Quantitative proteomics reveals neuronal ubiquitination of Rngo/Ddi1 and several proteasomal subunits by Ube3a, accounting for the complexity of Angelman syndrome

Quantitative proteomics reveals neuronal ubiquitination of Rngo/Ddi1 and several proteasomal... Abstract Angelman syndrome is a complex neurodevelopmental disorder caused by the lack of function in the brain of a single gene, UBE3A. The E3 ligase coded by this gene is known to build K48-linked ubiquitin chains, a modification historically considered to target substrates for degradation by the proteasome. However, a change in protein abundance is not proof that a candidate UBE3A substrate is indeed ubiquitinated by UBE3A. We have here used an unbiased ubiquitin proteomics approach, the bioUb strategy, to identify 79 proteins that appear more ubiquitinated in the Drosophila photoreceptor cells when Ube3a is over-expressed. We found a significantly high number of those proteins to be proteasomal subunits or proteasome-interacting proteins, suggesting a wide proteasomal perturbation in the brain of Angelman patients. We focused on validating the ubiquitination by Ube3a of Rngo, a proteasomal component conserved from yeast (Ddi1) to humans (DDI1 and DDI2), but yet scarcely characterized. Ube3a-mediated Rngo ubiquitination in fly neurons was confirmed by immunoblotting. Using human neuroblastoma SH-SY5Y cells in culture, we also observed that human DDI1 is ubiquitinated by UBE3A, without being targeted for degradation. The novel observation that DDI1 is expressed in the developing mice brain, with a significant peak at E16.5, strongly suggests that DDI1 has biological functions not yet described that could be of relevance for Angelman syndrome clinical research. Introduction Angelman syndrome (AS) is a rare neurodevelopmental disorder (OMIM #105830), with an estimated incidence of 1/15 000 births, characterized by a severe developmental delay, language impairment, ataxic movements, epilepsy, sleep disturbances and episodes of frequent laughter (1). In contrast to other complex syndromes that are caused by large genetic duplications/deletions, the underlying cause for AS is the loss of maternal expression in neurons of the brain of one single enzyme, UBE3A (2,3), a HECT-type ubiquitin E3 ligase (4–6). Although the deficiency of this paternally imprinted gene (7) is commonly originated by maternally inherited deletions on the 15q11-q13 chromosomal region (8), the syndrome is also caused by mutations affecting exclusively the UBE3A gene (9). Some of these UBE3A mutants lack the ubiquitin-ligase activity when tested in vitro (10,11), indicating that AS is caused by the lack of the ubiquitin ligase activity of UBE3A in neurons. Ubiquitinated substrates of UBE3A are therefore likely to be the effecting pathways of the resulting brain connectivity and/or function alterations. Interestingly, excess ligase activity of UBE3A has also been associated with autism spectrum disorders (12–14). Ubiquitin E3 ligase enzymes catalyze the covalent attachment of ubiquitin to the lysine residues on target proteins. According to in vitro studies, UBE3A catalyzes the preferential attachment of K48-linked poly-ubiquitin chains (15), presumably targeting its substrates for proteasomal degradation. Based on this premise, several UBE3A putative substrates (AIB1, Bak, Blk, Mcm7, Pbl) whose levels changed in the presence/absence of this enzyme were reported (16–20). Nevertheless, ubiquitination of those proteins by UBE3A has not yet been described. Similarly, the ubiquitination of other proposed neuronal UBE3A substrates was only validated in vitro (Arc, Na+/K+ ATPase, p27, Ring1B, Adrm1, Rpt5) or using non-denaturing immunoprecipitation approaches (Annexin A1, HHR23A, PSMD2, Ephexin5, p53) (21–29). We developed an ubiquitination assay for neuronal cell culture using a highly denaturing protocol, and showed that proteasome regulating proteins Rpn10 and Uch-L5 are ubiquitinated by Ube3a (30), both being reported to be essential for mammalian brain development (31,32). Recently, evidence is accumulating in regards to UBE3A regulating proteasomal activity (28–30,33), suggesting that UBE3A might indirectly be affecting the regulation of many other proteins targeted to the proteasome by other E3 ligases. Interaction between an ubiquitin ligase and its substrates is transient and hard to capture in vivo. Additionally, the low stoichiometry at which ubiquitin modified proteins are found within the cell hinders the identification of ubiquitination substrates in vivo. Antibodies that specifically recognize the ubiquitin diGlycine (diGly) signature have been employed to isolate, and subsequently identify putative ubiquitination sites by mass spectrometry (MS) (34–39). Such methodology has also been recently used in a screen for putative UBE3A substrates in HEK293 cells (29). However, this approach requires digestion of proteins by trypsin prior to their isolation, preventing any orthogonal validation, which is essential, as the diGly signature is also a remnant of other ubiquitin-like (UBL) modifications, such as Nedd8 or ISG15 (35), and can even be an experimental artefact under certain conditions (40). We have developed in our lab two methodologies that have proven to be suitable for the in vivo analysis of ubiquitinated proteins (41). The bioUb strategy, based on the in vivo biotinylation of ubiquitin (42), has recently been used in combination with quantitative shotgun proteomics to identify substrates of the E3 ligase Parkin involved in Parkinson’s disease (43). The second strategy favours the isolation of GFP-tagged proteins under denaturing conditions, and was first used to screen for Drosophila Ube3a substrates (30). In the present study, we have combined the bioUb strategy with the over-expression of Ube3a to identify 79 putative neuronal Ube3a substrates. Amongst those, we noted the presence of 13 proteasome subunits or proteasome interacting proteins. We validated that Ube3a ubiquitinates the proteasomal ubiquitin receptor Rings lost (Rngo) in Drosophila photoreceptor neurons in vivo. Furthermore, we have confirmed that ubiquitination of DNA damage-inducible protein 1 homologue 1 (DDI1), the human orthologue of Rngo, is enhanced upon UBE3A over-expression in neuroblastoma cells, without being targeted for degradation. The observation that Ddi1 is highly expressed in the developing mice brain suggests that this protein has a yet uncharacterized biological function in neuronal development. Results Unbiased identification of Ube3a substrates in Drosophila neurons in vivo In order to identify by MS analysis the proteins whose ubiquitination depends on Ube3a, we used the following fly lines: BirA, bioUb, bio15B and bioA3. bioUb flies express the (bioUb)6-BirA precursor (Fig. 1A) in the Drosophila photoreceptor neurons under the control of the eye-specific GMR-GAL4 driver, which has been shown to be the most suitable neuronal(-like) driver for identifying low abundance proteins, and for optimizing reproducibility across samples (44). BirA control flies express just the bacterial biotinylating enzyme BirA (44). bioA3 flies are bioUb flies over-expressing the Ube3a E3 ligase (Fig. 1B), as confirmed by immunoblotting (Fig. 1C). On the other hand, bio15B flies are bioUb flies carrying a loss of function Ube3a deletion (Ube3a15B allele) in heterozygosis. Homozygous mutant Ube3a15B flies lack any detectable Ube3a protein (Supplementary Material, Fig. S1A), but flies carrying this allele could not be expanded in homozygosis. The Ube3a15B heterozygous bio15B flies used for this study only show a partial reduction of Ube3a protein levels (Fig. 1C). Free BirA, indicating appropriate processing of the (bioUb)6-BirA precursor, was observed for all three genotypes (Fig. 1D); no undigested forms of the precursor were found above the expected molecular size of BirA (35 kDa). Biotin immunoblotting confirmed biotinylation and incorporation into conjugates of the GMR-GAL4-driven ectopic biotin-tagged ubiquitin in all bioUb, bioA3 and bio15B flies (Fig. 1E). Expression of the bioUb construct in those three fly lines did not significantly alter total ubiquitin levels, when compared to the BirA control (Supplementary Material, Fig. S1B). Figure 1. View largeDownload slide Drosophila Ube3a mutant and over-expressing flies expressing the biotin-tagged ubiquitin. (A) Schematic representation of the (bioUb)6-BirA construct, which is expressed as a poly-ubiquitin chain fused to BirA. This precursor polypeptide is digested by endogenous DUBs, so the ubiquitin moieties and the BirA enzyme are released. Each ubiquitin bears a 16 amino-acid long biotinylatable motif at their N-terminal part (bio) that is recognized by BirA. The sequence for biotinylation added to each ubiquitin is shown underlined, followed by the five amino-acid linker (italicized). The lysine where the biotin is attached is highlighted in bold. (B) Schematic representation of the domain structure of Drosophila Ube3a. Only two domains have been characterized for Ube3a so far: the AZUL domain (Amino-terminal Zn-finger of Ube3a E3 Ligase), which is thought to play a role in substrate recognition, and the HECT domain (Homologous to the E6-AP Carboxyl Terminus) that provides the E2-binding platform and a catalytic cysteine residue (C941) to which ubiquitin associates via a thioester linkage. Flies over-express Ube3a without any tag and under the control of a UAS sequence. (C) Anti-Ube3a immunoblot on head extracts. The specific band for Ube3a is indicated with an arrow. Unspecific bands are indicated with arrowheads. (D) Anti-BirA immunoblot on head extracts. Appropriate processing of the (bioUb)6-BirA precursor was observed for all three genotypes (bioUb, bioA3 and bio15B). Flies over-expressing just BirA were used as control. (D) Anti-biotin western blot performed for each of the genotypes. In the control sample (BirA) only endogenously biotinylated proteins are detected. Figure 1. View largeDownload slide Drosophila Ube3a mutant and over-expressing flies expressing the biotin-tagged ubiquitin. (A) Schematic representation of the (bioUb)6-BirA construct, which is expressed as a poly-ubiquitin chain fused to BirA. This precursor polypeptide is digested by endogenous DUBs, so the ubiquitin moieties and the BirA enzyme are released. Each ubiquitin bears a 16 amino-acid long biotinylatable motif at their N-terminal part (bio) that is recognized by BirA. The sequence for biotinylation added to each ubiquitin is shown underlined, followed by the five amino-acid linker (italicized). The lysine where the biotin is attached is highlighted in bold. (B) Schematic representation of the domain structure of Drosophila Ube3a. Only two domains have been characterized for Ube3a so far: the AZUL domain (Amino-terminal Zn-finger of Ube3a E3 Ligase), which is thought to play a role in substrate recognition, and the HECT domain (Homologous to the E6-AP Carboxyl Terminus) that provides the E2-binding platform and a catalytic cysteine residue (C941) to which ubiquitin associates via a thioester linkage. Flies over-express Ube3a without any tag and under the control of a UAS sequence. (C) Anti-Ube3a immunoblot on head extracts. The specific band for Ube3a is indicated with an arrow. Unspecific bands are indicated with arrowheads. (D) Anti-BirA immunoblot on head extracts. Appropriate processing of the (bioUb)6-BirA precursor was observed for all three genotypes (bioUb, bioA3 and bio15B). Flies over-expressing just BirA were used as control. (D) Anti-biotin western blot performed for each of the genotypes. In the control sample (BirA) only endogenously biotinylated proteins are detected. Comparison of the ubiquitinated proteome of bioA3 flies and bioUb flies should allow the identification of proteins whose ubiquitination is enhanced by Ube3a. Conversely, we would expect to have a reduction on the ubiquitination of Ube3a substrates on bio15B flies in respect to bioUb flies (Fig. 2A). Biotinylated ubiquitin conjugates formed within the fly photoreceptor neurons were isolated using neutravidin beads, those pulldowns being performed on three biological replicates for each of the three conditions. Despite collecting whole heads, the isolated material is expected to originate just from the GMR-GAL4 expressing cells. Similar amounts of ubiquitinated proteins were eluted from the three genotypes (Fig. 2B and Supplementary Material, Fig. S2A and B). After fractionation by SDS-PAGE, each gel lane, corresponding to one sample, was cut into several slices as indicated in Supplementary Material, Fig. S2B). Protein loads from each individual gel slice were in-gel digested with trypsin and subsequently analysed by LC-MS/MS. Figure 2. View largeDownload slide Isolation of candidate Ube3a substrates in Drosophila neurons. (A) Workflow for the identification of Drosophila Ube3a substrates. Flies over-expressing the (bioUb)6-BirA precursor in the photoreceptor cells under the control of the GMR-GAL4 driver (bioUb) were compared with heterozygous Ube3a mutant (bio15B) or with Ube3a gain of function flies (bioA3) in order to identify proteins whose ubiquitination increases in a Ube3a dose-dependent manner. Fly heads of each of the genotype were subjected to biotin pulldown and MS analysis. Proteins whose ubiquitination is regulated by Ube3a should be found in more abundance in bioA3 flies, as compared with bioUb and bio15B controls. (B). Silver staining of the eluted material from biotin pulldowns. Only endogenously biotinylated proteins are detected on the BirA control sample. (C) LFQ intensities of Ube3a obtained from MS analysis of eluted samples. One asterisk indicates P-value < to 0.05; three, P < to 0.0001. (D). Western blot to Ube3a indicates that it is mostly purified in its unmodified form (arrow), which is bound to the avidin beads due to ubiquitin bound to its active-site cysteine. This ubiquitin is removed from the active site by DTT-treatment of the samples on the elution step. A small fraction of Ube3a was also found conjugated to ubiquitin (asterisk). A non-specific band is observed in all input samples (arrowhead). Figure 2. View largeDownload slide Isolation of candidate Ube3a substrates in Drosophila neurons. (A) Workflow for the identification of Drosophila Ube3a substrates. Flies over-expressing the (bioUb)6-BirA precursor in the photoreceptor cells under the control of the GMR-GAL4 driver (bioUb) were compared with heterozygous Ube3a mutant (bio15B) or with Ube3a gain of function flies (bioA3) in order to identify proteins whose ubiquitination increases in a Ube3a dose-dependent manner. Fly heads of each of the genotype were subjected to biotin pulldown and MS analysis. Proteins whose ubiquitination is regulated by Ube3a should be found in more abundance in bioA3 flies, as compared with bioUb and bio15B controls. (B). Silver staining of the eluted material from biotin pulldowns. Only endogenously biotinylated proteins are detected on the BirA control sample. (C) LFQ intensities of Ube3a obtained from MS analysis of eluted samples. One asterisk indicates P-value < to 0.05; three, P < to 0.0001. (D). Western blot to Ube3a indicates that it is mostly purified in its unmodified form (arrow), which is bound to the avidin beads due to ubiquitin bound to its active-site cysteine. This ubiquitin is removed from the active site by DTT-treatment of the samples on the elution step. A small fraction of Ube3a was also found conjugated to ubiquitin (asterisk). A non-specific band is observed in all input samples (arrowhead). Similar number of ubiquitinated proteins and a high correlation of the label-free quantification (LFQ) intensity values were detected, both between replicas and across the different genotypes (Supplementary Material, Fig. S3A and B). Random LFQ values from a distribution meant to simulate expression below the detection limit (45) were imputed to those proteins for which LFQ values were not reported by the MaxQuant software on that given experiment (Supplementary Material, Fig. S3C). The experimental design was successful as evidenced from the LFQ intensity values obtained for Ube3a, which appeared highly enriched in the bioA3 sample, and significantly reduced in the bio15B sample (Fig. 2C). Western blot analysis confirmed those results and revealed that Ube3a is mostly isolated in an unmodified form (Fig. 2D), as reported previously (44). We compared the LFQ intensities of ubiquitinated proteins on the bioA3 sample with the corresponding bioUb values and plotted their fold-changes (X-axis) and significance P-values (Y-axis) as a Vulcano plot (Fig. 3). As expected, most of the proteins detected in this study displayed a ratio close to one, including endogenously biotinylated proteins acetyl-CoA carboxylase (ACC), pyruvate carboxylase (PCB) and CG2118 (46) (shown with filled squares in Fig. 3), indicating that the pull-down process was equally efficient in the different samples. From the 751 protein groups identified across all genotypes, 79 were significantly (P < 0.05) enriched at least 2-fold in the bioA3 sample relative to the bioUb control, and can therefore be defined as putative Ube3a substrates (Supplementary Material, Table S1). In order to focus on the