Effects of Deletions in the Regulatory Domain on the Stability and Enzymatic Characteristics of Tyrosine Hydroxylase

BACKGROUND: Various vectors have been developed and tried for the delivery of tyrosine hydroxylase (TH) in order to supplement dopamine, which is severely deficient in Parkinson's disease, however, none of the protocols tried have yielded fruitful results that can be applied directly to humans. One of the problems revealed from previous trials was a short duration of expression of the delivered gene, that is, tyrosine hydroxylase. METHODS: To extend the stability and to improve the enzymatic characteristics of the protein, part of the regulatory domain was deleted via PCR technique. The cDNA for regulatory domain-deleted THs (dTH) were sub-cloned into a retroviral vector and the resulting recom-binant retrovirus was used to infect NIH-3T3. After selection, expression levels of TH were determined by Western blot analysis and the enzymatic characteristics were examined. RESULTS: The deletion increased steady state expression level of TH protein by 7-fold for d19TH (TH with amino acids #2-19 are deleted) and 3-fold for d31TH (TH with amino acids #2-31 are deleted. The elevated expression level of d19TH is likely due to the enhanced stability of the protein as determined by a treatment of cycloheximide. The activity of d19TH was also increased approximately by 3-fold but no increase of the L-dopa production was observed. However, the production of L-dopa was dramatically increased when GTP cyclohydrolase I (GTPCH I) was co-transfected suggesting that the activity of d19TH is dependent on the presence of cofactor. d19TH seem to be free of feedback inhibition at low concentration of dopamine (10 nM~1 nM) but more sensitive to the inhibition at high concentration of dopamine (10 mM). CONCLUSIONS: The deletion of 18 amino acids on the regulatory domain increases the stability of the protein, reduces the activity, and frees it from the feedback inhibi-tion by the end product.

Ceramide is Involved in MPP+ -induced Cytotoxicity in Human Neuroblastoma Cells

To understand the cytotoxic mechanism of MPP+, we examined the involvement of ceramide in MPP+ -induced cytotoxicity to human neuroblastoma SH-SY5Y cells. When SH-SY5Y cells were exposed to MPP+, MPP+ induced dose-dependent cytotoxicity accompanied by 2-fold elevation of intracellular ceramide levels in SH-SY5Y cells. Three methods were used to test the hypothesis that the elevated intracellular ceramide is related to MPP+ -induced cytotoxicity: C2-ceramide was directly applied to cells, sphingomyelinase (SMase) was exogenously added, and oleoylethanolamine (OE) was used to inhibit degradation of ceramide. Furthermore, inhibition of ceramide-activated protein phosphatase (CAPP), the effector of ceramide, using okadaic acid (OA) attenuated cell death but treatment of fumonisin B1, the ceramide synthase inhibitor, did not alter the cytotoxic effect of MPP+. Based on these, we suggest that the elevation of intracellular ceramide is one of the important mediators in MPP+ -induced cell death.