Joost Van Durme

Vrije Universiteit Brussel, Bruxelles, Brussels Capital, Belgium

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Publications (26)124.51 Total impact

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    ABSTRACT: Motivation: Protein aggregation is associated with a number of protein misfolding diseases and is a major concern for therapeutic proteins. Aggregation is caused by the presence of aggregation-prone regions (APRs) in the amino acid sequence of the protein. The lower the aggregation propensity of APRs and the better they are protected by native interactions within the folded structure of the protein, the more aggregation is prevented. Therefore both the local thermodynamic stability of APRs in the native structure as well as their intrinsic aggregation propensity are a key parameter that needs to be optimised in order to prevent protein aggregation. The Solubis method presented here automates the process of carefully selecting point mutations that minimize the intrinsic aggregation propensity whilst improving local protein stability. All information about the Solubis plugin is available at http://solubisyasara.switchlab.org/. Contact: joost.schymkowitz{at}switch.vib-kuleuven.be or Frederic.Rousseau{at}switch.vib-kuleuven.be
    No preview · Article · Mar 2015 · Bioinformatics
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    ABSTRACT: Accurate prediction of amyloid-forming amino acid sequences remains an important challenge. We here present an online database that provides open access to the largest set of experimentally characterised amyloid forming hexapeptides. To this end, we expanded our previous set of 280 hexapeptides used to develop the Waltz algorithm with 89 peptides from literature review and by systematic experimental characterisation of the aggregation of 720 hexapeptides by transmission electron microscopy (TEM), dye binding and Fourier transform infrared spectroscopy (FTIR). This brings the total number of experimentally characterized hexapeptides in the WALTZ-DB database to 1089, of which 244 are annotated as positive for amyloid formation. Availability and implementation: The WALTZ-DB database is freely available without any registration requirement at http://waltzdb.switchlab.org. frederic.rousseau@switch.vib-kuleuven.be; joost.schymkowitz@switch.vib-kuleuven.be; © The Author (2015). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
    No preview · Article · Jan 2015 · Bioinformatics
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    ABSTRACT: Protein aggregation is sequence specific, favoring self-assembly over cross-seeding with non-homologous sequences. Still, as the majority of proteins in a proteome are aggregation prone, the high level of homogeneity of protein inclusions in vivo both during recombinant overexpression and in disease remains surprising. To investigate the selectivity of protein aggregation in a proteomic context, we here compared the selectivity of aggregation-determined interactions with antibody binding. To that purpose, we synthesized biotin-labeled peptides, corresponding to aggregation-determining sequences of the bacterial protein β-galactosidase and two human disease biomarkers: C-reactive protein and prostate-specific antigen. We analyzed the selectivity of their interactions in Escherichia coli lysate, human serum and human seminal plasma, respectively, using a Western blot-like approach in which the aggregating peptides replace the conventional antibody. We observed specific peptide accumulation in the same bands detected by antibody staining. Combined spectroscopic and mutagenic studies confirmed accumulation resulted from binding of the peptide on the identical sequence of the immobilized target protein. Further, we analyzed the sequence redundancy of aggregating sequences and found that about 90% of them are unique within their proteome. As a result, the combined specificity and low sequence redundancy of aggregating sequences therefore contribute to the observed homogeneity of protein aggregation in vivo. This suggests that these intrinsic proteomic properties naturally compartmentalize aggregation events in sequence space. In the event of physiological stress, this might benefit the ability of cells to respond to proteostatic stress by allowing chaperones to focus on specific aggregation events rather than having to face systemic proteostatic failure.
    Full-text · Article · Nov 2014 · Journal of Molecular Biology
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    ABSTRACT: Protein aggregation is geared by aggregation prone sequences (APRs) that self-associate by β-strand interactions. Charged residues and prolines are enriched at the flanks of APRs resulting in decreased aggregation. It is still unclear what drives the overrepresentation of these 'aggregation gatekeepers', i.e. whether their presence results from structural constraints determining protein stability or whether they constitute a bona fide functional class selectively maintained to control protein aggregation. As functional residues are typically conserved regardless of their cost to protein stability we compared sequence conservation and thermodynamic cost of these residues in 2659 protein families in E. coli. Across protein families we find gatekeepers to be under strong selective conservation while at the same time representing a significant thermodynamic cost to protein structure. This finding supports the notion that aggregation gatekeepers are not structurally determined but evolutionary selected to control protein aggregation.
