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. 2012 May 15;11(10):2006-21.
doi: 10.4161/cc.20423. Epub 2012 May 15.

Huntingtin Protein Interactions Altered by Polyglutamine Expansion as Determined by Quantitative Proteomic Analysis

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Free PMC article

Huntingtin Protein Interactions Altered by Polyglutamine Expansion as Determined by Quantitative Proteomic Analysis

Tamara Ratovitski et al. Cell Cycle. .
Free PMC article

Abstract

Huntington disease (HD) is a neurodegenerative disorder caused by an expansion of a polyglutamine repeat within the HD gene product, huntingtin. Huntingtin, a large (347 kDa) protein containing multiple HEAT repeats, acts as a scaffold for protein-protein interactions. Huntingtin-induced toxicity is believed to be mediated by a conformational change in expanded huntingtin, leading to protein misfolding and aggregation, aberrant protein interactions and neuronal cell death. While many non-systematic studies of huntingtin interactions have been reported, they were not designed to identify and quantify the changes in the huntingtin interactome induced by polyglutamine expansion. We used tandem affinity purification and quantitative proteomics to compare and quantify interactions of normal or expanded huntingtin isolated from a striatal cell line. We found that proteins preferentially interacting with expanded huntingtin are enriched for intrinsically disordered proteins, consistent with previously suggested roles of such proteins in neurodegenerative disorders. Our functional analysis indicates that proteins related to energy production, protein trafficking, RNA post-transcriptional modifications and cell death were significantly enriched among preferential interactors of expanded huntingtin. Expanded huntingtin interacted with many mitochondrial proteins, including AIFM1, consistent with a role for mitochondrial dysfunction in HD. Furthermore, expanded huntingtin interacted with the stress granule-associated proteins Caprin-1 and G3BP and redistributed to RNA stress granules under ER-stress conditions. These data demonstrate that a number of key cellular functions and networks may be disrupted by abnormal interactions of expanded huntingtin and highlight proteins and pathways that may be involved in HD cellular pathogenesis and that may serve as therapeutic targets.

