Sanyal S

Sanyal S., Menon A. a lack of activity. These results suggest that PRD is crucial for the function of the protein. The effects of the PRD deletion in hPLSCR1 and the addition of PRD to hPLSCR2 were characterized using various spectroscopic techniques. Our results clearly showed that hPLSCR1 and PRD-hPLSCR2 showed Ca2+-dependent aggregation and scrambling activity, whereas hPLSCR2 and PRD-hPLSCR1 did not show aggregation and activity. Thus we conclude that scramblases exhibit Ca2+-dependent scrambling activity by aggregation of protein. Our results provide a possible mechanism for phospholipid scrambling mediated by PLSCRs and the importance of PRD in its function and cellular localization. to humans (7). Although initially identified as scramblase, hPLSCR1 was found to be involved in many signal transduction pathways like IFN-mediated antiviral activity and PKC- mediated pathways and is also a substrate for cellular kinases (8, 9). hPLSCR3 localizes to mitochondria and is involved in intrinsic apoptotic pathway and cardiolipin translocation in mitochondria (10). Recent evidence suggests that hPLSCR4 also STAT3-IN-3 mediates bidirectional translocation of PLs across PL bilayer (11). hPLSCR2 is known to be localized to the nucleus; however, the structural and functional characterization of hPLSCR2 has not been performed yet (12). Homology studies of PLSCRs Rabbit Polyclonal to SFRS7 reveal that hPLSCR2, -3, and -4 share 59, 47, and 46% similarity with hPLSCR1 (5). PLSCRs are multidomain-containing proteins where each domain name has distinct functions that need to be elucidated. Major domains of PLSCRs include proline-rich domain name (PRD), DNA binding motif, palmitoylation motif, nuclear localization signal, putative EF-hand like calcium binding motif, and C-terminal helix (CTH) (5). Except for hPLSCR2, members of scramblase family contain an N-terminal PRD that possesses PDH5 and BL21 (DE3) strains were obtained from ATCC. cDNA of hPLSCR1 and -2 was purchased from Invitrogen, and pET-28b(+) was from Novagen. Isopropyl -d-1-thiogalactopyranoside, dithiothreitol (DTT), and EDTA were STAT3-IN-3 purchased from Himedia. SM-2 Biobeads and Chelex-100 resin were obtained from Bio-Rad. Nickel nitrilotriacetic acid STAT3-IN-3 was purchased from Qiagen. BL-21 (DE3) cells were transformed with the respective plasmids and grown in a selective media made up of kanamycin (50 g/ml). Post-induction, cells were pelleted and lysed in buffer A (20 mm Tris (pH 7.4), 200 mm NaCl) with 1 mm PMSF, 1 mm EDTA, and 1 mm DTT using a probe sonicator (Vibro cell ultrasonicator). Cell lysate was then clarified at 12,000 for 10 min, and the pellet (nuclear fraction) and supernatant (cytosolic + membrane fraction) were saved. Supernatant was then centrifuged at 21,000 for 30 min to separate the membrane and cytosolic fraction. The membrane and nuclear fraction were then solubilized using lysis buffer made up of 1% Nonidet P-40 detergent and used for Western blot analysis. Equal amounts of cytosolic, membrane, and nuclear proteins (50 g) was taken for Western blot analysis. Western Blot Analysis Transfected cells were lysed in lysis buffer (5 mm Tris (pH 7.4), 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mm EDTA, 1 mm PMSF, and protease inhibitors). Total protein was estimated by the BCA method using BSA as the standard. 50 g of total protein was loaded on 12% SDS-PAGE and transferred onto nitrocellulose membrane. Membrane was blocked using blocking buffer with BSA (10 mm Tris (pH 7.4), 150 mm NaCl, and 0.1% Tween 20 for 1 h at 25 C. Immunoblotting was done using hPLSCR1 and hPLSCR2 mouse monoclonal antibodies (Santa Cruz), and detection was performed using an ECL kit (Thermo Scientific kit). To check the protein expression levels of all four constructs, HEK 293T cells were transiently transfected with GFP-tagged gene constructs. After 18 h of transfection, cells STAT3-IN-3 were lysed in lysis buffer, and Western blots were developed as described above with rabbit monoclonal antibodies specific to GFP (Promega) and -actin (Sigma, mouse) with 1:5000 dilutions. The bands were visualized by Clarity Western ECL substrate (Bio-Rad). Ca2+ Binding Studies Stains-All, a cationic carbocyanine dye, was used to monitor the calcium binding properties of hPLSCR1 and -2 and mutant constructs. Stains-All was dissolved in 2 mm MOPS buffer (pH 7.2) containing 30% ethylene glycol. Stains-All produces a series of discrete spectra depending upon conversation and conformation of binding region (27). The free form of the dye produces two distinctive spectra at 535 nm (-band) and 575 nm (-band) that correspond to the monomeric and dimeric form.