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. 2019 May 3;10:534.
doi: 10.3389/fphys.2019.00534. eCollection 2019.

Detailed Dissection of UBE3A-Mediated DDI1 Ubiquitination

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Free PMC article

Detailed Dissection of UBE3A-Mediated DDI1 Ubiquitination

Nagore Elu et al. Front Physiol. .
Free PMC article

Abstract

The ubiquitin E3 ligase UBE3A has been widely reported to interact with the proteasome, but it is still unclear how this enzyme regulates by ubiquitination the different proteasomal subunits. The proteasome receptor DDI1 has been identified both in Drosophila photoreceptor neurons and in human neuroblastoma cells in culture as a direct substrate of UBE3A. Here, we further characterize this regulation, by identifying the UBE3A-dependent ubiquitination sites and ubiquitin chains formed on DDI1. Additionally, we found one deubiquitinating enzyme that is capable of reversing the action of UBE3A on DDI1. The complete characterization of the ubiquitination pathway of an UBE3A substrate is important due to the role of this E3 ligase in rare neurological disorders as Angelman syndrome.

Keywords: Angelman syndrome; GFP pull-down; UBE3A; mass-spectrometry; proteasome; ubiquitin E3 ligase.

Figures

FIGURE 1
FIGURE 1
Validation of DDI1 as a substrate of UBE3A in HEK293T cells. DDI1 ubiquitination was detected by western blot upon overexpression of wild-type UBE3A (UBE3AWT), ligase dead UBE3A (UBE3ALD), Parkin E3 ligase (PARKIN) and control vector (pCDNA3.1) in HEK293T cells. Anti-FLAG antibody (red) was used to detect the ectopically expressed ubiquitin, whereas the non-modified GFP-tagged DDI1 was detected by anti-GFP antibody (green). UBE3A overexpression was confirmed in whole cell lysates using anti-UBE3A antibody (Input UBE3A) while the FLAG signal in the inputs corroborated equivalent transfections in all samples [Input FLAG-(Ub)]. Quantification of western blots was performed with Image-J, by normalizing the FLAG intensities to the GFP signal. The analysis showed a statistically significant increase [one-way ANOVA, ∗∗∗p-value < 0.0001, (mean ± S.E.M., n = 3)] of GFP-DDI1 ubiquitination upon UBE3AWT overexpression in comparison to overexpression of UBE3ALD.
FIGURE 2
FIGURE 2
Schematic diagram of the MS-based procedure used to study ubiquitination sites and ubiquitin chain types on DDI1. HEK293T cells were co-transfected with DDI1-GFP and wild type UBE3A (UBE3AWT) or ligase dead UBE3A (UBE3ALD). Unmodified DDI1-GFP as well as its ubiquitinated forms were isolated by GFP pull-down, and separated by SDS–PAGE. The gel slice corresponding to ubiquitinated DDI1-GFP was excised and divided into two, basing on a previous western blot (a); a lower band corresponding for mono-ubiquitinated DDI1-GFP (black), and a band above it corresponding to poly-ubiquitinated DDI1-GFP (orange). Gel slices were digested with trypsin, and resulting peptides were extracted from the gel to further identify and quantify them by LC-MS/MS. Both bands were analyzed to infer DDI1 ubiquitination sites, whereas only the poly-ubiquitination band was used to detect the nature of the ubiquitin chain types formed on DDI1.
FIGURE 3
FIGURE 3
Identification and quantification of DDI1 ubiquitination sites. (A) Relative LFQ intensity of all the proteins detected by MS demonstrate that DDI1-GFP is the most abundant protein (green) in the GFP pull-down samples, followed by ubiquitin (orange). 