While membrane models now include the heterogeneous distribution of lipids, the impact of membrane charges on regulating the association of proteins with the plasma membrane is often overlooked. ...Charged lipids are asymmetrically distributed between the two leaflets of the plasma membrane, resulting in the inner leaflet being negatively charged and a surface potential that attracts and binds positively charged ions, proteins, and peptide motifs. These interactions not only create a transmembrane potential but they can also facilitate the formation of charged membrane domains. Here, we reference fields outside of immunology in which consequences of membrane charge are better characterized to highlight important mechanisms. We then focus on T cell receptor (TCR) signaling, reviewing the evidence that membrane charges and membrane-associated calcium regulate phosphorylation of the TCR-CD3 complex and discuss how the immunological synapse exhibits distinct patterns of membrane charge distribution. We propose that charged lipids, ions in solution, and transient protein interactions form a dynamic equilibrium during T cell activation.
Summary
Leukocytes play a critical role in recognizing and responding to infection and cancer. Central to this function is an array of cell‐surface receptors that lack sequence homology. Many of ...these receptors have in common the fact that their signaling involves phosphorylation of cytoplasmic domains by extrinsic tyrosine kinases. These non‐catalytic tyrosine‐phosphorylated receptors (NTRs) share a number of other features, including small size and optimal stimulation by surface–associated ligands. We argue here that NTRs are also likely to share the same kinetic‐segregation triggering mechanism, which involves segregation of the engaged NTR from receptor tyrosine phosphatases with large ectodomains such as CD45 and CD148. NTRs signal through tyrosine‐containing cytoplasmic motifs, which recruit distinct cytoplasmic signaling proteins when phosphorylated, transducing activatory or inhibitory signals. They have two features that make them uniquely well suited to their role in immune recognition of infection and cancer. Their modular structure enables the coupling of many rapidly evolving receptors with diverse ligand specificities to the same conserved signaling machinery. Their similarity in size and shared signaling machinery enables them to colocalize at cell‐cell interfaces when they engage ligands, facilitating the integration of activatory and inhibitory signals from multiple receptors at the cell surface.
S100A8 and S100A9 are generally considered proinflammatory. Hypohalous acids generated by activated phagocytes promote novel modifications in murine S100A8 but modifications to human S100A8 are ...undefined and there is no evidence that these proteins scavenge oxidants in human disease. Recombinant S100A8 was exquisitely sensitive to equimolar ratios of HOCl, which generated sulfinic and sulfonic acid intermediates and novel oxathiazolidine oxide/dioxide forms (mass additions, m/z +30 and +46) on the single Cys42 residue. Met78(O) and Trp54(+16) were also present. HOBr generated sulfonic acid intermediates and oxidized Trp54(+16). Evidence for oxidation of the single Cys3 residue in recS100A9 HOCl was weak; Met63, Met81, Met83, and Met94 were converted to Met(O) in vitro. Oxidized S100A8 was prominent in lungs from patients with asthma and significantly elevated in sputum compared to controls, whereas S100A8 and S100A9 were not significantly increased. Oxidized monomeric S100A8 was the major component in asthmatic sputum, and modifications, including the oxathiazolidine adducts, were similar to those generated by HOCl in vitro. Oxidized Met63, Met81, and Met94 were variously present in S100A9 from asthmatic sputum. Results have broad implications for conditions under which hypohalous acid oxidants are generated by activated phagocytes. Identification in human disease of the novel S100A8 Cys derivatives typical of those generated in vitro strongly supports the notion that S100A8 contributes to antioxidant defense during oxidative stress.
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► Recombinant human S100A8 and S100A9 scavenged hypohalous oxidants in vitro. ► HOCl oxidation of human S100A8 in vitro generated novel oxathiazolidine derivatives. ► Lung tissue and sputum from asthmatics contained S100A8, oxidized S100A8, and S100A9. ► S100A8 and S100A9 scavenged reactive oxygen species in vivo.
Single Molecule Localization Microscopy (SMLM) is an imaging method that allows for the visualization of structures smaller than the diffraction limit of light (~200 nm). This is achieved through ...techniques such as stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM). A large part of obtaining ideal imaging of single molecules is the choice of the right fluorescent label. An upcoming field of protein labeling is incorporating unnatural amino acids (UAAs) with an attached fluorescent dye for precise localization and visualization of individual molecules. For this technique, fluorescent probes are conjugated to UAAs and are introduced into the protein of interest (POI) as a label. Here we contrast this labeling method with other commonly used protein-based labeling methods such as fluorescent proteins (FPs) or self-labeling tags such as Halotag, SNAP-tags, and CLIP-tags, and highlight the benefits and shortcomings of the site-specific incorporation of UAAs coupled with fluorescent dyes in SMLM.
