Characterization of RNF5 - A Novel RING Finger Protein

一个复杂的质量控制系统(内质网相关降解；ERAD)降解错误折叠的蛋白质，作为内质网应激反应的一个组成部分，是细胞应对应激能力的核心。越来越多的证据表明内质网应激在肿瘤发生和发展中的重要性；然而，正常细胞和肿瘤细胞内质网应激控制的机制尚不清楚。这个应用程序提出了一个新的调控轴可能控制内质网应激。在目前的资助期内，我们表征了无名指E3连接酶RNF5，并证明RNF5在人类癌症中过度表达，特别是在乳腺癌和黑色素瘤中。在化疗药物治疗后，抑制乳腺癌细胞中的RNF5以p53依赖的方式重组细胞骨架并使肿瘤细胞对凋亡敏感。有趣的是，RNF5介导泛素化介导的降解和底物定位的改变，包括错误折叠的蛋白质。RNF5通过其c末端尾部锚定在内质网膜上，在那里它与新发现的7-跨膜蛋白JAMP相互作用。JAMP作为蛋白酶体募集到内质网的受体，受RNF5调控，表明内质网应激反应中存在新的调控轴。虽然RNF5限制了JAMP的活性，但它通过与ERAD中涉及的泛素连接酶Cl-HP或gp78合作，促进了错误折叠蛋白的降解。这些发现表明RNF5是ERAD的重要调节因子。基于我们的初步结果(i) RNF5在乳腺癌和黑色素瘤中过表达，（ii） RNF5以p53依赖的方式是乳腺癌增殖和细胞骨架组织所必需的，以及（iii） RNF5限制JAMP活性并调节内质网应激反应，我们提出了RNF5-JAMP调节内质网应激有助于乳腺癌和黑色素瘤发展所需的增殖和细胞骨架组织的假设。我们提出的研究将为RNF5-JAMP模块调控内质网应激及其对乳腺癌和黑色素瘤发展的影响提供机制见解。为了验证我们的假设，我们将：(1)确定乳腺癌和黑色素瘤中RNF5表达上调的机制。(2)确定RNF5在ERAD中不同功能的机制(i)通过与ER相关的泛素连接酶合作泛素化和降解错误折叠的蛋白质，以及（ii）通过控制JAMP向ER募集蛋白酶体。(3)利用我们培育的RNF5基因敲除（KO）和诱导转基因小鼠，评估RNF5在乳腺肿瘤发生中的作用，并将其与乳腺肿瘤模型小鼠杂交。了解rnf5通过JAMP对内质网应激的依赖性调节及其与乳腺癌和黑色素瘤中p53依赖性变化的关系，将为正常细胞和肿瘤中内质网应激的控制机制提供新的和重要的见解。

A complex quality control system (Endoplasmic Reticulum (ER) Associated Degradation; ERAD) degrades misfolded proteins as an integral part of the ER stress response and is central to a cell's ability to cope with stress. Growing evidence points to the importance of ER stress in tumor development and progression; yet, mechanisms underlying control of ER stress in normal and tumor cells are poorly understood. This application proposes to define a novel regulatory axis potentially controlling ER stress. In the current funded period we characterized the RING finger E3 ligase RNF5 and demonstrated that RNF5 is over-expressed in human cancer, specifically in breast cancer and melanoma. Inhibition of RNF5 in breast cancer cells reorganizes the cytoskeleton and sensitizes tumor cells to apoptosis following treatment with chemotherapeutic drugs, in a p53-dependent manner. Interestingly, RNF5 mediates both ubiquitination-mediated degradation and altered localization of substrates, including misfolded proteins. RNF5 is anchored via its C-terminal tail to the ER membrane where it interacts with a newly identified 7-transmembrane protein, JAMP. JAMP serves as a receptor for proteasome recruitment to the ER and is regulated by RNF5, indicating a novel regulatory axis in the ER stress response. While RNF5 limits JAMP activity, it promotes the degradation of misfolded proteins, by cooperating with Cl-HP or gp78, ubiquitin ligases implicated in ERAD. These findings position RNF5 as an important regulator of ERAD. Based on our preliminary results that (i) RNF5 is overexpressed in breast cancer and melanoma, (ii) RNF5 is required for breast cancer proliferation and cytoskeletal organization in a p53-dependent manner, and (iii) RNF5 limits JAMP activity and regulates the ER stress response, we have formulated the hypothesis that RNF5-JAMP regulation of ER stress contributes to breast cancer and melanoma proliferation and cytoskeletal organization which are required for their development. Our proposed studies will provide a mechanistic insight into the regulation of ER stress by the RNF5-JAMP module and its implications on breast cancer and melanoma development. To test our hypothesis, we will: (1) Determine the mechanism underlying upregulated RNF5 expression seen in breast cancer and melanoma. (2) Identify mechanism(s) underlying distinct RNF5 functions in ERAD (i) by cooperation with ER-associated ubiquitin ligases to ubiquitinate and degrade misfolded proteins and (ii) by control of JAMP recruitment of proteasomes to ER. (3) Assess the role of RNF5 in mammary tumor development using RNF5 knockout (KO) and inducible transgenic mice that we have generated and which will be crossed with mammary tumor model mice. Understanding RNF5-dependent regulation of ER stress via JAMP and its relationship to p53-dependent changes in breast cancer and melanoma should provide novel and important insight into mechanisms underlying control of ER stress in normal cells and in tumors.