Project description:The cell bodies of hypothalamic magnocellular neurones (MCNs) are confined to the hypothalamic supraoptic nucleus (SON) whereas their axons project to the anatomically discrete posterior pituitary gland (PP). We have taken advantage of this unique anatomical structure to document proteome and phosphoproteome dynamics in neuronal cell bodies and axonal terminals in response to neuronal activation induced by water deprivation (WD). We have found that proteome and phosphoproteome responses to WD are very different between cell bodies and axonal terminals, indicating the need of each cell domain to differentially adapt in response to stimuli. Whilst changes in the proteome and phosphoproteome in the cell body are involved in protein synthesis and cytoskeleton reorganisation, respectively, in the axonal terminals they regulate synaptic vesicle cycle and secretion. Further, by comparison with transcriptome data, we have identified peptides that are not synthesised in the SON, but are present as a consequence of afferent delivery.
Project description:Protein trafficking requires coat complexes that couple recognition of sorting motifs in transmembrane cargos with biogenesis of transport carriers. The mechanisms of cargo transport through the endosomal network are poorly understood. Here, we identify a sorting motif for endosomal recycling of cargos including the cation-independent mannose-6-phosphate receptor and semaphorin 4C by the membrane tubulating BAR domain-containing sorting nexins SNX5 and SNX6. Crystal structures establish that this motif folds into a β-hairpin that binds a site in the SNX5/SNX6 phox homology domains. Over sixty cargos share this motif and require SNX5/SNX6 for their recycling. These include cargos involved in neuronal migration and a Drosophila snx6 mutant displays defects in axonal guidance. These studies identify a sorting motif and provide molecular insight into an evolutionary conserved coat complex, the ‘Endosomal SNX-BAR sorting complex for promoting exit 1’ (ESCPE-1), which couples sorting motif recognition to BAR domain-mediated biogenesis of cargo-enriched tubulo-vesicular transport carriers.
Project description:Regular blood transfusion is the cornerstone of care for patients with red blood cell (RBC) disorders such as thalassemia or sickle cell disease. With repeated transfusion, alloimmunisation often occurs due to incompatibility at the level of minor blood group antigens. We use CRISPR-mediated genome editing of an immortalised human erythroblast cell line (BEL-A) to generate multiple enucleation competent cell lines deficient in individual blood groups. Edits are combined to generate a single cell line deficient in multiple antigens responsible for the most common transfusion incompatibilities: ABO (Bombay phenotype), Rh (Rhnull), Kell (K0), Duffy (Duffynull), GPB (S- s- U-). These cells can be differentiated to generate deformable reticulocytes, illustrating the capacity for coexistence of multiple rare blood group antigen null phenotypes. This study provides the first proof-of-principle demonstration of combinatorial CRISPR-mediated blood group gene editing to generate customisable or multi-compatible RBCs for diagnostic reagents or recipients with complicated matching requirements.
Project description:Obesity and its co-morbidities, such as diabetes and hypertension, can significantly reduce a person’s quality of life and place huge pressure on healthcare resources. When we eat a meal our gut and brain release hormones to control the amount of food and fluid we ingest to prevent overeating. One of these hormones is called glucagon-like peptide 1 (GLP-1) and is released from intestinal cells in response to food intake, but also produced and released in the brain. Drug analogues of GLP-1 are already in use in the clinic to treat both diabetes and obesity. The aim of this work was to obtain fundamental knowledge about a GLP-1 receptor population in nerve terminals of the posterior pituitary gland. We have investigated the pharmacological actions of GLP-1 using a selective receptor agonist called liraglutide, a drug that is approved for diabetes and obesity treatment in humans. Our work has focussed on the phosphoproteome of the neurointermediate lobe (posterior pituitary + intermediate lobe) of the rat pituitary gland 30 minutes after intraperitoneal injection of liraglutide (100 µg/kg) compared to vehicle controls (n = 6 animals per group). New understanding of this GLP-1 receptor population is essential for our knowledge of current treatments of diabetes and obesity that use stable peptide analogues in humans.
