#METABOLOMICS WORKBENCH KasparasP_20240820_065447 DATATRACK_ID:5129 STUDY_ID:ST003420 ANALYSIS_ID:AN005619 PROJECT_ID:PR002115 VERSION 1 CREATED_ON August 20, 2024, 7:04 am #PROJECT PR:PROJECT_TITLE TRAM–LAG1–CLN8 family proteins are acyltransferases regulating phospholipid PR:PROJECT_TITLE composition PR:PROJECT_SUMMARY The diversity of cellular phospholipids, crucial for membrane homeostasis and PR:PROJECT_SUMMARY function, arises from enzymatic remodeling of their fatty acyl chains. In this PR:PROJECT_SUMMARY work, we reveal that poorly understood TRAM–LAG1–CLN8 domain-containing PR:PROJECT_SUMMARY (TLCD) proteins are phospholipid remodeling enzymes. We demonstrate that TLCD1 PR:PROJECT_SUMMARY is an evolutionarily conserved lysophosphatidylethanolamine acyltransferase, PR:PROJECT_SUMMARY which regulates cellular phospholipid composition and generates novel fatty acid PR:PROJECT_SUMMARY and thiamine (vitamin B1) esters as its secondary products. Furthermore, we PR:PROJECT_SUMMARY establish that human TLCD protein CLN8, mutations in which cause fatal PR:PROJECT_SUMMARY neurodegenerative Batten disease, is a lysophosphatidylglycerol acyltransferase. PR:PROJECT_SUMMARY We show that CLN8 catalyzes the essential step in the biosynthesis of PR:PROJECT_SUMMARY bis(monoacylglycero)phosphate, a phospholipid critical for lysosome function. PR:PROJECT_SUMMARY Our study unveils a new family of acyltransferases integral to cellular membrane PR:PROJECT_SUMMARY phospholipid homeostasis and human disease. PR:INSTITUTE University of Cambridge PR:LAST_NAME Petkevicius PR:FIRST_NAME Kasparas PR:ADDRESS The Keith Peters Building, Cambridge, Cambridgeshire, CB2 0XY, United Kingdom PR:EMAIL kp416@mrc-mbu.cam.ac.uk PR:PHONE +447500233355 PR:PUBLICATIONS TRAM–LAG1–CLN8 family proteins are acyltransferases regulating phospholipid PR:PUBLICATIONS composition #STUDY ST:STUDY_TITLE Analysis of lipid composition of control, YPR114w and YJR116w yeast mutants ST:STUDY_TITLE grown under exponential phase ST:STUDY_SUMMARY Untargeted lipidomics experiments were conducted to analyze the differences in ST:STUDY_SUMMARY lipid composition between control, YPR114w and YJR116w yeast (Saccharomyces ST:STUDY_SUMMARY cerevisiae) mutants . See a reference publication (Sheokand et al) for details. ST:INSTITUTE University of Cambridge ST:LAST_NAME Petkevicius ST:FIRST_NAME Kasparas ST:ADDRESS The Keith Peters Building ST:EMAIL kp416@mrc-mbu.cam.ac.uk ST:PHONE 07500233355 ST:PUBLICATIONS TRAM–LAG1–CLN8 family proteins are acyltransferases regulating phospholipid ST:PUBLICATIONS composition #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Saccharomyces cerevisiae SU:TAXONOMY_ID 4932 #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 - Yeast process blank Genotype:Process blank | Sample source:Process blank | Replicate:Process blank RAW_FILE_NAME(Raw file name)=Yeast_process_blank.raw SUBJECT_SAMPLE_FACTORS - Wild-type replicate 1 Genotype:Wild-type | Sample source:Saccharomyces cerevisiae | Replicate:1 RAW_FILE_NAME(Raw file name)=Yeast_Wild-Type_exponential_phase_Replicate_1.raw SUBJECT_SAMPLE_FACTORS - Wild-type replicate 2 Genotype:Wild-type | Sample source:Saccharomyces cerevisiae | Replicate:2 RAW_FILE_NAME(Raw file name)=Yeast_Wild-Type_exponential_phase_Replicate_2.raw SUBJECT_SAMPLE_FACTORS - Wild-type replicate 3 Genotype:Wild-type | Sample source:Saccharomyces cerevisiae | Replicate:3 RAW_FILE_NAME(Raw file name)=Yeast_Wild-Type_exponential_phase_Replicate_3.raw SUBJECT_SAMPLE_FACTORS - YJR116w KO replicate 1 Genotype:YJR116w KO | Sample source:Saccharomyces