Identities of sequestered proteins in aggregates from cells with induced polyglutamine expression

Abstract

Proteins with expanded polyglutamine (polyQ) tracts have been linked to neurodegenerative diseases. One common characteristic of expanded-polyQ expression is the formation of intracellular aggregates (IAs). IAs purified from polyQ-expressing cells were dissociated and studied by protein blot assay and mass spectrometry to determine the identity, condition, and relative level of several proteins sequestered within aggregates. Most of the sequestered proteins comigrated with bands from control extracts, indicating that the sequestered proteins were intact and not irreversibly bound to the polyQ polymer. Among the proteins found sequestered at relatively high levels in purified IAs were ubiquitin, the cell cycle-regulating proteins p53 and mdm-2, HSP70, the global transcriptional regulator Tata-binding protein/TFIID, cytoskeleton proteins actin and 68-kD neurofilament, and proteins of the nuclear pore complex. These data reveal that IAs are highly complex structures with a multiplicity of contributing proteins.

Figure 1

PolyQ–GFP reporter constructs transiently and regulatably expressed in HEK293 cells. (a, left) Schematic of polyQ–GFP fusion proteins indicating Htt exon I–derived sequences (white), SV 40 NLS (gray), and eGFP sequences (black). (a, right) Schematic of CVBE and LPR Moloney murine leukemia virus–based retroviral vectors used in production of inducible polyQ cell lines. CVBE encodes flanking long terminal repeats (LTRs), a G418-resistance gene (G418), the immediate–early CMV promoter (CMV), and the VBE transactivator protein (VBE). LPR encodes long term repeats, a puromycin-resistance gene (Puro), a minimal CMV promoter with six tandem ecdysone response elements (RE), and a SfiI–PmeI polylinker for insertion of the polyQ transgenes. (b–f) Propidium iodide nuclear-stained (red) HEK293 cells transiently transfected with the expression construct 13Q–GFP (b), 13QN–GFP (c), 96Q–GFP (d), and 96QN–GFP (e). GFP produces the bright green fluorescence. (f) IAs in transfected cells revealing stellate fibrous appearance of IAs at high magnification. 72-h vehicle- (g) or ligand-treated (h) 13QN cells stained with the nuclear stain DAPI (red), vehicle- (i) or ligand-treated (j) 96Q cells, and vehicle- (k) or ligand-treated (l) 96QN cells. Bright GFP-positive profiles indicate IA formation. (m) Western blot analysis of induced 13QN (left) or 96QN (right) cells. Numbers at the top of each blot indicate the number of days of induction. WELL, bottom of the well; RES, bottom of the stacking gel and the beginning of the resolving gel. Bar: (b–e) 125 μm; (f) 20 μm; (g–l) 250 μm.

Figure 2

Morphological changes and cell death in induced polyQ-expressing cells. (a–d) 5-d induced 96QN cells matched fluorescence (a and c) and phase–contrast images (b and d), revealing multinucleated syncitia-like cell structures. In a and c, nuclei are red, and polyQ–GFP IAs are green. In b and d, red arrowheads indicate the syncitia boundaries. (e) Quantification of the number of syncitia-like structures/10³ cells in a typical experiment in each induced cell population at 0, 3, and 5 d. Numbers indicate fold change relative to day 0. (f–i) TUNEL positivity in induced 96QN cells. (f) Fluorescent view; (g) bright-field view reveling DAB-stained TUNEL-positive profiles; (h) overlay of both views; and (i) corresponding phase–contrast view. Arrows in panel g indicate TUNEL profiles that were scored as positive for quantification. Overlay and phase–contrast views reveal that the TUNEL positive cells are often rounded up and resting just above the monolayer of surviving cells below. (j) Quantification of the number of TUNEL-positive profiles in a typical experiment in each induced cell population at 0, 3, and 5 d. Numbers indicate fold change relative to day 0. The slight increase in apoptotic cells at 3 and 5 d for 13QN and 96Q cells is probably the consequence of high cell density near the end of the culture period. Bar: (a–d) 50 μm; (f–i) 250 μm.

