ANDROGEN RECEPTOR DEGRADATION

描述（由申请人提供）：人雄激素受体（AR）是核受体超家族的成员，是用于控制前列腺癌的药物的主要靶点。这种靶向是基于AR是前列腺生长和分化的决定因素。因此，重要的是要了解AR在细胞内是如何调节的。先前的研究表明，AR在其未配体状态下非常不稳定，这表明蛋白水解对调节AR稳态水平的重要性。我们的研究主要集中在AR降解的机制，以及这一过程本身是如何被调节的。在初步的研究中，我们发现，AR降解的泛素/蛋白酶体途径。这一过程进一步显示出受到一种称为Chip的蛋白质的影响，该蛋白质与分子伴侣如Hsp 7 O和Hsp 9 O相关联。我们还表明，转染芯片抑制前列腺癌细胞的生长，也表达AR。该项目的长期目标是充分表征AR降解的机制，以及Chip如何导致前列腺癌细胞生长抑制。在目标1中，我们将确定靶向AR降解的信号。 我们的假设是，这一事件的信号是基于部分AR构象，部分顺式作用序列。我们将首先确定蛋白质折叠在AR降解中的作用，然后研究一个假定的顺式作用信号（称为PEST序列）在AR降解中的作用。我们还将测试AR磷酸化作为其降解信号的作用。在目标2中，我们将确定AR泛素化的位点和催化该反应的分子组分。我们将确定是否有一个泛素化，sumoylation和PEST序列之间的功能关系。我们还将鉴定将泛素结合到AR的E2和E3酶。进一步的研究将证实这些酶在人前列腺上皮中表达。在目标3中，我们将确定芯片导致前列腺癌细胞生长停滞的机制。 我们将测试芯片对细胞生长的影响和降低AR水平之间是否存在相关性，并且我们将比较LNCaP细胞及其雄激素非依赖性衍生物（LNCaPAI）在凋亡、细胞周期停滞或坏死细胞死亡方面的生长停滞机制。

DESCRIPTION (Provided by applicant): The human androgen receptor (AR) is a member of the nuclear receptor superfamily and a major target of drugs used to control prostate cancer. This targeting is based on the AR being a determinant of prostate gland growth and differentiation. For this reason, it is important to understand how the AR is regulated inside cells. Previous studies showed that the AR is very unstable in its unliganded state, pointing to the importance of proteolysis to regulating AR steady state levels. Our studies are focused on the mechanisms underlying AR degradation, and how this process may itself be regulated. In preliminary studies, we show that the AR is degraded by the ubiquitin/proteasome pathway. This process is further shown to be affected by a protein called Chip that associates with molecular chaperones such as Hsp7O and Hsp9O. We also show that transfection of Chip inhibits growth of prostate cancer cells that also express AR. The long term goals of this project are to fully characterize the mechanisms governing AR degradation, and how Chip leads to prostate cancer cell growth inhibition In aim 1, we will determine the signals that target AR for degradation. Our hypothesis is that the signals for this event are based partly on AR conformation and partly on cis-acting sequences. We will first determine the role of protein folding in AR degradation, and then examine the role of a putative cis-acting signal, called a PEST sequence, in AR degradation. We will also test for the role of AR phosphorylation as a signal for its degradation. In aim 2, we will identify the sites of AR ubiquitinylation and the molecular components that catalyze this reaction. We will determine whether there is a functional relationship between ubiquitinylation, sumoylation, and the PEST sequence. We will also identify the E2 and E3 enzymes that conjugate ubiquitin to AR. Further studies will confirm that these enzymes are expressed in human prostate glandular epithelium. In aim 3, we will detrermine the mechanism by which Chip leads to arrest of prostate cancer cell growth. We will test whether there is a correlation between Chip effects on cell growth and decreasing AR levels, and we will compare the mechanisms of growth arrest in LNCaP cells and their androgen-independent derivative (LNCaPAI) in terms of apoptosis, cell cycle arrest or necrotic cell death.