{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002850","ANALYSIS_ID":"AN004668","VERSION":"1","CREATED_ON":"September 8, 2023, 9:11 am"},

"PROJECT":{"PROJECT_TITLE":"Bap1 Promotes Osteoclast Function by Metabolic Reprogramming","PROJECT_TYPE":"Untargeted Metabolomics","PROJECT_SUMMARY":"Treatment of osteoporosis most commonly diminishes osteoclast number which suppresses bone formation thus compromising fracture prevention. Bone formation is not suppressed, however, when bone degradation is reduced by retarding osteoclast functional resorptive capacity, rather than differentiation. We find deletion of deubiquitinase, BRCA1-associated protein 1 (Bap1), in myeloid cells (Bap1∆LysM), arrests osteoclast function but not formation. Bap1∆LysM osteoclasts fail to organize their cytoskeleton which is essential for bone degradation. Consequently, bone mass increases in the mutant mice. We find the deubiquitinase activity of Bap1 regulates osteoclast function by metabolic reprogramming. Bap1 deficient osteoclast lineage cells upregulate the cystine transporter, Slc7a11, by enhanced H2Aub occupancy of its promoter. SLC7A11 regulates cellular ROS levels and redirects the mitochondrial metabolites away from the TCA cycle, both of which are necessary for osteoclast function. Thus in osteoclasts, Bap1 appears to regulate epigenetic-metabolic axis and is a potential target to reduce bone degradation while maintaining osteogenesis in osteoporotic patients.","INSTITUTE":"Washington University in St. Louis","DEPARTMENT":"Pathology and Immunology, Medicine, Chemistry","LABORATORY":"Teitelbaum and Patti Laboratories","LAST_NAME":"Cho","FIRST_NAME":"Kevin","ADDRESS":"1 Brookings Drive, Campus Box 1134, St. Louis, MO, 63130, USA","EMAIL":"kevin.cho@wustl.edu","PHONE":"314-935-8813"},

"STUDY":{"STUDY_TITLE":"Bap1 Promotes Osteoclast Function by Metabolic Reprogramming","STUDY_TYPE":"Untargeted Metabolomics","STUDY_SUMMARY":"Treatment of osteoporosis most commonly diminishes osteoclast number which suppresses bone formation thus compromising fracture prevention. Bone formation is not suppressed, however, when bone degradation is reduced by retarding osteoclast functional resorptive capacity, rather than differentiation. We find deletion of deubiquitinase, BRCA1-associated protein 1 (Bap1), in myeloid cells (Bap1∆LysM), arrests osteoclast function but not formation. Bap1∆LysM osteoclasts fail to organize their cytoskeleton which is essential for bone degradation. Consequently, bone mass increases in the mutant mice. We find the deubiquitinase activity of Bap1 regulates osteoclast function by metabolic reprogramming. Bap1 deficient osteoclast lineage cells upregulate the cystine transporter, Slc7a11, by enhanced H2Aub occupancy of its promoter. SLC7A11 regulates cellular ROS levels and redirects the mitochondrial metabolites away from the TCA cycle, both of which are necessary for osteoclast function. Thus in osteoclasts, Bap1 appears to regulate epigenetic-metabolic axis and is a potential target to reduce bone degradation while maintaining osteogenesis in osteoporotic patients.","INSTITUTE":"Washington University in St. Louis","DEPARTMENT":"Pathology and Immunology, Medicine, Chemistry","LABORATORY":"Teitelbaum and Patti Laboratories","LAST_NAME":"Cho","FIRST_NAME":"Kevin","ADDRESS":"1 Brookings Drive, Campus Box 1134, St. Louis, MO, 63130, USA","EMAIL":"kevin.cho@wustl.edu","PHONE":"314-935-8813"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"Pos_WT_1",
"Factors":{"Genotype":"Wild-type"},
"Additional sample data":{"Polarity":"Positive","RAW_FILE_NAME":"Pos_WT_1.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"Pos_WT_2",
"Factors":{"Genotype":"Wild-type"},
"Additional sample data":{"Polarity":"Positive","RAW_FILE_NAME":"Pos_WT_2.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"Pos_WT_3",
"Factors":{"Genotype":"Wild-type"},
"Additional sample data":{"Polarity":"Positive","RAW_FILE_NAME":"Pos_WT_3.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"Pos_WT_4",
"Factors":{"Genotype":"Wild-type"},
"Additional sample data":{"Polarity":"Positive","RAW_FILE_NAME":"Pos_WT_4.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"Pos_WT_5",
"Factors":{"Genotype":"Wild-type"},
"Additional sample data":{"Polarity":"Positive","RAW_FILE_NAME":"Pos_WT_5.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"Pos_KO_1",
"Factors":{"Genotype":"Knockout"},
"Additional sample data":{"Polarity":"Positive","RAW_FILE_NAME":"Pos_KO_1.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"Pos_KO_2",
"Factors":{"Genotype":"Knockout"},
"Additional sample data":{"Polarity":"Positive","RAW_FILE_NAME":"Pos_KO_2.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"Pos_KO_3",
"Factors":{"Genotype":"Knockout"},
"Additional sample data":{"Polarity":"Positive","RAW_FILE_NAME":"Pos_KO_3.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"Pos_KO_4",
"Factors":{"Genotype":"Knockout"},
"Additional sample data":{"Polarity":"Positive","RAW_FILE_NAME":"Pos_KO_4.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"Pos_KO_5",
"Factors":{"Genotype":"Knockout"},
"Additional sample data":{"Polarity":"Positive","RAW_FILE_NAME":"Pos_KO_5.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"Neg_WT_1",
"Factors":{"Genotype":"Wild-type"},
"Additional sample data":{"Polarity":"Negative","RAW_FILE_NAME":"Neg_WT_1.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"Neg_WT_2",
"Factors":{"Genotype":"Wild-type"},
"Additional sample data":{"Polarity":"Negative","RAW_FILE_NAME":"Neg_WT_2.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"Neg_WT_3",
"Factors":{"Genotype":"Wild-type"},
"Additional sample data":{"Polarity":"Negative","RAW_FILE_NAME":"Neg_WT_3.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"Neg_WT_4",
"Factors":{"Genotype":"Wild-type"},
"Additional sample data":{"Polarity":"Negative","RAW_FILE_NAME":"Neg_WT_4.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"Neg_WT_5",
"Factors":{"Genotype":"Wild-type"},
"Additional sample data":{"Polarity":"Negative","RAW_FILE_NAME":"Neg_WT_5.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"Neg_KO_1",
"Factors":{"Genotype":"Knockout"},
"Additional sample data":{"Polarity":"Negative","RAW_FILE_NAME":"Neg_KO_1.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"Neg_KO_2",
"Factors":{"Genotype":"Knockout"},
"Additional sample data":{"Polarity":"Negative","RAW_FILE_NAME":"Neg_KO_2.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"Neg_KO_3",
"Factors":{"Genotype":"Knockout"},
"Additional sample data":{"Polarity":"Negative","RAW_FILE_NAME":"Neg_KO_3.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"Neg_KO_4",
"Factors":{"Genotype":"Knockout"},
"Additional sample data":{"Polarity":"Negative","RAW_FILE_NAME":"Neg_KO_4.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"Neg_KO_5",
"Factors":{"Genotype":"Knockout"},
"Additional sample data":{"Polarity":"Negative","RAW_FILE_NAME":"Neg_KO_5.mzML"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Primary mus musculus cells","SAMPLE_TYPE":"Osteoclast"},

