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Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, persistently infects over 90% of the human adult population and is associated with several human cancers. To establish life-long infection, EBV tampers with the induction of type I interferon (IFN I)-dependent antiviral immunity in the host. How various EBV genes help orchestrate this crucial strategy is incompletely defined. Here, we reveal a mechanism by which the EBV nuclear antigen 3A (EBNA3A) may inhibit IFNβ induction. Using proximity biotinylation we identify the histone acetyltransferase P300, a member of the IFNβ transcriptional complex, as a binding partner of EBNA3A. We further show that EBNA3A also interacts with the activated IFN-inducing transcription factor interferon regulatory factor 3 that collaborates with P300 in the nucleus. Both events are mediated by the N-terminal domain of EBNA3A. We propose that EBNA3A limits the binding of interferon regulatory factor 3 to the IFNβ promoter, thereby hampering downstream IFN I signaling. Collectively, our findings suggest a new mechanism of immune evasion by EBV, affected by its latency gene EBNA3A., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Gut microbiota of the gastrointestinal tract provide health benefits to the human host via bacterial metabolites. Bacterial butyrate has beneficial effects on intestinal homeostasis and is the preferred energy source of intestinal epithelial cells, capable of inducing differentiation. It was previously observed that changes in the expression of specific proteins as well as protein glycosylation occur with differentiation. In this study, specific mucin O-glycans were identified that mark butyrate-induced epithelial differentiation of the intestinal cell line CaCo-2 (Cancer Coli-2), by applying porous graphitized carbon nano-liquid chromatography with electrospray ionization tandem mass spectrometry. Moreover, a quantitative proteomic approach was used to decipher changes in the cell proteome. It was found that the fully differentiated butyrate-stimulated cells are characterized by a higher expression of sialylated O-glycan structures, whereas fucosylation is downregulated with differentiation. By performing an integrative approach, we generated hypotheses about the origin of the observed O-glycome changes. These insights pave the way for future endeavors to study the dynamic O-glycosylation patterns in the gut, either produced via cellular biosynthesis or through the action of bacterial glycosidases as well as the functional role of these patterns in homeostasis and dysbiosis at the gut-microbiota interface., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The type A glycan modification found in human pathogen Clostridioides difficile consists of a monosaccharide (GlcNAc) that is linked to an N-methylated threonine through a phosphodiester bond. This structure has previously been described on the flagellar protein flagellin C of several C. difficile strains and is important for bacterial motility. The study of post-translational modifications often relies on some type of enrichment strategy; however, a procedure for enrichment of this modification has not yet been demonstrated. In this study, we show that an approach that is commonly used in phosphoproteomics, Fe 3+ -immobilized metal affinity chromatography, also enriches for peptides with this unique post-translational modification. Using LC-MS/MS analyses of immobilized metal affinity chromatography-captured tryptic peptides, we observed not only type A-modified C. difficile flagellin peptides but also a variety of truncated/modified type A structures on these peptides. Using an elaborate set of mass spectrometry analyses, we demonstrate that one of these modifications consists of a type A structure containing a phosphonate (2-aminoethylphosphonate), a modification that is rarely observed and has hitherto not been described in C. difficile. In conclusion, we show that a common enrichment strategy results in reliable identification of peptides carrying a type A glycan modification, and that the results obtained can be used to advance models about its biosynthesis., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Histone post-translational modifications (PTMs) are critical for processes such as transcription. The more notable among these are the nonacetyl histone lysine acylation modifications such as crotonylation, butyrylation, and succinylation. However, the biological relevance of these PTMs is not fully understood because their regulation is largely unknown. Here, we set out to investigate whether the main histone acetyltransferases in budding yeast, Gcn5 and Esa1, possess crotonyltransferase activity. In vitro studies revealed that the Gcn5-Ada2-Ada3 (ADA) and Esa1-Yng2-Epl1 (Piccolo NuA4) histone acetyltransferase complexes have the capacity to crotonylate histones. Mass spectrometry analysis revealed that ADA and Piccolo NuA4 crotonylate lysines in the N-terminal tails of histone H3 and H4, respectively. Functionally, we show that crotonylation selectively affects gene transcription in vivo in a manner dependent on Gcn5 and Esa1. Thus, we identify the Gcn5- and Esa1-containing ADA and Piccolo NuA4 complexes as bona fide crotonyltransferases that promote crotonylation-dependent transcription., (© 2019 Kollenstart et al.)

