Evidence that the transcriptional repressor ICER is regulated via the N-end rule for ubiquitination.

ICER is a transcriptional repressor that is mono- or poly-ubiquitinated. This either causes ICER to be translocated from the nucleus, or degraded via the proteasome, respectively. In order to further studies the proteins involved in ICER regulation mass spectrometry analysis was performed to identify potential candidates. We identified twenty eight ICER-interacting proteins in human melanoma cells, Sk-Mel-24. In this study we focus on two proteins with potential roles in ICER proteasomal degradation in response to the N-end rule for ubiquitination: the N-alpha-acetyltransferase 15 (NAA15) and the E3 ubiquitin-protein ligase UBR4. Using an HA-tag on the N- or C-terminus of ICER (NHAICER or ICERCHA) it was found that the N-terminus of ICER is important for its interaction to UBR4, whereas NARG1 interaction is independent of HA-tag position. Silencing RNA experiments show that both NAA15 and UBR4 up-regulates ICER levels and that ICER's N-terminus is important for this regulation. The N-terminus of ICER was found to have dire consequences on its regulation by ubiquitination and cellular functions. The half-life of NHAICER was found to be about twice as long as ICERCHA. Polyubiquitination of ICER was found to be dependent on its N-terminus and mediated by UBR4. This data strongly suggests that ICER is ubiquitinated as a response to the N-end rule that governs protein degradation rate through recognition of the N-terminal residue of proteins. Furthermore, we found that NHAICER inhibits transcription two times more efficiently than ICERCHA, and causes apoptosis 5 times more efficiently than ICERCHA. As forced expression of ICER has been shown before to block cells in mitosis, our data represent a potentially novel mechanism for apoptosis of cells in mitotic arrest.

Improved osteogenesis in rat femur segmental defects treated with human allograft and zinc adjuvants.

Bone allograft is widely used to treat large bone defects or complex fractures. However, processing methods can significantly compromise allograft osteogenic activity. Adjuvants that can restore the osteogenic activity of processed allograft should improve clinical outcomes. In this study, zinc was tested as an adjuvant to increase the osteogenic activity of human allograft in a Rag2 null rat femoral defect model. Femoral defects were treated with human demineralized bone matrix (DBM) mixed with carboxy methyl cellulose containing ZnCl2 (0, 75, 150, 300 µg) or Zn stearate (347 µg). Rat femur defects treated with DBM-ZnCl2 (75 µg) and DBM-Zn stearate (347 µg) showed increased calcified tissue in the defect site compared to DBM alone. Radiograph scoring and µCT (microcomputed tomography) analysis showed an increased amount of bone formation at the defects treated with DBM-Zn stearate. Use of zinc as an adjuvant was also tested using human cancellous bone chips. The bone chips were soaked in ZnCl2 solutions before being added to defect sites. Zn adsorbed onto the chips in a time- and concentration-dependent manner. Rat femur defects treated with Zn-bound bone chips had more new bone in the defects based on µCT and histomorphometric analyses. The results indicate that zinc supplementation of human bone allograft improves allograft osteogenic activity in the rat femur defect model.