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.

摘要 虽然肿瘤抑制因子p53的主要功能是核转录调节因子， 涉及细胞凋亡、细胞周期调节和许多其他过程的不同基因组， 令人信服的证据还揭示了p53在细胞核外的、不依赖转录的活动。 在细胞质中，它诱导细胞凋亡并抑制自噬。然而，在这方面存在着重大差距。 细胞质中的p53蛋白。知识的缺乏至少部分是由于这样一个事实， 研究细胞质p53的方法主要是在人工环境中的体外研究。 在体内系统中，有必要对胞质p53的机制方面进行剖析，以澄清 细胞质p53发挥其核外、转录非依赖性功能的生物学事件。 最近对蝙蝠的基因组测序分析发现了蝙蝠和蝙蝠之间许多意想不到的差异， 其他哺乳动物其中包括蝙蝠特有的核定位信号（NLS）的改变 p53中的序列。预计这些改变将阻碍bat p53核进口， 细胞质积累为了探讨胞浆p53的意义，我们建立了小鼠 表达具有改变的NLS序列以模拟蝙蝠p53 NLS的突变型p53。我们发现p53 与乳酸脱氢酶B（LDHB）相互作用，后者在脂质代谢中发挥关键作用。我们也 发现细胞质p53通过破坏Beclin 1-Bcl-2/Bcl-xL相互作用促进自噬， 激活Beclin 1使用细胞质螯合的p53进行IP-质谱实验，并鉴定 CUL 9，一种细胞质cullin RING E3泛素连接酶，作为主要的p53结合伴侣。生化 实验显示CUL 9催化K63连接的p53的非蛋白水解的多泛素化。在这 因此，我们提出了一个假设，即细胞质p53发挥肿瘤抑制和抗衰老功能。 通过抑制糖酵解、增加脂肪生成和促进自噬。p53的这些功能是 由CUL 9催化的K63连接的p53多聚泛素化调节。