cerevisiae | Replicate:1 RAW_FILE_NAME(Raw file name)=YJR116w_KO_exponential_phase_Replicate_1.raw SUBJECT_SAMPLE_FACTORS - YJR116w KO replicate 2 Genotype:YJR116w KO | Sample source:Saccharomyces cerevisiae | Replicate:2 RAW_FILE_NAME(Raw file name)=YJR116w_KO_exponential_phase_Replicate_2.raw SUBJECT_SAMPLE_FACTORS - YJR116w KO replicate 3 Genotype:YJR116w KO | Sample source:Saccharomyces cerevisiae | Replicate:3 RAW_FILE_NAME(Raw file name)=YJR116w_KO_exponential_phase_Replicate_3.raw SUBJECT_SAMPLE_FACTORS - YPR114w KO replicate 1 Genotype:YPR114w KO | Sample source:Saccharomyces cerevisiae | Replicate:1 RAW_FILE_NAME(Raw file name)=YPR114w_KO_exponential_phase_Replicate_1.raw SUBJECT_SAMPLE_FACTORS - YPR114w KO replicate 2 Genotype:YPR114w KO | Sample source:Saccharomyces cerevisiae | Replicate:2 RAW_FILE_NAME(Raw file name)=YPR114w_KO_exponential_phase_Replicate_2.raw SUBJECT_SAMPLE_FACTORS - YPR114w KO replicate 3 Genotype:YPR114w KO | Sample source:Saccharomyces cerevisiae | Replicate:3 RAW_FILE_NAME(Raw file name)=YPR114w_KO_exponential_phase_Replicate_3.raw #COLLECTION CO:COLLECTION_SUMMARY Yeast cells were grown in synthetic medium (SC) containing 2% glucose, 0.17% CO:COLLECTION_SUMMARY yeast nitrogen base (Cat. No. 233520, Difco, BD, Franklin Lakes, NJ), 0.5% CO:COLLECTION_SUMMARY ammonium sulfate, and amino acid drop-out (60 mg/L leucine, 55 mg/L adenine, 55 CO:COLLECTION_SUMMARY mg/L uracil, 55 mg/L tyrosine, 20 mg/L of arginine, 10 mg/L histidine, 60 mg/L CO:COLLECTION_SUMMARY isoleucine, 40 mg/L lysine, 60 mg/L phenylalanine, 50 mg/L threonine, 10 mg/L CO:COLLECTION_SUMMARY methionine, 40 mg/L tryptophan). For lipidomics, cells were grown overnight to CO:COLLECTION_SUMMARY exponential phase (OD600 0.4-0.6), then were washed once in water and snap CO:COLLECTION_SUMMARY frozen in liquid nitrogen. For acyl-thiamine measurements, cultures were grown CO:COLLECTION_SUMMARY to the exponential phase overnight, then split in two; one half was left CO:COLLECTION_SUMMARY untreated while the second half was supplemented with 100 mM thiamine. All CO:COLLECTION_SUMMARY cultures were then grown for 2h 30 min. Cells were washed once in water and snap CO:COLLECTION_SUMMARY frozen in liquid nitrogen. CO:SAMPLE_TYPE Yeast cells CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Cells were untreated (collected under standard culturing conditions). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The extraction of total lipids from cellular matrices, immunoprecipitated SP:SAMPLEPREP_SUMMARY mitochondria and in vitro assays was conducted employing the butanol-methanol SP:SAMPLEPREP_SUMMARY (BUME) method, as described in detail (37). Note that the chloroform-methanol SP:SAMPLEPREP_SUMMARY lipid extraction methods result in the partitioning of acyl-thiamines into the SP:SAMPLEPREP_SUMMARY polar phase. 2 ml screw cap plastic tubes were used (3469-11, Thermo SP:SAMPLEPREP_SUMMARY Scientific), and a blank extraction was always performed in parallel to account SP:SAMPLEPREP_SUMMARY for the plastic-related contaminants. The extraction commenced with the SP:SAMPLEPREP_SUMMARY homogenization of frozen cell pellets, mitochondrial beads or in vitro assay SP:SAMPLEPREP_SUMMARY mixtures in a 0.5 ml ice-cold solution of butanol to methanol in a 3:1 ratio. SP:SAMPLEPREP_SUMMARY For lipidomics samples, BUME solution was enriched with SPLASH internal standard SP:SAMPLEPREP_SUMMARY mix (330707, Avanti Polar Lipids). For the extraction, a further 0.5 ml of 1% SP:SAMPLEPREP_SUMMARY acetic acid and 0.5 ml of a heptane:ethyl acetate 3:1 mixture were