The proline-rich motif of the proDer p 3 allergen propeptide is crucial for protease-protease interaction.

The majority of proteases are synthesized in an inactive form, termed zymogen, which consists of a propeptide and a protease domain. The propeptide is commonly involved in the correct folding and specific inhibition of the enzyme. The propeptide of the house dust mite allergen Der p 3, NPILPASPNAT, contains a proline-rich motif (PRM), which is unusual for a trypsin-like protease. By truncating the propeptide or replacing one or all of the prolines in the non-glycosylated zymogen with alanine(s), we demonstrated that the full-length propeptide is not required for correct folding and thermal stability and that the PRM is important for the resistance of proDer p 3 to undesired proteolysis when the protein is expressed in Pichia pastoris. Additionally, we followed the maturation time course of proDer p 3 by coupling a quenched-flow assay to mass spectrometry analysis. This approach allowed to monitor the evolution of the different species and to determine the steady-state kinetic parameters for activation of the zymogen by the major allergen Der p 1. This experiment demonstrated that prolines 5 and 8 are crucial for proDer p 3-Der p 1 interaction and for activation of the zymogen.Peer reviewe

CXCL14 preferentially synergizes with homeostatic chemokine receptor systems

Reflecting their importance in immunity, the activity of chemokines is regulated on several levels, including tissue and context-specific expression and availability of their cognate receptor on target cells. Chemokine synergism, affecting both chemokine and chemokine receptor function, has emerged as an additional control mechanism. We previously demonstrated that CXCL14 is a positive allosteric modulator of CXCR4 in its ability to synergize with CXCL12 in diverse cellular responses. Here, we have extended our study to additional homeostatic, as well as a selection of inflammatory chemokine systems. We report that CXCL14 strongly synergizes with low (sub-active) concentrations of CXCL13 and CCL19/CCL21 in in vitro chemotaxis with immune cells expressing the corresponding receptors CXCR5 and CCR7, respectively. CXCL14 by itself was inactive, not only on cells expressing CXCR5 or CCR7 but also on cells expressing any other known conventional or atypical chemokine receptor, as assessed by chemotaxis and/or β-arrestin recruitment assays. Furthermore, synergistic migration responses between CXCL14 and inflammatory chemokines CXCL10/CXCL11 and CCL5, targeting CXCR3 and CCR5, respectively, were marginal and occasional synergistic Ca2+ flux responses were observed. CXCL14 bound to 300-19 cells and interfered with CCL19 binding to CCR7-expressing cells, suggesting that these cellular interactions contributed to the reported CXCL14-mediated synergistic activities. We propose a model whereby tissue-expressed CXCL14 contributes to cell localization under steady-state conditions at sites with prominent expression of homeostatic chemokines.publishe

Structure prediction of GPCRs using piecewise homologs and application to the human CCR5 chemokine receptor: validation through agonist andantagonist docking

International audienceThis article describes the construction and validation of a three-dimensional model of the human CC chemokine receptor 5 (CCR5) receptor using multiple homology modeling. A new methodology is presented where we built each secondary structural model of the protein separately from distantly related homologs of known structure. The reliability of our approach for G-protein coupled receptors was assessed through the building of the human C-X-C chemokine receptor type 4 (CXCR4) receptor of known crystal structure. The models are refined using molecular dynamics simulations and energy minimizations using CHARMM, a classical force field for proteins. Finally, docking models of both the natural agonists and the antagonists of the receptors CCR5 and CXCR4 are proposed. This study explores the possible binding process of ligands to the receptor cavity of chemokine receptors at molecular and atomic levels. We proposed few crucial residues in receptors binding to agonist/antagonist for further validation through experimental analysis. In particular, our study provides better understanding of the blockage mechanism of the chemokine receptors CCR5 and CXCR4, and may help the identification of new lead compounds for drug development in HIV infection, inflammatory diseases, and cancer metastasis

Implication of AurA kinase in GBM cells chemotaxis in response to the production of CXCL12 in the subventricular zones

