16 research outputs found
Ube3a, the E3 ubiquitin ligase causing Angelman syndrome and linked to autism, regulates protein homeostasis through the proteasomal shuttle Rpn10
Ubiquitination, the covalent attachment of ubiquitin to a target protein, regulates most cellular processes and is involved in several neurological disorders. In particular, Angelman syndrome and one of the most common genomic forms of autism, dup15q, are caused respectively by lack of or excess of UBE3A, a ubiquitin E3 ligase. Its Drosophila orthologue, Ube3a, is also active during brain development. We have now devised a protocol to screen for substrates of this particular ubiquitin ligase. In a neuronal cell system, we find direct ubiquitination by Ube3a of three proteasome-related proteins Rpn10, Uch-L5, and CG8209, as well as of the ribosomal protein Rps10b. Only one of these, Rpn10, is targeted for degradation upon ubiquitination by Ube3a, indicating that degradation might not be the only effect of Ube3a on its substrates. Furthermore, we report the genetic interaction in vivo between Ube3a and the C-terminal part of Rpn10. Overexpression of these proteins leads to an enhanced accumulation of ubiquitinated proteins, further supporting the biochemical evidence of interaction obtained in neuronal cells.This work was supported by a Basque Government research grant (PI2011-24) and a March of Dimes Basil O′Connor Starter Scholar Research Award (5-FY12-16) to U.M. RB thanks the Spanish MICINN (grants BFU2008-01884, BFU2011-25986) and the Consolider Program (CSD2007-008-25120), the Basque Government (PI2009-16 and PI2012/42), and the Bizkaia County.Peer reviewe
Proteomic Analysis of the Ubiquitin Landscape in the <i>Drosophila</i> Embryonic Nervous System and the Adult Photoreceptor Cells
<div><p>Background</p><p>Ubiquitination is known to regulate physiological neuronal functions as well as to be involved in a number of neuronal diseases. Several ubiquitin proteomic approaches have been developed during the last decade but, as they have been mostly applied to non-neuronal cell culture, very little is yet known about neuronal ubiquitination pathways <i>in vivo</i>.</p><p>Methodology/Principal Findings</p><p>Using an <i>in vivo</i> biotinylation strategy we have isolated and identified the ubiquitinated proteome in neurons both for the developing embryonic brain and for the adult eye of <i>Drosophila melanogaster</i>. Bioinformatic comparison of both datasets indicates a significant difference on the ubiquitin substrates, which logically correlates with the processes that are most active at each of the developmental stages. Detection within the isolated material of two ubiquitin E3 ligases, Parkin and Ube3a, indicates their ubiquitinating activity on the studied tissues. Further identification of the proteins that do accumulate upon interference with the proteasomal degradative pathway provides an indication of the proteins that are targeted for clearance in neurons. Last, we report the proof-of-principle validation of two lysine residues required for nSyb ubiquitination.</p><p>Conclusions/Significance</p><p>These data cast light on the differential and common ubiquitination pathways between the embryonic and adult neurons, and hence will contribute to the understanding of the mechanisms by which neuronal function is regulated. The <i>in vivo</i> biotinylation methodology described here complements other approaches for ubiquitome study and offers unique advantages, and is poised to provide further insight into disease mechanisms related to the ubiquitin proteasome system.</p></div
Ubiquitin modified peptides identified in the <sup>bio</sup>Ub pulldown.
<p>Ubiquitin modified peptides for 35 different proteins, including ubiquitin itself, were identified. Peptides sequences, positions of di-gly (G-G) with each probability in brackets (when different from 1) and Posterior Error Probabilities (PEP Score) are reported. Oxidized methionine is indicated by an asterisk (*) and acetylation by (<i>ac</i>). Some of those ubiquitination sites were only identified upon Rpn10<sup>DN</sup> overexpression. Those ones are indicate by (r) in the position of di-glycine column. All peptides and intensities are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139083#pone.0139083.s010" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139083#pone.0139083.s011" target="_blank">S2</a> Tables.</p><p>Ubiquitin modified peptides identified in the <sup>bio</sup>Ub pulldown.</p
Biotinylated ubiquitin is incorporated into conjugates in the <i>Drosophila</i> adult eye.
