DEGRONOPEDIA:a web server for proteome-wide inspection of degrons

E3 ubiquitin ligases recognize substrates through their short linear motifs termed degrons. While degron-signaling has been a subject of extensive study, resources for its systematic screening are limited. To bridge this gap, we developed DEGRONOPEDIA, a web server that searches for degrons and maps them to nearby residues that can undergo ubiquitination and disordered regions, which may act as protein unfolding seeds. Along with an evolutionary assessment of degron conservation, the server also reports on post-translational modifications and mutations that may modulate degron availability. Acknowledging the prevalence of degrons at protein termini, DEGRONOPEDIA incorporates machine learning to assess N-/C-terminal stability, supplemented by simulations of proteolysis to identify degrons in newly formed termini. An experimental validation of a predicted C-terminal destabilizing motif, coupled with the confirmation of a post-proteolytic degron in another case, exemplifies its practical application. DEGRONOPEDIA can be freely accessed at degronopedia.com

DEGRONOPEDIA:a web server for proteome-wide inspection of degrons

E3 ubiquitin ligases recognize substrates through their short linear motifs termed degrons. While degron-signaling has been a subject of extensive study, resources for its systematic screening are limited. To bridge this gap, we developed DEGRONOPEDIA, a web server that searches for degrons and maps them to nearby residues that can undergo ubiquitination and disordered regions, which may act as protein unfolding seeds. Along with an evolutionary assessment of degron conservation, the server also reports on post-translational modifications and mutations that may modulate degron availability. Acknowledging the prevalence of degrons at protein termini, DEGRONOPEDIA incorporates machine learning to assess N-/C-terminal stability, supplemented by simulations of proteolysis to identify degrons in newly formed termini. An experimental validation of a predicted C-terminal destabilizing motif, coupled with the confirmation of a post-proteolytic degron in another case, exemplifies its practical application. DEGRONOPEDIA can be freely accessed at degronopedia.com

Kinetic parameters for control non-expressing <i>UAS-aS</i> flies (<i>w</i>; +; <i>UAS-Hsap</i>/+; CTRL) vs. aS-expressing flies (<i>w</i>; +; <i>UAS-Hsap/nSyb-Gal4</i>; AS) measured at 1, 7, 16, 21, 30 and 42 days of fly lifetime.

<p>(A, B) Mean velocity (mm/s). (C, D) Maximum velocity (mm/s). (E, F) Total walking duration (s). (G, H) Total walking distance (mm). (I, J) Percentage of time that flies are in motion (%). (K, L) Mean trajectory length (mm). (M, N) Mean trajectory length per episode (mm). (O, P) Sample tracings of fly trajectories for 1 day-young and 30-day old control and aS flies. Scattered line and bar diagrams represent the mean values. Error bars = ± SE. * <i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> <0.001.</p

ATR-FTIR spectra of amide-I region of recombinant human aS.

<p>Secondary structure analyses were performed for aS incubated at 37°C alone (A, C, E) or with FN075 (B, D, F). Spectra were collected at different points of incubation: 0h, 3h, 124h and 144h. Deconvolution and curve-fitting were used to determine the secondary structure composition.</p

Effects of 2-pyridones on level of soluble aS in fly brain extracts of 20 days old flies.

<p>Mean soluble aS levels were measured with antibody specific to human aS in ELISA test. Controls: vehicle-treated nsyb-Gal4 outcrossed with Oregon R flies (CTRL VEH) or aS expressing flies (AS VEH, white bars). Tested compounds were FN075 (AS FN075, magenta bars) or MS400 (AS MS400, green bars) or C10 (AS C10, blue bars) at 100μM concentration. Bars represent mean values (n = 2). Error bars indicate ± SD. * <i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> <0.001. Multivariate GLM followed by Fisher's post hoc showed <i>P</i> = 0.084 for AS FN075 vs AS VEH, <i>P</i><0.001 for AS MS400 vs AS VEH and <i>P</i> = 0.002 for AS C10 vs AS VEH. For raw data see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184117#pone.0184117.s012" target="_blank">S7 Table</a>.</p

Western blot analysis of fly head protein extracts probed with antibody to human aS.

<p>The protein extracts are divided in soluble and insoluble aS fractions prepared as described in SI. Tubulin (upper panel), aS (lower panel). Lanes: 1. Recombinant human aS (5 ng), 2. Molecular weight marker, 3. Soluble fraction of fly head extracted aS, 4. Insoluble fraction of fly head extracted aS.</p

Feeding effects of 2-pyridones on aS-expressing flies life span. Survival analysis is presented by Kaplan-Meier curves.

<p>(A) Cumulative survival of control flies (<i>w</i>⁺; +; +/<i>nSyb-GAL4</i>; CTRL VEH, dotted black line) and aS expressing flies (<i>w</i>; +; <i>UAS-Hsap/nSyb-Gal4</i>; AS) treated with either vehicle (AS VEH, black) or compounds: FN075 (AS FN075, magenta), MS400 (AS MS400, green line) or C10 (AS C10, blue line) at 100 μM concentration. Bar diagrams show (B) mean, (C) median and (D) maximum lifetime. Numbers in bars represent days of mean, median and maximum lifetime (n = 10). Error bars imply ± SE.</p