Characterization of the Cancer-Associated Meprin Βeta Variants G45R and G89R.

Meprin ß is a metalloprotease associated with neurodegeneration, inflammation, extracellular matrix homeostasis, transendothelial cell migration, and cancer. In this study, we investigated two melanoma-associated variants of meprin ß, both exhibiting a single amino acid exchange, namely, meprin ß G45R and G89R. Based on the structural data of meprin ß and with regard to the position of the amino acid exchanges, we hypothesized an increase in proteolytic activity in the case of the G45R variant due to the induction of a potential new activation site and a decrease in proteolytic activity from the G89R variant due to structural instability. Indeed, the G89R variant showed, overall, a reduced expression level compared to wild-type meprin ß, accompanied by decreased activity and lower cell surface expression but strong accumulation in the endoplasmic reticulum. This was further supported by the analysis of the shedding of the interleukin-6 receptor (IL-6R) by meprin ß and its variants. In transfected HEK cells, the G89R variant was found to generate less soluble IL-6R, whereas the expression of meprin ß G45R resulted in increased shedding of the IL-6R compared to wild-type meprin ß and the G89R variant. A similar tendency of the induced shedding capacity of G45R was seen for the well-described meprin ß substrate CD99. Furthermore, employing an assay for cell migration in a collagen IV matrix, we observed that the transfection of wild-type meprin ß and the G45R variant resulted in increased migration of HeLa cells, while the G89R variant led to diminished mobility.

Cancer-associated mutations in the canonical cleavage site do not influence CD99 shedding by the metalloprotease meprin ß but alter cell migration in vitro.

Transendothelial cell migration (TEM) is crucial for inflammation and metastasis. The adhesion molecule CD99 was shown to be important for correct immune cell extravasation and is highly expressed on certain cancer cells. Recently, we demonstrated that ectodomain shedding of CD99 by the metalloprotease meprin ß promotes TEM in vitro. In this study, we employed an acute inflammation model (air pouch/carrageenan) and found significantly less infiltrated cells in meprin ß knock-out animals validating the previously observed pro-inflammatory activity. To further analyze the impact of meprin ß on CD99 shedding with regard to cell adhesion and proliferation we characterized two lung cancer associated CD99 variants (D92H, D92Y), carrying point mutations at the main cleavage site. Interestingly, ectodomain shedding of these variants by meprin ß was still detectable. However the cleavage site shifted to adjacent positions. Nevertheless, expression of CD99 variants D92H and D92Y revealed partial misfolding and proteasomal degradation. A previously observed influence of CD99 on Src activation and increased proliferation could not be confirmed in this study, independent of wild-type CD99 or the variants D92H and D92Y. However, we identified meprin ß as a potent inducer of Src phosphorylation. Importantly, we found significantly increased cell migration when expressing the cancer-associated CD99 variant D92H compared to the wild-type protein.

Meprin Metalloproteases Generate Biologically Active Soluble Interleukin-6 Receptor to Induce Trans-Signaling.

Soluble Interleukin-6 receptor (sIL-6R) mediated trans-signaling is an important pro-inflammatory stimulus associated with pathological conditions, such as arthritis, neurodegeneration and inflammatory bowel disease. The sIL-6R is generated proteolytically from its membrane bound form and A Disintegrin And Metalloprotease (ADAM) 10 and 17 were shown to perform ectodomain shedding of the receptor in vitro and in vivo. However, under certain conditions not all sIL-6R could be assigned to ADAM10/17 activity. Here, we demonstrate that the IL-6R is a shedding substrate of soluble meprin α and membrane bound meprin ß, resulting in bioactive sIL-6R that is capable of inducing IL-6 trans-signaling. We determined cleavage within the N-terminal part of the IL-6R stalk region, distinct from the cleavage site reported for ADAM10/17. Interestingly, meprin ß can be shed from the cell surface by ADAM10/17 and the observation that soluble meprin ß is not capable of shedding the IL-6R suggests a regulatory mechanism towards trans-signaling. Additionally, we observed a significant negative correlation of meprin ß expression and IL-6R levels on human granulocytes, providing evidence for in vivo function of this proteolytic interaction.

Ectodomain shedding of CD99 within highly conserved regions is mediated by the metalloprotease meprin ß and promotes transendothelial cell migration.

