Calpain-mediated proteolysis of vimentin filaments is augmented in giant axonal neuropathy fibroblasts exposed to hypotonic stress.
Calpain-mediated proteolysis of vimentin filaments is augmented in giant axonal neuropathy fibroblasts exposed to hypotonic stress.
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DOI：
10.3389/fcell.2022.1008542
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发表时间：
2022
影响因子：
5.5
通讯作者：
Snider, Natasha T. T.
中科院分区：
文献类型：
--
作者：
Phillips, Cassandra L. L.;Fu, Dong;Herring, Laura E. E.;Armao, Diane;Snider, Natasha T. T.
巨轴突神经病 (GAN) 是一种小儿神经退行性疾病，由 KLHL16 基因编码的 E3 泛素连接酶接头 gigaxonin 功能丧失突变引起。 Gigaxonin 调节多种中间丝 (IF) 蛋白的降解，包括神经丝、GFAP 和波形蛋白，这些蛋白在 GAN 患者细胞中聚集。了解 IF 及其聚集体在压力下如何加工可以揭示新的 GAN 疾病机制和潜在的治疗靶点。在这里，我们测试了 GAN 中低渗应激诱导的波形蛋白水解受损的假设。在暴露于低渗应激的 GAN 和对照成纤维细胞中，我们观察到时间依赖性波形蛋白裂解，产生两个显着的~40-45 kDa 片段。然而，与未受影响的对照相比，GAN 细胞中波形蛋白水解发生得更快、更广泛，因为这两个片段生成得更早，且水平高出 4-6 倍。为了测试酶的参与，我们测定了钙敏感性钙蛋白酶-1和-2及其内源性抑制剂钙蛋白酶抑制素的表达水平和定位。虽然后者没有受到影响，但与对照细胞相比，GAN 细胞中两种钙蛋白酶的表达均高出 2 倍。此外，用 MDL-28170 或 MG-132 对钙蛋白酶进行药理抑制可减弱波形蛋白裂解。成像分析揭示了 GAN 成纤维细胞中大型核周波形蛋白聚集体和 calpain-2 之间的显着共定位。这种共定位因低渗应激而发生显着改变，GAN 细胞中细丝相对于聚集体的选择性分解迅速发生，并且与 calpain-2 细胞质重新分布同时发生。最后，基于质谱的蛋白质组学揭示，位于波形蛋白中央“杆”结构域和 C 端“尾”结构域之间连接处的 Ser-412 的磷酸化参与了这种应激反应。使用磷酸缺陷和磷酸模拟突变体的过表达研究表明，Ser-412 对于低渗应激时的丝组织、溶解度动力学和波形蛋白裂解非常重要。总的来说，我们的工作揭示了渗透压会诱导钙蛋白酶和蛋白酶体介导的波形蛋白降解和 IF 网络崩溃。当存在导致疾病的 KLHL16 突变（改变中间丝组织）时，这些影响会显着增强。虽然 GAN 细胞中钙蛋白酶生成的波形蛋白 IF 片段的具体作用仍有待确定，但这种蛋白水解途径与 GAN 具有翻译相关性，因为维持渗透压稳态对于神经系统功能至关重要。
Giant Axonal Neuropathy (GAN) is a pediatric neurodegenerative disease caused by loss-of-function mutations in the E3 ubiquitin ligase adaptor gigaxonin, which is encoded by the KLHL16 gene. Gigaxonin regulates the degradation of multiple intermediate filament (IF) proteins, including neurofilaments, GFAP, and vimentin, which aggregate in GAN patient cells. Understanding how IFs and their aggregates are processed under stress can reveal new GAN disease mechanisms and potential targets for therapy. Here we tested the hypothesis that hypotonic stress-induced vimentin proteolysis is impaired in GAN. In both GAN and control fibroblasts exposed to hypotonic stress, we observed time-dependent vimentin cleavage that resulted in two prominent ∼40–45 kDa fragments. However, vimentin proteolysis occurred more rapidly and extensively in GAN cells compared to unaffected controls as both fragments were generated earlier and at 4-6-fold higher levels. To test enzymatic involvement, we determined the expression levels and localization of the calcium-sensitive calpain proteases-1 and -2 and their endogenous inhibitor calpastatin. While the latter was not affected, the expression of both calpains was 2-fold higher in GAN cells compared to control cells. Moreover, pharmacologic inhibition of calpains with MDL-28170 or MG-132 attenuated vimentin cleavage. Imaging analysis revealed striking colocalization between large perinuclear vimentin aggregates and calpain-2 in GAN fibroblasts. This colocalization was dramatically altered by hypotonic stress, where selective breakdown of filaments over aggregates occurred rapidly in GAN cells and coincided with calpain-2 cytoplasmic redistribution. Finally, mass spectrometry-based proteomics revealed that phosphorylation at Ser-412, located at the junction between the central “rod” domain and C-terminal “tail” domain on vimentin, is involved in this stress response. Over-expression studies using phospho-deficient and phospho-mimic mutants revealed that Ser-412 is important for filament organization, solubility dynamics, and vimentin cleavage upon hypotonic stress exposure. Collectively, our work reveals that osmotic stress induces calpain- and proteasome-mediated vimentin degradation and IF network breakdown. These effects are significantly augmented in the presence of disease-causing KLHL16 mutations that alter intermediate filament organization. While the specific roles of calpain-generated vimentin IF fragments in GAN cells remain to be defined, this proteolytic pathway is translationally-relevant to GAN because maintaining osmotic homeostasis is critical for nervous system function.
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影响因子：
4
作者：
Balaban D;Miyawaki EK;Bhattacharyya S;Torre M
通讯作者：
Torre M
影响因子：
9.3
作者：
Early, Alexandria N.;Gorman, Amy A.;Morganti, Josh M.
通讯作者：
Morganti, Josh M.
DOI：
10.1093/brain/awab179
发表时间：
2021-11-29
期刊：
Brain : a journal of neurology
影响因子：
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作者：
Bharucha-Goebel, Diana X;Norato, Gina;Bonnemann, Carsten G
通讯作者：
Bonnemann, Carsten G
影响因子：
5.5
作者：
Aweida D;Cohen S
通讯作者：
Cohen S
DOI：
10.1073/pnas.1908263116
发表时间：
2019-08-20
影响因子：
11.1
作者：
Kedia, Niraja;Arhzaouy, Khalid;Bieschke, Jan
通讯作者：
Bieschke, Jan