#METABOLOMICS WORKBENCH tlhowellbray_20221012_062212 DATATRACK_ID:3505 STUDY_ID:ST002478 ANALYSIS_ID:AN004047 PROJECT_ID:PR001601 VERSION 1 CREATED_ON February 10, 2023, 7:14 am #PROJECT PR:PROJECT_TITLE The effect of prions on cellular metabolism: The metabolic impact of the [RNQ+] PR:PROJECT_TITLE prion and the native role of Rnq1p PR:PROJECT_SUMMARY Within the field of amyloid and prion disease there is a need for a more PR:PROJECT_SUMMARY comprehensive understanding of the fundamentals of disease biology. In order to PR:PROJECT_SUMMARY facilitate the progression treatment and underpin comprehension of toxicity, PR:PROJECT_SUMMARY fundamental understanding of the disruption to normal cellular biochemistry and PR:PROJECT_SUMMARY trafficking is needed. Here, by removing the complex biochemistry of the brain, PR:PROJECT_SUMMARY we have utilised known prion forming strains of Saccharomyces cerevisiae PR:PROJECT_SUMMARY carrying different conformational variants of the Rnq1p to obtain Liquid PR:PROJECT_SUMMARY Chromatography-Mass Spectrometry (LC-MS) metabolic profiles and identify key PR:PROJECT_SUMMARY perturbations of prion presence. These studies reveal that prion containing PR:PROJECT_SUMMARY [RNQ+] cells display a significant reduction in amino acid biosynthesis and PR:PROJECT_SUMMARY distinct perturbations in sphingolipid metabolism, with significant PR:PROJECT_SUMMARY downregulation in metabolites within these pathways. Moreover, that native Rnq1p PR:PROJECT_SUMMARY downregulates ubiquinone biosynthesis pathways within cells, suggesting that PR:PROJECT_SUMMARY Rnq1p may play a lipid/mevalonate-based cytoprotective role as a regulator of PR:PROJECT_SUMMARY ubiquinone production. These findings contribute to the understanding of how PR:PROJECT_SUMMARY prion proteins interact in vivo in both their prion and non-prion confirmations PR:PROJECT_SUMMARY and indicate potential targets for the mitigation of these effects. . We PR:PROJECT_SUMMARY demonstrate specific sphingolipid centred metabolic disruptions due to prion PR:PROJECT_SUMMARY presence and give insight into a potential cytoprotective role of the native PR:PROJECT_SUMMARY Rnq1 protein. This provides evidence of metabolic similarities between yeast and PR:PROJECT_SUMMARY mammalian cells as a consequence of prion presence and establishes the PR:PROJECT_SUMMARY application of metabolomics as a tool to investigate prion/amyloid-based PR:PROJECT_SUMMARY phenomena. PR:INSTITUTE Canterbury Christ Church University PR:LAST_NAME Howell-Bray PR:FIRST_NAME Tyler PR:ADDRESS 46 Canterbury Road, Kent PR:EMAIL t.l.howellbray@gmail.com PR:PHONE 07841631495 #STUDY ST:STUDY_TITLE The effect of prions on cellular metabolism: The metabolic impact of the [RNQ+] ST:STUDY_TITLE prion and the native role of Rnq1p ST:STUDY_SUMMARY Within the field of amyloid and prion disease there is a need for a more ST:STUDY_SUMMARY comprehensive understanding of the fundamentals of disease biology. In order to ST:STUDY_SUMMARY facilitate the progression treatment and underpin comprehension of toxicity, ST:STUDY_SUMMARY fundamental understanding of the disruption to normal cellular biochemistry and ST:STUDY_SUMMARY trafficking is needed. Here, by