KinFams: De-Novo Classification of Protein Kinases Using CATH Functional Units.

Protein kinases are important targets for treating human disorders, and they are the second most targeted families after G-protein coupled receptors. Several resources provide classification of kinases into evolutionary families (based on sequence homology); however, very few systematically classify functional families (FunFams) comprising evolutionary relatives that share similar functional properties. We have developed the FunFam-MARC (Multidomain ARchitecture-based Clustering) protocol, which uses multi-domain architectures of protein kinases and specificity-determining residues for functional family classification. FunFam-MARC predicts 2210 kinase functional families (KinFams), which have increased functional coherence, in terms of EC annotations, compared to the widely used KinBase classification. Our protocol provides a comprehensive classification for kinase sequences from >10,000 organisms. We associate human KinFams with diseases and drugs and identify 28 druggable human KinFams, i.e., enriched in clinically approved drugs. Since relatives in the same druggable KinFam tend to be structurally conserved, including the drug-binding site, these KinFams may be valuable for shortlisting therapeutic targets. Information on the human KinFams and associated 3D structures from AlphaFold2 are provided via our CATH FTP website and Zenodo. This gives the domain structure representative of each KinFam together with information on any drug compounds available. For 32% of the KinFams, we provide information on highly conserved residue sites that may be associated with specificity.

Role of germline variants in the metastasis of breast carcinomas.

Most cancer-related deaths in breast cancer patients are associated with metastasis, a multistep, intricate process that requires the cooperation of tumour cells, tumour microenvironment and metastasis target tissues. It is accepted that metastasis does not depend on the tumour characteristics but the host's genetic makeup. However, there has been limited success in determining the germline genetic variants that influence metastasis development, mainly because of the limitations of traditional genome-wide association studies to detect the relevant genetic polymorphisms underlying complex phenotypes. In this work, we leveraged the extreme discordant phenotypes approach and the epistasis networks to analyse the genotypes of 97 breast cancer patients. We found that the host's genetic makeup facilitates metastases by the dysregulation of gene expression that can promote the dispersion of metastatic seeds and help establish the metastatic niche-providing a congenial soil for the metastatic seeds.

Identification of New Toxicity Mechanisms in Drug-Induced Liver Injury through Systems Pharmacology.

Among adverse drug reactions, drug-induced liver injury presents particular challenges because of its complexity, and the underlying mechanisms are still not completely characterized. Our knowledge of the topic is limited and based on the assumption that a drug acts on one molecular target. We have leveraged drug polypharmacology, i.e., the ability of a drug to bind multiple targets and thus perturb several biological processes, to develop a systems pharmacology platform that integrates all drug-target interactions. Our analysis sheds light on the molecular mechanisms of drugs involved in drug-induced liver injury and provides new hypotheses to study this phenomenon.

Exploiting protein family and protein network data to identify novel drug targets for bladder cancer.

Bladder cancer remains one of the most common forms of cancer and yet there are limited small molecule targeted therapies. Here, we present a computational platform to identify new potential targets for bladder cancer therapy. Our method initially exploited a set of known driver genes for bladder cancer combined with predicted bladder cancer genes from mutationally enriched protein domain families. We enriched this initial set of genes using protein network data to identify a comprehensive set of 323 putative bladder cancer targets. Pathway and cancer hallmarks analyses highlighted putative mechanisms in agreement with those previously reported for this cancer and revealed protein network modules highly enriched in potential drivers likely to be good targets for targeted therapies. 21 of our potential drug targets are targeted by FDA approved drugs for other diseases - some of them are known drivers or are already being targeted for bladder cancer (FGFR3, ERBB3, HDAC3, EGFR). A further 4 potential drug targets were identified by inheriting drug mappings across our in-house CATH domain functional families (FunFams). Our FunFam data also allowed us to identify drug targets in families that are less prone to side effects i.e., where structurally similar protein domain relatives are less dispersed across the human protein network. We provide information on our novel potential cancer driver genes, together with information on pathways, network modules and hallmarks associated with the predicted and known bladder cancer drivers and we highlight those drivers we predict to be likely drug targets.

