Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

Finding a Needle in a Haystack: The Role of Electrostatics in Target Lipid Recognition by PH Domains

<div><p>Interactions between protein domains and lipid molecules play key roles in controlling cell membrane signalling and trafficking. The pleckstrin homology (PH) domain is one of the most widespread, binding specifically to phosphatidylinositol phosphates (PIPs) in cell membranes. PH domains must locate specific PIPs in the presence of a background of approximately 20% anionic lipids within the cytoplasmic leaflet of the plasma membrane. We investigate the mechanism of such recognition <em>via</em> a multiscale procedure combining Brownian dynamics (BD) and molecular dynamics (MD) simulations of the GRP1 PH domain interacting with phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P₃). The interaction of GRP1-PH with PI(3,4,5)P₃ in a zwitterionic bilayer is compared with the interaction in bilayers containing different levels of anionic ‘decoy’ lipids. BD simulations reveal both translational and orientational electrostatic steering of the PH domain towards the PI(3,4,5)P₃-containing anionic bilayer surface. There is a payoff between non-PIP anionic lipids attracting the PH domain to the bilayer surface in a favourable orientation and their role as ‘decoys’, disrupting the interaction of GRP1-PH with the PI(3,4,5)P₃ molecule. Significantly, approximately 20% anionic lipid in the cytoplasmic leaflet of the bilayer is nearly optimal to both enhance orientational steering and to localise GRP1-PH proximal to the surface of the membrane without sacrificing its ability to locate PI(3,4,5)P₃ within the bilayer plane. Subsequent MD simulations reveal binding to PI(3,4,5)P₃, forming protein-phosphate contacts comparable to those in X-ray structures. These studies demonstrate a computational framework which addresses lipid recognition within a cell membrane environment, offering a link between structural and cell biological characterisation.</p> </div

MD simulations of bound complex formation.

<p><b>A</b> An optimal configuration for subsequent membrane-binding extracted from the ensemble of BD simulations conducted using an uncharged, zwitterionic lipid bilayer containing PI(3,4,5)P₃. In this case <i>r</i> = 9 Å, <i>d</i> = 18 Å and <i>θ</i> = 27°. <b>B</b> A snapshot at the end of a subsequent 100 ns MD simulation. <b>C, D</b> Fingerprint plots showing the location of PH domain residues contacting the phosphorus atoms of PI(3,4,5)P₃ in <b>C</b> the X-ray structure (PDB 1FGY) and <b>D</b> the 100 ns snapshot from the MD simulation shown in <b>B</b>. In each plot the minimum distances between each residue and each phosphorus atom are shown.</p

Lipid bilayer models.

<p><b>A</b> Electrostatic potential isocontours of a POPC lipid bilayer (red = negative, blue = positive), with a central PI(3,4,5)P₃ molecule clearly visible as a region of negative electrostatic potential (indicated by a white circle). <b>B</b> Electrostatic potential isocontours after modifying lipids with an evenly distributed fractional charge. Each lipid headgroup of the upper (<i>i.e.</i> cytoplasmic) leaflet of the bilayer has a fractional charge of −0.4 <i>e</i>. <b>C</b> Isocontours after a randomly selected subset of 40% of the lipids in the upper leaflet were assigned a charge of −1.0 <i>e</i>.</p

Schematic diagram of the BD simulation setup.

<p>The <i>b</i>-sphere is truncated to form an open hemispherical surface situated on one side of the membrane, with the value of <i>q</i> set such that all trajectories are terminated before the protein is able to diffuse outside the perimeter of the membrane patch. The three coordinates <i>r</i>, <i>z</i> and <i>θ</i> specify the position and orientation of the protein relative to the PI(3,4,5)P₃ headgroup.</p

Positional steering of GRP1-PH using alternative lipid configurations.

<p><b>A</b> Distribution of radial locations, <i>r</i>, for the BD simulations based on randomly distributed, integer-valued negative charges on the lipid headgroups. The centre of mass of the ‘target’ PI(3,4,5)P₃ headgroup is located at <i>r</i> = 0. <b>B</b> Distribution of radial locations, <i>r</i>, for the BD simulations based on the lipid configurations generated from a CGMD simulation of a lipid bilayer containing 20% anionic lipids.</p

Structure of the GRP1 PH domain.

<p><b>A</b> The crystal structure of GRP1-PH (PDB 1FGY). The I(1,3,4,5)P₄ headgroup and the sidechains of two key residues (K279 and R284) are shown as van der Waals spheres. The protein is oriented such that the bilayer normal, as determined by previous MD simulations, is vertical (<i>i.e.</i> defines the <i>z</i> axis). <b>B</b> Electrostatic potential of GRP1-PH projected onto the solvent-accessible surface, showing the large positive electrostatic potential around the I(1,3,4,5)P₄ binding site. The electrostatic potential was calculated in the absence of I(1,3,4,5)P₄ using APBS as described in the main text and is coloured from −5 kT/<i>e</i> (red) to +5 kT/<i>e</i> (blue). The protein is shown in the same orientation as in <b>A</b> and the molecular dipole moment (calculated using the Protein Dipole Moments Server (<a href="http://bioinfo.weizmann.ac.il/dipol/" target="_blank">http://bioinfo.weizmann.ac.il/dipol/</a>; <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002617#pcbi.1002617-Felder1" target="_blank">[70]</a>) is indicated by a black arrow. <b>C</b> Snapshot from a BD simulation showing the protein solvent-accessible surface coloured by electrostatic potential, with the molecular dipole moment shown as a black arrow as in <b>B</b>. The POPC lipid bilayer is shown as a white surface with the single PI(3,4,5)P₃ molecule shown in black.</p

Orientational steering of GRP1-PH.

<p><b>A</b> Distribution of orientations, <i>θ</i>, of GRP1-PH over the course of the BD simulations for the case of the evenly spread, fractional charges from 0.0 to −1.0 <i>e</i>. The dotted line at <i>θ</i> = 56° corresponds to the angle, <i>θ</i>, that the molecular dipole moment makes with respect to the <i>z</i> axis in the membrane bound complex. <b>B</b> Two-dimensional distributions of <i>θ vs. r</i> from the BD simulations with the fractional charge distributions with all lipids (other than PI(3,4,5)P₃) given a charge of −0.2 <i>e</i>. Note that the origin corresponds to the configuration obtaining by manual docking plus simulations (see main text and <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002617#pcbi.1002617-Lumb1" target="_blank">[28]</a>). <b>C</b> Distribution of radial ‘first encounter’ locations for the BD simulations with evenly spread, fractional charges from 0.0 to −1.0 <i>e</i>.</p