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The metalloproteases meprin α and meprin β are highly expressed in the healthy gut but significantly decreased in inflammatory bowel disease, implicating a protective role in mucosal homeostasis. In the colon, meprin α and meprin β form covalently linked heterodimers tethering meprin α to the plasma membrane, therefore presenting dual proteolytic activity in a unique enzyme complex. To unravel its function, we applied N-terminomics and identified galectin-3 as the major intestinal substrate for meprin α/β heterodimers. Galectin-3–deficient and meprin α/β double knockout mice show similar alterations in their microbiome in comparison to wild-type mice. We further demonstrate that meprin α/β heterodimers differentially process galectin-3 upon bacterial infection, in germ-free, conventionally housed (specific pathogen–free), or wildling mice, which in turn regulates the bacterial agglutination properties of galectin-3. Thus, the constitutive cleavage of galectin-3 by meprin α/β heterodimers may play a key role in colon host-microbiome homeostasis.

Background The gastric microbiome and inflammation play a key role in gastric cancer (GC) by regulating the immune response in a complex manner and by inflammatory events supporting carcinogenesis. Meprin β is a zinc endopeptidase and participates in tissue homeostasis, intestinal barrier function and immunological processes. It influences local inflammatory processes, dysbiosis and the microbiome. Here, we tested the hypothesis that meprin β is expressed in GC and of tumor biological significance. Patients and methods Four hundred forty whole mount tissue sections of patients with therapy-naive GC were stained with an anti-meprin β antibody. The histoscore and staining pattern were analyzed for each case. Following dichotomization at the median histoscore into a “low” and “high” group, the expression was correlated with numerous clinicopathological patient characteristics. Results Meprin β was found intracellularly and at the cell membrane of GC. Cytoplasmic expression correlated with the phenotype according to Lauren, microsatellite instability and PD-L1 status. Membranous expression correlated with intestinal phenotype, mucin-1-, E-cadherin-, β-catenin status, mucin typus, microsatellite instability, KRAS mutation and PD-L1-positivity. Patients with cytoplasmic expression of meprin β showed a better overall and tumor-specific survival. Conclusions Meprin β is differentially expressed in GC and has potential tumor biological relevance. It might function as a tumor suppressor or promotor depending on histoanatomical site and context.

Dysregulation of proteolytic enzymes has huge impact on epidermal homeostasis, which can result in severe pathological conditions such as fibrosis or Netherton syndrome. The metalloprotease meprin β was found to be upregulated in hyperproliferative skin diseases. AP-1 transcription factor complex has been reported to induce Mep1b expression. Since AP-1 and its subunit fos-related antigen 2 (fra-2) are associated with the onset and progression of psoriasis, we wanted to investigate if this could partially be attributed to increased meprin β activity. Here, we demonstrate that fra-2 transgenic mice show increased meprin β expression and proteolytic activity in the epidermis. To avoid influence by other fra-2 regulated genes, we additionally generated a mouse model that enabled tamoxifen-inducible expression of meprin β under the Krt5-promotor to mimic the pathological condition. Interestingly, induced meprin β expression in the epidermis resulted in hyperkeratosis, hair loss and mottled pigmentation of the skin. Employing N-terminomics revealed syndecan-1 as a substrate of meprin β in skin. Shedding of syndecan-1 at the cell surface caused delayed calcium-induced differentiation and impaired adhesion of keratinocytes, which was blocked by the meprin β inhibitor fetuin-B.