Histamine, Metabolic Remodelling and Angiogenesis: A Systems Level Approach.

Histamine is a highly pleiotropic biogenic amine involved in key physiological processes including neurotransmission, immune response, nutrition, and cell growth and differentiation. Its effects, sometimes contradictory, are mediated by at least four different G-protein coupled receptors, which expression and signalling pathways are tissue-specific. Histamine metabolism conforms a very complex network that connect many metabolic processes important for homeostasis, including nitrogen and energy metabolism. This review brings together and analyses the current information on the relationships of the "histamine system" with other important metabolic modules in human physiology, aiming to bridge current information gaps. In this regard, the molecular characterization of the role of histamine in the modulation of angiogenesis-mediated processes, such as cancer, makes a promising research field for future biomedical advances.

A CATH domain functional family based approach to identify putative cancer driver genes and driver mutations.

Tumour sequencing identifies highly recurrent point mutations in cancer driver genes, but rare functional mutations are hard to distinguish from large numbers of passengers. We developed a novel computational platform applying a multi-modal approach to filter out passengers and more robustly identify putative driver genes. The primary filter identifies enrichment of cancer mutations in CATH functional families (CATH-FunFams) - structurally and functionally coherent sets of evolutionary related domains. Using structural representatives from CATH-FunFams, we subsequently seek enrichment of mutations in 3D and show that these mutation clusters have a very significant tendency to lie close to known functional sites or conserved sites predicted using CATH-FunFams. Our third filter identifies enrichment of putative driver genes in functionally coherent protein network modules confirmed by literature analysis to be cancer associated. Our approach is complementary to other domain enrichment approaches exploiting Pfam families, but benefits from more functionally coherent groupings of domains. Using a set of mutations from 22 cancers we detect 151 putative cancer drivers, of which 79 are not listed in cancer resources and include recently validated cancer associated genes EPHA7, DCC netrin-1 receptor and zinc-finger protein ZNF479.

Unique signalling connectivity of FGFR3-TACC3 oncoprotein revealed by quantitative phosphoproteomics and differential network analysis.

The FGFR3-TACC3 fusion is an oncogenic driver in diverse malignancies, including bladder cancer, characterized by upregulated tyrosine kinase activity. To gain insights into distinct properties of FGFR3-TACC3 down-stream signalling, we utilised telomerase-immortalised normal human urothelial cell lines expressing either the fusion or wild-type FGFR3 (isoform IIIb) for subsequent quantitative proteomics and network analysis. Cellular lysates were chemically labelled with isobaric tandem mass tag reagents and, after phosphopeptide enrichment, liquid chromatography-high mass accuracy tandem mass spectrometry (LC-MS/MS) was used for peptide identification and quantification. Comparison of data from the two cell lines under non-stimulated and FGF1 stimulated conditions and of data representing physiological stimulation of FGFR3 identified about 200 regulated phosphosites. The identified phosphoproteins and quantified phosphosites were further analysed in the context of functional biological networks by inferring kinase-substrate interactions, mapping these to a comprehensive human signalling interaction network, filtering based on tissue-expression profiles and applying disease module detection and pathway enrichment methods. Analysis of our phosphoproteomics data using these bioinformatics methods combined into a new protocol-Disease Relevant Analysis of Genes On Networks (DRAGON)-allowed us to tease apart pathways differentially involved in FGFR3-TACC3 signalling in comparison to wild-type FGFR3 and to investigate their local phospho-signalling context. We highlight 9 pathways significantly regulated only in the cell line expressing FGFR3-TACC3 fusion and 5 pathways regulated only by stimulation of the wild-type FGFR3. Pathways differentially linked to FGFR3-TACC3 fusion include those related to chaperone activation and stress response and to regulation of TP53 expression and degradation that could contribute to development and maintenance of the cancer phenotype.

Exploring the interactions of the RAS family in the human protein network and their potential implications in RAS-directed therapies.

