Bloom syndrome is acondition that affects multiple systems in the body and is associated withgrowth deficits, skin problems, compromised immune system, insulin resistanceand increased risk of developing diabetes and cancer (1). A preliminary analysiswas carried out to determine whether proteins are differentially expressed inpatients with Bloom syndrome. SILAC mass spectrometry analysis of wildtypefibroblasts and fibroblasts isolated from patients with Bloom syndrome showedthat several proteins are upregulated in affected individuals (Shastri andSchmidt, unpublished observation). Out of the different proteins that showed astatistically significant upregulation, the protein of interest in my study isTFAM.
Mitochondrial TranscriptionFactor A (TFAM) is a nuclear encoded mitochondrial DNA transcription factor (2).Specific binding of TFAM to the light strand promoter and the heavy strandpromoter regulates transcription and replication of mitochondrial DNA (3). TFAMalso functions in the compaction, organization and stabilization of the mtDNAthrough its high mobility group (HMG) box domain. By modulating multiplefunctions, TFAM can regulate mitochondrial copy number. Studies have shown thatTFAM protein levels directly correlate to mitochondrial copy number (4) (5) (6).From the initial screenit is evident that TFAM is upregulated in fibroblasts isolated from patientwith Bloom syndrome. Previous studies have indicated that oxidative stressbiomarkers are upregulated in Bloom syndrome (7) (8) (9).
The goal of myproject is to determine whether TFAM mediated increase in mitochondrial numberis the contributing factor to the oxidative stress phenotype that is observedin patients with Bloom syndrome. METHODSCell CultureGMO 0637, GMO 8505, KHS1452 and KHS 1453 fibroblast cell lines were cultured at 37°C and 5% CO2 inMinimal Essential Medium (DMEM) supplemented with 10% fetal bovine serum, 1%PSG. Western BlottingFor lysate preparation,cells were washed with ice cold PBS. Cells were harvested in ice cold RIPAlysis buffer using cell. The lysate was then vortexed continuously for 30 minat 4 ºC. The mixture was then centrifuged at 14000 rpm for 15 min and supernatant wascollected in fresh tube.The protein lysateswere then normalized for equal amounts of proteins using Pierce BCA proteinassay kit.
Samples were prepared using 2X Laemmli buffer containing 5%?-mercaptoethanol. It was then boiled at 96 ºC for 5 min and loaded onto 10%SDS-PAGE gels. The gels were run at constant voltage and the proteins weretransferred to 0.45µm PVDF membranes by wet transfer at 4 ºC for 90 min at320mA. The membrane was blocked for an hour in 5% of Skimmed milk prepared inTBST (Tris-Buffered Saline supplemented with 0.05% Tween 20) followed byincubation with primary antibody (prepared in 5% BSA in TBST, Dilution 1:1000)against protein of interest overnight at 4 ºC . Bound primary antibody wasrecognized using horseradish peroxidise coupled secondary antibody which wasincubated at room temperature for 1hr.
Following each step, the blots were washedthree times in TBST for 5 min each. Chemiluminescence was detected and theimages were acquired using ChemiDoc MP System (Bio-rad).ImmunofluorescencemicroscopyThecells were plated on sterile coverslips placed in 6 well plate. For TFAMstaining, cells were fixed using 4% Paraformaldehyde at 37 ºC for 20 min. Thecells were then permeabilized using 0.25% Triton X-100. Blocking was performedusing 5% BSA in TBST for 1 h at room temperature to prevent non-specificbinding of antibodies.
This was followed by incubation of the cells overnightat 4 ºC with antibody against TFAM (1:100 dilution) prepared in blockingbuffer. The primary antibodies were then incubated with Alexa Fluor conjugatedsecondary antibodies (1:500) for 1hr at room temperature. Cells were washedthrice with PBS following each step. The cells were finally mounted on a cleanglass slide using VECTASHIELD mounting medium containing DAPI and the imageswere acquired using confocal microscope.For mitotrackerstaining, cells were incubated with media containing mitotracker (finalconcentration: 50nM) at 37 ºC for 30min.
After this, cells were washed thricewith PBS, fixed and permeabilized using methanol at -20 ºC for 20min andmounted on glass slides.