Figures

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Figure 1. Htt expression and purification of Htt complexes and iTRAQ procedure. (A) Htt constructs, used in the study, comprising the first 586 amino acids of Htt with either 20 or 50 polyQ and two N-terminal tags (streptavidin, SBP and calmodulin, CBP) for affinity purification. (B) HEK293 cells were transfected with the TAP-Htt constructs, and Htt complexes were purified using the InterPlay mammalian TAP system (as described in the Materials and Methods); Protein complexes were eluted from the calmodulin agarose in two steps: first- with 2M NaCl, and then with 2% SDS and aliquots of the samples (~10%) were separated on SDS-PAGE and stained with Silver Stain; C-non-transfected cells. (C and D) STHdh Q7/Q7 cells were transiently transfected with normal (TAP-N586–20Q) or expanded (TAP-N586–50Q) Htt fragments: Highly expressed tagged normal or mutant Htt N586 fragments (migrating at 80 or 100 kDa respectively) were detected with both MAB2166 antibody to Htt (left panel) and a specific N586 neo-epitope antibody (right panel), the low levels of endogenous full-length Htt (migrating at 300 kDa) were also detected with MAB2166 (C). Immunostaining with Htt specific MAB2166 (green) and DAPI (blue) (D). (E) Purification of Htt protein complexes from STHdh cells. SDS-PAGE analysis of the aliquots of the samples prepared for MS. Normal (20Q) and expanded (50Q) Htt complexes from STHdh cells were expressed, purified and analyzed (as described in B). The inputs (before purification) are shown. (F) The iTRAQ workflow. Each sample was labeled with a unique tag, consisting of a reporter and a balance region, and then all samples were combined, fractionated and analyzed by LC-MS/MS.
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Figure 2. Quantitative analysis of normal and expanded Htt interactome. (A) Top cellular functions (as defined by IPA) most significantly enriched within Htt-50Q (dark blue) and Htt-20Q (light blue) preferential interactors, based on the protein data set shown in Table S1 (CV < 0.35 between duplicate samples). The graph shows the enrichment of particular functional categories based on the observed number of proteins for each group, relative to the number expected by chance. Log10 p-values are calculated by IPA. (B) The pie diagram, showing selected functional categories most significantly enriched within Htt-20Q and Htt-50Q preferential interactors, based on the IPA analysis of differential interactors with CV < 0.25 between duplicate samples. The numbers represent the percentage of differential interactors involved in a specific function, relative to the number of all differential interactors combined identified for all functional categories shown. (C) Top Canonical pathways (as defined by IPA) most significantly enriched within Htt-50Q (dark blue) and Htt-20Q (light blue) preferential interactors using the protein data set shown in Table S1. The graph shows the enrichment of particular canonical pathways based on the observed number of proteins for each pathway, relative to the number expected by chance. Log10 p-values are calculated by IPA. (D) Volcano plot showing the distribution of proteins differentially interacting with normal (20Q) and expanded (50Q) Htt based on iTRAQ ratios and p-values. The x-axis shows the Log2 of the median normalized iTRAQ ratios between 50Q and 20Q. The vertical lines indicate the threshold iTRAQ ratios required for a protein to be considered a differential interactor (> 1.2 or < 0.8).The x-axis shows the –Log10 of p-values, obtained using ANOVA approach, described in the Results. The horizontal line represents p-value of 0.05.
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Figure 3. AIFM1 interacts with Htt and mediates Htt toxicity. (A) STHdh Q7/Q7 cells were transiently transfected with normal (Htt-N586–20Q) or expanded (Htt-N586–82Q) Htt fragments, lysed 48 h after transfection, and Htt complexes were immunoprecipitated using a specific antibody to Htt (909). AIFM1 was detected in the IPs from transfected cells, but not in non-transfected cells or in control samples without the primary antibody (bottom panel). IPs were also analyzed for the presence of Htt using 1–82 antibody (middle panel). The inputs are shown on the top panels. (B) STHdh Q7/Q7 and Q111/Q111 cells were grown with and without FBS for 48 h, and AIFM1 complexes were immunoprecipitated using a specific antibody to AIFM1. Expanded Htt proteins were detected in the IPs from STHdh Q111/Q111 cells using MW1 antibody recognizing expanded polyQ, but not in control samples without the primary antibody (bottom panel). The inputs are shown on the top panels: expanded Htt is detected using MW1 antibody, both normal and expanded Htt are detected with 2166 Ab; NS - non-specific bands. (C) STHdh Q7/Q7 cells were transiently co-transfected with normal (Htt-N586–20Q) or expanded (Htt-N586–82Q) Htt fragments, AIFM1 and Bcl-2, as indicated. Cells were lysed 48 h after transfection, and AIFM1 complexes were immunoprecipitated using a specific antibody to AIFM1. Htt was detected in the IPs from transfected cells, but not in mock transfections or in control samples without the primary antibody (middle panel). IPs were also analyzed for the presence of AIFM1 (top panel). The inputs are shown on the bottom panels. (D) Sub-cellular