Thioredoxin is a metabolic rheostat controlling regulatory B cells.

Metabolic programming is important for B cell fate, but the bioenergetic requirement for regulatory B (Breg) cell differentiation and function is unknown. Here we show that Breg cell differentiation, unlike non-Breg cells, relies on mitochondrial electron transport and homeostatic levels of reactive oxygen species (ROS). Single-cell RNA sequencing analysis revealed that TXN, encoding the metabolic redox protein thioredoxin (Trx), is highly expressed by Breg cells, unlike Trx inhibitor TXNIP which was downregulated. Pharmacological inhibition or gene silencing of TXN resulted in mitochondrial membrane depolarization and increased ROS levels, selectively suppressing Breg cell differentiation and function while favoring pro-inflammatory B cell differentiation. Patients with systemic lupus erythematosus (SLE), characterized by Breg cell deficiencies, present with B cell mitochondrial membrane depolarization, elevated ROS and fewer Trx+ B cells. Exogenous Trx stimulation restored Breg cells and mitochondrial membrane polarization in SLE B cells to healthy B cell levels, indicating Trx insufficiency underlies Breg cell impairment in patients with SLE.

sciCSR infers B cell state transition and predicts class-switch recombination dynamics using single-cell transcriptomic data.

Class-switch recombination (CSR) is an integral part of B cell maturation. Here we present sciCSR (pronounced 'scissor', single-cell inference of class-switch recombination), a computational pipeline that analyzes CSR events and dynamics of B cells from single-cell RNA sequencing (scRNA-seq) experiments. Validated on both simulated and real data, sciCSR re-analyzes scRNA-seq alignments to differentiate productive heavy-chain immunoglobulin transcripts from germline 'sterile' transcripts. From a snapshot of B cell scRNA-seq data, a Markov state model is built to infer the dynamics and direction of CSR. Applying sciCSR on severe acute respiratory syndrome coronavirus 2 vaccination time-course scRNA-seq data, we observe that sciCSR predicts, using data from an earlier time point in the collected time-course, the isotype distribution of B cell receptor repertoires of subsequent time points with high accuracy (cosine similarity ~0.9). Using processes specific to B cells, sciCSR identifies transitions that are often missed by conventional RNA velocity analyses and can reveal insights into the dynamics of B cell CSR during immune response.

Pathogenic missense protein variants affect different functional pathways and proteomic features than healthy population variants.

Missense variants are present amongst the healthy population, but some of them are causative of human diseases. A classification of variants associated with "healthy" or "diseased" states is therefore not always straightforward. A deeper understanding of the nature of missense variants in health and disease, the cellular processes they may affect, and the general molecular principles which underlie these differences is essential to offer mechanistic explanations of the true impact of pathogenic variants. Here, we have formalised a statistical framework which enables robust probabilistic quantification of variant enrichment across full-length proteins, their domains, and 3D structure-defined regions. Using this framework, we validate and extend previously reported trends of variant enrichment in different protein structural regions (surface/core/interface). By examining the association of variant enrichment with available functional pathways and transcriptomic and proteomic (protein half-life, thermal stability, abundance) data, we have mined a rich set of molecular features which distinguish between pathogenic and population variants: Pathogenic variants mainly affect proteins involved in cell proliferation and nucleotide processing and are enriched in more abundant proteins. Additionally, rare population variants display features closer to common than pathogenic variants. We validate the association between these molecular features and variant pathogenicity by comparing against existing in silico variant impact annotations. This study provides molecular details into how different proteins exhibit resilience and/or sensitivity towards missense variants and provides the rationale to prioritise variant-enriched proteins and protein domains for therapeutic targeting and development. The ZoomVar database, which we created for this study, is available at fraternalilab.kcl.ac.uk/ZoomVar. It allows users to programmatically annotate missense variants with protein structural information and to calculate variant enrichment in different protein structural regions.

Predicting class switch recombination in B-cells from antibody repertoire data.

Statistical and machine learning methods have proved useful in many areas of immunology. In this paper, we address for the first time the problem of predicting the occurrence of class switch recombination (CSR) in B-cells, a problem of interest in understanding antibody response under immunological challenges. We propose a framework to analyze antibody repertoire data, based on clonal (CG) group representation in a way that allows us to predict CSR events using CG level features as input. We assess and compare the performance of several predicting models (logistic regression, LASSO logistic regression, random forest, and support vector machine) in carrying out this task. The proposed approach can obtain an unweighted average recall of 71 % $71\%$ with models based on variable region descriptors and measures of CG diversity during an immune challenge and, most notably, before an immune challenge.

