Tumour-associated antigenic peptides are present in the HLA class I ligandome of cancer cell line derived extracellular vesicles

Funding: Breast Cancer Now (Grant Number(s): 2018JulPR1086), Wellcome Trust (GrantNumber(s): 105621/Z/14/Z), Melville Charitable Trust.The recent success of monoclonal antibody checkpoint inhibitor therapies that enhance the ability of CD8+ T cells to detect cancer-related antigenic peptides has refocused the need to fully understand the repertoire of peptides being presented to the immune system. Whilst the peptide ligandome presented by cell surface human leucocyte antigen class I (HLA-I) molecules on cancer cells has been studied extensively, the ligandome of extracellular vesicles (EVs) remains poorly defined. Here we report the HLA-I ligandome of both the cell surface and EVs from eight breast cancer cell lines (MCF7, MDA-MB-231, MDA-MB-361, MDA-MB-415, MDA-MB-453, HCC 1806, HCC 1395, and HCC 1954), and additionally the melanoma cell line ESTDAB-056 and the multiple myeloma line RPMI 8226. Utilising HLA-I immunoisolation and mass spectrometry, we detected a total of 6574 peptides from the cell surface and 2461 peptides from the EVs of the cell lines studied. Within the EV HLA-I ligandome, we identified 150 peptides derived from tumour associated antigenic proteins, of which 19 peptides have been shown to elicit T cell responses in previous studies. Our data thus shows the prevalence of clinically relevant tumour-associated antigenic peptides in the HLA-I ligandome presented on EV.Publisher PDFPeer reviewe

Quantitative proteomic changes in LPS-activated monocyte-derived dendritic cells : a SWATH-MS study

We would like to thank Fiona Cooke for her help with collection of blood samples. We wish to thank the Wellcome Trust for funding the purchase of the TripleTOF 5600+ mass spectrometer (grant number 094476/Z/10/Z) and their Institutional Strategic Support Fund (grant number 097831/Z/11/Z) for funding a PhD studentship (to D.W.-M.). This work was also supported by Arthritis Research UK (grant number 21261).Dendritic cells are key immune cells that respond to pathogens and co-ordinate many innate and adaptive immune responses. Quantitative mass spectrometry using Sequential Window Acquisition of all THeoretical fragment-ion spectra-Mass Spectrometry (SWATH-MS) was performed here to determine the global alterations in monocyte-derived dendritic cells (moDCs) in response to stimulation with lipopolysaccharide (LPS). A moDC library of 4,666 proteins was generated and proteins were quantified at 0, 6 and 24 h post-LPS stimulation using SWATH-MS. At 6 h and 24 h post-LPS exposure, the relative abundance of 227 and 282 proteins was statistically significantly altered (p-value≤0.05), respectively. Functional annotation of proteins exhibiting significant changes in expression between the various time points led to the identification of clusters of proteins implicated in distinct cellular processes including interferon and interleukin signalling, endocytosis, the ER-phagosome pathway and antigen-presentation. Major histocompatibility complex (MHC) class I proteins were highly upregulated at 24 h, in SWATH-MS, whilst MHC class II proteins exhibited comparatively less change over this period. This study provides new detailed insight into the global proteomic changes that occur in moDCs during antigen processing and presentation and further demonstrates the potential of SWATH-MS for the quantitative study of proteins involved in cellular processes.Publisher PDFPeer reviewe

Characterization of the fast and promiscuous macrocyclase from plant PCY1 enables the use of simple substrates

H.L. is funded by the George and Stella Lee Scholarship and EPSRC. This project was funded by the European Research Council project 339367 NCB-TNT and by the BBSRC (J.H.N.). E.S.M. and M.A. are funded by EPSRC. S.A.S. is funded by BSRC mass spec facility.Cyclic ribosomally derived peptides possess diverse bioactivities and are currently of major interest in drug development. However, it can be chemically challenging to synthesize these molecules, hindering the diversification and testing of cyclic peptide leads. Enzymes used in vitro offer a solution to this; however peptide macrocyclization remains the bottleneck. PCY1, involved in the biosynthesis of plant orbitides, belongs to the class of prolyl oligopeptidases and natively displays substrate promiscuity. PCY1 is a promising candidate for in vitro utilization, but its substrates require an 11 to 16 residue C-terminal recognition tail. We have characterized PCY1 both kinetically and structurally with multiple substrate complexes revealing the molecular basis of recognition and catalysis. Using these insights, we have identified a three residue C-terminal extension that replaces the natural recognition tail permitting PCY1 to operate on synthetic substrates. We demonstrate that PCY1 can macrocyclize a variety of substrates with this short tail, including unnatural amino acids and nonamino acids, highlighting PCY1’s potential in biocatalysis.PostprintPeer reviewe

Structure, dynamics, and molecular inhibition of the Staphylococcus aureus m1A22-tRNA methyltransferase TrmK

