Biology subjects

Davis, S.

Publications and source records attributed to Davis, S..

At least 55 records · Page 3Linked to original sources

bioRxiv2022

SARS-CoV-2 mutations affect proteasome processing to alter CD8+ T cell responses

Viral CD8+ epitopes are generated by the cellular turnover of viral proteins, predominantly by the proteasome. Mutations located within viral epitopes can result in escape from memory T cells but the contribution of mutations in flanking regions of epitopes in SARS-CoV-2 has not been investigated. Focusing on two of the most dominant SARS-CoV-2 nucleoprotein CD8+ epitopes, we identified mutations in epitope flanking regions and investigated the contribution of these mutations to antigen processing and T cell activation using SARS-CoV-2 nucleoprotein transduced B cell lines and in vitro proteasomal processing of peptides. We found that decreased NP9-17-B*27:05 CD8+ T cell responses to the NP-Q7K mutation correlated with lower epitope surface expression, likely due to a lack of efficient epitope production by the proteasome, suggesting immune escape caused by this mutation. In contrast, NP-P6L and NP-D103N/Y mutations flanking the NP9-17-B*27:05 and NP105-113-B*07:02 epitopes, respectively, increased CD8+ T cell responses associated with enhanced epitope production by the proteasome. Our results provide evidence that SARS-CoV-2 mutations outside the epitope could have a significant impact on antigen processing and presentation, thereby contributing to escape from immunodominant T cell responses. Alternatively, mutations could enhance antigen processing and efficacy of T cell recognition, opening new avenues for improving future vaccine designs. One Sentence SummaryNatural mutations in the flanking regions of known immunodominant SARS-CoV-2 nucleoprotein epitopes can decrease CD8+ T cell responses leading to partial escape.

immunology↗

bioRxiv2022

Deep topographic proteomics of a human brain tumour

The spatial organisation of cellular protein expression profiles within tissue determines cellular function and is key to understanding disease pathology. To define molecular phenotypes in the spatial context of tissue, there is a need for unbiased, quantitative technology capable of mapping proteomes within tissue structures. Here, we present a workflow for spatially-resolved, quantitative proteomics of tissue that generates maps of protein abundance across tissue slices derived from a human atypical teratoid-rhabdoid tumour (AT/RT). We employ spatially-aware algorithms that do not require prior knowledge of the fine tissue structure to detect proteins and pathways with spatial abundance patterns. We identified PYGL, ASPH and CD45 as spatial markers for tumour boundary and reveal immune response-driven, spatially-organised protein networks of the extracellular tumour matrix. Overall, this work informs on methods for spatially resolved deep proteo-phenotyping of tissue heterogeneity, to push the boundaries of understanding tissue biology and pathology at the molecular level.

cancer biology↗

bioRxiv2022

Dynamic single-cell RNA sequencing reveals BCG vaccination curtails SARS-CoV-2 induced disease severity and lung inflammation

COVID-19 continues to exact a toll on human health despite the availability of several vaccines. Bacillus Calmette Guerin (BCG) has been shown to confer heterologous immune protection against viral infections including COVID-19 and has been proposed as vaccine against SARS-CoV-2 (SCV2). Here we tested intravenous BCG vaccination against COVID-19 using the golden Syrian hamster model together with immune profiling and single cell RNA sequencing (scRNAseq). We observed that BCG reduced both lung SCV2 viral load and bronchopneumonia. This was accompanied by an increase in lung alveolar macrophages, a reversal of SCV2-mediated T cell lymphopenia, and reduced lung granulocytes. Single cell transcriptome profiling showed that BCG uniquely recruits immunoglobulin-producing plasma cells to the lung suggesting accelerated antibody production. BCG vaccination also recruited elevated levels of Th1, Th17, Treg, CTLs, and Tmem cells, and differentially expressed gene (DEG) analysis showed a transcriptional shift away from exhaustion markers and towards antigen presentation and repair. Similarly, BCG enhanced lung recruitment of alveolar macrophages and reduced key interstitial macrophage subsets, with both cell-types also showing reduced IFN-associated gene expression. Our observations indicate that BCG vaccination protects against SCV2 immunopathology by promoting early lung immunoglobulin production and immunotolerizing transcriptional patterns among key myeloid and lymphoid populations.

