Relationships between predicted moonlighting proteins, human diseases, and comorbidities from a network perspective

International audienceMoonlighting proteins are a subset of multifunctional proteins characterized by their multiple, independent, and unrelated biological functions. We recently set up a large-scale identification of moonlighting proteins using a protein-protein interaction (PPI) network approach. We established that 3% of the current human interactome is composed of predicted moonlighting proteins. We found that disease-related genes are over-represented among those candidates. Here, by comparing moonlighting candidates to non-candidates as groups, we further show that (7 they are significantly involved in more than one disease, (ii) they contribute to complex rather than monogenic diseases, (iii) the diseases in which they are involved are phenotypically different according to their annotations, finally, (iv) they are enriched for diseases pairs showing statistically significant comorbidity patterns based on Medicare records. Altogether, our results suggest that some observed comorbidities between phenotypically different diseases could be due to a shared protein involved in unrelated biological processes

Integrated microscopy and metabolomics to test an innovative fluid dynamic system for skin explants in vitro

The in vitro models are receiving growing attention in studies on skin permeation, penetration, and irritancy, especially for the preclinical development of new transcutaneous drugs. However, synthetic membranes or cell cultures are unable to effectively mimic the permeability and absorption features of the cutaneous barrier. The use of explanted skin samples maintained in a fluid dynamic environment would make it possible for an in vitro experimentation closer to in vivo physiological conditions. To this aim, in the present study, we have modified a bioreactor designed for cell culture to host explanted skin samples. The preservation of the skin was evaluated by combining light, transmission, and scanning electron microscopy, for the histo/cytological characterization, with nuclear magnetic resonance spectroscopy, for the identification in the culture medium of metabolites indicative of the functional state of the explants. Our morphological and metabolomics results demonstrated that fluid dynamic conditions ameliorate significantly the structural and functional preservation of skin explants in comparison with conventional culture conditions. Our in vitro system is, therefore, reliable to test novel therapeutic agents intended for transdermal administration in skin samples from biopsies or surgical materials, providing predictive information suitable for focused in vivo research and reducing animal experimentation

NMR Structural and interaction studies of bile acid binding proteinscomplexed with physiological ligands and bile acid-derived contrastagents

Il progetto svolto durante il mio Dottorato di Ricerca riguarda essenzialmente la cartterizzazione strutturale di complessi proteinalegante tramite spettroscopia di Risonanza Magnetica Nucleare (NMR). Studiare le interazioni proteiche a livello molecolare \ue8 di fondamentale importanza per acquisire informazioni su molti processi biologici, quali lo sviluppo di malattie nell\u2019uomo o il relativo disegno di farmaci. La Risonanza Magnetica Nucleare \ue8 una tecnica particolarmente adatta per la caratterizzazione, a livello atomico, delle interazioni tra proteine e altre molecole in soluzione. La Risonanza Magnetica Nucleare pu\uf2 essere utilizzata per valutare gli aspetti strutturali, termodinamici e cinetici di una reazione di interazione proteina-legante e pu\uf2 rilevare anche le interazioni pi\uf9 deboli. In questo contesto, durante il mio Dottorato di Ricerca ho affrontato l'indagine strutturale e lo studio dei determinanti molecolari dell\u2019interazione di differenti proteine, le bile acid binding proteins (BABPs) in complesso sia con i loro leganti fisiologici sia legate a farmaci sintetitici. Le BABPs sono piccole proteine citosoliche che svolgono la loro funzione negli epatociti ed enterociti, dove agiscono come trasportatori intracellulari degli acidi biliari, permettendo il loro ricircolo tramite la circolazione enteroepatica. Nella prima prima parte di questa tesi viene descritta la determinazione strutturale tramite NMR della BABP di fegato di pollo (cL-BABP) in complesso con l\u2019addotto acido biliare-Gd-DTPA, un potenziale agente di contrasto epatospecifo per la Risonanza Magnetica per Immagine (MRI). Il razionale di questo studio deriva da una ricerca di nuovi agenti di contrasto epatospecifici per la discriminazione e la diagnosi di lesioni focali o tumori nel fegato, tramite la Risonanza Magnetica per Immagine. La seconda parte di questa tesi \ue8 focalizzata sulla caratterizzazione delle interazioni della BABP ileale di pollo (cI-BABP) con acidi biliari. Questo studio \ue8 stato svolto utilizzando diversi approcci, cos\uec come la spettroscopia NMR e misure calorimetriche. La terza parte del mio lavoro di tesi si basa sulla determinazione strutturale tramite NMR del complesso ternario tra la CI-BABP e due molecole di acido glicochenodeossicolico (GCDA). Infine, un singolo e doppio mutante della proteina CI-BABP (A101S e H99Q/A101S) sono stati prodotti per studiare i determinanti molecolari della cooperativit\ue0 di legame. I dati ottenuti permettono di capire le basi chimiche del trasporto intracellulare degli acidi biliari. Questa conoscenza pu\uf2 aprire nuove vie per l'esplorazione di strategie di prevenzione e trattamento delle malattie metaboliche.The research project of my PhD concerns essentially the Nuclear Magnetic Resonance (NMR) structural studies of protein-ligand complexes. Studying protein interactions at the molecular level is crucial to the understanding of many biological processes, such as human diseases and drug design. NMR spectroscopy is particularly well suited to the investigation, at the atomic level, of the interactions between proteins and other molecules in solution. NMR can be used to evaluate the structural, thermodynamic and kinetic aspects of a binding reaction, even for weak protein-ligand interactions. In this contest, during my PhD I addressed the structural investigation and the study of the molecular determinants of binding of different bile acid binding proteins (BABPs) and their native ligands or synthetic drugs. BABPs are small cytosolic proteins that display their function in the hepatocytes and enterocytes where they act as bile acids transporters participating to the enterohepatic circulation. The first part of this thesis is related to the structural determination of chicken liver-BABP (cL-BABP) in complex with a bile acid-based gadolinium(III)-chelate, a potential hepatospecific contrast agent for magnetic resonance imaging (MRI). The rationale of this study derived from a search for new hepatospecific MRI contrast agents for the discrimination and diagnosis of focal lesions or hepatic malignancies. The second part of this thesis is focused on the characterization of the interactions of chicken ileal BABP (cI-BABP) with bile acids. This study has been performed using different approaches, such as NMR spectroscopy and calorimetric measurements. The third part of the my thesis work is based on the NMR structural determination of the ternary complex between cI-BABP and two molecules of glycochenodeoxycholic acid (GCDA). Finally, a single and double mutant of cI-BABP (A101S and H99Q/A101S) were produced to investigate the molecular determinants of binding cooperativity. The obtained data allow shedding light on the chemical basis of intracellular bile acid transport. This notion may open new avenues for exploration of strategies for prevention and treatment of metabolic diseases

