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1.
Int J Mol Sci ; 18(9)2017 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-28841151

RESUMO

Chemokines are secreted proteins that direct the migration of immune cells and are involved in numerous disease states. For example, CCL21 (CC chemokine ligand 21) and CCL19 (CC chemokine ligand 19) recruit antigen-presenting dendritic cells and naïve T-cells to the lymph nodes and are thought to play a role in lymph node metastasis of CCR7 (CC chemokine receptor 7)-expressing cancer cells. For many chemokine receptors, N-terminal posttranslational modifications, particularly the sulfation of tyrosine residues, increases the affinity for chemokine ligands and may contribute to receptor ligand bias. Chemokine sulfotyrosine (sY) binding sites are also potential targets for drug development. In light of the structural similarity between sulfotyrosine and phosphotyrosine (pY), the interactions of CCL21 with peptide fragments of CCR7 containing tyrosine, pY, or sY were compared using protein NMR (nuclear magnetic resonance) spectroscopy in this study. Various N-terminal CCR7 peptides maintain binding site specificity with Y8-, pY8-, or sY8-containing peptides binding near the α-helix, while Y17-, pY17-, and sY17-containing peptides bind near the N-loop and ß3-stand of CCL21. All modified CCR7 peptides showed enhanced binding affinity to CCL21, with sY having the largest effect.


Assuntos
Quimiocina CCL21/metabolismo , Receptores CCR7/metabolismo , Tirosina/análogos & derivados , Sequência de Aminoácidos , Sítios de Ligação , Quimiocina CCL21/química , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Peptídeos/química , Peptídeos/metabolismo , Fosfotirosina , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Receptores CCR7/química , Tirosina/química , Tirosina/metabolismo
2.
J Biol Chem ; 288(14): 10024-10034, 2013 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-23408426

RESUMO

Chemokine receptors are commonly post-translationally sulfated on tyrosine residues in their N-terminal regions, the initial site of binding to chemokine ligands. We have investigated the effect of tyrosine sulfation of the chemokine receptor CCR2 on its interactions with the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). Inhibition of CCR2 sulfation, by growth of expressing cells in the presence of sodium chlorate, significantly reduced the potency for MCP-1 activation of CCR2. MCP-1 exists in equilibrium between monomeric and dimeric forms. The obligate monomeric mutant MCP-1(P8A) was similar to wild type MCP-1 in its ability to induce leukocyte recruitment in vivo, whereas the obligate dimeric mutant MCP-1(T10C) was less effective at inducing leukocyte recruitment in vivo. In two-dimensional NMR experiments, sulfated peptides derived from the N-terminal region of CCR2 bound to both the monomeric and dimeric forms of wild type MCP-1 and shifted the equilibrium to favor the monomeric form. Similarly, MCP-1(P8A) bound more tightly than MCP-1(T10C) to the CCR2-derived sulfopeptides. NMR chemical shift mapping using the MCP-1 mutants showed that the sulfated N-terminal region of CCR2 binds to the same region (N-loop and ß3-strand) of both monomeric and dimeric MCP-1 but that binding to the dimeric form also influences the environment of chemokine N-terminal residues, which are involved in dimer formation. We conclude that interaction with the sulfated N terminus of CCR2 destabilizes the dimerization interface of inactive dimeric MCP-1, thus inducing dissociation to the active monomeric state.


Assuntos
Quimiocina CCL2/metabolismo , Regulação da Expressão Gênica , Receptores CCR2/química , Tirosina/química , Sítios de Ligação , Cálcio/metabolismo , Dimerização , Células HEK293 , Humanos , Cinética , Espectroscopia de Ressonância Magnética/métodos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Químicos , Peptídeos/química , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Receptores CCR2/fisiologia , Enxofre/química
3.
J Immunol ; 189(5): 2365-73, 2012 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-22855709

RESUMO

The classical pathway of complement is crucial to the immune system, but it also contributes to inflammatory diseases when dysregulated. Binding of the C1 complex to ligands activates the pathway by inducing autoactivation of associated C1r, after which C1r activates C1s. C1s cleaves complement component C4 and then C2 to cause full activation of the system. The interaction between C1s and C4 involves active site and exosite-mediated events, but the molecular details are unknown. In this study, we identified four positively charged amino acids on the serine protease domain that appear to form a catalytic exosite that is required for efficient cleavage of C4. These residues are coincidentally involved in coordinating a sulfate ion in the crystal structure of the protease. Together with other evidence, this pointed to the involvement of sulfate ions in the interaction with the C4 substrate, and we showed that the protease interacts with a peptide from C4 containing three sulfotyrosine residues. We present a molecular model for the interaction between C1s and C4 that provides support for the above data and poses questions for future research into this aspect of complement activation.