    No preview · Article · Apr 2014 · Journal of Molecular Biology
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    ABSTRACT: Fabry disease is a lysosomal storage disorder caused by loss of α-galactosidase function. More than 500 Fabry disease mutants have been identified, the majority of which are structurally destabilized. A therapeutic strategy under development for lysosomal storage diseases consists of using pharmacological chaperones to stabilize the structure of the mutant protein, thereby promoting lysosomal delivery over retrograde degradation. The substrate analog 1-deoxygalactonojirimycin (DGJ) has been shown to restore activity of mutant α-galactosidase and is currently in clinical trial for treatment of Fabry disease. However, only ∼65% of tested mutants respond to treatment in cultured patient fibroblasts, and the structural underpinnings of DGJ response remain poorly explained. Using computational modeling and cell culture experiments, we show that the DGJ response is negatively affected by protein aggregation of α-galactosidase mutants, revealing a qualitative difference between misfolding-associated and aggregation-associated loss of function. A scoring function combining predicted thermodynamic stability and intrinsic aggregation propensity of mutants captures well their aggregation behavior under overexpression in HeLa cells. Interestingly, the same classifier performs well on DGJ response data of patient-derived cultured lymphoblasts, showing that protein aggregation is an important determinant of chemical chaperone efficiency under endogenous expression levels as well. Our observations reinforce the idea that treatment of aggregation-associated loss of function observed for the more severe α-galactosidase mutants could be enhanced by combining pharmacological chaperone treatment with the suppression of mutant aggregation, e.g. via proteostatic regulator compounds that increase cellular chaperone expression.
    Full-text · Article · Jul 2012 · Journal of Biological Chemistry
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    ABSTRACT: Single nucleotide variants (SNVs) are, together with copy number variation, the primary source of variation in the human genome and are associated with phenotypic variation such as altered response to drug treatment and susceptibility to disease. Linking structural effects of non-synonymous SNVs to functional outcomes is a major issue in structural bioinformatics. The SNPeffect database (http://snpeffect.switchlab.org) uses sequence- and structure-based bioinformatics tools to predict the effect of protein-coding SNVs on the structural phenotype of proteins. It integrates aggregation prediction (TANGO), amyloid prediction (WALTZ), chaperone-binding prediction (LIMBO) and protein stability analysis (FoldX) for structural phenotyping. Additionally, SNPeffect holds information on affected catalytic sites and a number of post-translational modifications. The database contains all known human protein variants from UniProt, but users can now also submit custom protein variants for a SNPeffect analysis, including automated structure modeling. The new meta-analysis application allows plotting correlations between phenotypic features for a user-selected set of variants.
    Full-text · Article · Nov 2011 · Nucleic Acids Research
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    ABSTRACT: A graphical user interface for the FoldX protein design program has been developed as a plugin for the YASARA molecular graphics suite. The most prominent FoldX commands such as free energy difference upon mutagenesis and interaction energy calculations can now be run entirely via a windowed menu system and the results are immediately shown on screen. AVAILABILITY AND IMPLEMENTATION: The plugin is written in Python and is freely available for download at http://foldxyasara.switchlab.org/ and supported on Linux, MacOSX and MS Windows.
    Full-text · Article · Jun 2011 · Bioinformatics
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    ABSTRACT: We here present a new method to measure the degree of protein-bound methionine sulfoxide formation at a proteome-wide scale. In human Jurkat cells that were stressed with hydrogen peroxide, over 2000 oxidation-sensitive methionines in more than 1600 different proteins were mapped and their extent of oxidation was quantified. Meta-analysis of the sequences surrounding the oxidized methionine residues revealed a high preference for neighboring polar residues. Using synthetic methionine sulfoxide containing peptides designed according to the observed sequence preferences in the oxidized Jurkat proteome, we discovered that the substrate specificity of the cellular methionine sulfoxide reductases is a major determinant for the steady-state of methionine oxidation. This was supported by a structural modeling of the MsrA catalytic center. Finally, we applied our method onto a serum proteome from a mouse sepsis model and identified 35 in vivo methionine oxidation events in 27 different proteins.
