tailieunhanh - Báo cáo Y học: Ubiquitination of soluble and membrane-bound tyrosine hydroxylase and degradation of the soluble form

Tyrosine hydroxylase (TH) demonstrates by two-dimen-sional electrophoresis amicroheterogeneitybothas a soluble recombinant humanTH(hTH1) andas amembrane-bound bovine TH (bTHmem). Part of the heterogeneity is likely due to deamidation of labile asparagine residues. Wild-type (wt)-hTH1was foundtobeasubstrate for theubiquitin(Ub) conjugating enzyme systemina reconstitutedin vitrosystem. | Eur. J. Biochem. 269 1561-1569 2002 FEBS 2002 Ubiquitination of soluble and membrane-bound tyrosine hydroxylase and degradation of the soluble form Anne P. Dùskeland and Torgeir Flatmark Department of Biochemistry and Molecular Biology University of Bergen Norway Tyrosine hydroxylase TH demonstrates by two-dimensional electrophoresis a microheterogeneity both as a soluble recombinant human TH hTH1 and as a membrane-bound bovine TH bTHmem . Part of the heterogeneity is likely due to deamidation of labile asparagine residues. Wild-type wt -hTH 1 was found to be a substrate for the ubiquitin Ub conjugating enzyme system in a reconstituted in vitro system. When wt-hTH1 was expressed in a coupled transcriptiontranslation TnTR-T7 reticulolysate system 35S-labelled polypeptides of the expected molecular mass of native enzyme as well as both higher and lower molecular mass forms were observed. The amount of high-molecular-mass forms increased by time and was enhanced in the presence of Ub and clasto-lactacystin b-lactone. In pulse-chase experiments the amount of full-length hTH1 decreased by first- order kinetics with a half-time of h and h in the absence and presence of an ATP-regenerating system respectively. The ATP-dependent degradation was inhibited by clasto-lactacystin b-lactone. Our Endings support the conclusion that hTH1 is ubiquitinated and at least partially degraded by the proteasomes in the reticulocyte lysate system. Finally it is shown that the integral TH of the bovine adrenal chromaffin granule membrane bTHmem is ubiquitinated most likely monoubiquitinated. Additional Ub-conjugates of this membrane detected by Western blot analysis have not yet been identified. Keywords tyrosine hydroxylase ubiquitin proteasome chromaffin granule membrane neuroendocrine cells. Tyrosine hydroxylase TH EC catalyzes the conversion of L-tyrosine to L-dihydroxyphenylalanine l-DOPA the rate-limiting step in the biosynthesis of dopamine and noradrenaline .

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