TMUB1 Inhibits BRL-3A Hepatocyte Proliferation by Interfering with the Binding of CAML to Cyclophilin B through its TM1 Hydrophobic Domain

Transmembrane and ubiquitin-like domain-containing 1 (Tmub1) encodes a protein (TMUB1) containing an ubiquitin-like domain and plays a negative regulatory role during hepatocyte proliferation, but its mechanism in this process is still unknown. Here, TMUB1 interfered with the binding of calcium-modulating cyclophilin ligand (CAML) to cyclophilin B, which may represent a key role in the negative regulatory process of TMUB1 in hepatocyte proliferation. Co-immunoprecipitation assays in rat BRL-3A cells confirmed the interaction between TMUB1 and CAML; significant regulation of the influx of Ca2+ ([Ca2+]i) and hepatocyte proliferation occurred following TMUB1 overexpression or knockout. Deletion of the TM1 hydrophobic domain of TMUB1 completely abolished this interaction and led to loss of TMUB1’s regulatory effects on cytological behavior. Furthermore, overexpression of TMUB1 completely abolished the interaction between CAML and its downstream protein cyclophilin B, which can act upstream of calcineurin by increasing [Ca2+]i during cell proliferation. Taken together, our results indicate that TMUB1 regulates BRL-3A hepatocyte proliferation by interacting with CAML and further interferes with the binding of CAML to cyclophilin B to decrease cellular [Ca2+]i.


Tmubl sequence：
Transmembrane and ubiquitin-like domain-containing protein 1, Rattus norvegicus (245 aa was tested repectively. BRL-3A rat hepatocytes were cultured in high-glucose DMEMsupplemented with 10% fetal bovine serum at 37˚C with 5% CO2. When the hepatocytesreached 80-90% confluence, they were passagedand placed in a 100-mm culture dish.After 24 hours, when the cells had reached 40-60% confluence, they were passaged and placed in DMEM without penicillin-streptomycin. A total of 21µl PEI or 72ulLipoFiterTM was mixed with 979ml DMEM (PEI) or 928ml DMEM (Lipo 2000) in a 100-mm culture dish and was not disturbed for 5 min. The final concentration of the plasmid was 7-8ug (PEI) or 24µg (Lipo 2000) DNA/1ml DMEM. The two abovementioned solutions were mixed, left undisturbedfor 20 min and added to DMEM in a 100-mm culture dish with a final volume of 12 ml. The cells were cultured at 37˚C with 5% CO2 for 7 hours and were then changed from DMEM to high-glucose DMEM supplemented with 10% fetal bovine serum. After 24 hours, the cells were collected, and protein was extracted for Western blot analysis with an anti-Flag antibody.
In this transfection experiment, we can see flag-tagged TMUB1 was tested in both kinds of transfection solution but with lower transfection efficiency. Hepatocytes suffered from extensive death. We thought it was due to long time incubation with transfection solution. So we operated a scond transfection experiments and decreased the incubation time to 4-6 hours.

Fig 2.
The second plasmid transfection with BRL-3A hepatocytes. PEI and Lipo 2000 was tested repectively. BRL-3A rat hepatocytes were cultured in high-glucose DMEMsupplemented with 10% fetal bovine serum at 37˚C with 5% CO2. When the hepatocytesreached 80-90% confluence, they were passagedand placed in a 100-mm culture dish.After 24 hours, when the cells had reached 40-60% confluence, they were passaged and placed in DMEM without penicillin-streptomycin. A total of 21µl PEI or 72ulLipoFiterTM was mixed with 979ml DMEM (PEI) or 928ml DMEM (Lipo 2000) in a 100-mm culture dish and was not disturbed for 5 min. The final concentration of the plasmid was 7-8ug (PEI) or 24µg (Lipo 2000) DNA/1ml DMEM. The two abovementioned solutions were mixed, left undisturbedfor 20 min and added to DMEM in a 100-mm culture dish with a final volume of 12 ml. The cells were cultured at 37˚C with 5% CO2 for 4-6 hours and were then changed from DMEM to high-glucose DMEM supplemented with 10% fetal bovine serum. After 24 hours, the cells were collected, and protein was extracted for Western blot analysis with an anti-Flag antibody.
In this transfection experiment, we can see flag-tagged TMUB1 was tested in both kinds of transfection solution with satified transfection efficiency. Hepatocytes suffered from extensive death. We thought it was due to long time incubation with transfection solution. So we operated a scond transfection experiments and decreased the incubation time to 4-6 hours. So we choosed lipo 2000 as the transfection solution and incubation time was 4-6 hours.

Fig3. PCR test for hypatocytes plas mid transfection.
The sense primers are located in the region of Flag and the anti-sense primers are licated in the region of Tmub1. The sense and antisense primers for pcDNA-FLAG-Tmub1 and its mutant amplification are as follows: 5'-AAGGATGACGACGATAAGAT-3' and 5'-GAGGTGTTGATGCTGTGA-3', respectively.  were tested by Westernblotting.

Fig5J.
The expression of β -actin after transfection of full-length TMUB1, its mutants and siRNA were tested by Westernblotting.