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. 2011 Mar 25;286(12):10457-65.
doi: 10.1074/jbc.M110.149633. Epub 2011 Jan 28.

Polyubiquitin linkage profiles in three models of proteolytic stress suggest the etiology of Alzheimer disease

Eric B Dammer  1 , Chan Hyun Na, Ping Xu, Nicholas T Seyfried, Duc M Duong, Dongmei Cheng, Marla Gearing, Howard Rees, James J Lah, Allan I Levey, John Rush, Junmin Peng
Affiliations

Polyubiquitin linkage profiles in three models of proteolytic stress suggest the etiology of Alzheimer disease

Eric B Dammer et al. J Biol Chem. .

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% (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.

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Figures

FIGURE 1.
FIGURE 1.
Mass spectrometry enables measurement of ubiquitin linkages in mammalian cells. A, workflow for LC-MS quantification using stable isotope-labeled proteins or peptides. After digestion, the labeled peptides and their unlabeled counterparts were co-eluted in reverse phase LC followed by MS analysis in the mode of SRM, also termed multiple reaction monitoring (MRM). SRM/multiple reaction monitoring settings monitor specific product ions for known precursor ions, where the light/heavy intensity ratio of product ions is a surrogate for relative peptide abundance in the mixture. B, quantification of total Ub and the level of conjugation in HEK293 cells. C, Western blotting shows that the majority of Ub conjugates was above 80 kDa, which were excised and further quantified by MS. Note that a direct comparison of the abundance of polyUb to monoUb using using Western blotting is not possible, as it is known that polyUb is preferentially detected and usually generates much stronger signals than monoUb during Western blotting. This may be due to the difference in epitope exposure because the ubiquitin monomer is a highly stable protein that readily refolds. The percentage calculation of different linkages was based on the absolute quantification using synthetic GG-tagged peptides as a reference. D, a pair of Lys11-specific product ions was monitored from HEK293 cell lysate. E, coelution of the light and heavy ion pairs of the Lys11 linkage peptide at a precise retention time during reverse phase LC. Retention time is strictly used to distinguish real native Lys11 peptide signal from noise signals (e.g. the small peak eluted at ∼39 min). The extracted ion currents in the LC-SRM ranged from targeted MS/MS ion monoisotopic m/z −0.5 unit to plus 1.5 unit (see the targeted ions in supplemental Table S2). F, light/heavy ion pairing for Lys29 linkages. G, matched column retention time for the Lys29 peptide pair.
FIGURE 2.
FIGURE 2.
PolyUb linkage profiles of mouse tissues. A, Coomassie Blue stained gel shows total protein of five tissue homogenates (15 μg) that were fully labeled by stable isotope. B, relative abundance of polyUb linkages and the proteasome subunit Rpn2, based on the relative intensity of conserved peptide pairs from unlabeled HEK293 lysate and labeled mouse samples by LC-MS analysis. The experiments were repeated once. All data are normalized according to the abundance of each peptide in HEK293 total cell lysate and is shown as mean and S.E. The level of Lys11 linkages in muscle was below the detection limit. The level of unmodified ubiquitin TLS peptide (supplemental Table S2, roughly reflecting the sum of ubiquitin in the region larger than the 80-kDa region) is 52, 59, 48, 69, and 41% in lung, cortex, muscle, liver, and kidney, respectively, compared with HEK293 cells (100%). Statistical significance (asterisk) was calculated between HEK293 cells and different tissues using Student's t test (p < 0.05).
FIGURE 3.
FIGURE 3.
Lys11, Lys48, and Lys63, but not Lys29 linkages accumulate in frontal cortex of AD or AD/LBD specimens. A–H, pathological staining of α-synuclein (Lewy body), amyloid β peptide (plaques), Tau (neurofibrillary tangles), and Lys63-linked polyUb in control and AD/LBD cases, respectively. Wide-field images were captured with a 10× objective, except a 40× objective was used for D and H. Scale bar is 150 μm for all panels but D and H (20 μm). I, elevated level of Ub in AD/LBD cases in Western blotting. J, analysis of four abundant polyUb linkages in total frontal cortex lysate of the matched control (n = 12), pure AD (n = 12), and AD/LBD cases (n = 12) by LC-MS. The asterisk indicates p value <0.05 according to Student's t test.
FIGURE 4.
FIGURE 4.
Proteasome deficiency increases all detected polyUb linkages, but Lys63 shows a delayed response. A, Western blot of Ub when HEK293 cells were treated with 10 μm epoxomicin. B, Ub linkage profile in a time course during epoxomicin treatment. The signal of Lys6 linkages was weak compared with others and might be subject to more variation. Rpn2 and E1 were also quantified as two internal loading controls. C, Ub linkage profile in cells treated with 10 μm MG-132. D, PolyUb linkage profile using mouse primary neuronal culture.
FIGURE 5.
FIGURE 5.
Lys48 and Lys63 linkages accumulate in inclusions (or inclusion-like structures) after proteasome inhibition. A, Lys48 polyUb was imaged by immunofluorescent staining at 0, 4, and 10 h of MG-132 treatment (10 μm). B, Lys63 polyUb (green) was analyzed together with LC3 (red) under the same conditions. The scale bars represent a distance of 10 μm.
FIGURE 6.
FIGURE 6.
Lysosomal deficiency increases Lys63 linkages and other polyUb linkages. A, Ub linkage profile of bafilomycin A1-treated (200 nm) HEK293 cells, shown as mean and S.E. Statistical significance (asterisk) was analyzed between the untreated samples and other samples based on Student's t test (p < 0.05). B, Ub linkage profile of bafilomycin A1-treated primary neurons in a similar time course. C, immunofluorescence of Lys48, Lys63 polyUb chains, and LC3 with varying bafilomycin A1 (200 nm) treatment time. The nuclei are shown by DAPI staining. Lysosome inhibition appeared to increase the size of LC3/Lys48/Lys63-positive structures in HEK293 cells over time. The scale bars represent a distance of 10 μm.
FIGURE 7.
FIGURE 7.
PolyUb linkage profiles during heat shock response in a cellular proteolytic stress model. A, the diagram of three types of treatment in a time course. HEK293 cells were treated by 6-AN, riluzole, or heat shock alone or were pretreated with 6-AN or riluzole prior to heat shock to increase the heat shock response capacity of cells. B, radioimmune precipitation assay buffer soluble and insoluble fractions for the six treatments shown as mean and S.E. Statistical significance (asterisk) was analyzed between the untreated samples and other samples based on Student's t test (p < 0.05). The protein concentrations were quantified to determine the percentage partitioning into the insoluble fraction from HEK293 cells. C, polyUb linkage profile of the total cell lysate. Statistical significance (asterisk) between heat shock treatment and the other two combined treatments (heat shock with 6-AN or riluzole) was based on the analysis of Student's t test (p < 0.05).
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