ANDROGEN RECEPTOR DEGRADATION

描述(申请人提供)：人雄激素受体(AR)是核受体超家族的成员，也是用于控制前列腺癌的药物的主要靶点。这个靶点是基于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是如何在细胞内调节的。以往的研究表明，AR在去连接状态下非常不稳定，这表明了蛋白分解对调节AR稳态水平的重要性。我们的研究集中在AR降解的潜在机制，以及这个过程本身是如何被调控的。在初步研究中，我们发现AR是通过泛素/蛋白酶体途径降解的。这一过程还被进一步证明受到一种名为Chip的蛋白质的影响，这种蛋白质与Hsp7O和Hsp9O等分子伴侣相关联。我们还发现，ChIP的转染抑制了同样表达AR的前列腺癌细胞的生长。这个项目的长期目标是充分描述AR降解的调控机制，以及芯片如何导致前列腺癌细胞生长抑制。在目标1中，我们将确定针对AR降解的信号。 我们的假设是，这一事件的信号部分基于AR构象，部分基于顺式作用序列。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这一反应的分子成分。我们将确定泛素化、苏莫化和PEST序列之间是否存在功能关系。我们还将确定将泛素与AR结合的E2和E3酶。进一步的研究将证实这些酶在人的前列腺腺上皮细胞中表达。在目标3中，我们将确定芯片导致前列腺癌细胞生长受阻的机制。我们将测试芯片对细胞生长的影响是否与AR水平降低之间存在相关性，并将从细胞凋亡、细胞周期停滞或坏死细胞死亡的角度比较LNCaP细胞及其雄激素非依赖性衍生物(LNCaPAI)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.