Tristan Zellmann

Processing, signaling, and physiological function of chemerin

Chemerin is an immunomodulating factor secreted predominantly by adipose tissue and skin. Processed by a variety of proteases linked to inflammation, it activates the G‐protein coupled receptor chemokine‐like receptor 1 (CMKLR1) and induces chemotaxis in natural killer cells, macrophages, and immature dendritic cells. Recent developments revealed the role of the nonsignaling chemerin receptor C‐C chemokine receptor‐like 2 (CCRL2) in inflammation. Besides further research establishing its link to inflammatory skin conditions such as psoriasis, functions in healthy skin have also been reported. Here, the current understanding of chemerin processing, signaling and physiological function has been summarized, focusing on the regulation of its activity, its different receptors and its controversially discussed role in diseases.

Proteolytic activation of prochemerin by kallikrein 7 breaks an ionic linkage and results in C-terminal rearrangement

The excessive accumulation of adipose tissue in obesity is associated with multiple inflammatory dermatological diseases. Chemerin, a chemoattractant adipokine, dependent on proteolytical activation, is highly expressed in skin. Different proteases have been reported to activate prochemerin, but none is inherently expressed in human skin. In the present study, we identified a tissue-specific protease and investigated the underlying mechanism of activation at the molecular level. We characterized human KLK7 (kallikrein 7) as a prochemerin processing protease in vitro converting prochemerin into active chemerinF(156). The activating truncation by the protease might trigger a structural rearrangement leading to an increased affinity of chemerin to CMKLR1 (chemokine-like receptor 1). Molecular modelling and experimental data suggest an underlying ionic interaction in prochemerin C-terminal domains. These findings provide a general molecular basis for the necessity of C-terminal processing of prochemerin. Moreover, immunohistochemistry was used to investigate prochemerin, KLK7 and the recently identified KLK7 inhibitor vaspin expression in human skin biopsies, and distinct co-localization in psoriatic biopsies was observed. On the basis of these results, it is hypothesized that KLK7 activity may contribute to the development of psoriatic lesions as a consequence of excessive chemerin activation and impaired protease activity regulation by vaspin. Therefore this interaction represents an interesting target for psoriasis therapy and treatment of other obesity-related diseases.

Unwinding of the C‐Terminal Residues of Neuropeptide Y is critical for Y2 Receptor Binding and Activation

Despite recent breakthroughs in the structural characterization of G-protein-coupled receptors (GPCRs), there is only sparse data on how GPCRs recognize larger peptide ligands. NMR spectroscopy, molecular modeling, and double-cycle mutagenesis studies were integrated to obtain a structural model of the peptide hormone neuropeptide Y (NPY) bound to its human G-protein-coupled Y2 receptor (Y2R). Solid-state NMR measurements of specific isotope-labeled NPY in complex with in vitro folded Y2R reconstituted into phospholipid bicelles provided the bioactive structure of the peptide. Guided by solution NMR experiments, it could be shown that the ligand is tethered to the second extracellular loop by hydrophobic contacts. The C-terminal α-helix of NPY, which is formed in a membrane environment in the absence of the receptor, is unwound starting at T(32) to provide optimal contacts in a deep binding pocket within the transmembrane bundle of the Y2R.

A Deep Hydrophobic Binding Cavity is the Main Interaction for Different Y2R Antagonists

The neuropeptide Y2 receptor (Y2R) is involved in various pathophysiological processes such as epilepsy, mood disorders, angiogenesis, and tumor growth. Therefore, the Y2R is an interesting target for drug development. A detailed understanding of the binding pocket could facilitate the development of highly selective antagonists to study the role of Y2R in vitro and in vivo. In this study, several residues crucial to the interaction of BIIE0246 and SF‐11 derivatives with Y2R were investigated by signal transduction assays. Using the experimental results as constraints, the antagonists were docked into a comparative structural model of the Y2R. Despite differences in size and structure, all three antagonists display a similar binding site, including a deep hydrophobic cavity formed by transmembrane helices (TM) 4, 5, and 6, as well as a hydrophobic patch at the top of TM2 and 7. Additionally, we suggest that the antagonists block Q3.32, a position that has been shown to be crucial for binding of the amidated C terminus of NPY and thus for receptor activation.

Comparison of agonist and antagonist binding sites of the neuropeptide Y receptor 2

e.g. appetite regulation, energy homeostasis, bone formation, circadian rhythm, memory retention, obesity, epilepsy, angiogenesis and cancer (Pedragosa-Badia et al., 2013; Walther et al., 2011). In various tumor tissues the hY2R is overexpressed and promotes tumor growth and vascularization (Lorner and Reubi, 2008). Therefore, the hY2R has great therapeutic potential and antagonists could represent promising drugs for the treatment of neuroblastoma or glioblastoma. Wewere interested in investigating the binding mode of BIIE0246, compound 40 and compound46 at the hY2R (Mittapalli et al., 2012).We generated receptor mutants and tested membrane localization by fluorescence microscopy and signal transduction by inositol phosphate accumulation assay in presence of pNPY and pNPY/antagonist. Here we report on suggested binding modes of three different antagonists on hY2R, which opens up the possibility for the development of more selective compounds.

Hydrophobic contacts specifically contribute to peptide binding at the neuropeptide Y2 receptor

assays, namelymobilization of intracellular calcium in the Fura-2/AM and in an aequorin-based assay with mitochondrially targeted photoprotein. Herewe present the results of investigations on the humanNPYY2 andY4 receptors. Native pNPY, K4hPP as well as newNPY Y4R ligands developed in our laboratory were studied on CHO cells, stably expressing the receptor of interest, the chimeric G protein qi5 and apoaequorin (Ziemek et al., 2006, 2007). Both peptides showed higher potency when studied with the label-free technique [EC50 (pNPY)= 3.80 nM, EC50 (K4hPP)= 2.95 nM] compared to the Fura-2/AM [EC50 (pNPY)= 4.57 nM, EC50 (K4hPP)= 11.22 nM] and the aequorin-based assay [EC50 (pNPY)= 14.45 nM, EC50 (K4hPP)= 75.86 nM]. Interestingly, the agonist-induced signals were completely suppressed after pretreatmentwith the selective Gαq/11 inhibitor UBO-QIC in the conventional assays, whereas the DMR signal was just partially reduced (approximately by 20%). Inversely, pertussis toxin almost completely prevented the DMR signal, but only partially inhibited the calcium response, suggesting additional cellular reactions to contribute to the holistic readout.