{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001954","ANALYSIS_ID":"AN003179","VERSION":"1","CREATED_ON":"October 25, 2021, 12:42 pm"},

"PROJECT":{"PROJECT_TITLE":"A pathogenic role for histone H3 copper reductase activity in a yeast model of Friedreich’s Ataxia","PROJECT_SUMMARY":"Disruptions to iron-sulfur (Fe-S) clusters, essential cofactors for a broad range of proteins, cause widespread cellular defects resulting in human disease. An underappreciated source of damage to Fe-S clusters are cuprous (Cu1+) ions. Since histone H3 enzymatically produces Cu1+ to support copper-dependent functions, we asked whether this activity could become detrimental to Fe-S clusters. Here, we report that histone H3-mediated Cu1+ toxicity is a major determinant of cellular functional pool of Fe-S clusters. Inadequate Fe-S cluster supply, either due to diminished assembly as occurs in Friedreich’s Ataxia or defective distribution, causes severe metabolic and growth defects in S. cerevisiae. Decreasing Cu1+ abundance, through attenuation of histone cupric reductase activity or depletion of total cellular copper, restored Fe-S cluster-dependent metabolism and growth. Our findings reveal a novel interplay between chromatin and mitochondria in Fe-S cluster homeostasis, and a potential pathogenic role for histone enzyme activity and Cu1+ in diseases with Fe-S cluster dysfunction.","INSTITUTE":"University of California, Los Angeles","DEPARTMENT":"Biomedical Sciences","LAST_NAME":"Matulionis","FIRST_NAME":"Nedas","ADDRESS":"615 Charles E Young Dr S, BSRB 354-05","EMAIL":"nmatulionis@mednet.ucla.edu","PHONE":"3302346450"},

"STUDY":{"STUDY_TITLE":"A pathogenic role for histone H3 copper reductase activity in a yeast model of Friedreich’s Ataxia","STUDY_SUMMARY":"Disruptions to iron-sulfur (Fe-S) clusters, essential cofactors for a broad range of proteins, cause widespread cellular defects resulting in human disease. An underappreciated source of damage to Fe-S clusters are cuprous (Cu1+) ions. Since histone H3 enzymatically produces Cu1+ to support copper-dependent functions, we asked whether this activity could become detrimental to Fe-S clusters. Here, we report that histone H3-mediated Cu1+ toxicity is a major determinant of cellular functional pool of Fe-S clusters. Inadequate Fe-S cluster supply, either due to diminished assembly as occurs in Friedreich’s Ataxia or defective distribution, causes severe metabolic and growth defects in S. cerevisiae. Decreasing Cu1+ abundance, through attenuation of histone cupric reductase activity or depletion of total cellular copper, restored Fe-S cluster-dependent metabolism and growth. Our findings reveal a novel interplay between chromatin and mitochondria in Fe-S cluster homeostasis, and a potential pathogenic role for histone enzyme activity and Cu1+ in diseases with Fe-S cluster dysfunction.","INSTITUTE":"University of California, Los Angeles","LAST_NAME":"Matulionis","FIRST_NAME":"Nedas","ADDRESS":"615 Charles E Young Dr S, BSRB 354-05","EMAIL":"nmatulionis@mednet.ucla.edu","PHONE":"3302346450"},

