title: N-terminal residues regulate proteasomal degradation of AANAT.
abstract: Serotonin N-acetyltransferase (AANAT) catalyzes the conversion of serotonin to N-acetylserotonin, which is the immediate precursor for formation of melatonin. Although it is known that AANAT is degraded via the proteasomal proteolysis, detailed mechanisms are not defined. In this paper, we tested the in vivo role of proteasome inhibition on AANAT activity and melatonin release and examined the amino acid residues in AANAT that contribute to the proteasome degradation. We have shown that inhibition of proteasome activities in vivo in the intact pineal gland fails to prevent the light-induced suppression of melatonin secretion. Furthermore, in cell lines stably expressing AANAT, inhibition of proteasomal proteolysis, which resulted in a large accumulation of AANAT protein, similarly failed to increase AANAT enzyme activity proportional to the amount of proteins accumulated. Site-directed mutagenesis analysis of AANAT revealed that the AANAT degradation is independent of lysine and the two surface cysteine residues. Deletion analysis of N-terminus identified the second amino acid leucine (L2) as the key residue that contributes to the proteasomal proteolysis of AANAT protein. These results suggest that rat AANAT protein is degraded via the N-end rule pathway of proteasomal proteolysis and the leucine at the N-terminus appears to be the key residue recognized by N-end rule pathway.
authors.forename: Zheping
authors.initials: Z
authors.lastname: Huang
authors.affiliation: Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, MI, USA.
authors.forename: Tiecheng
authors.initials: T
authors.lastname: Liu
authors.affiliation: Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, MI, USA.
authors.forename: Jimo
authors.initials: J
authors.lastname: Borjigin
mesh_terms: D000818:Animals; D047068:Arylalkylamine N-Acetyltransferase / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008027:Light; D008550:Melatonin / Q000378:metabolism*; D017551:Microdialysis; D016297:Mutagenesis, Site-Directed; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D061988:Proteasome Inhibitors; C520223:Aanat protein, rat; D047068:Arylalkylamine N-Acetyltransferase; D046988:Proteasome Endopeptidase Complex; D008550:Melatonin; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde