Heatmap visualization of protein and transcriptional Dube3a dependent changes.

<p><b>A)</b> Fold change for proteins identified as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061952#pone-0061952-g001" target="_blank">Figure 1</a> was calculated using fluorescent intensity as compared to <i>Heatshock</i>-GAL4 alone or w- extracts. The scale is from 30-fold decrease (red) to 30-fold increase (green) in intensity with yellow as an intermediate on the scale. Boxes represent individual proteins identified by mass spectrometry for that fraction (CE, cytoplasmic; NE, nuclear; and ME, membrane) and in that particular genotype. If a particular protein was identified several times in that fraction at different molecular weights or pH values, the average of these intensities was used for the purposes of the heatmap (for complete details on each protein see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061952#pone.0061952.s001" target="_blank">Table S1</a></b>). In three cases, a particular protein was determined to go up in one experiment, but down in another for a different isoform. These proteins are indicated with a slash through the box and the two opposing colors filled into each half. <b>B)</b> Average fold change by qRT-PCR for genes identified in the screen under varying levels of Dube3a protein expression. Although some of these genes have several predicted spliceforms, only common assays that could identify as many transcripts as possible were used for this analysis. In the case of Arginine kinase primer set 1 recognizes ArgK-RC and RF while set 2 recognizes RA and RB. For Myosin Heavy Chain set 1 includes RA-C, RF-RL and set 2 RB, RD-E, RK, RM and RJ. The scale is from 5-fold or greater decrease (red) to 5-fold or greater increase (green) in transcript vs. control (either <i>Heatshock</i>-GAL4 alone or <i>w¹¹¹⁸</i>). Note that some genes showed transcriptional changes greater than 5-fold, so they are on the upper or lower end of the color scale. A complete table of qRT-PCR results can be found in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061952#pone.0061952.s002" target="_blank">Table S2</a></b>, including error bars for the triplicate technical replicates.</p

String and phylogenetic analysis of putative Dube3a regulated proteins reveals a tight cluster of proteins involved in stress response, actin cytoskeleton and ATP binding.

<p><b>A)</b> All 43 proteins were submitted for STRING analysis and 27 of these proteins formed a single cluster of proteins related by function, pathway or literature search. The thickness of the blue line represents the predicted strength of the relationship between any two proteins. The tight cluster at the center of the diagram consists of 13 proteins enriched for involvement in stress response (Bonferroni corrected pval = 8.01E−03), molecular chaperones (Bonferroni corrected pval = 1.64E−05), ATP binding (Bonferroni corrected pval = 1.11E−04) and mitochondrial membrane proteins (Bonferroni corrected pval = 4.98E−01). <b>B)</b> Analysis of protein homology among common model organisms and humans for the Dube3a regulated protein set of 43 proteins from the proteomic screen. The gene name on the left is the fly gene and on the right of the (/) is the human gene name. The scale is from low homology (white) to high homology (black) with red being ∼50% protein sequence identity. Over half of the proteins (67%) were conserved by at least 50% identity from fly to human homologues. Many of these proteins were also conserved in other model organisms, indicating that these potential UBE3A targets could be studied in other systems to validate the results of our proteomic screen in flies. At least seven of the proteins (16%) showed almost no homology (less than 10%) to proteins in other non-insect organisms and are therefore of considerably less interest in terms of UBE3A involvement in human disease mechanisms.</p

Identification and quantification of fold change in potential Dube3a targets.