"SUBJECT":{"SUBJECT_TYPE":"Yeast","SUBJECT_SPECIES":"Saccharomyces cerevisiae","TAXONOMY_ID":"4932"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"cell culture",
"Sample ID":"cells-yeast-WT-01",
"Factors":{"Strain":"wild type"},
"Additional sample data":{"RAW_FILE_NAME":"cells-yeast-WT-01"}
},
{
"Subject ID":"cell culture",
"Sample ID":"cells-yeast-H3-A110C-01",
"Factors":{"Strain":"H3A110C"},
"Additional sample data":{"RAW_FILE_NAME":"cells-yeast-H3-A110C-01"}
},
{
"Subject ID":"cell culture",
"Sample ID":"cells-yeast-H3-H113N-01",
"Factors":{"Strain":"H3H113N"},
"Additional sample data":{"RAW_FILE_NAME":"cells-yeast-H3-H113N-01"}
},
{
"Subject ID":"cell culture",
"Sample ID":"cells-yeast-H3-dSGH1-01",
"Factors":{"Strain":"dGsh1"},
"Additional sample data":{"RAW_FILE_NAME":"cells-yeast-H3-dSGH1-01"}
},
{
"Subject ID":"cell culture",
"Sample ID":"cells-yeast-WT-02",
"Factors":{"Strain":"wild type"},
"Additional sample data":{"RAW_FILE_NAME":"cells-yeast-WT-02"}
},
{
"Subject ID":"cell culture",
"Sample ID":"cells-yeast-H3-A110C-02",
"Factors":{"Strain":"H3A110C"},
"Additional sample data":{"RAW_FILE_NAME":"cells-yeast-H3-A110C-02"}
},
{
"Subject ID":"cell culture",
"Sample ID":"cells-yeast-H3-H113N-02",
"Factors":{"Strain":"H3H113N"},
"Additional sample data":{"RAW_FILE_NAME":"cells-yeast-H3-H113N-02"}
},
{
"Subject ID":"cell culture",
"Sample ID":"cells-yeast-H3-dSGH1-02",
"Factors":{"Strain":"dGsh1"},
"Additional sample data":{"RAW_FILE_NAME":"cells-yeast-H3-dSGH1-02"}
},
{
"Subject ID":"cell culture",
"Sample ID":"cells-yeast-WT-03",
"Factors":{"Strain":"wild type"},
"Additional sample data":{"RAW_FILE_NAME":"cells-yeast-WT-03"}
},
{
"Subject ID":"cell culture",
"Sample ID":"cells-yeast-H3-A110C-03",
"Factors":{"Strain":"H3A110C"},
"Additional sample data":{"RAW_FILE_NAME":"cells-yeast-H3-A110C-03"}
},
{
"Subject ID":"cell culture",
"Sample ID":"cells-yeast-H3-H113N-03",
"Factors":{"Strain":"H3H113N"},
"Additional sample data":{"RAW_FILE_NAME":"cells-yeast-H3-H113N-03"}
},
{
"Subject ID":"cell culture",
"Sample ID":"cells-yeast-H3-dSGH1-03",
"Factors":{"Strain":"dGsh1"},
"Additional sample data":{"RAW_FILE_NAME":"cells-yeast-H3-dSGH1-03"}
},
{
"Subject ID":"cell culture",
"Sample ID":"cells-yeast-WT-04",
"Factors":{"Strain":"wild type"},
"Additional sample data":{"RAW_FILE_NAME":"cells-yeast-WT-04"}
},
{
"Subject ID":"cell culture",
"Sample ID":"cells-yeast-H3-A110C-04",
"Factors":{"Strain":"H3A110C"},
"Additional sample data":{"RAW_FILE_NAME":"cells-yeast-H3-A110C-04"}
},
{
"Subject ID":"cell culture",
"Sample ID":"cells-yeast-H3-H113N-04",
"Factors":{"Strain":"H3H113N"},
"Additional sample data":{"RAW_FILE_NAME":"cells-yeast-H3-H113N-04"}
},
{
"Subject ID":"cell culture",
"Sample ID":"cells-yeast-H3-dSGH1-04",
"Factors":{"Strain":"dGsh1"},
"Additional sample data":{"RAW_FILE_NAME":"cells-yeast-H3-dSGH1-04"}
},
{
"Subject ID":"cell culture",
"Sample ID":"cells-yeast-WT-05",
"Factors":{"Strain":"wild type"},
"Additional sample data":{"RAW_FILE_NAME":"cells-yeast-WT-05"}
},
{
"Subject ID":"cell culture",
"Sample ID":"cells-yeast-H3-A110C-05",
"Factors":{"Strain":"H3A110C"},
"Additional sample data":{"RAW_FILE_NAME":"cells-yeast-H3-A110C-05"}
