An Atypical Mechanism of SUMOylation of Neurofibromin SecPH Domain Provides New Insights into SUMOylation Site Selection

IF 4.7 2区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY
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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.

Abstract Image

神经纤维瘤蛋白 SecPH 结构域的非典型 SUMO 化机制为 SUMO 化位点选择提供了新的视角。
神经纤维瘤蛋白(Nf1)是一种由肿瘤抑制基因 NF1 编码的巨型多域蛋白。NF1 基因突变可导致一种常见的遗传疾病--I 型神经纤维瘤病 (NF1),也可导致多种癌症。该蛋白具有 Ras-GAP(GTPase activating protein,GTPase 激活蛋白)活性,但也通过其 SecPH 结构域与不同的信号通路相连,该结构域可与脂质和不同的蛋白伙伴相互作用。我们以前曾发现,Nf1 与 ProMyelocytic Leukemia(PML)蛋白部分共定位在 PML 核体(SUMOylation 的热点)中,从而表明 Nf1 可能存在 SUMOylation。 在这里,我们证明了 Nf1 的全长异构体 2 和 SecPH 片段是 SUMO 通路的底物,并确定了 SecPH 明确的 SUMOylation 特征,其中有两个主要修饰的赖氨酸。其中一个位点 K1731 是高度保守的表面暴露位点。尽管在 K1731 周围存在一个倒置的 SUMO 共识基序,而且在 SecPH 中存在一个潜在的 SUMO 相互作用基序 (SIM),但我们发现这两个元素都不是 K1731 SUMO 化所必需的,而且 K1731 的 SUMO 化也与 K14 上的 Ubc9 SUMO 化无关。以 K1731 为中心的 SecPH 与 Ubc9 之间相互作用的三维模型,结合定点突变,确定了 K1731 SUMOylation 所需的 SecPH 的特定结构元素,其中一些元素在已报道的 NF1 致病变体中受到了影响。这项工作提供了一个新的例子,说明SUMO酰化依赖于修饰位点周围的三级而非一级蛋白质结构,从而扩展了我们对SUMO酰化位点选择机制的认识。
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来源期刊
Journal of Molecular Biology
Journal of Molecular Biology 生物-生化与分子生物学
CiteScore
11.30
自引率
1.80%
发文量
412
审稿时长
28 days
期刊介绍: Journal of Molecular Biology (JMB) provides high quality, comprehensive and broad coverage in all areas of molecular biology. The journal publishes original scientific research papers that provide mechanistic and functional insights and report a significant advance to the field. The journal encourages the submission of multidisciplinary studies that use complementary experimental and computational approaches to address challenging biological questions. Research areas include but are not limited to: Biomolecular interactions, signaling networks, systems biology; Cell cycle, cell growth, cell differentiation; Cell death, autophagy; Cell signaling and regulation; Chemical biology; Computational biology, in combination with experimental studies; DNA replication, repair, and recombination; Development, regenerative biology, mechanistic and functional studies of stem cells; Epigenetics, chromatin structure and function; Gene expression; Membrane processes, cell surface proteins and cell-cell interactions; Methodological advances, both experimental and theoretical, including databases; Microbiology, virology, and interactions with the host or environment; Microbiota mechanistic and functional studies; Nuclear organization; Post-translational modifications, proteomics; Processing and function of biologically important macromolecules and complexes; Molecular basis of disease; RNA processing, structure and functions of non-coding RNAs, transcription; Sorting, spatiotemporal organization, trafficking; Structural biology; Synthetic biology; Translation, protein folding, chaperones, protein degradation and quality control.
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