Despite great improvement in standard therapies (i.e. surgery, radiotherapy and chemotherapy) of glioblastoma (GBM), the median survival rate is 15 months due to patient relapses. A major advance in the understanding of GBM recurrences has been the identification of GBM-initiating cells (GIC). GIC are thought to be deeply involved in GBM recurrences. Our lab designed a mouse model by grafting human GBM cells in the striatum. After the graft, we observed that tumors develop in the mouse striatum and that GIC specifically invade the subventricular zones (SVZ). SVZ are stem cells niches crucial for adult neurogenesis which seems particularly propitious for gliomagenesis since they are abundant in growth factors and permissive to proliferation. We therefore looked for soluble factors secreted by the SVZ environment and demonstrated that the local production of the CXCL12 chemokine in the SVZ is responsible for the GIC-directed migration. In this work, we aim to study the role GBM therapeutic resistance associated with the invasion of the SVZ. In this work, we identified a new actor of the CXCL12 pathway by the phosphoproteome analysis of U87MG cells stimulated with CXCL12: the mitotic kinase Aurora A (AurA) whose activity seems crucial for the CXCL12-dependent chemotaxis of GBM cellsImplication of AurA kinase in GBM cells chemotaxis in response to the production of CXCL12 in the subventricular zone

The Lys-Asp-Tyr Triad within the Mite Allergen Der p 1 Propeptide Is a Critical Structural Element for the pH-Dependent Initiation of the Protease Maturation.

The major house dust mite allergen, Der p 1, is a papain-like cysteine protease expressed as an inactive precursor, proDer p 1, carrying an N-terminal propeptide with a unique structure. The maturation of the zymogen into an enzymatically-active form of Der p 1 is a multistep autocatalytic process initiated under acidic conditions through conformational changes of the propeptide, leading to the loss of its inhibitory ability and its subsequent gradual cleavage. The aims of this study were to characterize the residues present in the Der p 1 propeptide involved in the initiation of the zymogen maturation process, but also to assess the impact of acidic pH on the propeptide structure, the activity of Der p 1 and the fate of the propeptide. Using various complementary enzymatic and structural approaches, we demonstrated that a structural triad K17p-D51p-Y19p within the N-terminal domain of the propeptide is essential for its stabilization and the sensing of pH changes. Particularly, the protonation of D51p under acidic conditions unfolds the propeptide through disruption of the K17p-D51p salt bridge, reduces its inhibition capacity and unmasks the buried residues K17p and Y19p constituting the first maturation cleavage site of the zymogen. Our results also evidenced that this triad acts in a cooperative manner with other propeptide pH-responsive elements, including residues E56p and E80p, to promote the propeptide unfolding and/or to facilitate its proteolysis. Furthermore, we showed that acidic conditions modify Der p 1 proteolytic specificity and confirmed that the formation of the first intermediate represents the limiting step of the in vitro Der p 1 maturation process. Altogether, our results provide new insights into the early events of the mechanism of proDer p 1 maturation and identify a unique structural triad acting as a stabilizing and a pH-sensing regulatory element

Orchestration of an uncommon maturation cascade of the house dust mite protease allergen quartet.

In more than 20% of the world population, sensitization to house dust mite allergens triggers typical allergic diseases such as allergic rhinitis and asthma. Amongst the 23 mite allergen groups hitherto identified, group 1 is cysteine proteases belonging to the papain-like family whereas groups 3, 6, and 9 are serine proteases displaying trypsin, chymotrypsin, and collagenolytic activities, respectively. While these proteases are more likely to be involved in the mite digestive system, they also play critical roles in the initiation and in the chronicity of the allergic response notably through the activation of innate immune pathways. All these allergenic proteases are expressed in mite as inactive precursor form. Until recently, the exact mechanisms of their maturation into active proteases remained to be fully elucidated. Recent breakthroughs in the understanding of the activation mechanisms of mite allergenic protease precursors have highlighted an uncommon and unique maturation pathway orchestrated by group 1 proteases that tightly regulates the proteolytic activities of groups 1, 3, 6, and 9 through complex intra- or inter-molecular mechanisms. This review presents and discusses the currently available knowledge of the activation mechanisms of group 1, 3, 6, and 9 allergens of Dermatophagoides pteronyssinus laying special emphasis on their localization, regulation, and interconnection