highest confidence Ube3a substrates, the 79 proteins regulated by Ube3a were analysed at the peptide level to comply with the following requirements: (1) Average ratio between common peptides identified in both conditions should be at least two after the subtraction of the standard error of the mean (S.E.M.); and (2) individual peptide intensities should globally follow the same tendency as the protein LFQ intensity and show in average a 2-fold enrichment. Out of the 79 proteins identified as enriched at the protein level, 39 candidate Ube3a substrates (labelled with filled circles in Fig. 3) appeared homogenously enriched also at the peptide level (Supplementary Material, Fig. S3D), most of them having human orthologues (Table 1). Among those 39 high confidence candidate Ube3a substrates, eight proteins regulate protein degradation through the ubiquitin-proteasome system, including proteasomal subunits Prosα1, Prosα3, Rpt2, Rpt4, Rpn3 and Rpn8, and the proteasomal shuttling proteins Rngo and Rpn10, the latest being previously identified as target of Ube3a in neuronal cell culture (30). Besides, two proteins related to autophagy, Atg8a and Ref(2)P (47), and three chaperone proteins, CCT3, CCT7 and CCT8 (48), were also found as high confidence substrates of Ube3a. In addition, proteasomal subunits Prosα4, Prosα7, Rpn2, proteasome activator REG and the proteasome-associated deubiquitinating (DUB) enzyme Uch-L5 were significantly enriched according to LFQ values, but did not pass the requirements at peptide level. Table 1. High confidence proteins whose ubiquitination is dependent on Ube3a over-expression Proteins whose abundance in the pulldowns is significantly altered by Ube3a, both at protein and at peptide level, are shown. The complete data set is available as Supplementary Material, Table S1. Proteasomal proteins are highlighted in bold. Cellular localization (CC) of the human proteins are indicated by graytones (dark grey rounded rectangle: nuclear; light grey cell: cytoplasmic; black surrounding square: plasma membrane). If a more specific localization within each compartment has been reported, it is further indicated with text (CT, cytoskeleton; E, endosome; ER, endoplasmic reticulum; P, peroxisomes; S, secreted; V, vesicles). a Given according to Flybase nomenclature. b Orthologues with the best Flybase score are provided. c Given according to HUGO Gene Nomenclature Committee. d Given according to Human Protein Atlas (www.proteinatlas.org) and Uniprot (www.uniprot.org). e Other M13 metallopeptidases are also considered orthologues (see Supplementary Material, Table S1). Table 1. High confidence proteins whose ubiquitination is dependent on Ube3a over-expression Proteins whose abundance in the pulldowns is significantly altered by Ube3a, both at protein and at peptide level, are shown. The complete data set is available as Supplementary Material, Table S1. Proteasomal proteins are highlighted in bold. Cellular localization (CC) of the human proteins are indicated by graytones (dark grey rounded rectangle: nuclear; light grey cell: cytoplasmic; black surrounding square: plasma membrane). If a more specific localization within each compartment has been reported, it is further indicated with text (CT, cytoskeleton; E, endosome; ER, endoplasmic reticulum; P, peroxisomes; S, secreted; V, vesicles). a Given according to Flybase nomenclature. b Orthologues with the best Flybase score are provided. c Given according to HUGO Gene Nomenclature Committee. d Given according to Human Protein Atlas (www.proteinatlas.org) and Uniprot (www.uniprot.org). e Other M13 metallopeptidases are also considered orthologues (see Supplementary Material, Table S1). Figure 3. View largeDownload slide Identification of candidate Ube3a substrates in Drosophila neurons. Comparison of the abundance, determined by their LFQ intensities, of the ubiquitinated proteins identified by MS upon Ube3a over-expression relative to bioUb flies. The Vulcano plot displays the LFQ bioA3/bioUb ratios in log2 scale (X-axis) and the t-test P-values in −log10 scale (Y-axis), determining the statistical significance (P < 0.05, horizontal grey lane) of the fold changes, for each protein. Labelled filled circles represent high confidence proteins found more ubiquitinated in the bioA3 sample than in the bioUb sample. Labelled empty circles are those found less ubiquitinated in bioA3 sample. Endogenously ubiquitinated proteins (ACC, CG2118 and PCB) are shown with filled squares, and ubiquitin with an empty square. The earlier reported putative Ube3a candidate Atpα, is shown with a triangle. bioUb: GMR-GAL4, UAS-(bioUb)6-BirA/CyO; bioA3: GMR-GAL4, UAS-(bioUb)6-BirA/CyO; UAS-Ube3aA3/TM6. Figure 3. View largeDownload slide Identification of candidate Ube3a substrates in Drosophila neurons. Comparison of the abundance, determined by their LFQ intensities, of the ubiquitinated proteins identified by MS upon Ube3a over-expression relative to bioUb flies. The Vulcano plot displays the LFQ bioA3/bioUb ratios in log2 scale (X-axis) and the t-test P-values in −log10 scale (Y-axis), determining the statistical significance (P < 0.05, horizontal grey lane) of the fold changes, for each protein. Labelled filled circles represent high confidence proteins found more ubiquitinated in the bioA3 sample than in the bioUb sample. Labelled empty circles are those found less ubiquitinated in bioA3 sample. Endogenously ubiquitinated proteins (ACC, CG2118 and PCB) are shown with filled squares, and ubiquitin with an empty square. The earlier reported putative Ube3a candidate Atpα, is shown with a triangle. bioUb: GMR-GAL4, UAS-(bioUb)6-BirA/CyO; bioA3: GMR-GAL4, UAS-(bioUb)6-BirA/CyO; UAS-Ube3aA3/TM6. On the other hand, a total of 55 proteins also appeared to be less ubiquitinated in bioA3 sample due to the over-expression of Ube3a (Supplementary Material, Table S1), displaying a 2-fold significant reduction in their abundance compared to bioUb (bioA3/bioUb < 0.5; P < 0.05). Following the same peptide analysis described before (Supplementary Material, Fig. S3E), we concluded that 14 proteins are clearly less ubiquitinated when Ube3a is over-expressed, as compared to the control sample (labelled with empty circles in Fig. 3). Interestingly, we reliably identified calcium/calmodulin-dependent protein kinase II (CaMKII)—which in Drosophila is coded by a single gene—as being less ubiquitinated upon over-expression of Ube3a. We also compared the LFQ intensities of the bio15B sample with the bioUb values; but, using the same criteria as above, Ube3a itself did not appear significantly enriched with high confidence in the bioUb sample relative to the bio15B ubiquitinated material. However, three proteins (Rdhb, Map2015 and Axo) appeared significantly less ubiquitinated in the bio15B sample, barely above the defined thresholds (data not shown). Ubiquitin LFQ values and ubiquitin chain linkages indicate proteasomal perturbation upon Ube3a over-expression Based on the experimental design, we expected ubiquitin levels to be unaltered by Ube3a over-expression. Furthermore, no significant differences were observed on the hard-to-quantify smears of the silver stained gels (Fig. 2B), as well as ubiquitin (Supplementary Material, Fig. S1B) and biotin (Supplementary Material, Fig. S2A) blots. However, based on LFQ values, ubiquitin levels were quantified to have a significant increase of 1.66-fold in the bioA3 flies, relative to the bioUb sample (Supplementary Material, Table S1), this is, a >60% increase of ubiquitin was detected on the collected ubiquitinated material of bioA3 flies. In order to elucidate the type of ubiquitin chains enriched upon over-expression of Ube3a, we compared the intensity of all detected diGly-containing peptides across the samples. Digestion of a complex mixture of ubiquitinated proteins is expected to result in a complex mixture of ubiquitin chain linkages. Even though the isolated ubiquitinated material is composed of substrates of hundreds of ligases, this analysis indicated that K48 and K63 linkages were significantly more abundant on the bioA3 sample, while K33 linkages were reduced (Supplementary Material, Fig. S3F). The 60% increase on ubiquitin levels together with the significant changes in chain linkages suggests a global perturbation of the ubiquitin proteasome system upon Ube3a over-expression. Rngo is ubiquitinated by Ube3a in vivo in Drosophila neurons Amongst the highest confidence candidates as Ube3a substrates (Table 1), eight of the identified proteins are proteasome integral or regulatory subunits. We decided to focus on Rngo, a predicted proteasomal shuttling factor for which working antibodies were available (49), and which was already observed in our first neuronal ubiquitome studies (42). According to the label-free quantitative MS-based analysis performed, Rngo is 5-fold more enriched in Ube3a over-expressing flies in respect to control flies. In agreement with that, western blot analysis showed that ubiquitination of Rngo is enhanced when Ube3a is over-expressed and reduced in its absence (Fig. 4A), making Rngo the first Ube3a substrate validated in any type of neuron in vivo. Interestingly, Rngo total levels were not altered, suggesting that it is not being targeted for degradation. As a control, the same membrane was used to detect the presence of Fax, a protein that we found by MS to be less ubiquitinated upon Ube3a over-expression (Fig. 3). Indeed, Fax displayed the opposite trend, and its ubiquitination was dramatically reduced by the over-expression of Ube3a (Fig. 4A), indicating that the increase ubiquitination seen for Rngo is specific to the protein and not to a more efficient general isolation of proteins in the bioA3 sample. We also analysed by western blot the ubiquitination of another proposed UBE3A substrate (27), the Na+/K+-ATPase α-subunit (Atpα), but as with the MS analysis (Fig. 3), western blot did not show any enhancement by Ube3a over-expression on its ubiquitination (Fig. 4B). Figure 4. View largeDownload slide Rngo is ubiquitinated by Ube3a in Drosophila photoreceptor neurons. (A) Immunoblot with anti Rngo-antibody confirmed the increase ubiquitination detected by MS in bioA3 flies as compared with bioUb flies, and more significantly to bio15B flies. This same membrane was reprobed with anti-Fax. Levels of ubiquitinated Fax were found reduced in bioA3 flies, which corroborated the MS results and confirmed that the increase ubiquitination seen for Rngo is specific to the protein and not to a more efficient general isolation of proteins in the bioA3 sample. Putative mono-, tri- and tetra-ubiquitinated forms are indicated with asterisks and unmodified or cysteine-ubiquitinated forms are indicated with an arrow. Equal levels of Avidin bands, which are non-specifically detected by Rngo antibody, are also shown. (B) Atpα is not a substrate of Ube3a in Drosophila photoreceptor cells. Western blot performed with anti-Atpα showed that its ubiquitination is not regulated by Ube3a, as levels of mono-ubiquitinated Atpα are similar, or even lower in bioA3 than in bio15B flies. The unmodified proteins are indicated with an arrow. Ubiquitinated forms are indicated with asterisks. Figure 4. View largeDownload slide Rngo is ubiquitinated by Ube3a in Drosophila photoreceptor neurons. (A) Immunoblot with anti Rngo-antibody confirmed the increase ubiquitination detected by MS in bioA3 flies as compared with bioUb flies, and more significantly to bio15B flies. This same membrane was reprobed with anti-Fax. Levels of ubiquitinated Fax were found reduced in bioA3 flies, which corroborated the MS results and confirmed that the increase ubiquitination seen for Rngo is specific to the protein and not to a more efficient general isolation of proteins in the bioA3 sample. Putative mono-, tri- and tetra-ubiquitinated forms are indicated with asterisks and unmodified or cysteine-ubiquitinated forms are indicated with an arrow. Equal levels of Avidin bands, which are non-specifically detected by Rngo antibody, are also shown. (B) Atpα is not a substrate of Ube3a in Drosophila photoreceptor cells. Western blot performed with anti-Atpα showed that its ubiquitination is not regulated by Ube3a, as levels of mono-ubiquitinated Atpα are similar, or even lower in bioA3 than in bio15B flies. The unmodified proteins are indicated with an arrow. Ubiquitinated forms are indicated with asterisks. Ddi1 and Ddi2 are expressed throughout development in the mouse brain Mammalians have two protein homologues to Drosophila Rngo: DNA damage-inducible protein 1 homologue 1 (DDI1) and homologue 2 (DDI2). Partial expression data for DDI2 are available at the Human Protein Atlas (www.proteinatlas.org; date last accessed March 19, 2018), but information about mammalian DDI1 is very scarce. We tried to assess the expression of DDI1 using commercially available antibodies, but, since DDI1 and DDI2 proteins have very high amino acid sequence identity (72%), all the commercially available antibodies recognize DDI2 protein, or do simply not work (data not shown). We therefore tested whether mouse Ddi1 and Ddi2 are expressed in the brain. Analysis of Ddi1 gene expression in mice brain at different ages performed by qRT-PCR revealed a drastic and significant increase of Ddi1 mRNA levels at embryonic stage E16.5 (Fig. 5A). The Ddi1 mRNA expression peak was rapidly reduced from E17.5, returning to basal levels at E19.5-P1, remaining relatively stable during all the tested adult time points. In contrast, Ddi2 mRNA levels, which were also detectable in the mice brain, only fluctuated slightly during development (Fig. 5B). Figure 5. View largeDownload slide Ddi1 and Ddi2 gene expression temporal profiles in the mouse brain. Changes in expression of Ddi1 (A) and Ddi2 (B) mRNA levels in brains of C57BL/6J mice during aging. RNA was isolated from the brains of E13.5 to 9-month-old mice and subjected to qRT-PCR. A significant increase of Ddi1 mRNA level was observed at embryonic stage E16.5. Ddi1 and Ddi2 mRNA levels were determined and adjusted by the signal intensity of GAPDH, and the average results (n = 3) were calculated and expressed with respect to the values obtained in E13.5. Figure 5. View largeDownload slide Ddi1 and Ddi2 gene expression temporal profiles in the mouse brain. Changes in expression of Ddi1 (A) and Ddi2 (B) mRNA levels in brains of C57BL/6J mice during aging. RNA was isolated from the brains of E13.5 to 9-month-old mice and subjected to qRT-PCR. A significant increase of Ddi1 mRNA level was observed at embryonic stage E16.5. Ddi1 and Ddi2 mRNA levels were determined and adjusted by the signal intensity of GAPDH, and the average results (n = 3) were calculated and expressed with respect to the values obtained in E13.5. Additionally, we analysed the expression profile of Ddi1 in the developing brain by performing RNA in-situ hybridization experiments in CD-1 mouse embryos at four different stages of development—E9.5, E10.5, E14.5 and E16.5. As shown in Figure 6, Ddi1 is expressed in all parts of developing brain (telencephalon, diencephalon, mesencephalon and rhombencephalon) at the two younger developmental stages. Similar Ddi1 expression pattern was observed for these two stages in both whole mount samples (Fig. 6A and C) and sagittal paraffin sections (Fig. 6B and D). At the stage E14.5, the expression is located in neurons of mesencephalic and telencephalic structures, with signal accumulation in upper hill (colliculus tectum) and ventricular zone of pallium (Fig. 6E–G). At further developmental stage E16.5, which shows highest mRNA level expression according to our qRT-PCR screen, Ddi1 is expressed in particular telencephalon parts, mainly the ventricular layer and cortical plate of isocortex and the ventricular layer of olfactory bulb (Fig. 6H and I). This could represent the dividing neuroblasts and their migration toward the superficial layer of isocortex. The positive staining of neuronal cells in the isocortex (Fig. 6J) is clear evidence of Ddi1 expression in neuronal tissue, however, up to date no single report has described the role DDI1 could exert in the brain. Figure 6. View largeDownload slide Spatial profile of Ddi1 gene expression in the mouse brain. Ddi1 is expressed in central nervous system during embryonic development according to RNA in-situ hybridization study on CD-1 mouse embryos. (A) and (B) Expression profile of Ddi1 in whole mount and paraffin sections of E9.5 embryo, respectively. (C) and (D) Ddi1 expression mapping in whole mount and paraffin section of E10.5 embryos. (E) Sagittal section of E14.5 embryonic brain with clearly located expression of Ddi1 in colliculus midbrain tectum and pallial part of telencephalon. (F) Detail of pallium: Ddi1 is expressed in isocortex and olfactory bulb. (G) Midbrain and hindbrain tissue shows expression of Ddi1 in culliculus tectum. (H) and (I) Ddi1 is specifically expressed in isocortex and ventricular layer of olfactory bulb of E16.5 mouse brain. (J) High resolution detail of the scan shown in (I). Red stars highlight tubular structures—probably capillaries of the central nervous system. Abbreviations: T, telencephalon; D, diencephalon; M, mesencephalon; R, rhombencephalon; C, cerebellum; CMT, colliculus