The adhesion molecule CD99 is essential for the transendothelial migration of leukocytes. In this study, we used biochemical and cellular assays to show that CD99 undergoes ectodomain shedding by the metalloprotease meprin ß and subsequent intramembrane proteolysis by γ-secretase. The cleavage site in CD99 was identified by mass spectrometry within an acidic region highly conserved through different vertebrate species. This finding fits perfectly to the unique cleavage specificity of meprin ß with a strong preference for aspartate residues and suggests coevolution of protease and substrate. We hypothesized that limited CD99 cleavage by meprin ß would alter cellular transendothelial migration (TEM) behavior in tissue remodeling processes, such as inflammation and cancer. Indeed, meprin ß induced cell migration of Lewis lung carcinoma cells in an in vitro TEM assay. Accordingly, deficiency of meprin ß in Mep1b-/- mice resulted in significantly increased CD99 protein levels in the lung. Therefore, meprin ß could serve as a therapeutic target, given that in a proof-of-concept approach we showed accumulation of CD99 protein in lungs of meprin ß inhibitor-treated mice.-Bedau, T., Peters, F., Prox, J., Arnold, P., Schmidt, F., Finkernagel, M., Köllmann, S., Wichert, R., Otte, A., Ohler, A., Stirnberg, M., Lucius, R., Koudelka, T., Tholey, A., Biasin, V., Pietrzik, C. U., Kwapiszewska, G., Becker-Pauly, C. Ectodomain shedding of CD99 within highly conserved regions is mediated by the metalloprotease meprin ß and promotes transendothelial cell migration.

Novel Potent Proline-Based Metalloproteinase Inhibitors: Design, (Radio)Synthesis, and First in Vivo Evaluation as Radiotracers for Positron Emission Tomography.

As dysregulation of matrix metalloproteinase (MMP) activity is associated with a wide range of pathophysiological processes like cancer, atherosclerosis, and arthritis, MMPs represent a valuable target for the development of new therapeutics and diagnostic tools. We herein present the chiral pool syntheses, in vitro evaluation, and SAR studies of a series of d- and l-proline- as well as of (4R)-4-hydroxy-l-proline-derived MMP inhibitors possessing general formula 1. Some of the synthesized hydroxamic acids were found to be potent MMP inhibitors with IC50 values in the nanomolar range, also demonstrating no off-target effects toward the other tested Zn2+-dependent metalloproteases (ADAMs and meprins). Utilizing the structure of the (2S,4S)-configured 4-hydroxyproline derivative 4, a selective picomolar inhibitor of MMP-13, the radiolabeled counterpart [18F]4 was successfully synthesized. The radiotracer's biodistribution in mice as well as its serum stability were evaluated for assessing its potential use as a MMP-13 targeting PET imaging agent.

Tetraspanin 8 is an interactor of the metalloprotease meprin ß within tetraspanin-enriched microdomains.

Meprin ß is a dimeric type I transmembrane protein and acts as an ectodomain sheddase at the cell surface. It was shown that meprin ß cleaves the amyloid precursor protein (APP), thereby releasing neurotoxic amyloid ß peptides and implicating a role of meprin ß in Alzheimer's disease. In order to identify non-proteolytic regulators of meprin ß, we performed a split ubiquitin yeast two-hybrid screen using a small intestinal cDNA library. In this screen we identified tetraspanin 8 (TSPAN8) as interaction partner for meprin ß. Since several members of the tetraspanin family were described to interact with metalloproteases thereby affecting their localization and/or activity, we hypothesized similar functions of TSPAN8 in the regulation of meprin ß. We employed cell biological methods to confirm direct binding of TSPAN8 to meprin ß. Surprisingly, we did not observe an effect of TSPAN8 on the catalytic activity of meprin ß nor on the specific cleavage of its substrate APP. However, both proteins were identified being present in tetraspanin-enriched microdomains. Therefore we hypothesize that TSPAN8 might be important for the orchestration of meprin ß at the cell surface with impact on certain proteolytic processes that have to be further identified.

Tetraspanin 8 is an interactor of the metalloprotease meprin ß within tetraspanin-enriched microdomains.

