New insight into a simple high-yielding method for the production of fully folded and functional recombinant human CCL5.

Chemokines are proteins important for a range of biological processes from cell-directed migration (chemotaxis) to cell activation and differentiation. Chemokine C-C ligand 5 (CCL5) is an important pro-inflammatory chemokine attracting immune cells towards inflammatory sites through interaction with its receptors CCR1/3/5. Recombinant production of large quantities of CCL5 in Escherichia coli is challenging due to formation of inclusion bodies which necessitates refolding, often leading to low recovery of biologically active protein. To combat this, we have developed a method for CCL5 production that utilises the purification of SUMO tagged CCL5 from E. coli SHuffle cells avoiding the need to reform disulfide bonds through inclusion body purification and yields high quantities of CCL5 (~ 25 mg/L). We demonstrated that the CCL5 produced was fully functional by assessing well-established cellular changes triggered by CCL5 binding to CCR5, including receptor phosphorylation and internalisation, intracellular signalling leading to calcium flux, as well as cell migration. Overall, we demonstrate that the use of solubility tags, SHuffle cells and low pH dialysis constitutes an approach that increases purification yields of active CCL5 with low endotoxin contamination for biological studies.

A systematic review reveals conflicting evidence for the prevalence of antibodies against the sialic acid 'xenoautoantigen' Neu5Gc in humans and the need for a standardised approach to quantification.

Despite an array of hypothesised implications for health, disease, and therapeutic development, antibodies against the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc) remain a subject of much debate. This systematic review of 114 publications aimed to generate a comprehensive overview of published studies in this field, addressing both the reported prevalence of anti-Neu5Gc antibodies in the human population and whether experimental variation accounts for the conflicting reports about the extent of this response. Absolute titres of anti-Neu5Gc antibodies, the reported prevalence of these antibodies, and the individual variation observed within experiments were analysed and grouped according to biological context ('inflammation', 'xenotransplantation', 'biotherapeutic use', 'cancer', and 'healthy populations'), detection method, target epitope selection, and choice of blocking agent. These analyses revealed that the experimental method had a notable impact on both the reported prevalence and absolute titres of anti-Neu5Gc antibodies in the general population, thereby limiting the ability to ascribe reported trends to genuine biological differences or the consequence of experimental design. Overall, this review highlights important knowledge gaps in the study of antibodies against this important xenoautoantigen and the need to establish a standardised method for their quantification if the extent of the importance of Neu5Gc in human health is to be fully understood.

Towards a 'clicked' PSMA targeting gene delivery bioconjugate-polyplex for prostate cancer.

Prostate cancer is the most common cancer in men in the UK with over 50 000 new cases diagnosed each year and although therapeutic advances in surgery, anti-androgens, radio- and chemotherapy have increased survival rates, there still remains a need for new treatments to combat the most aggressive forms of the disease. Gene therapy offers promise as an alternative approach but is reliant on selective targeting to the cancer cell surface. Herein we describe the novel construction of a prostate specific membrane antigen (PSMA) binding bioconjugate-polyplex, based on a glutamate-urea peptide scaffold using 'click' chemistry, which we demonstrate is capable of targeted delivery of a GFP gene to PSMA overexpressing prostate cancer cells, and therefore may have potential future application as part of a prostate cancer gene delivery therapy.

Exploring a Gemcitabine-Glucose Hybrid as a Glycoconjugate Prodrug.

Nucleoside analogues are established treatments for cancer and viral infection. Gemcitabine is a commonly employed nucleoside analogue displaying anticancer properties against a range of tumor types but is rapidly inactivated in vivo. Efforts to bolster its pharmaceutical profile include investigating prodrug forms. Herein, we explore the synthesis of a novel glucose-gemcitabine glycoconjugate, targeting uptake via glucose transport. We select a redox-reactive disulfide linker for conjugation of gemcitabine (through N4-cytosine) with glucose. Evaluation of this glycoconjugate reveals increased toxicity against androgen insensitive PC3 prostate cancer cells compared to LNCaP (which have lower levels of glucose transporter GLUT1). These preliminary results suggest that glycoconjugation of nucleosides may be an effective approach to targeting cells which display increased uptake and metabolism of glucose.

Cross aldol OPAL bioconjugation outcompetes intramolecular hemiaminal cyclisation of proline adjacent N-terminal α-oxo aldehydes at acidic pH.