midbrain tectum; TL, thalamus; P, pallium; SP, subpallium; HT, hypothalamus; BG, basal ganglia; OB, olfactory bulb; IC, isocortex; NC, nasal cavity. Figure 6. View largeDownload slide Spatial profile of Ddi1 gene expression in the mouse brain. Ddi1 is expressed in central nervous system during embryonic development according to RNA in-situ hybridization study on CD-1 mouse embryos. (A) and (B) Expression profile of Ddi1 in whole mount and paraffin sections of E9.5 embryo, respectively. (C) and (D) Ddi1 expression mapping in whole mount and paraffin section of E10.5 embryos. (E) Sagittal section of E14.5 embryonic brain with clearly located expression of Ddi1 in colliculus midbrain tectum and pallial part of telencephalon. (F) Detail of pallium: Ddi1 is expressed in isocortex and olfactory bulb. (G) Midbrain and hindbrain tissue shows expression of Ddi1 in culliculus tectum. (H) and (I) Ddi1 is specifically expressed in isocortex and ventricular layer of olfactory bulb of E16.5 mouse brain. (J) High resolution detail of the scan shown in (I). Red stars highlight tubular structures—probably capillaries of the central nervous system. Abbreviations: T, telencephalon; D, diencephalon; M, mesencephalon; R, rhombencephalon; C, cerebellum; CMT, colliculus midbrain tectum; TL, thalamus; P, pallium; SP, subpallium; HT, hypothalamus; BG, basal ganglia; OB, olfactory bulb; IC, isocortex; NC, nasal cavity. Human DDI1 is ubiquitinated by UBE3A, but is not targeted for degradation Once identified and confirmed that Ube3a ubiquitinates Rngo in flies, we aimed to test whether its homologs DDI1 and DDI2 are substrates of human UBE3A. For that purpose, we employed an in cellulo ubiquitination assay (30,50) in SH-SY5Y neuroblastoma cells. Wild-type UBE3A (UBE3AWT) induced the ubiquitination of DDI1-GFP in SH-SY5Y cells (Fig. 7 and Supplementary Material, Fig. S5A), but did not seem to increase the ubiquitination status of DDI2-GFP (Supplementary Material, Fig. S5B). We also confirmed the specificity of UBE3A-dependent DDI1 ubiquitination by proving that Parkin, the E3 ligase involved in Parkinson’s disease, could not mediate ubiquitination of DDI1 (Supplementary Material, Fig. S5B). Altogether, the data presented here demonstrates for the first time that Rngo human homologue DDI1 is a UBE3A substrate. Despite earlier reports suggested that UBE3A generates degradation-leading K48 ubiquitin linkages in vitro (15), at least in SH-SY5Y neuroblastoma cells, UBE3A-mediated ubiquitination of human DDI1 does not lead to its degradation, as indicated by the intensity of the DDI1-GFP bands, which is independent of the activity of UBE3A (Fig. 7). Figure 7. View largeDownload slide Human DDI1 is ubiquitinated by UBE3A in SH-SY5Y cells. (A) DDI1-GFP showed a significant increase in its ubiquitinated fraction in the presence of wild type UBE3A (UBE3AWT), as illustrated by western blot to FLAG-tagged ubiquitin, compared with control (pCDNA3.1) or to ligase dead UBE3A (UBE3ALD). The non-modified form of DDI1 was detected with anti-GFP antibody (green). The bottom panel shows levels of human UBE3A protein in the whole cell extract before the isolation of the GFP-tagged proteins. (B) Quantification of the ubiquitination of DDI1 was performed by calculating the FLAG: GFP ratio in panel A with Image-J. Statistical significance differences [***, P < 0.001 (mean ± S.E.M., n = 5)] were observed for the UBE3AWT sample relative to both control (pCDNA3.1) and UBE3ALD samples. Figure 7. View largeDownload slide Human DDI1 is ubiquitinated by UBE3A in SH-SY5Y cells. (A) DDI1-GFP showed a significant increase in its ubiquitinated fraction in the presence of wild type UBE3A (UBE3AWT), as illustrated by western blot to FLAG-tagged ubiquitin, compared with control (pCDNA3.1) or to ligase dead UBE3A (UBE3ALD). The non-modified form of DDI1 was detected with anti-GFP antibody (green). The bottom panel shows levels of human UBE3A protein in the whole cell extract before the isolation of the GFP-tagged proteins. (B) Quantification of the ubiquitination of DDI1 was performed by calculating the FLAG: GFP ratio in panel A with Image-J. Statistical significance differences [***, P < 0.001 (mean ± S.E.M., n = 5)] were observed for the UBE3AWT sample relative to both control (pCDNA3.1) and UBE3ALD samples. Discussion Our unbiased proteomic analysis for the identification of differentially ubiquitinated proteins in Drosophila photoreceptor neurons upon Ube3a over-expression has resulted in a list of 79 putative Ube3a substrates, out of which proteasomal proteins appear very highly enriched. To our knowledge, this is the first time a list of candidate Ube3a substrates, whose ubiquitinated fraction is enhanced upon Ube3a over-expression, is reported in neurons in vivo. Several of the putative Ube3a substrates identified (Arc1, Chc, Gclc, GlyRS, Path, Tig and SesB) play a role either in axon and dendrite morphogenesis (51–54) or synaptic transmission (55–57). Interestingly, upon Ube3a over-expression we also found a significant reduction of the ubiquitination of CaMKII, a key kinase known in humans to regulate neurotransmitter synthesis and release, modulation of ion channel activity, neurite extension, synaptic plasticity, learning and memory (58). Reduced activity and protein levels of CaMKII at the postsynaptic density have been described in a mouse model of AS (59). It still remains to be explained how a reduction of UBE3A levels enhances the ubiquitination and/or degradation of CaMKII. Analysis of the putative Ube3a substrates by g:Profiler analysis of GO terms and KEGG pathways indicate a highly significant enrichment of the proteasome (data not shown). Furthermore, comparison of the data obtained in this work to the dataset of Parkin substrates (43), further confirms a deregulation of the proteasome upon Ube3a over-expression. Indeed, UBE3A is known to be a proteasome-associated protein (28,60), as well as being capable of ubiquitinating several proteasomal subunits in cell culture (28–30). However, it is controversial whether UBE3A inhibits (28) or stimulates (33) the proteolytic activity of the proteasome. Ube3a over-expression results in an increase in total ubiquitin levels, while over-expression of Parkin did not. Having identified several proteasomal subunits as ubiquitinated by Ube3a, the simplest explanation is that Ube3a-driven proteasomal deregulation results in the accumulation of substrates ubiquitinated by Ube3a or other E3 ligases. This would also explain the reduction observed here in the ubiquitination of many other proteins. It is known that the specific deregulation of the proteasome by over-expressing a dominant negative Rpn10 subunit reduces the ubiquitination levels of Fax and other mono-ubiquitinated proteins (44). Deubiquitination of mono-ubiquitinated proteins is also seen when inhibiting the proteasome pharmacologically (35), and is explained by a reduction of the free ubiquitin available pool concomitant to the accumulation of proteasome targeted poly-ubiquitinated proteins (36,61–63). The decrease on Fax ubiquitination upon Ube3a over-expression, detected here by both MS (Fig. 3) and immunoblotting (Fig. 4A), could therefore be caused by an Ube3a-induced proteasomal inhibition. Significant changes in global ubiquitin chain linkages (Supplementary Material, Fig. S3F) are also in line with this interpretation. In this work, we have validated by western blot that Rngo is a direct Ube3a substrate in photoreceptor neurons. Rngo contains both an UBL and an Ubiquitin-binding domain (UBD), a hallmark of proteasomal shuttles (49), but can also bind directly another proteasomal shuttle protein, Rpn10 (49). The ubiquitination of Rpn10 is also increased in Ube3a over-expressing flies (Fig. 3), and was already identified as an Ube3a substrate in Drosophila cells, as well as being shown to interact genetically with Ube3a in neurons in vivo (30). Ubiquitination of such proteasomal regulators by Ube3a can be predicted to severely interfere with proteasomal function. Considering that the proteasome regulates dendritic development (32), long-term potentiation (64), long-term depression (65), synaptic plasticity (66), synaptic strengthening (67), memory consolidation (68), circadian rhythms (69) and many other aspects of neuronal function, an UBE3A-dependent proteasomal regulation could easily explain how a single E3 ligase mutation can cause a disorder as complex as AS. And indeed, it should be noted that UBE3A has been shown to regulate most aspects of neuronal function listed above (70–74). Non-degradative ubiquitination of proteasomal receptors could alter their function to a similar extent as their degradation, since the activity of those receptors is dependent on their UBDs. An ubiquitinated ubiquitin-binding subunit is likely to prioritirily bind its own ubiquitin moieties, therefore blocking its normal function. Our prediction would be that—based on the increased ubiquitination of those subunits upon UBE3A over-expression—proteasome activity should be increased in the brain of AS patients, on which UBE3A levels are reduced. Interestingly, Rngo is also the Drosophila homologue of yeast Ddi1/Vsm1, a protein that binds to several Snc-interacting t-SNAREs (75), negatively regulates exocytosis (76) and also regulates protein secretion (77). Similarly, the C. elegans homologue DDI-1/VSM-1 has also been proposed to regulate synaptic function, with vsm-1 mutants displaying a significant increase in synaptic density along the dorsal nerve cord (78). Thus, Rngo/DDI1 may have an additional role in synaptic transmission by controlling SNARE mediated exocytosis. In fact, over-expression of Ube3a, but not its ligase dead form, has been reported to alter neurotransmission at the neuromuscular junction in Drosophila (79). Two mammalian Rngo homologues have been described, DDI1 and DDI2, but neither of those proteins have to date been functionally characterized. We have found that both are expressed in the developing brain (Figs 5 and 6). Structurally, both proteins contain the characteristic Retroviral Protease-like domain that was recently reported to cleave/activate the Nrf1 transcription factor under proteasome inhibition (80,81). Further, they both contain an additional helical HDD domain (82,83) and an N-terminal UBL domain, but lack the well-defined Ubiquitin-associated (UBA) domain. Although, a weak Ubiquitin-binding motif (UIM) is present at the C-terminus of DDI2 (82), no such a motif can be found in DDI1. It is thus feasible that the DDI1 UBL domain, which in yeast Ddi1 is capable of binding ubiquitin (84), could substitute the role of the UBA domain, a mechanisms that would be facilitated by DDI1’s homodimeric conformation. Having confirmed the ubiquitination of Rngo in photoreceptor neurons by the Drosophila Ube3a and the expression of its mammalian homologues in the brain, we then tested whether any of Rngo’s human orthologues are ubiquitinated by UBE3A. We confirmed DDI1 to be an ubiquitination substrate of UBE3A in SH-SY5Y neuroblastoma cells. Different controls indicated that ubiquitination of DDI1 by UBE3A is specific, but does not lead to a reduction of DDI1 protein levels. It is not the first time that regulation of protein activity, even with formation of K48-linked chains, has been reported to be proteolysis independent (85). A UBD found in Met4 was proposed to cap its own K48-linked ubiquitin chain, inactivating the protein and protecting it from degradation (86), and something similar could be happening with Rngo/DDI1. Further work is required to elucidate what the functional role of DDI1 ubiquitination might be and whether the presence of UBD/UIM/UBA/UBL in ubiquitination substrates might interfere into the canonical role of K48 ubiquitin-linked chains. Up to now, no candidate ubiquitination substrates of UBE3A had been directly validated in neurons in vivo. We had earlier identified a proteasomal shuttling factor, Rpn10, to be regulated by Ube3a in Drosophila cells (30), which we now confirmed in vivo by MS. Further, we have validated that in Drosophila photoreceptor neurons Ube3a ubiquitinates another proteasomal shuttling factor, Rngo, becoming the first Ube3a substrate to be identified and validated in vivo in neurons within a whole organism. More importantly, UBE3A regulates the orthologue DDI1 protein in human neuroblastoma cells. Since UBE3A ubiquitination appears to be regulating several proteasomal-associated subunits, as already indicated by ourselves and others (29,30,33), it would not be surprising to see that a highly significant number of proteins regulated downstream the proteasome will display significant changes on their abundance upon UBE3A mutation or over-expression. It is now a challenge to elucidate which Ube3a substrates are direct, in addition to the proteasomal proteins themselves. DDI1 gene has been reported to be affected in siblings of a familial neurodegenerative disorder characterized clinically as a variant of Alzheimer’s disease (87). Having now described for the first time the temporal and spatial expression of Ddi1 in the mouse brain, we next need to perform a functional characterization to uncover the neuronal role of DDI1, as this might as well bring light to our understanding of how AS is regulated. Based on studies in yeast and C. elegans, it is likely that DDI1 is involved in both regulation of synapses and proteasomal function (75–78,84). Given the complexity of neuronal function, and previous work aiming to identify the mechanisms regulated by UBE3A, it is likely that both processes are actually misregulated during the genesis of AS. Finally, if we take into account that Ube3a regulates the proteasome, and that UBE3A expression declines with age (88), it would not be surprising that this E3 ligase has a further role in proteostasis not yet characterized. Materials and Methods Drosophila stocks and sample collection We have used in this work the BirA, bioUb, bioA3 and bio15B flies, all of which express their corresponding constructs in the Drosophila photoreceptor neurons under the control of the GMR-GAL4 driver. BirA and bioUb flies, expressing respectively the BirA enzyme alone and the (bioUb)6-BirA precursor, have been described previously (44). Their genotypes are respectively GMR-GAL4/CyO; UAS-BirA/TM6 and GMR-GAL4, UAS-(bioUb)6-BirA/CyO. Ube3a gain of function (UAS-Ube3aA3) and loss of function (Ube3a15B) flies (89) were a gift from Professor Janice Fisher. Both UAS-Ube3aA3 and Ube3a15B fly lines were independently mated to GMR-GAL4, UAS-(bioUb)6-BirA/CyO; TM2/TM6 flies to generate GMR-GAL4, UAS-(bioUb)6-BirA/CyO; UAS-Ube3aA3/TM6 and GMR-GAL4, UAS-(bioUb)6-BirA/CyO; Ube3a15B/TM6 lines. The Drosophila Ube3a mutants (Ube3a15B) had been reported to be viable and fertile in homozygosis (89). When combined with bioUb flies, it was, however, required to grow them in heterozygosis, as null Ube3a flies were viable but not fertile at 25°C (J. Ramirez and U. Mayor, unpublished data). GMR-GAL4 (BL 1104) and OregonR (BL 2376) flies were provided by the Bloomington Drosophila Stock Center (Bloomington, IN, USA). The bioUb abbreviation is used throughout the text to refer to the GMR-GAL4, UAS-(bioUb)6-BirA/CyO flies, and the bioA3 and bio15B (for Ube3aA3 and Ube3a15B alleles) to refer to GMR-GAL4, UAS-(bioUb)6-BirA/CyO; UAS-Ube3aA3/TM6 and GMR-GAL4, UAS-(bioUb)6-BirA/CyO; Ube3a15B/TM6 flies, respectively. Flies were grown at 25°C in 12 h light-dark cycles in standard Drosophila medium (0.9% agar, 7.5% dextrose, 6% corn flour, 8.5% yeast, 2.5% Nipagin, 0.4% propionic, 0.02% benzalkonium chloride in distilled H2O). Mixed-sex flies of 2–5 days old were flash-frozen in liquid nitrogen and shaken while still frozen to sever the heads. Frozen fly heads were then separated from the remaining body parts using a pair of sieves with a nominal cut-off of 710 and 425 µm, and then stored at −80°C. Head collections were typically performed in the morning. Plasmids Commercial pEGFP-N1 vector (Clontech) was used to generate DDI1-GFP and DDI2-GFP vectors (see Cloning procedures section). FLAG-UBE3A-pCMV (UBE3AWT) and FLAG-UBE3ALD-pCMV (UBE3ALD) plasmids, expressing N-terminally FLAG-tagged versions of the wild type and catalytically inactive human UBE3A protein (90), were a gift from Dr Vjekoslav Tomaić. FLAG-tagged ubiquitin (30) in pCDNA3.1 vector (FLAG-Ub) was generously provided by Dr Jose Antonio Rodriguez Pérez (University of the Basque Country-UPV/EHU, Spain). Untagged human Parkin plasmid has been described previously (43). Empty pCDNA3.1 vector (Invitrogen) was used as control. Cloning procedures DDI2-pEGFP-N1 (DDI2-GFP) plasmid was generated by amplifying DDI2 gene (Uniprot Q5TDH0) from DDI2-pET16b plasmid (82) with DDI2-Fw (5′-AAGGTACCATGCTGCTCACCGTG-3′) and DDI2-Rv (5′-AAGGATCCCCTGGCTTCTGACGCTCTGC-3′) primers and inserted between KpnI and BamHI restriction sites of pEGFP-N1 vector (Clontech). Gene for human DDI1 protein (Uniprot Q8WTU0) was synthesized by GenScript and further amplified using the DDI1-Fw (5′-TATAGGTACCATGCTGATCACCGTG-3′) and DDI1-Rv (5′-TATAACCGGTATGCTCTTTTCGTCC-3′) primers and inserted between the Acc65I and AgeI sites of the DDI2-pEGFP-N1 vector, after the DDI2 gene had been removed