},
{
"Subject ID":"cell culture",
"Sample ID":"cells-yeast-H3-H113N-05",
"Factors":{"Strain":"H3H113N"},
"Additional sample data":{"RAW_FILE_NAME":"cells-yeast-H3-H113N-05"}
},
{
"Subject ID":"cell culture",
"Sample ID":"cells-yeast-H3-dSGH1-05",
"Factors":{"Strain":"dGsh1"},
"Additional sample data":{"RAW_FILE_NAME":"cells-yeast-H3-dSGH1-05"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Please refer to Treatment and Sample Prep sections.","SAMPLE_TYPE":"Yeast cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"Yeast strains with wild type or mutant histone H3 were cultured in yeast Synthetic Complete Medium before collection and extraction. As a control and for comparison, a strain with GSH1 deleted is included. GSH1 encodes for gamma glutamylcysteine synthetase, which catalyzes the first step in glutathione biosynthesis."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"To extract metabolites, we washed the cells with ice-cold 150 mM ammonium acetate, pH 7.3, and then added 500 uL 80% methanol and incubated for 20 minutes at -80°C. Cells were then scraped off the plate, vortexed and centrifuged for 10 minutes at maximum speed. We dried 400 uL of the supernatant under vacuum and stored the dried metabolites at -80°C. Dried metabolites were reconstituted in 100 µL of a 50% acetonitrile(ACN) 50% dH20 solution. Samples were vortexed and spun down for 10 min at 17,000g. 70 µL of the supernatant was then transferred to HPLC glass vials."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Samples were run on a Vanquish (Thermo Scientific) UHPLC system with mobile phase A (20 mM ammonium carbonate, pH 9.7) and mobile phase B (100% Acetonitrile) at a flow rate of 150 µL/min on a SeQuant ZIC-pHILIC Polymeric column (2.1 × 150 mm 5 μm, EMD Millipore) at 35°C. Separation was achieved with a linear gradient from 20% A to 80% A in 20 min followed by a linear gradient from 80% A to 20% A from 20 min to 20.5 min. 20% A was then held from 20.5 min to 28 min.","CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Thermo Vanquish Horizon","COLUMN_NAME":"SeQuant ZIC-HILIC (150 x 2.1mm, 5um)","FLOW_GRADIENT":"100% Acetonitrile","FLOW_RATE":"150 µL/min","COLUMN_TEMPERATURE":"35°C","SOLVENT_A":"20 mM Ammonium carbonate, pH 9.7","SOLVENT_B":"100% Acetonitrile","INTERNAL_STANDARD":"10 nM Trifluoromethanesulfonate (extraction buffer)"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","LABORATORY_NAME":"UCLA","OPERATOR_NAME":"Nedas Matulionis"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"UNSPECIFIED","MS_COMMENTS":"The UHPLC was coupled to a Q-Exactive (Thermo Scientific) mass analyzer running in polarity switching mode with spray-voltage=3.2kV, sheath-gas=40, aux-gas=15, sweep-gas=1, aux-gas-temp=350°C, and capillary-temp=275°C. For both polarities mass scan settings were kept at full-scan-range=(70-1000), ms1-resolution=70,000, max-injection-time=250ms, and AGC-target=1E6. MS2 data was also collected from the top three most abundant singly-charged ions in each scan with normalized-collision-energy=35. Each of the resulting “.RAW” files was then