Genetic newborn screening and digital technologies: A project protocol based on a dual approach to shorten the rare diseases diagnostic path in Europe.

Since 72% of rare diseases are genetic in origin and mostly paediatrics, genetic newborn screening represents a diagnostic "window of opportunity". Therefore, many gNBS initiatives started in different European countries. Screen4Care is a research project, which resulted of a joint effort between the European Union Commission and the European Federation of Pharmaceutical Industries and Associations. It focuses on genetic newborn screening and artificial intelligence-based tools which will be applied to a large European population of about 25.000 infants. The neonatal screening strategy will be based on targeted sequencing, while whole genome sequencing will be offered to all enrolled infants who may show early symptoms but have resulted negative at the targeted sequencing-based newborn screening. We will leverage artificial intelligence-based algorithms to identify patients using Electronic Health Records (EHR) and to build a repository "symptom checkers" for patients and healthcare providers. S4C will design an equitable, ethical, and sustainable framework for genetic newborn screening and new digital tools, corroborated by a large workout where legal, ethical, and social complexities will be addressed with the intent of making the framework highly and flexibly translatable into the diverse European health systems

Towards a better understanding of xylanase inhibition by taxi-type proteins: a crystallographic study

De afbraak van plantaardig celwandmateriaal door middel van enzymen is b elangrijk in verschillende biologische processen. Ten eerste is het enzy matisch moduleren van de celwand een vereiste tijdens groei- en andere o ntwikkelingsstadia van planten. Daarnaast produceren tal van micro-organ ismen een ganse waaier van afbraakenzymen, die hen in staat moeten stell en een plant binnen te dringen. Tenslotte vormt de enzymatische depolyme risatie een sleutelproces in veel industriële toepassingen, alsook in de natuurlijke recyclage van organisch materiaal. De belangrijkste hemicellulose component in de complexe celwandmatrix va n graangewassen is de heteroxylanfractie die voornamelijk uit arabinoxyl anen bestaat. Endo-ß-1,4-xylanasen, de enzymen die het verbre ken van de suikerverbindingen in de (arabino)xylan-ketenstructuur kataly seren, kunnen daarom beschouwd worden als de xylanolytische enzymen met de grootste impact op zowel biologische als biotechnologische processen. In 1997 werd aan het Laboratorium voor Levensmiddelenchemie van de K.U. Leuven voor het eerst vastgesteld dat in graangewassen eiwitten aanwezig zijn die de normale werking van xylanasen verhinderen. Wat later slaagd e men erin een eerste type eiwitten met xylanase-inhiberende werking op te zuiveren uit graan (Triticum aestivum L.). Deze werden, verwijz end naar hun oorsprong, TAXIs (Triticum aestivum xylanase inhibito r) genoemd. In dit onderzoek werd proteïnekristallografie aangewend:&nbs p;i) om inzicht te verwerven in de driedimensionale structuur van deze recent ontdekte familie TAXI-type inhibitoren; ii) om zi cht te krijgen op de moleculaire interactie tussen xylanasen en TAXIs; e n iii) om de ruimtelijke structuur te bepalen van een gemodif iceerd xylanase dat niet langer door TAXIs wordt geïnhibeerd. Nauwkeurige structuren van TAXI-IA en TAXI-IIB, en structuren van TAXI-I A in complex met A. niger xylanase, TAXI-IA in complex met&nb sp;B. subtilis xylanase en TAXI-IIA geassocieerd met B. su btilis xylanase konden worden bepaald. Daarnaast werd de structuur va n een niet-geïnhibeerde B. subtilis xylanase mutant opgehelde rd, beide in vrije en ligand-gebonden toestand. De bevind ingen dat TAXIs in planten defensief kunnen optreden door het verhindere n van de afbraak van de celwandmatrix door microbiële xylanasen, samen m et het moleculaire inzicht bekomen met de drie modellen van TAXI·xy lanase complexen en resultaten van genetische en biochemische karakteris atie, laten toe om de fysiologische functie van xylanase activiteit en i nhibitie bij planten te evalueren. Binnen een pathogeen-gastheer relatie beïvloeden pathogeniciteit en resistentie elkaar door direkte interacti es tussen eiwitten van beide partijen. Een dergelijke wisselwerking vorm t de ideale voedingsbodem voor een moleculaire co-evolutie van de virule ntie en resistentie genen, die snel gaan diversifiëren met de kans een n ieuwe specificiteit te ontwikkelen. Voor de aminozuren betrokken in de T AXI-xylanase interactie kan inderdaad een voorgeschiedenis van positieve selectie en een aanpassend vermogen aangetoond worden. Een opbiedende w apenwedloop waarbij enerzijds pathogene micro-organismes meerdere xylana sen met een verschillende vatbaarheid tegenover TAXIs produceren en ande rzijds TAXIs zich aanpassen teneinde zoveel mogelijk xylanasen te inhibe ren, is bovendien perfect in overeenstemming te brengen met de criteria voor TAXI-specificiteit die we konden afleiden uit de bekomen structurel e informatie. De grondige kennis van de TAXI-xylanase wisselwerking die met dit onderzoek gegenereerd werd, samen met moleculair genetische tech nieken, laat toe om milieubelastende klassieke chemische processen te ve rvangen door de natuurlijke werking van recombinant aangemaakte gemodifi ceerde enzymen. Daarnaast vormt het inzicht in de TAXI·xylanase int eractie een goede basis om te screenen voor natuurlijk voorkomende xylan asen en/of inhibitoren die een groot potentieel in zich dragen om aan ee n welbepaald vatbaarheids- of specificiteitsprofiel te beantwoorden.status: publishe

