チロシン水酸化酵素の細胞内安定性

抄録

Wild-type human tyrosine hydroxylase (TH) type 1 and 4 mutants (del-52, the first 52 amino acid residues deleted; del-157, the first 157 amino acid residues deleted; RR-EE, Arg³⁷-Arg³⁸ replaced by Glu³⁷-Glu³⁸; and S40D, Ser⁴⁰ replaced by Asp⁴⁰) were expressed in AtT-20 mouse neuroendocrine cells in order to clarify how deeply the N-terminus of TH is involved in the efficient production of dopamine (DA) in mammalian cells. The amounts of DA that accumulated in AtT-20 cells expressing these human TH type 1 (hTH1) phenotypes were in the following order: del-52 = del-157 = RR-EE > S40D > wild-type, although the enzyme activities of del-52 and del-157 were lower than those of wild-type, RR-EE, and S40D. The observation on immunoblot analyses that the N-terminus-deleted hTH1 mutants were much more stable than wild-type can reconcile the discrepant results. Computer-assisted analysis of the spatial configuration of hTH1 identified 5 newly recognized PEST motifs, one of which was located in the N-terminus sequence of Met1-Lys12 and predicted that deletion of the N-terminus region would alter the secondary structure within the catalytic domain. We report that the high stability of the N-terminus-deleted hTH1 mutants can be generated by the loss of a PEST motif in their N-termini and the structural change in the catalytic domain, which would promise an efficient production of DA in mammalian cells expressing N-terminus deleted hTH1. [Jpn J Physiol 55 Suppl:S202 (2005)]