Virus-inspired designs of antimicrobial nanocapsules.

Antimicrobial resistance is becoming a serious burden for drug design. The challenges are in finding novel approaches for effectively targeting a number of different bacterial strains, and in delivering these to the site of action. We propose here a novel approach that exploits the assembly of antimicrobial peptidic units in nanocapsules that can penetrate and rupture the bacterial membrane. Additionally, the chemical versatility of the designed units can be tailored to specific targets and to the delivery of genetic material in the cell. The proposed design exploits a ß-annulus (sequence ITHVGGVGGSIMAPVAVSRQLVGS) triskelion unit from the Tomato Bushy Stunt Virus, able to self assemble in solution, and functionalised with antimicrobial sequences to form dodecahedral antimicrobial nanocapsules. The stability and the activity of the antimicrobial ß-annulus capsule is measured by molecular dynamics simulations in water and in the presence of model membranes.

Pan-cancer transcriptomic analysis dissects immune and proliferative functions of APOBEC3 cytidine deaminases.

APOBEC3 cytidine deaminases are largely known for their innate immune protection from viral infections. Recently, members of the family have been associated with a distinct mutational activity in some cancer types. We report a pan-tissue, pan-cancer analysis of RNA-seq data specific to the APOBEC3 genes in 8,951 tumours, 786 cancer cell lines and 6,119 normal tissues. By deconvolution of levels of different cell types in tumour admixtures, we demonstrate that APOBEC3B (A3B), the primary candidate as a cancer mutagen, shows little association with immune cell types compared to its paralogues. We present a pipeline called RESPECTEx (REconstituting SPecific Cell-Type Expression) and use it to deconvolute cell-type specific expression levels in a given cohort of tumour samples. We functionally annotate APOBEC3 co-expressing genes, and create an interactive visualization tool which 'barcodes' the functional enrichment (http://fraternalilab.kcl.ac.uk/apobec-barcodes/). These analyses reveal that A3B expression correlates with cell cycle and DNA repair genes, whereas the other APOBEC3 members display specificity for immune processes and immune cell populations. We offer molecular insights into the functions of individual APOBEC3 proteins in antiviral and proliferative contexts, and demonstrate the diversification this family of enzymes displays at the transcriptomic level, despite their high similarity in protein sequences and structures.

Structure and Dynamics of Nanoconfined Water Between Surfactant Monolayers.

The properties of nanoconfined water arise in direct response to the properties of the interfaces that confine it. A great deal of research has focused on understanding how and why the physical properties of confined water differ greatly from the bulk. In this work, we have used all-atom molecular dynamics (MD) simulations to provide a detailed description of the structural and dynamical properties of nanoconfined water between two monolayers consisting of an archetypal ionic surfactant, cetrimonium bromide (CTAB, [CH3(CH2)15N(CH3)3]+Br-). Small differences in the area per surfactant of the monolayers impart a clear effect on the intrinsic density, mobility, and ordering of the interfacial water layer confined by the monolayers. We find that as the area per surfactant within a monolayer decreases, the mobility of the interfacial water molecules decreases in response. As the monolayer packing density decreases, we find that each individual CTAB molecule has a greater effect on the ordering of water molecules in its first hydration shell. In a denser monolayer, we observe that the effect of individual CTAB molecules on the ordering of water molecules is hindered by increased competition between headgroups. Therefore, when two monolayers with different areas per surfactant are used to confine a nanoscale water layer, we observe the emergence of noncentrosymmetry.

Symmetry-breaking transitions in the early steps of protein self-assembly.

