Immunoregulatory effects of RGMb in gut inflammation.

Graft-versus-host disease (GvHD) is a major complication after allogeneic hematopoietic cell transplantation (HCT). Current strategies to prevent GvHD with immunosuppressive drugs carry significant morbidity and may affect the graft-versus-tumor (GVT) effect. Inflammatory bowel disease (IBD) is an intestinal inflammatory condition that affects more than 2 million people in the United States. Current strategies to prevent colitis with immunosuppressive drugs carry significant morbidity. Recently, Repulsive Guidance Molecule b (RGMb) has been identified as part of a signaling hub with neogenin and BMP receptors in mice and humans. In addition, RGMb binds BMP-2/4 in mice and humans as well as PD-L2 in mice. RGMb is expressed in the gut epithelium and by antigen presenting cells, and we found significantly increased expression in mouse small intestine after total body irradiation HCT conditioning. We hypothesized that RGMb may play a role in GvHD and IBD pathogenesis by contributing to mucosal inflammation. Using major-mismatched HCT mouse models, treatment with an anti-RGMb monoclonal antibody (mAb) that blocks the interaction with BMP-2/4 and neogenin prevented GvHD and improved survival compared to isotype control (75% versus 30% survival at 60 days after transplantation). The GVT effect was retained in tumor models. Using an inflammatory bowel disease dextran sulfate sodium model, treatment with anti-RGMb blocking monoclonal antibody but not isotype control prevented colitis and improved survival compared to control (73% versus 33% at 21 days after treatment) restoring gut homeostasis. Anti-RGMb mAb (9D1) treatment decreased IFN-γ and significantly increased IL-5 and IL-10 in the gut of the treated mice compared to the isotype control treated mice.

An efficient urine peptidomics workflow identifies chemically defined dietary gluten peptides from patients with celiac disease.

Celiac disease (CeD) is an autoimmune disorder induced by consuming gluten proteins from wheat, barley, and rye. Glutens resist gastrointestinal proteolysis, resulting in peptides that elicit inflammation in patients with CeD. Despite well-established connections between glutens and CeD, chemically defined, bioavailable peptides produced from dietary proteins have never been identified from humans in an unbiased manner. This is largely attributable to technical challenges, impeding our knowledge of potentially diverse peptide species that encounter the immune system. Here, we develop a liquid chromatographic-mass spectrometric workflow for untargeted sequence analysis of the urinary peptidome. We detect over 600 distinct dietary peptides, of which ~35% have a CeD-relevant T cell epitope and ~5% are known to stimulate innate immune responses. Remarkably, gluten peptides from patients with CeD qualitatively and quantitatively differ from controls. Our results provide a new foundation for understanding gluten immunogenicity, improving CeD management, and characterizing the dietary and urinary peptidomes.

Protein aggregation and the evolution of stress resistance in clinical yeast.

Protein aggregation, particularly in its prion-like form, has long been thought to be detrimental. However, recent studies have identified multiple instances where protein aggregation is important for normal physiological functions. Combining mass spectrometry and cell biological approaches, we developed a strategy for the identification of protein aggregates in cell lysates. We used this approach to characterize prion-based traits in pathogenic strains of the yeast Saccharomyces cerevisiae isolated from immunocompromised human patients. The proteins that we found, including the metabolic enzyme Cdc19, the translation elongation factor Yef3 and the fibrillarin homologue Nop1, are known to assemble under certain physiological conditions. Yet, such assemblies have not been reported to be stable or heritable. Our data suggest that some proteins which aggregate in response to stress have the capacity to acquire diverse assembled states, certain ones of which can be propagated across generations in a form of protein-based epigenetics. This article is part of the theme issue 'How does epigenetics influence the course of evolution?'

TagGraph reveals vast protein modification landscapes from large tandem mass spectrometry datasets.