using the same restriction enzymes. All PCR reactions were carried out with Phusion High-Fidelity DNA polymerase (Thermo Scientific). PCR product gel extractions and plasmid purifications were performed with the QIAGEN Gel Extraction Kit and QIAGEN plasmid mini and midi kits, respectively. Correct sequence for all plasmids was confirmed by sequencing either by the GATC Biotech Company (Köln, Germany) or the SGIKER Unit of Sequencing and Genotyping at the University of the Basque Country (Leioa, Spain). Western blotting and silver staining Both 4–12% Bolt Bis–Tris Plus pre-cast gels (Invitrogen) and 4–12% NuPAGE Bis–Tris gels (Invitrogen) were used for SDS-PAGE, then proteins were transferred to PVDF membranes using the iBlot system (Invitrogen). Following primary and secondary antibody incubation, membranes were developed with an ECL kit (Biorad Clarity). Dual-colour westerns were prepared by assigning independent colour channels to two independent westerns developed in the same membrane. The amount of material loaded for western blot analysis varied according to the tissue and the antibody employed. In the case of material obtained from fly biotin pulldowns, between 0.001% and 0.2% of the input samples and 5–10% of the elution samples were loaded. However, when material purified from cells was used, between 10–20% of inputs and 10–40% of the elution samples were loaded. The following primary antibodies were used: goat anti-biotin-horseradish peroxidase (HRP)-conjugated antibody (Cell Signalling; catalogue number 7075) at 1: 1000; chicken polyclonal anti-BirA antibody (Sigma; catalogue number GW20013F) at 1: 1000; mouse monoclonal anti-GFP antibody (Roche Applied Science; catalogue number 11814460001) at 1: 1000; mouse monoclonal anti-FLAG M2-HRP conjugated antibody (Sigma; catalogue number A8592) at 1: 1000; mouse monoclonal anti-Syx1A antibody (Developmental Studies of Hybridoma Bank; DSHB; catalogue number 8C3) at 1: 100; rabbit polyclonal anti-Fax antibody, a gift from Eric Liebl (Deninson University, OH, USA) at 1: 1000; mouse monoclonal anti-Atpα antibody (DSHB; catalogue number α5) at 1: 50; rabbit polyclonal anti-Ube3a antibody (91) for the detection of Drosophila Ube3a protein at 1: 1000; rabbit polyclonal anti-Rngo antibody (49) at 1: 500; rabbit polyclonal anti-ubiquitin antibody (Sigma; catalogue number U5379) at 1: 100; mouse monoclonal anti-UBE3A (clone E6AP-300) antibody (Sigma; catalogue number E8655) for the detection of human UBE3A protein at 1: 1000. The following secondary antibodies were used: goat anti-mouse-HRP-labelled antibody (Thermo Scientific; catalogue number 62-6520) at 1: 4000; goat anti-rabbit-HRP labelled antibody (Cell Signalling; catalogue number 7074) at 1: 4000 and donkey anti-chicken-HRP labelled antibody (Jackson ImmunoResearch; catalogue number 703-035-155) at 1: 2000. About 10% of the neat elution samples were used for silver staining analysis. Gels were fixed for 1 h at room temperature with 40% methanol and 10% acetic acid containing solution and then were stained using the SilverQuest kit from Invitrogen according to manufacturer’s instructions. Biotin pulldown Biotin pulldowns (42) from Drosophila heads were performed as described previously (43,44,92). About 500 mg of 2–5 days old fly heads of each genotype were homogenized in 2.9 mL of Lysis buffer (8 M urea and 1% SDS in PBS) supplemented with 50 mM N-ethylmaleimide (Sigma) and a complete protease inhibitor cocktail (Roche Applied Science). Lysates were centrifuged for 5 min at 16000g at 4°C and supernatant applied to a PD10 desalting column (GE Healthcare) previously equilibrated with 25 ml of binding buffer (3 M urea, 1 M NaCl, 0.25% SDS and 50 mM N-ethylmaleimide). Eluates, except 50 µl that were kept for monitoring the inputs, were then incubated with 250 µl of NeutrAvidin agarose beads suspension (Thermo Scientific). Unbound material (flow through) was separated by spinning the beads at 230g for 2 min. Beads were then subjected to stringent washes with six different washing buffers (WB): twice with WB1 (8 M urea, 0.25% SDS), thrice with WB2 (6 M guanidine-HCl), once with WB3 (6.4 M urea, 1 M NaCl, 0.2% SDS), thrice with WB4 (4 M urea, 1 M NaCl, 10% isopropanol, 10% ethanol, 0.2% SDS), once with WB1, once with WB5 (8 M urea, 1% SDS) and thrice with WB6 (2% SDS). All buffers were prepared in PBS. Beads were then heated at 95°C for 5 min in 125 µl of elution buffer (250 mM Tris–HCl pH 7.5, 40% glycerol, 4% SDS, 0.2% BPB, 100 mM DTT) and centrifuged for 2 min at 16 000g in a Vivaclear Mini 0.8 µm PES micro-centrifuge filter unit (Sartorius) to recover the eluted proteins. Finally, eluates were concentrated in Vivaspin 500 centrifugal filter units (Sartorius). Cell culture and transfection Human neuroblastoma SH-SY5Y cells were cultured under standard conditions (37°C, 5% CO2) in Dulbecco’s modified Eagle medium/nutrient mixture F-12 (DMEM/F-12) with GlutaMAX (Thermo Scientific), supplemented with 10% fetal bovine serum (Thermo Scientific), 100 U/ml of penicillin (Invitrogen) and 100 µg of streptomycin (Invitrogen). SH-SY5Y cells (3 × 105 cells) were seeded in six well-plates for transfection experiments. Overnight incubation under serum starvation was routinely performed prior to transfections. The following day OptiMEM serum-free medium (Thermo Scientific) was replaced by fresh DMEM/F-12 and cells were co-transfected with 1 μg of FLAG-Ub and 1 μg of DDI1-GFP, or DDI2-GFP, for 72 h using Lipofectamine 3000 transfection reagent (Invitrogen) according to manufacturer’s instructions. About 1 µg of either pcDNA3.1 (control), UBE3AWT, UBE3ALD or Parkin plasmids were additionally added to the transfection mixture to check the effect of UBE3A in DDI1 and DDI2 ubiquitination. Cells were washed twice in PBS and stored at −20°C until required. GFP beads pull-down assay Transfected SH-SY5Y cells were lysed with 500 μl of lysis buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% Triton, 1× protease inhibitor cocktail from Roche Applied Science and 50 mM N-ethylmaleimide from Sigma) and centrifuged at 14 000g for 10 min. Supernatants were mixed with 25 μl of GFP-Trap-A agarose beads suspension (Chromotek GmbH), which had been previously washed twice with a Dilution buffer (10 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 1× protease inhibitor cocktail, 50 mM N-ethylmaleimde). The mixture was then incubated at room temperature for 150 min with gentle rolling and centrifuged for 2700g for 2 min to separate the beads from the unbound material. GFP beads were subsequently washed once with the dilution buffer, thrice with washing buffer WB5 (8 M urea, 1% SDS in PBS) and once with 1% SDS in PBS. Bound GFP-tagged proteins were eluted in 25 µl of elution buffer (250 mM Tris–HCl pH 7.5, 40% glycerol, 4% SDS, 0.2% BPB, 100 mM DTT) by heating at 95°C for 10 min. In-gel trypsin digestion and peptide extraction Eluates from biotin pull-down assays were resolved by SDS-PAGE using 4–12% Bolt Bis–-Tris Plus pre-cast gels (Invitrogen) and visualized with Colloidal Blue following manufacturer’s instructions (Invitrogen). When processing biotin pull-down samples, each gel lane was cut into seven slices (see Supplementary Material, Fig. S2B). Based on earlier experiments and the BirA control, we excluded the intense bands corresponding to avidin monomers, dimers and an endogenously biotinylated protein from further analysis. The remaining four slices were subjected to in-gel digestion as described previously (93). Briefly, proteins were reduced and alkylated by incubating with DTT and chloroacetamide, respectively. Protein digestion was performed by saturation of the gel pieces with trypsin and overnight incubation at 37°C. Resulting peptides were extracted from the gel, dried down in a vacuum centrifuge and stored at −20°C. Peptide mixture was resuspended in 0.1% formic acid previous to the LC-MS/MS analysis. LC-MS/MS analysis Mass spectrometric analyses were performed on an EASY-nLC 1000 liquid chromatography system interfaced with a Q Exactive mass spectrometer (Thermo Scientific) via a nanospray flex ion source. Peptides were loaded onto an Acclaim PepMap100 pre-column (75 µm × 2 cm, Thermo Scientific) connected to an Acclaim PepMap RSLC (50 µm ×x 15 cm, Thermo Scientific) analytical column. Peptides were eluted from the column using a linear gradient of 2 to 40% acetonitrile in 0.1% formic acid at a flow rate of 300 nL min−1 over 45 min. The mass spectrometer was operated in positive ion mode. Full MS scans were acquired from m/z 300 to 1850 with a resolution of 70 000 at m/z 200. The 10 most intense ions were fragmented by higher energy C-trap dissociation with normalized collision energy of 28 and MS/MS spectra were recorded with a resolution of 17 500 at m/z 200. The maximum ion injection time was 120 ms for both survey and MS/MS scans, whereas AGC target values of 3 × 106 and 5 × 105 were used for survey and MS/MS scans, respectively. In order to avoid repeat sequencing of peptides, dynamic exclusion was applied for 45 s. Singly charged ions or ions with unassigned charge state were also excluded from MS/MS. Data were acquired using Xcalibur software (Thermo Scientific). Data processing and bioinformatics analysis Acquired raw data files were processed with the MaxQuant (94) software (version 1.5.3.17) using the internal search engine Andromeda (95) and searched against the UniProt database restricted to Drosophila melanogaster entries (release 2015_11; 43712 entries). Spectra originated from the different slices corresponding to the same biological sample were combined. Carbamidomethylation (C) was set as fixed modification whereas Met oxidation, protein N-terminal acetylation and Lys GlyGly (not C-term) were defined as variable modifications. Mass tolerance was set to 8 and 20 ppm at the MS and MS/MS level, respectively. Enzyme specificity was set to trypsin, allowing for cleavage N-terminal to Pro and between Asp and Pro with a maximum of two missed cleavages. Match between runs option was enabled with 1.5 min match time window and 20 min alignment window to match identification across samples. The minimum peptide length was set to seven amino acids. The false discovery rate for peptides and proteins was set to 1%. Normalized spectral protein label-free quantification (LFQ) intensities were calculated using the MaxLFQ algorithm. Data analysis and statistical tests MaxQuant output data was analysed with the Perseus module (version 1.5.6.0) (45). Initially, proteins only identified by site, contaminants, reverse hits and proteins with no unique peptides and/or no intensity were removed. Missing LFQ intensity values were replaced with values from a normal distribution (width 0.3 and down shift 1.8), meant to simulate expression below the detection limit (45). To determine statistically significant changes in protein abundance, as well as in ubiquitin diGly peptides, two-tailed Student’s t-test was used. In the analysis of the biotin pulldowns, two comparisons were carried out: bioA3 versus bioUb (i.e. Ube3a gain of function versus control) and bio15B versus bioUb (i.e. Ube3a loss of function versus control). Proteins displaying a LFQ fold change bigger than 2 with a P-value smaller than 0.05 were selected for further analysis. The selected proteins were further filtered based on the intensity pattern observed for their peptides. Statistical significance in western blotting semi-quantification was evaluated using an analysis of variance (ANOVA) complemented by Tukey’s honest significance difference test (Tukey’s HSD) performed in GraphPad PRISM software. RNA extraction, reverse transcription and real-time qPCR Total RNA was isolated from 30 mg of brain from embryos, young and adult wild-type mice from different ages. RNA was extracted and further purified by using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s protocol. Contaminating genomic DNA was removed by treatment with deoxyribonuclease I (QIAGEN), and cDNAs were synthesized from 1 μg RNA using the AffinityScript Multi Temperature cDNA Synthesis Kit (Agilent Technologies). Quantitative RT-PCR was performed on cDNA in the presence of Power SYBR Green PCR Master Mix (Applied Biosystems) containing preset concentrations of deoxynucleotide triphosphates and with specific primers, using the ABI Prism 7900 sequence Detection System (Applied Biosystems). PCR parameters were 50°C for 2 min, 95°C for 10 min, 40 cycles at 95°C for 15 s and 60°C for 1 min. The purity of the PCR products was assessed by dissociation curves. The amount of target cDNA was calculated by the comparative threshold (Ct) method and expressed by the 2-ΔΔCt method according to Applied Biosystems’ instructions, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. Expression of GAPDH mRNA was not affected by age, and the ratio of ΔCt value did not vary with the amount of cDNA. Each primer set was used at its optimal concentration (300 nM) with maximal efficacy. It was verified that one single specific product was amplified as shown by analysis of its melting temperature value. Primers GAPDH—Forward: 5′-ACCACAGTCCATGCCATCAC-3′  Reverse: 5′-TCCACCACCCTGTTGCTGTA-3′ Primers DDI1—Forward: 5′-TCACTGTGTATTGTGTGCGTAG-3′  Reverse: 5′-AGCTGTTCCATGTAAACGATCTG-3′ Primers DDI2—Forward: 5′-CCTCTCCGAGGTGACCTTTTC-3′  Reverse: 5′-GGCCTTTCTGCATAGACAATCT-3′ RNA in situ hybridization CD-1 mice were mated overnight, and the presence of a vaginal plug indicated embryonic day (E) 0.5. Unsexed embryos at E9.5 and E10.5 (the age 9.5 and 10.5 days post coitum) were fixed in 4% PFA and used for in situ hybridization, both in whole mount and in paraffin sections. Heads of 14.5 and 16.5 days post coitum old CD-1 embryos were fixed in 4% PFA and further processed for paraffin sections. Embryo samples for whole mount ISH were frozen in methanol at −20°C prior tissue hydration, proteinase K treatment, acetylation and the prehybridization and hybridization procedures. The embryos and heads used for sectioning were dehydrated, embedded in paraffin, cut to 7 µm sections and rehydrated prior further treatment. The experimental procedures were performed according to standard protocols (96). Murine Ddi1 coding sequence (NM_027942.1) was synthesized by GenScript and cloned into pGEM-T® easy plasmid. Linearized plasmid was purified with PCR Purification Kit (QIAGEN) and used for generation of digoxigenin labelled riboprobes with digoxigenin RNA labelling kit (Roche Applied Science) by in vitro transcription according to the provided manual. Probes were further cleaned by RNeasy Mini kit (QIAGEN) following RNA clean-up manual. Hybridization was performed overnight at 70°C with all probes for both whole mount and sectioned embryo samples. The DIG labelled probes were detected with anti-DIG antibody conjugated with alkaline phosphatase and BM purple AP substrate precipitating solution (Roche Applied Science) was used for signal development. All samples were postfixed in 4% PFA and the slides were mounted in Aquatex. Images were taken using Zeiss ApoTome microscope. Cell staining and microscopy SH-SY5Y cells in Supplementary Material, Fig S5C were transfected using Lipofectamine 3000 transfection reagent (Invitrogen) and grown over a coverslip glass. After 48 h, cells were washed twice with 2× PBS, fixed in 4% PFA for 20 min at room temperature, washed with PBST (0.1% Triton X-100 in 1× PBS) for three times and nuclei were stained with NucBlue Fixed Cell Stain Readyprobes reagent (Invitrogen). After staining, cells were washed with 1× PBS three times, the cover slips were mounted into slides with ProLong Diamond antifade reagent (Invitrogen), and samples were analysed in an inverted microscope ECLIPSE TS2-FL (Nikon). Supplementary Material Supplementary Material is available at HMG online. Acknowledgements The authors are grateful of the technical support provided by UPV/EHU SGIker and Animal house staff, which are supported with European funding (ERDF and ESF). In particular we thank Arantza Alejo and the Animal Unit at UPV/EHU. We thank Janice Fisher, Fen-Biao Gao, Eric Liebl, Jose Antonio Rodríguez Pérez, Vjekoslav Tomaić and Andreas Wodarz for antibodies, flies and plasmids. Conflict of Interest statement. None declared. Funding This work was supported by March of Dimes [Research Grant 1-FY15–339]; by ISCIII (grants PRB2 IPT13/0001 - ISCIII-SGEFI/ERDF and PRB3 IPT17/0019 - ISCIII-SGEFI/ERDF) and Spanish MINECO [grants SAF2013–44782-P and SAF2016–76898-P, both cofinanced with FEDER funds, to UM, BFU2014–52282-P and BFU2017–84653-P to RB]. UM and RB are also part of COST action Proteostasis. This work was also partially supported by Fondation Jérôme Lejeune grant 1381-MU2015A to UM. 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Quantitative proteomics reveals neuronal ubiquitination of Rngo/Ddi1 and several proteasomal subunits by Ube3a, accounting for the complexity of Angelman syndrome

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Abstract

