|Prof. Dr. Christoph Becker-Pauly|
|Unit for Degradomics of the Protease Web|
|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.|
|1A) Oligomeric structure of human meprins||1B) Cleavage specificity of meprin a and meprin ß|
|Click on an image for a larger representation|
|Figure 1A: 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).|
|Figure 1B: Cleavage specifity 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.|
2) Proteolytic interactions responsible for the shedding of APP by meprin β
(Click on image for a larger representation)
|Figure 2: Proteolytic interactions responsible for the shedding of APP by meprin β|
|The cartoon summarizes recent findings showing that meprin β generates Aβ peptides (Bien et al., 2012, J Biol Chem; Schönher et al., 2016, Mol Neurodeg) 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.|
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). 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.
| 3) De novo fibrillogenesis of type I collagen
after cleavage by meprin α or meprin β / In vivo analysis of collagen I maturation, deposition and mechanical strength in skin of wt, meprin α, and meprin β knockout mice
(Click on image for a larger representation)
|Figure 3: De novo fibrillogenesis of type I collagen after cleavage by meprin α or meprin β / In vivo analysis of collagen I maturation, deposition and mechanical strength in skin of wt, meprin α, and meprin β knockout mice|
De novo fibrillogenesis of type I collagen after cleavage by meprin α or meprin β.
(A) Transmission electron micrographs of negatively stained collagen fibrils assembled after cleavage of recombinant procollagen type I heterotrimer by meprin α and meprin β. 100 µg/ml of recombinant procollagen I was incubated in reaction buffer with either 15 nM BMP-1 (+ PCPE-1 equimolar to the substrate), meprin α or meprin β in a total volume of 10 µl at 37°C for 60 min. As a control, untreated recombinant procollagen I was visualized. (B) Cartoon summarizes procollagen processing by different proteases and subsequent assembly of collagen fibrils.
In vivo analysis of collagen I maturation, deposition, and mechanical strength in skin of wt, meprin α, and meprin β knockout mice.
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