The horseshoe crab Limulus polyphemus is one of few extant Limulus species, which date back to ∼250 million years ago under the conservation of a common Bauplan documented by fossil records. It possesses the only proteolytic blood-coagulation and innate immunity system outside vertebrates and is a model organism for the study of the evolution and function of peptidases. The astacins are a family of metallopeptidases that share a central ∼200-residue catalytic domain (CD), which is found in >1000 species across holozoans and, sporadically, bacteria. Here, the zymogen of an astacin from L. polyphemus was crystallized and its structure was solved. A 34-residue, mostly unstructured pro-peptide (PP) traverses, and thus blocks, the active-site cleft of the CD in the opposite direction to a substrate. A central `PP motif' (F35-E-G-D-I39) adopts a loop structure which positions Asp38 to bind the catalytic metal, replacing the solvent molecule required for catalysis in the mature enzyme according to an `aspartate-switch' mechanism. Maturation cleavage of the PP liberates the cleft and causes the rearrangement of an `activation segment'. Moreover, the mature N-terminus is repositioned to penetrate the CD moiety and is anchored to a buried `family-specific' glutamate. Overall, this mechanism of latency is reminiscent of that of the other three astacins with known zymogenic and mature structures, namely crayfish astacin, human meprin β and bacterial myroilysin, but each shows specific structural characteristics. Remarkably, myroilysin lacks the PP motif and employs a cysteine instead of the aspartate to block the catalytic metal., This study was supported in part by grants from Spanish and Catalan public and private bodies (grant/fellowship references PID2019-107725RG-I00 from MICIN/AEI/10.13039/ 501100011033 to FXG-R, TG and AR-B, 2017SGR3 and Fundacio´ La Marato´ de TV3 201815 to FXG-R, TG and AR-B). Further support was obtained from German funding bodies (grant SFB877, Project A9: ‘Proteolysis as a regulatory Event in Pathophysiology’ from the Deutsche Forschungsgemeinschaft to CB-P).

Membrane-type-I matrix metalloproteinase (MT1-MMP) is one of six human membrane-bound MMPs and is responsible for extracellular matrix remodelling by degrading several substrates like fibrillar collagens, including types I-III, or fibronectin. Moreover, MT1-MMP was described as a key player in cancer progression and it is involved in various inflammatory processes, as well as in the pathogenesis of Alzheimer's disease (AD). The membrane-tethered metalloprotease meprin β as well as a disintegrin and metalloproteinase 10 (ADAM10) and ADAM17 are also associated with these diseases. Interestingly, meprin β, ADAM10/17 and MT1-MMP also have a shared substrate pool including the interleukin-6 receptor and the amyloid precursor protein. We investigated the interaction of these proteases, focusing on a possible connection between MT1-MMP and meprin β, to elucidate the potential mutual regulations of both enzymes. Herein, we show that besides ADAM10/17, MT1-MMP is also able to shed meprin β from the plasma membrane, leading to the release of soluble meprin β. Mass spectrometry-based cleavage site analysis revealed that the cleavage of meprin β by all three proteases occurs between Pro

The metalloprotease meprin β (Mep1b) is capable of cleaving cell-adhesion molecules in different tissues (e.g. skin, kidney and intestine) and is dysregulated in several diseases associated with barrier breakdown (Alzheimer´s disease, kidney disruption, inflammatory bowel disease). In this study, we demonstrate that Mep1b is a novel regulator of tight junction (TJ) composition and blood–brain barrier (BBB) integrity in brain endothelium. In Mep1b-transfected mouse brain endothelial cells (bEnd.3), we observed a reduction of the TJ protein claudin-5, decreased transendothelial electrical resistance (TEER) and an elevated permeability to paracellular diffusion marker [14C]-inulin. Analysis of global Mep1b knock-out (Mep1b−/−) mice showed increased TJ protein expression (claudin-5, occludin, ZO-1) in cerebral microvessels and increased TEER in cultivated primary mouse brain endothelial compared to wild-type (wt) mice. Furthermore, we investigated the IgG levels in cerebrospinal fluid (CSF) and the brain water content as additional permeability markers and detected lower IgG levels and reduced brain water content in Mep1b−/− mice compared to wt mice. Showing opposing features in overexpression and knock-out, we conclude that Mep1b plays a role in regulating brain endothelial TJ-proteins and therefore affecting BBB tightness in vitro and in vivo.