added, SP:SAMPLEPREP_SUMMARY followed by vigorous vortexing for a total of 5 minutes. The mixture was then SP:SAMPLEPREP_SUMMARY centrifuged at 6000 g for 5 minutes, allowing for the separation of phases, SP:SAMPLEPREP_SUMMARY after which the upper organic phase was carefully decanted into glass vials. A SP:SAMPLEPREP_SUMMARY second extraction was conducted on the remaining aqueous phase, with the newly SP:SAMPLEPREP_SUMMARY acquired upper phase being combined in the same glass vials as the first. Post SP:SAMPLEPREP_SUMMARY extraction, the solvents were evaporated under a stream of nitrogen, and the SP:SAMPLEPREP_SUMMARY resultant dry lipid extracts were preserved at −70°C pending further SP:SAMPLEPREP_SUMMARY analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Dried samples were reconstituted in 100 μL of isopropanol, acetonitrile, and CH:CHROMATOGRAPHY_SUMMARY water (2:1:1 ratio) and thoroughly vortexed. Liquid chromatography was conducted CH:CHROMATOGRAPHY_SUMMARY using a Shimadzu HPLC System, with 10 μL of the sample introduced onto a Waters CH:CHROMATOGRAPHY_SUMMARY Acquity Premier UPLC® CSH column (1.7 μm pore size, 2.1 mm × 50 mm), which CH:CHROMATOGRAPHY_SUMMARY was maintained at 55 °C. The mobile phase A comprised a 6:4 ratio of CH:CHROMATOGRAPHY_SUMMARY acetonitrile to water with 10 mM ammonium formate, and mobile phase B consisted CH:CHROMATOGRAPHY_SUMMARY of a 9:1 ratio of isopropanol to acetonitrile with 10 mM ammonium formate. A CH:CHROMATOGRAPHY_SUMMARY flow rate of 500 μL per minute was maintained with a gradient protocol for CH:CHROMATOGRAPHY_SUMMARY mobile phase B as follows: 0.00 minutes_40% mobile phase B; 1.5 minutes_40% CH:CHROMATOGRAPHY_SUMMARY mobile phase B; 8.00 minutes_99% mobile phase B; 10.00 minutes_99% mobile phase CH:CHROMATOGRAPHY_SUMMARY B; 10.10 minutes_40% mobile phase B; 12.00 minutes_40% mobile phase. The sample CH:CHROMATOGRAPHY_SUMMARY injection needle was rinsed with a 9:1 isopropanol and acetonitrile solution CH:CHROMATOGRAPHY_SUMMARY (strong wash) and isopropanol, acetonitrile, and water (2:1:1, weak wash). CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Shimadzu 10A CH:COLUMN_NAME Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um) CH:SOLVENT_A 6:4 of acetonitrile to water with 10 mM ammonium formate CH:SOLVENT_B 9:1 of isopropanol to acetonitrile with 10 mM ammonium formate CH:FLOW_GRADIENT Gradient protocol for mobile phase B as follows: 0.00 minutes_40% mobile phase CH:FLOW_GRADIENT B; 1.5 minutes_40% mobile phase B; 8.00 minutes_99% mobile phase B; 10.00 CH:FLOW_GRADIENT minutes_99% mobile phase B; 10.10 minutes_40% mobile phase B; 12.00 minutes_40% CH:FLOW_GRADIENT mobile phase. CH:FLOW_RATE 500 μL per minute CH:COLUMN_TEMPERATURE 55 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE UNSPECIFIED MS:MS_COMMENTS Mass spectrometric detection was carried out on a ThermoFisher Scientific MS:MS_COMMENTS Q-Exactive Orbitrap equipped with a heated electrospray ionization source. The MS:MS_COMMENTS mass spectrometer was calibrated immediately before sample analysis using MS:MS_COMMENTS positive and negative ionization calibration solution (recommended by Thermo MS:MS_COMMENTS Scientific). The electrospray ionization parameters were optimized with a 50:50 MS:MS_COMMENTS mix of mobile phase A and B for spray stability, setting the capillary MS:MS_COMMENTS temperature at 300 °C, source heater temperature at 420 °C, with the sheath, MS:MS_COMMENTS auxiliary, and spare gas flows at specific arbitrary units (40, 15 and 3, MS:MS_COMMENTS respectively), and source voltage at 4 kV. The mass spectrometer operated at a MS:MS_COMMENTS scan rate of 4 Hz, yielding a resolution of 35,000 at m/z 200, over a full-scan MS:MS_COMMENTS range from m/z 120 to 1800, with continuous switching between positive and MS:MS_COMMENTS negative modes. