Temperature is a key determinant of alpha- and beta-synuclein membrane interactions in neurons.
Temperature is a key determinant of alpha- and beta-synuclein membrane interactions in neurons.
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DOI：
10.1016/j.jbc.2021.100271
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发表时间：
2021-01
期刊：
影响因子：
--
通讯作者：
Dettmer U
中科院分区：
文献类型：
--
作者：
Ramalingam N;Dettmer U
α-突触核蛋白（αS）的聚集导致帕金森病（PD）和相关突触核蛋白病的标志性神经病理学。αS被描述为以细胞溶质和膜结合形式存在，其相对丰度仍然不确定。为了通过定量方法研究最相关条件下的αS，我们培养并成熟啮齿动物原代皮质神经元超过17天，并通过基于弱离子去污剂毛地黄皂苷的无离心连续提取来确定αS胞浆：膜分布。我们注意到，在较低的温度（4 °C或室温）下，αS主要与膜相关。然而，在37 °C时，αS的溶解度显著增加。相反，对照蛋白质（GAPDH，胞质;钙连接蛋白，膜）的提取不受温度的影响。当我们在不同温度和毛地黄皂苷浓度（200-1200 μg/ml）的各种条件下比较突触核蛋白同源物αS和β-突触核蛋白（βS）的相对分布时，我们一致发现αS比βS更与膜相关。然而，这两种蛋白质，表现出温度依赖性的膜结合。在最相关的条件下（37 ℃和800 μg/ml毛地黄皂苷，即，αS和βS的胞质分布分别为49.8% ± 9.0%和63.6% ± 6.6%。发现PD连接的α SA 30 P主要存在于胞质中，证实了先前使用不同方法的研究。我们的工作突出了细胞突触核蛋白行为的动态性质，并对αS蛋白质稳定的蛋白质生物化学和细胞生物学研究具有重要意义，例如测试遗传和药理学操作的影响。
Aggregation of α-synuclein (αS) leads to the hallmark neuropathology of Parkinson’s disease (PD) and related synucleinopathies. αS has been described to exist in both cytosolic and membrane-associated forms, the relative abundance of which has remained unsettled. To study αS under the most relevant conditions by a quantitative method, we cultured and matured rodent primary cortical neurons for >17 days and determined αS cytosol:membrane distribution via centrifugation-free sequential extractions based on the weak ionic detergent digitonin. We noticed that at lower temperatures (4 °C or room temperature), αS was largely membrane-associated. At 37 °C, however, αS solubility was markedly increased. In contrast, the extraction of control proteins (GAPDH, cytosolic; calnexin, membrane) was not affected by temperature. When we compared the relative distribution of the synuclein homologs αS and β-synuclein (βS) under various conditions that differed in temperature and digitonin concentration (200–1200 μg/ml), we consistently found αS to be more membrane-associated than βS. Both proteins, however, exhibited temperature-dependent membrane binding. Under the most relevant conditions (37 °C and 800 μg/ml digitonin, i.e., the lowest digitonin concentration that extracted cytosolic GAPDH to near completion), cytosolic distribution was 49.8% ± 9.0% for αS and 63.6% ± 6.6% for βS. PD-linked αS A30P was found to be largely cytosolic, confirming previous studies that had used different methods. Our work highlights the dynamic nature of cellular synuclein behavior and has important implications for protein-biochemical and cell-biological studies of αS proteostasis, such as testing the effects of genetic and pharmacological manipulations.
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影响因子：
6
作者：
Galvin, JE;Giasson, B;Trojanowski, JQ
通讯作者：
Trojanowski, JQ
DOI：
10.1097/00005072-199608000-00004
发表时间：
1996-08-01
影响因子：
3.2
作者：
Irizarry, MC;Kim, TW;Hyman, BT
通讯作者：
Hyman, BT
影响因子：
5.3
作者：
Burre, Jacqueline;Sharma, Manu;Suedhof, Thomas C.
通讯作者：
Suedhof, Thomas C.
影响因子：
3.2
作者：
Brucale, Marco;Sandal, Massimo;Samori, Bruno
通讯作者：
Samori, Bruno
DOI：
10.1016/0169-328x(91)90043-w
发表时间：
1991-10-01
期刊：
MOLECULAR BRAIN RESEARCH
影响因子：
--
作者：
MAROTEAUX, L;SCHELLER, RH
通讯作者：
SCHELLER, RH