Despite the neurodegenerative disorder Alzheimer’s disease (AD) is the most common form of dementia in late adult life, there is currently no therapy available to prevent the onset or slow down the progression of AD. The progressive cognitive decline in AD correlates with a successive accumulation of cerebral amyloid-β (Aβ) due to impaired clearance mechanisms. A significant percentage is removed by low-density lipoprotein receptor-related protein 1 (LRP1)-mediated transport across the blood–brain barrier (BBB) into the periphery. Circulating proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to members of the low-density lipoprotein receptor protein family at the cell surface and targets them for lysosomal degradation, which reduces the number of functional receptors. However, the adverse impact of PCSK9 on LRP1-mediated brain Aβ clearance remains elusive. By using an established BBB model, we identified reduced LRP1-mediated brain-to-blood Aβ clearance due to PCSK9 across different endothelial monolayer in vitro. Consequently, the repetitive application of FDA-approved monoclonal anti-PCSK9 antibodies into 5xFAD mice decreased the cerebral Aβ burden across variants and aggregation state, which was not reproducible in brain endothelial-specific LRP1−/− 5xFAD mice. The peripheral PCSK9 inhibition reduced Aβ pathology in prefrontal cortex and hippocampus–brain areas critically involved in memory processing—and prevented disease-related impairment in hippocampus-dependent memory formation. Our data suggest that peripheral inhibition of PCSK9 by already available therapeutic antibodies may be a novel and easily applicable potential AD treatment.

Cardiometabolic diseases, such as type 2 diabetes and cardiovascular disease, have a high public health burden. Understanding the genetically determined regulation of proteins that are dysregulated in disease can help to dissect the complex biology underpinning them. Here, we perform a protein quantitative trait locus (pQTL) analysis of 248 serum proteins relevant to cardiometabolic processes in 2893 individuals. Meta-analyzing whole-genome sequencing (WGS) data from two Greek cohorts, MANOLIS (n = 1356; 22.5× WGS) and Pomak (n = 1537; 18.4× WGS), we detect 301 independently associated pQTL variants for 170 proteins, including 12 rare variants (minor allele frequency

Alzheimer's disease (AD) is the most commonly diagnosed dementia but its underlying pathological mechanisms still unclear. Neuroinflammation and secretion of cytokines such as interleukin-6 (IL-6) accompany the main hallmarks of the disease: amyloid plaques and neurofibrillary tangles. In this study, we analyzed the role of IL-6 trans-signaling in two mouse models of AD, Tg2576 and 3xTg-AD mice. The inhibition of IL-6 trans-signaling partially rescued the AD-induced mortality in females of both models. Before amyloid plaques deposition, it reversed AD-induced changes in exploration and anxiety (but did not affect locomotion) in Tg2576 female mice. However, after plaque deposition the only behavioral trait affected by the inhibition of IL-6 trans-signaling was locomotion. Results in the Morris water maze suggest that cognitive flexibility was reduced by the blocking of the IL-6 trans-signaling in young and old Tg2576 female mice. The inhibition of IL-6 trans-signaling also decreased amyloid plaque burden in cortex and hippocampus, and Aβ40 and Aβ42 levels in the cortex, of Tg2576 female mice. The aforementioned changes might be correlated with changes in blood vessels and matrix structure and organization rather than changes in neuroinflammation. 3xTgAD mice showed a very mild phenotype regarding amyloid cascade, but results were in accordance with those of Tg2576 mice. These results strongly suggest that the inhibition of the IL-6 trans-signaling could represent a powerful therapeutic target in AD.

Systemic inflammatory disorders (SIDs) comprise a broad range of diseases characterized by dysregulated excessive innate immune responses. Severe forms of SIDs can lead to organ failure and death, and their increasing incidence represents a major issue for the health care system. Protease-mediated ectodomain shedding of cytokines and their receptors represents a central mechanism in the regulation of inflammatory responses. The metalloprotease A Disintegrin And Metalloproteinase (ADAM) 17 is the best-characterized ectodomain sheddase capable of releasing TNF-α and soluble IL-6 receptor, which are decisive factors of systemic inflammation. Recently, meprin metalloproteases were also identified as IL-6 receptor sheddases and activators of the pro-inflammatory cytokines IL-1β and IL-18. In different mouse models of SID, particularly those mimicking a sepsis-like phenotype, ADAM17 and meprins have been found to promote disease progression. In this review, we summarize the role of ADAM10, ADAM17 and meprins in the onset and progression of sepsis and discuss their potential as therapeutic targets.

