A degradatory fate for CCR4 suggests a primary role in Th2 inflammation.

CCR4 is the sole receptor for the chemokines CCL22 and CCL17. Clinical studies of asthmatic airways have shown levels of both ligands and CCR4+ Th2 cells to be elevated, suggestive of a role in disease. Consequently, CCR4 has aroused much interest as a potential therapeutic target and an understanding of how its cell surface expression is regulated is highly desirable. To this end, receptor expression, receptor endocytosis, and chemotaxis were assessed using transfectants expressing CCR4, CCR4+ human T cell lines, and human Th2 cells polarized in vitro. CCL17 and CCL22 drove rapid endocytosis of CCR4 in a dose-dependent manner. Replenishment at the cell surface was slow and sensitive to cycloheximide, suggestive of de novo synthesis of CCR4. Constitutive CCR4 endocytosis was also observed, with the internalized CCR4 found to be significantly degraded over a 6-h incubation. Truncation of the CCR4 C-terminus by 40 amino acids had no effect on cell surface expression, but resulted in significant impairment of ligand-induced endocytosis. Consequently, migration to both CCL17 and CCL22 was significantly enhanced. In contrast, truncation of CCR4 did not impair constitutive endocytosis or degradation, suggesting the use of alternative receptor motifs in these processes. We conclude that CCR4 cell surface levels are tightly regulated, with a degradative fate for endocytosed receptor. We postulate that this strict control is desirable, given that Th2 cells recruited by CCR4 can induce the further expression of CCR4 ligands in a positive feedback loop, thereby enhancing allergic inflammation

A highly efficient method for the production and purification of recombinant human CXCL8

Chemokines play diverse and fundamental roles in the immune system and human disease, which has prompted their structural and functional characterisation. Production of recombinant chemokines that are folded and bioactive is vital to their study but is limited by the stringent requirements of a native N-terminus for receptor activation and correct disulphide bonding required to stabilise the chemokine fold. Even when expressed as fusion proteins, overexpression of chemokines in E. coli tends to result in the formation of inclusion bodies, generating the additional steps of solubilisation and refolding. Here we present a novel method for producing soluble chemokines in relatively large amounts via a simple two-step purification procedure with no requirements for refolding. CXCL8 produced by this method has the correct chemokine fold as determined by NMR spectroscopy and in chemotaxis assays was indistinguishable from commercially available chemokines. We believe that this protocol significantly streamlines the generation of recombinant chemokines

CXCR3 antagonist VUF10085 binds to an intrahelical site distinct from that of the broad spectrum antagonist TAK-779.

BACKGROUND AND PURPOSE: The chemokine receptor CXCR3 is implicated in a variety of clinically important diseases, notably rheumatoid arthritis and atherosclerosis. Consequently, antagonists of CXCR3 are of therapeutic interest. In this study, we set out to characterize binding sites of the specific low MW CXCR3 antagonist VUF10085 and the broad spectrum antagonist TAK-779 which blocks CXCR3 along with CCR2 and CCR5. EXPERIMENTAL APPROACH: Molecular modelling of CXCR3, followed by virtual ligand docking, highlighted several CXCR3 residues likely to contact either antagonist, notably a conserved aspartate in helix 2 (Asp-112(2:63) ), which was postulated to interact with the quaternary nitrogen of TAK-779. Validation of modelling was carried out by site-directed mutagenesis of CXCR3, followed by assays of cell surface expression, ligand binding and receptor activation. KEY RESULTS: Mutation of Asn-132(3.33) , Phe-207 and Tyr-271(6.51) within CXCR3 severely impaired both ligand binding and chemotactic responses, suggesting that these residues are critical for maintenance of a functional CXCR3 conformation. Contrary to our hypothesis, mutation of Asp-112(2:63) had no observable effects on TAK-779 activity, but clearly decreased the antagonist potency of VUF 10085. Likewise, mutations of Phe-131(3.32) , Ile-279(6.59) and Tyr-308(7.43) were well tolerated and were critical for the antagonist activity of VUF 10085 but not for that of TAK-779. CONCLUSIONS AND IMPLICATIONS: This more detailed definition of a binding pocket within CXCR3 for low MW antagonists should facilitate the rational design of newer CXCR3 antagonists, with obvious clinical potential.Arthritis Research UK. Grant Number: #1830

The protease associated (PA) domain in ScpA from Streptococcus pyogenes plays a role in substrate recruitment

Annually, over 18 million disease cases and half a million deaths worldwide are estimated to be caused by Group A Streptococcus. ScpA (or C5a peptidase) is a well characterised member of the cell enveleope protease family, which possess a S8 subtilisin-like catalytic domain and a shared multi-domain architecture. ScpA cleaves complement factors C5a and C3a, impairing the function of these critical anaphylatoxins and disrupts complement-mediated innate immunity. Although the high resolution structure of ScpA is known, the details of how it recognises its substrate are only just emerging. Previous studies have identified a distant exosite on the 2nd fibronectin domain that plays an important role in recruitment via an interaction with the substrate core. Here, using a combination of solution NMR spectroscopy, mutagenesis with functional assays and computational approaches we identify a second exosite within the protease-associated (PA) domain. We propose a model in which the PA domain assists optimal delivery of the substrate's C terminus to the active site for cleavage

Small molecule chemokine mimetics suggest a molecular basis for the observation that CXCL10 and CXCL11 are allosteric ligands of CXCR3.

