Same same, but different: exploring the enigmatic role of the pituitary adenylate cyclase-activating polypeptide (PACAP) in invertebrate physiology

Evidence has been accumulating that elements of the vertebrate pituitary adenylate cyclase-activating polypeptide (PACAP) system are missing in non-chordate genomes, which is at odds with the partial sequence-, immunohistochemical-, and physiological data in the literature. Multilevel experiments were performed on the great pond snail (Lymnaea stagnalis) to explore the role of PACAP in invertebrates. Screening of neuronal transcriptome and genome data did not reveal homologs to the elements of vertebrate PACAP system. Despite this, immunohistochemical investigations with an anti-human PAC1 receptor antibody yielded a positive signal in the neuronal elements in the heart. Although Western blotting of proteins extracted from the nervous system found a relevant band for PACAP-38, immunoprecipitation and mass spectrometric analyses revealed no corresponding peptide fragments. Similarly to the effects reported in vertebrates, PACAP-38 significantly increased cAMP synthesis in the heart and had a positive ionotropic effect on heart preparations. Moreover, it significantly modulated the effects of serotonin and acetylcholine. Homologs to members of Cluster B receptors, which have shared common evolutionary origin with the vertebrate PACAP receptors, PTHRs, and GCGRs, were identified and shown not to be expressed in the heart, which does not support a potential role in the mediation of PACAP-induced effects. Our findings support the notion that the PACAP system emerged after the protostome-deuterostome divergence. Using antibodies against vertebrate proteins is again highlighted to have little/no value in invertebrate studies. The physiological effects of vertebrate PACAP peptides in protostomes, no matter how similar they are to those in vertebrates, should be considered non-specific.</p

Novel linear motif filtering protocol reveals the role of the LC8 dynein light chain in the Hippo pathway

<div><p>Protein-protein interactions (PPIs) formed between short linear motifs and globular domains play important roles in many regulatory and signaling processes but are highly underrepresented in current protein-protein interaction databases. These types of interactions are usually characterized by a specific binding motif that captures the key amino acids shared among the interaction partners. However, the computational proteome-level identification of interaction partners based on the known motif is hindered by the huge number of randomly occurring matches from which biologically relevant motif hits need to be extracted. In this work, we established a novel bioinformatic filtering protocol to efficiently explore interaction network of a hub protein. We introduced a novel measure that enabled the optimization of the elements and parameter settings of the pipeline which was built from multiple sequence-based prediction methods. In addition, data collected from PPI databases and evolutionary analyses were also incorporated to further increase the biological relevance of the identified motif hits. The approach was applied to the dynein light chain LC8, a ubiquitous eukaryotic hub protein that has been suggested to be involved in motor-related functions as well as promoting the dimerization of various proteins by recognizing linear motifs in its partners. From the list of putative binding motifs collected by our protocol, several novel peptides were experimentally verified to bind LC8. Altogether 71 potential new motif instances were identified. The expanded list of LC8 binding partners revealed the evolutionary plasticity of binding partners despite the highly conserved binding interface. In addition, it also highlighted a novel, conserved function of LC8 in the upstream regulation of the Hippo signaling pathway. Beyond the LC8 system, our work also provides general guidelines that can be applied to explore the interaction network of other linear motif binding proteins or protein domains.</p></div

Structural organization and phylogenetic tree of the WWC family members.

<p>The tree branch lengths do not represent real values. The subtrees of the three vertebrate WWC paralogs are highlighted by colored rectangles. Red and yellow boxes indicate the domains and coiled coil regions, respectively. Small purple and dark green boxes mark the location of the LC8 and PDZ binding motifs, respectively.</p

GO enrichment in the high confidence set of LC8 binding partners.

<p>Molecular Function (blue), Biological Process (green) and Cellular Component (red) categories that are enriched in the high-confident LC8 binding partners compared to human background. The x-axis represents the log-odds ratio of each enriched GO category. Process names related to the Hippo pathway are colored in red.</p

Distribution of LC8 partners and predicted motifs.

<p>(A) Distribution of LC8 interaction partners from PPI databases over the number of experiments. Partners that contain neither known nor predicted motifs are shaded grey according to the interaction type. (B) The composition of the high confidence prediction set shown as a Venn diagram. PPI: motifs in partners that appear in PPI databases. SLIM: motifs that show island-like conservation with the SLiMPrints method.</p

Position specific scoring matrix of LC8 binding motifs.

<p>PSSM score of each position of the binding motif numbered from the central glutamine of the canonical “TQT” motif core. Amino acids corresponding to the ELM definition are framed. Values are color scaled as a heat map ranging from blue (negative) to red (positive) centered on zero (white). The Shannon entropy of each column is shown below in bitscore.</p

Experimentally validated LC8 binding motifs in the Hippo pathway.

<p>Experimentally validated LC8 binding motifs in the Hippo pathway.</p

Summarized evolutionary conservation results of known human LC8 binding partners.

<p>Protein names and motif start positions are indicated in the first and second columns. The colored boxes represent the presence of orthologues of the known partner at different evolutionary levels. The percentages and colour scheme of the boxes show the PSSM based motif conservation across all species. Conservation values increase from white (low motif conservation) to red (high motif conservation).</p

Filtering protocol to find true binding partners.

<p>(A) Schematic diagram of the binary filtering protocol we created utilizing information gain. A given attribute provides a binary split of the Parent group into the <i>C1</i> and <i>C2</i> child groups. The information gain (<i>I</i>) is then calculated as the difference of the Shannon entropy (<i>H</i>) of the Parent group minus the Shannon entropy of the Children groups weighted by their relative probabilities (<i>p</i>). These values were calculated over the dataset containing 40 known human binding partners and 10,000 random human segments from the proteome with a higher than zero PSSM score. (B) The information gain of the PSSM score (left panel) and four disorder prediction methods as a function of different cut-off values (right panel). The disorder prediction method used here were: IUPred (blue), Espritz Disprot (green) and VSL2 (red line), DISOPRED3 (cyan). Optimal cut-off values were obtained from the cut-off value corresponding to the maximum of the information gain, yielding 3.3 for the PSSM score, and 0.42 for IUPred disorder prediction score. (C) The outline of the final filtering protocol indicating the number of elements and percentage of cases in each Child group with the applied binary split.</p

Scattering curves of Ca²⁺-free, Ca²⁺-bound and MPT-bound S100A4.

<p>Differences in the WT (<i>cyan</i>) and the Ca²⁺-bound WT (<i>red</i>) are mainly observed at 0.15 Å⁻¹–0.25 Å⁻¹ (A). The scattering profile of the Δ13 mutant (<i>purple</i>) changes less upon Ca²⁺-binding (<i>orange</i>) (B). Typical one-dimensional ¹H NMR spectra for the studied systems acquired at 700.17 MHz, zoomed to the aliphatic proton region. From bottom to top: buffer; WT; WT-Ca²⁺; Δ13; Δ13-Ca²⁺ (C). Typical examples of EOM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097654#pone.0097654-Bernado1" target="_blank">[20]</a> extended (D) and compact S100A4 models (E). Distribution of the radii of gyration of the generated model ensemble (<i>black</i>) for ensemble optimization method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097654#pone.0097654-Bernado1" target="_blank">[20]</a>, the best ensemble fitting the Ca²⁺-free (<i>cyan</i>) and Ca²⁺-bound (<i>magenta</i>) WT S100A4 SAXS data (F). Scattering curve differences between the MPT-bound WT (<i>red</i>) and the MPT-bound Δ13 mutant (<i>orange</i>) are more diffuse (G).</p