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. 2024 Aug 6;121(32):e2319091121.
doi: 10.1073/pnas.2319091121. Epub 2024 Jul 29.

Huntingtin contains an ubiquitin-binding domain and regulates lysosomal targeting of mitochondrial and RNA-binding proteins

Gianna M Fote  1   2 , Vinay V Eapen  3   4 , Ryan G Lim  5 , Clinton Yu  6 , Lisa Salazar  7 , Nicolette R McClure  8 , Jharrayne McKnight  8 , Thai B Nguyen  8 , Marie C Heath  8 , Alice L Lau  7 , Mark A Villamil  1 , Ricardo Miramontes  7 , Ian H Kratter  9   10   11 , Steven Finkbeiner  9   12   13 , Jack C Reidling  5 , Joao A Paulo  3 , Peter Kaiser  1 , Lan Huang  6 , David E Housman  14 , Leslie M Thompson  1   5   7   8   15 , Joan S Steffan  5   7   15
Affiliations

Huntingtin contains an ubiquitin-binding domain and regulates lysosomal targeting of mitochondrial and RNA-binding proteins

Gianna M Fote et al. Proc Natl Acad Sci U S A. .

Abstract

Understanding the normal function of the Huntingtin (HTT) protein is of significance in the design and implementation of therapeutic strategies for Huntington's disease (HD). Expansion of the CAG repeat in the HTT gene, encoding an expanded polyglutamine (polyQ) repeat within the HTT protein, causes HD and may compromise HTT's normal activity contributing to HD pathology. Here, we investigated the previously defined role of HTT in autophagy specifically through studying HTT's association with ubiquitin. We find that HTT interacts directly with ubiquitin in vitro. Tandem affinity purification was used to identify ubiquitinated and ubiquitin-associated proteins that copurify with a HTT N-terminal fragment under basal conditions. Copurification is enhanced by HTT polyQ expansion and reduced by mimicking HTT serine 421 phosphorylation. The identified HTT-interacting proteins include RNA-binding proteins (RBPs) involved in mRNA translation, proteins enriched in stress granules, the nuclear proteome, the defective ribosomal products (DRiPs) proteome and the brain-derived autophagosomal proteome. To determine whether the proteins interacting with HTT are autophagic targets, HTT knockout (KO) cells and immunoprecipitation of lysosomes were used to investigate autophagy in the absence of HTT. HTT KO was associated with reduced abundance of mitochondrial proteins in the lysosome, indicating a potential compromise in basal mitophagy, and increased lysosomal abundance of RBPs which may result from compensatory up-regulation of starvation-induced macroautophagy. We suggest HTT is critical for appropriate basal clearance of mitochondrial proteins and RBPs, hence reduced HTT proteostatic function with mutation may contribute to the neuropathology of HD.

Keywords: Huntingtin; RNA-binding proteins; autophagy; ubiquitin; ubiquitin-binding domain.

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Conflict of interest statement