Abstract Angelman syndrome is a complex neurodevelopmental disorder caused by the lack of function in the brain of a single gene, UBE3A. The E3 ligase coded by this gene is known to build K48-linked ubiquitin chains, a modification historically considered to target substrates for degradation by the proteasome. However, a change in protein abundance is not proof that a candidate UBE3A substrate is indeed ubiquitinated by UBE3A. We have here used an unbiased ubiquitin proteomics approach, the bioUb strategy, to identify 79 proteins that appear more ubiquitinated in the Drosophila photoreceptor cells when Ube3a is over-expressed. We found a significantly high number of those proteins to be proteasomal subunits or proteasome-interacting proteins, suggesting a wide proteasomal perturbation in the brain of Angelman patients. We focused on validating the ubiquitination by Ube3a of Rngo, a proteasomal component conserved from yeast (Ddi1) to humans (DDI1 and DDI2), but yet scarcely characterized. Ube3a-mediated Rngo ubiquitination in fly neurons was confirmed by immunoblotting. Using human neuroblastoma SH-SY5Y cells in culture, we also observed that human DDI1 is ubiquitinated by UBE3A, without being targeted for degradation. The novel observation that DDI1 is expressed in the developing mice brain, with a significant peak at E16.5, strongly suggests that DDI1 has biological functions not yet described that could be of relevance for Angelman syndrome clinical research. Introduction Angelman syndrome (AS) is a rare neurodevelopmental disorder (OMIM #105830), with an estimated incidence of 1/15 000 births, characterized by a severe developmental delay, language impairment, ataxic movements, epilepsy, sleep disturbances and episodes of frequent laughter (1). In contrast to other complex syndromes that are caused by large genetic duplications/deletions, the underlying cause for AS is the loss of maternal expression in neurons of the brain of one single enzyme, UBE3A (2,3), a HECT-type ubiquitin E3 ligase (4–6). Although the deficiency of this paternally imprinted gene (7) is commonly originated by maternally inherited deletions on the 15q11-q13 chromosomal region (8), the syndrome is also caused by mutations affecting exclusively the UBE3A gene (9). Some of these UBE3A mutants lack the ubiquitin-ligase activity when tested in vitro (10,11), indicating that AS is caused by the lack of the ubiquitin ligase activity of UBE3A in neurons. Ubiquitinated substrates of UBE3A are therefore likely to be the effecting pathways of the resulting brain connectivity and/or function alterations. Interestingly, excess ligase activity of UBE3A has also been associated with autism spectrum disorders (12–14). Ubiquitin E3 ligase enzymes catalyze the covalent attachment of ubiquitin to the lysine residues on target proteins. According to in vitro studies, UBE3A catalyzes the preferential attachment of K48-linked poly-ubiquitin chains (15), presumably targeting its substrates for proteasomal degradation. Based on this premise, several UBE3A putative substrates (AIB1, Bak, Blk, Mcm7, Pbl) whose levels changed in the presence/absence of this enzyme were reported (16–20). Nevertheless, ubiquitination of those proteins by UBE3A has not yet been described. Similarly, the ubiquitination of other proposed neuronal UBE3A substrates was only validated in vitro (Arc, Na+/K+ ATPase, p27, Ring1B, Adrm1, Rpt5) or using non-denaturing immunoprecipitation approaches (Annexin A1, HHR23A, PSMD2, Ephexin5, p53) (21–29). We developed an ubiquitination assay for neuronal cell culture using a highly denaturing protocol, and showed that proteasome regulating proteins Rpn10 and Uch-L5 are ubiquitinated by Ube3a (30), both being reported to be essential for mammalian brain development (31,32). Recently, evidence is accumulating in regards to UBE3A regulating proteasomal activity (28–30,33), suggesting that UBE3A might indirectly be affecting the regulation of many other proteins targeted to the proteasome by other E3 ligases. Interaction between an ubiquitin ligase and its substrates is transient and hard to capture in vivo. Additionally, the low stoichiometry at which ubiquitin modified proteins are found within the cell hinders the identification of ubiquitination substrates in vivo. Antibodies that specifically recognize the ubiquitin diGlycine (diGly) signature have been employed to isolate, and subsequently identify putative ubiquitination sites by mass spectrometry (MS) (34–39). Such methodology has also been recently used in a screen for putative UBE3A substrates in HEK293 cells (29). However, this approach requires digestion of proteins by trypsin prior to their isolation, preventing any orthogonal validation, which is essential, as the diGly signature is also a remnant of other ubiquitin-like (UBL) modifications, such as Nedd8 or ISG15 (35), and can even be an experimental artefact under certain conditions (40). We have developed in our lab two methodologies that have proven to be suitable for the in vivo analysis of ubiquitinated proteins (41). The bioUb strategy, based on the in vivo biotinylation of ubiquitin (42), has recently been used in combination with quantitative shotgun proteomics to identify substrates of the E3 ligase Parkin involved in Parkinson’s disease (43). The second strategy favours the isolation of GFP-tagged proteins under denaturing conditions, and was first used to screen for Drosophila Ube3a substrates (30). In the present study, we have combined the bioUb strategy with the over-expression of Ube3a to identify 79 putative neuronal Ube3a substrates. Amongst those, we noted the presence of 13 proteasome subunits or proteasome interacting proteins. We validated that Ube3a ubiquitinates the proteasomal ubiquitin receptor Rings lost (Rngo) in Drosophila photoreceptor neurons in vivo. Furthermore, we have confirmed that ubiquitination of DNA damage-inducible protein 1 homologue 1 (DDI1), the human orthologue of Rngo, is enhanced upon UBE3A over-expression in neuroblastoma cells, without being targeted for degradation. The observation that Ddi1 is highly expressed in the developing mice brain suggests that this protein has a yet uncharacterized biological function in neuronal development. Results Unbiased identification of Ube3a substrates in Drosophila neurons in vivo In order to identify by MS analysis the proteins whose ubiquitination depends on Ube3a, we used the following fly lines: BirA, bioUb, bio15B and bioA3. bioUb flies express the (bioUb)6-BirA precursor (Fig. 1A) in the Drosophila photoreceptor neurons under the control of the eye-specific GMR-GAL4 driver, which has been shown to be the most suitable neuronal(-like) driver for identifying low abundance proteins, and for optimizing reproducibility across samples (44). BirA control flies express just the bacterial biotinylating enzyme BirA (44). bioA3 flies are bioUb flies over-expressing the Ube3a E3 ligase (Fig. 1B), as confirmed by immunoblotting (Fig. 1C). On the other hand, bio15B flies are bioUb flies carrying a loss of function Ube3a deletion (Ube3a15B allele) in heterozygosis. Homozygous mutant Ube3a15B flies lack any detectable Ube3a protein (Supplementary Material, Fig. S1A), but flies carrying this allele could not be expanded in homozygosis. The Ube3a15B heterozygous bio15B flies used for this study only show a partial reduction of Ube3a protein levels (Fig. 1C). Free BirA, indicating appropriate processing of the (bioUb)6-BirA precursor, was observed for all three genotypes (Fig. 1D); no undigested forms of the precursor were found above the expected molecular size of BirA (35 kDa). Biotin immunoblotting confirmed biotinylation and incorporation into conjugates of the GMR-GAL4-driven ectopic biotin-tagged ubiquitin in all bioUb, bioA3 and bio15B flies (Fig. 1E). Expression of the bioUb construct in those three fly lines did not significantly alter total ubiquitin levels, when compared to the BirA control (Supplementary Material, Fig. S1B). Figure 1. View largeDownload slide Drosophila Ube3a mutant and over-expressing flies expressing the biotin-tagged ubiquitin. (A) Schematic representation of the (bioUb)6-BirA construct, which is expressed as a poly-ubiquitin chain fused to BirA. This precursor polypeptide is digested by endogenous DUBs, so the ubiquitin moieties and the BirA enzyme are released. Each ubiquitin bears a 16 amino-acid long biotinylatable motif at their N-terminal part (bio) that is recognized by BirA. The sequence for biotinylation added to each ubiquitin is shown underlined, followed by the five amino-acid linker (italicized). The lysine where the biotin is attached is highlighted in bold. (B) Schematic representation of the domain structure of Drosophila Ube3a. Only two domains have been characterized for Ube3a so far: the AZUL domain (Amino-terminal Zn-finger of Ube3a E3 Ligase), which is thought to play a role in substrate recognition, and the HECT domain (Homologous to the E6-AP Carboxyl Terminus) that provides the E2-binding platform and a catalytic cysteine residue (C941) to which ubiquitin associates via a thioester linkage. Flies over-express Ube3a without any tag and under the control of a UAS sequence. (C) Anti-Ube3a immunoblot on head extracts. The specific band for Ube3a is indicated with an arrow. Unspecific bands are indicated with arrowheads. (D) Anti-BirA immunoblot on head extracts. Appropriate processing of the (bioUb)6-BirA precursor was observed for all three genotypes (bioUb, bioA3 and bio15B). Flies over-expressing just BirA were used as control. (D) Anti-biotin western blot performed for each of the genotypes. In the control sample (BirA) only endogenously biotinylated proteins are detected. Figure 1. View largeDownload slide Drosophila Ube3a mutant and over-expressing flies expressing the biotin-tagged ubiquitin. (A) Schematic representation of the (bioUb)6-BirA construct, which is expressed as a poly-ubiquitin chain fused to BirA. This precursor polypeptide is digested by endogenous DUBs, so the ubiquitin moieties and the BirA enzyme are released. Each ubiquitin bears a 16 amino-acid long biotinylatable motif at their N-terminal part (bio) that is recognized by BirA. The sequence for biotinylation added to each ubiquitin is shown underlined, followed by the five amino-acid linker (italicized). The lysine where the biotin is attached is highlighted in bold. (B) Schematic representation of the domain structure of Drosophila Ube3a. Only two domains have been characterized for Ube3a so far: the AZUL domain (Amino-terminal Zn-finger of Ube3a E3 Ligase), which is thought to play a role in substrate recognition, and the HECT domain (Homologous to the E6-AP Carboxyl Terminus) that provides the E2-binding platform and a catalytic cysteine residue (C941) to which ubiquitin associates via a thioester linkage. Flies over-express Ube3a without any tag and under the control of a UAS sequence. (C) Anti-Ube3a immunoblot on head extracts. The specific band for Ube3a is indicated with an arrow. Unspecific bands are indicated with arrowheads. (D) Anti-BirA immunoblot on head extracts. Appropriate processing of the (bioUb)6-BirA precursor was observed for all three genotypes (bioUb, bioA3 and bio15B). Flies over-expressing just BirA were used as control. (D) Anti-biotin western blot performed for each of the genotypes. In the control sample (BirA) only endogenously biotinylated proteins are detected. Comparison of the ubiquitinated proteome of bioA3 flies and bioUb flies should allow the identification of proteins whose ubiquitination is enhanced by Ube3a. Conversely, we would expect to have a reduction on the ubiquitination of Ube3a substrates on bio15B flies in respect to bioUb flies (Fig. 2A). Biotinylated ubiquitin conjugates formed within the fly photoreceptor neurons were isolated using neutravidin beads, those pulldowns being performed on three biological replicates for each of the three conditions. Despite collecting whole heads, the isolated material is expected to originate just from the GMR-GAL4 expressing cells. Similar amounts of ubiquitinated proteins were eluted from the three genotypes (Fig. 2B and Supplementary Material, Fig. S2A and B). After fractionation by SDS-PAGE, each gel lane, corresponding to one sample, was cut into several slices as indicated in Supplementary Material, Fig. S2B). Protein loads from each individual gel slice were in-gel digested with trypsin and subsequently analysed by LC-MS/MS. Figure 2. View largeDownload slide Isolation of candidate Ube3a substrates in Drosophila neurons. (A) Workflow for the identification of Drosophila Ube3a substrates. Flies over-expressing the (bioUb)6-BirA precursor in the photoreceptor cells under the control of the GMR-GAL4 driver (bioUb) were compared with heterozygous Ube3a mutant (bio15B) or with Ube3a gain of function flies (bioA3) in order to identify proteins whose ubiquitination increases in a Ube3a dose-dependent manner. Fly heads of each of the genotype were subjected to biotin pulldown and MS analysis. Proteins whose ubiquitination is regulated by Ube3a should be found in more abundance in bioA3 flies, as compared with bioUb and bio15B controls. (B). Silver staining of the eluted material from biotin pulldowns. Only endogenously biotinylated proteins are detected on the BirA control sample. (C) LFQ intensities of Ube3a obtained from MS analysis of eluted samples. One asterisk indicates P-value < to 0.05; three, P < to 0.0001. (D). Western blot to Ube3a indicates that it is mostly purified in its unmodified form (arrow), which is bound to the avidin beads due to ubiquitin bound to its active-site cysteine. This ubiquitin is removed from the active site by DTT-treatment of the samples on the elution step. A small fraction of Ube3a was also found conjugated to ubiquitin (asterisk). A non-specific band is observed in all input samples (arrowhead). Figure 2. View largeDownload slide Isolation of candidate Ube3a substrates in Drosophila neurons. (A) Workflow for the identification of Drosophila Ube3a substrates. Flies over-expressing the (bioUb)6-BirA precursor in the photoreceptor cells under the control of the GMR-GAL4 driver (bioUb) were compared with heterozygous Ube3a mutant (bio15B) or with Ube3a gain of function flies (bioA3) in order to identify proteins whose ubiquitination increases in a Ube3a dose-dependent manner. Fly heads of each of the genotype were subjected to biotin pulldown and MS analysis. Proteins whose ubiquitination is regulated by Ube3a should be found in more abundance in bioA3 flies, as compared with bioUb and bio15B controls. (B). Silver staining of the eluted material from biotin pulldowns. Only endogenously biotinylated proteins are detected on the BirA control sample. (C) LFQ intensities of Ube3a obtained from MS analysis of eluted samples. One asterisk indicates P-value < to 0.05; three, P < to 0.0001. (D). Western blot to Ube3a indicates that it is mostly purified in its unmodified form (arrow), which is bound to the avidin beads due to ubiquitin bound to its active-site cysteine. This ubiquitin is removed from the active site by DTT-treatment of the samples on the elution step. A small fraction of Ube3a was also found conjugated to ubiquitin (asterisk). A non-specific band is observed in all input samples (arrowhead). Similar number of ubiquitinated proteins and a high correlation of the label-free quantification (LFQ) intensity values were detected, both between replicas and across the different genotypes (Supplementary Material, Fig. S3A and B). Random LFQ values from a distribution meant to simulate expression below the detection limit (45) were imputed to those proteins for which LFQ values were not reported by the MaxQuant software on that given experiment (Supplementary Material, Fig. S3C). The experimental design was successful as evidenced from the LFQ intensity values obtained for Ube3a, which appeared highly enriched in the bioA3 sample, and significantly reduced in the bio15B sample (Fig. 2C). Western blot analysis confirmed those results and revealed that Ube3a is mostly isolated in an unmodified form (Fig. 2D), as reported previously (44). We compared the LFQ intensities of ubiquitinated proteins on the bioA3 sample with the corresponding bioUb values and plotted their fold-changes (X-axis) and significance P-values (Y-axis) as a Vulcano plot (Fig. 3). As expected, most of the proteins detected in this study displayed a ratio close to one, including endogenously biotinylated proteins acetyl-CoA carboxylase (ACC), pyruvate carboxylase (PCB) and CG2118 (46) (shown with filled squares in Fig. 3), indicating that the pull-down process was equally efficient in the different samples. From the 751 protein groups identified across all genotypes, 79 were significantly (P < 0.05) enriched at least 2-fold in the bioA3 sample relative to the bioUb control, and can therefore be defined as putative Ube3a substrates (Supplementary Material, Table S1). In order to focus on the highest confidence Ube3a substrates, the 79 proteins regulated by Ube3a were analysed at the peptide level to comply with the following requirements: (1) Average ratio between common peptides identified in both conditions should be at least two after the subtraction of the standard error of the mean (S.E.M.); and (2) individual peptide intensities should globally follow the same tendency as the protein LFQ intensity and show in average a 2-fold enrichment. Out of the 79 proteins identified as enriched at the protein level, 39 candidate