β-Site amyloid precursor protein (APP) cleaving enzyme-1 (BACE1) is the major described β-secretase to generate Aβ peptides in Alzheimer’s disease (AD). However, all therapeutic attempts to block BACE1 activity and to improve AD symptoms have so far failed. A potential candidate for alternative Aβ peptides generation is the metalloproteinase meprin β, which cleaves APP predominantly at alanine in p2 and in this study we can detect an increased meprin β expression in AD brain. Here, we report the generation of the transgenic APP/lon mouse model of AD lacking the functional Mep1b gene (APP/lon × Mep1b−/−). We examined levels of canonical and truncated Aβ species using urea-SDS-PAGE, ELISA and immunohistochemistry in brains of APP/lon mouse × Mep1b−/−. Additionally, we investigated the cognitive abilities of these mice during the Morris water maze task. Aβ1-40 and 1–42 levels are reduced in APP/lon mice when meprin β is absent. Immunohistochemical staining of mouse brain sections revealed that N-terminally truncated Aβ2–x peptide deposition is decreased in APP/lon × Mep1b−/− mice. Importantly, loss of meprin β improved cognitive abilities and rescued learning behavior impairments in APP/lon mice. These observations indicate an important role of meprin β within the amyloidogenic pathway and Aβ production in vivo.

Atherosclerosis is the underlying cause of heart attack, ischemic stroke and peripheral arterial disease, and genetic factors involved remain mostly unidentified. We previously identified a significant locus on mouse chromosome 17 for atherosclerosis, Ath49, in an intercross between BALB/c and SM strains. Ath49 partially overlaps in the confidence interval with Ath22 mapped in an AKR × DBA/2 intercross. Bioinformatics analysis prioritized Mep1a, encoding meprin 1α metalloendopeptidase, as a likely candidate gene for Ath49. To prove causality, Mep1a−/−Apoe−/− mice were generated and compared with Mep1a+/+Apoe−/− mice for atherosclerosis development. Mep1a was found abundantly expressed in atherosclerotic lesions but not in healthy aorta and liver of mice. Mep1a−/− Apoe−/− mice exhibited significant reductions in both early and advanced lesion sizes. Loss of Mep1a led to decreased necrosis but increased macrophage and neutrophil contents in advanced lesions, reduced plasma levels of CXCL5 and an oxidative stress biomarker. In addition, Mep1a−/− mice had significantly reduced triglyceride levels on a chow diet. Thus, Mep1a is a susceptibility gene for atherosclerosis and aggravates atherosclerosis partially through action on oxidative stress and inflammation.

Alzheimer's disease (AD) is the most common form of dementia, however incurable so far. It is widely accepted that aggregated amyloid β (Aβ) peptides play a crucial role for the pathogenesis of AD, as they cause neurotoxicity and deposit as so-called Aβ plaques in AD patient brains. Aβ peptides derive from the amyloid precursor protein (APP) upon consecutive cleavage at the β- and γ-secretase site. Hence, mutations in the APP gene are often associated with autosomal dominant inherited AD. Almost thirty years ago, two mutations at the β-secretase site were observed in two Swedish families (termed Swedish APP (APPswe) mutations), which led to early-onset AD. Consequently, APPswe was established in almost every common AD mouse model, as it contributes to early Aβ plaque formation and cognitive impairments. Analyzing these APPswe-based mouse models, the aspartyl protease BACE1 has been evolving as the prominent β-secretase responsible for Aβ release in AD and as the most important therapeutic target for AD treatment. However, with respect to β-secretase processing, the very rare occurring APPswe variant substantially differs from wild-type APP. BACE1 dominates APPswe processing resulting in the release of Aβ1-x, whereas N-terminally truncated Aβ forms are scarcely generated. However, these N-terminally truncated Aβ species such as Aβ2-x, Aβ3-x and Aβ4-x are elevated in AD patient brains and exhibit an increased potential to aggregate compared to Aβ1-x peptides. Proteases such as meprin β, cathepsin B and ADAMTS4 were identified as alternative β-secretases being capable of generating these N-terminally truncated Aβ species from wild-type APP. However, neither meprin β nor cathepsin B are capable of generating N-terminally truncated Aβ peptides from APPswe. Hence, the role of BACE1 for the Aβ formation during AD might be overrepresented through the excessive use of APPswe mouse models. In this review we critically discuss the consideration of BACE1 as the most promising therapeutic target. Shifting the focus of AD research towards alternative β secretases might unveil promising alternatives to BACE1 inhibitors constantly failing in clinical trials due to ineffectiveness and harmful side effects.

