Generation of biologically active interleukin-1beta by meprin B.
ABSTRACT Interleukin-1beta (IL-1beta) is a proinflammatory cytokine that is synthesized as an inactive precursor molecule that must be proteolytically processed to generate the biologically active form. Maturation of the precursor is primarily performed by caspase-1, an intracellular cysteine protease; however, processing by other proteases has been described. Meprins are cell surface and secreted metalloproteases expressed by renal and intestinal brush-border membranes, leukocytes, and cancer cells. In this study we show that purified recombinant meprin B can process the interleukin-1beta precursor to a biologically active form. Amino-terminal sequencing and mass spectrometry analysis of the product of digestion by activated meprin B determined that proteolytic cleavage resulted in an additional six amino acids relative to the site utilized by caspase-1. The biological activity of the meprin B-cleaved cytokine was confirmed by measuring the proliferative response of helper T-cells. These results suggest that meprin may play an important role in activation of this proinflammatory cytokine in various pathophysiological conditions.
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ABSTRACT: Inflammatory diseases are a significant burden on global healthcare systems, and tackling these diseases is a major focus of modern medicine. Key to many inflammatory diseases is the cytokine, Interleukin-1 (IL-1). Due to its apical role in initiating the inflammatory response, dysregulated IL-1 signalling results in a number of pathologies. Treatment of inflammatory diseases with anti-IL-1 therapies has offered many therapeutic benefits, however current therapies are protein based, with all the accompanying limitations. The non-conventional pathways involved in IL-1 signalling provide a number of potential therapeutic targets for clinical intervention and this has led to the exploitation of a number of model organisms for the study of IL-1 biology. Murine models have long been used to study IL-1 processing and release, but do not allow direct visualisation in vivo. Recently, fish models have emerged as genetically tractable and optically transparent inflammatory disease models. These models have raised questions on the evolutionary origins of the IL-1 family and the conservation in its processing and activation. Here we review the current understanding of IL-1 evolution in fish and discuss the study of IL-1 processing in these models.Developmental and comparative immunology 03/2014; DOI:10.1016/j.dci.2014.03.008 · 3.71 Impact Factor
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ABSTRACT: Zinc metalloproteinases meprin α and meprin β are implicated in a variety of diseases, such as fibrosis, inflammation and neurodegeneration, however, there are no selective small molecule inhibitors that would allow to study their role in these processes. To address this lack of molecular tools we have developed high throughput screening (HTS) assays to enable discovery of inhibitors of both meprin α and meprin β and screened a collection of well characterized pharmaceutical agents (LOPAC, n = 1,280 compounds). Two compounds (PPNDS, NF449) confirmed their activity and selectivity for meprin β. Kinetic studies revealed competitive (PPNDS) and mixed competitive/non-competitive (NF449) inhibition mechanisms suggesting that binding occurs in meprin β active site. Both PPNDS and NF449 exhibited low nanomolar IC50 and Ki values making them the most potent and selective inhibitors of meprin β reported to the date. These results demonstrate the ability of meprin α and β assays to identify selective compounds and discard artifacts of primary screening.Biopolymers 09/2014; 102(5). DOI:10.1002/bip.22527 · 2.29 Impact Factor
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ABSTRACT: Cleavable linkers, which are specifically cleaved by defined conditions or enzymes, are powerful tools that can be used for various purposes. Amongst other things, they have been successfully used to deliver toxic payloads as prodrugs into target tissues. In this work novel linker sequences targeting meprin β, a metalloprotease expressed in the kidney brush-border membrane, were designed and included in the sequence of three radiolabelled exendin-4 derivatives. As radiolabelled exendin-4 derivatives strongly accumulate in the kidneys, we hypothesised that specific cleavage of the radiolabelled moiety at the kidney brush-border membrane would allow easier excretion of the activity into the urine and therefore improve the pharmacological properties of the peptide. The insertion of a cleavable linker did not negatively influence the in vitro properties of the peptides. They showed a good affinity to the GLP-1 receptor expressed in CHL cells, a high internalisation and sufficiently high stability in fresh human blood plasma. In vitro digestion with recombinant meprin β rapidly metabolised the corresponding linker sequences. After 60 min the majority of the corresponding peptides were digested and at the same time the anticipated fragments were formed. The peptides were also quickly metabolised in CD1 nu/nu mouse kidney homogenates. Immunofluorescence staining of meprin β in kidney sections confirmed the expression of the protease in the kidney brush-border membrane. Biodistribution in GLP-1 receptor positive tumour-xenograft bearing mice revealed high specific uptake of the 111In-labelled tracers in receptor positive tissue. Accumulation in the kidneys, however, was still high and comparable to the lead compound 111In-Ex4NOD40. In conclusion, we show that the concept of cleavable linkers specific for meprin β is feasible, as the peptides are rapidly cleaved by the enzyme while retaining their biological properties.PLoS ONE 04/2015; 10(4):e0123443. DOI:10.1371/journal.pone.0123443 · 3.53 Impact Factor