Characterization of RNF5 - A Novel RING Finger Protein

RNF5（一种新型环指蛋白）的表征

• 批准号: 7582260
• 负责人: Ze'ev A Ronai
• 金额: $ 38.47万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7582260
• 关键词: Address; Affect; Apoptosis; Breast Cancer Cell; Breast Cancer Model; C-terminal; Caenorhabditis elegans; Cells; Complex; Cytoskeletal Modeling; Cytoskeletal Proteins; Cytoskeleton; Development; Dose; Endoplasmic Reticulum; Endoplasmic Reticulum Degradation Pathway; Enzymes; Fingers; Funding; Genes; Human; Integral Membrane Protein; Knock-out; MAPK8 gene; Malignant Neoplasms; Mammary Neoplasms; Mediating; Membrane; Membrane Proteins; Mouse Mammary Tumor Virus; Mus; Mutation; Neoplasm Metastasis; Non-Malignant; Normal Cell; Pharmaceutical Preparations; Phosphoric Monoester Hydrolases; Polyomavirus; Positioning Attribute; Post-Translational Protein Processing; Protein Binding; Quality Control; Recruitment Activity; Regulation; Role; Stress; System; Tail; Testing; Time; Transgenic Mice; Ubiquitin; Ubiquitination; base; biological adaptation to stress; cancer cell; coping; endoplasmic reticulum stress; human RBX1 protein; insight; malignant breast neoplasm; melanoma; mouse model; multicatalytic endopeptidase complex; neoplastic cell; novel; overexpression; prevent; protein misfolding; receptor; tumor; tumor autocrine motility factor receptor; tumorigenesis; ubiquitin ligase; ubiquitin-protein ligase

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.

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组成部分，对细胞应对应激的能力至关重要。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，正常和肿瘤细胞中内质网应激的潜在控制机制尚不清楚。本申请建议定义一个新的可能控制内质网应激的调节轴。在目前的资助期间，我们描述了环指E3连接酶RNF5的特征，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。抑制乳腺癌细胞中的RNF5以P53依赖的方式重组细胞骨架，并在化疗药物治疗后使肿瘤细胞对凋亡敏感。有趣的是，RNF5既介导了泛素化介导的降解，也介导了底物的改变定位，包括错误折叠的蛋白质。RNF5通过其C-末端锚定在内质网膜上，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有一个新的调节轴。虽然RNF5限制JAMP的活性，但它通过与参与ERAD的泛素连接酶Cl-HP或gp78合作，促进错误折叠蛋白的降解。这些发现使RNF5成为ERAD的重要调节因子。根据我们的初步结果，(I)RNF5在乳腺癌和黑色素瘤中过表达，(Ii)RNF5在乳腺癌和黑色素瘤的增殖和细胞骨架组织中以P53依赖的方式是必需的，以及(Iii)RNF5限制JAMP活性并调节ER应激反应，我们提出了这样的假设，即RNF5-JAMP调节ER应激有助于乳腺癌和黑色素瘤的生长和细胞骨架组织的发展。我们提出的研究将为RNF5-JAMP模块对ER应激的调节及其对乳腺癌和黑色素瘤发展的影响提供一个机械性的见解。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(S)：(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；(Ii)通过控制JAMP将蛋白酶体募集到内质网。(3)利用RNF5基因敲除(KO)和可诱导转基因小鼠与乳腺肿瘤模型小鼠杂交，评价RNF5在乳腺肿瘤发生发展中的作用。了解RNF5通过JAMP对ER应激的调节及其与乳腺癌和黑色素瘤中P53依赖变化的关系，将为正常细胞和肿瘤中控制ER应激的潜在机制提供新的和重要的见解。