Strategies to Investigate Ubiquitination in Huntington's Disease

Many neurodegenerative disorders including Huntington's Disease are hallmarked by intracellular protein aggregates that are decorated by ubiquitin and different ubiquitin ligases and deubiquitinating enzymes. The protein aggregates observed in Huntington's Disease are caused by a polyglutamine expansion in the N-terminus of the huntingtin protein (Htt). Improving the degradation of mutant Htt via the Ubiquitin Proteasome System prior to aggregation would be a therapeutic strategy to delay or prevent the onset of Huntington's Disease for which there is currently no cure. Here we examine the current approaches used to study the ubiquitination of both soluble Htt as well as insolubilized Htt present in aggregates, and we describe what is known about involved (de)ubiquitinating enzymes. Furthermore, we discuss novel methodologies to study the dynamics of Htt ubiquitination in living cells using fluorescent ubiquitin probes, to identify and quantify Htt ubiquitination by mass spectrometry-based approaches, and various approaches to identify involved ubiquitinating enzymes

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種別:卒業論文東京帝国大学工学

Global Proteome and Ubiquitinome Changes in the Soluble and Insoluble Fractions of Q175 Huntington Mice Brains

Huntington's disease is caused by a polyglutamine repeat expansion in the huntingtin protein which affects the function and folding of the protein, and results in intracellular protein aggregates. Here, we examined whether this mutation leads to altered ubiquitination of huntingtin and other proteins in both soluble and insoluble fractions of brain lysates of the Q175 knock-in Huntington's disease mouse model and the Q20 wild-type mouse model. Ubiquitination sites are detected by identification of Gly-Gly (diGly) remnant motifs that remain on modified lysine residues after digestion. We identified K6, K9, K132, K804, and K837 as endogenous ubiquitination sites of soluble huntingtin, with wild-type huntingtin being mainly ubiquitinated at K132, K804, and K837. Mutant huntingtin protein levels were strongly reduced in the soluble fraction whereas K6 and K9 were mainly ubiquitinated. In the insoluble fraction increased levels of huntingtin K6 and K9 diGly sites were observed for mutant huntingtin as compared with wild type. Besides huntingtin, proteins with various roles, including membrane organization, transport, mRNA processing, gene transcription, translation, catabolic processes and oxidative phosphorylation, were differently expressed or ubiquitinated in wild-type and mutant huntingtin brain tissues. Correlating protein and diGly site fold changes in the soluble fraction revealed that diGly site abundances of most of the proteins were not related to protein fold changes, indicating that these proteins were differentially ubiquitinated in the Q175 mice. In contrast, both the fold change of the protein level and diGly site level were increased for several proteins in the insoluble fraction, including ubiquitin, ubiquilin-2, sequestosome-1/p62 and myo5a. Our data sheds light on putative novel proteins involved in different cellular processes as well as their ubiquitination status in Huntington's disease, which forms the basis for further mechanistic studies to understand the role of differential ubiquitination of huntingtin and ubiquitin-regulated processes in Huntington's disease

Quantitative Proteomics Reveals Extensive Changes in the Ubiquitinome after Perturbation of the Proteasome by Targeted dsRNA-Mediated Subunit Knockdown in <i>Drosophila</i>

The ubiquitin–proteasome system (UPS), a highly regulated mechanism including the active marking of proteins by ubiquitin to be degraded, is critical in regulating proteostasis. Dysfunctioning of the UPS has been implicated in diseases such as cancer and neurodegenerative disorders. Here we investigate the effects of proteasome malfunctioning on global proteome and ubiquitinome dynamics using SILAC proteomics in <i>Drosophila</i> S2 cells. dsRNA-mediated knockdown of specific proteasome target subunits is used to inactivate the proteasome. Upon this perturbation, both the global proteome and the ubiquitinome become modified to a great extent, with the overall impact on the ubiquitinome being the most dramatic. The abundances of ∼10% of all proteins are increased, while the abundances of the far majority of over 14 000 detected diGly peptides are increased, suggesting that the pool of ubiquitinated proteins is highly dynamic. Remarkably, several proteins show heterogeneous ubiquitination dynamics, with different lysine residues on the same protein showing either increased or decreased ubiquitination. This suggests the occurrence of simultaneous and functionally different ubiquitination events. This strategy offers a powerful tool to study the response of the ubiquitinome upon interruption of normal UPS activity by targeted interference and opens up new avenues for the dissection of the mode of action of individual components of the proteasome. Because this is to our knowledge the first comprehensive ubiquitinome screen upon proteasome malfunctioning in a fruit fly cell system, this data set will serve as a valuable repository for the <i>Drosophila</i> community