RAS proteins are the founding members of the RAS superfamily of GTPases. They are involved in key signaling pathways regulating essential cellular functions such as cell growth and differentiation. As a result, their deregulation by inactivating mutations often results in aberrant cell proliferation and cancer. With the exception of the relatively well-known KRAS, HRAS and NRAS proteins, little is known about how the interactions of the other RAS human paralogs affect cancer evolution and response to treatment. In this study we performed a comprehensive analysis of the relationship between the phylogeny of RAS proteins and their location in the protein interaction network. This analysis was integrated with the structural analysis of conserved positions in available 3D structures of RAS complexes. Our results show that many RAS proteins with divergent sequences are found close together in the human interactome. We found specific conserved amino acid positions in this group that map to the binding sites of RAS with many of their signaling effectors, suggesting that these pairs could share interacting partners. These results underscore the potential relevance of cross-talking in the RAS signaling network, which should be taken into account when considering the inhibitory activity of drugs targeting specific RAS oncoproteins. This study broadens our understanding of the human RAS signaling network and stresses the importance of considering its potential cross-talk in future therapies.

Biocomputational resources useful for drug discovery against compartmentalized targets.

It has been estimated that the cost of bringing a new drug onto the market is 10 years and 0.5-2 billions of dollars, making it a non-profitable project, particularly in the case of low prevalence diseases. The advances in Systems Biology have been absolutely decisive for drug discovery, as iterative rounds of predictions made from in silico models followed by selected experimental validations have resulted in a substantial saving of time and investments. Many diseases have their origins in proteins that are not located in the cytosol but in intracellular compartments (i.e. mitochondria, lysosome, peroxisome and others) or cell membranes. In these cases, biocomputational approaches present limitations to their study. In the present work, we review them and propose new initiatives to advance towards a safer, more efficient and personalized pharmacology. This focus could be especially useful for drug discovery and the reposition of known drugs in rare and emergent diseases associated with compartmentalized proteins.

Insights into polypharmacology from drug-domain associations.

MOTIVATION: Polypharmacology (the ability of a single drug to affect multiple targets) is a key feature that may explain part of the decreasing success of conventional drug discovery strategies driven by the quest for drugs to act selectively on a single target. Most drug targets are proteins that are composed of domains (their structural and functional building blocks). RESULTS: In this work, we model drug-domain networks to explore the role of protein domains as drug targets and to explain drug polypharmacology in terms of the interactions between drugs and protein domains. We find that drugs are organized around a privileged set of druggable domains. CONCLUSIONS: Protein domains are a good proxy for drug targets, and drug polypharmacology emerges as a consequence of the multi-domain composition of proteins. CONTACT: amoyag@uma.es SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Bicyclic derivatives of L-idonojirimycin as pharmacological chaperones for neuronopathic forms of Gaucher disease.

New human ß-glucocerebrosidase (GCase) ligands with rigid 1,6-anhydro-ß-L-idonojirimycin cores have been designed with the aid of molecular modeling. Efficient pharmacological chaperones for the L444P (trafficking-incompetent) mutant GCase enzyme associated with type 2 and 3 Gaucher disease (GD) were identified.

Structural perspective on the direct inhibition mechanism of EGCG on mammalian histidine decarboxylase and DOPA decarboxylase.

Histidine decarboxylase (HDC) and l-aromatic amino acid decarboxylase (DDC) are homologous enzymes that are responsible for the synthesis of important neuroactive amines related to inflammatory, neurodegenerative, and neoplastic diseases. Epigallocatechin-3-gallate (EGCG), the most abundant catechin in green tea, has been shown to target histamine-producing cells and to promote anti-inflammatory, antitumor, and antiangiogenic effects. Previous experimental work has demonstrated that EGCG has a direct inhibitory effect on both HDC and DDC. In this study, we investigated the binding modes of EGCG to HDC and DDC as a first step for designing new polyphenol-based HDC/DDC-specific inhibitors.

The function of histamine receptor H4R in the brain revealed by interaction partners.

