Proteomic profiling of phosphoproteins and glycoproteins responsive to wild-type alpha-synuclein accumulation and aggregation.
Proteomic profiling of phosphoproteins and glycoproteins responsive to wild-type alpha-synuclein accumulation and aggregation.
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DOI：
10.1016/j.bbapap.2008.09.025
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发表时间：
2009-02
影响因子：
3.2
通讯作者：
Yen, Shu-Hui
中科院分区：
文献类型：
--
作者：
Kulathingal, Jayanarayan;Ko, Li-wen;Cusack, Bernadette;Yen, Shu-Hui
使用四环素诱导转染细胞系 (3D5)，在用视黄酸分化后能够产生可溶性和不溶性野生型人 α-突触核蛋白 (α-Syn) 组件，用于研究 α-Syn 积累对蛋白质磷酸化和糖基化的影响。通过二维凝胶电泳和用与蛋白质 (Sypro ruby)、磷蛋白 (Pro-Q Diamond) 和糖蛋白 (Pro-Q emerald) 结合的染料对凝胶进行染色，分析来自 3D5 细胞的可溶性蛋白质，无论是否诱导 α-Syn 表达。通过与固定化金属离子亲和柱结合进一步证实了磷蛋白。 α-Syn 积累分别导致 16 和 12 蛋白质的差异磷酸化和糖基化，其身份通过质谱法揭示。这些蛋白质，包括 HSP90，具有多种生物学功能，包括蛋白质折叠、信号转导、蛋白质降解和细胞骨架调节。重要的是，积累具有不同结合硫黄素S能力的α-Syn组装体的细胞在磷酸化和糖基化方面表现出不同的变化。与基于细胞的研究一致，我们证明与正常对照相比，帕金森病受试者黑质中磷酸化 HSP90 α/β 的水平降低。总之，结果表明 α-Syn 积累会引起复杂的细胞反应，如果持续存在可能会损害细胞活力。
A tetracycline inducible transfectant cell line (3D5) capable of producing soluble and sarkosyl-insoluble assemblies of wild-type human alpha-synuclein (α-Syn) upon differentiation with retinoic acid was used to study the impact of α-Syn accumulation on protein phosphorylation and glycosylation. Soluble proteins from 3D5 cells, with or without the induced α-Syn expression were analyzed by two-dimensional gel electrophoresis and staining of gels with dyes that bind to proteins (Sypro ruby), phosphoproteins (Pro-Q diamond) and glycoproteins (Pro-Q emerald). Phosphoproteins were further confirmed by binding to immobilized metal ion affinity column. α-Syn accumulation caused differential phosphorylation and glycosylation of 16 and 12, proteins, respectively, whose identity was revealed by mass spectrometry. These proteins, including HSP90, have diverse biological functions including protein folding, signal transduction, protein degradation and cytoskeletal regulation. Importantly, cells accumulating α-Syn assemblies with different abilities to bind thioflavin S displayed different changes in phosphorylation and glycosylation. Consistent with the cell-based studies, we demonstrated a reduced level of phosphorylated HSP90 α/β in the substantia nigra of subjects with Parkinson’s disease as compared to normal controls. Together, the results indicate that α-Syn accumulation causes complex cellular responses, which if persist may compromise cell viability.
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DOI：
10.1083/jcb.110.2.309
发表时间：
1990-02
期刊：
The Journal of cell biology
影响因子：
--
作者：
Brada D;Kerjaschki D;Roth J
通讯作者：
Roth J
影响因子：
4.2
作者：
Sáez-Valero, J;Fodero, LR;Small, DH
通讯作者：
Small, DH
DOI：
10.1093/jnen/61.3.245
发表时间：
2002-03-01
影响因子：
3.2
作者：
Kawamoto, Y;Akiguchi, I;Budka, H
通讯作者：
Budka, H
影响因子：
21.3
作者：
Fujiwara, H;Hasegawa, M;Iwatsubo, T
通讯作者：
Iwatsubo, T
影响因子：
4.3
作者：
Arendt, CW;Ostergaard, HL
通讯作者：
Ostergaard, HL