Gagnon Jonathan, Caron Véronique, Tremblay André
Research Center, CHU Sainte-Justine, 3175 Côte Ste-Catherine, Montréal, Québec, H3T 1C5, Canada.
Department of Biochemistry and Molecular Medicine, Faculty of Medicine, University of Montreal, Montréal, Québec, H3T 1J4, Canada.
Cell Biosci. 2024 Jul 13;14(1):91. doi: 10.1186/s13578-024-01273-x.
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.
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.
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.
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.
Nor1/NR4A3是核受体NR4A亚家族的成员，在调节与发育、细胞稳态和神经功能相关的基因表达中发挥重要作用。然而，Nor1仍被认为是一种孤儿受体，因为其介导转录激活的天然配体尚不清楚。不过，其他激活信号可能会调节Nor1的活性，尽管它们在神经系统发育和维持中的精确作用仍不清楚。
我们使用转录报告基因分析、基因表达谱分析、蛋白质周转测量和细胞生长分析来评估Nor1和SUMO缺陷变体在神经元细胞中的功能相关性。通过免疫沉淀评估SUMO1和SUMO2与Nor1的结合。通过乙酰化和聚合分析以及活细胞荧光显微镜确定微管蛋白的稳定性。
在此，我们证明Nor1在典型的ψKxE SUMO化基序内的赖氨酸89处发生SUMO1结合，这有助于形成Nor1 SUMO化的复杂模式，其中还包括赖氨酸137。赖氨酸89的破坏从而阻止SUMO1结合，导致Nor1转录能力和蛋白质稳定性降低，以及参与细胞生长和代谢的基因（如ENO3、EN1和CFLAR）以及参与微管细胞骨架动力学的基因（包括MAP2和MAPT）的下调，这导致神经元细胞存活率降低。有趣的是，响应于微管解聚药物诺考达唑，赖氨酸89的SUMO化增强，尽管尽管Nor1基因表达增强，但这不足以使细胞从微管破坏中恢复。相反，赖氨酸89的去SUMO化降低了微管切割基因（如KATNA1、SPAST和FIGN）的表达，并提高了α-微管蛋白的细胞水平、乙酰化和微丝组织，促进了微管稳定性和对诺考达唑的抗性。这些效应与赖氨酸137的SUMO化形成对比，表明基于特定Nor1输入SUMO化信号的不同调节机制。
我们的研究为Nor1转录信号传导能力提供了新的见解，并确定了一种分级机制，通过该机制选择性的Nor1 SUMO化可能控制神经元细胞骨架网络动力学和对微管干扰的抗性，微管干扰是一种与神经退行性疾病密切相关的情况。