Meprin β is a zinc-dependent metalloprotease exhibiting a unique cleavage specificity with strong preference for acidic amino acids at the cleavage site. Proteomic studies revealed a diverse substrate pool of meprin β including the interleukin-6 receptor (IL-6R) and the amyloid precursor protein (APP). Dysregulation of meprin β is often associated with pathological conditions such as chronic inflammation, fibrosis, or Alzheimer's disease (AD). The extracellular regulation of meprin β including interactors, sheddases, and activators has been intensively investigated while intracellular regulation has been barely addressed in the literature. This study aimed to analyze C-terminal phosphorylation of meprin β with regard to cell surface expression and proteolytic activity. By immunoprecipitation of endogenous meprin β from the colon cancer cell line Colo320 and subsequent LC-MS analysis, we identified several phosphorylation sites in its C-terminal region. Here, T694 in the C-terminus of meprin β was the most preferred residue after phorbol 12-myristate 13-acetate (PMA) stimulation. We further demonstrated the role of protein kinase C (PKC) isoforms for meprin β phosphorylation and identified the involvement of PKC-α and PKC-β. As a result of phosphorylation, the meprin β activity at the cell surface is reduced and, consequently, the extent of substrate cleavage is diminished. Our data indicate that this decrease of the surface activity is caused by the internalization and degradation of meprin β.

Meprin β is a membrane-bound metalloprotease involved in extracellular matrix assembly and inflammatory processes in health and disease. A disintegrin and metalloproteinase (ADAM)10 and ADAM17 are physiologic relevant sheddases of inactive promeprin β, which influences its substrate repertoire and subsequent biologic functions. Proteomic analysis also revealed several ADAMs as putative meprin β substrates. Here, we demonstrate specific N-terminal processing of ADAM9, 10, and 17 by meprin β and identify cleavage sites within their prodomains. Because ADAM prodomains can act as specific inhibitors, we postulate a role for meprin β in the regulation of ADAM activities. Indeed, prodomain cleavage by meprin β caused increased ADAM protease activities, as observed by peptide-based cleavage assays and demonstrated by increased ectodomain shedding activity. Direct interaction of meprin β and ADAM proteases could be shown by immunofluorescence microscopy and immunoprecipitation experiments. As demonstrated by a bacterial activator of meprin β and additional measurement of TNF-α shedding on bone marrow–derived macrophages, meprin β/ADAM protease interactions likely influence inflammatory conditions. Thus, we identified a novel proteolytic pathway of meprin β with ADAM proteases to control protease activities at the cell surface as part of the protease web.—Wichert, R., Scharfenberg, F., Colmorgen, C., Koudelka, T., Schwarz, J., Wetzel, S., Potempa, B., Potempa, J., Bartsch, J. W., Sagi, I., Tholey, A., Saftig, P., Rose-John, S., Becker-Pauly, C. Meprin β induces activities of A disintegrin and metalloproteinases 9, 10, and 17 by specific prodomain cleavage.

Alzheimer’s Disease (AD) is the sixth-leading cause of death in industrialized countries. Neurotoxic amyloid-β (Aβ) plaques are one of the pathological hallmarks in AD patient brains. Aβ accumulates in the brain upon sequential, proteolytic processing of the amyloid precursor protein (APP) by β- and γ-secretases. However, so far disease-modifying drugs targeting β- and γ-secretase pathways seeking a decrease in the production of toxic Aβ peptides have failed in clinics. It has been demonstrated that the metalloproteinase meprin β acts as an alternative β-secretase, capable of generating truncated Aβ2–x peptides that have been described to be increased in AD patients. This indicates an important β-site cleaving enzyme 1 (BACE-1)-independent contribution of the metalloprotease meprin β within the amyloidogenic pathway and may lead to novel drug targeting avenues. However, meprin β itself is embedded in a complex regulatory network. Remarkably, the anti-amyloidogenic α-secretase a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) is a direct competitor for APP at the cell surface, but also a sheddase of inactive pro-meprin β. Overall, we highlight the current cellular, molecular and structural understanding of meprin β as alternative β-secretase within the complex protease web, regulating APP processing in health and disease.

