HSP70-HSP90 chaperone networking in protein-misfolding disease

Molecular chaperones and their associated co-chaperones are essential in health and disease as they are key facilitators of protein-folding, quality control and function. In particular, the heat-shock protein (HSP) 70 and HSP90 molecular chaperone networks have been associated with neurodegenerative diseases caused by aberrant protein-folding. The pathogenesis of these disorders usually includes the formation of deposits of misfolded, aggregated protein. HSP70 and HSP90, plus their co-chaperones, have been recognised as potent modulators of misfolded protein toxicity, inclusion formation and cell survival in cellular and animal models of neurodegenerative disease. Moreover, these chaperone machines function not only in folding but also in proteasome-mediated degradation of neurodegenerative disease proteins. This chapter gives an overview of the HSP70 and HSP90 chaperones, and their respective regulatory co-chaperones, and explores how the HSP70 and HSP90 chaperone systems form a larger functional network and its relevance to counteracting neurodegenerative disease associated with misfolded proteins and disruption of proteostasis

AIPL1, FAT10 and NUB1 form a ternary complex.

<p>Cells were transfected with constructs as indicated followed by immunoprecipitation and immunoblot analysis. (A) AIPL1 and FAT10 both co-precipitate with NUB1 (top panel); NUB1 and FAT10 both co-precipitate with AIPL1 (bottom panel). MG132 increased FAT10 steady-state levels and the amount of co-precipitated AIPL1 (top panel) or NUB1 (bottom panel). (B) NUB1 and AIPL1 both co-precipitate with FAT10. The AIPL1 C239R pathogenic mutant immunoprecipitated FAT10, but did not affect its NUB1-mediated degradation profile. (C) The AIPL1 C239R mutant did not immunoprecipitate NUB1 but FAT10 expression promoted their interaction. Heavy (h) and light (l) immunoglobulin chains are indicated. The position of molecular weight markers is indicated in kilodalton (kDa).</p

The FAT10 E1 activating enzyme interacts with AIPL1.

<p>Cells were transfected as indicated and subject to immunoprecipitation and immunoblot analysis. (A) Expression of the FAT10 E1 activating enzyme UBA6 increases the amount of FAT10-conjugated proteins including the covalent AIPL1-FAT10 conjugate. AIPL1 altered the profile of UBA6-dependent FAT10 conjugation. (B) UBA6 co-precipitated with AIPL1. (C) Co-precipitation of UBA6 with AIPL1 is abrogated in the presence of FAT10. The position of molecular weight markers is indicated in kilodalton (kDa).</p

AIPL1 interacts with NUB1 to block the degradation of FAT10-DHFR.

<p>Cells were transfected with the indicated constructs, then treated 24 hours later with cycloheximide (CHX) to block protein synthesis and assess degradation of FAT10-DHFR over the indicated times. (A) AIPL1 blocked the NUB1-mediated degradation of FAT10-DHFR, and the effect was stronger in the presence of MG132. (B) AIPL1 delayed the degradation of FAT10-DHFR in the presence of NUB1. The percentage of FAT10 remaining was measured from 3 independent experiments (n = 3), and the level of significance calculated using the Wilcoxon signed-rank test. (C) Pathogenic AIPL1 mutants A197P and C239R were defective in blocking FAT10-DHFR degradation, while the G262S mutant was able to block degradation. (D) NUB1 co-precipitated with both WT and G262S AIPL1, but not with the A197P or C239R mutants. (E) FAT10-DHFR co-precipitated with WT AIPL1, and the mutants A197P, C239R and G262S.</p

AIPL1 binds to FAT10 and FAT10-modified proteins.

<p>(A) Monomeric FAT10 co-precipitates with AIPL1 from transfected SK-N-SH cell lysates. Cells were transfected with the indicated constructs and immunoprecipitation was performed followed by immunoblot analysis. (B) Recombinant purified GST-AIPL1 but not GST can pull down free FAT10 and FAT10-conjugated proteins from HA-FAT10-transfected cell lysates. (C) Recombinant purified GST-AIPL1, but not GST, pulls down recombinant purified His6-FAT10. The position of molecular weight markers is indicated in kilodalton (kDa).</p

AIPL1 alters the NUB1-mediated degradation of FAT10.

