Polyalanine expansions drive a shift into α-helical clusters without amyloid-fibril formation

Abstract

Polyglutamine (polyGln) expansions in nine human proteins result in neurological diseases and induce the proteins' tendency to form β-rich amyloid fibrils and intracellular deposits. Less well known are at least nine other human diseases caused by polyalanine (polyAla)-expansion mutations in different proteins. The mechanisms of how polyAla aggregates under physiological conditions remain unclear and controversial. We show here that aggregation of polyAla is mechanistically dissimilar to that of polyGln and hence does not exhibit amyloid kinetics. PolyAla assembled spontaneously into α-helical clusters with diverse oligomeric states. Such clustering was pervasive in cells irrespective of visible aggregate formation, and it disrupted the normal physiological oligomeric state of two human proteins natively containing polyAla: ARX and SOX3. This self-assembly pattern indicates that polyAla expansions chronically disrupt protein behavior by imposing a deranged oligomeric status.

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Figure 1: Distinct aggregation kinetics of recombinant polyAla and polyGln fusions to EGFP.
Figure 2: Expanded polyalanine self-associates via a spontaneous shift to large oligomers rather than through a nucleated mode of recruitment of monomers.
Figure 3: Expanded polyAla assembles into α-helical nonfibrillar aggregate clusters.
Figure 4: Expanded polyAla is pervasively homo-oligomerized in cells regardless of visible aggregation state.
Figure 5: Chronic disruption of SOX3 oligomeric state by polyAla expansion.
Figure 6: Increased granularity of nuclear-localized proteins containing a polyAla-expansion demonstrates an enhanced clustering of SOX3 and ARX in cells lacking visible nuclear aggregates.
Figure 7: Schematic summarizing how extended polyAla sequences drive an abnormally clustered state with multiple effects on structure and function.

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Acknowledgements

This work was funded by the Australian Research Council (Discovery Project Grant DP120102763 (D.M.H.) and Future Fellowships FT120100039 (D.M.H.), FT100100411 (T.B.) and FT100100560 (A.F.H.)) and by the Australian National Health and Medical Research Council (Project Grants APP1049458 (D.M.H.), 628946 (A.F.H.), 465401 (P.Q.T.) and APP1049459 (D.M.H. and T.B.)). The GFP-NLS vector was kindly provided by G. Mosely (University of Melbourne).

Author information

S.P., A.R.O., R.J.W., K.L., C.S., J.N.H., P.Q.T., M.G.W.D., Q.B. and T.B. performed experiments and/or analyzed data. S.P., A.R.O. and R.J.W. wrote parts of the manuscript. D.M.H. oversaw the project, analyzed data and wrote the manuscript. A.F.H., T.B., P.Q.T., C.S. and R.J.W. provided critical feedback and guidance in experimental design.

Correspondence to Danny M Hatters.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Analysis of high-mass aggregates by SVA at low speed (3,000 r.p.m.).

Fusion proteins (5 μM) were cleaved with TEV protease and incubated at 37 °C for the times shown. Samples were diluted to 0.8 μM immediately prior to sedimentation velocity analysis and assessed under the conditions shown on the panels. First scan is shown in red and subsequently scans in incrementing grayscale.

Supplementary Figure 2 Raw sedimentation scans and SVA fits for data in Figure 2.

Proteins were sedimented at 50,000 rpm. The first scan is shown in red with subsequent scans in incrementing grayscale. Fits of the data to the c(s) size distribution are shown in blue.

Supplementary Figure 3 Single-molecule imaging of polyAla-EGFP fusions expressed in AD293 cells.

(a) Images of polyAla-EGFP molecules/oligomers with 7 alanines (left) or 37 alanines (right) adhered non-specifically to a coverslip. Shown are representative images assessed on 3 different coverslips (but from one biological sample). (b) Measurement of the intensity of single EGFP molecules by photobleaching. Left: Example photobleaching traces of single EGFP molecules. The step height corresponds to the intensity of the molecule. Right: Histogram of single EGFP intensities. The mean of the distribution is the mean intensity of an EGFP molecule, which is used as a calibration value for determining the number of molecules per spot in the images above.

Supplementary Figure 4 FRET strategy to probe associations between different polyAla and polyGln lengths.

Data complements Fig 4c by coexpression of the indicated constructs.

Supplementary Figure 5 Effect of polyAla-expansion on cellular location and nuclear granularity of host proteins.

(a). Localization of EGFP-SOX3 co-transfected with mCherry in AD293 cells. Most cells exhibited a nuclear localized pattern; whereas in some cells proteins aggregated in the cytoplasm. (b) PolyAla-fusions to EGFP appended with a nuclear localization tag (NLS) cotransfected with mCherry in AD293 cells. Most cells exhibited a nuclear localized pattern; whereas in some cells proteins aggregated in the cytoplasm. (c) ARX transfected into HEK293T cells (WT, PA1 and PA2 mutants) and detected by immunofluorescence to ARX. Most cells exhibited a nuclear-localized ARX only.

Supplementary Figure 6 Raw scans, fits and analysis of SOX3 SVA experiments.

Data corresponds to data in Fig 5 and represents the fit to one of the 3 transfection replicates. (a) Low-speed sedimentation velocity analysis (SVA) on lysates supplemented with 2 M sucrose. The first scan is shown in red and subsequent scans in incrementing grayscale. Fits of the data to the c(s) size distribution are shown in blue. The orange bracket indicates the proportion of material allocated into the > 1500 S category. (b) Low-speed SVA on lysates without added sucrose. The first scan is shown in red and subsequent scans in incrementing grayscale. The abundance of SOX3 assigned to the 200–1500 S category was determined by the difference in plateau intensities between scan one and the final scan. (c) High-speed SVA data scans. Shown are the data divided into the early time points and later time points of data acquisition, which were fit separately to the c(s) size distribution. The first scan of the “early” subset is shown in red. The first scan of the “later” subset shown in magenta. Subsequent scans of each groups shown in incrementing grayscale, with the fits shown in blue. Abundances of different oligomeric states for the 1–15 S and 15–200 S categories were assessed by areas under the c(s) distribution curves.

Supplementary Figure 7 Images of the in vivo expression patterns of ARX.

Shown are the tiled images used to analyze the data in Fig 6d and show endogenous ARX in magenta (left panel), 4',6-diamidino-2-phenylindole (DAPI) stained nuclei in middle panel with the overlay in the right panel. Immunostaining of the ventrolateral mantle zone of telecephalon slices collected from 12.5 dpc embryo of wildtype (WT), compared to PA1 and PA2 knock-in mice.

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Supplementary Figures 1–7 and Supplementary Table 1 (PDF 919 kb)

Supplementary Data Set 1

Uncropped gel from Figure 1a (PDF 830 kb)

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Polling, S., Ormsby, A., Wood, R. et al. Polyalanine expansions drive a shift into α-helical clusters without amyloid-fibril formation. Nat Struct Mol Biol 22, 1008–1015 (2015). https://doi.org/10.1038/nsmb.3127

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