The SPRED family proteins act as negative regulators of the Ras-ERK pathway: the N-terminal EVH1 domain interacts with the Ras-GAP domain (GRD) of the NF1 protein, while the C-terminal Sprouty-related (SPR) domain promotes membrane localization of SPRED, thereby recruiting NF-1 to Ras. Loss-of-function mutations in the hSPRED1 cause Legius syndrome in an autosomal dominant manner. In this study, we investigated the effects of missense mutations in the SPR domain identified in patients with Legius syndrome. Among the 18 mutations we examined, six (C368S, M369L, V408E, P415A, P415L, and P422R) have defects in the palmitoylation of the SPRED1 protein, losing plasma membrane localization and forming cytoplasmic granular aggregates. To evaluate the in vivo effects of SPR mutations, knock-in (KI) mice with P415A and P415V substitutions or M417Afs∗4, a C-terminal 28 amino acid deletion, were generated. All these KI mice exhibited cranial malformations, a characteristic feature of Legius syndrome. However, both P415A and P415V mutants formed granular aggregates, whereas M417Afs∗4 showed a diffuse cytoplasmic distribution, and Spred1P415A and Spred1P415V mice, but not Spred1M417Afs∗4 mice, developed cerebellar ataxia and Purkinje cell loss with age. These data suggest that in addition to loss of palmitoylation, the C-terminal region is required for the granular aggregate formation and Purkinje cell loss. The autophagy inducer spermidine rescued the ataxia phenotypes and Purkinje cell loss in Spred1P415A mice. These results suggest that some, but not all, SPR mutations that lose lipid modification induce abnormal cytoplasmic aggregation, which could be a target for autophagic clearance, and potentially cause neurodegenerative diseases.

中文翻译：

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Ras 调节因子 SPRED1 中的 Legius 综合征突变会消除其膜定位并可能导致神经退行性变


SPRED 家族蛋白充当 Ras-ERK 通路的负调节因子：N 端 EVH1 结构域与 NF1 蛋白的 Ras-GAP 结构域 （GRD） 相互作用，而 C 端 Sprouty 相关 （SPR） 结构域促进 SPRED 的膜定位，从而将 NF-1 募集到 Ras。hSPRED1 中的功能丧失突变以常染色体显性方式导致 Legius 综合征。在这项研究中，我们调查了在 Legius 综合征患者中发现的 SPR 域中错义突变的影响。在我们检查的 18 个突变中，有 6 个（C368S、M369L、V408E、P415A、P415L 和 P422R）在 SPRED1 蛋白的棕榈酰化中存在缺陷，失去质膜定位并形成细胞质颗粒聚集体。为了评估 SPR 突变的体内效应，生成了具有 P415A 和 P415V 替换或 M417Afs∗4（C 端 28 个氨基酸缺失）的敲入 （KI） 小鼠。所有这些 KI 小鼠都表现出颅畸形，这是 Legius 综合征的一个特征。然而，P415A 和 P415V 突变体均形成颗粒聚集体，而 M417Afs∗4 表现出弥漫性细胞质分布，Spred1P415A 和 Spred1P415V 小鼠，而不是 Spred1M417Afs∗4 小鼠，随着年龄的增长而发展为小脑共济失调和浦肯野细胞丢失。这些数据表明，除了棕榈酰化的损失外，C 端区域也是颗粒聚集体形成和浦肯野细胞丢失所必需的。自噬诱导剂亚精胺挽救了 Spred1P415A 小鼠的共济失调表型和浦肯野细胞丢失。这些结果表明，一些（但不是全部）失去脂质修饰的 SPR 突变会诱导异常的细胞质聚集，这可能是自噬清除的靶标，并可能导致神经退行性疾病。