Novel methods to construct small molecule-protein bioconjugates are integral to the development of new biomedicines for a variety of diseases. C-C linked bioconjugates are increasingly desirable in this application due to their in vivo stability and can be accessed through cross aldol bioconjugation of reactive α-oxo aldehyde handles easily introduced at the N-terminus of proteins by periodate oxidation. We previously developed an organocatalyst-mediated protein aldol ligation (OPAL) for chemical modification of these reactive aldehydes, but the efficiency of this method was limited when a proline residue was directly adjacent to the N-terminus due to intramolecular hemiaminal formation. Herein we explore the competition between this cyclisation and the OPAL modification and demonstrate bioconjugation can be favoured through use of acidic pH for both oxidation and OPAL, and optimisation of reaction conditions and organocatalyst. We then showcase the utility of this acidic-OPAL in modification of the cholera toxin B-subunit (CTB), a homo-pentameric protein of biomedical promise.

Harnessing glycofluoroforms for impedimetric biosensing.

Glycans play a major role in biological cell-cell recognition and signal transduction but have found limited application in biosensors due to glycan/lectin promiscuity; multiple proteins are capable of binding to the same native glycan. Here, site-specific fluorination is used to introduce protein-glycan selectivity, and this is coupled with an electrochemical detection method to generate a novel biosensor platform. 3F-lacto-N-biose glycofluoroform is installed onto polymer tethers, which are subsequently immobilised onto gold screen printed electrodes, providing a non-fouling surface. The impedance biosensing platform is shown to selectively bind cancer-associated galectin-3 compared to control glycans and proteins. To improve the analytical capability, Bayesian statistical analysis was deployed in the equivalent circuit fitting of electrochemical impedance spectroscopy data. It is shown that Markov Chain Monte Carlo (MCMC) analysis is a helpful method for visualising experimental irreproducibility, and we apply this as a quality control step.

Blood immune profiles reveal a CXCR3/CCR5 axis of dysregulation in early sepsis.

We report on a pilot study exploring whether blood immune signatures can reveal early specific indicator profiles for patients meeting sepsis criteria upon hospital admission. We analysed samples of sepsis-suspected patients (N=20) and age-spanning healthy controls (N=12), using flow cytometry-based assays. We measured inflammatory markers from plasma fractions, and immunophenotyped freshly isolated unfixed PBMCs for leukocytes subsets representation and expression of activation markers, including chemokine receptors. We found that beside IL-6 and sCD14, CXCR3 ligands (CXCL9 and CXCL10) separated sepsis-suspected patients from healthy controls. The abundance of CD4+ T cells was significantly reduced in patients, while they displayed substantial losses of CCR5-expressing monocytes and CXCR3/CCR5 double positive T cells. Post-hoc subgrouping of patients according to their sepsis diagnosis on discharge, identified CXCR3/CCR5 double expression on T cells as a separating characteristic for confirmed cases. This work suggests a potential novel axis of dysregulation affecting CXCR3 and CCR5 in early sepsis.

TLR2-dependent pathway of heterologous down-modulation for the CC chemokine receptors 1, 2, and 5 in human blood monocytes.

During innate immune responses, the inflammatory CC chemokine receptors CCR1, CCR2, and CCR5 mediate the recruitment of blood monocytes to infected tissues by promoting cell migration in response to chemokines CCL2-5. Toll-like receptors also play an essential role, allowing pathogen recognition by the recruited monocytes. Here, we demonstrate that Toll-like receptor 2 (TLR2) stimulation by lipoteichoic acid (LTA) from Staphylococcus aureus leads to gradual down-modulation of CCR1, CCR2, and CCR5 from the plasma membrane of human blood-isolated monocytes and inhibits chemotaxis. Interestingly, LTA does not promote rapid desensitization of chemokine-mediated calcium responses. We found that the TLR2 crosstalk with chemokine receptors is not dependent on the Toll/interleukin-1 receptor domain-containing adaptor protein, but instead involves phospholipase C, the small G protein Rac1, and is phorbol ester sensitive. Activation of this pathway by LTA lead to ß-arrestin-mediated endocytosis of Ser349-phosphorylated CCR5 into recycling endosomes, as does CCL5 treatment. However, LTA-induced internalization of CCR5 is a slower process associated with phospholipase C-mediated and phorbol ester-sensitive phosphorylation. Overall, our data indicate that TLR2 negatively regulates CCR1, CCR2, and CCR5 on human blood monocytes by activating the machinery used to support chemokine-dependent down-modulation and provide a molecular mechanism for inhibiting monocyte migration after pathogen recognition.

Preparation and Application of an Inexpensive α-Formylglycine Building Block Compatible with Fmoc Solid-Phase Peptide Synthesis.

α-Formylglycine (fGly) is a rare residue located in the active site of sulfatases and serves as a precursor to pharmaceutically relevant motifs. The installation of fGly motifs into peptides is currently challenging due to degradation under the acidic and nucleophile-rich conditions accompanying resin cleavage during solid-phase peptide synthesis. We report the synthesis of acid- and nucleophile-tolerant α-formylglycine building blocks from vitamin C and use them to prepare callyaerin A, a macrocyclic peptide containing an fGly-derived motif.

Identification of a protein expression signature distinguishing early from organising diffuse alveolar damage in COVID-19 patients.

Diffuse alveolar damage (DAD) is the histological expression of acute respiratory distress syndrome and characterises lung pathology due to infection with SARS-CoV-2, and other respiratory pathogens of clinical significance. DAD reflects a time-dependent immunopathological process, progressing from an early/exudative stage through to an organising/fibrotic stage, yet within an individual these different stages of DAD may coexist. Understanding the progression of DAD is central to the development of new therapeutics to limit progressive lung damage. Here, we applied highly multiplexed spatial protein profiling to autopsy lung tissues derived from 27 patients who died from COVID-19 and identified a protein signature (ARG1, CD127, GZMB, IDO1, Ki67, phospho-PRAS40 (T246) and VISTA) that distinguishes early DAD from late DAD with good predictive accuracy. These proteins warrant further investigation as potential regulators of DAD progression.

Innate killing of Leishmania donovani by macrophages of the splenic marginal zone requires IRF-7.

Highly phagocytic macrophages line the marginal zone (MZ) of the spleen and the lymph node subcapsular sinus. Although these macrophages have been attributed with a variety of functions, including the uptake and clearance of blood and lymph-borne pathogens, little is known about the effector mechanisms they employ after pathogen uptake. Here, we have combined gene expression profiling and RNAi using a stromal macrophage cell line with in situ analysis of the leishmanicidal activity of marginal zone macrophages (MZM) and marginal metallophilic macrophages (MMM) in wild type and gene targeted mice. Our data demonstrate a critical role for interferon regulatory factor-7 (IRF-7) in regulating the killing of intracellular Leishmania donovani by these specialised splenic macrophage sub-populations. This study, therefore, identifies a new role for IRF-7 as a regulator of innate microbicidal activity against this, and perhaps other, non-viral intracellular pathogens. This study also highlights the importance of selecting appropriate macrophage populations when studying pathogen interactions with this functionally diverse lineage of cells.

Single-molecule and super-resolved imaging deciphers membrane behavior of onco-immunogenic CCR5.

The ability of tumors to establish a pro-tumorigenic microenvironment is an important point of investigation in the search for new therapeutics. Tumors form microenvironments in part by the "education" of immune cells attracted via chemotactic axes such as that of CCR5-CCL5. Further, CCR5 upregulation by cancer cells, coupled with its association with pro-tumorigenic features such as drug resistance and metastasis, has suggested CCR5 as a therapeutic target. However, with several conformational "pools" being reported, phenotypic investigations must be capable of unveiling conformational heterogeneity. Addressing this challenge, we performed super-resolution structured illumination microscopy (SIM) and single molecule partially TIRF-coupled HILO (PaTCH) microscopy of CCR5 in fixed cells. SIM data revealed a non-random spatial distribution of CCR5 assemblies, while Intensity-tracking of CCR5 assemblies from PaTCH images indicated dimeric sub-units independent of CCL5 perturbation. These biophysical methods can provide important insights into the structure and function of onco-immunogenic receptors and many other biomolecules.

Integrated miRNA/cytokine/chemokine profiling reveals severity-associated step changes and principal correlates of fatality in COVID-19.

Inflammatory cytokines and chemokines (CC) drive COVID-19 pathology. Yet, patients with similar circulating CC levels present with different disease severity. Here, we determined 171 microRNAomes from 58 hospitalized COVID-19 patients (Cohort 1) and levels of 25 cytokines and chemokines (CC) in the same samples. Combining microRNA (miRNA) and CC measurements allowed for discrimination of severe cases with greater accuracy than using miRNA or CC levels alone. Severity group-specific associations between miRNAs and COVID-19-associated CC (e.g., IL6, CCL20) or clinical hallmarks of COVID-19 (e.g., neutrophilia, hypoalbuminemia) separated patients with similar CC levels but different disease severity. Analysis of an independent cohort of 108 patients from a different center (Cohort 2) demonstrated feasibility of CC/miRNA profiling in leftover hospital blood samples with similar severe disease CC and miRNA profiles, and revealed CCL20, IL6, IL10, and miR-451a as key correlates of fatal COVID-19. These findings highlight that systemic miRNA/CC networks underpin severe COVID-19.

