Examination of Ornithine Decarboxylase Antizyme RNA Structure and Function from Various Organisms for the Development of Antibiological Agents

项目摘要 几乎所有的生物体都具有合成天然多胺的能力-腐胺，亚精胺和 精胺-这是细胞生长和分化所必需的。由于多胺的相互作用能力 几乎每一种生物分子-- DNA、RNA、磷脂、蛋白质和ATP，仅举几例--它们都发挥着许多作用。 在细胞内的作用，以支持细胞生长。已经有充分的文献证明， 哺乳动物细胞与细胞生长速率相关，高多胺浓度已被观察到， 快速增殖的细胞和低浓度已经在缓慢生长或静止的细胞中测量到。不 令人惊讶的是，多胺的转运和代谢受到复杂反馈的高度调节 机制等鸟氨酸脱羧酶（ODC）是多胺生物合成的关键调控酶。ODC 体内平衡影响细胞生长和癌症发展。ODC过度表达在许多人中被观察到。 肿瘤类型，包括前列腺癌、乳腺癌和皮肤癌。ODC和多胺的细胞摄取都是 被鸟氨酸脱羧酶抗酶（OAZ）抑制。从OAZ mRNA制备抗酶蛋白 需要在高度保守的位点进行翻译移码以绕过过早终止。哺乳动物 OAZ mRNAs还具有一个假结（PK）RNA 3，它位于移码位点，刺激+1移码。 此外，移码被多胺刺激，从而提供反馈机制，由此， 代谢产物的积累抑制生物合成。虽然OAZ假结RNA的作用 已经研究了多胺依赖性移码中的一种元素（进一步命名为OAZ-PK），但还没有 作为一种独特的多胺“传感器”进行了研究。核糖开关是在非编码区的元件， 直接与细胞代谢物结合并调节基因表达的mRNA。许多核糖开关提供了 同源物生物合成途径中基因产物的反馈调节机制 代谢物。核糖开关广泛存在于细菌中，还有一类存在于真菌和植物中， 在动物体内还没有发现核糖开关。提出OAZ-PK RNA作为核糖开关发挥功能， 并且在此提供了这种非编码RNA是多胺传感器的证据。这种RNA分子高度 在脊椎动物精胺生物合成所需的基因中保守。开发针对 因此，来自不同生物体的假定精胺核糖开关可能用于广泛的目的 例如抗癌剂、抗真菌剂或杀虫剂。该提案将审查结构， 来自各种生物体的OAZ-PK RNA的功能，包括具有生物医学相关性的生物体-人， 病原真菌和疾病窝藏昆虫-有以下具体目标：（1）检查特异性 多胺与不同生物体OAZ RNA结合的亲和力，（2）研究多胺与OAZ RNA结合的三维结构， OAZ RNA的结构，以及（3）探索来自各种生物体的抗酶OAZ RNA在控制 基因表达。

PROJECT SUMMARY Nearly all organisms possess the capability to synthesize the natural polyamines - putrescine, spermidine and spermine - which are essential for cell growth and differentiation. Due to the ability of polyamines to interact with nearly every biomolecule - DNA, RNA, phospholipids, proteins and ATP, to name a few - they play many roles within the cell in order to support cell growth. It has been well documented that polyamine levels in mammalian cells correlate with the rate of cell growth, high polyamine concentrations have been observed in rapidly proliferating cells and low concentrations have been measured in slow-growing or quiescent cells. Not surprisingly, the transport and metabolism of polyamines are highly regulated by complex feedback mechanisms. Ornithine decarboxylase (ODC) is the key regulatory enzyme in polyamine biosynthesis. ODC homeostasis affects cell growth and cancer development. ODC over-expression has been observed in many tumor types, including prostate, breast, and skin cancers. Both ODC and cellular uptake of polyamines is inhibited by Ornithine Decarboxylase Antizyme (OAZ). The making of Antizyme protein from OAZ mRNA requires translational frameshifting at a highly conserved site to bypass premature termination. Mammalian OAZ mRNAs further possess a pseudoknot (PK) RNA 3¢ to the frameshift site that stimulates +1 frameshifting. Moreover, frameshifting is stimulated by polyamines, thus providing a feedback mechanism whereby the accumulation of metabolic products inhibits biosynthesis. Although the role of the OAZ pseudoknot RNA element (further designated OAZ-PK) in polyamine-dependent frameshifting has been investigated, it has not been examined as a distinct polyamine “sensor”. Riboswitches are elements within noncoding regions of mRNAs that directly bind to cellular metabolites and modulate gene expression. Many riboswitches provide a mechanism of feedback regulation for gene products within the biosynthetic pathway of the cognate metabolite. Riboswitches are widespread among bacteria, and one class further resides in fungi and plants, but no riboswitches have been found in animals. It is proposed that the OAZ-PK RNA functions as a riboswitch, and herein evidence is provided that this noncoding RNA is a polyamine sensor. This RNA element is highly conserved among vertebrate genes required for spermine biosynthesis. Development of drugs that target putative spermine riboswitches from different organisms might therefore be used for wide-ranging purposes such as anticancer agents, antifungal agents, or pesticides. This proposal will examine the structure and function of the OAZ-PK RNA from various organisms, including those of biomedical relevance – human, pathogenic fungi and disease harboring insects - with the following specific aims: (1) examine the specificity and affinity of polyamine binding to OAZ RNAs from various organisms, (2) investigate the three-dimensional structure of OAZ RNAs, and (3) explore the role of Antizyme OAZ RNAs from various organisms in control of gene expression.