Ube3a substrates (labelled with filled circles in Fig. 3) appeared homogenously enriched also at the peptide level (Supplementary Material, Fig. S3D), most of them having human orthologues (Table 1). Among those 39 high confidence candidate Ube3a substrates, eight proteins regulate protein degradation through the ubiquitin-proteasome system, including proteasomal subunits Prosα1, Prosα3, Rpt2, Rpt4, Rpn3 and Rpn8, and the proteasomal shuttling proteins Rngo and Rpn10, the latest being previously identified as target of Ube3a in neuronal cell culture (30). Besides, two proteins related to autophagy, Atg8a and Ref(2)P (47), and three chaperone proteins, CCT3, CCT7 and CCT8 (48), were also found as high confidence substrates of Ube3a. In addition, proteasomal subunits Prosα4, Prosα7, Rpn2, proteasome activator REG and the proteasome-associated deubiquitinating (DUB) enzyme Uch-L5 were significantly enriched according to LFQ values, but did not pass the requirements at peptide level. Table 1. High confidence proteins whose ubiquitination is dependent on Ube3a over-expression Proteins whose abundance in the pulldowns is significantly altered by Ube3a, both at protein and at peptide level, are shown. The complete data set is available as Supplementary Material, Table S1. Proteasomal proteins are highlighted in bold. Cellular localization (CC) of the human proteins are indicated by graytones (dark grey rounded rectangle: nuclear; light grey cell: cytoplasmic; black surrounding square: plasma membrane). If a more specific localization within each compartment has been reported, it is further indicated with text (CT, cytoskeleton; E, endosome; ER, endoplasmic reticulum; P, peroxisomes; S, secreted; V, vesicles). a Given according to Flybase nomenclature. b Orthologues with the best Flybase score are provided. c Given according to HUGO Gene Nomenclature Committee. d Given according to Human Protein Atlas (www.proteinatlas.org) and Uniprot (www.uniprot.org). e Other M13 metallopeptidases are also considered orthologues (see Supplementary Material, Table S1). Table 1. High confidence proteins whose ubiquitination is dependent on Ube3a over-expression Proteins whose abundance in the pulldowns is significantly altered by Ube3a, both at protein and at peptide level, are shown. The complete data set is available as Supplementary Material, Table S1. Proteasomal proteins are highlighted in bold. Cellular localization (CC) of the human proteins are indicated by graytones (dark grey rounded rectangle: nuclear; light grey cell: cytoplasmic; black surrounding square: plasma membrane). If a more specific localization within each compartment has been reported, it is further indicated with text (CT, cytoskeleton; E, endosome; ER, endoplasmic reticulum; P, peroxisomes; S, secreted; V, vesicles). a Given according to Flybase nomenclature. b Orthologues with the best Flybase score are provided. c Given according to HUGO Gene Nomenclature Committee. d Given according to Human Protein Atlas (www.proteinatlas.org) and Uniprot (www.uniprot.org). e Other M13 metallopeptidases are also considered orthologues (see Supplementary Material, Table S1). Figure 3. View largeDownload slide Identification of candidate Ube3a substrates in Drosophila neurons. Comparison of the abundance, determined by their LFQ intensities, of the ubiquitinated proteins identified by MS upon Ube3a over-expression relative to bioUb flies. The Vulcano plot displays the LFQ bioA3/bioUb ratios in log2 scale (X-axis) and the t-test P-values in −log10 scale (Y-axis), determining the statistical significance (P < 0.05, horizontal grey lane) of the fold changes, for each protein. Labelled filled circles represent high confidence proteins found more ubiquitinated in the bioA3 sample than in the bioUb sample. Labelled empty circles are those found less ubiquitinated in bioA3 sample. Endogenously ubiquitinated proteins (ACC, CG2118 and PCB) are shown with filled squares, and ubiquitin with an empty square. The earlier reported putative Ube3a candidate Atpα, is shown with a triangle. bioUb: GMR-GAL4, UAS-(bioUb)6-BirA/CyO; bioA3: GMR-GAL4, UAS-(bioUb)6-BirA/CyO; UAS-Ube3aA3/TM6. Figure 3. View largeDownload slide Identification of candidate Ube3a substrates in Drosophila neurons. Comparison of the abundance, determined by their LFQ intensities, of the ubiquitinated proteins identified by MS upon Ube3a over-expression relative to bioUb flies. The Vulcano plot displays the LFQ bioA3/bioUb ratios in log2 scale (X-axis) and the t-test P-values in −log10 scale (Y-axis), determining the statistical significance (P < 0.05, horizontal grey lane) of the fold changes, for each protein. Labelled filled circles represent high confidence proteins found more ubiquitinated in the bioA3 sample than in the bioUb sample. Labelled empty circles are those found less ubiquitinated in bioA3 sample. Endogenously ubiquitinated proteins (ACC, CG2118 and PCB) are shown with filled squares, and ubiquitin with an empty square. The earlier reported putative Ube3a candidate Atpα, is shown with a triangle. bioUb: GMR-GAL4, UAS-(bioUb)6-BirA/CyO; bioA3: GMR-GAL4, UAS-(bioUb)6-BirA/CyO; UAS-Ube3aA3/TM6. On the other hand, a total of 55 proteins also appeared to be less ubiquitinated in bioA3 sample due to the over-expression of Ube3a (Supplementary Material, Table S1), displaying a 2-fold significant reduction in their abundance compared to bioUb (bioA3/bioUb < 0.5; P < 0.05). Following the same peptide analysis described before (Supplementary Material, Fig. S3E), we concluded that 14 proteins are clearly less ubiquitinated when Ube3a is over-expressed, as compared to the control sample (labelled with empty circles in Fig. 3). Interestingly, we reliably identified calcium/calmodulin-dependent protein kinase II (CaMKII)—which in Drosophila is coded by a single gene—as being less ubiquitinated upon over-expression of Ube3a. We also compared the LFQ intensities of the bio15B sample with the bioUb values; but, using the same criteria as above, Ube3a itself did not appear significantly enriched with high confidence in the bioUb sample relative to the bio15B ubiquitinated material. However, three proteins (Rdhb, Map2015 and Axo) appeared significantly less ubiquitinated in the bio15B sample, barely above the defined thresholds (data not shown). Ubiquitin LFQ values and ubiquitin chain linkages indicate proteasomal perturbation upon Ube3a over-expression Based on the experimental design, we expected ubiquitin levels to be unaltered by Ube3a over-expression. Furthermore, no significant differences were observed on the hard-to-quantify smears of the silver stained gels (Fig. 2B), as well as ubiquitin (Supplementary Material, Fig. S1B) and biotin (Supplementary Material, Fig. S2A) blots. However, based on LFQ values, ubiquitin levels were quantified to have a significant increase of 1.66-fold in the bioA3 flies, relative to the bioUb sample (Supplementary Material, Table S1), this is, a >60% increase of ubiquitin was detected on the collected ubiquitinated material of bioA3 flies. In order to elucidate the type of ubiquitin chains enriched upon over-expression of Ube3a, we compared the intensity of all detected diGly-containing peptides across the samples. Digestion of a complex mixture of ubiquitinated proteins is expected to result in a complex mixture of ubiquitin chain linkages. Even though the isolated ubiquitinated material is composed of substrates of hundreds of ligases, this analysis indicated that K48 and K63 linkages were significantly more abundant on the bioA3 sample, while K33 linkages were reduced (Supplementary Material, Fig. S3F). The 60% increase on ubiquitin levels together with the significant changes in chain linkages suggests a global perturbation of the ubiquitin proteasome system upon Ube3a over-expression. Rngo is ubiquitinated by Ube3a in vivo in Drosophila neurons Amongst the highest confidence candidates as Ube3a substrates (Table 1), eight of the identified proteins are proteasome integral or regulatory subunits. We decided to focus on Rngo, a predicted proteasomal shuttling factor for which working antibodies were available (49), and which was already observed in our first neuronal ubiquitome studies (42). According to the label-free quantitative MS-based analysis performed, Rngo is 5-fold more enriched in Ube3a over-expressing flies in respect to control flies. In agreement with that, western blot analysis showed that ubiquitination of Rngo is enhanced when Ube3a is over-expressed and reduced in its absence (Fig. 4A), making Rngo the first Ube3a substrate validated in any type of neuron in vivo. Interestingly, Rngo total levels were not altered, suggesting that it is not being targeted for degradation. As a control, the same membrane was used to detect the presence of Fax, a protein that we found by MS to be less ubiquitinated upon Ube3a over-expression (Fig. 3). Indeed, Fax displayed the opposite trend, and its ubiquitination was dramatically reduced by the over-expression of Ube3a (Fig. 4A), indicating that the increase ubiquitination seen for Rngo is specific to the protein and not to a more efficient general isolation of proteins in the bioA3 sample. We also analysed by western blot the ubiquitination of another proposed UBE3A substrate (27), the Na+/K+-ATPase α-subunit (Atpα), but as with the MS analysis (Fig. 3), western blot did not show any enhancement by Ube3a over-expression on its ubiquitination (Fig. 4B). Figure 4. View largeDownload slide Rngo is ubiquitinated by Ube3a in Drosophila photoreceptor neurons. (A) Immunoblot with anti Rngo-antibody confirmed the increase ubiquitination detected by MS in bioA3 flies as compared with bioUb flies, and more significantly to bio15B flies. This same membrane was reprobed with anti-Fax. Levels of ubiquitinated Fax were found reduced in bioA3 flies, which corroborated the MS results and confirmed that the increase ubiquitination seen for Rngo is specific to the protein and not to a more efficient general isolation of proteins in the bioA3 sample. Putative mono-, tri- and tetra-ubiquitinated forms are indicated with asterisks and unmodified or cysteine-ubiquitinated forms are indicated with an arrow. Equal levels of Avidin bands, which are non-specifically detected by Rngo antibody, are also shown. (B) Atpα is not a substrate of Ube3a in Drosophila photoreceptor cells. Western blot performed with anti-Atpα showed that its ubiquitination is not regulated by Ube3a, as levels of mono-ubiquitinated Atpα are similar, or even lower in bioA3 than in bio15B flies. The unmodified proteins are indicated with an arrow. Ubiquitinated forms are indicated with asterisks. Figure 4. View largeDownload slide Rngo is ubiquitinated by Ube3a in Drosophila photoreceptor neurons. (A) Immunoblot with anti Rngo-antibody confirmed the increase ubiquitination detected by MS in bioA3 flies as compared with bioUb flies, and more significantly to bio15B flies. This same membrane was reprobed with anti-Fax. Levels of ubiquitinated Fax were found reduced in bioA3 flies, which corroborated the MS results and confirmed that the increase ubiquitination seen for Rngo is specific to the protein and not to a more efficient general isolation of proteins in the bioA3 sample. Putative mono-, tri- and tetra-ubiquitinated forms are indicated with asterisks and unmodified or cysteine-ubiquitinated forms are indicated with an arrow. Equal levels of Avidin bands, which are non-specifically detected by Rngo antibody, are also shown. (B) Atpα is not a substrate of Ube3a in Drosophila photoreceptor cells. Western blot performed with anti-Atpα showed that its ubiquitination is not regulated by Ube3a, as levels of mono-ubiquitinated Atpα are similar, or even lower in bioA3 than in bio15B flies. The unmodified proteins are indicated with an arrow. Ubiquitinated forms are indicated with asterisks. Ddi1 and Ddi2 are expressed throughout development in the mouse brain Mammalians have two protein homologues to Drosophila Rngo: DNA damage-inducible protein 1 homologue 1 (DDI1) and homologue 2 (DDI2). Partial expression data for DDI2 are available at the Human Protein Atlas (www.proteinatlas.org; date last accessed March 19, 2018), but information about mammalian DDI1 is very scarce. We tried to assess the expression of DDI1 using commercially available antibodies, but, since DDI1 and DDI2 proteins have very high amino acid sequence identity (72%), all the commercially available antibodies recognize DDI2 protein, or do simply not work (data not shown). We therefore tested whether mouse Ddi1 and Ddi2 are expressed in the brain. Analysis of Ddi1 gene expression in mice brain at different ages performed by qRT-PCR revealed a drastic and significant increase of Ddi1 mRNA levels at embryonic stage E16.5 (Fig. 5A). The Ddi1 mRNA expression peak was rapidly reduced from E17.5, returning to basal levels at E19.5-P1, remaining relatively stable during all the tested adult time points. In contrast, Ddi2 mRNA levels, which were also detectable in the mice brain, only fluctuated slightly during development (Fig. 5B). Figure 5. View largeDownload slide Ddi1 and Ddi2 gene expression temporal profiles in the mouse brain. Changes in expression of Ddi1 (A) and Ddi2 (B) mRNA levels in brains of C57BL/6J mice during aging. RNA was isolated from the brains of E13.5 to 9-month-old mice and subjected to qRT-PCR. A significant increase of Ddi1 mRNA level was observed at embryonic stage E16.5. Ddi1 and Ddi2 mRNA levels were determined and adjusted by the signal intensity of GAPDH, and the average results (n = 3) were calculated and expressed with respect to the values obtained in E13.5. Figure 5. View largeDownload slide Ddi1 and Ddi2 gene expression temporal profiles in the mouse brain. Changes in expression of Ddi1 (A) and Ddi2 (B) mRNA levels in brains of C57BL/6J mice during aging. RNA was isolated from the brains of E13.5 to 9-month-old mice and subjected to qRT-PCR. A significant increase of Ddi1 mRNA level was observed at embryonic stage E16.5. Ddi1 and Ddi2 mRNA levels were determined and adjusted by the signal intensity of GAPDH, and the average results (n = 3) were calculated and expressed with respect to the values obtained in E13.5. Additionally, we analysed the expression profile of Ddi1 in the developing brain by performing RNA in-situ hybridization experiments in CD-1 mouse embryos at four different stages of development—E9.5, E10.5, E14.5 and E16.5. As shown in Figure 6, Ddi1 is expressed in all parts of developing brain (telencephalon, diencephalon, mesencephalon and rhombencephalon) at the two younger developmental stages. Similar Ddi1 expression pattern was observed for these two stages in both whole mount samples (Fig. 6A and C) and sagittal paraffin sections (Fig. 6B and D). At the stage E14.5, the expression is located in neurons of mesencephalic and telencephalic structures, with signal accumulation in upper hill (colliculus tectum) and ventricular zone of pallium (Fig. 6E–G). At further developmental stage E16.5, which shows highest mRNA level expression according to our qRT-PCR screen, Ddi1 is expressed in particular telencephalon parts, mainly the ventricular layer and cortical plate of isocortex and the ventricular layer of olfactory bulb (Fig. 6H and I). This could represent the dividing neuroblasts and their migration toward the superficial layer of isocortex. The positive staining of neuronal cells in the isocortex (Fig. 6J) is clear evidence of Ddi1 expression in neuronal tissue, however, up to date no single report has described the role DDI1 could exert in the brain. Figure 6. View largeDownload slide Spatial profile of Ddi1 gene expression in the mouse brain. Ddi1 is expressed in central nervous system during embryonic development according to RNA in-situ hybridization study on CD-1 mouse embryos. (A) and (B) Expression profile of Ddi1 in whole mount and paraffin sections of E9.5 embryo, respectively. (C) and (D) Ddi1 expression mapping in whole mount and paraffin section of E10.5 embryos. (E) Sagittal section of E14.5 embryonic brain with clearly located expression of Ddi1 in colliculus midbrain tectum and pallial part of telencephalon. (F) Detail of pallium: Ddi1 is expressed in isocortex and olfactory bulb. (G) Midbrain and hindbrain tissue shows expression of Ddi1 in culliculus tectum. (H) and (I) Ddi1 is specifically expressed in isocortex and ventricular layer of olfactory bulb of E16.5 mouse brain. (J) High resolution detail of the scan shown in (I). Red stars highlight tubular structures—probably capillaries of the central nervous system. Abbreviations: T, telencephalon; D, diencephalon; M, mesencephalon; R, rhombencephalon; C, cerebellum; CMT, colliculus midbrain tectum; TL, thalamus; P, pallium; SP, subpallium; HT, hypothalamus; BG, basal ganglia; OB, olfactory bulb; IC, isocortex; NC, nasal cavity. Figure 6. View largeDownload slide Spatial profile of Ddi1 gene expression in the mouse brain. Ddi1 is expressed in central nervous system during embryonic development according to RNA in-situ hybridization study on CD-1 mouse embryos. (A) and (B) Expression profile of Ddi1 in whole mount and paraffin sections of E9.5 embryo, respectively. (C) and (D) Ddi1 expression mapping in