Characterization of the degradation of recombinant rat urate oxidase in tetracycline controlled gene expression cells.
Our previous study has shown that uric acid is metabolized by recombinant rat urate oxidase (UOX) and generates hydrogen peroxide leading to DNA damage and cell transformation. However, in transformed cells protein levels of UOX were reduced compared with the control cell. To investigate the characterization and the mechanisms responsible for the degradation of UOX, a controllable gene expression system has been used to switch on/off controlled expression of rat UOX in vitro. Chinese hamster ovary cells were double transfected with regulatory and responsive plasmids, pCMV-tTA and pTRE-rUOX, respectively. The cells expressing rat UOX were subtly controlled by tetracycline (Tc). High levels of UOX mRNA and protein enzymatic activity were observed when the cells were cultured in the absence of Tc. The functional recombinant rat UOX was present in the form of crystalloid cores structures that localized within the peroxisomes of the cells, which was confirmed by transmission and immunoelectron microscopic studies. The addition of Tc into the medium led to the halting of rat UOX gene transcription. As a result, recombinant rat UOX mRNA was lost rapidly followed by loss of crystalloid cores structures and UOX protein degradation. Lysosomes assembled around the UOX specific structures indicating that they were involved in degradation of the protein. The observations suggest that the entire organelle rather than a single protein within the peroxisomes is degraded once the rat UOX gene expression is turned off, and the phagocytic vacuole/lysosome pathway (microautophagic process) may play an important role in degradation of the protein under the present situation.
Pan
Jie
J
College of Life Sciences, Shandong Normal University, No. 88 East Wenhua Avenue, Jinan 250014, People's Republic of China. nakayamapan@hotmail.com
Pan
Xin
X
Wang
Na
N
Ghazizadeh
Mohammad
M
Yeldandi
Anjana
A
D000818:Animals; D001343:Autophagy; D016466:CHO Cells; D006224:Cricetinae; D005786:Gene Expression Regulation; D008247:Lysosomes / Q000378:metabolism; D020675:Peroxisomes / Q000378:metabolism*; D012333:RNA, Messenger; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D013754:Tetracyclines / Q000494:pharmacology*; D014162:Transfection; D014503:Urate Oxidase / Q000235:genetics / Q000378:metabolism*; D014527:Uric Acid / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism
D012333:RNA, Messenger; D011994:Recombinant Proteins; D013754:Tetracyclines; D014527:Uric Acid; D014503:Urate Oxidase