14-3-3γ mediates the long-term inhibition of peripheral kappa opioid receptor antinociceptive signaling by norbinaltorphimine.

Long-term inhibition of kappa opioid receptor (KOR) signaling in peripheral pain-sensing neurons is a potential obstacle for development of peripherally-restricted KOR agonists that produce analgesia. Such a long-term inhibitory mechanism is invoked from activation of c-Jun N-terminal kinase (JNK) that follows a single injection of the KOR antagonist norbinaltorphimine (norBNI). This effect requires protein synthesis of an unknown mediator in peripheral pain-sensing neurons. Using 2D difference gel electrophoresis with tandem mass spectrometry, we have identified that the scaffolding protein 14-3-3γ is upregulated in peripheral sensory neurons following activation of JNK with norBNI. Knockdown of 14-3-3γ by siRNA eliminates the long-term reduction in KOR-mediated cAMP signaling by norBNI in peripheral sensory neurons in culture. Similarly, knockdown of 14-3-3γ in the rat hind paw abolished the norBNI-mediated long-term reduction in peripheral KOR-mediated antinociception. Further, overexpression of 14-3-3γ in KOR expressing CHO cells prevented KOR-mediated inhibition of cAMP signaling. These long-term effects are selective for KOR as heterologous regulation of other receptor systems was not observed. These data suggest that 14-3-3γ is both necessary and sufficient for the long-term inhibition of KOR by norBNI in peripheral sensory neurons.

The non-ELR CXC chemokine encoded by human cytomegalovirus UL146 genotype 5 contains a C-terminal ß-hairpin and induces neutrophil migration as a selective CXCR2 agonist.

Human cytomegalovirus (HCMV) is a major pathogen in immunocompromised patients. The UL146 gene exists as 14 diverse genotypes among clinical isolates, which encode 14 different CXC chemokines. One genotype (vCXCL1GT1) is a known agonist for CXCR1 and CXCR2, while two others (vCXCL1GT5 and vCXCL1GT6) lack the ELR motif considered crucial for CXCR1 and CXCR2 binding, thus suggesting another receptor targeting profile. To determine the receptor target for vCXCL1GT5, the chemokine was probed in a G protein signaling assay on all 18 classical human chemokine receptors, where CXCR2 was the only receptor being activated. In addition, vCXCL1GT5 recruited ß-arrestin in a BRET-based assay and induced migration in a chemotaxis assay through CXCR2, but not CXCR1. In contrast, vCXCL1GT1 stimulated G protein signaling, recruited ß-arrestin and induced migration through both CXCR1 and CXCR2. Both vCXCL1GT1 and vCXCL1GT5 induced equally potent and efficacious migration of neutrophils, and ELR vCXCL1GT4 and non-ELR vCXCL1GT6 activated only CXCR2. In contrast to most human chemokines, the 14 UL146 genotypes have remarkably long C-termini. Comparative modeling using Rosetta showed that each genotype could adopt the classic chemokine core structure, and predicted that the extended C-terminal tail of several genotypes (including vCXCL1GT1, vCXCL1GT4, vCXCL1GT5, and vCXCL1GT6) forms a novel ß-hairpin not found in human chemokines. Secondary NMR shift and TALOS+ analysis of vCXCL1GT1 supported the existence of two stable ß-strands. C-terminal deletion of vCXCL1GT1 resulted in a non-functional protein and in a shift to solvent exposure for tryptophan residues likely due to destabilization of the chemokine fold. The results demonstrate that non-ELR chemokines can activate CXCR2 and suggest that the UL146 chemokines have unique C-terminal structures that stabilize the chemokine fold. Increased knowledge of the structure and interaction partners of the chemokine variants encoded by UL146 is key to understanding why circulating HCMV strains sustain 14 stable genotypes.

The chemokine X-factor: Structure-function analysis of the CXC motif at CXCR4 and ACKR3.

