Purification of polyglutamine aggregates and identification of elongation factor-1alpha and heat shock protein 84 as aggregate-interacting proteins

Abstract

Aggregates of green fluorescent protein (GFP)-fused truncated N-terminal huntingtin containing abnormally long polyglutamine tracts (150 repeats of glutamine residue) were purified from an ecdysone-inducible mutant neuro2A cell line (HD150Q-28) by using a fluorescence-activated cell sorter. To analyze the aggregate-interacting proteins, we subjected the purified aggregates to SDS-PAGE; prominent protein bands in the gel were digested with Achromobactor lysyl endopeptidase, followed by a HPLC-mass spectrometry (MS) analysis. The resulting data of tandem MS analysis revealed that, in addition to ubiquitin and widely reported chaperone proteins such as heat shock cognate 70 (HSC70), human DNA J-1 (HDJ-1), and HDJ-2, the translational elongation factor-1alpha (EF-1alpha) and heat shock protein 84 (HSP84) also were recognized as aggregate-interacting proteins. Sequestration of these proteins to aggregates was confirmed further by several immunochemical methods. We confirmed that, in addition to the other known proteins, EF-1alpha and HSP84 also colocalized with the intracellular aggregates. An assay of the transient expression of EF-1alpha and HSP84 in HD150Q-28 cells revealed that both proteins improved cell viability. Moreover, the rate of aggregate formation decreased in both transfectants. Our study suggests that both EF-1alpha and HSP84 are involved in the neurodegenerative process of polyglutamine diseases.

Fig. 1.

Expression of tNhtt-16Q-GFP and tNhtt-150Q-GFP in differentiated N2A cells. HD16Q-23 (a, b) and HD150Q-28 (c, d) were treated with db-cAMP and ponasterone A. After 2 d of culture their morphology and tNhtt-GFP expressions were observed in phase contrast (a, c) and corresponding fluorescence (b, d) images. Scale bars, 20 μm.

Fig. 2.

Purification of tNhtt-150Q-GFP aggregates with a cell sorter. a, Sorting profiles of aggregates from HD150Q-28 lysate are shown in a scattergram [left; forward scattering (FS) vs side scattering (SS)] and in a histogram for the fluorescence at 535 nm (right). Aggregates with bright fluorescence are observed mostly in the particles within Region A of theleft panel. Numbers in theleft and right panels are the relative frequency of particles in Region A against the total particles and the frequency of bright particles in Region A, respectively. b, Results of the reanalysis of the sorted aggregates. Note that sorted particles are homogeneous with respect to the forward scattering, side scattering, and the fluorescence at 535 nm. c, Microscopic observation of purified aggregates presented by phase contrast image (left) and corresponding fluorescence image (right). Scale bars, 5 μm.

Fig. 3.

SDS-PAGE of purified aggregates and protein assignment to the CBB-stained bands. Approximately 17,000 purified aggregates were subjected to SDS-PAGE. Coomassie blue-stained bands in the gel were excised, followed by in-gel digestion with LysC. HPLC-MS/MS analysis of the enzyme-digested gel extracts assigned the indicated proteins to the corresponding bands. The white arrowheads with question marks indicate the protein bands that were applied to the HPLC-MS analysis but that could not be identified in this study.

Fig. 4.

Western blot analysis of AIP candidates. HD150Q-28 cell lysate (a) and purified aggregates (b) were subjected to SDS-PAGE, followed by Western blotting probed with antibodies against the proteins that are indicated on the top of each panel. HRP-conjugated secondary antibodies were used, and the signal was detected by ECL.

Fig. 5.

Immunocytochemical analysis of AIP candidates in HD150Q-28 cells. Cells of 2 d in culture after differentiation and tNhtt-150Q-GFP induction were used. Fixed cells were incubated with antibodies against the proteins that are indicated at theleft and then were labeled with Alexa Fluor 546-labeled anti-rabbit or anti-mouse secondary antibodies. Colocalization (a) and uncolocalization (b) of AIP candidates with aggregates are presented. Left column, Fluorescence of aggregates of tNhtt-150Q-GFP. Middle column, Alexa Fluor 546-labeled proteins. Right column, Merged images of the two signals. Scale bars, 20 μm.