"TREATMENT":{"TREATMENT_SUMMARY":"All in vitro experiments were performed at least 3 times. Primary bone marrow macrophages (BMMs) were prepared as described with slight modification. Marrow was extracted from femora and tibiae of 6- to 8-week-old mice with α minimum essential medium (α-MEM) and cultured in α-MEM containing 10% inactivated fetal bovine serum, 100 IU/mL penicillin, and 100 μg/mL streptomycin (α-10 medium) with 1:10 of mMCSF producing cell line, CMG 14-12 condition media on petri-plastic dishes. Cells were incubated at 37°C in 5% CO2 for 3 days and then washed with phosphate-buffered saline (PBS) and lifted with 1X trypsin/EDTA in PBS. A total of 1.2 × 104 BMMs were cultured in 500 μL α-MEM containing 10% heat-inactivated fetal bovine serum with glutathione-S transferase–RANKL and 30 ng/mL of mouse recombinant macrophage colony-stimulating factor (M-CSF) in 48-well tissue culture plates, some containing sterile bovine bone slices."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Cells were quenched with cold LC/MS-grade methanol, then scraped and transferred to Eppendorf tubes. Samples were dried in a SpeedVac. The samples were then reconstituted in 1 mL of cold methanol:acetonitrile:water (2:2:1) and subjected to three cycles of vortexing, freezing in liquid nitrogen, and 10 min of sonication at 25 °C. Samples were stored at −20 °C overnight and then centrifuged for 10 min at 14,000×g and 4 °C. Supernatants were transferred to new tubes and dried by a SpeedVac. The protein abundance of each sample was determined by using BCA. A quantity of 1 μl of acetonitrile:water (2:1) per each 2.5 μg of protein was used. Samples were subjected to two cycles of vortexing and 10 min of sonication at 25 °C. Next, samples were centrifuged for 10 min at 14,000×g and 4 °C, transferred supernatant to LC vials, and stored at −80 °C until MS analysis"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Thermo Vanquish Flex UHPLC Systems","COLUMN_NAME":"HILICON iHILIC-(P) Classic (100 x 2.1mm,5um)","SOLVENT_A":"20 mM ammonium bicarbonate, 0.1% ammonium hydroxideand 2.5 μM medronic acid in water:acetonitrile (95:5)","SOLVENT_B":"acetonitrile:water (95:5)","FLOW_GRADIENT":"0–1 min: 90% B, 1–12 min: 90-35% B, 12–12.5 min: 35-25% B, 12.5–14.5 min: 25% B","FLOW_RATE":"250 uL/min","COLUMN_TEMPERATURE":"45"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Orbitrap ID-X tribrid","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","MS_COMMENTS":"Data were collected with the following MS source settings: spray voltage, -2.8 kV; sheath gas, 50; auxiliary gas, 10; sweep gas, 1; ion transfer tube temperature, 300°C; vaporizer temperature, 200°C; mass range, 67 – 1000 Da; resolution, 120,000; maximum injection time, 200 ms; isolation window, 1.5 Da. XCMS and Skyline software were used for data processing","MS_RESULTS_FILE":"ST002850_AN004668_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Seconds"}

}