centroided and converted into two “.mzXML” files (one for positive scans and one for negative scans) using msconvert from ProteoWizard. These “.mzXML” files were imported into the MZmine 2 software package. Ion chromatograms were generated from MS1 spectra via the built-in Automated Data Analysis Pipeline (ADAP) chromatogram module and peaks were detected via the ADAP wavelets algorithm. Peaks were aligned across all samples via the Random sample consensus aligner module, gap-filled, and assigned identities using an exact mass MS1(+/-15ppm) and retention time RT (+/-0.5min) search of our in-house MS1-RT database. Peak boundaries and identifications were then further refined by manual curation. Peaks were quantified by area under the curve integration and exported as CSV files. If stable isotope tracing was used in the experiment, the peak areas were additionally processed via the R package AccuCor to correct for natural isotope abundance. Peak areas for each sample were normalized by the measured area of the internal standard trifluoromethanesulfonate (present in the extraction buffer) and by the number of cells present in the extracted well."},

"MS_METABOLITE_DATA":{
"Units":"Peak Area",

"Data":[{"Metabolite":"glycine","cells-yeast-H3-A110C-01":"1.70E+07","cells-yeast-H3-A110C-02":"1.57E+07","cells-yeast-H3-A110C-03":"2.07E+07","cells-yeast-H3-A110C-04":"1.72E+07","cells-yeast-H3-A110C-05":"2.34E+07","cells-yeast-H3-H113N-01":"9168753.594","cells-yeast-H3-H113N-02":"1.02E+07","cells-yeast-H3-H113N-03":"1.08E+07","cells-yeast-H3-H113N-04":"1.52E+07","cells-yeast-H3-H113N-05":"1.47E+07","cells-yeast-H3-dSGH1-01":"1.95E+07","cells-yeast-H3-dSGH1-02":"1.67E+07","cells-yeast-H3-dSGH1-03":"1.78E+07","cells-yeast-H3-dSGH1-04":"2.24E+07","cells-yeast-H3-dSGH1-05":"3.10E+07","cells-yeast-WT-01":"1.67E+07","cells-yeast-WT-02":"1.37E+07","cells-yeast-WT-03":"1.72E+07","cells-yeast-WT-04":"2.00E+07","cells-yeast-WT-05":"2.08E+07"},{"Metabolite":"alanine","cells-yeast-H3-A110C-01":"3.37E+08","cells-yeast-H3-A110C-02":"2.78E+08","cells-yeast-H3-A110C-03":"3.47E+08","cells-yeast-H3-A110C-04":"2.86E+08","cells-yeast-H3-A110C-05":"3.10E+08","cells-yeast-H3-H113N-01":"4.41E+08","cells-yeast-H3-H113N-02":"4.77E+08","cells-yeast-H3-H113N-03":"5.27E+08","cells-yeast-H3-H113N-04":"5.90E+08","cells-yeast-H3-H113N-05":"5.15E+08","cells-yeast-H3-dSGH1-01":"5.42E+08","cells-yeast-H3-dSGH1-02":"8.20E+08","cells-yeast-H3-dSGH1-03":"6.30E+08","cells-yeast-H3-dSGH1-04":"9.46E+08","cells-yeast-H3-dSGH1-05":"1.00E+09","cells-yeast-WT-01":"3.10E+08","cells-yeast-WT-02":"2.74E+08","cells-yeast-WT-03":"3.25E+08","cells-yeast-WT-04":"3.28E+08","cells-yeast-WT-05":"3.04E+08"},{"Metabolite":"choline","cells-yeast-H3-A110C-01":"8.30E+07","cells-yeast-H3-A110C-02":"1.24E+08","cells-yeast-H3-A110C-03":"1.55E+08","cells-yeast-H3-A110C-04":"7.68E+07","cells-yeast-H3-A110C-05":"6.25E+07","cells-yeast-H3-H113N-01":"6.77E+07","cells-yeast-H3-H113N-02":"8.02E+07","cells-yeast-H3-H113N-03":"7.67E+07","cells-yeast-H3-H113N-04":"1.17E+08","cells-yeast-H3-H113N-05":"4.95E+07","cells-yeast-H3-dSGH1-01":"2.40E+08","cells-yeast