Biological Roles of the Prolyl Isomerase, PIN1

描述（由申请人提供）：拟议研究的总体假设是Pin1脯氨酸异构酶功能的丧失可能导致对肿瘤发生的不同易感性。Pin1最初被确定为有丝分裂所需的真菌蛋白激酶的调节因子，因此被认为在细胞周期进程中起重要作用。现在已知Pin1在细胞周期的G1和G2中都起作用，并且已经确定了几种在细胞周期中也起重要作用的Pin1结合蛋白。Pin1与pSer/pThr-Pro基序上磷酸化的蛋白结合，脯氨酰胺键的异构化可以引起构象变化，从而以积极或消极的方式调节细胞中目标蛋白的丰度。Pin1在人类癌症中很重要，这不仅是因为它参与细胞周期调节，还因为一些研究报道了人类肿瘤标本中Pin1水平的增加或染色体19p13.3中含有Pin1的部分的杂合性的丧失。因此，根据遗传背景，Pin1的缺失是否会使细胞敏感或保护细胞免受转化的问题仍然是一个谜。在纯C57BL6/J遗传背景下对Pin1缺失小鼠的成纤维细胞进行评估，发现Pin1的两个新的靶蛋白是c-Myc和cyclin E1。Pin1调节c- Myc的降解。永生化小鼠成纤维细胞中Pin1的缺失增加了c-Myc和细胞周期蛋白E1的水平，导致基因组不稳定，并使这些细胞对ras介导的转化敏感。为了扩展这些引人注目的结果并解决这个令人着迷的难题，我们提出：1)通过使用NMR来评估Pin1的作用机制，以确定Pin1如何与c-Myc结合，并通过生物化学/细胞生物学来评估Pin1是否像c-Myc一样调节细胞周期蛋白E1的积累；2)研究Pin1的功能缺失如何导致永生化小鼠成纤维细胞的基因组不稳定；3)在基因工程小鼠癌症模型中验证Pint功能丧失可以改变肿瘤发生时间的假设，其中整个动物的自发重组是激活K-Ras致癌等位基因所必需的，这反过来导致100%的小鼠在300天内发生肿瘤。我们希望这些研究的完成将有助于阐明Pin1和/或其靶蛋白在癌变中的潜在作用。

DESCRIPTION (provided by applicant): The overarching hypothesis of the proposed research is that loss of function of the Pin1 prolyl isomerase can result in differential susceptibility to tumorigenesis. Pin1 was originally identified as a regulator of a fungal protein kinase required for mitosis and so was suggested to play important roles in cell cycle progression. It is now known that Pin1 functions in both G1 and G2 of the cell cycle and several Pin1 binding proteins that also play important roles in the cell cycle have been identified. Pin1 binds to proteins phosphorylated on pSer/pThr-Pro motifs and isomerization of the prolyl amide bond can cause a conformational change that regulates the abundance of the target protein in the cell either in a positive or negative manner. Pin1 has been implicated to be important in human cancers not only because it is involved in cell cycle regulation but also as several studies have reported either increased levels of Pin1 in human tumor specimens or Loss of Heterozygosity of the portion of chromosome 19p13.3 that contains Pin1. Thus, the question of whether the loss of Pin1 could, depending on the genetic background, either sensitize or protect cells from transformation remains enigmatic. Evaluation of fibroblasts from Pin1 null mice in a pure C57BL6/J genetic background revealed two novel target proteins for Pin1 to be c-Myc, and cyclin E1. Pin1 regulates the degradation of c- Myc. The absence of Pin1 from immortalized mouse fibroblasts increases c-Myc and cyclin E1 levels, results in genomic instability and renders these cells sensitive to Ras-mediated transformation. To extend these remarkably provocative results and address this fascinating conundrum, it is proposed to: 1) evaluate the mechanism of action of Pin1 by using NMR to determine how Pin1 binds to c-Myc and biochemistry/cell biology to evaluate whether Pin1 regulates accumulation of cyclin E1 as it does c-Myc; 2) examine how the loss of function of Pin1 contributes to genomic instability in immortalized mouse fibroblasts; and 3) test the hypothesis that loss of function of Pint can alter the timing of tumorigenesis in a genetically engineered mouse model of cancer wherein spontaneous recombination in whole animals is necessary to activate an oncogenic allele of K-Ras, which in turn leads to tumorigenesis in 100% of the mice by 300 days. We hope that completion of these proposed studies will help to clarify the potential role of Pin1 and/or its target proteins in carcinogenesis.