removing the complex biochemistry of the brain, ST:STUDY_SUMMARY we have utilised known prion forming strains of Saccharomyces cerevisiae ST:STUDY_SUMMARY carrying different conformational variants of the Rnq1p to obtain Liquid ST:STUDY_SUMMARY Chromatography-Mass Spectrometry (LC-MS) metabolic profiles and identify key ST:STUDY_SUMMARY perturbations of prion presence. These studies reveal that prion containing ST:STUDY_SUMMARY [RNQ+] cells display a significant reduction in amino acid biosynthesis and ST:STUDY_SUMMARY distinct perturbations in sphingolipid metabolism, with significant ST:STUDY_SUMMARY downregulation in metabolites within these pathways. Moreover, that native Rnq1p ST:STUDY_SUMMARY downregulates ubiquinone biosynthesis pathways within cells, suggesting that ST:STUDY_SUMMARY Rnq1p may play a lipid/mevalonate-based cytoprotective role as a regulator of ST:STUDY_SUMMARY ubiquinone production. These findings contribute to the understanding of how ST:STUDY_SUMMARY prion proteins interact in vivo in both their prion and non-prion confirmations ST:STUDY_SUMMARY and indicate potential targets for the mitigation of these effects. . We ST:STUDY_SUMMARY demonstrate specific sphingolipid centred metabolic disruptions due to prion ST:STUDY_SUMMARY presence and give insight into a potential cytoprotective role of the native ST:STUDY_SUMMARY Rnq1 protein. This provides evidence of metabolic similarities between yeast and ST:STUDY_SUMMARY mammalian cells as a consequence of prion presence and establishes the ST:STUDY_SUMMARY application of metabolomics as a tool to investigate prion/amyloid-based ST:STUDY_SUMMARY phenomena. ST:INSTITUTE Canterbury Christ Church University ST:LAST_NAME Howell-Bray ST:FIRST_NAME Tyler ST:ADDRESS 46 Canterbury Road, Kent ST:EMAIL t.l.howellbray@gmail.com ST:PHONE 07841631495 #SUBJECT SU:SUBJECT_TYPE Yeast SU:SUBJECT_SPECIES Saccharomyces cerevisiae #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - DeltaNIM1 Treatment:none | Ion mode:negative Genotype=Deltarnq-knockout; RAW_FILE_NAME=DeltaNIM1 .mzML SUBJECT_SAMPLE_FACTORS - DeltaNIM2 Treatment:none | Ion mode:negative Genotype=Deltarnq-knockout; RAW_FILE_NAME=DeltaNIM2 .mzML SUBJECT_SAMPLE_FACTORS - DeltaNIM3 Treatment:none | Ion mode:negative Genotype=Deltarnq-knockout; RAW_FILE_NAME=DeltaNIM3 .mzML SUBJECT_SAMPLE_FACTORS - DeltaNIM4 Treatment:none | Ion mode:negative Genotype=Deltarnq-knockout; RAW_FILE_NAME=DeltaNIM4 .mzML SUBJECT_SAMPLE_FACTORS - DeltaNIM5 Treatment:none | Ion mode:negative Genotype=Deltarnq-knockout; RAW_FILE_NAME=DeltaNIM5 .mzML SUBJECT_SAMPLE_FACTORS - DeltaNIM6 Treatment:none | Ion mode:negative Genotype=Deltarnq-knockout; RAW_FILE_NAME=DeltaNIM6 .mzML SUBJECT_SAMPLE_FACTORS - rnq-NIM1 Treatment:none | Ion mode:negative Genotype=74-D694; RAW_FILE_NAME=rnq-NIM1 .mzML SUBJECT_SAMPLE_FACTORS - rnq-NIM2 Treatment:none | Ion mode:negative Genotype=74-D694; RAW_FILE_NAME=rnq-NIM2 .mzML SUBJECT_SAMPLE_FACTORS - rnq-NIM3 Treatment:none | Ion mode:negative Genotype=74-D694; RAW_FILE_NAME=rnq-NIM3 .mzML SUBJECT_SAMPLE_FACTORS - rnq-NIM4 Treatment:none | Ion mode:negative Genotype=74-D694; RAW_FILE_NAME=rnq-NIM4 .mzML SUBJECT_SAMPLE_FACTORS - rnq-NIM5 