Phosphomimics and Serotonin Acetyltransferase Regulation

拟磷剂和血清素乙酰转移酶调节

• 批准号: 7110522
• 负责人: LAWRENCE M SZEWCZUK
• 金额: $ 4.88万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7110522
• 关键词: acetyl coA acetyltransferase; alanine; binding sites; bioassay; biomimetics; bioperiodicity; circadian rhythms; enzyme activity; fluorescence polarization; fluorescence resonance energy transfer; mass spectrometry; melatonin; microinjections; phosphoprotein phosphatase; phosphorylation; postdoctoral investigator; protein degradation; protein protein interaction; protein structure function; serotonin; tissue /cell culture

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.

描述（由申请人提供）：5-羟色胺乙酰转移酶（芳基烷基胺-N-乙酰转移酶，AANAT）控制褪黑激素的昼夜升降。在人类中，褪黑激素的节律性合成由AANAT的磷酸化状态控制。在夜间，AANAT在两个位点（N-和C-末端）磷酸化，而在白天，AANAT主要以其未修饰的形式存在。AANAT的磷酸化促进与14-3-3 γ的结合，这增加酶稳定性（例如对蛋白酶的抗性）和活性，从而促进褪黑激素合成。该建议的重点是使用不可水解的pSer/pThr模拟物，膦酰-二氟-亚甲基-L-丙氨酸（Pfa），和蛋白质半合成，以进一步探索AANAT的细胞调节。首先，将通过体外结合和动力学测定来检查Pfa作为磷酸模拟物的有效性。接下来，半合成制备的Pfa修饰的AANAT将用作诱饵以鉴定新的相互作用蛋白（例如磷酸酶）。最后，制备半合成荧光共振能量转移（FRET）标记的AANAT，并用于细胞显微注射测定，目的是测量真实的时间酶稳定性并鉴定影响酶稳定性的小分子和酶。这种半合成的方法将导致更好地理解AANAT调节和昼夜节律。