Biological network comparison using graphlet degree distribution

Analogous to biological sequence comparison, comparing cellular networks is an important problem that could provide insight into biological understanding and therapeutics. For technical reasons, comparing large networks is computationally infeasible, and thus heuristics such as the degree distribution have been sought. It is easy to demonstrate that two networks are different by simply showing a short list of properties in which they differ. It is much harder to show that two networks are similar, as it requires demonstrating their similarity in all of their exponentially many properties. Clearly, it is computationally prohibitive to analyze all network properties, but the larger the number of constraints we impose in determining network similarity, the more likely it is that the networks will truly be similar. We introduce a new systematic measure of a network's local structure that imposes a large number of similarity constraints on networks being compared. In particular, we generalize the degree distribution, which measures the number of nodes 'touching' k edges, into distributions measuring the number of nodes 'touching' k graphlets, where graphlets are small connected non-isomorphic subgraphs of a large network. Our new measure of network local structure consists of 73 graphlet degree distributions (GDDs) of graphlets with 2-5 nodes, but it is easily extendible to a greater number of constraints (i.e. graphlets). Furthermore, we show a way to combine the 73 GDDs into a network 'agreement' measure. Based on this new network agreement measure, we show that almost all of the 14 eukaryotic PPI networks, including human, are better modeled by geometric random graphs than by Erdos-Reny, random scale-free, or Barabasi-Albert scale-free networks.Comment: Proceedings of the 2006 European Conference on Computational Biology, ECCB'06, Eilat, Israel, January 21-24, 200

Molecular properties of human guanylate cyclase-activating protein 2 (GCAP2) and its retinal dystrophy-associated variant G157R