<p><b>A)</b> The middle 18 (out of 20) fractions from the first dimension liquid isoelectric focusing cells were run on 4–7% polyacrylaminde gels to separate proteins in each pH fraction by molecular weight and to determine what adjustments would be needed to normalize protein bands across fractions. Direct comparisons between matched pH samples were made between either wild type and <i>Dube3a^15b</i> loss of function extracts or <i>Heatshock</i>-GAL4 and <i>Heatshock-</i>GAL4>UAS-<i>Dube3a</i>, <i>Dube3a</i>-C/A or <i>hUBE3A</i> extracts. This figure illustrates proteins separated by isoelectric point (pI) and their measured average pH for each fraction from <i>Heatshock</i>-GAL4 alone on the top and <i>Heatshock</i>-GAL4>UAS-<i>Dube3a</i> on the bottom. Fractions that were approximately pH matched were then run on separate PAGE gels for direct comparisons. <b>B</b>) Direct comparison of <i>Heatshock</i>-GAL4 Alone pH 6.88 to <i>Heatshock</i>-GAL4>UAS-<i>Dube3a</i> pH 6.76 cytoplasmic protein extracts. Note the bands with the arrows indicating proteins that went up by 5-fold (∼51 kDa band) or 33-fold (∼37-kDa band) in the <i>Dube3a</i> over-expressing extracts. These bands, and others like it, were excised from the polyacrylamide gels and identified by MALDI-ToF ToF analysis. These particular proteins were identified as the predicted 53 kDa form of CG7430 and the predicted 39 kDa form of Arginine kinase (CG32031).</p

Gene Ontology (GO) characterization of potentially Dube3a regulated proteins.

<p>The GO classifications for each of the 43 proteins affected by changes in Dube3a expression or the expression of the ubiquitin defective Dube3a-C/A construct were extracted from Flybase (<a href="http://www.flybase.org" target="_blank">www.flybase.org</a>) and then input as a list into CateGOrizer (<a href="http://tinyurl.com/79kzqn9" target="_blank">http://tinyurl.com/79kzqn9</a>) for counting of the classification groups. The small pie chart in the upper left corner indicates the root GO categories represented in our data (18% cellular components, 46% involved in a biological process, and 36% involved in a particular molecular function). The larger pie chart illustrates the major categories of molecular function found in this data set. Most notable are the Catabolism, Carbohydrate Metabolism, Cytoskeleton, Nucleotide Binding, Energy Metabolism and Development categories which comprise ∼47% of the proteins identified in the screen. The colored slices with no percentage listed are all less than 2% of the total set of proteins. All of the categories are listed with a color-coded legend to the right of the pie chart.</p

Overview of bands excised, fractions and total number of novel proteins identified.

<p>Overview of bands excised, fractions and total number of novel proteins identified.</p

Ubquitination state of Eps15 and ATPα.

<p><b>A)</b> Ubiquitin enrichment from cytoplasmic extracts with elevated or decreased levels of Dube3a. Genotypes are listed below. Western blots were performed on ubiquitin-enriched extracts using Eps15 (top) and ATPα antibodies (bottom). Only the ∼100 kDa form of Eps15 could be detected in the elutions from the ubiquitin enrichment column for all genotypes. No ∼150 kDa form could be detected. The ∼100 kDa form appears to be ubiquitinated even in the <i>Dube3a^15b</i> mutant fractions. Ubiquitinated ATPα could be detected in all fractions except for the <i>Dube3a^15b</i> lane, indicating that ATPα may be ubiquitinated in a Dube3a dependent manner. <b>B)</b><i>In vitro</i> ubiquitin assays. Purified Dube3a and ATPα proteins were mixed with E1 and E2 proteins <i>in vitro</i> in an attempt to ubiquitinate ATPα using the Dube3a E3 ubiquitin ligase. The (+) and (–) symbols above the Western blot indicate the presence or absence of a component in the ubiquitination reaction run in that lane. ATPα protein could be detected in all of the appropriate lanes (red) and a Poly-Ub smear could be seen in the fourth lane from the left (the reaction which contained all components). <b>C)</b> The green signal (poly-Ub antibody) was used to quantify the level of ubiquitination detected above background (LANE 1) and above any Dube3a auto-ubiquitination (LANE 2). A strong smear detected in LANE 4 was 143%+/−15% above the background levels and was significantly higher than any signal that comes from Dube3a auto-ubiquitination (LANE 2). The (*) symbols indicates a p-value of less than 0.05 for these results by one way ANOVA analysis.</p

Proteomic Profiling in <i>Drosophila</i> Reveals Potential Dube3a Regulation of the Actin Cytoskeleton and Neuronal Homeostasis