-H3-dSGH1-02":"3.07E+08","cells-yeast-H3-dSGH1-03":"2.12E+08","cells-yeast-H3-dSGH1-04":"3.00E+08","cells-yeast-H3-dSGH1-05":"2.82E+08","cells-yeast-WT-01":"7.71E+07","cells-yeast-WT-02":"8.91E+07","cells-yeast-WT-03":"1.19E+08","cells-yeast-WT-04":"3.62E+07","cells-yeast-WT-05":"3.32E+07"},{"Metabolite":"serine","cells-yeast-H3-A110C-01":"1.94E+08","cells-yeast-H3-A110C-02":"2.17E+08","cells-yeast-H3-A110C-03":"2.14E+08","cells-yeast-H3-A110C-04":"2.16E+08","cells-yeast-H3-A110C-05":"2.38E+08","cells-yeast-H3-H113N-01":"1.38E+08","cells-yeast-H3-H113N-02":"1.66E+08","cells-yeast-H3-H113N-03":"1.52E+08","cells-yeast-H3-H113N-04":"2.18E+08","cells-yeast-H3-H113N-05":"2.08E+08","cells-yeast-H3-dSGH1-01":"1.88E+08","cells-yeast-H3-dSGH1-02":"2.28E+08","cells-yeast-H3-dSGH1-03":"1.84E+08","cells-yeast-H3-dSGH1-04":"2.53E+08","cells-yeast-H3-dSGH1-05":"2.94E+08","cells-yeast-WT-01":"1.92E+08","cells-yeast-WT-02":"1.89E+08","cells-yeast-WT-03":"1.82E+08","cells-yeast-WT-04":"2.36E+08","cells-yeast-WT-05":"2.40E+08"},{"Metabolite":"creatinine","cells-yeast-H3-A110C-01":"461138.5027","cells-yeast-H3-A110C-02":"120586.8742","cells-yeast-H3-A110C-03":"431188.8348","cells-yeast-H3-A110C-04":"215172.129","cells-yeast-H3-A110C-05":"201301.5588","cells-yeast-H3-H113N-01":"436931.9447","cells-yeast-H3-H113N-02":"177332.5271","cells-yeast-H3-H113N-03":"155538.4022","cells-yeast-H3-H113N-04":"532479.4642","cells-yeast-H3-H113N-05":"682773.6305","cells-yeast-H3-dSGH1-01":"149467.9284","cells-yeast-H3-dSGH1-02":"405557.3995","cells-yeast-H3-dSGH1-03":"218638.1903","cells-yeast-H3-dSGH1-04":"507630.0336","cells-yeast-H3-dSGH1-05":"424106.4812","cells-yeast-WT-01":"619691.2848","cells-yeast-WT-02":"617981.9907","cells-yeast-WT-03":"391534.2596","cells-yeast-WT-04":"544504.1731","cells-yeast-WT-05":"299347.2832"},{"Metabolite":"proline","cells-yeast-H3-A110C-01":"4.84E+09","cells-yeast-H3-A110C-02":"4.43E+09","cells-yeast-H3-A110C-03":"5.31E+09","cells-yeast-H3-A110C-04":"4.25E+09","cells-yeast-H3-A110C-05":"4.93E+09","cells-yeast-H3-H113N-01":"3.59E+09","cells-yeast-H3-H113N-02":"3.73E+09","cells-yeast-H3-H113N-03":"4.17E+09","cells-yeast-H3-H113N-04":"4.45E+09","cells-yeast-H3-H113N-05":"3.89E+09","cells-yeast-H3-dSGH1-01":"3.68E+09","cells-yeast-H3-dSGH1-02":"3.19E+09","cells-yeast-H3-dSGH1-03":"3.37E+09","cells-yeast-H3-dSGH1-04":"2.87E+09","cells-yeast-H3-dSGH1-05":"3.08E+09","cells-yeast-WT-01":"4.69E+09","cells-yeast-WT-02":"4.17E+09","cells-yeast-WT-03":"4.95E+09","cells-yeast-WT-04":"4.16E+09","cells-yeast-WT-05":"4.28E+09"},{"Metabolite":"betaine","cells-yeast-H3-A110C-01":"3.30E+07","cells-yeast-H3-A110C-02":"2.32E+07","cells-yeast-H3-A110C-03":"2.30E+07","cells-yeast-H3-A110C-04":"2.99E+07","cells-yeast-H3-A110C-05":"3.87E+07","cells-yeast-H3-H113N-01":"2.81E+07","cells-yeast-H3-H113N-02":"4.04E+07","cells-yeast-H3-H113N-03":"3.26E+07","cells-yeast-H3-H113N-04":"4.11E+07","cells-yeast-H3-H113N-05":"3.40E+07","cells-yeast-H3-dSGH1-01":"4.19E+07","cells-yeast-H3-dSGH1-02":"3.01E+07","cells-yeast-H3-dSGH1-03":"2.09E+07","cells-yeast-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}

}