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS nmol MS_METABOLITE_DATA_START Samples Yeast process blank Wild-type replicate 1 Wild-type replicate 2 Wild-type replicate 3 YJR116w KO replicate 1 YJR116w KO replicate 2 YJR116w KO replicate 3 YPR114w KO replicate 1 YPR114w KO replicate 2 YPR114w KO replicate 3 Factors Genotype:Process blank | Sample source:Process blank | Replicate:Process blank Genotype:Wild-type | Sample source:Saccharomyces cerevisiae | Replicate:1 Genotype:Wild-type | Sample source:Saccharomyces cerevisiae | Replicate:2 Genotype:Wild-type | Sample source:Saccharomyces cerevisiae | Replicate:3 Genotype:YJR116w KO | Sample source:Saccharomyces cerevisiae | Replicate:1 Genotype:YJR116w KO | Sample source:Saccharomyces cerevisiae | Replicate:2 Genotype:YJR116w KO | Sample source:Saccharomyces cerevisiae | Replicate:3 Genotype:YPR114w KO | Sample source:Saccharomyces cerevisiae | Replicate:1 Genotype:YPR114w KO | Sample source:Saccharomyces cerevisiae | Replicate:2 Genotype:YPR114w KO | Sample source:Saccharomyces cerevisiae | Replicate:3 PE_(32:1) 0 0.213276409 0.222070599 0.20444661 0.250931665 0.240778332 0.187420943 0.297076631 0.349799744 0.308327365 PE_(32:2) 0 0.538111654 0.543252812 0.525006262 0.612678723 0.577156982 0.495485451 0.437204547 0.459162065 0.411103778 PE_(34:1) 0 0.144757584 0.151616987 0.135784191 0.162962302 0.146185043 0.115896705 0.148333956 0.155132689 0.147414284 PE_(34:2) 0 0.292285491 0.278620632 0.263338387 0.344018988 0.308976599 0.268697549 0.22464156 0.211606061 0.204235539 PE_(36:1) 0 0.031090807 0.030556418 0.028334764 0.031523946 0.028605243 0.022156137 0.034917874 0.038214952 0.034698892 PE_(36:2) 0 0.103578863 0.101188183 0.092808515 0.128684416 0.169321634 0.093691 0.098126893 0.076156003 0.067123301 PC_(28:1) 0 0.098712744 0.106333834 0.092871078 0.138744216 0.122373396 0.120962497 0.094846558 0.098410761 0.087467723 PC_(30:1) 0 0.117496035 0.125758476 0.105958257 0.134306713 0.110458806 0.101342071 0.214260203 0.245950709 0.17935015 PC_(30:2) 0 0.045570283 0.049240054 0.047699065 0.070137645 0.058955199 0.052850628 0.068533207 0.073541438 0.06178775 PC_(32:1) 0 0.203062877 0.220610167 0.200993009 0.226342159 0.184110594 0.174916997 0.289978068 0.313179233 0.274730221 PC_(32:2) 0 1.170464959 1.246431758 1.204106606 1.595365724 1.320692309 1.279042226 1.205339489 1.314667576 1.187002393 PC_(34:1) 0 0.068362173 0.064846418 0.063532718 0.079023475 0.063466172 0.059496166 0.069913634 0.075685009 0.073746631 PC_(34:2) 0 0.528856665 0.605288516 0.555142484 0.790028983 0.646997488 0.570770225 0.484448021 0.491409979 0.447194154 PC_(36:1) 0 0.005630952 0.005515127 0.005505952 0.007940455 0.006158653 0.005823518 0.005672185 0.005257069 0.00514105 PC_(36:2) 0 0.029309574 0.027630282 0.027884154 0.033728701 0.025054793 0.022680225 0.051998066 0.051160199 0.048423948 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name m/z [M+H]+ Average Rt(min) PE_(32:1) 690.50623 4.229 PE_(32:2) 688.49121 3.639 PE_(34:1) 718.54077 4.784 PE_(34:2) 716.52209 4.303 PE_(36:1) 746.5697 4.751 PE_(36:2) 744.55377 4.265 PC_(28:1) 676.49152 2.281 PC_(30:1) 704.52283 3.296 PC_(30:2) 702.5069 2.494 PC_(32:1) 732.55371 4.075 PC_(32:2) 730.5379 3.44 PC_(34:1) 760.58527 4.666 PC_(34:2) 758.56903 4.156 PC_(36:1) 788.61652 5.136 PC_(36:2) 786.60046 4.709 METABOLITES_END #END