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Study on the cellular distribution and molecular function of ALS2, a product of the novel causative gene for famrlial ALS

家族性ALS新致病基因ALS2的细胞分布及分子功能研究

基本信息

批准号：
14380361
负责人：
HADANO Shinji
金额：
$ 9.47万
依托单位：
Tokai University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14380361/
关键词：
amyotrophic laterel sclerosis familial ALS ALS2 gene motor neuron neurodegenerative disease guanine nucleotide exchange factor small GTPase vesicle trafficking 細胞内膜・小胞移送タンパク質ドメイン

项目摘要

The aim of this study was to delineate molecular function of ALS2, a causative gene product underlying a number of recessive motor neuron diseases, thereby clarifying the molecular mechanisms for neuronal cell dysfunction and death resulted from loss of function mutations in the ALS2 gene. We have carried out the following four sets of molecular, biochemical, and cell biological studies; 1) immunocytochemical analysis of the subcellular localization of ALS2, 2) identification of the target small GTPases for ALS2, 3) identification of the ALS2 associating/binding factors, and 4) exploration of the biochemical signal pathways mediated by ALS2. We here demonstrate that ALS2 specifically binds to small GTPase Rab5 and functions as a GEF for Rab5. Ectopically expressed ALS2 localizes with Rab5 onto early endosomal compartments, and stimulates the enlargement of endosomes in the cultured neuronal and non-neuronal cells. The carboxy-terminus of ALS2 carrying the MORN/VPS9 domain mediates not only the activation of Rab5 via a guanine-nucleotide exchanging reaction, but also the endosomal localization for ALS2, thereby leading to endosome fusions by activating the Rab5-EEAI-SNARE molecular pathway. We have also shown that ALS2 forms a homophilic oligomer through its distinct C-terminal regions. This homo-oligomerization is crucial for the Rab5GEF activity and the ALS2-mediated endosome enlargement in the cells. Lastly, we have identified a novel ALS2 homologous gene, ALS2 C-terminal like (ALS2CL), which is a novel factor modulating the Rab5-mediated endosome dynamics in the cells. Taken together, a perturbation of the endosomal dynamics, which is resulted from a loss of ALS2 Rab5-GEF activity, might be a key molecular basis underlying neuronal dysfunction and degeneration in a number of motor neuron diseases caused by the ALS2 mutations.

本研究的目的是阐明als2的分子功能，这是一种导致许多隐性运动神经元病的基因产物，从而阐明als2基因功能缺失导致神经细胞功能障碍和死亡的分子机制。我们开展了以下四组分子、生化和细胞生物学研究：1)ALS2亚细胞定位的免疫细胞化学分析，2)ALS2靶标小GTP酶的鉴定，3)ALS2相关/结合因子的鉴定，以及4)ALS2介导的生化信号通路的探索。我们在这里证明了als2特异性地与小GTP酶Rab5结合，并作为Rab5的环境基金发挥作用。异位表达的als2与Rab5一起定位于早期的内小体隔室，并刺激培养的神经元和非神经元细胞内小体的增大。携带Morn/VPS9结构域的als2的羧基末端不仅通过鸟嘌呤-核苷酸交换反应介导Rab5的激活，而且还介导als2的内体定位，从而通过激活Rab5-EEAI-SNARE分子通路而导致内体融合。我们还证明了Als2通过其不同的C-末端区域形成了同亲的低聚物。这种同源寡聚对于Rab5gef的活性和als2介导的细胞内内体扩大是至关重要的。最后，我们发现了一个新的als2同源基因，als2C末端样蛋白(Als2CL)，它是一个新的调控Rab5介导的细胞内小体动力学的因子。综上所述，als2基因突变引起的内体动力学紊乱可能是导致神经元功能障碍和变性的关键分子基础。