whole mount and paraffin section of E10.5 embryos. (E) Sagittal section of E14.5 embryonic brain with clearly located expression of Ddi1 in colliculus midbrain tectum and pallial part of telencephalon. (F) Detail of pallium: Ddi1 is expressed in isocortex and olfactory bulb. (G) Midbrain and hindbrain tissue shows expression of Ddi1 in culliculus tectum. (H) and (I) Ddi1 is specifically expressed in isocortex and ventricular layer of olfactory bulb of E16.5 mouse brain. (J) High resolution detail of the scan shown in (I). Red stars highlight tubular structures—probably capillaries of the central nervous system. Abbreviations: T, telencephalon; D, diencephalon; M, mesencephalon; R, rhombencephalon; C, cerebellum; CMT, colliculus midbrain tectum; TL, thalamus; P, pallium; SP, subpallium; HT, hypothalamus; BG, basal ganglia; OB, olfactory bulb; IC, isocortex; NC, nasal cavity. Human DDI1 is ubiquitinated by UBE3A, but is not targeted for degradation Once identified and confirmed that Ube3a ubiquitinates Rngo in flies, we aimed to test whether its homologs DDI1 and DDI2 are substrates of human UBE3A. For that purpose, we employed an in cellulo ubiquitination assay (30,50) in SH-SY5Y neuroblastoma cells. Wild-type UBE3A (UBE3AWT) induced the ubiquitination of DDI1-GFP in SH-SY5Y cells (Fig. 7 and Supplementary Material, Fig. S5A), but did not seem to increase the ubiquitination status of DDI2-GFP (Supplementary Material, Fig. S5B). We also confirmed the specificity of UBE3A-dependent DDI1 ubiquitination by proving that Parkin, the E3 ligase involved in Parkinson’s disease, could not mediate ubiquitination of DDI1 (Supplementary Material, Fig. S5B). Altogether, the data presented here demonstrates for the first time that Rngo human homologue DDI1 is a UBE3A substrate. Despite earlier reports suggested that UBE3A generates degradation-leading K48 ubiquitin linkages in vitro (15), at least in SH-SY5Y neuroblastoma cells, UBE3A-mediated ubiquitination of human DDI1 does not lead to its degradation, as indicated by the intensity of the DDI1-GFP bands, which is independent of the activity of UBE3A (Fig. 7). Figure 7. View largeDownload slide Human DDI1 is ubiquitinated by UBE3A in SH-SY5Y cells. (A) DDI1-GFP showed a significant increase in its ubiquitinated fraction in the presence of wild type UBE3A (UBE3AWT), as illustrated by western blot to FLAG-tagged ubiquitin, compared with control (pCDNA3.1) or to ligase dead UBE3A (UBE3ALD). The non-modified form of DDI1 was detected with anti-GFP antibody (green). The bottom panel shows levels of human UBE3A protein in the whole cell extract before the isolation of the GFP-tagged proteins. (B) Quantification of the ubiquitination of DDI1 was performed by calculating the FLAG: GFP ratio in panel A with Image-J. Statistical significance differences [***, P < 0.001 (mean ± S.E.M., n = 5)] were observed for the UBE3AWT sample relative to both control (pCDNA3.1) and UBE3ALD samples. Figure 7. View largeDownload slide Human DDI1 is ubiquitinated by UBE3A in SH-SY5Y cells. (A) DDI1-GFP showed a significant increase in its ubiquitinated fraction in the presence of wild type UBE3A (UBE3AWT), as illustrated by western blot to FLAG-tagged ubiquitin, compared with control (pCDNA3.1) or to ligase dead UBE3A (UBE3ALD). The non-modified form of DDI1 was detected with anti-GFP antibody (green). The bottom panel shows levels of human UBE3A protein in the whole cell extract before the isolation of the GFP-tagged proteins. (B) Quantification of the ubiquitination of DDI1 was performed by calculating the FLAG: GFP ratio in panel A with Image-J. Statistical significance differences [***, P < 0.001 (mean ± S.E.M., n = 5)] were observed for the UBE3AWT sample relative to both control (pCDNA3.1) and UBE3ALD samples. Discussion Our unbiased proteomic analysis for the identification of differentially ubiquitinated proteins in Drosophila photoreceptor neurons upon Ube3a over-expression has resulted in a list of 79 putative Ube3a substrates, out of which proteasomal proteins appear very highly enriched. To our knowledge, this is the first time a list of candidate Ube3a substrates, whose ubiquitinated fraction is enhanced upon Ube3a over-expression, is reported in neurons in vivo. Several of the putative Ube3a substrates identified (Arc1, Chc, Gclc, GlyRS, Path, Tig and SesB) play a role either in axon and dendrite morphogenesis (51–54) or synaptic transmission (55–57). Interestingly, upon Ube3a over-expression we also found a significant reduction of the ubiquitination of CaMKII, a key kinase known in humans to regulate neurotransmitter synthesis and release, modulation of ion channel activity, neurite extension, synaptic plasticity, learning and memory (58). Reduced activity and protein levels of CaMKII at the postsynaptic density have been described in a mouse model of AS (59). It still remains to be explained how a reduction of UBE3A levels enhances the ubiquitination and/or degradation of CaMKII. Analysis of the putative Ube3a substrates by g:Profiler analysis of GO terms and KEGG pathways indicate a highly significant enrichment of the proteasome (data not shown). Furthermore, comparison of the data obtained in this work to the dataset of Parkin substrates (43), further confirms a deregulation of the proteasome upon Ube3a over-expression. Indeed, UBE3A is known to be a proteasome-associated protein (28,60), as well as being capable of ubiquitinating several proteasomal subunits in cell culture (28–30). However, it is controversial whether UBE3A inhibits (28) or stimulates (33) the proteolytic activity of the proteasome. Ube3a over-expression results in an increase in total ubiquitin levels, while over-expression of Parkin did not. Having identified several proteasomal subunits as ubiquitinated by Ube3a, the simplest explanation is that Ube3a-driven proteasomal deregulation results in the accumulation of substrates ubiquitinated by Ube3a or other E3 ligases. This would also explain the reduction observed here in the ubiquitination of many other proteins. It is known that the specific deregulation of the proteasome by over-expressing a dominant negative Rpn10 subunit reduces the ubiquitination levels of Fax and other mono-ubiquitinated proteins (44). Deubiquitination of mono-ubiquitinated proteins is also seen when inhibiting the proteasome pharmacologically (35), and is explained by a reduction of the free ubiquitin available pool concomitant to the accumulation of proteasome targeted poly-ubiquitinated proteins (36,61–63). The decrease on Fax ubiquitination upon Ube3a over-expression, detected here by both MS (Fig. 3) and immunoblotting (Fig. 4A), could therefore be caused by an Ube3a-induced proteasomal inhibition. Significant changes in global ubiquitin chain linkages (Supplementary Material, Fig. S3F) are also in line with this interpretation. In this work, we have validated by western blot that Rngo is a direct Ube3a substrate in photoreceptor neurons. Rngo contains both an UBL and an Ubiquitin-binding domain (UBD), a hallmark of proteasomal shuttles (49), but can also bind directly another proteasomal shuttle protein, Rpn10 (49). The ubiquitination of Rpn10 is also increased in Ube3a over-expressing flies (Fig. 3), and was already identified as an Ube3a substrate in Drosophila cells, as well as being shown to interact genetically with Ube3a in neurons in vivo (30). Ubiquitination of such proteasomal regulators by Ube3a can be predicted to severely interfere with proteasomal function. Considering that the proteasome regulates dendritic development (32), long-term potentiation (64), long-term depression (65), synaptic plasticity (66), synaptic strengthening (67), memory consolidation (68), circadian rhythms (69) and many other aspects of neuronal function, an UBE3A-dependent proteasomal regulation could easily explain how a single E3 ligase mutation can cause a disorder as complex as AS. And indeed, it should be noted that UBE3A has been shown to regulate most aspects of neuronal function listed above (70–74). Non-degradative ubiquitination of proteasomal receptors could alter their function to a similar extent as their degradation, since the activity of those receptors is dependent on their UBDs. An ubiquitinated ubiquitin-binding subunit is likely to prioritirily bind its own ubiquitin moieties, therefore blocking its normal function. Our prediction would be that—based on the increased ubiquitination of those subunits upon UBE3A over-expression—proteasome activity should be increased in the brain of AS patients, on which UBE3A levels are reduced. Interestingly, Rngo is also the Drosophila homologue of yeast Ddi1/Vsm1, a protein that binds to several Snc-interacting t-SNAREs (75), negatively regulates exocytosis (76) and also regulates protein secretion (77). Similarly, the C. elegans homologue DDI-1/VSM-1 has also been proposed to regulate synaptic function, with vsm-1 mutants displaying a significant increase in synaptic density along the dorsal nerve cord (78). Thus, Rngo/DDI1 may have an additional role in synaptic transmission by controlling SNARE mediated exocytosis. In fact, over-expression of Ube3a, but not its ligase dead form, has been reported to alter neurotransmission at the neuromuscular junction in Drosophila (79). Two mammalian Rngo homologues have been described, DDI1 and DDI2, but neither of those proteins have to date been functionally characterized. We have found that both are expressed in the developing brain (Figs 5 and 6). Structurally, both proteins contain the characteristic Retroviral Protease-like domain that was recently reported to cleave/activate the Nrf1 transcription factor under proteasome inhibition (80,81). Further, they both contain an additional helical HDD domain (82,83) and an N-terminal UBL domain, but lack the well-defined Ubiquitin-associated (UBA) domain. Although, a weak Ubiquitin-binding motif (UIM) is present at the C-terminus of DDI2 (82), no such a motif can be found in DDI1. It is thus feasible that the DDI1 UBL domain, which in yeast Ddi1 is capable of binding ubiquitin (84), could substitute the role of the UBA domain, a mechanisms that would be facilitated by DDI1’s homodimeric conformation. Having confirmed the ubiquitination of Rngo in photoreceptor neurons by the Drosophila Ube3a and the expression of its mammalian homologues in the brain, we then tested whether any of Rngo’s human orthologues are ubiquitinated by UBE3A. We confirmed DDI1 to be an ubiquitination substrate of UBE3A in SH-SY5Y neuroblastoma cells. Different controls indicated that ubiquitination of DDI1 by UBE3A is specific, but does not lead to a reduction of DDI1 protein levels. It is not the first time that regulation of protein activity, even with formation of K48-linked chains, has been reported to be proteolysis independent (85). A UBD found in Met4 was proposed to cap its own K48-linked ubiquitin chain, inactivating the protein and protecting it from degradation (86), and something similar could be happening with Rngo/DDI1. Further work is required to elucidate what the functional role of DDI1 ubiquitination might be and whether the presence of UBD/UIM/UBA/UBL in ubiquitination substrates might interfere into the canonical role of K48 ubiquitin-linked chains. Up to now, no candidate ubiquitination substrates of UBE3A had been directly validated in neurons in vivo. We had earlier identified a proteasomal shuttling factor, Rpn10, to be regulated by Ube3a in Drosophila cells (30), which we now confirmed in vivo by MS. Further, we have validated that in Drosophila photoreceptor neurons Ube3a ubiquitinates another proteasomal shuttling factor, Rngo, becoming the first Ube3a substrate to be identified and validated in vivo in neurons within a whole organism. More importantly, UBE3A regulates the orthologue DDI1 protein in human neuroblastoma cells. Since UBE3A ubiquitination appears to be regulating several proteasomal-associated subunits, as already indicated by ourselves and others (29,30,33), it would not be surprising to see that a highly significant number of proteins regulated downstream the proteasome will display significant changes on their abundance upon UBE3A mutation or over-expression. It is now a challenge to elucidate which Ube3a substrates are direct, in addition to the proteasomal proteins themselves. DDI1 gene has been reported to be affected in siblings of a familial neurodegenerative disorder characterized clinically as a variant of Alzheimer’s disease (87). Having now described for the first time the temporal and spatial expression of Ddi1 in the mouse brain, we next need to perform a functional characterization to uncover the neuronal role of DDI1, as this might as well bring light to our understanding of how AS is regulated. Based on studies in yeast and C. elegans, it is likely that DDI1 is involved in both regulation of synapses and proteasomal function (75–78,84). Given the complexity of neuronal function, and previous work aiming to identify the mechanisms regulated by UBE3A, it is likely that both processes are actually misregulated during the genesis of AS. Finally, if we take into account that Ube3a regulates the proteasome, and that UBE3A expression declines with age (88), it would not be surprising that this E3 ligase has a further role in proteostasis not yet characterized. Materials and Methods Drosophila stocks and sample collection We have used in this work the BirA, bioUb, bioA3 and bio15B flies, all of which express their corresponding constructs in the Drosophila photoreceptor neurons under the control of the GMR-GAL4 driver. BirA and bioUb flies, expressing respectively the BirA enzyme alone and the (bioUb)6-BirA precursor, have been described previously (44). Their genotypes are respectively GMR-GAL4/CyO; UAS-BirA/TM6 and GMR-GAL4, UAS-(bioUb)6-BirA/CyO. Ube3a gain of function (UAS-Ube3aA3) and loss of function (Ube3a15B) flies (89) were a gift from Professor Janice Fisher. Both UAS-Ube3aA3 and Ube3a15B fly lines were independently mated to GMR-GAL4, UAS-(bioUb)6-BirA/CyO; TM2/TM6 flies to generate GMR-GAL4, UAS-(bioUb)6-BirA/CyO; UAS-Ube3aA3/TM6 and GMR-GAL4, UAS-(bioUb)6-BirA/CyO; Ube3a15B/TM6 lines. The Drosophila Ube3a mutants (Ube3a15B) had been reported to be viable and fertile in homozygosis (89). When combined with bioUb flies, it was, however, required to grow them in heterozygosis, as null Ube3a flies were viable but not fertile at 25°C (J. Ramirez and U. Mayor, unpublished data). GMR-GAL4 (BL 1104) and OregonR (BL 2376) flies were provided by the Bloomington Drosophila Stock Center (Bloomington, IN, USA). The bioUb abbreviation is used throughout the text to refer to the GMR-GAL4, UAS-(bioUb)6-BirA/CyO flies, and the bioA3 and bio15B (for Ube3aA3 and Ube3a15B alleles) to refer to GMR-GAL4, UAS-(bioUb)6-BirA/CyO; UAS-Ube3aA3/TM6 and GMR-GAL4, UAS-(bioUb)6-BirA/CyO; Ube3a15B/TM6 flies, respectively. Flies were grown at 25°C in 12 h light-dark cycles in standard Drosophila medium (0.9% agar, 7.5% dextrose, 6% corn flour, 8.5% yeast, 2.5% Nipagin, 0.4% propionic, 0.02% benzalkonium chloride in distilled H2O). Mixed-sex flies of 2–5 days old were flash-frozen in liquid nitrogen and shaken while still frozen to sever the heads. Frozen fly heads were then separated from the remaining body parts using a pair of sieves with a nominal cut-off of 710 and 425 µm, and then stored at −80°C. Head collections were typically performed in the morning. Plasmids Commercial pEGFP-N1 vector (Clontech) was used to generate DDI1-GFP and DDI2-GFP vectors (see Cloning procedures section). FLAG-UBE3A-pCMV (UBE3AWT) and FLAG-UBE3ALD-pCMV (UBE3ALD) plasmids, expressing N-terminally FLAG-tagged versions of the wild type and catalytically inactive human UBE3A protein (90), were a gift from Dr Vjekoslav Tomaić. FLAG-tagged ubiquitin (30) in pCDNA3.1 vector (FLAG-Ub) was generously provided by Dr Jose Antonio Rodriguez Pérez (University of the Basque Country-UPV/EHU, Spain). Untagged human Parkin plasmid has been described previously (43). Empty pCDNA3.1 vector (Invitrogen) was used as control. Cloning procedures DDI2-pEGFP-N1 (DDI2-GFP) plasmid was generated by amplifying DDI2 gene (Uniprot Q5TDH0) from DDI2-pET16b plasmid (82) with DDI2-Fw (5′-AAGGTACCATGCTGCTCACCGTG-3′) and DDI2-Rv (5′-AAGGATCCCCTGGCTTCTGACGCTCTGC-3′) primers and inserted between KpnI and BamHI restriction sites of pEGFP-N1 vector (Clontech). Gene for human DDI1 protein (Uniprot Q8WTU0) was synthesized by GenScript and further amplified using the DDI1-Fw (5′-TATAGGTACCATGCTGATCACCGTG-3′) and DDI1-Rv (5′-TATAACCGGTATGCTCTTTTCGTCC-3′) primers and inserted between the Acc65I and AgeI sites of the DDI2-pEGFP-N1 vector, after the DDI2 gene had been removed using the same restriction enzymes. All PCR reactions were carried out with Phusion High-Fidelity DNA polymerase (Thermo Scientific). PCR product gel extractions and plasmid purifications were performed with the QIAGEN Gel Extraction Kit and QIAGEN plasmid mini and midi kits, respectively. Correct sequence for all plasmids was confirmed by sequencing either by the GATC Biotech Company (Köln, Germany) or the SGIKER Unit of Sequencing and Genotyping at the University of the Basque Country (Leioa, Spain). Western blotting and silver