<p>(A) Schematic illustration of the <i>in vivo</i> biotinylation of ubiquitin [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139083#pone.0139083.ref021" target="_blank">21</a>]. The construct is expressed as a polyubiquitin chain fused to the <i>E</i>.<i>coli</i> BirA enzyme, which is digested by the endogenous deubiquitinating enzymes (DUBs). While this clearly indicates that DUB enzymes are capable of deconstructing linear <sup>bio</sup>Ub chains, we have no evidence to support that E2/E3 enzymes are actually capable of constructing those linear <sup>bio</sup>Ub chains the same way they would do with the more abundant endogenous ubiquitin. Afterwards, the BirA enzyme recognizes the short motif incorporated at the N-terminus of each ubiquitin (sequence is indicated) and attaches a biotin molecule to its lysine residue (red). The biotinylated ubiquitins are then conjugated to the target proteins. (B) Western blot with anti-BirA antibody using total <i>Drosophila</i> head extracts confirmed the full digestion of the <sup>bio</sup>Ub precursor by endogenous DUBs. No undigested forms of the precursor were found above the expected molecular size of BirA (35 kDa). (C) Anti-biotin Western blot on the same total extracts confirmed the biotinylation and conjugation of the GMR<sup>GAL4</sup>–driven expressed ubiquitin. An endogenous protein known to be biotinylated (CG1516) appeared in all the samples (arrowhead). The expected smear corresponding to biotinylated ubiquitin conjugates was only present in the <sup>bio</sup>Ub sample. WT: <i>Oregon R</i>; BirA: <i>GMR</i><sup><i>GAL4</i></sup><i>/CyO</i>;<sup><i>UAS</i></sup><i>BirA/TM6</i>; <sup>bio</sup>Ub: <i>GMR</i><sup><i>GAL4</i></sup>,<sup><i>UAS</i></sup><i>(</i><sup><i>bio</i></sup><i>Ub)</i><sub><i>6</i></sub><i>-BirA/CyO</i>.</p
Strategy for pulldown of ubiquitinated material from <i>Drosophila melanogaster</i> tissues.
<p>(A) Schematic illustration of the strategy applied to purify ubiquitinated material from <i>Drosophila</i> embryo nervous system and adult eye. Both embryo and adult samples, expressing either the <sup>UAS</sup>(<sup>bio</sup>Ub)<sub>6</sub>-BirA or the <sup>UAS</sup>BirA (control) constructs, were homogenized, clarified and incubated with High Capacity NeutrAvidin agarose resin. Beads were then subjected to stringent washes to remove the non-biotinylated proteins. Afterwards, material bound to beads was eluted by applying a heat treatment. (B) Silver staining of the eluted material revealed no protein in the BirA samples except for the endogenously biotinylated proteins, particularly the most abundant (CG1516) (arrows), while in the <sup>bio</sup>Ub samples the typical smear of ubiquitinated material was detected (brackets). Monomer, dimer and tetramer forms of NeutrAvidin molecules leaking from the beads were also found in all the samples (arrowheads). Mass spectrometry analysis performed with embryo samples identified 37 and 234 proteins in the control and experimental samples, respectively. In adult the analysis identified 80 proteins in the control and 369 in the experimental samples. (C) Venn diagram showing the distribution of the identified ubiquitin conjugates. Identified ubiquitin carriers are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139083#pone.0139083.t001" target="_blank">Table 1</a>. Only 90 ubiquitin conjugates were found to be present in both data sets. The top 20 ubiquitin conjugates found only in embryo, only in adult and those found in both samples are listed below the Venn diagram. All peptides and intensities of the different analysis are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139083#pone.0139083.s010" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139083#pone.0139083.s011" target="_blank">S2</a> Tables.</p
Ubiquitin carriers identified in the <sup>bio</sup>Ub pulldown.
<p>Posterior Error Probabilities (PEP Score) and number of identifications in independent <sup>bio</sup>Ub pulldown experiments (n) are reported. All peptides and intensities are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139083#pone.0139083.s010" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139083#pone.0139083.s011" target="_blank">S2</a> Tables.</p><p><sup><i>a</i></sup> CG number, protein description and gen symbol given according to flybase nomenclature (<a href="http://www.flybase.org" target="_blank">www.flybase.org</a>).</p><p>Ubiquitin carriers identified in the <sup>bio</sup>Ub pulldown.</p
Western blot validation of identified ubiquitin conjugates and ubiqutin carriers.
