Wadas Brandon, Borjigin Jimo, Huang Zheping, Oh Jang-Hyun, Hwang Cheol-Sang, Varshavsky Alexander
From the Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125.
the Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109.
J Biol Chem. 2016 Aug 12;291(33):17178-96. doi: 10.1074/jbc.M116.734640. Epub 2016 Jun 23.
Serotonin N-acetyltransferase (AANAT) converts serotonin to N-acetylserotonin (NAS), a distinct biological regulator and the immediate precursor of melatonin, a circulating hormone that influences circadian processes, including sleep. N-terminal sequences of AANAT enzymes vary among vertebrates. Mechanisms that regulate the levels of AANAT are incompletely understood. Previous findings were consistent with the possibility that AANAT may be controlled through its degradation by the N-end rule pathway. By expressing the rat and human AANATs and their mutants not only in mammalian cells but also in the yeast Saccharomyces cerevisiae, and by taking advantage of yeast genetics, we show here that two "complementary" forms of rat AANAT are targeted for degradation by two "complementary" branches of the N-end rule pathway. Specifically, the N(α)-terminally acetylated (Nt-acetylated) Ac-AANAT is destroyed through the recognition of its Nt-acetylated N-terminal Met residue by the Ac/N-end rule pathway, whereas the non-Nt-acetylated AANAT is targeted by the Arg/N-end rule pathway, which recognizes the unacetylated N-terminal Met-Leu sequence of rat AANAT. We also show, by constructing lysine-to-arginine mutants of rat AANAT, that its degradation is mediated by polyubiquitylation of its Lys residue(s). Human AANAT, whose N-terminal sequence differs from that of rodent AANATs, is longer-lived than its rat counterpart and appears to be refractory to degradation by the N-end rule pathway. Together, these and related results indicate both a major involvement of the N-end rule pathway in the control of rodent AANATs and substantial differences in the regulation of rodent and human AANATs that stem from differences in their N-terminal sequences.
血清素N - 乙酰基转移酶（AANAT）将血清素转化为N - 乙酰血清素（NAS），NAS是一种独特的生物调节剂，也是褪黑素的直接前体，褪黑素是一种循环激素，可影响包括睡眠在内的昼夜节律过程。AANAT酶的N端序列在脊椎动物中各不相同。调节AANAT水平的机制尚未完全了解。先前的研究结果与AANAT可能通过N端规则途径降解来进行调控的可能性一致。通过不仅在哺乳动物细胞中，而且在酿酒酵母中表达大鼠和人类AANAT及其突变体，并利用酵母遗传学，我们在此表明大鼠AANAT的两种“互补”形式被N端规则途径的两个“互补”分支靶向降解。具体而言，N（α）末端乙酰化（Nt - 乙酰化）的Ac - AANAT通过Ac / N端规则途径识别其Nt - 乙酰化的N端甲硫氨酸残基而被破坏，而非Nt - 乙酰化的AANAT则被Arg / N端规则途径靶向，该途径识别大鼠AANAT未乙酰化的N端Met - Leu序列。我们还通过构建大鼠AANAT的赖氨酸到精氨酸突变体表明，其降解是由其赖氨酸残基的多聚泛素化介导的。人类AANAT的N端序列与啮齿动物AANAT不同，其寿命比大鼠AANAT长，并且似乎对N端规则途径的降解具有抗性。总之，这些及相关结果表明N端规则途径在啮齿动物AANAT的调控中起主要作用，并且啮齿动物和人类AANAT调控的显著差异源于它们N端序列的不同。