Mechanisms regulating chemokine receptor activity.

Co-ordinated movement and controlled positioning of leucocytes is key to the development, maintenance and proper functioning of the immune system. Chemokines and their receptors play an essential role in these events by mediating directed cell migration, often referred to as chemotaxis. The chemotactic property of these molecules is also thought to contribute to an array of pathologies where inappropriate recruitment of specific chemokine receptor-expressing leucocytes is observed, including cancer and inflammatory diseases. As a result, chemokine receptors have become major targets for therapeutic intervention, and during the past 15 years much research has been devoted to understanding the regulation of their biological activity. From these studies, processes which govern the availability of functional chemokine receptors at the cell surface have emerged as playing a central role. In this review, we summarize and discuss current knowledge on the molecular mechanisms contributing to the regulation of chemokine receptor surface expression, from gene transcription and protein degradation to post-translational modifications, multimerization, intracellular transport and cross-talk.

A Tale of Two Bioconjugations: pH Controlled Divergent Reactivity of Protein α-oxo-Aldehydes in Competing α-oxo-Mannich and Catalyst-Free Aldol Ligations.

Site-selective chemical methods for protein bioconjugation have revolutionized the fields of cell and chemical biology through the development of novel protein/enzyme probes bearing fluorescent, spectroscopic, or even toxic cargos. Herein, we report two new methods for the bioconjugation of α-oxo aldehyde handles within proteins using small molecule aniline and/or phenol probes. The "α-oxo-Mannich" and "catalyst-free aldol" ligations both compete for the electrophilic α-oxo aldehyde, which displays pH divergent reactivity proceeding through the "Mannich" pathway at acidic pH to afford bifunctionalized bioconjugates, and the "catalyst-free aldol" pathway at neutral pH to afford monofunctionalized bioconjugates. We explore the substrate scope and utility of both of these bioconjugations in the construction of neoglycoproteins, in the process formulating a mechanistic rationale for how both pathways intersect with each other at different reaction pH's.

Agonist-induced endocytosis of CC chemokine receptor 5 is clathrin dependent.

The signaling activity of several chemokine receptors, including CC chemokine receptor 5 (CCR5), is in part controlled by their internalization, recycling, and/or degradation. For CCR5, agonists such as the chemokine CCL5 induce internalization into early endosomes containing the transferrin receptor, a marker for clathrin-dependent endocytosis, but it has been suggested that CCR5 may also follow clathrin-independent routes of internalization. Here, we present a detailed analysis of the role of clathrin in chemokine-induced CCR5 internalization. Using CCR5-transfected cell lines, immunofluorescence, and electron microscopy, we demonstrate that CCL5 causes the rapid redistribution of scattered cell surface CCR5 into large clusters that are associated with flat clathrin lattices. Invaginated clathrin-coated pits could be seen at the edge of these lattices and, in CCL5-treated cells, these pits contain CCR5. Receptors internalized via clathrin-coated vesicles follow the clathrin-mediated endocytic pathway, and depletion of clathrin with small interfering RNAs inhibits CCL5-induced CCR5 internalization. We found no evidence for CCR5 association with caveolae during agonist-induced internalization. However, sequestration of cholesterol with filipin interferes with agonist binding to CCR5, suggesting that cholesterol and/or lipid raft domains play some role in the events required for CCR5 activation before internalization.

A correlative and quantitative imaging approach enabling characterization of primary cell-cell communication: Case of human CD4+ T cell-macrophage immunological synapses.