immunology↗

bioRxiv2022

ABPP-HT* - Deep meets fast for activity-based profiling of deubiquitylating enzymes using advanced DIA and DDA mass spectrometry methods

Activity-based protein profiling (ABPP) uses a combination of activity-based chemical probes with mass spectrometry (MS) to selectively characterise a particular enzyme or enzyme class. ABPP has proven invaluable for profiling enzymatic inhibitors in drug discovery. When applied to cell extracts and cells, challenging the ABP-enzyme complex formation with a small molecule can simultaneously inform on potency, selectivity, reversibility/binding affinity, permeability, and stability. ABPP can also be applied to pharmacodynamic studies to inform on cellular target engagement within specific organs when applied to in vivo models. Recently, we established separate high depth and high throughput ABPP (ABPP-HT) protocols for the profiling of deubiquitylating enzymes (DUBs). However, the combination of the two, deep and fast, in one method has been elusive. To further increase the sensitivity of the current ABPP-HT workflow we implemented state-of-the-art data-independent acquisition (DIA) and data-dependent acquisition (DDA) MS analysis tools. Hereby, we describe an improved methodology, ABPP-HT* (enhanced high-throughput-compatible activity-based protein profiling), that in combination with DIA MS methods allowed for the consistent profiling of 35-40 DUBs and provided a reduced number of missing values, whilst maintaining a throughput of 100 samples per day.

biochemistry↗

bioRxiv2021

Multiple metals influence distinct properties of the Arabidopsis circadian clock

Circadian rhythms coordinate endogenous events with external signals, and are essential to biological function. When environmental contaminants affect these rhythms, the organism may experience fitness consequences such as reduced growth or increased susceptibility to pathogens. In their natural environment plants may be exposed to a wide range of industrial and agricultural pollutants. Here, we investigate how the addition of various metal salts to the environment can impact plant-circadian rhythms, via the promoter:luciferase system. The consequences of these environmental changes were found to be varied and complex. Therefore, in addition to Fourier-based analyses, we apply novel wavelet-based spectral hypothesis testing and clustering methodologies to organize and understand the data. We are able to classify broad sets of responses to environmental contaminants, including pollutants which increase, or decrease, the period, or which induce a lack of precision or disrupt any meaningful periodicity. The methods are general, and may be applied to discover common responses and hidden structures within a wide range of biological time series data.

plant biology↗

bioRxiv2021

Non-additive QTL mapping of lactation traits in 124,000 sequence-imputed cattle reveals novel recessive loci

Deleterious recessive conditions have primarily been studied in a Mendelian disease context. Recently, several large effect, deleterious recessive mutations were discovered via non-additive GWAS of quantitative growth and developmental traits in cattle. This showed quantitative traits can be used as proxies of genetic disorders if they are indicative of whole animal health status and susceptible to underlying genetic conditions. Lactation traits might also reflect genetic disorders in cattle, given the increased energy demands of lactation and the substantial stresses imposed on the animal. Here, we report a screen of over 124,000 cows for recessive effects based on lactation traits. We discovered novel loci associated with five large recessive impacts on milk yield traits represented by missense variants (DOCK8, IL4R, KIAA0556, and SLC25A4) or premature stop variants (ITGAL, LRCH4, and RBM34) as candidate causal mutations. On milk composition traits, we identified several small effect dominance contributions to previously reported additive QTL. In contrasting analyses of milk yield and milk composition phenotypes, we note differing genetic architectures. Milk yield phenotypes presented lower heritabilities and fewer additive QTL, but higher non-additive genetic variance and a higher proportion of loci exhibiting dominance compared to milk composition phenotypes. Large-effect recessive QTL are segregating at surprisingly high frequencies in cattle. We speculate that the differences in genetic architecture between milk yield and milk composition phenotypes derive from underlying dissimilarities in the cellular and molecular representation of these traits. Lactation yields may act as a better proxy than milk composition traits for a wide range of underlying biological disorders affecting animal fitness

genetics↗

bioRxiv2021

Network analysis of large phospho-signalling datasets: application to Plasmodium-erythrocyte interactions

Phosphorylation based signalling is a complicated and intertwined series of pathways critical to all domains of life. This interconnectivity, though essential to life, makes understanding and decoding the interactions difficult. Large datasets of phosphorylation interactions through the activity of kinases on their numerous effectors are now being generated, however interpretation of the network environment remains challenging. In humans, many phosphorylation interactions have been identified across published works to form the known phosphorylation interaction network. We overlayed phosphorylation datasets onto this network which provided information to each of the connections. To analyse the datasets now mapped into a network, we designed a pathway analysis that uses random walks to identify chains of phosphorylation events occurring much more or much less frequently than expected. This analysis highlights pathways of phosphorylation that work synergistically, providing a rapid interpretation of the most critical pathways in a given dataset. Here we used datasets of human red blood cells infected with the notable stages of Plasmodium falciparum asexual development. The analysis identified several known signalling interactions, and additional interactions which could form the basis of numerous future studies. The network analysis designed here is widely applicable to any comparative phosphorylation dataset across infection and disease and can provide a rapid and reliable analysis to guide validation studies.

microbiology↗

bioRxiv2021

Fibrillar Aβ causes profound microglial metabolic perturbations in a novel APP knock-in mouse model

Microglial dysfunction is believed to play a pathogenic role in Alzheimers disease (AD). Here, we characterize the amyloid-{beta} related pathology and microglial responses in an engineered APP knock-in mouse model of familial AD. This model recapitulates key pathological features of AD such as a progressive accumulation of parenchymal amyloid plaques and vascular amyloid deposits, altered glial responses and neurodegeneration. Leveraging multi-omics approaches, we found lipid accumulation and an exacerbated disease-associated transcriptomic response in methoxy-X04-positive, phagocytic microglia. Together, these findings highlight the potential of this novel, open-access mouse model to investigate AD pathogenesis and demonstrate that fibrillar A{beta} triggers lipid dysregulation and immuno-metabolic perturbations in phagocytic microglia. HighlightsO_LINovel open-access APP KI mouse model shows salient AD pathological features C_LIO_LIDeep phenotyping of sorted microglia reveals profound lipidomic perturbations in line with Alois Alzheimers original descriptions of glial adipose inclusions C_LIO_LIImmunometabolic perturbations are exacerbated in microglia accumulating fibrillar A{beta} C_LI

neuroscience↗

bioRxiv2021

Age-dependent changes in protein incorporation into collagen-rich tissues of mice by in vivo pulsed SILAC labelling

Collagen-rich tissues have poor reparative capacity that is further impaired with age, predisposing to common age-related disorders such as osteoporosis and osteoarthritis. We used in vivo pulsed SILAC labelling to quantify new protein incorporation into cartilage, bone, skin and plasma of mice across the life course. We report highly dynamic matrisome turnover in bone and cartilage during skeletal maturation, which was markedly reduced after skeletal maturity. Comparing young adult with older adult mice, new protein incorporation was reduced in all tissues. STRING clustering revealed epigenetic modulation across all tissues, a decline in chondroprotective growth factors such as FGF2 and TGFb in cartilage, and clusters indicating mitochondrial dysregulation and reduced collagen synthesis in bone. Several of these pathways have been associated with age-related disease. Fewer changes were observed for skin and plasma. This methodology provides dynamic protein data at a tissue level, uncovering age-related molecular changes that may predispose to disease.

developmental biology↗

bioRxiv2020

Selective human tau protein expression in different clock circuits of the Drosophila brain disrupts different aspects of sleep and circadian rhythms

Circadian behavioural deficits, such as increased daytime naps and reduced night-time sleep, are common in Alzheimers disease and other tauopathies. But it has remained unclear whether these circadian abnormalities arise from tau pathology in either the master pacemaker or downstream neurons. Here we study this question by selectively expressing different human tau proteins in specific Drosophila brain circuits and monitoring locomotor activity under light-dark (LD) and in "free-running" dark-dark (DD) conditions. We show that expressing human tau proteins in the fly brain recapitulates faithfully several behavioural changes found in tauopathies. We identify discrete neuronal subpopulations within the clock network as the primary target of distinct circadian behavioural disturbances in different environmental conditions. Specifically, we show that the PDF-positive pacemaker neurons are the main site for night-activity gain and -sleep loss, whereas the non-PDF clock-neurons are the main site of reduced intrinsic behavioural rhythmicity. Bioluminescence measurements revealed that the molecular clock is intact despite the behavioural arrhythmia. Our results establish that dysfunction in both the central clock- and afferent clock-neurons jointly contribute to the circadian locomotor activity rhythm disruption in Drosophila expressing human tau. Significance StatementThis study directly links in vivo human tau protein expression in region-specific Drosophila clock-neurons with the resulting sleep and circadian rhythm deficits to extract new knowledge of how Alzheimers disease and other tauopathies perturb the balance of activity and sleep. We anticipate that this novel approach will provide a useful general template for other studies of neurodegeneration in model organisms, seeking to dissect the impact of neurodegenerative disease on circadian behaviour, and further deepening our understanding of how the clock-neuron network works.

neuroscience↗

bioRxiv2020

T cells exhibit unexpectedly low discriminatory power and can respond to ultra-low affinity peptide-MHC ligands

T cells use their T cell receptors (TCRs) to discriminate between peptide MHC (pMHC) ligands that bind with different affinities but precisely how different remains controversial. This is partly because the affinities of physiologically relevant interactions are often too weak to measure. Here, we introduce a surface plasmon resonance protocol to measure ultra-low TCR/pMHC affinities (KD ~ 1000 M). Using naive, memory, and blasted human CD8+ T cells we find that their discrimination power is unexpectedly low, in that they require a large >100-fold decrease in affinity to abolish responses. Interestingly, the discrimination power reduces further when antigen is presented in isolation on artificial surfaces but can be partially restored by adding ligands to CD2 or LFA-1. We were able to fit the kinetic proof-reading model to our data, yielding the first estimates for both the time delay (2.8 s) and number of biochemical steps (2.67). The fractional number of steps suggest that one of the proof-reading steps is not easily reversible.

immunology↗

bioRxiv2020

The urban-adapted underground mosquito, Culex molestus, maintains exogenously influenced circadian rhythms despite an absence of photoperiodically induced dormancy

In temperate climates, the mosquito Culex molestus lives almost exclusively in urban underground locations such as flooded basements, sewer systems and subway tunnels. Unlike most other mosquito taxa found at higher latitudes, Cx. molestus remains active and continues to breed throughout the winter. This is attributable to year-round above freezing temperatures in its preferred underground habitats combined with an inability to enter a state of arrested development ( diapause) in response to shortening photoperiods in autumn. Prior studies have shown that the genes associated with circadian rhythms (i.e. clock genes) also influence the photoperiodic induction of diapause in the closely related mosquito, Cx. pipiens. These results suggest that molecular changes in one or more clock genes could contribute to the absence of photoperiodically induced diapause in Cx. molestus. As Cx. molestus predominantly inhabits underground locations generally devoid of a predictable photoperiod, such mutations may not be removed by purifying selection as they would have minimal fitness consequences. To examine the possibility that Cx. molestus- specific genetic changes in one or more clock genes correlate with its inability to enter a photoperiodically induced dormant state, we first used genomic data to search for inactivating mutations or other structural variants in genes known to influence circadian rhythms in Diptera ( flies). We further investigated non-synonymous, derived genetic divergence in this same class of genes. Next, we generated transcriptome data from multiple life-stages of Cx. molestus to survey binary expression of annotated clock genes throughout this mosquitos lifecycle. Finally, we carried out experimental studies to assess the extent to which Cx. molestus retains exogenously influenced circadian rhythms, and whether it harbors any tendencies towards dormancy when exposed to a shortened photoperiod. Our results indicate that the gene Helicase domino (dom) has a nine-nucleotide, in-frame deletion specific to Cx. molestus. Previous work has shown that splice variants in this gene influence circadian behavior in Drosophila melanogaster. We also find derived, non-synonymous single nucleotide polymorphisms (SNPs) in eight genes that may also affect circadian rhythms and/or diapause induction in Cx. molestus. Four other circadian genes were found to have no quantifiable expression during any examined life stage, suggesting potential regulatory variation. Our experimental results confirm that Cx. molestus retains exogenously-influenced circadian rhythms but is not induced to enter a dormant state by a shortened photoperiod. Collectively, these findings show that the distinct, but potentially molecularly interconnected life-history traits of diapause induction and circadian rhythms are decoupled in Cx. molestus and suggest that this taxon may be a valuable tool for exploring exogenously influenced phenotypes in mosquitoes more broadly.

evolutionary biology↗

bioRxiv2020

The chaperonin CCT8 controls proteostasis essential for T cell maturation, selection, and function

T cells rely for their development and function on the correct folding and turnover of proteins generated in response to a broad range of molecular cues. In the absence of the eukaryotic type II chaperonin complex, CCT, T cell activation induced changes in the proteome are compromised including the formation of nuclear actin filaments and the formation of a normal cell stress response. Consequently, thymocyte maturation and selection, and T cell homeostatic maintenance and receptor-mediated activation are severely impaired. Additionally, Th2 polarization digresses in the absence of CCT-controlled protein folding resulting paradoxically in continued IFN-{gamma} expression. As a result, CCT-deficient T cells fail to generate an efficient immune protection against helminths as they are unable to sustain a coordinated recruitment of the innate and adaptive immune systems. These findings thus demonstrate that normal T cell biology is critically dependent on CCT-controlled proteostasis and that its absence is incompatible with protective immunity.

immunology↗

bioRxiv2020

A Humanized Antibody against LRG1 that Inhibits Angiogenesis and Reduces Retinal Vascular Leakage

Pathological angiogenesis contributes to morbidity in a number of diseases including cancer, diabetic retinopathy and the neovascular form of age-related macular degeneration, leading to significant efforts to develop effective anti-angiogenic therapeutics for these conditions. The field is dominated by inhibitors of vascular endothelial growth factor (VEGF), yet angiogenesis can also be driven and modified by other factors. We have previously demonstrated that leucine-rich alpha-2-glycoprotein 1 (LRG1) contributes to abnormal vessel growth by activating a TGF{beta} switch. Here we report the development and characterisation of a function-blocking fully humanised IgG4 and its Fab fragment, that bind to LRG1 with high affinity and specificity and inhibit vascular leakage in the mouse model of laser-induced choroidal neovascularisation. In summary, we have developed a therapeutic antibody that targets a VEGF-independent signalling axis, which may be effective in a number of conditions either as monotherapy or in combination with other vascular targeted therapies.

pathology↗

bioRxiv2020

Robust T-cell signaling triggered on soft polydimethylsiloxane-supported lipid-bilayers

The T-cell receptor (TCR) is thought to be triggered either by mechano-transduction or local tyrosine phosphatase exclusion at cell-cell contacts. However, the effects of the mechanical properties of activating surfaces have only been tested for late-stage T-cell activation, and phosphatase segregation has mostly been studied on glass-supported lipid bilayers that favor imaging but are orders-of-magnitude stiffer than typical cells. We developed a method for attaching lipid bilayers to polydimethylsiloxane polymer supports, producing soft bilayers with physiological levels of mechanical resistance (Youngs modulus of 4 kPa). Comparisons of T-cell behavior on soft and glass-supported bilayers revealed that early calcium signaling is unaffected by substrate rigidity, implying that early steps in TCR triggering are not mechanosensitive. Robust phosphatase exclusion was observed on the soft bilayers, however, suggesting it likely occurs at cell-cell contacts. This work sets the stage for an imaging-based exploration of receptor signaling under conditions closely mimicking physiological cell-cell contact.

biophysics↗

bioRxiv2020

A novel USP30 inhibitor recapitulates genetic loss of USP30 and sets the trigger for PINK1-PARKIN amplification of mitochondrial ubiquitylation.

