Chemokine CCL28 Is a Potent Therapeutic Agent for Oropharyngeal Candidiasis.

Candida albicans is a commensal organism that causes life-threatening or life-altering opportunistic infections. Treatment of Candida infections is limited by the paucity of antifungal drug classes. Naturally occurring antimicrobial peptides are promising agents for drug development. CCL28 is a CC chemokine that is abundant in saliva and has in vitro antimicrobial activity. In this study, we examine the in vivo Candida killing capacity of CCL28 in oropharyngeal candidiasis as well as the spectrum and mechanism of anti-Candida activity. In the mouse model of oropharyngeal candidiasis, application of wild-type CCL28 reduces oral fungal burden in severely immunodeficient mice without causing excessive inflammation or altering tissue neutrophil recruitment. CCL28 is effective against multiple clinical strains of C. albicans Polyamine protein transporters are not required for CCL28 anti-Candida activity. Both structured and unstructured CCL28 proteins show rapid and sustained fungicidal activity that is superior to that of clinical antifungal agents. Application of wild-type CCL28 to C. albicans results in membrane disruption as measured by solute movement, enzyme leakage, and induction of negative Gaussian curvature on model membranes. Membrane disruption is reduced in CCL28 lacking the functional C-terminal tail. Our results strongly suggest that CCL28 can exert antifungal activity in part via membrane permeation and has potential for development as an anti-Candida therapeutic agent without inflammatory side effects.

Structure-function analysis of CCL28 in the development of post-viral asthma.

CCL28 is a human chemokine constitutively expressed by epithelial cells in diverse mucosal tissues and is known to attract a variety of immune cell types including T-cell subsets and eosinophils. Elevated levels of CCL28 have been found in the airways of individuals with asthma, and previous studies have indicated that CCL28 plays a vital role in the acute development of post-viral asthma. Our study builds on this, demonstrating that CCL28 is also important in the chronic post-viral asthma phenotype. In the absence of a viral infection, we also demonstrate that CCL28 is both necessary and sufficient for induction of asthma pathology. Additionally, we present the first effort aimed at elucidating the structural features of CCL28. Chemokines are defined by a conserved tertiary structure composed of a three-stranded ß-sheet and a C-terminal α-helix constrained by two disulfide bonds. In addition to the four disulfide bond-forming cysteine residues that define the traditional chemokine fold, CCL28 possesses two additional cysteine residues that form a third disulfide bond. If all disulfide bonds are disrupted, recombinant human CCL28 is no longer able to drive mouse CD4+ T-cell chemotaxis or in vivo airway hyper-reactivity, indicating that the conserved chemokine fold is necessary for its biologic activity. Due to the intimate relationship between CCL28 and asthma pathology, it is clear that CCL28 presents a novel target for the development of alternative asthma therapeutics.

Fragment-based drug discovery of small molecule ligands for the human chemokine CCL28.

The mucosal chemokine CCL28 is a promising target for immunotherapy drug development due to its elevated expression level in epithelial cells and critical role in creating and maintaining an immunosuppressive tumor microenvironment. Using sulfotyrosine as a probe, NMR chemical shift mapping identified a potential receptor-binding hotspot on the human CCL28 surface. CCL28 was screened against 2,678 commercially available chemical fragments by 2D NMR, yielding thirteen verified hits. Computational docking predicted that two fragments could occupy adjoining subsites within the sulfotyrosine recognition cleft. Dual NMR titrations confirmed their ability to bind CCL28 simultaneously, thereby validating an initial fragment pair for linking and merging strategies to design high-potency CCL28 inhibitors.

Conformational selection guides ß-arrestin recruitment at a biased G protein-coupled receptor.

G protein-coupled receptors (GPCRs) recruit ß-arrestins to coordinate diverse cellular processes, but the structural dynamics driving this process are poorly understood. Atypical chemokine receptors (ACKRs) are intrinsically biased GPCRs that engage ß-arrestins but not G proteins, making them a model system for investigating the structural basis of ß-arrestin recruitment. Here, we performed nuclear magnetic resonance (NMR) experiments on 13CH3-ε-methionine-labeled ACKR3, revealing that ß-arrestin recruitment is associated with conformational exchange at key regions of the extracellular ligand-binding pocket and intracellular ß-arrestin-coupling region. NMR studies of ACKR3 mutants defective in ß-arrestin recruitment identified an allosteric hub in the receptor core that coordinates transitions among heterogeneously populated and selected conformational states. Our data suggest that conformational selection guides ß-arrestin recruitment by tuning receptor dynamics at intracellular and extracellular regions.