T cell activation is initiated when ligand binding to the T cell receptor (TCR) triggers intracellular phosphorylation of the TCR-CD3 complex. However, it remains unknown how biophysical properties ...of TCR engagement result in biochemical phosphorylation events. Here, we constructed an optogenetic tool that induces spatial clustering of ζ-chain in a light controlled manner. We showed that spatial clustering of the ζ-chain intracellular tail alone was sufficient to initialize T cell triggering including phosphorylation of ζ-chain, Zap70, PLCγ, ERK and initiated Ca
flux. In reconstituted COS-7 cells, only Lck expression was required to initiate ζ-chain phosphorylation upon ζ-chain clustering, which leads to the recruitment of tandem SH2 domain of Zap70 from cell cytosol to the newly formed ζ-chain clusters at the plasma membrane. Taken together, our data demonstrated the biophysical relevance of receptor clustering in TCR signaling.
Understanding the mechanisms behind T cell dysfunctions during chronic diseases is critical in developing effective immunotherapies. As demonstrated by several animal models and human studies, T cell ...dysfunctions are induced during chronic diseases, spanning from infections to cancer. Although factors governing the onset and the extent of the functional impairment of T cells can differ during infections and cancer, most dysfunctional phenotypes share common phenotypic traits in their immune receptor and biophysical landscape. Through the latest developments in biophysical techniques applied to explore cell membrane and receptor-ligand dynamics, we are able to dissect and gain further insights into the driving mechanisms behind T cell dysfunctions. These insights may prove useful in developing immunotherapies aimed at reinvigorating our immune system to fight off infections and malignancies more effectively. The recent success with checkpoint inhibitors in treating cancer opens new avenues to develop more effective, targeted immunotherapies. Here, we highlight the studies focused on the transformation of the biophysical landscape during infections and cancer, and how T cell biomechanics shaped the immunopathology associated with chronic diseases.
Due to recent technical developments in microscopy, huge advances have been made in our understanding of the architecture of the cell membrane. It is now well appreciated that nanoscale clustering is ...a common feature of membrane proteins. Many of these clusters have been implicated in signal initiation and integration platforms. However, the mechanisms that mediate the dynamic nanoscale arrangement of membrane proteins are not fully understood and could involve lipid domains, electrostatic interactions between proteins and lipid, protein scaffolding as well as purely mechanical processes. In this review we summarise these mechanisms giving rise to dynamic nanoscale protein reorganisation in the plasma membrane with reference to recent examples of immune receptor clustering to illustrate general principles.
The study of expressed proteins in neoplasia is undergoing a revolution with the advent of proteomic analysis. Unlike genomic studies where individual changes may have no functional significance, ...protein expression is closely aligned with cellular activity. This perspective will review proteomics as a method of detecting markers of neoplasia with a particular emphasis on lung cancer and the potential to sample the lung by exhaled breath condensate (EBC). EBC collection is a simple, new, and noninvasive technique, which allows sampling of lower respiratory tract fluid. EBC enables the study of a wide variety of biological markers from low molecular weight mediators to macromolecules, such as proteins, in a range of pulmonary diseases. EBC may be applied to the detection of lung cancer where it could be a tool in early diagnosis. This perspective will explore the potential of applying proteomics to the EBC from lung cancer patients as an example of detecting potential biomarkers of disease and progression.
Dose-response experiments are a mainstay of receptor biology studies and can reveal valuable insights into receptor function. Such studies of receptors that bind cell surface ligands are currently ...limited by the difficulty in manipulating the surface density of ligands at a cell-cell interface. Here, we describe a generic cell surface ligand system that allows precise manipulation of cell surface ligand densities over several orders of magnitude. These densities are robustly quantifiable, a major advance over previous studies. We validate the system for a range of immunoreceptors, including the T-cell receptor (TCR), and show that this generic ligand stimulates via the TCR at a similar surface density as its native ligand. We also extend our work to the activation of chimeric antigen receptors. This novel system allows the effect of varying the surface density, valency, dimensions, and affinity of the ligand to be investigated. It can be readily broadened to other receptor-cell surface ligand interactions and will facilitate investigation into the activation of, and signal integration between, cell surface receptors.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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