Biology subjects

Fisher, R.

Publications and source records attributed to Fisher, R..

4 recordsLinked to original sources

bioRxiv2021

Synthetic Antigen Presenting cells reveal the diversity and functional specialization of extracellular vesicles composing the fourth signal of T cell immunological synapses.

The T cell Immunological Synapse (IS) is a pivotal hub for the regulation of adaptive immunity by endowing the exchange of information between cells engaged in physical contacts. Beyond the integration of antigen (signal one), co-stimulation (signal two), and cytokines (signal three), the IS facilitates the delivery of T-cell effector assemblies including supramolecular attack particles (SMAPs) and extracellular vesicles (EVs). How these particulate outputs differ among T -cell subsets and how subcellular compartments and signals exchanged at the synapse contribute to their composition is not fully understood. Here we harnessed bead-supported lipid bilayers (BSLBs) as a tailorable and versatile technology for the study of synaptic particle biogenesis and composition in different T-cell subsets, including CART. These synthetic antigen-presenting cells (APCs) facilitated the characterisation of trans-synaptic vesicles (tSV) as a heterogeneous population of EVs comprising among others PM-derived synaptic ectosomes and CD63+ exosomes. We harnessed BSLB to unveil the factors influencing the vesicular release of CD40L, as a model effector, identifying CD40 trans presentation, T-cell activation, ESCRT upregulation/recruitment, antigen density/potency, co-repression by PD-1 ligands, and its processing by ADAM10 as major determinants. Further, BSLB made possible the comparison of microRNA (miR) species associated with tSV and steadily released EVs. Altogether, our data provide evidence for a higher specialisation of tSV which are enriched not only in effector immune receptors but also in miR and RNA-binding proteins. Considering the molecular uniqueness and functional complexity of the tSV output, which is also accompanied by SMAPs, we propose their classification as signal four. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/445691v3_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@100f3aorg.highwire.dtl.DTLVardef@57adfforg.highwire.dtl.DTLVardef@6052fcorg.highwire.dtl.DTLVardef@1e85c21_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIBead Supported Lipid Bilayers (BSLB) reconstituting antigen-presenting cells support synapse assembly by T cells and the release of effector particles. C_LIO_LIBSLB facilitate the dissection of the cellular machineries and synapse composition shaping the released tSV. C_LIO_LItSV and their steadily released counterparts have a different composition. TSV show a higher enrichment of effectors including immune receptors, miR, RNA- and other nucleic acid-binding proteins, than EVs. C_LI

immunology↗

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

ABPP-HT - high-throughput activity-based profiling of deubiquitylating enzyme inhibitors in a cellular context

The potency and selectivity of a small molecule inhibitor are key parameters to assess during the early stages of drug discovery. In particular, it is very informative for characterizing compounds in a relevant cellular context in order to reveal potential off-target effects and drug efficacy. Activity-based probes (ABPs) are valuable tools for that purpose, however, obtaining cellular target engagement data in a high-throughput format has been particularly challenging. Here, we describe a new methodology named ABPP-HT (high-throughput-compatible activity-based protein profiling), implementing a semi-automated proteomic sample preparation workflow that increases the throughput capabilities of the classical ABPP workflow approximately ten times while preserving its enzyme profiling characteristics. Using a panel of deubiquitylating enzyme (DUB) inhibitors, we demonstrate the feasibility of ABPP-HT to provide compound selectivity profiles of endogenous DUBs in a cellular context at a fraction of time as compared to previous methodologies.

biochemistry↗

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↗