The human chemokine family consists of 46 protein ligands that induce chemotactic cell migration by activating a family of 23 G protein-coupled receptors. The two major chemokine subfamilies, CC and CXC, bind distinct receptor subsets. A sequence motif defining these families, the X position in the CXC motif, is not predicted to make significant contacts with the receptor, but instead links structural elements associated with binding and activation. Here, we use comparative analysis of chemokine NMR structures, structural modeling, and molecular dynamic simulations that suggested the X position reorients the chemokine N terminus. Using CXCL12 as a model CXC chemokine, deletion of the X residue (Pro-10) had little to no impact on the folded chemokine structure but diminished CXCR4 agonist activity as measured by ERK phosphorylation, chemotaxis, and Gi/o-mediated cAMP inhibition. Functional impairment was attributed to over 100-fold loss of CXCR4 binding affinity. Binding to the other CXCL12 receptor, ACKR3, was diminished nearly 500-fold. Deletion of Pro-10 had little effect on CXCL12 binding to the CXCR4 N terminus, a major component of the chemokine-GPCR interface. Replacement of the X residue with the most frequent amino acid at this position (P10Q) had an intermediate effect between WT and P10del in each assay, with ACKR3 having a higher tolerance for this mutation. This work shows that the X residue helps to position the CXCL12 N terminus for optimal docking into the orthosteric pocket of CXCR4 and suggests that the CC/CXC motif contributes directly to receptor selectivity by orienting the chemokine N terminus in a subfamily-specific direction.

Comparative modeling and docking of chemokine-receptor interactions with Rosetta.

Chemokine receptors are a subset of G protein-coupled receptors defined by the distinct property of binding small protein ligands in the chemokine family. Chemokine receptors recognize their ligands by a mechanism that is distinct from other class A GPCRs that bind peptides or small molecules. For this reason, structural information on other ligand-GPCR interactions are only indirectly relevant to understanding the chemokine receptor interface. Additionally, the experimentally determined structures of chemokine-GPCR complexes represent less than 3% of the known interactions of this complex, multi-ligand/multi-receptor network. To enable predictive modeling of the remaining 97% of interactions, a general in silico protocol was designed to utilize existing chemokine receptor crystal structures, co-crystal structures, and NMR ensembles of chemokines bound to receptor fragments. This protocol was benchmarked on the ability to predict each of the three published co-crystal structures, while being blinded to the target structure. Averaging ensembles selected from the top-ranking models reproduced up to 84% of the intermolecular contacts found in the crystal structure, with the lowest Cα-RMSD of the complex at 3.3 Å. The chemokine receptor N-terminus, unresolved in crystal structures, was included in the modeling and recapitulates contacts with known sulfotyrosine binding pockets seen in structures derived from experimental NMR data. This benchmarking experiment suggests that realistic homology models of chemokine-GPCR complexes can be generated by leveraging current structural data.

Modeling the complete chemokine-receptor interaction.

Chemokines are soluble, secreted proteins that induce chemotaxis of leukocytes and other cells. Migratory cells can sense the chemokine concentration gradient following chemokine binding and activation of chemokine receptors, a subset of the G protein-coupled receptor (GPCR) superfamily. Chemokine receptor signaling plays a central role in cell migration during inflammatory responses as well as in cancer and other diseases. Given their important role in mediating essential pathologic and physiologic processes, chemokines and their receptors are attractive targets for therapeutic development. A better understanding of the molecular basis of chemokine-GPCR interactions will aid in the understanding of the mechanistic basis for chemokine function in disease-related processes, as well as aid in the design of new therapeutics. High resolution protein structures are critical for determining these mechanisms and investigating the interactions between approximately 50 chemokines and 20 chemokine receptors. Currently, three unique structures of chemokine-GPCR complexes have been determined and have greatly broadened our knowledge of this large protein-protein interaction. While these structures represent only a small fraction of clinically relevant chemokines and receptors, they can be exploited as scaffolds for homology modeling to understand the chemokine-GPCR interactions. This chapter presents a specialized methodology to construct and validate models of chemokine-GPCR complexes using the Rosetta software suite.

Design of artificial red blood cells using polymeric hydrogel microcapsules: hydrogel stability improvement and polymer selection.

PURPOSE: To improve the stability of pectin-oligochitosan hydrogel microcapsules under physiological conditions. METHODS: Two different approaches were examined: change of the cross-linker length and treatment of the hydrogel microcapsules with 150 Mm CaCl2. Replacement of pectin with alginate was also studied. RESULTS AND CONCLUSIONS: It was observed that the molecular weight of the cross-linker oligochiotsan had no significant improvement on microcapsule stability. On the other hand, the treatment of pectin-oligochitosan microcapsules with Ca2+ increased the microcapsule stability significantly. Different types of alginate were used; however, no red-blood-cell-shaped microcapsules could be produced, which is likely due to the charge-density difference between deprotonated pectin and alginate polymers.