Function and regulation of cytoplasmic p53

细胞质p53的功能和调节

• 批准号: 10567672
• 负责人: WILLIAM F. MARZLUFF
• 金额: $ 34.93万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-19 至 2028-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10567672
• 关键词: Apoptosis; Autophagocytosis; BCL2 gene; BCL2L1 gene; Binding; Biochemical; Biological; Biological Process; Body fat; CUL9 gene; Cell Cycle Regulation; Cell Line; Cell Nucleus; Chiroptera; Cullin Proteins; Cytoplasm; DNA Sequence Alteration; Development; Dissection; Enzymes; Event; Genes; Genetic; Genetic Transcription; Genomics; Glycolysis; Glycolysis Inhibition; Goals; In Vitro; Incidence; Induction of Apoptosis; Knowledge; Lactate Dehydrogenase; Link; Lipids; Longevity; Malignant Neoplasms; Mammals; Mass Spectrum Analysis; Mediating; Metabolic; Metabolism; Mitochondria; Mus; Mutant Strains Mice; Nuclear; Nuclear Export; Nuclear Import; Nuclear Localization Signal; Oxidative Phosphorylation; Peptide Signal Sequences; Physiological; Play; Polyubiquitination; Process; Proteins; Regulation; Research; Role; System; TP53 gene; Testing; Travel; Tumor Suppression; Ubiquitination; anti aging; comparative; disease diagnosis; experimental study; genome sequencing; human disease; improved; in vivo; inhibition of autophagy; insight; lipid biosynthesis; lipid metabolism; mitochondrial metabolism; mutant; oxidation; therapeutic development; trait; transcription factor; treatment strategy; tumor; ubiquitin-protein ligase

ABSTRACT While the principal function of tumor suppressor p53 is a nuclear transcription regulator that transcribes a diverse set of genes involved in apoptosis, cell cycle regulation, and numerous other processes, compelling evidences have also unraveled extra-nuclear, transcription-independent activities of p53 in the cytoplasm, where it induces apoptosis and inhibits autophagy. However, significant gaps exist in the knowledge of p53 in the cytoplasm. The lack of knowledge is due at least in part to the fact that approaches investigating cytoplasmic p53 have been primarily in vitro studies in artificial settings. Dissection of the mechanistic aspects of cytoplasmic p53 in an in vivo system is necessary to clarify the biological events by which cytoplasmic p53 exerts its extra-nuclear, transcription-independent functions. Recent genome sequencing analyses of bats discovered many unexpected variances between bats and other mammals. Among them are the bat-specific alterations in the nuclear localization signal (NLS) sequences in p53. The alterations are predicted to hamper bat p53 nuclear import, resulting in increased cytoplasmic accumulation. To explore the significance of cytoplasmic p53, we generated mice expressing a mutant p53 that has an altered NLS sequence to mimic bat p53 NLS. We identified that p53 interacts with lactate dehydrogenase B (LDHB), which plays a critical role in lipid metabolism. We also found that cytoplasmic p53 promotes autophagy through disrupting Beclin 1-Bcl-2/Bcl-xL interaction and activating Beclin 1. IP-mass spectrometry experiments using cytoplasm-sequestered p53 and identified CUL9, a cytoplasmic cullin RING E3 ubiquitin ligase, as a major p53 binding partner. Biochemical experiments revealed that CUL9 catalyzes K63-linked, non-proteolytic polyubiquitination of p53. In this proposal, we test the hypothesis that cytoplasmic p53 exerts tumor suppression and anti-aging functions through inhibiting glycolysis, increasing lipogenesis, and promoting autophagy. Such functions of p53 are regulated by CUL9 catalyzed, K63-linked p53 polyubiquitination.

抽象的 虽然抑制肿瘤p53的主要功能是转录A的核转录调节剂 参与凋亡，细胞周期调节和许多其他过程的各种基因集， 引人入胜的证据也揭示了p53在 细胞质，诱导凋亡并抑制自噬。但是，存在着很大的差距 对细胞质中p53的知识。缺乏知识至少部分是由于以下事实 研究细胞质p53的方法主要是在人工环境中进行体外研究。 必须在体内系统中对细胞质p53的机械方面进行解剖以阐明 细胞质p53通过其核外，转录独立的功能发挥的生物学事件。 最近对蝙蝠的基因组测序分析发现了蝙蝠和 其他哺乳动物。其中包括核定位信号（NLS）的蝙蝠特异性变化 p53中的序列。预测这些更改会妨碍蝙蝠p53核进口，从而增加 细胞质积累。为了探索细胞质p53的重要性，我们产生了小鼠 表达突变体p53，该突变体p53具有变化的NLS序列对模拟BAT P53 NLS。我们确定了p53 与乳酸脱氢酶B（LDHB）相互作用，在脂质代谢中起着至关重要的作用。我们也是 发现细胞质p53通过破坏Beclin 1-Bcl-2/bcl-XL相互作用和 激活Beclin1。使用细胞质再生P53的IP-MAS光谱实验并鉴定 CUL9，一种细胞质Cullin环E3泛素连接酶，是主要的p53结合伴侣。生化 实验表明，CUL9催化了p53的K63连接的非蛋白溶解多泛素化。在这个 提案，我们检验了细胞质p53施加肿瘤和抗衰老功能的假设 通过抑制糖酵解，增加脂肪生成并促进自噬。 p53的此类功能是 由CUL9催化，K63连接的P53多泛素化调节。