BACKGROUND AND PURPOSE: The chemokine receptor CXCR3 directs migration of T-cells in response to the ligands CXCL9/Mig, CXCL10/IP-10 and CXCL11/I-TAC. Both ligands and receptors are implicated in the pathogenesis of inflammatory disorders, including atherosclerosis and rheumatoid arthritis. Here, we describe the molecular mechanism by which two synthetic small molecule agonists activate CXCR3. EXPERIMENTAL APPROACH: As both small molecules are basic, we hypothesized that they formed electrostatic interactions with acidic residues within CXCR3. Nine point mutants of CXCR3 were generated in which an acidic residue was mutated to its amide counterpart. Following transient expression, the ability of the constructs to bind and signal in response to natural and synthetic ligands was examined. KEY RESULTS: The CXCR3 mutants D112N, D195N and E196Q were efficiently expressed and responsive in chemotaxis assays to CXCL11 but not to CXCL10 or to either of the synthetic agonists, confirmed with radioligand binding assays. Molecular modelling of both CXCL10 and CXCR3 suggests that the small molecule agonists mimic a region of the '30s loop' (residues 30-40 of CXCL10) which interacts with the intrahelical CXCR3 residue D112, leading to receptor activation. D195 and E196 are located in the second extracellular loop and form putative intramolecular salt bridges required for a CXCR3 conformation that recognizes CXCL10. In contrast, CXCL11 recognition by CXCR3 is largely independent of these residues. CONCLUSION AND IMPLICATIONS: We provide here a molecular basis for the observation that CXCL10 and CXCL11 are allosteric ligands of CXCR3. Such findings may have implications for the design of CXCR3 antagonists

Agarose Spot as a Comparative Method for in situ Analysis of Simultaneous Chemotactic Responses to Multiple Chemokines

yesWe describe a novel protocol to quantitatively and simultaneously compare the chemotactic responses of cells towards different chemokines. In this protocol, droplets of agarose gel containing different chemokines are applied onto the surface of a Petri dish, and then immersed under culture medium in which cells are suspended. As chemokine molecules diffuse away from the spot, a transient chemoattractant gradient is established across the spots. Cells expressing the corresponding cognate chemokine receptors migrate against this gradient by crawling under the agarose spots towards their centre. We show that this migration is chemokine-specific; meaning that only cells that express the cognate chemokine cell surface receptor, migrate under the spot containing its corresponding chemokine ligand. Furthermore, we show that migration under the agarose spot can be modulated by selective small molecule antagonists present in the cell culture medium

An Analysis of Factors Contributing to the Development of Total Parenteral Nutrition‐Induced Cholestasis

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141659/1/jpen0586.pd

Specifying enough light to feel reassured on pedestrian footpaths

This article discusses lighting for pedestrians and how investigation of reassurance might lead toward an understanding of the right amount of light. A conventional approach is to evaluate reassurance after dark under road lighting of different illuminance: this tends to show the trivial result that higher illuminances enhance reassurance, and that alone does not enable an optimum light level to be identified. One reason is that the category rating procedure widely used is prone to stimulus range bias; experimental results are presented that demonstrate stimulus range bias in reassurance evaluations. This article also recommends alternative methods for future research. One such method is the day–dark rating approach, which does not tend toward ever higher illuminances, and results are presented of two studies using this method

Structural Insights from Binding Poses of CCR2 and CCR5 with Clinically Important Antagonists: A Combined In Silico Study

Chemokine receptors are G protein-coupled receptors that contain seven transmembrane domains. In particular, CCR2 and CCR5 and their ligands have been implicated in the pathophysiology of a number of diseases, including rheumatoid arthritis and multiple sclerosis. Based on their roles in disease, they have been attractive targets for the pharmaceutical industry, and furthermore, targeting both CCR2 and CCR5 can be a useful strategy. Owing to the importance of these receptors, information regarding the binding site is of prime importance. Structural studies have been hampered due to the lack of X-ray crystal structures, and templates with close homologs for comparative modeling. Most of the previous models were based on the bovine rhodopsin and β2-adrenergic receptor. In this study, based on a closer homolog with higher resolution (CXCR4, PDB code: 3ODU 2.5 Å), we constructed three-dimensional models. The main aim of this study was to provide relevant information on binding sites of these receptors. Molecular dynamics simulation was done to refine the homology models and PROCHECK results indicated that the models were reasonable. Here, binding poses were checked with some established inhibitors of high pharmaceutical importance against the modeled receptors. Analysis of interaction modes gave an integrated interpretation with detailed structural information. The binding poses confirmed that the acidic residues Glu291 (CCR2) and Glu283 (CCR5) are important, and we also found some additional residues. Comparisons of binding sites of CCR2/CCR5 were done sequentially and also by docking a potent dual antagonist. Our results can be a starting point for further structure-based drug design

Implied Contribution Under the Federal Securities Laws: A Reassessment

Exposure to a strong T-helper 2 (Th2)-like environment during fetal development may promote allergy development. Increased cord blood (CB) levels of the Th2-associated chemokine CCL22 were associated with allergy development during the first 2 y of life. The aim of the present study was to determine whether CB Th1- and Th2-associated chemokine levels are associated with allergy development during the first 6 y of life, allowing assessment of respiratory allergic symptoms usually developing in this period. The CB levels of cytokines, chemokines, and total IgE were determined in 56 children of 20 women with allergic symptoms and 36 women without allergic symptoms. Total IgE and allergen-specific IgE antibody levels were quantified at 6, 12, 24 mo, and 6 y of age. Increased CB CCL22 levels were associated with development of allergic sensitization and asthma and increased CCL17 levels with development of allergic symptoms, including asthma. Sensitized children with allergic symptoms showed higher CB CCL17 and CCL22 levels and higher ratios between these Th2-associated chemokines and the Th1-associated chemokine CXCL10 than nonsensitized children without allergic symptoms. A pronounced Th2 deviation at birth, reflected by increased CB CCL17 and CCL22 levels, and increased CCL22/CXCL10 and CCL17/CXCL10 ratios might promote allergy development later in life