Meprin β 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 β, both exhibiting a single amino acid exchange, namely, meprin β G45R and G89R. Based on the structural data of meprin β 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 β, 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 β and its variants. In transfected HEK cells, the G89R variant was found to generate less soluble IL-6R, whereas the expression of meprin β G45R resulted in increased shedding of the IL-6R compared to wild-type meprin β and the G89R variant. A similar tendency of the induced shedding capacity of G45R was seen for the well-described meprin β substrate CD99. Furthermore, employing an assay for cell migration in a collagen IV matrix, we observed that the transfection of wild-type meprin β and the G45R variant resulted in increased migration of HeLa cells, while the G89R variant led to diminished mobility.

Metastasis is the major cause of death in cancer patients. Circulating tumor cells need to migrate through the endothelial layer of blood vessels to escape the hostile circulation and establish metastases at distant organ sites. Here, we identified the membrane-bound metalloprotease ADAM17 on endothelial cells as a key driver of metastasis. We show that TNFR1-dependent tumor cell–induced endothelial cell death, tumor cell extravasation, and subsequent metastatic seeding is dependent on the activity of endothelial ADAM17. Moreover, we reveal that ADAM17-mediated TNFR1 ectodomain shedding and subsequent processing by the γ-secretase complex is required for the induction of TNF-induced necroptosis. Consequently, genetic ablation of ADAM17 in endothelial cells as well as short-term pharmacological inhibition of ADAM17 prevents long-term metastases formation in the lung. Thus, our data identified ADAM17 as a novel essential regulator of necroptosis and as a new promising target for antimetastatic and advanced-stage cancer therapies.

Interleukin-6 (IL-6) signaling is a crucial regulatory event important for many biological functions, such as inflammation and tissue regeneration. Accordingly, several pathological conditions are associated with dysregulated IL-6 activity, making it an attractive therapeutic target. For instance, blockade of IL-6 or its &alpha, receptor (IL-6R) by monoclonal antibodies has been successfully used to treat rheumatoid arthritis. However, based on different signaling modes, IL-6 function varies between pro- and anti-inflammatory activity, which is critical for therapeutic intervention. So far, three modes of IL-6 signaling have been described, the classic anti-inflammatory signaling, as well as pro-inflammatory trans-signaling, and trans-presentation. The IL-6/IL-6R complex requires an additional &beta, receptor (gp130), which is expressed on almost all cells of the human body, to induce STAT3 (signal transducer and activator of signal transcription 3) phosphorylation and subsequent transcriptional regulation. In contrast, the IL-6R is expressed on a limited number of cells, including hepatocytes and immune cells. However, the proteolytic release of the IL-6R enables trans-signaling on cells expressing gp130 only. Here, we demonstrate a fourth possibility of IL-6 signaling that we termed joint reconstituted signaling (JRS). We show that IL-6R on extracellular vesicles (EVs) can also be transported to and fused with other cells that lack the IL-6R on their surface. Importantly, JRS via EVs induces delayed STAT3 phosphorylation compared to the well-established trans-signaling mode. EVs isolated from human serum were already shown to carry the IL-6R, and thus this new signaling mode should be considered with regard to signal intervention.

Mucus is covering the entire epithelium of the gastrointestinal tract (GIT), building the interface for the symbiosis between microorganisms and their host. Hence, a disrupted mucosal barrier or alterations of proper mucus composition, including the gut microbiota, can cause severe infection and inflammation. Meprin metalloproteases are well-known to cleave various pro-inflammatory molecules, contributing to the onset and progression of pathological conditions including sepsis, pulmonary hypertension or inflammatory bowel disease (IBD). Moreover, meprins have an impact on migration and infiltration of immune cells like monocytes or leukocytes during intestinal inflammation by cleaving tight junction proteins or cell adhesion molecules, thereby disrupting epithelial cell barrier and promoting transendothelial cell migration. Interestingly, both meprin α and meprin β are susceptibility genes for IBD. However, both genes are significantly downregulated in inflamed intestinal tissue in contrast to healthy donors. Therefore, a detailed understanding of the underlying molecular mechanisms is the basis for developing new and effective therapies against manifold pathologies like IBD. This review focuses on the regulation of meprin metalloproteases and its impact on physiological and pathological conditions related to mucosal homeostasis.

