背景:Nor1/NR4A3是核受体NR4A亚家族的成员,在调节与发育相关的基因表达中起着至关重要的作用,细胞稳态和神经功能。然而,Nor1仍然被认为是孤儿受体,因为其天然配体介导转录激活仍不清楚。还有其他激活信号可以调制Nor1活动,尽管它们在神经系统发育和维持中的确切作用仍然难以捉摸。
方法:我们使用了转录报告检测,基因表达谱分析,蛋白质周转测量,和细胞生长测定,以评估神经元细胞中Nor1和SUMO缺陷型变体的功能相关性。通过免疫沉淀评估SUMO1和SUMO2与Nor1的缀合。通过乙酰化和聚合试验确定微管蛋白的稳定性,和活细胞荧光显微镜。
结果:这里,我们证明Nor1在Lys-89上经历SUMO1缀合,有助于Nor1磺酰化的复杂模式,其中还包括Lys-137。破坏Lys-89,从而防止SUMO1共轭,导致Nor1转录能力和蛋白质稳定性降低,以及与细胞生长和代谢有关的基因的下调,如ENO3、EN1和CFLAR,在微管细胞骨架动力学中,包括MAP2和MAPT,导致神经元细胞的存活率降低。有趣的是,Lys-89SUMOylation对诺考达唑的反应增强,一种微管解聚药物,尽管尽管Nor1基因表达增强,但这不足以挽救细胞免受微管破坏。相反,Lys-89去SUMO化降低了微管切断基因的表达,如KATNA1,SPAST,FIGN,和增强的α-微管蛋白细胞水平,乙酰化,和微丝组织,促进微管稳定性和对诺考达唑的抗性。这些效果与Lys-137SUMO化形成对比,基于特定的Nor1输入磺酰化信号,提出不同的调节机制。
结论:我们的研究提供了对Nor1转录信号传导能力的新见解,并确定了一种分层机制,通过该机制,选择性的Nor1SUMO化可以控制神经元细胞骨架网络动力学和对微管干扰的抵抗力,与神经退行性疾病相关的疾病。
BACKGROUND: Nor1/NR4A3 is a member of the NR4A subfamily of nuclear receptors that play essential roles in regulating gene expression related to development, cell homeostasis and neurological functions. However, Nor1 is still considered an orphan receptor, as its natural ligand remains unclear for mediating transcriptional activation. Yet other activation signals may modulate Nor1 activity, although their precise role in the development and maintenance of the nervous system remains elusive.
METHODS: We used transcriptional reporter assays, gene expression profiling, protein turnover measurement, and cell growth assays to assess the functional relevance of Nor1 and SUMO-defective variants in neuronal cells. SUMO1 and SUMO2 conjugation to Nor1 were assessed by immunoprecipitation. Tubulin stability was determined by acetylation and polymerization assays, and live-cell fluorescent microscopy.
RESULTS: Here, we demonstrate that Nor1 undergoes SUMO1 conjugation at Lys-89 within a canonical ψKxE SUMOylation motif, contributing to the complex pattern of Nor1 SUMOylation, which also includes Lys-137. Disruption of Lys-89, thereby preventing SUMO1 conjugation, led to reduced Nor1 transcriptional competence and protein stability, as well as the downregulation of genes involved in cell growth and metabolism, such as ENO3, EN1, and CFLAR, and in microtubule cytoskeleton dynamics, including MAP2 and MAPT, which resulted in reduced survival of neuronal cells. Interestingly, Lys-89 SUMOylation was potentiated in response to nocodazole, a microtubule depolymerizing drug, although this was insufficient to rescue cells from microtubule disruption despite enhanced Nor1 gene expression. Instead, Lys-89 deSUMOylation reduced the expression of microtubule-severing genes like KATNA1, SPAST, and FIGN, and enhanced α-tubulin cellular levels, acetylation, and microfilament organization, promoting microtubule stability and resistance to nocodazole. These effects contrasted with Lys-137 SUMOylation, suggesting distinct regulatory mechanisms based on specific Nor1 input SUMOylation signals.
CONCLUSIONS: Our study provides novel insights into Nor1 transcriptional signaling competence and identifies a hierarchical mechanism whereby selective Nor1 SUMOylation may govern neuronal cytoskeleton network dynamics and resistance against microtubule disturbances, a condition strongly associated with neurodegenerative diseases.