Abstract：
NFE2L1 (also known as NRF1) is a member of the nuclear erythroid 2-like family of transcription factors and is critical for counteracting various types of cellular stress such as oxidative, proteotoxic or metabolic stress. This unique transcription factor is also known to undergo changes, including post-translational modifications, limited proteolysis or translocation into the nucleus, before it exerts full transcriptional activity. As a result, there are various molecular forms with distinct sizes for this protein, while the precise nature of each form remains elusive. In this study, the N-glycosylated status of NFE2L1 in cells was examined. The findings revealed that when NFE2L1 was deglycosylated by PNGase F, the size-shift on SDS-PAGE was minimal. This was in contrast to deglycosylation by Endo H, which resulted in a clear size-shift, even though N-linked GlcNAc residues remained on the protein. It was found that this unusual behavior of PNGase-deglycosylated NFE2L1 was dependent on the conversion of the glycosylated-Asn to Asp, resulting in the introduction of more negative charges into the core peptide of NFE2L1. We also demonstrate that NGLY1-mediated deglycosylation and DDI2-mediated proteolytic processing of NFE2L1 are not strictly ordered reactions. Our study will allow us to better understand the precise structures as well as biochemical properties of the various forms of NFE2L1.
摘要：
NFE2L1（也称为NRF1）是核红细胞2样转录因子家族的成员，对于抵抗各种类型的细胞应激（如氧化，蛋白毒性或代谢应激。这种独特的转录因子也会发生变化，包括翻译后修饰，有限的蛋白水解或易位到细胞核，在它发挥完全转录活性之前。因此,这种蛋白质有各种不同大小的分子形式，而每种形式的精确性质仍然难以捉摸。在这项研究中,检测细胞中NFE2L1的N-糖基化状态。研究结果表明，当NFE2L1被PNGaseF去糖基化时，SDS-PAGE上的大小偏移最小。这与EndoH的去糖基化相反，这导致了明显的规模转移，即使N-连接的GlcNAc残基保留在蛋白质上。发现PNGase去糖基化NFE2L1的这种异常行为取决于糖基化Asn向Asp的转化，导致在NFE2L1的核心肽中引入更多负电荷。我们还证明了NGLY1介导的去糖基化和DDI2介导的NFE2L1的蛋白水解加工不是严格有序的反应。我们的研究将使我们更好地了解各种形式的NFE2L1的精确结构和生化特性。