The mitochondrial deubiquitylase USP30 negatively regulates the selective autophagy of damaged mitochondria. It has been proposed as an actionable target to alleviate the loss of function of the mitophagy pathway governed by the Parkinsons Disease associated genes PINK1 and PRKN. We present the characterisation of a N-cyano pyrrolidine derived compound, FT3967385, with high selectivity for USP30. The compound is well tolerated with no loss of total mitochondrial mass. We demonstrate that ubiquitylation of TOM20, a component of the outer mitochondrial membrane import machinery that directly interacts with USP30, represents a robust biomarker for both USP30 loss and inhibition. We have conducted proteomics analyses on a SHSY5Y neuroblastoma cell line model to directly compare the effects of genetic loss of USP30 with selective inhibition in an unbiased fashion. We have thereby identified a subset of ubiquitylation events consequent to mitochondrial depolarisation that are USP30 sensitive. Within responsive elements of the ubiquitylome, several components of the outer mitochondrial membrane transport (TOM) complex are most prominent. Thus, our data support a model whereby USP30 can regulate the availability of ubiquitin at the specific site of mitochondrial PINK1 accumulation following membrane depolarisation. In this model, USP30 deubiquitylation of TOM complex components dampens the trigger for the Parkin-dependent amplification of mitochondrial ubiquitylation leading to mitophagy. Accordingly, PINK1 generation of phospho-Ser65 Ubiquitin proceeds more rapidly and to a greater extent in cells either lacking USP30 or subject to USP30 inhibition.

cell biology↗

bioRxiv2020

The deubiquitylase USP9X controls ribosomal stalling

When a ribosome stalls during translation, it runs the risk of collision with a trailing ribosome. Such an encounter leads to the formation of a stable di-ribosome complex, which needs to be resolved by a dedicated machinery. The initial stalling and the subsequent resolution of di-ribosomal complexes requires activity of Makorin and ZNF598 ubiquitin E3 ligases respectively, through ubiquitylation of the eS10 and uS10 sub-units of the ribosome. It is common for the stability of RING E3 ligases to be regulated by an interacting deubiquitylase (DUB), which often opposes auto-ubiquitylation of the E3. Here, we show that the DUB USP9X directly interacts with ZNF598 and regulates its abundance through the control of protein stability in human cells. We have developed a highly specific small molecule inhibitor of USP9X. Proteomics analysis, following inhibitor treatment of HCT116 cells, confirms previous reports linking USP9X with centrosome associated protein stability and reveals loss of ZNF598 and Makorin 2. In the absence of USP9X or following chemical inhibition of its catalytic activity, steady state levels of Makorins and ZNF598 are diminished and the ribosomal quality control pathway is impaired.

cell biology↗

bioRxiv2019

Intracellular Neutralization of Ricin Toxin by Single Domain Antibodies Targeting the Active Site Pocket

The extreme potency of the plant toxin, ricin, is due to its enzymatic subunit, RTA, which inactivates mammalian ribosomes with near perfect efficiency. Here we characterized, at the functional and structural levels, seven alpaca single-domain antibodies (VHHs) previously reported to recognize epitopes in proximity to RTAs active site. Three of the VHHs, V2A11, V8E6 and V2G10, were potent inhibitors of RTA in vitro and protected Vero cells from ricin when expressed as intracellular antibodies (\"intrabodies\"). Crystal structure analysis revealed that the complementarity-determining region 3 (CDR3) elements of V2A11 and V8E6 penetrate RTAs active site and interact with key catalytic residues. V2G10, in contrast, sits atop the enzymatic pocket and occludes substrate accessibility. The other four VHHs also penetrated/occluded RTAs active site, but lacked sufficient binding affinities to outcompete RTA-ribosome interactions. Intracellular delivery of high-affinity, single-domain antibodies may offer a new avenue in the development of countermeasures against ricin toxin.

immunology↗