|Prof. Dr. Christoph Becker-Pauly
|Unit for Degradomics of the Protease Web
Becker-Pauly group 2023
|The Degradome: Proteolytic enzymes, substrates, inhibitors, and regulatory proteins in health and disease
|Proteolysis is an irreversible posttranslational modification that affects every single cell of an organism.Over the last decades proteolytic enzymes have been identified to be master switches in the regulation of the immune system, in neuronal development and neurodegeneration, and in apoptosis and cancer progression. Substrates define protease roles. Therefore, the identification of single enzymes, their substrates and inhibitors, the “protease web”, is crucial to understand complex molecular events responsible for pathophysiological conditions, and subsequently also important for drug development.
|Amongst all 588 human proteases, the metalloproteases meprin α and meprin β exhibit unique structural and functional properties. We discovered structural details of meprins that explain the molecular basis for the unique cleavage specificity with high preference for acidic amino acid residues in P1' position, reflected by native substrates.
|Oligomeric structure of human meprins
(A) Meprin α and meprin β are multi-domain enzymes, building dimers linked by one intermolecular disulphide bond between the MAM (meprin A5 protein tyrosine phosphatase µ-like) domains. Meprins are expressed as zymogens with a propeptide (PRO) N-terminal to the protease domain (CAT) that must be cleaved off proteolytically to gain full activity. Only meprin α contains an inserted domain (I) which is cleaved by furin during the secretory pathway, resulting in secretion of the protein and further oligomerisation. Meprin α is the largest secreted protease known, up to 6 mDa in size, as visualized by electron microscopy of purified recombinant enzyme. Meprin β predominantly remains membrane bound but can be shed from the cell surface by ectodomain shedding through ADAM10 and ADAM17 activity. (B) Crystal structure of the ectodomain of dimeric human meprin β in different orientations (pdb accession numbers 4GWN, 4GWM). One monomer is displayed with a transparent surface, revealing the ribbon structure. (C) Model of the membrane bound form of meprin β and its orientation at the cell surface. For structural details see (Arolas et al., 2012, PNAS)
|Cleavage specificity of meprin α and meprin β
(A-D) With the help of proteomics approaches based on peptide libraries and native substrates, the cleavage specificity of human meprins has been determined. Results are displayed in the WebLogo style, where the most preferred amino acid residues are shown for positions P6 to P6’. Both proteases prefer negatively charged amino acids at the P1’ position. For detailed information see (Becker-Pauly et al., 2012, Mol Cell Proteomics). (E) Incubation of fluorogenic substrates consisting of only aspartate and glutamate residues demonstrates the ability of meprin β to cleave completely acidic peptides. (F) The unique specificity of meprin β is based on structural features of the active site cleft. Positively charged arginine (Arg) residues (dark blue) can interact with negatively charged amino acid residues of the substrate.
|Proteomics analyses enabled the identification of more than 100 new meprin substrates and revealed a possible link to neurodegeneration regarding the release of Aβ peptides by meprin β (summarized in Scharfenberg et al., 2020; CMLS).
|Extracellular regulation of meprin β activity with respect to β-site cleavage of APP. Meprin β is expressed as zymogen at the cell surface. A disintegrin and metalloprotease 10 and 17 (ADAM10/17) act as sheddases of pro-meprin β. Shed meprin β can be activated by tryptic proteases. When activated as soluble protein, shed meprin β does not cleave at the β-site of APP. Alternatively, inactive meprin β can be maturated by the membrane-bound serine protease matriptase-2 (MT-2). Once activated at the cell surface membrane-bound meprin β cannot be shed any more and acts as β-secretase thus generating amyloid-β (Aβ). (Scharfenberg et al., 2020; CMLS).
|Extracellular regulation of meprin β activity with respect to β-site cleavage of APP. Model of a membrane-bound ADAM10 based on the ectodomain (yellow, PDB: 6BE6) and pro-meprin β (blue). The pro-peptide of meprin β is shown in orange. The red arrow indicates the shedding site within pro-meprin β. E1/2: extracellular domains 1/2 ; Acd: acidic domain; JMR: juxtamembrane region ; AICD: APP intracellular domain. (Scharfenberg et al., 2020; CMLS).
|Cleavage of pro-inflammatory cytokines by meprins and genetic studies demonstrated contribution of these proteases to the progression of inflammatory bowel disease. This was further validated in DSS induced colitis in meprin α and meprin β knockout mice. Importantly, meprin β was demonstrated to be responsible for mucus detachment in the intestine, an important physiological function to prevent bacterial overgrowth (Schütte et al., 2014; PNAS; Wichert et al., 2017; Cell Rep).
ADAM-mediated meprin β shedding is required for mucus detachment, thereby regulating intestinal integrity.
Wichert et al. demonstrated that meprin β is exclusively shed in its pro-form. Activation of meprin β by
serine protease MT-2 or bacterial virulence factor RgpB abrogates its shedding, resulting in disturbed mucus
|Importantly, we discovered meprin α and meprin β as procollagen proteinases, capable of cleaving off the globular C- and N-terminal prodomains of fibrillar collagen type I and type III (Broder et al., 2013, PNAS; Kronenberg et al., 2010, J Invest Dermatol). This proteolytic process is sufficient to induce collagen fibril assembly as visualized by transmission electron microscopy. The biological relevance was demonstrated with the help of meprin α and meprin β knock-out mice, which exhibit decreased collagen deposition in skin resulting in impaired tensile strength. On the other hand, overexpression of meprin metalloproteases was found under fibrotic conditions in skin (keloids) and lung (pulmonary hypertension).
|Procollagen maturation by meprin metalloproteases. Meprins are secreted as zymogens and are activated by trypsin-like serine proteases (e.g. human kallikrein related peptidases, KLK). Meprin β is intrinsically membrane bound and can be shed from the cell surface by ADAM10/17. Several components of the ECM have been described as meprin substrates. These include, for example, MMP1, which is inactivated by meprins, or nidogen and fibronectin, which were shown to be cleaved in vitro. For procollagen type 1 it was demonstrated that cleavage of the prodomain leads to spontaneous fibril formation. Meprin deficient mice show a reduced tensile strength pointing towards an important in vivo function in collagen deposition. Left box shows the domain structure of meprin α and meprin β. Both consist of a propeptide (PRO), a catalytic domain (CAT), a MAM (meprin A5 protein tyrosine phosphatase μ) domain, a TRAF (tumour-necrosis-factor-receptor-associated factor) domain, an EGF (epidermal growth factor) like domain, a transmembrane region and a C-terminal part. Additionally, there is a so called inserted (I) domain found in meprin α between the TRAF and the EGF like domain. This inserted domain is cleaved by furin resulting in secretion into extracellular space. Meprin α forms large oligomers up to 6.4 MDa through a yet unknown oligomerisation site. This makes it the largest secreted protease known as depicted in the transmission electron microscopy image. (Arnold et al., 2014, Matrix Biology)
|Thus, regulation of meprin activity by specific inhibition to reduce collagen maturation might be a suitable approach for the treatment of certain pathological conditions. Further studies, including appropriate animal models, will elucidate the precise molecular pathways mediated by meprin α and meprin β activity in health and disease. The generation of specific meprin inhibitors is a crucial goal to develop potential therapeutics for the treatment of meprin-associated pathologies.
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|Letzte Änderung/Last change: Dezember 6, 2023