    No preview · Article · Mar 2011 · Molecular & Cellular Proteomics
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    ABSTRACT: Small heat shock proteins are molecular chaperones capable of maintaining denatured proteins in a folding-competent state. We have previously shown that missense mutations in the small heat shock protein HSPB1 (HSP27) cause distal hereditary motor neuropathy and axonal Charcot-Marie-Tooth disease. Here we investigated the biochemical consequences of HSPB1 mutations that are known to cause peripheral neuropathy. In contrast to other chaperonopathies, our results revealed that particular HSPB1 mutations presented higher chaperone activity compared with wild type. Hyperactivation of HSPB1 was accompanied by a change from its wild-type dimeric state to a monomer without dissociation of the 24-meric state. Purification of protein complexes from wild-type and HSPB1 mutants showed that the hyperactive isoforms also presented enhanced binding to client proteins. Furthermore, we show that the wild-type HSPB1 protein undergoes monomerization during heat-shock activation, strongly suggesting that the monomer is the active form of the HSPB1 protein.
    Full-text · Article · Feb 2010 · Journal of Biological Chemistry
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    ABSTRACT: Molecular chaperones are essential elements of the protein quality control machinery that governs translocation and folding of nascent polypeptides, refolding and degradation of misfolded proteins, and activation of a wide range of client proteins. The prokaryotic heat-shock protein DnaK is the E. coli representative of the ubiquitous Hsp70 family, which specializes in the binding of exposed hydrophobic regions in unfolded polypeptides. Accurate prediction of DnaK binding sites in E. coli proteins is an essential prerequisite to understand the precise function of this chaperone and the properties of its substrate proteins. In order to map DnaK binding sites in protein sequences, we have developed an algorithm that combines sequence information from peptide binding experiments and structural parameters from homology modelling. We show that this combination significantly outperforms either single approach. The final predictor had a Matthews correlation coefficient (MCC) of 0.819 when assessed over the 144 tested peptide sequences to detect true positives and true negatives. To test the robustness of the learning set, we have conducted a simulated cross-validation, where we omit sequences from the learning sets and calculate the rate of repredicting them. This resulted in a surprisingly good MCC of 0.703. The algorithm was also able to perform equally well on a blind test set of binders and non-binders, of which there was no prior knowledge in the learning sets. The algorithm is freely available at http://limbo.vib.be.
    Full-text · Article · Sep 2009 · PLoS Computational Biology
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    ABSTRACT: Caspase-3 and -7 are considered functionally redundant proteases with similar proteolytic specificities. We performed a proteome-wide screen on a mouse macrophage lysate using the N-terminal combined fractional diagonal chromatography technology and identified 46 shared, three caspase-3-specific, and six caspase-7-specific cleavage sites. Further analysis of these cleavage sites and substitution mutation experiments revealed that for certain cleavage sites a lysine at the P5 position contributes to the discrimination between caspase-7 and -3 specificity. One of the caspase-7-specific substrates, the 40 S ribosomal protein S18, was studied in detail. The RPS18-derived P6-P5' undecapeptide retained complete specificity for caspase-7. The corresponding P6-P1 hexapeptide still displayed caspase-7 preference but lost strict specificity, suggesting that P' residues are additionally required for caspase-7-specific cleavage. Analysis of truncated peptide mutants revealed that in the case of RPS18 the P4-P1 residues constitute the core cleavage site but that P6, P5, P2', and P3' residues critically contribute to caspase-7 specificity. Interestingly, specific cleavage by caspase-7 relies on excluding recognition by caspase-3 and not on increasing binding for caspase-7.
    Full-text · Article · Sep 2009 · Molecular & Cellular Proteomics
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    Full-text · Article · Feb 2009 · Molecular and Biochemical Parasitology
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    Full-text · Article · Jan 2009
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    ABSTRACT: 2,6-Dibromophenol (DBP) was reported as an auxin-like molecule using molecular quantum similarity measures. In this study, the auxin activity of this molecule and its chlorinated homologue is further determined using a bacterial biosensor: the auxin-inducible ipdC promoter of Azospirillum brasilense. We were able to demonstrate that DBP can induce gene expression, but to a lesser extent than the auxin indole-3-acetic acid (IAA) and that DBP is not an antagonist for the IAA signalling pathway. To investigate the role of the bromine groups, the molecule 2,6-dichlorophenol (DCP) was also tested for gene expression induction. However, no induction could be observed. In a second part, DBP and other molecules were modelled in the auxin-binding pocket of the plant auxin receptor TIR1 to evaluate theoretical binding energies. Both DBP and its chlorinated homologue DCP are not strong ligands compared to other known auxins such as IAA. The importance of a carboxylated side chain for optimal binding (and probably auxin activity) was demonstrated.