Improvement of ubiquitylation site detection by Orbitrap mass spectrometry

Ubiquitylation is an important posttranslational protein modification that is involved in many cellular events. Immunopurification of peptides containing a K-epsilon-diglycine (diGly) remnant as a mark of ubiquitylation combined with mass spectrometric detection has resulted in an explosion of the number of identified ubiquitylation sites. Here, we present several significant improvements to this workflow, including fast, offline and crude high pH reverse-phase fractionation of tryptic peptides into only three fractions with simultaneous desalting prior to immunopurification and better control of the peptide fragmentation settings in the Orbitrap HCD cell. In addition, more efficient sample cleanup using a filter plug to retain the antibody beads results in a higher specificity for diGly peptides and less non-specific binding. These relatively simple modifications of the protocol result in the routine detection of over 23,000 diGly peptides from HeLa cells upon proteasome inhibition. The efficacy of this strategy is shown for lysates of both non-labeled and SILAC labeled cell lines. Furthermore, we demonstrate that this strategy is useful for the in-depth analysis of the endogenous, unstimulated ubiquitinome of in vivo samples such as mouse brain tissue. This study presents a valuable addition to the toolbox for ubiquitylation site analysis to uncover the deep ubiquitinome. Significance: A K-epsilon-diglycine (diGly) mark on peptides after tryptic digestion of proteins indicates a site of ubiquitylation, a posttranslational modification involved in a wide range of cellular processes. Here, we report several improvements to methods for the isolation and detection of diGly peptides from complex biological mixtures such as cell lysates and brain tissue. This adapted method is robust, reproducible and outperforms previously published methods in terms of number of modified peptide identifications from a single sample. Indepth analysis of the ubiquitinome using mass spectrometry will lead to a better understanding of the roles of protein ubiquitylation in cellular event

NAP1 regulates SA phoshorylation levels by counteracting PP2A association with chromosomal cohesin during mitosis.

<p>(<b>A</b>) Colloidal blue staining of immunopurified, baculovirus expressed HA-tagged NAP1 from Sf9 cells. (<b>B–C</b>) NAP1 can displace PP2A from cohesin. The endogenous cohesin complex was immunopurified from embryo NE with antibodies against SA (<b>B</b>) or SMC1 (<b>C</b>) as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003719#pgen-1003719-g004" target="_blank">Figure 4B–C</a>. Next, increasing amounts of purified HA-NAP1 was added. Following extensive washes the binding of endogenous NAP1, HA-NAP1 and PP2A to the cohesin complex was analyzed by immunoblotting. (<b>D</b>) Western blot analysis of SA IPed from either mock-treated or NAP1 knockdown (KD) cells. Blots were probed with antibodies against SA, phosphorylated serine (phosphoSer), PP2A or NAP1. Note the increased PP2A binding to SA in the absence of NAP1. Concomitantly, SA phosphorylation levels decreased, as revealed by the antibodies against phosphoSer, which recognize a band corresponding to the migration of SA. A slower migrating form of SA, presumably due to phosphorylation, is indicated by an arrow. (<b>E</b>) NAP1 depletion does not affect cohesin complex stability or stoichiometry. In parallel to the immunoblotting in (<b>D</b>), we resolved the IPed SA by SDS-PAGE followed by colloidal blue staining. The identity of the cohesin subunits were determined by mass spectrometric analysis (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003719#pgen.1003719.s005" target="_blank">Figure S5A</a>). (<b>F</b>) Cell cycle profiles of mock-treated (Mock) or NAP1 depleted (KD) S2 cells arrested in mitosis by colhicine (red curves) as compared to asynchronously dividing cells (black curves). Cell cycle profiles were determined by FACS analysis. G1, S and G2/M phases are indicated. (<b>G</b>) PP2A dissociates from cohesin in mitosis, whereas NAP1 binding to SA is increased. Immunoblotting analysis of SA IPed from either mock or NAP1 depleted (KD) cells, treated (+) or untreated (−) with colhicine as in (<b>D</b>). Similar results were obtained for SMC1 IPs from colhicine-treated cells (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003719#pgen.1003719.s006" target="_blank">Figure S6</a>). (<b>H</b>) Immunopurification of SA from S2 cell extracts denatured by 6M Urea ((d)IP) to selectively identify phosphorylated SA with antibodies against phosphorylated serine (phosphoSer). Note that SMC1, NAP1 and PP2A dissociate from SA under these conditions. (<b>I</b>) Western blot analysis of SA IPed under denaturing conditions ((d)IP) from either mock- or NAP1 depleted (KD) cells, which were either treated (+) or untreated (−) with colchicine, confirmed the changes in SA phosphorylation caused by mitotic arrest or NAP1 depletion.</p

