Formation of morphologically similar globular aggregates from diverse aggregation-prone proteins in mammalian cells

Abstract

Huntington's disease is a progressive neurodegenerative disorder caused by a polyglutamine repeat expansion in the first exon of the huntingtin (Htt) protein. N-terminal Htt peptides with polyglutamine tracts in the pathological range (51-122 glutamines) form high-molecular-weight protein aggregates with fibrillar morphology in vitro, and they form discrete inclusion bodies in a cell-culture model. However, in some studies, formation of discrete Htt inclusions does not correlate well with cell death. We coexpressed N-terminal Htt fragments containing 91 glutamines fused to different affinity tags in HEK293 cells, and we isolated small aggregates by double sequential-affinity chromatography to assure the isolation of multimeric molecules. Transmission electron microscopy and atomic force microscopy revealed the isolated aggregates as globules or clusters of globules 4-50 nm in diameter without any detectable fibrillar species. Because small nonfibrillar oligomers, not mature fibrils, recently have been suggested to be the principal cytotoxic species in neurodegenerative disease, these Htt globular aggregates formed in cells may represent the pathogenic form of mutant Htt.

Fig. 1.

Isolation of HDQ91 aggregates under nondenaturing conditions. (a) Constructs. The GFP tag is indicated by a black box, and affinity tags (protein A and CBP) for isolation of HDQ91 aggregates are indicated by gray boxes. Q91, Htt exon 1 (3) protein with 91 glutamine repeats; TEV, TEV protease recognition sequence. (b) Diagram showing the isolation procedure for HDQ91 aggregates under nondenaturing conditions. (c) Immunoblotting (IB) of column fractions was performed using anti-GFP antibody (JL-8). Molecular mass markers are indicated in kDa at the left. The arrowhead indicates the bottom of the loading wells. (d) Silver staining of column fractions. The same amount of each indicated column fraction from c was applied to SDS/PAGE, and silver stained. (e) Fluorescence microscopy of column fractions. The individual fractions are as indicated in b. (Scale bar, 10 μm.)

Fig. 2.

Specificity of protein aggregation isolated by double-affinity method. The column fractions from a mixture of cells transfected with pQ91/GFP and pQ91/ProtA and cells transfected with only pQ91/CBP was immunoblotted by using anti-GFP antibody (JL-8). Molecular mass markers (in kDa) are indicated on the left. The naming of the individual fractions is the same as in Fig. 1b. The arrowhead indicates the bottom of loading wells.

Fig. 3.

Structure of isolated HDQ91 aggregates. (a and b) TEM of uranyl acetate-stained immunogold-labeled HDQ91 aggregates. The size of the gold particles was 10 nm. (Scale bar, 100 nm.) (c) AFM height image of HDQ91 aggregates. (d) Magnified image of the boxed area in c. (e and f) The AFM surface profile along A–B and C–D axes in d, respectively. The arrows in d indicate the globular aggregates, corresponding to the arrows in e and f.

Fig. 4.

Isolation of HDQ91 aggregates in the presence of urea. (a) Constructs. Affinity tags [(His)₆ tag and S·tag] for isolation of HDQ91 aggregates are indicated by gray boxes. Q91, Htt exon 1 (11) protein with 91 glutamine repeats; myc, myc tag; HA, HA tag; FLAG, FLAG tag. (b) Diagram showing the isolation step of HDQ91 aggregates in the presence of urea. (c) Immunoblotting of column fractions was performed by using anti-FLAG (M2) antibody. The individual fractions are as indicated in b. Molecular mass markers (in kDa) are indicated on the left. (d) TEM and AFM of HDQ91 aggregates isolated in the presence of urea. The size of the gold particles was 10 nm. (Scale bar, 100 nm.) The composition of the aggregates is indicated on the left.

Fig. 5.

Isolation of CFTRΔF508 aggregates under nondenaturing conditions. (a) Constructs. GFP tag is indicated by black box, and affinity tags (protein A and CBP) for isolation of CFTRΔF508 aggregates are indicated by gray boxes. TEV, TEV protease recognition sequence. (b) Immunoblotting of column fractions was performed by using anti-GFP antibody (JL-8). The individual fractions are designated by the convention introduced in Fig. 1b. Molecular mass markers (in kDa) are indicated on the left. (c and d) TEM of uranyl acetate-stained immunogold-labeled CFTRΔF508 aggregates. The size of the gold particles was 10 nm. (Scale bar, 100 nm.) (e–h) The AFM height images of CFTRΔF508 aggregates (e and f) and the surface profiles (g and h) along the A–B (e and g) and C–D (f and h) axes, respectively. The arrows in e indicate the globular aggregates, corresponding to the arrows in g. The arrows in f indicate the globular aggregates, corresponding to the arrows in h.