Cell-to-cell communication engages signaling and spatiotemporal reorganization events driven by highly context-dependent and dynamic intercellular interactions, which are difficult to capture within heterogeneous primary cell cultures. Here, we present a straightforward correlative imaging approach utilizing commonly available instrumentation to sample large numbers of cell-cell interaction events, allowing qualitative and quantitative characterization of rare functioning cell-conjugates based on calcium signals. We applied this approach to examine a previously uncharacterized immunological synapse, investigating autologous human blood CD4+ T cells and monocyte-derived macrophages (MDMs) forming functional conjugates in vitro. Populations of signaling conjugates were visualized, tracked and analyzed by combining live imaging, calcium recording and multivariate statistical analysis. Correlative immunofluorescence was added to quantify endogenous molecular recruitments at the cell-cell junction. By analyzing a large number of rare conjugates, we were able to define calcium signatures associated with different states of CD4+ T cell-MDM interactions. Quantitative image analysis of immunostained conjugates detected the propensity of endogenous T cell surface markers and intracellular organelles to polarize towards cell-cell junctions with high and sustained calcium signaling profiles, hence defining immunological synapses. Overall, we developed a broadly applicable approach enabling detailed single cell- and population-based investigations of rare cell-cell communication events with primary cells.

CCR5 susceptibility to ligand-mediated down-modulation differs between human T lymphocytes and myeloid cells.

CCR5 is a chemokine receptor expressed on leukocytes and a coreceptor used by HIV-1 to enter CD4(+) T lymphocytes and macrophages. Stimulation of CCR5 by chemokines triggers internalization of chemokine-bound CCR5 molecules in a process called down-modulation, which contributes to the anti-HIV activity of chemokines. Recent studies have shown that CCR5 conformational heterogeneity influences chemokine-CCR5 interactions and HIV-1 entry in transfected cells or activated CD4(+) T lymphocytes. However, the effect of CCR5 conformations on other cell types and on the process of down-modulation remains unclear. We used mAbs, some already shown to detect distinct CCR5 conformations, to compare the behavior of CCR5 on in vitro generated human T cell blasts, monocytes and MDMs and CHO-CCR5 transfectants. All human cells express distinct antigenic forms of CCR5 not detected on CHO-CCR5 cells. The recognizable populations of CCR5 receptors exhibit different patterns of down-modulation on T lymphocytes compared with myeloid cells. On T cell blasts, CCR5 is recognized by all antibodies and undergoes rapid chemokine-mediated internalization, whereas on monocytes and MDMs, a pool of CCR5 molecules is recognized by a subset of antibodies and is not removed from the cell surface. We demonstrate that this cell surface-retained form of CCR5 responds to prolonged treatment with more-potent chemokine analogs and acts as an HIV-1 coreceptor. Our findings indicate that the regulation of CCR5 is highly specific to cell type and provide a potential explanation for the observation that native chemokines are less-effective HIV-entry inhibitors on macrophages compared with T lymphocytes.

Analysis of chemokine receptor endocytosis and intracellular trafficking.

Chemokine receptors are G protein-coupled receptors (GPCRs) that, through their ability to regulate chemotaxis by responding to small chemoattractant peptides termed chemokines, are involved in the development, maintenance, and functional activities of the immune system. In addition, members of the chemokine receptor family have been implicated in a number of other physiological and pathological processes, including human immunodeficiency virus infection and malaria. These activities are dependent on receptor expression at the cell surface and cellular events that reduce the cell-surface expression of chemokine receptors can abrogate these activities. Moreover, internalization of chemokine receptors by endocytosis is necessary for both receptor degradation and recycling, key regulatory processes that determine cell-surface expression levels. Here we provide detailed methods for the quantitative analysis of CCR5 endocytosis and recycling by flow cytometry, as well as fluorescence and electron microscopic procedures to analyze the endocytosis and intracellular trafficking of CCR5 by immunolabeling of cells or cryosections. In principle, the same approaches can be used for analyzing other chemokine receptors and other GPCR or non-GPCR cell-surface proteins.

Dishevelled (Dvl-2) activates canonical Wnt signalling in the absence of cytoplasmic puncta.

Dishevelled family proteins are multidomain intracellular transducers of Wnt signals. Ectopically expressed mammalian Dishevelled 2 (Dvl-2) activates downstream signalling and localises to cytoplasmic puncta. It has been suggested that these Dvl-2-containing structures correspond to intracellular vesicles and may be involved in the Wnt signal transduction process. We report that cytoplasmic puncta are primarily formed in cells expressing Dvl-2 at high levels. Lower levels of expression can activate signalling without forming puncta. The structures do not localise with markers of the early or late endocytic pathway and time-lapse analysis demonstrates that Dvl-2 puncta move in a random fashion over short distances but do not originate from the plasma membrane. Based on our findings, we propose that Dvl-2 puncta are protein aggregates that are not required for signalling.