In murine and bovine photoreceptors, guanylate cyclase activating-protein 2 (GCAP2) activates retinal guanylate cyclases (GC) at low Ca2+ levels, thus contributing to the Ca2+/cGMP negative feedback on the cyclase together with its paralog GCAP1, which has the same function but different Ca2+ sensitivity. In humans, a GCAP2 missense mutation (G157R) has been associated with inherited-retinal degeneration (IRD) via an unknown molecular mechanism. Here, we characterized the biochemical properties of human GCAP2 and the G157R variant, focusing on its dimerization and the Ca2+/Mg2+-binding processes in the presence or absence of N-terminal myristoylation. We found that human GCAP2 and its bovine/murine orthologs significantly differ in terms of oligomeric properties, cation binding, and GC regulation. Myristoylated GCAP2 endothermically binds up to three Mg2+ ions with high affinity and forms a compact dimer that may reversibly dissociate in the presence of Ca2+. Conversely, non-myristoylated GCAP2 does not bind Mg2+ over the physiological range, and remains as a monomer in the absence of Ca2+. Both myristoylated and non-myristoylated GCAP2 bind Ca2+ with high affinity. At odds with GCAP1 and independently of myristoylation, human GCAP2 does not significantly activate retinal GC1 in a Ca2+-dependent fashion. The IRD-associated G157R variant is characterized by a partly misfolded, molten globule-like conformation with reduced affinity for cations, and is prone to form aggregates, likely mediated by hydrophobic interactions. Our findings suggest that GCAP2 in human photoreceptors might be mostly implicated in processes other than phototransduction, and suggest a possible molecular mechanism for G157R-associated IRD

Phospho3D: a database of three-dimensional structures of protein phosphorylation sites

Phosphorylation is the most common protein post-translational modification. Phosphorylated residues (serine, threonine and tyrosine) play critical roles in the regulation of many cellular processes. Since the amount of data produced by screening assays is growing continuously, the development of computational tools for collecting and analysing experimental data has become a pivotal task for unravelling the complex network of interactions regulating eukaryotic cell life. Here we present Phospho3D, , a database of 3D structures of phosphorylation sites, which stores information retrieved from the phospho.ELM database and is enriched with structural information and annotations at the residue level. The database also collects the results of a large-scale structural comparison procedure providing clues for the identification of new putative phosphorylation sites

Post-transcriptional regulatory patterns revealed by protein-RNA interactions

The coordination of the synthesis of functionally-related proteins can be achieved at the post-transcriptional level by the action of common regulatory molecules, such as RNA–binding proteins (RBPs). Despite advances in the genome-wide identification of RBPs and their binding transcripts, the protein–RNA interaction space is still largely unexplored, thus hindering a broader understanding of the extent of the post-transcriptional regulation of related coding RNAs. Here, we propose a computational approach that combines protein–mRNA interaction networks and statistical analyses to provide an inferred regulatory landscape for more than 800 human RBPs and identify the cellular processes that can be regulated at the post-transcriptional level. We show that 10% of the tested sets of functionally-related mRNAs can be post-transcriptionally regulated. Moreover, we propose a classification of (i) the RBPs and (ii) the functionally-related mRNAs, based on their distinct behaviors in the functional landscape, hinting towards mechanistic regulatory hypotheses. In addition, we demonstrate the usefulness of the inferred functional landscape to investigate the cellular role of both well-characterized and novel RBPs in the context of human diseases

pdbFun: mass selection and fast comparison of annotated PDB residues

pdbFun () is a web server for structural and functional analysis of proteins at the residue level. pdbFun gives fast access to the whole Protein Data Bank (PDB) organized as a database of annotated residues. The available data (features) range from solvent exposure to ligand binding ability, location in a protein cavity, secondary structure, residue type, sequence functional pattern, protein domain and catalytic activity. Users can select any residue subset (even including any number of PDB structures) by combining the available features. Selections can be used as probe and target in multiple structure comparison searches. For example a search could involve, as a query, all solvent-exposed, hydrophylic residues that are not in alpha-helices and are involved in nucleotide binding. Possible examples of targets are represented by another selection, a single structure or a dataset composed of many structures. The output is a list of aligned structural matches offered in tabular and also graphical format

Impact of observational incompleteness on the structural properties of protein interaction networks

The observed structure of protein interaction networks is corrupted by many false positive/negative links. This observational incompleteness is abstracted as random link removal and a specific, experimentally motivated (spoke) link rearrangement. Their impact on the structural properties of gene-duplication-and-mutation network models is studied. For the degree distribution a curve collapse is found, showing no sensitive dependence on the link removal/rearrangement strengths and disallowing a quantitative extraction of model parameters. The spoke link rearrangement process moves other structural observables, like degree correlations, cluster coefficient and motif frequencies, closer to their counterparts extracted from the yeast data. This underlines the importance to take a precise modeling of the observational incompleteness into account when network structure models are to be quantitatively compared to data.Comment: 17 pages, 7 figures, accepted by Physica

A disulfide bridge allows for site-selective binding in liver bile acid binding protein thereby stabilising the orientation of key amino acid side chains

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