Assuntos
Domínio Catalítico/imunologia , Ativação do Complemento/imunologia , Complemento C1s/metabolismo , Complemento C4/metabolismo , Via Clássica do Complemento/imunologia , Serina Proteases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação de Anticorpos/imunologia , Complemento C4/imunologia , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo
4.
J Biol Chem ; 287(18): 14692-702, 2012 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-22396538

RESUMO

Chemokine-receptor interactions regulate leukocyte trafficking during inflammation. CC chemokines exist in equilibrium between monomeric and dimeric forms. Although the monomers can activate chemokine receptors, dimerization is required for leukocyte recruitment in vivo, and it remains controversial whether dimeric CC chemokines can bind and activate their receptors. We have developed an obligate dimeric mutant of the chemokine monocyte chemoattractant protein-1 (MCP-1) by substituting Thr(10) at the dimer interface with Cys. Biophysical analysis showed that MCP-1(T10C) forms a covalent dimer with similar structure to the wild type MCP-1 dimer. Initial cell-based assays indicated that MCP-1(T10C) could activate chemokine receptor CCR2 with potency reduced 1 to 2 orders of magnitude relative to wild type MCP-1. However, analysis of size exclusion chromatography fractions demonstrated that the observed activity was due to a small proportion of MCP-1(T10C) being monomeric and highly potent, whereas the majority dimeric form could neither bind nor activate CCR2 at concentrations up to 1 µM. These observations help to reconcile previous conflicting results and indicate that dimeric CC chemokines do not bind to their receptors with affinities approaching those of the corresponding monomeric chemokines.


Assuntos
Quimiocina CCL2/metabolismo , Mutação de Sentido Incorreto , Multimerização Proteica , Substituição de Aminoácidos , Linhagem Celular , Quimiocina CCL2/química , Quimiocina CCL2/genética , Humanos , Receptores CCR2/química , Receptores CCR2/genética , Receptores CCR2/metabolismo
5.
Science ; 351(6269): 186-90, 2016 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-26657283

RESUMO

The addition of polysialic acid to N- and/or O-linked glycans, referred to as polysialylation, is a rare posttranslational modification that is mainly known to control the developmental plasticity of the nervous system. Here we show that CCR7, the central chemokine receptor controlling immune cell trafficking to secondary lymphatic organs, carries polysialic acid. This modification is essential for the recognition of the CCR7 ligand CCL21. As a consequence, dendritic cell trafficking is abrogated in polysialyltransferase-deficient mice, manifesting as disturbed lymph node homeostasis and unresponsiveness to inflammatory stimuli. Structure-function analysis of chemokine-receptor interactions reveals that CCL21 adopts an autoinhibited conformation, which is released upon interaction with polysialic acid. Thus, we describe a glycosylation-mediated immune cell trafficking disorder and its mechanistic basis.


Assuntos
Quimiocina CCL21/metabolismo , Quimiotaxia , Células Dendríticas/fisiologia , Linfonodos/fisiologia , Processamento de Proteína Pós-Traducional , Receptores CCR7/metabolismo , Ácidos Siálicos/metabolismo , Animais , Células da Medula Óssea/fisiologia , Glicosilação , Ligantes , Linfonodos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes
6.
ACS Chem Biol ; 8(9): 1955-63, 2013 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-23802178

RESUMO

Tyrosine sulfation is a post-translational modification that enhances protein-protein interactions and may identify druggable sites in the extracellular space. The G protein-coupled receptor CXCR4 is a prototypical example with three potential sulfation sites at positions 7, 12, and 21. Each receptor sulfotyrosine participates in specific contacts with its chemokine ligand in the structure of a soluble, dimeric CXCL12:CXCR4(1-38) complex, but their relative importance for CXCR4 binding and activation by the monomeric chemokine remains undefined. NMR titrations with short sulfopeptides showed that the tyrosine motifs of CXCR4 varied widely in their contributions to CXCL12 binding affinity and site specificity. Whereas the Tyr21 sulfopeptide bound the same site as in previously solved structures, the Tyr7 and Tyr12 sulfopeptides interacted nonspecifically. Surprisingly, the unsulfated Tyr7 peptide occupied a hydrophobic site on the CXCL12 monomer that is inaccessible in the CXCL12 dimer. Functional analysis of CXCR4 mutants validated the relative importance of individual CXCR4 sulfotyrosine modifications (Tyr21 > Tyr12 > Tyr7) for CXCL12 binding and receptor activation. Biophysical measurements also revealed a cooperative relationship between sulfopeptide binding at the Tyr21 site and CXCL12 dimerization, the first example of allosteric behavior in a chemokine. Future ligands that occupy the sTyr21 recognition site may act as both competitive inhibitors of receptor binding and allosteric modulators of chemokine function. Together, our data suggests that sulfation does not ubiquitously enhance complex affinity and that distinct patterns of tyrosine sulfation could encode oligomer selectivity, implying another layer of regulation for chemokine signaling.


Assuntos
Quimiocina CXCL12/metabolismo , Peptídeos/metabolismo , Receptores CXCR4/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Quimiocina CXCL12/química , Cricetulus , Humanos , Modelos Moleculares , Peptídeos/química , Ligação Proteica , Multimerização Proteica , Receptores CXCR4/química , Tirosina/química , Tirosina/metabolismo
7.
Chem Commun (Camb) ; 48(10): 1547-9, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22109100

RESUMO

Synthesis of sulfated and unsulfated (glyco)peptide fragments of Hirudin P6 (a potent anticoagulant from the leech Hirudinaria manillensis) is described. The effect of O-glycosylation and tyrosine sulfation on thrombin binding and peptidolytic activity was investigated, together with the inhibition of fibrinogen cleavage.


Assuntos
Peptídeos/farmacologia , Trombina/antagonistas & inibidores , Tirosina/química , Sítios de Ligação/efeitos dos fármacos , Glicosilação , Conformação Molecular , Peptídeos/síntese química , Peptídeos/química , Relação Estrutura-Atividade
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