Pro-Pro endopeptidases (PPEPs) belong to a recently discovered family of proteases capable of hydrolyzing a Pro-Pro bond. The first member from the bacterial pathogen Clostridium difficile (PPEP-1) cleaves two C. difficile cell-surface proteins involved in adhesion, one of which is encoded by the gene adjacent to the ppep-1 gene. However, related PPEPs may exist in other bacteria and may shed light on substrate specificity in this enzyme family. Here, we report on the homolog of PPEP-1 in Paenibacillus alvei , which we denoted PPEP-2. We found that PPEP-2 is a secreted metalloprotease, which likewise cleaved a cell-surface protein encoded by an adjacent gene. However, the cleavage motif of PPEP-2, PLP↓PVP, is distinct from that of PPEP-1 (VNP↓PVP). As a result, an optimal substrate peptide for PPEP-2 was not cleaved by PPEP-1 and vice versa. To gain insight into the specificity mechanism of PPEP-2, we determined its crystal structure at 1.75 Å resolution and further confirmed the structure in solution using small-angle X-ray scattering (SAXS). We show that a four-amino-acid loop, which is distinct in PPEP-1 and -2 (GGST in PPEP-1 and SERV in PPEP-2), plays a crucial role in substrate specificity. A PPEP-2 variant, in which the four loop residues had been swapped for those from PPEP-1, displayed a shift in substrate specificity toward PPEP-1 substrates. Our results provide detailed insights into the PPEP-2 structure and the structural determinants of substrate specificity in this new family of PPEP proteases., (© 2018 Klychnikov et al.)

Human leukocyte antigen (HLA) class I molecules generally present peptides (p) of 8 to 11 amino acids (aa) in length. Although an increasing number of examples with lengthy (>11 aa) peptides, presented mostly by HLA-B alleles, have been reported. Here we characterize HLA-A*02:01 restricted, in addition to the HLA-B*0702 and HLA-B*4402 restricted, lengthy peptides (>11 aa) arising from the B-cell ligandome. We analyzed a number of 15-mer peptides presented by HLA-A*02:01, and confirmed pHLA-I formation by HLA folding and thermal stability assays. Surprisingly the binding affinity and stability of the 15-mer epitopes in complex with HLA-A*02:01 were comparable with the values observed for canonical length (8 to 11 aa) HLA-A*02:01-restricted peptides. We solved the structures of two 15-mer epitopes in complex with HLA-A*02:01, within which the peptides adopted distinct super-bulged conformations. Moreover, we demonstrate that T-cells can recognize the 15-mer peptides in the context of HLA-A*02:01, indicating that these 15-mer peptides represent immunogenic ligands. Collectively, our data expand our understanding of longer epitopes in the context of HLA-I, highlighting that they are not limited to the HLA-B family, but can bind the ubiquitous HLA-A*02:01 molecule, and play an important role in T-cell immunity., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The Mycobacterium tuberculosis Beijing genotype, consisting of the more ancient (atypical) and modern (typical) emerging sublineage, is one of the most prevalent and genetically conserved genotype families and has often been associated with multidrug resistance. In this study, we employed a 2D-LC-FTICR MS approach, combined with dimethylation of tryptic peptides, to systematically compare protein abundance levels of ancient and modern Beijing strains and identify differences that could be associated with successful spread of the modern sublineage. The data is available via ProteomeXchange using the identifier PXD000931. Despite the highly uniform protein abundance ratios in both sublineages, we identified four proteins as differentially regulated between both sublineages, which could explain the apparent increased adaptation of the modern Beijing strains. These proteins are; Rv0450c/MmpL4, Rv1269c, Rv3137, and Rv3283/sseA. Transcriptional and functional analysis of these proteins in a large cohort of 29 Beijing strains showed that the mRNA levels of Rv0450c/MmpL4 are significantly higher in modern Beijing strains, whereas we also provide evidence that Rv3283/sseA is less abundant in the modern Beijing sublineage. Our findings provide a possible explanation for the increased virulence and success of the modern Beijing sublineage. In addition, in the established dataset of 1817 proteins, we demonstrate the pre-existence of several, possibly unique, antibiotic efflux pumps in the proteome of the Beijing strains. This may reflect an increased ability of Beijing strains to escape exposure to antituberculosis drugs., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Peptides presented by human leukocyte antigen (HLA) molecules on the cell surface play a crucial role in adaptive immunology, mediating the communication between T cells and antigen presenting cells. Knowledge of these peptides is of pivotal importance in fundamental studies of T cell action and in cellular immunotherapy and transplantation. In this paper we present the in-depth identification and relative quantification of 14,500 peptide ligands constituting the HLA ligandome of B cells. This large number of identified ligands provides general insight into the presented peptide repertoire and antigen presentation. Our uniquely large set of HLA ligands allowed us to characterize in detail the peptides constituting the ligandome in terms of relative abundance, peptide length distribution, physicochemical properties, binding affinity to the HLA molecule, and presence of post-translational modifications. The presented B-lymphocyte ligandome is shown to be a rich source of information by the presence of minor histocompatibility antigens, virus-derived epitopes, and post-translationally modified HLA ligands, and it can be a good starting point for solving a wealth of specific immunological questions. These HLA ligands can form the basis for reversed immunology approaches to identify T cell epitopes based not on in silico predictions but on the bona fide eluted HLA ligandome.