Protein misfolding and subsequent self-association are complex, intertwined processes, resulting in development of a heterogeneous population of aggregates closely related to many chronic pathological conditions including Type 2 Diabetes Mellitus and Alzheimer's disease. To address this issue, here, we develop a theoretical model in the general framework of linear stability analysis. According to this model, self-assemblies of peptides with pronounced conformational flexibility may become, under particular conditions, unstable and spontaneously evolve toward an alternating array of partially ordered and disordered monomers. The predictions of the theory were verified by atomistic molecular dynamics (MD) simulations of islet amyloid polypeptide (IAPP) used as a paradigm of aggregation-prone polypeptides (proteins). Simulations of dimeric, tetrameric, and hexameric human-IAPP self-assemblies at physiological electrolyte concentration reveal an alternating distribution of the smallest domains (of the order of the peptide mean length) formed by partially ordered (mainly ß-strands) and disordered (turns and coil) arrays. Periodicity disappears upon weakening of the inter-peptide binding, a result in line with the predictions of the theory. To further probe the general validity of our hypothesis, we extended the simulations to other peptides, the Aß(1-40) amyloid peptide, and the ovine prion peptide as well as to other proteins (SOD1 dimer) that do not belong to the broad class of intrinsically disordered proteins. In all cases, the oligomeric aggregates show an alternate distribution of partially ordered and disordered monomers. We also carried out Surface Enhanced Raman Scattering (SERS) measurements of hIAPP as an experimental validation of both the theory and in silico simulations.

RhoBTB1 interacts with ROCKs and inhibits invasion.

RhoBTB1 is an atypical Rho GTPase with two BTB domains in addition to its Rho domain. Although most Rho GTPases regulate actin cytoskeletal dynamics, RhoBTB1 is not known to affect cell shape or motility. We report that RhoBTB1 depletion increases prostate cancer cell invasion and induces elongation in Matrigel, a phenotype similar to that induced by depletion of ROCK1 and ROCK2. We demonstrate that RhoBTB1 associates with ROCK1 and ROCK2 and its association with ROCK1 is via its Rho domain. The Rho domain binds to the coiled-coil region of ROCK1 close to its kinase domain. We identify two amino acids within the Rho domain that alter RhoBTB1 association with ROCK1. RhoBTB1 is a substrate for ROCK1, and mutation of putative phosphorylation sites reduces its association with Cullin3, a scaffold for ubiquitin ligases. We propose that RhoBTB1 suppresses cancer cell invasion through interacting with ROCKs, which in turn regulate its association with Cullin3. Via Cullin3, RhoBTB1 has the potential to affect protein degradation.

BRepertoire: a user-friendly web server for analysing antibody repertoire data.

Antibody repertoire analysis by high throughput sequencing is now widely used, but a persisting challenge is enabling immunologists to explore their data to discover discriminating repertoire features for their own particular investigations. Computational methods are necessary for large-scale evaluation of antibody properties. We have developed BRepertoire, a suite of user-friendly web-based software tools for large-scale statistical analyses of repertoire data. The software is able to use data preprocessed by IMGT, and performs statistical and comparative analyses with versatile plotting options. BRepertoire has been designed to operate in various modes, for example analysing sequence-specific V(D)J gene usage, discerning physico-chemical properties of the CDR regions and clustering of clonotypes. Those analyses are performed on the fly by a number of R packages and are deployed by a shiny web platform. The user can download the analysed data in different table formats and save the generated plots as image files ready for publication. We believe BRepertoire to be a versatile analytical tool that complements experimental studies of immune repertoires. To illustrate the server's functionality, we show use cases including differential gene usage in a vaccination dataset and analysis of CDR3H properties in old and young individuals. The server is accessible under http://mabra.biomed.kcl.ac.uk/BRepertoire.

In silico identification of rescue sites by double force scanning.

Motivation: A deleterious amino acid change in a protein can be compensated by a second-site rescue mutation. These compensatory mechanisms can be mimicked by drugs. In particular, the location of rescue mutations can be used to identify protein regions that can be targeted by small molecules to reactivate a damaged mutant. Results: We present the first general computational method to detect rescue sites. By mimicking the effect of mutations through the application of forces, the double force scanning (DFS) method identifies the second-site residues that make the protein structure most resilient to the effect of pathogenic mutations. We tested DFS predictions against two datasets containing experimentally validated and putative evolutionary-related rescue sites. A remarkably good agreement was found between predictions and experimental data. Indeed, almost half of the rescue sites in p53 was correctly predicted by DFS, with 65% of remaining sites in contact with DFS predictions. Similar results were found for other proteins in the evolutionary dataset. Availability and implementation: The DFS code is available under GPL at https://fornililab.github.io/dfs/. Supplementary information: Supplementary data are available at Bioinformatics online.

