Functional interplays of proteins and proteases: AD13-VWF, TGFβ2-α2M, and the proteolysis of gliadin by neprosin

Abstract

[eng] The present thesis has established a new and state of the art mammalian protein expression system at the laboratory which has then contributed with high-yield expression for the purification and crystallization of three glycoproteins, namely TGFβ2, ADAMTS13 and Neprosin, which adopt important biomedical roles in human physiology, cardiovascular disorders, and celiac disease treatment, respectively.

Proteases are major players in the physiology and pathology of all living organisms. Their often highly sophisticated regulation mechanisms are therefore essential for proper function, and to prevent misdirected spatial and/or temporal proteolytic activity, which in turn may trigger disease. This regulation is achieved through a wide variety of mechanisms including protein-protein interaction, substrate allosteric activation, or their biosynthesis as inactive precursor, so called zymogens. In the present thesis a variety of regulatory mechanisms of proteases and inhibitors were studied by using a combination of biochemical, biophysical and structural techniques.

In the first project we comprehensively illuminate the biochemistry of the VWF:ADAMTS-13 axis. ADAMTS13 regulates the multimeric form of Von Willebrand Factor (VWF) in blood circulation by specific, shear-dependent proteolysis. Despite circulating in a seemingly latent form with a very long active plasma half-life, ADAMTS13 is resistant to plasma inhibitors and VWF is the only known substrate, and which involves an allosteric activation of ADAMTS13 by substrate binding. This unprecedented specificity has been attributed to extensive exosite interactions between the AD13-MDTCS domains, where the Disintegrin-like domain (D) approaches to Metalloprotease (M), Cysteine-rich (C), and Spacer (S) domain once the VWF is bound achieving a “tight” conformation. Structural analysis in solution revealed that the enzyme adopts a highly flexible unbound, latent structures and VWF peptide-bound, active structures that significantly differes from the AD13-MDTCS crystal structure. We integrate the experimental results with computational approaches. Thus, we hypothesize that the interaction culminates in a ‘fuzzy complex’ that follows a ‘dynamic zipper’ mechanism involving numerous reversible, weak but additive interactions that result in strong binding and ultimately in cleavage.

In the second project a novel family of glutamate peptidases was studied and the crystal structure determined of both the active and zymogen form, establishing the active site, catalytic mechanism and protease class annotation, and describing a unique catalytic dyad composed by two glutamates while showing a close structural homology with eqolisins. Neprosin was recently discovered in the fluid of the carnivorous pitcher plant Nepenthes ventrata. Given its high activity at low pH, thermic stability and great specificity, neprosin is especially useful in detoxifying gluten proteins including wheat gliadin and the 33-mer immunogenic fragment into non-toxic peptides in a human stomach environment. Additionally, we determined its inhibitory profile against a cohort of peptidase inhibitors. Future approaches will study the effect of neprosin processing of the 33-mer on inflammatory responses in cells and tissue explants to elucidate if neprosin indeed presents an effective and harmless supplementation approach for gluten intolerant patients.

In the third project the main protease inhibitor of the blood plasma has been studied; human α2 Macroglobulin (hα2Ms) has a great role on the human physiology by modulating the activity of cytokines such as TGFβ2. We characterised the interaction by a variety of biochemical, biophysical, and binding techniques. Intriguingly, during our crystallographic studies of the complex, we obtained a crystal structure of a TGFβ2 dimer in a novel dimeric assembly of biological unknown function.

Overall, the present thesis contributes substantially to the field of structural biochemistry, expanding previous knowledge at the molecular level and adding new regulatory mechanisms, which will hopefully pave the way for the design of specific drugs for therapeutic interventions as well as lead to new approaches for treating coeliac disease through an enzyme supplementation strategy.