Polyubiquitin Linkage Profiles in Three Models of Proteolytic Stress Suggest the Etiology of Alzheimer Disease
Polyubiquitin Linkage Profiles in Three Models of Proteolytic Stress Suggest the Etiology of Alzheimer Disease
复制标题
DOI：
10.1074/jbc.m110.149633
复制
发表时间：
2011-03-25
影响因子：
4.8
通讯作者：
Peng, Junmin
中科院分区：
文献类型：
--
作者：
Dammer, Eric B.;Na, Chan Hyun;Peng, Junmin
底物上的多聚泛素链通过7个赖氨酸残基或泛素（Ub）的N末端中的任何一个组装，在链结构中产生不同的连接。PolyUb连接调节修饰底物的命运，但它们在哺乳动物细胞中的丰度和功能尚未得到充分研究。我们提出了一种基于质谱的方法来直接从哺乳动物细胞的总裂解物中测量polyUb连接。在HEK293细胞中，发现polyUb键的水平为52%（Lys（48））、38%（Lys（63））、8%（Lys（29））、2%（Lys（11）），线性、Lys（6）、Lys（27）和Lys（33）键的polyUb键的水平为0.5%或更低。在重稳定同位素完全标记的小鼠（即SILAC小鼠）中检查这些连接的组织特异性。此外，我们分析了阿尔茨海默病患者的脑组织中的Ub连接，有或没有并发路易体病，以及蛋白水解应激的三种细胞模型：蛋白酶体缺乏症，溶酶体缺乏症和热休克。数据支持通过Lys（6）、Lys（11）、Lys（27）、Lys（29）和Lys（48）连接的polyUb链介导蛋白酶体降解，而Lys（63）链优先参与溶酶体途径。混合键，包括赖氨酸（48），也可能有助于溶酶体靶向，因为赖氨酸（63）和赖氨酸（48）键在LC 3标记的自噬体中共定位。有趣的是，热休克处理增加了赖氨酸（11），赖氨酸（48）和赖氨酸（63），但没有赖氨酸（29）的联系，这种独特的模式是类似的，在轮廓神经退行性疾病的情况下。我们的结论是，不同的polyUb连接发挥不同的作用下，三个蛋白水解应激条件下，和热休克反应途径中的蛋白质折叠能力可能会受到更大的影响，在阿尔茨海默病。
Polyubiquitin chains on substrates are assembled through any of seven lysine residues or the N terminus of ubiquitin (Ub), generating diverse linkages in the chain structure. PolyUb linkages regulate the fate of modified substrates, but their abundance and function in mammalian cells are not well studied. We present a mass spectrometry-based method to measure polyUb linkages directly from total lysate of mammalian cells. In HEK293 cells, the level of polyUb linkages was found to be 52% (Lys(48)), 38% (Lys(63)), 8% (Lys(29)), 2% (Lys(11)), and 0.5% or less for linear, Lys(6), Lys(27), and Lys(33) linkages. Tissue specificity of these linkages was examined in mice fully labeled by heavy stable isotopes (i.e. SILAC mice). Moreover, we profiled the Ub linkages in brain tissues from patients of Alzheimer disease with or without concurrent Lewy body disease as well as three cellular models of proteolytic stress: proteasome deficiency, lysosome deficiency, and heat shock. The data support that polyUb chains linked through Lys(6), Lys(11), Lys(27), Lys(29), and Lys(48) mediate proteasomal degradation, whereas Lys(63) chains are preferentially involved in the lysosomal pathway. Mixed linkages, including Lys(48), may also contribute to lysosomal targeting, as both Lys(63) and Lys(48) linkages are colocalized in LC3-labeled autophagosomes. Interestingly, heat shock treatment augments Lys(11), Lys(48), and Lys(63) but not Lys(29) linkages, and this unique pattern is similar to that in the profiled neurodegenerative cases. We conclude that different polyUb linkages play distinct roles under the three proteolytic stress conditions, and protein folding capacity in the heat shock responsive pathway might be more affected in Alzheimer disease.