Investigations of Mammalian Aminopropyltransferases

描述(由申请人提供)： 多胺是普遍存在的细胞成分，在细胞生理以及正常和肿瘤生长中发挥着许多作用。有很好的证据表明，多胺生物合成途径是药物设计的有效靶点。计划中的实验重点是研究哺乳动物体内精胺水平的合成、功能和调节。他们对上一次支持期间取得的几项主要调查结果采取后续行动。这些包括：用相关的多底物类似物抑制剂测定氨丙基转移酶的晶体结构；发现一种罕见的X连锁人类综合征(Snyder-Robinson综合征-SRS)是由于精胺合成酶基因的突变降低活性；我们观察到，大量过度表达精胺合成酶的CAG-SPMS转基因小鼠的精胺只有轻微的变化，表明代偿机制必须防止其积累；尽管饮食中存在精胺，但存在X染色体缺失的雄性GY小鼠没有精胺；初步结果表明，通过用CAG-SPMS小鼠饲养携带GY的雌性小鼠来恢复精胺来源可以恢复这些小鼠的正常生长和生育能力，尽管精胺合成酶表达的性质是不受调控的。有四个相互关联的特定目的：(1)了解氨丙基转移酶的作用机制和底物专一性。这些研究将使用结构数据，这些数据允许对氨丙基转移酶反应进行预测，并通过定点突变进行测试。他们将确定脱羧化的S-腺苷-蛋氨酸的氨丙基转移机制，将这一过程与S-腺苷蛋氨酸的甲基转移进行比较，并确定亚精胺和精胺合成酶显著专属底物的分子基础。(2)了解体内控制精胺水平的调节机制以及精胺产生过多或过少的后果。这些研究将使用CAG-SPM小鼠及其与过度表达多胺代谢其他酶的小鼠的杂交，以评估调节因素、控制精胺水平以及覆盖这些因素的效果。(3)研究从外源来源摄取精胺的情况，并设计程序以促进这种摄取，从而恢复辐射小鼠和SRS细胞的正常多胺水平和功能。(4)研究精胺含量的调节以及精胺的独特功能(S)在培养细胞中的作用。

DESCRIPTION (provided by applicant): Polyamines are ubiquitous cellular components that play many roles in cellular physiology and in normal and neoplastic growth. There is good evidence that the polyamine biosynthetic pathway is a valid target for drug design. The planned experiments focus on studies of the synthesis, function and regulation of spermine levels in mammals. They follow up on several major findings made during the last period of support. These include: determination of the crystal structure of an aminopropyltransferase with an associated multisubstrate analog inhibitor; the finding that a rare X-linked human syndrome (Snyder-Robinson syndrome-SRS) is due to a mutation reducing activity of the spermine synthase gene; our observation that CAG-SpmS transgenic mice that greatly overexpress spermine synthase have only modest alterations in spermine, indicating that compensatory mechanism must prevent its accumulation; the findings that male Gy mice, which have an X-chromosomal deletion in the SpmS gene, have no spermine despite the presence of spermine in their diets; and preliminary results indicating that restoring a source of spermine by breeding female Gy carrier mice with CAG-SpmS mice restores normal growth and fertility to these mice despite the unregulated nature of spermine synthase expression. There are four interrelated specific aims: (1) To understand the mechanism of action and substrate specificity of the aminopropyltransferase enzymes. These studies will use structural data, which allows predictions to be made concerning the aminopropyltransferase reaction and tested by site-directed mutagenesis. They will determine the mechanism of aminopropyl transfer from decarboxylated S-adenosyl-methionine, compare this process to methyl transfer from S-adenosylmethionine and determine the molecular basis of the strikingly exclusive substrate specificities of spermidine and spermine synthases. (2) To understand the regulatory mechanisms in vivo that control spermine levels and the consequences of overproduction or underproduction of spermine. These studies will use CAG-SpmS mice and crosses of them with mice that overexpress other enzymes of polyamine metabolism to evaluate the regulatory factors the control spermine levels and the effects of overriding these factors. (3) To examine the uptake of spermine from exogenous sources and devise procedures to facilitate such uptake and thus restore normal polyamine levels and function in Gy mice and SRS cells. (4) To investigate the regulation of spermine content and the unique function(s) of spermine in cultured cells using cells derived from Gy mice, and SRS cells that have a greatly reduced spermine synthase level due to a mutation in a splice site.