Structure-function guided modeling of chemokine-GPCR specificity for the chemokine XCL1 and its receptor XCR1.

Chemokines interact with their G protein-coupled receptors (GPCRs) through a two-step, two-site mechanism and, through this interaction, mediate various homeostatic and immune response mechanisms. Upon initial recognition of the chemokine by the receptor, the amino terminus of the chemokine inserts into the orthosteric pocket of the GPCR, causing conformational changes that trigger intracellular signaling. There is considerable structural and functional evidence to suggest that the amino acid composition and length of the chemokine amino terminus is critical for GPCR activation, complementing the size and amino acid composition of the orthosteric pocket. However, very few structures of a native chemokine-receptor complex have been solved. Here, we used a hybrid approach that combines structure-function data with Rosetta modeling to describe key contacts within a chemokine-GPCR interface. We found that the extreme amino-terminal residues of the chemokine XCL1 (Val1, Gly2, Ser3, and Glu4) contribute a large fraction of the binding energy to its receptor XCR1, whereas residues near the disulfide bond-forming residue Cys11 modulate XCR1 activation. Alterations in the XCL1 amino terminus changed XCR1 activation, as determined by assessing inositol triphosphate accumulation, intracellular calcium release, and directed cell migration. Computational analysis of XCL1-XCR1 interactions revealed functional contacts involving Glu4 of XCL1 and Tyr117 and Arg273 of XCR1. Subsequent mutation of Tyr117 and Arg273 led to diminished binding and activation of XCR1 by XCL1. These findings demonstrate the utility of a hybrid approach, using biological data and homology modeling, to study chemokine-GPCR interactions.

Decoding the chemotactic signal.

From an individual bacterium to the cells that compose the human immune system, cellular chemotaxis plays a fundamental role in allowing cells to navigate, interpret, and respond to their environments. While many features of cellular chemotaxis are shared among systems as diverse as bacteria and human immune cells, the machinery that guides the migration of these model organisms varies widely. In this article, we review current literature on the diversity of chemoattractant ligands, the cell surface receptors that detect and process chemotactic gradients, and the link between signal recognition and the regulation of cellular machinery that allow for efficient directed cellular movement. These facets of cellular chemotaxis are compared among E. coli, Dictyostelium discoideum, and mammalian neutrophils to derive organizational principles by which diverse cell systems sense and respond to chemotactic gradients to initiate cellular migration.

The Solution Structure of CCL28 Reveals Structural Lability that Does Not Constrain Antifungal Activity.

The chemokine CCL28 is constitutively expressed in mucosal tissues and is abundant in low-salt mucosal secretions. Beyond its traditional role as a chemoattractant, CCL28 has been shown to act as a potent and broad-spectrum antimicrobial agent with particular efficacy against the commensal fungus and opportunistic pathogen Candida albicans. However, the structural features that allow CCL28 to perform its chemotactic and antimicrobial functions remain unknown. Here, we report the structure of CCL28, solved using nuclear magnetic resonance spectroscopy. CCL28 adopts the canonical chemokine tertiary fold, but also has a disordered C-terminal domain that is partially tethered to the core by a non-conserved disulfide bond. Structure-function analysis reveals that removal of the C-terminal tail reduces the antifungal activity of CCL28 without disrupting its structural integrity. Conversely, removal of the non-conserved disulfide bond destabilizes the tertiary fold of CCL28 without altering its antifungal effects. Moreover, we report that CCL28 unfolds in response to low pH but is stabilized by the presence of salt. To explore the physiologic relevance of the observed structural lability of CCL28, we investigated the effects of pH and salt on the antifungal activity of CCL28 in vitro. We found that low pH enhances the antifungal potency of CCL28, but also that this pH effect is independent of CCL28's tertiary fold. Given its dual role as a chemoattractant and antimicrobial agent, our results suggest that changes in the salt concentration or pH at mucosal sites may fine-tune CCL28's functional repertoire by adjusting the thermostability of its structure.