Antizyme-mediated inhibition of polyamine transport

描述(由申请人提供)：本项目的长期目标是阐明抗酶(AZ)在细胞多胺运输的反馈调节中的作用。已有研究表明，癌细胞的生长和转移潜能与多胺的异常蓄积密切相关。多胺转运到癌细胞的活性增强，为针对肿瘤的细胞毒药物靶向提供了理想的系统。因此，许多基于多胺的抗癌化合物正在进行测试，其中一些已经进入临床试验。最近的研究表明，大多数多胺类似物还刺激AZ的产生，AZ是一种调节蛋白，通过抑制多胺转运蛋白来防止多胺的过度积累。这种由AZ介导的对潜在有用药物摄取的限制显然给这种运输系统的药理应用带来了严重的潜在问题。不幸的是，这种反馈系统的机制甚至最基本的特征都不为人所知。该项目中的四项调查旨在探索AZ参与阻止这一传送器的基本方面。这些研究将受益于最近的一项发现，即只存在于次要的全长AZ亚型上的短N-末端序列对于最大限度地抑制转运至关重要。具体地说，这些研究将：(A)确定两种AZ异构体在抑制多胺摄取和刺激出口方面的相对效果；(B)确定抗酶抑制剂(AZI)在逆转每种AZ异构体对多胺运输的反馈抑制方面的有效性；(C)探索AZ异构体与潜在的转运中间体分选Nexin-5相互作用的可能差异；以及(D)确定对这种反馈活性重要的N末端序列。运输抑制研究将使用稳定转染的CHO克隆进行，以控制不同的AZ亚型、AZ抑制剂和分类Nexin-5的表达。通过阐明AZ参与控制这一摄取系统，这项研究将促进利用这一有用的转运蛋白的化合物的设计和利用。

DESCRIPTION (provided by applicant): The long-term goal of this project is to clarify the role of antizyme (AZ) in feedback regulation of cellular polyamine transport. It has been well established that cancer cell growth and metastatic potential are closely associated with abnormal polyamine accumulation. The enhanced activity of polyamine transport into cancer cells provides an ideal system for targeting cytotoxic drugs specifically to tumors. Accordingly, many polyamine-based anticancer compounds are being tested and some have progressed to clinical trials. Recent studies have shown that most of these polyamine analogs also stimulate the production of AZ, a regulatory protein that prevents excessive accumulation of polyamines by inhibiting the polyamine transporter. This AZ-mediated limitation of the uptake of potentially useful drugs obviously presents a serious potential problem to the pharmacological use of this transport system. Unfortunately, the mechanisms and even the most basic characteristics of this feedback system are not known. The four investigations in this project are designed to explore fundamental aspects of AZ's involvement in blocking this transporter. The investigations will benefit from the recent discovery that a short N-terminal sequence, present only on the minor, full-length AZ isoform, is critical for maximal transport inhibition. Specifically these studies will (a) establish the relative effects of the two AZ isoforms on both inhibiting polyamine uptake and stimulating export; (b) determine the effectiveness of antizyme inhibitor (AZI) in reversing feedback inhibition of polyamine transport by each of the AZ isoforms; (c) explore possible differences in the interactions of the AZ isoforms with the potential transport intermediate sorting nexin-5; and (d) identify the N-terminal sequences that are important for this feedback activity. The transport inhibition studies will be conducted using CHO clones that have been stably transfected for controlled expression of the distinct AZ isoforms, AZ inhibitor and sorting nexin-5. By elucidating AZ's involvement in controlling this uptake system, this investigation will improve efforts to design and utilize compounds exploiting this useful transporter.