A cross-species spatiotemporal proteomic analysis identifies UBE3A-dependent signaling pathways and targets

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by the loss of neuronal E3 ligase UBE3A. Restoring UBE3A levels is a potential disease-modifying therapy for AS and has recently entered clinical trials. There is paucity of data regarding the molecular changes downstream of UBE3A hampering elucidation of disease therapeutics and biomarkers. Notably, UBE3A plays an important role in the nucleus but its targets have yet to be elucidated. Using proteomics, we assessed changes during postnatal cortical development in an AS mouse model. Pathway analysis revealed dysregulation of proteasomal and tRNA synthetase pathways at all postnatal brain developmental stages, while synaptic proteins were altered in adults. We confirmed pathway alterations in an adult AS rat model across multiple brain regions and highlighted region-specific differences. UBE3A reinstatement in AS model mice resulted in near complete and partial rescue of the proteome alterations in adolescence and adults, respectively, supporting the notion that restoration of UBE3A expression provides a promising therapeutic option. We show that the nuclear enriched transketolase (TKT), one of the most abundantly altered proteins, is a novel direct UBE3A substrate and is elevated in the neuronal nucleus of rat brains and human iPSC-derived neurons. Taken together, our study provides a comprehensive map of UBE3A-driven proteome remodeling in AS across development and species, and corroborates an early UBE3A reinstatement as a viable therapeutic option. To support future disease and biomarker research, we present an accessible large-scale multi-species proteomic resource for the AS community (https://www.angelman-proteome-project.org/)

SILAC-based quantitative mass spectrometry-based proteomics quantifies endoplasmic reticulum stress in whole HeLa cells

The unfolded protein response (UPR) involves extensive proteome remodeling in many cellular compartments. So far, a comprehensive analysis has been missing due to technological limitations. Here we employ Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)-based proteomics to quantify over 6200 proteins at increasing concentrations of tunicamycin in HeLa cells. We further compare the effects of tunicamycin (5 ug/ml) to those of thapsigargin (1 \ub5M) and DTT (2mM), both activating the UPR through different mechanisms. The systematic quantification of the proteome-wide expression changes following proteostatic stress is a resource for the scientific community, which enables the discovery of novel players involved in the pathophysiology of the broad range of disorders linked to proteostasis. We identified 38 proteins not previously linked to the UPR, whose expression increases, of which 15 likely remediate ER stress, and the remainder may contribute to pathological outcomes. Unexpectedly, there are few strongly downregulated proteins, despite expression of the pro-apoptotic transcription factor CHOP, suggesting that IRE1-dependent mRNA decay (RIDD) has a limited contribution to ER-stress mediated cell death in our system

Ultradeep Human Phosphoproteome Reveals a Distinct Regulatory Nature of Tyr and Ser/Thr-Based Signaling

Regulatory protein phosphorylation controls normal and pathophysiological signaling in eukaryotic cells. Despite great advances in mass-spectrometry-based proteomics, the extent, localization, and site-specific stoichiometry of this posttranslational modification (PTM) are unknown. Here, we develop a stringent experimental and computational workflow, capable of mapping more than 50,000 distinct phosphorylated peptides in a single human cancer cell line. We detected more than three-quarters of cellular proteins as phosphoproteins and determined very high stoichiometries in mitosis or growth factor signaling by label-free quantitation. The proportion of phospho-Tyr drastically decreases as coverage of the phosphoproteome increases, whereas Ser/Thr sites saturate only for technical reasons. Tyrosine phosphorylation is maintained at especially low stoichiometric levels in the absence of specific signaling events. Unexpectedly, it is enriched on higher-abundance proteins, and this correlates with the substrate KM values of tyrosine kinases. Our data suggest that P-Tyr should be considered a functionally separate PTM of eukaryotic proteomes

Comparison of phosphorylation variation of sites within different structural categories.

<p><b>A</b>) Sites within ordered regions (blue) show smaller variation of the phosphorylation fold change over the cell cycle than those within disordered regions (red). The significance of the observation has been tested with Kolmogorov-Smirnov test (p-value 6.6E-13). (<b>B</b>) The variation of phosphorylation changes over the cell cycle scales with the structural propensities of the phosphorylated residues: from lowest in regular structures (blue) to highest in disordered regions (red). The observed differences were found to be significant by ANOVA test (p-value 3.02E-09).</p

Enrichment of multi-phospho sites in disordered regions.

<p>The enrichment of additional phosphorylation sites at different distances from the central modified residues was computed. The odds ratios were calculated with the Fisher's Exact test implemented in R. Multiple phosphorylation sites were found significantly more often in disordered regions for any of the considered distances.</p

Temporal phosphorylation patterns of phospho-sites with distinct structural properties.

<p>Phosphorylation fold changes of three sites (UniProt accession number and residue identification number are given) during the six time points is shown together with their corresponding local structure. From left to right the phosphorylation variation over the six time points increases, together with the level of disorder: from (<b>A</b>) regular secondary structure (α-helix or β-sheet) through (<b>B</b>) irregular coils and loops to (<b>C</b>) disordered regions. Phospho-serine residues (pS) within regular regions and loops show small fluctuations in their phosphorylation levels, while larger changes occur in disordered regions.</p

Kinase motif decomposition based on phosphorylation variability and structural preferences.

<p>The preferences of various kinases for sites with specific structural background and phosphorylation variation were calculated by the 2D Annotation Enrichment technique (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002842#s4" target="_blank">Methods</a>). In general four classes can be distinguished: (i) tyrosine kinases (black squares), (ii) proline-directed kinases (red circles), (iii) non-proline directed kinases with charged residues in their substrate recognition motif (green and blue triangles corresponding to acidophilic and basophilic kinases respectively) and (iv) proline-oriented kinases, which contain a proline residue in their motif at position different from +1 relative to the modification site (red triangles and pentagons). The vertical axis separates the kinases according to their structural preferences. Tyrosine kinases favor sites within ordered regions with small phosphorylation variability. Serine/threonine kinases prefer more disordered regions, but span a larger space of phosphorylation variation. There is a tendency towards increasing disorderedness with higher phosphorylation variation, which clearly separates non proline-directed, proline-oriented and proline-directed kinases, the latter being characterized by the largest variation in phosphorylation. Examples for each class are shown and the data of all kinases can be found in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002842#pcbi-1002842-t002" target="_blank">Table 2</a>.</p

Two sample logo of flanking regions of phosphorylation sites of low <i>versus</i> high phosphorylation variation.

<p>Amino acids in the top and bottom parts (<b>A</b>) – central residue serine and <b>B</b>) – central residue threonine) represent residues, which are enriched or depleted correspondingly in the flanking regions of sites with small phosphorylation variation. Strong preferences are found for charged residues such as arginine, aspartate, and glutamate. In contrast, the majority of the amino acids that are more frequent in the negative set (i.e. variable phosphorylation set) are disorder-related e.g. proline, serine and glycine.</p

Conservation of phosphorylated sites versus conservation of control sites taking into account local structure.

<p>Lower values correspond to slower evolutionary rate and higher conservation. Phosphorylation sites predicted to lie within regular structures (in blue, pS/pT/pY regular) appeared to be more conserved than their equivalent non-phosphorylated residues from the same proteins (p-value 2.24e-16). The same tendency was present for modified sites in disordered regions (in red, pS/pT/pY disordered), which were also subjected to a statistically significant slower evolutionary rate than their control set (p-value 3.4E-03). Phosphorylation sites in regular structures showed higher conservation than that of phosphorylation sites in irregular structures (p-value 3.23E-120).</p