Identification of structural determinants for inhibition strength and specificity of wheat xylanase inhibitors TAXI-IA and TAXI-IIA

Triticum aestivum xylanase inhibitor ( TAXI)- type inhibitors are active against microbial xylanases from glycoside hydrolase family 11, but the inhibition strength and the specificity towards different xylanases differ between TAXI isoforms. Mutational and biochemical analyses of TAXI- I, TAXI- IIA and Bacillus subtilis xylanase A showed that inhibition strength and specificity depend on the identity of only a few key residues of inhibitor and xylanase [ Fierens K et al. ( 2005) FEBS J 272, 5872 - 5882; Raedschelders G et al. ( 2005) Biochem Biophys Res Commun 335, 512 - 522; Sorensen JF & Sibbesen O ( 2006) Protein Eng Des Sel 19, 205 - 210; Bourgois TM et al. ( 2007) J Biotechnol 130, 95 - 105]. Crystallographic analysis of the structures of TAXI- IA and TAXI- IIA in complex with glycoside hydrolase family 11 B. subtilis xylanase A now provides a substantial explanation for these observations and a detailed insight into the structural determinants for inhibition strength and specificity. Structures of the xylanase - inhibitor complexes show that inhibition is established by loop interactions with active- site residues and substrate- mimicking contacts in the binding subsites. The interaction of residues Leu292 of TAXI- IA and Pro294 of TAXI- IIA with the -2 glycon subsite of the xylanase is shown to be critical for both inhibition strength and specificity. Also, detailed analysis of the interaction interfaces of the complexes illustrates that the inhibition strength of TAXI is related to the presence of an aspartate or asparagine residue adjacent to the acid / base catalyst of the xylanase, and therefore to the pH optimum of the xylanase. The lower the pH optimum of the xylanase, the stronger will be the interaction between enzyme and inhibitor, and the stronger the resulting inhibition.status: publishe

Crystallization and preliminary X-ray diffraction study of two complexes of a TAXI-type xylanase inhibitor with glycoside hydrolase family 11 xylanases from Aspergillus niger and Bacillus subtilis

