Polyubiquitin Linkage Profiles in Three Models of Proteolytic Stress Suggest the Etiology of Alzheimer Disease

• Type of Material: Article
• Authors: Eric B Dammer, Emory University; Chan Hyun Na, Emory University; Ping Xu, Emory University; Nicholas Seyfried, Emory University; Duc Duong, Emory University; Dongmei Cheng, Emory University; Marla Gearing, Emory University; Howard D Rees III, Emory University; James J Lah, Emory University; Allan I Levey, Emory University; John Rush, Cell Signaling Technology, Inc.; Junmin Peng, Emory University
• Language: English
• Date: 2011-03-25
• Publisher: American Society for Biochemistry and Molecular Biology
• Publication Version: Final Published Version
• Copyright Statement: © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.
• Final Published Version (URL): http://www.jbc.org/content/286/12/10457
• Title of Journal or Parent Work: Journal of Biological Chemistry
• ISSN: 0021-9258
• Volume: 286
• Issue: 12
• Start Page: 10457
• End Page: 10465
• Grant/Funding Information: This work was also supported by Research Scholar Grant RSG-09-181 from the American Cancer Society.; This work was supported, in whole or in part, by National Institutes of Health Grants AG025688, NS055077, and RR025822 and Training Grant NS007480 (to E. B. D. and N. T. S.).
• Keywords: Alzheimer Disease; Lysosomes; Proteomics; Autophagy; Proteasome; Mass Spectrometry (MS); Ubiquitin; Parkinson Disease
• Research Categories: Biology, Neuroscience; Chemistry, Biochemistry; Biology, Genetics

Abstract

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% (Lys48), 38% (Lys63), 8% (Lys29), 2% (Lys11), and 0.5% or less for linear, Lys6, Lys27, and Lys33 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 Lys6, Lys11, Lys27, Lys29, and Lys48 mediate proteasomal degradation, whereas Lys63 chains are preferentially involved in the lysosomal pathway. Mixed linkages, including Lys48, may also contribute to lysosomal targeting, as both Lys63 and Lys48 linkages are colocalized in LC3-labeled autophagosomes. Interestingly, heat shock treatment augments Lys11, Lys48, and Lys63 but not Lys29 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.