36.sky.zip2025-06-25 18:12:201029029026,34537
Exp_CSF_MMCC_quant_all_adjBound_opttrans_nochick_2024-06-03_14-53-02.sky.zip2025-06-25 18:12:201,2058,3748,37424,93427
OT_GPF_PRM_survey_MMCC_boundaries_opttrans_nochick_2024-06-03_14-36-08.sky.zip2025-06-25 18:12:201,2801,9711,9715,80127
LIT_GPF_survey_newAlign_MMCC_boundaries_opttrans_nochick_2024-06-02_15-26-11.sky.zip2025-06-25 18:12:207982,0352,0355,99827
Aducanumab_Selectivity_2025-06-19_12-58-48.sky.zip2025-06-19 09:59:0644851236
hela_isolation_window_concentration_2000ng.sky.zip2025-06-02 18:36:1910,365188,947223,7653,101,14818
hela_isolation_window_experiment_200ng.sky.zip2025-06-02 18:36:1910,365188,947223,7653,101,14818
hela_isolation_window_experiment_500ng.sky.zip2025-06-02 18:36:1910,364188,947223,7653,101,14818
hela_isolation_window_experiment_1000ng.sky.zip2025-06-02 18:36:1910,365188,947223,7653,101,14818
2025-0405-MMCC-EV-Astral-noimpute_2025-05-29_11-52-05.sky.zip2025-06-02 18:36:194,90737,54742,331335,89142
2025-0405-MMCC-EV-Actis-noimpute_2025-05-29_11-46-45.sky.zip2025-06-02 18:36:195,18541,97847,810380,07242
SMTG-low-value-imputed_2025-05-01_14-22-15.sky.zip2025-06-01 21:38:1710,383187,220217,1092,214,24878
SMTG-MissForest-imputed_2025-05-01_11-41-41.sky.zip2025-06-01 20:04:5710,383187,220217,1092,214,24878
SMTG-nettle-imputed_2025-04-30_12-25-18.sky.zip2025-06-01 18:16:1410,639215,139251,4792,483,95978
SMTG-unimputed_2025-04-30_08-55-40.sky.zip2025-06-01 13:32:5610,639215,139251,4792,483,95978
TEI-REX-unimputed_2025-04-29_15-46-15.sky.zip2025-06-01 11:49:381,30749,85259,007585,98796
TEI-REX-nettle-imputed_2025-04-29_13-36-50.sky.zip2025-06-01 11:15:251,30749,85259,007585,98796
MagNet-holdout-nettle-imputed_2025-05-01_08-50-25.sky.zip2025-06-01 09:40:175,29914,11015,07460,28842
hela_isolation_window_concentration_2000ng.sky.zip2025-05-30 15:25:4610,365188,947223,7653,101,14818
ecoli_large_replicates_loaded_refined_2024-05-30_14-02-04.sky.zip2025-05-30 12:00:183669899894,9408
ecoli_large_replicates_loaded_2024-05-30_14-00-59.sky.zip2025-05-30 12:00:184611,3021,3027,5248
ecoli_large_replicates_2024-05-30_14-00-05.sky.zip2025-05-30 12:00:184611,3021,3027,5240
ecoli_subset_replicates_refined_cv_2024-05-30_13-58-18.sky.zip2025-05-30 12:00:187592,2662,26612,8072
ecoli_subset_replicates_refined_2024-05-30_13-57-25.sky.zip2025-05-30 12:00:187592,4372,43713,6022
ecoli_subset_replicates_2024-05-30_13-56-16.sky.zip2025-05-30 12:00:187592,4372,44130,8572
gpf_results_importer_2024-05-30_13-54-14.sky.zip2025-05-30 12:00:181,1925,3967,42785,6731
gpf_results_manual_2024-05-30_13-52-23.sky.zip2025-05-30 12:00:181,1925,3967,42785,6731
pq500_100spd_plasma_final_lightheavy_replicates_2024-05-30_13-50-10.sky.zip2025-05-30 11:58:415798181,62213,69910
