Structural and functional analysis of natural protein-based inhibitors and their protease targets
[eng] Several proteases and their antagonistic protein or peptide inhibitors sparked researchers’ interest due to their potential as therapeutic targets. The present thesis, which compiles results obtained in three independent projects, unravels new information regarding protease and protease inhibi...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2022 |
| País: | España |
| Institución: | Universidad de Barcelona |
| Repositorio: | Dipòsit Digital de la UB |
| OAI Identifier: | oai:diposit.ub.edu:2445/191298 |
| Acceso en línea: | https://hdl.handle.net/2445/191298 http://hdl.handle.net/10803/687191 |
| Access Level: | acceso abierto |
| Palabra clave: | Bioquímica Enzims proteolítics Inhibidors enzimàtics Proteòmica Biochemistry Proteolytic enzymes Enzyme inhibitors Proteomics |
| Sumario: | [eng] Several proteases and their antagonistic protein or peptide inhibitors sparked researchers’ interest due to their potential as therapeutic targets. The present thesis, which compiles results obtained in three independent projects, unravels new information regarding protease and protease inhibitor structures and their mechanisms of action. In the first project, we assessed the involvement of human reversion-inducing cysteine-rich protein with Kazal motifs (RECK) in embryogenesis and tumour suppression by investigating its involvement in the regulation of matrix metalloproteinases (MMPs). We developed bacterial and eukaryotic expression systems for the production of RECK variants and established an exhaustive purification protocol. The inhibitory activity of RECK variants towards MMP-2, MMP-7, MMP-9, and MMP-14 catalytic domain was assessed using fluorogenic peptides and natural protein substrates. Contrary to the published literature, no significant MMP inhibition was detected, suggesting that RECK is not a direct inhibitor of MMPs activity (Mendes et al., 2020). In the second project, we aimed to develop a specific and potent inhibitor of aureolysin, a protease and key virulence factor secreted by the human pathogen Staphylococcus aureus. The insect metallopeptidase inhibitor (IMPI) is a defensive molecule produced by the greater wax moth Galleria mellonella, which effectively and potently inhibits thermolysins from some pathogenic bacteria. In the thesis we evaluated IMPI inhibition of aureolysin activity in vitro and explored its mechanism of action through analysis of the crystallographic structure of the complex. Furthermore, in an attempt to obtain a more effective or specific aureolysin inhibitor, we designed a set of twelve mutants displaying single or multiple point mutations in the reactive-centre loop. The best aureolysin inhibition was achieved by the I57F mutant, with a calculated inhibition constant (Ki) of 346 nM. The work presented here provides extra input for the development of therapeutic proteins and peptide-based inhibitors based on IMPI and its inactivation mechanism for the treatment of infections caused by antibiotic resistant pathogens (Mendes et al., 2022). In the third project, a highly collaborative work was developed in order to explore the unique inhibitory mechanism of human plasma ⍺2M (h⍺2M), a homotetrameric protein of approximately 720 kDa, present in high concentrations in human plasma that performs several functions including the inhibition of peptidases. In this project we obtained eight ⍺2M structures by cryo electron microscopy (cryo-EM) elucidating the previously theoretical “Venus fly-trap” mechanism which allows the non- specific inactivation of up to two protease molecules independently of their catalytic type (Luque et al., 2022). Sparked by remarks during the review process of this paper, we then compared the function and biophysical properties of h⍺2M purified from fresh plasma with that of thawed frozen plasma through a range of experiments, providing light on this for the very first time, and demonstrating, that both h⍺2M preparations are indistinguishable (Mendes et al., in preparation). |
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