HLA-DQ2 and HLA-DQ8 are strongly predisposing haplotypes for type 1 diabetes (T1D). Yet HLA-DQ2/8 heterozygous individuals have a synergistically increased risk compared with HLA-DQ2 or HLA-DQ8 homozygote subjects that may result from the presence of a transdimer formed between the α-chain of HLA-DQ2 (DQA1*05:01) and the β-chain of HLA-DQ8 (DQB1*03:02). We generated cells exclusively expressing this transdimer (HLA-DQ8trans), characterized its peptide binding repertoire, and defined a unique transdimer-specific peptide binding motif that was found to be distinct from those of HLA-DQ2 and HLA-DQ8. This motif predicts an array of peptides of islet autoantigens as candidate T cell epitopes, many of which selectively bind to the HLA transdimer, whereas others bind to both HLA-DQ8 and transdimer with similar affinity. Our findings provide a molecular basis for the association between HLA-DQ transdimers and T1D and set the stage for rational testing of potential diabetogenic peptide epitopes.

Antibacterial proteins are components of the innate immune system found in many organisms and produced by a variety of cell types. Human blood platelets contain a number of antibacterial proteins in their alpha-granules that are released upon thrombin activation. The present study was designed to purify these proteins obtained from human platelets and to characterize them chemically and biologically. Two antibacterial proteins were purified from platelet granules in a two-step protocol using cation exchange chromatography and continuous acid urea polyacrylamide gel electrophoresis and were designated thrombocidin (TC)-1 and TC-2. Characterization of these proteins using mass spectrometry and N-terminal sequencing revealed that TC-1 and TC-2 are variants of the CXC chemokines neutrophil-activating peptide-2 and connective tissue-activating peptide-III, respectively. TC-1 and TC-2 differ from these chemokines by a C-terminal truncation of 2 amino acids. Both TCs, but not neutrophil-activating peptide-2 and connective tissue-activating peptide-III, were bactericidal for Bacillus subtilis, Escherichia coli, Staphylococcus aureus, and Lactococcus lactis and fungicidal for Cryptococcus neoformans. Killing of B. subtilis by either TC appeared to be very rapid. Because TCs were unable to dissipate the membrane potential of L. lactis, the mechanism of TC-mediated killing most probably does not involve pore formation.

A novel strategy combining peptide fingerprinting of single neurons by matrix-assisted laser desorption ionization mass spectrometry, molecular cloning, peptide chemistry, and electrospray ionization mass spectrometry was used to study the intricate processing pattern of a preprohormone expressed in identified neurons, the neuroendocrine light yellow cells (LYCs) of the gastropod mollusc, Lymnaea stagnalis. The cDNA encoding the precursor, named prepro-LYCP (LYCPs, light yellow cell peptides), predicts a straightforward processing into three peptides, LYCP I, II, and III, at conventional dibasic processing sites flanking the peptide domains on the precursor. However, matrix-assisted laser desorption ionization mass spectrometry of single LYCs revealed trimmed variant peptides derived from LYCP I and II. The variants were much more abundant than the intact peptides, indicating that LYCP I and II serve as intermediates in a peptide-processing sequence. Using the molecular masses of the peptides as markers to guide their isolation by well established purification methods, the structural identities of the peptides could be confirmed by amino acid sequencing. Furthermore, matrix-assisted laser desorption ionization mass spectrometry could detect colocalization of a novel peptide with the LYCPs.