Meprin ß is a dimeric type I transmembrane protein and acts as an ectodomain sheddase at the cell surface. It has been shown that meprin ß cleaves the amyloid precursor protein (APP), thereby releasing neurotoxic amyloid ß peptides and implicating a role of meprin ß in Alzheimer's disease. In order to identify non-proteolytic regulators of meprin ß, we performed a split ubiquitin yeast two-hybrid screen using a small intestinal cDNA library. In this screen we identified tetraspanin 8 (TSPAN8) as interaction partner for meprin ß. As several members of the tetraspanin family were described to interact with metalloproteases thereby affecting their localization and/or activity, we hypothesized similar functions of TSPAN8 in the regulation of meprin ß. We employed cell biological methods to confirm direct binding of TSPAN8 to meprin ß. Surprisingly, we did not observe an effect of TSPAN8 on the catalytic activity of meprin ß nor on the specific cleavage of its substrate APP. However, both proteins were identified as present in tetraspanin-enriched microdomains. Therefore we hypothesize that TSPAN8 might be important for the orchestration of meprin ß at the cell surface with impact on certain proteolytic processes that have to be further identified.

The intact Kunitz domain protects the amyloid precursor protein from being processed by matriptase-2.

Proteolytic processing of the amyloid precursor protein (APP) leads to amyloid-ß (Aß) peptides. So far, the mechanism of APP processing is insufficiently characterized at the molecular level. Whereas the knowledge of Aß generation by several proteases has been expanded, the contribution of the Kunitz-type protease inhibitor domain (KPI) present in two major APP isoforms to the complex proteolytic processing of APP is poorly understood. In this study, we have identified KPI-containing APP as a very potent, slow-binding inhibitor for the membrane-bound proteolytic regulator of iron homeostasis matriptase-2 by forming stable complexes with its target protease in HEK cells. Inhibition and complex formation depend on the intact KPI domain. By inhibiting matriptase-2, KPI-containing APP is protected from matriptase-2-mediated proteolysis within the Aß region, thus preventing the generation of N-terminally truncated Aß.

Metalloprotease meprin ß is activated by transmembrane serine protease matriptase-2 at the cell surface thereby enhancing APP shedding.

Increased expression of metalloprotease meprin ß is associated with fibrotic syndromes and Alzheimer's disease (AD). Hence, regulation of meprin activity might be a suitable strategy for the treatment of these conditions. Meprin ß is a type 1 transmembrane protein, but can be released from the cell surface by ectodomain shedding. The protease is expressed as an inactive zymogen and requires proteolytic maturation by tryptic serine proteases. In the present study, we demonstrate, for the first time, the differences in the activation of soluble and membrane bound meprin ß and suggest transmembrane serine protease 6 [TMPRSS6 or matriptase-2 (MT2)] as a new potent activator, cleaving off the propeptide of meprin ß between Arg(61) and Asn(62) as determined by MS. We show that MT2, but not TMPRSS4 or pancreatic trypsin, is capable of activating full-length meprin ß at the cell surface, analysed by specific fluorogenic peptide cleavage assay, Western blotting and confocal laser scanning microscopy (CLSM). Maturation of full-length meprin ß is required for its activity as a cell surface sheddase, releasing the ectodomains of transmembrane proteins, as previously shown for the amyloid precursor protein (APP).

Calcium negatively regulates meprin ß activity and attenuates substrate cleavage.

The meprin ß metalloproteinase is an important enzyme in extracellular matrix turnover, inflammation, and neurodegeneration in humans and mice. Previous studies showed a diminished cleavage of certain meprin ß substrates in the presence of calcium, although the mechanism was not clear. With the help of a specific fluorogenic peptide assay and the human amyloid precursor protein as substrate, we demonstrated that the influence of calcium is most likely a direct effect on human meprin ß itself. Analyzing the crystal structures of pro- and mature meprin ß helped to identify a cluster of negatively charged amino acids forming a potential calcium binding site. Mutation of 2 of these residues (D204A and D245A) led to severe differences in proteolytic activity and cellular localization of meprin ß. D245A was almost completely inactive and largely stored into intracellular vesicles, indicating severe misfolding of the protein. Astonishingly, D204A was not transported to the cell surface, but exhibited strong ß-secretase activity, resulting in massive accumulation of Aß-peptides. This could be explained by constitutive maturation of this meprin ß mutant already in the early secretory pathway. We hypothesize that lacking D204 abrogates the capability of binding calcium in the catalytic domain, an important step for proper folding of the propeptide and subsequent inhibition of the protease. This is supported by the inhibition constant of calcium for meprin ß (inhibitory constant 50 = 11 mM), which resembles the physiologic concentrations found in the endoplasmic reticulum. For instance, it was shown for amyotrophic lateral sclerosis that a loss of calcium in the endoplasmic reticulum leads to the misfolding of calcium-dependent proteins, which might also be relevant for proper function of meprin ß.