Pulmonary arterial hypertension (PAH) is a rare disease characterized by increased pulmonary pressure and vascular remodelling as a consequence of smooth muscle cell proliferation, endothelial cell dysfunction and inflammatory infiltrates. Meprin α is a metalloproteinase whose substrates include adhesion and cell–cell contact molecules involved in the process of immune cell extravasation. In this study, we aimed to unravel the role of meprin α in PAH-induced vascular remodelling. Our results showed that meprin α was present in the apical membrane of endothelial cells in the lungs and pulmonary arteries of donors and idiopathic PAH (IPAH) patients. Elevated circulating meprin α levels were detected in the plasma of IPAH patients. In vitro binding assays and electron microscopy confirmed binding of meprin α to the glycocalyx of human pulmonary artery endothelial cells (hPAECs). Enzymatic and genetic approaches identified heparan sulphate (HS) as an important determinant of the meprin α binding capacity to hPAEC. Meprin α treatment protected from excessive neutrophil infiltration and the protective effect observed in the presence of neutrophils was partially reversed by removal of HS from hPAEC. Importantly, HS levels in pulmonary arteries were decreased in IPAH patients and binding of meprin α to HS was impaired in IPAH hPAEC. In summary, our results suggest a role of HS in docking meprin α to the endothelium and thus in the modulation of inflammatory cell extravasation. In IPAH, the decreased endothelial HS results in the reduction of meprin α binding which might contribute to enhanced inflammatory cell extravasation and potentially to pathological vascular remodelling.

Highlights • Meprins subtly support epidermal and dermal skin wound healing. • Loss of both meprins reduces re-epithelialization and wound macrophage abundance. • Meprin α is a physiological maturing proteinase of collagen VII. • Meprins are reduced in recessive dystrophic epidermolysis bullosa skin., Astacin-like proteinases (ALPs) are regulators of tissue and extracellular matrix (ECM) homeostasis. They convey this property through their ability to convert ECM protein pro-forms to functional mature proteins and by regulating the bioavailability of growth factors that stimulate ECM synthesis. The most studied ALPs in this context are the BMP-1/tolloid-like proteinases. The other subclass of ALPs in vertebrates – the meprins, comprised of meprin α and meprin β – are emerging as regulators of tissue and ECM homeostasis but have so far been only limitedly investigated. Here, we functionally assessed the roles of meprins in skin wound healing using mice genetically deficient in one or both meprins. Meprin deficiency did not change the course of macroscopic wound closure. However, subtle but distinct contributions of meprins to the healing process and dermal homeostasis were observed. Loss of both meprins delayed re-epithelialization and reduced macrophage infiltration. Abnormal dermal healing and ECM regeneration was observed in meprin deficient wounds. Our analyses also revealed meprin α as one proteinase responsible for maturation of pro-collagen VII to anchoring fibril-forming-competent collagen VII in vivo. Collectively, our study identifies meprins as subtle players in skin wound healing.

// Tillmann Bedau 1 , Neele Schumacher 1 , Florian Peters 1 , Johannes Prox 1 , Philipp Arnold 2 , Tomas Koudelka 3 , Ole Helm 4 , Frederike Schmidt 1 , Bjorn Rabe 1 , Marlene Jentzsch 5 , Philip Rosenstiel 5 , Susanne Sebens 4 , Andreas Tholey 3 , Stefan Rose-John 1 and Christoph Becker-Pauly 1 1 Unit for Degradomics of the Protease Web, Institute of Biochemistry, University of Kiel, 24118 Kiel, Germany 2 Anatomical Institute, University of Kiel, 24118 Kiel, Germany 3 Institute of Experimental Medicine, University of Kiel, 24118 Kiel, Germany 4 Institute for Experimental Tumor Research, University of Kiel, 24118 Kiel, Germany 5 Institute of Clinical Molecular Biology, University of Kiel, 24118 Kiel, Germany Correspondence to: Christoph Becker-Pauly, email: cbeckerpauly@biochem.uni-kiel.de Keywords: CD99, meprin β, proteolytic shedding, lung cancer, inflammation Received: January 18, 2017 Accepted: June 17, 2017 Published: July 04, 2017 ABSTRACT 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 β promotes TEM in vitro . In this study, we employed an acute inflammation model (air pouch/carrageenan) and found significantly less infiltrated cells in meprin β knock-out animals validating the previously observed pro-inflammatory activity. To further analyze the impact of meprin β 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 β 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 β 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.