Project description:Bacterial and viral infections of the placenta are associated with inflammation and adverse pregnancy outcomes. Hofbauer cells (HBCs) are specialised fetal-origin macrophages in the placental villi and are proposed to protect the fetus from vertical transmission of pathogens; however, they are poorly understood. Here, we have performed quantitative proteomics on term HBCs under resting conditions and following exposure to bacterial and viral pathogen associated molecular patterns (PAMPs), and investigated the contribution of fetal sex to these responses. Resting HBCs expressed a plethora of proteins pertinent to macrophage function, including chemokines, cytokines, Toll-like receptors, and classical and non-classical major histocompatibility complex class I and II molecules. HBCs mounted divergent responses to bacterial versus viral PAMPs but exhibited protein expression changes suggestive of a switch towards a more pro-inflammatory phenotype. A comparison between male and female HBCs, showed that the latter mounted a much stronger and wider response. Sexual dimorphism in HBCs was primarily associated with lipid metabolism in males and cytoskeleton organisation in females. We provide a novel and comprehensive understanding regarding the phenotype of term placental macrophages and their sex-dependent responses to infectious triggers.
Project description:Protein expression by E. coli 26561 during the late-exponential phase of cultures under anaerobic conditions was examined. E. coli 26561 is a multidrug resistant (MDR) and shows an unusual hyper-mucoviscous phenotype. Resistance includes ESBL (CTX-M-14) and proteome was determined with and without exposure to sub-MIC concentrations of the 3rd generation cephalosporin ceftazidime. Ceftazidime exposure was at two sub-MIC levels, specifically 0.25x MIC (samples 5-7), 0.5x MIC (samples 8 - 10); samples 1-4 provided the unexposed Control. Both whole and phospho-enriched fractions for each sample were analysed. Quantification of peptides was assessed using 10-plex TMT labelling in conjunction with an Orbitrap Fusion Tribrid. Raw data produced by the Orbitrap were processed using Max Quant 1.5.4.7 using the included Andromeda search engine. Peptides were searched against our own database of E. coli 26561 proteins which was produced from a hybrid assembly of our reads obtained from MiSeq and PacBio sequencing platforms.
Project description:Retromer controls cellular homeostasis through regulating integral membrane protein sorting and transport and by controlling late-stage maturation of the endo-lysosomal network. Retromer dysfunction, which is linked to neurodegenerative disorders including Parkinson’s and Alzheimer’s diseases, manifests in complex cellular phenotypes, though the precise nature of this dysfunction, and its relation to neurodegeneration, remain unclear. Here, we perform the first integrated multiomics approach to provide precise insight into the impact of Retromer dysfunction on endo-lysosomal health and homeostasis within a human neuroglioma cell model. We quantify profound changes to the lysosomal proteome, indicative of broad lysosomal dysfunction and inefficient autophagic lysosome reformation, coupled with a reconfigured cell surface proteome and secretome reflective of increased lysosomal exocytosis. Through this global proteomic approach and parallel transcriptomic analysis, we provide an unprecedented integrated view of Retromer function in regulating lysosomal homeostasis and emphasise its role in neuroprotection.
Project description:Determination of the effect of the generic tyrosine kinase degrader TL12-186, and the BTK degraders DD-04-15 and DD-03-171 on human platelet protein content and function.
Project description:We are here presenting a new paracrine induction of RNA granules by viruses. Infection by viruses imposes major stress on the host cell. In response to this stress, infected cells can induce several defence mechanisms, which include the activation of stress response pathways and the innate immune response. These often result in an inhibition of translation culminating in the assembly of cytoplasmic granules called stress granules (SGs). SGs assembly follows from liquid phase separation of aggregation-prone proteins such G3BP1 and TIA-1, leading to the sequestration of mRNAs. Because this threatens viral gene expression, viruses need to evade these stress response pathways to propagate. Using feline calicivirus (FCV), surrogate for norovirus, the main virus responsible for gastroenteritis outbreaks worldwide, we previously showed that FCV impairs SGs assembly by cleaving the scaffold protein G3BP1. Interestingly, we observed that uninfected bystander cells assembled G3BP1 granules, suggesting a paracrine response trigged by the infection. We now present evidence that virus-free supernatant generated from infected cells can induce the formation of RNA granules. We have characterised the dynamic of the granules assembly via confocal microscopy. Moreover, we provide an understanding of paracrine granules function and specificity through their affinity purification followed by proteomics and RNAseq analysis of their proteins and mRNAs content. This helps to define rules of assembly and novel functions for paracrine granules highlighting fundamental differences with canonical stress granules.