The histamine H4 receptor is mainly expressed in haematopoietic cells, hence is linked to inflammatory and immune system conditions. It has been recently discovered that the receptor is expressed also in the mammalian central nervous system (CNS), but its role in the brain remains unclear. We address the potential functions of the histamine H4 receptor in the human brain using a 'guilty by association' logic, by close examination of protein-protein functional associations networks in the human proteome.

Analysis of Mammalian Histidine Decarboxylase Dimerization Interface Reveals an Electrostatic Hotspot Important for Catalytic Site Topology and Function.

Selective intervention of mammalian histidine decarboxylase (EC 4.1.1.22) could provide a useful antihistaminic strategy against many different pathologies. It is known that global conformational changes must occur during reaction that involves the monomer-monomer interface of the enzyme. Thus, the dimerization surface is a promising target for histidine decarboxylase inhibition. In this work, a rat apoenzyme structural model is used to analyze the interface of the dimeric active HDC. The dimerization surface mainly involves the fragments 1-213 and 308-371 from both subunits. Part of the overlapping surfaces conforms each catalytic site entrance and the substrate-binding sites. In addition, a cluster of charged residues is located in each overlapping surface, so that both electrostatic hotspots mediate in the interaction between the catalytic sites of the dimeric enzyme. It is experimentally demonstrated that the carboxyl group of aspartate 315 is critical for the proper conformation of the holoenzyme and the progression of the reaction. Comparison to the available information on other evolutionary related enzymes also provides new insights for characterization and intervention of homologous l-amino acid decarboxylases.

Molecular modeling and site-directed mutagenesis reveal essential residues for catalysis in a prokaryote-type aspartate aminotransferase.

We recently reported that aspartate (Asp) biosynthesis in plant chloroplasts is catalyzed by two different Asp aminotransferases (AAT): a previously characterized eukaryote type and a prokaryote type (PT-AAT) similar to bacterial and archaebacterial enzymes. The available molecular and kinetic data suggest that the eukaryote-type AAT is involved in the shuttling of reducing equivalents through the plastidic membrane, whereas the PT-AAT could be involved in the biosynthesis of the Asp-derived amino acids inside the organelle. In this work, a comparative modeling of the PT-AAT enzyme from Pinus pinaster (PpAAT) was performed using x-ray structures of a bacterial AAT (Thermus thermophilus; Protein Data Bank accession nos. 1BJW and 1BKG) as templates. We computed a three-dimensional folding model of this plant homodimeric enzyme that has been used to investigate the functional importance of key amino acid residues in its active center. The overall structure of the model is similar to the one described for other AAT enzymes, from eukaryotic and prokaryotic sources, with two equivalent active sites each formed by residues of both subunits of the homodimer. Moreover, PpAAT monomers folded into one large and one small domain. However, PpAAT enzyme showed unique structural and functional characteristics that have been specifically described in the AATs from the prokaryotes Phormidium lapideum and T. thermophilus, such as those involved in the recognition of the substrate side chain or the "open-to-closed" transition following substrate binding. These predicted characteristics have been substantiated by site-direct mutagenesis analyses, and several critical residues (valine-206, serine-207, glutamine-346, glutamate-210, and phenylalanine-450) were identified and functionally characterized. The reported data represent a valuable resource to understand the function of this enzyme in plant amino acid metabolism.

AMMO-Prot: amine system project 3D-model finder.

BACKGROUND: Amines are biogenic amino acid derivatives, which play pleiotropic and very important yet complex roles in animal physiology. For many other relevant biomolecules, biochemical and molecular data are being accumulated, which need to be integrated in order to be effective in the advance of biological knowledge in the field. For this purpose, a multidisciplinary group has started an ontology-based system named the Amine System Project (ASP) for which amine-related information is the validation bench. RESULTS: In this paper, we describe the Ontology-Based Mediator developed in the Amine System Project (http://asp.uma.es) using the infrastructure of Semantic Directories, and how this system has been used to solve a case related to amine metabolism-related protein structures. CONCLUSIONS: This infrastructure is used to publish and manage not only ontologies and their relationships, but also metadata relating to the resources committed with the ontologies. The system developed is available at http://asp.uma.es/WebMediator.