NanoLuX: a network-wide Nanoluciferase-based platform to monitor activation of classical and atypical chemokine receptors

The activity of chemokine receptors is dependent on G proteins that, upon chemokine binding, trigger various intracellular signalling cascades, as well as on b-arrestins that, following receptor phosphorylation by GRK, orchestrate its desensitization, endocytosis and trafficking. In addition to the 19 classical chemokine receptors, 4 receptors form a subfamily of atypical chemokine receptors (ACKR1-4) with ligand scavenging functions (Fig 1). These receptors are unable to couple to G proteins but their activity can be monitored via b-arrestins. In this study we describe the NanoLux platform, a network-wide profiling platform for chemokines and chemokines receptors based on the complementation of the nanoluciferase (NanoBiT). This platform allows to monitor the activation, modulation or bias of receptors or ligands by measuring the binding or the recruitment of effectors, regulators or parners such as G proteins, GRK or b-arrestin isoforms to the classical and the atypical chemokine receptors. For that purpose, we N-terminally fused the LgBiT portion of Nanoluciferase to the MiniGi protein, GRK2 or human b- arrestin 1 and 2, while the SmBiT was fused to the C terminus of all of the 23 human chemokine receptors (Fig 2). Using this approach, we are now able to assess and compare the activity of molecules at the chemokine-receptor network level

New paradigms in chemokine receptor signal transduction: Moving beyond the two-site model.

Chemokine receptor (CKR) signaling forms the basis of essential immune cellular functions, and dysregulated CKR signaling underpins numerous disease processes of the immune system and beyond. CKRs, which belong to the seven transmembrane domain receptor (7TMR) superfamily, initiate signaling upon binding of endogenous, secreted chemokine ligands. Chemokine-CKR interactions are traditionally described by a two-step/two-site mechanism, in which the CKR N-terminus recognizes the chemokine globular core (i.e. site 1 interaction), followed by activation when the unstructured chemokine N-terminus is inserted into the receptor TM bundle (i.e. site 2 interaction). Several recent studies challenge the structural independence of sites 1 and 2 by demonstrating physical and allosteric links between these supposedly separate sites. Others contest the functional independence of these sites, identifying nuanced roles for site 1 and other interactions in CKR activation. These developments emerge within a rapidly changing landscape in which CKR signaling is influenced by receptor PTMs, chemokine and CKR dimerization, and endogenous non-chemokine ligands. Simultaneous advances in the structural and functional characterization of 7TMR biased signaling have altered how we understand promiscuous chemokine-CKR interactions. In this review, we explore new paradigms in CKR signal transduction by considering studies that depict a more intricate architecture governing the consequences of chemokine-CKR interactions

G protein-receptor kinases 5/6 are the key regulators of G protein-coupled receptor 35-arrestin interactions

Human G protein–coupled receptor 35 is regulated by agonist-mediated phosphorylation of a set of five phospho-acceptor amino acids within its C-terminal tail. Alteration of both Ser300 and Ser303 to alanine in the GPR35a isoform greatly reduces the ability of receptor agonists to promote interactions with arrestin adapter proteins. Here, we have integrated the use of cell lines genome edited to lack expression of combinations of G protein receptor kinases (GRKs), selective small molecule inhibitors of subsets of these kinases, and antisera able to specifically identify either human GPR35a or mouse GPR35 only when Ser300 and Ser303 (orce; the equivalent residues in mouse GPR35) have become phosphorylated to demonstrate that GRK5 and GRK6 cause agonist-dependent phosphorylation of these residues. Extensions of these studies demonstrated the importance of the GRK5/6-mediated phosphorylation of these amino acids for agonist-induced internalization of the receptor. Homology and predictive modeling of the interaction of human GPR35 with GRKs showed that the N terminus of GRK5 is likely to dock in the same methionine pocket on the intracellular face of GPR35 as the C terminus of the α5 helix of Gα13 and, that while this is also the case for GRK6, GRK2 and GRK3 are unable to do so effectively. These studies provide unique and wide-ranging insights into modes of regulation of GPR35, a receptor that is currently attracting considerable interest as a novel therapeutic target in diseases including ulcerative colitis