The two metalloproteases meprin β and bone morphogenetic protein 1 (BMP-1) are both members of the astacin protease family. They share specificity for negatively charged residues around the scissile bond and they are expressed in overlapping compartments of the human body. One important proteolytic substrate they share is pro-collagen I. Ablation of one of the two proteases however leads to different collagen I associated phenotypes in vivo. Over the last years calcium emerged as a regulator for the proteolytic activity of both enzymes. For meprin β a reduction and for BMP-1 an increase in activity was reported under increasing calcium concentrations. Here we revisit different compartments that rely on pro-collagen I maturation and explore the crystal structure of both proteases to highlight possible calcium binding sites. With this we aim to emphasize a to date underestimated regulator that influences both proteases.

A crucial step for tumor cell extravasation and metastasis is the migration through the extracellular matrix, which requires proteolytic activity. Hence, proteases, particularly matrix metalloproteases (MMPs), have been discussed as therapeutic targets and their inhibition should diminish tumor growth and metastasis. The metalloproteases meprin α and meprin β are highly abundant on intestinal enterocytes and their expression was associated with different stages of colorectal cancer. Due to their ability to cleave extracellular matrix (ECM) components, they were suggested as pro-tumorigenic enzymes. Additionally, both meprins were shown to have pro-inflammatory activity by cleaving cytokines and their receptors, which correlates with chronic intestinal inflammation and associated conditions. On the other hand, meprin β was identified as an essential enzyme for the detachment and renewal of the intestinal mucus, important to prevent bacterial overgrowth and infection. Considering this, it is hard to estimate whether high activity of meprins is generally detrimental or if these enzymes have also protective functions in certain cancer types. For instance, for colorectal cancer, patients with high meprin β expression in tumor tissue exhibit a better survival prognosis, which is completely different to prostate cancer. This demonstrates that the very same enzyme may have contrary effects on tumor initiation and growth, depending on its tissue and subcellular localization. Hence, precise knowledge about proteolytic enzymes is required to design the most efficient therapeutic options for cancer treatment. In this review, we summarize the current findings on meprins' functions, expression, and cancer-associated variants with possible implications for tumor progression and metastasis.

Alzheimer’s disease (AD) is the most common type of dementia and characterized by tau hyperphosphorylation, oxidative stress, reactive microglia and amyloid-β (Aβ) deposits. A recent study revealed that Porphyromonas gingivalis infection is associated with amyloid β generation in Alzheimer’s disease. Increased Aβ levels, tau degradation and neuronal toxicity were observed as a consequence of ginigipain R (RgpB) activity, a cysteine protease constitutively secreted by P. gingivalis. Of note, we previously identified RgpB as a potent activator of the metalloproteinase meprin β. Interestingly, meprin β is an alternative β-secretase of the amyloid precursor protein (APP), which together with the γ-secretase leads to the generation of aggregation-prone N-terminally truncated Aβ2-x peptides. Importantly, identification of a risk gene variant of meprin β (rs173032) for Alzheimer’s disease using whole-exome sequencing of the BDR cohort further supports the impact of this alternative β-secretase. Thus, we wondered if increased Aβ levels as a consequence of P. gingivalis colonization into the brain might be due to meprin β activation by RgpB. Here, we demonstrate that i) upon incubation with RgpB the proteolytic activity of meprin β at the cell surface of transfected HEK cells or of endogenously expressed enzyme in SH-SY5Y neuroblastoma cells was significantly increased, and that ii) RgpB-mediated increase in meprin β activity leads to massive generation of Aβ-peptides. In conclusion, our findings would further explain the pathogenesis of P. gingivalis in AD brain.