Endo-beta-1,4-xylanases hydrolyze arabinoxylan, a major constituent of cereal cell walls, and are nowadays widely used in biotechnological processes. Purified complexes of family 11 xylanases from Aspergillus niger and Bacillus subtilis with TAXI I, a TAXI-type xylanase inhibitor from Triticum aestivum L., were prepared. In both cases the complex was crystallized using the hanging-drop vapour-diffusion method. The needle-like crystals of TAXI I in complex with A. niger xylanase belong to the trigonal space group P3(1) or P3(2), with unit-cell parameters a = b = 88.43, c = 128.99 A, and diffract to 1.8 A resolution. TAXI I in complex with B. subtilis xylanase crystallizes in the monoclinic space group C2, with a = 107.89, b = 95.33, c = 66.31 A, beta = 122.24 degrees. Complete data sets were collected for both crystal types using synchrotron radiation.journal: Acta Crystallographica Section D: Biological Crystallography content_type: crystallization papers peer_reviewed: Yes review_process: Single blind received: 27 October 2003 accepted: 18 December 2003 published_online: 25 February 2004 copyright: © 2004 International Union of Crystallographystatus: publishe

Structural basis for inhibition of Aspergillus niger xylanase by triticum aestivum xylanase inhibitor-I

Plants developed a diverse battery of defense mechanisms in response to continual challenges by a broad spectrum of pathogenic microorganisms. Their defense arsenal includes inhibitors of cell wall-degrading enzymes, which hinder a possible invasion and colonization by antagonists. The structure of Triticum aestivum xylanase inhibitor-I (TAXI-I), a first member of potent TAXI-type inhibitors of fungal and bacterial family 11 xylanases, has been determined to 1.7-A resolution. Surprisingly, TAXI-I displays structural homology with the pepsin-like family of aspartic proteases but is proteolytically nonfunctional, because one or more residues of the essential catalytical triad are absent. The structure of the TAXI-I.Aspergillus niger xylanase I complex, at a resolution of 1.8 A, illustrates the ability of tight binding and inhibition with subnanomolar affinity and indicates the importance of the C-terminal end for the differences in xylanase specificity among different TAXI-type inhibitors.status: publishe

Crystallization and preliminary X-ray diffraction study of a wheat (Triticum aestivum L.) TAXI-type endoxylanase inhibitor

A TAXI-type endoxylanase inhibitor from T. aestivum L. wheat flour has been crystallized using the hanging-drop vapour-diffusion method. The needle-like crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 49.92, b = 66.45, c = 106.42 A. From these crystals, a native data set and a gold-derivative data set were collected to 2.25 and 1.75 A resolution, respectively. The heavy-atom derivative of this crystal form was obtained by the soaking method and allowed determination of the initial phases.journal: Acta Crystallographica Section D: Biological Crystallography content_type: crystallization papers peer_reviewed: Yes review_process: Single blind received: 18 October 2002 accepted: 4 February 2003 published_online: 25 March 2003 copyright: © 2003 International Union of Crystallographystatus: publishe

Molecular identification of wheat endoxylanase inhibitor TAXI-II and the determinants of its inhibition specificity

Wheat grains contain Triticum aestivum xylanase inhibitor (TAXI) proteins which inhibit microbial xylanases, some of which are used in cereal based food industries. These inhibitors may play a role in plant defence. Among the TAXI isoforms described so far, TAXI-II displays a deviating inhibition specificity pattern. Here, we report on the molecular identity of TAXI-II and the basis of its inhibition specificity. Three candidate TAXI-II encoding sequences were isolated and recombinantly expressed in Pichia pastoris. To identify TAXI-II, the resulting proteins were tested against glycoside hydrolase family (GHF) 11 xylanases of Aspergillus niger (ANX) and Bacillus subtilis (BSX). One of these proteins (rTAXI-IB) inhibited both enzymes, like natural TAXI-I. The other candidates (rTAXI-IIA and rTAXI-IIB) showed an inhibition pattern typical for natural TAXI-II, only clearly inhibiting BSX. Comparative analysis of these highly similar sequences with distinct inhibition activity patterns, combined with information on the structural basis for ANX inhibition by TAXI-I [S. Sansen, C.J. De Ranter, K. Gebruers, K. Brijs, C.M. Courtin, J.A. Delcour, A. Rabijns, Structural basis for inhibition of Aspergillus niger xylanase by Triticum aestivum xylanase inhibitor-I, J. Biol. Chem. 279 (2004) 36022-36028], indicated a crucial role for Pro294 of TAXI-IIA and Gln376 of TAXI-IIB in determining the reduced inhibition activity towards ANX. Consequently, single point mutants rTAXI-IIA[P294L] and rTAXI-IIB[Q376H], both displaying the Leu/His combination corresponding to TAXI-I, were able to inhibit ANX. These results show that TAXI-II inhibition specificity bears on the identity of two key residues at positions 294 and 376, which are involved in the interaction at the -2 glycon subsite and the active site of GHF 11, respectively.status: publishe

