FUNCTIONS OF APOLIPOPROTEINS IN CANCER APOPTOSIS

载脂蛋白在癌症细胞凋亡中的功能

• 批准号: 7720457
• 负责人: Chien-An Andy Hu
• 金额: $ 9.46万
• 依托单位: NEW MEXICO STATE UNIVERSITY LAS CRUCES
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7720457
• 关键词: Adenoviruses; Apolipoproteins; Apoptosis; Autophagocytosis; BH3 Domain; Binding; Cell Death; Cell Line; Cell Survival; Cells; Cessation of life; Class; Computer Retrieval of Information on Scientific Projects Database; Cytosol; Electrons; Funding; Generations; Genes; Grant; Immunoblotting; Immunofluorescence Immunologic; Institution; Knowledge; Lipid Binding; Lipids; Localized; Malignant Neoplasms; Mass Spectrum Analysis; Microscopic; Phosphotransferases; Physiology; Process; Protein Overexpression; Proteins; Proteomics; Research; Research Personnel; Resources; Reverse Transcriptase Polymerase Chain Reaction; Role; Source; System; Tetanus Helper Peptide; United States National Institutes of Health; Vacuole; base; inhibitor/antagonist

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. To further investigate functions of apoplipoprotein L (ApoL) 2 and 3 in programmed cell death (PCD) and in normal physiology, in the fourth year of this NM-INBRE funded project, we utilized "Tet-off" inducible gene system in DLD-1 cells and adenovirus (AD)-based system to overexpress L2 or L3 in various cell lines as previously described. Interestingly, unlike their closely related proteins ApoL1 and ApoL6, neither ApoL2 nor ApoL3 induced PCD in cells examined. RT-PCR and immunoblot analyses confirmed the overexpression of L2 or L3 in transfected cells. However, we noticed that both L2 and L3 induce increase generation of autophagic vacuoles (AV), one of the hallmarks of autophagy. Unlike L1 which induces Autophagic cell death, overexpression of L2 or L3 seems to induce autophagic cell survival. It is known that autophagy can be either pro-survival or pro-death. We also noticed that a fraction of L2 might localize in AV. In a related research, we used electron microscopic, immunoblot and immunofluorescence analyses to show that overexpression of ApoL1 induces increase formation of AV, and activating translocation of LC3-II from cytosol to AV, two of the most significant hallmarks in autophagic processes. Inhibitors of class III phosphatidylinostol-3-kinase and autophagy, block ApoL1-induced AuCD. In addition, the BH3 domain deletion construct of ApoL1 failed to induce AuCD, demonstrating that ApoL1 is a bona fide BH3-only pro-death protein. To our knowledge, this is the first BH3-only protein with lipid binding and transport activity that induces AuCD. We will employ the same strategies to further study the subcellular localization and roles of L2 and L3 in the induction of autophagic cell survival. In addition, we plan to employ mass spectrometry (MS)-based proteomic and lipidomic strategies to analyze and characterize the binding partners, both protein and lipid species, of ApoL2 or ApoL3.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此，可以在其他清晰的条目中表示。列出的机构是 对于中心，这不一定是调查员的机构。 为了进一步研究程序性细胞死亡（PCD）和正常生理学中的腹膜蛋白L（Apol）2和3如前所述，1个细胞和基于腺病毒（AD）的系统在各种细胞系中过表达L2或L3。有趣的是，与它们密切相关的蛋白Apol1和apol6不同，在所检查的细胞中均未诱导Apol2和Apol3诱导PCD。 RT-PCR和免疫印迹分析证实了转染细胞中L2或L3的过表达。但是，我们注意到L2和L3都诱导自噬的自噬液泡的产生增加，这是自噬的标志之一。与诱导自噬细胞死亡的L1不同，L2或L3的过表达似乎诱导自噬细胞存活。众所周知，自噬可以是阳离子或亲死亡。我们还注意到，L2的一小部分可能本地化在AV中。在一项相关研究中，我们使用了电子显微镜，免疫印迹和免疫荧光分析，以表明APOL1的过表达诱导AV的形成增加，并激活LC3-II从cytosol到AV的易位，这是自噬过程中最重要的两个最重要标志。 III类磷脂酰基诺替洛尼诺斯特-3-激酶和自噬的抑制剂，阻断Apol1诱导的AUCD。另外，Apol1的BH3域缺失构建体无法诱导AUCD，这表明APOL1是一种真正的BH3仅BH3促pro死亡蛋白。据我们所知，这是第一个仅BH3蛋白具有脂质结合和诱导AUCD的转运活性的蛋白。我们将采用相同的策略来进一步研究L2和L3在诱导自噬细胞存活中的亚细胞定位和作用。此外，我们计划采用质谱（MS）的蛋白质组学和脂质组策略来分析和表征ApoL2或ApoL3的蛋白质和脂质物种的结合伴侣。