Figure 4

Protein and Western blot analyses of sequestered protein in concentrated IAs. (a) Coomassie-stained protein gel of isolated IAs revealing multiple bands from ∼10 kD (dye) up to the boundary of the stacking/resolving gel (stack). The four predominant bands used for MALDI analysis are labeled at the right. Lane organization is as described in the legend to Fig. 3. (b) Protein blot analysis of 68-kD neurofilament light polypeptide; (c) protein blot analysis of actin; (d) protein blot analysis with monoclonal antibody 414–detecting putative NPCP proteins Nup62, Nup153, Nup214, and Nup358 as labeled; (e) protein blot analysis of lamin revealing no detectable sequestration in purified IAs.

Figure 3

Western blot analysis of protein sequestration in purified IAs and control whole cell extracts. For all protein blots: lane 1, whole cell extracts from 5-d induced 13QN cells; lane 2, uninduced 96QN cells; lane 3, 5-d induced 96QN cells; and lane 4, isolated concentrated aggregates. Arrowheads at the right of autoradiograms indicate predicted molecular weights of individual protein species unless otherwise specified. (a) Processing for polyQ–GFP immunoreactivity. 96-P indicates the position of polyQN–GFP polymers within the stacking gel and near the top of the resolving gel (lanes 3 and 4), 96-M indicates a low level of 96QN monomers (lanes 2–4), and 13-M indicates the 13QN monomer band (lane 1). This autoradiogram was overexposed to reveal the 96QN monomer bands in lanes 3 and 4. (b) Ubiquitin; (c) HSP70; (d) MEF-2a; (e) Htt; (f) TBP; (g) Nck; (h) GRB-2; (i) caspase-9; (j) caspase-8 (putative procaspase-8, upper arrowhead; putative caspase-8 cleavage products, lower arrowheads); (k) caspase-3; (l) p53 and p50 immunoreactive species (upper and lower arrowheads, respectively); (m) mdm-2 antibody-1 (120-kD variant, upper arrowhead; p60 variant, lower arrowhead); and (n) mdm-2 antibody-2 immunoreactivity.

Figure 5

Immunohistochemical analyses of sequestered proteins in 96QN cells and human HD striatum. (a) Colocalization of ubiquitin (red) with IAs (green) resulting in uniform yellow color of all IAs in transfected 96Q cells. Nuclei are in blue. Arrows indicate colocalizing IAs. (b) Colocalization of HSP70 with IAs. Arrows indicate some colocalizing IAs. (c) Uninduced 96QN cells with infrequent intense TBP positivity (red) compared with 5-d induced 96QN cells (d) with frequent intensely TBP-positive cells. 96QN–GFP IAs are green. (e) Ubiquitin-positive profiles in the non-HD control striatum and in the HD striatum (f). Arrows indicate punctate ubiquitin-positive profiles. (g–i) Localization of TBP within punctate profiles in control striatum (g), HD striatum (h), and HD striatum after preabsorbtion with peptide (i). Arrows indicate TBP-positive profiles in HD tissue. (j–m) High magnification of TBP-positive profiles in HD striatum revealing immunoreactivity in a diffuse sphere (j), a dark intense sphere (k), or punctate profiles (l and m) with lighter surrounding immunopositivity.

Figure 6

Immunohistochemical fluorescent confocal microscopy of protein species predicted by MALDI analysis to associate with IAs from 96QN cells. Green fluorescence indicates the 96QN–GFP proteins and IAs, blue indicates the DNA stain DAPI, and red CY-3 fluorophore is used for each individual protein tested for localization. Yellow color indicates colocalization of IAs with the test protein antibody. (a) Immunostaining for NFL on 5-d induced 96QN cells. (b) Immunostaining for actin on 5-d induced 96QN cells. (c) Staining of 96QN cells with TRITC–phalloidin. (d and e) Magnification of a region from panel c with separation of the color channels to highlight colocalization by comparison of the red actin channel in panel d with matching signal in of 96QN–GFP green fluorescence in panel e. Arrows indicate some of the colocalizing IAs. (f) Staining of 96QN cells with monoclonal antibody 414 against NPCPs. Bright yellow signal indicates colocalization of NPCP signal with a subset of IAs. (g and h) Individual color channels to highlight colocalization of NPCPs with IAs. White arrows indicate colocalization of spherical monoclonal antibody 414 staining with a subset of IAs, blue arrows indicate areas of NPCP density on the nuclear envelope, and orange arrows indicate colocalization of 96QN–GFP with these perinuclear densities.