On the interaction between human IQGAP1 and actin

DM thanks the School of Biological Sciences, Queen’s University, Belfast for a summer studentship and EH thanks the Department of Employment and Learning, Northern Ireland for a postgraduate studentship. The work was funded in part by grants from the BBSRC (BB/D000394/1 To DJT) and by the Wellcome Trust [grant number GR06281AIA] which funded the purchase of the QStar XL mass spectrometer at the BBSRC Mass Spectrometry and Proteomics Facility, University of St Andrews and funded SLS.IQGAPs are eukaryotic proteins which integrate signals from various sources and pass these on the cytoskeleton. Understanding how they do this requires information on the interfaces between the proteins. Here, it is shown that the calponin homology domain of human IQGAP1 (CHD1) can be crosslinked with α-actin. The stoichiometry of the interaction was 1:1. A molecular model was built of the complex and associated bioinformatics analyses predicted that the interaction is likely to involve an electrostatic interaction between Lys-240 of α-actin and Glu-30 of CHD1. These residues are predicted to be accessible and are not involved in many intra-protein interactions; they are thus available for interaction with binding partners. They are both located in regions of the proteins which are predicted to be flexible and disordered; interactions between signalling molecules often involve flexible, disordered regions. The predicted binding region in CHD1 is well conserved in many eukaryotic IQGAP-like proteins. In some cases (e.g Dictyostelium discoideum and Saccharomyces cerevisiae) protein sequence conservation is weak, but molecular modelling reveals that a region of charged, polar residues in a flexible N-terminus is structurally well conserved. Therefore we conclude that the calponin homology domains of IQGAP1-like proteins interact initially through the electrostatic interaction identified here and that there may be subsequent conformational changes to form the final complex.PostprintPeer reviewe

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

Synthesis of Next Generation Maleimide Radical Labels

This work was funded by EPSRC, Grant Number EP/LO22044/1, a Royal Society University Research Fellowship to JEL , Royal Society Research Grant RG120645 and support from Active Spectrum Inc.The synthesis and characterization of four new nitroxide-radical-containing next-generation maleimides are presented. Each new label has a single leaving group which is either a phenoxyl or bromide. The linker between the maleimide and the nitroxide-containing framework is either a racemic mixture of a short chain or an achiral longer chain. These molecules have been designed to site-specifically label vicinal cysteines in proteins for magnetic resonance studies. The characterization of the final products includes crystallography and the labeling of sperm whale myoglobin protein.PostprintPeer reviewe

Spinal muscular atrophy patient iPSC-derived motor neurons have reduced expression of proteins important in neuronal development

This work was supported by The RJAH Institute of Orthopaedics, UK (H.F.), The SMA Trust, UK (H.F.), Cedars-Sinai Institutional startup funds (D.S), California Institute for Regenerative Medicine Grant RT-02040 (D.S.), National Center for Advancing Translational Sciences (NCATS), Grant UL1TR000124 (D.S.), and the Wellcome Trust [grant number 094476/Z/10/Z] which funded the purchase of the TripleTOF 5600 mass spectrometer at the BSRC Mass Spectrometry and Proteomics Facility, University of St Andrews (S.S. and C.B.). D.S. is also supported by funds from National Institute of Health (NINDS) grant U54NS091046. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Spinal muscular atrophy (SMA) is an inherited neuromuscular disease primarily characterized by degeneration of spinal motor neurons, and caused by reduced levels of the SMN protein. Previous studies to understand the proteomic consequences of reduced SMN have mostly utilized patient fibroblasts and animal models. We have derived human motor neurons from type I SMA and healthy controls by creating their induced pluripotent stem cells (iPSCs). Quantitative mass spectrometry of these cells revealed increased expression of 63 proteins in control motor neurons compared to respective fibroblasts, whereas 30 proteins were increased in SMA motor neurons versus their fibroblasts. Notably, UBA1 was significantly decreased in SMA motor neurons, supporting evidence for ubiquitin pathway defects. Subcellular distribution of UBA1 was predominantly cytoplasmic in SMA motor neurons in contrast to nuclear in control motor neurons; suggestive of neurodevelopmental abnormalities. Many of the proteins that were decreased in SMA motor neurons, including beta III-tubulin and UCHL1, were associated with neurodevelopment and differentiation. These neuron-specific consequences of SMN depletion were not evident in fibroblasts, highlighting the importance of iPSC technology. The proteomic profiles identified here provide a useful resource to explore the molecular consequences of reduced SMN in motor neurons, and for the identification of novel biomarker and therapeutic targets for SMA.Publisher PDFPeer reviewe

Investigation of the blood proteome in response to spinal cord injury in rodent models (dataset)

The dataset has been deposited on ProteomeXchange, but is not yet publicly released

Investigation of the blood proteome in response to spinal cord injury in rodent models (dataset)

The dataset has been deposited on ProteomeXchange, but is not yet publicly released