staining Both 4–12% Bolt Bis–Tris Plus pre-cast gels (Invitrogen) and 4–12% NuPAGE Bis–Tris gels (Invitrogen) were used for SDS-PAGE, then proteins were transferred to PVDF membranes using the iBlot system (Invitrogen). Following primary and secondary antibody incubation, membranes were developed with an ECL kit (Biorad Clarity). Dual-colour westerns were prepared by assigning independent colour channels to two independent westerns developed in the same membrane. The amount of material loaded for western blot analysis varied according to the tissue and the antibody employed. In the case of material obtained from fly biotin pulldowns, between 0.001% and 0.2% of the input samples and 5–10% of the elution samples were loaded. However, when material purified from cells was used, between 10–20% of inputs and 10–40% of the elution samples were loaded. The following primary antibodies were used: goat anti-biotin-horseradish peroxidase (HRP)-conjugated antibody (Cell Signalling; catalogue number 7075) at 1: 1000; chicken polyclonal anti-BirA antibody (Sigma; catalogue number GW20013F) at 1: 1000; mouse monoclonal anti-GFP antibody (Roche Applied Science; catalogue number 11814460001) at 1: 1000; mouse monoclonal anti-FLAG M2-HRP conjugated antibody (Sigma; catalogue number A8592) at 1: 1000; mouse monoclonal anti-Syx1A antibody (Developmental Studies of Hybridoma Bank; DSHB; catalogue number 8C3) at 1: 100; rabbit polyclonal anti-Fax antibody, a gift from Eric Liebl (Deninson University, OH, USA) at 1: 1000; mouse monoclonal anti-Atpα antibody (DSHB; catalogue number α5) at 1: 50; rabbit polyclonal anti-Ube3a antibody (91) for the detection of Drosophila Ube3a protein at 1: 1000; rabbit polyclonal anti-Rngo antibody (49) at 1: 500; rabbit polyclonal anti-ubiquitin antibody (Sigma; catalogue number U5379) at 1: 100; mouse monoclonal anti-UBE3A (clone E6AP-300) antibody (Sigma; catalogue number E8655) for the detection of human UBE3A protein at 1: 1000. The following secondary antibodies were used: goat anti-mouse-HRP-labelled antibody (Thermo Scientific; catalogue number 62-6520) at 1: 4000; goat anti-rabbit-HRP labelled antibody (Cell Signalling; catalogue number 7074) at 1: 4000 and donkey anti-chicken-HRP labelled antibody (Jackson ImmunoResearch; catalogue number 703-035-155) at 1: 2000. About 10% of the neat elution samples were used for silver staining analysis. Gels were fixed for 1 h at room temperature with 40% methanol and 10% acetic acid containing solution and then were stained using the SilverQuest kit from Invitrogen according to manufacturer’s instructions. Biotin pulldown Biotin pulldowns (42) from Drosophila heads were performed as described previously (43,44,92). About 500 mg of 2–5 days old fly heads of each genotype were homogenized in 2.9 mL of Lysis buffer (8 M urea and 1% SDS in PBS) supplemented with 50 mM N-ethylmaleimide (Sigma) and a complete protease inhibitor cocktail (Roche Applied Science). Lysates were centrifuged for 5 min at 16000g at 4°C and supernatant applied to a PD10 desalting column (GE Healthcare) previously equilibrated with 25 ml of binding buffer (3 M urea, 1 M NaCl, 0.25% SDS and 50 mM N-ethylmaleimide). Eluates, except 50 µl that were kept for monitoring the inputs, were then incubated with 250 µl of NeutrAvidin agarose beads suspension (Thermo Scientific). Unbound material (flow through) was separated by spinning the beads at 230g for 2 min. Beads were then subjected to stringent washes with six different washing buffers (WB): twice with WB1 (8 M urea, 0.25% SDS), thrice with WB2 (6 M guanidine-HCl), once with WB3 (6.4 M urea, 1 M NaCl, 0.2% SDS), thrice with WB4 (4 M urea, 1 M NaCl, 10% isopropanol, 10% ethanol, 0.2% SDS), once with WB1, once with WB5 (8 M urea, 1% SDS) and thrice with WB6 (2% SDS). All buffers were prepared in PBS. Beads were then heated at 95°C for 5 min in 125 µl of elution buffer (250 mM Tris–HCl pH 7.5, 40% glycerol, 4% SDS, 0.2% BPB, 100 mM DTT) and centrifuged for 2 min at 16 000g in a Vivaclear Mini 0.8 µm PES micro-centrifuge filter unit (Sartorius) to recover the eluted proteins. Finally, eluates were concentrated in Vivaspin 500 centrifugal filter units (Sartorius). Cell culture and transfection Human neuroblastoma SH-SY5Y cells were cultured under standard conditions (37°C, 5% CO2) in Dulbecco’s modified Eagle medium/nutrient mixture F-12 (DMEM/F-12) with GlutaMAX (Thermo Scientific), supplemented with 10% fetal bovine serum (Thermo Scientific), 100 U/ml of penicillin (Invitrogen) and 100 µg of streptomycin (Invitrogen). SH-SY5Y cells (3 × 105 cells) were seeded in six well-plates for transfection experiments. Overnight incubation under serum starvation was routinely performed prior to transfections. The following day OptiMEM serum-free medium (Thermo Scientific) was replaced by fresh DMEM/F-12 and cells were co-transfected with 1 μg of FLAG-Ub and 1 μg of DDI1-GFP, or DDI2-GFP, for 72 h using Lipofectamine 3000 transfection reagent (Invitrogen) according to manufacturer’s instructions. About 1 µg of either pcDNA3.1 (control), UBE3AWT, UBE3ALD or Parkin plasmids were additionally added to the transfection mixture to check the effect of UBE3A in DDI1 and DDI2 ubiquitination. Cells were washed twice in PBS and stored at −20°C until required. GFP beads pull-down assay Transfected SH-SY5Y cells were lysed with 500 μl of lysis buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% Triton, 1× protease inhibitor cocktail from Roche Applied Science and 50 mM N-ethylmaleimide from Sigma) and centrifuged at 14 000g for 10 min. Supernatants were mixed with 25 μl of GFP-Trap-A agarose beads suspension (Chromotek GmbH), which had been previously washed twice with a Dilution buffer (10 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 1× protease inhibitor cocktail, 50 mM N-ethylmaleimde). The mixture was then incubated at room temperature for 150 min with gentle rolling and centrifuged for 2700g for 2 min to separate the beads from the unbound material. GFP beads were subsequently washed once with the dilution buffer, thrice with washing buffer WB5 (8 M urea, 1% SDS in PBS) and once with 1% SDS in PBS. Bound GFP-tagged proteins were eluted in 25 µl of elution buffer (250 mM Tris–HCl pH 7.5, 40% glycerol, 4% SDS, 0.2% BPB, 100 mM DTT) by heating at 95°C for 10 min. In-gel trypsin digestion and peptide extraction Eluates from biotin pull-down assays were resolved by SDS-PAGE using 4–12% Bolt Bis–-Tris Plus pre-cast gels (Invitrogen) and visualized with Colloidal Blue following manufacturer’s instructions (Invitrogen). When processing biotin pull-down samples, each gel lane was cut into seven slices (see Supplementary Material, Fig. S2B). Based on earlier experiments and the BirA control, we excluded the intense bands corresponding to avidin monomers, dimers and an endogenously biotinylated protein from further analysis. The remaining four slices were subjected to in-gel digestion as described previously (93). Briefly, proteins were reduced and alkylated by incubating with DTT and chloroacetamide, respectively. Protein digestion was performed by saturation of the gel pieces with trypsin and overnight incubation at 37°C. Resulting peptides were extracted from the gel, dried down in a vacuum centrifuge and stored at −20°C. Peptide mixture was resuspended in 0.1% formic acid previous to the LC-MS/MS analysis. LC-MS/MS analysis Mass spectrometric analyses were performed on an EASY-nLC 1000 liquid chromatography system interfaced with a Q Exactive mass spectrometer (Thermo Scientific) via a nanospray flex ion source. Peptides were loaded onto an Acclaim PepMap100 pre-column (75 µm × 2 cm, Thermo Scientific) connected to an Acclaim PepMap RSLC (50 µm ×x 15 cm, Thermo Scientific) analytical column. Peptides were eluted from the column using a linear gradient of 2 to 40% acetonitrile in 0.1% formic acid at a flow rate of 300 nL min−1 over 45 min. The mass spectrometer was operated in positive ion mode. Full MS scans were acquired from m/z 300 to 1850 with a resolution of 70 000 at m/z 200. The 10 most intense ions were fragmented by higher energy C-trap dissociation with normalized collision energy of 28 and MS/MS spectra were recorded with a resolution of 17 500 at m/z 200. The maximum ion injection time was 120 ms for both survey and MS/MS scans, whereas AGC target values of 3 × 106 and 5 × 105 were used for survey and MS/MS scans, respectively. In order to avoid repeat sequencing of peptides, dynamic exclusion was applied for 45 s. Singly charged ions or ions with unassigned charge state were also excluded from MS/MS. Data were acquired using Xcalibur software (Thermo Scientific). Data processing and bioinformatics analysis Acquired raw data files were processed with the MaxQuant (94) software (version 1.5.3.17) using the internal search engine Andromeda (95) and searched against the UniProt database restricted to Drosophila melanogaster entries (release 2015_11; 43712 entries). Spectra originated from the different slices corresponding to the same biological sample were combined. Carbamidomethylation (C) was set as fixed modification whereas Met oxidation, protein N-terminal acetylation and Lys GlyGly (not C-term) were defined as variable modifications. Mass tolerance was set to 8 and 20 ppm at the MS and MS/MS level, respectively. Enzyme specificity was set to trypsin, allowing for cleavage N-terminal to Pro and between Asp and Pro with a maximum of two missed cleavages. Match between runs option was enabled with 1.5 min match time window and 20 min alignment window to match identification across samples. The minimum peptide length was set to seven amino acids. The false discovery rate for peptides and proteins was set to 1%. Normalized spectral protein label-free quantification (LFQ) intensities were calculated using the MaxLFQ algorithm. Data analysis and statistical tests MaxQuant output data was analysed with the Perseus module (version 1.5.6.0) (45). Initially, proteins only identified by site, contaminants, reverse hits and proteins with no unique peptides and/or no intensity were removed. Missing LFQ intensity values were replaced with values from a normal distribution (width 0.3 and down shift 1.8), meant to simulate expression below the detection limit (45). To determine statistically significant changes in protein abundance, as well as in ubiquitin diGly peptides, two-tailed Student’s t-test was used. In the analysis of the biotin pulldowns, two comparisons were carried out: bioA3 versus bioUb (i.e. Ube3a gain of function versus control) and bio15B versus bioUb (i.e. Ube3a loss of function versus control). Proteins displaying a LFQ fold change bigger than 2 with a P-value smaller than 0.05 were selected for further analysis. The selected proteins were further filtered based on the intensity pattern observed for their peptides. Statistical significance in western blotting semi-quantification was evaluated using an analysis of variance (ANOVA) complemented by Tukey’s honest significance difference test (Tukey’s HSD) performed in GraphPad PRISM software. RNA extraction, reverse transcription and real-time qPCR Total RNA was isolated from 30 mg of brain from embryos, young and adult wild-type mice from different ages. RNA was extracted and further purified by using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s protocol. Contaminating genomic DNA was removed by treatment with deoxyribonuclease I (QIAGEN), and cDNAs were synthesized from 1 μg RNA using the AffinityScript Multi Temperature cDNA Synthesis Kit (Agilent Technologies). Quantitative RT-PCR was performed on cDNA in the presence of Power SYBR Green PCR Master Mix (Applied Biosystems) containing preset concentrations of deoxynucleotide triphosphates and with specific primers, using the ABI Prism 7900 sequence Detection System (Applied Biosystems). PCR parameters were 50°C for 2 min, 95°C for 10 min, 40 cycles at 95°C for 15 s and 60°C for 1 min. The purity of the PCR products was assessed by dissociation curves. The amount of target cDNA was calculated by the comparative threshold (Ct) method and expressed by the 2-ΔΔCt method according to Applied Biosystems’ instructions, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. Expression of GAPDH mRNA was not affected by age, and the ratio of ΔCt value did not vary with the amount of cDNA. Each primer set was used at its optimal concentration (300 nM) with maximal efficacy. It was verified that one single specific product was amplified as shown by analysis of its melting temperature value. Primers GAPDH—Forward: 5′-ACCACAGTCCATGCCATCAC-3′  Reverse: 5′-TCCACCACCCTGTTGCTGTA-3′ Primers DDI1—Forward: 5′-TCACTGTGTATTGTGTGCGTAG-3′  Reverse: 5′-AGCTGTTCCATGTAAACGATCTG-3′ Primers DDI2—Forward: 5′-CCTCTCCGAGGTGACCTTTTC-3′  Reverse: 5′-GGCCTTTCTGCATAGACAATCT-3′ RNA in situ hybridization CD-1 mice were mated overnight, and the presence of a vaginal plug indicated embryonic day (E) 0.5. Unsexed embryos at E9.5 and E10.5 (the age 9.5 and 10.5 days post coitum) were fixed in 4% PFA and used for in situ hybridization, both in whole mount and in paraffin sections. Heads of 14.5 and 16.5 days post coitum old CD-1 embryos were fixed in 4% PFA and further processed for paraffin sections. Embryo samples for whole mount ISH were frozen in methanol at −20°C prior tissue hydration, proteinase K treatment, acetylation and the prehybridization and hybridization procedures. The embryos and heads used for sectioning were dehydrated, embedded in paraffin, cut to 7 µm sections and rehydrated prior further treatment. The experimental procedures were performed according to standard protocols (96). Murine Ddi1 coding sequence (NM_027942.1) was synthesized by GenScript and cloned into pGEM-T® easy plasmid. Linearized plasmid was purified with PCR Purification Kit (QIAGEN) and used for generation of digoxigenin labelled riboprobes with digoxigenin RNA labelling kit (Roche Applied Science) by in vitro transcription according to the provided manual. Probes were further cleaned by RNeasy Mini kit (QIAGEN) following RNA clean-up manual. Hybridization was performed overnight at 70°C with all probes for both whole mount and sectioned embryo samples. The DIG labelled probes were detected with anti-DIG antibody conjugated with alkaline phosphatase and BM purple AP substrate precipitating solution (Roche Applied Science) was used for signal development. All samples were postfixed in 4% PFA and the slides were mounted in Aquatex. Images were taken using Zeiss ApoTome microscope. Cell staining and microscopy SH-SY5Y cells in Supplementary Material, Fig S5C were transfected using Lipofectamine 3000 transfection reagent (Invitrogen) and grown over a coverslip glass. After 48 h, cells were washed twice with 2× PBS, fixed in 4% PFA for 20 min at room temperature, washed with PBST (0.1% Triton X-100 in 1× PBS) for three times and nuclei were stained with NucBlue Fixed Cell Stain Readyprobes reagent (Invitrogen). After staining, cells were washed with 1× PBS three times, the cover slips were mounted into slides with ProLong Diamond antifade reagent (Invitrogen), and samples were analysed in an inverted microscope ECLIPSE TS2-FL (Nikon). Supplementary Material Supplementary Material is available at HMG online. Acknowledgements The authors are grateful of the technical support provided by UPV/EHU SGIker and Animal house staff, which are supported with European funding (ERDF and ESF). In particular we thank Arantza Alejo and the Animal Unit at UPV/EHU. We thank Janice Fisher, Fen-Biao Gao, Eric Liebl, Jose Antonio Rodríguez Pérez, Vjekoslav Tomaić and Andreas Wodarz for antibodies, flies and plasmids. Conflict of Interest statement. None declared. Funding This work was supported by March of Dimes [Research Grant 1-FY15–339]; by ISCIII (grants PRB2 IPT13/0001 - ISCIII-SGEFI/ERDF and PRB3 IPT17/0019 - ISCIII-SGEFI/ERDF) and Spanish MINECO [grants SAF2013–44782-P and SAF2016–76898-P, both cofinanced with FEDER funds, to UM, BFU2014–52282-P and BFU2017–84653-P to RB]. UM and RB are also part of COST action Proteostasis. This work was also partially supported by Fondation Jérôme Lejeune grant 1381-MU2015A to UM. This work was supported by the Severo Ochoa Excellence Accreditation [grant SEV-2016–0644] and Consolider Programs [grant BFU2014–57703-REDC] to RB. Support was also provided from the Government of the Basque Country (Etortek Research Programs) and the Bizkaia County (Innovation Technology Department). This work was also supported by the Ministry of Education, Youth and Sports of the Czech Republic within the National Sustainability Program II [Project BIOCEV-FAR LQ1604] and by LM2015040 Czech Centre for Phenogenomics by MEYS, CZ.1.05/1.1.00/02.0109 Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University in Vestec (BIOCEV) by MEYS and ERDF, and CZ.1.05/2.1.00/19.0395—Higher quality and capacity for transgenic models by MEYS and ERDF. References 1 Buiting K. , Williams C. , Horsthemke B. ( 2016 ) Angelman syndrome - insights into a rare neurogenetic disorder . Nat. Rev. Neurol ., 12 , 584 – 593 . 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Human Molecular GeneticsOxford University Press

Published: Mar 21, 2018

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