Treatment:none | Ion mode:negative Genotype=74-D694; RAW_FILE_NAME=rnq-NIM5 .mzML SUBJECT_SAMPLE_FACTORS - rnq-StressNIM1 Treatment:0.2mM H202 | Ion mode:negative Genotype=74-D694; RAW_FILE_NAME=rnq-StressNIM1 .mzML SUBJECT_SAMPLE_FACTORS - rnq-StressNIM2 Treatment:0.2mM H202 | Ion mode:negative Genotype=74-D694; RAW_FILE_NAME=rnq-StressNIM2 .mzML SUBJECT_SAMPLE_FACTORS - rnq-StressNIM3 Treatment:0.2mM H202 | Ion mode:negative Genotype=74-D694; RAW_FILE_NAME=rnq-StressNIM3 .mzML SUBJECT_SAMPLE_FACTORS - rnq-StressNIM4 Treatment:0.2mM H202 | Ion mode:negative Genotype=74-D694; RAW_FILE_NAME=rnq-StressNIM4 .mzML SUBJECT_SAMPLE_FACTORS - rnq-StressNIM5 Treatment:0.2mM H202 | Ion mode:negative Genotype=74-D694; RAW_FILE_NAME=rnq-StressNIM5 .mzML SUBJECT_SAMPLE_FACTORS - rnq-StressNIM6 Treatment:0.2mM H202 | Ion mode:negative Genotype=74-D694; RAW_FILE_NAME=rnq-StressNIM6 .mzML SUBJECT_SAMPLE_FACTORS - RNQ+NIM1 Treatment:none | Ion mode:Positive Genotype=74-D694; RAW_FILE_NAME=RNQ+NIM1 .mzML SUBJECT_SAMPLE_FACTORS - RNQ+NIM2 Treatment:none | Ion mode:Positive Genotype=74-D694; RAW_FILE_NAME=RNQ+NIM2 .mzML SUBJECT_SAMPLE_FACTORS - RNQ+NIM3 Treatment:none | Ion mode:Positive Genotype=74-D694; RAW_FILE_NAME=RNQ+NIM3 .mzML SUBJECT_SAMPLE_FACTORS - RNQ+NIM4 Treatment:none | Ion mode:Positive Genotype=74-D694; RAW_FILE_NAME=RNQ+NIM4 .mzML SUBJECT_SAMPLE_FACTORS - RNQ+NIM5 Treatment:none | Ion mode:Positive Genotype=74-D694; RAW_FILE_NAME=RNQ+NIM5 .mzML SUBJECT_SAMPLE_FACTORS - RNQ+NIM6 Treatment:none | Ion mode:Positive Genotype=74-D694; RAW_FILE_NAME=RNQ+NIM6 .mzML SUBJECT_SAMPLE_FACTORS - RNQ+StressNIM1 Treatment:0.2mM H202 | Ion mode:Positive Genotype=74-D694; RAW_FILE_NAME=RNQ+StressNIM1 .mzML SUBJECT_SAMPLE_FACTORS - RNQ+StressNIM2 Treatment:0.2mM H202 | Ion mode:Positive Genotype=74-D694; RAW_FILE_NAME=RNQ+StressNIM2 .mzML SUBJECT_SAMPLE_FACTORS - RNQ+StressNIM3 Treatment:0.2mM H202 | Ion mode:Positive Genotype=74-D694; RAW_FILE_NAME=RNQ+StressNIM3 .mzML SUBJECT_SAMPLE_FACTORS - RNQ+StressNIM4 Treatment:0.2mM H202 | Ion mode:Positive Genotype=74-D694; RAW_FILE_NAME=RNQ+StressNIM4 .mzML SUBJECT_SAMPLE_FACTORS - RNQ+StressNIM5 Treatment:0.2mM H202 | Ion mode:Positive Genotype=74-D694; RAW_FILE_NAME=RNQ+StressNIM5 .mzML SUBJECT_SAMPLE_FACTORS - RNQ+StressNIM6 Treatment:0.2mM H202 | Ion mode:Positive Genotype=74-D694; RAW_FILE_NAME=RNQ+StressNIM6 .mzML SUBJECT_SAMPLE_FACTORS - DeltaPIM1 Treatment:none | Ion mode:negative Genotype=Deltarnq-knockout; RAW_FILE_NAME=DeltaPIM1 .mzML SUBJECT_SAMPLE_FACTORS - DeltaPIM2 Treatment:none | Ion mode:negative Genotype=Deltarnq-knockout; RAW_FILE_NAME=DeltaPIM2 .mzML SUBJECT_SAMPLE_FACTORS - DeltaPIM3 Treatment:none | Ion mode:negative Genotype=Deltarnq-knockout; RAW_FILE_NAME=DeltaPIM3 .mzML SUBJECT_SAMPLE_FACTORS - DeltaPIM4 Treatment:none | Ion mode:negative Genotype=Deltarnq-knockout; RAW_FILE_NAME=DeltaPIM4 .mzML SUBJECT_SAMPLE_FACTORS - DeltaPIM5 Treatment:none | Ion mode:negative Genotype=Deltarnq-knockout; RAW_FILE_NAME=DeltaPIM5 .mzML SUBJECT_SAMPLE_FACTORS - DeltaPIM6 Treatment:none | Ion mode:negative Genotype=Deltarnq-knockout; RAW_FILE_NAME=DeltaPIM6 .mzML SUBJECT_SAMPLE_FACTORS - rnq-PIM1 Treatment:none | Ion mode:negative