fractionation of STHdh Q7/Q7 and Q111/Q111 cells was performed as described in the Materials and Methods to obtain cytoplasmic (C), nuclear (N), outer mitochondrial membrane (OMM) and mitochondrial (M) fractions. Htt proteins were detected with 2166 MAB to Htt (top panel), or with MW1 antibody (middle panel); AIFM1 protein was detected exclusively in the mitochondrial fractions. Cytoplasmic (β tubulin, TUB), nuclear (PARP) and mitochondrial (COXIV) markers are shown. (E) AIFM1 knock-down in striatal cells attenuates toxicity of expanded Htt-N585–82Q fragment. STHdh Q7/Q7 cells were co-transfected with indicated plasmids and siRNAs, fixed 48 h later, co-stained with an antibody to Htt (1–82) and with Hoechst 33258, and the nuclei staining intensity was analyzed as described in Materials and Methods. The data are presented as percentage of surviving cells among transfected cells. About 250–600 transfected cells were counted for each condition, and the experiment was repeated 3 times (*n = 3, p = 0.01; **n = 3, p = 0.03). (F) blots demonstrating AIFM1 knock-down upon transfection with AIFM1 siRNA, but not with control siRNA
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Figure 4. Htt and AIFM1 partially co-localize in association with mitochondria. (A) STHdh Q7/Q7 (top) and Q111/Q111 (bottom) cells were fixed 48 h after plating. Confocal immunofluorescent detection of Htt with 2166 monoclonal antibody is shown in green (Alexa Fluor 488); detection of AIFM1 with polyclonal specific antibody is shown in red (Alexa Fluor 555); Yellow staining in merged images demonstrates partial co-localization. (B) Confocal immunofluorescent images of STHdh Q7/Q7 (top) and Q111/Q111 (bottom) cells. Detection of AIFM1 with polyclonal specific antibody and of Htt with rabbit polyclonal antibody to Htt epitope 1–17 are shown in red (Alexa Fluor 555); detection of Mn-SOD with mouse monoclonal antibody is shown in green (Alexa Fluor 488); The nuclear staining (DAPI) is shown in blue; Yellow staining in merged images demonstrates partial co-localization of Htt and AIFM1 with mitochondrial marker Mn-SOD.
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Figure 5. Confirmation of Caprin- 1/Htt interaction by IP-Western blotting. (A) STHdh Q7/Q7 cells were transiently transfected with normal (Htt-N586–20Q) or expanded (Htt-N586–82Q) Htt fragments, lysed 48 h after transfection, and Htt complexes were immunoprecipitated using a specific antibody to Htt (909). Endogenous Caprin -1 was detected in the IPs from transfected cells, but not in non-transfected cells or in control samples without the primary antibody (bottom panel). IPs were also analyzed for the presence of Htt using 1–82 antibody (middle panel). The inputs are shown on the top panels. (B) STHdh Q7/Q7 and Q111/Q111 cells were grown for 48 h with or without incubation with 10 μM thapsigargin for 50 min, and Caprin-1 complexes were immunoprecipitated using a specific antibody to Caprin -1. Expanded Htt proteins were detected in the IPs from STHdh Q111/Q111 cells using MW1 antibody recognizing expanded polyQ, but not in control samples without the primary antibody (bottom panel). NS- non-specific bands . The inputs are shown on the top panels: Normal and expanded Htt proteins were detected using 2166 antibody.
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Figure 6. Htt redistributes to cytoplasmic stress granules (SG) under the ER-stress conditions, where it co-localizes with Caprin-1 and a SG marker G3BP1. (A) STHdh Q7/Q7 (left) and Q111/Q111 (right) cells were fixed 48 h after plating. Confocal immunofluorescent detection of Htt with 2166 monoclonal antibody is shown in green (Alexa Fluor 488); detection of Caprin-1 with polyclonal specific antibody is shown in red (Alexa Fluor 555); The nuclear staining (DAPI) is shown in blue; Merged images demonstrates little or no co-localization. (B) STHdh Q7/Q7 and Q111/Q111 cells were treated with 10 μM thapsigargin for 50 min before fixing, to induce ER stress. Htt and Caprin-1 were detected as described above. Yellow dots in merged images demonstrate Htt and Caprin-1 co-localization in stress granules. (C) STHdh Q7/Q7 and Q111/Q111 cells were treated with 10 μM thapsigargin for 50 min before fixing, to induce ER stress. Confocal immunofluorescent detection of Htt with rabbit polyclonal antibody to epitope 1–17 is shown in red (Alexa Fluor 555); detection of G3BP1 with mouse monoclonal antibody is shown in green (Alexa Fluor 488); The nuclear staining (DAPI) is shown in blue; Yellow dots in merged images demonstrates Htt and G3BP1 co-localization in stress granules.
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Figure 7. STHdh Q111/Q111 cells expressing expanded Htt form stress granules more robustly than normal Q7/Q7 cells. (A) STHdh Q7/Q7 (top) and Q111/Q111 (bottom) cells were treated with 10 μM thapsigargin for 50 min before fixing, to induce ER stress. Caprin-1 and G3BP1 were detected as described in the legend to Figure 7. (B) Graph shows percentage of Q7/Q7 and Q111/Q111 cells containing stress granules (Caprin-1 and G3BP1- positive) upon treatment with 10 μM thapsigargin for 50 min before fixing, as determined by the presence of yellow dots in the merged images as shown in A. Total 150 cells were counted for each cell line (*n = 3, p = 0.02).

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