This work was supported by a Wellcome Trust Seed Award in Science [208980/Z/17/Z] to RGdS; a University of St Andrews/Scottish Funding Council St Andrews Restarting Research Fund to RGdS; and a Wellcome Trust Institutional Strategic Support Fund [204821/Z/16/Z] to the University of St Andrews. ES is the recipient of a Cunningham Trust PhD studentship (PhD-CT-18-41).The enzyme m1A22-tRNA methyltransferase (TrmK) catalyzes the transfer of a methyl group to the N1 of adenine 22 in bacterial tRNAs. TrmK is essential for Staphylococcus aureus survival during infection but has no homolog in mammals, making it a promising target for antibiotic development. Here, we characterize the structure and function of S. aureus TrmK (SaTrmK) using X-ray crystallography, binding assays, and molecular dynamics simulations. We report crystal structures for the SaTrmK apoenzyme as well as in complexes with methyl donor SAM and co-product product SAH. Isothermal titration calorimetry showed that SAM binds to the enzyme with favorable but modest enthalpic and entropic contributions, whereas SAH binding leads to an entropic penalty compensated for by a large favorable enthalpic contribution. Molecular dynamics simulations point to specific motions of the C-terminal domain being altered by SAM binding, which might have implications for tRNA recruitment. In addition, activity assays for SaTrmK-catalyzed methylation of A22 mutants of tRNALeu demonstrate that the adenine at position 22 is absolutely essential. In silico screening of compounds suggested the multifunctional organic toxin plumbagin as a potential inhibitor of TrmK, which was confirmed by activity measurements. Furthermore, LC-MS data indicated the protein was covalently modified by one equivalent of the inhibitor, and proteolytic digestion coupled with LC-MS identified Cys92 in the vicinity of the SAM-binding site as the sole residue modified. These results identify a cryptic binding pocket of SaTrmK, laying a foundation for future structure-based drug discovery.Publisher PDFPeer reviewe

Identification of putative adhesins and carbohydrate ligands of <em>Lactobacillus paracasei</em> using a combinatorial <em>in silico</em> and glycomics microarray profiling approach

Commensal bacteria must colonize host mucosal surfaces to exert health-promoting properties, and bind to gastrointestinal tract (GIT) mucins via their cell surface adhesins. Considerable effort has been directed towards discovery of pathogen adhesins and their ligands to develop anti-infective strategies; however, little is known about the lectin-like adhesins and associated carbohydrate ligands in commensals. In this study, an in silico approach was used to detect surface exposed adhesins in the human commensal Lactobacillus paracasei subsp. paracasei, a promising probiotic commonly used in dairy product fermentation that presents anti-microbial activity. Of the 13 adhesin candidates, 3 sortase-dependent pili clusters were identified in this strain and expression of the adhesin candidate genes was confirmed in vitro. Mass spectrometry analysis confirmed the presence of surface adhesin elongation factor Tu and the chaperonin GroEL, but not pili expression. Whole cells were subsequently incubated on microarrays featuring a panel of GIT mucins from nine different mammalian species and two human-derived cell lines and a library of carbohydrate structures. Binding profiles were compared to those of two known pili-producing lactobacilli, L. johnsonii and L. rhamnosus and all Lactobacillus species displayed overlapping but distinct signatures, which may indicate different abilities for regiospecific GIT colonization. In addition, L. paracasei whole cells favoured binding to α-(2 → 3)-linked sialic acid and α-(1 → 2)-linked fucose-containing carbohydrate structures including blood groups A, B and O and Lewis antigens x, y and b. This study furthers our understanding of host-commensal cross-talk by identifying potential adhesins and specific GIT mucin and carbohydrate ligands and provides insight into the selection of colonization sites by commensals in the GIT

Quantitative proteomic changes in LPS-activated monocyte-derived dendritic cells:a SWATH-MS study

Dendritic cells are key immune cells that respond to pathogens and co-ordinate many innate and adaptive immune responses. Quantitative mass spectrometry using Sequential Window Acquisition of all THeoretical fragment-ion spectra-Mass Spectrometry (SWATH-MS) was performed here to determine the global alterations in monocyte-derived dendritic cells (moDCs) in response to stimulation with lipopolysaccharide (LPS). A moDC library of 4,666 proteins was generated and proteins were quantified at 0, 6 and 24 h post-LPS stimulation using SWATH-MS. At 6 h and 24 h post-LPS exposure, the relative abundance of 227 and 282 proteins was statistically significantly altered (p-value≤0.05), respectively. Functional annotation of proteins exhibiting significant changes in expression between the various time points led to the identification of clusters of proteins implicated in distinct cellular processes including interferon and interleukin signalling, endocytosis, the ER-phagosome pathway and antigen-presentation. Major histocompatibility complex (MHC) class I proteins were highly upregulated at 24 h, in SWATH-MS, whilst MHC class II proteins exhibited comparatively less change over this period. This study provides new detailed insight into the global proteomic changes that occur in moDCs during antigen processing and presentation and further demonstrates the potential of SWATH-MS for the quantitative study of proteins involved in cellular processes

Lamin A/C dysregulation contributes to cardiac pathology in a mouse model of severe spinal muscular atrophy"

Cardiac pathology is emerging as a prominent systemic feature of spinal muscular atrophy (SMA), but little is known about the underlying molecular pathways. Using quantitative proteomics analysis, we demonstrate widespread molecular defects in heart tissue from the Taiwanese mouse model of severe SMA. We identify increased levels of lamin A/C as a robust molecular phenotype in the heart of SMA mice, and show that lamin A/C dysregulation is also apparent in SMA patient fibroblast cells and other tissues from SMA mice. Lamin A/C expression was regulated in-vitro by knockdown of the E1 ubiquitination factor UBA1, a key downstream mediator of SMN-dependent disease pathways, converging on β-catenin signalling. Increased levels of lamin A are known to increase the rigidity of nuclei, inevitably disrupting contractile activity in cardiomyocytes. The increased lamin A/C levels in the hearts of SMA mice therefore provide a likely mechanism explaining morphological and functional cardiac defects, leading to blood pooling. Therapeutic strategies directed at lamin A/C may therefore offer a new approach to target cardiac pathology in SMA