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Article:Croker, D.E., Halai, R., Kaeslin, G. et ReuseUnless indicated otherwise, fulltext items are protected by copyright with all rights reserved. The copyright exception in section 29 of the Copyright, Designs and Patents Act 1988 allows the making of a single copy solely for the purpose of non-commercial research or private study within the limits of fair dealing. The publisher or other rights-holder may allow further reproduction and re-use of this version -refer to the White Rose Research Online record for this item. Where records identify the publisher as the copyright holder, users can verify any specific terms of use on the publisher's website. TakedownIf you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request. ABSTRACTThe complement system is a major component of our innate immune system, in which the complement proteins C5a and C5a-des Arg bind to two G-protein coupled receptors (GPCR);namely the C5a receptor (C5a1) and C5a receptor like-2 receptor (C5a2, formerly called C5L2). Recently, it has been demonstrated that C5a, but not C5a-des Arg, up-regulates heteromer formation between C5a1 and C5a2, leading to an increase in IL-10 release from human monocyte derived macrophages (HMDM). A bioluminescence resonance energy transfer (BRET) assay was used to assess recruitment of -arrestins by C5a and C5a-des Arg at the C5a1 and C5a2 receptors. C5a demonstrated elevated -arrestin 2 recruitment levels in comparison to C5a-des Arg, while no significant difference was observed at C5a2. A constitutive complex that formed between -arrestin 2 and C5a2 accounted for half of the BRET signal observed. Interestingly, both C5a and C5a-des Arg exhibited higher potency for -arrestin 2 recruitment via C5a2, indicating preference for C5a2 over C5a1. When C5a was tested in a functional ERK1/2 assay in HMDM, inhibition of ERK1/2 was only observed at concentrations at or above the EC 50 for heteromer formation. This suggested that increased recruitment of the -arrestin-C5a2 complex at these C5a concentrations might have an inhibitory role on C5a1 signaling through ERK1/2. An improved understanding of C5a2 modulation of signaling in acute inflammation could be of benefit in the development of ligands for conditions such as sepsis.

Proline-rich antimicrobial peptides (PrAMPs) represent promising alternative therapeutic options for the treatment of multidrug-resistant bacterial infections. PrAMPs are predominantly active against Gram-negative bacteria by inhibiting protein expression via at least two different modes of action, i.e., blocking the ribosomal exit tunnel of 70S ribosomes (oncocin-type binding) or inhibiting the assembly of the 50S ribosomal subunit (apidaecin-type binding). The in vivo efficacy and favorable biodistribution of oncocins confirmed the therapeutic potential of short PrAMPs for the first time, whereas the in vivo evaluation of apidaecins is still limited despite the promising efficacy of apidaecin-analog Api88 in an intraperitoneal murine infection model. Here, the in vivo efficacy of apidaecin-analog Api137 was studied, which rescued all NMRI mice from a lethal intraperitoneal infection with E. coli ATCC 25922 when administered three times intraperitoneal at doses of 0.6 mg/kg starting 1 h after infection. When Api88 and Api137 were administered intravenous or intraperitoneal at doses of 5 and 20 mg/kg, their plasma levels were similarly low (<3 μg/mL) and four-fold lower than for oncocin-analog Onc72. This contradicted earlier expectation based on the very low serum stability of Api88 with a half-life time of only ~5 min compared to ~6 and ~3 h for Api137 and Onc72, respectively. Pharmacokinetic data relying on a sensitive mass spectrometry method utilizing multiple reaction monitoring and isotope-labeled peptides revealed that Api88 and Api137 were present in blood, urine, and kidney, and liver homogenates at similar levels accompanied by the same major metabolites comprising residues 1–16 and 1–17. The pretended discrepancy was solved, when all peptides were incubated in peritoneal lavage. Api137 was rapidly degraded at the C-terminus, while Api88 was rather stable despite releasing the same degradation products. Onc72 was very stable explaining its higher plasma levels compared to Api88 and Api137 after intraperitoneal administration illuminating its good in vivo efficacy. The data indicate that the degradation of therapeutic peptides should be studied in serum and further body fluids. Moreover, the high efficacy in murine infection models and the fast clearance of Api88 and Api137 within ~60 min after intravenous and ~90 min after intraperitoneal injections indicate that their in vivo efficacy relates to the maximal peptide concentration achieved in blood.

C5aR exerts detrimental functions in acute colitis, strongly supporting the idea that a C5aR-antagonist might be useful for IBD treatment. However, since the absence of C5aR was no longer protective and in some regards disadvantageous in chronic IBD, future studies should address the efficacy and the possible side effects of a sustained antagonist treatment.

BackgroundMelanoma cells are usually characterized by a strong proliferative potential and efficient invasive migration. Among the multiple molecular changes that are recorded during progression of this disease, aberrant activation of receptor tyrosine kinases (RTK) is often observed. Activation of matrix metalloproteases goes along with RTK activation and usually enhances RTK-driven migration. The purpose of this study was to examine RTK-driven three-dimensional migration of melanocytes and the pro-tumorigenic role of matrix metalloproteases for melanocytes and melanoma cells.ResultsUsing experimental melanocyte dedifferentiation as a model for early melanomagenesis we show that an activated EGF receptor variant potentiates migration through three-dimensional fibrillar collagen. EGFR stimulation also resulted in a strong induction of matrix metalloproteases in a MAPK-dependent manner. However, neither MAPK nor MMP activity were required for migration, as the cells migrated in an entirely amoeboid mode. Instead, MMPs fulfilled a function in cell cycle regulation, as their inhibition resulted in strong growth inhibition of melanocytes. The same effect was observed in the human melanoma cell line A375 after stimulation with FCS. Using sh- and siRNA techniques, we could show that MMP13 is the protease responsible for this effect. Along with decreased proliferation, knockdown of MMP13 strongly enhanced pigmentation of melanocytes.ConclusionsOur data show for the first time that growth stimuli are mediated via MMP13 in melanocytes and melanoma, suggesting an autocrine MMP13-driven loop. Given that MMP13-specific inhibitors are already developed, these results support the evaluation of these inhibitors in the treatment of melanoma.