<p>SK-N-SH Cells were transfected with NUB1-FLAG, HA-FAT10 and Myc-AIPL1 vectors in the presence and absence of the proteasome inhibitor MG132, as indicated. Cell lysates were harvested 24 hours post-transfection and immunoprecipitates were analyzed by immunoblotting to detect the protein indicated. (A) NUB1 interacts with FAT10 and accelerates the degradation of free FAT10 and FAT10-modified proteins. The change in levels of FAT10 was measured from 3 independent experiments (n = 3) of duplicate samples. Heavy (h) and light (l) immunoglobulin chains are indicated. (B) NUB1 co-precipitates with AIPL1. (C) AIPL1 enhances the steady-state levels of free FAT10 and FAT10 modified proteins, both alone and in the presence of NUB1. The change in levels of FAT10 was measured from 5 independent experiments (n = 5) of duplicate samples. (D) HA-FAT10 was visualised by immunocytochemical analysis with anti-HA and Cy2-conjugated secondary antibody. NUB1-mediated degradation of FAT10 is altered by the presence of AIPL1. Scale bar is 20 µM. (C) and (E) A small proportion of AIPL1 is itself covalently modified with FAT10, as detected by anti-HA, anti-AIPL1 and anti-FAT10 (rabbit polyclonal) antibodies. The percentage of AIPL1 modified by FAT10 was measured from 3 independent experiments (n = 3). Conjugation of FAT10 to AIPL1 is prevented using a FAT10 diglycine deletion mutant. The position of molecular weight markers is indicated in kilodalton (kDa).</p

AIPL1 localization overlaps with EB1 at the tips of microtubules: Immunofluorescent localization of AIPL1 (green) and EB1 (red) in SK-N-SH cells in the absence of nocodazole treatment (untreated), and following a recovery period of 5 min (5 min recovery) and 15 min (15 min recovery) after the removal of nocodazole.

<p>Nuclei are labelled with DAPI (blue). The zoomed images (zoom) are demarcated by the white squares in the merged images (merge). Split channel inserts for AIPL1 (green) and EB1 (red) are shown in the right hand column. Scale bars: 20 μm.</p

Endogenous AIPL1 co-localizes with ciliary markers and EB1 in human retinal cryosections.

<p><b>A</b>: Immunohistochemical localization of AIPL1 (green) and centrin-3 (red) in unfixed human retinal cryosections. Nuclei are labelled with DAPI (blue). The labelling on the DIC (differential interference contrast) image is: ganglion cell layer (GCL); inner plexiform layer (IPL); inner nuclear layer (INL); outer plexiform layer (OPL); outer nuclear layer (ONL); photoreceptor inner segments (IS) and photoreceptor outer segments (OS). <b>B</b>: Zoomed image of the immunohistochemical localization of AIPL1 (green) and centrin-3 (red) in photoreceptor cells. The white squares demarcate a further zoomed area (left hand column) highlighting the enrichment of AIPL1 (green) and centrin-3 in the cilia (white arrowheads). Nuclei are labelled with DAPI (blue). Inner segments (IS); connecting cilia (cc); outer segments (OS). Scale bar: 10 μm. <b>C</b>: Immunogold labelling of EB1 and AIPL1 in human retinal cryosections. 80 nm human retinal cryosections were double-labelled with antibodies against EB1 (PAG 10 nm, small arrow) and AIPL1 (PAG 15 nm, large arrowhead). Inner segments (IS); outer segments (OS); connecting cilium (cc); basal body (bb); microtubules (mt). Scale bar: 200 nm.</p

EB1, but not AIPL1, localizes to primary cilia in ARPE-19 cells.

<p><b>A</b>: Immunofluorescent localization of EB1 (green) with the cilia markers pericentrin (red) and ARL13b (red) in ARPE-19 cells. Nuclei are labelled with DAPI (blue). The white arrows demarcate the localization of EB1, pericentrin and ARL13b at the primary cilia. <b>B</b>: Immunofluorescent localization of AIPL1 (green) with the cilia markers γ-tubulin (red) and acetylated α-tubulin (red) in ARPE-19 cells. Nuclei are labelled with DAPI (blue). The white arrows demarcate the localization of γ-tubulin and acetylated α-tubulin at the primary cilia. The split channel inserts are shown in the right hand column. Scale bars: 20 μm.</p

JACoP analysis of co-localization.

<p>M1 = fraction A overlapping B, M2 = fraction B overlapping A</p><p>JACoP analysis of co-localization.</p