Phosphomimics and Serotonin Acetyltransferase Regulation

描述（由申请人提供）：5 -羟色胺乙酰转移酶（芳基烷基胺-a /-乙酰转移酶，AANAT）控制褪黑激素的昼夜升降。人类褪黑激素的节律性合成是由AANAT的磷酸化状态控制的。在夜间，AANAT在两个位点（N端和C端）磷酸化，而在白天，AANAT主要以未修饰的形式存在。AANAT的磷酸化促进与14-3-3 γ的结合，从而增加酶的稳定性（例如对蛋白酶的抗性）和活性，从而促进褪黑激素的合成。本课题拟利用不可水解的pSer/pThr模拟物、磷酸二氟亚甲基- l -丙氨酸（Pfa）和蛋白半合成进一步探讨AANAT的细胞调控。首先，Pfa作为磷模拟物的有效性将通过体外结合和动力学分析来检验。接下来，半合成制备的Pfa修饰的AANAT将作为诱饵，用于鉴定新的相互作用蛋白（如磷酸酶）。最后，制备半合成荧光共振能量转移（FRET）标记的AANAT，并将其用于细胞显微注射试验，旨在实时测量酶的稳定性，识别影响酶稳定性的小分子和酶。这种半合成方法将有助于更好地理解AANAT的调节和昼夜节律。

DESCRIPTION (provided by applicant): Serotonin acetyltransferase (arylalkylamine-A/-acetyltransferase, AANAT) governs the diurnal rise and fall of melatonin. In humans the rhythmic synthesis of melatonin is controlled by the phosphorylation state of AANAT. At night, AANAT is phosphorylated at two sites (N- and C- terminal) and during the day AANAT is mainly present in its unmodified form. Phosphorylation of AANAT promotes binding to 14-3-3 gamma which increases enzyme stability (e.g. resistance to proteases) and activity thus promoting melatonin synthesis. This proposal focuses on the use of the nonhydrolyzable pSer/pThr mimetic, phosphono-difluoro-methylene-L-alanine (Pfa), and protein semi-synthesis to further explore the cellular regulation of AANAT. First, the effectiveness of Pfa to serve as a phospho-mimetic will be examined by in vitro binding and kinetic assays. Next, semi- synthetically prepared Pfa modified AANAT will be used as bait to identify novel interacting proteins (e.g. phosphatases). Finally, semi-synthetic fluorescence resonance energy transfer (FRET) labeled AANAT will be prepared and used for cellular microinjection assays aimed at measuring real time enzyme stability and identifying small molecules and enzymes that effect enzyme stability. This semi-synthetic approach will lead to a better understanding of AANAT regulation and circadian rhythms.