Distantly related lipocalins share two conserved clusters of hydrophobic residues: use in homology modeling

BACKGROUND: Lipocalins are widely distributed in nature and are found in bacteria, plants, arthropoda and vertebra. In hematophagous arthropods, they are implicated in the successful accomplishment of the blood meal, interfering with platelet aggregation, blood coagulation and inflammation and in the transmission of disease parasites such as Trypanosoma cruzi and Borrelia burgdorferi. The pairwise sequence identity is low among this family, often below 30%, despite a well conserved tertiary structure. Under the 30% identity threshold, alignment methods do not correctly assign and align proteins. The only safe way to assign a sequence to that family is by experimental determination. However, these procedures are long and costly and cannot always be applied. A way to circumvent the experimental approach is sequence and structure analyze. To further help in that task, the residues implicated in the stabilisation of the lipocalin fold were determined. This was done by analyzing the conserved interactions for ten lipocalins having a maximum pairwise identity of 28% and various functions. RESULTS: It was determined that two hydrophobic clusters of residues are conserved by analysing the ten lipocalin structures and sequences. One cluster is internal to the barrel, involving all strands and the 310 helix. The other is external, involving four strands and the helix lying parallel to the barrel surface. These clusters are also present in RaHBP2, a unusual "outlier" lipocalin from tick Rhipicephalus appendiculatus. This information was used to assess assignment of LIR2 a protein from Ixodes ricinus and to build a 3D model that helps to predict function. FTIR data support the lipocalin fold for this protein. CONCLUSION: By sequence and structural analyzes, two conserved clusters of hydrophobic residues in interactions have been identified in lipocalins. Since the residues implicated are not conserved for function, they should provide the minimal subset necessary to confer the lipocalin fold. This information has been used to assign LIR2 to lipocalins and to investigate its structure/function relationship. This study could be applied to other protein families with low pairwise similarity, such as the structurally related fatty acid binding proteins or avidins.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Edgetic perturbation models of human inherited disorders

Cellular functions are mediated through complex systems of macromolecules and metabolites linked through biochemical and physical interactions, represented in interactome models as ‘nodes' and ‘edges', respectively. Better understanding of genotype-to-phenotype relationships in human disease will require modeling of how disease-causing mutations affect systems or interactome properties. Here we investigate how perturbations of interactome networks may differ between complete loss of gene products (‘node removal') and interaction-specific or edge-specific (‘edgetic') alterations. Global computational analyses of ∼50 000 known causative mutations in human Mendelian disorders revealed clear separations of mutations probably corresponding to those of node removal versus edgetic perturbations. Experimental characterization of mutant alleles in various disorders identified diverse edgetic interaction profiles of mutant proteins, which correlated with distinct structural properties of disease proteins and disease mechanisms. Edgetic perturbations seem to confer distinct functional consequences from node removal because a large fraction of cases in which a single gene is linked to multiple disorders can be modeled by distinguishing edgetic network perturbations. Edgetic network perturbation models might improve both the understanding of dissemination of disease alleles in human populations and the development of molecular therapeutic strategies

A reference map of the human binary protein interactome.

Global insights into cellular organization and genome function require comprehensive understanding of the interactome networks that mediate genotype-phenotype relationships(1,2). Here we present a human 'all-by-all' reference interactome map of human binary protein interactions, or 'HuRI'. With approximately 53,000 protein-protein interactions, HuRI has approximately four times as many such interactions as there are high-quality curated interactions from small-scale studies. The integration of HuRI with genome(3), transcriptome(4) and proteome(5) data enables cellular function to be studied within most physiological or pathological cellular contexts. We demonstrate the utility of HuRI in identifying the specific subcellular roles of protein-protein interactions. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms that might underlie tissue-specific phenotypes of Mendelian diseases. HuRI is a systematic proteome-wide reference that links genomic variation to phenotypic outcomes

Implication of a structural motif in the instability of a toxic protein: the prion

peer reviewe

Analysis of the C-Terminal Membrane Anchor Domains of Hepatitis C Virus Glycoproteins E1 and E2: toward a Topological Model