TITINdb-a computational tool to assess titin's role as a disease gene.

SUMMARY: Large numbers of rare and unique titin missense variants have been discovered in both healthy and disease cohorts, thus the correct classification of variants as pathogenic or non-pathogenic has become imperative. Due to titin's large size (363 coding exons), current web applications are unable to map titin variants to domain structures. Here, we present a web application, TITINdb, which integrates titin structure, variant, sequence and isoform information, along with pre-computed predictions of the impact of non-synonymous single nucleotide variants, to facilitate the correct classification of titin variants. AVAILABILITY AND IMPLEMENTATION: TITINdb can be freely accessed at http://fraternalilab.kcl.ac.uk/TITINdb. CONTACT: franca.fraternali@kcl.ac.uk. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Pooled sequencing of 531 genes in inflammatory bowel disease identifies an associated rare variant in BTNL2 and implicates other immune related genes.

The contribution of rare coding sequence variants to genetic susceptibility in complex disorders is an important but unresolved question. Most studies thus far have investigated a limited number of genes from regions which contain common disease associated variants. Here we investigate this in inflammatory bowel disease by sequencing the exons and proximal promoters of 531 genes selected from both genome-wide association studies and pathway analysis in pooled DNA panels from 474 cases of Crohn's disease and 480 controls. 80 variants with evidence of association in the sequencing experiment or with potential functional significance were selected for follow up genotyping in 6,507 IBD cases and 3,064 population controls. The top 5 disease associated variants were genotyped in an extension panel of 3,662 IBD cases and 3,639 controls, and tested for association in a combined analysis of 10,147 IBD cases and 7,008 controls. A rare coding variant p.G454C in the BTNL2 gene within the major histocompatibility complex was significantly associated with increased risk for IBD (p = 9.65x10-10, OR = 2.3[95% CI = 1.75-3.04]), but was independent of the known common associated CD and UC variants at this locus. Rare (<1%) and low frequency (1-5%) variants in 3 additional genes showed suggestive association (p<0.005) with either an increased risk (ARIH2 c.338-6C>T) or decreased risk (IL12B p.V298F, and NICN p.H191R) of IBD. These results provide additional insights into the involvement of the inhibition of T cell activation in the development of both sub-phenotypes of inflammatory bowel disease. We suggest that although rare coding variants may make a modest overall contribution to complex disease susceptibility, they can inform our understanding of the molecular pathways that contribute to pathogenesis.

Investigating Ebola virus pathogenicity using molecular dynamics.

BACKGROUND: Ebolaviruses have been known to cause deadly disease in humans for 40 years and have recently been demonstrated in West Africa to be able to cause large outbreaks. Four Ebolavirus species cause severe disease associated with high mortality in humans. Reston viruses are the only Ebolaviruses that do not cause disease in humans. Conserved amino acid changes in the Reston virus protein VP24 compared to VP24 of other Ebolaviruses have been suggested to alter VP24 binding to host cell karyopherins resulting in impaired inhibition of interferon signalling, which may explain the difference in human pathogenicity. Here we used protein structural analysis and molecular dynamics to further elucidate the interaction between VP24 and KPNA5. RESULTS: As a control experiment, we compared the interaction of wild-type and R137A-mutant (known to affect KPNA5 binding) Ebola virus VP24 with KPNA5. Results confirmed that the R137A mutation weakens direct VP24-KPNA5 binding and enables water molecules to penetrate at the interface. Similarly, Reston virus VP24 displayed a weaker interaction with KPNA5 than Ebola virus VP24, which is likely to reduce the ability of Reston virus VP24 to prevent host cell interferon signalling. CONCLUSION: Our results provide novel molecular detail on the interaction of Reston virus VP24 and Ebola virus VP24 with human KPNA5. The results indicate a weaker interaction of Reston virus VP24 with KPNA5 than Ebola virus VP24, which is probably associated with a decreased ability to interfere with the host cell interferon response. Hence, our study provides further evidence that VP24 is a key player in determining Ebolavirus pathogenicity.

AP1S3 mutations are associated with pustular psoriasis and impaired Toll-like receptor 3 trafficking.

