NUCLEAR HORMONE ACTION--ROLE OF UBIQUITIN PATHWAYS

类固醇、维生素、类维生素A和甲状腺激素通过其同源核受体对细胞生长、发育、分化、稳态和肿瘤发生产生深远影响，所述核受体构成结构相关的细胞内转录激活因子超家族。最近的证据表明，核激素受体（NHRs）的转录调控需要一组不同的蛋白质称为辅激活剂。辅激活因子描绘了一类以依赖于蛋白质的方式与受体相互作用的蛋白质，并且是受体最大化基因激活所需的。一般认为，共激活因子通过充当DNA结合受体与前起始复合物的基础转录因子之间的桥梁以及通过提供组蛋白乙酰转移酶活性HAT来增强受体功能，HAT破坏局部抑制性染色质结构并有助于增加转录活性。最近，我们的实验室和其他人已经克隆了泛素-蛋白酶体和泛素样（NEDD 8，神经前体细胞表达的发育下调）途径酶，E6相关蛋白（E6-AP），RSP 5/RPF 1，泛素缀合酶（UBC），SUG 1和E1泛素激活酶（UBA 3）作为NHR的共激活剂。这些共激活子具有其他酶活性，如泛素激活、泛素化缀合、泛素连接和蛋白酶活性，而不是HAT活性。这些观察结果表明，在核激素受体介导的基因激活的泛素和NEDD 8途径可能的调节作用。然而，这些途径调节核受体基因激活的确切机制目前还完全未知。基于我们的初步观察，我们假设泛素和NEDD 8途径是核激素受体共激活的调节剂。为了了解这些途径调节核激素受体基因激活的机制，我们提出了以下三个具体目标：A）鉴定和表征E6-AP相互作用蛋白并研究它们与E6-AP沿着在核激素受体共活化中的作用，B）为了鉴定和表征泛素的靶蛋白，蛋白酶体途径的研究，并了解这些途径调节核激素受体基因激活的机制，我们提出以下三个具体目标：A）鉴定和表征E6-AP相互作用蛋白并研究它们与E6-AP沿着在核激素受体共活化中的作用，B）为了鉴定和表征泛素的靶蛋白，蛋白酶体途径，并了解该途径调节核激素受体基因激活的机制，和C）表征E1泛素激活酶，UBA 3，. NEDD 8途径作为NHR的共激活剂，并表征其在核激素受体共激活中的作用。

Steroids, vitamins, retinoids, and thyroid hormones exert profound effects on cell growth, development, differentiation, homeostasis and tumorigenesis through their cognate nuclear receptors which make up a superfamily of structurally related intracellular hormone-activated transcription factors. Recent evidence suggests that transcriptional regulation by nuclear hormone receptors (NHRs) require a diverse group of proteins termed co-activators. Co-activators portray a growing class of proteins that interact with receptors in a hormone-dependent manner and are required for maximal gene activation by the receptors. It is the general belief that co-activators enhance receptor function by acting as a bridge between the DNA-bound receptor and basal transcription factors of the pre-initiation complex and also by providing histone acetyl transferase activity, HAT, which disrupts the local repressive chromatin structure and contributes to increase transcriptional activity. Recently, our laboratory and other have cloned ubiquitin-proteasome and ubiquitin-like (NEDD8, neural precursor cell-expressed developmentally down-regulated) pathway enzymes, E6-associated protein (E6-AP), RSP5/RPF1, ubiquitin- conjugating enzymes (UBCs), SUG1 and E1 ubiquitin-activating enzymes (UBA3) as co-activators of NHRs. These co-activators possess other enzymatic activities such as ubiquitin activation, ubiquitination conjugation, ubiquitin ligation and protease activities, instead of HAT activity. These observations suggest a possible regulatory role for the ubiquitin and NEDD8 pathways in nuclear hormone receptor-mediated gene activation. However, the exact mechanisms by which these pathways regulate nuclear receptor gene activation is completely unknown at this time. Based on our preliminary observations, we hypothesize that the ubiquitin and NEDD8 pathways are modulators of nuclear hormone receptor co-activation. In order to understand the mechanism by which these pathways modulate nuclear hormone receptor gene activation, we propose the following three specific aims: A) To identify and characterize the E6-AP interacting protein(s) and study their role along with E6-AP in nuclear hormone receptor co-activation, B) To identify and characterize target proteins of the ubiquitin-proteasome pathway and understand the mechanism by which these pathways modulate nuclear hormone receptor gene activation, we propose the following three specific aims: A) To identify and characterize the E6-AP interacting protein(s) and study their role along with E6-AP in nuclear hormone receptor co-activation, B) To identify and characterize target proteins of the ubiquitin-proteasome pathway and understand the mechanism by which this pathway modulates nuclear hormone receptor gene activation, and C) To characterize the E1 ubiquitin-activating enzyme, UBA3,. Of the NEDD8 pathway as a co-activator of NHRs and to characterize this role in nuclear hormone receptor co-activation.