The hepatitis C virus (HCV) glycoproteins E1 and E2 should be anchored in the viral membrane by their C-terminal domains. During synthesis, they are translocated to the endoplasmic reticulum (ER) lumen where they remain. The 31 C-terminal residues of the E1 protein and the 29 C-terminal residues of the E2 protein are implicated in the ER retention. Moreover, the E1 and E2 C termini are implicated in E1-E2 heterodimerization. We studied the E1 and E2 C-terminal sequences of 25 HCV strains in silico using molecular modeling techniques. We conclude that both C-terminal domains should adopt a similar and peculiar configuration: one amphipathic α-helix followed by a pair of transmembrane β-strands. Several three-dimensional (3-D) models were generated. After energy minimization, their ability to interact with membranes was studied using the molecular hydrophobicity potentials calculation and the IMPALA procedure. The latter simulates interactions with a membrane by a Monte Carlo minimization of energy. These methods suggest that the β-hairpins could anchor the glycoproteins in the ER membrane at least transiently. Anchoring could be stabilized by the adsorption of the nearby amphipathic α-helices at the membrane surface. The 3-D models correlate with experimental results which indicate that the E1-E2 transmembrane domains are involved in the heterodimerization and have ER retention properties

DIAGNOSIS OF HEMATOPOIETIC MALIGNANCIES WITH GERMLINE PREDISPOSITION : A FILTERED WHOLE EXOME SEQUENCING APPROACH

Background: In the frame of the development of new analytical methods for the diagnostic of patients suffering of suspected inherited malignancies with poor access to rapid diagnosis due to a restricted number of analytical sites or the current use of non-comprehensive targeted panels, we aimed at developing a new NGS panel for the analysis of several haematological diseases based on a filtered whole exome sequencing approach. Aims: Haematological diseases incorporated in the panel include myelodysplasic syndromes, acute myeloid leukemia, inherited bone marrow failure syndromes, Fanconi anemia, Diamond Blackfan anemia, dyskeratosis congenita, Telomeropathies and severe congenital neutropenia. Methods: Clinical exome is sequenced with the SureSelect Hulan Exome V6, recovering information for about 20k genes, representing ca. 180k exons. A newly designed variant filter based on an in-house pipeline tree (Alissa Interpret) for the flagging of retained variants, allows for the selection and labeling of ca. 100 variants per patient. Variants are categorized following the ACMG guidelines for the interpretation of inherited variants and reported in the context of the patient malignancies. The set of genes has been selected from a deep insight to the most recent literature and guidelines and covers 85 full (exonic) genes. The ncRNA gene TERC and intronic regions from RETL1 are also included in the set. The analytical pipeline considers the analysis of single patients, corroboration of relevant inherited germline mutations from fibroblasts and/or trio analysis including first or second relatives. Applications cover from simple diagnostic from single patients to familiar studies or donor compatibility analysis. Results: Inherited Haematological Malignancies are a heterogeneous group of malignancies not yet fully understood and in constant evolution. In this context, flexibility is a key concept to avoid analytical obsolescence. Due to the wide range of genes covered by the WES approach, significant genes could be further updated, with a minimal effort, to the panel. Summary/Conclusion: We therefore aim to provide physicians with a comprehensive diagnostic panel for the diagnostic work-up of patients affected (or suspected to be affected) by inherited haematological malignancies, and related diseases enhancing the patient’s chances to benefit from a personalized medicine approach; together with a view to increase the knowledge about those (rare) condition

Hematopoietic Malignancies with Germline Predisposition Diagnosis with Filtered Whole Exome Sequencing and in silico Panel Analysis.

In the frame of the development of new analytical methods for the diagnostic of patients suffering of suspected inherited malignancies with poor access to rapid diagnosis due to a restricted number of analytical sites or the current use of non-comprehensive targeted panels, we developed a new NGS panel for the analysis of several haematological diseases based on a filtered whole exome sequencing approach. We aim to provide physicians with a comprehensive diagnostic panel for the diagnostic work-up of patients affected (or suspected to be affected) by inherited haematological malignancies and related diseases enhancing the patient’s chances to benefit from a personalized medicine approach; together with a view to increase the knowledge about those (rare) conditions

Detection and identification of transgenic events by next generation sequencing combined with enrichment technologies.

Next generation sequencing (NGS) is a promising tool for analysing the quality and safety of food and feed products. The detection and identification of genetically modified organisms (GMOs) is complex, as the diversity of transgenic events and types of structural elements introduced in plants continue to increase. In this paper, we show how a strategy that combines enrichment technologies with NGS can be used to detect a large panel of structural elements and partially or completely reconstruct the new sequence inserted into the plant genome in a single analysis, even at low GMO percentages. The strategy of enriching sequences of interest makes the approach applicable even to mixed products, which was not possible before due to insufficient coverage of the different genomes present. This approach is also the first step towards a more complete characterisation of agrifood products in a single analysis