Gastrointestinal Physiology of PYY--Role of 3D Structure

PYY的胃肠生理学--3D结构的作用

• 批准号: 6727704
• 负责人: JOSEPH R REEVE
• 金额: $ 32.6万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6727704
• 关键词: CHO cells; circular dichroism; cyclic AMP; forskolin; gastrointestinal function; gastrointestinal pharmacology; high performance liquid chromatography; hormone regulation /control mechanism; laboratory rat; neuropeptide receptor; neuropeptides; nuclear magnetic resonance spectroscopy; protein structure function; receptor binding; stimulant /agonist; thyrotropin releasing hormone

DESCRIPTION (Applicant's Abstract): While stable tertiary structures have been demonstrated for several GI peptides, there is little experimental confirmation that this 3D structure influences the physiological response to any peptide. The investigators hypothesize that tertiary structure of the endocrine peptide PYY influences receptor subtype binding, activation and expression of biological activity. They will test this hypothesis in two ways: 1) Determine if change in tertiary structure alters biological activity and 2) determine if there are unique regions of tertiary structure for specific Y receptor agonists. For the first test, the investigators have designed a PYY analog where a minor primary structure change (formation of a disulfide bond in [Cys6,Cys23}PYY) produced a dramatic alteration in molecular conformation and a 33-fold decrease in Y1 receptor binding from the non-cross linked form. The reduced compound had less affinity than native PYY. A systematic design strategy will be used to identify di-cysteine PYY analogs that closely mimic PYY for Y1, Y2, and V5 receptor binding and 3D-conformation. The reduced analog will be oxidized to determine the consequences of tertiary structure alteration by disulfide bond formation. The influence of tertiary structure will be evaluated by measurements of receptor binding, second messenger activation, circulatory half-life, stability to digestion, and inhibition of pancreatic secretion in the rat. This unique coupling of peptide design strategy and evaluation of structure and multiple effectors of physiological response will provide the first direct quantitation of the role of tertiary structure in the expression of PYY bioactivity. The authors have determined the aqueous solution structure of PYY by NMR. They have also shown that Y1 and Y2 specific agonists ([Pro34]PYY and PYY(3 -3 6)) exhibit distinctly different secondary structures using circular dichroism (CD). For the second test, they will select the best 5-7 agonists that potently bind and activate Y1, Y2 or Y5 subtypes expressed in CHO cells. The tertiary structure will be determined for the selected agonists by NMR and used to predict structural elements that are specific for each receptor subtype. This structural analysis may lead to rational design strategies to produce therapeutic agents acting at these V receptor subtypes.

描述（申请人的摘要）：虽然稳定的三级结构已经 为几种GI肽证明，几乎没有实验确认 这种3D结构会影响对任何肽的生理反应。 研究人员假设内分泌肽的三级结构 PYY影响受体亚型结合，激活和表达 生物活性。他们将以两种方式检验这一假设：1）确定 如果三级结构的变化改变生物学活性，并且2）确定是否是否 特定Y受体有独特的三级结构区域 激动剂。对于第一次测试，研究人员设计了一个PYY类似物 其中一个次要的主要结构发生变化（形成了二硫键 [cys6，cys23} pyy）产生了分子构象和A的戏剧性改变 从非交叉链接形式的Y1受体结合中Y1受体结合的33倍。这 减少的化合物的亲和力比本机PYY少。系统设计 策略将用于识别紧密模仿的二半胱氨酸PYY PYY类似物 Y1，Y2和V5受体结合和3D构成的PYY。减少的类似物 将被氧化以确定三级结构改变的后果 通过二硫键形成。三级结构的影响将是 通过测量受体结合，第二信使激活评估， 循环半衰期，消化的稳定性和胰腺抑制 大鼠分泌。这种独特的肽设计策略和 评估结构和生理反应的多个效应因素将 提供第一个直接定量第三级结构在 PYY生物活性的表达。作者已经确定了水溶液 NMR的PYY结构。他们还表明Y1和Y2特定的激动剂 （[Pro34] Pyy和Pyy（3 -3 6））表现出明显不同的二级结构 使用圆形二色性（CD）。对于第二个测试，他们将选择最好的 5-7个激动剂，可有效结合并激活Y1，Y2或Y5亚型 CHO细胞。将确定针对选定激动剂的三级结构 通过NMR，用于预测针对每个特定的结构元素 受体亚型。这种结构分析可能导致理性设计 生产以这些V受体亚型作用的治疗剂的策略。