神经纤维蛋白（Neurofibromin）SecPH结构域SUMO化的非典型机制：对SUMO化位点选择的新见解
An Atypical Mechanism of SUMOylation of Neurofibromin SecPH Domain Provides New Insights into SUMOylation Site Selection
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影响因子:4.5
分区:生物学2区 / 生化与分子生物学3区
发表日期:2024 Nov 15
作者:
Mohammed Bergoug, Christine Mosrin, Amandine Serrano, Fabienne Godin, Michel Doudeau, Iva Dundović, Stephane Goffinont, Thierry Normand, Marcin J Suskiewicz, Béatrice Vallée, Hélène Bénédetti
DOI:
10.1016/j.jmb.2024.168768
摘要
神经纤维蛋白（Nf1）是由肿瘤抑制基因NF1编码的巨大多结构域蛋白。NF1在常见的遗传疾病——神经纤维瘤病I型（NF1）中发生突变，也与多种癌症相关。该蛋白具有Ras-GAP（GTP酶激活蛋白）活性，还通过其SecPH结构域与脂质及多种蛋白伙伴相互作用，连接多条信号通路。我们此前显示Nf1在PML核体中部分与ProMyelocytic Leukemia（PML）蛋白共定位，PML核体是SUMO化的热点区域，从而暗示Nf1可能被SUMO化。本研究证明，Nf1的全长变体2和SecPH片段是SUMO途径的底物，并鉴定出SecPH的两个主要修饰赖氨酸的SUMO化谱。一处修饰位点K1731高度保守且暴露在表面。尽管K1731周围存在倒置的SUMO共识基序和潜在的SUMO相互作用基序（SIM），我们发现这两个元素都不是K1731 SUMO化的必要条件，且此修饰也与Ubc9在K14上的SUMO化无关。结合K1731为中心的SecPH与Ubc9相互作用的三维模型和定点突变，鉴定出SecPH的特定结构元素对K1731 SUMO化的必需性，其中一些在已报道的NF1致病变体中受影响。这一工作提供了依赖三级而非一级蛋白质结构的SUMO化新例子，扩展了我们对调控SUMO化位点选择机制的理解。
Abstract
Neurofibromin (Nf1) is a giant multidomain protein encoded by the tumour-suppressor gene NF1. NF1 is mutated in a common genetic disease, neurofibromatosis type I (NF1), and in various cancers. The protein has a Ras-GAP (GTPase activating protein) activity but is also connected to diverse signalling pathways through its SecPH domain, which interacts with lipids and different protein partners. We previously showed that Nf1 partially colocalized with the ProMyelocytic Leukemia (PML) protein in PML nuclear bodies, hotspots of SUMOylation, thereby suggesting the potential SUMOylation of Nf1. Here, we demonstrate that the full-length isoform 2 and a SecPH fragment of Nf1 are substrates of the SUMO pathway and identify a well-defined SUMOylation profile of SecPH with two main modified lysines. One of these sites, K1731, is highly conserved and surface-exposed. Despite the presence of an inverted SUMO consensus motif surrounding K1731, and a potential SUMO-interacting motif (SIM) within SecPH, we show that neither of these elements is necessary for K1731 SUMOylation, which is also independent of Ubc9 SUMOylation on K14. A 3D model of an interaction between SecPH and Ubc9 centred on K1731, combined with site-directed mutagenesis, identifies specific structural elements of SecPH required for K1731 SUMOylation, some of which are affected in reported NF1 pathogenic variants. This work provides a new example of SUMOylation dependent on the tertiary rather than primary protein structure surrounding the modified site, expanding our knowledge of mechanisms governing SUMOylation site selection.