Genotype=74-D694; RAW_FILE_NAME=rnq-PIM1 .mzML SUBJECT_SAMPLE_FACTORS - rnq-PIM2 Treatment:none | Ion mode:negative Genotype=74-D694; RAW_FILE_NAME=rnq-PIM2 .mzML SUBJECT_SAMPLE_FACTORS - rnq-PIM3 Treatment:none | Ion mode:negative Genotype=74-D694; RAW_FILE_NAME=rnq-PIM3 .mzML SUBJECT_SAMPLE_FACTORS - rnq-PIM4 Treatment:none | Ion mode:negative Genotype=74-D694; RAW_FILE_NAME=rnq-PIM4 .mzML SUBJECT_SAMPLE_FACTORS - rnq-PIM5 Treatment:none | Ion mode:negative Genotype=74-D694; RAW_FILE_NAME=rnq-PIM5 .mzML SUBJECT_SAMPLE_FACTORS - rnq-PIM6 Treatment:none | Ion mode:negative Genotype=74-D695; RAW_FILE_NAME=rnq-PIM6 .mzML SUBJECT_SAMPLE_FACTORS - rnq-StressPIM1 Treatment:0.2mM H202 | Ion mode:negative Genotype=74-D694; RAW_FILE_NAME=rnq-StressPIM1 .mzML SUBJECT_SAMPLE_FACTORS - rnq-StressPIM2 Treatment:0.2mM H202 | Ion mode:negative Genotype=74-D694; RAW_FILE_NAME=rnq-StressPIM2 .mzML SUBJECT_SAMPLE_FACTORS - rnq-StressPIM3 Treatment:0.2mM H202 | Ion mode:negative Genotype=74-D694; RAW_FILE_NAME=rnq-StressPIM3 .mzML SUBJECT_SAMPLE_FACTORS - rnq-StressPIM4 Treatment:0.2mM H202 | Ion mode:negative Genotype=74-D694; RAW_FILE_NAME=rnq-StressPIM4 .mzML SUBJECT_SAMPLE_FACTORS - rnq-StressPIM5 Treatment:0.2mM H202 | Ion mode:negative Genotype=74-D694; RAW_FILE_NAME=rnq-StressPIM5 .mzML SUBJECT_SAMPLE_FACTORS - rnq-StressPIM6 Treatment:0.2mM H202 | Ion mode:negative Genotype=74-D694; RAW_FILE_NAME=rnq-StressPIM6 .mzML SUBJECT_SAMPLE_FACTORS - RNQ+PIM1 Treatment:none | Ion mode:Positive Genotype=74-D694; RAW_FILE_NAME=RNQ+PIM1 .mzML SUBJECT_SAMPLE_FACTORS - RNQ+PIM2 Treatment:none | Ion mode:Positive Genotype=74-D694; RAW_FILE_NAME=RNQ+PIM2 .mzML SUBJECT_SAMPLE_FACTORS - RNQ+PIM3 Treatment:none | Ion mode:Positive Genotype=74-D694; RAW_FILE_NAME=RNQ+PIM3 .mzML SUBJECT_SAMPLE_FACTORS - RNQ+PIM4 Treatment:none | Ion mode:Positive Genotype=74-D694; RAW_FILE_NAME=RNQ+PIM4 .mzML SUBJECT_SAMPLE_FACTORS - RNQ+PIM5 Treatment:none | Ion mode:Positive Genotype=74-D694; RAW_FILE_NAME=RNQ+PIM5 .mzML SUBJECT_SAMPLE_FACTORS - RNQ+PIM6 Treatment:none | Ion mode:Positive Genotype=74-D694; RAW_FILE_NAME=RNQ+PIM6 .mzML SUBJECT_SAMPLE_FACTORS - RNQ+StressPIM1 Treatment:0.2mM H202 | Ion mode:Positive Genotype=74-D694; RAW_FILE_NAME=RNQ+StressPIM1 .mzML SUBJECT_SAMPLE_FACTORS - RNQ+StressPIM2 Treatment:0.2mM H202 | Ion mode:Positive Genotype=74-D694; RAW_FILE_NAME=RNQ+StressPIM2 .mzML SUBJECT_SAMPLE_FACTORS - RNQ+StressPIM3 Treatment:0.2mM H202 | Ion mode:Positive Genotype=74-D694; RAW_FILE_NAME=RNQ+StressPIM3 .mzML SUBJECT_SAMPLE_FACTORS - RNQ+StressPIM4 Treatment:0.2mM H202 | Ion mode:Positive Genotype=74-D694; RAW_FILE_NAME=RNQ+StressPIM4 .mzML SUBJECT_SAMPLE_FACTORS - RNQ+StressPIM5 Treatment:0.2mM H202 | Ion mode:Positive Genotype=74-D694; RAW_FILE_NAME=RNQ+StressPIM5 .mzML SUBJECT_SAMPLE_FACTORS - RNQ+StressPIM6 Treatment:0.2mM H202 | Ion mode:Positive Genotype=74-D694; RAW_FILE_NAME=RNQ+StressPIM6 .mzML #COLLECTION CO:COLLECTION_SUMMARY Strain and cultivation conditions The S. cerevisiae strain used in this study CO:COLLECTION_SUMMARY were derivatives of 74-D694 (MATa ade1-14(UGA) trp1-289(UAG) ura3-52 his3-∆200 CO:COLLECTION_SUMMARY leu2-3, 112) (Chernoff et al. 1993). Yeast harbouring [RNQ+] and knockout strain CO:COLLECTION_SUMMARY Δrnq were kind gifts from the Kent Fungal Group. Yeast were grown at 30°C with CO:COLLECTION_SUMMARY shaking at 180 rpm in synthetic complete (SC) media (2 % (w/v) glucose, 0.17 % CO:COLLECTION_SUMMARY Yeast Nitrogen Base (without amino acids, without ammonium sulphate), 0.5% CO:COLLECTION_SUMMARY ammonium sulphate, the appropriate concentration of yeast synthetic complete CO:COLLECTION_SUMMARY supplement mixture or synthetic complete drop-out media supplement). Transient CO:COLLECTION_SUMMARY growth on SC media containing 3mM guanidine hydrochloride (GdnHCl) was used as a CO:COLLECTION_SUMMARY curing agent in the media of S. cerevisiae cells that required a [prion-] CO:COLLECTION_SUMMARY status. Mild oxidative stresses were achieved by the addition of H2O2 (final CO:COLLECTION_SUMMARY concentration 0.2 mM) to the appropriate culture mediums. Cultures were grown CO:COLLECTION_SUMMARY using the filter culture method (as described by rabinowiz 2008) CO:SAMPLE_TYPE Yeast cells CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Mild oxidative stresses were achieved by the addition of H2O2 (final TR:TREATMENT_SUMMARY concentration 0.2 mM) #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolite sample preparation Analytical grade standards were supplied by Sigma SP:SAMPLEPREP_SUMMARY Aldrich. Quenching was achieved by adaption of cold methanol protocol (56), via SP:SAMPLEPREP_SUMMARY submersion of entire filter membrane. Metabolite extraction was performed on the SP:SAMPLEPREP_SUMMARY resultant cell pellets using the boiling ethanol technique (57). Briefly, each SP:SAMPLEPREP_SUMMARY tube was taken from the −80°C and 5 ml 75% (v/v) boiling ethanol was added SP:SAMPLEPREP_SUMMARY (pre-heated). Each tube was immediately vortexed and placed in a water bath at SP:SAMPLEPREP_SUMMARY 80°C. After 5 min each tube was cooled on ice for 3 min, followed by SP:SAMPLEPREP_SUMMARY centrifugation (5000xg, 5 minutes, -20°C, precooled). Extracts were then stored SP:SAMPLEPREP_SUMMARY at -80°C until further use. Immediately prior to mass spectrometry SP:SAMPLEPREP_SUMMARY experimentation all extracts were concentrated by speed vacuum at 35° C for ≈ SP:SAMPLEPREP_SUMMARY 3 hours. Following resuspension in 500µL of LC/MS grade water samples were SP:SAMPLEPREP_SUMMARY lyophilised overnight. Lyophilised samples were then resuspended in 200µL of SP:SAMPLEPREP_SUMMARY 0.1M formic acid, vortexed and loaded into vials. SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACTION_METHOD Boiling ethanol #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME ACQUITY SYNAPT G2-Si Mass Spectrometer CH:COLUMN_NAME 1.7 µm C18 BEH column CH:SOLVENT_A 95% Acetronitrile/5% water, 0.1% formic acid CH:SOLVENT_B 100% water; 0.1% formic acid CH:FLOW_GRADIENT 10-minute gradient from 0% to 50% acetonitrile (0.1% formic acid) CH:FLOW_RATE 500nL/min CH:COLUMN_TEMPERATURE 35 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Synapt G2 Si QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Data is labeled for negative or positive ionization mode MS:MS_RESULTS_FILE ST002478_AN004047_Results.txt UNITS:m/z values Has m/z:Yes Has RT:Yes RT units:Minutes #END