Adaptor protein complex 1 (AP-1) is an evolutionary conserved heterotetramer that promotes vesicular trafficking between the trans-Golgi network and the endosomes. The knockout of most murine AP-1 complex subunits is embryonically lethal, so the identification of human disease-associated alleles has the unique potential to deliver insights into gene function. Here, we report two founder mutations (c.11T>G [p.Phe4Cys] and c.97C>T [p.Arg33Trp]) in AP1S3, the gene encoding AP-1 complex subunit σ1C, in 15 unrelated individuals with a severe autoinflammatory skin disorder known as pustular psoriasis. Because the variants are predicted to destabilize the 3D structure of the AP-1 complex, we generated AP1S3-knockdown cell lines to investigate the consequences of AP-1 deficiency in skin keratinocytes. We found that AP1S3 silencing disrupted the endosomal translocation of the innate pattern-recognition receptor TLR-3 (Toll-like receptor 3) and resulted in a marked inhibition of downstream signaling. These findings identify pustular psoriasis as an autoinflammatory phenotype caused by defects in vesicular trafficking and demonstrate a requirement of AP-1 for Toll-like receptor homeostasis.

Surface Accessibility and Dynamics of Macromolecular Assemblies Probed by Covalent Labeling Mass Spectrometry and Integrative Modeling.

Mass spectrometry (MS) has become an indispensable tool for investigating the architectures and dynamics of macromolecular assemblies. Here we show that covalent labeling of solvent accessible residues followed by their MS-based identification yields modeling restraints that allow mapping the location and orientation of subunits within protein assemblies. Together with complementary restraints derived from cross-linking and native MS, we built native-like models of four heterocomplexes with known subunit structures and compared them with available X-ray crystal structures. The results demonstrated that covalent labeling followed by MS markedly increased the predictive power of the integrative modeling strategy enabling more accurate protein assembly models. We applied this strategy to the F-type ATP synthase from spinach chloroplasts (cATPase) providing a structural basis for its function as a nanomotor. By subjecting the models generated by our restraint-based strategy to molecular dynamics (MD) simulations, we revealed the conformational states of the peripheral stalk and assigned flexible regions in the enzyme. Our strategy can readily incorporate complementary chemical labeling strategies and we anticipate that it will be applicable to many other systems providing new insights into the structure and function of protein complexes.

PinSnps: structural and functional analysis of SNPs in the context of protein interaction networks.

UNLABELLED: We present a practical computational pipeline to readily perform data analyses of protein-protein interaction networks by using genetic and functional information mapped onto protein structures. We provide a 3D representation of the available protein structure and its regions (surface, interface, core and disordered) for the selected genetic variants and/or SNPs, and a prediction of the mutants' impact on the protein as measured by a range of methods. We have mapped in total 2587 genetic disorder-related SNPs from OMIM, 587 873 cancer-related variants from COSMIC, and 1 484 045 SNPs from dbSNP. All result data can be downloaded by the user together with an R-script to compute the enrichment of SNPs/variants in selected structural regions. AVAILABILITY AND IMPLEMENTATION: PinSnps is available as open-access service at http://fraternalilab.kcl.ac.uk/PinSnps/ CONTACT: franca.fraternali@kcl.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Plasticity and conformational equilibria of influenza fusion peptides in model lipid bilayers.

Membrane fusion is critical to eukaryotic cellular function and crucial to the entry of enveloped viruses such as influenza and human immunodeficiency virus. Influenza viral entry in the host cell is mediated by a 20-23 amino acid long sequence, called the fusion peptide. In the last years, possible structures for the fusion peptide and their implication in the membrane fusion initiation have been proposed; these ranging from an inverted V shaped α-helical structure to an α-helical hairpin, or to a complete α-helix. Here we develop a coarse grained approach to describe effectively the plasticity of the fusion peptide and the explored conformational states. We describe also a trimeric assembly for the fusion peptide and analyse the explored states in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine model membrane. For the single fusion peptide systems the kink angle observed experimentally for the V shaped structure shows a strong correlation with the orientation of the fusion peptide within the lipid bilayer. The trimeric fusion peptide model also experiences different conformational states and represents a more realistic model for the anchoring mechanism of one influenza haemagglutinin molecule. This article is part of a Special Issue entitled: Viral Membrane Proteins - Channels for Cellular Networking.