Phosphomimics and Serotonin Acetyltransferase Regulation

拟磷剂和血清素乙酰转移酶调节

• 批准号: 7278322
• 负责人: LAWRENCE M SZEWCZUK
• 金额: $ 1.44万
• 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 2007-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7278322
• 关键词: Acetyltransferase; Alanine; Amides; Antibodies; Arylalkylamine N-Acetyltransferase; BRCA1 gene; Binding; Biological Assay; Buffers; C-terminal; Catalysis; Cells; Chemicals; Chinese Hamster Ovary Cell; Circadian Rhythms; Coenzyme A; Consensus; Effectiveness; Endopeptidases; Enzyme Stability; Enzymes; Fluorescein; Fluoresceins; Fluorescence Anisotropy; Fluorescence Resonance Energy Transfer; Furuncles; Head; Human; In Vitro; Investigation; Kinetics; Label; Lead; Length; Ligands; Ligation; Measures; Melatonin; Microinjections; N-terminal; Peptide Hydrolases; Peptides; Phosphoric Monoester Hydrolases; Phosphorylation; Pineal gland; Polo-Box Domain; Production; Protein Binding Domain; Protein Dephosphorylation; Protein phosphatase; Proteins; Regulation; Resistance; Rhodamine; Rhodamines; Role; Sepharose; Site; Techniques; Tertiary Protein Structure; Time; Tryptamines; base; carbene; cell growth regulation; day; falls; follow-up; inhibitor/antagonist; interest; mimetics; novel; phosphatase inhibitor; protein degradation; small molecule; synthetic enzyme; tandem mass spectrometry; tryptamine

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(申请人提供)：5-羟色胺乙酰转移酶(芳基烷胺-A/-乙酰转移酶，AANAT)控制褪黑素的每日升降。在人类中，褪黑素的有节奏的合成是由AANAT的磷酸化状态控制的。在夜间，AANAT在两个位置(N-末端和C-末端)被磷酸化，而在白天，AANAT主要以其未修饰的形式存在。AANAT的磷酸化促进了与14-3-3伽马的结合，从而增加了酶的稳定性(如对蛋白酶的抗性)和活性，从而促进了褪黑素的合成。本研究的重点是利用非水解性pSer/pThr模拟物、膦-二氟-亚甲基-L-丙氨酸(PFA)和蛋白质半合成来进一步探讨AANAT的细胞调控。首先，将通过体外结合和动力学试验来检验PFA作为磷化模拟物的有效性。接下来，半合成的PFA修饰的AANAT将被用作诱饵来鉴定新的相互作用蛋白(例如磷酸酶)。最后，将制备半合成荧光共振能量转移(FRET)标记的AANAT，并用于细胞微注射分析，旨在测量实时酶稳定性，并识别影响酶稳定性的小分子和酶。这种半合成的方法将有助于更好地理解AANAT的调节和昼夜节律。