Fig. 6.

Immunogold labeling of AIP candidates on purified aggregates. The ultrastructure of purified aggregates was observed by electron microscope (a). For immunogold labeling the aggregates were treated with antibodies against HDJ-2 (b), EF-1α (c), HSP84 (d), actin (e), and tubulin (f), followed by probing with immunogold-conjugated anti-IgG antibodies. Black dots on the aggregate fibers represent the labeled gold particles. Scale bars, 30 nm.

Fig. 7.

Immunohistochemical analysis of aggregate-interacting candidates in an R6/2 transgenic mouse brain. Frozen brain sections of an R6/2 transgenic mouse were double labeled with anti-ubiquitin antibody, together with the antibodies against the indicated proteins at the left. Anti-ubiquitin antibody was probed with Alexa Fluor 546-labeled secondary antibody, and the antibodies against the indicated proteins were probed with Alexa Fluor 488-labeled secondary antibody. Left column, The Alexa Fluor 488-labeled proteins. Middle column, The localization of Alexa Fluor 546-labeled ubiquitin. Right column, The merged images. Scale bars, 20 μm.

Fig. 8.

Reduction of polyQ-mediated cellular toxicity in HSP84- and EF-1α-overexpressing mutant N2A cells. a, HD150Q-28 cells were transfected with expression plasmids encoding LacZ (control), HDJ-1, EF-1α, or HSP84. After 24 hr the cells were replated to the 48-well plates at a density of 5 × 10⁴/well and then differentiated by the addition of 5 mm db-cAMP (light gray bars), or the cells were differentiated and induced tNhtt-150Q-GFP by 5 mmdb-cAMP plus 1 μm ponasterone A (dark gray bars). Cell viability on the fourth day of induction was measured by MTT assay and presented as a percentage of differentiated-only LacZ-overexpressing control (indicated asCtrl). Values are the means ± SEM;n = 12. *p < 0.01, compared by Student's t test with a ponasterone A-treated LacZ-overexpressing experiment. b, Expressions of LacZ (lane 1), HDJ-1 (lane 2), EF-1α (lane 3), and HSP84 (lane 4) in the transfected HD150Q-28 cells were confirmed by Western blotting at 2 d after the treatment with db-cAMP and ponasterone A. Bands were detected with anti-v5 antibody. c, Western blot analysis to check the effect of transient overexpression of HDJ-1, EF-1α, and HSP84 on the expression of tNhtt-polyQ-GFP from differentiated and induced HD150Q-28 and HD16Q-23 cell lines. The expression level of tNhtt-polyQ-GFP was detected by anti-N-terminal huntingtin antibody, showing no differences among those transfected cells.

Fig. 9.

Effect of EF-1α and HSP84 on aggregate formation. a, HD150Q-28 cells overexpressing LacZ, HDJ-1, EF-1α, and HSP84, followed by differentiation and induction with 5 mm db-cAMP plus 0.1 μm ponasterone A, were probed with anti-v5 antibody and stained with Alexa Fluor 546-labeled secondary antibody. The panels show the typical immunofluorescent images. Red, green, andyellow indicate v5, tNhtt-150Q-GFP, and the coexistence of both molecules, respectively. b, The frequency of aggregate formation in the v5-positive cells was measured by counting the numbers of aggregate-positive cells. The data are presented as the percentage of aggregate and v5 double-positive cells in total v5-positive cells. Values are the means ± SEM;n = 10. *p < 0.01, compared by Student's t test with a control experiment that used LacZ-expressing cells. c, Filter retardation assay for comparing aggregate formation in control (LacZ-), HDJ-1-, EF-1α-, and HSP84-overexpressing cells. The aggregated form of tNhtt-150Q-GFP in the cells was trapped on the cellulose acetate membrane; the tNhtt-immunoreactive spots were detected (top), and the density was quantified (bottom) as described in Materials and Methods. The significant reduction of SDS insoluble aggregates was observed in HDJ-1-, EF-1α-, and HSP84-overexpressing cells. Values are the means ± SEM; n = 4. *p < 0.01, compared by Student's ttest with a control experiment that used LacZ-expressing cells.