Competing interests statement:The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Proteostasis is disrupted in 8988T TMEM192-HA cells by HTT KO. (A) Lysotracker red staining was performed on six wells of parental and HTT KO cell lines, and three images were averaged per well. Spots were quantified using Imaris software and compared using a two-tailed t test. (B) LC3 abundance and ratio of LC3II to LC3I were evaluated by western blot with or without 4 h of 50 nM Baf treatment. Whole protein levels were quantified using revert protein stain from LI-COR Biosciences. (C) Abundance of autophagic receptor p62 was evaluated by western blot with and without Baf treatment. (D) Lipid droplet staining was performed using Bodipy following overnight oleic acid (200 nM) treatment on six wells of each cell line. Three images were averaged per well. For all statistical analyses, three HTT KO cell lines were grouped. (E) 20S proteasome activity was analyzed using a fluorescence-based assay with and without Baf treatment at 50 nM for 4 h using HTT KO clone 10. (F) Ubiquitin puncta were quantified using Imaris software from HTT KO clone 10. Confocal microscopy was used to take three 20× images per well, three wells per cell line. In all statistical analyses, replicates from two or three HTT KO clonal lines were analyzed together as biological replicates.
Fig. 2.
Fig. 2.
HTT KO alters the contents of lysosomes. LysoIP was performed in 8988T cells expressing TMEM192-HA using magnetic HA pulldown beads. All cells were treated with Baf for 4 h prior to harvest in order to preserve lysosomal proteins for analysis. Proteomic contents of purified lysosomes were analyzed by mass spectrometry. Scatterplot of log2 fold changes comparing HTT KO to WT starvation-induced macroautophagy (turquoise = significant in both lists, blue = RNA- or mRNA-binding protein, red line = perfect correlation, blue line = LOESS curve fit, labeled proteins are exemplary). (A). Gene ontology pathways significantly altered by HTT KO were identified using Enrichr. Threshold for significant enrichment was FDR < 0.1. (B) Cellular composition enrichment of proteins that were decreased in the lysosomes of HTT KO cells compared to parental cells. (C) Molecular function enrichment of proteins that were increased in the lysosomes of HTT KO cells compared to parental cells. (D) Molecular function enrichment of proteins that were increased in the lysosomes of parental cells with HBSS treatment for 4 h prior to harvest, for stimulation of macroautophagy. (E) Cellular composition enrichment of proteins that were decreased in the lysosomes of parental cells with HBSS treatment for 4 h prior to harvest did not include mitochondrial proteins, unlike what we observed with HTT KO basally.
Fig. 3.
Fig. 3.
HTT copurifies with potentially ubiquitinated RNA-binding proteins. 17Q-502-HIS-HA-HA-HIS and FLAG-ubiquitin were coexpressed in St14A cells, crosslinked with formaldehyde, tandem copurified, and subjected to mass spectrometry analysis. (A) Plasmids encoding 17Q N-terminal 502 or 586 amino acid HTT fragments C-terminally tagged with HIS-HA-HA-HIS were transfected into St14A cells together with FLAG-ubiquitin. Cells were subjected to crosslinking with formaldehyde 2 d post-transfection, then lysed. Lysates were incubated overnight with an anti-FLAG matrix, washed then eluted with FLAG peptide, then incubated overnight with an anti-HA matrix, washed, and eluted with glycine pH 2. Samples from each copurification step were subjected to western analysis with anti-FLAG MAB (red) and polyclonal anti-HA antibody (green). FT denotes flow through of proteins that did not bind matrix. The final HA eluate shows bands the size of unmodified HTT fragment (green), and also a monoubiquitinated species (both red and green), together with copurifying ubiquitinated proteins (red). (B) Molecular function analysis of the ubiquitinated proteins associated noncovalently with HTT showed them to be most significantly RBPs. (C) 205 proteins copurified significantly above vector control in at least 2 of 3 tandem purifications. These proteins were found in protein databases for the nucleus (61%), stress granules (SG, 61%), defective ribosomal products (DRiPs, 50%), brain-derived autophagosomes (auto, 75%), and mitochondria (mito, 14%).
Fig. 4.
Fig. 4.
Mutation of amino acids within HTT’s 235-367 amino acid ubiquitin-binding domain destabilizes the in vitro interaction with I44A ubiquitin. (A, B) HTT GST-235-502 fragment was incubated in vitro with I44A ubiquitin. DISUBM (Y288G F289G Y290G S291A W292G) and UBM1 (L261A K262A), UIM1 (A259G S263A), UIM2 (I279G S283A), and UBM3 (V324A P325A) mutations reduced the in vitro interaction between I44A ubiquitin and HTT, while S421A enhanced the interaction as analyzed by One-Way ANOVA with Bonferroni’s multiple comparisons test and compared with wt control. (C) Cryo-EM structure from PDB:6X90 (46) or 6EZ8 (47) demonstrates the domain (residues 261 to 325, black circle) and amino acids involved in the in vitro HTT interaction with I44A ubiquitin.
Fig. 5.
Fig. 5.
Higher levels of ubiquitinated proteins coimmunoprecipitate with mutant HTT fragment compared with wt control, and HTT KO alters RBP-containing stress granule clearance and mitochondrial health. (A) St14A cells were cotransfected with FLAG-ubiquitin and with 17Q-502-HIS-HA-HA-HIS (wt) vs. 136Q-502-HIS-HA-HA-HIS HTT (mutant) fragment. Whole-cell (WC) lysate was used for anti-HA MAB immunoprecipitation. Western analysis demonstrates that ubiquitinated proteins coimmunoprecipitate with wt HTT fragment, and the expansion of the polyQ repeat significantly enhances HTT binding to ubiquitinated proteins. Statistical analysis was done with an unpaired t test (P = 0.0108). (B) FLAG-ubiquitin does not stick nonspecifically to the protein G dynabeads used for immunoprecipitation. Whole-cell lysates of St14A cells expressing FLAG-ubiquitin together with either 136Q- or 17Q- wt, S421D, G429V, or S421A 502 HTT-HIS-HA-HA-HIS fragment were subjected to immunoprecipitation with mouse anti-FLAG antibody (+) or zero (−) antibody control. While FLAG-ubiquitin was easily purified with this anti-FLAG immunoprecipitation, FLAG-ubiquitin did not bind nonspecifically to protein G dynabeads used to capture the antibody. Western immunoblot (IB) was incubated with mouse anti-FLAG and with rabbit anti-HA antibodies for detection. (C) HTT KO reduces the number of arsenite-induced G3BP1-positive stress granules in 8988T TMEM192-HA cells. Cells were treated with acute sodium arsenite stress (125 μm NaAsO2) for 90 min, then fixed and stained with G3BP1 (green) and Hoechst (cyan). For each condition, quantitation of cells with G3BP1-positive stress granules (SGs) and Hoechst markers were captured. SG and nuclei quantification were completed using Cell Profiler. A parameter was created which normalized the SG count to nuclei in a single image to help control for differences in cellular confluency attributed to stressors. Data from the parental (P) and each HTT KO clone (clone 10 and clone 59) were statistically analyzed using a One-Way ANOVA (F(2, 24) = 23.34, P < 0.0001; n = 9, mean ± SEM; ****P < 0.0001). (D) HTT KO significantly increases the MitoTimer red/green ratio indicative of increased mitochondrial oxidative stress and reduced mitochondrial health. 8988T parental or clone 10 HTT KO cells were transfected with pMitoTimer and images were analyzed by Imaris software using the surface module to measure the sum of green intensity and the sum of red intensity of each cell. Statistical analysis was done by an unpaired t test.
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References

    1. Bates G. P., et al. , Huntington disease Nat. Rev. Dis. Primers 1, 15005 (2015). - PubMed
    1. Barnat M., et al. , Huntington’s disease alters human neurodevelopment. Science 369, 787–793 (2020). - PMC - PubMed
    1. Saudou F., Humbert S., The biology of huntingtin. Neuron 89, 910–926 (2016). - PubMed
    1. Eshraghi M., et al. , Mutant Huntingtin stalls ribosomes and represses protein synthesis in a cellular model of Huntington disease. Nat. Commun. 12, 1461 (2021). - PMC - PubMed
    1. Ochaba J., et al. , Potential function for the Huntingtin protein as a scaffold for selective autophagy. Proc. Natl. Acad. Sci. U.S.A. 111, 16889–16894 (2014). - PMC - PubMed

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