Although mass spectrometry is well suited to identifying thousands of potential protein post-translational modifications (PTMs), it has historically been biased towards just a few. To measure the entire set of PTMs across diverse proteomes, software must overcome the dual challenges of covering enormous search spaces and distinguishing correct from incorrect spectrum interpretations. Here, we describe TagGraph, a computational tool that overcomes both challenges with an unrestricted string-based search method that is as much as 350-fold faster than existing approaches, and a probabilistic validation model that we optimized for PTM assignments. We applied TagGraph to a published human proteomic dataset of 25 million mass spectra and tripled confident spectrum identifications compared to its original analysis. We identified thousands of modification types on almost 1 million sites in the proteome. We show alternative contexts for highly abundant yet understudied PTMs such as proline hydroxylation, and its unexpected association with cancer mutations. By enabling broad characterization of PTMs, TagGraph informs as to how their functions and regulation intersect.

Antigen presentation profiling reveals recognition of lymphoma immunoglobulin neoantigens.

Cancer somatic mutations can generate neoantigens that distinguish malignant from normal cells. However, the personalized identification and validation of neoantigens remains a major challenge. Here we discover neoantigens in human mantle-cell lymphomas by using an integrated genomic and proteomic strategy that interrogates tumour antigen peptides presented by major histocompatibility complex (MHC) class I and class II molecules. We applied this approach to systematically characterize MHC ligands from 17 patients. Remarkably, all discovered neoantigenic peptides were exclusively derived from the lymphoma immunoglobulin heavy- or light-chain variable regions. Although we identified MHC presentation of private polymorphic germline alleles, no mutated peptides were recovered from non-immunoglobulin somatically mutated genes. Somatic mutations within the immunoglobulin variable region were almost exclusively presented by MHC class II. We isolated circulating CD4+ T cells specific for immunoglobulin-derived neoantigens and found these cells could mediate killing of autologous lymphoma cells. These results demonstrate that an integrative approach combining MHC isolation, peptide identification, and exome sequencing is an effective platform to uncover tumour neoantigens. Application of this strategy to human lymphoma implicates immunoglobulin neoantigens as targets for lymphoma immunotherapy.

Genetic dissection of Flaviviridae host factors through genome-scale CRISPR screens.

The Flaviviridae are a family of viruses that cause severe human diseases. For example, dengue virus (DENV) is a rapidly emerging pathogen causing an estimated 100 million symptomatic infections annually worldwide. No approved antivirals are available to date and clinical trials with a tetravalent dengue vaccine showed disappointingly low protection rates. Hepatitis C virus (HCV) also remains a major medical problem, with 160 million chronically infected patients worldwide and only expensive treatments available. Despite distinct differences in their pathogenesis and modes of transmission, the two viruses share common replication strategies. A detailed understanding of the host functions that determine viral infection is lacking. Here we use a pooled CRISPR genetic screening strategy to comprehensively dissect host factors required for these two highly important Flaviviridae members. For DENV, we identified endoplasmic-reticulum (ER)-associated multi-protein complexes involved in signal sequence recognition, N-linked glycosylation and ER-associated degradation. DENV replication was nearly completely abrogated in cells deficient in the oligosaccharyltransferase (OST) complex. Mechanistic studies pinpointed viral RNA replication and not entry or translation as the crucial step requiring the OST complex. Moreover, we show that viral non-structural proteins bind to the OST complex. The identified ER-associated protein complexes were also important for infection by other mosquito-borne flaviviruses including Zika virus, an emerging pathogen causing severe birth defects. By contrast, the most significant genes identified in the HCV screen were distinct and included viral receptors, RNA-binding proteins and enzymes involved in metabolism. We found an unexpected link between intracellular flavin adenine dinucleotide (FAD) levels and HCV replication. This study shows notable divergence in host-depenency factors between DENV and HCV, and illuminates new host targets for antiviral therapy.

Substituent effects on the binding of natural product anthocyanidin inhibitors to influenza neuraminidase with mass spectrometry.

The binding of three closely related anthocyanins within the 430-cavity of influenza neuraminidase is studied using a combination of mass spectrometry and molecular docking. Despite their similar structures, which differ only in the number and position of the hydroxyl substituents on the phenyl group attached to the chromenylium ring, subtle differences in their binding characteristics are revealed by mass spectrometry and molecular docking that are in accord with their inhibitory properties by neuraminidase inhibition assays. The cyanidin and delphinidin, with the greatest number of hydroxyl groups, bind more strongly and are better inhibitors than pelargonidin that contains a lone hydroxyl group at the 4' position. The study demonstrates, for the first time, the sensitivity of the mass spectrometry based approach for investigating the molecular basis and relative affinity of antiviral inhibitors, with subtly different structures, to their target protein. It has broader application for the screening of other protein interactions more generally with reasonable high-throughput.