pq500_60spd_plasma_final_lightheavy_replicates_2024-05-30_13-48-26.sky.zip2025-05-30 11:58:415798181,62213,87610
pq500_100spd_plasma_final_replicates_2024-05-30_13-47-38.sky.zip2025-05-30 11:58:415798188187,08110
pq500_60spd_plasma_final_replicates_2024-05-30_13-46-43.sky.zip2025-05-30 11:58:415798188186,97710
pq500_100spd_plasma_multireplicate_results_refined_2024-05-30_13-46-07.sky.zip2025-05-30 11:58:415798188187,0811
pq500_60spd_plasma_multireplicate_results_refined_2024-05-30_13-45-40.sky.zip2025-05-30 11:58:415798188186,9771
pq500_100spd_plasma_multireplicate_results_2024-05-30_13-45-07.sky.zip2025-05-30 11:58:415798188187,6512
pq500_60spd_plasma_multireplicate_results_2024-05-30_13-44-21.sky.zip2025-05-30 11:58:415798188187,6012
pq500_100spd_neat_multireplicate_results_refined_2024-05-30_13-43-42.sky.zip2025-05-30 11:58:415798188187,6891
pq500_60spd_neat_multireplicate_results_refined_2024-05-30_13-43-05.sky.zip2025-05-30 11:58:415798188187,6011
pq500_100spd_neat_multireplicate_results_2024-05-30_13-42-26.sky.zip2025-05-30 11:58:4157981881811,0881
pq500_60spd_neat_multireplicate_results_2024-05-30_13-41-26.sky.zip2025-05-30 11:58:4157981881811,0881
pq500_60spd_neat_multireplicate_2024-05-30_13-40-53.sky.zip2025-05-30 11:58:4157981881811,0880
hela_isolation_window_experiment_200ng.sky.zip2025-05-30 01:29:3910,365188,947223,7653,101,14818
hela_isolation_window_experiment_500ng.sky.zip2025-05-30 00:53:5310,364188,947223,7653,101,14818
hela_isolation_window_experiment_1000ng.sky.zip2025-05-29 23:15:1110,365188,947223,7653,101,14818
2025-0405-MMCC-EV-Astral-noimpute_2025-05-29_11-52-05.sky.zip2025-05-29 12:13:324,90737,54742,331335,89142
2025-0405-MMCC-EV-Actis-noimpute_2025-05-29_11-46-45.sky.zip2025-05-29 12:03:295,18541,97847,810380,07242
20240709_OBIPhA.sky.zip2025-05-16 09:25:513333723512
Swaney-IPMS-Data-DIANN_2025-05-15_11-53-43.sky.zip2025-05-15 12:02:265,18545,78152,836422,66115
Vial Comparison Post 01152025 v1 (not daily)_2025-04-29_15-52-29.sky.zip2025-04-30 21:22:2310242419
Vial Comparison Pre v2_2025-04-29_15-49-25.sky.zip2025-04-30 21:22:2310242423
ATI_Samples_2023_2025-04-26_13-39-03.sky.zip2025-04-28 10:30:1413244828042
ATI_Response_2023_2025-04-26_13-25-06.sky.zip2025-04-28 10:30:1413244828018
ATI_Samples_2022_2025-02-20_23-31-07.sky.zip2025-04-28 10:30:1012234626848
ATI_Samples_2021_2025-02-20_23-21-52.sky.zip2025-04-28 10:30:0812234626830
2023_ATIsamples_circe_2025-02-20_13-24-11.sky.zip2025-04-25 14:58:1713224325096
2022_ATIsamples_circe_2025-02-20_13-17-06.sky.zip2025-04-25 14:58:0912224325097
2021_ATIsamples_circe_2025-02-20_13-06-53.sky.zip2025-04-25 14:58:0212224325094
deamidation_localization_gS_7-18_2025-04-24_20-52-46.sky.zip2025-04-25 12:05:18144181
synthetic_heavy_gS_peptides_4-21-25_2025-04-21_16-42-22.sky.zip2025-04-25 12:05:1516116632