NAP1 and PP2A act antagonistically in cohesin cycle.

<p>(<b>A</b>) Analysis of mitotic chromosomes from colchicine-treated S2 cells after knockdown of NAP1, PP2A or both factors. We quantified the frequency of resolved (blue), unresolved (red) sister chromatids and loss of centromeric cohesion (Cen. Loss; green). Concomitant depletion of NAP1 and PP2A resulted in a statistically significant increase of the frequency of resolved chromatids compared to the NAP1 knockdown, as determined by χ²-test (n>30, from 3 biological replicates). For the corresponding Western blot analysis see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003719#pgen.1003719.s007" target="_blank">Figure S7A</a>. (<b>B</b>) Representative example of mitotic chromosomes from colhicine-treated S2 cells depleted for NAP1, PP2A or for both proteins. DNA visualized by DAPI staining is shown in red. Centromers are indicated by arrowheads, whereas loss of centromeric cohesion is indicated by full arrows. (<b>B′</b>) The localization of SA (green) on mitotic chromosomes same as in (<b>B</b>) was determined by indirect immunofluorescence. (<b>C</b>) RAD21 (green) localization on mitotic chromosomes. (<b>D</b>) MeiS332 (green) localization on mitotic chromosomes. (<b>E</b>) Depletion of PP2A restores SA phosphorylation in cells lacking NAP1. Western blot analysis of SA IPed from either mock-treated S2 cells or after knockdown (KD) of NAP1, PP2A or both proteins under normal (top panel) or denaturing (middle panel, (d)IP) conditions from asynchronously dividing cells (− colhicine) or colhicine treated cells (bottom panel, + colhicine). Blots were probed with antibodies against SA, phosphorylated serine, PP2A or NAP1. After NAP1 knockdown, SA phosphorylation levels drop substantially. Whereas depletion of PP2A alone does not affect bulk SA phosphorylation, concomitant knockdown of PP2A and NAP1 neutralized the effect of NAP1 depletion, leading to restored levels of phosphorylated SA. Antibodies against phosphoSer recognize a band corresponding to the migration of SA. A slower migrating form of SA, presumably due to phosphorylation, is indicated by an arrow. (<b>F</b>) Analysis of mitotic chromosomes from colchicine-treated S2 cells after over-expression (OE) of GFP (Mock), NAP1, PP2A, both NAP1 and PP2A or the catalytic mutant PP2A^H59Q. Quantification of mitotic phenotypes was as described above (A). Overexpression of PP2A, but not PP2A^H59Q, resulted in significant increase of the frequency of unresolved chromatids. The PP2A over-expression phenotype was rescued by co-expression of NAP1, as determined by χ²-test (n>30, from 3 biological replicates). For the corresponding Western blot analysis see . Representative examples of mitotic chromosomes are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003719#pgen.1003719.s007" target="_blank">Figure S7C</a>–D.</p