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Prof. Dr. Christoph Becker-Pauly

Christoph Becker-Pauly
Christoph Becker-Pauly
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Unit for Degradomics of the Protease Web
Research
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.
 
Meprin Scheme 1 Meprin Scheme 2
Oligomeric structure of human meprins Cleavage specificity of meprin a and meprin ß
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Figure 1A and 1B: 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. 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.
 
Scheme - Shedding of APP
Proteolytic interactions responsible for the shedding of APP by meprin β
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The cartoon summarizes recent findings showing that meprin β generates Aβ peptides (Bien et al., 2012, J Biol Chem) and specific N-APP fragments (Jefferson et al., 2011, J Biol Chem). We further identified activation of ADAM10, the constitutive α-secretase, by meprin β (Jefferson et al., 2013, Cell Mol Life Sci). On the other hand, shedding by ADAM10 leads to soluble meprin β, which is no longer capable of cleaving at the β-secretase site, but still releases N-APP fragments that lack neurotoxic capacity and are rather protective against death receptor 6 (DR6) mediated neuronal death. Human tissue kallikrein-related peptidases (KLK) are activators of meprin β.
 
The knockdown of meprin expression in zebrafish revealed important functions of these enzymes during embryonic development. Meprin α, for instance, was shown to be a pro-angiogenic enzyme, and therefore might contribute to tumor progression in colon carcinoma, where the protease is highly expressed.
 
Scheme 3 - Meprin
Meprin α promotes blood vessel formation
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To analyse the biological function of meprins, morpholino knockdowns of meprin α1, meprin α2, and meprin β were done in zebrafish embryos. Microangiography with fluorogenic TRITC-Dextran revealed severe defects in the formation of blood vessels when meprin α2 was reduced. Compared to wild type (WT) embryos, meprin α2 morphants (MO) showed nearly complete absence of the head vascular system and the posterior cardinal vein (PCV), indicating a pro-angiogenic function of this protease. PCeV: posterior cerebral vein; ISV: intersegmental vessels; DLAV: dorsal longitudinal anastomotic vessel; CA: caudal artery; DA: dorsal aorta CV: caudal vein; PCV: posterior cardinal vein; L: liver; H: heart; hpf: hours post fertilization; scale bar: 250 µm. (For detailed information see (Schütte et al., 2010, PLoS One).
 
Scheme 4 - Meprin
Activity of human meprin α and meprin β in skin homeostasis and fibrosis
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(A) Meprin α and meprin β are expressed in separate layers of human epidermis, in the stratum basale and the s. granulosum, respectively. Meprin β was shown to be involved in terminal differentiation of keratinocytes, whereas meprin α promotes cell proliferation by activating the EGF receptor. A proteomics screen identified desmogleins as substrates of meprin β (Jefferson et al., 2013, Cell Mol Life Sci), adhesion molecules that are cleaved during desquamation. Immunogold staining of human epidermis revealed co-localization of meprin β (black dots) and desmoglein containing desmosomes (white arrows). Red arrow indicates possible degradation of desmosomal proteins by meprin β. (B) In the dermis both meprins are overexpressed in fibroblasts of fibrotic skin, as demonstrated by immunofluorescence microscopy (Kronenberg et al., 2010, J Invest Dermatol). Here, meprins might contribute to the formation of fibrillar collagen. In vitro it was shown that meprins can cleave off the N- and C-propeptides of procollagen III, thereby releasing mature triple helical collagen.
 
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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