Evolution of influenza neuraminidase and the detection of antiviral resistant strains using mass trees.

A new approach employing mass trees is described and implemented which enables the evolution of influenza neuraminidase across all subtypes (N1-N9) in human and animal hosts to be monitored and charted without gene or protein sequencing. These mass trees are shown to be congruent with sequence based trees. Such trees can be built solely from the masses of the proteolytic peptide ions of viral proteins recorded by a mass spectrometer. They are shown to be able to correctly chart the evolutionary history of human pandemic influenza viruses, which originated in animal hosts, and can also resolve antiviral resistant from sensitive strains. Furthermore, experimental mass map data recorded for a circulating strain is correctly positioned onto a mass tree so as to quickly establish its evolutionary history and identify whether it is resistant or sensitive to the antiviral inhibitor oseltamivir. This new computational approach is expected to find wider application for evolutionary studies of organisms more generally.

Inhibition of influenza hemagglutinin with the antiviral inhibitor arbidol using a proteomics based approach and mass spectrometry.

A proteomics gel electrophoresis based approach has been applied to study the effect of arbidol on the proliferation of influenza virus in vitro through quantitation of hemagglutinin levels. An arbidol concentration of 20µg/ml was required to achieve a 50% reduction in virus proliferation and hemagglutinin levels. The use of a MALDI mass spectrometry approach to study the binding of arbidol to influenza hemagglutinin revealed it bound solely to residues 104-120 of the HA2 subunit, a region known to contain an arbidol resistance mutation. Parallel molecular docking results revealed that this binding site was favoured in which the arbidol molecule binds in two possible orientations approximately 180° to one another at HA2 residues 118-123. The combined studies support the recognized potential of arbidol as an effective and targeted antiviral agent against the influenza virus.

Binding of a natural anthocyanin inhibitor to influenza neuraminidase by mass spectrometry.

The binding of a natural anthocyanin to influenza neuraminidase has been studied employing mass spectrometry and molecular docking. Derived from a black elderberry extract, cyanidin-3-sambubiocide has been found to be a potent inhibitor of sialidase activity. This study reveals the molecular basis for its activity for the first time. The anthocyanin is shown by parallel experimental and computational approaches to bind in the so-called 430-cavity in the vicinity of neuraminidase residues 356-364 and 395-432. Since this antiviral compound binds remote from Asp 151 and Glu 119, two residues known to regulate neuraminidase resistance, it provides the potential for the development of a new class of antivirals against the influenza virus without this susceptibility.

Mass trees: a new phylogenetic approach and algorithm to chart evolutionary history with mass spectrometry.

A new phylogenetics approach and algorithm with which to chart the evolutionary history of organisms is presented. It utilizes mass spectral data produced from the proteolytic digestion of proteins, rather than partial or complete gene or translated gene sequences. The concept and validity of the approach is demonstrated herein using both theoretical and experimental mass data, together with the translated gene sequences of the hemagglutinin protein of the influenza virus. A comparison of the mass trees with conventional sequenced-based phylogenetic trees, using two separate tree comparison algorithms, reveals a high degree of similarity and congruence among the trees. Given that the mass map data can be generated more rapidly than gene sequences, even when next generation parallel sequencing is employed, mass trees offer new opportunities and advantages for phylogenetic analysis.

Anti-viral inhibitor binding to influenza neuraminidase by MALDI mass spectrometry.

A matrix-assisted laser desorption ionization (MALDI) mass spectrometry-based approach is applied to identify active site domains within influenza neuraminidase that bind the antiviral inhibitors zanamivir (ZANA) and 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA). Combined data from the tryptic and Glu-C endoproteinase digests of neuraminidase-inhibitor complexes have identified binding peptides that contain the active site residues Arg118, Glu119, Arg156, Glu276, and Tyr406. The binding of these residues was confirmed from the analysis of available X-ray crystal structures. The ability to identify peptides within the active sites of proteins and likely binding residues provides both a rapid and relatively high throughput approach with which to screen protein-drug interactions by MALDI mass spectrometry.