Analysis of the decarboxylation step in mammalian histidine decarboxylase. A computational study.

We report a hybrid quantum mechanics/molecular mechanics theoretical study on the reaction mechanism of mammalian histidine decarboxylase that allows us to obtain valuable insights on the structure of the cofactor-substrate adduct (external aldimine) in the active site of rat histidine decarboxylase. By means of molecular dynamics simulations, we traced the potential of mean force corresponding to the decarboxylation reaction of the adduct both in the active site of the enzyme and in aqueous solution. By comparing this process in both media, we have identified the key electrostatic interactions that explain the lowering of the free energy barrier in the enzyme. Our analysis also offers a validation of Dunathan's hypothesis (Dunathan, H. (1966) Proc. Natl. Acad. Sci. U. S. A. 55, 712-716) regarding the role of stereoelectronic effects in the enzymatic decarboxylation process.

Mammalian histidine decarboxylase: from structure to function.

Histamine is a multifunctional biogenic amine with relevant roles in intercellular communication, inflammatory processes and highly prevalent pathologies. Histamine biosynthesis depends on a single decarboxylation step, carried out by a PLP-dependent histidine decarboxylase activity (EC 4.1.1.22), an enzyme that still remains to be fully characterized. Nevertheless, during the last few years, important advances have been made in this field, including the generation and validation of the first three-dimensional model of the enzyme, which allows us to revisit previous results and conclusions. This essay provides a comprehensive review of the current knowledge of the structural and functional characteristics of mammalian histidine decarboxylase.

Mapping of catalytically important residues in the rat L-histidine decarboxylase enzyme using bioinformatic and site-directed mutagenesis approaches.

HDC (L-histidine decarboxylase), the enzyme responsible for the catalytic production of histamine from L-histidine, belongs to an evolutionarily conserved family of vitamin B6-dependent enzymes known as the group II decarboxylases. Yet despite the obvious importance of histamine, mammalian HDC enzymes remain poorly characterized at both the biochemical and structural levels. By comparison with the recently described crystal structure of the homologous enzyme L-DOPA decarboxylase, we have been able to identify a number of conserved domains and motifs that are important also for HDC catalysis. This includes residues that were proposed to mediate events within the active site, and HDC proteins carrying mutations in these residues were inactive when expressed in reticulocyte cell lysates reactions. Our studies also suggest that a significant change in quartenary structure occurs during catalysis. This involves a protease sensitive loop, and incubating recombinant HDC with an L-histidine substrate analogue altered enzyme structure so that the loop was no longer exposed for tryptic proteolysis. In total, 27 mutant proteins were used to test the proposed importance of 34 different amino acid residues. This is the most extensive mutagenesis study yet to identify catalytically important residues in a mammalian HDC protein sequence and it provides a number of novel insights into the mechanism of histamine biosynthesis.

Local changes in the catalytic site of mammalian histidine decarboxylase can affect its global conformation and stability.

Mature, active mammalian histidine decarboxylase is a dimeric enzyme of carboxy-truncated monomers (approximately 53 kDa). By using a biocomputational approach, we have generated a three-dimensional model of a recombinant 1/512 fragment of the rat enzyme, which shows kinetic constants similar to those of the mature enzyme purified from rodent tissues. This model, together with previous spectroscopic data, allowed us to postulate that the occupation of the catalytic center by the natural substrate, or by substrate-analogs, would induce remarkable changes in the conformation of the intact holoenzyme. To investigate the proposed conformational changes during catalysis, we have carried out electrophoretic, chromatographic and spectroscopic analyses of purified recombinant rat 1/512 histidine decarboxylase in the presence of the natural substrate or substrate-analogs. Our results suggest that local changes in the catalytic site indeed affect the global conformation and stability of the dimeric protein. These results provide insights for new alternatives to inhibit histamine production efficiently in vivo.