<div><p>The molecular defects associated with Angelman syndrome (AS) and 15q duplication autism are directly correlated to expression levels of the E3 ubiquitin ligase protein UBE3A. Here we used <i>Drosophila melanogaster</i> to screen for the targets of this ubiquitin ligase under conditions of both decreased (as in AS) or increased (as in dup(15)) levels of the fly Dube3a or human UBE3A proteins. Using liquid phase isoelectric focusing of proteins from whole fly head extracts we identified a total of 50 proteins that show changes in protein, and in some cases transcriptional levels, when Dube3a fluctuates. We analyzed head extracts from cytoplasmic, nuclear and membrane fractions for Dube3a regulated proteins. Our results indicate that Dube3a is involved in the regulation of cellular functions related to ATP synthesis/metabolism, actin cytoskeletal integrity, both catabolism and carbohydrate metabolism as well as nervous system development and function. Sixty-two percent of the proteins were >50% identical to homologous human proteins and 8 have previously be shown to be ubiquitinated in the fly nervous system. Eight proteins may be regulated by Dube3a at the transcript level through the transcriptional co-activation function of Dube3a. We investigated one autism-associated protein, ATPα, and found that it can be ubiquitinated in a Dube3a dependent manner. We also found that Dube3a mutants have significantly less filamentous actin than wild type larvae consistent with the identification of actin targets regulated by Dube3a. The identification of UBE3A targets is the first step in unraveling the molecular etiology of AS and duplication 15q autism.</p></div

Dube3a regulated proteins identified in this study that are ubiquitinated in the nervous system according to Franco <i>et al.</i> 2011 [36].

<p>Dube3a regulated proteins identified in this study that are ubiquitinated in the nervous system according to Franco <i>et al.</i> 2011 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061952#pone.0061952-Franco1" target="_blank">[36]</a>.</p

Dopamine (DA) levels are increased in striatum, midbrain and frontal cortex under conditions of increased and decreased <i>Ube3a</i> expression.

<p>A significant increase was detected in DA levels in <i>Ube3a</i> deficient mice in both striatum and frontal cortex (p≤0.05) and a clear trend was detected in midbrain that did not reach significance. Animals with a 7p duplication encompassing the <i>Ube3a</i> gene showed a significant increase in DA levels in all three regions when the duplication was paternally inherited, but only showed a significant increase in striatum and frontal cortex when maternally inherited, although a trend towards increased DA is detected here as well. In all cases, DA levels for each genotype were compared to wild type littermates from each cross (100%). Significant differences were found at p≤0.05 for all groups (n = 6). * p≤0.05 compared to wild type littermate.</p

Altered Serotonin, Dopamine and Norepinepherine Levels in 15q Duplication and Angelman Syndrome Mouse Models

<div><p>Childhood neurodevelopmental disorders like Angelman syndrome and autism may be the result of underlying defects in neuronal plasticity and ongoing problems with synaptic signaling. Some of these defects may be due to abnormal monoamine levels in different regions of the brain. <em>Ube3a</em>, a gene that causes Angelman syndrome (AS) when maternally deleted and is associated with autism when maternally duplicated has recently been shown to regulate monoamine synthesis in the <em>Drosophila</em> brain. Therefore, we examined monoamine levels in striatum, ventral midbrain, frontal cerebral cortex, cerebellar cortex and hippocampus in <em>Ube3a</em> deficient and <em>Ube3a</em> duplication animals. We found that serotonin (5HT), a monoamine affected in autism, was elevated in the striatum and cortex of AS mice. Dopamine levels were almost uniformly elevated compared to control littermates in the striatum, midbrain and frontal cortex regardless of genotype in <em>Ube3a</em> deficient and <em>Ube3a</em> duplication animals. In the duplication 15q autism mouse model, paternal but not maternal duplication animals showed a decrease in 5HT levels when compared to their wild type littermates, in accordance with previously published data. However, maternal duplication animals show no significant changes in 5HT levels throughout the brain. These abnormal monoamine levels could be responsible for many of the behavioral abnormalities observed in both AS and autism, but further investigation is required to determine if any of these changes are purely dependent on Ube3a levels in the brain.</p> </div