    No preview · Article · Jan 2009 · European Journal of Soil Biology
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    ABSTRACT: Using a targeted peptide-centric proteomics approach, we performed in vitro protease substrate profiling of the apoptotic serine protease granzyme B resulting in the delineation of more than 800 cleavage sites in 322 human and 282 mouse substrates, encompassing the known substrates Bid, caspase-7, lupus La protein, and fibrillarin. Triple SILAC (stable isotope labeling by amino acids in cell culture) further permitted intra-experimental evaluation of species-specific variations in substrate selection by the mouse or human granzyme B ortholog. For the first time granzyme B substrate specificities were directly mapped on a proteomic scale and revealed unknown cleavage specificities, uncharacterized extended specificity profiles, and macromolecular determinants in substrate selection that were confirmed by molecular modeling. We further tackled a substrate hunt in an in vivo setup of natural killer cell-mediated cell death confirming in vitro characterized granzyme B cleavages next to several other unique and hitherto unreported proteolytic events in target cells.
    Full-text · Article · Nov 2008 · Molecular & Cellular Proteomics
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    ABSTRACT: The transthyretin-like (ttl) gene family is one of the largest conserved nematode-specific gene families, coding for a group of proteins with significant sequence similarity to transthyretins (TTR) and transthyretin-related proteins (TRP). In the present study, we investigated the ttl family in Ostertagia ostertagi (a nematode of the abomasum of cattle). Mining of expressed sequence tag (EST) databases revealed the presence of at least 18 ttl genes in O. ostertagi (Oo-ttl), most of which are constitutively transcribed from the free-living, third larval stage onwards. The full-length cDNA of one of these genes (Oo-ttl-1) was amplified and cloned for recombinant expression. Western blot analysis using a specific antiserum showed that the native protein Oo-TTL-1 was highly present in the excretory-secretory (ES) products of adults of O. ostertagi. The protein was immunolocalized to the pseudocoelomic fluid of adult worms. A phylogenetic-bioinformatic analysis of all amino acid sequence data for TTL proteins from a range of strongylid nematodes showed that they could be divided into at least five different classes. This classification was based on conserved amino acids in the first TTL signature domain and the number and location of cysteine residues. The biological role(s) of the TTLs in nematode biology is still unclear. A theoretical three-dimensional model of Oo-TTL-1 indicated that it had a similar structure to TTRs (i.e., containing β-sheets, arranged in a β-sandwich). In contrast to TTRs, competitive binding studies using recombinant Oo-TTL-1 indicated that the protein was devoid of any hydrophobic ligand- or thyroid hormone-binding properties. Finally, combinatorial analysis by double-stranded RNA interference of five ttl genes in the free-living nematode Caenorhabditis elegans did not reveal any visible phenotypes. More information on the transcription profile and tissue distribution of TTLs in nematodes is needed to provide new insights into the biological role of this gene family.
    No preview · Article · Jun 2008 · International journal for parasitology
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    ABSTRACT: Previous vaccination trials with calves have shown that intramuscular immunization with natively purified activation-associated secreted proteins (ASPs) of Ostertagia ostertagi induces protection against a homologous challenge infection with a 74% reduction in cumulative faecal egg counts and a significant reduction in worm length. Recently, O. ostertagi ASP1 was recombinantly expressed using a baculovirus system and tested in a vaccination trial. However, immunized calves failed to recognize native ASPs and no protection was observed. These results suggest an important structural difference between the baculo r-ASP1 and its native counterpart. Therefore, we investigated whether glycans and/or structural epitopes are key features in the induction of a protective immune response. The results show that ASPs carry two hybrid N-glycosylations with a complex alpha-1,3-arm, an unprocessed alpha-1,6-arm and an alpha-1,6-fucose core. While removal of these glycans had little effect on antibody recognition by vaccinated animals, denaturing and reducing the proteins dramatically reduced recognition, suggesting the importance of conformational protein backbone epitopes.