<p>(A) Western blot performed with antibodies against Parkin (upper panel) or Ube3a E3 ligases (bottom panels). The ability of the HECT-type, as well as RING between RING-type E3 ligases to form thioester linkages with the ubiquitin before they transfer it to the substrates allow us to trap them while they are carrying ubiquitin. Since the reducing agents used to perform the elution from the beads breaks this type of linkage, there is no increase in their molecular weight relative to the inputs (*). A fraction of both E3s appears also conjugated with ubiquitin despite the DTT (arrows). This is probably due to auto-ubiquitination at some lysine residue. Parkin antibody non-specifically recognized several proteins in the inputs. The appropriate Parkin band is the one at 55 kDa. (B) Western blots with specific antibodies to some of the proteins identified in the adult pulldown revealed the expected increase of their molecular weight in the <sup>bio</sup>Ub sample relative to the inputs. Covalent attachment of ubiquitin should increase the protein’s molecular weight by about 10 kDa for each ubiquitin attached. Therefore, the increase shown in the Western blots (arrows) reflects their ubiquitinated status. Endogenous biotinylated CG1516 protein, non-specifically identified by some antibodies, is marked with an arrowhead. All Western blots were performed with adult samples, except Ube3a that was also test in embryo. BirA: <i>GMR</i><sup><i>GAL4</i></sup><i>/CyO</i>;<sup><i>UAS</i></sup><i>BirA/TM6</i>; <sup>bio</sup>Ub: <i>GMR</i><sup><i>GAL4</i></sup>,<sup><i>UAS</i></sup><i>(</i><sup><i>bio</i></sup><i>Ub)</i><sub><i>6</i></sub><i>-BirA/CyO</i>; A3 (Ube3a overexpression): <i>GMR</i><sup><i>GAL4</i></sup>,<sup><i>UAS</i></sup><i>(</i><sup><i>bio</i></sup><i>Ub)</i><sub><i>6</i></sub><i>-BirA/CyO</i>;<sup><i>UAS</i></sup><i>Dube3A/TM6B</i>; 15B (Ube3a deletion mutant): <i>GMR</i><sup><i>GAL4</i></sup>,<sup><i>UAS</i></sup><i>(</i><sup><i>bio</i></sup><i>Ub)</i><sub><i>6</i></sub><i>-BirA/CyO</i>, <i>Dube3A</i><sup><i>15B</i></sup><i>/TM6B</i>.</p
Ubiquitination sites of nSyb in neuronal cell line.
<p>(A) Ilustration of the peptides and the ubiquitination sites found in <i>Drosophila melanogaster</i> nSyb. The region of the protein where the modified lysines (red) were found is conserved among different species. The conserved aminoacids are shown in gray. (B) nSyb double mutant (DM) showed a significant reduction in its ubiquitinated fraction, as shown with anti-Flag antibody Western blot (red) compared to the wild type (WT) or the single lysine mutated forms (K71R or K78R). The non-modified form of nSyb was detected by GFP antibody (green). Quantification of the ubiquitination status of nSyb mutants relative to the non-modified form (Y axis: relative ubiquitination) was performed with Image-J. The plot shows relative levels of nSyb ubiquitination normalized to the GFP levels (average intensity ± SD). A t-test analysis for every pair of conditions was performed with GraphPad. One asterisk indicates p-value < to 0.05; two, p < to 0.01; three, p < to 0.0001.</p
Functional interpretation of the identified ubiquitin conjugates from embryo and adult samples.
<p>Ubiquitin conjugates present either in embryo or in adult pulldowns were further analysed with GO Term mapper. The analysis provided a list of broad GO terms (GO Slim) for the Biological Process (72 categories), Cellular Compartment (30 categories) and Molecular Function (44 categories) domains, which were additionally grouped into fewer categories to make their representation more visual and understandable. In the Biological Process pie chart categories representing less than 3% were grouped into “others”. In the Molecular Function pie chart biggest groups are provided. In the Cellular Compartment pie chart only “extracellular matrix”, “plasma membrane”, “nucleus” and “cytoplasm” categories are depicted.</p