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

Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 03/2011; 286(12):10457-65. DOI: 10.1074/jbc.M110.149633
Source: PubMed


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.

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Available from: Eric B Dammer, Oct 02, 2015
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    • "Are Captured by TRABID K29 linkages can be detected in proteomic studies (Dammer et al., 2011). Therefore, we hypothesized that the selective recognition of these linkages by TRABID could be exploited to capture K29 chains from cells. "
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    ABSTRACT: Polyubiquitin chains regulate diverse cellular processes through the ability of ubiquitin to form chains of eight different linkage types. Although detected in yeast and mammals, little is known about K29-linked polyubiquitin. Here we report the generation of K29 chains in vitro using a ubiquitin chain-editing complex consisting of the HECT E3 ligase UBE3C and the deubiquitinase vOTU. We determined the crystal structure of K29-linked diubiquitin, which adopts an extended conformation with the hydrophobic patches on both ubiquitin moieties exposed and available for binding. Indeed, the crystal structure of the NZF1 domain of TRABID in complex with K29 chains reveals a binding mode that involves the hydrophobic patch on only one of the ubiquitin moieties and exploits the flexibility of K29 chains to achieve linkage selective binding. Further, we establish methods to study K29-linked polyubiquitin and find that K29 linkages exist in cells within mixed or branched chains containing other linkages. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Molecular cell 03/2015; 58(1). DOI:10.1016/j.molcel.2015.01.041 · 14.02 Impact Factor
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    • "Chains that are linked through K48 are the principal signal for degradation by the proteasome [30] [31]. Recent studies, based on mass spectrometry have shown that homogeneous chains consisting of K29, K11, K27 and K6- linkages, heterogeneous chains with mixed lysine linkages, as well as multiple nearby monoubiquitination and, in cases of substrates up to 150 amino acids, even monoubiquitination can promote proteasomal degradation [32] [33]. Chain elongation of ubiquitinated substrates is mediated via another class of ubiquitin ligases, "
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    ABSTRACT: Posttranslational modification of proteins often controls various aspects of their cellular function. Indeed, over the past decade or so, it has been discovered that posttranslational modification of lysine residues plays a major role in regulating translesion DNA synthesis (TLS) and perhaps the most appreciated lysine modification is that of ubiquitination. Much of the recent interest in ubiquitination stems from the fact that proliferating cell nuclear antigen (PCNA) was previously shown to be specifically ubiquitinated at K164 and that such ubiquitination plays a key role in regulating TLS. In addition, TLS polymerases themselves are now known to be ubiquitinated. In the case of human polymerase η, ubiquitination at four lysine residues in its C-terminus appears to regulate its ability to interact with PCNA and modulate TLS. Within the past few years, advances in global proteomic research have revealed that many proteins involved in TLS are, in fact, subject to a previously underappreciated number of lysine modifications. In this review, we will summarize the known lysine modifications of several key proteins involved in TLS; PCNA and Y-family polymerases η, ι, κ and Rev1 and we will discuss the potential regulatory effects of such modification in controlling TLS in vivo. Copyright © 2015. Published by Elsevier B.V.
    DNA Repair 02/2015; 29. DOI:10.1016/j.dnarep.2015.02.011 · 3.11 Impact Factor
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    • "Moreover, they show that the K11 specificity of the APC/C is not essential for the turnover of anaphase substrates , whereas K48-linked chains are not a prerequisite for substrate targeting to the proteasome. Our observations thus support studies that found most linkages to accumulate in response to proteasome inhibition, indicative of a proteolytic role for the majority of chain topologies (Dammer et al., 2011; Peng et al., 2003; Xu et al., 2009). "
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    ABSTRACT: Posttranslational modification of cell-cycle regulators with ubiquitin chains is essential for eukaryotic cell division. Such chains can be connected through seven lysine residues or the amino terminus of ubiquitin, thereby allowing the assembly of eight homogenous and multiple mixed or branched conjugates. Although functions of homogenous chain types have been described, physiological roles of branched structures are unknown. Here, we report that the anaphase-promoting complex (APC/C) efficiently synthesizes branched conjugates that contain multiple blocks of K11-linked chains. Compared to homogenous chains, the branched conjugates assembled by the APC/C strongly enhance substrate recognition by the proteasome, thereby driving degradation of cell-cycle regulators during early mitosis. Our work, therefore, identifies an enzyme and substrates for modification with branched ubiquitin chains and points to an important role of these conjugates in providing an improved signal for proteasomal degradation.
    Cell 05/2014; 157(4):910-21. DOI:10.1016/j.cell.2014.03.037 · 32.24 Impact Factor
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