Properties of TAXI-type endoxylanase inhibitors

Two types of proteinaceous endoxylanase inhibitors occur in different cereals, i.e. the TAXI [Triticum aestivum endoxylanase inhibitor]type and XIP [endoxylanase inhibiting protein]-type inhibitors. The present paper focuses on the TAXI-type proteins and deals with their structural characteristics and the identification, characterisation and heterologous expression of a TAXI gene from wheat. In addition, to shed light on the mechanism by which TAXI-type endoxylanase inhibitors work, the enzyme specificity, the optimal conditions for maximal inhibition activity, the molar complexation ratio and the inhibition kinetics of the inhibitors are explained and the effect of mutations of an endoxylanase on the inhibition by TAXIs is discussed. (C) 2003 Elsevier B.V. All rights reserved.status: publishe

Targeted molecular engineering of a family 11 endoxylanase to decrease its sensitivity towards Triticum aestivum endoxylanase inhibitor types

The Bacillus subtilis endoxylanase XynA (BSXY) is frequently used to improve the functionality of arabinoxylan-containing material in cereal based industries. The presence of endogenous Triticum aestivum xylanase inhibitors (TAXI-I and TAXI-II) in wheat is a real concern as they have a direct negative impact on the efficiency of this enzyme. Here, we used the recently determined structure of the complex between TAXI-I and an endoxylanase of Aspergillus niger to develop inhibitor-insensitive BSXY variants by site-directed mutagenesis of strategically chosen amino acids. We either induced steric hindrance to reject the inhibitors or interrupted key interactions with the inhibitors in the endoxylanase substrate-binding groove. The first strategy was successfully applied to position G12 where G12W combined inhibition insensitivity with unharmed catalytic performance. Variants from the second strategy showed altered inhibitor sensitivities concomitant with changes in enzyme activities and allowed to gain insight in the binding-mode of both TAXI-I and TAXI-II with BSXY.status: publishe

His374 of wheat endoxylanase inhibitor TAXI-I stabilizes complex formation with glycoside hydrolase family 11 endoxylanases

Wheat endoxylanase inhibitor TAXI-I inhibits microbial glycoside hydrolase family 11 endoxylanases. Crystallographic data of an Aspergillus niger endoxylanase-TAXI-I complex showed His374 of TAXI-I to be a key residue in endoxylanase inhibition. Its role in enzyme-inhibitor interaction was further investigated by site-directed mutagenesis of His374 into alanine, glutamine or lysine. Binding kinetics and affinities of the molecular interactions between A. niger, Bacillus subtilis, Trichoderma longibrachiatumendoxylanases and wild-type TAXI-I and TAXI-I His374 mutants were determined by surface plasmon resonance analysis. Enzyme-inhibitor binding was in accordance with a simple 1 : 1 binding model. Association and dissociation rate constants of wild-type TAXI-I towards the endoxylanases were in the range between 1.96 and 36.1 x 10(4)m(-1) x s(-1) and 0.72-3.60 x 10(-4) x s(-1), respectively, resulting in equilibrium dissociation constants in the low nanomolar range. Mutation of TAXI-I His374 to a variable degree reduced the inhibition capacity of the inhibitor mainly due to higher complex dissociation rate constants (three- to 80-fold increase). The association rate constants were affected to a smaller extent (up to eightfold decrease). Substitution of TAXI-I His374 therefore strongly affects the affinity of the inhibitor for the enzymes. In addition, the results show that His374 plays a critical role in the stabilization of the endoxylanase-TAXI-I complex rather than in the docking of inhibitor onto enzyme.status: publishe