神经纤维蛋白 (Nf1) 是一种由肿瘤抑制基因 NF1 编码的巨型多域蛋白。 NF1 在常见遗传病 I 型神经纤维瘤病 (NF1) 和各种癌症中发生突变。该蛋白具有 Ras-GAP（GTP 酶激活蛋白）活性，但也通过其 SecPH 结构域与多种信号通路相连，该结构域与脂质和不同的蛋白伴侣相互作用。我们之前表明，Nf1 与原粒细胞白血病 (PML) 蛋白部分共定位于 PML 核体中，即 SUMO 化的热点，从而表明 Nf1 可能存在 SUMO 化。在这里，我们证明 Nf1 的全长亚型 2 和 SecPH 片段是 SUMO 途径的底物，并鉴定了 SecPH 具有两个主要修饰赖氨酸的明确 SUMO 化谱。其中一个位点 K1731 高度保守且表面暴露。尽管 K1731 周围存在反向 SUMO 共有基序，并且 SecPH 内存在潜在的 SUMO 相互作用基序 (SIM)，但我们表明这些元件对于 K1731 SUMO 化来说都不是必需的，这也独立于 K14 上的 Ubc9 SUMO 化。以 K1731 为中心的 SecPH 和 Ubc9 之间相互作用的 3D 模型，结合定点诱变，确定了 K1731 SUMOylation 所需的 SecPH 特定结构元件，其中一些在已报道的 NF1 致病变异中受到影响。这项工作提供了 SUMO 化依赖于修饰位点周围的三级而非一级蛋白质结构的新例子，扩展了我们对 SUMO 化位点选择机制的了解。版权所有 © 2024 作者。由爱思唯尔有限公司出版。保留所有权利。

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.Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.