No description available.

No description available.

No description available.

No description available.

Chemokines are important mediators of leukocyte migration. Chemokines bind to G protein–coupled receptors (GPCR) and cause conformational changes that trigger intracellular signaling pathways involved in inflammation, injury healing, cancer, metastasis, and HIV infections. No direct structural information about any chemokine receptor is available, but the structure of chemokines has been well studied. Structural studies of chemokines coupled with cell-biological investigations may lead to a better understanding of the mechanisms of chemokine-receptor interactions. In this Ph.D. project, I studied the structural and functional relationship between chemokines and chemokine receptors using NMR, X-ray crystallography, and mutagenesis approaches, coupled with several different cell-biology assays. We found that the conserved “chemokine fold” can support different dimerization types in the chemokines family, although changing the dimers from CC- to CXC-type fold is not readily accomplished. I also used an engineered covalently-bound dimer of the MIP-1β mutant, MIP-1β-A10C, to study the relationship between dimerization of chemokines and their interaction with the CCR5 receptor. My results suggest that MIP-1β dimer neither bind nor activate the CCR5 receptor. I also studied the biophysical properties of one N-terminal awkward mutant of P2-RANTES, which was originally selected by others from a phage display using CCR5-expressing cells. Although the NMR and X-ray crystal studies revealed that the wild type RANTES is a tight homodimer, analytical ultracentrifugation reveals that P2-RANTES is a monomer in solution, the 1.7 Å resolution X-ray crystal structure of P2-RANTES was found to be a packed tetramer. The mutated N-terminal residues play a very important role in the tetramerization in the X-ray crystal structure. Finally I used the HIV-1 env mediated cell-cell fusion assay to study the combination of chemokines or chemokine variants with anti-HIV peptides C37 or/and T-20. A surprisingly synergistic effect was found between P2-RANTES and C37 or T-20. This combination stratagem may lead to further useful drug combinations or drug delivery for more potent anti-HIV treatments.

Chemokines (chemotactic cytokines) comprise a large family of proteins that recruit and activate leukocytes, giving chemokines a major role in both immune response and inflammation-related diseases. Viral CC chemokine inhibitor (vCCI) is a poxvirus encoded protein that has been shown to bind tightly and inhibit the action of many CC chemokines. This function suggests that vCCI could be explored as an antiinflammatory therapeutic, a possibility that has been supported in mouse studies. The structure of vCCI in unbound form was determined by others, but to date no structure has been reported of bound vCCI. We report the NMR structure of vCCI in complex with the human CC chemokine MIP-1[beta]. The non-aggregating MIP-1[beta] variant MIP-1[beta] 45AASA48 was used in this complex to allow sufficiently high concentration at pH 7 to carry out the solution structure determination. A combination of NOE distance restraints, torsion angle restraints, and residual dipolar coupling were used to determine the structure of the complex, which also required protein deuteration due to its relatively large size (34kDa). The structure shows that MIP-1[beta] binds to vCCI with 1:1 stoichiometry, forming a complex of 311 amino acids. vCCI uses residues from its [beta]- sheet II to interact with a surface of MIP-1[beta] that includes residues adjacent to its Nterminus, as well as residues in the 20's region, and the 40's loop. The structure of the MIP-1[beta]-vCCI complex reveals for the first time the regions of each protein involved in the interaction, and allows a greater understanding of the strategy used by vCCI to tightly bind numerous chemokines, while retaining selectivity for the CC chemokine subfamily.

Universiẗat, Diss., 2007--Giessen.

Darmstadt, Techn. Univ., Diss., 2005.

This research focuses on the antimicrobial activity of the mouse chemokine CCL28. In addition to their well characterized chemotactic activity, many chemokines have been shown to be antimicrobial in vitro, including the mucosally expressed chemokine CCL28. I have investigated the primary sequence features required for antimicrobial activity, salt sensitive nature of killing/binding mechanism, and in vivo microbial interactions of CCL28. Through the use of protein mutation and expression techniques, I have shown that the holoprotein (108 amino acids) is necessary for full antimicrobial activity of CCL28. Furthermore, the C terminal region of CCL28 is essential for microbial killing as an almost complete loss of antimicrobial activity is seen following the removal of the C terminal 24 amino acids. The positively charged amino acids of the C-terminus directly contributed to the antimicrobial activity of CCL28. These experiments are the first to investigate the role of primary structure on the killing activity of an antimicrobial chemokine. Using flow cytometry analysis, I found that the salt-sensitive nature of CCL28 killing activity corresponds to its binding ability. Additionally, I have shown direct evidence for in vivo interaction between commensal bacteria and endogenously expressed CCL28 in the mouse large intestine. This interaction may directly correlate to the in vivo antimicrobial activity of CCL28. Lastly, I have begun to generate a CCL28 knockout mouse model to directly address the in vivo antimicrobial activity of CCL28. Vector construction and ES cell targeting by the vector has been completed, chimeric mouse generation remains to be done. This work represents the first systematic study of antimicrobial chemokine function. This work extends our understanding of antimicrobial proteins and their role in innate immune protection of the host and provides guidance for making better alternative antimicrobials.

Chemokines triggered by Toll-like receptors (TLRs) are small chemoattractant proteins, which mainly regulate leukocyte trafficking in inflammatory reactions via interaction with G protein-coupled receptors. Forty-two chemokines and 19 cognate receptors have been found in the human genome. Prior to this study, only 11 chicken chemokines and 7 receptors had been reported. The objectives of this study were to identify systematically chicken chemokines and their cognate receptor genes in the chicken genome and to annotate these genes and ligand-receptor binding by a comparative genomics approach. Twenty-three chemokine and 14 chemokine receptor genes were identified in the chicken genome. The number of coding exons in these genes and the syntenies are highly conserved between human, mouse, and chicken although the amino acid sequence homologies are generally low between mammalian and chicken chemokines. Chicken genes were named with the systematic nomenclature used in humans and mice based on phylogeny, synteny, and sequence homology. The independent nomenclature of chicken chemokines and chemokine receptors suggests that the chicken may have ligand-receptor pairings similar to mammals. The TLR family represents evolutionarily conserved components of the patternrecognizing receptors (PRRs) of the innate immune system that recognize specific pathogen-associated molecular patterns (PAMPs) through their ectodomains (ECDs). TLR's ECDs contain 19 to 25 tandem copies of leucine-rich repeat (LRR) motifs. TLRs play important roles in the activation of pro-inflammatory cytokines, chemokines and modulation of antigen-specific adaptive immune responses. To date, nine TLRs have been reported in chicken, along with a non-functional TLR8. Two non-mammalian TLRs, TLR21 and TLR22, have been identified in pufferfish and zebrafish. The objectives of this study were to determine if there is the existence of chicken genes homologous to fish-specific TLRs, and if possible ligands of these receptors exist. After searching the chicken genome sequence and EST database, a novel chicken TLR homologous to fish TLR21 was identified. Phylogenetic analysis indicated that the identified chicken TLR is the orthologue of TLR21 in fish. Bioinformatic analysis of potential PAMP binding sites within LRR insertions showed that CpG DNA is the putative ligand of this receptor.