    No preview · Article · Jun 2008 · Molecular and Biochemical Parasitology
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    ABSTRACT: Single nucleotide polymorphisms (SNPs) are, together with copy number variation, the primary source of variation in the human genome. SNPs are associated with altered response to drug treatment, susceptibility to disease and other phenotypic variation. Furthermore, during genetic screens for disease-associated mutations in groups of patients and control individuals, the distinction between disease causing mutation and polymorphism is often unclear. Annotation of the functional and structural implications of single nucleotide changes thus provides valuable information to interpret and guide experiments. The SNPeffect and PupaSuite databases are now synchronized to deliver annotations for both non-coding and coding SNP, as well as annotations for the SwissProt set of human disease mutations. In addition, SNPeffect now contains predictions of Tango2: an improved aggregation detector, and Waltz: a novel predictor of amyloid-forming sequences, as well as improved predictors for regions that are recognized by the Hsp70 family of chaperones. The new PupaSuite version incorporates predictions for SNPs in silencers and miRNAs including their targets, as well as additional methods for predicting SNPs in TFBSs and splice sites. Also predictions for mouse and rat genomes have been added. In addition, a PupaSuite web service has been developed to enable data access, programmatically. The combined database holds annotations for 4,965,073 regulatory as well as 133,505 coding human SNPs and 14,935 disease mutations, and phenotypic descriptions of 43,797 human proteins and is accessible via http://snpeffect.vib.be and http://pupasuite.bioinfo.cipf.es/.
    Full-text · Article · Feb 2008 · Nucleic Acids Research
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    ABSTRACT: In G protein-coupled receptors (GPCRs), the interaction between the cytosolic ends of transmembrane helix 3 (TM3) and TM6 was shown to play an important role in the transition from inactive to active states. According to the currently prevailing model, constructed for rhodopsin and structurally related receptors, the arginine of the conserved "DRY" motif located at the cytosolic end of TM3 (R3.50) would interact with acidic residues in TM3 (D/E3.49) and TM6 (D/E6.30) at the resting state and shift out of this polar pocket upon agonist stimulation. However, 30% of GPCRs, including all chemokine receptors, contain a positively charged residue at position 6.30 which does not support an interaction with R3.50. We have investigated the role of R6.30 in this receptor family by using CCR5 as a model. R6.30D and R6.30E substitutions, which allow an ionic interaction with R3.50, resulted in an almost silent receptor devoid of constitutive activity and strongly impaired in its ability to bind chemokines but still able to internalize. R6.30A and R6.30Q substitutions, allowing weaker interactions with R3.50, preserved chemokine binding but reduced the constitutive activity and the functional response to chemokines. These results indicate that the constitutive and ligand-promoted activity of CCR5 can be modified by modulating the interaction between the DRY motif in TM3 and residues in TM6 suggesting that the overall structure and activation mechanism are well conserved in GPCRs. However, the molecular interactions locking the inactive state must be different in receptors devoid of D/E6.30.
    Full-text · Article · Aug 2007 · Cellular Signalling
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    ABSTRACT: The glycoprotein-hormone receptor information system (GRIS) presents a comprehensive view on all available molecular data for the lutropin/choriogonadotropin receptor, follitropin receptor, and thyrotropin receptor G protein-coupled receptors. It features a mutation database presently containing 696 point mutations, combined with all sequences and the associated homology models. The mutation information was automatically extracted from the literature and manually augmented with respect to constitutivity, surface expression, sensitivity to hormones, and binding affinity. All information in this integrated system is presented in a G protein-coupled receptor specialist-friendly way. A series of interactive tools such as rotamer analysis, mutation prediction, or cavity visualization aids with the design and interpretation of experiments. A universal residue numbering system has been introduced to ease database searches as well as the use of the information in conjunction with literature data from diverse origins. Users can upload new mutations. GRIS is freely accessible at http://gris.ulb.ac.be/.
    Full-text · Article · Oct 2006 · Molecular Endocrinology

Publication Stats

835 Citations
124.51 Total Impact Points

Institutions

  • 2006-2015
    • Vrije Universiteit Brussel
      • Department of Applied Biological Sciences
      Bruxelles, Brussels Capital, Belgium
  • 2005-2008
    • University Hospital Brussels
      Bruxelles, Brussels Capital Region, Belgium
  • 2004-2006
    • Université Libre de Bruxelles
      • Institute of Interdisciplinary Research in human and molecular Biology (IRIBHM)
      Bruxelles, Brussels Capital, Belgium
  • 2003-2004
    • Radboud University Nijmegen
      Nymegen, Gelderland, Netherlands