287 more proteins (blue) were identified with marginal intensities. (B) Comparison of the intensities recorded for the two most abundant proteins in UBE3AWT and UBE3ALD reveal that whereas the levels of DDI1 detected are similar in both conditions, more ubiquitin was detected in the presence of UBE3AWT overexpression [t-test, ∗∗p-value < 0.05, (mean ± S.E.M., n = 3)]. (C) Schematic illustration of DDI1-GFP sequence, its domains (blue, UBL, ubiquitin-like domain; orange, HDD, helical-double domain; red, RVP, retroviral protease domain; green, GFP, GFP-tag of DDI1 protein) and the localization of the ubiquitination sites detected by mass spectrometry. Additionally, the diGly-modified peptides identified, within the ubiquitinated lysine marked in red, and the intensity values measured in each condition (WT, UBE3AWT and LD, UBE3ALD) are also indicated. “∙” indicates that the ubiquitination site is not reported in PhosphoSitePlus database. DiGly peptides encompassing K133 and K161 were statistically more abundant upon UBE3AWT overexpression [t-test, p-value < 0.05, (mean ± S.E.M., n = 3)]. (D) UBE3A-dependent ubiquitination of three distinct DDI1-GFP mutants (K77R, K133R, or K161R) was monitored by western blot after isolating by GFP-pulldown. In red it is illustrated the ubiquitination pattern of DDI1, and in green the unmodified version of DDI1-GFP. Quantification and statistical analysis of the ubiquitination was performed with Image-J after normalizing FLAG intensities to GFP levels. Mutation on DDI1 lysine 133 significantly [one-way ANOVA, p-value < 0.05, (mean ± S.E.M., n = 3)] abolishes its ubiquitination by UBE3AWT.
FIGURE 4
FIGURE 4
Analysis of the ubiquitin linkages formed in DDI1. Four ubiquitination sites on ubiquitin were confidently identified and quantified by mass spectrometry. The intensity ratios for each ubiquitin chain type are given as the ratio of the UBE3AWT overexpressing condition over the intensity recorded for the UBE3ALD sample. The intensity ratio for ubiquitin is also indicated for reference. As K29 was not detected in neither of the UBE3ALD samples, its missing values were substituted by the lowest intensity value found in each replica.
FIGURE 5
FIGURE 5
Screen of 41 human DUBs indicates USP9X to be the counteracting DUB of UBE3A. (A) 41 different human DUBs were silenced using 10 nM siRNAi. After GFP-pulldown of DDI1-GFP, FLAG/GFP intensity ratios were determined. Those ratios were normalized to the average of three lowest values on each 6-well experimental dataset. Upon silencing of most DUBs, the ubiquitination of DDI1 did not change quantitatively (0.5 < fold-change < 2, white). However, silencing of some DUBs decreased DDI1 ubiquitination (fold-change < 0.5, red), while some others increased it (fold-change > 2, gray). Black bars highlight the DUBs that affect more severely DDI1 ubiquitination (UCHL-5, USP7, USP9X, and USP42). (B) siRNAs were used to silence UCHL-5, USP7, USP9X, and USP42 DUBs and scramble siRNAi was used as a control. After GFP-pulldown, DDI1 ubiquitination was detected by western blot analysis using anti-FLAG (red) and anti-GFP (green) antibodies. Equivalent amounts of ectopically overexpressed ubiquitin was detected in the cell lysates [Input FLAG-(Ub)], while USP9X silencing was corroborated by anti-USP9X antibody (USP9X). Quantification was performed with Image-J after normalizing FLAG/GFP intensities. USP9X inhibition significantly enhanced DDI1 ubiquitination [one-way ANOVA, ∗∗p-value < 0.05, (mean ± S.E.M., n = 3)]. DDI1 ubiquitination also increased upon USP42 silencing, but not significantly, while no changes were observed for the UCHL-5 and USP7 experiments.
FIGURE 6
FIGURE 6
USP9X inhibition by WP1130 enhances DDI1 ubiquitination. Cells expressing GFP-DDI1 and FLAG-Ub were treated with 5 μM WP1130 or DMSO for 1 h. GFP-DDI1 was pulled down and its ubiquitination monitored by western blot. Anti-GFP antibody was used to detect unmodified GFP-DDI1 levels whereas anti-FLAG antibody was used to detect ubiquitination. The signal of FLAG was normalized to the GFP signal. Equivalent amount of transfected ubiquitin was detected in the cell lysates by measuring FLAG-Ub levels [Input FLAG-(Ub)]. Quantification using Image-J showed a statistically significant increase in DDI1 ubiquitination after WP1130 treatment [t-test, p-value < 0.05, (mean ± S.E.M., n = 3)].

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